Listeria Listeriosis and Food Safety Third Edition

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Cover photo: An immunofluorescent image of L. monocytogenes (red) showing cell-to-cell spread via polymerized actin
tails (green). Photo courtesy of Dr. Pascale Cossart, Institut Pasteur, Paris.
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Library of Congress Cataloging-in-Publication Data
Listeria, listeriosis, and food safety / editors, Elliot T. Ryser and Elmer H. Marth. -- 3rd ed.
p. ; cm. -- (Food science and technology ; 160)
Includes bibliographical references and index.
ISBN-13: 978-0-8247-5750-2 (hardcover : alk. paper)
ISBN-10: 1-4200-1518-4 (hardcover : alk. paper)
1. Listeriosis. 2. Listeria monocytogenes 3. Foodborne diseases. 4. Food--Microbiology. I. Ryser,
Elliot T., 1957- II. Marth, Elmer H. III. Title. IV. Series: Food science and technology (Taylor & Francis)
; 160.
[DNLM: 1. Listeria Infections--etiology. 2. Food Contamination--prevention & control. 3. Food
Microbiology. 4. Listeria monocytogenes--pathogenicity. W1 FO509P v.160 2007 / WC 242 L773
2007]
QR201.L7R9 2007
615.9’52937--dc22 2007000412
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IN MEMORIAM
After conceiving the idea for a third edition of Listeria, Listeriosis and Food Safety in June 2002,
Elmer Marth and I were in biweekly contact by phone to ensure continued progress toward its
completion. This daunting project involved 33 contributors, 3 of whom in addition to myself (Robert
E. Brackett, Jeffrey L. Kornacki, and Ahmed E. Yousef) earned their doctorates in the laboratory
of Elmer Marth. As the book was rapidly nearing completion in Spring 2006, I was deeply saddened
to learn that Elmer had become seriously ill. On June 19, 2006, Emeritus Professor Elmer H. Marth
died in Madison, Wisconsin at age 78 while I was editing the chapter proofs for the book.
Consequently, it is only fitting that the third edition of Listeria, Listeriosis and Food Safety be
dedicated in memory of Dr. Elmer H. Marth who grew up on a small dairy farm in Grafton,
Wisconsin to become one of the most preeminent dairy microbiologists of our time.
Elliot T. Ryser
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Preface to the Third Edition

Since the second edition of Listeria, Listeriosis and Food Safety was published in 1999, the United
States has seen a 40% decrease in the incidence of listeriosis, with the current annual rate of illness
rapidly approaching the 2010 target of 2.5 cases per million population. This reduction in the rate
of listeriosis would not have been possible without the combined effort of researchers, academicians,
commodity organizations, and various regulatory authorities. We would like to believe that the first
and second editions of our book also played a role in this outcome by providing needed information
to concerned persons.
Despite considerable progress in understanding the sources, spread, control, and pathogenicity
of Listeria monocytogenes, research on this foodborne pathogen has continued unabated, with more
than 5,000 publications on Listeria and foodborne listeriosis appearing in the scientific literature
since the second edition of this book was published. A portion of this work was fueled by a series
of widely publicized outbreaks of listeriosis involving ready-to-eat meat products in the United
States that began in the late 1990s. Over the last 5 years, increasing emphasis has been given to
development of risk assessments that can be used to focus limited financial resources on certain
high-risk foods, such as soft cheeses, ready-to-eat meats, and smoked fish, that support growth of
the pathogen. Efforts ultimately have had an impact on public policies regarding allowable levels
of L. monocytogenes in these and other foods.
The third edition of Listeria, Listeriosis and Food Safety again summarizes much of the newly
published literature and integrates this information with earlier knowledge to present readers with
a complete and current overview of foodborne listeriosis. The 17 chapters in the second edition
have been retained; all are updated and expanded as appropriate. A total of 33 authors have lent
their expertise to preparing this book, with new authors contributing to Chapters 1, 2, 4, 6, 7, 8,
10, 13, and 17. Sometimes these authors have collaborated with the original authors to develop the
revised chapter. Two new chapters, Chapter 18 and Chapter 19, have been added to the book.
Chapter 18 deals with risk assessment, cost of foodborne listeriosis outbreaks, and regulatory control
of the Listeria problem in various countries. In Chapter 19, four experts point out specific data
gaps and where, in their view, research efforts should be directed.
As was true for the earlier editions, this book will be useful to advanced undergraduate students
in food science, microbiology, and public health; graduate students in these same disciplines; and
practitioners in food or dairy microbiology, food or dairy science, bacteriology or microbiology,
public health, epidemiology, risk assessment, meat science, animal science, and veterinary medicine.
It will also be helpful to personnel in the food and dairy industries and the food service industry,
and to researchers in industrial, governmental, and university laboratories.
Elliot T. Ryser
Elmer H. Marth
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Preface to the Second Edition

Listeriosis and Listeria monocytogenes continue to be of worldwide interest to the food industry
and regulatory agencies, scientists in various disciplines, and consumers of food. Such interest is
prompted by the occasional appearance of L. monocytogenes in ready-to-eat foods, leading to the
removal of these products from the marketplace. Furthermore, sporadic cases of listeriosis continue
to occur and several food-associated outbreaks of the disease have occurred since the first edition
of this book was published.
Scientists in several disciplines are still studying different aspects of the listeriosis problem.
Their efforts have resulted in the development of much new information that has appeared in
hundreds, if not thousands, of papers published since the first edition of this book was completed
in 1990. This explosion of information warranted publication of a second edition.
The second edition differs markedly from the 1991 edition. Whereas we were the authors of
the earlier edition, chapters in this edition have been prepared by various experts in the field. We
now serve as editors, although one of us (ETR) has revised several chapters. The contributions of
this edition’s authors have resulted in an improved book that contains timely topics.
The chapters in the first edition have been retained; each has been revised and expanded with
new information when appropriate. Two new chapters deal with typing methods and pathogenesis.
Thus, this book contains 17 chapters addressing the following topics:
description of L. monocytogenes
occurrence and behavior of this pathogen in various natural environments
animal and human listeriosis
pathogenesis of L. monocytogenes
characteristics of L. monocytogenes important to food processors
conventional and rapid methods to isolate, detect, and identify L. monocytogenes
strain-specific typing of L. monocytogenes
foodborne listeriosis
incidence of behavior of L. monocytogenes in unfermented and fermented dairy products,
meat, poultry (including eggs), fish and seafood, and products of plant origin
incidence and control of this pathogen within various types of food-processing facilities
This book will be useful to advanced undergraduate students, graduate students, and practitio-
ners in food or dairy microbiology, food or dairy science, bacteriology or microbiology, public
health, dietetics, meat science, poultry science, and veterinary medicine. It also will be helpful to
personnel in the food and dairy industries and regulatory agencies, as well as researchers in
industrial, governmental, and university laboratories.
Elliot T. Ryser
Elmer H. Marth
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Preface to the First Edition

Interest in the occurrence of Listeria in food, particularly Listeria monocytogenes, escalated rapidly
during the 1980s and continues unabated as a result of several major outbreaks of foodborne
listeriosis. The first of these occurred during 1981 and involved consumption of contaminated
coleslaw. In 1983, the reputation of the American dairy industry for producing safe products suffered
when epidemiological evidence showed that 14 of 49 people in Massachusetts died after consuming
pasteurized milk that was supposedly contaminated with L. monocytogenes. Two years later,
consumption of contaminated Mexican-style cheese manufactured in California was directly linked
to more than 142 cases of listeriosis, including at least 40 deaths.
Heightened public concern regarding the prevalence of L. monocytogenes in food prompted
the United States Food and Drug Administration to initiate a series of Listeria surveillance programs.
Subsequent discovery of this pathogen in many varieties of domestic and imported cheese, in ice
cream, and in other dairy products prompted numerous product recalls, which in turn have led to
staggering financial losses for the industry, including several lawsuits. These listeriosis outbreaks,
together with a subsequent epidemic in Switzerland involving consumption of Vacherin Mont d’Or
soft-ripened cheese and discovery of L. monocytogenes in raw and ready-to-eat meat, poultry,
seafood, and vegetables, have underscored the need for additional information concerning foodborne
listeriosis.
In 1961, Professor H. P. R. Seeliger, now retired from the University of Würzburg, published
his time-honored book, Listeriosis. His monograph has provided scientists, veterinarians, and the
medical profession with much needed information regarding Listeria and human and animal
listeriosis, as well as pathological, bacteriological, and serological methods to diagnose this disease.
However, documented cases of foodborne listeriosis were virtually unknown 30 years ago. Although
much information in his book is still valid today, some of the knowledge regarding media and
methods used to isolate, detect, and identify L. monocytogenes in clinical and, particularly, non-
clinical specimens is now largely out of date.
The emergence of L. monocytogenes as a serious foodborne pathogen together with the virtual
flood of Listeria-related papers that have appeared in scientific and trade journals as well as
numerous conference proceedings, prompted us to review and summarize the current information
so that food industry personnel, public health and regulatory officials, food microbiologists, veter-
inarians, and academicians have a ready source of information regarding this now fully emerged
foodborne pathogen.
This book consists of 15 chapters that address the following topics:
L. monocytogenes as the causative agent of listeriosis

occurrence and survival of this pathogen in various natural environments
human and animal listeriosis
characteristics of L. monocytogenes important to food processors
conventional and rapid methods for isolating, detecting, and identifying L. monocytogenes
in food
recognition of cases and outbreaks of foodborne listeriosis
incidence and behavior of L. monocytogenes in fermented and unfermented dairy products,
meat, poultry (including eggs), seafood, and products of plant origin
incidence and control of this pathogen within various types of food-processing facilities
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It is evident that major emphasis has been given to information directly applicable to food
processors. Information concerning the bacterium and the disease has been admirably reviewed by
Professor Seeliger and others, so our discussion of these topics should not be considered exhaustive.
Thus, the first four chapters of this book supply only pertinent background information to comple-
ment our discussion of foodborne listeriosis.
Although many in the scientific community must be commended for the extraordinary progress
made since 1985 toward understanding foodborne listeriosis, the continuing “explosion” of infor-
mation concerning Listeria and foodborne listeriosis has made the 3-year task of compiling an up-
to-date review of this subject quite difficult. Therefore, to produce as current a document as possible,
we have included a bibliography of references that have appeared since the book was completed.
We acknowledge with gratitude the many investigators whose findings made this book necessary
and possible. Special thanks go to individuals who shared unpublished information with us so that
we could make the book as up to date as possible. Our thanks also go to the scientists who provided
photographs or drawings; each person is acknowledged when the appropriate figure appears in the
book. We thank Barbara Kamp, Pat Gustafson, Beverly Scullion, and Judy Grudzina for typing
various parts of the manuscript. Illustrations were prepared by Jennifer Blitz and Suzanne Smith;
their help is acknowledged and appreciated. Special thanks to Dr. Ralston B. Read, Jr., former
director of the Microbiology Division of the Food and Drug Administration and now deceased,
who in 1984 encouraged development of a research program on foodborne Listeria at the University
of Wisconsin–Madison, and to Dr. Joseph A, O’Donnell, formerly with Dairy Research, Inc. and
now director of the California Dairy Foods Research Center, for his early interest in and support
of research on behavior of L. monocytogenes in dairy foods.
Research done in the Department of Food Science at the University of Wisconsin-Madison and
described in this book was supported by the U.S. Food and Drug Administration; National Cheese
Institute; the National Dairy Promotion and Research Board; the Wisconsin Milk Marketing Board;
Kraft, Inc.; Carlin Foods; Chr. Hansen’s Laboratory, Inc.; the Aristotelian University of Thessal-
oniki, Greece; the Cultural and Educational Bureau of the Egyptian Embassy in the United States;
the Malaysian Agricultural Research and Development Institute; the Korean Professors Fund; and
the College of Agricultural and Life Sciences, the Center for Dairy Research, and the Food Research
Institute—all of the University of Wisconsin. We thank these agencies for their interest in and
support of research on L. monocytogenes.
Our book is dedicated to all persons who have contributed to a better understanding of foodborne
listeriosis so that control of this disease is facilitated.
Elliot T. Ryser
Elmer H. Marth
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Editors
Elmer H. Marth, Ph.D., (1927–2006), a native of Jackson, Wisconsin, was emeritus professor of
food science and bacteriology at the University of Wisconsin–Madison. He earned his B.S. (1950),
M.S. (1952), and Ph.D. (1954) from the University of Wisconson–Madison in bacteriology with
an emphasis on food and dairy bacteriology. After 3 years as instructor of bacteriology, he joined
the R&D Division of Kraft Foods in Geneva, Illinois, in 1957. He rose through the ranks and in
1966 was named associate manager of the microbiology laboratory. Also in 1966, Dr. Marth returned
to the University of Wisconsin–Madison as associate professor of food science with joint appoint-
ments in bacteriology and food microbiology and toxicology. He was promoted to professor in
1971 and, upon retirement in 1990, was named emeritus professor. In 1981, Dr. Marth was a visiting
professor at the Swiss Federal Institute of Technology in Zürich. From 1967 to 1987, he was editor
of the Journal of Food Protection.
At the University of Wisconsin–Madison, Dr. Marth taught courses in food sanitation, food
fermentations, farm bacteriology, and writing scientific reports; he lectured in seven other courses
and in five short courses. His research program included studies on food spoilage, food fermentation,
and foodborne disease organisms, including Listeria monocytogenes. During his career, he was the
author, co-author, editor, or co-editor of more than 660 scientific publications, including research
papers, review papers, books, chapters in books, patents, and abstracts of papers given at meetings
of professional organizations. Dr. Marth served as major professor for 32 students who received
M.S. degrees and 32 students who earned Ph.D. degrees; in addition, he supervised 17 postdoctoral
researchers who worked in his laboratory.
Dr. Marth was named a fellow of the Institute of Food Technologists (IFT) (1983), the Inter-
national Association for Food Protection (IAFP) (1998), and the American Dairy Science Associ-
ation (ADSA) (1998). He was also a member of the American Society for Microbiology and the
Council of Science Editors. From the ADSA he received Pfizer (1975), Dairy Research Foundation
(1980), Borden (1986), and Kraft Teaching (1988) awards. The IAFP honored him with Educator
(1977), Citation (1984), Honorary Life Member (1987), and NFPA Food Safety (2000) awards.
The IFT presented him with the Nicolas Appert (1987) and Babcock–Hart (1989) awards. In 2002,
the Institute for Scientific Information designated Dr. Marth as a highly cited researcher, worldwide,
in the agricultural sciences. The National Cheese Institute presented its highest honor, the Laureate
Award, to Dr. Marth in 2004.
Elliot T. Ryser, Ph.D., a native of Milwaukee, Wisconsin, is an associate professor in the Depart-
ment of Food Science and Human Nutrition and the National Food Safety and Toxicology Center
at Michigan State University. He earned his B.S. (1979) in biology from Carroll College, Waukesha,
Wisconsin, and his B.S. (1980) in bacteriology, and M.S. (1982) and Ph.D. (1990) in food science,
from the University of Wisconsin–Madison, with an emphasis on microbial safety of food and dairy
products. Following a 1-year appointment as a research scientist at Institute National de la Recherche
Agronomique, Station de la Recherches Laitieres, Jouy-en-Josas, France, Dr. Ryser joined Silliker
Laboratories Group, Inc. in Chicago Heights, Illinois, where he worked for 2 years as a research
project manager. In 1994 he left Silliker and began his academic career as a research associate in
the Department of Animal and Food Sciences at the University of Vermont, working in the laboratory
of Dr. Catherine Donnelly. Dr. Ryser joined Michigan State University as an assistant professor in
1998 and was promoted to associate professor in 2004. He teaches courses on foodborne diseases,
food safety, and HACCP.
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Dr. Ryser’s research program is focused on the incidence, survival, transfer, and eradication of
Listeria monocytogenes and other foodborne pathogens from various foods, including dairy prod-
ucts. He has authored, co-authored, or co-edited more than 140 scientific publications, including
research papers, review papers, books, chapters in books, patents, and abstracts of work presented
at professional meetings. He has served thus far as the major professor for four Ph.D. and six M.S.
students and supervised the work of four postdoctoral researchers. Dr. Ryser is a member of the
International Association for Food Protection, Institute of Food Technologists, American Society
for Microbiology, and American Dairy Science Association. He received the National Milk Pro-
ducers Federation Richard M. Hoyt Award in 1988. In addition, he served as a scientific editor for
the Journal of Food Science from 2000 to 2005 and is currently a scientific editor for the Journal
of Food Protection.
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Contributors
Robert E. Brackett Werner Goebel
Centers for Food Safety and Applied Nutrition Department of Microbiology
U.S. Food and Drug Administration University of Würzburg
College Park, Maryland Würzburg, Germany
Lewis M. Graves
Christopher R. Braden
Enteric Diseases Laboratory Response Branch
Enteric Diseases Epidemiology Branch
Division of Foodborne, Bacterial and Mycotic
Division of Foodborne, Bacterial and Mycotic
Diseases
Diseases
National Center for Zoonotic, Vectorborne and
National Center for Zoonotic, Vectorborne and
Enteric Diseases
Enteric Diseases
Centers for Disease Control and Prevention
Centers for Disease Control and Prevention
Atlanta, Georgia
Atlanta, Georgia
Joshua Gurtler
Byron F. Brehm-Stecher Department of Food Science
Department of Food Science and Human University of Georgia
Nutrition Griffin, Georgia
Iowa State University
Ames, Iowa Susan B. Hunter
Coordinating Center for Infectious Diseases
Carmen Buchrieser Centers for Disease Control and Prevention
Laboratoire de Genomique des Atlanta, Georgia
Microorganismes Pathogenes
Karen C. Jinneman
Institut Pasteur
Seafood Products Research Center, Pacific
Paris, France
Regional Laboratory—Northwest
Office of Regulatory Affairs
Catherine W. Donnelly U.S. Food and Drug Administration
Department of Nutrition and Food Science Bothell, Washington
University of Vermont
Burlington, Vermont Eric A. Johnson
Departments of Food Microbiology and
Mel W. Eklund Toxicology and Bacteriology
U.S. National Marine Fisheries Service (retired) University of Wisconsin–Madison
Mel Eklund and Associates, Inc. Madison, Wisconsin
Seattle, Washington
Sophia Kathariou
Department of Food Science
Jeffrey M. Farber
North Carolina State University
Microbiology Research Division
Raleigh, North Carolina
Bureau of Microbial Hazards
Food Directorate
Health Canada Jeffrey L. Kornacki
Banting Research Centre Kornacki Microbiology Solutions, LLC
Ottawa, Ontario, Canada McFarland, Wisconsin
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Michael Kuhn Brian D. Sauders

Biozentrum Department of Food Science
University of Würzburg Cornell University
Würzburg, Germany Ithaca, New York
Beatrice H. Lado Chris Scherf

Nestle Research and Development Centre National HIV and Retrovirology Laboratory
Shanghai Ltd., China Public Health Agency of Canada
Ottawa, Ontario, Canada
Elmer H. Marth Laurence Slutsker

University of Wisconsin–Madison Foodborne and Diarrheal Diseases Branch
Madison, Wisconsin Centers for Disease Control and Prevention
Atlanta, Georgia
Dawn M. Norton
Enteric Diseases Epidemiology Branch Bala Swaminathan
Division of Foodborne, Bacterial and Mycotic Foodborne and Diarrheal Diseases Branch
Diseases Division of Bacterial and Mycotic Diseases
National Center for Zoonotic, Vectorborne and National Center for Infectious Diseases
Enteric Diseases Centers for Disease Control and Prevention
Centers for Disease Control and Prevention Atlanta, Georgia
Atlanta, Georgia
Ewen C.D. Todd
National Food Safety and Toxicology Center
David G. Nyachuba
Michigan State University
Department of Food Science
East Lansing, Michigan
University of Massachusetts
Amherst, Massachusetts R. Bruce Tompkin
Food Safety Consultant
Franco Pagotto LaGrange, Illinois
Listeriosis Reference Service
Bureau of Microbial Hazards Marleen M. Wekell
Health Products and Food Branch Center for Veterinary Medicine
Health Canada U.S. Food and Drug Administration
Ottawa, Ontario, Canada Laurel, Maryland
Irene V. Wesley
John Painter Enteric Diseases and Food Safety Research
Foodborne and Diarrheal Diseases Branch Unit
Centers for Disease Control and Prevention National Animal Disease Center
Atlanta, Georgia Agricultural Research Service, USDA
Ames, Iowa
Jocelyn Rocourt
Institut Pasteur Martin Wiedmann
Paris, France Department of Food Science
Cornell University
Ithaca, New York
Elliot T. Ryser
Department of Food Science and Human Ahmed E. Yousef
Nutrition Department of Food Science and Technology
Michigan State University The Ohio State University
East Lansing, Michigan Columbus, Ohio
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Contents
Chapter 1
The Genus Listeria and Listeria monocytogenes: Phylogenetic Position,
Taxonomy, and Identification.............................................................................................................1
Jocelyn Rocourt and Carmen Buchrieser
Chapter 2
Ecology of Listeria Species and L. monocytogenes in the Natural Environment..........................21
Brian D. Sauders and Martin Wiedmann
Chapter 3
Listeriosis in Animals ......................................................................................................................55
Irene V. Wesley
Chapter 4
Listeriosis in Humans ......................................................................................................................85
John Painter and Laurence Slutsker
Chapter 5
Molecular Virulence Determinants of Listeria monocytogenes ....................................................111
Michael Kuhn and Werner Goebel
Chapter 6
Characteristics of Listeria monocytogenes Important to Food Processors...................................157
Beatrice H. Lado and Ahmed E. Yousef
Chapter 7
Conventional Methods to Detect and Isolate Listeria monocytogenes .........................................215
Catherine W. Donnelly and David G. Nyachuba
Chapter 8
Rapid Methods for Detection of Listeria .....................................................................................257
Byron F. Brehm-Stecher and Eric A. Johnson
Chapter 9
Subtyping Listeria monocytogenes ................................................................................................283
Lewis M. Graves, Bala Swaminathan, and Susan B. Hunter
Chapter 10
Foodborne Listeriosis.....................................................................................................................305
Dawn M. Norton and Christopher R. Braden
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Chapter 11
Incidence and Behavior of Listeria monocytogenes in Unfermented Dairy Products .....................357
Elliot T. Ryser
Chapter 12
Incidence and Behavior of Listeria monocytogenes in Cheese and Other
Fermented Dairy Products .............................................................................................................405
Elliot T. Ryser
Chapter 13
Incidence and Behavior of Listeria monocytogenes in Meat Products ........................................503
Jeffrey M. Farber, Franco Pagotto, and Chris Scherf
Chapter 14
Incidence and Behavior of Listeria monocytogenes in Poultry and Egg Products ......................571
Elliot T. Ryser
Chapter 15
Incidence and Behavior of Listeria monocytogenes in Fish and Seafood....................................617
Karen C. Jinneman, Marleen M. Wekell, and Mel W. Eklund
Chapter 16
Incidence and Behavior of Listeria monocytogenes in Products of Plant Origin ........................655
Robert E. Brackett
Chapter 17
Incidence and Control of Listeria in Food Processing Facilities .................................................681
Jeffrey L. Kornacki and Joshua Gurtler
Chapter 18
Listeria: Risk Assessment, Regulatory Control, and Economic Impact ......................................767
Ewen C.D. Todd
Chapter 19
Perspectives on Research Needs....................................................................................................813
Elmer H. Marth, Robert E. Brackett, R. Bruce Tompkin,
Sophia Kathariou, and Ewen C.D. Todd
Index ..............................................................................................................................................843
DK3089_C001.fm Page 1 Tuesday, February 20, 2007 6:47 PM
1 monocytogenes
The Genus Listeria and Listeria
: Phylogenetic
Position, Taxonomy,
and Identification
Jocelyn Rocourt and Carmen Buchrieser
CONTENTS
History ................................................................................................................................................1
Phylogenetic Position of the Genus Listeria .....................................................................................3
Numerical Taxonomy ...............................................................................................................3
Chemotaxonomy.......................................................................................................................3
rRNA Sequencing.....................................................................................................................4
Whole Genome Sequencing .....................................................................................................4
Conclusion ................................................................................................................................4
Taxonomy of the Genus Listeria .......................................................................................................5
L. monocytogenes, L. ivanovii, L. welshimeri, and L. seeligeri ..............................................6
L. grayi (and “L. murrayi”)......................................................................................................7
L. denitrificans/Jonesia denitrificans .......................................................................................8
Present State of the Taxonomy of the Genus Listeria ............................................................8
Identification of Bacteria of the Genus Listeria................................................................................9
Genus Characteristics ...............................................................................................................9
Morphology ..................................................................................................................9
Culture ..........................................................................................................................9
Nutritional Requirements ...........................................................................................10
Metabolism and Biochemical Characteristics............................................................10
Species Identification..............................................................................................................10
Conclusion........................................................................................................................................12
References ........................................................................................................................................12
HISTORY
Murray, Webb, and Swann first published a description of Listeria monocytogenes in 1926 [112].
Several earlier reports may have described Listeria isolation [62,156]; the most plausible is certainly
that by Hulphers [73]. However, the authors of these reports did not deposit their isolates in a permanent
collection, so no subsequent investigations or comparisons with further strains were possible.
Murray and colleagues observed six cases of sudden death of young rabbits in 1924 in the
animal breeding establishment of the Department of Pathology at Cambridge and many more in
1
2 Listeria, Listeriosis, and Food Safety
the succeeding 15 months. The interesting characteristics presented by the disease and the increasing
mortality prompted investigation. The authors wrote at that time [112]:
Both the natural and the experimental disease have interesting and characteristic features and their
consideration has forced us to the conclusion that the causative organism either has not been described
previously, or has been inadequately described and so cannot be traced in the literature. In either case,
we feel justified in naming it. Its salient character is the production of a large mononuclear leucocytosis.
This is far the most important and most striking character we have discovered and we name the
microorganism we shall describe in this paper “Bacterium monocytogenes.” The question of the generic
name is more difficult and we have not succeeded in associating our organism with many other genera
proposed in Bergey’s Manual of Determinative Bacteriology (1925). We propose for the present to use
the undefined Bacterium ([...], for, if the present chaos is to be resolved and if the classification adopted
by the American Society of Bacteriologists is to be improved, it will be achieved only by co-operation
and with this end in view we cannot use the term Bacillus.
In 1927, during investigations of unusual deaths observed in gerbils near Johannesburg, South
Africa, Pirie [130] discovered a new microorganism, agent of what he called “the Tiger River
disease.” He named this new agent Listerella hepatolytica 1: “The causative organism is a Gram-
positive bacillus for which, from its most striking pathogenic effect, I propose the specific name
hepatolytica, and the generic name Listerella, dedicating it in honour of Lord Lister, one of the
most distinguished of those concerned with bacteriology whose name has not been commemorated
in bacteriological nomenclature.”2
Discoverers Murray and Pirie [130] sent their strains to the National Type Collection at the
Lister Institute in London. Dr. Leningham, the director, was struck by the similarity of the two
microorganisms and put Murray and Pirie into contact. Because the identity was clear, they decided
to call this bacterium Listerella monocytogenes [111,130,159].
However, in 1939, the Judicial Commission of the International Committee on Systematic
Bacteriology rejected the generic name Listerella because it had previously been used for a mycetozoan
in 1906 in honor of Arthur Lister (younger brother of Lord Lister) and for a species of foraminifer
in 1933 in honor of Joseph Jackson Lister (father of Lord Lister). As noted by Gibbons in 1972
[54], it is certainly unique that the same name was chosen for three quite different groups of
microorganisms to honor contributions of a father and his two sons. In 1940, Pirie proposed the
name Listeria [129]. Before and even after this date, numerous names were used to designate
L. monocytogenes:
Bacterium monocytogenes hominis and later Listerella hominis by Nyfeldt, who considered
that it was the agent of infectious mononucleosis [116,117]
Corynebacterium parvulum by Schultz et al. in 1934 [154]
Listerella ovis by Gill in 1937 [55]
Listerella bovina, L. gallinaria, L. cunniculi, and L. gerbilli by Nyfeldt [117,118]
Erysipelothrix monocytogenes by Wilson and Miles in 1946 [187]
Corynebacterium infantisepticum by Potel (1951) during his first observations of fetal and
neonatal listeriosis in Germany [132]
Unlike some pathogenic agents responsible for large outbreaks that have marked the history
of humans for centuries, the history of L. monocytogenes and listeriosis is recent: It begins
officially in 1924. The first confirmed diagnosis in a human was that of a soldier suffering from
meningitis at the end of World War I (retrospective identification of the strain [28]). Before this
case, there are no validated observations. Interestingly, however, a historian has suggested that
L. monocytogenes could have been the cause of Queen Anne’s 17 unsuccessful pregnancies
[150].
The Genus Listeria and Listeria monocytogenes 3
PHYLOGENETIC POSITION OF THE GENUS LISTERIA

The relationships of Listeria to other bacteria remained obscure until the 1970s. Absent from the
three first editions of Bergey’s Manual of Determinative Bacteriology published in 1923, 1925, and
1930, the genus Listeria was included in the tribe Kurthia of the Corynebacteriaceae family in the
next edition in 1934. In the sixth and seventh editions (published in 1948 and 1957, respectively),
Listeria was still a member of the Corynebacteriaceae; however, in the next edition (1974), it was
considered a genus of uncertain affiliation and placed with Erysipelothrix and Caryophanon after
the family of Lactobacillaceae [3–6]. Finally, Listeria was classified with Lactobacillus, Erysipelo-
thrix, Brochothrix, Renibacterium, Kurthia, and Caryophanon in the section of “regular, nonsporing,
Gram-positive rods” in Bergey’s Manual of Systematic Bacteriology [7].
How can these repeated reclassifications be explained? On the basis of morphological resem-
blances (Gram-positive, non-spore-forming rod), Listeria has long been associated with the coryne-
form group of bacteria. However, with the successive introduction and development of numerical
taxonomy, chemotaxonomy, DNA/DNA hybridization, and, more recently, rRNA (ribosomal RNA)
and DNA sequencing, the phylogenetic position of Listeria has been more clearly determined.
NUMERICAL TAXONOMY
Numerical taxonomy provided the first attempts to investigate in depth the phylogenetic position
of Listeria among Gram-positive bacteria. In the first studies, Listeria was included among coryne-
form bacteria and actinomycetes and, consequently, was located with the corynebacteria [13,31]
or in an indefinite position [18,71]. In contrast, from 1969, more natural relationships were described
when Listeria was compared to various representatives of lactic acid bacteria [33,172,173]. In 1975,
the close relatedness with these microorganisms was clearly demonstrated by the broader numerical
taxonomic survey of Jones, who studied 173 characteristics of 233 strains of various genera,
including coryneform and lactic acid bacteria [79].
The refined position of Listeria was later investigated by Wilkinson and Jones in 1977 and
Feresu and Jones in 1988 [43,186]. From these works, it became clear that Listeria is distinct from
other known genera, including Erysipelothrix and Brochothrix thermosphacta (formerly Microbac-
terium thermosphactum) and is closely related to Lactobacillus and Streptococcus. Consequently,
Wilkinson and Jones [186] suggested that Listeria, Gemella, Brochothrix, Streptococcus, and
Lactobacillus be classified in the family Lactobacillaceae. In spite of some imprecision over the
exact position with regard to the higher taxonomic relationships—especially with Brochothrix,
certain lactobacilli, and Carnobacterium [43,186]—conclusions based on numerical analysis of
data for large numbers of phenotypic features were precursors of the current phylogenetic classi-
fication of the genus Listeria.
CHEMOTAXONOMY
A number of chemotaxonomic markers have been especially useful for solving the phylogenetic
position of the genus Listeria, reinforcing its distinctness from coryneform bacteria and its relat-
edness to the lactic acid bacteria. The G + C percent DNA content of L. monocytogenes isolates
ranges from 36 to 42% [43,142,172], indicating that Listeria belongs to the low G + C percent
DNA content (<55%) group of Gram-positive bacteria. Lipoteichoic acids (amphilic polymers of
the cytoplasmic membranes) have been isolated from L. monocytogenes [68,148,178]. These acids
consist of hydrophilic poly(glycerophosphate) chains covalently attached to glyco- or phosphati-
dylglycolipids (the hydrophilic moieties of the molecules) and exhibit structural analogies with
lipoteichoic acids from other bacteria. Although lipoteichoic acids show a distinct structural diver-
sity in their hydrophilic and lipophilic portions, a given lipoteichoic acid is known to be a fairly
stable characteristic and may be used as a taxonomic marker.
In the case of Listeria, the presence of particular lipoteichoic acids provides further evidence
that the genus is a biochemically coherent taxon [148]. In addition, lipoteichoic acids are absent
from coryneform bacteria but are found in Bacillus, Staphylococcus, Streptococcus, and Lactoba-
cillus, indicating that Listeria should be grouped with these microorganisms [148]. With the
exception of one report [106], free mycolic acids, which are specific for high G + C percent DNA
content Gram-positive bacteria, have not been detected in Listeria [43,81]. The presence of respi-
ratory menaquinones (seven isoprene units) of predominantly methyl-branched cellular fatty acids
and meso-DAP as major peptidoglycan diamino acid support the close relatedness of Listeria and
Brochothrix and the greater distance from lactobacilli [24,26,43,46,134]. Analysis of low-molecular-
weight RNA profiles also supports the independent identity of L. monocytogenes among other
Gram-positive taxa [171].
rRNA SEQUENCING
Analysis of the 16S and 23S rRNA of L. monocytogenes has further clarified the position of Listeria
with regard to other genera of Gram-positive bacteria. In 1986, using the 16S rRNA cataloguing
approach (partial sequencing), Ludwig et al. unambiguously demonstrated that Listeria forms with
Brochothrix thermosphacta one of the several sublines within the Clostridium subdivision [100].
They did not detect any relationship with the coryneform organisms (except for highly conserved
16S RNA sequences universal to all eubacteria and those of Gram-positive bacteria, respectively).
Reverse transcriptase sequencing of 16S rRNA data confirmed this phylogenetic position of Listeria
and indicated that the Listeria–Brochothrix subline is approximately equidistant from the Bacillus
and Enterococcus–Carnobacterium sublines [26].
The close relationship with Bacillus was later confirmed [38]. On the basis of rRNA sequencing
data and chemotaxonomic properties, Collins et al. [26] considered that Listeria is phylogenetically
remote from Lactobacillus and should not be included in the Lactobacillaceae family and that the
Listeria–Brochothrix subline probably merits a separate family, the Listeriaceae. This great distance
between Lactobacillus and Listeria has been recently confirmed by sequencing of the 23S rRNA
Listeria exhibiting the highest similarity with Bacillus and Staphylococcus [149].
WHOLE GENOME SEQUENCING

Recently, the complete genome sequences of L. monocytogenes and L. innocua have been deter-
mined [56]. Comparison of the gene content and organization of the two sequenced Listeria
genomes, as well as comparison with that of other genomes, allows insight into phylogenetic
relationships (Figure 1.1). The two Listeria genomes show a perfect conservation of the order and
the relative orientation of orthologous genes, indicating high stability in genome organization and
a close phylogenetic relationship. When the Listeria genomes were compared to those of B. subtilis
and B. halodurans, 1,428 and 1,296 orthologous genes, respectively, were predicted (on the basis
of Blastp comparisons) [104]. Furthermore, high synteny in genome organization was observed
between Listeria and Bacillus genomes.
The same results were obtained when the genome organization of L. monocytogenes and
Staphylococcus aureus were compared. In contrast, comparison of the L. monocytogenes genome
with Streptococcus agalactiae (another phylogenetically closely related member of the group of
Gram-positive, low GC content bacteria) or Lactococcus lactis showed no synteny [17]. Thus,
recent data from complete genome comparisons confirmed that Listeria exhibits the highest simi-
larity with Bacillus and Staphylococcus.
CONCLUSION
Data accumulated during the last three decades clearly demonstrate that Listeria is a well-
defined taxon that possesses a number of features distinguishing it from neighboring taxa. It is not
Origin
2 944 528 bp
Terminus
FIGURE 1.1 Circular genome map of L. monocytogenes strain EGDe showing position and orientation of
genes. From the outside: circle 1: L. monocytogenes genes on the + and – strands, respectively; color code:
red = L. monocytogenes genes; black = rRNA operons; circle 2: genes present in L. monocytogenes but absent
in L. innocua; circle 3: genes present in L. innocua but absent in L. monocytogenes; circle 4: G + C content
of L. monocytogenes with <32.5% G + C in light yellow, between 32.5 and 43.5% in yellow, and >43.5%
G + C in greenish yellow. The putative origin of replication and terminus are indicated on the outside.
a coryneform bacterium as evidenced by numerical phenetic studies, chemotaxonomic properties,

and various rRNA and DNA sequencing analyses. However, the exact phylogenetic position of this
genus still remains controversial. Although it is generally agreed that its nearest neighbor is
Brochothrix, its relationships with other members of the low G + C percent DNA content
Gram-positive bacteria, especially with Lactobacillus, need further clarification.
TAXONOMY OF THE GENUS LISTERIA

For many years after its discovery, the genus Listeria was monospecific, containing only the
L. monocytogenes species. Because of its ability to reduce nitrates, L. denitrificans was added in
1948 [172]; L. grayi was added in 1966 in honor of M. L. Gray, an American microbiologist [98].
L. murrayi was added in 1971 (in honor of E. G. D. Murray, a Canadian microbiologist) [185] and
L. innocua (named thusly because of its harmlessness) in 1981 [155]. In 1985, L. ivanovii was
added in honor of I. Ivanov, a Bulgarian microbiologist [161]; L. welshimeri was added in 1983
(in honor of H. J. Welshimer, an American microbiologist) and L. seeligeri (in honor of H. P. R.
Seeliger, a German microbiologist) in 1983 [143].
As for the phylogenetic analysis of Listeria, the introduction of molecular biology methods
allowed a better appreciation of the diversity within the genus Listeria, which now contains six
species: L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, and L. grayi.
L. MONOCYTOGENES, L. IVANOVII, L. WELSHIMERI, AND L. SEELIGERI
Serotyping was the first approach used to elucidate the infrageneric structure of the genus Listeria.
The first antigenic scheme was devised by Paterson [123,124], who described the first four serovars.
This scheme was later extended by Donker-Voet and Seeliger with the addition of new serovars
[36,160]. Until 1960, Listeria was nearly exclusively isolated from pathological samples—thus the
isolation of nearly only L. monocytogenes strains. In 1962, Ivanov observed atypical L. monocytogenes
strains isolated from aborted sheep and proposed to allocate them to a new species, L. bulgarica,
on the basis of their strong hemolytic activity and their new antigenic structure (serovar 5)
[75,76].
Years later, with development of selective media, numerous strains were isolated from various
environmental sources. Seeliger serotyped hundreds of strains collected between 1965 and 1980
and observed that they were nonhemolytic, characterized by particular antigenic factors (serovars
6a and 6b [formerly 4f and 4g] and undesignated serovars), and apparently nonpathogenic. He
proposed to name them L. innocua in 1981 [155]. Thus, simple phenotypic methods, serotyping
and hemolysis, led to the demonstration that the species L. monocytogenes as defined in the eighth
edition of Bergey’s Manual of Determinative Bacteriology [6] was heterogenous, covering a number
of different species.
Early DNA/DNA hybridizations (filter study) by Stuart and Welshimer in 1974 showed L.
monocytogenes to be heterogenous [175]. However, the number of DNA hybridization groups in
their collection of strains could not be ascertained because only one DNA from L. monocytogenes
was labeled; moreover, serovars were not indicated. Further DNA/DNA hybridization studies (S1
method) that aimed to resolve the genomic heterogeneity of the so-called L. monocytogenes and
evaluate the validity of the new species L. bulgarica and L. innocua (not officially validated at that
time) were undertaken in 1982 with many strains of various origins [142]. Five DNA relatedness
groups were found among strains formerly identified as L. monocytogenes:
Genomic group 1 contained the type strain of L. monocytogenes, thus corresponding to L.

monocytogenes, sensu stricto; it included strains belonging to serovars 1/2a, 1/2b, 1/2c,
3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7.
Genomic group 2 was constituted by strongly hemolytic strains, all belonging to serovar 5
(for which Ivanov suggested the name L. bulgarica) and deserved species rank [75]; they
were named L. ivanovii in 1985 [161]. Further investigations of strains of this species
using multilocus enzyme electrophoresis, DNA/DNA hybridizations, and rRNA gene
restriction patterns described two subspecies: L. ivanovii subsp. ivanovii (ribose positive)
and L. ivanovii subsp. londoniensis (ribose negative) [10].
Genomic group 3 contained nonhemolytic and nonpathogenic (for mice) strains of serovars
4ab, 6a, 6b, and undesignated serovars, including the two strains that Seeliger previously
proposed as reference strains for L. innocua [155]. Therefore, this group corresponded to
L. innocua. Based on these data, this species was officially validated in 1983 [179].
Genomic group 4 contained nonhemolytic strains of serovars 6a and 6b; these strains produced
acid from D-xylose and were nonpathogenic for mice [139,144]; they therefore corre-
sponded to a group of strains previously described by Groves and Welshimer in 1977 [65].
The group was given species status and called L. welshimeri in 1983 [143].
Genomic group 5, an unexpected group, included hemolytic and nonpathogenic strains of
various serovars (1/2b, 4c, 4d, 6b, and undesignated serovars) [139,144]. It was subse-
quently named L. seeligeri [143].
Numerical taxonomic surveys confirmed that L. monocytogenes, as defined in the eighth edition
of Bergey’s Manual of Determinative Bacteriology, was not a single taxon. However, this method
is of limited sensitivity for bacteria that differ by a small number of characteristics and these studies
were of little help in resolving the heterogeneity [43,82]. DNA/DNA homology experiments (optical
method) in 1993 supported these results and confirmed that L. monocytogenes, L. innocua,
L. ivanovii, L. welshimeri, and L. seeligeri each corresponded to a single cluster with no overlaps
among them [67]. This classification was later sustained by data using a multilocus enzyme
electrophoresis study, two-dimensional protein mapping, and sequencing of the 16S rRNA gene
and 16S-23S rRNA operon intergeneric spacer region [11,57,60,181].
More recently, DNA/DNA hybridization of macroarrays containing Listeria-specific genes
from the genome sequence of L. monocytogenes and L. innocua were used to predict absence
and presence of genes studied in 110 Listeria strains [37]. A matrix of binary data (absence/
presence) was analyzed by hierarchical clustering and neighbor joining to illustrate possible
phylogenetic relationships among the different listeriae. Important gene conservation within
each species of the genus Listeria and within distinct groups of L. monocytogenes strains was
identified. Furthermore, this analysis revealed that, without exception, the tested strains of L.
monocytogenes, L. innocua, L. ivanovii, L. welshimeri, and L. seeligeri clustered according to
their species definition. Each species was defined by a combination of genes specifically present
or absent [37].
Furthermore, DNA/DNA hybridization showed that serogrouping of the different Listeria
species as well as of the species L. monocytogenes is reflected in the genetic content and evolution
of the different serotypes. Ninety-three L. monocytogenes strains tested by DNA/DNA hybridization
of macroarrays were grouped into three evolutionary lineages correlated with serovars. Lineage I
comprised serovars 1/2a, 1/2c, 3a, and 3c, lineage II contained strains of serovars 1/2b, 3b, 4b, 4d,
4e, and 7, and lineage III contained serovars 4a and 4c. In addition, this allowed two subdivisions
within lineages I, II, and III to be distinguished clearly.
Lineage I was subdivided into two groups. One consisted of the strains of serovars 1/2a and
3a; the other consisted of the strains of serovars 1/2c and 3c. Lineage II was subdivided into two
groups. One group consisted of the strains of serovars 4b, 4d, and 4e; the other group was made
up of strains of serovars 1/2b, 3b, and 7. Lineage III was subdivided into lineage IIa, containing
serovar 4a strains, and lineage IIIb, which contained serovar 4c strains. Each lineage and subgroup
is characterized by highly conserved genes representing specific markers for L. monocytogenes
strains belonging to lineages I, II, or III [37]. Similar results are obtained by comparison of almost
full-length iap (invasion-associated protein) gene sequences that grouped the L. monocytogenes
strains into the same three distinct genotypes, correlating well with distinct serotypes. These results
were corroborated by further comparative sequence analysis of genes encoding two phospholipases,
pIcA and pIcB [151].
L. GRAYI (AND “L. MURRAYI”)
The long controversy about the taxonomic position of L. grayi and L. murrayi started when
DNA/DNA homology studies of Stuart and Welshimer in 1974 demonstrated low DNA relatedness
between L. monocytogenes on the one hand and L. grayi and L. murrayi on the other and high
genomic homology between L. grayi and L. murrayi. These data were supported by a numerical
phenetic analysis [174,175]. The authors proposed to transfer L. grayi and L. murrayi to a new
genus, L. murraya, with L. grayi as the type species, divided into two subspecies: L. grayi subsp.
grayi and L. grayi subsp. murrayi.
L. grayi, L. murrayi, and L. monocytogenes share a number of similarities: They cluster in all
numerical taxonomic studies [79,173,175,186] and possess lipoteichoic acid [148], teichoic acid of
the polyribitol phosphate type [47], peptidoglycan of the A1 gamma variation [46], nonhydrogenated
menaquinones of the MK-7 type [24,43], and the same cytochromes (a1bdo) [43]. However, other
features support the distinctness of the L. grayi and L. murrayi pair within the genus Listeria:
slight difference in G +C percent DNA content (42 vs. 36 to 38) [43,175]

small number of biochemical reactions [144]
nature of substitution of the lipoteichoic acids [148]
slight differences of protein electrophoregrams [97]
cellular fatty acid composition [114]
antigenic structure [160]
low DNA homology values [174]
Finally, the 16S rRNA oligonucleotide cataloguing of L. murrayi placed it close to L. mono-
cytogenes. These data, together with the substantial phenotypic similarity with L. monocytogenes,
provided no support for exclusion of L. murrayi (and the closely related species L. grayi) from the
genus Listeria [145]. This was later confirmed by 16S rRNA gene sequencing [181].
Various investigations offered evidence of the close relationship between L. grayi and L. murrayi.
They fall into a single distinct cluster in numerical taxonomic analysis [43,186], in multilocus
enzyme electrophoresis analysis [11], and in DNA/DNA hybridization studies [174]; in addition,
they share a number of chemotaxonomic properties that distinguish them from the other Listeria
species: the same DNA base composition values [43,174], the same substitution of lipoteichoic
acids [148], the same patterns of cellular fatty acids and fatty aldehydes [84], and a common
antigenic pattern structure in spite of small differences [160,182]. They have been distinguished
from each other only on the basis of nitrate reduction [185].
Reexamination of the genomic relatedness of L. grayi and L. murrayi using DNA/DNA hybrid-
izations and multilocus enzyme electrophoresis indicated that they should be considered members
of a single species: L. grayi [140]. These data are consistent with 16S and 23S rRNA sequencing
and cellular protein electrophoretic pattern data [26,88,149] as well as with DNA/DNA macroarray
studies in which L. grayi and the former L. murrayi exhibit a closely related hybridization profile
distinct from the other Listeria species [37].
L. DENITRIFICANS/JONESIA DENITRIFICANS
Although only a single isolate is currently known for this species, an amazing number of papers
have dealt with its taxonomic position. As early as 1966, a numerical taxonomic study showed that
L. denitrificans clustered with certain coryneform bacteria; this was later confirmed [18,79,173,174].
The results of chemotaxonomic studies and DNA/DNA hybridization further emphasized the
phenotypic differences between L. denitrificans and other members of the genus Listeria
[24,25,46,47,148,175]. In 1987, 16S rRNA cataloguing confirmed that this species is not a member
of the genus Listeria and belongs to the coryneform group of bacteria [146]. It was later demon-
strated that Jonesia differs from members of the family Cellulomonadaceae and that its closest
relatives are Dermobacter hominis and Brachybacterium faicium [136].
PRESENT STATE OF THE TAXONOMY OF THE GENUS LISTERIA

Finally, the genus Listeria currently contains six species—L. monocytogenes, L. ivanovii, L. innocua,
L. welshimeri, L. seeligeri, and L. grayi—as evidenced by DNA homology values, 16S rRNA and
DNA sequencing, chemotaxonomic properties, and multilocus enzyme analysis. Based on
DNA/DNA hybridization, DNA/DNA macroarray hybridization, 16S rRNA cataloguing, reverse
transcriptase sequencing of 16S and 23S rRNA, sequencing of 16S–23S rRNA operon intergenic
spacer region, and protein mapping, the genus embraces two closely related but obviously distinct
lines of descent. One contains L. grayi and the other L. monocytogenes, L. ivanovii, L. innocua,
L. welshimeri, and L. seeligeri. Within this line, the species can be divided into two groups:
L. monocytogenes and L. innocua, on the one hand, and L. ivanovii, L. seeligeri, and L. welshimeri
on the other [26,37,59,142,145,149,181].
IDENTIFICATION OF BACTERIA OF THE GENUS LISTERIA

GENUS CHARACTERISTICS
Morphology
Listeria is a small (0.5 µm in diameter and 1 to 2 µm in length), regular Gram-positive rod with rounded
ends. Cells are found as single units or in short chains, or they may be arranged in V and Y forms or
in palisades. Sometimes, cells are coccoid, averaging about 0.5 µm in diameter and may be confused
with streptococci. In old cultures, some cells lose the ability to retain Gram stain and may be occa-
sionally mistaken for Hemophilus. Long, thin, filamentous cells appear in old and rough culture and
after osmotic shock [66,83,93]. Listeria does not produce spores and capsules are not formed [159].
Listeria is motile from its few peritrichous flagella when cultured at 20 to 25°C (not or very
weakly motile at 37°C) [51]. Hanging-drop preparations of a fresh culture in tryptose phosphate
broth incubated at 20°C show a characteristic tumbling motility: Cells start with twisting and
wriggling movements that increase to fast, eccentric rotations before they suddenly move quickly
in various directions. Stab cultures in semisolid motility medium produce a typical picture of an
“umbrella” or inverted “pine tree” about 0.5 cm below the surface because of the microaerophilic
nature of the organism. A recent report indicates that L. monocytogenes and L. innocua differ
markedly in motility and flagellin production at 37°C: L. monocytogenes strains are virtually
nonmotile and produce little or no detectable flagellin, whereas strains of L. innocua are frequently
motile and produce substantial amounts of flagellin [89].
Culture
On nutrient agar, colonies are 0.2 to 0.8 mm in diameter, smooth, punctiform, bluish gray, trans-
lucent, and slightly raised with a fine surface texture and entire margin after 24 h of incubation.
After 5 to 10 days, well-separated colonies may be 5 mm or more in diameter. When cultures of
Listeria grown for 18 to 24 h at 37°C on a clear medium are examined with a binocular microscope
under obliquely transmitted light, the smooth colonies exhibit a typical blue-green iridescence
[61,95,110]. Even when the population of contaminants is rather high and that of Listeria is low,
Listeria can be recognized because of this characteristic [61,99]. Rough colonies may occasionally
be observed [63]. Conversion of smooth colonies to rough colonies is not reversible [156]. Differ-
ences in virulence between rough and smooth colonies have been observed [70,93,96]. Petite colony
formation by strains grown on esculin-containing agar has been described [167].
Listeria usually grows well on most commonly used bacteriological media. The growth rate is
increased by the presence of fermentable sugar, particularly glucose. On plate culture, Listeria has
a particularly penetrating acid odor that may result from formation of carboxylic acids, hydroxy
acids, and alcohols [32]. In broth, the medium becomes turbid after 8 to 24 h of incubation at 37°C.
Profuse growth is always observed slightly below the clear area near the surface of the medium,
indicating the propensity for Listeria to grow better at oxygen tensions lower than that in air [156].
The temperature limits of growth are 1–2°C to 45°C [85,158]. The ability to grow at very low
temperature was first used by Gray [64] for selective enrichment of a contaminated sample. In
broth, Listeria grows between pH 4.5 and pH 9.2, optimally at pH 7 [53,119,125]. It can grow in
10% (w/v) NaCl and survive at higher concentrations [158,163]. Survival at low pH and high salt
concentration depends strongly on temperature [23]. Listeria is one of the few foodborne pathogens
that can grow at aw below 0.93 [40,125].
Nutritional Requirements
According to published data, growth factors include cystine, leucine, isoleucine, arginine, methion-
ine, valine, cysteine, riboflavin, biotin, thiamine, and thioctic acid [133,164,184]. Growth is stim-
ulated by Fe3+ and phenylalanine [133,164]. In some experiments, virulent strains grew faster in
the presence of iron than did the avirulent strains [29]. Glucose and glutamine are required as
primary sources of carbon and nitrogen [133,158]. Chemically defined media have been described
for Listeria [133,135,165].
Metabolism and Biochemical Characteristics
Listeria is aerobic, microaerophilic, facultatively anaerobic, catalase positive (rare catalase-negative

strains have been observed), and oxidase negative. Although Feresu and Jones [43] reported
cytochrome a1bdo, presence of cytochrome is controversial [122,177]. Listeria is homofermentative
and oxidizes glycolytic intermediate compounds [28]. It possesses glucose oxidase and NADH
oxidase activities [122].
All strains grow on glucose, forming lactate, acetate, and acetoin under aerobic conditions as
main end products [127,147]. Acetoin is not produced under anaerobic conditions. Only hexoses
and pentoses support growth anaerobically; maltose and lactose support growth of some strains
aerobically, but sucrose does not [127]. Catabolism of glucose proceeds by the Embden-Meyerhof
pathway aerobically and anaerobically [158]. L. monocytogenes imports glucose by a high-affinity
phosphoenolpyruvate-dependent phosphotransferase system and a low-affinity proton motive force-
mediated system [19,120]. All strains are methyl red and Voges-Proskauer test positive.
Acid is also produced from amygdalin, cellobiose, fructose, mannose, salicin, maltose, dextrin,
alpha-methyl-D-glucoside, and glycerol. Acid production from galactose, lactose, melezitose, sor-
bitol, starch, sucrose, and trehalose is variable. Acid is almost never produced from adonitol, arabinose,
dulcitol, erythritol, glycogen, inositol, inulin, melibiose, raffinose, or sorbose. Phenylalanine-
deaminase, ornitine-, lysine-, and arginine-decarboxylases are not produced; H2S is not produced.
Urea is not hydrolyzed and indole is not produced. Additional information on biochemical tests is
available [43,87,141,158,162,166,186].
SPECIES IDENTIFICATION
All Listeria species are phenotypically very similar, but can be distinguished by combinations
of the following tests: hemolysis, acid production from D-xylose, L-rhamnose, alpha methyl-
D-mannoside, and mannitol [144]. Phenotypic similarities are consistent with high genomic homol-
ogies between the different species [26,144,149].
Hemolysis is a key characteristic for allocating an isolate to a species and is obviously the most
difficult characteristic of the identification gallery. During a collaborative study on Listeria identi-
fication, Higgins and Robinson [69] noted that a large percentage of errors in the identification of
L. seeligeri and L. ivanovii was caused by inaccurate reading of the CAMP test and hemolysis.
Isolates of L. monocytogenes and L. seeligeri show narrow, slight clearing zones of beta-hemolysis.
L. ivanovii shows wide, clearly delineated zones of beta-hemolysis. In contrast, L. innocua,
L. welshimeri, and L. grayi are not hemolytic. L. innocua can produce a green zone of hemolysis
according to the medium used [131,169]. L. monocytogenes is hemolytic for blood of various
origins: sheep, horse, cow, guinea-pig, piglet, and human [152,156,168,180].
Various methods have been developed to determine hemolytic activity, especially for weakly
hemolytic strains (L. seeligeri and some L. monocytogenes isolates): examination of hemolysis
below the colonies, prolonged incubation (48 h), incubation for a few hours at 4°C, thin layer
blood agar plates, several media [50], tube tests and microplate techniques with erythrocyte
suspension [34,35,176], addition of an exosubstance from Rhodococcus equi, S. aureus, or
L. ivanovii to the blood agar [107,168,180], and the CAMP test with Staphylococcus aureus or
R. equi [15,48,65,74,75,108,109,113,144,170]. Positive CAMP tests are indicated by an enhanced

zone of beta-hemolysis at the intersection of the test strains L. monocytogenes, L. ivanovii, and L.
seeligeri with S. aureus and L. ivanovii with R. equi.
Conflicting readings of the CAMP test with R. equi have been reported; some authors consider
L. monocytogenes to be positive [44,153,183] and others consider it negative [144,158]. The typical
positive CAMP test with R. equi, as observed with L. ivanovii, gives a shovel-like shape. In contrast,
when this test is positive with L. monocytogenes, the shape is that of an onion. This could reflect
different abilities of R. equi strains to interact with L. monocytogenes because of different amounts
of listeriolysin O secreted by L. monocytogenes strains or to variation in the capability of R. equi
strains to secrete cholesterol oxidase [45,183]. However, whether or not positive, this test is not
essential because L. monocytogenes and L. ivanovii can be easily distinguished by acid production
from D-xylose, L-rhamnose, and alpha-methyl-D-mannoside. The L. monocytogenes exosubstance
involved in the CAMP reaction with S. aureus and R. equi is listeriolysin O [137]. The exosubstances
of S. aureus and R. equi are sphingomyelinase C and cholesterol oxidase, respectively [45,107,137].
Hemolysin is a major virulence factor of L. monocytogenes (see the chapter on L. monocyto-
genes virulence factors). Three species—L. monocytogenes, L. ivanovii, and L. seeligeri—are
hemolytic and possess the virulence gene cluster as recently demonstrated [58]; however, only two,
L. monocytogenes and L. ivanovii, are naturally and experimentally pathogenic [103,139]. L.
ivanovii is mainly responsible for abortion in animals. Therefore, pathogenicity should not be
presumed on the observation of hemolysis alone. Few nonhemolytic L. monocytogenes isolates
have been observed. The best known example is the type strain of this species [70,80,90]. Disso-
ciation between hemolytic and nonhemolytic colonies is rarely observed [128]. Nonhemolytic and
a number of weakly hemolytic strains are not or are weakly pathogenic [12,27,34,42,70,94,126,176].
In spite of these atypical strains, routine pathogenicity testing for L. monocytogenes is not recom-
mended [99].
Additional tests, especially to distinguish L. monocytogenes from L. innocua, have been pro-
posed, such as detection of phospolipase C activity [22,115], hydrolysis of D-alanine-p-nitroanilide
[20,87], hydrolysis of DL-alanine-beta-naphthylamide [20], and hydrolysis of a naphthylamide
substrate (API Listeria [8]).
Several commercially miniaturized culture or enzyme multitest assays have been tested or used
for Listeria identification because conventional culture procedures for identification are tedious and
time consuming. They include API 5O CH [92,141], APl-ZYM [141], API 20 STREP [102], API
Listeria [8,9,50,121], API Coryne [91], Micro-ID Listeria [2,8,69,138], Mast ID [92], RAPID
CORYNE [52], RAPID ID 32 Strep [49], and a microtiter plate method [167]. Information provided
by API ZYM, API 20 STREP, and RAPID ID 32 Strep distinguishes isolates at the genus level,
whereas API Listeria, Mast-ID, API Coryne, and API 50 CH are more appropriate for genus and
species identifications.
Phenotypic markers are often used for routine identification of Listeria isolates in food and
clinical laboratories. More sophisticated methods, often based on genotypic markers, have been
proposed; according to the goal, some of them can be used for rapid identification of isolates—
alone or in association with other methods—or can help allocate atypical isolates to known species.
These methods include
16S rRNA sequencing [30]

sequence analysis of the 16S-23S internal transcribed spacer loci [39,59]
automated RNA probe [101]
amplification of the amplified product of a small 16S rRNA gene fragment [105]
ribotyping [77]
pulsed-field gel electrophoresis (PFGE) [14]
random amplification of polymorphic DNA [40]
repetitive element sequence-based PCR [78]
multiplex PCR based on a single reaction [16]

denaturing gradient gel electrophoresis (DGGE) based on the PCR amplification of the iap
gene [21]
multilocus enzyme electrophoresis [11]
cellular protein electrophoretic pattern analysis [88]
enzymatic profiling using fluorogenic substrates [86]
analysis of fermentation products by frequency-pulsed electon-capture gas-liquid chromatog-
raphy [32]
Fourier transform infrared spectroscopy analysis [72]
thermogram determination [1]
Genome-based typing using macro- or microarray techniques is not used for routine identification.
However, it is a very promising approach and first results indicate its power and usefulness.
CONCLUSION
Studies on the phylogenetic position of Listeria started when numerical phenetic studies were
applied to Gram-positive bacteria. These first studies were of primary importance in demonstrating
that Listeria was not a coryneform bacterium. The data were confirmed by 16S rRNA cataloguing
and whole genome comparisons. The refined location of Listeria within the low G + C percent
DNA content Gram-positive bacteria were later determined by reverse transcriptase 16S and 23S
sequencing data; this was later confirmed. Numerical taxonomic studies revealed a certain hetero-
geneity within this genus and the exact species content was determined by DNA/DNA hybridization,
rRNA sequencing, and, more recently, by genome sequencing. On the basis of this genomic
dissection, the genus contains six species divided into two sublines of descent.
The present state of Listeria taxonomy is the result of studies over more than 25 years by
various laboratories in different countries, using as many methods as possible. The distinction
between L. monocytogenes and nonpathogenic species was already defined when foodborne liste-
riosis became a public health problem with a major economic impact on the food industry. This
allowed restricting efforts to food contaminated with L. monocytogenes because food contaminated
by other Listeria species was of no concern.
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2 and
Ecology of Listeria Species
L. monocytogenes in
the Natural Environment
Brian D. Sauders and Martin Wiedmann
CONTENTS
Introduction ......................................................................................................................................21
Methods to Study the Ecology of Listeria Species ........................................................................22
Traditional Enrichment and Isolation Methods .....................................................................22
Enumeration Methods ............................................................................................................23
Limitations and Pitfalls of Current Environmental Characterization
Methods for Understanding the Ecology of Listeria.............................................................24
Molecular Detection and Subtyping Methods .......................................................................25
Ecology of Listeria Species in Different Environments .................................................................27
Survival and Multiplication of Listeria spp. under Environmentally
Relevant Stress Conditions ..............................................................................................................27
Listeria spp. in Soil and Vegetation.......................................................................................28
Listeria spp. in Sewage ..........................................................................................................30
Listeria spp. in Water .............................................................................................................31
Listeria spp. in Animal Feeds ................................................................................................32
Regulation of Listeria Gene Expression during Environmental
Survival and Multiplication....................................................................................................34
Transmission Dynamics ...................................................................................................................35
Natural Environment and Transmission of Human Listeriosis .............................................35
Transmission of Listeria between Natural and Food-Processing
Environments ..............................................................................................................36
Ecology of Human Outbreak-Associated L. monocytogenes Strains........................37
Ecology of L. monocytogenes Strains Associated with Sporadic
Human Listeriosis Cases ............................................................................................38
Natural Environment and Transmission of Listeria in Animals............................................40
Transmission of Listeria within Natural Environments ........................................................42
Defining Global Listeria Transmission Pathways and Reservoirs ........................................42
Acknowledgments ............................................................................................................................44
References ........................................................................................................................................44
INTRODUCTION
Most members of the genus Listeria, including L. monocytogenes, appear to be widely distributed
in many different environments. Due to the importance of L. monocytogenes as a human foodborne
and animal pathogen most studies on Listeria in the environment have focused on food processing
21
and farm environments. Because the presence and ecology of Listeria in food processing environ-
ments is covered extensively in other chapters of this book, this chapter will focus on the ecology
of Listeria in environments outside food processing and preparation operations. A broad under-
standing of the distribution and ecology of Listeria spp. and L. monocytogenes in the natural
environment will be critical to provide for better control of this and other foodborne and environ-
mental pathogens.
METHODS TO STUDY THE ECOLOGY OF LISTERIA SPECIES

Studies on the ecology of Listeria spp. critically depend on our ability to detect and characterize
these organisms in different environments. Other chapters in this book will provide a detailed
overview of detection and characterization methods for Listeria spp. and L. monocytogenes from
food, food-processing environments, and clinical specimens. Here, we will critically review selected
detection and characterization methods, highlighting their application for isolation and character-
ization of Listeria spp. from samples collected from natural environments.
TRADITIONAL ENRICHMENT AND ISOLATION METHODS

Despite wide distribution in nature, L. monocytogenes and other Listeria spp. occur in small numbers
in most natural habitats. Detection methods for Listeria spp. thus generally include selective
enrichment, followed by plating on selective and differential media. Commonly used selective and
differential plating media (e.g., Oxford, lithium-chloride-phenylethanol-moxalactam [LPM]) allow
for isolation of Listeria spp., but do not provide for specific identification of L. monocytogenes.
Listeria-like colonies must be further characterized biochemically or genetically to determine the
species isolated.
Because Listeria can survive and multiply at refrigeration temperatures (1 to 8°C), cold enrich-
ment at 5°C represented the selective enrichment methodology of choice for many years. This
technique required incubation and subsequent periodic plating for several months to one year [85].
Newer media use selective agents to allow for enrichment of Listeria spp. at higher temperatures
(30 and 35°C). Selective agents widely used in different enrichment media include acriflavin (to
inhibit many Gram-positive bacteria) and nalidixic acid (to inhibit Gram-negative bacteria), among
others. A wide variety of different one-step and two-step enrichment and isolation media and
protocols for Listeria have been described (see Chapter 7); three commonly used isolation methods
are briefly described here.
All three methods covered here were originally developed for detection of Listeria spp. and L.
monocytogenes from foods and food-processing environments; however, these methods (or adap-
tations of these methods) also have been used successfully for isolation of L. monocytogenes from
environmental samples [12,206]. All three methods use selective enrichment but the concentrations
of the selective agents vary to strike a balance between recovery of injured cells and suppression
of other microorganisms. For example, the U.S. Food and Drug Administration (FDA) uses primary
enrichment for 4 h at 30°C without selective agents to increase recovery of potentially injured L.
monocytogenes. After 4 h of incubation, acriflavin, nalidixic acid, and cycloheximide are added,
followed by incubation for an additional 44 h (48 h total). Enrichments are plated at 24 and 48 h
onto Oxford (OX) agar, and LPM or polymyxin-acriflavin-lithium chloride-ceftazidime-esculin-
mannitol (PALCAM) agar [186].
The U.S. Department of Agriculture (USDA) uses primary enrichment in University of Vermont
(UVM) broth for 24 h at 30°C, with subsequent plating onto modified Oxford agar (MOX) plates
and transfer of the primary enrichment to Fraser broth (FB) for secondary enrichment. FB enrich-
ments are plated onto MOX plates after 24 h at 30°C [36]. The Netherlands Government Food
Inspection Service (NGFIS) method [187] uses PALCAM-egg yolk broth incubated at 30°C for
24 to 48 h with subsequent plating onto PALCAM agar at 24 and 48 h. The U.S. Centers for
Ecology of Listeria Species and L. monocytogenes in the Natural Environment 23
Disease Control and Prevention reported that utilizing the USDA and NGFIS methods provided
for 90% sensitivity for isolating L. monocytogenes from food and fecal samples [23].
Although many media, including those mentioned in the previous paragraphs, have been widely
evaluated and used for isolation of Listeria spp. and L. monocytogenes from foods, food-processing
environments, and fecal materials [23,57,62,95,113], very few studies have evaluated different enrichment
procedures for their ability to detect Listeria spp. and L. monocytogenes in the natural environment. Initial
work in the 1970s by Weis [196] compared the use of an early selective enrichment medium (potassium
thiocyanate medium with and without 10 µg/mL of acriflavin per milliliter) with cold enrichment in
tryptose broth and in brain–heart infusion broth for isolation of Listeria spp. and L. monocytogenes from
plant and soil materials. The study concluded that selective enrichment provided a suitable method for
isolation of Listeria from environmental samples.
A more recent study by van Renterghem [187] compared four different enrichment procedures
for their ability to detect L. monocytogenes in manure samples and samples from natural environ-
ments and concluded that cold-enrichment procedures were unsatisfactory for isolation of L.
monocytogenes from natural environments. This is consistent with an evaluation of cold enrichment
and the USDA selective enrichment procedure on food samples, which showed that the selective
enrichment procedure provided significantly better results for isolation of L. monocytogenes than
cold enrichment [92]. Many publications [32,34,58] report the use of cold enrichment for detection
of Listeria and L. monocytogenes from samples collected from natural environments (e.g., silage);
however, selective enrichment likely provides a better method for isolation of Listeria and L.
monocytogenes from these sample types. The most commonly used enrichment procedure for
samples from natural environments includes selective enrichment in LEB (although with varying
concentrations of selective agents), followed by plating on selective and differential media (most
often Oxford or LPM) [102,206].
Definitive recommendations on the most effective protocols for detection of Listeria and L.
monocytogenes in natural environments are not possible until comparative evaluations of different
detection protocols for different sample types (silage, sewage, soil, etc.) are performed. Based on
experiences with development and evaluation of enrichment protocols for food samples, it is
unlikely that any single method will allow for sensitive detection of Listeria in different sample
types. A combination of different enrichment and plating procedures will likely provide for the
most sensitive detection in natural environments, but may not be feasible for many larger studies
on Listeria in the natural environment.
Although maceration and suspension of samples from natural environments (e.g., soil, silage,
plant materials) in selective enrichment media represent a common and suitable approach for
detection of Listeria, certain sample types may require additional bacterial cell concentration steps.
For water samples, filtration is often used to concentrate bacterial cells before culture. For example,
Watkins and Sleath [195] used filtration combined with cold enrichment and subculturing onto
selective media for detection of Listeria in water samples. Colburn et al. [34] also used filtration
and cold enrichment to isolate Listeria spp. from water samples. Immunomagnetic separation (IMS)
using magnetic beads with antibodies specific to Listeria provides another approach to concentrating
Listeria cells [174]. For example, IMS has been used to concentrate Listeria cells after incubation
in enrichment broth and before plating [174]. IMS has also been used to concentrate Listeria cells
after primary enrichment of human [86] and animal fecal specimens [16].
ENUMERATION METHODS
Quantification of Listeria spp. and L. monocytogenes is sometimes critical to understanding the
ecology and transmission of Listeria in natural environments. Although a most-probable-number
(MPN) approach using selective enrichment methods must be used for quantification of Listeria
populations <100 CFU/g, direct plating can be utilized if Listeria populations >100 CFU/g are
present [36]. MPN methods have been used to quantify Listeria spp. and L. monocytogenes in food
samples [211], although few have used this approach to quantify Listeria spp. and L. monocytogenes
in samples from natural environments [20].
Enumeration of Listeria spp. in samples contaminated with >100 CFU/g can easily be achieved
by direct plating on selective and differential media, which allow specific detection of Listeria spp.
(e.g., Oxford). On the other hand, specific enumeration of L. monocytogenes by direct plating is
not practically feasible unless plating media are available that allow for specific detection of
L. monocytogenes. Vazquez-Boland et al. [188] demonstrated that a method using direct plating on
Listeria selective agar followed by 37- to 48-h incubation at 37°C with a subsequent overlay with
blood agar allowed for quantification of hemolytic Listeria in silage samples. This method can thus
be used to enumerate L. monocytogenes if the presence of hemolytic L. seeligeri and L. ivanovii
can be excluded.
More recently, several selective and differential chromogenic agars were developed to allow
specific detection of L. monocytogenes. L. monocytogenes plating medium (LMPM; Biosynth)
contains the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate to allow
detection of phosphatidylinositol phospholipase C (PI-PLC) activity, which, among the Listeria
spp., is unique to L. monocytogenes and L. ivanovii [157]. In combination with rapid biochemical
tests for differentiation of L. ivanovii and L. monocytogenes (e.g., L. monocytogenes confirmation
media, LMCM, Biosynth), this selective and differential medium thus provides a tool for more
rapid quantification of L. monocytogenes by direct plating or MPN procedures.
LIMITATIONS AND PITFALLS OF CURRENT ENVIRONMENTAL CHARACTERIZATION

METHODS FOR UNDERSTANDING THE ECOLOGY OF LISTERIA
Despite the continued development of improved selective and sensitive isolation procedures for
Listeria spp. and L. monocytogenes, culture-based methods show certain limitations that may affect
our ability to understand the ecology of Listeria. There is considerable evidence that use of a single
enrichment method may limit recovery of injured Listeria cells (if the medium is too selective) or
may not allow for effective recovery of Listeria cells in the presence of high levels of other
microorganisms (if less selective compounds are used). Although two-step enrichment procedures
may ameliorate these problems to a certain extent, recovery of injured Listeria cells in samples
with high levels of competing microorganisms represents a particular challenge. Furthermore, a
putative viable but not culturable (VBNC) state was recently proposed for L. monocytogenes [21];
cells in this state are unlikely to be recovered by any currently used enrichment procedures.
There also is considerable evidence that many environments as well as samples collected from
these environments can contain multiple Listeria species and/or multiple Listeria strains
[151,161,162]. For example, Ryser et al. [162] performed enrichment of ground beef, pork sausage,
ground turkey, and chicken, followed by ribotyping of up to 10 isolates per sample. Over half of
all positive samples contained more than one L. monocytogenes ribotype and the choice of enrich-
ment medium affected the subtypes recovered from a given sample. In some instances, detection
of certain L. monocytogenes ribotypes was only possible when 10 isolates from a sample were
typed. This clearly shows the challenging nature of characterizing the Listeria microbial diversity
using current detection methods.
Although it is increasingly recognized that collection and characterization of multiple isolates
may be necessary to evaluate the Listeria diversity in a given sample, enrichment methods appear
to affect recovery of different Listeria species and strains selectively. In addition to the fact that
L. innocua may outcompete L. monocytogenes during enrichment [51,126], it also appears that
different enrichment media lead to recovery of different bacterial subtypes from the same sample
[161]. Future application of molecular methods will provide an opportunity to improve our ability
to probe and understand the ecology of Listeria in natural environments. Similar to commonly used
16S rDNA-based approaches to characterize bacterial population structures, Listeria- and/or
L. monocytogenes-specific PCR primers [30,166,194] could be used to detect and characterize
Listeria isolates and their diversity directly, including through construction and sequencing of clone
libraries. The full power of these molecular methods can only be realized as sensitive, inexpensive,
and rapid methods for isolation from natural environments of DNA suitable for PCR amplification
are developed and applied [39,42,107,155,169].
MOLECULAR DETECTION AND SUBTYPING METHODS

Chapter 9 provides a comprehensive summary of subtyping methods for L. monocytogenes; appli-
cation of molecular subtyping methods is critical to our ability to understand the ecology and
transmission of L. monocytogenes in natural environments. Methods commonly used to subtype L.
monocytogenes include ribotyping, pulsed-field gel electrophoresis (PFGE), and, more recently,
multilocus sequence-based typing (MLST). Ribotyping [204,206] and PFGE [122,190] have been
applied in studies of the diversity of L. monocytogenes in natural environments. These DNA-based
methods define bacterial subtypes using restriction digestion of bacterial DNA to generate DNA
fragment banding patterns.
DNA fragment size-based subtyping methods have significant drawbacks. For example, despite
the existence of software packages for data normalization and analyses (e.g., Bionumerics, Applied
Maths, Sint-Martens-Latem, Belgium), these subtyping methods are often difficult to standardize.
As a consequence, the ease of exchanging and comparing subtype data among laboratories can be
severely limited. Although DNA fragment size-based subtyping methods have been used for cluster
analyses, they generally do not provide information amenable to inference of primary genetic
characteristics (i.e., nucleotide sequences) for evolutionary analyses. Long-term studies on the
ecology and evolution of Listeria require subtyping data that can be used to infer and quantify the
genetic relatedness of isolates, so DNA fragment size-based subtyping methods have limited utility
for these applications.
DNA sequencing-based methods are being developed and increasingly used for subtyping and
characterizing L. monocytogenes [30,165]. With these methods, complete or partial nucleotide
sequences are determined for one or more bacterial genes or chromosomal regions, thus providing
unambiguous and discrete data. Sequencing can target a single gene (single locus approach) or
multiple genes. Advantages of sequencing methods over DNA fragment-sized typing methods
include their ability to generate unambiguous data that are portable through Web-based databases
and that can be used for phylogenetic analyses [54].
Although a variety of DNA sequence-based subtyping strategies targeting virulence genes,
housekeeping genes, or other chromosomal genes and regions is feasible, multilocus sequence
typing (MLST), which is an extension of multilocus enzyme electrophoresis (MLEE), represents
a widely used strategy [30,48,53,54]. Ultimately, MLST may lead to integration of PCR-based
detection and subtyping in a single format, eliminating the need for culturing for certain applications
[53]. Molecular subtyping methods have been widely used and developed for L. monocytogenes
[78,202], but only limited information is available on molecular subtyping methods for Listeria
spp. [154,176,193]. Further development, particularly of MLST-based subtyping methods, is necessary
to allow comprehensive studies of the ecology of Listeria spp. in natural environments.
Our knowledge of the transmission and ecology of Listeria spp. and L. monocytogenes has
critically relied on application of classical and molecular subtyping methods for strain differenti-
ation. For the purpose of studying the ecology of L. monocytogenes in the natural environment or
otherwise, it is particularly important to bear in mind the limitations of all subtyping methods
currently used for L. monocytogenes strain differentiation and also to consider the advantages and
disadvantages of any specific subtyping method. As outlined later, subtyping data using a variety
of different methods have, for example, shown that human disease-associated strains can also be
found in farm and natural environments (see Figure 2.1 for examples); however, one must bear in
mind the relative discriminatory power of different subtyping methods when interpreting these
findings.
A
Location
Strain No. Source Date collected (city, state) Ribotype Ribotype Pattern
FSL J1-119 human epidemic 1985 Los Angeles, CA DUP-1038B Will be run 4/15/03
FSL C1-057 human sporadic Jul 1998 Cortland, NY DUP-1038B
FSL E1-128 animal (bovine) Jul 2001 Brooktondale, NY DUP-1038B
FSL N3-080 animal (caprine) Jan 2002 Burdette, NY DUP-1038B
FSL J1-179 food (hummus) Jun 1997 Albany, NY DUP-1038B
FSL L4-170 processing plant drain Oct 2002 Unknown, US DUP-1038B
FSL N3-796 farm (water trough) May 2002 Perry, NY DUP-1038B
FSL S4-049 urban lake water Jun 2001 Syracuse, NY DUP-1038B
FSL S4-169 urban playground soil Aug 2001 Rochester, NY DUP-1038B
FSL S4-257 urban park A soil Nov 2001 Albany, NY DUP-1038B
FSL S4-628 urban park B soil Jun 2002 Albany, NY DUP-1038B
FSL S4-774 urban sidewalk Aug 2002 Syracuse, NY DUP-1038B
FSL S4-780 urban drain water Aug 2002 Syracuse, NY DUP-1038B
FSL S4-926 urban stream water Oct 2002 Rochester, NY DUP-1038B
FSL S6-096 urban park vegetation Nov 2002 Albany, NY DUP-1038B
FSL S6-116 urban sidewalk Nov 2002 Albany, NY DUP-1038B
FSL S6-136 urban runoff water Nov 2002 Albany, NY DUP-1038B
FSL S4-941 pristine vegetation Oct 2002 Tompkins county, NY* DUP-1038B
B
Location
Strain No. Source Date collected (city, state) Ribotype Ribotype Pattern
FSL C1-103 human epidemic Oct 1998 Troy, NY DUP-1044A

FSL J1-193 human sporadic Jun 1997 Manhasset, NY DUP-1044A
FSL E1-054 animal (bovine) Mar 1999 Unknown, NY DUP-1044A
FSL N3-038 animal (caprine) Oct 2001 Lake Ariel, PA DUP-1044A
FSL F2-008 food (hispanic cheese) Jul 1999 New York, NY DUP-1044A
FSL H1-139 processing plant drain Mar 2000 Unknown, US DUP-1044A
FSL N3-243 farm (haylage) Mar 2002 Cazenovia, NY DUP-1044A
FSL S4-728 urban sidewalk Jul 2002 Albany, NY DUP-1044A
FSL S6-134 urban river water Nov 2002 Albany, NY DUP-1044A
FIGURE 2.1 Ribotypes of Listeria monocytogenes isolated from epidemic and sporadic listeriosis, foods, food-
processing environments, and the natural environment. Examples demonstrate that ribotypes that have caused
human epidemic listeriosis are temporally and geographically widely distributed among animal cases of listeriosis,
foods, food-processing environments, and the natural environment. Ribotype and associated isolate information
were obtained from the PathogenTracker database (www.pathogentracker.net). (A) Ribotype DUP-1038B was
implicated as cause of a human listeriosis outbreak in Los Angeles, California (1985), linked to consumption of
Mexican-style cheese. (Linnan, M. J. et al., 1988, N. Engl. J. Med. 319:823–828.) (B) Ribotype DUP-1044A
was implicated as the cause of a multistate human listeriosis outbreak in the United States (1998 to 1999) linked
to consumption of contaminated hotdogs. (Anonymous, 1999, MMWR 47:1117–1118.) (*) = Sample collected
from the Connecticut Hill Wildlife Management area; county location is provided because city was not applicable.
Although use of less discriminatory methods (e.g., MEE, single enzyme ribotyping) to characterize
a given set of isolates may show that the same strains are found among isolates from human clinical
cases and natural environments, application of more discriminatory subtyping methods (e.g., PFGE)
may further differentiate strains. By no means does this make MEE or ribotyping data meaningless;
rather, it indicates different levels of relatedness. We do not yet understand the specific levels of
relatedness determined by each subtyping method. However, less discriminatory methods will
generally indicate that two indistinguishable isolates may share a less recent common ancestor as
compared to two indistinguishable isolates using a very discriminatory method.
Subtype characterization by a less discriminatory method thus can still provide a very
powerful tool to probe the distribution of different clonal groups (i.e., a group of genetically
closely related isolates) and to differentiate broadly distributed clonal groups (e.g., clonal groups
found in natural environments, among human and animal clinical cases and foods) from clonal
groups that occupy a specific niche (e.g., cause disease in only a single specific host species).
The power of less discriminatory subtyping methods has been demonstrated by the broad
contributions of MEE studies to our understanding of bacterial population genetics [17,24,62,63,
113,142,160].
ECOLOGY OF LISTERIA SPECIES IN DIFFERENT ENVIRONMENTS

Listeria spp. are generally considered ubiquitously distributed in the natural environment. Listeria
spp. and L. monocytogenes have specifically been isolated from many different environments
including soil [196], water [195], vegetation [196,199], sewage [3,4,34,67,195], animal feeds
[32,58,60,121,162,175,188,190,203,205], farm environments [45,63,101,102,179,184,192], and
food-processing environments [43,89,95,105,112,151,160].
Although many reports indicate that L. monocytogenes and L. innocua may represent the most
common Listeria spp. in many natural environments, others have reported considerable prevalence
of other Listeria spp. in specific natural environments. For example, a survey of an urban setting
showed that L. ivanovii and L. seeligeri represented the most common Listeria spp. isolated from
soil samples [127]. Cox et al. (1989) isolated L. monocytogenes and L. innocua from environmental
samples collected from a sawmill; however, L. ivanovii represented the most prevalent Listeria spp.
isolated [37]. Interestingly, although L. ivanovii, as well as L. welshimeri and L. seeligeri (both of
which were not isolated in their study) ferment xylose, a common decomposition product of wood,
L. monocytogenes and L. innocua do not ferment xylose.
Further studies on the distribution of different Listeria spp. and subtypes in different natural
environments will be needed to develop a true understanding of the ecology of Listeria and L.
monocytogenes, including the reservoirs and niches of the different Listeria spp. and different clonal
groups. This knowledge will also be necessary to understand transmission pathways of the patho-
genic Listeria and the sources and spread of human and animal infections.
SURVIVAL AND MULTIPLICATION OF LISTERIA SPP. UNDER

ENVIRONMENTALLY RELEVANT STRESS CONDITIONS
L. monocytogenes is distinguished among foodborne pathogens in that it can tolerate high (up to 20%)
salt concentrations, can multiply over a wide range of temperatures (1 to 45°C), and can adapt to
and survive acid stress. In contrast to other non-spore-forming bacteria that cause foodborne illness,
L. monocytogenes appears to be able to survive longer under adverse environmental conditions
[195]. The ability of L. monocytogenes to colonize, multiply, and persist in the food-processing
environment and on food-processing equipment likely also reflects its ability to survive in the
natural environment for extended periods.
Data available on stress resistance and survival characteristics of Listeria spp. are more limited
than those available for L. monocytogenes. In general, it is hypothesized that other Listeria spp.
show resistance to environmental stress (acid, salt, temperature, etc.) similar to that observed for
L. monocytogenes because L. innocua and other Listeria spp. are commonly found in foods
[9,31,149,209] and food-processing environments [152,181]. Experimental studies comparing stress
resistance between L. monocytogenes and other Listeria spp., including survival and growth of
temperature stress [147], salt stress [18], low water activity [140]; in the presence of various
antimicrobial agents [183]; and under various combined stress conditions [114,156,163], also
support that all Listeria spp. share similar stress resistance characteristics.
Recent sequencing of the genome of one L. monocytogenes strain and one L. innocua strain
[76] also showed that these two species share a large number of predicted genes that encode various
components of potential stress response systems including regulatory proteins, transporters, and
transcriptional regulators [29,76]. Further comparative genomic analyses on presence and transcrip-
tion of various stress response genes in different Listeria spp. and subtypes will provide a unique
opportunity to increase our understanding of the stress response pathways that facilitate Listeria
survival in the environment and under stress conditions. Laboratory studies on survival under
controlled environmental stress conditions can help to understand the biology of Listeria, but data
on Listeria survival in natural environments also critically contribute to our understanding of Listeria
transmission and ecology. The following sections attempt to summarize our current knowledge on
distribution, prevalence, and survival of L. monocytogenes and Listeria spp. in different natural
environments. In addition, Table 2.1 provides a summary of data from various studies on the
persistence of L. monocytogenes in different natural and farm environments.
LISTERIA SPP. IN SOIL AND VEGETATION

L. monocytogenes as well as other Listeria spp. appears to be commonly present in soil and vegetation
in the natural environment. Most studies on Listeria in the natural environment conducted to date
have almost exclusively focused on farm environments and associated croplands. Interestingly, most
studies on Listeria in natural environments not associated with farms indicate that prevalence of L.
monocytogenes is lower than that of other Listeria spp. [127,166]. MacGowan et al. [127] specifically
reported that L. seeligeri was more frequently isolated from soils than L. innocua or L. monocytogenes.
In early work Weiss and Seeliger (1975) isolated Listeria spp. from plant samples collected
from cornfields (9.7% of samples positive), grain fields (13.3%), cultivated fields (12.5%), uncul-
tivated fields (44%), meadows and pastures (15.5%), forests (21.3%), and wildlife feeding areas
(23.1%) in southern Germany [197]. Soil samples taken at a depth of 10 cm showed a significantly
lower Listeria spp. prevalence as compared to surface soil samples. Although the original paper
by Weiss and Seeliger (1975) reported L. monocytogenes prevalence, only 37 of 103 Listeria isolates
elicited disease consistent with listeriosis in a mouse bioassay. This indicates that as many as 64%
of these isolates may have been Listeria spp. other than L. monocytogenes or L. ivanovii, thus
suggesting that these isolates represented a variety of Listeria spp.
Welshimer and Donker-Voet [199] did not isolate L. monocytogenes from soil or dead vegetation
sampled from agricultural sites in early autumn, yet soil and decayed vegetation sampled the
following spring were nearly all positive for the organism. Listeria isolates from this study were
also evaluated for virulence in mice and not all were found to be virulent [199], again suggesting
the occurrence of species other than L. monocytogenes. Clearly, changes in the Listeria taxonomy
that have occurred over the years may require careful interpretation of older studies. These studies
may have used a broader definition of a L. monocytogenes sensu lato, which might include Listeria
spp. other than those currently classified as L. monocytogenes. Only very limited recent data are
available on the presence and distribution of L. monocytogenes and other Listeria spp. in soils,
limiting our understanding of the soil ecology of these organisms [127].
More recently, Fenlon et al. [63] examined samples of grass, leaves, stems, and roots/stems
from two crops of growing sward before harvesting. L. monocytogenes was not detected in any of
the samples, but L. innocua and L. seeligeri were isolated from 3 of 10 samples from the root/stem
area. L. monocytogenes was detected though in 9 of 10 samples of cut grass from the same crops
that had wilted for 24 h before ensiling. Whittenbury [201] had demonstrated the importance of
the sheath area as a source of lactic acid bacteria, so Fenlon [63] hypothesized that the higher
incidence of L. monocytogenes in harvested (processed) grass compared with other plant products
could be attributed to the presence of a sheath of decaying plant material at the base of the plant
that might act as an inoculum at harvest. Although this process may contribute to contamination
of grass with Listeria during the ensiling process, Listeria contamination of plant-based feeds and
foods probably more likely results from direct deposition of animal feces, spreading of animal
waste and sewage sludge as fertilizer, or from indirect contamination via feces-contaminated soil.
TABLE 2.1
Survival of L. monocytogenes in Various Environmental Samples
Sample Storage Temperature (°C) Survival (days)
Soil
Sterile soil (I) Outside: winter/spring 154
Clay soil (I) 24–26 225
Sealed tubes 24–26 67
Fertile soil (I)
Sealed tubes 24–26 295
Cotton-plugged tubes 24–26 67
Top soil (I)
Exposed to sunlight NG 12
Not exposed to sunlight NG 182
Moist soil NG ~497
Dry soil NG >730
Soil 4–12 240–311
Soil 18–20 201–271
Fecal material
Cattle feces (NC) 5 182–2190
Moist horse/sheep feces (I) Outside 347
Dry horse/sheep feces (I) Outside 730
Sheep feces Outside 242
Liquid manure Summer 36
Liquid manure Winter 106
Sewage
Sewage sludge cake (NC)
Surface 28–32 35
Interior 48–56 49
Sprayed on field Outside >56
Water
Sterilized pond water (I) Outside 7
Unsterilzed pond water (I) Outside <7–63
Pond water 35–37 346
Pond water 15–20 299
Pond water/ice 2–8 790–928
Pond/river water 37 325
Pond/river water 2–5 750
Water Outside 140–300
Distilled water (I) 4 <9
Animal feed
Silage (NC) 4 450
Silage (NC) 5 180–2190
Mixed feed Outside 188–275
Oats (I) Outside 150–300
Hay (I) Outside 145–189
Straw (NC/I) ca. 22 365
Straw (I) Outside 47–207
Straw Outside: summer 23
Straw Outside: winter 135
Notes: (I) = inoculated; NG = not given; (NC) = naturally contaminated.
Sources: Adapted from al-Ghazali, M. R. and S. K. al-Azawi, 1988, J. Appl. Bacteriol.
65:203–208 and 209–213; Amtsberg, G., 1979, Dtsch. Tierarztl. Wochenschr.
86:253–257; and Mitscherlich, E. and E. H. Marth, 1984.
Supporting the importance of animals as sources of L. monocytogenes in soil and on plants,

Fenlon et al. [63] did not find any L. monocytogenes in the few soils examined that were associated
with vegetable crops, but isolated L. monocytogenes from soil collected from fields where cattle
or sheep on silage diets had been kept. Although lettuce and vegetables generally show low
L. monocytogenes and Listeria spp. prevalence [57,63,77], preharvest contamination of vegetables
with L. monocytogenes can provide a source for human listeriosis infections. For example, an
outbreak involving 42 human cases in Nova Scotia in 1981 was linked to consumption of coleslaw.
This coleslaw was produced from cabbage harvested from fields fertilized with untreated sheep
manure—obtained from a farm with a history of ovine listeriosis [168].
Some studies have focused on describing the occurrence of Listeria in the natural environment;
others have taken a more experimental approach to evaluating the ability of Listeria to survive in
soil. Survival of L. monocytogenes in soil likely depends on a variety of factors, including soil
type, moisture content, and water activity. Welshimer [198] used cotton wool-plugged tubes con-
taining clay or fertile soils inoculated with L. monocytogenes and stored for 67 days at 24 to 26°C,
to show that cell numbers decreased in both by seven orders of magnitude as the soils dried out.
When these experiments were repeated with tubes sealed to prevent water loss, L. monocytogenes
numbers in clay soil still decreased by seven orders of magnitude, but numbers in fertile soil
decreased by only two orders of magnitude.
In a study using autoclaved soils inoculated with L. monocytogenes at approximately 5 × 10–2
CFU/g and stored at ambient winter temperatures ranging from –15 to +18°C, Botzler [25] found
an increase in L. monocytogenes numbers to 1 × 107 CFU/g over a 154-day period. It is not known
what effect the autoclaving process had on Listeria growth by increasing availability of nutrients
and eliminating competing organisms. Watkins and Sleath [195] demonstrated that there was little
decrease in L. monocytogenes numbers in soil on land to which sewage sludge was applied; an
initial inoculum of 170 CFU/100 g was recovered consistently over an 8-week period. Salmonellae,
applied at a similar rate, decreased to undetectable levels in less than 4 weeks.
L. monocytogenes appears to be able to survive and possibly even multiply in some or many
soil types; damp surface soil and the presence of decaying vegetation appear to provide a particularly
suitable environment for L. monocytogenes and Listeria spp. However, there appears to be
no convincing evidence that soil or certain soil-associated niches serve as true reservoirs for
L. monocytogenes. Similarly, our understanding of the soil ecology of other Listeria spp. is also
extremely limited.
LISTERIA SPP. IN SEWAGE

Sewage appears to be contaminated commonly with L. monocytogenes and other Listeria spp. For
example, Watkins and Sleath [195] reported L. monocytogenes levels >18,000 CFU/L in trade
effluents associated with animals and sewage sludge after treatment. Levels of the organism in
settled raw sewage from primary tanks ranged from 700 to 18,000 CFU/L. Much higher levels of
Listeria spp. were reported [72] in a West German sewage treatment plant; untreated waste and
filtered effluent contained 103 to 105 CFU Listeria spp./mL. The fact that there was only a 10-fold
reduction in numbers between untreated and treated waste suggests that biological oxidation may
not be an effective method for eliminating viable Listeria in sewage.
Studies in northeastern Scotland [63] showed that L. monocytogenes numbers ranged from 120
CFU/mL in untreated sewage to 2–21 CFU/mL in treated effluent. Anaerobic digestion reduced numbers
to 1.1 CFU/g, and lime-treated sludge had no culturable Listeria species. Al-Ghazali and al-Azawi
[3,4] specifically studied survival of L. monocytogenes during sewage treatment and in stored sewage
sludge cake in Iraq. A decrease of 85 to 97% in viable Listeria numbers occurred during activation
and digestion stages of the sewage treatment process [3] and <3 to 15 L. monocytogenes CFU/mL
were recovered from final effluent and sludge cake. These same authors [4] also demonstrated that
the listeriae were inactivated when sludge was stored in direct sunlight for at least 8 weeks.
Inactivation was slower in the interior of the sludge piles.
Studies in the United Kingdom showed that when L. monocytogenes–contaminated sewage
sludge was spread on land, numbers of the pathogen failed to decrease over an 8-week period
[195]. In 2003, Garrec et al. investigated the impact of sludge treatments on five types of sludge
in three different treatment plants and found that only liming the sludge reduced Listeria spp.
loads to less than detectable levels [71]. They found that 87% of dewatered sludge contained
Listeria spp., while 73% contained L. monocytogenes. In addition, 96% of sludge stored in
tanks contained Listeria spp. and 80% contained L. monocytogenes. Concentrations of L.
monocytogenes found in the dewatered sludge were lower (0.15 to 20 MPN/g dry matter) than
concentrations found in sludge stored in tanks (1 to 240 MPN/g dry matter) [71]. In a survey
of five types of sludge, Italian investigators also found the highest concentrations of Listeria
spp. (L. monocytogenes, L. innocua, L. welshimeri, and L. grayi) in activated sludge (2,743
MPN/g dry matter); the lowest concentration was found in digested sludge (6 MPN/g dry matter)
[41]. Both authors note that sludge from these treatment plants is used to fertilize agricultural
fields and thus may represent a constant source of Listeria contamination of crops bound for
human or animal consumption [41,71]. Because fecal contamination has been linked to one
foodborne outbreak of human listeriosis associated with coleslaw [168], caution should thus be
taken when applying potentially contaminated sludge and animal wastes on crops that may
subsequently be consumed raw.
Using isolates from a defined geographic region, Lozniewski et al. used serotyping, phage
typing, and PFGE with two enzymes to further probe for links between L. monocytogenes present
in sewage sludge and human listeriosis cases [121]. In this study, reported in 2001, all sludge
isolates except one were distinguishable from human isolates; two human isolates were found to
be indistinguishable from one sludge isolate, although no epidemiological data were available to
further evaluate the relevance of these findings.
Thus, although it has been shown that sewage used as fertilizer often contains L. monocytogenes
and other Listeria spp. and that one human listeriosis outbreak has been linked to contamination
by manure application [168], further studies are needed to define the importance of sewage as a
source of human and animal listeriosis cases. Optimized sewage treatment processes and appropriate
sludge and sewage application guidelines may help to minimize Listeria dissemination into the
environment and possibly subsequent introduction into the human or animal food chain.
LISTERIA SPP. IN WATER

The ubiquitous nature of L. monocytogenes and Listeria spp., in conjunction with use of surface
waterways for discharge of sewage effluents, inevitably results in the presence of these organisms
in a wide range of surface waters, including lakes, rivers, and streams. For example, Dijkstra [47]
found L. monocytogenes in 21% of the surface water samples in the northern Netherlands. Although
widely distributed, L. monocytogenes has been reported to be present in varying numbers in water.
Watkins and Sleath isolated L. monocytogenes from seven rivers in the United Kingdom at levels
ranging from 3 to >180/L [195]. A study of the course of the River Don in northeastern Scotland
[63] showed that between 42 and 53% of sites were positive for L. monocytogenes (with levels
ranging from 10 to 350 CFU/L) with no measured factor, seasonal or otherwise, related to presence
or numbers of L. monocytogenes. However, a survey of 128 fresh-water samples from four rivers
and one lake in Greece showed a rather low prevalence (4%) of L. monocytogenes [12] and a
smaller survey of 15 groundwater samples in Belgium yielded only one L. monocytogenes–positive
sample [187].
Few reports have examined occurrence of Listeria spp. other than L. monocytogenes in surface
waters. One study of surface waters (canals, rivers, ponds, and lakes) in the United Kingdom
[67] analyzed 30 samples (100 mL) from 21 sites, finding 8 sites positive for L seeligeri (27%),
1 for L. innocua, and 1 for L. welshimeri, and no sites positive for L. monocytogenes. In another
study, Bernagozzi et al. [20] cultured 15 samples from two rivers in Italy for presence of Listeria
and found that 47% of the samples contained at least one Listeria sp. From their positive cultures
they isolated three different species, including six L. monocytogenes, six L. innocua, and two
L. grayi [20].
In addition to studies on Listeria spp. in fresh waters, some data are also available on Listeria
spp. in marine environments. Soonthoranant and Garland [177] found L. monocytogenes in 35 to
100% of samples collected from discharges of a sewage treatment pond and from fish-processing
factory effluents, which also contained sewage. Inshore marine waters, which eventually received
these discharges, contained L. monocytogenes in 6.6% of samples. They also found L. monocytogenes
in 15% of Pacific oysters and blue mussels, a fact of interest because these are bottom-dwelling
filter-feeders that may naturally concentrate bacterial cells as a result of their feeding behavior.
Colburn et al. [34] found that 81% of fresh water and 62% of low-salinity waters harbored Listeria
spp., including L. monocytogenes. Specifically in bay water, one of three samples was found to
contain three Listeria spp. including L. monocytogenes, L. innocua, and L. welshimeri. They also
found L. innocua in 1 of 35 oysters sampled [34]. Furthermore, Motes [134] isolated L. monocyto-
genes from shrimp caught off the U.S. Gulf Coast, and Destro et al. [43] showed that some strains
associated with shrimp can persist in processing plants and enter the final product.
At present no epidemiological data suggest the occurrence of direct infection of humans through
L. monocytogenes–contaminated water. However, Gray et al. [84] successfully infected two sheep,
four goats, and one cow with L. monocytogenes–contaminated water. Based on quantitative esti-
mates from natural water samples [20,63,195], it seems unlikely that L. monocytogenes contami-
nation in water could reach sufficient numbers to cause human infections, unless gross fecal
contamination occurs. It is conceivable, however, that L. monocytogenes–contaminated water might
serve as an indirect source of human infections from marine or freshwater fish or seafood harvested
and stored for extended times under refrigeration [98]. Appropriate thermal processing and/or
application of listeriocidal treatments can prevent Listeria contamination of ready-to-eat products
though, if post-processing contamination is prevented [180].
LISTERIA SPP. IN ANIMAL FEEDS

Most formulated animal feeds have low levels of available water, which restricts multiplication
of Listeria spp. and L. monocytogenes. The same is true of hay and cereal grains; although
L. monocytogenes has been reported in such materials [70], the numbers are unlikely to reach levels
that present a serious risk to animals. Many formulated feeds are sold in pellet form and will have
received a degree of heat treatment capable of killing a high proportion, if not all, Listeria present.
The animal feed most closely linked with animal listeriosis is silage, and this association is well
documented. Olafson [145] noted the link between “Listerella” encephalitis in ruminants and silage
in 1940, and in 1960, Gray [82] reported isolating L. monocytogenes from the fetus of a pregnant
mouse fed poor-quality silage implicated in death and abortion in cattle. Identical serotypes
of L. monocytogenes were isolated postmortem from the mice and cattle.
Today, numerous reports link the feeding of silage with listeriosis outbreaks in sheep and cows
[60,65,74,75,88,120,138,206]. Much of this problem can be attributed to the high numbers of L.
monocytogenes present in contaminated silage [59,60] as compared with other animal feeds. In
good-quality silage prepared from grass, maize, whole crop cereals, or leguminous plants, which
may or may not be wilted (dried to optimum moisture content) in the field before ensiling, the
onset of anaerobic conditions stimulates the indigenous or inoculated lactic acid bacteria to multiply
quickly. As these bacteria expand to populations as high as 109 CFU/g within 48 h, they convert
the plant sugars to lactic acid, causing a rapid decrease in pH [129] (well-preserved silage generally
has a pH < 4.5). Such acidic conditions inhibit the growth of spoilage microorganisms and Listeria.
Higher dry-matter silage tends to have a higher pH, but lower levels of available water in these
silages may help minimize Listeria growth.
Grass silage in cooler, wetter climates tends to have lower sugar levels and higher moisture
contents resulting in a poorer, slower fermentation. These silages may be more susceptible to
L. monocytogenes contamination and growth than grass and maize silage in countries with warmer
climates. Although Listeria die in well-fermented silage, if the pH increases before all bacterial
cells are killed, the surviving Listeria will multiply. Dijkstra [44] showed that L. monocytogenes
can survive 4 to 6 years in naturally contaminated silage. Fenlon et al. [63] also noted that
L. monocytogenes could survive over 1 year in bags used to wrap big bales and stored for reuse.
Fortunately, much of the L. monocytogenes contamination in silage occurs in visibly moldy areas,
and if these are removed and discarded before feeding, exposure of animals to high levels of
L. monocytogenes can be considerably reduced [61].
In addition to commonly fed types of silages, more obscure feed sources of animal listeriosis
have also been reported, including silage made from orange peel and artichokes [191]. Several
outbreaks in Canada and the northern United States have also reportedly been caused by cattle
feeding on ponderosa pine needles [2].
A variety of studies have confirmed that L. monocytogenes contamination is most frequently
associated with poor-quality silage. In 1979, Grønstol [88] analyzed 291 grass silage samples from
113 farms and isolated L. monocytogenes from 22% of samples with a pH <4.0, from 37% of
samples with a pH of 4.0 to 5.0, and from 56% of samples with a pH >5.0. In a study of clamp
silage implicated in an outbreak of listeriosis in cattle [60], levels of L. monocytogenes in excess
of 12,000 CFU/g were found in the surface layer (pH 8.3 to 8.5), whereas no Listeria spp. were
detected 15 cm into the silage mass where the pH was 4.5. In another study [64], the extent of
Listeria contamination in silage was directly related to its hygienic quality as measured by the
numbers of Enterobacteriaceae present. This case indicates that management of postharvest pro-
cessing of the grass can markedly affect numbers of Listeria present.
Gitter [74] noted that from 1975 to 1985, the incidence of listeriosis in sheep in Great Britain
increased from less than 50 cases per year to over 250. During the same period, only a small
increase in the number of cattle listeriosis cases was observed. In addition, the pattern changed
from isolated single cases to much larger flock-wide outbreaks. This was attributed to a change
in the conserved forage used to feed sheep. In Great Britain, sheep are mainly kept on upland
areas and before this time were traditionally fed hay. The wet climate of these hill farms is not
conducive to the making of good-quality hay, and silage was not an economic option before the
mid-1970s because it required expensive capital outlay for silos. The invention of the big-baler
and the half-ton round bale made silage feeding feasible by baling grass and sealing it in large
plastic bags to make silage. Unfortunately, some of the early attempts to make silage in this way
were not very successful, and baled silage was of poorer quality than clamp silage [58]. L.
monocytogenes appears to be predominantly a surface problem and big bales have a much greater
surface area than clamp or silo silage, so the potential for contamination to develop is much
greater in incorrectly prepared bale silage and if aerobic deterioration takes place. Fenlon [59]
reproduced the problem in 500-g laboratory bales ensiled in plastic bags held for 2 to 3 weeks
before testing. L. monocytogenes was detected at levels of ≥1.1 × 106 CFU/g silage in moldy
areas near the tie end of the bag where air infiltrated. In the center of the bags pH remained at
3.8, silage appeared to be of good visual quality, and no Listeria were isolated. Donald et al.
[50] specifically demonstrated the relationship among oxygen tension, pH, and L. monocytogenes
growth by infusing laboratory silos with gas mixtures containing from 0.1 to 5.0% oxygen and
showing that the greater the oxygen level was, the more rapidly the pH rose with subsequent
multiplication of Listeria.
In addition to systemic listeriosis, silage has also been implicated as a cause of inflammation
of the iris (iritis) by L. monocytogenes [194], particularly in cattle. This condition appears to occur
when cattle and sheep burrow their heads into bale silage in self-feeding systems and the eye
becomes infected via abrasions caused by stems of grass contaminated with the organism. Modi-
fying feeding practices to prevent eye contact with silage is the best preventative measure [119].
Although only limited studies are available on the molecular ecology of L. monocytogenes in
most natural environments, a variety of subtyping studies on L. monocytogenes in silage have been
published [17,120,190,203,205,206,208]. Almost all studies were specifically performed to track
the sources of animal listeriosis cases, but the data available provide some interesting insight
into the molecular ecology of Listeria. Many studies showed that a considerable diversity of
L. monocytogenes can be found in an individual silage sample, a single silo, and on a given farm.
Wiedmann et al. reported two outbreaks of epizootic listerial encephalitis, one in sheep and
one in goats [206]. In both outbreaks, L. monocytogenes was isolated from silage and brain tissue
samples and subtyped using random amplified polymorphic DNA analysis. Subtype analysis
revealed two distinct L. monocytogenes strains, one that was identical to the sheep brain isolate,
in the silage associated with the outbreak in sheep. In the goat outbreak, three brain isolates and
one silage isolate all demonstrated different subtypes. Wiedmann et al. further reported an additional
analysis of the previous goat and sheep outbreaks in addition to two bovine listeriosis outbreaks
using ribotyping [205]. For all but one outbreak, L. monocytogenes strains represented by the same
ribotype were found in clinical and silage samples. Additional L. monocytogenes strains with
ribotypes different from those of the respective clinical samples were isolated from all silage samples,
except for the outbreak in goats. On one farm five different ribotypes were isolated from silage
samples collected from a single bunker silo, and on another farm three different L. monocytogenes
ribotypes were isolated from a single 25-g silage sample.
Similarly, Baxter isolated three different MEE types of L. monocytogenes from silage collected on
a single farm with an outbreak of listerial encephalitis; interestingly, all of the silage isolates were
distinct from the strain isolated from clinical specimens collected from infected animals on this
farm [17]. Low et al. [120] also found six distinct phage types among 45 L. monocytogenes isolates
from baled silage. These studies provide clear evidence that a diverse population of L. monocyto-
genes strains exists in farm environments, of which some may be more likely than others to cause
disease [205].
Investigations of an epizootic of listerial encephalitis in a flock of 655 sheep showed that all
seven clinical L. monocytogenes isolates had ribotypes identical to those for isolates from farm
equipment used to transport silage. Corn silage, which was not fed to the sheep, also contained L.
monocytogenes of the same pattern type as defined by ribotyping. L. monocytogenes was not isolated
from the stored haylage designated for feeding the sheep (the cut-off point for isolation was <102 CFU/g).
Based on ribotype results, it appears that corn silage cross-contaminated the haylage destined for
the sheep during handling with a front-end loader, providing possible evidence for environmental
transmission. In this case, suspension of silage feeding coincided with cessation of listeriosis cases.
In another investigation of listeriosis among goats, Wiedmann et al. (1999) found that during
a 16-month period, 10 goats with listeriosis were identified in two herds that shared three bucks,
including one that died of listeriosis [208]. Analysis revealed that a single genetically unique
L. monocytogenes strain had infected all goats from which isolates were available. Silage was not
fed to either herd, and L. monocytogenes was not isolated from vaginal or rectal swab specimens
obtained from healthy goats or from samples of feed. Three bucks were the only common elements
between the two herds. The authors hypothesized a venereal route of transmission in this outbreak,
but environmental contamination other than silage could not be excluded as a source [208]. These
case studies further support that the environment can also represent a direct source for animal infection.
REGULATION OF LISTERIA GENE EXPRESSION DURING ENVIRONMENTAL SURVIVAL

AND MULTIPLICATION
Studies on the presence and persistence of Listeria spp. and L. monocytogenes in different natural
environments have significantly advanced our understanding of the ecology of these bacterial
species. Studies on gene expression by Listeria spp. and L. monocytogenes in the environment
outside a host and under conditions typically encountered in the environment may provide critical
information on the biology of these organisms. Although the genetics of virulence and regulation
of virulence gene expression in L. monocytogenes are covered in more detail in chapter 5, it is
important to emphasize that negative regulation of virulence gene expression during survival outside
a host may be critical for a saprophytic pathogen such as L. monocytogenes.
Expression of virulence genes and synthesis of virulence factors outside the host may represent
a burden compromising the ability of L. monocytogenes to survive in the natural environment and
limiting its potential for transmission [189]. Evidence is accumulating that L. monocytogenes has
evolved signal-sensing and signal-transducing mechanisms to sense environmental signals to adapt
virulence gene expression to the particular needs of saprophytic versus parasitic lifestyles [189].
Although temperature appears to be one environmental signal controlling virulence genes, other
signals also appear to be contributing to down-regulation of virulence gene expression outside a
host [189]. For example, some studies suggest that the main L. monocytogenes virulence gene
regulator, prfA, is down-regulated by the presence of the disaccharide cellobiose, a product of plant
decomposition found ubiquitously in the environment [146].
In addition, the presence of bioavailable iron also appears to down-regulate virulence gene
expression [35]. The availability and application of microarrays for studying global gene expres-
sion patterns in L. monocytogenes [133] will provide unique opportunities to further probe and
compare Listeria gene expression under different environmental conditions. This will thus help
us to improve our understanding of strategies Listeria uses to facilitate environmental survival
and transmission.
TRANSMISSION DYNAMICS
As outlined earlier, considerable data are available on prevalence of L. monocytogenes in different
environments; however, we still lack a good understanding of overall transmission dynamics of
L. monocytogenes. Even though L. monocytogenes is widely distributed in the natural environment
and humans and animals are likely to come in frequent contact with L. monocytogenes through
a variety of sources, food or feedborne exposure, respectively, seem to be the most common
sources of infection. Considering the apparently frequent exposure of humans and animals to
L. monocytogenes, the rare occurrence of clinical disease raises the intriguing question of the
importance of human and animal infections for survival of L. monocytogenes. On the one hand,
human and/or animal infections may represent a critical part of L. monocytogenes survival by
providing a mechanism for rapid multiplication to high numbers and wide environmental dispersal,
e.g., through fecal shedding.
On the other hand, L. monocytogenes may be an “accidental” pathogen with humans or farm
ruminants representing dead-end hosts that do not critically contribute to survival of the species L.
monocytogenes. In the latter case, survival in the natural environment, including possibly some yet
undefined host species (e.g., protozoans, lower vertebrates, or mammals other than the apparent
human and ruminant hosts), may be critical to the evolutionary success of this bacterial species.
Continued research on the ecology and evolution of L. monocytogenes and Listeria spp. will be
necessary to define the transmission of this pathogen and to control and reduce human and animal
infections. This section reviews our current knowledge on the transmission of L. monocytogenes
with a particular focus on the role of the natural environment in the transmission of this pathogen.
NATURAL ENVIRONMENT AND TRANSMISSION OF HUMAN LISTERIOSIS

Overall, there is strong evidence that most human listeriosis cases are caused by foodborne
infections, usually from contaminated ready-to-eat foods [93]. Although it is conceivable that
L. monocytogenes can enter the food chain at many and possibly even at nearly any point, food-
processing environments appear to be of particular importance as sources for introduction of L.
monocytogenes into the food system [181]. Food contamination incidences can often be tracked
back to postprocessing contamination in food-processing plants; however, the contribution of
contamination at retail and in-home or at restaurants to human foodborne listeriosis infections has
not been clearly defined.
The role of contaminated raw animal-based agricultural products (e.g., milk, meat) as a direct
source of L. monocytogenes contamination of ready-to-eat food products is likely to be minimal
because commercially applied heat treatments generally kill L. monocytogenes effectively enough
to provide an appropriate margin of safety. Although infected animals and contaminated agricultural
environments rarely appear to be a direct cause of human infections, animal sources can play an
important role in animal-derived food products that are not processed before consumption (e.g.,
raw milk). In addition, manure from infected or shedding animals may represent a source of food
contamination, such as in the listeriosis outbreak in Nova Scotia in 1981, which was linked to
consumption of coleslaw [168].
Transmission of Listeria between Natural and Food-Processing Environments
Considerable numbers of cross-sectional and longitudinal studies on the subtype diversity and
ecology of L. monocytogenes in food-processing environments have been performed, but only a few
studies have specifically probed the transmission of Listeria between natural and food-processing
environments. Longitudinal studies in different types of food-processing facilities have demonstrated
that a large diversity of L. monocytogenes strains exists in different foods and food-processing
environments.
In many food-processing plant environments persistent and transient L. monocytogenes strains
appear to be present [97,143,159,173], although some plants appear to contain only transient strains
[143]. Persistent strains can be present in processing plants for months and years, up to 7 to 12
years [109]. For example, Harvey and Gilmour [89] used MEE and RFLP analysis to compare L.
monocytogenes isolates from four milk-processing centers and two dairy farms in Northern Ireland
with food and clinical isolates. They found that recurrent strains, specific to each dairy processor,
colonized plants over long periods. When isolates from a poultry-processing environment were
examined with RAPD analysis, Lawrence and Gilmour [113] showed that a single RAPD type was
predominant in the raw processing environment over the 6-month period of the study and survived
the clean-in-place schedules. In a follow-up study 12 months later, the same RAPD type was again
isolated from final cooked products.
Norton et al. showed that specific L. monocytogenes ribotypes persisted over time in the
environments of two of the three smoked-fish processing plants studied [143]. Similar findings
were reported by a variety of groups, which also showed the persistence of specific L. monocyto-
genes subtypes in different food-processing plants, including smoked fish, poultry, meat, and dairy
plants [1,13,97,113,137,160,173]. Although it is clear that a large diversity of L. monocytogenes
strains exists and persists in food-processing environments, information on how these strains enter
that environment is limited. In one of the few studies probing the linkages between L. monocyto-
genes and Listeria in the natural environment, Arimi et al. used automated ribotyping to probe for
links between on-farm sources of Listeria contamination (dairy cattle, raw milk, and silage) and
contamination of dairy-processing environments [10]. Among a total of 475 Listeria isolates from
20 different dairy-processing facilities and different farms, 8 L. monocytogenes and 12 non-L.
monocytogenes ribotypes were found in dairy-processing and farm environments [10]. Although
the study design did not allow for conclusions on the directionality of transmission, these data
support that Listeria clonal groups found in farm environments can also be present in dairy-
processing environments.
Ecology of Human Outbreak-Associated L. monocytogenes Strains
Until the 1980s, when several large outbreaks of human listeriosis occurred, listeriosis was con-
sidered a rare and sporadic human disease. As outbreak and case investigations implicated contam-
inated foods as the most likely sources of human L. monocytogenes infections, increased efforts
were placed on linking human listeriosis cases to presence of this pathogen in foods and in the
environment. Although most human listeriosis cases have been thought to be sporadic, multiple
large human listeriosis outbreaks have been reported over the last 20 to 25 years. In addition, recent
evidence suggests that a higher number of human listeriosis cases than previously assumed might
represent outbreaks or single source clusters [168].
Most large human listeriosis outbreaks have been associated with L. monocytogenes serotype
4b strains, including outbreaks linked to contaminated coleslaw [169], soft cheese [22,104], p â té
[73], and pork tongue [103]. Additional outbreaks caused by serotype 1/2b strains have been linked
to milk [40], rice salad [164], and imitation crabmeat [56]. Only few outbreaks have been linked
to other serotypes, including an outbreak in Finland caused by a serotype 3a strain and linked to
contaminated butter [125] as well as a L. monocytogenes serotype 1/2a outbreak in the United
States linked to sliced turkey [69]. L. monocytogenes serotype 1/2b and 4b strains as well as 3b
and 3c strains have been recognized to form a distinct evolutionary lineage of L. monocytogenes
(lineage I), while serotypes 1/2a, 1/2c, and 3a [135] form lineage II. Serotypes 4a and 4c form
lineage III; strains classified in this lineage appear to be rarely isolated from foods and human
listeriosis cases [106].
Interestingly, subtyping efforts have also shown that the serotype 4b strains responsible for
human listeriosis outbreaks can be grouped into three “epidemic clones,” each of which has caused
at least two large human listeriosis outbreaks [28,103,109]. The three subtypes associated with
multiple human listeriosis outbreaks, include ribotype DUP-1038B, linked to outbreaks in Anjou
(France, 1976), Nova Scotia (Canada, 1981), Los Angeles (United States, 1985), and Switzerland
[106]; ribotype DUP-1042B, linked to outbreaks in Boston (United States, 1979), Massachusetts
(United States, 1983) [106], and ribotype DUP-1044A, linked to two outbreaks in the United States
in 1998 to 1999 and in 2002 caused by consumption of contaminated hot dogs [6] and sliced turkey
[8], respectively. These epidemic clonal groups have also been confirmed by other subtyping
methods [109,148].
With the large diversity of molecular subtypes observed among L. monocytogenes strains from
various sources, it is noteworthy that the larger outbreaks have almost all been attributed to lineage
I strains and the three clonal groups described in the preceding paragraph. Investigations of many
of these outbreaks have included or precipitated investigations to identify the origin and distribution
of these outbreak strains. For example, Norton et al. found that the epidemic clones represented
by ribotypes DUP-1038B, DUP-1042B, and DUP-1044A were also present in smoked-seafood
processing plants not linked to any of the outbreaks caused by these strains [143].
A study using MEE for subtype differentiation showed that the electrophoretic type (ET) of
the serotype 4b strain that caused the soft-cheese outbreak in Switzerland was also widely distributed
in bovine milk, bovine feces, minced meat, silage, and soil as well as in human and animal clinical
cases [24]. Although MEE typing reportedly has good discrimination for serotype 1/2 isolates,
MEE may not adequately discriminate between serotype 4b strains [80], such as the strain respon-
sible for the Swiss listeriosis outbreak. For example, Donachie et al. [49] showed that PFGE typing
could further differentiate serotype 4 isolates of the same ET obtained from a variety of sources
and that all but one of the human isolates could be grouped into exclusive human PFGE types.
Thus, the data on prevalence of outbreak-associated clonal groups always need to be evaluated
carefully along with the discriminatory power of typing methods used.
In a retrospective subtyping study of clinical, environmental, and food-processing plant isolates
from the 1985 Mexican-style soft-cheese outbreak in Los Angeles [116], Wesley and Ashton [200]
showed that the strain isolated from clinical specimens was also recovered from samples of curd,
the pasteurizer, cooler water, a floor drain, and insects caught in the factory. The widespread
distribution of this strain in the plant environment was linked to poor hygiene in the plant.
McLauchlin and Nichols [131] also proposed a relationship between poor hygiene, as measured
by total viable bacterial counts and the presence of Listeria spp. in 4,405 samples of seafood. A
similar relationship between food-processing hygiene and Listeria was shown when pâté samples
were tested in an outbreak in the U.K. [73]. Poor processing plant hygiene likely increases the risk
of L. monocytogenes persistence and survival in food-processing environments as well as the risk
of consistent introduction of L. monocytogenes from natural environments into the processing plant
with subsequent possible contamination of the foods produced.
In 2000, a human listeriosis outbreak in the United States was linked with consumption of deli-
style sliced turkey [7] and provided intriguing information on the ecology of L. monocytogenes.
The unique L. monocytogenes subtype responsible for this outbreak was also responsible for a
single listeriosis case linked to consumption of hot dogs produced in the same facility in 1988 [14].
This indicates that this subtype persisted for more than 10 years in this facility [109], while
maintaining its ability to cause human disease.
In conclusion, subtypes and clonal groups linked to human listeriosis outbreaks may be widely
distributed in many environments, including food-processing and natural environments (see fig. 2.1).
It thus appears that epidemic L. monocytogenes clones do not represent highly host-adapted
subtypes, but rather have maintained their ability to survive in various environments. Most human
listeriosis outbreaks likely follow one of two scenarios. In scenario 1, errors in food handling
(e.g., poor hygiene) lead to high levels and/or high prevalence of contamination of one or a few
lots of foods from a given producer, which subsequently cause infections in multiple susceptible
individuals. In scenario 2, a highly virulent L. monocytogenes strain, which persists in a food-
processing facility, contaminates multiple lots, possibly over days, months, and years with possible
widespread occurrence of human infections (depending on food distribution) [181]. In scenario 1,
the immediate source of L. monocytogenes may include the natural environment or the food-
processing plant environment; in scenario 2, the food-processing environment will be the immediate
source (even though the original source responsible for introduction of a given subtype in a plant
is usually not known and may include the natural environment). Although most “outbreak strains”
thus appear to have the ability to survive in natural environments, the immediate source of many
human listeriosis outbreaks is likely to be the processing plant environment.
Ecology of L. monocytogenes Strains Associated with Sporadic Human

Listeriosis Cases
Although a number of listeriosis outbreaks have been reported, most human listeriosis cases likely
represent sporadic cases (and possibly small outbreaks) caused by a wide variety of L. monocyto-
genes strains [130,167]. Similar to the findings described for epidemic outbreaks, most sporadic
cases appear to be caused by strains representing serotypes 4b and 1/2b, which can be grouped
into L. monocytogenes lineage I [202,204]. For example, using L. monocytogenes isolates collected
over a 30-year period from human listeriosis cases and foods, McLauchlin showed that the serotype
distribution among human listeriosis cases differed from that among food isolates [130]. Charac-
terization of human isolates showed that serotype 4b was most common (60%), and serotypes 1/2a
(17%), 1/2b (11%), and 1/2c (4%) were less common. Among food isolates, serotype 1/2a was
most common (32%), and serotypes 4b (22%), 1/2b (15%), and 1/2c (21%) were less common.
Similarly, Norton et al. found a significantly higher proportion of human isolates (69.1%) than
isolates from three smoked-seafood processing plants classified as lineage I; lineage II strains were
more common among smoked seafood processing plant isolates [144]. Jeffers et al. [106] also
showed that lineage I strains (which include serotypes 4b and 1/2b) were significantly more common
among isolates from human clinical cases as compared to isolates from animal cases. In conjunction
with tissue culture studies, which showed that lineage I strains formed larger plaques as compared
to lineage II strains [144,204], these data may indicate that serotype 4b and/or lineage I strains
may be characterized by increased human virulence as compared to some or all lineage II and or
serotype 1/2a strains. The latter may have increased environmental survival capabilities or may
have adapted to survival in foods and the food-processing environment.
Although some comparative studies on subtype diversity of environmental and food L. monocy-
togenes isolates and diversity of human sporadic cases are available, most reports have focused on
comparing L. monocytogenes strains associated with human sporadic cases with strains isolated from
food samples and food-processing plants [10,24,142,144,182]. For example, Norton et al. [144]
specifically compared the frequency of L. monocytogenes strains (as determined by EcoRI ribotyping)
isolated from three smoked-seafood processing plants with their frequency among human disease
associated isolates. There were significant differences in distribution of ribotypes among human and
food isolates; however, 14 ribotypes isolated from samples collected in the three smoked-fish pro-
cessing plants were also represented among human clinical isolates. Only extremely limited data are
available on frequency of sporadic case-associated strains in the natural environment. For example,
Arimi et al. [10] reported that human clinically associated ribotypes were also found among samples
collected from silage (four ribotypes) and dairy processing plant environments.
Although surveys of L. monocytogenes subtypes associated with human and animal clinical
cases, foods and food-processing facilities, and natural environments provided information on the
associated L. monocytogenes strain diversity, subtyping alone does not provide information on the
virulence potential of different L. monocytogenes subtypes and strains. Virulence mechanisms
of L. monocytogenes are well described in Chapter 5 of this book; however, much of our
understanding comes from the intensive study of relatively few L. monocytogenes strains. Surveys
of virulence-associated characteristics of clinical and/or environmental L. monocytogenes strains
have been performed using mouse bioassays, a chicken embryo test, and/or characterization in
tissue culture assays.
Most comparative mouse virulence studies of human clinical and food isolates have not iden-
tified consistent differences between human clinical and food isolates, but have shown considerable
strain variation in virulence [109]. Some studies have revealed apparent differences between human
clinical and food isolates though, including a study using the chick embryo test [141]. Character-
ization of selected L. monocytogenes isolates from food-processing environments representative of
different ribotypes showed that some of these environmental isolates had an impaired ability to
invade and/or replicate in tissue culture cells, indicating that at least some L. monocytogenes
subtypes present in food-processing environments may have limited human-pathogenic potential.
Similarly, other groups have also shown that some food isolates are virulence attenuated in
mouse models of infection [27,33,109]. Based on these findings, it appears appropriate to hypoth-
esize that a considerable proportion of L. monocytogenes present in natural environments is likely
to have the ability to cause human and/or animal infections, although some strains found in natural
environments may be virulence attenuated. Further studies using appropriate animal and tissue
culture models to characterize L. monocytogenes isolates are required to understand the virulence
potential of the L. monocytogenes strains found in natural environments.
Although Chapter 4 summarizes current knowledge on human fecal carriage of L. monocytogenes,
it is important to consider human fecal shedding and carriage in the ecology of L. monocytogenes.
Early studies reported a relatively high percentage (up to 62%) of positive fecal specimens from
human volunteer surveys; however, these studies involved cohorts most likely exposed to higher
levels of environmental L. monocytogenes, including slaughterhouse and farm workers. Recent
work has shown that carriage rates among healthy humans may be much lower than was
previously assumed. Grif et al. show the rate of carriage among randomly selected human volunteers
to be very low using culture-based methods (0.2%) [86], although a slightly higher percentage of
specimens tested positive using PCR (3.6%). In another study, Grif and colleagues followed three
human subjects longitudinally for a 1-year period for L. monocytogenes by PCR detection,
standardized culture, and IMS culture [87]. In this study they found that each volunteer intermit-
tently shed L. monocytogenes serotypes 1/2a or 1/2b with no coincidental overt clinical disease.
The three volunteers in this study averaged two episodes of shedding over a maximum 4-day period
[87]. Patients with diarrheal listeriosis manifestations also shed L. monocytogenes [40,69] and there
are also some indications that humans with clinical listeriosis symptoms may shed L. monocytogenes
in their feces [168]. Although it is likely that fecal shedding in humans with and without clinical
symptoms contributes to dispersal of L. monocytogenes, including into the natural environment,
the relative importance of this dispersal mechanism for the ecology of this organism remains to be
determined.
Overall, a variety of studies have established that a diversity of L. monocytogenes subtypes are
responsible for sporadic human listeriosis cases. Many or some of the human clinical disease-
associated subtypes have also been isolated from food-processing and natural environments.
Because only very limited subtype studies on L. monocytogenes in the natural environment have
been published, we can only extrapolate from studies on L. monocytogenes subtypes in processing
plants as to the presence and survival of human disease-associated L. monocytogenes subtypes in
the natural environment. It appears likely that subtypes associated with human sporadic cases are
present and also survive and multiply in the natural environment.
The natural environment may thus serve as a source (although not necessarily a reservoir) of
these pathogenic strains, but it is unlikely that the environment serves as a direct source for human
infection. Rather, natural and farm environments are likely to serve as sources of L. monocytogenes
subtypes introduced into food-processing environments. Subsequent food contamination by
L. monocytogenes introduced from environments outside a processing plant and multiplication in
contaminated RTE refrigerated foods may then provide L. monocytogenes numbers required for
infection. There is evidence that the L. monocytogenes populations causing human disease and the
populations present in natural environments overlap; however, some observations also support the
hypothesis that specific L. monocytogenes strains may be predominantly environmental
[24,63,144,182] and have limited ability to cause human disease.
NATURAL ENVIRONMENT AND TRANSMISSION OF LISTERIA IN ANIMALS

As outlined earlier, listeriosis infections in farm animals and particularly in cattle and other farm
ruminants (e.g., goats and sheep) are often linked to consumption of contaminated silage. In
addition, animal listeriosis cases sometimes occur in animals that are not fed silage and environ-
mental sources have been speculated to be responsible for at least some of these cases [108,208].
The agricultural environment thus may serve as an important source for contamination of silage
and may also be a direct source of animal infection in some cases.
As also summarized in one of the previous sections, a variety of studies have found a consid-
erable diversity of L. monocytogenes subtypes in feed (primarily silage) samples as well as in other
environmental samples collected on farms with listeriosis cases in the animals kept on a given
premise. Linking presence of a specific L. monocytogenes subtype in feeds to animal infections
may be challenging due to the possibly long incubation periods for animal listeriosis [120,207].
Nevertheless, in many case and outbreak investigations, identical L. monocytogenes strains have
been recovered from animals with clinical disease and from silage fed to them [65,120,
188,190,200,203,205].
However, silage may contain a diverse array of L. monocytogenes strains [63,162,190,205].
Although animals may thus be exposed to multiple L. monocytogenes subtypes, only a single
subtype generally appears to be isolated from a given animal with clinical symptoms, even if
multiple specimen types are tested (e.g., blood and cerebrospinal fluid). Interestingly, although in
some outbreaks multiple distinct L. monocytogenes subtypes were responsible for multiple animal
cases that occurred on a single farm at a given time [17,118,205], in others a single subtype was
responsible for all cases observed [17,118,203]. In a survey of PFGE types among L. monocytogenes
isolated from clinical samples and feedstuffs in 21 flocks of sheep, Vela et al. also showed that, in
most cases, clinical strains from different animals of the same flock had identical PFGE types and
L. monocytogenes strains with PFGE types identical to those of clinical strains were isolated from
silage, potatoes, and maize stalks [190].
These studies indicate that listeriosis epizootics may differ with regard to the causative strains
and their spread. In some epizootics, a single, possibly highly virulent, strain may be responsible
for infection of all involved animals, whereas in others distinctive strains (possibly with lower
virulence and/or transmission potential) may be responsible for individual infections [203]. In many
of the listeriosis outbreaks in farm animals a considerable proportion, and sometimes all, of the L.
monocytogenes subtypes found on a given farm were only isolated from environmental samples
and not from infected animals [17,118]. These findings could indicate that some environmental
isolates may have a limited ability to cause disease and thus further supports that L. monocytogenes
strains may differ in their virulence characteristics.
In addition to clinical cases, L. monocytogenes has been found in the feces of a wide variety
of healthy animal species. Gray and Killinger [83] listed 37 mammals from whose feces the
organism had been isolated. Given that L. monocytogenes is distributed so widely on vegetation in
nature, it is not surprising that its presence in fecal materials from grazing animals such as healthy
sheep [88,120,172], goats [117,210], and cattle [62,94,210] is well documented. However, presence
of L. monocytogenes in fecal materials also has been reported for pigs [52,152], chickens
[15,81,136], turkeys [19,90,136], pheasants [171], gulls [58], rooks [58], pigeons [150], fish [11],
and crustaceans [11]. L. monocytogenes appears to be a food-related pathogen; naturally occurring
listeriosis has been recorded in many other animals, including mice [172], voles [139], rats [172],
rabbits [139], guinea pigs [153], chinchillas [66], lemmings [150], hyraxes [150], mink [111],
skunks [153], horses [197], dogs [178], cats [139], foxes [139], deer [139], buffalo [52], giraffes
[38], bats [96], ducks [150], partridges [150], eagles [150], parrots [150], canaries [150], starlings
[111], frogs [26], turtles [26], ticks [5], and flies [5]. Thus, it is reasonable to suspect that these
animals also excrete the organism in their feces.
Various reports support that the diet of healthy animals may have a considerable effect on
excretion of L. monocytogenes. For example, Low et al. [120] reported a low incidence of L.
monocytogenes excretion in a flock of 100 grazing sheep. Similar to previous findings by Husu
[99] that the tendency is for L. monocytogenes excretion rates to be lower in grazing animals, the
prevalence of L. monocytogenes in fecal samples increased significantly (P < 0.00001) to between
10 and 33% once silage feeding commenced. Phage typing of fecal isolates from the 100 sheep
fed silage revealed 10 different phage types as compared to 6 phage types identified among the
isolates from the silage fed to these animals. Several strains present in feces were absent from
silage. Sheep consume large amounts of silage (indeed, the authors noted that a single sheep may
consume 100 to 1000 times more silage than the amount of silage tested for Listeria); thus, Listeria
strains present in excreta may be more likely to be representative of the total silage population.
A report by Fenlon et al. [63] also supports that grazing animals show a lower prevalence of
L. monocytogenes in fecal samples as compared to silage-fed animals. While grazing, none of the
cattle tested (n = 10 and 13) among two groups of animals excreted L. monocytogenes. When tested
after silage feeding commenced, 4 of 14 (28.6%) in one group and 4 of 13 (30.8%) in the other
excreted L. monocytogenes. Numbers of Listeria in the excreta were low, ranging from present in
25 g to 11 CFU/g. In the same study, examination of feces of sheep on a hay diet showed no
detectable Listeria, presumably because the moisture level in hay is too low to support Listeria
growth. Studies on fecal and litter samples from other food animal species, such as chickens and
pigs, generally found low prevalence of L. monocytogenes and Listeria spp. [63].
In a larger study on the presence of Listeria spp., Dijkstra [46] examined the intestines of 2,373
broilers from 146 farms and showed that 4.1% were contaminated with Listeria. Husu et al. [100]
noted that most 2-day-old chicks dosed orally with L. monocytogenes had eliminated the organism
within 9 days, indicating that chickens are unlikely reservoirs of the organism and that any carriage
is probably transient. Overall, it thus appears that L. monocytogenes can be isolated from many
farm animals, although the organism appears most prevalent in silage-fed ruminants. In addition,
shedding of L. monocytogenes in ruminant stool can occur in higher numbers when animals are
subjected to stressful conditions (see Chapter 4 for a comprehensive discussion).
Clearly, L. monocytogenes appears to be widely distributed in agricultural environments and
silage, and infection of farm ruminants appears to be more common than infection of many other
animal species. Oral infection appears to be the most common route of infection for ruminants,
although alternative transmission pathways have sometimes been suggested [194]. Although L.
monocytogenes is also often found in fecal material collected from farm ruminants, particularly
those fed silage, it is not clear whether farm animals can be true carriers or whether presence in
fecal materials generally represents a transient “pass-through” phenomenon.
Although we have developed some understanding on the sources of animal listeriosis infections, the
overall transmission dynamics of L. monocytogenes in farm environments is not well understood. For
example, although it is likely that clinically apparent or unapparent animal infections are critical to L.
monocytogenes survival in farm environments, one might also hypothesize that presence of manure (and
other nutrient-rich environments) alone, even in the absence of infection, may be sufficient to allow for
L. monocytogenes survival and multiplication. Detailed longitudinal studies of on-farm transmission of
L. monocytogenes combined with mathematical modeling efforts will be required to truly understand
the ecology and transmission of L. monocytogenes in farm animals and farm environments.
TRANSMISSION OF LISTERIA WITHIN NATURAL ENVIRONMENTS

A variety of efforts have been made to better understand transmission of L. monocytogenes in farm
animals and farm environments and from foods to humans; however, only very limited data are
available on the transmission and spread of L. monocytogenes in natural environments, including
infection of wild mammals and nonmammalian or invertebrate hosts. Based on a variety of case
reports (see preceding discussion) that indicate a broad host range for L. monocytogenes among
predominantly captive and farm animals, it appears likely that wild animals may at least occasionally
experience L. monocytogenes infections and clinical disease.
In addition, we are only beginning to understand the complex dynamics of the relationships
between microorganisms and between microorganisms and multicellular organisms. Aside from
bacteria–bacteria interactions, protozoa play a large role in marshalling bacterial populations
through predation and grazing. Because L. monocytogenes is an intracellular pathogen found in
many different environments, it has been hypothesized that protozoa may provide a potential host
for this pathogen similar to the intracellular human pathogen, Legionella pneumophila, which
multiplies intracellularly in Tetrahymena thermophila [110].
Many other bacteria, including other human and animal pathogens, can survive within a variety
of protozoan species [13a]. In preliminary studies, Ly and Müller demonstrated that L. monocyto-
genes may be able to survive and multiply within the protozoans Tetrahymena pyriformis and
vegetative Acanthamoeba sp. [123,124]. It is thus possible that protozoa may represent a reservoir
of pathogenic Listeria and serve to maintain virulence genes during environmental survival; it may
be possible that Listeria-protozoan interactions played a critical role in evolution of Listeria
intracellular survival mechanisms and virulence. It has also been hypothesized that protozoans
or other alternate nonhuman hosts may serve as a host for L. seeligeri, which carries many of
the typical virulence genes found in L. monocytogenes and L. ivanovii [78], but is not able to
cause infections in animal or tissue culture models that have been successfully used to study
L. monocytogenes pathogenesis [68,128].
DEFINING GLOBAL LISTERIA TRANSMISSION PATHWAYS AND RESERVOIRS

The natural environment harbors a highly diverse range of Listeria spp. and L. monocytogenes
strains, including some with the potential to cause clinical listeriosis in humans and animals.
The presence of these organisms in natural environments appears to be characterized generally by

low prevalence and low numbers, although a high prevalence of positive samples and the presence
of high numbers of organisms have been described in some instances and for some environments
and sample types. Although it is apparent that the species within the genus Listeria are all widely
distributed in nature and that food- and feedborne transmission is critical for human and animal
infections, we have been unable to define the global Listeria transmission pathways and the true
reservoirs of pathogenic and nonpathogenic Listeria species.
Haydon et al. [91] define a reservoir as “one or more epidemiologically connected populations
or environments in which the pathogen can be permanently maintained and from which infection
is transmitted to the defined target population.” Although it seems possible that the farm environment
(e.g., soil, plant materials, feed) may indeed represent a reservoir for some Listeria subtypes that
can cause animal infections, it is unclear whether ruminants represent a true target population or
whether Listeria could be permanently maintained in this environment without causing animal
infections. It is conceivable that for human infections, the transmission cycle may draw on a wider
dynamic, possibly involving farm and food-processing environments. Most evidence indicates that
at the processing stage of food and feedstuffs (e.g., silage preparation) amplification of numbers
or persistent contamination occurs, leading to potentially serious human and animal health hazards.
Stringently enforced good manufacturing and hygiene practices in the food-production and food-
processing environments are thus critical to prevent introduction of pathogenic Listeria spp. into
the feed and food chain from environmental sources.
In this context, it is noteworthy that poultry products [132,185] may be more commonly
contaminated with L. monocytogenes than beef [55,115,175], even though the environment in which
beef cattle are reared appears to show a higher prevalence of this organism than that of the intensively
reared broiler chicken [63]. However, in processing, the chicken may be exposed to greater risk of
contamination from other carcasses and mechanical equipment than beef carcasses [158]. In con-
junction with many other data on L. monocytogenes in the food-processing environment, it thus
appears that the environment of food-processing establishments represents a major direct source
of L. monocytogenes found in foods destined for human consumption. In addition, some human
listeriosis cases may be caused by direct introduction of L. monocytogenes into a food item from
nonfarm and nonprocessing environments, but this contamination is likely to be of a low level and
sporadic. Direct infection of humans from human environmental sources is rare; however, one
listeriosis outbreak report from Costa Rica implicates bathing in mineral oil contaminated with a
L. monocytogenes 4b strain as the source of illness in nine newborns [170].
Although some transmission cycles proposed here may seem compelling, very few data could
be used to quantify and define the importance of different environments as sources or reservoirs
of virulent strains of L. monocytogenes that can enter and pass along the food chain and cause
human infections. For example, no quantitative data are available to be used to evaluate the true
significance of L. monocytogenes present in farm animals or in farm environments as a direct or
indirect source of human infections. Defining the reservoirs and transmission of L. monocytogenes
thus represents a formidable challenge, and improved knowledge on the transmission pathways
of this organism will be critical to improve our ability to reduce human listeriosis cases and
outbreaks.
Even more limited than our knowledge of the transmission of L. monocytogenes is our under-
standing of the ecology of other Listeria spp. This is evidenced by the fact that we have no
indications as to the biological role of the virulence genes present in L. seeligeri, which does not
cause human or animal infections in any known model systems [78]. Although food microbiologists
often use presence of Listeria spp. as an indictor for the presence of L. monocytogenes, it is
becoming increasingly clear that the physiology and ecology of L. monocytogenes and other Listeria
spp. differ considerably. Further studies and data on the ecology of Listeria spp. other than L.
monocytogenes will thus likely provide additional knowledge that will help us to understand the
natural history and ecology of L. monocytogenes.
In addition, development of large standardized repositories of Listeria subtyping data (such as

http://www.pathogentracker.net) will allow comparative studies on the genetic diversity of Listeria
and L. monocytogenes strains from human and animal disease, food and food-processing environ-
ments, and the natural environment. In combination with longitudinal studies and mathematical
modeling efforts, these data will likely improve our understanding of the ecology and transmission
dynamics of Listeria spp. in general and the human pathogen L. monocytogenes in particular.
ACKNOWLEDGMENTS
We thank and acknowledge David Fenlon for allowing adaptation of his previous chapter from the
second edition of Listeria, Listeriosis, and Food Safety for this edition. This work was supported
in part by National Institutes of Health Award No. R01GM63259 (to M.W.). The Pathogen Tracker
2.0 online public subtyping database is supported by USDA Special Research Grants 2001-34459-
10296 and 2002-34459-11758 (to M.W.)
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3 Listeriosis in Animals
Irene V. Wesley
CONTENTS
Introduction ......................................................................................................................................55
Incidence ..........................................................................................................................................56
Predisposing Factors ........................................................................................................................56
Transmission to Humans..................................................................................................................57
Transmission to Animals..................................................................................................................57
Sheep.......................................................................................................................................57
Goats .......................................................................................................................................60
Cattle.......................................................................................................................................62
Swine ......................................................................................................................................66
Fowl ........................................................................................................................................67
Minor Species.........................................................................................................................69
Fish and Crustaceans..............................................................................................................71
Treatment..........................................................................................................................................72
References ........................................................................................................................................73
INTRODUCTION
Numerous animal species are susceptible to listeric infection, with a large proportion of healthy
asymptomatic animals shedding Listeria monocytogenes in their feces. Although most infections
are subclinical, listeriosis in animals can occur sporadically or as epidemics and often leads to fatal
forms of encephalitis. Clinical listeriosis in livestock presents as encephalitis, septicemia and
abortions during the last trimester of gestation. Virtually all domestic animals are susceptible to
listeriosis; sheep [7,103,204,206,239, 285], cattle [103,204,208,217,226], goats [166,167,243], and,
less frequently, birds [100,199,204,223] succumb to infection. Several comprehensive reviews have
detailed the distribution and pathology of L. monocytogenes in food animals [7,56,102,
123,158,173,175,186,219,229]. Not all serotypes are pathogenic to livestock and may be divided
into at least three lineages based on DNA profiles. Interestingly, isolates of lineage I predominate
in human infections, whereas animal isolates cluster in lineage III [294,295].
L. monocytogenes enters the animal usually through ingestion. Following entry into the intes-
tinal epithelium via M cells in Peyer’s patches or epithelial cells [215,229], a bacteremic or
septicemic phase or latent infection may develop, depending on the immune status of the host.
L. monocytogenes subsequently colonizes the viscera, gravid uterus, or medulla oblongata [152].
In pregnant animals, the organism can localize in the placentomes and enter the amniotic fluid.
The fetus aspirates the pathogen, which multiplies and kills the fetus late in gestation [267].
A single flock may experience abortion, septicemia, and encephalitis [176].
55
INCIDENCE
Listeriosis in domestic livestock is recognized worldwide despite significant differences in climate
and livestock management practices [101,279]. In addition, most infections in livestock are sub-
clinical and therefore go undiagnosed [152]. Because listeriosis is not a reportable disease, the
exact incidence of infections in domestic livestock remains unknown, thus precluding comparison
of prevalence data between countries [221].
Livestock losses attributed to L. monocytogenes may be substantial. During the early 1970s,
the agricultural economies of Australia and Norway were adversely affected by the loss of approx-
imately 1 million and 2,000 to 2,500 sheep, respectively, from listeric infection. Before a vaccine
became available and lowered the incidence of listeriosis, infections in Norwegian livestock
remained relatively constant with approximately 1,900 to 2,300 sheep herds, 90 to 160 goat herds,
and 3 to 17 cattle herds affected during the 10-year period between 1977 and 1986 [304]. In the
United Kingdom, approximately 30,000 ovine abortions occur annually with 0.05 to 0.13% attrib-
uted to listeriosis [7]. In The Netherlands, between 1970 and 1985, the annual percentage of bovine
abortions attributed to L. monocytogenes in cattle ranged between 0.7 and 8.7%, with an average
of 3.2%, which represented between 234 and 928 cases [69].
Prevalence studies may be based on microbiological surveys of healthy cattle as well as sero-
surveys. In a 1995 survey of domestic and companion animals in Germany [288], L. monocytogenes
was found in fecal samples of healthy bovines (33%), sheep (8%), birds (8%), pigs (5.9%), horses
(4.8%), and dogs (0.9%). In addition to relatively small numbers of acutely infected sheep, goats,
and cattle, substantially larger proportions of animals within a herd may be asymptomatic carriers
of L. monocytogenes and shed the organism in feces and milk [221,239]. The role of the symptomless
carrier was clearly demonstrated in another report in which 30 of 44 listeriosis outbreaks on sheep
farms involved introduction of clinically healthy animals from known infected herds [239].
Although the humoral immune response may not be pivotal, serum antibodies are useful for
serodiagnosis of Listeria carriage in healthy animals [27,29,192]. Sindoni et al. reported Listeria
antibodies in apparently healthy sheep (18.5%), cows (10.4%), goats (17.3%), and swine (13.3%),
suggesting previous exposure to L. monocytogenes [247]. Although these data estimate the distri-
bution of L. monocytogenes infections in healthy carrier animals, they should be interpreted with
caution because antibodies to a number of Gram-positive microbes may cross-react with Listeria
antigens. In contrast, antibody titers to listeriolysin O (LLO) and actA proteins, which are regarded
as virulence proteins, are a more specific indicator of infection and do not cross-react with other
Listeria species [32]. LLO antibodies have been used to monitor a variety of livestock, including
cattle, buffalo, sheep, and goats. To illustrate, in experimentally infected goats, an increase in LLO
antibodies was correlated with rapid clearance of L. monocytogenes from the gastrointestinal tract
and thus predicted a favorable outcome [193].
PREDISPOSING FACTORS
A number of factors contribute to susceptibility to infection: season; immune status, such as
pregnancy; on-farm feeding; and management practices, including feeding silage as well as breed
of livestock species. Seasonal variation in the number of cases of animal listeriosis has often been
observed. To illustrate, in the Northern Hemisphere (England, Bulgaria, Hungary, United States,
France, and Germany), cases in domestic animals generally occur from late November to early
May with the greatest incidence during February and March [2,102].
Consumption of contaminated silage contributes significantly to infection in livestock. For
example, listeriosis cases increased when animals were fed silage during periods of extreme cold,
whereas sharp decreases in numbers of reported cases were observed as soon as pasture was
available. Data from The Netherlands [69] indicate that most cases of listeric abortion in cattle occurred
between December and May. Approximately 40% of these cases were linked to consumption of
Listeriosis in Animals 57
contaminated silage. Recent changes in production methods have reduced levels of L. monocytogenes
in silage, which in turn has decreased the incidence of listeriosis in silage-fed animals. Yet an
increase in ovine listeriosis with conversion to big bale silage production has been documented in
the United Kingdom [299]. Although in Norway listeriosis can be diagnosed year-round in sheep
and goats, the illness is more prevalent from October to June and also appears to be influenced by
housing and feeding conditions [221].
TRANSMISSION TO HUMANS
Transmission of L. monocytogenes from livestock to humans occurs by (1) direct contact with
infected animals, especially during calving or lambing; and (2) consumption of contaminated raw
milk. To illustrate, primary cutaneous listeriosis is regarded as an occupational disease of veteri-
narians and farmers who have attended deliveries of stillborn or aborting bovine fetuses
[4,44,188,206,210,275]. A case of septicemia and ultimately fatal meningitis was reported in a
Dutch farmer who developed cutaneous lesions after assisting in the delivery of a stillborn calf [197].
Apart from human infections acquired as a direct result of contact with infected animals or
consumption of foods from an infected animal, the connections, if any, between human and animal
listeriosis are unclear. The springtime peaks of animal listeriosis and the autumn seasonality of
human cases suggest that cases are not causally related. The source of contamination for human
foods and animal feed is usually environmental [173,187]. The rise in cases of human listeriosis
may result, in part, from changes in food manufacturing, postprocessing contamination [173], and
improved diagnostic methods.
The WHO concluded that foodborne listeriosis is predominantly transmitted by nonzoonotic means
and that L. monocytogenes is an environmental organism whose primary route of transmission to humans
is via foods contaminated during production [293]. More specifically, sensitive molecular tools, such
as multilocus enzyme electrophoresis and pulsed field gel electrophoresis, suggest that L. monocytogenes
strains isolated from meat or raw milk may have originated mainly from the processing environment
rather than animals [31,113]. However, it is possible that live animals brought into slaughter may
introduce Listeria into the processing plant. Although an animal origin of contamination was inferred
for the three major epidemics occurring in North America, in the absence of documented evidence, the
role of direct animal involvement in human foodborne outbreaks remains speculative [290].
TRANSMISSION TO ANIMALS
SHEEP
Ovine listeriosis is caused by L. monocytogenes serotypes 1/2, 3, and 4 as well as by L. ivanovii
[171]. Although “listeric-like” infections were previously observed in sheep [239], Gill is credited
with the first isolation of L. monocytogenes from domestic farm animals [94]. In 1929, he observed
an illness in sheep in New Zealand that he called “circling disease.” This name is still used today
to describe listeric encephalitis, encephalomyelitis, and meningoencephalitis [239].
Attack rates are higher in sheep (up to 30%) than in cattle (up to 15%), suggesting the greater
susceptiblity of ovines to listeriosis [173,234]. As in other livestock species, the clinical manifes-
tations of ovine listeriosis are (1) encephalitis; (2) placentitis, with abortions occurring in the last
trimester [103,140,178,280]; and (3) gastrointestinal septicemia with hepatitis, splenitis, and pneu-
monitis [154]. Encephalitis is the most common form diagnosed in sheep [152]. Lambs as young
as 5 weeks may develop septicemia with older feedlot lambs (4 to 8 months) manifesting enceph-
alitis. All sheep are probably exposed to the same contaminated feed, indicating a high natural
resistance with the total of 5 to 10% of exposed animals exhibiting clinical signs [152].
Meningoencephalitis caused by L. monocytogenes is the most common bacterial infection of
the central nervous system of adult sheep [237]. After an incubation period of approximately
3 weeks, clinical symptoms of ovine encephalitis appear. These include elevated temperature and
refusal to eat or drink followed by neurological disturbances, which include grinding of teeth,
paralysis of masticatory muscle, and excessive salivation caused by the animal’s inability to swallow
resulting from damage to the cranial nerve. At this point the animal moves in circles to the right
or left, depending on the direction in which the head is bent. This characteristic movement accounts
for the name “circling disease.” In advanced stages, muscular incoordination develops and is
followed by inability of the animal to walk. Death usually occurs within 2 to 3 days after onset of
clinical symptoms, with the illness seldom lingering beyond 10 days [239]. In the brainstem, Listeria
antigens are characteristically variable but always sparse. Perivascular microabscesses with L.
monocytogenes and microgranulomas in histopathologic specimens of brain stems are characteristic.
T lymphocytes (CD8+ and CD4+) and B lymphocytes contribute to the inflammatory process [157].
Direct entry via abrasions or lesions of the buccal mucosa, lips and nostrils, or conjunctiva
may lead to encephalitis. Because entry into dental terminals of the trigeminal nerve in sheep can
cause an ascending neuritis and encephalitis [49,50], listeric encephalitis is most common in winter
and early spring in sheep that are losing and cutting teeth [17]. L. monocytogenes penetrates the
buccal epithelium, accesses endings of the trigeminal (V) and hypoglossal (XII) cranial nerves,
and enters the brain stem where replication and dissemination to the medulla and pons occur.
In severe cases, respiratory failure and death follow within 1 month of infection [152,171].
Abrasions of the eye by contaminated silage lead to ophthalmitis without any other clinical
manifestations [283].
After ingestion and hematogenous spread to the gravid uterus, L. monocytogenes appears within
48 h in the amniotic fluid and fetus [171]. Listeric infections in pregnant sheep often result in
premature birth and infectious abortions [103,178,280]. This seldom occurs concurrently with
encephalitis [131]. Initially, pregnant ewes contract purulent metritis, from which most recover.
Intrauterine transmission of L. monocytogenes results in septic infection of the fetus, which in turn
gives rise to abortion or premature delivery; most fatalities occur as stillbirths. Clinical symptoms
are resolved following expulsion of the fetus, after which the ewes recover. Morbidity in ewes
ranges from 1 to 20% [272] with mortality of lambs usually high [154]. Experimental injections
of pregnant ewes with L. monocytogenes led to abortion in 10% of inoculated animals and retar-
dation of bone growth in lambs [98].
Septicemia is most frequent in neonates and lambs and follows 2 to 3 days after oral infection,
though congenital and navel infection can also occur [171]. Septicemia is characterized by an
elevated temperature, loss of appetite, and diarrhea. Although death may eventually occur as a
result of extensive liver damage and focal pneumonia, the mortality rate is much lower for the
septicemic than for the encephalitic form of listeriosis.
Most importantly, silage feeding, followed by season, breed, and on-farm management prac-
tices, and stress caused by overcrowding, contributes to ovine listeriosis. The quality of silage may
influence the incidence of ovine listeriosis [82]. In an outbreak of ovine listeriosis in the United
Kingdom, the D value of silage, which measures digestibility, was below the optimum of 65 to
70% [299]. In Scotland, listeriosis caused by serotype 1/2 occurred in sheep that were reluctant to
eat poor-quality silage [176]. Silage analysis indicated pH > 4.0 and a high ash content reflecting
soil contamination. Despite antibiotic treatment, 19 ewes died; more than 60 developed vaginal
discharges and at lambing 94 were barren [176].
A British study linked silage feeding and development of ovine listeric encephalitis (relative
risk of 3.8). The excretion of L. monocytogenes by sheep was linked to diet: animals fed entirely
on hay or manufactured diets did not excrete detectable levels of L. monocytogenes; animals fed
on silage commonly excreted the organism [297]. An outbreak in a flock consisting of 450 sheep
(attack rate 11.8%) with a case fatality rate of 94.3% was traced to feeding poor-quality silage
(pH 7.8) that was highly contaminated (106 L. monocytogenes/g) [272]. The serovar and phagovar
of the L. monocytogenes strains isolated from two silage samples and the brains of 3 of the 53
affected sheep were indistinguishable [272]. Another outbreak, with a mortality of 2%, in a flock
of 650 sheep was traced to contaminated silage. The rise and fall of cases coincided with the silage
feeding practices [297].
Whereas the incidence of L. monocytogenes in sheep and cattle has declined in The Netherlands
(attributed in part to the change of silage production), in Great Britain modifications in silage
production may have led to an increase in ovine listeriosis [96]. To illustrate, in 1975, listeriosis
was reported in a modest number of cattle (n = 37) and sheep (n = 44). By 1984, sheep listeriosis
cases had increased (n = 269) with little change in the number of bovine listeriosis cases [96]. In
parallel with the rise in the incidence in the United Kingdom there was also a change in the disease
pattern, with encephalitis and abortion occurring in the same flock [96,176].
Ovine listeriosis occurs most frequently in late autumn, winter, and early spring. Stress factors,
such as abrupt changes in feed, introduction of new animals into the flock and resultant overcrowd-
ing, concurrent disease, changes in dentition, and physical or viral damage to the epithelial lining
of the digestive tract may predispose to infection [151,152]. Climatic changes, such as heavy rains
[303], especially following a drought, which may spoil feed [228], or periods of wintry cold weather,
which may cause animals to be housed indoors [190,285], are also determinants. In Saudi Arabia,
an outbreak occurred in a flock of 2,100 Naimi sheep during the winter rainy season with a resultant
morbidity rate of 7.1% and mortality rate of 2.4%. Encephalitis was observed in pregnant ewes,
but no abortions were seen. Sheep were grazed during the day and housed in the evening with
access to grass rolls, which were most likely the source of infection [2].
A prospective study conducted on early lambing flocks in southwest England (1989–) monitored
4,413 lambs from birth to slaughter for listerial meningoencephalitis. Two of three flocks developed
clinical disease attributed to serotype 1/2. Six weeks before lambing, ewes on the two affected
premises were bedded in straw; ewes on the farm without clinical listeriosis were on softwood slats
[104]. In the single flock with no cases, preventive measures after lambing included replacing
bedding and silage daily and regular cleaning of the silage feeders, which were on concrete floors.
In the affected flocks, silage was replaced on alternate days and the silage feeders were on soil-
based floors and thus easily contaminated. Before weaning, the lambs were eating more silage
because ewes were producing less milk. Although all lambs were exposed to these risk factors,
only an estimated 1.3% developed clinical infection and death occurred in 0.56% (21/4413) of
lambs from 4 to 32 days after weaning [103].
Multiple strains of the same or different serotypes may be isolated in an outbreak in the same
flock. When isolates of the same serovar are recovered from a single outbreak, they may be further
differentiated by DNA fingerprinting, phage typing [10], or pyrolysis mass spectrometry [172]. To
illustrate, DNA fingerprinting by random amplified polymorphic DNA (RAPD) analysis and
ribotyping affirmed the identity of L. monocytogenes strains from silage, farm equipment, and sheep
brain [296,298]. In contrast, examination of outbreaks of ovine listeriosis in Scotland indicated
multiple strains of L. monocytogenes serovar 1/2 recovered from the silage incriminated in the
outbreak.
By multilocus enzyme electrophoresis (MEE), the L. monocytogenes strains for each of the
affected animals in an outbreak were identical. Yet by MEE, none of the strains from the silage
matched those recovered from brains. This could reflect bias during sampling from the bales, thus
missing a small virulent population of L. monocytogenes in vegetation which, upon entry and
replication into the ovine host, became the dominant strain [21].
Reports on the incidence of L. monocytogenes in ewes’ milk are limited. In Spain, L. mono-
cytogenes was present in 2.2% of 1,052 ewes’ milk samples representing 283 farms. Yet L.
monocytogenes was recovered from 18% of milk tanker samples in Spain. Farm ewes’ milk samples
indicated contamination by L. monocytogenes was significantly higher on premises where cows
were also reared than on farms where only ewes were maintained [230]. The data suggest envi-
ronmental contamination on farms from L. monocytogenes excretion in cows’ feces or common
exposure to contaminated ensilage on the premises shared by sheep and cattle [230]. Interestingly,
no seasonal variation in milk contamination was evident in that study.
Although not widely practiced, most likely because of limited cost benefits, vaccination with
live attenuated strains of L. monocytogenes may effectively reduce ovine listeriosis [109,110,
165,201,282]. In Norway, immunization of sheep with a commercially available live attenuated
vaccine of serovars 1/2a and 4b lowered the incidence of listeriosis and abortions when compared
with unvaccinated control farms [109,110]. In this study, half of the sheep in 70 flocks (total of
3,130 sheep) with a history of listeriosis received two attenuated strains of L. monocytogenes
serotypes 1/2 and 4b, whereas the remaining half of the sheep served as unvaccinated controls
[109]. Both groups of animals were then housed together in the same pens. Results of this study
showed listeriosis incidences of 1 and 3% in vaccinated and unvaccinated sheep, respectively. The
incidence of abortions was 0.7% in vaccinated compared to 1.1% in unvaccinated flocks [110].
In another study, an experimental polyvalent vaccine, consisting of serovars 1/2a and 4b, which
were attenuated via metabolic drift mutations, was tested in sheep, lambs, and ewes [165]. The
results of field tests indicated that vaccinated ewes delivered more lambs free of listeriosis (93.4
vs. 69.7%) and of higher birth weight (2.2 vs. 1.8 kg) than lambs from control unvaccinated ewes.
In addition, L. monocytogenes was not isolated from milk samples of vaccinated ewes, in contrast
to controls in which 32% of milk samples yielded Listeria [165]. Although vaccination produced
few adverse side effects, economic constraints suggest that it should be restricted to flocks to achieve
maximum cost benefit.
Preferential breed susceptibility may occur [201]. In a survey conducted in the Midwest in
which consumption of contaminated silage was a key factor, unvaccinated Rambouillet ewes were
more at risk (odds ratio 4.6) than other ewes; yearling ewes were more at risk than older animals
(odds ratio 4.1). Interestingly, although use of a bacterin did not decrease the risk of L. monocy-
togenes in Rambouillet ewes (odds ratio 0.8), it did among ewes of other breeds (odds radio 0.1).
This indicates that the inherent susceptibility of Rambouillet ewes to L. monocytogenes cannot be
modified by vaccination [201].
As in other livestock species, serosurveys have been used to estimate the occurrence of listeriosis
in sheep [250]. However, antibodies to other Gram-positive microbes cross-react with Listeria antigens,
thus giving rise to false positive results. Berche and coworkers [26] developed a highly specific test for
L. monocytogenes based on detection of antibodies to listeriolysin O (LLO). Antibodies to LLO have
been detected in lambs experimentally infected with L. monocytogenes [13,163,174].
Listeria ivanovii was first described in association with ovine abortion and accounts for up to
8% of all animal listeriosis cases [10,240]. Listeria ivanovii is a recognized cause of ovine abortions,
stillbirths, and unthrifty lambs [36,62,126,133,177,178,186]. Interestingly, goats may be resistant
to L. ivanovii. To illustrate, L. ivanovii and L. monocytogenes were reported in two migratory flocks
of sheep and goats in Himachal Radesh, India. Whereas L. monocytogenes was isolated from sheep
and goats, L. ivanovii was cultured only from sheep [244]. Factors that predispose livestock to L.
ivanovii abortions are similar to those described for L. monocytogenes: lowering of ewes’ resistance
to infection following general immune suppression as well as to nutritional stress, periods of cold
and wet weather, feeding of poor-quality silage, and exposure to carrier animals [73,95,219].
Outbreaks of L. ivanovii have been reported to affect up to 45% of pregnant ewes [133]. One
outbreak in New South Wales involved a total of 110 animals that aborted or died shortly after birth.
Heavy grazing by sheep (120 sheep per hectare for 18 days) on pastures cut for hay that had not been
baled and had spoiled because of heavy rains was presumed to be the source of initial contamination
of L. ivanovii [242]. Multiple hepatic foci were seen in aborted lambs. In addition, L. ivanovii was
cultured from fetal liver, lung, and stomach contents [242]. A report of bovine abortions caused by
L. ivanovii associated the grazing of cattle on pastures previously used by sheep [3].
GOATS
Clinical listeriosis in goats as in sheep presents as encephalitis, septicemia, and abortion. [37].
As is true for other livestock species, asymptomatic infections also have been noted in goats [239].
After ingestion, L. monocytogenes penetrates the intestinal tract, sets up a transient bacteremia
and spreads to the central nervous system, viscera, or placenta. Depression, loss of appetite, a drop
in milk yield, and elevated body temperature (up to 41°C) are the first indications of septicemia.
The animals may have diarrhea [107]. In the pregnant doe, L. monocytogenes may penetrate the
placenta and enter the fetus, where it replicates and causes late-term abortion.
In experimental studies in pregnant goats, localization of L. monocytogenes in the placenta led
to an elevation in prostaglandin F2 and a decline in progesterone levels. A slight decrease in
secretion of estrone sulfate by the fetal-placental unit prompted myometrial contraction and abortion
[75]. As with other species of livestock, goats excrete Listeria in feces and milk during and after
septicemia and may contaminate the environment. Thus, newborn kids housed with does may be
infected through the navel or through sucking on soiled teats [108].
Meningoencephalitis is the most frequently reported form of listeriosis in goats with fatalities
achieving 60% [107]. Early signs of listerial encephalitis mirror lesions to the respective cranial
nerves: drooping ears, marked drooling, tongue protrusion, and cud retention because of difficulty
in swallowing [37]. Goats may be more susceptible than sheep to listeric encephalitis based on the
severity of brain damage in goats [16]. In an outbreak of encephalitis and abortion in a mixed herd of
goats and sheep in Iraq, the morbidity (30 vs. 16.7%) and mortality (21 vs. 15%) was higher in goats
than in sheep [303]. Heavy rains, cold weather, and susceptibility of pregnant animals may have
contributed to high numbers of encephalitis cases [303]. More frequent recovery of L. monocytogenes
serotype 1/2b from goats than from sheep has also been documented in Sudan [246].
Listeriosis is linked to the practice of feeding silage [167,171]. However, in a study of 355
goat herds in Missouri, encephalitic listeriosis was correlated with browsing on woody plants and
location of the herd in areas with a preponderance of alkaline soil [141]. Heavy browse consumption
may have abraded oral tissues followed by penetration by L. monocytogenes into the dental pulp
or buccal cavity, which may have led to encephalitis [141].
Although it is not widely practiced, vaccination has limited the number of goat listeriosis cases
[65,87,201]. Before vaccination of goats in Norway with a live attenuated strain, the abortion rate
in the test herd was 20 to 25%. After vaccination, the incidence rate of abortions decreased to 3%
[155]. The optimal time for vaccination of goats as well as sheep may be shortly before mating
season [155].
Few studies describe distribution of L. monocytogenes in raw goats’ milk [1]. Gaya et al.
cultured L. monocytogenes from 2.6% of 1,445 samples from 405 Spanish goat milk farms located
in mountain and plateau regions [92]. Overall, recoveries were higher in the autumn (5.7%) than
in the winter (4%) or in spring (0.3%). Interestingly, the higher recovery was traced to farms located
on the plateau where goats graze on stubble and germinating vegetation and thus consume more
soil in autumn. A seasonal trend was not apparent in goats on mountain farms where vegetation
was available throughout the year, thus minimizing soil consumption [92].
In one report [105], L. monocytogenes was detected in 3.6% of raw milk from cows with
considerably lower recovery of L. monocytogenes from raw milk from goats (1%) and ewes (2%).
Yet in a comparable study of 450 raw goat-milk samples from 39 goat farms in the United States,
the 3.8% Listeria isolation rate paralleled earlier data for cows’ milk [1]. In addition, a seasonal
trend was noted with isolations higher during winter (14.3%) and spring (10.4%) versus autumn
(5.3%) and summer (0.9%) [2]. In Scandinavia, summer visits to local farms resulted in a small
cluster of human listeriosis cases resulting in febrile gastroenteritis linked to consumption of cheese
[46]. Listeria concentrations in cheeses produced from goat’s milk (~106 CFU/g) and blended milk
(~106 CFU/g) were notably higher than in cheeses manufactured from cow’s milk (5 × 102 CFU/g).
This may represent the higher concentrations shed in goats versus cows or the result of bacterial
replication during refrigeration of the cheese.
Goats have been linked to human listeriosis based on DNA fingerprinting. Specifically, an
isolate of L. monocytogenes serovar 1/2b from the brain of a goat with listeriosis exhibited the
identical DNA profile by ribotyping as an isolate from cheese made from that goat’s milk and an
isolate from the refrigerator in which the cheese was stored. This suggested that the cheese made
from the infected goat’s milk may have contaminated refrigerator shelves, thus serving as a reservoir
for L. monocytogenes [72]. In contrast, a human endocarditis fatality with a recent history of
exposure to goats showed that the isolates from humans and animals were of the same serogroup.
However, the goat and human strains were clearly different via DNA analysis, thus reducing the
likelihood of zoonotic transmission [58].
CATTLE
The distribution of L. monocytogenes undoubtedly reflects the available pool of susceptible animals.
In North America, most listeriosis cases occur in cattle (82%); a smaller percentage is found in
sheep (17%) and an even lower percentage in pigs [22]. From 1993 to 2000, listeriosis cases
(n = 346) submitted to the Iowa State University Veterinary Diagnostic Laboratory [292] were from
cattle (85.8%), sheep (9.5%), goats (3.8%), a llama (0.3%), and two horses (0.6% each). In Missouri
[143], from 1986 through 1994, encephalitic listeriosis cases were from cattle (67%), goats (30%),
and sheep (13%). In Iowa, 88% of listeriosis cases were diagnosed as encephalitis [292]. In marked
contrast, from 1975 to 1984, listeriosis cases in Great Britain were from sheep (63%) and cattle
(32%), with pigs, goats, fowls, and other species constituting less than 1% each of the total
submissions [299].
The symptoms of bovine listeriosis include encephalitis, abortion, and septicemia with miliary
abscesses [144]. In 1928, Matthews detailed an outbreak of encephalitis of unknown origin in cattle
which, in retrospect, was probably bovine listeriosis [181]. Since then, listeric encephalitis has been
well documented [60,66,143,221,226,239]. However, even in acute outbreaks, generally no more
than 8 to 10% of a herd succumbs to infection. In Switzerland, bovine listeriosis is the most
frequently diagnosed neurological disease [120] and thus may be the most common cause of
bacterial infection of the central nervous system in adult cattle [226].
Foodborne transmission is the main mode of infection in naturally occurring listeriosis in cattle
with silage most frequently implicated. As in sheep, following ingestion, Listeria is disseminated
via hematogenous spread to the viscera, brain, and gravid uterus.
In addition, because L. monocytogenes is present in soil, fecal material, or other vegetation, it
may enter via abrasions of the nostrils or the conjunctiva while grazing or via the teat of a lactating
cow [139]. Direct injection of the conjunctiva, resulting in keratoconjunctivitis [214], has occurred
as a result of contaminated silage particles abrading the faces of browsing cattle [194]. By travel
along peripheral nerves, especially the hypoglossal (XII) and trigeminal (V) cranial nerves inner-
vating the buccal cavity, L. monocytogenes enters the central nervous system and localizes in the
pons and medulla.
Damage to the cranial nerves (indicated by Roman numerals in parentheses) underlies the
clinical presentation. For example, lesions of the fifth (V) cranial and mandibular nerves lead to
inability to eat or drink or retain food in the mouth. Excessive salivation because of difficulty in
swallowing (IX and X); protrusion of the tongue (XII); ataxia or circling (VIII); facial paralysis,
including unilateral drooping of the lip, ear, and eyelid (VII); and strabismus (VI), reflect damage
to the respective cranial nerve [225]. In the advanced stage, as vision and locomotion are impaired
and the animal becomes increasingly irritable, the illness may be confused with rabies or lead
poisoning. Finally, the animal lapses into a coma and generally dies within 1 to 2 days [239].
Histopathologic lesions of the brainstem consist of foci of necrosis infiltrated with neutrophils,
macrophages, and bacteria [138]. Perivascular cuffing with mononuclear cells is evident [267].
Unlike listeric encephalitis in sheep and goats, most cattle survive at least 4 to 14 days after the
initial onset of symptoms, with a few reports of spontaneous recovery [221].
Listeriosis in cattle is associated with abortion [99,208,221,239] during the last trimester
of pregnancy. However, as demonstrated by Dora, an 11-year-old cow with atypical mastitis,
healthy calves are born to chronic carriers that shed the pathogen in milk [71]. As was true for
sheep, L. monocytogenes is transmitted to the placenta and enters the fetus. Meningitis in
neonates may follow intrauterine infection with the septicemic young animals dying shortly
after birth [241].
In the U.S. Midwest, L. monocytogenes is the third most frequently encountered bacterium
from bovine late trimester abortion cases. L. monocytogenes accounted for 1.35% of bovine
abortions and stillbirth submissions received by the South Dakota Animal Disease Research and
Diagnostic Laboratory from 1980 to 1989 [153]. Ten years earlier, 2.2% of 2,544 bovine abortion
cases examined in the Northern Plains region of the United States were attributed to listeriosis
[154]. Similarly, in Germany from 1984 to 1989, L. monocytogenes serotype 1/2 was recovered in
1.2% (122/9931) and 1.8% (122/9931) of bovine abortions [30].
L. ivanovii is most often associated with sheep [21,38,62,126,133,177,178,242] and is some-
times reported from cattle [3,95]. In California, during a 3-year period, five cases of listeric bovine
abortions of a total of 243 fetuses submitted for evaluation were diagnosed. L. ivanovii was
recovered from four cases and L. monocytogenes was isolated from only a single bovine abortion.
The pathologic findings in these five listeriosis cases were similar [3].
L. monocytogenes may enter a herd by contaminated feeds, introduction of new stock, and
rodents. Bovine abortions and stillbirths occur shortly after contaminated silage is eaten [5].
Improvement in silage production and hay making reduced (from 8.7 to 1.2%) the number of listeric
bovine abortion cases in The Netherlands [63]. A change in silage production also decreased the
percentage of carrier animals (from 15 to 0.8%) based on fecal sampling [68]. As expected, the
number of healthy carriers is lower on premises without overt disease than on premises with clinical
listeriosis. To illustrate, L. monocytogenes was cultured from 2.0% of cows on farms without
L. monocytogenes and in 6.7% of healthy animals on farms on which listeriosis had occurred [63].
This may indicate exposure to a common source of infection. Rodents are known carriers of
L. monocytogenes and fecal contamination of animal feed is a potential source of contamination
[5,101,151].
Normal healthy cattle may intermittently shed Listeria in their feces; prevalence rates range
from a few percent up to 52%, with some seasonal differences [81,127,271,288]. Fecal shedding
may reflect levels of L. monocytogenes in feed. Shedding of L. monocytogenes (52%) was related
to feeding wet feed (e.g., silage of beet tops, oat, and pea straw), 67% of which was contaminated
[252]. L. monocytogenes was isolated from the feces of 8.7% of nearly 4,000 randomly selected
dairy cows in Finland over a 2-year interval [129]. Again, L. monocytogenes was recovered more
frequently from bovine feces on farms where L. monocytogenes was in feed than on premises where
feed (silage or pasture grass) was negative for L. monocytogenes [127,129].
Switching cattle from grazing to a diet of silage increased fecal shedding of L. monocytogenes.
Distribution was seasonal: L. monocytogenes was recovered from feces more frequently during
the indoor season (9.2%) than when animals were on pasture (3.1%). As expected, the seasonal
occurrence of L. monocytogenes in milk reflected the frequency of L. monocytogenes in feces
but not in grass silage and pasture grass; thus, fecal contamination during milking could be
inferred [127].
Although not particularly common, generalized listeric infections can give rise to mastitis
[41,60,71,97,145,216,258,274,281]. Beginning in 1938, Schmidt and Nyfeldt postulated that a small
outbreak of human listeriosis in Denmark may have been caused by drinking milk from mastitic
cows. However, the role of Listeria in mastitic infections was not clearly identified until 1944 when
Wramby [302] isolated L. monocytogenes from milk and udders of mastitic cows in Sweden. In
1956, de Vries and Strikwerda [60] described another case of bovine mastitis in which a penicillin-
resistant strain of L. monocytogenes was cultured from one quarter of a 6-year-old dairy cow.
Following acute onset, the condition soon became chronic with shedding of L. monocytogenes in
the milk for 3 months. Prolonged excretion of L. monocytogenes in milk [64,65,131, 208,209, 221],
apparently normal appearance of the milk, and consumption of raw milk on farms could be important
in transmission and epidemiology of Listeria infection [103].
L. monocytogenes strain Scott A (serotype 4b), a clinical isolate recovered from the New
England outbreak, has produced experimental mastitis in dairy cows [39,291]. Following repeated
intramammary inoculation of 34 Holstein cows with 103 to 107 L. monocytogenes cells, 75% of
the animals became chronically infected and shed Listeria in milk (ca. 103 to 105 L. monocytogenes
CFU/mL of milk) intermittently for up to 8 months. The intramammary route of inoculation
simulated infection from contaminated bedding directly into the teat canal [291]. Interestingly, one
of these experimentally infected cows delivered a normal, healthy bull calf. Bourry et al. [34]
induced mastitis also via intramammary inoculation with a single dose of 300 L. monocytogenes
serotypes 4b and 1/2. L. monocytogenes was recovered from the supramammary lymph node, but
not from the spleen or liver. DNA profiles of isolates recovered throughout experimental infection
confirmed persistence of the inoculated strain [34].
As with sheep [7] and goats [166], L. monocytogenes also is shed in milk by healthy dairy
cattle with no indication of mastitis [63,80,97,123,130,131,198,278]. Schultz [235] collected milk
samples from 1,004 cows and isolated L. monocytogenes from the milk of 10 animals (0.1%), 7
of which appeared perfectly healthy. Shedding of Listeria in milk from these animals was inter-
mittent, but continued for as long as 12 months. Examination of dairy herds in Yugoslavia [247]
also has demonstrated that clinically healthy cows are asymptomatic carriers of L. monocytogenes
and secrete the organism in their milk for months over several lactation periods. In one such survey,
L. monocytogenes was detected in milk from 3.2% of 845 clinically normal cows on seven farms
in which listeriosis had been previously diagnosed [156]. Earlier, Kampelmacher [148] reported
that dairy cattle shed L. monocytogenes at levels of 10,000 to 20,000 CFU/mL of milk.
Surveys of the raw milk supply prompted by dairy-related outbreaks of human listeriosis
in 1983, 1985, and 1987 confirmed that asymptomatic cattle are carriers for L. monocytogenes.
L. monocytogenes has been reported in raw cow’s milk with a distribution generally ranging from
0.1 to 45% [22,70,80,81,88,105,118,170,254,260,265]. In the United States and Canada, the inci-
dence is reported to be no more than ~5% [135], although in the northeastern United States, Hayes
et al. [118] reported L. monocytogenes in 12% of raw milk samples. L. monocytogenes was isolated
from 4.1% of raw milk samples from Tennessee [231]. Interestingly, in that U.S. study, consumption
of raw bulk milk was reported by 35% of dairy producers [231].
Jayaro et al. also reported that nearly 60% of dairy farmers consumed raw milk with few
reporting clinical illness [135]. The recoveries varied depending on whether individual cows, bulk
tanks maintained on the farm, or milk tankers serving multiple premises were sampled [114,230].
A 23-year survey involving 36,200 dairy herds in Denmark indicated that the incidence of
L. monocytogenes-infected cows varied from 0.01 to 0.1% and of herds with an infected cow
ranging from 0.2 to 4.2%. Yet, 8.5% of bulk milk samples (n = 445) were contaminated with
L. monocytogenes [6].
Differences in recovery may also result from variations in farm management practices and
regional, seasonal differences, as well as variations in isolation protocols. Indirect contamination
of bulk milk occurs under unhygienic milking conditions, if L. monocytogenes is present in feed,
feces, udder surface, or bedding [81], or if an animal is recovering from a recent infection [98,192].
On-farm risk management factors have been reported for New York dairy farms [15]. Specif-
ically, the observed risk (OR) was lower for farms that used milking parlors (OR = 0.21, P = 0.01)
than for premises using a bucket system. Premilking teat disinfection (OR = 0.26, P = 0.001),
premilking examination for abnormal milk (OR = 0.4, P = 0.01), and dry-cow therapy for
>6 months (OR = 0.34, P = 0.04) were protective factors. In contrast, use of Escherichia coli J5
vaccine (OR = 3.3, P = 0.03) was linked to higher incidence.
Regional differences have been documented despite relatively low recovery rates (~5%) of L.
monocytogenes in milk. Yet Dominguez-Rodriguez et al. [70] found L. monocytogenes in 45.3%
of 95 raw milk samples from a single bulk tank (80,000-liter capacity). The dairy received raw
milk from a number of small farms in western and central Spain over a 16-month interval. Seasonal
distribution of L. monocytogenes in raw milk, mirroring numerous determinants including a change
of diet or weather-related stress, has been observed. Lovett et al. [170] surveyed raw milk at various
times from three different regions in the United States and found L. monocytogenes in 4.2% of the
overall samples. However, the recovery of L. monocytogenes from Massachusetts samples was
seasonal with the incidence highest during cooler months and lowest in hot-weather months.
A more recent survey of 404 New York dairy farms revealed higher recoveries (odds ratio = 1.8) in
spring (18%) than other seasons [115]. In the Pacific Northwest, prevalence varied from 4.7% in
November to 7% in June. In that study, PFGE profiles suggested relatively stable clones, which
may reflect selection and survival of strains that have adapted to the bovine host [198]. Interestingly,
no seasonal trend was exhibited by samples recovered from Ohio, Kentucky, and Indiana [173]. A
study of L. monocytogenes in raw milk in Nebraska, a state that experiences severe climatic changes,
indicated a seasonal distribution with 6% of raw milk samples harboring L. monocytogenes in
February and 2% of samples positive in July [164].
Seasonal variation may be related to silage feeding during the winter. L. monocytogenes was
cultured from 4.9% of raw milk samples collected from 70 Irish farms with the incidence higher
in the winter when cows were housed indoors than in the summer [224]. In Scotland, a seasonal
distribution was indicated for L. monocytogenes, which was present overall on 25 premises of
the 160 farms surveyed (16%). Contamination was sporadic, with bacterial titers generally less
than one L. monocytogenes per milliliter. Although more raw milk samples were positive for
L. monocytogenes in January than at other sampling dates throughout the year, the authors caution
that no link to farm management practices was evident [84]. In Ontario, Canada, raw milk
recoveries indicated seasonal and geographical differences, with the incidence higher in the
eastern region [85,254].
Serosurveys have monitored distribution of L. monocytogenes in dairy cows [71,198]. Infection
in cattle can be estimated by measuring antibodies to whole cells and antibodies specifically
targeting antibodies to listeriolysin (LLO) [12,32,33] as well as internalin A [32]. Because up to
33% of dairy cattle continued to shed L. monocytogenes despite high serological titers, antibody
levels do not appear to modulate recovery of L. monocytogenes from milk [71,257,276]. As in
humans [26], sheep [13,163,174], and goats [193], antibodies to listeriolysin O (LLO) have con-
firmed previous or current infection with L. monocytogenes in cattle [12,32,33].
When antibody titers to LLO and internalin A were correlated with dairy farm practices, a
positive correlation was found between feeding corn silage (OR = 6.5). In contrast, use of rubber
bedding mats (OR = 0.2) and an electrical device to train cows to defecate and urinate off the
bedding area (OR = 0.36) were correlated with low LLO and internalin A titers, suggesting low
exposure to L. monocytogenes. Serum levels also imply differences in breed susceptibility [32].
Stress-related immunosuppression associated with change of diet, weather, transportation [83],
pregnancy, parturition, and lactation may lower resistance to bovine listeriosis [239]. Dexametha-
sone mimics the stress-related release of glucocorticoids. In cattle, dexamethasone elevates total
white blood neutrophil counts and decreases eosinophil and lymphocyte populations. When admin-
istered to cows experimentally infected with L. monocytogenes, dexamethasone increased the
shedding by up to 100-fold of L. monocytogenes in milk [291]. The increased levels of L. mono-
cytogenes in the milk may reflect impairment of cell-mediated immune mechanisms and phagocytic
cell functions that underlie listerial immunity [269]. Likewise, transport of live animals over long
distances may increase the level of fecal excretion of L. monocytogenes.
A major concern of bovine listeriosis is the potential risk posed to humans. In Denmark, a
case-control study indicated that human listeriosis was frequently linked to consumption of unpas-
teurized milk (risk factor of 8.6), although other factors, such as immunosuppression and underlying
diseases, were regarded as more significant [137]. Furthermore, a comparison of 33 isolates from
bovine mastitis and 27 human clinical isolates in Denmark recovered during 1993 was made by
sero- and ribotyping. Serotyping showed that all bovine and 63% of human isolates belonged to
serogroup 1, whereas 37% of the human isolates were of serogroup 4. DNA fingerprinting by
ribotyping indicated that a low but constant percentage of Danish dairy herds have cows infected
with L. monocytogenes strains similar to human clinical strains [141]. L. monocytogenes ribotypes
common to dairy processing and the farm environment (dairy cattle, raw milk, silage) were also
reported in the United States. This indicated that the farm may serve as a reservoir for L. mono-
cytogenes strains capable of entering the dairy-processing facility [8]. Alternatively, it verifies the
ubiquitous distribution of the pathogen.
Although foodborne listeriosis in humans is more frequently linked to consumption of
contaminated dairy products than to beef consumption, L. monocytogenes was recovered from
3% of composite fecal samples representing 224 feedlot beef cattle [249]. In a limited study of
experimentally infected Holstein cows (n = 4), L. monocytogenes was cultured from muscle,
organ, and lymphoid tissues at 2 days after infection; none was recovered at 6 or 54 days after
inoculation [142]. This indicates that cattle may be an insignificant source of L. monocytogenes
contamination in meat [168]. Epizootics have been observed in feedlot cattle and in beef cattle
herds [5,301].
Transport of cattle over long distances increased the level of fecal excretion of L. monocyto-
genes, but not contamination of carcasses [83]. Yet in this study L. monocytogenes was detected
in 91% (21 of 23) of minced beef samples, again demonstrating that postharvest processing
significantly increases the level of contamination compared to that of the whole carcass [83]. This
hypothesis is further strengthened by tracking L. monocytogenes strains by multilocus enzyme
electrophoresis. L. monocytogenes strains of electrophoretic type (ET) I have been implicated in
major human foodborne epidemics and are found coincidentally in livestock. L. monocytogenes
strains of ET I predominate in cattle at the beginning of slaughter, but are not detected on the
carcasses at the end of processing or in the environment of the abattoir [31].
In contrast, environmental strains, such as ET 19, contaminate the carcass during processing.
Likewise, ET 19 strains were found on the carcasses of pigs at the end of processing in two
slaughterhouses, but not on live animals or at the beginning of slaughter. Taken together, these
studies indicate that contamination of meat during processing occurs from L. monocytogenes strains
that reside in the packing plant rather than from strains indigenous to animals [31].
SWINE
Porcine listeriosis manifests primarily as septicemia. Encephalitis is reported less frequently and
abortions are rare [29]. Clinical septicemia is usually observed in the neonate where hepatic necrosis
may be a characteristic feature [112,191]. Unlike its frequent occurrence in ruminants (cattle, sheep,
and goats), listeriosis is rare in monogastric swine. Slabospits’Kii [253] first reported Listeria
infection in young swine raised on a Russian farm and designated the organism as L. suis [29].
The first description of porcine listeriosis in the United States occurred when Biester and Schwarte
[28] reported it in Iowa in swine with encephalitis. Later, Kerlin and Graham [150] recovered
Listeria from the liver of a pig with no clinical signs of encephalitis. In Norway, Hessen [121]
reported listeric septicemia in piglets raised on a farm where sheep had died of listeriosis several
weeks earlier. Whether transmission was from the sheep to the pigs or the result of common
exposure is unknown.
In natural and experimental infections, listeriosis is more severe in young animals [30,42,148].
Piglets succumb to infection whereas adults generally survive. In the neonate, L. monocytogenes
may originate from the tonsils of the sow, penetrate the intestinal tract of the piglet, and become
systemic [267]. Neonatal listeriosis may be seasonal with cases peaking in early winter [169]
and spring.
Listeric encephalitis seldom occurs in pigs. Symptoms of central nervous system disturbance,
including incoordination and progressive weakness followed by death, are characteristic of listeri-
osis of the younger animal. Meningoencephalitis in swine begins with a sudden refusal to eat and
is typically followed by various neurological disorders including trembling, partial paralysis, incoor-
dination, circling movements, and convulsions. Histopathological findings from meningoencephalitis
include severe monocytic infiltration. Numerous blood vessels, particularly those in the pons, reveal
perivascular cuffing [29,102,219,239]. Although listerial meningoencephalitis in swine is infre-
quent, several such outbreaks have been reported, including one in India in which 27 of 75
pigs died [179,220].
In England, the Veterinary Investigation Center reported only 14 listeriosis cases in swine
between 1975 and 1982 as compared to 666 cases in sheep and 472 cases in cattle [96]. Listeriosis
in pigs is also reported to be uncommon in The Netherlands [200]. Porcine listeriosis comprised
1% of listeriosis cases in Western Canada [22]. In Iowa, a major hog-producing state, from 1993
to 2000, of a total of 253 listeriosis submissions to the state veterinary diagnostic laboratory, none
were from pigs. In that same interval, 87% of listeriosis cases in Iowa were from cattle [292].
Earlier, Blenden reported that cattle and sheep accounted for 274 of 281, or 98%, of listeriosis
cases submitted to the Missouri Diagnostic Laboratory; only 1 case was from pigs [29]. Missouri,
like Iowa, is a major hog-producing state.
Because few surveys are available describing the prevalence of L. monocytogenes in healthy
pigs, its distribution (0 to 16%) may be estimated from surveys of fecal excretors and recoveries
from tonsils and carcass swabs collected at slaughter [86,125,203,277]. In a study in the United
States of 300 market-weight hogs, L. monocytogenes was recovered overall from 2.4% of hog
tissues, including tonsils (7.0%) as well as thoracic (3.5%) and superficial inguinal (1.9%) lymph
nodes [146]. For cull sows (n = 181)—animals raised primarily for reproductive purposes and under
less dense animal housing conditions—L. monocytogenes was cultured from a single tonsil sample
(0.6%) and from none of the rectal content samples (Wesley, unpublished). In a limited study of
131 wild boars in Japan, L. monocytogenes serotype 4b was cultured from 2% of fecal samples.
The lower frequency of recovery may be attributed to low population density in the wild. No
attempts were made to recover the pathogen from other sites, including tonsils [117].
Asymptomatic carriers of Listeria may be more prevalent in Eastern Europe [221,222]. Ralovich
[221] reviewed studies in which fecal recovery rates of 47% in individual animals and in 11 of 12
farms were described. A high infection rate in pigs has led to speculation that swine may be
important reservoirs of L. monocytogenes in Eastern Europe [102].
Husbandry practices such as feeding pigs dry feed or silage, rearing in closed houses, main-
taining specific-pathogen-free (SPF) herds as well as differences in sampling sites (tonsils vs. feces)
and seasonality may account for variation in the incidence of healthy porcine carriers reported. To
illustrate, in Yugoslavia, L. monocytogenes was recovered more frequently from tonsils of pigs
raised on silage (61%) than animals raised on dry feed (29%).
Recoveries from meat generally exceed isolation from live animals, suggesting postslaughter
contamination during processing. Skovgaard et al. [252] reported that although only 1.7% of pig
fecal samples yielded L. monocytogenes, the pathogen was detected in 12% of ground pork samples
in Denmark, indicating dissemination of Listeria during processing—an observation made by other
investigators [40]. In France, an outbreak involving 279 human cases incriminated pickled pork
tongue as a major vehicle of transmission, although other highly processed, ready-to-eat delicatessen
items subject to environmental contamination were also implicated [59,134]. Live hogs were
thought to have introduced contamination into the processing plant.
Although limited epidemiological data are provided, two cases of human neonatal listeriosis
may have been linked indirectly to contact with pigs [256]. Alternatively, they may reflect exposure
to a common source of contamination.
FOWL
Avian listeriosis was first described in 1935 [238], 3 years after TenBroeck isolated L. monocyto-
genes (then Bacterium monocytogenes) from diseased chickens. Wild [35,227,288] and domestic
avians, including turkeys [24,116,204], ducks [100,239], geese [100,239], and pheasants [100],
may be asymptomatic carriers. Up to 33% of all healthy chickens may asymptomatically shed
L. monocytogenes in fecal material [66,67,252]. Birds most likely become infected by pecking
Listeria-contaminated soil, feces, or dead animals; however, contaminated fecal material also may
pose a hazard to other livestock. Bovine encephalitic listeriosis developed in four cows housed in
stables where chicken litter was used as flooring. L. monocytogenes serogroup 4b was recovered
from the bovine brains, litter, and the intestinal contents of 4.1% of the donor birds (n = 2.3 million)
[68]. In another study, the increased incidence of L. monocytogenes in rooks coincided with nesting
season and the peak of ovine listeriosis, which in turn was linked to consumption of contaminated
silage [82].
Despite the many sporadic cases of avian listeriosis that have been documented over the last
60 years, this disease is far less common in birds than in sheep, goats, and cattle [100–102,
199,211,236].
Listeriosis in birds may be a secondary infection associated with viral infections [57] as well
as salmonellosis, Newcastle disease, fowl pest, coryza, coccidiosis, worm infestations, mites,
enteritis, lymphomatosis, ovarian tumors, and other immunocompromising conditions [102]. In
1988, an encephalitic form of listeriosis was reported in broiler chickens in California [54].
Predisposing conditions that may have precipitated the outbreak included recent debeaking and vacci-
nation with a modified live viral arthritis vaccine given subcutaneously in the neck. L. monocytogenes
serotype 4b was recovered from a liver and multiple brain samples.
Three years later, a second outbreak occurred in breeder replacement birds, affecting 0.3% of
the 54,000 birds in the flock. L. monocytogenes was recovered from soil samples collected near an
adjacent dairy, but not from other sites on the premises. The stress associated with the unusually
cold climate described in the report coupled with vaccine stress of the 7- to 10-day-old chicks may
have led to this outbreak [54].
Septicemia, the most frequent manifestation of listeriosis in domestic fowl, is characterized by
focal necrosis within the viscera, particularly the liver and spleen [102]. Although not present in
all cases [199], cardiac lesions frequently develop and, in turn, lead to engorgement of cardiac
vessels, pericarditis, and increased amounts of pericardial fluid [102]. Other conditions produced
by the septicemic form of avian listeriosis have included splenomeglia, nephritis, peritonitis,
enteritis, ulcers in the ileum and ceca, necrosis of the oviduct, generalized or pulmonary edema,
inflammation of the air sacs, and conjunctivitis. In acute cases, lesions resulting from these condi-
tions may be partially obscured by congestion and hemorrhages throughout the viscera [102].
Unfortunately, domestic fowl that suffer from listeric septicemia normally exhibit few overt signs
of disease other than progressive emaciation and usually die 5 to 9 days after infection.
Although far less common than the septicemic form of listeriosis, L. monocytogenes also can
produce meningoencephalitis in domestic fowl. Domestic birds suffering from listeric meningoen-
cephalitis exhibit several striking behavioral changes, including incoordination, tremors, torticollis,
unilateral/bilateral toe paralysis, and dropped wings, all of which directly relate to disturbances of
the central nervous system [18]. Such infections are virtually always fatal. Postmortem examination
often reveals congestion and necrotic foci in the brain along with many of the aforementioned
conditions that are characteristic of listeric septicemia. Microscopically, gliosis and satellitosis in
the cerebellum and microabscesses containing Gram-positive bacteria are found in the midbrain
and medulla of birds with encephalitic listeriosis [55].
L. monocytogenes colonizes chick embryos and young birds; older birds appear to be more
resistant [11,61,102,111,266]. Following oral challenge of chickens with 102 or 106 L. monocytogenes
cells, Bailey et al. [15] detected the pathogen more frequently in ceca, spleen, liver, and cloacal
swab samples from 1- rather than 14- or 35-day-old chickens. Diarrhea and emaciation have been
noted in experimental infection, thus facilitating spread via feces and nasal secretions. In a later
study, 2-day-old chicks were experimentally infected with L. monocytogenes. Although most of
the inoculated chicks appeared healthy, depression, ruffled feathers, dullness, and diarrhea followed
by death were noted 2 to 5 days after inoculation. Milder symptoms such as anorexia and drowsiness
were also observed in a few of the animals. At 5 days after infection, 100% of the cecal samples
yielded L. monocytogenes. However, the percentage of L. monocytogenes-positive birds decreased

and by day 28, L. monocytogenes was recovered from the caeca of only 10% of experimentally
infected birds [128].
In another report using a smaller number of birds, L. monocytogenes was only found on the
first day following infection in 15% of fecal samples [183]. Taken together, these data suggest that
L. monocytogenes is cleared rapidly, indicating that birds are at best transiently infected and not
likely reservoirs of L. monocytogenes [305]. Interestingly, following artificial infection, Pustovaia
[218] found that Columbiformes, Passeriformes (perching birds), and Galliformes were susceptible,
while Falconiformes and Strigiformes (owls) were resistant [124].
L. monocytogenes has been used to investigate macrophage function in retrovirus infection and
the cell-mediated immune response in susceptible and resistant chickens exposed to Marek’s disease
virus [45,57]. Viral infection depressed the resistance of 10-day-old chickens to experimental
infection with an avian osteopetrosis virus [57]. When compared to virus-free chickens, the dually
infected birds were less efficient in clearing L. monocytogenes from their spleens [57].
L. monocytogenes is present in healthy birds from nondetectable levels to up to 33%
[66,67,93,123,132,288]. Yet contamination in retail poultry ranges from 17 to 70%, again suggesting
postharvest contamination [14,31,86,93,183,207]. A link between transport stress and fecal shed-
ding of L. monocytogenes has been suggested. In one study L. monocytogenes was found in 33%
of pooled fecal samples collected from cages, suggesting recrudescence of L. monocytogenes
because of transport stress. But no data on the L. monocytogenes status of these birds before
shipment are provided [252].
In an effort to trace the source of L. monocytogenes present in retail poultry, low levels of
natural carriage (5%) were reported in cecal samples of parent flocks providing broilers. Yet, L.
monocytogenes was not cultured from cecal samples from over 2,000 broilers from 90 flocks in
Denmark [208]. However, L. monocytogenes was found in processed poultry. Comparison of DNA
fingerprinting patterns by pulsed-field gel electrophoresis (PFGE) indicated that live birds contrib-
uted little to the total contamination of the product [207,232].
MINOR SPECIES
L. monocytogenes has been diagnosed in a number of minor wildlife and livestock species, such
as horses, llamas, animals raised commercially for pelts, companion animals, deer, and primates
[195]. The routes of transmission and symptoms parallel those of cattle, sheep, and goats.
As with other livestock species, Listeria infection in horses can cause abortion [289], septicemia
[25,51,74,106], and encephalitis as well as keratoconjunctivitis [233]. In contrast to cattle and
sheep, few cases of equine listeriosis are reported [160,182,185,239,263,264,268]. A survey of
fecal samples of 400 German horses indicated a carrier rate of 4.8% for L. monocytogenes, 6% for
L. innocua, and 1.5% for L. seeligeri; less than 1% harbor L. welshimeri [288]. The results of a
limited number of surveys describing L. monocytogenes-seropositive horses should be interpreted
cautiously in light of possible cross-reactivity of antibodies of other bacterial species to Listeria.
Previous history of contact with cattle and feeding on silage may explain sporadic cases of equine
listeriosis [184]. L. monocytogenes was reported from four Welsh and two Shetland ponies housed
together with cattle, one of which was diagnosed with listeriosis, and given poor-quality silage [74].
At necropsy, L. monocytogenes was cultured from the equine liver, spleen, heart, kidneys, and lungs
[74]. In Tasmania, abortions occurred in two mares allowed to graze on pasture that had previously
been a sheep farm but had most recently served as a dairy farm. L. monocytogenes serogroup 1 was
cultured from the lung and stomach of one fetus. Following antibiotic therapy, the mare was
bred and later gave birth to a normal live foal, indicating again that L. monocytogenes infection
may not impair fertility [180].
Equine abortion, preceded by mild respiratory tract infection, caused by L. monocytogenes
serotype 4 was reported in a mare that had wintered with cattle and been on ensilage [289].
L. monocytogenes was cultured from the equine fetal liver, lung, spleen, and stomach. Neonatal
septicemia was documented in a 3-day-old foal whose mare was housed indoors and fed poor-
quality, contaminated hay [122].
As is true in other species, the origin of infection in equine listeriosis cases may be unknown
[233]. To illustrate, L. monocytogenes was recovered from the brainstem of a 16-year-old Welsh
pony gelding with signs of ataxia, weakness, and deficits of cranial nerves. No immunological deficit
was detected and there was no history of contact with ruminants or access to silage. A 21-day-old
Appaloosa filly was examined because of diarrhea of 2 weeks’ duration [284]. Septicemia was
diagnosed based on presence of L. monocytogenes cultured from blood. No sources of infection
were evident, thus leading to speculation that L. monocytogenes was transmitted to a foal via
contaminated mare’s milk.
As described in humans and livestock, listeriosis occurs in immunocompromised hosts with
defects in the humoral and cell-mediated immune components [269]. Listeriosis has been described
in an Arabian foal with combined immunodeficiency [51]. The 1-month-old foal was ataxic,
lethargic, failed to nurse, and spent most of the time with its head down. Most strikingly, hoofs
were dragged when the animal was exercised. At necropsy widespread lesions were present in the
viscera and central nervous system.
In the llama, listeriosis occurs as a septicemia with meningitis in neonates, but more commonly
causes asymmetric vestibular disease in adults [19]. Most animals affected are weaned and grazing
or consuming roughage, but are not on silage. Multifocal suppurative encephalitic listeriosis was
diagnosed in two adult llamas that were pregnant. L. monocytogenes was cultured from one of two
animals and was visualized in the brainstem lesions of both llamas by fluorescein-conjugated antibody
to L. monocytogenes [43]. In another report, L. monocytogenes caused fatal meningo-encephalomy-
elitis in a 3- to 5-month-old llama. The animal displayed unilateral peripheral disease progressing to
encephalitis [270]. The source of infection for cases detailed in these two reports is unknown [19,43].
Listeriosis may have an economic impact in commercial pelt farms. An outbreak of disseminated
visceral listeriosis in chinchillas in Nova Scotia [89] incriminated consumption of contaminated
sugar beet pulp, although L. monocytogenes was not isolated from the feed. The outbreak occurred
in a colony with 23% mortality of breeding chinchillas [300]. Approximately 4 days before death,
animals were anorexic and hunched and some had torticollis (twisted necks). However, many
animals were found dead without clinical signs [300]. Hay contaminated with rodent, bird, or
ruminant feces has been implicated in previous outbreaks of chinchilla listeriosis and removal of
contaminated feed often interrupts the cycle of transmission [47,89]. L. monocytogenes could have
been transmitted by coprophagia because animals defecated in dust bath pans and the pans were
transferred from cage to cage [298]. In an enzootic of listeriosis in a rabbitry, L. monocytogenes
1/2a was cultured from feed samples and from a doe that had died of septic metritis [213].
The early literature describes L. monocytogenes in nondomesticated ruminants, including rein-
deer, roe deer, antelope, and a Grant’s gazelle with previous contact with listeric sheep [20,23,76,
78,149,204,286]. L. monocytogenes has been recovered from ruminants housed in zoological parks
[9,76,78,179,286]. Meningoencephalitis occurred in 42 deer in a flock of 1,800 head in a park
during the winter and early spring in Denmark. This was preceded in the previous spring by the
death of six deer that exhibited circling and appeared to be blind. The following year, the first
sign of illness was a drooping ear, due to paralysis of the facial nerve, and a slight inability to
follow the herd. No external source of L. monocytogenes was evident and stress because of a
poor beech-mast crop, increased stocking rate of animals resulting in overcrowding with possible
introduction of healthy carrier animals, and sudden change in weather were all potential con-
tributing factors [76].
Listeriosis is rarely reported in dogs and has caused encephalitis, including circling, and abortion
[261]. In a survey of domestic animals, L. monocytogenes was detected in 1.3% of dog (n = 300)
and 0.4% of cat (n = 275) fecal samples [288]. The low recovery may indicate that companion
animals are not important in the epidemiology of listeriosis in humans [287] or that shedding is
sporadic. In contrast, serosurveys indicating that up to 90% of dogs may be seropositive [251]
should be interpreted cautiously because of the cross-reactivity inherent in agglutination tests. A
single report on the possible transmission to L. monocytogenes from humans to dogs [262] warrants
re-evaluation because the isolates were later identified as L. innocua. Nevertheless, recovery of a
single species of Listeria from humans and dogs in close proximity could reflect substantial
environmental contamination rather than human-to-dog passage.
Listeriosis occurs in nonhuman primates, where it manifests as septicemia [48,303], menin-
goencephalitis [61], and stillbirths [185,273], as documented in a large outdoor breeding colony
in California [212]. Transmission may occur by consumption of contaminated foods [273]. Exper-
imental infections were attempted by exposure to aerosols [147] as well as by feeding [79,255].
Cynomolgus monkeys (Macaca fascicularis) shed L. monocytogenes in their feces for up to 21
days after receiving an oral dose of 109 Listeria. Neither septicemia nor encephalitis was reported,
indicating that the normal healthy primate is resistant to L. monocytogenes. In contrast, abortion
and stillbirths were reported in rhesus monkeys fed 108 CFUs in a high-fat vehicle [255].
FISH AND CRUSTACEANS

Consumption of seafood is growing in popularity, and the aquaculture industry is responding by
providing fish raised in controlled fish farms rather than depending on the availability of fresh
caught products. In 1980 in New Zealand, Lennon et al. [160] reported a cluster of 22 perinatal
human listeriosis cases. A weak association between these cases and the consumption of contam-
inated raw fish and shellfish was established. DNA fingerprinting determined that fish in the patient’s
refrigerator were identical.
The first report of the occurrence of listeriosis in fish was in 1957 from Romania [259]. In that
study, L. monocytogenes was isolated from the viscera of pond-reared rainbow trout that presumably
became infected after consuming contaminated donkey meat. Results of current surveys indicate
that Listeria is absent from live seawater fish, but is present in live freshwater species. In contrast,
L. monocytogenes was recovered from two of five intestinal tracts of market-purchased fresh water
fish in India [119].
Channel catfish (Ictalurus punctatus) is the most widely cultured species in the United States;
most commercial ponds are located in the southeastern region of the country. A study of catfish,
water, and feed collected from university ponds in Alabama indicated presence of L. monocytogenes
on the skin and viscera (mean presumptive count = 1.99 log CFU/wet weight). L. monocytogenes
was not detected in water or feed. Unfortunately, the authors provide no data on the percentage of
L. monocytogenes–positive fish, but conclude that the bacterial concentrations in the viscera suggest
that cross-contamination is possible during evisceration [162].
A survey of three rainbow-trout farms in Switzerland showed that L. monocytogenes was present
in the feces (40%) and on the skin (33%, 5/15) of fish from one of the farms. Yet L. monocytogenes
was detected on the finished product in only 6% of the fish from this farm. In contrast, L.
monocytogenes was not found in feces, skin of fish, or the finished product of two of the farms
where fish were raised in concrete ponds and starved 3 to 7 days before harvest [136]. Similarly,
L. monocytogenes was not recovered from the skin, gills, intestines, tank water, or diet of striped
bass grown in recirculating water tanks [202]. Experimental infection of zebrafish (Brachydanio
rerio) indicated the LD50 was higher in fish than in mice and that L. monocytogenes did not multiply
in fish [189]. Experimental infection stimulated an increase in granulocyte and monocytes. In
contrast to L. monocytogenes, strains of L. welshimeri, L. innocua, and L. seeligeri killed more
than 50% of fish 7 days postinfection [189].
Brackett [36] proposed that contamination of fish and shellfish through their ambient waters
may influence distribution of L. monocytogenes. Surface waters, sewage effluents, and agricultural
runoff all may potentially contribute Listeria spp. to the aquatic environment. In addition, presence
of L. monocytogenes in sea gulls may be another source of shellfish contamination [82]. In 1959,
L. monocytogenes was detected in crustaceans gathered from a Russian stream [248].
A more recent survey conducted on the Gulf Coast of the United States examined shrimp,
oysters, and estuarine waters for L. monocytogenes [196]. L. monocytogenes was detected in 11%
of unprocessed shrimp (n = 74), but not in oysters (n = 75), although some of the oysters were
harvested from prohibited shellfish-growing sites. In a parallel study conducted in fresh water
tributaries off the Humboldt–Arcata Bay in Northern California, L. monocytogenes was detected
in the freshwater samples (61%), some of which received runoff from nearby farms. However, L.
monocytogenes was not found in oysters in that study [52] or in oysters kept in live holding tanks
in seafood markets in Seattle [53].
Taken together, these data indicate that live fish and shellfish are not likely carriers of L.
monocytogenes. PCR tests have been used to detect L. monocytogenes in experimentally contam-
inated marinated rainbow trout [77]. With the increased interest in L. monocytogenes in fish and
seafood [77], this sensitive technique may be useful for the rapid screening of live shellfish and
fish for L. monocytogenes. Nevertheless, the paucity of reports documenting L. monocytogenes in
live freshwater fish and shellfish suggests that Listeria spp. cultured from the retail product are
most likely from postharvest contamination.
TREATMENT
Poor husbandry, consumption of contaminated feed, and stress are important in initiating the disease.
Thus, identifying and eliminating these factors are critical to preventing recurrences. In general,
because antemortem diagnosis is rarely made, treatment is seldom attempted.
Because listeric encephalitis is a rapidly debilitating disease in ruminants, treatment must be
initiated early during the course of infection if there is to be any reasonable hope of survival. L.
monocytogenes is resistant to many drugs but is sensitive to chlortetracycline. The intravenous
injection of chlortetracycline (10 mg/kg body weight per day for 5 days) is effective in meningoen-
cephalitis of cattle but less so in sheep [219]. If penicillin is used, high doses are required because
of the difficulty of maintaining therapeutic levels in the brain. Penicillin G should be given at
44,000 U/kg body weight, intramuscularly daily for 1 to 2 weeks [90]. If signs of encephalitis are
severe, death usually occurs in spite of treatment.
Supportive therapy, which is usually reserved for valuable animals, includes fluid and electrolyte
replacement and is indicated for animals having difficulty eating and drinking as a result of neural
damage. Excessive salivation leads to acidosis, which is remedied by intravenous replacement of
bicarbonate ions. Permanent neurological damage often occurs in ruminants, despite proper therapy.
In view of the severe economic losses from listeric encephalitis in sheep, it may be prudent to
consider vaccinating animals against listeriosis, particularly if they are raised in areas prone to
listeric infection [202].
In birds, tetracyclines (5 to 10 mg/kg body weight daily for 1 week) are efficacious in acute
and subacute forms. Treatment of the chronic form is unsuccessful. As with other livestock species,
rigid sanitation and disinfection procedures with culling and isolation of affected birds may be
helpful [91].
A benefit of early treatment of animals with listeriosis has been demonstrated. However,
timeliness is most important and recognition of the disease depends upon observation of clinical
signs. In cattle and sheep the appearance of clinical signs is an indication of neurological damage
and thus of a guarded prognosis for treatment. In all cases, the economics of the attempted treatment
must be considered along with the alternative of humane euthanasia.
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4 Listeriosis in Humans
John Painter and Laurence Slutsker
CONTENTS
Introduction ......................................................................................................................................85
Human Infection and Clinical Manifestations of Listeriosis ..........................................................87
Listeriosis during Pregnancy ..................................................................................................87
Neonatal Disease: Early Onset...............................................................................................87
Neonatal Disease: Late Onset ................................................................................................88
Invasive Disease in Nonpregnant Adults .........................................................................................88
Noninvasive Disease: Gastrointestinal Illness .................................................................................90
Asymptomatic Carriage..........................................................................................................91
Epidemiological Patterns of Listeriosis...........................................................................................94
Epidemic Listeriosis ...............................................................................................................94
Sporadic Disease—Incidence.................................................................................................97
Sporadic Disease—Dietary Risk Factors...............................................................................98
Sporadic Disease—Possible Other Sources.........................................................................100
Diagnosis ........................................................................................................................................100
Treatment........................................................................................................................................101
Prevention.......................................................................................................................................101
References ......................................................................................................................................102
INTRODUCTION
Listeria monocytogenes has been recognized as a human pathogen since 1929 [114], but the route
of transmission was unclear until the 1980s when a series of outbreaks indicated that L. monocy-
togenes was transmitted by food [13,43,59,70,89,102,123,130]. It is now recognized that nearly all
cases of human listeriosis are foodborne [1,104,112,132,136]. Although listeriosis is a very small
fraction of all illness due to known foodborne pathogens, it is an important cause of severe illness,
accounting for 3.8% of foodborne disease hospitalizations and 27.6% of foodborne disease deaths
[104]. Development of improved laboratory techniques to detect and subtype L. monocytogenes
has also contributed to an improved understanding of human listeriosis [9,11,14,125,126]. Listeria
monocytogenes is found in multiple ecological sites throughout the environment, including soil
[152], water, and decaying vegetation [151,153]. Control of human listeriosis, therefore, relies on
improving the understanding of how to control Listeria contamination of food.
Human disease caused by L. monocytogenes occurs most frequently in women of childbearing
age, infants, and the elderly (Figure 4.1). The risk of listeriosis is greatest among certain well-
defined high-risk groups, including pregnant women, neonates, and immunocompromised adults
but may occasionally occur in persons who have no predisposing underlying condition (Table 4.1).
The ongoing epidemic of acquired immunodeficiency syndrome (AIDS), as well as widespread use
of immunosuppressive medications for treatment of malignancy and management of organ trans-
plantation, has expanded the immunocompromised population at increased risk of listeriosis.
85
35%
30%
Percent of Total
25%
FEMALE
20%
MALE
15%
10%
5%
0%
<1 >=1-2 >=2-5 >=5-15 >=15-25 >=25-35 >=35-45 >=45-55 >=55-65 >=65-75 >=75
age group
FIGURE 4.1 Percent of reported laboratory-confirmed listeriosis within age groups for males and females,
United States, 2000 to 2004. Isolation of Listeria monocytogenes reported to CDC through the Public Health
Laboratory Information System (PHLIS).
Unlike infection with other common foodborne pathogens such as Salmonella, which rarely
result in fatalities, listeriosis is associated with a mortality rate of approximately 20% [53]. The
high case-fatality rate, growing awareness of listeriosis as a foodborne disease, and increasing
clinical concern about illness in the expanding population of highly susceptible persons have
resulted in increased attention to the importance of L. monocytogenes as a human pathogen.
In this chapter, we will consider various aspects of listeriosis in humans, including infection
and clinical manifestations of disease, epidemiological patterns of disease, diagnosis, treatment,
and prevention. Information on the microbiology, ecology, pathogenesis, detection, subtyping,
manifestations of infection in other animals, and occurrence of L. monocytogenes in various foods
is presented elsewhere in this book. Although some foodborne outbreaks of listeriosis will be
discussed here, a more exhaustive treatment of foodborne listeriosis outbreaks can be found in
Chapter 10.
TABLE 4.1
Clinical Syndromes Associated with Infection with Listeria monocytogenes
Predisposing Condition
Population Clinical Presentation Diagnosis or Circumstances
Pregnant women Fever ± myalgia ± diarrhea Blood culture ± amniotic fluid culture
Preterm delivery
Abortion
Stillbirth
Newborns:
<7 days old Sepsis, pneumonia Blood culture Prematurity
≥7 days old Meningitis, sepsis Cerebrospinal fluid culture
Nonpregnant adults Sepsis, meningitis, focal Culture of blood, cerebrospinal fluid, Immunosuppresion,
infections or other normally sterile site advanced age
Healthy adults Diarrhea and fever Stool culture in selective enrichment Possibly large innoculum
broth
Listeriosis in Humans 87
HUMAN INFECTION AND CLINICAL MANIFESTATIONS OF LISTERIOSIS

LISTERIOSIS DURING PREGNANCY
Infection with L. monocytogenes in pregnant women may result in fetal loss, stillbirth, premature
delivery, or neonatal infection. Listeriosis may occur at any time during pregnancy, but is most
frequently documented during the third trimester [18,109]. Because bacterial cultures are not
routinely obtained from spontaneously aborted fetuses or stillborn neonates, it is difficult to estimate
accurately the proportion of fetal loss that may be attributable to infection caused by L. monocy-
togenes during pregnancy. In one study, L. monocytogenes was cultured from placental and fetal
samples in 1.6% of spontaneously aborted pregnancies [55]; other estimates have varied [4]. Among
pregnant women diagnosed with listeriosis, the rate of fetal mortality is between 16 and 45%
[54,100,138].
In a recent review of the literature, 20% of listeriosis cases during pregnancy resulted in
spontaneous abortion or stillbirth; of the remaining pregnancies, 63% resulted in neonatal infection
[109]. Preterm delivery resulted from 6 (55%) of 11 listeriosis cases identified at four hospitals in
the United States during a 10-year period [109] and 4 (36%) of 11 at two hospitals in Israel during
a similar 10-year period [12].
Most cases of listeriosis during pregnancy occur in otherwise healthy women. Among cases
identified by literature review, only 4% had a possible predisposing condition (corticosteroid use,
diabetes mellitus, systemic lupus erythematosus, or HIV infection) [109]. Women pregnant with
multiple gestations may be at increased risk for listeriosis compared with singleton pregnancies.
In Los Angeles from 1985 through 1992, rates of listeriosis were approximately four times higher
among women with multiple rather than singleton pregnancies [98].
Although most cases of neonatal listeriosis present with severe illness, symptoms in women
with listeriosis during pregnancy may be nonspecific, and they often manifest as a mild illness. In
a case-control study of sporadic listeriosis, women who delivered infants with listeriosis were
significantly more likely than controls to report histories of fever, headache, myalgia, or gastrointes-
tinal symptoms [134]. Approximately two thirds of pregnant women with listeriosis report fever
or flu-like illness lasting approximately 6 days (range: 1 to 21 days) [18,103,109]. These symptoms
are associated with the bacteremic phase of infection and represent the optimal time to obtain
diagnostic blood cultures. In a recent case-series of pregnant women with listeriosis, none of whom
had evidence of severe systemic illness, 37% yielded a positive blood culture for Listeria [109].
Infection of the fetus with L. monocytogenes is thought to result from transplacental transmis-
sion following maternal bacteremia, although some infections could also occur through ascending
spread from vaginal colonization. Intrauterine infection may result in preterm labor, amnionitis,
spontaneous abortion, stillbirth, or early-onset neonatal infection. In one case-series, there was
evidence of chorioamnionitis in all cases of early-onset neonatal infection [109].
Neonatal infection can be prevented by antibiotic treatment during pregnancy. Case reports
suggest that fetal- or early-onset neonatal infection does not always follow maternal listeriosis for
which treatment has been delayed or not given [49,78,89]. However, given the potentially adverse
consequences of maternal listeriosis and the availability of effective treatment, it is prudent to
evaluate all febrile episodes during pregnancy with blood cultures. It is not recommended that
women with a history of pregnancy-associated listeriosis undergo routine microbiological screening
or antimicrobial prophylaxis during subsequent pregnancies. However, dietary counseling on avoid-
ing high-risk foods should be given.
NEONATAL DISEASE: EARLY ONSET

In contrast to the mild clinical illness seen in maternal listeriosis, neonatal infection caused by L.
monocytogenes is a serious and often fatal disease. Neonatal listeriosis is divided into two clinical
forms: early onset and late onset. These clinical forms parallel the pattern of the disease seen in
neonates with infection from group B streptococci and as such suggest different modes of trans-
mission for the two forms.
Early-onset neonatal listeriosis occurs in infants infected in utero, and it results in illness at
birth or shortly thereafter, usually defined as occurring within the first week of life. Between 45
and 70% of neonatal listeriosis is of early onset [48,103]; this disease often presents with sepsis
rather than meningitis [54]. Signs and symptoms include respiratory distress, fever, and neurologic
abnormalities. Listeria monocytogenes may be isolated from blood, cerebrospinal fluid, oropharyn-
geal secretions, placenta, amniotic fluid, urine, or external sites including conjuctiva, ear, nose, or
throat [109].
Less frequently, infants with early-onset disease may present with granulomatosis infantiseptica,
a syndrome characterized by disseminated abscesses or granulomas in multiple internal organs,
including the liver, spleen, lungs, kidney, and brain [64]. In this syndrome, evidence of amnionitis
or meconium-stained fluid may be present, and the infant may appear obviously ill; in some
instances, however, the infant may merely appear weak and may develop respiratory or circulatory
insufficiency. Early-onset disease in the neonate may be complicated by aspiration of meconium
fluid with resultant respiratory complications, including cyanosis, apnea, and pneumonia.
NEONATAL DISEASE: LATE ONSET

In contrast to early-onset disease, the late-onset type may occur from one to several weeks after
birth [54,148]. Infants are usually born healthy and full term to mothers who have had uncomplicated
pregnancies. Similarly to late-onset neonatal disease caused by infection with group B streptococci,
listeriosis in these neonates presents as meningitis more frequently than in early-onset disease. In
a review of neonatal listeriosis cases from 1967 to 1985 in Britain, 39 of 42 (93%) infants with
late-onset disease had meningitis [103]. Listeria monocytogenes accounted for nearly 7% of all
neonatal meningitis in England and Wales during a similar time-period [142]. Active surveillance
for listeriosis in the United States in 1986 documented meningitis as the presenting syndrome in
88% of late-onset cases [54]. Mortality rates for early- and late-onset disease are usually 20
to 30% [19,91,103].
Although transplacental transmission of L. monocytogenes is the presumed source of infection
in early-onset disease, the route of infection in late-onset neonatal disease is not well understood.
Acquisition of infection during passage through the birth canal is likely, although cases of late-
onset disease have been reported following cesarean delivery. Because most listeriosis is foodborne,
contaminated foods likely contribute to transmission through direct or indirect contact with high-
risk foods. Clusters of late-onset disease have been identified in newborn nurseries, suggesting that
some nosocomial transmission also occurs [81,87,110,139]. In one outbreak of late-onset disease
in Costa Rica, infection was linked to contaminated mineral oil used to bathe newborns [133].
INVASIVE DISEASE IN NONPREGNANT ADULTS

Listeria is an important cause of bacterial meningitis in nonpregnant adults and the case-fatality
rate is approximately 30% [108]. In a recent review of 103 cases of acute bacterial meningitis in
adults, Listeria monocytogenes was the second most frequently isolated bacteria, accounting for
10% of cases [79]. In an older study in the United States, Listeria was the fifth most common
cause of meningitis in 1986 [154]. Listeria may be an increasing cause of meningitis in adults as
a result of improved vaccination for pneumococcal and meningococcal disease and should be
considered when adults present with acute meningitis.
Nonpregnant adults with listeriosis most frequently present with sepsis, meningitis, or menin-
goencephalitis. Presenting symptoms in nonpregnant adults with central nervous system listeriosis
may include fever, malaise, ataxia, seizures, and altered mental status. Interestingly, in contrast
with meningitis due to other pathogens, approximately one third of patients with listerial meningitis
initially present without meningeal signs [108]. Fever is generally present in patients with bacter-
emia; other nonspecific symptoms such as malaise, fatigue, and abdominal pain may also occur.
The cerebrospinal fluid may exhibit a pleocytosis; the Gram stain may show Gram-positive bacilli
but is often unrevealing. Because the spinal fluid white cell count and differential, glucose, and
protein levels can vary widely, the spinal fluid profile cannot be used to differentiate listerial
meningitis from meningitis caused by other bacteria.
Difficulties in diagnosing listerial meningitis were highlighted in a review of bacterial meningitis
in adults [79]. Most patients with listerial meningitis had negative CSF Gram stains and normal
CSF glucose. CSF Listeria antigen detection was positive in only a third of cases. However, 80%
of cases had a positive blood culture. CT scan or MRI of the brain may reveal focal lesions and/or
hydrocephalus in half of Listeria cases with clinical evidence of central nervous system (CNS)
abnormalities [108]. Listerial brain stem encephalitis (rhombencephalitis) occurs infrequently and
is characterized by asymmetrical cranial nerve deficits, cerebellar signs, and hemiparesis or
hemisensory deficits [5,140,145].
In addition to cases with central nervous system listeriosis, many listeriosis patients present
with bacteremia without evidence of meningitis. In the United States, active surveillance in an
aggregate population of 34 million persons in 1986 found that 66% of 179 nonpregnant adults had
bacteremia without meningitis, 19% had meningitis with concurrent bacteremia, and 12% had
meningitis without documented bacteremia [54]. Focal infections are rare and usually result from
seeding during a preceding bacteremic phase. Endocarditis from L. monocytogenes occurs primarily
in patients with an underlying cardiac lesion, including prosthetic or porcine valves, and is clinically
indistinguishable from other causes of endocarditis [10,52,96].
Other sites of involvement have been reported, including endophthalmitis [8,90], septic arthritis
[34,111], osteomyelitis [76], pleural infection [101], and peritonitis [112]. Cutaneous infections
without bacteremia have been reported in persons handling infected animals [118] and in acciden-
tally exposed laboratory workers [3].
Various clinical conditions are associated with listeriosis in nonpregnant persons; these include
malignancy, organ transplants, immunosuppressive therapy, infection with the human immunodefi-
ciency virus (HIV), and advanced age [6,54,119,131]. A frequent feature of these conditions is a
decreased level of cell-meditated immunity. Cancer, especially lymphoreticular malignancy, has been
associated with listeriosis in several reviews [92,103,108,113]. In a review of 458 patients with listerial
CNS infections between 1966 and 1997, the most frequent underlying conditions were [108]
malignancy (24%, of which 67% of known malignancies were hematologic)

transplants (21%, of which 90% were solid organ and 10% bone marrow)
alcoholism and/or liver disease (13%)
miscellaneous immunosuppressive therapy (11%)
HIV/AIDS (8%)
diabetes (7%)
autoimmune disorder (4%)
hemochromatosis (1%)
Cases of listeriosis associated with transplantation [56,75,122,124] and other conditions

[53,67,85,106,112,113,131] are well documented.
Despite the strong association between decreased immunity and invasive listeriosis in nonpreg-
nant adults, many cases do not involve a predisposing condition. In one report, 30% of patients
with meningitis and 11% of those with bacteremia caused by L. monocytogenes had no recognized
predisposing condition [112]. In another, between 24 and 37% of patients with Listeria infections
of the CNS had no known preexisting condition [108]. In these studies, however, cases were largely
identified through a literature review and thus may not be comparable to those identified through
population-based surveillance.
In a study of sporadic listeriosis conducted from 1988 to 1990 in diverse geographical popu-
lations in the United States, 98% of 98 cases that were not associated with pregnancy occurred in
persons with at least one underlying illness, although some had conditions such as heart disease
that are not traditionally considered immunosuppressive [132]. The most frequently identified
diseases or conditions were heart disease (33%), corticosteroid therapy (31%), cancer (29%), renal
disease (24%), and diabetes (24%); several patients had more than one underlying condition.
Malignancy, corticosteroid use, and HIV infection or AIDS were the most common immunosup-
pressive conditions, and at least one of these three conditions was present in 69% of nonpregnant
adult patients.
There have been many reports of patients with HIV infection or AIDS who contracted listerial
meningitis or bacteremia [15,36,38,68,99]. Although listeriosis does not appear to be a common
opportunistic infection among persons with HIV infection or AIDS, it nonetheless occurs far more
frequently among these individuals than among the general population. In 1989, in a prospective
population-based 2-year study in San Francisco, the incidence of listeriosis among AIDS patients
was estimated to be 280 times the baseline incidence of listeriosis in the general population [132].
Using similar methodology in the early 1990s, studies in Atlanta [83] and Los Angeles [41]
estimated that incidence of listeriosis was 9 to 62 times higher for those with HIV infection and
62 to 145 times higher for those with AIDS.
Recent articles have advanced the idea that listeriosis is associated with inflammatory bowel
disease (IBD) [20]. The “cold-chain” hypothesis suggests that the incidence of Crohn’s disease
parallels the availability of refrigerated foods, and that ingestion of psychrophilic bacteria such as
Listeria and Yersinia may lead to an abnormal immunologic response in susceptible persons [77].
Investigators have documented increased serologic response among IBD patients to several enteric
pathogens including Listeria [16]; however, attempts to identify L. monocytogenes DNA from
enteric biopsies have not demonstrated a significant difference between cases and controls [31,32].
Published experiments have yet to demonstrate a causal role for Listeria in IBD.
NONINVASIVE DISEASE: GASTROINTESTINAL ILLNESS

Information from several outbreak investigations suggests that L. monocytogenes causes a febrile
gastroenteritis in normal hosts [129]. The first such outbreak of gastroenteritis and fever was linked
to consumption of chocolate milk served at a picnic [37]. Picnic attendees drank chocolate milk
that was later found to be heavily contaminated with L. monocytogenes (109 CFU/mL); 79% of 58
persons who consumed implicated milk from the picnic reported diarrhea and 72% reported fever.
The median incubation period was 20 hours (range: 9 to 32 hours). The same subtype of L.
monocytogenes was isolated from the stools of ill persons and the chocolate milk. Ill persons were
more likely than well persons to have elevated antilisteriolysin O levels and to have stool cultures
that yielded L. monocytogenes. None of the individuals involved in this outbreak had a chronic
illness or immunodeficiency and none had evidence of invasive listeriosis. Similar outbreaks of
febrile gastroenteritis due to delicatessen meats [50], cheese [22], smoked fish [105], corn [7], and
rice salad [128] have been described.
A study of seven febrile gastroenteritis outbreaks caused by L. monocytogenes since 1989
suggests a consistent syndrome among otherwise healthy persons [117]. The incubation period is
1 day (range 6 hours to 10 days). Fever is the most frequently reported symptom. Diarrhea (median
of 12 stools per 24 hours of maximal diarrhea) with nonbloody stool is the most frequently reported
gastrointestinal sign. One characteristic of febrile gastroenteritis outbreaks is unusual compared
with other foodborne outbreaks: over half of patients reported somnolence and three quarters
reported fatigue.
The factors that lead to febrile gastroenteritis versus invasive illness are unclear. Susceptible
persons are at higher risk for invasive listeriosis but no host risk factors have been associated with
noninvasive listeriosis. Strain difference could account for different syndromes, but no strain
differences have been observed to date. Strains causing noninvasive gastroenteritis also cause
invasive illness. In the chocolate milk outbreak, three cases of sporadic invasive listeriosis were
linked to consumption of the implicated chocolate milk; the same subtype of L. monocytogenes as
the outbreak strain was isolated from these patients [37].
In studies of the pathogenic characteristics of Listeria strains, no differences have been found
between strains linked to invasive illness and those linked to gastroenteritis [47]. Because there is
no indication of a Listeria entertoxin, it is hypothesized that febrile gastroenteritis is due to limited
invasion of the gut mucosa. The infectious dose of L. monocytogenes necessary to cause listeriosis
or febrile gastroenteritis is not fully understood. Quantitatively cultured foods recovered from
Listeria outbreaks provide some evidence but the number of organisms ingested is difficult to
determine. The density of Listeria in foods implicated in febrile gastroenteritis outbreaks has ranged
from 3 × 101 CFU/g to 1.6 × 109 CFU/g with a median of 105 CFU/g. The dose of Listeria sufficient
to cause invasive illness in susceptible persons is also poorly understood but is thought to be much
lower [95].
The incidence of febrile gastroenteritis caused by L. monocytogenes is difficult to establish
because stool specimens from persons with diarrhea are rarely cultured for L. monocytogenes.
Clinicians and public health officials should consider examining stool cultures for L. monocytogenes
in outbreaks of illness characterized by fever, diarrhea, headaches, and myalgia if stool cultures
for other more common enteric pathogens have been negative. When such an outbreak is suspected,
care should be taken to notify the laboratory that L. monocytogenes is suspected so that appropriate
special culture media are used. The organism is frequently cultured from food, but because the
organism is commonly found in many foods, recovery of L. monocytogenes from foods is seldom
sufficient to verify the source of infection unless a matching strain is identified from ill persons
epidemiologically linked to the food.
ASYMPTOMATIC CARRIAGE
L. monocytogenes is distributed throughout the environment and can be frequently recovered from
a broad spectrum of foods [42,120] and sometimes from the gastrointestinal tracts of healthy people.
Numerous fecal carriage studies of L. monocytogenes among different populations have been done
with widely varying rates reported (Table 4.2). Some of this variation may be attributed to differ-
ences in populations studied, culture techniques, numbers of specimens obtained from each indi-
vidual, specimen handling, and whether results were reported as a point or cumulative prevalence.
Among surveys reviewed, point prevalence for healthy persons with unknown exposure to
Listeria is between 0.6 and 3.4%. A large stool survey conducted in Germany found that fecal
specimens yielded L. monocytogenes at a similar rate specimen yield from persons with diarrhea
(0.6%) and from healthy food handlers (0.8%) [107]. Among studies of persons exposed to Listeria,
fecal carriage rates are much higher. In Denmark, fecal specimens from 26% of 34 household
contacts of persons with listeriosis yielded L. monocytogenes [17]. In this study, among 14 house-
holds sampled, 5 (36%) had at least one household member with a positive stool culture; however,
only two family members had the same serotype of L. monocytogenes as the patient. Up to eight
specimens were collected from each household contact, suggesting that the carriage rate in this
study may not be directly comparable to that in others.
In the United States, 82 household contacts of 28 patients with invasive listeriosis were identified
through active surveillance and investigated [135]. Twenty-one percent of these individuals (and
households) were positive for L. monocytogenes; 88% of 17 isolates were of the same serotype
and enzyme type as the strain from the index patient. The rate of carriage was significantly higher
among persons less than 30 years of age than among older persons.
In Austria, Grif et al. found that 0.2% of healthy volunteers in a 1-year study were shedding
L. monocytogenes by conventional culture; up to 3.6% were positive by PCR techniques [66]. In
a related study, three healthy adults provided stool samples over a 1-year period [66]. Stool culture
TABLE 4.2
Reported Fecal Carriage Rates of Listeria monocytogenes
% with
Year Population No. Studied L. monocytogenes Comment Ref.
Point Prevalence Studies among:
Persons with Unknown Exposure to Listeria

1986 Healthy nonpregnant women 59 3.4 86
Healthy pregnant women 51 2.0
1990 Healthy food handlers 2,000 0.8 107
Persons with diarrhea 1,000 0.6
1991 Home hemodialysis patients 80 2.5 94
Outpatients with gastroenteritis 171 1.8
2001 Healthy volunteers 505 0.2 Traditional culture 65
3.6 PCR
Persons Likely Exposed to Listeria

1972 Slaughterhouse workers 1,147 4.8 Cold enrichment 17
Hospitalized adults 1,034 1.2 culture
Hospitalized children 195 0
Hospitalized adults with diarrhea 595 1.0
1992 Pregnant women with listeriosis 18 5.6 99
Household contacts of pregnant 60 8.3
women with listeriosis
Age-, race-, and hospital- 7 0
matched controls
Cheese plant employees 31 9.7
Household contacts of cheese 94 10.6
plant employees
1993 Household contacts of patients 82 21 133
with listeriosis (persons)
Households of patients with 28 21
listeriosis with at least one
carrier
2003 Healthy adults 3 1.2 One-year period; 66
between 242 and
363 samples per
person; average of
two episodes/
person/year
Cumulative Prevalence Studies among:
Persons with Unknown Exposure to Listeria

1972 Office workers with no L. 26 62.0 8-week period; up to 84
monocytogenes contact eight cultures/person
1991 Renal transplant patients 177 5.6 Mean 2.5 94
specimens/patient
1993 Healthy pregnant women 147 2.7 Mean 2.4 63
specimens/patient
(continued)
TABLE 4.2 (CONTINUED)

Reported Fecal Carriage Rates of Listeria monocytogenes
% with
Year Population No. Studied L. monocytogenes Comment Ref.
Persons Likely Exposed to Listeria

1972 Household contacts of persons 341 26.0 6-month period; up 17
with listeriosis to eight
cultures/person
1972 Laboratory workers handling L. 26 77.0 8-week period; up to 84
monocytogenes eight cultures/person
yielded L. monocytogenes in 10 (1.2%) of 868 stool specimens. Based on subtyping and the
dates of collection, there were five independent exposures to L. monocytogenes—an average of
two per person per year; none of the episodes caused illness. All isolates were serotypes 1/2a
and 1/2b.
Elevated rates of fecal carriage have been demonstrated among those with underlying medical
conditions. In a survey of renal transplant recipients, 5.7% of 177 had a positive stool culture over
a 1-year period and this was associated with ranitidine use or consumption of three or more types
of cheese since the beginning of the year [94]. The same investigators reported fecal carriage rates
of 1.8% among 171 patients with gastroenteritis attending a general practice and 2.5% of 80 home
hemodialysis patients. Among all three patient groups, Listeria isolations were highest during the
months of July and August.
Although pregnancy is a strong risk factor for clinical illness due to L. monocytogenes, fecal
carriage rates do not appear to be higher among pregnant compared with nonpregnant women.
Lamont and Postlethwaite reported a fecal carriage rate of 2% among 51 women early in pregnancy
(10 to 16 weeks), similar to the 3.4% rate observed among 59 nonpregnant women attending the
same clinic [86]. During a large foodborne listeriosis epidemic in Los Angeles, Mascola et al.
compared fecal carriage rates in pregnant women with listeriosis to age-, sex-, and hospital-matched
controls; carriage rates were not significantly different between the two groups (1 of 18 versus 0
of 7, respectively, p-value = NS) [97].
Timing of carriage was examined among 147 women attending an antenatal clinic in the United
Kingdom [63]. One fecal specimen was obtained during each trimester. Among the four (2.7%)
women whose fecal specimens yielded L. monocytogenes, one was in the first, two in the second,
and one in the third trimester. The prevalence rate among 60 household contacts of 18 pregnant
women with listeriosis in Los Angeles was 8.3%, whereas no Listeria were isolated from 30
household contacts of age-, sex-, and hospital-matched controls [97].
Elevated rates of fecal carriage have also been demonstrated among those with occupational
exposure. Kampelmacher et al. also reported high rates of fecal carriage among laboratory workers
having daily contact with L. monocytogenes (77% of 26), but rates were also high among office
workers who had no contact with the organism (62% of 26). Because stool cultures were collected
weekly for 8 weeks, figures from this study represent cumulative rather than point prevalence
estimates [84]. Subjects had L. monocytogenes isolated an average of 1.3 times out of the eight
serial specimens collected. Investigators in Denmark determined that 4.8% of 1,147 healthy slaugh-
terhouse workers had stool cultures yielding L. monocytogenes [17]. Similar surveys by the same
researchers documented fecal carriage rates of 1.2% among 1,034 hospitalized adults, none of 195
hospitalized children, and 1% of 595 hospitalized adults with diarrhea.
EPIDEMIOLOGICAL PATTERNS OF LISTERIOSIS

Our understanding of listeriosis as a foodborne disease and risk factors for illness caused by
infection with L. monocytogenes have increased greatly over the last 25 years through epidemio-
logical studies of epidemic (Table 4.3) and sporadic illness. Compared with other bacterial food-
borne illnesses, several characteristics of listeriosis, including the relatively low incidence and the
long incubation period, make identifying outbreaks difficult. Improved laboratory-based surveil-
lance is helping to overcome those difficulties.
Beginning in 1998, PulseNet [62] has performed molecular subtyping of L. monocytogenes
isolates by pulsed-field gel electrophoresis (PFGE) on a routine basis for isolates submitted to
public health laboratories. The results have enhanced the ability of public health officials to detect
and investigate outbreaks of listeriosis. In the 20 years before PulseNet, five Listeria outbreaks
were identified in the United States; 12 Listeria outbreaks have been identified since, despite a
decreasing incidence of listeriosis overall (Figure 4.1). Of those outbreaks recognized through
PulseNet surveillance, four were multistate outbreaks caused by widely distributed foods, demon-
strating the importance of linking geographically dispersed illnesses. Ready-to-eat meats
[24,25,116] and unpasteurized cheese [26,93] have been the most frequently identified causes of
Listeria outbreaks in the United States from 1998 to 2006.
EPIDEMIC LISTERIOSIS
The first convincing evidence that listeriosis can be a foodborne disease came from a 1981 outbreak
in Nova Scotia [130]. Thirty-four pregnancy-associated cases and seven cases in nonpregnant adults
occurred over a 6-month period in the Maritime Provinces. Of the infants born alive, 27% died.
No patient had evidence of underlying immunosuppression. Case-patients were significantly more
likely than controls to have consumed locally produced coleslaw in the 3 months before illness
onset. The epidemic strain was subsequently isolated from coleslaw in the refrigerator of one patient
and later from two unopened packages of the product.
Review of the production process determined that cabbage used in the coleslaw came from a
farm where cases of listeriosis in sheep had occurred and that the cabbage fields had been fertilized
with raw sheep manure. Harvested cabbage was stored over the winter and spring in an unheated
shed, potentially enhancing growth of L. monocytogenes. As well as establishing listeriosis as a
foodborne disease, this outbreak also highlighted the potential for uncooked vegetables to be a
source of infection.
Another outbreak of listeriosis, in Massachusetts in 1979, may also have involved raw produce
[70]. Twenty patients with serotype 4b infection were hospitalized during a 2-month period during
1979; only nine cases had been detected in the previous 26 months. Ten of the patients were
immunosuppressed adults and five died. Fifteen patients were thought to have acquired their
infection in the hospital. Patients were more likely than controls to have consumed tuna fish, chicken
salad, or cheese, but no single brand was implicated. It was postulated that raw celery and lettuce,
served as a garnish with these foods, may have been the vehicle of infection. Although a definitive
source of infection was not identified in this outbreak, information suggested that gastrointestinal
tract conditions might be important in acquiring infection. Case-patients were significantly more
likely than controls to have taken cimetidine or antacids, raising the possibility that, like salmo-
nellosis, decreased gastric acidity might increase the chance that L. monocytogenes could survive
passage through the stomach.
Pasteurized milk was identified as the most likely vehicle of infection in another large outbreak
of listeriosis in Massachusetts in 1983 [43]. Over a 2-month period, 49 cases occurred; 42 were in
immunosuppressed adults and 7 in pregnant women. The overall case-fatality rate was 29%. Case-
patients were more likely than neighborhood-matched controls to have consumed a specific brand
of 2% fat pasteurized milk; other evidence supporting this milk as the vehicle of infection included
TABLE 4.3
Selected Foodborne Outbreaks of Invasive Listeriosis
L. monocytogenes
Listeriosis in Humans
Year Place No. of Cases (deaths) Implicated (or Likely) Vehicle % Perinatal Cases Serotype Ref.
1979 Massachusetts, U.S.A. 20 (5) (Raw vegetables) 0 4b 44

1981 Nova Scotia, Canada 41 (18) Coleslaw 83 4b 85
1983 Massachusetts, U.S.A. 49 (14) Pasteurized milk 14 4b 27
1985 California, U.S.A. 142 (48) Mexican-style cheese 66 4b 56
1983–1987 Switzerland 122 (34) Soft cheese 53 4b 10
1988–1989 United Kingdom — P â té — 4b 66

1989 Connecticut, U.S.A. 10 (0) (Shrimp) 33 4b 80
1992 France 279 (85) Pork tongue in jelly 0 4b 38
1993 Italy 18 (0) Rice salad 0 1/2b 84
1994 Illinois, U.S.A. 48a (0) Pasteurized chocolate milk 0 1/2b 23
1998 Multiple states, U.S.A. 105 Hot dog — 4b 26
1999 Florida, U.S.A. 2 Turkey, ham, and roast beef deli meat — 26
1999 Multiple states, U.S.A. 11 Pate — 26
1999 Minnesota, U.S.A. 5 Deli meats — 26
1999 New York, U.S.A. 4 Hot dog — 1/2a 26
1999 New York, U.S.A. 6 — — 26
2000 Multiple states, U.S.A. 30 Turkey deli meat 27 1/2a 116
2000 North Carolina, U.S.A. 13 Queso fresco 92 4b 93
2001 California, U.S.A. 6 Deli sandwich — 26
2002 Multiple states, U.S.A. 54 Turkey deli meat — 4b 25
2003 Texas, U.S.A. 12 Queso fresco, unpasteurized — 4b 26
2003 New York, U.S.A. 3 — 26
a
Includes 45 cases of diarrhea and 3 cases of invasive disease.
95
a dose–response effect, a protective effect of drinking low-fat milk (1% or skim), an association
between the implicated brand of milk and cases of listeriosis in another state, and the linking of a
specific phage type of L. monocytogenes with infection in the 2% milk drinkers.
The 2% milk came from farms where cows were known to have had listeriosis, and multiple
serotypes (but not the epidemic strain) of L. monocytogenes were isolated from raw milk at the
implicated dairy. No defects in the pasteurization process were noted at the dairy. Although
this outbreak initially raised the question of whether pasteurization was adequate to eliminate
L. monocytogenes from milk, subsequent investigations have shown that L. monocytogenes is
inactivated by proper pasteurization [27]. In this outbreak, contamination likely occurred during
postpasteurization handling.
The largest North American outbreak of listeriosis occurred in Los Angeles County, California,
in 1985; over an 8-month period, 142 cases were detected [89]. Pregnant women accounted
for 93 cases and nonpregnant adults for 49 cases; 48 of the nonpregnant adult cases had predisposing
conditions for listeriosis. Among the pregnancy-associated cases, 87% occurred in Hispanic women.
The case-fatality rate was 32% among the perinatal cases (all were fetal or neonatal deaths) and
37% in the nonpregnant adults. A case-control study implicated a particular brand of Mexican-style
soft cheese produced locally in California as the vehicle, and the epidemic serotypes and phage
type were isolated from unopened packages of this product. Inadequate pasteurization and mixing
of pasteurized and unpasteurized milk likely contributed to the contamination.
This outbreak provided valuable data on the incubation period of invasive listeriosis because
food histories were available for four patients who had a single known exposure to the implicated
cheese. These patients’ median incubation period of 31 days (range: 11 to 70 days) is far longer
than that observed for most common foodborne pathogens, and it highlights the difficulty in
obtaining a relevant food history when investigating cases of sporadic listeriosis.
This outbreak was detected quickly through public health surveillance because many infections
occurred in an ethnic minority group that sought care primarily at one medical facility. However,
it is likely that the outbreak would not have been as readily detected had the product been distributed
over a larger geographical area or eaten by a more diverse group of consumers. Detection of
outbreaks can be improved by active surveillance and timely reporting and by serotyping and
molecular subtyping L. monocytogenes isolates.
Another soft cheese-related outbreak occurred in Switzerland during 1983 to 1987, with 122
cases of listeriosis affecting 65 pregnant women (and their infants) and 57 nonpregnant adults [13].
Over half of the nonpregnant adult cases had no underlying predisposing condition; the case-fatality
rate for nonpregnant patients was 32% [21]. Increasing age and clinical presentation with menin-
goencephalitis were independently associated with an increased risk of death. Neurological sequelae
were present in 30% of survivors at follow-up 5 months to 3 years later. Although early case-control
studies failed to incriminate a particular food, in 1987 investigators implicated a locally produced
soft cheese as the vehicle of infection. Two epidemic strains of L. monocytogenes serotype 4b with
a particular phage type were isolated from the product and led to an international recall.
Unpasteurized cheese continues to be a source of outbreaks. In one outbreak in 2000, 13 cases
of listeriosis were attributed to consumption of Mexican-style cheese made from contaminated raw
milk [93]. Eleven cases involved pregnant women, in whom infection resulted in five stillbirths,
three premature deliveries, and three infected neonates. Investigators isolated L. monocytogenes
serotype 4b from patients, locally available fresh cheese, and raw milk from a local dairy; all
isolates were indistinguishable on PFGE. This outbreak renewed attention on raw milk Mexican-
style cheese (e.g., Queso fresco) intended for sale to Latin American consumers in the United
States, increasing concern that consumption of such cheese may account for the higher proportion
of listeriosis among Hispanic women [88].
Outbreaks associated with ready-to-eat meats have been identified in Europe and North Amer-
ica. In the United States, deli meats or hot dogs were implicated in seven Listeria outbreaks reported
to CDC (Table 4.3). In one example of a multistate outbreak involving 30 patients in 11 states over
an 8-month period, delicatessen turkey meat was implicated [116]. Among those ill, 22 (73%) were
men or nonpregnant women; eight (27%) were pregnant women or mother–infant pairs. Infection
resulted in deaths of four persons, two miscarriages, and one stillbirth.
L. monocytogenes was isolated from product samples that matched those isolated from cases.
Subsequent tracing back of contaminated delicatessen turkey meat led to two plants in different
states. However, one plant served as a copacker for the other plant, and it was determined that
all of the implicated meat could have come from one of the plants. Interestingly, meat (turkey
franks) from the same plant was identified as a source of listeriosis in 1989 [28,155]. Molecular
subtyping indicated that isolates from 1989 and 2000 were indistinguishable. This outbreak
indicates that Listeria may persist in a meat plant and could intermittently contaminate food.
The source of most infections is not identified; this plant may have been a source of human
illness for over 12 years.
In England, Wales, and Northern Ireland, the annual total of listeriosis cases approximately
doubled from 1987 to 1989 compared with the annual totals for the 3 previous years. Of the 823
isolates reported during 1987 to 1989, 30 to 54% were of two subtypes of L. monocytogenes
serotype 4b (4bX and 4b phage type 6, 7) that had occurred less commonly before and after this
period [102]. A microbiological survey showed that L. monocytogenes contamination in meat pâté
from one manufacturer was more frequent (48% of 107 samples of pâté from the implicated
manufacturer versus 4% of 781 samples of pâté from other manufacturers) and heavy (11% of
samples of pâté from manufacturer A with ≥1000 organisms/g versus 0.6% of samples of pâté from
other manufacturers with ≥1000 organisms/g).
Of pâté isolates from manufacturer A, 96% were 4bX or 4b phage type 6, 7, compared with
19% of isolates from other manufacturers. Patients infected with either of these two subtypes were
significantly more likely to have eaten pâté than patients infected with other strains. Warning about
pâté consumption and removal of manufacturer A’s pâté from sale in late 1989 resulted in a dramatic
decrease in the incidence of listeriosis. This investigation illustrated how subtyping can help clarify
surveillance data and ultimately lead to public health action.
In 1992, a different ready-to-eat meat product was implicated in an outbreak of 279 cases of
listeriosis in France [59]. Ninety-two cases (33%) were pregnancy related and 187 occurred in
nonpregnant adults; 73 (39%) of the nonpregnant adults had no known predisposing condition for
listeriosis. A case-control study implicated one brand of pork tongue in jelly as the major vehicle;
however, other ready-to-eat meats, cross-contaminated by the implicated meat at retail stores where
the implicated brand of pork tongue in jelly was sold, were thought to have been responsible for
some infections. The epidemic strain was isolated from samples of food, and subtyping analysis
helped to confirm the findings of the epidemiological investigation that implicated the pork tongue
in jelly [80].
Heightened surveillance efforts in France have also led to detection of two smaller outbreaks
of listeriosis. In 1993, an outbreak of 39 cases was associated with rilletes (pork pâté) [60]. In
1995, 20 cases were traced to Brie de Meaux cheese made from raw milk. Implicated cheese was
removed from sale based on results of the epidemiological investigation [60].
SPORADIC DISEASE—INCIDENCE
Although much has been learned about epidemic listeriosis, most cases of human listeriosis occur
sporadically. The incidence of listeriosis is difficult to estimate and estimates may vary considerably
depending on the methods used to identify sporadic cases. In the United States, voluntary disease
reporting and hospital discharge data have been used to estimate the number of sporadic listeriosis
cases [35]. However, such methods are generally insensitive and result in underestimates of the
true incidence of listeriosis in the population. In 1997, only 373 cases were reported through passive
surveillance to U.S. public health officials, but estimates from active surveillance at sentinel sites
indicated that 1,259 cases occurred [104].
Mead et al. assumed the true number of listeriosis cases to be two times the number of reported
cases: 2,518 estimated cases per year (9.4 cases per million persons per year) resulting in 2,322
hospitalizations and 504 deaths [104]. Adak estimated 194 cases per year of listeriosis occurred in
England and Wales (3.7 cases per million persons per year), resulting in 194 hospitalizations and
68 deaths per year in 2000 [1]. In France, the incidence of listeriosis was estimated from three
different sources by Goulet et al. to be between 6.3 and 16.7 per million in 1987 and between
4.1 and 4.4 per million in 1997 [58]. In Denmark, an estimated six to seven cases per million per
year occurred during the 1980s [82]. Japan has estimated an average incidence of 0.65 per million
between 1996 and 2002 [115].
Differences in national rates should be interpreted in the context of the methods used for case
ascertainment and reporting in each country. Without a consistent methodology to estimate inci-
dence, it is difficult to evaluate trends or compare rates between countries.
Efforts have been under way in the United States to establish annual incidence estimates for
listeriosis. In 1986, active surveillance for listeriosis was initiated by the Centers for Disease Control
and Prevention (CDC). Surveillance officers systematically contacted infection control practitioners
at all acute-care hospitals and clinical microbiology laboratories in the study areas to collect infor-
mation on all patients from whom L. monocytogenes was isolated from a normally sterile site [54,132].
In 1986, the annual incidence of listeriosis was 7 cases per million; in 1989, 7.9 per million; and in
1993, 4.4 per million. The declining incidence since 1989 was distributed uniformly throughout the
different geographical areas and was attributed to regulatory and educational efforts [143].
Beginning in 1996, the Foodborne Disease Active Surveillance Network (FoodNet) of the
CDC’s Emerging Infections Program collected data from several well-defined populations in the
United States in representing different geographical areas. To identify cases, FoodNet contacted
clinical laboratories weekly or monthly depending on lab size. The incidence of listeriosis was five
per million persons in 1996 and 1997 [149]. Comparing 2004 to the average between 1996 and
1998, the incidence of listeriosis declined 40% to 2.7 per million persons [30], which suggests a
real and continued reduction in illness (see section on prevention). Based on these data, the projected
number of deaths per year due to listeriosis would have declined from 504 in 1997 to 302 in 2004.
In the United States, rates have varied by geographical site. Rates in San Francisco (9.3 per
million) were twice those in Oklahoma (4.8 per million) [132]. In 1986, perinatal listeriosis rates
were highest in Los Angeles (24.3 per 100,000 live births), whereas nonperinatal rates did not vary
significantly among the study sites. No clear seasonal trends were noted [54]. Of the 246 cases
reported in 1986, 67 (27%) were perinatal and 179 (73%) were nonperinatal. Between 1989 and
1993, pregnancy-associated cases accounted for about one third of all cases each year [143].
Serotypes 4b (43%), 1/2b (35%), and 1/2a (20%) accounted for almost all infections.
Surveillance data reported to the CDC from state public health laboratories from 2000 to 2004
indicate the distribution of illness by age group, sex, and ethnicity (Figure 4.2 and Figure 4.3) [40].
The greatest number of cases occurs among infants and the elderly (Figure 4.2). For adults age 15
to 35 years, listeriosis occurs in a higher percentage of women than men of the same age group.
The excess numbers of cases in women of childbearing age compared with men, combined with
the number of infant cases, represent the burden of pregnancy and perinatal listeriosis. From 2000
to 2004, a far greater percentage of listeriosis occurred in Hispanics from 15 to 35 years of age
than in non-Hispanics (Figure 4.3). This difference is largely due to pregnancy and perinatal
infections in Hispanic women, who represent an important target group for prevention programs.
SPORADIC DISEASE—DIETARY RISK FACTORS

Dietary risk factors for sporadic listeriosis were assessed through case-control studies conducted in the
CDC active surveillance project. Patients identified through surveillance were enrolled and matched
by age, underlying disease, and healthcare provider (as an indirect measure of geographical location
and socioeconomic status). In the 1986 to 1987 study, 82 patients and 239 controls were enrolled.
10
9 Single state outbreak
8
Multistate outbreak
7
6
5
4
3
2
1
0
1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004
FIGURE 4.2 Incidence of reported cases (per one million persons; data from active surveillance systems
[2004 data are preliminary]) (From Centers for Disease Control and Prevention. 2005. Morbidity Mortality
Wkly. Rep. 54:352–356; Gellin, B. G. et al. 1991. Am. J. Epidemiol. 133:392–401; Tappero, J. W. et al. 1995.
JAMA 273:1118–1122) and outbreaks of Listeria (outbreaks of confirmed Listeria monocytogenes reported
to CDC’s Foodborne Outbreak Reporting System. From Centers for Disease Control and Prevention. 2005.
U.S. Foodborne Disease Outbreaks. Published on the World Wide Web at http://www.cdc.gov/foodborneout-
breaks/us_outb.htm), United States, 1973 to 2004.)
Case-patients were significantly more likely than controls to have eaten uncooked or nonreheated
hot dogs (frankfurters) or undercooked chicken. An estimated 20% of the overall risk of listeriosis
was thought to be attributable to consumption of these foods [136].
The first human case of listeriosis that was microbiologically linked to consumption of
ready-to-eat poultry products occurred in 1989. A strain of L. monocytogenes serotype 1/2a with
an identical multilocus enzyme elctrophoretic type was isolated from the blood of a patient with
cancer, an open package of turkey frankfurters and other opened foods in the patient’s refrigerator,
and unopened packages of turkey frankfurters at a retail store [28]. Subsequent investigation of the
35%
30%
Percent of Total
HISPANIC (N=228)
25%
20% NON-HISP (N=918)
15%
10%
5%
0%
<1 >=1-2 >=2-5 >=5-15 >=15-25 >=25-35 >=35-45 >=45-55 >=55-65 >=65-75 >=75
Age Group
FIGURE 4.3 Percent of reported laboratory-confirmed listeriosis by age groups for Hispanic and non-Hispanic
ethnicity, United States, 2000 to 2004. Isolation of Listeria monocytogenes reported to the CDC through the
Public Health Laboratory Information System (PHLIS).
turkey frankfurter production facility found that cultures from a conveyor belt transporting finished
frankfurters yielded the case strain of L. monocytogenes [155]. Systematic culturing at various
points in the production process identified likely points where L. monocytogenes was introduced
into the product and suggested appropriate control points for reducing contamination in such food-
processing facilities.
From 1988 to 1990, a larger case-control study of 165 patients and 376 controls was conducted
that included microbiological assessment of foods eaten by patients [132]. Case-patients were
significantly more likely than controls to have eaten soft cheeses or delicatessen-counter foods. In
a separate analysis examining dietary risks among a subset of patients defined as highly immuno-
suppressed (persons with malignancy, AIDS, or organ transplants or who had received corticoster-
oids or chemotherapy), consumption of undercooked chicken was associated with a threefold
increased risk of listeriosis. Other exposures associated with an increased risk of sporadic disease
included recent use of antacids, laxatives, or H2-blocking agents.
In the microbiological component of this study, foods were collected from the refrigerators of
123 patients [120]. L. monocytogenes was isolated from at least one food in the refrigerators of
64% of patients. Highest contamination rates among the 2,013 food specimens were seen in beef
(36%) and poultry (31%) with 7.6% of ready-to-eat foods (processed meats, raw vegetables,
leftovers, and cheeses) also yielding L. monocytogenes. One third of refrigerators contained food
isolates of L. monocytogenes that were the same multilocus enzyme elctrophoretic type as those
isolated from the patient. In multivariate analysis, foods that were ready to eat, foods that contained
high numbers of L. monocytogenes, and foods that yielded serovar 4b were associated with disease.
Dietary risk factors for sporadic listeriosis were also examined in a recent study in Denmark;
drinking unpasteurized milk or eating pâté were the only risk factors identified [82]. However, one
third of cases reported during the study period could not be included in the risk analysis for sporadic
disease because the ill persons were infected with an outbreak strain epidemiologically linked to
Danish blue-mold cheeses.
SPORADIC DISEASE—POSSIBLE OTHER SOURCES

Transmission by routes other than food may play a role in a few cases of sporadic listeriosis. Sexual
transmission of L. monocytogenes has been hypothesized as a possible route in perinatal listeriosis;
however, there is no evidence to support this [121]. Because L. monocytogenes can cause asymp-
tomatic bacteremia and survives refrigeration, it is theoretically possible that transmission through
donated blood could occur. Such transmission has been documented for Yersinia enterocolitica but
has not yet been described for L. monocytogenes [144]. Nosocomial transmission of L. monocyto-
genes has been documented in a variety of settings [34,61,70,73,74,81] in addition to the transmis-
sion among newborns in a nursery mentioned earlier.
DIAGNOSIS
Diagnosis of invasive listeriosis depends on isolation of L. monocytogenes from a normally sterile
site such as blood or cerebrospinal fluid. Because the organism may be mistaken for a diphtheroid
contaminant on Gram stain, complete bacteriological evaluation should be done. Recovery of the
organism from stool samples is helpful when febrile gastroenteritis is suspected, but isolation from
stool by itself is not diagnostic because asymptomatic carriage occurs. To document an outbreak
of febrile gastroenteritis, the isolation rate among symptomatic persons should be significantly
higher than among asymptomatic persons.
L. monocytogenes strains isolated from sterile-site specimens usually grow well in routinely
used media. The specimen is directly plated on tryptic soy agar containing 5% sheep, horse, or
rabbit blood. The organism is usually identified within 36 hours. Isolation of the organism from
other sources such as stool specimens that contain large numbers of competing microorganisms is
more difficult; these specimens should be selectively enriched for Listeria spp. before being plated
on Listeria-selective media. Identification of L. monocytogenes by use of fluorescent antibody
methods or approaches that use DNA probes coupled with PCR technology may prove useful for
some specimens. Experimental assays for antibody to listeriolysin O have been useful in some
epidemiological investigations [37] and have been used to support the diagnosis in culture-negative
listeriosis of the central nervous system [51].
TREATMENT
Controlled trials to determine the optimal antibiotic therapy for listeriosis have not been done. The
treatment of choice is high doses of amoxicillin I.V. (2 to 3 g three to four times a day), with an
additional dose of gentamicin (360 mg once a day) for nonpregnant adults [72]. The role of ami-
noglycosides is poorly understood because they penetrate cells poorly and may be ineffective in the
living host. L. monocytogenes has grown in cells despite high extracelluar concentrations of ami-
noglycosides [69]. However, the majority of Listeria may be extracellular in cases of meningitis [71].
Trimethoprim-sulfamethoxazole readily enters cells and kills L. monocytogenes. This drug
combination has proved effective in patients with listeriosis who have hypersensitivity to penicillin
[5]. Compared with the aminopenicillins, newer quinolone antimicrobials may have increased
bacteridicidal activity, cross the blood–brain barrier into the CNS, and accumulate within host cells,
but clinical studies have not been done [72].
The ability of L. monocytogenes to grow and survive within cells probably explains the poor
response to bacteriostatic drugs and the slow response to penicillin [141]. Bacteriostatic drugs such
as chloramphenicol or tetracycline have been associated with high treatment failure rates, so they
are not recommended [141]. Cephalosporins are not recommended for treatment because they have
a low affinity for the penicillin-binding protein of Listeria [147].
Relapses have been reported in immunosuppressed patients after 2 weeks of penicillin therapy
[150]. Because many immunosuppressed patients have a decreased ability to clear infected cells,
antibiotic treatment for 3 to 6 weeks may be prudent [6]. Optimal length of therapy for other groups
of patients has not been established. A prudent treatment course may be 2 weeks for listeriosis in
pregnancy, 2 to 3 weeks for neonatal listeriosis, 2 to 4 weeks for nonimmunosuppressed adults
with meningitis and bacteremia, and longer for complicated infections such as endocarditis.
PREVENTION
Recognition that most human listeriosis is foodborne has led to control measures that have reduced
the incidence of listeriosis. Healthy People 2010 goals established for the United States called for
a reduction of foodborne listeriosis by 50% by the end of the year 2005; by 2004, those goals were
nearly met [29]. The observed decrease in perinatal and nonperinatal cases since 1989 is likely the
result of enhanced listeriosis prevention efforts by the U.S. food industry, including enforcement
by regulatory agencies of a zero-tolerance policy for processed meat and intensified clean-up
programs in meat-processing facilities.
In 2001 and 2003, the Food and Drug Administration (FDA), CDC, and the U.S. Department
of Agriculture (USDA) released a national Listeria Action Plan to help guide control efforts by
industry, regulators, and public health officials [45,46]. Those plans called for multiple points of
action, including increased regulatory guidance over the manufacture of ready-to-eat foods. Also
in 2003, following a large outbreak linked to deli turkey meat, the USDA issued new regulations
aimed at further reducing L. monocytogenes contamination of ready-to-eat meat and poultry prod-
ucts [39]. Published dietary recommendations for consumers may also have contributed to the
decreased disease incidence [29,44,137]. Noncommercial sources of food, such as raw milk cheese,
continue to be sources of listeriosis, especially among Latin American women [33,146].
For persons who are at increased risk for listeriosis, including those who are pregnant or
immunocompromised, specific dietary measures can be taken to decrease risk [23]. Such persons
should avoid high-risk foods such as hot dogs; deli meats or luncheon meats (particularly deli
sliced), unless they are reheated until steaming hot; soft cheeses (such as feta, Brie, and Camembert,
blue-veined cheeses, or Mexican-style cheeses such as queso blanco, queso fresco, and Panela),
unless they have labels that clearly state they are made from pasteurized milk; unpasteurized (raw)
milk; refrigerated p â tés or meat spreads (canned or shelf-stable p â tés and meat spreads may be
eaten). In addition, they should avoid getting fluid from hot dog or other deli meat packages on
other foods or food preparation surfaces and wash hands after handling hot dogs and deli meats.
Dietary and food preparation measures have been recommended to the general public; these
should decrease the risk not only of listeriosis but also of other common foodborne diseases, such
as salmonellosis and campylobacteriosis. These measures include thorough cooking of raw food
from animal sources; washing raw vegetables thoroughly before eating; keeping uncooked meats
separate from vegetables, cooked foods, and ready-to-eat foods; avoiding raw (unpasteurized) milk
or foods made from raw milk; and washing hands, knives, and cutting boards after handling
uncooked foods [29].
In addition to individual advice for consumers, control of listeriosis requires action from public
health agencies and the food industry. Important control strategies from public health agencies
include developing and maintaining timely and effective disease surveillance programs, promptly
investigating clusters of listeriosis cases, and enforcing current regulations designed to minimize
L. monocytogenes in foods consumed without further cooking. A survey in the United States found
that 1.8% of ready-to-eat foods were contaminated with L. monocytogenes [57]. For foods such as
ready-to-eat salad vegetables, only rare servings may be contaminated but the level of contamination
may be high [127]. It is unlikely that such contamination would be found during routine product
testing. It is imperative therefore that the food industry develop an understanding of how contam-
ination occurs and then implement hazard analysis critical control point (HACCP) programs to
minimize the presence of L. monocytogenes at important points in the processing, distribution, and
marketing of processed foods [2].
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5 Molecular Virulence
Determinants of Listeria
monocytogenes
Michael Kuhn and Werner Goebel
CONTENTS
Introduction ....................................................................................................................................111
Molecular Aspects of the Invasion of Mammalian Cells..............................................................113
Invasion of Nonprofessional Phagocytic Cells ....................................................................114
Cellular Adhesion .................................................................................................................118
Uptake by Macrophages and Dendritic Cells......................................................................118
Escape from the Phagocytic Vacuole.............................................................................................120
Growth in the Host Cell Cytoplasm ..............................................................................................125
Intracellular Motility and Cell-to-Cell Spread ..............................................................................126
Bile Salt Hydrolase—A Novel Virulence Factor ..........................................................................129
Accessory Virulence Factors..........................................................................................................130
p60, Product of the iap Gene...............................................................................................130
Superoxide Dismutase and Catalase ....................................................................................131
Stress Response Mediators...................................................................................................131
Iron Uptake Systems ............................................................................................................132
PrfA and Regulation of Virulence Gene Expression in L. monocytogenes ..................................132
The Positive Regulatory Factor A (PrfA) ............................................................................132
PrfA-Dependent Promoters, Transcripts, and Mechanism
of Temperature-Dependent Virulence Gene Expression......................................................134
Environmental Signals Affecting Virulence Gene Expression ............................................136
Two-Component Systems and Regulation of Virulence Gene Expression .........................137
Lessons Learned from Genome Sequence of L. monocytogenes .................................................137
Evolutionary Aspects .....................................................................................................................138
Open Questions ..............................................................................................................................139
Acknowledgments ..........................................................................................................................139
References ......................................................................................................................................139
INTRODUCTION
Studies that aimed to unravel the mechanisms of Listeria monocytogenes pathogenicity and its
interaction with hosts on the cellular, molecular, and genetic levels were initiated about two decades
ago. The early studies used transposon mutagenesis and infection of primary and established cell lines
to obtain insights into the interaction of L. monocytogenes with eucaryotic host cells. The recent
sequencing of the genomes of L. monocytogenes and Listeria innocua together with development of
genetic tools now allows manipulation of L. monocytogenes, which has, together with cell culture
111
and transgenic animal models, greatly broadened our understanding of the molecular and cell biology
of L. monocytogenes infections.
Most of the early studies on the cell biology of L. monocytogenes infections used epithelia-
like and macrophage-like cell lines [103,229,315]. Macrophages actively ingest L. monocytogenes,
but internalization of the bacterium by normally nonphagocytic cells is triggered by L. monocytogenes
-specific products. Aside from the internalization step, the intracellular life cycle of the bacteria in
phagocytes or normally nonphagocytic mammalian cells is, however, very similar. The pathogen
first appears in a vacuole, which is subsequently lysed by some of the ingested bacteria allowing
L. monocytogenes to escape into the cytoplasm. Whereas most of the bacteria begin to replicate in
the cytoplasm, those remaining in the phagosome are killed and digested.
Concomitant with the onset of intracellular replication, L. monocytogenes induces nucleation of
host actin filaments arranged to a polar tail. Formation of a tail at one pole of the bacterial cell
produces a propulsive force that moves the bacteria through the cytoplasm. Bacteria that reach the
surface of the infected host cell induce the formation of pseudopode-like structures with the bacterium
at the tip and the actin tail behind it. These pseudopods are taken up by neighboring cells. The bacteria
thus entering the neighboring cells are within a vacuole surrounded by a double membrane, which is
subsequently lysed to release the bacteria into the cytoplasm of the newly infected host cell. The line
drawing shown in Figure 5.1 summarizes this intracellular life cycle. The different steps and the
listerial virulence factors involved in this life cycle are discussed in detail later.
FIGURE 5.1 Schematic drawing of the intracellular life cycle of Listeria monocytogenes (A) and represen-
tative electron micrographs showing adhesion (B), entry (C), bacteria inside vacuoles (D), bacteria free in the
cytoplasm (E), moving bacteria in protrusions (F and G), and bacteria in a double membrane vacuole formed
during cell-to-cell spread (H). (Reprinted with permission from Vazquez-Boland, J. A. et al. 2001. Clin.
Microbiol. Rev. 14:584–640.)
Molecular Virulence Determinants of Listeria monocytogenes 113
FIGURE 5.2 Organization of the central virulence gene cluster of L. monocytogenes and structure of the
locus in other Listeria species. Genes belonging to the virulence gene cluster are in gray, and the flanking loci
are in black. The virulence gene cluster is inserted in a chromosomal region delimited by the prs and ldh
genes. In the plcB-ldh intergenic region, two ORFs, orfA and orfB, are found in all Listeria species, indicating
that the insertion point of the cluster is between the prs and orfB loci. In the plcB–orfB intergenic region of
L. monocytogenes, there are two small ORFs, orfX and orfZ (stippled), which delimit the putative excision
point of the cluster in L. innocua. In the plcB–orfB intergenic region of L. ivanovii, two small ORFs, orfX
(encoding a homologue of the orfX product from L. monocytogenes) and orfL (stippled), are also present,
which, like those in L. monocytogenes, delimit the deletion point of the cluster in the nonpathogenic species
L. welshimeri. The additional ORFs found in the L. seeligeri virulence gene cluster are hatched. (Reprinted
with permission from Vazquez-Boland, J. A. et al. 2001. Clin. Microbiol. Rev. 14:584–640.)
Some of the known virulence genes whose products are involved in the intracellular life cycle
of L. monocytogenes are clustered on the chromosome in the so-called PrfA-dependent virulence
gene cluster. The cluster comprises six well-characterized genes, prfA, plcA, hly, mpl, actA, and
plcB (Figure 5.2) along with four small open reading frames (ORFs) of unknown functions
downstream of plcB, called orf X, Z, B, and A. The ends of the gene cluster are defined by genes
coding for housekeeping enzymes. Distal from prfA, defining the “left” border of the gene cluster,
the prs gene is located, encoding a phosphoribosyl-pyrophosphate synthetase [117,127,185]. The
ldh gene coding for lactate dehydrogenase together with the orfs A and B [45,117,127,319] mark
the “right” border of the gene cluster downstream from plcB and the small orfs X and Z.
The products of these virulence genes are listeriolysin (LLO, encoded by hly), a phosphatidylinositol-
specific phospholipase C (PI-PLC, encoded by plcA), a phosphatidylcholine-specific phospholipase
C (PC-PLC, encoded by plcB), a metalloprotease (Mpl, encoded by mpl), ActA, a protein involved
in actin polymerization (encoded by actA), and the positive regulatory factor PrfA (encoded by
prfA). The well-studied internalins internalin A (InlA) and InlB are encoded by the inlAB operon
[102]. Many other internalin-like genes are found dispersed around the L. monocytogenes chromo-
some [38,117]. Several other genes suggested to play a role in virulence are located outside the
virulence gene cluster [320]. Some of them are, however, connected to the virulence cluster genes
because they are also regulated by the transcriptional activator PrfA (discussed later).
MOLECULAR ASPECTS OF THE INVASION OF MAMMALIAN CELLS

Uptake of L. monocytogenes by macrophages of different origin is well documented [183,208,253].
Invasion of L. monocytogenes into different, normally nonphagocytic mammalian cell types,
including murine and human fibroblasts [87,142,183,253], murine and human epithelial cells
[8,87,103,253], murine hepatocytes [76,332], human endothelial cells [82,130,131,293,294], and

mouse and human dendritic cells [135,173,174,247], has also been described.
INVASION OF NONPROFESSIONAL PHAGOCYTIC CELLS

Transposon mutagenesis and an appropriate in vitro invasion assay using Caco-2 epithelial cells
resulted in the identification of internalin (InlA), a surface protein of L. monocytogenes, to
mediate bacterial invasion into epithelial cells [102]. The mutants identified exhibit a lower
invasive capacity than the wild-type strain when tested on different cells. Transposon insertions
occur in a chromosomal region, which represents an operon consisting of the inlA and inlB genes.
Expression of inlA in L. innocua, a noninvasive Listeria species closely related to L. monocytogenes,
renders this species invasive (at least to some extent). This experiment shows that the inlA gene
product is necessary and sufficient to mediate invasion. A large number of internalin homologues
have since been identified in L. monocytogenes [38,78,89,117,257]. Common to all internalins
is an element of several leucine-rich repeats with leucine residues at a fixed position in a typical
22 amino acid (aa) unit.
Internalin is an acidic protein of 800 amino acids [77,102] that possesses two extended repeat
domains. Domain A consists of 15 leucine-rich repeats, whereas domain B consists of 2.5 repeats
of about 70 amino acids (aa) each. The InlA protein has a typical N-terminal transport signal
sequence and a cell wall anchor in the C-terminal part comprising the sorting motif LPXTG followed
by a hydrophobic membrane-spanning region of 20 aa and a few positively charged aa (Figure 5.3)
[77]. This distal LPXTG motif, like similar motifs in other surface proteins covalently linked to
the peptidoglycan in Gram-positive bacteria, is responsible for the attachment of InlA to the bacterial
cell envelope in a process mediated by the enzyme sortase [21,68,109].
InlB, a 630-amino acid protein also carries an N-terminal transport signal sequence, eight
leucine-rich repeats and three C-terminal GW modules; however, in contrast to InlA, it has no
LPXTG motif and no cell wall-spanning region (Figure 5.3) [77]. Nevertheless, InlB is a listerial
surface protein targeted to the bacterial surface via the interaction of the GW modules with
lipoteichoic acid in the listerial cell wall. This type of association appears to be relatively weak
because significant amounts of InlB are found in the supernatant fluid [27,158].
To get more insight into the molecular details of InlA- and InlB-mediated cellular invasion,
three-dimensional structures of important parts of both proteins were solved at the atomic level
[211,284,285]. In InlA and InlB (and also InlH [284]), three N-terminal parts in each protein are
combined to form a contiguous internalin domain. In this internalin domain, a central LRR region
is flanked contiguously by a truncated EF-hand-like cap and an immunoglobulin-like fold
(Figure 5.3). The extended beta-sheet, resulting from the distinctive fusion of the LRR and the
immunoglobulin-like folds, constitutes an adaptable concave interaction surface proposed to interact
with the respective mammalian receptor molecules during infection. In the case of InlB it was
shown that four surface-exposed aromatic amino acids along its concave face are essential for host
cell invasion and binding to its receptor Met (discussed later) [206].
Four eucaryotic receptors for internalin and InlB were recently identified [28,159,219,290].
Human E-cadherin was identified as the internalin receptor by a biochemical approach using matrix-
bound purified InlA to isolate the internalin ligand from epithelial membrane proteins [219].
A member of the cadherin family, E-cadherin is mainly expressed at the basolateral site of entero-
cytes and is a major constituent of adherence junctions, where it connects adjacent cells by
homophilic interactions of its extracellular domains [232]. It binds internalin directly and its location
on the basolateral membrane of epithelial cells is in line with previous observations suggesting the
basolateral membrane as the entry site for L. monocytogenes [311]. Antibodies directed against the
leucine-rich repeat region of internalin block entry of L. monocytogenes into cells expressing
E-cadherin, thereby underlining the importance of the repeat regions of internalin for its function
as an invasin [219].
(A)
(B)
FIGURE 5.3 A: Structure of the members of the internalin multigene family present in L. monocytogenes
strain EGD-e. S: signal peptide; B: B-repeats; C: Csa domain repeats C-repeat; D: D-repeats. See text for
details. (Reprinted with permission from Vazquez-Boland, J. A. et al. 2001. Clin. Microbiol. Rev. 14:584–640.)
B: Crystal structure of the N-terminal part of InlA and the distal domain of its receptor E-cadherin. (Reprinted
with permission from Heesemann, J. et al. 2003. Biospektrum 9:486–489.)
The first InlB receptor to be isolated also by a biochemical approach was the complement
receptor for the globular part of the C1q-fragment (gC1q-R). Direct interaction of the ubiquitously
expressed gC1q-R and InlB was demonstrated and it could be shown that soluble C1q or antibodies
against gC1q-R block InlB-mediated entry of L. monocytogenes [28]. The receptor tyrosine kinase
Met was recently identified as a second InlB receptor required for InlB-dependent entry of L.
monocytogenes into various cells [290]. Treatment of mammalian cells with InlB protein or infection
with L. monocytogenes induces rapid tyrosine phosphorylation of Met, a receptor tyrosine kinase
for which the only other known ligand is hepatocyte growth factor (HGF). Like HGF, InlB binds
to the extracellular domain of Met and induces “scattering” of epithelial cells.
Finally, glucosaminoglycans (GAGs) were identified as a third type of InlB ligand [159]. GAGs
are present on the surface of mammalian cells, where they decorate the proteoglycans; they promote
the oligomerization of growth factors such as HGF. InlB binds to GAGs through its C-terminal
GW repeats, which anchor the protein to the bacterial cell surface. GAGs are hence believed to
detach InlB from the bacterial surface, thus allowing its interaction with the Met receptor at the
contact site of L. monocytogenes on the host cell surface [210].
L. monocytogenes uptake by macrophages and other mammalian cells depends on functional
actin microfilaments because invasion requires membrane extensions formed by rearrangement of
actin filaments and is hence blocked by treatment with actin depolymerizing drugs such as cytoch-
alasins [103,183]. L. monocytogenes is taken up in a way described as a zipper mechanism, which
means that the host cell membrane is in close contact with the bacterium without surface changes
in the vicinity. Entry can also be blocked by tyrosine kinase inhibitors such as genistein
[272,310,322] and the tyrosin phosphatase inhibitor vanadate [180], which shows that signal
transduction events involving various cellular kinases and phosphatases are needed.
Epithelial cell invasion by L. monocytogenes is initiated by binding of InlA to its receptor
E-cadherin. The structural basis for this highly specific interaction became obvious recently, when
the crystal structure of the functional InlA domain, bound to the N-terminal domain of its receptor
E-cadherin, was solved [285]. The concave interaction domain formed mainly by the LRR surrounds
and specifically recognizes human E-cadherin. Individual amino acid residues in InlA were probed
for their role in the InlA-E-cadherin interaction. These studies explained the tremendous specificity
of InlA for human E-cadherin [191] because the aa proline at position 16 in human E-cadherin
(which is changed to glutamic acid in the mouse homologue) represents a very exposed aa in the
molecule responsible for the intimate contact with InlA [285]. The cytoplasmic domain of E-
cadherin directly interacts with β-catenin, which in turn recruits α-catenin [193]. α-Catenin directly
binds to actin filaments and hence completes the direct link of the listerial receptor to the host cell
cytoskeleton. Other proteins normally present in the adherence junction complex are also recruited
to the site of L. monocytogenes entry [54], but their precise role in the uptake process, and the
rearrangement of the actin network particularly, is unknown.
Originally proposed to be a specific factor for hepatocyte invasion [76], InlB was recently
shown to be the only relevant internalin for endothelial cell invasion [17,129,130,244]. InlB-specific
invasion of human brain microvascular endothelial cells is very sensitive to the presence of adult
human serum, which regularly harbors anti-Listeria antibodies [145]. InlB is also involved in
epithelial cell invasion [17,203], but InlA and InlB play no role in fibroblast invasion [203]. As
mentioned before, three mammalian receptors for InlB have been characterized and a complex
series of intracellular signal transduction events are triggered by the interaction of InlB with Met
(reviewed in detail by Bierne and Cossart [19] and Cossart et al. [54]), the only one of the three
receptors that has a cytoplasmic domain.
The PI-3 kinase is in the center of the signal transduction pathway leading to actin rearrangement
[150] and is most likely activated upon interaction with adaptor proteins recruited to the phosphory-
lated cytoplasmic domain of Met [151,290]. The generation of PIP3 by the PI-3 kinase [150] then
leads to an activation of the downstream kinases Rac, PAK, and the LIM-kinase [20]. It is currently
believed that these events finally lead to the activation of the Arp2/3 complex (see later discussion),
which is necessary to induce the actin polymerization involved in rearrangement of the actin
cytoskeleton during the uptake process [54]. Additionally, the InlB-Met interaction triggers
signals leading to activation of the Ras-mitogen-activated protein kinase pathway [52], the
activation of phospholipase Cγ, protein kinase C, and Akt, which in turn activate the central
transcription factor NF-κB [19].
For a long time, the significance of the listerial surface proteins InlA and InlB in in vivo host
cell invasion was less clear. An inlAB mutant is only slightly impaired in virulence in the mouse
model upon intravenous infection and the mutant was only transiently impaired in persistence in
the liver and behaved like the wild-type in spleens and lymph nodes of infected mice [76]. In a
different study inlAB mutants were only rarely found inside hepatocytes compared with the wild-
type strain, indicating a role for the inlAB locus in hepatocyte invasion in vivo [105]. Most
surprisingly, penetration of the intestinal barrier in orally infected mice was very inefficient and
totally independent of the presence of InlA or InlB [191]. This finding is in sharp contrast to the
unequivocally proven role of InlA in epithelial cell invasion in vitro.
The identification of a single nucleotide exchange in mouse E-cadherin compared to human
E-cadherin (Pro16Glu), which renders the mouse homologue unable to bind InlA [191], gave a
first hint to clarify the unexpected findings in the mouse model of listeriosis. A recent publication
by Lecuit et al. [194] elegantly used a transgenic mouse model to demonstrate convincingly the
role of internalin in the penetration of the intestinal barrier. They constructed transgenic mice
expressing human E-cadherin in their enterocytes and showed that, in these mice, L. monocytogenes
mutants lacking InlA were significantly less able to cross the intestinal barrier because they were
severely impaired in promoting their uptake by the enterocytes forming the intestinal barrier
[192,194].
A large number of other internalins are present in L. monocytogenes as deduced from the
genome sequence [38,117]; some, including InlC, InlE, InlF, InlG, and InlH [78,89,256], were
already described in the pregenomic era. None of these proteins seems to be able to induce
phagocytosis in mammalian cells and their roles in the infection process are largely unknown. By
the construction of various combinations of in-frame deletions in most of the respective internalin
genes it could recently be shown that—in contrast to InlB—InlA by itself triggers uptake into
Caco-2 epithelial cells poorly and needs the help of other internalins [17]. This additional trigger
function can be supplied by InlC alone or by InlC together with InlG, InlH, and InlE, or by InlB.
Because no cellular receptors for internalins other than InlA and InlB are known, it is presently
unclear whether these internalins trigger host cell function via the known InlB receptors or via
other mammalian surface molecules.
Several other L. monocytogenes surface structures have been described as involved in invasion.
LpeA (Lipoprotein promoting entry) [262], a listerial lipoprotein with homology to a Streptococcus
pneumoniae adherence factor, was implicated in the invasion of hepatocytes (and to a lesser extent
of epithelial cells) because the respective mutant shows clearly diminished capacity of cellular
invasion, but not of adhesion or intracellular growth. LpeA is the first listerial lipoprotein involved
in invasion to be identified. The major extracellular protein p60 of L. monocytogenes [182,249]
was initially postulated to be involved in fibroblast invasion, but its role in mediating cellular uptake
is still under debate (see later discussion).
The listerial surface protein ActA, a major virulence factor allowing actin-based intracellular
motility [74,168] (see following), also was suggested recently to play a role in internalin-independent
uptake of L. monocytogenes by epithelial cells [7]. Analysis of the invasive capacity of strains
lacking or overexpressing ActA suggests that ActA may function as an invasion-mediating
protein—at least when overexpressed [307]. Such an ActA-promoted attachment and invasion of
CHO epithelia-like cells as well as IC-21 murine macrophages was shown to be mediated by
interaction of the listerial surface protein ActA with a heparan-sulfate proteoglycan receptor [7].
It is supposed that electrostatic interactions between heparan sulfate and positively charged residues
in the N-terminal part of ActA could lead to low-stringency binding to the cell surface proteoglycan
receptors widely distributed in mammalian cells [7]. Whether the proposed low-stringency binding
of L. monocytogenes to heparan sulfate proteoglycan receptors directly triggers uptake or results
in adequate presentation of other bacterial factors to the host cell membrane that ultimately lead
to phagocytosis remains to be clarified.
CELLULAR ADHESION
The mechanisms allowing L. monocytogenes adhesion to various mammalian cells without follow-
ing uptake are not well understood. InlA can clearly mediate binding to the surface of epithelial
cells because mutants lacking the inlA gene bind less well to Caco-2 epithelial cells [17,262]. In
contrast, InlB, which by itself triggers high-efficiency invasion of endothelial cells, is obviously
not involved in cell adhesion. Mutants lacking the inlB gene adhere to human brain microvascular
endothelial cells essentially as the wild-type strain [17,129]. Because even the nonpathogenic
species L. innocua (which lacks the internalins of L. monocytogenes [117]), binds to endothelial
cells, one must assume that common cell wall structures mediate the interaction of L. monocytogenes
and L. innocua with the endothelial cells [129].
One of the listerial surface structures that may mediate internalin-independent binding to
mammalian cells are the lipoteichoic acids found in listerial cells walls [91]. These polymers are
known to interact with mammalian pattern recognition receptors of the scavenger- and toll-receptor
families [85]. It was shown that purified lipoteichoic acid from L. monocytogenes can trigger host
cell responses in macrophages [141] and such interactions may also confer cell binding. A recent
paper by Abachin et al. [1] has now shown that an L. monocytogenes mutant with an altered
lipoteichoic acid lacking D-alanine is severely impaired in binding to epithelial cells, hepatocytes,
and even macrophages. It is believed that the mutation increases the electronegativity of the bacterial
surface, which then interferes with cell binding even in the presence of internalin and InlB.
Various extracellular bacterial pathogens exploit the extracellular matrix protein fibronectin
as a bridging receptor for cell binding [92]. Five fibronectin binding proteins were identified in
L. monocytogenes, one of which was a 55-kDa cell surface protein [114]. Additionally, the gene
of one fibronectin binding protein of 25 kDa was cloned and sequenced [115]. However, the role
of these listerial proteins in virulence in general and specifically in cell binding is not known.
As mentioned before, the L. monocytogenes surface protein ActA can act as an invasion—at
least when overexpressed. However, it is possible that in the situations tested, ActA actually acts
as an adhesin allowing more efficient contact of the bacteria to the cell surface followed by
interaction of the invasion-promoting molecules with the host cell [7]. Another surface protein,
called p104, probably involved in listerial adhesion to Caco-2 epithelial cells was recently identified
by transposon mutagenesis [243]. The mutants that lack p104 expression are reduced about tenfold
in adhesion to the Caco-2 cells and antibodies against the protein also inhibit adhesion.
Up to now, the only listerial protein clearly shown to mediate L. monocytogenes adhesion
without promoting uptake is the surface molecule Ami [224,225]. Ami is a 102-kD autolysin with
the catalytic activity in the N-terminal part of the molecule. The C-terminal cell wall anchor region
is made up of repeated modules containing a GW dipeptide as also found in InlB [27]. Ami confers
cell binding to epithelial cells and hepatocytes in a ∆inlAB background. It was shown by comple-
mentation that the C-terminal GW modules are responsible for mediating cell adhesion and the
purified C-terminal part binds to epithelial cells in vitro [224].
UPTAKE BY MACROPHAGES AND DENDRITIC CELLS

Macrophages of different origin were used in in vitro studies to analyze the mechanisms of
L. monocytogenes uptake by professional phagocytes, which are generally assumed to take up
L. monocytogenes by conventional phagocytosis involving actin-polymerization. If present, complement
factors C1q and C3 are deposited on the bacterial surface and stimulate L. monocytogenes uptake
by binding the bacteria to the respective receptors [4,59,80].
Macrophages ingest L. monocytogenes very rapidly and intracellular killing starts shortly after
phagocytosis and leads to destruction of most of the ingested bacteria [64,66,259,325]. In a single
macrophage, killed bacteria inside acidified phagosomes and phagolysosomes and growing bacteria
that have escaped into the cytoplasm can be detected. These findings suggest competition between
phagosome–lysosome fusion followed by killing of the bacteria and their escape from the acidified
phagosome before phagosome–lysosome fusion occurs. The result of this competition is a popu-
lation of cytoplasmic bacteria able to grow inside the macrophage. The route of uptake by the
macrophages may also be important for the fate of the invading bacteria. As demonstrated by
Drevets et al. [81], the mode of uptake is critical for subsequent survival because L. monocytogenes
taken up in the presence of complement C3 leads to enhanced killing of the bacterium.
Whether the surface protein InlA substantially contributes to the triggering of phagocytosis of L.
monocytogenes by macrophages is still under debate. Using bone marrow-derived macrophages, InlA
had only a slight effect on invasion because an inlAB mutant still showed more than 60% invasion
when compared with the wild-type strain [146]. Uptake of L. monocytogenes by the mouse macrophage
-like cell line J774A.1 was inhibited by at least 50% by the pretreatment of L. monocytogenes with
anti-InlA antibodies and that recombinant InlA specifically bound to the macrophages [281]. It was
suggested recently that the listerial protein p60 might enhance phagocytosis by macrophages because
a Salmonella typhimurium strain that expresses and secretes p60 seems to be more invasive in
phagocytic cells but not in enterocytes [146]. In line with this assumption is that pretreatment of L.
monocytogenes with a polyclonal anti-p60 antiserum inhibits uptake of the bacteria by a macrophage-
like cell line [146]. Another factor that could be involved in attachment and invasion of L. monocy-
togenes in macrophages is the listerial cell wall polymer lipoteichoic acid. L. monocytogenes binds
to the macrophage scavenger receptor most likely via lipoteichoic acid [85,128]. This interaction may
also trigger conventional receptor-mediated phagocytosis of L. monocytogenes.
The mechanisms specifically underlying L. monocytogenes uptake by macrophages are not well
understood. As expected, a functional actin cytoskeleton is necessary as deduced from inhibitor
studies with cytochalasins [103,183]. In addition, microtubules also seem to be involved because
the drugs nocodazole and cholchicin, which depolymerize microtubules, inhibit L. monocytogenes
uptake by different macrophages [181]. The early signaling events associated with L. monocytogenes
uptake by J774 macrophages were analyzed by Goldfine and coworkers (summarized in Goldfine
and Wadsworth [125]).
They demonstrated that very rapidly and before attachment and invasion, the addition of the
bacteria to the macrophages results in three peaks of calcium mobilization within 10 min [323].
Whereas the first two peaks result from calcium influx, the third results from the mobilization of
intracellular calcium stores. Linked to the calcium peaks is the mobilization of several protein
kinase C isoforms that are translocated to the cell membrane [324]. Calcium signaling and protein
kinase C activation depend on the listerial virulence factors LLO and PI-PLC (see later discussion)
and modulate the uptake of L. monocytogenes into the macrophages. It appears that calcium
mobilization and protein kinase C activation negatively influence the speed of the uptake process
because mutants lacking LLO and PI-PLC are taken up more rapidly as the wild-type strain and
specific inhibitors of PKC also induce the speed of uptake [323].
Another class of professional phagocytic cells important in the innate immune response against
L. monocytogenes is the dendritic cells. They can be found in different tissues where they take up foreign
material and present it to T-cells to stimulate adaptive responses [264]. Dendritic cells of mouse and
human origin efficiently take up L. monocytogenes [135,173,174,247]. Invasion of dendritic cells seems
to be independent of internalin and InlB [135,173], but requires a functional cellular cytoskeleton.
Furthermore, the uptake is significantly improved in the presence of human serum and specifically
enhanced by antibodies against the listerial protein p60, which act as opsonins [174]. At least some of
the intracellular bacteria are released into the cytoplasm, but intracellular replication seems to be very
low in dendritic cells. Because dendritic cells are able to migrate over long distances toward lymphoid
tissue, they might also be important vectors during the early dissemination of the bacteria [255].
ESCAPE FROM THE PHAGOCYTIC VACUOLE

Hemolytic activity detected around colonies of L. monocytogenes growing on blood-agar plates
was long supposed to represent a major virulence determinant because all clinical isolates of
L. monocytogenes show this hemolytic phenotype. The hemolytic activity is due to the action of a
cytolysin, called listeriolysin O (LLO). In experimental infections all virulent strains were found
to be hemolytic, whereas nonhemolytic strains were avirulent. Nonhemolytic mutants obtained
after transposon mutagenesis using the conjugative transposons Tn1545 or Tn916 [104,163,253]
always proved to be avirulent in the mouse model. Virulence is restored in hemolytic revertants
that have lost the transposon insertion or by the introduction of the cloned hly gene into a
nonhemolytic L. monocytogenes transposon mutant [55].
Listeriolysin O, a secreted protein of 58 to 60 kDa, belongs to a family of thiol-activated,
cholesterol-dependent, pore-forming toxins (CDTX) for which streptolysin O is the prototype [242].
All members of this family are inhibited by low concentrations of cholesterol and oxygen and
activated by reducing agents like DTT. Cholesterol is the receptor for these cytolysins because only
membranes containing cholesterol are attacked and this component inhibits pore formation and
toxicity [242]. Upon addition to erythrocytes, toxin monomers oligomerize in the target cell
membrane to form stable pores that can be visualized by electron microscopy (Figure 5.4) [246].
Listeriolysin O has been purified to homogeneity and its toxicity, determined by intraperitoneal
injection into the mouse, shows LD50 of 1.7 µg per mouse. Optimal hemolytic activity is found at
pH 5.5, a pH value much lower than that determined for the other CDTX [111], a property in
agreement with the function of LLO in the acidified phagosome (see later discussion).
The gene encoding LLO, hly, was cloned from strains of different serovars of L. monocytogenes
and sequenced [71,216,220]. The deduced amino acid sequence for LLO yielded 529 amino acids
including an N-terminal signal sequence of 25 amino acids. As expected, the sequence shows
extended homologies with the protein sequences of other CDTX. The highest homology is observed
in the C-terminal part and includes a highly conserved undecapeptide containing the unique cysteine
thought to be essential for cytolytic activity. Site-directed mutagenesis revealed, however, that the
cysteine is not essential for hemolytic activity.
In contrast, a tryptophan residue, in close vicinity to the cysteine, appears to be required for
hemolytic activity and virulence [222] as it was also shown for an alanine residue also located in
that motif [152]. The three-dimensional structure of LLO is not known. However, the structure of
the closely related toxin perfringolysin O (PFO) was solved. This toxin—and hence most likely
also LLO—is composed of four domains that form an L-shaped molecule (Figure 5.4) [273]. The
expression of domain 4 alone or of domains 1 to 3 of LLO showed that domain 4 is responsible
for membrane binding and oligomerization, whereas the first three domains are necessary for full
hemolytic activity [83,172]. Strikingly, when expressed simultaneously, the two secreted domains
LLO-d123 and LLO-d4 reassembled into a hemolytically active form [83].
The role of LLO in virulence was determined by injection, intravenous and intraperitoneal, of
wild-type and nonhemolytic mutants of L. monocytogenes into mice and by following the fate of
the bacteria in liver and spleen. In contrast to the wild-type strain, the nonhemolytic mutants are
eliminated from these organs within a few hours without eliciting protective immunity
[55,104,163,253]. The role of LLO in the intracellular survival was determined using different
mouse and human cell lines. In the human enterocyte-like cell line Caco-2 [103], mouse 3T6
fibroblasts [183], and mouse CL.7 fibroblasts [253], nonhemolytic L. monocytogenes mutants are
as invasive as the isogenic wild-type strains.
The nonhemolytic mutants are, however, incapable of survival and intracellular growth within
these host cells and also in mouse peritoneal macrophages [183], mouse bone marrow-derived
macrophages [253], and the mouse macrophage-like cell line J774 [253]. Electron microscopy of
infected macrophages and epithelial cells revealed that nonhemolytic L. monocytogenes mutants
found inside the cells are unable to open the phagosome and hence unable to escape into the
FIGURE 5.4 A: Pore-forming activity of LLO. Sheep erythrocyte ghost after treatment with purified LLO,
showing the ring-shaped oligomeric structures of the toxin attached to the membrane (bar, 100 nm). (Reprinted
with permission from Jacobs, T. et al. 1998. Mol. Microbiol. 28:1081–1089.) B: Crystal structure of PFO, a
close relative of LLO. (Reprinted with permission from Rossjohn, J. et al. 1997. Cell 89:685–692.)
cytoplasm of the host cells [103,315]. Additional evidence that LLO is essential for lysis of the
phagosomal membrane was obtained by infection of macrophages with a Bacillus subtilis strain
expressing LLO [18]. This engineered strain escapes from the phagosome into the cytoplasm,
whereas the nonhemolytic B. subtilis parental strain stays in the phagosome as do the nonhemolytic
L. monocytogenes mutants.
The low pH optimum of LLO is in agreement with its function in the acidified phagosome.
Bafilomycin or ammonium chloride treatment inhibits vacuolar acidification and inhibits the
escape of L. monocytogenes from phagosomes of infected epithelial cells or macrophages. These
findings sustain the importance of the low pH activity optimum of LLO for its role as a vacuole
opener [15,51]. Recently, Portnoy and coworkers [156,157] analyzed the role of LLO (and
especially the role of its low pH optimum) by constructing L. monocytogenes strains that secrete
the closely related extracellular cytolysin perfringolysin instead of LLO. Such a strain escaped
from the vacuole, but damaged the host cell. An elegant selection procedure was used to isolate
mutants that do not damage the host cell upon perfringolysin expression in the cytoplasm. The
mutated perfringolysins were reduced in activity at neutral pH, had a generally reduced hemolytic
activity, or showed a shorter half-life in the cytoplasm. These results show that the low activity
of LLO at neutral pH values and its short half-life in the cytoplasm are critical parameters for
its suitability as a phagosome opener without concomitant cytotoxicity. The strains expressing
mutated perfringolysins allowing intracellular growth without cell damage are totally avirulent,
however [157].
Further study [118] now showed that a single amino acid exchange in LLO (Leu461Thr) results
in an LLO variant with ten times higher hemolytic activity at neutral pH and an L. monocytogenes
strain expressing this LLO variant is about 100-fold less virulent. The reduction in virulence is
most likely associated with a higher toxicity of the molecule to the host cells, which are killed by
membrane damage resulting from activity of LLO [118,119].
The importance of restriction of LLO activity to the phagosomal compartment is further
supported by other lines of evidence. A so-called PEST sequence was recently identified in
the N-terminal region of LLO [65,202]. PEST sequences target proteins to the proteasome degra-
dation pathway [260], hence dramatically reducing their cytoplasmic half-life. LLO variants lacking
the PEST sequence demonstrate normal hemolytic activity and allow vacuolar escape, but are toxic
to their host cells. Their expression results in a decrease in virulence in the mouse model [65]. A
recent publication by Lety et al. [201], however, showed that rather than the actual PEST sequence,
some amino acids immediately downstream of PEST could be responsible for the correct function
of LLO, thus questioning the results mentioned earlier. The expression of LLO in the infected host
cell is tightly controlled; expression is induced when the bacteria are taken up by mammalian cells
and seems to be highest in the phagosome [36], although it also continues in the cytoplasm [227].
A recent paper by Dancz et al. [61] finally showed that induction of LLO expression (with an
IPTG-inducible expression system) at different time points after infection allows bacteria trapped
in the cytosol to escape into the cytoplasm of macrophages and then grow intracellularly, again
demonstrating the crucial role of LLO in phagosomal escape.
Fusion of L. monocytogenes-containing phagosomes with endosomes has been observed in
electron microscopy studies [315]. However, it is not known whether such an event is necessary
for L. monocytogenes to progress through its intracellular life cycle [325]. The recent description
of Rab5-regulated fusion of L. monocytogenes-containing phagosomes with endosomes and results
that indicate that Rab5a controls early phagosome–endosome interactions and governs the matu-
ration of the early phagosome leading to phagosome–lysosome fusion [6] show that phagosome
maturation events take place upon ingestion of L. monocytogenes into macrophages [3].
On the other hand, live L. monocytogenes delays phagosome maturation and subsequent deg-
radation by yet unknown mechanisms. It is believed that this allows the bacteria to prolong their
survival inside the phagosome/endosome, assuring their viability as a prelude to escape into the
cytoplasm [5]. Prolonged intraphagosomal survival of L. monocytogenes in macrophages was
recently demonstrated [61]; this could be nicely explained by the data on an L. monocytogenes-
induced delay in phagosome maturation.
Listeriolysin O-independent escape of L. monocytogenes from primary vacuoles in human
epithelial cells [253] is mediated by two listerial phospholipases and a metalloprotease [213] that
also contribute to vacuole escape in other cells like bone marrow-derived macrophages [42].
Phospholipase activity of L. monocytogenes was first discovered as a zone of opacity surrounding
colonies on egg yolk agar [100]. Transposon mutants of L. monocytogenes lacking phospholipase
activity were identified by formation of small plaques on fibroblast cell monolayers [309] and by
reduced hemolysis on blood-agar plates [164], indicating a participation of phospholipase activity
in hemolysis. The gene encoding a phosphatidylinositol-specific phospholipase C (PI-PLC), called
plcA [41,195,215], encodes a protein of 34 kDa that exhibits high homology to several Gram-
positive phospholipases and contains a typical transport signal sequence.
This enzyme was purified from culture supernatant fluid of an overexpressing L. monocytogenes
strain [124] and was highly specific for phosphatidylinositol with no detectable activity on
phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. It also does not cleave
phosphatidylinositol-4-phosphate or phosphatidylinositol-4,5-bisphosphate, but is active, albeit with
low specific activity, on glycosylated phosphatidylinositol-anchored proteins [108]. The crystal struc-
ture of PI-PLC was recently determined (Figure 5.5) [228]. The enzyme consists of a single
(βα)8-barrel domain with the active site located at the C-terminal side of the β-barrel. Unlike other
FIGURE 5.5 Ribbon diagram of the structure of PI-PLC viewing toward the active site pocket where the
bound myoinositol molecule (labeled Ins in yellow) is shown. α-Helices (A to G) are colored in red, β-strands
(I—VIII) in blue, loops in green. β-Strands are labeled with roman numerals. The N-terminus is labeled with
N′, the C terminus with C. (Reprinted with permission from Moser, J. et al. 1997. J. Mol. Biol. 273:269–282.)
(βα)8-barrels, the barrel in PI-PLC is open. Interaction between the substrate and the active site pocket
is made by specific hydrogen bonds with a number of charged amino acid side-chains. Two histidine
residues (His38 and His86) not present in the active site are, however, important for the activity of the
enzyme because their mutagenesis results in PI-PLC variants with activity reduced 100-fold [11].
L. monocytogenes strains expressing these variants behave like plcA-deletion mutants in cell culture assays.
In addition to the highly specific PI-PLC, L. monocytogenes produces a second phospholipase
C, which hydrolyzes phosphatidylcholine (lecithin), and is thus a phosphatidylcholine-specific
phospholipase C (PC-PLC) or lecithinase [112], also called broad-spectrum phospholipase C. A
32-kDa protein was detected in the supernatant liquid of an L. monocytogenes culture that showed
phospholipase activity on egg yolk overlays [164]. The protein was purified to homogeneity
[112,123] and is a zinc-dependent phospholipase C of 29 kDa. The pH optimum of the enzyme is
between pH 6 and 7 and its activity is stimulated by 0.5 M NaCl and 0.05 mM ZnSO4. In addition
to phosphatidylcholine, it also hydrolyzes phosphatidylethanolamine, phosphatidylserine, and, with
lower efficiency, sphingomyelin. Phosphatidylinositol is not a suitable substrate. The purified protein
exhibits weak hemolytic activity but is not toxic to mice [112].
The gene plcB, encoding PC-PLC, is part of the lecithinase operon, which consists of mpl,
actA, plcB, and the small orfs X and Z [319]. PC-PLC is a protein of 289 amino acids with a
25-amino-acid N-terminal transport signal and a putative propeptide of 26 amino acids. Maturation
of the 32-kDa precursor of PC-PLC occurs after secretion because both forms of the protein
can be found in the supernatant liquid; it is obviously accomplished by the metalloprotease
of L. monocytogenes [254,258].
Use of in-frame deletions in the plcA gene enabled clear demonstration that PI-PLC is required
for efficient escape of L. monocytogenes from the phagosome of mouse bone marrow-derived
macrophages. However, the mutation in plcA has only a slight effect on virulence [42]. It is assumed
that PI-PLC acts in concert with listeriolysin inside the acidified phagosomal vacuole of the host
cell to mediate lysis of the vacuolar membrane. The broad pH optimum of PI-PLC, ranging from
pH 5.5 to 7.0, is consistent with its postulated function in the acidified phagocytic vacuole of
infected cells. To further assess the role of PI-PLC, the plcA gene was expressed in L. innocua,
which lacks the prfA-dependent virulence gene cluster and is therefore unable to escape from the
host cell vacuole. The PI-PLC expressing L. innocua strain cannot escape from the phagosome of
J774 macrophages, but shows limited intracellular growth inside the vacuoles, which appear to be
structurally intact [286].
The role of PC-PLC in escaping from the primary vacuole is not clear and differs from cell type
to cell type. In mouse bone marrow-derived macrophages, PC-PLC has no role in lysis of the vacuole
[299]. However, in the human Henle 407, HEP-2, and HeLa epithelia-like cell lines, where escape
of L. monocytogenes occurs at low efficiency independently from LLO [133,213,253], PC-PLC is
required for lysis of the phagocytic vacuole together with the metalloprotease. PI-PLC is not required
in this system, but the efficiency of escape was reduced in a hly, plcA double mutant [213].
The metalloprotease Mpl of L. monocytogenes indirectly contributes to pathogenicity and
intracellular replication of the bacteria. Transposon mutants with insertions in the mpl gene are less
virulent but grow normally inside mammalian cell lines [258]. The reduced virulence was attributed
to lack of proteolytic processing and hence activation of the 32-kDa PC-PLC proform [254,305].
This also seems the way in which Mpl contributes to lysis of the vacuole in Henle 407 cells, where
vacuolar escape is independent of LLO but depends on PC-PLC and Mpl [258].
Located immediately downstream of the hly gene, the mpl gene [72,218] encoding Mpl is the
first gene of the lecithinase operon [72,218,319]. The deduced amino acid sequence of this protease
shows high homology to several zinc-dependent metalloproteases from Bacillus species and yields
510 amino acids with a typical N-terminal signal sequence and a putative internal cleavage site.
Like other metalloproteases, the enzyme is activated by proteolytic maturation resulting in a mature
35-kDa protein [72,218]. A 60-kDa protein is detected with an antiserum raised against Bacillus
stearothermophilus thermolysin, which probably represents the proform of the metalloprotease.
Only small amounts of the mature 35-kDa form of the protein were detected in the supernatant
liquid of an L. monocytogenes culture [72].
Recently, the protein Mpl was purified and biochemically characterized and shown to be an
enzyme active at a wide range of temperatures and pH values. The protein exhibits a high thermal
stability and shows a narrow substrate specificity cleaving, besides the pro-PC-PLC, also casein
and actin [50]. The translocation through and release of PC-PLC from the bacterial cell wall occurs
most efficiently upon a decrease in pH as regularly encountered in primary and secondary phago-
somes. The release coincides with the proteolytic activation of PC-PLC by Mpl, which both
colocalize at the cell wall–membrane interface [302].
Listeria monocytogenes encodes a large number of putative lipoproteins [38,117] with largely
unknown functions. By deleting the gene encoding a putative lipoprotein-specific signal peptidase,
Reglier-Poupet et al. [261] recently got first insights into the role of at least some members of this
protein family. The deletion mutant failed to process several lipoproteins and showed reduced
virulence in the mouse model. Expression of the signal peptidase is strongly induced while the
bacteria reside in the phagosome and mutant bacteria are clearly impaired in phagosomal escape.
The mechanism of how listerial lipoproteins contribute to lysis of the phagosomal membrane is,
however, still unknown.
GROWTH IN THE HOST CELL CYTOPLASM

As mentioned earlier, phagosomal escape is a prerequisite for L. monocytogenes to replicate
intracellularly and is hence a critical virulence mechanism of this species. In principle, intracellular
bacteria have two possibilities for intracellular multiplication: They replicate in a membrane-bound
vacuole as exemplified by many pathogens, including Salmonella Typhimurium, Legionella pneu-
mophila, Mycobacterium tuberculosis, and Coxiella burnetii or they escape into the host cell
cytoplasm. This second intracellular niche is only chosen by L. monocytogenes, L. ivanovii, Shigella
flexneri, and Rickettsia spp. [92]. Because the vacuole is a potentially hostile environment, bacteria
that stay there have evolved many different ways to interfere with phagosome maturation that allow
them to live in an intracellular compartment fulfilling their needs [138].
L. monocytogenes starts intracellular multiplication shortly after escape from the vacuole with
intracellular generation times of 40 to 60 min—close to the approximately 40-min generation time
observed in rich broth culture [253]. The host cell cytoplasm hence allows listerial growth with
high efficiency. However, the cytosol is poorly characterized as a substrate supporting bacterial
growth and the relative abundance of nutrients is unknown. Whereas various auxotrophic mutants
of L. monocytogenes are able to grow intracellularly [212], expression of several metabolic genes
is increased intracellularly [166], indicating that at least some metabolites may be limiting in the
cytosol, but there is no general upregulation of expression of stress proteins in intracellularly
growing L. monocytogenes [139].
Early studies addressing the question whether even nonpathogenic bacteria not adapted to an
intracytoplasmic lifestyle can grow in the host cell cytoplasm used B. subtilis [18] or L. innocua
[90] strains engineered to express listeriolysin to allow phagosomal escape after uptake. Results
of these studies showed that nonpathogenic bacteria were able to multiply—at least to some
extent—in the host cell cytoplasm and hence implied that this compartment may be favorable for
bacterial growth. A recent study by Goetz et al. [122] addressed this question differently by directly
microinjecting L. monocytogenes and other bacteria into cytoplasm of Caco-2 epithelial cells and
J774 macrophages. Intracellular multiplication of the bacteria was followed microscopically
because they were constructed to express the green fluorescent protein.
In contrast to the studies just mentioned that used this method, only bacteria naturally capable
of intracytoplasmic growth (L. monocytogenes, S. flexneri, and enteroinvasive Escherichia coli)
grew in the cells; others such as B. subtilis, L. innocua, and S. Typhimurium did not [122]. Furthermore,
an L. monocytogenes mutant lacking the central virulence regulator PrfA (discussed later) did not
multiply upon microinjection, clearly pointing to the need of specific virulence determinants
necessary for efficient intracytoplasmic multiplication. The obvious discrepancies in the studies
concerning the ability of bacteria to use host cell cytosol for growth have been discussed in detail
elsewhere [120,240,318], but further investigation is needed before a clear decision can be made.
Probably some cytosolic compartments permit bacterial growth and others do not, depending on
conditions of the infected cell and infecting bacterium.
In search of specific bacterial factors allowing intracellular growth, a bacterial homologue of
the mammalian glucose-6-phosphate translocase (Hpt) [48] and a lipoate protein ligase (LplA1)
[239] were identified that are necessary for efficient intracellular proliferation of L. monocytogenes.
Expression of the Hpt permease is tightly controlled by the central virulence regulator PrfA, which
induces a set of virulence factors required for listerial intracellular parasitism upon entry into host
cells. Loss of Hpt resulted in impaired listerial intracytosolic proliferation and attenuated virulence
in mice but did not affect bacterial growth in a rich medium like BHI broth [48]. However, Hpt
alone is not sufficient for cytoplasmic growth because L. innocua expressing Hpt together with
LLO cannot grow intracytoplasmically upon infection of macrophages [298].
Lack of LplA1 results in bacteria that cease intracellular replication after about five rounds of
replication and are clearly defective in mouse virulence assays. A major target for LplA is the E2
subunit of the pyruvate dehydrogenase enzyme (PDH) complex; in intracellularly grown lplA1
mutants, PDH is no longer lipolyated. Studies of lipoic acid metabolism have shown that little free
lipoic acid exists in mammalian cytosol [241]. Thus, LplA1 may not be important in replication
of L. monocytogenes when free lipoic acid is available, but it is required in the host cell wherein
lipoic acid may be scavenged by host molecules [239].
INTRACELLULAR MOTILITY AND CELL-TO-CELL SPREAD

Intracellular movement of L. monocytogenes inside the host cell cytoplasm as well as intercellular
spread mediated by actin polymerization were initially described by Mounier et al. [229] and
Tilney and Portnoy [315]. Their studies were followed by a series of analyses describing the
cell biology of the process. It was shown that L. monocytogenes moves rapidly through the
cytoplasm with the help of the formed actin tails with a speed of up to 1.5 µm/sec. Quite
surprisingly, L. monocytogenes rotates around its long axis as it is propelled by actin polymerization
[271]. The rate of actin assembly, which occurs at the barbed ends of the actin filaments near
the bacterial surface, equals the rate of actin-based motility; actin polymerization provides the
propulsive force for intracellular movement, which can also take place in cytoplasmic extracts
from Xenopus oocytes [60,280,312,313,314].
Mutants defective in intracellular motility were obtained by transposon mutagenesis. These
mutants have lost the ability to initiate actin polymerization [309] because of insertion into a gene
called actA or still induce actin polymerization but are unable to rearrange actin filaments to actin
tails [184]. The gene actA is located downstream of mpl in the lecithinase operon and codes for a
proline-rich protein (ActA) of 639 amino acids (Figure 5.6). Its apparent molecular weight deter-
mined by SDS-PAGE is 92 kDa [74,319]. ActA is a surface protein consisting of three domains:
the N-terminal domain with the transport signal sequence, the central proline-rich repeat region,
and the C-terminal part, which includes a membrane anchor [74,168,319]. Mutations in the actA
gene result in loss of virulence in mice [74], lack of intracellular actin polymerization around
bacteria, and inability of intracellular movement [74,168]. Inside the host cells, actA mutants form
microcolonies located near the nucleus [74].
Several assays were used to prove that ActA alone is sufficient to stimulate F-actin assembly
and promote intracellular movement: First, the nonmotile species L. innocua was engineered to
express ActA; the recombinant bacteria induce formation of actin tails and move in cytoplasmic
extracts as the wild-type L. monocytogenes strain [170]. Second, actA was transfected into mam-
malian cells [99,250,251] where the ActA protein (including the membrane anchor) was targeted
FIGURE 5.6 Diagram of the molecular components required for actin-based motility of L. monocytogenes.
A: Interactions between host-cell proteins and ActA at the bacterial surface. Two domains of ActA are required
for normal motility. The amino-terminal domain activates actin filament nucleation through Arp2/3. The central
proline-rich domain binds VASP and profilin interacts with VASP, enhancing filament elongation. B: Host
protein functions throughout the comet tail. In addition to the factors that act at the bacterial surface, capping
protein binds to the barbed end of actin filaments to prevent elongation of older filaments, α-actinin cross-
links filaments to stabilize the tail structure, and ADF/cofilin disassembles old filaments. (Reprinted with
permission from Cameron, L. A. et al. 2000. Nat. Rev. Mol. Cell Biol. 1:110–119.)
to mitochondria, which subsequently assemble F-actin on their surface. Third, polystyrene beads
were coated with purified ActA that induced F-actin polymerization in in vitro systems that also
support listerial movement [39].
The ActA protein is distributed asymmetrically on the surface of L. monocytogenes. After cell
division, it is concentrated at the old pole [169,311] and absent or present only at low concentrations
at the new bacterial pole. This asymmetric distribution of the ActA protein is required and sufficient
to direct actin-based motility by coating streptococci asymmetrically with genetically engineered
ActA protein [300]. In a cell-free system, these streptococci, but not uniformly coated ones, moved
efficiently in cytoplasmic extracts [300]. Additionally, only polystyrene beads coated with ActA
asymmetrically induce true F-actin tails and move in extracts [39]. ActA seems to be an elongated
protein [49] that thus far resists crystallization efforts. It was postulated that ActA is present on
the bacterial surface as a dimer [230], but these data were questioned recently by demonstration
that functional ActA is a monomeric protein [207].
Elucidation of the precise mechanisms by which ActA allows actin recruitment and intracellular
movement by stimulating F-actin polymerization is in the center of research interests of several
laboratories and has been reviewed in detail elsewhere [14,40,53]. Expression of mutated forms of
ActA in mammalian cells [99, 250] or in L. monocytogenes [49,186–188,252,297,301,306] made
it possible to define regions of the ActA protein with specific functions in recruitment of cellular
proteins and hence in actin polymerization and movement. Deletion of the whole N-terminal domain
of ActA was followed by total abolishment of actin polymerization and intracellular movement in
both systems, showing the absolute necessity of this domain in ActA function [186]. Within the
N-terminal part of ActA, two smaller regions were identified that are required for filament elongation
(aa 117 to 121) or for continuity of the actin tail (aa 21 to 97); their deletion led to discontinuous
actin tail formation [187,188,252]. In contrast, deletion of the C-terminal domain did not inhibit
actin assembly [186]. The actin tails produced for L. monocytogenes strains expressing ActA without
the central proline-rich repeats were significantly shorter and movement speed was drastically
reduced [301].
The protein composition of the F-actin tails was analyzed using different methods and several
actin binding proteins and proteins regulating the actin dynamics were colocalized with tails,
including α-actinin, tropomyosin, vinculin, talin, fimbrin, villin, ezrin/radixin, cofilin, coronin, Rac,
capZ, profilin, VASP, Mena, and the Arp2/3 complex [44,60,63,70,113,167,311,313,328]. From
these proteins only profilin, Mena, the Arp2/3 complex, and the vasodilator-stimulated phosphoprotein
(VASP) are associated with the surface of moving bacteria and colocalize with ActA [44,113,313]; only
VASP, Mena, and the Arp2/3 complex directly bind to ActA [44,113,252,334]. Another cellular protein,
called LaXp180, directly binds to ActA-expressing intracellular L. monocytogenes. This resulted in
recruitment of the cellular phosphoprotein stathmin [13,248]. However, the role of these proteins in
intracellular motility, if any, is unknown.
The Arp2/3 complex, named after two of its members (Actin related proteins 2 and 3), consists
of seven host cell proteins and was initially characterized as a profilin-binding protein complex
[205]. Arp2/3 initiates F-actin polymerization because of its nucleation activity [231], which is
activated significantly by the presence of ActA [328,329]. Studies with ActA mutants clearly
demonstrated that the N-terminal region of ActA is sufficient for this activation [297]. The regions
of the ActA protein that directly interact with proteins from the Arp2/3 complex during activation
were mapped to two regions spanning the acidic aa 41 to 46 and the basic aa 146 to 150 [26,252,334].
In this interaction, ActA is thought to mimic the activity of proteins of the WASP family, which
are natural ligands and activators of Arp2/3 [26]. Arp2/3 can also bind to the side of pre-existing
actin filaments and initiate a new filament at this point, creating branched structures found through-
out the comet tails associated with moving L. monocytogenes [327]. In summary, current data point
to a central role of the Arp2/3 complex in initiating actin-based listerial intracellular movement.
However, a recent study showed that ActA can also generate new actin structures in an Arp2/3-
independent but VASP-dependent manner [94]. This clearly shows that a complex protein like ActA
can interfere with host cell actin polymerization machinery in multiple ways.
The phosphoprotein VASP binds directly to the proline-rich repeats of ActA [44] via its
Ena/VASP-homology domain [238]. On the other hand, VASP is a natural ligand of profilin [263]
and could stimulate actin assembly by binding to ActA and enhancing profilin concentration in
the vicinity of the bacterium. In this respect it is important to note that ActA can interact
simultaneously with four Ena/VASP homology domains [207] and thus recruit at least four
profilins to the bacterium. Mena, which is closely related to VASP, also binds ActA and profilin
directly and might function in concert with VASP to recruit profilin–actin complexes to the site
of actin polymerization [113].
However, profilin is dispensable, at least in vitro, because profilin-depleted cytoplasmic extracts
still supported actin assembly and bacterial movement [209]. ActA can bind G-actin with a region
in its N-terminal part, but deletion of this region does not interfere with actin tail formation in
infected cells [297]. It is believed that, inside cells, the VASP-mediated profilin/G-actin recruitment
can bypass defects in actin binding of ActA [296]. Another function recently attributed to Ena/VASP
proteins is control of temporal and spatial persistence of bacterial actin-based motility [9]. Further-
more, purified VASP binds to F-actin [189] and enhances actin-nucleating activity of wild-type
ActA and the Arp2/3 complex while also reducing frequency of actin branch formation. The ability
of VASP to contribute to actin filament nucleation and to regulate actin filament architecture
highlights the central role of VASP in actin-based motility [279,296].
The 92-kDa ActA protein on the bacterial surface is cleaved by the listerial metalloprotease
Mpl, resulting in a major 72-kDa degradation product and, depending on the Listeria strains tested,
additional smaller degradation products [121,237]. These products are found on the bacterial surface
or in the supernatant liquid as 65- and 30-kDa fragments [237]. ActA is also degraded inside the
cytoplasm of the infected host cell [226]. However, this type of degradation seems to be mediated
by the proteasome of the host cell because it can be blocked by proteasome inhibitors. Additionally,
ActA becomes phosphorylated inside the host cell as shown by the presence of three distinct forms
of the protein with slightly different motilities in SDS-PAGE found in infected cells [33]. A
genetically engineered ActA variant, which is fully functional but lacks the C-terminal region, is
no longer phosphorylated inside host cells, suggesting that phosphorylation may not be necessary
for movement [186]. The roles of Mpl- or proteasome-mediated ActA degradation as well as the
phosphorylation inside the host cell are currently not understood, but one could imagine that
different variants of ActA could interact differently with host cell proteins regulating the dynamics
of the F-actin cytoskeleton.
Expression of ActA is controlled by PrfA (discussed later) as are the other genes of the virulence
gene cluster. However, different virulence factors are required at different steps of the intracellular
life cycle and in different quantities. These necessities are reflected by complex regulatory networks
governing expression of the virulence genes; this is far from being completely understood. ActA
expression is maximally induced when bacteria have reached host cell cytoplasm and the expression
level is increased more than 200-fold in cytosolic bacteria in comparison to broth-grown cultures
[291]. ActA expression in intracellularly located bacteria was analyzed by reporter gene fusions
(β-galactosidase, gfp, or β-glucuronidase) to actA [36,95,217,291] or by direct measurement of
actA-specific transcripts [36]. In all these studies, the principle finding was identical, showing a
marked induction of ActA expression in intracytoplasmically located L. monocytogenes.
L. monocytogenes can spread from cell to cell without leaving the cytoplasm by forming
microvilli-like protrusions on the host cell surface that are phagocytosed by neighboring cells
[229,315]. The mechanisms of microvilli formation and induction of phagocytosis by the neigh-
boring cell are largely unknown. Listeria randomly move through the cytoplasm; bacteria are finally
propelled into the host’s distended plasma membrane and long protrusions are formed (Figure 5.1).
In a monolayer of cells, the protrusions enter neighboring cells; they are subsequently taken up
and a secondary vacuole with a double membrane is formed. This secondary vacuole is disrupted,
which requires the action of LLO and the listerial phospholipase PC-PLC. Electron micrographs
of plcB mutants inside mammalian cells [319] show numerous bacteria followed by actin tails and
trapped in vacuoles surrounded by double membranes, indicating that plcB mutants are unable to
lyse the double membrane of the vacuole.
Careful examination of the plaque formation capacity of different mutants, which is thought
to be a good correlate for intercellular spread, revealed that in addition to the broad spectrum
phospholipase PC-PLC, PI-PLC and the metalloprotease contribute to plaque formation, most likely
by supporting lysis of the double membrane vacuole [214,299]. The listerial metalloprotease Mpl
probably supports this lysis by proteolytic activation of PC-PLC, but host cell proteases may also
cleave and hence activate PC-PLC in the absence of Mpl [214]. The pivotal role of LLO in escape
from the secondary vacuole was shown in an elegant study using an L. monocytogenes hly mutant
coated with recombinant LLO, which allowed bacteria to escape from the primary vacuole and
spread into neighboring cells once. However, dilution of the recombinant LLO through bacterial
replication inhibited further spread of bacteria [110].
The predominant role of LLO in lysis of the secondary vacuole and hence in cell-to-cell spread
was confirmed by microinjection of hly mutants into the cytosol [122] and use of strains allowing
temporal control of LLO expression [61]. Listerial factors other than ActA, LLO, Mpl, and PC-
PLC are not known to contribute to intercellular spread. The timeline and mechanics of cell-to-
cell spread were analyzed in some detail by Robbins et al. [270], who presented a model for this
process. Upon membrane contact, bacteria continue to move and form the protrusion. The fitful
bacterial movement stops after several minutes during which the protrusion is formed. After about
20 min, the protrusion suddenly collapses and the double membrane vacuole is formed and rapidly
acidifies to activate LLO and PC-PLC. Upon lysis of the membrane, motility recovers after one to
two bacterial generations.
BILE SALT HYDROLASE—A NOVEL VIRULENCE FACTOR

Comparison of L. monocytogenes and L. innocua genomes [117] has revealed the presence of an
L. monocytogenes-specific putative gene, termed bsh, encoding a bile salt hydrolase (BSH) [86].
Bile salts are end products of cholesterol metabolism in the liver; they are stored in the gall bladder
and released into the duodenum, helping fat digestion. In addition, bile salts are known to have
antimicrobiol activity because they are amphipathic molecules that can attack and degrade lipid
membranes. Some intestinal microorganisms have hence evolved mechanisms to resist the detergent
action of bile, including synthesis of porins, efflux pumps, and transport proteins [134]. Others
produce bile salt hydrolases that transform and inactivate bile salts. Deletion of the bsh gene
from the L. monocytogenes chromosome results in increased bile sensitivity, reduced virulence,
and reduced liver colonization after infection of mice. This demonstrates that BSH is a novel
L. monocytogenes virulence factor involved in the intestinal and hepatic phases of listeriosis.
In addition, the bsh gene is positively regulated by the central listerial virulence regulator PrfA
(see later discussion), further demonstrating that it is a true virulence factor.
ACCESSORY VIRULENCE FACTORS

The listerial proteins reviewed so far are true virulence factors because their sole function is
specifically connected to colonization of the vertebrate host; they are found exclusively in patho-
genic Listeria species. Other listerial factors involved in virulence have been identified that also
contribute to successful colonization of the host. They have functions outside the pathogenic lifestyle
and are present in nonpathogenic Listeria species. Such factors are sometimes called accessory
virulence factors.
P60, PRODUCT OF THE IAP GENE

Protein p60 is a major protein secreted by all L. monocytogenes isolates [34,182]. It is also found
on the cell surface of the bacteria [278]. Because it possesses murein hydrolase activity that appears
to be involved in a late step of cell division, p60 is an important enzyme for cell metabolism of L.
monocytogenes cells [333]. The gene for this protein, initially called iap (invasion associated
protein), codes for an extremely basic protein of 484 amino acids, with a 27-amino-acid signal
sequence and an extended repeat domain consisting of 19 threonine-asparagine units separated by
a proline–serine–lysine motif. A single cysteine found in the C-terminal part of p60 is probably
essential for its enzymatic activity [171,333] and amino acids in the N-terminal region of the protein
define its intracellular stability [295]. p60 is a member of a protein family in L. monocytogenes
with three members: p45, which is a peptidoglycan lytic protein [283] encoded by the spl gene;
the putative protein encoded by lmo394 [117]; and p60.
At least one type of rough mutant of L. monocytogenes characterized by expression of reduced
amounts of p60 shows significantly reduced uptake by 3T6 fibroblast cells [182]. These mutants
form long cell chains that possess double septa between the individual cells. Treatment of L.
monocytogenes rough mutants with partially purified p60 protein disaggregates the cell chains to
normally sized single bacteria, which again become invasive for fibroblasts. Ultrasonication leads
to physical disruption of the cell chains, producing similar single cells that are noninvasive. The
reduced invasiveness of the p60 mutants is only observed with certain mammalian host cells. Cell
chains of p60 mutants adhere normally to Caco-2 epithelial cells and are perfectly invasive for
these cells upon disruption of bacterial cell chains by ultrasonication without addition of p60 [34].
Other rough mutants of L. monocytogenes that show normal or even increased levels of p60
expression have been isolated [277]. These mutants are adherent and invasive like WT bacteria
despite formation of long filaments. The genetic basics of these phenotypes are unknown.
p60 was long regarded as an essential protein [333]. However, viable mutants with in-frame
deletions in the iap gene recently demonstrated that p60 is not an essential protein for L. monocy-
togenes [199,249]. Detailed characterization of one of the deletion mutants showed that the mutant
forms cell chains but is nearly as invasive as the WT strain. However, the mutant grows in
microcolonies inside host cells and does not form F-actin tails; it only induces actin clouds around
the bacteria. A defect in polar ActA distribution caused by impaired cell division is the reason for
lack of intracellular motility in this p60-lacking strain. According to these findings, p60, renamed
CwhA for cell wall hydrolase A, seems not to be linked directly to cell invasion; rather, it indirectly
modifies bacterial behavior via its impact on cell division [249]. Another L. monocytogenes pepti-
doglycan hydrolase, called MurA or p66, was recently identified [43]. Deletion of the gene encoding
MurA, which shows homology with p60 in its C-terminal domain, also results in the formation of
long cell chains. It is currently believed that p60 and p66 may act in concert to control proper cell
separation during the last step of bacterial division [43].
Two recent papers by Portnoy and coworkers [199,200] also shed light on genetic and bio-
chemical bases of reduced p60 expression and the rough phenotype. They described an additional
secA gene in L. monocytogenes, called secA2, located immediately upstream of the iap gene and
involved in the smooth–rough transition. They found that rough mutants with reduced p60 expres-
sion have a deletion in the secA2 gene or express truncated SecA2 proteins. Furthermore, deletion
of the secA2 gene resulted in reduced p60 expression and conversion to the rough phenotype. Rough
mutants displaying normal p60 levels are not affected in their secA2 gene. A number of additional
proteins, the secretion of which is secA2 dependent, have been identified [199]; it has been shown
that the hydrolytic activity of p60 is crucial for L. monocytogenes virulence through generation of
glucosaminylmuramyl dipeptide, which modifies host inflammatory responses.
SUPEROXIDE DISMUTASE AND CATALASE

Possible roles of catalase and superoxide dismutase in the virulence of L. monocytogenes have
recently been reviewed [137,320]. Both enzymes act in concert to detoxify potentially harmful
superoxide radicals. Generated by the oxidative burst in a phagocytic cell, these radicals are
converted into hydrogen peroxide by action of superoxide dismutase, which is then cleaved by
catalase into water and molecular oxygen. Bacterial catalases and superoxide dismutases have long
been suspected to be important virulence factors of intracellular bacteria, but no correlation of
superoxide dismutase expression with virulence was found in L. monocytogenes [326]. The gene
for superoxide dismutase from L. monocytogenes [29], called lmsod, reveals an ORF coding for a
protein of 202 amino acids with high homology to manganese-containing superoxide dismutases
from other organisms.
Catalase mutants obtained by transposon mutagenesis show wild-type virulence in infected
mice [190]. Whereas catalase-negative L. monocytogenes mutants are killed by mouse resident
macrophages already at low serum concentrations, killing wild-type bacteria requires high serum
concentrations, suggesting that resistance to fully activated macrophages is partially mediated by
catalase activity [316]. Isolation of catalase-negative L. monocytogenes strains from listeriosis
patients supports the notion that catalase does not seem to be necessary for intracellular growth of
L. monocytogenes [35,88]. Also in line with these data is the finding that all species of Listeria
produce the same type of SOD, which is constitutively expressed and not regulated by environmental
factors [317], as true virulence factors are.
STRESS RESPONSE MEDIATORS

Bacterial survival under stress conditions requires an adaptive response mediated by a set of
conserved proteins that are upregulated upon exposure to many different stress conditions and when
bacterial growth is restricted. However, in contrast to other facultative intracellular bacteria, L.
monocytogenes does not induce expression of general stress proteins during intracellular prolifer-
ation in macrophages [139]. Nevertheless, stressful conditions are likely to be encountered by L.
monocytogenes during transient residence in the phagosome upon uptake by macrophages. In that
stage, expression of the hly gene encoding LLO is induced as it is under other stress conditions
like heat treatment [303–305].
A group of virulence-associated stress mediators involved in phagosomal escape and intracel-
lular multiplication were identified in L. monocytogenes. The first was ClpC, an ATPase belonging
to the heat-shock protein 1000 family [282]. The gene encoding the ClpC ATPase, called clpC,
was identified by Tn917 mutagenesis and a selection for mutants depending on iron [274]. The
clpC mutants are highly susceptible to stress, including iron limitation, elevated temperatures, and
high osmolarity. Virulence of these mutants is severely impaired in the mouse with restricted
capacity to grow in bone marrow-derived macrophages [275,276]. Electron microscopy of infected
macrophages showed that clpC mutants remained inside the phagocytic vacuole, indicating that
ClpC is involved in phagosomal lysis or helps the bacteria to survive in the harsh conditions of
this environment to allow production of sufficient amounts of LLO and PI-PLC to destroy the
vacuolar membrane [275]. Expression of ClpC is not directly controlled by PrfA and no PrfA-
boxes (discussed later) are found in the promoter region of the clpC gene. In contrast, overexpression
of the PrfA regulon leads to a loss of ClpC expression, showing that expressional cross-talk between
PrfA and ClpC is most likely mediated by a negative regulator activated by PrfA [269]. Recent
evidence also points to a role for ClpC in expression of internalin, InlB and ActA, because ClpC
is required for efficient uptake into several cell lines [235].
ClpE is closely related to ClpC and is also involved in virulence of L. monocytogenes [234].
L. monocytogenes mutants with deletions in the clpC and clpE genes are totally avirulent in mice;
it is believed that both proteins have redundant functions in stress tolerance because the absence
of one is compensated by the transcriptional upregulation of the other [234].
ClpP is a stress-induced L. monocytogenes serine protease of 22 kDa belonging to a highly
conserved protein family. ClpP is required for growth under stress conditions and for survival in
macrophages and infected animals [107]. ClpP seems to be involved in stress-induced upregulation
of LLO expression because clpP-mutants secrete only small amounts of functional LLO [106].
Expression of ClpC, ClpE, and ClpP is negatively controlled by ctsR, the first gene of the
operon containing ClpC [233]. L. monocytogenes CtsR is homologous to the B. subtilis CtsR
repressor of stress response genes. Consistent with the function of CtsR as a repressor is the fact
that a ctsR deletion mutant showed enhanced survival under stress conditions and normal virulence,
whereas overproduction of CtsR resulted in a significantly attenuated virulence in the mouse model
of infection [233].
IRON UPTAKE SYSTEMS

Iron is a key element for all bacteria, serving as a cofactor in many proteins involved in electron
transport processes. Inside the host organism, however, iron is not freely available because it is
tightly bound to transferrin or ferritin. Pathogenic bacteria therefore had to evolve specific mech-
anisms to capture iron from host tissues and these uptake mechanisms play an important role in
virulence. In L. monocytogenes, different iron-uptake mechanisms have been described [32]: The
first uses the direct transport of ferric citrate [2] and the second involves an extracellular ferric-
iron reductase that uses iron-loaded catecholamines as a substrate [12,57,67]; the third system may
use a surface-located transferrin-binding protein [140]. Unlike many other pathogenic bacteria, L.
monocytogenes does not secrete siderophores to capture extracellular iron [58]. The genetic basis
of different iron uptake systems has not been studied and there is no experimental evidence whether
these systems directly contribute to pathogenesis of L. monocytogenes infection.
PRFA AND REGULATION OF VIRULENCE GENE EXPRESSION

IN L. MONOCYTOGENES
THE POSITIVE REGULATORY FACTOR A (PRFA)
The first indications for coordinate regulation of virulence genes in L. monocytogenes by a trans
acting factor were obtained from analysis of spontaneously occurring nonhemolytic mutants of L.
monocytogenes, which carry deletions in a region upstream of the hly gene [126,197]. Cloning and
sequencing of the locus affected by the deletion led to identification of the prfA (positive regulatory
factor A) gene. Its product, PrfA, a cytoplasmic protein of 27 kDa [198,217], regulates all virulence
genes of the virulence gene cluster. The prfA deletion mutants can be complemented in trans by
introduction of the cloned prfA gene to yield a wild-type phenotype again [198]. Site-specific
mutations or transposon insertions in the prfA promoter or the prfA coding region block transcription
of the entire gene cluster, i.e., plcA, hly, mpl, actA, and plcB [47,217]. This indicates that the prfA
gene encodes a transcriptional activator required for expression of the L. monocytogenes virulence
gene cluster. Additional evidence for this presumptive role of PrfA was provided by transcriptional
activation of the cloned hly gene by PrfA in B. subtilis [98].
Also present in the closely related species L. ivanovii and L. seeligeri are prfA-like genes with
high sequence similarity to prfA from L. monocytogenes [178,185]. In L. seeligeri the prfA gene
is silenced [178] and activity of the protein is not known; however, PrfA from L. ivanovii also
controls sets of virulence genes similar to those of L. monocytogenes [185]. PrfA is a member of
the Crp/Fnr family of transcriptional activators, which have been identified so far mainly in Gram-
negative bacteria. Like all members of this family, PrfA contains a conserved helix–turn–helix
motif in its C-terminal part. In addition, adjacent to this motif, PrfA carries a sequence with features
of a leucine zipper and a second helix–turn–helix motif at its N-terminus [30,185,288] (Figure 5.7).
In hyperhemolytic strains of L. monocytogenes, an altered PrfA, termed PrfA*, has been
identified and characterized and it was shown that all these PrfA variants carry a Gly145Ser
FIGURE 5.7 Structure of PrfA (A) and its target DNA sequences in PrfA-regulated promoters (B). In PrfA,
amino acid coordinates correspond to the peptide sequence deduced from the prfA gene (position 1 corresponds
to the Met residue of the first triplet), whereas in Crp they correspond to the actual amino acid position in
the primary structure of the native protein, which lacks the residue encoded by the first triplet. Therefore, the
amino acid numbering of Crp is shifted one position with respect to that of PrfA and hence position 144 of
Crp, where the Crp* mutation Ala144Thr lies, aligns exactly with position 145 of PrfA, where the Gly145Ser
substitution leading to the PrfA* mutant phenotype is located. In Crp, A to F designate the α-helical stretches
of the protein and AR1 is activating region 1 (two other activating regions [AR2 and AR3] are embedded
within the β-roll structure). In PrfA, structures specific for this protein are in light gray. (B) N indicates any
nucleotide. (Reprinted with permission from Vazquez-Boland, J. A. et al. 2001. Clin. Microbiol. Rev. 14:584–640.)
substitution [23,267]. Furthermore, all genes of the virulence gene cluster are highly expressed in
these strains and do not respond to conditions under which expression of virulence genes is induced
in wild-type strains [267]. Interestingly, Crp mutants are known that carry a mutation at similar
positions and lead to a transcriptionally active conformation without the need of the cofactor cAMP
(Figure 5.7). This suggests that wild-type PrfA also needs a conformational change for activity and
that the Gly145Ser substitution results in a constitutively active form of PrfA [321]. Three other
mutations in PrfA were recently reported to increase PrfA function: a Gly155Ser mutation that
appears to be similar in nature to Gly145Ser and a Glu77Lys substitution located in the β-roll
structure in the N-terminal part of the protein [292]. A Ser183Ala substitution located in the second
HTH motif that also resulted in increased PrfA activity was described by Sheehan et al. [288].
On the other hand, it was shown that inactive PrfA can be present in large quantities in L.
monocytogenes [266]. In addition, PrfA mutants were selected with single point mutations rendering
the protein partially or totally inactive [144]. These mutants were unable to bind to DNA and
harbored mutations in the leucin zipper or the HTH motif or they still bound to the target DNA
but were unable to form a stable complex with RNA polymerase and were located in the β-roll
structure. Other studies suggested that PrfA-mediated activation of gene expression requires
presence of a coactivator protein [22, 69] the nature of which is still unknown.
PRFA-DEPENDENT PROMOTERS, TRANSCRIPTS, AND MECHANISM

OF TEMPERATURE-DEPENDENT VIRULENCE GENE EXPRESSION
A 14-bp palindromic sequence first identified in the promoter region of the hly gene [221] was
found to be present in promoters of all PrfA-dependent genes, located about 40 bp upstream of the
transcriptional start site. However, the 14-bp palindrome, called PrfA-box, is not perfectly conserved
in all promoters and the differences could contribute to differential regulation of the adjacent genes
by PrfA (Figure 5.7) [30,176,289,331]. Meanwhile, it was shown that PrfA directly binds to these
palindromic sequences [22,24,69,288], thereby activating the virulence genes. Purified PrfA alone
is able to bind specifically to the target sequence as shown by gel motility shift assays. However,
addition of PrfA-free cell extracts results in formation of additional PrfA-containing DNA-binding
complexes that also contain RNA polymerase [24,69].
Whether PrfA first binds to the PrfA-box and enhances binding activity for RNA polymerase
or whether the PrfA/RNA polymerase complex may already form before interaction with DNA is
still unknown. The observation that PrfA can exist in an inactive form and the striking similarity
of the cAMP-independent mutant Crp* and the constitutively active PrfA* strongly suggest involve-
ment of a cofactor in regulation of PrfA activity. Despite extensive investigations, such a cofactor
remains unknown [178].
The transcriptional organization of most PrfA-regulated genes—especially the virulence gene
cluster—is complex. Three transcripts of the prfA gene have been identified: a long (2.1 kb)
transcript cotranscribed with the plcA gene and autoregulated by PrfA, and two shorter ones (0.8 and
0.9 kb) transcribed from three distinct promoters located in front of the prfA gene [97]. The
listeriolysin gene, hly, is the only gene in the virulence gene cluster transcribed in a monocistronic
mRNA from two PrfA-dependent promoters, P1 and P2, located in the intragenic region between
hly and plcA [221]. A third hly promoter, P3, downstream from P1 and P2, was recently identified
and shown to be PrfA independent; it results in low-level transcription of the hly gene [73].
The three genes of the lecithinase operon are transcribed from at least two PrfA-regulated
promoters: One, located in front of the mpl gene, yields a 5.4- to 5.7-kb transcript comprising mpl,
actA, and plcB, as well as an additional mRNA of 1.8 kb comprising mpl alone [24]. A second
promoter, located directly in front of the actA gene, leads to a 3.6-kb bicistronic transcript com-
prising actA and plcB [24]. The inlAB operon is transcribed from multiple PrfA-dependent and
-independent start sites upstream of inlA. In different studies, three or four start sites were mapped
in the promoter region upstream of inlA that harbors a degenerated PrfA-box with two mismatches
(Figure 5.7) [79,203]. In addition, an inlB monocistron is detectable and a putative transcription
terminator is located between inlA and inlB. Overall, transcriptional control of inlAB expression is
complex and not fully understood.
Full expression of PrfA-dependent virulence genes requires synthesis of monocistronic prfA
transcripts and the bicistronic plcA-prfA transcript [96,42]. In an initial step of the infectious process,
it is believed that transcription of prfA via the prfA promoters results in synthesis of a limited
amount of PrfA sufficient to activate the high-affinity PrfA-dependent hly and plcA promoters.
This, in turn, would result in synthesis of the plcA-prfA transcript, which leads to enhanced PrfA
synthesis. The higher cellular level of PrfA activates the mpl and actA promoters, which seem to
have a lower affinity for PrfA because of base mismatches in their palindromic PrfA boxes [53].
As in many other instances [149,175], PrfA-dependent virulence gene expression in L. mono-
cytogenes is thermoregulated and the shift from 30 to 37°C results in a dramatic increase in
expression of virulence genes [196,217]. Detailed recent analysis of this phenomenon culminated
in identification of an RNA thermosensor controlling temperature-dependent gene expression [155].
Low expression of virulence genes at temperatures below 30°C coincides with absence of PrfA
protein. However, quite surprisingly, the prfA gene is still transcribed under these conditions from
its own promoter, resulting in a monocistronic prfA transcript [196,266]. At 37°C, prfA is transcribed
from the prfA promoter and the PrfA-dependent plcA promoter, resulting in monocistronic and
bicistronic plcA-prfA messengers [196,217,266].
Johannson et al. [155] could now demonstrate that, at 30°C, the monocistronic prfA messenger
is not translated because the upstream untranslated mRNA (UTR) preceding prfA forms a secondary
structure that masks the ribosome-binding region. This secondary structure is thermosensitive and
hence unstable at higher temperatures (37°C), which then allow efficient translation resulting in an
approximately fivefold increase in PrfA levels. As mentioned earlier, in the presence of PrfA, the
bicistronic plcA-prfA messenger is transcribed from the PrfA-dependent promoter upstream of plcA,
further increasing the amount of PrfA in the bacterial cell and finally allowing virulence gene
expression from low-affinity promoters. L. monocytogenes has hence evolved a sophisticated two-
step mechanism to control PrfA-dependent virulence gene expression (reviewed in Johansson and
Cossart [154] and Newman and Weiner [236]).
Before completion of the genomic sequence of L. monocytogenes [117], knowledge of the PrfA
regulon was very limited. At that time, the only known genes that did not belong to the virulence
gene cluster but were also regulated by PrfA were inlAB, [79] and inlC [75,89]. The availability
to sequence the complete genome of L. monocytogenes allowed in silico screening of the sequence
for genes preceded by putative PrfA-boxes. This screen identified four additional previously
unknown genes harboring PrfA-boxes. One of the genes, later called hpt, encodes for a putative
hexose permease, expression of which was shown to depend strictly on PrfA [48]. The other three
genes preceded by PrfA-boxes are genes of unknown function.
A recent systematic approach to elucidate the complete PrfA regulon used a whole genome
macroarray based on the complete genome sequence of L. monocytogenes [223]. With this mac-
roarray, expression profiles of the wild-type strain and a prfA-deletion mutant were compared upon
growing the bacteria in standard BHI medium or in media known to induce or to repress virulence
gene expression. With this approach, three groups of differently regulated genes were identified.
In addition to the 10 already known PrfA-regulated genes, Group I comprises 2 new genes, positively
regulated and preceded by a PrfA box. Group II comprises eight negatively regulated genes: One
is preceded by a PrfA box and the others form an operon. Group III comprises 53 genes of which
only 2 are preceded by a PrfA box and that are activated or repressed under different conditions;
most of the genes in this group are transcribed from sigma B-dependent promoters. Taken together,
the results suggest that PrfA positively regulates a core set of 12 genes preceded by a PrfA box
that is probably expressed from a sigma A-dependent promoter and negatively regulates 8 genes.
A second set of PrfA-regulated genes lacks PrfA boxes and is expressed from sigma B-dependent
promoters. The data presented reveal that PrfA can act as an activator or a repressor and suggest
that PrfA may directly or indirectly activate sets of genes in association with different sigma factors
[223]. The important role of sigma B in expression of L. monocytogenes virulence genes was also
confirmed by recent studies [165,308] showing that sigma B regulates not only stress response
genes but also several well-known virulence genes like those encoding the internalins and the bile
salt hydrolase.
ENVIRONMENTAL SIGNALS AFFECTING VIRULENCE GENE EXPRESSION

Pathogenic bacteria that can also live in the free environment are forced to sense their surroundings in
order to regulate expression of genes needed for living inside or outside their host. Facultative intracellular
pathogens additionally should be able to know whether they are inside or outside their individual
mammalian host cell.
An increasing number of signals have been shown to affect virulence gene expression in L.
monocytogenes (reviewed in Brehm et al. [30] and Kreft and Vazquez-Boland [178]). The signals
can be classified into physicochemical signals (temperature, iron, glucose, cellobiose, salt, pH,
activated charcoal) or stress conditions (heat shock, oxidative stress, nutritional stress, growth inside
host cells). The mechanisms of altered gene expression under such conditions are unknown or only
poorly understood. However, in all systems analyzed, PrfA plays a role in regulation of environ-
mentally modulated gene expression.
At temperatures below 30°C, the PrfA-dependent genes are not transcribed because of a
lack of translation of the monocistronic prfA transcript (see earlier discussion) [155]. A shift
in temperature to 37°C results in onset of prfA expression followed by transcription of the
virulence cluster genes [79,196]. Treatment of culture medium with activated charcoal probably
depletes a signal molecule from the medium; this would result in increased transcription of
prfA and the PrfA-dependent genes [268]. Carbohydrates modulate virulence gene expression
in a complex and poorly understood manner. Glucose directly influences prfA gene expression
and thereby interferes with PrfA regulation. Addition of larger amounts of glucose to the
medium results in acidification, which reduces LLO expression by unknown mechanisms
[62,97]. The disaccharide cellobiose results in inhibition of hly and plcA expression without
reduction in monocistronic prfA mRNA levels and with only slightly decreased amounts of
PrfA protein. This indicates that the inhibitory mechanism exerted by cellobiose leads to a
change in PrfA activity [16,166,245].
Two genomic loci have been identified [31,148] that contribute to cellobiose-mediated virulence
gene repression; one of these, the bvrABC system, most likely encodes for a sensor system for
extracellular cellobiose [31]. The mechanisms of stress-mediated altered gene expression are even
less understood. Heat-shock conditions increase hly, plcA, and actA expression. P60 expression is
inhibited by heat shock and also by oxidative stress (H2O2) [303–305]. Shift of L. monocytogenes
from a rich medium into a minimal essential medium (MEM) induces expression of the virulence
cluster genes as well as other surface-associated proteins [268].
Phagocytosis and intracellular localization are supposed to be natural stress factors. It has been
shown that expression of numerous proteins is selectively induced during phagocytosis of L.
monocytogenes by macrophages [139]. An insertional mutagenesis study [166] and two IVET
studies [84,101] tried to identify genes preferentially expressed inside mammalian cells using the
hly gene as a reporter. The first study resulted in identification of genes involved in nucleotide
biosynthesis, an arginine transporter, and plcA [166]; the IVET studies only identified some of the
known virulence genes and some unknown genes whose function is unknown in the intracellular
life cycle. Experiments directly measuring bacterial mRNA levels inside host cells by RT-PCR or
gfp-reporter gene assays revealed that the genes hly, actA, and inlC are heavily expressed inside
the mammalian cell [24,36,89,95,291]. It was also shown that PrfA is upregulated during interaction
of L. monocytogenes with host cells [265]. The complexity of regulation of gene expression inside
the host cell is, despite all progress, far from understood.
TWO-COMPONENT SYSTEMS AND REGULATION OF VIRULENCE GENE EXPRESSION

Signal transduction mechanisms allowing bacteria to modulate gene expression in response to
diverse stimuli often involve two-component systems (TCSs) composed of a sensor kinase and a
response regulator [132]. The sensor kinase is often localized in the cytoplasmic membrane to sense
outside signals and contains a highly conserved cytoplasmic kinase domain which, upon activation,
phosphorylates the cognate response regulator. The phosphorylated response regulator then binds
to specific DNA sequences and activates transcription of its target genes.
TCSs have not been studied intensively in L. monocytogenes; lisR/K [52], cheY/A [93], agrA/C
[10], and cesRK [161] were characterized in some detail and shown to contribute to virulence of
L. monocytogenes. Kallipolitis et al. [160] identified a total of seven putative TCSs in a PCR-based
approach including the already known lisR/K. Availability of the complete genomic sequence of
L. monocytogenes strain EGD-e allowed the in silico identification of 16 putative two-component
systems in addition to a large array of other putative regulatory systems belonging to different
families. The largest of these are GntR-like regulators and BglG-like antiterminators, many of which
are associated with PTS. Surprisingly, only 1 of 16 putative TCSs identified had no counterpart in
the closely related but totally apathogenic species L. innocua. This points to a minor role of TCSs
for virulence gene regulation in L. monocytogenes [117].
To study the role of the listerial TCS for in vitro and in vivo survival and growth of L.
monocytogenes more systematically, in-frame deletion mutants in 15 of 16 TCSs were constructed
by introducing large deletions in the individual response regulator genes, removing most of the
open reading frames [330]. The mutants are currently characterized by testing them in various in
vitro growth assays and several cell culture assays and by assessing their virulence potential in
experimentally infected mice. At least one of the previously undescribed response regulators (degU)
(lmo2515) also contributes to virulence because the L. monocytogenes degU mutant is severely
impaired in colonizing experimentally infected mice [330].
LESSONS LEARNED FROM GENOME SEQUENCE OF L. MONOCYTOGENES

Availability of complete genomic sequences of L. monocytogenes and its close nonpathogenic relative
L. innocua [117] opens new possibilities for investigation of listerial virulence. Sequencing of the
2,944,528 base pairs of the circular chromosome of L. monocytogenes revealed the presence of 2,853
protein-coding genes, from which about 35% are without a predicted function. Comparison of this
sequence with that of L. innocua and the closely related species B. subtilis revealed some special features
of L. monocytogenes [37,117].
The first surprise was the presence of many putative surface proteins belonging to six families (19
internalins, 21 other LPXTG proteins, 9 GW module-containing proteins, 11 hydrophobic-tail proteins,
4 p60-like proteins, and up to 68 putative lipoproteins). The high number of related members in each
family seems to be—at least in part—because of extensive gene duplications. Of particular interest are
surface proteins characterized by the common LPXTG motif in their C-terminus necessary for covalent
linkage of the proteins to the cell wall peptidoglycan. L. monocytogenes harbors a total of 41
genes encoding for LPXTG proteins (more than any other bacterial species analyzed). These can
be further grouped into the family of internalins and internalin-like proteins (characterized by
the leucine-rich repeat motif) and the LPXTG proteins lacking this leucine-rich repeat. The
function of the vast majority of these genes is unknown, but one may speculate that internalins
and internalin-like proteins confer host-species and cell-type specificity during infection. They
may also mediate attachment of bacteria to other surfaces during their life outside the mammalian
host [38,117].
Another surprising feature of L. monocytogenes revealed by analysis of the genome was the
high number of transport proteins (331 in total), of which 88 are devoted to carbohydrate transport
by phosphoenolpyruvate-dependent phosphotransferase systems (PTS). Hence, L. monocytogenes
has nearly three times as many PTS systems as B. subtilis, which probably gives L. monocytogenes
the ability to take up, and therefore grow on, a large number of carbohydrates. As mentioned earlier,
one specific hexose-phosphate transporter is necessary for intracellular multiplication of L. mono-
cytogenes and further studies will most likely reveal the importance of other uptake systems for
adaptation to different environmental conditions. As anticipated, because of the wide range of
different conditions faced by L. monocytogenes during extracellular and intracellular growth, a high
number of transcriptional regulators (209 in total) have also been identified. This number is second
only to that of Pseudomonas aeruginosa, another ubiquitous opportunistic pathogen [117].
EVOLUTIONARY ASPECTS
Based on in vitro data, all Listeria species are regarded as normally noncompetent [25]. It was
therefore totally unexpected to find genes in L. monocytogenes and L. innocua coding for putative
DNA uptake systems homologous to B. subtilis competence genes [25,117]. The uptake apparatus
may no longer be functional, or its regulation or signals that induce competence may differ from
those of B. subtilis and correct conditions to induce competence have not yet been found during
laboratory culture. Nevertheless, the possibility of gene transfer by transformation could well
explain genomic differences between sequences of the two Listeria species characterized on a
genomic level to date.
These differences are mainly found in blocks dispersed around the chromosome, resulting in a
mosaic genome structure. Furthermore, the collinearity identified for L. monocytogenes and L.
innocua chromosomes (see Figure 1.1) also extends to numerous regions of the B. subtilis chromo-
some. The hundreds of insertions found in the three chromosomes are best explained by multiple
independent transformation events followed by DNA integration at various sites in the chromosomes.
The origin of the known virulence genes in Listeria is still unclear. The large family of
internalins seems to have evolved in Listeria after initial combination of the LPXTG membrane
anchor motif with a leucine-rich repeat motif (both of unknown origin) to form a proto-internalin.
This then (probably) duplicated several times and evolved further by recombinations and by point
mutations. The virulence gene cluster was obviously acquired a long time ago because traces of
the transfer event are barely detectable, or it evolved in Listeria. Of the individual genes present
in the virulence gene cluster, homologues of the listeriolysin gene [242], the metalloprotease gene
[72,218], and the two phospholipase genes [195, 215, 319] are present in many related species but
never found in a cluster as in L. monocytogenes. The origin of the actA gene is unknown because
no bacterial protein with significant homology has yet been isolated.
It has been speculated, however, that the actA gene may be of eukaryotic origin because parts
of the protein show some homology to eukaryotic cytoskeletal proteins [45]. At present, fully
functional virulence clusters are found in L. monocytogenes and in the animal pathogen L. ivanovii;
a similar cluster with additional genes is present in the nonpathogenic species L. seeligeri
(Figure 5.2) [45,127]. Interestingly, one of the ORFs with unknown function at the right border of
the cluster shows some weak homology to genes of Listeria phages [45,204]. This might point to
phage transduction events involved in early evolution of this gene cluster.
Analysis of the sequences flanking the virulence gene cluster in different Listeria species indicates
that it is ancestral to the genus Listeria because it is present in all isolates exactly at the same
chromosomal position [45,46,179]. Most likely, the virulence gene cluster was lost by two independent
events from the nonpathogenic species L. innocua and L. welshimeri as indicated by the presence of
short DNA sequences believed to belong originally to the virulence gene cluster [179]. The situation
in L. grayi is still unknown, but current sequencing efforts [116] will soon resolve the situation in
this more distantly related species. Availability in the near future of genome sequences of L. ivanovii,
L. welshimeri, L. grayi, and L. seeligeri—in addition to those of L. monocytogenes and L. innocua
(thus, the complete genome sequences of the whole Listeria genus) [46,116]—and their comparison
will certainly greatly enlarge understanding of the evolution of the genus Listeria.
OPEN QUESTIONS
The last years have seen an enormous increase in understanding of the molecular basis of infectious
diseases. Knowledge of the genes determining virulence of L. monocytogenes and the role played
by virulence gene products in the infectious process is rapidly expanding. However, many problems
concerning the virulence of L. monocytogenes remain unsolved.
For instance, L. ivanovii, a species largely apathogenic for humans [287], resembles L. mono-
cytogenes in its intracellular life cycle [162]. Genes homologous to most of the known virulence
genes of L. monocytogenes are also detectable in this species [127,136,177] and the complete PrfA-
regulated gene cluster identified in L. monocytogenes is also present in L. ivanovii [127]. However,
L. ivanovii is virulent for animals and avirulent for humans; an experimental infection in mice
yielded a different outcome than that of L. monocytogenes [147]. What is the molecular explanation
for this obvious difference in the pathogenic potential of these two Listeria species? Is it the result
of a different mechanism in regulation of known virulence genes inside the infected cells or
differences in specific activity of the known virulence gene products? What is the role of the many
small internalins present in L. ivanovii but absent in L. monocytogenes? The availability of the
genomic sequence of L. ivanovii in the near future [116] will probably help to answer at least some
of these questions.
Expression of L. monocytogenes virulence genes inside infected mammalian host cells and
tissues is another important but unsolved problem. The expression pattern of known L. monocyto-
genes virulence determinants is complex under in vitro growth conditions and regulated by PrfA-
dependent and PrfA-independent mechanisms. Very little is known of how PrfA and other putative
regulatory factors control these genes while the bacteria reside inside host cells and tissues.
Application of available high-throughput methods like macro- and microarrays to measure gene
expression on the mRNA level [223] or two-dimensional gel electrophoresis on the protein level
[257] of many genes in parallel under different conditions will certainly dramatically increase
understanding of gene expression regulation of L. monocytogenes. These data, together with others,
will greatly add to understanding of how this fascinating human pathogen thrives under very
different conditions inside and outside the human host.
ACKNOWLEDGMENTS
We thank T. Williams and J. Kreft for carefully and critically reading this manuscript. We apologize
to all those who contributed to our current knowledge on the biology of L. monocytogenes but were
not mentioned in this chapter. Work from this group was supported by the Deutsche Forschungs-
gemeinschaft through the grants SFB 479-B1 (WG) and SFB 479-B5 (MK), the European Union
through the grants BMH4-CT96-0659, BIO4-CT98-0036, and QLG2-CT1999-00932 (WG), and
the Fonds der Chemischen Industrie (WG).
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6 Characteristics of Listeria
monocytogenes Important
to Food Processors
Beatrice H. Lado and Ahmed E. Yousef
CONTENTS
Introduction ....................................................................................................................................158
Temperature....................................................................................................................................159
Growth Temperature.............................................................................................................159
Range and Optimum ................................................................................................159
Growth Kinetics........................................................................................................160
Cold Tolerance..........................................................................................................161
Stress Adaptation at Elevated Sublethal Temperatures............................................163
Variations in Virulence with Temperature ...............................................................163
Freezing ................................................................................................................................163
Lethal Temperature...............................................................................................................164
Mechanisms of Thermal Inactivation.......................................................................164
Kinetics of Thermal Inactivation..............................................................................165
Extrinsic Factors in Resistance to Heat ...................................................................167
Intrinsic Factors in Resistance to Heat ....................................................................167
Surrogate Microorganisms for Thermal Studies..................................................................168
Acidity ............................................................................................................................................169
Survival at Low pH ..............................................................................................................169
Inactivation at Low pH.............................................................................................169
Factors Influencing Acid Tolerance .....................................................................................170
Mechanisms of Acid Damage and Tolerance ......................................................................170
Consequences of Enhanced Acid Tolerance ........................................................................171
Water Activity ................................................................................................................................171
Water Activity and Growth or Survival ...............................................................................171
Osmotolerance Factors .........................................................................................................172
Antimicrobial Components in Food ..............................................................................................174
Salt ........................................................................................................................................174
Survival at Extreme Salt Concentrations .................................................................174
Physiology at High Salt Concentrations ..................................................................174
Organic Acids and Their Salts .............................................................................................175
Lactate.......................................................................................................................176
Sodium Diacetate......................................................................................................176
Sodium Propionate ...................................................................................................177
Potassium Sorbate ....................................................................................................177
157
Sodium Benzoate......................................................................................................178
Parabens and Other Benzoic Acid Derivatives ........................................................178
Fatty Acids and Related Compounds...................................................................................178
Free Fatty Acids .......................................................................................................178
Fatty Acid Monoesters .............................................................................................179
Sodium Nitrite ......................................................................................................................180
Antioxidants..........................................................................................................................180
Smoke ...................................................................................................................................181
Spices, Herbs, and Plant Extracts ........................................................................................181
Lysozyme..............................................................................................................................182
Hydrogen Peroxide...............................................................................................................183
Lactoperoxidase System.......................................................................................................183
Lactoferrin ............................................................................................................................184
Biocontrol.......................................................................................................................................185
Live Fermentate....................................................................................................................185
Bacteriocins ..........................................................................................................................185
Modified Atmosphere.....................................................................................................................186
Alternative Processing Technologies .............................................................................................187
Irradiation .............................................................................................................................187
Ionizing Radiations...................................................................................................187
Ultraviolet Radiation and High-Intensity Pulsed Light...........................................188
High-Pressure Processing.....................................................................................................189
Pulsed Electric Field Processing..........................................................................................189
Attachment and Biofilm Formation...............................................................................................190
Sanitizers ........................................................................................................................................192
Chlorine and Chlorinated Compounds.................................................................................193
Quaternary Ammonium Compounds ...................................................................................194
Acid Sanitizers......................................................................................................................194
Ozone....................................................................................................................................194
Miscellaneous Sanitizing Agents .........................................................................................195
Active Packaging............................................................................................................................196
Multiple Antimicrobial Treatments................................................................................................197
References ......................................................................................................................................198
INTRODUCTION
The goal of food processing is to produce a safe, wholesome product that has a suitable shelf life
and is acceptable to the consumer. Food manufacturers rely on a variety of processing and preser-
vation methods to reach this goal. These methods inactivate or inhibit growth of spoilage and
pathogenic microorganisms, suppress undesirable chemical and biochemical changes and hence
ensure food’s safety, and maintain its desirable physical and sensory properties. Methods currently
used in food preservation involve physical, chemical, or biological factors. Physical preservation
factors include heating, cooling, freezing, radiation, high-pressure processing, and packaging.
Chemical treatments include addition of antimicrobial agents (e.g., benzoate, propionate, and
sorbate), acidifying agents (e.g., acetic and lactic acids) or curing agents (e.g., sodium chloride and
sodium nitrite). Preservation by biological means (biopreservation) includes fermentations that
control spoilage and pathogenic microorganisms through competition for substrate, gradual lower-
ing of pH, and release of antimicrobial metabolites.
Success of a preservation technology depends on meeting the processing goal described earlier.
Heat is the most reliable and commonly used preservation factor, but thermal processing alters
Characteristics of Listeria monocytogenes Important to Food Processors 159
quality of food and decreases availability of nutrients. Alternative technologies, such as radiation
and high-pressure processing, may maintain the critical balance between food’s safety and its
quality. Similarly, treatment combinations are used in food processing and the value of using
multiple preservation factors is best expressed as the hurdle concept.
Growth, inhibition, or inactivation of Listeria monocytogenes in response to food-processing
and preservation techniques will be detailed in this chapter. To eliminate any ambiguities, some basic
concepts will be defined, and every effort will be made to use these terms uniformly in this chapter.
The expression “log” or “log count” refers to the microbial count in “log10 CFU/mL or g.” When
measurable increase in count is encountered, it is described as such, unless multiplication of the
microorganism is reported; in this instance, the increase will be described as “growth.” Growth of
microorganisms is enhanced when the lag phase decreases, generation time decreases (i.e., maximum
specific growth rate increases), or the gain in count attained after a given growth period increases.
Conversely, “growth inhibition” or simply “inhibition” is the condition when opposite change
in the growth parameters just described occurs. Growth inhibitors may also be described as
bacteriostatic and, for inhibition of Listeria, as listeriostatic agents; such agents do not usually
cause measurable inactivation. “Inactivation” refers to a decrease in cell population; it will be
expressed as a decrease in log count. An agent that causes microbial inactivation is described as
bactericidal, but may also be reported as listericidal when it is active against Listeria. The D-value
is the time of exposure to a lethal factor (e.g., heat or radiation) required to inactivate 90%
(i.e., one log) of the population of a given microorganism at a given dose of the deleterious factor.
D-value is a measure of resistance of the microorganism to the deleterious (lethal) factor; the larger
the D-value is, the greater is the resistance. When the count does not change appreciably, the status
of the microorganism is best described as survival. The word “survival” also refers to ability of the
microorganism to maintain its viability during the treatment.
Some generalizations and conclusions in this chapter should be viewed with caution. These are
based on published research using bacterial strains available to researchers at the time of the study.
Recent reports show that strains of L. monocytogenes vary considerably in resistance to processing and
new processing-resistant strains are occasionally discovered [190,310]. If highly resistant pathogenic
strains are discovered in the future, the conclusions in this chapter will need to be modified accordingly.
TEMPERATURE
The temperature to which food is exposed may have growth-conducive, preserving, or lethal effects
on microorganisms in the product. At ~0 to 45°C, L. monocytogenes grows to various extents when
present in a suitable medium. Temperatures below 0°C freeze the culture or food and preserve or
moderately inactivate the pathogen. Temperatures greater than 50°C are lethal to the pathogen.
These three ranges of temperature will be addressed separately.
GROWTH TEMPERATURE
Range and Optimum
The temperature range that permits growth of L. monocytogenes is of particular interest to food
processors because this pathogen is a psychrotrophic bacterium. Listeria monocytogenes was reported
to grow at temperatures between –1.5 and 45°C [148,261]. In contrast, L. innocua (19 strains),
L. murrayi (1 strain), and L. grayi (1 strain) failed to grow at temperatures below 1.7 (± 0.4), 2.8, and
3.0°C, respectively [161]. Difficulty in determining experimentally the minimum temperature for
growth of L. monocytogenes led Tienungoon et al. [316] to use mathematical modeling to estimate
this value. According to these investigators, the estimated minimum growth temperature, under
optimum pH and aw, was –1.6 and 0.41°C for L. monocytogenes Scott A and L5, respectively. Limits
of growth at refrigeration temperature depended strongly on medium pH [316].
Microorganisms grown at optimum incubation conditions exhibit a short lag phase, short
generation time during the exponential growth phase, and high cell count or density at the stationary
phase. Interestingly, incubation conditions producing the shortest generation time do not always
result in the shortest lag phase or the largest cell density. Therefore, reported “optimum” growth
temperatures are only estimated determinations. Optimum temperature for growth of L. monocy-
togenes, as frequently reported in publications, occurs between 30 and 37°C.
Growth Kinetics
This section addresses the growth rate of L. monocytogenes, related parameters, and influence of
temperature on parameters. Specific growth rate (µ), a commonly used kinetic parameter, reaches
a maximum at the midexponential phase of growth. Specific growth rate and generation time
(or doubling time) are inversely related as follows:
Generation time = 0.693/µ
Bacteria at the lag phase show no detectable growth; however, duration of this phase is a useful
indicator of subsequent growth.
Researchers from the U.S. Department of Agriculture, Eastern Regional Research Center (USDA-
ERRC) modeled growth of L. monocytogenes in broth culture at different incubation conditions [88].
According to these models, lag phase and generation time were smallest (1.7 and 0.3 h, respectively)
when the bacterium was incubated aerobically at 37°C, and the medium has 0.997 water activity
(aw) and pH 7.0 initially. Decreasing incubation temperature progressively increased these two
growth parameters. Robinson et al. [277] used a different model to estimate growth parameters of
L. monocytogenes at different incubation temperatures. These authors observed a shorter lag phase
at 15°C than at 20 or 25°C. Zaika and Fanelli [351] reported minor variation in generation time
of L. monocytogenes when the bacterium was grown in brain–heart infusion at temperatures ranging
from ~30 to 42°C; the corresponding value was ~0.6 h.
Determination of L. monocytogenes growth kinetics in food produced different results. The
generation time of L. monocytogenes decreases substantially as temperature increases from –1.5 to
30°C. The pathogen grew at –1.5°C in vacuum-packaged sliced roast beef with a calculated generation
time of 100 h [148]. Generation times of 62 to 131 h in chicken broth and pasteurized milk were
observed during extended incubation at –0.1 to –0.4°C [330]. Generation time was 28.5 to 46 h, 1.8
h, and 0.7 h when the pathogen was incubated at 4, 21, or 35°C in dairy products, respectively [280].
Therefore, food storage at refrigeration temperature retards but does not prevent growth of Listeria
contaminants. In the absence of antimicrobials, holding food at 20 to 43°C supports rapid growth of
the pathogen.
A relationship between incubation temperature and generation time of Listeria would be useful
in risk assessment studies, but large variations have been observed at a given temperature. Analysis
of some published data (e.g., [280]) shows a linear relationship between the square root of the
maximum specific growth rate ( µmax ) and incubation temperature (Figure 6.1). Other studies pro-
duced a nonlinear relationship between these two variables, with ( µmax ) showing a plateau after
the optimum growth temperature is reached [277,351]. Medium composition contributes to the
large variations in generation time at a given temperature. High salt (4.5 to 7.5%) or high EDTA
(0.1 to 0.3 mM) concentration, for instance, may mask the effect of temperature on growth by
increasing generation time, therefore contributing to deviation from linearity [351]. The listeriostatic
activity of 7.5% NaCl or 0.3 mM EDTA was higher at 37 to 42°C than at 19°C [351].
Strains of L. monocytogenes vary considerably in growth characteristics. Lag phase durations
for 39 strains varied from 70 to 270 h at 4°C and from 36.5 to 70 h at 10°C in trypticase soy
broth–yeast extract [15]. Scott A, a strain extensively used in Listeria-related research, had the
longest (209 h) and the second longest (62.8 h) average lag phase at 4 and 10°C, respectively.
0.8
Square root of max
0.6
0.4
0.2
0
0 10 20 30 40
Incubation Temperature (°C)
FIGURE 6.1 Linear relationship between incubation temperature and the square root of the maximum specific
growth rate ( µmax ) of L. monocytogenes in various foods. Note that µmax values were calculated from minimum
generation times (h) as follows: µmax = 0.693/generation time. (From Hudson, J. et al. 1994. J. Food Prot.
57:204–208; Iturriaga, M. H. et al. 2002. J. Food Prot. 65:1745–1749; and Rosenow, E. M. and E. H. Marth.
1987. J. Food Prot. 50:452–457.)
However, correlation between growth parameters and strains’ serotype was not established [15].
Variability among strains shows the importance of selecting a target strain for use in challenge
studies. Selecting a fast growing and process-resistant target strain would be particularly valuable
for food processing optimization research. Such a strain could be used to illustrate the worst-case
scenario for lethality of a process, as well as survivability or growth during subsequent storage.
Cold Tolerance
The ability of L. monocytogenes to grow at refrigeration temperature is problematic to food

processors. Mechanistic studies on bacterial cell membrane and osmoprotectants help explain how
this pathogen maintains its physiological functions in a cold environment. Membrane phospholipids
must remain in a liquid–crystalline state to maintain membrane fluidity and therefore growth at
low temperature. The fatty acid composition determines whether membrane phospholipids are in
the liquid–crystalline state.
Membranes of L. monocytogenes contain >95% branched-chain fatty acids [12]. When grown
at 37°C, major fatty acids are anteiso-C15:0 (41 to 52%), anteiso-C17:0 (24 to 51%), and iso-C15:0
(2 to 18%) (Figure 6.2). When grown at 5°C, the anteiso-C15:0 form becomes a strongly predominant
group, representing 65 to 85% of total membrane fatty acids [12]. This reduction in the proportion
of long aliphatic chains (C17:0) and the increase in asymmetric branching reduce van der Waals
CH3 O
C
H3C OH
Iso-C 15:0
O
C
OH
H3C
CH 3
Anteiso-C 15:0
O
C
OH
H3C
CH 3
Anteiso-C 17:0
FIGURE 6.2 Structure of the major membrane fatty acids in L. monocytogenes. (From Annous, B. A. et al.
1997. Appl. Environ. Microbiol. 63:3887–3894.)
bonds among membrane constituents. Tight packing of membrane phospholipids at low temperature
is therefore reduced; this helps maintain the pathogen’s membrane fluidity.
Food-grade agents interfering with the biosynthesis of anteiso-C15:0 would be useful in control-
ling growth of L. monocytogenes in refrigerated foods. Highest anteiso-C15:0 fatty acid proportions
in the membrane were found when the pathogen was grown in the presence of glycine betaine [12].
Growth of L. monocytogenes at low temperature is stimulated by presence of glycine betaine and
carnitine [10,226]. When L. monocytogenes was grown at 4°C, addition of 130 µM glycine betaine
nearly doubled the specific growth rate [181]. Listeria does not synthesize glycine betaine and
carnitine, but imports these compounds from the environment. Plants are rich in betaine, and meat
is rich in choline, a precursor of betaine [26].
Processed meat contains approximately 340 to 480 nmol/g glycine betaine [297]. Abundant
levels of carnitine are found in foods of animal origin; processed meats (sausages and ham) and
skim milk contain 230 to 950 and 120 to 140 nmol/g free carnitine, respectively [11,297]. The
ATP-dependent glycine betaine porter II (Gbu) and, to a lesser extent, the glycine betaine-Na+
symporter (BetL) allow accumulation of glycine betaine into the cytoplasm at refrigeration tem-
perature, even when the osmolyte concentration is very low in the growth medium [226]. Uptake
is higher in the late exponential phase than in the stationary phase [11], but this uptake does not
appear to be under σB control [180]. Carnitine was transported into the pathogen cytoplasm via an
ATP-dependent transporter (OpuC) [120].
Prior treatment of a microorganism affects its subsequent physiology and growth kinetics.
Temperature downshift, from 25 to 4°C, increased σB transcription [17]; expression of this alter-
native sigma factor is known to contribute to resistance of L. monocytogenes to heat, carbon
starvation, acid, and osmotic and oxidative stresses [17,109]. Another study showed that growth of
L. monocytogenes at 10°C, compared to 37°C, upregulated genes involved in cold-adaptive response
(flaA and flp), regulatory adaptive response (rpoN, lhkA, yycJ, bglG, adaB, and psr), general
microbial stress responses (groEL, clpP, clpB,flp, and trxB), amino acid metabolism (hisJ, trpG,
cysS, and aroA), cell surface alteration (fbp, psr, flaA), and degradative metabolism (eutB, celD,
and mleA) [201]. An oligopeptide permease (OppA) and a high-affinity potassium uptake system
(Kdp) were required for growth at low temperature [29,37].
Stress Adaptation at Elevated Sublethal Temperatures
Physiological changes caused by exposure to elevated, yet sublethal, temperature has been exten-
sively studied for its consequences on stress resistance, or heat-shock response. Heat-shock response
of L. monocytogenes has been triggered at temperatures ranging from 43 to 52°C, i.e., ~8 to 17°C
above the pathogen’s optimal growth temperature [105,124,296]. Temperature upshift, from 25 to 48°C,
increased transcription of the σB general stress regulon [17]. Expression of major molecular
chaperones DnaK, DnaJ, GrpE, GroEL, and GroES also increased [124,136]. These molecular
chaperones are referred to as heat-shock proteins because they fold newly synthesized proteins,
repair misfolded proteins, and prevent protein aggregation upon heat shock [124]. In general, heat-
adapted L. monocytogenes cells are more resistant to stress than cells grown at optimal temperature.
Holding food at temperatures in the heat-shock range should be avoided because the pathogen may
gain resistance to subsequent processing treatments.
Variations in Virulence with Temperature
Virulence of Listeria increases when the pathogen is grown at refrigeration rather than optimum
temperature. Durst [84] reported that 7 of 36 weakly virulent L. monocytogenes strains became
markedly virulent to mice by intraperitoneal injection after maintaining the cultures on agar slants
for 6 months at 4°C. Similarly, Wood and Woodbine [340] found a strain of L. monocytogenes that
was more virulent to chick embryos when grown at 4°C, instead of 37°C. Cold storage, therefore,
may enhance virulence of some L. monocytogenes strains. In contrast, activity of listeriolysin O
appeared lost after several weeks of storage at 4°C [47]. The decrease in activity of listeriolysin O
was more pronounced at pH 7.0 than at pH 5.5. However, pathogenicity was recovered in ≤ 24 h
by incubating these refrigerated cells at 37°C [47]. Listeriolysin and other PrfA-regulated virulence
genes are thermoregulated and expressed only in Listeria cultures grown above 30°C [192].
Heat shock and other environmental stresses affect the virulence of L. monocytogenes. When
L. monocytogenes was heat shocked at 48°C for 2 h, listeriolysin O was almost totally lost; however,
subsequent growth of the heat-shocked cells at 37°C resulted in production of listeriolysin 40 times
greater than that present immediately after heat shock [174]. Restoration of virulence after heat
shocking reinforces the importance of eliminating L. monocytogenes from minimally processed,
ready-to-eat food.
FREEZING
Although L. monocytogenes does not grow below –1.5°C, this pathogen can readily survive at much
lower temperatures. The Listeria population decreased < 1 log over 3 months’ storage at –18 to –20°C
in inoculated samples of fish, shrimp, ground beef, ground turkey, frankfurters, corn, and ice-cream
mix [140,250]. Populations of the pathogen in tomato soup (pH 4.7) decreased ~3 logs of L.
monocytogenes under similar inoculation and frozen storage conditions [250]. The high inactivation
rate for Listeria in tomato soup was attributed to freeze–thaw injury [250]. Freezing–thawing ruptured
the cell wall, altered plasma membrane integrity, and caused leakage of cytoplasmic contents [92].
Survival and injury of L. monocytogenes during storage at frozen temperature vary with the
temperature, freezing rate, and freezing menstruum [91,92,94]. A low freezing temperature and
rapid freezing rate were most favorable to bacterial survival [94]. No evidence of cell death was
observed when L. monocytogenes was frozen and stored at –198°C in liquid nitrogen. Freezing
and storage at –18°C inactivated 1 to 2 logs and injured >50% of the pathogen population.
Similar ranges of inactivation were observed in five foods (spinach, cheese, fish, chicken, and beef)
during initial quick freezing to –50°C in 57 min and subsequent storage at –18°C for up to 300
days [128]. The quick freezing and subsequent storage only inactivated 0.1 to 1.6 and 0.0 to 1.0
logs, respectively. Injury of the surviving population ranged from a nondetectable level to 90%.
Multiple freeze–thaw cycles are more detrimental to survival of Listeria than is a single cycle
[94]. Such treatment is also far more damaging to the pathogen when frozen at –18°C than at
–198°C. Four freeze–thaw cycles in phosphate buffer inactivated > 2 logs L. monocytogenes and
caused no detectable injury when the freezing temperature was –18°C. In phosphate buffer lethality
was only 34% and injury was 15% when the pathogen was subjected to four freeze–thaw cycles
and the freezing temperature was –198°C [94]. Freezing and subsequent storage cause limited
inactivation of L. monocytogenes, so contamination of frozen food should be prevented through
good manufacturing practices and, whenever possible, by subjecting the food or ingredients to
listericidal processes (e.g., pasteurization) before freezing.
Some food ingredients may protect Listeria against freeze–thaw injury. Addition of 2 to 4%
glycerol or 2% milk to phosphate buffer markedly decreased the extent of cell death and injury
during freezing at –18°C [91]. Survival of Listeria over 5 months of frozen storage is higher in
the presence of 2% glycerol than in 2% milk [91]. Casein, lactose, and fat were identified as the
main milk fractions protecting Listeria against freeze injury. Cryoprotection by lactose and milk
fat was observed during the first month of storage at –18°C. Cryoprotection by glycerol and casein
exceeded 5 months under similar storage conditions and was, therefore, longer lasting than that of
lactose or milk fat [91]. Simulated milk ultrafiltrate, compared to phosphate buffer, caused almost
no change in death rate, but decreased cell injury during the first 24 h of frozen storage at –18°C [91].
Adaptation of L. monocytogenes to sublethal levels of environmental stress from acid, ethanol,
sodium chloride, heat, or starvation increases survival of the pathogen during freezing, frozen
storage, and freeze–thaw cycles [204]. Although freezing and frozen storage may cause a limited
decrease in viability of L. monocytogenes, such treatments can cause injury and thus sensitize L.
monocytogenes cells to listeriostatic or listericidal agents. After frozen storage, viability of the
pathogen decreased in the presence of acid [23,250], lysozyme, or lipase [93]. Cross-contamination
of meat during freezing by immersion in nitrogen was reduced, although not fully prevented, by
2% lactic acid wash before freezing [23].
LETHAL TEMPERATURE
Thermal processing, such as pasteurization, is the most widely used method to preserve food. These
processes target microorganisms of concern in a given food, thus rendering the product safe for
human consumption. Listeria monocytogenes is ubiquitous in the environment, and it has been
increasingly associated with foodborne diseases and food recalls [312]. The pathogen appears
relatively resistant to processing compared to other nonsporing bacteria; processors consider L.
monocytogenes to be the primary pathogen of concern in minimally processed and ready-to-eat
products. Researchers, therefore, have been actively pursuing new intervention strategies and
evaluating efficacy of conventional preservation processes, particularly those involving heat.
Mechanisms of Thermal Inactivation
Elevated temperature causes multiple irreversible cellular damage in Listeria that results in cell
death. Heating L. monocytogenes at temperatures above 56°C causes ribosomal damage, protein
unfolding and denaturation, and, consequently, enzyme inactivation [8,49]. Upon heating, ribosomes
lose Mg2+, which results in dissociation of the 30S and 50S ribosomal subunits [233,342]. The 30S
is more heat labile than the 50S subunit, which is in turn more heat labile than the entire 70S
ribosome [79]. Ribosomal damage, particularly denaturation of the 30S ribosomal subunit, has been
associated with thermal inactivation of vegetative bacteria and is believed to be the main cause of
bacterial death [233].
Superoxide dismutase and catalase activities in L. monocytogenes decreased at temperatures
higher than 45 to 50°C and 55 to 60°C, respectively. Higher processing temperatures may completely
inactivate these enzymes [72], sensitizing the pathogen to aerobic storage conditions [177,254].
However, no apparent correlation between these enzymes and the pathogen’s heat resistance was
found [72]. Heating probably releases Mg2+ from teichoic acids in the cell wall of Gram-positive
bacteria [8]. Mild heat (56°C), however, did not damage the membrane permeability of Listeria [49].
Recovery of thermally injured Listeria requires synthesis of mRNA and repair proteins [49].
Kinetics of Thermal Inactivation
Measurement
Measuring thermal inactivation kinetics involves heating an inoculated medium uniformly at the
desired temperature and monitoring survivors during the course of the treatment. Small thickness
of samples maximizes the rate of heat transfer, minimizes temperature come-up and cooling down
times, and ensures treatment homogeneity [18,99]. Researchers used capillary tubes, sealed bags,
sealed tubes, or open tubes to measure Listeria thermal inactivation kinetics [18,78,99], but variations
among these methods led to conflicting results [78]. For fluid products, capillary tubes are generally
recommended to establish reliable survivor plots [18,99]. Data are analyzed and inactivation rates
or thermal death times are calculated. Before implementation in food processing, it is recommended
that the kinetic results be confirmed in pilot-scale pasteurizers, using inoculated food; operating
parameters, pasteurizer design, and treated matrix may affect the efficacy of thermal processes.
D-Value and z-Value
The relationship between thermal treatment time and the log count of survivors is commonly
referred to as a “survivor plot.” If this relationship is linear, thermal resistance parameters can be
readily calculated. Time required to inactivate one log of the microbial population at a given
temperature (i.e., D-value) is a popular expression of its thermal resistance. If the survivor plot is
nonlinear, the D-value cannot be determined accurately. Implications of the nonlinearity of inacti-
vation data have been addressed in a recent publication [142].
Pooled data from multiple sources (411 data points) show a log-linear relationship between
treatment temperature and D-values of L. monocytogenes (Figure 6.3). Thermal inactivation
rates at any given temperature varied considerably among studies and when the pathogen was
heated in different media. Using raw and reconstituted nonfat dry milk, for example, produced
D63°C and D71.7°C values of ~0.33 and 0.015 min, respectively [32,99]. In contrast, the pathogen
appeared more resistant to heat when present in ice-cream mix; D-values at 68.3, 73.9, and
79.4°C were 3.9, 0.53, and 0.043 min, respectively [33]. Although Figure 6.3 shows large
variability in thermal resistance of L. monocytogenes in different studies, these data were pooled
and used to estimate the temperature change necessary to vary the D-value of L. monocytogenes
by one log (i.e., z-value); this calculation resulted in a z-value of 7.6°C. Recently, a large
number of published inactivation studies were reviewed and D- and z-values of L. monocyto-
genes were compared for various laboratory media, and dairy, meat, egg, seafood, and vegetable
products [79]. The authors reported average and median z-values of 7.1 and 6.4°C, respectively
(minimum: 4.3°C; maximum: 29.3°C).
Heat treatment of low-acid refrigerated food should be sufficient to decrease the target
pathogen ≥ 5 logs. Manufacturers of fruit juice and other acid foods also adopt this principle.
Presence of L. monocytogenes in ready-to-eat food is not permitted in the United States. This
zero-tolerance policy, in addition to the pathogen’s ubiquity and resistance to adverse condi-
tions and mild processing, makes L. monocytogenes a suitable target for thermal processing.
100
10
D-value (min)
0.1
0.01
50 54 58 62 66 70 74
Temperature (°C)
FIGURE 6.3 Decrease of L. monocytogenes D-values with heating temperature of inoculated meat products,
dairy products, seafood, fruits, juices, and vegetables. (From Chhabra, A. T. et al. 1999. J. Food Prot.
62:1143–1149; Doyle, M. E. et al. 2001. J. Food Prot. 64:410–429; Juneja, V. K. and B. S. Eblen. 1999. J.
Food Prot. 62:986–993; Mazzotta, A. S. 2001. J. Food Prot. 64:315–320; and Murphy, R. Y. et al. 2002. J.
Food Prot. 65:53–60.)
The following are examples of thermal treatments that successfully inactivated L. monocytogenes
in various foods.
Cooking ham and brined salmon to internal temperatures of 65 and 82.8°C, respectively,
eradicates Listeria from these foods [79]. Pasteurization of apple cider at 71.1°C for 11 sec inactivates
> 5 logs L. monocytogenes. When processed at 68.1°C for 14 sec, low numbers of injured Listeria
were recovered from artificially contaminated apple cider with relatively high pH (4.1) and low
sugar content (11°Brix); however, survivors died when the processed cider was stored at 4°C for 24
h [216]. Minimum temperature-time pasteurization processes required by the U.S. Food and Drug
Administration (FDA) for dairy products are sufficient to eliminate ≥ 105 CFU/g L. monocytogenes
[13,51,99,341]. Processing food at a different temperature should achieve equivalent microbial
inactivation [114].
The hardiness of L. monocytogenes to mild thermal processes has frequently been observed. At
temperatures < 60°C, Listeria spp. had a substantially higher D-value than Salmonella spp. in meat
products, especially in chicken [239]. Equal or slightly lower D-values for Listeria were observed at
70°C, indicating that the z-value for Listeria was probably lower than that for Salmonella in these
products. In fruit juices processed at 56 to 60°C, Listeria was more heat resistant than Salmonella, but
less heat resistant than Escherichia coli O157:H7 [220]. The z-value of the latter pathogen, however,
was smaller than that of Listeria [220]. Populations of L. monocytogenes decreased only 1.6 and 0.38
logs during heat treating egg white (56.7°C for 3.5 min) and salted/sweetened egg yolk (63.3°C for
3.5 min), respectively [230]. Because low populations of the pathogen are typically found in contam-
inated egg products, current pasteurization protocols for whole egg and egg yolk may be sufficient to
eliminate the organism, even though these thermal processes do not provide a wide safety margin [230].
Extrinsic Factors in Resistance to Heat
Food Composition
Some food components may protect L. monocytogenes against heat. Listeria monocytogenes was more
resistant in presterilized or repasteurized milk than in raw milk, but the reason for this increased
resistance has not been identified [34]. Resistance of L. monocytogenes to mild heat increases with the
food’s pH [160], fat content [210], salt concentration [160], freedom from antimicrobials [215], high-
fructose corn syrup solids concentration [146], and presence of stabilizers such as guar gum and
carrageenan [266]. However, acid, salt, and phosphate in beef gravy caused only a minor increase, if
any, in D-values at temperatures higher than 62.5°C [160]. High salt concentration increased the
denaturation temperature of Listeria’s 30S ribosomal subunit, which contributes to heat tolerance of
the pathogen [302]. The fat fraction from sheep milk protected L. monocytogenes against heat [210].
Increased viscosity in the presence of gum decreased the rate of heat transfer in the food; this may have
been sufficient to induce heat adaptation [266]. Variations in heat resistance because of food composition
may be associated with availability of nutrients that support growth of Listeria. Starvation of Listeria
can trigger a stress-adaptive response and thereby increase the pathogen’s tolerance to heat [205].
Listeria monocytogenes Engulfment

Most in vitro inactivation studies processed freely suspended cells of L. monocytogenes. Animals
suffering from listeric mastitis produce milk in which L. monocytogenes is usually entrapped within
phagocytic leukocytes. Bunning et al. [52] reported average D71.7°C values of 5.0 and 3.1 sec for
intracellular (zintracellular = 8.0°C) and freely suspended (zsuspension = 7.3°C) L. monocytogenes, respectively.
The higher thermal resistance of intracellular compared to freely suspended Listeria was also observed
by Doyle et al. [80], except when contaminated milk was stored ≥ 4 days at refrigeration temperature.
During cold storage of milk, heat resistance of Listeria decreased in parallel to leukocyte breakdown;
therefore, the researchers speculated that leukocytes protected the engulfed pathogen against thermal
inactivation [80]. Homogenization of raw milk disrupts phagocytes; this frees the pathogen, which loses
thermal protection during the subsequent thermal process. As infected cows develop fever, L.
monocytogenes grown at these elevated temperatures may become adapted to heat [80]. Consistent with
this hypothesis, heat shocking (48°C, 15 min) Listeria in milk increased the D71.7°C value from 3.0 to
4.6 sec [51]. The latter value is comparable to that measured for intracellular L. monocytogenes [52].
Intrinsic Factors in Resistance to Heat
Strain
Listeria monocytogenes strains vary in resistance to heat [99]. Strains from serovar 4b tend to
be slightly more heat resistant than those from serovar 1/2a [46]. This trend, however, is not
always confirmed. One of the most commonly studied strains, Scott A (serovar 4b), has low heat
resistance, compared to V7 (serovar 1a) [99]. Sporadic survival of hardy strains may be encoun-
tered if process parameters are based on inactivation studies using heat-sensitive strains, such as
Scott A. Process validation, therefore, should aim at destruction of a target Listeria strain selected
for its high resistance to heat. Some studies have assessed thermal resistance using mixtures of
strains (“cocktails”). Presence of at least one problematic (target) strain and strains from various
serovars is recommended in these cocktails to minimize the risk of underestimating the pathogen’s
thermal resistance.
Physiological State
As frequently observed in bacteria, Listeria is more heat resistant in the stationary than in the
exponential phase of growth. When L. monocytogenes is exposed to sublethal stress, it may develop
an adaptive response to subsequent thermal treatments. Environmental and processing stresses that
may cause this phenomenon include sublethal heat, acids, oxidants, starvation, and high osmolarity
[158,205]. Prewarming, slow heating, or cooking food; hot water washing; underprocessing; and
holding food in warm trays (as may happen in food service establishments) are examples of sublethal
heat shock. Holding L. monocytogenes at sublethal temperatures (e.g., 45 to 48°C, for 15 min to
1 h) induces adaptive thermotolerance [124,206]. The D52 to 71.1°C-values of heat-adapted cells were
1.5 to 4 times greater than that observed in cells incubated at optimal growth temperature (30 to 38°C)
before the thermal process [51,175,199,296].
A nearly twofold increase in D62°C was observed when the pathogen was heated in ground
pork at 1.3°C/min compared to 8°C/min [175]. The z-value of heat-adapted L. monocytogenes,
however, remained similar to that of unadapted cells [199]. The average D56°C of L. monocy-
togenes suspended in a phosphate buffer (pH 7.0) was 4.1, 8.8, and 2.9 min, when cells had
been previously exposed to pH 4.5, 4 to 8% (v/v) ethanol, and 500 ppm hydrogen peroxide,
respectively. For comparison, the corresponding nonadapted L. monocytogenes strain had a
D56°C of 1.0 min [205]. Starvation at 30°C for up to 163 h in phosphate buffer increased the
D56°C value of the remaining viable cells to 13.6 min [205]. The pathogen’s thermotolerance
at 60°C increased 1.3- to 8-fold in the presence of 0.5 to 1.5 mol/L NaCl, compared to cells
exposed to 0.09 mol/L NaCl [211].
Adaptive thermotolerance is a transient, nonheritable property [157,158]. Acquired thermotol-
erance of L. monocytogenes lasted at least 24 h at 4°C in a sausage mix [105], <1 h at 35°C
(optimum temperature for growth) in tryptic soy—0.6% yeast extract broth (TSB-YE) [50], and
≥4 h at 42°C in TSB-YE (heat-shock temperature) [50]. Thermotolerance in liquid media was lost
in <5 min after osmotic downshock [158]. Potential heat adaptation, therefore, should be taken into
account when establishing thermal-processing parameters.
SURROGATE MICROORGANISMS FOR THERMAL STUDIES
Because of safety concerns, researchers look for nonpathogenic indicator microorganisms that may
replace a pathogen such as L. monocytogenes in thermal inactivation trials. A suitable indicator
microorganism, called a surrogate, should be at least as resistant to the thermal process as the most
heat-resistant L. monocytogenes strain. Listeria innocua, which is nonpathogenic to humans, is
frequently used as a surrogate for L. monocytogenes because of the genetic relatedness of the two
species. Listeria innocua has growth rates and resistance to common food preservation factors
similar to or higher than those of L. monocytogenes [81,112,260].
Listeria innocua M1 is a potential surrogate for thermal process validation. This strain is
resistant to rifampin and streptomycin, which aids in its enumeration in food [102]. When
determining survivor plots, Fairchild and Foegeding [102] showed the importance of enumer-
ating uninjured and injured cells to prevent underestimation of Listeria counts after processing.
In spite of the merits of using L. innocua as a surrogate, it is judicious to select Listeria
surrogates from different bacterial genera. Screening protocols used in food processing facilities
commonly target Listeria spp. rather than L. monocytogenes specifically. Because using L.
innocua in lieu of L. monocytogenes may lead to accidental release of the bacterium in the
processing environment, detection of this surrogate by screening tests may raise unwarranted
concerns. Generally recognized as safe (GRAS) bacteria, such as those used in food fermenta-
tions, are ideal surrogates for pathogens.
ACIDITY
Like many other bacteria, L. monocytogenes grows optimally at a pH close to neutrality. Growth
of L. monocytogenes has been reported at pH values ranging from 4.0 to 9.6 [261,263]. In the
absence of other growth-limiting factors, highest final populations were reached at pH 6.0 to 8.0
[42,261,284]. Lag phase and generation time increase considerably as pH decreases to below 6.5
[88,284]. Listeria will grow in some low-acid foods, including fermented products depending on
water activity, SOH content, and other intrinsic factors. Camembert [283], brick [286], and white
pickled cheese [1] have a pH ≥ 5.9 and support growth of L. monocytogenes.
SURVIVAL AT LOW PH
Low pH is one of the most critical safety and quality determinants of fermented foods. Although
growth of L. monocytogenes at pH < 4.0 has not yet been documented, this organism appears to
be fairly acid tolerant under such conditions, with the pathogen surviving 1 to 4 days in orange
juice (pH 3.6) stored at 4°C [252] and > 200 days and >365 days in cheddar cheese (pH 5.1) stored
at 13 and 6°C, respectively [282]. The effect of pH on cell viability, however, depends strongly on
other environmental factors and on the physiological state of the microorganism.
Inactivation at Low pH
Absence of growth and decrease in cell viability may be observed at pH ≤ 5.5, when other
environmental conditions (e.g., temperature) are not optimal for survival of L. monocytogenes. The
Pathogen Modeling Program from the USDA estimated the decrease in count in nutrient broth at
pH 3.2 to 4.4 [88]. Within this pH range, the predicted D-value decreased with pH in a log-linear
manner (Figure 6.4). This decrease in D-value was also more marked as the temperature increased
from 4 to 35°C.
10000
3 x D-value (hours)
1000
4.
4
100
4.
lue
0
va
3.
6
pH
10
3.
2
5 10 15 20
0 2
25 30 35
5
Temperature (°C)
FIGURE 6.4 Exposure time, under lethal acidic conditions, needed to decrease L. monocytogenes populations
3 logs (i.e., 3 × D-values) at different storage temperatures, as determined by the USDA Pathogen Modeling
Program. (From Eastern Regional Research Center. 2003. USDA. Pathogen Modeling Program [version 7.0].
http://ars.usda.gov/services/docs.htm?docid=6786.)
During prolonged storage in orange serum at pH ≤ 4.8, counts of L. monocytogenes were first
constant (~40- and 20-day lag periods at pH 4.8 and 4.0, respectively), then decreased at a rate of
approximately 1 log/5 to 8 days [252]. When pH of the orange serum decreased, the lag period
decreased and the subsequent inactivation rate increased [252]. At 5°C, the pathogen population
decreased < 2 logs after 49 days and >4 logs after <10 days in cabbage juice acidified to pH 4.8
and 4.4, respectively [68]. The specific listeriostatic and listericidal activity of organic acids is
reviewed later in this chapter. Listeria counts decreased during ripening of parmesan [349] and
stretching and storage of mozzarella cheese [39,328]. Inactivation was likely related to the combined
antilisterial effect of the cheeses’ low pH (pH ≤ 5.2) and elevated processing temperature.
FACTORS INFLUENCING ACID TOLERANCE

Refrigeration inhibits growth, but favors survival of L. monocytogenes in acid food. This bacterium,
for example, survived well in hard salami (pH ~4.4) during refrigerated storage [156]. When milk
was inoculated with Listeria and made into yogurt, the pathogen survived in the product for up to
27 days at 4°C [62]. The pathogen persisted in cottage cheese (pH 5.0) more than 35 days at 5°C
[265]. This also suggests that refrigerated acid products should be considered as possible vehicles
of infection in epidemiological investigations of human listeriosis.
Temperature influences the growth behavior of L. monocytogenes under acidic condition. When
L. monocytogenes is incubated at 5 to 35°C in growth-permitting acidic media (i.e., pH 4.5 to 6.5);
greatest inhibition occurs at lowest pH and temperature [88]. Listeria monocytogenes populations
(104 CFU/mL-inoculum) increased to 109 CFU/mL in cabbage juice (pH 5.2) incubated at 30°C
for 3 days [68]. In similarly processed juice, the population remained unchanged during 22
days of storage at 5°C [68]. Rapid growth at 30°C, however, was followed by equally rapid bacterial
death, and the pathogen was no longer detectable after 15 days’ storage. Although the pathogen’s
viability in acidic food is expected to decrease as storage temperature increases from refrigeration
to ambient, such storage is likely to lessen overall quality of perishable low-acid foods and is
therefore inadvisable. Sensitivity of L. monocytogenes to high acidity (pH <4.5) increases with
temperature (Figure 6.4), especially when the medium is not conducive to growth [88].
Acid resistance varies substantially with physiological state of the pathogen. Exposure to some
environmental stresses, for example, may elicit an adaptive response that increases tolerance of L.
monocytogenes to acid. Counts of L. monocytogenes in a culture medium at pH 3.5 were 3.1 to 6.4
times higher when the population was pre-exposed to mild acidity (pH 4.5 to 5.5), a stepwise pH
decrease to 4.5, or 5% ethanol, compared to the unadapted control [206]. Prolonged survival of acid-
adapted cells compared to unadapted cells was confirmed in cottage cheese (pH 4.7), cheddar cheese
(pH 5.2), yogurt (pH 3.9), salad dressing (pH 3.0), and orange juice (pH 3.8) stored at refrigeration
temperature [123]. Acid-adapted survivors were detected in these foods when stored at refrigeration
temperatures for ≥ 15 days, 70 days, 2 days, 7 h, and 7 h, respectively [123]. Lactic and acetic acid
are more effective than hydrochloric acid in inducing acid adaptation in L. monocytogenes [246].
Acid-tolerant spontaneous mutants also have been isolated after prolonged incubation at pH 3.5 [246].
MECHANISMS OF ACID DAMAGE AND TOLERANCE

Viability of bacteria depends on the cell’s ability to maintain an intracellular pH close to neutrality,
regardless of environmental pH. The cell can cope with mild acidity by eliminating excess protons
from the cytoplasm. Below a threshold pH, cells are unable to expel protons fast enough and a
decrease in intracellular pH becomes inevitable. Cytoplasm acidification reduces the proton motive
force of membranes, thus depriving the cell of essential energy for its metabolism. Excess H+ alters
enzymatic activity and may denature proteins [304,344]. Therefore, depending on food composition,
processing, and storage conditions, low pH can be listeriostatic or listericidal.
Constitutive and induced mechanisms increase the pathogen’s acid tolerance. The glutamate
decarboxylase system of L. monocytogenes contributes to the pathogen’s survival in acid foods that
contain ≥ 0.22 mM free glutamate (e.g., salad dressing, mayonnaise, fruit juices, yogurt) [70]. It is
believed that internalized glutamate acts as a proton sink and therefore prevents cytoplasm acidifica-
tion. A shift in gene expression characterizes induction of acid tolerance. Cells in the stationary phase
are naturally more acid tolerant than those in the exponential phase, regardless of the nature of the
acidifying agent [246]. Transition to the stationary phase and a mildly acidic environment increases
expression of σB factor. This activates a general stress regulon, which increases survival to various
growth-limiting factors, such as high acidity or elevated temperature [109]. Increased expression of
the following proteins was observed in acid-adapted L. monocytogenes: molecular chaperones, ATP
synthase, a thioredoxin reductase homologue of B. subtilis, and a ferric uptake regulator [264].
Molecular chaperones refold damaged bacterial proteins and therefore contribute to acid resistance.
CONSEQUENCES OF ENHANCED ACID TOLERANCE

Acid adaptation cross-protects L. monocytogenes against a variety of deleterious environments;
these include lethal doses of hydrogen peroxide, heat, NaCl, and ethanol [205,206]. Therefore,
prolonged storage of mildly acidic food (pH ≥ 4.5) may harden L. monocytogenes to processes
designed to inactivate the pathogen. Strains with constitutive acid resistance are more likely to
persist in plant environments than are other strains.
To cause foodborne listeriosis, L. monocytogenes must cross the intestinal epithelial cells, and
survive defense by phagocytes. Acid adaptation increased adhesion, entry, and growth in an intes-
tinal cell line, Caco-2 cells [69]. Entry efficiency in phagocytes was similar in acid-adapted and
nonadapted cells. However, acid-adapted cells and those of an acid-tolerant mutant multiplied
approximately four times faster than nonadapted wild-type cells [69]. Mice infected intraperito-
neally died faster when injected with an acid-tolerant mutant than with the corresponding wild-
type L. monocytogenes strain [246]. Virulent L. monocytogenes excrete listeriolysin O, which has
a maximum activity at pH 4.0 to 5.0 [224]. Growth rate is not proportional to virulence of the
pathogen. Some weak organic acids enhance pathogenicity of the bacterium, while others reduce
it. Citrate, acetate, and lactate increased secretion of listeriolysin O, whereas sorbate inhibited
secretion of this hemolysin [182,183].
WATER ACTIVITY
Availability of moisture for microbial growth is best expressed as water activity (aw), which is
defined as the ratio of the water vapor pressure of a food substrate to the vapor pressure of pure
water at the same temperature [88]. High osmolarity, i.e., low water activity, decreases turgor
pressure in a bacterial cell. Bacterial turgor pressure provides the force that expands the cell and
therefore contributes to cell wall growth and cell division. Reduction in turgor pressure inhibits
bacterial growth [6]. Most fresh foods have a water activity > 0.98 [88]. Drying and addition of
salt (e.g., during sausage making) or sugar (e.g., in fruit preserves) are traditional methods to lower
water activity of food and therefore enhance its shelf life. Listeria monocytogenes survives or even
grows in foods with relatively low water activity [242]. Tolerance of L. monocytogenes to low aw
should be considered when developing food applications that use minimal processing or involve
dehydration or brining.
WATER ACTIVITY AND GROWTH OR SURVIVAL

Like most bacteria, L. monocytogenes grows optimally at aw ≥ 0.97 [261]. However, when compared
to other common infectious foodborne pathogens, this bacterium has a rather unique ability to
multiply at aw values as low as 0.90. The lowest water activity values allowing growth to ≥ 6.5 log
1000.0
Lag phase (h) 100.0
0.930
10.0
0.950
0.970
ty
tivi
0.990
1.0
c
0.997
5 10 15 20 25 30 35
te ra
Temperature (°C) Wa
B
Generation time (h)
100.0
10.0
0.930
1.0
0.950
0.970
0.990
0.1 ity
ctiv
0.997
5 10 15 20 25 30 35 a
ter
Temperature (°C) Wa
FIGURE 6.5 Changes in lag phase (A) and generation time (B) of L. monocytogenes at different water
activities and incubation temperatures (pH 7.0), as determined by the USDA Pathogen Modeling Program.
(From Eastern Regional Research Center. 2003. USDA. Pathogen Modeling Program [version 7.0].
http://ars.usda.gov/services/docs.htm?docid=6786.)
CFU/mL in presence of glycerol, sucrose, NaCl, and propylene glycol were 0.90, 0.92, 0.92, and
0.97, respectively [232,242]. These water activities correspond to approximately 30% glycerol,
39.4% sucrose, 11.5% NaCl, and 16.7% (w/w) propylene glycol. Lag phase and generation time
increase as water activity of the medium decreases (Figure 6.5).
Although L. monocytogenes does not appear to grow at aw < 0.90, the bacterium survives in
these environments, particularly under refrigeration, for long periods. Listeria monocytogenes
survived in fermented hard salami (aw: 0.79 to 0.86) at 4°C for at least 84 days [156]. When
commercial cheese brines were inoculated with L. monocytogenes (2 × 104 to 2 × 105 CFU/mL)
and stored at 4°C, the pathogen was detectable for up to 259 days [191]. Low aw (< 0.90) is
listeriostatic, but rapid growth may resume as aw increases. Cheese brines, therefore, should be
considered as potential sources for cross-contamination [191].
OSMOTOLERANCE FACTORS
The combined effect of aw and incubation temperature on growth of L. monocytogenes is profound;
the greatest inhibition is observed when both aw and temperature are minimum (Figure 6.5). Survival
of L. monocytogenes at reduced moisture levels depends on the dominant solute in the medium.
Survival in media of equal aw, but containing different solutes, was in the following increasing
order: propylene glycol, NaCl, sucrose, and glycerol [232,242]. The capacity of L. monocytogenes
to grow on processed meats was related to accumulation of high levels of glycine betaine, carnitine,
and glycine- and proline-containing peptides (200 to 1,000 nmol/mg cell protein) [6,297]. These
osmoprotectants are essential to maintain intracellular turgor pressure [6].
Glycine betaine is preferentially used upon cell exposure to severe osmotic or cold stress [6].
Glycine betaine transporters (BetL and Gbu) and carnitine uptake-transporter (OpuC) pump osmo-
protectants into the cytoplasm at high environmental osmolarity [10]. The BetL symporter instan-
taneously accumulates glycine betaine upon osmotic upshock, at a rate of 2 nmol/min/mg cellular
protein. The Gbu transporter is essential to long-term osmotolerance or to survival under high
(≥ 6%) salinity; this transporter activity started a few minutes after upshock, increased progressively,
and accumulated 8 nmol glycine betaine/min/mg cell protein after 4-h exposure to 4%NaCl [226].
Dimethyl glycine, trigonelline, triethylglycine, and γ-butyrobetaine are glycine betaine analogs and
potent inhibitors of glycine betaine and/or carnitine uptake (Figure 6.6) [226].
Osmotic upshift induces expression of the σB factor in L. monocytogenes [17]. As observed in heat-
or acid-adapted cells, induction of the σB regulon increases resistance of osmotically adapted cells to
mild stresses, such as acid, heat, and freeze injury [109,160,206]. The extent of cross-protection is
minimized when combining mild hurdle factors adequately. A combination of stresses was probably
responsible for the rapid demise of Listeria in parmesan cheese [349]; this cheese curd had a
relatively low pH, was cooked at 51°C for ~45 min, and had low moisture content (30.1 to 31.4%).
Glycine betaine Carnitine

CH3 CH3 OH
| O | | O
CH3 — N + — CH2 — C CH3 — N + — CH2 — CH — CH2 — C
| O- | O-
CH3 CH3
•Triethylglycine
•Dimethylglycine •Dimethylglyc ine • -butyrobetaine
•Trigonelline •Triethylglycine
Na+
BetL Gbu Gbu OpuC
OpuC
CELL
MEMBRANE
Na+ ATP ADP + Pi ATP ADP + Pi ATP ADP + Pi
CH3 CH3 OH
CYTOPLASM
| O | | O
CH3 — N + — CH2 — C CH3 — N + — CH2 — CH — CH2 — C
| O- | O-
CH3 CH3
CH3 O CH3CH2 O
NH + — CH2 — C CH3CH2 — N + — CH2— C
CH3 O- CH3CH2 O-
Dimethylglycine Triethylglycine
O CH3 O
—C CH3 — N + — CH2 — CH2 — CH2 — C
CH3 — N+ O- CH3 O-
Trigonelline -Butyrobetaine
FIGURE 6.6 Glycine betaine and carnitine transport across the cell membrane and uptake impairment by
glycine betaine analogs. Figure insert shows the structure of glycine betaine analogs that were found to inhibit
uptake of the two osmoprotectants. (From Mendum, M. L. and L. T. Smith. 2002. Appl. Environ. Microbiol.
68:5647–5655.)
ANTIMICROBIAL COMPONENTS IN FOOD

Antimicrobial ingredients are frequently added to control foodborne microbiota, with some (e.g.,
salt and spices) contributing to the food flavor profile. Antilisterial activity of salt, organic acids,
fatty acids, nitrite, antioxidants, smoke, plant extracts, and related compounds has been thoroughly
investigated over the past two decades.
SALT
Salt (i.e., sodium chloride) is an important food ingredient. High salt concentrations reduce the growth
of L. monocytogenes by decreasing the food’s water activity [261] and electrochemical potential across
the cell membrane [253]. Influence of water activity on microbial growth, survival, and death of L.
monocytogenes was described earlier, with NaCl-specific interactions reported in this section.
Survival at Extreme Salt Concentrations
Listeria monocytogenes tolerates extremely high salt concentrations. The pathogen survived in com-
mercial cheese brine (23.8% NaCl, pH 4.9) stored at 4°C for 259 days [191]. Consequently, use of
concentrated brine solutions in food applications, such as in cheese manufacturing and salmon
processing, should not be considered a reliable means to eliminate L. monocytogenes. Survival of
Listeria in the presence of >12% NaCl decreases with increasing salt concentration or incubation
temperature. Presence of 16% NaCl was listeriostatic for at least 33 days at ≤4°C. Presence of 26%
NaCl decreased Listeria populations 2 and 3.5 logs when the pathogen was incubated at 0 and 4°C,
respectively, for a similar storage period [147]. Incubation of L. monocytogenes in the presence of
14% NaCl for 36 days at 10 and 25°C decreased populations ~2 and ≥ 6 logs, respectively [299].
Physiology at High Salt Concentration
Listeria formed colonies with a rough surface and irregular borders on agar media containing ≥6%
NaCl [152]. Cell morphology changed from short rod to filamentous and deformed shapes [22,351],
with a strongly hydrophilic surface [22]. Morphological changes were less pronounced when
incubating the cells at refrigeration temperature [38]. Interestingly, elongated cells were also
observed when L. monocytogenes was grown in media
adjusted to pH 5.0 to 6.0 with citric acid

adjusted to pH > 9.0 with NaOH
containing ≥ 1.75 mM H2O2
containing ≥ 0.3 mM EDTA
This suggests that filamentous morphology contributes to adaptation to adverse conditions

[152,351]. Changes in cell morphology, in response to medium salinity, altered the adhesion
properties of the pathogen [22]. Whether these changes facilitate persistence of the pathogen in food-
processing environments or on food and equipment surfaces has not been reported.
Viability of cells requires maintenance of an electrochemical potential across the membrane as
well as a low cytoplasmic Na+ concentration regardless of the extracellular NaCl concentration.
Regulation of potassium uptake (kdp), Na+ efflux (gbu and ykpA), and multidrug efflux (mdrL)
were overexpressed under high salinities, presumably to expel excess Na+ ions from cytoplasm
[37,126]. An increase in anteiso-C15:0 and decrease in anteiso-C17:0 fatty acid levels in the Listeria
membrane have been observed upon exposure to high salinities [61]. A similar membrane fatty
acid profile has been observed when the pathogen is stored at refrigeration temperature. Reasons
for this change in membrane fatty acid profile have not been determined and may be linked to
increased osmoprotectant (e.g., glycine betaine) concentration in the cell cytoplasm. Activation of
the osmotic stress response was discussed in the previous section.
Up to 6% sodium chloride protected L. monocytogenes against thermal inactivation in beef

gravy (pH 4.0) [160]. This thermoprotective effect was particularly evident at treatment temperatures
≤ 60°C. The highest D55°C-value was observed in gravy containing 1.5 to 4.5% NaCl. When heating
the gravy at 55°C, increasing the salt concentration to 4.5% decreased the rate of thermal inactivation
of L. monocytogenes. Furthermore, thermotolerance of the pathogen increased slightly with NaCl
concentration when sodium pyrophosphate was included in the gravy formulation. As explained
earlier, high salt concentration increases the temperature of denaturation of the pathogen’s ribo-
somes and hence increases its thermotolerance [302].
ORGANIC ACIDS AND THEIR SALTS

Organic acids and their salts (Table 6.1) are frequently incorporated into foods as acidulants (e.g.,
acetic acid) or preservatives (e.g., sodium diacetate). Acid sprays and acid dips are used to inactivate
TABLE 6.1
Organic Acids Commonly Used as Acidulants or Antimicrobial Agents in Food
Name Molecular Weight (g/mol) pKa Structure
Acetic acid 60.05 4.74
Sodium diacetate 142.09
Citric acid 192.1 3.13

4.76
6.40
Benzoic acid 122.12 4.19
Lactic acid 90.08 3.79
Parabens: 152.14 8.47

e.g., methylparaben
Para-aminobenzoic acid 4.65

137.13
4.80
Propionic acid 74.08 4.87
Sorbic acid 112.12 4.76
Source: Budavari, S. 1989. The Merck index. Rahway, NJ: Merck & Co., Inc.
L. monocytogenes on food surfaces. According to the USDA Pathogen Modeling Program [88],
bactericidal activity of acid increases with temperature. When listeriostatic doses of organic acids
are used, storage at refrigeration temperature is essential to prevent further growth of the pathogen
[151]. Under listericidal conditions, however, refrigeration diminishes acid lethality (Figure 6.5).
The growth rate of L. monocytogenes in the presence of organic acids varies markedly with
type and concentration of acid and medium pH. Acetic and lactic acids (50 mM) inhibited growth
of the pathogen at 37°C when the medium pH was 4.7, but not when it was 6.0 [344]. Growth of
Listeria on turkey frankfurters was retarded when frankfurters were dipped into 15 to 25% sodium
diacetate or sodium benzoate and to a lesser extent when they were dipped into 15 to 25% potassium
sorbate or sodium propionate [151].
At equal pH and equimolar total acid, growth inhibition of L. monocytogenes was in the
following order: acetic > lactic > citric [344]. However, at equal pH and equimolar undissociated
acid concentration, listeriostatic activity was in the following order: citric > lactic > acetic. These
organic acids were more potent bacteriostatic agents than was HCl [344]. Although relatively high
concentrations of citric acid (>0.5 M ) have listeriostatic activity, smaller concentrations promoted
growth of L. monocytogenes; these data suggest that citric acid contributes to the pathogen’s
metabolism [41,344]. When multiple organic acids are applied simultaneously, these acids may act
additively or synergistically against targeted microorganisms. A combination of 1.5 to 2.5% sodium
lactate and 0.15% sodium diacetate successfully prevented growth of L. monocytogenes in cured
meat products (wieners, ham, light bologna, and Cotto salami) at 4°C during 18 weeks [292].
It is widely accepted that antimicrobial activity of weak organic acids, such as acetic acid, is
associated with the acid’s undissociated form. The undissociated acid freely permeates the cyto-
plasmic membrane and dissociates inside the cytoplasm, thus accumulating protons and anions
within the cell. This decreases the proton motive force and interferes with microbial metabolism
[304,344]. Concentration of the undissociated form of a weak organic acid at a given pH can be
calculated according to the Henderson–Hasselbalch equation:
pH = pKa + log ([dissociated form]/[undissociated form])
Concentration of the undissociated (uncharged) organic acid form increases as the pH decreases.
Contrary to the weak-acid theory, some researchers believe that hydrophobicity of sorbic acid is the
cause of its antimicrobial properties [304]. According to these researchers, weak organic acids are
membrane-active agents that act to disrupt cytoplasmic membrane function, producing cell death.
Lactate
Salts of lactic acid (Table 6.1) are used at 1 to 4% as additives in baked goods, meat, and poultry
products. When added to food, lactates do not change pH of the products [59]. Sodium or potassium
lactate (4%) is listeriostatic at refrigeration temperature [59]. Sodium, potassium, and calcium
lactates were equally effective in inhibiting growth of L. monocytogenes in cooked strained beef
stored at 20°C [59]. Calcium lactate, however, was most antilisterial in pork-liver sausage [335].
Application of NaCl (2 to 3%), nitrate (125 ppm), or low temperature enhanced the listeriostatic
effect of lactate against L. monocytogenes in meat and smoked salmon [259,335]. A combination
of lactate (4%) and nisin (400 IU/mL) is listericidal at pH 5.5 and 4°C, and the inactivation rate
increases with addition of polyphosphate (0.5%) [48]. Lactate–nisin and lactate–nisin–polyphosphate
combinations caused ~2.3- and 4.2-log reductions in 28 and 20 days, respectively; nisin alone only
resulted in an initial 1.1-log decrease and did not prevent subsequent growth of L. monocytogenes
[48]. Addition of 0.3% sorbate did not improve antilisterial activity of lactate (4%) [48].
Sodium Diacetate
Sodium diacetate (Table 6.1) is a GRAS additive [115]. The additive is used as an acidulant,
flavoring compound, and antimicrobial agent in foods. When sodium diacetate was added to brain
heat infusion (BHI) broth at the 18- to 35-mM level (mixture pH of 6.3 to 5.25), a small inoculum
of L. monocytogenes (103 CFU/mL) was inhibited in a concentration-dependant manner [293]. A
low incubation temperature (5°C, compared to 35°C) enhanced inhibitory action of the diacetate.
Minimum inhibitory concentrations of diacetate in the broth were 35, 32, and 28 mM at 35, 20,
and 5°C, respectively. Based on equal levels of undissociated acetic acid at different pH values,
sodium diacetate was more effective and had lower minimum inhibitory concentrations at 35°C
than did acetic acid; minimum inhibitory concentrations were 5, 20, 30, 40, and >100 mM for
sodium diacetate and 5, 20, >50, >100, and >150 mM for acetic acid, at pH 4.7, 5.0, 5.5, 6.0, and
6.5, respectively.
Dipping turkey frankfurters into 15 to 25% sodium diacetate (pH 4.6) at 22°C for 1 min
decreased L. monocytogenes populations 1.7 to 2.0 logs [151]. Sodium acetate on the surface of
these frankfurters inhibited Listeria growth ≥14 days when the treated product was stored at 4 to
13°C. The pathogen population increased 2.5 logs over the same time period when frankfurters
were stored at 22°C [151]. Sodium diacetate also reportedly enhanced listericidal activity of gamma
irradiation [298] and 5,000 AU/mL of pediocin [289].
Sodium Propionate
Propionic acid (Table 6.1) and its salts are useful antimycotic agents, and their potential role as
antilisterial preservatives has been investigated. Broth media containing >2,000 ppm sodium propi-
onate inhibited growth of L. monocytogenes at pH 5 [96]. Generation times for L. monocytogenes in
tryptose broth at pH 5.6 and without sodium propionate decreased from 68 to 49 min as the incubation
temperature increased from 4 to 35°C. When 3,000 ppm sodium propionate was added to the medium,
generation times decreased from 3.0 days to 4.5 h as the incubation temperature increased from 4 to
35°C [96]. Sodium propionate (3,000 ppm) at pH 5.0 was listeriostatic at 4°C and listericidal at 35°C
in tryptose broth incubated for 67 days. Combinations of propionate and acetic acid in growth media
produced strong antilisterial action [98]. Lowering the incubation temperature from 35 to 13°C not
only diminished the rate of growth of L. monocytogenes, but also decreased maximum populations
of the bacterium in the presence of propionate and other organic acids [98].
Sodium propionate (3,000 ppm) was less effective than sorbic acid in eliminating L. monocy-
togenes from cold-pack cheese at pH 5.2 to 5.5 [285]. Growth of the pathogen on the surface of
frankfurters dipped into 15 to 25% sodium propionate (pH ~9.0) was inhibited at 4°C for ≥14 days
[151]. Refrigerated storage is essential for long-term listeriostatic activity of sodium propionate at
pH >5.5. When contaminated frankfurters were stored at 13 and 22°C, growth was observed after
~7 and 3 days, respectively [151].
Potassium Sorbate
Sorbic acid (Table 6.1) is primarily active against yeasts and molds, but this antimicrobial agent
also inhibits a wide range of bacteria, particularly aerobic, catalase-positive organisms. Hence,
several investigators have assessed the ability of potassium sorbate and sorbic acid to inhibit L.
monocytogenes in laboratory media and various foods. Sorbate had a minimum inhibitory concen-
tration of 400 to 600 and >5,000 mg/L at pH 5.0 and 6.0, respectively, when L. monocytogenes
was cultured in BHI broth at 35°C [234]. Antilisterial activity of sorbate was enhanced by other
organic acids, with acetic and tartaric acids being most effective [97]. The lower the storage
temperature and pH of the medium were, the greater was the effectiveness of sorbate against L.
monocytogenes. Strong listericidal effects were observed when 0.3% sorbate was used in combi-
nation with 125 ppm sodium nitrite, 0.5% polyphosphate, or 400 IU/mL nisin [48].
Potassium sorbate is widely used to extend the shelf life of many foods, including butter, cheese,
meat, cereals, and bakery items. Listeria did not grow on turkey frankfurters (pH ~ 9.0) dipped for
1 min in 15 to 25% potassium sorbate when the frankfurters were subsequently stored at 4°C for
≥14 days [151], with any decrease in viability being minimal (<0.6 log CFU/mL). Even though
potassium sorbate retarded the pathogen, growth was observed on frankfurters stored at ≥13°C with
the growth rate increasing with storage temperature.
Sodium Benzoate
Benzoic acid (Table 6.1) is a potent antimicrobial agent in acid foods; optimum activity is in the pH
range of 2.5 to 4.0. Listeriostatic activity of this antimicrobial agent at pH value near neutrality is
more pronounced at refrigeration than ambient temperature. Growth of the pathogen on the surface
of frankfurters dipped into 15 to 25% sodium benzoate (pH ~7.8) was inhibited at 4°C for ≥14 days
[151]. When contaminated frankfurters were stored at 13 and 22°C, slow growth was observed after
~10 and 3 days, respectively. Listeria cells sublethally injured in the presence of benzoate have
impaired mRNA synthesis [40]; therefore, processes requiring mRNA synthesis for survival of the
pathogen (e.g., high acidity) would likely enhance listericidal action of benzoate synergistically.
Parabens and Other Benzoic Acid Derivatives
Parabens are esters of p-hydroxybenzoic acids (Table 6.1). Of these esters, methyl, propyl, and heptyl
parabens are approved in several countries for direct addition to food. Parabens exhibit antimicrobial
activity in low-acid (pH ≥4.6) and acid (pH <4.6) foods. Antilisterial activity of parabens increases
with decreasing pH and temperature [257]. Parabens (1,000 ppm) were listeriostatic in milk [257],
but their action was questionable in processed meat products [28]. Synergistic listericidal activity was
observed when methylparaben was used in the presence of anise, basil, or fennel oil [122].
Para-aminobenzoic acid had greater inhibitory activity against L. monocytogenes than propionic,
formic, acetic, citric, and lactic acids, in order of decreasing activity [276]. Highest activity of
p-aminobenzoic acid against this pathogen was observed as pH decreased. The high potency of
p-aminobenzoic acid may result from dual damage to the pathogen; this antimicrobial agent
interferes with peptidoglycan synthesis and alters the cell’s metabolism. The minimum inhibitory
concentration of p-aminobenzoic acid against L. monocytogenes in BHI (pH ~6.7) at 37°C after
24 h was 9 to 12 mmol/L [276].
FATTY ACIDS AND RELATED COMPOUNDS

Antilisterial activity of free fatty acids, particularly those of medium chain length, has been
demonstrated. Some fatty acid esters, such as monoacylglycerols (monoglycerides) and esters of
sucrose, inhibit a wide spectrum of microorganisms, including L. monocytogenes, in addition to
their primary function as food emulsifiers.
Free Fatty Acids
Lauric (C12:0), linoleic (C18:2), and linolenic (C18:3) acids (Table 6.2) exhibited listeriostatic to
listericidal activity and reduced the invasiveness of Listeria [262,332]. Presence of these fatty acids
at ≥ 200 µg/mL was listericidal in a storage-conditioning solution (1% NaCl) for mozzarella cheese
[262]. The potassium salt of conjugated linoleic acid (100 to 300 µg/mL) was listeriostatic in whole
milk [332]. Duration of the L. monocytogenes lag phase increased with an increase in fatty acid
concentration [332]. Listeriostatic activity of lauric, linoleic, and linolenic acids varied with pH,
presence of other organic acids, and antioxidants. These fatty acids were more active against Listeria
at pH 5.0 than at pH 6.0 or 7.0 [262,332]. Listeriostatic activity of K-conjugated linoleic acid
(200 µg/mL) in whole milk was enhanced by addition of 2,000 µg/mL citrate, 200 µg/mL butylated
hydroxyanisol, or 200 µg/mL ascorbate [332].
Other fatty acids have variable impact on the pathogen’s growth or viability. Counts of L.
monocytogenes decreased below the detection level when incubated at 37°C in a storage-conditioning
TABLE 6.2
Minimal Listeriostatic Concentration of Lauric, Linoleic and Linolenic Acids
in Brain Heart Infusion Broth Incubated at 37°C for 24 h and These Fatty
Acids’ Effect on Invasion Efficiency of Caco-2 Cells by Listeria monocytogenes
Fatty Acid Minimum Inhibitory Concentration (µg/mL) Invasion Efficiency (%)a
pH 5.0 pH 6.0
Lauric acid 10 20 0.1

Linoleic acid 50 200 1.26
Linolenic acid 20 100 0.04
aIn absence of fatty acids (control), 23% of inoculated L. monocytogenes were internalized in Caco-2 cells.
Sources: Petrone, G. et al. 1998. Lett. Appl. Microbiol. 27:362–368, and Wang, L. L. and E. A. Johnson.
1992. Appl. Environ. Microbiol. 58:624–629.
solution of mozzarella (pH 5.0) supplemented with 200 µg/mL butyric (C4:0), caproic (C6:0), caprylic
(C8:0), capric (C10:0), or myristic (C14:0) acid for 24 h. Survival (in the presence of capric or myristic
acid) or growth (in the presence of butyric, caproic, or caprylic acid) of the pathogen was observed
when the pH of the solution was 7.0 [262]. Medium supplementation with 200 µg/mL palmitic (C16:0),
stearic (C18:0), or oleic (C18:1) acid did not inhibit growth of the pathogen [262,332].
Fatty Acid Monoesters
Monoacylglycerols (i.e., monoglycerides) are active against L. monocytogenes. Minimum concen-

trations inactivating ~103 to 104 L. monocytogenes CFU/mL in BHI broth (pH 6.0) after 24 h at
37°C were 25, 75, and 50 µg/mL for monolaurin, monocaprin, and monomyristin, respectively
[334]. At concentrations ≥ 200 µg/mL, monocaprin was more effective than monolaurin in inacti-
vating L. monocytogenes in milk, possibly because of the higher water solubility of monocaprin
[334]. Listeria monocytogenes was not inhibited by 300 µg/mL monopalmitin, monostearin,
monoolein, or monolinolein [334].
Monoacylglycerols were listericidal in beef frank slurries and seafood salad stored at 4°C [333].
These compounds were listeriostatic in turkey frank slurries, cooked shrimp, imitation crabmeat,
and Camembert cheese stored at 4°C [333]. Monolaurin showed stronger antilisterial activity in
2% chocolate milk than in 2% milk [334]. The listericidal activity of monolaurin has been observed
in media with low pH and low fat content stored at refrigeration temperature [332,333]. Although
100 µg/mL and ≥ 200 µg/mL monolaurin in skim milk at 4°C were listeriostatic and listericidal,
respectively, 200 µg/mL monolaurin showed no antilisterial activity in whole milk [332].
Listericidal activity is enhanced when combining monolaurin with organic acids, chelating
agents, high salinity, antioxidants, or other monoacylglycerols [272,333]. Propyl gallate (200 µg/g)
and lactic acid (0.2%) enhanced the bacteriostatic activity of monolaurin and monocaprin against
L. monocytogenes in seafood and Camembert cheese, respectively, stored at 4°C [333]. Monomyris-
tin (200 µg/mL) and monocaprin (200 µg/mL) synergistically inactivated L. monocytogenes in skim
milk at 4°C [334]. Coconut oil contains high levels of monolaurin and monomyristin. Synergistic
interaction between these monoacylglycerols may have accounted for higher listericidal activity of
coconut oil compared to that of monolaurin [334]. Monolaurin has been proposed for decontami-
nation of food contact surfaces [325].
Poor water solubility and the soapy flavor resulting from a high concentration of monolaurin
limit its use in food. Monoacylglycerols and derivatives of monolaurin with greater solubility
compared to monolaurin were investigated. Listeria was inhibited by triglycerol-1,2-laureate
(380 µg/mL) in the presence of 380 µg/mL EDTA [272]. Sucrose monolaurate (400 µg/mL) was
listericidal in tryptose phosphate broth at 30°C [235]. The listeriostatic activity of 200 µg/mL
sucrose monolaurate decreased as temperature increased from 4 to 15°C [235]. Presence of EDTA
(50 to 100 µg/mL) enhanced synergistically the listericidal activity of sucrose monolaurate [235].
Sucrose monolaurate alone, or in combination with EDTA, synergistically increased the thermal
inactivation rate for L. monocytogenes [235]. Sucrose fatty acid esters (e.g., 0.01% sucrose palm-
itate) also reportedly acted synergistically with nisin (250 IU/mL) against L. monocytogenes [315].
SODIUM NITRITE
Sodium nitrite is frequently used to preserve meat and fish and occasionally as a preservative in
certain cheeses. This curing agent slightly inhibits growth of L. monocytogenes [88]. Lag phase
and generation time increased as the nitrite concentration increased from 0 to 150 ppm in nutrient
broth. Inhibition increases when the addition of nitrite is combined with low temperature, pH, or
oxygen level, or when the concentration of sodium chloride in the medium increases [43]. Nitrite
antilisterial activity has mainly been reported at pH ≤ 5.5. At pH 6.3, combining 103 ppm sodium
nitrite and 3.5% sodium chloride in meat did not control growth of L. monocytogenes at 32°C
[132]. Nitrite (30 ppm) did not increase the listeriostatic activity of sodium diacetate in turkey
slurries (pH 6.2) [289].
Growth of L. monocytogenes was suppressed for ≥12 weeks in hard salami sausage (aw = 0.79
to 0.86) that contained sodium nitrite (156 ppm before meat fermentation) and NaCl (5.0 to 7.8%),
had a pH ~4.4, and was stored at 4°C [156]. However, the contribution of low water activity and low
pH was probably more important in preventing growth than was the presence of sodium nitrite.
Addition of EDTA (0.9 g/L) or nisin (0.45 g/L) enhanced the listeriostatic activity of sodium nitrite
(0.18g/L) in BHI (pH 6.0) [131]. Viability of L. monocytogenes at 4°C decreased up to 3.7 logs in
12 days in BHI (pH 5.5) supplemented with nitrite (125 ppm) and one or several of the following
compounds: sorbate (0.3%), lactate (4%), nisin (400 IU/mL), and polyphosphate (0.5%) [48].
The mechanism of nitrite action against L. monocytogenes in processed food is unclear. Filter-
sterilized nitrite at 50 µg/mL and pH 5.0 prevented visible growth of Listeria within 48 h at ≤
20°C, whereas 200 µg/mL autoclaved nitrite at the same conditions did not retard Listeria growth
[223]. Listeriostatic activity of nitrite, however, may be linked to reactive species derived from
nitrite rather than nitrite itself [55]. These compounds may be formed during food processing.
Production of low levels of nitrous acid may contribute to growth inhibition at pH < 6.0. Under
aerobic conditions, peroxynitrite is formed upon reaction of nitric oxide with superoxide or nitroxyl
ion with oxygen. Iron–sulfur–nitrosyl complexes are formed during heating. Minimum inhibitory
concentration of Roussin’s black salt (NH4+[Fe4S3(NO)7]–), an iron–sulfur–nitrosyl complex, against
L. monocytogenes was 3 µmol/L. Nitric oxide and nitrosothiols break DNA strands, impair ribo-
nucleotide reductase activity, and damage the cell membrane.
ANTIOXIDANTS
Antioxidants such as butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), tertiary
butylhydroquinone (TBHQ), and propyl gallate comprise an important category of food additives
[116]. Primarily used to prevent oxidation of fat, some of these antioxidants also possess antimi-
crobial activity. TBHQ is the most inhibitory to L. monocytogenes, followed by BHA, propyl gallate,
and BHT. Minimum inhibitory concentrations of these antioxidants were ~64, 128, 256, and > 512
ppm, respectively, when they were used in tryptose phosphate agar and the inoculated medium was
incubated at 35°C for 18 h [257]. Listeria monocytogenes exhibited an increasingly longer lag
phase and generation time as well as a lower maximum population in tryptose broth incubated at
35°C that contained BHA at 100 to 200 ppm, added as a DMSO solution [346]. Concentrations of
BHA ≥300 ppm were listericidal [346]. Increasing the concentration of TBHQ from 10 to 30 ppm
in the medium increased the duration of the lag phase from 6.5 to 34 h at 35°C, but did not appreciably
affect generation time or maximum population. Presence of TBHQ (150 µg/mL) in milk was listericidal
and listeriostatic after 24 h at 35°C when this medium was inoculated with 1 × 101 CFU/mL
and 1 × 103 CFU/mL, respectively [257]. Propyl gallate inhibited growth of L. monocytogenes in
cabbage juice at room temperature, but ellagic acid, the hydrolytic product of propyl gallate, failed
to inhibit the bacterium [63]. Although these findings appear promising for the food industry, FDA
regulations stipulate that BHA and similar additives can only be used as antioxidants and cannot
be added indiscriminately to foods for other purposes.
Phenolic antioxidants are believed to damage bacterial cell membranes [2,321]. Carvacrol, an
antioxidant naturally found in oregano and thyme, is structurally related to BHA. Carvacrol (1 mM)
depletes bacterial membrane potential and alters membrane permeability to cations [322]. Exposure
to BHT sensitized bacteria to osmotic shock [321]. Development of BHA resistance was observed
in the presence of glycerol and was attributed to the increase in lipid content of the cell wall and
membrane of the pathogen [2].
SMOKE
Smoking has traditionally been used to preserve meat and fish and enhance their sensory quality.
Addition of 0.2 and 0.6% (v/v) of a commercial liquid smoke to wiener exudates inactivated
L. monocytogenes with D-values of 36 and 4.5 h, respectively; Listeria in the smoke-free exudate
grew from an initial population of 105 CFU/mL to ~108 CFU/mL after 3 days at 25°C [103]. Hot
smoking (core temperature at 65°C for 20 min) also inactivates this pathogen [154]. Smoking time
was proportional to the reduction in Listeria counts [287]. Cold smoking of fish was recommended
at a temperature < 21°C [279]. The combination of 20 ppm phenols and 4% NaCl was listericidal
at 4 to 12°C in nutrient broth [225]. Nisin (4,000 to 6,000 IU/mL) and sodium lactate (60%) can
be injected before smoking to further reduce the viability of Listeria in processed fish [245]. Despite
numerous studies showing listericidal activity of smoke compounds, L. monocytogenes may be
detected in smoked fish [159]. Therefore, observing good hygiene and good manufacturing practices
during processing and refrigerated storage is essential to minimize risks of contamination with this
pathogen.
Liquid smoke flavorings owe their antimicrobial activity to the presence of phenolic compounds
and acetic acid, both of which are bacteriostatic at relatively low concentrations [305]. Isoeugenol,
for example, is a phenolic compound that retarded growth of Listeria in exudates of smoked wieners
[103]. Listeriostatic activity of 100 ppm isoeugenol was higher at pH 5.8 than at pH 7.0 [103].
This compound seemed to interact with the bacterial membrane [331].
SPICES, HERBS, AND PLANT EXTRACTS

Although primarily used as flavoring and seasoning agents, many spices contain chemicals or
essential oils that inactivate or inhibit growth of pathogenic and spoilage organisms. Consequently,
surveys were made to identify spices with activity against L. monocytogenes, especially those
suitable for use in minimally processed foods. Cilantro (6%), rosemary (1%), sage (1%), oregano
(0.1 to 0.7%), clove (0.5%), thyme (0.1%), cinnamon (0.1%), pimento (0.1%), and pelargonium
(0.05%) showed listericidal activity [14,129,141,200,251,317]. Sensitivity to spices varied among
strains of Listeria [141].
Listeriostatic and listericidal activity of plant oils have been observed in food. Essential oil of
oregano reduced Listeria counts in packed meat, regardless of the composition of the atmosphere
within the packet [320]. Cilantro oil (6%) was bacteriostatic against Listeria in vacuum-packed
ham [129]. Pelargonium oil prevented growth of Listeria in quiche filling at 25°C for ≥24 h [200].
Adding thyme’s essential oil to minced pork at a level of 0.08% decreased the population of L.
monocytogenes ~1 log during 8 days’ storage at 4 and 8°C. The organism grew in the untreated
controls from 1 × 107 to 6 × 107 and 2 × 108 CFU/g, respectively [14].
TABLE 6.3
Concentration of Compounds from Plant Oils That
Inactivated 50% of L. monocytogenes Population in
Phosphate-Buffered Saline Solution after Incubation at
37°C for 1 h (BA50%)
Active Compound Oil Source BA50%
Benzaldehyde Bitter almond 0.36–0.46
Carvacrol Thyme, oregano 0.083–0.086
Carvone S Dill seed 0.17–0.35
Cinnamaldehyde Cinnamon 0.008–0.019
Citral Lemon grass 0.099–0.200
Estragole Tarragon 0.35–0.36
Eugenol Clove 0.061–0.081
Geraniol Rose 0.28–0.51
Menthol Peppermint 0.48–0.57
Perillaldehyde Mandarin peel 0.30–0.35
Salicylaldehyde Almond 0.43–0.45
Thymol Thyme, oregano 0.077
Source: Friedman, M. et al. 2001. J. Food Prot. 65:1545–1560.
Antioxidant extract from rosemary (0.3%) or encapsulated rosemary oil (5%) substantially
retarded growth of L. monocytogenes in pork liver sausage [251]. Listeria populations in these
sausages increased from 103 CFU/g to ~2 × 105 CFU/g during 50 days’ storage at 5°C in the
presence of the antioxidant extract from rosemary (0.3%) or encapsulated rosemary oil (5%). In
the absence of rosemary extract, maximum counts of 109 CFU/mL were reached after 25 days at
5°C [251]. Fat from food and an emulsifier reduced the antimicrobial activity of plant oil [138].
Partitioning plant oil (hydrophobic) in the fat component of food may have decreased availability
of the antimicrobial agent for bacterial cells [129].
Compounds from plant oil that express listericidal (Table 6.3) or listeriostatic activity have
been identified. Carvacrol, cinnamaldehyde, eugenol, geraniol, and thymol, for instance, were
listericidal within 30 to 60 min of exposure [121]. Pinene was responsible for the listeriostatic
activity of rosemary oil, and 0.1 µl α-pinene/100 mL was sufficient to suppress growth of Listeria
in BHI at 35°C for >24 h [251]. These active compounds (e.g., thymol) damage the cell membrane
and may interfere with peptidoglycan synthesis [271].
Because of the potential link between consumption of chocolate milk and listeriosis, Pearson
and Marth [258] studied the effect of caffeine and theobromine, two methylxanthine compounds
in cocoa, against L. monocytogenes (103 CFU/mL, initially) in skim milk at 30°C. Limited antil-
isterial activity was observed with 2.5% theobromine; however, the authors found that 0.5% caffeine
exhibited antilisterial activity in skim milk through extending the lag phase ~3 to 6 h, increasing
the generation time 1 h, and decreasing the final maximum population ~1.4 log. The combination
of 2.5% theobromine and 0.5% caffeine slightly increased antilisterial activity when compared with
0.5% caffeine alone.
LYSOZYME
Lysozyme is an antimicrobial enzyme naturally present in foods of animal origin, including hen’s
eggs and milk. The enzyme is active against many Gram-positive bacteria [267] and has been
proposed for use in cheese [113]. Lysozyme is particularly attractive as a food preservative because
this enzyme is active between 4 and 95°C and over the pH range generally encountered in food
[155]. The mechanism of inactivation of Listeria by lysozyme is unclear, but appears to be nonlytic;
cell leakage but no peptidoglycan hydrolysis was observed in lysozyme-treated L. innocua [218].
Inhibition of L. monocytogenes by lysozyme in tryptic soy broth increased as temperature and pH
decreased [155].
The presence of lysozyme in food, on food surfaces, or on packaging surfaces contributes to
control of L. monocytogenes [31,130,169]. Listeriostatic or listericidal activity has been reported
in vegetables, fresh meat, processed meat products (e.g., ham), fish fillets, mayonnaise, milk, and
cheese [101,149,150,169]. Lysozyme activity in food, however, is diminished in the presence of
minerals and meat [150,169]. Addition of EDTA (1 to 5 mM) or lipase (12.5 IU/mL) enhances
lysozyme (0.02 to 0.1 mg/g) activity against the pathogen [150,198]. Lysozyme sensitized the
pathogen to mild heat [267]. Combinations including 0.05% potassium sorbate, 5 mM sodium
acetate, 0.95% ethanol, or 10 mM ascorbic acid did not substantially increase antilisterial activity
of lysozyme [149].
HYDROGEN PEROXIDE
The United States permits the use of hydrogen peroxide solution (< 35% H2O2) to sterilize multilayer
packaging materials in aseptic processing systems. According to FDA regulations, residual hydrogen
peroxide must be < 0.5 ppm [117] immediately after packaging. Hydrogen peroxide is used as a
preservative, particularly with raw milk, in several parts of the world. Use of this agent as a direct
food additive in the United States is very limited. The FDA permits adding 0.05% (w/w) hydrogen
peroxide to raw milk for making cheese [115]. Addition of 0.02% hydrogen peroxide to cheese
brine inactivates L. monocytogenes, when the brine is held at 12°C; lower levels were sufficient as
the storage temperature decreased [191].
Hydrogen peroxide (0.05%) was relatively ineffective in decreasing the Listeria count in raw
milk or milk artificially contaminated with equal numbers of S. aureus and S. faecalis [77]. It was
listericidal when combined with mild heat [327], lactic acid [327], or peroxyacetic acid [179]. The
population of L. monocytogenes decreased >5 logs when inoculated produce was sprayed or dipped
in a solution containing a mixture of hydrogen peroxide and lactic acid, at 1.5% each [327].
Alternatively, Listeria may be controlled when lactic acid bacteria that produce hydrogen peroxide
are immobilized on the surface of produce [138].
Hydrogen peroxide causes oxidative damage in bacterial DNA, RNA, protein, and lipids [271].
Catalase from L. monocytogenes detoxifies low levels of hydrogen peroxide. A high concentration
of this antimicrobial agent, however, overwhelms a cell’s catalase and hence overcomes the natural
resistance of this pathogen. Resistance of L. monocytogenes to 0.1% hydrogen peroxide was
observed after the bacterium was adapted to pH 4.5 to 5.0, 500 ppm H2O2, 5% ethanol, 7% NaCl,
or 45°C heat for 1 h [206]. Adaptation of L. monocytogenes to H2O2 increased the D56°C value of
this pathogen 2.9-fold [205].
LACTOPEROXIDASE SYSTEM
The lactoperoxidase system is a naturally occurring antimicrobial mechanism in milk. Activation
of this system has been proposed to extend the shelf life of raw milk in countries with inadequate
refrigeration. A functional lactoperoxidase system in raw milk may inactivate or inhibit growth
of Listeria. The lactoperoxidase system extended the lag phase of L. monocytogenes in the presence of
35 mg/L thiocyanate, 0.2 g/L glucose, and 1 mg/L glucose oxidase [30,295]. Listericidal activity
of the lactoperoxidase system was reported in ultrahigh temperature-treated milk supplemented
with potassium thiocyanate (84 mg/L), glucose (10 g/L), and glucose oxidase (2 mg/L) [87].
The lactoperoxidase system enhanced destruction of Listeria during thermal processing [166],
high-pressure processing [125], and nisin treatment [30]. However, processors should consider
the biphasic inactivation patterns of L. monocytogenes when the lactoperoxidase system is

activated [166].
Lactoperoxidase catalyzes oxidation of thiocyanate (SCN–) by hydrogen peroxide to hypothio-
cyanate (OSCN–) and hypothiocyanous acid (HOSCN) [187] as follows:
2SCN − + H 2O 2 + 2H + Lactoperoxidase
→(SCN)2 + 2H 2O
(SCN)2 + H 2O → HOSCN + H + + SCN −
HOSCN OSCN − + H + ( pKa = 5.3)
The hypothiocyanate and hypothiocyanous acid oxidize sulfhydryl residues (R-SH) in mem-
brane proteins of bacteria, generating sulphenic acid derivatives (R-SOH). When the lactoperoxidase
system is activated, leakage of potassium ions, amino acids, and peptides was first observed. Uptake
of glucose, amino acids, purines, and pyrimidines was subsequently inhibited. Consequently,
bacterial replication and protein synthesis decreased [187].
Efficacy of the lactoperoxidase system depends on sufficient quantities of lactoperoxidase and
reactants in milk. Lactoperoxidase represents 1% of whey proteins [274], which is adequate for a
functional lactoperoxidase system. Bovine milk naturally contains 1 to 7 ppm thiocyanate. Supple-
mentation of milk with thiocyanate (>35 mg/L, final concentration) is necessary for satisfactory
listeriostatic activity of the lactoperoxidase system [87]. Hydrogen peroxide must also be added
exogenously or generated by exogenous enzymes such as glucose oxidase [75]. This enzyme
oxidizes glucose to gluconic acid and hydrogen peroxide. Production of gluconic acid decreases
the pH; this may increase the bactericidal effect of the lactoperoxidase system beyond what would
have been observed in similar model systems with pH values near neutrality [75]. Listeria mono-
cytogenes was inhibited and inactivated in milk containing ≥0.75% gluconic acid during extended
incubation at 13 and 35°C, respectively [95]. The lactoperoxidase system in TSBYE was more
effective at 5 to 10°C than at 20 to 30°C; corresponding increases in lag time were 32 to 98 h and
2.8 to 8.9 h [295].
LACTOFERRIN
Lactoferrin is an iron-binding glycoprotein found in mammalian milk. At 23 to 46 mg/mL, lacto-
ferrin was slightly listeriostatic in milk stored at 35°C for 18 h [255]. Its listericidal action is
localized in the N-terminus domain of the protein and not at its iron-binding site [20,76].
Dialysis reduces the degree of lactoferrin saturation with iron, and the resulting product is
known as apo-lactoferrin. At 15 and 30 mg/mL, apo-lactoferrin was listeriostatic and listericidal
in milk stored at 35°C for 18 h, respectively [255]. Lactoferricin B is a small antimicrobial
peptide (25 amino-acid residues) resulting from hydrolysis of the N-terminal region of bovine
lactoferrin by gastric pepsin [76]. At 1 to 3 µg/mL and ≥15 µg/mL, respectively, lactoferricin B
was listeriostatic and listericidal in peptone glucose yeast extract broth at 30°C [19].
Storage of milk at refrigeration temperature was recommended for optimal lactoferrin hydroly-
sate activity [237]. Activity of lactoferrin and related compounds strongly depends on composition
of the medium. Lactoferricin maintained its antilisterial activity over a pH range of 5.5 to 7.5. The
listericidal activity of lactoferrin or lactoferricin decreased as cation concentrations in the medium
increased [19]. Listeria monocytogenes grew in milk supplemented with ferric ammonium citrate
(0.125 M) and apo-lactoferrin (30 mg/mL) after incubation at 35°C for 18 h. In this study, Listeria
counts decreased 1.5 logs in the absence of ferric ammonium citrate [255]. A combination of 15
mg/mL apo-lactoferrin and 150 µg/mL lysozyme retarded growth of L. monocytogenes in milk at
36°C [256].
BIOCONTROL
The terms “biopreservation” and “biocontrol” refer to use of microorganisms or their metab-
olites to inhibit or inactivate undesirable microorganisms in food. Biocontrol may be attributed
to the starter microorganisms, their metabolites, and fermentation end products. Biocontrol of
L. monocytogenes in food is achieved by adding a bacteriocin-producing microorganism,
bacteriocin-containing fermentate, bacteriocin crude extract, or purified bacteriocin [238].
Bacteria that produce antilisterial bacteriocins include strains of Lactococcus lactis, Lactoba-
cillus bavaricus, Lb. reuteri, Lb. acidophilus, Lb. curvatus, Lb. saké, Lb. plantarum, Leuconos-
toc carnosum, Leuconostoc mesenteroides, Carnobacterium piscicola, Pediococcus acidilac-
tici, Propionibacterium thoenii, and Enterococcus spp. [186,209,238,347]. Numerous studies
listed potential applications of identified bacteriocins in food. This section gives an overview
of the most potent bacteriocins against Listeria and regulatory issues regarding applications
of bacteriocins in food. To list all bacteriocins with known efficacy against Listeria is beyond
the scope of this review.
Bacteria that have been used traditionally in food fermentation (i.e., most lactic acid bacteria)
include bacteriocin-producing strains. Starter cultures, including these bacteriocin-producing
strains, may be used to ferment food or food ingredients. Legal issues arise, however, when purified
bacteriocins are applied to foods. Approval of U.S. regulatory agencies, mainly the FDA, is required
before commercial use of purified bacteriocins [110]. A company can self-affirm whether a bacte-
riocin of interest is GRAS; however, the company is required to justify application of the bacteriocin
if requested by the FDA [110]. Among purified bacteriocins, FDA has approved only nisin, for use
in pasteurized cheese spreads [115].
LIVE FERMENTATE
Live fermentate is a preparation with high microbial biomass that is generally the product of
fermentation. The microorganism of interest is used as starter culture, but the exact composition
of the live fermentate is unknown because it contains microbial metabolites and the unused portion
of substrates. Live fermentate used for antimicrobial purposes may be incorporated in the food,
distributed over the food surface, or immobilized on the packaging surface in contact with the food
[138,238,303]. Lactic acid bacteria are most suitable for biocontrol purposes because these bacteria
are used in many traditional fermented foods and generally recognized as safe. These bacteria
compete with other microorganisms for nutrients or produce antimicrobial compounds, such as
weak acids, hydrogen peroxide, and bacteriocins [303].
Biocontrol of L. monocytogenes in refrigerated storage requires the use of suitable antagonistic
psychrotrophic bacteria. Nisin-producing lactococci are poor biocontrol microorganisms because
they do not grow well at refrigeration temperature or in meat products. Pediocin-producing pedi-
ococci were not suitable meat biopreservatives because they were effective against L. monocyto-
genes only at abuse temperatures [347]. The psychrotrophs C. piscicola L103 [290], Leuconostoc
carnosum 4010 [45], and several strains of Lb. bavaricus [339] were proposed as alternative
bacteriocin producers to prevent growth of Listeria at refrigeration temperature. Growth of the
pathogen may also be retarded in ready-to-eat, fresh-cut vegetables inoculated with Lb. delbrueckii
[138].
BACTERIOCINS
Bacteriocins are polypeptides synthesized by bacteria; these agents are generally active against
strains related to the producer [238]. Bacteriocins are suitable as processing aids, complementing
other preservation methods. Heat, freezing–thawing, acid [238], high hydrostatic pressure [163],
and pulsed electric field [163] were more listericidal in the presence of bacteriocins than they were
in their absence. Nisin and pediocin are the most investigated bacteriocins against L. monocytogenes.
Nisin is produced by a limited number of Lc. lactis subsp. lactis strains [67]. This bacteriocin is
useful in preventing outgrowth of Clostridium spp., including C. botulinum [64]. Pediocins are
produced by strains of Pediococcus spp. [74,197].
Dipping meat [104,352] or salmon [306] in a nisin solution (1.3 × 103 to 5.0 × 103 IU/mL)
inhibited growth of L. monocytogenes during refrigerated storage. Listeria populations decreased ~1.5
logs on packaged cheese stored at 4°C for 84 days when nisin (61.4 mg/cm2) was immobilized on
the package [288]. Nisin activity, however, varies with environmental conditions. Listeriostatic activity
of nisin increased with decreasing temperature [306] and pH [139]. Addition of 2% sodium chloride
enhanced the listericidal activity of nisin in laboratory media, particularly at levels < 10 µg nisin/mL
[139]. Stearic acid (≥10%) decreased the antimicrobial efficacy of nisin (1.0 × 105 IU/mL) [67] and
carbon dioxide sensitized the pathogen to nisin [241]. After 10 days, L. monocytogenes decreased ~4
logs in the presence of 2.5 µg/mL nisin in phosphate buffer (pH 6.2) with 100% carbon dioxide
atmosphere at 4°C [241]. Maximum counts were reached after 35 days’ incubation; in the absence
of nisin or when aerobic incubation was used, maximal counts were reached after 10 to 15 days [241].
Listeria monocytogenes strains vary in their sensitivity to nisin [21].
Listeriostatic activity of pediocin has been demonstrated in numerous foods, including meat
and dairy products [74,134,197]. Conflicting results were reported on pediocin activity in milk and
liquid eggs, likely because compounds in the crude extract offset pediocin activity [197]. Antilis-
terial activity of this bacteriocin in food slurries was improved when pediocin was encapsulated in
phosphatidylcholine-based liposomes or when 0.1% Tween 80 was added to the product [74].
Unlike nisin, pediocin is not in the FDA’s list of food-grade GRAS substances. Food supplemen-
tation with purified pediocin, therefore, requires regulatory approval in the United States. Pediocin
bound to heat-killed producer cells [134] or in dehydrated fermented whey permeate [197] was
recommended for food applications.
Bacteriocins such as nisin and pediocin disrupt the bacterial membrane, thereby depleting the
proton motive force of the membrane [134]. Acid adaptation or starvation increased resistance of
L. monocytogenes to nisin and pediocin [204]. Resistant Listeria subpopulations were not detected
when nisin was immobilized on edible cellulose-based packaging material [288]. Relatively stable
populations of bacteriocin-resistant mutants have been isolated from bacterial suspensions exposed
to a bacteriocin for extended periods [202]. Application of bacteriocin cocktails and rotation of
bacteriocins could ensure their efficacy against Listeria over extended processing times. Incorpo-
ration of bacteriocins in food, however, should not preclude use of proper sanitary practices.
MODIFIED ATMOSPHERE
“Modified atmosphere” refers to an altered gaseous environment that aims to extend shelf life of
a food. Proper packaging maintains the food under this modified atmosphere. A defined mixture
of nitrogen (N2), carbon dioxide (CO2), or oxygen (O2) commonly replaces air in modified-
atmosphere packaging [65]. A vacuum replaces air in vacuum-packaged food. Sous vide-processed
foods are vacuum packaged, then cooked, chilled, and finally stored at refrigeration temperature.
Modified-atmosphere packaging is frequently used to maintain the quality of fresh meat, vegetables,
and fruits at refrigeration temperatures [65]. Listeria monocytogenes grows well under aerobic and
anaerobic conditions and at refrigeration temperatures. These properties make L. monocytogenes
a potential threat to the safety of foods packaged under vacuum or modified atmospheres [65].
Growth of L. monocytogenes is not inhibited in food that has been packaged under vacuum [148]
or superatmospheric oxygen (>5 kPa O2) [4]. Presence of ≥80% carbon dioxide or 100% nitrogen
gas increased the lag phase of L. monocytogenes in Stilton cheese stored at 4°C by 2 to 3 weeks
[338]. The presence of 10% oxygen in a nitrogen-rich (e.g., 80% nitrogen) environment did not
satisfactorily inhibit growth of the pathogen [338]. An increase in the amount of carbon dioxide
increases the length of the lag phase and the generation time [106,338]. The presence of > 80% carbon
dioxide is recommended to retard growth of Listeria [106,184,338]. Growth inhibition by carbon
dioxide (≥ 50%) was more pronounced at 4°C than at 10°C, and at pH 5.5 than at pH 6.5 [106].
Incorporation of organic acids or bacteriocins increases the safety of modified-atmosphere
packaged foods. Inhibition of L. monocytogenes was increased by incorporating 0.5% sodium acetate,
2% sodium lactate, or 0.26% potassium sorbate into vacuum-packaged bologna stored at 4°C [336].
The presence of nisin in vacuum or modified-atmosphere packaging (gaseous mixture not described
by the authors) was slightly listericidal during storage of cheese and cooked ham at 4°C [288].
ALTERNATIVE PROCESSING TECHNOLOGIES

Heat is most commonly used for food preservation, but alternative methods are gradually being
implemented in food processing. Processors hope these alternative technologies will preserve
nutritional quality and fresh taste and texture while decreasing the risk of disease transmission.
Some alternative technologies may be applied to packaged food to overcome accidental postprocess
contamination of the product with L. monocytogenes. Physical alternative processes include radi-
ation, high hydrostatic pressure, pulsed electric field, and high-intensity pulsed light [176,189,281].
These alternative technologies are gradually gaining acceptance from regulatory agencies as
well as from consumers. For example, the United States has approved irradiation for control of
foodborne pathogens in fresh or frozen meat, dehydrated spices, seeds for sprouting, and whole
eggs [118]. Alternative technologies are perceived as nonthermal, although some heat may be
generated during their application [82,190]. However, processing conditions are generally selected
so that heat is minimal and quickly dissipated [189]. Control of pathogens is therefore accomplished
mainly by nonthermal means.
IRRADIATION
Microwaves, radio frequencies, high-intensity pulsed light, ultraviolet, electron beam, and
gamma are the forms of radiation of interest to food manufacturers. These radiations differ in
wavelength and penetrating power [27]. Gamma radiation and electron beams are the most
effective and applicable forms for decontamination of food [66,309]. Ultraviolet radiation has
poor penetrating power and is therefore limited to decontamination of surfaces, water, and juices
[118]. Pulsed light treatment controls microorganisms on food surfaces only [118]. Microwave
penetrates food, but treatment uniformity is questionable: Listeria spp. survived on chickens
cooked in microwave ovens [107]. Because microwave lethality is caused by heating, it will
not be addressed in this section.
Ionizing Radiations
Food irradiation, i.e., processing food with ionizing radiation, generally refers to treatment with
gamma radiation or electron beam. Cobalt-60 or cesium-137 emits gamma rays for food treatment.
Accelerators (<10 MeV) generate electron beams [118]. Contrary to gamma irradiation, electron
beam technology does not result in nuclear waste, and the treatment can be switched on and off.
Irradiation with x-rays for elimination of foodborne pathogens is not commonly reported in the
literature, even though the FDA allows its use [118]. Irradiation technology allows in-package
decontamination of refrigerated and frozen foods and thus results in safer, fresh-like, nonthermally
treated, ready-to-eat food. Packaging products before processing is preferable to prevent postprocess
contamination with pathogens such as L. monocytogenes. A radiation dose is frequently limited to
2 to 3 kGy to prevent development of off-odors, off-colors, and aftertaste in food [66,165].
Inactivation of L. monocytogenes by gamma radiation varies with the treated product (Table 6.4).
Successive irradiation treatments were more listericidal than an equivalent single treatment at 20°C,
TABLE 6.4
Gamma Irradiation Dose Necessary to Inactivate 90%
of an L. monocytogenes Populationa in Selected
Food Products
Food D-Value (kGy) Temperature (°C) Ref.
Ground beef 0.51–1.00 21 100

Raw turkey nuggets 0.56 4 313
Cooked turkey nuggets 0.69 4 313
Sandwich 0.71–0.81 –40 66
Ice cream 0.38 –72 165
Broccoli 0.51 –5 240
Corn 0.61 –5 240
Lima beans 0.77 –20 240
Peas 0.92 –20 240
a
Radiation D-value.
but not at refrigeration (4°C) or freezing (–80°C) temperatures [9]. Listeria innocua NADC 2841
in ground pork was substantially more resistant to electron beam irradiation than were three
L. monocytogenes strains [309]. This L. innocua strain is therefore a potential surrogate for process
validation studies [309]. Extrinsic and intrinsic resistance factors caused variations in the inactiva-
tion rate of L. monocytogenes by irradiation. Resistance of L. monocytogenes decreases with
treatment temperature [240]. Some food ingredients scavenge cell-damaging free radicals produced
during irradiation and therefore contribute to the resistance of pathogens to radiation treatment.
Resistance of L. monocytogenes to irradiation varied among strains [100] and decreased with
initial counts [9]. The reduced recovery of the pathogen on selective compared to nonselective
media shows that substantial numbers of Listeria were sublethally injured during exposure to
irradiation [100,309]. Irradiation breaks DNA strands and this disrupts replication. Radiation-
induced radicals may alter other cell components, such as the bacterial membrane [309]. Sublethally
irradiated cells are therefore sensitized to strong selective agents that may be present in agar
media [100,309].
Ultraviolet Radiation and High-Intensity Pulsed Light
The FDA permits food processing with ultraviolet radiation at 253.7 nm [118]. A population of
L. monocytogenes decreased ≥ 5 logs when treated with ultraviolet irradiation (100 µW/min/cm2)
for 2 min [348]. Survivor plots, however, indicated the presence of small Listeria subpopulations
that survived extended exposure to ultraviolet radiation [348]. Causes of tailing in this survivor
plot have not been identified. A population of L. monocytogenes attached to stainless steel decreased
~4 logs when exposed to 750 µW/min/cm2 ultraviolet radiation at 25°C [176]. This radiation dose
was not sufficient to eliminate the pathogen from the surface of chicken. Titanium dioxide (1 mg/mL)
generates free radicals when exposed to ultraviolet light (< 385 nm) and therefore synergistically
enhances ultraviolet activity against foodborne pathogens, including L. monocytogenes [170].
Pulsed light processing is based on emission of radiation of broad spectral range (200 to 1,100 nm)
by xenon flash lamps [118]. In the United States, total treatment permitted in food must be ≤ 12.0 J/cm2
[118]. Pulsed light (1 J/cm2) inactivated ~6 logs L. monocytogenes on the surface of nutrient agar
[83]. The population of L. monocytogenes decreased > 3 logs when exposed to white light (200 to
900 nm, with peak emission at 500 to 650 nm) for 100 pulses (100 nsec/pulse) at a frequency
TABLE 6.5
High-Pressure Processing Treatments Decreasing L. monocytogenes
4 Logs in Selected Foods
Food Pressure (MPa) Temperature (°C) Time (min) Ref.
Fruit juices 250 30 5.0 5

Jams 300 20 5.0 269
Ovine milk (6% fat) 300 5 15 127
Fresh pork loin 414 2 5.1 7
Fresh pork loin 414 25 8.7 7
Frankfurters 300 25 6.7 207
of 1 Hz [281]. In this study, no inactivation was observed when cells were treated with white light
that was low in ultraviolet rays (350 to 900 nm), which suggests that pulsed light inactivation
results from the bactericidal action of ultraviolet radiation [281].
HIGH-PRESSURE PROCESSING
Treating food with high hydrostatic pressure may improve a product’s safety without damaging its
quality. Pressures ≥ 300 MPa are commonly used and food pressure processors able to deliver up to
900 MPa are currently available. The pressure is transferred instantaneously and homogeneously
throughout the treated product, thus making this technique suitable for surface and in-depth decon-
tamination of food in flexible packages [350]. Adiabatic heating is generated as pressure increases
[270]. The treatment temperature refers to the average temperature during the pressure-holding time.
Listeria is particularly susceptible to inactivation at pressures ≥250 MPa (Table 6.5). Inactivation
increases with pressure [207]. D-values are difficult to estimate because survivor plots have a sigmoid
shape with considerable tailing during prolonged processing time [222,310]. With respect to pressure
resistance, heterogeneity among cells within a bacterial population may explain the tailing [324].
The efficacy of high pressure against L. monocytogenes varies with treatment conditions,
medium composition, and different strains of the pathogen. High-pressure inactivation of Listeria
follows a second-order relationship with processing temperature [7,127]. Inactivation of Listeria
in milk was minimal at around 20°C, intermediate at ~5°C, and highest at 50°C [127]. Food with
high levels of proteins or glucose favored survival of the pathogen during the pressure treatment
[294]. The level of fat in food did not consistently affect survival of Listeria [127,294]. Food
formulations containing bacteriocins [162], the lactoperoxidase system [125] or carvacrol [167]
synergistically enhanced the action of high-pressure processing against Listeria. Sensitivity to high
pressure varied among Listeria strains [125,310]. Interestingly, strains resistant to high pressure
[310] also expressed superior resistance to pulsed electric field and heat [188,190].
Pressure ≥ 200 MPa causes irreversible protein denaturation, rupture of cytoplasmic membrane,
and leakage of cell contents [189]. This range of pressure also induces autolysis of Listeria [218].
Expression of cold-shock proteins is induced in survivors of high-pressure processing [218]. During
storage, expression of cold-shock proteins also enhances the cell’s resistance to high pressure [337].
PULSED ELECTRIC FIELD PROCESSING

Pulsed electric field (PEF) treatment involves exposing food to pulses ≥20 kV/cm for a total
treatment time of microseconds to milliseconds [82]. The continuous PEF process is well adapted
to decontamination of liquids. The PurePulse system received a no-objection letter from the FDA
and can be used for acid and low-acid foods. Other systems are currently being developed for
industrial-scale processing of acid foods.
TABLE 6.6
Pulsed Electric Field Processing Parameters for Decreasing Listeria
spp. 2 Logs in Selected Foods
Food Electric Field (kV/cm) Treatment Time (µs) Ref.
Whole milk 35 300 273

50% Acid whey (pH 4.2) 25 ≤48 190
Liquid whole egg 40 64 53
Pulsed electric field rapidly inactivates Listeria in fluid products (Table 6.6). Numerous processing
parameters influence the bactericidal efficacy of the process. Inactivation of Listeria spp. with PEF
increases with the electric field intensity, pulse length, pulse frequency, gap between electrodes, number
of product passages between electrodes, and process temperature [82]. Process efficacy decreases as a
product’s flow rate increases [82]. The efficacy also varies with food composition. Inactivation of Listeria
spp. increases with decreasing pH and conductivity [82]. The population of L. monocytogenes Scott A
decreased ~0.2 and 3.4 logs in sweet whey (pH 6.8; 0.46 S/m) and sweet whey acidified with lactic acid
(pH 4.2; 0.46 S/m), respectively, when processed with a PEF at 25 kV/cm and 23°C for 48 µsec [188].
Cell injury was detected when treated cells were grown on acidified agar [343].
Inactivation of L. monocytogenes by PEF varies among strains. Listeria monocytogenes OSY-
8578, a meat isolate with superior resistance to the process compared to other strains, is a potential
target strain for PEF process optimization [190]. Lactobacillus plantarum ATCC 8014 has greater
resistance to the process than does L. monocytogenes OSY-8578 in acid whey; therefore, it is a
potential surrogate microorganism for PEF process validation in this medium [188].
The electric field permeabilizes the bacterial membrane, resulting in leakage of intracellular
contents [54]. Expression of molecular chaperones (DnaJ, GroEL, and GroES) decreases ~5 to 20
min after the PEF process [188]. Cell permeabilization [342] and chaperone decrease [188] are
more pronounced in process-sensitive than process-resistant strains. When the input energy and
maximum treatment temperature exceeded ~80 J/mL and 60°C, respectively, damage of Listeria
cells resulted from the combined effect of heat and electric field [82]. Nonthermal treatment with
mild electric pulses strongly sensitized L. monocytogenes to mild heat [188]. The population of L.
monocytogenes decreased 3.3 to 6.1 logs when treated with heat (55°C for 10 min) 10 min after
PEF treatment (27.5 kV/cm for 144 µsec). Treatments with PEF or heat killed ~0.5 log only. Heat
sensitization subsequent to PEF treatment paralleled the decrease in expression of molecular
chaperones [188].
ATTACHMENT AND BIOFILM FORMATION

Listeria monocytogenes attaches to numerous surfaces, including stainless steel, glass, wood,
porcelain, iron, plastic, polyester, propylene, rubber, waxed cardboard, and paper [85,185,
212,236,301]. Consequently, equipment surfaces, conveyor belts, floor sealants, and drains are
potential reservoirs for Listeria spp. in food-processing plants and may lead to secondary food
contamination. Floor drains should therefore not be located next to filling and packaging equipment,
and using high-pressure hoses to clean these drains should be avoided to prevent formation of an
L. monocytogenes aerosol [300].
The ability of Listeria to attach to objects with different surface properties suggests that packaging
material is a potential source of contamination of food with this pathogen. Different materials,
however, were not equivalent in hosting the pathogen. Attachment was stronger on polyvinyl chloride
and polyurethane than on stainless steel [231]. Stainless steel was more supportive of surface colo-
nization by Listeria at refrigeration to ambient temperatures than was polytetrafluoroethylene [58].
1. Planktonic cells
2. Cell deposition on surface

• Hydrophilic interactions
• Flagella
3. Cell adhesion to surface

• Hydrophilic interactions
• ±Fibrils
• Synthesis of exopolymers
4. Surface colonization
• Cells monolayer
5. Biofilm formation
• Layers with variable cell density
• Homogeneous cell distribution horizontally
6. Biofilm development
• Biofilm growth
• Presence of capillary water channels (3-D structure)
FIGURE 6.7 Formation of L. monocytogenes biofilms under static conditions, on a flat nonporous surface.
Deviations from this representation may be observed among strains and with the properties of attachment
surface. (From Chae, M. S. and H. Schraft. 2000. Int. J. Food Microbiol. 62:103–111; Chavant, P. et al.
2002. Appl. Environ. Microbiol. 68:728–737; Kalmokoff, M. L. et al. 2001. J. Appl. Microbiol.
91:725–734; Mafu, A. A. et al. 1990. J. Food Prot. 53:742–746; Midelet, G. and B. Carpentier. 2002.
Appl. Environ. Microbiol. 68:4015–4024; and Vatanyoopaisarn, S. et al. 2000. Appl. Environ. Microbiol.
66:860–863.)
Attachment and biofilm formation of L. monocytogenes on solid surfaces progress in the

following sequence: cell deposition on the surface, adhesion to the surface, surface colonization,
biofilm formation, and biofilm development (Figure 6.7) [56,58,164,212,231,326]. Adhesion of
Listeria to surfaces has been attributed to hydrophilic interactions, presence of flagella, fibrils, and
synthesis of exopolysaccharides. Listeria monocytogenes is a hydrophilic microorganism with
surface free energy ~66 mJ/m2 [213]. Therefore, cell attachment and surface colonization are rapid
(≤ 2 h at 20 and 37°C) on hydrophilic substrata [58].
Because of its flagella, Listeria movement overcomes electrostatic repulsion forces of the surface
thereby facilitating attachment of the pathogen [58,326]. Strongest negative electrophoretic mobility
of L. monocytogenes (3.95 µm/s) was observed at pH 6.0 and 20°C [229]. Listeria produces flagella
at 20°C but not at 37°C; these are rich in negatively charged groups (COO–) [229]. The net charge
of the pathogen was neutral at pH 2.0 to 3.5 [213,229]. Listeria flagella also act as adhesive structures
during early stages of cell attachment [326]. Highly adherent strains synthesize fibrils [164]. Fibril
formation was observed at ≤ 21°C and pH 6.0 to 9.0, after 9-h incubation on an iron surface [300].
Exopolysaccharide fiber secretion, after a 1-h incubation at 20°C, seemed to aid attachment of
the pathogen to polypropylene and glass surfaces [212]. Attachment to stainless steel, polyvinyl
chloride, or polyurethane at 25°C was greater for Listeria, compared to Staphylococcus sciuri,
Pseudomonas putida, and Comamonas sp. [231]. Attachment of L. monocytogenes to surfaces varies
in the presence of other bacteria. The adherence of L. monocytogenes was enhanced in the presence
of Flavobacterium spp. [35], but decreased when the pathogen was cocultured with S. sciuri [195]
or with a nisin-producing strain of Lc. lactis [196].
The three-dimensional structure of L. monocytogenes biofilms changes with time, and it is
influenced by incubation conditions (i.e., static vs. constant agitation). After 2 to 3 days’ incubation
at 37°C, dense bacterial mats (i.e., biofilm) were formed on the colonized surface [56,58]. The
bottom layer of the biofilm had a higher cell density than the upper layer, after 72-h incubation at
37°C on a glass slide [56]. Cell distribution horizontally appeared homogeneous [56]. Water
channels are clearly observed with scanning electron microscopy as the biofilm develops [58,217].
Water channels are important to circulate nutrients and dissolved gases inside the biofilm, as well
as to eliminate metabolic waste products [58].
The structure and physiology of adherent cells are different from those of planktonic (i.e., suspended)
cells. Listeria monocytogenes in the stationary phase in a biofilm changed from a rod to a coccoid
shape as the population aged [319]. The pathogen grew more slowly when immobilized than in
a planktonic mode. Strains of L. monocytogenes varied in their ability to adhere and form biofilms,
even when exhibiting a growth pattern similar to that of planktonic cell populations [56,164].
The ability to mount a stringent response, via induction of the relA gene, and physiological
adaptation to nutrient deprivation were essential for growth of adhered Listeria [311].
Microscopic analysis shows an increase in cell density within the Listeria biofilm over time,
while enumeration on agar indicates a constant colony count [319]. This difference in count by
these two enumeration methods may have been caused by an increase in noncultivable or dead L.
monocytogenes as the biofilm aged [319]. During biofilm development, synthesis of the following
proteins was upregulated: pyruvate dehydrogenase, 6-phosphofructokinase, the 30S ribosomal
proteins YvyD and rpsB, superoxide dismutase, a sensing protein CysK, a DNA repair protein
RecO, and a cell division initiation protein DivIVA. These proteins are associated with carbohydrate
metabolism, stress response, quorum sensing regulation, DNA repair, and cell multiplication [319].
Synthesis of flagellin, a flagellar protein, decreased during biofilm growth [319]. This decrease was
expected because flagella help during early stages of cell attachment [326], but they may impair
formation of a structured biofilm.
Listeria in a biofilm is harder to remove and inactivate than when it is present as freely suspended
(planktonic) cells. Planktonic Listeria is less resistant to inimical treatments than is adherent
Listeria. Resistance to antimicrobial agents increased with the initial inoculum size [196], age of
the biofilm [247], and when the environmental temperature decreased [249]. Food soils protected
biofilms against removal by cleaning [143]. Proteins enhanced attachment to silica surfaces in the
following decreasing order: bovine serum albumen < β-casein and α-lactoglobulin < β-lactoglobulin
[3]. Active packaging is proposed as a means to prevent growth and attachment of the pathogen to
surfaces in direct contact with food. Coating surfaces with nisin, an antagonistic starter culture
(e.g., bacteriocin producer), egg white lysozyme, or bacteriophages was proposed to prevent
bacterial attachment and colonization of surfaces [31,196]. Surfactants also decrease adhesion of
the pathogen to surfaces [229].
SANITIZERS
Cleaning the food-processing environment is generally a multistep process [308]. A first rinse
removes loose soils. Detergents, followed by a second rinsing step, are then applied to eliminate
residual soils. The washed surface is then sanitized to inactivate residual microbial contaminants.
A final rinsing step with potable water removes the sanitizer and microbial residues. Sanitization
is a critical step that decreases pathogenic and spoilage microorganisms in food-processing
facilities and therefore prevents cross-contamination of food. Water may also disseminate micro-
organisms, unless a suitable sanitizer is added to the processing water. To be deemed effective,
sanitizing agents must reduce at least 5 logs of a given test organism population within 30 sec
of exposure at ambient temperatures [171]. Most sanitizing agents active against Listeria spp.
belong to one of five categories:
chlorine-containing compounds
quaternary ammonium compounds
acid sanitizers
ozone
iodophors
Efficacy of sanitizers decreases with temperature [214], increasing surface porosity [214], and
as an L. monocytogenes biofilm develops [247]. Plasmid-mediated resistance to multiple sanitizers
is a problematic phenomenon because it can be transferred at high frequency (8 × 10–6 to 1 × 10–3
transconjugant CFU per one donor CFU) between Listeria spp. and S. aureus [194].
CHLORINE AND CHLORINATED COMPOUNDS

Chlorine and its derivatives are used extensively in food, water, and surface decontamination.
Chlorinated compounds include hypochlorite solution, chloramines, and chlorine dioxide (gaseous
or aqueous). According to the Grade-A Pasteurized Milk Ordinance, utensils and equipment are
preferably sanitized with ≥ 50 ppm available chlorine at 24°C for ≥ 1 min [86]. A survivor plot of
Listeria exposed to 1 ppm available chlorine follows a biphasic pattern. The pathogen population
decreased rapidly during the first 2 min of exposure, whereas a subpopulation survived for > 60
min [90]. Listericidal efficacy of active chlorine is strongly impaired by organic loads, phosphate,
temperatures close to 25°C, and pH > 6 [89,90,307]. Although a solution containing 100 ppm
available chlorine was effective in broth [203], 200 ppm decreased populations only 0.6 log CFU
on tomatoes after 3 h [25]; 1,000 ppm inactivated 2 logs when the pathogen formed a biofilm [243].
Sensitivity of L. monocytogenes to chlorine varies among strains [90] and with the physiological
state of the Listeria population [89].
High chlorine concentrations may alter the taste of food and increase the risk of chlorine gas
release into the processing environment, thus increasing safety hazards for workers, and necessi-
tating the removal of chlorine residuals from wastewater. After considering chlorine’s advantages
and adverse effects, 120 to 200 ppm active chlorine is recommended for treatment of fresh fruits
and vegetables [25,307]. Addition of 10 to 100 ppm sodium hypochlorite to cheese brine will also
inactivate Listeria in this product [191].
Addition of chlorine or hypochlorite to water generates hypochlorous acid (HOCl) and
hypochlorite ion (OCl–) [86]:
Cl2 + H2O → HOCl + H+ + Cl−

Ca(OCl)2 + H2O → Ca2+ + H2O + 2 OCl–
Ca(OCl)2 + 2 H2O → Ca(OH)2 + 2 HOCl
HOCL H + + OCl −
The bactericidal activity of chlorine increases with the concentration of undissociated hypochlo-
rous acid [86]; therefore, chlorine activity increases with decreasing pH. Its dissociated form (OCl–)
has weak bactericidal activity [86]. Undissociated hypochlorous acid diffuses into the bacterial cell,
where this acid induces formation of toxic oxidative species and combines with proteins. This leads
to inhibition of mRNA, protein synthesis, and oxidative phosphorylation [86,271].
QUATERNARY AMMONIUM COMPOUNDS

Quaternary ammonium compounds are noncorrosive cationic surfactants frequently used to sanitize
equipment surfaces [208,318]. Application of 100 to 200 ppm active compounds on surfaces is
listericidal [203]. Listeria monocytogenes is slightly more resistant to quaternary ammonium com-
pounds than to other sanitizers [214]. However, when the pathogen was exposed to cleaning com-
pounds (4°C for 30 min) before treatment with quaternary ammonium compounds (25°C for 30 sec),
>7 logs were inactivated [308].
Quaternary ammonium compounds react with bacterial carboxylic groups. Consequently, the
cytoplasmic membrane is permeabilized and the cytoplasmic content may coagulate [318].
Enhanced resistance (≤10-fold) of L. monocytogenes to sanitizers has been reported after 2-h
exposure to sublethal concentrations of quaternary ammonium compounds [208]. Adaptation to
quaternary ammonium compounds involves a decrease in efflux pump activity [318] and modifi-
cations in cell wall structure [228]. Cells adapted to benzalkonium chloride were more elongated
and filamentous than unadapted cells [318]. Adapted cells doubled their contents of C17:0 (iso and
anteiso) fatty acids in the membrane and increased activity of the efflux pump system.
The multidrug efflux pump, MdrL, which prevents accumulation of chemicals in bacterial cells,
was present in L. monocytogenes adapted to sanitizers [278]. The gene encoding for this pumping
system in L. monocytogenes can be chromosomal and plasmid encoded [278]. The contribution of
MdrL to adaptation to sanitizers, however, is unclear [228,278]. Adaptation to multiple sanitizers
may contribute to persistence of the pathogen in food-processing environments. However, the
minimal inhibitory concentration of sanitizers against L. monocytogenes adapted to quaternary
ammonium compounds is below the concentration of sanitizers generally used in food-plant
sanitation [208].
ACID SANITIZERS
Acid sanitizers are nonvolatile agents that retain bactericidal activity at <100°C. They are frequently
used in conjunction with rinsing agents in automated cleaning (i.e., clean in place) systems. Acetic,
peracid, and peroctanoic acids, combined or not with other antimicrobials, are active against L.
monocytogenes. When L. monocytogenes adhered for 24 h to stainless steel and then was treated
with a combination of acetic acid (1%) and monolaurin (100 µg/mL) at 25°C, the population of the
pathogen decreased > 5 logs in 25 min [247]. At 80 ppm, peracid and peroctanoic acids were more
potent than chlorine against a Listeria biofilm in the presence of milk residues on stainless steel [108].
When fruits were inoculated with L. monocytogenes and washed with lactic acid (1.5%) and
hydrogen peroxide (1.5%) at 40°C for 15 min, pathogen populations decreased ≥ 6 logs [327].
Mixtures of peroxyacetic acid and hydrogen peroxide (120 ppm) were proposed as alternatives to
a chlorine wash for lettuce [307]. Inclusion of sodium lactate and sodium acetate in wiener or
bratwurst formulations inhibited growth of L. monocytogenes during extended storage at refriger-
ation temperature [133]. Dipping these sausages in lactate–diacetate solution was less effective
than including this mixture in product formulations [133].
OZONE
Ozone (O3) has been used in European countries for decades and has recently been approved in
the United States by the FDA for treatment, storage, and processing of foods, including meat and
poultry, unless use is precluded by a standard of identity [119]. Aqueous ozone reduces the microbial
load on surfaces of food, packaging material, and equipment [172]. Gaseous ozone minimized
TABLE 6.7
Inactivation of L. monocytogenes in Suspensions or on Food Surfaces after
Exposure to Ozonated Water
Temperature Ozone Inactivationa
Medium (°C) (ppm) Time (min) (log CFU/mL) Ref.
Water (pH 6.1) 20 0.2 2 5.0 275

Soluble starch solutionb 20 0.2 2 5.0 275
BSA solutionb,c 20 0.2 2 3.7 275
Phosphate buffer (pH 5.9) 25 1.8 0.5 7.0 173
Alfalfa sprouts 4 23 2 0.9 329
Beef 4 3 5 1.1 244
a Decrease in log count compared to control.
b 20 ppm, pH 6.0.
c Bovine serum albumin.
growth of microbial contaminants during storage of fresh produce [172]. In pure cell suspensions,
L. monocytogenes is inactivated rapidly by small concentrations of ozone. The pathogen population
decreased ~7 logs in 50 mM phosphate buffer (pH 5.9) after 30-sec exposure to 1.8 ppm ozone at
25°C [173]. Inactivation of L. monocytogenes increases with ozone concentration [111].
Listericidal activity of ozone varies with temperature, medium composition, strain, and phys-
iological status. At an equivalent ozone concentration and exposure time (0.25 ppm; 2-min expo-
sure), the inactivation rate of the pathogen almost doubled as temperature decreased from 37 to
4°C [111]. Processing at refrigeration temperature is therefore desirable to optimize the listericidal
activity of ozone. Organic compounds such as proteins react with ozone [275], thus reducing the
concentration of active ozone available for microbial inactivation in food. Consequently, efficacy
of ozone decreases when L. monocytogenes is present in food, compared to deionized water or
phosphate buffer (Table 6.7). Listeria is more sensitive to ozone than are other bacteria [173,275].
Sensitivity to ozone varied among strains of L. monocytogenes [111]. Resistance to ozone
increased as L. monocytogenes entered the stationary phase.
Ozone decomposes very rapidly in an aqueous solution; this generates numerous free radical species,
such as hydroxyl (⋅OH) and superoxide (⋅O2–) [172]. These reactive species have high oxidizing power
and initiate the oxidation of organic compounds. As expected from these findings, ozone causes oxidative
damage in bacteria. Cell wall, membrane, intracellular enzymes, and DNA are possible sites of damage
by ozone. Catalase and superoxide dismutase may protect L. monocytogenes against ozone; superoxide
dismutase is more important than catalase for this protection [111]. Sublethal treatment with ozone did
not sensitize L. monocytogenes to mild heat, alkali, or 6% NaCl [244].
MISCELLANEOUS SANITIZING AGENTS

Iodophors, such as octylphenoxy-polyethoxy ethanol iodine complexes, are used to sanitize surfaces
in contact with food. Iodophors are polymeric organic molecules that complex iodine species
(I–, I2, and I3–) [135]. Listericidal activity of 25 ppm available iodophor is equivalent to that of 200
ppm chlorine [203]. Listeria monocytogenes is ~10 times more resistant to iodophors when the
pathogen is attached to the surface of polypropylene or rubber than to stainless steel or glass [214].
The activity of these sanitizers against L. monocytogenes attached to stainless steel was 2 to 13
times higher at 20°C than at 4°C [212]. No inactivation of Listeria, however, was detected when
an iodophor (80 ppm I2) was used in the presence of milk or human serum [24].
Iodophors cause multiple damage in bacterial cells. The bacterial cell wall is permeable to
iodine, which interacts with double bonds of membrane phospholipids; oxidizes free sulfhydryl
groups; and combines with tyrosine, histidine, cytosine, and uracil [135]. Therefore, death of cells
treated with iodine was attributed to loss of membrane integrity, protein inactivation, and DNA
denaturation [135].
The population of L. monocytogenes decreased >7 logs CFU on tomatoes when surface-
inoculated fruit was sprayed with a 0.5% calcinated calcium solution (HYCEA-S, Kaiho Ltd.,
Tokyo, Japan) [16]. Addition of 100 ppm hydrogen peroxide inactivated the pathogen in contam-
inated cheese brines [191]. Suspended in peptone water, the population of L. monocytogenes
decreased >6 logs when incubated at 37°C for 8 h in the presence of a packaging material coated
with nisin (188 µg/mL) and lauric acid (200 mg/mL) [145]. This type of active packaging successfully
decreased L. monocytogenes by 1 log on the surface of contaminated turkey bologna, and no growth
was reported for >21 days at 21°C [73]. Compounds that have profound listericidal efficacy on
nonfood surfaces include sodium dichloroisocyanurate [24], glutaraldehyde [24], chitosan [179],
carvacrol [179], monolaurin [325], and listeriophages [144].
Listeria monocytogenes is ubiquitous in nature and prevention of cross-contamination during
handling of ready-to-eat food is essential. Regular use of listericidal hand sanitizers can contribute
to prevention of cross-contamination with Listeria during food handling. Kerr et al. [168] estimated
that 12 and 7% of food workers carried Listeria spp. and L. monocytogenes, respectively. Thimothe
et al. [314] detected Listeria spp. on 5.1% of employee-contact surfaces (gloves and apron) in
seafood-processing plants. Thorough hand rubbing for 30 sec with nonmedicated soap reduced the
aerobic mesophilic flora <1 log on hands contaminated by raw chicken [137]. Medicated soap
containing chlorhexidine achieved ~3 logs reduction under similar experimental conditions. A
peroxide-based powder (Ultra-Kleen) effectively decontaminated gloves previously dipped in
cooking water contaminated with Listeria spp. [221].
Image analysis of palm imprints made on agar plates (24.5 × 24.5 cm) can be used to predict
hand hygiene status [137]. In this method, subjects placed the palm of each hand on the surface
of agar plates for 15 sec, and imprints were allowed to dry at ambient temperature for 30 min
before incubation at 33°C and 60% relative humidity for 48 h [57]. Photographs of the palm imprint
were taken, scanned, and the degree of contamination of palms from food calculated by image
analysis (Image-Pro Plus, version 4.1.0.12) [137].
ACTIVE PACKAGING
Packaging materials containing agents with activity against L. monocytogenes are increasingly
investigated as a means to improve food safety after processing. Listeriostatic or listericidal agents
of active packaging are applied as coatings to surfaces in contact with food [71,145] or incorporated
into packaging materials [36]. Immobilizing antimicrobial agents such as organic acids, bacterio-
cins, spice extracts, lysozyme, chitosan, listeriophages, or EDTA onto packaging helps prevent
growth of Listeria on food surfaces [31,36,145].
Nisin (1 mg/mL) adsorbed on silanized silica inhibited some, but not all, strains of L. mono-
cytogenes [31]. Effectiveness of adsorbed nisin increased with bacteriocin concentration [31].
Adsorbed antimicrobials are generally more effective against Listeria when used in combination
rather than individually [145]. The listericidal activity of a nisin (4%)–lauric acid (8%) mixture,
however, was dramatically reduced when the pathogen was at the surface of turkey bologna rather
than suspended in 1% peptone water [73]. Organic acid anhydrides form an effective antimicrobial
coating on nonpolar polymers such as low-density polyethylene [36]. The anhydride hydrolyzes in
aqueous environments (e.g., moist food), and the resulting free acid migrates from the polymer
surface into the food [36].
A mixture of 2.5% silver ion and 14% zinc ion zeolite (sodium aluminosilicate) on the surface
of stainless steel inactivated >6 logs L. monocytogenes [71]. Similar listericidal activity was reported
when this zeolite was wet-process or powder coated [71]. When coextruded in plastic film, silverion
zeolite showed strong antimicrobial action, even though it was not tested specifically against Listeria
[36]. Bactericidal activity of silver ion zeolite was associated with transfer of silver ions to the cell,
which led to formation of reactive oxygen species [219].
MULTIPLE ANTIMICROBIAL TREATMENTS

Controlling L. monocytogenes in food may require application of multiple hurdles. Thermal pas-
teurization alone is generally considered adequate for eliminating this pathogen. This heat treatment,
however, may damage heat-sensitive nutrients and flavor compounds. Thermal treatments less than
pasteurization require additional hurdles (i.e., treatment combinations) to achieve the desired lethal-
ity [193]. Treatments that are commonly combined include mild heating (normally, 55 to 75°C),
addition of antimicrobial agents (e.g., nitrites), acidification, and nonthermal alternative processing
(e.g., ultrahigh pressure). These treatments may be applied simultaneously or sequentially to
maximize antimicrobial efficacy and retention of product quality.
When milk and liquid egg were experimentally treated with mild heat, limited inactivation of
Listeria spp. was observed. Combining heat with nisin resulted in greater listericidal action
(Table 6.8) compared to heat alone. Hurdles with lethal or growth-inhibitory action against food-
borne microorganisms may be combined, depending on food and processing conditions. Compat-
ibility between hurdles is essential and a synergistic or additive antimicrobial action is expected.
Synergistic action is most desirable; this is noticeable in combinations producing inhibition or
lethality greater than the additive actions of individual hurdles [53]. Satisfactory hurdle treatments
cause multiple cell injury, such as physical cell damage, disruption of cell homeostasis, or metabolic
exhaustion [193].
Multiple hurdles are particularly useful in eliminating L. monocytogenes in ready-to-eat meat
and poultry products [323]. Listeriosis is increasingly associated with consumption of these products;
TABLE 6.8
Inactivation of Listeria by Heat, Pulsed Electric Field (PEF), and High-Pressure
Processing (HPP) in Combination with Nisin
Food Processing Parameters Nisin (µg/mL) Log CFU/mL Decreased Ref.
Milk Heat: 60°C, 2 min 0 0.5–0.7 215

Heat: 60°C, 2 min 7 2.2–4.0 215
PEF: 40 kV/cm, 64 µs, 28°C 0 2.0 54
PEF: 40 kV/cm, 64 µs, 28°C 37 3.0 54
No treatment ≤ 37 0 54
Liquid whole egg Heat: 54°C, 16 min 0 1.9 178
Heat: 54°C, 16 min 10 3.0 178
HPP: 300 MPa, 20°C, 10 min
+ 0 day storage at 4°C 0 0.8 268
+ 0 day storage at 4°C 5 0.7 268
+ 18 days storage at 4°C 0 0 268
+ 18 days storage at 4°C 5 3.5 268
PEF: 40 kV/cm, 64 µs, 31°C 0 2.1 53
PEF: 40 kV/cm, 64 µs, 31°C 100 4.7 53
No treatment
+ 0 day storage at 4°C 100 0 291
+ 18 days storage at 4°C 100 0 291
recent recalls of contaminated batches have caused major economic losses to the industry [312].
Ready-to-eat meat and poultry products may be formulated to contain antilisterial agents (e.g.,
diacetate), are thermally treated, and occasionally may be stored in contact with antimicrobial
packaging materials [36,73]. Increasing awareness of possible postprocessing contamination of
ready-to-eat meat prompted processors to apply lethal treatments at late stages of production. These
are referred to as “post-lethality treatments” because they are applied after the traditional critical
control point, e.g., the main heating step during production of frankfurters [323]. In-package thermal
pasteurization and high-pressure processing are examples of post-lethality treatments that target
primarily environmental food-surface contaminants.
Careful integration of hurdles is crucial to the success of the combined treatment. Consideration
of hurdle–hurdle interactions is essential to avoid antagonism. Inactivation of L. monocytogenes
increased when high-pressure treatment increased from 300 to 700 MPa [310]. Using pressures
much lower in magnitude than those just described, Pagan et al. [248] observed some antagonism
between pressure and ultrasonic treatments. When ultrasonic waves of 117 µm and pressurization
were applied simultaneously, D40°C of L. monocytogenes decreased to a greater extent when pressure
increased from 0 to 100 kPa than from 300 to 400 kPa. It is presumed that ultrasonic treatments
inactivate bacteria through intracellular cavitation and high pressure counteracts this mechanism.
Improper application of hurdles may have a negative impact on the safety of treated food.
Treating L. monocytogenes with commonly used hurdles, such as mild heat and acid, induced stress-
adaptive responses and protected the pathogen against lethal preservation factors [17,206,345].
Simultaneous exposure to multiple hurdles, therefore, may be necessary to minimize the risk of
stress hardening of foodborne pathogens. Unlike conventional preservation technologies, emerging
alternative methods, such as high-pressure processing, target a limited number of loci in a microbial
cell [189]. These technologies cause limited microbial lethality and considerable stress and injury.
Adaptation of pathogens to stresses caused by application of these technologies may protect
pathogens from subsequent lethal treatments and thus compromise the safety of food.
Alternative processing technologies may benefit from the hurdle concept. Addition of bacteri-
ocins, for example, improved the lethality of high-pressure processing against L. monocytogenes
[268]. Treating L. monocytogenes with PEF decreased levels of molecular chaperones for a short
period after treatment [188]. When heat was applied to PEF-treated cells to coincide with maximum
depression in the level of chaperones, great lethality was observed. Applying heat before PEF
processing resulted only in a limited additive lethal effect. Careful choice of treatment combinations,
therefore, makes alternative technologies feasible in today’s food applications.
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DK3089_C007.fm Page 215 Monday, February 19, 2007 12:31 PM
7 Conventional Methods
to Detect and Isolate
Listeria monocytogenes
Catherine W. Donnelly and David G. Nyachuba
CONTENTS
Introduction ....................................................................................................................................216
Cold Enrichment ............................................................................................................................217
Selective Enrichment and Plating at 30 to 37°C...........................................................................219
Selective Agents ...................................................................................................................219
Potassium Tellurite ...................................................................................................219
Lithium Chloride/Phenylethanol ..............................................................................220
Nalidixic Acid...........................................................................................................221
Trypaflavine/Acriflavine ...........................................................................................221
Potassium Thiocyanate .............................................................................................222
Thallous Acetate .......................................................................................................222
Polymyxin B.............................................................................................................222
Moxalactam ..............................................................................................................223
Ceftazidime...............................................................................................................223
Selective Media for Enrichment and Isolation of Listeria............................................................223
Selective Enrichment Media ................................................................................................223
UVM Broth...............................................................................................................225
Fraser Broth ..............................................................................................................226
Isolation Media.....................................................................................................................227
McBride Listeria Agar (MLA).................................................................................227
LPM Agar .................................................................................................................228
Oxford Agar (OXA) and Modified Oxford Agar (MOX)........................................228
PALCAM Agar .........................................................................................................228
Other Selective Plating Media .................................................................................229
Comparative Evaluation of Direct Plating Media for Recovery
of Listeria from Foods .............................................................................................230
Incubation Conditions ..............................................................................................232
Oblique Illumination ............................................................................................................232
β-Hemolysis ............................................................................................................233
Official Methods for Isolating L. monocytogenes from Food .............................................234
FDA Method.............................................................................................................234
International Dairy Federation Method ...................................................................238
USDA–FSIS Method................................................................................................239
The Netherlands Government Food Inspection Service..........................................243
Research Advances.........................................................................................................................244
215
Considerations for Recovery of Injured Listeria ..........................................................................244

Conclusions ....................................................................................................................................248
References ......................................................................................................................................249
Die Methode ist alles.

Ralovich, German proverb [122]
INTRODUCTION
Listeria monocytogenes is a nonfastidious organism that can be subcultured on most common
bacteriological media such as tryptose agar, nutrient agar, and blood agar. However, attempted
isolation or re-isolation of Listeria from inoculated or naturally contaminated food and clinical
specimens by use of nonselective media is often challenging. Difficulties encountered in isolating
L. monocytogenes date back to initial characterization of this pathogen in 1926 when Murray and
his coworkers [127] stated, “The isolation of the infecting organism is not easy and we found this
to remain true even after we had established the cause of the disease.”
Although efforts to isolate L. monocytogenes from blood and cerebrospinal fluid of infected
patients have met with considerable success (mainly because of the presence of Listeria in pure
culture), obvious difficulties arise when food and clinical specimens (tissue biopsies and autopsy
specimens) contain small populations of L. monocytogenes in combination with large numbers of
other organisms. The first isolation methods were generally based on direct inoculation of samples
on simple agar media. Isolation was problematic in cases of low numbers of viable Listeria
cells, and inoculation into test animals (for example, embryonated eggs) was recommended. Upon
conducting experiments with guinea pigs and mice, Larsen concluded that this biological method
could be of value for detecting small numbers of Listeria in samples without competitive microflora
[104]. However, in the presence of other microorganisms, especially Gram-negative mixed flora,
test animals died of septicemia.
Direct plating, cold enrichment, selective enrichment, and several rapid methods can be used
in various combinations to detect L. monocytogenes in food, clinical, and environmental samples.
In 1948, Gray et al. [76] introduced the cold enrichment procedure as an alternative method to
isolate L. monocytogenes from highly contaminated samples. Although this method has contributed
much to present-day knowledge concerning the epidemiology of listeriosis, the prolonged incuba-
tion period necessary to obtain positive results is a serious disadvantage. Major improvements in
selective enrichment and plating media have since decreased analysis times from several months
to less than 1 week. Outbreaks of foodborne listeriosis coupled with the high mortality rates
associated with sporadic cases of illness and the advent of mandatory hazard analysis critical control
point (HACCP) programs have underscored the need for faster and more efficient methods to detect
small numbers of Listeria in a wide range of foods.
The purpose of this chapter is to review and update development of various enrichment broths,
as well as plating media and methods, used to isolate Listeria spp. (including L. monocytogenes)
from food, environmental, and clinical samples. Numerous enrichment broth and plating media
formulations have been developed and used during the past five decades for selective cultivation
of Listeria. Detection and isolation of Listeria remain complicated tasks. Unfortunately, researchers
have not yet been able to identify a single procedure sensitive enough to detect L. monocytogenes
in all types of foods within a reasonable time. Furthermore, many selective enrichment broths and
plating media fail to allow repair or growth of sublethally injured Listeria frequently present in
semiprocessed and processed foods [32] or food-processing environments.
Conventional Methods to Detect and Isolate Listeria monocytogenes 217
Despite these inherent shortcomings, research efforts in response to foodborne listeriosis outbreaks
have led to development of numerous regulatory procedures, including the U.S. Department of Agri-
culture Food Safety and Inspection Service (USDA–FSIS) and the U.S. Food and Drug Administration
(FDA) procedures [86,88,91,178]. Both of these methods have been adopted in the United States as
“standard methods” to isolate L. monocytogenes from a wide variety of foods and food-processing
environments. However, in an effort to detect more rapidly and reliably healthy and sublethally injured
Listeria in the wide range of foods currently examined, these methods and others have proved to be
somewhat insensitive and do undoubtedly require further modifications and refinement.
COLD ENRICHMENT
Difficulties in detecting and isolating L. monocytogenes typically arise when small numbers of
Listeria are present in environmental and clinical or food samples containing large numbers of
indigenous microorganisms. Hence, numbers of Listeria must be increased significantly, relative
to that of the background flora, before the bacterium can be detected. Thirteen years after the first
description of L. monocytogenes by Murray et al. [127], Biester and Schwarte [17] observed that
Listerella (Listeria) could be frequently isolated from naturally infected sheep organs that were
held refrigerated in 50% glycerol for several months. Although the organism was only rarely isolated
after initial plating of diluted specimens, Biester and Schwarte failed to comment on the significance
of cold storage.
Following similar chance observations, a young graduate student, M. L. Gray, recognized the
benefits of low-temperature incubation for recovering L. monocytogenes from clinical specimens.
In 1948, Gray et al. [76] reported that in three of five bovine listeriosis cases, L. monocytogenes
was only isolated after brain tissue was diluted in tryptose broth, stored for 5 to 13 weeks at 4°C
and then plated on tryptose agar. Although a few Listeria colonies were observed after directly
plating the remaining two brain tissue samples on tryptose agar, the bacterium was more readily
isolated following cold enrichment. These results clearly demonstrated the ability of L. monocyto-
genes to multiply to detectable levels in the presence of other microbial contaminants during
extended storage at refrigeration temperature (4°C).
Gray’s cold enrichment method, in which samples homogenized in tryptose broth were incu-
bated at 4°C and plated weekly or biweekly on tryptose agar during 3 months of storage, was soon
adopted as the standard procedure for recovering L. monocytogenes. Normally only a few weeks of cold
enrichment are required before Listeria can be detected; however, in one instance [74], 6 months of
refrigerated storage was necessary before L. monocytogenes could be isolated from calf brains. Although
the cold enrichment procedure is clearly slow and laborious, this method greatly enhances the likelihood
of isolating Listeria (if present) from a variety of specimens, including food.
In 13 studies summarized by Bojsen-Møller [21], Listeria was identified in 995 tissue and
organ specimens from naturally and experimentally infected domestic animals. Using direct plating
and cold enrichment procedures, Listeria was isolated from 684 of 995 (68.7%) specimens, whereas
307 of 995 (30.8%) specimens required cold enrichment before the bacterium could be detected.
Furthermore, cold enrichment failed to detect Listeria in only 4 of 684 (0.6%) samples that were
previously positive by direct plating.
A study by Ryser et al. [156] stressed the importance of cold enrichment for recovery of
L. monocytogenes from cottage cheese manufactured from milk inoculated with this pathogen.
Using direct plating, L. monocytogenes was recovered from 43 of 112 (38.4%) cottage cheese
samples stored at 3°C for up to 28 days, whereas cold enrichment of the same samples in tryptose
broth for up to 8 weeks yielded Listeria in 59 of 112 (52.7%) samples. Thus, cold enrichment was
necessary to detect this pathogen in 16 of 112 (14.3%) cheese samples. Ryser and Marth also found
cold enrichment to be of great value in detecting low levels of L. monocytogenes in cheddar [157],
Camembert [158], and brick cheese [160] manufactured from pasteurized milk inoculated with the
bacterium.
Lewis and Corry [109] compared cold enrichment and the FDA method [86] for isolating
L. monocytogenes and Listeria spp. from ready-to-eat (RTE) foods at retail in the United Kingdom.
Of the 57 food samples examined using cold enrichment, 5 yielded L. monocytogenes, and 2
L. innocua while the FDA method yielded 3 samples positive for L. monocytogenes only.
Despite the proven success of cold enrichment, the mechanism by which numbers of L.
monocytogenes are enhanced during prolonged incubation at 4°C is not fully understood. Although
cold enrichment exploits the psychrotrophic nature of L. monocytogenes and simultaneously sup-
presses growth of indigenous nonpsychrotrophic organisms, Gray and Killinger [74] indicated that,
at times, growth of Listeria was too rapid to attribute enhanced growth of this pathogen to mere
multiplication. When this procedure was first described in 1948, Gray et al. [76] suggested possible
involvement of an inhibitory factor in bovine brain tissue that suppressed growth of competing
organisms. However, this theory has since been dispelled by subsequent studies that demonstrated
enhanced growth of Listeria during cold enrichment of such diverse samples as mouse liver [144],
oat silage [73], feces [139], sewage [56], cabbage [78], raw milk [170], and cheese [156–160].
A more plausible explanation is that, in many clinical specimens, Listeria may exist within
monocytes, macrophages, or other phagocytic cells, with cold storage facilitating release of the
intracellular organism. More recent research on the role of cold-shock proteins, cold-acclimating
proteins, and other mechanisms that enable psychrotrophic growth of L. monocytogenes may help
further explain the preferential growth of Listeria during cold enrichment [11,98]. For instance,
anteiso-C15:0 fatty acid reportedly plays a critical role in adaptation of L. monocytogenes to cold
temperatures [4]; mutants deficient in this fatty acid have been shown to be cold sensitive.
As previously reported by Ryser and Marth [161], over 20 media formulations have been
successfully used to cold enrich a diverse group of samples naturally or artificially contaminated
with L. monocytogenes. Incubation at 4°C is partially selective for growth of L. monocytogenes,
so nonselective broths such as tryptose broth and Oxoid nutrient broth no. 2 (ONB2) rapidly
emerged as media of choice; tryptose broth was generally recognized as superior. In earlier studies,
cold enrichment was used as the sole enrichment procedure and was followed by plating a portion
of the enriched sample on tryptose agar at intervals during 2 to 12 months [164]. Following
incubation, plates were examined under oblique lighting for typical bluish green, Listeria-like
colonies. Cassiday and Brackett also have briefly reviewed the methods and media used by various
researchers [22a,35,36].
Although growth of L. monocytogenes is favored at 4°C, other organisms, including Proteus,
Hafnia, Pseudomonas, enterococci, and certain lactic acid bacteria are also able to multiply in
nonselective media at refrigeration temperatures [2], thus making detection of Listeria more diffi-
cult. To prevent overgrowth by non-Listeria organisms, investigators began adding selective agents
to various nonselective cold enrichment broths in an effort to inhibit non-Listeria microbes. In
1972, Bojsen-Møller [21] recognized that supplementing tryptose phosphate broth with polymyxin
B substantially reduced populations of Gram-negative rods (i.e., Escherichia coli, Pseudomonas
aeruginosa, and Proteus spp.) and enterococci while at the same time allowing rapid growth of L.
monocytogenes. Unfortunately, certain species of lactic acid bacteria resistant to polymyxin B can
ferment lactose to lactic acid and reduce the pH to the point where L. monocytogenes fails to grow
at 4°C. Attempts at maintaining a pH of 7.2 by adding 0.1 M MOPS (3-N-morpholino propane
sulfonic acid) to cold-enriched raw milk samples were unsuccessful [80].
Recovery of L. monocytogenes is enhanced when cold enrichment is used as a secondary
enrichment following selective primary enrichment at 30 to 37°C. Bannerman and Bille [10]
subjected numerous cheese and cheese factory environmental samples to secondary cold enrichment
in the FDA Listeria enrichment broth (LEB) following primary enrichment at 30°C for 48 h. After
plating enrichments on two selective agars, 34 and 62 of 96 isolates were obtained using warm
and cold enrichment, respectively. Thus, cold enrichment for 28 days resulted in a 29.2% (28 of
96) increase in recovery of L. monocytogenes from cheese and cheese factory samples. However,
with the advent of improved selective media and methods, most investigators have concluded that
cold enrichment offers no advantages over selective enrichment [82]. In addition, the lengthy
incubation period necessary for cold enrichment makes this procedure impractical for routine
regulatory analysis of foods that most often require quick reporting of results.
SELECTIVE ENRICHMENT AND PLATING AT 30 TO 37 °C

The principle of enrichment at elevated temperatures (30 to 37°C) is based on selective inhibition
of indigenous microflora through addition of inhibitory agents while at the same time allowing
unhindered growth of Listeria. Given the many months required for cold enrichment, the scientific
community soon became aware of the need for a shorter incubation period. In 1950, Gray et al.
[75] isolated L. monocytogenes from contaminated material that was inoculated into nutrient broth
containing 0.05% potassium tellurite and incubated at 37°C for 6 to 8 h before being plated on
tryptose agar with or without 0.05% potassium tellurite. Even though subsequent studies showed
both potassium tellurite-containing media to be partially inhibitory to Listeria [96,108,133,145],
Gray and his colleagues can still be credited with introducing the first cold enrichment procedure
and the first warm enrichment media for selective isolation of L. monocytogenes.
Since 1950, various combinations of selective agents have been added to basal media (i.e., tryptose
broth, ONB2, and tryptose phosphate broth) to obtain media suitable for selective enrichment of
Listeria at 30 to 37°C. Mavrothalassitis [120] reported an optimum incubation temperature of 30°C
for enrichment of L. monocytogenes from heavily contaminated samples. Results from at least two
additional studies [41,130] also showed that laboratory cultures of L. monocytogenes, L. seeligeri, or
L. ivanovii were more susceptible to commonly used Listeria selective agents (i.e., ceftazidime,
cefotetan, laxamoxef, and fosfomycin) when incubated at 37 rather than 30°C. Hence, most Listeria
enrichments are done at 30°C. Ryser and Marth [161] previously reviewed the wide range of media
formulations developed for selective enrichment of L. monocytogenes from environmental, clinical,
and food specimens.
SELECTIVE AGENTS
Modest, nonspecific nutritional requirements of L. monocytogenes have led to difficulties in formu-
lating media that enhance growth of this pathogen. Consequently, efforts have primarily focused on
inhibition of the indigenous bacterial flora by taking advantage of the resistance of L. monocytogenes
to various selective agents, including chemicals, antimicrobials, and dyes. The major advances that
have contributed to present-day ability to isolate Listeria from heavily contaminated environments
are shown in Table 7.1.
Although many inhibitory agents have proven to be at least somewhat useful for selective
isolation of L. monocytogenes from naturally and artificially contaminated biological specimens,
others have demonstrated very little value when added to basal media, as previously reviewed by
Ryser and Marth [161]. Throughout the following discussion of selective agents, one must keep in
mind that formulating media that are selective and now differential for L. monocytogenes is not a
straightforward process; many selective agents may partially or completely inhibit growth of this
pathogen, particularly when the organism is sublethally injured [32,46,131,167].
Potassium Tellurite
Many selective media, including the early formulation by Gray et al. [75], contain inhibitory
substances that are now of questionable value. As previously described, in 1950, Gray et al. [75]
examined the potential usefulness of potassium tellurite and sodium azide in Listeria-selective
media. Sodium azide prevented growth of L. monocytogenes in tryptose broth, whereas potassium
tellurite was quite selective for the pathogen. However, shortly after these findings were published,
Olson et al. [133] observed that potassium tellurite prevented growth of numerous L. monocytogenes
TABLE 7.1
Recognition of Selective Agents Useful in Isolation of Listeria
Year Compound Role in Selective Media Ref.
1950 Potassium tellurite Selective/differential for 24,75,96,100,108,121,133,145,171

Listeria that reduces tellurite
to tellurium, producing black
colonies
1960 Lithium Amplification of Listeria in 47,59,77,78,80,106,113,121,157,158,170
chloride/phenylethanol the presence of Gram-
negative bacteria
1966 Nalidixic acid Inhibitory to Gram-negative 1,20,52,55,71,92,96,134,135,147,166
bacteria through interference
with DNA gyrase
1971 Acriflavin(e)/trypaflavin(e) Inhibitory to Gram-positive 3,19,44,51,52,57,77,89,93,94,134,135,
cocci 145,146,148,149,152
1971 Polymyxin B Prevents growth of Gram- 21,43,47,113,134,152,168
negative rods and
streptococci
1986 Moxalactam Broad spectrum; inhibitory to 86,106,124,134
many Gram-positive and
Gram-negative
contaminants, including
Staphylococcus, Proteus,
and Pseudomonas
1988 Ceftazidime Broad-spectrum 10,114,117,130
cephalosporin antibiotic
strains. Other investigators [96,100,108,121,145] have substantiated these findings and discouraged
the use of potassium tellurite as a selective agent.
The advantage of adding potassium tellurite to selective media is that the resulting Listeria colonies
appear black from reduction of potassium tellurite to tellurium. Unlike the typical black-yellowish
and gray colonies produced by Gram-positive cocci, the marginal zone of Listeria colonies appears
green when the organism is grown on media containing potassium tellurite and viewed with oblique
illumination [164]. A modification of Vogel Johnson agar (MVJA) was evaluated by Buchanan
et al. [24] for isolating Listeria from foods. Selective agents, including moxalactam, nalidixic acid,
bacitracin, and potassium tellurite, permitted growth of Listeria while suppressing background
contaminants. Furthermore, the ability to distinguish colonies readily was not predicated on the
need for obliquely transmitted light.
Buchanan et al. [27] also found that lithium chloride-phenylethanol-moxalactam agar (LPM)
and MVJA generally gave comparable recovery of Listeria from naturally contaminated samples
of fresh meat, cured meat, poultry, fish, and shellfish. Adding tellurite and mannitol to MVJA
greatly aided in differentiating Listeria colonies from those formed by naturally occurring contam-
inants, including various species of enterococci and staphylococci. However, Smith and Archer
[171] reported that potassium tellurite prevented repair of heat-injured L. monocytogenes.
Lithium Chloride/Phenylethanol
Using the combination of phenylethanol and lithium chloride, McBride and Girard [121] suc-
ceeded in amplifying numbers of L. monocytogenes in the presence of Gram-negative bacteria.
The usefulness of phenylethanol and lithium chloride as Listeria-selective agents has since been
confirmed by other investigators, resulting in the earlier widespread use and acceptance of
McBride Listeria agar (MLA) as a plating medium for L. monocytogenes [38,59,77,78,80,106,
113,121,157,158,170]. A modification of MLA (omission of sheep blood and addition of cyclo-
heximide as an antifungal agent) was once recommended by the FDA for analyzing food samples
suspected of harboring Listeria [113,114]. Ryser and Marth [159,160] and Yousef and Marth
[186] reported that increasing the lithium chloride concentration to 0.5% (0.05% lithium chloride
in the original formulation [121]) increased selectivity of the medium without appreciably
decreasing recovery of healthy Listeria [159,160,183].
Nalidixic Acid
Beerens and Tahon-Castel [14] were first to report the usefulness of nalidixic acid in isolating
L. monocytogenes from heavily contaminated pathological specimens. Increased isolation of
Listeria using media containing nalidixic acid primarily resulted from inhibition of indigenous
Gram-negative bacteria [71]. The benefits of adding nalidixic acid to otherwise noninhibitory
media were soon confirmed in many laboratories [20,52,92,96,134,135,166]. After discovering
the benefits of adding nalidixic acid to enrichment broth [14], Ralovich et al. [147] effectively
used serum agar containing nalidixic acid to isolate L. monocytogenes from feces, organs, and
other clinical specimens.
Although the microbial background flora was largely inhibited on this medium, streptococci
and other nalidixic acid-resistant organisms occasionally persisted. Nalidixic acid was eventually
recognized as one of the most important selective agents, and it is now used alone or more commonly
in combination with other selective agents for isolating L. monocytogenes from food and clinical
specimens. Farber et al. [55] developed an improved Listeria-selective plating medium by combin-
ing the positive attributes of McBride Listeria agar and LPM agar. In their formula for Farber
Listeria agar, oxolinic acid was substituted for nalidixic acid. Both agents function by interfering
with the activity of DNA gyrase, an enzyme needed to maintain proper DNA structure and resealing
of chromosomal nicks [71].
Trypaflavine/Acriflavine
Despite successful use of nalidixic acid, Ralovich et al. [148,149] found that growth of certain
Gram-positive cocci and Gram-negative rods in the presence of this selective agent complicated
the isolation of Listeria. Such difficulties led to inclusion of trypaflavine, a known inhibitor of
Gram-positive cocci, in media containing nalidixic acid. This medium soon became known as trypafla-
vine nalidixic acid serum agar (TNSA). The end result was the selective inhibition of virtually all other
bacteria; growth of L. monocytogenes was only slightly decreased [19,133].
Following successful use of this medium in many European studies [19,93,134,135,148],
Ralovich et al. [145] endorsed TNSA as the plating medium of choice for isolating L. monocytogenes
from contaminated materials. Additional work revealed that contaminating organisms, predomi-
nantly streptococci, grew infrequently on clear media containing both antibiotics and were generally
discernible from L. monocytogenes with the naked eye. In 1972, Seeliger [165] reported that
combined use of acriflavine and nalidixic acid greatly suppressed Gram-negative organisms and
fecal streptococci without apparently affecting recovery of L. monocytogenes. These findings were
subsequently confirmed by Bockemühl et al. [20], who reported easy recovery of L. monocytogenes
from enriched fecal samples using an agar medium that contained nalidixic acid and acridine dye.
Confirmation of these findings in other European laboratories [52,57,77,94] led to widespread
use of trypaflavine/nalidixic acid as Listeria-selective agents. In 1974, Hofer [89] proposed using
a medium prepared from tryptose agar containing nalidixic acid, trypaflavine, and thallous acetate.
Trypaflavine can be replaced by other acridine dyes, including xanthacridine, acriflavine, or profla-
vinehemisulfate [146]. According to Gregorio et al. [77], use of nalidixic acid together with
acriflavine or trypaflavine gave rise to media that were equally inhibitory to background microflora,
suggesting that similar results can be obtained by substituting acriflavine for trypaflavine. Based
on results from European laboratories [44,51,93,146], a serum agar- or blood agar-based medium
containing trypaflavine, acriflavine, and nalidixic acid appeared to be satisfactory for selective
isolation of L. monocytogenes from samples containing a mixed microbial flora.
In 1984, Rodriguez et al. [152] developed a blood agar medium containing acriflavine and nalidixic
acid (Rodriguez isolation medium [RIM]) that was far superior to the earlier formulations of Ralovich
et al. [147,149]. During the last decade, numerous media containing acriflavine and nalidixic acid
with or without other antibiotics have been developed for selective isolation/enrichment of Listeria
from food and environmental samples, including Merck Listeria agar [22,79], which is commercially
available in Europe.
Potassium Thiocyanate
In 1961, Fuzi and Pillis [65] proposed a medium containing 0.35% potassium thiocyanate for
selective enrichment of L. monocytogenes. Although it was reported useful by some researchers
[52,107,166], others found that potassium thiocyanate inhibited L. monocytogenes [100,108,148].
Despite these reports, several studies demonstrated that an enrichment broth containing this selective
agent in combination with nalidixic acid was useful in isolating L. monocytogenes from cabbage
[78], milk [80,170], and other dairy products [102]. In 1972, Ralovich et al. [148] endorsed
Levinthal’s broth and Holman's medium, which contain nalidixic acid and trypaflavine, for selective
enrichment of Listeria. Results obtained by Slade and Collins-Thompson [170] demonstrated that
growth of L. monocytogenes in ONB2 containing nalidixic acid and potassium thiocyanate can be
improved by adding acriflavine.
Thallous Acetate
During the early 1950s, thallous acetate was employed as a selective agent for lactic acid bacteria;
however, it was not until 1969 that Kramer and Jones [100] recommended the combined use of
thallous acetate and nalidixic acid in Listeria-selective media. Three years later, Khan et al. [96]
found that, unlike potassium tellurite, thallous acetate used alone or together with nalidixic acid
did not adversely affect recovery of L. monocytogenes from biological specimens and silage
samples. In 1979, Leighton [108] demonstrated that the combined use of thallous acetate and
nalidixic acid completely suppressed growth of E. coli strains previously resistant to nalidixic acid.
Greater inhibition of Gram-positive bacteria also occurred when both selective agents were used
together rather than separately.
Although Leighton [108] recommended a medium composed of tryptose phosphate broth,
thallous acetate, and nalidixic acid for recovery of L. monocytogenes from mixed bacterial popu-
lations, thallous acetate (as well as potassium thiocyanate, potassium tellurite, and lithium chloride)
altered the colonial morphology of L. monocytogenes from the smooth to rough form. In view of
this experience, most currently used formulations of Listeria-selective media omit thallous acetate.
Polymyxin B
In 1971, Despierres [43] reported that the combination of polymyxin B and nalidixic acid was useful
for recovering L. monocytogenes from feces and that these antibiotics prevented growth of many
background organisms, including Enterococcus faecalis. That same year, Ortel [134] proposed another
medium containing polymyxin B and bacitracin to isolate L. monocytogenes from stool samples. Accord-
ing to Bojsen-Møller [21], Gram-negative rods and enterococci failed to grow in tryptose phosphate
broth containing polymyxin B, but growth of L. monocytogenes was relatively unaffected. After exam-
ining six different enrichment and isolation media, Rodriguez et al. [152] concluded that little if any
benefit was gained by adding polymyxin B to media already containing nalidixic acid and acriflavine.
Doyle and Schoeni [47] successfully isolated L. monocytogenes from milk and clinical and
fecal samples after enrichment in a selective broth containing polymyxin B, acriflavine, and nalidixic
acid that resembled isolation medium II developed by Rodriguez et al. [152]. Although the selective
enrichment broth developed by Doyle and Schoeni gained some attention [113], the necessity for
polymyxin B in this medium remains somewhat questionable. Siragusa and Johnson [168] success-
fully isolated L. monocytogenes from yogurt using a medium containing polymyxin B, nalidixic
acid, and acriflavine. Their medium reportedly prevented growth of Streptococcus thermophilus
and Lactobacillus delbrueckii subsp. bulgaricus, thus making it particularly suitable for isolating
L. monocytogenes from certain fermented dairy products.
Moxalactam
Results from antibiotic susceptibility tests [134] led Lee and McClain [106] to add moxalactam
(a broad-spectrum antibiotic that is inhibitory to many Gram-positive and Gram-negative bacteria,
including Staphylococcus, Proteus, and Pseudomonas) to MLA containing 0.25% phenylethanol
and 0.5% lithium chloride. The result was a highly selective medium for recovery of L. monocy-
togenes from raw beef and many other foods. This medium, LPM agar, is recommended by the
USDA–FSIS for isolating L. monocytogenes from raw meat and poultry [124] and also has been
incorporated into the current FDA procedure as a second selective plating medium [86].
Ceftazidime
Bannerman and Bille [10] used Columbia agar base in combination with acriflavine and ceftazidime
(AC agar), a broad-spectrum cephalosporin antibiotic, to isolate L. monocytogenes from cheese
samples. AC agar was superior to FDA-modified McBride Listeria agar (MMLA) [114,117]; it
recovered approximately 50% more L. monocytogenes isolates from soft cheese and cheese man-
ufacturing environments than did FDA-MMLA. Except for a few enterococci, the combination of
acriflavine and ceftazidime inhibited all other non-Listeria organisms, including yeasts and molds.
However, van Netten et al. [130] reported that PALCAM agar, which contains polymyxin B
and lithium chloride along with half or less the concentration of acriflavine and ceftazidime found
in AC agar, was superior to the latter medium. After comparing 13 different plating media, these
authors also concluded that media containing ceftazidime and 1.5% lithium chloride afforded more
selectivity than did phenylethanol alone. However, increased selectivity results in decreased recov-
ery of stressed or sublethally injured cells that are frequently present in foods.
SELECTIVE MEDIA FOR ENRICHMENT AND ISOLATION OF LISTERIA

SELECTIVE ENRICHMENT MEDIA
Frequent outbreaks of foodborne illness caused by L. monocytogenes in the recent past and the high
mortality rate associated with listeriosis have highlighted the need for more sensitive, reliable, and
rapid detection methods for the pathogen. The logical approach was to use some of the previously
described enrichment broths containing selective agents and to incubate samples at an elevated
temperature, generally 30°C. In response to numerous requests from the food industry, several enrich-
ment schemes have been developed that include primary or secondary selective enrichments.
An outbreak of listeriosis epidemiologically linked to consumption of pasteurized milk [63]
led Hayes et al. [80] to develop a two-stage enrichment procedure for isolating L. monocytogenes
from raw milk. Primary cold enrichment in ONB2 followed by secondary enrichment at 35°C in
ONB2 containing potassium thiocyanate (KSCN) and nalidixic acid and plating on GBNA yielded
the highest number of positive milk samples. No statistically significant difference in recovery of
Listeria was observed using Stuart transport medium or selective enrichment broth containing
potassium thiocyanate and nalidixic acid. Although 15 milk samples were positive when plated on
GBNA medium as compared with 11 on MLA2 without blood, the difference was not statistically
significant. The authors concluded that primary cold enrichment in ONB2 followed by secondary
selective enrichment at 35°C and plating on GBNA medium were the most useful for identifying
positive raw milk samples.
Slade and Collins-Thompson [170] developed a somewhat shorter two-stage enrichment pro-
cedure to isolate Listeria from foods. Their method was tested using raw milk inoculated to contain
approximately 100 L. monocytogenes CFU/mL. Results showed that tryptose broth was superior
to ONB2 as a primary cold enrichment medium. In addition, diluting milk samples 1:10, rather
than 1:5, increased the number of Listeria isolations on selective media. The more dilute samples
probably maintained a higher pH (≥6) during cold enrichment as a result of fewer lactic acid bacteria
and little lactose being present; this in turn led to faster growth and increased detection of Listeria
on solid media.
Original MLA without blood was the only medium tested that proved to be useful for plating
primary cold enrichments because tryptose agar and trypaflavine nalidixic acid agar were typically
overgrown by competing microflora. Favorable results were, however, obtained using tryptose agar
after secondary enrichment at 37°C. Addition of acriflavine to thiocyanate nalidixic acid broth
proved beneficial for recovery of L. monocytogenes. Thus, following 7 to 14 days of cold enrichment
in tryptose broth, L. monocytogenes was most frequently isolated after plating samples enriched in
thiocyanate nalidixic acid broth on MLA with blood or tryptose agar.
A “shortened” enrichment procedure and a two-stage cold/selective enrichment procedure were
developed in Canada by Farber et al. [54] for isolating Listeria spp. from raw milk. In the shortened
enrichment procedure, milk samples underwent primary and secondary enrichment at 30°C as well
as primary cold enrichment in two selective media (FDA enrichment broth and University of
Vermont medium [UVM]). Although no single step within the procedure was completely satisfac-
tory for isolating Listeria from raw milk, the two steps that were most helpful involved surface
plating the primary FDA enrichment broth culture on MLA2 with blood after 1 day of incubation
at 30°C, and surface plating the 30-day-old cold enriched FDA enrichment broth culture (initially
incubated 7 days at 30°C) on MLA2 with blood.
Collectively, these steps detected Listeria spp. in 31 of 51 (60.8%) positive raw milk samples.
Although 11 isolations were made after 1 day but not 7 days of primary selective enrichment at
30°C, 6 isolations were only possible after 7 days of primary selective enrichment. Thus, incubating
the primary selective enrichment at 30°C for 7 days before plating on MLA2 with blood markedly
enhanced recovery of Listeria from raw milk.
The two-stage cold/warm enrichment method, which was the second of two procedures devel-
oped by Farber et al. [54], also detected Listeria spp. in raw milk samples. Using this procedure,
Listeria spp. were isolated from 12 samples that tested negative using the shortened enrichment
procedure. Similarly, 10 samples that tested positive for Listeria spp. using the shortened enrich-
ment procedure were negative with the two-stage cold/warm enrichment method. Thus, when
used alone, neither procedure detected Listeria in all positive samples. Following cold enrichment,
similar numbers of samples were positive for Listeria spp. after enrichment in FDA enrichment
broth and UVM.
However, eight raw milk samples were only positive after 2 weeks of cold enrichment as
compared with three samples in which Listeria was only detected after 4 weeks of cold enrichment.
These results are similar to those of Doyle and Schoeni [47], who also observed that Listeria spp.
could be more readily isolated from raw milk and soft, surface-ripened cheese [48] during the first
2 weeks of cold enrichment.
Food-associated outbreaks of listeriosis along with discovery of L. monocytogenes in many
European varieties of soft- and smear-ripened cheese prompted two Swiss investigators, Bannerman
and Bille [10], to develop a two-stage selective/cold enrichment procedure to recover Listeria spp.
from cheese and dairy plant surfaces. Their isolation method is similar to the shortened enrichment
procedure just described [54] with the exception that the secondary selective enrichment step has
been eliminated and AC agar has been included as an additional selective plating medium. Using
this method, Listeria spp. were isolated from 157 of 1,099 (14.3%) cheese and environmental
samples. A total of 99 samples were positive for Listeria using both plating media. Following
selective enrichment, 56 of 99 (57%) and 35 of 99 (35%) samples were positive after surface-
plating enrichment cultures on AC agar and FDA-MMLA, respectively. Increased selectivity of AC
agar was presumably responsible for detection of approximately 50% more Listeria isolates as
compared with FDA-MMLA.
Important information concerning presence of Listeria spp. in food and environmental samples
can be gained using the three procedures just described as well as procedures developed by Hayes
et al. [80] and Slade and Collins-Thompson [170]; however, the need for cold enrichment in these
procedures increased the length of analysis to 30 to 40 days. Hence, the time constraints of this
method negate its use in any isolation procedure that is to be adopted by the food industry as a
“standard” enrichment method.
Rodriguez et al. [154] developed a complicated scheme to isolate Listeria from raw milk that
more importantly paved the way for subsequent development of several widely used enrichment
media, including UVM enrichment broth [33,45]. Their protocol included three noninhibitory
collection (primary enrichment) media, three selective (secondary) enrichment media, and one
selective plating medium, RIM III, all of which were previously described by Rodriguez et al. [152].
The three selective enrichment media used in this protocol contained nalidixic acid and trypan
blue with or without polymyxin B; nalidixic acid and acriflavine were used as selective agents in
the plating medium. Milk was added to all three collection media, with collection medium B
streaked onto RIM III after 7 and 15 days of storage at 4°C. Collection medium A was incubated
at 4°C for 24 h, subcultured in all three secondary enrichment media, which were incubated at
22°C until a color change occurred, and then samples were streaked onto plates of RIM II. A
portion of collection medium A also was diluted in collection medium C, which was streaked on
to RIM III following 7 and 15 days at 4°C. According to these authors, 11 L. monocytogenes
isolates were obtained after primary cold enrichment, with collection medium C accounting for 9
of 11 isolations.
Although results for collection medium C appear impressive, the increased number of isolations
using this medium may have resulted from a more dilute sample: approximately 1:40 as compared
with approximately 1:8 in collection media A and B. Under these conditions, collection medium
C should have maintained a higher pH during cold enrichment because fewer lactic acid bacteria and
less lactose were likely present, thereby enhancing the growth environment for L. monocytogenes. In
contrast to cold enrichment, 49 L. monocytogenes isolates were obtained following secondary
enrichment at 22°C with 16, 32, and 1 colonies originating from Rodriguez enrichment media
1, 2, and 3, respectively.
Recovery of only one Listeria isolate using Rodriguez enrichment medium 3 is not surprising
considering that collection medium A was diluted approximately 1:68 in collection medium C after
only 24 h of enrichment at 4°C. Transfer of the culture after 24 h of cold enrichment provides little
opportunity for appreciable growth of L. monocytogenes, so the organism was likely diluted out of
the sample. Overall, primary cold enrichment of milk samples diluted approximately 1:8 followed
by secondary enrichment in Rodriguez enrichment media 1 and 2 at 22°C and plating on an isolation
medium containing nalidixic acid and acriflavine provided the best opportunity for detecting
L. monocytogenes in raw milk.
UVM Broth
Selective media originally recommended by the FDA [114,117] and USDA–FSIS [123,124] for
enrichment of food samples containing L. monocytogenes were modifications of media proposed by
Ralovich et al. [149] and Rodriguez et al. [152] as modified by Donnelly and Baigent [45], respectively.
Donnelly and Baigent [45] explored the use of several selective enrichment media to inhibit growth
of raw milk contaminants and select for L. monocytogenes. The most successful medium for this
application was a modification of Rodriguez enrichment medium III [152]. This medium, designated
LEB by Donnelly and Baigent [45], consisted of proteose peptone (5.0 g/L), tryptone (5.0 g/L),
Lab-Lemco powder (5.0 g/L), yeast extract (5.0 g/L), sodium chloride (20.0 g/L), disodium phosphate-
2-hydrate (12.0 g/L), potassium phosphate monobasic (1.35 g/L), esculin (1.0 g/L), nalidixic acid
(40 mg/L), and acriflavine HCl (12 mg/L).
McClain and Lee [123] modified this formula to contain 20 mg/L nalidixic acid, and this
formulation was known as USDA LEB I. These authors further modified LEB I to contain 25 mg/L
acriflavine and used this medium, LEB II, for secondary enrichment of meat and poultry samples.
USDA–FSIS currently recommends use of UVM broth (LEB I) for primary enrichment of meat,
poultry, egg, and environmental samples [33,91,178].
Fraser Broth
Fraser broth [64] is a modification of USDA LEB II which contains lithium chloride (3.0 g/L) and
ferric ammonium citrate (0.5 g/L). This medium reportedly was advantageous for detecting Listeria
spp. in enriched food and environmental samples. Because Listeria will turn Fraser broth black
from esculin hydrolysis within 48 h of incubation [23], this broth has now replaced USDA LEB
II in the USDA protocol as the preferred secondary enrichment medium for meat, poultry, and
environmental samples [91,178].
In 1986, Doyle and Schoeni [47] used the microaerophilic nature of L. monocytogenes in
developing a shortened one-step enrichment procedure to isolate this organism from milk as well
as fecal and biological specimens. In their protocol, the sample was placed inside an Erlenmeyer
flask equipped with a side arm and then diluted 1:5 in Doyle and Schoeni selective enrichment broth
(DSSEB). Following 24 h of incubation at 37°C in an atmosphere of 5% O2:10% CO2:85% N2, a
portion of the sample was streaked onto plates of MLA (original formulation with blood), which
were similarly incubated under microaerobic conditions. Using DSSEB, L. monocytogenes was
consistently isolated from raw milk samples inoculated to contain 10 L. monocytogenes CFU/mL.
In addition, about two and five times as many L. monocytogenes isolates were recovered from fecal
and biological specimens, respectively, using DSSEB rather than cold enrichment and direct plating.
Another enrichment procedure, which is partially based on microaerobic incubation, was devel-
oped by Skovgaard and Morgen [169] to isolate Listeria spp. from heavily contaminated samples,
including feces, silage, minced meat, and poultry. In this two-step enrichment procedure, microaerobic
incubation (24 h/30°C/95% air: 5% CO2) of the sample in USDA LEB I is followed by aerobic
secondary selective enrichment in USDA LEB II, after which untreated and KOH-treated samples
are surface plated on LPM agar.
Using this isolation scheme, which, with the exception of microaerobic incubation, closely
resembles the original USDA procedure, numerous fecal, silage, minced beef, and poultry samples
were positive for Listeria spp., including L. monocytogenes. Based on these results, the authors
concluded that their method was suitable for detecting Listeria in heavily contaminated materials,
including samples of raw ground beef and poultry. Although both procedures just described decrease
the Listeria detection time to approximately 3 days, incubating enrichment cultures under microaer-
obic conditions is particularly awkward and not feasible for large-scale testing programs.
A large listeriosis outbreak in which coleslaw was implicated as the vehicle of infection
prompted Hao et al. [78] to compare various media and methods to detect L. monocytogenes in
cabbage. Preliminary results clearly demonstrated a need for some type of enrichment procedure
before L. monocytogenes could be isolated from inoculated samples. After comparing results from
various plating and enrichment media, these investigators proposed a two-step enrichment procedure
for isolating L. monocytogenes from cabbage. A cold enrichment period of 14 or 30 days at 5°C
in ONB2 or brain heart infusion broth (BHI) led to increased recovery of Listeria from cabbage
following secondary enrichment (30°C/48 h) in FDA enrichment broth or ONB2 containing potas-
sium thiocyanate and nalidixic acid.
A comparison of nine selective plating media, with and without an additional 5 mg of Fe3+/L,
led to the recommendation of modified Doyle/Schoeni selective agar II and MLA with glycine
anhydride rather than glycine (MLA2) for isolating L. monocytogenes from cabbage. Both media
contained 5% sheep blood, which was beneficial for picking Listeria-like colonies. As was true for
the cold enrichment broths, several popular plating media, including FDA-MMLA and LPM agar,
were not examined in this study. However, their efficacy in isolating L. monocytogenes from cabbage
and other vegetables needs to be determined before recommending this procedure for use in routine
analysis of such products.
Despite repeated efforts toward developing an effective enrichment medium for recovery of L.
monocytogenes, no one single selective enrichment broth has proven to be totally reliable for
analysis of food products containing Listeria. Nevertheless, several enrichment broths have moved
to the forefront, including the FDA enrichment broth [86], UVM broth [91,178], and Fraser broth
[91], all of which are commercially available from BBL, Difco Laboratories, and other manufac-
turers. Truscott and McNab [174] developed a selective enrichment medium called Listeria test
broth (LTB) as an alternative to UVM broth for detecting L. monocytogenes in meat products. After
primary and/or secondary enrichment of 50 frozen ground beef samples in both enrichment broths,
L. monocytogenes was detected in 19 of 50 (38%) and 16 of 50 (32%) samples using UVM and
LTB, respectively.
Although Listeria recovery rates for these two broths are not appreciably different, neither
medium alone was able to detect the pathogen in all 29 samples that were positive. In addition, L-
PALCAMY broth, which was developed by van Netten et al. [130], has shown superior results to
USDA LEBs I and II as well as the tryptose broth-based antibiotic medium of Beckers et al. [13]
for detecting L. monocytogenes in naturally contaminated cheese, minced meat, fermented sausage,
raw chicken, and mushrooms. However, given wide variations in the type and the number of
naturally occurring microbial contaminants in the food supply, development of a single enrichment
broth for truly optimal recovery of Listeria from all types of food appears improbable.
ISOLATION MEDIA
McBride Listeria Agar (MLA)
MLA was the first widely used plating medium for selective isolation of L. monocytogenes. This
medium, introduced by McBride and Girard [121] in 1960, is prepared from phenylethanol agar
to which lithium chloride, glycine, and sheep blood are added. At least seven subsequent changes
in the original formulation of MLA have led to considerable confusion as to the exact composition
of this medium. Ironically, the first reported modification of MLA by Bearns and Girard [12] dates
back to 1959, nearly 1 year before the original formulation appeared in the literature [121]. This
medium, named modified McBride medium (MLA2) by the authors and known today as one of
several modified MLAs, is similar to the original formulation except that sheep blood is omitted
and glycine anhydride is substituted for glycine [106].
In most instances, MLA2 was more Listeria selective than nalidixic acid agar [59,134], acri-
flavine nalidixic acid agar [170], or acridine nalidixic acid agar [59]. The selectivity of MLA2 can
be further improved, without affecting recovery of Listeria, by increasing the lithium chloride
content to 0.5%. With the further addition of sheep blood, this medium became partially differential
and hence was better suited than MLA2 for recovering L. monocytogenes from brick [160], feta
[137], and blue cheese [138], as well as cold-pack cheese food [159].
Following an earlier report in which glycine was found to partially inhibit L. monocytogenes
[106], many individuals began to replace glycine with glycine anhydride, which is far less
inhibitory to Listeria. Nevertheless, two widely used formulations of the original MLA containing
glycine have been commercially available since 1985 from Difco Laboratories and BBL (now
Becton Dickinson).
Although addition of blood provides one means of identifying possible L. monocytogenes
colonies (virtually all are at least somewhat β-hemolytic) and enhances growth of the pathogen
in certain B vitamin- and/or amino acid-deficient media, many workers preferred to omit blood
from the various formulations of MLA and examine the plates under oblique illumination for
blue to bluish green Listeria-like colonies. In 1987, Lovett et al. [117] added cycloheximide to
blood-free MLA2 and named this particularly useful medium FDA-modified McBride Listeria
agar (FDA-MMLA). Although one earlier study claimed that TNSA was superior to MLA2,
subsequent data indicated that FDA-MMLA [113,114,115,117] and MLA2 [69,78,80,117,
149,170], which contain glycine anhydride, were the MLA formulations of choice for isolating
Listeria spp. from foods, particularly dairy, vegetable, and seafood products. The FDA formu-
lation previously served as one of two plating media (the other is LPM agar) in the FDA procedure
[116].
LPM Agar
In 1986, Lee and McClain [106] added 4.5 g of lithium chloride and 20 mg of moxalactam to
MLA2 and named their new medium lithium chloride-phenylethanol-moxalactam (LPM) agar.
Although this selective medium (commercially available from Becton Dickinson is particularly
well suited for isolating Listeria from raw meat and poultry—as evidenced by its inclusion as the
medium of choice in an earlier version of the USDA procedure—LPM agar has since been
replaced by modified Oxford agar [33], which produces black Listeria colonies, each with a
black halo following 24 h of incubation. However, LPM plus esculin and ferric iron is still used
as one of the selective isolation agars in the FDA procedure [88].
Oxford Agar (OXA) and Modified Oxford Agar (MOX)
In 1989, Curtis et al. [42] developed an agar medium that eliminated the need for oblique
illumination. Their medium, Oxford agar (OXA), was prepared from Columbia agar base to
which several selective agents, including colistin sulfate (20 mg/L), fosfomycin (10 mg/L),
cefotetan (2 mg/L), cycloheximide (400 mg/L), lithium chloride (15 g/L), and acriflavine (5 mg/L),
were added. Esculin and ferric ammonium citrate also were added as differential agents to produce
black Listeria colonies from esculin hydrolysis. This medium was slightly modified by McClain
and Lee by incorporating moxalactam; this new medium was designated modified Oxford agar
(MOX) [33].
In May 1989, the USDA–FSIS procedure was changed to incorporate MOX as the recom-
mended plating medium. Late in 1990, the FDA modified its procedure by replacing FDA-
MMLA with Oxford agar (OXA). In the present version of the FDA method [88], one of the
following selective media must now be used: PALCAM, OXA, MOX, or LPM fortified with
esculin and Fe3+.
PALCAM Agar
In 1988, van Netten et al. [129] reported that RAPAMY agar, a modification of TNSA developed
by Ralovich et al. [149] that includes acriflavine, phenylethanol, esculin, mannitol, and egg yolk
emulsion, was suitable for enumerating Listeria spp. Virtually identical populations were observed
when overnight broth cultures of L. monocytogenes, L. seeligeri, and L. ivanovii were surface-
plated on RAPAMY and nonselective agar; growth of all non-Listeria organisms tested, except
Enterococcus faecalis and Enterococcus faecium, was completely inhibited on the selective medium.
Like OXA [42], RAPAMY agar also produced distinctive black Listeria colonies surrounded
by a dense black halo from esculin hydrolysis. Although such characteristic colonies were present
against a deep red background (inability to utilize mannitol) on RAPAMY agar, E. faecalis and E.
faecium generally produced colonies with blue-green halos. Although attempts to eliminate growth
of these two species of enterococci by adding cefoxitin (moxalactam) to this medium failed, results
suggested that RAPAMY agar could be used to quantify Listeria spp. in thermally processed and
dried foods with total aerobic plate counts of ≤106 CFU/g and Enterococcus counts of ≤102 CFU/g.
However, as might be expected, high populations of enterococci severely hampered detection of
Listeria spp. in chicken, minced meat, and mold-ripened cheese.
Further attempts by van Netten et al. [128] to eliminate growth of enterococci by adding
fosfomycin (20 mg/L) to RAPAMY agar met with only limited success. Addition of lithium chloride
(1.5%) to RAPAMY agar inhibited many Listeria spp.; however, an improved selective and differ-
ential medium was obtained by adding lithium chloride to RAPAMY agar and omitting nalidixic
acid. The resultant medium was named ALPAMY agar because it contains acriflavine, lithium
chloride, phenylethanol, esculin, mannitol, and egg yolk emulsion agar.
In a study with pure cultures, ALPAMY agar allowed uninhibited growth of all 10 L. monocytogenes
strains tested but completely prevented growth of single strains of L. seeligeri and L. ivanovii. Selectivity
tests showed that ALPAMY agar supported growth of only 2 of 41 non-Listeria organisms—one strain
each of Staphylococcus aureus and Micrococcus spp., both of which were readily differentiated from
Listeria colonies. Subsequent studies indicate that ALPAMY agar is far superior to RAPAMY agar for
detecting Listeria in raw milk and soft cheeses manufactured from raw milk, as well as in raw vegetables
and chicken. This medium is the forerunner to PALCAM agar [130], which contains polymyxin B and
lithium chloride along with half or less the concentration of acriflavine and ceftazidime found in AC
agar. It is recommended that PALCAM agar plates be incubated for 40 to 48 h at 30°C under
microaerobic conditions (5% oxygen, 7.5% carbon dioxide, 7.5% hydrogen, and 80% nitrogen). This
medium, along with L-PALCAMY enrichment broth, is the basis for the Netherlands Government
Food Inspection Service (NGFIS) method for Listeria detection and isolation.
Other Selective Plating Media
Interest in foodborne listeriosis during the 1980s led to development of many additional Listeria-
selective media for examining milk and dairy products. In 1984, Martin et al. [119] developed gum
base nalidixic acid medium (GBNA), a synthetic agar-free solid medium superior to the MMLA
of Bearns and Girard [12] for isolating L. monocytogenes from raw milk [80]. Bailey et al. [8] also
found that a modified version of this medium containing lithium chloride and moxalactam was
suitable for isolating L. monocytogenes from raw chicken. A selective agar medium [78] based on
the enrichment broth of Doyle and Schoeni [47], from which acriflavine was omitted and Fe3+ was
added, compared favorably with the original formulation of MLA [121]. Supplementation of
selective [78] and nonselective [38] media with Fe3+ enhances growth of L. monocytogenes and
may be beneficial for isolating sublethally injured cells from food samples containing a mixed
microbial flora.
Attempts to isolate L. monocytogenes from food products have focused on enhancing the selec-
tivity of currently available blood-free plating media, as well as development of media that incorporate
differential agents other than blood to aid microbiologists in differentiating Listeria and L.
monocytogenes colonies in mixed cultures. In 1987, Buchanan et al. [24] found the combination of
moxalactam, nalidixic acid, and bacitracin to be effective in allowing growth of Listeria spp. while
preventing growth of most other foodborne organisms, including micrococci and streptococci. These
selective agents were used to formulate MVJ on which L. monocytogenes colonies appear entirely
black (reduction of tellurite) on a red background (due to the microbe’s inability to use mannitol).
Thus, suspect Listeria colonies could be readily identified on MVJ without using oblique illumination.
Adding the same three selective agents to the MMLA of Bearns and Girard [12] resulted in
Agricultural Research Service-modified McBride Listeria agar (ARS-MMLA) which could be used
in conjunction with oblique lighting to quantitate Listeria in a wide range of dairy and meat products.
In a subsequent study, Buchanan et al. [26] found that MVJ was slightly superior to ARS-MMLA
for recovery of L. monocytogenes from inoculated samples of milk, dairy products, meat, and
coleslaw. Although ARS-MMLA was more selective than MVJ, the black Listeria-like colonies
that appeared on MVJ were more readily discernible. Initial comparisons of ARS-MMLA and MVJ
with LPM agar indicated that both of the new media functioned well.
In a follow-up study, Buchanan et al. [25] assessed the ability of MVJ and LPM Agar to detect
Listeria in retail samples of raw meat, fish, and shellfish. Listeria populations were generally too
low to be detected by direct plating on either medium. However, using USDA Listeria enrichment
broth I (USDA LEB I) in a three-tube/24-h most probable number (MPN) method, comparable
isolation rates were obtained for MVJ and LPM agar. The differential capability of MVJ was again
extremely useful in selecting presumptive Listeria colonies.
Comparative Evaluation of Direct Plating Media for Recovery

of Listeria from Foods
The need for reliable media in routine food analysis precipitated several studies to identify the
most suitable direct plating media. Rijpens and Herman [151] conducted a study to compare
selective and nonselective primary enrichments for detection of L. monocytogenes in cheese. A
completely selective enrichment procedure was compared with two partially nonselective protocols.
After enrichment for approximately 48 h, the enrichment media were streaked on selective agars
and presumptive Listeria colonies were confirmed using PCR. In some instances, PCR was also
done directly on the enrichment broth. The conventional, completely selective enrichment procedure
was not always the best choice to detect stressed L. monocytogenes in cheeses. The methods that
incorporated a nonselective enrichment step gave better results than the completely selective
method. However, for mold-ripened soft cheeses, best results were obtained using the completely
selective enrichment procedure.
In another study by Johansson et al. [90], enrichment in half-Fraser broth for 24 h at 30°C,
followed by plating onto L. monocytogenes blood agar (LMBA) and PALCAM medium combined
with additional streaking proved to be the most rapid and specific method to detect indigenous L.
monocytogenes populations from soft mold-ripened cheese in comparison with standard methods.
With a high sensitivity (93%) and a low detection limit (1 to 10 CFU/25 g–1), this procedure provided
negative and presumptive positive results within 2 to 3 days. Differences among LMBA, PALCAM,
and Oxford medium were highly significant (at 99% significance level). Overall, plating on LMBA
after standard enrichment protocols gave the best results.
An improvement in detection was also obtained by modifying the confirmation procedure. A
loopful of culture (an additional streak) from PALCAM or Oxford medium was streaked on a
nonselective medium in addition to streaking only separate colonies as specified in the standards.
A year-long survey of two Northern Ireland milk-processing plants for L. monocytogenes was
carried out by Kells and Gilmour [95]. Sample sites included the milk-processing environment
(walls, floors, drains, and steps), processing equipment, and raw and pasteurized milk. The FDA
Listeria-selective enrichment procedure was used to process samples and an additional agar
medium, LMBA, was utilized as part of the isolation procedure to compare its performance to that
of the recommended Oxford and PALCAM agars. LMBA was able to isolate L. monocytogenes
from 94.1% of sites compared to isolation rates of 76.5 and 79.4% using Oxford and PALCAM
agars, respectively.
Duarte et al. [50] examined four secondary enrichment protocols (conventional methods: UVM
II, Fraser 24 h and Fraser 48 h; and an impedimetric method: Listeria electrical detection medium)
for their ability to detect Listeria spp. and L. monocytogenes in fish and environmental samples
collected along the processing chain of cold-smoked fish. From all methods, Listeria spp. and L.
monocytogenes were present respectively in 56 and 34 of the 315 samples analyzed. Fraser broth
incubated for 48 h gave the fewest false-negative Listeria spp. results (4/56; [7.1%]), but concurrently
only 15/34 (44.1%) samples were correctly identified as containing L. monocytogenes. Listeria
electrical detection (LED) medium detected only 36/56 (64.3%) Listeria spp.-positive samples.
Despite this lower isolation rate, LED identified 20/34 (58.8%) L. monocytogenes-positive samples
correctly and gave fewer false positive results. The overall conclusion of these authors, similar to
those of many others, was that more than one isolation method is needed to estimate L. monocytogenes
contamination rates accurately.
Golden et al. [68], Hao et al. [78], and Cassiday et al. [34] collectively compared 20 selective
plating media for their ability to recover uninjured cells of L. monocytogenes from samples of
pasteurized milk, Brie cheese, ice cream mix, raw cabbage, dry-cured/country-cured ham, and/or
raw oysters inoculated to contain approximately 102, 104, and 106 L. monocytogenes colony-forming
units per gram or milliliter. Gum base nalidixic acid tryptose soya medium (GBNTSM), MLA2,
FDA-MMLA, and modified Despierres agar (MDA) were consistently superior to nine other media
used by Golden et al. [68] for enumerating all three inoculum levels of Listeria in samples of
pasteurized milk and ice cream mix. Ability to recover low levels of Listeria from both products
was facilitated by the lack of significant levels of non-Listeria contaminants.
Five of fourteen plating media used in this study failed to recover L. monocytogenes from
inoculated samples of pasteurized milk as well as Brie cheese and were therefore omitted for
analysis of ice cream and raw cabbage. Examination of Brie cheese containing approximately 102
and 104 L. monocytogenes CFU/g indicated that none of the nine remaining direct plating media
was sufficiently selective to prevent overgrowth of Listeria by molds, yeasts, and Gram-positive
cocci. Despite these inherent difficulties in detecting small numbers of Listeria, modified Rodriguez
isolation medium III (MRIM III), MLA2, FDA-MMLA, and MDA were judged to be satisfactory
when Brie cheese contained ≥106 Listeria CFU/g.
However, subsequent results from the same laboratory [35] indicate that LPM agar was superior
to these four media for isolating L. monocytogenes from Brie cheese. With raw cabbage, enumer-
ation of Listeria was a problem only at the lowest inoculum level where large populations of
microbial contaminants (i.e., Gram-positive and Gram-negative rods as well as Gram-positive cocci)
typically interfered with recovery. At the two higher inoculum levels, L. monocytogenes was readily
quantitated by direct plating on MDA, GBNTSM, and MLA2. However, this same investigative
team [35] later obtained even better results using LPM agar.
One year earlier, Hao et al. [78] successfully recovered L. monocytogenes from inoculated
samples of cabbage using GBNA, Doyle and Schoeni selective enrichment agar (DSSEA), DSSEA
+ ferric citrate, DSSEA + acriflavine + ferric citrate, thiocyanate nalidixic acid agar (TNAA) +
glucose + ferric citrate, and MLA2, but concluded that DSSEA + acriflavine + ferric citrate and
MLA2 outperformed the other media tested. When results from the previous three studies are
combined, LPM agar, GBNTSM, MLA2, FDA-MMLA, and MDA generally emerged as the plating
media of choice for detecting uninjured Listeria in dairy and vegetable products.
Overall, these findings agree with those of at least four other studies [66,84,102,110] in which
LPM agar outperformed other popular plating media, including FDA-MMLA, RIM III, and/or MVJ
for recovery of L. monocytogenes from raw milk, ice cream, yogurt, soft cheese, and/or vegetables
inoculated with the pathogen. In addition, Rodriguez et al. [153] found that RIM III containing 6
rather than 12 g of acriflavine hydrochloride was superior to the original formulation of MLA for
isolating L. monocytogenes from artificially contaminated raw milk and hard cheese. Although the
best media for recovering Listeria from dairy products and vegetables remain to be defined, OXA,
MOX, LPM, and PALCAM agar appear to be the present plating media of choice in the United
States for selective isolation of Listeria from such products as evidenced by their inclusion in the
FDA and USDA procedures [83,86,91,114,115].
Given the inherent differences that exist between the natural microflora found in various foods,
one can easily surmise that Listeria-selective plating media best suited for dairy products and
vegetables might be somewhat less than ideal for analysis of meat, poultry, and seafood. Conse-
quently, Cassiday et al. [34] evaluated 10 selective plating media for their ability to enumerate
L. monocytogenes in artificially contaminated dry- and country-cured ham as well as raw oysters.
According to their results, MDA, FDA-MMLA, and LPM agar recovered approximately equal
numbers of uninjured Listeria from dry-cured ham. However, ease in differentiating L. monocyto-
genes colonies from those formed by background contaminants led these authors to recommend
LPM agar for analysis of dry-cured ham.
Not surprisingly, LPM agar also was equal or superior to three other plating media (i.e., MRIM
III, MVJ, and UVM) deemed acceptable for isolating Listeria from country-cured ham. Unlike
both types of ham, high populations of indigenous microflora in raw oysters greatly complicated
detection of Listeria on virtually all 10 plating media. Although MRIM III and MVJ supported
less growth of Listeria than other marginally acceptable plating media (including MLA2, FDA-
MMLA, and GBNTSM), MRIM III and MVJ were somewhat more reliable for differentiating L.
monocytogenes from background contaminants. Therefore, these authors hesitantly recommended
MRIM III and MVJ for examination of raw oysters.
Several less extensive studies also have dealt with the ability of various plating media to recover
Listeria from meat, poultry, and seafood. According to a 1988 report by Loessner et al. [110],
recognition of L. monocytogenes in inoculated samples of raw ground beef and scallops was only
possible using LPM agar. Among the three other plating media tested, RIM III and the original
formulation of MLA proved to be insufficiently selective, whereas MVJ was inhibitory to the L.
monocytogenes strain tested. Unlike these findings, Garayzabel and Genigeorgis [66] indicated that
LPM agar and RIM III were acceptable for detecting Listeria in raw meat and both media were
superior to FDA-MMLA. Bailey et al. [8] found that LPM agar and GBNA fortified with lithium
chloride and moxalactam were superior to unfortified GBNA and MLA for recovering L. monocy-
togenes as well as other Listeria spp. from naturally contaminated raw poultry.
Incubation Conditions
Most plating media used to isolate Listeria are normally incubated aerobically at 30 to 37°C. Plates
containing popular selective media such as LPM agar or MOX agar normally are incubated for 48
h; plates containing pure or near-pure cultures of Listeria on nonselective media can generally be
examined after 24 h. Because growth of L. monocytogenes is reportedly enhanced under conditions
of reduced oxygen [164], inoculated plates [47,128,129,156–158,160] as well as selective enrich-
ment broths [38] have been incubated under microaerobic conditions (5% O2:10% CO2:85% N2).
Microaerobic conditions are especially recommended when using PALCAM agar.
OBLIQUE ILLUMINATION
Except for plating media that contain esculin, xylose, mannitol, or other differential agents, most
formulations of Listeria-selective plating media can be classified into one of two categories based
on presence or absence of blood. Recognition of Listeria-like colonies on blood-free media such
as MMLA, TNSA, and GBNA is greatly facilitated when colonies are observed under oblique
illumination with a binocular scanning microscope. When the Henry technique [85], in which plates
are examined under obliquely transmitted white light at an angle of 45° (Figure 7.1), is used,
Listeria colonies are small, round, finely textured, bluish green to bluish gray with an entire margin.
In 1984, Martin et al. [119] compared the appearance of L. monocytogenes on nalidixic acid
agar and tryptone soya gum base nalidixic acid medium and found that the uniformly transparent
nature of the gum-base medium greatly enhanced the bluish-green color of Listeria colonies when
observed under oblique illumination, as described by Henry [85]. Noting that the angle of trans-
mission in the Henry method is 135°, Lachica [101] found that the bluish-green hue of Listeria
colonies was more easily observed if plates were viewed from the backside at an angle of 45° with
a 5× magnification hand lens while colonies were directly illuminated with a high-intensity beam
of light that traveled perpendicular to the bench surface (Figure 7.2). This latter method has
FIGURE 7.1 Oblique illumination technique developed by Henry. Angles of reflected light (β) and transillu-
mination (α) equal 45 and 135°, respectively. (From Henry, B. S. 1933. J. Infect. Dis. 52:374–402.)
eliminated many of the problems (i.e., reproducibility and convenience) associated with the classical
technique developed by Henry [85] more than 70 years ago.
Given enough experience, either of these two lighting techniques can be used easily to differ-
entiate probable Listeria colonies from background organisms, even on heavily contaminated plates.
However, these procedures are time consuming and not readily adaptable for routine use in large
testing laboratories.
β-Hemolysis
Addition of blood to solid media also can be used to differentiate Listeria, including L. monocytogenes,
from other microorganisms. When grown on media containing blood, such as MLA, L. monocytogenes
colonies are typically surrounded by a narrow zone of β-hemolysis. In some instances, β-hemolytic
activity is so weak that the clearing zone cannot be observed until the colony is gently removed from
the agar surface.
FIGURE 7.2 Modified Henry technique developed by Lachica [101]. Angle of transillumination (α) equals
135°. (From Lachica, R. V. 1989. Annu. Meeting, Soc. Ind. Microbiol., Seattle, Washington, August 13–18,
Abstr. P-44.)
In 1989, Blanco et al. [18] proposed overlaying previously inoculated plates of blood-free
Listeria selective agar with a thin layer of blood agar so that the β-hemolytic activity associated
with pathogenic Listeria could be directly observed after reincubation. According to these authors,
hemolysis was more readily observed using this procedure than when blood was incorporated into
plating media before incubation. However, further work using highly contaminated samples such
as raw milk showed that the success of this procedure primarily depended on selectivity of the
initial plating medium, with highly selective media yielding the best results.
OFFICIAL METHODS FOR ISOLATING L. MONOCYTOGENES FROM FOOD

Heightened worldwide interest in foodborne listeriosis coupled with the advent of mandatory
HACCP programs for meat and seafood products in the United States has led to development of
more reliable commercial screening methods for Listeria. Two protocols developed in the United
States by the FDA and USDA–FSIS have emerged as “standard methods” to isolate L. monocytogenes
from dairy foods, seafoods, vegetables and meat, poultry, and egg products. Despite widespread
use of these methods in the United States, Canada, and Western Europe, both procedures are still
plagued with difficulties that include the inability to isolate Listeria from all positive samples as
well as difficulties in recovering sublethally injured cells.
In response to these concerns, the USDA–FSIS and FDA protocols have been modified to
enhance their ability to recover injured Listeria. Working in cooperation with the International
Dairy Federation (IDF), other official European agencies have developed somewhat similar proto-
cols partially based on current FDA methodology. In this section, positive and negative aspects of
the most widely used Listeria testing protocols will be discussed, along with identification of some
of the most critical steps involved in isolating L. monocytogenes from different foods.
FDA Method
The FDA method, originally developed by Lovett et al. [114,117], is the most frequently used
procedure in the United States for detecting and enumerating L. monocytogenes in milk, milk
products (particularly ice cream and cheese), seafood, and vegetables. The original protocol [114]
has been modified several times since it was developed [86,88,114,117,177]. The most current
standard FDA methodology [88] and permitted alternative rapid methodologies recommended to
detect and isolate L. monocytogenes from foods are as follows.
Presumptive contaminated food lots are sampled. Generally, subsamples are composited, if
required, according to FDA field laboratory instructions. Analytical portions (25 g) are pre-
enriched for Listeria species at 30°C for 4 h in buffered Listeria enrichment broth (BLEB M52),
equivalent [87] to AOAC/IDF dairy products enrichment broth [5,175] base containing sodium
pyruvate. Four hours after nonselective incubation, the selective agents (acriflavin, 10 mg/L;
sodium nalidixate, 40 mg/L; optional antifungal, e.g., cycloheximide 50 mg/L) are added. Incu-
bation for selective enrichment is continued at 30°C for a total of 48 h. The enrichment culture
is streaked at 24 and 48 h on one of the prescribed differential selective agars to isolate Listeria
species or L. monocytogenes.
Surveillance enumeration of L. monocytogenes levels in contaminated food is now required for
regulatory samples that test positive for the pathogen [88]. Detection may be done first and if
contamination is detected, a reserve sample portion can be tested for numbers. This is probably
the preferable method because, generally, only a small percentage of samples can be expected to
be positive and most often at low levels approaching 1 CFU/25 g. However, the option of combining
regulatory detection and enumeration is permitted.
Enumeration of L. monocytogenes in positive samples is performed on reserve samples by
colony count on L. monocytogenes differential selective agar in conjunction with MPN enumeration
using selective enrichment in BLEB with subsequent plating on ALOA (a diagnostic, chromogenic
isolation medium) [179a] or BCM [50,167] differential selective agar. Most samples are likely to
be negative for Listeria and most positive samples will only contain a few colony-forming units
per 25 g. Hence, it is efficient to delay enumeration of reserve samples until the Listeria detection
stage is completed.
Nevertheless, it may sometimes be more convenient to detect and enumerate simultaneously.
To accomplish this, the enrichment homogenate is prepared as described earlier and 0.1 mL is
immediately spread on ALOA, BCM, or an equivalent L. monocytogenes selective agar. Plates are
incubated at 35°C for 24 to 48 h. The combined minimal method will allow the cell number of
presumptive L. monocytogenes to be categorized as <0.04 CFU/g, 0.04 to 100 CFU/g, 100 to 25,000
CFU/g, or >25,000 CFU/g. More replica plates and more decimal dilutions in trypticase soy broth
with 0.6% yeast extract (TSBye M157) are optional to obtain more precise enumeration. Five
representative colonies are tested for ability to ferment L-rhamnose by the conventional fermentation
method, by the BCM rhamnose confirmatory agar, or by a rapid L. monocytogenes identification
kit to rule out definitively the uncommon occurrence of L. ivanovii in foods.
Alternatively, prescribed rapid detection kits with their respective enrichment media may be condi-
tionally used to screen for presence of Listeria contaminants. Putative Listeria isolates on selective agars
from standard or screen-positive enrichments are purified on nonselective agar and confirmed by con-
ventional identification tests or by a battery of such tests in kit form. Isolates may be rapidly confirmed
as L. monocytogenes (or not) by using specific test kits. Subtyping of L. monocytogenes isolates is
optional except for FDA isolates, which must be typed serologically, and strain typed by pulsed-field
gel electrophoresis (PFGE) and ribotyping. Nonobligatory pathogenicity testing of L. monocytogenes
isolates is described. The major changes in the revised FDA methodology (Figure 7.3) include:
Certain prescribed rapid detection kits and their enrichments are now authorized screening
alternatives to the standard selective enrichment.
It is now necessary to use only one instead of two of the several prescribed selective isolation
agars (Oxford agar, PALCAM, LPM plus esculin and ferric iron, MOX). Oxford agar is
still the preferred standard selective isolation medium. MOX has been added to the list of
prescribed selective agars and LPM without added esculin and ferric iron has been
removed. Use of the new chromogenic differential selective agars, like BCM, ALOA,
CHROMagar Listeria, and Rapid L’mono, is encouraged as long as it is in parallel with
one of the prescribed selective agars. The new agar media differentiate L. monocytogenes
and L. ivanovii colonies from those of other Listeria spp. and will greatly facilitate choosing
L. monocytogenes colonies when colonies of more than one species are present on a plate.
The Henry illumination technique is de-emphasized because only differential selective
isolation agars are prescribed.
The current enrichment medium, which resembles the step-1 enrichment of the internation-
ally harmonized method proposed by Asperger et al. [6], is basically unchanged. However,
pimaricin (natamycin), a much less toxic compound than cycloheximide, is introduced as
the alternative antifungal compound in the Listeria enrichment medium.
If L. monocytogenes is detected in a food sample, enumeration of the level of contamination
in the food is required.
The isolation procedure involves streaking BLEB culture onto one of the following esculin-
containing selective isolation agars: OXA or PALCAM or MOX or LPM fortified with esculin and
Fe3+. These esculin-containing media are listed in order of preferred use, subject to their availability.
OXA, PALCAM, or MOX plates are incubated at 35°C for 24 to 48 h and fortified LPM plates at
30°C for 24 to 48 h. It is strongly recommended that one of the L. monocytogenes–L. ivanovii
differential selective agars, such as BCM, ALOA, RapidL’mono, or CHROMagar Listeria be
streaked at 48 h (optionally at 24 h) in addition to the chosen esculin-containing selective agar.
This will reduce the problem of masking of L. monocytogenes by L. innocua.
Add 25 g or 25 mL sample to 225 mL BLEB
Stomach or blend Screening with

rapid detection kits
Incubate 24–48 h at 35°C
Spread 0.1 mL on ALOA M10a, BCM M17a or an

equivalent L. monocytogenes selective agar, and if
enumeration is required, more replica plates and
more decimal dilutions in TSBye
Streak onto
OXA/MOX and LPM (with or w/o

Esculin & Fe3+) or PALCAM
OXA/MOX LPM PALCAM
35° C 30° C 35° C

24–48 h 24–48 h 24–48 h
Examine Listeria-like colonies
FIGURE 7.3 FDA procedure for isolating L. monocytogenes from foods. (From Hitchins, A. D. 2003. In Food
and Drug Administration bacteriological analytical manual, 8th ed. AOAC International, pp. 10.01–10.13.)
BCM has been collaboratively validated by FDA [87]. An ISO TC34 SC9 comparative validation
showed that all the media (and a selective blood agar—LMBA, Sifin, Germany) inhibited Listeria
competitors more or less equally well. ALOA was preferred only because its formulation is public.
Another differential selective medium, chromogenic Listeria agar (M40b) is now available.
Listeria colonies are black with a black halo on esculin-containing media. Certain other bacteria
can form weakly brownish-black colonies, but color development takes longer than 2 days. Five or
more typical colonies are transferred from OXA and PALCAM or modified LPM or MOX to TSAye,
streaking for purity and typical isolated colonies. If BCM plates are streaked as recommended
previously and blue colonies are observed, they are presumptive L. monocytogenes colonies because
L. ivanovii is not often reported in foods. L. monocytogenes and L. ivanovii colonies on ALOA are
blue and have a zone of lipolysis around them.
Purification on TSAye is a mandatory step in the conventional analysis because isolated colonies
on selective agar media may still be in contact with an invisible weak background of partially
inhibited competitors. At least five isolates are necessary because more than one species of Listeria
may be isolated from the same sample. BCM and ALOA plates are used to help in reducing the
number of colonies that need to be picked. L. monocytogenes and L. ivanovii can be distinguished
using a commercial confirmatory medium (Biosynth International, Inc.) or by conventional
TABLE 7.2
Serotypes of Listeria Species
L. monocytogenes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a,
4ab, 4b 4c, 4d, 4e, “7”
L. ivanovii 5
L. innocua 4ab, 6a, 6b, Una
L. welshimeri 6a, 6b
L. seeligeri 1/2b, 4c, 4d, 6b, Un
a Un = undefined.
Source: von Koenig, C. H. et al. 1983. Infect. Immun.

40:1170–1177.
rhamnose/xylose fermentation broths or agars. TSAye plates are incubated at 30°C for 24 to 48 h.
The plates may be incubated at 35°C if colonies will not be used for wet-mount motility observations
(one of the many classical identification tests). For the approved rapid methods [88], the selective
isolation agar recommended by the manufacturer must be used but auxiliary use of the new
L. monocytogenes–L. ivanovii differential agars is also recommended.
Isolates should be typed serologically and genetically. For serological typing, commercial sera
are used to characterize isolates as type 1 or 4 or not type 1 or 4 (types 3, 5, 6, etc.) [88]. Table 7.2
exhibits the serological relationships of Listeria spp. Most L. monocytogenes isolates obtained from
patients and the environment are type 1 or 4. More than 90% of L. monocytogenes isolates can be
serotyped with commercially available sera. All nonpathogenic species, except L. welshimeri, share
one or more somatic antigens with L. monocytogenes. Serotyping alone without thorough charac-
terization, therefore, is not adequate for identification of L. monocytogenes.
The BCM L. monocytogenes detection system (LMDS), mentioned earlier [50,167], consists
of a selective pre-enrichment broth (LMPEB), selective enrichment broth (LMSEB), selective/
differential plating medium (LMPM), and identification on a confirmatory plating medium
(LMCM). Restaino et al. [150] explored the efficacy of the BCM LMDS using pure cultures and
naturally and artificially contaminated environmental sponges. The BCM LMPEB allowed growth
of Listeria and resuscitation of heat-injured L. monocytogenes. The BCM LMSEB, which contains
the fluorogenic substrate 4-methylumbelliferyl-myo-inositol-1-phosphate and detects phosphati-
dylinositol phospholipase C (PI-PLC) activity, provided a presumptive positive test for the presence
of pathogenic Listeria (L. monocytogenes and L. ivanovii) after 24 h at 35°C.
An initial inoculum of 10 to 100 CFU/mL of L. monocytogenes in BCM LMSEB yielded a
fluorogenic response after 24 h. On BCM LMPM, L. monocytogenes and L. ivanovii were the two
Listeria species forming turquoise convex colonies (1.0 to 2.5 mm in diameter) from PI-PLC activity
on the chromogenic substrate, 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate. L. monocy-
togenes was distinguished from L. ivanovii by its fluorescence on BCM LMCM or acid production
from rhamnose. False-positive organisms (Bacillus cereus, Staphylococcus aureus, Bacillus thur-
ingiensis, and yeasts) were eliminated by at least one of the media in the BCM LMDS. Using a
pure culture system, the BCM LMDS detected one to two L. monocytogenes cells from a sponge
rehydrated in 10 mL of DE neutralizing broth.
In an analysis of 162 environmental sponges from facilities inspected by the USDA, the values
for identification of L. monocytogenes by BCM LMDS and the USDA method were 30 and 14
sites, respectively, with sensitivity and specificity values of 85.7 and 100.0% with BCM LMDS
vs. 40.0 and 66.1% with the USDA method. No false-positive organisms were isolated by BCM
LMDS, whereas 26.5% of the sponges tested by the USDA method produced false-positive results.
Serology is useful when epidemiological considerations are crucial. A TSBye culture is used
to inoculate tryptose broth. This is incubated for 24 h at 35°C, the temperature at which flagella
(H) antigen expression is reduced. The culture is transferred to tryptose agar slants and incubated
for 24 h at 35°C. Both slants are washed in a total of 3 mL Difco fluorescent antibody (FA)
buffer and transferred to a sterile 16- × 125-mm screw-cap tube. These slants are heated in a
water bath at 80°C for 1 h. Cells are sedimented by centrifugation at 1600 g for 30 min, 2.2 to
2.3 mL of supernatant fluid are removed, and the pellet is resuspended in the remaining buffer.
The manufacturers usually provide recommendations for sera dilution and agglutination proce-
dures to be followed.
Genetic subtyping involves submission of data from pulsed-field gel electrophoresis (PFGE)
of DNA restriction fragments of FDA isolates to PulseNet (CDC, Atlanta, Georgia). Isolates should
also be ribotyped or sent to a ribotyping laboratory [184,185].
Present versions of the FDA procedure have greatly shortened and simplified isolation of
Listeria spp. from many foods compared with earlier methods developed to detect the pathogen in
clinical specimens; revised procedures have afforded many improvements over the original FDA
protocol [114,117]. In 1987, Doyle and Schoeni [48] compared the original FDA classic cold
enrichment and shortened enrichment procedures for their ability to recover L. monocytogenes from
90 samples of commercially produced, soft, surface-ripened cheese that was previously identified
as likely to contain L. monocytogenes. Although L. monocytogenes was isolated from 41 of 90
(46%) cheeses, no single procedure detected the pathogen in all positive samples. A total of 21
samples were positive after cold enrichment as compared with only 16 and 13 samples that were
positive using the FDA and shortened enrichment procedures, respectively. Thus, the latter two
protocols failed to recover L. monocytogenes from 5 of 21 (23.8%) and 8 of 21 (38.1%) samples
that were positive following cold enrichment.
Furthermore, because Listeria was never isolated from the same positive sample by all three
protocols, it appears that the original FDA method was inferior to cold enrichment. Similar results
were obtained by Doyle et al. [49] when these same three enrichment procedures were used to
isolate L. monocytogenes from milk samples after HTST pasteurization. Researchers in Canada
[55] and England [141] found negligible differences between numbers of Listeria recovered from
naturally contaminated samples of raw milk and soft cheeses analyzed by the FDA and cold
enrichment procedures, although both methods again failed to detect Listeria in all positive samples.
These variable findings for the original FDA and cold enrichment procedures have been attributed
to nonuniform distribution of Listeria within samples.
However, Doyle and Schoeni [48,49] found cold enrichment superior to the FDA method for
analysis of soft, surface-ripened cheese, when nonuniform distribution of Listeria is expected,
as well as in pasteurized milk. Hence, variations in the ability of the FDA and cold enrichment
procedures to detect Listeria in dairy products probably result from inherent differences between
the two methods (media, incubation conditions) and/or presence of microbial competitors rather
than nonuniform distribution of Listeria in the product. Although these results indicate that cold
enrichment was generally superior to the original FDA protocol, the time-consuming nature
of cold enrichment makes this procedure unacceptable as a commercial screening method for
L. monocytogenes.
International Dairy Federation Method
Using the original FDA method as a starting point, the IDF initiated development of a “reference”
method in 1988 [173] to recover L. monocytogenes from dairy products. Development of the IDF
method essentially followed that of the FDA protocol as previously reviewed by Ryser and Marth
[161] with the eventual elimination of pre-enrichment (for detecting sublethally injured Listeria)
and the KOH treatment of the enrichment broths before plating on Listeria-selective media. The
present IDF method [7] received AOAC approval in 1993 based on results from an AOAC
Add 25 g or 25 mL into 225 mL IDF Selective Enrichment Medium
Blend or stomach for about 2 min
If necessary, adjust pH
48 h/30°C
Streak onto Oxford Agar
48 h/37°C
Pick 5 presumptive Listeria colonies

(black with black halos) for confirmation
FIGURE 7.4 IDF procedure for isolating L. monocytogenes from milk and dairy products. (From Association
of Official Analytical Chemists. 1996. In Official methods of analysis of the association of official analytical
chemists. Gaithersburg, Md.: AOAC International, 17.10.01.)
collaborative study [175] assessing the ability of this method to recover L. monocytogenes from
inoculated samples of raw milk, ice cream, Camembert cheese, Limburger cheese, and skim milk
powder.
The AOAC-approved IDF method (Figure 7.4) closely resembles the FDA protocol (see Figure 7.3);
the sample is enriched in IDF enrichment broth that contains the same concentrations of selective agents
found in LEB [88]. Following 48 h of incubation at 30°C, enrichments are plated on Oxford agar as
opposed to the FDA procedure, which calls for Oxford agar and LPM without esculin/Fe3+ or PALCAM.
This method requires a minimum of 4 days to obtain presumptive results and continues to be popular
in Europe for detecting Listeria in dairy products.
USDA–FSIS Method
The USDA–FSIS devised a method for detecting L. monocytogenes in meat and poultry products
(Figure 7.5) [91,176,178]. The original USDA protocol developed in 1986 by Lee and McClain
[106,124] differs from the original and revised FDA procedures in that primary and secondary
enrichment steps are included for detecting Listeria. The original USDA procedure enabled Listeria
detection within 3 days compared with 9 to 11 or 5 to 6 days using the original and revised FDA
methods, respectively.
The original USDA–FSIS procedure was revised in May 1989 [33] and differed from the
original method in that LEB II was replaced by Fraser broth [64] as the secondary enrichment
medium; LPM agar was replaced by MOX; and the regulatory sample size was increased to 25 g.
Fraser broth and modified Oxford agar will blacken during incubation because Listeria spp. and
other contaminants can hydrolyze esculin; colonies of Listeria will exhibit black halos on modified
Oxford agar following 24 to 48 h of incubation. However, MOX is more selective than LPM or
Oxford agar [42], and staphylococci and streptococci are generally unable to grow on it.
Add 25 g meat sample to 225 mL UVM broth,

stomach 2 min
Incubate at 30°C
0.1 mL + 10 mL Fraser broth
Incubate 26 ±2 h Incubate 48 h
35° C 35°C
Streak onto MOX
Examine for black colonies If negative

(presumptive Listeria)
Streak onto MOX
Incubate 35°C, 48 h
Examine for black colonies
FIGURE 7.5 USDA procedure for isolating L. monocytogenes from red meat, poultry, egg products, and environ-
mental samples. (From USDA/FSIS. 2002. In Microbiology laboratory guidebook, 3rd ed., revision 3, chapter 8.)
Reported inadequacies in the previous [33] USDA–FSIS procedure were related to use of Fraser
broth for secondary enrichment. False-negative results caused by reliance on Fraser broth darkening
and a 24-h secondary enrichment have been reported by several laboratories [9,99]. Kornacki et al.
[99] compared recovery of L. monocytogenes from Fraser broth incubated for 26 vs. 48 h. L.
monocytogenes was isolated from 60 of 1,088 meat product and environmental swab samples from
meat and dairy plants. False-negative rates as high as 6.7% were attributed to the inability of L.
monocytogenes to be detected in Fraser broth at 26 h but not at 48 h, and to the failure of Fraser broth
to blacken. Furthermore, investigators failed to detect L. monocytogenes in eight Fraser broth enrich-
ments that were positive by primary enrichment. These findings clearly stress the importance of
incubating Fraser broth enrichments for 48 h.
The USDA–FSIS has recommended several modifications to its original procedure. The latest
USDA–FSIS method to isolate and identify L. monocytogenes from red meat, poultry, egg, and
environmental samples has been in effect since April 2002 [178]. This revised method includes use
of rapid screening tests and has a sensitivity of <1 CFU/g in a 25-g sample. In this revised protocol,
media required for enrichment, plating, and preliminary confirmation tests include modified Uni-
versity of Vermont broth (UVM, also known as UVM1), Fraser broth (FB), modified Oxford agar
(MOX), horse blood overlay agar (HL, also known as HBO), trypticase soy agar with 5% sheep
blood (TS-SBA, also known as CAMP test agar), and BHI broth. Additional media for environ-
mental samples include Dey–Engley (D/E) neutralizing broth (e.g., Nasco cat# B01256WA or
equivalent) and an optional medium, trypticase soy agar-yeast extract (TSA-YE).
At least one L. monocytogenes-positive control strain and one L. innocua-negative culture are
required; other Listeria spp., such as L. seeligeri, L. grayi, and L. ivanovii, may be necessary as
controls for additional confirmatory testing. If the β-lysin CAMP factor test is not employed, Staphy-
lococcus aureus and Rhodococcus equi are required to do the traditional CAMP test. A 25- ± 1.0-g
portion is used for raw and processed red meat, poultry, and egg product testing. Primary enrichment
in UVM broth is done as follows:
For all meat, poultry, and egg product samples, 225 ± 5 mL of UVM broth is added to 25-
± 1.0-g test portion, stomached or blended for 2 min, and then incubated at 30 ± 2°C for
22 ± 2 h.
For environmental sponge samples, 200 ± 5 mL of UVM broth are added to each bagged
sponge sample, stomached 2 ± 0.2 min, and incubated at 30 ± 2°C for 22 ± 2 h.
For environmental aqueous chilling solutions (which may include water, brine, and propylene
glycol solutions) and surface rinse solutions, about 100 mL of sample solution is filtered
through one or more 0.45-µm hydrophobic grid membrane filters. Using sterile forceps,
the membrane is aseptically removed from the plastic housing and transferred to a stoma-
cher bag. About 200 mL or additional volume of UVM broth sufficient to cover the filters
completely is added, stomached 2 min, and incubated at 30 ± 2°C for 22 ± 2 h.
Secondary enrichment is achieved by transferring 0.1 ± 0.02 mL of the UVM enrichment to 10

± 0.5 mL of FB (containing appropriate supplements, per media preparation instructions). Inoculated
FB tubes are incubated at 35 ± 2°C for 22 ± 2 h. During secondary enrichment, a loopful or a drop
approximating 0.1 mL of UVM is streaked over the surface of a MOX plate. Alternatively, a sterile
cotton-tipped applicator or equivalent is dipped into the UVM and 25 to 50% of the surface of a
MOX plate swabbed. A loop is then used for isolation from the swabbed area onto the remainder
of the plate. The MOX plate is incubated at 35 ± 2°C for 26 ± 2 h.
UVM-streaked MOX plates are examined for colonies having morphology typical of Listeria
spp. At 24 h, suspect colonies are typically small (ca. 1 mm) and are surrounded by a zone of
darkening from esculin hydrolysis. If suspect colonies are present on MOX, they are transferred
to HL agar (description follows). If no suspect colonies are evident, the MOX plate is reincubated
until a total incubation time of 48 ± 2 h has been achieved. Then the FB is examined after 26 ± 2 h
of incubation for the potential presence of L. monocytogenes by visual examination of the broth
for darkening from esculin hydrolysis.
If any degree of FB darkening is evident, 0.1 mL of FB is aseptically dispensed onto a MOX
plate. About 25 to 40% of the surface of MOX is swabbed or streaked with the FB inoculum
and a loop is used to streak for isolation from the initial swab/streak quadrant onto the remainder
of the plate. The MOX plate is incubated at 35 ± 2°C for a minimum of 24 h. Upon incubation,
suspect colonies are examined and selected from any MOX agar plate pending analysis (i.e.,
MOX plates streaked from 26 ± 2 h FB and/or UVM) as described earlier. If no FB darkening
is evident, the FB is reincubated at 35 ± 2°C and re-examined for evidence of darkening until a
total incubation time of 48 ± 2 h has been achieved. If any degree of darkening is evident, a
MOX plate is swabbed, streaked, and incubated as already described. If no darkening is evident
and no suspect MOX and/or HL colonies have been demonstrated, the sample is considered
negative for L. monocytogenes.
Isolation and purification of presumptive Listeria in the current USDA–FSIS [178] method is
accomplished as follows. If suspect colonies are present on MOX from any source, a loop or
equivalent sterile device is used to contact a minimum of 20 (if available) suspect colonies that are
collectively streaked for isolation on one or more HL plates. The streaked HL plates are incubated
at 35°C for 22 ± 4 h. After incubation, the HL plates are examined against backlight for translucent
colonies surrounded by a small zone of β-hemolysis. If at least one suspect colony is clearly isolated,
one should proceed to confirmatory testing, while holding all HL plates containing suspect colonies
(at room temperature or refrigerated) until confirmatory testing is complete.
If suspect colonies or β-hemolytic growth are present on HL but not clearly isolated, represen-
tative suspect colonies/growth should be restreaked onto one or more fresh HL plates and incubated
at 35°C for 22 ± 4 h. If, however, no suspect isolates are present on HL, follow-up of MOX and/or
HL isolates from other branches of analysis should be done (e.g., FB follow-up vs. UVM direct
streak follow-up). If no branch of analysis produces suspect β-hemolytic colonies on HL, the sample
may be reported as negative for L. monocytogenes.
Confirmatory identification of L. monocytogenes consists of preliminary confirmation tests such
as the tumbling motility test followed by biochemical tests. The CAMP/CAMP factor test and
genetic tests may be required in certain circumstances.
To facilitate rapid screening of processed meat and poultry products, USDA–FSIS has approved
the use of a commercial PCR-based procedure, L. monocytogenes BAX screening test [178]. All
samples identified as presumptively positive for L. monocytogenes by these tests are subject to
cultural confirmation. For this method, L. monocytogenes detection limits are determined to be <1
CFU/g in a 25-g sample. Sample preparation and enrichment are similar to those described for the
USDA–FSIS isolation and identification of L. monocytogenes from red meat, poultry, egg, and
environmental samples [178].
Secondary enrichment involves transferring 0.1 mL of the UVM enrichment to 10 mL to
MOPS–BLEB (prepared using a complete powdered LEB medium [DIFCO–BBL] to which the MOPS
free acid and sodium salt are added before autoclaving) and incubating at 35°C for 18 to 24 h. A MOX
plate is streaked with a loopful of UVM over the surface and a loop is used to streak for isolation from
the swabbed area onto the remainder of the plate; the plate is incubated at 35 ± 2°C for 26 ± 2 h. The
BAX screening test is then done according to the current BAX User’s Guide. Cultural confirmation of
presumptive positive samples is obtained by streaking a MOX plate using a loopful of the MOPS–BLEB,
incubating the MOX at 35 ± 2°C for a minimum of 24 h before proceeding with all isolation and
purification procedures as described earlier in the USDA–FSIS method.
In their study on comparative evaluation of culture- and BAX polymerase chain reaction PCR-
based detection methods for Listeria spp. and L. monocytogenes in environmental and raw fish
samples, Hoffman and Wiedmann [89a] evaluated two commercial PCR-based Listeria detection
systems, the BAX for screening L. monocytogenes and the BAX for screening genus Listeria, and
a culture-based detection system, the Biosynth L. monocytogenes detection system (LMDS), for
their ability to detect L. monocytogenes and Listeria spp. in raw ingredients and the processing
environment.
For detection of L. monocytogenes from raw fish, enrichment was done in Listeria enrichment
broth (LEB), followed by plating on OXA and LMDS L. monocytogenes plating medium (LMPM).
Detection of Listeria and L. monocytogenes from environmental samples was done using LMDS
enrichment medium, followed by plating on OXA and LMPM. A total of 512 environmental and 315
raw fish samples were taken from two smoked-fish processing facilities and screened using these
molecular and cultural Listeria detection methods. The BAX method for L. monocytogenes was used
to screen raw fish and was 84.8% sensitive and 100% specific. The BAX method for the genus Listeria
was evaluated on environmental samples and had 94.7% sensitivity and 97.4% specificity.
In conjunction with enrichment in LEB, LMPM had a sensitivity and specificity of 97.8 and
100%, respectively, for detection of L. monocytogenes in raw fish. Use of LMDS enrichment medium
followed by plating on LMPM yielded sensitivity and specificity rates of 94.8 and 100%, respec-
tively, for detection of L. monocytogenes from environmental samples. This shows that both BAX
systems and LMPM allow for reliable and rapid detection of Listeria spp. and L. monocytogenes.
However, BAX systems are slightly faster; they provide screening results in about 3 days compared
to 4 to 5 days when LMPM is used.
A study by Lammerding and Doyle [103] found that the USDA protocol yielded better recov-
eries of L. monocytogenes from dairy products than did the FDA method. Warburton [181] reported
that, with a limited number of samples, the USDA–FSIS method proved to be slightly more efficient
in isolating L. monocytogenes than the FDA method in a comparative study of the FDA and
USDA–FSIS methods for detection of this organism in foods and environmental samples. Nineteen
laboratories across Canada took part in this study. Similarly, Ferron and Michard [58] compared
the FDA and USDA–FSIS enrichment procedures using 300 pastry samples provided by 100
different suppliers in western France. The USDA procedure was superior, detecting 69% of all
positive samples compared with the FDA procedure, which detected only 34%. The USDA–FSIS
method appears to be more efficient in quantitative recovery of L. monocytogenes from contaminated
food and environmental samples.
The Netherlands Government Food Inspection Service
Using the NGFIS protocol, food samples are enriched in L-PALCAMY enrichment broth for 48 h
at 30°C. After 24 and 48 h, 0.1 mL of L-PALCAMY enrichment broth is streaked onto PALCAM
agar. Plates are incubated at 30°C for 48 h under microaerophilic conditions (5% oxygen, 7.5%
carbon dioxide, 7.5% hydrogen, and 80% nitrogen) [109], after which presumptive Listeria colonies
are black and surrounded by a dense black hole from esculin hydrolysis.
Lund et al. [118] examined 300 raw milk samples for presence of Listeria using three primary
enrichment media. A total of 84 positive samples were identified by one or more of these media.
PALCAMY was the most effective medium, identifying 50 of 84 positive samples, followed by
UVM and LEB, which identified 46 and 42 Listeria-positive samples, respectively. Given that the
best of these primary enrichment broths identified only 50 of 84 (59.5%) Listeria-positive samples,
the use of two or more primary enrichment broths identified an additional 34 samples and increased
the overall incidence of Listeria by almost 41%. These results once again highlight the inadequacy
of relying on a single primary enrichment broth for Listeria detection.
Noah et al. [132] evaluated the impact of more than one test procedure on recovery of Listeria
species from naturally contaminated seafood and seafood products. A total of 211 samples were
evaluated using five different protocols. The FDA procedure [116] was used as a control against
which the efficacy of the other procedures was evaluated. A total of 60 samples were identified
as Listeria-positive by at least one of the procedures. Of these samples, the FDA procedure
missed seven samples that were subsequently found to harbor Listeria when other procedures
were employed. The overall incidence of Listeria increased 11.7% using more than one testing
procedure.
Hayes et al. [81] assessed the USDA–FSIS and cold enrichment procedures for recovery
of L. monocytogenes from suspect food samples. Both procedures identified L. monocytogenes in
28 of 51 positive samples. The USDA–FSIS procedure identified 21 samples missed by cold
enrichment, whereas the cold enrichment procedure identified an additional 2 samples that the
USDA–FSIS procedure missed. Three enrichment methods were also compared by Hayes et al.
[82] during an examination of foods obtained from the refrigerators of patients with active
clinical cases of listeriosis. Of 2,229 food samples examined in this study, 11% were positive
for L. monocytogenes.
Overall, the USDA–FSIS [33], FDA [116], and NGFIS [130] methods were not statistically
different in their ability to isolate Listeria from 899 samples included in the comparative evaluation.
The FDA procedure [116] identified 65% of all L. monocytogenes-positive foods, whereas the
USDA–FSIS and NGFIS procedures detected L. monocytogenes in 74% of foods shown to be
positive. Although none of these widely used Listeria detection methods proved to be highly
sensitive when used independently, use of any two methods improved detectability from 65 to 74%
for individual protocols to 87 to 91% for combined protocols.
RESEARCH ADVANCES
Peng and Sheref [140] have developed a simple, automated method to detect Listeria in foods. It
consists of a 6-h pre-enrichment step followed by overnight incubation in selective Listeria broth
(SLB) at 35°C. Changes in light transmittance in the selective broth are registered continuously by
an optical sensor of the BioSys instrument, now Soleris (Neogen Corp., Lansing, MI) and recorded
in the computer. Esculin hydrolysis by Listeria results in black coloration of the medium that causes
a sharp drop in light transmittance, whereas transmittance in negative samples remains unchanged.
Confirmation of L. monocytogenes is carried out only on esculin-positive samples and is completed
within 6 h. This method is able to detect as few as 10 to 50 cells of Listeria in 25 g of food.
CONSIDERATIONS FOR RECOVERY OF INJURED LISTERIA

Most conventional and rapid detection procedures for Listeria use highly selective enrichment
media to facilitate growth over competitive background flora. However, these procedures will not
generally recover sublethally injured Listeria, which could exist in various heated, frozen, or
acidified foods or within heated, frozen, and sanitized areas/surfaces of food-processing facilities.
Sublethal injury in Listeria as a result of heating, freezing, drying, irradiation, or exposure to
chemicals (i.e., sanitizers, preservatives, acids) has been extensively studied [1,15,30–32,34,39,41,
66,69,70,122,155,157,163] and was reviewed in Chapter 6.
Under ideal conditions, such injury is reversible; Listeria is capable of repairing sublethal damage
in foods where growth conditions are favorable. Repair of heat-injured L. monocytogenes has been
reported in whole and 2% milk stored at 4°C [126]. Ngutter and Donnelly [131] have documented
nitrite injury of L. monocytogenes in frankfurters. The injury was completely reversible or growth
of uninjured Listeria occurred in LRB when injury was between 98.5 and 98.7%. However, total
recovery was not observed in Listeria repair broth (LRB) when injury exceeded 99%. Modified
UVM was unable to reverse the effects of nitrite-injured L. monocytogenes. Generally, LRB was
found to be consistently superior to UVM at recovering L. monocytogenes from frankfurters.
Several investigators have attempted to improve the sensitivity of current detection systems by
focusing on recovery of injured Listeria that may be present in food products and food-processing
environments. Most research is focusing on RTE foods because they usually do not undergo any
further treatment sufficient to kill Listeria before consumption. All current detection procedures,
with the exception of cold enrichment, involve selective enrichment and/or selective plating. The
main limitation of selective enrichment or selective plating is that they often do not allow for growth
of sublethally injured Listeria [46]. Cold enrichment, despite its superiority in recovering injured
Listeria, is not feasible for routine testing because several months of incubation may be necessary
to obtain positive results.
By failing to consider recovery of injured Listeria, current methodologies undoubtedly under-
estimate the true incidence of this organism. Several previous studies have reported on the ability
of commonly used plating media to recover injured Listeria. Among the most commonly used
selective agents examined, phenylethanol, acriflavine, polymyxin, and sodium chloride were found
to inhibit recovery of thermally stressed and nonstressed Listeria [39,105,171,180,181,183]. Fur-
thermore, when examined for ability to quantitatively recover thermally stressed Listeria, LEB
agar, modified McBride’s agar (MMA), LPM agar [106], and FDA enrichment broth agar showed
significantly impaired recovery [26].
Warburton et al. [182] examined the ability of the modified FDA and USDA–FSIS methods to
recover stressed cells and low levels of L. monocytogenes in food and environmental samples. Although
the modified FDA and USDA–FSIS methods were comparable in their abilities to isolate stressed
and low-level populations of L. monocytogenes, these authors failed to assess the extent of injury
within bacterial populations following exposure to sublethal stress. The percentage of injury existing
within a population of bacterial cells can profoundly affect comparative results of media performance.
Thus, it is difficult to determine whether valid conclusions can be drawn from such studies.
Busch and Donnelly [32] developed an enrichment medium capable of resuscitating heat-injured
Listeria. This medium, LRB, permits complete repair of injured Listeria within 5 h at 37°C after
which selective agents can be added to inhibit the growth of competing microflora during continued
incubation. In studies comparing the efficacy of LRB in promoting repair/enrichment of heat-injured
Listeria with that of existing selective enrichment media, repair was not observed in FDA enrichment
broth [116], phosphate-buffered Listeria enrichment broth (PEB; Gene-Trak Systems, Framingham,
Massachusetts), or UVM enrichment broth [103]. Final Listeria populations in selective enrichment
media after 24 h of incubation at 30°C were 1.7 × 108 to 9.1 × 108 CFU/mL compared with
populations in LRB, which consistently averaged 2.5 × 1011 to 8.2 × 1011 CFU/mL [32].
Knabel [97] developed a one-step, recovery-enrichment broth—optimized Penn State University
(oPSU) broth—that consistently detected low levels of injured and uninjured L. monocytogenes cells
in RTE foods. The oPSU broth contains special selective agents that inhibit growth of background
flora without inhibiting recovery of injured Listeria cells. After recovery in the anaerobic section of
oPSU broth, Listeria cells migrate to the surface, forming a black zone. This migration separates
viable from nonviable cells and the food matrix. The high Listeria-to-background ratio in the black
zone results in consistent detection of low levels of L. monocytogenes in pasteurized foods by cultural
and molecular methods, greatly reducing false-negative and false-positive results. One other advan-
tage of oPSU broth is that it does not require transfer to a secondary enrichment broth; this makes
it less laborious and less subject to external contamination than two-step enrichment methods.
Studies with LRB were extended to examine the potential for repair of freeze-injured and
sanitizer-injured L. monocytogenes [60,163]. Although variation in susceptibility of L. monocyto-
genes to freeze injury was recorded, in general, L. monocytogenes is not severely injured by freezing
[53,69,136]. Percentage of injury ranged from only 40 to 60% after Listeria populations were
frozen at –9 to –11°C for 24 h [60]. As storage time increased, the percentage of injury increased
to a maximum of only 70 to 80%. To examine reversibility of freeze injury, low-level populations
of freeze-injured L. monocytogenes cells were added to UVM enrichment broth, FDA enrichment
broth, and LRB. Repair of freeze-injured populations occurred quickly, probably because of the
low initial degree of injury; the pathogen again attained high populations in LRB.
Sallam and Donnelly [163] examined the ability of four commonly used dairy plant sanitizers
to induce injury in L. monocytogenes when exposed to sublethal concentrations. UVM broth failed
to support growth of sanitizer-injured cells, whereas LRB permitted their recovery. Flanders et al.
[62] examined the efficacy of using a repair step to increase recovery of injured Listeria from
environmental sponge samples obtained from dairy-processing plant environments. The
USDA–FSIS Listeria isolation protocol using UVM-modified Listeria enrichment broth was com-
pared with a modified USDA–FSIS format that utilized LRB as the primary enrichment medium.
UVM and LRB broths also were used in conjunction with a rapid DNA hybridization (Gene-Trak)
and ELISA (Organon Teknika, Durham, North Carolina) assay. Of 80 sites found positive by any
method, UVM and LRB showed similar recovery rates (87.5 and 88.8%, respectively). However,
combining the cultural methods with either rapid method for each broth increased detection to 97.5
to 98.8% [62].
Flanders et al. [61] also evaluated the abilities of LRB, LRB containing ceftazidime (LRBC),
and UVM to enhance recovery of Listeria from dairy plant environmental samples. Although no
single broth could detect all Listeria-positive sites, LRBC identified 67 of 89 positive sites (75.3%),
and LRB and UVM each detected 60 of 90 positive sites (66.7%). Combining results from any
two broths increased recovery from 66.7–75.3% to 82.2–94.4%. The combination of LRBC and
UVM detected 94.4% of positive samples, whereas LRB and LRBC identified 91.1% of positive
samples.
Pritchard et al. [144] also compared the ability of UVM, LRB, and LRBC to isolate Listeria
from dairy plant environments. Of 80 positive samples identified, 54 samples came from UVM
medium, 56 were from LRB, and 57 came from LRBC. A total of 26 samples (32.5% of positive
samples) were identified by LRB or LRBC but not by UVM media. Combining UVM with
LRB or LRBC again substantially increased the number of positive samples identified. When
results from UVM and LRB were combined, 65 to 80 (81.3%) positive samples were identified.
Using UVM and LRBC, 74 of 80 (92.5%) positive samples were identified. These results
illustrate the severe limitations associated with current regulatory procedures used to assure
absence of Listeria in foods and food-processing environments.
Ryser et al. [162] evaluated the ability of UVM and LRB to recover different strain-specific
ribotypes of L. monocytogenes from meat and poultry products. Forty-five paired 25-g retail samples
of ground beef, pork sausage, ground turkey, and chicken underwent primary enrichment in UVM
and LRB (30°C/24 h) followed by secondary enrichment in Fraser broth (35°C/24 h) and plating
on modified Oxford agar. A 3-h nonselective enrichment period at 30°C was used with LRB to
allow repair of injured Listeria before adding selective agents. Listeria spp. were detected in 73.8
and 69.4% of the 180 meat and poultry samples tested using LRB and UVM, respectively. Although
these differences were not statistically significant, combining UVM and LRB results increased
overall Listeria recovery rates to 83.3%. Thus, enrichment in LRB for repair of injured cells in
conjunction with the USDA–FSIS method has potential to improve recovery of Listeria from meat
and poultry products.
In this study, following 24 h of incubation at 35°C, Listeria colonies were biochemically
confirmed and selected isolates were ribotyped using the automated Riboprinter Microbial Char-
acterization System, (E.I. du Pont de Nemours and Co., Inc., Wilmington, DE). A total of 36
different Listeria strains comprising 16 L. monocytogenes (including 4 known clinical ribotypes),
12 L. innocua, and 8 L. welshimeri ribotypes were identified from selected positive samples (15
samples of each product type, 2 UVM and 2 LRB isolates/sample). Using both UVM and LRB,
26 of 36 (13 L. monocytogenes) ribotypes were detected; whereas 3 of 36 (1 L. monocytogenes)
and 7 of 36 (3 L. monocytogenes) Listeria ribotypes were observed using only UVM or LRB,
respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. monocyto-
genes), 21 (12 L. monocytogenes), 20 (9 L. monocytogenes), and 19 (11 L. monocytogenes) different
Listeria ribotypes, respectively; some Listeria ribotypes were confined to a particular product.
More importantly, striking differences in the number and distribution of Listeria ribotypes,
including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were
observed when 10 UVM and 10 LRB isolates from five samples of each product were examined.
When a third set of six samples per product type was examined from which two Listeria
isolates were obtained using only one of the two primary enrichment media, UVM and LRB
failed to detect L. monocytogenes (clinical and nonclinical ribotypes) in two and four samples,
respectively (Table 7.3). These findings stress the complex microbial ecology of Listeria in
foods and limitations of existing detection procedures to fully characterize the total Listeria
population. Furthermore, two of the L. monocytogenes ribotypes missed using UVM were
known clinical ribotypes linked to sporadic and epidemic cases of human listeriosis in England
and Scotland [125].
Continuing work [143] on enrichment of dairy environmental samples in UVM and LRB has
shown that combining these two primary enrichment media into a single tube of Fraser broth for
secondary enrichment yields a significantly higher (P < 0.05) percentage of Listeria-positive
samples than when LRB or UVM is used alone. These findings, combined with reports of L. innocua
being able to outgrow L. monocytogenes in UVM (and Fraser broth) [40,142] suggest that different
ribotypes of L. monocytogenes may vary somewhat in nutritional requirements or their ability to
compete with other ribotypes of L. monocytogenes or other Listeria spp. Refinement of existing
Listeria recovery methods should consider the nutritional needs associated with those specific
genetic types widely distributed in foods.
Silk et al. [167] examined five selective (Food and Drug Administration Bacteriological
Analytical Manual Listeria enrichment broth base with selective agents [BAMS]), BCM Listeria
TABLE 7.3
Ribotypes of Listeria spp. Recovered from 10 Samples of Raw
Chicken Following Primary Enrichment in UVM or LRB
and Secondary Enrichment in Fraser Broth
Ribotype Listeria spp. No. of Isolates
UVM LRB
1-909-3 L. innocua
5-418-3 L. monocytogenes 0 1
5-415-4 L. innocua 2 0
2-864-3 L. welshimeri 2 0
1-916-1a L. monocytogenes 2 0
1-909-4 L. innocua 2 0
1-910-7 L. innocua 5 5
5-426-1 L. innocua 0 1
0 1
aRecognized clinical ribotypes associated with known epidemic or sporadic cases
of human listeriosis.
Source: Adapted from Ryser, E. T. et al. 1996. Appl. Environ. Microbiol.

62:1781–1787.
pre-enrichment broth [BCM, Biosynth International, Inc., Naperville, Illinois], Fraser broth, Listeria
repair broth with selective agents [LRBS], and University of Vermont-modified Listeria enrichment
broth) and three nonselective (Food and Drug Administration Bacteriological Analytical Manual
Listeria enrichment broth base [BAM], Listeria repair broth [LRB], and trypticase soy broth [TSB])
media with respect to the growth of healthy and repair of heat-injured L. monocytogenes (strain
F5069 [ATCC 51414]).
After inoculation of approximately 2 × 102 CFU healthy cells/mL, each test broth was incubated
at 30°C for 72 h. During the incubation period, 12 samples concentrated in the growth transition
regions of lag-log-stationary phase were removed from the broth, diluted in Butterfield phosphate
buffer, plated onto trypticase phosphate agar (TPA; trypticase phosphate broth [TPB] with 1.5%
agar; Difco) and incubated at 35°C for 48 h before enumeration. L. monocytogenes was injured by
placing 200 mL of 56°C preheated TPB in a 500-mL Erlenmeyer flask followed by addition of a
1:100 dilution of an 18-h-old culture of L. monocytogenes F5069. This suspension was heated for
20 min in a shaking water bath before being transferred into a 250-mL centrifuge bottle and spun
at 10,000 × g for 10 min at 25°C. The supernatant fluid was discarded and the pellet washed with
test medium. The test broths were incubated at 30°C for 72 h and sampled as described earlier for
healthy cells. Heat-injured cells were plated onto TPA and TPA supplemented with 4% NaCl
(TPAN) and incubated at 35°C for 48 h before enumeration. The percentage of injury was calculated
using the following equation: (1 – [counts of TPAN/counts on TPA]) × 100.
These researchers also modeled growth of this strain of L. monocytogenes with the Gompertz
equation [29,37,67] using a nonlinear regression procedure (PROCLIN) of the SA system for
Windows version 8.2 (SAS Institute Inc., Cary, North Carolina). Gompertz parameter values were
then used to calculate the exponential growth rate (EGR), lag-phase duration (LPD), generation
time (GT), and maximum population density (MPD) [28]. For heat-injured cell populations, the
Gompertz equation was used to model L. monocytogenes growth on TPA and TPAN. The time
required for the repair was determined by graphing the models and determining the time point at
which bacterial counts on nonselective (TPA) and selective (TPAN) media were equal [37].
Growth curves were generated using healthy and heat-injured L. monocytogenes strain F5069
in the three nonselective and five selective enrichment broths mentioned previously. The Gompertz
equation was successfully used to model the growth of L. monocytogenes. Gompertz parameters
were used to calculate EGR, LPD, GT, MPD, and time required for repair of injured cells. Statistical
differences (P < 0.05) in broth performance were noted for LPD and MPD when healthy and injured
cells were inoculated into the broths.
With the exception of Fraser broth, no significant differences were found in the time required for
repair of injured cells. These results suggest that the distinction between selective and nonselective
broths in their ability to grow healthy Listeria and to repair sublethally injured cells is not solely an
elementary issue of presence or absence of selective agents. Heat shock proteins produced by Listeria
during heating [172], for example, may be conferring some cross-protective properties [112], making
the injured cells less sensitive to selective agents than they were previously thought to be.
Roth and Donnelly [155] assessed survival of acid-injured L. monocytogenes in four different
acidic foods and also examined the efficacy of LRB and UVM to recover acid-injured Listeria
from such foods. L. monocytogenes was injured in lactic (pH 3.0) and acetic (pH 3.5) acids. Two
levels of injury were produced and monitored: one population with 99.99% injury and the second
with approximately 95% injury. The four acidic food systems studied at 4 and 30°C included fresh
apple cider (pH 3.3), plain nonfat yogurt (pH 4.2), fresh coleslaw (pH 4.4), and fresh salsa (pH 3.9).
Acid-injured Listeria was added to each acidic food and monitored by selective and nonselective
plating. Simultaneously, samples were enriched in LRB and UVM followed by standard isola-
tion/identification procedures with survival of healthy L. monocytogenes also monitored.
Although acid-injured cells failed to repair in the acidic foods tested, the pathogen did survive
for more than a week. Storage temperatures did affect the survival rate of acid-injured cells; storage
at 4°C had a bacteriostatic effect and at 30°C was bacteriocidal. Parameters involved in survival of
acid-injured Listeria include the degree to which the bacterial population is injured (percentage of
injury), storage temperature, and the pH of the food. At time points where differences were detected,
LRB proved to be superior (22 of 54) in its ability to detect injured Listeria compared with UVM
(3/54). Hence, use of LRB is recommended when examining acidic foods for L. monocytogenes.
CONCLUSIONS
Standard enrichment and plating procedures for Listeria detection will continue to enjoy widespread
use because they are economical when compared to rapid methods. Disadvantages of standard
procedures include their time-consuming and cumbersome nature, as well as inability to detect
injured Listeria in processed RTE foods. Advances in media development and formulation have
increased recovery and decreased potential for false positive results.
The ability to recover a broad diversity of Listeria spp. from foods is critical in epidemiological
investigations where traceback to contaminating foods needs to be done rapidly and accurately.
Outbreaks of foodborne listeriosis coupled with the high mortality rates associated with sporadic
cases of illness and the advent of mandatory HACCP programs have underscored the need for
faster and more efficient methods to detect small numbers of Listeria in a wide range of foods.
There is no doubt that continued work to refine optimal enrichment and detection procedures for
Listeria in foods will occur in the future.
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8 Rapid Methods for Detection

of Listeria
Byron F. Brehm-Stecher and Eric A. Johnson
CONTENTS
Introduction ....................................................................................................................................257
The “Ideal” Detection Method.............................................................................................258
Traditional Methods for Detection of Listeria ....................................................................258
Rapid Methods for Detection of Listeria.............................................................................258
Commercially Available Rapid Test Kits and Systems .......................................................258
Automation ...........................................................................................................................259
Detection of Generic Listeria versus Listeria monocytogenes ............................................259
Diagnostic Targets ................................................................................................................259
Nucleic Acid–Based Methods........................................................................................................259
The Polymerase Chain Reaction..........................................................................................260
Potential Pitfalls of PCR ......................................................................................................261
DNA Microarrays .................................................................................................................262
Fluorescence in Situ Hybridization ......................................................................................263
Recombinant Bacteriophage.................................................................................................264
Antibody-Based Methods ..............................................................................................................264
Potential Pitfalls of Antibody-Based Methods ....................................................................265
Advances in Antibody-Based Technologies.........................................................................265
Cell Separation and Concentration ......................................................................................266
Additional Methods........................................................................................................................267
Flow Cytometry....................................................................................................................267
Biosensors.............................................................................................................................269
Chip-Based Microanalytical Systems ..................................................................................271
Spectroscopic Methods.........................................................................................................271
Conclusions ....................................................................................................................................274
References ......................................................................................................................................275
INTRODUCTION
Members of the genus Listeria are found in association with soil, water, and vegetation and can
grow at refrigeration temperatures. Foods involved in outbreaks of listeriosis include ready-to-
eat (RTE) products such as milk, soft-ripened cheeses, coleslaw, and vacuum-packaged meats
[117]. Although listeriosis is relatively rare, it is characterized by a high mortality rate (~25–30%).
The seriousness of this disease, and the efficiency and volume of today’s food production and
distribution networks highlight the need to develop rapid methods for detecting L. monocytogenes.
This chapter provides a broad overview of methods and technologies for rapid detection of
257
Listeria or L. monocytogenes that have been reported in the literature. Some methods, such as
those employed by commercially available test kits, have received relatively wide acceptance
and are used routinely in laboratory analyses. Other methods described here are not yet widely
used and may be considered “emerging technologies.” The benefits and drawbacks of each
approach for rapid detection, and in some instances, characterization, of Listeria or L. monocy-
togenes will be considered.
THE “IDEAL” DETECTION METHOD

Although no ideal detection method exists for Listeria (or for any analyte), consideration of
characteristics that such a system would have may be useful in evaluation of existing methods or
in development of new ones. The ideal detection method would be: specific for the target analyte
(Listeria or L. monocytogenes), sensitive (able to detect 1 CFU in a 25-g sample), rapid (substan-
tially faster than cultural methods alone), reproducible, simple to use (with easily interpreted
results), capable of direct detection in foods with minimal or no interference from the food matrix,
able to distinguish between live and dead (or injured) cells, inexpensive (relative to expenses
associated with traditional methods of detection), validated against standard techniques, automat-
able, and scalable according to testing needs. Additional properties may include continuous online
operation and operation over a wide dynamic range. Although no single method fulfills all of these
criteria, food processors can prioritize which characteristics of the ideal are required for their
specific testing needs and choose an available method that best meets these needs.
TRADITIONAL METHODS FOR DETECTION OF LISTERIA

“Classical” or culture-based methods for the detection of Listeria are limited primarily by their
heavy demands on time and labor [9,33,99]. As an extreme example, the original cold-enrichment
method described by Gray and colleagues [47] used incubation periods ranging from several weeks
to months, a time frame clearly at odds with today’s rapid pace of food processing and distribution.
A wide variety of selective enrichment broths and agars have since become available for detection
of Listeria or for differentiation of L. monocytogenes from other members of the genus [9,111].
Additional formulations have been described for the recovery of thermally or chemically injured
listeriae [32,127]. Still, at least four sequential steps are generally required for cultural methods of
detection: pre-enrichment, selective enrichment, selective plating, and biochemical screening. As
a result, positive detection of Listeria in food or environmental samples using cultural methods
alone may take up to 5–7 days [99].
RAPID METHODS FOR DETECTION OF LISTERIA

Using the time required for cultural methods as a benchmark, a rapid method can be broadly defined
as any approach yielding comparable results in less time. The potential benefits of rapid methods
include reduced likelihood that contaminated product will be released for sale, reduction in costs
associated with media, labor, or storage of product pending microbiological results, and increased
time on shelves for products cleared for sale. The ability to rapidly detect listeriae in foods and in
the food-processing environment may also enable more timely monitoring of critical control points,
contributing to our ability to control L. monocytogenes in these environments and, ultimately, the
incidence of disease [150].
COMMERCIALLY AVAILABLE RAPID TEST KITS AND SYSTEMS

The benefits of commercially supported rapid test kits or systems include test availability, standardiza-
tion, independent validation, simplicity, cost-effectiveness, and the availability of technical support
[99]. Test kits and systems for rapid detection of both generic Listeria and L. monocytogenes are
Rapid Methods for Detection of Listeria 259
commercially available, and many of these have received AOAC approval. Assay formats include
colorimetric DNA probe, latex bead-based lateral flow immunoassay, enzyme-linked immunosorbent
assay (ELISA), enzyme-linked immunofluorescence assay (ELFA), immunomagnetic separation
(IMS), fluorescence in situ hybridization (FISH), and polymerase chain reaction (PCR). Most methods
require selective enrichment for up to 48 h. One exception may be IMS-based methods, which can be
used to selectively concentrate generic Listeria or L. monocytogenes from a sample without enrichment.
AUTOMATION
A drawback of many assay formats is that they are relatively complex and may involve repetitive
steps, which can lead to operator fatigue and error. A number of rapid methods are amenable to
automation. Benefits of assay automation include reduced labor, increased throughput, better repro-
ducibility and faster time-to-results [99,100]. Automated methods reported for rapid detection or
characterization of Listeria include enzyme-linked immunoassays, DNA or RNA probe assays,
ribotyping, biochemical screening, and impedance testing [28,37,67,83,100,104,105,137].
DETECTION OF GENERIC LISTERIA VERSUS LISTERIA MONOCYTOGENES
The decision to test for generic Listeria or for L. monocytogenes has legal, ethical, and practical
implications. This decision is further complicated by limited knowledge of exactly which factors
contribute to the pathogenicity of L. monocytogenes [101,149]. Apart from a few isolated cases,
most human cases of listeriosis are caused by L. monocytogenes, arguing for use of tests that are
specific for this species. However, testing for generic Listeria has been suggested as a more robust
means of environmental monitoring than testing for L. monocytogenes alone [130]. Additionally,
recent regulatory directives now require certain sectors of the food industry to conduct routine
environmental testing for generic Listeria [4]. It is likely that this new regulatory environment will
lead to an increased demand for rapid methods to detect generic Listeria.
DIAGNOSTIC TARGETS
A diagnostic target is any unique molecule whose detection signals the presence of a specific
organism in a sample. Potential diagnostic targets for Listeria or L. monocytogenes include evolu-
tionarily distinct nucleic acid sequences found in rRNA, mRNA, or chromosomal DNA. Additional
targets may include structural components such as flagellar, somatic, or capsular antigens, or
proteinaceous virulence factors such as ß-hemolysin or phospholipase C [10,110,136]. The presence
of these targets may be detected using a number of techniques, including PCR, DNA, or RNA
hybridization, antibody-based approaches, or phenotypically, using diagnostic media. As a practical
matter, it may not be possible to demonstrate that a given sequence, molecule, or enzymatic activity
is truly unique among all microorganisms. However, for it to be diagnostically useful, a target must
only be unique within a certain environmental niche. If necessary, microorganisms that could
potentially lead to false-positive results may also be excluded from the sample before testing using
selective enrichment. In choosing a diagnostic target, any potential pitfalls regarding its use must
be identified. For example, expression of diagnostic epitopes may depend on media used to grow
cells, and virulence factor mRNAs may be expressed only at certain temperatures [63,93,98].
NUCLEIC-ACID-BASED METHODS
Availability of complete genome sequences of Listeria monocytogenes (serotypes 1/2a, 4b, 6a) and
L. innocua has provided new insights into molecular features contributing to the pathogenesis of
L. monocytogenes [16,44,145] (http://www.tigr.org/tdb/mdb/mdbcomplete.html). In addition to
providing a greater understanding of the pathogenesis of L. monocytogenes, these efforts may also
yield new diagnostic targets for detection of this organism. Several nucleic-acid-based approaches
are available to detect L. monocytogenes and other Listeria spp., including amplification-based
strategies such as the ligase chain reaction [148], nucleic-acid-sequence-based amplification
(NASBA) [11], and PCR. PCR is discussed in more detail in the following subsection.
THE POLYMERASE CHAIN REACTION

PCR provides a method for exponential amplification of specific DNA sequences present in a
sample. Key components of the buffered PCR reaction mixture include oligonucleotide primers,
deoxyribonucleotide triphosphates, the DNA template to be amplified, and a thermostable DNA
polymerase. The primers are designed to hybridize on either side of the target sequence, defining
the region to be amplified [99,116,153]. Additional components such as enzyme cofactors
(e.g., Mg2+) or additives intended to increase the specificity of the reaction or the thermal stability
of the enzyme may also be present. PCR reactions are characterized by a three-step cycle involving
(1) denaturation of the double-stranded template, (2) hybridization or annealing of primers to
complementary regions of the target template, and (3) primer-directed synthesis of new DNA, also
termed extension [99,116,153]. DNA fragments generated in each cycle serve as templates for
subsequent rounds of amplification and the number of templates present doubles with each cycle,
leading to an exponential accumulation of the product [99,116,153]. Typically, denaturation is
carried out at 95°C, annealing at 55°C, and extension at 72°C, the temperature optimum for Taq
polymerase. However, extension will still occur at nonoptimal temperatures, and annealing and
extension can sometimes be combined into a single step [6].
PCR is by far the most widely reported rapid method for detection of Listeria, particularly
L. monocytogenes, and a number of authoritative reviews have been written on this topic
[76,99,153]. Once available only to a handful of specialist laboratories, PCR has since become
accessible to a much wider user base. Factors responsible for easing PCR further into the mainstream
testing environment include the commercial availability of prepackaged reagents and advances in
automated detection of PCR products, which obviate the need for labor-intensive postamplification
handling steps. Still, some barriers exist to routine adoption of PCR by the food-testing community,
including the relatively high cost of instrumentation and a lack of standardized and validated
methods for PCR in foods [85].
Several primers available for PCR-based detection of L. monocytogenes have been tabulated
in recent reviews [76,153]. Commonly targeted genes in L. monocytogenes include the listeriolysin
O gene, hly [5,6,14,20,54,56,59,69,96,112,146], and the gene for the invasion-associated protein
p60, iap [14,15,22,53,69,118]. Other targets include genes for aminopeptidase C [152], internalins
A and B [65], phospholipase C [24], a fibronectin-binding protein [42], and lmaA (also referred
to as Dth-18), whose product is responsible for delayed-type hypersensitivity reactions in L.
monocytogenes-immune mice [76,115]. Genus or species-specific 16S rRNA [48,142,143,146]
and 23S rRNA [59,112] sequences have also been targeted. Apart from detection, several PCR-
based methods have also been developed for characterization or typing of L. monocytogenes
isolates [38,62,69,140].
Real-time PCR represents an important development in the field of PCR technology. Real-time
refers to the ability to detect a PCR product as it forms from cycle to cycle, in contrast to endpoint
detection approaches such as gel electrophoresis. Advantages of real-time methods include their
wide dynamic ranges, quantitative nature, and the fact that both amplification and detection take
place in the same closed tube, reducing the chance of contamination between reactions [56,98].
Because detection of PCR products is automated, the need for time-consuming postreaction han-
dling is eliminated [98]. Several different chemistries are available for real-time PCR, including
probe-based methods such as the 5′ nuclease (TaqMan) reaction and the use of fluorescence resonance
energy transfer (FRET) probes or molecular beacons. These approaches use detection probes that
hybridize to unique sequences within target amplicons. Advantages of probe-based methods include
the ability to detect target amplicons in the presence of nonspecific PCR products and simultaneous
detection of multiple amplicons in the same reaction. Cycle-to-cycle production of PCR products
can also be monitored using fluorescent dyes that stain double-stranded DNA [64]. Although this
approach is less expensive, it lacks the specificity of probe-based systems.
In the 5′ nuclease reaction, the detection probe is labeled at the 5′ end with a reporter dye and
at the 3′ end with a quencher molecule. During the extension step of PCR, Taq DNA polymerase
displaces the detection probe from its target on the template and digests it, using the inherent
5′ to 3′ exonuclease activity of this enzyme [25,96]. In an intact probe, fluorescence from the reporter
dye is effectively quenched. Upon digestion of the probe, the reporter dye is released into solution,
away from the quencher, increasing the overall fluorescence of the system [25,96]. Signal generation
in the 5′ nuclease reaction is very specific, as only those probes that are hybridized to their targets
will be available for digestion by Taq polymerase. Design and operational factors affecting the
performance of 5′ nuclease assays have been systematically examined using L. monocytogenes as
a model target organism [82]. Use of more than one reporter dye enables multiplexing of the 5′
nuclease reaction. An example is the assay for the simultaneous and quantitative detection of both
L. monocytogenes and generic Listeria developed by Rodríguez-Lázaro et al. [112]. Food or
beverage systems in which L. monocytogenes–specific 5′ nuclease assays have been applied include
water, cabbage, and various dairy products, including raw or pasteurized milk, yogurt, cottage
cheese, and ice cream [25,96,56]. Reported sensitivities, per 25-g sample of food, range from 5
CFU after 20-h enrichment [25] to 1.42 × 102 CFU after direct extraction of DNA from spiked
samples [56]. A non-real-time (endpoint) 5′ nuclease assay using an “asymmetric” set of singly
labeled probes has also been suggested as a less expensive alternative to real-time, dual-probe
approaches for detection of L. monocytogenes [69].
POTENTIAL PITFALLS OF PCR

Errors that can affect the specificity of the final test can be introduced at various points during
PCR assay development. These include inaccuracies in the original sequences used for alignments
during primer design and use of misidentified reference strains to validate assay performance
[5]. Additionally, target sequences must be carefully chosen to ensure they are present only in
the group to be detected [76,99]. Primer specificity should also be validated experimentally
against an extensive panel of well-characterized reference strains consisting of both target and
nontarget organisms [5,99].
Foods contain a number of components that can interfere with the PCR reaction. These PCR
inhibitors include proteins, fats, polyphenolic compounds, target or primer-degrading nucleases,
and competitors of Mg2+ [99,116]. Additionally, certain components of selective media used to
enrich for Listeria may also have inhibitory activity, including acriflavin, bile salts, esculin, and
ferric ammonium citrate [116]. Apart from dilution, approaches for removal of PCR inhibitors
include centrifugation, filtration, immunomagnetic capture of target cells, adsorption of cells to
hydroxyapatite or metal hydroxides, surface adhesion of cells to polycarbonate membranes, spotting
of food washes onto filters impregnated with chelators and denaturants, and chaotropic precipitation
of DNA [8,33,59,72,84].
The extreme sensitivity of PCR can become a liability when amplicons produced in previous
reactions are present as contaminants. One approach for reducing false-positives from such
carryover contamination involves substitution of dTTP with dUTP in the reaction mix. A short
incubation with the enzyme uracil-N-glycosylase (UNG) at the beginning of each new reaction
leads to degradation of contaminant amplicons (e.g., those containing uracil residues). Ideally,
this enzyme would be inactivated during PCR cycling. However, residual activity after cycling
has been reported for UNG isolated from Escherichia coli. Such activity could result in unwanted
degradation of uracil-containing products before endpoint analysis. Use of a more heat-labile
UNG, such as that described from a psychrophilic marine bacterium, may help circumvent this
potential problem [120].
The inability of PCR to distinguish between live and dead cells has often been cited as a key
drawback of this method. Knowing whether a positive result derives from live cells or from
“environmental” DNA is of significant practical value to food processors. At least two strategies
have been devised for the PCR-based detection of live L. monocytogenes cells. One approach,
reverse transcription-PCR (RT-PCR), takes advantage of the inherent lability of mRNA, which has
a half-life on the order of a few minutes [68]. Because mRNA should be quickly degraded following
cell death, its detection can be used as a sensitive indicator of the presence of viable cells [68,98].
Klein and Juneja [68] examined the suitability of three mRNA targets for RT-PCR. Using Southern
hybridization to digoxigenin-labeled probes, these authors were able to detect an iap-specific
product from between 10 and 15 L. monocytogenes cells inoculated from pure culture into nonse-
lective media, with a total assay time of 54 h. Detection of hly or prfA messages was substantially
less sensitive. Using iap-specific RT-PCR, low numbers of L. monocytogenes were also detected
in an artificially contaminated cooked meat system after nonselective enrichment [68]. Norton and
Batt [98] adapted the 5′ nuclease assay to detect hlyA mRNA, raising the possibility of automated
detection of viable L. monocytogenes by real-time RT-PCR. Drawbacks of these mRNA-based
approaches include difficulties in extracting target RNA and interference from DNA [68,98].
A second approach to PCR-based detection of viable cells is based on use of the DNA-binding
dye ethidium monoazide (EMA), a reporter of membrane integrity [97,114]. This dye is excluded
from intact (e.g., viable) cells, but readily enters those having damaged cell membranes. Once
inside the cytoplasm, EMA binds to cellular DNA. Subsequent exposure to visible light results in
photolytic activation of bound EMA and its covalent linkage to DNA. Unbound dye also undergoes
photolysis and is no longer available for covalent binding. Nogva et al. [97] found that covalent
linkage of EMA to pure DNA inhibited the 5′ nuclease reaction, suggesting the use of this approach
for PCR-based discrimination of cell viability. An optimized EMA treatment gave good differen-
tiation between live and dead cells for isopropanol-killed L. monocytogenes. EMA-mediated reduc-
tions in PCR signals also correlated well with plate counts when other methods of preparing killed
cells (heat, benzalkonium chloride) were used [97]. Further work is needed to determine how well
the EMA method will work with natural populations and if it can be applied in food systems.
DNA MICROARRAYS
DNA microarrays are powerful tools for global genetic analyses of microorganisms. A single high-
density microarray can be used to simultaneously measure gene expression and identify species-
specific polymorphisms for every gene in an organism. This enables researchers to identify genes
associated with virulence or to examine physiological responses to stress and changes in the
environment [27]. Alternatively, a single microarray could be used to detect multiple pathogens,
and contain several pathogen-specific probes per organism [119,145]. In the analysis of Listeria,
microarrays have been used to assess the genetic diversity among different strains of L. monocy-
togenes, enabling identification of lineage and serotype-specific differences in the genome content
of these strains [17,114,155]. Host cell responses to infection with L. monocytogenes have also
been characterized using microarray technology [23].
Apart from these more sophisticated applications, DNA microarrays have also found practical
application as a means of endpoint detection in PCR [18,114,138]. In this latter application, DNA
microarrays provide an alternative to gel electrophoresis, allowing the unambiguous detection of
target amplicons [138]. This involves using them essentially as dense arrays of dot-blots against
which PCR products are hybridized [18]. In conventional PCR, successful amplification is visual-
ized by electrophoresis, with the appearance of a band correlated to the expected size of the
fragment. Resolution of similarly sized fragments requires use of more complicated techniques
such as denaturing gradient gel electrophoresis (DGGE) [22]. Using microarrays, a specific product
can be demonstrated on the basis of its hybridization to the array. The high density of microarrays
enables simultaneous detection of several genetic markers in a single experiment, even in the
presence of nonspecific PCR products that might confound gel-based analyses [18,138]. In addition
to planar arrays, microsphere-based arrays have also been developed for multiplex detection of
pathogen-specific amplicons, including those from L. monocytogenes [34].
FLUORESCENCE IN SITU HYBRIDIZATION

First described for bacteria in 1989 [29], Fluorescence in situ hybridization (FISH) is a rapid nucleic
acid–based method for identification of specific cells. In this technique, fluorescently labeled nucleic
acid probes are hybridized to complementary rRNA sequences located on ribosomes within whole,
permeabilized cells. Actively growing cells may contain several thousand ribosomes and the aggre-
gate signal arising from multiple probe/ribosome binding events leads to sequence-specific fluo-
rescence of target cells [2]. A typical FISH assay involves at least four different steps: fixation,
hybridization, removal of nonspecifically bound probe by washing, and detection [3]. Although
fixation alone is enough to permeabilize some cell types to probe entry, the thick cell wall of
Listeria requires additional enzymatic or chemical digestion before hybridization with DNA-based
probes [139].
FISH probes may be designed against either the 16S or the 23S ribosomal subunits. The 23S
subunit is larger and contains several potentially useful variable regions, but the 16S subunit has
been more thoroughly characterized, and is therefore more frequently used [2]. A number of reports
have described development of Listeria or L. monocytogenes–specific probes targeting the 16S
rRNA [13,48,88,121,142]. However, these probes were developed for PCR or membrane-based
assays, rather than for detection of whole cells. The three-dimensional structure of the ribosome
is complex, and potentially diagnostic sequences may be “buried” within this structure or located
under protein binding sites. As a result, not all rRNA-targeted probes will bind to their comple-
mentary targets in whole bacterial cells.
Two FISH-suitable DNA probes targeting the 16S rRNA of Listeria spp. have recently been
described: Lis-637, which reacts with all Listeria species except for L. grayi, and Lis-1255, which
reacts with all six Listeria species as well as with members of the closely related genus Brochothrix
[118]. The specificities of these probes reflect the difficulty of designing FISH-suitable probes that
react with all six species, yet are restricted to the genus Listeria. A FISH-based testing kit containing
probes said to be specific for Listeria spp. or for L. monocytogenes is now commercially available
(VIT-Listeria, vermicon, A.G., Munich). According to the manufacturer, Listeria spp. and L. mono-
cytogenes can be detected in a single test with this slide-based kit. The test is said to require about
30 min of preparative time, and to be complete within 3 h. In addition to the sample, a positive
and a negative control are done with each test. After hybridization, L. monocytogenes is visually
differentiated from other Listeria spp. according to the color of probe-conferred fluorescence, with
L. monocytogenes fluorescing red and Listeria spp. fluorescing green. This probe system was
evaluated against cultural methods for detection of Listeria spp. and L. monocytogenes in 195 food
samples (mainly minced meat) and 103 swabs from food-processing environments [124]. For
standard cultural detection, samples were pre-enriched for 24 h in Half Fraser broth, transferred to
Fraser broth for another 24 h, plated onto PALCAM or Oxford agars, and incubated for 48 h before
biochemical testing. For rapid FISH-based detection, FISH analysis was done after the second
liquid enrichment. For detection of L. monocytogenes, the rapid method yielded the same results
as culture, but 48 h earlier. Detection of generic Listeria was also as rapid, but in some samples,
strong green autofluorescence from nontarget bacteria reduced the sensitivity of the test [124].
Although most reports of FISH-based detection of Listeria spp. have involved DNA-based
methods, peptide nucleic acid (PNA) probes have been shown to have substantial practical and
functional advantages for detection of this genus [12]. PNAs are synthetic DNA mimics made
by attachment of natural or modified nucleobases onto a repeating backbone of amide-linked
N-(2-aminoethyl) glycine units [95]. As with DNA probes, PNAs hybridize to complementary DNA
or RNA sequences through Watson–Crick base pairing, but they also exhibit several advantageous
properties that can be ascribed to the uncharged, hydrophobic nature of the PNA backbone [123].
Some of these properties include accelerated hybridization kinetics, intrinsic resistance to nucleases
and proteases, and the ability to penetrate recalcitrant biological structures such as the Gram-positive
cell wall [123]. PNA probes are also able to bind their target sequences under high-stringency,
denaturing conditions (e.g., low salt, high temperature, high pH). As a result, PNA probes are able
to bind to portions of the ribosome that are inaccessible to DNA probes because of the higher-
order structure of the ribosome [123]. Together, these properties make PNA probes uniquely suited
for use as FISH probes, with important applications in detection of Listeria spp. (see later section
titled “Flow Cytometry”).
FISH has been used to great effect for cultivation-independent characterization of complex
microbial communities of interest to environmental microbiologists [2]. Similar approaches may
also be useful to describe microbial ecosystems occurring in foods [43]. For example, methods
have recently been described for the FISH-based analyses of thinly sectioned, resin-embedded
cheeses, including Stilton and Brie [36]. In this way, valuable information may be collected on the
spatial arrangements and physical interactions of different cell types occurring within these foods,
including pathogens such as L. monocytogenes.
RECOMBINANT BACTERIOPHAGE
Phage-based diagnostic schemes take advantage of host specificities displayed by these viruses.
Phage-bacterium recognition is mediated by specific receptors displayed on the host cell surface.
For L. monocytogenes, known phage receptors include teichoic-acid-associated carbohydrate resi-
dues and peptidoglycan [73,79,132]. Infection begins with recognition and attachment to host
surface receptors, followed by injection of phage nucleic acids into the host cell. As with other
viruses, the machinery of the host cell is then subverted to make more virus particles [87]. The
efficiency of phage-mediated delivery of nucleic acids to specific host cells forms the basis of their
use as diagnostic agents.
Development and application of a recombinant phage-based method for identification of Listeria
spp. has been described [79,80]. In this work, a recombinant derivative of the Listeria-specific
bacteriophage A511 was constructed. The recombinant phage, A511::luxAB, carried the gene for
a Vibrio harveyi LuxAB fusion protein immediately downstream from the gene for the major capsid
protein (cps) [79]. Bacterial luciferase catalyzes a light-producing reaction involving oxidation of
reduced flavin mononucleotide (FMNH2) and long-chain aliphatic aldehydes by molecular oxygen.
An aldehyde substrate (nonanal) was added to the system and postinfection transcription of the
luxAB fusion within target cells resulted in Listeria-specific luminescence [79]. Because the reduc-
ing power needed to regenerate FMNH2 is derived from cellular metabolism and because dead cells
do not support phage replication, only live host cells are detected with this method [91,125].
This phage-based detection approach was comparable in sensitivity to standard plating methods
for Listeria, yet detection of L. monocytogenes was possible in food and environmental samples
after 20 h of enrichment, compared to the 4 days required for the culture method [80]. Apart from
selective identification of live target cells, an advantage of phage-based detection methods is that
phage can be produced in relatively large quantities, an attractive feature for any diagnostic reagent
[87]. Drawbacks include the inability to detect phage-resistant strains and the potential for con-
tamination of certain foods (e.g., seafood) with naturally bioluminescent microflora that might
confound the analysis [80,87,132].
ANTIBODY-BASED METHODS
Antibodies play several essential roles in host defense against intracellular pathogens such as
L. monocytogenes. These include preventing entry into host cells through binding of key surface
molecules and neutralization of toxins and opsonization, leading to activation of the complement
cascade [35]. The molecular selectivity that antibodies display in these natural roles also forms the
basis for their use as diagnostic reagents. Antibodies are versatile and can be used to detect both
whole cells and isolated cellular components [75]. A comprehensive review of L. monocytogenes-
specific antigens and antibodies reported for their detection has been published [10]. Apart from
detection, antibodies can also be used as capture agents for selective concentration of target cells
(see later section titled “Cell Separation and Concentration”).
Certain properties make antibodies uniquely suitable for detecting live target cells. These
include the ability of some antibodies to bind epitopes present only in living cells [122,131] and
their capacity to bind under mild, physiologically compatible conditions. Additionally, antibodies
directed against surface antigens do not require cell permeabilization to reach their targets, and can
therefore be used on unfixed (living) cells. These properties enable antibody-based labeling to be
combined with cell analysis and sorting techniques such as flow cytometry, with subsequent cultural
analyses of sorted cells.
POTENTIAL PITFALLS OF ANTIBODY-BASED METHODS

In comparison to amplification-based detection methods such as PCR, antibody-based methods are
relatively insensitive. Typical detection limits for ELISA are ~105 to 106 CFU/mL [93,99]. Antibody
concentrations can be varied to saturate available target molecules, but in some assay formats high
concentrations can lead to deleterious effects such as cell clumping and precipitation [30]. Anti-
bodies may also suffer from substantial cross-reactivity, and expression of antigens may vary under
different cultural conditions or as a result of environmental stress [31,40,93]. Finally, genes coding
for antigenic determinants can be acquired from closely related species through lateral transfer.
As an example of this, strains of L. innocua expressing cell surface antigens characteristic of
L. monocytogenes serogroup 4 have recently been isolated [73].
ADVANCES IN ANTIBODY-BASED TECHNOLOGIES

Typical methods of generating antibodies include immunization of animals for production of
polyclonal antisera or use of monoclonal-producing hybridoma technology [60]. With the advent
of phage display techniques, recombinant antibody fragments can now be selected from highly
diverse libraries consisting of billions of clones [106]. In a phage display library, a large repertoire
of single-chain or other antibody fragments is displayed on the surface of filamentous phage such
as M13, fused to the phage’s coat proteins. Phage displaying antibody fragments that bind to a
given cell type can be isolated from the library through sequential affinity selection or “biopanning.”
In this process, phage are mixed in vitro with whole target cells, which are bound to a solid support.
Phage having an affinity for target cells are captured, and nonbinding phage are washed away.
Bound phage can then be eluted, propagated in a susceptible host, and subjected to further rounds
of affinity selection [7,60,102,106]. Subtractive panning against cells of closely-related species may
be used to remove phage that cross-react with nontarget cells [102]. The same overall approach
may also be followed using a purified antigen instead of whole cells. Multiple panning iterations
can result in selection of antibody fragments that are highly specific for target cells or antigen.
Advantages of this technique include its speed, simplicity, and ability to detect antigens in their
native (e.g., nondenatured) form [7,60,106]. Recently, Paoli et al. [102] isolated single-chain
antibodies (scFvs) that react with L. monocytogenes, but not with other Listeria spp. Benhar et al.
[7] used the same library (“Griffin.1”) to isolate scFvs for use in a Listeria-specific biosensor.
Biopanning against a phage display library of llama single-domain antibodies has also been reported
for selection of antilisteriolysin binding fragments, although affinities of these fragments were
reported to be low [21].
An interesting variation on the immunoassay format used a superconducting quantum interfer-
ence device (SQUID) for label-free detection of L. monocytogenes in solution [49]. Cells of
L. monocytogenes were reacted with antibody-labeled magnetic spheres, and a pulsed magnetic
field was applied. Differences in the magnetic relaxation signals of bound or unbound magnetic
spheres, as measured with a SQUID microscope, allowed indirect detection of L. monocytogenes
in solution. This technology is still in the early developmental stage and the reported sensitivity
(5.6 × 106 L. monocytogenes cells per mL) was low. However, this approach differs from a typical
immunoassay in that it is label free, and there is no need for immobilization of the target cells or
for removal of unbound microspheres before taking measurements [49].
Through molecular imprinting (MIP), artificial antibodies or biological receptor mimics can be
formed within a polymer matrix. Imprinted polymers capable of recognizing specific molecular
substrates, viruses, or even whole cells have been demonstrated [51,52]. Some of the earliest work
on whole-cell imprinting was done using L. monocytogenes as a template organism. In this work,
chemically functional (lectin-reactive) “lithographic prints” of this organism were made on surfaces
of polymer beads [1]. Future improvements in MIP-based recognition of whole cells may lead to
new Listeria-specific bioaffinity surfaces for use in biosensing applications. Unlike natural molec-
ular recognition elements (e.g., antibodies), molecularly imprinted polymers can be designed to
have high physical and chemical stabilities, can bind target molecules under nonaqueous conditions,
and may be reused multiple times [107].
CELL SEPARATION AND CONCENTRATION

Separation of low numbers of target cells from food matrices represents a major obstacle to rapid
detection of specific foodborne bacteria, including Listeria [94]. Contamination can occur in a wide
variety of foods, and may occur unevenly within a given food, resulting in “hot spots” or localized
regions of higher bacterial concentration [33]. Effective methods for capture and concentration of
cells from foods could potentially shorten enrichment times or even eliminate the need for growth-
based enrichment completely [144]. Concentration techniques may also be useful for removal of
food components that could interfere with assay performance. Examples include removal of partic-
ulate matter that could interfere with light scattering in flow cytometry, or removal of PCR inhibitors.
Common physical means of separating cells from food matrices include filtration and centrif-
ugation [8,69]. Methods available for nonspecific capture of bacteria based on cell surface properties
include phase partitioning using aqueous polymers, surface adhesion to polycarbonate membranes,
and immobilization on hydroxyapatite or metal hydroxide particles [8,81,103]. Dielectrophoresis
provides a means for effective separation of bacteria from other biological particles having different
dielectric properties [57] (see later section titled “Chip-Based Microanalytical Systems”).
Immunomagnetic separation (IMS) enables specific capture and concentration of Listeria spp.
directly from foods or environmental samples [59,90,96,134]. This approach uses commercially
available paramagnetic beads whose surfaces are functionalized with Listeria-specific antibodies.
Typically, samples are homogenized in buffer and large food particles are removed via centrifuga-
tion. Antibody-coated beads are then combined with the resulting supernatant liquid, and the sample
is incubated with gentle mixing to facilitate cell binding [59]. Once cells are bound to the surface
of the beads, a magnetic field is applied, trapping the beads in place. At this point, the supernatant
liquid may be removed and the beads washed to allow more complete removal of food components.
Immunomagnetically isolated cells may then be added to liquid or solid media for further growth
or enumeration, or processed for molecular analyses. Other variations of the method include
application of IMS after an initial enrichment, or the use of DNA-reactive beads to isolate DNA
before PCR [66,96]. Problems reported for this technique include reduced recovery of beads from
high-fat foods, and poor cell recovery when multiple wash steps were used [33,59].
The relatively small fraction of the original sample typically subjected to IMS has also been
cited as a potential source of reduced assay sensitivity [66]. Use of less expensive nonmagnetic
beads, combined with centrifugal recovery, has been suggested as an economically feasible approach
to scaling up the immunoseparation of L. monocytogenes from foods to larger sample volumes [66].
As another approach to this problem, one commercially available IMS-based system (PATHATRIX,
Matrix MicroScience, Ltd., Cambridgeshire, UK) operates by continuously circulating an entire
1:10 dilution (225-mL + 25-g food sample) over a magnetic bead-based antibody “capture phase.”
Heating of the sample during circulation allows simultaneous capture and enrichment, increasing
the sensitivity of the method. Once cells are captured and washed, the magnet is removed and the
beads are eluted for further cultural or molecular analyses. The system’s test for Listeria spp. is
designated as an AOAC “Performance Tested” method.
Another immunocapture system that addresses the need for concentration of low numbers of
Listeria spp. and other target bacteria from large sample volumes has been described [94,144].
This system employs a fluidized bed of relatively large (~ 3-mm diameter) antibody-coated glass
beads through which a liquefied food or environmental sample is passed at flow rates exceeding
100 mL/min. After capture, bound cells are detected via bead-based ELISA or with molecular
methods. The system is able to detect as few as 100 CFU/mL in as little as 30 min and is volume
independent, being able to process samples ranging from milliliters to several liters.
ADDITIONAL METHODS
FLOW CYTOMETRY
Flow cytometry is a basic diagnostic tool that facilitates the rapid analysis of complex cell popu-
lations according to single cell fluorescence characteristics. Data on multiple cellular parameters
may be collected simultaneously, including information on cell count, size, or response to diagnostic
fluorescent probes. Fluorescent probes interacting with a variety of cellular targets are commercially
available, and range from nucleic acid–binding dyes and respiratory substrates to fluorescently
labeled antibodies or rRNA-targeted nucleic acid probes. In flow cytometry, cells suspended in a
liquid sample are passed individually through an intense light source, such as a laser or an arc
lamp. Data on cellular characteristics, such as light scattering and probe-conferred fluorescence, are
collected and saved as a computer file for subsequent offline analysis. The information-rich nature
of these files enables detailed and quantitative analyses of specific populations and subpopulations
of interest. Software for data analysis is available commercially or as freeware and provides flexible
options for data presentation. Different types of data displays can be used to highlight particular
features of the dataset. In general, data collected on several thousand events are displayed as dot
plots, contour plots, or histograms, using a four-decade log scale.
Until recently, most commercially available flow cytometers were large, expensive machines
optimized for immunological studies on mammalian cells. However, a handful of smaller, less
expensive machines, some of which are designed specifically for microbial analysis are now
available. These include “turnkey” or “pushbutton” systems incorporating automated reagent-
dispensing and sample-handling capabilities. Many of these systems also offer precise, metered
sample injection, enabling the absolute enumeration of target cells as a function of sample volume.
Combined with commercial availability of a wide variety of fluorescent stains and probes, microbial
cytometry is now more accessible than ever before.
As a general tool, flow cytometry can be used in a wide variety of fluorescence-based applications.
This method has been used to study several aspects of the biology of L. monocytogenes, including
its uptake and survival in peripheral blood leukocytes [109], identification of potential virulence
genes induced during the infection process [151], characterization of bacteriocin-mediated cell
damage [108,126], and fluorescence-based viability assessment of both L. monocytogenes and other
Listeria spp. [61,108]. However, very little has been published regarding use of flow cytometry for
rapid detection of L. monocytogenes or other Listeria spp. In their pioneering work, Donnelly et
al. combined use of fluorescent antibodies and nucleic acid staining with flow cytometry to detect
L. monocytogenes in raw milk [30,31]. This work demonstrated the potential of flow cytometry for
detecting specific microorganisms in foods. Because the antibodies used in this study showed
FIGURE 8.1 Combined PNA-FISH and flow cytometry for rapid detection of Listeria spp. in hot dogs.
Commercially purchased hot dogs were contaminated with L. monocytogenes, vacuum-packaged, and stored
for 1 week under conditions of mild temperature abuse (8°C). Samples were then opened, washed with a
small amount of a dilute, nonselective nutrient medium (1/4-strength MRS broth), and either processed
immediately for hybridization and cytometry or incubated for up to 70 min at 35°C before processing. Whereas
L. monocytogenes was detected directly in washes from contaminated samples without incubation (panel B),
brief exposure to dilute nutrient media allowed further growth of the cells, leading to brighter hybridizations
with the rRNA-targeted PNA probes (panel D). Panels A and C represent noninoculated controls without
incubation, or with 70-min exposure to dilute nutrient media, respectively. Samples were analyzed using a
Becton Dickinson FACSCalibur.
substantial cross-reactivity, especially with Staphylococcus aureus, a selective enrichment step was
done before cytometric analysis. After enrichment, it was possible to differentiate Listeria from
nontarget microflora on the combined basis of light scatter, nucleic acid staining, and immunoflu-
orescence characteristics [31].
In recent work, we have combined PNA-FISH with flow cytometry for rapid detection of
Listeria spp. in media or in washes from vacuum-packaged RTE meats (Figure 8.1). Using
this approach, L. monocytogenes was detected directly from contaminated packages of hot dogs
held for up to a week under conditions of mild temperature abuse (Figure 8.1B). Brief postsampling
incubation in dilute nutrient media yielded further cell growth and rRNA production, leading to
brighter hybridizations with the rRNA-targeted probes (Figure 8.1D). Compared to hot dogs, sliced
turkey samples contained high levels of particulate matter, likely caused by enhanced microbial
degradation of their open surfaces, with associated release of meat fragments and production of
fat micelles. However, detection of L. monocytogenes in these samples was still possible even
without use of additional methods for sample cleanup, such as filtration (Figure 8.2). For both meat
systems, hybridizations were very brief (10–15 min) and cell fixation and permeabilization steps
FIGURE 8.2 Combined PNA-FISH and flow cytometry for rapid detection of Listeria spp. in sliced turkey.
Commercially purchased sliced turkey meat was contaminated, processed, and analyzed as described for hot
dogs in Figure 8.1. Background noise from particulate matter (population A) was high in these unfiltered
washes, but did not interfere with detection of L. monocytogenes. In this figure, two discrete subpopulations
of L. monocytogenes can be seen: the main population (B) and a smaller, more active population containing
higher amounts of rRNA (C).
were accomplished simultaneously with resuspension of pelleted wash material in 50% ethanol.
For RTE meat samples contaminated with high numbers of listeriae, combined incubation, fixation,
hybridization, and cytometric analysis steps were accomplished in as little as 2 h. Without optimi-
zation, we have been able to detect approximately 105 cells/mL of stationary phase L. monocyto-
genes against a background of 107 cells/mL nontarget bacteria in spiked hot dog wash. Approaches
for improving the sensitivity of detection include “gating out” or ignoring signals from smaller
food particles, fat micelles, or unlabeled (nontarget) microflora and removal of larger food particles
via filtration. The use of a cytometer optimized for microbial detection should further improve the
sensitivity of this assay, as some such systems are able to detect fewer than 100 well-labeled target
cells per milliliter.
BIOSENSORS
Biosensors are compact analytical devices capable of translating the binding or recognition of a target
analyte into an output signal that is proportional to the concentration of analyte in the sample [133].
The three major components of a biosensor are a recognition element or bioaffinity agent that is bound
to the surface of the biosensor, a means of generating an electrical signal from the binding of the
target to this recognition element (a transducer), and an output device [75]. The types of recognition
FIGURE 8.3 Detection of L. monocytogenes using a surface plasmon resonance-based biosensor. Output from
a flow-through surface plasmon resonance (SPR) biosensor is shown. The presence of L. monocytogenes in
suspension was detected through shifts in resonant wavelength upon binding of target cells to antilisterial
antibodies immobilized on the sensor’s surface. The magnitude of each shift was proportional to the number
of Listeria present. Although the sensitivity of this system was low (106 CFU/mL), it was comparable to
ELISA using the same antibodies. Advantages over standard ELISA include label-free detection of target cells
and the ability to follow the capture reaction in real time. Replacement of solutions, addition of various levels
of L. monocytogenes, and changes in flow rate are indicated by arrows. Key: tw = 0.05% Tween in PBS; LM
106 = 106 CFU/mL L. monocytogenes in the same buffer; f20 = flow rate of 20 µL/min). (Reprinted from
Koubová, V., B. Brynda, L. Karasová, J. Skvor, J. Homola, J. Dostálek, P. Tobiska, and J. Rosicky, 2001.
Detection of foodborne pathogens using surface plasmon resonance biosensors. Sens. Actuators B Chem. 74:
100–105. With permission from Elsevier.)
elements that could be incorporated into biosensor design include nucleic acids (e.g., single-stranded
DNA, peptide nucleic acids, DNA or RNA aptamers, etc.), antibodies or recombinant antibody
fragments, enzymes, lectins, peptides, or even whole cells [60,71,133,154]. Methods for transducing
a specific binding reaction into a useful signal include optical, acoustic, electrochemical, or thermal
approaches [71,75,133]. For example, surface plasmon resonance (SPR), an optical approach, detects
minute changes in refractive index at the surface of a functionalized waveguide upon binding of the
target analyte (Figure 8.3). The waveguide can be functionalized with either antibodies or nucleic
acids, and changes in refractive index are proportional to the amount of analyte that binds to these
bioaffinity agents [129]. Acoustic sensors, such as quartz crystal microbalances (QCMs), operate on
the principle that binding of an analyte to the surface of a functionalized crystal will increase its mass
and modulate the frequency at which the crystal resonates [75,133]. Again, changes in resonant
frequency are proportional to the amount of analyte bound.
Attractive features of biosensors include their potential to provide rapid, label-free detection
with minimal sample preparation, the capacity to detect multiple analytes, and the ability to
follow binding reactions in real time. Drawbacks include questions about their long-term stability
under “real-world” processing conditions, whether or not the bioaffinity agent can be successfully
regenerated, and the small volumes that are typically sampled [75]. Additionally, the specificity
or sensitivity of a biosensor is ultimately limited by the binding properties or quality of the
bioaffinity reagent used [70,128].
Several antibody-based biosensors to detect whole L. monocytogenes cells have been described.
Depending on the experimental setup and the antibodies used, reported detection limits (in buffer)
vary from 500 CFU/mL to 4 × 108 CFU/mL, with total assay times ranging from minutes to hours.
To date, there are few reports detailing the use of biosensors to detect L. monocytogenes directly
in foods. However, biosensor-based detection of L. monocytogenes in enrichment cultures could
potentially shorten the time-to-result compared with traditional media-based detection. Biosensor
formats that have been investigated to detect L. monocytogenes include QCM [89,135], enzyme-
linked amperometric detection using screen-printed electrodes functionalized with L. monocytogenes-
specific antibodies [7,26], evanescent wave fiber-optic systems [41,128], and SPR [70].
CHIP-BASED MICROANALYTICAL SYSTEMS

Microscale bioanalytical systems (or biochips) are a result of the convergence of materials science
and biology and may combine electrical, mechanical, chemical, and/or microfluidic approaches for
analysis of biological materials. Steps such as cell manipulation, addition, mixing and washing of
reagents, temperature cycling, and analyte detection can be done sequentially within the same
device [39,58]. Although the amount of analytical material handled by a single biochip device is
small, so are the amounts of potentially expensive reagents used. Multiple devices may be operated
in parallel, and they are amenable to automation. Analytical methods that have been successfully
translated to the microscale and could potentially be incorporated within chip-based analytical
systems include flow cytometry and cell sorting [39], impedance spectroscopy [45,46], the poly-
merase chain reaction, and various isothermal methods for nucleic acid amplification [58]. Move-
ment of biological particles (molecules or cells) on or through a biochip can be directed through
application of an electrical field. Examples include use of a DC electrical field for electrophoretic
movement of charged bioparticles or the use of an AC field for the dielectrophoretic manipulation
of polarized (neutral or charged) bioparticles [57]. Dielectrophoresis is especially useful, as it can
be used to move, trap, separate, or concentrate cells based on their dielectric properties, which may
differ according to cell volume, structure, composition, or viability status [19,77]. An example of
dielectrophoresis for on-chip separation of L. monocytogenes from red blood cells is given in
Figure 8.4. Other chip-based approaches have been described to detect and characterize Listeria
spp. [19,45,46,77]. Bacterial metabolism of a low-conductivity medium alters its electrolyte con-
centrations, allowing detection of bacterial growth over time by measuring changes in impedance.
Gómez et al. [46] constructed a microfluidic biochip able to measure impedance of bacterial
suspensions in a very small volume of medium (~5 nL) over a 100 Hz–1 Mhz range. The small
volume of the test chamber increases the effective concentration of bacterial cells, so that presence
of only a few metabolizing cells can lead to rapid changes in impedance within a short time [46].
After off-chip incubations of 2 h, L. innocua or L. monocytogenes could be detected to a level of
~100–200 cells. Among limitations of the present work is that there is no mechanism for ensuring
that impedance signals are derived from the activity of target cells. Antibody-based capture of
Listeria spp. or use of Listeria-selective conductance media may be potential options for addressing
this need. Microfabricated devices have also been described for distinguishing live Listeria from
heat-killed Listeria based on either electrophoretic mobility or dielectrophoretic properties [19,77].
In one study, dielectrophoretic separation of mixed populations of live and dead L. innocua in water
was possible with a reported efficiency of 90% [77].
SPECTROSCOPIC METHODS
Vibrational methods such as Fourier transform infrared (FT-IR) and Raman spectroscopies provide
a means of obtaining unique spectral “fingerprints” of specific cell types and can discriminate
FIGURE 8.4 Dielectrophoretic separation of Listeria monocytogenes from red blood cells. A mixture of
L. monocytogenes (LM) and red blood cells (RBC) was introduced onto a microelectronic array of platinum
electrodes fabricated on the surface of a silicon wafer (A). A 10-KHz AC voltage was applied in a “checkerboard”
mode, with adjacent electrodes along either vertical or horizontal lines receiving signals of the opposite polarity.
In response to the applied field, LM cells accumulated on the circular electrodes and RBC accumulated at
positions between the electrodes (B). Stronger dielectrophoretic forces acting on LM cells allowed the selective
removal of RBC from the system by washing (C and D). The captured LM cells could then be released from
the array by turning off the electric field. Alternatively, captured cells may be subjected to on-chip identification
via electric field–mediated immunoassay or nucleic acid–based methods. (Example kindly provided by Y.
Huang and D. Hodko, Nanogen, San Diego, California.)
between bacteria at the genus, species, or strain level [86,113]. These spectral signatures are a
function of the intrinsic biochemical composition (lipids, proteins, carbohydrates, and nucleic acids)
of each cell type and do not depend on the use of labels or dyes [86]. Whole-cell FT-IR and Raman
spectroscopies are nondestructive techniques. Destructive methods such as matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-MS) may also be used [86,92,113,141]. Typi-
cally, FT-IR analyses of bacteria are done by drying liquid cultures or resuspended colonies onto
an IR-transparent substrate, such as a zinc selenide crystal [55,74,92,113]. Alternatively, disposable
optical polymer films may be used. These provide a reproducible, archivable substrate—attributes
that would be beneficial if FT-IR spectroscopy were to be used for routine identification of
pathogenic bacteria [92]. During spectral collection, multiple scans (hundreds to thousands) may
be averaged to enhance signal-to-noise ratios [86,92]. Total collection times vary according to
experimental setup, but range from less than 1 min to 45 min [86]. Several multivariate statistical
techniques can be used for analysis of the resulting complex spectra, including principal component
analysis (PCA) and hierarchical component analysis (HCA) [86,92,113]. In addition to whole cells,
preparations of cellular components, such as fatty acid methyl esters, may also be analyzed with
spectroscopic techniques [147]. Advantages of spectroscopic methods are that they are fast, accurate,
require minimal sample preparation, and can be coupled with objective means for data analysis
[55,74,113]. Drawbacks are that cells must be isolated in pure culture before analysis and that high
cultural densities (~108–109 CFU/mL) are often needed to obtain reproducible spectra [86,113].
However, recent advances include methods for analysis of mixed cultures using MALDI-MS [141]
and the ability to collect spectra from microcolonies or individual cells using microspectroscopic
methods [50,86].
A number of studies have focused on the use of spectroscopy to identify Listeria or L. monocytogenes.
Holt et al. [55] used FT-IR spectrometry to discriminate between all six species of Listeria, with
an additional distinction made between L. grayi and L. grayi subsp. murrayi. Figure 8.5 shows the
graphical output of transformed spectral data for the Listeria strains examined in this work. Lefier
et al. [74] examined effects on spectra of different cell growth conditions and sample preparation
procedures and found the method to be robust and relatively unaffected by changes in these variables.
10
L. grayi
L. ivanovii
5
L. grayi subsp. murrayi

L. innocua
Canonical Variate 1
L. monocytogenes
L. welshimeri
–5
L. seeligeri
–10
–10 –5 0 5 10
Canonical Variate 2
FIGURE 8.5 Differentiation of Listeria spp. using Fourier transform infrared (FT-IR) spectroscopy. Listeria
spp. grown in liquid culture were pelleted, washed, and resuspended in distilled water. Portions of these
suspensions were dried (50°C, 45 min) on the surface of a ZnSe optical plate and infrared spectra were
collected. Spectral data were transformed for canonical variate (CV) analysis. A projection of two of the
resulting canonical variates is shown. All Listeria species (including L. grayi subsp. murrayi) could be
distinguished from each other using this method. Although spectral clusters for L. monocytogenes and
L. seeligeri are superimposed in this two-dimensional projection, the two species could be clearly differentiated
using additional canonical variates (not shown). (Reprinted from Holt, C., D. Hirst, A. Sutherland, and F.
MacDonald. 1995. Discrimination of species in the genus Listeria by Fourier transform infrared spectroscopy
and canonical variate analysis. Appl. Environ. Microbiol. 61: 377–378. With permission from the American
Society for Microbiology.)
These authors also found that spectral analysis could be used to differentiate between two serotype
groups of L. monocytogenes—one containing serotypes 4 and 4b and the other containing
serotypes 1, 1/2b, and 1/2c. Using near-infrared Fourier transform (FT-NIR) spectroscopy, Rodriguez-
Saona et al. [113] were able to clearly differentiate L. innocua from other bacteria, including
Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa. Lin et al. [78] used FT-IR
spectroscopy to differentiate between Listeria spp. and also found differences in spectra collected
from “healthy” cells and those subjected to injury via sonication. Grow et al. [50] described a
chip-based system for capture and on-chip spectral fingerprinting of microorganisms using
surface enhanced Raman scattering (SERS) microscopy. Capture agents investigated for generic
Listeria or L. monocytogenes included polyclonal antibodies, various lectins, and fibronectin.
Once bound to the surface of the chip, Listeria and other target cells were identified using whole-
organism Raman spectra.
CONCLUSIONS
To maintain a safe and wholesome food supply, the food industry needs access to rapid, reliable, and
sensitive methods of detecting bacteria, including Listeria spp. and L. monocytogenes. Other desirable
attributes include simplicity, cost-effectiveness, quantitative capacity, and online or real-time capabil-
ities. Although no single method can meet these ideal demands, rapid microbial detection is a
constantly evolving field characterized by continuous technological advances. Future developments
will undoubtedly bring the state of the art closer to these ideals. This chapter has reviewed several
analytical methods reported for detection of Listeria. These include both established methods (PCR,
antibody-based methods) and more experimental technologies (biosensors, chip-based systems,
spectroscopy). Rapid methods are valuable tools for routine screening of foods and environmental
samples, but will not entirely replace standard culture in the near term. At present, negative results
by rapid methods are considered definitive, but a positive result is viewed as presumptive and must
be confirmed through culture [99]. Changes in this outlook will require both advances in rapid
detection technology and regulatory acceptance of alternative methods. Apart from the detection step
itself, sample preparation and adequate sampling procedures remain key challenges in development
of effective rapid detection techniques [94]. The sheer diversity of food matrices and the fact that
Listeria may be present at very low levels are particularly daunting challenges [32,94,114]. As a result,
most rapid detection technologies still require a cultural enrichment step, although recent advances
in immunological separation techniques have sought to circumvent this need [144]. Data regarding
the performance of rapid detection methods are most often collected on laboratory-grown cells cultured
under optimal conditions. Listeria present in foods or in the food-processing environment may be
subjected to a variety of stressful or injurious conditions, including heating, freezing, exposure to
sanitizers, and high acid or salt concentrations [32,40]. Rapid methods capable of discriminating
between living, dead, or sublethally injured cells are needed. Finally, advances in technologies for
microbial inactivation such as use of high pressure, pulsed electric fields, pulsed light, ohmic heating,
or ozonolysis are not well characterized in terms of their physiological effects on bacterial cells.
Further studies should address how use of such alternative processing methods may impact the ability
of rapid methods to detect Listeria spp. in foods.
ACKNOWLEDGMENTS
This work was supported in part by a grant from the North American Branch of the International
Life Sciences Institute (ILSI N.A.). The opinions expressed herein are those of the authors and do
not necessarily represent the views of ILSI. Additional support was provided by grants from
sponsors of the Food Research Institute, and by the College of Agricultural and Life Sciences,
University of Wisconsin–Madison.
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9 monocytogenes
Subtyping Listeria
Lewis M. Graves, Bala Swaminathan,

and Susan B. Hunter
CONTENTS
Introduction ....................................................................................................................................283
Conventional Methods ...................................................................................................................284
Serotyping.............................................................................................................................284
Bacteriophage Typing (Phage Typing).................................................................................285
Bacteriocin Typing ...............................................................................................................286
Antimicrobial Susceptibility Testing....................................................................................286
Molecular Methods ........................................................................................................................287
Multilocus Enzyme Electrophoresis ....................................................................................287
Chromosomal DNA Restriction Endonuclease Analysis (REA).........................................287
Restriction Fragment Length Polymorphism Analysis:
Ribotyping and Other Methods............................................................................................288
DNA Macrorestriction Analysis by PFGE...........................................................................291
Random Amplification of Polymorphic DNA (RAPD).......................................................292
Repetitive Element-Based Subtyping...................................................................................294
Amplified Fragment Length Polymorphism ........................................................................294
DNA-Sequence-Based Subtyping Strategies .......................................................................295
Comparison of Methods Used to Subtype L. monocytogenes ......................................................297
References ......................................................................................................................................299
INTRODUCTION
Most bacteria at the species level have sufficient phenotypic and genotypic diversity to allow for
identification of different subtypes. Therefore, phenotyping and genotyping systems—singly or
in combination—provide useful subtyping schemes for pathogenic bacteria. The various subtyp-
ing systems reviewed in this chapter provide different degrees of discrimination among Listeria
monocytogenes isolates. Distinguishing individual strains or groups of strains using these systems
in epidemiologic studies has allowed researchers to obtain information on relationships between
isolates, identify disease outbreaks, identify source of infections in outbreaks and sporadic disease
settings, and determine modes of transmission for the organism. We present a broad overview
Note: Use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the
U.S. Department of Health and Human Services. The manuscript for this chapter was submitted to the editors on December
2, 2003. The information provided in this chapter and the references cited herein are current as of December 2003.
283
of the subtyping methods that have been applied to L. monocytogenes and where appropriate,
discuss briefly the strengths and weaknesses of each.
Since publication of the second edition, the genome of Listeria monocytogenes serotype 1/2a
(EGD strain) has been sequenced [45]; the genome sequence of L. monocytogenes serotype 4b has
been completed, and BLAST searches against the genome sequence can be made at the Web site
of The Institute of Genomic Research (available at http://www.tigr.org). Consequently, development
of more sophisticated molecular techniques are forthcoming or are awaiting evaluation and vali-
dation. However, in this chapter we will continue to separate the most commonly used typing
methods for L. monocytogenes into two major categories: conventional methods and molecular
methods. At present, each of these methods has some utility; however, with extraordinary devel-
opments in nucleic acid technology and wide availability of these technologies, some conventional
methods may not be used in the future.
L. monocytogenes is among the first of the pathogenic bacteria for which a concerted and
internationally coordinated attempt has been made to critically evaluate various available subtyping
methods and to standardize the more useful methods. In 1996, J. Bille and J. Rocourt organized
the World Health Organization (WHO) Multicentre Listeria monocytogenes Subtyping Study. The
results of Phase I of this study were published in a special issue of the International Journal of
Food Microbiology [39] and will be referenced throughout this chapter.
CONVENTIONAL METHODS
SEROTYPING
Serotyping has been one of the classic tools for epidemiologic and sporadic case studies of
L. monocytogenes [41,58], and many scientists consider it the “gold standard” for typing L. monocytogenes.
Strains of L. monocytogenes differ in antigenic determinants expressed on the cell surface. Such
antigenic variations are produced by many different surface structures, including lipoteichoic acids,
membrane proteins, and extracellular organelles (e.g., flagella and fimbriae). These differences can
be identified by serologic typing (serotyping). Strains of Listeria species are divided into serotypes
based on somatic (O) and flagellar (H) antigens [101]. Flagellar antigens as well as somatic antigens
must be identified to type strains of serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, and 3c. The remaining
serotypes all have the same flagellar antigens (A, B, and C). Serotypes 1/2a, 1/2b, 1/2c, 3a, 3b,
and 3c can be identified with two O antisera (one with antibodies to factor I and the other with
antibodies to both factors I and II) and three H antisera (one with antibodies to factors A and B,
one reacting with C, and one reacting with D). With antisera for O factors V and VI, VII and IX,
VIII, X, XI, and XV, strains of serotypes 4a, 4b, 4c, 4d, 5, 6a, and 6b can be typed [116]. Serotype
4bX is a variant of serotype 4b and was implicated in an outbreak of listeriosis in the United
Kingdom that was traced to contaminated pâté [73].
Most (95%) human infections are caused by strains of L. monocytogenes belonging to serotypes
1/2a, 1/2b, and 4b. Therefore, serotyping alone is of limited value in epidemiologic investigations.
In the WHO Multicentre L. monocytogenes Subtyping Study, Schönberg et al. [99] found that all
of 80 strains tested by serotyping were typeable. However, there was complete agreement between
the six participating laboratories on the serotype of only 49 (61%) of the tested strains (21 of
serotype 1/2a and 28 of serotype 4b). The intralaboratory reproducibility, assessed on 11 duplicate
strains, ranged from 82 to 100%, with a median value of 91%. Interlaboratory reproducibility varied
from 64 to 95%; no laboratory correctly identified the two serotype 4bX strains in the set. Schönberg
et al. [99] concluded that there is a critical need for good-quality antisera prepared by using
standardized strains. Also, they emphasized the need to completely and efficiently absorb these
antisera to produce good-quality factor sera.
Subtyping Listeria monocytogenes 285
During the 14th International Symposium on Problems of Listeriosis in Mannheim, Germany,

Lehmann and Schönberg [62] reported the results of the WHO Phase III Serotyping Study of
L. monocytogenes. Six strains of L. monocytogenes (serotype 1/2a, 3a, 3c, 4b, 4c, and 4d) were
used representing all of the important O- and H-factors. Seven laboratories from various countries
were selected for serotyping these strains using two methods: their in-house method and a suggested
simple procedure that required commercially produced Listeria Antisera (Denka Seiken, Tokyo).
Of seven laboratories, five sent in results to be analyzed. Four of the five laboratories using their
in-house method identified the expected serotype of the six strains and one identified the serotype
for five strains. Using the suggested simple method, four out of five laboratories were able to
identify the expected serotype of the six strains; one identified the serotype for four strains. They
concluded that it is possible to use the simple method for the identification of O- and H- antigenic
factors of L. monocytogenes and that the six strains used in the evaluation are suitable as reference
strains for serotyping L. monocytogenes.
Serotyping has poor discriminating power when compared to other subtyping methods. Isolates
from foods and the environment are frequently nontypeable with standard typing antisera. Never-
theless, serotyping provides valuable information for rapid screening of groups of strains isolated
during suspected outbreaks. Serotype information allows elimination of isolates that are not part
of an outbreak and facilitates the efficient application of other more sensitive but time-consuming
subtyping methods.
BACTERIOPHAGE TYPING (PHAGE TYPING)

Numerous lytic bacteriophages have been identified for Listeria spp. [64]. Listeria monocytogenes
isolates can be characterized by their patterns of resistance or susceptibility to a standard set of
phages as demonstrated by Rocourt et al. [90]. Until the recent advent of molecular subtyping,
phage typing was often used in conjunction with serotyping for epidemiologic investigations
because of its high discriminating power [7,71,90,98]. The WHO Multicentre L. monocytogenes
Subtyping Study on phage typing was done using an international phage set in five laboratories
and unique phage sets in two laboratories [70]. When typed using the international phage set,
20–51% of the isolates were found to be nontypeable. Nontypeability was a greater problem
among strains of serogroups l/2 and 3 (28–72%) than for serogroup 4 (11–22%) when these
isolates were typed using the international phage set. The two laboratories that used unique phage
sets had fewer problems with nontypeable strains. One of these laboratories was able to type all
strains of serogroup 4 and 81% of strains of serogroups 1/2 and 3. The reproducibility of phage
typing among the participating laboratories was 79% when laboratorians used criteria for interpre-
tation previously proposed for the international phage set [72].
Based on findings of this WHO Multicentre Study, McLauchlin et al. [72] recommended that
researchers review phages in the international set and consider additions to increase typeability of
strains. Lemaitre et al. [63] have proposed a method that facilitates detection of induced phages;
this procedure may be useful for identifying additional typing phages. McLauchlin et al. [70]
suggested that better interlaboratory reproducibility may be achieved by standardization of phage
suspensions, propagation strains, and methodology. Marquet-van der Mee et al. [68] used a panel
of 395 L. monocytogenes isolates to evaluate seven experimental phages for inclusion in the
international phage set for epidemiological typing of L. monocytogenes. Results of their evaluation
showed that inclusion of five of the experimental phages contributed greatly to the overall typeability
and discriminating power of the system, especially for strains within serogroup 1/2. Using centrally
propagated phages—as does the Central Public Health Laboratory, London—for phage typing of
Salmonella serotypes may be helpful.
Despite its high discriminating power and easy applicability to large numbers of strains, phage
typing is available only at selected national and international reference laboratories because this
method necessitates maintaining stocks of biologically active phages and control strains. Although
the procedure is technically not very demanding, it suffers from considerable experimental as well
as biologic variability. The percentage of nontypeable strains may vary with the standard phage set
used. Nevertheless, phage typing remains the only practical method that can be rapidly applied to
type strains in massive outbreaks. Rocourt et al. [91] phage-typed more than 16,000 isolates in 1
year during the investigation of an outbreak in France in 1993 in which “pork tongue in jelly” was
implicated as the vehicle of infection.
BACTERIOCIN TYPING
Bacteriocins (monocins) were first isolated from L. monocytogenes by Sword and Pickett [111]
and Hamon and Peron [55]. Monocins are resistant to trypsin, sensitive to heating at 56°C for 30
min and stable at 4°C. In monocin typing, an isolate is assessed for susceptibility to a set of
bactericidal peptides produced by selected strains [80,121]. Curtis and Mitchell [31] studied mono-
cin interactions of 97 strains of L. monocytogenes using an improved production method and
standardization of the monocins against the type strain of Listeria ivanovii. Only serotype 4 strains
acted as indicators. A typing system using 8 producer and 11 indicator strains showed poor
discrimination. Bacteriocin typing has limitations similar to those of phage typing.
Bannerman et al. [5] typed 100 strains of L. monocytogenes from sporadic cases and
epidemic outbreaks by a combination of monocin typing and phage receptor/reverse phage
receptor methods. The combination monocin-phage receptor subtyping method had a discrim-
ination index of 0.99 for 87 epidemiologically unrelated strains, which was the highest of seven
subtyping methods evaluated. The authors suggested that the combination monocin-phage
reversal method was simple enough to be done in a nonspecialized laboratory, highly discrim-
inatory, and reproducible, but they cautioned that the method and the indicator test strains must
be rigorously standardized.
ANTIMICROBIAL SUSCEPTIBILITY TESTING

Antimicrobial susceptibility testing has relatively limited utility for typing Listeria species simply
because for many years L. monocytogenes susceptibility patterns have all but remained constant.
However, resistance plasmids conferring resistance to chloramphenicol, macrolides, and tetracy-
clines have been found in L. monocytogenes [54,84]. Resistance to other antibiotics such as
streptomycin, erythromycin, kanamycin, sulfamethoxazole, and rifampin have been observed in
Listeria species [30,37,66,104]. In a minireview, Charpentier and Courvalin [28] give an overview
of the mechanisms for recent emergence of antibiotic resistance in L. monocytogenes and Listeria
spp. by acquisition of three types of mobile genetic elements: self-transferable plasmids, mobilizable
plasmids, and conjugative transposons [2,29,30,54]. They conclude by suggesting that in all prob-
ability the current favorable situation of quasiuniform susceptibility of L. monocytogenes and
Listeria spp. to the antibiotics used in clinical practice will deteriorate under the selective pressure
exerted by mis- or overuse of the drugs. Recently, Godreuil et al. [46] screened 488 L.
monocytogenes isolates from human cases of listeriosis for antibiotic resistance; they found that
five isolates were resistant to fluoroquinolones. Fluoroquinolone resistance was attributed to active
efflux of the drug. The authors’ observations indicate that increasing use of fluoroquinolones can
select resistant mutants in nontarget species.
Because antimicrobial susceptibility testing may be used as a method of typing bacteria
according to antibiotic susceptibilities, with the emergence of resistance in L. monocytogenes
strains antimicrobial susceptibility testing may provide an additional tool in epidemiologic
investigations.
MOLECULAR METHODS
MULTILOCUS ENZYME ELECTROPHORESIS
The characterization of prokaryotes and eukaryotes by multilocus enzyme electrophoresis (MEE)
is based on differences in electrophoretic mobility of their metabolic enzymes. The electrophoretic
mobility differences of these enzymes are a result of charge differences caused by amino acid
substitutions in the polypeptide sequence; these charge differences, in turn, reflect changes in the
nucleotide sequence of the DNA encoding the polypeptide [102]. In MEE, cell extracts containing
the soluble metabolic enzymes are electrophoresed in nondenaturing starch gels. After electrophore-
sis is completed, the gel is sliced, and each slice is treated with a specific chromogenic substrate
for a specific enzyme (e.g., aldolase) to render the enzyme band visible. Mobility variants of each
enzyme are considered to be different electromorphs and are subjectively designated by different
numbers. Combinations of a set of electromorphs (usually 10–20) constitute an electrophoretic type
(ET). Thus, each ET represents a multilocus genotype. Some isolates may present nulls (absence
of activity for specific enzymes); this complicates the analysis of MEE data.
In the early 1990s, MEE was used in the United States and Europe for epidemiologic investi-
gations of listeriosis outbreaks [6,49,77] and to determine the extent of involvement of contaminated
foods in sporadic listeriosis [83]. Also, MEE has been useful for taxonomic and genetic character-
ization studies of L. monocytogenes [11,12,82]. Boerlin et al. [12] used MEE to analyze the genus
Listeria and to estimate the genetic relatedness among Listeria species. The MEE data allowed not
only identification of different genotypes within a population, but also provided an estimation of
the genetic relatedness between strains. Although MEE is a very powerful tool for population
genetic, taxonomic, and evolutionary studies, it is only moderately discriminatory for use as a
subtyping tool in epidemiologic investigations.
Caugant et al. [24] coordinated evaluation of MEE for the WHO Multicentre L. monocytogenes
Subtyping Study. Seven laboratories participated in the study, assaying a total of 24 enzymes.
Reproducibility and discriminating power of the method varied greatly among the seven laboratories.
Five of the seven laboratories reported null alleles. In some instances, the nulls could be attributed
to less-than-optimal activity of the enzyme in the cytoplasmic extracts applied to gels, whereas in
other instances it was clearly the characteristics of the strains. Caugant et al. [24] concluded that to
ascertain the immediate epidemiologic relationships of L. monocytogenes strains, the laboratorian
will need, in some instances, to supplement MEE with other methods that provide further discrim-
ination. Similar conclusions were reached by Norrung and Gerner-Smidt [78], who reported an
overall discrimination index (DI) of 0.83 for MEE. When results of MEE were combined with those
of restriction endonuclease analysis, the DI increased to 0.92. Further, MEE is a labor-intensive
method that requires techniques and equipment that are available in relatively few laboratories. For
these reasons, this method presently has relatively limited application in epidemiologic studies.
CHROMOSOMAL DNA RESTRICTION ENDONUCLEASE ANALYSIS (REA)

Several authors have demonstrated the usefulness of chromosomal DNA restriction endonuclease
analysis (REA) using frequently cutting restriction endonucleases for typing L. monocytogenes
[38,44,76,119]. The number and sizes of restriction fragments generated by digesting a given piece
of DNA are influenced by both the recognition sequence of the enzyme and composition of the
DNA. Thus, investigators expect enzymes with a four-base recognition sequence to yield more and
smaller restriction fragments than enzymes with six-base recognition sequences. However, an
enzyme whose recognition sequence is composed of only guanine (G) and cytosine (C) will cut
DNA with a low G + C content less frequently and consequently will generate fewer and larger
restriction fragments than an enzyme that recognizes sequences of adenine and thymine. Selection
of an appropriate restriction endonuclease is based on both the recognition sequence of a restriction

endonuclease and the G+C content of the test organism [42,81]. Because of the high specificity of
restriction endonucleases, complete digestion of a given DNA with a specific enzyme provides a
reproducible array of fragments. Within a size range from 30 to 1 kb, the fragment can be separated
by agarose gel electrophoresis to obtain DNA “fingerprint” patterns.
For L. monocytogenes, the advantages of REA are that it is universally applicable, sensitive
(because the entire genome is evaluated), cost effective, and relatively easy to perform. Gerner-
Smidt et al. [44] evaluated the REA method for the WHO Multicentre L. monocytogenes Subtyping
Study. They reported that of the restriction endonucleases tested, HaeIII, HhaI and CfoI were the
most useful enzymes for REA typing of L. monocytogenes. The 80 test strains were divided into
27 REA types with HaeIII, 24 with HhaI, and 25 with CfoI. DNA fragment patterns obtained with
HhaI and CfoI were nearly identical to each other and were the easiest to interpret by visual
examination; EcoRI divided the strain set into only 14 REA types and the patterns were difficult
to compare.
The major limitation of REA is the difficulty of comparing the complex profiles, which consist
of hundreds of bands that may be unresolved and overlapping. Patterns generated by some restriction
enzymes may not be stable. Investigators need to clearly establish the differentiation criteria (the
minimum number of band differences that are indicative of differences between the isolates) for
each enzyme before they can reliably evaluate the discrimination [44]. Consequently, REA is not
the method of choice for large-scale comparisons and for building a dynamic database of DNA
patterns for comparative evaluation of new patterns.
RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS:

RIBOTYPING AND OTHER METHODS
Ribotyping is one type of Southern hybridization analysis in which strains are characterized
for the restriction fragment length polymorphism (RFLP) associated with the ribosomal oper-
ons. Southern blot analyses detect only the particular restriction fragments associated with
specific chromosomal loci, thereby significantly reducing the number of DNA fragments to be
analyzed [106]. Ribotyping involves transferring chromosomal DNA restriction fragments that
have been electrophoretically separated on a gel matrix onto a nitrocellulose or nylon membrane.
After immobilizing the DNA fragments, laboratorians probe the membrane with an appropri-
ately labeled 16 + 23S ribosomal RNA (rRNA) or rDNA probe [51,108]. Because the genes
coding for rRNA are highly conserved, E. coli rRNA [108] or a cloned ribosomal operon
(rrnB) of E. coli [17] can be used to probe L. monocytogenes. In general, ribotype patterns
appear to be stable and reproducible after in vitro and in vivo passage of strains [17]. Therefore,
this method may be best suited for long-term epidemiologic or phylogenetic studies. Figure 9.1
shows a gel containing genomic DNA restriction fragments of L. monocytogenes. Figure 9.2
shows a typical nylon membrane containing ribotype patterns obtained when the strains are
Southern-blotted and probed with a cloned E. coli rrnB operon (plasmid pKK3535) labeled
with digoxigenin.
Several investigators have used ribotyping for subtyping L. monocytogenes [4,18,32,49,
50,59,75,78]. Most investigators have used EcoRI for ribotyping of L. monocytogenes. Baloga and
Harlander [4] compared HaeIII and HindIII with EcoRI and concluded that EcoRI was the most
discriminating enzyme for subtyping L. monocytogenes. Nocera et al. [75] reported that 69 of 96
serotype 4b isolates clustered in two closely related EcoRI ribotypes; they found bacteriophage
typing to be more discriminating than ribotyping. Norrung and Gerner-Smidt [78] also found
ribotyping to be less discriminating than bacteriophage typing, REA, and MEE for subtyping 99
clinical, food, and slaughterhouse isolates of L. monocytogenes.
FIGURE 9.1 Genomic fingerprints of EcoR1-digested DNA of L. monocytogenes strains isolated from human
and food sources. Lanes 2 and 14, human isolates (serotype 1/2a); lanes 3–13 and 15–20, food isolates
(serotype 1/2a); lane 21, human isolate (serotype 4b); lanes 22 and 23, food isolates (serotype 4b); lanes 1 and
24, molecular size marker.
Swaminathan et al. [110] evaluated ribotyping and another probe derived from repeat
sequences of L. monocytogenes DNA for the WHO Multicentre L. monocytogenes Subtyping
Study. Six laboratories did ribotyping using EcoRI enzyme to restrict the L. monocytogenes DNA
and rRNA or DNA as the probe for Southern hybridization. A seventh laboratory used NciI to
restrict the DNA, and two probes, one randomly cloned and the other containing repeat sequences
cloned from L. monocytogenes DNA. Their results showed that the overall Simpson’s index of
diversity (DI) for five of the six laboratories that ribotyped most or all study strains ranged from
0.83 to 0.88. A DI value of 0.91 was obtained for the combination of two probes used by one
laboratory. Also, DI values for strains that were serotypes 1/2a or 1/2c were greater than DI
values for strains of serotypes 1/2b, 3b, 4b, or 4bX for ribotyping as well as for the randomly
cloned probes used by one laboratory. Swaminathan et al. [110] concluded that although ribotyp-
ing satisfies two requirements for a good subtyping method, namely typeability and reproduc-
ibility, its discriminating ability for serotype 4b strains may not be adequate for epidemiologic
investigations. They recommended that ribotyping be supplemented by other methods such
as pulsed-field gel electrophoresis (PFGE) for determining the molecular epidemiology of
L. monocytogenes serotype 4b.
FIGURE 9.2 Ribotype profiles obtained by Southern blot analysis of genomic fingerprints of EcoR1-digested
DNA of L. monocytogenes from Figure 9.1.
Probes other than rRNA or rDNA have been used to type L. monocytogenes. Saunders et al.
[97] selected cloned DNA fragments of L. monocytogenes from a bacteriophage lambda gene library
to type 64 isolates of serogroup 1/2 using restriction enzyme NciI and found good discrimination
among epidemiologically unrelated isolates. Ridley [89] evaluated the same probe’s ability to type
862 isolates representing serogroups 1/2, 3, and 4. Although the utility of the method for subtyping
serogroup l/2 isolates was demonstrated in the evaluation, Ridley observed that the method did not
adequately discriminate among serogroup 4 isolates. The cloned probe evaluated by Ridley [88]
and another probe derived from repeat sequences of L. monocytogenes DNA were used in the WHO
Multicentre L. monocytogenes Subtyping Study because of their abilities to subtype L. monocytogenes
[110]. Similar to ribotyping, using the two cloned probes did not provide adequate discrimination
between epidemiologically unrelated serotype 4b isolates. Also, these probes did not discriminate
between a serotype 4d isolate and other serotype 4b isolates associated with a listeriosis outbreak.
However, the repeat sequence probe discriminated between serotype 4b isolates and serotype 4bX
isolates [110].
The RiboPrinter is an automated ribotyping system that generates, analyzes, and stores ribo-
print patterns of bacteria. The first version of the RiboPrinter was configured to generate ribotype
patterns using only EcoRI and was used to generate a database of patterns for 1346 isolates of
L. monocytogenes [18,57]. Ryser et al. [93] used the RiboPrinter to demonstrate that different
ribotypes of L. monocytogenes were favored by different selective enrichment protocols. Wiedmann
et al. [120] characterized 133 isolates of L. monocytogenes by automated ribotyping using the
RiboPrinter and examined them for polymorphisms in virulence-associated genes. They concluded
that the isolates could be separated into three distinct phylogenetic lineages; human isolates were
found in lineages one and two but lineage three was composed of exclusively animal isolates.
Recently, Aarnisalo et al. [1] subtyped 486 L. monocytogenes isolates originating from 17
Finnish food-processing plants using the RiboPrinter and compared the results to those of DNA
macrorestriction analysis. Although the automated RiboPrinter base typing was not as discriminatory
as PFGE typing, the author suggested that automated ribotyping would be appropriate for
screening large numbers of isolates from food processing plant environments. Gendel et al. [43]
studied the temporal and spatial distribution patterns of L. monocytogenes strains from smoked
salmon by ribotyping 72 isolates recovered over a 3-year period. No geographical or temporal
stratification of strains was observed; 12.6% of the food samples yielded multiple ribotypes of
L. monocytogenes.
Polymerase chain reaction-ribotyping (PCR-ribotyping) is a method that uses oligonucleotide
primers designed to be complementary to conserved regions of the 5S, 16S, and 23S regions of
the rRNA genes. These primers are amplified with a purified or crude preparation of template DNA
by PCR. The resulting PCR products may be digested with a restriction endonuclease of choice or
added to an agarose gel, electrophoresed, and visualized by ethidium bromide staining.
The potential of PCR-ribotyping for discriminating among and within various species of
Listeria, as well as among strains of L. monocytogenes, has been explored [106]. Sontakke and
Farber [105] analyzed 49 strains of L. monocytogenes and 12 isolates of other Listeria species.
They subjected genomic DNA isolated from bacteria to PCR amplification using the region of DNA
encoding 16S and 5S rRNA. They found that PCR-ribotyping distinguished between L. monocy-
togenes serotypes 1/2a and 1/2b with no overlap in composite profiles. The sensitivity of this method
for differentiating serotype 1/2a and 1/2b isolates appears to be as good as that of other molecular
methods. However, the PCR-ribotyping method was less discriminatory for serotype 4b strains
[40]. Sontakke and Farber [106] concluded that PCR-ribotyping could be considered as an alternate
molecular subtyping technique. However, they recommended combining the PCR-ribotyping
method with another highly discriminatory molecular subtyping method for serotype 4b isolates
for confirmation or association between similar isolates.
DNA MACRORESTRICTION ANALYSIS BY PFGE

DNA macrorestriction analysis by PFGE has revolutionized precise separation of DNA fragments
greater than 40 kb. Schwartz and Cantor [100] developed PFGE, a variation of agarose gel elec-
trophoresis in which the orientation of the electric field across the gel is changed periodically
(“pulsed”) rather than kept constant as it is in conventional agarose gel electrophoresis used for
the REA. This technology separates large fragments of unsheared microbial chromosomal DNA
obtained by embedding intact bacteria in agarose gel plugs, enzymatically lysing the cell wall and
digesting the cellular proteins. The intact DNA is digested with an infrequently cutting restriction
endonuclease. Subsequent RFLP analysis allows differentiation of clonal isolates from unrelated
ones. PFGE analysis has been used for epidemiological subtyping of L. monocytogenes by several
investigators [15,16,19,23,56].
Brosch et al. [15] first demonstrated the usefulness of PFGE for subtyping L. monocytogenes
by applying the method to type serotype 4b strains. Using ApaI, SmaI, and NotI, they showed
that PFGE can distinguish between closely related strains that are indistinguishable by other
typing methods. The ability of PFGE to subtype L. monocytogenes serotypes l/2 and 3 was
subsequently demonstrated [20]. The applicability of PFGE for outbreak investigations was
demonstrated by Buchrieser et al. [19], who typed 75 L. monocytogenes strains isolated during
six major and eight smaller listeriosis outbreaks. PFGE divided these strains into 20 subtypes.
Strains within each major epidemic (Switzerland [1983–1987], California [1985], and Denmark
[1985–1987]) demonstrated indistinguishable patterns, whereas strains responsible for other

outbreaks were characterized by specific combinations of patterns. Variations within PFGE
patterns occurred more frequently within epidemiologically unrelated isolates. PFGE was used
to demonstrate the link between contaminated chocolate milk and febrile gastroenteritis among
a group of people who attended a cattle show in Illinois [33,85]. Destro et al. [34] found that
RAPD and PFGE were the most useful methods for tracing dissemination of L. monocytogenes
in a shrimp processing plant.
Brosch et al. [14] evaluated the PFGE method for the WHO Multicentre L. monocytogenes
Subtyping Study. Four laboratories participated in evaluating PFGE by analyzing 80 coded strains
of L. monocytogenes. Two restriction endonucleases (Apa1 and SmaI) were used by all laboratories;
one laboratory used an additional restriction endonuclease (Asc1). Agreement among the four
laboratories ranged from 79 to 90%. Sixty-nine percent of the strains were placed in exactly the
same genomic group by all four laboratories; most of the epidemiologically related strains were
correctly identified by all four laboratories. This study validated the previous claims that PFGE is
a highly discriminating and reproducible method for subtyping L. monocytogenes and is particularly
useful for subtyping serotype 4b isolates, which are not typed satisfactorily by most other typing
methods [14].
Currently, PFGE is widely used for molecular subtyping of L. monocytogenes for listeriosis
cluster detection, epidemiologic investigations, and monitoring food production facilities. The
method has been standardized for subtyping L. monocytogenes and is used internationally
[1,3,48,65,95,109,115]. In the United States, the Centers for Disease Control and Prevention
has established a network (PulseNet) of public health and food regulatory laboratories that
routinely subtype foodborne pathogenic bacteria to rapidly detect foodborne disease clusters
that may have a common source. PulseNet laboratories use highly standardized protocols for
subtyping bacteria by PFGE and are able to quickly compare PFGE patterns of foodborne
pathogens from different locations within the country via the Internet. Routine and timely
subtyping of L. monocytogenes by participating PulseNet laboratories has significantly
enhanced investigators’ ability to recognize and investigate outbreaks of listeriosis [9,25–27].
PFGE could possibly be considered the current “platinum standard” for molecular subtyping.
Figure 9.3 shows the patterns obtained when genomic DNA of L. monocytogenes was digested
with restriction endonucleases (RE) ApaI and AscI using the PulseNet’s L. monocytogenes
standardized protocol.
The major disadvantages of PFGE are the time required to complete the procedure (1 day), the
requirement for large quantities of expensive restriction endonucleases, and the need for relatively
expensive, specialized equipment for electrophoresis.
RANDOM AMPLIFICATION OF POLYMORPHIC DNA (RAPD)

Arbitrarily primed polymerase chain reaction (AP-PCR) and random amplified polymorphic DNA
(RAPD) analysis are polymerase-chain-reaction-based methods in which researchers allow a single
arbitrarily selected primer to anneal to nearly complementary sequences on the target DNA by doing
the annealing step at a very low temperature (37°C). Typically, the primer anneals to several locations
on the target and amplifies an array of DNA fragments of different sizes, yielding a DNA pattern suitable
for typing. RAPD uses primers of 10-bp length, whereas AP-PCR uses longer primers [117,122]. RAPD
was first applied to subtyping of L. monocytogenes by Mazurier et al. [70]. They used a 10-mer primer
(HLWL 74) to analyze 104 L. monocytogenes isolates that included representative strains from six
outbreaks. All but one of the outbreak-associated isolates were classified by RAPD in complete agree-
ment with phage typing. Mazurier et al. [69] suggested that RAPD offers an attractive alternative to
phage typing. Lawrence et al. [61] used a different 10-mer primer to type 91 isolates from raw milk,
food, veterinary, environmental, and clinical sources. They obtained 33 different patterns. Farber and
FIGURE 9.3 Pulsed-field gel electrophoresis separation of AscI (lanes 2–5) and ApaI (lanes 7–9) macrorestric-
tion fragments of L. monocytogenes genomic DNA test isolates for certifying PulseNet participating laboratories.
Lanes 1, 6, and 10, XbaI-digest of Salmonella ser. Braenderup standard reference strain (CDC. no. H 9812).
Addison [40] applied RAPD to type 52 L. monocytogenes isolates representing 11 serotypes and
concluded that the method offered much promise as a subtyping method for L. monocytogenes.
Niederhauser et al. [74] used a 19-mer primer to subtype 57 L. monocytogenes isolates and reported
that the method allowed them to trace back L. monocytogenes contamination in several food outlets to
a food-processing plant. Boerlin et al. [10] did an extensive evaluation of RAPD by typing 100 L.
monocytogenes isolates that had been characterized by serotyping, phage typing, MEE analysis, REA,
and ribotyping. They found RAPD to be a highly discriminating method for subtyping. O’Donoghue
et al. [79] found RAPD to be useful for typing serogroup 1/2; also they found that the method distin-
guished the serotype 4bX strains involved in a pâté-associated outbreak from other serotype 4b isolates.
Wernars et al. [118] evaluated RAPD for the WHO Multicentre L. monocytogenes Subtyping
Study. Six laboratories participated in the study. Using three different 10-mer primers, the six
participating laboratories obtained a median reproducibility of the RAPD results of 86.5%
(range 0–100%). Any failure in reproducibility was mainly attributable to results obtained with
one particular primer. Wernars et al. [118] concluded that RAPD analysis is a rapid and relatively
simple technique for epidemiologic typing of L. monocytogenes isolates and that reproducible
useful results can be obtained.
Despite the simplicity and high discriminating ability of RAPD, much more work is needed
to make RAPD typing a standard technique for general and widespread use. Its primary drawback
is the inconsistent reproducibility of patterns. Although some investigators claim that the method
is reproducible, there is a need to identify all steps in the procedure that are critical for obtaining
consistently reproducible results. Because RAPD conditions are less stringent than those of other
typing methods to facilitate initiation of the polymerization reaction at sites having one or more
sequence mismatches, the polymerization is initiated with various efficiencies. The final quantities of
DNA produced may vary widely among the different fragments amplified from a given isolate. Such
variation is inherent in RAPD analysis and introduces two specific problems. First, comparison and
interpretation of patterns with differences in intensity become quite difficult. Second, because some of
the products may represent relatively inefficient reactions, the actual fragments obtained from a single
isolate may vary in different amplification reactions. It is, therefore, very important that a well-standard-
ized protocol be followed in RAPD analysis and used consistently for reliable results.
REPETITIVE ELEMENT-BASED SUBTYPING

Repetitive element-based (REB) typing is a PCR typing method that incorporates use of primers
based on short extragenic repetitive sequences [113] or generic rRNA intergenic spacer oligonu-
cleotides [47]. Sequences are typically present at many sites around the bacterial chromosome such
that when two sequences are located near enough to each other the DNA fragment between those
sites is effectively amplified. Because the number and location of the repetitive sequences are quite
variable, the number and size of the interrepeat fragments generated can similarly vary from strain
to strain. Ericsson et al. [36] used an REB analysis to investigate 133 strains of L. monocytogenes
serotype 4b. A segment of 2,916 bp containing parts of the two genes inlA and inlB in L. mono-
cytogenes was amplified by the PCR technique. The PCR product obtained was digested with the
restriction enzyme Alu1. The fragments generated provided two distinct groups—one containing
37 types and the other, 96 types. These results indicate that REB analysis may be a useful tool for
subtyping L. monocytogenes serotype 4b strains.
AMPLIFIED FRAGMENT LENGTH POLYMORPHISM

Amplified fragment length polymorphism (AFLP) is a DNA fingerprinting technique based on
the selective PCR amplification of restriction fragments from a total digest of genomic DNA.
Vos et al. [114] developed AFLP; the technique involves restriction of the DNA and ligation
of oligonucleotide adapters, selective amplification of sets of restriction fragments, and gel
analysis of the amplified fragments. Investigators can use the technique to generate fingerprints
from DNA of any origin and from any complexity without prior sequence knowledge. The method
allows the specific coamplification of a high number of restriction fragments. The number of
fragments that can be analyzed simultaneously is dependent on the resolution of the detection
system. The optimal number of fragments (50–100) are amplified and detected on denaturing
polyacrylamide gels.
Ripabelli et al. [89] used AFLP to analyze a set of 33 L. monocytogenes isolates collected from
patients and from foods implicated in outbreaks, from human sporadic cases, or from foods. Of
four selective primers used, one generated 20 different-sized DNA fragments with the AFLP
technique. The 33 cultures segregated into 14 different patterns, each comprising 7–12 different
fragments. They concluded that AFLP analysis reconfirmed the observation that L. monocytogenes
comprises two major genetic groups.
In a recent study Guerra et al. [52] evaluated 84 EcoRI digests of L. monocytogenes DNA using
AFLP. The results were compared with those obtained with serotyping, phage-typing, and cad-
mium/arsenic resistance typing. They found that the results of the AFLP technique were reproducible
and that 14 different banding patterns comprising between five and eight DNA fragments were
produced. There were associations with AFLP results and those from phage-typing and cad-
mium/arsenic resistance typing, although each method showed some independence. The evaluation
suggests that the AFLP method may be useful for epidemiologic studies.
The advantages of AFLP are high reproducibility, high PCR multiplex ratio, the fact that this
method is not hampered by genome complexity, the possibility of generating a virtually infinite
number of markers, and the fact that no prior sequence information is required. Disadvantages of
AFLP include the requirement for an automated DNA sequencer and the complexity of the proto-
cols. Also, the epidemiologic relevance of AFLP has not been firmly established.
DNA-SEQUENCE-BASED SUBTYPING STRATEGIES

Much work has been done on DNA-sequence-based subtyping and several approaches have been
suggested; however, much work remains to be done. Some potential targets have been identified.
Listeria monocytogenes is an attractive candidate for implementation of DNA-sequence-based
subtyping for the following reasons. There is an excellent set of well-characterized, epidemiolog-
ically related and unrelated strains of L. monocytogenes that have been subtyped by all available
phenotypic, protein-based, and DNA-RFLP-based subtyping methods. These strains will be invalu-
able for developing a database of DNA sequences for L. monocytogenes. Also, the extensive
characterization of virulence-associated genes of L. monocytogenes that has been accomplished
over the past 15 years provides valuable information on virulence-associated genes of L.
monocytogenes and the sequence heterogeneities in these genes [35,86,87,97,103,123]. The virulence-
associated genes of L. monocytogenes that have now been sequenced include iap (gene encodes
an invasion-associated protein), inlA (a family of genes that are involved in the internalization
of the organism into the host cell), hlyA gene (encodes a β-hemolysin), the plcA gene (encodes
a phosphatidyl inositol-specific phospholipase C, which is involved in lysis of the membrane
during cell-to-cell spread of L. monocytogenes), the mpl gene (encodes a metalloprotease), the
actA gene (encodes factors involved in actin polymerization), and the lma operon (associated with
inducing delayed-type hypersensitivity reactions in L. monocytogenes-immune mice). In addition,
the flaA gene that encodes the flagellin protein, the flaR gene that appears to modulate DNA
topology, and the genes encoding 16S and 23S ribosomal RNA in L. monocytogenes have been
sequenced. Some virulence-associated genes such as hlyA are highly conserved and may not be
suitable targets for strain identification. Other virulence-associated genes such as the inlA operon
and the genes encoding cell surface structures such as cell membrane and flagella may be more
polymorphic and, hence, are likely to be more useful for discrimination of strains.
Rasmussen et al. [87] sequenced internal fragments of the flaA, iap, hly, and 23S rDNA genes
of isolates of L. monocytogenes from clinical, food, and environmental sources. These isolates
represented different serotypes. A 150-bp region of the hly gene was sequenced in 75 strains.
A total of 27 strains were sequenced for the other genes. Although the DNA sequence data for hly,
iap, and flaA were useful for identifying three lineages, the genetic diversity within the sequencing
targets was insufficient to provide adequate sensitivity for subtyping.
However, because DNA insertions, deletions, and rearrangements are very frequently encoun-
tered, the targeting of a single gene for subtyping may provide misleading answers. Also, targeting
a single gene, even a hypervariable one, may not provide adequate discrimination for epidemi-
ologic subtyping, and will not necessarily provide reliable estimates of genetic relatedness
between strains.
Multilocus sequence typing (MLST) is a novel typing method in which alleles for multiple
housekeeping enzyme loci are assigned directly by nucleotide sequencing. This method differs
from MEE, in which alleles are inferred and assigned indirectly from the electrophoretic mobilities
of gene products [67,107]. Complete or partial nucleotide sequences are determined for several
bacterial genes or chromosomal regions, thereby providing unambiguous and discrete data. MLST
provides excellent assessment of genetic relatedness between strains; therefore, it is very useful
for the study of genetic structure, evolution, and population biology of living organisms including
pathogenic bacteria.
Salcedo et al. [94] described an MLST-based subtyping method for L. monocytogenes that uses
the sequence diversity data of seven housekeeping genes (abcZ, bglA, cat, dapE, dat, ldh, and
lhkA). Two additional loci (pgm and sod) that were examined in preliminary tests were not
considered further because of low sequence diversity. Salcedo et al. [94] evaluated the MLST
method against 62 strains of L. monocytogenes that had been previously characterized by PFGE.
Although quite a bit of congruence was found among groupings obtained using sequence analysis
of housekeeping genes and those obtained using PFGE, PFGE was much more discriminating than
MLST (29 MLST sequence types [ST] versus 46 PFGE types). Five STs included multiple PFGE
types (ST4:2; ST 11; ST14: 4; ST6:6 and ST2:7). In contrast, only one PFGE type (pattern 31) was
shared between two STs (ST6 and ST8).
Cai et al. [21] used a different approach to selecting multiple genomic targets on L. monocytogenes.
They selected two housekeeping genes (prs and recA), one stress response gene (sigB), two
virulence genes (actA and inlA), and two intergenic regions (plcA-hly and hly-mpl) and
sequenced their complete open reading frames in 15 well-characterized isolates. The virulence
genes and the two intergenic regions provided higher discrimination than did the housekeeping
and stress-response-related genes; actA exhibited the highest sequence variability with 14.3%
of the nucleotides being polymorphic, whereas the phosphoribosyl synthetase gene showed the
lowest sequence variability (4.9%). Cai et al. [21] also used the complete sequence information
for these genes and regions to define the most discriminatory 600-bp fragments within them
that could be used for rapid sequencing-based subtyping. This approach needs to be further
evaluated and validated using a larger set of epidemiologically and microbiologically well-
characterized strains.
Rudi et al. [92] used another variation of MLST employing a DNA-array-based approach in
which only informative genetic changes in multiple genetic loci were investigated by sequence-
specific labeling of oligonucleotide probes. They identified 29 such informative regions in the
following virulence-associated genes of L. monocytogenes: hlyA, iap, flaA, actA, and inlA. They
used a set of 32 L. monocytogenes strains to type by multilocus array hybridization and compared
the results with AFLP. Although the strains were tightly clustered by multilocus array hybridization,
in many instances they also clustered by AFLP. However, the two techniques were less congruent
for distantly related strains. It is difficult to assess the utility of multilocus array hybridization for
subtyping L. monocytogenes from their data [92] because AFLP is not extensively used for typing
L. monocytogenes in public health laboratories and its utility in the context of public health
surveillance and outbreak investigations has not been demonstrated.
Borucki et al. [13] constructed a mixed-genome microarray using a genomic library based
on 10 strains of L. monocytogenes. They evaluated the microarray using 24 isolates that were
from humans, ovine/bovine brain, bulk milk, and the environment. The four major serotypes
of L. monocytogenes were represented. The isolates were separated into two major clusters.
This result was in agreement with serotype-based clustering and previously described phylo-
genetic lineages. Later, the investigators interrogated the mixed-genome microarray with an
expanded set of 50 L. monocytogenes isolates [22]. They concluded that the major serotypes
could be discerned by using the data from only four probes in the mixed-genome microarray.
Further, they were able to distinguish between isolates that clustered within a serotype. The
potential of this approach for molecular epidemiologic investigations of listeriosis remains to
be demonstrated.
Multilocus variable number tandem repeats analysis (MLVA) is a powerful subtyping tech-
nique for bacterial pathogens; it has been successfully used for subtyping even those bacterial
pathogens that do not show much diversity by other methods. In this method, tandem repeats on
the bacterial genome are determined by scanning the whole-genome sequence with specialized
software [53,60] developed specifically for this purpose. Gene sequences that encompass these
repeat sequences are targeted for amplification by PCR and are sized by separation on gel-based
DNA sequencing systems. Multiple repeat sequences on the genome are evaluated for diversity in
number of repeats in each target using a small set of carefully selected strains of the pathogen to
identify the highly polymorphic loci. Multiple polymorphic loci that allow amplification from
different strains are selected for further evaluation with a larger set of previously characterized
strains. Although there are no reports of the application of MLVA to L. monocytogenes, we anticipate
publication of such reports in the near future given the fact that whole-genome sequences for
L. monocytogenes serotypes 1/2a and 4b, and L. innocua are now available.
A third approach is to assess the diversity among strains of L. monocytogenes by assessing
single-nucleotide polymorphisms (SNP) at multiple locations on the genome. An attempt to develop
SNP-based subtyping was made by Unnerstad et al. [112], who used pyrosequencing to catalog
variations in positions 1575 and 1578 of the inlB gene of L. monocytogenes. Using this approach,
they were able to categorize 106 L. monocytogenes strains into four groups: serogroups 1/2a and
1/2c were in one group, 1/2b and 3b were clustered in another group, and serotype 4b strains were
separated into two groups. By extending this approach to additional genomic targets, investigators
may be able to develop a sensitive subtyping method.
COMPARISON OF METHODS USED TO SUBTYPE L. MONOCYTOGENES

Several studies report comparisons of various methods for subtyping L. monocytogenes.
Researchers compared different methods using different sets of isolates. For example, Baloga
and Harlander [4] used genomic DNA fingerprints, ribotyping, serotyping, and MEE to study
28 strains of L. monocytogenes. DNA fingerprinting was more discriminating than ribotyping
in this study. Norrung and Gerner-Smidt [78] compared MEE, ribotyping, REA, and phage
typing. In this study, phage typing was the most discriminatory with a DI of 0.88. REA, MEE,
and ribotying had DIs of 0.87, 0.83, and 0.79, respectively. Norrung and Gerner-Smidt [78]
also saw differences in discrimination depending on O serotype. For serotype 1, REA gave the
best discrimination, and for serotype 4, phage typing gave the best discrimination. Nocera et al.
[75] also characterized Listeria strains from an outbreak using ribotyping and four other typing
methods (serotyping, phage typing, MEE, and REA). They studied 134 isolates, of which 96
were serotype 4b. Within 4b isolates, phage typing gave the highest DI, followed by REA,
MEE, and ribotyping. They also showed that combining methods could give a higher DI. Graves
et al. [49] compared ribotyping and MEE for 305 isolates of L. monocytogenes. Overall, they
found MEE to be more discriminating with this set of isolates than ribotyping. They concluded
that these methods did not provide adequate discrimination for serotype 1/2b and 4b.
At the beginning of this chapter, we discussed the WHO Multicentre Listeria monocytogenes
Subtyping Study. Phase I of this study used a set of 80 coded L. monocytogenes strains that included
11 sets of duplicates. Bille and Rocourt [8] published an overview of the study. Because several
different methods were compared using a well-defined set of isolates, this study is very useful in
comparing the utility of the various subtyping methods. Table 9.1 shows a comparison of the
methods used in the study. On the basis of these results, serotyping, phage typing, REA, PFGE,
and RAPD were selected for standardization in Phase II. Currently, antisera are commercially
available for serotyping, ribotyping can be done using automated technology, and PFGE method-
ology is standardized. These methods provide a selection of standardized subtyping that can
be employed in listeriosis cluster detection and epidemiologic investigation using multicenter
comparison of data. Novel DNA-sequence-based subtyping methods (MLST, multilocus array
hybridization, MLVA, and SNP) must be evaluated and validated.
298
TABLE 9.1
Characteristics of Phenotypic and Molecular Subtyping Methods Used in the WHO Multicentre L. monocytogenes
Subtyping Study
Percentage Intralaboratory Interlaboratory Discriminatory
Method Typeability Reproducibility (%) Reproducibility (%) Power (DI) Recommendations Reference
Serotyping 100 82–100 83 0.68 For standardization, reagents should be produced 99

and distributed by one laboratory. Serotyping is
not very discriminatory, but is a useful
prerequisite to other methods, especially in
outbreak investigations.
Phage typing 49–80* NA 79 ND Centrally propagate phages and standardize 72
phage suspensions, propagation strains, and
methodology.
MEE 100 27–91 ND 0.83–0.93 Not sufficiently discriminating to be used alone 24
for epidemiologic investigations.
REA 100 88–97 98 0.93–0.98 Develop standardized nomenclature for types. 44
Ribotyping 100 80–100 ND 0.83–0.88 Not sufficiently discriminating to be used alone 110
for epidemiologic investigations.
PFGE 100 ND 84 0.95–0.96 Highly discriminating and useful for 14
epidemiologic investigations; standardize
method.
RAPD 100 0–100 0–100, 0.75–0.95 for Highly discriminating and useful for 119
for 3 primers median 86.5 3 primers epidemiologic investigations. One primer gave
serious reproducibility problems. Standardize
method and address problems with
reproducibility.
Note: DI = Simpson’s index of diversity; ND = not done; NA= not available; * = the remaining isolates did not give a strong reaction with any of the phages in the
international phage set.
Listeria, Listeriosis, and Food Safety
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10 Foodborne Listeriosis
Dawn M. Norton and Christopher R. Braden
CONTENTS
Identification of Listeria monocytogenes as a Foodborne Pathogen ............................................308

Role of Molecular Subtyping in Listeriosis Outbreak Detection and Investigation ....................309
Dairy Products................................................................................................................................310
Soft, Unripened “Mexican-Style” Cheese, Los Angeles County ........................................310
Vacherin Mont d’Or, Western Switzerland ..........................................................................312
Brie de Meaux, France.........................................................................................................313
Blue-Mold or Hard Cheese, Denmark .................................................................................314
Raw-Milk Soft or Semihard Cheese, Quebec, Canada .......................................................314
Illicitly Produced or Distributed Raw-Milk Fresh, Unripened Cheese...............................315
Fluid Milk.............................................................................................................................317
Other Dairy Products............................................................................................................318
Meat and Poultry Products ............................................................................................................320
Ready-to-Eat Meat and Poultry Products in the United States:
Frankfurters and Deli Meat ..................................................................................................321
Other Processed Meats .........................................................................................................327
Reduction of Meat and Poultry Product–Associated Invasive Listeriosis ..........................333
Seafood Products............................................................................................................................333
Smoked Mussels...................................................................................................................334
Cold-Smoked Fish Products.................................................................................................334
Vegetables.......................................................................................................................................335
L. monocytogenes–Associated Febrile Gastroenteritis ..................................................................336
Potential Association with a Mild Clinical Syndrome:
Shrimp, 1989, United States ................................................................................................337
Rice Salad, 1993, Northern Italy .........................................................................................338
Chocolate Milk, 1994, United States...................................................................................338
Corn and Tuna Salad, 1997, Northern Italy ........................................................................340
Cold-Smoked Rainbow Trout, Finland ................................................................................340
Ready-to-Eat Meat and Poultry Products, New Zealand and United States ......................341
Fresh, Raw-Milk Cheese, Sweden .......................................................................................342
Cheese, Japan .......................................................................................................................343
Control of L. monocytogenes–Associated Febrile Gastroenteritis ......................................343
Surveillance of Listeriosis in the United States ............................................................................344
Foodborne Outbreak Surveillance........................................................................................344
Surveillance of Sporadic Listeriosis ....................................................................................344
Improved Estimates of the Burden of Listeriosis in the United States ..............................348
References ......................................................................................................................................349
305
Although listeriosis is a well-documented veterinary infectious disease, it was historically thought to

occur only rarely in humans and primarily as a result of contact with ill animals or animal reservoirs
(35). Indeed, food has been recognized as a primary mode of transmission for human disease only
within the last 25 years. Hypotheses regarding the potential transmission of Listeria monocytogenes
to humans through food can be found as early as 1926, when Murray et al. described a disease in
rabbits caused by a bacterium subsequently identified as L. monocytogenes [115]. Results of feeding
trials indicated successful infection via the oral route, leading the authors to suggest that the gastrointes-
tinal tract may serve as a route of infection. Strong evidence for foodborne transmission to humans
was first reported by Potel in the 1950s [123]. He described a marked increase in stillbirths in Halle,
Germany, with up to 100 instances documented at a single clinic from ~1945 to 1952. Investigation
efforts led to isolation of the same L. monocytogenes serotype from the milk of a cow with atypical
mastitis and stillborn twins of a mother who had consumed the cow’s raw milk. Today, non-foodborne
transmission of L. monocytogenes to humans is considered to occur only rarely. Clusters of late-onset
neonatal listeriosis have been identified in newborn nurseries, suggesting that nosocomial transmission
is possible [116,134]. It is also possible that late-onset neonatal listeriosis in infants born healthy and
at full term may have been acquired during passage through the birth canal, though the route of infection
in this circumstance is not well understood. Nonetheless, the proportion of listeriosis cases due to
foodborne transmission has been recently estimated to be 99% [110].
The epidemiologic and laboratory-based investigation of foodborne listeriosis outbreaks has
contributed significantly to our understanding of this disease. This chapter will thus place significant
emphasis on outbreaks. However, invasive listeriosis outbreaks are often very difficult to identify and
investigate. A discussion of challenges unique to their detection and investigation will facilitate a
better understanding of investigative findings and conclusions. Many outbreaks occur among geo-
graphically dispersed populations over long time periods and affect relatively few exposed individuals,
making their recognition difficult and often delayed. Surveillance for listeriosis and identification of
cases potentially associated with outbreaks is also limited, for example, by the lack of routine testing
to diagnose listeriosis among cases of spontaneous abortion. Long periods between exposure and
diagnosis hamper the ability to identify the vehicle of infection. In the 1985 outbreak of listeriosis
due to Mexican-style soft cheese in Los Angeles, reviewed in detail in this chapter, the median
incubation period was 31 days, with a range of 11 to 70 days [97]. Investigators typically assess food
exposures for patients in listeriosis outbreaks for the entire four to six weeks prior to the patient’s
diagnosis, and patients often have difficulty recalling their specific food exposures. The narrow range
of susceptible persons makes it difficult to identify and enroll appropriate control persons in case–
control studies. Although approximately 20% of listeriosis cases may occur in healthy adults, most
cases occur among pregnant women, neonates, the elderly, or immunocompromised adults [135].
Limited selection of comparable control persons potentially exposed to the food vehicle but not infected
by L. monocytogenes or, specifically, the outbreak-C associated subtype may compromise the study.
Successful identification of the food vehicle in an outbreak is further confounded by microbiological
aspects of food contamination. L. monocytogenes is common in the natural environment (see Chapter 2),
and multiple subtypes may contaminate foods. Subtyping L. monocytogenes isolates from patients and
food is a critical tool for linking foods to human infections. However, the finding of multiple subtypes
in food vehicles and among patients, some of which may not match, can add a layer of complexity to
identification of cases and potential food vehicles. Last, food vehicles responsible for listeriosis are
often those with prolonged shelf lives, and growth of L. monocytogenes can occur at refrigerator
temperatures. Thus, the increased opportunities for cross-contamination of multiple foods during food
production and storage can make identification of the original source vehicle in outbreaks difficult.
Despite these obstacles in surveillance, outbreak detection, and investigations, we have learned
much about the foods associated with listeriosis. In this chapter, we will review the current
knowledge gained from listeriosis outbreaks and surveillance. Table 10.1 summarizes the reported
invasive listeriosis outbreaks and the associated food vehicles.
Foodborne Listeriosis 307
TABLE 10.1
Foodborne Invasive Listeriosis Outbreaks with Ten or More Cases
Year Location Suspect/Implicated Vehicle No. Cases a Reference
1945–1952 Halle, East Germany Raw milk, sour milk, cream, ~100 [123]
cottage cheese
1954 Jena, East Germany Unknown 26 [146]
1956 Soviet Union Pork, mouse 19 [79]
1960–1961 Bremen, West Germany Unknown 81 [62]
1966 Halle, East Germany Unknown 279 [121]
1969 Auckland, New Zealand Unknown 13 [27]
1975–1976 Anjou, France Unknown 162 [45]
1977–1978 Johannesburg, South Africa Unknown 14 [88]
1978–1979 Western Australia Raw vegetables 12 [141]
1979 Massachusetts Raw vegetables 20 [82]
1979–1980 Auckland, New Zealand Unknown 10 [124]
1980 Auckland, New Zealand Shellfish, raw fish suspected 22 [96]
1981 East Cambria, England Cream 11 [106]
1981 Slovakia Unknown 49 [124]
1981 Maritime Provinces, Canada Coleslaw 41 [132]
1981–1982 Christchurch, New Zealand Unknown 18 [60]
1983 Houston, Texas Unknown 10 [43]
1983 Saxony, West Germany Unknown 25 [117]
1983 Massachusetts Pasteurized milk 49 [63]
1983–1987 Vaud, Switzerland Vacherin Mont d’Or cheese 122 [41]
1985 Los Angeles Mexican-style cheese 142b [97]
1985–1987 Denmark Unknown 35 [131]
1986 Linz, Austria Raw milk, vegetables 20 [2, 145]
1986–1987 Los Angeles Raw eggs 33 [136]
1986–1987 Philadelphia Unknown, multiple vehicles likely 36 [137]
1987 Los Angeles Butter 11 [103]
1987 England Unknown 23 [107]
1987–1989 England, Wales, North Ireland Pâté 366 [108]
1989 New York Shrimp 10 [125]
1989–1990 Denmark Blue-mold or hard cheese 26 [91]
1990 Western Australia Processed meats or pâté 11 [94, 148]
1992 France Pork tongue in aspic (jelly) 279 [89, 130]
1993 France Rillettes 38 [74]
1995 France Brie de Meaux cheese 33 [72]
1997 France Pont l’Évêque cheese 14 c
1998–1999 United States, multistate (n = 24) Processed meats 108 [109]

1999 Finland Butter, pasteurized 25 [98]
1999 Connecticut, Maryland, NewYork Pâté 11 [11]
1999–2000 France Rillettes 10 [52]
1999–2000 France Pork tongue in aspic 32 [52]
2000 United States, multistate Delicatessen turkey meat 30 [119]
(n = 11)
2000 North Carolina Homemade Mexican-style cheese 13 [99]
2002 United States, multistate Delicatessen turkey meat 54 [70]
(n = 9)
2002 Quebec, Canada Raw milk cheese 17 [9]
a Number of cases reflects laboratory-confirmed and epidemiologically linked cases.
b 63 cases from the epidemic subtype.
c Goulet, 1998. Personal communication to E.T. Ryser.
IDENTIFICATION OF LISTERIA MONOCYTOGENES

AS A FOODBORNE PATHOGEN
In 1981, the investigation of a large epidemic of adult and perinatal listeriosis in the Maritime
Provinces of Canada provided overwhelming evidence for foodborne transmission of L. monocy-
togenes to humans [132]. First detected from the occurrence of perinatal listeriosis in 1.3% of births
at a Nova Scotia hospital over a 3-month period, this outbreak of serotype 4b infections resulted
in 7 adult and 34 perinatal cases (including 5 fetal deaths and 4 stillbirths, Figure 10.1). The case-
fatality rate among live-born infants was 27%, and 2 of 6 nonpregnant adults who developed
meningitis died. The magnitude of the outbreak provided a unique opportunity to investigate risk
factors for infection. Investigators conducted two case–control studies, in which prior exposures
of case-patients are compared to an appropriate control population of persons without the disease;
differences in exposure between the groups indicates an association between exposure and the
disease [53]. For this study, information on potential exposures was collected from case-patients
and a healthy control population. The first study primarily explored hypotheses of transmission by
exposure to ill persons, animals, and environmental sources based on the body of knowledge at
that time, but did not identify potential risk factors. However, collection of a comprehensive food
history for the 3 months before illness and a second case–control study implicated coleslaw as the
most likely vehicle of infection. Case-patients were 2.3 times more likely to have consumed coleslaw
than controls (P = 0.04). The vehicle was laboratory-confirmed when coleslaw obtained from a
patient’s refrigerator yielded L. monocytogenes serotype 4b, the same serotype associated with the
human infections. This serotype was also isolated from unopened packages of coleslaw prepared by
the same manufacturer. The isolates were further characterized by molecular means in subsequent
studies [21,40,49,77,150], and were found to share molecular profiles. In an effort to find the source
of contamination, investigators traced ingredients back to their suppliers. Carrots and cabbage were
obtained from several regional farms. One also raised sheep and routinely fertilized with raw and
composted sheep manure. Environmental samples did not yield L. monocytogenes. However, reports
of listeriosis among the sheep and long-term storage of cabbage in a cold-storage shed, conditions
that would allow growth of this organism in the cabbage, support the hypothesis that contaminated
cabbage from this farm was the source of L. monocytogenes. On the other hand, because this organism
16 Adult cases
14
Perinatal cases
12
10
Cases
0
J F MAM J J A S O N D J F MAM J J A S O N D J F MAM J J A S O N D
1979 1980 1981
FIGURE 10.1 Nonpregnant adult and perinatal listeriosis cases — Maritime Provinces, Canada, 1979–1981.
(Adapted by E.T. Ryser from Schlech, W.F.I., P.M. Lavigne, R.A. Bortolussi, A.C. Allen, E.V. Haldane, A.J.
Wort, A.W. Hightower, S.E. Johnson, S.H. King, E.S. Nicholls, and C.V. Broome 1983. Epidemic listeriosis—
evidence for transmission by food. N Engl J Med 308: 203–206.)
is often found in food processing environments (see Chapter 17), product contamination during or
after preparation is also possible. In addition to bringing listeriosis to international attention, this
outbreak definitively documented foodborne transmission of L. monocytogenes to humans.
Since the recognition of food as the primary mode of transmission of L. monocytogenes to
humans, subsequent investigations of many outbreaks and sporadic listeriosis cases have implicated
a wide variety of food types. A common characteristic of many implicated foods is that they are
eaten without cooking by the consumer; that is, they are “ready to eat.” Many of these foods are
cured, pasteurized or cooked in the production process, indicating that postprocessing contamination
from the production environment is an important route of food contamination. These products also
have a prolonged shelf life compared to fresh foods. As L. monocytogenes grows well at refrigeration
temperatures (see Chapter 6), even cold-storage of products allows conditions favorable for growth
of this organism to levels posing a high risk for human infection and disease, especially in
susceptible individuals. In the following sections, we review epidemiological and laboratory inves-
tigations implicating different types of foods. We will first discuss invasive listeriosis, and then
turn the focus to febrile gastroenteritis. As molecular subtyping has become a critical component
of epidemic investigations, we first discuss its important role and provide an example of a molecular
surveillance system in place in the United States and several other countries—PulseNet.
ROLE OF MOLECULAR SUBTYPING IN LISTERIOSIS

OUTBREAK DETECTION AND INVESTIGATION
Historically, foodborne disease outbreaks were of local scale, often linked to a single restaurant or
social event and attributed to improper food handling. Today, outbreaks often involve food products
that are centrally produced and widely distributed to many states and, occasionally, other countries.
Cases of illness are often geographically diffuse, making it more difficult to detect, investigate, and
control the outbreak. Subtyping methods have proven to be a valuable tool during investigations
of foodborne disease outbreaks, beginning with the use of methods including serotyping, plasmid
profiling, and phage typing over 20 years ago [84,85]. A number of highly discriminatory subtyping
methods have since been developed (see Chapter 9 for a detailed discussion). However, their
widespread adoption and the ability to compare results from different laboratories have been
hindered by issues including cost, reproducibility, and time to completion [142].
Pulsed-field gel electrophoresis (PFGE) (detailed in Chapter 9) is a highly discriminatory and repro-
ducible DNA-based subtyping method for several foodborne pathogens, including L. monocytogenes. In
1993, PFGE was applied for characterization of clinical and food isolates during the investigation
of an Escherichia coli O157:H7 outbreak in the western United States. Subtyping facilitated
investigation of the extent of the outbreak and confirmed the link between patient clinical isolates
and hamburger patties served by a regional restaurant chain, thus demonstrating the utility of PFGE
in outbreak investigations [25]. Over the next several years, the Centers for Disease Control and
Prevention (CDC) standardized the methodology for PFGE and collaborated with the Association
of Public Health Laboratories to develop PulseNet, a national public health laboratory network for
molecular surveillance of foodborne disease and rapid outbreak response. Participating laboratories
are certified to perform PFGE analysis and rapidly compare the resulting patterns. Beginning in
1996 with ten laboratories, PulseNet has grown to include all state public health laboratories in the
United States, several large county public health laboratories, the U.S. Department of Agriculture’s
(USDA) Food Safety and Inspection Service (FSIS), and the U.S. Food and Drug Administration’s
(FDA) Center for Food Safety and Applied Nutrition (CFSAN) and Center for Veterinary Medicine
(CVM). PulseNet routinely monitors five foodborne bacterial pathogens, including L. monocytogenes.
Canadian provincial public health laboratories have also joined together to form PulseNet Canada,
and share PFGE results with PulseNet USA. Other international PulseNet networks are forming in
Europe, Latin America, the Pacific Rim, and China.
6 PulseNet begins
subtyping clinical
Number of outbreaks
5 Listeria isolates
4
0
’78 ’80 ’82 ’84 ’86 ’88 ’90 ’92 ’94 ’96 ’98 ’00 ’02
Year
FIGURE 10.2 Outbreaks of listeriosis detected in the United States by public health officials, 1978–2003.
Arrow denotes initiation of subtyping and routine surveillance by the National Molecular Subtyping Network
(PulseNet).
The severity of listeriosis and potential for geographically diffuse and prolonged outbreaks
underscored the need for continued vigilance for listeriosis clusters and the ability to rapidly
compare isolate subtypes. PulseNet scientists developed a standardized protocol enabling isolate
analysis within 30 h [76], and routine surveillance was initiated in 1998. PulseNet surveillance has
revolutionized the ability to detect listeriosis outbreaks. Figure 10.2 shows the number of known
outbreaks of listeriosis in the United States by year, from 1978 through 2003. From 1978 to 1997,
five outbreaks were detected and reported by health officials. Following initiation of subtyping and
surveillance of Listeria by PulseNet in 1998, the number of outbreaks detected increased dramat-
ically. A total of five outbreaks were detected in 1999 alone. This increase can be largely attributed
to enhanced surveillance, as opposed to an increase in listeriosis, as surveillance data have shown
an overall national decrease in listeriosis in recent years (discussed later in this chapter).
DAIRY PRODUCTS
SOFT, UNRIPENED “MEXICAN-STYLE” CHEESE, LOS ANGELES COUNTY
In 1985, a large epidemic occurred in Los Angeles County, which brought listeriosis to the forefront
of public health and regulatory concern [97]. First recognized as an increase in perinatal listeriosis,
142 cases of listeriosis were eventually confirmed in Los Angeles County over an 8-month period.
A striking feature was the predominance of perinatal infections (65%), of which 87% occurred
among Hispanic women. In contrast, only 29% of cases in nonpregnant adults occurred among
Hispanic persons. The case-fatality rate among 87 perinatal or early fetal infections was 63%, and
the overall case-fatality rate was 34%. Predisposing factors or conditions among nonpregnant adult
patients placed 98% in a higher risk category for infection due to immunosuppression, and included
cancer, chronic illness such as diabetes, acquired immune deficiency syndrome (AIDS), taking
steroids, and being over 65 years of age. A total of 5 L. monocytogenes serotypes were among the
105 available clinical isolates, of which 82% were serotype 4b. Among the serotype 4b isolates,
73% were the same phage type and were defined as epidemic-associated cases (Figure 10.3). Of
the remaining 42 non-epidemic-associated cases, 73% occurred predominantly among nonpregnant
adults, and likely represented sporadic cases.
Two case–control studies among perinatal listeriosis patients resulted in the implication of a
specific brand of soft, unripened Mexican-style cheese as the most likely vehicle of infection.
Results of the first study, which explored dietary, occupational, health, and personal behaviors
14
Epidemic
12 Nonepidemic
10
8
Cases
0
Jan Feb Mar Apr May Jun Jul Aug
Week of Positive Culture
FIGURE 10.3 Cases of listeriosis according to phage type—Los Angeles County, 1985. Arrow designates the
time of product recall. (Adapted by E.T. Ryser from Linnan, M.J., L. Mascola, X. Dong Lou, V. Goulet, S. May,
C. Salminen, D.W. Hird, M.L. Yonekura, P.S. Hayes, R. Weaver, A. Audurier, B.D. Plikaytis, S. Fannin, L. A.
Kleks, and C.V. Broome 1988. Epidemic listeriosis associated with Mexican-style cheese. N Engl J Med 319:
823–828.)
found a statistical association between illness and consumption of Mexican-style cheese (odds ratio
[OR] = 5.5, 95% confidence interval [CI] = 1.2–24.8]. Case-patients were also significantly more
likely than controls to have consumed the root vegetable jicama; however, this food item is a less
likely vehicle as it was consumed by only 41% of cases. In a second case–control study, the
investigators focused upon brands of Mexican-style cheese common to the Los Angeles County
area; case-patients were significantly more likely than controls to have consumed Brand A cheese
(OR = 8.5, 95% CI = 2.4–26.2). Isolation of L. monocytogenes serotype 4b from Brand A cheese
collected from patients’ homes and from unopened packages purchased at supermarkets prompted
a Food and Drug Administration (FDA) recall of this product, which was subsequently expanded
to encompass approximately 500,000 lb of products distributed by the manufacturer to 26 states
and the U.S. Protectorates of Guam, American Samoa, and the Marshall Islands [10]. These cheese
isolates were confirmed to be of the same phage type that characterized most cases caused by
serotype 4b, thus providing laboratory confirmation of the vehicle of infection for epidemic-
associated cases. That epidemic-associated cases continued to occur following the product recall
can be explained by the long incubation period for listeriosis, and reports that some stores continued
to sell the product up to a week after the announcement.
Further investigation at the Brand A production facility identified raw milk as the likely source
of Brand A cheese contamination. Although the milk was reportedly pasteurized on-site, FDA
investigators documented deficiencies in the pasteurization process, including the delivery of quan-
tities of raw milk that would overwhelm the capacity of the pasteurizer by 10% and the presence
of high levels of alkaline phosphatase in samples of cheese produced over a 6-month period. These
findings indicated that either the milk was inadequately pasteurized or that raw milk was added to
pasteurized milk. Postprocessing contamination of the cheese by L. monocytogenes residing in
the facility environment was also possible, as investigators isolated the epidemic phage type of
L. monocytogenes from the environment of the Brand A production facility. However, recovery of
the epidemic phage type from Mexican-style sour cream and cottage cheese produced at a different
production facility, which received raw milk from the same supplier as used for Brand A cheese,
supports the hypothesis that raw milk was the source of L. monocytogenes in Brand A cheese.
Reanalysis of data from the aforementioned studies revealed that case-patients were more likely
than controls to have consumed the Mexican-style sour cream product (OR = 4.0, 95% CI =
1.1–14.2) [97]. The potential association between consumption of cottage cheese and illness was
not explored, however, as the epidemiological investigation focused exclusively on perinatal cases
and foods that the patients routinely consumed.
Occurrence of the majority of cases in pregnant women of a single ethnic group who sought
care at a single facility in Los Angeles County enabled investigators to detect this outbreak, launch
a successful investigation, and intervene such that further transmission was prevented. Without this
focused cluster of cases to herald the outbreak, it may not have been detected. Such clustering is
less common today as more outbreaks are geographically diffuse and are related to centrally
produced, widely distributed and commonly consumed products. This underscores the importance
for enhanced surveillance, in place in the United States and other countries via notifiable disease
reporting systems and standardized, routine subtyping of L. monocytogenes isolates. This epidemic
also provided early indication of significant ethnic disparities in the incidence of listeriosis. Last,
as mentioned earlier, this epidemic brought L. monocytogenes to the forefront of public health and
regulatory attention. In 1986, CDC initiated enhanced surveillance for listeriosis in several U.S.
sites and launched epidemiological studies to facilitate a better understanding of dietary risk factors
for listeriosis (see Chapter 6 for a detailed discussion). FDA began a program to monitor dairy
products for L. monocytogenes, which was later expanded to include other ready-to-eat foods. In
1989, the FDA established a zero-tolerance policy (i.e., no detectable levels of viable organisms
permitted) for L. monocytogenes in FDA-regulated ready-to-eat foods [138].
VACHERIN MONT D’OR, WESTERN SWITZERLAND

An outbreak of L. monocytogenes serotype 4b, electrophoretic type (ET) I infections occurred in
western Switzerland over a 5-year period from 1983 to 1987, and was associated with consumption
of a soft cheese produced with pasteurized milk [12,30,31,33,36,41,102]. This outbreak, the first
known to have occurred in Switzerland, was also recognized due to an increase in listeriosis cases.
Over a 15-month period, 25 cases were diagnosed at a single medical facility (14 in nonpregnant
adults and 11 perinatal infections), compared to a mean of 3 cases per year at that facility from
1974 to 1982 [102]. Fifteen additional cases were identified in neighboring facilities during the
same time period. The cases were of uniform geographic distribution by patient residence and
peaked during winter months. A similar increase in cases during winter months was observed in
the following years. Investigators suspected a common source, but results of two case–control
studies addressing a variety of food, occupational, and household exposures were inconclusive
[12,30]. In addition, attempts to isolate L. monocytogenes from several hundred food items did
not identify a potential source. In an independent effort following the 1985 Los Angeles County
outbreak in the United States, Swiss health officials conducted studies to determine the preva-
lence of L. monocytogenes in cheese and other dairy products. These efforts resulted in isolation
of L. monocytogenes from regionally produced Vacherin Mont d’Or soft cheese, including the two
predominant phage types found in patients [36]. In 1987, a third case–control implicated this cheese
as the most likely vehicle of transmission, with 84% of 37 case-patients having consumed Vacherin
Mont d’Or cheese, compared to 39% of 51 controls (OR = 8.0, 95% CI = 2.8–22.6, P < 0.05)
[33,41]. Further, investigators isolated the subtypes representing most clinical isolates from a
patient’s open package of cheese.
A total of 122 cases of listeriosis occurred in the Swiss canton of Vaud from 1983 to 1987; 65
(53%) occurred in pregnant women and neonates and 57 (47%) occurred in nonpregnant adults
[41]. The overall case-fatality rate was 28% (32% among 57 nonpregnant adults). Among 120
clinical isolates available for characterization, 93% were serotype 4b. Among these, 85% were one
of two phage types also recovered from the cheese, later confirmed to match by a variety of other
subtyping methods [32,34,40,122]. PFGE analysis using two restriction enzymes later distinguished
a total of four subtypes each among the human epidemic and cheese isolates [34]. Three subtypes
matched between the two groups, lending further support to epidemiological findings. These
analyses highlight the importance of a combined laboratory-based and epidemiological approach
to outbreak investigations, and the utility of highly discriminatory subtyping methods such as PFGE.
This outbreak had notable clinical features. First, underlying conditions associated with immu-
nosuppression were present in 42% of nonpregnant adults, a proportion much lower than that typical
of invasive listeriosis outbreaks [41]. Second, this outbreak was characterized by an unusually high
proportion of cases with central nervous system (CNS) involvement (79% of cases among non-
pregnant adults), with 39% of patients developing meningoencephalitis. Evaluation of cases among
nonpregnant adults found an independent association between increasing age and presentation with
meningoencephalitis and an increased risk of death. Neurological sequelae were observed among
30% of surviving patients upon evaluation after 5 months to 3 years. Interestingly, a higher pro-
portion of CNS listeriosis cases were caused by the two epidemic-associated subtypes (although
not statistically significant). This finding suggests a role for subtype-specific virulence properties.
Upon implication of Vacherin Mont d’Or cheese, Swiss officials initiated an international recall
and halted production of this cheese. The cheese production process is instructive with regard to
epidemiological features of this outbreak, potential routes of contamination, and the effect of a
rigorous cleaning and sanitation program. This regional cheese has been produced during winter
months both in western Switzerland and in the neighboring area in France (Vacherin Du Haut-
Doubs/Mont d’Or) for up to two centuries [104]; this correlates well with the observed seasonal
increase in epidemic cases. Although produced from raw milk in France, the Swiss began producing
this cheese from pasteurized milk in 1983 in an effort to better control production and ripening.
The cheese associated with this outbreak was reportedly made by forty producers [31,33]. Following
coagulation of the milk, the curd was dipped into wooden hoops and allowed to drain for 1 to 2
days. The cheese was then allowed to ripen in one of 12 caves for 3 weeks prior to packaging for
sale, where ripening continued. It was not uncommon for cheese to be transferred among different
cellars during ripening and distribution or for hoops to be returned to different producers and reused
without disinfection, facilitating dissemination of potential contaminants. Indeed, follow-up studies
indicated that half of the cellars were contaminated with one or both epidemic subtypes of L.
monocytogenes. Implementation of a rigorous cleaning and sanitation program, along with place-
ment of more easily cleaned equipment, was highly effective in ending the outbreak. Only two
cases of listeriosis were reported in western Switzerland during the months of January to September
1988 [31].
BRIE DE MEAUX, FRANCE

The French National Reference Centre for Listeriosis, which has conducted routine surveillance
for L. monocytogenes in humans and in food since 1987, facilitated detection of a geographically
diffuse listeriosis outbreak associated with the soft, raw milk cheese Brie de Meaux in 1995. This
was the first outbreak in France to be associated with a raw milk cheese [72,127]. The outbreak
was first identified as a cluster of 6 cases caused by an unusual phage type, which had represented
only 33 cases in France since 1987; this was distinct from subtypes identified in other European
and North American outbreaks. Twenty cases were identified over approximately 2 months. Four
of 11 perinatal infections resulted in fetal death. Nine cases were among nonpregnant adults; all
recovered except for one patient, who remained in a coma at the time of the report.
Routine food surveillance data provided evidence for a potential source of the infections, as
the epidemic subtype had also been isolated from four surveillance samples of Brie de Meaux.
A case–control study, along with Ministry of Agriculture investigations, implicated a specific
production batch of this cheese. Health officials initiated a recall and enforced disinfection and
control measures at the production facilities. Targeted health education messages regarding pre-
vention were also disseminated. In total, 33 cases caused by the epidemic phage type were
identified during 1995 [127].
BLUE-MOLD OR HARD CHEESE, DENMARK

During a case–control study to identify risk factors for sporadic listeriosis in Denmark, laboratory-
based surveillance efforts identified an outbreak that occurred over a 21-month period, from 1989 to
1990 [91]. A total of 26 outbreak-related cases were identified, caused by a serotype 4b phage type
(the epidemic subtype) matching that implicated in foodborne listeriosis outbreaks due to Vacherin
Mont D’Or cheese in Switzerland from 1983 to 1987 and pork tongue in aspic in France in 1992
(both discussed elsewhere in this chapter). Meningitis was more common (65%) among persons
infected with the epidemic subtype than in sporadic cases (40%) that occurred over the same time
period; perinatal cases occurred among 12% of epidemic cases. Among nonpregnant adults, 57%
had underlying conditions associated with immune suppression, including leukemia, lymphoma,
and renal transplant. The overall case-fatality rate was 26%. Subanalyses of data collected for the
larger case–control study, in which data for patients infected with the epidemic subtype were
compared to data for listeriosis patients infected with non-epidemic subtypes and control persons
matched by hospitalization date and department, age, and gender implicated a specific brand of
blue-mold cheese as the most likely vehicle of infection (OR = 4.6, 95% CI = 1.0–21, P = 0.03).
The epidemic subtype was isolated from seven brand types of a different, popular hard cheese type
during routine dairy product screening conducted during the outbreak period; however, it was
consumed by over 90% of both listeriosis patients and controls and, therefore, could not be
epidemiologically linked to the outbreak. This epidemic is unique in that it was identified and
investigated during an effort to identify dietary risk factors for listeriosis in a nonoutbreak setting;
case-patients were interviewed upon report of the diagnosis using a questionnaire developed for
that purpose. A limitation of the outbreak investigation component was that a hypothesis was not
established before interview of associated case-patients, resulting in a greater chance of missing
similar exposures among them. On the other hand, there are also advantages to be gained by
administration of a standardized food consumption questionnaire upon diagnosis, before an outbreak
is recognized. As discussed earlier, the severity of invasive listeriosis and delays of up to several
months due to the incubation period and time until outbreak recognition contribute to poor food
consumption recall among patients. The longer time period also results in delay of interventions
to prevent additional listeriosis cases.
In an effort to improve the ability to respond quickly to listeriosis outbreaks, particularly those
that are geographically diffuse, public health officials in the United States are currently piloting a
standardized, hypothesis generation questionnaire that is administered to all listeriosis patients upon
disease reporting to public health agencies. This allows rapid comparison of food exposures between
patients infected with a specific subtype and patients infected with other subtypes, expedites the
launch of focused epidemiological studies, and results in more rapid intervention measures. A
similar approach was successfully applied during the investigation of an outbreak associated with
rillettes in France [52]. Used in conjunction with routine molecular subtyping of human L.
monocytogenes isolates, this strategy may help to substantially increase the timeliness of outbreak
investigations. In turn, appropriate intervention measures can be implemented sooner, reducing the
number of outbreak-associated cases and outbreak duration.
RAW-MILK SOFT OR SEMIHARD CHEESE, QUEBEC, CANADA

The first listeriosis outbreak to be documented in Quebec, Canada, was linked to consumption of
a raw-milk soft or semihard cheese that had undergone <60 days aging [9]. The outbreak was
recognized due to an increase in cases caused by the same PFGE subtype across several areas from
April to November 2002, with a peak in the number of cases in September and October. Among
17 cases, 5 perinatal infections resulted in 3 infected neonates and 2 premature births. Twelve cases
occurred in nonpregnant adults, and no deaths were reported. Although interviews of patients with
listeriosis did not provide hypotheses for potential sources of infection, an investigation of unas-
sociated reports of gastroenteritis following consumption of cheese from a specific producer led to
isolation of the outbreak-associated L. monocytogenes from 56 samples among 16 batches of cheese.
Sixty-one percent of 13 listeriosis patients re-interviewed later recalled consuming the cheese prior
to illness. A recall conducted in October 2002 was an effective intervention, as only 1 additional
case was identified thereafter. Not all of the cases with isolates matching the outbreak-associated
PFGE subtype could be explained by cheese consumption, as production did not begin until May
2002. However, the September/October peak in the number of cases, isolation of the same subtype
of L. monocytogenes from the cheese, and cessation of the outbreak following the recall provide
convincing evidence that this cheese was the source of infection for most of cases.
L. monocytogenes was not isolated from the processing environment or bulk milk from the producer’s
herd during the investigation. The producer was said to have begun operating in May 2002, following
renovations, which may have facilitated the introduction of L. monocytogenes into the production
environment. L. monocytogenes was isolated from soil samples outside the facility, but the isolates
were not available for characterization. Contamination of the processing area due to renovation
and construction is highly possible, given the organism’s ubiquity in the environment [135]. Indeed,
a similar hypothesis resulted from the investigation of a contaminated hot dog–associated outbreak
that occurred following production facility renovations [109].
ILLICITLY PRODUCED OR DISTRIBUTED RAW-MILK FRESH, UNRIPENED CHEESE

Several outbreaks highlight a unique risk factor for listeriosis among members of the Hispanic
community, namely, consumption of fresh, unripened cheese (often referred to as queso fresco,
queso blanco, Hispanic-style, or Mexican-style soft cheese) made from unpasteurized milk. The
cheeses, produced locally by unlicensed manufacturers or in people’s homes and internationally
(e.g., Mexico), reach community members in the United States in a variety of ways, including
distribution by family and acquaintances or sale at flea markets, by unlicensed street vendors, from
people’s vehicles, from door to door, and in small markets. In addition to production from unpas-
teurized milk, often under conditions of suboptimal sanitation, this cheese style has a relatively
high moisture content and pH (>5.6) [127] and is consumed without additional aging. Thus, if
contaminated, there are no barriers inherent to the food matrix to prevent growth of L. monocytogenes.
Early indication of health risks associated with consumption of this style of cheese occurred in
1997, when three outbreaks of multidrug-resistant Salmonella Typhimurium DT104 infections
among Hispanic persons in Northern California and Washington were linked to consumption of
Mexican-style soft, raw-milk cheeses [48,147]. In one outbreak, the cheese was purchased predom-
inately from small Hispanic markets [147]. In the other two, unlicensed vendors or family members
produced the cheese with raw milk purchased from local dairies. The laboratory investigations
supported implication of contaminated raw milk as the source of contamination. The intrastate sale
of raw milk is legal in California and Washington; however, the sale of milk to persons who intend
to resell the milk or milk products without a license to do so is illegal. These outbreaks provided
evidence that this practice was still occurring, emphasizing the importance of a continued focus
on enforcement of existing regulations. Both states launched educational campaigns among pro-
ducers and members of the Hispanic community regarding the risks of fresh, unpasteurized-milk
cheese consumption.
In 2000, an outbreak of listeriosis linked to illicitly produced Mexican-style soft cheese occurred
among Mexican immigrants in North Carolina [99]. First detected as a small cluster of perinatal cases
caused by serotype 4b, this outbreak resulted in 13 cases, including 11 perinatal infections and
5 stillbirths. Results of a case–control study implicated Mexican-style soft cheese as the most likely
vehicle of transmission (matched OR = 17.8, 95% CI = 1.9–69.6). Patients reportedly purchased

cheese in unlabeled packages from small Hispanic markets, street vendors, and by door-to-door sales.
Two cheese makers, identified by a patient and store owner, reported purchase of raw milk from a
local dairy; one person available for further interview reported not pasteurizing the milk during
preparation. Personnel from one of three area dairies visited by investigators confirmed selling raw
milk to unlicensed persons, despite a warning from the state’s Dairy and Food Protection Branch that
the sale of raw milk is illegal in North Carolina and can pose a health risk.
L. monocytogenes serotype 4b was isolated from unlabeled fresh, Mexican-style soft cheese
obtained from the home of one patient and from two area Hispanic grocery stores, and from bulk
tank raw milk samples from a local, manufacturing-grade dairy. Clinical, cheese, and raw-milk
isolates were indistinguishable by serotyping (4b), ribotyping, and two-enzyme PFGE analysis.
The PFGE pattern was rare, representing only seven isolates from three states among more than
2,000 human clinical isolates in the PulseNet database, further supporting the epidemiological link
between consumption of the illicitly produced cheese and listeriosis cases. As a result of this
outbreak, listeriosis was made a reportable disease in North Carolina.
The source of L. monocytogenes in the bulk tank milk was not identified; milk samples from
each of 49 cows were repeatedly culture-negative. Following initiation of a program emphasizing
proper teat preparation before milking and thorough cleaning and sanitation of equipment and the
facility, however, bulk milk samples no longer yielded L. monocytogenes.
In 2003, another outbreak of listeriosis linked to Mexican-style soft, unripened cheese occurred
in southern Texas, again characterized by perinatal infections [81]. Among six cases, one infection
resulted in fetal death and one in the death of a neonate. Five of six women reported consuming
Mexican-style soft cheese (queso fresco) during the month before illness. Further investigation by
Texas health officials indicated that the queso fresco was made in Mexico from unpasteurized milk
and sold illegally in the state at flea markets and by unlicensed street venders [18]. In a local health
advisory, health officials warned against consumption of the illegally imported cheese and educated
consumers about how to identify properly manufactured and labeled cheese made from pasteurized
milk and sold in reputable stores and markets.
Fresh, soft cheese was classified as posing a moderate risk for listeriosis to consumers in the
2003 USDA–FSIS/FDA joint risk assessment [16] (available at http://www.foodsafety.gov/
~dms/lmr2-toc.html). Placement in the moderate-risk category was attributed predominately to
reduced contamination rates, facilitated by improved control measures such as use of pasteurized
milk. However, officials acknowledge that this classification may not be representative of the risk
among members of the Hispanic community. Data used in the risk assessment were derived in part
from several studies of contamination of soft, fresh cheeses, and not specifically related to cheeses
of this style made from unpasteurized milk. Soft, unripened cheese made from unpasteurized milk
is a traditional part of the Hispanic diet and presents a unique challenge for reducing the risk for
listeriosis in this community. Indeed, in a qualitative study investigating knowledge, attitudes, and
practices regarding consumption of unpasteurized milk products among 76 members of the Hispanic
community in the state of Georgia, most believed that unpasteurized milk products were “healthier”
than those made from pasteurized milk [87]. None had heard of listeriosis, other infections, or
pregnancy complications associated with consumption of unpasteurized milk or milk products.
Most (including pregnant women) reported regular consumption of homemade cheese. Studies
addressing knowledge, attitudes, and practices regarding consumption of fresh, soft cheese made
from unpasteurized milk, along with outbreak data, indicate that the cheese is often produced or
sold illicitly by unlicensed vendors, rather than by licensed commercial establishments. It is
therefore difficult to obtain estimates for the prevalence of L. monocytogenes in these cheeses, and
regulations regarding their production and sale are difficult to enforce.
In addition to continued regulatory enforcement efforts, a key strategy for prevention of
listeriosis and other infections associated with consumption of fresh cheese made from unpasteur-
ized milk in this community may lie in health education. Following the outbreak of Salmonella
Typhimurium DT104 infections in Yakima County, WA, health officials collaborated with University
of Washington Cooperative Extension to launch the Abuela Project among Hispanic community
members [28]. Educators worked with abuelas (grandmothers), who are highly respected and
traditionally make the cheese, to develop an organoleptically acceptable recipe and produce the
cheese using pasteurized milk and improved safety measures. The abuelas, in turn, trained 15
additional community members. In total, more than 225 persons were trained; positive behavior
changes were maintained for at least 6 months, and the incidence of Salmonella Typhimurium
DT104 infections in Yakima County decreased dramatically. This project may serve as a model
component of culturally appropriate educational intervention programs aimed at reducing the
incidence of foodborne listeriosis in Hispanic communities [5].
FLUID MILK
Few outbreaks of invasive listeriosis have been associated with consumption of contaminated fluid
bovine milk. Aside from the raw milk–associated epidemic in Halle, Germany (late 1940s–1957),
discussed earlier, we are aware of only one other documented invasive listeriosis outbreak poten-
tially associated with raw-milk consumption. Austrian officials reported an outbreak in 1986 that
resulted in 24 perinatal infections and 4 cases in nonpregnant adults [2]. Case histories indicated
that all 4 nonpregnant adults, along with 5 of 20 pregnant women, had consumed raw milk.
Consumption of vegetables produced using biologic agriculture by 11 patients was suggested as a
second potential risk factor.
In 1983, health officials linked an outbreak of serotype 4b infections in Massachusetts to
consumption of a specific brand of 2% and whole pasteurized milk [63]. A dramatic increase in
listeriosis occurred from June to August, with an incidence 4 to 5 times that for previous summers
in the state. Of 49 cases, 42 (86%) occurred among nonpregnant adults, all with underlying conditions
causing immunosuppression. Seven infections were perinatal. The overall case-fatality rate was 29%,
with 2 cases resulting in fetal death. One case-patient acquired the infection in a hospital. Forty of
49 isolates were available for further characterization; 32 were serotype 4b. The first of two case-
control studies, in which case-patients and controls were matched for age, gender, and neighborhood
of residence, showed that case-patients were more likely than controls to have shopped at a specific
grocery chain (Chain A). Comparison of a list of items purchased by 5 patients during the month
before illness, obtained during store visits with the patients, helped to narrow the focus to dairy
products. Further interviews regarding dairy product consumption showed that case-patients were
significantly more likely to have consumed Chain A whole or 2% milk (OR = 9.3, P < 0.01).
Pasteurized milk was an unexpected vehicle, and investigators were concerned that the association
may have resulted from comparison of patients to healthy neighbors less susceptible to L. monocytogenes
infection. Results of a second study, in which patients’ food consumption exposures were compared
to those for persons with similar underlying health conditions, confirmed the findings of the first;
patients were significantly more likely to have consumed Chain A whole or 2% milk than controls
(OR = 11.5, 95% CI = 2.7–48.8, P < 0.001). Additional epidemiologic and laboratory studies
supported these findings. Evaluation of the volume of Chain A milk consumed per day among
patients and controls suggested a dose–response effect, and consumption of skim milk (which would
decrease the chance of exposure to whole or 2% milk) was significantly protective against illness
(OR = 0.25, 95% CI = 0.08–0.75, P < 0.02). Listeriosis cases in a neighboring state may have also
been associated with this outbreak; an investigation of 12 cases found that 3 patients had consumed
milk from, among 2600 total stores in the state, 1 of only 2 stores that sold milk from the implicated
facility (P < 0.0001). Last, further characterization of clinical isolates found that one phage type
was isolated significantly more often from patients who had reported drinking Chain A whole or
2% milk than from those who did not (P < 0.001). The epidemic-associated phage type was later
shown to be genetically similar to subtypes implicated in European listeriosis outbreaks associated
with pâté in the United Kingdom (1987–1989) [108] and France (rillettes, 1993) [74,127].
The route of milk contamination was not determined; both improper pasteurization and post-
pasteurization contamination seemed unlikely based on environmental investigation findings. List-
eriosis among cows at supplier dairy farms was reported to have occurred during the human
epidemic period. However, time/temperature logs were consistent with proper pasteurization, and
no equipment defects that would have compromised the pasteurization process were noted. Facility
hygiene practices were appropriate, and skim milk (not associated with the epidemic) and 2% and
whole milk were processed with the same equipment; this argued against long-term postpasteur-
ization contamination that would not also affect skim milk. Investigation following the epidemic
period resulted in isolation of several L. monocytogenes serotypes (including 4b) from milk filters
and from raw-milk samples (12% of 124) collected from the facility, the cooperative that supplied
raw milk, and from farms. However, the epidemic-associated phage type was not recovered.
These findings, together with reports describing high heat resistance of L. monocytogenes during
pasteurization [26] and postulations of enhanced heat resistance among organisms that had invaded
bovine leukocytes led investigators to hypothesize that some organisms may have survived proper
pasteurization. This called into question the efficacy of FDA pasteurization guidelines with regard
to inactivation of L. monocytogenes in milk. However, results of subsequent, carefully designed
studies addressing this issue found recommended pasteurization procedures to be efficacious for
inactivation of L. monocytogenes [42,54,55].
OTHER DAIRY PRODUCTS

Two outbreaks of invasive listeriosis, one in Southern California and one in Finland, have been linked
to consumption of contaminated butter. Enhanced surveillance in Los Angeles County, initiated
following the large 1985 outbreak associated with Mexican-style soft cheese, facilitated detection of
a cluster of 11 perinatal cases in 1987 [103]. A case–control study implicated butter as the most likely
vehicle of transmission (OR = 4.0, 95% CI = 0.9–16.7). Results also indicated a potential association
of salad (OR = 6.0, 95% CI = 0.5–66.7) and carrots (OR = 4.5, 95% CI = 0.8–24.3).
An outbreak of 25 listeriosis cases caused by an unusual serotype, 3a, occurred primarily
among patients of a tertiary care hospital in Finland during 1998–1999 [98,100]. Twenty patients
were diagnosed with sepsis, four with meningitis, and one with an abscess; six (24%) patients
died. Their clinical isolates were indistinguishable by PFGE (designated as the outbreak-associated
subtype). Patients infected with the outbreak-associated subtype were more likely to suffer severe
immunosuppression from underlying conditions including malignancy or to have been admitted
to the tertiary care hospital than patients infected with other subtypes (P = 0.02 and P < 0.001,
respectively), and were also more likely to have been admitted to a specific tertiary care facility.
The same serotype had been isolated previously from a sample of butter produced at the Finnish
dairy that served as the exclusive supplier to the facility; the isolates were subsequently shown
to match the clinical isolates by PFGE analysis.
Samples of butter collected from the hospital kitchen yielded the outbreak subtype, prompting
national and local food authorities to expand sampling (the dairy also supplied other hospital
kitchens and a wholesaler) and conduct an environmental investigation at the dairy. Deficiencies
in pasteurization were not identified; however, the outbreak subtype was isolated from butter
packages of several sizes collected from the dairy and wholesaler, as well as from processing and
packaging equipment at the dairy.
A case–control study among outbreak-associated cases at the tertiary care facility and control
patients matched by age and underlying condition did not statistically implicate butter as the likely
vehicle of transmission, perhaps because of the availability of only seven outbreak-associated
patients for interview and the frequency of butter consumption among all patients. Although not
statistically significant, investigators found that case-patients consumed over four times as much
butter as control patients during their stay. Further, hospital units with cases received significantly more
butter than units with no cases (1378 vs. 181 g/100 patient-days, P = 0.01). The median length of the
hospital stay before collection of clinical specimens that yielded L. monocytogenes was significantly
longer among case-patients than controls (31 vs. 10 days, P = 0.008).
This outbreak provided a unique opportunity to estimate probable levels of exposure among
at-risk tertiary care facility patients, as packages of the implicated butter were available at the time
of outbreak detection [100]. Using quantitative contamination data (obtained by screening samples
from the hospital kitchen) and daily consumption data (obtained from five case-patients and
accounting for length of stay), the estimated continuous daily dose ranged from 101 to 103 CFU/day;
the dose ranged from 104 to 105 CFU/day if the higher level (105) detected in a wholesale sample
was used. Using maximum contamination levels, the single dose (i.e., number of organisms con-
sumed at one meal) causing illness was estimated to be 7.7 × 104. The data supported two
hypotheses, each having a different impact on dose–response predictions from these data. For the
first hypothesis, consumption of a single, high-level dose resulted in listeriosis. Based on sample
screening results, occasional contamination (and therefore consumption in a single meal) of a
package of butter with a disease-producing dose was possible. However, the same brand of butter,
and likely the same dose of L. monocytogenes, was consumed in other units and hospitals without
incident. Dose–response curves for a single high dose further predicted a low probability of
listeriosis in the population. For the second hypothesis, prolonged exposure to a lower level of
L. monocytogenes in the butter resulted in disease as opposed to a single exposure. This was also
possible, as the majority of samples were contaminated at <100 CFU/g and as case-patients were found
to have significantly longer stays. This hypothesis underscores the potential risk for listeriosis posed by
consumption of foods with lower-level contamination (<100 CFU/g) among susceptible populations.
Although dose–response curves must be interpreted with caution, as the unusual serotype,
vehicle, and highly susceptible populations are not generalizable to all exposure scenarios or at-
risk populations, these data provide a valuable contribution to risk assessment in humans, and were
used for exposure assessment (dose–response estimates for persons with immunosuppression) in
the USDA–FSIS/FDA joint risk assessment [16].
An unusual invasive listeriosis outbreak involving several different serotypes and, probably,
several independent vehicles occurred in the Philadelphia, PA metropolitan area from December
1986 to April 1987 (Figure 10.4) [137]. The incidence during this period was 2.0/100,000 area
16
14
12
Number of Cases
10
0
J F M A M J J A S O N D J F M A
Month, 1986–1987
FIGURE 10.4 Listeriosis cases reported between January 1986 and April 1987—Philadelphia Standard
Metropolitan Statistical Area, Pennsylvania. (Adapted from Schwartz, B., D. Hexter, C.V. Broome, A.W.
Hightower, R.B. Hischhorn, J.D. Porter, P.S. Hayes, W.F. Bibb, B. Lorber, and D.G. Faris 1989. Investigation
of an outbreak of listeriosis: new hypotheses for the etiology of epidemic Listeria monocytogenes infections.
J Infect Dis 159: 680–685. With permission.)
population, compared to 0.3/100,000 during the preceding 11 months. Among 36 cases identified
during the epidemic period, 32 occurred in nonpregnant adults (75% had underlying conditions
resulting in immuno suppression) and 4 were perinatal. The case-fatality rate was higher than that
for most outbreaks; 44% of patients, including 2 neonates, died. Also uncharacteristic of other
outbreaks of invasive listeriosis, 35% of patients reported having diarrhea during the week before
their illness was diagnosed.
A case–control study among patients (or surrogates) and neighborhood- and underlying
condition-matched controls identified several potential dietary risk factors for illness. Case-
patients were significantly more likely than controls to have shopped for food at a specific
grocery chain (OR = 5.0, 95% CI = 1.5–16.4), to have consumed ice cream (94% of case-
patients compared to 75% of controls, OR = 9.1, 95% CI = 1.6–51.4), and to have consumed
salami (22% of case-patients compared to 5% of controls, OR = 6.3, 95% CI = 1.2–33.3). Case-
patients were also more likely to have consumed Brie cheese, although this association did not
reach statistical significance. However, specific brands of these foods were not epidemiologically
associated with illness.
Molecular characterization identified 4 serotypes and 11 electrophoretic types (ET) among
clinical isolates, of which ET 1 and ET 4 were most prevalent. ET 1 was isolated from three of
four patients who had consumed a specific brand of ice cream (P = 0.04), but no other subtype
was significantly associated with specific food types. An ET 4 isolate was recovered from Brie
obtained from a patient’s refrigerator; this ET matched that of the patient’s clinical isolate. Samples
of ice cream, cheese and other dairy products, vegetables, and meat products, some of which were
obtained from patients’ homes, did not yield L. monocytogenes.
The time and geographic clustering of cases in this area support the hypothesis that an outbreak
occurred. The diversity of clinical subtypes and case–control study results, however, argue against
a common source. This outbreak is illustrative of the utility of molecular characterization for
interpretation of epidemiological data. It is possible, albeit an unusual scenario, that more than one
cluster occurred simultaneously, as each food was a plausible vehicle (see Chapters 11 to 13 for
discussion of the prevalence of L. monocytogenes in these foods). Also, more than one subtype of
L. monocytogenes may be present in a contaminated food item [41,109,128], potentially contributing
to the diversity of subtypes associated with an outbreak. Case–control study results showed use of
antidiarrheal medication and family members who were ill during the month before illness among
significantly more case-patients. These data, along with the presence of gastrointestinal symptoms
among ~30% of patients led investigators to hypothesize that gastrointestinal carriage may have
resulted from consumption of a contaminated item, with transition to invasive disease facilitated
by coinfection by another enteric pathogen such as an enteric virus circulating in the community.
MEAT AND POULTRY PRODUCTS

The first laboratory-confirmed association of meat/poultry products with invasive listeriosis
occurred in 1988, when a case was linked to consumption of contaminated turkey franks (13).
Coincidentally, results from the first case–control study to identify dietary risk factors for sporadic
listeriosis were published; persons who had consumed undercooked chicken and uncooked hot
dogs were more likely to develop listeriosis [136]. The patient, hospitalized with sepsis caused by
L. monocytogenes serotype 1/2a, reported daily consumption of turkey franks that were heated in
a microwave oven. L. monocytogenes serotype 1/2a was isolated from opened packages of turkey
franks obtained from the patient’s refrigerator (MPN >1100/g) and from unopened packages of the
same brand obtained from a local store (MPN < 3/g) (149). The patient and food isolates were of
the same electrophoretic type. Based on these data, the USDA initiated a product recall. The
following day, USDA–FSIS and CDC began an environmental investigation at the processing
facility to identify potential routes of contamination and strategies to prevent product contamination
[149]. L. monocytogenes was isolated from finished product samples representing six of seven
lots tested, with isolates from five lots matching the case isolate by serotyping and multilocus
enzyme electrophoresis (MEE). Production line sampling revealed an increased contamination
frequency of samples after mechanical removal (peeling) of the cellulose casing, and a swab sample
taken from the conveyer belt onto which the peeled franks dropped yielded the same subtype.
Another L. monocytogenes serotype, 1/2b, was isolated from product and environmental samples
in the room in which the cooked franks were cooled prior to peeling. The environmental investi-
gation results provided strong evidence that the source of contamination was the processing envi-
ronment, and that contamination occurred at the peeling step—after the cook step, which would
inactivate L. monocytogenes. Isolation of this subtype from the plant environment over 4 months
after the case occurred suggested its persistence in the processing area. The presence and persistence
of L. monocytogenes in processing areas has proven to be a significant challenge to regulators and
the food industry in the United States, as the contamination of ready-to-eat meat and poultry
products after preparation of the finished product but before packaging led to three large, multistate
listeriosis outbreaks over the following 12 years. This investigation provided early evidence that
prevention of even low-level finished product contamination is critical to prevention of listeriosis,
especially since L. monocytogenes grows well during storage of processed poultry products [69]
and appeared to have grown by several logs during home refrigeration of the frankfurters implicated
here. In 1987, USDA–FSIS responded to increased knowledge of the association of L. monocyto-
genes with deli meats and other processed foods by initiating a monitoring program for ready-to-
eat meat and poultry products produced in federally inspected establishments (4). In 1989,
USDA–FSIS established a zero tolerance policy for L. monocytogenes and other pathogens in ready-
to-eat meat and poultry products [3].
READY-TO-EAT MEAT AND POULTRY PRODUCTS

IN THE UNITED STATES: FRANKFURTERS AND DELI MEAT
The largest listeriosis outbreak in the United States occurred during 1998–1999 [109]. In November,
1998, PFGE data submitted to PulseNet of serotype 4b isolates from contemporaneous listeriosis
clusters in Connecticut, New York, Ohio, and Tennessee revealed that they shared an unusual pattern.
Concerned about the potential for a multistate outbreak, CDC and collaborating state public health
officials initiated an epidemiologic and laboratory-based investigation. Investigators defined a case
as infection by L. monocytogenes with one of three closely related PFGE patterns (indistinguishable
by AscI restriction and differing by no more than 2 in 26 bands on ApaI restriction [75]). A total
of 447 clinical isolates from cases with illness onset from January 1998 to July 1999 were
characterized by PFGE. Among them, 108 (Figure 10.5) from 24 states (Figure 10.6) met the
case criteria, with most cases occurring between August 1998 and January 1999. Thirteen cases
resulted from perinatal infection. Among 43 nonpregnant patients (infected with both outbreak-
and nonoutbreak-associated subtypes) for whom information regarding underlying conditions was
available, all but 2 were either >65 years of age or had a condition that would result in immuno-
suppression. Hypertension, diabetes, and malignancy were most common, and there were no notable
differences between the outbreak- and nonoutbreak-associated groups. The median age among
nonpregnant adults infected with an outbreak-associated subtype was 70 years.
Results from two case–control studies implicated frankfurters as the most likely vehicle of
infection. In the second study and for the first time in a listeriosis investigation, patients with
contemporaneous L. monocytogenes infection caused by a nonoutbreak-associated PFGE subtype
served as the control population. Although a rather unconventional approach, this method allows
for efficient identification of a comparable control group. Selection of control persons has histor-
ically been a challenge as the at-risk population for listeriosis is not representative of the general
population. In the first case–control study, case patients were more likely to have consumed cooked
frankfurters during the month preceding illness (89% of 18 case patients vs. 32% of 19 controls,
OR = 17.3, 95% CI = 2.4–160.0, P < 0.0004). The second case–control study, which included
322
20
18 Nonfatal
Fatal
16
14
12
10
Cases
8
4
Ju
Ju
M
M
M
Se
Fe
Fe
De
Oc
Ap
Au
No
Jan
Jan
ar
ar
n-
ay
p-
b-
b-
l-9
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-9
-9
9
t-9
r-9
c-9
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9
-9
-9
8
9
-9
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98
98
99
8
8
8
8
8
9
8
Date
FIGURE 10.5 Epidemic curve showing listeriosis cases caused by outbreak-associated subtypes, United States, January 1998–April
1999. Light arrow indicates removal of refrigeration unit from processing facility A production area. Dark arrow indicates date of
recall of frankfurters and deli meats. (Adapted from Mead, P., E. F. Dunne, L. Graves, M. Weidmann, M. Patrick, S. Hunter,
E. Salehi, F. Mostashari, A. Craig, P. Mshar, T. Bannerman, B. Sauders, P. Hayes, W. Dewitt, P. Sparling, P. M. Griffin, D. Morse,
L. Slutsker, and B. Swaminathan 2006. Nationwide outbreak of listeriosis due to contaminated meat. Epidemiol Infect 134(4):
744–751. With permission.)
Foodborne Listeriosis
FIGURE 10.6 States with listeriosis cases in an outbreak associated with contaminated ready-to-eat meat products — United States, 1998–1999.
(Reprinted from Mead, P., E. F. Dunne, L. Graves, M. Weidmann, M. Patrick, S. Hunter, E. Salehi, F. Mostashari, A. Craig, P. Mshar, T. Bannerman,
B. Sauders, P. Hayes, W. Dewitt, P. Sparling, P. M. Griffin, D. Morse, L. Slutsker, and B. Swaminathan 2006. Nationwide outbreak of listeriosis
due to contaminated meat. Epidemiol Infect 134(4): 744–751. With permission.)
323
questions about deli meat products, also implicated frankfurters as the most likely vehicle of
transmission (OR = 6.0, 95% CI = 1.2 – 38.6, P = 0.01). Case patients were also more likely to
have consumed turkey cold cuts sliced at a deli; however, the association was not statistically
significant. Eleven of 14 case patients from the first study for whom purchase information was
available reported consumption of products made by a specific company, and one available package
bore the processor establishment code (processing facility A). The company, however, did not issue
a recall at that time. Among case patients in both studies, 13 of 44 (30%) recalled consumption of
brands produced at processing facility A, and an additional 9 recalled consumption of frankfurters
or deli meats from retailers who received products from this facility. At the same time, a review
of isolates obtained by academic researchers identified two deli meat isolates that matched the
outbreak-associated subtype by PFGE and automated ribotyping (38,39). The isolates were from
two brands of deli meat consumed by members of one family, of which three members developed
febrile gastroenteritis. A specific diagnosis was not made; however, convalescent serum from one
ill person indicated high titers of α-listeriolysin O antibodies. This is consistent with acute illness
caused by L. monocytogenes. Processing facility A also produced one brand of this deli meat.
An investigation at processing facility A revealed that construction had occurred in July 1998,
shortly before the outbreak began, with the replacement of a refrigeration unit in the room where
the frankfurters were collected on conveyer belts after their casings were removed. In-plant records
indicated that the frequency of isolation of psychrotrophic microorganisms (organisms that can
grow between 0°C and 7°C and produce visible colonies within 7 to 10 days [90]), which can be
indicative of the presence of L. monocytogenes, sharply increased among samples collected from
retail frankfurter processing lines during the months following construction. L. monocytogenes had
likely colonized areas of the refrigeration unit, and the environmental disruption resulted in con-
tamination of processing equipment and finished product. Whereas L. monocytogenes was not
recovered from the processing environment at the time of the investigation, the outbreak strain was
isolated from opened and unopened packages of frankfurters and deli meats. Following isolation
of this subtype from an opened package of frankfurters, the company voluntarily recalled specific
production lots of various brands of frankfurters and deli meats and issued a public notification
[14]. As shown in Figure 10.5, the outbreak abruptly ended after the recall.
This outbreak had several informative features. The rapid decrease in cases that followed identifi-
cation and recall of the product suggested that the incubation period for invasive listeriosis may be
shorter than historical estimates of 3 to 4 weeks, data which were derived primarily from studies
involving perinatal infection. In this outbreak, the onset of cases in nonpregnant adults ended within
5 days of the recall. One person had illness onset within 48 hours of a single exposure. Thus, it is
important to consider even recent exposures during investigations of invasive listeriosis. Another
interesting feature was the isolation of a second serotype (1/2a) from deli meat at a much higher level
(3,000 CFU/g) than the outbreak-associated subtype, serotype 4b (<0.3 CFU/g). This second subtype
was not epidemiologically linked to any outbreak-associated cases and was not represented among any
of the sporadic case isolates characterized for this outbreak. These data are supportive of the growing
body of evidence for differences in pathogenic potential among different subtypes of L. monocytogenes
[95,122,151]. However, such interpretations must be made with caution. As reported by Graves et al.
[75], quantitative data for the outbreak-associated subtype were obtained from analysis of opened
packages of frankfurters collected from patient refrigerators more than 3 weeks after illness onset,
while unopened packages of deli meat obtained from the processor yielded serotype 1/2a. It is possible
that the outbreak-associated subtype had entered the stationary or death phase by the time of sample
collection; thus, the data may not be representative of the dose consumed by patients. In a recent study,
the serotype 4b subtypes associated with this outbreak (designated EC II) were genetically compared
with other serotype 4b isolates [59]. Analyses revealed genetic fragments that were conserved in the
ECII subtypes but not conserved among other 4b isolates. The serotype 4b specific region in which
these fragments were located lies adjacent to inlA, which codes for a virulence factor that has a critical
role in invasion of several classes of mammalian cells. Whereas any association with host–pathogen
interaction characteristics of this subtype remains to be determined, the divergent region provides
potential targets for DNA-based tools designed for specific detection and monitoring of this subtype.
In 2000, the processor linked to the 1988 listeriosis case discussed earlier (turkey franks [113])
was the source of contaminated turkey deli meat implicated in an outbreak involving 30 cases in
11 states [119]. The outbreak was first recognized as a cluster of nine serotype 1/2a infections that
occurred over a 2-month period in New York City. The isolates matched by PFGE analysis and
ribotyping, prompting health officials to notify CDC and share the PFGE pattern with other public
health laboratories via the PulseNet Webboard. Comparison to patterns in the national PulseNet
database revealed matches in New York State, Michigan, and Georgia. CDC initiated a multistate
investigation; investigators posted a message on the PulseNet message board asking state labora-
tories to perform real-time PFGE analysis on all clinical L. monocytogenes isolates and requested
case history information from epidemiologists at state health departments. Thirty cases in 11 states
with matching PFGE patterns and ribotypes were identified, with culture dates ranging from May
to December 2000; 90% occurred after mid-July (Figure 10.7). A case–control study, with patients
contemporaneously infected with a nonoutbreak-associated subtype of L. monocytogenes serving
as the control population, identified consumption of turkey deli meat from a delicatessen (76% of
17 case patients vs. 21% of 24 controls, OR = 12.4, 95% CI = 2.3–72.5) and lettuce (94% of 18
case-patients vs. 57% of 23 controls, OR = 13.1, 95% CI = 1.4–600.0). When the data were analyzed
controlling for consumption of turkey deli meat, lettuce did not remain significantly associated
with illness. However, turkey deli meat consumption remained significantly associated with illness
when controlling for consumption of lettuce. Purchase information available from nine patients
10
9
8
Number of Cases
7
6
5
4
3
2
1
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0
/1
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1
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/2
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9/
–7
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–6
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7/
–1
1
–8
–5
12
–6
3–
–9
–7
10
11
9–
6–
6–
9–
25
28
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5–
2–
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11
14
/1
9/
17
23
6/
/2
5/
8/
/2
7/
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5/
/1
7/
9/
/1
/1
10
11
12
10
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Date of Culture (2000)
FIGURE 10.7 Epidemic curve showing listeriosis cases caused by the outbreak-associated subtype, United
States, May–December 2000. Arrow indicates date of recall of processed turkey and chicken meats, December
14, 2000. (Adapted from Olsen, S.J., M. Patrick, S.B. Hunter, V. Reddy, L. Kornstein, W.R. MacKenzie,
K. Lane, S. Bidol, G.A. Stoltman, D. M. Frye, I. Lee, S. Hurd, T. F. Jones, T. N. LaPorte, W. E. Dewitt,
L. Graves, M. Wiedmann, D. Schoonmaker-Bopp, A.J. Huang, C. Vincent, A. Bugenhagen, J. Corby, E.R.
Carloni, M.E. Holcomb, R.F. Woron, S.M. Zansky, G. Dowdle, F. Smith, S. Ahrabi-Fard, A. Rae Ong,
N. Tucker, N.A. Hynes, and P. Mead 2005. Multistate outbreak of Listeria monocytogenes infection linked to
delicatessen turkey meat. Clin Infect Dis 40: 962–967. With permission.)
was used in a traceback investigation to determine the source of the turkey deli meat. Investigators
visited the places of purchase to collect environmental swab samples and identify turkey deli meat
brands and production establishment codes. Swabs from four stores yielded L. monocytogenes, and
isolates from three stores matched the outbreak strain. Twenty-seven production facilities supplied
turkey deli meat to stores where the nine patients reported purchasing the meat. However, one
establishment (plant A) supplied turkey deli meat to stores named by at least six (potentially eight,
based upon brand) patients, and a second (plant B) supplied to retailers named by five. Combined,
products from these two establishments could explain up to eight of these nine cases. Further
investigation revealed that plant B was a copacker for plant A, meaning that product could be
shipped to plant A to be processed further and packaged under the plant A label. Based upon this
information, USDA–FSIS investigated the two processing facilities. Product and environmental
samples did not yield L. monocytogenes; however, plant B initiated a voluntary recall of 16 million
pounds of processed turkey and chicken meat, based upon results of the epidemiologic investigation.
This outbreak highlighted the critical role of molecular subtyping and efficient information
sharing in detection and investigation of outbreaks in the age of centrally produced, widely dis-
tributed food products. With a proportionately small at-risk population and long incubation period,
listeriosis outbreak investigations present a unique challenge to public health officials. These tools
enabled investigators to better determine the scope, magnitude, and source of the outbreak, such
that the appropriate intervention prevented further illness.
In 2002, contaminated turkey deli meat was implicated as the source of yet another large,
multistate outbreak in the United States [70]. This outbreak was detected as a cluster of PFGE-
matched serotype 4b isolates among 22 cases reported by Pennsylvania health officials. CDC
initiated a collaborative, multistate investigation, asking that health officials in northeastern states
conduct active case finding, perform real-time PFGE analysis of all clinical isolates, and submit
the patterns to PulseNet for rapid comparison. Among 188 cases identified in nine states from July
to November 2002, 54 clinical isolates shared the outbreak-associated subtype. This PFGE pattern
had only been identified 20 times previously in the national database, which contained approxi-
mately 2000 isolates from the previous 6 years. Among the 54 patients infected with the outbreak-
associated subtype, 8 (15%) died and 3 women lost their fetuses (Figure 10.8). Among the case-
patients were 8 pregnant women, 4 neonates, 30 patients with medical conditions associated with
immunosuppression, and 4 patients aged >65 years with no underlying medical condition.
A case–control study, in which controls were listeriosis patients in states where case-patients
resided with contemporaneous infection by a nonoutbreak-associated subtype, found that case-patients
were significantly more likely than controls to have consumed turkey deli meat more than one to two
times during the 4 weeks preceding illness (55% of 38 case-patients vs. 29% of 53 controls, OR =
4.5, 95% CI = 1.3–17.1). Consumption of precooked turkey breast purchased at grocery stores or
restaurants was statistically associated with infection by the outbreak-associated subtype (P = 0.008).
Based upon this information, health and regulatory officials launched a traceback investigation. Case-
patients were unable to recall specific brands; however, 80 purchase sites were identified by the first
29 patients interviewed. Among 57 purchase sites investigated, turkey deli meat products were supplied
by more than 50 processors. USDA–FSIS officials systematically evaluated the 15 most frequently
identified processors, addressing factors including in-plant Listeria sampling results, food safety
violations, and construction projects. Results from four processors warranted further investigation.
The outbreak-associated subtype was isolated from the processing environment of one processor
(processor C, designations differ from original reference), from unopened packages of turkey deli
meat from another (processor D), and from opened packages of deli meats produced at both processors
which were obtained during purchaser investigations and from patients. Notably, in a similar scenario
to that observed in the 1998–1999 outbreak linked to ready-to-eat meats, a construction project had
occurred at processor C in the room where cooked turkey was handled. Coincidentally, the frequency
of Listeria-positive environmental samples increased in this processing area. In response to investi-
gation findings, which provided strong evidence for the association of processor C and D products
12
10 Died
Number of Patients Had fetal death
8
0
7/1 7/15 7/29 8/12 8/26 9/9 9/23 10/7 10/21 11/4 11/18
Week of Culture, 2002
FIGURE 10.8 Epidemic curve showing listeriosis cases caused by outbreak-associated subtypes, United
States, July–November 2002. Dark arrow indicates approximate date of initial recall of turkey deli meat
products (October 9, 2002). (Adapted from Gottlieb, S.L., C.E. Newbern, P.M. Griffin, L.M. Graves, R.M.
Hoekstra, N.L. Baker, S.B. Hunter, K.G. Holt, F. Ramsey, M. Head, P. Levine, G. Johnson, D. Schoonmaker-
Bopp, V. Reddy, L. Kornstein, M. Gerwel, J. Nsubuga, L. Edwards, S. Stonecipher, S. Hurd, D. Austin, M.A.
Jefferson, S.D. Young, K. Hise, E.D. Chernak, J. Sobel, and the Listeriosis Outbreak Working Group 2006.
Multistate outbreak of listeriosis linked to turkey deli meat and subsequent changes in United States regulatory
policy. Clin Infect Dis 42: 29–36. With permission.)
to this outbreak, both processors voluntarily recalled a combined >30 million pounds of ready-to-eat
poultry products. Investigation findings further informed recent USDA–FSIS policy changes, detailed
in the closing discussion for this section.
OTHER PROCESSED MEATS

Two outbreaks of invasive listeriosis have been linked to pâté [11,108]. The first occurred in
England, Wales, and northern Ireland, when trends in the epidemiology of listeriosis in 1988 and
1989 suggested the occurrence of one or more common source epidemics [108]. As shown in
Figure 10.9, the number of laboratory-confirmed cases in 1987–1989 was nearly double the number
of cases in 1983–1986. Consistent seasonal peaks in late summer and early fall disappeared during
this period, and the percentage of perinatal infections increased to 52% in 1989, compared to 38%
in 1983–1987. Isolate characterization revealed two frequent subtypes: a specific serotype 4b phage
type (phage type 6,7) and serotype 4bX accounted for 30 to 54% of the cases annually. In 1989,
routine public health follow-up of a listeriosis case resulted in isolation of L. monocytogenes from
pâté obtained from the patient’s refrigerator. This prompted a comprehensive survey of pâté products
for this organism, and survey of listeriosis patients with regard to their food exposures during the
3 weeks before illness. Of 1,698 pâté samples screened in 1989, 9.5% were positive. Notably,
samples produced by one manufacturer (manufacturer Y) were contaminated at a higher frequency
(48% of 107 samples positive, 11% at >1000 CFU/g) than samples produced by other manufacturers
(4% of 781 samples). Further, serotype 4b phage type 6,7 and serotype 4bX accounted for 96% of
isolates from manufacturer Y’s pâté samples, compared to 19% of samples from other producers.
Patients infected with the two predominant subtypes were significantly more likely to have con-
sumed pâté during the 3 weeks before illness, compared to patients infected by other subtypes
(87% of 15 vs. 35% of 17, χ2 test P = 0.0095).
350
Total Cases
Non-pregnancy
Pregnancy
300
250
Number of Cases
200
150
100
50
0
1983 84 85 86 87 88 89 90 91 92 93 94
Year
FIGURE 10.9 Listeriosis in England, Wales, and Northern Ireland, 1983–1994. (Adapted by E.T. Ryser from
McLauchlin, J. and L. Newton 1995. Human listeriosis in England, Wales and Northern Ireland: a changing
pattern of infection. Proc. XIIth International Symposium on Problems of Listeriosis, Perth, Western Australia,
October 2–6, pp. 177–181.)
The chance finding of L. monocytogenes in pâté obtained from a patient’s refrigerator also
prompted a government health warning (May–June 1989) to at-risk consumers regarding pâté
consumption. Following this warning and suspension of sales of pâté from manufacturer Y, the
incidence of listeriosis declined dramatically (Figure 10.9). Further, a concurrent decline in infection
by the two predominant subtypes was also observed. Last, in a 1990 survey of 626 pâté samples, the
frequency of L. monocytogenes serotype 4b phage type 6,7 and 4b X-positive samples declined to 4%.
In 1999, a small outbreak of invasive listeriosis in three U.S. states (New York, Connecticut,
and Maryland) was attributed to consumption of pâté [11]. Eleven cases caused by an unusual
subtype of serotype 4b were identified. Two patients had consumed the same brand of pâté, and a
third had consumed an unknown brand of pâté. Based upon USDA–FSIS investigation findings,
the company recalled approximately 400 pounds of pâté and other similar products produced from
poultry meat [17]. The recall was subsequently expanded to encompass approximately 80,000
pounds of product [17].
One of the largest documented outbreaks of invasive listeriosis occurred in France in 1992
[72a,73,89,130] and was associated with consumption of pork tongue in aspic (jelly). Detection
of this outbreak was facilitated by two National Reference Centers (NRC), components of a
surveillance system for listeriosis established in 1987. Clinical isolate serotyping at the Nantes
facility, followed by phage typing at the Pasteur Institute, revealed that 279 of 758 captured cases
(37%) were caused by a specific subtype of serotype 4b (Figure 10.10), compared to only 6 to 27 cases
during previous years [89]. Among the 279 outbreak-associated cases, 182 (65%) outbreak-associated
cases occurred in nonpregnant adults, 5 occurred in children (2%), and 92 (33%) in pregnant women.
120
Epidemic Non-epidemic
100
Number of Cases
80
60
40
20
0
J F M A M J J A S O N D
Month
FIGURE 10.10 Listeriosis in France, 1992. (Adapted by E.T. Ryser from Jacquet, C., B. Catimel, R. Brosch,
C. Buchrieser, P. Dehaumont, V. Goulet, A. Lepoutre, P. Veit, and J. Rocourt 1995. Investigations related to
the epidemic strain involved in the French listeriosis outbreak in 1992. Appl Environ Microbiol 61: 2242–2246.)
This outbreak resulted in 63 deaths in nonpregnancy-associated cases, 22 fetal deaths, and 7 neonate
deaths, for an overall case fatality rate of 33% [72a].
Health officials launched epidemiologic and laboratory-based investigations to identify risk
factors for infection and potential control measures [72a, 73]. The first case–control study, conducted
among 144 case-patients and 288 matched controls, did not identify risk factors for infection.
However, a second case-control study among women with perinatal infection found that case-
patients were more likely than controls to have consumed pork tongue in aspic (60% of case-
patients vs. 6.1% of controls, OR = 9.2) and, to a lesser extent, other delicatessen items. A third
study implicated a specific brand of pork tongue in aspic (OR = 14.8). In the contemporaneous
laboratory-based based investigation, the clinical isolates were further characterized by PFGE and
ribotyping along with a subset of >14,000 nonhuman isolates submitted to the NRC and isolates
from samples collected in conjunction with this investigation [89]. A total of 247 (89%) human
clinical isolates shared the same 3-enzyme PFGE profile, designated as the epidemic subtype sensu
stricto. Notably, this subtype is closely related to that implicated in several U.S. and European
outbreaks described in this chapter (California, 1985; Switzerland, 1983–1987; Denmark,
1989–1990). The sensu stricto subtype of L. monocytogenes was recovered from 154 food items
collected during various stages of production and distribution, including abattoirs, processing
facilities, retailers, and patient refrigerators. Among these food items were pork tongue in aspic
from delicatessens (n = 112), other meat products (n = 19), cheeses (n = 12), and other food items
with lower frequency. This subtype was further isolated from opened and unopened packages of
the brand of pork tongue in aspic cited by patients, from environmental samples collected at the
facility that produced that brand, and from retail stands. Thus, results of the laboratory-based
investigation strongly supported the epidemiologic investigation findings. In another report describ-
ing the investigation of six processors suspected to have produced contaminated product, L.
monocytogenes was isolated from 35% of 270 samples collected throughout the processing area
[130]. Among these, 33% were from cooked product contact surfaces, correlating well with envi-
ronmental investigation findings from other ready-to-eat meat and poultry products. The epidemic-
associated phage type was isolated from raw brine in a facility producing pork tongue in aspic. In
follow-up investigations in two plants after cleaning and disinfection, L. monocytogenes was isolated
from 17% of raw product surfaces and 7% of finished product contact surfaces, most of which
remained visibly soiled or covered by a biofilm; these results highlight the difficulty of eliminating
this organism from the food processing environment (see Chapter 19 for a detailed discussion).
Two invasive listeriosis outbreaks in France were linked to consumption of contaminated
rillettes, a ready-to-eat pâté-like product produced by cooking ham meat in grease [52,74]. In July
1993, the National Reference Center notified Ministry of Health officials of a cluster of 10 listeriosis
cases caused by an unusual phage type [73]. This serotype 4b subtype had accounted for <1% of
cases from 1987 to 1992, and was later found to be closely related to that implicated in outbreaks linked
to pâté (England, Wales, Northern Ireland) [108] and pasteurized milk (Massachusetts) [89]. Upon
identification of eight additional cases in the following weeks, an investigation was launched to assess
the magnitude of the outbreak, identify risk factors for infection, and implement control measures.
As shown in Figure 10.11, 38 cases caused by the epidemic-associated phage type were
identified from June to October 1993. This figure highlights the importance of routine subtyping
in timely outbreak recognition, as the overall incidence of listeriosis did not increase significantly
during the epidemic period. Most patients resided in western France. This outbreak was character-
ized by a high percentage of perinatal cases (82%), with seven cases occurring in nonpregnant
adults. Twelve women gave birth prematurely. The overall case fatality rate was 32%, and included
nine fetal deaths and one death in a neonate. Similar to observations in the 1998–1999 frankfurter-
associated outbreak, the incubation period appeared to be shorter among three nonpregnant patients
(11 to 19 days) than in six pregnant women (median 33 days).
In the case control study, a standardized questionnaire addressing consumption history of >100
food items was administered to case-patients and controls matched by gestational age or age and
underlying condition as appropriate. Within 6 days, preliminary analyses implicated rillettes
16
Sporadic cases
Epidemic cases (Materno-neonatal)
14 Epidemic cases (Non-pregnant)
12
10
Cases
0
17 24 31 7 14 21 28 5 12 19 26 2 9 16 23 30 6 13 20 27 4 11 18 25 1 8
May June July August September October November
FIGURE 10.11 Listeriosis in France, 1993. (From Goulet, V., J. Rocourt, I. Rebiere, C. Jacquet, C. Moyse, P.
Dehaumont, G. Salvat, and P. Veit 1998. Listeriosis outbreak associated with the consumption of rillettes in France
in 1993. J Infect Dis 177: 155–160. With permission.)
purchased at a specific retail chain as the most likely vehicle of transmission (OR = 18.0, CI =
2.2–208.0). The retail chain was supplied with a specific brand (designated “Brand A”) of meat
products, including rillettes, produced at a single plant. Based upon these results and isolation of
the epidemic phage type from a raw product sample from the implicated establishment, the producer
recalled the product and ceased production.
Further analysis of case–control study data resulted in the implication of additional Brand A
meat products, including pâtés and sausages. Two Brand A products remained significantly asso-
ciated with illness in a multivariate statistical model, and included rillettes (OR = 10.9, CI =
2.1–54.4) and country pâté (OR = 5.0, CI = 1.0 –24.1). Each of 35 case-patients interviewed reported
exposure to Brand A products; 30 had consumed rillettes, 4 had consumed other meat products,
and 1 had a family member who had consumed rillettes, suggestive of the potential for cross-
contamination of other items during storage. Further interview of case-patients and controls who
had consumed rillettes revealed that case-patients were more likely than controls to have stored
the rillettes longer (> 6 days, 48% of case patients vs. 28% of controls, P = 0.12) and to have eaten
them over more meals (> 4 meals, 48% of case patients vs. 23% of controls, P = 0.05).
Results of the environmental investigation correlated well with the epidemiologic investigation
findings. The epidemic-associated subtype was isolated from 15 of 508 containers of Brand A
rillettes sampled prior to their sell-by dates, 20 containers removed from retail shelves, 11 containers
returned to retailers in response to the recall, and 2 containers sampled from patients’ refrigerators
and from several other Brand A meat products. Sell-by dates indicated contamination of at least
14 batches. The majority of unopened containers contained L. monocytogenes concentrations of
<100 CFU/g. Two opened containers returned prior to their sell-by dates were contaminated at
>10,000 CFU/g, demonstrative of the growth potential during home storage. An intensive environ-
mental investigation at the processing facility revealed several potential sources of contamination.
Records indicated isolation of coliforms from the rillettes production line. Production of rillettes
had increased during that time, and time of disinfection had been shortened to the apparent detriment
of product safety. Further, the epidemic-associated subtype of L. monocytogenes was isolated from
smoked pork breast, the hood over the frankfurter processing line, and, two months after the
outbreak ended, from one of the filling and packaging machines for rillettes. These results support
both raw materials and the processing environment as potential sources of product contamination.
As a result of these findings, raw materials and cooked product areas were better separated, a
Hazard Analysis Critical Control Point (HACCP) plan was developed and implemented, bacterio-
logical monitoring throughout the processing chain was reinforced, and the processor initiated post-
packaging pasteurization of rillettes.
Analysis of data from hospital surveys and two primary surveillance systems for invasive
listeriosis in France indicated a dramatic estimated 68% decline in the incidence of listeriosis from
1987 to 1997 [71]. Data from a network of general and regional hospitals (EPIBAC) indicated a
decline from 12.3 bacteremia and 3.4 meningitis cases per million population to 3.5 and 0.9 cases
per million, respectively, whereas data from the National Reference Center showed a decline from
6.3 to 4.1 cases of listeriosis per million population. Contemporaneous declines among retail-level
food product samples evidenced the contribution of aggressive prevention efforts implemented
during that decade, including enhanced surveillance, intensified microbiological monitoring of food
products throughout the production and distribution chains, improved hygiene at the retail level
(particularly at delicatessen counters), and targeted health education messages for high-risk popu-
lations. Due to the severity of disease and high case-fatality rate, invasive listeriosis remained a
public health priority. In 1998, health officials mandated notification of all laboratory-confirmed
listeriosis cases, along with standardized collection of food exposure history (complete with pur-
chase information). Detection and investigation of the two coincidental outbreaks described below
were greatly facilitated by these initiatives, in addition to the molecular surveillance system already
in place, which also enabled their differentiation.
Between October 1999 and February 2000, health officials identified 10 cases of invasive
listeriosis throughout France caused by the same serotype 4b subtype; clinical isolates were of the
same phage type, and matched by two-enzyme PFGE analysis [52]. Six cases occurred in nonpreg-
nant adults with underlying conditions known to be associated with increased risk of listeriosis,
three cases were perinatal, and one occurred in a previously healthy adult. Two adults with
underlying conditions and one infant died. A food history review for the first five recognized cases
revealed that all patients had reported consumption of rillettes during the 2 months prior to illness,
with four of five having purchased the products from the same market chain. In a review of
nonclinical isolates, 21 matched the epidemic-associated subtype; 7 of those had been isolated from
rillettes produced by the manufacturer that supplied the market named by the patients. In-plant
records indicated isolation of L. monocytogenes from finished product stock during the outbreak
period, and isolates from rillettes were later shown to match the epidemic-associated subtype. The
manufacturer recalled its products (rillettes, pork tongue in aspic) in January, and the outbreak
ended several weeks later. In response to this outbreak, French manufacturers reduced the shelf
life for rillettes to 28 days to help reduce the potential growth of inadvertent contaminants to high
levels during storage.
A second, simultaneous outbreak occurred from November 1999 to February 2000 [52]. This
outbreak involved a total of 32 cases and was caused by a subtype of serotype 4b with a different
PFGE pattern than that associated with the first outbreak. Nine perinatal cases, 11 cases in non-
pregnant adults with underlying conditions, and 12 cases in previously healthy nonpregnant adults
(all male, median age 52.5 years, all with central nervous system listeriosis) were distributed
throughout France. This outbreak had an overall case fatality rate of 31% and included death in
five adults with underlying conditions, four premature neonates, and one fetus. A case–control
study, in which patients infected with nonoutbreak-associated L. monocytogenes subtypes served
as controls, found that case-patients were significantly more likely to have consumed pork tongue
in aspic (OR = 28.0, 95% CI = 3.2–1,222.1), along with several ready-to-eat items including cervelas
sausage, cooked ham, and pâté de campagne. Having consumed at least one meat product purchased
from a delicatessen counter was also significantly associated with illness. On multivariate analysis,
consumption of pork tongue in aspic (OR = 75.5, 95% CI 4.7–1,216.0) and pâté de campagne (OR
= 8.9, 95% CI = 1.7–46.1) remained significantly associated with illness. It is likely that more than
one vehicle of transmission was associated with this outbreak, as consumption of pork tongue in
aspic explained 14 of 29 cases (48%) and all other case patients reported consumption of pâté
and/or ham. Patients reported a variety of purchase sites. A review of distribution records allowed
identification of two processors that had supplied the majority of purchase sites named by patients.
Available records, extensive product sampling, and in-plant environmental investigations did not,
however, enable investigators to implicate a single brand of product or processor. At the time of
this outbreak, L. monocytogenes was permissible in ready-to-eat meat products manipulated
between the microbial inactivation and packaging steps if studies were available to show that the
concentration of L. monocytogenes would not exceed 100 CFU/g at the end of the product shelf
life. In response to this outbreak, the food safety agency advised a production level zero-tolerance
policy for these products.
In 2000, investigators used PCR-based detection methods and automated ribotyping to quickly
link a cluster of three cases of invasive listeriosis (serotype 1/2) in Western Australia, one of which
resulted in fetal death, to contaminated cooked chicken products [86]. The efficient turn-around
time (8 hours for ribotyping) resulted in data that guided generation of hypotheses for illness and
allowed the investigators to make efficient recommendations for intervention. The products were
recalled, and isolation of high levels of L. monocytogenes from unopened packages of cooked
chicken pieces prompted implementation of a more rigorous cleaning and sanitation program and
additional heat treatment at the end of production. Other isolated listeriosis cases linked to con-
sumption of meat or poultry products have been reported and include (1) isolation of >106 CFU/g
of L. monocytogenes serogroup 4 from a homemade sausage consumed by a man prior to
development of listeriosis caused by the same serogroup [44]; (2) in the context of a study to
identify risk factors for listeriosis, isolation of the same electrophoretic enzyme type from two
patients and retail packages of pork sausage and ground beef [133]; and (3) isolation of the same
phage type from cooked-chilled chicken and from fetal tissue obtained from a patient who had
consumed the chicken prior to development of listeriosis [93].
REDUCTION OF MEAT AND POULTRY PRODUCT–ASSOCIATED INVASIVE LISTERIOSIS

Findings from the investigation of outbreaks associated with meat and poultry products strongly
support the USDA–FSIS/FDA joint risk assessment, in which analysis of per serving and per annum
relative risk rankings placed deli meats and frankfurters (not reheated) in the highest risk category
(very high risk) and pâté and meat spreads in the second highest category (high risk) [16]. The
categorization of unreheated frankfurters and deli meats reflects several factors, including (1) a
relatively high rate of contamination, (2) rapid growth of L. monocytogenes under refrigerated
storage temperatures, (3) likely storage for extended periods, and (4) frequent consumption by
members of the U.S. population. Supportive data indicated a relatively low annual consumption
rate for pâté and meat spreads (<1 × 109 annual servings) and a moderate level of contamination
at retail; however, the high growth rate during home storage and likely long home storage time (>6
to 10-day range) contributed to the high risk ranking. Thus, these products warranted particular
attention with respect to reducing the incidence of listeriosis. Outbreak investigation findings, in
conjunction with risk assessment results, have led to an aggressive USDA–FSIS regulatory policy.
Following the 2002 turkey deli meat-associated outbreak in the United States, USDA–FSIS issued
a final rule mandating that establishments producing ready-to-eat meat and poultry products have
in their HACCP plans or Sanitation Standard Operating Procedures (SSOPs) controls to prevent
product contamination by L. monocytogenes from the processing environment [8]. These controls
must be scientifically validated, and are stratified in accordance with the number of control steps
taken (i.e., a post-lethality step treatment and/or a growth inhibitor). Controls could include a post-
packaging thermal step or the addition of growth inhibiting compounds. Irradiation would also be
an effective measure; however, FDA has not yet approved it for use with these products. The final
rule also states that processors relying solely upon sanitation for prevention of contamination would
be subject to intensified testing by USDA–FSIS. Establishments would also be mandated to share
in-plant control measure verification data. Last, this final rule clarified that isolation of L.
monocytogenes from processing equipment would provide sufficient justification for product recall,
regardless of whether finished product yielded this organism or not. In addition to efforts to reduce
the frequency and level of L. monocytogenes contamination, another critical prevention strategy
for reduction of listeriosis associated with ready-to-eat meat and poultry products lies in effective
consumer education programs.
SEAFOOD PRODUCTS
Several small outbreaks of listeriosis have been linked to seafood items, including imitation crab
meat, smoked mussels, gravad, and cold-smoked fish. Two 1996 cases in Canada were suspected to
have been caused by consumption of imitation crab meat produced from the Alaskan Pollock fish
[61]. A healthy adult female was hospitalized with severe gastrointestinal symptoms including
explosive diarrhea and projectile vomiting. L. monocytogenes serotype 1/2b was isolated from her
blood and stool. Her husband experienced similar but less severe symptoms; L. monocytogenes
serotype 1/2b was isolated from his stool. Both reported consuming imitation crab meat during the
18 hours before onset of illness. Samples of imitation crab meat and four other food items obtained
from the patients’ refrigerator yielded L. monocytogenes isolates that matched the patient isolates
by serotyping, PFGE, and randomly amplified polymorphic DNA (RAPD) analysis. The imitation crab
meat, along with two other items, was contaminated at a high level (2 × 109 CFU/g). Retail samples
of imitation crab meat also yielded matching isolates, although at low levels, suggestive of growth
during subsequent home storage. Based upon these findings, along with information indicating that
two well meal companions had not consumed this item, investigators concluded that the imitation
crab meat was the most likely vehicle of infection. Although one case was invasive, the predomi-
nance of gastrointestinal symptoms following consumption of a high inoculum correlates well with
reports of noninvasive L. monocytogenes-associated gastroenteritis [120]. This outbreak also high-
lights the importance of consumer education regarding safe food storage, as food sampling results
demonstrated the likelihood of cross-contamination of several other food items.
SMOKED MUSSELS
Investigators in New Zealand linked three cases that occurred from October to December 1992 to
consumption of contaminated smoked mussels [37]. Initially, two perinatal cases caused by serotype
1/2a were detected. A food exposure history indicating consumption of the same brand of mussels
prior to development of listeriosis prompted investigators to analyze all 1991 and 1992 serotype
1/2a clinical isolates by PFGE. Two additional patient isolates, along with L. monocytogenes isolates
from an unopened package of mussels obtained from a patient’s refrigerator, were indistinguishable
by PFGE. One patient with illness in October 1992 reported consumption of mussels, but the fourth
patient, with illness in May 1991, did not. Isolates from three patients reporting mussel consumption
and the mussel isolates were indistinguishable by phage typing, sensitivity to arsenic and cadmium,
and restriction fragment length polymorphism (RFLP). The fourth patient’s isolate was untypable
via phage typing. This is an oft-encountered caveat of this subtyping method, and likely means
that the isolate was of a type not represented in the phage set [105]. These laboratory findings
highlight the higher discriminatory power offered by characterization with more than one typing
method when feasible.
Confirmation of “Brand X” mussels as the vehicle of transmission prompted officials to screen
additional Brand X products, along with samples collected from the processing environment.
Of 27 isolates from Brand X products, 26 matched the human clinical isolates, along with 4 of 7
from environmental samples taken from the processing facility. Notably, this subtype had been
isolated from the processing environment 3 years earlier. These data are supportive of long-term
colonization of the processing environment, as has been observed in other studies focused upon
the smoked seafood industry [23,24,58,64,83,118,126], which can then serve as an ongoing source
of contamination.
Another small outbreak with four cases of L. monocytogenes serotype 1/2b associated gastro-
enteritis, which occurred in Tasmania in 1991, was also microbiologically confirmed to have been
caused by mussels produced in New Zealand [112,113]. A high concentration of L. monocytogenes
(1.6 × 107 CFU/g) was isolated from both open and unopened packages of mussels. Traceback
investigations identified three batches for which the shelf life had been overestimated by at least
3 months, prompting a recall. The isolates were indistinguishable by RFLP and phage typing
(J. McLauchlin, unpublished data) but did not match those isolated from Brand X products or the
respective processing environment.
COLD-SMOKED FISH PRODUCTS

Despite frequent isolation of L. monocytogenes in cold-smoked fish products [29,57,92,118], few
listeriosis outbreaks have been attributed to these food items. In a 1995 report from Australia,
smoked salmon was implicated as the vehicle of transmission for two cases of perinatal listeriosis
which resulted in fetal death [20]. Another outbreak, associated with cold-smoked rainbow trout
and gravad (made by curing a raw fillet with sugar, salt, pepper, and dill), occurred in the province
of Värmland, Sweden from August 1994 to June 1995 [58,144]. Of nine cases due to serotype 4b, three
were perinatal infections and six cases were in elderly or immune-compromised nonpregnant adults.
Two patients, including one neonate, died. All patients reported consumption of gravad or smoked
rainbow trout or salmon, five of whom recalled definitely or possibly consuming the same brand.
Four subtypes were identified among patient isolates using restriction enzyme analysis (REA) with
three enzymes and phage typing (designated clonal types A–D); six isolates were clonal type B,
seen only once previously in Sweden.
Investigators launched an environmental investigation, in which samples from the producer pro-
cessing area along with gravad and smoked fish collected from patient refrigerators, retailers, and the
producer were screened. Samples from the processing area and from fish yielded L. monocytogenes.
Molecular subtyping results linked environmental and food isolates to eight of nine cases. Type B
was recovered from samples of gravad fish from the home of one patient and from the producer,
along with residue found in the packing machine, providing laboratory confirmation of the vehicle
and identifying the likely environmental source of product contamination. Similar to several other
investigations, the same subtype had been isolated from a product from this producer 6 months earlier,
indicating that this subtype was likely among the resident environmental microflora. In addition, L.
monocytogenes types C and D were isolated from product sampled at the processing plant, explaining
two additional cases. One sample yielded two subtypes, highlighting the utility of characterizing more
than one L. monocytogenes colony from a given sample during investigations.
In the 2003 USDA–FSIS/FDA joint risk assessment, smoked fish was categorized as a high risk
food on a per serving basis [16]. The high risk classification resulted largely from several character-
istics: (1) a high frequency of contamination (12.9%), as indicated by data compiled from 30 studies
evaluating prevalence; (2) a high estimated contamination level at retail (>0.6% of servings containing
103–106 CFU); (3) a moderate growth rate during storage; and (4) a typical storage duration of 3 to
5 days. Given the risk ranking, the infrequency of association of these products with listeriosis
outbreaks is intriguing. Differences in pathogenic potential among L. monocytogenes subtypes com-
mon to these products do not provide an explanation, as several studies have reported the isolation
of human epidemic- and sporadic-case–associated subtypes from smoked fish and smoked fish pro-
cessing environments [23,29,118,144]. Potential contributors to this phenomenon could include the
relatively low frequency of consumption by a large segment of the population or an inherent feature
of the food matrix or production process that affects the organisms’ ability to cause disease once they
are consumed. It is also possible that outbreaks have occurred but have gone undetected.
Control of L. monocytogenes in the cold-smoked fish-processing environment and products
presents a unique and significant challenge to the industry. This organism is ubiquitous in the
environment, thus easily introduced into production facilities. Further, persistence of L. monocy-
togenes in smoked fish processing facilities, thereby establishing an environmental reservoir that
is difficult to eliminate, has been well documented [23,24,58,64,83,118,126]. The production
process, which typically involves brining via rubbing with salt, cure injection, or soaking in 3 to 5%
aqueous phase NaCl followed by smoking at 25 to 30°C [144], does not include a step that would
inactivate L. monocytogenes or prevent growth during refrigerated storage. As a result of these
factors, sporadic contamination with low levels of viable L. monocytogenes may be difficult to
control. Strategies to minimize L. monocytogenes contamination should include a continued focus
on rigorous in-plant cleaning and sanitation programs, verified and modified in accordance with
data obtained from routine environmental screening, and exploration of measures effective against
established microflora. Options to inhibit growth of sporadic contaminants include exploration into
process modification to employ potential inhibitory properties of lactic acid bacteria, salt and smoke,
post-packaging pasteurization (i.e., irradiation), and frozen storage [5].
VEGETABLES
Compared to other food categories, few invasive listeriosis outbreaks have been linked to consump-
tion of contaminated vegetables or vegetable products. The large coleslaw-associated outbreak that
occurred in the Canadian Maritime Provinces, discussed earlier in this chapter, was among the first
to provide convincing evidence for transmission of L. monocytogenes by food. Although reported

after this outbreak, an outbreak likely associated with raw vegetables occurred among hospitalized
patients in Massachusetts a few years earlier in 1979 [82]. Twenty-three patients hospitalized during
September and October had invasive listeriosis. Of these cases, 20 (87%) were caused by serotype
4b (identified as the epidemic serotype), compared to 33% of cases over the previous 26 months.
Fifteen of these 20 patients acquired their infection while hospitalized. All cases occurred among
nonpregnant adults, half of whom had underlying conditions contributing to immune suppression.
Five patients died, although two deaths were attributed to other causes. Epidemiologic studies
among patients and controls matched by age, gender, and hospitalization date did not result in
identification of specific dietary risk factors. Case-patients were more likely than controls to have
consumed tuna fish, chicken salad, and hard cheese; the common factor was the inclusion of these
items in salads that also contained lettuce, celery, and/or tomatoes.
This investigation also revealed intriguing clinical findings. Outbreak-associated case patients
were more likely than patients with sporadic listeriosis to have had gastrointestinal symptoms at
the time of fever onset (85% of outbreak-associated case-patients, compared to 22% of patients
with sporadic listeriosis), a phenomenon similar to that observed in the 1987 outbreak that occurred
in Philadelphia [137]. They were also more likely to have received antacids or histamine-blocking
drugs prior to the onset of listeriosis (60% compared to 17%). These results were corroborated by
case–control study findings; 60% of case-patients took antacids or histamine blockers before onset
of listeriosis, compared to 25% of controls, and were also more likely to have undergone gas-
trointestinal procedures. Decreased stomach acidity may have resulted in improved survival of
L. monocytogenes during passage through the stomach, as has been observed for other enteric
pathogens [66]. In addition, underlying gastrointestinal lesions, for which these medications may
have been used, may have resulted in an increased risk for invasive disease.
A small outbreak of serotype 4b infections that occurred among patients subsequently admitted
to a hospital on the Texas–Mexico border was epidemiologically associated with frozen vegetables
[140]. There were five cases (four perinatal, one in an immunocompromised nonpregnant adult)
detected over a 5-week period, with two additional cases identified over the following 2 months.
A case–control study implicated frozen broccoli and cauliflower, and L. monocytogenes serotype
4b was later isolated from opened and unopened packages of these frozen vegetables. Human
clinical and food isolates were indistinguishable by PFGE subtyping.
In the recent USDA–FSIS/FDA risk assessment, vegetables were classified in the moderate
risk category, due largely to the high number of annual servings and moderate frequency of
contamination by L. monocytogenes (about 2 to 5% of samples). However, vegetables remain
infrequently associated with outbreaks of listeriosis. Potential differences in pathogenic potential
among different subtypes of L. monocytogenes do not offer a conclusive explanation for this
phenomenon, as both outbreak and sporadic-case–associated subtypes have been isolated from
vegetables (see Chapter 16 for a detailed discussion). The overall low growth rate during storage
[16], along with the relatively short shelf life of unpreserved vegetable products may contribute to
the infrequent association with invasive disease, compared to other food categories. Continued emphasis
on processing facility hygiene, along with careful handling of raw vegetables and ready-to-eat vegetable
products by individuals in high-risk populations, will help to further reduce the risk of listeriosis
from consumption of vegetables.
L. MONOCYTOGENES–ASSOCIATED FEBRILE GASTROENTERITIS

A second, increasingly well-described disease state resulting from infection by L. monocytogenes
is febrile gastroenteritis (for a recent review with a focus on clinical practice, see Ooi and Lorber
[120]). As shown in Table 10.2, at least eight outbreaks of foodborne gastroenteritis have been
attributed to this organism. These outbreaks have largely been recognized as point-source outbreaks
among otherwise healthy members of a cohort and are characterized by consumption of a high-level
TABLE 10.2
Listeria monocytogenes-Associated Febrile Gastroenteritis Outbreaks
Year Location Serotype No. Cases Implicated Vehicle Reference
1993 Northern Italy 1/2b 18 Rice salad [129]

1994 Illinois 1/2b 45 Chocolate milk [50]
1997 Northern Italy 4b 1,566 Cold corn and tuna salad [22]
1998 Finland 1/2a 5 Cold-smoked rainbow trout [110a]
2000 New Zealand 1/2 31 Ready-to-eat meat products [139]
2001 Los Angeles 1/2a 16 Turkey delicatessen meat [65]
2001 Sweden 1/2a 48 Fresh, raw-milk cheese [46,51]
2001 Japan 1/2b 38 Cheese [101]
inoculum followed by onset of fever, gastrointestinal symptoms including watery diarrhea and
nausea, headaches, and joint and muscle pain within 24 hours. Symptoms typically resolve within
a few days. Here, we discuss several well-characterized outbreaks.
POTENTIAL ASSOCIATION WITH A MILD CLINICAL SYNDROME:

SHRIMP, 1989, UNITED STATES
The suggestion of milder illness in otherwise healthy adults was reported by Riedo et al. following
an investigation that grew from follow-up of two perinatal cases of L. monocytogenes bacteremia
[125]. After learning that the patients’ only common exposure was attendance at a party, public
health officials launched a retrospective cohort investigation among 36 attendees to evaluate the
potential for mild illness and determine risk factors for illness. In this type of study, food and/or
other exposures are collected from all or a representative sample of persons in a well-defined
population among whom disease occurred (e.g., attendees of an event after which illness occurred
among some attendees), allowing the calculation of attack rates of illness in those who did or did not
report a certain exposure [53]. The attack rates can then be compared to determine the food item or
other exposure associated with the greatest increase in risk. For this investigation, based upon the
body of knowledge regarding foodborne listeriosis at that time, a case of illness was defined as
isolation of L. monocytogenes from blood or stool or illness with any two concurrent gastrointestinal
or musculoskeletal symptoms in a party attendee within 6 weeks after the party. Illness in 10
patients met the case definition, 9 met the symptom criteria, and L. monocytogenes was isolated
from 1 of 25 stool samples collected from attendees. Symptom onset and/or isolation of
L. monocytogenes from a clinical specimen occurred throughout the 6 weeks after the event. The
3 clinical isolates (2 from the blood of the women with perinatal infection and the stool isolate)
were serotype 4b and matched by multilocus enzyme electrophoresis.
Among 24 food items served at the party, persons who consumed greater amounts of shrimp (boiled
and chilled, served with a sauce) and, to a lesser extent, nonalcoholic beverages, Camembert cheese,
and cauliflower were at significantly increased risk for illness (relative risk [RR] for shrimp consumption
7.0, 95% CI = 2.3–21.5). After controlling for consumption of other foods, only consumption of shrimp
and cauliflower remained significantly associated with illness. Only three ill persons, however, recalled
consumption of cauliflower. No leftover foods were available for screening, and foods purchased from
suppliers approximately 6 weeks after the party did not yield L. monocytogenes.
Although a matching L. monocytogenes subtype was isolated from a person with noninvasive
illness, the longer incubation period (19 to 23 days for index cases) is not representative of other
L. monocytogenes-associated outbreaks of foodborne gastroenteritis. It is possible that another organ-
ism was responsible for the mild gastrointestinal illness observed in the patients who subsequently
developed invasive listeriosis and that the syndrome observed in others was not associated with
food consumed at the party. However, this report highlighted the potential for a milder illness
caused by L. monocytogenes in otherwise healthy adults and raised awareness of this hypothesis
for consideration in subsequent investigations.
RICE SALAD, 1993, NORTHERN ITALY

In 1993, an outbreak of febrile gastroenteritis occurred among 39 attendees of a supper [129]. Four
attendees were hospitalized during the 24 hours following the event with high fever (average 39.6 °C),
diarrhea, nausea, abdominal pain, arthromyalgia, headache, and sore throat. Several days later,
blood cultures yielded L. monocytogenes serotype 1/2b, whereas stool cultures were negative for
enteric pathogens including Salmonella and Shigella. Routine public health follow-up revealed that
the outbreak occurred among nonpregnant, otherwise healthy persons. Based upon these unusual
findings, health officials launched epidemiologic and laboratory investigations to better characterize
the outbreak.
Investigators defined a case as onset of fever plus diarrhea, nausea, vomiting, and/or arthromyalgia
within 3 days of attending the supper. Among the 39 attendees, 18 met the case definition, for an attack
rate of 46%. Ill persons ranged in age from 17 to 54 years; their median age was significantly higher
than that of well attendees (36 years compared to 22 years, P < 0.001). Their clinical syndrome largely
mirrored that of hospitalized attendees, with the exception of a flu-like syndrome (arthromyalgia, sore
throat, headache) in four persons in the absence of gastrointestinal symptoms. Among patients with
gastrointestinal symptoms, onset occurred a median of 18 hours following the supper. None of the
stool specimens collected from all attendees approximately 1 month after the event yielded L.
monocytogenes. Among convalescent serum tested from 18 ill attendees, however, 39% showed α-
somatic antigen type 1, compared to 1 of 4 well persons. None had α-type 4b antibodies, and none of
11 community members who served as a comparison group had detectable antibody titers against either.
Consumption of rice salad was significantly associated with illness. Eighteen of 20 persons
(90%) who consumed rice salad developed illness, compared to none who did not consume that
item (RR undefined, P < 0.001). A food preparation review revealed that, due to limited refrigeration
space, the rice salad had been held at ambient temperature during the 24 hours before the supper.
Samples of rice salad and other available food items did not yield pathogens including pathogenic
Escherichia coli, Salmonella, Bacillus cereus, or Staphylococcus aureus. Although there was no
rice salad left for further screening, samples of several other food items, along with environmental
samples collected from the blender and freezer in the home of the cook, yielded L. monocytogenes.
The clinical, food, and freezer isolates all matched by MEE and phage typing.
Although it is possible that the gastrointestinal syndrome characterizing this outbreak may have
been caused by an organism not detected in clinical specimens, which were collected approximately
1 month after the event, several factors support L. monocytogenes as the etiologic agent: (1) isolation
of the same subtype from the blood of two patients, food, and environmental samples; (2) several
ill persons with high titers of α-Listeria antibodies, compared to a low percentage among well
attendees and nondetectable titers in a comparison population; and (3) a median incubation period
longer than that typically observed for toxin-mediated illness, yet shorter than that typical for viral
etiologies. This outbreak highlights the importance of proper food handling and storage in non-
commercial settings, as L. monocytogenes was isolated at >103 CFU/g from two food items. Further,
investigation findings are supportive of considering L. monocytogenes as a potential etiology in
outbreaks of febrile gastroenteritis.
CHOCOLATE MILK, 1994, UNITED STATES

The investigation of a 1994 outbreak linked to contaminated chocolate milk provided overwhelming
evidence for the association of L. monocytogenes with febrile gastroenteritis [50]. Again recognized
as a point-source outbreak, illness occurred among approximately 92 persons who attended a picnic
at a Holstein cow show in Illinois. Of 82 persons interviewed, 45 (55%) met the case definition of
onset of symptoms from two to four symptom complexes including (1) fever; (2) diarrhea, nausea,
or vomiting; (3) myalgia or arthralgia; and (4) headache within a week of attending the picnic or
consuming food from it. As observed in the previous outbreak, the median age of ill persons was
higher than that of well persons (31 years vs. 24 years). None of the attendees reported underlying
conditions; one pregnant woman at 40 weeks gestation had diarrhea the day after the picnic and
delivered a healthy baby several days later. Common symptoms among ill persons included diarrhea,
fatigue, fever, chills, headache, myalgia, and abdominal cramps. The median incubation period was
20 hours (range 9 to 32 hours), and diarrhea lasted a median of 42 hours (range 3 to 50 hours).
Four patients were hospitalized as a result of their illness, and others reported loss of work time
due to the severity of their symptoms. L. monocytogenes was isolated from 11 stool specimens.
All positive specimens were from persons with illness meeting the case definition. Analysis of
convalescent serum collected from 48 attendees and a control group with nonoutbreak-associated
enteric infections showed a significantly higher median α-Listeriolysin O antibody titer (143 ELISA
units) in persons with illness meeting the case definition than in controls (63 ELISA units, P < 0.001).
Lower titers were also reported among attendees who were not ill and persons with mild illness.
The strong association of higher α-Listeriolysin O antibody titers with illness highlights the
diagnostic utility of this serologic assay.
Among 60 persons who consumed chocolate milk, 45 (75%) became ill, compared to none of
22 persons who did not consume this item (RR undefined, 95% CI = 13.6 – ∞). Consumption of
Swiss cheese was also significantly associated with illness; however, fewer attendees had consumed
the cheese, and all ill persons who consumed cheese had also consumed chocolate milk. Culture of
a carton of milk leftover from the picnic and a carton collected from the dairy yielded L. monocytogenes
at 1.2 × 109 and 8.8 × 108 CFU/mL, respectively. An 8-ounce carton of milk would, therefore,
potentially provide an inoculum of 2.9 × 1011 CFU. These and all but one clinical isolate were
indistinguishable by MEE, ribotyping, and PFGE analysis. The PFGE pattern of one clinical isolate
differed by one band. Inspection of the dairy and review of handling during transport to the picnic
provided a clear explanation for the presence of high levels of L. monocytogenes in the chocolate
milk. After flavor addition and milk pasteurization, the chocolate milk was held in a jacketed tank
for 2 hours before being pumped to a filler line over a 7-hour period. The refrigerated jacket had
been in disrepair, thus unrefrigerated, for 3 years. Inspection revealed a breach in the tank lining,
which allowed milk to leak into the insulation jacket and pool there. When milk was pumped out,
the pooled milk could leak back into the tank. Sanitizer spray nozzles were clogged, thus inhibiting
the flow of sanitizer into the tank lining. A total of 180 cartons of chocolate milk for the picnic were
in transport for over 2 hours without refrigeration. Upon delivery, they were held in a noncommercial
refrigerator overnight. They were placed in an unrefrigerated cooler on the morning of the picnic
and were available throughout the day. Based upon these data, it is likely that postpasteurization
contamination of the milk occurred due to poor facility hygiene practices, with subsequent temper-
ature abuse allowing rapid growth of L. monocytogenes.
Records further indicated that chocolate milk had been distributed to three additional states.
Consumption of chocolate milk from the implicated dairy was associated with similar illness in a
family of five traveling through a neighboring state. A review of clinical isolates submitted to health
officials in Illinois, Michigan, and Wisconsin resulted in identification of three additional cases of
invasive listeriosis with some association with chocolate milk from the implicated dairy. Two
patients had consumed chocolate milk from the implicated dairy, and the third had consumed
chocolate milk purchased at a store that sold milk from the implicated dairy. Further, each clinical
isolate was indistinguishable from the outbreak-associated subtype. Recommendations resulting
from this investigation included diagnostic screening for L. monocytogenes in outbreaks with similar
clinical features, enhanced monitoring of food products for pathogens including L. monocytogenes,
and rigorous cleaning and sanitation programs.
CORN AND TUNA SALAD, 1997, NORTHERN ITALY

A massive outbreak of L. monocytogenes-associated febrile gastroenteritis occurred in northern
Italy in 1997 [22]. On a single day, local health units in three different towns received unusually
high reports of febrile illness and gastroenteritis among students and staff of two primary schools
and students of a university. All had dined at cafeterias supplied by the same local caterer, which
prepared approximately 8,000 meals daily. Health officials immediately launched epidemiologic
and laboratory-based investigations to investigate the scope, magnitude, and potential source of
illness. Investigators determined that a cohort of 2,930 persons had consumed cafeteria meals on
the day before the illnesses began. Of 2,189 primary school students and teachers enrolled in a
cohort study, 93 adults and 1,473 students reported having at least one flu-like or gastrointestinal
symptom, for an overall attack rate of 72%. Headache (88% in adults, 86% in children), abdominal
pain (72% in both groups), and fever (68% in adults, 86% in children) were most commonly
reported, and the median incubation period was 24 hours. Fever and vomiting occurred with
significantly more frequency in children, whereas diarrhea, arthralgia, and myalgia were signifi-
cantly more frequent in adults. A total of 292 persons (19%) were hospitalized as a result of their
illness for a median of 3 days, underscoring the severity of associated symptoms. Of 292 stool
specimens screened by 9 hospital laboratories, 290 were negative for bacterial and viral enteric
pathogens and two yielded different Salmonella serotypes. L. monocytogenes was isolated from
the blood of one patient. Among 141 stool specimens subsequently screened for L. monocytogenes,
123 (87%) were positive. All isolates were serotype 4b, and were indistinguishable by PFGE and
RAPD analysis.
Persons who consumed a salad made from corn and tuna were over six times more likely to
become ill than those who did not (84% vs. 14%, RR = 6.2, 95% CI = 4.8–8.0, P < 0.001). No
other food items were significantly associated with illness. Investigators conducted an environmental
investigation at the catering facility and tested food samples obtained there. Although unopened
cans of corn and tuna did not yield microorganisms, a sample of the leftover corn and tuna salad
yielded L. monocytogenes at >106 CFU/g. These, along with L. monocytogenes isolates from facility
sink drains and a meal preparation surface, were indistinguishable from the clinical isolates by
PFGE and RAPD analysis. In a food preparation review, caterers reported opening the cans of tuna
and corn during early morning on the day of preparation and allowing them to drain at ambient
temperature. They were then mixed (no additional ingredients were added), portioned, and trans-
ported to the schools for lunch service. A corn salad without tuna was prepared later that day for
dinner service at the university that had reported gastrointestinal illness among students. Studies
with inoculated corn showed the potential for growth of L. monocytogenes to high concentrations
(>106 CFU/g) during storage at 25°C.
Based upon the environmental and laboratory-based investigation results, salad ingredients or
the prepared salad were likely cross-contaminated with L. monocytogenes at the catering facility.
Storage at ambient temperatures for several hours prior to serving could then allow the contaminants
to grow to high levels. This outbreak highlights the importance of rigorous cleaning and sanitation
programs in food production areas and proper food handling practices in reducing illness caused
by L. monocytogenes. The high hospitalization rate underscores the potential severity of febrile
gastroenteritis.
COLD-SMOKED RAINBOW TROUT, FINLAND

A small outbreak in Finland was linked to consumption of cold-smoked rainbow trout [110a]. Four
adults and one child developed a gastrointestinal illness with nausea, abdominal cramps, and
diarrhea within 27 hours of sharing a meal together. Fever was reported by three adults, and other
symptoms included vomiting, headache, fatigue, and arthralgia. The child was hospitalized over-
night as a result of the illness. Information from patient interviews identified cold-smoked rainbow
trout as the most likely vehicle of infection. No leftover fish was available; however an unopened package
from the same lot obtained from the retailer yielded 1.9 × 105 L. monocytogenes CFU/g. Retail inspection
findings indicated that the storage temperature of the cold-smoked trout was higher than that recom-
mended by the producer.
Stool samples collected from the patients did not yield enteric pathogens. However, subsequent
stool swabs obtained from two ill persons yielded L. monocytogenes. Clinical and smoked trout
isolates were both serotype 1/2a, and were indistinguishable by two-enzyme PFGE analysis.
READY-TO-EAT MEAT AND POULTRY PRODUCTS, NEW ZEALAND AND UNITED STATES
In 2000, investigators in New Zealand linked several independent foodborne illness reports to
consumption of contaminated ready-to-eat meats produced by the same manufacturer [139]. The
first two reports of febrile gastroenteritis on South Island were associated with corned beef. Two
additional reports of a similar syndrome indicated consumption of products from the same manu-
facturer. Leftover corned beef samples yielded L. monocytogenes at concentrations of >2.5 × 105
CFU/g, prompting the manufacturer to issue a product recall. On that date and shortly afterward,
two foodborne outbreaks were reported among 7 residents of South Island and among 21 of a
cohort of 24 residents of North Island; both meals had included ready-to-eat products produced by
the same manufacturer. In the latter case, the retailer had failed to remove the product from sale
despite the product recall.
The two reported outbreaks were characterized by different clinical syndromes; flu-like symp-
toms including fever, myalgia, and headache predominated in South Island cases, whereas the
majority of persons associated with the North Island outbreak also experienced gastrointestinal
symptoms including diarrhea, vomiting, and abdominal cramps. Most experienced symptom onset
within 24 hours of meal consumption. L. monocytogenes serotype 1/2 was isolated from stool
specimens collected from 2 of the 4 ill persons from the initial reports, 2 ill persons associated
with the South Island outbreak, and from 20 of 21 specimens collected from 17 ill and 3 well
persons associated with the North Island outbreak.
Several of the manufacturer’s food items were available for screening. L. monocytogenes
serotype 1/2 was isolated from leftover corned beef (as noted above) and ham (North Island
outbreak, 1.8 × 107 CFU/g), luncheon ham (<100 CFU/g) obtained from retailers cited by patients,
and unopened retail packages of bulk corned beef and roast pork (102 to 104 CFU/g from each of
six samples). All clinical and food isolates were indistinguishable by two-enzyme PFGE analysis.
A review of the manufacturing process revealed two potential sources for contamination: (1) cross-
contamination of cooked product prior to repackaging for retail and (2) raw meat, due to the
identified potential for an inadequate heat treatment. Investigators further found that the estimated
shelf life for corned beef (90 days) was erroneous, and it was subsequently reduced to 6 weeks.
Overall, the results of this investigation support the relatedness of these reports of febrile
gastroenteritis and the likelihood of a specific subtype of L. monocytogenes serotype 1/2 as the
etiology. Although many outbreaks of invasive listeriosis have been linked to contaminated ready-
to-eat meat products, this report provides evidence for an association with febrile gastroenteritis.
This underscores the importance of efforts to reduce the frequency of L. monocytogenes contami-
nation in these products, along with proper food handling practices at the retail and consumer levels.
In 2001, an outbreak of febrile gastroenteritis occurred among approximately 60 persons who
attended a birthday party in Los Angeles [65]. Food served at the party had come from a variety
of sources, including a delicatessen, a bakery, a local farm, and a grocery store. Among 44 attendees
interviewed, 16 (36%) had experienced at least one systemic symptom (fever, headache, body ache)
and one gastrointestinal symptom (diarrhea, cramps, vomiting, nausea), thus meeting presumptive
case criteria. Ill persons were a median of 15.5 years of age (range 7 to 66 years), and half were
female. Similar to other outbreaks described here, predominant symptoms included fever, head-
ache, diarrhea, and vomiting. Symptom onset occurred a median of 25 hours after the party.
Stool specimens collected initially from 6 attendees were negative for enteric pathogens; however,
L. monocytogenes serotype 1/2a was isolated from 6 of 8 additional specimens requested from ill
persons with presumptive cases. The isolates were indistinguishable by PFGE analysis.
Consuming a portion of 1 of 3 kinds of sandwiches served—a 6-foot-long half-turkey, half-
vegetable sandwich with pepper-jack cheese—was significantly associated with illness (RR = 5.6,
P < 0.002). In analyses addressing specific ingredients, consumption of turkey (RR undefined,
P < 0.0001) and pepper-jack cheese (RR = 6.3, P = 0.0002) were significantly associated with
illness. Consumption of pepper-jack cheese explained fewer cases, however, and only turkey
remained significantly associated with illness when controlling for consumption of each food item.
Leftover, home-refrigerated portions of the sandwich yielded the outbreak-associated subtype; a
sample of turkey from the sandwich, obtained 14 days after the party, contained Listeria spp. at
1.6 × 109 CFU/g. Inspection of the delicatessen revealed multiple retail food code violations,
including inadequate sanitation procedures and inadequate cold-holding temperatures in a walk-in
refrigerator. Although traceback efforts allowed identification of a processor, it was determined that
an in-plant investigation was unfeasible due to the lack of a specific lot number and the apparent
limited nature of the outbreak. Nonetheless, based upon investigation findings, the likely source of
this outbreak was contaminated turkey delicatessen meat.
FRESH, RAW-MILK CHEESE, SWEDEN

During 2001, health officials in Sweden were alerted to an increase in gastrointestinal illness among
residents of one county who had visited a summer farm prior to their illness [46,51]. Patient
interviews rapidly identified consumption of on-farm–produced dairy products, including fresh,
raw-milk cheeses and butter, as the likely source of illness. Health officials initiated active case-
finding and launched an investigation to determine the etiology, specific source, and evidence for
a dose–response effect between the amount of each food item consumed and development of illness.
Among a composite cohort consisting of 42 persons in 2 groups who had visited the farm and
50 additional community members identified by active case-finding efforts, 48 persons had illness
meeting the case definition. All had consumed a dairy product from the farm during the study
period and had developed 2 or more symptoms consistent with febrile gastroenteritis (fever, diarrhea,
vomiting, arthralgia, headache, or body pain) within the 2 weeks following consumption. Approx-
imately half of all persons were female, and their median age was 52 years (range 2 to 85 years).
Predominant symptoms included diarrhea (88%), fever (60%), and stomach cramps (54%), and
onset occurred a median of 31 hours following product consumption. Ill persons reported a symptom
duration of 1 to 15 days. One person who had been on immunosuppressive therapy was hospitalized
upon development of a joint abscess due to L. monocytogenes. Stool samples collected from ill
persons were negative for bacterial and viral pathogens, with the exception of the detection of
genetic markers for verotoxigenic E. coli (VTEC) in 6 of 35 specimens and enterotoxigenic E. coli
(ETEC) in 1 specimen. L. monocytogenes was isolated from 27 (84%) of 32 stool specimens
collected from persons whose illness met the case definition, compared to 9 (82%) of 11 specimens
from persons whose illness did not, indicating common exposure but varying clinical symptoms.
Results of the epidemiologic study indicated that consumption of raw-milk cheeses produced
from cow’s milk (RR = 2.23, 95% CI = 1.49–3.34) or cheese of an unknown type (RR = 2.23,
95% CI = 1.49–3.34) was significantly associated with illness. Although not statistically significant,
the attack rate increased as consumption of dairy products increased, suggesting a potential
dose–response effect. Samples of goat’s milk cheese, cow’s milk cheese, whey cheese, and butter
yielded L. monocytogenes serotype 1/2a at concentrations of 101 to 107 CFU/g. However, it is
difficult to estimate the dose consumed as some samples had been in home-storage for 1 to 2 weeks
before collection. Further isolation of high concentrations of coagulase-positive Staphylococcus
aureus and presumptive E. coli was indicative of compromised product quality. Notably, all clinical
isolates from ill persons, isolates from persons who did not have illness meeting the case definition,
and food L. monocytogenes isolates matched by restriction enzyme analysis (REA) and by PFGE.
The potential contribution of pathogenic E. coli these illnesses is uncertain.
Further investigation at the farm revealed significant food hygiene issues and a lack of process
controls. L. monocytogenes was isolated from environmental samples including a wooden bench
in the production area and brine used for cheese storage prior to sale [51]. Stool specimens from
5 of 19 lactating cows, along with milk samples from 10 of 14 goats and 1 fecal sample from a
goat yielded L. monocytogenes. Samples of milk from one goat collected twice over the following
month remained positive, leading to a diagnosis of subclinical mastitis. Isolates from the environ-
mental specimens, milk from the mastitic goat, and a subset of additional fecal and milk specimens
(number unspecified) matched the human clinical and food isolates by PFGE analysis.
This outbreak of febrile gastroenteritis was likely caused by consumption of on-farm–produced
dairy products contaminated with L. monocytogenes. The data support the milk of a goat with
subclinical mastitis as a source, although isolation of the outbreak-associated subtype from the
production environment demonstrated the likelihood of cross-contamination of other products. The
data also supported carriage of the outbreak-associated subtype by other animals whose milk was
used for production. The summer farm was subsequently closed to visitors. This outbreak contrib-
utes to the enormous body of evidence regarding the risks associated with consumption of raw
milk and raw milk products, and emphasizes the importance of enhanced food safety programs for
summer farms and other similar venues such that visitors can continue to enjoy these historical
cultural traditions.
CHEESE, JAPAN
Investigators recently reported the first identified outbreak caused by L. monocytogenes in Japan
[101]. This gastroenteritis outbreak was identified and investigated after routine sampling resulted
in isolation of high levels of L. monocytogenes serotype 1/2b from several soft and semi-hard
cheeses produced at a single facility. Further investigation revealed extensive contamination of the
processing environment. The same subtype, along with serotypes 4b and 1/2a, was also isolated
from samples collected throughout the facility. Following isolation of L. monocytogenes from the
cheese, investigators identified persons who had consumed it and collected fecal specimens for
screening, along with health information. L. monocytogenes was isolated from 60% of fecal
specimens, a proportion much higher than expected for fecal carriage [78]. Among persons from
whom L. monocytogenes was isolated, 38 reported a gastrointestinal or “cold-like” illness within
several days of cheese consumption, and 38% sought medical care. A subset of human clinical and
cheese isolates was further characterized by PFGE, ribotyping, mouse bioassay, cellular virulence
assays, and PCR-based subtyping. Human and cheese isolates showed >85% similarity by PFGE,
and were genetically similar according to other characterization methods.
CONTROL OF L. MONOCYTOGENES–ASSOCIATED FEBRILE GASTROENTERITIS

Strategies outlined for the continued reduction of invasive listeriosis would serve this goal well.
As described in the examples above, the sources and route of contamination often parallel scenarios
observed for invasive listeriosis outbreaks, with growth to high levels facilitated by subsequent
temperature abuse or long periods of storage. In addition to efforts to reduce the frequency and
level of L. monocytogenes contamination at the farm, producer, distribution, and retail levels,
prevention strategies should include educational programs focused on proper handling and storage
of products during preparation and prior to consumption. Such messages should target the general
population in addition to persons at higher risk for invasive listeriosis.
L. monocytogenes-associated gastroenteritis has been well characterized only recently. Out-
breaks are likely underrecognized because of a lack of awareness of this syndrome and because
stool samples are infrequently screened for L. monocytogenes. Healthcare workers and public health
officials should consider L. monocytogenes as a cause of febrile gastroenteritis, particularly in cases

where routine screening fails to identify an enteric pathogen. The factors leading to febrile gastro-
enteritis as opposed to invasive disease are currently poorly understood. As with invasive listeriosis,
a better understanding of factors resulting in development of this illness along with the disease
process may contribute to improved control and prevention strategies.
SURVEILLANCE OF LISTERIOSIS IN THE UNITED STATES

FOODBORNE OUTBREAK SURVEILLANCE
In the United States, the CDC has collected reports of foodborne outbreaks since 1973. These
reports comprise summary information concerning outbreaks including the number of illnesses,
hospitalizations, and deaths, the etiologic agent for illnesses, implicated food vehicles, and other
outbreak-associated factors. Officials in all states currently report foodborne outbreaks in this
surveillance system, now called the electronic Foodborne Outbreak Reporting System (eFORS).
This system was recently enhanced by efforts to ensure complete and accurate reports, including
verification of the number and content of reports with state officials and the implementation of
Internet-based data entry into a national database. The eFORS system receives between 1,200 and
1,500 outbreak reports annually.
Among 6,647 foodborne outbreaks reported to eFORS from 1998 to 2002, 2,168 had an
identified etiology; 11 were due to L. monocytogenes, including a total of 256 cases and 38 (14.8%)
deaths (CDC unpublished data). These deaths accounted for 48% of all deaths among outbreak-
associated cases reported. The number of cases in listeriosis outbreaks ranged from 2 to 101. Four
outbreaks occurred in multiple states. Four outbreak reports indicated delicatessen meats as the
implicated vehicle (two sliced turkey, one multiple delicatessen meats, and one unspecified deli-
catessen meat), three implicated hot dogs, one implicated deli sandwiches, one implicated pâté,
one implicated queso fresco (cheese), and one implicated potato salad.
Foodborne outbreak surveillance provides an opportunity to construct statistical models to
assess the burden of illnesses due specific food categories (i.e., poultry). These models incorporate
data from case surveillance to estimate the total number of illnesses associated with various foods,
as outbreak-related cases comprise only a fraction of the total burden. Investigators in the United
Kingdom have published one such model [1]. From 1996 to 2000, an estimated 221 cases and 78
deaths were attributed to listeriosis. Though the authors provided estimates of the total estimated
number of illness attributed to food categories due to all etiologies, they did not provide estimates
due to listeriosis specifically. Several groups in the United States and other countries are conducting
similar analyses; these efforts will undoubtedly enhance our knowledge of the burden of listeriosis
due to specific food categories.
SURVEILLANCE OF SPORADIC LISTERIOSIS

Although we have learned much about the epidemiology of listeriosis through outbreak investiga-
tion, timely, effective surveillance of listeriosis is critical to the overall goals of control and
prevention of this disease. Reliable surveillance data are critical in estimating the burden of
listeriosis, determining disease trends, guiding development of targeted intervention strategies,
providing a framework for their assessment, and developing risk assessment models used to guide
regulatory actions. Furthermore, determining the expected incidence of sporadic listeriosis in a
population through surveillance is critical for efficient outbreak detection and response, as both
local and diffuse outbreaks are often detected by an increase of cases over the baseline rate.
Prior to the mid-1980s, when the Los Angeles County outbreak solidified the public health
significance of foodborne listeriosis, the incidence of sporadic human disease in the United States
was poorly understood. Listeriosis was monitored exclusively by passive surveillance, which relies
upon voluntary reporting by physicians and clinical laboratories to state health officials, who then
voluntarily report to CDC [114], and by analysis of hospital discharge data [47]. The low sensitivity
of these methods likely greatly underestimated its true incidence in the population. The 1985
outbreak underscored the importance of improved surveillance for listeriosis in the United States.
An active surveillance program, for which public health officials routinely contacted personnel at
all clinical laboratories and acute-care hospitals in five states and Los Angeles County to ascertain
listeriosis cases, was initiated in 1986 in an effort to more precisely estimate the incidence of
laboratory-confirmed disease [68,133]. The resulting estimates are detailed in Chapter 4. In an
effort to improve nationwide reporting in the United States, listeriosis was made a nationally
notifiable disease (one for which routine and timely reporting is considered necessary for prevention
and control measures) in 2000. Although disease reporting is mandated only at the state level, and
laws and regulations regarding which diseases and conditions must be reported to public health
officials vary slightly from state to state, most currently require reporting of listeriosis. CDC
provides uniform case criteria to increase specificity and data comparability [7], and reports the
data weekly in its Morbidity and Mortality Weekly Report. Comprehensive annual summaries of
nationally notifiable diseases, including listeriosis, present data stratified by important risk markers
including geographic distribution, gender, age group, and race/ethnicity. The incidence and trends
in listeriosis, as estimated by this surveillance program, along with international listeriosis estimates,
are detailed in Chapter 4. Last, as discussed earlier in this chapter and in Chapter 9, participating
PulseNet laboratories routinely subtype L. monocytogenes isolates by PFGE to facilitate rapid
detection of clusters of listeriosis that may have a common source.
In an effort to address emerging infectious diseases in the United States, CDC launched the
Emerging Infections Program (EIP) in collaboration with selected state health departments, local
health departments, academic institutions, and additional partner agencies in 1994. The Foodborne
Diseases Active Surveillance Network (FoodNet), a collaboration among CDC, the USDA, FDA,
and the EIP sites, is the principle foodborne disease component of the EIP [111]. FoodNet’s
primary goals include (1) determining more precisely the burden of foodborne diseases in the
United States, (2) monitoring trends in foodborne diseases, (3) determining the proportion of
foodborne disease attributable to specific foods, and (4) developing and assessing interventions to
reduce the burden of foodborne illness. In 1996, FoodNet began active, laboratory-based surveil-
lance of selected foodborne diseases including listeriosis. As described for the active surveillance
program initiated in 1986, which was continued and expanded as a component of the EIP, public
health officials in FoodNet sites contact laboratory directors frequently to ascertain new cases of
laboratory-confirmed foodborne disease. As a result, the burden of specific foodborne diseases in
the United States can be more precisely estimated over time. Surveillance began in 5 geographically
diverse sites with a catchment population of 14.3 million people. By 2003, FoodNet had expanded
considerably to include 10 sites, more than 650 clinical laboratories, and a catchment population
of 41.9 million people (14.4% of the U.S. population) [15]. By 2004, the catchment population
had expanded to 44.5 million (15.1% of the U.S. population) (Figure 10.12). The FoodNet popu-
lation under surveillance is comparable to the U.S. population, with few limitations [80]. A
demographic comparison of the FoodNet population under surveillance and the U.S. population,
conducted in 2000, found little variation in age and gender distributions. However, among race/
ethnic groups, the Hispanic population in FoodNet was underrepresented at 6% compared to 12%
of the U.S. population as indicated by census data.
Figure 10.13, in which relative rates of listeriosis are shown in comparison with a 1996–1998
baseline estimate, summarizes trends in the incidence of listeriosis since the initiation of FoodNet
surveillance. The use of an average annual incidence for the 3 years (1996 to 1998) as the baseline
period provides the most stable, precise relative rate estimates [15]. To account for the increase in the
number of FoodNet sites, the corresponding increase in the population under surveillance and variations
in the incidence of listeriosis among sites, a main effects, log-linear Poisson regression (negative
binomial) model was used to estimate statistically significant changes in incidence. As shown in
346
FIGURE 10.12 Map showing Emerging Infections Program sites () participating in the Foodborne Diseases Active Surveillance Network, United
States, 2004. The population under surveillance is 44.5 million persons, representing 15.1% of the total population.
Figure 10.13, the relative rate (RR) of listeriosis among the FoodNet population declined significantly
by 45% from the 1996–1998 baseline period to 2002 (95% CI = 31%–57%, RR = 0.55). The relative
rate increased in 2003 compared to 2002 (RR = 0.71, 29% decrease from 1996 to 1998, 95% CI =
12%–42%). These data may indeed reflect a true increase in the incidence of listeriosis in 2003.
However, the widely publicized, multistate outbreak that occurred during late 2002 [70] may have
resulted in an increased awareness of listeriosis among health providers, leading to enhanced diagnosis
and reporting. This would, in turn, contribute to an apparent increase in incidence. Health officials
hypothesized that a similar phenomenon may have contributed to statistically significant geographic
differences in 1986 rates of perinatal listeriosis [68], estimated using active surveillance data collected
following the large, highly publicized 1985 outbreak in Los Angeles County. The rate in Los Angeles
County (24.3/100,000 live births) was over three times the combined rate in other surveillance areas.
As shown in Figure 10.13, the relative rate of listeriosis declined again in 2004 to rates similar to 2002
(RR = 0.60, 40% decrease from 1996 to 1998, 95% CI = 25% to 52%) [15]. The incidence of 0.27
cases/100,000 persons approaches the 2005 National Health Objective of 0.25 cases/100,000 persons.
Analyses of FoodNet data have revealed ethnic disparities in the incidence of listeriosis, which
correlate well with nationwide trends discussed in Chapter 4. Among 523 listeriosis cases reported
from 1996 to 2003 among individuals of known race/ethnicity, the incidence among Hispanics was
significantly higher than in non-Hispanics (RR = 1.7, 95% CI = 1.2–2.4) (56). Among Hispanic case-
patients, 58% were female and 63% were of childbearing age (15 to 39 years). These findings
correspond well with the findings from outbreak investigations in which Hispanics were overrepresented
among outbreak cases and infections were associated with the consumption of Hispanic-style cheeses.
The substantial overall decline in the incidence of listeriosis can be attributed to several factors.
The FDA and USDA have focused significant effort on regulatory measures, industry guidance,
and initiatives on L. monocytogenes, including FDA and USDA–FSIS’s 1989 zero-tolerance rulings
for ready-to-eat foods, the joint USDA–FSIS/FDA risk assessment focused on ready-to-eat foods,
and the USDA’s more recent completion of a risk assessment for deli meats [67]. In October 2003,
the USDA published an interim final rule (detailed in the discussion of meat and poultry product-
associated outbreaks) requiring processors to enhance and verify measures to reduce the prevalence
of L. monocytogenes in ready-to-eat products [8]. The food industry has responded with significant
2
Relative Rate
–29% (–42% –12%)

0.8
0.7
0.6
0.5 –40% (–52% –25%)

–45% (–57% –31%)
1996–1998 1999 2000 2001 2002 2003 2004 2005

Year
FIGURE 10.13 Relative rates of laboratory-diagnosed Listeria infections in humans compared with 1996–1998
baseline period, by year—Foodborne Diseases Active Surveillance Network, 1996–2004.
effort to develop strategies to reduce the levels of L. monocytogenes in the food processing
environment and prevent post-processing contamination of food items. For example, subsequent
to the 2003 USDA interim rule, a survey of more than 2,900 establishments producing ready-to-
eat meat and poultry products found that more than 87% had made improvements to L. monocy-
togenes controls [6]. Improvements in the timeliness of listeriosis outbreak detection, greatly
facilitated by the routine molecular characterization of L. monocytogenes human isolates, allows
public health and regulatory officials to investigate and control outbreaks more efficiently. Enhanced
consumer education efforts, such as the “Fight BAC” educational campaign (www.fightbac.org)
developed by the Partnership for Food Safety Education in conjunction with the President’s National
Food Safety Initiative and programs of the Food Safety Training and Education Alliance have likely
contributed to a reduction in listeriosis incidence as well.
FoodNet data have been used to provide the most accurate estimates of the incidence of listeriosis.
However, these and estimates derived from passive surveillance data must be interpreted with limi-
tations in mind. First, FoodNet captures exclusively laboratory-confirmed infections. A primary
outcome of listeriosis during pregnancy is fetal loss; however, bacterial cultures are not routinely
requested for spontaneously aborted fetuses or stillborn neonates. This results in reduced laboratory
diagnosis [19]. Thus, whereas FoodNet surveillance provides the most robust estimates available, the
true incidence of listeriosis in the United States is most certainly higher. Second, it is notable that the
Hispanic population is underrepresented in the population under surveillance by FoodNet. Although
it is clear that the incidence of listeriosis is disproportionately high among Hispanics compared to
other racial-ethnic groups, it is likely that FoodNet data underestimate the true incidence for this
population. This, in turn, would contribute to underestimation of listeriosis overall.
IMPROVED ESTIMATES OF THE BURDEN OF LISTERIOSIS IN THE UNITED STATES

In an ongoing effort to provide more accurate estimates of foodborne diseases including listeri-
osis, which are critical to guide prevention efforts and assess the effectiveness of food safety
regulations, Mead et al. reported foodborne disease estimates in 1999 that were derived and
validated using data from multiple sources [110]. Although L. monocytogenes causes only about
2,500 of the estimated 76,000,000 annual cases of food-related disease in the United States, it
is responsible for an estimated 500 deaths annually, or one-third of the deaths caused by known
foodborne pathogens. The hospitalization rate was estimated to be 92%, with a case fatality rate
of 20%.
Several data sources contributed to the listeriosis estimates. The annual number of food-related
cases was calculated using an average of the 1996–1997 FoodNet incidence applied to the total U.S.
population, and previous estimates resulting from a comparable sentinel surveillance system [143].
A multiplier of two was applied to account for underreporting based upon the assumption that the
severity of listeriosis results in medical attention for most cases and, in turn, a higher rate of diagnosis
and reporting as compared to a disease with less severity. Similarly, hospitalization rates were drawn
from FoodNet data indicating that nearly 90% of listeriosis cases result in hospitalization. The case
fatality rate was estimated using FoodNet data, previous estimates of the incidence of listeriosis in
the United States [143], and outbreak data. Accounting for the demonstrated potential for nosocomial
transmission [134], calculations were performed based upon the estimate that 99% of listeriosis
cases are transmitted by food. Although some assumptions were required for generation of these
estimates, such as those used to arrive at the multiplier accounting for underreporting, they are
generally accepted as the most precise estimates of the burden of listeriosis currently available.
L. monocytogenes is a foodborne pathogen of significant public health concern. Though rela-
tively rare compared to other foodborne pathogens, it is responsible for almost one-third of food-
borne disease-related deaths caused annually by known pathogens. Despite the challenges encoun-
tered during the investigation of listeriosis outbreaks, much has been learned from investigations
resulting in the successful implication of food vehicles. These implicated foods tend to be those
commercially prepared and consumed without cooking by the consumer. Environmental contami-
nation of the processing facilities, which can be sustained for prolonged periods, often leads to
contamination of the implicated processed foods.
The incidence of listeriosis has been decreasing in the United States and other countries. Results
of surveillance and successful outbreak investigations, as described in this chapter, have focused
regulatory and food industry actions to control food contamination. However, despite documented
progress, foodborne listeriosis outbreaks and sporadic cases leading to severe disease and death
continue to occur. Further efforts are required to enhance surveillance, outbreak identification,
investigation, regulatory interventions, industry controls, and consumer education to achieve food
safety objectives nationally and internationally.
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11 Incidence and Behavior

of Listeria monocytogenes
in Unfermented Dairy Products
Elliot T. Ryser
CONTENTS
Introduction ....................................................................................................................................357
Incidence of Listeria spp. in Unfermented Dairy Products ..........................................................358
Raw Cow’s Milk...................................................................................................................358
Raw Ewe’s and Goat’s Milk ................................................................................................366
Pasteurized Milk and Other Unfermented Dairy Products..................................................367
Behavior of L. monocytogenes in Unfermented Dairy Products ..................................................376
Raw Milk ..............................................................................................................................377
Pasteurized and Intensively Pasteurized Milk .....................................................................379
Autoclaved Milk, Cream, and Chocolate Milk....................................................................380
Sweetened Condensed and Evaporated Milk.......................................................................387
Ultrafiltered Milk..................................................................................................................388
Growth of L. monocytogenes in Mixed Cultures ..........................................................................388
Nonfluid Dairy Products ................................................................................................................391
Ice Cream..............................................................................................................................391
Butter ....................................................................................................................................392
Nonfat Dry Milk...................................................................................................................393
References ......................................................................................................................................394
INTRODUCTION
Recognition of raw milk as a potential source of Listeria monocytogenes led to speculation that
consumption of such milk was at least partly responsible for the previously described listeriosis
outbreak in post–World War II Germany. After this listeriosis epidemic, sporadic reports of indi-
viduals drinking raw milk, along with assurances that raw milk was being properly pasteurized,
virtually eliminated the threat of any further outbreaks of milkborne listeriosis. Consequently,
research in this area also decreased. However, in 1983, concerns about the possibility of milkborne
listeriosis were rekindled when consumption of pasteurized milk was epidemiologically linked to
an outbreak of listeriosis in Massachusetts. Two events, namely, publication of an article in the
New England Journal of Medicine detailing this outbreak in Massachusetts and a report in June of
1985 that as many as 300 people in California had acquired listeriosis after eating Mexican-style
cheese contaminated with L. monocytogenes, caused considerable concern in the United States
about the presence of Listeria in dairy products. This problem subsequently took on international
proportions with the 1987 report of another cheese-related outbreak in which consumption of tainted
357
Vacherin Mont d’Or soft-ripened cheese was directly linked to numerous cases of listeriosis in
Switzerland. Years later, one outbreak of Listeria gastroenteritis was traced to consumption of
contaminated chocolate milk in the United States [201]. Despite considerable progress, L. mono-
cytogenes outbreaks continue to plague the dairy industry, with soft cheeses, particularly the
Mexican and surface-ripened varieties, being responsible for continued sporadic listeriosis out-
breaks in North America and Europe.
In response to questions raised by milk producers, dairy processors, health officials, and the
general public, a plethora of work has been conducted worldwide since 1983 to determine the
incidence and behavior of L. monocytogenes in unfermented (raw milk, pasteurized milk, chocolate
milk, cream, butter, ice cream, other frozen dairy desserts), as well as fermented (cheese, yogurt,
cultured milk) dairy products. The incidence and behavior of L. monocytogenes in unfermented
dairy products will be dealt with in this chapter; similar information about fermented dairy products
appears in Chapter 12.
INCIDENCE OF LISTERIA SPP. IN UNFERMENTED DAIRY PRODUCTS

The dairy-related listeriosis outbreaks reported during the mid-1980s (see Chapter 10) prompted
scientists worldwide to determine the extent of Listeria contamination in raw milk and in pasteurized
dairy products including milk, ice cream, ice cream novelties, frozen desserts, nonfat dry milk, and
casein. Listeria monocytogenes can readily enter dairy processing facilities in the raw milk supply,
which can in turn lead to contamination of the factory environment. The occasional appearance of
listeriae in pasteurized dairy products nearly always has been associated with contamination of the
product after pasteurization. Thus, it is fitting to begin this discussion by examining the incidence
of Listeria spp. in raw milk, which is a major source of this bacterium in dairy factory environments.
RAW COW’S MILK

As you will recall from the discussion of animal listeriosis in Chapter 2, dairy cattle can intermit-
tently shed L. monocytogenes in their milk as a consequence of listerial mastitis, encephalitis, or
a Listeria-related abortion. Although milk from animals showing obvious signs of listeriosis is
unlikely to reach consumers, the scientific literature contains numerous accounts in which mildly
infected and apparently healthy dairy cattle, sheep, and goats have shed L. monocytogenes inter-
mittently in their milk for many months. Thus, it appears that such asymptomatic carriers of listeriae
pose the greatest threat to public health.
The 1983 listeriosis outbreak in Massachusetts that was supposedly associated with drinking
a particular brand of pasteurized milk raised numerous milk safety questions. The well-publicized
outbreak of 1985 in which consumption of contaminated Mexican-style cheese was directly linked
to at least 40 deaths in California prompted additional concerns about the safety of dairy products
manufactured in the United States. Because raw milk is a potential source of L. monocytogenes,
recalls of Listeria-contaminated pasteurized dairy products (i.e., milk, chocolate milk, ice cream)
and imported soft-ripened cheeses prompted more than 80 surveys worldwide to determine the
extent of Listeria contamination in raw milk. Results of these surveys, which will now be described
in some detail, have been summarized in Table 11.1 and Table 11.2.
The first large-scale survey of raw milk for Listeria spp. was prompted by the 1983 listeriosis
outbreak in Massachusetts [127]. During the 3-week period immediately following the outbreak,
Hayes et al. [151] examined 121 raw milk samples collected from milk trucks (40 samples),
milk cooperatives (72 samples), and bulk tanks from four farms on which bovine listeriosis was
diagnosed (9 samples), as well as 14 milk socks used to remove debris but not leukocytes from
milk. All samples were analyzed for L. monocytogenes using a multiple two-stage enrichment
procedure. Although investigators at the US Centers for Disease Control and Prevention (CDC)
isolated the epidemic serotype along with other serotypes of L. monocytogenes from 15 of 121 (12.4%)
Incidence and Behavior of Listeria monocytogenes in Unfermented Dairy Products 359
TABLE 11.1
Incidence of Listeria spp. in Raw Milk Produced in the United States and Canada
Number Number of Positive Samples (%)

Location of Samples L. monocytogenes L. innocua L. welshimeri Others Reference
United States
Northwest 948 56 (5.9) ND ND ND 186
West 176 7 (4.0) ND ND ND 236
Midwest 361 18 (5.0) ND ND ND 236
Northeast 363 32 (8.8) ND ND ND 236
Southeast 61 6 (9.8) ND ND ND 236
California 200 14 (7.0) 19 (9.5) 0 0 168
100 0 4 (4.0) 0 1 (1.0)a 168
Massachusetts 121 15 (12.4) ND ND ND 151
Massachusetts, Vermont 939 15 (1.6) ND ND ND 101
Minnesota 300 9 (3.0) 77 (25.7) 5 (1.7) 0 171
84 0 6 (7.1) 1 (1.2) 0 193
Nebraska 200 8 (4.0) 10 (5.0) 0 0 165
South Dakota /Minnesota 131 12 (9.2) ND ND ND 158
Ohio, Kentucky, 350 13 (3.7) 27 (7.7) 6 (1.5) 3 (0.9)a 168
and Indiana
Pennsylvania 2511 79 (3.1) ND ND ND 102
Tennessee 292 12 (4.1) ND ND ND 210
Wisconsin 50 0 ND ND ND 103
55 0 0 0 0 237
Total 7227 288 (4.0) 143 (11.1) 12 (0.9) 4 (0.3)
Canada
Alberta 426 8 (1.9) ND ND ND 123
252 4 (1.6) ND ND ND 235
Manitoba 256 4 (1.6) ND ND ND 93
Ontario 1720 47 (2.7) ND ND ND 227
Ontario 445 6 (1.3) 43 (9.7) 6 (1.3) 0 112
315 17 (5.4) 26 (8.2) 1 (0.3) 0 223
Total 3414 86 (2.5) 69 (9.1) 7 (0.9) 0 (0)
Note: ND = Not determined (omitted from total).

a Two L. ivanovii and one L. seeligeri.
(including 1 of 9 bulk tank samples) [150] and 2 of 14 (14%) raw milk and milk sock samples,
respectively, the epidemic phage type was never detected.
Between October 1984 and August 1985, US Food and Drug Administration (FDA) officials
surveyed 650 raw milk samples that were collected from bulk tanks in Massachusetts, Vermont,
California, and the tristate area of Kentucky, Ohio, and Indiana. The samples were examined for
Listeria spp. using the original FDA method [168]. Low levels of various Listeria spp., including
L. monocytogenes, were detected in raw milk samples obtained from all states except California.
Overall, 82 of 650 (12.6%) samples contained Listeria spp., with L. monocytogenes being found
in 27 of 650 (4.2%) samples. Of the 27 L. monocytogenes strains isolated from raw milk, 16 were
serotype 1, 10 were serotype 4, and 1 was nontypeable. In addition, only 2 of the 27 L. monocy-
togenes strains proved to be nonpathogenic to mice, and both were of serotype 4.
In 1988, Donnelly et al. [101] used flow cytometry to analyze 939 raw milk samples obtained
from 54 farms in California. Unlike the FDA study just described [168], string samples (milk pooled
TABLE 11.2
Incidence of Listeria spp. in Raw Cow’s Milk Collected outside North America
Number of Positive Samples (%)
Location Number of Samples L. monocytogenes L. innocua L. welshimeri Others Reference
Europe
Austria 201 0 2 (1.0) 0 2 (1.0) 107
Belgium 143 9 (6.3) ND ND ND 97
Czechoslovakia 177 6 (3.3) ND ND ND 181
123 4 (3.3) ND ND ND 182
Denmark 1,227,053 278 (0.02) ND ND ND 200
Finland 256 13 (5.1) ND ND ND 200
59 1 (1.7) ND ND ND 200
France 1787 60 (3.4) ND ND ND 180
1409 85 (6.0) ND ND ND 45
561 21 (3.8) ND ND ND 51
337 14 (4.2) 5 (1.5) 0 0 139, 140
51 10 (19.6) 10 (19.6) 18 (35.3) 9 (17.6) 139, 140
Germany 635 2 (0.3) ND ND ND 133, 232
Hungary 80 3 (3.8) ND ND ND 207
50 2 (4.0) ND ND ND 207
Ireland 589 29 (4.9) 20 (3.4) ND ND 206
50 4 (8.0) ND ND ND 157
Italy 290 8 (2.8) 16 (5.6) 0 2 (0.7) 96
142 0 1 (0.6) 0 0 226
98 2 (2.0) ND ND ND 164
85 0 0 0 0 136
50 0 1 (2.0) 0 0 234
40 0 0 0 0 175
32 0 2 (6.3) ND ND 130
The Netherlands 137 6 (4.4) ND ND ND 78
Poland 134 2 (1.5) ND ND ND 161
81 6 (7.4) ND ND ND 211
Portugal 54 3 (5.6) ND ND ND 146
Spain 340 23 (6.8) 24 (7.0) 1 (0.3) 6 (1.8) 240
774 28 (3.6) 21 (2.7) 0 0 135
Sweden 294 3 (1.0) 7 (2.3) 0 0 241
Switzerland 4046 14 (0.4) ND ND ND 73
340 2 (0.6) ND ND ND 73
Turkey 77 14 (18.2) ND ND ND 220
United Kingdom
Great Britain 350 13 (3.6) ND ND ND 137
England/Wales 2009 102 (5.1) ND ND ND 188
361 13 (3.6) ND ND ND 145
Scotland 640 90 (14.1) ND ND ND 125
560 14 (2.5) 7 (1.3) 0 1 (0.2) 124
540 14 (2.6) ND ND ND 124
Northern Ireland 176 27 (15.3) 18 (10.2) 0 5 (2.8)a 149
113 6 (5.3) ND ND ND 138
Total 18,271 653 (3.6) 134 (3.4) 19 (0.6) 35 (1.0)
(continued)

Incidence of Listeria spp. in Raw Cow’s Milk Collected outside North America
Location Number of Samples L. monocytogenes L. innocua L. welshimeri Others Reference
Elsewhere
Argentina 208 1 (0.5) 5 (2.4) 1 (0.5) 0 162
Australia 169 1 (0.6) ND ND ND 110
150 0 4 (2.7) 0 0 154
Brazil 220 11 (4.8) 21 (9.5) 2 (0.9) 1 (0.4) 185
20 0 ND ND ND 83
12 1 (8.3) 2 (16.7) 0 0 129
Costa Rica 220 0 10 (4.5) 3 (1.4) 1 (0.5)b 70
Egypt 236 7 (3.0) 20 (8.5) 0 0 108
Iran 190 4 (2.1) ND ND ND 205
Japan 362 3 (0.8) ND ND ND 244
120 0 ND ND ND 231
150 6 (0.4) 2 (1.3) 0 0 219
Jordan 170 2 (7.1) ND ND ND 121
Korea 50 1 (2.0) ND ND ND 147
45 2 (4.4) ND ND ND 74
Malaysia 930 18 (1.9) 20 (2.1) 6 (0.6) 0 85
Mexico 1300 162 (12.5) 9 (0.7) 0 129 (9.9) 238
100 0 7 (7.0) 2 (2.0) 0 169
Morocco 30 3 (10.0) ND ND ND 109
New Zealand 71 0 10 (14.1) 1 (1.4) 7 (9.9) 229
South Africa 982 67 (6.8) ND ND ND 243
Taiwan 80 5 (6.3) ND ND ND 84
Total 5815 304 (5.2) 110 (3.1) 15 (0.4) 138 (3.8)
Note: ND = Not determined (omitted from total).

a L. seeligeri.
b L. grayi.
from 25 to 40 cows), combination samples (milk pooled from 200 cows), and samples of raw milk
from bulk tanks were tested for L. monocytogenes. Using this method, L. monocytogenes was
detected in 15 of 939 (1.5%) samples.
Researchers in Minnesota [193] and Wisconsin [103,237] failed to detect L. monocytogenes in
raw milk during three small surveys. However, the pathogen was found in 4.0 and 2.8% of raw
milk samples obtained from bulk storage tanks and tank trucks in Nebraska [166] and Pennsylvania
[102], respectively, with approximately equal numbers of L. monocytogenes isolates being classified
as serotype 1, 4, or nontypeable (non–serotype 1 or 4) in the latter study. More recent findings
indicate a relatively stable incidence for L. monocytogenes in bulk tank raw milk, with 3.0 and
4.1% of such samples from Minnesota [171] and Tennessee [210], respectively, being positive. In
the Minnesota survey, Listeria-positive samples also tended to have higher bacterial and somatic
cell counts, which are indicative of less stringent sanitation and mastitis control practices.
As part of the National Animal Health Monitoring System, a large-scale national survey was
conducted in 2002 during which 861 raw milk bulk tank samples were analyzed for various
microbial contaminants including L. monocytogenes [236]. The incidence of L. monocytogenes
contamination was 4.0, 5.0, 8.8, and 9.8% for milk samples collected in the West, Midwest,
Northeast, and Southeast (average of 6.5%), respectively, with 93% of the isolates belonging to
serotypes 1/2a, 1/2b, and 4b. One year earlier, Muraoka et al. [186] reported an overall incidence
rate of 5.9% for L. monocytogenes in bulk tank samples from the Pacific Northwest with strains
of serotype 1/2a predominating. In one additional survey [149], milk filters rather than samples
of bulk tank milk were collected from 404 farms throughout New York State from April 1998 to
March 1999 and assessed for L. monocytogenes. Fifty-one (12.6%) of these milk filters through
which an average of 1351 kg of milk had passed yielded L. monocytogenes with this substantially
higher contamination rate compared to bulk tank milk related to presence of debris in the filters
at the time of sampling. Additional observations from this study included regional differences in
percentage of positive filters (6.5 to 19.0%), with the highest contamination rate found in central
New York State and a higher isolation rate during spring (18%) compared to summer, fall, and
winter (6–8%).
The overall findings from Table 11.1 indicate that L. monocytogenes was present in 0–12.4%
of the US raw milk samples examined. When the results are averaged, 4.0% of all raw milk processed
in the United States can be expected to contain low levels (i.e., <10 CFU/mL) of L. monocytogenes
at any given time, with the incidence of L. innocua being appreciably higher at 11.1%. Hence, it
is imperative that all milk processors take special precautions to prevent the spread of L. monocy-
togenes from raw milk to production, packaging, and other sensitive areas within the factory.
Turning to the Canadian raw milk supply (Table 11.1), Farber et al. [112] and Slade et al. [223],
respectively, isolated L. monocytogenes from 6 of 445 (1.3%) and 17 of 315 (5.4%) raw milk
samples obtained from bulk tanks located throughout the province of Ontario. Davidson et al. [93]
also reported that a similar percentage of raw milk samples collected from four local dairies and
48 farms in Manitoba contained L. monocytogenes. In the study by Slade et al. [223], 14 of 17
(82%) L. monocytogenes isolates belonged to serotype 1, the remaining three strains being classified
as serotype 4. Although subsequent work [126] demonstrated that none of these L. monocytogenes
strains harbored plasmid DNA, 8 of 22 (36%) L. innocua strains did carry plasmids ranging in size
from 10 to 44 MDa. Variable plasmid profiles among these isolates further suggest that contami-
nation of raw milk on the farm is an ongoing process. Whereas these authors did not quantitate
L. monocytogenes in any of the samples examined, Slade and Collins-Thompson [222] reported that
positive raw milks from bulk tanks in southwestern Ontario always contained <5 L. monocytogenes
CFU/mL when samples were analyzed by direct plating and a most probable number (MPN)
enrichment procedure. Thus, the positive raw milk samples encountered in three Canadian studies
likely contained only low levels of L. monocytogenes. In two subsequent surveys, L. monocytogenes
was identified in 1.6 [235] and 1.9% [123] of all raw milk bulk tank samples examined in the
province of Alberta. However, both surveys also indicated substantially higher contamination rates
for commingled raw milk before processing with milk in 5 of 72 (6.9%) tank trucks [123] and in
4 of 15 (26.6%) milk silos [235] testing positive for L. monocytogenes. Finally, in the largest
Canadian survey of raw milk for pathogens [227], L. monocytogenes was identified in 2.7% of
1720 bulk tank samples collected from dairy farms in Ontario. More importantly, these same authors
estimated that the likelihood of L. monocytogenes contamination would increase to 8.0, 12.9, and
24.1% from the pooling of 3, 5, and 10 bulk tank samples, respectively, during multiple pickups.
Consequently, all dairy facilities can expect to have low levels of L. monocytogenes present in the
incoming raw milk. As was true for the three U.S. surveys [168] just discussed, L. innocua also
was the most common Listeria sp. isolated from Canadian raw milk, with 8.2 and 9.7% of the
samples being reported as positive. Overall, 2.5 and 9.1% of all Canadian raw milk samples
contained L. monocytogenes and L. innocua, respectively, as compared to 4.0 and 11.1% of raw
milk samples examined in the United States. Thus, the incidence of listeriae in raw milk from both
countries appears to be similar.
Public concern and economic hardships brought about by several recalls of French Brie cheese
imported into the United States prompted French scientists to begin surveying raw milk for
L. monocytogenes. Results from three such surveys [45,51,139] (see Table 11.2) conducted between
1986 and 1988 indicated that 85 of 1409 (6.0%), 21 of 561 (3.8%), and 14 of 337 (4.2%) raw milk
samples from French bulk tanks were positive for L. monocytogenes, with L. innocua being detected
in about one-third as many samples in the latter study [139]. During January of 1986, 51 raw milk
samples were submitted to the French Central Laboratory of Food Hygiene. Both L. monocytogenes
and L. innocua were found in 19.6% of these samples. The unusually high incidence of listeriae
observed in this survey, as compared to other studies described thus far, may be the result of nonrandom
sampling or seasonal variations, the latter of which will be discussed shortly. Meyer-Broseta et al.
[180] sampled 1787 raw milk tankers from 1997 to 2001 and reported an L. monocytogenes
prevalence rate of 2.4 to 4.4% (average of 3.4%). This milk originated from 50 different dairy
farms with up to six farms positive for L. monocytogenes during any given month. Levels of
L. monocytogenes in positive bulk tank samples were low and never exceeded 210 CFU/mL.
Frequent isolation of L. monocytogenes from dairy products prompted additional surveys of the raw
milk supply throughout Europe and later elsewhere, the primary focus being given to countries with
sizeable dairy industries (see Table 10.2). Results from two comprehensive year-long surveys in the
United Kingdom [137] and Scotland [124] revealed an incidence of L. monocytogenes in raw milk
similar to that observed in the United States and Canada, with actual Listeria populations in positive
samples also estimated to be extremely low. As in North America, L. monocytogenes strains belonging
to serotype 1/2 appear to predominate in European raw milk [124,125,145,212]. Although four
subsequent surveys from the United Kingdom yielded similar findings [124,125,145,188], L. mono-
cytogenes contamination rates of 14 to 15% have been reported from both Scotland [125] and
Northern Ireland [148], suggesting considerable local variability. Harvey and Gilmour [148] also
found that 33% of raw milk samples collected from processing centers harbored L. monocytogenes,
which again emphasizes the impact of commingling milk. However, levels of listeriae in such milk
again appear to be quite low, with actual numbers of L. monocytogenes typically being <10 CFU/mL
in positive samples from Scotland [125].
In 1987, Beckers et al. [78] reported culturing L. monocytogenes from 6 of 137 (4.4%) raw
milk samples obtained from farms in the Utrecht region of The Netherlands. As was true for raw
milk tested in the United States and Canada, milk samples from The Netherlands again contained
<100 L. monocytogenes CFU/mL. Similar findings have been reported from most other European
surveys, with 3–5% of raw milk samples harboring low levels of L. monocytogenes. However, two
nationwide surveys conducted in Switzerland during and after the outbreak involving Vacherin
Mont d’Or cheese indicated a far lower incidence, with only 0.4 and 0.6% of the raw milk samples
being positive for L. monocytogenes [73]. Several years earlier, Terplan [232] detected L. monocy-
togenes in only 2 of 635 (0.3%) raw milk samples obtained from farm bulk tanks in Würtemburg,
Germany. Subsequent attempts to isolate this pathogen from 448 quarter-milk and 30 separator
sludge samples failed, along with attempts to culture this organism from raw milk samples obtained
from tank trucks and storage tanks. Findings from an exhaustive survey in Denmark of over
1 million raw milk samples demonstrated an L. monocytogenes contamination rate of only 0.2%,
with several additional small-scale Italian surveys yielding negative results. Thus, when the Danish
results are excluded, 3.6% of all European raw milk would be expected to contain low levels of
L. monocytogenes as compared to 3.3% of the raw milk produced in North America with these
contamination rates having remained relatively unchanged since the 1980s.
In response to the aforementioned surveys and continued sporadic outbreaks of listeriosis
traced to consumption of soft and surface-ripened cheeses, investigators in Australia, New Zealand,
the Middle East, South America, South Africa, and the Orient also have begun assessing the
incidence of L. monocytogenes contamination in their own raw milk supplies. In the first of these
surveys, workers in New Zealand [229] collected and analyzed 71 raw milk bulk tank samples
between August 1986 and March 1987 for listeriae using both warm and cold enrichment.
Although L. monocytogenes was apparently absent from this milk as well as from milk examined
in a later Australian survey, isolation of other Listeria spp. from these samples strongly suggests
that such milk is unlikely to be completely free of L. monocytogenes. Similar negative findings
have been obtained from surveys conducted in Brazil [83] and Costa Rica [70], with Arias et al.
[70] also citing another survey in which 20% of hand-milked samples harbored L. monocytogenes.
According to Vasquez-Salinas [238], 162 of 1300 raw milk samples obtained from four different
dairy farms near Mexico City, Mexico, yielded L. monocytogenes. These findings support current
safety concerns regarding the consumption of soft Hispanic-style cheeses prepared from raw milk.
Working in Japan, Takai et al. [231] reported the absence of Listeria spp. from 120 raw milk
samples, whereas L. monocytogenes was recovered from 6 of 150 (4.0%) and 3 of 362 samples in
subsequent Japanese surveys [219,244]. Three more recent surveys have shown L. monocytogenes
contamination rates of 2.0–4.4 [74,147] and 1.9% [85] for raw milk sampled in Korea and Malaysia,
respectively. Given these findings, and additional reports of L. monocytogenes in raw milk from
Egypt [1,108], Jordan [121], Iran [205], Turkey [143,220], Morocco [109], and South Africa [243],
it is now clear that milkborne listeriosis constitutes a worldwide threat. However, confirmation of
dairy-related listeriosis cases in such developing countries will likely remain very difficult given
their lack of resources and understandable preoccupation with far more immediate health concerns.
Examination of data summarized in Table 10.1 and Table 10.2 indicates that 4.0, 2.5, 3.6, and 5.2%
of the raw milk produced in the United States, Canada, Europe, and elsewhere, respectively, can
be expected to contain low levels of L. monocytogenes, with similar contamination rates also likely
occurring in other parts of the world. Except for the European samples, L. innocua was isolated
from raw milk more frequently than L. monocytogenes, with the former found in 11.1, 9.1, 3.4,
and 3.1% of the samples analyzed for all Listeria spp. in the United States, Canada, Europe, and
elsewhere, respectively.
Although L. innocua is nonpathogenic, isolation of this organism and other listeriae from dairy
products and dairy processing facilities is taken very seriously in the United States, because
nonpathogenic listeriae and L. monocytogenes are assumed to occur in similar environmental niches.
Use of L. innocua as a potential indicator of L. monocytogenes in the United States and elsewhere
is supported by data from the raw milk surveys just described (see Table 10.1) in that isolation of
listeriae other than L. monocytogenes from dairy products and processing facilities suggests that
the factory environment may be contaminated with raw milk that can be expected to contain L.
monocytogenes 3–4% of the time. Although long theorized, the spread of identical L. monocytogenes
strains from the farm environment into the raw milk supply and ultimately to dairy processing
facilities now has been confirmed using various strain-specific typing methods, including restriction
fragment length polymorphism [148] and automated ribotyping [71].
In addition to assessing the general incidence of listeriae in raw milk, several of the surveys
just described also dealt with seasonal variations in the incidence of L. monocytogenes and
L. innocua in raw milk produced in the United States [102,165,168] and Canada [112]. However,
because all the aforementioned studies differ in numbers and sizes of samples analyzed as well as
the Listeria isolation procedures employed, it is difficult to make any definitive statement concerning
the seasonal occurrence of Listeria in raw milk [124,223]. Nonetheless, several distinct trends can
be observed from selected data in Figure 10.1. First, the overall incidence of L. monocytogenes in
raw milk was highest in spring (5.8%), followed by winter (4.5%), fall (2.8%), and summer (2.5%).
Second, a somewhat similar seasonal variation also can be observed for the incidence of L. innocua
in raw milk, with the highest overall percentage of positive samples again occurring in winter
(11.7%), followed by spring (10.2%), summer (6.0%), and fall (4.2%). These findings again suggest
that L. innocua can be used as a potential indicator for the presence of L. monocytogenes.
Although not fully understood, current herd management and feeding practices may be at least
partly responsible for seasonal differences observed in isolation rates for L. monocytogenes and
possibly L. innocua. During cold winter months, silage comprises a major component of the diet.
While investigating one listeriosis outbreak, Donnelly [99] observed that 8 of 44 Holstein cows
fed Listeria-contaminated silage shed the organism in their milk. Furthermore, milk from these
animals was free of L. monocytogenes 1 month after feeding of contaminated silage ceased.
Ruminants that ingest contaminated silage may either succumb to infection or carry L. monocytogenes
A = Massachusetts, Vermont NS= Not Sample

B = Ohio, Kentucky, Indiana LA= Listeria Absent
C = Pennsylvania = L. monocytogenes
20 D = Nebraska = L. innocua
E = Ontario, Canada
F = England/Wales
16
Positive Samples (%)
12
NS NS NS LA
A B C D E F A BC D E F A B CD E F A BC D E F
Spring Summer Fall Winter
FIGURE 11.1 Seasonal variation in the incidence of L. monocytogenes and L. innocua in raw milk from the
United States (from Doores, S. and J. Amelang. 1990. Personal communication; Hayes, P. S., J.C. Feeley,
L.M. Graves, G.W. Ajello, and D.W. Fleming. 1986. Isolation of Listeria monocytogenes from raw milk. Appl.
Environ. Microbiol. 51: 438–440; Liewen, M.B. and M.W. Plautz. 1988. Occurrence of Listeria monocytogenes
in raw milk in Nebraska. J. Food Prot. 51: 840–841; Lovett, J., D.W. Francis, and J.M. Hunt. 1987. Listeria
monocytogenes in raw milk: detection, incidence, and pathogenicity. J. Food Prot. 50: 188–192), Canada (from
Farber, J.M., G.W. Sanders, and S.A. Malcom. 1988. The presence of Listeria spp. in raw milk in Ontario.
Can. J. Microbiol. 34: 95–100), and England/Wales (from O’Donnell, E.T. 1995. The incidence of Salmonella
and Listeria in raw milk from farm bulk tanks in England and Wales. J. Soc. Dairy Technol. 48: 25–29).
asymptomatically; however, if the animal lives, the organism can be shed for many months in feces
and in milk from lactating animals. Extended survival of L. monocytogenes in fecal material, soil,
and grass can perpetuate the infectious cycle shown in Figure 11.2, particularly when animals are
wintered in cramped quarters. Once dairy cattle resume grazing on pastures during late spring,
summer, and early fall, L. monocytogenes becomes dispersed over a wide area, which, in turn,
weakens the infectious cycle by decreasing the likelihood that animals will come in contact with
contaminated material.
Survival and/or growth

of L. monocytogenes in
silage
Ingestion
Survival in
by ruminants
soil and grass
Winter
confinement
L. monocytogenes
excreted in feces
L. monocytogenes
secreted in milk
FIGURE 11.2 Infective cycle for maintaining L. monocytogenes in ruminants.

Seasonal differences in the incidence of Listeria spp. in raw milk also may be related to breeding
practices. Dairy cattle typically bear their young in late winter or early spring. During winter
gestation, dairy cattle develop a weakened immune system as a direct result of pregnancy, which,
in turn, makes these animals more susceptible to listerial infections and abortions. These events
can then culminate in the shedding of L. monocytogenes in milk and fecal material. Increased
environmental stress and changes in habitat that occur during winter, along with increased diffi-
culties in providing proper herd hygiene, all can serve to decrease the natural defense system in
dairy cattle, which again increases the likelihood of listerial infections. Once an asymptomatic
animal begins shedding L. monocytogenes in feces, the organism is likely to spread quickly to other
animals that are housed in close proximity to the shedder. In this way, confinement of dairy cattle
may play an important role in increasing the number of animals that shed L. monocytogenes in
their milk during late winter and early spring.
RAW EWE’S AND GOAT’S MILK

Surveys in Europe, Australia, and the United States also have demonstrated that raw milk from
ewes and goats can occasionally contain L. monocytogenes (Table 11.3) with incidence rates ranging
from 0 to 3.0% (average of 1.6%) and 0 to 4.0% (average 2.4%), respectively. These contamination
levels, and that reported in India for buffalo milk, are similar to those seen for cow’s milk. Working
in Vermont, Abou-Eleinin et al. (2) recovered Listeria spp. from 35 of 445 (7.9%) bulk tank samples
of raw goat’s milk using three different enrichment methods. Seventeen samples contained L.
monocytogenes and 8 contained both L. monocytogenes and L. innocua. Listeria contamination
TABLE 11.3
Incidence of L. monocytogenes in Raw Ewe’s and Goat’s Milk
Type of Milk Location Number of Samples Number of Samples Positive (%) Reference
Ewe England/Wales 56 (1.8) 145

England/Wales 26 0 167
India 23 0 76
Italy 40 0 81
Italy 34 0 88
Portugal 119 2 (1.7) 146
Spain 1052 23 (2.2)a 208
Spain 202 6 (3.0) 240
Turkey 302 0 91
Total 1854 32 (1.7)
Goat United States 450 17 (3.8)b 2
England/Wales 480 4 (0.8) 145
England/Wales 100 0 167
Italy 60 0 129
India 64 1 (1.6) 76
Italy 24 0 81
Portugal 39 0 146
Portugal 25 1 (4.0) 92
Spain 1445 37 (2.6) 134
Total 2756 61 (2.2)
Buffalo India 64 4 (6.3) 75
a
Also 21 L. innocua, 4 L. welshimeri, 3 L. seeligeri, 3 L. grayi, and 2 L. ivanovii.
b
Eight L. monocytogenes and L. innocua, and twenty-six L. innocua.
rates were higher in winter (14.3%) and spring (10.4%) than in fall (5.3%) and summer (0.9%),
with similar observations reported for cow’s milk. Overall, 62.6 and 37.4% of those L. monocyto-
genes that were further characterized belonged to serovars 1 and 4, respectively. Automated ribotyp-
ing of selected isolates also indicated five distinct ribotypes, two clinically important ribotypes of
which were eventually traced back to the farm environment.
PASTEURIZED MILK AND OTHER UNFERMENTED DAIRY PRODUCTS

The dairy industry has long been considered as the most regulated food industry in the United
States. The FDA was given responsibility under the Food, Drug and Cosmetic Act and the Public
Health Service Act to assure the public that this country’s milk supply is both uniformly safe and
wholesome. Dairy sanitation laws and regulations, including microbiological criteria for some dairy
products, enforced by the FDA and state agencies are based almost exclusively on the Public Health
Service/FDA Grade A Pasteurized Milk Ordinance.
In the United States, pasteurized milk and other unfermented dairy products prepared from
pasteurized milk, including ice cream, butter, and nonfat dry milk, generally have earned a reputation
for being both safe and nutritious, with dairy products accounting for <1.5% of all foodborne
illnesses [77]. Pasteurized milk is responsible for about 5% of all reported dairy-related illnesses
[178]. Despite these impressive findings, public confidence in the safety of pasteurized milk began
to erode in July 1982 following an outbreak of yersiniosis in Tennessee, Arkansas, and Mississippi
[230] and again in 1983 when the CDC claimed that consumption of pasteurized milk was respon-
sible for 49 cases of listeriosis in Massachusetts. In 1985, the dairy industry was dealt another blow
when at least 16,000 culture-confirmed cases of salmonellosis were associated with drinking a
particular brand of pasteurized milk produced in the Chicago area [216].
These three outbreaks, along with the previously discussed listeriosis outbreak in California
linked to consumption of contaminated Mexican-style cheese, prompted the FDA to take corrective
action in the form of a large-scale testing program commonly referred to as the FDA Dairy Initiative
Program [159] (Figure 11.3). This program, begun in April of 1986 in cooperation with individual
June to August, 1983: March to April, 1985: January to June, 1985:

Listeriosis outbreak in Salmonellosis outbreak Listeriosis outbreak in
Massachusetts associated in Chicago linked to California linked to
with pasteurized milk pasteurized milk Mexican-style cheese
April 1986: Begin dairy iniciatives. Portion of IMS and non-IMS

inventory sampled for Listeria, Salmonella, Yersinia and
Campylobacter. Plant inspections in cooperation with
state agencies. 1174 samples analyzed during fiscal 1986.
October 1986: Continue sampling except omit Campylobacter.

1444 samples analyzed during fiscal 1987.
October 1987: Continue sampling remainder of inventory and the

following composite samples – Fluid milk: Listeria,
Salmonella, and Staphylococcus aureus.
FIGURE 11.3 FDA Dairy Initiatives Program. IMS, interstate milk shipment. (Adapted from Kozak, J.J.
1986. FDA’s dairy program initiatives. Dairy Food Sanit. 6: 184–185.)
state agencies and members of the National Conference on Interstate Milk Shipments, was designed
to examine every interstate milk shipment (IMS) pasteurization facility in the United States for
potential safety problems related to pasteurization, postpasteurization contamination, cleaning and
sanitizing regimens, equipment maintenance, and educational/training programs for dairy factory
personnel. As part of the FDA Dairy Initiative Program, the agency also established the Microbi-
ological Surveillance Program, which was designed to detect L. monocytogenes, Salmonella spp.,
Yersinia enterocolitica, Campylobacter jejuni, and C. coli (Campylobacter omitted in 1987) as well
as Staphylococcus aureus, which was added in 1987 for dry and fluid milk [18]. Dairy products
tested under this program included fluid milk, nonfat dry milk, cream, butter, ice cream, ice milk,
and other dairy commodities over which the FDA has jurisdiction. Analysis of cheeses (except cottage
cheese) was covered under a series of separate programs, which will be discussed in Chapter 12
concerning fermented dairy products. Under the provisions of this program, FDA inspectors col-
lected 30 retail-sized containers of as many as five different products available from dairy factories
at the time of inspection. Duplicate 25-g or 25-mL samples obtained after combining 30 retail-
sized samples per product were then analyzed for L. monocytogenes and other listeriae using the
original and later versions of the FDA procedure.
Because raw milk containing L. monocytogenes will periodically enter virtually every dairy
factory in the United States, it is logical to assume that finished products also may become
contaminated with this pathogen. During the first 2 years of the FDA Dairy Initiative Microbio-
logical Surveillance Program (Table 11.4), L. monocytogenes was isolated from 2 of 350
samples of pasteurized whole milk and 5 of 415 samples of chocolate milk, which suggests that
TABLE 11.4
Incidence of Listeria spp. in Unfermented Dairy Products Manufactured in
the United States during 1986 and 1987
Product Number of Samples Tested L. monocytogenes L. innocua
Whole milk 350 2 (0.57) 0

Low-fat milk 182 0 2 (1.10)
Skim milk 98 0 0
Chocolate milk 415 5 (1.20) 1 (0.24)
Cream 52 0 0
Half-and-half 42 0 0
Ice milk 99 3 (3.03) 0
Ice cream 659 23 (3.03) 12a (1.82)
Novelty ice cream 351 30 (8.55) 10b (2.85)
Butter 30 0 0
Nonfat dry milk 44 0 0
Casein/Milk protein hydrolysate 15 0 0
Other productsc 171 0 0
Total 2518 63 (2.50) 25 (0.99)
a L. seeligeri also detected in 7 of 659 (1.06%) samples.
b L. grayi also detected in 1 of 351 (0.28%) samples.
c Dairy blend whey, eggnog.
Source: Archer, D.L. 1988. Review of the latest FDA information on the presence of Listeria in foods.
WHO Working Group on Foodborne Listeriosis, Geneva, February 15–19; Kozak, J., T. Balmer, R. Byrne,
and K. Fisher. 1996. Prevalence of Listeria monocytogenes in foods: incidence in dairy products. Food
Control 7: 215–221.
approximately 0.67% of the pasteurized milk available in the United States could contain this
pathogen unless factories take corrective action to reduce this value. In contrast, L. monocytogenes
was isolated from 3 of 99, 23 of 659, and 30 of 351 samples of ice milk, ice cream, and novelty
ice cream, respectively. Only two of the positive ice cream samples were analyzed quantitatively
for L. monocytogenes. One contained an average of 15 L. monocytogenes CFU/g, whereas the other
sample contained between 1 and 5 CFU/g. Thus, during the time of this survey, approximately
5% of frozen dairy products manufactured in the United States presumably contained low levels
of L. monocytogenes.
Furthermore, L. innocua was isolated from three of four product categories (see Table 11.4) that
also contained samples positive for L. monocytogenes. Because both organisms likely occupy similar
niches in the natural environment and dairy processing facilities, isolation of L. innocua from a dairy
product should raise immediate concerns about the possible presence of L. monocytogenes.
Greater success in isolating L. monocytogenes from chocolate rather than whole milk is likely
related to the organism’s ability for enhanced growth in this product as compared to other fluid
dairy products. Reasons for increased growth of Listeria in chocolate milk will be discussed shortly
in conjunction with the behavior of listeriae in autoclaved fluid dairy products.
The higher incidence of L. monocytogenes in frozen rather than fluid dairy products coincides
with the relatively complex handling of ice milk and ice cream, particularly ice cream novelties,
during manufacture and packaging. This, in turn, suggests that these products are most likely
contaminated after pasteurization through either direct or indirect contact with listeriae within the
dairy factory environment. This hypothesis is supported by frequent isolations of L. monocytogenes
from many areas within dairy factories, including floors, ceilings, drains, and coolers. In addition,
this organism also has been found in air and condensate and on various pieces of equipment,
including conveyor belts. A detailed discussion of the incidence of L. monocytogenes in food-
processing facilities, including dairy factories, can be found in Chapter 17.
Inability of the FDA to detect L. monocytogenes in skim and low-fat milk as well as half-and-
half, cream, and butter may have resulted from the separation processes used for adjusting milk-
fat content that these products undergo. Such centrifugal separation processes tend to decrease
levels of listeriae, particularly if leukocytes containing the organism are still present after initial
clarification of the milk.
Failure to isolate L. monocytogenes from nonfat dry milk and casein/protein hydrolysates may
be partly related to the heat treatments necessary to manufacture these products. This theory is
supported by the work of Doyle et al. [104], who demonstrated that populations of L. monocytogenes
decreased by greater than 90% during conversion of skim milk into nonfat dry milk via spray
drying. However, failure to isolate L. monocytogenes from dried dairy products also may result
from the generally recognized inability of the FDA method to detect cells of L. monocytogenes
that have been sublethally injured during thermal processing. Thus, the methodology employed to
detect L. monocytogenes will predetermine whether or not the organism can be isolated from a
particular food.
According to the Food, Drug and Cosmetic Act of 1938, a food may be considered adulterated
and therefore unfit for human consumption if the product contains poisons or other harmful
substances (e.g., pathogenic microorganisms) at detrimental concentrations. Although the oral
infective dose for L. monocytogenes is known to vary widely based on individual susceptibility,
evidence from the California listeriosis outbreak involving Mexican-style cheese demonstrated that
the number of L. monocytogenes cells needed to induce this life-threatening illness can in some
instances be quite low—perhaps as few as several hundred to a few thousand total cells for certain
segments of the population. Several independent studies involving immunocompromised mice
demonstrated LD50 values (the dose of cells that is lethal to 50% of a given population) in the
range of approximately 10 [142] and 4 to 480 [89,228] L. monocytogenes cells when the
pathogen was administered orally and intraperitoneally, respectively. However, more recent
work with rhesus monkeys suggests an oral infectious dose generally >106 L. monocytogenes
cells (225). Although the FDA is morally obligated to uphold its policy of “zero tolerance” for L.
monocytogenes in ready-to-eat foods, results from several risk assessments to be discussed
in Chapter 18 indicate that the current zero tolerance policy will likely be modified to allow
L. monocytogenes at levels up to 100 or 1000 CFU/g in certain products such as ice cream in which
the organism is unable to grow.
In accordance with Title 21 of the United States Code of Federal Regulations, Section 7.40
[128], the FDA can request that firms voluntarily recall any product that contains, or is suspected
of containing, L. monocytogenes. These recalls can be classified into one of three categories: Class I,
Class II, or Class III. A Class I recall is the most serious and is defined by the FDA as “a situation
in which there is a reasonable probability that the use of or exposure to a violative product will
cause serious adverse health consequences or death.” Thus far, all recalls issued for products
contaminated with L. monocytogenes have been categorized as Class I. In the unlikely event that
a firm fails to comply with the FDA’s request to recall a product containing L. monocytogenes,
FDA officials can (1) initiate a seizure request in the US District Court to have the product removed
from commerce (Title 21 Code of Federal Regulations, Section 334) or (2) obtain a legal injunction
to halt production and distribution of the contaminated product (Title 21 Code of Federal Regulations,
Section 332). In addition, the FDA also can take criminal action against individuals of a company
who are responsible for commercial distribution of a contaminated product.
Adoption of the FDA Dairy Initiative Microbiological Surveillance Program in April 1986
prompted the first of at least 58 recalls for milk and unfermented dairy products contaminated with
L. monocytogenes (Table 11.5). Just 1 month after the surveillance program began, a California
firm voluntarily recalled an unknown quantity of ice milk mix contaminated with L. monocytogenes.
During the same month, approximately 1 million gal of fluid dairy products that comprised milk,
chocolate milk, half-and-half, whipping cream, ice milk mix, ice milk shake mix, and ice cream
mix also were recalled in Texas [13,14]. However, other than this single incident, the remaining
recalls have been primarily confined to frozen dairy products such as ice cream, ice cream novelties,
ice milk, and sherbet, with only six additional recalls thus far being traced to nonfrozen dairy
products (i.e., butter).
In July 1986, a large recall of Listeria-contaminated ice cream bars received considerable
media attention, which in turn did much to enhance the state of hysteria concerning the presence
of L. monocytogenes in dairy products [7]. The following month, another nationwide recall was
issued for frozen dairy products. As a result of this recall, approximately 1 million gal of products
possibly contaminated with L. monocytogenes and including ice cream (132 flavors), ice milk
(16 flavors), sherbet (9 flavors), and gelati-da products (6 flavors) were reportedly buried at a
Minnesota landfill site. One year later, a similar recall was issued for contaminated ice cream, ice
milk, and sherbet manufactured in Iowa [28,32]. By the end of 1987, well over 500 Listeria-
contaminated dairy products were voluntarily recalled in the United States at a total cost to the
dairy industry of well over $70 million [31,177]. Although corrective measures instituted by the
dairy industry in response to governmental pressure sharply reduced both the incidence of Listeria
contamination in processing facilities and the number of Class I recalls issued for L. monocytogenes-
contaminated dairy products since 1988 [51], recalls involving frozen desserts—and most recently
butter—have continued to negatively impact the industry. During the 6-year period from 1990 to
1995, 17 of 39 (44%) dairy-related L. monocytogenes recalls involved unfermented dairy products,
with Listeria being responsible for 31% of all dairy-related recalls issued for any reason [217].
Unfortunately, product losses are also being substantially underreported, because, under certain
circumstances manufacturers can retrieve their own product without issuing a formal Class I recall.
In one such instance, a manufacturer of Listeria-contaminated ice cream was not required to issue
a formal recall, because the product had not yet reached the consumer [36]. Additional cases also
likely occurred in which contaminated products moved only as far as the company’s warehouse
and were recalled internally by the manufacturer.
TABLE 11.5
Chronological List of Voluntary Class I Recalls Issued in the United States from 1986 to 2005
for Milk and Unfermented Dairy Products Contaminated with L. monocytogenes
Type of Dairy Month/Year State of
Product of Recall Manufacture Distribution Quantity Reference
Ice milk mix 5/86 California Arizona, Nevada, California Unknown 11,13
Milk (2 and 1/2% fat), 5/86 Texas Texas ∼1 million gal 13,14
chocolate milk,
chocolate milk (2%
fat), half-and-half,
whipping cream,
vanilla and
chocolate ice
cream mix, ice
milk shake mix,
and ice cream mix
Ice cream bars 7/86 Virginia Eastern United States Large but unknown 7
Ice cream, sherbet, 7/86 Wisconsin Minnesota, Wisconsin 8,000 gal 10
glacee
Ice cream (132 8/86 Minnesota Illinois, Indiana, Iowa, Kentucky, ∼1 million gal 12,15
favors), ice milk Michigan, Minnesota, Missouri,
(16 flavors), North Dakota, Ohio,
sherbet (9 flavors), South Dakota, Wisconsin
gelati-da products
(6 flavors)
Ice cream and ice 8/86 Iowa Illinois, Iowa, Minnesota, Unknown 8
cream Missouri, North Dakota,
novelties—bars, Wisconsin
drumsticks, slices,
sundaes
Ice cream 9/86 Wisconsin Wisconsin Unknown 9
Ice cream 12/86 New York New York ∼835 gal 27,32
Ice cream 12/86 West Virginia West Virginia, Ohio 450 gal 17,28
Ice cream, ice milk, 1/87 Iowa Arkansas, Delaware, Illinois, ∼1 million gal 17,21,30,
sherbet Iowa, Kansas, Maryland, 32
Minnesota, Missouri, Nebraska,
Oklahoma, Pennsylvania,
South Dakota, Wisconsin
Ice cream 4/87 New York New York, Pennsylvania ∼316 gal 29,32
Ice cream 7/87 California California, Oregon ∼60,000 gal 22,42
(48 flavors), ice
milk (6 flavors),
sherbet (5 flavors)
Ice cream nuggets 7/87 Maryland Connecticut, Florida, Maryland, 20,400 boxes 23,30
New Jersey, New York, North
Carolina, Ohio, Pennsylvania,
Virginia
Ice cream, ice milk 7/87 Nebraska Colorado, Iowa, Kansas, ∼30,000 gal 19
Nebraska, North Dakota,
South Dakota, Wyoming
(continued)

Ice cream sundae 8/87 Florida Alabama, Arizona, British West Unknown 24
cones Indies, Florida, Louisiana,
Mississippi, North Carolina,
Ohio, Puerto Rico,
South Carolina, Tennessee,
Virginia, West Virginia
Chocolate ice cream 8/87 Kentucky Florida, Puerto Rico ∼956 gal 16
Ice cream nuggets 9/87 Maryland Nationwide Unknown 25
Ice cream 9/87 Ohio Nationwide Unknown 26
novelties—bars,
sandwiches,
pieces, slices,
sundae cones
Ice cream bars 10/87 Ohio Michigan, Ohio, Pennsylvania, 51,780 bars 20
West Virginia
Chocolate ice cream 2/88 Georgia Georgia, North Carolina Unknown 34,37
Ice cream, sherbet, 7/88 Ohio Ohio >1083 gal 33
ice
Ice cream 8/88 Connecticut Connecticut, New York, 5.6–8.4 gal 39
Massachusetts
Ice cream 8/88 Connecticut Connecticut 30 gal 38
Ice cream pies 9/88 Connecticut Connecticut, New York, 1700 pies 35
Massachusetts
Ice cream 9/88 Pennsylvania Pennsylvania 215 gal 40
Ice cream bars 12/88 New York New York ∼128 gal 43
Ice cream 2/89 Connecticut Connecticut Unknown 44
Ice cream bars 11/89 Wisconsin Wisconsin ∼365 gal 46
Ice cream bars 2/90 New Mexico Alabama, Illinois, Pennsylvania, ∼1000 gal 48
Texas
Sherbet, ice milk, 6/90 Ohio Indiana, Kentucky, Michigan, Unknown 50
ice cream Ohio
Ice cream 8/90 Ohio Indiana, Kentucky, Ohio ∼778 gal 49
Ice cream novelties, 5/91 Tennessee Alabama, Georgia, Kentucky, Unknown 47
frozen novelties Tennessee, Virginia, West Virginia
Ice cream, ice milk 6/91 Illinois Illinois, Indiana, Wisconsin >2275 gal 53
Butter 6/91 North Pennsylvania ∼18,165 lb 52
Carolina,
Tennessee
Butter 6/91 North Pennsylvania ∼18,428 lb 52
Carolina,
Tennessee
Butter, butterine 3/92 Wisconsin Arizona, California, Florida, Unknown 54
Illinois, Maryland, Massachusetts,
Michigan, Minnesota, Mississippi,
Missouri, New Jersey, Ohio,
Wisconsin
Ice milk, ice cream 10/92 Wisconsin Wisconsin ∼1125 gal 55
(continued)

Ice cream bars 6/93 Ohio Ohio, Kentucky, New England 34,752 bars 56
Chocolate milk 7/94 Wisconsin Michigan, Wisconsin Unknown 168
Whipping cream 8/94 Wisconsin Michigan, Wisconsin Unknown 168
Butter 8/94 California Maine, Michigan, 36 lb 57
New Hampshire, Wisconsin
Butter 9/94 Wisconsin Pennsylvania, Wisconsin Unknown 168
Ice cream bars 9/94 Wisconsin Michigan, Wisconsin 167 gal 58
Ice cream novelties 10/95 Ohio Illinois, Indiana, Iowa, Kentucky, Large but unknown 59
Maryland, Michigan, Ohio,
North Carolina, Pennsylvania
Ice cream, frozen 10/95 Ohio Georgia, Indiana, Kentucky, ∼420,000 gal 62
yogurt, sherbet, Michigan, North Carolina, Ohio,
sorbet, ice cream Pennsylvania, South Carolina,
mix Tennessee, Virginia
Ice cream 12/95 California Arizona, Hawaii, California, >500 gal 63
New Mexico, Nevada
Ice cream 12/95 Michigan Michigan Unknown 63
Frozen yogurt 12/95 Ohio Connecticut, Delaware, Florida, ∼12,381 gal 60
Maine, Maryland,
Massachusetts, Michigan, New
Hampshire, New Jersey, New
York, Ohio, Pennsylvania,
Rhode Island, Vermont, Virginia
Ice cream, sherbet 1/96 Ohio Ohio 2,864 gal 61
Chocolate ice cream 7/97 Michigan Michigan Unknown 64
Frozen strawberry 7/97 Pennsylvania Pennsylvania ∼42 gal 65
yogurt
Ice cream bars 10/97 Texas Arkansas, California, Florida, 29,814 cases 66
Georgia, Illinois, Kansas,
Maryland, New York, Ohio,
Oregon, Texas, Washington State
Ice cream 1/98 California California Unknown 67
sandwiches
Ice cream 3/98 North Delaware, Florida, Georgia, 130,000 gal 68
Carolina Kentucky, Maryland,
North Carolina, Pennsylvania,
South Carolina, Tennessee,
Virginia, West Virginia
Heavy whipping 1/99 Minnesota Nationwide 500,000 gal 122
cream, half-and-
half, milk,
chocolate milk
Chocolate ice cream 2/99 Ohio Ohio 244 gal 122
Cream flan dessert 4/01 Minnesota Florida, Illinois, Kentucky, 375 lb 122
Michigan, Minnesota,
New York, Oklahoma,
Pennsylvania, Washington State
Butter 2/04 Wisconsin Nationwide 4,000,000 lb 122
Despite millions of gallons of frozen dairy products that have been recalled both formally and
internally, it must be stressed that only one case of listeriosis has been positively linked to
consumption of a contaminated frozen dairy product—in Belgium [6] (see Chapter 10). On this
basis, the International Ice Cream Association and the Milk Industry Foundation contended that a
Class I recall is too harsh a response for a frozen dairy product containing presumably very low
levels of L. monocytogenes.
The US government has developed one of the most stringent policies regarding presence of
L. monocytogenes in ready-to-eat foods, whereas most other countries have adopted more relaxed
policies (i.e., not >100 or 1000 CFU/g or mL), particularly for products in which Listeria is unable
to grow. Until recently, the Canadian government confined its recalls to only those foods that have
been linked to major outbreaks of listeriosis, with the role of pasteurized milk in foodborne listeriosis
still being highly debated [157]. Hence, no recalls were issued when investigators at the Health
Protection Branch of Health and Welfare Canada (analogous to the U.S. FDA) identified L. monocy-
togenes in 1 of 394 (0.25%) and 1 of 51 (2.0%) samples of ice cream and ice cream novelties,
respectively [111], during their own federal inspection program. Although subsequent investigations
were presumably conducted to identify (1) the source of contamination, (2) proper corrective measures,
and (3) possible links to human illness, Canadian officials maintained that recalling the two contam-
inated lots would be inappropriate without proof that consumption of Listeria-contaminated ice cream
could lead to listeriosis. Many individuals and most manufacturers have argued in favor of the more
relaxed Canadian position. When one considers the numerous recalls of Listeria-contaminated ice
cream in the United States, the fact that worldwide only one case of listeriosis has been positively
linked to ice cream containing unusually high numbers of listeriae, the inability of L. monocytogenes
to grow in this product during frozen storage, and the normal exposure rate of the human population
to listeriae, it is clear that the risk of contracting listeriosis from contaminated ice cream is extremely
low, as will be discussed later in regard to several risk assessments. Although current regulations
mandate immediate removal of fluid dairy products and cheeses that support growth of L. mono-
cytogenes, a scientifically valid argument can now be made against recalling certain dairy products
in which listeriae will not proliferate, such as ice cream and dried goods which, if contaminated,
typically contain very low numbers of listeriae as postpasteurization contaminants.
As a result of several large recalls of French Brie cheese and a listeriosis outbreak in Switzerland
that was traced to consumption of Vacherin Mont d’Or soft-ripened cheese, European scientists
have logically focused their attention on the incidence of listeriae in soft cheese. However, numerous
recalls of unfermented dairy products in the United States also have heightened public health
concerns about the presence of listeriae in pasteurized dairy products manufactured outside North
America [41].
In one of the first European surveys of finished products reported in 1988, researchers in
Germany [232] failed to isolate Listeria spp. from pasteurized milk (39 samples), nonfat dry milk
(11 samples), casein/caseinate (30 samples), and various dried products, including baby food
(Table 10.6). During the same year, investigators in Hungary [115] and The Netherlands [79] also
failed to recover L. monocytogenes from samples of pasteurized milk, with similar negative findings
being obtained from most other subsequent surveys of pasteurized milk and cream produced
elsewhere (Table 11.6). However, L. monocytogenes was eventually demonstrated in 11 of 1039
(1.1%), 4 of 115 (3.5%), and 1 of 95 (1.1%) pasteurized milk samples examined in the United
Kingdom [138,145,215] for a combined contamination rate of 1.3%, these findings generally being
similar to those observed in the United States. According to Garayzabal et al. [131], 21.4, 89.2,
10.7, and 3.6% of pasteurized milk samples from one particular milk processing facility in Madrid
contained L. monocytogenes, L. grayi, L. innocua, and L. welshimeri, respectively. These authors
[132,209] previously reported similar Listeria contamination rates for raw milk entering the same
processing facility. Further, after pasteurization these same samples had a total mesophilic aerobic
plate count of 2.5 × 107 CFU/mL, which is well above the maximum allowable limit of 1 × 104
CFU/mL for properly pasteurized milk in the United States. Hence, improper pasteurization caused
TABLE 11.6
Incidence of Listeria spp. in Pasteurized Dairy Products Produced outside the United
States and Canada
Number of L. monocy- L. welshi-
Product Country of Origin Samples togenes L. innocua meri Other Reference
Milk Australia 77 0 0 0 0 144

33 0 ND ND ND 72
Brazil 220 0 2(0.9) 0 0 185
20 0 0 0 0 83
Czechoslovakia 30 0 ND ND ND 181
15 0 ND ND ND 182
Germany 39 0 0 0 0 232
Hungary 100 0 ND ND ND 155
50 0 0 0 0 115
Italy 348 0 0 0 0 136
Korea 26 0 ND ND ND 74
50 0 0 0 0 234
Morocco 20 0 ND ND ND 109
The Netherlands 41 0 0 0 0 79
Poland 73 0 0 0 7a 211
Portugal 28 0 ND ND ND 179
Turkey 22 0 ND ND ND 220
United Arab 182 0 0 0 0 141
Emirates
United Kingdom
England/Wales 1039 11 (1.1) ND ND ND 145
Scotland 115 4 (3.5) ND ND ND 215
Northern Ireland 95 1 (1.1) ND ND ND 138
Chocolate milk Hungary 60 7 (11.6) 0 0 0 155
Flavored milk Australia 206 1 (0.5) ND ND ND 239
Ice cream Australia 166 23 (13.9) ND ND ND 72
Chile 603 21 (3.5) ND ND ND 90
Costa Rica 50 1 (2.0) ND ND ND 184
England/Wales 40 0 ND ND ND 137
Korea 132 8 (6.1) ND ND ND 74
Turkey 50 5 (10.0) 6 (12.0) 0 0 86
Cream Australia 12 0 0 0 0 144
England/Wales 40 0 ND ND ND 145
Hungary 15 0 ND ND ND 155
Morocco 20 0 ND ND ND 109
Butter Hungary 15 1 (6.7) 0 0 1b 155
Italy 130 5 (3.8) ND ND ND 202
Nonfat dry milk Germany 11 0 0 0 0 232
Casein/caseinate Germany 30 0 0 0 0 232
Dry infant Germany 120 0 0 0 0 232
formula
Note: ND = Not determined.

a
Seven non-L. monocytogenes isolates.
b
One non-L. monocytogenes.
by leaking pasteurizer plates, as suggested by Northolt et al. [187], or postpasteurization contam-

ination from the factory environment appear to be most likely responsible for the unusually high
incidence of listeriae in “pasteurized” milk samples from this particular dairy factory. Although
results from these aforementioned surveys of pasteurized milk, cream, and dried products are very
encouraging, the isolation methods used in these studies were generally unable to detect sublethally
injured listeriae. Hence, the true incidence of listeriae in pasteurized milk, cream, and dried products
may well be somewhat higher. To enhance recovery of injured cells, the International Dairy
Federation has recommended that such dairy products undergo preenrichment in a nonselective
medium (i.e., buffered peptone water) before primary enrichment in various selective broths and
plating on Listeria-selective media [39,233]. Further details concerning recovery of sublethally
injured listeriae can be found in Chapter 7.
Results from a 1989 International Dairy Federation survey [157] indicated that public health
issues regarding the presence of listeriae in pasteurized milk were clearly spreading beyond the
continental boundaries of Europe and North America, with the many aforementioned surveys from
Table 10.6 attesting to these concerns. More recently, the safety of several additional dairy products,
including flavored milks, chocolate milk, ice cream, and particularly butter, has attracted international
attention with the FDA Initiatives Program, the many Class I recalls of Listeria-contaminated dairy
products, and fears of international trade embargoes fueling these concerns. Following the 1987
discovery of L. monocytogenes in Australian ricotta cheese, New Zealand and Australian officials
instituted Listeria-monitoring programs for casein/caseinate products as well as high-moisture cheese,
pasteurized milk, ice cream, and milk powders. Results from one 10-month survey begun in April
1988 [239] revealed the presence of L. monocytogenes in 1 of 206 (0.48%) samples of pasteurized
flavored/unflavored milk processed in and around Melbourne. Subsequent identification of heat-labile
alkaline phosphatase in the contaminated product (pasteurized milk to which a pasteurized flavored
syrup was added) suggested that improper pasteurization was most likely responsible for the presence
of L. monocytogenes in the final product. However, unsatisfactory storage of the flavored syrup also
may have contributed to contamination. In keeping with Listeria policies developed in the United
States and Canada, Australian officials withdrew the affected product from the marketplace and
prohibited the sale of all subsequently produced product until 12 consecutive lots of Listeria-free
pasteurized flavored milk could be produced from the same product line.
As in the United States, recent foreign surveys also have shown a higher incidence of
L. monocytogenes in chocolate milk (11.6%), ice cream (2.0–13.9%), and butter (3.8–6.7%) as
compared to pasteurized milk and dried products that are seldom contaminated (Table 11.6). The
increased incidence of listeriae in ice cream and butter is clearly the result of postpasteurization
contamination during handling and packaging, as evidenced by the highest contamination rates in
ice cream bars and novelties. The fact that Listeria spp. are more commonly found in chocolate
milk, as opposed to unflavored milk, is also not surprising given that the added ingredients can
serve as another source of listeriae.
BEHAVIOR OF L. MONOCYTOGENES IN UNFERMENTED

DAIRY PRODUCTS
Although the psychrotrophic nature of L. monocytogenes and the ability of both normal and diseased
animals to shed this pathogen in their milk have been recognized for many years, behavior of
L. monocytogenes in raw milk and unfermented dairy products did not receive serious attention until
1983 when an outbreak of “milkborne” listeriosis was reported in Massachusetts. Research efforts
prompted by this and two other dairy-related outbreaks in the United States and Switzerland have
given us an understanding of the behavior of L. monocytogenes in raw and pasteurized milk as well
as in chocolate milk, cream, nonfat dry milk, and butter. The remainder of this chapter will describe
results from these studies along with information concerning behavior of this organism in ultrafiltered
milk and ice cream mix.
RAW MILK
Despite longstanding recognition of L. monocytogenes as a raw milk contaminant, relatively few
studies assessing the behavior of this organism in raw milk can be found in the literature. In 1958,
Dedie [95] found that L. monocytogenes survived 210 days in naturally contaminated raw milk stored
in an ice chest. Thirteen years later, Dijkstra [98] reported results from a much longer storage study
in which 36 samples of naturally contaminated raw milk (obtained from cows that experienced
Listeria-related abortions) were held at 5°C and examined for viable L. monocytogenes over a period
of 9 years. Although 4 of 36 (11%) samples were free of L. monocytogenes within 6 months, the
pathogen was still detected in 16 of 36 (44%) samples following 2 years of refrigerated storage. The
number of samples from which listeriae could be isolated continued to decrease, with 9 of 36 (25%)
samples being positive after 4 years of storage. However, the pathogen was still present in 4 of 36
(11%) raw milk samples after 8–9 years of storage. These early findings emphasize the importance
of establishing proper cleaning and sanitizing programs for all phases of milk production. If routinely
used, such programs will likely prevent this organism from finding an appropriate niche within the
farm or dairy factory environment and greatly reduce the threat of this pathogen surviving long term.
The studies just described adequately demonstrate that L. monocytogenes can persist in raw milk
for long periods; however, until several outbreaks of milkborne and cheeseborne listeriosis were
reported in the 1980s, little attention had been given to the potential for growth of L. monocytogenes
in raw milk.
In 1988, Northolt et al. [187] examined the behavior of listeriae in samples of freshly drawn
raw milk that were inoculated to contain approximately 500 L. monocytogenes CFU/mL and
incubated at 4 and 7°C. As shown in Figure 11.4, Listeria populations decreased approximately
4- and 8.5-fold in raw milk during the first 2 days of incubation at 4 and 7°C, respectively. These
authors suggested that naturally occurring antibacterial substances in raw milk (i.e., lactoperox-
idase and lysozyme) may have partially inhibited growth of listeriae during the first 2 days of
incubation as has been more recently confirmed by two other investigators [199,245]. However,
in a Canadian study that will be discussed shortly [113], no such decrease was observed when
incubated samples of naturally contaminated raw milk were surface-plated on FDA Modified
McBride Listeria Agar. Hence, a more likely explanation is that the plating medium—Trypaflavine
Nalidixic Acid Serum Agar—used by Northolt et al. [187] was less than ideal for recovering listeriae,
as also was observed during concurrent work with pasteurized milk. Although L. monocytogenes
failed to grow in raw milk samples incubated at 4°C for up to 7 days, Listeria populations increased
approximately 10-fold during this period when the incubation temperature was raised to 7°C.
Following 3 days of incubation at 4 and 7°C, Listeria populations began doubling every 3.5 and
1.0 days, respectively. Two years later, Wenzel and Marth [242] reported that populations of
L. monocytogenes strain V7 remained constant in inoculated raw milk during 5 days of storage at
4 and 7°C, with numbers of listeriae also being unaffected by the presence of a commercial
raw milk lactic acid bacteria inoculant designed to suppress growth of primarily Gram-negative
psychrotrophic bacteria.
L. monocytogenes failed to grow during 3–5 days of incubation at 7°C. It appears that the
3-day period during which raw milk is sometimes held in farm bulk tanks is insufficient to allow
growth of the organism. However, the temperature of raw milk in farm bulk tanks fluctuates every
time freshly drawn raw milk at 37°C is commingled with bulk tank milk at 4°C from previous
milkings. In 1985, Oz and Farnsworth [190] found that raw milk in farm bulk tanks attained
temperatures of 30–31°C, 10–14°C, 12°C, and 9°C when freshly drawn raw milk was added after
the first, second, third, and fourth milking periods, respectively. Moreover, 6 h were generally
needed for the milk to cool to 4°C after each milking period. In view of these findings, it appears
that temperatures obtained after adding warm milk to farm bulk tanks may be sufficient to allow
at least limited growth of L. monocytogenes, particularly when raw milk from early milkings enters
the bulk tank. Although the temperature of bulk tank milk will eventually decrease to 4°C, exposure
104
7°C
L. monocytogenes CFU/mL 103
4°C
102
Raw Milk
0
0 2 4 6
Days
FIGURE 11.4 Growth of L. monocytogenes strains in raw milk incubated at 4 and 7°C (enumerated on
Trypaflavine Nalidixic Acid Serum Agar). (Adapted from Northolt, M.D., H.J. Beckers, U. Vecht, L. Toepoel,
P.S.S. Soentoro, and H.J. Wisselink. 1988. Listeria monocytogenes: heat resistance and behavior during storage
of milk and whey and making of Dutch types of cheese. Neth. Milk Dairy J. 42: 207–219.)
to temperatures as high as 9°C when raw milk is trucked to processing facilities during summer
[114] also may lead to some multiplication of the pathogen.
Discovery of a naturally infected cow in Canada that shed freely suspended and phagocytized
cells of L. monocytogenes in milk (maximum of 104 CFU/mL in milk from one of four quarters
of the mammary gland) continuously for nearly 3 years provided Farber et al. [113] with a unique
opportunity to study growth of L. monocytogenes in naturally rather than artificially contaminated
raw milk during extended storage. When raw milk from this cow was analyzed for numbers of
L. monocytogenes, no appreciable growth of the pathogen was observed during the first 3 days and
1 day of incubation at 4 and 10°C, respectively (Figure 11.5). The delay in onset of growth was
less than 1 day at 15°C. Immunological staining of milk smears indicated that some multiplication
of L. monocytogenes had occurred within macrophages after 1 and 2 days of incubation at 15 and
10°C, respectively, with 10–50% of the macrophages containing 1–20 intracellular listeriae. None-
theless, as previously noted by Doyle et al. [105], rapid deterioration of macrophages shortly
thereafter was followed by appearance of freely suspended listeriae in milk with few intact mac-
rophages remaining after 5 days, regardless of incubation temperature. Following the lag phase,
L. monocytogenes entered a period of logarithmic growth, with generation or doubling times of
25.3, 10.8, and 7.4 h being calculated for raw milk samples held at 4, 10, and 15°C, respectively.
7.0
L. monocytogenes log CFU/mL

10
6.0
4°C
5.0 10°C
15°C
4.0
0 2 4 6 8 10 12 14
FIGURE 11.5 Growth of L. monocytogenes in naturally contaminated raw milk during incubation at 4, 10,
and 15°C. (Adapted from Farber, J.M., G.W. Sanders, and J.I. Speirs. 1990. Growth of Listeria monocytogenes
in naturally-contaminated raw milk. Lebensm. Wiss. Technol. 23: 252–254.)
Although maximum L. monocytogenes populations were approximately 2 × 107 CFU/mL after 10,
7, and 3 days of incubation at 4, 10, and 15°C, respectively, the highest achievable population in
raw milk was independent of incubation temperature (Figure 11.6). As in the previous study by
Northolt et al. [187], these findings again stress the importance of maintaining raw milk at 4°C
during storage and transport to milk processing facilities.
Despite an increasing market for both ewe’s and goat’s milk and the use of these milk types
in a wide range of ethnic and specialty cheeses and, to a lesser extent, yogurt, most work on
growth and survival of Listeria in raw milk has been conducted using cow’s milk. Reports of
ovine listeriosis in Europe dating back to the 1940s prompted an early study by Ikonomov and
Todorov [156] to examine the behavior of L. monocytogenes in raw ewe’s milk inoculated with
the pathogen. In their work, L. monocytogenes remained viable for long periods and persisted in
the milk even after coagulation at 10 and 20°C. Information on the behavior of L. monocytogenes
in goat’s milk is limited to one 2002 report by Leuchner et al. [164a] in which samples of raw
and pasteurized goat’s milk were inoculated with a 3-strain “cocktail” of L. monocytogenes at 103
CFU/mL and then stored at 4, 10, and 15°C for 58 days. The pathogen survived at least 58 days
when raw goat’s milk was held at 4 and 10°C and up to 44 days when the same milk was stored
at 15°C. Populations of Listeria in pasteurized milk increased about 3 logs after 58 days of storage
at 4°C and about 4 logs after 58 days at 10 and 15°C. Given these findings and the fact that many
varieties of ethnic- and specialty-type cheeses are now being manufactured by many small cheese
makers from ewe’s or goat’s milk, the safety of these cheeses is likely to receive increased attention
in the future.
PASTEURIZED AND INTENSIVELY PASTEURIZED MILK

In addition to defining the growth pattern of L. monocytogenes in artificially contaminated raw
milk (Figure 10.4), Northolt et al. [187] also examined behavior of this organism in pasteurized
(72°C/15 sec) and intensively pasteurized whole milk (Figure 11.6). Although L. monocytogenes
failed to grow in raw milk incubated at 4°C (Figure 11.4), Listeria populations in pasteurized
milk increased nearly 10-fold during 7 days of incubation at the same temperature. The organism
also grew markedly faster in pasteurized than in raw milk when both products were incubated
7°C
7°C 4°C
L. monocytogenes CFU/mL
2 4°C
Intensively
HTST-Pasteurized Milk Pasteurized Milk
0 2 4 6 0 2 4 6
FIGURE 11.6 Growth of L. monocytogenes in high-temperature, short-time (HTST)-pasteurized and inten-

sively pasteurized milk incubated at 4 and 7°C. —: Enumerated from samples at 4°C on Trypaflavine Nalidixic
Acid Serum Agar; ---: enumerated on Nutrient Agar. (Adapted from Northolt, M.D., H.J. Beckers, U. Vecht,
L. Toepoel, P.S.S. Soentoro, and H.J. Wisselink. 1988. Listeria monocytogenes: heat resistance and behavior
during storage of milk and whey and making of Dutch types of cheese. Neth. Milk Dairy J. 42: 207–219.)
at 7°C. In contrast to their data for raw and pasteurized milk, lag times for L. monocytogenes
were reduced considerably when the organism was grown in intensively pasteurized milk incu-
bated at 4 and 7°C. Further, numbers of listeriae in intensively pasteurized milk increased
approximately 100-fold following 3 and 6 days of incubation at 7 and 4°C, respectively. When
L. monocytogenes was later grown in ultrahigh temperature (UHT) sterilized milk, Rajikowski
et al. [204] reported generation times of 4.7, 1.7, 1.0, and 0.9 h for samples incubated at 12, 19,
28, and 37°C, respectively. Hence, these findings suggest that the growth rate for L. monocyto-
genes in milk is directly related to the degree of heat applied to milk, as was also reported by
Mathew et al. [176]. Further work is needed to define more clearly the effect of competing
microorganisms on growth of listeriae in raw and pasteurized milk as compared to intensively
pasteurized and UHT-sterilized milk, with biochemical changes that occur in milk during thermal
processing (i.e., protein denaturation, enzyme inactivation, carmelization) also likely influencing
listeriae growth in these products. However, the aforementioned studies all indicate the potential
for L. monocytogenes to reach potentially hazardous levels in pasteurized milk during the now
normal 2-week refrigerated shelf life, with at least one risk assessment to be discussed in Chapter 18
also suggesting that consumption of pasteurized milk is likely responsible for the largest per-
centage of listeriosis cases.
AUTOCLAVED MILK, CREAM, AND CHOCOLATE MILK

Except for the three studies just mentioned [113,187,176] and an initial attempt by Pine et al. [198]
to quantify growth of L. monocytogenes in inoculated samples of pasteurized milk, all remaining
Strain CA log10 CFU/mL

7
Skim Milk
5
Whole Milk
4 Chocolate Milk
Cream
3
2
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
Days
FIGURE 11.7 Growth of L. monocytogenes strain California in fluid dairy products at 4°C. (Adapted from
Rosenow, E.M. and E.H. Marth. 1987. Growth of Listeria monocytogenes in skim, whole and chocolate milk,
and in whipping cream during incubation at 4, 8, 13, 21, and 35°C. J. Food Prot. 50: 452–459.)
work dealing with behavior of Listeria in fluid dairy products has been done using autoclaved
samples. Although using such sterile products as growth media for listeriae offers several major
advantages, including the ability to accurately quantify both stressed and unstressed listeriae on
nonselective plating media in the absence of other microbial competitors, readers should keep in
mind that growth rates of L. monocytogenes are somewhat faster in autoclaved than in pasteurized,
and especially in raw milk, products. Nevertheless, L. monocytogenes clearly can grow to danger-
ously high levels in all three types of milk during extended refrigeration.
In 1987, Rosenow and Marth [213] published results from a definitive study in which autoclaved
(121°C/15 min) samples of whole, skim, and chocolate milk, as well as whipping cream, were
each inoculated separately with four strains of L. monocytogenes (Scott A, V7, V37CE, or Cali-
fornia), incubated at 4, 8, 13, 21, or 35°C, and examined for numbers of listeriae at suitable intervals
by surface-plating appropriate dilutions on Tryptose Agar. Growth rates for L. monocytogenes were
generally similar in all four products at a given temperature and increased with an increase in
incubation temperature. At 4°C, listeriae grew after an initial delay of approximately 5–10 days
depending on the bacterial strain and type of product (see Figure 11.7). All four strains generally
attained maximum populations of 107 CFU/mL after 30–40 days of incubation, with little change
in numbers occurring after 30–40 days of additional storage. Overall, chocolate milk supported
development of the highest Listeria populations followed by skim milk, whole milk, and whipping
cream. Generation times at 4°C ranged between 28.16 and 45.55 h with average generation times
for L. monocytogenes in all four products shown in Table 11.7. Although these results clearly
demonstrate the ability of L. monocytogenes to reach potentially hazardous levels in fluid dairy
products held at 4°C, more recent data suggest that slow growth of this organism can even occur
in milk held at 0°C. Thus, the only way to avoid a public health problem with fluid dairy products
is to prevent L. monocytogenes from entering such products before, during, and after manufacture.
Increasing the incubation temperature from 4 to 8°C decreased the lag period to 1.5–2 days
(Figure 11.8) and nearly tripled the growth rate for L. monocytogenes in all four products
(Table 11.7) [213,214]. After 10–14 days of incubation, the growth curves at 4 and 8°C were similar,
TABLE 11.7
Generation Times for L. monocytogenes in Autoclaved
Samples of Various Dairy Products
Generation Time (h) at
Product 4°Ca 8°Ca 13°Ca 21°Cb 35°Cb
Whole milk 33.27 13.06 5.82 1.86 0.692
Skim milk 34.52 12.49 6.03 1.92 0.693
Chocolate milk 33.46 10.56 5.16 1.72 0.678
Whipping cream 36.30 11.93 5.56 1.80 0.683
a
Average generation times for four strains of L. monocytogenes.
b
Strain V7 only.
Source: Adapted from Meyer-Broseta, S., A. Diot, S. Bastian, J. Riviere, and O.

Cerf. 2003. Estimation of low bacterial concentration: Listeria monocytogenes in
raw milk. Int. J. Food Microbiol. 80: 1–15.
with highest Listeria populations again being found in chocolate milk. Theoretical calculations
based on these data indicate that Listeria populations could increase from 10 to 4.2 × 106 organ-
isms/qt (947 mL) of milk during 10 days of storage at 8°C (46°F), a temperature that commonly
occurs in some home and commercial refrigerators. These findings, which have since been
confirmed by Siswanto and Richard [221] using skim milk, raise additional safety concerns
about reclaiming and reprocessing returned products that have likely undergone some degree
of temperature abuse.
As is true for 8°C, 13°C (55°F) also represents a temperature that dairy products occasionally
encounter during transportation and storage. Following a 12-h lag period, all four Listeria strains
grew nearly twice as fast at 13°C as at 8°C (see Table 11.7) and generally attained levels of
9 .
8.
Strain CA log10 CFU/mL
7.
6.
Skim Milk
5.
Whole Milk
4. Chocolate Milk
Cream
3.
2.
0 2 4 6 8 10 12 14 16 18 20 22
Days
FIGURE 11.8 Growth of L. monocytogenes strain California in fluid dairy products at 8°C. (Adapted from
Rosso, L., S. Bajard, J.P. Flandrois, C. Lahellec, J. Fournaud, and P. Veit. 1996. Differential growth of Listeria
monocytogenes at 4 and 8°C: Consequences for the shelf life of chilled products. J. Food Prot. 59: 944–949.)
106 CFU/mL in all four products by the third day [213]. These generation times are somewhat
longer than those observed by Farber et al. [113] when naturally contaminated raw milk was
incubated at 4 (25.3 h), 10 (10.8 h), and 15°C (7.4 h). L. monocytogenes also attained maximum
populations that were approximately 10-fold lower in raw than in sterile milk, which in turn suggests
possible depletion of essential nutrients by raw milk contaminants or production of substances
inhibitory to growth of the pathogen. Maximum Listeria populations of 109 CFU/mL were again
observed in chocolate milk, with numbers generally being 10-fold lower in skim milk, whole milk,
and whipping cream [213]. Increasing the incubation temperature to 21°C doubled the growth rate
(see Table 10.7) and led to maximum Listeria populations of 108–109 CFU/mL within 48 h. As
expected, L. monocytogenes grew most rapidly at 35°C, with populations of 108–109 CFU/mL being
observed after only 24 h of incubation.
In another study examining the influence of temperature and milk composition on growth of
listeriae, Donnelly and Briggs [100] found that five L. monocytogenes strains began growing in
inoculated samples of autoclaved (121°C/10 min) whole, skim, and reconstituted nonfat dry milk
(11% total solids) after approximately 24–48, 2–24, 4–12, and 0.5–4.0 h of incubation at 4, 10,
22, and 37°C, respectively. Although growth rates for all Listeria strains were primarily determined
by the incubation temperature, two strains of L. monocytogenes serotype 4b grew considerably
faster in whole rather than skim or reconstituted nonfat dry milk during incubation at 4 and 10°C.
These observations led Donnelly and Briggs [100] to suggest a possible relationship between levels
of milk fat and growth rate of L. monocytogenes in milk during refrigerated storage. Furthermore,
these authors suggested that enhanced psychrotrophic growth in whole milk may be related to a
listerial lipase produced by both hemolytic strains of L. monocytogenes serotype 4b. Unlike both
of these strains, the three remaining L. monocytogenes strains of serotypes 1 and 3 failed to exhibit
enhanced growth in whole milk at 10°C and had little if any hemolytic activity on McBride Listeria
Agar containing sheep blood.
In contrast to what might be expected from the study just described, Rosenow and Marth [213]
failed to observe any significant difference in growth rates among four strains of L. monocytogenes
(two serotype 4b, two serotype 1) when they were incubated in autoclaved samples of whole and
skim milk at 4, 8, 13, 21, and 35°C. The pathogen also attained lower maximum populations in
whipping cream than in whole, skim, or chocolate milk at all incubation temperatures. In support
of these findings, Marshall and Schmidt [174] failed to observe enhanced growth of L. monocyto-
genes strain Scott A (serotype 4b) in whole rather than skim milk during 8 days of incubation at
10°C. Finally, in a study to be discussed in greater detail in Chapter 12 [218], four strains of
L. monocytogenes (three serotype 4b and one serotype 1) frequently attained higher maximum
populations in whey samples that were defatted by centrifugation, filter sterilized, and incubated
at 6°C than would be expected to occur in autoclaved skim milk, whole milk, or whipping cream
after prolonged incubation at 8°C. Thus, although some L. monocytogenes strains are lipolytic as
reported by Marshall and Schmidt [174], one must presently conclude that psychrotrophic growth
of L. monocytogenes is not generally enhanced by the normal level of milk fat found in fluid milk.
Recognizing the vital importance of carbohydrates in microbial metabolism, researchers at the
CDC [198] attempted to define growth of Listeria spp. in terms of sugar utilization. An initial
experiment using aerobically incubated broth media indicated that five strains of L. monocytogenes
and one strain each of L. innocua, L. seeligeri, and L. ivanovii utilized only the glucose moiety of
lactose, whereas single strains of L. grayi and L. murrayi utilized both the glucose and galactose
of lactose. Overall, maximum cell populations, as determined by optical density, were directly
proportional to the concentration of glucose (0.125%) in the growth medium. However, marked
differences were observed in the ability of L. monocytogenes and L. innocua to utilize lactose, with
three strains of L. monocytogenes (isolated from Mexican-style cheese in connection with the 1985
listeriosis outbreak in California) unable to grow in a medium containing lactose as the only
carbohydrate. Although these observations agree with several reports [117,173,174] indicating that
the pH of fluid milk is unaffected by L. monocytogenes growth, Quinto et al. [203] did report a
sharp pH decrease in such milk after 16 and 24 days of incubation at 14 and 7°C, respectively,
these differences being most likely related to strain variation.
Growth of L. monocytogenes in autoclaved samples of whole and skim milk was generally
similar to that previously observed by Rosenow and Marth [213], with maximum populations of
5 × 108 CFU/mL developing after extended incubation at 5 and 25°C. Except for L. seeligeri, the
behavior of L. innocua and L. ivanovii did not differ markedly from that of L. monocytogenes in
these samples (Figure 11.9). However, as noted by Northolt et al. [187], higher maximum popula-
tions and increased survival rates were again observed when these organisms were grown in
autoclaved rather than pasteurized whole milk. Examination of milk by gas-liquid chromatography
indicated that lactic, acetic, isobutyric, isovaleric, and 2-hydroxy isocaproic acids were formed
during incubation. Because this milk initially contained 81–85 mg of glucose/L, the aforementioned
acids likely resulted, at least in part, from fermentation of glucose. Considerably lower populations
of L. monocytogenes as well as L. innocua, L. grayi, and L. murrayi also developed in glucose-
oxidase-treated (an enzyme that degrades glucose) rather than untreated milk during both aerobic
and anaerobic incubation, and so it is evident that glucose is one of the major substrates for growth
of listeriae in milk. However, when incubated anaerobically in glucose-oxidase-treated milk, two
lactose-negative L. monocytogenes isolates from Mexican-style cheese still attained final popula-
tions of 108 CFU/mL, thus suggesting the involvement of other as yet unidentified growth factors.
L. monocytogenes
L. seeligeri
L. ivanovil
L. innocua
9.0
Listeria log10 CFU/mL
8.0
7.0
6.0
0 4 8 12 16 20 24
Days
FIGURE 11.9 Growth of Listeria spp. in pasteurized (open symbols) and autoclaved whole milk (solid
symbols) incubated at 5°C. (Adapted from Pine, L., G. B. Malcolm, J.B. Brooks, and M.I. Daneshvar.
1989. Physiological studies on the growth and utilization of sugars by Listeria species. Can. J. Microbiol.
35: 245–254.)
10
8
Strain V7 log10 CFU/mL
7
6
2% milk (m)
5 2%m+sugar (s)
4 2%m+cocoa(c)+carr.
2%m+c+s+carr.
3
2
0 20 40 60 80 100 120 140 160 180 200
Hours
FIGURE 11.10 Growth of L. monocytogenes strain V7 in 2% fat milk with added sugar, cocoa, and carrag-
eenan (carr.) at 13°C. (Adapted from Rosenow, E.M. and E.H. Marth. 1987. Addition of cocoa powder, cane
sugar, and carrageenan to milk enhances growth of Listeria monocytogenes. J. Food Prot. 50: 726–729, 732.)
In the aforementioned study by Rosenow and Marth [213], maximum populations of L. mono-
cytogenes were typically about 10-fold higher in chocolate milk than in other fluid dairy products.
To explain the enhanced growth of L. monocytogenes in chocolate milk, several investigators at
the University of Wisconsin examined the effect of major chocolate milk constituents (i.e., cocoa,
sugar, and carrageenan) on growth of this organism in autoclaved skim milk and laboratory media.
Rosenow and Marth [212] found that growth of L. monocytogenes at 13°C was only slightly
enhanced in skim milk containing 5% cane sugar, and that the organism attained higher final
populations when commercial cocoa power (1.3%) and carrageenan stabilizer (0.5%) were used in
place of cane sugar (Figure 11.10). Carrageenan also enhanced the growth rate of L. monocytogenes
in the presence of cocoa; however, the organism attained similar maximum populations regardless
of the presence or absence of carrageenan. These findings suggest that carrageenan may be more
important in increasing contact between cocoa particles and Listeria than as a source of nutrients.
Highest final populations and shortest generation times were observed when L. monocytogenes was
grown in skim milk containing cocoa, sugar, and carrageenan. In addition, maximum Listeria
populations obtained in skim milk containing all three ingredients (see Figure 11.10) were similar
to populations observed in initial work with commercially produced chocolate milk (see Figure 11.7
and Figure 11.8).
Subsequently, Pearson and Marth [194] examined growth of L. monocytogenes strain V7 at
13°C in skim milk containing various concentrations of cocoa, sugar, and carrageenan. Because
some Listeria strains can utilize sucrose, it is not surprising that L. monocytogenes developed
significantly higher final populations (see Figure 11.11) and had shorter generation times (5.05 vs.
5.17 h) as the concentration of cane sugar (sucrose) in skim milk was increased from 0 to 12%.
(Peters and Liewen [197] also reported that addition of 7% sucrose to ultrafiltered (concentrated)
skim milk caused maximum L. monocytogenes populations to increase rather than decrease.) A
near-linear relationship between increasing sugar concentration and maximum attainable popula-
tions of L. monocytogenes also was observed for all but one combination of sugar, cocoa, and
8.90
8.80
Maximum population
8.70
(log10 CFU/mL)
8.60
8.50
8.40
8.30
0 3 6 9 12
Cane Sugar (%, W/V)
FIGURE 11.11 Maximum L. monocytogenes populations in skim milk alone (), skim milk + carrageenan
(), skim milk + cocoa (), and skim milk + cocoa + carrageenan () with 0, 6.5, and 12.0% cane sugar
after 36 h of incubation at 13°C. Any two points differing by 0.07 log10 CFU/mL are significantly different
(P < .05). (Adapted from Pearson, L.J. and E.H. Marth. 1990. Behavior of Listeria monocytogenes in the
presence of cocoa, carrageenan, and sugar in a milk medium incubated with and without agitation. J. Food
Prot. 53: 30–37.)
carrageenan tested; that was 12% sugar and 0.03% carrageenan (see Figure 11.11). Although
addition of 0.03% carrageenan significantly lengthened generation times and decreased maximum
populations compared to those observed in skim milk without carrageenan, L. monocytogenes
achieved highest populations in skim milk containing 0.75% cocoa with or without carrageenan,
which in turn indicates that the apparent ability of cocoa to stimulate growth of this organism in
skim milk containing 0–12% sugar is independent of carrageenan. Because cocoa contains only
trace amounts of fermentable carbohydrates, these authors theorized that cocoa enhanced growth
of L. monocytogenes in skim milk by providing increased levels of peptides and amino acids,
particularly valine, leucine, and cysteine, which are reportedly essential for growth. Additional
work showed that agitation, combined with the presence of cocoa, sugar, and/or carrageenan in
skim milk, enhanced growth of the pathogen at 30°C when compared to growth in the same medium
that was incubated quiescently. However, growth of Listeria in skim milk alone was better without
rather than with agitation. Thus, agitation most likely increased the availability of extractable
nutrients from cocoa, which in turn led to enhanced growth of the pathogen.
In 1968, anthocyanins in cocoa were reported to inhibit growth of salmonellae in laboratory
media; however, the inhibitory effect of cocoa could be neutralized with casein [82]. These early
findings prompted Pearson and Marth [196] to investigate the effect of cocoa with and without
casein on growth of L. monocytogenes strain V7. Using Modified Tryptose Phosphate Broth
containing 0.2% tryptose, addition of 0.75–10% cocoa increased the generation time for L. mono-
cytogenes at 30°C (1.02–1.12 h) as compared to samples without cocoa (0.94 h). However, the
pathogen generally attained higher populations when grown in media with (1.1–1.5 × 109 CFU/mL)
rather than without (6.4 × 108 CFU/mL) cocoa. Interestingly, when the same medium was inoculated
to contain 105 L. monocytogenes CFU/mL and agitated, the pathogen decreased to nondetectable
levels in samples containing 5–10% cocoa after 15–24 h of incubation at 30°C. Nevertheless, the
organism readily grew in the presence of 0.75% cocoa and attained higher maximum populations
in media with (1.9 × 109 CFU/mL) rather than without (7.6 × 108 CFU/mL) cocoa during agitated
incubation at 30°C.
As previously reported for salmonellae, the presence of 1.5 or 3.0% casein neutralized the
inhibitory effect of cocoa toward L. monocytogenes, the pathogen exhibiting shorter lag phases and
higher maximum populations in media containing both casein and 5.0% cocoa rather than cocoa
alone, and incubated quiescently at 30°C. However, results obtained during agitated incubation
of cultures containing 5% cocoa were far more dramatic, with L. monocytogenes populations of
2.9 × 109 rather than <10 CFU/mL developing in samples with rather than without 2.5% casein.
Hence, these findings suggest that the behavior of L. monocytogenes in laboratory media containing
cocoa partially depends on the concentration of one or more inhibitory substances that can be neutralized
by casein and that are more readily extracted during agitated rather than quiescent incubation.
Next, Pearson and Marth [195] determined if theobromine and caffeine (i.e., two methylxanthine
compounds in cocoa that reportedly possess different degrees of antimicrobial activity) were
responsible for the previously observed antilisterial activity of cocoa. Overall, addition of 2.5%
theobromine to both Modified Tryptose Phosphate Broth and autoclaved skim milk with and without
0.5% caffeine did not markedly influence the behavior of L. monocytogenes during incubation at
30°C. This suggests that theobromine is not responsible for suppressing or enhancing growth of
listeriae in chocolate milk. Unlike theobromine, addition of 0.5% caffeine to Modified Tryptose
Phosphate Broth doubled or tripled the length of the organism’s lag phase, nearly doubled the
organism’s generation time, and led to maximum Listeria populations approximately 10-fold lower
than those obtained in caffeine-free media. Similar trends also were observed when autoclaved
skim milk instead of Modified Tryptose Phosphate Broth served as the growth medium. Thus,
although caffeine in cocoa may contribute to inhibition of L. monocytogenes in a broth medium,
failure of casein in skim milk to neutralize the inhibitory effect of cocoa indicates that caffeine
also is not responsible for inhibition of listeriae as observed in the previous study. Such efforts to
identify Listeria-active components within cocoa should be continued to better understand the
behavior of this pathogen in chocolate milk.
SWEETENED CONDENSED AND EVAPORATED MILK

Thus far Listeria spp. have not yet been isolated from commercially produced sweetened condensed
milk (i.e., a nonsterile concentrated fluid milk product containing approximately 64% sucrose or
glucose in the water phase, 8.5% milk fat, and 28% total milk solids) or evaporated milk (an
unsweetened commercially sterile concentrated fluid milk product containing approximately 7.9%
milk fat and 25.9% total milk solids). However, given the widespread incidence of Listeria in food-
processing facilities, it is conceivable that listeriae could enter both of these products as postprocessing
contaminants. Such concerns prompted Farrag et al. [118] to examine the fate of three L. monocyto-
genes strains in samples of commercially produced sweetened condensed and evaporated milk that
were inoculated to contain three different levels (103–107 CFU/mL) of the pathogen.
Regardless of initial inoculum, Listeria populations in sweetened condensed milk decreased
1.2 and 1.6–3.4 orders of magnitude following 42 days of storage at 7 and 21°C, respectively. This
behavior was not surprising because addition of sugar to this product during manufacture reduces
its water activity (aw) to 0.83, which is well below the minimum aw value of 0.90 reported for
growth of L. monocytogenes. Unlike sweetened condensed milk, the relatively high aw value for
evaporated milk (0.986) allowed profuse growth of listeriae, with lowest inoculum levels increasing
approximately 4 orders of magnitude after 7 and 14 days of incubation at 21 and 7°C, respectively.
In addition, no decrease in numbers of listeriae was noted during continued incubation at either
temperature. Thus, because L. monocytogenes can survive >42 days in sweetened condensed milk
and grow rapidly in evaporated milk, special precautions should be taken to prevent listeriae from
entering these products during packaging, storage, and subsequent use.
ULTRAFILTERED MILK
Ultrafiltration, a mechanical process by which milk is filtered under pressure and concentrated,
results in major compositional changes in the finished product when compared to the starting
material. During ultrafiltration, 94–100% of the milk proteins and protein-bound vitamins
(i.e., vitamin B12 and folic acid) remain in the retentate along with milk fat, whereas lactose is
equally divided between the retentate and permeate. Increased use of ultrafiltered milk in cheese-
making prompted El-Gazzar et al. [106] to investigate the growth characteristics of L. monocytogenes
in 2X and 4X retentate as well as the corresponding permeate obtained from ultrafiltered pasteurized
milk. When samples were inoculated to contain about 104 CFU/mL of L. monocytogenes strain V7
or CA and incubated at 4°C, growth of both organisms was enhanced 10- to 100-fold in retentate
as compared to unfiltered skim milk. Increasing the concentration of ultrafiltered skim milk retentate
from 2X to 5X also resulted in faster growth, the pathogen attaining a population of 106 CFU/mL
in 5X and 2X retentate after approximately 7 and 10–12 days of refrigerated storage, respectively.
L. monocytogenes also grew to dangerous levels in permeate with maximum levels of 106 CFU/mL
as compared to 108 CFU/mL in retentate following 30 days of incubation. When identical tyndalized
samples were incubated at 32 and 40°C, both Listeria strains grew similarly in skim milk and
retentate, with populations of 108 CFU/mL generally being reported after 24 h of incubation.
However, as was true for samples incubated at 4°C, maximum numbers of listeriae were again 10-
to 100-fold lower in permeate than in retentate and unfiltered skim milk. Hence, the same care
should be given to production of unfiltered milk to prevent contamination and subsequent growth
of Listeria in the product during cold storage.
GROWTH OF L. MONOCYTOGENES IN MIXED CULTURES

Except for several early works assessing the behavior of L. monocytogenes in raw and pasteurized
milk, all studies described thus far have dealt with Listeria growth in the absence of competitive
microorganisms. Although results from these studies have been of great value to the dairy industry,
one should remember that pasteurized dairy products are not sterile. Psychrotrophic bacteria
belonging to the genera Pseudomonas and Flavobacterium are typically present in raw milk
and, similar to L. monocytogenes, can grow in milk at refrigeration temperatures both before
and after milk is pasteurized. Although readily destroyed during pasteurization, these organisms
universally appear in pasteurized dairy products as postpasteurization contaminants, often at
levels >100 CFU/mL. Because L. monocytogenes is thought to enter dairy products primarily
after pasteurization, products that contain low levels of listeriae (probably <10 CFU/mL) will
likely contain higher populations of other psychrotrophs. Although the ability of psychrotrophic
pseudomonads to stimulate growth of nonpathogenic as well as pathogenic bacteria in dairy products
has been recognized for more than 25 years, data concerning the behavior of L. monocytogenes in
mixed cultures are of more recent origin.
After initial work [116] with Tryptose Broth demonstrated that growth of L. monocytogenes
was slightly inhibited by the presence of Pseudomonas fluorescens, Farrag and Marth [117] exam-
ined associative growth of L. monocytogenes (strains Scott A, CA, and V7) with P. fluorescens
(strains P26 and B52) in autoclaved (121°C/15 min) skim milk that was inoculated to contain equal
populations (105 CFU/mL) of both organisms and incubated at 7 or 13°C for 56 days. Growth of
L. monocytogenes was generally enhanced by the presence of P. fluorescens after 7 days of
incubation, the pathogen attaining populations of 107 CFU/mL in mixed cultures. However, con-
tinued incubation at 7°C led to lower numbers of listeriae in mixed rather than pure cultures,
populations of strain V7 being inhibited approximately 8-fold by P. fluorescens B52 following 56
days of storage. Farrag and Marth [117] later showed that inactivation of strain CA was affected
by initial levels of P. fluorescens P26, with highest populations being most detrimental to Listeria
survival. However, populations of L. monocytogenes strains Scott A and V7 remained unaltered in

samples that were initially inoculated to contain P. fluorescens P26 at levels of 103–106 CFU/mL.
When Farrag and Marth [117] increased the incubation temperature to 13°C, growth of L. mono-
cytogenes was neither enhanced nor inhibited by either Pseudomonas strain during the first 7 days
of incubation. However, after 56 days of incubation, final populations of Listeria were as much as
20-fold lower in mixed rather than pure culture. Although these findings and those of Quinto et al.
[203] indicate that P. fluorescens was more detrimental to survival of listeriae in skim milk stored
at 13 than 7°C, growth and survival of P. fluorescens was not appreciably affected by the presence
of L. monocytogenes, with pseudomonads consistently reaching populations of 108–109 CFU/mL.
In a similar study, Marshall and Schmidt [173] found that growth of L. monocytogenes strain Scott
A (used in the previous study) in autoclaved skim and whole milk was not enhanced by the presence
of Pseudomonas fragi during 8 days of incubation at 10°C. Similarly, growth of P. fragi also was
unaffected by L. monocytogenes.
Some researchers have speculated that psychrotrophic pseudomonads may be able to utilize some
of the nutrients in milk faster than L. monocytogenes, thus suppressing growth of listeriae in milk
during refrigerated storage. Marshall and Schmidt [173] investigated this theory by inoculating
samples of autoclaved whole milk, skim milk, and reconstituted nonfat dry milk (10% solids) with
P. fragi or P. fluorescens (strain T25, P26, or B52); incubating the samples for 3 days at 10°C to
obtain 106–107 P. fragi CFU/mL or 104–106 P. fluorescens CFU/mL; inoculating these Pseudomonas
cultures with L. monocytogenes; and then incubating the samples for an additional 8 days at 10°C.
Throughout this study, addition of listeriae to all milks preincubated with P. fluorescens or P. fragi
did not significantly affect growth or survival of either pseudomonad. However, as shown in
Figure 11.12, L. monocytogenes grew faster and attained higher final populations in samples of whole
milk that were preincubated with either of the two pseudomonads than in whole milk that was not
treated with pseudomonads. L. monocytogenes behaved similarly in both whole and skim milk, with
average generation times of approximately 7 and 8 h in milks preincubated with P. fluorescens and
P. fragi, respectively (Figure 11.13). Although accelerated growth of Listeria was observed in recon-
stituted nonfat dry milk preincubated with either pseudomonad, generation times for listeriae in either
of the two mixed cultures generally did not differ significantly. As was true for whole and skim
milk, L. monocytogenes attained populations of 1 × 107 to 5 × 107 CFU/mL in reconstituted nonfat
dry milk, with highest numbers occurring in milk preincubated with P. fluorescens rather than P. fragi.
Flavobacterium is another genus of Gram-negative psychrotrophic bacteria that is frequently
recovered from raw milk, pasteurized milk, and butter. Hence, Farrag and Marth [119] also examined
behavior of L. monocytogenes in the presence of flavobacteria in skim milk at 7 and 13°C. Growth
of L. monocytogenes strains Scott A, CA, and V7 in autoclaved skim milk was enhanced by presence
of F. lutescens during 14–42 days of a 56-day incubation period at both 7 and 13°C, with these
higher populations again being attributed to proteolysis of milk proteins by F. lutescens. However,
Flavobacterium sp. ATCC 21429 failed to impact growth of L. monocytogenes at 7°C and proved
to be slightly inhibitory to the same three Listeria strains when samples were held at 13°C. One
strain of Bacillus spp. [170] also prevented Listeria growth in raw milk.
These results dispel the previous theory and indicate that L. monocytogenes can readily compete
with P. fragi, P. fluorescens, and certain Flavobacterium spp. for nutrients in milk and at the same
time can outgrow these organisms at refrigeration temperatures even if the ratio of L. monocytogenes
to pseudomonads or flavobacteria is on the order of 1:100,000. Enhanced growth of microorganisms,
including L. monocytogenes, in the presence of these psychrotrophs is related to increased levels
of nutrients that occur in milk as a result of proteolytic enzymes produced by these organisms
[119,174]. Because many of these enzymes are heat-stable and able to survive pasteurization, raw
milk must be handled properly and pasteurized within a reasonable time (i.e., 3–4 days) to prevent
conditions that may favor growth of listeriae. As previously noted [187], enhanced growth of
listeriae in intensively pasteurized as compared to HTST-pasteurized and raw milk also might be
related to this phenomenon.
6
L. monocytogenes log10 CFU/mL
5
L. monocytogenes
L.monocytogenes
+ P. fragi
3
2 L. monocytogenes
+ P. fluorescens
0
0 1 2 3 4 5 6 7 8
Days
FIGURE 11.12 Growth of L. monocytogenes at 10°C in whole milk preincubated for 3 days with selected
Pseudomonas spp. (Adapted from Marshall, D.L. and R.H. Schmidt. 1988. Growth of Listeria monocytogenes
at 10°C in milk preincubated with selected pseudomonads. J. Food Prot. 51: 277–282.)
17
16 L. monocytogenes
L. monocytogenes
P. fluorescens
15
L. monocytogenes
14 P. fragi
Generation Time (hours)
13
12
11
10
6
Whole Skim Nonfat Milk Solids
Product
FIGURE 11.13 Generation times of L. monocytogenes at 10°C in various milks preincubated for 3 days with
P. fragi or P. fluorescens spp. (Adapted from Marshall, D.L. and R.H. Schmidt. 1988. Growth of Listeria
monocytogenes at 10°C in milk preincubated with selected pseudomonads. J. Food Prot. 51: 277–282.)
NONFLUID DAIRY PRODUCTS

Although the aforementioned studies demonstrate the ability of L. monocytogenes to grow to
potentially hazardous levels in fluid dairy products held at refrigeration temperatures, concern about
the behavior of this organism in dairy products extends well beyond fluid milks and cream. As you
will recall from Table 10.5, nearly 50 recalls have been issued in the United States for Listeria-
contaminated ice cream. These recalls, along with FDA reports suggesting that about 3.5% of the
ice cream and 8.5% of the ice cream novelties produced in the United States may be contaminated
with presumably low levels of L. monocytogenes, prompted research on the extent of Listeria
survival in frozen dairy products. Additionally, behavior of Listeria during manufacture and storage
of nonfat dry milk and butter also has been investigated in the event that these products are
inadvertently prepared from contaminated skim milk and cream, respectively.
ICE CREAM
Pasteurized milk that has not been sold in retail stores is sometimes returned to dairy factories and
reprocessed into ice cream. Because large commercial refrigeration units often fail to maintain a
constant temperature of 4°C, virtually all reclaimed milk has undergone some degree of temperature
abuse during the period in which the product was on sale. In addition to possible growth of
L. monocytogenes during this 2-week period of “cold enrichment,” pseudomonads also can grow
in milk and produce an environment that is more favorable for growth of Listeria. However, proper
pasteurization of the ice cream mix would be expected to eliminate L. monocytogenes, with any
listeriae in the final product most likely coming from the surrounding environment during subse-
quent product handling and packaging. As indicated from the studies summarized in the following
text and the lack of listeriosis outbreaks involving commercial ice cream, Listeria is unable to grow
in the final product during frozen storage, with similar lack of growth and partial inactivation
reported for ewe’s milk during frozen storage [192].
The numerous Class I recalls issued since the late 1980s for Listeria-contaminated ice cream
prompted Berang et al. [80] to investigate the behavior of L. monocytogenes in inoculated samples
of chocolate ice cream (and chocolate milk as discussed earlier) prepared from fresh skim milk
and commercial skim milk that was held beyond the expiration date. Although growth of listeriae
was certainly not expected in ice cream held at −18 to −24°C, the pathogen survived equally well
in both types of chocolate ice cream. Hence, use of returned milk in chocolate ice cream did not
appear to enhance Listeria survival. Long-term survival of L. monocytogenes was also confirmed
in a later study [191] in which the pathogen persisted for 14 weeks in ice cream stored at −18°C
with no apparent cell death or injury. In 1996, Dean and Zottola [94] assessed the fate of L.
monocytogenes V7 in full-fat (10%) and reduced-fat (3%) soft-serve ice cream prepared with and
without 14-ppm nisin. Regardless of fat content, L. monocytogenes populations remained constant
in ice cream during freezing and 3 months of storage at −18°C. However, nisin effectively reduced
Listeria survival in both full- and reduced-fat ice cream during manufacture, with Listeria popula-
tions generally decreasing 2 and 3 orders of magnitude in full- and low-fat ice cream, respectively,
following 1 month of frozen storage at −18°C. Although not currently approved in the United States
as an ice cream ingredient, incorporation of nisin into ice cream formulations appears to be an
effective, albeit costly, means of inactivating listeriae in this product during frozen storage.
In 1989, Amelang and Doores [4,5] determined generation times for L. monocytogenes in nine
formulations of commercially produced ice cream mix that varied in type and level of fat (cream,
butter), sugar (cane sugar, corn sweetener), and milk solids (condensed milk, skim milk, whey
powder). To simulate postprocessing contamination, all samples were inoculated to contain 103
L. monocytogenes strain Scott A or V7 CFU/mL and incubated at 4, 21, and 35°C. Overall,
L. monocytogenes had average generation times of 21.6, 1.08, and 0.79 h in ice cream mixes
incubated at 4, 21, and 35°C, respectively, with similar growth rates occurring in mixes containing
10, 14, and 15% fat and held at the same temperature. It is noteworthy that these generation times
are markedly shorter than those calculated by Rosenow and Marth [213] for growth of the same
strains in whole milk, skim milk, chocolate milk, and whipping cream (see Table 11.5). Although
L. monocytogenes generally behaved similarly in all ice cream mixes incubated at 4 and 21°C,
differences in generation times were noted at 35°C when the pathogen was cultured in ice cream
mixes made with alternative fat and milk solids. At 35°C, growth of listeriae was somewhat
enhanced in mixes containing butter rather than cream, skim milk powder, or whey powder rather
than condensed skim milk, and egg yolk as additional sources of solids. Although the pathogen
grew most rapidly in ice cream mix containing a 50:50 ratio of cream to butter, partial replacement
of cane sugar (sucrose: glucose + fructose) with corn sweetener (glucose and maltose) or high
fructose corn syrup failed to significantly shorten generation times.
BUTTER
In 1988, Olsen et al. [189] examined the fate of L. monocytogenes during manufacture and storage
of butter in the event that the product is prepared from contaminated cream. According to their
report, pasteurized cream was inoculated to contain 104–105 L. monocytogenes CFU/g and churned
into butter. After removing the buttermilk, washed butter grains were salted to a level of 1.2% and
resultant butter was analyzed weekly for listeriae during 10 weeks of storage at −18, 4–6, and
13°C. During manufacture 95% of the L. monocytogenes population was lost in buttermilk, with
the remaining 5% of the population appearing in butter. The pathogen was present at levels of 1.7 × 104
to 1.8 × 105 CFU/g in cream as compared with 1.5 × 103 to 1.6 × 104 CFU/g in butter, indicating
that similar to Staphylococcus aureus [183], L. monocytogenes also favors the water rather than
lipid phase during butter making. As shown in Figure 10.14, Listeria populations increased 1.9
and 2.7 orders of magnitude in butter stored at 4–6 and 13°C, with maximum numbers being
7.00
6.00
10 CFU/g
13 °C
5.00
L. monocytogenes log
4 to 6 °C
4.00
3.00
–18 °C
2.00
0 20 40 60 80
Days
FIGURE 11.14 Survival of L. monocytogenes in butter manufactured from artificially contaminated cream
and stored at 12, 4–6, and −18°C. Each line represents the average of 4 trials. (Adapted from Olsen, J.A.,
A.E. Yousef, and E.H. Marth. 1988. Growth and survival of Listeria monocytogenes during making and storage
of butter. Milchwissenschaft 43: 487–489.)
observed after 49 and 42 days of storage, respectively. These findings along with similar results
by Lanciotti et al. [163] for commercially prepared light butter stored at 4 and 20°C indicate that
enough milk solids were trapped in the water phase (containing 6% salt) to support growth of
listeriae during storage. Numbers of listeriae then began to decrease; however, the organism was
still present at levels >104 CFU/g following 70 days of refrigerated storage. Although freezing the
contaminated butter prevented growth of L. monocytogenes, the organism was still present at levels
of 103 CFU/g after 70 days of storage at −18°C, as was also reported by Slavchev et al. [224].
Following a 2000 report from Finland linking consumption of butter to 25 listeriosis cases
including 6 fatalities [172], researchers at the University of Georgia [152,153] assessed the fate of
L. monocytogenes in butter and other related fat spreads as a postmanufacturing contaminant. In
the first of these studies [153], sweet cream whipped unsalted butter (pH 4.51), salted light butter
(pH 4.58), two yellow fat spreads (pH 4.05 and 5.37), and a light margarine (pH 5.34) were either
surface-inoculated with a 6-strain cocktail of L. monocytogenes and then stored at 4.4 or 21°C
under high relative humidity for 21 days, or uniformly contaminated by mixing the inoculum into
the product followed by storage at 4.4 or 21°C for 14 days. For surface-inoculated samples, growth
of L. monocytogenes was only seen on sweet cream whipped salted butter (pH 6.40) with populations
increasing < 1 log during 21 days of storage. On all other surface-inoculated samples, numbers of
Listeria decreased 1.7 to > 5 logs and > 5 logs during storage at 4.4 and 21°C, respectively. In
uniformly contaminated samples of sweet cream whipped salted butter (pH 6.40), Listeria popu-
lations remained relatively constant during 14 and 7 days of storage at 4.4 and 21°C, respectively,
with decreases of 1.5 to > 2.0 logs reported for the remaining products.
Holliday and Beuchat [152] later assessed the impact of storage temperature on L. monocytogenes
growth in seven different yellow fat spreads. In this study, one margarine, one butter–margarine blend,
and five dairy/nondairy spreads and toppings were inoculated with a 6-strain cocktail of L. monocy-
togenes at ~106 CFU per 3.5 mL or 4.0 g and then stored at 4.4, 10 and 21°C for up to 94 days.
Overall, L. monocytogenes persisted 10–14 to >94 days in the seven fat spreads examined, with longer
survival seen in samples stored at 4.4 as opposed to 10 and 21°C. The only growth of Listeria was
seen when the butter–margarine blend (pH 6.66) was stored at 21°C with numbers of Listeria
increasing ~1.5 logs after 2 weeks and then slowly decreasing to populations near the original
inoculation level after 94 days of storage. These findings are also consistent with those of Cirigliano
and Keller [87] who reported that L. monocytogenes failed to grow in margarine, yellow fat spreads,
or topping held at 5, 10, or 23°C. Lack of Listeria growth in these non-butter-containing yellow fat
spreads has been attributed to acidity (pH < 5.60) along with the presence of preservatives (potassium
sorbate, sodium benzoate, salt) and the unfavorable growth environment provided by some emulsions.
Addition of garlic to butter is also reportedly somewhat effective in reducing Listeria survival when
the product is stored at 10 and 37 but not at refrigeration temperatures [3].
Thus far L. monocytogenes has not been isolated from pasteurized cream manufactured in the
United States; however, given the massive Listeria recall of Texas-produced fluid dairy products,
including half-and-half and whipping cream, in May 1986 (see Table 11.5), one cannot assume
that all pasteurized cream and butter manufactured in the United States and elsewhere will be
universally free of listeriae. Hence, because at least six Class I recalls have been issued for
L. monocytogenes-contaminated butter, and also because growth of L. monocytogenes has been
demonstrated experimentally in both cream and butter during refrigerated storage, it is necessary
to ensure that cream is pasteurized and that recontamination of pasteurized cream is prevented
before and during its churning into butter. However, margarine and other non-dairy-based fat spreads
are generally unable to support growth of Listeria and therefore pose a lesser risk for consumers.
NONFAT DRY MILK

Dried dairy products, including nonfat dry milk, whey, and casein, also may become contaminated
with pathogenic microorganisms both before and after drying. Such concerns have been raised
recently in Australia and New Zealand [157]. Although all dry dairy products examined thus far
have been Listeria-free, methods used to detect listeriae in these surveys were generally unable to
recover cells that may have been injured during the drying process.
Two factors, namely, the unusual thermal resistance of L. monocytogenes and the report of a
milkborne listeriosis outbreak in Massachusetts during 1983, prompted Doyle et al. [104] to examine
behavior of L. monocytogenes during manufacture and storage of nonfat dry milk. Samples of
concentrated (30% solids) and unconcentrated (10% solids) skim milk were inoculated to contain
105–106 L. monocytogenes (strain Scott A or V7) CFU/mL and dried to moisture contents of
3.6–6.4% in a gas-fired pilot-plant-sized spray dryer with inlet and outlet air temperatures of
165 ± 2 and 67 ± 2°C, respectively. All samples of nonfat dry milk were stored at 25°C for up to
16 weeks and periodically analyzed for listeriae, using both direct plating on McBride Listeria
Agar (detects uninjured cells) and cold enrichment in Tryptose Broth (detects injured and uninjured
cells). Listeria populations decreased approximately 1.0–1.5 orders of magnitude during spray
drying regardless of whether or not nonfat dry milk was prepared from concentrated or unconcen-
trated skim milk. Strain V7 was generally hardier than strain Scott A during both spray drying and
storage of nonfat dry milk. Twelve to 16 weeks of storage at room temperature were required to
decrease populations of strain V7 >1000-fold in nonfat dry milk, whereas only 6 weeks of storage
were necessary to obtain similar decreases in numbers of strain Scott A. Overall, strains Scott A
and V7 survived a maximum of 8 and 12 weeks in nonfat dry milk, respectively. Although strain
Scott A generally survived equally well in nonfat dry milk prepared from concentrated and uncon-
centrated skim milk, strain V7 survived 2 weeks longer in nonfat dry milk manufactured from
concentrated rather than unconcentrated skim milk. The higher moisture content of nonfat dry milk
(i.e., 5.7 and 6.4%) prepared from concentrated skim milk may have enhanced survival of listeriae
in this product during extended storage. Overall, populations of L. monocytogenes decreased
>10,000-fold in nonfat dry milk during 16 weeks of storage at room temperature. Hence, if
commercially produced nonfat dry milk is ever found to contain L. monocytogenes, presumably at
very low levels, it may be possible to eliminate this pathogen by holding the product at room
temperature for several months.
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in Cheese and Other
Fermented Dairy Products
Elliot T. Ryser
CONTENTS
Introduction ....................................................................................................................................406
U.S. Surveillance Programs and Recalls for L. monocytogenes
in Domestic and Imported Cheese ................................................................................................407
Domestic Cheese ..................................................................................................................407
Imported Cheese...................................................................................................................414
France .......................................................................................................................414
Other Western European Countries..........................................................................418
Surveys and Monitoring Programs for Listeria spp. in Cheese Produced outside
the United States ............................................................................................................................419
Canada ..................................................................................................................................419
France ...................................................................................................................................421
Germany ...............................................................................................................................428
Italy .......................................................................................................................................430
Switzerland ...........................................................................................................................430
Other European Countries....................................................................................................432
Other Countries ....................................................................................................................435
Behavior of L. monocytogenes in Fermented Milks .....................................................................436
Starter Cultures, Cultured Milks, and Cream ......................................................................436
Mesophilic Starter Cultures......................................................................................436
Cultured Buttermilk..................................................................................................439
Cultured Cream ........................................................................................................441
Thermophilic Starter Cultures..................................................................................441
Yogurt .......................................................................................................................443
Kefir ..........................................................................................................................445
Traditional Fermented Milk Products ..................................................................................445
Behavior of L. monocytogenes in Cheese .....................................................................................446
Coagulants ............................................................................................................................447
Coloring Agents and Starter Distillates ...............................................................................448
Mold-Ripened Cheeses.........................................................................................................449
Camembert Cheese...................................................................................................449
Blue Cheese ..............................................................................................................453
405
Bacterial Surface–Ripened Cheeses.....................................................................................454

Brick Cheese.............................................................................................................455
Taleggio Cheese........................................................................................................456
Tilsiter Cheese ..........................................................................................................456
Trappist Cheese ........................................................................................................456
Soft Italian Cheeses..............................................................................................................457
Mozzarella ................................................................................................................457
Semisoft and Hard Cheeses .................................................................................................457
Gouda and Maasdam Cheeses .................................................................................458
Colby Cheese............................................................................................................458
Cheddar Cheese ........................................................................................................459
Swiss Cheese ............................................................................................................462
Parmesan Cheese ......................................................................................................462
Hard Italian-Type Cheese.........................................................................................463
Hispanic Cheeses..................................................................................................................464
Queso Blanco Cheese...............................................................................................464
Queso de los Ibores Cheese .....................................................................................464
Mexican Manchego ..................................................................................................464
Chihuahua .................................................................................................................465
Pickled Cheeses ....................................................................................................................465
Feta ...........................................................................................................................465
Turkish White-Brined Cheese ..................................................................................467
Domiati Cheese ........................................................................................................467
Bulgarian White-Pickled Cheese..............................................................................467
Sudanese White-Pickled Cheese ..............................................................................467
Yugoslavian White-Brined Cheese...........................................................................468
Ewe’s and Goat’s Milk Cheese ............................................................................................468
Kachkaval Cheese.....................................................................................................468
Manchego Cheese.....................................................................................................469
Goat’s Milk Cheese ..................................................................................................469
Soft Unripened Cheese.........................................................................................................471
Cottage Cheese .........................................................................................................471
Cream Cheese...........................................................................................................474
Whey Cheeses ......................................................................................................................474
Cold-Pack Cheese Food ...........................................................................................476
Pasteurized Processed Cheese..............................................................................................477
Behavior of L. monocytogenes in Cheese as Affected by Cheese Composition ................478
Feasibility of Preparing Cheese from Raw Milk.................................................................481
Whey.....................................................................................................................................482
Brine Solutions .....................................................................................................................483
References ......................................................................................................................................486
INTRODUCTION
On June 14, 1985, Listeria monocytogenes emerged from relative obscurity to the front page of
many American newspapers because of a large listeriosis outbreak in California that was directly
linked to consumption of Mexican-style cheese manufactured in metropolitan Los Angeles. By
the time this outbreak subsided in August 1985, as many as 300 cases of listeriosis had been
reported, including 85 deaths—at least 40 of which were traced to the tainted cheese. In response
Incidence and Behavior of Listeria monocytogenes 407
to this foodborne outbreak of listeriosis, U.S. Food and Drug Administration (FDA) officials
added L. monocytogenes to their list of pathogenic organisms that should be of concern to cheesemakers
and began surveying various soft domestic cheeses for listeriae.
Approximately 6 months later, isolation of L. monocytogenes from several imported Brie
cheeses purchased at a supermarket led to the eventual recall of approximately 300,000 tons of
Brie cheese imported from France and to a real concern about the incidence of this pathogen in
other European cheeses. Recall of this cheese prompted two corrective measures: (1) adoption of
a cheese certification program by the United States and France to prevent importation of Listeria-
contaminated cheese and (2) initiation of numerous large-scale surveys to determine the extent of
Listeria contamination in virtually all types of cheese manufactured in the United States, Canada,
and Western Europe.
Throughout 1986 and most of 1987, the impact of Listeria on European cheesemakers was
primarily in the form of economic losses from destruction of contaminated product. However,
L. monocytogenes struck again late in 1987 with the report of a large listeriosis outbreak in
Switzerland (see Chapter 11) in which Vacherin Mont d’Or soft-ripened cheese was incriminated
as the vehicle of infection. This pathogen has continued to plague consumers of cheese with a
series of smaller outbreaks traced to commercially produced European soft cheeses and, most
recently, several small outbreaks in the United States that involved soft homemade Mexican-style
cheese prepared from raw milk.
These cheeseborne listeriosis outbreaks have prompted ongoing worldwide efforts to better
understand the incidence of and behavior of L. monocytogenes during manufacture and storage of
numerous cheese varieties as well as other fermented dairy products. The first half of this chapter
summarizes Listeria-related recalls of cheese in the United States and results from surveys dealing
with the incidence of listeriae in domestic and imported fermented dairy products. The second half
of this chapter addresses the fate of L. monocytogenes during manufacture and storage of buttermilk,
yogurt, and various cheeses (including whey) and the potential for cheese ingredients, such as
rennet, salt brine, and coloring agents, to serve as vehicles of contamination during cheesemaking.
U.S. SURVEILLANCE PROGRAMS AND RECALLS FOR L. MONOCYTOGENES

IN DOMESTIC AND IMPORTED CHEESE
DOMESTIC CHEESE
The concept of listeriosis as a foodborne illness is not new. Consumption of contaminated raw milk
was believed to have caused several cases of listeriosis in post–World War II Germany. In 1961,
Seeliger [358] also suggested sour milk, cream, and cottage cheese as possible vehicles of infection
in this outbreak. Although results from two Yugoslavian studies concerned with behavior of L.
monocytogenes in various fermented dairy products (i.e., cultured cream, unsalted skim milk cheese,
Kachkaval cheese, and yogurt) were published in 1964 [246] and 1981 [367], no surveys dealing
with the incidence of listeriae in fermented dairy products were made before contaminated Mexican-
style cheese was linked to the California listeriosis outbreak in June 1985.
Public health concerns about presence of L. monocytogenes in domestic and imported fermented
dairy products as well as other foods, such as meat, poultry, seafood, fruits, and vegetables, can be
traced either directly or indirectly to the 1985 listeriosis outbreak in California. Less than 1 month
after the first nationwide Class I Listeria-associated recall was issued for 22 varieties (∼500,000 lb)
of Mexican-style cheese contaminated with L. monocytogenes (Table 12.1), the FDA developed a
series of programs designed to prevent the recurrence of such an outbreak [365] (Figure 12.1).
The Domestic Soft Cheese Surveillance Program—the first of the dairy factory initiative
programs—was instituted by the FDA in July 1985 and involved on-site inspection of firms
manufacturing soft cheese [15]. Priority was given to manufacturers of Mexican-style soft cheese,
followed by firms producing other ethnic-type soft cheeses, such as Edam, Gouda, Liederkranz,
408
TABLE 12.1
Chronological List of Class I Recalls in the United States for Domestic Cheese Contaminated with L. monocytogenes
Date Recall Reference FDA
Type of Cheese Initiated Origin Distribution Quantity (lb) Enforcement Report
Jalisco-brand soft Mexican-style: Cotija, Queso fresco, 6/13/1985 California Arizona, Arkansas, California, Colorado, ~500,000 11,12
and 20 other varieties Georgia, Guam, Hawaii, Idaho, Illinois,
Kansas, Louisiana, Marshall Islands,
Massachusetts, Nevada, New Jersey,
New Mexico, New York, Oklahoma,
Oregon, Rhode Island, Samoa, Texas, Utah,
Washington State
Liederkranz (Brie,a Camembert) 8/14/1985 Ohio Nationwide, Puerto Rico ~10,000 9,10,13
Soft Mexican-style: Queso fresco and 5 other varieties 3/5/1986 California Arizona, California, Oregon, Texas 127,607 21,39
Semisoft Salvador-style white 9/11/1986 Virginia Virginia, Washington, DC 10,850 43,55

Soft-ripened: Old Heidelberg 4/17/1987 Illinois Illinois, North Carolina, Ohio, Pennsylvania 1150 53
Soft-ripened: Bonbel and Gouda 5/6/1987 Kentucky Nationwide ~13,800 52
Raw milk sharp Cheddar 8/21/1987 Wisconsin California, Washington State ~1400 54,365
Soft Mexican-style: Cotija, Queso fresco, and 8 other 1/29/1988 California Arizona, California, Florida, Texas, Unknown 65
varieties; Baby Jack and Monterey Jack Washington State
Mexican-style soft cheese 11/6/1990 California Arizona, California, Idaho, Nevada, Oregon, 500,000 80
Washington State
Cheese spread 2/1/1991 Florida Southeastern United States ~1362 83
Mozzarella 2/14/1991 Wisconsin Connecticut, Georgia, Illinois, Michigan, >89,000 82
New York, Ohio, Pennsylvania, Texas,
West Virginia, Wisconsin
Ricotta 7/11/1991 New York Florida, New York 1109 84
Jack 10/28/1991 Wisconsin Iowa, Minnesota, Wisconsin 12,500 81
Cold-pack cheese food 3/10/1992 Wisconsin Arizona, California, Colorado, Florida, Unknown 83 85
Georgia, Illinois, Indiana, Maryland,
Michigan, Minnesota, New York, Ohio,
Pennsylvania, Tennessee, Texas, Vermont,
Virginia, Wisconsin
Queso fresco 10/14/1992 Washington Oregon, Washington State Unknown 87
Limburger 12/18/1992 Wisconsin Wisconsin 1500 86,88
Cheese spread 3/4/1993 Tennessee Alabama, Illinois, Indiana, Kentucky, 11,789 86
Mississippi, Tennessee
Cream cheese 10/19/1993 Wisconsin California, Florida, Georgia, Illinois, 3075 90
Indiana, Iowa, Minnesota, Nebraska, North
Carolina, Ohio, South Carolina, South
Dakota, Tennessee, Wisconsin
Queso Prensado 4/15/1994 Wisconsin Florida, New Jersey, Wisconsin 1429 96
Cream cheese and lox 5/11/1994 Massachusetts Connecticut, Georgia, Massachusetts 20 91
Mexican-style soft white 5/20/1994 Texas Texas Unknown 94
Queso blanco 5/24/1994 Wisconsin New Jersey 1220 95
Goat milk cheese 6/15/1994 California California, Colorado, Georgia, Illinois, ~5682 92
Massachusetts, Michigan, New York,
Oregon, Texas
Torte loaf cheese 8/11/1994 Missouri Illinois, Indiana, Kansas, Louisiana, 301 98
Missouri, Texas
Incidence and Behavior of Listeria monocytogenes
Swiss cold-pack cheese food 8/11/1994 Wisconsin Missouri, Ohio 510 97

Swiss 10/28/1994 Ohio Pennsylvania 2270 99
Gorgonzola 2/2/1996 Wisconsin California, Colorado, Florida, Georgia, 4500 100
Illinois, Minnesota, New Jersey, New York,
North Carolina, Pennsylvania, Tennessee,
Washington State, Wisconsin
Cream cheese with vegetables 10/30/1997 Massachusetts Connecticut, Maine, Massachusetts, New 7340 103
Hampshire, New Jersey, New York, Rhode
Island, Pennsylvania, Vermont
Cream cheese 11/14/1997 Massachusetts Connecticut, Maine, Massachusetts, New Unknown 104
Hampshire, New Jersey, New York, Rhode
Island, Vermont
Queso fresco 2/4/1998 Wisconsin Nationwide 248,938 107,108
(continued)
409
410

Chronological List of Class I Recalls in the United States for Domestic Cheese Contaminated with L. monocytogenes
Date Recall Reference FDA
Type of Cheese Initiated Origin Distribution Quantity (lb) Enforcement Report
Queso blanco 3/20/1998 Wisconsin Nationwide 248,938 389

Queso fresco 3/23/1998 Domestic Alabama, Florida, Georgia, North Carolina, Unknown 109
South Carolina, Tennessee, Virginia
Queso blanco 3/26/1998 Wisconsin Florida Unknown 389
Blue cheese 3/27/1998 Wisconsin Nationwide 123,000 389
Mozzarella 4/10/1998 California California, Colorado, Nevada Unknown 389
Blue cheese 4/11/1998 Wisconsin Nationwide Unknown 103
Blue cheese salad dressing 5/1/1998 Louisiana Nationwide Unknown 106
Queso fresco 5/26/1998 New York New York Unknown 389
Spanish white 5/26/1998 New York New York Unknown 389

Mexican-style fresh white 7/27/1998 New York New York Unknown 389
Shredded Monterey Jack 3/29/1999 Iowa Texas 6000 389
Raw milk Colby 5/22/1999 Missouri Arizona, California, Colorado, Georgia, 135 389
Illinois, Maryland, Missouri, New York,
Pennsylvania, South Carolina, Tennessee,
Utah
Queso fresco 6/1/1999 New York New Jersey, New York 500 389
Raw milk mild Cheddar 6/11/1999 Missouri Alabama, Arkansas, Florida Georgia, 228 389
Michigan, Missouri, North Carolina,
Pennsylvania, Tennessee, Washington State
Cheddar 7/24/2000 Oregon Alaska, California, Oregon, Washington State 1260 389
Cheddar, Monterey Jack, and Muenster (19 varieties) 10/17/2000 Wisconsin Nationwide 1,325,000 389
Shredded Colby and Monterey Jack 10/27/2000 Wisconsin Nationwide 62,180 389
Cheddar 11/1/2000 Wisconsin Nationwide 33,963 389
Cheddar 11/1/2000 Wisconsin Georgia, Oklahoma, Wisconsin 159,850 389
Jack, cheese sticks 11/2/2000 Wisconsin States east of the Rocky Mountains 74,302 389
Cheddar 11/4/2000 Mississippi Alabama, Florida, Louisiana, Mississippi 17,362 389
Colby and Monterey Jack 11/6/2000 Wisconsin Nationwide (except far western United 70,216 389
States)
Cheddar 11/6/2000 Wisconsin Alabama 18,000 389
Colby and Monterey Jack 11/6/2000 Wisconsin Nationwide 1,431,539 389
Sharp Cheddar 11/8/2000 Wisconsin California, Florida, Illinois, Maryland, North 17,712 389
Carolina, Tennessee, Virginia
Mozzarella string cheese 12/8/2000 Wisconsin Minnesota, Ohio, Oklahoma, Wisconsin 8688 389
Cheddar, Jack, and spread cheese 5/1/2001 California Nationwide Unknown 389
Cheddar, Colby, and American cheese cubes 12/3/2001 Ohio Maryland, Pennsylvania 6200 389
Ackawi 2/27/2002 Michigan Michigan Unknown 389
Queso fresco 4/25/2003 New York New York, New Jersey 350 389
Blue cheese 9/12/2003 Colorado Nationwide 360 389
Queijo Minerio 10/3/2003 Pennsylvania New Jersey, New York 4758 389
Queso fresco 10/16/2003 North Carolina Illinois 4266 389
Raw milk Cheddar 10/22/2003 Oregon Oregon 46 389
Sharp Cheddar, cold-pack cheese food 11/9/2003 Wisconsin Illinois, Minnesota, Wisconsin 2736 389
Queso fresco 12/12/2003 New York New Jersey, New York Unknown 389
Manouri Greek cheese 12/17/2004 New Jersey California, Illinois, Michigan, New Jersey, 32 389
New York, Pennsylvania, Virginia
Queso seco 8/22/05 Florida Florida 2000 389
American pasteurized processed cheese 12/10/05 Wisconsin Alabama, Illinois, Minnesota, Missouri, 550,254 389
Texas, Washington State
Bleu cheese 12/14/05 Massachusetts Massachusetts 36 389
aLater found to contain only L. innocua.
411
California listeriosis outbreak —

DOMESTIC January–August 1985
Domestic soft cheese surveillance

program — begun July 1985
L. monocytogenes accidentally isolated
IMPORTED
from French Brie cheese — January 1986
Imported soft cheese surveillance
Aged and ripened cheese survey —
program — begun March 1986
begun January 1986
Temporary FDA/French soft-ripened

Continuation of soft cheese survey cheese testing program — begun April 1986
— March 1987
French certification program — begun
April 1987
Survey of cheese manufactured L. monocytogenes isolated from Italian Italian cheese surveillance program —
from raw milk — begun April 1987 Romano cheese — June 1987 begun July 1987
Survey of import cheese in domestic

Cheese under general pathogen
status — begun December 1987
surveillance program — 1988
FIGURE 12.1 Surveillance programs for Listeria spp. in domestic and imported cheese. (Adapted from
Archer, D. L. 1988. Review of the latest FDA information on the presence of Listeria in foods. WHO Working
Group on Foodborne Listeriosis. Geneva, Switzerland, February 15–19.)
Limburger, Monterey Jack, Muenster, and Port du Salut, made from raw, heat-treated (<71.7°C
[161°F]/15 sec) or pasteurized (≥71.7°C [161°F]/15 sec) milk. In addition to determining the firm’s
compliance with good manufacturing practices (i.e., use of proper pasteurization, cleaning, and
sanitizing procedures), FDA inspectors collected and analyzed cheese samples for L. monocytogenes
using the original FDA procedure. Cheese samples also were tested for the presence of entero-
pathogenic strains of Escherichia coli and for phosphatase activity, which, if present, generally
indicates improper pasteurization of cheesemilk and/or subsequent contamination with raw milk.
However, suitability of the phosphatase test for cheese has since been questioned.
Less than 2 months into this program, FDA officials isolated a pathogenic strain of L. mono-
cytogenes from one sample of domestically produced Liederkranz cheese (see Table 12.1). The
manufacturer subsequently recalled the product nationwide. Following preliminary FDA reports of
further Listeria contamination, this recall was extended to include all lots of Brie and Camembert
cheese manufactured at the same facility [9,13]. However, final laboratory reports indicated that
both Brie and Camembert cheese were contaminated with Listeria innocua, which is nonpathogenic,
rather than L. monocytogenes. Although the Domestic Soft Cheese Surveillance Program also
was responsible for temporarily closing two soft cheese factories in California that produced
phosphatase-positive cheese [10], it must be stressed that L. monocytogenes was never isolated
from cheeses produced at either facility.
In general, FDA inspections of other soft cheese factories uncovered problems similar to those
encountered during inspections of Grade A fluid milk factories: (1) potential bypasses of the
pasteurizer, (2) postpasteurization blending of product, and (3) a general lack of education and
training of plant personnel [282]. Items of particular concern to cheesemakers and that were not
generally found during visits to Grade A milk factories included defects in the pasteurization
process, discrepancies in pasteurization/production records, and a higher incidence (than in Grade
A milk factories) of pathogenic microorganisms (including L. monocytogenes) on environmental
surfaces in production and storage areas.
Inspections of domestic cheese factories continued throughout 1986, 1987, and 1988 under
four separate programs (see Figure 12.1), with FDA officials reaching nearly half of the 400 soft
cheese factories in the United States by April 1986 and the remaining factories (including follow-up
inspections of problem factories) by late 1987 [47]. According to FDA records [113], L. monocytogenes
was confirmed in 12 of 658 (1.82%) domestic cheese samples analyzed during 1986. During these
inspection programs, six Class I recalls were issued for various ethnic-type soft and semisoft cheeses
containing L. monocytogenes. In response to (1) a 1987 report of a woman who developed listeriosis
in San Bernadino, CA, after consuming illegally produced Mexican-style cheese and (2) the
widespread availability of uninspected, unbranded Mexican-style cheese illegally produced from
raw milk in metropolitan Los Angeles [73], Genigeorgis et al. [216], in conjunction with the
California Department of Food and Agriculture, U.S. Department of Agriculture’s Food Safety and
Inspection Service (USDA–FSIS), the Immigration and Naturalization Service, and the Los Angeles
District Attorney’s Office, surveyed 100 California-produced soft Hispanic-style cheeses that were
either seized or purchased undercover between June and November of 1988. Overall, two samples
each were positive for L. monocytogenes and L. innocua. These four Listeria-contaminated cheeses
had a pH of 6.2–6.5 and were presumably prepared from raw milk, as evidenced by a positive
alkaline phosphatase test. Given the ability of L. monocytogenes to grow in such cheeses during
refrigerated storage and marketing, Hispanic-style cheeses continue to constitute a significant
public health threat. Hispanic-style cheeses manufactured commercially in the United States
accounted for 23 of 68 (33.8%) recalls issued through 2005 including one large recall in June
1990 involving approximately 500,000 lb of product. However, of greatest concern are those
Hispanic-style cheeses that have been illegally manufactured from raw milk, as evidenced by
continued sporadic cases of listeriosis among the Hispanic population, with Gombas et al. [230]
recovering L. monocytogenes from only 5 of 2,931 (0.17%) commercially produced soft Hispanic-
style cheeses recently purchased in Maryland and California over a 50-week period. As part of
this same survey, L. monocytogenes was also isolated from 23 of 1,623 (1.42%) and 14 of 1,347
(1.04%) retail samples of blue-veined and mold-ripened cheese, respectively, with some of these
cheeses also having been imported.
As previously mentioned, although all products containing L. monocytogenes must be retrieved
from the marketplace, formal Class I recalls do not have to be issued for contaminated products
that have not yet reached retail stores. Because such situations typically lead to nonpublished
“internal recalls” issued by the manufacturer, far more cheese was likely destroyed during this
20-year period than has actually been reported. Several such informal recalls involved a part-skim
milk cheese manufactured in California [58] as well as ricotta, Parmesan, and mozzarella cheese
of uncertain origin [297].
Following a report by Ryser and Marth [340] that L. monocytogenes can survive more than
1 year in Cheddar cheese (i.e., well beyond the mandatory 60-day aging period for Cheddar cheese
manufactured from raw milk), the FDA modified its Domestic Cheese Program in August 1987 to
include cheese prepared from unpasteurized milk [47]. Between April and October of 1987, 181
samples of domestic aged (held a minimum of 60 days at ≥1.7°C [35°F]) natural cheese manufac-
tured from raw milk, as well as similar imported cheeses in domestic status, were collected from
retail stores by FDA field personnel and analyzed for L. monocytogenes (Table 12.2). These efforts
uncovered one positive sample—a sharp Cheddar cheese manufactured in Wisconsin, which was
subsequently recalled from the market in July 1987 (see Table 12.1). Thus far, 17 of the 68 (25%)
Listeria-related cheese recalls have involved Cheddar or Colby, including four cheeses prepared
from raw milk, with 14 of these recalls issued since 2000.
Late in 1987, the FDA announced plans for a 2-year pathogen surveillance program [44] that
was designed to examine domestic and imported cheese as well as other high-risk foods (i.e., milk,
vegetables, and seafood) for the presence of L. monocytogenes and other selected pathogens,
including Vibrio cholerae, V. parahaemolyticus, E. coli, enteropathogenic E. coli, Staphylococcus
aureus, Salmonella spp., Yersinia enterocolitica, Campylobacter jejuni, and C. coli. Under this
program, samples of soft-ripened and raw milk cheese as well as imported hard and artificial
blended cheese were examined for all of the aforementioned organisms except Vibrio spp. Domestic
cheeses were collected at the wholesale level, whereas samples of imported cheese were obtained
TABLE 12.2
Incidence of L. monocytogenes in “Domestic” Cheese Manufactured
from Raw Milk—FDA 1987a
Number of Samples Number of Positive Samples
Type of Cheese Analyzed (%)
Blue 18 0
Brick 5 0
Cheddar 71 1b (1.4)
Colby 8 0
Edam 4 0
Goat 6 0
Gouda 1 0
Monterey Jack 9 0
Swiss 42 0
Other 17 0
Total 181 1 (0.55)
a
Includes imported cheese in domestic status.
b
Two samples with L. innocua.
Source: Adapted from Archer, D. L. 1988. Review of the latest FDA information on the presence
of Listeria in foods. WHO Working Group on Foodborne Listeriosis. Geneva, Switzerland,
February 15–19.
from retail stores. Although this program prompted only one Listeria-related recall of domestic
cheese during 1989 and 1990, five separate recalls of Anari and Halloumi cheese imported from
Cyprus were reported during this same 2-year period along with one additional recall of Italian
soft-ripened/semisoft cheese. However, because additional cheese-related recalls after 1990 have
been limited to eight imported cheeses, the present FDA inspection program in combination with
increased vigilance by commercial cheesemakers appears to be highly effective in limiting consumer
exposure to both domestic and imported Listeria-contaminated cheese.
Several other fermented dairy products also were examined for L. monocytogenes in conjunction
with the FDA Dairy Initiative Program [113] (see Chapter 11). In 1986, 10 samples of cottage
cheese were found to be free of listeriae. Other than cheese and cheese food, 1% fat cultured
buttermilk [34,36] and frozen yogurt [72] are the only other domestically produced, fermented
dairy products known to have been contaminated with L. monocytogenes. The first of these products
was included in a 1986 Class I recall involving approximately 1 million gallons of dairy products
(fluid milk, chocolate milk, half-and-half, whipping cream, ice milk, ice milk mix, ice milk shake
mix, ice cream, and ice cream mix), all of which presumably contained L. monocytogenes. Three
years later, officials from the Wisconsin Department of Agriculture, Trade and Consumer Protection
issued a statewide recall for one particular brand of frozen yogurt after routine testing revealed the
presence of L. monocytogenes in one sample of mandarin orange frozen yogurt [75]. Both these
products were retrieved from the marketplace without incident.
IMPORTED CHEESE
France
International concern over the potential health hazard of consuming Listeria-contaminated cheese
also stems from the California listeriosis outbreak of 1985. This outbreak and an earlier link between
consumption of French Brie and/or Camembert cheese and several outbreaks of foodborne illness in
the United States and Europe caused by enterotoxigenic and/or enteropathogenic E. coli [275,298,385]
prompted a meeting in September 1985 between FDA officials and representatives of the French
Embassy/French Delegation on Food Safety and Food Distribution to discuss the FDA’s plans for
inspecting imported soft cheese [14]. Late in September, representatives from the Codex Committee
on Food Hygiene and the International Dairy Federation agreed with FDA officials that a “Code of
Hygienic Practices” should be developed for manufacturing fresh and soft cheese [8]. Use of raw
milk (a known source of L. monocytogenes) to improve organoleptic properties of certain cheeses
was cited as a particular area of concern. Although a basic certification program for soft cheese
produced in France was in operation for some time, agreement on a general Code of Hygienic Practices
for manufacture of soft cheese was not reached during the remainder of 1985.
In January 1986, as part of a research effort to enhance recovery of listeriae from cheese, FDA
officials inadvertently isolated a pathogenic strain of L. monocytogenes from two uninoculated
control samples of French Brie cheese purchased at a local supermarket [20,113]. Ironically, both
cheeses were prepared from pasteurized milk in a cheese factory certified by the French government
under the existing soft-ripened cheese agreement. In response to these findings, the first in a series
of nationwide recalls was issued in February 1986 for Listeria-contaminated French Brie cheese
(Table 12.3). The following week, the French firm that manufactured the tainted cheese agreed to
halt all production [20]. Shipment of additional cheese that was previously certified “Listeria free”
by an independent French laboratory (certification by the French Ministry of Agriculture began on
January 1, 1986) was also stopped pending identification of the contamination source. In addition,
all suspect lots of French Brie cheese en route to the United States were detained on entry and
tested for Listeria by the FDA before being released. Shortly thereafter, sampling was extended to
include virtually all lots of French Brie cheese produced by this manufacturer.
The French cheese industry was dealt its most serious blow in March 1986, when approximately
660 million pounds of Brie cheese produced by five different manufacturers were recalled in the
United States (Table 12.3). This recall, which involved nearly 60% of all Brie cheese marketed in
the United States, immediately raised the possibility of block-listing French firms that produced
contaminated cheese [22]. Consequently, FDA officials immediately began testing all shipments of
French soft-ripened cheese as well as 20% of French soft/semisoft cheese and 20% of all other
imported soft cheeses for Listeria, E. coli, and phosphatase (see Figure 12.1) [25].
Recognizing the danger of contracting listeriosis from consuming contaminated French soft-
ripened cheese, FDA officials drafted the following proposal to detain, test, and certify all French
soft-ripened cheeses exported to the United States [74]:
FDA intends to detain any entry that is found to be Listeria-positive, regardless of the species of Listeria
found. French soft-ripened cheese without a certificate indicating negative results for the Listeria
analysis or with a positive analysis for Listeria will be detained. … In addition, all French soft-ripened
cheeses are to be sampled and analyzed for the presence of Listeria. [Under the previous agreement,
cheeses were shipped with a certificate of analysis that only indicated the level of E. coli and the absence
of phosphatase.] The analysis may be carried out by a private laboratory after the cheese has arrived
in the United States, or alternatively the analysis may be conducted so that the availability of the results
will coincide with the arrival of the shipment in the United States. The timing of the analysis is important
to ensure that the nature of the cheese tested, particularly the pH, is identical to that examined by the
FDA, if we decide to perform an audit on the entry. Thus, importers of French soft-ripened cheese
should provide FDA with certificates indicating the dates testing was initiated and completed. Cheese
shipments will not be released without a certificate indicating negative results for Listeria analysis. …
In this proposal, FDA officials stressed that other methods used to detect Listeria in cheese should
conform to the 7-day FDA method, and also suggested that 1/16-in.-thick slices from the cheese surface
(sample with highest pH) be analyzed for listeriae rather than cross-sectional plugs of cheese.
Following FDA threats to halt importation of soft-ripened cheese, the French Ministry of
Agriculture agreed to begin lot-by-lot testing in April 1986, as outlined in the March FDA proposal [26].
416
TABLE 12.3
Chronological List of Class I Recalls in the United States for Imported Cheese Contaminated with L. monocytogenes
Date Recall Country of
Type of Cheese Initiated Manufacture Distribution Quantity Ref.
Brie 2/12/1986 France Bermuda, Nationwide 57,000 2–6 lb 18,20,27,30

wheels
Brie 2/14/1986 France Georgia, New Jersey 40 cases 17
Brie 2/14/1986 France Colorado, Connecticut, Florida, Louisiana, Maryland, Massachusetts, New 100 cases 17
Jersey, New York, Ohio, Washington, DC
Brie 2/14/1986 France Florida, New York, Washington, DC 10 cases 17
Brie 2/14/1986 France Nationwide Unknown 17,38
Brie 2/21/1986 France Oregon, Washington State Unknown 16,38
Brie 2/24/1986 France Illinois, Minnesota, New Jersey 909 cases 18,40
Brie 3/14/1986 France Nationwide ~660 million 37,41

Brie 4/1/1986 France Colorado, Connecticut, Georgia, New Jersey, New York, North Carolina, Texas, ~230 28,37
Washington, DC
Soft-ripened: Tourre de l’Aubier and 6/23/1986 France New York, Ohio, Pennsylvania Unknown 29,32
Fromage des Burons
Soft-ripened: Tourte de l’Aubier 8/13/1986 France California, Illinois, Maine, Massachusetts, New Jersey, New York, Oregon 1056 33,29,31,35
Semisoft: Morbier Rippoz 8/18/1986 France Illinois, Massachusetts, Michigan ~1600 29,31,35
Soft-unripened, full fat 4/16/1987 France New Jersey, New York, Texas 15 wheels 48, 49
Semisoft 1/27/1988 Italy Nationwide 410 cartons 60
Semisoft: L’Amulette Danish Estron 2/11/1988 Denmark California Unknown 57,58,61
Semisoft: L’Amulette Danish Estron 2/12/1988 Denmark East, Midwest, North, South ~11,500 57,58,62
Semisoft: L’Amulette Danish Estron 2/18/1988 Denmark Florida, New Jersey, New York, Massachusetts ~1,150 57–59,63
Blue 4/6/1988 Denmark California, Florida, Illinois, Massachusetts, Michigan, Minnesota, New Jersey, ~5,000 56, 57
New York, North Carolina, Oregon, Pennsylvania, Texas
Anari 5/26/1989 Cyprus New York 50 cases 69
Anari 7/27/1989 Cyprus Illinois, Texas 79 cases 70
Anari 8/9/1989 Cyprus New York 80 cases 71
Halloumi 9/15/1989 Cyprus Florida, New Jersey, New York 14,400 77
Italian soft-ripened and semisoft 7/27/1990 Italy California, Connecticut, New Jersey, New York, Pennsylvania Unknown 78
Fontina 4/6/1993 Sweden California, Connecticut, Maryland, Massachusetts, Minnesota, New 85,080 89
Hampshire, New York, North Carolina, Pennsylvania, Rhode Island,
Washington State
Limburger 2/29/1996 Germany Florida, Indiana, Maine, Massachusetts, New Jersey, New York, Ohio, Utah, 813 102
Virginia
Jarlsberg 6/7/1996 Norway Alaska, California, Guam, Hawaii, Idaho, Montana, Nevada, Oregon, Utah, 30,727 101
Washington State
Ricotta 5/25/1999 Italy Connecticut, Florida, Delaware, Georgia, Indiana, Maryland, Massachusetts, 164 cases 389
Nebraska, New Hampshire, New Jersey, New York, North Carolina,
Pennsylvania, South Carolina, Tennessee, Virginia
St. Nectaire 6/20/2002 France California, Illinois, Ohio, New York 236 389
Havarti 11/21/2002 Unspecified Nationwide 52,998 389
Ricotta 11/26/2002 Italy Nationwide 6702 389
Manouri 12/9/2004 Greece California, Illinois, Michigan, New Jersey, New York, Pennsylvania, Virginia 1–7 cases 389

417
However, French authorities stressed that such a program would not be practical on a long-term
basis and hoped that the FDA would accept an expansion of the existing factory/product certification
program to include Listeria testing in the near future. Beginning in May 1986, FDA officials
announced that all shipments of French soft-ripened cheese lacking certification of analysis for
Listeria would be detained [24]. Inspections during the next 2 months uncovered L. monocytogenes
in two French cheeses—a noncertified Brie and a 6-lb certified lot of Muenster [24]—both of which
were presumably recalled internally.
Continued problems with Listeria-contaminated French soft-ripened cheeses prompted FDA
officials to revise the imported cheese surveillance program in August 1986 [19]. These changes
allowed immediate detention of French cheeses that were (1) manufactured at a non-government-
certified factory, (2) unaccompanied by a Listeria-free government certificate, (3) positive for
phosphatase, or (4) manufactured by one of several firms that were block-listed for Listeria.
Although these changes made importation of French cheeses more difficult, sampling of cheeses
that were manufactured at certified factories and accompanied by Listeria-free certificates was
decreased to the 20% level.
Between June and August 1986, three additional Class I recalls were issued for French semisoft/
soft-ripened cheese contaminated with L. monocytogenes (see Table 12.3). Two of three firms
involved in these recalls were previously block-listed by the FDA [19]. An additional Class I recall
issued for semisoft Morbier Rippoz cheese (see Table 12.3) was accompanied by the following
press release [35]:
Although Listeria is a rare cause of human illness, it can be life-threatening to pregnant women and
their fetuses, frail elderly persons, or other persons with weakened immune systems. In healthy adults,
it is a transient illness with such mild-to-moderate flu like symptoms as fever, headaches, and/or
gastrointestinal tract distress.
The language used in such press releases also has received considerable attention. These
messages to the public must be firm enough to accomplish the goals of the recall but not so alarming
as to create undue panic.
After considerable consultation, the governments of France and the United States reached
agreement on a French certification program for soft cheese [50,113]. Under this program, which
began on February 15, 1987, cheeses were tested before shipping using methods that were mutually
acceptable by both governments. French cheeses manufactured at certified factories would be
sampled at the 5% level, whereas other French cheeses (and cheeses manufactured in other countries
without certification programs) would be analyzed at the 20% level. In the event of a Listeria-
positive shipment, personnel at the French cheese factory would be required to investigate the
potential source of contamination and analyze every lot of cheese for listeriae in at least the next
20 consecutive shipments destined for the United States. Although a positive finding would not
automatically result in suspension of the certified status for a cheese factory under this program,
FDA officials reserved the right to initiate detentions and recalls if a product was found to contain
L. monocytogenes. After this certification program was accepted, only two additional recalls involv-
ing a French soft-ripened full-fat cheese have been reported (see Table 12.3).
Other Western European Countries
Despite the adverse publicity that the French cheese industry received throughout 1986 and 1987,
it must be recognized that the problem of Listeria-contaminated cheese was not limited to France.
Between October and December 1986, FDA inspectors isolated Listeria spp. from 4 of 74 (5.4%)
cheeses imported from Italy, 2 cheeses of which also contained high levels of phosphatase [46].
After finding similar percentages of positive samples during January, February, and March 1987,
FDA officials told representatives of the Italian government to either submit a draft for a certification
program (or recommend an alternate solution) or face a ban on importation of potentially hazardous
cheeses into the United States. As of April 30, 1987, only 13 of all Italian cheese samples analyzed
complied with current FDA safety standards: free of Listeria, phosphatase, and enteropathogenic
strains of E. coli. Additionally, 144 cheese samples examined as part of an import alert were
suspected to contain L. monocytogenes [45].
After isolating listeriae from Italian Pecorino Romano cheese prepared from goat’s milk
(see Figure 12.1) in June 1987 [365], the previous import alert was extended to include both soft
and hard varieties of Italian cheese [51]. (This was the first instance in which L. monocytogenes
was isolated from hard cheese.) Subsequently, the FDA ordered intensified sampling of soft and
hard cheese for the next 2 months [45].
Late in 1987, FDA officials also increased the number of cheeses sampled from Austria,
Denmark, Germany, Italy, and Switzerland as part of the agency’s ongoing imported cheese sur-
veillance program (see Figure 12.1) [58]. Although this action prompted the recall of several Danish
cheeses in early 1988 (see Table 12.3), no additional Class I recalls were issued during the remainder
of 1988 for imported cheese contaminated with L. monocytogenes. Heightened concern over the
presence of this pathogen in European cheeses (which stems from the 1987 cheeseborne outbreak
of listeriosis in Switzerland) and subsequent initiation of corrective action are probably both
responsible for the lack of Class I recalls issued during the remainder of 1988 and early 1989.
However, during the latter half of 1989, FDA officials issued (1) four separate Class I recalls for
Listeria-contaminated soft cheeses manufactured in Cyprus (see Table 12.3) and (2) an import alert
for contaminated soft and hard cheeses produced by two Italian firms [202]. The overall situation
regarding presence of L. monocytogenes in imported cheese has greatly improved since 1986 [79],
with only nine additional recalls of imported cheese issued during the 15-year period since 1990.
However, continued detection of listeriae in imported cheeses, as evidenced by the importation
refusal of 24 cheeses (23 from France and 1 from Colombia) from July 2004 to May 2005 [389],
confirms that ongoing surveillance of the soft and semisoft surface-ripened varieties, in particular,
is still necessary to safeguard public health, with these cheese categories having been documented
as important sources of L. monocytogenes in recent risk assessments.
SURVEYS AND MONITORING PROGRAMS FOR LISTERIA SPP. IN CHEESE

PRODUCED OUTSIDE THE UNITED STATES
In response to the 1985 cheeseborne listeriosis outbreak in California, scientists worldwide began
testing many types of cheese for Listeria spp. Given the possible ramifications of selling Listeria-
contaminated cheese and the fact that increasingly large quantities of European specialty cheeses
are exported to the United States and Canada, high priority was given to determining the incidence
of listeriae in Western European cheeses.
Since 1986, over 100 surveys have dealt with the incidence of Listeria spp. in various cheeses.
However, because these surveys now span a 20-year period and differ in design (i.e., number and
size of sample, site of sample collection [cheese factory or retail store], age of sample, portion of
cheese analyzed [surface, interior, or both]), and methods for detecting and identifying listeriae,
many of the Western European studies and those conducted elsewhere need to be interpreted with
some caution.
CANADA
Reports from federal monitoring programs in both Canada and the United States [58,247] indicate
that the incidence of listeriae in Canadian cheese (and nonfermented dairy products as described
in Chapter 11) is relatively low. However, one outbreak was reported in Quebec during 2002 in
which 17 cases of listeriosis were traced to four types of soft or semihard raw milk cheese that
were aged at least 60 days before consumption [149].
In the only Canadian survey thus far reported, Farber et al. [203] examined 182 samples of soft
and semisoft cheese for listeriae using the original FDA method. The cheeses analyzed in this survey
were produced at 61 different factories, most of which were located in the provinces of Ontario and
Quebec. Although all cheeses examined were Listeria-free, 19 of 79 samples (24%) were positive for
phosphatase, which suggests that these cheeses may have been prepared, at least in part, from raw
milk. In addition to this survey, the literature also contains one report of the unconfirmed isolation of
L. monocytogenes from Cheddar and Colby cheeses [260], both of which were manufactured from
raw milk and held a minimum of 60 days at ≥1.7°C (≥35°F) as required by the Canadian government.
Although the incidence of L. monocytogenes in Canadian-produced cheese appears to be low,
the pathogen has been detected in cheese exported to Canada from several Western European
countries, including Denmark, France, Switzerland, and Germany [67,247]. During April 1997 to
June 2005, the Canadian Food Inspection Service issued health hazard alerts for eight primary soft
or semisoft cheeses, of which four were domestic, with the remainder imported from France,
Greece, Italy, and Portugal [150]. In conjunction with the Canadian survey just discussed, Farber
et al. [203] also examined 187 samples of Western European soft and semisoft cheese (98 different
brands from 12 different countries) that were regularly exported to Canada. Three soft and semisoft
cheeses produced by the same manufacturer in France were positive for Listeria spp. (Table 12.4).
Two cheeses contained L. monocytogenes alone, whereas the third contained both L. monocytogenes
and L. innocua. In keeping with the previously described 1988 policy regarding foods that have
been directly linked to major listeriosis outbreaks, Canadian officials immediately recalled the
contaminated cheese. Although some of the tainted cheese was likely consumed before the recall,
no cases of listeriosis linked to consumption of this cheese were reported. Despite being labeled
as “manufactured from pasteurized milk,” the three French cheeses from which listeriae were
isolated yielded positive results with the phosphatase test, as did other cheeses imported from
Denmark, Finland, and Switzerland. Such findings, along with unpublished reports of phosphatase
in pasteurized dairy products, have raised serious questions as to the validity of the phosphatase
test. Results from one study [318] demonstrated that certain heat-labile, microbially produced
TABLE 12.4
Incidence of Listeria spp. in Soft/Semisoft European Cheeses Exported to Canada
between October 1985 and March 1987
Number of Samples Number of Positive Samples (%)

Country of Origin Analyzed L. monocytogenes L. innocua Other Listeria spp.
Austria 2 0 0 0
Denmark 43 0 0 0
Finland 1 0 0 0
France 104 3 (2.9) 1 (1.0) 0
Germany 16 0 0 0
Greece 2 0 0 0
Italy 3 0 0 0
The Netherlands 2 0 0 0
Norway 7 0 0 0
Portugal 2 0 0 0
Sweden 2 0 0 0
Switzerland 3 0 0 0
Total 187 3 (1.6) 1 (0.5) 0
Source: Adapted from Farber, J.M., M.A. Johnston, U. Purvis, and A. Loit. 1987. Surveillance of soft and
semi-soft cheeses for the presence of Listeria spp. Int. J. Food Microbiol. 5: 157–163.
TABLE 12.5
Incidence of Listeria spp. in French Cheese Destined for Domestic (France) and Foreign
Markets during 1986 and 1987
Market Type of Cheese Number of Samples Analyzed L. monocytogenes Other Listeria spp.
Domestica Soft 192 2 (1.0) 3 (1.6)

Other 135 0 1 (0.7)
Foreignb Soft (pasteurized milk) 736 11 (1.5) 10 (1.4)
Foreignb Soft (pasteurized milk) 736 11 (1.5) 10 (1.4)
Soft (raw milk) 355 6 (1.7) 5 (1.4)
Total 1418 19 (1.34) 19 (1.34)
a
Results from January 1987 to November 1987.
b
Results from January 1986 to September 1987.
Source: Adapted from Bockemuhl, J., G. Schulze, G. Marcy, and H.P.R. Seeliger. 1992. Number and distribution of
Listeria monocytogenes in soft cheese: how relevant is the natural contamination for causing disease? In Proceedings of
the 3rd World Congress on Foodborne Infections and Intoxications, Berlin, Germany, June 16–19, pp. 496–500.
alkaline phosphatases can mimic the natural phosphatase found in milk and produce false-positive
results in the Scharer test. Hence, the ability of the phosphatase test to determine whether or not
a dairy product such as cheese was made from pasteurized milk (or from pasteurized milk
contaminated with raw milk) needs to be reexamined.
FRANCE
Beginning in early 1986, sporadic Listeria-contamination problems have been associated with French
soft cheeses exported to the United States and Canada as well as England [247], Germany [76], The
Netherlands [128], Norway [247,402], Sweden [247], and Australia [241]. Hence, in an effort to
bolster public confidence in the safety of cheeses produced in France, the French government, in
cooperation with the Veterinary Service for Food Hygiene in France, conducted a series of systematic
surveys to determine the incidence of Listeria spp. in French cheeses destined for domestic and foreign
markets (Table 12.5) [136,225]. Overall, 1.34% of predominantly soft, 30-day-old French cheeses
examined during 1986 and 1987 contained detectable levels of both L. monocytogenes and other
Listeria spp., with few differences observed between cheeses destined for domestic or foreign con-
sumption. It also is noteworthy that comparable levels of contamination were seen in soft cheeses
prepared from raw and pasteurized milk. These findings agree with those of most other surveys and
suggest that soft cheeses are most likely to become contaminated with L. monocytogenes during the
latter stages of manufacture and ripening.
Although L. monocytogenes also was recovered from 10.3% of soft/semisoft French cheeses
marketed in Sweden from 1989 to 1993 [268], most surveys have suggested contamination rates
of <10%, with 108 of 2425 (4.5%) French cheeses surveyed (Table 12.6) reportedly harboring
L. monocytogenes. Based on the six independent surveys in Table 12.6, 4.7% of soft/semisoft and
4.5% of other cheese types yielded L. monocytogenes with contamination rates of 9.7 and 1.7%
for soft cheese prepared from raw/heat-treated and pasteurized milk, respectively. Thus, soft
cheeses prepared from raw milk were 5.7 times more likely to be contaminated as the same cheeses
prepared from pasteurized milk. Contamination rates of 46.9 and 87.0% have been reported for
soft surface-ripened cheeses prepared from raw milk [364], with L. monocytogenes populations
as high as 106 CFU/g detected on the cheese surface.
TABLE 12.6
Incidence of Listeria spp. in Cheeses Manufactured outside the United States
Number of
Samples Other Listeria
Country Type of Cheese Analyzed L. monocytogenes L. innocua spp. Ref.
Europe
Austria Soft red smear 4 1 (25.0) ND ND 339
Semisoft red smear 4 0 ND ND 339
Hard red smear 2 0 ND ND 339
Belgium Soft 886 62 (6.9) ND ND 268
Unspecified 929 214 (23.0) ND ND 182
Unspecified 262 35 (13.4) ND 68 (25.9) 393
Raw milk 71 2 (2.8) ND ND 328
Unspecified 37 0 0 0 139
Croatia Hard sheep’s milk 38 0 0 0 349
Czechoslovakia Soft-ripened 77 6 (7.8) ND ND 291
Sheep’s milk 10 0 ND ND 291
Hard 33 0 ND ND 291
Denmark Soft/semisoft 46 0 ND ND 268
Soft red smear 3 0 ND ND 339
Unspecified 25 8 (32.0) ND 8 (32.0) 136
France Soft-ripened 330 3 (0.9) 6 (1.8) 1 (0.3) 224
(raw milk)
Soft, surface-ripened 23 20 (87.0) ND ND 290
(raw milk
Soft (raw milk) 32 15 (46.9) 13 (40.6) 1 (3.1)a 198
Soft (heat-treated 5 0 2 (40.0) 0 198
milk)
Soft-ripened 873 12 (1.4) 5 (0.6) 3 (0.3) 224
(pasteurized milk)
Soft (pasteurized 32 3 (9.4) 5 (15.6) 0 198
milk)
Soft red smear 124 5 (4.0) ND ND 339
Semihard 289 10 (3.5) 1 (0.3) 1 (0.3) 224
Blue 126 0 0 0 224
Cottage 149 2 (1.3) 0 0 224
Unspecified 242 20 (8.3) ND 61 (25.2) 336
Germany Soft 712 33 (4.6) 58 (8.1) 4 (0.6)a 374
Soft 248 3 (1.2) 16 (6.4) 0 356
Soft 166 7 (4.2) ND ND 377
Soft (raw milk) 22 2 (9.1) 2 (9.1) 0 198
Soft (unripened) 8 0 0 0 396
Soft (mold-ripened) 117 4 (3.4) 5 (4.3) 0 396
Soft red smear 52 6 (11.5) ND ND 339
Soft (smear-ripened) 41 3 (7.3) 5 (12.2) 0 396

Number of
Soft/semisoft 256 23 (9.0) ND 7 (2.7) 136

Soft/semisoft 31 1 (3.2) ND ND 268
Semisoft 268 9 (3.4) 7 (2.6) 8 (3.0)a 374
Semisoft 45 0 1 (2.2) 0 356
Semisoft 89 4 (4.5) 1 (1.1) 3 (3.4)a 396
(unripened)
Semisoft (mold- 12 0 0 0 396
ripened)
Semisoft red smear 42 4 (9.5) ND ND 339
Semisoft (smear- 7 2 (28.6) 0 0 396
ripened)
Semihard 237 7 (3.0) 14 (5.9) 0 374
Semihard 108 6 (5.6) ND ND 377
Hard red smear 26 1 (3.8) ND ND 339
Hard 42 2 (4.8)b 0 1 (2.4)a 376
Acid curd 41 2 (4.8) 8 (19.5) 0 338
Acid 61 2 (3.3) 22 (36.1) 1 (1.6)a 376
Acid 48 1 (2.1) ND ND 377
Fresh 149 0 0 0 376
Processed 21 0 0 0 376
Unspecified 89 8 (9.0) 17 (19.0) 0 357
Greece/Crete Pichtogalo Chanion 62 4 (6.5) ND ND 310
Hungary Soft 25 0 ND ND 331
Soft 15 0 ND 4 (26.7)c 245
Semisoft 25 0 ND ND 331
Molded 10 2 (20.0) ND ND 331
Mold-ripened 10 0 ND ND 331
Hard 15 1 (6.7) ND 4 (26.7)c 245
Hard 10 0 ND ND 331
Curd 15 0 0 0 245
Processed 20 0 ND ND 331
Processed 15 0 0 0 245
Ireland Soft 40 1 (2.5) ND ND 247
Soft/semisoft 17 0 ND ND 169
Italy Soft 1,284 65 (5.1) 140 (10.9) 218 (17.0)d 163
Soft 400 8 (2.0) ND ND 160
Soft 54 2 (3.7) 4 (7.4) 0 176
Soft 29 0 ND ND 215
Soft 21 2 (1.6) 2 (1.6) 0 280
Soft 30 0 1 (3.3) 0 381
Soft 12 1 (8.3) ND ND 316
Soft-unripened 69 6 (8.7) ND ND 326
Soft-unripened 18 0 ND ND 162
Soft-ripened 136 9 (6.6) ND ND 326
Soft surface-ripened 16 0 ND ND 176
(mold)
(continued)

Number of
Soft surface-ripened 90 1 (1.1) 10 (11.1) 1 (1.1)c 381

Soft red smear 5 0 ND ND 339
Fresh 239 0 0 0 163
Fresh 38 2 (5.3) 2 (5.3) 0 176
Fresh 17 0 ND ND 215
Soft/semisoft 36 1 (2.8) ND ND 268
Semisoft red smear 14 3 (21.4) ND ND 339
Semisoft 118 0 5 (4.2) 0 163
Ripened 50 1 (2.0) 0 0 335
Hard red smear 4 1 (25.0) ND ND 339
Hard 99 0 0 0 163
Hard 40 0 0 0 176
Hard 10 0 ND ND 162
Goat’s milk 24 0 ND ND 143
Goat’s milk 21 1 (4.8) ND ND 162
Sheep’s milk 40 0 ND ND 143
Unspecified 1,846 24 (1.3) ND ND 123
Unspecified 373 22 (5.9) 50 (13.4) 70 (18.8)b 164
Unspecified 115 6 (5.2) 3 (2.6) 0 320
Unspecified 75 0 ND ND 303
Unspecified 52 4 (7.7) 0 0 136
Asiago 12 0 ND ND 143
Caciocavallo 12 0 0 0 386
Corleonese
Crescenza 212 0 ND ND 143
Crescenza 15 0 0 0 139
Gorgonzola 67 6 (9.0) 28 (41.8) 1 (1.1)a 320
Gorgonzola 58 3 (5.2) 21 (36.2) 0 315
Gorgonzola 44 4 (9.1) ND ND 316
Gorgonzola 40 2 (5.0) 4 (10.0) 0 176
Gorgonzola 40 2 (5.0) 2 (5.0) 0 139
Gorgonzola 25 2 (8.0) ND ND 215
Mozzarella 94 15 (16.0) 10 (10.6) 1 (1.1)a 320
Mozzarella— 94 0 ND ND 219
buffalo’s milk
Mozzarella 74 0 2 (2.7) 2 (2.7)e 176
Mozzarella 50 0 0 0 116
Mozzarella 30 0 0 0 198
Mozzarella 29 4 (13.8) 2 (6.9) 1 (3.4)e 315
Mozzarella 24 0 ND ND 143
Mozzarella 20 2 (10.0) ND ND 316
Mozzarella 14 0 ND ND 215
Pecorino 8 0 ND ND 143

Number of
Ricotta 50 2 (4.0) ND ND 144

Ricotta 32 0 0 0 167
Ricotta 11 0 ND ND 153
Ricotta—buffalo’s 18 0 ND ND 219
milk
Talleggio 45 0 7 (15.6) 2 (4.4) 315
Talleggio 38 0 ND ND 316
Taleggio 15 0 0 0 139
Taleggio 12 0 ND ND 143
Tosone—raw milk 45 0 2 (4.4) ND 266
The Netherlands Soft (raw milk) 938 43 (4.6) ND ND 382
Soft (pasteurized 484 10 (2.0) ND ND 382
milk)
Norway Soft (domestic) 850 0 ND NDg 333
Soft (imported) 90 10 (11.0) ND ND 333
Portugal Semihard raw 9 4 (44.4) ND ND 237
sheep’s milk
Hard pasteurized 12 2 (16.7) ND ND 237
sheep/goat’s milk
Hard pasteurized 11 0 ND ND 237
sheep/cow’s milk
Hard raw 2 2 (100) ND ND 237
sheep/cow’s milk
Hard/semihard 68 8 (11.8) 5 (7.4) 4 (5.9) 236
Spain Fresh 23 ND ND 1 (4.3)b 278
Soft 99 1 (1.0) 6 (6.1) 1 (1.0) 392
Soft 14 ND ND 1 (4.3)g 278
Semihard/hard 20 0 0 0 278
Blue (raw milk) 11 0 ND ND 269
Unspecified ~100 0 ND ND 247
Unspecified (raw 49 1 (2.0) 0 1 (2.0)a 321
milk)
Unspecified (raw 42 10 (23.8) ND ND 156
milk)
Unspecified 21 0 ND ND 156
(pasteurized milk)
Carneros—soft 18 1 (5.6) ND ND 300
goat’s milk
Cebrero 49 1 (2.0) ND ND 322
Queso 91 7 (7.7) 4 (4.4) 2 (2.2)a 137
fresco/cottage
Sweden Soft/semisoft 27 0 ND ND 268
Soft 24,954 324 (1.3) ND ND 304
Soft 604 40 (6.6) 38 (6.3) 0 142
Soft (mold-ripened) 54 0 0 ND 42
(continued)

Number of
Soft (smear-ripened) 18 4 (22.2) 3 (16.7) ND 42

Semisoft 205 4 (1.9) 0 0 142
Semisoft (mold- 261 7 (2.7) ND ND 142
ripened)
Semisoft (smear- 343 33 (9.6) ND ND 142
ripened)
Semisoft (smear- 69 6 (8.7) 13 (18.8) ND 42
ripened)
Switzerland Hard/semihard 36,379 2,692 (7.4) ND ND 304
Hard 88 0 0 0 142
Unspecified 17 0 ND 1 (5.9) 136
Turkey Kashor 30 0 ND ND 232
White 82 11 (13.4) ND ND 231
White 50 2 (4.0) 6 (12.0) 0 114
White 30 0 ND ND 232
Unspecified 224 4 (1.8) 7 (3.1) 0 238
Unspecified (raw 40 2 (6.1) ND ND 232
milk)
Yugoslavia White-brined 170 9 (5.3) ND ND 182
United Kingdom
England Soft 251 1 (0.4) 9 (3.6) 0 271
England/Wales Soft 1,437 16 (1.1) ND ND 218
Soft 251 10 (4.0) ND ND 271
Soft 222 23 (10.4) ND ND 314
Soft 131 0 ND ND 135
Soft-ripened 769 63 (8.2) ND ND 234
Soft-unripened 366 4 (1.1) ND ND 234
Hard 66 1 (1.5) ND ND 234
Ewe’s milk 141 1 (0.7) ND ND 234
Goat’s milk 476 22 (4.6) ND ND 234
Northern Ireland Soft 33 0 0 1 (3.3)a 241
Scotland Soft 27 3 (11.1) ND ND 337
Unspecified 305 3 (1.0)c 0 1 (3.3)a 124
Elsewhere
Australia Soft 437 15 (3.4) ND 24 (5.5)c 115
Soft 28 1 (0.2) 0 0 115
Unspecified 126 1 (0.8) 1 (0.8) 0 233
Brazil Minas Frescal 20 0 0 0 155
Coalho 43 1 (2.3) ND ND 205
Gorgonzola, Brie, 53 3 (5.7) 7 (13.1) 1 (1.9) 172
Roquefort

Number of
Minas Frescal 12 0 1 (8.3) 0 362

Homemade Minas 17 7 (41.2) 3 (17.7) 0 172
Frescal
Commercial Minas 33 1 (3.0) 3 (9.0) 6 (18.2) 172
Frescal and Ricotta
Chile Soft 256 2 (0.8) ND ND 167
Hard 155 0 ND ND 167
Costa Rica Soft 20 9 (45.0) ND ND 293
Egypt Damietta 50 1 (2.0) 1 (2.0) 0 204
Kareish 100 1 (1.0) 3 (3.0) 1 (1.0)h 204
Japan Domestic fresh 92 0 ND ND 296
Domestic 94 0 ND ND 296
soft/semisoft
Domestic 105 0 ND ND 296
semihard/hard
Imported fresh 94 0 ND ND 296
Imported 418 16 (3.8) ND ND 296
soft/semisoft
Imported 403 0 ND ND 296
semihard/hard
Imported other 55 0 ND ND 296
Jordan White brined 67 0 ND ND 192
Korea Pasteurized 45 0 0 0 372
processed
Mexico Chihuahua— 40 0 0 0 372
pasteurized milk
Fresh Panela—raw 40 6 (15.0) 6 (15.0) 14 (35.0) 348
and pasteurized milk
Manchego— 40 0 0 0 372
pasteurized milk
Morocco Domestic fresh 20 0 ND ND 190
Imported mold- 45 0 ND ND 190
ripened
United Arab Domestic 53 0 0 0 228
Emirates
Imported 196 2 (1.0) 2 (1.0) 0 228

aL. seeligeri.
bIsolated from sheep’s milk cheese.
cNon–L. monocytogenes.
d185 L. welshimeri, 18 L. ivanovii, 15 L. murrayi.
eL. grayi
f63 L. welshimeri, 6 L. ivanovii, 1 L. murrayi.
gListeria sp.
hL. welshimeri.
In another French survey [224], workers at the Veterinary Service for Food Hygiene recovered
L. monocytogenes as well as L. innocua and other Listeria spp. from 0.3 to 3.5% of cottage, soft-
ripened, and semihard cheeses examined (Table 12.6). However, in contrast to other surveys,
comparable contamination rates were observed for soft-ripened cheese prepared from raw and
pasteurized milk.
Additional efforts in France have focused on characterizing listeriae isolates from cheese and
other milk products. Listeria spp. recovered from French dairy products during 1986 included
L. monocytogenes (370 strains), L. innocua (134 strains), L. seeligeri (17 strains), and L. ivanovii
(1 strain), with 299 of 370 (80%) and 48 of 370 (13%) L. monocytogenes strains belonging to
serovars 1/2 and 4b, respectively. Additional surveys conducted in France [136,225] and Belgium
[116] from 1985 to 1990 indicated that a disproportionately large number of L. monocytogenes
strains isolated from cheese and other dairy products were serovar 1/2. This situation appears to
be reversed in the United States, with isolates of serovar 4b typically outnumbering those of
serovar 1/2.
In one of the earlier studies, phage typing was used to characterize particular L. monocytogenes
strains isolated from dairy products. Although only 33.8% of all L. monocytogenes strains isolated
from French dairy products during 1986 and 1987 were typeable using the available set of phages,
some phage types were unique to particular regions within France [136]. In some instances, excellent
correlations were observed between specific phage types and certain cheese varieties, with some
phage types even being specific to a particular dairy. Such findings have led to better control of
the listeriosis problem within the dairy industry.
The inadvertent isolation of L. monocytogenes from French soft-ripened cheese by FDA officials
in January 1986 prompted several additional surveys of French cheese exported to other Western
European countries. Working in The Netherlands, Beckers et al. [128,129] examined 69 samples
of French soft cheese (i.e., Brie and Camembert) for L. monocytogenes using both direct plating
and cold enrichment. The pathogen was recovered from 7 of 69 (10.1%) cheeses at levels ranging
between 103 and 106 CFU/g. Cold enrichment uncovered three additional cheeses with L. mono-
cytogenes for a total of 10 positive samples. Although all 10 Listeria-positive cheeses were prepared
from raw milk, comparable rates of contamination have been reported for cheese manufactured
from raw and pasteurized milk [136,224].
GERMANY
Germany experienced a major outbreak of listeriosis shortly after World War II. This outbreak,
which may have resulted from consumption of contaminated raw milk, led to an increased interest
in listeriosis research, and this in turn prompted Professor H. P. R. Seeliger to publish his time-
honored monograph Listeriosis in 1961. During the last 40 years, the late Professor Seeliger emerged
as one of the world’s leading authorities on listeriosis. In addition, he operated a listeriosis research
center at the Institut für Hygiene und Mikrobiologie der Universität Würzburg to which Listeria
isolates could be sent for biochemical and serological confirmation. Hence, it is not surprising that
the incidence of listeriae in cheese has received considerable attention in Germany.
Since 1990, German officials have been enforcing a policy similar to that adopted in Canada
in which only contaminated foods previously associated with foodborne listeriosis outbreaks are
recalled from the marketplace. Although spared the heavy economic losses experienced by the
United States and France, Germany and most other European countries have not escaped the
Listeria problem completely unscathed. Despite rigorous testing, Listeria-laden German blue-
veined cheese was recalled from France [247], with a similar recall being issued for sour milk
cheese exported to Canada [67,247] and The Netherlands [247]. In 2000, Germany also recalled
one hard and one unspecified cheese because of L. monocytogenes contamination. Consequently, a
series of Listeria-monitoring programs now continues for German soft, semisoft, semihard, and hard
cheeses as well as cultures, cheese byproducts, and the general environment within cheese factories.
Results from various surveys made since 1986 (see Table 12.6) indicate that 0–11% (average of
4.4%) and 0–28.6% (average of 2.9%) of the soft and semisoft cheeses marketed in Germany
contained L. monocytogenes, respectively, with the highest incidence of listeriae generally occurring
in smear-ripened varieties. With few exceptions, L. innocua was isolated more frequently from soft
and semisoft cheese than was L. monocytogenes. Although somewhat similar average percentages
were reported for the incidence of L. monocytogenes in semihard (3.8%) and hard cheese (4.8%),
two of the three hard cheeses that contained L. monocytogenes were reportedly manufactured from
ewe’s rather than cow’s milk. Overall, it appears that the Listeria contamination rate for hard
cheeses prepared from cow’s milk may still be relatively low, as also was observed in Switzerland
(see Table 12.6). Hence, these results from Germany generally agree with those from other
surveys in that L. monocytogenes was found more frequently in high- rather than low-moisture
cheese, particularly smear- and surface-ripened varieties.
Of the three remaining categories of German cheese shown in Table 12.6, only acid curd cheese
was positive for listeriae. The apparent absence of Listeria spp. from samples of fresh (i.e., cottage)
and processed cheese is expected, because relatively severe heat treatments are used in their
manufacture. Even if a few listeriae survived cheesemaking, most, if not all, of the survivors would
have been sublethally injured during exposure to heat and/or acid and would therefore be unable
to grow in most selective enrichment broths that are commonly used for examining cheese.
In the only other study thus far reported, Weber et al. [396] examined various German cheeses,
including 11 types manufactured from ewe’s and goat’s milk, for listeriae (Table 12.7). Although
all cheeses prepared from ewe’s or goat’s milk were free of L. monocytogenes, L. innocua was
detected in one sample of fresh goat’s milk cheese.
In addition to cows, lactating sheep and goats can also shed L. monocytogenes in their milk.
However, information concerning the incidence of L. monocytogenes in cheese prepared from
nonbovine milk is far less plentiful. From the survey data presented in Table 12.6, 29 of 575 (5.0%)
samples of goat’s milk cheese from Italy and the United Kingdom yielded L. monocytogenes, as
did 5 of 228 (2.2%) sheep’s milk cheeses analyzed in Italy, the United Kingdom, and Croatia, with
112 samples of Italian buffalo milk Mozzarella testing negative. In addition to the aforementioned
survey of German hard cheese produced from ewe’s milk [374], Tham [378] also reported isolating
TABLE 12.7
Incidence of Listeria spp. in Domestic and Imported Cheese
Analyzed in Germany between October 1987 and June 1988

Type of Cheese/Milka Analyzed L. monocytogenes L. innocua
Fresh/cow 21 2 (9.5) 0
Fresh/goat 1 0 1 (100.0)
Soft/cow 307 8 (2.6) 11 (3.6)
Soft/goat 1 0 0
Soft/ewe 3 0 0
Semisoft/cow 144 19 (13.2) 8 (5.6)
Semisoft/ewe 6 0 0
Semihard/cow 22 0 11 (50.0)
Hard/cow 4 0 0
a Milk from which cheese was manufactured.
Source: Adapted from Weber, A. von, C. Baumann, J. Potel, and H. Friess. 1988.
Nachweis von Listeria monocytogenes und Listeria innocua in Käse. Berl. Münch.
Tierärztl. Wochenshr. 101: 373–375.
L. monocytogenes from one sample of 8-week-old goat cheese marketed in Sweden. Given similar
L. monocytogenes contamination rates for cheeses prepared from cow’s milk, the cheesemaking
procedure as well as the moisture content and other aspects of the cheese that can enhance growth
and/or survival of Listeria are of greater importance than the source of milk.
The incidence of L. monocytogenes in the remaining cheeses prepared from cow’s milk was
similar to those values obtained in other studies (see Table 12.6), with the pathogen being detected
in 2.6 and 13.2% of the soft and semisoft (presumably mold- and smear-ripened varieties) cheeses
examined, respectively. The unusually high incidence of L. monocytogenes in fresh curd cheese
(9.5%) is probably the result of contamination during later stages of manufacture or packaging as
well as the small number of samples examined. As was true for surveys of French soft-ripened
cheese discussed earlier, most L. monocytogenes isolates were serovar 1/2 (22 strains), as also
reported by Schönberg et al. [357], with the remaining seven isolates being classified as 4ab or 4b.
However, because most clinical L. monocytogenes isolates from Germany are serovar 4b, it appears
that some questions remain concerning the ability of present isolation methods to recover
L. monocytogenes serovar 4b as compared with other serovars from various foods, including cheese.
ITALY
Public health concerns raised in the United States and elsewhere following the isolation of L.
monocytogenes from various cheeses have now prompted nearly 30 surveys of cheeses manufactured
in Italy (see Table 12.6). Overall, L. monocytogenes was recovered from 204 of 6655 (3%) Italian
cheeses surveyed, with this pathogen being most prevalent in Gorgonzola (6.9%), followed by
mozzarella (4.9%) and various soft cheeses (4.3%). In another study, Cantoni et al. [151] reportedly
isolated L. monocytogenes from 14 of 375 (3.7%), 14 of 216 (6.5%), and 5 of 95 (5.3%) samples
of Gorgonzola (blue-veined), Tallegio (soft, surface-ripened), and other Italian cheeses, respectively.
Although a follow-up study demonstrated L. monocytogenes at levels of <100 to 12,000 CFU/g in
Gorgonzola and Taleggio cheese, respectively, the pathogen was never recovered from 1150 samples
of soft, semisoft, semihard, or hard cheese or from 72 samples of pasta filata-type cheese such as
provolone and mozzarella. When present, however, L. monocytogenes serovar 1 typically predom-
inated [351], as has been reported for other European cheeses.
Between January 1987 and September 1988, Massa et al. [280] also examined 54 soft rindless
(i.e., Mascarpone, mozzarella, Crescenza) and 67 soft thin-rind (i.e., Italico, Caciotta) cheeses
produced by both large and small northern Italian factories for listeriae and E. coli. Listeria
monocytogenes was detected in only 2 of 47 (4.2%) thin-rind cheeses manufactured by one small
factory, with core samples from these two positive cheeses being negative for the pathogen. Although
all other thin-rind and rindless soft cheeses were free of L. monocytogenes, two mozzarella cheeses
contained detectable levels of L. innocua. According to these investigators, E. coli populations in
these cheeses ranged from <10 to 8 × 105 CFU/g, with the two L. monocytogenes-positive cheeses
containing 104 E. coli CFU/g. However, because 14 similar Listeria-free cheeses also contained
103 E. coli CFU/g, E. coli is clearly a poor indicator organism for possible presence of listeriae.
SWITZERLAND
Most Western European countries have experienced various degrees of economic loss from Listeria-
contaminated cheese; however, thus far only Switzerland, France, and the United States have been
forced to deal with major outbreaks of cheeseborne listeriosis. Well before the 1987 listeriosis
outbreak in Switzerland (linked to consumption of Vacherin Mont d’Or soft-ripened cheese), Swiss
officials began examining various cheeses for listeriae. Although these surveys apparently were
prompted by the 1985 listeriosis outbreak in California, an unusually high incidence of unexplain-
able listeriosis cases in certain areas of Switzerland may have provided added incentive to initiate
these surveys. A two-stage Listeria-monitoring program was later established for cheese and other
dairy products with random testing of 10-g samples obtained at both the factory and retail levels
[247]. According to the Federal Bureau of Health, such samples must be completely free of L.
monocytogenes before the product is deemed acceptable. Based on results from a 10-year-long
survey of more than 60,000 cheeses conducted by the Swiss Dairy Research Station during
1990–1999, 7.4 and 1.3% of all hard/semihard and soft cheeses yielded L. monocytogenes, respec-
tively. In this study, serotype 1/2b was more frequently isolated from hard/semihard cheese, whereas
serotype 1/2a predominated in soft cheese.
Working in Switzerland, Breer [142] examined 799 domestic and imported cheese samples for
listeriae during the winters of 1985 and 1986. Various Listeria spp. were detected in 19.2% of the
soft surface-ripened cheeses, all of which were traced to 10 Swiss and a few foreign manufacturers.
During follow-up investigations of these 10 cheese factories in Switzerland, Listeria spp. were
isolated from surfaces of various cheeses and also from curing and smearing brines, wastewater
sinks, and surfaces of wooden boards used in cheese ripening. In addition, identical serovars
of L. monocytogenes (1/2b and/or 4b) and L. innocua were isolated repeatedly from the same
cheese factories. These findings demonstrate that ample opportunity existed for cheese to become
contaminated with listeriae during the later stages of manufacture and ripening.
Subsequently, Breer [142] reported that 4.9 and 4.7% of all cheeses sold in Switzerland
contained L. monocytogenes and L. innocua, respectively (see Table 12.6). Of equal importance is
the fact that both Listeria spp. were isolated more frequently from soft (6.6, 6.3%) than semisoft
cheese (1.9, 0%) and that neither organism was detected in 88 samples of hard cheese.
During 1986, Breer [141] also found that 12.9 and 10.0% of soft surface-ripened cheeses
manufactured in Switzerland were contaminated with L. monocytogenes and L. innocua, respectively
(Table 12.8). The incidence of both Listeria spp. was generally twice as high in smear- rather than
mold-ripened cheese. As in previous studies [339], the rate of Listeria contamination was typically
independent of the type of milk (raw or pasteurized) from which smear-ripened cheeses were
manufactured.
These Swiss studies, along with several of the aforementioned German surveys, indicate a
greater likelihood of isolating L. monocytogenes and L. innocua from high- rather than low-moisture
cheese, with special emphasis on mold- and smear-ripened varieties. In support of this observation,
Bannerman and Bille [125] found that 110 of 449 (24.5%) rinds from soft cheese produced in
TABLE 12.8
Incidence of Listeria spp. in Soft Mold- and Smear-Ripened
Cheeses Manufactured in Switzerland during 1986
from Pasteurized and Raw Milk

Type of Cheese Analyzed L. monocytogenes L. innocua
Mold-ripened
Raw milk 22 2 (9.1) 0
Pasteurized milk 9 0 1 (11.1)
Smear-ripened
Raw milk 17 3 (17.6) 4 (23.5)
Pasteurized milk 22 4 (18.2) 2 (9.1)
Total 70 9 (12.9) 7 (10.0)
Source: Adapted from Breer, C. 1986. The occurrence of Listeria spp. in cheese. In
Proceedings of 2nd World Congress on Foodborne Infections and Intoxications,
Institute of Veterinary Medicine, Robert von Ostertag Institute, Berlin, pp. 230–233.
Switzerland were contaminated with Listeria spp., including a high percentage of samples with
L. monocytogenes. During an additional survey made between October 1986 and September 1987
[42], L. monocytogenes was detected in 4 of 18 (22.2%) and 6 of 67 (8.7%) smear-ripened soft
(i.e., Limburger, Romadur, Muenster, Reblochon) and semisoft (i.e., St. Paulin, Tilsiter, Mutschli,
Raclette) cheeses, respectively, with many cheeses also containing L. innocua. From the apparent
widespread distribution of Listeria within some cheese factory environments, it follows that cheeses
prepared from raw and pasteurized milk are equally likely to contain listeriae. Additional informa-
tion concerning the incidence and control of listeriae in dairy factories and other food processing
facilities is given in Chapter 18.
OTHER EUROPEAN COUNTRIES

As already implied, ongoing Listeria problems that have affected the European cheese industry are
not limited to France, Germany, Italy, and Switzerland. Other European nations, including Austria,
Belgium, Denmark, England, Finland, Greece, Hungary, The Netherlands, Spain, and Sweden, also
expressed concern in a March 1989 poll conducted by the International Dairy Federation [247].
According to the survey, L. monocytogenes was isolated from domestic cheese sold in Austria (soft
cheese), Belgium (rind-type cheese), Denmark (various soft cheeses), England (various cheeses),
Ireland (soft farm-house cheese), The Netherlands (young farm-house Gouda), and Sweden (goat’s
milk cheese), with this pathogen later being identified in cheeses from Czechoslovakia (soft-ripened)
[291], Greece (Feta, Pichtogalo Chanion) [182,310], Hungary (mold-ripened) [245], Ireland (soft)
[247], Norway (soft imported) [333], Portugal (hard/semihard) [237], Turkey (soft, white)
[232,238], and Yugoslavia (white brined) [182] (see Table 12.6). Denmark, England, and Sweden
have also experienced problems with soft or semisoft cheeses imported from Denmark, France,
Germany, and Italy (Table 12.9). In contrast, Norway [124,399] and Spain [247] have been primarily
TABLE 12.9
Incidence of L. monocytogenes in Cheeses Marketed in Sweden from 1989 to 1993
Country of Type of Cheese Type of Cheese Milk

Origin White Mold Green/Blue Mold Smear-Ripened Other Heat-Treated Raw
Austria — 0/1a — — 0/1 —

Denmark 0/19 0/27 — — 0/46 —
England — 0/12 — — 0/12 —
France 15/119 (12.6) 0/23 2/18 (16.7) 0/14 5/144 (3.5) 13/30 (43.3)
Germany 0/8 1/19 (5.3) 0/1 0/3 1/31 (3.2) —
Greece — — — 0/1 0/1 —
Italy 0/5 0/25 1/2 (50.0) 0/4 1/36 (2.8) —
The Netherlands — — 0/2 — 0/2 —
Norway — 0/1 — — 0/1 —
Romania — — — 0/1 0/1 —
Spain — 0/1 — — 0/1 —
Sweden 0/3 0/13 0/3 0/8 0/26 0/1
Total 15/154 (9.7) 1/122 (0.8) 4/26 (15.4) 0/31 7/302 (0.4) 13/31 (41.9)
a Number of positive samples/number of samples analyzed (%).
Source: Adapted from Loncarevic, S., M.-L. Danielsson-Tham, and W. Tham. 1995. Occurrence of Listeria monocytogenes
in soft and semi-soft cheeses in retail outlets in Sweden. Int. J. Food Microbiol. 26: 245–250.
affected by imported soft and blue-veined cheeses. Thus, in addition to France, Italy, Switzerland,
and Germany, other EU member countries including Austria, Belgium, Denmark [68], England,
Finland, Greece, Hungary, The Netherlands, and Sweden also have developed programs to actively
monitor the incidence of listeriae in domestic/imported cheese (especially soft surface-ripened
varieties) and manufacturing environments within cheese factories.
As of March 1989 [247], Austria, Denmark, France, Germany, Greece, Hungary, Italy, Sweden,
and Switzerland had regulations regarding the sale of cheese and other foods contaminated with
L. monocytogenes, with most European countries now attempting to prevent distribution and sale
of cheese (and in some instances other ready-to-eat foods) containing ≥100 L. monocytogenes
CFU/g. Because these observations along with the 1987 listeriosis outbreak in Switzerland involving
Vacherin Mont d’Or cheese both support the widespread notion that L. monocytogenes poses a
significant health threat to certain segments of the population, the European Economic Community
Consumers Association published a list of soft and semisoft cheeses manufactured in France and
Switzerland that should be avoided by susceptible individuals—pregnant women, immunocompro-
mised adults, and the elderly. This highly controversial list of cheeses (and brands when applicable)
included Brie (La Renommee), Muenster (Ermitage), Crème de Bleu (Diapason), Lys Bleu, Cam-
embert (I Signy), Tilsit, Fourme de Bresse, Bleu de Bresse, Reblochon, Pont L’Eveque, Gruyère,
and Vacherin Mont d’Or.
In February 1988, a case of cheeseborne listeriosis was reported in England in which a
40-year-old woman contracted meningitis shortly after consuming Anari-type soft goat’s milk
cheese that contained L. monocytogenes at levels >107 CFU/g. During follow-up investigations at
the factory [224], the same L. monocytogenes strain also was isolated from 8 of 11 and 4 of 8
factory and retail samples of Halloumi and Cheddar cheese, respectively, as well as single samples
of Gjestost and soft chive cheese. In addition, L. innocua also was recovered from several samples
of Halloumi and Cheddar cheese. As in the previously described studies by Pini and Gilbert [283]
and Massa et al. [280], no clear relationship was observed between the presence of L. monocytogenes/
L. innocua and coliforms/E. coli. According to several additional surveys, some Costa Rican [293]
and Turkish cheeses [179] contained 104 L. monocytogenes and ≥102 coliforms CFU/g. Hence,
coliforms appear to be relatively poor indicators of Listeria contamination.
The Public Health Laboratory Service in London coordinated a large-scale survey in which
various dairy products marketed in England and Wales were sent to 46 laboratories throughout the
country for Listeria testing. Results from this comprehensive survey (see Table 12.6) indicated that
8.2, 1.1, 1.5, and 4.1% of the soft-ripened, soft-unripened, hard, and goat’s milk cheese manufac-
tured in England and Wales contained L. monocytogenes; 75, 42, and 7 isolates classified as serovar
1/2, 4b, and 4, respectively. Among the soft-ripened varieties, 13 cheeses harbored >103 L. mono-
cytogenes CFU/g with 3 samples exceeding 105 CFU/g. Of these 13 cheeses, 7 were prepared from
raw milk, with only 1 being manufactured in the United Kingdom. In contrast, only 2 of 33 cheeses
prepared from ewe’s or goat’s milk contained >500 L. monocytogenes CFU/g. Overall, the incidence
of this pathogen was similar in imported (7.4%) and U.K.-produced cheese. These incidence rates
and serovar distribution patterns for L. monocytogenes in soft-ripened and unripened cheese are
generally similar to those observed in most other European studies [265,268].
As just suggested, numerous surveys for incidence of listeriae in cheese also have been
completed in many of these aforementioned countries which, with the exception of Denmark and
France, have not experienced major economic problems associated with Listeria-contaminated
cheese. Following the 1986 report of an English woman who contracted listeriosis after consuming
French soft cheese [126], two English researchers [314] examined 45 domestic soft cheeses as well
as 177 soft cheeses imported from France, Italy, Cyprus, Germany, Denmark, and Lebanon for
Listeria spp. and E. coli. (Table 12.10). Overall, L. monocytogenes was isolated from 2 of 45 (4.4%)
English cheeses and 21 of 177 (11.9%) soft cheeses imported from France, Italy, and Cyprus.
Populations of L. monocytogenes in contaminated cheese ranged from <102 to 105 CFU/g, with
9 of 12 French cheeses containing ≥104 CFU/g. Despite differences in media and methods used in
TABLE 12.10
Incidence of L. monocytogenes and E. coli in Soft Cheese Sampled
in England during 1987
Number of L. monocytogenes Number of Samples

Samples Number of Positive with >10 E. coli
Country of Origin Analyzed Samples (%) Level/g CFU/g (%)
England 85 12 (14.1) <102–105 32 (37.6)

France 45 2 (4.4) <102 14 (31.1)
Italy 44 7 (15.9) <102–104 12 (27.3)
Cyprus 20 2 (10.0) <102 3 (15.0)
West Germany 17 0 ND 9 (52.9)
Denmark 6 0 ND 2 (33.3)
Lebanon 5 0 ND 1 (20.0)
Total 222 23 (10.4) ND–105 73 (32.9)
Note: ND = not detected.
Source: Adapted from Pini, P.N. and R.J. Gilbert. 1988. The occurrence in the U.K. of Listeria species in
raw chickens and soft cheese. Int. J. Food Microbiol. 6: 317–326.
various surveys, the contamination rate of 14.1% for soft French cheeses calculated in this study
was close to the 14.5% previously observed for French soft cheese exported to The Netherlands.
As was true of previous surveys of French dairy products, all strains of L. monocytogenes (except
one nontypeable strain) were of serovar 1/2 or 4b, with the former predominating. Listeria innocua,
the only other Listeria sp. detected during this survey, was isolated from 9 of 85 (10.6%), 7 of 44
(15.9%), 2 of 45 (4.4%), and 1 of 6 (16.7%) soft cheeses produced in France, Italy, England, and
Denmark, respectively, with 6 of 222 (2.7%) cheeses containing both Listeria spp. Although E.
coli populations exceeded 10 CFU/g in 73 of 222 (32.9%) cheeses examined, no correlation was
again observed between the presence of L. monocytogenes or L. innocua and contamination with
E. coli. In fact, E. coli was detected at >10 CFU/g in only 10 of 23 (43.5%) cheeses that contained
the pathogen. In this study, 10 of 23 (43.5%) cheeses contaminated with L. monocytogenes were
prepared from pasteurized milk, whereas 2 and 11 of the remaining positive cheeses were manu-
factured from raw milk and milk of undetermined processing, respectively. Thus, as in previous
studies, the type of milk (i.e., raw or pasteurized) from which cheese is made appears to be a poor
indicator of possible Listeria contamination.
Problems regarding the occasional presence of listeriae in soft cheese also have surfaced in the
Scandinavian countries, with L. monocytogenes being recovered from 0.3% of Norwegian cheeses
[151] and also identified in Danish Esrom and Blue Costello cheese that was exported to Norway
[196,308], Sweden [196], and the United States. Although four Class I recalls were issued for
Danish Esrom and Blue cheese in the United States (see Table 12.3), both of these cheeses
(~20% of which were contaminated) were on sale for up to 2 months in Norway before being
removed from the market, apparently without incident [399]. Danish officials also took steps to
prevent unsold cheese from reaching consumers and have since developed a Listeria surveillance
program [68] similar to that instituted in the United States, with routine testing of various cheeses
as well as cheesemaking facilities.
Additional concern over the microbiological safety of various cheeses also led to isolation of
L. monocytogenes serovar 1/2b from two presumably French soft-ripened cheeses (one prepared
from raw milk and the other from pasteurized milk) that were exported to Norway and Sweden
[379]. Surface and interior samples from the raw milk cheese contained 7.5 × 105 and 1.0 × 102
L. monocytogenes CFU/g, respectively, whereas corresponding samples from the pasteurized milk
cheese contained 4.0 × 106 and 1.0 × 106 L. monocytogenes CFU/g. The reasons for nonuniform
distribution of listeriae in soft-ripened cheese will be explored in the second half of this chapter.
Although cheese prepared from raw milk contained 3 × 106 to 7 × 106 coliforms CFU/g, coliform
tests indicated that the remaining cheese manufactured from pasteurized milk was fit for con-
sumption. These findings reinforce the fact that coliform-free cheese may not necessarily be free
OTHER COUNTRIES
Reports of Listeria-contaminated cheese in countries beyond North America and Europe also are
beginning to surface (see Table 12.6). In 1987, L. monocytogenes was recovered from ricotta cheese
manufactured in Melbourne, Australia [390], and again in 2002 in ricotta cheese from New Zealand
[111]. This earlier event, along with identification of L. monocytogenes in the same imported brands
of Danish blue and French brie cheese that were recalled in the United States [241], prompted
Venables [390] to determine the incidence of listeriae in Camembert, blue vein, ricotta, cottage,
pasta filata, high-moisture, low-acid, and other cheese varieties manufactured in and around
Melbourne. Overall, L. monocytogenes was recovered from 6 of 338 (1.8%) cheeses produced by
five different manufacturers, with the pathogen identified as being present in pasta filata (three
samples), ricotta (two samples), and shredded (one sample) cheese. One cheese also contained
L. seeligeri. Simultaneous identification of L. monocytogenes in environmental samples from all
factories producing Listeria-positive cheese strongly suggests that these cheeses were contaminated
during manufacture or ripening. In keeping with U.S. policies, attempts were made to remove
tainted cheese from the marketplace. Furthermore, after thoroughly cleaning and sanitizing the
factory, government officials required that Listeria-free cheese be produced for 12 consecutive days
before being released to the public. Recent discovery of nonpathogenic listeriae in raw milk
from neighboring New Zealand and later L. monocytogenes in several cheeses manufactured in
New Zealand [110] also has prompted the New Zealand government to develop the Food Safety
Authority Certificate of Listeria Clearance for shipment of products to Australia.
Information regarding the presence of listeriae in dairy products produced elsewhere is scarcer
still, with results from a March 1989 IDF Survey [247] indicating that L. monocytogenes had not
yet been isolated from any dairy products manufactured in South Africa, Israel, or the former Soviet
Union. Most cheese-related surveys from other countries have yielded negative results, with detec-
tion of L. monocytogenes largely being limited to a few high-moisture domestic cheeses produced
in Egypt [204] as well as Chile [166], Costa Rica [293], and Venezuela [177]. However, notably
higher isolation rates have been reported for soft and Hispanic-style cheeses from Brazil and
Mexico—particularly for those cheeses that were homemade or prepared from raw milk. In the
Brazilian survey by DaSilva et al. [172], homemade Minas Frescal (a fresh white soft cheese) had
an L. monocytogenes contamination rate nearly 14 times greater than commercially produced cheese
of the same type, with isolates belonging to serotype 1/2a being most common followed by serotype
4b—the serotype most often associated with outbreaks of foodborne listeriosis. Similar findings
were reported in the Mexican survey by Saltijeral et al. [348], with L. monocytogenes being more
prevalent in raw milk Fresh Panela cheese sold by street vendors as compared to the same cheese
manufactured commercially from pasteurized milk. Such homemade cheeses clearly pose a greater
public health risk than do commercially produced cheeses, as has been reported in regard to several
illegally produced Hispanic-style cheeses that were linked to cases of listeriosis in the United States.
Given the enormous volume of dairy products exported to other countries and the fact that
L. monocytogenes has been isolated from the natural environment of all seven continents except
Antarctica, it appears that developing countries are unlikely to remain completely untouched by
the problems associated with Listeria-contaminated foods. Consequently, interest in the incidence
of listeriae in dairy products and other ready-to-eat foods will likely continue in the years ahead.
BEHAVIOR OF L. MONOCYTOGENES IN FERMENTED MILKS

Before the well-known 1985 outbreak of cheeseborne listeriosis occurred in California, very little
information was available about the behavior of L. monocytogenes in fermented milks and cheese. In
fact, at the time of this outbreak, a search of the scientific literature uncovered only four such studies,
which were reported from Bulgaria [246,383] and Yugoslavia [363,367] between 1965 and 1979.
Hence, in addition to prompting numerous surveys for Listeria spp. in cheese, confirmation of cheese
as an important vehicle in foodborne listeriosis has led to several hundred publications addressing
the fate of L. monocytogenes in fermented dairy products during manufacture and storage.
As described elsewhere in this book, cows, sheep, and goats can shed L. monocytogenes
naturally in their milk during lactation. According to results from recent environmental surveys of
dairy processing facilities, ample opportunity exists for this pathogen to enter pasteurized milk as
a postpasteurization contaminant before the fermentation process begins as well as afterward as a
contaminant of the finished product. Thus far most studies have dealt with behavior of L. mono-
cytogenes in fermented dairy products inoculated with the pathogen either before or after fermen-
tation, with relatively few studies addressing the fate of listeriae in fermented dairy products
manufactured from naturally contaminated raw milk.
Although the extent to which L. monocytogenes survives in cultured dairy products is partly
dictated by whether or not the pathogen enters the product before or after fermentation, viability
of Listeria in fermented dairy products, particularly cheese, depends on the type of product in
which the pathogen is found as well as the degree of acid tolerance possessed by the contaminating
strain [172]. Hence, to better understand the complex interactions between the various factors that
affect viability of listeriae in cheese (i.e., amount, activity and type of starter culture, aw, pH, salt
content, temperature during manufacture and storage), it is appropriate to begin this section by first
discussing the behavior of L. monocytogenes in milk fermented with mesophilic and thermophilic
lactic starter cultures. Commingled with this information will be data concerning the fate of this
foodborne pathogen during manufacture and storage of cultured buttermilk, cream, and yogurt. The
viability of L. monocytogenes in coagulants (e.g., calf rennet, microbial rennet, and bovine-pepsin
rennet extract), coloring agents (e.g., annatto), and starter distillates (e.g., natural flavor compounds
derived from cultured milk) used in cheesemaking also will be considered before our discussion
of the many cheese varieties. Two additional areas of concern to cheesemakers, namely, the fate
of L. monocytogenes in whey and salt brine solutions, will be examined at the end of this chapter.
STARTER CULTURES, CULTURED MILKS, AND CREAM

Fermented or cultured buttermilk, cream, and yogurt were among the first dairy products to be mass-
produced commercially using pure bacterial starter cultures. Today, two mesophilic (optimal growth
at 30°C) lactic acid bacteria starter cultures, namely, Lactococcus lactis subsp. lactis and Lactococcus
lactis subsp. cremoris (formerly Streptococcus lactis and Streptococcus cremoris, respectively), are
commonly used either alone or in combination to manufacture cultured buttermilk and cream, whereas
a mixture of two thermophilic (optimal growth at or above 37°C) lactic acid bacteria, namely, Strep-
tococcus salivarius subsp. thermophilus and Lactobacillus delbrückii subsp. bulgaricus (formerly
Streptococcus thermophilus and Lactobacillus bulgaricus, respectively), is used to produce yogurt.
These same mesophilic and thermophilic starter cultures also are used to produce over 400 varieties
of cheese that were recognized by the U.S. Department of Agriculture [387] in 1978, with over 1200
cheese varieties now manufactured worldwide. The variety of cheese to be produced depends, in part,
on which of the lactic acid bacteria are used, either alone or in combination, as the starter culture.
Mesophilic Starter Cultures

Viability of L. monocytogenes in the presence of mesophilic lactic acid bacteria was first examined by
Schaack and Marth [353]. Samples of autoclaved skim milk were inoculated to contain approximately
=0.1% =1.0%
Change in Listeria Population (log10 CFU/mL)

=0.5% =5.0%
1.50
1.25
1.0
0.75
0.50
0.25
0
21°C 30°C 21°C 30°C
S. cremoris S. lactis
FIGURE 12.2 Changes in populations of L. monocytogenes in skim milk following a 15-h fermentation at
21 and 30°C with 0.1, 0.5, 1.0, or 5.0% (v/v) added S. cremoris or S. lactis. (Adapted from Schaack, M.M.
and E.H. Marth. 1988. Behavior of Listeria monocytogenes in skim milk during fermentation with mesophilic
lactic starter cultures. J. Food Prot. 51: 600–606.)
103 L. monocytogenes CFU/mL along with 0.1, 0.5, 1.0, or 5.0% of a S. cremoris or S. lactis milk culture
and then fermented at 21 or 30°C for 15 h to simulate preparation of a conventional bulk starter culture
for cheesemaking.
As shown in Figure 12.2, L. monocytogenes grew to some extent in all samples during fermen-
tation regardless of the species of lactic acid bacterium, inoculum level, or incubation temperature.
Maximum Listeria populations after 15 h of incubation at 21°C were ~1.0–2.3 orders of magnitude
lower in skim milk containing 0.1–5.0% of either starter culture than in control samples without
starter culture. Increasing the incubation temperature to 30°C led to final Listeria populations that
were ~2.5–4.0 orders of magnitude lower than in controls. As expected, growth of L. monocytogenes
increased as the starter culture inoculum level decreased from 5.0 to 0.1%. Acid production by the
starter culture played a major role in limiting multiplication of Listeria, with the pathogen never
growing at pH <5.0. However, in two instances, the organism was almost completely inhibited at
pH 5.6–6.0—well above the minimum pH value generally required for growth.
Twelve years later Pitt et al. [317] conducted an abbreviated study in which samples of
pasteurized rather than autoclaved milk were inoculated to contain approximately 103 L. monocy-
togenes CFU/mL followed by Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris,
or Lactobacillus plantarum at a level of approximately 106 CFU/mL. After 72 h of incubation at
30°C, L. monocytogenes populations decreased 89, 98, and 100% using L. lactis, L. cremoris, and
L. plantarum, respectively; these findings confirm the earlier work by Schaack and Marth [353].
Buttermilk, yogurt, sour cream, and other similar fermented milk products must be prepared
from pasteurized milk, whereas raw and/or heat-treated milk can be used in place of pasteurized
milk for the manufacture of many aged cheeses including Cheddar, Colby, and brick provided that
the cheese is aged for at least 60 days at ≥1.7°C before consumption. Recognizing the ability of
Listeria to become sublethally injured in heat-treated milk processed at <71.6°C for 15 sec and
survive in certain aged cheeses well beyond this 60-day ripening period, Mathew and Ryser [285]
assessed the competition of thermally injured L. monocytogenes with a mesophilic lactic acid bacteria
starter culture in milks exposed to various heat treatments. The study design was similar to that of
TABLE 12.11
Percentage Increase in Injury of Healthy, Low-Heat-Injured, and High-
Heat-Injured L. monocytogenes Cells in Different Milks Following a
6-h/31.1°C Fermentation with a 1.0% (v/v) Commercial L. lactis
subsp. lactis/L. lactis subsp. cremoris Starter Culture
Percentage Increase in L. monocytogenes Injury
Type of Milk Uninjured Low-Heat Injured High-Heat Injured
Raw 60.58a 46.76a 52.54a

Low-heat-treated 56.38b 46.32a 50.00b
High-heat-treated 55.31b 46.10a 48.09c
Pasteurized 53.82b 45.92a 47.39c
Ultrapasteurized 48.00c 45.01a 45.72d
Means (n = 3) in the same column with different superscripts are significantly different ( p < 0.05).
Source: Adapted from Mathew, F.P. and E.T. Ryser. 2002. Competition of thermally injured Listeria
monocytogenes with a mesophilic lactic acid starter culture in milk of various heat treatments. J.
Food Prot. 65: 643–650.
Schaack and Marth [353] except for the use of (1) a commercial starter culture comprising L. lactis
subsp. Lactis/L. lactis subsp. cremoris, (2) a three-strain cocktail that was sublethally injured by
heating in Tryptose Phosphate Broth at 56°C for 20 min (low-heat-injured) or at 64°C for 2 min
(high-heat-injured) before inoculating the milk, and (3) milk that was raw, low-heat-treated
(56°C/20 min), high-heat-treated (64°C/2 min), pasteurized (72°C/25 sec), or commercially sterile
ultra-high-temperature–pasteurized milk.
In the starter-free controls, 76 to 81% and 59 to 69% of the low- and high-heat–injured cells,
respectively, repaired after 8 h of incubation at 31.1°C. Increased injury was observed for previously
healthy L. monocytogenes cells using starter culture inoculum levels of 1 and 2% as opposed to
0.5%, with the extent of injury related to acid production. However, less injury was seen for the
low- and high-heat–injured cells, with sublethal injury affording some protection against the drop
in pH, as was previously discussed in Chapter 6. Furthermore, raw and subpasteurized milk were
less conducive to recovery of uninjured and high-heat–injured cells of L. monocytogenes as com-
pared to pasteurized and ultrapasteurized milk (Table 12.11). Given that most fermentation studies
have used pasteurized milk inoculated with Listeria, the less favorable environment associated with
raw and subpasteurized milk would suggest a decreased likelihood for survival in cheeses prepared
from raw or heat-treated as opposed to pasteurized milk.
Although still in use today, conventionally prepared bulk starter cultures have one major
disadvantage in that excessive acid development from an inherent lack of buffering capacity
frequently leads to some cell damage and partial loss of starter culture activity. Consequently, in
industrially prepared bulk starter cultures, the final pH is now normally held constant at 5.1–5.2
by either adding (manual or automated) a neutralizer during the fermentation process (i.e., external
pH control) or by using a specially prepared growth medium containing chemical buffers that
solubilize as the pH decreases (i.e., internal pH control).
Given the popularity of internal-pH-controlled media for starter culture preparation, researchers
at the University of Wisconsin investigated the ability of L. monocytogenes strains Scott A, V7,
and CA to compete with S. lactis [397] and S. cremoris [398] in one commercially available starter
culture medium having internal pH control. In both studies, the starter culture medium was inoc-
ulated to contain 103 L. monocytogenes CFU/mL together with either a 0.25 or 1.0% inoculum of
S. lactis or S. cremoris and incubated at 21 or 30°C for 30 h. Growth of the pathogen was only
partially inhibited by S. lactis and S. cremoris when compared to starter-free controls, with the
greatest inhibition occurring at the higher inoculum level and higher temperature. However, Listeria
populations of 105–106 and 104–105 CFU/mL developed in samples fermented with S. lactis and
S. cremoris, respectively, when these cultures were ready for use (pH 5.5) after 15 to 18 h of
incubation. Because neither conventional bulk starter technology nor internal-pH-controlled media
will completely inhibit this pathogen, manufacturers of cheese and other fermented dairy products
should not discount the starter culture as a possible source for Listeria but rather should adopt
rigorous sanitation standards and Hazard Analysis Critical Control Point (HACCP) programs to
minimize Listeria contamination and potential product loss. Ultrafiltered milk, a type of concen-
trated milk sometimes used for commercial manufacture of certain cheeses including mozzarella,
ricotta, cottage, and Cheddar, also possesses a higher buffering capacity than that of unfiltered milk
because of higher concentrations of proteins and insoluble salts. Consequently, listeriae may have
greater opportunity to grow in ultrafiltered as opposed to unfiltered milk during fermentation. El-
Gazzar et al. [189] examined this question by inoculating samples of unfiltered skim milk as well
as retentate (concentrated twofold and fivefold by volume) and permeate from unfiltered skim milk
to contain 103–105 L. monocytogenes CFU/mL together with 107–108 L. lactis subsp. cremoris
CFU/mL. In contrast to the aforementioned studies involving skim milk and internal-pH-controlled
bulk starter media, Listeria failed to grow in ultrafiltered milk containing starter culture, with
populations remaining constant in unfiltered skim milk and decreasing up to 10- and 100-fold in
2-fold retentate and permeate, respectively, after 36 h of incubation at 30°C. Increased inactivation
of L. monocytogenes in the permeate as compared to retentate and unfiltered skim milk is again
related to the lower buffering capacity of the permeate that results from ultrafiltration. When these
samples were refrigerated at 4°C, L. monocytogenes persisted 4–6 weeks in skim milk (pH 4.2),
3–5 weeks in retentate (pH 4.6), and 1 week in permeate (pH 4.1). Thus, fermentation of ultrafiltered
milk at 30°C will not guarantee complete inactivation of Listeria even after the finished product is
moved to refrigerated storage.
Cultured Buttermilk
Cultured buttermilk is essentially pasteurized skim milk that has undergone a 12- to 15-h fermen-
tation at 20°C with S. cremoris or S. lactis (0.5% initial inoculum) and certain flavor-enhancing
lactic acid bacteria such as Leuconostoc cremoris or L. dextranicum. After fermentation, the final
product is packaged and refrigerated until consumed.
As part of a follow-up study [354], all 15-h-old fermented milk samples from the aforemen-
tioned study by Schaack and Marth [353] were stored at 4°C and examined for L. monocytogenes.
Survival of the pathogen ranged from an average of 5 weeks in skim milk fermented at 30°C with
a 5.0% inoculum of S. cremoris to 12.5 weeks in skim milk fermented at 21°C with a 0.1% inoculum
of S. cremoris (Table 12.12). Similarly, Listeria viability averaged 2.5–13.0 weeks in skim milk
previously fermented at 30 and 21°C with 5.0 and 0.1% S. lactis, respectively. Slower inactivation
of the organism in skim milks fermented at 21 rather than 30°C may be related to the rate of acid
production during fermentation, because pH values for fermented milks ranged between 4.3–6.0
and 4.2–4.6 immediately after 15 h of incubation at 21 and 30°C, respectively. The fact that L.
monocytogenes can survive 10.5 weeks in this refrigerated product (fermented 15 h at 21°C with
0.5% S. cremoris) emphasizes the importance of maintaining sanitary conditions during manufac-
ture of cultured buttermilk.
In addition to entering pasteurized skim milk as a postpasteurization contaminant, L. monocytogenes
also can be introduced into cultured buttermilk as a postfermentation contaminant. Choi et al. [159]
studied the second of these scenarios. Samples of commercially produced, cultured buttermilk
(pH 4.21) were inoculated separately with each of four strains to contain an average of 3.5 × 103
L. monocytogenes CFU/mL and stored at 4°C. Under these conditions, the pathogen survived an
average of 22.8 days (Table 12.13), with populations of three of four Listeria strains decreasing
TABLE 12.12
Weeks of Survival of L. monocytogenes in Skim
Milks Fermented with S. cremoris or S. lactis at
21 and 30°C for 15 h and Then Stored at 4°C
S. cremoris S. lactis
Inoculum (%) 21°C 30°C 21°C 30°C
0.1 12.5a 6.5 13.0 2.0

0.5 10.5 6.0 ND ND
1.0 8.0 4.5 8.5 3.0
5.0 9.0 5.0 6.0 2.5

a
Average of two trials.
Source: Adapted from Schaack, M.M. and E.H. Marth. 1988.

Survival of Listeria monocytogenes in refrigerated cultured milks
and yogurt. J. Food Prot. 51: 848–852.
more than 100-fold during the first 8–12 days of refrigerated storage. It is important to realize that
although L. monocytogenes was inactivated faster when added directly to cultured buttermilk than
when skim milk was fermented into a “buttermilk-like” product in the previous study [354], the
pathogen still survived throughout the normal shelf life of the product. Furthermore, results from
an earlier study [226] demonstrating that E. coli and Enterobacter aerogenes are inactivated
faster than L. monocytogenes in cultured buttermilk imply that coliform-free buttermilk may not
TABLE 12.13
Survival of L. monocytogenes in Inoculated Samples of Commercially
Produced Buttermilk and Yogurt Stored at 4–5°C
L. monocytogenes Inoculum PH
Product (log10 CFU/mL) Initial Final Survival (days)
Buttermilk 3.55 4.21 4.38 22.8

Plain yogurt
Brand Da 4.26 4.02 4.08 21.2
Brand Yb 4.36 4.03 4.09 24.7
Vanilla-flavored yogurt
Brand D 4.21 4.03 4.10 24.7
Brand Y 4.70 4.03 4.10 22.3
Plain low-fat yogurt 2.10 4.10 4.10 3
Plain low-fat yogurt 7.10 4.10 4.10 9
aCustard-style.
bFluid-style.
Source: Adapted from Choi, H.K., M.M. Schaack, and E.H. Marth. 1988. Survival of Listeria
monocytogenes in cultured buttermilk and yogurt. Milchwissenschaft 43: 790–792; and Siragusa,
G.R. and M.G. Johnson. 1989. Persistence of Listeria monocytogenes in yogurt as determined by
direct plating and cold enrichment methods. Int. J. Food Microbiol. 7: 147–160.
necessarily be free of listeriae. These findings again emphasize the importance of good sanitation
in producing Listeria-free buttermilk.
Cultured Cream
Unlike cultured buttermilk, far less is known about the viability of L. monocytogenes in cultured
cream. In the only study reported thus far, Stajner et al. [367] manufactured cultured cream from
naturally contaminated raw cow’s milk containing approximately 5 × 105 L. monocytogenes
CFU/mL. According to these Yugoslavian authors, viable listeriae were detected in the finished
product throughout 7 days of storage at 3–5°C.
Thermophilic Starter Cultures
Practically speaking, thermophilic fermentations used to produce yogurt and certain cheeses (e.g.,
Swiss, Parmesan, mozzarella, and Romano) are not normally continued beyond 4–6 h. The only
two exceptions are in production of Bulgarian buttermilk and acidophilus milk, which require
thermophilic fermentations of 10–12 and 18–24 h, respectively. Therefore, primary emphasis will
be placed on behavior of Listeria during the first 6 h of fermentation.
In addition to determining the fate of L. monocytogenes in the presence of mesophilic starter
cultures [353], Schaack and Marth [352] also investigated the ability of this organism to grow during
fermentation of skim milk with thermophilic lactic acid bacteria. As in the previous study, samples
of autoclaved skim milk were inoculated to contain 103 L. monocytogenes CFU/mL. After adding
0.1, 1.0, or 5.0% S. thermophilus, L. bulgaricus, or a mixture of the two species, all samples were
examined for numbers of listeriae during 15 h of incubation at 37 and 42°C.
Limited growth of L. monocytogenes occurred in all samples, with the organism generally
attaining maximum populations after 6 h of incubation at either temperature (Figure 12.3). At this
Change in Listeria Population (log10 CFU/mL)
=0.1%
1.75
=1.0%
1.50
=5.0%
1.25
1.0
0.75
0.50
0.25
0
37°C 42°C 37°C 42°C 37°C 42°C
S. thermophilus L. bulgaricus L. bulgaricus and
S. thermophilus
FIGURE 12.3 Changes in populations of L. monocytogenes in skim milk following a 6-h fermentation at
37 and 42°C with 0.1, 1.0, and 5.0% (v/v) added Lactobacillus bulgaricus, Streptococcus thermopilus, or
L. bulgaricus + S. thermopilus. (Adapted from Schaack, M.M. and E.H. Marth. 1988. Behavior of Listeria
monocytogenes in skim milk and in yogurt mix during fermentation by thermophilic lactic acid bacteria.
J. Food Prot. 51: 607–614.)
point, Listeria populations were generally 1.0–1.5 orders of magnitude lower in fermented than in
unfermented control samples, as was also reported by Lukasova [270], indicating that growth of
the pathogen was markedly suppressed by the thermophilic starter culture, particularly when used
at inoculum levels of 5%. In addition, greater inhibition of listeriae was consistently observed in
milks fermented at 42 rather than 37°C.
Listeria monocytogenes behaved similarly in milks fermented with S. thermophilus, L. bulgar-
icus, and a mixture of both starter cultures during the initial 6 h of incubation; however, viability
of the pathogen in milks fermented beyond 6 h varied with the species of lactic acid bacterium
used in the fermentation. Although populations of listeriae remained relatively unchanged in all
milks fermented 6–15 h with S. thermophilus (final pH of 4.55–4.90), the pathogen frequently
survived only 9–15 h in milks fermented with L. bulgaricus alone, with similar findings reported
12 years later by Pitt et al. [317], who used pasteurized rather than autoclaved milk. Rapid
inactivation of the pathogen coincided with pH values near 4.0 which developed in milks fermented
9–15 h with L. bulgaricus. The combination of L. bulgaricus and S. thermophilus was more
inhibitory to Listeria than was S. thermophilus alone but less inhibitory than L. bulgaricus alone.
Although populations of listeriae failed to decrease in milks fermented at 37°C with the mixed
starter culture, some inactivation was noted in all corresponding samples incubated at 42°C. As
was true when L. bulgaricus was used alone, inactivation of listeriae by the mixed starter culture
again was most pronounced in samples having pH values near 4.0.
In addition to the mesophilic starter culture work by Pitt et al. [317] discussed earlier, these
investigators also assessed the ability of L. monocytogenes to compete with thermophilic starter
cultures during the fermentation of pasteurized as opposed to autoclaved milk. Using commercial
cultures of L. bulgaricus and S. thermophilus separately at inoculation levels of approximately
106 CFU/mL, populations of L. monocytogenes in the milk increased from 103 to 105 and from 103
to 106 after 12 and 20 h of incubation at 37°C, respectively, and thereafter declined. In the presence
of S. thermophilus and L. bulgaricus, Listeria growth was inhibited 93 and 100%, respectively,
with Schaack and Marth [352] reporting similar findings 12 years earlier.
In the previous study by Schaack and Marth [354], following 15 h of incubation all fermented
milk samples were stored at 4°C and monitored for listeriae. Using S. thermophilus alone,
L. monocytogenes survived 21–32 and 5–15 weeks in milks fermented at 37 and 42°C, respectively
(Table 12.14). Failure of these milks to attain pH values near 4.0 after fermentation with S.
thermophilus helps explain the unusually long survival of listeriae. As expected, L. bulgaricus was
most detrimental to listeriae, with the pathogen surviving beyond 15 h only in samples fermented
with the lowest inoculum. Using a 0.1% L. bulgaricus inoculum the pathogen was eliminated from
TABLE 12.14
Weeks of Survivala of L. monocytogenes in
Skim Milks Fermented with S. thermophilus or
S. thermophilus + L. bulgaricus (LBST) at 37
and 42°C for 15 h and Then Stored at 4°C
S. thermophilus LBST
Inoculum (%) 37°C 42°C 37°C 42°C
0.1 28.5 15.0 7.5 1.5

1.0 32.0 8.5 1.5 12 h
5.0 21.0 5.0 1.0 15 h
aAverage of two trials.
Source: Adapted from Ref. 352.
milks fermented at 37 and 42°C following 7 and 3 days of refrigerated storage, respectively. Milks
cultured with a combination of S. thermophilus and L. bulgaricus yielded results that were between
both extremes observed when the two starter cultures were used separately (Table 12.14). Once
again, L. monocytogenes survived longer in refrigerated milks fermented at 37 than 42°C, with
slower inactivation in the former being attributed to slightly higher pH values. These findings are
similar to those of Zuniga-Estrada et al. [403], who found that Listeria persisted for only 8 h when
milk containing 103 L. monocytogenes and 106 S. salivarius subsp. thermophilus/L. delbrueckii
subsp. bulgaricus CFU/mL was fermented at 42°C to pH 4.4.
Yogurt
As previously mentioned, L. monocytogenes can enter yogurt either before the fermentation as a
milk contaminant or afterward as a contaminant of the finished product. Schaack and Marth
[352,354] examined the behavior of Listeria under the first of these conditions by inoculating yogurt
mix to contain ~103–104 L. monocytogenes (strains V7, OH, Scott A, or CA) CFU/mL and 2% of
a commercial starter culture containing S. thermophilus, L. bulgaricus, and L. acidophilus. Yogurt
mix was fermented at 45°C for 5 h and then stored at 4°C. Populations of all four Listeria strains
increased an average of 2.5- to 10.0-fold in yogurt mix during the fermentation. After finished
yogurt at pH 4.75 was refrigerated at 4°C, numbers of listeriae decreased, with strains V7, OH,
Scott A, and CA surviving 7–12, 7–12, 4–12, and 1–5 days, respectively. The pH of yogurts in
which listeriae were last detected ranged from 3.88 to 4.11. Subsequent work has shown Listeria
survival to be somewhat more variable than first reported, with L. monocytogenes persisting 2–7 days
[279], 4 days [5], 3–5 days, 18–24 days [255], 10 days [239], and 14–25 days [329] in yogurts of
pH 4.2, 4.0–4.3, 4.1–4.7, 3.8–3.9, 4.1, and 4.5–5.0, respectively, during refrigerated storage, with
growth of the pathogen also generally not being observed during fermentation. However, these
survival times can reportedly be shortened by fermenting the yogurt mix with a bacteriocin-
producing strain of S. thermophilus or by adding purified enterocin—a bacteriocin produced by
Enterococcus faecium—to the mix after the start of fermentation [264]. In one additional French
study [168] involving highly acidic yogurt (pH 3.5) prepared from yogurt mix inoculated to contain
102–107 L. monocytogenes CFU/mL, the pathogen was eliminated after only 1–2 days of storage
at 4°C. Although acid development and differences in acid tolerance/injury during manufacture
[211] play major roles in determining the fate of listeriae in yogurt, other factors including various
starter culture metabolites and antilisterial compounds (bacteriocins) produced by certain strains
of L. acidophilus [323] also may contribute to the rapid death of Listeria in yogurt, provided this
lactic acid bacterium is in the product, with addition of 100 mg/L lysozyme to yogurt mix before
fermentation also reportedly decreasing Listeria survival in the finished product [329].
Although nearly all yogurt in the United States is now prepared from cow’s milk, this product
is sometimes manufactured from ewe’s and goat’s milk, particularly in Europe and the Middle East.
In a 1964 Bulgarian study [246], L. monocytogenes viability in yogurt prepared from naturally
contaminated ewe’s milk was markedly influenced by the storage temperature, with the patho-
gen surviving <24–48 h and >6 days in yogurt held at 18–22 and 10°C, respectively. Faster
demise of listeriae at the higher rather than lower storage temperature was attributed to increased
acid production by S. thermophilus and L. bulgaricus. When Abdel-Gawad et al. [personal
communication] prepared yogurt from cow’s, ewe’s, and goat’s milk inoculated to contain 103–104
L. monocytogenes CFU/mL, 97–99.9% of the population was sublethally injured within 24 h of
manufacture with no cells repairing or surviving in any yogurt samples (pH 4.0–4.3) beyond 5 days
of cold storage.
Many dairy industry personnel believe that contamination of yogurt is more likely to occur
after rather than before fermentation. Choi et al. [159] simulated postfermentation contamination
of yogurt by inoculating two commercial brands of plain and vanilla-flavored custard- and fluid-
style yogurt to contain 104–105 L. monocytogenes CFU/mL. The pathogen survived an average of
21.2–24.7 days in yogurt held at 4°C (Table 12.13), with most listeriae being inactivated during
the first 8–12 days of refrigerated storage. Khattab et al. [255] reported similar findings, with
listeriae persisting 18–24 days in experimentally produced Egyptian yogurt (pH 3.8–3.9) inoculated
to contain 106 L. monocytogenes CFU/mL. After inoculating various formulations of experimentally
produced yogurt to contain 106 L. monocytogenes CFU/mL, Griffith and Deibel [235] also detected
the pathogen in yogurt samples having a pH value of 4.3 following 28 days of storage at 4°C. More
recently, Tipparaju et al. [380] inoculated retail samples of low-fat and nonfat plain (pH 4.3–4.4)
as well as low-fat and nonfat vanilla yogurt (pH 4.1–4.5) to contain approximately 104 L. mono-
cytogenes CFU/mL and stored the products at 8°C. Listeria populations decreased 2.0 to 3.5 logs during
31 days of storage. Greatest reductions were seen after 14 to 21 days with populations also
significantly lower in vanilla-flavored yogurt containing vanillin. Based on these studies, the point
at which yogurt becomes contaminated greatly influences Listeria survival, with the pathogen more
vulnerable to inactivation as a pre- than a postfermentation contaminant.
Siragusa and Johnson [364] conducted a similar study in which three different brands of commer-
cial, unflavored, low-fat yogurt were inoculated to contain approximately 102 and 107 L. monocytogenes
CFU/g and then stored at 5°C. Listeriae survived <3 days in yogurt inoculated with low levels of the
pathogen even though pH values of yogurt were similar to those in the study by Choi et al. [159]
(Table 12.13). Using the high inoculum, viable listeriae were found for only 9 days, with populations
decreasing approximately 100-fold each after 3 and 6 days of refrigerated storage. Ayre et al. [120]
reported similar results using an L. monocytogenes inoculum level of 107 CFU/g. In contrast, Ribeiro
and Carminati [329] examined the effect of yogurt pH on Listeria survival by inoculating experimentally
produced (pH 4.5), commercial fruit (pH 4.05), and plain yogurt (pH 3.76) to contain ~104 L. mono-
cytogenes CFU/mL. Overall, the pathogen survived 2–3 days, 3–4 days, and 12–14 days in refrigerated
yogurts having pH values of 3.76, 4.05, and 4.5, respectively, thereby verifying the impact of pH on
Listeria survival. However, Griffith and Deibel [235] reported that L. monocytogenes populations
decreased approximately 4 orders of magnitude in artificially acidified (pH 4.2) rather than fermented
yogurt during the first 6 days of storage at 4°C. Hence, decreased tenacity of L. monocytogenes in
inoculated yogurt samples in these studies as compared with the work by Choi et al. [159] again
demonstrates that factors other than pH also are contributing to the death of Listeria.
Many yogurts and cultured buttermilks marketed today have pH values of approximately
4.0 and 4.3, respectively. Because large populations of L. monocytogenes were inactivated faster in
yogurt than in buttermilk during refrigerated storage, one would expect a lower incidence of listeriae
in commercial yogurt. Evidence from FDA surveys discussed earlier in this chapter supports this
view, because thus far the pathogen has been detected in commercial buttermilk but not yogurt,
with 100 retail and farm-produced samples also negative for L. monocytogenes in the United
Kingdom [254]. However, as was true for buttermilk, E. coli and Enterobacter aerogenes are also
inactivated faster than L. monocytogenes in yogurt during refrigerated storage [226]. Hence,
coliform-free yogurt may not necessarily be free of Listeria. This is important to remember when
results of coliform tests on these products are interpreted. It is evident from this discussion that
good sanitation practices are of utmost importance in producing Listeria-free yogurt, buttermilk,
and other fermented milk products.
Because yogurt is occasionally used as an ingredient in other foods, Sikes [361] investigated
the fate of L. monocytogenes in low-moisture (1.9% water), medium-acid (pH 4.9) yogurt-based
dairy bars supplied to the U.S. military. These bars, which contained ~34% heavy cream, 27.5%
yogurt, 27.5% cream cheese, and 11% of other ingredients (i.e., sugar, sunflower oil, whey), were
inoculated to contain approximately 1 × 105 L. monocytogenes strain Scott A CFU/g and then
periodically examined for numbers of listeriae during extended storage at 25°C. Results indicated
that Listeria populations decreased only approximately 100-fold after 40 days of incubation, thus
demonstrating the ability of this organism to persist in low-moisture, medium-acid foods. As will
soon be discussed, similar behavior has been reported for L. monocytogenes in semihard cheeses,
such as Cheddar, Colby, and Gouda, which also have pH values near 5.0.
Kefir
Kefir, an alcoholic milk-based beverage that was originally produced in the Caucasian Mountains
of Russia near the Black Sea, is now commercially produced in Europe, Asia, and North America.
In the manufacture of kefir, whole cow’s milk is heated to 85°C for 30 min, cooled to 22°C, and
then seeded with reusable kefir grains—gelatinous, walnut-sized irregularly shaped granules made
up of yeasts (Saccharomyces kefir, Torula spp., or Candida kefir ), lactic acid bacteria (Lacotoba-
cillus kefir, leuconostocs, and lactococci), and polysaccharides. Following overnight fermentation,
lactic acid is produced along with ethanol (about 1.0%), diacetyl, acetone, and carbon dioxide.
After straining the curdled milk to recover the kefir grains, the product is bottled, with a good kefir
foaming much like beer.
Working in Turkey, Gulmez and Guven [240] conducted two studies to assess the fate of
L. monocytogenes in kefir as both a pre- and postfermentation contaminant. In the first of these
studies, milk for kefir manufacture was inoculated to contain about 104 L. monocytogenes
CFU/mL and then fermented at 30°C for 24 h. Listeria populations increased about 10-fold
during the first 3 h and then slowly decreased during the remainder of the fermentation period as
well as during subsequent storage at 4°C, with the pathogen surviving between 5 and 10 days
in the final product at pH 4.2. Subsequently, Gulmez and Gowen [240] prepared. Listeria-
contaminated kefir as just described, except that the milk was fermented at 30°C for 24 (pH 4.5–4.7)
or 48 h (pH 4.2). In addition, Listeria-free kefir that had been similarly prepared was pasteurized
at 85°C, cooled, inoculated with L. monocytogenes and then stored at 4°C. Based on their
results, the pathogen increased about 100-fold to 106 CFU/mL in samples fermented for 24 h
and then decreased about 10-fold after 10 days of refrigerated storage. In contrast, Listeria
populations were at or near the original inoculation level after the 48-h fermentation period,
with the numbers slowly decreasing to less than 102 CFU/mL at day 10. When L. monocytogenes
was introduced into pasteurized kefir that had been previously fermented for 24 and 48 h,
populations remained relatively stable and decreased about 100-fold, respectively, after 10 days
of storage at 4°C, indicating that this pathogen poses a greater risk in kefir as a prefermentation
contaminant.
TRADITIONAL FERMENTED MILK PRODUCTS

In certain African and Middle Eastern countries, traditionally fermented ethnic milk products such
as ergo (Ethiopia) and labneh (Middle East) comprise a significant portion of the daily diet. Although
produced from commercially pasteurized milk under generally adequate hygienic conditions, these
products are also frequently prepared at home (particularly in rural areas) from raw milk that is
allowed to undergo a natural souring (i.e., fermentation) at ambient temperature without addition
of starter cultures. Hence, the likelihood for the presence of Listeria and other bacterial pathogens
in raw milk, combined with an often questionable fermentation and a short product shelf life, makes
the microbiological safety of such home-fermented milks particularly suspect.
Working in Zimbabwe, Dalu and Feresu [142] examined the fate of L. monocytogenes in
commercially fermented milk prepared from both raw and pasteurized milk. Commercial fer-
mented milk was prepared from pasteurized milk that was inoculated to contain 104 L. monocy-
togenes CFU/mL together with an active mesophilic starter culture and then packaged and
fermented 24 h at ambient temperature, whereas both traditionally fermented milks were prepared
by allowing raw milk to sour naturally in earthenware pots during 24 h of incubation at ambient
temperature. Overall, Listeria populations increased ≤10-fold in all three milks during fermen-
tation, with the final product attaining a pH of 4.3–4.6. After 5 days of storage at 4 and 20°C,
numbers of listeriae generally decreased about 10- and 100-fold, respectively. Consequently,
survival of L. monocytogenes beyond the normal 3–7 day shelf life of these traditionally fermented
milks poses a legitimate public health concern.
In 1994, Ashenafi [117] prepared ergo, a traditional Ethiopian fermented milk, from boiled
milk that was inoculated to contain 103 L. monocytogenes CFU/mL along with Lactobacillus and
Streptococcus starter organisms from a previous batch of product. After the first 12 h of a 24-h
fermentation at 25°C, numbers of listeriae generally increased 10- to 100-fold in ergo (pH 4.5–5.5),
with the highest populations being observed in samples fermented in unsmoked rather than olive
wood-smoked glass containers. During the next 12 h, the pathogen was slowly inactivated as the
pH of the product decreased to ≤4.0. Continued ambient storage led to complete demise of Listeria
in ergo fermented in wood-smoked and unsmoked containers within 36–48 and 48–60 h, respec-
tively. Consequently, traditional preparation of ergo in rural areas using wood-smoked fermenting
vessels appears to be beneficial in minimizing Listeria survival in this product that is typically
prepared from raw milk and consumed immediately after the 24-h fermentation period.
Labneh, a Middle Eastern yogurt-like product, is produced using a normal yogurt starter culture
of L. delbreuckii subsp. bulgaricus/S. salivarius subsp. thermophilus. Following approximately 5 h
of incubation at 42°C, the product is salted (1% NaCl), poured into muslin bags, and hung in a
cooler for 48 h. Gentle mixing to obtain a smooth consistency follows, after which the finished
product (pH 3.8) is packaged for sale. These manufacturing steps, which afford many opportunities
for contamination, prompted Gohil et al. [229] to assess the fate of L. monocytogenes in labneh as
a postfermentation contaminant. When commercially produced labneh was inoculated to contain
104 L. monocytogenes CFU/g and stored at 4°C, the pathogen survived 2 and 7 days in products
of pH 3.8 and 4.5, respectively, with the addition of 1% NaCl not appreciably altering Listeria
survival. Raising the holding temperature to 10, 20, or 30°C led to more rapid demise of listeriae,
with samples generally being free of L. monocytogenes after 2–3 days of storage.
Several years later, Issa and Ryser [248] examined the behavior of L. monocytogenes in labneh
as both a pre-and postfermentation contaminant. In this study, pasteurized milk was heated at
85–87°C for 30 min, cooled to 43°C, and inoculated to contain 103 L. monocytogenes CFU/mL.
Following a 4-h fermentation at 43°C with a 2% commercial yogurt starter culture, the fermented
milk was stored at 6°C for 12–14 h and then centrifuged to obtain a labneh-like product containing
about 22% total solids. Alternatively, traditional labneh containing 0 and 1% salt was prepared
using a 3% starter culture, inoculated to contain 103 L. monocytogenes CFU/g, and then stored at
6 and 20°C. During fermentation, growth of Listeria was suppressed as was previously reported
by Schaack and Marth [352]. When L. monocytogenes was inoculated into both salted and unsalted
labneh as a postfermentation contaminant, the pathogen decreased to nondetectable levels in 9 of 12
lots after 4 days of storage at 4 and 20°C. However, regardless of storage temperature,
L. monocytogenes persisted in one lot each of salted and unsalted labneh for at least 15 days.
Based on these findings, both labneh and yogurt pose minimal risk for consumers and are unlikely
to be associated with cases of listeriosis.
BEHAVIOR OF L. MONOCYTOGENES IN CHEESE

Considerable work has been done worldwide to define the behavior of L. monocytogenes during
manufacture and ripening of various types of cheese. Most of these studies describe what might
happen if cheese is prepared from contaminated milk. Because North American and European
surveys have indicated that soft/semisoft cheeses ripened with mold or bacteria are most frequently
contaminated with L. monocytogenes, research dealing with such varieties will be discussed first,
followed by data on ripened cheeses of progressively lower moisture content, goat’s milk cheese,
unripened cheese, whey cheese, and cold-pack cheese food. Whereas this chapter concludes with
a discussion of L. monocytogenes behavior in whey and brine solutions, this section begins with
an examination of the viability of listeriae in coagulants, coloring agents, and starter distillates, all
three of which are commonly used in cheesemaking.
COAGULANTS
To produce cheese curd, milk must first be coagulated or clotted, which can be done either by
acidification or addition of a coagulating enzyme. In the first method, an active lactic starter culture
is used to lower the pH of the milk to 4.6–4.7 (isoelectric point of casein), at which point the casein
micelles in the milk precipitate and form a coagulum. Alternatively, coagulation is occasionally
accomplished by adding food-grade acids directly to milk. Coagulation of milk by either means of
acidification is primarily confined to the manufacture of cottage cheese and a few ethnic varieties
of fresh cheese.
The second method, in which a coagulating enzyme is added to destabilize the casein micelles
and clot milk at a near-neutral pH, is used to manufacture virtually all other types of cheese.
Coagulants presently used in cheesemaking include calf rennet extract, chymosin, bovine pepsin-
rennet extract, and microbial rennet. Traditionally, calf rennet is extracted from the lining of the
abomasum (fourth stomach) of suckling calves and contains two enzymes—pepsin and chymosin
(the latter is most important for coagulation of milk). A shortage of calf rennet following World
War II led to the use of bovine pepsin-rennet, an extract obtained from the abomasum of somewhat
older calves, which can be substituted for calf rennet. Increased production costs of both calf rennet
and bovine pepsin-rennet have in turn prompted development of several rennets of microbial origin.
Thus far enzyme preparations obtained from molds belonging to the genus Mucor (particularly M.
miehei) have proved to be the most satisfactory substitutes for animal rennet.
Because two of four coagulants used in cheesemaking are of animal origin and because these
animals sometimes carry L. monocytogenes, this pathogen might occasionally appear in both crude
enzyme preparations and finished coagulant at the time of shipping. Although microbial rennet
should be free of Listeria spp. when manufactured, listeriae within the rennet-manufacturing facility
or the cheese factory environment could contaminate any of these products if mishandled. Given
the recovery of an apparent sodium benzoate-resistant strain of L. monocytogenes from commer-
cially produced calf rennet extract [188], the possible presence of L. monocytogenes in coagulants
should be of concern to cheesemakers, with the International Dairy Federation also having con-
templated the addition of rennet to its list of cheesemaking ingredients to be examined for Listeria
spp. [375].
During 1988 and 1989, El-Gazzar and Marth published results from three studies examining
the viability of listeriae in calf [184], bovine pepsin [185], and microbial rennet [187]. In each of
these studies, commercially produced, Listeria-free rennet was inoculated to contain approximately
103, 104, 105, or 106 L. monocytogenes CFU/mL and analyzed for listeriae during 56–70 days of
storage at 7°C, using both direct plating and cold enrichment.
All samples of calf and bovine pepsin-rennet inoculated with the two lowest levels of Listeria
were free of the pathogen after 14–28 days of storage at 7°C (Table 12.15). Even though 42–56
days of storage were required to eliminate the pathogen from samples containing initial inocula of
approximately 105 and 106 L. monocytogenes CFU/mL, one must remember that all four inoculum
levels used in these studies were many times greater than levels that might occur naturally in
commercially produced coagulants. Hence, barring contamination in the cheese factory, these
findings suggest that calf and bovine pepsin rennet are normally held long enough in distribution
channels to ensure cheesemakers that both coagulants are Listeria-free. Inactivation of L. monocy-
togenes in calf rennet and pepsin rennet probably results from the combined effects of 5% propylene
glycol, 2% sodium propionate, 0.1% (or more) sodium benzoate, 14–21% salt, and a relatively low
pH of 5.6. Results from several studies assessing the viability of L. monocytogenes in the presence
of benzoic acid and sodium propionate are discussed in Chapter 6.
Unlike calf and bovine pepsin-rennet, more than 70 days of storage were required to eliminate
L. monocytogenes at even the lowest inoculum level from microbial rennet (see Table 12.15).
Enhanced survival of listeriae in microbial rennet may be related to the nature of the coagulant
itself as well as the presence of fewer preservatives. Although L. monocytogenes is unlikely to enter
TABLE 12.15
Survival of L. monocytogenes Strain CA in Three Milk Coagulants Stored at 70°C
Number/mL after Days of Storage
Product 0 7 14 28 42 56 70
Calf rennet extract 2.5 × 103 1.5 × 102 (+)a <10 (−)b <10 (−) <10 (−) — —
1.1 × 104 3.5 × 102 (+) <10 (−) <10 (−) <10 (−) — —
3.0 × 105 6.0 × 104 (+) 1.2 × 103 (+) 30 (+) <10 (−) — —
2.0 × 106 6.0 × 104 (+) 4.5 × 103 (+) 2.0 × 102 (+) <10 (−) — —
Bovine pepsin-rennet 9.5 × 103 10 <10 (−) <10 (−) <10 (−) <10 (−) —
extract
2.0 × 104 30 <10 (−) <10 (−) <10 (−) <10 (−) —
7.5 × 105 1.0 × 103 1.0 × 102 <10 (+) <10 (+) <10 (−) —
1.0 × 106 2.0 × 104 1.0 × 102 <10 (+) <10 (+) <10 (−) —
Microbial rennet 6.0 × 103 1.5 × 103 7.6 × 102 2.8 × 102 3.3 × 102 1.2 × 102 40
7.0 × 103 1.7 × 103 1.0 × 103 2.2 × 102 4.4 × 102 1.4 × 102 90
2.0 × 105 1.7 × 104 2.3 × 104 2.5 × 104 2.3 × 104 1.6 × 104 8.5 × 103
1.0 × 1.06 1.5 × 105 4.0 × 104 3.6 × 104 9.3 × 104 7.0 × 103 9.0 × 103
a (+) Positive result by cold enrichment.
b (−) Not detected after 6 weeks of cold enrichment.
Source: Adapted from El-Gazzar, F.E. and E.H. Marth. 1988. Loss of viability by Listeria monocytogenes in commercial
calf rennet extract. J. Food Prot. 51: 16–18; El-Gazzar, F.E. and E.H. Marth. 1988. Loss of viability of Listeria
monocytogenes in commercial bovine pepsin-rennet extract. J. Dairy Sci. 72: 1098–1102; and El-Gazzar, F.E. and E.H.
Marth. 1989. Loss of viability by Listeria monocytogenes in commercial microbial rennet. Milchwissenschaft 44: 83–86.
microbial rennet during manufacture, the relatively high incidence of listeriae in cheese factories
may lead to inadvertent contamination of the coagulant during cheesemaking. Considering the
tenacity of L. monocytogenes in microbial rennet and the long shelf life of this product, it may be
prudent for cheesemakers to periodically verify that the microbial rennet they are using is indeed
Listeria-free.
COLORING AGENTS AND STARTER DISTILLATES

Depending on local preference, various yellow-orange colorants such as annatto (an extract from
annatto seed [Bixia orellana]) and turmeric (an extract from the turmeric root [Curcuma longa])
can be added to milk at the beginning of cheesemaking, with annatto most commonly being used
in the manufacture of Cheddar, Colby, Muenster, and brick cheese. Although freshly prepared
colorants are unlikely to contain microbial pathogens, inadvertent exposure of these coloring agents
to L. monocytogenes in the cheese factory could lead to production of a contaminated product.
Thus, in addition to the aforementioned coagulants, El-Gazzar and Marth [186] also investigated
the fate of listeriae in five commercially available annatto/turmeric extracts that were inoculated
to contain approximately 103–107 L. monocytogenes strain CA/mL and stored at 22°C. Regardless
of the initial inoculum level, populations of listeriae immediately decreased ≥4 orders of magnitude
in all colorants, with the pathogen being completely inactivated immediately after addition to three
of five extracts. The almost instantaneous death of Listeria in these three colorants was attributed
to the presence of propylene glycol and a pH of 13.3 (one extract). Although Listeria populations
of ≤800 CFU/mL were observed in the two remaining colorants immediately after inoculation, with the
highest level of listeriae, these samples were free of the pathogen following 7 days of ambient storage.
Working with a double-strength annatto extract, Galindo-Cuspinera et al. [212] recently reported
that a 1:40 dilution of this concentrated extract was inhibitory to L. monocytogenes. Thus, these
findings indicate that the length of time that these colorants spend at ambient temperatures during
distribution and before use at the cheese factory is more than adequate to inactivate small numbers
of listeriae that might enter as chance contaminants.
Small levels of starter distillates, that is, mixtures of natural flavor compounds such as diacetyl
obtained by distilling specially cultured milks, are frequently used to enhance the flavor of cottage
and processed cheese as well as ice cream, margarine, butter, yogurt, snack foods, and certain types
of candy. Hence, in connection with the study just described, El-Gazzar and Marth [186] also examined
the fate of listeriae in a commercially available starter distillate that was inoculated to contain 102–106
L. monocytogenes strain CA, Scott A, or V7 CFU/mL and held at 7°C. Overall, strain CA decreased
to nondetectable levels in all samples after 2–7 days of storage depending on the initial inoculum,
whereas 7–28 days of incubation were required to eliminate strains Scott A and V7 from similar
samples. Therefore, barring inadvertent contamination in the cheese factory, the time involved in
shipping and distributing these starter distillates as well as the aforementioned coagulants and colorants
should be more than sufficient to eliminate any inadvertent Listeria contaminants.
MOLD-RIPENED CHEESES
Mold-ripened cheeses can be divided into two categories: (a) white mold cheeses, which are surface-
ripened by Penicillium camemberti, P. caseicolum, or P. candidum (i.e., Brie and Camembert) and
(b) blue-mold or blue-veined cheeses in which ripening results from growth of Penicillium roqueforti
or P. glaucum throughout the cheese (i.e., Roquefort, blue, Gorgonzola). The relatively high
moisture content of these surface-ripened cheeses, along with a nearly neutral pH in fully ripened
cheese, allows rapid growth of L. monocytogenes as well as other foodborne pathogens that would
normally be inhibited in more acidic cheeses. Because mold-ripened cheeses also are highly
susceptible to surface contamination during ripening, it is not surprising that Brie and Camembert
were among the first cheese varieties in which L. monocytogenes was detected and the behavior
of the organism studied.
Camembert Cheese
In one of the first studies to examine behavior of L. monocytogenes during cheese manufacture and
storage, Ryser and Marth [341] prepared Camembert cheese from pasteurized milk inoculated to
contain approximately 500 L. monocytogenes strains Scott A, V7, CA, or OH CFU/mL. Following
10 days of storage at 15°C/95% relative humidity (RH) to permit proper growth of P. camemberti
on the cheese surface, all cheeses were wrapped in foil and ripened at 6°C. Wedge (pie-shaped),
surface, and interior samples of cheese were diluted in Tryptose Broth and analyzed for listeriae
at appropriate intervals using both direct plating and cold enrichment.
Populations of L. monocytogenes increased 5- to 10-fold during the first 24 h of manufacture;
however, this increase probably did not result from growth of the organism during cheesemaking.
Numerous studies have shown that bacterial populations typically increase 5- to 10-fold during
curd formation as a direct result of entrapment of organisms in the curd matrix, with the extent of
this increase dependent on the moisture content of the cheese. In all likelihood, L. monocytogenes
was similarly concentrated during formation of Camembert cheese curd. Entrapment of L. mono-
cytogenes in the curd is further supported by the fact that in this study only 1.3% of the original
Listeria inoculum in the milk was lost in the whey. Yousef et al. [402] later demonstrated that the
failure to observe L. monocytogenes population increases of approximately 5- to 10-fold after
formation of Camembert as well as Cheddar and cottage cheese curd was probably related to the
method of sample preparation. Their improved procedure in which curd samples were homogenized
in warm (45°C) Tryptose Broth containing 2% trisodium citrate was subsequently used to examine
Strain CA log10 CFU/g
FIGURE 12.4 Behavior of L. monocytogenes strain CA (solid symbols) and pH (open symbols) during
ripening of Camembert cheese. Solid symbols at <1.0 log10 strain CA/g indicate results for cold enrichment.
Numbers indicate the week at which strain CA was found, whereas an “x” signifies that the pathogen was
not detected after 8 weeks of cold enrichment. (Adapted from Ryser, E.T. and E.H. Marth. 1987. Fate of
Listeria monocytogenes during manufacture and ripening of Camembert cheese. J. Food Prot. 50: 372–378.)
behavior of L. monocytogenes during manufacture and storage of brick [345], Colby [400], feta
[307], blue [306], and Parmesan cheese [401].
During the initial 17 days of cheese ripening, the first 10 days of which occurred at ~15°C,
populations of three of four L. monocytogenes strains decreased 10- to >1000-fold, with lowest
numbers generally being observed in surface samples (Figure 12.4). On further ripening at 6°C, all
four Listeria strains grew (particularly between 25 and 30 days of storage) and attained maximum
populations of 106–108 CFU/g in wedge and surface samples from fully ripened cheese; however,
maximum listeriae populations were generally 10- to 100-fold lower in interior samples from the same
cheeses. Although growth of L. monocytogenes clearly paralleled the increase in pH of the cheese
during ripening, with growth usually commencing after the cheese attained a pH value of 5.75–6.25,
decreased viability of three of four Listeria strains in surface samples having pH values of 6.25–6.50
suggests that factors other than pH, including the presence of potentially inhibitory surface bacteria
and yeasts, may also be involved in controlling growth of this pathogen in Camembert cheese.
At least six additional studies have addressed the fate of L. monocytogenes during manufacture
and ripening of Camembert cheese [122,286,325,368,370,376]. Regardless of the method of inoc-
ulation (i.e., milk, surface, brine), the basic conclusions from these reports were similar to those
reached by Ryser and Marth [341] years earlier in that (a) L. monocytogenes failed to grow during
cheesemaking, (b) low numbers of listeriae were recovered during the period of rapid growth
of P. candidum on the cheese surface, (c) populations of listeriae in cheese increased rapidly after
21 to 28 days of ripening, and (d) maximum Listeria populations of >106 CFU/g were detected in
surface slices from fully ripened cheese. However, Terplan et al. [376] found that interior samples
from 4- to 56-day-old Camembert cheeses ripened at 5°C consistently contained <100 L. monocy-
togenes CFU/g and never attained pH values >5.6 even after 56 days of ripening. Hence, under
these conditions, growth of the pathogen was probably suppressed or severely retarded. However,
some cells may have been sublethally injured during continuous exposure to this acidic environment,
which in turn would have probably decreased the number of Listeria colonies observed on selective
plating media. Two of these studies also addressed the influence of cheese ripening temperature on
Listeria growth [122,370]. As expected, growth of L. monocytogenes was enhanced as the cheese storage
temperature was increased from 3 to 15°C in response to more rapid ripening of the cheese and a
concomitant increase in pH.
Results from a subsequent study by Ryser and Marth [343] showed greater growth of L. mono-
cytogenes in filter-sterilized Camembert cheese whey previously cultured with P. camemberti than in
uncultured whey adjusted to pH values of 5.60–6.80 and thus suggest that P. camemberti is not
involved in reducing Listeria populations on the surface of Camembert cheese. In support of these
findings, Geisen et al. [214] also failed to observe any antilisterial activity among several strains of
P. camemberti that were tested against L. monocytogenes in vitro. Although not yet recognized when
this work was published in 1987, a wide range of non-lactic-acid bacteria (i.e., entrococci, staphylo-
cocci) [154,195] and yeasts [178] that possess antiliserial activity and colonize the surface of smear-
ripened cheese may also be initially present on the surface of Camembert, with these organisms likely
responsible for partial inhibition of Listeria during the initial stages of ripening.
To simulate contamination of cheese in the ripening room, Ryser and Marth [341] also inoculated
surfaces of 10-day-old wheels of Listeria-free Camembert cheese to contain 2–40 L. monocytogenes
(four strains tested separately) CFU/20 cm2. All cheeses were then ripened at 6°C for 60 days, during
which time 10-g surface samples were analyzed for listeriae. Three of four L. monocytogenes strains
grew on the surface of the cheese and attained maximum populations of ~103–105 CFU/g (Figure 12.5).
Although the remaining Listeria strain failed to grow on the cheese surface after 60 days of storage,
Strain log10 CFU/g
FIGURE 12.5 Growth and survival of L. monocytogenes on the surface of Camembert cheese. Half-solid and
solid symbols at <1 log10 Listeria/g indicate that the organism was detected in one of two or two of two
samples, respectively, using cold enrichment. Numbers indicate the week at which L. monocytogenes was
found. (Adapted from Ryser, E.T. and E.H. Marth. 1987. Fate of Listeria monocytogenes during manufacture
and ripening of Camembert cheese. J. Food Prot. 50: 372–378.)
the pathogen was routinely detected throughout the ripening period using cold enrichment. These
findings, along with the unfortunate recall of over 300,000 tons of French Brie cheese, stress the
importance of manufacturing surface-ripened soft cheeses from high-quality, Listeria-free milk and
observing good sanitary practices in the ripening room. Nonetheless, even under ideal manufacturing
and ripening conditions, such cheeses may still inadvertently become contaminated with listeriae, as
is now well recognized from the high incidence of Listeria in surface-ripened cheese.
Because Vacherin Mont d’Or and Brie de Meaux (a raw milk cheese) were directly involved
in two major outbreaks of listeriosis in Europe, scientists in both Europe and North America have
been exploring various means of eliminating this pathogen from such cheeses that are surface-
ripened with mold and bacteria. Not surprisingly, Banks [124] reported that L. monocytogenes grew
rapidly in Camembert cheese prepared from raw milk, reaching a population of 106 CFU/g in fully
ripened cheeses. Although heat-treating this Listeria-contaminated milk at subpasteurization
temperatures (i.e., 62.8 or 65.6°C) led to markedly lower Listeria populations in the cheese
immediately after manufacture, the pathogen was not completely inactivated, with some growth
being reported during 7 weeks of cheese ripening.
Using a different approach, Helloin et al. [242] inoculated a chemically defined medium
resembling Camembert cheese (pH 5.0 and 7.0) with healthy and heat-injured (56°C/30 min)
cultures of L. monocytogenes and then incubated these samples at 4, 12, or 21°C for up to 14 days.
Compared to the uninjured controls, growth of the heat-injured culture was retarded at pH 7.0
during incubations at 4, 10, and 25°C with no growth and faster demise of the heat-injured culture
at pH 5.0. Thus, these findings agree with those of Banks [110] and suggest that sublethally heat-
injured cells in heat-treated milk are less likely to increase to potentially hazardous levels in
Camembert cheese during ripening than are healthy cells.
Although initial attempts by Asperger et al. [118] failed, results from several subsequent studies
yielded more promising findings concerning the use of nisin and nisin-producing starter cultures
to eliminate chance Listeria contaminants from soft surface-ripened cheese. When Maisner-Patin
et al. [274] used a nisin-producing strain of L. lactis subsp. lactis to manufacture Camembert cheese
from milk inoculated to contain 101, 103, or 105 L. monocytogenes strain V7 CFU/mL, numbers of
listeriae decreased dramatically 6–9 h into cheesemaking because of the presence of 700 IU nisin/g
of curd. Richard [330] also reported that inactivation of L. monocytogenes can be further enhanced
by directly adding as little as 25 IU nisin/mL to the milk at the start of cheesemaking.
Inhibition of Listeria continued during the first 2 weeks of ripening; however, regrowth of
survivors was then reported in the presence of 250–300 IU nisin/g, first on the cheese surface and
later in the interior, with the rate and extent of regrowth again paralleling the increase in cheese
pH during ripening. Addition of other inhibitory organisms, including Enterococcus faecalis and
Lactobacillus paracasei, to milk failed to arrest L. monocytogenes growth in Camembert cheese
during extended ripening. However, a difference of 2.4 log CFU/g between numbers of L. mono-
cytogenes in cheeses made with and without nisin-producing starter cultures was maintained
throughout 6 weeks of ripening. Nisin was most effective when the milk for cheesemaking contained
101 L. monocytogenes CFU/mL, with the pathogen being absent in 25-g samples even after 6 weeks
of ripening. These findings agree with those of Sulzer and Busse [371], who used a different nisin-
producing strain of L. lactis subsp. lactis.
In contrast to the previous work, Wan et al. [394] added piscicolin (2048 AU/mL)—a bacteriocin
produced by Carnobacterium piscicola—to pasteurized milk containing about 100 L. monocyto-
genes CFU/mL for manufacture of Camembert cheese. Although piscicolin inhibited growth of
Listeria during both manufacture and the first week of cheese ripening, the pathogen was again
detected at day 21 and increased to 3.8 log CFU/g as opposed to 6.8 log CFU/g in piscocolin-free
cheese after 47 days of ripening. Although both nisin and piscicolin have similar limits to preventing
regrowth of Listeria during cheese ripening, these bacteriocins can minimize Listeria growth during
Camembert cheese manufacture provided that the cheese milk is of good hygienic quality and
contains <103 L. monocytogenes CFU/mL.
In another report [175], addition of lactoperoxidase system components to the surface of

soft bacterial smear-ripened French cheese containing 102–106 L. monocytogenes CFU/g led to
complete inactivation of the pathogen following 4 days of storage at 15°C. In 1989, Hughey
et al. [244] showed that lysozyme was only bacteriostatic to L. monocytogenes in Camembert
cheese. Incorporating 2–10% carrot juice into homogenized Brie cheese was also effective in
minimizing growth of L. monocytogenes in refrigerated samples [124]. Although several addi-
tional reports also attest to the usefulness of x-ray [140] and gamma irradiation [196] in
eliminating high populations of L. monocytogenes from Camembert and other soft surface-
ripened cheeses, such treatments do not appear to be very practical, because ripening of the
cheese will be adversely affected.
Current evidence suggests that L. monocytogenes behaves similarly in naturally contaminated,
commercially produced soft and semisoft mold-ripened cheese. While conducting a survey of
soft/semisoft cheese sold in Canada, Farber et al. [166] discovered eight 4-month-old French
cheeses, presumably of the Brie/Camembert variety, that contained ~104–105 L. monocytogenes
CFU/g. Following 1 year of continuous storage at 4°C, Listeria populations remained constant in
one cheese and decreased only 10- to 100-fold in the seven remaining cheeses. In view of these
results, it is easy to understand why this pathogen has been most frequently detected in soft/semisoft
cheeses that have been surface ripened by molds.
Blue Cheese
Blue-veined cheeses such as blue, Roquefort, and Gorgonzola that are ripened internally and
sometimes externally with P. roqueforti or P. glaucum also have been examined for their ability to
support growth and survival of listeriae. Papageorgiou and Marth [306] used the modified Iowa
method to manufacture blue cheese from pasteurized milk inoculated to contain approximately
1000 L. monocytogenes (strains Scott A or CA) CFU/mL. All cheeses were ripened 84 days at
9–12°C (90–98% RH) and then held an additional 36 days at 4°C.
Numbers of listeriae increased by an average of 1.50 log10 CFU/g during the first 24 h of
manufacture, with increases of 0.62 and 0.71 log10 CFU/g being attributed to entrapment of the
organism within the curd matrix and growth, respectively. Growth of L. monocytogenes occurred
primarily during the first 9 h of manufacture and ceased when the pH of the cheese dropped
below 5.0. As expected, somewhat less growth occurred in two lots of cheese with particularly
rapid acid production.
Unlike the behavior of L. monocytogenes in Camembert cheese, the pathogen not only failed
to grow during ripening of blue cheese, but decreased in numbers by 2 to nearly 3 orders of
magnitude during the first 56 days of storage at 5°C (Figure 12.6). These decreases, which occurred
despite favorable pH values that developed during ripening from growth of P. roqueforti, were most
likely caused by formation of free fatty acids [355], with listeriostatic/listeriocidal levels of caproic,
caprylic [257], lauric, and other medium-chain fatty acids [258] produced as by-products of
P. roqueforti growth during ripening of blue-veined cheeses. However, at least eight different
P. roqueforti strains can also produce listeriocins in laboratory media [214]. Nonetheless, combined
effects of a relatively high pH and low storage temperature were probably responsible for both
Listeria strains surviving at least 120 days in all lots of blue cheese. Although additional tests
showed that strain Scott A was evenly distributed throughout blocks of 120-day-old blue cheese,
strain CA was far less tolerant to environmental conditions on the cheese surface and was detected
in such samples only after cold enrichment. The lengthy survival of L. monocytogenes in blue
cheese, coupled with the recall of Danish blue cheese and isolation of L. monocytogenes from
Italian Gorgonzola cheese, all stress the importance of preparing blue-mold cheeses from properly
pasteurized milk under good hygienic conditions to prevent a possible public health problem
involving Listeria.
L. monocytogenes log10 CFU/g
FIGURE 12.6 Changes in population of L. monocytogenes strain Scott A and pH during ripening of blue
cheese. (Adapted from Papageorgiou, D.K. and E.H. Marth. 1989. Fate of Listeria monocytogenes during the
manufacture and ripening of blue cheese. J. Food Prot. 52: 459–465.)
BACTERIAL SURFACE–RIPENED CHEESES

This group of cheeses consists of soft and semisoft varieties that are ripened under conditions that
induce a progression of microbial growth on the cheese surface. Examples of such cheeses include
brick from the United States, Pont l’Evêque and Saint Paulin from France, Tilsiter from Germany,
Trappist from Yugoslavia, Havarti from Denmark, Bel Paese and Taleggio from Italy, and Limburger
from Belgium. Differences between these varieties result from the shape of the cheese as well as
the amount and type of surface growth. Microorganisms are not normally added as pure cultures
but rather develop naturally on the cheese surface, because ripening conditions promote growth of
organisms that are normally present in the ripening room.
Proper aging of these surface- or smear-ripened cheeses results from the sequential growth
of halotolerant yeast, lactic-acid-metabolizing bacteria (Micrococcus spp.), and Brevibacterium
linens, the last-named organism being essential for proper flavor development. As in mold-ripened
cheeses, a pH gradient also develops during aging of bacterial surface-ripened cheeses, which
in turn creates a more favorable environment for growth of contaminating microorganisms,
including Listeria [119].
Brick Cheese
Using the washed-curd procedure, Ryser and Marth [345] prepared brick cheese from pasteurized
milk inoculated to contain ~102–103 L. monocytogenes (strain OH, Scott A, V7, or CA) CFU/mL.
Following manufacture, cheeses were smeared with a culture of B. linens and ripened at 15°C/95%
RH for 2, 3, and 4 weeks to simulate production of mild, aged, and Limburger-like brick cheese,
respectively. Because a natural pH gradient develops as brick cheese ripens, three types of cheese
samples—surface, interior, and slice (surface and interior)—were analyzed for numbers of listeriae
during 20–22 weeks of additional storage at 10°C.
Populations of strains OH, Scott A, CA, and V7 increased approximately 64.6- , 37.2- , 7.4- ,
and 6.8-fold, respectively, on completion of brining approximately 32 h after the start of cheese-
making. Because a population increase of approximately 10-fold can be attributed to entrapment
of listeriae within the curd matrix, with relatively few organisms appearing in the whey, growth of
L. monocytogenes during the latter stages of cheesemaking before brining was confined to strains
OH and Scott A. Numbers of listeriae remained relatively stable at 103–104 CFU/g of cheese during
brining; however, a few organisms leached from the cheese into the 22% NaCl brine solution.
Information on behavior of Listeria in salt brine solutions appears at the end of this chapter.
Strains OH (isolated from Liederkranz, a bacterial surface-ripened cheese formerly produced
in Ohio) and Scott A grew rapidly during the initial 2 weeks of smear development required to
manufacture mild brick cheese and generally attained maximum populations of approximately 6.20
and 6.60, 6.90 and 7.0, and 5.10 and 5.60 log10 CFU/g in 4-week-old slice (pH 6.0–6.5), surface
(pH 6.5–6.9), and interior (pH 5.6–6.2) samples, respectively. During the remaining 20 weeks of
ripening at 10°C, numbers of strains OH and Scott A generally decreased only 1- to 7-fold in mild
brick cheese. Both strains also behaved similarly in aged and Limburger-like cheese during smear
development and extended storage at 10°C.
In contrast, strains CA and V7 failed to grow appreciably during or after smear development,
despite favorable pH values of 6.8–7.4 in fully ripened cheese. Although strains CA and V7 were
detected only sporadically in 4- to 26-week-old samples of mild, aged, and Limburger-like cheese
at levels ranging between 2.7 and 4.6 log10 CFU/g, both strains were routinely recovered from
24- to 26-week-old slice, surface, and interior samples after cold enrichment. Hence, all four
L. monocytogenes strains survived beyond the normal shelf life of brick cheese. Subsequent
experiments [342] dealing with possible antilisterial effects of several sulfur compounds (i.e., methyl
sulfide, dimethyl disulfide, and methyl trisulfide) produced during ripening of brick cheese failed
to explain the inability of strains CA and V7 to grow in mild, aged, and Limburger-like brick
cheese. Additional possibilities include (a) inhibition of strains CA and V7 by smear-ripening
organisms such as Geotrichum candidum [178,273], Lactobacillus plantarum [194,197,267],
B. linens [199,276,295], enterococci [195,208,220,221,263,347], coryneform bacteria [302,347],
and/or certain staphylococci [154,347], all of which can reportedly produce bacteriocin-like sub-
stances active against listeriae or (b) heightened sensitivity of these L. monocytogenes strains to
the inhibitory effects of certain listeriocidal fatty acids (i.e., linoleic) and monoglycerides [395]
produced during cheese ripening.
In conjunction with the previously mentioned European study involving Camembert cheese,
Terplan et al. [376] also assessed the behavior of Listeria during manufacture and ripening of red
smear-ripened (“bricklike”) cheese. When this cheese was produced from pasteurized milk inocu-
lated to contain 95 L. monocytogenes CFU/mL, numbers of listeriae increased ≤10-fold after the
coagulum was cut as a result of entrapment within the curd matrix; however, no growth of the
pathogen was detected during the remainder of cheese manufacture. In fact, unlike the study by
Ryser and Marth [345], Listeria populations decreased 10-fold by the time the cheese (pH 4.9)
was ready for brining, with the cheese containing only 9 L. monocytogenes CFU/g after brining.
Following 8 days of smear development at 16.5°C/93% RH, all cheeses were ripened at 5°C for
an additional 62 days. Listeria populations close to the cheese surface increased from 2.5 × 101 CFU/g
immediately after smear development to 1.5 × 104 CFU/g in 14-day-old cheese, during which
time the pH increased from 4.9 to 5.1. Continued ripening of bricklike cheese at 5°C led to
development of stable L. monocytogenes populations of 2.5 × 105 CFU/g in 1-cm thick surface
slices of 42-day-old cheese. However, unlike the study by Ryser and Marth [345], the pathogen
was never detected in interior cheese samples that were more than 4 days old, despite pH values
of 5.7 in interior samples of 56-day-old cheese. Although results of Ryser and Marth [264] suggest
that L. monocytogenes should at least have been isolated occasionally from interior samples of
bricklike cheese, the FDA procedure used in this study was unable to detect listeriae in these
samples, possibly because of acid injury that may have occurred during exposure to pH values 5.2
for as long as 6 months.
Taleggio Cheese
First manufactured in the Taleggio Valley in Lombardy, Italy, following World War I, Taleggio is
a soft smear-ripened cheese that is particularly prone to contamination during smearing and weekly
washing with a brine solution. Hence, several investigators have assessed the fate of Listeria
on Taleggo cheese as a postmanufacturing contaminant during cheese ripening. Working in
Italy, Marchisio et al. [284] surface-inoculated ripened 35- to 40-day-old Talleggio cheeses
with L. monocytogenes and reported that Listeria populations on the cheese surface remained
relatively unchanged after 30 to 60 days of additional storage at 6°C. Failure of Listeria to grow
on the surface of Taleggio despite the increase in pH during ripening is related to the composition
of the surface microflora, with some cultures of Corynebacterium and micrococci [220] as well
as bacteriocin-producing strains of Enterococcus faecium [302] being inhibitory to Listeria, with
the greatest inhibition coming from well-developed bacterial smears during the later stages of
ripening [152].
Tilsiter Cheese
In 1995, Bachmann and Spahr [121] manufactured Tilsiter cheese (a semifirm, slightly yellow,
smear-ripened cheese similar to brick that originated in the vicinity of Tilsit in East Prussia) from
milk inoculated to contain 104 L. monocytogenes CFU/mL. Overall, their findings were similar to
those observed for brick cheese containing strains L. monocytogenes strains CA and V7 [345], with
Listeria populations varying between 103 and 104 CFU/g in Tilsiter cheese during 90 days of
ripening at 10–13°C.
Trappist Cheese
First manufactured at a monastery in Bosnia during the 1880s, Trappist is another soft surface-
ripened cheese similar to Port du Salut in France that undergoes frequent washing to prevent the
growth of surface mold. The ability of L. monocytogenes to survive the Trappist cheesemaking
process and persist during 90 days of ripening was investigated by Kovincic et al. [259]. Cheeses
were prepared from pasteurized milk inoculated to contain 102–105 L. monocytogenes CFU/mL and
a 1% starter culture inoculum of L. lactis subsp. lactis and L. lactis subsp. cremoris. After rennet
coagulation, the curd was cooked at 39°C for 45 min, hooped, drained, and pressed for 10–12 h.
Thereafter, the cheese was brine-salted (18% NaCl, 4 h), dried (5 days at 16–18°C), waxed, and
aged at 10°C for up to 90 days. Populations of L. monocytogenes increased ~10-fold in the finished
cheese during the first 30 days of ripening, stabilized over the next 30 days, and then gradually
decreased to levels approaching the original inoculum after 90 days of storage. Similar results were
also obtained when L. monocytogenes was added to the curd/whey mixture rather than the pasteurized
milk during cheesemaking. The limited growth and extended survival of L. monocytogenes in Trappist
cheese (pH 4.9, 30% moisture, 1.4% NaCl) are generally similar to what has been observed for
several common varieties of semisoft/hard cheeses to be discussed shortly.
SOFT ITALIAN CHEESES

Most of the Italian soft cheeses are classified as pasta filata or “plastic curd” cheeses and include
such varieties as mozzarella, Provolone, Caciocavallo, and Scamorze, with mozzarella clearly being
the most economically important. Traditionally, fresh mozzarella has been produced from the high-
fat milk of the water buffalo, whereas mozzarella cheese for use on pizza is manufactured from
cow’s milk and aged several weeks to develop proper elasticity and meltability. Production of these
pasta filata-type cheeses is similar in that the resulting curd is always stretched in hot (65–85°C)
water and then kneaded and molded into a characteristically shaped mass of curd which is hardened
in cold water, brine-salted, and ripened for various times.
Mozzarella
The severe heat treatment that the cheese curd receives and the reported thermal tolerance of listeriae
led Buazzi et al. [145] to assess the fate of L. monocytogenes during manufacture of mozzarella
cheese. When this cheese was prepared from pasteurized milk inoculated to contain 104–105
L. monocytogenes strain OH, CA, or V7 CFU/mL, Listeria populations of 104–105 CFU/mL were
reported in the curd after cutting, cooking, and cheddaring. However, immersing and stretching the
curd 3 to 4 min in hot water (77°C) led to the complete demise of the pathogen. In subsequent
work, Kim et al. [256] reported that L. monocytogenes populations decreased more than 3 logs
after mozzarella curd was stretched at 66°C for 3 min or 77°C for 1 min. Because the curd
temperature was maintained at 65°C for at least 2 min and L. monocytogenes has a reported
D-value of 28.1 sec at 65°C [272], mozzarella cheese should be Listeria-free even if small numbers
of the pathogen are present in the curd before stretching. These findings are generally similar to
those reported during manufacture of traditional mozzarella cheese from buffalo milk [391], with
L. monocytogenes populations decreasing at least 100-fold during brief stretching of the curd at
90–95°C. Although a few survivors remained after curd stretching and molding, all cheeses prepared
from milk inoculated to contain 103 and 105 L. monocytogenes CFU/mL were free of this pathogen
after 24 and 48 h of refrigerated storage, respectively.
The heat treatment given to mozzarella cheese curd is clearly sufficient to inactivate small
numbers of listeriae that might be present. However, ample opportunity exists for postprocessing
contamination as evidenced by recent surveys and at least three Class I recalls of Listeria-contaminated
mozzarella cheese. Stecchini et al. [369] addressed the issue of postprocessing contamination by
inoculating the surface and packaging fluid of mozzarella cheese with L. monocytogenes and then
storing the product at 5°C for up to 21 days. Under these conditions, numbers of listeriae increased
about 10,000-fold during 21 days of storage, with inclusion of a crude heat-treated bacteriocin
preparation from L. lactis subsp. lactis yielding final populations only 10-fold lower as compared
to untreated controls. Thus, manufacturers of mozzarella cheese must adhere to good manufacturing
and sanitary practices to prevent contamination of the curd after stretching and growth of
L. monocytogenes to potentially hazardous levels during storage.
SEMISOFT AND HARD CHEESES

By definition, hard cheeses are those that contain ≤40% moisture. Cheeses in this category include
such varieties as Edam and Gouda (which can also be classified as semisoft cheeses) as well as
Colby, Cheddar, Swiss, Emmentaler, Gruyère, Romano, and Parmesan, the last two of which are
very hard grating cheeses. Transformation of chalky, acid-tasting curd into a ductile, full-flavored
cheese is accomplished during ripening through the action of milk enzymes, rennet, and various
microorganisms in the cheese, including the starter culture. The biochemical changes that occur
during cheese ripening are complex and involve hydrolysis of fats and proteins with subsequent
decarboxylation, deamination, and dehydrogenation as well as production of carbonyls, nitrogenous
compounds, fatty acids, and sulfur compounds, all of which contribute to the overall flavor of the
final product. The amount of aging needed to obtain a fully ripened cheese is directly related
to moisture content, with a minimum of 2 and 10 months of ripening being required for Edam
(~40% moisture) and Parmesan cheese (~30% moisture), respectively. The current popularity
of many of these cheeses, along with the ability of L. monocytogenes to survive in acidic
environments during refrigerated storage, has prompted a series of studies examining the
behavior of L. monocytogenes during manufacture and storage of at least seven semisoft and
hard cheese varieties.
Gouda and Maasdam Cheeses
Beginning with semisoft/hard cheeses, Northolt et al. [299] prepared Gouda and Maasdam cheese
in The Netherlands from pasteurized milk inoculated to contain approximately 500 L. monocyto-
genes CFU/mL. Gouda cheese was manufactured according to standard procedures, with the
exception that one lot was prepared using 0.3 rather than 0.6% starter culture to obtain a cheese
with an unusually high moisture content of ~45%. Maasdam cheese was prepared using a culture
of propionic acid bacteria in combination with 0.6% mesophilic lactic starter. After brine salting,
Gouda cheese was ripened 6 weeks at 13°C, whereas Maasdam cheese was ripened 2 weeks at
13°C, 2 weeks at 18°C, and then stored at 4°C for an additional 2 weeks.
As in previous studies, entrapment of listeriae within the curd matrix during cheesemaking
resulted in population increases of approximately 10-fold as compared to the original level in
pasteurized milk. However, some Listeria growth was noted during manufacture, with populations
increasing an additional fourfold in normal Gouda and Maasdam cheese before brining. Six hours
after manufacture, slightly higher Listeria populations were detected in Gouda cheese of high rather
than normal moisture. Although numbers of listeriae in interior samples from both Gouda and
Maasdam cheese remained relatively constant at ~104 CFU/g during the first 2 weeks of ripening,
the pathogen was not detected in cheese samples taken at or near the surface. After 6 weeks
of ripening, L. monocytogenes reappeared in surface samples from all cheeses at levels between
102 and 104 CFU/g. In contrast, numbers of listeriae in interior samples from 6-week-old cheese
were only four- to eightfold lower than populations in the same cheeses immediately after brining.
Although L. monocytogenes survived best in high-moisture Gouda cheese that had a pH of 6.0, the
selective plating medium used in this study, Trypaflavine Nalidixic Acid Serum Agar, was subop-
timal for recovery of stressed or acid-injured listeriae that probably were present in fully ripened
Gouda and Maasdam cheese having pH values of 5.48 and 5.44, respectively.
Colby Cheese
Yousef and Marth [400] prepared Colby cheese from pasteurized milk inoculated to contain 102–103
L. monocytogenes (strain V7 or CA) CFU/mL. Following manufacture, all blocks of cheese were
held at 4°C for 140 days.
During cheesemaking, most Listeria cells were trapped in the curd matrix, with an average of
only 2.4% of the original inoculum escaping in whey. Populations of L. monocytogenes in cheese
increased an average of 1.27 orders of magnitude after pressing—about 29 h after the start of
manufacture (Figure 12.7). Because an increase of no more than one order of magnitude can be
attributed to entrapment of listeriae within the curd matrix after cutting, these findings suggest that
slight growth of the organism did occur, particularly during the later stages of cheesemaking and
pressing. Numbers of both Listeria strains remained relatively constant in cheese during the first
40 days of ripening, after which populations decreased almost linearly (Figure 12.7). Viability of
L. monocytogenes was strongly influenced by moisture content, with strain V7 decreasing more
than twice as fast in cheese containing 38.5% (D-value of 54 days) rather than 42.3% (D-value of
124 days) moisture, which is well above the maximum allowable moisture content of 40.0% for
Colby cheese.

4
3
V7, 42.3% moisture
V7, 38.5% moisture
CA, 39.5% moisture
2
0 1 2 3 4 0 50 100 150
Hours Days
Time
FIGURE 12.7 Behavior of L. monocytogenes during manufacture and ripening of Colby cheese. (Adapted
from Yousef, A.E. and E.H. Marth. 1988. Behavior of Listeria monocytogenes during the manufacture and
storage of Colby cheese. J. Food Prot. 51: 12–15.)
Behavior of L. monocytogenes in cheese of normal moisture content also was strain dependent,
with strain CA being less stable than strain V7. However, strains V7 and CA were still detected in
140-day-old Colby cheese by direct plating, with cold enrichment results from a follow-up study
[402] indicating that both strains were still viable in 5- to 8-month-old Colby cheese stored at 4°C.
According to the FDA, Colby and other selected cheeses can be manufactured from raw or heat-
treated (subpasteurization) milk provided that the finished cheese is held a minimum of 60 days at
or above 1.7°C (35°F) before sale in an attempt to eliminate pathogenic microorganisms. These
results (and those for Cheddar cheese to follow) have prompted the FDA to reconsider the adequacy
of this aging requirement for cheeses prepared from raw milk.
Cheddar Cheese
Normal stirred-curd Cheddar cheese, which has a moisture content only slightly less than Colby
cheese (i.e., 36–38%), was manufactured by Ryser and Marth [340] from pasteurized whole milk
inoculated to contain approximately 5 × 102 L. monocytogenes (strain Scott A, V7, or CA) CFU/mL.
The resulting 10-lb blocks of cheese were ripened at 6 and 13°C and assayed for numbers of
listeriae at appropriate intervals.
All curd samples examined during manufacture contained approximately 5 × 102 L. monocy-
togenes CFU/g, which suggests that the organism was only minimally concentrated in the curd
and failed to grow during cheesemaking. However, because only 6.4% of the initial Listeria
inoculum was recovered in the whey, the expected 10-fold increase from entrapment of the organism
in the curd matrix probably went unnoticed because of inadequate sample preparation methods
that have since been improved [402]. Numbers of listeriae increased slightly in cheese during
pressing, with all three strains attaining maximum populations of ~3.50–3.75 log10 CFU/g after
14–35 days of ripening at 13 (Figure 12.8) and 6°C (Figure 12.9). This population increase, which
was approximately 10-fold higher than that of the original inoculum in milk, probably occurred
because of enhanced recovery of the pathogen from older cheese that was easier to homogenize
4.0
Trial 4
3.0 Trial 5
Strain V7 log10 CFU/g
Trial 6
2.0
1.0
0
0 25 50 75 100 125 150 175 200 225 250 275 300 325
Days
FIGURE 12.8 Survival of L. monocytogenes strain V7 in Cheddar cheese ripened at 13°C. Open symbols at
<1 log10 Listeria/g indicate that the organism was not detected after 8 weeks of cold enrichment, whereas
half-solid or solid symbols at <1 log10 Listeria/g indicate that the pathogen was detected in one of two or two
of two samples, respectively, using cold enrichment. Numbers indicate the week at which L. monocytogenes
was found. (Adapted from Ryser, E.T. and E.H. Marth. 1987. Behavior of Listeria monocytogenes during the
manufacture and ripening of Cheddar cheese. J. Food Prot. 50: 7–13.)
4
Trial 4
Trial 5
3 Trial 6
Strain V7 log10 CFU/g
4/6
6 6
0
0 50 100 150 200 250 300 350 400
Days
FIGURE 12.9 Survival of L. monocytogenes strain V7 in Cheddar cheese ripened at 6°C. See Figure 12.8 for
explanation of symbols. (Adapted from Ryser, E.T. and E.H. Marth. 1987. Behavior of Listeria monocytogenes
during the manufacture and ripening of Cheddar cheese. J. Food Prot. 50: 7–13.)
rather than from actual growth, as shown by Yousef et al. [402]. After 35 days of storage at either
temperature, Listeria populations in cheese began to decrease, with all cheeses maintaining pH
values of 5.04–5.09 throughout ripening. Strains Scott A, V7, and CA survived 70–224, 126–196,
and 70–126 days in cheese ripened at 13°C, respectively, whereas the same strains remained viable
for 70–154, 126–434, and 70–154 days in cheese aged at 6°C. Thus, except for strain V7, which
was still present in one block of 434-day-old cheese at a level of 30 CFU/g, the remaining two
strains survived equally well in cheeses ripened at either temperature. These findings suggest
different acid tolerances among the L. monocytogenes strains tested, as has also been reported by
Gahan et al. [211]. Additional experiments with strains V7 and CA demonstrated that L. monocy-
togenes was uniformly distributed in Cheddar cheese during at least the first 98 days of ripening
at 6°C. Working in England, Banks [124] also reported that L. monocytogenes persisted 8–9 months
and up to 7 months in Cheddar cheese prepared from Listeria-contaminated raw and subpasteurized
(i.e., 62.8 or 65.6°C) milk, respectively. Hence, these data provide some of the strongest evidence
for the inadequacy of the 60 day/≥1.7°C minimum holding period for cheeses manufactured from
raw milk.
Several subsequent studies examined the effect of Cheddar cheese compositional changes
on Listeria survival during cheese ripening. Mehta and Tatini [287] assessed the behavior of
L. monocytogenes strains Scott A and V7 in stirred-curd Cheddar cheese containing 1.3 or 2.5%
NaCl or an equal molar mixture of NaCl and KCl. Lowering the level of NaCl enhanced
destruction of Listeria, with 1.3% NaCl, 2.5% Na/KCl, and 2.5% NaCl decreasing populations
4.3-, 2.3- and 0.4-orders of magnitude in cheese, respectively, after 10 weeks of aging at 7°C.
Thus, in addition to being healthier, low-sodium Cheddar cheese appears to be safer in regard
to listeriae. When these same investigators [288] prepared stirred-curd Cheddar cheese from
whole milk and reduced-fat milk (1.55 or 2.0% milk fat), L. monocytogenes strains Scott A and V7
persisted in the finished cheese during 20 months of storage at 7°C, with no survival differences
being observed between full-fat (28.9% fat) and reduced-fat (20.7%) cheese. However, Listeria
survival is strongly influenced by milk fat composition and release of free fatty acids during
cheese ripening [312]. Schaffer et al. [355] increased the levels of both long-chain (C18, C18:2)
and unsaturated fatty acids in milk by feeding cows a diet of extruded soybeans or sunflower seeds
and then used this milk to manufacture stirred-curd Cheddar cheese containing L. monocytogenes
strains Scott A and V7 as previously described. During manufacture, numbers of listeriae
increased 1.0–1.5 orders of magnitude as previously reported by Ryser and Marth [340]. More
important, after 120 days of ripening at 7°C L. monocytogenes populations were 5 to 8 orders
of magnitude lower in cheeses prepared from modified-fat milk as compared to unmodified milk.
Although some inconsistences were noted in performance of sunflower- and soybean-modified
milk, inactivation of L. monocytogenes always occurred most rapidly in cheeses containing the
highest levels of free fatty acids, with oleic, linoleic, lauric, and myristic acids shown to be major
contributors to Listeria destruction during cheese ripening.
In addition to modification in milk and cheese composition, addition of various bacteriocins as
well as various bacteriocin-producing starter and nonstarter cultures to milk during cheesemaking has
also proved beneficial in reducing survival of Listeria in Cheddar cheese during ripening. Benech
et al. [130,131] compared the effectiveness of adding liposome-encapsulated nisin Z (300 IU/g) to
milk with the use of a nisin Z L. lactis subsp. diacetylactis starter culture for inactivation of L. innocua
during manufacture and ripening of Cheddar cheese. When cheeses were prepared from milk inocu-
lated to contain 105 to 106 L. innocua CFU/mL, the encapsulated form of nisin Z was more effective
than the nisin Z-producing starter culture, with Listeria populations decreasing 3 and 1.5 logs
immediately after cheesemaking, respectively. Most importantly, L innocua populations in 6-month-
old cheeses prepared with encapsulated nisin Z decreased to <10 CFU/g compared to about 104 CFU/g
for cheeses using the nisin Z-producing starter culture. The greater effectiveness of encapsulated nisin
was related to both nisin stability and location with 90 and only 12% of encapsulated and starter-
produced nisin Z remaining active at the fat/casein interface and in the fat of the cheese, respectively.
In other work, Buyong et al. [148] used a pediocin PA-I-producing strain of L. lactis subsp.
lactis in production of Cheddar cheese from pasteurized milk that was inoculated to contain about
103 L. monocytogenes CFU/mL. Overall, pathogen populations were about 3 logs lower in cheese
prepared with rather than without the pediocin-producing starter culture. Based on these findings,
use of encapsulated nisin and other bacteriocin-producing starter cultures affords an added degree
of protection against long-term survival of Listeria and should be considered, particularly if Cheddar
and other similar cheeses are to be prepared from raw milk.
Swiss Cheese
Unlike the aforementioned cheeses, manufacture and ripening of Swiss cheese involves several
decidedly different steps, including cooking of the curd at 50–53°C and ripening the finished cheese
at an elevated temperature for “eye” development. These observations prompted Buazzi et al. [146]
to examine the fate of L. monocytogenes during manufacture and ripening of Swiss cheese. When
rindless Swiss cheese was prepared from pasteurized milk inoculated to contain 104–105 L. mono-
cytogenes strain V7, CA, or OH CFU/mL, the pathogen was generally unable to grow during
cheesemaking, with populations increasing 43% during the early stages of cooking owing to
physical concentration and curd shrinkage. Thereafter, about 57% of the population in the curd
was inactivated after 30–40 min of cooking at 50°C. After pressing, the curd contained 50% fewer
listeriae, with this population decreasing most sharply after 30 h of brining at 7°C. Storing the
finished cheese (pH 5.2–5.4) 10 days at 7°C reduced the Listeria population to very low numbers.
Complete inactivation of the pathogen occurred after 66–80 days of ripening at 24°C, with pro-
duction of propionate by eye-forming bacteria likely contributing to the death of listeriae. Two
studies conducted in Switzerland [121,253] demonstrated that the environments within Emmen-
thaler and Gruyère cheese (i.e., other varieties of Swiss cheese) also are not conducive to Listeria
survival, with the pathogen no longer being present in 24-hour-old cheeses (pH 5.2–5.4) prepared
from raw milk inoculated to contain 104 L. monocytogenes CFU/mL.
Parmesan Cheese
Parmesan cheese, a hard grating cheese that undergoes a high-temperature cook resulting in a very
low moisture content, was prepared by Yousef and Marth [401] from pasteurized milk inoculated
to contain ~104–105 L. monocytogenes (strain V7 or CA) CFU/mL. Unlike the cheeses discussed
previously, a lipolytic enzyme (lipase) is often added to cheesemilk to produce the characteristic
flavor of fully ripened Parmesan cheese. In addition, the coagulum is cut into very small particles
that are cooked at ~52°C (125°F) for 45 to 60 min until the pH decreases to 6.1. This step serves
to expel whey, thus producing a dry, ricelike curd that can be pressed to form a very dense,
low-moisture cheese. Following manufacture, the cheese produced in this study was brine-salted
(22% NaCl) for 7 days at 13°C, dried 4–6 weeks in a humidity-controlled chamber at 13°C, vacuum-
packaged, and ripened at 13°C for a minimum of an additional 9 months.
During the first 2 h of cheesemaking, populations of both Listeria strains increased approxi-
mately 6- to 10-fold, largely from entrapment of the organism within the curd matrix (Figure 12.10).
Although Listeria counts remained relatively stable during cooking of the curd at 52°C (125°F)
for 45 min, populations decreased appreciably during pressing of the curd. During brining, drying,
and ripening at 13°C numbers of both Listeria strains decreased almost linearly, with estimated
D-values ranging between 8 and 36 days. Using direct plating, strains V7 and CA were no longer
detected in cheese after 21–112 and 14–63 days of ripening at 13°C, respectively. Despite large
differences in survival of L. monocytogenes between different batches of cheese, both Listeria
strains decreased at a faster rate in Parmesan than in Gouda, Maasdam, Cheddar, and Colby cheese
during ripening. Decreased viability of the pathogen in Parmesan cheese is probably related to a
combination of factors, including (a) action of lipase added to the milk, (b) heat treatment that the
107
Manufacture
L. monocytogenes log10 CFU/g 106
105
Ripening
104
Batch #1
Batch #3
103
Batch #6
102
10
0 1 2 3 0 40 80 120
Hours Days
FIGURE 12.10 Survival of Listeria monocytogenes strain V7 during manufacture and ripening of Parmesan
cheese. (Adapted from Yousef, A.E., and E.H. Marth. 1990. Fate of Listeria monocytogenes during the
manufacture and ripening of Parmesan cheese. J. Dairy Sci. 73: 3351–3356.)
curd receives during cheesemaking, and (c) lower moisture content (and water activity) of the fully
ripened cheese. To decrease the moisture content and develop proper flavor, the present regulation
in the United States requires that Parmesan cheese be aged a minimum of 10 months regardless of
whether the cheese is prepared from raw or pasteurized milk. According to the results of this study,
such an aging process should be sufficient to produce Listeria-free Parmesan cheese.
Hard Italian-Type Cheese
Working in Italy, Comi and Valenti [161] inoculated the surface and interior of three freshly prepared
hard Italian-type cheeses (aw 0.95–0.98, pH 5.2–5.5) to contain 104–105 L. monocytogenes CFU/g.
As was true for Parmesan cheese, the pathogen failed to grow in this relatively hard cheese, with
Listeria populations decreasing 10- to 100-fold in both surface and interior samples from all three
cheeses during the first 28 days of ripening at 4°C. Although the pH of surface and interior samples
increased to 5.4–5.5 and 5.8–6.0, respectively, after 35 days ripening, Listeria populations continued
to decrease, with the pathogen only being detected by cold enrichment (i.e., populations <102
CFU/g) in samples from 35-day-old cheeses. Compositional analysis of these cheeses suggested
that the amount of moisture lost after 35 days of ripening (aw 0.95–0.96) may have offset the benefit
for Listeria growth caused by the increase in pH.
HISPANIC CHEESES
Traditional Hispanic-type cheeses comprise a wide range of white cheeses produced in Mexico and in
Central and South America. Some of the most popular varieties, including Queso blanco, Queso fresco,
and Queso de Puna, are high-moisture fresh cheeses consumed shortly after manufacture, whereas
others, such as Queso Anejo, Queso de Bola, Queso de Crema, Queso de los Ibores, and Queso de
Prensa, are lower in moisture and undergo various degrees of aging. Although 30 min of heating at
80–85°C is more than adequate to inactivate L. monocytogenes during manufacture of Queso blanco
cheese [179], typical production practices for Hispanic-type cheeses involve extensive curd manipula-
tions, including hand stirring, salting, and molding, any of which can easily lead to product contami-
nation. Based on recent risk assessments that are discussed elsewhere and over 20 Class I recalls of
Hispanic-style cheeses, Queso blanco, Queso fresco, and other soft varieties pose a greater risk to
consumers than most other cheeses, particularly when illegally prepared from raw milk.
Queso Blanco Cheese
In 1995, Glass et al. [222] reported on the behavior of L. monocytogenes in starter culture-free
Queso blanco cheese containing citric, malic, or acetic acid as acidulants and ALTA (a commercial
bacteriocin preparation resembling pediocin AcH) as an antilisterial agent. After the finished product
(pH 5.2) was inoculated to contain 106 L. monocytogenes CFU/g, populations increased about 10-
fold in cheeses containing citric or malic acid during 42 days of storage at 4°C, whereas numbers
of listeriae decreased slightly in cheeses prepared with acetic acid. These findings are consistent
with those of other investigators who used various laboratory media acidified with malic, citric, or
acetic acid (see Chapter 6). Addition of 0.6% ALTA to these cheeses yielded slightly lower Listeria
counts as compared to cheeses without ALTA. Using an L. monocytogenes inoculum level of
102 CFU/g, these workers concluded that acetic acid was significantly more effective than malic
or citric acid in reducing numbers of L. monocytogenes in Queso blanco cheese and that addition
of ALTA provided added protection against this pathogen. Similar benefits also were reported when
Queso blanco cheese was prepared using a nisin-producing starter culture, with L. monocytogenes
populations being about 1000-fold lower in such cheeses (pH 5.3) after 21 days of storage at 4 or
12°C than in nisin-free controls [144]. Although direct addition of Nisaplin (1000 AU/mL) to the
cheese milk yielded Listeria populations 100-fold lower in 1-day-old cheeses than in Nisaplin-free
controls, the pathogen recovered to control levels within 21 days at 12°C. Hence, incorporating a
nisin-producing starter culture was superior to direct addition of Nisaplin for minimizing survival
of L. monocytogenes in Queso blanco cheese during storage.
Queso de los Ibores Cheese
The fate of L. monocytogenes in Queso de los Ibores cheese (a hard, ripened cheese of pH ~5) also
was indirectly determined by Mas and Gonzalez-Crespo [277] using commercially available cheeses
of various ages. Overall, detecting Listeria spp. in 5 of 10, 2 of 10, and 1 of 10 cheeses that had
been aged for 7, 30, and 60 days, respectively, suggests that this hard, low-moisture cheese will
not support long-term survival of listeriae.
Mexican Manchego
Unlike Spanish Manchego cheese, which is a hard aged variety traditionally produced from ewe’s milk
(see the section on ewe’s and goat’s milk cheese), Mexican Manchego is a high-moisture cheese that
is produced from pasteurized cow’s milk and is ready for consumption within 5 days of manufacture.
Solano-Lopez and Hernandez-Sanchez [366] prepared Mexican Manchego cheese from pasteurized
cow’s milk inoculated to contain 106 L. monocytogenes CFU/mL along with a 1% mesophilic lactic
acid bacteria starter culture. After the curd was cooked at 40°C, salted, and pressed into hoops, numbers
of listeriae decreased almost 10-fold. An additional 6-fold reduction was seen following 5 days of
ripening at 12°C and 85% RH, with the final cheese having a pH of 5.4 and a moisture content of 42.1%.
Chihuahua
In addition to Mexican Manchego cheese, Solano-Lopez and Hernandez-Sanchez [366] also

assessed the fate of L. monocytogenes in Chihuahua. Except for a cheddaring step after curd cooking,
manufacture of this semihard aged cheese is similar to Mexican Manchego. When Chihuahua cheese
was prepared from pasteurized cow’s milk inoculated to contain 106 L. monocytogenes CFU/mL
along with a 1% mesophilic lactic acid bacteria starter culture, numbers of Listeria decreased about
10-fold during cheesemaking and an additional 10-fold in the finished cheese (pH 5.8, 36.4%
moisture) after 6 weeks of ripening at 12°C and 85% RH. Overall, these findings were similar to
those for Cheddar cheese [340] and indicate that Listeria could also persist for more than 60 days
if Chihuahua cheese is prepared from contaminated raw milk.
PICKLED CHEESES
The terms pickled and white-brined are often used to describe a group of soft/semisoft, white curd
cheeses to which large quantities of salt are added as a preservative. Cheeses belonging to this
group are principally manufactured in countries bordering the Mediterranean Sea and include such
varieties as feta (Greece), Turkish white-brined cheese (Turkey), Teleme (Bulgaria), Domiati
(Egypt), and Kareish (Egypt). Some of these cheeses are frequently prepared from ewe’s, goat’s,
or buffalo’s milk. Depending on the cheese variety, salt either can be added directly to the milk or
curd, or the finished cheese can be stored in salt brine, salted whey, salted skim milk, or dry salt.
The extreme tolerance of L. monocytogenes to high concentrations of salt, along with the organism’s
ability to grow at refrigeration temperature, has made these cheeses of particular interest to food
microbiologists working with Listeria.
Feta
In 1989, Papageorgiou and Marth [307] described the fate of L. monocytogenes during manufacture,
ripening, and storage of feta cheese. During the course of this work, there was an unconfirmed
report of a woman in New York who delivered a stillborn infant in December 1987 after consuming
feta cheese contaminated with L. monocytogenes. Hence, this study, which will now be discussed,
took on added importance.
According to these authors, cow’s milk was inoculated to contain approximately 5 × 103 L.
monocytogenes (strain Scott A or CA) CFU/mL. After warming the milk to 35°C, a 1% commercial
starter culture of S. thermophilus/L. bulgaricus was added. Forty min after addition of rennet, the
coagulum was cut and the resulting curd was transferred to metal hoops. Following 6 h of draining,
cheeses were removed from the hoops and placed in a 12% salt brine solution for 24 h. The
following day, all cheeses were transferred to 6% salt brine at 22°C for 4 days until the cheese
attained a pH of 4.3–4.4. Finally, cheese in the same 6% brine solution was moved to storage at 4°C.
Cells of L. monocytogenes were entrapped in the curd matrix during cheesemaking, with
populations nearly 10-fold greater in curd than in inoculated milk. Only about 3.2% of the original
inoculum was lost in the whey. During whey drainage and the first 1–2 days of ripening at 22°C,
numbers of listeriae increased 1.5 log10 CFU/g, with both strains attaining maximum populations
of approximately 1 × 106 CFU/g (Figure 12.11). Although growth of both Listeria strains generally
ceased at pH values between 4.6 and 5.0, numbers of listeriae remained virtually constant in cheese
during 2–5 days of storage at 22°C in 6% brine solution. Both salt brines in which feta cheese was
ripened and/or stored were positive for listeriae (these details are discussed with brine solutions at
the end of this chapter). Although all feta cheeses older than 5 days maintained a pH of 4.3, both
L. monocytogenes strains survived >90 days in finished cheese stored at 4°C (Figure 12.12).
7 7
Average pH
L. monocytogenes log10 CFU/mL or g

6 6
pH
5 5
4 Trial 1 4
Trial 2
Trial 3
3 3
–20 0 20 40 60 80 100 120 140
Hours
FIGURE 12.11 Fate of L. monocytogenes strain Scott A during manufacture and early brining of feta cheese.
(Adapted from Papageorgiou, D.K. and E.H. Marth. 1989. Fate of Listeria monocytogenes during the manu-
facture, ripening and storage of feta cheese. J. Food Prot. 52: 82–87.)
However, differences between the two Listeria strains were noted, with populations of strains Scott
A and CA decreasing 1.28 and 3.07 log10 CFU/g in 90-day-old as compared with 2-day-old feta
cheese, respectively.
Years later, Papageorgiou et al. [309] used the same protocol to produce feta cheese from
pasteurized milk inoculated to contain L. monocytogenes strains Scott A and CA at a level of
1.2 × 106 CFU/mL. After manufacture, the finished cheese containing 3.5 × 107 and 8.0 × 106
CFU/g of strains Scott A and CA, respectively, was periodically analyzed for numbers of listeriae
7
4
Trial 1
Trial 2
3 Trial 3
2
0 20 40 60 80 100
Days
FIGURE 12.12 Survival of L. monocytogenes strain Scott A during storage of feta cheese. (Adapted from
Papageorgiou, D.K. and E.H. Marth. 1989. Fate of Listeria monocytogenes during the manufacture, ripening
and storage of feta cheese. J. Food Prot. 52: 82–87.)
during 7.5 months of frozen storage at −38 and −18°C. Regardless of storage temperature, popu-
lations of strain CA decreased 9- to 10-fold in cheese samples taken from the surface and interior
after 2 to 6 weeks of storage and remained at these levels thereafter. However, strain Scott A was
far hardier, decreasing only about 2- and 4- to 6-fold in cheese samples from the surface and
interior, respectively, during 7.5 months of frozen storage at either temperature, thus indicating that
extended freezing is insufficient to inactivate substantial numbers of Listeria.
Turkish White-Brined Cheese
Sarumehmetoglu and Kaymaz [351] reported that L. monocytogenes behaved similarly in Turkish
white-brined cheese prepared from artificially contaminated raw milk, with numbers of listeriae
generally decreasing >100-fold in the finished cheese during 90 days of refrigerated storage.
In similar cheesemaking trials by Erkmen [200,201], L. monocytogenes exhibited D-values of 15.7
to 22.4 days in Turkish white-brined cheese stored at 4°C with the shorter D-values associated with
cheeses prepared with rather than without a starter culture as well as with cheese brined using a
20 rather than 15% salt solution. Although feta and Turkish white-brined cheese can be prepared
from raw milk, ripening such cheese at or above 1.7°C for 60 days will not in any way guarantee
that the final product is Listeria-free, with long-term survival of this pathogen highly probable.
Hence, it would appear prudent to manufacture these cheeses only from pasteurized milk under
good hygienic conditions to decrease the chance of a public health problem involving Listeria.
Domiati Cheese
Domiati cheese, a popular fresh white-brined cheese most commonly consumed in Egypt and other
parts of the Middle East, was prepared by Tawfik [373] using a 1:1 mixture of pasteurized
cow:buffalo milk to which 7.5% NaCl, 0.5% Lactobacillus casei, and 106 L. monocytogenes were
added. Listeria populations increased approximately 10-fold in the finished cheese as a result of
entrapment within the curd matrix and then decreased over time, with the pathogen surviving 4–8
weeks when the cheese was stored in salted whey at 20–25°C. Similar findings were reported by
Ahmed et al. [6], with L. monocytogenes strain V7 being inactivated in Domiati cheeses (pH 4.5–5.5)
containing 5 and 10% NaCl during 4 weeks of ripening at 30°C. According to Abou-Donia and
Al-Medhagi [3], L. monocytogenes also persisted no more than 8 weeks in naturally contaminated
retail samples of Domiati cheese. A decrease in cheese pH from 6.1 to 3.8 and an increase in salt
content from 6.8 to 11.4% are clearly responsible for limiting growth and survival of L. monocy-
togenes in this product.
Bulgarian White-Pickled Cheese
As early as 1964, Ikonomov and Todorov [246] reported manufacturing white-pickled cheese
from ewe’s milk containing 102–103 L. monocytogenes CFU/mL. A mixture of 0.1% S. lactis
and L. casei served as the starter culture. Although L. monocytogenes persisted 15–30 days in
white-brined cheese ripened at 18–22°C, the pathogen survived twice as long when the same
cheese was ripened at 12–15°C. In addition to storage temperature, Listeria viability also was
partly dependent on the amount of acid produced in the cheese during ripening, with pH values
of approximately 4.3 and 4.6 being reported as lethal to listeriae in cheese ripened at 12–15 and
18–22°C, respectively.
Sudanese White-Pickled Cheese
Working in the United States, Abdalla et al. [1] prepared a traditional Sudanese white-pickled
cheese from Lactococcus starter and Lactococcus starter-free pasteurized milk that was inoculated
to contain 105 L. monocytogenes Scott A CFU/mL. During cheesemaking, Listeria populations
increased approximately 10-fold to 106 CFU/g in the cheese, with growth and acid production by
the starter culture being prevented by addition of 8% NaCl to the milk during cheesemaking.
Consequently, the finished product had a pH of 6.5–7.0, which is optimal for Listeria growth.
Relatively stable Listeria populations of approximately 108 CFU/g developed in the cheese after
30–65 days of refrigerated storage in brine containing 8.6% NaCl. Similar Listeria populations
were observed when the same starter-free cheese was prepared from pasteurized milk containing
4% NaCl and preserved in a 4% brine solution with addition of 1% potassium sorbate, nisin
(25 Mg/mL), or 0.1% hydrogen peroxide to the milk not affecting Listeria survival [2]. Ineffec-
tiveness of these antimicrobial agents was attributed to several factors, including loss during
processing, degradation during the early stages of cheese ripening, and suboptimal environmental
conditions. In contrast, when a 1% lactic starter inoculum was used, L. monocytogenes populations
decreased more than 6 orders of magnitude, with the pathogen no longer being detected in cheese
after 50 days of ripening (pH 4.6) at 4°C. As was true for the other pickled cheeses, large numbers
of listeriae again leached from the cheese into the brine solution; these findings are discussed in
detail at the end of this chapter.
Yugoslavian White-Brined Cheese
In 1974 Sipka et al. [363] published results from another study in which white-brined cheese was
prepared from naturally infected cow’s milk containing 240 L. monocytogenes CFU/mL. Following
manufacture, the pathogen grew rapidly in cheese, reaching populations of 7.8 × 105 and 1.0 × 106
CFU/g after 7 and 14 days of brining, respectively. Although maximum Listeria populations were
similar to those observed in feta cheese [307], the pathogen appeared to be less hardy in white-
brined than in feta cheese, with populations decreasing to 1.3 × 105 and 1.2 × 102 CFU/g in
24- and 42-day-old fully ripened cheese, respectively. Increased inactivation of listeriae in white-
brined rather than feta cheese apparently is not entirely related to acid development, because the
pH of the former cheese, 4.5–5.1, was higher than that of the latter, 4.3.
Twenty-one years later, Katic [252] prepared a similar white-brined cheese from artificially
contaminated milk containing 0.8% starter culture and found that after 40 days of storage at 4°C, L.
monocytogenes populations had increased 100- and 43-fold in cheeses stored in 10 and 16% brine,
respectively. In contrast, numbers of listeriae increased only 4- and 12-fold in identical cheeses that
were stored in 10 and 16% brine solutions at 8°C, respectively. Thus, given the same brining
conditions, higher storage temperatures were, as expected, more detrimental to Listeria survival.
EWE’S AND GOAT’S MILK CHEESE

In areas of the world where cows are not plentiful (i.e., northern Scandinavia, Eastern Europe,
Mediterranean, Middle East), ewe’s and goat’s milks have been adapted for use either alone or in
combination with cow’s milk to manufacture different types of cheese. Representative cheeses in
this group include such well-known varieties as French Roquefort and Greek feta cheese, both of
which are traditionally prepared from ewe’s milk. Lesser-known cheeses typically prepared from
ewe’s and goat’s milk include Egyptian Kachkaval, Italian Fontina, Spanish Manchego, and Italian
Pecorino Romano as well as many varieties of white-brined and ethnic goat’s milk cheese, the
latter being often produced in mountainous areas of Central Europe and Scandinavia. The occasional
presence of L. monocytogenes in milk from healthy ewes and goats has prompted several studies
dealing with behavior of this pathogen during manufacture and ripening of some of these less
common cheeses.
Kachkaval Cheese
Work with this group of cheeses dates back to 1964 when Ikonomov and Todorov [246] examined
the behavior of L. monocytogenes in Kachkaval cheese (a relatively soft, brine-salted cheese
manufactured from ewe’s milk in Eastern Europe and the Middle East, pH 5.0–5.8) prepared from
raw ewe’s milk inoculated with the pathogen. According to these investigators, L. monocytogenes
survived in curd immersed in 5–6% salt brine at 71–76°C during cheesemaking and was still present
in Kachkaval cheese (pH 5.0–5.4) after 30–50 days of ripening at 18–22°C.
Manchego Cheese
The next such study did not appear in the literature until 1987 when Dominguez et al. [180] reported
manufacturing four lots of Manchego cheese (a hard aromatic cheese traditionally prepared from
ewe’s milk in Spain, pH ~5.8) from a blend of pasteurized ewe’s, goat’s, and cow’s milk (15:35:50)
inoculated to contain either 4.0 × 103 or 1.9 × 105 L. monocytogenes CFU/mL. To assess growth
of listeriae in cheeses having different rates of acid development, milks were inoculated to contain
either 0.1 or 1.0% of a mesophilic lactic acid bacteria starter culture. The coagulum was cut
approximately 45 min after addition of rennet; the resulting curd was drained, hooped, and pressed
for approximately 10 h. The finished cheese was then brine-salted overnight, ripened 10 days at
15°C/85–90% RH, covered with paraffin, and aged an additional 50 days at 15°C.
Numbers of L. monocytogenes increased 10-fold in all cheeses during the first 10 h of manu-
facture, primarily from entrapment of the organism within curd matrix. The fact that cheeses
prepared with either 0.1 or 1.0% starter culture contained similar Listeria populations indicates
that behavior of the pathogen was not greatly influenced by the rate of acid production during
cheesemaking. After brining the cheese overnight, populations of listeriae decreased approximately
3- to 100-fold, with additional small decreases being observed during ripening of unparaffined
cheese at 15°C. Numbers of listeriae remained relatively constant in cheese at pH 5.1–5.8 after
paraffining, with populations in 60-day-old cheese approximating the original inoculum in milk
from which the cheese was manufactured. However, Rodriguez et al. [332] more recently showed
that using a nisin-producing L. lactis subsp. lactis starter culture could markedly shorten survival
of L. innocua in Manchego cheese during ripening.
Goat’s Milk Cheese
Working in Sweden, Tham [291] examined the viability of Listeria in cheese prepared from raw
goat’s milk inoculated to contain 105–106 L. monocytogenes CFU/mL. A mixture of mesophilic
lactic acid bacteria served as the starter culture. Approximately 45 min after addition of rennet, the
coagulum was cut into cubes that were cooked at 37°C, drained, hooped, pressed for 1 h, and brine-
salted for 10 h. The finished cheese was then ripened at 12°C for 22 weeks.
Actual numbers of L. monocytogenes could be determined in cheeses from only two of six lots
using a blood agar/pour plate method. As shown in Figure 12.13, Listeria populations decreased
approximately 10-fold in goat’s milk cheese during the first 14 weeks of ripening at 12°C. Extended
survival, along with slight growth of listeriae in cheeses ripened longer than 14 weeks, probably
is related to an increase in pH from 5.55 to 6.20 during ripening as well as a decrease in numbers
of competitive microorganisms that were initially present in the raw milk. Although large
numbers of enterococci and other microbial competitors interfered with the quantitative recovery
of L. monocytogenes in the remaining four cheeses, the pathogen survived 10–16 weeks in three
of these cheeses as determined by cold enrichment. The impact of an active starter culture on the
fate of Listeria in Swedish raw milk goat cheese was also later confirmed by Eilertz et al. [183],
with L. monocytogenes populations increasing 10- and 10,000-fold in cheeses prepared with and
without starter culture, respectively, after 4 weeks of ripening at 4°C.
In 2001, Morgan et al. [294] prepared a soft surface-ripened cheese from raw goat’s milk
inoculated to contain 102 L. monocytogenes CFU/mL with cultures of Kluyvermyces lactis and
Geotrichum candidum used for surface ripening. During cheesemaking, numbers of listeriae
increased to 3.3 log CFU/g, with half of this increase resulting from physical entrapment in the
L. monocytogenes in cheese VI
L. monocytogenes in cheese V
Total aerobic count in cheese VI
9.0 Total aerobic count in cheese V
10 CFU/g 8.0 Added L. monocytogenes per mL milk

L. monocytogenes log
7.0
6.0
5.0
0 2 4 6 8 10 12 14 16 18
Weeks
FIGURE 12.13 Survival of L. monocytogenes and total aerobic flora during manufacture and ripening of raw
goat’s milk cheese. (Adapted from Tham, W. 1988. Survival of Listeria monocytogenes in cheese made of
unpasteurized goat milk. Acta Vet. Scand. 29: 165–172.)
curd matrix, as previously discussed. After 7 days of ripening, cheese samples taken from the
surface and interior contained about 2.0 and 3.0 L. monocytogenes log CFU/g, respectively. How-
ever, this difference was no longer evident in 28-day-old cheeses with surface and interior samples
both yielding about 1.5 log CFU/g. Although the pH of cheese samples taken from the surface and
the interior increased from 4.25 to 6.52 and 4.25 to 5.23 after 42 days of ripening, respectively,
numbers of listeriae remained at levels near 1.5 log CFU/g. These results are in sharp contrast to
those reported for Camembert and most other surface-ripened cheeses, with G. candidum perhaps
involved in suppressing the growth of Listeria during cheese ripening as has been previously
reported by Dieuleveux and Gueguen [178]
A middle-aged English woman developed listerial meningitis in February 1988 after consuming
2–3 oz of commercially produced Anari-type goat’s milk cheese containing 107 L. monocytogenes
CFU/g [64]. During a series of follow-up investigations [283], L. monocytogenes populations of
<10 CFU/g were discovered in one 2- to 3-day-old Anari cheese and in three 2- to 3-day-old
Halloumi cheeses produced by the same manufacturer. After these naturally contaminated cheeses
were stored at 4°C for 4–5 weeks, Listeria populations as high as 8 × 104 and 1 × 106 CFU/g
developed in Anari (pH 5–6) and Halloumi cheese (pH 6), respectively. Assuming a lag time of
zero and an original L. monocytogenes population of 1–9 CFU/g, these authors calculated generation
times of 47–56 and 32–37 h for this pathogen in Anari and Halloumi cheese, respectively. Both of
these generation times are similar to those previously reported for L. monocytogenes in refrigerated
fluid milks (see Chapter 11, Table 11.7). Assuming that these cheeses were on sale for up to 3
months after distribution, potentially hazardous levels of listeriae easily could have developed in
such products during retail storage. Although a recent attempt to minimize Listeria growth in soft
goat cheese by modified atmosphere packaging under increased levels of CO2 failed [301], high-
pressure processing of Listeria-contaminated raw goat’s milk cheese at 450 MPa for 10 min or
500 MPa for 5 min reportedly decreased Listeria populations >5.6 log CFU/g without adversely
affecting appearance, texture, or flavor [213]. Although a costly microbial reduction strategy, high-
pressure processing may be one means of enhancing the safety of goat’s milk and other specialty
cheeses that command a premium.
SOFT UNRIPENED CHEESE

Soft unripened cheeses include such high-moisture, white-curd varieties as cottage, baker’s, cream,
and American-type Neufchâtel cheese. Unlike the groups of cheeses discussed thus far, milk to be
manufactured into soft unripened cheese is coagulated through production of acid by the starter
culture (or alternatively, by direct acidification of milk to pH 4.6–4.7 with gluconic acid, glucono-
delta-lactone, or a mineral acid plus the lactone) rather than by the addition of a coagulant. Hence,
these products are sometimes referred to as acid-curd cheese. Because the refrigerated shelf life of
most soft unripened cheeses is typically less than 60 days, these varieties must be prepared from
pasteurized milk or cream in the case of cream cheese. Although pH values of 4.6–5.0 provide an
unfavorable environment for microorganisms that may contaminate soft unripened cheese before,
during, or after manufacture, the fact that these cheeses can be consumed immediately after
production may pose a public health risk, particularly if psychrotrophic, acid-tolerant organisms
such as L. monocytogenes are present.
Cottage Cheese
Nearly 1 year before the famed cheeseborne listeriosis outbreak in California, Ryser et al. [346]
used the short-set procedure to prepare cottage cheese from pasteurized skim milk inoculated to
contain 104–105 L. monocytogenes CFU/mL. Following manufacture, half the curd was creamed
to contain ≥4% milk fat and half remained uncreamed. Both products were examined for numbers
of listeriae during 28 days of storage at 3°C.
Numbers of L. monocytogenes remained relatively constant during the initial 5–6 h of cheese-
making, during which the pH of milk decreased from 6.65 to 4.70. These findings agree with those
of Schaack and Marth [353], who later demonstrated that growth of L. monocytogenes in skim
milk at 30°C is completely suppressed by a 5% inoculum of S. cremoris. After increasing the
temperature of the curd/whey mixture to 57.2°C (135°F) over 90 min and cooking the curd at this
temperature for an additional 30 min, L. monocytogenes was not detected in samples of curd or
whey that were directly plated on McBride Listeria Agar. However, following cold enrichment in
Tryptose Broth, L. monocytogenes was detected in four of eight, two of eight, one of eight, and
two of eight samples of cooked curd, whey, wash water, and washed curd, respectively, which
suggests that some Listeria cells were only sublethally injured during cooking of the curd at pH
4.6–4.7. Such injury may preclude growth on McBride Listeria Agar, which contains both lithium
chloride and phenylethanol as selective agents.
Examination of the finished product indicated that L. monocytogenes survived in both creamed
and uncreamed cottage cheese at levels generally <100 CFU/g during 28 days of refrigerated
storage. Although there was no evidence for growth of listeriae in either cheese during storage,
probably because of pH values generally <5.5, the pathogen was recovered more frequently and
at higher numbers in creamed rather than uncreamed cottage cheese. The higher pH of 3-day-old
creamed (pH 5.32–5.45) rather than uncreamed (pH 5.12–5.22) cottage cheese may have been
responsible for increasing the repair rate of injured cells, thereby increasing recovery of listeriae.
Although behavior of L. monocytogenes in cheese failed to gain widespread attention until
1985, a search of the scientific literature uncovered an earlier study by Stajner et al. [367] that
examined the viability of Listeria in unsalted small-curd skim milk cheese (similar to uncreamed
cottage cheese) manufactured from naturally infected milk containing approximately 5 × 105
L. monocytogenes CFU/mL. Results from these Yugoslavian investigators support the findings of
Ryser et al. [346] in that the pathogen survived at least 7 days in finished cheese (pH 4.55–4.75)
stored at 3–5°C.
More recently, El-Shenawy and Marth [191] studied the behavior of listeriae in cottage cheese
prepared from pasteurized skim milk that was inoculated to contain 106 L. monocytogenes CFU/mL
and then coagulated over a period of 3 h using hydrochloric acid, gluconic acid, or bovine rennet
L. monocytogenes log10 CFU/g or mL 5
3
Rennet Curd
Rennet Whey
2 HC1 Curd
HC1 Whey
GA Curd
1 GA Whey
0
A B C D E F
FIGURE 12.14 Survival of L. monocytogenes in curd and whey obtained during preparation of cottage cheese
made with rennet, HCl, or gluconic acid (GA). A: Immediately after cutting; B: after temperature was increased
to 48.9°C; C: after temperature was increased to 54.4°C; D: after temperature was increased to 57.2°C;
E: after 15 min of cooking; and F: after 30 min of cooking. (Adapted from El-Shenawy, M.A. and E.H. Marth.
1990. Behavior of Listeria monocytogenes in the presence of gluconic acid and during preparation of cottage
cheese curd using gluconic acid. J. Dairy Sci. 73: 1429–1438.)
rather than a lactic acid bacteria starter culture, during which time the temperature of the milk was
gradually increased from 2 to 32°C. The resulting coagulum was then cut and cooked using the
aforementioned procedure of Ryser et al. [346].
As might be expected, acidification of the milk to pH 4.7–4.8 followed by heating was again
detrimental to survival of listeriae during manufacture of cottage cheese. Overall, L. monocytogenes
populations decreased ~4.5 and > 6.0 orders of magnitude in fully cooked (57.2°C/30 min) curd
obtained by adding hydrochloric and gluconic acid, respectively (Figure 12.14). However, using
cold enrichment, Listeria was recovered from samples of fully cooked gluconic acid curd. Numbers
of listeriae decreased faster in acidified whey than curd, with cold enrichment results indicating
that the pathogen was eliminated from gluconic but not hydrochloric acid whey after 30 min of
cooking at 57.2°C. Nonetheless, direct acidification of milk (pH 4.7–4.8) for cottage cheesemaking
followed by cooking the resultant curd at 57.2°C for 30 min should be more than sufficient to
eliminate expected numbers of listeriae (<10 CFU/mL) that might inadvertently enter pasteurized
milk as postprocessing contaminants.
In contrast to acid curd and whey, populations of listeriae in freshly cut rennet curd and whey
were virtually identical to those initially observed in milk (Figure 12.14). Further, slight increases
in numbers of listeriae were noted midway through manufacture, with fully cooked rennet curd
(pH 6.6) and whey still containing 104 and 103 L. monocytogenes CFU/g or CFU/mL, respectively.
Thus, listericidal effects associated with cooking were greatly enhanced under acidic condi-
tions. Subsequent experiments with selective plating media confirmed that substantial numbers of
L. monocytogenes cells were sublethally injured during manufacture of cottage cheese, as was
suggested by Ryser et al. [346] five years earlier. Sublethal injury was far more evident in whey
rather than curd samples, probably because curd afforded some thermal protection to listeriae. Not
surprisingly, the degree of sublethal injury was also closely related to coagulant type (i.e., acidity)
and cooking temperature, with less injury being observed in rennet rather than acid curd and whey,
and partially rather than fully cooked samples of curd and whey. Heat alone was primarily respon-
sible for rennet-associated injury, whereas the combined effects of heat and acid led to injury of
listeriae in acid curd and whey.
With the exception of cottage cheese prepared from milk acidified with gluconic acid, both of
these studies demonstrated limited survival of L. monocytogenes in cottage cheese. However, the
fact that the pathogen failed to grow in the product and decreased drastically in numbers during
manufacture suggests that cottage cheese poses far less of a public health threat than do those
varieties that are surface-ripened with molds or bacteria. Lower health risks associated with con-
sumption of cottage cheese are also supported by the extremely low incidence of L. monocytogenes
in commercially produced cottage cheese examined in American and European surveys.
The likelihood of L. monocytogenes entering cottage cheese during creaming or packaging of
the product is far greater than having listeriae present in pasteurized milk at sufficiently high levels
to survive in the cooked curd. Although most recently published work has addressed the fate of
L. monocytogenes in cottage cheese as a postmanufacturing contaminant, results from these efforts
have been somewhat conflicting. Based on the findings of three studies conducted in the United
States [313] and England [207,243], L. monocytogenes failed to grow in artificially contaminated,
commercially prepared creamed cottage cheese (pH 4.5–5.1), with populations generally decreasing
0.5–1.5 orders of magnitude during 1–5 weeks of storage at refrigeration or abusive temperatures.
According to Moir et al. [292], numbers of L. monocytogenes remained relatively stable in com-
mercial, Australian-produced creamed cottage cheese during 1 month of storage at 15°C. This
behavior is similar to that observed by Hicks and Lund [243] when creamed cottage cheese was
inoculated with an acid-adapted strain of L. monocytogenes previously cultured in Tryptose Phos-
phate Broth at pH 5.5. In contrast to these findings, at least four additional studies attest to growth
of L. monocytogenes [158,171,217] and L. innocua [206] in similar samples of creamed cottage
cheese, with populations increasing 0.5–3.0 orders of magnitude during refrigerated storage.
Although not readily apparent, such behavioral differences in creamed cottage cheese may be
related to differences between Listeria strains as well as differences in acid tolerances [211] and
abilities to readily compete with the native microflora.
Because L. monocytogenes can persist beyond the normal shelf life of cottage cheese, several
options also have been examined for minimizing growth and/or survival of Listeria in this product
during refrigerated storage. A few chemical additives, including sorbate [313], 3% sodium lactate
[313], 3% calcium lactate [313], and 0.04% lysozyme, were shown to be, at best, only minimally
effective, with Listeria populations in inoculated creamed cottage cheese decreasing ≤10-fold during
the product’s normal refrigerated shelf life.
Bacteriocin-producing starter cultures appear to be a far more promising means of inactivating
any listeriae that may inadvertently contaminate the curd after cooking. One approach for intro-
ducing these bacteriocins into cottage cheese is the use of a bacteriocin-producing starter culture
for cheese manufacture. Applying this strategy, McAuliffe et al. [281] prepared cottage cheese from
skim milk using a lacticin 3147-producing strain of L. lactis at an inoculum level of 1%. After
manufacture, the curd was inoculated to contain about 104 L. monocytogenes strain Scott A CFU/g
at the time of creaming. Following 5 days of storage at 4°C, L. monocytogenes populations decreased
below the detectable limit of 10 CFU/g in cottage cheese prepared with the lacticin-producing
starter culture compared to populations of 5 × 103 in cheese prepared with a non-lacticin-producing
strain of L. lactis.
Alternatively, purified or partially purified bacteriocin powders can be directly incorporated
into cottage cheese at the time of creaming. Using commercially prepared dry cottage cheese curd
to which 7.5 × 103 L. monocytogenes CFU/g were added during creaming, Pucci et al. [319] found
that the product (pH 5.1) still contained 1 × 102 L. monocytogenes CFU/g after 7 days of storage
at 4°C. However, addition of purified bacteriocin PA-1 powder from Pediococcus acidilactici to
identical samples led to complete inactivation of the pathogen within 24 h. Similarly, El-Ziny and
Debevere [193] found that addition of reuterin (a bacteriocin produced by Lactobacillus reuteri)
to the creaming mixture at concentrations of 100, 150, and 250 units/g reduced L. monocytogenes
populations 2, 5, and >5 log CFU/g, respectively, after 7 days of storage at 7°C.
Using a different brand of dry cottage cheese curd that was inoculated to contain 3 × 105
L. monocytogenes CFU/g during creaming, Benkerroum and Sandine [132] detected very few viable
listeriae in the product (pH 4.9–5.0) after 1 or more days of storage at 4°C. This antagonistic effect
of cottage cheese (presumably from natural flora or by-products in cottage cheese) toward listeriae
was enhanced by adding nisin (2.5 × 103 IU/g), with viable listeriae completely being eliminated
from such cheese after 1 day of refrigerated storage. The efficacy of nisin has since been confirmed
by Ferreira and Lund [207], who reported that L. monocytogenes populations decreased about
1000-fold in creamed cottage cheese (pH 4.6–4.7) containing 2000 IU nisin/g after 3 days of
storage at 20°C.
In these studies, nisin, bacteriocin PA-1, and reuterin remained active in creamed cottage cheese
during prolonged storage. Thus, it appears that these bacteriocins may also prove useful in limiting
Listeria survival in other fermented dairy products, including natural cheeses, cold-pack cheese
food, and various cheese spreads [66,74] during the normal shelf life. However, decreased efficacy
of nisin as well as PA-1 and reuterin in commercially prepared cheese sauce (pH 6.0), half-and-
half (pH 6.6), and skim milk (pH 6.6) shows that listericidal activity of these bacteriocins is strongly
pH dependent.
Alternatively, modified atmosphere packaging also has been explored as a means of both
extending the refrigerated shelf life of cottage cheese and minimizing Listeria growth. According
to Chen and Hotchkiss [158], L. monocytogenes populations in creamed cottage cheese (pH 5.1)
packaged under 35% CO2 remained stable during 9 weeks of storage at 4°C, whereas the pathogen
increased 1000-fold in aerobically packaged cheese. At 7°C, numbers of listeriae failed to change
in CO2-packaged cheese during the first 4 weeks of storage. However, rapid growth occurred within
16 days in aerobically packaged cheese. Fedio et al. [206] also reported that L. innocua failed to
grow in creamed cottage cheese (pH 5) during 28 days of storage at 5°C when the product was
packaged under 50 or 100% CO2. However, this organism began growing rapidly in identical
samples packaged aerobically or under 100% N2 after 7 days of incubation. Although modified
atmosphere packaging appears to be useful in minimizing Listeria growth in cottage cheese,
inactivation of the pathogen will not occur under such conditions, with the results of Chen and
Hotchkiss [158] also indicating that the effectiveness of CO2 is strongly temperature dependent.
Cream Cheese
In the only other study dealing with the behavior of listeriae in soft unripened cheese, Cottin et al.
[168] prepared cream cheese from a chemically acidified mixture of milk and cream that was
inoculated to contain 101–107 L. monocytogenes CFU/mL. Using the lowest inoculum, Listeria
grew in the finished product (pH ≤6) and attained a stable population of ~103 CFU/g within 2 days
of storage at 4°C. Thus, unlike cottage cheese, the pH and moisture content of cream cheese are
both sufficiently high to permit limited growth of listeriae in the product during refrigeration.
WHEY CHEESES
A few cheeses such as ricotta, Broccio, Ricotone, and Anthotyros are prepared from sweet whey
derived from the manufacture of mozzarella, Cheddar, Swiss, Tilsiter, and feta cheese. Manufacture
of these whey cheeses is based on the direct acidification of whey, whey and milk, and whey–cream
mixtures to pH 5.9–6.0 using food-grade acids (i.e., citric, acetic), lactic starter cultures, or acid
whey powder, followed by cooking at 180–190°F to precipitate the whey protein. The fine precipitate
that eventually rises to the vat surface is then removed, drained, matted, and marketed either as
whey cheese or a dairy ingredient. Several additional extremely low-moisture (13 to 18%) whey
cheeses, including Gjetost, Mysost, and Gudbrandsdalsost, are unique to Norway and are prepared
by thermally concentrating and then boiling a blend of goat’s and cow’s milk whey until the mixture
carmelizes and becomes viscous. This plastic mass is then cooled, extruded, and cut into extremely
dense blocks for marketing.
As was true for mozzarella cheese, L. monocytogenes will be completely inactivated during
manufacture of these whey cheeses. However, the potential still exists for contamination during
packaging, as is evidenced by at least one Class I recall of ricotta cheese in July 1991. In limited
work, Davies et al. [173] manufactured a ricotta-type cheese from raw milk to which 500 ppm
potassium sorbate and 50 or 100 ppm nisin were added before the start of cheesemaking. After
coagulating the milk by heating at 90°C for 30 min with or without direct acidification to pH 5.9
with acetic acid, the precipitated curd was drained and inoculated with a 5-strain L. monocytogenes
cocktail at a level of 2.5 × 102 CFU/g. During 63 days of storage at 6–8°C, Listeria populations
in nisin-free control cheeses prepared without and with direct acidification of the milk steadily
increased to about 107 and 109 CFU/g, respectively. In contrast, addition of 125 ppm nisin resulted
in lag times of 11 and 26 days when the milk was coagulated by heating without and with
acidification, respectively, with Listeria reaching maximum populations that were 1 to 2 logs lower
than those in the control samples after 63 days of storage. However, regardless of the method of
milk coagulation, the Listeria population was held constant for at least 55 days using 250 ppm
nisin, indicating that addition of nisin is a viable intervention strategy for preventing growth of
listeriae during long-term refrigerated storage of ricotta cheese.
Following the report of a small cluster of listeriosis cases traced to Anari whey cheese produced
in Cyprus, Papageorgiou et al. [308] examined the fate of L. monocytogenes as a postprocessing
contaminant in Greek Myzithra (identical to Anari cheese), Anthotyros, and Manouri cheese, all
of which are starter culture-free, soft (50–70% moisture), low-acid (pH 6.0–6.5) whey cheeses.
Immediately after commercial manufacture, these cheeses were inoculated to contain ~500 L.
monocytogenes strain Scott A or CA CFU/g. Regardless of cheese type or Listeria strain, the
pathogen grew rapidly and attained maximum populations of 107–108 CFU/g after 24–30 days,
5–12 days, and 56–72 h of storage at 5, 12, and 22°C, respectively.
The study just described prompted investigators in Greece to assess the efficacy of nisin and
irradiation for minimizing growth and survival of Listeria in Anthotyros cheese. In the first of these
studies, Samelis et al. [350] evaluated nisin as a biopreservative to control L. monocytogenes as a
postprocessing contaminant. Anthotyros cheese was prepared from naturally or directly acidified
whey to which 100 or 500 IU nisin/mL was added before the start of cheesemaking, with the
finished cheese inoculated to contain 104 L. monocytogenes strain Scott A CFU/g. Alternatively,
100 or 500 IU nisin/g was added to the final cheese at the time of Listeria inoculation rather than
to the whey used for cheesemaking. Following initial decreases of 1 to 2 logs, Listeria attained
levels of 107 to 108 CFU/g in the cheese after 42 days of storage at 4°C when nisin was added
directly to the finished cheese or when 250 IU nisin/mL was added to the whey at the start of
cheesemaking. However, addition of 500 IU nisin/mL to the whey at the start of cheesemaking
effectively prevented Listeria from exceeding the initial inoculum level of 104 CFU/g after 45 days
of storage. Thus, nisin was most effective when added to the whey rather than to the finished cheese.
In the only other study thus far reported involving Anthotyros cheese, Tsiotsias et al. [384] found that
irradiation at 4 kGy decreased populations of L. monocytogenes strain Scott A about 3 logs in freshly
made cheese. However, surviving cells were able to recover from the effects of irradiation, with the
pathogen increasing 2 to 3 logs in the cheese during 42 days of storage at 4 and 10°C. Thus, irradiation
dose not appear to be effective in controlling Listeria in dairy products, with this treatment also
negatively impacting the ripening of aged cheeses such as Cheddar and Camembert. Consequently,
addition of nisin during cheesemaking and strict hygienic practices during packaging appear to be
the most practical means of minimizing the presence and growth of Listeria in Anthotyros cheese.
3.0
2.5
2.0
Log10 Strain/g
1.5
Scott A
1.0 V7
CA
.5
OH
.0
0 20 40 60 80 100 120 140 160 180 200
Days
FIGURE 12.15 Survival of four strains of L. monocytogenes strains in nonacidified cold-pack cheese food
manufactured without preservatives. (Adapted from Ryser, E.T. and E.H. Marth. 1988. Survival of Listeria
monocytogenes in cold-pack cheese food during refrigerated storage. J. Food. Prot. 51: 615–621, 625.)
Cold-Pack Cheese Food
Unlike the natural cheeses discussed thus far, cold-pack cheese food is typically prepared by
comminuting and blending aged Cheddar cheese (or another variety) with nonfat dry milk, dried
whey, water, cream, plastic cream (composition similar to butter), salt, acidulants (i.e., lactic or
acetic acid), preservatives (i.e., potassium sorbate or sodium propionate), and other optional ingre-
dients into a homogeneous mass without heating. Because all the dairy ingredients and some of
the optional ingredients used in manufacturing cold-pack cheese food can potentially harbor
Listeria Ryser and Marth [344] investigated the behavior of four L. monocytogenes strains in nine
different formulations of cold-pack cheese food inoculated to contain approximately 500 L.
monocytogenes CFU/g.
During 182 days of storage at 4°C, populations of all four Listeria strains decreased less than
10-fold in nonacidified (pH 5.20) preservative-free cheese food, with the pathogen surviving through-
out the product’s entire 6-month shelf life (Figure 12.15). In sharp contrast to these findings, addition
of preservatives with or without acidifying agents led to the eventual demise of listeriae in cheese
food stored at 4°C (Figure 12.16). In nonacidified cheese food (pH 5.20) preserved with 0.3% sodium
propionate, L. monocytogenes survived an average of 142 days as compared to 118, 103, and 98 days
in the same product adjusted to pH 5.0–5.1 with lactic, acetic, and lactic plus acetic acid, respectively.
Using 0.3% sorbic acid in place of sodium propionate, the pathogen survived an average of 130 days
in nonacidified cheese food (pH 5.45) as compared to 112, 93, and 74 days in cheese food acidified
to pH 5.0–5.1 with lactic, lactic plus acetic, and acetic acids, respectively. Thus, sorbic acid was
consistently more antagonistic to listeriae than sodium propionate. In addition, antilisterial effects of
both preservatives were more pronounced in cheese food acidified with acetic rather than lactic acid.
Because organic acids are far more bactericidal in the undissociated than dissociated state, increased
inactivation of listeriae in the presence of acetic acid probably resulted from the higher proportion of
undissociated acetic (~36%) rather than lactic acid (~5.9%) in cheese food acidified to pH 5.0–5.1.
These findings indicate that it would be prudent to consider (a) adding preservatives, particularly
150
140
S Sorbic Acid
130
P Na Propionate
L Lactic Acid
120
a A Acetic Acid
Survival (Days)
110
100
90
80
70
60
50
P S P+L S+L P+A P+L+A S+L+A S+A
Additive
FIGURE 12.16 Maximum duration of survival of L. monocytogenes in acidified and nonacidified cold-pack
cheese food containing preservatives. Each bar represents the average maximum length of survival of all four
Listeria strains in one of eight different formulations of cheese food manufactured in duplicate. Any two
differing by >20.24 days (length of bar) are significantly different (p < 0.05). (Adapted from Ryser, E.T. and
E.H. Marth. 1988. Survival of Listeria monocytogenes in cold-pack cheese food during refrigerated storage.
J. Food. Prot. 51: 615–621, 625.)
sorbic acid, to cold-pack cheese food and (b) reducing the pH of the product to 5.0 by adding small
amounts of lactic or acetic acid to minimize L. monocytogenes survival in the finished product.
Additional information on conditions leading to inhibition and/or inactivation of this pathogen by
sorbic, propionic, lactic, and acetic acids can be found in Chapter 6.
PASTEURIZED PROCESSED CHEESE

American pasteurized processed cheese was first developed at the turn of the twentieth century and
is made by grinding and mixing different natural cheeses with citrate and phosphate-based emulsifiers,
with this cheese mixture then heated for 30 sec or more at 150°F to produce a homogeneous mass.
This molten cheese is then quickly cooled to form slices or blocks of cheese upon cooling that are
ready for sale. Owing to the extreme heat treatment that this cheese receives during manufacture,
pasteurized processed cheese continues to have an excellent safety record, with very few foodborne
outbreaks of any type associated with this product over the past 50 years. Further, this type of cheese
has not been implicated in any Listeria-related recalls or outbreaks of listeriosis, with the pathogen
also thus far absent from samples of pasteurized processed cheese analyzed in limited surveys.
However, given the relatively common occurrence of L. monocytogenes in food processing environ-
ments, Glass et al. [223] chose to assess the ability of L. monocytogenes to proliferate on pasteurized
processed cheese as a postprocessing contaminant. In their study, retail slices of pasteurized processed
cheese from six different lots (pH 5.61–5.84, 2.35–2.62% salt, 0.918–0.929 aw) were surface-inocu-
lated to contain 103 CFU/g of a 3-strain L. monocytogenes cocktail that included strain Scott A. Overall
no growth was evident, with Listeria decreasing 0.6 to 0.7 log CFU/g on these cheese slices after
4 days of storage at an abusive temperature of 30°C. Thus, given these findings, pasteurized processed
cheese poses a very minimal health risk to consumers.
BEHAVIOR OF L. MONOCYTOGENES IN CHEESE AS AFFECTED BY CHEESE COMPOSITION

As suggested earlier, the fate of L. monocytogenes and other foodborne pathogens during cheese
ripening is determined by the microbiological, biochemical, and physical properties of the particular
cheese. Thus, cheese is a very complex system, with the following factors acting simultaneously
to determine the behavior of L. monocytogenes during ripening: (a) type, amount, and activity of
the starter culture; (b) pH as determined by concentrations of lactic, acetic, formic, and other acids;
(c) presence of hydrogen peroxide, diacetyl, and various antimicrobial agents (i.e., nisin and other
bacteriocins); (d) levels of nutrients, salt, moisture, and oxygen; and (e) the temperature at which
the cheese is ripened.
All these factors act together to produce a particular outcome; however, a few conclusions
concerning the ability of L. monocytogenes to grow and survive in some of the aforementioned cheeses
prepared from contaminated milk can be drawn by examining the behavior of this pathogen in relation
to the combined effects of moisture content, water activity (aw), and salt in the moisture phase as well
as the pH of the cheese and the temperature at which the cheese is ripened (Table 12.16).
Although fully ripened Camembert and feta cheese have widely differing pH values of 7.5 and 4.4,
respectively, both cheeses are very similar in terms of moisture content, water activity, percentage
of salt in the water phase, and ripening temperature. Thus, rapid growth of L. monocytogenes in
Camembert cheese can be largely attributed to the increase in pH of the cheese during ripening,
whereas a pH value of 4.4 appears to be responsible for preventing growth of the bacterium during
ripening of feta cheese. Inability of L. monocytogenes to multiply in blue cheese during ripening
and storage is probably related to the high concentration of salt in the water phase (which results
in a low aw), because other workers have confirmed that this organism will not grow in laboratory
media [359] and skim milk [303] containing >10 and 12% salt, respectively. As with Camembert
cheese, growth of two of four L. monocytogenes strains in brick cheese appears to be directly
related to the high pH that the cheese attained during extended ripening. However, inability of the
remaining two strains to grow in brick cheese of similar composition is as yet unexplained.
When comparing the behavior of L. monocytogenes in Cheddar and Colby cheese, the initial
inactivation rate for the pathogen was somewhat slower in the latter cheese. At first glance, it
appears that increased viability of Listeria in Colby cheese during the early stages of ripening may
be related to the lower percentage of salt in the water phase in this cheese than in Cheddar cheese.
However, data in Table 12.16 show that L. monocytogenes was inactivated at similar rates in Colby
cheese and cold-pack cheese food, the latter being compositionally similar to Colby cheese except
for a higher concentration of salt in the water phase. Hence, factors other than a low concentration
of salt in the water phase also must be involved in enhancing the viability of listeriae during ripening
of Colby cheese.
Lack of growth and decreased survival of L. monocytogenes in Parmesan cheese and in an
unidentified hard Italian cheese as compared to other varieties in Table 12.16 correlate well with
the lower moisture content/aw of these cheeses during ripening. Barring thermal or acid injury of
L. monocytogenes, which is likely to occur during manufacture of cottage cheese and other varieties
that undergo substantial heat treatments during manufacture (i.e., mozzarella, Swiss), factors out-
lined in Table 12.16 are useful in predicting whether or not this pathogen will grow in other cheeses
having similar microbiological, biochemical, and physical characteristics.
In addition to being present in milk at the time of cheesemaking, Listeria also can easily
contaminate the finished cheese during packaging, ripening, and storage. Consequently, Genigeorgis
et al. [217] evaluated the fate of Listeria as a postprocessing contaminant by inoculating 49 retail
cheeses (24 types/28 brands) with L. monocytogenes and then storing these cheeses at 4–30°C for
up to 36 days. As expected, Listeria growth was primarily confined to high-moisture varieties,
including Brie, Camembert, ricotta, and the soft Hispanic cheeses, all of which had a pH ≥5.9 and
low to moderate levels of salt in the moisture phase (Table 12.17). According to Razavilar [327],
L. monocytogenes populations in inoculated Queso fresco cheese curd of pH 6.6 also increased
TABLE 12.16
Behavior of L. monocytogenes during Cheese Ripening as Affected by Cheese Composition
Estimated
Salt in
Moisture Estimated Water Phase pH Ripening Log10 of L. monocytogenes CFU/g Survival
Cheese (%) aw (%) Initiala Final Temperature (°C) Initiala Maximum Final (days) Ref.
Camembert 54.4 0.975 4.72 4.6 7.5 15/6 3.1–3.6 6.7–7.5 6.7–7.5 65 341
Blue 38.9 0.950 11.52 4.6 6.3 9–12/4 4.0–5.0 4.0–5.0 1.0–2.3 120 306
Brick 43.0 0.990 1.89 5.3 7.3 15/10 3.0–4.7 4.6–6.7 2.7–6.1 168 345
Feta 54.7 0.975 4.57 4.7 4.4 22/4 5.2–6.2 5.7–6.2 2.8–4.6 90 307
Cheddar 37.2 0.975 4.61 5.1 5.1 6 2.5–3.2 2.6–3.8 0–1.5 70–434 340
Cheddar 37.2 0.975 4.61 5.1 5.1 13 2.6–3.4 3.0–3.7 0 70–224 340
Colby 40.0 0.975 3.91 5.1 5.1 4 3.5–4.5 3.6–4.6 2.3–4.1 112–140 460
Parmesan 32.0 0.935 4.96 5.1 5.1 13 3.3–4.3 3.3–4.3 1.0–1.3 21–112 401
Hard Italian NRc 0.950 2.12d 5.3 5.7 4 4.5–5.1 4.5–5.6 2.0 35 161
Cold-pack cheese 41.4 0.975 4.90 5.3 5.1 4 2.4–2.8 2.4–2.8 1.1–2.0 180 344
foodb
a Approximately 24 h after the start of cheesemaking.

b Prepared without preservatives or acidifying agents.
c Not reported.
d Percentage of salt in solid and water phase.

479
TABLE 12.17
Growth and Inactivation of L. monocytogenes in Surface-Inoculated Retail
Cheeses during Storage at 4–30°C
% NaCI in
Cheese Category and Type pH Moisture Phase Growth
Soft mold-ripened
Brie 6.0–7.7 2.5–3.6 +
Camembert 7.3 2.5 +
Blue 5.1 6.1 −
Bacterial surface-ripened
Limburger 7.2 4.8 −
Muenster 5.5 3.8 −
Soft Italian
Provolone 5.6 4.6 −
String cheese 5.5 4.4 −
Semisoft and hard-ripened
Monterey Jack 5.0–5.2 1.0–3.0 −
Colby 5.5 4.9 −
Cheddar 4.9–5.6 2.6–5.4 −
Swiss 5.5 2.7 −
Hispanic
Queso fresco 6.5–6.6 4.5–6.6 +/−
Queso Ranchero 6.2 4.1 +
Queso Panella 6.2–6.7 2.5–3.9 +
Cotija 5.5–5.6 9.6–12.5 −
Pickled cheese
Feta 4.2–4.3 2.2–7.5 −
Ewe’s milk cheese
Kesseri 4.8–5.3 5.5–5.87 −
Soft unripened
Cottage cheese 4.9–5.1 1.0–1.2 +/−
Cream cheese 4.8 <1.9 −
Whey cheeses
Ricotta 5.9–6.1 <0.7 +
Processed cheese
American 5.7 2.1 −
Monterey Jack 5.7 4.4 −
Piedmont 6.4 5.1 −
Source: Adapted from Genigeorgis, C., M. Carniciu, D. Dutulescu, and T.B. Farver. 1991. Growth and
survival of Listeria monocytogenes in market cheeses stored at 4 to 30°C. J. Food Prot. 54: 662–668.
from 104 to 107 CFU/g following 6 and 19 days of storage at 8 and 4°C, respectively. Back et al.
[122] also reported temperature-dependent surface growth of L. monocytogenes on several addi-
tional retail European soft cheeses including Cambazola, English Brie, Blue Lymeswold, and White
Lymeswold, with Listeria populations remaining relatively stable on Blue Stilton, White Stilton,
Mycella, and Chaume cheese during short-term storage. In addition, commercial [157] and exper-
imentally produced [209] Arzua cheese (a soft, low-acid Spanish cheese prepared from raw cow’s
milk) supported growth of Listeria as evidenced by populations >1000 CFU/g in the finished
product. All these findings again point to the high-moisture, low-acid cheeses as being of primary
public health importance.
Going one step further, Bolton and Frank [138] developed a predictive model for growth/no
growth of L. monocytogenes in Mexican-style soft cheeses based on salt, pH, and moisture content.
In their study, a soft Mexican-style cheese was prepared by direct acidification and subsequently
frozen. After thawing, the cheese composition was adjusted to four different moisture contents
(42–60%), four different salt contents (2.0–8.0%), and six different pH levels (5.0–6.5), giving 96
treatment combinations. Thereafter, each sample was inoculated with a four-strain cocktail of
L. monocytogenes at about 104 CFU/g and analyzed for numbers of Listeria after 21 and 42 days
of storage at 10°C. Using a binary and ordinal regression model, none of 60 samples having a 5%
probability of supporting Listeria growth (pH 5.0–6.0, brine concentration 8.17–16.00%) supported
growth, whereas Listeria grew in 20 of 30 samples having a 50% probability of supporting growth
(pH 5.5–6.5, brine concentration 3.23–12.50%) of the pathogen. Furthermore, when applied to the
data of Genigeorgis et al. [216] both models provided reasonable predictions for growth/no growth
of Listeria in Mexican-style as well as non-Mexican-style cheese, with these models likely to be
quite useful in refining current Listeria risk assessments for different cheeses.
FEASIBILITY OF PREPARING CHEESE FROM RAW MILK

According to current FDA regulations, milk pasteurization or use of a similar heat treatment during
cheesemaking is required for the manufacture of 16 cheese varieties, including Brie, cottage, cream,
Neufchâtel, Monterey, mozzarella, Scamorza, Muenster, Gammelost, Koch Kaese, and Sapsago
[249,388]. Seven varieties of manufacturing cheese (i.e., for use in pasteurized processed cheese,
cheese food, cheese spread) require neither pasteurization of the cheesemilk nor a 60-day minimum
ripening at ≥1.7°C (≥35°F), whereas the 34 remaining varieties of cheese recognized under current
standards of identity must either be manufactured from pasteurized milk or held a minimum of 60
days at ≥1.7°C (≥35°F) to eliminate pathogenic microorganisms. Although statistics on milk
pasteurization for cheesemaking are scarce, available evidence indicates that most natural cheese
sold in the United States is prepared from pasteurized milk.
The mandatory holding period of 60 days at ≥1.7°C (≥35°F) for cheeses manufactured from
raw milk was adopted in 1949 [7,249] after researchers demonstrated that viable Brucella abortus,
the causative agent of brucellosis, was eliminated from cheese by such an aging process. Although
this 60-day holding period was generally deemed adequate to eliminate most foodborne pathogens,
later studies demonstrated that Salmonella Typhimurium and other hazardous microorganisms can
survive such a cheese-ripening process [227,250]. Furthermore, results in Table 12.16 indicate that
L. monocytogenes can survive well beyond 60 days in many natural cheeses held at ≥1.7°C (≥35°F).
In keeping with the grave nature of listeriosis as compared to most other foodborne illnesses,
the FDA has continued to maintain a policy of “zero tolerance” for L. monocytogenes in all ready-
to-eat foods. Thus far, no well-documented cases of listeriosis have been associated with consump-
tion of cheeses that were legally prepared from raw milk and held a minimum of 60 days at ≥1.7°C
(≥35°F). However, because 4% of the raw milk supply can be expected to contain L. monocytogenes,
it would be prudent to manufacture cheeses from pasteurized milk whenever possible. Although
Yousef and Marth [401] demonstrated that ripening Parmesan cheese for 10 months, as legally
required, is sufficient to produce a high-quality, Listeria-free product, desirable flavor and texture
characteristics are not easily attainable in sharp Cheddar and Swiss cheese prepared from pasteur-
ized milk. Hence, alternative means should be developed to enhance the safety of these products.
Such methods might include cold sterilization of the milk via microfiltration or addition of various
flavor- and texture-enhancing enzymes (or microorganisms) to pasteurized milk, which would allow
the cheesemaker to obtain a higher-quality product [251]. However, as important as it is to manu-
facture cheese from Listeria-free milk, it is equally important to prevent contamination of the
product during manufacture, ripening, and storage by using good manufacturing practices. Infor-
mation concerning problem areas and safeguards during manufacture of dairy products and other
foods can be found in Chapter 18.
TABLE 12.18
Number of Listeriae Recovered from Whey during
Manufacture of Various Cheeses Prepared from
Pasteurized Milk Inoculated to Contain ~500–5000
L. monocytogenes CFU/mL
L. monocytogenes Percentage of Original
Cheese CFU/mL of Whey Inoculum in Whey Ref.
Camembert 8 1.3 261

Blue 43 3.6 237
Brick 12 2.5 264
Feta 15 3.2 238
Gouda 5 1.0 235
Colby 21 2.4 306
Cheddar 22 5.0 259
Average 18 21.7
WHEY
Listeria monocytogenes has not yet been isolated from commercially produced cheese whey.
However, studies have shown that when various cheeses were experimentally produced from
pasteurized milk inoculated with L. monocytogenes, between 1 and 5% of the original inoculum
was lost in the whey during cheesemaking (Table 12.18). These findings again demonstrate that
the pathogen is concentrated 8- to 10-fold in curd during milk coagulation. Unlike other wheys,
populations of L. monocytogenes in acid whey (pH 4.6) obtained from the manufacture of cottage
cheese were reduced more than 10,000- fold after cooking the curd–whey mixture at 57.2°C (135°F)
for 30 min. However, as previously noted, Listeria was detected in several whey samples after
6 weeks of cold enrichment [346], which, in turn, suggests that some cells were only sublethally
injured during cooking of the curd–whey mixture.
These observations prompted Ryser and Marth [343] to examine the behavior of L. monocy-
togenes in wheys from Camembert cheese that were filter-sterilized and adjusted to pH values of
5.0–6.8. All whey samples were then inoculated to contain ~100–500 L. monocytogenes (strains
Scott A, V7, CA, or OH analyzed separately) CFU/mL and incubated at 6°C.
Although the four L. monocytogenes strains failed to grow in wheys having pH values ≤5.4,
the pathogen survived in all samples with populations decreasing ≤10-fold during 35 days of
refrigerated storage. In contrast, L. monocytogenes grew in all remaining samples after a 3-day lag
period and attained average maximum populations of 7.48, 7.87, and 7.84 log10 CFU/mL in wheys
adjusted to pH 5.6, 6.2, and 6.8, respectively, following 35 days of incubation. As previously noted,
these Listeria populations in whey were slightly higher than those that would be expected to develop
in skim or whole milk during refrigerated storage. Generation times for L. monocytogenes in wheys
adjusted to pH 5.6, 6.2, and 6.8 ranged between 25.2–31.6 h, 14.8–21.1 h, and 14.0–19.4 h, respec-
tively, depending on the individual strain. Although doubling times were similar for all strains
at the same pH, generation times were significantly longer at pH 5.6 than at pH 6.2 and 6.8.
Interestingly, Hughey et al. [244] observed that L. monocytogenes could be inactivated in similar
samples of demineralized whey by adding lysozyme. However, this enzyme did not decrease
viability of the pathogen in normal whey, which in turn suggests that lysozyme activity is neutralized
by whey minerals and/or proteins.
Using a different approach to examine the behavior of Listeria in whey, Northolt et al. [299]
inoculated heat-treated (68°C/10 s) wheys (pH 6.5) to contain 500–1000 L. monocytogenes CFU/mL
and incubated the samples at temperatures between 7 and 30°C. Following a 6- to 24-h lag period,
the pathogen grew in all samples with doubling times of 12 h, 6 h, 4 h, and 40 min in wheys
incubated at 7, 12, 20, and 30°C, respectively. Although incubation of all whey samples was
terminated before L. monocytogenes reached the stationary growth phase, the pathogen did attain
populations of 104–106 CFU/mL at the conclusion of the experiment.
In the only study thus far reported dealing with nonsterile whey, researchers in France [127]
produced whey containing 2–7 L. monocytogenes CFU/mL from previously inoculated milk and
examined samples for listeriae after 101, 156, and 251 days of storage at 4°C. Moderate growth
and extended survival of the pathogen were observed in wheys collected immediately after coag-
ulation of the milk (pH 5.4), with 156- and 251-day-old whey samples at pH 4.8 containing 2.5 × 104
and 7.0 × 102 L. monocytogenes CFU/mL, respectively. As predicted by Ryser and Marth [343],
the pathogen also failed to grow in more acidic wheys (pH 5.2–5.3) collected after hooping with
listeriae no longer observed in 101- and 156-day-old wheys having pH values of 3.28 and 4.26,
respectively. Not surprisingly, increasing the incubation temperature to 6, 14, and 20°C led to faster
demise of listeriae in 21 similar wheys (pH 3.75–5.72) initially containing 60–96 L. monocytogenes
CFU/mL, with the pathogen being eliminated from all but one sample (pH 5.72) examined after
114 days of incubation at 6°C. These findings demonstrate that L. monocytogenes can grow to high
numbers in fluid wheys having pH values >5.4 and remain viable in more acidic wheys during
many months of refrigerated storage.
Given results of a study described in Chapter 11 in which numbers of L. monocytogenes
decreased only 1–5 orders of magnitude during manufacture of nonfat dry milk [181], it appears
that this organism also is likely to survive the spray-drying process used in converting fluid whey
into whey powder. However, to our knowledge, no Listeria spp. have yet been isolated from dried
whey manufactured commercially in Europe or the United States. Although Gabis et al. [210] failed
to find any Listeria spp. in 23 environmental samples from whey-processing factories, these authors
did isolate L. monocytogenes from a floor drain that was located within a raw-milk-receiving room
of a dry-milk-processing factory. Additionally, Listeria spp. other than L. monocytogenes were
isolated from several drains and trenches in the powder production area of a second factory
that manufactured dry milk products. Considering the widespread use of dried whey (and
nonfat dried milk) as an ingredient in numerous products including cheese food, ice cream,
sherbet, candy, beverages, and baked goods, strict enforcement of good manufacturing practices
for dried whey and nonfat dry milk should be continued to prevent a possible public health
problem involving listeriae.
BRINE SOLUTIONS
Because L. monocytogenes is quite halotolerant, it is not surprising to learn that brine solutions in
which cheeses are salted or ripened also can serve as potential reservoirs for this organism. Current
evidence suggests that these brine solutions may become contaminated with L. monocytogenes
through direct or indirect contact with the cheese factory environment (i.e., equipment, condensate
on walls, floors, and ceilings) as well as actual shedding of L. monocytogenes into the brine solution
from Listeria-contaminated cheese [119]. Breer [142] and Terplan [290] reported isolating
L. monocytogenes from commercial brine solutions in Europe. In one instance, the pathogen was
detected in brine tanks 4 days after soft/semisoft cheeses were removed from the salt brine. Because
several recalls in the United States [82] have presumably been traced to contaminated brine tanks
[82], interest in the incidence of listeriae in brine solutions has increased with Larson et al. [262]
having examined the survival of L. monocytogenes in 38 commercial cheese brines (5.6–24.7%
NaCl, pH 4.4–6.8) from Wisconsin and northern IIlinois. In their study, 23 brine samples (16.9–24.7
NaCl, pH 4.9–55.4) were collected from nine different manufactures of mozzarella cheese along
with 5, 4, 3, and 3 brine samples from factories producing other Italian, brick, brick/Hispanic, and
feta cheese. Each brine sample was inoculated with a six-strain cocktail of L. monocytogenes strains
Scott A, OH, CA V7, 87189, and 2811M so as to contain 104 to 105 CFU/mL and then stored at
4 and 12°C. Overall, 15 of 38 and 3 of 38 samples were still positive for Listeria after more than
200 days of storage at 4 and 12°C, respectively, with length of survival unrelated to the level of
background microflora, mineral content, or nitrogen content. However, Listeria survival was sub-
stantially shortened in brines containing 10 to 100 ppm sodium hypochlorite, 0.01 to 0.05%
hydrogen peroxide, 1% potassium sorbate or 1% sodium benzoate. Because these levels of sodium
hypochlorite did not yield any residual sodium hypochlorite in mozzarella cheese after brining,
addition of sodium hypochlorite at levels ≤100 ppm should be useful for minimizing Listeria
survival in cheese brines.
Migration of L. monocytogenes into brine solutions and salted whey during salting of artificially
contaminated cheese has been well documented. Ryser and Marth [345] brine-salted brick cheese
containing ~103–104 L. monocytogenes CFU/g at 10°C. Cold enrichment of membrane filters
through which 50-mL portions of 22% brine solution were filtered indicated that the pathogen
leached from the cheese into the salt solution during 24 h of brining. Further, viable listeriae were
detected in samples of 22% brine-stored at 10°C at least 5 days after blocks of cheese were removed
from the brine. When Abdalla et al. [1] brine-salted Sudanese white pickled cheese in whey
containing 8% NaCl, L. monocytogenes once again leached from the cheese into the salted whey,
with populations increasing 2.5–3.5 orders of magnitude during 65 days of storage at 4°C.
In conjunction with their study on the fate of L. monocytogenes during manufacture, ripening,
and storage of feta cheese, Papageorgiou and Marth [307] also examined Listeria viability in the
brine solution in which cheese was salted and stored. After 1 day of salting, a 12% brine solution
contained an average Listeria population of 2.63 log10 CFU/g, which again indicates that the
pathogen leached from cheese into the brine (Figure 12.17). However, no growth of L. monocyto-
genes was observed in this brine despite ample migration of cheese nutrients into salt brine as well
as favorable values for temperature and pH. After transferring feta cheese to a 6% brine solution,
the pathogen leached from cheese into salt brine and grew rapidly, with similar populations being
observed in both the cheese and brine after 6 days of incubation at 22°C. Although numbers of
listeriae decreased in both cheese and salt brine during 90 days of refrigerated storage, the pathogen
was inactivated slower in 6% salt brine than in feta cheese.
6.0
Strain CA log10 CFU/mL or g
5.0
4.0 Brine Solution

Feta Cheese
3.0
12%/20°C 6%/22°C 6%/4°C

2.0
0 1 2 3 4 5 6 20 34 48 62 76 90
Days
FIGURE 12.17 Average populations of L. monocytogenes strain CA in 12 and 6% salt brine during ripening
and storage of feta cheese. (Adapted from Papageorgiou, D.K. and E.H. Marth. 1989. Fate of Listeria
monocytogenes during the manufacture, ripening and storage of feta cheese. J. Food Prot. 52: 82–87.)
Larsen et al. [261] reported that L. monocytogenes began growing in salt-free whey (pH 5.6)
and whey containing 3.6% NaCl and/or KCl after 7–14 days of storage at 4°C, with Listeria growth
generally being unrelated to the type of salt used. However, L. monocytogenes populations remained
unchanged in identical samples containing 4.8% NaCl and/or KCl. When similar whey samples
were incubated at 25°C, L. monocytogenes populations increased 3–4 orders of magnitude, with
more rapid growth being observed in wheys containing 3.6 rather than 4.8% NaCl and/or KCl.
However, Listeria growth at 25°C could be prevented by adding a L. lactis subsp. lactis or L. lactis
subsp. cremoris starter culture before incubation. In 1994, Rajkowski et al. [324] also reported that
addition of 4.5% NaCl to ultrahigh temperature (UHT)-pasteurized milk decreased the growth rate
of L. monocytogenes in samples incubated at 12–37°C. However, fortification of these same samples
with 0.5 or 1.0% polyphosphate did not alter Listeria growth characteristics.
Because feta and other white-pickled cheeses such as Teleme, Halloumi, and Domiati are
frequently cured in whey or skim milk containing 6 or 12% salt, Papageorgiou and Marth [305]
examined behavior of Listeria in salted whey and skim milk. Autoclaved samples of skim milk
(pH 6.0–6.2) and deproteinated whey (pH 5.5–5.7) containing 6 and 12% NaCl were inoculated
to contain 103 L. monocytogenes (strains Scott A or CA) CFU/mL and incubated at 4 and 22°C.
After a lag period of 5–10 days, L. monocytogenes grew rapidly in 6% salted whey and 6%
salted skim milk, with the pathogen attaining maximum populations of 107–108 CFU/mL following
50–55 days of refrigerated storage (Table 12.19). In the study discussed earlier, Ryser and Marth
[343] reported that these same Listeria strains had shorter lag periods and generation times but
achieved similar maximum populations in unsalted, filter-sterilized whey (pH 5.6) after 24 days of
incubation at 6°C. Hence, these findings suggest that addition of NaCl or KCl to whey and milk
plays a major role in decreasing the growth rate of Listeria.
Increasing the incubation temperature to 22°C resulted in lag periods of 6–12 h for both Listeria
strains in whey and skim milk containing 6% salt. Generation times were similarly reduced, with
both strains exhibiting faster growth rates and higher maximum populations in salted whey rather
than salted skim milk (see Table 12.19). These findings agree with those of other researchers, who
TABLE 12.19
Generation Times (GTs) and Maximum Populations (MPs) of
L. monocytogenes Strains Scott A and CA in 6% Salted Whey
and Skim Milk Incubated at 4 and 22°C
MP
Strain Product GT (h) (log10 CFU/mL)
Incubation at 4°C
Scott A Whey 46.81 7.97
Scott A Skim milk 45.23 7.58
CA Whey 37.49 8.04
CA Skim milk 49.43 7.69
Incubation at 22°C
Scott A Whey 3.67 8.02
Scott A Skim milk 4.31 7.70
CA Whey 3.56 8.10
CA Skim milk 4.42 7.89
Source: Adapted from Papageorgiou, D.K. and E.H. Marth. 1989. Behavior of Listeria
monocytogenes at 4 and 22°C in whey and skim milk containing 6 and 12% sodium
chloride. J. Food Prot. 52: 625–630.
5
Scott A Whey

4 Scott A Skim milk
1 CA Whey
CA Skim milk
0
–25 0 25 50 75 100 125 150
Days
FIGURE 12.18 Behavior of L. monocytogenes strains Scott A and CA in 12% salted whey and skim milk
during extended storage at 22°C. (Adapted from Papageorgiou, D.K. and E.H. Marth. 1989. Behavior of
Listeria monocytogenes at 4 and 22°C in whey and skim milk containing 6 and 12% sodium chloride. J. Food
Prot. 52: 625–630.)
also observed that L. monocytogenes grew faster and attained higher maximum populations in
unsalted whey [343] than in skim milk [334].
Unlike the previous findings obtained with 6% salt, growth of L. monocytogenes was completely
inhibited in 12% salted whey and skim milk, with populations of both strains decreasing ≤10-fold
during 130 days of storage at 4°C. Although strain Scott A also persisted ≥130 days in 12% salted
whey and skim milk incubated at 22°C, strain CA proved to be less salt tolerant, surviving only
80 and 105 days in 12% salted skim milk and whey, respectively (Figure 12.18). Increased
destruction of L. monocytogenes in salt solutions held at ambient rather than refrigeration temper-
atures has been well documented. Results from several of these studies dealing with viability of
listeriae in salted Tryptose Broth [360] and cabbage juice [165] are discussed elsewhere. Based on
these observations, acidification of brine solutions used in cheesemaking to pH values <5.0 has
been recommended to prevent growth of L. monocytogenes, particularly if such solutions contain
≤10% salt. In 1989, Hughey et al. [244] also noted that addition of 0.35% H2O2 to a 23% brine
solution caused numbers of L. monocytogenes to decrease by 6 orders of magnitude within 24 h.
However, unlike organic acids, the bactericidal activity of H2O2 dissipates fairly rapidly. Hence,
addition of H2O2 to salt brine provides only temporary protection against listeriae that might be
present within the cheesemaking environment.
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in Meat Products
Jeffrey M. Farber, Franco Pagotto, and Chris Scherf
CONTENTS
Introduction ....................................................................................................................................504
Incidence of Listeria in Meat Products .........................................................................................504
Results from USDA–FSIS Listeria-Monitoring Programs..................................................504
Raw Meat..................................................................................................................505
Cooked and RTE Meat Products..............................................................................505
Recalls and Other Regulatory Actions.....................................................................507
Results from Canadian Listeria monocytogenes Monitoring Programs..............................508
Cooked and RTE Meat Products..............................................................................508
Recalls and Other Regulatory Actions.....................................................................514
Incidence of Listeria spp. in Raw Meat ..............................................................................514
North America ..........................................................................................................515
Non–North American Countries ..............................................................................516
Other Countries ........................................................................................................522
Incidence of Listeria spp. in Sausage and RTE Meat Products..........................................523
North America ..........................................................................................................523
Europe.......................................................................................................................523
Other Countries ........................................................................................................527
Behavior of L. monocytogenes in Meat Products .........................................................................527
Listeriosis in Domestic Livestock........................................................................................528
Localization in Tissues .........................................................................................................528
Raw Beef ..............................................................................................................................530
Growth and Survival.................................................................................................530
Raw Lamb and Pork.............................................................................................................533
Cooked and RTE Meats .......................................................................................................534
Cured Ham................................................................................................................534
Cooked Roast Beef...................................................................................................535
Luncheon Meats .......................................................................................................535
Unfermented Sausage...........................................................................................................538
Fresh Sausage ...........................................................................................................538
Cooked Smoked Sausage .........................................................................................540
Uncooked Smoked Sausage .....................................................................................543
Cooked Meat Specialty Items ..................................................................................543
Fermented Sausage...................................................................................................544
503
Semidry Fermented Sausage ....................................................................................544

Dry Fermented Sausage ...........................................................................................545
Modified-Atmosphere (MA) Packaging...............................................................................547
Beef...........................................................................................................................548
Sausages....................................................................................................................549
Lamb .........................................................................................................................549
Pork...........................................................................................................................550
Thermal Inactivation in Meats .................................................................................552
Bacteriocins for Controlling Listeriae in Meat....................................................................557
Raw Ground Meat ....................................................................................................557
Fermented Sausages .................................................................................................558
References ......................................................................................................................................560
Internet-Based References .............................................................................................................570
INTRODUCTION
Only within the past 15 years have there been cases of human listeriosis traced to meat, with
evidence for transmission of listeriosis through consumption of contaminated meat and meat
products (see Chapter 3).
It is generally accepted among all listeriologists that all Listeria monocytogenes strains should be
considered as potentially pathogenic. With this in mind, and given the ubiquity of this pathogen within
slaughterhouse and meat-packing environments, it is not surprising that the incidence and behavior of
L. monocytogenes in meat products have received increased attention worldwide. This is especially so
given that a number of recent foodborne listeriosis outbreaks have been linked to meat products (see
Table 10.1, Chapter 10), with products such as pâté, frankfurters, and deli meats being implicated [172].
INCIDENCE OF LISTERIA IN MEAT PRODUCTS

RESULTS FROM USDA–FSIS LISTERIA-MONITORING PROGRAMS
The USDA–FSIS monitoring and verification program for L. monocytogenes in meat products
began in September 1987 with sampling of domestic corned beef, cooked corned beef, and massaged
corned beef, as well as imported cooked meats. This program was later expanded to include a wider
range of products with meat or poultry salads and spreads added in 1988 [63,111].
During the 1980s, L. monocytogenes began to emerge as a problem in processed meat and
poultry products. In the 1990s, state health departments and the Centers for Disease Control and
Prevention (CDC) investigated an outbreak of foodborne illness in which hot dogs, and possibly
deli (luncheon) meats, were implicated. CDC and FSIS investigators isolated the outbreak strain,
a strain of L. monocytogenes, from an opened and a previously unopened package of hot dogs
manufactured by a single plant. In total, 101 illnesses were reported, with 15 adult deaths and
6 stillbirths or miscarriages. By 1999, an especially virulent strain of L. monocytogenes emerged.
The agency then informed establishments that they should reassess their hazard analysis and critical
control point (HACCP) plans. As a consequence, FSIS published a notice advising manufacturers
of ready-to-eat (RTE) meat and poultry products of the need to reassess their HACCP plans to
ensure that they were adequately addressing L. monocytogenes contamination risks.
Data gathered during an outbreak of Listeria-related illnesses during the summer of 2002,
combined with other food safety investigations and in-depth verification reviews, led FSIS
to conclude that some establishments were not adequately addressing the potential for bacterial
contamination in their HACCP plans, sanitation standard operating procedures (sanitation SOP),
or other control measures.
Incidence and Behavior of Listeria monocytogenes in Meat Products 505
Then, in December 2002, FSIS implemented a directive outlining additional steps to be taken
by USDA inspectors to ensure that establishments producing RTE meat and poultry products were
preventing L. monocytogenes contamination [156]. Under this directive, plants producing deli meats
and hot dogs without validated Listeria programs to eliminate L. monocytogenes on the product,
on food contact surfaces, and in the environment were subject to an intensified FSIS testing program.
This intensified program included increased product and food contact surface testing, environmental
testing in the plant, and more frequent reviews of plant records and data.
Subsequently, in February 2003, FSIS released its draft risk assessment on Listeria in RTE
meat and poultry products. A public meeting was held on February 26, 2003, to discuss the results.
The risk assessment, in conjunction with a previously released FDA/FSIS risk ranking and public
comment gathered on the topic (see Chapter 18), provided important data enabling FSIS to design
a final L. monocytogenes rule [246].
Raw Meat
The USDA–FSIS monitoring and sampling program was not designed to initiate regulatory action
against particular firms, but rather to provide the agency with critical background information.
During the 26-month period from January 1987 to February 1990, L. monocytogenes was isolated
from 122 of 1726 (7.1%) 25-g monitoring samples of domestically produced raw beef [75]. In
1995, the FSIS reported on the levels of bacteria, including L. monocytogenes, in ground beef [25].
The survey, based on 600 1-lb samples of ground beef from 661 plants, reported an 18% incidence
of the organism.
Cooked and RTE Meat Products
Interest in the extent of L. monocytogenes contamination in both domestic and imported cooked
and RTE meat products also began in the mid-1980s. The list of these products now includes beef
jerky, cooked sausage (both large- and small-diameter), cooked, roast, and corned beef, meat salads
or meat spreads, and sliced canned ham and luncheon meat. Each sample collected, consisting of
one to six subsamples, represents one lot of product. A portion of each subsample is then pooled
to form a composite sample, which is analyzed. If a positive result is obtained for a lot, the composite
sample is not reanalyzed to determine the number of positive subsamples in the lot.
Results from this program for the years 1993–2000 (Table 13.1) showed that, in general, there
was a low incidence of L. monocytogenes in cooked and RTE meat products in the United States,
with overall incidences from 0 to 8.1%. The organism was usually absent from beef jerky, as its
presence was reported in this product only twice: in 1994 at a 2.2% incidence and in 1998 at a
1.6% incidence. Cooked large-diameter sausage showed a higher prevalence of the organism than
cooked small-diameter sausage, with the incidences ranging from 1.8 to 5.3% and 0.4 to 2.1%,
respectively. L. monocytogenes was present in only 2.1 to 3.4% of samples of domestic or imported
cooked beef, roast beef, and cooked corn beef over this 7-year period. In most instances, these
products were removed from their packages after cooking and then repackaged for sale, suggesting
that the pathogen most likely entered the product through direct contact with the factory environ-
ment, knives, or gowns worn by workers [128]. At least two other firms handled raw and finished
product in the same production area of the factory but at different times, which, in turn, indicates
the possibility of cross-contamination between raw and finished product.
In December 1987, USDA–FSIS officials added sliced canned ham and sliced canned luncheon
meat to the monitoring and verification program for the presence of L. monocytogenes. From 1993
to 2000, the pathogen was detected in 4.6 to 8.1% of these samples, which were among the higher
incidences among the RTE meat products examined (Table 13.1). In June 1988, monitoring of meat
or poultry salads and spreads was begun, with incidences of 1.2 to 4.7% being reported from 1993
to 2000 (Table 13.1).
506
TABLE 13.1
Incidence of Listeria monocytogenes in USDA–FSIS Monitoring Samples of Cooked and RTE Meat Products, 1993–2000a
1993 1994 1995 1996 1997 1998 1999 2000
Meat Product # Lotsb (% pos) # Lots (% pos) # Lots (% pos) # Lots (% pos) # Lots (% pos) # Lots (% pos) # Lots (% pos) # Lots (% pos)
Beef jerky 39 0 45 2.2 50 0 43 0 40 0 192 1.5 278 0 0.8

Cooked sausage—large diam 328 2.1 438 1.1 438 1.1 420 1.0 371 1.6 506 1.2 1,167 0.4 0.5
Cooked sausage—small diam 472 5.3 602 4.8 611 4.1 561 3.7 621 2.7 746 3.5 2,162 1.8 1.3
Cooked/roast/corned beef 426 3.0 479 2.1 560 2.7 507 3.4 530 2.1 511 2.2 922 2.7 2.2
Salads/spreads 273 2.2 580 2.4 597 4.7 554 2.2 206 2.4 225 3.1 435 1.2 1.0
Sliced ham/sliced luncheon meats 149 8.1 232 5.5 99 5.0 91 7.7 286 4.2 263 4.2 960 4.6 3.1
Fermented sausages NAc NA NA NA NA NA NA NA 108 9.3 244 2.9 478 2.1 1.5
aRef. 248.
bEach sample, consisting of one to six subsamples, represents one lot of product.
cNA = Not available.
TABLE 13.2
USDA/FSIS Microbiological HACCP Verification Testing Program for RTE Meat and
Poultry Products Produced in USDA-Inspected Establishments
Product Category Examples (Not All-Inclusive)
Whole sausage-type product, peeled Hot dogs, frankfurters, knockwurst, and other products cooked in a casing
that is removed by a peeling process after the lethality step and before
final packaging
Whole sausage-type product, unpeeled Hot dogs, bologna, andouille sausage, pepperoni, salami, and similar
products that are shipped in the same casing that exists during the lethality
step
Large mass, chopped and formed Turkey roll, loaves, cooked ham, and other products that have been
processed before lethality in a manner where exterior bacteria could be
transferred into the internal tissues
Large mass, whole muscle Cooked roast beef, whole chickens, cooked corned beef, cooked turkey
breast, bone in ham, prosciutto, dry cured ham, that is, products with only
external bacteria before the lethality step.
Small mass, chopped and formed Meatballs, chicken nuggets, patties, breakfast sausage
Small mass, whole muscle Chicken tenders, whole muscle cutlets, chicken breasts
Salads, pâtés, and spreads Chicken salad, ham salad, liverwurst, pâté de foie gras
Sliced, diced, shredded (with or without Sliced ham, sliced turkey, diced cooked chicken, beef barbeque, sliced
sauce) pepperoni, chipped beef
Multi-component products Dinners, entrees, wraps, pocket sandwiches, egg rolls, pizza
Other Products that cannot be categorized into the other nine categories
In December 2000, the FSIS microbiological monitoring projects based on selected product
types were discontinued and a HACCP verification program based on the HACCP process was
initiated. Thus, samples were scheduled based on the different RTE HACCP processes used in
USDA-inspected establishments and were randomly scheduled in the establishments in which the
RTE processes existed (Table 13.2). Therefore, the results for 2001 and 2002 are not directly
comparable to those published for previous years (Table 13.3). However, in general, the incidence
of L. monocytogenes in these types of products appeared to decrease.
A survey of commercially prepared frankfurters from 12 producers over a 2-year period showed
that L. monocytogenes contamination can be highly variable from plant to plant, and that seasonality
has no effect on the incidence of this pathogen in frankfurters. Recovery rates of L. monocytogenes
from two plants were 1.5 and 2.2% for plants producing frankfurters that were made from meat.
In total, 104 of 27,300 packages (0.38%) of samples contained L. monocytogenes [231].
Recalls and Other Regulatory Actions
After a request from the food industry that the FDA reconsider its policy of “zero tolerance” for
presence of L. monocytogenes in foods, the FDA stated that it would need the support of scientific
data on infectious levels of the organism to justify any change in its regulation that the pathogen
should not be present in RTE foods [15]. Later, the FDA announced that its pathogen-monitoring
program would concentrate on selected high-risk foods, including prepared sandwiches [26]. The
agency stated that if a prepared sandwich sample tested positive for L. monocytogenes, then a
comprehensive follow-up inspection of the firm would be warranted, including sampling of raw
materials, plant surfaces, and finished product.
TABLE 13.3
Prevalence of L. monocytogenes in USDA–FSIS Microbiological HACCP Verification
Samples of RTE Meat Products, Cumulative Years (CY) 2001 and 2002
Summary by Product Type Summary by Product Type
CY 2001 % Incidence CY 2002 % Incidence
Product (Positives/Sample Number) (Positives/Sample Number)
Peeled sausage type product 0.93 (6/646) 1.72 (13/758)

Unpeeled sausage type product 0.71 (12/1689) 0.63 (11/1758)
Large mass chopped & formed type product 2.34 (5/214) 0.93 (3/322)
Large mass whole muscle type product 2.42 (12/495) 0.49 (3/609)
Small mass chopped and formed type product 0.85 (5/585) 1.05 (6/570)
Small mass whole muscle type product 0.67 (6/896) 0.77 (7/914)
Salads/pâtés/spreads type product 1.33 (3/226) 0.42 (1/237)
Sliced, diced, and shredded type product 2.59 (25/966) 1.96 (25/1274)
Multi-component products 1.48 (10/675) 0.94 (8/851)
Other type product 1.42 (2/141) 0.33 (1/299)
Summary by HACCP process type 1.32 (86/6533) 1.03 (78/7592)
Between January 1994 and October 2006 at least 175 separate Class I or voluntary recalls were
issued for cooked and RTE meat contaminated with L. monocytogenes in the United States,
including 74 for deli meats, 42 for sausages, 37 for hot dogs, and 22 for other products (Table 13.4).
Since 1994 a number of these recalls have involved prepared sandwiches; these recalls have resulted
in at least 50,000 sandwiches being removed from the marketplace. In these recalls of prepared
sandwiches, the distribution usually involved several states (Table 13.4). Product losses may be
higher than indicated because firms holding tested products until results of Listeria analyses become
known can “recall” all contaminated lots internally, thereby avoiding a formal Class I recall.
Considering the wide variety of tainted sandwiches that have been withdrawn from sale, it appears
that L. monocytogenes entered these products during cutting, slicing, or packaging, rather than
being initially present in the many different types of sandwich fillings.
RESULTS FROM CANADIAN LISTERIA MONOCYTOGENES MONITORING PROGRAMS

Cooked and RTE Meat Products
Agriculture and Agri-Food Canada initiated and passed on to the Canadian Food Inspection Agency
(CFIA) Canada’s L. monocytogenes-monitoring program on processed and RTE meat products from
registered processing plants in 1987. At the same time, the Health Products and Food Branch
(HPFB), Health Canada, started a similar testing program for L. monocytogenes in RTE meat
products from nonregistered establishments [84]. Five subsamples of 150 g (or a convenient package
size) each were collected. The quantity analyzed depended on the product; for meats supporting
the growth of Listeria spp., 25 g was tested, whereas for meats not supporting growth of Listeria
spp., 5 g was tested.
During 1989–1990, the incidence of L. monocytogenes in the RTE meat samples tested by the
Agriculture Canada monitoring program was high, i.e., an average of about 24% (Table 13.5). During
this time, samples taken at establishments with Listeria-positive products showed an incidence of
46% for meat products (data not shown), and an average of 12% for environmental samples. The
incidence dropped sharply during 1991–1992 from 0 to 3% for meat products, but contamination in
the plant environment remained about the same. After 1992, only environmental samples were taken
for testing domestic products, but a more rigorous testing procedure involving three phases was instituted.
TABLE 13.4
Chronological List of Recalls (Class I or Voluntary) Issued in the United States for Cooked
and RTE Meat Products Contaminated with Listeria monocytogenes, 1991–November 2006a
Date Recall Initiated
Product (mm/dd/yy) Origin Quantity in lbs
Sandwichesb 1991 LA NA
Ham saladb 1991 MN 460
Sandwichesb 07/03/91 LA NA
Skinless hot dogsc 1991 MI 3,700
Ham saladd 1991 WV 600
Frankfurterse 03/20/92 CT 3,578
Sandwichesf 06/18/92 LA NA
Sandwichesg 09/25/92 TN NA
Beef frankfurters 01/25/94 CA 1,220
Beef frankfurters 02/09/94 MI 1,600
Beef cooked salami 02/23/94 NY 1,268
Linguica sausage 03/21/94 RI 100
Ham, sliced 03/28/94 NE 3,950
Ham salad 04/12/94 IN 1,105
Ham, cooked, sliced 05/05/94 MI 844
Smoked sausage 05/15/94 MS 250
Wieners 10/03/94 NE 5,500
Hot dogs (franks) 14/14/94 NY 432
Ham, sliced 10/20/94 NM 3,920
Smoked Polish sausage 01/10/95 MN 36
Beef franks 01/18/95 PA 1,000
Buffalo franks 03/03/95 CO 260
Frankfurters 05/30/95 NY 3,780
Hot dogs (franks) 07/06/95 BC, Canada 11,420
Bologna ring 07/24/95 PA 200
Sandwiches — ham and cheese, ham salad, etc.b 09/08/95 MI NAb
Beef wieners 10/27/95 AB, Canada 3,510
Hot dogs, pork 11/06/95 AB, Canada 4,320
Sandwiches — roast beef, hot dog, burgers, sausage, 02/16/96 LA NAb
meatball, ham and cheese, etc.b
Cooked beef roast 03/13/96 NE 487
Roast beef, sliced 04/03/96 IL 720
Cooked beef 06/14/96 TX 2,608
Extra hot beef jerky 06/20/96 BC, Canada 360
Ham, chunked and formed 10/24/96 OH 400
Sliced ham 12/09/96 NH 52
Sandwiches — hot dog, ham and cheese, sausage, beef, 01/22/97 MS NAh,i
salami, etc.h
Dry sausage (kayseri soujouk) 10/03/97 NY 347
Filzette salami 12/18/97 MO 507
Frankfurters 03/19/98 NY 1,440
Ham steaks 04/17/98 MO 635
Dry sausage 06/04/98 CA 272
Frankfurters 10/22/98 FL 1,734,002
Smoked beef strips 10/29/98 AR 3,600
Hot dogs/packaged meats 12/22/98 MI 35,000,000
Luncheon meat 01/15/99 WI 28,312
Sliced ham 01/22/99 OH 348
(continued)

and RTE Meat Products Contaminated with Listeria monocytogenes, 1991–November 2006
Various 01/22/99 AR 35,000,000

Mortedella 02/05/99 ON, Canada 456
Hot dogs, bockwurst 02/05/99 WA 1,545
Head cheese 02/17/99 IL 2,586
Frankfurters 02/18/99 GA 4,460
Pasta with sausage 03/02/99 IL 1,923
Frankfurters 03/18/99 NJ 18
Luncheon meats, various 05/14/99 NC 16,392
Weisswurst 05/28/99 PA 60
Wieners, frankfurters 06/01/99 ND 150
Franks, skinless 06/04/99 IL 1,285
Frankfurters 06/18/99 HI 9,620
Bacon chips 07/27/99 IN 126,739
Ham, sliced/whole 07/29/99 MI 200
Roasting sausage 07/30/99 NJ 200
Hot dogs 07/30/99 PA 200
Chorizos 08/26/99 PR 1,640
Sausage 08/27/99 MA 3,720
Beef frankfurters 10/13/99 NY 2,100,000
Smoked sausage 11/12/99 LA 1,270
Hot dogs 11/12/99 HI 312
Hot dogs 11/18/99 BA 1,020
Polish sausage 11/19/99 MD 800
Beef franks 12/09/99 PA 4
Luncheon meats 12/14/99 NJ 900
Pâtés and mousses 12/17/99 NY 10,064
Roast beef 01/14/00 AZ 600
Dry sausage 01/28/00 Il 200
Cooked corned beef and ham 03/01/00 MI 80
Hot dogs 03/15/00 NY 400
Franks 03/24/00 PA 34,500
Roast beef 04/06/00 UT 13,351
Sausage, various 04/13/00 OH 850
Sliced cooked meats 04/26/00 MI 180
Sliced cooked meats 05/03/00 MI 215
Sliced cooked meats 05/05/00 MD 450
Pork cretons spread 05/10/00 ME 210
Frankfurters 05/11/00 HI 1,125
Sausage, various 05/12/00 MS 5,900
Salami, Westphalian ham 05/19/00 BC, Canada 400
Franks 05/24/00 MI 2,870
Sliced cooked meats 05/24/00 AL 45
Franks 05/29/00 NC 15,000
Sliced ham 06/07/00 MO 60
Beef bologna 06/07/00 NY 2,200
Ham 06/14/00 CA 1,800
Sliced luncheon meat 06/14/00 HI 270
(continued)

Beef jerky 07/06/00 CT 125

Hot dogs, RTE deli meats 08/03/00 NY 19,000
Sliced luncheon meats 08/08/00 ID 380
Wieners 10/03/00 TN 900,000
Polish sausage 10/04/00 MN 240
Country ham 12/20/00 KY 10,400
Mexican-style meat products 01/30/01 IL
Hungarian salami 02/28/01 NJ 3,700
Sliced, cooked beef 03/22/01 IL 1,570
RTE beef sausage 03/28/01 NY 7,800
RTE meat and poultry products 04/12/01 OK 14,500,000
RTE bratwurst 07/01/01 WA 100
Cooked smoked ham 07/05/01 IA 70
RTE pork andouille sausage 09/06/01 WA 20
Cooked pork and corned beef 09/26/01 IN
Cooked beef products 10/05/01 IL 5,600
Cooked beef products 10/10/01 IL ∼5,000
Luncheon meat 10/31/01 IA 189,000
Cooked roast beef products 12/12/01 IL 700
Bratwurst 12/19/01 MO 115
Sausage patties 12/20/01 NC 3,300
Beef and pork sausage products 01/15/02 TX 2,500
Pork sausage products 01/18/02 FL 150
RTE semi-dry sausage 02/06/02 NY 1,800
Dried, seasoned beef 02/12/02 CA 22
Cooked, chopped ham 02/18/02 CA 200
Beef and pork sausage 03/05/02 TX 360
RTE souses loaf 04/17/02 VA 190
Frankfurters and hot dogs 04/25/02 OH 140,000
RTE ham 05/07/02 NV 300
Frankfurters and bologna 05/23/02 NY 77,000
Sliced ham 06/17/02 MO 10
Cured ham 06/21/02 NY 2,300
Sausage products 06/25/02 MN 250
Sliced pork hocks 07/03/02 NY 100
RTE braunschweiger 07/19/02 NY 65
Sausage 07/27/02 PA 22
RTE Italian loaf 08/10/02 NY 1,300
Sliced corned beef 08/14/02 FL 19
Sausage product 08/16/02 FL 500
Cooked, boneless hams 08/28/02 AR 2,200
Cooked, boneless hams 08/30/02 AR 6,525
Imported RTE sausage 09/04/02 PA 1,035
RTE Prosciutto ham 09/04/02 CA 510
Cooked and cured beef loaf 09/17/02 PA 185
RTE ham 10/08/02 IN 95
Pork dumplings 10/11/02 HI 150
(continued)

RTE pork shoulder 11/20/02 PR 6,800

RTE pork luncheon meat 11/26/02 IN 210
Cooked pork products 11/27/02 VA 540
RTE souse products 12/03/02 MS 500
Pork sausage 12/04/02 FL 8,600
Pork sausage 12/12/02 FL 200
Chicken frankfurters 01/03/03 NY 26,400
Cooked beef products 01/22/03 SD 2,100
Cooked pork shoulder 01/28/03 PR 490
Cooked pork shoulder 01/30/03 PR 590
Smoked pork chops 03/06/03 PA 11
RTE fully cooked pork and veal bologna 03/22/03 NY 330
Cooked beef sausage 05/05/03 NC 180
RTE chicken salad 05/05/03 IL 400
RTE Thai-style noodle salad containing chicken 10/04/03 NJ 270
RTE luncheon meat 10/10/03 CA 550
RTE meat and poultry 10/12/03 MA 9,230
Beef products 10/24/03 WA 200
RTE sausage and ham products 11/05/03 AR 4,500
Cooked roast beef 11/18/03 NE 110
Cooked diced chicken breast 11/25/03 GA 7,500
Chicken salad 12/11/03 FL 2,700
Frozen beef 01/11/04 IL 5,190
RTE meat products 01/28/04 PA 52,000
Beef and pork frankfurters 02/17/04 NY 540
Fully cooked boneless hams 03/16/04 GA 713
RTE fully cooked bologna 04/05/04 PA 100
Fresh deli meat and cheese trays 04/12/04 IL 135
Scrapple 06/03/04 DE 350
Beef jerky 06/29/04 PA 130
Frozen, fully cooked chicken products 07/01/04 NC 404,730
Frozen, fully cooked chicken products 07/21/04 GA 36,980
Beef and pork products 07/27/04 NJ 500
Wieners 08/04/04 UT 5,360
Fully cooked ham 08/10/04 NY 422
RTE chicken products 11/03/04 MD 1,275
Chicken products 01/31/05 NY 5,760
RTE ham 02/09/05 TN 47
Cooked pork products 02/28/05 LA 1,120
Chicken salad 03/15/05 NY 250
Chicken products 03/25/05 CA 12,500
Chicken wrap sandwiches 04/05/05 FL 3,316
Various sausage products 04/05/05 MI 5,117
Smoked turkey and pork products 04/11/05 NY 39,000
Various sausage products 04/11/05 GA 10,700
Pork blood sausage 04/19/05 CA 40
Various RTE meat products 04/21/05 MO 1,077
Deli meat wraps “turkey and cooked ham capicolla club 04/26/05 TX 191
wrap”
(continued)

Chicken breast wraps 04/28/05 NY 385

RTE ham 04/29/05 KY 29,000
Various RTE meat products 04/30/05 Nationwide 363,332
RTE chicken salads 06/15/05 NY 5,065
Spanish brand sausage (Primera Chorizos) 06/26/05 NJ 720
Natural proportion cooked chicken meat 07/21/05 GA 170
Meat and poultry products 07/29/05 Nationwide 93,200
Barbeque beans with beef and chicken salad 08/31/05 OK 23,435
Chorizo, blood sausage and blood pudding 09/15/05 NY 890
Chicken frankfurters 09/20/05 NY 23,040
Cooked country hams 09/28/05 VA 165
Cooked chicken sausage products and beef wieners 10/02/05 IL 1,000
RTE meat and poultry products 10/22/05 MA 11,200
RTE beef products 10/31/05 NY 2,263
RTE chicken product 11/08/05 CA 275
Chicken salad products 11/10/05 PA 5,523
Turkey, ham, bologna, and chicken lunch makers meals 12/01/05 MO 2,800,000
Pork barbeque 02/18/06 NC 30
Dried beef products 03/23/06 TX 100
Ham salad 04/05/06 ME 92
aReference 247, unless otherwise indicated.
bReference 20.
cReference 18.
dReference 19.
eReference 21.
fReference 22.
gReference 23.
hNA = Not available.
iReference 27.
Phase I of the program consisted of taking 10 post-process environmental samples from product
contact surfaces. These 10 samples were composited into one analysis. Any positive Phase I analysis
triggered entrance into Phase II, consisting of 10 repeat environmental samples, analyzed individually,
and a review of Good Manufacturing Practices (GMP) throughout the plant. Phase III is entered if
one or more analyses of Phase II samples is found to be positive, in which instance a thorough review
of all plant procedures is undertaken before collection of further environmental samples for individual
analysis. Between 1992 and 1994, the incidence of L. monocytogenes in the plant environment dropped
sharply, indicating the value of this approach [85]. Selected data from three major regions of Canada,
the provinces of Ontario, Quebec, and the provinces on the Atlantic coast of Canada, grouped as
“Atlantic,” showed a slight overall increase in the incidence of L. monocytogenes from the period
1997–2000 (Figure 13.1). However, it has been shown that the incidence of L. monocytogenes in
Canada had decreased to around 10% in 2001–2002 (Table 13.5).
The presence of L. monocytogenes in imported RTE meat products during this period remained
low, with incidences ranging from 0 to 5% (Table 13.5). The importance to the Canadian consumer
of imports from the United States compared to other countries is evident from the relatively large
number of American samples tested.
35
Ontario
Quebec
30
Atlantic
25
% of Unsatisfactory Samples
20
15
10
0
97/98 98/99 99/00
Work Specification M-205 Sampling Years Assessed
FIGURE 13.1 Change over time of the prevalence of L. monocytogenes in Canadian federally regulated ready-
to-eat meat establishments, 1997–1998 to 1999–2000, using protocol M-205 for sampling. (From Health
Canada Food Safety Assessment Program, Assessment Report of the Canadian Food Inspection Agency Activities
Related to Domestic Ready-to-Eat Meat Products, 2002.)
Recalls and Other Regulatory Actions
The current Canadian policy on control of L. monocytogenes in foods is risk-based. Thus, for high-
risk meats that have been causally linked to listeriosis, such as liver pâté and jellied pork tongue,
a Class I recall to the retail level would be put into place. However, all other RTE meats that support
growth of L. monocytogenes and that have a refrigerated shelf life of >10 days would only be
subject to a Class II recall. The largest number of recalls (20) for RTE meat products in Canada
between 1989 and 1997 was for dry, fermented sausages, some of which were smoked (Table 13.6).
An almost equal number of recalls were initiated for sandwiches, including substitutes, containing
meat. Sliced cold meats of various types were also found contaminated with L. monocytogenes
during this period, with contamination likely coming from retail slicers. Wieners and miscellaneous
products made up the balance of the recalls. A dramatic decrease in the number of recalls due to
meat contaminated with L. monocytogenes since 1997 was revealed through the CFIA food recall
database. There were 11 recalls of meat products due to contamination with L. monocytogenes. In
particular, there were no such recalls in 1998 or 2000 (CFIA Web site, product recalls, 1997–2002).
INCIDENCE OF LISTERIA SPP. IN RAW MEAT

Slaughter animals are a recognized reservoir of human pathogens, including L. monocytogenes.
Studies indicate an incidence of listeriae ranging from 0 to 9%, both in feedlot cattle [209] and in
pork carcasses [2,96,184,197]. However, a 96% incidence of listeriae was reported on pork carcasses
from one plant [96], showing the results of poor sanitation. The incidence of listeriae on slaughter
plant equipment was also low (0–3%) [96,190], except for the plant mentioned previously, in which
TABLE 13.5
Incidence of Listeria monocytogenes in Canadian Food Inspection Agency Testing
Samples of Domestic and Imported RTE Meat Products, and in Processing
Plants, 1989–2002a
Domestic Products Imported Products
Number of Samples Number of Samples Plant Environment
(% positive) (% positive) Number of Samples (% positive)
Year U.S.A. Others

1989 396 (12) 50 (4) 5 (0) 677 (17)
1990 66 (35) 1279 (2) 3 (0) 2267 (8)
1991 19 (0) 984 (2) 14 (0) 3314 (13)
1992 35 (3) 469 (3) 9 (0) Phase 1b Phase II Phase III
3808 (10) 430 (9) 164 (13)
1993 NDc 417 (3) 1 (0) 2522 (6) 124 (19) 20 (0)
1994 ND 382 (5) 1 (0) 3629 (2) 138 (6)
1999–2000 77 (9)
2000–2001 416 (11.5)
2001–2002 431 (10)
aRef. 12.
bPhase I – 10 composited post-process samples of product contact surfaces.
Phase II – if Phase I positive, 10 individual environmental samples.
Phase III – if Phase II positive, review of plant procedure before further individual sampling.
cND = not determined. After 1982, environmental testing only was done for domestic products.
the incidence was reported at 42% [96]. Levels of the organism were low, ranging from 10 to
100 CFU/g [96]. The only areas where L. monocytogenes was demonstrated at a plant were cold
rooms maintained at 5°C, at which temperature this psychrotrophic organism is able to grow.
North America
In addition to the government monitoring programs in North America, there have been several
reports of nonregulatory surveys of both raw and RTE meat products (Table 13.7). Surveys on raw
meat products such as roasts and steaks sampled in the United States indicate that the incidence
of Listeria spp. ranges from 0 to 6% (Table 13.7). Comminuted raw meats, however, show a much
higher incidence of listeriae, from 24 to 100%, with L. monocytogenes ranging from 0 to 25%.
Reported levels of listeriae ranged from 4 to 2.1 × 104 CFU/g.
Most beef comes from cattle that are well treated before slaughtering. However, there are times
when the stresses of mixing cattle, transportation over long distances, as well as weather and time
of year, can reduce the amount of muscle glycogen. The beef that results from this is called dark,
firm, dry (DFD) beef, or dark cutting beef. Hooper-Kinder et al. [121] studied susceptibility of this
type of beef to growth of L. monocytogenes Scott A. Inoculation of 0, 50, and 100% DFD ground
meat samples with L. monocytogenes showed that despite the lower glycogen content, there was
no difference between this and normal beef available at the retail level with regard to growth of
L. monocytogenes.
In raw meat products sampled in Canada, usually ground meats, the incidence of Listeria spp.
is high, ranging from 66 to 100%. The incidence of L. monocytogenes ranges from 44 to 100%,
indicating that contamination of raw meats with this pathogen is relatively common (Table 13.7).
TABLE 13.6
Class II Recalls Issued in Canada for Processed and RTE Meat Products Contaminated
with Listeria monocytogenes, 1983–November 2002a
Number of Recalls
Dry Fermented Sliced Roast Cooked
Year Wieners Sausages Sandwiches/Subs Beef Ham Breastb Misc.C
1983 2 1
1994 1 1
1995 1 2 4 3
1996 15
1997 2 1 1 2
1998
2000 1 2
2001 45 1 1 12 3 12
2002 3 2 4
2003 2 2 1
2004 4
2005 2 7 1 5 1 3
2006
Totals 51 6 26 7 25 6 29
aRef. 13, 28, 248.
bTurkey or chicken.
cPizza, burrito, cretons, pork ribs, ham salad, sliced turkey, bacon, bologna, salami, meat loaf, pepperoni, smoked beef
steak nuggets chicken fried rice, goose rillettes, infant foods.
These results may be due to contaminated equipment at either the wholesale or retail level. It is
noteworthy that there is a much lower incidence of listeriae in the wild animal population than in
domesticated animals. Amoril and Bhunia [5] took 20 beef and 20 poultry samples from local
grocery outlets to determine the level of L. monocytogenes in raw meats. L. monocytogenes was
found in 13 (32.5%) of the samples, which is lower than reported by other researchers [85a,136].
Combined with research data generated by the scientific community, it is surmised that the lower
incidence rate is due to efforts of regulatory agencies and industries to decrease the presence of
microorganisms in foods.
Heredia et al. [117], investigating incidence of Listeria spp. in Mexican ground meat samples,
found Listeria spp. and L. monocytogenes in 62 and 16%, respectively, of the 88 samples taken.
Non–North American Countries
Since 1990, many reports have been published on the prevalence of Listeria spp. and of L. monocytogenes
in raw meat and raw meat products prepared in various countries (Table 13.8). In the United
Kingdom, MacGowan et al. [162] reported incidences of Listeria spp. ranging from 59 to 88% in
beef, lamb, and pork, with L. monocytogenes present in 28–40% of these samples. Wilson [236],
based on a larger sampling of beef, lamb, and pork in Northern Ireland, reported an incidence of
about 4% for listeriae. Gilbert [93] reported an incidence of L. monocytogenes in raw pork sausage
of 49%, whereas MacGowan et al. [162] found a similar incidence of 35%. In a study in Ireland,
Sheridan et al. [205] reported high incidences of listeriae, ranging from 45 to 85% in 20 samples
TABLE 13.7
Incidence of Listeria monocytogenes in Raw Meat and RTE Meat Products
in the United States and Canada, 1990–2003
Number of Percentage of Positive Levels of

Samples Samples Listeria spp.
Product Analyzed Listeria spp. LM (CFU/g) Reference
Raw meats—U.S.A.
Beef roast 50 6 NDa 137
Pork roast 50 6 5 10b 137
Lamb roast 10 0 ND 137
Ground beef 39 41 8 4–560 244
Ground pork 20 30 25 4–240 244
Pork sausage 17 24 6 240–21,000 244
Lamb patty 2 100 0 4–56 244
Beef 658 ND 6 132
Pork loin 135 ND 0 197
Whole-muscle, store-packaged 96 28.1 14.6 76
pork
Whole-muscle, enhanced pork 96 25 14.6 76
Store-ground fresh pork and/or 96 61.5 22.9 76
pork sausage
Prepackaged ground pork 96 53.1 27.1 76
and/or pork sausage
Ground pork and/or pork 120 47.5 26.7 76
sausage
Luncheon meats, 2000–2001, 45,994/4,600 ND 1.2/0.6 104
Maryland/California
Deli salads, 2000–2001, 4,293/4,256 ND 2.4/2.3 104
Maryland/California
Ground pork at packing 340 50.2 51.9 141
plants/retail grocery
Chitterlings (small intestines) 300 9.3 9.3 141
at packing plants/retail
grocery
Swine tissues 1,849 2.4 2.8 141
Raw meats—Canada
Ground beef 22 100 77 87
Ground pork 19 100 95 87
Ground veal 3 100 100 87
Ground meat 11 100 63 160
Meat cuts 18 65 44 160
Wild animal meat—moose, 10 10 10 160
deer, bear
RTE products—U.S.A.
Wieners 93 10 8 232
Wieners 24 83 71 232
Wieners 30 20 17 24
Large sausage, 1998/1999 1.2/0.4 247
Small sausage, 1998/1999 3.5/1.8 247
Salads/spreads, 1998/1999 3.1/1.1 247
Sliced ham/pork, 1998/1999 4.2/4.6 247
(continued)

Incidence of Listeria monocytogenes in Raw Meat and RTE Meat Products
in the United States and Canada, 1990–2003
Number of Percentage of Positive Levels of

Samples Samples Listeria spp.
Product Analyzed Listeria spp. LM (CFU/g) Reference
Cooked/roast/corned beef, 2.2/1.4 247

1998/1999
Fermented sausage, 1998/1999 2.9/2.1 247
Jerky, 1998/1999 1.6/0.0 247
Cooked meat products 369 65 9.2 3
Luncheon meats, 2000–2001, 4,599/4,600 ND 1.2/0.6 104
Maryland/California
Deli salads, 2000–20001, 4,293/4,256 ND 2.4/2.3 104
Maryland/California
Frankfurters 27,300 0.4 231
RTE products—Canada
Fermented sausages 30 33 20 88
Cooked meat 16 0 0 216
Wieners 38 26 21 216
Luncheon meats 67 13 13 216
Sausages 9 0 0 216
Note: LM = L. monocytogenes; ND = not determined; RTE = ready-to-eat.
each of beef, pork, and lamb, with L. monocytogenes present in 15–50% of the samples. Interestingly,
the incidence of the organism was lower in comminuted meats (94 samples of frozen beef burgers,
85 samples of ground beef, and 20 samples of sausage), with L. monocytogenes present in 5 to
18% of the samples.
An incidence of 5% for L. monocytogenes in ground meat was found in Norway [195], all the
organisms belonging to serotype 1.
Raw beef products in Germany have a reported incidence of L. monocytogenes of about 50%
[132]. As raw minced meat is a popular dish in Germany, the German Ministry of Health decided
to restrict sales of the contaminated product [11]. If a sample contained less than 100 CFU/g, there
would be further surveillance of the factory or retail outlet, and if the number of listeriae reached
1,000 CFU/g, there would be a public warning.
Workers in Italy have reported on the incidence of listeriae in many types of raw meat. According
to Maini et al. [164], the incidences of listeriae in 19 samples each of calf, horse, pork, and mutton
were 63, 26, 53, and 56%, respectively. Cantoni et al. [49] showed that prevalence of the organism
on both sausages and their casing surfaces were roughly equal, 60 and 47% for listeriae and 13
and 12% for L. monocytogenes, respectively. Other samplings of fresh pork and beef for the
organism showed incidences ranging from 21 to 47% for listeriae and from 13 to 22% for L.
monocytogenes. Serotype 4 was found in 7% of these samples [49]. Comminuted meats and sausages
showed higher incidences for Listeria spp. (44–54%), but in the same range for L. monocytogenes
(9–19%) [60,61,155]. The main serotype isolated was 1/2c [60]. When present, the organism was
at a level of less than 100 CFU/g.
Breer and Schöpfer [44] reported that of 209 samples of beef, pork, and pork products collected
in Switzerland, listeriae were recovered from 11 to 45% of the samples, with an incidence ranging
from 0 to 15% for L. monocytogenes. In comminuted meat products, the corresponding incidences
were 40–65% and 8–15%, respectively.
TABLE 13.8
Incidence of Listeria monocytogenes in Raw Meat in European and Other Countries
(1990–1997)
Percentage of Positive Levels of
Samples Listeria spp.
Country Product Number Listeria spp. LM (CFU/g) Reference
Australia Beef 50 34 24 131

Lamb 50 40 16 131
Pork 50 30 10 131
Belgium Prepacked blood sausages, 137 8.8 222
25 g
Other prepacked meats, 25 g 199 8 222
Not prepacked meats, 406 5.2/0.3a 222
25 g/0.1 g
Not prepacked pâtés, 25 g 130 4.6 222
Not prepacked aspic, 25 g 67 1.5 222
Not prepacked blood 18 11.1 222
sausages, 25 g
Other not prepacked meats, 132 6.8 222
25 g
Raw cured meats
Whole muscles (sliced, 516 14.9/6.8a 222
prepacked), 25 g/0.1 g
Beef, 25 g 43 4.7 222
Horse, 25 g 17 5.9 222
Loin, 25 g 121 19.8 222
Raw ham, 25 g 169 11.8 222
Pork bacon, 25 g 153 18.3 222
Minced muscles (sliced or 308 11.7/4.22a 222
not), dry fermented
sausages, etc., 25 g/0.1 g
Raw cured meat products, 824 13.7/5.8a 222
25 g/0.1 g
Bosnia/Herzegovina Beef 20 20 10 158
Pork 50 18 8 158
Brazil Dressed lamb carcasses 69 26 3 7
Bulgaria Beef and pork 234 8 186
China Pork 25 60 28 233
Beef 10 70 0 233
Lamb 14 43 0 233
Denmark Preserved meat products (not 335 10.8 187
heat-treated), 25 g
Heat-treated meat products, 772 5 187
25 g
Raw meat, 25 g 343 30.9 187
Preserved meat products (not 132/225b 14.4/16.5b 187
heat-treated), 1997/1998
Heat-treated meat products 3,180/3, 7.8/10.1b 187
handled after heat 629b
treatment, 1997/1998
(continued)

(1990–1997)
Germany Ground meat 21 52 132

Rinderhack (ground beef) 59 46 132
Greece Sliced, vacuum-packed 26 27 8 198
cooked meats
Frankfurters 8 0 0 198
Country-style sausages 10 40 10 198
Emulsion-type sausages 12 0 0 198
heated in packs
Dry-fermented sausages 4 0 0 198
India Cattle 54 6 42
Buffalo 54 6 42
Sheep 54 4 42
Goat 54 0 42
Ireland Beef 20 85 5 206
Ground beef 85 68 4 5–500 206
Pork 20 45 3 206
Lamb 20 60 10 206
Sausage 20 65 1 206
Frozen beef burgers 94 97 17 5–10,000 206
Italy Calf 19 63 164
Horse 19 26 164
Pork 19 53 164
Mutton 18 56 164
Sausage 156 60 13 < 100 49
Casing surface 116 47 12 < 100 49
Pork 67 33 16 228
Beef 99 21 13 228
Ground beef 148 53 9 LM < 100 60
Pork 153 35 16 LM < 100 60
Ground meat 308 54 19 61
Beef 174 47 12 61
Raw meat 82 44 22 155
Sausage 30 44 17 155
Japan Beef 15 40 13 196
Pork 18 61 39 196
Korea Beef 43 11 1 56
Pork 20 4 1 56
Frozen pork cutlet patties 52 9 1 56
and dumplings
Fermented sausage 15 2 2 56
Malaysia Beef 12 50 30
Spain Ground meat 88 62 16 117
Norway Ground meat 40 5 195
(continued)

(1990–1997)
Poland Pork 245 19 11 149

Beef 114 9 7 149
Spain Ground meat 168 80 17 69
Pork, raw 5 0 211
Switzerland Ground meat 85 40 15 44
Beef 18 33 15 44
Pork 31 45 3 44
Sausage meat 102 65 8 44
Dried meat 44 16 9 44
Ham (uncooked) 19 11 0 44
Taiwan Beef steak 25 24 239
Pork 34 59 239
Trinidad Beef 76 7 3 1
Mutton 66 0 0 1
Goat meat 70 10 4 1
Pork 71 1 0 1
United Arab Beef 15 7 0 103
Emirates
Goat 17 6 0 103
Sheep 24 21 0 103
Camel 14 0 0 103
United Kingdom Pork sausage 59 49 93
Beef 26 88 35 162
Lamb 20 80 40 162
Pork 32 59 28 162
Sausage 23 87 35 162
Meat 15 93 53 115
Beef 1295 3 236
Lamb 37 3 236
Pork 794 4 236
Note: LM = L. monocytogenes.
aThese values are for 2-g and 0.1-g samples.
bThese values are for 1997/1998.
In Denmark, Norrung et al. [187] surveyed retail foods for L. monocytogenes during 1997–1998.
For heat-treated meat products, 3180 and 3629 samples were tested in 1997 and 1998, respectively. The
percentage of unsatisfactory samples (L. monocytogenes between 10 and 100 per g) rose from 7.6 to
9.6% and the percentage of unacceptable samples (L. monocytogenes >100 per g) increased from 0.2
to 0.5% from 1997 to 1998. In the same study 132 and 225 samples of non-heat-treated preserved meats
were examined from 1997 and 1998, respectively. The percentage of unsatisfactory samples increased
from 21 to 21.5% over the 2 years, and the percentage of samples that were unacceptable rose from 0.8
to 1.8% during the same time.
In a survey of five French pork slaughtering and cutting plants, Giovanacci et al. [99] recovered
287 isolates of L. monocytogenes from various locations, including live animals, carcasses imme-
diately after slaughter and after chilling, pork cuts (rind and meat), and the environment, including
work surfaces, equipment, and various areas of the slaughterhouses. These 287 isolates were
grouped into 19 genotypes by three molecular typing methods: random amplification of polymor-
phic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and PCR-restriction enzyme analysis
(PCR-REA). Contamination was present over a period of 1 year in two of the plants, even after
cleaning and disinfection procedures.
Chasseignaux et al. [55] used molecular typing methods to track L. monocytogenes in two poultry
and pork processing plants, and found that 502 isolates of the pathogen could be separated into
50 combined genotypes by ApaI and SmaI digest profiles. Uyttendaele et al. [222] surveyed cooked
meat products, raw cured meat products, and other foodstuffs to determine incidence of Listeria
contamination at the retail level in Belgium. Incidence of Listeria spp. was significantly higher in
raw cured meat products as compared to cooked meat products (13.7 and 4.9%, respectively).
Incidence of Listeria spp. in whole cooked meats was higher after slicing than before (6.6 vs. 1.6%),
and the incidence rate for cooked minced meats was higher than for whole cooked meats (6.1 vs. 4.0%).
Reports from Spain showed that, of 251 samples of ground meat, 63–80% contained listeriae,
with L. monocytogenes being recovered from 17 to 29% of the samples [69,165]. A survey by
Soriano et al. [211] of 103 samples of raw and RTE foods from restaurants in Spain found an
incidence of 2.9% for L. monocytogenes, whereas other species were found more frequently, i.e.,
L. grayi 13.6%, L innocua 1.9%, L. ivanovii 5.8%, L. seeligeri 3.9%, and L. welshimeri 1.9%.
In countries of Eastern Europe, reports have demonstrated similar levels (8–20%) of listeriae
to those of Western Europe in 613 samples of fresh beef and pork [149,158,186]. The incidence
of L. monocytogenes in these samples ranged from 7 to 10%, with a high prevalence (94%) of
serotypes 1 to 3, the remainder being serotype 4 [149].
Other Countries
Reports from other countries worldwide indicate that the problem of Listeria-contaminated meat
is widespread. In general, the prevalence of Listeria spp., and more specifically L. monocytogenes,
is in the same range as that already discussed for North America and Europe.
In Trinidad, Adesiyun [1] reported listeriae in 0–10% of samples of fresh beef, mutton, and
goat meats, with L. monocytogenes present in 0–4% of the samples. The organisms were prevalent
in ground beef samples at similar levels, 11 and 6%, respectively. L. monocytogenes serotypes
1/2c and 4 were found in both local and imported meat. A later study on slaughter pigs
demonstrated 7 (5%) of 139 rectal swabs and 3 (1.9%) of 155 carcass samples to be positive for
Similar incidences of listeriae have been reported in Asian countries. In the United Arab
Emirates, Gohil et al. [103] reported that in fresh beef (15 samples), goat (17 samples), sheep (24
samples), and camel (14 samples), listeriae were present in 0–21% of the samples, and that no L.
monocytogenes was found. In Australia, of 50 samples each of fresh beef, lamb, and pork, listeriae
were found in 34, 40, and 30%, and L. monocytogenes in 24, 16, and 10%, respectively [131]. In
Malaysia, L. monocytogenes was found in 50% of 12 samples of beef [30]. Fifty-four samples each
of cattle, buffalo, sheep, and goat were examined by Brahmbhatt and Anjaria [42] in India, with a
low incidence of L. monocytogenes (4 to 6%) being found.
Ryu et al. [196] found a high incidence of listeriae in Japanese meats, with fresh beef and pork
having an incidence rate of 40 and 61% for Listeria spp., and 13 and 39% for L. monocytogenes,
respectively. In ground beef and pork samples, the prevalence was higher, with 80 and 100%, and
60 and 67% of the samples being positive, respectively. A study done by Wang et al. [233] in China
showed a high incidence (from 43 to 70%) of listeriae in pork, beef, and lamb samples. However,
the incidence of L. monocytogenes was much lower, from 0 to 28%. Serotypes found were 1/2a,
1/2b, and 1/2c. Meats from Taiwan, however, showed a high incidence of this organism in beef
steak and pork, namely, 24 and 59%, respectively [239].
Choi et al. [56] studied the incidence of Listeria spp. in Korean foods. A total of 410 samples were
collected, of which 130 were beef, pork, fermented sausage, or frozen foods (frozen pork cutlet
patties and dumplings). An overall incidence of Listeria spp. of 20% was found, with 5 of the 26
isolates identified as L. monocytogenes. The individual meat products with incidences of Listeria
spp. and L. monocytogenes found and total number of samples, respectively, in parentheses were
as follows: beef (11/1/43), pork (4/1/20), frozen foods (9/1/52), and fermented sausage (2/2/15).
Only these four food types and chicken contained L. monocytogenes, with this pathogen not found
on raw vegetables or in ice cream. Rho et al. [193] looked at the microbial hazards on farms, in
slaughterhouses, and in processing lines of swine in Korea. L. monocytogenes was detected in
processing rooms in three provinces, with the final pork products being manufactured in the
Kyonsang province. Although the aerobic plate count (APC) was low (3.0 log10 CFU/g) at the
outset, the final pork products showed relatively high levels of overall contamination at the retail
level (data not given).
INCIDENCE OF LISTERIA SPP. IN SAUSAGE AND RTE MEAT PRODUCTS

The prevalence of L. monocytogenes in processed RTE meats is of greater concern than contami-
nation of raw meats. An examination of retail meat slicers revealed a contamination rate of 13%
with L. monocytogenes; hence, such machines could be a source of cross-contamination [128]. Two
factors, namely, development of proper methods to isolate Listeria spp. from samples containing
a diverse microflora and a heightened concern about foodborne listeriosis, have led scientists to
examine sausage, pâté, and other RTE meat products for listeriae. Consequently, numerous world-
wide surveys have been made since 1990 to determine the incidence of these organisms in such
products.
North America
Since the association of sporadic cases and outbreaks of listeriosis with consumption of uncooked
frankfurters [202], there have been several nonregulatory U.S. studies on the incidence of listeriae
in packages of retail wieners (Table 13.7). Wang and Muriana [232] reported that, in a survey of
20 brands of retail wieners, 19 brands (93 samples) showed a 10% incidence of listeriae, with an
8% incidence of L. monocytogenes. However, brand no. 20 contained listeriae in 83% of its 24 packages,
with a 71% incidence of L. monocytogenes. They reported that listeriae were found in the liquid
exudate (purge) at a level of 1–3 CFU/mL, but not in the meat itself, indicating the likelihood of
postprocessing contamination. A survey commissioned by the Los Angeles Times found that, in a
sample of 30 packages of retail wieners, there was a 20% incidence of listeriae with a 17% incidence
of L. monocytogenes [24].
A Canadian study [216] reported that wieners (38 samples) and sliced meats (67 samples)
showed an incidence of Listeria spp. of 13–26%, and of L. monocytogenes of 13–21% (Table 13.7).
In contrast, Listeria spp. were not isolated from cooked meats (16 samples) and sausages (9 samples).
However, fermented sausages (30 samples) studied by Farber et al. [87] showed a 20% incidence
Europe
Numerous studies have investigated the incidence of listeriae in RTE meat and sausage products
produced in Europe (Table 13.9). Public Health Laboratory Service workers in England and Wales
conducted surveys on a large number of samples to determine the incidence of listeriae in
TABLE 13.9
Incidence of Listeria monocytogenes in Sausages and RTE Meat Products in European
and Other Countries (1990–2003)
Levels of
Number of Percentage of Positive Samples Listeria
Country Product Analyzed spp. LM (CFU/g) Reference
Australia Pâté 7 0 0 204

Luncheon meat 28 11 7 204
Pâté 25 8 118
Processed meat 25 4 118
VP salami 19 5 0 109
VP corned beef 72 83 72 109
VP ham 71 41 34 109
VP luncheon meat 13 23 15 109
Salami 132 31 4 225
Ham 90 39 17 225
Corned beef 39 41 10 225
Pâté 7 0 0 225
Belgium Cooked meat products 3405 4.9 222
Whole muscles, 25 g/0.1 g 2194 4.0/0.5 222
Cooked ham, 25 g/0.1 g 1069 1.4/ND 222
Cooked loin, 25 g 87 3.5 222
Minced muscles, 25 g/0.1 g 1107 6.1/0.g (RTE) 222
Minced muscles, 0.1g 1107 0.6 222
Prepacked meats, 25 g/0.1 g 701 6.7/0.7 (RTE) 222
Prepacked pâtés, 25 g 217 2.8 222
Prepacked aspic, 25 g 48 8.3 222
Salads 222
Meat (ham) salad, 25-g 159 20.75 222
sample
Denmark Sliced ham 80 10 192
Sliced rolled sausage 80 10 192
Sliced smoked pork loin 78 23 192
Frankfurters 67 6 192
France Sausages, pâtés, ham 990 14 151
Hungary Dry-cured sausage 136 27 10 147
Fermented sausage 21 33 10 147
Smoked sausage 23 65 13 147
Italy Pork sausage 55 87 24 157
Sausage—mixed meat 20 90 60 157
Ground beef rissoles 45 93 33 157
Sausage 82 88 28 53
Würstel 118 39 5 LM— 53
10–224
Pâté 48 27 2 53
New Zealand RTE pork 34 50 3 124
(continued)

Incidence of Listeria monocytogenes in Sausages and RTE Meat Products in European
and Other Countries (1990–2003)
Levels of
Number of Percentage of Positive Samples Listeria
Country Product Analyzed spp. LM (CFU/g) Reference
RTE beef 18 50 0 124
RTE lamb 3 67 0 124
Norway VP processed meat 35 11 195
South Africa Vienna sausage 47 4 0 229
Ham 43 14 0 229
Cervelat 44 7 0 229
Spain Cured sausage 17 18 12 34
Cooked ham or sausage 15 7 0 34
Luncheon meat 60 42 27 1–100 150
Pâté 36 14 14 1100 150
Pork, ready-to-eat 5 0 211
Beef, ready-to-eat 5 0 211
Switzerland Sliced cured dried beef 26 15 4 LM—20 218
Salami 59 49 5 LM—20 218
Mettwurst 14 93 0 218
United Kingdom Pâté 696 20 17 173
RTE meat and poultry 3939 15 8 173
Sliced meat—ham 303 8 3 226
Sliced meat—salami 128 17 9 226
Sliced meat—tongue 28 7 0 226
Sliced meat—corned beef 27 15 4 226
Sliced meat—mixed meats 119 5 0 226
Prepared sandwiches 91 17 129
Cooked meats 1551 13 5 <20–1000 16
Pâté 239 11 7 <20–100 16
Salad with meat 15 33 13 <20–100 16
Sandwiches with meat 237 20 8 20–100 16
Cook-chill—meat and poultry 736 2 93
Salami 67 16 93
Pâté 216 35 93
Pâté 1834 10 LM— 94
<200–106
Pâté 626 4 LM— 94
<200–105
Cook-chill food—meat 992 5 3 LM— 122
<10–103
Cook-chill food—meat 854 9 LM— 122
<10–103
Cured or smoked meat 29 7 14
Cooked tripe 44 9 130
Cold meats 2894 0.06 0.02 LM— 78
104–105
Pâté 1184 0.04 0.02 78
Yugoslavia Fermented sausage 21 80 19 46
Hot-smoked sausage 15 28 0 46
VP hot-smoked sausage 14 35 21 46
Note: LM = L. monocytogenes; RTE = ready-to-eat; VP = vacuum-packed.
various foods. McLauchlin and Gilbert’s report [173] on a study of pâté (696 samples) and RTE
meat and poultry (3939 samples) showed listeriae present in 20 and 15%, and L. monocytogenes
in 17 and 8% of the samples, respectively. A report on 605 samples of sliced meats showed a low
incidence of 6% for listeriae and 3% for L. monocytogenes, with levels of <200 to <500 CFU/g
[177]. Most (91%) of the L. monocytogenes isolates were serotype 1/2, with the remainder being
serotype 4. An extensive study done in Yorkshire [16] showed that cooked meats (1551 samples),
pâté (239 samples), meat salad (15 samples), and meat sandwiches (237 samples) had incidences
for listeriae of 13, 11, 33, and 20%, and for L. monocytogenes of 5, 7, 13, and 8%, respectively.
Levels of the organism in this study were 20–1000 CFU/g. Another report on 91 samples of
preprepared sandwiches showed a 17% incidence of L. monocytogenes [129]. Of 13 strains
typed, 10 were serotype 1/2, and 3 were serotype 4. A study of pâté, with 1834 and 626 samples
in 1989 and 1990, showed incidences of L. monocytogenes of 10 and 4%, with levels of the organism
up to 106 CFU/g [94]. Serotyping revealed that both serotypes 1/2 and 4 were present. L. mono-
cytogenes was also present in 16% of 67 salami samples [93]. Cooked tripe, which is often eaten
without further heating, showed a 9% incidence of L. monocytogenes [130]. In cook-chill catering,
often employed in hospitals, food is prepared and cooked in a traditional manner, cooled very
rapidly, and maintained chilled (0–3°C) for up to 5 days until reheated for use. In three surveys of
cook-chill food containing meat (736, 992, and 854 samples), the incidence of L. monocytogenes
was 2, 3, and 9%, respectively [93,122]. Of 28 strains typed, 7 were serotype 1/2, 17 were serotype
3, and 4 were serotype 4 [122]. These results must cause concern, in light of the vulnerability of
a hospital population. Gillespie et al. [98] found that of 3494 samples of cold, RTE sliced meats
collected from 2579 catering establishments in the United Kingdom, Listeria spp. and L. monocy-
togenes were present in 13 (0.3%) and 5 samples (0.1%), respectively.
In Denmark, 305 samples of wieners and sliced meats showed similar incidences, with 10% of
the samples containing L. monocytogenes at a level <100 CFU/g [192]. A study in Norway of vacuum-
packaged processed meat showed 11% of 35 samples to be contaminated with L. monocytogenes [195].
In France, a sampling of 18 dry sausages showed that 22% contained L. monocytogenes, whereas
in Germany, 9% of 11 mettwurst samples contained the organism [132].
Several studies on sausages in Italy showed similar incidences to those just mentioned. Levre
et al. [157], reporting on 120 samples of pork sausage/mixed chicken, turkey, and pork sausage/beef
rissoles, found that, overall, as high as 90% of these samples contained listeriae, with 33% positive
for L. monocytogenes. Casolari et al. [116], in a study carried out over a 2-year period (1990–1991),
demonstrated similar incidences in sausage, with 88 and 28% of 82 samples positive for Listeria
and L. monocytogenes, respectively. In 166 samples of meat products (würstel, pâté), overall, 36%
contained listeriae, with L. monocytogenes present in 4% of the samples at a level of 10–224 CFU/g.
A survey in Switzerland taken during production of cured and air-dried meat products [218]
uncovered a substantial number of bündnerfleisch (cured, air-dried beef), salami, and mettwurst that
contained listeriae. Overall, of samples taken during production, bündnerfleisch (19 samples), salami
(30 samples), and mettwurst (3 samples) had an incidence of 26, 50, and 100% listeriae, and 11, 10,
and 0% for L. monocytogenes, respectively (data not shown). The end products showed listeriae at
incidences ranging from 15 to 93%, in contrast to incidences ranging from 0 to 5% for L. monocy-
togenes. The organism was present at levels of <20 CFU/g. Listeriae were isolated only from the
surface of bündnerfleisch. L. monocytogenes isolates were of serotype 1/2 (86%), and serotype 4
(14%). Because most L. monocytogenes isolates from human listeriosis patients in Switzerland are
of serotype 4b, these data suggest that transmission of the pathogen via meat is relatively uncommon.
Recent surveys of RTE meat products in Spain showed that cured or cooked sausages and ham
(a total of 32 samples) had a 13% incidence of Listeria spp. [34]. Twelve percent of the cured
sausages contained L. monocytogenes, whereas the organism was not isolated from ham or cooked
sausage samples [34]. Sliced meat (60 samples) and pâté (36 samples) contained 42 and 14% of
Listeria spp., and 22 and 3% of L. monocytogenes, respectively [150]. The organisms were found
at a level of 1–100 CFU/g.
In 1991, a Hungarian researcher, Kovácsné Domjan [147], reportedly recovered listeriae from
37 of 136 (27%), 7 of 21 (33%), and 15 of 23 (65%) samples of dry-cured, fermented, and smoked
pork sausage, respectively. L. monocytogenes was present in 10, 10, and 13% of the samples,
respectively. Buni [47] in Yugoslavia found that 21 samples of fermented sausage showed a 28 and
19% incidence of listeriae and L. monocytogenes, respectively. In contrast, listeriae were not
recovered from hot, smoked sausage. Of 14 surface samples of vacuum-packaged hot-smoked
sausage, 35 and 21% contained listeriae and L. monocytogenes, respectively, indicating recontam-
ination before and during vacuum packaging (VP).
Overall, the results in Table 13.9 show that a substantial portion of European processed meats
are contaminated with listeriae, including L. monocytogenes. The presence of listeriae other than
L. monocytogenes in processed as well as raw meats may be indicative of possible contamination
with L. monocytogenes.
Other Countries
A recent report by Vorster et al. [229] in South Africa showed a low prevalence of listeriae in 134
retail samples of processed, RTE meats, with an overall incidence of 8% in Vienna sausage, ham,
and cervelat (Table 13.9). L. monocytogenes was not found in any of the samples. A number of reports
from Australian workers show, in general, the same incidence of listeriae in processed meats as in
North America and Europe. Robertson and co-workers [204, 217] reported that 1 of 20 (5%) and 3
of 28 (11%) samples of sliced meats contained Listeria spp., with L. monocytogenes recovered at
rates of 0 and 7%, respectively. No Listeria spp. were recovered from 7 samples of pâté. Hobson
et al. [118] reported that in 25 samples each of pâté and processed meat, L. monocytogenes was
present in 8 and 4% of the samples, respectively. In a study on 175 samples of vacuum-packaged
processed meats, Grau and Vanderlinde [110] reported that 60 of 72 (88%) samples of corned beef,
29 of 71 (41%) samples of ham, 3 of 13 (23%) samples of luncheon meats, and 1 of 19 (5%) samples
of salami were contaminated with listeriae, with 72, 34, 15, and 0% of these samples contaminated
with L. monocytogenes, respectively. Sixteen of the corned beef samples had listeriae counts >50
CFU/g, and, of these, six counts were >1000 CFU/g. In salami, counts of listeriae were <50 CFU/g,
and no L. monocytogenes was found. In an extensive study of 284 samples of RTE meat products,
Varabioff [225] found that except for pâté, in which no listeriae were found, the incidence of listeriae
ranged from 5 to 41%, with L. monocytogenes ranging from 4 to 19%. Of the isolates recovered,
62% were serotype 1 and 36% were serotype 4. Further investigation of the occurrence of listeriae
on cutting utensils and cutting boards showed that most of the contamination occurred at the retail level.
A survey of 55 samples of RTE meat products in New Zealand [124] showed incidences of
contamination with listeriae ranging from 23 to 67%. Interestingly, mixed-source products had a
lower incidence (23%) of listeriae than products made from a single meat (50–67%), whereas the
organism was seldom found in single-meat products. The authors also found that significantly more
smoked products were contaminated with L. monocytogenes than meats preserved by other methods.
Additional information concerning habitats, niches, and relative incidence of listeriae in all
facets of the meat industry is needed, as it is now evident that the presence of L. monocytogenes
in processed meat products can pose a serious health hazard to certain individuals. Therefore, it is
necessary to control all listeriae in meat-processing facilities and to design procedures and treat-
ments that will eliminate L. monocytogenes from RTE processed meat products (see Chapter 17).
BEHAVIOR OF L. MONOCYTOGENES IN MEAT PRODUCTS

Although L. monocytogenes was reported during the 1950s in European meat products destined
for human consumption, a general failure to positively link cases of human listeriosis to foods other
than raw milk provided little incentive to examine the behavior of listeriae in meat. This situation
was further complicated by a lack of reliable and convenient methods to selectively isolate listeriae
from heavily contaminated samples of animal origin, including organ as well as muscle tissue.
Thus, the first definitive studies on the behavior of L. monocytogenes in raw meat were not begun
until the 1970s. Even then, to accurately quantitate this organism in meat products during extended
storage, it was generally deemed necessary to use specially treated “sterile” meat rather than raw
meat products containing an expected (i.e., “normal”) microbial background flora.
Outbreaks of foodborne listeriosis linked to the consumption of contaminated cheese prompted
concerns about the microbiological safety of raw and, particularly, RTE meat products marketed
in North America and Europe. The outbreaks also demonstrated an urgent need for methods to
rapidly and accurately detect listeriae in a wide range of foods. Subsequent development of the
USDA procedure to detect listeriae in meat and poultry products provided researchers with a method
to determine the growth and survival of L. monocytogenes in raw and RTE products. Recent meat-
oriented epidemiological studies along with large listeriosis outbreaks linked to meat products
suggest that the behavior and control of L. monocytogenes in meat products are likely to remain
an active area of research for some time to come. As in the previous section of this chapter,
information concerning the behavior of L. monocytogenes in raw meat will be presented first,
followed by a discussion of the fate of this pathogen in processed meat products.
LISTERIOSIS IN DOMESTIC LIVESTOCK

Domestic farm animals such as cows, sheep, and pigs can succumb to listerial infections, as well
as asymptomatically shed L. monocytogenes in their feces for many months (see Chapter 2).
Although virtually all meat from animals exhibiting obvious signs of listeriosis will be condemned
and destroyed immediately after slaughter, meat from subclinically infected animals will likely be
passed by inspectors as being fit for consumption. Because between 2 and 16% of all healthy cows,
sheep, and pigs passively shed L. monocytogenes in their feces, there is ample opportunity for
contamination of muscle tissue during slaughter, evisceration, and dressing of the animals.
It is likely that meat from domestic livestock will first be exposed to listeriae in the slaughter-
house environment. However, various organs from apparently healthy animals can also occasionally
contain L. monocytogenes. In 1972, Höhne [119] reported that approximately 13% of the parotid
glands from clinically healthy pigs contained L. monocytogenes. Three years later, Höhne et al.
[120] detected L. monocytogenes in intestinal lymph nodes from eight apparently healthy slaugh-
tered animals (five small ruminants, two pigs, and one cow) destined for human consumption.
Subsequently, Cottin et al. [62] identified L. monocytogenes in 15 of 514 (3.1%) spleen and lung
tissue samples obtained from apparently healthy cattle. According to Amtsberg et al. [6], L.
monocytogenes was isolated from the spleen and muscle tissue of an apparently healthy animal.
These findings suggest that L. monocytogenes might be transported to tissue via the bloodstream
in animals suffering from symptomatic as well as asymptomatic septicemic listerial infections.
Hence, to understand the behavior of L. monocytogenes in meat products, it is fitting to begin this
section by first examining the localization of L. monocytogenes in organ tissue, and particularly
muscle tissue, from infected animals.
LOCALIZATION IN TISSUES
In 1988, Johnson et al. [135] reported results from a study of samples of muscle, organ, and
lymphoid tissue, as well as feces and blood were obtained from several Holstein cows previously
inoculated intravenously with 1010–1011 L. monocytogenes. Samples were examined for the
pathogen 2, 6, and 54 days after inoculation, using a combination of direct plating and cold
enrichment. As expected, recovery of listeriae varied among animals and was strongly influenced
by the time that elapsed between inoculation and slaughter. Overall, 94% of all samples obtained
from cows slaughtered 2 days after inoculation contained L. monocytogenes, with 23 of 32 (72%)
samples positive by direct plating. More important, the pathogen was routinely detected in muscle
tissue from the same animals, frequently at levels of 120–280 CFU/g. Despite a marked decrease
in recovery of L. monocytogenes from animals examined 6 and 54 days postinoculation, the
pathogen was still present at levels of ~140–675 CFU/g in kidney tissue as well as in mesenteric
and mammary lymph nodes, with populations about 2 orders of magnitude lower in liver and spleen
tissue. Following 2 weeks of cold enrichment, the pathogen also was detected in plate-flank tissue
taken from an animal 6 days after slaughter. These findings suggest that consumption of animal
organs may constitute a greater health hazard than consumption of muscle tissue, and also readily
explains how evisceration of domestic animals can lead to surface contamination of muscle tissue.
Assuming that most contamination occurs during handling of carcasses after slaughter, Chung
et al. [57] investigated the ability of L. monocytogenes to attach to (or become entrapped) and
proliferate on muscle and fat tissue both alone and in the presence of Pseudomonas aeruginosa.
Immersion of lean muscle and fat tissue in broth cultures containing 105 or 108 L. monocytogenes
CFU/mL for various times followed by thorough rinsing resulted in large numbers of listeriae being
attached to (or entrapped in) both types of samples within the first 10 min. Despite large differences
in hydrophobicity, pliability, and surface qualities, L. monocytogenes adhered (entrapped) equally
well to lean muscle and fat tissue during 50–60 min of incubation at ambient temperature. According
to an earlier report by Herald and Zottola [116], attachment of L. monocytogenes to stainless steel
and presumably meat surfaces is related to flagellae, fibrils, and exopolymeric substances (i.e.,
polysaccharides), all of which are readily produced by Listeria during extended incubation at room
temperature. However, lean muscle tissue supported faster growth of the pathogen than fat tissue
when samples were stored 1 day at ambient temperature, followed by 7 days at refrigeration
temperature. Following attachment of L. monocytogenes and P. aeruginosa to lean meat at a con-
centration of ~106 CFU/4 cm2, Listeria populations increased approximately 100-fold during 24 h
of incubation at room temperature and remained at this level during 7 days of refrigerated storage,
regardless of the presence or absence of P. aeruginosa. In contrast, populations of P. aeruginosa on
lean meat increased >100-fold during initial storage at room temperature, but then decreased, with
levels frequently 10 times lower than the Listeria population following 7 days of refrigerated storage.
Dickson [71] simulated contamination of raw beef during processing, handling, and storage by
placing surfaces of heavily inoculated lean and fat beef tissue (~2 × 106 L. monocytogenes CFU/cm2)
in direct contact with uninoculated tissue. Overall, transfer of listeriae was largely dependent on the
type of tissue, with minimum and maximum transfer observed from fat-to-fat and lean-to-fat tissue,
respectively. However, bacterial transfer was also influenced by adsorption time of the original
inoculum and contact time with uninoculated tissue. Adsorption times of <60 min generally led to
the highest Listeria transfer rates, particularly between inoculated and uninoculated lean beef tissue.
These findings indicate that most listeriae are likely to be in suspension in water films on tissue
surfaces shortly after inoculation. Following an adsorption time of 30 min, approximately 30 and
50% of the original Listeria inoculum migrated from lean and fat tissue, respectively, to lean tissue
after 5 min of direct contact at ambient temperature. When contamination between fat and lean tissue
was simulated using shorter contact times of 15–60 sec, a greater percentage of listeriae migrated
from inoculated fat to uninoculated lean tissue, reflecting transfer of cells in unadsorbed water from
hydrophobic fat to hydrophilic lean tissue. More important, the fact that bacterial transfer also occurred
at 5°C, with 0.6–9.5% of the original Listeria population migrating from inoculated to uninoculated
lean and/or fat tissue after an 18-h adsorption period, provides a reasonable explanation for spread
of this pathogen to Listeria-free meat during storage in walk-in coolers.
These findings attest to the hardy nature of L. monocytogenes on the surface of raw meats and
to the need for effective means of reducing surface contamination on carcasses. Regarding the
latter, Chung et al. [58] reported that wash solutions containing nisin effectively delayed growth of
L. monocytogenes on surfaces of raw meats, particularly when such products were incubated at
refrigeration, rather than ambient temperatures. Although nisin-producing bacterial starter cultures have
been used in the dairy industry for many years, with the exception of certain types of cheese spread,
present laws in North America still prevent direct addition of nisin to most foods, including raw meat.
Warriner et al. [234] examined attachment of various bacteria to beef from steam-pasteurized
carcasses. There was no significant difference in attachment of bacteria to steam-pasteurized
carcasses versus loin cuts that were not given any treatment. Furthermore, there did not appear to
be any significant difference in attachment strength between the different bacteria tested (Pseudomo-
nas fragi NCTC 10689, L. monocytogenes BL5/2, Salmonella typhimurium LT2, and Escherichia
coli JM109).
Populations of enteric pathogens (i.e., Salmonella spp., enteropathogenic E. coli, and Yersinia spp.)
on raw meats can be sharply reduced by exposing the water phase of meat surfaces to 0.2 M lactic acid
(pH 2.5) at 21°C [135]. Although L. monocytogenes is generally recognized as being more acid
tolerant, it can be inactivated at pH values less than 4.0. Hence, provided that L. monocytogenes
is exposed to lactic acid for sufficient time, acid washes may be somewhat helpful in decreasing
Listeria populations on the surface of animal carcasses.
As noted by Johnson et al. [135], L. monocytogenes was routinely detected in muscle tissue
from cows that were killed 2 days after being inoculated intravenously with the pathogen. Although
some contamination of muscle tissue might have occurred during sampling, results from this study
suggest that L. monocytogenes can enter muscle tissue via the bloodstream.
To further investigate this hypothesis, Johnson et al. [133] examined muscle, liver, and spleen
tissue from two lambs and one calf that had been inoculated intravenously with L. monocytogenes.
Microscopic examination of tissues stained with immunoperoxidase or Azure A revealed L. mono-
cytogenes cells at levels of 103–104 CFU/g in muscle tissue and 103–106 CFU/g in both liver and
spleen tissue. Although L. monocytogenes appeared to be associated with phagocytes in liver and
spleen tissue, the pathogen was observed in loose connective tissue between muscle fibers and also
within the muscle fibers themselves.
Using the USDA enrichment method, Johnson et al. [137] subsequently detected L. monocy-
togenes serotype 1/2a at 10 CFU/g in aseptically removed interior samples from 2 of 50 (4%) and
3 of 50 (6%) retail whole-muscle beef and pork roasts, respectively. One beef sample also was
positive for L. innocua and L. welshimeri. Although the presence of these two nonpathogenic (i.e.,
noninvasive) Listeria spp. within a whole-muscle roast suggests possible contamination during
sampling, other results from Johnson et al. [135–137] strongly support at least limited transmission
of L. monocytogenes from the bloodstream into muscle tissue.
Niebuhr and Dickson [185] studied the impact of pH enhancement, i.e., raising the pH to ~9.6,
on survival of L. monocytogenes on boneless beef trimmings. Populations were reduced by 3 log10
cycles after exposure to ammonia gas, which was used to raise the pH [176].
RAW BEEF
Growth and Survival
Interest in behavior of Listeria in raw meat products dates back to at least 1966 when Sielaff [208]
inoculated beef, pork, and rabbit with L. monocytogenes immediately after slaughter and examined
these samples for listeriae during extended storage at 3–4°C. Under these conditions, the pathogen
survived at least 15 days in all three products.
Although this study was among the first to examine the ability of L. monocytogenes to survive
in raw beef, information concerning actual growth of this pathogen in raw beef was not available
until 1978. In that year, Gouet et al. [105] reported results from a study that examined multiplication
of L. monocytogenes in “sterile” minced beef alone and in combination with a defined microflora.
L. monocytogenes failed to grow alone in minced beef (pH 5.8) stored at 8°C, and populations
decreased <10-fold during 17 days of incubation. In contrast, numbers of listeriae decreased
approximately 100-fold in samples of minced beef (pH 5.8) that were simultaneously inoculated
with Lactobacillus plantarum and held at 8°C for 17 days. These researchers also found that higher
concentrations of L. monocytogenes (106 CFU/g) enhanced growth of L. plantarum. When samples
of minced beef were simultaneously inoculated to contain equal numbers of L. monocytogenes,

P. fluorescens, and E. coli, Listeria populations decreased approximately 10-fold after 24 h of
incubation at 8°C, with numbers rapidly increasing after day 7. Rapid growth of listeriae during
the latter half of incubation was likely prompted by proteolysis of meat proteins by P. fluorescens,
which, in turn, led to a gradual increase in pH of the meat from 5.8 to 6.8. With a complex microflora
consisting of ~103 CFU/g each of L. plantarum, P. fluorescens, E. coli, Micrococcus sp., Clostridium
perfringens, and Streptococcus faecalis, behavior of L. monocytogenes was similar to that previously
observed for the pathogen in the presence of P. fluorescens and E. coli. Listeriae populations reached
approximately 6 × 105 CFU/g in minced beef following 17 days at 8°C. A rapid increase in numbers
of P. fluorescens before growth of L. plantarum again appeared instrumental in raising the pH from
5.8 to 7.2, which, in turn, stimulated growth of listeriae.
In a similar investigation completed 11 years later, Kaya and Schmidt [143] also found that
growth of L. monocytogenes in artificially contaminated sterile minced meat during extended
incubation at 8–20°C was suppressed by the addition of 106 Lactobacillus CFU/g, but was unhin-
dered in the presence of 106 Pseudomonas sp. CFU/g. However, in contrast to the previous study
by Gouet et al. [105], this strain of L. monocytogenes readily grew in the absence of other
microorganisms, with populations increasing approximately 2 and 4 orders of magnitude in sterile
minced meat after 10 and 1–5 days of storage at 4 and 8–20°C, respectively. In this particular
instance, proteolysis of meat proteins by pseudomonads apparently was not a prerequisite for growth
of L. monocytogenes in sterile minced meat with an initial pH value of 5.8–6.0.
During 1988 and 1989, three additional studies were done to determine the behavior of listeriae
in ground beef. However, unlike previous investigations, the meat was not pretreated to eliminate
the normal background flora. When ground beef at pH 5.6–5.9 was inoculated to contain 105 and
106 CFU/g of L. monocytogenes Scott A or V7, packaged in oxygen-permeable or oxygen-impermeable
film, and held at 4°C, Johnson et al. [113] found that the numbers of organisms as well as the pH
remained relatively constant during 14 days of incubation, regardless of the film’s degree of
permeability. In contrast to the previous study by Gouet et al. [105], in which the pH of minced
beef containing a complex microflora increased from 5.8 to 7.2 during extended incubation at 8°C,
the pH of all packaged ground beef samples in this study remained in the range of pH 5.6–5.9
throughout storage. Thus, the lower pH of the meat in this study, along with a lower incubation
temperature (4 vs. 8°C) are likely to have been at least partly responsible for inhibiting growth of
listeriae in packaged samples of “retail-like” ground beef.
In keeping with these findings, Shelef [205] also reported that L. monocytogenes failed to
grow in artificially contaminated ground beef or ground liver during 1 and 40 days of incubation
at 25 and 4°C, respectively. However, in contrast to results of the previous study by Johnson
et al. [134], the pathogen failed to grow in ground beef despite a final pH value of 7.8. Although
the reasons for this behavior remain obscure, inability of L. monocytogenes to multiply in ground
beef under certain conditions is likely related to the type and load of inherent microflora in the
product.
This hypothesis regarding the microflora in foods is supported by a West German study in
which Kaya and Schmidt [143] showed that an increase in the natural bacterial flora of ground
beef from 105 to 107 CFU/g led to increased inhibition of L. monocytogenes in artificially contam-
inated meat. When ground beef was inoculated to contain ~105 L. monocytogenes CFU/g, the
pathogen grew after 1–5 days at 7–20°C in samples harboring ~105 non-Listeria contaminants per
g but failed to multiply in corresponding samples containing higher levels of naturally occurring
organisms. Although a similar growth pattern was observed for samples naturally contaminated
with 102 Listeria per g and 105 non-Listeria per g, growth was markedly hindered in naturally
rather than artificially contaminated samples, with Listeria populations remaining constant in the
former during 14 days at 8°C. However, behavioral differences were no longer observed at lower
temperatures, with the organism failing to grow during 14 days of incubation at 4°C in both
artificially and naturally contaminated ground beef containing similar background populations.
Work by Barbosa et al. [33] with vacuum-packaged ground beef indicates that the organism
grows better in beef with a pH >6.0. The study also showed that in addition to pH, strain type
can affect the fate of the organism in ground beef. The authors used four different strains of
L. monocytogenes for inoculation and observed slightly different responses with each. For example,
in normal pH (5.47) ground beef L. monocytogenes serotypes 3a and 3b increased 2.3 and 1.8 logs,
respectively, after 35 days of storage at 4°C, whereas after 56 days the levels of strain 1/2a remained
constant. Interestingly, the Scott A strain (serotype 4b) decreased by 1 log in numbers. In general,
the organism multiplied slowly on all vacuum-packaged samples, but growth was better on ground
beef of pH 6.14 than of pH 5.47. For example, after 28 days of storage at 4°C, serotypes 3b, 3a,
and 1/2a had increased by 2.87, 2.64, and 2.24 logs, respectively, in high-pH ground beef, whereas
the Scott A strain did not change significantly in numbers [33]. The authors felt that the antimicrobial
effect of low pH may also be an indirect one, altering the natural microbial flora while promoting
growth of bacteriocin-producing lactic acid bacteria.
L. monocytogenes appears to behave in a similar manner on surfaces of fresh intact beef muscle.
In two studies conducted shortly before L. monocytogenes emerged as a serious foodborne pathogen,
Lee et al. [153,154] dealt indirectly with the incidence and subsequent behavior of Listeria spp.
along with many other psychrotrophic as well as mesophilic organisms present on the surface of
hot-boned and conventionally boned beef. In their study, hot-boned beef was obtained from five
steers 2 h after slaughter, and then was vacuum-packaged and cooled from 32 to 21°C. Conven-
tionally processed beef was obtained from carcasses that were hung in a cold room at 2°C for 2
days after slaughter. Both types of beef were then examined for mesophilic and psychrotrophic
organisms at day 0 when the surface temperature had decreased to 21°C (<1 h for conventionally
processed beef) and again following 14 days of storage at 2°C. Nearly 1250 bacterial isolates were
subsequently identified by computer analysis of 116 miniaturized tests/isolate. Although Listeria
spp. were never isolated from slow or moderately chilled, hot-boned beef at day 0, 9.1 to 13.5%
of all microorganisms present on the surface of such beef after 14 days were identified as Listeria
spp., some isolates of which were likely L. monocytogenes. Overall, only one isolate from conven-
tionally processed beef was identified as belonging to the genus Listeria. From these data one can
infer that Listeria spp. can grow on the surface of vacuum-packaged hot-boned beef, but not on
the surface of unpackaged conventionally processed beef during 2 weeks of storage at 2°C. Because
the high water-binding properties of hot-boned beef make this product particularly well suited for
sausage making, widespread use of hot-boned beef by the processed-meat industry may be partially
related to the relatively high incidence of listeriae in RTE meat products.
In a more definitive Australian study reported in 1988, Grau and Vanderlinde [108] examined
the ability of L. monocytogenes to grow on the surface of artificially contaminated (102–103
CFU/cm2) vacuum-packaged, nonsterile beef striploin during extended incubation at 0 and 5.3°C.
Although L. monocytogenes populations increased in all samples (and in the purge that developed
in packages) during storage, the extent of Listeria growth was markedly influenced by incubation
temperature, pH of the sample (5.6 vs. 6.0), and type of tissue (lean vs. fat). Overall, higher Listeria
populations consistently developed on fat tissue, with growth also being more rapid at the higher
of the two incubation temperatures and pH values. Numbers of listeriae on fat tissue of pH 5.6
increased from 5 × 103 to 3 × 107 CFU/cm2 during 16 days of incubation, whereas the pathogen
was just beginning to grow on corresponding samples after 7 to 14 days of storage at 0°C. These
researchers also noted that Listeria populations increased <10- and ~1000-fold on vacuum-packaged
meats of pH 5.6 and 6.0, respectively, after 10 to 11 weeks of storage at 0°C. Thus, it appears that
two conditions, (a) a storage temperature of 0°C and (b) a product pH value of 5.6, must be met
simultaneously to prevent significant growth of L. monocytogenes in vacuum-packaged raw meats.
Grau and Vanderlinde [110] extended their studies by using two models, the modified Arrhenius
and square-root models, to examine aerobic growth of L. monocytogenes on lean and fatty raw
beef tissue. For both lean and fatty tissue, the modified Arrhenius model gave better fits and estimates
of growth rates. The effect of temperatures between 0 and 30°C on the growth rate of the organism
could be described by a modified Arrhenius equation: Ln (gen/h) = −205.73 +1.2939 × 105/K −

2.0298 × 107/K2, where K = Kelvin. The combined effect of temperature and pH on the growth
rate of the organism on lean beef was best described by the following equation: Ln (gen/h) =
−232.64 + 1.4041 × 105/K − 2.1908 × 107/K2 + 1.1586 × 102/pH − 4.0952 × 102/pH2. For lean
meat at pH values of about 5.5–5.6 and 6.0–7.0, the latter equation applied between approximately
2.5 and 35°C, and 0 and 35°C, respectively. There was considerable scatter in the measured lag
periods for both types of meat, and therefore, a poor fit was observed for both models. In two trials,
both models predicted growth on lean tissue where no growth was observed experimentally. In the
first, L. monocytogenes failed to grow on lean tissue with a mean pH of 5.61 during 13 weeks of
storage at 0°C, whereas in the other, no growth was observed after 48 h of incubation at 43.2°C,
pH 5.46. However, growth of the organism was observed on lean tissue having a mean pH of 5.61
at 2.5°C, on lean meat of pH values 6.0, and all fatty tissue (pH 5.5–5.7), regardless of storage
temperature. One should be aware, however, that in this study the investigators used only one strain
of L. monocytogenes (which was not a meat strain), and that meats in which the contaminating
flora exceeded 10% of the Listeria count were discarded, thus partially eliminating the effects of
background microflora on growth of the organism.
Growth rates predicted by the best equations for lean tissue were compared with the literature
data for aerobic growth of L. monocytogenes on several foods. Growth rates predicted for lean meat
were higher than those found for corn, clarified cabbage juice, and milk, whereas foods supporting
similar growth rates to the lean beef included UHT milk, raw and cooked chicken, and cooked
ground meat [37,110].
Other researchers have found that some of the models derived from growth of L. monocytogenes in
broth cannot reliably predict growth of the organism in raw pork [97]. For example, L. monocytogenes
grew on pork fat tissue without lag at −0.3°C and higher temperatures at rates that were greater
than those at lower temperatures in unacidified tryptic soy broth (TSB). The better growth of the
organism on fat tissue as compared to TSB suggests that fat tissue may contain micronutrients that
are not present in TSB [97]. In addition, the organism grew on pork muscle tissue only at temper-
atures of ≥15.4°C at a slower rate than in acidified (pH 5.5) TSB, implying that pork muscle tissue
may contain growth inhibitors that are either absent or present in very low levels both in TSB and
fat tissue. All published models done to date in broth predict that the organism will grow at pH
5.5 at 5°C, in contrast to the failure of L. monocytogenes to grow on normal pH raw pork muscle
even at temperatures as high as 15°C. There has only been one published study showing that the
organism can grow on raw meat (beef muscle) of normal pH at low temperatures [110].
RAW LAMB AND PORK

Most investigations have focused on behavior of Listeria in raw beef; however, several reports exist
on the fate of this pathogen in raw lamb and pork. As early as 1973, Khan et al. [144] published
results from a study in which aseptically obtained “sterile” raw lamb meat was inoculated to contain
~105 CFU/g of L. monocytogenes, packaged in gas-permeable or gas-impermeable film, and exam-
ined for survival of listeriae during extended storage at 0 and 8°C. Although Listeria populations
remained relatively constant in lamb meat packaged in gas-permeable film during 20 days at 0°C,
numbers of listeriae decreased approximately 10-fold in corresponding samples that were packaged
in a gas-impermeable film. These results are similar to those obtained by Johnson et al. [134], who
concluded that L. monocytogenes also was unable to grow in refrigerated ground beef that was
packaged in gas-permeable or gas-impermeable film. Following 12 days of storage at 8 rather than
0°C, populations of listeriae in lamb increased >1000-fold; however, unlike meat packaged in
gas-permeable film, the pathogen exhibited a 2-day lag period and grew markedly slower in
gas-impermeable packages. Various physical differences of the meat, combined with an increased
concentration of CO2 (and presumably a lower pH) in gas-impermeable packages, may have been
responsible for partially inactivating the pathogen at 0°C and delaying the onset of growth at 8°C.
Two years later, Khan et al. [145] examined the behavior of L. monocytogenes in preparations
of sarcoplasmic pork, beef, and lamb protein inoculated to contain ~104 CFU/mL of L. monocytogenes
and held at 4°C. According to these investigators, the pathogen grew readily in preparations of
pork and beef protein, reaching levels of approximately 105 and 107 CFU/mL, respectively, after
12 days of refrigerated storage. Although Listeria populations remained relatively constant in
corresponding preparations of lamb protein held at 4°C, the pathogen grew readily in lamb meat
stored at 8°C. Hence, it appears that raw pork, beef, and lamb can support rapid growth of listeriae,
particularly when these products have undergone temperature abuse, as frequently occurs at the
retail level.
These early observations were confirmed by Lovett et al. [159], who found that L. monocytogenes
reached levels of at least 108 CFU/g in inoculated samples of retail lamb, pork, and beef after 14
days at 7°C. L. monocytogenes exhibited both a 2-day lag period and a slower rate of growth in
beef and pork than in lamb. Such variations in growth rate might be related to differences in
concentrations of various amino acids (particularly lysine, serine, and valine, reportedly essential
for growth of L. monocytogenes), as first suggested by Khan et al. [144] in 1973. A survey of
microbiological contamination of retail pork products by Duffy et al. [76] revealed that L. monocytogenes
was detected in 26.7 and 19.8% of plant and retail samples, respectively, with contamination was
found more often in ground products.
In comparing methodologies for detecting Listeria spp. and L. monocytogenes in pork, Kanuganti
et al. [141] compared two methods, i.e., an enrichment and subculturing method (method I, UVMII)
versus an enrichment followed by plating on PALCAM agar (method II, PALCAM). Results showed
that PALCAM was slightly more sensitive for detecting L. monocytogenes in hog intestine (chitterlings)
samples (8.3% in method I vs. 9.3% for method II). In a sampling of 1849 swine tissues, 16 and 44
samples were found to be contaminated with L. monocytogenes using methods I and II, respectively.
The researchers also used these methods for detecting L. monocytogenes in ground pork and from
small intestines (raw chitterlings, 300 samples) from three plants and one retail grocery store. Out
of 340 samples of ground pork, 152 (45%) and 171 (50.2%) yielded L. monocytogenes, using
methods I and II, respectively. Interestingly, although one plant had a high rate of contamination
(73 and 80% contamination according to the respective methods), the highest rate was at the retail
level, i.e., 85 and 92%, for methods I and II. Raw chitterlings were not sampled from retail
establishments.
COOKED AND RTE MEATS

Cured Ham
Investigators in Europe and the United States determined behavior of L. monocytogenes in ham
during processing and extended storage. Working in The Netherlands, Stegeman et al. [212]
examined the thermal resistance of listeriae in experimentally produced hams to which 2 or 3%
NaCl and 120 or 180 ppm sodium nitrite were added during manufacture. After inoculating the
product with ~104 CFU/g of L. monocytogenes, all hams were canned, heated to an internal
temperature of 68.9–71.0 (or 64.0°C) within 5 h to simulate normal and underprocessing conditions,
respectively, cooled, and sampled after 5 days of storage at 4°C. Three different enrichment
procedures failed to recover viable listeriae from any samples.
Results from Stegeman et al. [212] appear to indicate that standard thermal treatments are more
than sufficient for producing Listeria-free ham. However, once removed from protective packaging,
all cooked/RTE meats can become contaminated with listeriae during slicing and further handling.
To simulate postprocessing contamination, Glass and Doyle [100] inoculated the surface of com-
mercially produced ham slices as well as five other meat products (further discussion follows) to
contain approximately 0.2 or 500 CFU/g of L. monocytogenes. All samples were then vacuum-
packaged and periodically examined for numbers of listeriae during prolonged incubation at 4.4°C.
Regardless of the original inoculum, L. monocytogenes attained populations of 105–106 CFU/g on
organoleptically acceptable ham (pH 6.3–6.5) after 4 weeks of refrigerated storage, indicating that
manufacturers cannot rely on the combination of VP and refrigeration for control of listeriae on ham.
Samelis and Metaxopoulos [198] examined the incidence and main sources of Listeria spp.
and L. monocytogenes in processed meats and a meat processing plant. The results showed that
many of the tumblers were heavily contaminated, because the need to operate continuously pre-
vented proper cleaning and disinfection. Listeriae were found to survive in tumbled meats that were
cooked in boilers, where the core temperature was below 70°C.
Cooked Roast Beef
Glass and Doyle [100] evaluated the potential for growth of L. monocytogenes on the surface of
vacuum-packaged samples of cooked roast beef having initial pH values of ∼5.9. Unlike sliced
ham, cooked roast beef supported far less growth of listeriae, with populations increasing 2 orders
of magnitude on organoleptically acceptable product after 4 weeks of refrigerated storage. Slower
growth of the pathogen on precooked roast beef correlated well with a decrease in product pH to
values of ≤5.15 in 4-week-old samples.
Luncheon Meats
Because previous studies found that luncheon meat, cooked ham, and cooked turkey breast meat were
the most frequently contaminated cooked meat products in The Netherlands, these products were used
in a study to determine survival and growth of L. monocytogenes [38]. Products were inoculated with
low levels (10 CFU/g) of the organism and stored under vacuum or in an atmosphere of 30% CO2/70%
N2 at 7°C for 4–6 weeks. Growth on vacuum-packaged product was similar to that on modified-
atmosphere-packaged stored meats, with counts increasing up to 108 CFU/g after 35 days. High
numbers of lactic acid bacteria were present, but did not affect growth of the pathogen. However,
Listeria decreased in number on saveloy (fermented sausage, pH 5.5–5.7) and raw Coburger ham
(pH 4.3–4.5), most likely from the acidity of these products.
Grau and Vandelinde [109] examined growth of L. monocytogenes on both naturally contam-
inated and artificially inoculated processed corned beef and ham. For corned beef stored at 0°C,
L. monocytogenes grew at about half the rate of the other microflora, whereas at 9°C, both groups
of organisms grew at similar rates. Similarly, on ham stored at 5°C, the organism grew at one-third
the rate of the other flora. The composition of the meats, i.e., pH, salt, and residual nitrite, played
a role in determining the growth potential of the organism. For example, at 0°C, the organism did
not grow on ham containing 170 ppm residual nitrite, but did grow on ham with 11 ppm nitrite.
Similarly, although the organism grew at similar rates on ham stored at 15°C, as the storage
temperature decreased, the organism grew slower on ham containing the higher level of residual
nitrite. The organism grew fastest on corned beef of pH 6.2, aw 0.97, and <5 ppm nitrite, and slowest
on ham of pH 6.6, aw 0.97, and 170 ppm nitrite. From inoculated studies, equations were developed
to describe the growth of both L. monocytogenes and other flora, i.e., lactic acid bacteria and
Brochothrix thermosphacta on both ham and corned beef. For naturally contaminated corned beef,
good agreement was obtained between predicted and actual growth of the organism. As predicted,
L. monocytogenes was not able to grow on naturally contaminated ham stored at 0.1°C, although
slight growth (i.e., from approximately 0.3 CFU/g to 6.2 CFU/g) of the organism was observed on
product stored at 4.8°C.
Juneja et al. [138] examined the potential for outgrowth of various foodborne pathogens from
cooked ground beef during cooling from 54.4 to 7.2°C within 6, 9, 12, 15, 18, or 21 h. Ground
beef was first inoculated with L. monocytogenes at levels of around 103 CFU/g; the meat was then
heated in a linear fashion to 60°C within 1 h, and then cooled as described earlier. The organism
was not detected in any of the beef samples examined. Presumably, the slight heating and cooling
regime was sufficient to reduce levels of the organism below the detectable level, levels at which
the organism remained during the duration of the cooling period [138].
The fate of L. monocytogenes on unirradiated and irradiated cook-chill roast beef and gravy
was examined at 5 and 10°C [107]. The organism grew well on both products, increasing 5 logs
in number on unirradiated beef and gravy over 15 days at 5°C, and by 6 logs in irradiated products
stored at 10°C for 23 days. Although the observed lag phase of L. monocytogenes was longer on
irradiated as compared to unirradiated product, the specific growth rates were similar at each storage
temperature, suggesting that the background microflora on the beef and gravy did not interfere with
growth of L. monocytogenes. The authors concluded that there would be no increased risk of
listeriosis if cook-chill roast beef and gravy were to be irradiated with 2 kGy [107]. The organism
grows well on cooked beef (Table 13.10) [123]. Interestingly, the organism had similar growth rates
aerobically and anaerobically, although the authors claimed that samples stored under aerobic
conditions probably became anaerobic during the course of the experiment [123].
L. monocytogenes was able to survive pasteurization (e.g., 91 and 96°C for 3 or 5 min) of
precooked beef roasts and then grow when products were stored at 4 and 10°C for up to 56 and
12 days, respectively [113]. Products stored at 10°C supported much better growth of the surviving
listeriae than those stored at 4°C, i.e., at 10°C L. monocytogenes reached levels in 12 days that
took up to 8 weeks to attain at the lower storage temperature. It is well known that the organism
can repair itself much better at higher temperatures. Van Laack et al. [223] examined the effect of
three packaging treatments, i.e., vacuum-packaged directly after hot boning (hot-packaged),
TABLE 13.10
Generation (GT) and Lag Times (LT) for L. monocytogenes in Meats
Food Temperature (°C) GT (h) LT (h) Reference
Roast beef −1.5a 100.0 173.7 125

3a 26.7 59.0 125
3b 80.9 477.1
Corned beef 0 110.0 109
Cooked meat 5 44–61 210
Ham 5 33.2 109
10 13.4 109
15 6.1 109
Cooked beef 5 18.6–22.6 80.6–83.4 123
10 8.5–9.0 22.6–30.4 123
Pâté 7 19.7 48 68
12.5a 1.6 24
Pâté 10a 9.12 27.6 89
Pork belly, water pH 7.1–7.3 5 15.2 <1 224
Pork belly, 1% LADc pH 7.0 5 17.1 <1 224
Pork belly, 2% LADc pH 7.0 5 23.4 1 224
Pork belly, 5% LADc pH 7.0 5 27.6 4 224
aVacuum packs.
bCO2 packs.
cLactic acid decontaminated.
Source: Adapted from Carlin, F., C. Nguyen-the, and A.A. Da Silva. 1995. Factors affecting the
growth of Listeria monocytogenes on minimally processed fresh endive. J. Appl. Bacteriol. 78:
636–646; Hudson, J.A., S.J. Mott, and N. Penney. 1994. Growth of Listeria monocytogenes,
Aeromonas hydrophila, and Yersinia enterocolitica on vacuum and saturated carbon dioxide
controlled atmosphere-packaged sliced roast beef. J. Food Prot. 57: 204–208; Snyder, O.P. 1996.
Use of time and temperature specifications for holding and storing food in retail food operations.
Dairy, Food, Environ. Sanit. 16: 374–388.
vacuum-packaged after chilling for 1 day (cold-packaged), or unpackaged, on survival of L.

monocytogenes on pork loins stored at 1°C for up to 9 days (Table 13.11). Numbers of the
organism increased by about 1 log, demonstrating the ability of the organism to grow on raw
refrigerated pork in the presence of numerous competitors. Although a statistical analysis of the
data was not done, the type of packaging did not appear to greatly influence growth.
The issue of whether L. monocytogenes can grow on raw or cooked meats is complex. It is
evident that factors such as pH, aw , background microflora, sodium nitrite and NaCl, length and
temperature of storage, strain characteristics, and product history all play a role in determining the
fate of Listeria on a meat surface. Another seemingly important factor that is often overlooked is
the initial inoculum on the product. Although early work from modeling experiments done in broth
suggested that the initial numbers of organisms present on a food surface had little or no influence
on subsequent outgrowth and growth rate, some research with naturally contaminated product does
not substantiate this theory. For example, Farber and Daley [84] found that when L. monocytogenes
was present in very low numbers on meats such as sliced ham, turkey breasts, wieners, and pâté
stored at 4°C, the pathogen did not increase in number. Similar results have been observed with
foods other than meats and poultry [Farber, unpublished results].
TABLE 13.11
Influence of Packaging Treatment (Hot Packaging [HP], Cold
Packaging [CP], and Cold Unpackaged [CU]) on Numbers of
L. monocytogenes on Pork Loins during 9 Days of Storage at 1 ± 1°C
Average log10 CFU/cm2
Sampling Day Packaging Treatment Trial 1 Trial 2
0 HP 2.68a 2.42 (8/8)

(5/8)b
CP 2.48 (5/8) 2.86 (8/8)
CU 2.65 (6/8) 2.86 (8/8)
1 HP 2.98 (6/8) 2.86 (8/8)

CP 2.48 (5/8) 2.86 (8/8)
CU 2.91 (7/8) 2.73 (8/8)
2 HP 3.36 (8/8) 3.11 (8/8)

CP 2.68 (5/8) 2.77 (7/8)
CU 2.64 (6/8) 2.91 (7/8)
5 HP 3.77 (7/8) 3.11 (8/8)

CP 2.92 (7/8) 3.36 (8/8)
CU 2.73 (8/8) 3.19 (7/8)
9 HP 3.73 (8/8) 3.23 (8/8)

CP 3.19 (7/8) 3.61 (7/8)
CU 3.48 (5/8) 3.23 (5/8)
aAverage of MPN values from positive samples.
bThe number between parentheses indicates the proportion of samples from which L. mono-
cytogenes was recovered.
Source: Adapted from Van Laack, R.L.J.M., J.L. Johnson, C.J.N.M. Van der Palen, F.J.M.
Smulders, and J.M.A. Snijders. 1993. Survival of pathogenic bacteria on pork loins as influ-
enced by hot processing and packaging. J. Food Prot. 56: 847–851.
Midelet and Carpentier [178] examined the transfer of microorganisms from various materials
to beef and discovered that L. monocytogenes biofilms had a higher attachment strength to polymers
than other bacteria, and it is these biofilms that are the highest source of contamination. The higher
the biofilm population, the higher the CFU detached. L. monocytogenes appears to have the ability to
persist on meat processing equipment, as evidenced by Lunden et al. [161]. A dicing machine that
diced cooked meat products was the source of L. monocytogenes contamination, as revealed through
molecular typing with pulsed-field gel electrophoresis (PFGE). The contamination was traced from
one plant to another, and the dicing machine that was transferred between plants was implicated
as the source.
UNFERMENTED SAUSAGE
Even though potentially contaminated raw meats find their way into enormous quantities of sausage
products, with over 200 varieties manufactured in the United States alone, no information pertaining
to behavior of L. monocytogenes during manufacture and storage of these popular meat products
appeared in the scientific literature before 1988. Although the California listeriosis outbreak of
1985 eventually led to the aforementioned surveys in which Listeria was detected in raw and
processed meats, including sausage, the early consensus was that consumption of such products
did not pose a serious threat of contracting listeriosis, as shown by a lack of any confirmed cases
of meatborne listeriosis. However, this situation changed in December 1988, following the report
of a breast cancer patient who developed listerial meningitis and eventually died after consuming
turkey frankfurters that were contaminated with L. monocytogenes.
Unfermented sausages are best classified according to the following five categories, which are
based on the method of manufacture: (1) fresh sausage (e.g., fresh pork sausage, bratwurst), (2)
cooked sausage (e.g., liver sausage, Braunschweiger), (3) cooked smoked sausage (e.g., frankfurters,
bologna), (4) uncooked smoked sausage (e.g., Mettwurst, smoked country-style pork sausage,
Kielbasa), and (5) cooked meat specialty items (e.g., head cheese). Research efforts have dealt
primarily with the behavior of Listeria in sausages belonging to the first three categories.
Fresh Sausage
Although fresh pork sausage is by far the most widely manufactured fresh sausage, this category
also includes other well-known varieties such as fresh Italian, breakfast, and beef sausage as well
as fresh bratwurst, Thuringer, and bockwurst. The latter two are most popular in Germany. All
varieties of fresh sausage are normally prepared from coarse or finely comminuted pork, beef, or
veal to which water is added along with an array of spices that varies with the type of sausage to
be produced. In the United States, certain varieties of fresh sausage also may contain binders and/or
extenders (e.g., cereal, vegetable starch, nonfat dry milk, and dried whey) at levels not exceeding
3.5% by weight. After being stuffed into natural or artificial casings, the product is twisted and cut
to form individual sausage links, which are cooled rapidly to preserve freshness and flavor. Unlike
cooked and fermented sausages, fresh sausages have a short shelf life and must be constantly
refrigerated to prevent growth of spoilage organisms, including lactic acid bacteria and micrococci.
When commercially prepared fresh bratwursts were surface-inoculated to contain approxi-
mately 0.1 or 600 L. monocytogenes CFU/g, vacuum-packaged, and stored at 4.4°0 C, Glass and
Doyle [100] found that the pathogen attained populations of 106 CFU/g on organoleptically accept-
able 4-week-old bratwursts, regardless of the initial inoculum. As with ham, profuse growth of
listeriae on fresh bratwurst was attributed to a pH value >6.0, which was maintained by the product
throughout the first 4 weeks of refrigerated storage.
In another study involving fresh sausage, Hughey et al. [127] investigated the ability of lysozyme
to prevent growth of L. monocytogenes in bratwurst prepared from coarsely ground pork. After
addition of commercial bratwurst spice, distilled water was added with or without 100 ppm
lysozyme and 5 mM EDTA, a generally recognized as safe chelating agent that enhances antibac-
terial activity of lysozyme. This meat mixture was then inoculated to contain ~4 × 103 CFU/g of
L. monocytogenes and stuffed into natural hog casings that were subsequently linked, separated,
vacuum-packaged, and stored at 5°C for 45 days.
As expected, based on the previous study, Listeria populations increased rapidly in fresh
bratwurst (pH ≅ 6.0) without added lysozyme or EDTA, reaching levels of >106 CFU/g following
10 days of refrigerated storage. L. monocytogenes also behaved similarly in bratwurst containing
lysozyme alone; however, the presence of EDTA alone resulted in a 15-day lag period, thus
preventing the pathogen from reaching populations of 106 CFU/g until nearly 30 days of storage.
In contrast, lysozyme and EDTA acted synergistically to retard growth of L. monocytogenes in
fresh bratwurst. Under these conditions, the pathogen exhibited a lag period of nearly 21 days, i.e.,
approximately 7 days beyond the normal shelf life of the product, and reached populations of
<105 CFU/g following 44 days of refrigerated storage. Although only listeriostatic, the combined
use of lysozyme and EDTA appears to be an effective means of controlling Listeria growth during
the normal shelf life of fresh bratwurst. Furthermore, once growth is prevented, low levels of
L. monocytogenes that occasionally appear in fresh bratwurst (<103 CFU/g) should be readily
eliminated by proper cooking. Glass et al. [102] looked at the use of sodium lactate and sodium
diacetate as antilisterial agents on wieners and cooked bratwurst. The results showed that dipping
wieners in lactate-diacetate solutions was not effective in controlling L. monocytogenes, but addition
of these inhibitors to weiner and bratwurst formulations limited growth for up to 12 weeks on cured,
smoked bratwurst containing 3.4% lactate and 0.1% diacetate. Sodium lactate levels of 3% and
combinations of sodium lactate and sodium diacetate prevented listerial growth on wieners stored
for 60 days at 4.5° C (Figure 13.2).
Seman et al. [203] modeled the growth of L. monocytogenes in different RTE meats by
manipulating levels of sodium chloride, sodium diacetate, potassium lactate, and product moisture
content. The cured meat products used were light bologna, wieners, smoked or cooked ham, and
cotto salami. Sodium lactate and sodium diacetate treatments (1.5 or 2.5% sodium lactate with
0.15% sodium diacetate) limited growth of the pathogen at 4°C throughout the 18-week test period
11
1.3% Lactate
2.0% Lactate
10 2.5% Lactate
3.0% Lactate
Log10 CFU/Package
9 3.5% Lactate
1% Lactate, 0.1% Diacetate
1% Lactate, 0.25% Diacetate
8 2% Lactate, 0.1% Diacetate
4
0 7 14 30 45 60
Time (d)
FIGURE 13.2 Inhibition of L. monocytogenes by sodium lactate and sodium diacetate on cured, smoked
weiners stored at 4.5°C. Population numbers reported are averages for triplicate packages. (Adapted from
Glass, K.A., D.A. Granberg, A.L. Smith, A.M. McNamara, M. Hardin, J. Mattias, K. Ladwig, and E.A.
Johnson. 2002. Inhibition of Listeria monocytogenes by sodium diacetate and sodium lactate on wieners and
cooked bratwurst. J. Food Prot. 65: 116–123.)
Lm Count (log10 CFU/g)
Time (weeks)
FIGURE 13.3 Plot of L. monocytogenes growth on slices of light bologna inoculated with a five-strain L.
monocytogenes cocktail over 18 weeks of storage at 4°C. Controls contain no potassium lactate or sodium
diacetate. T2 contains 1.5% potassium lactate solids and 0.15% sodium diacetate. T3 contains 2.5% potassium
lactate solids and 0.15% sodium diacetate. (Adapted from Seman, D.L., A.C. Borger, J.D. Meyer, P.A. Hall,
and A.L. Milkowski. 2002. Modelling the growth of Listeria monocytogenes in cured ready-to-eat processed
meat products by manipulation of sodium chloride, sodium diacetate, potassium lactate and product moisture
content. J. Food Prot. 65: 651–658.)
(Figure 13.3). The model provided a good approximation of the actual situation, but the authors
acknowledge that these results are very specific to these products, and there would likely be different
results in other meat systems.
Cooked Smoked Sausage
This group of sausages, which includes the ever-popular frankfurter (hot dog) as well as bologna
and various luncheon meats, is prepared from mixtures of comminuted beef and/or pork to which
salt, sugar, sodium nitrite, and spices are normally added. When making frankfurters, this meat
and ingredient mixture, commonly referred to as the sausage emulsion, is stuffed into natural or
artificial casings, which are then twisted to form sausage links. This string of frankfurter links is
cooked to an internal temperature of 71.1°C (160° F) to (1) coagulate protein, (2) fix the color,
and (3) pasteurize the product. Although not absolutely required, frankfurters and other similar
sausages are frequently hung in smoking rooms either before or after cooking. Alternatively,
commercially available liquid smoke products can be added to the sausage emulsion or applied
directly to the surface of frankfurters before or during heating. In either event, besides imparting
a pleasant smoked flavor to the finished product, some smoke components (i.e., formaldehyde,
acetic acid, creosote, and phenols with high boiling points) are actually bacteriostatic and/or
bactericidal toward many microbial contaminants. After cooking, frankfurters are carefully cooled,
packaged, and refrigerated during shipment to wholesale and retail markets. Skinless frankfurters,
which are very popular, are produced in a similar manner except that the casing is mechanically
peeled from the sausage after cooking or smoking.
Epidemiological data from the Centers for Disease Control and Prevention (CDC) showing an
apparent association between listeriosis and undercooked frankfurters prompted several studies
examining the thermal resistance of L. monocytogenes in this sausage. Zaika et al. [245] prepared
frankfurters from a sausage emulsion inoculated to contain ~108 CFU/g of L. monocytogenes. After
stuffing, all frankfurters were thermally processed (without smoke) according to a standard com-
mercial heating schedule. These USDA officials found that L. monocytogenes populations decreased
approximately 1000-fold in frankfurters that were heated to an internal temperature of 71.1°C
(160°F). Based on these data, cooking frankfurters to an internal temperature of 71.1°C would
probably eliminate maximum levels of L. monocytogenes (<103 CFU/g) that could conceivably occur
in raw frankfurter emulsions. Data gathered by the American Meat Institute in 1988 pointed to
frankfurters as being likely carriers of L. monocytogenes and also suggested that poor environmental
conditions before packaging could play a major role in contaminating the finished product [8].
Moreover, Glass and Doyle [100] reported that this pathogen can proliferate on vacuum-packaged,
artificially contaminated (~0.01 L. monocytogenes CFU/g) retail frankfurters during incubation at
4.4°C, with populations 2 to 5 orders of magnitude higher on organoleptically acceptable samples
after 4 weeks of refrigerated storage. Similarly, L. monocytogenes increased in number from 5 × 102
to 2.1 × 105 MPN/g on vacuum-packaged frankfurters stored at 4°C for 20 days. Interestingly,
noninoculated control products contained 1.2 × 102 MPN/g after the 20-day storage period, after
initially being negative for the organism [46]. In a more detailed study examining growth of L.
monocytogenes on vacuum-packaged all-beef, poultry, or beef/pork wieners at 5°C for up to 28 days,
McKellar et al. [171] found that of a total of 61 wieners analyzed, 40 (65.6%) supported growth of
L. monocytogenes. For those samples supporting growth, an average increase of 1.26 logs was
observed within a 14-day period. Initial lactic acid bacteria concentrations, as well as phenol and
nitrite levels varied considerably during storage, unlike NaCl levels. In addition, average pH levels
decreased significantly during storage by 0.19 pH units. Several statistical models were derived in
an attempt to adequately describe growth and death of the organism in all wiener samples. Although
no one model was sufficient, the best model obtained implicated initial and final lactic acid bacteria
counts and initial pH as factors that influence growth of L. monocytogenes. Prevention of Listeria
contamination and inhibition of growth is further complicated by present consumer demands for
reduced amounts of salt and preservatives, along with longer shelf life, smaller packages, and greater
convenience. All these will require the processed meat industry to develop even stricter requirements
for processing, cooking, handling, packaging, and refrigeration of products.
Several studies were initiated by the food industry to examine the feasibility of using heat to
eliminate L. monocytogenes from the surface of finished frankfurters. In one such study [10],
frankfurters were dipped in a broth culture of L. monocytogenes (106–108 CFU/mL) to simulate
postprocessing contamination. Listeria populations on the surface of the frankfurters decreased
only 100-fold after 8 min of heating at 86.1–87.8°C (187–190°F). Furthermore, this heat treatment
rendered the sausages organoleptically unacceptable for most consumers. Hence, “postprocess
pasteurization” may not be a viable means of eliminating L. monocytogenes from the surface of
frankfurters that have been contaminated after manufacture.
Additional efforts to control Listeria contamination on the surface of frankfurters have focused
on the bactericidal properties of commercially available liquid smoke products. In one study [177],
beef frankfurters were immersed in a culture containing 1 × 103 CFU/mL of L. monocytogenes,
removed, thoroughly air-dried, and then dipped in full-strength commercially available liquid smoke
solution (CharSol C-10). Although Listeria populations remained unchanged in control frankfurters
that were dipped in phosphate buffer, vacuum-packaged, and analyzed after 72 h at 4°C, numbers
of listeriae decreased 60 to ≥99.9% 15 min after the frankfurters were treated with liquid smoke.
Furthermore, the pathogen was never detected in smoke-treated sausage following 72 h of refrig-
erated storage. Even though dipping frankfurters in full-strength liquid smoke eliminated L.
monocytogenes, this treatment produced an extremely intense smoky-flavored product that would
likely be organoleptically unacceptable to most consumers.
Preliminary results from additional experiments dealing with the antilisterial effects of less
concentrated liquid smoke solutions were reported by Wendorff [235]. Initially, beef frankfurters
were dipped into a concentrated broth culture of L. monocytogenes, thoroughly dried, dipped into
aqueous liquid smoke solutions containing 10–40% CharSol C-10 or Poly-10 (concentrations
normally used in frankfurter production) and then analyzed for listeriae after 72 h of refrigerated
storage, using the Gene-Trak DNA Hybridization assay. Although L. monocytogenes was eliminated
from the surface of frankfurters dipped in 40% solutions of CharSol C-10 or Poly-10, the pathogen
was still detected on frankfurters treated with 10 and 25% solutions of CharSol C-10.
Demonstrating that these liquid smoke compounds lost their activity against Listeria on the
surface of frankfurters when added directly to sausage emulsion before stuffing, Wendorff [235]
examined the ability of liquid smoke compounds to inactivate listeriae on surface-inoculated skinless
frankfurters that were sprayed with five levels of CharSol Poly-10 and CharSol Supreme (twice
the strength of CharSol C-10) just before VP (Table 13.12). When used at organoleptically accept-
able concentrations, CharSol Supreme was more effective than CharSol Poly-10, with Listeria
populations on the surface of frankfurters decreasing >40% following 72 h of refrigerated storage.
When this study was repeated with a more realistic L. monocytogenes inoculum level (~103 CFU/g),
approximately 89% of the Listeria population was inactivated on frankfurters treated with CharSol
or CharSol Poly-10 at levels of 2.0 and 4.0 oz/100 lb of frankfurters, thus indicating that none of
these treatments can guarantee a Listeria-free product. Hence, to avoid contamination of finished
product, efforts must be made to develop microbial monitoring, sampling, and HACCP programs
that can effectively address problems pertaining to a lack of separation between raw and finished
processing areas, as well as procedures used to clean and sanitize the factory environment and
equipment such as grinders, mixers, and particularly sausage peelers.
In the only other reported study involving cooked smoked sausage, Glass and Doyle [100]
examined behavior of L. monocytogenes on vacuum-packaged, artificially contaminated slices of
commercially produced bologna. As was true of ham and bratwurst, the pathogen also grew well
on bologna (pH 6.1–6.4), with populations generally 3 to 4 orders of magnitude higher than initially
on organoleptically acceptable samples held 4 weeks at 4.4°C. These findings stressed the importance
TABLE 13.12
Fate of L. monocytogenes on the Surface of Beef Frankfurters Sprayed with CharSol
Poly-10 or CharSol Supreme Liquid Smoke and Stored at 4°C for 72 h
L. monocytogenes
Treatment Level (oz/100 lb Frankfurters) Initial Inoculum (CFU/g) Inactivation after 72 h (%)
Control 0 5.28 0
CharSol Poly-10 1.7a 5.30 Growth
3.4a 5.28 0
5.1a 5.16 23.6
8.5 4.84 63.1
12.0 4.67 75.2
CharSol supreme 1.8a 5.02 44.7
3.6a 5.04 42.1
5.4 4.73 71.5
9.0 4.41 86.3
12.6 4.30 89.5
aOrganoleptically acceptable concentration of liquid smoke.
Source: Adapted from Wendorff, W.L. 1989. Effect of smoke flavorings on Listeria monocytogenes in skinless franks.
Seminar presentation, Department of Food Science, University of Wisconsin–Madison, January 13.
of following good manufacturing practices, which will, in turn, greatly reduce the possibility of
listeriae contaminating RTE meats during slicing and packaging.
Uncooked Smoked Sausage
According to incidence data (Table 13.9), one variety of West European uncooked smoked sausage,
namely Mettwurst, appears to be particularly prone to contamination with Listeria spp., including
L. monocytogenes. These observations prompted a 1989 study by Trüssel and Jemmi [219], in
which an experimentally produced Mettwurst emulsion (pH 5.3) containing ~2.6% NaCl and 100 ppm
sodium nitrate was artificially contaminated with L. monocytogenes at levels of 103 or 107 CFU/g
and examined for numbers of listeriae during manufacture, after 7 days of ripening at 14°C, and
after 3 weeks of subsequent storage at 4°C. Overall, Listeria populations remained relatively
constant during manufacture and ripening, which, in turn, reflects the apparent inability of this
organism to multiply in RTE meat products having pH values <5.5. As expected from the results
of European surveys, this pathogen also survived well in Mettwurst during the product’s entire
refrigerated shelf life, with 4-week-old samples still containing approximately 102 and 106 CFU/g
of L. monocytogenes. Given these findings, it would be prudent for Mettwurst producers to review
their manufacturing practices and develop procedures for decreasing Listeria contamination during
all stages of production.
Cooked Meat Specialty Items
A significant amount of beef jerky is consumed annually in the United States. It is popular because
it is easy to prepare, lightweight, and stable without refrigeration. However, some health concerns
have arisen with this product, and outbreaks of salmonellosis have been reported [54]. A recent study
examined the fate of several foodborne pathogens, including L. monocytogenes, during preparation
and storage of beef jerky [114]. Half of the inoculated beef loin strips tested were marinated at 4°C
overnight and then dried at 60°C for 10 h. The remaining half of the samples were first heated in
marinade to 71.7°C, and then dried. L. monocytogenes populations decreased by 1.8 and 6.0 logs
after 3 and 10 h of drying, respectively. Cooking to 71.7°C before drying led to a 4.5-log decrease
in numbers of the organism, with a further 2-log reduction in numbers occurring during the 10-day
drying period. After 8 weeks of storage at 25°C, none of the pathogens (E. coli O157:H7, Salmonella
Typhimurium, and L. monocytogenes) were recovered from the beef jerky [114].
Much attention has been drawn to pâté as it has been incriminated in at least two foodborne
outbreaks. There is still controversy in the literature regarding the growth potential of L. monocy-
togenes on pâté. De Boer and van Netten [68] found inoculated retail pâté to be a good menstruum
for growth of the organism, which reached levels as high as >8.3 log CFU/cm2 after 7 days at
12.5°C, when the background microflora was low (<2.3 CFU/cm2). There was an inverse correlation
between growth of the organism and presence of lactic acid bacteria, i.e., when high numbers of
lactic acid bacteria were present, the pH was low (average of 4.9), and L. monocytogenes increased
slightly or decreased in numbers during 1 week of storage at 7° or 12.5°C. Morris and Ribeiro
[180] also found that L. monocytogenes could grow on some naturally contaminated pâtés with
refrigerated storage for 21 days, yielding levels as high as 2 × 108 CFU/g, whereas on other samples,
growth was not evident. Other investigators found that L. monocytogenes could not multiply on
retail inoculated pâté stored at 4°C for up to 3 weeks [84]. To more fully assess the health hazard
posed by L. monocytogenes in liver pâté products, multifactorial design experiments were conducted
to examine the influence of temperature (4 and 10°C), NaCl (1 and 3%), sodium nitrite (0 and
200 ppm), sodium erythrobate (0 and 550 ppm), and spice (0 and 0.4%) on growth of the organism
on experimental pâté [89]. A total of 16 different liver pâté formulations made experimentally were
stored at 4 and 10°C for various periods of time. Analysis of variance was used to determine the
effect of the various factors on maximum growth rate. L. monocytogenes grew well on the exper-
imental pâtés, with temperature being the only factor exerting an effect on the growth rate.
The generation and lag times for L. monocytogenes on pâté are shown in Table 13.10. Potential
growth of L. monocytogenes is related to pâté composition and pH, the numbers of lactic acid
bacteria present, and the storage temperature and time.
Fermented Sausage
Sausages classified as fermented undergo a controlled lactic-acid-type fermentation, usually through

the action of a commercially produced starter culture added to the meat. Although all fermented
sausages can be further classified according to moisture content as either semidry or dry, manu-
facturing procedures for both types are generally similar until the point of drying. Fermented
sausages are normally prepared from comminuted beef and/or pork to which sugar and various
spices are added along with sodium or potassium nitrate and/or nitrite. This meat preparation,
known as a mix rather than an emulsion, is inoculated with a commercial mixture of lactic acid
bacteria, which frequently includes species of Pediococcus (particularly P. cerevisiae and P.
acidilactici), Lactobacillus, and Leuconostoc. After stuffing the inoculated sausage mix into natural
or artificial casings, the strings of sausage links are hung in ripening or “green rooms” at 27–40°C
with 80 to 90% relative humidity (RH). Within 2–3 days, sugar added to the mix is fermented to
lactic acid by the starter culture, which, in turn, decreases the pH to ~5.1 and produces the
characteristic tangy flavor found in fermented sausages. As in cheese making, controlled lowering
of sausage pH to levels near the isoelectric point of meat protein is important for proper removal
of water during later stages of sausage manufacture. Following fermentation, sausages destined to
become semidry varieties containing ~50% moisture (e.g., Cervelat-type sausages and Lebanon
bologna) are normally placed in smokehouses where they are smoked and cooked to internal
temperatures of 60–68°C. In contrast, dry sausages which will ultimately contain ~35% moisture
(e.g., pepperoni, Genoa, and Milano salamis) are moved to drying rooms (10–17°C/65–80% RH),
where they remain for various times, depending on the type and size of sausage. Some varieties
also may be exposed to cool smoke before drying; however, unlike semidry varieties, dry sausages
are never cooked. Although fermented sausages keep well because of their relatively high salt
content, low pH, low aw, and moisture content, both varieties, particularly semidry, should be stored
at refrigeration temperatures.
Semidry Fermented Sausage
The relatively severe heat treatment that semidry sausages receive during manufacture generally
has been regarded as sufficient to eliminate most commonly encountered non-spore-forming food-
borne pathogens. Hence, despite concerns regarding presence of listeriae in meat products, behavior
of L. monocytogenes during manufacture and storage of semidry fermented sausage has received
relatively little attention.
Although L. monocytogenes is unlikely to survive during the manufacture of semidry sausage,
ample opportunity exists for this pathogen to contaminate the finished product during slicing
and packaging. To simulate postprocessing contamination, Glass and Doyle [100] inoculated
slices of commercially produced, fermented semidry sausage to contain approximately 0.01 or 100
L. monocytogenes CFU/g, followed by vacuum packaging. Quantitative examination for listeriae
during prolonged incubation at 4.4°C was undertaken. Unlike ham, bologna, and frankfurters,
the pathogen failed to grow on fermented semidry sausage of pH 4.8–5.2, with populations
generally decreasing ≤10-fold on organoleptically acceptable samples after 6 to 12 weeks of
refrigerated storage.
Recognizing the likelihood of listeriae being introduced into semidry sausage during slicing or
packaging and surviving throughout the normal shelf life of the product, Cirigliano et al. [59]
investigated the possibility of eliminating L. monocytogenes from inoculated slices of German- and
Polish-type beef sausage (104–105 CFU/g L. monocytogenes strain Scott A or V7) by exposing
vacuum-packaged product to temperatures of 32.2–51.7°C (90–125°F) for up to 72 h. According

to the authors, L. monocytogenes populations failed to change in product held at 32.2°C (90°F);
however, numbers of both Listeria strains decreased approximately 100-fold after product was held
at 37.8°C (100°F) for 72 h, with a slightly faster rate of inactivation observed in Polish- than
German-type sausage. Although increasing the temperature to 43.3°C (110°F) led to elimination
of strain V7 from Polish- and German-type sausage after 8 and 48 h, respectively, strain Scott A
was not eliminated from either product until completion of a 72-h heat treatment. When exposed
to 48.9 and 51.7°C (120 and 125°F), strain V7 was inactivated in Polish- and German-type sausages
within 4 and 24 h, respectively. Although somewhat more heat resistant, strain Scott A was
eliminated from both products after 24 h at 51.7°C (125°F). In some instances, objectionable fat
losses were observed for both product types; however, the authors concluded that mild heat
treatments could be used to salvage Listeria-contaminated German- or Polish-type sausage without
seriously affecting product quality. In fermented “tea” sausages inoculated with 8 × 106 MPN/g of
L. monocytogenes, the organism decreased about 1.5 logs in number during the initial 4-day ripening
period when the pH dropped from 5.47 to 4.80, decreased another 1.5 logs in number during
the 9-day drying period, and then remained constant in number during the 20-day storage period
at 18 to 22°C [47]. Similar results were obtained for sausages inoculated with lower levels of
the organism. The initial aw value, as well as those after drying and after storage were 0.974,
0.933, and 0.861, respectively.
Dry Fermented Sausage
Unlike semidry varieties, dry fermented sausages are never exposed to temperatures that would
be expected to inactivate even small numbers of listeriae. Consequently, dry sausages have
attracted more attention, as shown by several recent studies that examined the fate of L. monocytogenes
during the fermentation, drying, and storage of hard salami, pepperoni, and different varieties
of sausage.
In the first such study, Johnson et al. [135] prepared hard salami from naturally contaminated
(i.e., meat from cows inoculated intravenously with L. monocytogenes) as well as artificially
inoculated ground beef, both of which contained ~104 CFU/g of L. monocytogenes. Glucose, spices,
sodium nitrite, and salt were added to the ground beef along with a glucose-fermenting strain of
Pediococcus acidilactici. After stuffing the mix into casings, all sausages were fermented at 40°C
for 24 h, dried at 13°C for 9 days, vacuum-packaged in gas-impermeable film, and stored at 4°C
for 12 weeks. Listeriae populations decreased approximately 10- and 100-fold during fermentation
(40°C/24 h) of hard salami prepared from naturally contaminated and artificially inoculated ground
beef, respectively. Inactivation of listeriae during the fermentation appeared to be primarily attrib-
utable to production of lactic acid (or other metabolites) by the starter culture, with pH values
decreasing from approximately 5.7 to 4.4 by the end of fermentation. Although numbers of listeriae
remained relatively constant in naturally contaminated hard salami following 9 days of drying
(13°C/65% RH), listeriae populations decreased nearly 100-fold during drying of product prepared
from artificially inoculated ground beef. L. monocytogenes was detected in both products during
8 weeks of refrigerated storage. Higher levels of listeriae were recovered from naturally contami-
nated rather than artificially inoculated hard salami, suggesting that the behavior of this pathogen
is best studied using sausage prepared from naturally contaminated rather than artificially inoculated
ground beef. Compositionally, both products were very dry, having aw values of 0.79–0.81 as
compared to ~0.91 for commercially produced hard salami. Although L. monocytogenes might be
expected to survive more readily in higher-moisture commercial products, growth of the pathogen
in retail hard salami appears unlikely given the presence of 5–7% NaCl and 100–150 ppm sodium
nitrite, combined with a pH of 4.3–4.5 and a relatively low storage temperature.
Trüssel and Jemmi [219] reported a different finding, in that L. monocytogenes populations in
salami prepared from a mix inoculated to contain approximately 103 or 107 CFU/g of L. monocytogenes
decreased ≤10-fold in product of pH ≤5.6 during 7 days of ripening at 12–22°C/82–95% RH. After
8 weeks of drying at 10–17°C/78–82% RH, numbers of listeriae in salami of pH 5.4–5.7 decreased
to <10 CFU/g, regardless of the initial inoculum. However, when an enrichment procedure was used,
the pathogen still could be detected in these sausages after an additional 6 to 11 weeks of drying at
10–17°C/35–50% RH to aw values of 0.68–0.69. Thus, as was true of certain fermented dairy products
(see Chapter 12), small numbers of L. monocytogenes cells can also persist in fermented dry sausages
for at least 14–19 weeks. Farber et al. [88] examined the fate of L. monocytogenes during production
of uncooked German, American, and Italian-style fermented sausages. Similar results were obtained
with both the German and American sausages, in which levels of the organism decreased about 2
to 3 logs after fermentation and smoking and a further 1 to 2 logs after drying. This contrasted with
how L. monocytogenes behaved in Italian-style fermented sausages, the only types not made with
starter culture. In these latter sausages, L. monocytogenes increased slightly during the fermentation
period, remained constant in number during drying, and then decreased slightly during the 4-week
holding period at 4°C. This study points to the importance of using starter cultures during production
of dry fermented sausages, and also emphasizes the necessity of having a well-designed and operating
HACCP plan (plus GMPs) for each style of sausage made.
Work also has been done to examine the fate of L. monocytogenes in fermented sausage made
from poultry meat inoculated with approximately 104 CFU/g followed by fermentation at 20°C.
The sausages were formulated to contain two levels (2.0 and 2.5%) of sodium nitrite and glucono-
delta-lactone (GdL). Starter cultures were added to some formulations. L. monocytogenes survived
the 28-day ripening period, with numbers decreasing by about 1 log in those formulations containing
2.5% sodium nitrite plus either GdL or starter culture, and staying constant in the other batches
(one exception was the batch containing 2% sodium nitrite, where a 2-log decrease in numbers
was observed). It would appear that the hurdles, consisting of preservatives, starter culture, aw
(0.90–0.92), and pH (final 4.8–5.9) were not sufficient to inactivate the listeriae cells present [168].
Recognizing that L. monocytogenes may survive the typical process used to manufacture hard
salami, Glass and Doyle [101] attempted to identify various heat treatments that could be used to
inactivate L. monocytogenes during manufacture of dry fermented sausage. Work with “beaker
sausage” prepared from ground beef/pork containing 3.5% salt, 103 ppm sodium nitrite, and
approximately 5 × 103 CFU/g of L. monocytogenes indicated that numbers of listeriae decreased
>10-fold following an active fermentation (32.2°C/16 h) with P. acidilactici during which the pH
decreased from approximately 6.3 to 4.8. Prolific growth of L. monocytogenes in a similar lot of
beaker sausage prepared without P. acidilactici confirms the importance of an active starter culture
in preventing growth of listeriae in fermented sausage. Furthermore, these findings suggest that
3.5% salt and 103 ppm sodium nitrite are of virtually no value in preventing growth of listeriae in
sausage mix. Subsequent holding of fermented beaker sausage at 46.1°C for 8 h or heating to an
internal temperature of 51.7 or 57.2°C failed to eliminate listeriae, with the pathogen still present
in all samples examined according to the USDA enrichment method. Although holding samples at
51.7°C for 8 h or 57.2°C for 4 h reduced Listeria populations >100-fold, enrichment data indicated
that the pathogen was still present in either of two samples that received both heat treatments. Only
after heating beaker sausage to an internal temperature of 62.8°C was the pathogen no longer
detected either by direct plating or enrichment.
Subsequently, Glass and Doyle [101] investigated the fate of. L. monocytogenes in pepperoni
during normal processing and storage and during heating to an internal temperature of 51.7°C for
4 h immediately after fermentation or drying. After inoculating commercially prepared pepperoni mix
to contain 104 CFU/g of L. monocytogenes, populations of listeriae decreased approximately 100-fold
following fermentation (35.6°C/12 h) by P. acidilactici, which caused the pH to decrease from 6.0
to 4.7. These findings are similar to those of Johnson et al. [135], who found that Listeria populations
decreased 10- to 100-fold during fermentation of hard salami. Following 5 days of drying at 12.8°C,
numbers of listeriae decreased to <10 CFU/g in normally processed pepperoni; however, with the
USDA enrichment procedure, L. monocytogenes could still be detected in 82-day-old refrigerated
samples of vacuum-packaged pepperoni. Heating the same pepperoni to an internal temperature of

51.7°C between fermentation and drying had relatively little effect on L. monocytogenes, with viable
populations decreasing only about 10-fold. Although holding pepperoni for 4 h at 51.7°C reduced
Listeria populations to undetectable levels (using direct plating and enrichment), the pathogen was
sporadically recovered with the USDA enrichment procedure from 5- to 22-day-old sausage. Subse-
quent holding of the same pepperoni (pH 4.6) at an internal temperature of 51.7°C for 4 h immediately
after 26 days of drying at 12.8°C led to complete inactivation of the pathogen as determined by direct
plating and enrichment procedures. Additional experiments conducted on pepperoni containing
5.3 × 103 CFU/g of L. monocytogenes after 19 days of drying verified that a minimum heat treatment
of 4 h at 51.7°C was required to obtain a Listeria-free product. Thus, although normal processes used
to manufacture pepperoni are not sufficient to eliminate L. monocytogenes from heavily contaminated
product, holding pepperoni and possibly other dry sausages at an internal temperature of 51.7°C for
at least 4 h may prove to be a viable means of salvaging contaminated product.
The antibotulinal properties of nitrate, and particularly nitrite, have been recognized for years,
yet current scientific literature contains little information concerning the effect of these preservatives
on Listeria behavior in dry fermented sausage. Junttila et al. [140] examined the ability of
L. monocytogenes to survive in dry Finnish sausage containing various levels of potassium nitrate,
sodium nitrite, and salt. All sausage was prepared from a mixture of ground beef and pork to
which sugar, spices, and 3 or 3.5% salt were added along with 50 to 1000 ppm potassium nitrate
and/or sodium nitrite. After inoculation to contain approximately 105 CFU/g of L. monocytogenes
and a starter culture consisting of Staphylococcus carnosus and Lactobacillus plantarum, the
sausage mix was stuffed into casings. All sausage links were fermented 2 days at 23°C, smoked 5
days at 20–22°C and dried 1 week each at 18 and 10°C. Listeria monocytogenes populations in
sausage containing commonly used levels of salt (3.0%) and sodium nitrite (120 ppm) decreased
1.14 orders of magnitude over 21 days. Similar findings also were reported with 3.5% salt.
Increasing levels of sodium nitrite (200 ppm) and potassium nitrate (330 ppm) to those commonly
used 30 years ago led to a somewhat faster inactivation of listeriae in dry fermented sausage, with
inactivation again most pronounced during the later stage of drying. Over the same 21-day period,
Listeria populations decreased approximately 3.3 orders of magnitude in sausage containing 3.5%
salt and 1000 ppm potassium nitrite; however, this concentration of potassium nitrate is no longer
permitted in dry fermented sausage. Growth of L. monocytogenes in this product was apparently
suppressed by the combination of salt, sodium nitrite, and a pH of 4.7; however, given the pathogen’s
known tolerance to salt, acid, and low temperatures, addition of commonly used levels of sodium
nitrite to fermented sausage was only marginally effective in inactivating listeriae.
Samelis et al. [200] did a microbiological ecology study regarding the stability and safety of
traditional Greek salami. All of the batches of fermented sausages had naturally occurring Listeria
spp., which disappeared over time. The results showed that although 60% of the incoming materials
had listeriae, L. monocytogenes was completely eliminated from the sausages 7 days after fermen-
tation. The study used local traditional starter cultures rather than the premanufactured cultures as
they are typically produced in northern Europe, which may be appropriate for that climate, but the
climate in Greece is very different, and consequently the microbiological components of the starter
culture would be different. Thus, although this study and others have provided valuable information
concerning the behavior of L. monocytogenes in sausage products, an understanding of inter-
actions between various factors such as starter cultures, food additives, and various heat treat-
ments is still needed to develop suitable methods to eliminate L. monocytogenes from fermented
sausage and other processed meat products.
MODIFIED-ATMOSPHERE (MA) PACKAGING

Modified-atmosphere packaging (MAP) can extend the shelf life of many perishable foods including
meats and poultry. The CO2-enriched atmosphere that is created within a meat pack can inhibit the
normal spoilage flora and select for organisms such as the lactic acid bacteria [82]. Concerns have
been raised about the ability of L. monocytogenes to outgrow the normal spoilage flora on MA-
packaged foods. In addition, the relatively long shelf life of MA-packaged foods can allow psy-
chrotrophic foodborne pathogens such as L. monocytogenes to grow to high levels. As seen earlier,
the organism can grow on vacuum-packaged meats including beef, lamb, and pork. The effects of
intermediate to high levels of CO2 on the survival and growth of the organism on meats and poultry
are not clear [91].
Beef
Growth of L. monocytogenes was observed on samples of vacuum-packaged high-pH (>6.0) beef

stored at 0, 2, 5, and 10°C, but not on those vacuum packs stored at −2°C. In general, rather long
lag periods were observed, and the organism grew at a slower rate than spoilage flora [81]. The
organism only grew in CO2 packs stored at 10°C, and not at any of the lower storage temperatures
tested. However, when normal ultimate pH beef (pH 5.3–5.5) was used as the food menstruum, L.
monocytogenes was not able to grow on beef stored in CO2 packs at 5 or 10°C [27]. It is likely
that the lower pH of normal as compared to DFD meat, combined with the high-CO2 environment,
was sufficient to cause inhibition and a slight decline in numbers of the organism. As in the findings
of Grau and Vanderlinde [109], the organism was able to grow well on vacuum-packaged meat
stored at 5 and 10°C. It was interesting that in the study by Avery et al. [31], L. monocytogenes
grew at a faster rate than the spoilage flora on vacuum-packaged beef, which is in contrast to the
results obtained by Gill and Reichel [95]. Perhaps L. monocytogenes can compete better with the
spoilage microflora at a lower pH. A follow-up study by Avery et al. [32] was designed to study
the effects of previous high CO2 exposure of L. monocytogenes to its subsequent growth during
abusive retail display. Beef steaks of normal pH were first inoculated with the organism, individually
packaged in CO2 packs, and then stored at −1.5°C for <3 h, 5 or 8 weeks. At each of the latter
three time intervals, samples were removed, overwrapped, and placed on retail display at 12°C for
up to 140 h. Even after only a brief (<3 h) exposure to CO2 , L. monocytogenes grew slightly or
not at all during retail display, with demonstrated lag phases of >75 h. There were no comparative
controls, however, to show the growth of the organism when inoculated into normal-pH beef and
then stored at 12°C. Experiments were also done whereby steaks were removed from storage, and
then rinsed to remove some cells of L. monocytogenes, which were reinoculated into freshly cut
beef steaks to simulate cross-contamination. In this instance, inocula from steaks stored in CO2
packs for 5 or 8 weeks did not grow on the cross-contaminated steaks. It was concluded that
(1) prior exposure of beef steaks contaminated with L. monocytogenes to high-CO2 environments
will not increase the risk of growth of the organism when steaks are placed on retail display and
possibly temperature-abused, and (2) there is not a large risk of growth when cross-contamination
occurs from high-CO2 stored beef to fresh raw beef before retail display. Experiments have also
been done to examine survival and growth of the organism on sliced roast beef stored under vacuum
or CO2 at −1.5 and 3°C. The organism was not able to grow under CO2 at −1.5°C, but did grow
under all other test conditions. At 3°C, the organism grew three times faster on vacuum-packaged
as compared to CO2-stored roast beef (see Table 13.10). When growth occurred, maximum numbers
of the organism were attained only at the end of shelf life of the product.
Tsigarda et al. [220] examined behavior of L. monocytogenes as well as autochthonous flora
on sterile and naturally contaminated beef fillets stored under aerobic, vacuum, and modified-
atmosphere packaging (MAP; 40% CO2, 30% O2, and 30% N2) conditions at 5°C, with or without
the presence of oregano essential oil (Figure 13.4). In this particular study, the type of packaging
film was the critical factor for L. monocytogenes growth and survival, and for determining the
dominant microflora. L. monocytogenes increased in number when the pseudomonads dominated,
which was under aerobic storage and under MAP/VP conditions using a high-permeability film.
B. thermosphacta comprised the majority of the microflora when MAP/VP low-permeability
3.5
Log CFU/g−1 2.5
2 0% Oregano Oil
0.8% Oregano Oil
1.5
0.5
0
0 2 3 4 6 7 9 11 14 16
Time (d)
FIGURE 13.4 Growth and survival of L. monocytogenes on naturally contaminated beef fillets with 0 and 0.8%
oregano essential oil, packaged in 40% CO2, 30% O2, and 30% N2 within low-permeability film at 5°C. Each
number is the mean of two samples taken from different experiments. Each sample was analyzed in duplicate
(coefficient of variation <5%). (Adapted from Tsigarda, E., P. Skandamis, and G.-J.E. Nychas. 2000. Behaviour
of Listeria monocytogenes and autochthonous flora on meat stored under aerobic, vacuum and modified
atmosphere packaging conditions with or without the presence of oregano essential oil at 5°C. J. Appl.
Microbiol. 89: 901–909.)
films were used and there was no growth of L. monocytogenes. Oregano essential oil (0.8% v/w)
reduced the initial bacterial population by 2–3 logs, with limited aerobic growth and survival or
death of L. monocytogenes observed under both MAP and VP, in both types of film at 5°C. These
researchers discovered that use of low-permeability film controlled growth of L. monocytogenes
throughout storage, regardless of the gaseous atmospheres.
Sausages
Experiments were done to determine survival of L. monocytogenes on sliced frankfurters incubated

under 20–80% CO2 at 4, 7, and 10°C for up to 6 weeks of storage [148]. Within the commercial
minimum shelf life of 3 weeks at 4°C, L. monocytogenes populations increased in vacuum packs, 20%
CO2, and 30% CO2 by 2.5, 1, and 0.5 logs, respectively, but growth was inhibited in the presence of
both 50 and 80% CO2. Upon continued storage for another 3 weeks, the organism grew in the presence
of 50%, but not 80% CO2. However, increasing the storage temperature to 7°C allowed for growth of
the organism within the 3-week storage period even in the presence of 80% CO2. Therefore, under
commercial conditions (4 to 10°C, 3-week shelf life), only 80% CO2 was able to inhibit growth of the
organism. However, this level of CO2 caused undesirable organoleptic changes in the product, and it
was suggested that as a compromise, perhaps CO2 levels between 50 and 80% should be used [148].
Lamb
Sheridan et al. [207] examined growth of L. monocytogenes on both raw and minced lamb pieces
stored under various gas atmospheres (vacuum; 80% O2:20% CO2; 50% O2:50% CO2; and 100%
TABLE 13.13
Mean Total Aerobic (TA) and L. monocytogenes (LM) Counts
(Log10 CFU/g) at 42 Days on Lamb Pieces and Mince Packaged
in Different Gas Atmospheres at 5°C
Meat Type Pieces Mince
TA LM TA LM
Gas atmosphere
Air 8.86 6.07 9.2 5.37
Vacuum pack 7.53 3.81 8.65 1.74
80% O2/20% CO2 8.91 4.91 8.97 2.68
50% CO2/50% N2 8.15 4.22 7.75 3.68
100% CO2 7.55 1.35 8.07 0.58
Although not clearly stated, the initial total count appears to be 2.8 × 101 CFU/g.
a
Source: Adapted from Sheridan, J.J., A. Doherty, P. Allen, D.A. McDowell, I.S. Blair, and
D. Harrington. 1995. Investigations on the growth of Listeria monocytogenes on lamb
packaged under modified atmospheres. Food Microbiol. 12: 259–66.
CO2) at 0 and 5°C. In all instances, the organism did not grow on lamb stored at 0°C. Moreover,
at 5°C under vacuum, L. monocytogenes grew on lamb pieces, but not on minced lamb (Table 13.13).
After 42 days of storage at 5°C, besides the air-stored samples, the organism grew best on lamb
and mince pieces stored under 80% O2:20% CO2 and 50% O2:50% CO2, respectively. Again, L.
monocytogenes did not grow on lamb stored under 100% CO2.
Pork
In studies done to examine the microbial ecology of fresh MAP pork stored at various temperatures,
listeriae were found to be one of the predominant organisms on product stored at −1°C, but not on
samples stored at 4.4 or 10°C [174]. In fact, most of the flora, producing bacteriocins with a wide
spectrum of activity, were isolated from samples stored at the two higher temperatures. Growth of
the organism on fresh pork longissimus dorsi was examined. No growth of the organism was
observed at 1°C, regardless of storage atmosphere. At 7°C storage, L. monocytogenes grew on the
air-stored samples, but not on those stored under 100% N2, 80% O2:20% CO2, or 60% O2:40%
CO2 [166]. No additional hazards could be observed by using modified atmosphere (MA) for
packaging pork of normal pH. Although no results were actually presented, Davies [67] found that
under an atmosphere of 80% O2:20% CO2, growth of L. monocytogenes on cooked ham was not
greater than that in the aerobically stored controls. Manu-Tawiah et al. [167] examined the influence
of CO2 levels of 20 and 40% on growth of L. monocytogenes and Yersinia enterocolitica on fresh pork
chops stored at 4°C for 35 days. In general, levels of L. monocytogenes in air- or vacuum-packaged
pork were not significantly different from numbers in chops packaged in the gas atmospheres,
with levels increasing about 1.5 to 2.0 logs to close to 106 CFU/cm2 (initial count of approximately
3.7 CFU/cm2) after 35 days. The growth rate of the organism, and of the aerobic psychrotrophic
spoilage flora, was faster in air as compared to the gas atmospheres, and in all instances was slower
than that of the aerobic psychrotrophic spoilage flora. In contrast to the results of Wimpfheimer et al.
[237], no differences were observed in numbers of the organism on chops packed in 60% N2, 0%
O2:40% CO2, or 50% N2:10% O2:40% CO2. Y. enterocolitica grew much better than L. monocytogenes
in all gas atmospheres tested, increasing to close to 108 CFU/cm2 after 35 days at 4°C. The fact that
Yersinia grew much better in MA-packaged than air-stored chops is disconcerting from a public health
standpoint.
The application of additional hurdles in conjunction with MAP is a strategy that will be more
commonly used in the future. In an interesting study, the effect of nisin and MAP on growth and
survival of L. monocytogenes on cooked pork tenderloin stored at 4 and 20°C was examined [80].
It was found that the organism was able to grow on pork tenderloin stored under 100% CO2 at both
storage temperatures. However, when nisin (104 IU/mL) was added, the combination proved to be
bactericidal (Table 13.14). A lower concentration of nisin (103 IU/mL) also proved to be effective
for pork stored at 4°, but not at 20°C. At the same time, growth of Pseudomonas fragi, a spoilage
organism, was observed. In general, numbers of pseudomonads were reduced in MA-stored samples,
but growth of the organism was not affected by nisin [79]. The authors used the general concept
of a “safety index,” which compares relative numbers of spoilage organisms to pathogens. In MA-
packaged plus nisin-treated samples, numbers of P. fragi increased with time relative to growth of
L. monocytogenes. However, this was not observed in those samples not containing nisin. MAP
has also been used in conjunction with irradiation in an attempt to control the growth of foodborne
pathogens on raw pork [106]. In nonirradiated pork stored under an atmosphere of 75% N2:25%
CO2, the organism increased about 2 logs in number in 9 days at 10°C, whereas the irradiated
(1.75 kGy) control was at undetectable levels. Using a higher initial inoculum (106 vs. 103 CFU/g),
similar results were obtained; that is, after 9 days at 10°C, total Listeria counts were around
4.5 × 104 and 1 × 108 CFU/g in the irradiated and nonirradiated treated samples, respectively. The
benefits of using irradiation after meat packaging were evident, as during storage the lactic acid
microflora outgrew L. monocytogenes as well as some other pathogens tested [106]. It was theorized
that because Lactobacillus saké usually predominates in irradiated MA-packaged pork, sakacin A,
a bacteriocin produced by this organism, may have been responsible for inhibiting the growth of
L. monocytogenes. However, in both irradiated and nonirradiated samples, L. monocytogenes
increased in number by about the same amount. Devlieghere et al. [70] found that growth of L.
monocytogenes was not influenced when the level of lactic acid bacteria was <107 CFU/mL, but once
TABLE 13.14
Cumulative Changea in log CFU/g for L. monocytogenes on Cooked Tenderloin Stored
in MAP at 4 and 20°C
MAP
100% Air 100% CO2 100% CO2
TimeC with Nisinb with Nisin 100% Air with Nisinb with Nisin
(days)
0 104 0 104 Timed (days) 0 104 0 104
6 2.78 −2.11 0.65 –2.18 1 4.2 –2.1 1.9 –2.1

12 4.28 −2.1 2.16 –2.18 2 5.24 –2.11 4.32 –2.11
18 5.03 –2.12 4.97 –2.19 4 5.36 –2.13 5.57 –2.1
24 4.97 –2.15 4.74 –2.18 7 5.64 –2.14 5.67 –2.13
30 4.81 –2.18 4.67 –2.18 10 6.07 –2.15 5.53 –2.14
a(Log CFU/g at day x − Log CFU/g at day zero) for two samples per treatment per day. The concentration of
L. monocytogenes on the cooked tenderloin surface at day zero was 3.18 log10CFU/g.
bThe unit for nisin activity was IU/mL.
cStorage temperature of 4°C.
dStorage temperature of 20°C.
Source: Adapted from Fang, T.J., and L.-W. Lin. 1994. Growth of Listeria monocytogenes and Pseudomonas fragi on
cooked pork in a modified atmosphere packaging/nisin combination system. J. Food Prot. 57: 479–485.
numbers reached greater than 107 CFU/mL, the concentration of lactic acid increased with a
concurrent significant decrease in pH (data not shown). In this study, the researchers also compared
mathematical models for prediction of growth and actual results.
One can conclude that in most instances, L. monocytogenes will be able to grow at levels of
CO2 up to 50%. Growth of the organism at levels greater than this will depend mainly on the
interplay between the gas atmosphere, pH, temperature, and microbial competition.
Thermal Inactivation in Meats
Interest in the heat resistance of listeriae in raw meat products dates back to at least 1980 when
Karaioannoglou and Xenos [142] reported on survival of Listeria in grilled meatballs. In their
experiments, minced beef inoculated to contain 102, 103, 104, or 105 L. monocytogenes CFU/g, was
combined with eggs, bread, onion, garlic, salt, and spices, and then fashioned into meatballs
weighing 35–40 g each. Meatballs were then placed on a coal-fired grill at 110 to 120°C and cooked
for 15 min until they attained an internal temperature of 78–85°C. After grilling, L. monocytogenes
was isolated from all meatballs that originally contained 104–105 listeriae per g. However, the
pathogen was discovered in only one of four meatballs inoculated to contain 103 listeriae per g and
was absent from meatballs that originally contained 102 listeriae per g. Because data from the
European surveys indicate that retail raw beef occasionally may contain up to 103 Listeria CFU/g
(some of which are likely to be L. monocytogenes), thorough cooking of raw meat is presently
advised to eliminate L. monocytogenes as well as salmonellae, pathogenic strains of E. coli, and
other organisms that have been associated with foodborne illness. Although these findings attest to
the hardy nature of listeriae in fresh ground beef, L. monocytogenes also was equally tenacious in
artificially contaminated frozen ground beef, with populations remaining unchanged at 105 CFU/g
during 6 months of storage at −18°C.
Concern about the possible resistance of L. monocytogenes to pasteurization of milk, along
with detection of this pathogen in cooked meats, prompted interest in the possibility of its surviving
thermal processing steps commonly used to convert raw meat into RTE products. Boyle et al. [41]
investigated the thermal destruction of L. monocytogenes strain Scott A in ground beef (approxi-
mately 20% fat) by submerging sealed tubes containing ground beef with 108 listeriae per g in a
water bath at 75°C until the internal temperature of samples was 50, 60, 65, or 70°C. Samples then
were examined for listeriae by direct plating and selective and cold enrichment. According to these
researchers, Listeria levels did not decrease in samples heated to an internal temperature of 50°C
over 6.2 min. Numbers of listeriae decreased 4.4 to 6.1 orders of magnitude in samples of ground
beef during 8.4 and 10.6 min of heating to 60 and 65°C, respectively; similar results also were
reported in 1988 by Farber et al. [86]. Ground pork heated to 62°C over 25 min was sufficient to
inactivate 5.8–7.35 logs/g of L. monocytogenes, depending on the pork formulation. In general, it
was found that most of the additives such as kappa-carrageenan, sodium lactate, and algin or
calcium binders used in the ground pork formulations did not influence thermal inactivation of the
organism [240]. Similarly, Yen et al. [242] found that sodium phosphates, sodium erythorbate, and
added water had little or no effect on survival of various strains of L. monocytogenes during heating
of ground pork. Interestingly, the same authors found that although, as noticed previously [83,241],
addition of cure resulted in 2.0–2.2 logs less inactivation of the organism in ground pork cooked
to 62°C, this protective effect was only seen at temperatures below 67°C.
There have been several studies done to determine the D- and z-values of L. monocytogenes
in various ground and whole-meat products (Table 13.15). D60°C values for most products are in
the range of 1.8 to 8.3 min. Carlier et al. [51] examined destruction of L. monocytogenes during
the cooking of whole hams to an internal temperature of 58.8°C. During storage of the hams for
2 months at 9°C, survivors were found among the hams inoculated with around 4 × 105 CFU/g,
but not in those inoculated with <10 CFU/g. It was suggested that a minimum core temperature of
65°C be attained in these products, with an F70°C-value of at least 40 min. Survival of L. monocytogenes
TABLE 13.15
Heat Resistance of L. monocytogenes in Meats
Temperature D-value z-Value
Product (°C) (°C/min) (°C) Reference
Ground beef 60 3.12 5.3 86

Fermented sausage mix 60 16.7 4.6
Ground beef roast 60 4.47 201
Ground beef 60 1.62
Beaker sausage 60 9.13
Meat slurry 60 2.54 41
70 0.23
Meat slurry 60 7.3 6.8 191
Beef 60 3.8 7.2 163
70 0.14
Beef steak 60 8.32, 6.27 5.98, 5.98 92
70 0.20, 0.14
Liver sausage slurry 60 2.42 6.2 39
Lean ground beef 62.8 0.6 9.3 81
Fatty ground beef 62.8 1.2 11.4
Meats (predicted value) 60 3.82 6.8 163
70 0.13
Ham 60 1.82 5.05 50
60 3.48a 6.74 146
Ground pork 62 6.5–7.7
Ground pork 60 1.14–1.7 5.05–5.45 188
Minced beef, in vacutainer 60 0.31 40
Minced beef, in vacuum pack 60 0.15
Vacuum-packed minced beef, 60 2.87/4.39 112
late logarithmic LM pH 5.6/6.2
Vacuum-packed minced beef, 60 8.23/8.79
late stationary LM pH 5.6/6.2
Hot dog batter, composite culture, stationary phase 62 3.2 ± 0.5 5.9 169
Hot dog batter, composite culture, starvation 62 3.3 ± 0.3 6.0 169
Hot dog batter, outbreak strain, stationary phase 62 1.8 ± 0.5 5.6 169
Hot dog batter, outbreak strain, starvation 62 1.8 ± 0.6 5.2 169
Beef, pH 5.4, log, no heat shock 60 3.07 74
Beef, pH 5.4, log, heat-shocked 60 3.83 74
Beef, pH 5.4, stationary, no heat shock 60 8.65 74
Beef, pH 5.4, stationary, heat-shocked 60 7.88 74
Pork, heating 1.3°C/min 60 9.2 74
Sausage, 23% beef, 77% pork 60 9.13 10.0 74
Sausage, pork only 60 7.3 6.8 74
Minced beef in vacutainers 60 0.33 72
Minced beef in vacutainers 60 0.31 40
Minced beef in vacuum pack 60 0.15 40
Minced beef with LM NCTC11994, no prior 55 6.66 230
heat treatment
Minced beef with LM NCTC11994, prior heat 55 5.57 230
treatment
(continued)

Heat Resistance of L. monocytogenes in Meats
Temperature D-value z-Value
Product (°C) (°C/min) (°C) Reference
Minced beef with LM M63, no prior heat treatment 55 4.54 230

Minced beef with LM M63, prior heat treatment 55 3.98 230
Minced beef with LM NCTC 12480, no prior 55 5.13 230
heat treatment
Minced beef with LM NCTC 12480, prior heat 55 4.58 230
treatment
Minced beef with LM M102, no prior heat treatment 55 5.53 230
Minced beef with LM M102, prior heat treatment 55 4.57 230
Beef with 0% sodium lactate and 0.0% sodium 60 4.67 139
diacetate
Beef with 2.4% sodium lactate and 0.1% sodium 60 10.37 139
diacetate
Beef with 4.8% sodium lactate and 0.1% sodium 60 12.31 ND 139
diacetate
Franks (L. innocua) 60 5.52 183
Beef patties (L. innocua) 60 3.25 183
Beef–turkey patties 60 6.66 183
aCells heat-shocked at 42°C for 1 h.
in vacuum-packaged, nitrite-free beef roasts prepared with brines containing selected antimicrobial
agents was examined [221]. The brines used included sodium chloride, sodium tripolyphosphate,
Brifisol 414™, acetic acid, sodium lactate, Lauralac™, and potassium sorbate. Meats were cooked
in a bag once or twice to 62.8°C under conditions simulating product contamination from pumping
brines or from postcooking slicing or cutting. Some of the salient findings from this study were
that (1) survival of L. monocytogenes on the surface of the beef roasts was surprisingly high,
considering that cells had possibly been exposed to temperatures in the 80°C range for more
than 30 min; (2) regardless of the number of cookings, the survival rates for listeriae were similar
in internally cooked inoculated roasts for all brine treatments except NaCl-sodium tripolyphosphate,
giving rise to the possibility that sublethal heating may have induced a heat shock response in these
organisms; (3) the highest incidence of listeriae-positive samples was seen in those roasts processed
to most closely resemble product currently being marketed, i.e., standard NaCl-phosphate brines;
and (4) greatest destruction of the organism was observed with brines containing a phosphate blend
and sodium lactate or glycerol monolaurin in combination with one cooking, and more so with
two cookings [221].
Steam surface pasteurization of meats is gaining acceptance as a means of reducing levels of
pathogenic microorganisms on meats. Some advantages of using steam pasteurization include the
following: (1) the entire surface of a carcass can be uniformly heated, (2) irregularly shaped surfaces
can be uniformly covered, (3) wastewater accumulation is not an issue, and (4) the process is not
subject to operator misuse. A study was designed in which frankfurters inoculated with L. innocua
were steam-pasteurized in a small pasteurizer designed in-house. The heating chamber was evac-
uated for 15 sec, and then the product was exposed to steam at a set pressure until the desired
treatment time was reached. Treatment times of 32 and 40 sec at 136 and 115°C, respectively, led
to a 4-log reduction in counts of L. innocua on the meat surface, with only a slight effect on color
or weight [66]. Another study compared steam pasteurization (S; 15 sec) to traditional methods of
reducing pathogens on meat surfaces [189]. The latter methods, which were tested both individually
TABLE 13.16
Effectiveness of Combination and Individual Decontamination Treatments in Reducing
L. monocytogenes on Surfaces of Freshly Slaughtered Beef
Treatmenta Initialb Mean Red.c Treatmenta Initialb Mean Red.c
TW 5.52 ± 0.22 4.96 ± 0.34d T 5.26 ± 0.33 2.54 ± 0.33f

TWS 5.57 ± 0.15 4.56 ± 0.34de W 5.27 ± 0.35 1.28 ± 0.33g
WS 5.46 ± 0.15 4.40 ± 0.34def V 5.39 ± 0.20 3.33 ± 0.33ef
VW 5.56 ± 0.12 3.49 ± 0.34f S 5.38 ± 0.16 3.44 ± 0.33ef
VWS 5.49 ± 0.04 3.84 ± 0.34ef VWLS*5 5.18 ± 0.35 4.51 ± 0.33d
TWLS 5.51 ± 0.19 5.07 ± 0.34d VWLS*10 5.21 ± 0.33 4.23 ± 0.33de
VWLS 5.54 ± 0.13 5.01 ± 0.34d
aOrder of treatment within abbreviation indicates order of application. T = trim; W = 35°C water wash; S = 15-sec
steam pasteurization; V = hot water/steam vacuum spot cleaning; L = 2% lactic acid spray; S*5 and S*10 = 5- and 10-
sec exposure time, respectively, for steam pasteurization.
bMean initial pathogen population (log CFU/cm2) from four replications ± standard error of mean.
cMean reduction in pathogen population (log CFU/cm2) from four replications ± standard error of mean.
d,e,f,gMeans having the same superscript within columns are not significantly different (P < 0.05).
Source: Adapted from Phebus, R.K., A.L. Nutsh, D.E. Schafer, R.C. Wilson, M.J. Rieman, J.D. Leising, C.L. Kastner,
J.R. Wolf, and R.K. Prasai. 1997. Comparison of steam pasteurization and other methods for reduction of pathogens
on surfaces of freshly slaughtered beef. J. Food Prot. 60: 476–484.
and in combination, included knife trimming (T), water washing (W; 35°C), hot water/steam vacuum
spot cleaning (V), and spraying with 2% vol/vol lactic acid of pH 2.25 at 54°C (L). All combination
treatments were effective in reducing the numbers of L. monocytogenes on the meat surface, with
TWLS, VWLS, and TW treatments (the order of treatment within the abbreviation indicates the
order of application) being the most and the VW and VWS the least effective (Table 13.16). The
authors concluded that higher levels of pathogen reduction can be attained through use of combi-
nations of decontamination treatments, with knife trimming and/or steam vacuum spot cleaning (to
remove visible contamination), followed by steam pasteurization, being a very effective intervention
strategy that can be used by the meat industry [189]. Dorsa et al. [73] also examined effects of
steam vacuuming (SV) and a hot water spray wash of 74 ± 2°C (W) on L. innocua present on the
surface of beef carcass tissue. Initial reductions of 2.0, 2.2–2.5, and 2.6–2.7 CFU/cm2 were observed
after the SV, W, and SV + W treatments, respectively. However, after storage of the meat for 21 days
at 5°C, numbers of L. innocua on all treated meat samples were the same as those of the untreated
control. It should be mentioned, however, that the initial loads of listeriae on meat were high
(106 CFU/cm2) and that with lower initial levels, outgrowth of the organism may not have occurred
or may have been minimal.
Bréand et al. [43] studied the influence of time and mild temperature on L. monocytogenes
survival curves in trypticase soy broth. They applied 11 different temperatures to stationary-phase
L. monocytogenes CIP 7831 (ATCC 35152) ranging from 53 to 60°C for 0 to 250 min for
temperatures between 53 and 55°C, and 0 to 120 min for temperatures between 56 and 60°C.
Results showed that the linear relationship between the logarithm of the death rate and the
temperature inducing death was also valid for mild temperatures. The study also demonstrated
that mathematical modeling permitted building a new strategy to determine the best mild heat
treatment in which the pasteurization tables based on the Bigelow law [39a] are no longer
appropriate.
Doherty et al. [72] performed studies on heat inactivation of L. monocytogenes in minced beef
and minced beef homogenate in vacuum bags or vacuum containers. Results showed no significant
differences in the D-values at 50, 55, or 60°C. However, these D-values were lower than previously
reported for L. monocytogenes in ground meat by Mackey et al. [163].
Studies by several researchers [72,182,194,230] have shown that postpackage pasteurization
of meats proved effective in reducing or eliminating L. monocytogenes from inoculated samples.
Muriana et al. [182] looked at the effect of heat on inactivation of L. monocytogenes on roast beef
and smoked ham deli products. Inactivation rates differed, depending on the temperature and
substrate (Figure 13.5). Miller et al. [179] used a cold shock treatment of 0 to 15°C for 1 to 3 h
to increase the thermal sensitivity and thermal inactivation of L. monocytogenes. Taormina and
Beuchat [214] found that the alkaline-stress response in L. monocytogenes may induce a response
which leads to greater resistance to otherwise lethal thermal processing conditions, and that exposure
to chlorine increased thermal sensitivity. In a later study, these researchers discovered that alkali-
stressed cells that are not alkali-adapted have lower D59°C-values than alkaline-cleaner-exposed cells
[215]. Growth on frankfurters at 4 and 12°C was more delayed with alkali-adapted cells than with
cells exposed to an alkaline cleaner [215]. Doyle et al. [74], in a review of the heat resistance of
L. monocytogenes, mention that heat resistance varies depending on the strain, age of culture,
growth conditions, recovery media, and characteristics of the particular food, such as salt content,
aw , pH, and presence of inhibitors. They also describe L. monocytogenes having a higher heat
resistance than most other foodborne pathogens that are non-spore-forming and that criteria
aimed at the elimination of E. coli or Salmonella spp. may not be sufficient for elimination of
L. monocytogenes.
In summary then, several points regarding the heat resistance of L. monocytogenes in meat can
be stated: (1) the organism appears to be about four times more heat resistant than salmonellae in
meats; (2) attention should be paid to those meats that are heated slowly, a process that may induce
the production of heat shock or stress proteins in these and other organisms; (3) D-values obtained for
Roast Beef Smoked Ham

0 0
-1 -1
-2 -2
Log reduction
Log reduction
-3 -3
-4 -4
-5 -5
-6 -6
-7 -7
0 10 20 30 40 50 60 70 80 90 100 110 120 0 10 20 30 40 50 60 70 80 90 100 110 120
Time (s) Time (s)
D145F= 95.2 seconds 62.8 65.6 68.3 71 D145F= 67.6 seconds

D150F= 54.1 seconds D150F= 29.6 seconds
---------- ----------
Zroastbeef = 10.3°F (5.7°C) Zham = 14.2°F (7.9°C)
FIGURE 13.5 Regression trend lines and associated D- and z-values for L. monocytogenes heated in purge
from ready-to-eat (RTE) roast beef and smoked ham deli products. Heating regimens were performed at 145°F
(62.8°C), 150°F (65.6°C), 155°F (68.3°C), and 160°F (71°C). (Adapted from Muriana, P.M., W. Quimby,
C.A. Davidson, and J. Grooms. 2002. Postpackage pasteurization of ready-to-eat deli meats by submersion
heating for reduction of Listeria monocytogenes. J. Food Prot. 65: 963–969.)
meats can vary quite widely, depending on the prior history of the food and organism, recovery
methodology, and strain type; (4) in general, the D-values for L. monocytogenes in meat are higher
than those for dairy products; (5) cured meats require greater heating than noncured products to ensure
the same level of protection; (6) combinations of decontamination treatments work best for eliminating
the organism from carcass surfaces; and (7) heat-in-bag thermal processes which avoid the possibilities
of in-plant postprocessing contamination should be encouraged.
BACTERIOCINS FOR CONTROLLING LISTERIAE IN MEAT

There have been several review articles describing the potential for using bacteriocins and/or lactic
acid bacteria for controlling Listeria spp. in foods [181], and more specifically in meats
[177,199,213]. The general use of bacteriocins to control listeriae has been dealt with in other
chapters of this book. Therefore, a summary of the information related specifically to meat products
follows.
Raw Ground Meat
Buchanan and Klawitter [45] were among the first investigators to look at the application of
bacteriocin-producing cultures to control L. monocytogenes in foods, investigating the potential for
using Carnobacterium piscicola strain LK5 to inhibit growth of the organism in various foods.
Foods were inoculated with 103 CFU/g L. monocytogenes, either with or without 104 CFU/g of the
LK5 strain. In sterile raw ground beef, strain LK5 inactivated the organism at 5°C and prevented
its growth at 19°C. C. piscicola did not have any effect on the organism in nonsterile ground beef
(or chicken roll); however, L. monocytogenes did not grow on the control products. In general, the
bacteriocin-producing strain was most effective when the background microflora on the foods was
low. Similar studies with sterile and nonsterile ground beef were done with another lactic acid
bacterium, Lactobacillus casei as well as its associated bacteriocin, lactocin 705 [227]. Meat was
inoculated with either the pure bacteriocin or the L. casei strain and then stored at 20°C for 24 h.
In general, the reduction in numbers of L. monocytogenes was greatest with the highest levels of
lactocin 705 tested (800 AU/mL) [16], with the fewest numbers of L. monocytogenes present in
the meat slurry, and with autoclaved ground beef.
Starter cultures also have been assessed for their ability to inhibit L. monocytogenes in minimally
heat-treated vacuum-packed beef cubes either with or without gravy and/or glucose. For these exper-
iments, Lactobacillus bavaricus strain MN was inoculated into beef at 105 or 103 CFU/g, along with
102 CFU/g L. monocytogenes, then vacuum-sealed, and stored at 4 or 10°C [238]. Several results
were obtained: (1) strain MN grew and produced bacteriocin at refrigeration temperatures, although
it was much more effective at inhibiting L. monocytogenes at the lower storage temperature; (2) the
higher inoculum level used was significantly more effective in reducing levels of L. monocytogenes;
(3) bacteriocin production was independent of the presence of glucose in the meat; (4) addition of
sugar enhanced the antilisterial activity even though the pH was not greatly reduced in those meats
with gravy and glucose; (5) the antilisterial activity was greater in those meats containing gravy with
glucose, suggesting that the gravy may have enhanced diffusion of the bacteriocin; and (6) bavaricin
production occurred during the early stages of growth of strain MN. The authors concluded that the
food industry may find the use of bacteriocinogenic lactic acid bacteria attractive.
Bacteriocin-producing lactic acid bacteria have also been used to control growth of L. mono-
cytogenes on frankfurters. In these experiments, either high (107 CFU/g) or low (103–104) levels
of a bacteriocin-producing strain of Pediococcus acidilactici, as well as its plasmid-cured,
bacteriocin-negative derivative (bac−), were inoculated separately onto wieners along with a five-
strain cocktail of L. monocytogenes. Frankfurters were stored both aerobically and anaerobically
at 4 and 15°C. In general, presence of P. acidilactici on the frankfurters inhibited listeriae to various
degrees, depending on the pediococci levels, storage temperature, and package atmosphere [36].
Yousef et al. [243] examined the ability of L. monocytogenes to grow in wiener exudates. The organism
grew in two of the five exudates tested, with the best growth being observed in those exudates
containing the lowest amount of phenols, the lowest indigenous levels of lactic acid bacteria, and
the highest pH. However, exudates stored at 4°C did not support growth of the organism. Those
exudates that did not support growth of the organism proved to be listericidal. Previous work done
by Glass and Doyle [101] showed that L. monocytogenes could grow on wieners stored at 4.4°C.
Differences in results between the two studies could have resulted from loss of wiener exudate
during manipulation, uneven distribution of exudate on the wiener surface, or variations in the
intensity of smoking within and among different brands [243]. Both P. acidilactici H and pure
pediocin AcH inactivated listeriae that were inoculated into one brand of wiener exudate which
had been stored at 25°C for 8 days. In the control samples, L. monocytogenes increased in number
from approximately 104 to 107 CFU/g within 4 days. Following up on this work, the latter authors
used the same lactic acid bacteria in an attempt to control the growth of L. monocytogenes in
temperature-abused vacuum-packaged wieners stored at 4 and 25°C. At 25°C, the presence of P.
acidilactici strain LB42 inhibited growth of L. monocytogenes, but was not listericidal. In contrast,
P. acidilactici strain JBL1095 was listericidal, decreasing counts of L. monocytogenes by 2.7 logs.
This inactivation was not caused solely by pH, as pH values in wieners inoculated with both strains
of pediococci were similar. The only difference between the two strains was that one (JBL1095)
produced pediocin AcH and the other did not, strongly suggesting that this bacteriocin enhanced
the antilisterial activity of lactic acid bacteria in vacuum-packaged wieners.
Amezquita and Brashears [4] examined the use of lactic acid bacteria for competitive inhibition
of L. monocytogenes in RTE meat products. Three cultures of lactic acid bacteria (LAB) were used
as they were shown to exert the greatest effect on L. monocytogenes growth, exhibiting not only
antilisterial activity, but an inhibitory capacity as well. With cooked ham, LAB cultures proved to
be bacteriostatic, but with frankfurters, LAB were antilisterial. In frankfurters, LAB had an
antilisterial effect whether they were used singly or in combined cultures.
Several researchers [102,170,203] have examined the potential antilisterial effects of sodium
lactate and sodium diacetate in meat, wieners, and RTE meats. Overall, there was a direct correlation
between concentration of inhibitory substances and inhibition of L. monocytogenes. In addition,
there was a synergistic effect when these additives were used in combination (Figure 13.3).
Fermented Sausages
A number of studies have examined the effects of starter cultures and their bacteriocins on survival
and growth of L. monocytogenes on sausages. Foegeding et al. [90] used pediocin-producing strains
of P. acidilactici (along with an isogenic mutant as a control) to control growth of L. monocytogenes
on dry-fermented American-style sausages. Recovery of the organisms during the fermentation was
made easier by using antibiotic-resistant strains of both the pediococci and listeriae. The study was
unique in showing that in combination with other fermentation end products, inhibition of L.
monocytogenes was enhanced by bacteriocin production in situ during fermentation and drying.
Berry et al. [35] had also previously shown the benefits of using P. acidilactici as a starter culture
to control growth of listeriae during sausage fermentation. According to these authors, a commercial
summer sausage mix was inoculated to contain ~106 CFU/g of L. monocytogenes and fermented
with either bacteriocin-producing or non-bacteriocin-producing strains of P. acidilactici. Following 12
to 14 h of fermentation at 37.8°C, populations of listeriae decreased approximately 100- and
10-fold in summer sausage fermented with bacteriocin-producing and non-bacteriocin-producing
strains of P. acidilactici, respectively. The pathogen also was inactivated in sausages with slower
acid production (pH > 5.5), which suggests that bacteriocin production occurred independent of
carbohydrate fermentation. However, L. monocytogenes was still detected in 9 of 90 (10.0%)
sausage samples that had been heated to an internal temperature of 64°C and then refrigerated for
up to 2 weeks. Thus, although use of this bacteriocin-producing starter culture led to a dramatic
decrease in numbers of viable listeriae in summer sausage, it did not completely eliminate the
pathogen in finished product prepared from sausage mix containing 106 CFU/g of L. monocytogenes.
However, because the presence of >103 L. monocytogenes/g in commercially prepared sausage mix
is highly unlikely, it appears that current heat treatments are adequate to produce a Listeria-free
semidry sausage. Another starter culture, L. saké, was found to control growth of L. monocytogenes
on German-style fresh sausages. Results varied depending upon the initial pH of the “fresh
Mettwurst,” i.e., at initial pH values of 6.3 and 5.7, and bacteriostatic and bacteriocidal (1-log
reduction) effects on L. monocytogenes, respectively, were observed. In another study using the
same starter culture [126], a bacteriocin-producing (sakacin K) strain of L. saké, isolated from a
naturally fermented sausage, was able to reduce numbers of L. monocytogenes in dry-fermented
sausages by 1.25 logs, as compared to the isogenic bac− control strain. Lactobacillus plantarum
has also been used as a starter culture to study the fate of L. monocytogenes in naturally and
artificially contaminated salami [48]. In this study, the starter cultures prevented growth of listeriae,
but were not listericidal. In inoculated samples, little difference was observed between samples
inoculated with bac+ or bac− strains of L. plantarum. However, in naturally contaminated samples,
the only samples that were free of listeriae were those inoculated with the bacteriocin-producing
strain of L. plantarum.
Lahti et al. [152] examined survival and detection of E. coli and L. monocytogenes during
manufacture of dry sausage, using two different starter cultures. Starter culture A contained
Staphylococcus xylosus DD-3, bacteriocin-producing P. acidilactici PA-2 and Lactobacillus bavar-
icus MI-401, and starter culture B contained S. carnosus MIII and L. curvatus Lb3. Sausage
batter (35% pork, 35% beef, 30% fat) was inoculated with high (5.05–5.41) or medium (2.92–3.35)
log10 CFU/g levels of L. monocytogenes serovar 4b, manufactured (fermented and dried) in a smoke
chamber at 17–23°C for 15 days, and then stored at 15–17°C for 35 days. The sausages were
smoked for 30 min during days 2–5. L. monocytogenes decreased more rapidly in the high-inoculum
sausages produced with starter A than with starter B, but no significant difference was found between
the starters in the medium-inoculated sausages. This pathogen was eliminated from the medium-
inoculated sausages after 49 days (Figure 13.6).
Bacteriocins have also been used in studies aimed at inactivating or preventing the attachment
of L. monocytogenes to muscle surfaces. El-Khateib et al. [77] investigated the ability of lactic
acid, nisin, and pediocin to inactivate L. monocytogenes on beef muscle, as well as to prevent its
attachment after short-term exposure and during 48 h of refrigerated storage. The latter experiments
were done to simulate secondary contamination of meat during refrigerated storage. Lactic acid
(2%), nisin (4 × 104 IU/mL), and pediocin (3.2 × 103 AU/mL) decreased numbers of the organism
on the meat surface by 1.7, 1.1, and 0.6 log10 CFU/6 cm2 (cubical piece), respectively. Interestingly,
in comparison to the control, lactic acid treatment caused a greater percentage of listeriae cells to
attach to meat surfaces. This was partially attributed to acidic conditions that can sometimes enhance
adhesion of bacteria to surfaces [77]. In general, the percentage of L. monocytogenes cells attached
to beef muscle in the presence of the two bacteriocins did not change significantly or slightly
decreased, although after 1 h, nisin- and pediocin-treated samples had 30 and 17% cells, respec-
tively, attached, as compared to the respective controls with 6.3 and 12%. Other work done by
Cutter and Siragusa [64,65] has shown nisin to be effective in reducing (by 2.0 to 2.83 log10
CFU/cm2) numbers of L. innocua in beef. The latter study showed the combination of vacuum-
packaging and nisin spraying to be effective in suppressing growth of L. innocua enough so that
at the end of the 4-week incubation period at 4°C, counts were lower than the corresponding
nontreated controls.
In summary, bacteriocins, especially pediocin, appear to be good candidates for controlling
growth of listeriae in meats, as they have proved to be effective in controlling growth of the organism
in wieners and wiener exudates, semidry and dry sausages, and fresh beef and pork. The main
obstacles at present to their use in foods are development of bacteriocin resistance, activity loss
with time, inactivation by proteases, and adsorption to meat and fat particles resulting in inactivation,
limited diffusion (especially in minced meats and poultry), and finally regulatory acceptance.
1000000 High-inoculum starter A

Medium-inoculum starter A
High-inoculum starter B
Medium-inoculum starter B
100000
10000
CFU/g 1000
100
10
1
0 3 7 14 21 35 49
Time (d)
FIGURE 13.6 Survival of L. monocytogenes in dry sausage produced using starter cultures A (Flora-Carn
LC) and B (Müller RM 52). Counts are given as CFU/g. High-inoculum starter A(♦), medium-inoculum
starter A (•), high-inoculum starter B(), medium-inoculum starter B(). Values of high inoculum are means
of six subsamples and values of medium inoculum are means of three subsamples. The value 1 CFU/g
represents counts below the enumeration limit (<3 CFU/g) but detected by enrichment. The value 0 CFU/g
represents counts below enumeration limit (3 CFU/g) and not detected by enrichment. (Adapted from Lahti,
E., T. Johansson, T. Honkanen-Buzalski, P. Hill, and E. Nurmi. 2001. Survival and detection of Escherichia
coli O157:H7 and Listeria monocytogenes during the manufacture of dry sausage using two different starter
cultures. Food Microbiol. 18: 75–85.)
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in Poultry and Egg Products
Elliot T. Ryser
CONTENTS
Introduction ....................................................................................................................................572
USDA–FSIS Listeria-Testing Program for Cooked/Ready-to-Eat Poultry Products ...................572
Listeria Contamination in Cooked/Ready-to-Eat Poultry Marketed outside
the United States ............................................................................................................................574
Incidence of Listeria spp. in Raw Poultry Marketed in the United States ..................................577
Chicken .................................................................................................................................577
Turkey ...................................................................................................................................579
Incidence of Listeria spp. in Poultry Products Marketed in Europe and Elsewhere ...................580
Raw Poultry ..........................................................................................................................580
United Kingdom .......................................................................................................580
Continental Europe...................................................................................................584
Elsewhere..................................................................................................................585
Cooked/Ready-to-Eat Poultry ..............................................................................................586
Behavior of L. monocytogenes in Raw and Cooked Poultry Products.........................................587
Growth: Raw Chicken ..........................................................................................................587
Antimicrobial Additives ...........................................................................................589
Susceptibility to Gamma Radiation .........................................................................589
Growth: Cooked/Ready-to-Eat Poultry Products.................................................................590
Thermal Inactivation During Cooking .................................................................................593
USDA Guidelines for Control of Listeria in Ready-to-Eat Poultry .............................................597
Postpackaging Pasteurization ...............................................................................................598
Antimicrobial Agents for Listeria Growth Suppression......................................................598
Egg Products ..................................................................................................................................599
Incidence...............................................................................................................................600
Growth ..................................................................................................................................601
Thermal Inactivation.............................................................................................................604
References ......................................................................................................................................606
571
INTRODUCTION
Avian listeriosis was first recognized in 1932 when TenBroeck isolated Listeria monocytogenes
(then Bacterium monocytogenes) from diseased chickens [186,187]. Chickens have remained a
common avian host for L. monocytogenes since avian listeriosis was first recognized. Listeriosis
also has been observed in at least 22 other avian species, including such frequently consumed fowl
as turkeys [32,93,158], ducks [84,188], geese [84,162], and pheasants [84]. Large outbreaks of
listeriosis in domestic fowl are relatively rare [157]; however, sporadic cases occur much more
frequently and are often accompanied by asymptomatic shedding of Listeria in feces. According
to one report, 4.7% of cecal samples from Danish broiler chickens harbored L. monocytogenes
[162]. Hence, as was true of beef, pork, and lamb, poultry meat destined for human consumption
also is at risk of becoming contaminated with L. monocytogenes from the abattoir environment
during defeathering, evisceration, and further processing. Several early studies suggested that poultry-
and egg-processing workers could contract Listeria infections by handling contaminated birds
[65,108,109]. Additionally, several reports from England during the 1970s indicated that L.
monocytogenes could be isolated with some frequency from raw chickens as well as turkeys, ducks,
and pheasants. An added concern with poultry relates to eggs that can become contaminated with
this pathogen during collection and processing.
Emergence of L. monocytogenes as a bona fide foodborne pathogen following the Mexican-
style cheese outbreak of 1985 prompted immediate concern about the presence of Listeria in dairy
products and also generated a parallel interest in the incidence and behavior of L monocytogenes
in meat and poultry products; the latter is the topic of this chapter. Interest in this area has increased
dramatically since 1988 as a result of four major listeriosis outbreaks that were directly linked to
consumption of turkey frankfurters [83] and ready-to-eat delicatessen turkey meat [49,70,163] in
the United States. In this chapter, information concerning the incidence, behavior, and control of
L. monocytogenes in poultry products will be reviewed first, followed by a discussion of L.
monocytogenes in egg products.
USDA–FSIS LISTERIA-TESTING PROGRAM FOR COOKED/READY-TO-EAT

POULTRY PRODUCTS
Public health concerns regarding Listeria-contaminated raw and, particularly, processed ready-to-
eat poultry products sold in the United States also stem directly from the 1985 listeriosis outbreak
in California that was associated with consumption of Mexican-style cheese. Soon thereafter, U.S.
Department of Agriculture-Food Safety Inspection Service (USDA–FSIS) officials announced their
intention to develop a Listeria-monitoring/verification program for cooked and ready-to-eat meat
as well as poultry products based on product type. Because the monitoring/verification programs
for meat and poultry products developed in parallel and were both similar in terms of sampling
scheme, methodology, and action taken when L. monocytogenes was found, the following discussion
focuses on the various poultry products tested and pertinent results rather than on an in-depth
analysis of these programs.
A Listeria-monitoring/verification program for cooked/ready-to-eat poultry was first suggested
in December 1985 and was to cover all such products prepared in federally inspected establishments
as well as those produced by certified foreign manufacturers [94]. However, actual testing of poultry
sausage, that is, the first category of ready-to-eat poultry examined, did not begin until September
1988, 1 year after the Listeria-monitoring/verification program was first instituted for cooked beef,
roast beef, and cooked corned beef.
Before April 1989, the USDA’s Listeria policy, which gave firms the opportunity to clean
up their facility before additional verification samples were analyzed under hold-test procedures,
Incidence and Behavior of Listeria monocytogenes in Poultry and Egg Products 573
was consistent with the fact that listeriosis had not yet been linked to consumption of poultry
products. However, the official USDA–FSIS position regarding the presence of L. monocytogenes
in cooked ready-to-eat poultry products changed radically on April 14, 1989, when Centers for
Disease Control and Prevention (CDC) investigators directly linked consumption of contaminated
turkey frankfurters to an instance of listerial meningitis in a breast cancer patient in Oklahoma
[15]. After isolating L. monocytogenes serotype 1/2a of the same electrophoretic enzyme type
from the woman and opened, as well as unopened, retail packages of turkey frankfurters,
USDA–FSIS officials requested that the manufacturer issue an immediate Class I recall for
approximately 600,000 pounds of Texas-produced turkey frankfurters that were marketed by
retail and institutional establishments in Alaska, Arizona, Arkansas, California, Florida, Georgia,
Idaho, Illinois, Indiana, Kentucky, Louisiana, Mississippi, Missouri, New Jersey, New York, Ohio,
Oklahoma, Pennsylvania, Tennessee, Texas, Utah, and Washington. As expected, this recall
immediately prompted an intensified monitoring/verification program to determine the extent of
Listeria contamination in a far wider range of cooked/ready-to-eat poultry products marketed in
the United States.
Despite initial attempts by the National Turkey Association to develop tolerance levels for
L. monocytogenes in cooked and ready-to-eat poultry products [10], USDA–FSIS officials main-
tained that because an “acceptable level” of L. monocytogenes in such products could at that time
not be determined, the only acceptable alternative was to adopt a “zero tolerance” for this pathogen
in cooked/ready-to-eat poultry and meat products [11]. Consequently, under this program [56],
which was identical to that developed for cooked and ready-to-eat red meat products, USDA–FSIS
officials requested that firms issue a Class I recall for all lots of cooked and ready-to-eat poultry
products in which L. monocytogenes was detected in monitoring samples taken from intact packages
of product. However, Listeria-positive lots under direct control of the manufacturer could be
recalled internally, thus avoiding adverse publicity. If the pathogen was initially detected in
monitoring samples from unpackaged products, USDA–FSIS officials did not request that firms
immediately recall the sampled lot. Instead, government officials analyzed subsequent lots and,
if necessary, initiated further steps (i.e., hold-test programs) to prevent distribution of contam-
inated products.
Our knowledge concerning the incidence of Listeria in cooked/ready-to-eat poultry products
marketed in the United States initially came from the USDA–FSIS Listeria-monitoring/verifica-
tion program, with the first results from 1987 to 1990 indicating that 1.5–2.0% of all such products
were contaminated with L. monocytogenes [8,19,47]. Poultry products in which L. monocytogenes
had been found during 1989 and 1990 included chicken patties [8], chicken salad [8], diced
poultry [8], poultry salad [19], poultry spread [19], poultry frankfurters [8], poultry bologna [8],
and turkey sausage [8]. In all likelihood, the pathogen entered these products during the latter
stages of manufacture and/or packaging. By 1990, USDA–FSIS established nine different regu-
latory programs in which ready-to-eat meat and poultry products were collected at federally
inspected establishments and tested for L. monocytogenes [119,210]. Four of these nine programs
were entirely meat based. The remaining programs included poultry products; however, only one
of these programs, entitled “Cooked Poultry Products—Uncured,” was solely devoted to poultry.
Based on results from this USDA–FSIS program, 0.95 to 3.17% of all uncured cooked poultry
products tested from 1990 to 2000 yielded L. monocytogenes with an average of 1.97% of the
samples positive during this 11-year period (Figure 14.1). In December 2000, USDA–FSIS ceased
testing according to product type and began collecting samples based on Hazard Analysis and
Critical Control Point (HACCP) processing categories as defined in the Code of Federal Regu-
lations for ready-to-eat products, with these new categories no longer definable in terms of meat
or poultry.
In one remaining report available, the Oregon State Department of Agriculture [164] surveyed
a wide range of meat and poultry products that included breaded chicken (22 samples), ready-to-eat
FIGURE 14.1 USDA–FSIS monitoring program for L. monocytogenes in cooked poultry products:
1990–2000. (From Levine, P., B. Rose, S. Green, G. Ransom, and W. Hill. 2001. Pathogen testing of
ready-to-eat meat and poultry products collected at federally inspected establishments in the United
States, 1990 to 1999. J. Food Prot. 64: 1188–1193; USDA–FSIS. 2005. Electronic reading room:
microbiological testing program. Available at: http://www.fsis.usda.gov/ophs/rtetest/rettable6.htm.)
chicken (72 samples), sliced turkey breast (118 samples), turkey ham (8 samples), turkey pastrami
(7 samples), and ready to-eat turkey (4 samples). From this list of products, L. monocytogenes was
only recovered from 3 samples—2 (2.8%) ready-to-eat chicken and 1 (14.3%) turkey pastrami—
with all others negative for L. monocytogenes by enrichment.
Formal Class I recalls for ready-to-eat poultry products were quite limited during the first few
years of the USDA–FSIS Listeria-monitoring/verification program and include the aforementioned
recall of turkey frankfurters [15], two recalls of chicken salad [5,7], two recalls of deli sandwiches
containing turkey and /or chicken [12,18], and one additional incident involving 13,000 lb of chicken
spread produced by a Virginia-based firm [16]. Beginning in 1994, recall information is more
complete, with 9 recalls totaling less than 25,000 lb of product issued through September 1998
(Table 14.1). However, this situation changed dramatically on December 22, 1998 when 35 million
pounds of turkey frankfurters and deli meats were recalled in response to a nationwide listeriosis
outbreak involving 108 cases, including 14 deaths and 4 miscarriages/stillbirths, in 22 states [83].
This widely publicized outbreak prompted increased testing of ready-to-eat poultry which, in turn,
greatly increased the number of Listeria-related Class I recalls. In 2000 and 2002, two additional
recalls involving 16.9 and 27.4 million pounds of delicatessen turkey meat, respectively, were issued
when both products were traced to two separate nationwide outbreaks of listeriosis [49,163]. All
totaled, 76 Class I recalls have been issued from 1994 to October 2005 for 124.6 million pounds
of ready-to-eat poultry products contaminated with L. monocytogenes. Of these 76 recalls, 48 (63%)
involved cooked chicken products, with the remaining 14 (18%), 6 (8%), 5 (7%), and 3 (4%) traced
to manufacturers of ready-to-eat turkey, turkey and chicken, duck, and unspecified poultry products,
respectively.
LISTERIA CONTAMINATION IN COOKED/READY-TO-EAT POULTRY

MARKETED OUTSIDE THE UNITED STATES
Since 1997, several small European surveys that included samples of various ready-to-eat
poultry products have been conducted. Working in Belgium, Rijpens et al. [175] reportedly
recovered L. monocytogenes from 7 of 36 (29%) samples of ready-to-eat sliced turkey and
TABLE 14.1
Class I Recalls Issued in the United States for Ready-to-Eat Poultry Products
Contaminated with L. monocytogenes during 1994–2005
Product Date of Recall State of Manufacture Quantity (lb)
Chicken salad 3/23/94 California 278

Chicken salad 5/24/94 Tennessee 1,643
Smoked chicken sausage 7/1/94 California 2,894
Chicken salad 8/31/94 North Carolina 1,953
Duck confit 10/26/94 New York 88
Chicken salad 5/5/95 North Carolina 105
Duck rillettes 5/19/95 New Jersey 270
Chicken breast and broccoli 6/5/95 Pennsylvania 50
Cooked chicken 6/2/97 West Virginia 17,434
Chicken and turkey frankfurters 10/22/98 Florida 1,734,002
Turkey frankfurters and deli meats 12/22/98 Michigan 35,000,000
Poultry hot dogs and deli meats 1/22/99 Arkansas 35,000,000
Chicken burritos 2/2/99 Illinois 26,975
Turkey franks 8/27/99 California 33,710
Chicken salad 9/9/99 Pennsylvania 240
Sliced turkey and other luncheon meats 12/14/99 New Jersey 900
Roast chicken meat, ground 1/28/00 Michigan 4,500
Chicken nuggets, patties 2/23/00 Alabama 160,760
Sliced turkey breast and other meats 5/3/00 Michigan 215
Chicken nuggets 5/9/00 Puerto Rico 7,083
Sliced turkey breast and other meats 5/24/00 Alabama 45
Chicken enchiladas 5/30/00 New Mexico 45
Chicken salad 7/6/00 Alabama 5,376
Chicken salad 7/17/00 Massachusetts 3,300
Turkey breast and other meats 8/8/00 Idaho 380
Chicken salad spread 9/27/00 Minnesota 80
Chicken caesar salad 10/10/00 Pennsylvania 90
Cooked turkey and chicken 12/14/00 Texas 16,895,000
Duck 1/16/01 Indiana 4,400
Chicken wings 1/24/01 New Jersey 2,100
Mexican-style poultry products 1/30/01 Illinois Unknown
Duck 2/9/01 Indiana 6,000
Duck 4/4/01 California 780
Turkey 6/20/01 Pennsylvania 75
Frozen chicken dinners 8/10/01 California 650
Chicken fajitas 8/14/01 California 950
Frozen chicken 10/4/01 New Jersey 7,500
Turkey ham 11/8/01 Iowa 11,800
Fajita chicken strips 2/26/02 Alabama 30,700
Smoked turkey and turkey ham 3/15/02 Minnesota 23,000
Chicken/pork hot dogs 4/25/02 Ohio 140,000
Turkey bologna 5/23/02 New York Unspecified
Turkey frankfurters 8/2/02 New York 3,400
Chicken salad 8/4/02 North Carolina 2,200
(continued)

Class I Recalls Issued in the United States for Ready-to-Eat Poultry Products
Contaminated with L. monocytogenes during 1994–October 2006
Product Date of Recall State of Manufacture Quantity (lb)
Turkey and chicken breast 10/9/02 Pennsylvania 295,000

Turkey and chicken breast 10/12/02 Pennsylvania 27,400,000
Turkey and chicken breast 11/2/02 New Jersey 200,000
Turkey and chicken breast 11/20/02 New Jersey 4,200,000
Chicken frankfurters 1/3/03 New York 26,400
Chicken salad 5/5/03 Illinois 400
Turkey, ham, roast beef 9/10/03 California 550
Cooked poultry 9/12/03 Massachusetts 9,230
Turkey and ham 11/18/03 Florida 1,135
Cooked diced chicken breast 11/25/03 Georgia 7,500
Chicken salad 12/11/03 Florida 2,700
Cooked chicken 7/1/04 North Carolina 404,730
Cooked chicken 7/21/04 North Carolina 36,980
Chicken products 11/3/04 Maryland 1,275
Chicken products 1/31/05 New York 5,760
Chicken salad 3/15/05 New York 250
Chicken dumplings 3/16/05 California 12,090
Chicken products 3/28/05 California 12,500
Chicken wrap sandwiches 4/5/05 Florida 3,316
Smoked turkey and pork products 4/11/05 New York 39,000
Turkey and ham wraps 4/26/05 Texas 191
Chicken breast wraps 4/28/05 New York 385
Chicken salad 6/15/05 New York 5,065
Cooked chicken meat 7/21/05 Georgia 170
Chicken products 7/28/05 New York 3,200
Chicken salad 8/31/05 Oklahoma 23,435
Chicken frankfurters 9/20/05 New York 23,040
Chicken sausages 10/2/05 Illinois 1,000
Poultry products 10/21/05 Massachusetts 11,200
Chicken products 11/8/05 California 275
Chicken salad 11/10/05 Pennsylvania 5,523
Turkey and chicken products 12/1/05 Missouri 2,800,000
Source: From USDA–FSIS. 2005. FSIS Recalls. Available at: http: //www.fsis.usda.gov/Fsis Recalls/index.asp.
chicken purchased locally for sandwiches. The fact that none of seven similarly packaged
samples contained L. monocytogenes suggests that contamination most likely occurred during
slicing of the product at retail, as suggested in recent surveys of prepackaged and sliced deli
meats. This hypothesis is further supported by the work of Vorst et al. [217] that demonstrated
extensive transfer of L. monocytogenes between delicatessen slicer blades and roast turkey
breast during slicing. However, if the Belgian data for unpackaged product are excluded, then
the overall L. monocytogenes contamination rate of 1.1% for ready-to-eat poultry products
marketed in Europe is similar to that reported by USDA–FSIS for similar products available
in the United States.
INCIDENCE OF LISTERIA SPP. IN RAW POULTRY MARKETED

IN THE UNITED STATES
Immediately after the 1985 listeriosis outbreak in California was announced, public concerns were
raised about safety of dairy products and other potentially contaminated foods, including meat and
poultry products. Interest in the safety of raw poultry soon escalated after a nationwide telecast
informing the general public that 50% of all raw chicken marketed in the United States was
contaminated with Salmonella. Beginning in the late 1980s, several surveys were initiated to
determine the extent to which raw chicken and turkey meat are contaminated with Listeria and
other foodborne pathogens.
CHICKEN
The aforementioned concerns prompted USDA–FSIS officials to initiate a poultry back/neck testing
program for L. monocytogenes, Salmonella, and Escherichia coli serotype O157:H7 in January of
1989. L. monocytogenes and Salmonella were detected in 508 of 2686 (18.9%) and 792 of 2739
(28.9%) samples, respectively, with E. coli O157:H7 being absent from 2696 samples [19]. About
the same time, Bailey et al. [25] assessed the incidence of L. monocytogenes and Listeria spp. on the
surface of broiler carcasses processed in the southeastern United States and also compared L.
monocytogenes serotypes isolated from raw chicken with those associated with human cases of
listeriosis. Using an enrichment procedure together with three selective plating media, Listeria spp.
were detected in rinse samples from 34 of 90 (37.8%) broiler carcasses; however, recovery of Listeria
varied widely with three lots of 10 birds each being reported as negative. More important, 21 of 90
(23.3%) carcasses yielded L. monocytogenes, with 64, 18, 6, and 12% of the isolates being identified
as serotypes 1/2b, 1/2c, 3b, and nontypeable strains, respectively. Although only 7 of 115 (6.1%) L.
monocytogenes isolates from clinical listeriosis cases in the United States were of serotype 1/2b or
1/2c, the fact that most L. monocytogenes strains isolated from chickens were pathogenic to mice
suggested chicken meat as a possible vehicle of listeriosis in humans.
Between June 1988 and May 1989, Genigeorgis et al. [72,73] conducted two large surveys that
examined the incidence of Listeria spp. on fresh and/or semifrozen chicken and turkey parts obtained
from retail sources and slaughterhouse. According to their results for chicken, 12.5% of fresh wings,
16.0% of fresh legs, and 15.0% of fresh livers purchased at three supermarkets in northern California
contained detectable levels of L. monocytogenes (Table 14.2). Furthermore, with the exception of
fresh chicken liver, L. innocua was generally two to three times more prevalent in the remaining
samples than was L. monocytogenes. Overall, the highest incidence of Listeria spp. was observed
for fresh legs (54.0%), followed by fresh wings (42.5%) and fresh livers (32.5%). In contrast to
fresh products, only 10% of semifrozen chicken wings, legs, and livers contained Listeria spp.
However, finding L. monocytogenes alone in 1 of 10 semifrozen legs and livers attests to the ability
of this pathogen to survive in semifrozen raw chicken and turkey, as was also observed by Palumbo
and Williams [166].
In addition to these efforts, Genigeorgis et al. [72] also attempted to trace the route of Listeria
contamination on fresh chicken wings, legs, and livers by examining samples at various stages
of production and storage. Although all chicken parts at the start of production were free of
L. monocytogenes, results in Table 14.3 indicate that most contamination occurred during the latter
stages of production when carcasses came in direct contact with Listeria-laden fecal material,
because at the time of packaging, 70, 30, and 33% of chicken wings, legs, and livers contained
L. monocytogenes, respectively. Not surprisingly, L. innocua, which was virtually absent from
chicken parts at the beginning of production, also was routinely isolated from wings, livers, and
particularly legs at the end of production. Despite these relatively high contamination rates, both
Listeria spp. failed to grow on all three packaged products during the first 4 days of refrigerated storage.
Wimpfheimer et al. [222] observed a 3- to 4-day lag phase for L. monocytogenes when inoculated
TABLE 14.2
Incidence of Listeria spp. on Fresh and/or Semifrozen Chicken and Turkey Parts
Purchased from Three California Supermarkets between June 1988 and May 1989
Number Number (%) of Positive Parts

Type and Part of Parts L. L. L. Total
of Poultry Examined monocytogenes innocua welshimeri Listeria spp.
Chicken
Wings Fresh 40 5 (12.5) 14 (35.0) 1 (2.5) 17a (42.5)
Semifrozen 10 0 1 (10.0) 0 1 (10.0)
Legs Fresh 50 8 (16.0) 19 (38.0) 0 27 (54.0)
Semifrozen 10 1 (10.0) 0 0 1 (10.0)
Livers Fresh 40 6 (15.0) 8 (20.0) 1 (2.5) 13a (32.5)
Semifrozen 10 1 (10.0) 0 0 1 (10.0)
Turkey
Wings Fresh 60 12 (20.0) 3 (5.0) 12 (20.0) 27 (45.0)
Legs Fresh 60 8 (13.3) 0 9 (15.0) 17 (28.3)
Tails Fresh 60 7 (11.7) 0 7 (11.7) 14 (23.2)
aSome chicken parts contained both L. monocytogenes and L. innocua or L. innocua and L. welshimeri.
Source: Adapted from Genigeorgis, C.A., D. Dutulescu, and J. F. Garayzabal. 1989. Prevalence of Listeria spp. in
poultry meat at the supermarket and slaughterhouse level. J. Food Prot. 52: 618–624; Genigeorgis, C.A., P. Oanca,
and D. Dutulescu. 1990. Prevalence of Listeria spp. in turkey meat at the supermarket and slaughterhouse level. J.
Food Prot. 53: 282–288.
samples of raw minced chicken were held at 4°C. Given this information, the failure of Genigeorgis
et al. [72] to detect growth of L. monocytogenes and L. innocua on naturally contaminated packaged
chicken parts is not surprising.
In a 1997 study by Cox et al. [55] to determine how often L. monocytogenes enters poultry
processing plants on live chickens, L. monocytogenes was infrequently found in hatchery samples
and on the exterior of fully grown birds. Although L. monocytogenes was not recovered from the
intestinal tract of broiler chickens at the time of slaughter, 25% of postprocessing and retail-level
carcasses contained L. monocytogenes. In another study involving perorally dosed chicks, Husu
et al. [99] reported that L. monocytogenes was generally eliminated within 9 days, which again
suggests that intestinal carriage of L. monocytogenes is most often transient.
The scientific literature relating to pathogens commonly associated with processed poultry
is extensive, and a review of this literature has been published [218]. In another review paper
[106] covering the years 1971–1994, the prevalence of L. monocytogenes in meats was highly
variable, with approximately 16% of the products being positive. In general, the highest numbers
of L. monocytogenes have been found in processed meat and poultry products, with fresh meats
generally containing much lower numbers. In raw chicken, serotypes 1/2a, 1/2b, and 1/2c are
encountered more often than serotype 4b, with the latter most often associated with outbreaks
of foodborne listeriosis [216]. Thus, chicken, in contrast to turkey, has remained an uncommon
vehicle for infection. However, in 1996, Ryser et al. [182] identified Listeria spp. in 34 of 45
retail raw chicken pieces, with 11 different L. monocytogenes ribotypes, including three ribotypes
associated with previous foodborne listeriosis cases, being detected among the Listeria-positive
samples. These findings suggest the presence of multiple incoming sources of contamination
and/or heavily contaminated single sites within the poultry processing environment, including
floors, drains, and conveyor belts [33,50,138]. Hence, routine disinfection procedures are essential
to minimize the presence of both transient and persistent strains of Listeria that may colonize
the processing environment.
It is important to remember that, similar to other meats, poultry products also can be used for
purposes other than human consumption. Al-Sheddy and Richter [2] determined the incidence of
L. monocytogenes in frozen ground meat that contained raw chicken together with chopped beef
by-products. Although not conclusive, recovery of L. monocytogenes from all five samples exam-
ined, as well as the fact that this pathogen is more commonly found in chicken rather than beef
products, suggests that raw chicken was the most likely source of contamination. Hence, considering
the high incidence of L. monocytogenes in raw chicken, it may be prudent to eliminate raw poultry
products from the diet of zoo animals to curb the number of listeriosis cases occurring in zoological
parks.
TURKEY
Chickens and other types of domesticated fowl are similarly processed. Hence, one would also
expect to isolate various Listeria spp., including L. monocytogenes, from the surface of other raw
poultry products, such as turkey [161], ducks [20], and pheasants [113].
After completing the aforementioned chicken part survey in California [72], Genigeorgis et al.
[73] initiated a similar study to determine the prevalence of various Listeria spp. on fresh turkey
parts obtained from retail sources and slaughterhouses. Listeria contamination rates for fresh turkey
were generally similar to those previously observed for fresh chicken, with 45.0% of turkey wings,
28.3% of legs, and 23.3% of tails obtained from three northern California supermarkets harboring
various Listeria spp. (see Table 14.2). Although their findings further demonstrate that L. monocy-
togenes is equally common on fresh chicken and turkey parts, with isolation rates of 10.0–16.0%
and 11.7–20.0%, respectively, the same cannot be said of L. innocua and L. welshimeri. In fact,
L. innocua, the Listeria sp. most commonly detected on fresh chicken in this study, as well as
several others [85,167,225], was recovered from only 3 of 180 (1.7%) fresh turkey parts. Similarly,
L. welshimeri, the dominant Listeria sp. on fresh turkey, was only rarely observed on fresh chicken.
Although both surveys were confined to fresh chicken and poultry parts available from three local
supermarkets, these findings still suggest that chickens and turkeys may be preferential hosts for
L. innocua and L. welshimeri, respectively, with the latter most likely to harbor L. monocytogenes
strains belonging to serotype 4b—the serotype that continues to be most frequently associated with
foodborne listeriosis outbreaks, including those traced to turkey [83,163].
In a subsequent survey [53], 9 of 42 turkey skin samples harbored L. monocytogenes, with the
contamination rate apparently unrelated to the incidence in flocks before processing or after
defeathering. As was true for fresh chicken, additional testing at a local slaughterhouse once again
demonstrated that fresh turkey parts are most likely to become contaminated with Listeria during
the latter stages of processing (i.e., evisceration and chilling) (see Table 14.3). This scheme,
mentioned earlier as the route by which fresh poultry becomes contaminated, was further confirmed
by identifying various Listeria spp., including L. monocytogenes, in 4 of 15 (26.7%) samples of
mechanically deboned raw turkey meat obtained from the same slaughterhouse. In a similar study,
Wesley et al. [221] recovered L. monocytogenes and other Listeria spp. from 57 (38%) and 29
(19%) of 150 samples of mechanically separated turkey meat that were obtained from one mid-
western turkey processor. Among the 57 isolates serotyped, 29 (51%) and 25 (44%) belonged to
serotypes 1 and 4, respectively. Ryser et al. [182] further stressed the importance of postprocessing
contamination during separation and grinding when they reported that 33 of 45 (73%) retail samples
of ground turkey contained Listeria spp., including a diverse group of L. monocytogenes strains
belonging to nine different ribotypes.
TABLE 14.3
Incidence of Listeria spp. on Commercially Produced Fresh Chicken
and Turkey Parts before and after Being Packaged and/or Stored at 4°C
Type and Part Production Line 4-Day-Old Packaged

of Poultry Listeria sp. Beginning End Product Stored at 4°C
Chicken L. monocytogenes 0/20a 21/30 (70) 18/25 (72)

Wings L. innocua 0/20 6/30 (20) c 4/25 (16)
Legs L. monocytogenes 0/20 11/30 (37) 13/25 (52)
L. innocua 0/20 19/30 d (67) 17/25 (68)
Livers L. monocytogenes 0/31 5/15 (33) 6/15 (40)
L. innocua 2/31 (6.5)b 4/15 (27) 4/15 (27)
Turkey L. monocytogenes 1/30 (3.3) 0/30 ND
Wings L. welshimeri 1/30 (3.3) 4/30 (13.3) ND
Legs L. monocytogenes 0/30 2/30 (6.7) ND
L. welshimeri 1/30 (3.3) 1/30 (3.3) ND
Livers L. monocytogenes 0/30 0/30 ND
L. welshimeri 1/30 (3.3) 5/30 (16.7) ND
ND, not determined.

aNumber of positive parts/number of parts examined.
bPercentage positive.
cStrain of L. welshimeri also detected.
Source: Adapted from Refs. 72 and 73.
INCIDENCE OF LISTERIA SPP. IN POULTRY PRODUCTS MARKETED

IN EUROPE AND ELSEWHERE
RAW POULTRY
European scientists have been aware of the possible relationship between listeriosis in fowl and humans
for nearly 50 years, as evidenced by several reports in which infected poultry was found in the
immediate vicinity of human cases. Considering the fecal carriage rate for L. monocytogenes in
domestic and wild fowl, as well as the mechanized slaughtering practices that result in fecal contam-
ination of carcasses during defeathering, evisceration, and subsequent chilling in “spinchillers,” it is
not surprising that interest in the incidence of Listeria in poultry has increased. However, given that
the first European case of avian listeriosis was diagnosed in England during 1936 [169] and that no
additional cases were recorded in England over the next 22 years [84], one would probably be surprised
to learn that our first knowledge concerning the incidence of Listeria in European raw poultry products
originates from surveys made in England and Wales during the 1970s.
United Kingdom
After identifying L. monocytogenes in human stool samples from 32 of 5100 (0.6%) asymptomatic
individuals who resided in an area of Wales in which clinical cases of listeriosis had not been
identified for 15 years, Kwantes and Isaac [112] postulated that the fecal carriage rate may be
related to food consumption and began surveying both fresh and frozen chicken for L. monocyto-
genes. Following a 1971 preliminary report [112], Kwantes and Isaac [113] published final results
of their study in 1975 when they reported detecting L. monocytogenes on the internal/external
surface of 27 of 51 (52.9%) raw chickens obtained from a local processor (Table 14.4), with 23
(85.2%) and 4 (14.8%) L. monocytogenes isolates being identified as serotype 1 and 4b, respectively.
To determine actual public exposure to contaminated poultry, these investigators went to homes of
poultry consumers in Wales and swabbed the external/internal surfaces of locally purchased fresh
and frozen chicken, turkey, duck, and pheasant carcasses. Overall, L. monocytogenes was isolated
from 50% of fresh chickens sampled from home refrigerators, as well as from 64% of frozen
chickens stored in home freezers, thus demonstrating the ability of this pathogen to persist on
frozen carcasses. However, unlike chickens obtained directly from processors, L. monocytogenes
isolates of serotype 4b outnumbered those of serotype 1 on fresh and, particularly, on frozen chickens
obtained from consumers’ homes. Although relatively few samples were examined, isolation of
L. monocytogenes from the internal/external surface of one turkey, three ducks, and one wild
pheasant (see Table 14.4) indicates that improperly handled poultry products other than chicken
may also pose a potential threat to consumers.
One year later, Gitter [78] published results from a similar study, which examined the incidence
of L. monocytogenes on surfaces of various “over-ready” poultry products purchased at 26 different
shops and supermarkets in southern England. Using a combination of direct plating and cold
enrichment, L. monocytogenes was identified on 7 of 56 (12.5%) frozen and 2 of 6 (33.3%)
fresh chickens, as well as on 1 of 2 frozen ducks (see Table 14.4). Although the incidence of
L. monocytogenes on raw poultry products was markedly lower than that previously found by
Kwantes and Isaac [113], L. monocytogenes isolates identified as serotype 4 again outnumbered
those of serotype 1/2.
Following emergence of L. monocytogenes as a serious foodborne pathogen in June 1985, Pini
and Gilbert [173] determined the prevalence of this pathogen in uncooked fresh and frozen chickens
obtained from retail outlets throughout London. Unlike previous studies that relied on swab samples
from carcasses, these researchers examined two different samples from each chicken carcass
whenever possible—one sample consisting of edible offal (trimmings and/or viscera) and the other
a composite sample of skin and carcass remnants. Using cold enrichment in conjunction with the
Food and Drug Administration (FDA) procedure, L. monocytogenes was recovered from 33 of 50
(66%) fresh and 27 of 50 (54%) frozen chickens. These results are similar to those reported from
other 1987–1994 surveys [77,97,125,131,174] in which L. monocytogenes was detected on 10 of
16 (62.5%), 60 of 100 (60%), and 21 of 32 (66%) fresh chicken carcasses marketed in England
and Wales (see Table 14.4). According to a second report by Pini and Gilbert [172], other Listeria
spp., including L. innocua, L. seeligeri, and L. welshimeri, also were detected either alone or
together with L. monocytogenes in 26 and 30% of the fresh and frozen chicken samples tested,
respectively. Overall, 74 L. monocytogenes strains representing serotypes 1/2, 3a, 3b, 3c, 4b, 4d,
and two nontypeable strains, with serotype 1/2 predominating, were isolated from 60 edible offal
and 100 composite samples. Composite samples yielded more isolates of L. monocytogenes (57%)
as well as a higher percentage of other Listeria spp. (23%) than did edible offal samples (22 and
15%), respectively.
In another survey from 1994, 91% of cooked poultry samples contained listeriae. Although
L. monocytogenes was isolated from 59% of raw samples, all cooked samples were negative [116].
Among the chicken parts examined (e.g., drumsticks, breasts, wings, and livers), drumsticks were
most frequently contaminated with listeriae and also harbored the highest populations. From January
1997 to May 1998, Uyttendaele et al. [212] surveyed fresh poultry carcasses and poultry products
that were marketed in Belgium for Listeria. Among 44 samples originating from the United
Kingdom, 6 and 28 yielded L. monocytogenes at levels of >1 CFU/100 cm2 or 25 g and >1 CFU/cm2,
respectively, these levels being significantly higher than those detected in similar samples from
Belgium and France. In the only other survey reported to date, Soultos et al. [199] recovered L.
monocytogenes from 14 of 80 (17.5%) packages of chicken pieces that were purchased at various
supermarkets in Northern Ireland from September to December 2002.
TABLE 14.4
Incidence of L. monocytogenes in Raw Poultry Products Marketed in Europe
and Elsewhere
Number of Number of Positive
Country Type of Poultry Samples Analyzed Samples (%) Reference
Europe
Belgium Fresh chicken carcasses 279 111 (39.8) 212
and pieces
Belgium and France Fresh chicken carcasses 635 152 (23.9) 213
Fresh chicken parts 421 113 (26.8) 213
Fresh turkey parts 429 22 (5.3) 213
Denmark Fresh chicken 17 8 (47.1) 196
Fresh turkey parts 46 8 (17.4) 161
England/Wales Fresh chicken 100 60 (60.0) 174
Fresh chicken 64 41 (64.0) 113
Fresh chicken 50 33 (66.0) 172,173
Fresh chicken 50 27 (54.0) 172,173
Turkey 4 1 (25.0) 113
Frozen turkey 3 0 78
Fresh turkey 1 0 78
Duck 3 3 (100.0) 113
Frozen duck 2 1 (50.0) 78
Wild pheasant 2 1 (50.0) 113
Finland Fresh chicken pieces 61 38 (62.0) 132
France Fresh chicken carcasses 434 156 (65.9) 212
and pieces
Germany Fresh/frozen chicken 100 85 (85.0) 185,201
Unspecified 30 6 (20.0) 165
Unspecified 11 3 (27.3) 201
Italy Poultry 1,269 24 (1.9) 40
Fresh chicken 200 0 54
Fresh chicken carcasses 13 0 212
and pieces
Nordic countries Fresh chicken 18 4 (22.2) 85
Northern Ireland Fresh chicken 80 14 (17.5) 199
(supermarkets)
Norway Fresh chicken 150 75 (50.0) 179
Fresh chicken pieces 95 48 (50.5) 179
Portugal Fresh chicken 65 16 (24.6) 86
(continued)

Incidence of L. monocytogenes in Raw Poultry Products Marketed in Europe
and Elsewhere
Number of Number of Positive
Country Type of Poultry Samples Analyzed Samples (%) Reference
Spain Fresh chicken and turkey 158 57 (36.1) 216

Fresh chicken 5 0 198
Sweden Fresh chicken 45 0 202
Switzerland Fresh chicken 24 5 (20.8) 38
Turkey Fresh minced chicken 26 3 (11.5) 225
United Kingdom Fresh chicken carcasses 44 28 (63.6) 212
and pieces
Total 5478 1316 (24.8)
Elsewhere
Brazil Fresh and frozen chicken 90 4 (4.4) 114
Fresh chicken carcasses at 21 3 (14.3) 28
packaging
Egypt Frozen squab 50 0 (0) 1
India Fresh chicken 37 3 (8.1) 29
Poultry 50 0 (0) 167
Fresh chicken 9 0 (0) 60
Japan Minced chicken 46 17 (37.0) 103
Korea Fresh chicken 86 26 (30.2) 24
Malaysia Fresh chicken 16 4 (25.0) 74
South Africa Fresh chicken carcasses 15 2 (13.3) 214
(butcher)
Frozen chicken carcasses 17 6 (35.3) 214
(butcher)
Fresh chicken carcasses 45 9 (20.0) 214
(supermarket)
Frozen chicken carcasses 16 1 (6.3) 214
(supermarket)
Fresh chicken carcasses 6 1 (16.7) 214
(street vendor)
United Arab Fresh chicken 30 1 (3.3) 81
Emirates
Total 564 80 (14.2)
Despite wide differences in time, sample collection, sampling techniques, Listeria isolation meth-
odologies, and types of samples analyzed in the aforementioned studies, averaging the results in
Table 14.4 indicates that 196 of 447 (43.8%) fresh chickens and 75 of 170 (44.1%) frozen chickens
marketed in the United Kingdom from 1971 to 2002 contained L. monocytogenes. That the 1986
findings of Pini and Gilbert [172,173] are similar to those obtained in both American and European
surveys as far back as the mid-1970s underscores the continuing need for proper kitchen hygiene,
cooking of raw poultry products, and continuous inspection of carcasses (e.g., identification of liver
and heart lesions), along with use of good manufacturing and sanitizing practices in poultry
processing facilities. Because 152 of 214 (71%) clinical L. monocytogenes isolates obtained from
British patients between November 1986 and 1987 [130] were of serotype 4b, poultry products, in
which L. monocytogenes serotype 1/2 predominates, may be a less common vehicle for listeriosis
than other foods. However, pâtés, which are in essence poultry spreads prepared from chicken or
goose liver, may be an exception.
Continental Europe
Concern about the incidence of L. monocytogenes in raw poultry products marketed outside the
United Kingdom dates back to at least 1982 when two Swedish workers, Ternstrom and Molin
[202], examined 45 chickens obtained from two local slaughterhouses (see Table 14.4). Although
these researchers failed to isolate L. monocytogenes from any of the chickens examined, it appears
that the Listeria isolation /detection methods used in this study were primarily responsible for their
lack of success, because listeriosis in Swedish poultry is relatively common, with 112 cases being
diagnosed in the 10-year period between 1948 and 1957 [158]. In support of this view, more recent
surveys have yielded L. monocytogenes contamination rates of 47.1, 62.0, and 50.0 for fresh
chickens marketed in Denmark [196], Finland [132], and Norway [179]. Given the high incidence
of L. monocytogenes in raw poultry sold in the United States and the United Kingdom, inadequate
isolation/detection methods of the early 1980s also were likely responsible for the inability of Comi
and Cantoni [54] to recover this pathogen from approximately 200 chicken samples (i.e., carcass,
skin, and entrails) obtained from slaughterhouses in northern Italy.
After the 1985 cheeseborne listeriosis outbreak in California, Western European scientists began
to determine the incidence of Listeria in a wide range of foods, including fresh poultry products.
In the first of these studies, which was published in 1988, Skovgaard and Morgen [196] visited
two large Danish poultry slaughterhouses and examined chilled chicken carcasses for evidence of
Listeria contamination. According to these authors, Listeria spp. were detected in neck-skin samples
from 16 of 17 (94.1%) chicken carcasses, with L. innocua being identified in all but two Listeria-
positive samples. Although most of the poultry processed at these facilities was heavily contami-
nated with L. innocua, 8 of 17 (47.1%), 1 of 17 (5.9%), and 2 of 17 (11.8%) carcasses also
contained detectable levels of L. monocytogenes (see Table 14.4), L. innocua, and other Listeria spp.
(L. welshimeri, L. murrayi, and/or L. denitrificans), respectively. Thus the L. monocytogenes
contamination rate for chickens processed in Denmark closely reflected the average (43.8%) for
fresh chicken carcasses marketed in England and Wales. These Danish researchers also identified
Listeria spp., including L. monocytogenes, in chicken feces and transport cage material, further
supporting the widespread belief that poultry carcasses most likely become contaminated with
Listeria during evisceration and subsequent handling. This theory was later confirmed by Ojeniyi
et al. [162], who surveyed seven Danish abattoirs for L. monocytogenes. The pathogen was absent
in fecal samples from over 2000 broilers representing 90 randomly selected flocks. However, L.
monocytogenes was isolated from 0.3 to 18.7% of all poultry processing environmental samples
examined. Based on serotyping, phage typing, pulsed-field gel electrophoresis, and ribotyping, 62
distinct L. monocytogenes strains were identified from the 247 isolates tested, several of which
predominated in poultry processing samples. Interest in the incidence of Listeria in European fresh
poultry again intensified following reports that 34 individuals in Switzerland died after consuming
contaminated Vacherin Mont d’Or soft-ripened cheese. Breer [38] isolated L. monocytogenes,
L. innocua, and L. seeligeri from 5 of 24 (20.8%), 6 of 24 (25.0%), and 1 of 24 (4.2%) raw chickens
purchased in Switzerland (see Table 14.4), respectively. Using a modified version of the FDA
procedure, German investigators [185,200] recovered Listeria spp. and L. monocytogenes from 94
of 100 (94%) and 85 of 100 (85%) chicken carcasses, respectively. However, results from two
smaller surveys of German poultry [165,200] suggested far lower L. monocytogenes contamination
rates, with only 20.0–27.3% of unspecified fresh poultry carcasses (presumably chicken) containing
this pathogen (see Table 14.4).
The incidence of Listeria in raw poultry products has received increased attention in Belgium
and France, with L. monocytogenes recognized in French poultry since 1983 [34]. In the first large
survey of poultry carcasses collected from 33 abattoirs in France and Belgium, Uyttendaele et at.
[213] reported that the L. monocytogenes contamination rate decreased from 32.1% in 1992 to
9.2% in 1995, with 87–98% of the carcasses tested yielding L. monocytogenes at levels of <1
CFU/cm2. Contamination of poultry parts also decreased from 25.8 to 3% during this same 4-year
period, with L. monocytogenes-positive samples primarily being confined to chicken legs and wings.
In a follow-up study, Uyttendaele et al. [212] examined 772 poultry carcasses marketed in Belgium
during January 1997 to May 1998, most of which originated from Belgium and France with a few
samples also coming from Italy, The Netherlands, and the United Kingdom. Overall, 12.3 and
38.2% of these samples yielded L. monocytogenes at levels of >1 CFU/100 cm2 or 25 g and >1
CFU/cm2 or g, respectively, with generally similar contamination rates seen for samples coming
from Belgium and France. However, L. monocytogenes was more prevalent in a subset of samples
without skin (17.6%) rather than with skin (6.9%), suggesting contamination from the environment
during processing. In one additional subset, L. monocytogenes was recovered from 34 of 78 (43.6%)
and 41 of 82 (50.0%) French samples classified as free range and non–free range, respectively,
with this difference not statistically significant.
In 1995, Franco et al. [69] identified Listeria spp. on 96, 84, 80, and 0% of fresh chicken legs,
wings, breasts, and livers, respectively, that were processed at a Spanish facility, with Listeria
counts ranging from <2.0 to 2.8 log CFU/g on chicken wings. Follow-up testing of the processing
environment again showed that most of the contamination occurred during the latter stages of
production as carcasses entered the quartering room and exited on conveyor belts. Several additional
surveys revealed L. monocytogenes contamination rates of 0 to 36.0, 24.6 to 41.0, and 11.5% for
fresh chicken and turkeys marketed in Italy [40,71,126], Portugal [21,86], and Turkey [225],
respectively. Overall, these findings from continental Europe (Table 14.4) show that fresh poultry
is a common source of L. monocytogenes, with 1031 of 4898 (21.1%) samples taken primarily
from fresh chicken carcasses testing positive.
Elsewhere
At least 12 small-scale surveys for Listeria in raw poultry products from the Middle East, Asia,
Africa, and South America are now recorded in the scientific literature (see Table 14.4). In one of
these studies, 1 of 30 domestically grown fresh chickens from the United Arab Emirates [81]
reportedly contained Listeria spp., with one of these carcasses being positive for L. monocytogenes.
However, Listeria spp. were detected on 32 of 39 (82%) imported frozen chickens, 18 (46%) of
which contained L. monocytogenes. Similarly, Rusul et al. [74] identified L. monocytogenes in
4 of 16 (25%) poultry samples collected from six local Malaysian markets, with one of these
markets subsequently yielding L. monocytogenes in 15 of 24 (62.5%) samples. Limited work with
fresh poultry carcasses or pieces in India and Korea indicated L. monocytogenes contamination
rates of 0 to 8.1 and 10 to 30.2%, respectively, with 37.0% of minced chicken from Japan yielding
the pathogen. In the first survey of poultry processing facilities in South Africa [61], L. innocua
was recovered from the rubber fingers of a defeathering machine and on all neck-skin samples
after evisceration. L. monocytogenes was also detected on the rubber fingers, packaging funnel,
and all neck-skin samples after chilling. Following two studies that assessed microbial contamina-
tion in six raw poultry samples collected from street vendors in Johannesburg [135,136], Van Nierop
et al. [214] conducted a more extensive survey to determine the incidence of L. monocytogenes on
fresh and frozen chickens available from butchers, supermarkets, and street vendors in Gauteng, a
densely populated province in South Africa. Overall, 19.2, 19.2, and 32.3% of the 99 carcasses
were culture positive for L. monocytogenes, Salmonella spp., and Campylobacter, respectively.
However, 41 of 99 carcasses tested positive for L. monocytogenes using a PCR method. Furthermore,
12 of 66 (19.2%) fresh and 7 of 33 (21.2%) frozen carcasses rinse samples yielded L. monocytogenes,
with the highest and lowest contamination rates seen on frozen carcasses from butchers (35.3%)
and supermarkets (6.3%). Interestingly, only 1 of 6 chickens slaughtered and plucked by street
vendors at the time of purchase was positive for L. monocytogenes. In the last of these Listeria
surveys, Barbalho et al. [28] analyzed chicken carcass, chill water, and worker hand/glove samples
from one poultry processing facility in Bahia, Brazil that were collected over a 9-month period.
Listeria innocua was recovered from carcasses at bleeding, scalding, defeathering, and eviscer-
ation with 76.2% of the packaged carcasses positive for L. innocua. Only 3 of 21 carcasses at
packaging yielded L. monocytogenes (3.3%), with both L. monocytogenes and L. innocua orig-
inating from the hands and gloves of the workers. Overall, the data from Table 14.4 indicate
incidence rates of 24.8% and 14.2% for L. monocytogenes in fresh and frozen poultry marketed
in Europe and elsewhere, respectively, with the lower incidence of L. monocytogenes in products
of non-European origin most likely related to differences in sample collection methods and
isolation techniques.
COOKED/READY-TO-EAT POULTRY
Overwhelming evidence now points to the meat processing environment as the primary source for
Listeria contamination of deli meats, including turkey and chicken. With this premise in mind,
Lawrence and Gilmour [116] examined the incidence of Listeria spp., including L. monocytogenes,
in one poultry processing facility, along with raw and cooked chicken at this same facility, in
Northern Ireland between March and August 1992. Within the raw and cooked poultry processing
environments, 36 of 79 (46%) and 51 of 173 (29%) samples harbored Listeria spp., whereas 21 of
79 (26%) and 27 of 173 (15%) yielded L. monocytogenes, respectively. Contamination rates were
fairly uniform, with several environmental sites yielding L. monocytogenes throughout the study.
Among raw and cooked products tested, 53 of 58 (91%) and 8 of 96 (8%) contained Listeria spp.,
whereas 34 of 58 (59%) and none of 96 cooked samples yielded L. monocytogenes, respectively.
Although L. monocytogenes was absent from all cooked products tested, the presence of other
Listeria spp. and a subsequent report by the same authors [117] attesting to isolation of identical
L. monocytogenes strains from raw poultry, cooked poultry, and the processing environment (some
environmental strains of which persisted for up to 1 year) confirm the importance of minimizing
postprocessing contamination.
The association between human listeriosis and consumption of cooked/cooked-chilled/ready-
to-eat poultry products that are frequently held refrigerated for at least 5 days before being consumed
without further heating is now well documented as a result of four widely publicized outbreaks in
the United States that were directly linked to consumption of turkey frankfurters [83] and ready-
to-eat delicatessen turkey meat [49,70,163]. However, interest in the incidence of Listeria in poultry
originated in the United Kingdom 20 years ago. In the first survey of ready-to-eat poultry products
conducted by the Public Health Laboratory Service in London [76,77,174], L. monocytogenes was
present in 63 of 127 (12.0%) precooked ready-to-eat poultry products collected from London-area
retail establishments between November 1988 and January 1989. Little information is available
concerning actual numbers of L. monocytogenes present in cooked poultry products; however,
14 samples that were examined quantitatively contained <100 CFU/g. In addition L. monocytogenes
was isolated from 13 of 74 (18%) retail chilled meals, most of which were poultry products given
to hospital patients. The pathogen was also discovered in 6 of 24 (25%) cook-chilled poultry
products [14], 7 cook-chilled poultry dishes at levels up to 700 L. monocytogenes CFU/g, and
2 cooked chicken products labeled “ready-to-eat” that contained up to 400 L. monocytogenes CFU/g.
Working at the Cardiff Public Health Laboratory Service in Wales, Morris and Ribeiro [134]
determined the potential Listeria-related risks associated with consumption of pâté, an appetizer-
type poultry spread that is typically prepared from chicken or goose liver. According to their report,
14 varieties of pâtés (primarily imported from Belgium) were obtained in bulk from area
delicatessen counters or in unopened packages from supermarket refrigerators and examined for
L. monocytogenes using established methods. Overall, this pathogen was isolated from 37 of 73
(50.4%) pâtés, with 28 (75.7%) and 9 (24.3%) of these positive samples originating from
delicatessens and supermarket refrigerators, respectively. These pâtés were subsequently withdrawn
from the market after officials discovered dangerously high L. monocytogenes populations of
≥104–105 CFU/g in seven samples [9]. L. monocytogenes serotype 4b, the serotype responsible for
~80% of all human listeriosis cases in England and Wales, was isolated from 36 of 37 positive
samples, with one strain of L. monocytogenes serotype 4b matching clinical isolates from a 1987
cluster of listeriosis cases in which the exact origin of illness could never be determined.
These findings prompted Public Health Laboratory officials to greatly expand their survey of
pâté. By March 1990 [75,174], workers at 48 of 53 (90.6%) participating laboratories in England
and Wales isolated L. monocytogenes from 187 of 1834 (10.2%) samples of imported and domestic
pâté. As in the previous survey, 10% of all positive samples contained >104−106 L. monocytogenes
CFU/g, with over half of all isolates belonging to serotype 4. Following this pâté-related outbreak,
the number of listeriosis cases reported in England and Wales decreased to approximately half the
level reported in 1988. Although some individuals expected to see a further decrease [75,174], the
incidence of human listeriosis in the United Kingdom has since stabilized, with about 100–125
cases being reported annually over the last 5 years.
In another study, Lieval et al. [121] isolated L. monocytogenes, L. seeligeri, and L. innocua
from one of nine, two of nine, and one of nine chicken sandwiches obtained from cafes in and
around Paris. Although identical efforts to recover Listeria from 20 fast-food fried chicken items
failed, the ability of such foods to harbor Listeria, including L. monocytogenes, has been well
established by the previously discussed surveys in England. More recently, Rijpens et al. [175]
reportedly recovered Listeria spp. and L. monocytogenes from 35.5 and 15.5%, respectively, of
poultry samples examined in Belgium, with the incidence of Listeria spp. markedly higher in
unpackaged (41.7%) than in prepackaged (11.1%) poultry.
BEHAVIOR OF L. MONOCYTOGENES IN RAW AND COOKED

POULTRY PRODUCTS
Although L. monocytogenes was first detected on European raw chicken nearly 30 years ago, the
behavior of Listeria in raw and processed poultry products received no attention until this organism
was recognized as a bona fide foodborne pathogen in the mid-1980s. Initial research efforts provided
the poultry industry with valuable information concerning growth of L. monocytogenes in raw and
cooked chicken products, including levels of heat and microwave/gamma irradiation needed to
destroy this pathogen in raw chicken. However, our present-day knowledge of Listeria behavior in
raw and processed chicken products has greatly expanded since 1998, when the first of four poultry-
related listeriosis outbreaks in the United States was directly linked to consumption of turkey
frankfurters [83] that became surface-contaminated after processing. Results from these studies
should add much to our knowledge about Listeria behavior in poultry products and aid in the
development of improved microbial reduction strategies that will decrease the incidence of this
pathogen in raw and ready-to-eat poultry products.
GROWTH: RAW CHICKEN

Wimpfheimer et al. [222] were among the first to examine the behavior of L. monocytogenes in
raw chicken. In their study, raw minced chicken meat was inoculated to contain 102 L. monocyto-
genes CFU/g, packaged anaerobically (75% CO2:25% N2), microaerobically (72.5% CO2:22.5%
N2:5% O2), or aerobically (air) and examined for numbers of L. monocytogenes as well as aerobic
spoilage organisms during storage at 4, 10, or 27°C. Neither L. monocytogenes nor aerobic spoilage
organisms grew in anaerobically packaged raw chicken during extended storage at any of the three
temperatures, as also reported by Hart et al. [92], with both populations decreasing to <10 CFU/g
Bacteria log10 CFU/g
FIGURE 14.2 Growth of L. monocytogenes and aerobic spoilage organisms in aerobically (O), microaerobi-
cally (), and anaerobically () packaged raw chicken during incubation at 4°C. (Adapted from Wimpfheimer,
L., N.S. Altaian, and J.H. Hotchkiss. 1990. Growth of Listeria monocytogenes Scott A, serotype 4 and
competitive spoilage organisms in raw chicken packages under modified atmospheres and in air. Int. J. Food
Microbiol. 11: 205–214.)
after 6 days of storage at 4°C (Figure 14.2). When packaged aerobically or microaerobically under
conditions more closely simulating commercial practices, numbers of L. monocytogenes in raw
chicken increased rapidly during extended storage at 4°C, whereas growth of aerobic spoilage
organisms was strongly inhibited (see Figure 14.2). Under these conditions, L. monocytogenes can
rapidly proliferate in normal unspoiled raw chicken during refrigerated storage, as also reported
by two other investigators [92,115]. Zeitoun and Debevere [226] also later reported on the successful
use of 10% lactic acid/sodium acetate buffer (pH 3) in conjunction with modified atmosphere
packaging (90% CO2:10% O2) to prevent growth of L. monocytogenes on uncooked chicken legs
and extend the product’s shelf life at 6°C to 17 days. According to Wimpfheimer et al. [222], the
ability of L. monocytogenes to grow in microaerobically packaged raw chicken was not affected
by initial levels of Listeria (<101 or 102 CFU/g) or aerobic spoilage organisms (104 or 108 CFU/g).
However, the ratio of Listeria to spoilage organisms was strongly temperature dependent, with both
organisms reaching populations of 107–108 and 109–1010 CFU/g in microaerobically packaged
chicken following < 2 and 8 days of storage at 10 and 27°C, respectively. Neither L. monocytogenes
nor aerobic spoilage organisms were inhibited in aerobically packaged raw chicken, with Listeria
and spoilage organisms attaining populations >107 and 109 CFU/g, respectively, in products stored
at 4, 10, and 27°C. Thus, except for microaerobically packaged product, raw chicken would likely
become overtly spoiled before L. monocytogenes could proliferate to the point at which the pathogen
might be detectable in minimally cooked chicken. Nevertheless, it is important to remember that
consumers must take special precautions to prevent cross-contamination between raw chicken that
may contain L. monocytogenes, Salmonella, and/or Campylobacter and ready-to-eat products,
including cooked chicken.
Antimicrobial Additives
L. monocytogenes is now considered a microbiological hazard that is reasonably likely to occur in

any poultry processing facility in the United States and must now be addressed in USDA-mandated
HACCP programs. Various food additives have been evaluated for their ability to inactivate Listeria
spp. on fresh poultry. In the first of such studies, dipping inoculated chicken wings in 10% trisodium
phosphate (TSP) at 10°C for 15 sec and hot water (95°C) for 5 sec resulted in a 79.5% reduction
in numbers of L. monocytogenes, with minimal changes in subcutaneous temperature [177]. Hwang
and Beuchat [100] found that washing chicken skin in 1% TSP or 1% lactic acid significantly
reduced viable populations of L. monocytogenes compared with washing in water. According to
Shelef and Yang [191], growth of L. monocytogenes in sterile comminuted chicken was slowed
during refrigerated storage by adding 4% sodium or potassium lactate. When used at levels ≥ 0.3%,
sodium diacetate also was inhibitory to L. monocytogenes in poultry slurries [184], with the
antilisterial activity of sodium diacetate being further enhanced by supplementing the product with
0.25% ALTA, a commercially available microbially produced shelf life extender. More recently,
several investigators have shown that a 15-min treatment in 12% TSP [44,45,46,176] or
chlorine-based sanitizers [82,181] just before packing can greatly minimize or suppress growth
of L. monocytogenes, Salmonella, and other surface contaminants on raw poultry carcasses
and pieces during refrigerated storage.
Susceptibility to Gamma Radiation
Survey results indicate that up to ~60% of all raw poultry products sold in retail stores may be
contaminated with L. monocytogenes, Salmonella, and Campylobacter. These statistics have prompted
development of various processes to eliminate such pathogens from raw poultry. Exposure to the
bactericidal effects of gamma radiation appears to be among the most effective means of reducing
populations of both pathogenic and spoilage organisms. Although presently allowable, gamma radiation
doses of 2.5–7.0 kGy in England [138] and <3.0 kGy in the United States [17] are generally regarded
as sufficient for eliminating these organisms from raw poultry [203]. Readers should be aware that
exposing cooled poultry to levels >2.5 kGy may adversely affect product odor, color, and flavor [88].
In 1989, Patterson [171] examined sensitivity of Listeria to gamma radiation using radiation-
sterilized raw minced chicken meat that was inoculated to contain ~106 L. monocytogenes CFU/g.
After exposing the product to gamma radiation doses of 0, 0.5, 1.0, 1.5, 2.0, and 2.5 kGy, L.
monocytogenes exhibited a D10-value (i.e., radiation dose required to decrease the population 10-
fold) of 0.42 to 0.55 kGy, depending on bacterial strain and type of plating medium used to quantify
the pathogen in minced poultry. In support of these findings, Huhtanen et al. [98] also reported
that a gamma radiation dose of 2 kGy was sufficient to inactivate an L. monocytogenes population
of ~104 CFU/g (average D-value of 0.45 kGy) in artificially contaminated, mechanically deboned
chicken. Similar D-values also have been published for Salmonella spp. in fresh poultry [137].
Although all of the aforementioned studies support the use of 2.5 kGy of gamma radiation to
eliminate L. monocytogenes at levels of <104 CFU/g from raw poultry meat, researchers in England
[131] recovered this pathogen from 1 of 12 (8.3%) fresh chicken carcasses that had been surface-
inoculated to contain L. monocytogenes at approximately 102 or 104 CFU/cm2 and exposed to 2.5
kGy of gamma radiation. More important, after extended storage at 5–10°C, the pathogen was recovered
from 1 of 18 (5.6%) and 7 of 18 (38.8%) irradiated carcasses that originally contained low and high
inoculum levels, respectively. When poultry carcasses were inoculated to contain >104 L. monocytogenes
CFU/g, several investigators [120,215] also confirmed that small numbers of the pathogen survived
irradiation at 0.5 kGy and eventually grew, particularly in the absence of air, in samples stored at
4°C. However, Shamsuzzaman et al. [189] reported that combined use of 3.1 kGy irradiation and
sous-vide cooking to an internal temperature of 71.1°C was sufficient to reduce L. monocytogenes
populations in chicken breast meat from 106 CFU/g to undetectable levels, with no growth of the
pathogen being observed during 5 weeks of storage at 8°C. Hence, in the absence of multiple
treatments, these findings suggest that small numbers of Listeria may either escape sublethal injury
during irradiation or undergo repair and grow on these carcasses during refrigerated storage.
From this information, it appears that a gamma radiation dose of 2.5 kGy may be only
marginally sufficient to inactivate levels of Listeria that one might reasonably expect to find on
naturally contaminated raw poultry. Nevertheless, provided that irradiated poultry products are
properly packaged to prevent recontamination (and subsequent growth) with Listeria and other
foodborne pathogens, this procedure should markedly decrease the risk of contaminating ready-to-
eat foods (e.g., salads and raw vegetables) during consumer preparation in the kitchen. Unfortu-
nately, although the scientific community generally contends that foods exposed to such low levels
of radiation are safe for human consumption, irradiated foods have not yet gained full acceptance
by consumers. Perhaps the continued outpouring of scientific evidence will eventually curb the
remaining unfounded fear regarding the safety of irradiated foods in the mind of the general public.
GROWTH: COOKED/READY-TO-EAT POULTRY PRODUCTS

Confirmation of cooked-chilled chicken and turkey frankfurters as vehicles of listeriosis in England
and the United States during 1988 and 1989 prompted immediate international efforts to assess the
potential hazards associated with growth of L. monocytogenes in a wide range of retail cooked/ready-
to-eat poultry products. Six of these early studies are summarized in Table 14.5. Because all these
studies differ in experimental design, sampling times, and initial inoculum levels of L. monocytogenes,
these findings cannot be compared directly. However, it is evident that numbers of Listeria increased
1−6 orders of magnitude in all six artificially contaminated products after 6−28 days of storage at 3−7°C.
Higher populations were observed in aerobically packaged as opposed to vacuum-packaged or mod-
ified-atmosphere-packaged products as reported by Murphy et al. [145], who also found that an
atmosphere containing 15−30% CO2 decreased L. monocytogenes populations about 1 log on cooked
chicken-breast patties during extended storage at 8°C. Equally important, sensory acceptability of
these products was not altered by growth of this pathogen. Overall, L. monocytogenes grew most
abundantly in vacuum-packaged sliced chicken and in one brand of sliced turkey, both of which were
similar in pH (6.3 and 6.4) and in contents of moisture (71.3 and 74.0%), protein (18.9 and 22.6%),
carbohydrate (1.3 and 0.9%), and salt (1.7 and 1.4%). These authors attributed decreased growth of
the pathogen in a second brand of sliced turkey to higher levels of salt (2.7%) and carbohydrate
(1.7%); the latter was largely responsible for the eventual decrease in pH of this product to 4.97.
These findings are in general agreement with several predictive models for growth of L. monocytogenes
in different poultry products [35,59,160].
In a 1990 report by Ingham et al. [102] cooked/sterilized chicken loaf was inoculated to contain
103 L. monocytogenes and 103 Pseudomonas fragi CFU/g, packaged aerobically (air), microaero-
bically (10% O2), or anaerobically and examined for both organisms during 6 days of incubation
at 3, 11, and 37°C. Regardless of incubation temperature, P. fragi attained maximum populations
of approximately 4 × 109 CFU/g in all aerobic samples after 6 days of storage (Figure 14.3).
However, growth of Listeria was totally or partially suppressed in similar samples stored at 3°C
for up to 15 days [101]. Although both organisms attained lower maximum populations in cooked
chicken loaf following 6 days of microaerobic or anaerobic incubation at all three temperatures,
these conditions led to Listeria populations that were 1−2 orders of magnitude higher than those
attained by P. fragi. Thus, as was true of raw poultry (see Figure 14.2), microaerobic and anaerobic
refrigerated storage both appear to selectively favor growth of L. monocytogenes over P. fragi and
TABLE 14.5
Populations of L. monocytogenes (log10 CFU/g) in Artificially Contaminated
Cooked/Ready-to-Eat Poultry Products of Acceptable Organoleptic Quality
during Extended Refrigerated Storage
Incubation Length of Incubation (days)
Temperature Initial
Product (°C) Inoculum 3 6 8 10 14 20 28 Reference
Sliced chickena 4.4 2.8 —b — — — 6.9 — — 79

4.4 0c — — — — 5.9 — — 79
Sliced turkey 4.4 3.0 — — — — 5.0 — 6.2 79
(brand A)a 4.4 –1.3 — — — — 2.4 — 3.7
Sliced turkey 4.4 2.9 — — — — 6.7 — — 79
(brand B)a 4.4 –1.7 — — — — 4.8 — 7.7
Chicken homogenate 4 6.7 — — — — — 9.4 — 195
4 2.7 — — — — — 7.9 —
Breaded chicken fillets 5 2.7 — 3.6 — — — — — 194
5 1.7 — 3.6 — — — — —
Chicken casserole 3 2.7 2.9 3.5 3.3 3.6 — — — 77
6 2.7 3.6 5.3 6.3 7.5 — — —
Chicken nuggets 3 4.8 4.8 5.2 5.7 6.1 6.3 — 127
7 4.8 5.3 6.0 6.1 7.4 — —
Chicken nuggetsd 3 4.7 4.8 4.8 5.0 5.2 6.0 — 127
7 4.7 4.9 5.1 5.3 5.3 — —
aVacuum-packaged.
bNot tested.
c1 CFU/g.
dModified atmosphere—80% CO : 20% N .

2 2
possibly other spoilage organisms, which, in turn, could potentially yield an organoleptically
acceptable product with dangerously high numbers of Listeria.
Identification of turkey frankfurters as the infectious vehicle in a 1988 case of listeriosis
involving an Oklahoma breast-cancer patient eventually led to a safety assessment of poultry
sausage. According to McKellar et al. [129], L. monocytogenes grew on the surface of 13 of 27
(48%) artificially contaminated retail poultry wieners, with populations on some vacuum-packaged
samples increasing nearly 4 orders of magnitude during 21 days of storage at 5°C. Wederquist et al.
[220] reported similar growth of L. monocytogenes when vacuum-packaged slices of turkey bologna
were stored at 4°C. However, incorporating 0.5% sodium acetate, 2.0% sodium lactate, or 0.26%
potassium sorbate into the product completely suppressed growth of the pathogen during the first
35 days of refrigerated storage, with populations in 3-month-old samples remaining 2−5 orders of
magnitude lower than those in the controls. Two additional studies on fermented summer sausage
prepared from ground chicken [23] and turkey [122] also demonstrated the importance of an active
starter culture in limiting growth of L. monocytogenes during fermentation. When these sausages
were prepared from a chicken or turkey batter (pH 6.6) containing a pediococcal starter culture
and L. monocytogenes at a level of 104−107 CFU/g, numbers of Listeria decreased 0.9−1.8 orders
of magnitude after an 11-h fermentation (pH 5). Replacing this pediococcal starter culture with a
pediocin-producing strain of Pediococcus acidilactici essentially doubled the inactivation rate of
L. monocytogenes during fermentation. However, regardless of the starter culture used, all remaining
listeriae were subsequently inactivated during 45 min of cooking to an internal temperature
10
9 L. monocytogenes
P. fragi
8
7
Bacteria log10 CFU/g
0 1 4 6
Days
FIGURE 14.3 Growth of L. monocytogenes and Pseudomonas fragi in aerobically (O), microaerobically (),
and anaerobically () packaged cooked chicken loaf during incubation at 7°C. (Adapted from Ingham, S.C.,
J.M. Escude, and P. McCown. 1990. Comparative growth rates of Listeria monocytogenes and Pseudomonas
fragi on cooked chicken loaf stored under air and two modified atmospheres. J. Food Prot. 53: 289–291.)
of 66.5°C. Thus, an active fermentation combined with normal thermal processing before packaging
should result in a Listeria-free product.
Several investigations also assessed behavior of Listeria in inoculated samples of chicken
gravy and chicken broth during cooling and/or refrigerated storage. According to Huang et al. [95],
L. monocytogenes populations in individual 1000-g samples of artificially contaminated chicken gravy
(prepared from poultry stock, spices, waxy maize wash, and chicken base) increased by 2 orders of
magnitude as the product cooled from 40 to 9°C during 24 h of storage at 7°C (Figure 14.4). Even
though generation times for L. monocytogenes approximately doubled after the chicken gravy stabi-
lized at 7°C, the pathogen still attained a maximum population of 109 CFU/g in 8-day-old gravy.
Huang et al. [96] subsequently reported a reduction in L. monocytogenes when similar chicken gravy
samples were held at 65°C for 1.3 min; however, such a heat treatment is clearly inadequate to inactivate
higher Listeria populations that can develop in chicken gravy during prolonged refrigerated storage.
Walker et al. [219] also demonstrated the ability of three L. monocytogenes strains to multiply in
artificially contaminated sterile chicken broth (pH ∼ 6.4) held at near-freezing temperatures, with
Listeria populations in this product increasing 100-fold during extended incubation at 0.8°C
(Figure 14.5). In fact, growth of this pathogen was also evident in samples of chicken broth that were
held at temperatures as low as −0.4°C, below which the broth froze and was no longer sampled.
Results from these investigations stress the importance of cooling foods as rapidly as possible
and show that hazardous situations can easily develop if refrigerated foods are subjected to mild
temperature abuse, that is, holding at temperatures above 4°C. In an effort to increase the safety
of cooked, cooked-chilled, and ready-to-eat poultry, USDA officials lowered the recommended
long-term storage temperature for such products from 4.4 (40) to 1.7°C (35°F) and also have
developed stricter guidelines that require faster (than previously recommended) cooling of warm
products at the end of manufacture [4]. Continued attention to rapid cooling of finished products
10
50
L. monocytogenes
9 Temperature 45
40
8
35
Temperature (°C)
7 30
25
6 20
15
5
10
4 0
0 1 2 3 4 5 6 7 8
Days
FIGURE 14.4 Behavior of L. monocytogenes in chicken gravy during cooling to 7°C and extended storage.
(Adapted from Huang, D., A.E. Yousef, M.E. Matthews, and E.H. Marth. 1993. Growth and survival of Listeria
monocytogenes in chicken gravy during cooling and refrigerated storage. J. Food Serv. Syst. 7: 185–192.)
and avoidance of postprocessing contamination are both essential to decrease the incidence of this
psychrotrophic pathogen in cooked/ready-to-eat poultry products.
THERMAL INACTIVATION DURING COOKING

Heating is the most obvious means of destroying L. monocytogenes and other foodborne pathogens
in any raw food, including poultry. However, numerous early reports attesting to unusual thermal
8
Log10 CFU/mL
0 10 20 30 40 50
Days
FIGURE 14.5 Growth of L. monocytogenes in sterile chicken broth during extended incubation at 8.7 (),
3.5 (), 1.5 (), and 0.8°C (). (Adapted from Walker, S.J., P. Archer, and J.G. Banks. 1990. Growth of
Listeria monocytogenes at refrigeration temperatures. J. Appl. Bacteriol. 68: 157–162.)
tolerance of L. monocytogenes in various foods, coupled with discovery of L. monocytogenes in

several cooked poultry products that were directly linked to cases of listeriosis in the late 1980s,
have raised questions about the exact thermal processing times and temperatures required to
eliminate this pathogen from raw poultry products. Beginning in 1989, Carpenter and Harrison
reported results from three studies in which raw chicken breasts were surface-inoculated to contain
∼105−107 L. monocytogenes CFU/g and cooked to internal temperatures of 65.6, 71.1, 73.9, 76.7,
or 82.2°C using dry heat [48], moist heat [90], and microwave radiation [91]. All cooked chicken
breasts were then vacuum-packaged or wrapped in oxygen-permeable film and analyzed for
numbers of Listeria during 4 weeks of storage at 4 and 10°C.
Overall, L. monocytogenes was recovered from chicken breasts cooked to all five internal
temperatures using dry heat, moist heat, and microwave radiation. As expected, the magnitude of
lethality was directly related to cooking temperature. Because chicken breasts contained somewhat
different levels of L. monocytogenes before heating, one cannot directly compare the effectiveness
of the three cooking methods used in these studies. However, assuming that L. monocytogenes
populations in these chicken breasts decreased linearly during heating (admittedly, some “tailing”
of the survivor curve likely occurred at the three highest temperatures using dry heat, moist heat,
and microwave irradiation), then the number of survivors in chicken breasts that contained any
initial inoculum can be estimated. Thus, if Carpenter and Harrison had used an initial population
of 1.0 × 106 L. monocytogenes CFU/g in all three studies, one would expect their results to have
been similar to the estimated number of survivors shown in Table 14.6. Considering these approx-
imations, it appears that L. monocytogenes was generally more tolerant of microwave radiation
than dry or moist heat, with numbers of Listeria decreasing less than 4 orders of magnitude on
chicken cooked to an internal temperature of 82.2°C. Of greater importance is the fact that Listeria
populations decreased < 2 orders of magnitude on chicken breasts cooked to an internal temperature
of 71.1°C, the minimum internal temperature to which poultry was previously required to be heated
to designate the product as fully cooked in the United States [3]. Although numbers of Listeria
decreased approximately 5 orders of magnitude when chicken was cooked to higher internal
temperatures using either dry or moist heat, these authors [89] later demonstrated that moist heating
of surface-inoculated chicken breasts to an internal temperature of 73.9°C also failed to completely
inactivate more realistic L. monocytogenes populations of ~102−104 CFU/g. Overall, microwave
TABLE 14.6
Estimated Decrease in Numbers of L. monocytogenes on the Surface of Chicken Breasts
Inoculated to Contain 6.00 log10 CFU/g and Cooked to Various Internal Temperatures
Using Dry Heat, Moist Heat, or Microwave Radiation
Decreasea in L. monocytogenes Population (log10 CFU/g) after Cooking to Various
Internal Temperatures
Cooking method 65.6°C 71.1°C 73.9°C 76.7°C 82.2°C

Dry heat 2.42 2.00 5.05 5.24 5.04
Moist heat 2.08 1.83 3.46 5.08 4.96
Microwave radiation 0.82 1.95 2.50 3.77 3.26
aInitial inoculum of 6 log10 L. monocytogenes CFU/mL.
Source: Adapted from Carpenter, S.L. and M.A. Harrison. 1989. Survival of Listeria monocytogenes on processed
poultry. J. Food Sci. 54: 556–557; Harrison, M.A. and S.L. Carpenter. 1989. Survival of large populations of Listeria
monocytogenes on chicken breasts processed using moist heat. J. Food Prot. 52: 376–378; Harrison, M.A. and S.L.
Carpenter. 1989. Survival of Listeria monocytogenes on microwave cooked poultry. Food Microbiol. 6: 153–157.
heating was less effective than either dry or moist heating, with Listeria populations decreasing
less than 4 orders of magnitude on chicken breasts cooked to an internal temperature of 82.2°C
(see Table 14.6). In 1989, researchers in England [123] also reported that microwave heating was
less effective than other forms of cooking for eliminating L. monocytogenes from the surface and
interior (stuffing) of whole stuffed chickens (~1.7 kg each) inoculated to contain 106 and 107 Listeria
CFU/g of skin and stuffing, respectively. These findings were further substantiated by Farber et al.
[64] using naturally contaminated raw whole broilers. As a result of uneven heating, which is an
inherent problem in microwave cooking, 20 min of additional standing time was required after
38 min of cooking (final skin temperature of 80–99°C) to completely inactivate the pathogen on
the surface of whole chickens. However, low levels of L. monocytogenes (<10 CFU/g) were still
detected in stuffed samples from one of two similarly treated whole chickens that attained temperatures
of 72–85°C after 20 min of standing. Thus, these findings serve as a warning to people who regularly
cook large stuffed birds (particularly turkeys) in microwave ovens, and they also stress the impor-
tance of postcooking standing time for further inactivation of Listeria and other foodborne pathogens
after microwave cooking.
Not surprisingly, follow-up work by Carpenter and Harrison [48,90,91] demonstrated that
L. monocytogenes survivors (most likely sublethally injured during heating) can persist and multiply
on both oxygen-permeable film-wrapped and vacuum-packaged cooked chicken during extended
storage at 4 and 10°C. As shown in Figure 14.6, growth of Listeria on chicken breasts packaged
7
Dry heat (D)
Moist heat (M)
6 Microwave heat
(M)
1 (D)
(D)
0 1 2 3 4
Weeks
FIGURE 14.6 Effects of three different heating methods on behavior of L. monocytogenes on inoculated
chicken breasts that were cooked to internal temperatures of 71.1 (), 76.7 (), or 82.2°C (), packaged in
oxygen-permeable film, and stored at 4°C. (Adapted from Carpenter, S.L. and M.A. Harrison. 1989. Survival
of Listeria monocytogenes on processed poultry. J. Food Sci. 54: 556–557; Harrison, M.A. and S.L. Carpenter.
1989. Survival of large populations of Listeria monocytogenes on chicken breasts processed using moist heat.
J. Food Prot. 52: 376–378; Harrison, M.A. and S.L. Carpenter. 1989. Survival of Listeria monocytogenes on
microwave cooked poultry. Food Microbiol. 6: 153–157.)
in oxygen-permeable film was most evident after 2 weeks of refrigerated storage, with larger
populations generally developing on products that were cooked using microwave radiation, followed
by those given moist or dry heat. Most important, L. monocytogenes was recovered via direct
plating from all 4-week-old aerobically packaged chicken breasts except those that were cooked
to an internal temperature of 82.2°C using moist heat. In addition, higher numbers of Listeria also
developed on aerobically packaged chicken breasts that were exposed to less severe heat treatments.
As expected, increasing the incubation temperature also led to much faster growth of Listeria, with
the pathogen generally attaining a level of 106–107 CFU/g on aerobically packaged chicken breasts
after only 7 days at 10°C.
Behavior of L. monocytogenes on chicken breasts was influenced by the heating method/treatment,
temperature at which the cooked product was ultimately stored, and type of packaging material.
According to these same authors, Listeria populations were generally 1–2 orders of magnitude
lower in vacuum-packaged than in aerobically wrapped product following 4 weeks of storage at
4°C; however, the pathogen was present in all 4-week-old samples except those that were originally
cooked to an internal temperature of 82.2°C using moist or dry heat (Figure 14.7). Although numbers
of Listeria were again generally 1–2 orders of magnitude lower in vacuum-packaged than in
aerobically wrapped chicken breasts following 1 week of storage at 10°C, populations were as
much as 5 orders of magnitude lower in vacuum-packaged chicken breasts that were cooked to an
internal temperature of 82.2°C using moist heat.
7
Dry heat (D)
Moist heat (M)
Microwave heat
6
5
(M) (D)
1 (M)
(D)
(D)
(D)
0 1 2 3 4
Weeks
FIGURE 14.7 Effects of three different heating methods on behavior of L. monocytogenes on inoculated
chicken breasts cooked to internal temperatures of 71.1 (), 76.7 (), or 82.2°C (), vacuum-packaged, and
stored at 4°C. (Adapted from Carpenter, S.L. and M.A. Harrison. 1989. Survival of Listeria monocytogenes
on processed poultry. J. Food Sci. 54: 556–557; Harrison, M.A. and S.L. Carpenter. 1989. Survival of large
populations of Listeria monocytogenes on chicken breasts processed using moist heat. J. Food Prot. 52:
376–378; Harrison, M.A. and S.L. Carpenter. 1989. Survival of Listeria monocytogenes on microwave cooked
poultry. Food Microbiol. 6: 153–157.)
Because raw chicken normally contains <1000 L. monocytogenes CFU/g [131] and Listeria
populations generally decrease approximately 3–5 orders of magnitude in fully cooked poultry
heated to an internal temperature of 63.9°C, as previously specified in the U.S. Code of Federal
Regulations, such cooking temperatures were, at best, only marginally adequate to eliminate this
pathogen from raw poultry products. In fact, Harrison and Carpenter [89] reported that moist heating
of inoculated chicken breasts to an internal temperature of 73.9°C failed to completely inactivate
L. monocytogenes surface populations of 102–104 CFU/g, with the pathogen reestablishing itself at
levels equal to or greater than the original inoculum level after 4 weeks at 4°C. Nonetheless, the
adequacy of this poultry processing method was maintained by another report [6], which indicated
that a turkey meat emulsion (containing salt, sodium tripolyphosphate, carrageenan, and water)
inoculated to contain 5.78 L. monocytogenes log10 CFU/g was free of the pathogen after holding
the product at 71.1°C for 2 min (estimated D71.1° C = 0.28 min).
Following mandatory HACCP implementation by all meat and poultry processors in the
United States, USDA–FSIS changed its cooking requirement in 1999 from specific end-point
temperatures to minimum required reductions for pathogens. This latter lethality-based perfor-
mance standard requires a minimum Salmonella reduction of 7.0 logs for fully cooked poultry
[207] and other ready-to-eat poultry products [208]. FSIS has supported use of Salmonella
cocktails containing relatively heat-resistant strains to verify the adequacy of various time/temperature
treatments [208]. Using multistrain cocktails of Salmonella and L. monocytogenes, Murphy and
colleagues at the University of Arkansas [148] reported that Salmonella was more heat resistant
than L. monocytogenes in ground chicken thigh/leg meat. However, cooking chicken leg quarters
in an air-impingement oven to an internal temperature of 73.9°C, as previously required by
FSIS, sometimes failed to decrease L. monocytogenes 7 logs as now required by USDA–FSIS
[154]. Using these same cocktails of Salmonella and L. monocytogenes, Murphy et al. [147]
demonstrated greater heat resistance of L. monocytogenes in previously cooked chicken breast,
turkey breast, duck muscle, and duck skin. When the same Salmonella cocktail was compared
to L. innocua M1—a heat-resistant surrogate for L. monocytogenes, Listeria was somewhat
more heat resistant than Salmonella in ground chicken breast meat [143,144] and ground turkey
[153], with similar resistance reported for chicken patties [149] and chicken tenders [149]. Con-
sequently, certain poultry products that meet the 7-log reduction for Salmonella may not
necessarily be free of Listeria.
USDA GUIDELINES FOR CONTROL OF LISTERIA

IN READY-TO-EAT POULTRY
Despite the findings just discussed that suggest possible inadequacies in the lethality-based performance
standards in regard to Listeria, overwhelming evidence from the more than 80 recalls to date points
to postprocess contamination (rather than contamination during processing) as the root of the problem.
In response to the 1998 turkey frankfurter outbreak and three others that were traced to ready-to-eat
delicatessen turkey meat [49,70,163], various postcook thermal and nonthermal pasteurization strategies
were investigated for packaged products along with product formulations that would suppress growth
of Listeria during extended storage. The end result in October 2003 was issuance of the USDA–FSIS
rule on L. monocytogenes in ready-to-eat meat and poultry products [209] that applied to any product
exposed to the processing environment between cooking and packaging. This rule mandated that
manufacturers of ready-to-eat meat and poultry products adopt one of the following three L.
monocytogenes control alternatives: (1) use of a postlethality treatment to reduce or eliminate L.
monocytogenes and an antimicrobial agent or process that suppresses or limits L. monocytogenes
throughout shelf life, (2) use of a postlethality treatment that reduces or eliminates L. monocytogenes
or an antimicrobial agent or process that suppresses or limits L. monocytogenes throughout shelf life,
or (3) use of sanitation measures in conjunction with regular environmental sampling to prevent
L. monocytogenes contamination. The various Listeria postprocessing pasteurization treatments and

Listeria growth inhibitors that can be used to satisfy these alternatives will now be presented.
POSTPACKAGING PASTEURIZATION
Alternative 1 is partially based on exposing the fully processed product to some type of postlethality
treatment, typically employing steam or hot water, to inactive small numbers of Listeria that may
have contaminated the product surface from the surrounding environment before packaging. Any
such postprocessing pasteurization step must be verified as being effective and included as a critical
control point in the HACCP plan.
Working at the University of Arkansas, Murphy and colleagues conducted a series of studies
to assess the ability of different steam and hot water postprocessing pasteurization treatments
to decrease populations of L. monocytogenes or L. innocua M1 (a heat-resistant surrogate for
L. monocytogenes) ≥7 logs on surface-inoculated fully cooked poultry products. When chicken
breast strips were vacuum-packaged in 0.2-mm-thick bags and steam pasteurized at 88°C using
either a continuous or batch process, a 7-log reduction was achieved after 28 and 34 min, respec-
tively [155]. In contrast, 22 min of steaming at 96°C was required for similar inactivation of L.
monocytogenes on chicken leg quarters [151]. As expected, thermal inactivation of Listeria is also
dependent on the amount of product being pasteurized, with 5, 25, and 35 min of steaming at 90°C
required to achieve a 7-log reduction in packages containing single cooked chicken breast fillets
(120 g), and 227 and 454 g of chicken strips, respectively [156]. Similar inactivation of L. innocua
M1 on vacuum-packaged cooked chicken breast fillets was obtained using hot water rather than
steam [156], with the weight of product [150,142] and thickness of the packaging film [150]
impacting lethality. Although both hot water and steam pasteurization led to slight decreases in
expressible total moisture and product yield [141], these thermal pasteurization treatments have
gained wide acceptance in the poultry industry, with at least one process lethality model now
published for hot water pasteurization [146].
Hot water postpackage pasteurization is also the preferred method to inactivate Listeria on the
surface of ready-to-eat delicatessen turkey products. Muriana et al. [139,140] reported Listeria
reductions of 2 to 3.5 logs when commercial chubs of various ready-to-eat deli-style turkey products
were surface-inoculated with a 4-strain cocktail of L. monocytogenes, vacuum-packaged, and then
submerged in a 96.1°C water bath for 3 to 4 min. However, these reductions were significantly
lower than those seen in inoculated purge, with these differences attributed to product surface
imperfections. The impact of surface roughness (e.g., crevices, dents, cuts, folds, wrinkles, and
netting marks), packaging-film thickness and pasteurizer design/water circulation velocity on heat
transfer, and thermal inactivation of Listeria was later confirmed by Murphy et al. [152]. When
surface-inoculated, vacuum-packaged ready-to-eat turkey breast was hot water pasteurized at 96°C,
these researchers reported that 20 and 50 min of heating was required to raise the product temper-
ature to 71°C at depths of 8 and 15 mm, respectively, with this treatment producing a 7-log reduction
in L. monocytogenes. Nonthermal pasteurization strategies, including gamma radiation
[68,87,197,204] and high-pressure processing [192], have thus far proven to be more costly and/or
less effective than thermal pasteurization in reducing numbers of Listeria on packaged ready-to-
eat meat and poultry products, with gamma radiation doses of 2.5 kGy or higher also negatively
impacting product quality. Thus, although time consuming, with the end result being strongly
dependent on surface characteristics of the product, hot water postpackage pasteurization remains
the most effective treatment and has been widely adopted by poultry processors seeking to satisfy
the requirements of USDA–FSIS alternatives 1 and 2.
ANTIMICROBIAL AGENTS FOR LISTERIA GROWTH SUPPRESSION

In lieu of a thermal or nonthermal postlethality treatment that reduces or eliminates L. monocyto-
genes on the product surface, manufacturers that wish to operate under alternative 2 can incorporate
one or more antimicrobial agents into their product to suppress or limit growth of L. monocytogenes
throughout normal shelf life. Among the numerous food additives investigated, combined use of
lactate and diacetate has proven to be most effective, with these two preservatives acting synergis-
tically to minimize Listeria growth. According to Zhu et al. [227], populations of L. monocytogenes
in inoculated turkey hams increased <1 log during 42 days of refrigerated storage when the
product was formulated to contain 2% sodium lactate with either 0.1% sodium diacetate or 0.1%
potassium benzoate.
Rather than incorporating these generally-recognized-as-safe additives into the product formu-
lation at the time of manufacture, these same antimicrobial agents can be directly applied to the
product surface at the time of packaging to minimize Listeria growth. When retail chicken luncheon
meat was surface-inoculated to contain about 104 L. monocytogenes CFU/g, Islam et al. [105]
reported initial Listeria reductions of 1.32, 1.19, 0.41, and 0.25 logs after spraying the product
with a 25% solution of sodium diacetate, sodium benzoate, potassium sorbate, or sodium propionate,
respectively, with populations decreasing an additional 0.2 to 0.5 logs for all treatments during 2
weeks of subsequent storage at 4°C. At lower concentrations of 15 and 20%, growth of Listeria
was also suppressed about 2–3 logs compared to the unsprayed controls, with sodium diacetate
and sodium benzoate again proving to be most efficacious. These same researchers [104] reported
similar results after inoculated turkey frankfurters were held in 15, 20, or 25% solutions of sodium
diacetate, sodium benzoate, potassium sorbate, or sodium propionate, with Listeria populations
3.5 to 4 logs lower in treated as compared to untreated samples after 2 weeks of refrigerated storage.
Despite the high antimicrobial concentrations used, uptake of these solutions by the frankfurters
was only about 0.2 to 0.4%. Because all 3-day-old samples were acceptable to consumers based
on a sensory evaluation, dipping and or spraying ready-to-eat poultry products in sodium benzoate
appears to be a promising means for minimizing Listeria growth on these products during refrig-
erated storage. However, use of several bacteriocins, including enterocin [22] and sakacin P [110],
and different plant extracts [88] also has been suggested.
As an alternative approach to dipping and spraying, various types of antimicrobial edible films
or coatings have been proposed for inactivating Listeria on the surface of ready-to-eat foods, with
these films potentially functioning as antimicrobial packaging materials [41]. Cagri et al. and Capita
et al. [42,43] developed a whey protein-based hot dog casing containing 1.0% p-aminobenzoic acid
that successfully inhibited growth of L. monocytogenes on surface-inoculated hot dogs during
42 days of refrigerated storage, with this film also likely to be suitable for ready-to-eat poultry
products. In another study, Lungu and Johnson [124] reported that L. monocytogenes generally
failed to grow on surface-inoculated turkey frankfurters during 28 days of storage at 4°C if the
frankfurters were first coated with a zein solution containing various combinations of nisin, sodium
diacetate, or sodium lactate. Additional studies have also shown that growth of L. monocytogenes
can be prevented on turkey bologna during extended refrigerated storage by packaging the product
in wheat gluten films containing nisin [128] or soy-based films containing nisin and/or lauric acid
[57]. Thus, antimicrobial packaging films and coatings may provide another alternative for meeting
the goals of the USDA–FSIS final rule on L. monocytogenes in ready-to-eat meat and poultry
products [209].
EGG PRODUCTS
L. monocytogenes is most frequently isolated from the heart, liver, and spleen tissue of fowl suffering
from listeriosis; however, based on early scientific literature, this pathogen also has been detected
in necrotic oviduct lesions of several infected hens [84] and in follicles of one artificially infected
chicken [109]. These observations prompted a large-scale survey in 1958 [107] in which 600 intact
hen’s eggs were examined and found to be negative for L. monocytogenes. Similarly, Busani et al.
[40] were also unable to isolate L. monocytogenes from 431 eggs collected in Italy during 2001
and 2002. In keeping with these findings, consumption of eggs and egg products also has not yet
been linked to a single case of listeriosis. However, the possible presence of L. monocytogenes on
egg shells that may contain minute cracks, along with the ability of this pathogen to survive
90 and >14 days on eggs stored at 5 [27] and 10°C [30], respectively, persist on inoculated eggs
treated with sodium hypochlorite containing 100 ppm available chlorine [30], and grow in artificially
contaminated eggs stored at refrigeration as well as ambient temperatures [27] suggests that
although unlikely, eggs cannot be completely ignored as a possible source of listerial infection.
Hence, it is not surprising that recent concerns surrounding contaminated poultry products also
have prompted interest in reducing the numbers of listeriae on intact whole eggs and better defining
both the incidence and behavior of L. monocytogenes in eggs and egg products, including pasteur-
ized liquid and dried egg.
INCIDENCE
As mentioned in the preceding paragraph, isolation of L. monocytogenes from the interior of intact
whole eggs has not yet been documented. However, the outer eggshell surface can harbor Listeria,
as suggested by Farber et al. [63], who recovered L. innocua from 6 of 119 egg wash water samples
collected in Quebec and Ontario, Canada. Given these findings, several intervention strategies have
been explored to reduce the likelihood of Listeria contamination during egg washing. When Park
et al. [168] inoculated intact shell eggs to contain about 107 L. monocytogenes CFU/egg, populations
decreased about 0.25, 4, and 5 logs after 5 min of washing in deionized water and water containing
45 and 200 ppm available chlorine, respectively, with acidic electrolyzed water yielding reductions
of 3–4 logs. In support of these findings, Russell [180] concluded that spraying of electrolyzed
water is likely to eliminate the normal numbers of L. monocytogenes that might be expected to
contaminate the surface of shell eggs, with Brackett and Beuchat [37] reporting that L. monocytogenes
populations on surface-inoculated eggs decreased 1–2 logs after 1 week of storage at 5 or 20°C.
In the only other inactivation work thus far reported, Wong and Kitts [223] found that L. monocytogenes
was reduced to nondetectable levels following a 3-min exposure to 3 kGy of electron beam
irradiation. However, this treatment adversely affected albumin quality.
Although the incidence of Listeria is very low on the surface of intact shell eggs, the same is
not true of broken eggs. According to Leasor and Foegeding [118], Listeria spp. were isolated from
15 of 42 (36%) previously frozen samples of raw commercially broken solids-adjusted liquid whole
egg (21 samples), natural-proportion liquid whole egg (20 samples), and yolk (1 sample) obtained
on several occasions from 6 of 11 (54%) commercial manufacturers located throughout the United
States. On closer examination of the data, L. innocua and L. monocytogenes were identified in 15
of 15 (100%) and 2 of 15 (13.3%) positive samples, respectively. Moreover, 12 of 15 (80%) and
3 of 15 (20%) positive samples, including 1 sample each with L. monocytogenes, were classified
as solids-adjusted and natural-proportion liquid whole egg, respectively. Increased handling during
manufacture and, as suggested by the authors, a higher solids content which may enhance growth
and survival of Listeria are just two of many possible reasons why a higher incidence of Listeria
was observed in solids-adjusted rather than natural-proportion liquid whole egg. Although results
from direct plating indicated that the two L. monocytogenes-positive samples contained approxi-
mately 1 and 8 L. monocytogenes CFU/g at the time of analysis, the fact that these samples were
held frozen for up to 4.5 months and subjected to two freeze-thaw cycles suggests that numbers
of Listeria most likely decreased by at least 50% during storage. Hence, both samples probably
contained <100 L. monocytogenes CFU/g before being frozen.
Working in the United Kingdom, Moore and Madden [133] recovered Listeria from 125 of
173 (72%) inline filters used to remove shell fragments from raw blended whole egg, 78 and 47
samples of which contained L. innocua and L. monocytogenes, respectively, generally at levels
<400 CFU/mL. However, because 500 pasteurized egg samples yielded negative results for listeriae,
pasteurization at 64.4°C for 2.5 min, as required in the United Kingdom, appears to afford a high
degree of safety. During a subsequent survey of three Australian egg factories in New South Wales,
Desmarchelier et al. [58] identified L. innocua in 9 of 13 (69%) samples of unpasteurized liquid

whole egg, with no other Listeria spp. being observed. Floors, drains, and mobile equipment in
raw processing areas of these factories were later confirmed as major sources of contamination
through strain-specific typing of environmental Listeria isolates. Although 7 samples of raw sugared
yolk and 26 peeled boiled eggs were Listeria free, L. innocua was recovered from 1 of 51 samples
of pasteurized liquid egg, with this organism also being detected in 2 of 14 peeled boiled eggs
following 3 weeks of modified-atmosphere storage at 4°C. Hence, under certain conditions, Listeria
can multiply to detectable levels in egg products during extended cold storage.
GROWTH
The ability of Listeria to grow in hen’s eggs was first recognized in 1940 when Paterson [170]
inoculated a laboratory culture of L. monocytogenes into the chorioallantoic membrane of a
chicken embryo. This procedure was traditionally used to determine virulence of L. monocyto-
genes isolates [200]. Information concerning growth of this pathogen in nonfertile eggs and egg
products is limited. A search of the scientific literature has uncovered only two studies pertaining
to growth of L. monocytogenes in raw whole eggs or egg components. According to results from
the first such paper published in 1955, Urbach and Schabinski [205] found that populations of
L. monocytogenes in intact experimentally infected nonfertile eggs increased nearly 6 orders of
magnitude during 10 days of storage at ambient temperature. Following this study, 20 years
passed until viability of L. monocytogenes was again examined in artificially contaminated raw as
well as cooked (121°C/15 min) whole egg, albumen, and egg yolk during extended storage at 5
and 20°C [111]. Results for raw whole and separated egg showed that growth of L. monocytogenes
was primarily confined to egg yolk (Figure 14.8), with the pathogen exhibiting respective
generation times of 1.7 days and 2.4 h at 5 and 20°C. Overall, Listeria populations in raw whole
egg generally varied less than 1 order of magnitude from the original inoculum during extended
storage at either temperature; however, numbers of Listeria in raw albumen (pH 8.9) decreased
8
5
Raw
4 Cooked
2
0 10 20 30
Days
FIGURE 14.8 Behavior of L. monocytogenes in raw and cooked whole egg (), albumen (), and egg yolk
() during extended incubation at 5°C. (Adapted from Khan, M.A., I.A. Newton, A. Seaman, and M.
Woodbine. 1975. Survival of Listeria monocytogenes inside and outside its host. In M. Woodbine, Ed.,
Problems of Listeriosis. Surrey, U.K., Leicester University Press, pp. 75–83.)
3 and 5 orders of magnitude during prolonged incubation at 5 and 20°C, respectively. Despite
the reported ability of L. monocytogenes to grow in laboratory media having pH values as high
as 10 [188], loss of Listeria viability in raw albumen was pH related, with numbers of Listeria
decreasing less than 2 orders of magnitude in samples that were preadjusted to pH 7 and held
at 5°C. Unlike raw whole egg, albumen, and egg yolk, Listeria grew rapidly in corresponding
cooked samples. Generation times for the pathogen in cooked whole egg, egg yolk, and albumen
were 1.9, 2.3, and 2.4 days, respectively, at 5°C, and 2.6, 2.6, and 3.5 h, respectively, at 20°C.
These authors speculated that loss of the aforementioned antilisterial properties of raw albumen
resulted from inactivation of one or more binding proteins (i.e., ovotransferrin, ovoflavoprotein,
and avidin) during heating. Because L. monocytogenes can grow rapidly in cooked whole as well
as separated eggs, investigators of foodborne outbreaks should not completely overlook these
products as potential vehicles of infection.
In 1990, Sionkowski and Shelef [193] provided the first information concerning growth of L.
monocytogenes in pasteurized egg products. To simulate postpasteurization contamination, pasteur-
ized (64.4°C/2.5 min) samples of liquid egg and reconstituted dried egg were inoculated to contain
~104–105 L. monocytogenes CFU/mL and examined for numbers of Listeria during 7 days of storage
at 4°C. As shown in Figure 14.9, the pathogen grew similarly in both products, reaching populations
of ~107 CFU/mL after 7 days of refrigerated storage. Although transmission of L. monocytogenes
by pasteurized egg products has not yet been documented, these findings suggest that a possible
108
107
106
105
Liquid egg
Reconstituted dried egg
104
0 1 2 3 4 5 6 7
Days
FIGURE 14.9 Growth of L. monocytogenes in liquid and reconstituted dried egg incubated at 4°C. (Adapted
from Sionkowski, P.J. and L.A. Shelef. 1990. Viability of Listeria monocytogenes strain Brie-1 in the avian
egg. J. Food Prot. 53: 15–17, 25.)
public health problem could develop if L. monocytogenes enters pasteurized liquid egg, since
the shelf life of some of these refrigerated products now has been extended to several months.
Growth of L. monocytogenes in commercially processed, liquid whole egg was first assessed
by Foegeding and Leasor [67]. In this study, commercially broken, liquid whole egg was ultrapas-
teurized (68°C/118 sec), homogenized, inoculated to contain one of five L. monocytogenes strains
(Scott A [clinical isolate], F5069 [milk isolate], ATCC 19111 [poultry isolate], NCF-U2K3, or
NCF-F1 KKr [raw liquid whole-egg isolate]) at a level of 5 × 102 to 1 × 103 CFU/mL, overlaid
with mineral oil to prevent oxygen transfer, and examined for numbers of Listeria during extended
incubation at 4, 10, 20, and/or 30°C.
Generation times and maximum populations were generally similar to those previously observed
in fluid milk products (see Chapter 11) except for strain Scott A, which failed to grow in liquid
whole egg during prolonged incubation at 4 and 10°C. Although growth of strain Scott A in fluid
milk, cheese, and cabbage juice during refrigeration is well documented, Buchanan et al. [39]
reported that this strain failed to grow in certain meat and poultry products incubated at 4°C. As
shown in Table 14.7, generation times for the five L. monocytogenes strains ranged from 24.0 to 51.0,
8.0 to 31.0, 7.5 to 26.0, and 4.3 to 15.0 h at 4, 10, 20, and 30°C, respectively. Maximum populations
ranged from 5.00 to 7.00, 5.48 to 8.48, 6.85 to 8.00, and 7.00 to 8.00 L. monocytogenes log10
CFU/g in liquid whole eggs incubated at 4, 10, 20, and 30°C, respectively. However, Sheldon and
Schuman [190] reported that when stored at 4°C, L. monocytogenes populations in an inoculated
commercially available reduced-cholesterol liquid whole-egg product could be reduced as much
as 3.9 orders of magnitude by adjusting the pH of the product to 6.6 with citric acid and adding
nisin at a level of 1000 IU/mL. Considering current distribution and marketing practices, it is likely
that perishable products such as liquid whole egg will occasionally encounter periods of temperature
abuse. Hence, from these data, it follows that even brief exposure to temperatures ≥10°C can lead
TABLE 14.7
Generation Times and Maximum Populations of L. monocytogenes in
Ultrapasteurized Liquid Whole Egg Incubated at 4, 10, 20, and 30°C
Strain of Incubation Temperature (°C)

L. monocytogenes 4 10 20 30
Generation Time (h)

F5069 24 12 7.5 4.3
Scott A NG NG 26 7.1
ATCC 19111 51 31 22 12
NCF-U2K3 26 7.8 ND ND
NCF-FIKK4 25 8.0 ND ND
Maximum Population (log10 CFU/g)

F5069 6.70 7.00 8.00 8.00
Scott A NG ND 7.00 7.85
ATCC 19111 5.00 5.48 6.85 7.00
NCF-U2K3 7.00 8.48 ND ND
NCF-FIKK4 6.48 8.30 ND ND
Note: ND, not determined; NG, no growth.
Source: Adapted from Foegeding, P.M. and S.B. Leasor. 1990. Heat resistance and growth of Listeria
monocytogenes in liquid whole egg. J. Food Prot. 53: 9–14.
to a dramatic increase in both growth rate (i.e., decreased generation time) and maximum
populations of L. monocytogenes attained in ultrapasteurized liquid whole egg. Because most L.
monocytogenes strains can proliferate in ultrapasteurized liquid egg products at refrigeration tem-
peratures, postpasteurization contamination should be avoided and the product should be stored at
temperatures at or preferably below 0°C.
The behavior of L. monocytogenes has been assessed in several additional egg products. Brackett
and Beuchat [36] showed that L. monocytogenes could survive throughout the normal shelf life of
powdered and frozen egg products. The survival characteristics of L. monocytogenes on egg shells
as well as during and after cooking raw, whole, scrambled eggs by frying were also determined
by Brackett and Beuchat [37]. On the egg shell surface, L. monocytogenes populations decreased
from 104 to <10 CFU/egg after 6 days of storage at 5 and 20°C. Frying scrambled eggs
reduced L. monocytogenes populations >3 orders of magnitude, whereas frying eggs sunny-side
up did not significantly reduce levels of L. monocytogenes. Similarly prepared fried scrambled eggs
that were inoculated with L. monocytogenes failed to support growth of the pathogen during 36 h
of storage at 5°C or 12 h of storage at 18–22°C [224]. However, when hard-boiled eggs were
inoculated by Claire et al. [52] to contain 102 L. monocytogenes CFU/g, the pathogen increased to
a maximum population of 109 CFU/g following 7, 14, and 21 days of storage at 4, 8, and 12°C,
respectively, suggesting possible public health concerns if hard-boiled eggs become contaminated
during subsequent peeling and handling.
Looking at other egg-related products, Erickson and Jenkins [62] showed that L. monocytogenes
inactivation rates in commercial mayonnaise were directly correlated with aqueous phase acetic
acid concentration as follows: sandwich spread ≥ real mayonnaise > cholesterol-free reduced
calorie mayonnaise dressing > reduced calorie mayonnaise dressing. Higher antilisterial activity
in the cholesterol-free formulation was attributed to egg white lysozyme. Additionally, Glass, and
Doyle [80] reported that L. monocytogenes populations in two types of commercially produced low-
calorie mayonnaise containing 0.7% acetic acid in the aqueous phase decreased from an initial
inoculum of ~106 CFU/g to nondetectable levels following 10–14 days of ambient storage. These
studies document that commercial mayonnaise products represent a negligible consumer safety risk.
In the only other egg-related growth study thus far reported, Notermans et al. [159] examined the
viability of several foodborne pathogens, including L. monocytogenes, in an eggnog-like product
prepared from raw whole egg and sugar (25% w/v) with or without ethanol (7% v/v). When samples
of ethanol-free product were inoculated to contain ~104–105 L. monocytogenes CFU/g, numbers of
Listeria generally decreased 10-fold during the first 2 days of incubation at 4°C and then slowly increased
to levels near or slightly above the original inoculum level after 5 additional days of refrigerated storage.
Although L. monocytogenes generally exhibited similar behavior patterns in nonalcoholic samples
incubated at 22°C, initial population decreases were far more abrupt, with the pathogen then increasing
to populations 1 to 3 orders of magnitude lower than the original inoculum after 7 days of incubation.
Unlike alcohol-free samples, L. monocytogenes was slowly inactivated in samples containing 7%
ethanol, with populations typically 1–2 and more than 4 orders of magnitude lower in 7-day-old samples
held at 4 and 22°C, respectively, than were present initially. Hence, given the normal 2-week refrigerated
shelf life of similar commercially available nonalcoholic eggnog-like products, recontamination of these
beverages during packaging could lead to potential public health problems involving Listeria and other
foodborne pathogens, with Salmonella enteritidis and Salmonella Typhimurium reportedly also remain-
ing viable in artificially contaminated samples during 63 days of refrigerated storage.
THERMAL INACTIVATION
Interest in possible heat resistance of L. monocytogenes in eggs is of recent origin; however, concerns
raised by European scientists regarding potential transmission of Listeria through eggs prompted
a 1955 study by Urbach and Schabinski [205], which examined the ability of this pathogen to
survive in artificially infected eggs that were fried. According to these authors, L. monocytogenes
was isolated from fried eggs (congealed white and soft yolk) prepared from inoculated raw eggs
in which the pathogen had previously grown to levels >5 × 105 CFU/g.
Whereas the aforementioned work appears to be fairly crude by current standards and is now
primarily only of historical interest, Foegeding and Leasor [67] conducted a more sophisticated study
in which D-values were determined for five strains of L. monocytogenes (Scott A [clinical isolate],
F5069 [milk isolate], ATCC 19111 [poultry isolate], NCF-U2K3 and NCF-F1KK4 [raw liquid whole-
egg isolates]) in sterile raw egg. Inoculated samples of raw liquid whole egg were added to glass
capillary tubes that were then heat-sealed and immersed in a water or oil bath at 51.0, 55.5, 60.0, and
66.0°C. After various times, tubes were removed and contents examined for survivors. Numbers of
Listeria decreased linearly in raw egg during all four heat treatments, with D-values for the five L.
monocytogenes strains ranging from 14.3 to 22.6, 5.3 to 8.2, 1.3 to 1.7, and 0.06 to 0.20 min at 51.0,
55.5, 60.0, and 66.0°C, respectively. Strain Scott A was generally less heat resistant than the other four
strains, particularly at the two lower temperatures; however, strain F5069 and the two isolates from
raw egg exhibited moderate thermal tolerance at all four temperatures. Muriana et al. [138] subsequently
reported similar D-values at 60°C for L. monocytogenes strain Scott A when inoculated samples of
liquid whole egg were tested using either capillary tubes (D-values of 1.8 min) or a flow injection
system (D-value of 1.95 min). Although this pathogen appears to exhibit a similar degree of heat
resistance in both raw whole milk (see Chapter 11) and raw liquid whole egg, survival of L. monocy-
togenes is significantly enhanced by supplementing liquid whole egg or egg yolk with 10% NaCl
[31,133]. At 64°C, L. monocytogenes exhibited D-values of ~10 and 10.5 min in salted liquid whole
egg and egg yolk, respectively, as compared with ~1.0 and 1.8 min for unsalted samples, with increased
thermal tolerance attributed to a decrease in water activity. In contrast, adding 10% sucrose to unsalted
samples neither increased thermal resistance of listeriae nor altered the product’s water activity.
In more practical terms, USDA officials currently require that liquid whole egg be pasteurized
at a minimum of 60°C for 3.5 min to effect a 9-D reduction of Salmonella spp. [206]. Although
results from the study just discussed indicate that minimum pasteurization of liquid whole egg
would yield only a 2.1- to 2.7-D kill of L. monocytogenes, one must remember that current
estimates place L. monocytogenes populations in liquid whole egg at <100 CFU/g [67]. Hence,
as is true of milk pasteurization, current minimum pasteurization requirements for raw liquid
whole egg appear adequate to inactivate normal levels of Listeria that might be present in the
product. However, it is important to stress that current minimum pasteurization requirements for
liquid whole egg, as specified in the USDA Egg Pasteurization Manual, indicate that the margin
of safety is approximately 6 orders of magnitude lower for L. monocytogenes than for most
Salmonella spp. Furthermore, such pasteurization treatments appear to be inadequate for salted
liquid whole egg and egg yolk.
In 1987, Ball et al. [26] documented that ultrapasteurization (i.e., pasteurization at >60°C for
<3.5 min) in combination with aseptic processing and packaging can be used to produce liquid
whole egg with a refrigerated shelf life of 3–6 months. After results from this study were published,
FDA officials issued a temporary permit allowing a North Carolina firm to market ultrapasteurized
liquid whole egg [13,178]. Although two of the four objectives of the process were to render the
product free of Salmonella and L. monocytogenes, the exact time/temperature requirements to
completely inactivate L. monocytogenes in liquid whole egg were not specified in the FDA
temporary permit.
Based on extrapolations from the aforementioned survivor curves that showed no evidence of
tailing, Foegeding and Leasor [67] predicted that the ultrapasteurization processes used by Ball et
al. [26] would effect a 1- to 34-D (average of 14-D) kill of L. monocytogenes in liquid whole egg.
Assuming that the ultrapasteurization times and temperatures used in conjunction with the tempo-
rary FDA permit are the values that were previously determined by Ball et al. [26], Foegeding and
Leasor [67] went on to speculate that four of the ten thermal treatments used by Ball and coworkers
may not conform to the definition of ultrapasteurization in the temporary permit, depending on
how one views the necessity for a 9-D reduction in numbers of Listeria. However, it appears that
Listeria-free ultrapasteurized liquid whole egg having a refrigerated shelf life of 1 to several months
can be produced, provided that the raw product is processed using one of the six more severe
time/temperature treatments proposed by Ball et al. [26] and then is aseptically packaged to eliminate
postpasteurization contamination.
Foegeding and Stanley [66] verified the previous predictions concerning heat resistance of
Listeria by determining the thermal death time (F-value) for L. monocytogenes strain F5069 in
liquid whole egg. Using their previously described submerged capillary tube method [62], they
found that L. monocytogenes was eliminated from inoculated samples (1.0 × 108 to 4.0 × 108
CFU/mL) of sterile liquid whole egg after processing at 62, 64, 66, 69, and 72°C for 16.0, 8.0,
4.5, 1.6, and 0.6 min, respectively.
Although results from this study confirm that minimum pasteurization (60°C/3.5 min) will not
result in a Listeria-free product if initial populations are large, the need for a 9-D kill as currently
required by the USDA may not be appropriate for L. monocytogenes, because present estimates
place the population of this pathogen at <100 CFU/g in raw liquid whole egg. Hence, based on
maximum expected L. monocytogenes levels in raw liquid whole egg, pasteurization by current
standards should render such products free of Listeria. The situation regarding ultrapasteurization
appears to be somewhat different because the thermal death-time data obtained by Foegeding and
Stanley [66] indicate that 4 of the 10 ultrapasteurization processes proposed by Ball et al. [26]
(63.7°C/26.2 sec, 63.8°C/92.0 sec, 67.7°C/9.2 sec, and 71.5°C/2.7 sec) would likely fail to produce
a 9-D kill in raw liquid whole egg. Nonetheless, the 9-D kill effected by the remaining six
ultrapasteurization treatments proposed by Ball et al. [26] indicates that ultrapasteurization pro-
cesses can be designed to produce Listeria-free liquid whole egg with an anticipated refrigerated
shelf life of 3–6 months.
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in Fish and Seafood*
Karen C. Jinneman, Marleen M. Wekell, and Mel W. Eklund
CONTENTS
Introduction ....................................................................................................................................617
FDA Surveys of L. monocytogenes in Domestic and Imported Seafood .....................................618
Other Surveys for L. monocytogenes in Fish and Seafood Products ...........................................626
Crustaceans ...........................................................................................................................626
Shellfish ................................................................................................................................630
Finfish ...................................................................................................................................630
Smoked Fish Products..........................................................................................................630
Lightly Processed Fish Products..........................................................................................631
Human Listeriosis Associated with Fish and Seafood Products...................................................632
Regulatory Aspects of L. monocytogenes in Fish and Seafood....................................................634
Risk Assessment.............................................................................................................................635
Hazard Identification ............................................................................................................635
Exposure Assessment ...........................................................................................................635
Hazard Characterization .......................................................................................................635
Risk Characterization ...........................................................................................................635
Behavior of Listeria in Fish and Seafood .....................................................................................636
Modes of Transmission ........................................................................................................636
Growth and Survival.............................................................................................................638
Inhibition...............................................................................................................................640
Inactivation ...........................................................................................................................643
Summary ........................................................................................................................................646
References ......................................................................................................................................647
INTRODUCTION
Listeria monocytogenes is ubiquitous in nature. Therefore, the many aquatic creatures including
finfish, oysters, shrimp, crabs, lobsters, squid, and scallops harvested from natural environments
can be potential sources of Listeria in the human diet. Many of these products are also subjected
to a variety of processing methods that can inactivate Listeria on the raw product. Listeria can also
* The views expressed here are those of the authors and are not necessarily endorsed by the U.S. Food and Drug
Administration, National Marine Fisheries, or the Government of the United States.
617
be introduced during processing by poor sanitation conditions or manufacturing practices or by

postprocess contamination. The psychrotrophic nature of L. monocytogenes allows its survival or
even multiplication during refrigerated storage or temperature abuse situations. This is of special
concern for those products receiving minimal or no heat treatment before consumption. Since L.
monocytogenes was first isolated from imported cooked crabmeat in 1987, at least 126 Class I
recalls (where reasonable probability exists that the use of or exposure to a violative product would
cause serious adverse health consequences or death) have been issued by the U.S. Food and Drug
Administration (FDA) for more than 266,070 lb of ready-to-eat (RTE) domestic or imported fish
and seafood; this pathogen is routinely found in 7.6% of all such products marketed in the United
States. The first of several cases of listeriosis positively linked to consumption of fish or seafood
was not reported until 1989, when a 54-year-old woman in Italy contracted listeric meningitis
4 days after consuming steamed fish from which L. monocytogenes was later isolated [43]. This
case and the potential hazard associated with consumption of other Listeria-contaminated RTE
food such as cooked crabmeat, cooked shrimp, and smoked salmon have prompted studies on
determining the incidence and control of Listeria in various seafoods.
In this chapter, data reviewed are from a series of FDA surveys from 1987 to 2003. These were
designed to determine the incidence of L. monocytogenes in domestic and imported shrimp, crab,
smoked seafood, and various other fish and seafood products. As in previous chapters, Class I
recalls that have been issued for Listeria-contaminated fish and seafoods also will be mentioned.
The following topics will also be included: (1) international surveys of fish and seafood products
for Listeria, (2) an overview of human listeriosis cases and outbreaks associated with fish and
seafood, (3) discussion of regulatory aspects of L. monocytogenes in fish and seafoods and use of
risk assessments, (4) behavior of L. monocytogenes in these foods, including growth and thermal
resistance data for L. monocytogenes in seafoods, and (5) mitigation strategies such as the
application of lactic acid for controlling growth of Listeria in seafood.
FDA SURVEYS OF L. MONOCYTOGENES IN DOMESTIC

AND IMPORTED SEAFOOD
Immediately after the June 1985 outbreak of cheeseborne listeriosis in California, FDA officials
focused their attention on immediate problems that confronted the entire dairy industry. Despite a
lack of evidence linking consumption of meat and poultry products to cases of human listeriosis
before 1988, as early as December 1985 USDA–FSIS officials began taking an active interest in
determining the incidence of L. monocytogenes in meat and poultry products.
Increased concern about the potential hazard of Listeria-contaminated seafood to public
health began in the spring of 1987 after a private testing laboratory in the United States isolated
L. monocytogenes from frozen cooked crabmeat obtained from a Mexican supplier [3]. L. mono-
cytogenes was confirmed in this product by the FDA in Baltimore, MD, in May 1987. In maintaining
FDA’s “zero tolerance policy” for L. monocytogenes in RTE foods, the first in a series of Class I
recalls for nearly 4 tons of tainted crabmeat marketed in four states was issued. These events also
prompted an import alert on June 17, 1987 [4], calling for automatic detention and testing for Listeria
and Escherichia coli in all frozen crabmeat shipped to the United States from Mexico.
Less than 1 month after this product was recalled, the FDA in Seattle, WA, detected L.
monocytogenes in samples of imported frozen raw shrimp [2] and lobster tails [14]. Although no
recalls were issued for these products, which are almost invariably cooked before consumption,
confirmation of Listeria in these seafoods together with the recall of cooked crabmeat noted
previously prompted the FDA to initiate two surveys in July 1987.
In the first of these surveys, every month six imported samples of frozen raw shrimp were
collected and examined at each FDA district laboratory for Listeria. The samples represented as
many different countries as possible (Table 15.1) [2]. Additionally, each district also was requested
to collect three domestic samples of frozen raw shrimp per month at the wholesale or retail level.
Incidence and Behavior of Listeria monocytogenes in Fish and Seafood 619
TABLE 15.1
Results from an FDA Survey of Imported Frozen Raw Shrimp, July to October 1987
Country of Origin Analyzed L. monocytogenes Other Listeria spp.
Brazil 4 1 (25) 1 (25)

Ecuador 8 1 (12.5) 2 (25)
Guyana 1 1 (100) 0
Honduras 5 1a (20) 2 (40)
Hong Kong 1 0 0
India 4 0 1 (25)
Indonesia 1 0 0
Macau 1 0 0
Mexico 10 0 1 (10)
Nigeria 3 0 1 (33.3)
Norway 1 0 0
Pakistan 4 0 0
Panama 7 0 0
People’s Republic of China 4 0 1 (25)
Peru 1 0 0
Philippines 3 0 0
Taiwan 9 0 4 (44.4)
Thailand 4 0 2 (50)
Venezuela 3 0 0
Total 74 4 (5.4) 15 (20.3)
aOne sample contained L. monocytogenes and other Listeria spp.
Source: Adapted from FDA. 1987. Program 7303.030 Pathogen monitoring of selected high risk foods
(FY 88/89). In Department of Health and Human Services. Public Health Service. FDA, U.S. Food and Drug
Administration Compliance Program Guidance Manual. U.S. Government Printing Office, Pittsburgh, PA.
Listeria spp. were detected in 18 of 74 (24.3%) samples of frozen raw shrimp imported from 10
different countries between July and October 1987 (Table 15.1) using the original FDA method
[89]. L. monocytogenes was also isolated from 4 of 74 (5.4%) imported samples of frozen raw
shrimp, with all positive samples originating from Central or South American countries. Subse-
quently, three lots of raw shrimp imported from Ecuador and Honduras also were found to contain
103–105 L. monocytogenes or L. innocua CFU/g [95]. However, because shrimp are normally not
consumed raw in the United States, FDA officials did not request the recall of any of these
contaminated lots. The FDA conducted a 2-year nationwide survey in fiscal years 1989 and 1990
to determine the incidence of filth and microbiological (Salmonella and Listeria spp.) contamination
in domestic and imported fresh and frozen shrimp [63]. Listeria spp. were detected in 14 samples
(6.8%), and L. monocytogenes was recovered from 9 (4.4%) of the 205 samples examined [63].
L. monocytogenes and Salmonella were isolated from 1 of 11 samples in which decomposition was
also detected by sensory evaluation [63].
In the second FDA survey, domestic and imported samples of cooked, frozen, and refrigerated
crabmeat (i.e., picked or extracted) were examined for presence of L. monocytogenes, Staphylococcus
aureus, Vibrio cholera, V. parahaemolyticus, V. vulnificus, and Yersinia enterocolitica and numbers of
E. coli [2]. Again, samples of imported crabmeat from as many different countries as possible were
collected. As of January 1988, 6 of 98 (6.1%) domestic samples of cooked crabmeat contained Listeria,
with L. monocytogenes and L. innocua being recovered from 4 and 2 samples, respectively
(Table 15.2). Similarly, Listeria spp. were detected in 3 of 24 (12.5%) imported samples of cooked
crabmeat, with L. monocytogenes in 2 of 24 (8.3%) samples of product marketed in the United States.
TABLE 15.2
Results from an FDA Survey of Domestic/Imported Refrigerated
or Frozen Cooked Crabmeat, July 1987 to January 1988
Number of Number of Positive Samples (%)
Country of Samples
Origin Analyzed L. monocytogenes Other Listeria spp.
United States 98 4 (4.1) 2 (2.0)

Canada 3 0 1 (33.3)
Chile 2 0 0
Korea 11 2 (18.2) 2a (18.2)
Japan 2 0 0
Mexico 3 0 0
Venezuela 3 0 0
Total (imported) 24 2 (8.3) 3 (12.5)
a
One sample contained L. monocytogenes and L. innocua.
Source: Adapted from Archer, D.L. 1988. Review of the latest FDA information on the
presence of Listeria in foods. WHO Working Group on Foodborne Listeriosis, Geneva,
Switzerland, February 15–19.
Weagant et al. [138] formally published the first results of a survey dealing with the incidence
of Listeria spp. in imported and domestic frozen seafood products analyzed at the FDA District
Laboratory in Seattle, WA, during the second half of 1987; 31 of 50 (62%) imported and 4 of 7
(57%) domestic samples of frozen seafood tested positive for Listeria spp. using the FDA method
[89]. The only Listeria spp. detected were L. monocytogenes (15 of 57, 26.3%) and L. innocua
(26 of 57, 45.6%); both L. monocytogenes and L. innocua were isolated from several samples.
Although the number of samples examined from various product categories was limited, results
suggested that frozen seafood more frequently contains L. innocua than L. monocytogenes. Hence,
as was true for raw milk, meat, and poultry products, it appears that both organisms may also
occupy similar niches in seafood processing environments. Therefore, presence of L. innocua in
raw, and particularly in cooked, seafood should not be ignored but rather should be viewed as an
indicator of possible contamination with L. monocytogenes.
Discovery of Listeria in raw shrimp, crabmeat, and other seafood products, coupled with an
increased concern about the general safety of seafood, prompted FDA officials in October 1987 to
include analysis for L. monocytogenes in a compliance program for domestic and imported shrimp
[6] and to increase testing of many other domestically produced seafoods for Listeria spp. under the
pathogen monitoring program for select high-risk foods (CPGM 7303.030) [10,14,50]. This increased
sampling effort was intended to determine the geographic distribution of Listeria in domestic and
imported seafood and to identify the incidence of Listeria spp. in such products. In March 1988, a
processed seafood assignment was issued [53]. The purpose of the Processed Seafood Compliance
Program (CPGM 7303.036) was to conduct microbiological analyses for several bacterial pathogens,
including Listeria, of imported and domestic processed seafood that is minimally processed or
consumed raw. Products selected for Listeria analyses under the CPGM 7303.036 program included
the following: crabmeat (cooked or pasteurized), crayfish/crawfish, lobster, langostinos (cooked,
parboiled), molluscan shellfish, processed imitation seafood (surimi), seafood salads, shrimp (cooked),
smoked or salted fish, and other processed seafood. In addition, the National Advisory Committee
on Microbiological Criteria for Foods (NACMCF) in April 1988 began the laborious task of developing
microbiological criteria for cooked shrimp and crabmeat [7]. During the FDA surveys from October
1988 through September 1990, L. monocytogenes was recovered from domestic samples of crabmeat,
lobster, shrimp, smoked salmon, and surimi. Imported fish, lobster, shellfish, shrimp, smoked fish,
squid, and surimi also tested positive for L. monocytogenes during the same time period [53].
Three FDA compliance programs in effect since 1991–1996 have surveyed the incidence of
Listeria in fish and seafood products and reported analytical data into the FDA Microbiological
Information System. These data have been reported into the FDA Field Accomplishments and
Compliance Tracking System (FACTS) since late 1999. The Domestic Fish and Fisheries Products
Compliance Program (CPGM 7303.842) provided coverage for investigations and sampling of
domestic fish and fishery products [56]. Imported seafood and seafood products were surveyed
for the presence of Listeria under the Import Seafood Products Compliance Program (CPGM
7303.844) [55]. The CPGM 7303.036 program was in existence through 1994 and covered both
domestic and imported processed seafood and seafood products [53,54]. Since 1994, the items
covered by this program have been incorporated into the CPGM 7303.842 and CPGM 7303.844
programs for domestic and imported products, respectively. RTE foods that require no further
or minimal processing by the consumer or products collected as a follow-up to a suspected case
of foodborne illness are identified for Listeria analyses in each of these compliance programs.
The CPGM 7303.842 program also includes Listeria analyses of in-line and swab samples
collected during processor establishment investigation reviews. Some domestic and imported
RTE products containing seafood are also collected under the Food Safety/Microbiological
Program (CPGM 7303.803) and the Imported Foods Program (CPGM 7303.819), respectively
[57, 58].
Between these three compliance programs a total of 7158 samples of fish, seafood products,
or seafood processing in-line samples were analyzed by the FDA between 1991 and 1996. L.
monocytogenes was detected in 622 of these samples, for an overall incidence of 8.7%. The overall
incidence between 2000 and February 2003 was 5.07% with L. monocytogenes isolated from 157
of 3101 samples. The breakdown of samples analyzed and those in which L. monocytogenes was
detected by year and origin (domestic or import) are presented (Table 15.3 and Table 15.4). The
incidence found by FDA is comparable to the 4–12% incidence of L. monocytogenes in seafood
TABLE 15.3
Seafood Product and Seafood Processing In-Line Samples Analyzed for Listeria
monocytogenes by the FDA from 1991 through 1996
1991 1992 1993 1994 1995 1996 Overall
Domestic product (CPGM) [1,3] [1,3] [1,3] [1,3] [1] [1]

Positive 20 63 89 94 67 41 374
Negative 403 501 558 623 481 382 2948
Total 423 564 647 717 548 423 3322
Positive (%) 4.7 11.2 13.8 13.1 12.2 9.7 11.3
Imported product (CPGM) [3] [2,3] [2,3] [2,3] [2] [2]
Positive 32 51 65 42 41 17 248
Negative 362 643 801 737 589 456 3588
Total 394 694 866 779 630 473 3836
Positive (%) 8.1 7.3 7.5 5.4 6.5 3.6 6.5
Overall
Positive 52 114 154 136 108 58 622
Negative 765 1144 1359 1360 1070 838 6536
Total 817 1258 1513 1496 1178 896 7158
Positive (%) 6.4% 9.1% 10.2% 9.1% 9.2% 6.5% 8.7%
Note: Data compiled from the FDA Microbiological Information System. Samples collected from the following
compliance programs (CPGM) [40,41,42]: (1) CPGM 7303.842 Domestic Fish and Fisheries Products (1991–1996),
(2) CPGM 7303.844 Import Seafood Products (1992–1996), (3) CPGM 7303.036 Processed Seafood (1991–1994).
TABLE 15.4
Seafood Product and Seafood Processing In-Line Samples Analyzed for Listeria
monocytogenes by the FDA from October 1999 through February 2003
2000 2001 2002 2003 Overall
Domestic product (CPGM)
Positive 40 31 18 10 99
Negative 434 467 246 65 1212
Total 474 498 264 75 1311
Positive (%) 8.44 6.22 6.82 13.33 7.55
Imported product (CPGM)
Positive 13 18 18 9 58
Negative 298 430 749 255 1732
Total 311 448 767 264 1790
Positive (%) 4.18 4.02 2.35 3.41 3.24
Overall
Positive 53 49 36 19 157
Negative 732 897 995 320 2944
Total 785 946 1031 339 3101
Positive (%) 6.75 5.18 3.49 5.60 5.06
Note: Data compiled from the FDA FACTS System. Samples collected from the following compliance programs
(CPGM) [40,41,42]: (1) CPGM 7303.842 Domestic Fish and Fisheries Products and CPGM 7303.803 Food
Safety/Microbiological samples and (2) CPGM 7303.844 Import Seafood Products and CPGM 7303.819 Imported
Foods.
and seafood products from temperate areas as reported by Embarek [38]. Surveys of other food
products have indicated a 4–60% incidence in raw meat, 23–60% in fresh poultry, and 2.2% in raw
milk [38,49,80].
Samples found positive for L. monocytogenes in FDA compliance programs represent a wide
range of fish and seafood products. During the 1991–1996 period a total of 218 crustacean products
(crab, shrimp/prawns, lobster, and crawfish) were positive, with crab accounting for the largest
number with 142 positive samples. Fifteen samples were positive for L. monocytogenes from the
shellfish category, which includes mussels, oysters, clams, scallops, and snails. The finfish category
had 231 samples positive for L. monocytogenes, with 164 of these from smoked seafood products.
The remaining positive L. monocytogenes samples represent a diverse group of products including
the following: squid/calamari, 3 samples; eel, 9 samples; roe/caviar, 19 samples; imitation seafood,
17 samples; seafood salad/spread/pâté or mousse, 13 samples; and processing in-line or swabs, 94
samples.
Many of the seafood products and related samples analyzed by the FDA with results reported
into FACTS from 2000 to February 2003 are included among the food categories identified in the
FDA/FSIS/CDC 2003 Quantitative Assessment of Relative Risk to Public Health from Foodborne
Listeria monocytogenes among Selected Categories of Ready-to-Eat Foods [133,134] (Table 15.5).
These include food categories for smoked seafood, raw seafood, preserved fish, cooked RTE
crustaceans, and sometimes, deli salads. The food categories with greater than 50 samples analyzed
and in which L. monocytogenes was recovered more frequently than the overall average (5.07%)
included the following: smoked seafood–cold-smoked fish, 14.92%; smoked fish (smoke process
not determined), 11.69%; prepared fish–roe/caviar, 8.62%; combination foods (containing sea-
food)–deli salads, 5.79%; and other deli items containing seafood, 11.32% (Table 15.5).
Smoked seafood ranked fifth for predicted relative risk among the 23 food categories in the
FDA/FSIS/CDC 2003 Quantitative Assessment of Relative Risk to Public Health from Foodborne
TABLE 15.5
Seafood Product and Seafood Processing In-Line Samples Analyzed for Listeria monocytogenes
by the FDA and Reported into FACTS from October 1999 through February 2003
Smoked Seafood
2000 2001 2002 2003 Overall
Total Positive Total Positive Total Positive Total Positive Total Positive Positive (%)
Fish—Hot 109 4 113 3 62 2 24 308 308 9 2.92

Fish—Cold 68 15 80 11 73 5 27 248 248 37 14.92
Fish—N/A 21 3 27 3 24 2 5 77 77 9 11.69
Shellfish and 5 0 2 0 5 0 1 13 13 0 0.00
other—hot
Shellfish and 0 0 0 0 1 0 0 1 1 0 0.00
other—cold
Shellfish and 2 0 1 0 1 0 0 4 4 0 0.00
other—N/A
Raw Seafood
2000 2001 2002 2003 Overall
Total Positive Total Positive Total Positive Total Positive Total Positive Positive ( %)
Raw 4 1 1 1 2 1 2 0 9 3 33.33
Preserved Seafood Products

2000 2001 2002 2003 Overall
Marinated/cultured 7 0 6 0 4 0 4 1 21 1 4.76
Dried 35 1 11 0 36 0 14 0 96 1 1.04
Roe/caviar 25 2 13 2 15 1 5 0 58 5 8.62
Mollusks 47 2 62 1 72 2 34 0 215 5 2.33
Prepared 49 5 56 3 98 2 36 0 239 10 4.18
RTE Crustaceans
2000 2001 2002 2003 Overall
Crab 168 6 211 1 206 5 47 3 632 15 2.37

Shrimp 61 1 114 15 173 2 39 0 387 18 4.65
Crawfish 10 0 18 0 39 3 12 0 79 3 3.80
Lobster 9 2 11 1 7 0 3 0 30 3 10.00
Spiny lobster 1 0 4 0 2 0 1 0 8 0 0.00
Langostinos 1 0 7 1 5 1 0 0 13 2 15.38
Other 0 0 1 0 1 0 1 0 3 0 0.00
Combination Foods Containing Seafood

2000 2001 2002 2003 Overall
Deli salads 43 4 44 3 31 0 3 0 121 7 5.79

Deli sandwiches 13 0 21 0 7 0 5 0 46 0 0.00
(continued)

Seafood Product and Seafood Processing In-Line Samples Analyzed for Listeria monocytogenes
by the FDA and Reported into FACTS from October 1999 through February 2003
Deli 15 1 15 1 13 0 3 0 46 2 4.35
spreads/pâté/dips
Other deli items 12 0 14 0 17 1 10 5 53 6 11.32
(i.e., sushi)
Imitation seafood 25 0 25 0 54 0 22 0 126 0 0.00
Other combination 39 1 69 0 71 2 38 1 217 4 1.84
foods
Other
2000 2001 2002 2003 Overall
Environ. 16 5 20 3 12 7 3 2 51 17 33.33
Overall 785 53 946 49 1031 36 339 19 3101 157 5.06
Listeria monocytogenes Among Selected Categories of Ready-to-Eat Foods on a per-serving basis

for all subpopulations, intermediate age, elderly, and perinatal [133]. In the FDA studies, a total
of 1210 smoked finfish products were analyzed for Listeria, with 164 (13.6%) samples positive for
L. monocytogenes between 1991 and 1995. Of those smoked seafood samples for which the smoke
process was known, the incidence of L. monocytogenes was higher in cold-smoked (21.3%, 51 of
240) compared to hot-smoked (8.8%, 19 of 215) samples [70]. Between FY 2000 and February
2003, the overall incidence of L. monocytogenes in smoked seafood was 8.45%, with 55 positive
samples among 651 samples analyzed (Table 15.5). Cold-smoked fish had the highest L. monocy-
togenes incidence at 14.92% (37 of 248 samples), compared to hot-smoked fish, which had an
incidence of 2.92% (9 of 308 samples). Smoked fish in which the type of smoking process was
not identified yielded L. monocytogenes from 9 of 77 samples (11.69%). Several other investigations
have evaluated prevalence of L. monocytogenes in smoked finfish products, with incidences ranging
from 0 to 75 % [38]. In those surveys with greater than 100 samples, the incidence of L.
monocytogenes also tended to be greater in cold-smoked finfish products (11.3 to 24%) [76–78]
compared to hot-smoked finfish products (8.4 to 8.9%) [76–78]. Postprocess contamination most
likely accounts for the presence of L. monocytogenes in hot-smoked finfish products. However,
with cold-smoked finfish, while the process may not eliminate L. monocytogenes present on the
raw product, contamination could also occur during or after processing of the product [35].
Cooked RTE crustaceans, preserved fish, and combination foods–deli salads ranked 6th, 12th,
and 19th, respectively, among the 23 food categories for relative risk based on a per-serving basis
[133]. During 1991–1996, 1886 RTE crab samples were analyzed for Listeria and 142 (7.5%)
tested positive for L. monocytogenes. The incidence rate for RTE crab fell to 2.37% (15 of 632
samples) during October 1999 to February 2003 (Table 15.5). The overall L. monocytogenes
incidence rate for all RTE crustaceans was 3.56% (41 of 1152 samples) during this same period.
Several other surveys [22,37,44,105,120,138] have reported L. monocytogenes incidence rates of
0 to 29.2%. It is highly likely that the presence of L. monocytogenes in RTE crab is the result of
postprocess contamination. The preserved fish category had an overall L. monocytogenes incidence
of 3.5% (22 of 629 samples), with roe/caviar having the highest positive rate in this category.
Among the combination foods containing seafood, the overall incidence of L. monocytogenes was
3.12% (19 of 609 samples). Products in this category with the highest L. monocytogenes incidence
were other deli items, i.e., sushi, and deli salads (11.32%).
Since 1987, at least 126 Class I recalls have been issued in the United States for domestic and
domestic/imported RTE seafood products contaminated with L. monocytogenes [13,51,52]. Recalls
have only been issued when L. monocytogenes was found in RTE seafood or seafood products that
would receive no subsequent or minimal heat treatment by the consumer before consumption. The
number of recalls by product category is shown (Table 15.6). The products with the greatest number
TABLE 15.6
Class I Recalls Issued in the United States for Domestic and Domestic/Imported
Ready-to-Eat Seafood Products Contaminated with L. monocytogenes since 1987a
Number of Class I Pounds (lb)
Product Recalls since 1987 Affected Location of Manufacturer
Crustacean
Crab 46 >114457 AL, FL, GA, ME, NC, OR, TX,
VA, WA, Chile, Mexico
Shrimp 7 >31332.5 FL, GA, MA, NY, WA
Lobster, langostinos 3 >264 Canada, Chile
Shellfish
Mussels (marinated) 1 Unknown MA
Mussels (smoked) 1 Unknown New Zealand
Finfish
(Hot-smoked) 7 >253 CA, KY, MD, ME, NY, WA
(Cold-smoked) 22 >93722 CA, MA, ME, NJ, NY, OR, WA,
United Kingdom
(Smokedb) 22 >10142 FL, IL, MD, ME, MN, NC, NY,
VA, WA, Denmark
(Salted) 1 Unknown Canada
Other
Imitation seafood 4 >11793 VA, WA, Japan, Korea
Seafood salad or spread 12 >4107 FL, ID, MA, ME, NY, VA, WA
aData compiled from FDA enforcement reports: Anonymous. 1995. Misdeclaration and Recall of Smoked Salmon
from the United Kingdom. U.S. FDA Import Bulletin 16-B86; FDA. Recalls and Field Corrections. In FDA
Enforcement Reports for June 10, 1987; December 16,1987; December 23, 1987; July 20, 1988; August 10, 1988;
October 5, 1988; October 20, 1988; December 28, 1988; January 18, 1989; May 24, 1989; October 4, 1989;
November 1, 1989; November 8, 1989; January 24, 1990; July 11, 1990; August 8, 1990; October 31, 1990;
November 28, 1990; December 19, 1990; January 9, 1991; March 27, 1991; August 14, 1991; September 4, 1991;
November 6, 1991; December 4, 1991; December 11, 1991; February 19, 1992; June 10, 1992; July 2, 1992; July 29,
1992; August 5, 1992; August 19, 1992; September 2. 1992; September 9, 1992; September 23, 1992; September 30,
1992; October 7, 1992; October 28, 1992; November 25, 1992; January 27, 1993; February 17, 1993; March 10, 1993;
May 19, 1993; June 9, 1993; August 25, 1993; July 28, 1993; August 4, 1993; August 25, 1993; October 27, 1993;
November 10, 1993; November 17, 1993; December 15, 1993; January 19, 1994; April 13, 1994; June 1, 1994; July
20, 1994; July 27, 1994; September 21, 1994; October 5, 1994; December 7, 1994; February 22, 1995; March 8,
1995; March 15, 1995; April 5, 1995; April 12, 1995; April 26, 1995; August 9, 1995; August 30, 1995; September
27, 1995; December 13, 1995; May 8, 1996; June 12, 1996; July 24, 1996; September 4, 1996; October 23, 1996;
January 2, 1997. U.S. Government Printing Office, Pittsburgh, PA; FDA. Recalls and Field Corrections. In FDA
Enforcement Reports for April 30, 1997; August 6, 1997; September 17, 1997; July 29, 1998; September 2, 1998;
December 23, 1998; November 5, 1999; January 10, 2000, March 10, 2000; April 5, 2000; August 9, 2000; August
29, 2000; August 31, 2000; January 4, 2001; January 10, 2001; September 6, 2001; May 22, 2002; July 3, 2002;
July 30, 3002; September 4, 2002; September 17, 2002. www.fda.gov/OC/po/firmrecalls/archive.html.
b Hot- or cold-smoking process not identified.
of recalls reflected the types of products most frequently identified as positive for L. monocytogenes
in compliance program surveys. Crab accounted for 46 and smoked finfish accounted for 51 of the
126 recalls.
OTHER SURVEYS FOR L. MONOCYTOGENES IN FISH

AND SEAFOOD PRODUCTS
The documented presence of Listeria in fish and seafood and subsequent product recalls prompted
several studies to determine the incidence of Listeria spp. in a variety of products from many
geographic locations (Table 15.7). Results of many of these studies have been extensively reviewed
[12,32,38,78]. Sampling strategies, number of samples analyzed, and detection methods varied, so
that although it is useful to note the results from these studies, the data from them cannot always
be directly compared.
CRUSTACEANS
Listeria spp. including L. monocytogenes, have been recovered from cooked and picked RTE
crabmeat, and as noted earlier this product has been among those most frequently recalled. Because
this product is thermally processed, which should eliminate or reduce microorganisms, presence
of L. monocytogenes on the finished product most likely represents postprocessing contamination.
Several studies have included a limited number of crab samples. L. monocytogenes was detected
in 7 and L. innocua in 12 of 24 cooked imported crab products [138]. Although no L.
monocytogenes was detected in another study, L. welshimeri was found in 1 of 2 cooked crab
samples [22]. Listeria spp. were recovered in 2 of 5 crab samples collected as part of a survey in
Alexandria, Egypt [37], and L. monocytogenes was isolated from 1 of 7 crab samples from the
United States and China [44]. Two larger U.S. studies of cooked and processed crab have also been
published. Although 31 of 138 (22.5%) processed crab samples contained Listeria spp., no attempt
was made for further speciation [105]. In a second study, 126 cooked and picked blue crab samples
were analyzed; 10 samples (7.9%) were positive for L. monocytogenes and 3 samples (2.4%)
positive for L. innocua [120]. No L. monocytogenes was detected in 154 crab samples analyzed
in Canada [47]. Very few studies have quantified L. monocytogenes in naturally contaminated
cooked and processed crab. Among the 10 samples positive for L. monocytogenes in one study
[120], 1 sample contained 1100 Listeria/g but in the remaining samples <100 Listeria/g were
detected, indicating a generally low level of contamination.
Listeria spp. have been detected in fresh or raw shrimp with two of seven samples
containing L. monocytogenes [138], and one of four samples containing L. innocua [22] in two
U.S. studies. In a survey of fresh shrimp in Japan, Listeria spp. were recovered from 6 of 70 samples
(8.6%) with one (1.4%) of these positive for L. monocytogenes [94]. In France, L. monocytogenes
was isolated from 2 of 17 (11.8%) uncooked shrimp samples and Listeria spp. from 4 samples
(23.5%) [119]. In Trinidad, where shrimp are consumed in a nearly raw state, Listeria spp. were
recovered from 2 of 41 (5%) fresh, uncooked shrimp samples [1]. L. monocytogenes also has been
detected in 6.7% of raw shrimp from China, Ecuador, and Mexico [16].
Despite cooking and other heat processing steps, which should eliminate Listeria on raw
product, Listeria spp. have been recovered from cooked and RTE shrimp/prawns by several inves-
tigators. L. monocytogenes was detected in 2 of 8 (25%) cooked and processed shrimp in a U.S.
study [138], 4 of 49 (8.2%) RTE shrimp, 4 of 287 (1.5%) cooked shrimp, 0 of 4 cooked shrimp
in Canada [44,47], and 1 of 38 (2.6%) [128] RTE shrimp products in Japan. No L. monocytogenes
was recovered from 40 retail samples of cooked prawns, shrimp, and cockles sold in England and
Wales between 1987 and 1989 [122]. Brined RTE shrimp had L. monocytogenes in 3 of 18 (16.7%)
samples in Norway [126].
TABLE 15.7
Incidence of Listeria spp. in Fresh, Frozen, and Processed Seafood
Number of (%) of Positive Samples

Product (country) Samples Listeria spp. L. monocytogenes Reference
Crab
Crab (c) (multiple countries) 24 29.2 138
Crab (c) (U.S.A.) 2 50 0 22
Crab (r) (China) 7 14 44
Crab (c or p) (U.S.A.) 138 22.5 105
Crab (Egypt) 5 40 0 37
Blue crab (c) (U.S.A.) 126 10.3 7.9 120
Crab (c) (multiple countries) 154 0 47
Shrimp/prawns
Shrimp (f), (multiple countries) 7 28.6 138
Shrimp (f and fr) (U.S.A.) 4 25 0 22
Shrimp (f) (Japan) 70 8.6 1.4 94
Shrimp (f) (Trinidad) 41 5 1
Shrimp (raw, fr) (France) 17 23.5 11.8 119
Shrimp (c and p) (multiple countries) 8 25 138
Shrimp (r) (multiple countries) 49 8.2 44
Shrimp (f and p) (India) 19 10.5 0 93
Shrimp (Canada) 20 20 44
Shrimp in brine (r) (Norway) 16 18 126
Shrimp (c and f) (Iceland) 11 9 9 69
Prawn (r) (Japan) 38 15.8 2.6 128
Shrimp (Egypt) 5 40 20 37
Prawns/shrimp/cockles (c) (U.K.) 40 0 122
Shrimp (c) (multiple countries) 287 1.5 47
Shrimp (peeled) (multiple countries) 4 0 47
Shrimp (Brazil) 178 50 18 28
Shrimp (r) (China, Ecuador, Mexico) 6.7 16
Lobster
Lobster tail (fr) (multiple countries) 2 50 138
Lobster (c) (Canada) 70 0 47
Mussels
Mussels (sm) (New Zealand) 14 35.7 73
Mussels (f) (Spain) 40 22.5 7.5 130
Oysters
Oysters (fr) (multiple countries) 1 0 0 138
Oysters (p) (U.S.A.) 2 0 0 22
Oysters (f) (Japan) 84 0 0 94
Oysters (f) (Egypt) 2 0 0 37
Clams
Clam (f) (India) 1 0 0 62
Clam (f) (U.S.A.) 1 0 0 22
Clam (surf and c) (multiple countries) 59 0 47
Scallops
Scallops (fr) (multiple countries) 2 50 0 139
Scallops (raw) (U.S.A.) 1 0 0 22
Scallop (Canada) 1 0 47
(continued)


Other shellfish and invertebrates

Shellfish (c) (Iceland) 11 0 0 69
Non-oyster shellfish (f) (Japan) 147 11.6 1.4 94
Shellfisha 25 44 25 73
Donax spp. (coquina) (f) (Egypt) 6 16.7 16.7 37
Ruditapes spp. (clam) (f) (Egypt) 4 50 25 37
Crayfish (c) (multiple countries) 6 0 47
Finfish
Fish (f) (U.S.A.) 4 50 50 22
Catfish (f) (U.S.A.) 1 100 0 22
Fish (f) (Trinidad) 61 14.8 2 1
Minced fish (f) (Norway) 8 12 126
Fish (f) (Japan) 382 12.6 2.4 94
Fish (f) (India) 51 3.9 0 93
Fish (f) (Egypt) 39 25.6 12.8 37
Fish (fr) (Egypt) 17 17.6 5.9 37
Fish (f) (India) 4 25 62
Fish (fr) (India) 10 20 62
Fish (r) (New Zealand) 25 52 32 73
Minced fish (raw, r) (Japan) 37 43.2 8.1 128
Fish (trout) (f) (Iceland) 2 0 0 69
Fish (dried haddock) (Iceland) 5 0 0 69
Fish (fr) (multiple countries) 4 25 138
Fish (ceviche) (Peru) 32 75 9 61
Fish (preserved not heat-treated) (Denmark) 335 10.8 108
Fish (preserved not heat-treated) (Denmark) 282 22.3 108
Fish (r) (Denmark) 232 14.2 108
Fish (lightly pickled) (Switzerland) 89 25.8 77
Fish (Gravad) (Iceland) 22 63.6 22.7 69
Fish (cold sm, salmon) (Switzerland) 100 24 76
Fish (cold sm, salmon) (Switzerland) 64 6.3 65
Fish (cold sm, fish) (Switzerland) 324 13.6 77
Fish (cold sm, fish) (Switzerland) 434 11.3 78
Fish (cold sm, salmon) (Norway) 33 9 126
Fish (sm, salmon) (Iceland) 31 29 3.2 69
Fish (cold sm, salmon) (Canada) 32 31.2 44
Fish (cold sm, salmon) (Italy) 37 0–80 0 136
Fish (cold sm, salmon) (New Zealand) 12 75 73
Fish (cold sm, salmon) (U.S.A.) 61 78.7 35
Fish (sm) (New Zealand)b 12 66.7 73
Fish (smoked and cold-salted) (Finland) 110 20 81
Fish (smoked) 380 7.1 23
Fish (cold sm, salmon) (Denmark) 340 20.9 74
Fish (cold sm, salmon) (Denmark) 190 33.7 82
Fish (sm, salmon) (Japan) 92 5.4 75
Fish (gravad or sm, salmon/rainbow trout) 51 39 88
Fish (gravad or sm, salmon/rainbow trout) 32 9.4 88
(continued)



Fish (gravad or sm, salmon/rainbow trout) 94 17 88
Fish (sm fish) (Canada) 71 11.3 33
Fish (sm fish) (Canada) 20.4 4.4 31
Fish (hot sm fish) (Switzerland) 496 8.9 77
Fish (hot sm fish) (Switzerland) 691 8.4 78
Fish (sm and/or salted) (Egypt) 11 18.2 5.6 37
Fish (Canada import) 3 33.3 47
Other fish and seafood products
Seafood (smk) (Canada import) 64 1.6 64
Seafood (smk) (Canada) 130 0 47
Seafood (eel) (Canada import) 14 0 47
Seafood (squid), langostinos) (multiple 2 100 0 138
countries)
Seafood (f and fr) (Taiwan) 57 10.5 139
Seafood (f and p) (Iceland) 26 3.9 3.9 69
Seafood (India) 200 8 0 83
Seafood (f) (U.S.A.) 59 49.2 105
Seafood (p) (U.S.A.) 14 0 105
Seafood (other) (Iceland) 5 20 20 69
Seafood raw (r) (Japan) 28 7.1 10.7 128
Seafood (r) (New Zealand) 50 48 26 73
Seafood (c) (Japan) 5 0 0 128
Seafood (other) (Japan) 6 0 0 128
Seafood (r) (Japan) 213 3.3 75
Seafood salad (p) (U.S.A.) 2 0 0 22
Fish salads (r) (Iceland) 37 32 16 69
Seafood (Pasta with minced fish) (Iceland) 3 0 0 69
Seafood (surimi) (multiple countries) 7 28.6 139
Seafood (surimi) (U.S.A.) 1 0 0 22
Seafood (surimi) (Canada) 46 2 44
Kamaboko (surimi) (Canada import) 335 0 47
Pâté (Canada import) 20 0 47
Seafood salad (Canada) 4 0 47
Processed seafood (Canada) 11 0 47
Note: (c) = cooked; (f) = fresh; (fr) = frozen; (p) = processed; (r) = ready-to-eat.
aIncludes the 14 smoked mussel samples listed in mussels category.
bIncluded in the 25 ready-to-eat fish samples in the New Zealand study list given earlier.
Source: Adapted in part from Embarek, P.K.B. 1994. Presence, detection and growth of Listeria monocytogenes in
seafoods: a review. Int. J. Food Microbiol. 23: 17–34.
As with the surveys of cooked and processed crab, few studies have attempted to quantify
L. monocytogenes in cooked shrimp. However, low levels of L. monocytogenes in three lots of
naturally contaminated RTE shrimp (0.54, 5.5, and 0.04 MPN/g) and lobster (2.0, 0.23, and 0.4
MPN/g) were reported in one study [44].
SHELLFISH
Smoked mussels have been associated with several listeriosis cases in Australia and New Zealand
[42,97] and the subject of recalls in the United States. L. monocytogenes was isolated from 5 of 14
smoked mussel samples in a survey [73] in New Zealand, whereas for fresh mussels from Spain,
L. monocytogenes was recovered from 3 and other Listeria spp. from 9 of 40 samples [130].
The incidence of Listeria in other shellfish products, including oysters, clams, and scallops has
been remarkably low. No Listeria spp. were isolated from oysters in two studies in the United
States and one in Egypt that included one or two samples [22,37,138]. In a Japanese study, no
Listeria spp. were recovered from 84 oyster samples [94]. No Listeria spp. were found in clams
in two studies with a single sample in each [22,62]; however, in Egypt, Listeria spp. were found
in two of four Ruditapes spp. (clam) samples, one of which yielded L. monocytogenes [37]. Scallops
are another category for which only a limited number of samples have been analyzed. L. innocua
was isolated from one of two scallop samples [138]. No Listeria spp. were recovered from a single
sample in a later study [22]. Similarly, no listeriae were found in 11 cooked shellfish samples from
Iceland [69]. In a survey of fresh seafood purchased from markets in Alexandria, Egypt, one of
six Donax spp. (coquina) samples was positive for L. monocytogenes [37]. In Japan, L. monocyto-
genes was isolated from 2 samples and Listeria spp. were recovered from 17 samples of a total of
147 non-oyster shellfish samples [94,101].
FINFISH
In the United States raw fresh or frozen fish are generally not consumed without further processing;
therefore, surveys there have included very few fresh or frozen fish samples. L. monocytogenes
was isolated from two of four fresh fish samples, L. innocua from one catfish sample [22], and L.
monocytogenes from one of four frozen fish samples [138] in surveys conducted in the United
States. In India, Fuchs and Surendran [62] reported Listeria spp. in 1 of 4 fresh and 2 of 10 frozen
fish samples. In a larger survey of fresh fish in India, Listeria spp. were isolated from 2 of 51
(3.9%), samples but no L. monocytogenes was recovered [93]. In Alexandria, Egypt, Listeria spp.
were recovered from 10 of 39 (25.6%) fresh and 3 of 17 (17.6%) frozen fish samples, with L.
monocytogenes isolated from 5 of 39 (12.8%) and 1 of 17 (5.9%) of these fresh and frozen fish
samples, respectively [37]. In Norway, L. monocytogenes was isolated from 1 of 8 (12.5%) minced
fresh fish samples [126]. The practice of consuming fresh seafood in an almost raw state is common
in Trinidad, where Listeria spp. were detected in 9 of 61 (14.8%) fresh fish samples [1]. In Japan,
3 of 37 (8.1%) raw RTE minced fish samples yielded L. monocytogenes with 16 of 37 (43.2%)
containing Listeria spp. [128]. In another study in Japan, L. monocytogenes was found in 9 (2.4%)
and Listeria spp. in 48 of 382 (12.6%) fresh fish samples [94]. It is not clear if all these samples
were RTE. A more recent study in Japan reported that 7 of 213 (3.3%) raw seafood samples were
positive for L. monocytogenes [75]. However, only one of the seven positive samples contained
>100 L. monocytogenes CFU/g.
SMOKED FISH PRODUCTS

The prevalence of Listeria in smoked and lightly processed fish products is often a concern
because many of these products are commonly eaten without further heating. The cold-smoking
process does not generate sufficient heat to inactivate Listeria organisms that may be present
on fish [35,66,91]. In Switzerland, L. monocytogenes was isolated from 24 and 6% of cold-
smoked salmon [65,76] and 13.5 and 11.3% of cold-smoked fish samples [77,78]. In studies
in Norway [123], Canada [44], and New Zealand [73], the organism was isolated from 9, 31,
and 66% of the cold-smoked salmon samples examined (Table 15.7). The level of L. monocytogenes
in smoked fish tends to be low. In a Swedish survey, 12 of 17 samples positive for L.
monocytogenes had less than 10 L. monocytogenes CFU/g; 4 samples had levels between 10
and 1000 CFU/g, and only one had more than 10,000 CFU/g [88]. A second Scandinavian
study reported 6 of 16 positive samples had less than 10 L. monocytogenes CFU/g, 6 samples
with 10–1000 CFU/g, 2 samples with 1000–10,000 L. monocytogenes CFU/g, and 2 samples
with more than 10,000 L. monocytogenes CFU/g [88]. In a third study 19 of 27 positive samples
had less than 100 L. monocytogenes CFU/g, 4 samples had between 100 and 10,000 L.
monocytogenes CFU/g, and more than 10,000 L. monocytogenes CFU/g was recovered from
the remaining 4 positive samples [23]. In comprehensive studies of one processing plant,
Eklund et al. [35] identified incoming raw salmon (36% combined incidence and 65% skins
from a frozen storage warehouse) as the initial source of L. monocytogenes. During filleting
and brining, the pathogen was transferred to other fish fillets with exposed flesh. As the product
moved through the different stages of processing, equipment, personnel, and other contact
surfaces became contaminated and served as additional sources of contamination. If these
secondary sources of contamination are not properly eradicated, they can become a continuous
source of contamination. In more recent studies by Hoffman et al. [71], a 29.5% incidence of
L. monocytogenes in U.S. West Coast salmon and a lower incidence in other fish species were
reported. Autio et al. [15], Rørvik et al. [125], and Vogel et al. [137] also reported a low incidence
of L. monocytogenes in raw fish and concluded that the primary source for L. monocytogenes
contamination was from equipment and the processing environment. Differences in the reported
incidence of L. monocytogenes in raw fish could in part be related to methodology. Because
of the low incidence of L. monocytogenes and possible harboring of the bacterium under scales
of the fish, Eklund et al. [35] removed and enriched the entire skin of raw fish in both Listeria
enrichment broth (EB) and University of Vermont modified (UVM) broth. In comparison, other
investigators have used smaller samples (sections of fish, swabbed areas, and scrapings). In
many instances, the incidence in raw fish may be low; however, even if only a small number
of raw fish are initially contaminated, they can easily contaminate the hundreds of other fish
in a day’s production. As an example, Autio et al. [15] reported that only 1 out of a total of
60 fish contained L. monocytogenes. They also reported that the water, salt, and sugar used in
the brine were not contaminated. However, the frequency of fish containing L. monocytogenes
clearly increased after brining, which was the most critical point of L. monocytogenes con-
tamination. In addition, gloves of employees working on the product after brining were positive
for L. monocytogenes.
Recent studies using molecular subtyping also indicate that raw materials rarely seem to be
responsible for finished product contamination in the production of cold-smoked seafood. Instead,
the processing environment seemed to be responsible for most contamination of smoked fish
[15,81,109,125,137]. In these instances, L. monocytogenes appeared to have become established
on equipment and in processing areas with sanitation procedures inadequate to eliminate Listeria.
In those studies that specifically identified hot-smoked fish samples, L. monocytogenes was also
recovered despite the heat processing that these products received, with the pathogen present in
8.9 and 8.4% of samples [77,78].
LIGHTLY PROCESSED FISH PRODUCTS

Other lightly processed RTE fish products have also harbored L. monocytogenes. These include
lightly pickled fish from which L. monocytogenes was isolated from 25.8% of samples surveyed
in Switzerland [77]. Ceviche is a lightly acidified RTE fish product, which is popular in several
South American countries. Listeria spp. were recovered from 75% and L. monocytogenes from
9.4% of ceviche samples in Peru [61]. In Iceland, seafood and fish salads yielded Listeria spp. in
32.4% and L. monocytogenes in 16.2% of the samples [69]. No Listeria spp. were recovered from
two seafood salad samples included in a U.S. survey [22] or from three pasta salads containing
minced fish in the Iceland study [69]. Imitation seafoods made from surimi had an L. monocytogenes
incidence of 28.6% in the United States [138] and 2.2% in Canada. No Listeria were recovered
from a single sample in a second U.S. survey [22].
HUMAN LISTERIOSIS ASSOCIATED WITH FISH

AND SEAFOOD PRODUCTS
There have been several reported human listeriosis outbreaks associated with consumption of fish
and seafoods. As early as 1980, an epidemic of 22 perinatal L. monocytogenes infections during
an 11-month period was reported in Auckland, New Zealand [86]. Although the cause was not
confirmed, an epidemiological survey suggested that shellfish and raw fish consumption may have
played a role [86].
The first case of listeriosis positively linked to consumption of fish or seafood was not reported
until 1989, when a 54-year-old woman in Italy contracted listeric meningitis 4 days after consuming
steamed fish from which L. monocytogenes was later isolated [43]. Two L. monocytogenes isolates,
one from the patient’s cerebrospinal fluid and the other from a leftover portion of the fish, were
both of serotype 4 and were identical in terms of phage type and restriction analysis of chromosomal
DNA, indicating that the fish was the most likely vehicle of infection in this case of listeric meningitis.
However, how this fish became contaminated remains a mystery.
In 1991, three healthy people, aged 83, 37, and 10, became ill in two separate incidents in the
state of Tasmania, Australia, after consuming smoked mussels [97]. Symptoms included malaise,
chills, fever, and headache followed by diarrhea. Samples of the implicated mussels contained over
one million L. monocytogenes CFU/g with this pathogen also recovered from stool samples. The
implicated mussels were exported to Australia, repackaged illegally by a retail outlet, and labeled
with a code date that overestimated their shelf life by 3 months or more. Patient isolates and those
from associated refrigerated packages of mussels were identified as L. monocytogenes serovar 1/2b
and were indistinguishable by both phage typing and restriction fragment length polymorphism
(RFLP) analysis [21].
Newborn twins died as a result of an L. monocytogenes infection in Auckland, New Zealand, in
1992. Their deaths were attributed to consumption of contaminated smoked mussels by their mother.
Reports from New Zealand indicated that the company producing the smoked mussels had Listeria
in their product several months before the twins died. In 1993, the company owner and a consultant
to the company were charged with manslaughter by New Zealand police [42]. The two patient isolates
of L. monocytogenes were indistinguishable by serotype and pulsed-field gel electrophoresis (PFGE)
analysis. In addition, 2 more clinical isolates of the same serotype and PFGE pattern were identified
among 15 clinical listeriosis cases in New Zealand during 1991 and 1992. These 4 patient isolates
along with 26 isolates from 15 retail packages of the implicated mussels, the processing factory, the
refrigerator of one patient, and a wholesaler in the United Kingdom and 7 isolates from environmental
swabs taken in the processing factory environment were analyzed by various subtyping methods [21].
These included serotyping, phage typing, DNA-RFLP analysis, cadmium and arsenic sensitivity
testing, and PFGE analysis. Isolates from 3 of the patients as well as 26 from the product and 4 from
factory environmental swabs were all indistinguishable by all of the subtyping systems [21]. This
linked the patient isolates with the implicated smoked mussels and the processing environment.
These isolates were also distinct from the patient cultures in the previously mentioned Tasmanian
outbreak [21].
Since these outbreaks, the Australian National Health and Medical Research Council [102,103]
has issued two bulletins recommending special dietary advice for pregnant women, transplant
patients, and other immunocompromised patients and providing information for medical practitio-
ners on the diagnosis, treatment, and other advice for patients.
Shrimp was identified as a significant risk factor for illness during an epidemiological investigation
in Connecticut [121]. The investigation was initiated after two pregnant women who attended a common
party developed Listeria bacteremia. The case definition of Listeria infection, including isolation of L.
monocytogenes from a blood or stool sample, or the occurrence of two or more symptoms of mild
listeriosis including fever, musculoskeletal problems, nausea, vomiting, or diarrhea was used to evaluate
the possibility of mild disease and foods associated with the illnesses. The case definition of listeric
infection was met for 10 (28%) of the 36 party attendees. Consumption of shrimp, nonalcoholic
beverages, Camembert cheese, and cauliflower were identified as being associated with illness, but
only shrimp remained after controlling for consumption of other foods among the party attendees.
Two cases of listeriosis in previously healthy adults were linked to consumption of imitation
crabmeat in Ontario, Canada [48]. L. monocytogenes was isolated from several items from the
patient’s refrigerator, including imitation crabmeat, canned black olives, macaroni and vegetable
salad, spaghetti sauce with meatballs, and mayonnaise. The imitation crabmeat contained L.
monocytogenes at a level of 2.1 × 109 CFU/g. In addition, L. monocytogenes was also isolated
from unopened packages of the imitation crabmeat. The isolates from the patients’ opened and
unopened imitation crabmeat were serotype 1/2b, indistinguishable by random amplified polymor-
phic DNA (RAPD) and PFGE analyses. The ability of imitation crabmeat to support growth of L.
monocytogenes in challenge studies emphasizes the concern about refrigerated products with a long
shelf life and no additional control factors to prevent growth of L. monocytogenes.
Gravad or cold-smoked rainbow trout was identified as the source of a listeriosis outbreak in
Värmland, Sweden [41,132]. Nine people became ill, with two deaths due to listeriosis in this small
province between August 1994 and June 1995. L. monocytogenes isolated from six of the patients,
a package of gravad rainbow trout in one patient’s refrigerator, and an unopened package of gravad
rainbow trout from the same manufacturer were indistinguishable from each other using PFGE. The
level of L. monocytogenes in the fish from the patient’s refrigerator was reported to be 6200 CFU/g.
The levels in four unopened packages from the same manufacturer were less than 100 CFU/g for
three samples and 120 CFU/g for the fourth sample. A fifth sample was delayed during shipment
to the laboratory, resulting in a level of 2.5 × 106 CFU/g when analyzed, indicating that this product
does support growth of L. monocytogenes to high levels when temperature-abused. Gravad fish is
prepared from raw fillets rubbed with sugar, salt, and pepper, covered with dill, refrigerated 2 days,
and then vacuum packaged either whole or sliced in oxygen-impermeable film. The cold-smoked
fish products from this same manufacturer were prepared by either rubbing raw fillets with salt, or
injecting the cure followed by smoking at 25–30°C for 2–3 h, and then packaging either whole or
sliced under vacuum. L. monocytogenes of the same clonal group associated with the outbreak was
isolated from unopened packages of gravad and cold-smoked rainbow trout and from the packaging
machine in the processing plant. This observation, and the fact that the packaging machine was
difficult to clean, led investigators to hypothesize that the packaging machine was the likely source
of contamination.
Consumption of cold-smoked rainbow trout was also implicated in an outbreak involving five
previously healthy adults who developed febrile gastroenteritis in Finland [96]. They consumed the
same cold-smoked fish at a meal and all experienced nausea, abdominal cramps, and diarrhea within
27 h. An unopened package from the same lot of implicated product obtained from the same retail store
contained 1.9 × 105 L. monocytogenes CFU/g. The temperature in the retail store display cabinet varied
from 4.6°C at the bottom to 11.6°C at the top, whereas the manufacturer recommended a storage
temperature of 0–3°C. The elevated number of L. monocytogenes organisms in this product was likely
due to the higher storage temperature. The L. monocytogenes isolates from the five patients and the
cold-smoked rainbow trout were serotype 1/2a and indistinguishable by PFGE analysis.
REGULATORY ASPECTS OF L. MONOCYTOGENES

IN FISH AND SEAFOOD
Although a detailed discussion of proposed criteria for L. monocytogenes and other pathogens in
foods has been reserved for Chapter 18, the reader should be aware that the NACMCF has
recommended a “zero-tolerance” for L. monocytogenes in cooked shrimp and crabmeat [9,11]. The
policies of different countries vary for the regulation of L. monocytogenes in food products. In
1994, Madden stated “The policy of the (U.S.) FDA remains what has been commonly referred to
as the ‘zero tolerance’ policy, which is very conservative. No L. monocytogenes organisms are
permitted in a food which was not intended for further heat treatment.” [92]. The detectable presence
of L. monocytogenes in RTE foods is considered to be a hazard to health under current FDA policy
[36]. Elliot and Kvenberg state, “Currently, FDA requests recall of any ready-to-eat food in which
L. monocytogenes is detected using present methodology, if it is determined that the cooking or
heating instruction would not provide for lethality. A Class I recall [CFR 7.3 (m) (1)] is initiated
when there is a reasonable probability that the use of, or exposure to, a violative product will cause
serious adverse health consequences or death.” They noted a quantitative L. monocytogenes risk
assessment was underway in the United States as a joint effort between HHS and USDA and
concluded that a thorough review of this risk assessment should be made before making further
risk management decisions about L. monocytogenes policy.
Canadian regulations regarding the presence of L. monocytogenes in RTE foods have been
based on the ability of L. monocytogenes to grow on a given food product [45]. Three categories
of foods have been established based on assessed health risk. Category 1 includes foods linked to
outbreaks and receives the highest regulatory priority. The second level of regulatory priority
includes RTE foods that can support growth of L. monocytogenes and have a shelf life greater than
10 days. RTE foods that either do not support growth of L. monocytogenes or have a shelf life of
less than 10 days are classified as category 3 and receive the lowest priority in terms of inspection
and compliance actions [47].
Complete agreement among the countries that make up the European Economic Community
(EEC) for the criteria of L. monocytogenes in all foods [131] has not to date been achieved.
Criteria established for L. monocytogenes by the EEC are only within the Milk Hygiene Directive.
Several approaches have been used by individual European countries, including addressing
industry groups about HACCP-based hygiene plans and education of the most susceptible groups,
such as pregnant women and immunocompromised individuals. A quantitative approach setting
limits at the point of sale or end of product shelf life has also been explored by several countries.
For example, German regulations categorize foods into four risk-level categories and set specific
L. monocytogenes action levels based on the risk category. Group I foods have the most restrictive
limit (absence of L. monocytogenes in 25 g or mL of food). Under German regulations, seafood
products such as heat-treated shrimp or prawns would be categorized as Group III. For products
in this category, low-level contamination (<100 L. monocytogenes CFU/g) would require a food
establishment hygiene check. Higher levels of contamination would cause the product to be
classified as “unfit for human consumption” or a health hazard in substantiated cases [131].
Danish policy, introduced in 1998, is based on inspection and testing of RTE foods to ensure
that levels of L. monocytogenes in food do not exceed 100 CFU/g at the point of consumption
[108]. Foods are placed into one of six categories. No L. monocytogenes is permitted in a 25-g
sample for foods in three of the categories: those that receive a heat treatment in the final package,
those that are heat-treated and handled after heat treatment and could support growth during the
shelf life of the product, and those foods that are lightly preserved but not heat-treated. The final
three categories are treated similarly and include products that are handled after heat treatment
but are stabilized again to prevent L. monocytogenes growth; lightly preserved, non-heat-treated
RTE foods that are stabilized; and raw RTE foods. For these latter three categories, L.
monocytogenes at a level of ≤10 CFU/g is acceptable, whereas those between 10 and 100 CFU/g
are unsatisfactory. Action may be taken based on consideration of possible growth within the
product shelf-life time, and an evaluation of the processor’s HACCP system may be requested.
The authorities would consider the prohibition of sale and recalls for products that contain more
than 100 CFU/g.
RISK ASSESSMENT
Risk assessment is likely to be used more often as the basis for regulatory risk management and
policy decisions. Application of risk assessment was one of the elements reviewed at the May 1999
Food and Agriculture Organization (FAO) of the United Nations expert consultation on the trade
impact of Listeria in fishery products [46]. A risk assessment is composed of the following four
parts: (1) hazard identification, (2) exposure assessment, (3) hazard characterization/dose–response,
and (4) risk characterization.
HAZARD IDENTIFICATION
The presence of L. monocytogenes in foods is generally well established. Infections caused by
L. monocytogenes are infrequent, fewer than 10 cases/million, and the population at greatest risk
is the immunocompromised; however, the severe consequences, 20–30% mortality rate that
increases to 75% for the most highly susceptible individuals, make this organism an important
hazard and its presence in foods a significant public health concern [107,110].
EXPOSURE ASSESSMENT
Hazard exposure is more difficult to specify. The ability of L monocytogenes to grow over a wide
range of temperatures, 0–45°C, at pH 4.5–9.2, at water activity values > 0.92, and as a facultative
aerobe contributes to the ubiquitous nature of this organism in the environment and many foods
and suggests that human exposure is common [107]. In addition, L. monocytogenes has found an
ecological niche for growth in refrigerated RTE foods having an extended shelf life [110]. Foods
that do not have a listericidal step or could become contaminated before consumption are of
particular concern [127]. Hazard exposure is also directly related to consumption patterns for RTE
foods, which can be difficult to assess and vary with cultural dietary preferences in different
countries. Consumption patterns of 23 different RTE foods in the 2003 FDA/FSIS/CDC Listeria
monocytogenes Risk Assessment [133] were based on The Continuing Survey of Food Intakes by
Individuals (CSFII 1994–1996) and the Third National Health and Nutrition Examination Survey
(NHANES III) (1988–1994) and reflect U.S. dietary patterns.
HAZARD CHARACTERIZATION
Although three main serotypes of L. monocytogenes (4b, 1/2a, and 1/2b) are associated with
most human epidemics and sporadic cases, there are no extensive data demonstrating systematic
differences between food and clinical isolates. Therefore, at this time all L. monocytogenes
isolates are generally considered pathogenic [107]. Dose–response data for humans are not well
established. Epidemiological investigations usually suggest that elevated levels of organisms and
consumption of foods that support growth of L. monocytogenes are associated with illness [107].
Although several attempts have been made to establish dose–response curves, large variation in
dose–response data has limited their usefulness [110].
RISK CHARACTERIZATION
Risk assessment studies for L. monocytogenes are being undertaken by authorities from several
countries, the European Union, WHO, and FAO [110]. Development of a model to characterize
risk is complicated by lack of complete information and differences in how the variables are
weighted. There is a need for more quantitative information about survival and growth of L.
monocytogenes in fish and seafood during handling and processing to more fully assess health
risk [127] from these products. Ideally, a risk assessment model would include all the variables
that may affect growth, survival, and inactivation of L. monocytogenes in fish products, such
as pH, organic acids, a w , gaseous atmosphere, smoke components, and the ability of L.
monocytogenes to compete among bacterial flora present on the product (Jameson effect) [127].
The FDA/FSIS/CDC 2003 Quantitative Assessment of Relative Risk to Public Health from
Foodborne Listeria monocytogenes Among Selected Categories of Ready-to-Eat Foods [133]
used exposure and dose–response models to predict the relative risk of illness attributable to
each of 23 food categories. Although a substantial amount of uncertainty exists around these
models, several categories were identified as part of the FDA/FSIS/CDC Quantitative Risk
Assessment. Food categories containing seafood that were designated “high risk” and warranted
identification of new approaches for reducing potential L. monocytogenes contamination
included smoked seafoods. Food categories containing seafood designated “moderate risk” included
cooked RTE crustaceans and deli salads. These foods generally use bactericidal treatments during
manufacture or limit growth of L. monocytogenes; therefore, consistent application of proven
control measures for these foods is recommended. Foods with low predicted relative risk due
to inherent characteristics less likely to be associated with listeriosis cases or outbreaks include
preserved and raw seafood.
BEHAVIOR OF LISTERIA IN FISH AND SEAFOOD

Before 1987, very little was known about the incidence and behavior of Listeria spp. in fish and
seafoods. Since this time, a number of studies have focused on growth, inhibition, and thermal
resistance of L. monocytogenes in different products. More recent studies have incorporated molec-
ular typing methods to trace dissemination of L. monocytogenes strains in seafood processing
facilities. The results from these studies and the means by which this pathogen may be transmitted
to various forms of aquatic life are discussed in this section.
MODES OF TRANSMISSION
Current data point to cross-contamination as the major source of Listeria in cooked or otherwise
processed seafood, as evidenced by recovery of healthy, noninjured cells of L. monocytogenes
from the surface of many heat-processed/RTE seafood products. Raw fish contaminated in the
natural environment cannot be completely ruled out as the source of contamination in a seafood
processing environment that could subsequently affect the final product. Even if initial contami-
nation of the processing environment is due to incoming raw product, often only a few strains
become established in the processing environment and are recovered from final products, indicating
that adherence to proper sanitation in the processing environment is necessary for control of L.
monocytogenes in the final product. The salt-tolerant nature of L. monocytogenes and presence of
this pathogen in sewage effluent entering the North Sea [30] and also from crustaceans that were
harvested from stream water in which L. monocytogenes was previously identified [112] indicate
that it is present in the marine environment and can contaminate fish and other aquatic animals.
In 1989, Fuad et al. [60] evaluated the ability of L. monocytogenes to survive in the estuarine
environment. Because there appears to be a higher incidence of Listeria in chitinous seafood (i.e.,
shrimp, crab, and lobster), samples of filtered and unfiltered seawater with and without chitin and
chitin-free filtered and unfiltered stream water were inoculated with various strains of L. monocytogenes,
many of which possessed chitinase activity. Although preliminary results indicate that Listeria
populations decreased in chitin-free filtered and unfiltered seawater, addition of chitin to both
types of seawater stimulated growth of Listeria. Moreover, the pathogen also grew in filtered
stream water. These findings, along with those from a report in which L. monocytogenes was
found on the exoskeleton but not in the digestive tract of shrimp that were exposed to high levels
of L. monocytogenes in aquaculture tanks [136], suggest that this pathogen may be ecologically
adapted to chitin. If this is true, then it is imperative that holding tanks for chitinous marine
animals be properly set up and maintained to avoid potential microbiological problems involving
L. monocytogenes and other foodborne pathogens, including Vibrio spp. and Aeromonas hydro-
phila. It is well established that Listeria spp. are often associated with wild animals and birds,
both of which can serve as reservoir hosts. Fenlon et al. [59] demonstrated the role that scavenging
birds can play in the Listeria cycle. In that study a definite association was seen between gulls
feeding on sewage and fecal carriage of Listeria (26.3% positive), which compared to a Listeria
carriage rate of only 8.0% for gulls feeding in less polluted areas. This higher carrier rate could
lead to the conclusion that L. monocytogenes is part of the normal microflora of the near estuarine
environment [12]. Two studies of estuarine waters, shrimp, and oysters along the northern Gulf
of Mexico [100] and of freshwater tributaries, sediments, bay water, and oysters in the Humboldt–
Arcata Bay of Northern California [25] reached similar conclusions. In one study [99], 5% of 78
salt water samples were positive for Listeria spp. In comparison, 11% of 74 shrimp samples were
positive for L. monocytogenes and no Listeria were isolated from the oysters. Listeria species and
L. monocytogenes were found in 81 and 62% (37 samples), respectively, of fresh or low-salinity
waters in tributaries draining into Humboldt–Arcata Bay. The incidence of Listeria spp. and L.
monocytogenes in sediment (46 samples) from the same tributaries was 30.4 and 17.4%, respec-
tively. One of three bay water samples contained Listeria spp. (including L. monocytogenes),
whereas L. innocua was recovered from only 1 of 35 oyster samples [25]. Both these studies
indicated that estuarine environments are continuously subjected to potential contamination with
Listeria spp. from processing effluents, agricultural runoff, and sewage effluents. Listeria spp. can
be recovered from nonpolluted environments, and the source of this bacterium may very well be
from avian species, especially seagulls [99].
The issue of whether the same strains recovered from seafood products are also isolated from
human clinical cases has been studied by several groups. An investigation by Rørvik et al. [125]
in a Norwegian smoked salmon processing plant identified a single L. monocytogenes strain that
had colonized the processing environment and contaminated the smoked fish. This strain was also
one of the most frequently recovered from patients in Norway, indicating that smoked fish could
be a source of illness. In this study, L. monocytogenes was infrequently isolated from the raw
product, and these strains were indistinguishable from those found in the processing environment
or final product. An additional study by Rørvik et al. [124] characterized 305 L. monocytogenes
isolates from patients, seafood products, seafood processing plants, seawater, and a transport
terminal by MLEE and REA. Patient isolates belonged to 11 electrophoretic groups, with 4 groups
accounting for 77% of the isolates. There was much greater genetic diversity among isolates from
seafood products which also included strains isolated from patients. Despite the greater diversity
of strain patterns among seafood and environmental isolates, as a whole individual processing
plants tended to be colonized by a specific L. monocytogenes strain. These observations were
further supported by Norton et al. [109] who used several molecular typing methods (ribotyping,
PCR-RFLP typing of hylA and actA genes) to characterize 392 isolates from patients, smoked
fish, raw materials, fish during processing, and the processing environment. Significantly more
human clinical isolates (69%) belonged to lineage group 1 than industrial isolates (36.8%). All
epidemic strains of L. monocyotgenes belong to lineage group 1, indicating that strains in this
lineage may possess enhanced virulence characteristics. Boerlin et al. [17] concluded that isolates
from human illness and fish products imported into Switzerland belonged to separate subpopula-
tions of the species.
It is generally recognized that contamination of finished seafood can occur within the pro-
cessing environment. One of the first comprehensive studies of L. monocyotgenes contamination
throughout a cold-smoked rainbow trout (Oncorhynchus mykiss) processing plant was conducted
by Autio et al. [15]. The incidence of L. monocytogenes on raw fish was very low, and the
frequency of fish contamination clearly increased after the brining step. In addition, the brining
and postbrining areas of the plant were the most contaminated. PFGE was used to characterize
303 isolates from raw fish, finished product, and the environment. The predominant L. monocytogenes
PFGE patterns associated with isolates from the finished product were associated with brining
and slicing operations but not isolates from the raw fish. Adoption of a rigorous eradication
program using hot steam, hot air, and hot water eliminated the presence of L. monocytogenes in
the processing environment and for the remainder of the study (5 months). Isolates from a Finnish
survey of an RTE vacuum-packed smoked and cold-salted fish manufacturer and fish farms
providing raw materials were characterized by serotyping and PFGE [81]. No L. monocytogenes
strains were isolated from raw product, but a single predominant PFGE clone of L. monocytogenes
was isolated from the salting, skinning, and slicing machines and the finished product. Imple-
mentation of a HACCP program at this plant resulted in samples negative for L. monocytogenes
for at least 9 months. A total of 3585 samples were analyzed over a 4-year period from products
and the processing environments of two Danish cold-smoked salmon processing plants [137].
RAPD was used to subtype 429 isolates. Strains isolated from the final product were indistin-
guishable from strains from processing equipment and the processing environment. The brining
and slicing areas were identified as the most likely areas of product contamination. This suggested
that contamination of final product was due to processing rather than from incoming raw fish,
but raw fish as a source of contamination could not be excluded. There were several differences
noted between the two processing plants. L. monocytogenes was isolated more frequently from
finished product from plant 1 than from plant 2. The same strain of L. monocytogenes was isolated
over a 4-year period from plant 1, indicating that it had become established in the processing
area and was not eliminated by routine cleaning. In contrast, no predominant single strain of L.
monocytogenes persisted for a long period of time in plant 2. The authors of this study concluded
that a rigorous cleaning and sanitation program in plant 2 reduced the incidence of persistent
strains, but sporadic contamination could still occur. A study by Hoffman et al. [71] sought to
determine the prevalence of L. monocytogenes in raw fish and in the processing environments of
two US cold-smoked fish processors. They characterized 512 environmental isolates and 315
isolates from raw fish by automated ribotyping. Consistent with the previous studies [15,137],
they concluded that contamination of the smoked-fish processor environmental was separate from
incoming raw materials and that some strains could become established and persist in the plant
environment for long periods of time. Both plants in this study had similar L. monocytogenes
incidence rates on incoming raw materials but different prevalence rates for the environmental
sites, indicating that operational and sanitation procedures can have a significant impact on
contamination in processing environments.
Molecular subtyping methods, including RAPD and PFGE, were used to trace L. monocytogenes
contamination in a Santos, SP, Brazil, shrimp processing plant [29]. Many different sources and
subtyping group profiles of L. monocytogenes were identified within the plant. Only two strain
profile groups were observed on frozen finished product, and these were among nine different
profile groups recovered from processing areas and food handlers. Interestingly, none of the profile
groups recovered from the environment, water, or utensils were observed on shrimp.
GROWTH AND SURVIVAL

Raw and processed seafoods have long been regarded as excellent substrates for growth of most common
agents of foodborne disease, particularly if seafoods are held at improper temperatures; the behavior of
L. monocytogenes specifically in the products has also been studied [38]. According to data gathered by
Lovett et al. [90] in 1988, L. monocytogenes grew readily (generation time ≅ 12 h) in inoculated samples
of raw shrimp, crab, surimi, and whitefish, with the pathogen attaining maximum populations greater
than 108 CFU/g in all four products following 14 days of storage at 7°C. Two years later, Brackett and
Beuchat [18] also reported that L. monocytogenes grew and retained similar levels of pathogenicity on
artificially contaminated crabmeat during 14 days of storage at 5 to 10°C.
In similar studies, Rawles et al. [120] determined both the incidence and growth of L. monocy-
togenes in blue crabmeat during refrigeration. Of the 126 samples analyzed, 10 were positive for L.
monocytogenes and 3 were positive for L. innocua. The levels of Listeria cells found in fresh picked
blue crabmeat were usually less than 100 CFU/g. Based on these data, commercially pasteurized
crabmeat was inoculated to contain 50 CFU/g after which the growth rate was determined at 1.1, 2.2,
and 5°C. Generation times were calculated as 68.7 h at 1.1°C, 31.4 h at 2.2°C, and 21.8 h at 5°C. At
5°C there was a 7 log10 increase in L. monocytogenes populations and only a 2.5 log10 increase at 1.1°C
after 21 days. The authors therefore concluded that blue crabmeat should be maintained below 1.1°C.
However, in contrast to what Lovett et al. [90] observed for shrimp and whitefish, Harrison et al. [67]
and Shineman and Harrison [129] found that L. monocytogenes failed to grow in overwrapped/
vacuum-packaged raw shrimp and finfish, with numbers of Listeria generally decreasing by approx-
imately 1 log10 after 21 days of storage in an ice chest. When catfish were stored at 4°C, L. monocy-
togenes populations increased slowly (1–1.5 log10) during the first 12 days and then decreased 1.5
log10 by day 16 [87]. During storage, psychrotrophic populations increased from 103 to >107 CFU/g,
thus reinforcing the notion that L. monocytogenes can readily survive in refrigerated raw foods, even
when greatly outnumbered by other natural contaminants. Because L. monocytogenes was recovered
from laboratory-contaminated shrimp (initial inoculum ≅ 105 CFU/g) after 90 days of storage at
−20°C [71], it is evident that this pathogen also is fairly resistant to subfreezing temperatures.
Unlike the aforementioned products, preliminary results from Kaysner et al. [84] suggest that
L. monocytogenes was unable to grow in artificially contaminated oysters, with Listeria populations
remaining constant in shucked oysters after 21 days at 4°C. The apparent inability of Listeria to
grow in raw oysters may partially explain the difficulties in isolating Listeria from retail raw oysters.
According to Farber [44], L. monocytogenes (inoculum level of 2 × 103 CFU/mL) grew fairly well
on cooked lobster, shrimp, crab, and smoked fish and in most instances increased about 2–3 log10
within 7 days at 4°C. When these same products were temperature-abused for a short time (6 h)
at room temperatures, the levels of L. monocytogenes increased by 1 log on shrimp, crab, and
lobster, and only 0.2 log on smoked salmon. During a survey of the incidence of this pathogen on
shrimp and lobster meat at the wholesale level, 13 of 113 samples were positive and were contam-
inated at a level of less than 10 MPN/g. Storage of these naturally contaminated products at 4°C
resulted in L. monocytogenes populations below 100 MPN/g after 1 week with numbers increasing to
2.5 × 103 MPN/g after 2 weeks [44]. In these studies, the growth rate of different L. monocytogenes
strains was comparable in shrimp, crab, and cooked lobster. The difference in growth rate of two
strains in smoked salmon, however, probably reflected the greater tolerance of one strain to sodium
chloride or possibly to the antimicrobial compounds present in smoke.
The behavior of L. monocytogenes in cold-smoked salmon has been studied at several different
laboratories. Guyer and Jemmi [65] and Jemmi [78] determined the growth of L. monocytogenes
during fabrication and storage of cold-smoked salmon. Populations increased to 2.3 × 106 MPN/g
at 10°C and up to 1.5 × 105 at 4°C during 20 days of storage. After 30 days, L. monocytogenes
populations increased to 107 MPN/g in samples stored at both temperatures. A pH of 5.8 and aw of
0.93 had no influence on growth of L. monocytogenes. During fabrication of cold-smoked salmon,
the numbers of L. monocytogenes inoculated onto the surfaces did not change. In contrast, Eklund,
et al. [35] demonstrated that during injection of recirculated brines into the interior of cold-smoked
salmon, L. monocytogenes was also inoculated into these sites. During processing at temperatures
between 17.2 and 21.1°C, L. monocytogenes increased 2- to 6-fold; when the processing temperature
was increased to 22.2–30.6°C, this pathogen increased up to 100-fold. These authors therefore
emphasized the importance of eliminating or reducing the L. monocytogenes population on the
outside of the fish before they are filleted. In addition, they also recommended that brines draining
off fillets during the injection process not be collected, recirculated, and injected into the product.
The incidence and behavior of L. monocytogenes in three lots of naturally contaminated vacuum-
packed sliced smoked salmon from different processors and stored at 2 and 10°C were studied
by Cortesi et al. [26]. L. monocytogenes was isolated from 20 of 100 packages stored at 2°C
and 12 of 65 packages stored at 10°C [26]. In a similar study by Hudson and Mott [72], L.
monocytogenes increased rapidly on cold-smoked salmon within the shelf life of the product. At
10°C, L. monocytogenes growth was comparable in samples packaged in either oxygen-permeable
or oxygen-impermeable films. However, at 5°C the lag phase was longer in the vacuum-packaged
sample, but once growth was established, the generation times were again comparable. The effect
of inoculum level on growth of L. monocytogenes in cold-smoked salmon stored at 4°C was reported
by Rørvik et al. [123]. Starting with an inoculation level of 6 or 600 CFU/g, levels increased about
4.8 log10 for the higher inoculum and 2.1 log10 for the 6 CFU/g inoculum. In these same studies,
the growth rate of L. monocytogenes was compared in products with different initial bacterial
counts. When the inoculum level was 6 CFU/g, L. monocytogenes populations increased faster in
samples with the lower initial total bacterial populations.
INHIBITION
Of the processes used to prepare smoked fishery products, the cold-smoking operation has been of
special interest because the temperatures used are not lethal to L. monocytogenes. The following
interventions therefore have been recommended to reduce the risks from L. monocytogenes in these
products: (1) elimination or reduction of L. monocytogenes on the outside surfaces of frozen or
fresh fish before filleting, (2) prevention of recontamination and growth of L. monocytogenes during
all stages of processing, and (3) inhibition of any possible survivors or recontaminants during
processing and distribution [35].
Several papers have been published on the inhibition of L. monocytogenes in cold-smoked fish
processed with sodium chloride, sodium nitrite, and sodium lactate. In these studies, smoke was
not applied to the products, so that the efficacy of the different inhibition treatments could be
addressed. Peterson et al. [116] studied the behavior of L. monocytogenes (150 CFU/15 g) in cold-
processed salmon containing 3.5 or 6% water-phase sodium chloride. The products were packaged
in either oxygen-permeable film or vacuum-sealed in impermeable film and stored at 5 and 10°C.
By the second week at 10°C, L. monocytogenes increased to the range of 106–108 CFU/g, with no
difference attributed to the sodium chloride concentration. Vacuum packaging suppressed growth
of L. monocytogenes by 10- to 100-fold in samples with 3 or 5% sodium chloride. Inhibition related
to salt concentration was most apparent at 5°C, and L. monocytogenes populations were held below
102 CFU/g by 6% water-phase salt, but increased to 104 CFU/g in products with 5% water-phase
salt and to 105 CFU/g with 3% water-phase salt. Brown sugar is often used in processing cold-
smoked salmon. The use of sugar in the product, however, did not influence growth of L. mono-
cytogenes. In these same studies, growth of the clinical isolate Scott A and two L. monocytogenes
strains isolated from salmon were found to be comparable in cold-smoked salmon at 5 and 10°C.
Because of the salt tolerance of L. monocytogenes and the consumer unacceptability of smoked-
fish products with water-phase NaCl concentrations much above 3 or 4%, it was concluded that
other inhibitors in addition to NaCl were needed to control the growth of this bacterium. Pelroy
et al. [115] therefore studied the behavior of L. monocytogenes (150 or 4900 CFU/15 g sample) in
relation to sodium nitrite (190–200 ppm) in combination with sodium chloride in cold-processed
salmon during storage at 5 and 10°C. The combination of NaCl and NaNO2 was most effective at
5°C. L. monocytogenes with an initial inoculum of 150 CFU/15 g was held below 15 CFU/g by a
combination of 190–200 ppm NaNO2 and 3% water-phase salt and below 20 CFU/g with NaNO2
× 5% NaCl. Packaging in oxygen-permeable or vacuum-sealed in oxygen-impermeable films had
little effect on the growth of L. monocytogenes when NaNO2 was included in the process. Increasing
the storage temperatures to 10°C markedly reduced the efficacy of both NaCl and NaCl + NaNO2
treatments. There was little difference in inhibition between 3 or 5% water-phase NaCl at 10°C,
with the combination of NaCl and NaNO2 only slightly more effective than NaCl alone. The
packaging method, however, had the most pronounced effect on growth of L. monocytogenes at
10°C. Growth was consistently higher in samples packaged in oxygen-permeable film as compared
to vacuum-sealed impermeable film. L. monocytogenes populations increased from a 10 CFU/g
inoculum to the range of 108 CFU/g in samples packaged in oxygen-permeable and 106 CFU/g in
oxygen-impermeable films. Differences in the inhibition attributable to inoculum level and NaCl
+ NaNO2 concentrations were obvious in products stored at 5°C. In comparison, when the initial
inoculum level was increased to 327 CFU/g, L. monocytogenes reached a population of 106 to
107 CFU/g after 20 days of storage.
Of the different inhibitors studied by Pelroy et al. [114], sodium lactate used in combination
with salt or salt plus sodium nitrite was most effective in controlling the growth of L. monocytogenes
(150 CFU/15 g) on vacuum-packaged cold-smoked salmon stored at 5 and 10°C [114]. A concen-
tration of 3% lactate in combination with 3% salt or 3% salt + 125 ppm nitrite prevented any
increase in the L. monocytogenes population during 40 to 50 days storage at 5 or 10°C (Figure 15.1).
FIGURE 15.1 Growth of L. monocytogenes (150 CFU of Scott A/15-g sample) in comminuted salmon
containing 0, 2, or 3% water-phase NaCl, with or without 125 ppm NaNO2, during storage at 10° or 5°C in
vacuum-sealed impermeable film packages. Baseline symbols indicate that L. monocytogenes was detected by
enrichment only. (Adapted from Pelroy, G.A., M.E. Peterson, P.J. Holland, and M.W. Eklund. 1994. Inhibition
of Listeria monocytogenes in cold-process (smoked) salmon by sodium lactate. J. Food Prot. 57: 108–113.)
Addition of 2% sodium lactate prevented L. monocytogenes growth in all samples stored at 5°C.
At 10°C storage, however, a combination of sodium lactate (2%) and NaCl (3%) inhibited growth
for 14 days, after which L. monocytogenes reached populations within 1 to 2 logs of those in the
control samples with NaCl only. When the products contained NaNO2 (125 ppm), sodium lactate
(2%), and NaCl (3%), growth of L. monocytogenes was totally suppressed at 10°C except for one
of four samples in which populations reached 9.3 × 102 CFU/g. The inhibitory effects of nisin,
sodium lactate, and their combination on L. monocytogenes in smoked rainbow trout were studied
by Nykanen et al. [111]. Although both nisin and lactate were inhibitory, the combination had a
synergistic inhibitory effect. Nisin reduced growth of L. monocytogenes by 1.1 log compared to
the control, lactate prevented the growth of L. monocytogenes, and the combination resulted in a
decrease from 3.33 to 1.8 log10 CFU/g in vacuum-packed smoked rainbow trout after 17 days of
storage at 8°C [111]. Nilsson et al. [104] studied inhibition of L. monocytogenes on cold-smoked
salmon using a combination of nisin, carbon dioxide, NaCl, and low temperature. At 10°C nisin
resulted in various degrees of inhibition; however, the effect was more pronounced in the presence
of 100% CO2 and increasing salt concentration (0.5–5%) [104].
Lactic acid bacterium fermentation products have been studied to determine their efficacy in
controlling L. monocytogenes in blue crabmeat [27]. In these studies crabmeat, sterilized by steam,
was inoculated with 5.5 log 10 CFU/g of a three-strain mixture of L. monocytogenes and then washed
with various lactic acid bacterium fermentation products at levels of 2,000 to 20,000 arbitrary units
(AU) per mL of wash. L. monocytogenes populations remained relatively constant in control
samples stored for 6 days at 4°C. In comparison, crabmeat washed with Perlac 1911 or Micro-Gard
(10,000 to 20,000 AU) initially decreased (0.5 to 1.0 log10 units/g) but recovered to original levels
within 6 days. When crabmeat was washed with 10,000 to 20,000 AU of Alta 2341, enterocin 1083
or nisin per mL, the L. monocytogenes population initially decreased by 1.5 to 2.7 log10 units/g.
Thereafter, counts increased by 0.5 to 1.6 log10 units within 6 days.
Degnan et al. [27] also showed that when crabmeat was washed with food-grade sodium acetate
(4 M), sodium diacetate (0.5 to 1 M), and sodium lactate (1 M) or sodium nitrate (1.5 M), there
was only a modest reduction in L. monocytogenes population (0.4 to 0.8 log10 CFU/g). However,
Listeria counts decreased 2.6 log10 CFU/g within 6 days when the sodium diacetate concentration
was increased to 2 M. In addition, trisodium phosphate (1 M) reduced L. monocytogenes counts
from 1.7 to greater than 4.6 log10 CFU/g within 6 days. These results demonstrated that L.
monocytogenes populations on crabmeat may be reduced by washing with selected antimicrobial
agents. The combination of lactic acid and various gas environments on the growth of L.
monocyotgenes in crayfish tail meat during refrigerated storage was studied by Pothuri et al.
[117]. Growth of L. monocytogenes was more inhibited using 0 or 1% lactic acid in the presence
of modified atmosphere packaging than air or vacuum packaging. When the crayfish tail meat
was treated with 2% lactic acid, L. monocytogenes levels decreased regardless of the packaging
atmosphere.
The potential use of strains of Enterococcus faecium to control L. monocytogenes was suggested
by Embarek et al. [40]. E. faecium isolates from sous-vide cooked fish fillets were tested on different
strains of L. monocytogenes and other pathogenic bacteria in brain heart infusion broth with CaCO3
to avoid decreases in pH during the growth of E. faecuim. Of the 19 isolates tested, 14 produced
proteinaceous substances inhibitory to the different strains of L. monocytogenes. An inoculum of
107 CFU E. faecium reduced L. monocytogenes populations of 102 CFU per mL to 10 per mL after
14 days at 3°C and to 1 CFU/mL after 35 days. Using a lower E. faecium inoculum of 104 CFU/mL,
L. monocytogenes was only slightly inhibited at 15°C and not at 3 or 5°C. However, after 11 days
at 15°C a spontaneous resistance phenomenon was observed, and L. monocytogenes reached
1 × 108 CFU/mL with an inhibitory effect no longer seen. When tested for sensitivity to E. faecium,
all of these L. monocytogenes isolates were resistant.
The antibacterial effects of 209 psychrotrophic Pseudomonas spp. strains isolated from spoiled
iced fish and newly caught fish were assessed by screening L. monocytogenes and other organisms
using the agar diffusion assay [64]. Only eight strains inhibited the growth of L. monocytogenes.
Inhibitory action was most pronounced among Pseudomonas strains producing siderophores (an
iron chelator), with the addition of iron eliminating the antibacterial effect of some strains. However,
some strains of Pseudomonas enhanced the growth of L. monocytogenes; dense growth was
observed around the wells containing these Pseudomonas strains, and these strains may have created
a more advantageous nutritional environment either by supplying of iron or by increasing the
availability of low-molecular-weight nutrients.
INACTIVATION
As was true for dairy, meat, and poultry products, the rash of Class I recalls during the past decade
involving RTE seafoods has prompted concerns about the adequacy of thermal processing treatments
currently used for raw seafood. In early 1988 Pace et al. [113] reported results from a study in
which freshly shucked oysters were exposed to 150 ppm chlorine for 30 min, pasteurized at an
internal temperature of 72–74°C for 8 min, and then periodically examined for major bacterial
groups during 5 months of refrigerated storage. According to these authors, chlorination reduced
initial aerobic plate counts of 4.5 × 105 CFU/g by 40–90%, with pasteurization reducing the
population by an additional 99.9%. Despite these large reductions in microbial flora, survivors
classified in eight different genera of aerobic or facultatively anaerobic bacteria were present in
the product. However, at this point the oysters were unfit for consumption, as evidenced by profuse
gas production and swelling of plastic pouches in which the product was pasteurized.
In response to public and industry concerns, FDA [95] also examined the thermal resistance
of Listeria in raw shrimp tails that were inoculated internally to contain approximately 104–105
L. monocytogenes CFU/g. Using a combination of cold (with/without broth) and warm (selective)
enrichment, these investigators failed to recover the pathogen from shrimp tails that were boiled
longer than 5 min. Although appreciable numbers of heat-stressed cells were detected in inocu-
lated shrimp tails that were boiled for 3 min, frozen storage of the product at −20°C eventually
led to complete inactivation of the pathogen. More important, when this study was repeated using
naturally contaminated frozen shrimp from Ecuador and Honduras in which 103–105 L. mono-
cytogenes or L. innocua CFU/g were presumably present only on the chitinous exoskeleton, all
listeriae were eliminated after 1 min of boiling. Hence, because shrimp are more likely to be
contaminated externally than internally with relatively low levels of Listeria, these preliminary
findings suggest that current cooking methods are adequate to eliminate these organisms from
raw shrimp.
Since these initial studies, the thermal death time of L. monocytogenes has been determined in
different seafoods and fish. Results of these studies are summarized in Table 15.8.
In an effort to determine whether presence of L. monocytogenes in processed lobster could
result from undercooking or postcooking contamination, Budu-Amoako et al. [24] determined the
thermal death time of L. monocytogenes in 25-g samples inoculated to contain 107 CFU/g. The
observed decimal reduction times (D-values) at 51.6, 54.4, 57.2, 60.0, and 62.7°C were 97.0, 55.0,
8.3, 2.39, and 1.06 min, respectively, with a z-value of 5.0°C (Table 15.8). After isolating this
pathogen from facilities that used good manufacturing practices, the authors speculated that the
presence of L. monocytogenes in the product was probably due to underprocessing.
Blue crab is a popular seafood item with more than 50% of the picked meat being pasteurized
to offset seasonal fluctuations in some regions. Several recalls of unpasteurized picked blue crabmeat
have been issued due to contamination with L. monocytogenes. Based on the lack of information
about growth and survival of this pathogen in blue crabmeat, Harrison and Huang [68] determined
the heat resistance of L. monocytogenes strain Scott A in this product. The crabmeat was
inoculated to contain 107 CFU/g after which 7.5-g samples were distributed into sausage casings
(1.6 cm × 4 cm). D-values were 40.43, 12.00, and 2.61 min at 50, 55 and 60°C, respectively,
with a z-value of 8.40°C as determined by using trypticase soy agar (Table 15.8). The D-values
were less, 34.48, 9.18, and 1.31 min, respectively, with a z-value of 6.99°C for the same heating
temperatures when Vogel Johnson agar was used.
Heightened interest in foodborne listeriosis has also led to intense efforts toward determining
whether current industry practices of cooking crawfish are adequate to inactivate L. monocytogenes.
In 1993, Dorsa et al. [34] first determined the growth rate of L. monocytogenes in crawfish tail
meat at 0.6 and 12°C; exponential growth began with no apparent lag phase and 109 CFU/g were
observed after 10 and 4 days, respectively. Rapid growth of the bacterial pathogen at these tem-
peratures further emphasized the need for additional information on thermal resistance. In crawfish
tail meat observed D-values at 55, 60, and 65°C were 10.23, 1.98, and 0.19 minutes, respectively,
with a z-value of 5.5°C (Table 15.8). Dorsa et al. [34] suggested that industry consider the use of
postpackaging heat treatments based on their thermal inactivation data.
Based on the psychrotrophic characteristic of L. monocytogenes and the lack of information
on minimum effective dose, the New Zealand Department of Health has taken the conservative
approach and recommended that for RTE seafood, L. monocytogenes should not be present in a
25-g sample. Bremer and Osborne [20] therefore determined the thermal death time of 7 strains
of L. monocytogenes in green shell mussels that were brined in preparation for hot smoking. The
brined mussels were blended and inoculated to contain 106 CFU/g with heat resistance determined
in plastic pouches. The D-values at 56, 58, 59, 60, and 62°C were 48.09, 16.25, 9.45, 5.49, and
1.85 minutes, respectively, with a z-value of 4.25°C (Table 15.8). When D-values for the different
seafoods were compared at 60°C, values for lobster meat, crawfish tail meat, and crabmeat were
similar. The D-values for brined mussels, however, were two to three times higher (Table 15.8).
The authors indicated that the addition of salt and brown sugar used in the smoked products may
have enhanced the thermal resistance of L. monocytogenes in mussels. Based on these differences,
product form and composition should be considered when determining the heat resistance of a pathogen.
TABLE 15.8
Thermal Death Times of L. monocytogenes in Different Seafoods and Fish
Product Mussels
Temperature Lobster Meat Blue Crab Crawfish Tail Brine
(°C) (19) Meat (51) Meat (26) Soaked (17) Salmon (30) Cod (30)
D-value (min)
50.0 40.43
51.6 97.0
54.4 55.0
55.0 12.00 10.23
56.0 48.09
57.2 8.3
58.0 16.25 10.73 7.28
59.0 9.45
60.0 2.39 2.61 1.98 5.49 4.48 1.98
62.0 1.85 2.07 0.87
62.7 1.06
65.0 0.19 0.87 0.28
68.0 0.15 0.15
70.0 0.07 0.03
Note: Z-values = lobster meat 5.0°C; blue crabmeat 8.40°C in trypticase soy agar; crawfish tail meat 5.5°C; mussels
4.25°C; salmon 5.6°C; cod 5.7°C.
In addition, these data emphasize the importance of determining the D-value for each product
experimentally.
Minimally processed foods, also called refrigerated pasteurized foods, of extended dura-
bility [98] using sous-vide technology are relatively new, and concern has been expressed about
survival of L. monocytogenes. Embarek and Huss [39] studied the heat resistance of two L.
monocytogenes strains (062 and 057) in a fatty fish (salmon) and a nonfatty fish (cod). The
strain 062 was slightly more heat resistant. In the processing of sous-vide cooked fish, the raw
ingredients were vacuum-packaged before heating at 58, 60, 62, 65, 68, and 70°C. For the cod,
D-values were 7.28, 1.98, 0.87 0.28, 0.15, and 0.03 min, respectively, with a z-value of 5.7°C
for L. monocytogenes strain 062 (Table 15.8). The two strains of L. monocytogenes were one
to four times more heat resistant in salmon than in cod. The D-values for strain 062 in salmon
were 10.73, 4.48, 2.07, 0.87, 0.2, and 0.07 min, respectively, with a z-value of 5.6°C (Table
15.8). The authors attributed the protective effect in salmon to the higher fat content. They also
emphasized the importance of product form and ingredients in determining the heat resistance
If the time and temperatures previously described in this section for different seafoods had
been followed, then the recent Class I recalls likely resulted from postprocessing contamination,
as has already been implied by Kvenburg [8,85] and other FDA officials [5,136]. However, con-
sidering the possibility of an error during thermal processing, members of the seafood working
group of NACMCF agreed it would be prudent to consider certifying or licensing persons who are
directly involved in thermal processing of different seafoods, as has been done for many years in
the canned food industry [8].
Recognizing that L. monocytogenes is more likely to contaminate the surface rather than the
interior of most fish products, several studies have assessed the use of different treatments to
inhibit or inactivate L. monocytogenes on the surface of processed products. Noel et al. [106]
investigated the possibility of using various lactic acid treatments to inactivate L. monocytogenes
on processed peeled and unpeeled shrimp. The shrimp were immersed in a broth culture of L.
monocytogenes, drained, immersed in an aqueous solution of 1.5, 3.0, and 6.0% lactic acid for
1, 10, or 120 min, and then examined for survivors during 28 days of storage at −20°C. Although
all lactic acid treatments decreased the numbers of Listeria, exposure to 1.5% lactic acid for 10 min
was deemed most appropriate because the treatment did not adversely affect the product’s
organoleptic quality. Overall, initial L. monocytogenes populations of 2.6 × 103 CFU/g on
inoculated shrimp decreased to less than 3.2 × 102 CFU/g following 10 min of exposure to 1.5%
lactic acid. Although Listeria populations continued to decrease in all samples, fewer Listeria were
observed in lactic acid treated (~10 CFU/g) as compared to the untreated shrimp (~40 CFU/g) after
28 days of frozen storage.
In a similar investigation, D-values were determined for a mixture of seven strains of L.
monocytogenes exposed to marinades in the presence and absence of green shell mussels [19].
With an acetic acid marinade (1.5% W/V), calculated D-values in the presence and absence of
mussels were 77.3 and 33.3 h, respectively. When acetic acid–glucono-delta-lactone (1.5 and 0.2%,
respectively)-based marinades were used, D-values increased to 86.3 h in the presence of mussels
and decreased to 19.3 h without the mussels. Likewise, for acetic acid (0.75%) and lactic acid
(0.75%) marinade, D-values increased to 125.5 h in the presence of mussels and 26.9 h in their
absence. Through enrichment experiments, L. monocytogenes was detected after 26 days for acetic
acid (1.5%) and after 53 days for the acetic acid–lactic acid (0.75% each) combination, but no
survivors were found after 29 days with the acetic acid–glucono-delta-lactone (1.5 and 0.2%,
respectively) combination. Based on these findings, the authors emphasized that care must be taken
in extrapolating from the results of a model broth system to a “real food” system. Thus, despite
the inability of organic acids to completely eliminate L. monocytogenes from seafoods, storage of
marinated products before release is an effective method for further decreasing the possibility of
L. monocytogenes being associated with these products. These studies also indicate that dipping
products such as shrimp, mussels, lobster, crab, and scallops in organic acids could prove useful
in decreasing the levels of Listeria before and during frozen storage.
During the past decade there have been several recalls of smoked fish products because of
L. monocytogenes contamination. To estimate the potential health hazard associated with consump-
tion of such products, Jemmi and Keusch [79] determined the heat resistance and growth of
L. monocytogenes in artificially inoculated heat-smoked trout. In these experiments, two strains of
L. monocytogenes were surface-inoculated (106 MPN/g) onto raw trout and kept in a salt and spice
marinade (10% NaCl and 0.7% spices, pH 6.4) for 12 h prior to smoking in a kiln. The trout were
surface-dried at 60°C for 30 min; then the kiln was heated to 110°C and the fish were held at this
temperature for 20 min after reaching an internal temperature of 65°C. The product was then
smoked for 45 min, cooled, and stored at 8–10°C for up to 20 days. After the hot-smoking process
and during storage, L. monocytogenes could no longer be detected. In these same studies, smoked
trout was inoculated to contain 30 or 45 MPN/g after processing and stored at 4 and 8–10°C; L.
monocytogenes increased to 107 MPN/g.
It has generally been assumed that presence of L. monocytogenes on hot-smoked fishery
products is the result of postprocessing contamination. Poysky et al. [118] therefore focused their
studies on defining the processing parameters required to inactivate L. monocytogenes during the
hot-smoked process. Based on previous research [35] that L. monocytogenes contamination is most
likely to occur on the surface of raw fish fillets or steaks, the effectiveness of using a combination
of heat and smoke to inactivate L. monocytogenes on the surface of brined salmon steaks was
investigated. When the brined salmon steaks were heat processed without smoke, L. monocytogenes
survived on steaks processed to an internal temperature of 181°F (82.8°C) for 30 min. Application
of generated smoke reduced the minimum lethal temperature to 153°F (67.2°C). In contrast, when
smoke was applied during the last half of the process after surface drying and formation of the
surface pellicle, L. monocytogenes was recovered from steaks heated to an internal temperature of
176°F (80.0°C) for 30 min.
Poysky et al. [118] also reported that use of undiluted liquid smoke applied at the beginning
of the process lowered the inactivation temperature to as low as 138°F (58.9°C). Diluting liquid
smoke to 50% reduced the effectiveness, and the lethal temperature was increased to 150°F (65.5°C).
The oil-soluble fraction of CarSol C-10, CharOil, was less effective, and L. monocytogenes survived
in samples processed to an internal temperature of 166°F (74.4°C), the highest temperature tested
with this liquid smoke fraction. In these studies, a pre-enrichment technique was used for enhanced
recovery of injured cells. The processed product was stored at 5°C for 4 days and then pre-enriched
in trypticase soy broth for 6 h at 30°C before the addition of selective UVM and selective EB.
Incubation time in EB also played an important role in the detection of survivors. Of the 245
samples tested, 57 were negative for L. monocytogenes after 1 day at 30°C, but were positive after
extended incubation for 6 and 9 days. These studies demonstrated the interaction of heat and smoke
in reactivation of L. monocytogenes and that smoke must be applied before the pellicle is formed
on the surface of the fish. Otherwise, fewer smoke components are absorbed and the pellicle also
serves as a protection blanket for L. monocytogenes from the smoke.
SUMMARY
The topic of the incidence and behavior of L. monocytogenes in fish and seafood has also been
addressed in several other reviews [12,32,38,49]. Risk assessment and, ultimately, implementation
of risk management and identification of appropriate measures to control L. monocytogenes in
fish and seafood products will benefit from a more complete understanding of the growth
behaviors as well as the effectiveness of inhibition and inactivation of L. monocytogenes in fish
and seafood products.
ACKNOWLEDGMENTS
The authors thank Cecilia Wolyniak (FDA, CFSAN), Pat Pinkerton (FDA, Seattle District),
Stephanie Dalgliesh (FDA, Seattle District), and Daryl Thompson (FDA, Atlanta Regional Office,
retired) for retrieval and assistance with the FDA recall information. Maxine Heinitz (FDA, Midwest
Laboratory for Microbiological Investigations, FDA Microbiological Information Systems Man-
ager, retired) and Jan Johnson (FDA, PRL/NW) provided invaluable assistance with accessing data
from the FDA Microbiological Information System and Betsy Bower assisted in accessing the data
in the FDA FACTS System. Literature searches were conducted by Walter E. Hill (now with USDA,
FSIS) and Jim Hungerford (FDA, Seattle District) for which we are grateful. We would also like
to thank Walter E. Hill and Nancy Hill (FDA, Seattle District) for assistance with building and
finalizing a database for the reference list.
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in Products of Plant Origin
Robert E. Brackett
CONTENTS
Introduction ....................................................................................................................................655
Risk of Listeriosis from Products of Plant Origin ........................................................................656
Incidence of Listeria in Raw Vegetables.......................................................................................657
United States.........................................................................................................................658
Canada ..................................................................................................................................660
Western Europe ....................................................................................................................660
Africa ....................................................................................................................................662
Asia and the Middle East.....................................................................................................662
Behavior of L. monocytogenes in Vegetables................................................................................663
Growth and Survival.............................................................................................................663
Modified Atmosphere Storage..............................................................................................666
Inactivation ...........................................................................................................................667
Heat...........................................................................................................................667
Chemical Sanitizers ..................................................................................................668
Plant Components.....................................................................................................669
Processing Techniques..............................................................................................671
Incidence of Listeria in Fruits .......................................................................................................671
Behavior and Inactivation of L. monocytogenes in Fruit and Fruit Juices...................................672
Behavior of L. monocytogenes in Other Products of Plant Origin...............................................675
References ......................................................................................................................................676
INTRODUCTION
Use of adequate isolation procedures, enough time, and a little perseverance by investigators make
it possible to isolate Listeria spp., including Listeria monocytogenes, from most forms of animal
life. A similar situation also exists with products of plant origin. An apparent association between
consumption of silage and occurrence of an illness resembling listeriosis in ruminants was observed
as early as 1922; however, this link between silage consumption and listeriosis in domestic livestock
was not confirmed until 1960 [54]. Although several papers published during the next 15-year
period documented the presence of L. monocytogenes in vegetation grown primarily for consump-
tion by animals [103,104,105], scientists at the time were generally unconcerned about the incidence
of listeriae in produce destined for human consumption, primarily because such products had not
been positively linked to human listeriosis. In fact, the only instance in which listeriae were
655
reportedly recovered from raw retail produce before 1981 occurred in 1975, when investigators
isolated three untypeable Listeria strains from lettuce marketed in Brazil [62].
Although 18 of 41 Canadians died of listeriosis in 1981 after consuming coleslaw from which
L. monocytogenes was isolated and positively identified [92], it was not until 1985—when a
listeriosis outbreak in California was associated with cheese—that the research community began
to understand the public health significance of L. monocytogenes in foods including vegetables,
fruits, and other products of plant origin. Nevertheless, with the exception of one isolated listeriosis
case in Finland involving homemade salted mushrooms [66] and a cluster of five cases traced to
frozen broccoli and cauliflower in Texas [75], no additional cases of listeriosis have been positively
linked to consumption of plant products produced in North America or elsewhere. Despite the lack
of confirmed cases of listeriosis arising from consumption of contaminated fruits and vegetables,
the potential for such outbreaks has prompted significant attention to be paid to the presence of
L. monocytogenes in these products since the previous edition of this book was published. Indeed,
fruits and vegetables were chosen for evaluation of their relative risk of contribution to foodborne
listeriosis in the “United States Food and Drug Administration (FDA)/United States Department
of Agriculture (USDA) Listeria monocytogenes Risk Assessment” [7], the results of which will be
discussed later in this chapter.
This chapter will specifically address the incidence of Listeria species in raw retail vegetables
and fruits. As in earlier chapters, information concerning behavior of L. monocytogenes in fresh
produce and other plant-based products (orange juice/serum, soy milk, pasta, and beet pigment)
will also be presented, along with some possible means by which listeriae can be inactivated in
some of these products.
RISK OF LISTERIOSIS FROM PRODUCTS OF PLANT ORIGIN

Raw fruits and vegetables would normally be expected to be free of most human and animal
enteric pathogens unless somehow contaminated by human or animal waste. Although few
cases of foodborne disease had traditionally been associated with consumption of fresh pro-
duce, such outbreaks have been recognized in recent years to occur with greater frequency
than previously thought. Although not among the organisms most often associated with illness
resulting from consumption of produce, L. monocytogenes is among the foodborne pathogens
most often associated with these foods. This may result, in part, from the variety of ways in
which L. monocytogenes can contaminate fresh vegetables, as shown in Figure 16.1 [19,20].
Consequently, the fact that L. monocytogenes can find its way into fresh produce makes its potential
presence on such products a public health issue.
In 2003, the FDA and USDA published a quantitative risk assessment (LMRA) [7] to determine
the public health impact of L. monocytogenes in various foods, including fruits and vegetables;
other plant-based products discussed in this chapter were not addressed as such. The LMRA
determined both qualitatively and quantitatively the relative likelihood that various foods would
result in listeriosis, taking into consideration such factors as (1) the frequency with which a food
is likely to be contaminated with L. monocytogenes, (2) potential for growth or survival of
L. monocytogenes in the food, (3) historical involvement of particular foods in cases of listeriosis,
and (4) the frequency and quantity of foods consumed (and therefore the probability that a
consumer would be exposed to a contaminated product). Among the most sought-after results
of the assessment were the relative risk of various categories of foods with respect to one another,
and the estimated number of listeriosis cases associated with the food on a per-serving and on
a per-annum basis.
Information published on the number of listeriosis outbreaks generally supports the predictions
of the LMRA in that relatively few outbreaks of the disease have been associated with consumption
of plant products. Nevertheless, there have been some cases. As mentioned earlier, the first case of
Incidence and Behavior of Listeria monocytogenes in Products of Plant Origin 657
FIGURE 16.1 Potential pathways for transmission of L. monocytogenes to humans via vegetables. (From
Beuchat, L.R. and M.P. Doyle. 1995. Survival and growth of Listeria monocytogenes in foods treated or
supplemented with carrot juice. Food Microbiol. 12: 73–80.)
listeriosis actually traced to a food was associated with cabbage [92]. Subsequently, other outbreaks
have come to light.
One outbreak of febrile listeriosis in Italy has been cited as having been associated with
consumption of corn; however, the actual food involved was a corn salad [13]. The investigation
of this outbreak was important in establishing unequivocally that L. monocytogenes is capable
of causing a febrile gastrointestinal form of listeriosis. However, it failed to demonstrate that
corn was the source of the organism. Rather, the illness was associated with consumption of a
cold salad prepared with canned corn and canned tuna in which the two ingredients were allowed
to drain in trays before preparation. Samples of the corn salad yielded very high populations
of L. monocytogenes (>106 CFU/g), but corn from unopened cans was reported by the authors
to be virtually sterile. Hence, L. monocytogenes likely came from environmental sources or
food handlers, although none of the workers reported symptoms of classic listeriosis or febrile
gastroenteritis.
INCIDENCE OF LISTERIA IN RAW VEGETABLES

Despite less attention being paid to fruits and vegetables as a source of listeriosis, the original
Canadian outbreak [92], which resulted in 18 fatalities, prompted increased surveillance and
regulatory programs focused on these commodities. This increased surveillance has resulted in
numerous recalls of fresh produce. During 1997 and the first half of 1998, six Class I recalls were
issued in the United States for fresh frozen coconut [10], hummus with red peppers and vegetables
[4,6,9], sprouts [5], and potato salad [3], the last of which involved over 5.5 million lb of product.
From 1999 to 2003, the FDA was made aware of at least 86 Class I recalls for the presence of
L. monocytogenes. Among the types of products of plant origin recalled were cut fruits and
vegetables, salad mixes, sprouts, bay leaves, lettuce, and bell peppers [107].
Although the presence of L. monocytogenes in fresh produce has been a concern, the LMRA
revealed that foods in the vegetable category had a low predicted relative risk of causing listeriosis
in the United States on a per-serving basis and only a slightly higher risk on a per-annum basis.
The increase in the per-annum risk over the per-serving risk reflects the large volume of vegetables
TABLE 16.1
Vegetables from Which L. monocytogenes Has Been Isolated
Country Food
Australia Lettuce
Brazil Cabbage, lettuce, parsley, watercress
Canada Broccoli florets, cabbage salad, celery, coleslaw mix, green peppers, lettuce, chopped
lettuce, radishes, salad mix, tomatoes, vegetables
Costa Rica Cabbage salad
Czech Republic Vegetables
Germany Legumes, mushrooms, minimally processed fresh vegetables, salads, vegetable
foods
India Beetroot, cabbage, capsicum, carrot, coriander, cucumber, lettuce, vegetable leaves
and roots, radish roots, ready-to-eat salads
Italy Celery, lettuce, fennel
Japan Vegetables
Korea Raw vegetables
Netherlands Fresh and cut vegetables
Saudi Arabia Cabbage, carrot, cucumber, lettuce
Spain Celery, cilantro, fresh vegetables, laurel, lettuce, onion, parsley, thyme, spinach,
winter sweet
Switzerland Salads, vegetables
Taiwan Vegetables
United Kingdom Individual salad ingredients (bean sprouts, cabbage, carrot, celery, cress, cucumber,
lettuce, mushroom, peppers, radish, spring onions, tomato, vegetables, watercress),
mixed vegetables, mixed and prepacked salads, raw salad vegetables, vegetables
and salads
United States Broccoli, cabbage, celery, cilantro, cucumbers, green beans, jalapeño, kelp, kidney
beans, lettuce, mixed vegetable salads, mung beans, mushrooms, onion, pea, potato,
radish, spinach, sprouts, tomato, vegetable, vegetable salads, yam
United States Salad (leafy bagged and precut)
(California)
United States Salad (leafy bagged and precut)
(Maryland)
Source: Adapted from Anonymous. 2003. Quantitative Assessment of Relative Risk to Public Health from Foodborne
Listeria monocytogenes Among Selected Categories of Ready-to-Eat Foods. http: //www.foodsafety.gov/~dms/lmr2-
toc.html, pp. 146–151.
eaten per year. It estimated that the median number of cases of listeriosis from vegetables likely
occurring in the United States was less than one case per year.
In response to heightened concern about foodborne listeriosis, many surveys have been initiated
to determine the extent of Listeria contamination in raw fresh and frozen vegetables destined for
human consumption. Table 16.1, adapted from the LMRA [7], lists the many types of vegetable
products and the geographical regions from which L. monocytogenes has reportedly been isolated.
The discussions that follow provide more details on some of the more pertinent reports.
UNITED STATES
During 1986 and 1987, Petran et al. [85] attempted to isolate Listeria spp. from 23 retail samples
of vegetables, including fresh beet peels, broccoli, cabbage (outer leaves), carrot peels, cauliflower
stems, corn husks, head lettuce, leaf lettuce, mushroom stems, potato peels, and spinach as well
as frozen green beans, pea pods, green peas, and spinach. Using FDA and Centers for Disease
Control and Prevention (CDC) procedures along with direct plating, officials at the CDC [11] tried
to isolate listeriae from 22 samples of broccoli, carrots, celery, lettuce, green peppers, and potatoes
in conjunction with several clusters of listeriosis cases in Los Angeles County, California, and
Philadelphia, Pennsylvania. Finally, as part of a much larger survey dealing with the incidence of
Listeria spp. in retail meat, poultry, and seafood products, Buchanan et al. [32] used an MPN (most
probable number) method to examine two samples of potato salad for listeriae. As already implied,
no Listeria spp. were recovered from any samples examined in the three surveys just described.
However, when one considers the small number of samples examined, these findings should not
indicate that these products will always be free of listeriae. As a result of an extensive review of
both published and unpublished surveys, the LMRA reported that the percentage of vegetable
samples with detectable L. monocytogenes contamination was about 3.6% [7].
In the first truly definitive survey reported, Heisick et al. [60] determined the incidence of
various Listeria spp., including L. monocytogenes, in 10 different varieties of raw unwashed
vegetables (total of 1000 samples) obtained from two Minneapolis-area supermarkets between
October 1987 and August 1988. Listeria spp. were detected in one or more samples of cabbage,
cucumbers, lettuce, mushrooms, potatoes, and radishes, but were never found in broccoli, carrots,
cauliflower, or tomatoes. Listeria monocytogenes, L. innocua, L. welshimeri, and L. seeligeri were
recovered from 5.0, 2.6, 0.8, and 1.3% of all raw produce examined, respectively, with 41 of 50
(82%) and 9 of 50 (18%) L. monocytogenes strains classified as serotypes la and 4a/4ab, respectively.
However, the overall incidence of Listeria spp. as well as L. monocytogenes was markedly higher
in radishes and potatoes than in other types of vegetables. Given that carrots possess some inherent
antilisterial activity [15,21,23,77,78], it appears that root crops such as potatoes and radishes more
frequently carry viable listeriae than other vegetables, ostensibly because of their close association
with soil. Interestingly, contamination rates for most raw vegetables were fairly consistent through-
out the year, and this reinforces the belief that listeriae populations remain relatively constant in
soil. These findings are supported by the study of Heisick et al. [60], who used four different
procedures to ultimately identify Listeria spp. in 19 of 70 (27.1%) and 25 of 68 (36.8%) potato
and radish samples, respectively. No listeriae were detected in mushrooms, carrots, cabbage,
broccoli, cauliflower, lettuce, tomatoes, or cucumbers obtained from the same two Minneapolis
supermarkets.
Use of an adequate isolation procedure and sufficient time to examine large numbers of samples
have made it clear that a small percentage of raw vegetables marketed in the United States are
likely to harbor Listeria spp., including L. monocytogenes, with the incidence of this pathogen
being highest in root crops. Hence, the inability to detect listeriae in raw vegetables examined in
the aforementioned surveys was probably because insufficient numbers of samples were examined.
Although the presence of low levels of L. monocytogenes in retail raw vegetables appears to
contribute only minimally to the overall risk of foodborne listeriosis in the United States, it is
prudent that all produce be carefully handled and washed thoroughly, particularly when it is to be
consumed by pregnant women, the elderly, and other individuals at greater than normal risk of
developing listeriosis.
Others in the United States have reportedly found L. monocytogenes infrequently in fresh
vegetables. Lin et al. [74] determined the occurrence of L. monocytogenes and other foodborne
pathogens in vegetable salads served in 31 food service establishments in Florida. Of the 63
vegetable salad samples tested, L. monocytogenes was recovered from only one (1.6%) salad
consisting of iceberg lettuce, red cabbage, carrots, cucumbers, and tomatoes. Interestingly, this
salad and others yielding potentially pathogenic bacteria other than Listeria were obtained from
only 5 of the 31 establishments. Several of these facilities had apparently sold contaminated salads
on more than one occasion, with contamination most likely a result of product mishandling by
workers.
More recently, Thunberg et al. [96] surveyed retail samples of produce in the Washington, DC
area for several foodborne pathogens, including Listeria species. About half of the food types and
20% of the samples analyzed yielded Listeria spp. Products yielding listeriae included celery, field
cress, lettuce, mung bean sprouts, potatoes, soybean sprouts, watercress, and yams. However, only
field cress (2 of 11 samples) and potatoes (4 of 8 samples) yielded L. monocytogenes. Interestingly,
the observation that a relatively high percentage (50%) of potato samples contained L. monocyto-
genes appears similar to the findings of Heisick et al. [59], suggesting that root crops may be
particularly vulnerable to L. monocytogenes contamination.
The importance of proper sanitation and handling in minimizing L. monocytogenes contamination
of salads and vegetables was mentioned by Harvey and Gilmour [58]. They speculated that systematic
contamination of vegetable salads by L. monocytogenes was more likely a result of improper handling
by food service workers rather than from natural contamination of the raw product.
CANADA
Although several instances of L. monocytogenes in Canadian vegetables have been documented
(Table 16.1), only one formal Canadian publication on the subject of L. monocytogenes in produce
is recorded in the scientific literature. Farber et al. [47] failed to recover any Listeria spp. from
lettuce (50 samples), celery (30 samples), or tomatoes (20 samples) purchased in Ottawa during
1988. However, L. innocua was detected in 1 of 10 radish samples, which again suggests that the
incidence of listeriae may be somewhat higher in root crops than in other vegetables.
WESTERN EUROPE
When the first edition of this book appeared in 1991, knowledge concerning the incidence of
listeriae in raw vegetables marketed in Western Europe was confined to a few scattered reports.
Since then much more information has been published.
An increase in the number of listeriosis cases in England, along with the possibility that
some of these cases may have been food related, prompted several surveys to determine the
incidence of listeriae in various foods including dairy, meat, poultry and seafood products, raw
vegetables, and prepackaged salads. Working at Cambridge, Sizmur and Walker [93] examined
10 different varieties of prepackaged salads obtained from two leading area supermarkets. Overall,
L. monocytogenes serotype 1/2 was isolated from 4 of 60 (6.7%) samples, with L. monocytogenes
serotype 4b also being present in one of these positive samples. Prepackaged salads from which
the pathogen was recovered consisted of two varieties that contained either (a) cabbage, celery,
sultanas, onions, and carrots or (b) lettuce, cucumbers, radishes, fennel, watercress, and leeks.
Both salad varieties contained cabbage, cucumbers, and/or radishes—three of four raw vegetables
from which L. monocytogenes (predominantly serotype la) was isolated in the United States.
Although no Listeria spp. were recovered from plain bean sprout salads or those that contained
nuts, possibly because of a low pH, L. innocua was detected in 13 of 60 (21.7%) samples
representing five different varieties of mixed vegetables and fruit salad. In addition to these
findings, English investigators [52] isolated L. monocytogenes from 12.2% of vegetables sampled;
the organism was only recovered from lettuce, Chinese cabbage, and green onions. On further
characterization, all vegetable isolates were of serotypes other than 1 and 4. In contrast, more than
90% of isolates from turkey and beef were serotype 1, with these strains and those from seafood
having greater hemolytic activity than isolates obtained from vegetables. Consequently, these
authors hypothesized that characteristics of L. monocytogenes might be related to the food of
origin.
Several surveys of retail foods have been conducted by Danish regulatory authorities to deter-
mine approximate levels of L. monocytogenes in various food categories sold in that country [79].
Danish regulatory policy specifies tolerances for L. monocytogenes, depending on the ability of the
food to support growth of the organism and whether the food receives a terminal heat treatment or
is otherwise preserved. Results of the surveys conducted during 1997 and 1998 indicated that
vegetables (including sprouts) were among those foods frequently contaminated by higher popu-
lations of L. monocytogenes. Between 64.9 (1998) and 87.4% (1997) of sprouts and sliced vege-
tables contained less than 10 L. monocytogenes cells/g (acceptable), and 12.1 (1997) and 34.5%
(1998) contained L. monocytogenes at levels between 10 and 100 cells per g (not satisfactory).
Less than 1% of these products contained populations of L. monocytogenes in excess of 100 cells/g,
the level that Danish regulatory policy considers unacceptable.
A large study on the microbiological quality of produce and its risk to public health was
conducted in Norway by Johannessen et al. in 2000 and 2001 [65]. The authors analyzed 890
samples of fresh produce for presence of L. monocytogenes and several other foodborne
pathogens. The produce analyzed was varied, including lettuce, precut salads, herbs, parsley,
dill, mushrooms, and strawberries. Despite the wide variety of items analyzed, the authors
were able to isolate L. monocytogenes from only one sample of champignons (mushrooms)
and two samples of Chinese cabbage and strawberries. While infrequent detection of L.
monocytogenes suggests that this bacterium is not widespread, good hygienic practices are
still warranted.
Robertson et al. [88] examined alfalfa and mung bean sprouts in Norway for the presence of
various pathogens. The authors were unable to detect L. monocytogenes on either type of sprout,
or in irrigation water used during the sprouting process.
Soriano et al. [94] surveyed and characterized Listeria spp. from raw and ready-to-eat restaurant
foods in the Barcelona area. Overall Listeria species were recovered from up to 30% of lettuce
and 10% of spinach samples, with L. monocytogenes present in only 1 of 10 samples each of raw
and ready-to-eat lettuce.
Bendig and Strangeways [17] proposed that a 74-year-old postoperative patient in a London
hospital may have acquired listerial septicemia and meningitis from consuming contaminated
lettuce. Although different serotypes of L. monocytogenes (l/2a and l/2c) were isolated from
the patient and 1 of 11 (9.1%) samples of washed English round lettuce prepared in the hospital’s
kitchen, with the pathogen absent from 44 other food samples examined, the authors concluded
that consumption of washed raw vegetables may pose a potential health threat to hospital
patients, many of whom are debilitated and immunocompromised.
Consumption of homemade uncooked salted mushrooms containing 106 L. monocytogenes
serotype 4b CFU/g was positively linked to a nonfatal case of listerial septicemia in an 80-year-
old apparently healthy Finnish man [66]. During this investigation, L. monocytogenes was not
detected in any vegetables. However, they isolated L. innocua from two samples each of imported
and locally grown vegetables. Working in The Netherlands, van Netten et al. [99] also isolated L.
monocytogenes from 2 of 20 raw mushroom samples obtained from area markets.
Although their goal was not to determine the prevelance of L. monocytogenes in vegetables
per se, Aguado et al. [1] reported that this pathogen was uncommon in frozen Spanish vegetables
and vegetable processing machinery. Their original intent was to characterize L. monocytogenes in
these locations. However, the number of frozen vegetable samples yielding L. monocytogenes was
insufficient for the authors to accomplish their research as planned. Nevertheless, they were able
to establish a low incidence of L. monocytogenes in frozen vegetables with 1.2% of 906 samples
positive over 23 months. The bacterium was mainly found in green beans and tomato products,
although occasional samples of cauliflower, peas, and artichokes also yielded L. monocytogenes.
Most strains belonged to serotype 1/2a. The authors concluded that although the incidence of L.
monocytogenes in frozen vegetables and on processing equipment was low, concurrent isolation
of other Listeria species indicated the need for measures to further prevent establishment of
L. monocytogenes.
AFRICA
Most surveys on presence of listeriae in foods have been conducted in Europe and North America.
However, interest in L. monocytogenes contamination of foods has also developed in other regions
not typically associated with listeriosis, including Africa. Mosupye and von Holy [76] attempted
to recover L. monocytogenes from foods sold by street vendors in Johannesburg. Despite the often
questionable conditions under which the street-vended foods were prepared and sold, the authors
were unable to detect L. monocytogenes in any of the foods analyzed, including salad consisting
of tomato and onion mixtures. The authors concluded that adequate cooking and short holding
times apparently compensated for poor hygienic and handling practices.
ASIA AND THE MIDDLE EAST

Other countries not typically associated with listeriosis include those within Asia and the Middle
East. In contrast to Africa, however, several studies have been published documenting the
frequency with which fruits and vegetables are contaminated with L. monocytogenes. One of the
earliest surveys of occurrence of L. monocytogenes in foods sold in Tokyo was published by
Ryu et al. in 1992 [89]. Their survey included a variety of plant products, including fresh
vegetables, potato salad, and pickled vegetables. L. monocytogenes was frequently isolated from
meat (34%) and fish products (6.1%); however, no listeriae were recovered from vegetable
products or from ready-to-eat vegetable foods such as fermented soybeans, cooked bamboo
shoots, or coleslaw.
In 1996, Kaneko et al. [69] conducted a similar survey of ready-to-eat foods, including fresh
produce, in retail establishments and food production facilities in the Tokyo area. Only 7 of 134
samples of produce or produce-containing products contained detectable listeriae, and none contained
L. monocytogenes.
In contrast to the relatively low frequency of L. monocytogenes contamination of produce
reported in Japan, Arumugaswamy et al. [12] surveyed various fresh and ready-to-eat foods in
Malaysia and found a high incidence of L. monocytogenes contamination. About 22% of leafy
vegetables analyzed contained the bacterium, with similar proportions of bean cakes and peanut
sauces also reported as being positive. Although these values are higher than those reported for
other countries, they were low when compared with other Malaysian vegetable products tested.
Eighty percent of ready-to-eat cucumber slices and 80% of bean sprouts tested positive for L.
monocytogenes. The authors suggested that a high percentage of positive samples may have resulted
because many of the samples came from street vendors or small processors that often employed
nonstandard sanitation practices. As was mentioned by Mosupye and von Holy [76] for South
African street markets, Arumugaswamy et al. [12] likewise stressed the need for health agencies
to put greater priority on foods marketed by street vendors.
In stark contrast to the relatively low frequency of contamination in most other surveys,
Pingulkar et al. [86] found that virtually all vegetables analyzed from a local market in India
contained Listeria spp. with 73% of ready-to-eat salads from several local restaurants also positive
for listeriae. Although most Listeria isolates from produce were species other than L. monocyto-
genes, 11% of tomatoes, 50% of coriander leaves and spinach, and 25% of cabbage samples
contained L. monocytogenes.
Salamah [90] conducted an extensive survey for presence of L. monocytogenes in various fresh
market vegetables sold in Riyadh, Saudi Arabia. In general, the different types of produce analyzed
contained L. monocytogenes, at incidence rates varying from 1.3 to 16.3%. Similar to Heisick
et al. [59], Salamah found that root crops were more frequently contaminated with L. monocytogenes
than vegetables grown above the ground.
Conversely, Gohil et al. [53] examined 183 imported and locally grown vegetable samples in
the United Arab Emirates for presence of L. monocytogenes. Unlike other surveys, they were unable
to detect any L. monocytogenes in vegetables. However, they isolated L. innocua from two samples
each of imported and locally grown produce.
Although consumption of coleslaw prepared from contaminated cabbage was linked to a large
Canadian outbreak of listeriosis in 1981 [91], in retrospect, it appears that this outbreak could have
been avoided if the coleslaw manufacturer had realized that the cabbage farmer had fertilized the
cabbage with sheep manure from a flock that was previously diagnosed as having listeriosis. Years
later, van Renterghem et al. [100] suggested that L. monocytogenes dies quickly in fecal matter,
and therefore animal manure may not be as important in the spread of L. monocytogenes as once
thought. However, they were able to demonstrate that L. monocytogenes could be transferred from
contaminated soil to vegetables. In these experiments, carrots and radishes were planted in soil that
had been inoculated with L. monocytogenes (105 CFU/g soil). They found that three of six radishes
but none of the carrots grown in the inoculated soil contained L. monocytogenes.
BEHAVIOR OF L. MONOCYTOGENES IN VEGETABLES

Despite the 1981 listeriosis outbreak in Canada being directly linked to consumption of contami-
nated coleslaw [92] and an earlier cluster of listeriosis cases in Massachusetts that appears to have
been epidemiologically linked to raw celery, lettuce, and tomatoes [73], until recently scientists
were generally unconcerned about behavior of L. monocytogenes in raw produce.
This renewed interest also prompted a series of investigations to determine behavior of
L. monocytogenes on raw vegetables. Results from these studies will now be reviewed along with
some data concerning thermal inactivation of L. monocytogenes in cabbage and the effect of
modified atmospheric storage, chlorine, and lysozyme on growth and survival of this pathogen in
various raw vegetables.
GROWTH AND SURVIVAL

Coleslaw was the first vegetable product that was directly linked to an actual outbreak of listeriosis
in humans. Consequently, it is not surprising that growth and survival of L. monocytogenes in
cabbage was initially investigated. In the first such study, Beuchat et al. [27] determined behavior
of L. monocytogenes strains Scott A (clinical isolate) and LCDC 81-861 (Canadian cabbage isolate)
on inoculated samples of shredded raw and autoclaved (121°C/20 min) cabbage as well as in
autoclaved (121°C/15 min) salted and unsalted cabbage juice during extended storage at 5 and
30°C. Both test strains exhibited similar patterns of behavior on sterile cabbage, with populations
decreasing from approximately 107 to 104–105 CFU/g during 42 days of refrigerated storage.
This apparent inability of L. monocytogenes to grow on heat-sterilized cabbage at 5°C suggests
that heating either decreases the availability of essential nutrients or leads to development of toxic
and/or inhibitory constituents in cabbage. In sharp contrast, L. monocytogenes competed well with
the normal aerobic flora and lactic acid bacteria of raw cabbage, with Listeria populations increasing
approximately 4 orders of magnitude on raw cabbage during the first 25 days of refrigerated storage
(Figure 16.2). Thereafter, numbers of listeriae remained unchanged on raw cabbage stored up to
64 days. Similar results were observed in subsequent studies by Hao et al. [55] and Lovett et al.
[75]. Thus, L. monocytogenes can grow under conditions normally encountered during shipping
and distribution of cabbage. Although both Listeria strains failed to grow in autoclaved cabbage
juice containing >5% NaCl during 2 weeks of storage at 30°C, L. monocytogenes increased to 106
CFU/g in the aforementioned homemade salted mushrooms (~7.5% NaCl) during 5 months of cold
storage [66]. Hence, behavior of listeriae in raw vegetables appears to be greatly affected by
incubation temperature as well as concentration of salt and various growth constituents [84].
Subsequently, Conner et al. [38] more closely examined the influence of temperature, NaCl, and
pH on growth of L. monocytogenes in autoclaved (121°C/15 min) clarified and unclarified cabbage
10
7
Bacteria log10 CFU/mL
0
0 16 32 48 64
Days
FIGURE 16.2 Growth of monocytogenes (•), lactic acid bacteria (n n ), and total aerobic microorganisms
(▲) on raw cabbage incubated at 5°C. (Adapted from Bhagwat, A.A., R.A. Saftner, and J.A. Abbott. 2004.
Evaluation of wash treatments for survival of foodborne pathogens and maintenance of quality characteristics
of fresh-cut apples. Food Microbiol. 21: 319–326.)
juice. As in the previous study, salt-free unclarified cabbage juice was an excellent growth medium
for L. monocytogenes, with initial populations of ~104 CFU/mL increasing to ~109 CFU/mL after
8 days of incubation at 30°C. Beyond 8 days, populations in cabbage juice containing low levels
of salt decreased rapidly, and viable cells were no longer detected after 20 days of incubation at
30°C. Growth rates of both Listeria strains at 30°C decreased markedly in cabbage juice containing
low levels of salt, with inactivation of strains Scott A and LCDC 81-861 occurring in the presence
of >1.5 and 2.5% NaCl, respectively. As expected, behavior of both strains was strongly influenced
by acid production, with the pH of samples in which growth had occurred decreasing from 5.6 to
less than 4.3 after 8 days of incubation at 30°C. Although populations of both Listeria strains failed
to increase in salted and unsalted cabbage juice when the experiment was repeated at 5°C, popu-
lations in cabbage juice containing 3.5–5.0% NaCl remained relatively stable, generally decreasing
only 10- to 100-fold during 70 days of refrigerated storage. Results from another study [37] suggest
that viability of Listeria in similar samples of salted and unsalted cabbage juice can be reduced by
adding extracts from several Chinese medicinal plants.
Concern about behavior of L. monocytogenes in fresh produce has extended beyond cabbage
and now includes an ever-increasing variety of fresh salad vegetables. In 1988, Steinbruegge et al.
[95] first reported results of a study that examined the ability of L. monocytogenes to survive and
grow on inoculated (103–105 CFU/g) samples of washed retail head lettuce during storage in sealed
and unsealed plastic bags at 5, 12, and 25ºC. Although behavior of L. monocytogenes on lettuce
was somewhat variable, the pathogen generally grew under conditions simulating proper refriger-
ation, normal handling, and ambient serving temperatures, with the pathogen increasing 1–4 orders
of magnitude following 2 weeks of storage. Similar results were observed when inoculated samples
of fresh lettuce juice were held at 5°C for 2 weeks. Salamah [90] reported similar increases in
lettuce juices held at 26°C and also observed up to a 2-log increase at 4°C.
Several more recent reports have appeared regarding the fate of L. monocytogenes in various
types of salad greens. Butterhead lettuce (Lactuca sativa L.) supported growth of L. monocytogenes
better than did endive (Cichorium endivia L.), but the bacterium was unable to grow on lamb’s
lettuce (Valerianella olitoria L.). In contrast, populations of total aerobic microorganisms were
unaffected by salad type. Carlin and Nguyen-the [33] offered no hypothesis as to why lamb’s lettuce
failed to support growth of L. monocytogenes.
Carlin et al. [34] followed up on their previous work by more closely examining several factors
that affected growth of L. monocytogenes on endive, with emphasis on storage temperature, age, and
quality of the endive leaves, role of epiphytic microflora, and strains and initial concentration of L.
monocytogenes present. Overall, the growth rate of L. monocytogenes was essentially the same as
that of the natural aerobic microflora at 10 and 20°C but slower than the native microflora at 30 and
6°C. Furthermore, the bacterium grew faster when initially present at lower (10–1000 CFU/g) rather
than at higher (105 CFU/g) populations. In accordance with earlier results of Beuchat and Brackett
[24], Carlin et al. [34] detected no differences in growth among the various strains tested.
Carlin and co-workers [35] subsequently investigated in detail the role of indigenous microflora
on growth of L. monocytogenes in unsanitized endive leaves and on leaves treated with 10%
hydrogen peroxide to reduce or eliminate the indigenous microflora. These investigators also
challenged L. monocytogenes with individual strains of pseudomonads and Enterobacteriaceae
isolated from endive. The authors observed that reducing the native microflora by disinfection
resulted in higher populations of L. monocytogenes on endive leaves. Moreover, they also observed
that high populations (106–107 CFU/g) of some strains of indigenous microorganisms reduced
growth of L. monocytogenes on endive. A complex mixture of various microorganisms isolated
from endive completely inhibited growth of L. monocytogenes in a medium composed of endive
leave exudate.
Processing treatments can influence indigenous microflora and have subsequent impact on
growth of L. monocytogenes. Li et al. [73] investigated the impact of heating lettuce on the
development of L. monocytogenes during subsequent storage. They demonstrated that although the
heat treatment was effective in reducing browning and discoloration associated with cut lettuce, it
also resulted in development of higher L. monocytogenes populations than in unheated lettuce. The
authors hypothesized that the enhanced growth of L. monocytogenes in heated lettuce resulted from
a reduction in competitive microflora. Alternatively, they offered the notion that the effects from
heating may have also contributed to more favorable growth conditions for the bacterium. Regard-
less of the specific reason for enhanced growth, this study clearly demonstrated that treatments
designed to maintain sensory quality of products can have a detrimental effect on microbiological
safety.
Although botanically a fruit, tomatoes are typically used as vegetables and are among the most
popular ingredients in fresh salads. Beuchat and Brackett [25] demonstrated that tomatoes are not
a good substrate for growth of L. monocytogenes, probably because of their acidity. Although some
growth of Listeria was evident on whole tomatoes after 10–21 days of storage at 10 and particularly
21°C, the pathogen was inactivated in chopped tomatoes (~pH 4.1) held at these temperatures.
Additionally, when commercial tomato products were inoculated to contain ~106 L. monocytogenes
CFU/g, populations remained reasonably stable in tomato sauce and tomato juice during 14 days
of storage at 21 and particularly 5°C. However, the pathogen survived only 4 and 8 days in samples
of ketchup held at 21 and 5°C, respectively, with Listeria inactivation being attributed to higher
levels of acetic acid in ketchup as compared to the other tomato products. In contrast, Pingulkar
et al. [86] observed that L. monocytogenes populations decreased in tomato slurry regardless of
storage temperature, with inactivation increasing with temperatures.
Information regarding the fate of L. monocytogenes in or on other types of salad vegetables also
is limited; however, results from several studies indicate that with the exception of raw carrots
[23,31], fennel, red cabbage, and Savoy cabbage [31], and beets [30], this pathogen will grow and/or
survive on most other types of fresh vegetables including asparagus [17], broccoli [17], cauliflower
[17], corn [63], green beans [63], lettuce [24,63], certain types of cabbage [38,31], celery [30], potato
juice [90], and radishes [75] during the normal refrigerated shelf life of the product. Gianfranceschi
and Aureli [51] noted that survival of L. monocytogenes in spinach was essentially unaffected by
freezing at −50°C and extended storage at −18°C. In addition, Sizmur and Walker [93] reported that
L. monocytogenes populations in several naturally contaminated vegetable salads purchased from
two supermarkets in England increased approximately twofold after 4 days of refrigerated storage.
Given the apparent ability of L. monocytogenes to survive and/or grow on most raw salad vegetables
and the possible presence of this pathogen on many types of raw produce, health officials need to
consider raw vegetables as a potential source of listerial infections.
The observation that L. monocytogenes can thrive in various fresh vegetables also led to
questions regarding its growth and survival in products prepared from these vegetables. In 1995,
Lee et al. [72] published a study on growth and survival of L. monocytogenes in kimchi, a traditional
Korean fermented vegetable product. This product can be prepared from various ingredients, but
the most common type contains Chinese cabbage and various flavoring agents such as red pepper,
garlic, ginger, NaCl, and pickled seafood. These ingredients are mixed and subjected to a natural
lactic acid fermentation, with the product ultimately reaching a mildly acidic pH [12,13]. The
authors found that populations of L. monocytogenes Scott A increased during the first 2 days of
kimchi fermentation, but then decreased. Although eventually inactivated by kimchi ingredients
and low pH, the pathogen still persisted after 10 days of fermentation. However, the authors
concluded that kimchi could be safely produced by using ingredients of good microbiological
quality.
MODIFIED ATMOSPHERE STORAGE

The term modified atmosphere usually refers to systems in which the atmosphere in which a product
is stored is intentionally changed to a desired gas composition. The widespread practice of packaging
and storing fresh produce in a modified atmosphere has led to dramatic increases in types of produce
available to consumers and in their shelf life so that most fresh vegetables (and fruits) are now available
throughout the year. However, given the occasional presence of L. monocytogenes on raw produce,
the ability of this pathogen to multiply relatively rapidly under microaerobic conditions at refrigeration
temperatures and the present popularity of modified-atmosphere packaging, legitimate questions have
been raised about the safety of refrigerated produce during long-term storage with modified
atmosphere [106].
In response to these concerns, Berrang et al. [18] investigated behavior of L. monocytogenes
on inoculated (103–105 CFU/g) samples of fresh asparagus, broccoli, and cauliflower during
extended refrigerated storage in glass jars containing (a) 15% O2:6% CO2:79% N2, (b) 11% O2:10%
CO2:79% N2, (c) 18% O2:3% CO2:79% N2, or (d) air. L. monocytogenes behaved similarly on each
vegetable when the product was stored in a modified atmosphere or air. All three vegetables
supported growth of L. monocytogenes at 15°C, with the pathogen attaining populations of 106 to
nearly 109/g when these products were first deemed to be unfit for human consumption 6–10 days
after the start of incubation. Although storage in a modified atmosphere at 15°C did not appreciably
affect growth of listeriae on any of the three vegetables examined, the ability of such storage
conditions to increase the shelf life of these products by 2–4 days beyond that of products packaged
in air led to higher Listeria populations when these vegetables were first declared inedible by
subjective evaluation. In contrast, only asparagus supported growth of L. monocytogenes at 4°C,
with initial numbers being approximately 10- to 100-fold higher at the end of the product’s 21-
day shelf life. However, numbers of listeriae remained relatively constant on broccoli and cauli-
flower during 21 days at 4°C regardless of the storage atmosphere. Because these findings and
those of the previous study indicate that L. monocytogenes is basically unaffected by controlled-
atmosphere storage, the extended shelf life gained with such storage conditions provides additional
time for growth of this pathogen which, in turn, increases the public health hazard associated with
consumption of raw vegetables. Similarly, Garcia-Gimeno et al. [50] and Kallander et al. [67]
observed that use of modified atmosphere with salads neither enhanced nor repressed growth of
L. monocytogenes but that the extended shelf life afforded by modified-atmosphere packaging
increased the risk of foodborne illness.
Subsequently, Beuchat and Brackett observed that L. monocytogenes behaved similarly when
inoculated samples of iceberg lettuce [23] and tomatoes [25] were stored at 5 or 10°C (lettuce) or
10 and 21°C (tomatoes) in 3% O2:97% N2 or air. As with cabbage and asparagus, the pathogen
grew on lettuce, reaching populations of 108–109 CFU/g after 10 days of storage at 10°C, with
only slight growth observed in identical samples held at 5°C. However, the bacterium only achieved
populations of about 105–106 CFU/g in tomatoes regardless of storage atmosphere.
Vacuum packaging is another means by which produce can be held under a modified atmo-
sphere. This practice has the effect of reducing O2 and thereby slowing respiration and senescence.
Aytac and Gorris [14] investigated the effect of a moderate vacuum on growth of L. monocytogenes
in chicory endive and mung bean sprouts; the organism responded differently depending on the
product in question. Growth of L. monocytogenes at 5.6°C was enhanced by 400 mB of vacuum
in the endive but was repressed in sprouts. The authors pointed out the need for additional barriers
when such techniques are used to extend the shelf life of fresh vegetables.
The atmosphere also can be changed as a result of metabolic processes of fresh fruits and
vegetables. In this instance, gas-permeability characteristics of the packaging material can often
drastically affect the atmosphere within the package and, consequently, the microflora in the food.
Omary et al. [80] investigated the influence of packaging material on growth of L. monocytogenes
in shredded cabbage packaged in films having oxygen transmission ratios (OTR) of 5.6, 1500,
4000, and 6000 cc O2/m2 per 24 h. They found that the type of packaging material used had a
significant effect on growth of L. innocua (as a substitute for L. monocytogenes) in cabbage.
Populations of L. innocua decreased in all samples after 14 days of storage regardless of packaging
film used. However, populations of L. innocua then increased 3.5 logs CFU/g in cabbage packaged
in all but the film with the highest OTR, in which populations only increased by about 2 logs
CFU/g. In the latter sample, CO2 concentrations had equilibrated at near-ambient concentrations,
whereas concentrations reached from 30 to 90% when films of lower OTR were used.
Although most publications to date indicate that only low populations of L. monocytogenes
infrequently contaminate fresh produce, the chance of this pathogen multiplying in products with
extended shelf life is significant. Therefore, it appears prudent for handlers of raw produce that is
intended to be held in a modified atmosphere to institute sanitation and quality control programs
that will decrease the incidence of listeriae in incoming raw vegetables. In addition, holding produce
at the coldest tolerable temperature will also reduce the risk of L. monocytogenes attaining high
populations. It also may be necessary to shorten the marketable period for such products even
though the food may appear to be acceptable.
INACTIVATION
Among the most active areas of food microbiology research in the past decade has been identifi-
cation of practical means of eliminating or greatly reducing populations of foodborne pathogens.
Considerable research has been done to characterize the effectiveness of heat, chlorine, and
lysozyme, and these will be discussed in the following text. In addition, information concerning
the effect of other methods such as ozone, chlorine dioxide, high hydrostatic pressure, and irradiation
has generated interest.
Heat
In response to the 1981 listeriosis outbreak in Canada involving consumption of contaminated

coleslaw, Beuchat et al. [27] investigated thermal inactivation of L. monocytogenes in cabbage juice.
Flasks of sterile, clarified cabbage juice adjusted to pH 4.0, 4.6, and 5.6 were inoculated to contain
approximately 4 × 106 L. monocytogenes CFU/mL, placed in a shaking water bath at 50, 52, 54,
56, or 58°C, and sampled for listeriae at 10-min intervals for up to 60 min. As expected, thermal
inactivation rates for Listeria in cabbage juice at 50, 52, 54, and 56°C were faster at lower pH
values, with D-values of 25, 14, 6.7, and 3.6 min at pH 4.6 as compared to values of 60, 34, 8.4,
and 6.8 min at pH 5.6, respectively. No viable cells were detected in cabbage juice held at 58°C
for 10 min. Although inactivation rates were unaffected by addition of 1 or 2% NaCl to cabbage
juice, sublethally injured cells were more sensitive to NaCl on a nonselective plating medium
(Tryptic Soy Agar) than were uninjured cells, indicating increased sensitivity to NaCl. As shown
by data in Figure 16.2, L. monocytogenes can multiply in raw cabbage during extended refrig-
erated storage; however, results from the study just discussed suggest that normal pasteurization
treatments given to cabbage juice and sauerkraut are probably sufficient to eliminate any viable
listeriae that may be present. Hence, unlike unfermented raw vegetables, the risk of contracting
listeriosis from sauerkraut and other pasteurized fermented vegetable products appears to be
minimal.
Chemical Sanitizers
Brackett [29] investigated the possibility of using hypochlorite solutions to inactivate L. monocytogenes
on the surface of Brussels sprouts. In this study, fresh retail Brussels sprouts were inoculated to
contain ~106 L. monocytogenes CFU/g, immersed in a hypochlorite solution containing 200 mg of
chlorine/L, removed, air-dried for 30 min, and examined for numbers of surviving listeriae. The
procedure just described decreased populations of L. monocytogenes on Brussels sprouts approx-
imately 100-fold. However, because dipping inoculated Brussels sprouts in sterile chlorine demand-
free water reduced the number of viable listeriae approximately 10-fold, the author concluded that
many cells were simply washed from the surface rather than inactivated by chlorine. In a follow-
up study with fresh retail lettuce, Beuchat and Brackett [24] also found that L. monocytogenes was
still present at levels of 105–106 and 107–108 CFU/g after extended storage at 5 and 10°C, respec-
tively, regardless of whether or not the lettuce was pretreated with a sodium hypochlorite solution
to reduce the population of naturally occurring microflora.
Zhang and Farber [108] evaluated several sanitizers and sanitizer combinations, including
sodium hypochlorite, chlorine dioxide, trisodium phosphate, and lactic and acetic acids, for inac-
tivation of L. monocytogenes on lettuce and cabbage. Their results generally were similar to those
of Brackett [29] in that disinfectants were ineffective at reducing populations of L. monocytogenes.
None of the treatments tested provided more than a 1.7-log reduction and most were under a
1-log reduction. Trisodium phosphate had no effect on listeriae, and surfactants actually reduced
efficacy of sanitizers. Hypochlorite can be used very effectively to inactivate L. monocytogenes in
water supplies and on the surface of previously cleaned equipment. However, current evidence
indicates that chlorine dips are relatively ineffective for eliminating L. monocytogenes from
contaminated raw vegetables.
Similar to chlorine, ozone has also been used as an effective disinfectant for water. Conse-
quently, it should not be surprising that it would also be considered a candidate for a practical
means by which to disinfect fresh produce. Fisher et al. [49] investigated this possibility with
respect to L. monocytogenes inactivation and found that ozone could effectively reduce populations
of L. monocytogenes in distilled water and phosphate-buffered saline solution, and that the
efficacy increased as temperatures decreased. In addition, efficacy differed among strains of L.
monocytogenes. The authors reported that treating cabbage with 1% ozone for 5 min virtually
eliminated all listeriae present. However, the authors did not indicate what effect this treatment had
on sensory properties of the cabbage.
Delaquis et al. [40] attempted to use gaseous acetic acid to inactivate several foodborne
pathogens, including L. monocytogenes, on mung bean seeds. Unfortunately, treatment of mung bean
seeds with 242 µl of acetic acid per L of air for 12 h at 45ºC yielded only sporadic reduction of L.
monocytogenes, but apparently did not adversely impact germination rates. In contrast, treatment
with 0.1 or 0.5% of the quaternary ammonium sanitizer cetylpyridinium chloride reportedly
reduced populations of L. monocytogenes on fresh-cut broccoli, cauliflower, and radishes by as
much as 4-log CFU/g [102].
After demonstrating that egg white lysozyme, a GRAS (generally recognized as safe) food
additive, inhibited growth of L. monocytogenes in laboratory media and phosphate buffer [63],
Hughey et al. [64] investigated the possibility of using this enzyme during refrigerated storage to
inactivate L. monocytogenes on various retail vegetables, including fresh lettuce, cabbage, sweet
corn, green beans, and carrots as well as previously frozen corn and green beans. In this study,
1.8-kg portions of coarsely shredded or fresh-cut and thawed frozen vegetables were treated to
contain 100 mg of lysozyme/kg of vegetables and/or 5 mM of ethylenediamine tetraacetic acid
(EDTA), inoculated to contain ~103–104 L. monocytogenes CFU/g, mixed by hand, and examined
for numbers of listeriae during extended storage at 5°C.
Overall, lysozyme was fairly effective in decreasing populations of L. monocytogenes on the
surface of fresh vegetables, particularly when used in conjunction with EDTA, which presumably
facilitated cell lysis by increasing contact between lysozyme and peptidoglycan in the cell wall.
Listericidal effects from the combined use of lysozyme and EDTA were most pronounced on lettuce,
with the pathogen no longer being detected after 12 days of refrigerated storage. Although lysozyme
alone was listeriostatic, use of EDTA alone failed to prevent growth of L. monocytogenes, with
the pathogen eventually attaining levels only slightly lower than those observed in untreated lettuce.
Listeria behaved similarly on fresh green beans and sweet corn with two exceptions: (a) growth
occurred on lysozyme-treated sweet corn and (b) combined use of lysozyme and EDTA never
completely eliminated the pathogen from either product. Unlike fresh lettuce, green beans, and
sweet corn, numbers of listeriae on EDTA- and lysozyme-treated raw cabbage increased during
the first 20 days of refrigerated storage and then decreased 4–5 orders of magnitude during 28 days
of incubation at 5°C. Although use of lysozyme combined with EDTA again was most listericidal,
41 days of refrigerated storage were required to rid this lettuce of listeriae. Unlike other fresh
vegetables, the pathogen was eliminated within 9 days from untreated raw carrots as well as
from those that were treated with lysozyme alone or in combination with EDTA. Hence, these
findings support the notion that carrots probably contain one or more naturally occurring listericidal
substances [24].
In contrast to fresh vegetables, numbers of listeriae remained relatively constant on previously
inoculated frozen green beans and corn that were treated with lysozyme alone or in combination with
EDTA. This apparent failure of lysozyme to inactivate listeriae on frozen vegetables may be related
to loss of certain lysis-enhancing substances during processing of vegetables. These findings, together
with the current use of lysozyme to prevent growth of gas-producing spore-forming bacteria in certain
European cheeses, suggest that commercial use of lysozyme in combination with other previously
discussed measures should help inhibit Listeria and other foodborne pathogens on fresh vegetables.
Plant Components
Various plant components have long been known to possess antimicrobial properties with the
essential oils of herbs and spices having been studied most extensively. Kim et al. [70] demonstrated
the antilisterial activity of various essential oil components in vitro and suggested that these
components might also be incorporated into foods as barriers to microbial growth. Hao and Brackett
[57] and Hao et al. [56] tested this suggestion by determining the efficacy of plant extracts in
inhibiting growth of L. monocytogenes in beef and chicken, respectively. They found that eugenol
and pimento extracts significantly inhibited growth of the bacterium on cooked chicken breasts
during refrigerated storage [57]. However, they also noted that the type of food in which extracts
were used was important. Unlike chicken, none of the spice extracts tested effectively inhibited
growth of L. monocytogenes in refrigerated, cooked beef [56]. Moreover, some extracts contributed
strong odors and flavors that would need consumer approval if actually used for commercial
products.
As mentioned previously, carrots reportedly possess some inhibitory antilisterial factors.
Beuchat and Brackett [23] were the first to document this when they attempted to artificially
inoculate carrots by dipping in suspensions of L. monocytogenes. They noted that populations of
the bacterium decreased on both raw whole and shredded carrots but not cooked carrots. Moreover,
populations of L. monocytogenes decreased in the inoculating suspensions after dipping of shredded
carrots. Based on these results, they suggested that the antilisterial components of carrots are heat
sensitive and released when carrot tissue is damaged. The authors suggested that phytoalexin 6-
methoxymellein might be one potential compound responsible for the antilisterial action. The results
of Beuchat and Brackett [23] were later confirmed by Nguyen-the and Lund [77], who also noted
that maceration of carrots in a high-speed blender or in liquid nitrogen also destroyed the antilisterial
activity. The obvious potential for using carrot juice or its antilisterial components as a natural
antimicrobial agent in foods prompted these two groups of researchers to investigate the mechanism
of antilisterial activity. Nguyen-the and Lund [78] found that the antilisterial effect of carrots was
suppressed by anaerobiosis, thiol compounds, bovine serum, and the free-radical scavengers histi-
dine and diazabenzocyclooctane. However, the activity was not affected by sodium ascorbate, propyl
gallate, catalase, superoxide dismutase, or chelating agents but was enhanced by Tween 20. Despite
these results, these authors were unable to determine a specific compound or compounds responsible
for the antilisterial activity.
Beuchat et al. [26] also attempted to characterize the antilisterial components of carrots.
They found that the lethal and inhibitory effects were greatest in the pH range of 5.0–6.4 and
that the optimum concentration of carrot juice needed to inhibit L. monocytogenes growth was
about 10%. In addition, they observed that NaCl at concentrations of up to 5% protected listeriae
from the antimicrobial action of carrot juice, especially in 10% juice incubated at 5 or 12°C.
Despite these observations, they were unable to identify the compounds responsible for antilis-
terial activity or predict the effectiveness of carrot juice as an antilisterial ingredient in food.
However, Babic et al. [14] suggested that dodecanoic acid and methyl esters of dodecanoic and
pentadecanoic acids identified in purified active extracts of carrots may be responsible for the
observed antimicrobial activity.
Looking at the potential use of carrot juice as an antilisterial treatment for foods, Beuchat and
Doyle [21] determined the influence of dipping shredded lettuce in 20 or 50% carrot juice or adding
up to 10% carrot juice to Brie cheese and frankfurter homogenates. Overall, both concentrations
of carrot juice significantly repressed growth of L. monocytogenes in shredded lettuce stored at 5
and 12°C but not at 20°C. It is also noteworthy that the carrot juice was rather specific in its activity
in that it had no discernible effect on growth of other aerobic microorganisms. In contrast to lettuce,
addition of carrot juice to Brie cheese was less effective and was completely ineffective in frankfurter
homogenates. Hence, it appears that carrot juice may only be of value as an antilisterial agent in
some foods.
One of the most well-recognized classes of antimicrobial agents arising from plant products
are various sulfur compounds, particularly those associated with certain Brassica species. Kyung
and Fleming [71] investigated the relative antilisterial effect of some selected sulfur compounds
derived from cabbage. Of the compounds tested, they found isothiocyanate and methyl methane
thiosulfonate to have the smallest minimal inhibitory concentration. However, the authors did not
attempt to assess the effectiveness of these compounds to inhibit growth of L. monocytogenes in
fresh produce. Moreover, the authors did point out that despite the presence of such compounds,
other researchers have reported that L. monocytogenes grew well in cabbage samples. Kyung and
Fleming suggested that this observation may have been due to different concentrations of these
compounds in cabbage or a decrease in the levels of these components in cabbage as a result of
heating.
TABLE 16.2
Survival of Listeria spp. on Coriander Leaves Treated with
Gamma Radiation and/or Chlorine and Stored at 8–10°C
Storage Time
Treatment Day 0 Day 10 Day 20
Control + + +
1 kGy +
Chlorine-treated + + +
1 kGy + Chlorine
Source: Adapted from Lovett, J., D.W. Francis, and J.G. Bradshaw. 1988. Outgrowth
of Listeria monocytogenes in foods. In A.J. Miller, J.L. Smith, and G.A. Somkuti.
Foodborne Listeriosis. Elsevier, New York, pp. 183–187.
Processing Techniques
New or newly applied food processing techniques have also offered promise for reducing or
eliminating populations of L. monocytogenes in some plant-based foods. In particular, high-
pressure treatments can significantly reduce populations of L. monocytogenes in some semifluid
or viscous products. For example, Raghubeer et al. [87] were able to reduce populations of L.
monocytogenes in fresh salsa from 107 CFU/g to <0.3 CFU/g by applying 545 MPa pressure for
at least 0.5 min. Irradiation also has been proposed as a means to eliminate pathogens, including
L. monocytogenes from various foods. Kamat et al. [68] found that treatment of coriander leaves
with 1 kGy or greater of gamma irradiation eliminated detectable levels of L. monocytogenes,
whereas viable listeriae were recovered from leaves treated with 250 mg/L of sodium hypoclorite
for 10 min (Table 16.2). However, in neither case was the population of L. monocytogenes in
untreated leaves provided.
INCIDENCE OF LISTERIA IN FRUITS

In contrast to raw vegetables, less information is available concerning the incidence of Listeria
species on raw fruit (Table 16.3). The first documentation was a preliminary report [11] in which
CDC officials failed to recover listeriae from seven fruit samples (e.g., cherries, pears, peaches,
avocados, and tomatoes) while investigating two clusters of presumed foodborne listeriosis in
Los Angeles County, California, and Philadelphia, Pennsylvania. Later, Schlech [91] mentioned
that blueberries, strawberries, and nectarines were implicated in outbreaks of listeriosis. One of the
few culture-confirmed cases of listeriosis resulting from consumption of a fruit product occurred
in Italy. In this case, DNA fingerprinting was used to link consumption of pickled olives with a
sporadic neonatal case of listeriosis [36].
Although scientific evidence is lacking, two observations, namely, infrequent association between
consumption of fruit and listeriosis and the fact that most fruits grow well above the ground and are
therefore not subject to frequent contact with Listeria-contaminated soil or feces, lead one to speculate
that the incidence of listeriae on fruit may well be as low or lower than that observed for raw vegetables.
Despite these assumptions, however, the recent Listeria monocytogenes risk assessment (see Chapter
18) [7] ranked fruits as carrying a slightly higher risk than vegetables, although still ranking it as a
low predicted relative risk and contributing less than a median of 1 case per year. Although the exact
reasons for the higher risk are somewhat unclear, one important factor was the higher percentage of
TABLE 16.3
Fruit and Fruit Products from Which L. monocytogenes Has Been Recovered
Country Food
United States Apples, blueberries, cantaloupe, dried fruit, fruit salad, fruit products, gelatin
dessert with fruit, melons, pears, pineapples, watermelon
Czech Republic Dried fruit
Germany Fresh fruit, fruit product
Source: Adapted from Anonymous. 2003. Quantitative Assessment of Relative Risk to Public Health from Foodborne
Listeria monocytogenes among Selected Categories of Ready-to-Eat Foods. http: //www.foodsafety.gov/~dms/lmr
2-toc.html, p. 465.
samples (11.8 vs. 3.6% for vegetables) with detectable contamination. In addition, the ability of certain
fruits, such as melons, to support rapid growth of L. monocytogenes together with the absence of data
required for more precise calculation of risk lead to a calculated high risk for fruits.
FDA officials prompted an Oregon firm to issue a Class I recall for over 500,000 flavored
frozen ice and juice bars that were apparently contaminated with L. monocytogenes during the
latter stages of manufacture [8], which suggests postpasteurization contamination. Because raw
milk also was routinely processed into frozen dairy products at this same facility, L. monocytogenes
was most likely introduced into the factory environment through the raw milk supply rather than
fruit juice. If this is true, then it follows that the incidence of listeriae in highly acidic fruit juice
is likely to be extremely low. This view is further supported by results from a survey in which
Parish and Higgins [81] failed to detect any Listeria spp. in 100 retail samples of reconstituted
single-strength orange juice (pH 3.63–3.84) that were pasteurized at 30 geographically distinct
dairy and nondairy facilities located across the United States and Canada.
BEHAVIOR AND INACTIVATION OF L. MONOCYTOGENES

IN FRUIT AND FRUIT JUICES
Although there has been a significant increase in information on the growth and survival of
L. monocytogenes in vegetables, the same cannot be said for fruits. Because we currently lack
sufficient information on the incidence of listeriae in raw fruits, it is not surprising that our
knowledge of Listeria behavior in these products remains limited.
In 1989, Parish and Higgins [82] published data from the first of two studies in which Listeria
failed to grow in refrigerated orange serum adjusted to pH <4.6 and was completely eliminated
from these samples after 18 to 70 days of storage, with lowest pH values proving most detrimental
to survival of Listeria. However, modest growth of listeriae was observed in orange serum samples
at pH 5, with the pathogen still being present at levels of 102–103 CFU/mL in the two least acidic
samples after 90 days of refrigerated storage. Incubation at 30°C led to Listeria increases in
serum that was adjusted to pH values of 3.6–5.0 with hydrochloric acid, inoculated to contain ~106
L. monocytogenes CFU/mL, and examined for numbers of listeriae during prolonged incubation
at 4 and 30°C. As was true for cabbage juice [38], behavior of listeriae in orange serum also
was markedly influenced by incubation temperature and pH. Overall, L. monocytogenes popu-
lations increased approximately 10- and 100-fold in orange serum samples adjusted to pH values
of 4.8 and 5.0, respectively. As was true for unclarified cabbage juice [39], overall viability of
listeriae again was greatly reduced by raising the incubation temperature, with the pathogen
generally being eliminated from orange serum samples at pH 3.6–4.0 and 4.2–5.0 after 5 and 8 days
of incubation at 30°C, respectively.
The same authors [81] subsequently used several enrichment procedures to determine viability
of listeriae in single-strength reconstituted samples of commercial frozen concentrated orange juice
that were inoculated to contain ~l–10 L. monocytogenes CFU/mL. Although numbers of inherent
microorganisms (primarily lactic acid bacteria and yeast) increased from ~102 to 108 CFU/mL after
4 weeks of incubation at 4°C, L. monocytogenes was eliminated from reconstituted frozen orange
juice (average pH of 4.06) after 42 days of refrigerated storage. These findings appear to be
consistent with those from the previous study involving orange serum.
Acidic pH minimizes the growth and survival of L. monocytogenes on many citrus and pome
fruits. However, melons and tropical fruits are often less acidic. Consequently, one would expect
greater likelihood for survival and growth of the bacterium on such fruits. Behrsing et al. [16]
investigated survival of L. innocua on skins of several fruits, with the assumption that L.
monocytogenes would behave similarly. Indeed, they found that L. innocua survived well on
bananas, passionfruit, and honeydew melon held at 8ºC for 7 days, but actually grew on the
rind of cantaloupe under the same conditions. Although Ukuku and Fett [97] did not observe
growth of L. monocytogenes on cantaloupe rinds, they were able to demonstrate that the cells
present on the rind could be transferred to the flesh during cutting. Hence, this stresses the
importance of decontaminating the exterior of such fruits, and particularly cantaloupe, before
cutting.
Although survival and growth of L. monocytogenes on inedible skins and rinds increases the
potential for eventual consumption, the behavior of L. monocytogenes on fruit flesh is of more
immediate concern with regard to listeriosis. L. monocytogenes grew well in the pulp of papaya
(pH 4.87), watermelon (pH 5.50), and melon (Cucumis melo L. var. “Valenciano amarelo,” pH 5.87),
achieving concentrations in excess of 108 CFU/g in the latter [82]. These results show that cut fruits
could serve as a vector for L. monocytogenes and that proper sanitation and temperature control
are important.
As with vegetables, various microbial reduction strategies, primarily chlorine-based sani-
tizers, have been evaluated for efficacy against L. monocytogenes on fruits. However, thus far
generally only minor reductions in numbers of listeriae have been achieved. One example for
which greater reduction was reported was when apple slices were treated with a solution
containing isoascorbic acid, calcium ascorbate, calcium propionate, and N-acetylcysteine [28].
Depending on pH of the solution, the authors were able to achieve L. monocytogenes reduction
of <1 to >5 logs.
Venkitanarayanan et al. [101] evaluated the efficacy of a combination of 1.5% lactic acid and
1.5% hydrogen peroxide to reduce populations of several foodborne pathogens on the surface of
apples, oranges, and tomatoes. Exposing the fruits to this solution for 15 min at 40°C effected a
>5-log reduction in populations of L. monocytogenes (Table 16.4). Moreover, the authors observed
no adverse effects on sensory characteristics of the products.
High-pressure processing and irradiation have also been evaluated as antilisterial treatments
for fruits. Alpas and Bozoglu [2] evaluated the efficacy of high-pressure treatment for destruction
of L. monocytogenes in apple, apricot, cherry, and orange juices. They were able to attain an
approximately 4- and 7-log reduction in populations of L. monocytogenes when the juices were
treated at a hydrostatic pressure of 250 and 350 MPa, respectively (Table 16.5). Later, Dogan and
Erkmen [41] reported that the D-values for L. monocytogenes in peach juice exposed to 200, 400,
and 600 MPa of hydrostatic pressure were 1.52, 3.39, and 6.17 min, respectively. The process was
even more effective for orange juice, with D-values of 0.87, 1.80, and 2.87 min for the same
pressure treatments, respectively.
Gamma irradiation was less effective in eliminating L. monocytogenes from frozen avocado
pulp [98] than from coriander leaves [68]. In the former case, irradiation was successful in reducing
populations of L. monocytogenes by only 1 to 4 logs.
TABLE 16.4
Listeria monocytogenes Populations on Apple, Orange, and Tomato Surfaces
(after Treatment with 1.5% Hydrogen Peroxide at 40°C for 15 min)
Product
Treatment Apple Orange Tomato
Baselinea 6.04 ± 0.92 7.01 ± 0.09 7.20 ± 0.25

Treated productb ND 0.97 ± 0.03 ND
Treated solutionc ND ND ND
Control productd 4.07 ± 0.24 4.92 ± 0.17 6.11 ± 0.65
Control solutione 5.20 ± 0.31 6.08 ± 0.48 7.02 ± 0.30
aCount (log10 CFU per product) of inoculated pathogen dried on fruit.
bCount (log10 CFU per treated product) of inoculated pathogen after treatment with 1.5% hydrogen peroxide
at 40ºC for 15 min.
cCount (log CFU/mL) of inoculated pathogen in a solution of 1.5% lactic acid plus 1.5% hydrogen peroxide
10
in which pathogen-inoculated apples were immersed at 40ºC for 15 min.
dCount (log CFU per product) of inoculated pathogen after immersion in sterile deionized water at 40ºC
10
for 15 min.
eCount (log CFU/mL) of inoculated pathogen in deionized water in which pathogen-inoculated products
10
were immersed at 40ºC for 15 min.
Source: Adapted from Brackett, R.E. 1987. Antimicrobial effect of chlorine on Listeria monocytogenes.
J. Food Prot. 50: 999–1003.
TABLE 16.5
Effect of High-Pressure Processing on Viability Loss
of L. monocytogenes CA in Fruit Juices
Log10 CFU mLa
Pressurized
250 MPa, 350 MPa, 350 MPa,

Product Control 30°C, 5 min 30°C, 5 min 40°C, 5 min
Apple juice 8.20 4.30 1.40 NDb

Apricot juice 8.20 4.15 1.26 ND
Orange juice 8.70 4.45 1.44 ND
Cherry juice 8.34 4.00 0.90 ND
a Values represent mean (n = 8) log10 CFU mL−1 of unpressurized control
samples.
b ND, no CFU detected in 1 mL of cell suspension from each of the samples
tested.
Source: Adapted from Alpas, H. and F. Bozoglu. 2003. Efficiency of high

pressure treatment for destruction of Listeria monocytogenes in fruit juices.
FEMS Immunol. Med. Microbiol. 25: 269–273.
BEHAVIOR OF L. MONOCYTOGENES IN OTHER PRODUCTS

OF PLANT ORIGIN
Increased consumer demand for precooked, long-shelf-life, ready-to-eat foods containing a mini-
mum of preservatives is making development of microbiologically safe products increasingly
challenging. Not too many years ago, it was thought that no foodborne pathogen could multiply
in properly refrigerated food. However, this belief has proven to be invalid following the
emergence of L. monocytogenes and Yersinia enterocolitica, in particular, as causes of food-
borne illness. Thus, public health officials have become concerned about the safety of many cook-
chilled and ready-to-eat foods of animal origin, including fermented and unfermented dairy prod-
ucts, luncheon meats, sausage, and precooked chicken as well as peel-and-eat shrimp. These
concerns have since spread to products of plant origin, including soy milk, precooked delicatessen
products such as ravioli (prepared in part from flour), and food colorants derived from red beets.
Increased use of soy milk by individuals who cannot tolerate cow’s milk prompted Ferguson
and Shelef [48] to inoculate commercially available pasteurized and sterile soy milk to contain
102–104 L. monocytogenes CFU/mL and incubate the products at 5 and 22°C. The pathogen
attained maximum populations similar to those previously observed in cow’s milk, reaching levels
of 7 × 108–3 × 109 and 8 × 108 CFU/mL of soy milk following 3 and 30 days of incubation at 5
and 22°C, respectively. Not surprisingly, generation times for L. monocytogenes in soy milk, 1.55
h at 22°C and 37.68 h at 4°C, are similar to those previously reported for the same strain of L.
monocytogenes in cow’s milk. No one has yet reported the incidence of Listeria spp. on soybeans;
however, because listeriae are relatively common in soil, it is possible for these organisms to find
their way into soy milk processing factories and also into the finished product as a postpasteur-
ization contaminant. If this is true, then rapid growth of L. monocytogenes in soy milk at refrig-
eration temperatures suggests that this product should not be overlooked as a possible vehicle of
human listerial infection.
Concern about behavior of Listeria in delicatessen products marketed in England and the United
States prompted Beuchat and Brackett [22] to investigate viability and thermal inactivation of
L. monocytogenes in commercially prepared meat, cheese, and egg ravioli purchased from Atlanta-
area delicatessens. The growth portion of this study involved quantification of L. monocytogenes
in inoculated (~104 and 106 CFU/g) samples of ravioli during 14 days of incubation at 5°C. For
thermal inactivation tests, the three types of ravioli were inoculated to contain 3 × 105 L. monocy-
togenes CFU/g, stored for 0 or 9 days at 5°C, and then boiled for up to 7 min using home cooking
procedures. Overall, numbers of viable listeriae decreased <10-fold during the 9-day refrigerated
shelf life of the three types of ravioli. Results of thermal inactivation studies indicated that normal
cooking procedures (7 min of boiling) were adequate to destroy L. monocytogenes populations of ~105
CFU/g in all three types of ravioli regardless of whether or not ravioli was refrigerated for 0 or
9 days before cooking. Although this study provides valuable information concerning behavior
of Listeria in ravioli, there appears to be an urgent need for more work of this type to address
the microbiological safety of precooked and ready-to-eat delicatessen products such as sand-
wiches, filled rolls, pizza, garlic bread, desserts, confectionery products, and chocolate, because
work in England [52] and elsewhere has shown that all these products can harbor L. monocyto-
genes.
Increased use of plant-based food colorants prompted El-Gazzar and Marth [45] to investigate
behavior of Listeria in a commercial aqueous extract from the red beet root (Beta vulgaris) to
which vitamin C, citric acid, and sodium propionate (1.5%) were added as preservatives. As in
their previous work with milk coagulants [43,44] and annatto colorants [42], samples of beet extract
were inoculated to contain 103–107 L. monocytogenes strain CA, V7, or Scott A CFU/mL and
examined for numbers of survivors during prolonged storage at 7°C. Not surprisingly, the combined
effect of a relatively low pH of 4.3–4.8 and sodium propionate prevented growth of listeriae in all
samples of beet colorant. However, although 42–56 days of incubation at 7°C was sufficient to rid
these extracts of 103–104 strain CA CFU/mL, this strain was still detected in 56-day-old samples
that contained larger initial populations. In contrast, strains V7 and Scott A were far more resistant
to the listericidal action of beet extract, with both strains being recovered at levels of 101–104
CFU/mL, depending on the initial inoculum, following 56 days of storage. Hence, unlike highly
alkaline annatto extracts in which L. monocytogenes was inactivated almost instantaneously, pro-
longed survival of listeriae in beet colorants makes it imperative that these extracts be processed
and handled carefully to prevent contamination with this pathogen.
The sporadic nature of listeriosis suggests that L. monocytogenes can be an infrequent problem
in unusual foods of plant origin as well as fruits and vegetables. Although this viewpoint is supported
by one sporadic case of listeriosis in Canada that was linked to alfalfa tablets [39,46], the fact that
L. monocytogenes can be present in virtually any ecological niche suggests that it is possible for
occasional cases of listeriosis to be associated with unusual as well as common plant-based foods.
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17 Incidence and Control

of Listeria in Food
Processing Facilities
Jeffrey L. Kornacki and Joshua B. Gurtler
CONTENTS
Introduction ....................................................................................................................................683
Development of Microbial Growth Niches and Biofilms: General Principles.............................685
Maintenance and Repair Practices .......................................................................................693
Factory and Equipment Design............................................................................................697
Traditional Approaches to Listeria Control...................................................................................704
Control of Food Contamination through Effective Cleaning and Sanitation......................707
Cleaning Dry Areas ..................................................................................................707
Cleaning Wet Processing Areas ...............................................................................707
Sanitization ...............................................................................................................709
Other Approaches to Sanitization ............................................................................709
Other Factors to Consider for Control of Listeria in Food Processing Environments ................710
Traffic Patterns......................................................................................................................710
Monitoring the Finished Products, Ingredients, and Processing Environment...................711
Hazard Analysis Critical Control Point (HACCP) Concept .........................................................711
Sampling Plans for L. monocytogenes in Foods ...........................................................................713
Establishment of Barriers between Raw and Finished Product
and Production Areas ...........................................................................................................715
HACCP Plan Development............................................................................................................716
The HACCP Team................................................................................................................716
Principle 1: Conduct a Hazard Analysis..................................................................716
Principle 2: Determine CCPs ...................................................................................717
Principle 3: Establish Critical Limits.......................................................................717
Principle 4: Establish Monitoring Procedures .........................................................717
Principle 5: Establish Corrective Actions ................................................................718
Principle 6: Establish Verification Procedures.........................................................718
Principle 7: Establish Record-Keeping and Documentation Procedures ................718
Incidence of Listeria spp. in Various Types of Food Processing Facilities
in the United States........................................................................................................................719
Dairy Processing Facilities...................................................................................................720
Meat Processing Facilities....................................................................................................724
Poultry Processing Facilities ................................................................................................730
Egg Processing Facilities .....................................................................................................732
681
Seafood Processing Facilities...............................................................................................732

Vegetable and Fruit Processing Facilities ............................................................................738
Incidence of Listeria spp. in Western European and Australian Food Processing Facilities.......739
Western Europe ....................................................................................................................739
Dairy Production Facilities.......................................................................................739
Meat, Poultry, and Seafood Production Facilities ...................................................741
Chocolate Production Facilities ...............................................................................741
England and the United Kingdom .......................................................................................742
English Poultry Processing Facilities.......................................................................742
English Chocolate Production Facilities ..................................................................742
United Kingdom: Miscellaneous Production Facilities ...........................................742
France ...................................................................................................................................743
Cheese Production Facilities ....................................................................................743
Poultry and Pork Production Facilities ....................................................................743
Smoked-Salmon Processing Facilities .....................................................................743
Finland ..................................................................................................................................744
Meat Processing Facilities........................................................................................744
Seafood Processing Facilities...................................................................................744
Italy .......................................................................................................................................744
Meat Processing Facilities........................................................................................744
Spain .....................................................................................................................................745
Vegetable Processing Facility...................................................................................745
The Netherlands....................................................................................................................745
Miscellaneous Production Facilities.........................................................................745
Sweden..................................................................................................................................746
Dairy Production Facilities.......................................................................................746
Switzerland ...........................................................................................................................747
Australia................................................................................................................................747
Incidence of Listeria in Household Kitchens................................................................................748
Industry-Specific Equipment, Processing Methods, and Products ...............................................749
Dairy Industry.......................................................................................................................750
Farm Environment ....................................................................................................750
Clarifiers and Separators ..........................................................................................750
Pasteurization............................................................................................................750
Pipeline and Cross-Connections ..............................................................................752
Filling and Packaging...............................................................................................752
Reclaimed and Reworked Product ...........................................................................753
Frozen Dairy Products..............................................................................................753
Fermented Dairy Products........................................................................................754
Meat Industry........................................................................................................................754
Roast Beef, Corned Beef, and Other Rebagged Products.......................................755
Frankfurters and Other Link Products .....................................................................755
Luncheon Meats .......................................................................................................756
Poultry Industry ....................................................................................................................756
Egg Industry .........................................................................................................................757
Fish and Seafood Industry ...................................................................................................757
Fruit and Vegetable Industry ................................................................................................758
References ......................................................................................................................................759
Incidence and Control of Listeria in Food Processing Facilities 683
INTRODUCTION
Overwhelming evidence indicates that contamination of commercially processed foods with Listeria
monocytogenes and other Listeria spp. occurs in the postprocessing environment rather than because
these organisms survived heat treatments that normally render the product safe. This view is strongly
supported by widespread distribution of the organism and lack of scientific evidence indicating
minimum required heat treatments given to dairy, meat, poultry, seafood, and other products are
inadequate to inactivate listeriae that might be reasonably expected to occur in such products before
heat processing. To date, no recall of commercially prepared, Listeria-contaminated products has
been unequivocally linked to the inadequacy of minimum required heat treatments, despite the
microbe’s greater heat resistance as compared to many vegetative organisms. However, the clearest
indication that L. monocytogenes and other Listeria spp. enter commercially processed foods as
postprocessing contaminants arises because apparently healthy, nonthermally injured cells have
been routinely recovered from many thermally processed dairy, meat, poultry, and seafood products
and these organisms have been found in the working environments of virtually all processing
facilities that have produced foods involved in Listeria-related recalls. In fact, frequent isolation
of Listeria spp. from meat processing environments has resulted in a viewpoint that complete
elimination of these organisms from ready-to-eat meat and poultry processing environments may
not be possible with current technology [150].
Refrigerated food factories, in particular, provide conditions which allow for survival and
growth of L. monocytogenes. The organism can adhere to food-contact surfaces and form a biofilm
or coating which impedes effectiveness of sanitation procedures [56,69]. The refrigerated, moist
environment, coupled with organic soil deposition, allows L. monocytogenes to survive and grow.
Greater survival of L. monocytogenes occurred in the presence of pork serum on stainless steel,
acetal-polymer, fiber-reinforced plastic, and mortar as compared to unsoiled surfaces (Figure 17.1
to Figure 17.4). L. monocytogenes is also a frequent contaminant of raw materials used in processing
plants, so there is constant reintroduction of the organism into the plant environment [55]. To
control this pathogen, every potential avenue of entry and cross-contamination must be controlled.
This daunting task requires careful thought, continual observation, frequent and routine factory
environmental sampling, and documented corrective actions.
Listeria monocytogenes survival on stainless steel:

Soil effect
6
A
Log CFU/coupon
5 B A A A
A
4 soil
A A
3 B no soil
2 B
B
B
1
0
0 3 6 9 12 15
Days after attachment
FIGURE 17.1 Survival of Listeria monocytogenes on stainless steel: soil effect. (Adapted from Yan, Z., J.L.
Kornacki, C.M. Lin, and M. Doyle. 2004. Fate of Aersolized Listeria monocytogenes in a Closed Bioaersol
Chamber. International Association for Food Protection. Annual Meeting. Abstract P058.)
Listeria monocytogenes survival on acetal resin:

Soil effect
7
A
6 B A
Log CFU/coupon
A B A
5 A A
4 soil
B B
3 B no soil
2 B
1
0
0 3 6 9 12 15
FIGURE 17.2 Survival of Listeria monocytogenes on acetal-polymer: soil effect. (Adapted from Yan, Z., J.L.
Kornacki, C.M. Lin, and M. Doyle. 2004. Fate of Aersolized Listeria monocytogenes in a Closed Bioaersol
Chamber. International Association for Food Protection. Annual Meeting. Abstract P058.)
Listeria monocytogenes survival on FRP:

Soil effect
7
A
6 A A B A A A
Log CFU/coupon
A
5
soil
4 B
B B no soil
3 B
2
1
0
0 3 6 9 12 15
FIGURE 17.3 Survival of Listeria monocytogenes on fiber-reinforced plastic: soil effect. (Adapted from Yan,
Z., J.L. Kornacki, C.M. Lin, and M. Doyle. 2004. Fate of Aersolized Listeria monocytogenes in a Closed
Bioaersol Chamber. International Association for Food Protection. Annual Meeting. Abstract P058.)
Survival of Listeria monocytogenes on mortar:

Soil effect
6 A
5 B A
Log CFU/coupon
B AA A
4 soil
A
3 A no soil
B A
2 B A
1 B B B AA
AA
0
0 3 6 9 16 24 48 72 96 120
Time after attachment (h)
FIGURE 17.4 Survival of Listeria monocytogenes on mortar: soil effect. (Adapted from Yan, Z., J.L. Kornacki,
C.M. Lin, and M. Doyle. 2004. Fate of Aersolized Listeria monocytogenes in a Closed Bioaerosol Chamber.
International Association for Food Protection. Annual Meeting. Abstract P058.)
This chapter has been specifically designed for plant managers, sanitation workers, quality-control
and quality-assurance personnel, regulatory field representatives, and others who desire or have a
need to understand how, where, and why Listeria establishes itself in the factory. This chapter will
progress from general principles and move into specific food processing industries and products.
General principles for understanding the development of microbial growth niches and biofilms
in factory environments and guidelines for monitoring and reducing the presence of listeriae and
other microbial contaminants in working areas that are common to many food processing facilities
will be described and illustrated. No two factories are exactly alike in terms of design, equipment,
maintenance, product flow, sanitation practices and procedures, distribution patterns, and managerial
policies. Consequently, only general principles of cleaning and sanitation will be described for
control of this organism. There will be a brief discussion of how Good Manufacturing Practices
(GMPs), prerequisite programs, and Hazard Analysis Critical Control Point (HACCP) programs
can all be used to sharply decrease the microbial content in any food, thereby reducing the possibility
of producing a product contaminated with L. monocytogenes or any other foodborne pathogen.
Specific problem areas within selected processing plants, such as pasteurizers, fillers, sausage
peelers, etc., associated with the manufacture of particular products will also be identified.
DEVELOPMENT OF MICROBIAL GROWTH NICHES

AND BIOFILMS: GENERAL PRINCIPLES
The potential for Listeria contamination of food in the postprocessing environment theoretically
exists whenever such food is not biocidally treated in the end-use container. Factory conditions
that promote the growth of Listeria dramatically increase the risk of postprocessing product
contamination. Many factors affect the growth of microorganisms in food processing environments,
including moisture, nutrients, pH, oxidation-reduction potential, temperature, presence or absence
of inhibitors, interactions between microorganisms in a population, and time. Moisture is the most
critical among these as it is essential for microbial growth [63] and is often the most readily
controlled of these factors. The transfer of microbes present in a nonsterile factory environment
into niches that are inaccessible for cleaning and sanitation can occur via air, water, tools, workers,
traffic, and other means. Attachment of Listeria cells to environmental surfaces with cell-membrane
bound structures (proteins, polysaccharides, glycoproteins, etc.) occurs, given enough contact time
between the cells and the surface. Furthermore, if appropriate conditions exist (sufficient nutrients,
water, and time) biofilms can also develop. These biofilms further entrap Listeria in a mucilaginous
matrix that can shield these organisms from cleaners and sanitizers [49,52]. Attached cells have
also been associated with increased heat resistance [52,69].
The water activity in niches also impacts the type of microflora that develops therein [63].
Disruption of these niches can result in direct or indirect contamination of the product stream [63].
The probability of product contamination is affected by several variables including, but not limited
to (1) proximity of microbial growth niches to the product stream, (2) number of niches, (3) spatial
relationship of niches to the product stream, (4) microbial populations in niches, (5) extent of niche
disruption, and (6) exposure of the product stream to the environment [63]. The chemical and
physical nature of these microenvironments and the degree to which they can bind to surfaces and
become entrapped in food residue is likely to play an important role in the growth and/or survival
of Listeria in the factory environment.
Factory structures, including equipment, as well as maintenance, repair, and practices that entrap
moisture often result in microbial growth niche development [63]. Operating conditions that may
release, entrap, or cause accumulations of moist residues (thus providing an environment for a
potential microbial growth niche and increasing the potential for cross-contamination) in the factory
environment observed by the first author have included accumulations of dust or powder (e.g., in
FIGURE 17.5 Disassembled equipment and tool on moist floor after cleaning.
corners, on ledges, and angle iron supports, etc.), water drain lines that are not tapped to floor
drains, accumulation of fatty residues on equipment surfaces, high humidity resulting in conden-
sation, floors that are not sloped to drains, cleaning water hoses dragged from one room to another,
failure to segregate forklifts used in wet raw areas from those used in finished product areas, reuse
of soiled and wet cleaning “green pads,” failure to clean and sanitize tools used to repair equipment,
failure to clean and sanitize equipment after repair, soiled aprons, soiled gloves, product or ingre-
dient storage containers, packaging films stored on end in standing water, movement of soiled
pallets into finished product areas, etc. (Figures 17.5 to 17.15).
FIGURE 17.6 Failure to execute captive-shoe policy.

FIGURE 17.7 Hoses on floor. Left: Not hung to dry after use; right: moved from room to room during use.
FIGURE 17.8 Pipe cleaner (rotations promote aerosolization).

FIGURE 17.9 Use of floor scrubbers (aerosol creation potential).
Controversy exists over the importance of aerosol-borne contamination to direct measurable

product contamination. Bioaerosols could be described as solid or liquid microscopic particles
suspended in air which carry microorganisms. Yan et al. [160] performed a number of trials in
which various levels of L. monocytogenes were released into a 315-L bioaerosol chamber. They
were unable to recover a five-strain cocktail of L. monocytogenes from settling plates exposed to
200 CFU/L for 15 min. A settling rate of about 1 log10 CFU per h was determined after release
and mixing of 5 × 106 CFU/L into the chamber. However, the preceding 20-min mixing period
resulted in about a 3 log10 CFU reduction of this organism at both 38 and 75% relative humidity. There
was no significant difference in survival of L. monocytogenes in the air at either relative humidity
FIGURE 17.10 Inappropriate storage of rusted implements, cleaning implements, miscellaneous fittings,
pipes, gloves, etc.
FIGURE 17.11 Accumulation of moist product residue under processing equipment.
FIGURE 17.12 Open back-motor fan covers (bioaerosol potential if not covered during wet cleaning and
sanitation procedures).
FIGURE 17.13 Moist leather tool belts (cross-contamination potential).
(Figure 17.16). In one trial, 5.5 log10 CFU of L. monocytogenes was released and agar strips were
used to trap airborne microbes, using a centrifugal sampler. The agar was removed from the sampler,
diluted, and plated onto selective (modified Oxford medium, MOX), nonselective (trypticase soy
yeast extract agar, TSAYE), and TSAYE overlaid on MOX. No recovery was found on MOX;
however, TSAYE and MOX overlaid with TSAYE recovered 5.8 and 5.4 log10 CFUs, respectively.
The data suggest a profound level of cell injury associated with use of the centrifugal sampler.
However, differences in recovery were not nearly as profound when sedimentation plates of the
same medium were used. In this instance, only 0.5 to 2 log10 CFU of cell injury occurred as
compared to recovery on a nonselective medium. These findings suggest that the mixing of air such
as occurs in the centrifugal sampler may have resulted in greater cell injury. This is most likely
the result of the impact of desiccation on the cells. Hence, it is likely that factories with high air
flow may also have greater injury of liquid aerosol-borne Listeria than those with quiescent air
flow. Yan et al. [160] also exposed 100 cm2 of pre-heat-treated (71°C for 5 min) ham samples to
various levels of L. monocytogenes at both 38 and 75% relative humidity (Table 17.1). Plates were
opened for 15-min periods over 3 to 4 h. Levels greater than 100 CFU/L were required to
contaminate ham to detectable levels when the entire ham sample was enriched and tested. The
ham product used had been treated with sodium lactate and sodium diacetate by the producer.
These data suggest that fluid aerosols may not be a significant source of direct measurable
L. monocytogenes contamination of food treated with Listeria-inhibiting antimicrobials. De Roin
et al. [50] presented evidence to suggest that frankfurters exposed to dust contaminated with
L. monocytogenes could result in measurable detection. Tompkin [151] reported that in 14 years of
investigational work, the air in a room has never been found to be a chronic source of contamination
FIGURE 17.14 Wheeled bin raw-receiving area of a meat factory (cross-contamination and aerosolization
risk from wheels when moved between raw and finished areas over wet areas).
FIGURE 17.15 High-pressure hoses (aersolization) and use of bristled broom to move fluids (aersolization,
cross-contamination, and microbial growth niche potential).
4.0
3.5
3.0 38%
Log CFU/plate
2.5 75%
2.0
1.5
1.0
0.5
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Time after releasing (h)
FIGURE 17.16 Comparison of settling rates of a five-strain cocktail of Listeria monocytogenes at 38 and
75% relative humidity (time 0.0 is after mixing of the air).
of product contact surfaces. Nevertheless, injured Listeria could contaminate products from the air.
However, moist areas in a factory environment in close proximity to product or product contact
surfaces could provide a means to revive injured Listeria from the air, resulting in a microbial
growth niche thus resulting in a greater risk of measurable product contamination. Contaminated
air from compressed air in close proximity to the product (e.g., growth in a filter) has been implicated
(and traced to a niche near the point of use), and exhaust from a small pump near the floor was
suspected as the source [151]. The first author has observed moisture accumulation in low areas
of compressed air lines, likely originating from condensate formation, which also resulted in
microbial growth. This shows the need for use and routine replacement of point-of-use filters for
compressed air lines in food processing environments. Thus, the air supply within the factory must
be considered as a potential source of Listeria and other microbial contaminants. Hence, all heating,
ventilation, and air conditioning (HVAC) ducts and accompanying air filters should be kept in good
repair and cleaned regularly to eliminate excessive dust and dirt. Compressed air lines and filters
should also be inspected regularly and be free of moisture, oil, and debris.
TABLE 17.1
Recovery from Exposed Ham of L. monocytogenes Aerosolized into a Bioaerosol Chamber
at 38% Relative Humidity
Listeria Released Exposure Time (min)

Trial (CFU/L) Con 5 30 60 120 180 240
1 44 0/3a 0/3 0/3 0/3 0/3 0/3 0/3

2 2.2 × 103 0/3 0/3 0/3 0/3 0/3 0/3 1/3
3 1 × 105 0/3 2/3 3/3 3/3 3/3 3/3 3/3
4 6.6 × 105 0/3 3/3 3/3 3/3 3/3 3/3 3/3
a
Positive samples/samples assayed.
Source: From Yan, Z., J.L. Kornacki, C.M. Lin, and M. Doyle. 2004. Fate of Aersolized Listeria monocytogenes in a
Closed Bioaersol Chamber. International Association for Food Protection. Annual Meeting. Abstract P058.
FIGURE 17.17 Failure to inspect and remove wet residue from air boxes.
MAINTENANCE AND REPAIR PRACTICES

Maintenance and repair practices (or failures) that may result in release, entrapment, or accumu-
lation of moisture (if present) in the factory environment observed by the first author have included
torn insulation, duct tape repairs of leaking fluid lines, torn hoses allowing entrapment of moisture
between the torn outer and unbroken inner skin, broken or cracked floors or floor tiles, failure to
repair stress cracks in food contact surfaces, clogged air handling unit drains resulting in fluid
backup, rusted valves, roof leaks, bolt penetrations into hollow structures, torn gaskets, torn, abraded
or leaking product pump gaskets, clogged drains, drains under negative pressure, inadequately
vented drains, torn rubber seals around doors or ports, poorly maintained filters for point-of-use
compressed air lines, etc. (Figures 17.17 to 17.25).
FIGURE 17.18 Exposed and water-soaked insulation over and adjacent to finished-product conveyor belt to freezer.
FIGURE 17.19 Rusted electrical boxes (from failure to maintain door gasket—top photo).
FIGURE 17.20 Ill-fitting and protruding gasket in finished-product tank bottom (see Figure 17.21 for outside view).
FIGURE 17.21 Unchanged gasket at bottom of finished-product tank (with entrapped residues—see
Figure 17.20 for inside view).
FIGURE 17.22 Torn gasket.

FIGURE 17.23 Leaking and bowed ceiling with water marks suggesting entrapped fluid.
FIGURE 17.24 Failure to effectively maintain drainage from roof.

FIGURE 17.25 Unsealed penetrations in bowed ceiling tiles permitting leakage of fluids into factory.
FACTORY AND EQUIPMENT DESIGN

In addition to the building itself, all equipment within the factory should be designed to minimize
cross-contamination between the factory environment and product and should also be constructed
of stainless steel or other easily cleaned and sanitized nonabsorbent, nontoxic materials, such as
certain types of bonded rubber and plastic. All piping in food processing facilities should be free-
draining and designed to eliminate entrapment of food and cleaning and sanitizing solutions used
in clean-in-place (CIP) systems. It is also important that equipment such as product conveyors is
positioned high enough above the floor to minimize cross-contamination from floors and drains.
Equipment designs that may result in release, entrapment, or accumulation of moisture (if present)
as observed by the first author include moistened cloth conveyor belts, hollow portions of conveyor-
belt rollers, sandwiched or hollow portions of slicer assemblies (e.g., shuttles, grippers, housings),
moisture or steam exhaust vents resulting in condensate reflux, product accumulation, excessive
surface area for equipment support pads, rotating pipe brushes, filler assemblies, brine chill solu-
tions, open bearings, condensate in compressed air lines, motor housings, ice makers, crevices
inside spiral freezers, smokehouses that have a common entrance and exit for raw and finished
product, respectively, etc. (see also Figure 17.24, Figure 17.25, Figure 17.28, Figures 17.34 to
17.36, Figure 17.39, Reference 29, and Reference 133). An extensive discussion of sanitary equip-
ment design replete with drawings and photographs can be found in Engineering for Food Safety
and Sanitation: A Guide to the Sanitary Design of Food Plants and Food Plant Equipment [86].
Factory designs that may result in release, entrapment, or accumulation of moisture (if present)
observed by the first author include penetrated double wall construction, flat roofs with improperly
sealed penetrations, hollow areas resulting from newer construction added to older construction,
negative pressure inside finished-product packaging rooms, unwise location of rest room facilities
such that individuals must traverse from highly contaminated regions to finished product areas,
inadequate ventilation, etc. (see Figures 17.37 to 17.39).
Design features that are widely considered to be essential for all types of food processing
facilities include: (1) a raw product receiving area that is completely isolated from processing and
packaging areas of the factory; (2) tight-fitting exterior windows and doors that will prevent animals
and insects from entering processing and packaging areas; (3) easily cleaned and sanitized walls,
floors, and ceilings that are constructed of tile, metal, or appropriately sealed concrete and not
FIGURE 17.26 Failure to maintain a gasket on an overhead light resulting in moisture entrapment and mold growth.
FIGURE 17.27 Common drain line from multiple liquid-product vessels.

FIGURE 17.28 Angle conveyor with sandwiched surfaces and numerous bolts (entraps product residue and
difficult to disassemble for cleaning and sanitization).
FIGURE 17.29 Penetrations in hollow support structures (can collect moisture and grow microbes).
FIGURE 17.30 Entrapment of fluid between hose clamps (microbial growth and cross-contamination potential).
FIGURE 17.31 Steam vent positioned near and toward floor (introduces moisture and aerosols into factory
environment).
FIGURE 17.32 Bottom openings on an electrical control box over a product filler.
porous materials such as wood; (4) floors designed to drain rapidly and prevent pooling of water;
(5) floor drains located away from packaging equipment, especially if processed foods are exposed
to factory air; (6) proper screens, debris baskets, and traps on floor drains; (7) a quality control or
quality assurance laboratory that is well isolated from other areas of the factory; and (8) proper
means of waste disposal outside the factory to discourage congregation of insects, rodents, birds,
and other animals that may harbor Listeria and other pathogenic microorganisms.
FIGURE 17.33 Drainage line untapped to drain resulting in standing water (potential to create microbial
growth niches in resultant wet areas).
FIGURE 17.34 Unsealed sewerage cover under positive pressure in production area (likely aerosolization of
contaminants into production area).
In addition to these concerns, the HVAC system also must be properly designed to minimize
airborne contamination [129]. Features considered to be essential for such a system include (1) intake
air vents on the roof of the buildings that are located upwind from prevailing air currents but away
from dumpsters, raw product receiving areas, and vents that discharge factory air; (2) installation of
screens and filters inside incoming air vents to remove particulate matter and condensate; (3) easily
cleanable HVAC systems; and (4) proper location of dehumidifiers and air conditioning systems so
FIGURE 17.35 Ball valves (difficult to CIP clean, entrapment of fluid and food residue within valve
assembly—between ball and housing—can entrap microbes and allow growth of microorganisms).
FIGURE 17.36 Air handling/refrigeration units (some may entrap moisture in catch pans resulting in micro-
bial growth and contaminant release via aerosols or dripping depending upon maintenance, design, and relative
humidity—the catch pans on these were appropriately tapped to drains).
FIGURE 17.37 Large metal support pads bolted to floor (readily entrap wet residue and very difficult to clean).
FIGURE 17.38 Drain opening above cleaning implements.
that these units drain away from processing and packaging areas. All HVAC systems should be
designed to produce a higher positive air pressure in processing and packaging rather than in raw
receiving areas. This design readily prevents movement of airborne contaminants from raw product
areas to the cleanest areas of the factory where foods are processed and packaged. The first author
has isolated Listeria from cooling coils and catch pans of some HVAC systems. Tube and fin cooling
coils provide a likely environment for the growth of Listeria spp. and were shown to harbor bacteria
to 5 log10 to 7 log10 CFU per m2 in a well-maintained HVAC system [83]. It has been reported that
air movement of 4 m/sec across a quiescent aqueous surface may be adequate to begin aerosolization
of the fluid [112]. Catch-pan drain traps must be maintained because clogged traps may result in
collection of moisture and growth of microbes. Hugenholtz and Fuerst [83] found that draining fluid
in a condensate drip pan had counts of up to 7 log10 CFU/mL. Some HVAC systems are designed
with the final filter located before, and others with the final filter located after cooling coils. In the
first author’s opinion, final filters should be located after cooling coils and catch pans to prevent
aerosolization of contaminants into the factory environment. Care should also be taken to periodically
inspect and change filters as wet filters may become a source of microbial growth [136], which may
be exhausted into the processing environment.
TRADITIONAL APPROACHES TO LISTERIA CONTROL

The traditional approaches to controlling microbiological hazards associated with food products
involve the simultaneous use of employee education and training programs, frequent inspection
and monitoring of facilities and operations, and extensive microbiological testing of raw ingredients
and unfinished and finished products.
Food safety is everyone’s responsibility. Hence, employee education and training programs
should be directed toward a thorough understanding of food hygiene, factory cleaning and sanitation
requirements, and various causes of microbial contamination, including growth and survival patterns
of potential contaminants such as listeriae. Trained employees should also be able to select and
apply control methods that will provide consumers with safe, high-quality products. New employees
should be trained and refresher courses or continuing education of all employees should occur at
routine intervals. Creative approaches to motivating employees to attend such courses have been
developed in some companies.
Factory managers and supervisors must stress good employee hygiene and also set a good
example for other workers. All individuals with obvious illnesses, infected cuts, or abrasions need
to be excluded from working in processing areas or from doing other tasks that may lead to
contamination of food, food contact surfaces, packaging materials or equipment. Furthermore, the
use of tobacco and chewing gum, as well as the consumption of food should be banned in processing
areas along with the wearing of hairpins, rings, earrings, watches, and other jewelry. Employees
should always wash their hands thoroughly before starting work, on returning to work, and after
touching floors, walls, light switches, any other unclean surface, and garbage. To further promote
their use, hand-washing facilities should be properly designed and conveniently located near work-
stations. All factory workers need to be provided with hair and beard nets, as well as clean clothes,
suitable footwear, and disposable gloves. Special attention is also needed to ensure that street clothes
do not enter processing areas and that factory clothing, including footwear, remains inside the factory.
All factory clothing should be changed daily, or more often if soiled, with the responsibility of
laundering being left to the employer. These recommendations may, in some instances, be difficult
for food processors to follow and enforce; however, this task will be made much easier if management
can instill in workers the conviction that each employee is personally responsible for both the quality
and safety of the foods that are produced and ultimately consumed by the public.
The second means of controlling microbiological hazards, routine and frequent inspection
and monitoring (e.g., by taking factory environmental samples for microbiological evaluation)
of facilities, equipment, and operations, is necessary to ensure that GMPs (i.e., hygienic procedures
that minimize the potential for production of unsafe or low-quality products) are being followed.
GMPs to produce specific foods have been outlined in both advisory and regulatory documents
such as GMP guidelines and the various codes of hygienic practice developed by the Codex
Alimentarius Committee on Food Hygiene. Environmental samples are typically taken and analyzed
for the presence of Listeria (when that is the organism of concern). However, pathogen testing in
factory environments is usually limited to non-food-contact surfaces (e.g., walls, drains, floors,
forklift tires, etc.). It is advisable to monitor product contact surfaces for the presence of indicator
organisms (e.g., aerobic plate count, coliforms, Listeria-like or esculin-hydrolyzing bacteria) to
determine the efficacy of sanitation. Some factories use ATP bioluminescence assays, available
from several manufacturers [65], on environmental samples to determine the efficacy of cleaning
as this can be done in real time. Microbiological test results are typically not available until days
later and, therefore, often after the surface has been soiled again from subsequent production. Such
samples are tested after cleaning and sanitation and preoperation. These have routinely been used
to validate a sanitation break in production. Acceptable preoperational contact surface data, in
conjunction with extensive finished-product testing and other investigational data, could be helpful
in limiting the scope of contamination investigations to selected lots by providing evidence that
the manufactured product was previously not contaminated. Routine sampling of postsanitation/pre-
operation food contact surfaces can save the factory much trouble by focusing recall-related efforts
on selected product lots as opposed to all product lots.
A variety of techniques have been developed for taking microbiological samples in the factory
environment. Traditional techniques include swab, contact plate, and sponge-based approaches [61].
Each approach offers advantages and disadvantages, depending upon the nature of the specific
sample area (Table 17.2).
TABLE 17.2
Performance Capabilities of Environmental Sampling Techniques
Sample Sample Heavily Soiled
Sampling Technique Irregular Surfaces Qualitative Assays Surfaces
Traditional swabs Yes Yes No

Sponges Yes Yes Yes
Contact plates Yes Noa No
Tongue blades Yes Yes Yesb
a Possible with nontraditional approaches.

b Biofilm removal possible.
Environmental samples are most easily collected using swabs or sponges. Only polyurethane
or expanding cellulose sponges should be used for this purpose, because other types, including retail
cellulose sponges, contain inhibitory agents that not only prevent recovery of L. monocytogenes and
Staphylococcus aureus but also interfere with recovery of Brochothrix thermosphacta, Aeromonas
hydrophila, Pseudomonas putrefaciens, and P. fluorescens, as well as Escherichia coli, Serratia
marcescens, and Enterobacter cloacae [63,104]. It is important to stress that laboratory personnel
should never attempt to isolate pathogenic microorganisms from such samples unless the laboratory
is completely separate from the factory. Analysis of environmental and, if necessary, food samples
for microbial pathogens is best left to outside commercial testing laboratories that are ISO 17025
compliant or otherwise certified. However, it is strongly recommended that coliform and standard
aerobic plate counts be obtained for samples from the factory environment and the food during all
stages of production to monitor the extent of postprocessing contamination and thus to quickly
identify any problems associated with inadequate cleaning and sanitizing. Coliform organisms are
commonly regarded as being indicators of postprocessing or postsanitation contamination and the
possible presence of pathogens; however, the presence or absence of coliforms in food or environ-
mental samples does not guarantee the presence or absence of foodborne pathogens. In fact, little,
if any, correlation has often been observed between the presence of coliforms and Listeria in
finished products. Therefore, routine testing of environmental samples for Listeria spp. and other
foodborne pathogens by outside laboratories remains a critical component of any sanitation
verification program.
It is a common misconception that only one assay can be done per sponge, swab, or other
similar environmental surface sample. This has resulted in some individuals sampling the same
surface more than once and testing each of these samples, ostensibly from the same surface, for a
different organism or assay of interest. However, this need not and should not be done. Each time
a sample is taken off a surface, that surface is altered. Some microbes are removed although others
may remain. Hence, the microbes recovered from the same surface should not be expected to yield
the same results. Appropriate aliquots of diluent from a single sample can be subdivided for
quantitative or qualitative (enrichment) analysis of each organism or group of organisms of interest.
One example of this occurred when the first author and others took 49 duplicate samples in a dry
factory environment for the analysis of Enterobacter sakazakii. In this instance, each of the 49
areas was sampled twice. The first author reported recovery of the organism from 12 sites whereas
the other laboratory reported recovery from 11 sites. However, only 7 sites were common. In addition,
the first author reported that 7 total ribotypes of the organism were recovered from 49 samples, whereas
analysis of the duplicate set resulted in detection of 14 ribotypes and only four ribotype patterns in
common for a total of 17 distinct ribotypes of the organism recovered [96,99]. The same phenomenon
is likely to be true for L. monocytogenes, as multiple and varied ribotypes were found in factory
environmental samples depending upon the enrichment medium used [132].
Many believe that it is important to sample a defined surface area (e.g., 4 in.2); however, it is the
first author’s conviction that this approach, which may have some value in routine product contact
surface testing, is detrimental to investigational sampling of the factory environment, particularly
relating to Listeria or other pathogen surveys. Numerous areas are unnecessarily precluded from
meaningful pathogen sampling because of rigid adherence to sampling a defined surface area. Impor-
tant, but precluded, areas may include product-pump-bearing seals, small penetrations in walls, and
equipment that may entrap moisture, irregular surfaces, etc. (Figures 17.5 to 17.15; 17.17 to 17.38).
CONTROL OF FOOD CONTAMINATION THROUGH EFFECTIVE

CLEANING AND SANITATION
Any discussion related to control of L. monocytogenes in food processing facilities would not be
complete without mention of appropriate approaches to cleaning and sanitation. However, a detailed
discussion of cleaning and sanitation is beyond the scope of this chapter, and many excellent
resources exist [79,84,107,109]. However, some general principles will be described.
Cleaning can be defined as the physical removal of visible dirt, impurities, and other extraneous
matter commonly referred to as soil. Such soil may entrap and provide nutrients to microbes,
including Listeria, although profound differences exist in cleaning and sanitization of wet processes
(such as meat, poultry, and fluid dairy factories) as opposed to dry facilities (e.g., chocolate, dry
blend factories, dry areas of dry milk processing facilities, etc.) or dry areas.
Cleaning Dry Areas
Dry approaches to cleaning (e.g., appropriate vacuum removal of residues, brushing, scraping, and
damp mopping) should be used in dry facilities processing dry products. The use of unfiltered portable
dry vacuum devices is discouraged as the exhaust may simply redistribute the residue to be removed.
However, some factories have excellent central vacuum systems. The general absence of moisture in
these environments suppresses the growth of microbes, preventing the development of microbial
growth niches. Application of water to such environments is strongly discouraged, as this may result
in creation of microbial growth niches and greater potential for product contamination.
Cleaning Wet Processing Areas
The first step in any cleaning procedure should be physical removal of visible residues. This can
be accomplished by appropriate brushing, sweeping, and application of hot water (in wet processes).
Wet cleaning is done through proper use of solutions of soaps, detergents, surfactants, and abrasive
agents. In contrast, sanitizing causes inactivation of most microorganisms left on cleaned surfaces
by exposing them to heat or chemical agents such as chlorine, iodine (iodophor), acid anionic, or
quaternary ammonium compounds. Hence, the routine use of good cleaning and sanitizing practices
is of utmost importance in controlling microbiological safety and quality of finished products. In
establishments such as those that produce fluid milk and ice cream, adherence to good cleaning
and sanitation practices that involve both equipment and the factory environment may be the only
means of preserving product quality beyond initial pasteurization of ingredients.
Each food processing facility needs to institute and enforce an effective cleaning and sanitizing
program that will ensure manufacture of safe products. Management personnel need to develop
standard operating procedures for every job in the factory as part of this program along with master
schedules with the frequency of cleaning and sanitizing procedures, so that the workers will
recognize their individual responsibilities and maintain accurate records regarding routine sanitation
practices. Management personnel also need to instill the great importance of good cleaning and
sanitizing practices in their employees through the use of continuing education programs that deal
with current issues such as Listeria. Such cleaning and sanitizing responsibilities should never be
assigned to new untrained employees.
Floors, drains, walls, ceilings, and each piece of equipment in the factory should be cleaned
and sanitized on a regular basis, with the frequency of cleaning and sanitizing being dependent on
the extent to which the particular item becomes contaminated during normal operation and whether
or not a product is likely to come in contact with the item during processing and packaging. The
extent to which a particular area becomes contaminated can be determined by careful observation
and appropriate sampling and testing of that area. All food contact surfaces such as tables, peelers,
slicers, collators, overhead shielding, conveyors, conveyor belts, chain rollers, supports, and other
intricate equipment directly associated with processing, filling, and packaging operations need to
be cleaned and sanitized daily, sometimes more often, particularly around filling and packaging
operations. A regular cleaning and sanitizing schedule must also be adopted for non-food-contact
surfaces such as floors, walls, ceilings, floor drains, pipes, blowers, HVAC ducts, coils and pans
from dehumidifying and air conditioning units, light fixtures, material-handling equipment, and
wet and dry vacuum canisters. Listeria spp., including L. monocytogenes, have been most frequently
isolated from floor drains and floors, as will be shown, thus suggesting that these areas may function
as reservoirs for listeriae in food processing facilities. All floors and drains, including drain covers
and baskets, in production and refrigerated storage areas should be thoroughly cleaned and sanitized
daily; however, high-pressure hoses should never be used in these areas, because such practices
readily promote the spread of listeriae to nearby equipment and other areas of the factory through
splashing and the production of aerosols.
Managers of food processing facilities must be sure that proper equipment is available for daily
cleaning and sanitizing operations. Absorbent articles such as sponges and rags should never be used
in the factory environment because these items, when moistened, can support the growth of high
numbers of diverse microflora. Various types of metal scrapers can be used for removing hard mineral
deposits, with disposable paper towels being best suited for eliminating excess moisture and accidental
spills. Brushes are readily cleaned and sanitized, unlike sponges and rags, and are therefore suitable
for widespread use in the factory. However, to avoid cross-contamination, separate color-coded brushes
with nonporous plastic or metal handles should be used for scrubbing (1) exterior and interior surfaces
of equipment, (2) raw and finished product areas, (3) food contact and non-food-contact equipment
surfaces, and (4) floor drains. Brushes, particularly those used to scrub floor drains, are best cleaned
and stored in an effectively maintained sanitizing solution after use.
Water temperatures sufficient to liquefy fats and solubilize proteins should be employed. There
is disagreement as to the appropriate water temperatures to use to accomplish this purpose and required
temperatures can vary depending upon the nature of the soil and the method of cleaning (e.g., CIP
vs. manual cleaning, cleaning chemicals used, etc.). It should be recognized that stainless steel and
metal surfaces in factories act as heat sinks, and water hose exit temperatures may need to be
appreciably higher (e.g., often 20–30°F) than the area’s targeted surface temperature. This is especially
important to recognize in those factories where the environment is refrigerated. If water must be
applied, then use of low-pressure high-volume delivery is to be preferred over high pressure. Moisture
from a high-pressure hose may project food residue and moisture to places inaccessible for cleaning
and result in microbial growth niche development. In many instances (e.g., meat processing facilities),
surfaces soiled with fatty residues will need to be warmed to 140°F to liquefy the fat and move it
away from the surface. Temperatures as high as 160–170°F may need to be used to achieve a surface
temperature of 140°F. Data supporting these temperatures can be found [117,138,152]; however, it is
best to check with one’s supplier of cleaning agents and sanitizer chemicals and seek advice related
to proper water temperatures specific to the factory environment, equipment, and soil type.
Second, application of suitable cleaning agents should be used. It is important to understand
the nature of the soil in the factory to best understand which cleaner to use. Relevant literature and
commercial suppliers of cleaning and sanitizing chemicals can provide guidance. Bohner and
Bradley [32] published a very useful chart in this regard. Cleaning should be in the direction of
product flow and from top to bottom. Subsequent to cleaning, scrubbing may be necessary depend-
ing upon the cleaning approach. This is typically followed by rinsing. Moisture on the floor and
walls should be squeegied to appropriately trapped drains. Sanitizing is the final step in eliminating
L. monocytogenes, other foodborne pathogens, and the myriad spoilage organisms present in the
production environment. Because the presence of organic debris, particularly if proteinaceous,
readily decreases the effectiveness of sanitizing agents against most microorganisms, including
listeriae [30], it is important to remember that every item must first be thoroughly cleaned before
being sanitized.
Sanitization
In those relatively rare instances in which application of aqueous sanitizer in dry factory environ-
ments is necessary, it can sometimes be done by carefully damp-wiping surfaces with a clean cloth
previously immersed in appropriate concentrations of sanitizer. Damp mopping of floors with
sanitizer may also be done. Thorough drying of the factory equipment and environment is critical
and should be done after application of sanitizer to both wet and dry factories. The Code of Federal
Regulations [68] specifies concentrations of sanitizers that may be used in food processing facilities.
Research has demonstrated that L. monocytogenes is sensitive to sanitizing agents commonly
employed in the food industry. According to several authors [105,122], chlorine-based, iodine-based,
acid anionic, and quaternary ammonium-type sanitizers were effective against L. monocytogenes when
used at concentrations of 100 ppm, 25–45 ppm, 200 ppm, and 100–200 ppm, respectively. However,
21 CFR 178.1010 indicates that these can be used at the 200-ppm level on finished product contact
surfaces without the requirement of a subsequent water rinse, with the exception of iodine-based
sanitizers where the maximum level on product contact surfaces not receiving a subsequent water
rinse is 25 ppm. These concentrations may have to be adjusted to compensate for in-plant use, as
well as oxidation and reduction factors relating to water quality and hardness. However, recom-
mended concentrations should not be markedly exceeded, because the use of extremely concentrated
sanitizing solutions heightens the danger to employees, increases the risk of chemical contamination
of food and, in some instances, causes corrosion of equipment. Because foaming chlorine-based
sanitizers are corrosive, their use should be primarily confined to floors, floor drains, walls, and
ceilings. These areas can, alternatively, be flooded or foamed with quaternary ammonium-type
sanitizers; however, fogging exterior surfaces with quaternary ammonium-type sanitizers is fre-
quently regarded as being ineffective and dangerous for employees. Quaternary ammonium-based
sanitizers are also not recommended for use on food contact surfaces and should never be used in
cheese or sausage factories, because lactic acid starter culture bacteria are rapidly inactivated by
small residues of these sanitizers. In contrast, acid anionic and iodine-type sanitizers are best suited
for equipment surfaces, with the former readily neutralizing excess alkalinity from cleaning com-
pounds and preventing formation of alkaline mineral deposits.
Application of sanitizer should be done in the order of the process flow and from the bottom
to the top of equipment to prevent recontamination of sanitized equipment surfaces from the floor.
Many factories employ frequent applications of sanitizer to the wet processing environment.
However, establishment of application frequencies should be based upon appropriate in-factory
research related to their efficacy. Thorough drying of cleaned and sanitized surfaces before start-
up is essential. Sanitizers are typically inactivated over time by organic matter and the remaining
moisture may become a microbial growth niche.
Other Approaches to Sanitization
Sometimes pieces of equipment cannot be adequately or efficiently broken down for complete
and effective cleaning and sanitization. Examples of such equipment include some slicers [29],
sausage peelers, angle conveyors, etc. Some companies have relied upon novel applications of
moist heat or steam [150]. However, observations and validation of such approaches should be
undertaken to ensure that they are appropriate (i.e., do not add excessive moisture to the
environment or damage the equipment) and effective. The use of steam should be confined to
closed systems because of potential hazards associated with aerosol formation, liberation of more
moisture in the factory environment for future microbial growth, and burn risks associated with
human exposure.
Custom-designed CIP systems have been installed in many food processing facilities,
particularly dairies, for automated cleaning and sanitizing of pipelines, tanks, vats, heat
exchangers, homogenizers, and other equipment in processing lines. Presumably adequate by
design, CIP systems should also be reviewed for proper timing, flow rate, temperature, pressure,
and sanitizer strength as recommended by chemical suppliers. Proper operation of the entire
system should be verified from data collected on recording charts, which can be stored for
future reference.
It is important that equipment be designed for ease of disassembly for cleaning and sanitization.
Alternative approaches to effectively clean and sanitize equipment in which sandwiched surfaces
cannot be broken down for cleaning should be researched.
OTHER FACTORS TO CONSIDER FOR CONTROL OF LISTERIA IN FOOD

PROCESSING ENVIRONMENTS
TRAFFIC PATTERNS
Employee movement within food processing facilities can also have a major impact on the micro-
biological quality of finished products. Therefore, traffic patterns need to be developed that restrict
or preferably eliminate movement of workers between raw, processing, filling, packaging, and
shipping areas. Managers need to educate employees about the spread of Listeria and other
microbial contaminants from clothing, boots, and tools to all areas of the factory, and they need
to situate locker rooms, changing areas, and lunch and break rooms to minimize traffic through
production areas. Issuing different-colored outer garments to workers in various areas of the factory
has proved helpful in monitoring employee movement. Because L. monocytogenes and other
microbial pathogens are commonly associated with raw products of both plant and animal origin,
employees working in raw product receiving areas (including maintenance personnel) and individ-
uals who deliver raw products, particularly milk haulers, should be denied access to all processing
areas. When necessary, employee movement between raw product and processing areas of the
factory should only be allowed after completely changing outer garments, as well as scrubbing and
disinfecting boots. In many factories workers are encouraged to use disinfectant-containing foot-
baths that are placed in all doorways leading into the factory, as well as between raw product and
production areas. However, if disinfectant footbaths are used, routine monitoring of sanitizer
concentration should be done; otherwise, these will become a source of moisture and a potential
Listeria growth niche. Experience demonstrates that the undersides of these footbaths can entrap
wet residue and become microbial growth niches as well. Hence, they should be cleaned, sanitized,
and thoroughly dried before use on a daily basis. These footbaths need to be monitored throughout
the day for sanitizer strength and cleanliness. These are not recommended for use in dry processing
areas as they contribute moisture to such environments. Instead, sanitary shoe covering or boot-
changing areas should be provided at key areas of the plant. Because a great variety of microor-
ganisms are carried on street clothing, it may also be prudent for managers to consider limiting
the number of visitors and tour groups going through the factory. Large glass observation windows
provide ample opportunity for visitors to view processing areas and simultaneously prevent intro-
duction of additional microbial contaminants.
MONITORING THE FINISHED PRODUCTS, INGREDIENTS,

AND PROCESSING ENVIRONMENT
The final means of controlling microbial hazards in finished products is through rigorous micro-
biological testing of ingredients, as well as the unfinished and finished product. Analysis of samples
for pathogens or, more commonly, indicator (aerobic plate count, coliforms, yeasts, and molds) or
spoilage organisms is crucial to ascertaining that good manufacturing, handling, and distribution
practices are being followed. If such analysis records are maintained in an appropriate database,
then trend analysis can be a powerful tool for solving problems [57]. However, the data are only
as valid as the accuracy of the assay, the sanitary manner in which the sample was taken and
analyzed, and the statistical confidence associated with the sampling plan (see section titled “Sam-
pling Plans for L. monocytogenes in Foods”).
HAZARD ANALYSIS CRITICAL CONTROL POINT (HACCP) CONCEPT

Traditional approaches for controlling microbial contaminants are being used by many food com-
panies around the world; however, cases of foodborne illnesses still occur. The need for a modified
approach to food-safety assurance led to the development of the HACCP concept, which can be
used to identify and control biological, chemical, and physical hazards in foods from raw material
production, procurement, and handling to manufacturing, distribution, and consumption of the
finished product. A detailed discussion of HACCP is beyond the scope of this chapter; however, a
brief explanation will assist the reader in understanding how the concept, along with GMPs and
prerequisite programs, can be used to reduce the level of L. monocytogenes in food processing
facilities and subsequently in cooked, ready-to-eat (RTE) foods.
The HACCP concept was developed by the Pillsbury Company with the cooperation and
participation of the National Aeronautics and Space Administration (NASA), the Natick Labora-
tories of the U.S. Army, and the U.S. Air Force Space Laboratory Project Group [28]. Development
of the HACCP system began in 1959, when Pillsbury was asked to produce a food that could be
used in the space program. There needed to be assurance (as close to 100% as possible) that the
food produced for the space program would not be contaminated with bacterial or viral pathogens,
toxins, and chemical or physical hazards that could cause illness or injury. The HACCP concept
was developed and first presented to the scientific community at the 1971 Conference for Food
Protection. The HACCP concept was first used in the acidified and low-acid canned-food industry
and was then adopted by a number of companies during the 1970s and early 1980s. The food
industry expressed considerable interest in the application of HACCP after an important 1985
National Academy of Sciences publication strongly recommended the HACCP concept. Con-
sequently, in 1988 the National Advisory Committee on Microbiological Criteria for Foods
(NACMCF) was established and embraced the HACCP concept. In 1989, the committee developed
a HACCP document as a guide for maintaining uniformity of the principles and definitions of
terminology [113]. Since then, the NACMCF has made several refinements and improvements in
the HACCP concept and published revisions in 1992 [114] and 1997 [115]. The 1997 document,
entitled “HACCP Principles and Guidelines” [115], contains many additions and includes a section
on prerequisite programs. Prerequisite programs are essential to successful development and imple-
mentation of a HACCP plan [141] and form the foundation upon which the plan is built. Many of
the prerequisite programs are based on the current GMPs in the Code of Federal Regulations [67]
and in the Codex Alimentarius General Principles of Food Hygiene [91] for foods intended for
international trade. In addition to specific items in the GMPs, prerequisite programs can include
other activities such as ingredient specifications, supplier approval programs, ingredient-to-product
traceability, and consumer-complaint-management programs. A summary of prerequisite program
activities is presented in Table 17.3 [141].
TABLE 17.3
Summary of Prerequisite Program Activities
Facilities
Adjacent properties
Building exterior
Building interior
Traffic flow patterns
Ventilation
Waste disposal facilities
Sanitary hand-washing facilities
Water, ice, culinary steam
Lighting
Raw materials controls
Specifications
Supplier approval
Receipt and storage
Temperature control
Testing procedures
Sanitation
Master schedules
Pest control program
Environmental surveillance activities
Chemical
Training
Personal safety
GMPs
HACCP
Production equipment
Sanitary design and installation
Cleaning and sanitation
Preventive maintenance
Calibration of equipment
Production controls
Product zone controls
Foreign material control
Metal protection program
Allergen control
Glass control
Storage and distribution
Temperature control
Transport vehicle cleaning and inspection
Product controls
Labeling
Product traceability
Customer and consumer complaint investigations
Source: Adapted from Sperber, W.H., K.E. Stevenson, D.T. Bernard, K.E. Deibel, L.J. Moberg, L.R. Hontz, and V.N.
Scott. 1998. The role of prerequisite programs in managing a HACCP system. Dairy Food Environ. Sanit. 18: 418–423.
Prerequisite programs are not part of the formal HACCP system and are established and
maintained separately. There are some circumstances where the existence of a prerequisite program
does not preclude use of specific activities with a HACCP system [141]. For example, sanitation
procedures are normally part of a prerequisite program; however, some manufacturers manage
selected sanitation procedures as critical control points (CCPs) in their HACCP systems. This has
been done frequently in the meat and dairy industries in which sanitation procedures for meat
slicers, ice cream fillers, and other pieces of equipment were established as CCPs to help prevent
recontamination of processed products by L. monocytogenes [141].
The existence and effectiveness of prerequisite programs should be assessed during the design
and implementation of each HACCP plan. Well-developed and consistently performed prerequisite
programs can simplify the HACCP plan, so it is imperative that all food processors establish,
document, and maintain effective prerequisite programs to support their HACCP plans [141]. In
the absence of appropriate prerequisite programs, it would be wrong to assume that a company
with a HACCP plan, even one that is well designed, is immune from having serious product
contamination events. The first author has observed factories with seemingly acceptable HACCP
plans that have had serious food contamination problems [97]. Listeria and many other microor-
ganisms can establish themselves in niches with potential to inoculate the product stream because
of unsanitary operating practices, unsanitary maintenance and repair practices, or unsanitary
equipment or factory design (see Figures 17.5 to 17.37). If such niches are in the post-CCP
environment, serious contamination can occur, as evidenced by a Michigan food factory that
distributed products across the country from which 80 people developed listeriosis, and 21 died
in 22 states [38,39,110].
SAMPLING PLANS FOR L. MONOCYTOGENES IN FOODS

Prevention of microbiological hazards is clearly of considerable importance when one considers
the serious health problems that may develop in certain individuals who consume Listeria-contaminated
foods. Consequently, the International Commission on Microbiological Specifications for Foods
(ICMSF) [87] initially proposed that L. monocytogenes be placed in the same category with agents
that pose severe health hazards to the general population, such as Brucella (strains abortus, suis,
melitensis), Burkholderia cocovenans, the neurotoxin produced by Clostridium botulinum, C.
butyricum, and C. baratti, C. perfringens type C, enterohemorrhagic E. coli O157:H7 and
O111:NM, Mycobacterium bovis, Salmonella typhi (serotypes paratyphi A, B, and C), Shigella
dysenteriae I, Vibrio cholera 01 and 0139, aflatoxins, vCJD/BSE, the parasite Taenia solium,
and hepatitis A virus [10,87].
In February 1988, the ICMSF considered application of its sampling plans to assess accept-
ability of foods with respect to L. monocytogenes. (The reader must be cautioned from the start
that no microbiological sampling plan other than one that involves total destructive sampling of all
products manufactured can ever provide complete consumer protection.) According to terminology
developed previously by the ICMSF, sampling plans for L. monocytogenes in RTE foods would
follow the recommendations made for cases 13, 14, and 15 [87,88]. These cases include those
considered a “severe hazard for (a) the general population or (b) restricted populations, causing
life-threatening or substantial chronic sequelae or illness of long duration [88].” Listeria monocy-
togenes is now considered to fit subcategory 2 in relation to immunocompromised individuals [88]
along with Campylobacter jejuni serovar O:19 and serotypes associated with Guillain-Barre syn-
drome, enteropathogenic E. coli (EPEC and ETEC strains) in food intended for infants, Clostridium
botulinum types A and B (infants), Enterobacter sakazakii (infants), C. perfringens type C (enteritis
necrotans) for protein-deficient persons, Vibrio vulnificus and hepatitis A virus for patients with
liver disease, and the parasite Cryptosporidium parvum with immunocompromised persons.
Case 13 applies when conditions under which the product is normally handled and consumed
after sampling reduce the degree of hazard associated with the product, whereas cases 14 and 15
refer to situations in which hazard levels remain constant or increase, respectively. Using a statis-
tically based two-class attribute sampling plan, n (i.e., number of sample units to be examined from
a particular lot) would equal 15, 30, or 60 for cases 13, 14, and 15, respectively, and c (i.e., the
maximum allowable number of sample units containing L. monocytogenes) would equal 0 for all
three cases. However, it is possible to reduce the number of actual tests done through use of validated
sample compositing schemes [47].
The FDA [80] has a compositing scheme that involves taking 10 × 50 g (or mL) subsamples.
These are then divided into two sets of 5 × 50 g samples, which are combined and stomached in
250 mL of buffered Listeria broth each. Aliquots of 50 g (or mL) from each of the two stomached
preparations are added to two separate 200-mL BLEB enrichments and the assay continued. The
assay, when performed in this manner, is effectively on two 25-g (mL) portions of the sample.
An attribute sampling plan was also proposed in the United States by a working group of
the NACMCF [20]. According to this plan, which was developed for RTE shrimp and crabmeat,
n (i.e., number of samples for foods produced in facilities employing HACCP and GMP
systems) would equal 10, whereas c (i.e., mandatory standard for L. monocytogenes that should
not be exceeded) would equal “negative.” Thus, with the exception of n, this plan is similar to
the two-class attribute plan proposed by the ICMSF.
Before recommending any Listeria-sampling plan, there must be good epidemiological or risk
assessment data indicating that the product or product group to be sampled has been implicated in
foodborne listeriosis. In addition, there must be good reason to believe that introduction of a sampling
program will substantially reduce the risk of contracting listeriosis by consumption of such products.
The number of samples required to detect a contaminated lot increases as the incidence of food
contamination decreases. A food production lot contaminated at 10% incidence would require
30 × 25 g samples to be taken to detect with 95% confidence whether or not the lot was contaminated
(e.g., at least 1 positive in the 30 samples assayed). However, this assumes a random distribution of
contaminants and a 100% accurate test [23]. A lot contaminated at a 1% level would require 299
such samples (Table 17.4). This is a time-consuming, costly, and ineffectual approach to food safety,
if used alone. This is also an approach of diminishing returns as factory processes move toward
safety. This illustrates the importance of a comprehensive approach to food safety, including GMPs,
monitoring of the factory environment with documented corrective actions, and HACCP.
TABLE 17.4
Test Number Needed to Detect One or More Positives per Lot
Percent Positives Number of Analytical Units to Be Tested (n)

% Positive 90% Confidence 95% Confidence 99% Confidence
100 3 4 4
10 23 30 46
1 230 299 461
0.1 2,303 2,996 4,605
0.01 23,026 29,963 46,052
Source: Adapted from APHA. 2001. Sampling plans, sample collection, shipment,
and preparation for analysis, in F.P. Downes and K. Ito (eds.), Compendium of
Methods for the Microbiological Examinations of Foods, 4th ed. American Public
Health Association, Washington, DC, chap. 2.
The International Dairy Foods Association (IDFA) made a series of recommendations concerning
sampling plans for listeriae in milk, soft cheeses, and vegetables based on information collected
in 1988 [10]. L. monocytogenes is commonly found in raw milk; however, minimum required
pasteurization (71.7°C/15 sec) should eliminate this hazard. Therefore, the IDFA recommended
that manufacturers institute monitoring programs to prevent postpasteurization contamination
rather than routine sampling plans of pasteurized end products as the most appropriate means of
protecting the consumer.
Many varieties of Listeria-contaminated cheese have also been identified since 1985, with
contamination most frequently being reported in surface-ripened cheeses. Because L. monocytogenes
can grow rapidly in Brie and Camembert cheese during the late stages of ripening, a two-class
attribute-sampling program should be considered if such cheeses are destined for consumption by
pregnant women, immunocompromised adults, or the elderly. However, because no sampling
program can ensure that such products are completely free of L. monocytogenes, public health
interests are far better served by application of HACCP principles during cheese manufacture
and ripening. The ICMSF recognized that raw vegetables may also become contaminated with
L. monocytogenes, but routine testing of raw vegetables is unlikely to markedly reduce the risk of
contracting listeriosis. Hence, consumers of raw vegetables are urged to wash all such products
vigorously before consumption.
End-product sampling programs are clearly not the answer to protecting consumers from
listeriosis or other types of foodborne illness. However, microbiological sampling is recommended
by many regulatory agencies and the World Health Organization as part of the HACCP approach
to prevent opportunities for contamination, survival, and growth of L. monocytogenes as well as
other microbial pathogens in raw materials, factory environments, and food products during manu-
facture, storage, distribution, sale, and use. The statistical principles for taking appropriate micro-
biological samples can be found in a variety of sources [23,88,139].
ESTABLISHMENT OF BARRIERS BETWEEN RAW AND FINISHED PRODUCT

AND PRODUCTION AREAS
Food processors must strive to prevent the spread of microbial contaminants from heavily
contaminated raw product receiving areas to processing and packaging areas because air, water,
waste products, and anything else that comes in contact with the finished product must be
considered as a potential source of contamination. In addition to construction of physical barriers
between such areas in food processing plants, all incoming cases, pallets, containers, forklifts,
and cleaning materials such as brushes and other equipment must be assumed to harbor listeriae
along with other microbial contaminants and, therefore, should never be allowed to enter pro-
cessing and packaging areas. Ideally, separate equipment, including tools employed by mainte-
nance personnel, should be available for use in raw and finished product areas. If this is not
possible, then all equipment should be cleaned and sanitized before entering processing and
packaging areas.
As previously stressed, all areas within food processing facilities should be kept dry and as
free as possible from processing waste to minimize microbial growth. Also, floor drainage problems
that lead to pooling of water must be eliminated, as well as cracks and holes in floor tiles and
grouting in which water and food particles can accumulate. Because L. monocytogenes has been
recovered from condensate in many factories, it is imperative to keep all processing and packaging
equipment and walls, floors, and ceilings as condensate-free as possible. In the event that dripping
condensate cannot be prevented by manipulation of temperature and humidity in processing and
packaging areas, deflector shields should be installed to prevent direct contact between the exposed
product and dripping condensate.
HACCP PLAN DEVELOPMENT

HACCP is a process management system that is designed for use in all segments of the food
industry from production agriculture to consumption of the finished product. The HACCP approach
for controlling biological hazards in food is based on seven principles [115]:
1. Conduct a hazard analysis

2. Determine the critical control points (CCPs)
3. Establish critical limits
4. Establish monitoring procedures
5. Establish corrective actions
6. Establish verification procedures
7. Establish record-keeping and documentation procedures
THE HACCP TEAM

A HACCP team should be formed before development of a HACCP plan. The HACCP plan should
be a team effort involving representatives from a wide variety of factory responsibilities. This will
ensure emotional buy-in necessary if the plan is to have wholehearted support of the factory. The
insights gained from a wide diversity of backgrounds will also be invaluable. One individual fully
versed in the principles of HACCP should be appointed as the team leader. This individual can,
but need not, be an outside consultant, and should begin the process of introducing HACCP by
educating team members in its basic principles. Once educated, the team is ready to begin devel-
oping the plan.
Principle 1: Conduct a Hazard Analysis
Hazard analysis is the key element in developing an effective HACCP plan. Oddly enough, this is
often the most neglected or inadequately developed aspect of many HACCP plans.
The purpose of hazard analysis is to determine which of the potential hazards associated with
a food or a manufacturing process presents a reasonable risk to consumers. The hazard analysis
involves two stages. The first stage, hazard identification, involves the review of ingredients used
in the product, the activities conducted at each step in the process, the equipment used, the final
product and its method of storage and distribution, and intended use and consumers of the product.
Hazard identification focuses on developing a list of potential food hazards associated with each
process step. In stage two, hazard evaluation, each potential hazard is evaluated based on severity
and its likelihood of occurrence.
It is important that food processors conduct a hazard analysis on all existing and any new
products to be manufactured, because microbiological safety of processed foods not subjected
to a microbicidal treatment (e.g., heating, irradiation) is directly related to the quality of raw
materials. Furthermore, contaminated raw materials, even when given an adequate microbicidal
treatment, may introduce pathogens into the food processing environment. Any hazard analysis
must begin with identification of hazards associated with raw materials, with particular attention
being given to raw products of animal origin (i.e., milk, meat, poultry, and seafood), all of which
may harbor L. monocytogenes and other foodborne pathogens. Heat treatments, acidulation,
fermentation, salting, and drying are designed to destroy or inhibit growth of pathogenic and
spoilage microorganisms; nevertheless, other operations such as slicing and dicing, cooling of
cooked products, and filling and packaging may allow pathogenic organisms to contaminate the
final product. Therefore, all hazards associated with manufacturing procedures and postprocessing
contamination, as previously discussed, must be fully understood along with consequences of
processing failures and errors. A thorough review of operations, facilities, and processes should
be done to ensure that this occurs. It is advisable for a thorough in-factory risk assessment to
be made involving inspection of the premises for potential microbial, chemical, or physical
hazards. Inspection or sampling with a view to understanding the microbial ecology of the
ingredients, process, equipment, and overall factory environment should be done. It will be
necessary to seek counsel from those trained to engage in such activities (e.g., factory quality-
assurance personnel, experienced food safety scientists or outside consultants, etc.). Food pro-
cessors should also be familiar with the effect of various physicochemical factors (e.g., pH, water
activity, preservatives, and type of packaging with or without modified atmosphere) on the behavior
of pathogenic organisms, including L. monocytogenes, in the product during processing, distribu-
tion, storage, and use by the consumer. The National Advisory Committee HACCP document [115]
contains a series of questions regarding ingredients, intrinsic factors of the food during and after
processing, processing procedures, microbial content of the food, factory and equipment design
and use, and packaging when conducting a hazard analysis. It should be noted that any change in
raw materials, product formulation, processing, packaging, distribution, or intended use of the
product should prompt an immediate reassessment of hazards, as these changes have the
potential to adversely affect product safety. Careful hazard analysis should lead to the ready
identification of CCPs.
Principle 2: Determine CCPs
A CCP is a step at which control can be applied and is essential to prevent or eliminate a food safety
hazard or reduce it to an acceptable level. Complete and accurate identification of CCPs is fundamental
to controlling food safety hazards, and CCPs must be carefully developed and documented. Examples
of CCPs may include thermal processing, chilling, and product formulation control.
Principle 3: Establish Critical Limits
A critical limit is a boundary of safety and is used to distinguish between safe and unsafe operating
conditions at a CCP. Each CCP will have one or more control measures to ensure that the identified
hazards are prevented, eliminated, or reduced to acceptable levels. Critical limits may be based on
extrinsic factors such as temperature, humidity and time or intrinsic factors such as physical
dimensions, moisture level, water activity (aw), pH, titratable acidity, salt concentration, available
chlorine, viscosity, or preservatives. Critical limits must be scientifically based and may be obtained
from regulatory standards and guidelines, scientific literature, experimental results, and experts.
However, care should be taken when establishing a critical limit because exceeding a critical limit
is an indication that the food produced may be unsafe for consumption. Hence validation of critical
limits should be done (see subsection titled “Validation”).
Principle 4: Establish Monitoring Procedures
Monitoring is a planned sequence of observations or measurements to assess whether a CCP is

under control and is used to produce an accurate record for future use in verification. Monitoring
facilitates the tracking of an operation and is used to control the process. Monitoring is also used
to determine when there is loss of control and deviation at a CCP (i.e., exceeding or not meeting
a critical limit). Monitoring procedures must be effective to determine deviations and then docu-
mented corrective actions must be taken. Most monitoring procedures need to be rapid and often
include visual observations and measurement of temperature, time, pH, and moisture level. Micro-
bial tests are seldom effective for monitoring owing to their time-consuming nature and problems
with ensuring detection of contaminants (e.g., because of statistics associated with sampling
plans—see section titled “Sampling Plans for L. monocytogenes in Foods”).
Principle 5: Establish Corrective Actions
Corrective actions are necessary when there is a deviation from an established critical limit. Foods
that may be hazardous are prevented from reaching consumers through the establishment of
corrective actions. When there is a deviation from critical limits, corrective actions are needed to:
• Determine and correct the cause of noncompliance

• Determine the disposition of noncompliant product
• Record the corrective actions that are taken
Specific corrective actions should be developed for each CCP and included in the HACCP plan.
Plans should avoid vague language related to future decisions that may or may not be made related
to disposition of the product. The time to determine the specific corrective actions is during the
HACCP plan development, rather than waiting until there is a deviation from a critical limit during
production of a product. Corrective actions should be clearly and precisely stated.
Principle 6: Establish Verification Procedures
Verification determines the validity of the HACCP plan and is used in evaluating whether the
facility’s HACCP system is functioning according to the HACCP plan. An effective HACCP system
requires little end-product testing, because sufficient validated safeguards are built in early in the
process. However, verification of the efficacy of the HACCP plan can and has been done using
finished product testing. Nevertheless, firms should rely on frequent reviews of their HACCP plan,
verification that the plan is being correctly followed, and review of CCP monitoring and corrective
action records.
Validation
Another important aspect of HACCP plan development is validation that the initial plan is scien-
tifically and technically sound, that all hazards have been properly identified, and that if the HACCP
plan is properly implemented, these hazards will be effectively controlled. Subsequent validations
are done and documented by the HACCP team or independent experts as needed. Validations are
conducted when there is an unexplained system failure, when a significant product, process, or
packaging change occurs, or when new hazards are recognized. Incorporation of CCPs into a
HACCP plan implies that they have been validated. Validation is so important that it is being
considered as a candidate worthy of the status of an eighth HACCP principle [140].
Validation of critical limits at CCPs has been done in a variety of ways. These include tradition
(not advised), regulations, published data, and challenge studies. However, some multivariable
processes are difficult to completely control in the factory or cost prohibitive to model in a laboratory
study. In these instances, harmless surrogates may be used to validate (or verify) processes [98].
Surrogates avoid the risk associated with introduction of pathogens and offer the advantage of
assessing the efficacy of the process under actual use conditions. Parameters for selection of
appropriate surrogates were listed by USFDA-CFSAN [22]. In recent times, USDA–FSIS has
stressed the importance of validation [59,60].
Principle 7: Establish Record-Keeping and Documentation Procedures
Establishment of an effective record-keeping system is an integral part of a HACCP system. Records

are the only reference available to trace the production history of a finished product. If questions
arise concerning the safety of a product, a review of records may be the only way to prove that
the product was prepared and handled in a safe manner in accordance with the company’s HACCP
plan [85].
A well-developed and implemented HACCP plan built on a strong foundation of GMPs and
prerequisite programs can reduce the incidence of L. monocytogenes in food processing facilities
and in cooked RTE foods.
In conclusion, to reduce the incidence of L. monocytogenes in the food supply, food processors
must develop and implement HACCP plans that are built on a strong foundation of GMPs and
prerequisite programs which in turn will decrease the incidence of listeriosis.
INCIDENCE OF LISTERIA SPP. IN VARIOUS TYPES OF FOOD PROCESSING

FACILITIES IN THE UNITED STATES
Interest in the extent of Listeria contamination in various food processing facilities has grown over
the past 2 decades, because L. monocytogenes was not identified as a serious foodborne pathogen
until 41 cases of listeriosis in Canada, including 17 deaths, were linked to consumption of con-
taminated coleslaw in 1981 [134]. Despite further evidence 2 years later suggesting possible
involvement of pasteurized milk in an outbreak of listeriosis in Massachusetts, public health officials
in the United States and elsewhere did not yet regard the presence of L. monocytogenes in food as
a major threat to public health. However, this situation changed dramatically in June 1985 when
up to 300 cases of listeriosis, including 85 deaths, were eventually linked to consumption of
contaminated Mexican-style cheese in southern California. This listeriosis outbreak, along with
America’s major outbreak of salmonellosis, in which over 16,000 individuals in the Chicago area
became ill during March and April 1985 after consuming a particular brand of pasteurized milk
contaminated with Salmonella Typhimurium [103], prompted U.S. Food and Drug Administration
(FDA) officials to begin testing various types of domestic and imported cheese for Listeria. FDA
officials also developed the Dairy Safety Initiative Program, which included microbiological sur-
veillance of finished dairy products and the factory environment in which they were produced along
with in-depth inspection of fluid milk factories and eventually all types of dairy processing facilities
located throughout the United States. The subsequent discovery of L. monocytogenes in cooked
RTE meat, poultry, and seafood products by FDA and U.S. Department of Agriculture (USDA)
officials led manufacturers of foods other than dairy products to become concerned about the
incidence of listeriae in their products and processing facilities.
Once FDA and USDA officials announced their plans to review GMPs that were presumably
being used by most American firms, the food industry launched a Herculean effort to identify
Listeria spp. and eliminate such problems within the food processing environment before
governmental inspectors arrived. Considering the adverse publicity and potential monetary losses
that could result from discovery of L. monocytogenes in the finished product and the factory
environment, it is not surprising that very little information concerning the incidence of listeriae
and other microbial contaminants in food (except Class I recalls) and food processing facilities
has been released to the scientific community. Vast amounts of data have been generated since 1985 by
local, state, and federal government inspectors, as well as private microbiological testing and
consulting laboratories and the food manufacturers themselves; however, much of the information
which now follows is either of a general nature describing particular niches within food processing
facilities from which listeriae have been isolated or consists of limited results from academic
surveys which describe the actual incidence of Listeria contamination in a relatively small number
of food processing facilities. This is because many industry-generated data are kept proprietary,
at least in part because of concerns about liability-related issues associated with misuse of this
information by others. Food companies should be encouraged, not discouraged, to generate such
proprietary information, so they have the courage to find real problem areas and correct them.
Requiring companies to make results of environmental testing public creates an incentive against
responsible rigorous inspectional sampling to find and correct real problem areas. Food processors
should be encouraged to thoroughly monitor, understand, and control the microbial ecology
within their factories.
DAIRY PROCESSING FACILITIES

Following several dairy-related outbreaks of salmonellosis and listeriosis, FDA officials, in coop-
eration with state governments and the dairy industry, intensified surveillance of various types of
dairy processing facilities under the Dairy Safety Initiative Program which began April 1, 1986
[100]. Under this program, state officials were requested to sponsor a series of statewide meetings
to discuss foodborne illness associated with Grade A and non–Grade A dairy products and to
intensify their surveillance and inspection efforts in dairy processing facilities. Nationally, FDA
officials vowed to (1) conduct intensified check ratings in every interstate milk shipment (IMS)
milk pasteurization plant, (2) conduct similar inspections at non–Grade A (non-IMS) milk pasteur-
ization plants, (3) initiate a microbiological surveillance program designed to detect pathogenic
microorganisms in finished products, (4) intensify and upgrade training and standardization prac-
tices for federal and state milk specialists, rating officers, and sanitarians, and (5) regularly prepare
national reports which summarize the status of the U.S. dairy industry.
In the first of these reports [4] covering the 6-month period from April to September 1986, 9
of 357 (2.5%) milk pasteurization factories examined produced various dairy products contaminated
with L. monocytogenes. A subsequent report in February 1987 indicated generally similar contam-
ination rates with 16 of 620 (2.6%) and 3 of 620 (0.48%) dairy processing facilities manufacturing
finished products containing L. monocytogenes and L. innocua, respectively [5]. Eight months later,
FDA officials reported that 11 of 604 (1.8%) IMS and 18 of 412 (4.4%) non-IMS milk pasteurization
factories had produced products contaminated with Listeria spp., principally L. monocytogenes [6].
Extensive follow-up efforts in milk processing plants producing Listeria-contaminated products
uncovered various defects in factory design and pasteurization equipment. Nevertheless, FDA
officials have maintained that listeriae entered these products as postpasteurization contaminants.
This view is strongly supported by FDA’s success in isolating Listeria from numerous floor drains
in processing and other areas, wooden (porous) walls, floors and ceilings, wooden pallets, external
surfaces of milk cartons, and sweetwater (refrigerated water) from leaking pasteurizer plates.
Although not clearly identified in FDA’s list of environmental samples that harbored Listeria, FDA
officials [66] noted the following problem areas related to environmental, postpasteurization con-
tamination of dairy products with listeriae: (1) improperly operating high-temperature short-time
(HTST) and vat pasteurizers, (2) leaking or cracked storage tanks, jacketed vessels, and valves,
(3) inadequate sanitizing regimens, (4) cross-connecting pipes which allow commingling of raw and
pasteurized product, (5) use of contaminated rags and sponges, (6) exposure to contaminants in
unfiltered air and condensate, (7) filling and packaging operations, (8) conveyor belts, (9) use of returned
product and reclaiming operations, (10) walls, floors, and ceilings, particularly in walk-in refrigerators,
(11) formation of aerosols, (12) traffic patterns within the factory, (13) entrances and floor mats, and
(14) personal cleanliness of employees and others in the factory. In reality, L. monocytogenes and other
foodborne pathogens have been detected in environmental samples from many of these problem areas,
as indicated in the following surveys of dairy factories in California and Vermont.
In response to these federal programs, officials of the Milk and Dairy Foods Control Branch
of the California Department of Food and Agriculture published [41] results from a statewide survey
in which 597 environmental samples were collected from 156 milk processing facilities during the
first half of 1987 and analyzed for listeriae. Overall, Listeria spp. were identified in the working
environment of 46 (29.5%) milk-processing facilities with 31 of these 46 (67.4%) Listeria-positive
factories being contaminated with L. monocytogenes. Furthermore, L. monocytogenes and other
Listeria spp. were most frequently observed in factories producing fluid milk products, followed
by those that manufactured frozen dairy products (i.e., ice cream and novelty desserts) and cultured
milk products (i.e., yogurt and cottage cheese), with lowest contamination rates being associated with
production of miscellaneous products and cheese. In all likelihood, this unusually low incidence of
listeriae in California cheese factories was a direct result of massive cleanup efforts that were instituted
following the 1985 listeriosis outbreak in the Los Angeles area involving Mexican-style cheese.
TABLE 17.5
Evaluation of Listeria Species Isolates Based on Area of the Processing Plant
Area Designation Number of Sites Number of Positive % Positive
Processing 145 52 35.9

Entrance (to processing) 53 21 39.6
Entrance (other than processing) 37 11 29.7
Raw milk receiving/storage 33 13 39.4
Coolers and freezers 30 14 46.7
Dry storage 16 7 43.8
Othera 31 4 12.9
Total 346 122 —
aIncludes areas such as common hallways, testing laboratories, and wheels of forklifts.
Source: Adapted from Pritchard, T.J., C.M. Beliveau, K.J. Flanders, and C.W. Donnelly. 1994. Increased
incidence of Listeria species in dairy processing plants having adjacent farm facilities. J. Food Prot. 57:
770–775.
Pritchard et al. [127] sampled 30 dairy processing plants in Vermont. Of the 346 sites tested,
122 (35.3%) contained one or more species of Listeria. Coolers and freezers had the highest rate,
with 14 of 30 sites (46.7%) being positive for Listeria (Table 17.5). Other sites that resulted in
high positive rates included dry storage areas (39.6%) and raw milk receiving and storage areas
(39.4%). Pritchard et al. [127] also noted that plants producing dairy ingredients, frozen milk
products, or fluid milk had significantly higher incidence rates of Listeria than expected. Facilities
producing cultured dairy foods or a combination of cultured dairy foods and fluid milk were found
to have significantly lower incidence rates of Listeria than expected. These researchers also observed
that when dairy farms were contiguous to the processing facilities, these plants were more likely
to be contaminated than plants without on-site dairy farms.
More recently, Pritchard et al. [128] surveyed 21 dairy processing factories, including 5 fluid
milk, 12 cultured diary, 3 frozen dairy, and 1 fluid milk/cultured product processing facilities,
respectively. These factories ranged from small, single-room farmstead fluid milk operations to
facilities producing millions of pounds of cheese per year. Environmental and processing equipment
(non–product-contact surfaces) were evaluated by the sponge sampling technique of Silliker and
Gabis [137]. Three different enrichments recovered Listeria species in 21.2% (80) of samples,
including 26 more samples than if a single enrichment had been used. L. innocua, L. monocytogenes,
and L. seeligeri were recovered in 59 (73.8%), 35 (43.8%), and 5 (6.3%) of samples, respectively.
Among the 21 factories surveyed, Listeria-positive equipment samples were obtained from 6
(28.6%) plants. However, 19 of the 21 plants (90.5%) had positive environmental sites. Listeria
species were found in 17 (17.9%) of the 215 equipment samples taken. L. monocytogenes was
recovered from 11 of these 17 Listeria-species-positive sites, including three holding tanks, two
table tops, three conveyor/chain systems, a pasta filata wheel, a pint milk filler, environmental
samples and a brine prefilter machine. Listeria species were recovered from 63 of 163 (41.1%) of
which 24 were positive for L. monocytogenes.
A comparison of the incidence of listeriae in different milk processing areas and sample sites
(Table 17.6) supports the widespread belief that listeriae most frequently enter products after,
rather than before, pasteurization, with the prevalence of these organisms in the factory environ-
ment increasing as the product passes through processing, filling, packaging, and storage areas.
This apparent movement of listeriae through milk processing facilities is most readily seen in
TABLE 17.6
Incidence of Listeria-Indifferent Milk Processing Areas and Sample Sites
Facility Working Area Number of Number (%) of Positive Samples

and Sample Site Samples Examined L. monocytogenes All Listeria spp.
Raw milk receiving room

Drain 30 1 (3.3) 1 (3.3)
Condensate 32 0 1 (4.5)
Other a 1 0 0
Processing room
Drain 150 4 (2.7) 14 (9.3)
Condensate 76 1 (1.3) 3 (3.9)
Other a 21 3 (14.3) 6 (28.6)
Filling/packaging room
Drain 60 7 (11.7) 12 (20.0)
Condensate 36 1 (2.8) 1 (2.8)
Conveyor 15 5 (33.3) 7 (20.0)
Other a 10 0 2 (20.0)
Cold storage room
Drain 105 12 (11.4) 17 (16.2)
Condensate 44 0 1 (2.3)
Conveyor 14 4 (28.6) 9 (64.3)
aIncludes wooden blocks, pallets, case dollies, and utility tables.
Source: Adapted from Charlton, B.R., H. Kinde, and L.H. Jensen. 1990. Environmental survey for
Listeria species in California milk processing plants. J. Food Prot. 43: 198–201.
results obtained from sampling conveyor belts and floor drains. However, sporadic isolation of
listeriae from condensate, as well as wooden blocks, pallets, case dollies, and utility tables points
to additional routes by which these organisms can be disseminated in dairy processing plants.
The low incidence of listeriae in raw milk receiving rooms as compared to other areas of the
factory may at first seem surprising; however, these findings most likely reflect difficulties
encountered in adequately cleaning and sanitizing equipment in processing, filling, and packaging
areas of factories rather than what could be interpreted as a near absence of listeriae in California
raw milk.
In an environmental survey of 39 frozen milk product plants in California, Walker et al. [158]
collected 922 samples and found 111 (12%) positive for Listeria spp. L. monocytogenes was the
only specie recovered from 5 (12.8%) plants and L. innocua was the only specie recovered from
13 (33.3%) plants. Both species were isolated from 9 (23.1%) plants. The highest recovery rates
of Listeria were found in the batch flavoring, freezing, ingredient blending, and packaging and
filling areas of plants surveyed.
Working at the University of Vermont, Klausner and Donnelly [94] performed a large-scale
survey to identify sources of Listeria (and Yersinia) contamination in fluid milk, cheese, and non-
cheese processing facilities. Overall, 66.7, 9.5, and 23.8% of samples collected from floors and
other non-food-contact surfaces at 34 fluid milk, cheese, and non-cheese factories were positive
for Listeria spp., with L. monocytogenes and L. innocua being identified in 1.4 and 16.1% of 361
samples examined, respectively. As expected, the percentage of Listeria-positive samples was higher
among those from floors (12.0–27.9%) than from other non-food-contact surfaces (8.1%)
(Table 17.7) and wet (85.7%) rather than dry (14.3%) areas. According to these investigators, paper-
filler beds, whey drainage pans on cheese presses, and case-washing areas were particularly prone
TABLE 17.7
Incidence of Listeria and Yersinia on Floors and Non-Food-Contact Surfaces
of 34 Fluid Milk, Cheese, and Non-Cheese Factories in Vermont
Number of Samples Number (%) of Positive Samples
Type of Sample Analyzed Listeria Yersinia
Floor areas
Coolers 43 12 (27.9) 9 (20.9)
Processing 117 21 (17.9) 14 (12.0)
Entrance 64 11 (17.2) 18 (27.7)
Mats/footbaths 25 3 (12.0) 1 (4.0)
Other areas 38 10 (26.3) 6 (15.8)
Non-food-contact surfaces 74 6 (8.1) 4 (5.4)
Total 361 63 (17.5) 52 (14.4)
Source: Adapted from Klausner, R.B. and C.W. Donnelly. 1989. Personal communication.
to contamination with Listeria and Yersinia. The fact that 20.9% of all positive samples contained
both Listeria and Yersinia suggests that yersiniae might be somewhat useful as a potential indicator
of Listeria contamination within the dairy processing environment.
Kabuki et al. [91a] examined L. monocytogenes contamination patterns in Latin-style fresh-
cheese processing plants in the United States. Three plants were evaluated over a 6-month period
by collecting 246 environmental samples. L. monocytogenes was found in 11.0% of environmental
samples and in 6.3% of cheese samples tested. L. monocytogenes was found to be most prevalent
in crates, drains, and floors at 55.6, 30.0, and 20.6%, respectively. However, only one of three plants
tested positive for L. monocytogenes on finished product contact surfaces. The most common subtype
detected in two plants (20/36 isolates) was ribotype DUP-1044A, linked in 1998 to a multistate
listeriosis outbreak. In one plant, this subtype was widespread and found in finished products.
As noted, L. monocytogenes, Yersinia spp., and most other foodborne pathogens are more
commonly found in wet rather than dry processing areas. However, the fact that listeriae (1) were
recovered from whey drainage pans, (2) were routinely shed in whey during cheese-making
experiments, (3) grew in samples of refrigerated milk and whey, and (4) survived the typical spray-
drying process used to manufacture nonfat dry milk suggests that these organisms should be of
concern to manufacturers of dry dairy products.
In light of these concerns, Gabis et al. [70] determined the incidence of Listeria in the working
environment of 18 dry milk and whey processing facilities throughout the United States. The authors
supplied environmental sampling kits containing sterile cellulose sponges, fabric-tipped swabs, and
other necessities to all firms participating in the survey along with instructions as to how and where
to collect samples. All samples were then sent to a central laboratory and, within 48 h of collection,
were analyzed for listeriae according to the FDA procedure. Overall, only 2 of 410 (0.24%) samples
examined were positive for Listeria spp., with L. monocytogenes and a Listeria species other than L.
monocytogenes being isolated from floor drains in a raw milk receiving area and from a composite
sample from several floor drains and trenches in a powder production area, respectively (Table 17.8).
Allowing factory employees to choose specific sampling sites, as well as the number of samples to
be analyzed, may have somewhat biased these results; however, the incidence of listeriae and hence
the risk of postprocessing contamination appears to be many times lower in dry rather than wet dairy
processing facilities. Nevertheless, since manufacturers of nonfat dry milk and dry whey are not
immune to the Listeria problem, they should take appropriate action to eliminate this organism from
the processing environment, thereby greatly reducing the chance of producing a contaminated product.
TABLE 17.8
Incidence of Listeria in the Working Environment of 18 Nonfat Dry Milk
and Whey Processing Facilities Located throughout the United States

Work Area Analyzed L. monocytogenes Other Listeria spp.
Raw milk receiving 62 1 (1.6) 0

Wet processing 151 0 0
Cheese factory 22 0 0
Whey factory 23 0 1 (4.3)
Dryer 38 0 0
Bagging room 27 0 0
Heating, ventilating, and 53 0 0
air conditioning system
Miscellaneous 34 0 0
Total 410 1 (0.24) 1 (0.24)
Source: Adapted from Gabis, D.A., R.S. Flowers, D. Evanson, and R.E. Faust. 1989. A survey
of 18 dry dairy product processing plant environments for Salmonella, Listeria and Yersinia. J.
Food Prot. 52: 122–124.
MEAT PROCESSING FACILITIES

Unlike dairy processing facilities in which raw milk is pumped into the factory, pasteurized, and
then either packaged immediately or pumped to closed vats for processing into cream, butter, ice
cream, cheese, or other dairy products, raw meat processing factories are actually open-air disas-
sembly line operations in which animals are slaughtered, eviscerated, and broken down to obtain
various cuts of meat, hides for leather, and other items of commercial value. Considering that
domestic cattle, sheep, and pigs frequently shed L. monocytogenes asymptomatically in fecal
material, it is not surprising that surveys have shown this pathogen to be not only ubiquitous but
also endemic to slaughterhouses and meat-packing facilities.
Rivera-Betancourt et al. (2004) reported the prevalence of L. monocytogenes, E. coli O157:H7,
and Salmonella in two geographically distant commercial beef processing plants in the United
States. Plant sampling took place during the 5-month period of April, May, July, August, and
October at two beef processing plants, one in the northern (plant A) and one in the southern (plant B)
U.S. region. Detection of Listeria spp. was reported as being accomplished by the AOAC official
method 996.14 and specific species confirmed biochemically using the AOAC official method
992.19. Cattle hides and environmental samples were taken as an indicator of Listeria residing in
and entering the plant. The prevalence of both Listeria spp. and L. monocytogenes on hides was
higher in plant B. The prevalence of both Listeria spp. on holding-pen fence panels was significantly
different between the two plants A and B, at 14.7 and 40%, respectively. L. monocytogenes
prevalence during each month of sampling was 2.0% for plant A, but it varied between 3.8 and
48.6% for plant B. The authors attributed this difference to regional differences in Listeria preva-
lence on animal hides as the cattle came from a 150-mi radius of each plant. Environmental samples
(812 and 797, respectively) other than those obtained from cattle pens were gathered from 11
environmental sites sampled at each plant. Samples were taken from the slaughter floor, fabrication
floor, and locker rooms at various times during processing (Table 17.9 and Table 17.10). Significant
findings included L. monocytogenes recovery from floor drains toward the end of operation in plant
B and before operation on the brisket saw. L. monocytogenes was also recovered from one source
TABLE 17.9
Prevalence of Listeriae in Samples from the Slaughter Floor Environment
of Two Beef Processing Plants
Sampling Site Listeria spp. Prevalence (%) L. monocytogenes Prevalence (%)
Floor drains, before operation

Plant A 0/50 (0.0) 0/50 (0.0)
Plant B 0/50 (0.0) 0/50 (0.0)
Floor drains, late in operation
Plant A 2/74 (2.7) 0/74 (0.0)
Plant B 4/75 (5.3) 1/75 (1.3)
Product contact surfaces,
before operation
Plant A 0/49 (0.0) 0/49 (0.0)
Plant B 0/50 (0.0) 0/50 (0.0)
Product contact surfaces, late in
operation
Plant A 0/74 (0.0) 0/74 (0.0)
Plant B 1/75 (1.3) 0/75 (0.0)
Brisket saw, before operation
Plant A 0/20 (0.0) 0/20 (0.0)
Plant B 1/11 (9.1) 1/11 (9.1)
Brisket saw, taken during lunch
break
Plant A 0/30 (0.0) 0/30 (0.0)
Plant B 0/17 (0.0) 0/17 (0.0)
Splitting saw, taken before
operation
Plant A 0/20 (0.0) 0/20 (0.0)
Plant B 0/20 (0.0) 0/20 (0.0)
Splitting saw, taken during
lunch break
Plant A 0/27 (0.0) 0/27 (0.0)
Plant B 0/29 (0.0) 0/29 (0.0)
Trolleys, late in operation
Plant A 1/73 (1.4) 0/73 (0.0)
Plant B 2/73 (2.7) 0/73 (0.0)
Source: Adapted from Rivera-Betancourt, M., S.D. Shackelford, T.M. Arthur, K.E. Westmoreland, G. Bellinger,
M. Rossman, J.O. Reagan, and M. Koohmaraie. 2004. Prevalence of Escherichia coli O157: H7, Listeria
monocytogenes, and Salmonella in two geographically distant commercial beef processing plants in the United
States. J. Food Prot. 67: 295–302.
on the fabrication floor in plant B, the conveyor belts, where the incidence of contamination late
in operation was as high as 14.2%. L. monocytogenes prevalence in locker rooms of plants A and
B (145 and 150 samples, respectively) was 3.3% for plant B (recovered only from floor drains)
and 0% for plant A. The authors hastened to point out that prevalence reports for all three pathogens
(L. monocytogenes, E. coli O157:H7, and Salmonella) occurring concurrently in beef receiving and
processing plants in the United States are scarce or nonexistent. The authors concluded that the
level of L. monocytogenes recovered from animal hides and holding pens is an excellent means of
determining plant carcass-contamination patterns.
TABLE 17.10
Prevalence of Listeriae in Fabrication Floor Environmental Samples in Two Beef
Processing Plants
Sampling Site Listeria spp. Prevalence (%) L. monocytogenes Prevalence (%)
Product contact surfaces, prior to

operation
Plant A 0/100 (0.0) 0/100 (0.0)
Plant B 6/99 (6.1) 6/99 (6.1)
Product contact surfaces, late in operation
Plant A 0/150 (0.0) 0/150 (0.0)
Plant B 25/148 (16.9) 21/148 (14.2)
Source: Adapted from Rivera-Betancourt, M., S.D. Shackelford, T.M. Arthur, K.E. Westmoreland, G. Bellinger,
M. Rossman, J.O. Reagan, and M. Koohmaraie. 2004. Prevalence of Escherichia coli O157: H7, Listeria
monocytogenes, and Salmonella in two geographically distant commercial beef processing plants in the United
Initiation of the USDA–FSIS testing program for listeriae in cooked and RTE meat products
in September 1987 prompted immediate action by the meat industry. However, even before
government testing began, meat processors became concerned about the incidence of listeriae in
the working environment. In June 1987, results from a large-scale survey were reported in which
nearly 2,300 environmental samples were collected from over 40 meat processing facilities nation-
wide and analyzed for listeriae [11]. Fourteen processing areas within these factories yielded
evidence of being contaminated with L. monocytogenes or other Listeria spp. Overall, listeriae
were recovered from 21% of the environmental samples examined. (These results also compare
favorably with those of a much smaller survey [14] in which Listeria spp. were detected in 9 of
27 (33%) meat processing environmental samples.) Problem areas in which >20% of the samples
were positive included drains, trenches, floors, exhaust hoods, cleaning aids (sponges, brooms,
hoses, and mops), product contact surfaces (peelers, conveyors, and slicers), and wash areas.
Sampling of surfaces in contact with sliced luncheon meat revealed Listeria contamination rates
of 9.3, 32.3, and 23.6% before, during, and after production, respectively. Similarly, listeriae were
recovered from 2.8, 14.5, and 25.5%, respectively, of food contact surfaces examined before, during,
and after production of frankfurters.
From September 1987 to October 1991, USDA–FSIS inspectors sampled over 15,000 processed
meat products, including cooked beef, sliced hams from cans, cooked sausage, jerky, cooked poultry,
salads and spreads, and imported meats [149]. The overall incidence of L. monocytogenes during
this sampling period was 1.6%, with 235 products testing positive for L. monocytogenes This led
to 25 recalls of product from the market during 1989–1991 [149].
During the USDA’s microbiological monitoring of over 13,000 lots of RTE meat products, from
January 1, 1993 to September 30, 1997, 2.9% of the lots tested positive for L. monocytogenes (Levine,
personal communication 1998). Results from a large-scale 1987 survey sponsored by the American
Meat Institute [3,14] support the notion that Listeria spp. are widely distributed within the environment
of many meat-processing facilities, and as in the earlier study by Flowers [11], also point to floors,
drains, cleaning aids, wash areas, sausage peelers, and food contact surfaces as significant problem
areas, with between 20 and 37% of such samples harboring listeriae. In addition, Listeria or Listeria-
like organisms have been reported in RTE meat and poultry factories in a variety of areas, including
those associated with brine chillers, ceiling refrigeration units, peelers, conveyors, slicers, dicers, can
openers, spiral freezers, and even packaging machines (Table 17.11). Identification of listeriae in
condensate and compressed air and on walls and ceilings strongly indicates that these organisms are
also airborne and very widespread in at least some meat processing facilities.
Recently, the USDA published its interim final rule [153]. According to this rule, RTE meat
processing facilities were given three alternatives with respect to Listeria testing of the plant environ-
ment. Factories that must test according to alternative 3 are those which have no postprocessing
antimicrobial or microbicidal treatment (e.g., after the smokehouse or other typical treatment). Fac-
tories which must conform to alternative 3 must test the environment for Listeria or an appropriate
indicator (e.g., presence of the genus Listeria, esculin-hydrolyzing bacteria, Listeria-like organisms,
etc.) at a level relative to the size of the facility. Factories that have one additional postprocessing
antimicrobial treatment (e.g., use of an antimicrobial substance in the product formulation that is still
active after processing) or process must test according to alternative 2. Factories that fall into alternative
2 must test their environment, but at a reduced level as compared to alternative 3. Factories using two
antimicrobial processes or ingredients, in addition to the antimicrobial process normally used, are
considered alternative 1 factories and are not required to test the production environment.
Recognizing the potential opportunity for Listeria to contaminate meat during packaging, one
major manufacturer of processed meat products attempted to obtain “near–operating room conditions”
in its packaging room by cleaning the area for 3 days and then fogging the entire packaging room with
200 ppm quaternary ammonium compound [9,11]. Despite these efforts, listeriae were still detected
in 1 of 19 environmental samples obtained from the packaging room. After this exercise, the firm
packaged processed meat products in this room over a 2-week period. Despite adherence to normal
cleaning and sanitizing procedures at the end of each workday, the overall incidence of listeriae in the
packaging room increased, with 3 of 20 (15%), 6 of 20 (30%), and 8 of 20 (40%) samples testing
positive for Listeria spp. 3, 6, and 8 days after the room was initially cleaned and fogged, respectively.
Owing to the increased concern about L. monocytogenes in meat products, there has been a
concerted effort to minimize the risk of postprocess contamination during the production of pro-
cessed meats. In one study, cited by Tompkin et al. [149], swab samples were collected from
packaging lines and floors where exposed RTE products were transported, chilled, stored, or
packaged. The incidence of Listeria at these locations, from August 1989 to January 1992, is
summarized in Figure 17.39. The overall trend is toward improved control of Listeria with fewer
positive samples being evident following the inception of a Listeria-control program. The results
show a strong seasonal effect for the presence of Listeria in finished product environments, with
fewer positive samples being detected during the winter months. In addition, Tompkins et al. [149]
also reported results of a 3-year study in which about 100 packaging lines were tested for Listeria
(Table 17.12). The percentage of Listeria-negative samples increased from 44 in 1989 to 64 in
1991. The percentage of lines that exceeded the company’s established criterion of 5% of samples
positive for Listeria decreased from 29 in 1989 to 13 in 1991. As improvements were made, attention
was given to chronically positive lines. Examples of contaminated sites on packaging lines included
hollow rollers for conveyors, on and off valves and switches, rubber seals around doors, fibrous
conveyor belts, and areas of equipment that were inaccessible to cleaning. Tompkin [151] listed
many such places (see Table 17.11). The authors also noted that occasional lapses in cleaning and
sanitizing procedures resulted in a fairly rapid loss of control. They observed that a certain sequence
of events can lead to periodic contamination of packaging lines. The floor is particularly difficult
to render Listeria-negative, and this situation provides a ready source of organisms to contaminate
the packaging line during production or while cleaning, allowing the establishment of sites for
microbial multiplication. This sequence of events can be prevented by striving for Listeria-negative
floors, effectively cleaning and sanitizing packaging lines at the end of each day’s production,
eliminating inaccessible sites in the equipment, and by providing adequate preventive maintenance
of the equipment. The authors summarized their report with the comment, “. . . for the present, it
must be concluded that existing technology cannot eliminate Listeria from the cooked product
TABLE 17.11
Sites Yielding Listeria or Listeria-Like Organisms in Ready-to-Eat Meat/Poultry Factories
Sites Source Product
Brine chiller
Continuous chamber Chill unit doors Frankfurters/similar linked products
Rubber door seals Frankfurters/similar linked products
Rubber coated fabric Frankfurters/similar linked products
Hinges extending width of door Frankfurters/similar linked products
Bump guards Frankfurters/similar linked products
Uncleanable areas in metal-to- Frankfurters/similar linked products
metal sandwiched steel framing
Ammonia cooling unit Brine-saturated insulation on Frankfurters/similar linked products
ammonia line to brine chilling unit
Exit Hoses and spray nozzles used to Frankfurters/similar linked products
spray franks for easier peeling
Tunnel for product on hanging racks Rubber seals on stainless steel door at Hams
exit end of tunnel
Ceiling refrigeration unit (in staging Condensate from the unit Frankfurters/similar linked products
cooler for peeler)
Peeler or peeler area Gears in peeler (personal observation) Frankfurters/similar linked products
On/off valves for steam and water Frankfurters/similar linked products
lines near peeling equipment
Hopper (post-peeler) for catching Wet insulation (from condensate) Frankfurters/similar linked products
product behind a fiberglass wall
Vacuum-based casing removal Frankfurters/similar linked products
system
Inclined conveyers-peeler to packaging Meat entrapped between two-ply Frankfurters/similar linked products
Plexiglas shield guard—under
conveyer
Fluid entrapped in a hollow split Frankfurters/similar linked products
sprocket
Conveyers (other) Hollow rollers Cooked products
Fibrous/fabric conveyor belts Cooked products
Wheel bearings Cooked products (personal
observation) and breaded products
Buildup inside safety cover over gear Sliced meat products (lunch meats,
and drive belt and over finished canned ham, pepperoni)
product conveyer
Slicers Worn hydraulic seals at base of slicer Sliced meat products (lunch meats,
canned ham, pepperoni)
Slicer blade guides (personal Cooked products
observation)
Slicer blade housing (R. Behling [29], Cooked products
personal communication)
Product grippers for slicers (personal Cooked products
observation)
Can opener Sliced meat products (lunch meats,
canned ham, pepperoni)
Dicer Product residue inside dicer blade Diced cooked meat/poultry
guides (personal observation)
Hollow support rods Cooked products
(continued)

Sites Yielding Listeria or Listeria-Like Organisms in Ready-to-Eat Meat/Poultry Factories
Sites Source Product
Spiral freezer Wet insulation at entrance over Cooked products

product (personal observation)
Entrapped product residues in belt Cooked products
(personal observation)
Electrical control box/wet residue Cooked products
over product inside spiral conveyor
(personal observation)
Standing water inside spiral freezer Cooked products
after defrosting (personal
observation)
Packaging machine Crack in stainless steel covering on Cooked products
top edge of packaging machine
Stainless steel rods for pushing Cooked products
product into carton
Air duct at bottom for blowing bags Cooked products
open prior to insertion of product
Source: Adapted from Tompkin. R.B. 2002. Control of Listeria in the food-processing environment. J. Food Prot. 65(4):
709–723.
FIGURE 17.39 Incidence of Listeria on packaging lines and in the environment (floors) from August 1989
to January 1992, inclusive. (From Tompkin, R.B., L.N. Christiansen, A.B. Shaparis, R.L. Baker, and J.M.
Schroeder. 1992. Control of Listeria monocytogenes in processed meats. Food Aust. 44: 370–376.)
TABLE 17.12
Listeria Contamination on Packing Lines from 1989 to 1991
Year Number of Lines Percentage of Lines Positive for Listeria at
0% ≤5% >5%a
1989 96 44 27 29
1990 106 59 27 14
1991 97 64 23 13
aExceeds company guideline.
Source: Adapted from Tompkin, R.B., L.N. Christiansen, A.B. Shaparis, R.L. Baker,
and J.M. Schroeder. 1992. Control of Listeria monocytogenes in processed meats. Food
Aust. 44: 370–376.
environment of processing plants.” However, this does not mean that this organism’s growth or
prevalence in the factory is beyond reasonable control.
Since Listeria spp., including L. monocytogenes, have been found in up to 50% of raw beef,
pork, and lamb marketed in the United States [132], complete elimination of listeriae from meat
processing environments appears highly improbable. However, the American Meat Institute has
developed a series of interim guidelines [3], which, if followed, will reduce the incidence of listeriae
and decrease the overall microbial load in the working environment. A detailed description of these
guidelines appears later in this chapter.
POULTRY PROCESSING FACILITIES

Reports have shown that up to 50% of all raw poultry sold in the United States contains various
Listeria spp., including L. monocytogenes, with fecal material from infected flocks cited most
frequently as the source of contamination. Researchers at the University of California–Davis
investigated the prevalence of listeriae in processing samples from one chicken [73] and one
turkey slaughterhouse [74] during three or four separate visits. According to these investigators,
no Listeria spp. were isolated from feathers, incoming chiller water, or scalding water, the latter
of which aids in feather removal (Table 17.13). Nonetheless, L. monocytogenes and L. innocua
were identified in samples of overflow chiller water and feather picker drip water obtained from
the chicken slaughterhouse, with both organisms being detected in recycled water used to clean
gutting equipment. Incidence rates for L. monocytogenes in chicken and turkey processing
facilities were generally similar, with the percentage of Listeria-positive samples increasing
approximately 2- to 2.5-fold during the latter stages of processing. However, L. welshimeri and
L. innocua were absent from most chicken and turkey processing samples, respectively. Only
two poultry slaughterhouses were examined in this survey; however, the inability of these
researchers to detect L. welshimeri in fresh chicken meat and L. innocua in fresh turkey meat
routinely processed at these facilities suggests that L. welshimeri and L. innocua might be able
preferentially to colonize the gastrointestinal tract of turkeys and chickens, respectively. These
findings, along with the ability of these investigators to further demonstrate an increasing
incidence of Listeria spp. on the gloves and hands of poultry workers from the beginning to the
end of processing (Table 17.14) confirm that these contaminants move along the processing line
with the raw product.
TABLE 17.13
Incidence of Listeria spp. in One Chicken and One Turkey Slaughterhouse in California
Number of
Chicken/Turkey
Slaughterhouse Number (%) of Positive Samples
Sample Samples Analyzed L. monocytogenes L. innocua L. welshimeri Total
Scalding water overflow 16/15 0/0 0/0 0/0 0/0

Feather picker drip water 16/15 0/1 (6.7) 3 (18.8)/0 0/1 (6.7) 3 (18.8)/2 (13.3)
Incoming chiller water 16/0 0/0 0/0 0/0 0/0
Overflow chiller water 16/15 2 (12.5)/0 0/0 0/1 (6.7) 2 (12.5)/1 (6.7)
Recycled water for 16/15 1 (6.3)/2 (13.3) 5 (31.3)/0 0/3 (20.0) 6 (37.5)/5 (33.3)
cleaning gutters
Source: Adapted from Genigeorgis, C.A., D. Dutulescu, and J.F. Garayzabal. 1989. Prevalence of Listeria spp. in poultry
meat at the supermarket and slaughterhouse level. J. Food Prot. 52: 618–624, 630; Genigeorgis, C.A., P. Oanca, and D.
Dutulescu. 1990. Prevalence of Listeria spp. in turkey meat at the supermarket and slaughterhouse level. J. Food Prot. 53:
282–288.
Considering the fecal carriage rate for listeriae in domestic birds, the current assembly-line
methods for processing poultry, and the fact that Listeria spp. (including L. monocytogenes) and
salmonellae have been isolated from up to about half of all raw chickens marketed in the United
States, one can speculate that the poultry and meat industries face similar problems in controlling
the spread of listeriae and other organisms in the work environment. If one draws a parallel between
methods used to process meat and poultry, then floors, drains, cleaning aids, wash areas, and food
contact surfaces emerge as likely niches for Listeria spp., including L. monocytogenes, in poultry
processing facilities. Berrang et al. [29a] uncovered L. monocytogenes in drains on the raw
side of a poultry processing plant. These isolates appeared to be identical on a molecular level to
TABLE 17.14
Incidence of L. monocytogenes and L. innocua on the Hands and Gloves of Poultry Meat
Processors Assigned to Three Different Stations in a Slaughterhouse
Number of
Chicken/Turkey
Slaughterhouse Number (%) of Positive Samples
Sample Samples Analyzed L. monocytogenes L. innocua L. welshimeri Total
Postchilling handlers 20/30 2 (10.0)3 (10.0) 2 (10.0)/0 0/2 (6.7) 4 (20.0)/5 (16.7)
Leg/wing cutters 11/30 4 (36.4)/3 (10.0) 1 (9.1)/0 0/7 (23.3) 5 (45.5)/10 (33.3)
Leg/wing packers 44/30 20 (45.5)/5 (16.7) 11 (25.0)/0 0/7 (23.3) 31 (70.5)/12 (40.0)
Source: Adapted from Genigeorgis, C.A., D. Dutulescu, and J.F. Garayzabal. 1989. Prevalence of Listeria spp. in poultry
meat at the supermarket and slaughterhouse level. J. Food Prot. 52: 618–624, 630; Genigeorgis, C.A., P. Oanca, and D.
Dutulescu. 1990. Prevalence of Listeria spp. in turkey meat at the supermarket and slaughterhouse level. J. Food Prot. 53:
282–288.
L. monocytogenes isolated from fully cooked products in the same plant, suggesting environmental
cross-contamination from the raw to the finished areas of the processing facility.
EGG PROCESSING FACILITIES

The discovery of L. innocua and, to a lesser extent, L. monocytogenes in 15 of 42 (36%) samples
of frozen, raw, commercial liquid whole egg obtained from 6 of 11 manufacturers located throughout
the United States suggests that listeriae- as well as salmonellae-laden poultry feces may contaminate
the surface of eggs before breaking, and that these organisms in turn may be spread to various
areas within the egg processing environment. Fortunately, the Egg Products Inspection Act of 1970
led to regulations, which now require that all egg products be pasteurized to eliminate salmonellae
(and L. monocytogenes). However, as is true for fluid milk, there is ample opportunity for recon-
tamination of liquid egg products in the factory environment (e.g., with listeriae, salmonellae, and
nonpathogenic organisms) after pasteurization, which can greatly decrease the shelf life, microbial
quality, and acceptability of the finished product. Prudent producers of such products should be
certain that floors, drains, cleaning aids, wash areas, and food contact surfaces, as well as egg-
breaking and egg-separating, pasteurization, and packaging equipment are thoroughly cleaned and
sanitized on a regular basis to eliminate potential problems involving listeriae, salmonellae, and
high levels of spoilage organisms. Special care should be taken to control listeriae in the postpas-
teurization environment and on equipment. Effective cleaning and sanitation of these environments
should include the complete breakdown of positive displacement pumps, including the back plate,
for effective cleaning and sanitation. Gaskets should be checked for leaks and kept on a preventative
maintenance program.
SEAFOOD PROCESSING FACILITIES

FDA officials began testing a wide range of domestic and imported fish and seafood products for
listeriae and other organisms of public health significance after L. monocytogenes was recovered
from fresh frozen crabmeat in May 1987. The results from these analyses led to numerous Class
I recalls of Listeria-contaminated products, and government officials also released additional find-
ings that were obtained during visits to various seafood processing facilities.
The literature has well established the link between environmental contamination and cross-
contamination of seafood, especially RTE seafood products during processing [54,64,118].
Between January and April 1988, inspectors from the Oregon Department of Agriculture ana-
lyzed 480 environmental swab samples from 17 seafood processing facilities located throughout
Oregon [12,66,157]. Only 4% of all samples were positive for Listeria spp.; however, 10 of 17
(60%) factories yielded evidence of Listeria contamination in the work environment. Specific
locations from which listeriae were isolated included the following: (1) a fiberglass tote in a
walk-in cooler, (2) a drain in a walk-in cooler, (3) a phosphate recirculation system on a shrimp
processing line, (4) an ice tote in a cold room, (5) a floor gutter near a shrimp peeler, (6) a
wooden door frame in a crab freezing room, (7) tires on heavy machinery, (8) a cold saturated
brine solution, (9) the framework of a fish dumpster, (10) floor and wall junctions in a cooler,
and (11) seagull droppings on an office manager’s window. Additional environmental niches
within processing plants that are strongly suspected of harboring listeriae include walls, floors,
ceilings, condensate, pooled water, and processing wastes. Hence, this information, along with
other observations that virtually all Listeria cells recovered from processed seafoods have been
healthy rather than thermally or otherwise injured, suggest that the presence of listeriae in
processed seafood is almost exclusively the result of recontamination after processing. L.
monocytogenes and other Listeria species have been isolated from different types of raw and
processed seafood. Several studies [51,58] have been done to detect potential sources of this
pathogen in seafood processing plants so product contamination could be minimized. Eklund
et al. [58] surveyed cold-smoked salmon processing plants to determine occurrence and sources
of L. monocytogenes. These authors observed that cleaning and sanitizing procedures adequately
eliminated L. monocytogenes from the processing line and equipment, but recontamination
occurred soon after processing was resumed. They also identified the external surfaces of fresh
and frozen fish as the primary source of L. monocytogenes in cold-smoked fish processing plants
(Table 17.15). During filleting, rinsing, and brining operations, the bacterium is transferred to
the exposed flesh and, as the product moves through the processing steps, equipment, personnel,
and other surfaces which the product contacts become contaminated. These then serve as
secondary sources of contamination.
Destro et al. traced the transmission of L. monocytogenes in a shrimp processing plant [51],
using two molecular typing methods: random amplified polymorphic DNA (RAPD) analysis and
pulsed-field gel electrophoresis (PFGE). The breakdown of 115 L. monocytogenes isolates exam-
ined follows: 25 were recovered from the plant environment (floors, walls, and pipes); 15 were
from equipment and utensils, including tables, plastic boxes, knives, and trays; 9 were found in
water used in shrimp processing; 7 were isolated from the hands of employees; and 59 were
from the shrimp. The results from this study indicated that environmental strains all fell into
composite groupings unique to the environment, whereas strains from both water and utensils
shared another composite profile group. The L. monocytogenes isolates from fresh shrimp belonging
to one profile group were found in different areas of the processing line. This same profile group
was also present on the hands of employees from the processing and packaging areas of the plant.
TABLE 17.15
Incidence of Listeria in a Cold-Smoked Salmon Processing Plant
Area in Plant L. monocytogenes L. innocua
Raw Product and Processing Area

Thawing water for fish (from tank) 11/59 15/59
Rack from bottom of thawing tank 2/2 0/2
Filleting table 1/9 0/9
Rinse water 6/6 3/6
Skins from raw salmon 3/7 4/7
Slime from raw salmon 4/4 3/4
Drip from raw salmon 8/9 3/9
Trimming from raw salmon 15/26 14/26
Finished Product and Processing Area

Salmon sides from smokehouse 9/9 7/9
Trim table 1/8 0/8
Trim machine 6/15 2/15
Skins from skinning machine 29/30 8/30
Fillet midline trimmings 8/20 0/20
Product trimmings from slicers 17/35 20/35
Source: Adapted from Eklund, M.E., F.T. Poysky, R.N. Paranjpye, L.C. Lashbrook, M.E. Peterson, and G.A. Pelroy.
1995. Incidence and sources of Listeria monocytogenes in cold-smoked fishery products and processing plants.
J. Food Prot. 58: 502–508.
This study showed that there were many different sources of L. monocytogenes in shrimp pro-
cessing plants.
Lappi et al. (2004) monitored two RTE crawfish processing plants over a period of 2 years to
determine the efficacy of Listeria control strategies including targeted sanitation procedures and
worker education 1 [102]. Environmental, as well as raw material and finished product samples,
were taken weekly during the months of April to June and tested for L. monocytogenes in addition
to Listeria spp. Before implementation of the control strategies, Listeria spp. prevalence stood at
5.2% in the processing environment, 29.5% in raw crawfish, and 0% in the finished product.
However, 1 year after implementing the control strategies, Listeria spp. prevalence jumped to 10.8%
in the environment, 57.5% in raw crawfish, and 1% in finished product. L. monocytogenes preva-
lence increased from 3.9 to 10.9% in the raw materials and remained static in the environment.
Thimothe et al. (2004) conducted a longitudinal study using processing plants in the smoked-
fish industry as a model to examine the sources and dissemination of L. monocytogenes in RTE
food processing plant environments [148]. During regular monthly sampling (February–December
of one calendar year) contamination rates of environmental sources were assessed along with
those for raw and finished seafood products. The sampling methodology was coupled with
molecular subtyping to determine the extent of L. monocytogenes transmission in the plants. The
researchers chose to examine two plants on the east coast and two on the west coast, each producing
a variety of products including hot- or cold-smoked fish and shellfish, as well as smoked-fish
salads and retorted (shelf stable) smoked products in some instances. The authors stated that
although the varying prevalence of L. monocytogenes in processing plants is well established
[58,64,81], there is only limited information available on L. monocytogenes transmission from
raw materials and environmental sources to finished products. Twelve to fourteen environmental
samples were taken on each sampling date and included drains in the raw, in-process, and finished
product area, floors, cart wheels, underneath tables, knives, slicing machines, scales, skinning
machines, employee hands, gloves, and aprons, as well as door knobs. Flat surface areas were
swabbed in 24 × 24 in. areas, and all accessible surfaces of a given drain were swabbed. All plants
were relatively new and had been remodeled in the past 4 to 10 years, except for plant 1, which
was located in a building almost 100 years old. Results compiled from all plants confirmed that
71 of 553 environmental samples (12.8%) tested positive for L. monocytogenes in all four plants,
with 151 of the 553 samples (27.3%) testing positive for all presumptive Listeria spp. (Table 17.16).
The prevalence of L. monocytogenes and Listeria spp. differed significantly among the four plants
(P < 0.0001). The environmental samples also displayed a statistically significant positive asso-
ciation (P = 0.0005) between L. monocytogenes and Listeria spp. prevalence. Drain samples were
the most common source of L. monocytogenes and Listeria spp., although prevalence varied from
plant to plant (Table 17.16). L. monocytogenes was isolated from drain samples in both the raw
and finished areas of plants 1, 2, and 3, but not in plant 4 (Table 17.16 and Table 17.17). The
second highest source of isolation was from non-food-contact surfaces (e.g., floors, floor mats,
wheels of rolling carts, trash containers, and the undersides of tables). L. monocytogenes was
found in these areas in plants 1 and 3, but not in plants 2 and 4, although plant 2 had a 38.9%
prevalence of Listeria spp. in these areas. Other common sources of contamination included wheels
of rolling carts, floors, and stress mats. Seven of 10 swabs from wheels of rolling carts in plant
1 were positive for L. monocytogenes and the stress mats in the finished product handling area of
plant 3 were positive 5 of 11 times. Two of the four plants also had waste-material containers that
tested positive for L. monocytogenes.
Employee contact surfaces (e.g., gloves, aprons, door handles, and switches) tested positive for
L. monocytogenes and Listeria spp. at 10 and 16%, respectively. Plant 2 had the highest prevalence
(21.2%) of L. monocytogenes–positive employee samples, although six of seven samples were from
a raw material employee’s apron. Bacterial prevalence on food contact surfaces was relatively low,
TABLE 17.16
Prevalence of Listeriae in Four Smoked-Fish Processing Plants; Percentage
of Positive Samples (Total Samples Taken)
Plants
Product Sampled/ Sampling Site Plant 1 Plant 2 Plant 3 Plant 4 Total
Unprocessed fish
L. monocytogenes 10.0 (60) 3.0 (66) 0 (66) 2.4 (42) 3.8 (234)
Listeria spp. (including L. 18.3 (60) 9.0 (66) 21.2 (66) 19.0 (42) 16.7 (234)
monocytogenes)
Finished product
L. monocytogenes 3.3 (60) 0 (66) 1.5 (65) 0 (42) 1.3 (233)
Listeria spp. (including L. 11.7 (60) 0 (66) 15.4 (65) 9.5 (42) 9.0 (233)
monocytogenes)
Environmental surfaces
L. monocytogenes 29.8 (131) 7.0 (143) 14.4 (153) 0 (126) 12.8 (553)
monocytogenes)
Food contact samples
L. monocytogenes 6.1 (33) 0 (33) 12.5 (32) 0 (27) 4.8 (125)
Listeria spp. (including L. 12.1 (33) 27.3 (33) 21.9 (32) 0 (27) 16.0 (125)
monocytogenes)
Employee contact samples
L. monocytogenes 16.1 (31) 21.2 (33) 4.5 (44) 0 (27) 10.4 (135)
monocytogenes)
Non-food-contact samples
L. monocytogenes 37.8 (37) 0 (36) 13.6 (44) 0 (45) 12.3 (162)
monocytogenes)
Floor drains
L. monocytogenes 60.0 (30) 7.3 (41) 30.3 (33) 0 (27) 23.7 (131)
monocytogenes)
Source: Adapted from Thimothe, J., K.K. Nightingale, K. Gall, V.N. Scott, and M. Wiedmann. Tracking
of Listeria monocytogenes in smoked fish processing plants. J. Food Prot. 67: 328–341.
with plants 2 and 4 testing negative for L. monocytogenes, and plant 4 also testing negative for
Listeria spp. One finished product meat-and-bone separator in plant 1, used to make smoked-fish
salad, was positive for L. monocytogenes. Other finished product food contact areas that were
positive for L. monocytogenes included two samples from a slicing machine and two samples from
a scale in plant 3. The authors did regression analysis and determined that there was a correlation
between L. monocytogenes prevalence in the environment and raw materials (P = 0.027). The corre-
lation was even stronger (P < 0.0001) between L. monocytogenes prevalence in environmental and
finished product samples, although no positive correlation existed between Listeria spp. prevalence
in the environmental samples vs. finished product samples. A positive statistical association was found
TABLE 17.17
Listeria monocytogenes Prevalence Among Environmental Samples in Four Smoked-Fish
Processing Plants (Positive Samples/Total Samples Taken)
Plant 1 Plant 2 Plant 3 Plant 4
Food contact Meat/bone separator (1/10); Slicer faceplate (2/11);

samples hand utensil (1/1) 0 scale (2/11) 0
Employee contact Aprons, raw area (4/10); Aprons, raw area (6/11); Aprons, raw area (1/11);
samples gloves, finished area (1/10) door handle, finished gloves, finished area (1/11) 0
area (1/11)
Non-food- Floors (4/10); Floor mats (5/11);

contact samples cart wheels (7/10); waste totes (1/11) 0
tubs (3/4)
Drain samples Cold-smoking Finished area

ovens (5/10); Raw area (2/11); (e.g., slicing) (5/11);
raw area (8/10); finished area raw area (e.g., racking) (2/11); 0
finished area trench (1/11) raw area (e.g., skinner) (3/11)
(e.g., slicing) (5/10)
Source: Adapted from Thimothe, J., K.K. Nightingale, K. Gall, V.N. Scott, and M. Wiedmann. 2004. Tracking of Listeria
monocytogenes in smoked fish processing plants. J. Food Prot. 67: 328–341.
between Listeria spp. prevalence in the environment and L. monocytogenes prevalence in the envi-
ronment (P = 0.0005) and in the finished product (P = 0.031).
Molecular examination of the 71 L. monocytogenes isolates from environmental samples elu-
cidated 14 distinct ribotypes. Only two of these ribotypes were found in the environment of more
than one plant. Eight different ribotypes were uncovered from environmental samples of plant 1.
One of these ribotypes was isolated at 8 of 10 sample-collection times representing 44% of all
environmental samples recovered. Plant 2 yielded four different ribotypes from environmental
samples, with one ribotype being isolated seven times and the three other ribotypes being isolated
only one time each. One ribotype isolated from raw fish later the same day was isolated from the
apron of a raw material employee. Four ribotypes were also isolated from environmental samples
in plant 3 with one ribotype being isolated 18 times and the other three ribotypes being isolated
only 1 or 2 times. The authors concluded that the variance of L. monocytogenes prevalence in
environmental samples (0–30%) is consistent with previous studies that have reported L.
monocytogenes prevalences ranging from 5 to 30%.
Each of three plants had at least one predominant L. monocytogenes strain isolated throughout
the study. The authors concluded that it is difficult, however, to differentiate between a persistent
subtype in a processing plant and the persistent reintroduction of a specific subtype, since L.
monocytogenes can enter the plant through numerous routes, including equipment, employees, and
raw material [150]. Three persistent strains in plants 1, 2, and 3 were never uncovered in the raw
product. Two ribotypes found on raw materials were not isolated from environmental samples in
the same plant. Plant 1 harbored a persistent strain (determined via ribotyping) that was isolated
throughout the plant and on the wheels of carts and conveyers on 4 of 10 sampling dates. This
plant also had the most lax policies to prevent movement of raw materials, equipment, and
employees between various areas of the plant. One persistent strain in plant 3 represented 82% of
all L. monocytogenes–positive environmental samples. The authors concluded that their data support
the hypothesis that L. monocytogenes on raw material represents a separate population from
persistent plant subtypes and that L. monocytogenes on raw material does not always become
established in the plant environment. They left open the possibility that some of the persistent
subtypes may have been introduced into the environment via raw materials before the study or
were being introduced to the plant concurrent with the study through other sources (e.g., employees,
equipment).
Thimothe et al. (2002) examined the prevalence of L. monocytogenes and Listeria spp. in
two crawfish processing plants in southwest Louisiana. Each plant was 2 to 3 decades old and
processed between 60,000 and 100,000 lb of fresh and frozen crawfish meat per year. Seasonal
production is from January to July and the authors chose to take samples between April and
June. A total of 130 samples were taken from plant I on five occasions, and on eight occasions
a total of 207 samples were collected from plant H. Samples including both crawfish and the
environment remained consistent throughout the study. Samples were taken between the middle
and end of the processing day. The sample pool of 337 samples from both plants yielded 31
positive (9.2%) for Listeria spp. whereas the incidence of Listeria spp. in whole crawfish stood
at 29.5% (23 of 78 positive). Four Listeria spp. (L. innocua, L. welshimeri, L. grayi, and L.
monocytogenes) were identified in the crawfish samples. Although 18% of crawfish sampled were
positive for L. innocua, only 3.8% tested positive for L. monocytogenes. In stark contrast, only
8 of 181 (4.4%) environmental samples tested positive for L. monocytogenes and Listeria spp.
The drains tested positive for Listeria spp. in 6 of 39 instances (15.4%), and gloves and aprons
tested positive in 2 of 39 instances (5.1%), with 38 food contact surfaces consistently testing
negative (Table 17.18). Drains in the peeling room and boiling room tested positive one time
each for L. innocua and L. monocytogenes, respectively. The L. monocytogenes isolate in the
boiling room was subsequently identified as ribotype DUP-1045B, the same ribotype previously
reported as a contaminant in smoked-fish processing plants [119]. A drain in the peeling room
of plant H tested positive for L. innocua on three occasions. These results corroborate other
reports of recovering Listeria spp. and L. monocytogenes most frequently from food processing
plant drains [118,131]. All samples in the crawfish picking room were negative. L. innocua was
TABLE 17.18
Listeriae Prevalence in Two Crawfish Processing Plants; Percentage of Listeriae
(Listeriae-Positive Samples/Total Samples Taken)
Plant H Plant I Plants Total
Product samples (Total) 13.5 (13/96) 16.7 (10/60) 14.7 (23/156)

Raw product samples 27 (13/48) 33.3 (10.30) 29.5 (23/78)
Finished products samples 0 (0/48) 0 (0/30) 0 (0/78)
Environmental samples (Total) 4 (4/111) 6 (4/70) 4.4 (8/181)

Food contact samples 0 (0/23) 0 (0/15) 0 (0/38)
Employee contact samples 4.2 (1/24) 6.7 (1/15) 5.1 (2/39)
Non-food-contact samples 0 (0/40) 0 (0/25) 0 (0/65)
Drain samples 12.5 (3/24) 20 (3/15) 15.4 (6/39)
isolated once in each plant on employee contact surfaces in the boiling rooms. These were pooled
composite swab samples that included swabs of aprons and gloves of employees in contact with
raw material. All finished products also tested negative for listeriae. This study stands in stark
contrast to a previous report suggesting worker or environmental cross-contamination in 17% of
frozen partially cooked crawfish tail meat with 3% of whole boiled crawfish testing positive for
VEGETABLE AND FRUIT PROCESSING FACILITIES

Consumption of coleslaw prepared from contaminated cabbage was directly linked to the first
documented outbreak of foodborne listeriosis in 1981; however, the incidence of listeriae in raw
vegetables and fruits and, particularly, the prevalence of these organisms in work environments of
vegetable and fruit processing facilities have received relatively little attention. Nevertheless, the
long-recognized association of listeriae with soil and the discovery of Listeria spp., including
L. monocytogenes, on raw vegetables suggest that these organisms are almost certainly present in
vegetable and fruit processing facilities. The presence of Listeria spp. in vegetable processing
facilities has also been observed by the first author. Soil and production-area samples from one
potato processing factory in The Netherlands have yielded L. monocytogenes, L. innocua, and
L. seeligeri (see Table 17.19 and Table 17.20).
TABLE 17.19
Incidence of L. innocua in Working Environments of 15 Food Processing Facilities
in The Netherlands
Number of Positive Samples/Sample Analyzed (%)
Fluid Dairy Ice Cream Italian-Style Frozen Food Potato Processing
Factory Factory Cheese Factory Factory Factory
Environmental sample na = 5 n=1 n=5 n=3 n=1

Drains 2/4 (50.0) 4/4 (100.0) 19/42 (45.2) 2/3 (66.7) 7/13 (53.8)
Condensed/stagnant 2/5 (40.0) 4/8 (50.0) 7/20 (35.0) NA 7/10 (70.0)
water
Floors 0/2 8/16 (50.0) 14/44 (31.8) 2/4 (50.0) 9/13 (69.2)
Residues NAb 4/12 (33.3) 16/71 (22.5) NA 5/15c (33.3)
Processing equipment 0/10 7/20 (35.0) 6/68 (8.8) 1/6 (16.7) NA
Miscellaneous 0/13 2/8d (25.0) 12/103e (11.7) 15/78 (19.2) 4/17f (23.5)
Total 4/34 (11.8) 29/68 (42.6) 74/348 (21.3) 20/91 (22.0) 32/68 (47.1)
Note: NA = not applicable.

aNumber of factories analyzed.
bNot analyzed.
cIncludes one sample positive for L. seeligeri.
dConveyor belt (two of two positive).
eRaw milk (two of two positive), untreated effluent.
fPotato delivery soil (two of three positive), sand from effluent treatment (two of two positive).
Source: Adapted from Cox, L.J., T. Kieiss, J.L. Cordier, C. Cordellana, P. Konkel, C. Pedrazzini, R. Beumer, and A. Siebenga.
1989. Listeria spp. in food processing, non-food and domestic environments. Food Microbiol. 6: 49–61.
TABLE 17.20
Incidence of L. monocytogenes in Working Environments of 15 Food Processing Facilities
in The Netherlands
Number of Positive Samples/Sample Analyzed (%)
Italian-Style Frozen Food Potato Processing
Environmental Fluid Dairy Ice Cream Factory Cheese Factory Factory Factory
Sample Factory (na = 5) (n = 1) (n = 5) (n = 3) (n = 1)
Drains 0/4 0/4 2/20 (4.8) 1/3 (33.3) 0/13

Condensed/ 0/5 0/8 0/20 NA 0/10
stagnant water
Floors 0/2 1/16 (6.3) 2/44 (4.5) 0/4 1/13 (7.7)
Residues NAb 0/12 7/71 (9.9) NA 0/15
Processing 0/10 6/20 (30.0) 2/68 (2.9) 0/6 NA
equipment
Miscellaneous 1/13 (7.7) 1/8c (12.5) 0/103 2/78 (2.6) 1/17d (5.9)
Total 1/34 (2.9) 8/68 (11.8) 13/348 (3.7) 3/91 (3.3) 2/67 (3.0)

aNumber of factories analyzed.
bNot analyzed.
cSponge (one of one positive).
dPotato delivery soil (one of three positive).
Source: Adapted from Cox, L.J., T. Kieiss, J.L. Cordier, C. Cordellana, P. Konkel, C. Pedrazzini, R. Beumer, and A. Siebenga.
INCIDENCE OF LISTERIA SPP. IN WESTERN EUROPEAN AND AUSTRALIAN

FOOD PROCESSING FACILITIES
Information concerning the extent of Listeria contamination in European food processing facilities
is limited. However, existing information indicates that European and American food companies
are experiencing similar problems regarding listeriae in the manufacturing environment. Further-
more, because similar food production, processing, and packaging methods and cleaning and
sanitation practices are employed in both Western Europe and North America, much of the following
information regarding the incidence of Listeria contamination within Western European food-
processing facilities is probably applicable to manufacturers of similar products in the United States
and Canada. The following are selected examples of Listeria recovery in other processing facilities
in other parts of the world.
WESTERN EUROPE
Dairy Production Facilities
In 1988, Cox [44,45] presented some preliminary data concerning prevalence of Listeria spp. within
one blue and six soft cheese factories in Western Europe, as well as in one ice cream factory and
eight chocolate factories. As expected, listeriae generally occupied similar environmental niches in
both soft (Table 17.21) and blue cheese factories; however, Listeria contamination was far more
common in ripening than production areas of the one blue cheese factory examined (Table 17.22).
Ripening practices for blue cheese, including maintenance of a relatively moist environment, appear
TABLE 17.21
Prevalence of L. monocytogenes and Nonpathogenic Listeria spp. within the
Working Environment of German Factories Producing Soft Smear-Ripened Cheese
Number of Number (%) of Positive Samples

Samples Nonpathogenic
Environmental Sample Analyzed L. monocytogenes Listeria spp. Total
Smear liquid and smearing machine 210 2 (0.9) 33 (15.7) 35 (16.7)

Other machinery 251 12 (4.8) 31 (12.3) 43 (17.1)
Ripening boards 69 0 2 (2.9) 2 (2.9)
Condensate and cooling water 36 1 (2.8) 2 (5.6) 3 (8.3)
Floor drains 74 3 (4.1) 29 (39.2) 32 (43.2)
Source: Adapted from Terplan, G. 1988. Listeria in the dairy industry—situation and problems in the Federal
Republic of Germany. Foodborne Listeriosis—Proceedings of a Symposium, Wiesbaden, West Germany,
to be the likely reason for higher rates of Listeria contamination in ripening than production areas.
Some environmental niches in this blue cheese factory were not sampled; however, results for soft
cheese factories point to walls, air coolers, stagnant water, and condensate as possible problem
areas in blue cheese factories as well.
TABLE 17.22
Incidence of Listeria spp. in Several Western European Blue
and Soft Cheese Factories
Percentage of Samples Yielding Listeria spp.
Environmental Sample Soft Cheese Factory Blue Cheese Factory
Drains 22 71a/80b
Floors 20 5/83
Residues NAc 23/46
Equipment 0 0/NA
Walls 33 NA/NA
Air coolers 22 NA/NA
Stagnant water 14 NA/NA
Condensate 5 NA/NA
Brine NA 0/NA
Miscellaneous 19 NA/NA

aProduction areas.
bRipening areas.
cNot analyzed.
Source: Adapted from Cox, L.J. 1988. Listeria monocytogenes—a European viewpoint.
General Assembly of IOCCC, Hershey, PA, April 28–30; Cox, L.J. 1988. Prevention of
foodborne listeriosis—the role of the food processing industry. WHO Informal Working
Group on Foodborne Listeriosis, Geneva, Switzerland, February 15–19.
TABLE 17.23
Incidence of Listeria spp. in the Production Environment of One
Western European Ice Cream Factory
Percentage of Samples Yielding Listeria Populations
Environmental Sample Listeria spp. (CFU/g or mL)
Drains 100 ≥106

Conveyors 75 102
Stagnant water 66 NR
Floors 63 10–106
Residues/waste products 50 10–104
Note: NR = Not reported.
Source: Adapted from Cox, L.J. 1988. Listeria monocytogenes—a European viewpoint.
General Assembly of IOCCC, Hershey, PA, April 28–30; Cox, L.J. 1988. Prevention of
foodborne listeriosis—the role of the food processing industry. WHO Informal Working
Group on Foodborne Listeriosis, Geneva, Switzerland, February 15–19.
Samples from at least half of the drains, conveyors, stagnant water, floors, and residue and
waste products from one Western European ice cream factory contained populations of Listeria
spp. ranging from 10 to >106 CFU/g or mL (Table 17.23). This factory manufactured all of its
ice cream from commercially produced reconstituted powdered milk (a product from which
Listeria has not yet been isolated) rather than fresh milk. These findings strongly suggest that
Listeria contamination in dairy processing facilities is not always linked to incoming raw milk
or milk haulers.
Meat, Poultry, and Seafood Production Facilities
Gudbjornsdottir et al. [77] reported results of 36 surveys taken for presence of L. monocytogenes
in 13 meat processing plants (two poultry plants, five seafood plants, and six meat plants) in the
Faroe Islands, Finland, Iceland, Norway, and Sweden. A combined sum of 2,522 samples were
taken from the environment, personnel, raw material, and raw or RTE products. Environmental and
personnel samples were taken before process start-up and then 2 h after start-up. Cleaning resulted
in 11.5 and 8.3% of 661 samples positive for Listeria spp. and L. monocytogenes, respectively.
However, the incidence increased to 26.3% for Listeria spp. and 14.9% for L. monocytogenes 2 h
after start-up. The incidence of L. monocytogenes in the plants ranged from 0 to 34.6% after
cleaning. Drains and floors in 11 of 13 plants had detectable levels of L. monocytogenes. L.
monocytogenes was not detected in any air samples. The incidence of L. monocytogenes in
personnel samples was roughly the same after cleaning (7.6%) and during processing (6.3%). Three
of five seafood plants and five of six meat plants had no L. monocytogenes detected on personnel.
This is in contrast to 42.3% of personnel samples in poultry plants testing positive for Listeria spp.
One positive personnel sample of note was from a quality-assurance individual who moved between
various areas of the plant.
Chocolate Production Facilities
A 1988 report by Cox [44] indicated that 8 of 32 (25%) and 10 of 59 (17%) samples obtained
from damp, wet, and dry areas of eight Western European chocolate factories were positive for
Listeria spp. Growth of listeriae in chocolate is very unlikely given its low water activity; however,
contamination of the finished product during packaging is clearly possible. The relatively low
risk of producing Listeria-contaminated chocolate can be further reduced by development of
adequate cleaning and sanitation programs and by maintaining production and packaging areas as
dry as possible.
ENGLAND AND THE UNITED KINGDOM

English Poultry Processing Facilities
In one Western European survey, Hudson and Mead [82] determined the incidence of Listeria spp.
at 10 different sites within one large English poultry processing facility. According to these authors,
scald water, feathers, and chill water, as well as swab samples from defeathering machines and
conveyors leading to the chiller were free of listeriae; however, L. monocytogenes was routinely
isolated from automatic carcass openers and was also present in samples from evisceration-line
drains, neck-skin trimmers, and conveyors on which carcasses travel to the packing area
(Table 17.24). Only one to three samples from each site were analyzed in three successive visits;
however, the areas from which L. monocytogenes was recovered in this poultry processing facility
were generally similar to those observed by Genigeorgis et al. [73,74] for chicken and turkey
slaughterhouses in California (see Table 17.13).
English Chocolate Production Facilities
Listeria spp., including L. monocytogenes, have also been detected in commercially produced
chocolate that was marketed in England [75].
United Kingdom: Miscellaneous Production Facilities
Evans et al. (2004) examined refrigeration systems of chilled rooms in 15 food processing plants
in the United Kingdom [62]. The plants processed a variety of foods including vegetarian meals,
ready-prepared meals, pies, cooked meats, ready-prepared Chinese meals, raw and cooked poultry,
raw meat, dairy products, mechanically recovered meat, salads, and ready-prepared pasta meals.
The study examined 336 locations on chilled-room walls, evaporators, and drip trays for Listeria
spp. The investigators determined that the chilled rooms were rarely cleaned and temperatures in
chilled rooms ranged from −1 to +16.9°C. A total of 891 sites examined for aerobic plate count
TABLE 17.24
Incidence of Listeria spp. in the Working Environment of One
Poultry Processing Facility in England

Type of Sample Analyzed L. monocytogenes L. innocua
Transport crates 9 0 1 (11.1)

Automatic carcass opener 3 3 (100) 0
Evisceration line drain 3 2 (66.7) 0
Neck skin trimmer 3 2 (66.7) 0
Conveyor to packing area 3 1 (33.3) 0
Source: Adapted from Hudson, W.R. and G.C. Mead. 1989. Listeria contamination at a
poultry processing plant. Lett. Appl. Microbiol. 9: 211–214.
resulted in 25% of samples with bacterial counts greater than 105 CFU/cm2. However, all 336
samples were negative for Listeria spp.
FRANCE
Cheese Production Facilities
Large quantities of French Brie cheese were contaminated with L. monocytogenes in 1986, as noted
in a previous chapter. Therefore, emphasis was first placed on determining prevalence of listeriae
in cheese factories. Results of one small-scale environmental survey of French cheese factories
[27] identified L. monocytogenes in one floor sample, and L. innocua was recovered from boards,
wheels, and equipment (7 of 22 samples), brushes (1 of 6 samples), and filtered air (1 of 19 samples).
A French cheese factory was sampled for Listeria contamination from 1988 to 1990; of the 344
samples collected and analyzed for Listeria, 61 isolates (44 L. monocytogenes and 17 L. innocua)
were recovered from four varieties of cheese, cheese brines, processing equipment, and the plant
environment [90]. All L. monocytogenes–positive samples were from the ripening and rind-washing
stages and not before, suggesting that the cheese contamination occurred at these points in the
manufacturing process [90]. Terplan [146] surveyed German factories producing soft smear-ripened
cheese and isolated nonpathogenic Listeria spp. from smear liquid, various pieces of machinery
(especially smearing machines), and floor drains, with L. monocytogenes being detected far less
frequently than other listeriae (see Table 17.21). Hence, opportunity exists for contamination of
both mold and bacterial surface-ripened cheese during the later stages of manufacture and storage.
Poultry and Pork Production Facilities
Chasseignaux et al. [42] determined the incidence of L. monocytogenes in the processing environ-
ments and finished products from a poultry (plant A) and pork production plant (plant B) in France
over a period of 1 year and 4 months, respectively. A total of 232 samples from plant A and 116
samples from plant B were taken from the environment and equipment. Finished products in plants
A and B, respectively, tested positive 40 and 35.7% of the time, whereas environmental and
equipment samples were only positive 18.3 and 16.4% of the time. In another study, Chasseignaux
et al. [43] reported three pork and two poultry processing plants in France for L. monocytogenes.
The pathogen was found in 23.7% of 497 samples (263 samples taken during production and 234
taken after cleanup and sanitation). From a total of 100 samples taken during production, 38%
were positive for L. monocytogenes. The authors reported this level as being higher than the 26%
reported by Lawrence et al. (1994) in poultry production facilities and lower than the 55% in a
pork processing plant reported by Salvat et al. [133].
SMOKED-SALMON PROCESSING FACILITIES

Dauphin et al. (2001) reported the incidence and PFGE characterization of L. monocytogenes in
three cold-smoked salmon processing plants in France [48]. Samples were taken from the products
in all three plants but only from the processing environment in two of three plants. A total of 44
environmental samples taken from plant 1 came from food contact areas, transport boxes, equip-
ment, floors, and worker’s hands. A combined total of 59 of 141 samples (42%) tested positive for
L. monocytogenes from all three plants. However, 64 and 84% of the salmon and environmental
samples, respectively, tested positive for the bacterium in plant 1. Swabs from workers’ hands in
plant 2 tested positive for L. monocytogenes on 2 of 2 occasions. Following PFGE typing, one
pulsotype was shown to predominate and persist in plant 1. The results indicated that the finished
product was most likely contaminated from persistent environmental strains rather than from the
raw product.
FINLAND
Meat Processing Facilities
Lunden et al. (2004) assessed the incidence of L. monocytogenes in three RTE pork and beef
processing plants and one poultry processing plant in Finland [106]. Overall, 596 L. monocyto-
genes isolates were recovered from the processing plants over several years. Samples were taken
from walls, floors, drains, equipment, products, and raw materials. Plant A yielded L. monocy-
togenes isolates (18 total) from the environment (2), equipment (9), and product (7), whereas
plant B yielded isolates (92 total) from the environment (24), equipment (49), product (18), and
raw material (1). In plant C L. monocytogenes isolates (307 total) were recovered from the
processing environment (43), equipment (199), product (63), and raw material (2). Plant D (the
poultry processing facility) yielded L. monocytogenes isolates (179 total) from the environment
(38), equipment (104), and product (37). Contaminated environmental surfaces included slicing
machines, spiral freezers, packing machines, conveyers, dicing machines, peeling machines,
weigh scales, packing machines, and various components of said equipment (e.g., control panel,
motor, lubricant, chamber, funnel, etc.). These processing machines were persistently contami-
nated in all four plants. Multiple isolates recovered from finished products had identical PFGE
patterns to those from the processing machines. The 596 isolates uncovered were subdivided
into 47 groupings based on PFGE typing. Persistent L. monocytogenes strains were classified as
those that were found five or more times over a period of ≥3 months. Nonpersistent strains were
those that were found fewer than five times, or only within a limited time frame (<3 months).
Overall, 19 of the 47 PFGE groups were determined to be persistent, with the remaining 28
being nonpersistent.
Seafood Processing Facilities
Autio et al. [25] were unable to prove the spread of L. monocytogenes through heavily contaminated
air in a food processing plant in Finland. Furthermore, neither environmental nor smoked-salmon
products tested positive for L. monocytogenes for 5 months after comprehensive disinfection of a
processing plant with hot water, hot air, and hot steam.
ITALY
Meat Processing Facilities
Peccio et al. (2003) examined the occurrence and characteristics of L. monocytogenes in two
meat producing plants in northeast Italy [123]. Over a 20-month period, 72 and 68 samples,
respectively, were taken from a cattle slaughterhouse and a swine meat processing plant. All of
the 46 environmental samples taken from the bovine slaughterhouse were L. monocytogenes
negative except for three samples (6.5%), all from knives. However, in the same plant, 4 out of
26 raw product samples (15.4%) were positive for L. monocytogenes. Other environmental
samples that were all negative for L. monocytogenes included swabs taken from a refrigerated
room, tables, saws, and floor drains, as well as other unspecified areas. Results from the pork
processing plant were similar, with 2 of 51 environmental samples (3.9%) positive for L.
monocytogenes, including one kneader and one mincer sample. However, the pathogen was
recovered from 5 of 7 (29.4%) samples of raw pork products. L. monocytogenes was not recovered
from tables, a meat stuffer, or other environmental samples. All seven L. monocytogenes–positive
samples from the beef processing facility were identical when they were characterized via PFGE.
However, in the pork processing plant, six different PFGE profiles were found among the seven
isolates.
SPAIN
Vegetable Processing Facility
Aguado et al. [2] reported the incidence of L. monocytogenes in a vegetable processing facility
during a 23-month study in Spain. The 906 sampler taken included environmental and RTE
vegetable samples. Listeriae incidence in frozen vegetables was 1.2, 8.5, 0.2, and 0.1% for L.
monocytogenes, L. innocua, L. welshimeri, and L. seeligeri, respectively. Of 166 environmental
samples taken, 11 of 166 (6.6%) were positive for L. innocua and 2 of 166 (1.2%) were positive for
L. monocytogenes. These isolates were taken from various points along the processing chain (e.g.,
washing tunnels, conveyer belts, floors, and machinery).
THE NETHERLANDS
Miscellaneous Production Facilities
In one large European survey during the latter half of 1986, Cox et al. [46], investigated the incidence
of Listeria spp. in the processing environment of 17 establishments in The Netherlands that produced
fluid dairy products, ice cream, Italian-style cheese, frozen food, potato products, and dry culinary
foods. A total of 608 samples were collected from drains, floors, condensed and stagnant water,
residues, processing equipment, and other areas and were analyzed for listeriae using the original
USDA or FDA method with or without modification. All presumptive Listeria isolates were then
speciated according to results from conventional biochemical tests.
Despite use of GMPs in these factories, Listeria spp. were recovered from all types of food
processing facilities examined, with the exception of two that produced dry culinary products.
Overall, 181 of 608 (29.8%) samples yielded Listeria spp. with L. innocua, L. monocytogenes, and
L. seeligeri being identified in 87.3, 14.9, and 0.5% of all positive samples, respectively. Only five
samples contained both L. monocytogenes and L. innocua; however, the actual number of such
samples is probably somewhat greater, because a limited number of presumptive Listeria isolates
from each sample were chosen for confirmation. L. innocua was most prevalent in establishments
that produced processed potato products followed by those that produced ice cream, frozen food,
Italian-style cheese, and fluid dairy products, with the organism generally being isolated from
drains, floors, and condensed and stagnant water (Table 17.19).
In contrast, L. monocytogenes was detected in 11.8% of all environmental samples obtained
from one ice cream factory, but was found in 2.9, 3.0, 3.3, and 3.7% of similar samples from
establishments that manufactured fluid dairy products, potato products, frozen food, and Italian-
style cheese, respectively (Table 17.20). Only one ice cream factory was examined in this survey;
however, the results are as expected when one recalls that Cox [44,45] previously found that listeriae
were widespread in another Western European ice cream factory and also were present in very
large numbers, particularly in floor drains (see Table 17.23). Given such populations of listeriae in
ice cream factories and the current extruding, molding, and freezing methods used to produce ice
cream, particularly ice cream novelties, one can easily postulate many routes whereby listeriae may
recontaminate the finished product, as has been reported in the United States.
Results concerning the incidence of Listeria spp. as well as L. innocua and L. monocytogenes
in various work environments of 15 food processing facilities are summarized in Table 17.25.
Overall, these findings are comparable to what has been previously noted for similar food processing
facilities in the United States; for example, Listeria spp. and L. innocua were most frequently
recovered from drains, followed by condensed and stagnant water, floors, residues, and processing
equipment. This same trend is readily apparent for all five types of food processing facilities listed
in Table 17.19 with a few minor exceptions, which probably resulted from the number of samples
analyzed. Thus a logical pattern emerges in which L. innocua moves from floor drains to pools of
condensed and stagnant water, which then come into direct contact with floors and residues.
TABLE 17.25
Overall Incidence of Listeria spp. in Working Environments of 15 Food Processing
Facilities in The Netherlands
No. of Samples No. (%) of Positive Samplesa

Environmental Sample Analyzed Listeria spp. L. innocua L. monocytogenes
Drains 66 36 (54.5) 34 (51.5) 3 (4.5)

Condensed/stagnant water 43 20 (46.6) 20 (46.5) 0
Floors 79 36 (45.6) 33 (41.8) 4 (5.1)
Residues 97 32a (33.0) 24 (24.7) 7 (7.2)
Processing equipment 104 20 (19.2) 14 (13.5) 8 (7.7)
Miscellaneous 219 37 (16.9) 33 (15.1) 5 (2.3)
Total 608 181b (29.8) 158 (26.0) 27 (4.4)
a One sample yielded L. seeligeri.
b Five samples yielded both L. monocytogenes and L. innocua.
Source: Adapted from Cox, L.J., T. Kieiss, J.L. Cordier, C. Cordellana, P. Konkel, C. Pedrazzini, R. Beumer, and A.
Siebenga. 1989. Listeria spp. in food processing, non-food and domestic environments. Food Microbiol. 6: 49–61.
Once present in open areas of the work environment, employees can spread L. innocua by various
means, to processing equipment that comes into direct contact with the product. L. monocytogenes,
unlike L. innocua, was far less prevalent in all types of food processing facilities and was
distributed fairly evenly within the factory environment, with incidence rates ranging between
2.3 and 7.7%. L. innocua is by definition nonpathogenic; however, as L. innocua and L.
monocytogenes (and possibly other Listeria spp.) occupy similar environmental niches, it follows
that detection of listeriae anywhere within the manufacturing environment should prompt immediate
corrective action.
SWEDEN
Dairy Production Facilities
Waak et al. [156] reported the incidence of Listeria spp. in raw whole milk from farm bulk tanks,
as well as from raw milk stored in a Swedish dairy. Raw whole milk samples (25 mL) were taken
from 153 farms between the months of December and March and then again from August to
September, minus 12 farms that were excluded from the study because of cessation of production.
Raw whole milk samples (295 total) at the dairy processing plant were taken from three storage
silos with respective capacities of 40,000, 40,000, and 100,000 L each. These samples were taken
in the months of November to April and July. Equipment at the dairy plant was also sampled for
the prevalence of Listeria spp. Two plate coolers, eight different-sized gaskets from the plate cooler
and two silo pumps, and rinse water from silo sampling cocks were microbiologically examined.
Weekly sampling of milk products and dairy plant drains resulted in 440 surface samples from
blue mold cheese, 70 whey samples, 149 swab samples from pasteurized-product-area exposed
drains, and 116 cheese-washing liquid samples.
Only 1% (3 samples) of the farm milk tank samples tested positive for L. monocytogenes,
with Waak et al. [156] reporting that these results are similar to findings from other countries
[53]. The three samples contained L. monocytogenes at levels of <10, 60, and <10 CFU/mL,
respectively. Listeria prevalence in dairy plant silos was significantly higher than on the farm
because of the high probability of cross-contamination from previously positive bulk milk loads.
The fact that one silo had nearly twice the prevalence of L. monocytogenes as the remaining two
silos was attributed to either persistent contamination within the silo or a greater number of L.
monocytogenes–positive shipments going into that particular silo. The incidence was also higher
from January to March than in other months of the year as is now well documented. Farm milk
was more frequently contaminated with L. innocua (3.4%) than L. monocytogenes (2.3%);
however, just the opposite was true in sampling dairy plant silos, which had levels of 19.6% and
8.5%, respectively. The authors concluded that because silo milk was stored longer than farm milk,
the lactoperoxidase system of raw milk may have been more effective against L. innocua than L.
monocytogenes [125]. However, these results are in contrast to those of Canillac and Mourney [37]
who found no L. monocytogenes in raw milk whereas 25% of the samples were L. innocua positive,
and Harvey and Gilmour [78], who reported L. monocytogenes in 33.3% of raw milk samples from
dairy silos and Listeria spp. in 54% of samples.
All processed milk product and environmental samples (e.g., rinse water, surface swabs, gaskets,
and floor drains) tested negative for Listeria spp. The authors concluded that the lack of Listeria
detection might be the result of a rigorous in-house sanitation program, as well as the fact that an
alternative non-enrichment-based Listeria method was used for processed milk products and envi-
ronmental tests in contrast to traditional enrichment protocol for milk. These findings differ from
previous studies that have reportedly recovered Listeria spp. in large numbers from dairy-processing-
plant floors, drains, presses, conveyer belts, cheese-ripening areas, and brushing machines
[40,78,95,111].
SWITZERLAND
Swiss officials tracing the source of contamination in the 1987 listeriosis outbreak involving
consumption of Vacherin Mont d’Or soft-ripened cheese recovered the epidemic strain of L. monocy-
togenes from smear brine, curing brine, wastewater sinks, wooden cheese hoops, and wooden boards
used in 10 different cheese factories that manufactured Listeria-contaminated cheese [36]. Addi-
tionally, nearly half of the 12 cellars used to ripen cheese contained listeriae, with the pathogen
being detected on 6.8% of the wooden shelves and 19.8% of the brushes used in the ripening
cellars. Although not noted in the report, one would suspect that L. monocytogenes was also present
in commonly recognized environmental niches such as drains, floors, stagnant water, and various
food contact surfaces within the cheese factories and ripening cellars. Thus, brushing cheese with
saltwater and ripening hooped cheese on wooden shelves appear to be two important means for
dissemination of listeriae within cheese factories.
AUSTRALIA
Information concerning the prevalence of listeriae in food processing facilities located in other
parts of the world is currently limited to a few Australian studies. Following the isolation of
L. monocytogenes from ricotta cheese in 1987, the Victorian Dairy Industry Authority and the
Department of Agriculture and Rural Affairs conducted a joint survey to determine the extent of
Listeria contamination in the working environments of 52 Melbourne-area factories producing
pasteurized milk and different types of cheese [155]. Overall, various Listeria spp. were detected
in 141 of 763 (18.5%) environmental samples from 21 of 52 (40.4%) factory environments, with
L. monocytogenes, L. seeligeri, and L. ivanovii being identified in 132 (93.6%), 8 (5.7%), and 1 (0.7%)
of these Listeria-positive samples, respectively. More important, L. monocytogenes was present in all
but one of the Listeria-positive factories. As expected from other surveys conducted in the United
States and Western Europe, factory sites most frequently contaminated with listeriae once again
included drains and floors in coolers, surfaces of manufacturing and packaging equipment, and
conveyors. Even though strict cleaning and sanitizing programs were implemented at many of these
facilities, Listeria spp. were very difficult to eliminate from the working environment, with these
organisms being continuously isolated from one factory over a period of 5 months.
TABLE 17.26
Three-Year Study in 12 Australian Dairy Processing Plants to Determine Environmental
Diversity and Identify Major Environmental Niches of L. monocytogenes
Number of Number of Listeria spp. L. monocytogenes L. innocua L. grayi Mixed
Factory Type Factories Samples (%) (%) (%) (%) (%)
Cheese 7 319 34 (11) 26 (8) 9 (3) 2 (1) 3 (1)

Milk 2 87 51 (59) 20 (23) 42 (48) — 11 (13)
Ice cream 1 53 9 (17) 3 (6) 7 (13) — 1 (20)
Mixed produce 2 106 22 (22) 19 (18) 8 (8) — 5 (5)
Total 12 565 116 (21) 68 (12) 66 (12) 2 (0.4) 20 (4)
Source: Adapted from Australian Dairy Authorities’ Standards Committee. 1994. Australian Manual for Control of Listeria
in the Dairy Industry, pp. 1–35.
Sutherland and Porritt [145] conducted a 3-year study in 12 Australian dairy processing facilities
to assess the environmental diversity and identify the major environmental niches for L. monocy-
togenes. A total of 565 environmental samples were collected and tested. The overall incidence of
Listeria-positive samples was 21% (Table 17.26), with approximately half of these samples (12%)
positive for L. monocytogenes.
Cheese, ice cream, and mixed-product plants all had similar incidences of L. monocytogenes and
Listeria spp. The incidence of L. monocytogenes in mixed-product factories (18%) was comparable
to the higher levels found in milk factories. Sutherland and Porritt [145] also highlighted the following
four major routes by which L. monocytogenes enters a dairy processing facility:
1. Ingredients: especially raw milk

2. Inward goods: including milk crates and crate washers, vehicles (trucks, road and rail
tankers), and wooden pallets
3. Environment: including air and internal air quality
4. Personnel: especially outside contractors and visitors
Once L. monocytogenes is inside the processing plant, these authors [145] found numerous
areas in which this organism can survive, grow, and potentially contaminate products. Conveyor
systems, drains, and floors were the most common isolation sites. Other areas of concern were
traffic flow, cooking units, and internal air quality.
Complete elimination of listeriae from dairy processing facilities may, in some instances, be nearly
impossible; however, the likelihood of producing Listeria-contaminated products can be greatly
reduced by following GMPs, which include implementation of rigorous cleaning and sanitizing
programs for equipment used at critical points during manufacture and packaging of these foods.
INCIDENCE OF LISTERIA IN HOUSEHOLD KITCHENS

Thus far, this chapter has dealt exclusively with Listeria contamination in commercial food pro-
cessing facilities; however, because of the relatively high incidence of Listeria spp. (including L.
monocytogenes), salmonellae, and other foodborne pathogens in fresh beef, pork, lamb, and poultry
available to the general public at butcher shops and supermarkets, safe home preparation of these
foods must be reemphasized. In 1989, Cox et al. [46] isolated nine strains of listeriae from 7 of
35 (20%) household kitchens surveyed in The Netherlands. Overall, L. monocytogenes was recov-
ered from four dishcloths and one refrigerator, with two dishcloths and two dustbins from two
other households yielding L. innocua and L. welshimeri, respectively. Considering results from
commercial food processing facilities, one might expect to recover Listeria spp. from household
kitchen areas such as drains, U-tubes, and drain boards. If this is true, then garbage disposal systems
could conceivably lead to problems resulting from production of aerosols. Further work is needed
to clarify the public health significance of listeriae in the kitchen environment; however, you may
recall from the previous chapter that L. monocytogenes was found in many refrigerated foods
belonging to an Oklahoma woman who contracted listeriosis after consuming contaminated turkey
frankfurters that were eventually recalled nationwide.
Centers for Disease Control and Prevention (CDC) officials also isolated L. monocytogenes
from 15 of 25 (60%) refrigerators that were used by apparent victims of foodborne listeriosis [21].
Hence, consumers should regularly clean and sanitize kitchen areas, sinks, and refrigerators. Such
efforts can help prevent potential problems involving listeriosis and other forms of foodborne illness
in the home.
INDUSTRY-SPECIFIC EQUIPMENT, PROCESSING

METHODS, AND PRODUCTS
The discovery of Listeria spp., including L. monocytogenes, in various fermented and unfermented
dairy products, raw and RTE meats, poultry products, seafoods, and vegetables has prompted food
manufacturers to renew their concern for factory hygiene and product safety. Failsafe procedures
for production of Listeria-free foods largely do not yet exist; however, specific guidelines have
been developed for controlling listeriae and other microbial contaminants within American
dairy [4,16,35,76,100,129,144,], meat [1,3,136], poultry [18,136], and seafood [66,72,157] pro-
cessing facilities, with Denmark [19], England [89,101,126], France [17], and Australia [24] also
addressing the elimination of listeriae from fluid milk and cheese operations during all facets of
production, distribution, and retail sale.
In response to the discovery of L. monocytogenes in RTE foods and delicatessen products,
European public health officials have expressed particular concern about contamination of these
products during retail slicing and storage. They have also warned grocery store managers to give
particular attention to storage temperatures for refrigerated foods in display cases and the potential
sale of products beyond their normal code dates. Most of these guidelines stress the need to
(1) decrease the possibility that raw products will contain listeriae, (2) minimize environmental
contamination in food processing facilities, and (3) use processing methods that will eliminate
listeriae from food. Following these proposed guidelines, which will be discussed in detail shortly,
will decrease the possibility of producing foods contaminated with L. monocytogenes and other
foodborne pathogens. In addition, diligent attention to cleaning and sanitation and overall GMPs
will lead to lower microbial populations in processed foods, which will, in turn, increase the shelf
life of the finished product.
Any approach to controlling the spread of listeriae and other microorganisms in food processing
facilities is complicated by the enormous variety of foods being processed today along with the
variable quality of incoming raw products, design of the factory, sanitary design of the processing
and packaging equipment, and processing methods. However, this subject can be simplified by first
focusing on problem areas such as factory design, general factory environment, heating and air
conditioning systems, traffic patterns, and personnel cleanliness that are common to all food
processing facilities described earlier in this chapter. Once Listeria control measures for these
problem areas are understood, attention can be given to specific processing steps which are unique
to the dairy, meat, poultry, seafood, and vegetable industries.
It is now appropriate to briefly examine some of the industry-specific equipment and pro-
cessing methods, many of which have been cited as critical control points for the production of
Listeria-free dairy, meat, poultry, seafood, and vegetable products. This information will be useful
to enhance the effectiveness of preexisting cleaning and sanitation programs; however, the reader
is reminded that food processing facilities, even though they manufacture similar products, are
all unique in terms of factory design, raw product quality, product flow, handling, and processing
methods. Therefore, no universally acceptable cleaning and sanitation program can be developed
for the safe production of a given product.
DAIRY INDUSTRY
Farm Environment
Listeriae are widespread in the environment. Therefore, any quality control program should first
contain a plan to minimize contamination of raw milk with Listeria and other microorganisms on
the dairy farm. Along with good animal husbandry practices, including the use of only high-quality
feed and silage, farm workers should also give attention to cleanliness of the milk house and milking
equipment. Most important, teats and udders of all cows should be properly sanitized and dried
before milking equipment is attached. Bulk tanks in which raw milk is stored also need to be
properly maintained and inspected regularly.
Clarifiers and Separators
All raw milk should be filtered and subsequently clarified and separated by centrifugation to remove
extraneous matter and somatic cells (i.e., leukocytes) before pasteurization. As L. monocytogenes
is sometimes found in leukocytes, clarifiers and separators should be well isolated from the
pasteurizer and all finished product areas of the factory. Sealed containers should be used to dispose
of all clarifier and separator waste, both of which may contain high levels of listeriae. Special care
should also be taken to clean and sanitize separators, clarifiers, and surrounding areas.
Pasteurization
Proper pasteurization using a vat or high-temperature short-time (HTST) pasteurizer is the only
commercially practical means by which all non-spore-forming pathogens, including L. monocytogenes,
can be inactivated in raw milk. Thus, it is imperative that all pasteurization equipment be designed,
installed, maintained, and operated properly.
Continuous-flow HTST pasteurization is used to process virtually all fluid milk and ice cream
mix; however, vat (or batch) pasteurization is employed by many smaller firms, particularly those
involved in cheese making, when the volume of incoming raw milk is too small to justify the use
of a continuous-flow HTST system. If vat pasteurization is used, raw milk must be heated to a
minimum of 62.8°C (145°F) and then maintained at that temperature for at least 30 min. In theory,
vat pasteurization is a relatively simple process, with raw milk being pumped into a steam- or hot-
water-jacketed vat and held for the prescribed time. However, FDA inspections conducted as part
of the Dairy Initiative Program mentioned earlier have uncovered numerous problems with vat
pasteurizers, including improper equipment design, the absence of proper outlet valves and air-
space thermometers, and improperly operated air-space heaters. The latter problem is particularly
critical, because the air-space temperature above the product in the vat must be at least 2.8°C (5°F)
higher than that of the product at all times to ensure proper pasteurization. Operators of such
pasteurizers should be made accountable for proper performance, as well as proper cleaning and
sanitizing of the equipment. In addition, recording charts showing time and temperature relation-
ships along with other data for each vat of product pasteurized should be kept for at least 3 months.
As mentioned earlier, continuous-flow HTST pasteurization at 71.7°C (161°F) for a minimum
of 15 sec is the principal method for processing raw milk. An in-depth discussion of the many
intricate problems associated with HTST-pasteurization equipment is beyond the scope of this book;
however, a basic knowledge of HTST pasteurization is essential to appreciate the seriousness of
some of the recently identified problems that have been linked to faulty maintenance and operation
of the equipment. Interested readers may consult the “HTST Pasteurizer Operation Manual” [8]
for more detailed information on HTST pasteurization.
All HTST pasteurizers consist of five basic components, as shown in Figure 17.40: (1) plate
heat exchanger—a series of thin, gasketed stainless-steel plates divided into three sections (heater,
regenerator, and cooler) for heating incoming raw milk and cooling outgoing pasteurized milk;
(2) constant level tank—provides a constant level of raw milk to the HTST system; (3) timing
pump—a positive displacement pump that establishes the holding time and temperature relationship
for pasteurization; (4) holding tube—a length of pipe in which fully heated milk is held for the
required holding time; and (5) flow diversion valve—a three-way valve that will allow properly
pasteurized milk to enter the regenerator section of the plate heat exchanger or divert improperly
pasteurized milk to the constant level tank for repasteurization. In addition to these five components,
a source of steam or hot water is required to heat incoming raw milk, a safety thermal limit recorder
is needed to activate the flow diversion valve in the event of improper pasteurization, and a cold
milk recorder is required to record the temperature of outgoing pasteurized milk. Finally, auxiliary
components that may be added to HTST units for additional processing of milk or milk products
include a booster pump, homogenizer as a timing pump, stuffing pump, and flavor treatment or
vacuum units.
Inspections of HTST pasteurizers conducted in conjunction with the FDA Dairy Initiative
Program uncovered numerous problems relating to proper installation and maintenance of these
units. Problems most commonly associated with HTST pasteurization equipment have included
stress cracks and pinholes in the heat-exchanger plates, leaking gaskets, improper flow diversion
valves, and inadequate cleaning and sanitizing of the pasteurization unit. Positive pressure should
be maintained between the product and heating medium, as well as the product and cooling medium
(sweetwater), to prevent Listeria-contaminated raw milk or sweetwater from mixing with the
pasteurized product in the event that some of the heat-exchanger plates contain stress cracks or
pinholes. Operators should examine all pasteurization plates for defects every 6 months using the
standard dye test. Sweetwater and glycol solutions should also be routinely examined for microbial
contaminants, as these coolants may harbor L. monocytogenes, Yersinia enterocolitica, and Salmonella
typhimurium for extended periods along with large populations of psychrotrophs [16,124]; the latter
Raw Milk
Pasteurized Milk Diversion Line
Flow-Diversion Valve
Cold Past.
Milk Out Holding Tube
Cooler Regenerator Heater

Timing Pump
Raw Milk In
Homogenizer
Raw Milk Constant

Level Tank
FIGURE 17.40 Schematic diagram of milk flow through an HTST pasteurizer. (Adapted from Anonymous.
1987. HTST Pasteurizer Operation Manual. Oregon Association of Milk, Food and Environmental Sanitarians,
Oregon State University, Corvallis, OR.)
are particularly detrimental to product shelf life. Operators of HTST pasteurizers must be respon-
sible for proper operation of these units and retain accurate records and chart recordings for each
lot of pasteurized product for at least 3 months. The inability of L. monocytogenes to survive the
minimum allowable HTST heat treatment given to commercially available raw milk (71.7°C/15
sec) is now generally accepted; however, most fluid milk processors in the United States are
pasteurizing milk at 76.7°C for 20 sec, which is well above the minimum requirement established
in the Pasteurized Milk Ordinance. This more severe heat treatment markedly extends the shelf life
of the finished product by inactivating larger numbers of spoilage organisms than minimal HTST
pasteurization. However, the psychrotrophic nature of L. monocytogenes increases the need to
prevent introduction of listeriae into the product after pasteurization.
Pipeline and Cross-Connections
Many large dairy processors have installed up to several miles of pipeline in the factory to handle
movement of raw milk from storage tanks to the pasteurizer and pasteurized milk from the
pasteurizer to various holding tanks, mixing tanks, and product areas located throughout the factory.
Considering the enormous quantities of product (sometimes in excess of 1 million lb) that can be
manufactured at such facilities during one production period, careful attention must be given to
each stage of manufacture, because an error made during these operations could adversely affect
thousands of people, as was true for the 1985 outbreak of milkborne salmonellosis in Chicago.
FDA inspections have uncovered numerous violations related to pipelines, including cross-
connections between raw and pasteurized milk lines and storage and holding tanks, as well as
cross-connections between CIP and product lines and other potentially hazardous circuits. Many
of these lines allow easy bypass of raw product around the pasteurizer, thus permitting postpas-
teurization contamination in the event of equipment failure or operator error. Therefore, factory
managers, engineers, or other qualified people need to walk through the factory and construct an
up-to-date detailed blueprint of raw and pasteurized product flow throughout the entire factory.
Once the blueprint is constructed, any unwanted piping, dead ends, illegal cross-connections, or
unauthorized changes made to initial installations should be promptly identified and eliminated.
Most important, all pasteurized product lines need to be separated from raw and CIP lines by a
physical break. In many plants, pipes are physically labeled with the type of product (raw or
pasteurized) that flows through them. Blueprints must be routinely updated and reviewed for
accuracy to be of continued use by “walking” the blueprints through the factory. Finally, no piping
changes should ever be made without prior review by qualified authorities.
Filling and Packaging
Postpasteurization contamination frequently occurs during filling and packaging operations when
products are exposed to difficult-to-clean surfaces on equipment, the manufacturing environment,
and airborne contaminants [92]. Areas associated with product contamination have included man-
drels, drip shields, bottom and top breakers, prefilling coding equipment, deflector bars, and cutting
blades, as well as overhead shielding, conveyors, conveyor belts, chain rollers, supports, and
lubricants. Product extruder heads are particularly prone to contamination and therefore should be
sanitized frequently during filling operations. Such practices will lead to the production of safe
products with markedly increased shelf lives.
Aerosols provide another ready means for disseminating listeriae and other microbial contam-
inants throughout critical areas of food processing facilities [93], with L. monocytogenes surviving
3.42 h in experimentally produced aerosols of reconstituted skim milk [142]. Therefore, high-
pressure sprays should never be used in processing and packaging areas for cleaning floors or
drains, because both are major sources of listeriae and other microbial contaminants and resulting
aerosols can contaminate food contact surfaces of equipment. Operation of unshielded centrifugal
pumps in such areas is also discouraged.
Reclaimed and Reworked Product
Salvage programs, by their nature, are high-risk operations that can put an entire company in
jeopardy if not done in a sanitary manner. Potential hazards associated with such salvage operations
include (1) failure to repasteurize a returned product before reuse; (2) inadvertently pumping
returned but not repasteurized product through pasteurized product lines without proper cleaning
and sanitizing between use; (3) accidental reuse of outdated product; (4) reuse of product returned
from retail stores that may have been temperature abused, tampered with, or exposed to chemical
or biological contamination; and (5) the use of product from contaminated, leaking, or otherwise
damaged containers. Therefore, any product that left the possession and control of the processor
or has been mishandled, inadequately protected from contamination, or exposed to temperatures
of >7.2°C (45°F) should be discarded. Dairy processors should also seriously consider confining
the use of reclaimed and repasteurized milk to dairy products prepared from non–Grade A milk.
The Pasteurized Milk Ordinance indicates that American dairies involved in reclaiming programs
must now have separate areas or rooms isolated from Grade A milk operations for receiving,
handling, and storing all returned products. Outdated products and those which have left the control
of the processor and are later returned to the dairy for disposal should never reenter the factory.
Given the isolation of L. monocytogenes and other microbial contaminants from the external surface
of cartons containing returned product, along with the proven ability of L. monocytogenes and
Salmonella spp. to survive up to 14 days on the external surface of both waxed cardboard and
plastic-type milk containers [143], the process of opening containers and emptying reclaimed
product into vats for reprocessing will likely introduce many new unwanted microbial contaminants
into the factory environment. Therefore, it is imperative that all returned products be handled similar
to raw milk and be repasteurized, preferably using times and temperatures well above the required
minima. After reprocessing, all equipment, including tanks, pumps, and pipelines used in the
reclaiming operation should be thoroughly cleaned and sanitized. In view of the problems associated
with salvage operations, each dairy processor needs to reevaluate the advantages and disadvantages
involved in reclaiming products and then decide whether or not the monetary benefits gained by
such practices will outweigh the potential public health and other risks.
Frozen Dairy Products
Few bacterial species can grow at temperatures below 0°C, but most microorganisms, including listeriae,
can survive for long periods in frozen dairy products such as ice cream, ice cream novelties, and sherbet.
Frozen dairy products are particularly susceptible to microbial contamination during freezing and filling
operations. All barrel freezers used to make frozen dairy products should be thoroughly sanitized before
use, because hand assembly of the many intricate freezer parts is likely to introduce numerous con-
taminants. The source of air for the barrel freezer is another likely source of contamination. Hence, in
addition to maintaining positive air pressure in this area and keeping the surrounding area as clean and
sanitary as possible, all air lines connected to the barrel freezer should be equipped with dryers and
bacterial filters to prevent airborne contaminants from entering the product.
Ingredient feeders are perhaps the greatest source of contaminants in frozen dairy products.
Therefore, fruits, nuts, candy, and other ingredients that are added directly to frozen ice cream mix
need to be closely monitored for coliforms, pathogens, and other microbial contaminants. Exposure
of ingredients to the factory environment should also be minimized. Strict adherence to GMPs is
necessary during the production of molded, extruded, or dipped ice cream novelties, as many such
products have been recalled because of contamination with L. monocytogenes. Condensate in and
around hardening rooms, as well as conveyor belts, appears to be a likely source for such contaminants.
Finally, handling of product rerun exiting the freezer needs to be assessed at each factory. Rerun
product should never be added directly back to tanks containing unfrozen mix; however, frozen rerun
product can be reclaimed by blending it with fresh mix, which is then repasteurized. Any rerun that
is not reclaimed should be clearly separated from reclaimable material and properly disposed.
Fermented Dairy Products
Fortunately, the incidence of Listeria contamination in yogurt, cultured cream, cultured buttermilk,
and other fermented fluid milk products appears to be quite low, with very few recalls being issued
for these products. The species of lactic acid bacteria used in manufacturing these products, as well
as the bacteriostatic and bactericidal effects of various organic acids produced during fermentation
and the resultant lowering of pH are undoubtedly responsible for the near absence of such recalls.
However, because bacterial pathogens (including L. monocytogenes) and various spoilage organisms
may inadvertently contaminate fermented milk products during any stage of manufacture, producers
of such products need to follow GMPs and be readily aware of potential problems regarding
improper cleaning and sanitizing of equipment and the processing-plant environment, as well as
potential sources of postpasteurization contamination (e.g., filling and packaging areas) discussed
earlier in this chapter.
Production of Listeria-free cheese, particularly soft and semisoft varieties surface-ripened with
mold (e.g., Brie, Camembert) or bacteria (e.g., brick, Limburger), is difficult, because environmental
conditions required for proper cheese ripening also promote growth of L. monocytogenes and other
unwanted organisms. Swiss officials who investigated the 1987 listeriosis outbreak involving con-
sumption of Vacherin Mont d’Or soft-ripened cheese eventually isolated the epidemic strain of
L. monocytogenes from wooden shelves and cheese hoops found in over half of the caves used to
ripen the tainted cheese. Thus, the basic problem associated with soft-cheese manufacture is to
prevent postprocessing contamination by eliminating L. monocytogenes from the ripening room
and particularly the shelves on which such cheese must be ripened. Considering the ability of
L. monocytogenes to grow very rapidly both inside and on the surface of Brie, Camembert, brick,
Limburger, and other similar cheeses during ripening, manufacturers of such products should test
a portion of each lot for listeriae before releasing the product for sale.
In addition to these concerns, several studies have demonstrated that L. monocytogenes can
survive well beyond 60 days in brick, Cheddar, and other varieties of cheese that were prepared
from pasteurized milk inoculated with the pathogen. Certain cheeses, primarily hard and semihard
varieties, can be manufactured from raw milk in the United States and elsewhere if the finished
product is aged a minimum of 60 days at or above 1.7°C (35°F) to eliminate pathogenic microor-
ganisms. However, since experimental evidence has indicated that this process is inadequate to free
contaminated cheese from viable cells of L. monocytogenes, cheesemakers should consider prepar-
ing cheese from pasteurized milk whenever possible.
MEAT INDUSTRY
Because Listeria spp., including L. monocytogenes, are virtually endemic to slaughterhouse environ-
ments, meat processors are faced with an almost impossible challenge of producing Listeria-free raw
meats. Direct application of lactic or acetic acid to animal carcasses is one of the few economically
feasible means by which meat processors can effectively reduce populations of listeriae and other
surface contaminants, including common spoilage organisms [15,22,110,116]. Nevertheless,
although adopting this procedure and following the general guidelines for controlling listeriae in food
processing establishments will benefit slaughterhouse operators, it appears unlikely that rigid enforce-
ment of even the most stringent slaughter, dressing, cleaning, and sanitizing procedures will completely
eliminate L. monocytogenes from wholesale and retail cuts of raw beef, pork, and lamb. Therefore,
consumers of such products need to understand the potential health hazards associated with consump-
tion of less-than-thoroughly cooked meats and must also follow appropriate hygienic practices in the
kitchen to prevent the spread of listeriae from raw meats to RTE foods.
Firms producing processed meat products must assume that all incoming raw meat is poten-
tially contaminated with listeriae, including L. monocytogenes. Because most Listeria contami-
nation of finished product appears to result from postprocessing contamination rather than from
the organism surviving various processing treatments, it is essential to segregate raw and finished
products, as well as employees working in raw and finished-product areas of the factory. There
is no “magic bullet” for Listeria control; however, the incidence of listeriae in all areas of the
factory can be greatly reduced through conscientious enforcement of a stringent cleaning and
sanitation program. One six-step program that has been recommended for cleaning food contact
surfaces [7] includes (1) an initial dry cleanup step to remove as much product residue as possible,
followed by (2) a warm water rinse (with minimum splashing) to mobilize fat and remove product,
(3) cleaning with an appropriate foaming detergent, (4) warm or hot water rinse with minimum
splashing, (5) disinfecting with an appropriate sanitizing agent (i.e., chlorine or quaternary ammo-
nium compound), and finally (6) thorough drying of the cleaned and sanitized area (see also the
section entitled “Control of Food Contamination Through Effective Cleaning and Sanitation” in
this chapter). According to Boyle et al. [33,34], L. monocytogenes populations in inoculated
samples of carcass-rinse fluid, Hobart meat-grinder-rinse fluid, and floor-drain waste water
obtained from a beef and lamb processing facility increased one to four orders of magnitude
during 24 h of incubation at 8 and 35°C, with the pathogen exhibiting shorter generation times
in waste fluids containing 3.1 rather than <1.4% protein. The procedure just described may seem
adequate; however, routine random testing for Listeria and coliforms, as well as an estimation
of the general microbial load on cleaned and sanitized food contact surfaces, should be done as
an integral part of any sanitation program.
In 1987, the American Meat Institute published some interim guidelines for controlling the
incidence of listeriae and other pathogenic and nonpathogenic microbial contaminants during
production of RTE meat products [3]. Since then, others have published similar guidelines
[150,151]. The recommendations in these reports regarding facility requirements, factory environ-
ment, food contact and non-food-contact surfaces, cross-contamination, airborne contamination,
condensation control, cleaning and sanitizing, traffic patterns, and personnel cleanliness are gen-
erally similar to those already presented in this chapter as general guidelines; however, this report
also outlined some of the critical operations associated with production of specific categories of
RTE meat products.
Roast Beef, Corned Beef, and Other Rebagged Products
Products such as roast beef and corned beef that are repackaged after cooking are particularly prone
to contamination with listeriae and other microorganisms. Therefore, attention must be given to
proper sanitation and prevention of cross-contamination when these products are removed from
bags in which they were cooked. The outside surface of all bags should be thoroughly washed and
sanitized before the bags are opened. In addition to a sanitary working environment, repackaging
of cooked product requires use of clean clothing, as well as frequently sanitized utensils and gloves.
Trimming and cutting of cooked product just before rebagging are two additional critical steps
where listeriae and other contaminants can enter and compromise the integrity of the final product.
Therefore, contact between cooked product and unsanitized surfaces must be avoided during
rebagging operations. As repackaging is by nature a wet process, this operation also needs to be
well isolated from other processing areas to reduce cross-contamination.
Frankfurters and Other Link Products
Sausages such as frankfurters and other link varieties are typically prepared from a finely ground
mixture (or emulsion) of beef or pork, which is stuffed into artificial or natural casings. After
twisting the casing at approximately 6-in. intervals, the links are cooked using steam or hot water
and then hung for smoking. To obtain skinless frankfurters, the artificial casing must be mechan-
ically peeled from the congealed meat mixture. Prompt attention to cleaning, sanitizing, and cross-
contamination problems is required during all stages of frankfurter production; however, the product
is particularly vulnerable to contamination with listeriae and other microorganisms during the
peeling process. It is imperative to keep the area around peeling machines as dry and free from
meat scraps and juices as possible. Peeling machine operators also need to change protective
garments and gloves frequently. Hoods on peeler machines have been cited as a source of listeriae
and should therefore be eliminated, if possible.
Manufacturing practices should also be reviewed to ensure that losses from floor contamination
and reworked product are minimized. Unpeeled frankfurters that touch the floor or other unclean
surfaces can be reworked (i.e., appropriately washed and peeled after all other frankfurters have
been peeled). However, any peeled frankfurters that come in contact with the floor or other unclean
surfaces should be destroyed. This latter recommendation is supported by data indicating that
L. monocytogenes is difficult to destroy on the surface of frankfurters during cooking without
making the product organoleptically unacceptable [13]. In addition to these concerns, brine chillers
have also been cited as a potential source of listeriae, thus leading to contamination of casings and
product surfaces. Some studies have suggested that reformulation of frankfurters with various
antimicrobial agents or immersion in organic acid solutions (i.e., in brine) may be an effective way
to reduce Listeria contamination and prevent growth [26]. Finally, all packaging and heat-shrinking
equipment should be cleaned and sanitized daily to avoid spreading contaminants from steam and
water to packaging lines.
Luncheon Meats
Concerns regarding control of listeriae and other contaminants in luncheon meats are generally similar
to those just discussed for frankfurters and other link products. However, in addition, slicing equipment
should be kept dry and free of scraps and juices that may serve as potential nutrients for microbial
contaminants, including listeriae. A variety of technologies have been studied to reduce or control
listeriae on RTE meats, including chemical-antimicrobial treatments, irradiation (not yet approved for
RTE meats), and thermal processes such as pre- and postpackage thermal treatments [71].
POULTRY INDUSTRY
Potential sources of listeriae contamination during processing of raw poultry are in many ways
similar to those just discussed for the meat industry. Since a substantial percentage of birds harbor
Listeria spp. (including L. monocytogenes) and Salmonella in their intestinal tract, enforcement of
proper cleanup (i.e., elimination of water, condensate, and waste) and cleaning and sanitizing
programs will likely decrease the incidence of contamination but will never completely eliminate
these pathogens from raw poultry processing facilities or the raw product.
Most modern poultry processing facilities are continuous line operations in which incoming
birds are shackled, electrically stunned, bled, scalded to facilitate feather removal, plucked of
feathers, eviscerated, inspected, washed, chilled, dried, and packaged for sale. Processing steps
during which L. monocytogenes, Salmonella spp., and other pathogens are most likely to contam-
inate the product include scalding, defeathering, evisceration, and chilling [120,121]. USDA offi-
cials have proposed processing changes that may be helpful in decreasing the incidence of Salmo-
nella (and presumably Listeria) in raw poultry [18]. These changes included (1) segregating and
processing pathogen-infected flocks at different times from noninfected flocks; (2) examining the
potential benefits of adding bactericidal concentrations of organic acids or other approved antimi-
crobials to chill water tanks; (3) experimentation with different scalding methods (e.g., hot-water
sprays, steam scalders, or scald additives); (4) routine sanitizing of all equipment and utensils with
hot water or bactericidal agents; (5) reemphasis of employee hygiene programs with routine hand
washing and sanitizing required by all evisceration line workers; (6) elimination of offline process-
ing; and (7) installation of equipment designed automatically to transfer carcasses from the picking
line to the evisceration line. Additional work is needed to streamline further processing of poultry
carcasses and minimize cross-contamination during their processing.
Increasing consumption of poultry both in and outside the home mandates that persons preparing
these products must take special precautions to prevent the spread of L. monocytogenes, Salmonella
spp., and other foodborne pathogens from raw poultry to other products (e.g., fruits and vegetables)
that are frequently consumed without heating. The common practice of washing and rinsing raw
poultry before cooking has been questioned, because this step fails to markedly reduce microbial
populations on poultry skin and also leads to increased contamination of kitchen sinks, faucets,
and other food preparation areas [159]. As all foodborne pathogens commonly associated with
raw poultry (including L. monocytogenes) are readily susceptible to heat, thorough cooking
appears to be the best means of ensuring that such products are free of hazardous microorganisms.
An Oklahoma breast cancer patient contracted listeriosis in December 1988 after consuming
Listeria-contaminated turkey frankfurters. Thus, producers of processed poultry products (e.g., turkey
and chicken frankfurters and rolls) need to take precautions similar to those previously described for
the manufacture of roast beef, corned beef, frankfurters, link sausage, and luncheon meats with special
attention being given to the cleanliness of rebagging operations and sausage peelers.
EGG INDUSTRY
As stated earlier, the contents of intact whole eggs are normally sterile unless the laying hen infects
the yolk with Salmonella enteritidis. Foodborne pathogens, including L. monocytogenes and
S. enteritidis, have frequently been isolated from commercially broken, raw liquid whole egg, with
contamination most likely coming from the manufacturing environment or the eggshells. Pasteur-
ization as required for commercially broken, raw liquid whole egg is likely sufficient to eliminate
normally encountered populations of L. monocytogenes and salmonellae in raw liquid egg; however,
all egg breaking operations need to be well isolated from pasteurization and filling and packaging
areas to minimize recontamination of the finished product. L. monocytogenes and other foodborne
pathogens probably enter egg processing facilities as eggshell contaminants; therefore, egg receiving
and washing sections of the factory also should be segregated from other processing areas. Con-
sidering the potential for postpasteurization contamination, many of the previously described
guidelines for cleaning and sanitizing dairy factories also appear to be applicable to manufacturers
of pasteurized liquid egg products.
FISH AND SEAFOOD INDUSTRY

L. monocytogenes and other foodborne pathogens such as Vibrio, Salmonella, Shigella, Staphylo-
coccus aureus, Clostridium botulinum, Aeromonas hydrophila, and certain strains of Escherichia
coli have been isolated from raw or cooked finfish, shrimp, crab, lobster, oysters, and scallops. An
integrated approach to product safety is needed to minimize contamination of seafood from harvest
to the time of consumption.
The FDA adopted final regulations to ensure the safe and sanitary processing of fish and fishery
products on December 18, 1997 [154]. These regulations mandate the application of HACCP
principles to the processing of seafood. Seafood processors and importers need to evaluate the
kinds of hazards that could affect their products, institute controls to keep these hazards from
occurring, or significantly minimize their occurrence, monitor the performance of those controls,
and maintain records of this monitoring as a matter of routine practice.
Limiting postharvest contamination of freshly caught fish and seafood is the first step toward
producing a safe, high-quality end product. Adherence to good sanitation and hygienic practices
aboard fishing vessels is imperative. Contact between freshly caught seafood and waterfowl,
such as pelicans and seagulls, should be minimized because these birds are intestinal carriers
of L. monocytogenes and other foodborne pathogens. All seafood should be either frozen or
refrigerated immediately after harvest to stop or retard growth of microbial contaminants,
including spoilage organisms.
The general guidelines that were discussed previously regarding factory design, processing
environment, proper cleaning and sanitizing, employee traffic patterns, and personnel cleanliness
are also valid for the seafood industry. In addition to these recommendations, seafood processors
are also urged to: (1) eliminate processing waste, pooled water, and condensate from walls, floors,
and ceilings, as well as from processing and refrigerated areas; (2) eliminate use of high-pressure
sprays; (3) reduce airborne contamination; (4) cover outside dumpsters to decrease problems
involving seagulls and other wildlife; (5) assign specific equipment (i.e., product totes) for use in
either raw or cooked product areas of the factory; and (6) if possible, replace wooden totes with
fiber totes, which can be easily cleaned and sanitized.
Listeria spp., including L. monocytogenes, have been isolated most frequently from crabmeat
and cooked and peeled shrimp, as stated earlier in this book. This observation is not surprising if
one considers how these products are processed and packaged for the consumer. Processing of
Dungeness crab generally begins by immersing and cooking either sections of or the entire crab
in boiling water for approximately 7–9 or 17–20 min, respectively. Current information indicates
that such a heat treatment is sufficient to destroy listeriae [66]; however, underprocessing may lead
to survivors. After cooking, the crab is cooled in a water bath and either “picked” immediately or
iced and refrigerated in a walk-in cooler until the meat can be hand picked from the shell. Extensive
handling of the product by workers during picking, subsequent inspection, and packaging affords
many opportunities for postprocessing contamination. Lactic acid dips appear to be somewhat
useful in reducing populations of L. monocytogenes and other microorganisms on the surface of
crabmeat and fresh and frozen shrimp; however, such treatments will not completely eliminate
listeriae from the finished product [66]. Therefore, strict adherence to GMPs, which include proper
employee hygiene and cleaning and sanitizing of picking equipment, must be observed in and
among picking areas to avoid negating the benefits of cooking.
Unfortunately, crab processing varies widely with the species of crab—Dungeness, blue, stone,
king, and golden crab. Hence, some of the critical control points discussed for Dungeness crab are
not applicable to other species. For example, blue crabmeat is typically removed from the animal
in the raw state, placed in sealed containers, and then pasteurized (85°C/1 min) to eliminate
L. monocytogenes and other microbial pathogens. As pasteurization of blue crabmeat becomes a
critical control point in processing, it may be prudent to certify or license crabmeat pasteurization
operators or their supervisors, as has been required for operators of retorts in the canning industry
for many years.
Problems regarding postprocessing contamination are also encountered during production of
cooked and peeled shrimp. After shrimp are cooked, those destined for breading are mechanically
peeled and sometimes deveined by splitting and removing the veinlike intestine. Unfortunately,
many mechanical shrimp peelers have design flaws which necessitate almost continuous movement
of the operator between both raw and cooked sides of the equipment, thus affording ample
opportunity for postprocessing contamination. Proper cleaning and sanitizing of the equipment
(particularly protective covers over flumes and gutters) and the surrounding area are essential for
producing high-quality microbiologically safe products.
Raw seafood such as crab, shrimp, lobster, clams, oysters, and the myriad of finfish currently
available to consumers will probably never be completely free of L. monocytogenes or other
foodborne pathogens, even when handled under the best possible conditions. Considering that many
individuals are not “seafood smart,” processors and marketers of seafood have an obligation to
educate the general public and provide consumers with proper handling and cooking instructions.
Individuals who insist on consuming unprocessed fish (e.g., sushi) and seafood (e.g., oysters) should
also be made aware of potential health problems associated with consumption of such products.
FRUIT AND VEGETABLE INDUSTRY

There is limited information concerning the incidence of L. monocytogenes in fruits; however, it
has been widely recovered from plant material and vegetables, including cabbage, cucumbers,
mushrooms, potatoes, leafy vegetables, bean sprouts, and radishes [32]. Well-documented outbreaks
from fruits and vegetables have been limited. In addition to the 1981 Canadian listeriosis outbreak
involving coleslaw [134], one isolated case in Finland was linked to consumption of raw salted
mushrooms. Evidence suggests the implication of raw vegetables (including salads) in other out-
breaks. L. monocytogenes can persist on endive, lettuce, tomatoes, asparagus, broccoli, cauliflower,
and cabbage [31]. Historically, the scientific community and the public at large have been somewhat
less concerned about Listeria contamination in vegetables than in dairy, meat, poultry, and seafood
products; however, this may be changing as our culture moves to consumption of more raw fruits
and vegetables. Routine examination of raw vegetables for L. monocytogenes and other food-
borne pathogens is unlikely to reduce the risk of foodborne illness to any great extent. However,
because raw sheep manure was the probable source of L. monocytogenes in the Canadian coleslaw
outbreak, vegetable processors should have some assurance that incoming raw vegetables have
been grown, irrigated, fertilized, harvested, packaged, and transported to the firm using good
agricultural practices. Consumption of vegetables that will be adequately cooked before eating is
of little concern, because L. monocytogenes and other non-spore-forming pathogens are destroyed
during cooking. A wide range of produce sanitizers (e.g., chlorine, peroxyacetic acid, ozone,
chlorine dioxide) is available for commercial disinfection of fresh produce, but their effectiveness
varies and none is able to ensure complete destruction of pathogens. Thus good hygienic practices
(GHPs) and HACCP should be implemented along the entire process from production to consump-
tion of raw vegetables. Vegetable processors should consider rejecting raw vegetables that are likely
to be consumed without cooking if the grower clearly fails to demonstrate the use of good
agricultural practices [135]. Routine washing of raw vegetables in potable water is recommended
for commercial establishments and homes; however, this practice generally fails to reduce the micro-
bial load on raw vegetables by more than 10-fold. Therefore, persons handling and preparing raw
produce and salad vegetables should follow good hygienic practices during slicing, dicing, chopping,
and grating operations to prevent the spread of potentially hazardous microorganisms to other foods.
Finally, all knives, cutting boards, and other food contact surfaces should be thoroughly cleaned and
sanitized after use to inactivate organisms inadvertently introduced into the kitchen environment during
preparation of raw produce.
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18 Listeria: Risk Assessment,

Regulatory Control, and
Economic Impact
Ewen C.D. Todd
CONTENTS
Risk Analysis..................................................................................................................................768
Definitions.............................................................................................................................770
The Risk Assessment Process ..............................................................................................771
Risk Assessments on a Country or International Basis ................................................................773
Australia................................................................................................................................773
Canada ..................................................................................................................................775
China (Hong Kong) ..............................................................................................................775
France ...................................................................................................................................775
Germany ...............................................................................................................................777
The Netherlands....................................................................................................................777
Sweden..................................................................................................................................777
United States.........................................................................................................................778
International..........................................................................................................................781
Codex Alimentarius Commission.............................................................................783
Standards and Criteria....................................................................................................................790
Australia and New Zealand..................................................................................................791
North America ......................................................................................................................793
United States.............................................................................................................793
Canada ......................................................................................................................794
European Countries ..............................................................................................................796
Austria.......................................................................................................................796
Denmark ...................................................................................................................796
France .......................................................................................................................797
Germany ...................................................................................................................797
Italy ...........................................................................................................................798
The Netherlands........................................................................................................798
Slovenia ....................................................................................................................798
United Kingdom .......................................................................................................798
European Union (EU) ..........................................................................................................799
International Activities .........................................................................................................800
Costs .....................................................................................................................................801
References ......................................................................................................................................807
767
RISK ANALYSIS
The significant increase in listeriosis, particularly outbreaks from contamination of ready-to-eat
(RTE) processed food, over the last 20 years has indicated the need for better control using more
effective risk management methods. Currently, there is no international agreement on the acceptable
levels for Listeria monocytogenes in foods, and the importance of harmonizing microbiological
criteria for L. monocytogenes in foods for trade at the international level based on principles of
risk assessment is seen as critical. Several countries have begun developing risk-based approaches
to manage L. monocytogenes better, along with initiatives at the international level, supported by
these countries. The establishment of the World Trade Organization (WTO) in 1995 and the related
Sanitary and Phytosanitary Agreement (SPS) between member countries have given new impetus
to the Codex Alimentarius Commission (CAC) to develop international standards for all foodborne
hazards. The primary purpose of the Joint FAO/WHO Food Standards Programme of the CAC is
to protect the health of consumers and ensure fair practice in the food trade. CAC formally adopts
Codex standards, guidelines, and other recommendations that have been developed by its subsidiary
bodies, such as the Codex Committee on Food Hygiene (CCFH). In addition, CAC provides
guidance to these subsidiary bodies, including that related to risk management. The work of these
committees is supported by expert advisory groups, such as the Joint FAO/WHO Expert Committee
on Food Additives (JECFA) and the Joint FAO/WHO Meeting on Pesticide Residues (JMPR), as
well as other expert bodies, such as the International Commission on Microbiological Specifications
for Food [68]. However, no specific advisory committee had been established for microbiological
issues until the Joint FAO/WHO Expert Meeting on Microbiological Risk Assessment (JEMRA)
was formed in 2000.
Food safety risk analysis is an emerging discipline, particularly for microbial hazards, and
the methodological basis for assessing and managing risks associated with food hazards is still
developing. Risk assessment is one method by which science can be used to address food
safety, but this is only the first of several key steps in managing hazards in foods. Risk
assessment is a part of risk analysis, which includes two other components: risk management
and risk communication [33] (Figure 18.1). However, the managers decide when and how a
risk assessment is to be done, and they use the risk characterization results from the risk
assessment process to strategize the best management options. Most government agencies try
and keep the risk assessment process separate from the management, but frequent consultation
is necessary. In addition to data arising from risk characterization, risk management also
includes recommendations for Good Manufacturing Practices (GMPs) and Good Hygienic
Practices (GHPs), which may result in criteria for microbial hazards in food. An overall
framework is needed to help managers develop a consistent approach that will lead to all
relevant inputs and uncertainties, and appropriate decisions. Regulatory authorities need to
better balance risks and benefits across a society in an acceptable way, and to make the decision-
making process more explicit by being more open and transparent. The FAO/WHO approach
is to have four steps: risk evaluation, option assessment, option implementation, and monitoring
and review [33]. Another such framework prepared by the Presidential/Congressional Commis-
sion on environmental health risks in the United States [26] consists of a series of six inter-
connected and interrelated steps. These are listed as follows, with issues to be considered in
each step:
1. Defining the problem and putting it in context:

a. Characterization of the problem or a potential problem caused by microorganisms or
their toxins.
b. Identification of the appropriate risk managers with the authority and involvement of
stakeholders.
Listeria: Risk Assessment, Regulatory Control, and Economic Impact 769
Risk Assessment Risk Management

(science based) (policy based)
• Hazard Identification • Risk Evaluation
• Hazard Characterization • Option Assessment
• Exposure Assessment • Option Implementation
• Risk Characterization • Monitoring and Review
Risk Communication
(Interactive exchange of
information and opinions
concerning risk)
FIGURE 18.1 Example of a schematic diagram of the interactions between risk assessment, risk management,
and risk communication. (From FAO. 1997 Risk management and food safety—FAO Food and Nutrition
Paper 65, Report of a Joint FAO/WHO Consultation, Rome, Italy, January 27–31. http://www.fao.org/docrep/
W4982E/w4982e00.htm. Accessed August 7, 2006.)
2. Analyzing the risks associated with the problem in context:

a. Risk assessment with hazard identification, dose response assessment, exposure
assessment, and risk characterization.
b. Risk perceptions and local or regional information, if the problem is specific to one
area, need to be considered.
c. This process should generate enough information for managers to make decisions.
3. Examining options for addressing the risks:
a. Considering regulatory or other options for managing the risk.
b. Exploring the feasibility of carrying out these options.
c. Analyses for cost effectiveness and cost distributions among the affected parties.
d. Determination of any possible new risks that might arise from the proposed manage-
ment actions.
4. Making decisions about which options to implement:
a. Decisions are based on the best scientific, economic, and other technical information.
b. Actions take into account all sources of the hazard and impacts of the problem to be
solved.
c. Management options are chosen that are feasible and have benefits that are reasonably
related to costs.
d. A prioritization of options preventing or reducing risks, not just controlling them.
e. Alternatives to the command and control approach to regulation are important.
f. Political, social, legal, and cultural considerations have to be considered.

g. Incentives for innovation, evaluation, and research are long-term options.
5. Taking actions to implement the decisions:
a. Implementation is carried out by one group of managers in one agency but coordi-
nating as required with other agencies.
b. Compliance will be better if all stakeholders agree with the decision-making process
and actions taken.
6. Conducting an evaluation of the actions’ results to determine the following:
a. Whether decisions resulted in intended actions, whether cost-benefit estimates were
accurate.
b. What information gaps prevented full implementation of the actions.
c. Whether new information has emerged during the process that would be helpful in
improving the decision-making process.
d. Whether the risk management framework and policy were effective or needed to be
changed.
e. How stakeholders reacted to the process and how valuable was their input.
f. What overall lessons were learned to help other risk management decisions.
To these can be added less measurable parameters that are critical for decision makers to be
aware of in any management process, which include benefits and constraints of taking action or
not, risk perception by stakeholders, involving a variety of value judgments, and the contentious
precautionary principle in which a very conservative approach to management is adopted. Although
this management structure has not been universally adopted, it is clear that risk assessment is only
a part of the process, but a critical one. Risk communication is probably the least understood of
the three components of risk analysis because it is difficult to incorporate into a structure. Latest
WHO thinking is that risk communication encompasses both risk assessment and risk management
and is therefore broader in scope than simply relating results of the assessment and management
processes. In any new policy on Listeria, risk communication strategies will be essential to explain
what the impact and benefits will be. Even explaining what is a “zero-tolerance policy” is not an
easy task (it is certainly not zero tolerance in a total sense, but only absence, by the analytical
methods used, in a portion of the food).
To this end, the WTO encouraged the CAC to establish quantitative risk assessments regarding
pathogenic microorganisms and develop appropriate tools.
DEFINITIONS
In any risk assessment process, definitions of terms are important. The following are definitions
listed in Principles and Guidelines for the Conduct of Microbiological Risk Assessment [34].
A hazard is a biological, chemical, or physical agent in, or condition of, food with the potential
to cause an adverse health effect (harm).
In contrast, risk is a function of the probability of an adverse health effect and the severity of
that effect, consequential to hazards in food. Understanding the association between a reduction in
hazards that may be associated with a food and reduction in the risk to consumers of adverse health
effects is of particular importance in development of appropriate food safety controls [115].
Risk analysis—A process consisting of three components: risk assessment, risk management,
and risk communication.
Risk assessment—A scientifically based process consisting of the following steps: (1) hazard
identification, (2) hazard characterization, (3) exposure assessment, and (4) risk characterization.
Quantitative risk assessment—A risk assessment that provides numerical expressions of risk
and indication of the attendant uncertainties.
Qualitative risk assessment—A risk assessment based on data, which, although forming an
inadequate basis for numerical risk estimations, permits risk ranking or separation into descriptive
categories of risk when conditioned by prior expert knowledge and identification of attendant
uncertainties.
Hazard identification—The identification of biological, chemical, and physical agents that
are capable of causing adverse health effects and may be present in a particular food or group of
foods.
Hazard characterization—The qualitative and/or quantitative evaluation of the nature of
adverse health effects associated with the hazard, and this can include a dose–response assessment.
For the purpose of microbiological risk assessment (MRA) the concerns relate to microorganisms
and/or their toxins.
Dose–response assessment—The determination of the relationship between the magnitude of
exposure (dose) to a chemical, biological, or physical agent and severity or frequency of associated
adverse health effects (response).
Exposure assessment—The qualitative and/or quantitative evaluation of the likely intake of
biological, chemical, and physical agents via food as well as exposures from other sources if
relevant.
Risk characterization—The process of determining qualitative and quantitative estimation,
including attendant uncertainties, of the probability of occurrence and severity of known or potential
adverse health effects in a given population based on hazard identification, hazard characterization,
and exposure assessment.
Risk estimate—Output of risk characterization.
Risk management—The process of weighing policy alternatives in light of results from the
risk assessment and, if required, selecting and implementing appropriate control options, including
regulatory measures.
Risk communication—The interactive exchange of information and opinions concerning risk
and risk management among risk assessors, risk managers, consumers, and other interested parties.
Sensitivity analysis—A method used to examine the behavior of a model by measuring the
variation in its outputs resulting from changes to its inputs.
Transparent—Characteristics of a process in which rationale, logic of development, con-
straints, assumptions, value judgments, decisions, limitations, and uncertainties of the expressed
determination are fully and systematically stated, and documented, and are accessible for review.
Uncertainty analysis—A method used to estimate uncertainty associated with model inputs,
assumptions, and structure/form.
THE RISK ASSESSMENT PROCESS

Control of L. monocytogenes in foods provides an example of the need to consider a structured
risk management approach. Listeria is frequently consumed in small amounts by the general
population without apparent ill effects. It is believed by many that only higher levels of Listeria
have caused severe disease problems. It is also believed that Listeria is a pathogen that will always
be present in the environment. Therefore, the critical issue may be how to control its survival and
growth to minimize the potential risk, rather than how to prevent Listeria from contaminating foods.
Despite a zero-tolerance policy in a few countries, many CAC members feel that the complete
absence of Listeria in all RTE foods is unattainable, and trying to achieve this goal can limit trade
without having any appreciable benefit to public health. One risk management option, therefore,
is to focus on foods that have historically been associated with listeriosis, and foods that support
growth of Listeria to high levels, rather than those that do not. Thus, establishment of tolerable
low levels of Listeria in specific foods may be one food safety objective established by risk managers
after a rigorous and transparent risk analysis. Such an approach is now being considered by CAC
and its committees [33–35].
In its general principles of microbiological risk assessment document [34], the CAC so far has
preferred a quantitative approach to microbiological risk assessment, although a qualitative approach
may be valid depending on the manager’s needs. This is mainly because no expert committee has tackled
the various issues of when and how qualitative assessments should be considered. Not all the published
assessments adhere to the following principles and guidelines completely, partly because they are more
generic models than those used for a particular Listeria problem:
1. Microbiological risk assessment should be soundly based on science.

2. There should be a functional separation between risk assessment and risk management.
3. A microbiological risk assessment should be conducted according to a structured
approach that includes hazard identification, hazard characterization, exposure assess-
ment, and risk characterization.
4. A microbiological risk assessment should clearly state the purpose of the exercise,
including the form of risk estimate that will be the output.
5. The conduct of a microbiological risk assessment should be transparent.
6. Any constraints that impact the risk assessment, such as cost, resources, or time, should
be identified and their possible consequences described.
7. The risk estimate should contain a description of uncertainty in the estimate and where
the uncertainty arose during the risk assessment process.
8. Data should be such that uncertainty in the risk estimate can be determined; data and
data collection systems should, as far as possible, be of sufficient quality and precision
that uncertainty in the risk estimate is minimized.
9. A microbiological risk assessment should explicitly consider the dynamics of microbi-
ological growth, survival, and death in foods and the complexity of the interaction
(including sequelae) between human and agent following consumption as well as the
potential for further spread.
10. Wherever possible, risk estimates should be reassessed over time by comparison with
independent human illness data.
11. A microbiological risk assessment may need reevaluation, as new relevant information
becomes available.
Risk assessments can be used to rank or measure hazards, assess exposure, and characterize
risks involved with any food–pathogen combination, or even a multiple food or pathogen situation.
For L. monocytogenes, most risk assessments follow the four stages of hazard identification, hazard
characterization, exposure assessment, and risk characterization. The purpose of hazard identifica-
tion is to identify the types of adverse health effects that may occur from its transmission through
one or more foods. Information can be obtained from published and unpublished scientific literature,
including food industry, government, and international organization sources, and through solicita-
tion of expert opinions. Relevant information includes data in areas such as clinical studies,
epidemiological studies and surveillance, laboratory animal studies, investigations of the charac-
teristics of microorganisms, interaction between microorganisms and their environment through
the food chain from primary production up to and including consumption, and studies on analogous
microorganisms and situations.
An ideal hazard characterization should include a dose–response assessment with end points
such as infection or illness; these may use animal models and human outbreak scenarios. The
effects on different populations, particularly those at most risk, should be attempted. Factors such
as virulence, possibility of secondary transmission (less likely for L. monocytogenes), and attributes
of a food that may alter microbial pathogenicity, e.g., high fat content of a food vehicle, are
important to identify. Where data are missing, experts may be able to devise ranking systems
so that they can be used to characterize severity and/or duration of disease. For L. monocytogenes,
several different dose–response assessments have been derived, so that any risk characterization
process becomes much more uncertain. For instance, from a manager’s point of view, is one
dose–response assessment chosen, a combination for different populations at risk, or a median
value of several assessments, if such exist?
Exposure assessments estimate the level, within various levels of uncertainty, of L. monocyto-
genes in specific foods at the time of consumption, usually on a serving size or per-gram basis.
The assessment can start with data on all ingredients going into the food, or later in the process.
With Listeria there is the complication that different strains of the pathogen may enter at different
steps in the process, and it may be the rare final contamination in the processing plant that is the
most critical. Most assessments have started at the processing stage, partly because of lack of data
at the earlier steps. Ideally, assessors attempt to model changes in populations as they go through
production and processing, with some kind of measures for growth, decline, and recontamination.
Consumer practices are important since long-term storage in refrigerators can greatly impact growth
of the pathogen at home. Also, consumption patterns need to be estimated that vary by gender,
age, nationality, season, and consumer preferences. Such consumption data are usually limited with
expert opinions often used, although some national surveys can prove valuable for some foods.
Risk characterization brings together all the qualitative or quantitative information of the
previous steps to provide an estimate of risk for a given population. If there is not enough
information following the integration of quantitative and qualitative data, only a qualitative estimate
of risk may be possible. Typically, in a quantitative risk assessment, an estimate will be expressed
in risks per serving (meal size) or per population (region or country), but both of these should be
accompanied by some estimate of uncertainty.
RISK ASSESSMENTS ON A COUNTRY OR INTERNATIONAL BASIS

AUSTRALIA
The Government of New South Wales has outlined its use of risk analysis in managing food safety
risks [84]. Safefood NSW was established in 1998 and it became the NSW Food Authority in
April 2004 [85]. Safe food legislation [46] requires that a food safety scheme be based on risk
assessment to ensure that all known hazards are addressed and that appropriate resources are
allocated. Moreover, it was agreed that an assessment of food safety risks in the food industry
should be in accordance with national and international standards of risk assessment. The principles
that should guide the achievement of these objectives are that (1) protection of public health is
paramount; (2) risk assessment principles should be used to systematically identify public health
issues of concern and develop effective solutions; (3) implementing reforms should involve gov-
ernment, industry, and consumers in partnership; and (4) although efficiency is to be promoted,
costs and benefits should be taken into account [84]. Assessments have been carried out for seafood
and meat products. Sumner and Gallagher [94] prepared a semiquantitative risk assessment of ten
seafood hazard–product combinations to generate a risk estimate which was scaled from 0 to 100,
where 0 represents no risk and 100 represents all meals containing a lethal dose of the hazard. The
highest ranking (>48) was for seafood toxins (ciguatera, scombroid, and algal toxins in bivalve
mollusks) and viruses in oysters. Risks for listeriosis arising from RTE cold-smoked fish products
had a score of 39–45, depending on the population affected.
Another unpublished study was to assess the public health risk to Australian consumers caused
by L. monocytogenes in Australian-made processed meat products [90]. The project aimed to (1)
characterize the nature and size of the microbial food safety risk from Listeria in processed red meat
products relative to that of other foodborne pathogens; (2) identify areas in which critical data and/or
knowledge are lacking; (3) characterize factors that contribute most significantly to the risk; and (4)
recommend management strategies to reduce the microbial food safety risk. No specific constraints
on performance of the assessment were identified. The assessment considered processed meats in
three categories, including luncheon meats (hams, etc.), pâtés, and cooked sausages intended to be
reheated. The risk from fermented meats was demonstrated to be insignificantly small and was not
of further concern because no additional risk management actions were considered necessary. With
cooked products, it was assumed that the cooking step was completely listericidal and that contami-
nation arose after processing, which was the starting point for the risk assessment. The effects of all
storage and transport steps on concentration and prevalence of L. monocytogenes in RTE processed
meat products were considered, including consumer handling. The effect of contamination at various
steps in the factory-to-fork chain was also considered, although quantitative data to describe the
numbers of L. monocytogenes transferred were almost nonexistent. Prevalence and concentration data
were obtained from published sources, government surveys, and industry-funded studies. In addition,
total count data at the point of production were obtained for inclusion in the model to account for
interactions affecting the growth potential of L. monocytogenes during the life of the product. Large
amounts of data were obtained from producers. Serving sizes and frequencies were determined from
several government-sponsored food consumption studies. However, the product groupings in these
studies were not fully relevant to product categories described in the assessment. To estimate the
amount of each product consumed within the product groupings used in the nutrition surveys, national
sales volumes for individual products were obtained from a large supermarket chain. From these data,
the proportions of each product type in the nutrition survey groupings were calculated. Production
volumes were also estimated from published data as an additional verification that the overall con-
sumption estimates were realistic.
The demography of the Australian population was considered. The age and gender structure
of the population was determined from 0 to 6 months, and in 1-year age intervals thereafter. Surveys
also identified consumption by age and gender. The proportion of pregnant women estimated by
two methods was 1.3%. For some product categories, reduced consumption was evident in women
of childbearing age and in the elderly. The number of cancer, transplant, AIDS, and diabetes sufferers
was also determined. Relative susceptibility by age was determined from several sources of U.S.
data for the age of listeriosis victims divided by the proportion of the population in those age classes.
Susceptibility was expressed relative to adolescence, which experience the lowest relative risk. Apart
from the age and gender data, no differentiation of consumption rate and frequency for the other
at-risk groups could be determined.
Time and temperature data for transport and storage of meat products were obtained from
Australian surveys, but also compared to U.S. data. These data included retail display and domestic
refrigeration and transport. Distributions of average temperatures and times during these stages
were determined and used in subsequent modeling. Predictive microbiology models for growth rate
of L. monocytogenes, including generic and product-specific models, were collated. Where possible,
performance and applicability of the models were assessed, including accounting for lag times,
with suitable models selected for inclusion in the exposure assessment model. Interactions between
the background microbial flora and its effect on potential growth of L. monocytogenes (i.e., the so-
called Jameson effect) were also modeled.
These data and models were integrated into a process risk model that was used to assess the
effectiveness of several putative risk management options and to assess the relative contribution of
contamination at the plant compared to contamination at retail. Variability and uncertainty were identified
and their effects discussed. In addition, the Monte Carlo simulation model was run several times with
alternative assumptions in which uncertainty existed. From this method, the importance of uncertain
values was estimated. The exposure assessment was relatively complex, and was a major component of
the overall risk assessment, modeling the prevalence and concentration of L. monocytogenes from
production to consumption. The effects of temperature, time, product formulation, and competing
microorganisms in packages are all considered. Some differentiation of exposure levels among at-risk
population subgroups was possible. The dose–response model developed by FAO/WHO [37] was
incorporated into the risk assessment without change. The final risk characterization was developed but
the results only privy to the meat industry, which commissioned the study.
CANADA
Farber et al. [39] published an assessment focused on RTE foods, particularly pâté and soft cheese,
consumed in Canada. The authors recognized that L. monocytogenes may grow or be inactivated,
and from a predictive model, they calculated that levels could increase from 1 CFU to 105 CFU in
about 40 days at 4°C or 15 days at 8°C. Although it was noted that postprocess contamination of
cooked meat products probably results in higher levels of L. monocytogenes at the surface of the
product, and also higher levels in or near the rind of soft cheeses because of the higher pH, this
information was not used in the assessment. The exposure assessment was limited because there
were no quantitative links developed between the different steps in food production, retailing, and
storage by the consumer. Unlike other L. monocytogenes risk assessments, the Weibull-Gamma
model was chosen for the hazard characterization step. In this model, they used ID10 and ID90 values
(doses causing illness in 10 and 90% of the population, respectively), i.e., 107 and 109 L. monocytogenes
for normal individuals, and 105 and 107 for high-risk persons. The purpose of the assessment was
to help Canadian heath officials set policy direction, allocate resources, and determine research and
regulatory surveillance priorities.
Todd et al. [99] developed a quantitative risk assessment for L. monocytogenes in cabbage in
Canada. The objective, addressed in the extensive production module, was to show the prevalence
and concentration of L. monocytogenes in the product after harvest, processing, and packaging under
a modified atmosphere, during transportation, and retail and consumer storage. Although worldwide
data were used for prevalence and concentration estimates, the geographic range of the assessment
was limited to Canada. Distributions were used for all the prevalence, concentration, growth, and
reduction scenarios. Consumption characteristics were derived from Canadian surveys. Uncertainty
and variation were addressed by use of Monte Carlo simulations. In conclusion, this exposure
assessment was relatively complex and modeled the prevalence and concentration of L. monocytogenes
from harvest to consumption. The effects of temperature, time, disinfection, growth, pH, and atmo-
sphere in packages were all considered, along with serving volumes and sizes at home and away from
home. These were incorporated into the dose–response assessment step using both the Farber et al.
[39] Weibull-Gamma and the Buchanan et al. [13] exponential models to determine the probability
of infection and the number of illnesses in Canada for normal and susceptible (high-risk) populations.
From results of the risk characterization, the likelihood of listeriosis from consumption of shredded
cabbage in Canada was considered to be very low. For individuals in the high-risk group, the median
probability of illness was 10 −8, with 5th and 95th percentiles 2 × 10 −13 and 9 × 10 −4, giving an estimate
of one case every 5 years.
CHINA (HONG KONG)

One report of a microbiological risk assessment for salads in Hong Kong is really more of a risk profile
than a risk assessment, with several microbiological hazards and indicator organisms examined
[67]. L. monocytogenes was only occasionally found in routine surveillance studies of restaurants and
other premises (1 in 169 samples in 1999, 6 in 194 samples in 2000, and none in 210 samples in 2001).
No specific determination of risk was made, but general hygienic recommendations were given for
reducing contamination. The elderly, children, pregnant women, and persons with lowered immunity
were advised to be careful choosing salads, considered a high-risk food.
FRANCE
Bemrah et al. [11] undertook a quantitative risk assessment of L. monocytogenes from consumption
of a generic soft cheese made from unpasteurized milk. L. monocytogenes may be introduced into
raw milk either from environmental contamination or from mastitic cows. Although subsequent
contamination was recognized, this was not included in the modeling. A key aim was to use the
QRA model to simulate the consequences of alternative risk management options on the risk of
illness from consumption of raw-milk soft cheese. The authors constructed a process risk model
from milk production and storage through transport, storage, and cheese production to consumption
with data on the prevalence and concentration of L. monocytogenes at the different stages, mainly from
French sources. Time and temperature parameters were used to estimate growth in the milk, but it
was assumed that no growth would take place after production. The authors assumed a uniform
distribution of the pathogen in the cheese, although they recognized that there would likely be a
lack of homogeneity. The probability of consuming a contaminated cheese serving was estimated
to be 0.653, with concentrations in contaminated servings of 100 CFU (median) with 5 and 95
percentiles of 0 and 450 CFU. The authors noted that assessment was limited by the lack of useful
available data. The assessment approach is consistent with the CAC framework for microbial risk
assessment [115] and uses a probabilistic approach using distributions and Monte Carlo techniques
to produce the model. Environmental contamination resulted in an average of 2.25 L. monocytogenes
CFU/mL milk. In addition, individual cows’ milk contaminated at higher levels (104 CFU/mL)
could come from infected udders, yet this event occurred in only about 1 of 2000 infected cows.
Assumptions were made that (1) there was no net growth of L. monocytogenes during cheese
ripening and distribution, (2) the dose–response relationships chosen for normal and at-risk popu-
lations were those of Farber et al. [39], and (3) the per capita yearly consumption of such soft
cheeses was 50 servings of 31 g each. A Monte Carlo simulation on these data gave an annual
cumulative risk of listeriosis ranging from 1.97 × 10 −9 to 6.4 × 10 −8 in the low-risk population,
and 1.04 × 10 −6 to 7.19 × 10 −5 in a high-risk population. This is the equivalent to 74 cases of severe
listeriosis per year in a population of 50 million, with 20% of these being susceptible individuals
(mean 50) at the 99th percentile of incidence. If milk from excreting mastitic cows is not used for
cheese production, this percentile could be reduced by a factor of 3.7 (mean could be reduced by
5.2). Model simulation showed that this measure had almost no influence on the proportion of
servings with low contamination level (i.e., <10 L. monocytogenes CFU/g). Thus, the risk assess-
ment provided useful information for managing of the risk of listeriosis from raw-milk cheese.
A more specific assessment was built on the work of Bemrah et al. [11], being a quantitative
risk assessment of human listeriosis associated with consumption of Camembert from Normandy
and Brie of Meaux, both soft cheeses made from raw milk in France [91]. The estimated L. monocyto-
genes concentration in raw milk was on average 0.8 and 0.3 CFU/L, respectively, where Camembert
and Brie were produced. A Monte Carlo simulation was used to account for the time–temperature
history of the milk and cheeses from farm to table. It was assumed that viable cells would not
spread within the solid cheese matrix. Cheese rind was assumed to represent 10% of the cheese
volume, and the L. monocytogenes populations in the rind and core were modeled separately.
Interaction between pH and temperature was included in the growth model. The simulated
proportion of servings with no L. monocytogenes cells was 88% for Brie and 82% for Camembert.
The 99th percentile of L. monocytogenes cell numbers in 27-g servings was 131 for Brie and 77
for Camembert at the time of consumption, corresponding, respectively, to 3–5 L. monocytogenes
CFU/g. For the 17 million and 480 million annual servings of Brie and Camembert consumed in
France, the number of expected listeriosis cases would be ≤5.10 × 10−4 and ≤2.5 × 10−3 per year,
respectively, based on an exponential dose–response model. These low probabilities are borne out
by effective monitoring throughout the process. The authors argue that improved hygiene at the
farms has demonstrated low prevalence levels in raw milk, and heightened testing by cheese
producers has resulted in very few milk batches containing the pathogen. Cheese samples are also
tested for L. monocytogenes at the end of shelf life in the plant when cheeses are stored at 8°C.
Other sampling is done at retail. For example, in 2000, none of 850 cheeses analyzed 21 days after
milk coagulation, and none of 450 cheeses kept for 70 days at 8°C in the Brie cheese-making plant
was positive, with a detection threshold of 10 L. monocytogenes CFU/g. Similarly, no positive
samples were found in 2000 at the Camembert cheese-making plant. They also argue that the predicted
probability of severe listeriosis from the two French cheeses made from raw milk does appear lower
than the probability of listeriosis cases from soft cheeses made from pasteurized milk consumed in the
United States based on data in the FDA/FSIS/CDC [42] draft relative risk ranking.
GERMANY
Buchanan et al. [13] derived a conservative dose–response relationship for L. monocytogenes based
on data from Van Schothorst [110,111]. The exposure assessment in each instance was based on
German data from a 1991–1992 retail survey for the presence of L. monocytogenes in 25-g and
1-g samples. Consumption frequency and meal size estimates were combined with the contamina-
tion and prevalence survey data to estimate the annual number of exposures to 100-g servings of
RTE meat and smoked fish containing between 104 and 106 L. monocytogenes CFU/g and those
containing greater than 106 L. monocytogenes CFU/g. The Buchanan et al. [13] study only used
the smoked-salmon data with about 20 servings per capita per year in Germany. No attempt was made
to model the change in numbers between the time of sampling and consumption. These studies
were useful to help prepare later assessments, e.g., in Sweden.
THE NETHERLANDS
Notermans et al. [83] used mice to develop dose–response data. The oral ID50 for nonprotected
mice was 6.5 log cells compared with >9.0 log cells for immunologically protected mice with a
1–2 log difference between the ID50 and LD50. The dose for infection through intravenous injection
was lower. The results showed that both the intestinal barrier and the specific immune defense
mechanism were highly effective in preventing infection of mice orally exposed to L. monocytogenes
and support the epidemiological findings that listeriosis is a rare disease in humans, despite frequent
exposure to the organism.
SWEDEN
Gravad or hot- or cold-smoked salmon and rainbow trout are popular RTE foods in Sweden. After
processing, the fish is packaged and stored for varying times at refrigerator temperatures before
consumption without prior heat treatment. Packaged products made from both types of fish have
been identified as potential sources of human listeriosis. A quantitative risk assessment model
developed by Lindqvist and Westöö [78] showed the risk per serving and the annual cumulative
individual risk based on number of exposures and two different dose–response relationships from
the literature. Growth or inactivation was not included in the model. The constant r is specific for
each pathogen and helps define the shape of the dose–response curve. In one dose–response study,
with an exponential dose–response model, a conservative r value for L. monocytogenes was estimated
from the levels in smoked fish and epidemiological data in Germany (GR) [13]. The authors made
the assumption that foods with a concentration over 104 L. monocytogenes CFU/g cause illness
only in the high-risk population. The second dose–response model chosen was the flexible Weibull-
Gamma (WG) model suggested by Farber et al. [39], which was less conservative. Using these
relationships the estimated number of cases in the population was calculated and compared to the
annually reported number of listeriosis cases in Sweden. The estimated mean risk per serving was
2.8 × 10 −5 (GR, high-risk group), 2.0 × 10−3 (WG, low-risk group), and 1.6 × 10−2 (WG, high-risk
group), respectively. The average number of reported listeriosis cases in Sweden is 37 per year.
In comparison, the mean number of annual cases predicted by the risk assessment model was 168
(range 47 to 2800, GR, high-risk group), and 95,000 (range 3.4 × 104 to 1.6 × 10 6, WG, high-risk
group), respectively. If 1 to 10% (uniform distribution) of strains, instead of all, were considered
virulent, the mean number of predicted cases would decrease to 9 (GR) and 5200 (WG), respectively.
Even with underreporting of actual cases, this assessment shows the difficulty in estimating a
reasonable number compared to what is expected. The WG model predicted far greater risks than
the exponential model, and thus, choosing the dose–response model that best fits the data is critical.
However, that model may not yet exist. Lindqvist and Westöö [78] stated that apart from
dose–response issues, the most important parameter determining the risk was the concentration of
L. monocytogenes at the time of consumption.
UNITED STATES
Peeler and Bunning’s [88] assessment of L. monocytogenes during processing of bovine milk was
one of the earliest publications in microbial risk assessment. However, this study was more a limited
exposure assessment rather than a complete risk characterization setup to help establish a hazard
analysis and critical control point (HACCP) plan for bovine milk up to and including pasteurization.
The authors aimed to demonstrate how determination of critical control points could be more
objective using quantitative modeling methods. Survey or experimental data were used wherever
possible to model pathogen incidence, potential growth during storage and transport, and death
during pasteurization. In comparing their predictions to available data for the probability of
contamination of retail units of pasteurized milk in North America, the authors found that the
incidence of contamination was much higher than their model predicted, probably because of
contamination after pasteurization. Cassin et al. [20] considered that this assessment had overly
compounded conservatism into the model.
Hitchins [66] used survey data on the frequency of foodborne occurrence and dietary exposure
to L. monocytogenes to estimate the minimal mean per person annual rate of exposure in the
United States during the late 1980s. The mean amount of each food type per L. monocytogenes
occurrence was calculated for about 100 sources, and dietary intake data from published U.S.
literature were used to calculate the mean number of occurrences of L. monocytogenes consumption
per person per year. The mean number of occurrences of exposure to L. monocytogenes consumed
annually per person was determined to be 10 to 100, when the proportion of RTE food of the total
dietary intake was estimated at 2 to 20%, respectively.
The FDA conducted a risk ranking for L. monocytogenes in collaboration with USDA and
CDC [42]. This draft risk assessment compared the risks of illness and death arising from L.
monocytogenes in 20 RTE categories. Risk ranking assessments are useful to estimate which foods
present the greatest and the least risks per serving or in a population to help prioritize control actions.
They are most useful not only to guide regulatory activities, but also to determine data gaps, research
needs, educational activities, and predominant risk factors. The working assumption used was that
all listeriosis cases were attributed to different categories of foods, so that the risk assessment could
be “anchored” to the U.S. public health statistics. A newer version of the risk assessment incorporated
changes in response to the submitted comments and newly available data for 23 RTE food categories,
including data submitted by the National Food Processors Association (NFPA, see the following text)
[43]. This version also contained improved modeling techniques, as well as changes to the model
inputs and the use of “what if” scenarios. The assessment was based on foods at the retail level and
the overall burden of listeriosis on public health and considered three age-based groups of people
(perinatal, i.e., fetuses and newborns, elderly and intermediate age, i.e., remaining population, mostly
healthy). The risk assessment used 2078 cases of listeriosis and 390 fatalities as the baseline values
for the national annual public health impact of the disease, based on values projected from an average
of CDC’s surveillance data from 1997 to 2000. An assumption made was that there were 2460 cases
of listeriosis in the United States every year, and it was the rankings within this by food category that
were the most important outputs. Much of the exposure assessment modeled changes in contamination
levels during refrigerated storage and reheating in the home. The dose–response model was internally
designed by FDA. The results of the exposure and dose–response assessments were combined to
provide an estimate of risk on a per-serving and annual basis. Although the analysis contained
considerable variability and uncertainty, this risk assessment indicates that in spite of consumers being
regularly exposed to low levels of L. monocytogenes, relatively few people become seriously ill.
For the total U.S. population the predicted median risk per serving for foods ranged from a high
of 7.7 × 10 −8 (deli meats) to a low of 4.5 × 10−15 (aged hard cheese). The total predicted median number
of annual cases in the United States was 1598.7 for deli meats, 90.8 for pasteurized milk, 56.4 for high-
fat and other dairy products, 30.5 for frankfurters not reheated, 7.7 for soft unripened cheeses, 3.8 for
pâté and meat spreads, 3.1 for unpasteurized milk, 2.8 for cooked RTE crustaceans, 1.3 for smoked
seafood, and less than 1 for all other categories. The differential between per-serving risks associated
with deli meats (relative risk rank of 1) and hard cheeses (relative risk rank of 23) is almost 10,000,000-
fold. Values were also determined for each subpopulation (perinatal, elderly, and intermediate age).
Scenarios were also run to allow comparison of baseline calculations to new situations that might arise
as a result of potential risk reduction strategies. The predicted annual number of listeriosis cases would
be reduced from 2105 to 656 (69%) if all home refrigerators operated at 45°F, and to 28 (>98%)
annually if temperatures were at 41°F. If the storage time for deli meats was reduced from the maximum
of 28 days to 14 days, the median number of listeriosis cases in the elderly population would drop
from 228 to 197 (13.6% reduction), and a further benefit (total of 154 cases) would result if the storage
time was decreased to 10 days (32.5% reduction). Conversely, increasing the maximum storage time
for smoked seafood from 30 to 45 days more than doubled the predicted median number of listeriosis
cases for the elderly subpopulation (0.8 vs. 2.1). The “what if” scenarios also predict that inclusion of
any treatment resulting in a 1-log reduction in contamination of deli meats at retail would reduce the
number of predicted cases in the elderly population by 50%, from 227 to 120, and a 2-log treatment
would result in a 74% reduction. Data incorporated into the risk assessment largely came from com-
mercially produced Hispanic soft cheeses, whereas the cheeses linked to the disease have most often
been associated with noncommercially produced cheese made from raw milk. Consequently, when the
exposure model was constructed using contamination data representative of raw milk, the estimated
risk per serving was 43 times greater for the neonatal population and 36 times greater for the elderly
population when these cheeses were made from unpasteurized as opposed to pasteurized milk.
The highest risk categories on both per-serving and per-annum bases were deli meats and
frankfurters that have not been reheated. This is because they have relatively high rates of contam-
ination, support the relatively rapid growth of L. monocytogenes under refrigerated storage, are
stored for extended periods, and are widely consumed. These products also have been directly
linked to outbreaks of listeriosis. They should be given particular attention for development of new
control strategies and consumer education programs to help achieve the national goal for reducing
the incidence of foodborne listeriosis. Pâté and meat spreads, smoked seafood, and unpasteurized
fluid milk have relatively high rates of contamination and thus high predicted per-serving relative
risks, but are consumed in small quantities by a relatively small portion of the population. However,
all three products have been associated with outbreaks or sporadic cases, which justifies new control
measures or recommendations for avoidance.
The assessment made some general conclusions:
• The risk assessment reinforces past epidemiological conclusions that foodborne listeriosis
is a moderately rare, although severe, disease. U.S. consumers are exposed to low to
moderate levels of L. monocytogenes on a regular basis.
• The risk assessment supports the findings of epidemiological investigations of both
sporadic illness and outbreaks of listeriosis that certain foods are more likely to be
vehicles for L. monocytogenes.
• Three dose–response models were developed that relate exposure to different levels of
L. monocytogenes in three age-based subpopulations (i.e., perinatal [fetuses and new-
borns], elderly, and intermediate age) with the predicted number of fatalities. These
models were used to describe the relationship between levels of L. monocytogenes
ingested and the incidence of listeriosis. The dose of L. monocytogenes necessary to
cause listeriosis depends greatly upon the immune status of the individual, and strategies
for control and prevention need to be designed for these populations.
• The dose–response models developed for this risk assessment considered, for the first
time, the range of virulence observed among different isolates of L. monocytogenes. The
dose–response curves suggest that the relative risk of contracting listeriosis from low-
dose exposures could be less than previously estimated.
• The exposure models and the accompanying “what-if” scenarios identify five broad
factors that affect consumer exposure to L. monocytogenes at the time of food
consumption.
These five factors affecting consumer exposure to L. monocytogenes at the time of food
consumption are (1) amount and frequency of food consumption, (2) frequency of contamination
and levels of L. monocytogenes in RTE food, (3) potential of the food to support growth of L.
monocytogenes during refrigerated storage, (4) refrigerated storage temperature, and (5) duration
of refrigerated storage before consumption. Any of these factors can affect potential exposure to
L. monocytogenes from a food category. These factors are “additive” in the sense that foods in
which multiple factors favor high levels of L. monocytogenes at the time of consumption are
typically more likely to be riskier than foods in which a single factor is high. These factors also
suggest that several broad control strategies could be used to reduce the risk of foodborne listeriosis,
such as product reformulation to reduce the potential for L. monocytogenes growth or encouraging
consumers to keep refrigerator temperatures at or below 40°F and reduce refrigerated storage times.
To eliminate some of the data gaps in the FDA/FSIS/CDC [42] assessment, the NFPA conducted
a survey of over 31,000 RTE retail food samples, representing eight product categories, that showed
an overall prevalence rate of 1.82%, with these data then used to conduct a risk assessment [24,61].
The rationale for this risk assessment was to develop more effective management strategies for the
pathogen because the existing zero-tolerance policy makes no distinction between foods containing
high and low levels. The confidence interval for prevalence of L. monocytogenes was 1.68 to 1.97%,
which ranged from 2 to 6 log CFU/g, and could be fitted to a beta distribution (0.29, 2.68, 1.69, 6.1).
An exponential model with an r value (the probability of a single cell causing illness) of 1.76 × 10−10
for the population at highest risk was used for the dose–response assessment. Based on this
assessment, the NFPA (now Food Products Association) argued that management strategies focusing
on levels of L. monocytogenes rather than its presence alone should have a greater impact on
improving public health by facilitating development of control measures to limit maximum levels
of L. monocytogenes in foods.
The Food Safety and Inspection Service (FSIS) risk assessment [58] was initiated in February
2002 in response to public comments on the FSIS-proposed rule: Performance Standards for the
Production of Processed Meat and Poultry Products [57] and incorporated material from the
FDA/FSIS/CDC [42] draft risk ranking. This ranking indicated that deli meats posed the greatest
public health risk for listeriosis of all the RTE foods. Several comments indicated that the proposal
should require testing and sanitation of food contact surfaces for Listeria species, where the
relationship between the prevalence and levels of Listeria species in the plant environment and
RTE meat and poultry products is not well understood. This model was originally released for
public comment and review in January 2001. Based on review and comments, the exposure
assessment for deli meats (and hot dogs) and the dose–response relationship were updated. The
FSIS Listeria risk assessment was designed to simulate RTE food production within the processing
plant and predicts the L. monocytogenes concentrations at retail. The updated FDA/FSIS exposure
assessment for deli meats and the updated dose–response relationship were used to model the
distributions of L. monocytogenes concentrations in RTE products at retail through consumption
and estimate the subsequent annual number of deaths and illnesses. The model assumes that a
Listeria reservoir exists in the plant and is capable of contaminating the food contact surface. These
reservoirs include harborage sites, floor drains, air-conditioning ducts, etc. The model assumes that
Listeria species move from this reservoir onto the food contact surface during what is termed a
contamination event. The key parameters defining a contamination event are as follows: (1) the
time between initialization of events (i.e., how often is a food contact surface contaminated?), (2)
the duration of the event, and (3) the number of Listeria transferred from the in-plant reservoir to
various food contact surfaces. Once on the food contact surface, these organisms can be transferred
to the lot of RTE product being produced, removed through sanitation after producing each lot of
product, or remain on the surface. If the contamination event is continuous, new organisms trans-
ferred from the reservoir will be added to those already on the food contact surface. For each lot
produced, the food contact surface can also be tested for Listeria species, with various mitigation
steps taken if the surface tests positive. A positive food contact surface test can trigger a required
lot of RTE product to be tested for L. monocytogenes. It can also trigger more intensive sanitation
(i.e., enhanced sanitation) of the food contact surface at the end of production. Some fraction of
the Listeria organisms on the food contact surface is transferred to the lot. This fraction is the
transfer coefficient, which can range from 0 to 1. A transfer coefficient of 0 indicates that none of
the Listeria cells are transferred. A transfer coefficient of 1 indicates that all the organisms present
are transferred to the product lot being manufactured. Based on limited data not involving Listeria,
transfer coefficients were assumed to be log normally distributed (normally distributed on the log
scale) with a mean of –0.14 and a standard deviation of 1. The assessment data generally show
that L. monocytogenes concentrations in RTE product decline at retail as food contact surface testing
and sanitation efforts increase. The most effective strategy is postprocess intervention or control
(90–95% effective) combined with growth-inhibiting packaging or product reformulation (90–95%
effective). However, this assumes 100% adoption and compliance by industry. Contamination from
the baseline model would drop from 2.06 × 105 to 1.67 × 103 under the most effective scenario.
The baseline model was calibrated to 310 deaths per year among the elderly, 67 intermediate age
deaths per year, and 16 neonatal (newborn) deaths per year in the U.S. population. These would
drop to 59, 13, and 3.3, respectively, if the best intervention and control practices were introduced.
The main findings of the assessment were as follows:
1. Food contact surfaces found to be positive for Listeria species greatly increased the
likelihood of finding positive lots for L. monocytogenes.
2. The duration of Listeria contamination on food contact surfaces is about a week.
3. Increased frequency of testing food contact surfaces and subsequent sanitation leads to
a proportionally lower risk of listeriosis.
4. A combination of intervention procedures (e.g., sanitation, pre- and postprocessing
interventions, and use of growth inhibitors or product reformulation) appears to be more
effective than any one intervention alone in reducing L. monocytogenes contamination
and the number of illnesses and deaths.
A slightly modified draft risk assessment for deli meats was released for comment a few months
later [59] to incorporate changes in the final FDA/FSIS/CDC [43] risk assessment compared with
the draft FDA/FSIS/CDC [42] report. Transfer coefficients are being generated to improve risk
assessments such as those by FSIS and FDA [74,113], which will further improve the model. (See
Figure 18.2.)
INTERNATIONAL
The Report of the FAO Expert Consultation on the Trade Impact of Listeria in Fish Products [35]
was developed to assist in the process of assessing the risks of listeriosis from fishery products,
and also identify data gaps relating to L. monocytogenes in fish and fish products. The risk
assessment was not considered to be quantitative because of time constraints. Products having the
potential to contain and not contain high levels of L. monocytogenes at the time of consumption
were included. Growth limits and maximum growth rates under different conditions were tabulated.
A predictive mathematical model for growth under conditions representative of fish products was
FIGURE 18.2 Proposed decision tree for the establishment of L. monocytogenes criteria in foods. (From FAO.
1999. Report of the FAO Expert Consultation on the Trade Impact of Listeria in Fish Products, Amherst, USA,
May 2000. FAO Fisheries Report No. 604. (FIIU/ESNS/R604). Food and Agriculture Organization, Rome.)
mentioned with some predictions listed, but the model was not used in the exposure assessment.
In fact, many of the data discussed were not used in the assessment or referenced. Missing
information for an exposure assessment was clearly noted, especially those from tropical regions.
Their calculations suggest that per capita human exposure to L. monocytogenes doses exceeding
1000 CFU is likely to occur several times each year. Despite this exposure, the total incidence of
invasive listeriosis is estimated to be only 2–10 cases per million population per annum. The
document primarily dealt with risk management issues and recommended that fishery products
have a “use by date,” such that the product will contain <100 L. monocytogenes CFU/g at the end
of shelf life. Furthermore, health authorities should ensure that the stated shelf life for RTE products
that support growth of L. monocytogenes is within safe limits and that highly susceptible individuals
are informed and provided with guidelines about safe food handling practices.
Because of a lack of EU standards for L. monocytogenes in foods other than dairy products, a
study was conducted to evaluate the risks of various RTE foods and reported as Opinion of the
Scientific Committee on Veterinary Measures Relating to Public Health on Listeria monocytogenes
[28]. This study was considered particularly important because member countries have different or
no standards for this pathogen in foods, and this issue has complicated intra-EU trade. Their work
was a review of some of the earlier risk assessments which the authors admit did not constitute a
full risk assessment. The hazard characterization approach discussed most completely was the
mouse dose–response model of Notermans et al. [83] where there was a protective effect of the
physical barrier in the gastrointestinal tract along with prior immunity. In addition, growth of L.
monocytogenes would be different in different RTE foods based on limiting factors of pH, water
activity, temperature, salt and other chemical preservatives, and competitive flora. Examples were
given using UK Food MicroModel and USDA Pathogen Modeling Program software to predict
growth of the pathogen in five RTE foods. Very little consumption data were available for the
different EU countries. The report states that L. monocytogenes levels <100 CFU/g represent a
very low risk to consumers, but consumption patterns, as yet not known, may influence any
management policy. The authors recommend criteria based on groupings of RTE foods with
different control potentials (see the section titled “Standards and Criteria”).
Codex Alimentarius Commission
The CAC requested that FAO and WHO collaborate with world experts to assess a variety of microbial
foodborne risks. The FAO/WHO program initiated work on various pathogen–commodity combina-
tions, one of them being L. monocytogenes in RTE foods to respond to CAC and member country
needs. This L. monocytogenes assessment resulted in an extensive monograph and interpretive sum-
mary [36,37]. The current risk assessment was undertaken, in part, to determine how previously
developed risk assessments done at the national level could be adapted or expanded to address concerns
related to L. monocytogenes in RTE foods at an international level. In addition, the risk assessors
were asked to consider three specific questions related to RTE foods. These were the issues addressed:
1. Estimate the risk from L. monocytogenes in food when the number of organisms ranges
from 0 in 25 g to 1000 CFU per g or mL, or does not exceed specified levels at the point
of consumption.
2. Estimate the risk for consumers in different susceptible population groups (elderly,
infants, pregnant women, and immunocompromised patients) relative to the general
population.
3. Estimate the risk from L. monocytogenes in foods that support growth and foods that do
not support growth at specific storage and shelf life conditions.
RTE Foods Selected

Four RTE foods were selected as examples where L. monocytogenes contamination would demonstrate
different risks. Pasteurized milk is widely consumed and has very low frequencies and levels of
contamination but allows growth during storage. Ice cream is similar to milk but does not permit
growth during storage. Fermented dried sausages are often contaminated and are produced without
any lethal processing step, but the final composition prevents growth during storage. Cold-smoked
fish is frequently contaminated, has no lethal processing step, and permits growth during extended
storage. Both fermented meats and smoked fish have low consumption rates compared with milk and
ice cream. The four selected foods illustrate how different factors interact to affect the risk of listeriosis
per one million servings and the risk per 100,000 people per year. The four foods were modeled with
the same general structure: contamination frequency and level at retail; growth or inactivation until
consumption using storage temperatures, storage times, exponential growth rates or death rates, lag
phases, maximum growth, and consideration of spoilage; frequency and amount of consumption; and
dose–response relationship for healthy and susceptible populations. However, available data and
approaches to the modeling process were not always the same for each of the four foods.
Initial Contamination at Retail

Data from published scientific papers, government surveys, and the FDA/FSIS/CDC [42] risk
assessment were collected by the risk assessment team. Data from all countries and years that were
found in the literature were included in this assessment. Most of the data related to prevalence,
usually presence or absence of the organism, with determinations based on an analytical sensitivity
of 0.04 L. monocytogenes/g (1 microorganism per 25-g sample). An estimate of uncertainties about
presence or absence data was made with a Beta distribution, thereby including the effect of
the number of samples in a data set. Only a small portion of the available data sets stated levels
of L. monocytogenes/g in positive samples. These quantitative data were arrayed as a cumulative
frequency distribution. After uncertainty ranges were assigned, these distributions were used for
estimating the levels of L. monocytogenes in foods evaluated at the point of purchase.
Growth before Consumption

A survey of 939 home refrigerators conducted by Audits International [10] in the United States
provided data for considering the impact of home storage temperatures on the levels of L. monocytogenes
at consumption. A cumulative distribution of the data was used without any model fitting. The 5th,
50th, and 95th percentile temperatures were 0.5, 3.4, and 6.9°C, respectively. Other published home
refrigerator temperature studies in the United Kingdom, United States, and Greece were converted
to distributions but were not used in the final analysis; all these would generate a warmer range of
temperatures, particularly the Greek information, that would increase the potential for growth. Total
storage times at retail and in homes were based on expert opinion. Triangular distributions for the
variation in storage times were defined by minimum, most frequent, and maximum times, e.g.,
milk was given values of 1, 5, and 12 days, respectively. To further emphasize and explore the
uncertainty associated with storage time values, the most likely and maximum values were given
uniform distributions, e.g., for milk, the most likely value was assigned an uncertainty range of
4 to 6 days. Storage times and temperatures were not considered by the risk assessors to be
independent, and models allowed spoilage or growth of lactic acid bacteria (in smoked salmon)
to be limiting factors.
The storage time and temperature data were used in combination with information on
growth rates of L. monocytogenes to estimate how levels in foods were likely to change between
point of purchase and time of consumption. Most growth rates in the selected foods were from
published inoculated pack studies where foods with their normal spoilage flora were inoculated
with L. monocytogenes. The inoculated foods were stored at various temperatures and sampled at
various times with L. monocytogenes enumerated, and the exponential growth rate determined.
Except for ice cream, predictive models were used to estimate growth and inactivation rates, and
growth limits for L. monocytogenes in foods. Whenever possible, the growth model also considered
the effect of temperature on the maximum growth level.
Consumption
Unlike prevalence and concentration data, consumption patterns are often regionally specific.
Thus, examples of serving size and consumption frequencies were either taken from Canadian
Federal-Provincial Nutrition Survey’s databases or were estimated globally from national con-
sumption statistics. Data were used to represent the total population or an estimated susceptible
(at-risk) population. Serving frequencies were calculated as both the probability of consumption
during a day and the total number of servings per year for 100,000 people. The survey data were
not complete for all ages, nor were there any seasonal patterns of consumption.
Dose–Response Assessment
The outputs from the exposure assessment were fed into the dose–response models. Distribution
at consumption was characterized as a cumulative frequency of log10 CFU/serving of contaminated
food. Uncertainty estimates accompanied each percentile value to estimate confidence in the
accuracy of the percentiles. Other output values were the Beta distribution for contamination
frequency, the number of servings per year, and size of servings. Twelve dose–response models
based on outbreak data, animal dose studies, and expert opinion were reviewed. Most models
were exponential, but one was combined with a Beta-Poisson model and another was a Weibull-
Gamma model. Each of these had different r values (see the following text). The exponential
dose–response model was chosen because it was a simple single-parameter model, it fitted well
when modeling severe listeriosis, and it could be extrapolated to the low-dose ranges of interest.
The equation is
P=1−e −r*N
where P is the probability of severe illness, N is the number of L. monocytogenes cells consumed,
and r is the parameter that defines the dose–response relation for the population being considered.
The r value is considered to be a constant for a specified population, but its accuracy is dependent
on size and inclusiveness of the population being considered, accuracy of listeriosis surveillance
data, reliability of the frequency, and level of L. monocytogenes contamination in the food. The
exponential model is a nonthreshold model that implies that there is no minimum infectious dose.
Instead the model assumes that a single L. monocytogenes cell has a very small but finite probability
of causing illness. The maximum levels of L. monocytogenes encountered in foods have a large
impact on the calculated maximum ingested doses. The r values were estimated for 4-point estimates
of the maximum doses of 7.5, 8.5, 9.5, and 10.5 log CFU, respectively. The lower the maximum
dose assumed, the larger is the estimated r value. The larger the r value, the greater is the assumed
virulence of L. monocytogenes.
The available contamination and epidemiological data for L. monocytogenes did not permit a
clear choice of the most appropriate r values for different populations. Different r values were chosen
to address the questions posed by the CCFH. For CCFH Question 1, an r value of 5.85 × 10 −12 was
used for the susceptible population. This was the most “conservative” (i.e., the greatest assumed
virulence for L. monocytogenes) dose–response curve used in the current risk assessment and was
calculated under the assumption of a maximum dose of 7.5 log CFU per serving. For illustrating
how to estimate r values based on relative risks for different susceptible subpopulations in
Question 2, an r value of 5.34 × 10 −14 was selected as the reference value for the general healthy
population. This r value was calculated based on the assumption of an intermediate maximum dose,
8.5 log10 CFU per serving, in food. For the food examples described in the risk assessment and
CCFH Question 3, the r values used were based on the use of Monte Carlo simulation techniques in
combination with a discrete uniform distribution wherein the maximum number of L. monocytogenes
consumed varied from log10 7.5 to 10.5 CFU per serving. For the median population with increased
susceptibility, the median r value used with its distribution was 1.06 × 10−12 and for the healthy
population this was 2.37 × 10−14.
Risk Characterization
Exposure assessment outputs and dose–response relationships were combined in the risk char-
acterization portion of the risk assessment to calculate the probability of contracting listeriosis.
Distributions of prevalence and level of L. monocytogenes in contaminated food at consumption
and dose–response relationships led to estimates of risk per million servings for healthy and
susceptible populations. The risk per serving and number of servings were used to estimate the
number of illness per 100,000 people per year (Table 18.1). It is clear that the risks for fermented
meats and ice cream are much less than for smoked fish and pasteurized milk. Not only are initial
prevalence rates and concentration levels important, but so also are the potential for growth during
storage and amounts of these foods typically consumed.
TABLE 18.1
Estimated Risk of Listeriosis for Selected Foods Using
a Range of r Values for Both Healthy and Susceptible
Populations
Cases of Listeriosis per Cases of Listeriosis per
Food 10 Million People 1 Million Servings
Pasteurized milk 9.1 0.005

Ice cream 0.012 0.000014
Fermented meats 0.00066 0.0000025
Smoked fish 0.46 0.021
Source: FAO/WHO. 2004. R. Buchanan, R. Lindqvist, T. Ross, M. Smith, E.

Todd, and R. Whiting. Risk Assessment of Listeria monocytogenes in Ready-to-
Eat Foods—Technical Report. Microbiological Risk Assessment Series 5. 304
pp. http://www.fao.org/es/esn/food/risk_mra_listeria_report_en.stm. Accessed
October 1, 2004.
Response to Specific Codex Questions
Codex Question 1: Estimate the risk from L. monocytogenes in food when the number
of organisms ranges from 0 in 25 g to 1000 CFU/g or mL or does not exceed specified
levels at the point of consumption.
The most conservative dose–response model was used; i.e., maximum virulence of L. monocytogenes
was assumed. The r value for this relationship was 5.85 × 1012. The dose ingested is a function
of the pathogen level in the food (CFU/g) multiplied by the serving size. Thus, the equation
for calculating the probability of listeriosis was
P=1−e (5.85 × 10−12) (31.6g × n)
where n is the number of L. monocytogenes CFU/g.
By substituting different values for n, the likelihood of listeriosis at levels between 0.04 and
1000 CFU/g was calculated. The total number of RTE servings was assumed to be 6.41 × 1010,
and the corresponding number of listeriosis cases for the susceptible population was considered to
be 2130, both based on the FDA/FSIS/CDC [42] draft risk assessment. The results in Table 18.2
show a large number of predicted cases for servings containing higher contamination levels.
Another approach would be to employ a known or estimated distribution of L. monocytogenes
levels in foods when consumed. The example chosen was the overall distribution of L. monocytogenes
levels in 20 classes of RTE foods from the FDA/FSIS/CDC [42] risk assessment. This, then, could
be combined with the dose–response assessment to estimate the number of cases (Table 18.3). The
large difference in estimated number of cases (shown in Tables 18.2 and 18.3) is not only a factor of
the dose–response model chosen, but also of distribution of contaminated servings consumed. As
either the frequency of contamination or the level of contamination increases, so does the risk and
the predicted number of cases. Thus, if L. monocytogenes increased in all RTE foods from 1 to 1,000
CFU per serving, the risk of listeriosis would increase 1,000-fold if the serving size remained the same.
TABLE 18.2
Probability of Illness and the Estimated Number of Cases per Year in the United
States for Different Levels of L. monocytogenes at the Time of Consumption
Level Dosea Log10 Dose Probability of Estimated Number
(CFU/g) (CFU) (log10 CFU/serving) Illness per Serving Relative Riskb of Cases per Yearc
< 0.04 1 0 7.39 × 1012 1 0.54 d

0.1 3 0.5 1.85 × 1011 2.5 1
1 32 1.5 1.85 × 1010 25 12
10 316 2.5 1.85 × 109 250 118
100 3160 3.5 1.85 × 108 2,500 1,185
1000 31600 4.5 1.85 × 107 25,000 11,850
a Serving size of 31.6 g. A fixed serving size of 31.6 g was assumed to simplify calculations because it approximates
to a typical serving size and because dose levels were estimated in 0.5 log10 increments (100.5 = 3.16).
b Using the risk from a dose of 1 CFU as reference.
cA total of 6.41 × 1010 serving per year assumed.
d<1 per year.
Source: From FAO/WHO. 2004. R. Buchanan, R. Lindqvist, T. Ross, M. Smith, E. Todd, and R. Whiting. Risk
Assessment of Listeria monocytogenes in Ready-to-Eat Foods—Technical Report. Microbiological Risk Assess-
ment Series 5. 304 pp. http: //www.fao.org/es/esn/food/risk_mra_listeria_report_en.stm. Accessed October 1,
2004.
TABLE 18.3
Predicted Annual Number of Listeriosis Cases When the Level of L. monocytogenes
Was Assumed Not to Exceed a Specified Maximum Value and the Levels in
L. monocytogenes in the Food Are Distributed as Indicated in Table 18.2
Percentage of Servings When Estimated Number of Listeriosis
Level (CFU/g) Maximum Dosea (CFU) at Maximum Levelb Cases per Yearc
0.04 1 100.0 0.5d

0.1 3 3.6 0.5d
1 32 1.7 0.7d
10 316 0.8 1.6
100 3,160 0.4 5.7
1000 31,600 0.2 25.4
aServing size of 31.6 g.
bNumber of servings in the highest L. monocytogenes level assumed divided by 6.41 × 1010 times 100.
cLevels of L. monocytogenes per serving used to calculate predicted number of cases based on the overall distribution
from the FDA/FSIS/CDC. From FDA/FSIS/CDC. 2001. Center for Food Safety and Applied Nutrition, Food and
Drug Administration/Food Safety and Inspection Service, USDA and Centers for Disease Control and Prevention.
Draft assessment of the relative risk to public health from foodborne Listeria monocytogenes among selected categories
of ready-to-eat foods. http: //www.foodsafety.gov/~dms/lmrisk.html. Accessed October 1, 2004) risk assessment. A
total of 6.41 × 1010 serving per year was assumed.
d <1 case per year.
TABLE 18.4
Hypothetical “What-if” Scenario Demonstrating the Effect of Different
Defect Rates for Servings on the Number of Predicted Listeriosis Cases
Assumed Percentage Predicted Listeriosis Cases Predicted Listeriosis Cases
of Defect Servings a Where the Limit Is 0.04 CFU/g b Where the Limit Is 100 CFU/g b
0.0 0.5 5.7

0.00001 1.7 6.9
0.0001 12.3 17.4
0.001 119 124
0.01 1185 1191
0.018 2133 2133
0.1 11,837 11,848
1.0 117,300 117,363
a
All defective servings are assumed to contain 10 CFU/g.
r value of 5.85 × 1012 and serving size of 31.6 g.
b
Conversely, the effect of introducing into the food supply 10,000 servings contaminated with L.
monocytogenes at a level of 1,000 CFU/g would, in theory, be compensated by removing from the
food supply a single serving contaminated at a level of 107 CFU/g. These scenarios are made on the
assumption that they occur without any deviation from the set limits. In fact, the zero tolerance policy
for the United States is equivalent to the first scenario in Table 18.3 (a maximum of 0.04 CFU/g or
a dose of 1 CFU). If compliance were 100%, less than one case of listeriosis per year would occur
in the United States, compared with the estimated 2,130 baseline level in the FDA/FSIS/CDC [42]
risk assessment. If a microbiological limit of 0.04 CFU/g with a defect rate of 0.018% (2,133 cases)
is replaced by a 100 CFU/g limit and a 0.001% defect rate (124 cases), the predicted result would
reduce the number of listeriosis cases by about 95%. (Table 18.4). Compliance, therefore, is a critical
issue in reducing illnesses and is more important than whether the regulatory limit is 0.004 or
100 CFU/g.
Codex Question 2. Estimate the risk for consumers in different

susceptible population groups.
The basic approach taken to developing the requested dose–response relations was to take
advantage of epidemiological estimates of the relative rates of listeriosis for different subpopulations.
These relative susceptibility values were generated by taking the total number of listeriosis
cases for different subpopulations and dividing them by the estimated number of people in
the total population with that condition. This value is then divided by a similar value for the
general population. The r values for an exponential dose–response curve can be estimated for
a subpopulation using a relative susceptibility ratio and a reference r value for the general
population, with epidemiological data from France (transplant, AIDS, dialysis, cancer, liver
disease, diabetes, alcoholic, >65-year-old patients, and <65 years, no other condition) and the
United States (perinatal, >60 years, general population). The relative susceptibility values and
corresponding r values increase as the immune system becomes increasingly compromised.
The most compromised group, transplant patients, is 2584 times more susceptible to infection
than individuals <65 years old with no other medical conditions (the reference population),
with r values of 1.41 × 10 −10 and 5.34 × 10−14, respectively. This is followed by leukemia and
AIDS patients, respectively, 1364 and 865 (times more susceptible.) Pregnant women/newborns
at the perinatal stage were 14 times and the elderly <10 times more susceptible. In the two
outbreaks in which r values were able to be determined, the r value for the Los Angeles Hispanic
cheese outbreak involving pregnant women was very close to that estimated (3 × 10−11 vs.
4.51 × 10−11). The r value for an outbreak in Finland that was traced to hospitalized transplant
patients who consumed butter was much higher than the values for transplant patients (3 × 10−7
vs. 1.41 × 10−10). This was explained by the smaller number of individuals exposed in the
outbreak, the extremely compromised and highly variable immunological status of the popu-
lation, or the involvement of a highly virulent strain of L. monocytogenes.
Codex Question 3. Estimate the risk from L. monocytogenes in foods that support growth
and foods that do not support growth at specific storage and shelf-life conditions.
The risk characterization has shown that the ability of a product to support growth can increase
substantially the risk of its being a vehicle for foodborne listeriosis (Table 18.1). To show how
much growth is important, the mathematical models in the exposure assessment for pasteurized
milk were modified to ignore any increase in numbers; i.e., the milk was consumed immediately
after purchase at retail (Table 18.5). The results suggest an approximately 1,000-fold increase
in risk can be attributed to predicted growth of L. monocytogenes in pasteurized milk in risk
per 100,000 total population. The healthy population has an increased risk over the susceptible
population if growth is permitted, because higher numbers of L. monocytogenes are required
to infect healthy people. Another scenario was to let the contamination levels of milk be
truncated at 100 CFU/g at retail, but with growth still allowed (this only reduces the incidence
of listeriosis by about two-thirds). Two other scenarios relate to growth: increased storage
temperature from a median of 3.4 to 6.2°C (mean number of illnesses increased over 10-fold
for both populations), and increased storage time from a median of 5.3 to 6.7 days (4.5-fold
and 1.2-fold for the healthy and susceptible populations, respectively).
A similar approach was taken with smoked fish for not only time and temperature but also the
impact of lactic acid bacteria. The risk per serving and instances per 100,000 population increased
700- to 1000-fold in the 80 to 100% suppression model, and 67- to 85-fold in the 95% suppression
model from no L. monocytogenes growth to baseline (growth) scenarios. The predicted effect of
reducing the shelf life of smoked fish by 50% (1–28 days to 1–14 days) reduced the predicted
increase in risk from growth by 80%.
TABLE 18.5
Three Scenarios That Illustrate the Impact of Growth,
Contamination, and Storage of Pasteurized Milk on the Estimated
Risks of Listeriosis per 100,000 Population
Cases of Listeriosis per 100,000
Milk People (Mean Value)
Milk baseline 9.1 × 102

No growth 6.7 × 105
Truncate contamination at 100 CFU/g 2.8 × 102
Increase storage temperature from 3.4 to 6.2°C 1.2 × 100
Increase storage time from 5.3 to 6.7 days 2.0 × 101
Summary of the Assessment

The public health impact of an RTE food can be evaluated by both risk per serving and number
of cases per population per year, and risks may be different. Models developed predict that nearly
all cases of listeriosis result from consumption of high numbers of the pathogen. There are
differences in frequency of contamination and distribution of contamination levels within different
types of RTE food, although only four RTE foods were chosen for illustration. Differences in
manufacturing, storage practices, and consumption patterns in various countries may affect con-
tamination level and, therefore, risk per serving for a food. High levels of contamination at retail
are relatively rare, but cases could be reduced if vigilance in food processing facilities keeps
sources of L. monocytogenes entering the manufacturing environment to a minimum and there is
effective cleanup in the plant. Control measures that reduce the frequency of contamination will
proportionaly reduce the rate of illness, provided the proportion of high contamination is similarly
reduced. All cases of foodborne listeriosis in the United States and other countries with a similar
zero-tolerance policy occur from foods with more than 1 CFU in 25 g. From this assessment, it
would appear that many RTE foods exceed this level at manufacturing, although in many instances
not by much, with subsequent growth permitting levels to rise to those sufficient to cause illness.
However, it is also clear that raising a zero-tolerance standard to a higher value (e.g., 1 CFU/25 g
to 100 CFU/g) would increase the incidence of listeriosis unless new or improved mitigation
strategies were effective, such as reducing levels of L. monocytogenes in RTE food servings. In
foods that permit growth, control measures, such as better temperature control or limiting the
length of the storage period, will reduce the increases in risk from higher levels of L. monocytogenes.
Specific populations at more risk than others may need specific control conditions not necessary
for the general population.
STANDARDS AND CRITERIA

Although food safety is mainly improved through efficient implementation of GMP and HACCP
systems, including prerequisite programs, microbiological criteria may be used to help producers
verify that the HACCP plan has been correctly carried out. In addition, for trade purposes, the
burden of microbial load in foods is often required and this applies particularly to Listeria in RTE
foods. Currently there is no international agreement on what numbers of L. monocytogenes in foods
are acceptable to protect the consumer. In several countries, different criteria or recommendations
for tolerable levels of L. monocytogenes in RTE foods have been established [35]. Some countries
like the United States, Austria, Australia, New Zealand, and Italy require absence of L. monocytogenes
in ≥25 g of food (referred to as zero tolerance). Other European countries, for example, Germany,
The Netherlands, and France, have a tolerance of <100 CFU/g at the point of consumption. Others,
such as Canada and Denmark, have a tolerance of <100 CFU/g for some foods and a zero tolerance
for other foods, especially those with extended shelf lives that can support growth of L. monocytogenes.
In the United Kingdom, the standard only officially applies to dairy products. In addition to
differences in criteria for L. monocytogenes, several countries use different sampling plans and
methodologies that make comparisons difficult for trade purposes. The EU is at the discussion stage
to try and harmonize standards and cover all RTE foods that are potentially hazardous.
Therefore, there is agreement for the need to develop microbiological criteria for L. monocytogenes
in foods at the international level based on the principles of risk assessment, both for protecting
public health and for facilitating trade in various products. For regulatory purposes, it has been
assumed explicitly or implicitly that all strains of L. monocytogenes are potentially pathogenic,
although this may not be true, and discussions on standards have often focused on the dose–response
for individuals both in the general population and those at high risk. At the international level, the
CAC is encouraging the CCFH to develop guidelines for L. monocytogenes in RTE foods to be used
by member countries [19]. Where standards are applied, there is often a sampling plan and
recommended analytical method to be used. The two- or three-class sampling plans are usually
based on ICMSF [68], where n = the number of sampling units from the lot and c = the maximum
allowed number of sample units that exceed the microbiological criterion. The three-class plan is
used to designate acceptable, marginally acceptable, or unacceptable food where m = the maximum
number or level of L. monocytogenes CFU/g or CFU/mL (threshold value), and M = the maximum
number used to separate marginally acceptable and unacceptable quality food (maximum permitted
level). In the two-class plan, used to designate a batch or lot of food, either acceptable or unac-
ceptable, m = the maximum level of L. monocytogenes CFU/g or CFU/mL.
AUSTRALIA AND NEW ZEALAND

The approach to food safety in both Australia and New Zealand is undergoing change as the two
countries try to harmonize their regulations. Although both countries and the individual states in
Australia have their own food codes and regulations, the Australia/New Zealand Food Authority
(ANZFA), created in 1996 to facilitate trade, is attempting to form a unified food safety program
[48]. This authority will develop an agreed, uniform legal framework governing food in Australia
and New Zealand. Transitional arrangements were negotiated to cover the period as new standards
are produced over time. Food Standards Australia New Zealand [55] now takes over responsibility
for ANZFA.
The transitional arrangements are as follows:
• New Zealand’s agreement to adopt Australian standards

• Ordering the work of the current Australian review of the Australian Food Standards
Code (AFSC) to give priority consideration to those standards seen as impeding trade
between Australia and New Zealand
• The adoption by New Zealand of the entire AFSC thereby establishing dual standards
for that country
• Recognition by Australia of New Zealand food regulations for New Zealand food sold
in Australia during the period, i.e., dual standards in Australia also
During the review of microbiological criteria in developing the joint Australia New Zealand
Food Standards Code (ANZFSC), a number of policy principles were established by ANZFA [3].
It was stated that microbiological criteria should be established and applied only where there is a
definite need and where its application is practical. A definite need may be identified through a
risk assessment. To fulfill the purposes of a microbiological criterion, consideration should be given
to the following:
• Evidence of actual or potential hazards to health

• Microbiological status of the raw materials
• Effect of processing on microbiological status of the food
• Likelihood and consequences of microbial contamination and/or growth during subsequent
handling, storage, and use
• Categories of consumers
• Cost–benefit ratio associated with application of the criteria
• Intended use of the food
In Australia, the number of listeriosis cases reported averages about 50–70 per year for persons
of all ages. In the Australian Capital Territory, nine cases were reported since the disease became
notifiable in June 1992 until 1999 [52]. There have been relatively few incidents of listeriosis in
these countries with the exception of pâté. In Western Australia in 1990, there were six stillbirths
in a cluster of nine cases after contaminated pâté was eaten. A listeriosis warning was issued in
1995 by the Health and Community Services Department in Victoria after a middle-aged woman
became ill after consuming contaminated pâté [47]. In New Zealand in 1992, delivery of stillborne
twins was linked to a mother who consumed contaminated smoked mussels during her pregnancy
[52]. The safety of foods imported into Australia is evaluated by laboratory testing for microbio-
logical and chemical hazards under the Imported Food Program. The program, operating under the
Imported Food Control Act of 1992, is jointly administered by the Australian Quarantine Inspection
Service and ANZFA, now FSANZ [14]. Of the 17,685 microbiological tests done on so-called risk
foods from 1995 to 1999, there were 486 failures (2.7%), with individual yearly failure rates ranging
from 2.3% to 4.0%. Foods tested for Listeria included smoked vacuum-packed fish, soft cheeses,
chicken, and mussels. The percentage of total Listeria failures increased sharply until 1998 and
then declined in 1999. Of all risk foods tested, smoked vacuum-packed fish had the highest failure
rate of 8.6% for Listeria contamination (388 samples tested). Soft cheeses had a 1.1% failure rate
of 1140 samples tested. Thus, there was some concern about the pathogen in both countries.
In the AFSC, microbiological standards for L. monocytogenes specify the following: not
detected in soft cheeses, cheeses manufactured from thermized milk, smoked fish products, smoked
mussels, meat paste, and pâté. These standards apply to products sampled at the processing factory
or wholesale level, but do not apply to products at the retail level [4]. Microbiological standards
for L. monocytogenes are also contained in Standard 1.6.1 Microbiological Limits for Food [5] and
the User Guide [6]. These standards for L. monocytogenes include the following: no detection in
five 25-g samples (n = 5, c = 0, m = 0) for unpasteurized milk, butter made from unpasteurized
milk and/or unpasteurized milk products, soft and semisoft cheese (moisture content >39%) with
pH >5.0, all raw milk cheese (cheese made from milk not pasteurized or thermized), unpasteurized
milk, packaged cooked cured/salted meat, packaged heat-treated meat paste and packaged heat-
treated pâté, cooked crustaceans, and mollusks that have undergone processing other than depura-
tion. For RTE processed finfish other than fully retorted finfish, one out of five samples may contain
up to 100 CFU/g of L. monocytogenes (n = 5, c = 1, m = 100 CFU/g).
Because of industry concerns, FSANZ undertook to review the L. monocytogenes limits set
for cooked crustaceans and RTE processed finfish just described. Based on a scientific risk
assessment [7], FSANZ recommended that existing microbiological limits for Listeria in finfish be
retained, along with zero tolerance for L. monocytogenes in cooked crustaceans, since these products
can potentially compromise public health and safety, particularly for vulnerable subgroups. The
implications of a zero tolerance are that any product recall would affect the importer or manufacturer,
as well as the entire seafood industry, costing millions of dollars (one submitter estimated this cost
to be $10 million in terms of business revenue alone, based on a $1/kg reduction in price). Estimates
for the cost of compliance vary from A$200,000 to A$530,000 p.a. for the prawn industry, and $60,000
for the rock lobster industry. ANZFA was aware that this regulation comes with a substantial cost to
the food industry. However, the potential benefit is a reduction in the number of listeriosis cases in
Australia and New Zealand. ANZFA recognized that it is difficult to compare the costs to industry
with potential benefits to the community, particularly when some of the community benefits are less
tangible, such as quality of life and longevity. However, when setting regulatory policy, ANZFA took
a precautionary approach where the public health and safety consequences are severe. Nevertheless,
the exclusion position for crustaceans was upheld in the 2003 review [53,54]. This report claimed
that modeling of a worst-case scenario, which assumed that cooked prawns would be stored in a
domestic refrigerator for a maximum of 3 days after purchase allowing growth, would yield one case
of listeriosis from cooked crustaceans every 2.5 years, a risk considered to be acceptable. The report
recommended a variation to Standard 1.6.1 by deleting the microbiological limit for L. monocytogenes
in cooked crustaceans, because (1) risk assessment work undertaken for P239 shows that L. mono-
cytogenes in cooked crustaceans presents a low risk to public health and safety, and (2) a stringent
microbiological standard requiring an absence of L. monocytogenes in cooked crustaceans cannot be
justified on public health and safety grounds given the low risk presented by this pathogen–commodity
combination. In a similar assessment ANZFA proposed not to amend the microbiological criteria for
L. monocytogenes in processed RTE finfish (other than fully retorted finfish) of n = 5, c = 1, m = 100
CFU/g in Standard 1.6.1. The assessors concluded that smoked salmon posed a moderate-to-high risk
because the shelf life of smoked salmon is 4 to 6 weeks, allowing growth of the pathogen. These
actions show that the ANZFA management policy is based on a risk assessment approach.
However, these standards apply to specific product types. For foods not mentioned, recall
guidelines are similar to the Canadian policy. Category 1 RTE foods are those requiring refrigerated
storage that are able to support growth of L. monocytogenes, and RTE foods that have been
implicated in human listeriosis (e.g., soft and semisoft cheeses, pâté, cooked cold chicken, cold-
smoked fish) and/or which may be consumed by at-risk groups, especially infants. Although the
pathogen should not be detected in 25 g, the ANZFSC has a sampling plan for cold-smoked fish
that allows one out of five samples to contain L. monocytogenes at levels up to 100 CFU/g (see
preceding text). Category 2 includes all other packaged RTE foods and has a limit of 100 CFU/g.
In New Zealand, because Listeria contamination of fishery products is seen as a threat to its
continued success both domestically and internationally, standards for monitoring L. monocytogenes
in the environment and product were developed under the 1995 Fish Export Processing
Regulations [86]. A decision tree approach to finding positive environmental testing applies (pos-
itives increase the amount of testing until the pathogen is at acceptable levels). Positive product is
reprocessed or destroyed. The requirements of industry-agreed standards and FIICC circulars, the
1981 Meat Act and the Fish (Packing for Export) Regulations [87], are mandatory and must be
followed. It is recommended that all fish-processing operations in New Zealand use these guidelines
to minimize the risk of Listeria contamination of their products.
In addition to the policies with standards, FSANZ and ANZFA have produced an educational
brochure on Listeria and pregnancy with updated information over several years [8,52,56] listing
which foods are safe to eat and which to avoid.
NORTH AMERICA
United States
The first large listeriosis outbreak in the United States occurred in 1985 when Mexican-style soft
cheese in Los Angeles, California caused over 142 cases with 48 fatalities, mainly in perinatal
cases. L. monocytogenes was present in several cheese samples, most likely coming from the raw-milk
supply when the pasteurizer was bypassed. Surveys done the same year by the FDA found L.
monocytogenes in both imported and domestic soft cheeses. It was recognized that L. monocytogenes
has caused foodborne disease from food products regulated by FDA and USDA, or had the potential
to do so. Therefore, the FDA established a policy of zero tolerance for L. monocytogenes in RTE
foods [93]. This means that detection of any L. monocytogenes in either of two 25-g samples of a
food renders the product adulterated as defined by the Federal Food, Drug, and Cosmetic Act. This
policy was affirmed in the 1995 U.S. District court decision in United States v. Union Cheese Co.
In another decision in 2001, a motion was granted to FDA to enjoin a fish processor from violating
adulteration provisions of the Food, Drug, and Cosmetic Act [40]. It reemphasized that the respon-
sibility for protecting public health was on the producer, not consumers. The company challenged
the adulteration charge based on the presence of L. monocytogenes in its fish products by arguing
that this pathogen is not an added substance and does not ordinarily present a risk to health because
it affects only some segments of the population. The processor argued that if the court found
L. monocytogenes to be an added substance, the FDA’s zero-tolerance policy violates provisions of
the Food, Drug, and Cosmetic Act. The court rejected these arguments, finding that (1) L. monocytogenes
is an added substance, (2) L. monocytogenes is injurious to the health of significant populations of
consumers, and (3) the FDA is not required to set a tolerance level for L. monocytogenes. The
USDA also considers L. monocytogenes to be an adulterant in RTE products and enforces a zero
tolerance (no detectable level permitted) for this pathogen in RTE meat and poultry products [105].
Since 1989, FSIS analyzed approximately 3500 samples for L. monocytogenes each year. The
following product categories are included in the monitoring program: (1) beef jerky, (2) roast beef,
cooked beef, and cooked corned beef, (3) sliced ham and luncheon meat, (4) small-diameter
sausage, (5) large-diameter sausage, (6) cooked, uncured poultry, (7) salads and spreads, and (8)
dry and semidry fermented sausage. Of 3547 samples of RTE meat and poultry products analyzed
in 1998, approximately 2.5% tested positive for L. monocytogenes. FSIS sampling of hot dogs from
1993 to 1996 showed that approximately 4.4% of the samples were positive for L. monocytogenes.
Action by both the FDA and FSIS, together with compliance by industry, seemed to be on track
in reducing L. monocytogenes contamination in food and the number of listeriosis cases. However,
two major outbreaks have caused renewed concern about this pathogen and its control in the
United States. From August 1998 to February 1999 an outbreak, which affected at least 100
people and caused 20 deaths, was associated with the consumption of hot dogs and deli meats
produced in a Michigan plant [22,95]. In 2002, another major outbreak occurred with 46 culture-
confirmed cases, seven deaths, and three stillbirths or miscarriages in eight states that had been
linked to eating sliceable turkey deli meat [23]. The responsible company in Pennsylvania recalled
27.4 million pounds of cooked sandwich meat [89]. This came less than 3 months after a beef
supplier recalled nearly 19 million pounds of ground beef because of an E. coli O157:H7
contamination in Colorado. Government agencies are responding to these concerns by encour-
aging more research, continuing dialogue with industry, and initiating risk assessments. HACCP
and GHPs still seem to be a logical way to reduce contamination, but from the information
revealed during the outbreak investigations, it seems that compliance with in-house and govern-
ment policies still needs more attention.
Fifteen trade associations submitted a citizen petition on December 24, 2003, requesting
that the FDA amend the regulations for the agency to establish a regulatory limit of 100 CFU/g
for L. monocytogenes in foods that do not support growth of the microorganism [41]. The
petitioners assert that such a regulatory limit would establish a science-based standard for
presence of L. monocytogenes in such foods, noting that their request is based on new and
emerging evidence that consumer protection is a function of the organism’s cell numbers in food,
and not its mere presence, and that there is general scientific agreement that low levels of L.
monocytogenes are not uncommon in the food supply and that such low levels are regularly
consumed without apparent harm. The agency is requesting comment on the petition. On December 1,
2004, a report outlining the impact of the interim final rule designed to further reduce the
incidence of L. monocytogenes in RTE meat and poultry products and making recommendations
for possible future actions was released for public comment by FSIS. The report shows that the
overall safety of these products has improved in response to the Listeria interim final rule because
establishments have strengthened their control procedures, increased testing, and taken additional
steps to eliminate the pathogen [109].
Canada
Health Canada developed its policy independently of other countries, and not in response to major
outbreak problems, unlike the United States. It is considered a risk-based approach with different
requirements for different types of RTE foods. The highest priority is given to those RTE foods
that have been linked causally to listeriosis and those with a shelf life greater than 10 days [38].
The policy is based on a combination of inspection, environmental sampling, and product testing.
Because of changes in government policy in 2000, the Canadian Food Inspection Agency is now
responsible for any regulatory action, and Health Canada is required to set policy and standards,
and conduct risk assessments.
In October 2004, a revised Policy on Listeria monocytogenes in Ready-To-Eat Foods was
developed by Health Canada [64]. RTE foods have been placed in three categories, based upon
health risk. In the establishment of the Category 1 food list, considerations were given to RTE
foods that have been causally linked to documented outbreaks of listeriosis and/or have been
placed in the high-risk category in the recent FDA/FSIS/CDC [43] assessment. Products in
Category 1 should receive the highest priority for inspection and compliance activities. Presence
of L. monocytogenes in these RTE foods will trigger a Health Risk 1 concern with consideration
of a public alert. Category 2 contains all other RTE foods that are capable of supporting growth
of L. monocytogenes and have a shelf life exceeding 10 days. Presence of L. monocytogenes in
these products will trigger a Health Risk 2 concern, with possible consideration of a public alert,
and receives the second highest priority in inspection and compliance activity. Category 3 contains
two types of RTE food products: those supporting growth with a ≤10-day shelf life and those not
supporting growth. These products receive the lowest priority in terms of inspection and compli-
ance action. For Category 3 RTE foods, factors such as adherence to GMPs, levels of L. mono-
cytogenes in the food (action level, 100 CFU/g), and/or a health risk assessment, should all be
considered in determining the compliance action taken. RTE foods contaminated with L. mono-
cytogenes produced primarily for consumption by individuals who might be considered as most
susceptible to listeriosis (neonates, pregnant women, the elderly, and immunocompromised indi-
viduals) would be considered a Health Risk 1 or 2 (Table 18.6), but would not be considered a
Health Risk 3, regardless of product type. Health risk 1 represents a situation in which there is a
reasonable probability that consumption of or exposure to a food will cause adverse health
consequences which are serious or life threatening, or where the probability of a foodborne
TABLE 18.6
Compliance Criteria for L. monocytogenes in Ready-to-Eat (RTE) Foods in Canada
Action Level
Category for LM GMPs Status Nature of Concern
1. The list presently includesa soft cheese, Detected in 50 g NA Health Risk 1

liver pâté, unacidified jellied pork tongue,
hot dogs/wieners, cold-smoked rainbow
trout, and processed deli turkey meat
2. All other RTE foods supporting growth of Detected in 25 g NA Health Risk 2
LM with a refrigerated shelf life >10 days
(e.g., vacuum-packaged meats, modified-
atmosphere-packaged sandwiches,
refrigerated sauces)
3. RTE foods supporting growth of LM with ≤100 CFU/g Adequate GMPs Health Risk 3
a refrigerated shelf life of 10 ≤days (e.g., ≤100 CFU/g Inadequate, absent, Health Risk 2
packaged salads) and all RTE foods not or no information on
supporting growthb (e.g., ice cream, hard GMPs
cheese, dry salami, salted fish, breakfast, >100 CFU/g NA Health Risk 2
and other cereal products)
Note: LM = L. monocytogenes, NA = not available, GMP = Good Manufacturing Practices

aRTE foods causally linked to documented outbreaks of listeriosis and/or to RTE foods rated as “high risk” in the
FDA/FSIS risk assessment (from FDA/FSIS/CDC. 2003. Center for Food Safety and Applied Nutrition, Food and
Drug Administration/Food Safety and Inspection Service, USDA and Centers for Disease Control and Prevention.
Quantitative assessment of the relative risk to public health from foodborne Listeria monocytogenes among selected
categories of ready-to-eat foods. http: //www.foodsafety.gov/~dms/lmr2-toc.html. Accessed October 1, 2004.).
bA refrigerated RTE food not supporting growth of L. monocytogenes includes the following: pH 5.0–5.5 and a
w
<0.95; pH <5.0 regardless of aw; aw ≤0.92 regardless of pH; or frozen foods.
outbreak situation is considered high. Health Risk 2 represents a situation in which there is a
reasonable probability that consumption of or exposure to a food will cause temporary or non–life
threatening health consequences, or the probability of serious adverse health consequences is
considered remote. Health Risk 3 represents a situation in which no health hazard has been
identified and there is a reasonable probability that consumption of or exposure to the food is not
likely to result in any adverse health consequence.
EUROPEAN COUNTRIES
Austria
Austria has a zero-tolerance policy for most foods, such as fish, and not only those that are RTE
products [2,35]. This presents some problems for the industry. For instance, all raw meat is required
by the existing law to be free of L. monocytogenes in 25 g, which could result in many lots being
recalled; therefore, these products are not routinely tested for the pathogen (personal communica-
tion, Franz Allerberger, Institute for Hygiene and Social Medicine, Bereich Hygiene, University of
Innsbruck, Austria). There are exceptions to this policy, brought about through individual court
actions. These are raw sausage, e.g., salami and mettwurst (spreadable, pâté-like), raw pork products
cured with nitrate, other meat and fish products with added chemical preservatives, and RTE
salads with mayonnaise. All these are permitted to have up to 100 CFU/g. Following an appeal
against food standards introduced by the Government of Austria, the European Court decided
that European law does not preclude the imposition by a member state of a zero tolerance for
L. monocytogenes in fishery products, even if it is not technically possible to eliminate that
hazard [44,45]. This would seem to indicate that some fishery products might not be sold in
Austria without risking prosecution. Therefore, it appears that the European Court has decided
that zero-tolerance standards for L. monocytogenes are lawful.
Denmark
In 1999, several thousand samples of RTE foods at the retail level were tested and less than 1%
were found positive [25,82]. The Danish regulatory policy on L. monocytogenes in foods, built on
the principles of HACCP and developed using a health risk assessment approach, is based on a
combination of inspection and product testing [35,82]. RTE foods are placed in six categories
(I–VI), absence of L. monocytogenes in 25 g being required in the first three. This level is necessary
in foods capable of supporting growth, unless there is evidence of a listericidal treatment in
category I, not to exceed 100 L. monocytogenes per gram at the point of consumption. In the other
three categories, a level between 10 and 100 L. monocytogenes per gram is not satisfactory and a
level above 100/g is not acceptable, depending on the sampling scheme. In 1994 and 1995, 1.3%
of RTE food samples (heat-treated meat products, preserved meat and fish products) were contam-
inated with L. monocytogenes at a level above 100 CFU/g. The samples included in this survey
were primarily foods produced by approved companies and were comprised mainly of vacuum-
packed products or products packed in modified atmosphere and with long shelf lives, typically
above several weeks. The corresponding percentages of positive samples primarily processed in
the retail outlets (heat-treated meat products, preserved meat and fish products) in 1997 and 1998
were 0.3 and 0.6%, respectively. These results suggest that RTE meat and fish products with
extended shelf lives produced by companies are more likely to contain high numbers (>100 CFU/g)
of L. monocytogenes than products processed in the retail sector, which often have a shorter shelf
life. In 1997, preserved meat and fish products, and to a lesser extent, vegetables and meat or
vegetable mayonnaise were more likely to contain >100 L. monocytogenes CFU/g than other food
commodities. In 1998, preserved meat products, but also heat-treated meat products, vegetables,
and meat or vegetable mayonnaise had the highest frequency of samples with >100 L. monocytogenes
per gram. In the Danish categorization of foods heat treatment, storage time, and stabilization are
key factors. The means of stabilizing foods include freezing, pH < 4.5, and aw < 0.9. The categories
are as follows:
• Foods heat treated in the final package (absence in 25 g).

• Heat-treated foods that are handled after heat treatment. The products support growth of
L. monocytogenes during shelf life. Typically, shelf life of these products is above 1
week. Examples of products: packed meat products such as cooked ham and wiener
sausages, and hot-smoked fish (absence in 25 g).
• Lightly preserved non-heat-treated RTE products. The products support growth of
L. monocytogenes during shelf life. Typically, shelf life of these products is above 3
weeks. Examples of products: cold-smoked or gravad fish and meat (absence in 25 g).
• Heat-treated foods handled after heat treatment. The products are stabilized against
growth of L. monocytogenes within the shelf life. Products that have a shelf life less than
1 week are regarded as stabilized. Examples of products: packed or unpacked meat
products such as cooked ham and wiener sausages, and hot-smoked fish (sampling plan:
n = 5, c = 1, m = 100).
• Lightly preserved non-heat-treated RTE products. The products are stabilized against
growth of L. monocytogenes during the shelf life. Products with a shelf life less than
3 weeks are regarded as stabilized. Examples of products: cold-smoked or gravad fish
and meat (sampling plan: n = 5, c = 1, m = 100).
• Raw RTE foods. Examples of products: steak tartar (tatar), sliced vegetables, sprouts
(sampling plan: n = 5, c = 1, m = 100).
France
Outbreaks have occurred in France in the past, but the incidence is lower today [79,102]. In France,
hygiene and sanitary regulations require the absence of any pathogen in foodstuffs for human
consumption. For dairy products, the Community Directive 92/46 of 16 June 1992 is more precise
because it requires the absence of L. monocytogenes in 25 g of soft cheese, and in 1 g of hard
cheese and other dairy products, at the manufacturing stage [79]. France conformed to this directive
in 1999 [62]. The CSHPF (French High Council for Public Hygiene) established the criterion
of 100 L. monocytogenes per gram as the maximum admissible threshold at consumption, a
threshold that applies to all other foodstuffs [79].
Germany
Listeriosis in Germany is not a notifiable disease, so the total number of cases is unknown, but the
estimate is at least 50–80 cases per year [101]. Germany has developed recommendations to be
used by food inspection authorities. Four different categories of food have been developed along
with action levels [35]. Group I consists of foods for infants and small children as well as dietary
foods, with the action level being the presence of L. monocytogenes in 25 g of product. Group II
contains foods such as pasteurized milk and aseptically packaged products, with the same action
level as Group I foods. Foods in Group III include frozen meals, cheese (but not raw milk cheese),
prepacked sausages, and shrimp, with an action level of >100 CFU/g. Low-level contamination
would precipitate follow-up action in the plant, and higher contamination would render a food
“unfit for human consumption.” The last group (Group IV) consists of three different food categories
that are “otherwise stabilized foods,” such as smoked fish and fermented sausages, raw foods
consumed in the raw or unprocessed state, and raw food that is heated before consumption. For
Group IV foods, L. monocytogenes levels of <100 CFU/g usually are permissible, but in cases in
which higher levels are observed, the food plant or shop would be inspected. The upper limit of
L. monocytogenes allowable in any food product is 104 CFU/g or CFU/mL (action level, not
tolerance level), but authorities are considering lowering this to 103 CFU/g or CFU/mL.
Italy
A general food law [70] prohibits the marketing of food containing harmful microbes, which is inter-
preted as zero tolerance for pathogenic organisms. There was also a more specific order of the Ministry
of Health [71,73]. This requires that the label must bear “to be consumed after heat treatment.” The
order allows different limits in three groupings for all foods except milk and dairy products.
• Raw foods that have not been heat treated, 3 sample units—not more than 11 CFU/g
in 1 sample unit and not more than 110 CFU/g in 2 sample units; n = 3, c = 2, m = 11,
M = 110
• Frozen or quick-frozen foods, 5 sample units—not more than 11 CFU/g in 2 sample
units and not more than 110 CFU/g in 3 sample units; n = 5, c = 3, m = 11, M = 110
• Precooked or pasteurized foods, 5 sample units—not more than 11 CFU/g in 4 sample
units and not more than 110 CFU/g in 1 sample unit; n = 5, c = 1, m = 11, M = 110
In addition, a more recent decree [72] covers dairy products in a similar way to regulations in The
Netherlands and the U.K.; no L. monocytogenes in 25 g of soft cheeses (“not hard”), and no L.
monocytogenes in 1 g of milk and all other dairy products (c = number of sample units giving values
between m and M).
The Netherlands
Under The Netherlands National Food and Commodities Law, L. monocytogenes should be <100/g
for all food types except raw food, based on EU draft regulations [29]. There is, however, absence
of specific standards for milk-based products based on EC Council Directive 92/46 [27]: absence
of L. monocytogenes in 25 g of cheese other than hard cheese and absence in 1 g of other milk-
based products (n = 10, c = 0, m = 0). L. monocytogenes was not identified in HACCP plans of
two establishments in which a hazard was likely to occur.
Slovenia
A recent study in Slovenia showed that L. monocytogenes was found in samples of raw meats, raw
sausage, mechanically deboned chicken meat, and fast fermented sausages [80]. Recommendations
were to improve hygiene and to ensure that sausages were fully matured before sale, but did not
include any proposed regulations.
United Kingdom
Specific legislation only exists for dairy products [103]. These state that L. monocytogenes must be
absent in 25 g cheese other than hard cheese, where n = 5 and c = 0. For all other milk-based products,
the pathogen must be absent in 1 g. This legislation is similar to that in The Netherlands except
that the sampling plans are different. However, this regulation is broadly interpreted to cover other
RTE foods besides dairy products, in which L. monocytogenes may or may not grow. In 2001,
62,209 laboratory-confirmed instances of food poisoning were documented, including 113 from
infection by L. monocytogenes [1]. The Food Standards Agency wants to see a 20% decrease in
the number of all foodborne illnesses, including listeriosis, by 2006 [50,51]. The agency established
a Foodborne Disease Strategy Consultative Group involving a cross-section of stakeholders to help
achieve its goals in 2002 [51]. Most of the efforts used so far employ farm-to-fork approach, with
the aim of reducing contamination of foods during production and processing and of promoting
good food hygiene practice in the commercial and home kitchen. It will involve both sector-specific
measures and measures that will have impact across all food sectors, including training and
education of food handlers, promotion of HACCP, and a media food hygiene campaign. There is
nothing specific to Listeria.
EUROPEAN UNION (EU)

In Europe, most member states report on human listeriosis, but the disease is only notifiable in
Sweden and Denmark, whereas in other member states reporting is based on laboratory findings
[28,31]. The EU reported 665 cases of listeriosis in 1999, but with no information on fatalities
[30]. There are also animal listeriosis monitoring programs. Many member countries conduct
periodic sampling of foods; however; the sampling schemes and analytical methods used are not
harmonized. Belgium continually monitors for L. monocytogenes in meats, and The Netherlands
monitors its prevalence in cheese. European countries have different policies for Listeria control,
including two with strict zero-tolerance policies. In June 2002, a parliamentary question was asked
in the EU on zero-tolerance standards in some member states for L. monocytogenes in smoked
salmon. It was recognized that elimination of this hazard from this product is not possible with
some member states (notably Italy) maintaining a zero tolerance compared to a limit of 100 CFU/g
in some other states [44]. The Commission had no plans to harmonize standards and did not appear
unduly concerned at the anomaly. However, the European Court decided in late 2002 that zero-
tolerance standards for L. monocytogenes are lawful, which will probably have repercussions in
the form of legal action, because this may conflict with some existing policies [45].
Countries exporting meat to the United States also have periodic inspections of selected
establishments. Audit reports have indicated some deficiencies, indicating that even if regulations
are promulgated, they are not necessarily followed. For instance, reports in Austria, Germany, Italy,
and The Netherlands indicated that there were some areas that needed corrections [104,106–108].
Two establishments were delisted for noncompliance with HACCP by the Government of Austria.
Most problems relating to L. monocytogenes involved the lack of a testing schedule, particularly
relating to HACCP programs, and improper hygienic controls. Two of these countries (Austria and Italy)
have zero-tolerance policies for L. monocytogenes.
In the opinion of a scientific committee [28], control of Listeria in Europe must be based on
knowledge of the pH, water activity, presence of preservatives, and shelf life of the different RTE
products. Food groupings used by Germany, Denmark, and Canada were presented as examples.
The opinion suggested that the groupings can be summarized in three areas: (1) the capacity of
food technology to kill L. monocytogenes, (2) the capacity of food technology to prevent recon-
tamination with L. monocytogenes, and (3) the potential for L. monocytogenes to grow in the food
commodity. The opinion favored a grouping almost identical to that used in Denmark (A–E vs.
I–VI). For foods in Groups A, B, and C, L. monocytogenes should not be detected in 25 g at the
time of production. For foods in Groups D, E, and F, L. monocytogenes levels should be <100
CFU/g at the time of consumption and, therefore, throughout the shelf life of the commodity. Apart
from these microbiological criteria, adoption of HACCP, displaying production dates on all product
packages, paying attention to temperature and time of storage, and exploring the potential to limit
shelf life are issues that should be considered. Risk communication strategies need to be imple-
mented to advise consumer groups about increased risk. In its opinion of 22 June 2000, The
Scientific Committee on Food (SCF) agreed with the opinion that the concentration of L. mono-
cytogenes in food should be kept below 100 CFU/g. Discussions on how microbiological criteria
can be derived in the EU are in progress. One possible set of criteria shown in Table 18.7 was
presented for consideration of member countries in 2003 [32]. These came into force on January
1, 2006, and should be adopted by all EU member nations.
TABLE 18.7
Proposed EU Criteria for Listeria monocytogenes in RTE Foods
Sampling Plana Stages at Which the Criterion
Food Categories n c Limit m Applies
RTE foods intended for infants and for 10 0 Absence in 25 g End of the manufacturing process
special medical purposes and products on the market
RTE foods able to support growth of L. 5 0 Absence in 25 gb End of the manufacturing process
monocytogenes other than those
intended for infants and for special
medical purposes
RTE foods able to support growth of L. 5 0 <100 CFU/gc End of the manufacturing process
monocytogenes other than those and products on the market
medical purposes
RTE foods unable to support growth of 5 0 <100 CFU/g Products during the shelf life
L. monocytogenes other than those
medical purposes
an = number of units comprising the sample, c = number of sample units giving values over m.
bThis criterion applies if the food manufacturer is not able to demonstrate, to the satisfaction of competent authority, that
the product will meet the limit <100 CFU/g throughout shelf life.
cThis criterion applies if the food manufacturer is able to demonstrate, to the satisfaction of competent authority, that the
product will meet the limit <100 CFU/g throughout shelf life. The exact value of the limit to be implemented depends
on characteristics of the product and conditions during shelf life; the limit shall be low enough to guarantee that the limit
100 CFU/g is not exceeded at the end of shelf life.
For all RTE foods, the microbiological quality of the batch tested shall be considered unsatisfactory
if L. monocytogenes is present in any one of the sample units for the different limits of m. Where the
criteria are exceeded the lot or batch shall not be released to the market or it shall be withdrawn from
the market. The aforementioned criteria for L. monocytogenes apply to all RTE foods, but regular
testing is usually not useful for foodstuffs such as those that have received heat treatment or other
effective processing to eliminate L. monocytogenes, when recontamination is not possible after this
treatment, e.g., packaged products and UHT milk; fresh, uncut, and unprocessed vegetables and fruits,
excluding sprouted seeds; bread and biscuits and similar products; bottled or packed waters, soft
drinks, beer, cider, wine, spirit drinks, and similar products; sugar, honey and confectionery, including
cocoa and chocolate products; and live bivalve mollusks. The keys to setting criteria are the analytical
methods used and an appropriate sampling plan.
INTERNATIONAL ACTIVITIES
The International Commission for Microbiological Specifications for Foods (ICMSF) recommends
that some testing is useful as part of the HACCP verification program, and there are specific
protocols that apply to L. monocytogenes [35]:
• In-pack, heat-treated products—no testing is necessary (documentation for the heat

treatment process).
• Raw products and/or products that are to be heat-treated before consumption—no testing
is necessary.
• RTE products unable to support growth of L. monocytogenes—10 samples should be

taken and the lot should be rejected if any sample contains >100 L. monocytogenes/g.
• RTE products able to support growth of L. monocytogenes—20 samples should be taken
and the lot rejected if any sample contains >100 L. monocytogenes/g.
The CAC has also been studying international criteria for L. monocytogenes through a Working
Group of the Codex Committee for Food Hygiene [34]. This working group agreed to propose to
the Codex Committee on Food Hygiene to recognize that numbers of L. monocytogenes not
exceeding 100 CFU/g in food at the time of consumption are of low risk to consumers. Furthermore,
with respect to imported foods, it agreed to recommend that lower levels may need to be applied
at the port-of-entry for foods that support growth of L. monocytogenes, so as not to exceed 100
CFU/g at the point of consumption. The recent FAO/WHO risk assessment for L. monocytogenes [36,37]
is evidence of the need to harmonize the criteria for this pathogen around the world based on sound
science. Because L. monocytogenes contamination can come from multiple sources, a comprehensive
control program may involve a combination of strategies that involve HACCP and GHP. A decision
tree approach was suggested by FAO [35] to discover which category of RTE foods has the
commodity of interest fit. There is also an ongoing discussion through working groups in the CCFH
to make recommendations on management of processes by adopting GHPs specifically to reduce
L. monocytogenes contamination and growth [19], and the International Life Sciences Institute is
preparing a report on a risk-based approach to achieving continuous improvement in reduction of
foodborne listeriosis [69], which includes education strategies.
COSTS
Relatively few economic analyses of foodborne disease for countries or regions have been made,
and even fewer for listeriosis, even though these are valuable in three main ways: (1) evaluating
the economic impact of foodborne diseases in a country, (2) helping target pathogen reduction
efforts toward the most costly diseases, and (3) comparing benefits and costs of control efforts to
determine the most cost-effective interventions. These analyses cannot be made without some
understanding of the degree of morbidity and mortality of the pathogen, which are not easily
available. In the United States, only the more severe hospitalized cases and deaths are estimated,
although outbreaks involving milder cases have been investigated and reported. For most other
foodborne pathogens, all cases are considered in determining the burden of disease [81]. Attempts
at costing all foodborne disease for the United States started in the mid-1980s, although costs of
a few individual outbreaks had been published earlier [9,12,60,96–98].
In 1994, the Council for Agriculture and Science Technology [21] reviewed the literature and
used expert opinion to indicate that microbial foodborne pathogens cause 6.5–33 million cases of
human illnesses and 9000 deaths in the United States each year. Based on these data, the Economic
Research Service (ERS) of the USDA estimated that the annual cost of human illnesses for six
foodborne bacteria and one parasite from all food sources was $5.6–$9.4 billion [17,18]. Many
costs are involved in estimating the impact of a disease (Table 18.8), but most are hard to measure
or determine. The ERS approach focuses on the cost-of-illness (COI) and willingness-to-pay (WTP)
methods. The COI method measures the combination of medical expenses, lost earnings of affected
individuals, and productivity losses to employers of affected individuals on paid sick leave. The
WTP method attempts to estimate what individuals place on the value to society of publicly provided
risk reduction strategies. The strength of using a COI approach is that it uses available, calculable
data, and such measures have been widely used for many years. The weakness is that these costs
are a small portion of the many experienced by individuals and society, and exclude, for instance,
costs to the industry when an outbreak or recall occurs. The foregone earnings associated with
premature deaths, which vary with age and gender, usually dominate COI estimates, and higher-
paid members of society will be assigned higher values of life, e.g., a statistical life of $11,867 to
TABLE 18.8
Societal Costs of Foodborne Illness
Costs to individuals/householdsa
Human illness costs
Medical costs
Physician visits
Laboratory costs
Hospitalization or nursing home
Drugs and other medications
Ambulance or other travel costs
Income or productivity loss for—
Ill person or person dying
Caregiver for ill person
Other illness costs
Travel costs to visit ill person
Home modifications
Vocational/physical rehabilitation
Child care costs
Special educational programs
Institutional care
Lost leisure time
Psychological (psychic) costs (pain and other psychological suffering)
Risk aversion
Averting behavior costs
Extra cleaning/cooking time costs
Extra cost of refrigerator, freezer, etc.
Flavor changes from traditional recipes (especially meat, milk, egg dishes)
Increased food cost when more expensive but safer foods are purchased
Altruism (willingness to pay for others to avoid illness)
Industry costsb
Costs of food production
Morbidity and mortality of animals on farms, loss of crops, etc.
Reduced growth rate/feed efficiency and increased time to market for farmed animals
Costs of disposal of contaminated animals or crops
Illness among workers because of handling contaminated animals or ingredients
Control costs for pathogens at all links in the food chain
New farm practices (GAPs, HACCP, etc.)
New wholesale/retail practices (pathogen tests, employee training, procedures)
Risk assessment modeling by industry for all links in the food chain
Price incentives for pathogen-reduced product at each link in the food chain
Outbreak costs
Product recall
Plant closings and cleanup
Regulatory fines
Product liability suits from consumers and other firms
Reduced product demand because of outbreak
Generic animal product—all firms affected
Reduction for specific firm at wholesale or retail level
Increased advertising or consumer assurances following outbreak
(continued)

Societal Costs of Foodborne Illness
Regulatory and public health sector costs for foodborne pathogens
Disease surveillance costs to
Monitor incidence/severity of human disease by foodborne pathogens
Monitor pathogen incidence in the food chain
Develop integrated database from farm to table for foodborne pathogens
Research to
Identify new foodborne pathogens for acute and chronic human illnesses
Establish appropriate production and consumption practices for high-risk products
Identify which consumers are at high risk for specific pathogens
Develop cheaper and faster pathogen tests
Risk assessment modeling for all links in the food chain
Outbreak costs
Costs of investigating outbreak
Testing to contain an outbreak
Costs of cleanup
Legal suits to enforce regulations that may have been violatedc
Other considerations
Distributional effects in different regions, industries, etc.
Equity considerations, such as special concern for children
aWTP estimate for reducing risks of foodborne disease is a comprehensive estimate of all
these categories (assuming that individuals have included employer-funded sick leave and
medical programs in their estimates). The estimate is comprehensive and covers reduced
risks for everyone—those who will become ill as well as those who will not.
bSome industry costs may decrease with better pathogen control, such as reduced product
spoilage, possible increases in product shelf life, and extended shelf life permitting shipment
to more distant markets or lowering shipment costs to nearby markets.
cIn adding up costs, care must be taken to ensure that product liability costs to firms are
not already counted in the estimated cost of pain and suffering to individuals. However,
legal and court expenses incurred by all parties are societal costs.
Source: USDA, Economic Research Service, based on Todd, E.C.D. and T. Roberts. 1996.
Approaches to estimating the benefits and costs of foodborne disease control choices. In
WHO Consultation on Costs and Preharvest Treatment of Animals, June 8–10, 1995,
Washington, DC.
$1,584,605 in 1993 dollars (based on Landefeld and Seskin, [76]). This is compared with wage
studies suggesting that employed people would be willing to pay between $3 million and $7 million
(1990 dollars) to reduce the risks generating each additional death [112]. Because the COI method
is likely to give extremely conservative benefit estimates for publicly provided risk reduction, the
COI has been recommended as a lower bound estimate for WTP estimates [63]. Kuchler and Golan [75]
have reviewed strengths and weaknesses of different methods, with suggestions regarding how the
results should be interpreted.
Different data sources are used to determine the number (or range) of instances and deaths for
COI evaluations of foodborne diseases. According to CAST [21], these sources are: (1) national
surveys conducted by national heath departments, (2) passive and active surveillance of foodborne
disease, (3) risk models based on prevalence of pathogens in foods, and on infectious doses,
(4) case histories of individuals who have been infected, and (5) extrapolations by experts to obtain
estimates of the total number of instances and the disease severity distributions to account for
undiagnosed or unreported instances. Productivity losses include ill workers, poorly performing
workers because of discomfort and frequent medical checks, and those who die prematurely. Current
legal incentives to produce safer food in the United States are limited, and less than 0.01% of
instances are litigated and even fewer are paid compensation [16]. However, a settlement is more
likely in situations resulting from outbreaks in which foodborne illness can be more easily traced
to individual firms, and some settlements can be high.
In 2000, the CDC revised its estimates of foodborne disease, with 76 million cases and 5000
deaths each year in the United States [81]. Of these, 2298 cases requiring hospitalization and 499
deaths were caused by L. monocytogenes, and 62 of the nonfatal cases developed chronic compli-
cations. The ERS calculated that costs of these were $2.3 billion annually (Table 18.9). The
estimated costs include chronic disability or impairment resulting from congenital and newborn
infections, but exclude other chronic complications, less severe cases not requiring hospitalization
(the numbers were not estimated by CDC), time lost from work caring for sick children, travel to
obtain medical care, lost leisure time, and pain and suffering. Thus, $2.3 billion can be considered
a conservative estimate, and the annual costs from foodborne listeriosis would be substantially
increased if WTP to avoid disability, pain, and suffering were also taken into account.
The risk of contamination of RTE food by L. monocytogenes is of great concern to the food
industry, not only because of possible legal suits but because of recalls following the isolation of
the pathogen at any level by a regulatory agency. However, Hewitt [65] argues that there are weak
economic incentives for industry to improve because information on pathogen contamination or its
likelihood is hidden from the purchasing public. Because industry cannot earn a monetary premium
for safer products, food producers have little incentive to conduct research and development that
might enhance safety. Hewitt [65] suggests some economic incentives to reduce the incidence of
foodborne disease such as listeriosis:
1. Publishing more information on the inspection history and pathogen levels by plant.
2. Creating a consumer label for use on products produced meeting superior “pathogen
control standards.” This could be implemented by a joint industry–government body that
oversees approval and enforcement.
3. Creating special tax breaks for industry investing in new food safety inventions, or for
their adoption.
4. Increasing funding of epidemiological research to discover risks associated with various
production and consumption practices and behaviors.
5. Creating a mechanism for industry to have an incentive to share food safety information
with researchers. This might be done through an insurance mechanism that protects
industry from costs associated with an outbreak. Plants that share auditing information
and pathogen test results with researchers could participate in the insurance program at
a lower cost than plants that do not share information.
6. Better enforcement, higher fines, and more frequent pathogen testing may increase the
economic incentives to reduce the incidence of foodborne disease.
Items 1, 2, and 6 are already being carried out to some extent. National governments occasion-
ally carry out surveys of food products or processing plants and may release information on
prevalence levels of pathogens, but quantitative data are rarely published. One study by Gombas
et al. [61] does give such information, which was generated to provide input to a quantitative risk
assessment for L. monocytogenes in RTE products. So-called superior pathogen control standards
could be built upon HACCP plans, and performance criteria and food safety objectives established
[68]. Fines are probably less of an incentive compared with costs of a recall triggered by detection
of L. monocytogenes in a food product.
TABLE 18.9
Cost Estimates for Listeriosis
Cases Estimated Annual Costs
(Number) (Million U.S. Dollars)
Acute a
Medical
No medical care Unknown Not estimated
Physician visit Unknown Not estimated
Hospitalized and survived — —
Maternal 311 3
Newborn/fetal 368 14.1
Other adult — —
Moderate illness 58 0.6
Severe illness 1,062 30.6
Hospitalized and died — —
Maternal 0 0
Other adult 422 12.1
Subtotal 2,298 60.5
Productivity loss/premature deaths
No medical care Unknown Not estimated
Physician visit Unknown Not estimated
Maternal 311 0.6
Newborn/fetal 0 0
Other adult — —
Moderate disability 58 0.1
Severe disability 1,062 2.2
Hospitalized and diedb — —
Maternal 0 0
Other adult 422 1,514.30
Subtotal 1,930 2,187.10
Subtotal—Acute 2,298 2,247.60
Chronic (all newborn/fetal)c — —
Medical — —
Mild disability 11 0.5
Severe disability 11 6.5
Hospitalized and died 0 0
Subtotal 56 11.3
Productivity loss/premature deaths
Mild disability 11 4.8
Severe disability 11 17.9
(continued)

Cost Estimates for Listeriosis
Cases Estimated Annual Costs
Hospitalized and died 0 0

Subtotal 56 74.3
Subtotal—Chronic 56 85.6
Total 2,298 2,333.20

aData from the Centers for Disease Control and Prevention (from Mead, P.S., L. Slutsker, V. Dietz, L.F. McCaig,
J.S. Bresee, C. Shapiro, P.M. Griffin, and R.V. Tauxe. 1999. Food-related illness and death in the United States,
Emerg. Infect. Dis. 5) except for physician visits and hospitalized case subcategories, which were estimated by ERS.
bThese estimated cases and costs are for the congenital and newborn infections resulting in chronic disability or
impairment.
cCost calculations are based on the labor market approach for valuing the cost of premature deaths.
Source: Buzby, J.C. 2000. Estimated annual costs due to foodborne listeriosis. Economic Research Service, USDA.
http://www.ers.usda.gov/Briefing/FoodborneDisease/listeria/. Accessed December 23, 2004.
In 2001–2002, the New South Wales (NSW) Department of Health and the Commonwealth
Department of Health and Ageing funded a National Risk Validation Project [49]. The aims of the
project were to identify potentially high-risk food industry sectors, use risk assessment principles
to validate the categorization of selected sectors as high risk, and include costs and benefits of
implementing food safety programs in high-risk food industry sectors. The most frequently encoun-
tered hazards based on all the material reviewed were faulty temperature control of hazardous
foods, poor worker hygiene, cross-contamination, and contaminated raw materials. Food businesses
that ranked as high risk in order of priority were as follows:
1. Food service for sensitive populations

2. Producers, harvesters, processors, and vendors of RTE seafood
3. Catering operations serving food to the general population
4. Eating establishments
5. Producers of manufactured and fermented meats
Other areas of concern were processed raw foods not treated listericidally by heat, and processed
foods treated listericidally by heat but subject to potential recontamination during subsequent
handling. Scott et al. [92] adopted a value of NZ$2 million for the cost of a fatality in New Zealand
(approximately equivalent to A$2 million) but provided no details on how this figure was deter-
mined. In view of problems interpreting results when using the COI methodology for valuing a
statistical life, the National Risk Validation Project chose the WTP approach.
Costs were estimated for the top five high-risk businesses. For the 36 cases (33 hospitalized
and 7.1 dead) resulting from catering operations supplying food with L. monocytogenes to sensitive
populations, costs were estimated at A$39,098, of which a large proportion was attributed to the
deaths (A$35,722) and cost per meal of A$0.1108, higher than in any other category, served to
sensitive populations. For the 42 cases (39 hospitalized and 8.4 dead) resulting from contaminated
fermented and manufactured meats, costs were estimated at A$46,206, of which a large proportion
was also attributed to the deaths (A$42,217) and cost per meal of A$0.2310, the highest cost per
meal served by any of the businesses estimated.
In New Zealand an estimated 119,320 episodes of foodborne infectious disease occur annually
[92]. The total cost of these cases is NZ$55.1 million (NZ$462 per case) made up of direct medical
costs of NZ$2.1 million, direct nonmedical costs of NZ$0.2 million, indirect cost of lost productivity
of NZ$48.1 million, and intangible cost of loss of life of NZ$4.7 million. Campylobacteriosis
generated most of these costs. Lost productivity was the major cost component for all diseases.
Infectious intestinal disease in England and Wales occurs in one of every five people each year,
of whom one in six presents to a general practitioner [114]. The proportion of cases not recorded
by national laboratory surveillance is large and varies widely by microorganism. Listeriosis was
not included in organism-specific incidences based on cases for which a stool specimen was obtained
and analyzed. The U.K. Food Standards Agency (which spends 12% of its budget on foodborne
illness) is committed to reducing foodborne illness by 20% by 2006 [77]. The Food Standards
Agency estimates that foodborne illness currently costs the economy £350 million a year, whereas
other estimates have suggested the true cost may be as much as £1 billion per year. Another study
found that the care cost to the National Health Service for 100,000 people treated for food poisoning
in 1991–1994 was £83 million. These types of costs can be seen in relation to advertising costs of
almost £600 million per annum in the United Kingdom, the budget of which grossly outweighs
the promotion of improved diets and safety.
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19 Perspectives
Needs
on Research
Elmer H. Marth, Robert E. Brackett, R. Bruce Tompkin,

Sophia Kathariou, and Ewen C.D. Todd
CONTENTS
Introduction ....................................................................................................................................814
References.............................................................................................................................815
Research Needs: An FDA Perspective ..........................................................................................815
Introduction...........................................................................................................................815
Research Needs ....................................................................................................................816
Exposure of Consumers to L. monocytogenes.........................................................816
Behavior of L. monocytogenes in Foods..................................................................817
Pathogenicity ............................................................................................................817
Detection and Enumeration of L. monocytogenes ...................................................818
Elimination or Destruction of L. monocytogenes ....................................................818
References.............................................................................................................................819
Research Needs: An Industry Perspective.....................................................................................819
Significance of the FAO/WHO and FDA/FSIS Risk Assessments
to the Development of Control Strategies ...........................................................................819
Outbreaks vs. Sporadic Cases ..............................................................................................820
Reducing Consumer Exposure .............................................................................................821
Preventing Contamination ........................................................................................821
Postpasteurization .....................................................................................................823
Preventing Growth....................................................................................................824
Estimating Consumer Exposure ...............................................................................826
Concluding Remarks ............................................................................................................827
Acknowledgment ..................................................................................................................827
References.............................................................................................................................827
Research Needs: An Academic Perspective ..................................................................................830
What the Organism Has and Expresses: The Genomic and Proteomic
Endowment of L. monocytogenes ........................................................................................831
How, Where, and When L. monocytogenes Expresses Special Genome Components:
Listerial Pathogenesis, Ecology, and Stress Responses Further Considered ......................833
Concluding Thoughts ...........................................................................................................834
References.............................................................................................................................835
Research Needs: A Food Safety Perspective.................................................................................835
Regulatory Tolerance............................................................................................................836
Control Options ....................................................................................................................837
Risks Associated with Processed Food................................................................................838
813
Deli Meats ................................................................................................................838

More Focus on Retail Product .................................................................................838
Other Foods of Concern...........................................................................................839
Labeling and Consumer Education......................................................................................839
References.............................................................................................................................840
INTRODUCTION
ELMER H. MARTH
The bacterium later named Listeria monocytogenes [9] was first isolated from diseased laboratory
animals, characterized, and a description published in 1926 [8]. This publication did not result in
an immediate groundswell of research interest in the organism. Instead, for years afterward the
research consisted largely of reports of different species of animals and birds that suffered from
listeriosis. Transmission of the disease to humans who were exposed to infected animals was also
described. Other limited studies on the pathogen and the disease it caused were reported.
It was not until the 1940s through 1960s that a sustained research effort on the organism was
established by M.L. Gray and associates at Montana State College (now University). From this
work we learned, among many other things, about methods to isolate the organism [5,6] and that
it is widespread in the environment [4]. Sufficient research was done at Montana State College and
elsewhere, so at least two conferences, with published proceedings, were organized by Gray and
his co-workers.
Meanwhile, H.P.R. Seeliger began to do a lifetime of research on L. monocytogenes at the
University of Berlin and continued the work when he moved to the University of Würzburg.
Although Seeliger’s work emphasized disease aspects, he also studied other properties of the
bacterium, and in 1961 published his book, Listeriosis, in English [11].
In the late 1940s, there was some evidence in Germany of the possible role of food in human
listeriosis. However, at the time no one became sufficiently concerned to do research on this potential
cause of foodborne illness. This changed in the 1980s when there were three major outbreaks of
listeriosis in North America attributed to contaminated food.
The first outbreak involved coleslaw and prompted food microbiologists Robert Brackett and
Larry Beuchat of the University of Georgia–Griffin to initiate research on behavior of L. monocy-
togenes in vegetable products and on methods to isolate the bacterium from various foods [1,3].
The next outbreak involved pasteurized milk. Soon after this outbreak occurred, Ralston B. Read,
then of the FDA, encouraged food microbiologists Elmer H. Marth and Michael P. Doyle of the
University of Wisconsin–Madison to determine the behavior of L. monocytogenes during manufacture
and subsequent storage of nonfat dry milk and cottage cheese. Thus, a research program was initiated
in 1984 with results reported in 1985 [2,10]. These reports were among the first, worldwide, to describe
behavior of the pathogen during manufacture and subsequent storage of food products.
The third outbreak occurred in 1985 and involved Mexican-style cheese made and distributed
in California. This outbreak received considerable news media attention and also prompted the
scientific community to organize research programs to explore various aspects of the bacterium
and the disease. Thus, research efforts were undertaken by persons in such fields as food, veterinary
and medical microbiology, immunology, epidemiology, cell and molecular biology, clinical medi-
cine, pathology, and microbial taxonomy [7].
With this much effort being exerted, it was believed by the Society for Industrial Microbiology
that a conference to share the new information was needed. This conference was held in October
1988 in Sonoma Valley, California. Proceedings of the conference appeared in 1990 in a book titled
Foodborne Listeriosis [7]. A year later, in 1991, the first edition of the present book was published,
and it was followed by the second edition in 1999. The current edition again describes results of
recent and not-so-recent research.
Perspectives on Research Needs 815
After about 20 years of concentrated effort by researchers, do we still need more information
about the pathogen and the disease? We have all read or heard the expression, “further studies are
needed.” Does this apply to L. monocytogenes and listeriosis? Four experts were invited to shed
some light on this question. After you read their contributions, you, too, will agree that further
studies are needed.
REFERENCES
of Listeria monocytogenes in cabbage and cabbage juice. Can. J. Microbiol. 32: 791–795.
3. Golden, D.A., L.R. Beuchat, and R.E. Brackett. 1988. Evaluation of selective direct plating media
for their suitability to recover uninjured, heat-injured and freeze-injured Listeria monocytogenes from
foods. Appl. Environ. Microbiol. 54: 1451–1456.
4. Gray, M.L. 1963. Epidemiological aspects of listeriosis. Am. J. Pub. Health 53: 554–563.
5. Gray, M.L., H.J. Stafseth, and F. Thorp. 1950. The use of potassium tellurite, sodium azide and acetic
acid in a selective medium for the isolation of Listeria monocytogenes. J. Bacteriol. 59: 443–444.
6. Gray, M.L., H.J. Stafseth, F. Thorp, Jr., L. B Shell, and W.F. Riley, Jr. 1948. A new technique for
isolating Listerellae from the bovine brain. J. Bacteriol. 55: 471–476.
7. Miller, A.L., J.L. Smith, and G.A. Somkuti. 1990. Foodborne Listeriosis. Elsevier, Amsterdam.
8. Murray, E.G.D., R.A. Webb, and M.B.R. Swann. 1926. A disease of rabbit characterized by a large
mononuclear leucocytosis, caused by a hitherto undescribed bacillus Bacterium monocytogenes (n. sp.).
J. Pathol. Bacteriol. 29: 407–439.
9. Pirie, J.H.H. 1940. The genus Listerella Pirie. Science 91: 383.
10. Ryser, E.T., E.H. Marth, and M.P. Doyle. 1985. Survival of Listeria monocytogenes during manufacture
and storage of cottage cheese. J. Food Prot. 48: 746–750.
11. Seeliger, H.P.R. 1961. Listeriosis. Hafner Publishing Co., New York.
RESEARCH NEEDS: AN FDA PERSPECTIVE

ROBERT E. BRACKETT
INTRODUCTION
Although much has been learned about the many facets of the Listeria monocytogenes problem
over the past decade, it has also become clear that we still have much to learn about this organism,
how to detect it in foods, and its role in foodborne disease.
In 2003, the FDA and USDA published a quantitative L. monocytogenes risk assessment
(LMRA) [2] to determine the public health impact from L. monocytogenes in various foods.
An important benefit of conducting a risk assessment is the identification of knowledge, gaps
in available data, and research needs. In the course of collecting and evaluating the data for
the LMRA, it became apparent that additional data could enhance the certainty and reduce
variability in the risk assessment results. As a result, new data were generated specifically for
this risk assessment, and data were obtained from the published literature on levels of L.
monocytogenes in food, growth in deli salads, home storage practices, and other data. These
new data significantly improved the predicted risk estimates and reduced the amount of
uncertainty associated with those estimates. New data and information would also facilitate
development of commodity- or product-specific risk assessments. The research needs summa-
rized in the following paragraphs were identified as important in providing data needed to
continue filling existing gaps to facilitate future L. monocytogenes risk assessment work and
to improve efforts to reduce listeriosis.
RESEARCH NEEDS
Research needs are driven by the type of information needed for the FDA to fulfill its public health
mission. When dealing with L. monocytogenes, the type of information for which research is needed
falls primarily into five general categories, each of which will be discussed:
• Exposure of consumers to L. monocytogenes

• Behavior of L. monocytogenes in foods
• Pathogenicity of L. monocytogenes
• Detection and enumeration of L. monocytogenes
• Elimination or destruction of L. monocytogenes
Exposure of Consumers to L. monocytogenes
Before one can assess the potential public health impact of L. monocytogenes, it is necessary to
determine the concentration of the organism to which consumers are exposed and which they
ultimately consume, and the frequency of those exposures. Among the more obvious factors influ-
encing exposure is the level of contamination in the food itself. Although a variety of studies and
reports have been conducted to determine the presence of L. monocytogenes in foods, accurate and
precise data on concentration of this bacterium in foods are still lacking, particularly at concentrations
below 100 CFU/g. As will be discussed later in this subsection, the lack of good enumeration methods
for L. monocytogenes in foods has also hindered collection of enumeration data by the scientific
community. Moreover, “zero tolerance” (no detectable L. monocytogenes) regulatory policies have
resulted in a situation in which there is little practical need for the food industry to enumerate L.
monocytogenes in foods. Demonstration of its mere presence was sufficient information on which
to judge the acceptability of the product. Consequently, additional research is needed on precisely
how many viable L. monocytogenes cells are present in foods at the time of consumption.
Another important factor that affects exposure is how the food is actually handled and prepared
before consumption because preparation practices can affect survival of the bacterium. Estimating
survival of L. monocytogenes in foods intended to be eaten in a raw state is relatively straightforward.
However, the same cannot be said for all foods at all stages of preparation. Various surveys have
been done to determine estimates of specific foods that are being consumed, but these surveys
typically do not determine how the foods are prepared. Consequently, data on consumer food
preparation and eating practices are limited. A survey on home storage times for deli meats and
frankfurters was conducted [1], but additional information is needed for other food categories. Because
L. monocytogenes can grow during refrigerated storage, storage time and temperature are major factors
in the degree of hazard. Related factors include the time after opening the original package (particularly
if it is a vacuum or modified-atmosphere package) and likely subsequent cross-contamination, such
as might occur at a deli counter, or in the home refrigerator, or kitchen. More research into how
consumers purchase, store, and prepare foods would enable better estimates of the numbers of viable
L. monocytogenes consumed.
Finally, it is important to know the actual amount of specific foods consumed if one is to
estimate the potential contribution of those foods to overall listeriosis. The LMRA attempted to
quantify risk and in doing so had to determine the relative quantities of various foods that consumers
normally ate. Although this task on the surface appears simple and straightforward, risk assessors
very quickly found that this was not true. Indeed, there is a paucity of information on consumption
of specific foods in a form that lends itself for use in a microbial risk assessment.
The LMRA relied primarily on two food consumption surveys to provide estimates of food
consumption. However, these surveys were designed primarily for nutritional purposes rather than
for a microbiological exposure assessment. They were not designed to collect information on aspects
of food consumption related to food safety questions. Examples of such questions include whether
a cheese was made from unpasteurized fluid milk; whether the milk or juice that was consumed
was pasteurized or unpasteurized; whether smoked seafood is hot- or cold-smoked; whether peas
put into a pasta salad were freshly cooked, frozen, or canned; and whether steamed shrimp or crabs
and fried chicken were eaten freshly cooked or allowed to cool before eating.
Specific dietary information was limited or lacking for many of the susceptible subpopulations.
For example, data are not available to characterize consumption by elderly people living in nursing
homes or other forms of assisted living out of the home. In addition, better information is needed
about the health status of consumers. Future food consumption surveys that collect consumption
information of the type just described would greatly enhance the utility of their results for addressing
food safety as well as human nutrition issues. However, such information is also among the most
difficult to obtain.
Behavior of L. monocytogenes in Foods
One of the most well-recognized areas of research for which additional data are needed is behavior
of L. monocytogenes in different types of foods. Additional inoculated pack studies are needed on
selected foods to determine survival, growth rates, and maximum growth in the presence of normal
spoilage flora. Essential information from these studies should include the physical properties (such
as pH, aw, or NaCl concentration) of the food studied. In addition, future studies should be conducted
using a group of well-characterized L. monocytogenes strains, to allow for both direct comparisons
across foods and information on the diversity of strain responses. The growth and survival charac-
teristics of L. monocytogenes in certain foods is a related category of information that is needed
for better assessment of the potential role of foods in listeriosis. The risk of contracting foodborne
listeriosis is greatly reduced for foods that do not support growth of L. monocytogenes. Hence,
knowing which foods do and which do not support growth of the bacterium would allow industry
and government to better prioritize resources toward those of highest risk.
Pathogenicity
Public health and regulatory policies have typically been developed based on the assumption that
all L. monocytogenes isolates are essentially equally virulent and pathogenic. However, microbi-
ologists and clinicians have recognized for decades that this simple assumption is usually not true.
Listeriosis, as most other foodborne diseases, is a function of the genotype of the bacterium,
concentration of organism consumed, immunological state and age of the host, and a variety of
other known and unknown factors. The immunological response of an individual is critical in
determining whether an exposure to L. monocytogenes will result in clinical signs of illness.
Objective measures of the immune status of symptomatic and asymptomatic individuals would help
to provide a better assessment of an individual’s vulnerability to listeriosis. Likewise, information
about the role of the human immune system in preventing listeriosis is also limited. Most of the
information on resistance to L. monocytogenes infection has been obtained based on studies utilizing
laboratory animals, primarily mice. The relevance of these studies to immune mechanisms important
in human infection, particularly in pregnancy, should be investigated more thoroughly.
As suggested in the previous paragraph, much is still unknown about how and why L. monocytogenes
strains differ in pathogenicity. In animal models, there is at least a 5-log range in virulence among L.
monocytogenes strains. The traditional serotyping system (1/2a, 4b, etc.) is often cited as being
indicative but is not related to or based on specific virulence mechanisms. Development of
methodologies to rapidly quantify the virulence of strains would allow more effective assessment
of the public health threat of L. monocytogenes found in foods, and allow both food processors
and regulatory agencies to focus on those strains of greatest pathogenicity.
More also needs to be known about the effect of the food matrix and factors such as stomach
acidity, achlorhydria, and use of antacids on development of listeriosis. Such information would
be useful in understanding differential susceptibility in humans and would allow physicians to
provide specific advice to at-risk individuals on how to minimize their odds of contracting listeriosis.
Finally, more and better epidemiological studies are needed. A collection of data on attack rates
and consumption of L. monocytogenes would aid in the development of better dose–response
models. In addition, animal and biochemical tests need to be correlated to the epidemiological data
to enable assessment of L. monocytogenes isolates and to establish relevant biomarkers of human
susceptibility.
Detection and Enumeration of L. monocytogenes
Methods to detect L. monocytogenes in foods have improved greatly over the past 2 decades, with
detection times decreasing from 30 days to 48 h. Newer techniques are capable of detecting very low
populations of L. monocytogenes in many different types of foods. However, the relative sensitivity,
selectivity, and time frame for detection are still often dependent on the specific food matrix. To be of
practical use for regulatory purposes, methods must be precise and reproducible in addition to being
sensitive. Consequently, not only is additional research needed to improve existing methods of achieving
these characteristics, but also much more effort needs to be expended on properly validating the methods
in the wide variety of food matrices for which FDA has regulatory authority.
There is a growing trend internationally toward supporting the use of performance standards
or regulatory limits that would allow very low populations of L. monocytogenes in certain foods.
Such policies would require some use of enumeration to verify that the limits are being achieved.
However, despite the improvements in detection methods for L. monocytogenes in foods, the same
cannot be said about enumeration methods. The primary options for estimating populations of
L. monocytogenes in foods are much the same as they were in the 1980s, that is, the direct plating
and most probable number (MPN) techniques. Direct plating is relatively simple but is only capable
of providing population estimates exceeding about 100 CFU/g. In contrast, MPNs are capable of
providing estimates at lower concentrations, but this technique is very imprecise. Because typical
contamination levels in foods are less than 100 CFU/g, neither method is acceptable for reliable
and precise enumeration of the bacteria. Consequently, sensitive and precise enumeration methods
still need to be developed and validated for both research and regulatory actions requiring accurate
estimates of L. monocytogenes populations in foods.
Sampling strategies for detection and enumeration of L. monocytogenes in various foods
comprise another research need overlooked or ignored. Techniques for concentrating very low
populations of L. monocytogenes cells distributed throughout large volumes of samples would
improve sensitivity of methods. A related problem that needs to be addressed is how to develop
proper sampling plans that would ensure detection of L. monocytogenes in foods in which distri-
bution is nonrandom. Such research would likely require a more multidisciplinary approach than
is typically seen in microbiology, and require participation by food technologists, mathematicians,
statisticians, microbiologists, and professionals from even more unrelated sciences, such as physical
chemists.
Finally, there is a great need for research to determine appropriate surrogates and indicators for
L. monocytogenes in the food processing and natural environment. A thorough knowledge of the
microbial ecology of the bacterium could lead to identification of nonpathogenic indicators of con-
tamination or presence of ecological niches in which L. monocytogenes is likely to become established.
Likewise, such information would allow for better evaluation of testing programs often conducted as
part of Good Manufacturing Practices and Hazard Analysis Critical Control Point programs.
Elimination or Destruction of L. monocytogenes
Finally, there is still a great need for techniques that are capable of destroying or eliminating
L. monocytogenes on environmental harborages, food contact surfaces, and in foods. In particular,
innovative techniques to destroy L. monocytogenes in ready-to-eat foods that support their growth,
while still maintaining food quality, could greatly advance efforts to reduce the number of cases
of listeriosis. Although it is not generally the role or within the purview of regulatory agencies to
develop such techniques, their development in the academic or private sector would benefit
regulatory efforts by allowing regulatory agencies to shift their focus to foods and environments
in which L. monocytogenes would remain the highest risk. The FDA will likely continue to view
listeriosis as a significant public health threat into the foreseeable future. However, successfully
addressing the research needs just described would accelerate the agency’s efforts to eliminate
listeriosis associated with the foods it regulates.
REFERENCES
1. American Meat Institute. 2001. Consumer Handling of RTE Meats. (Unpublished data submitted to
Docket No. 99N–1168.)
2. Anonymous. 2003. Quantitative Assessment of Relative Risk to Public Health from Foodborne Listeria
monocytogenes Among Selected Categories of Ready-to-Eat Foods. http://www.foodsafety.gov/~dms/
lmr2-toc.html.
RESEARCH NEEDS: AN INDUSTRY PERSPECTIVE

R. BRUCE TOMPKIN
From an industry perspective, Listeria research generally falls into four categories: (1) elucidating
factors that lead to foodborne listeriosis, (2) preventing contamination of ready-to-eat (RTE) foods,
(3) developing lethal postpackaging treatments, and (4) preventing growth of Listeria monocyto-
genes on food. Through this research, control measures are developed and implemented to ensure
consumer protection and regulatory compliance. Prevalence and persistence of L. monocytogenes
in certain food processing environments indicate that long-term strategies are needed for consumer
protection. The strategies should lead to continuous improvement in reduction of listeriosis when
measured in cases per 100,000 population per year. Progress should be measured by an epidemi-
ologic system that is consistent from year to year and sufficiently sensitive to detect trends. Other
measurements can involve data from product samples collected at manufacturing plants, for example,
in the Food Safety and Inspection Service (FSIS) monitoring program, and/or at retail [18,31].
SIGNIFICANCE OF THE FAO/WHO AND FDA/FSIS RISK ASSESSMENTS

TO THE DEVELOPMENT OF CONTROL STRATEGIES
The Food and Agriculture Organization/World Health Organization (FAO/WHO) risk assessment
[19] evaluated the risk per serving and predicted the number of annual cases of listeriosis using a
distribution of L. monocytogenes levels in foods. For levels less than 100 CFU/g in all servings of
RTE foods eaten during an entire year in the United States, the model predicted 5.7 cases of
listeriosis per year. This outcome of risk assessment is important to industry because manufacturers
can strive to develop strategies to ensure that L. monocytogenes in RTE foods will not exceed a
specified level, as may be stated in a performance objective or food safety objective [13,36].
The FAO/WHO and Food and Drug Administration/Food Safety and Inspection Service
(FDA/FSIS) risk assessments both conclude that foods in which L. monocytogenes can multiply
are of greatest concern [19,21]. Thus, frozen, acidified, or dried foods or foods containing
additives to inhibit growth can be placed into a low-risk category. An important strategy for industry,
then, is to conduct research on medium- and high-risk foods to shift them to a lower-risk category.
Management strategies that focus on limiting the maximum concentration of L. monocytogenes in
food at the consumer level should have considerable public health impact toward reducing
listeriosis [15,19].
Research is needed to more clearly define the risk from specific types of foods within the
FDA/FSIS categories. For example, deli meats are considered high risk, but epidemiologic data
have identified only cooked noncured poultry breast products as an important source of foodborne
listeriosis. Numerous other deli meats (e.g., cured poultry breast products, ham, bologna, corned
beef, pastrami, roast beef, souse, headcheese, dry sausage, and loaf items) sold through the deli
counter have not been implicated. Thus, research (e.g., challenge studies) may be necessary to
better discriminate the level of risk associated with various products within these categories.
Research also is needed at the retail level, with an emphasis on the deli counter, to better understand
the risk factors leading to contamination of foods [18,56]. It is significant that the list of implicated
foods has changed very little over the past 10 years and, thus, consumer guidance from the Centers
for Disease Control and Prevention (CDC), FSIS, and FDA has changed very little. Is it possible
that the major food sources have been identified?
Smoked seafood has been categorized as a high-risk food [21] despite very little evidence
linking this product to listeriosis. Large quantities are consumed annually throughout the world. A
retail survey in two regions of the United States found the prevalence of L. monocytogenes in
smoked seafood of all types to be 4.31%, with 3 of 114 positive samples (2.6%) in the range of
103 to 106 CFU/g [31]. Similar or higher prevalence rates in RTE fish products have been reported
in other countries at the retail level, with some samples exceeding 103 CFU/g [17,39,40]. Why has
a stronger epidemiological link not been made with this food group? Does the ecology of cold-
smoked salmon operations, in particular, lead to strains that are less prone to cause illness or are
there other reasons? Even more broadly, why are certain strains found in food more virulent,
especially those few that have been responsible for major outbreaks in different regions of the
world [56,61]? To date, very few foods containing sodium nitrite have been implicated in listeriosis.
Is this because cured products normally contain a moderate amount of salt and the combination
creates a less favorable environment for growth? Frankfurters are an exception, but this might
reflect the greater risk of contamination because of the complexity of processes used for chilling,
casing removal, sorting, packaging, and the many potential harborage sites that have been detected
[61]. Industry has made considerable progress between 1990 and 2004 toward reducing the risk of
contamination of frankfurters, but exceptions remain [26,66].
OUTBREAKS VS. SPORADIC CASES

If continuous progress is to be made in reducing the incidence of listeriosis, conditions that lead
to sporadic cases must be resolved. Since 1988, CDC has conducted three case control studies to
evaluate the role of food in sporadic listeriosis. The foods identified have been undercooked chicken
and non-reheated frankfurters [58], soft cheeses, and foods purchased at deli counters [57] and,
more recently, Camembert cheese, hummus, and sorbet [65]. Experience has verified the signifi-
cance of frankfurters, certain soft cheeses, and certain foods purchased at the deli counter. Further-
more, it has been estimated that 99% of all listeriosis is foodborne [45]. However, do all cases of
listeriosis result from commercially manufactured foods? Will existing regulatory policies and
industry efforts be adequate to prevent sporadic cases? If industry were to succeed in consistently
providing foods with ≤100 CFU per serving, would the baseline of listeriosis actually decrease to
fewer than 10 cases per year, as has been estimated for the United States? Commercial facilities
have large kitchens and special equipment for preparing foods on a large scale. Because the presence
of L. monocytogenes in commercially prepared RTE foods is primarily caused by environmental
contamination, could similar contamination occur in other locations (e.g., restaurants, retail outlets,
catering, facilities for special events, and homes)? To what degree are such foods involved in
listeriosis? One example of this is the 1993 outbreak of gastroenteritis in Italy, which was most
likely caused by rice salad [55]. The salad of boiled rice, Swiss cheese, picked vegetables, hard-
boiled eggs, and mixed frozen vegetables was prepared 24 h in advance of a party and allowed to
remain at ambient temperature until serving. Average daily temperatures in that region during June
are 27–28°C. This dramatic event demonstrates the potential significance of food handling at the
home level. A few studies have suggested home refrigerators as a source of contamination, but
other potential sites also may exist in these locations (e.g., drains in sinks; seams along countertops;
can openers; blenders; slicers; cloth used for cleaning dishes, countertops, and refrigerators; and
other sites where food residue and moisture accumulate). If other food-handling locations are found
to be a source, what can be done to reduce illness?
REDUCING CONSUMER EXPOSURE

Preventing Contamination
The goal here is to minimize or prevent contamination during the time RTE foods are exposed between
a kill step and when the food is packaged for distribution. Considerable progress has been made in
environmental control. This is directly the result of industry’s willingness to share best practices and
treat pathogen control as a noncompetitive issue. Through workshops, publications, and other means,
continued progress has been made as new interventions have been identified, shared, and implemented
[1,28,35,63]. This practice should be continued to achieve further improvement.
Food manufacturers must work closely with equipment manufacturers to ensure that equipment
is cleanable, free of harborage sites, and performs as expected. Although there have been standards
for the design of dairy and egg processing equipment, this has not been true of meat and poultry
equipment since FSIS discontinued its equipment approval program. Dairy and egg processing
typically involves closed systems with equipment that must meet rigid standards of hygiene design.
Meat and poultry processing typically involves open systems with equipment that has lacked the
level of hygiene design necessary to control L. monocytogenes. This situation has been changing
through a joint effort involving equipment manufacturers and meat and poultry processors, and is
leading toward improved control [2,3]. Continued research and development is needed to eliminate
harborage sites in equipment as a source of L. monocytogenes [33].
Floors can be a significant source of listeriae in food manufacturing facilities unless an effective
program of control has been established. New sanitizers are being developed that have improved
microbial control and maintain their effectiveness over time on difficult surfaces. Less corrosive
sanitizers (e.g., granulated quaternary ammonium compounds and hydrogen peroxide in a powdered
form) have become available and offer two advantages. One is that floors can be maintained in a
drier state, and the other is that they replace granulated citric acid, an effective but corrosive sanitizer
that necessitates periodic floor resurfacing. New systems for delivering sanitizers have been devel-
oped and implemented (e.g., floor foamers at entrances of rooms and hallways). Some manufacturers
strive to control L. monocytogenes by maintaining dry floors during production. Others maintain
wet floors by applying a liquid sanitizer periodically during production. Some manufacturers have
installed elaborate systems for washing and sanitizing footwear as employees enter certain rooms,
whereas others avoid using footbaths. Some manufacturers fog a sanitizer into rooms as a final
sanitization step. The decisions to maintain dry or wet floors, install permanent footbaths, apply
sanitizer in the form of a fog, and adopt many other control measures are often made at the corporate
level and may not reflect the uniqueness of each establishment or the food that is being produced.
Data from the environmental sampling program should be evaluated and used to determine the best
approach for each plant.
An early observation was that the ratio of L. monocytogenes to other listeriae in the food
processing environment appears to be unique to each facility and tends to be relatively stable [62].
In any particular facility, for example, the ratios seem to be relatively stable over time. Why do
some facilities harbor a lower proportion of L. monocytogenes? Could it result from a decreased
ability to compete with the normal flora of the facility? This has given rise to the idea of using
competitive exclusion to reduce the presence of L. monocytogenes in food processing environments.
Is it really possible to manage the ecology of a food processing environment and control the presence
of a pathogen such as L. monocytogenes?
The significance of L. monocytogenes in drains remains highly debatable. Many companies
now maintain an aggressive program of cleaning and sanitizing floor drains. Some have replaced
conventional drains with new stainless steel drains for improved cleanability. Others assume that
L. monocytogenes will likely be present and merely clean and sanitize the basket and cover of the
drains. Many companies use solid drain rings containing a quaternary ammonium sanitizer that
slowly dissolves and is released. The significance of drains is further complicated by the FSIS
assumption that drains reflect the microflora in the surrounding area and finding L. monocytogenes
is evidence that the isolated strains likely came from nearby equipment and that product may have
been contaminated. This interpretation has led to regulatory action against some facilities. The
likelihood that a drain can be a harborage site in which L. monocytogenes can become established
and multiply should be considered in any investigation. It may be established that a drain is a
harborage site and the surrounding equipment is not involved or, when properly managed, the risk
of contaminating the product contact surfaces is low and controllable. In each instance, the likeli-
hood that L. monocytogenes can be dispersed from a positive drain to contaminate exposed food
should be investigated and proved, not assumed.
Experience has shown that equipment positioned close to a drain (e.g., the bottom of an incline
conveyor) is more likely to become contaminated. Some of this may be the result of splashing and
aerosols created during cleaning. Unclear, however, is whether drains “breathe” and contaminate
the immediate environment during production as headspace in the drains fluctuates when large
volumes of liquid flow through the drainage system of the plant. This could be evaluated by sampling
the immediate environment around drains to determine if the frequency of positives increases over
time and whether the frequency of positives decreases as distance from the drain increases. Although
this may provide better information on the significance of drains in the environment in which RTE
products are exposed, this information alone would not be sufficient to close the gap between the
significance of a positive drain and the probability of product contamination. Very little research
has been published on the role of drains in the overall hygiene of food operations [34].
The relevance of airborne contamination needs additional investigation. Efforts to recover L.
monocytogenes from the air in food operations have yielded negative results [5,33,37,41]. This has
been corroborated by unpublished industry results. Yet, regulatory action has been taken when an
agency has concluded that the air of a facility is a source of product contamination even in the absence
of supporting data. It is highly unlikely that a negative air pressure could account for equipment
surfaces or product samples testing positive repeatedly over days or months with the same PFGE type
of L. monocytogenes. Existing knowledge of food processing environments indicates that a variety
of strains would more likely be detected. Additional research is needed to clarify the significance of
ambient air. This question must not be confused with compressed air that can be a direct source of
contamination, for example, when an air filter is moist, dirty, and has become a harborage site.
Research is needed to better understand the diversity of L. monocytogenes strains found in the
food processing environment and on food from different facilities throughout the food industry.
One study found 10 pulsotypes were common to two or more types of food and 17 pulsotypes
were detected in foods of more than one producer having no apparent association with each other.
In addition, similar pulsotypes were recovered from foods in different European countries over
several years [6]. Other studies have found the same PFGE types of L. monocytogenes in different
facilities [39,44,54]. During an investigation of isolates from dairy farms and sporadic cases in the
U.S. Pacific Northwest, some strains matched those associated with previous epidemics (e.g.,
outbreak in 1983 in Massachusetts, Mexican-style soft cheese outbreak in 1985 in California, pâté
outbreak in 1988–1990 in the United Kingdom, sliced turkey deli meat in 2000 in the United
States). It was suggested that these strains might have an on-farm reservoir [10]. This information
demonstrates that certain strains of great public health significance are more widely spread
geographically and in nature than was previously thought.
Unfortunately, the existing regulatory climate in the United States that borders on “zero presence”
for L. monocytogenes in the environment in which RTE foods are exposed during processing and
packaging discourages such investigation. An untapped body of data, however, does exist within
the FSIS database for L. monocytogenes isolates from meat and poultry products. This database
should be used to help answer the diversity question. Because industry does not have access to the
FSIS data, this evaluation must be conducted by the agency. A study of 149 isolates collected by
the FDA from late 1999 to early 2003 found several strains in samples from different sources. The
most common ribotype pattern was found in samples from Chile, Germany, Spain, and seven
different states in the United States [30].
Monitoring programs by control authorities and others must consider whether the analytical
method may yield biased results for the strains detected. Analysis of food and environmental samples
typically involves an enrichment step before isolation. Enrichment of samples having more than
one strain in a selective medium (e.g., University of Vermont medium) is biased against detection
of those strains more likely to cause illness [11]. This bias can have great impact on epidemiologic
studies to identify a specific food or processing plant as the source of illness. The bias also raises
questions about programs such as the FSIS monitoring program for L. monocytogenes in RTE meat
and poultry products. Each isolate is subjected to PFGE analysis and the results shared with public
health agencies to determine if matches occur with clinical isolates. Research is needed to better
understand and correct the bias in the analytical method. Direct plating may be more productive
when analyzing foods in which growth may have occurred (e.g., food collected from retail or home
refrigerators) [11,43].
Existing methodology for assessing control of L. monocytogenes in food operations appears to
be adequate to detect a problem, if one exists. The sponge method has proved very sensitive provided
sufficient samples are collected from the environment and at frequent intervals (e.g., weekly) in
higher-risk environments. More rapid and/or more sensitive methods may be desirable, but this is
not a high priority for L. monocytogenes research. There is a greater need for a cost-effective system
that smaller manufacturers can use to assess control. In addition, when investigating the source of
a contamination that is detected infrequently but is cause for concern, hundreds of samples may
need to be collected over several weeks before the source is detected and corrections can be
implemented. Low-cost analytical methods are necessary for producers of all sizes when a difficult,
elusive problem must be resolved.
Two approaches that are being used for sampling the environment deserve comment. One involves
the zone concept, in which the emphasis is on detecting L. monocytogenes in zones of lower risk to
product contamination. Some proponents of this strategy suggest that if L. monocytogenes can be
controlled in lower-risk zones, then the likelihood that product contact surfaces and product will
become contaminated is reduced. This strategy does not ensure that a harborage site will not develop
in close proximity to product contact surfaces. There is a continuing need to sample product contact
surfaces at a frequency that can provide confidence that the focus on lower-risk zones continues to
be effective. A strategy that offers greater confidence is to routinely sample higher-risk zones
(i.e., product contact surfaces) and include a small number of samples from lower-risk zones.
The second approach is to create a list of sample sites associated with a processing line or
area. Sites are randomly selected from the list for each sampling interval. An inherent weakness
of this approach is that infrequent sampling of critical sites (i.e., product contact surfaces) may
not permit an assessment of the risk of product contamination. Site selection is a very important
attribute of this approach. The list of sites should consist of those that permit a short- and long-
term assessment of the likelihood that the food may be contaminated. Each manufacturer of
medium- and high-risk food should be able to assess on an ongoing basis whether the foods
being packaged on each line are likely to have become contaminated between the lethality step
and final packaging. For the present, each plant must develop the best sampling plan for its
situation and for the level of risk associated with the food that is being produced. Of equal or,
perhaps, greater concern is the urgency and effectiveness of the response by the manufacturer
when positive sample sites are detected.
Postpasteurization
The goal here is to ensure the absence of viable cells of L. monocytogenes on medium- and high-
risk RTE foods. With categorization of foods according to risk, food manufacturers are applying
newer technologies to shift their products from higher- to lower-risk categories. One method is to
apply a listericidal treatment after the food is in its final package. Many foods have traditionally
received a listericidal treatment immediately before (i.e., aseptically packaged foods) or after
packaging. Examples include canned shelf-stable foods, canned perishable hams and luncheon
meat, certain fluid dairy and egg products, puddings, and juices.
New equipment and packaging materials have been developed to expand the options available
for delivering listericidal treatments after final packaging, while retaining quality of product in an
acceptable package at a cost that is competitive. The lethality treatments generally fall into three
methods: heat, high pressure, and irradiation. Heat has been used commercially to postpasteurize
meat and poultry products for control of L. monocytogenes since at least 1990 and is now widely
applied throughout the world. Most of the systems involve passing the packaged product through
hot water or steam. Research and improvement in such systems continue [15,46,48,49], and more
should be encouraged. Similarly, high hydrostatic pressure is being used commercially for a variety
of foods. This technology shows great promise of broader acceptance if the cost and certain
performance issues can be resolved. Additional research on the use of high hydrostatic pressure,
alone or in combination with other control options, is needed [7]. Although extensive research on
irradiation has been done over the past 50 years and still continues [8,15, 16, 27, 60], this is not
an approved process for medium- and high-risk RTE products of concern. Application will require
approval by control authorities responsible for food protection at the national level and appropriate
labeling and education of consumers. Additional research and development is needed to improve upon
these technologies to provide more choices, greater flexibility in processing, minimal space require-
ments, reliable performance, minimal cost, and optimal product quality. Research is needed to learn
whether combinations (e.g., heat, high pressure, additives, changes in pH, etc.) can be more effective
than single treatments. Can synergism be demonstrated for certain combinations? In addition, con-
siderable research is necessary to validate the efficacy of these systems to ensure destruction of L.
monocytogenes in a variety of foods. For RTE meat and poultry products manufactured in the United
States, FSIS has not specified the log10 reduction required to validate the effectiveness of a postpas-
teurization process [23], but this is likely to change. Research is needed to understand the number
and distribution of cells that occur when RTE foods become contaminated before final packaging.
This information is critical to design of validation studies for control measures (e.g., lethality treat-
ments and additives to prevent growth).
Preventing Growth
The goal here is to manage the intrinsic and extrinsic factors to prevent multiplication of L. monocyto-
genes in foods between final packaging and when foods are eaten. Manufacturers of medium- and
high-risk foods should be informed that consumer risk is significantly reduced if L. monocytogenes
growth is delayed or, ideally, prevented, so that the concentration of L. monocytogenes remains low
throughout the code date on the package. This result of the Listeria risk assessments should drive
research to identify effective, approved additives that reduce consumer risk and manufacturer liability.
Intrinsic factors (e.g., pH, aw) of some foods prevent L. monocytogenes growth. Examples of
foods in which L. monocytogenes cannot multiply include citrus juices, yogurt, hard cheeses, and
dried foods. Foods that have been implicated as vehicles for listeriosis typically are high in pH
(e.g., >5.5), high in free moisture, and require refrigeration because of their perishable nature.
A high priority in the minds of some has been the relationship between samples from product
contact surfaces that test positive and the probability of product contamination. This should be
expanded to include an estimate of the numbers and distributions of L. monocytogenes that can
occur when product becomes contaminated. When antimicrobial agents are added to control growth
of L. monocytogenes, they should be validated for efficacy and the supporting data should be
available for review by control authorities [22–25]. One means of generating realistic data is to set
aside positive production lots and determine the initial level of contamination and the rate of growth
during storage. The information also could be used to understand whether inoculated pack studies
truly reflect the risk associated with the low level of contamination that likely occurs with natural
contamination. To what degree is the physiology of natural contaminants the same as laboratory
cultures? Are natural contaminants able to compete with the other flora that occurs on freshly
packaged product? Intrinsic and extrinsic factors associated with each food further complicate this
issue and may result in different responses in different foods.
There is some evidence that natural contaminants of L. monocytogenes may not grow on certain
foods stored under commercial conditions. This is suggested by a study on frankfurters in which
the rate of positive packages did not increase during extended storage at 4 and 10°C [66]. Further-
more, when unopened packages from lots that tested negative in the FSIS monitoring program were
held at 4°C for 6 weeks, only 18 of 1984 (0.91%) packages tested positive [66]. For cold-smoked
salmon, many packages from contaminated lots do not show multiplication of L. monocytogenes
during the normal shelf life. One reason appears to be the presence of other microorganisms (e.g.,
Carnobacterium spp.) that inhibit outgrowth of L. monocytogenes [12,32,51,52]. At very low
inoculum levels, an increasing salt concentration can become increasingly inhibitory. Stationary-
phase and sublethally injured cells have a lower probability of growth from low populations [53].
Additional research is needed to better understand conditions when growth from naturally contam-
inated foods will not occur.
Extensive research has been conducted on a wide variety of additives (e.g., chemicals and
bacteriocins), but very few (e.g., sodium and potassium lactate and sodium diacetate) have been
used commercially. A variety of antimicrobial agents coupled with other hurdles (e.g., reduced pH)
are being used to control pathogens such as L. monocytogenes in perishable foods. Many trade
associations (e.g., manufacturers of prepared salads) have sponsored research, some of which
has not been published, that is used as guidance for its members. A summary of research on the
use of additives to control growth of L. monocytogenes in RTE meat and poultry products is
included in the FSIS guidelines [24]. To this information should be added research on additives
in cold-smoked fish [38] and other foods. Research by Seman et al. [59] as well as Legan et al.
[42] has been used to generate software that enables manufacturers to formulate their RTE meat
and poultry products to control growth of L. monocytogenes. The fact that this information has
been made available to others in the industry is an excellent example of the commitment that
has evolved within many companies to help competitors enhance the safety of their products.
A variation on adding antimicrobial agents to food as an ingredient during formulation is to apply
a listericidal treatment, such as heat [29] and/or an antimicrobial agent, to the surface of a food product
(e.g., frankfurters and deli meat, or poultry) just before the food is packaged. Combinations of
pasteurization before and after packaging also have been explored [47]. Such treatments can have the
effect of killing microbial contaminants, such as L. monocytogenes, at the surface of the product and,
in combination with an additive, retard or prevent growth of surviving cells during subsequent storage.
Research should continue to identify additional listericidal treatments, additives, and combina-
tions of listericidal treatments and additives that kill or prevent growth of L. monocytogenes. In
addition to the research is the need to pursue regulatory approval for use of the treatments and
additives in a wide variety of foods. This would enable selection of one or more options for control
that provide the best fit for each specific type of food. Freezing, of course, is an option to prevent
growth of L. monocytogenes in certain foods.
Code-dating practices can influence consumer exposure to high concentrations of L. monocy-
togenes. The risk associated with perishable foods that may be exposed to contamination before
packaging can be reduced by using shorter code dates. The National Advisory Committee on
Microbiological Criteria for Foods has developed guidance that could be used to validate the safety
of “use by” dates that are applied by industry [50]. Similar guidance has been developed to ensure
that product shelf life is assessed in a standardized manner by European food producers and retailers
[9]. This guidance could be used to ensure compliance with European directives that require
establishment of appropriate shelf life dates for food.
Numerous issues must be resolved through research to arrive at an acceptable, standardized

method of validation. Among the issues is whether a laboratory-prepared culture of L. monocytogenes
will behave in the same manner as a natural contaminant of L. monocytogenes. The source of
the strains (e.g., food or clinical isolates), methods of preparing the inoculum (e.g., 24-h broth
culture), and method of inoculation (e.g., on the surface of the product) should be defined. What
is an appropriate level of inoculum? What levels (i.e., prevalence and concentration) actually
occur when a food is contaminated in a processing plant? Can strains having little or no virulence
be used to minimize risk among laboratory personnel? What constitutes a sufficient change in
a food, process, or package that would necessitate revalidation? What temperatures should be
used to represent the temperatures occurring throughout the food chain? What maximum con-
centration of L. monocytogenes is acceptable for code-date validation, i.e., what is the food
safety objective for L. monocytogenes in RTE foods? Some of these questions are being
investigated [64].
Estimating Consumer Exposure

Research is needed to better estimate consumer exposure to contaminated foods and, ideally,
estimate the number of cells consumed. As foods are identified as the source of listeriosis, a
statistically based sampling plan should be used to examine representative lots to develop estimates
of prevalence, concentration, and distributions. This is seldom possible, because the food is often
no longer available or too few samples remain. Occasionally, however, opportunities occur, such
as the 1998 multistate outbreak when approximately 100 illnesses were recognized. The recall of
all frankfurters and other cooked meat and poultry products with “sell-by” dates before
February 1999 involved about 35 million lb. Because only 5.9 million lb were recovered, it can be
assumed the remainder had been consumed. Although the quantity of frankfurters was not specified,
it is reasonable to assume that the amount of potentially contaminated frankfurters consumed
exceeded 5 million pounds. The plant reportedly produced 250,000 lb of frankfurters per day [4]
and, if true, the quantity of frankfurters produced between July 4, 1998, and February 1999 should
have been far greater than 5 million lb.
During 1998 antimicrobial agents were not added to frankfurters and many companies in
the United States used sell-by or use-by dates of 60 days or greater from the date of production.
Growth to levels exceeding 103 CFU/frankfurter (i.e., serving) would have been expected in
some portion of the packages. The potential for product contamination existed over an extended
period (≥6 months). During the 6 weeks before the July 4th weekend, about 25% of the plant’s
environmental samples tested positive for Listeria spp. After the July 4th weekend, when a cooling
unit was removed and replaced, the rate of positive samples reportedly increased during the
following 6 weeks to 92% and remained at nearly 70% over the subsequent 9 weeks of testing [4].
The contamination was attributed to construction dust; however, during removal of the refrigeration
unit, the room temperature would have significantly increased, allowing for a substantial increase
in the microbial population. Subsequent cleaning and sanitizing may not have been adequate to
cope with the increased population. The possibility of harborage sites from which a persistent strain
of L. monocytogenes could be dispersed over a period of weeks or months was not recognized at
the time by public health officials.
Retail frankfurters are normally manufactured to yield 8 or 10 per pound. It can be assumed that
more than one individual would have eaten frankfurters from many of the packages and, in many
instances, the frankfurters would have been eaten on multiple occasions until none in the package
remained. The recall notice does not provide production dates or number of production lots. The available
information suggests that consumer exposure was frequent and would have involved packages with high
numbers of cells. It is surprising that only approximately 100 instances of illness were recognized. Why
were so few cases recognized, given the probable extent of exposure? If a more complete analysis had
been made of the product and information made available to the agencies, an assessment, for example,
of the relative virulence of the unique strain may have been possible.
Although the foregoing description includes many assumptions, the information is provided
to illustrate the opportunity for expanding our knowledge of consumer exposure and the risk
of illness. More complete investigation and information gathering during such events could
enhance our knowledge of dose–response and exposure assessment for L. monocytogenes in
RTE foods.
CONCLUDING REMARKS
The road to controlling L. monocytogenes since the early 1980s has been long and difficult. The
food industry had no previous experience with controlling a human pathogen that was capable of
multiplying in refrigerated rooms in which perishable RTE foods may be exposed to contamination.
For many foods, research at the processing plant level and in the laboratory was necessary to, first,
understand the issues involved, second, develop strategies for control, and third, implement effective
controls appropriate for the foods of concern. Enormous changes have occurred throughout the
food industry since the year 2000, particularly in the U.S. meat and poultry industry, the source of
several major outbreaks. Additional improvements toward control of L. monocytogenes continue
to be developed through research, and it is anticipated that future risk assessments will have to
consider the many changes that have occurred within the medium- and high-risk food categories.
A conclusion of the FAO/WHO risk assessment is that compliance by industry to control contam-
ination and ensure low numbers of cells when foods are consumed is one of the keys to reducing
the incidence of listeriosis. This represents an important challenge to the food industry, but it also
clarifies the goal that must be achieved. Another outcome was the importance of temperature
throughout the life of perishable RTE foods. This will require an effective educational program to
achieve the desired results. Validation of the educational program’s effectiveness will be as important
as validation of other control measures applied throughout the food chain. Additional research and
application of the results are needed to achieve the desired level of control with optimal, cost-
effective systems.
ACKNOWLEDGMENT
Comments from Randy Huffman, American Meat Institute, and Jenny Scott, Food Products Association,
are gratefully appreciated and acknowledged by the author.
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RESEARCH NEEDS: AN ACADEMIC PERSPECTIVE

SOPHIA KATHARIOU
After an intense and close scrutiny of its physiology, genomic composition, and its interactions
with mammalian model systems during the past two decades, Listeria monocytogenes remains both
intriguing and interesting, and yet threatening. It is intriguing to those who see it as a multilayered
system with complex, sophisticated adaptations for impressively diverse environments, be it mam-
malian host cells, food processing plants, or the organism’s other habitats in nature; interesting to
those who see it as a paradigm for foodborne pathogens constantly responding to shifts in their
available habitats, often made possible through human, social, and economic trends that impact the
way food is processed, distributed, and handled; and threatening to those humans and animals for
whom it can cause frequently devastating illness. It also remains threatening in a related sense to
the food industry, the economic well-being of which can be seriously compromised when contam-
inated ready-to-eat products are recalled or implicated in outbreaks of listeriosis.
In the next series of research studies to come, I expect that we, as students of the organism,
will continue to pursue further understanding of the organism’s attributes that render it, in the
aforementioned sense, intriguing, interesting, and threatening. New generations of scientists,
equipped with an increasingly rich collection of the tools of genomics, proteomics, and biomath-
ematics, will carry the frontiers of Listeria research further, posing new questions. Interesting and
novel answers will be pursued to address fundamental problems of the sort that have occupied the
Listeria community heretofore; these can be summarized as questions of what virulence and other
features the organism has and expresses, as well as how, where, and when it does so.
Following are thoughts on some projected trends in studies along these lines, from the point
of view of one researcher involved in some of these issues, and what these trends might look like
during the next several years.
WHAT THE ORGANISM HAS AND EXPRESSES: THE GENOMIC AND PROTEOMIC
ENDOWMENT OF L. MONOCYTOGENES
A milestone in the study of the organism was the first complete genome sequence of L. monocy-
togenes (strain EGD-e), along with the sequence of the genome of L. innocua, a nonpathogenic
species closely related to L. monocytogenes [3].
L. monocytogenes was found to have a remarkably conserved genomic backbone compared to
its nonpathogenic relative. Underlying the identification and analysis of genomic segments found
in L. monocytogenes, but absent from L. innocua, was the realization that some of these genomic
segments would be specific to the strain or serotype of the strain that was chosen for genome
sequencing, strain EGD, of serotype 1/2a. Earlier genomic surveys had suggested that indeed strains
of other serotypes harbor genomic fragments unique to them and absent from the genome of serotype
1/2a strains [5,6]. The complete genome sequencing of three additional strains (one of serotype
1/2a and two of serotype 4b) confirmed and extended these findings [8].
The clonal population structure of L. monocytogenes suggests that detailed genomic analysis
of strains of diverse serotypes and clonal groups would be valuable, and indeed necessary, for an
adequate understanding of the genomic makeup of the species. Genomic initiatives that will reveal
the complete genomic sequence of several additional strains are now anticipated during the next
few years.
How will the choice be made of which new strains will have their complete genome sequenced?
This is an issue of considerable importance, because complete genome sequencing, even though it
currently can be accomplished in a relatively short period of time, remains a large, and expensive,
undertaking and should be designed to maximize use of its findings by scientists of as many diverse
research interests, backgrounds, and program objectives as may be possible. Ideally, funding
agencies would make such choices, following open discussion and evaluation of feedback by the
Listeria research community as a whole.
Leading in priority would be strains implicated in outbreaks. At this time, genome sequence
information has been reported for only three strains implicated in outbreaks of human listeriosis
[8]. A number of additional outbreak strains may be chosen for complete genome sequencing,
including (a) strain Scott A (serotype 4b) used in many food microbiology studies, (b) a strain of
serotype 3a implicated in a butter-associated outbreak, (c) and strains implicated in febrile gastro-
enteritis outbreaks.
Even though outbreaks receive most of our attention, the public health burden of clinical
listeriosis primarily involves sporadic cases. None of the four strains, the genomes of which have
been already sequenced, was from sporadic cases of human illness. The genome sequence of
selected strains from sporadic cases is now eagerly awaited. Choosing among isolates from sporadic
cases will be demanding and, again, should ideally be done in consultation with the Listeria
community. Special efforts should be made to utilize feedback from sources that can evaluate
temporal and geographical trends in sporadic illness incidence of isolates with specific genomic
fingerprints, such as the Centers for Disease Control and Prevention, the PulseNet database, and
equivalent agencies or databases in other countries. It would be valuable to include strains with
genomic fingerprints common among isolates from sporadic cases, as well as selected strains with
fingerprints that are unique, or rarely encountered. It would also be valuable to include represen-
tatives of the prevalent clinical serotypes (1/2a, 1/2b, and 4b) as well as of those serotypes rarely
encountered in human illness (1/2c, 3a, 3b, and 3c). Comparative genomic analysis of such isolates
would yield a wealth of information that may be critical to our understanding of how (and whether)
human virulence may be reflected at the genomic level of the microbe. In addition, genome
sequencing of selected isolates from a poorly understood group (Lineage III), primarily consisting
of strains of serotype 4a and 4c, would be valuable for furthering our understanding of the evolution
of L. monocytogenes. Lineage III strains appear to be significantly divergent from other members
of the species and were reported to be significantly more frequent among animal listeriosis isolates
than among those implicated in human illness [9].
In addition to strains chosen on the basis of their epidemiological significance, there is still a
keen need to know the genomic composition of isolates frequently implicated in colonization of
food processing plants and consequently in contamination of food products. Of interest would be
recently identified isolates from foods that harbor the genetic fragments typical of epidemic-
associated strains [10] as well as isolates that are frequently encountered in foods and processing
environments but are relatively rare in illness, for example, isolates of serotype 1/2c. An additional
major contribution in Listeria genomics would be the genome sequence of isolates from the
organism’s potential reservoirs in nature (animals, soil, vegetation, and others as yet unidentified).
These genome sequencing projects are expected to provide further information of the genomic
attributes that characterize different serotypes and clonal groups and may be unique to isolates with
special epidemiological or ecological (plant colonization, reservoir persistence) features. They
would ideally identify targets of special relevance for subsequent functional analysis. Such analysis
will further our understanding of the mechanisms that contribute to differential prevalence of certain
strains of the organism in illness, environmental (including processing plant) contamination, or
persistence in selected reservoirs. In addition, they have the potential to provide us with the
molecular tools for subsequent design of sensitive detection and monitoring systems not just for
L. monocytogenes as a species but of specific subpopulations within the species.
Current trends in technology suggest that design for such systems would likely employ array-
type formats that incorporate entire genomic sequences or selected signature sequences of diverse
serotypes and clonal groups. In addition to detection and monitoring of known clonal groups,
these systems would facilitate detection of emergent strain types, with genomic fingerprints that
deviate from those previously encountered. Clearly, successful application of such systems would
require construction and careful maintenance of a molecular signature database, for which
successful prototypes such as PulseNet already exist. Molecular subtyping with such high-resolution
systems would be greatly aided by suitable equipment for automated processing of the arrays.
Proteomic analysis will continue to be invaluable for characterization of bacterial responses to
different conditions and identification of likely targets for subsequent functional analysis. Although
proteomics-based tools can also be used for high-resolution subtyping of the organism, stringent
conformity would be required in conditions for growth of the bacterium, because the proteomic
profile (unlike the genomic content) would be highly condition dependent. Reaching an agreement
on conditions most suitable for proteomic analysis would therefore be crucial. Because regulation
of expression of a certain gene may differ among strains, and may be sensitive to even slight
changes in environmental conditions, conclusions that are related to strain-specific proteins would
need to be quite carefully formulated. Furthermore, regulatory changes within the genome that alter
the expression of specific genes may be selected in the process of growth and serial passage in the
laboratory, potentially creating further sources of artifacts and difficulties in use of proteomic
signatures for molecular epidemiology purposes.
Agreed-upon criteria on what differences actually are allowed for two isolates to be considered
“different” will need to be carefully thought through by the Listeria community for all software-
dependent applications that might involve large collections of data at the genomic or proteomic
level. Hopefully, development of such criteria would only occur following careful studies that
examine results obtained from a carefully selected library of strains. Such a library would include,
among others, strains that represent lineages that may differ in genomic stability, those that are
epidemiologically related isolates (multiple patient and food isolates from a confirmed common-
source outbreak), and finally, those isolates that are obtained following repeated transfers in the
laboratory. For proteomic analysis, it would be crucial to determine intra- and interlaboratory
reproducibility of proteomic profiles, and the possible perturbations of proteomic signatures by
unavoidable or subtle changes in growth conditions, such as those resulting from different lots of
the same growth medium.
HOW, WHERE, AND WHEN L. MONOCYTOGENES EXPRESSES SPECIAL GENOME

COMPONENTS: LISTERIAL PATHOGENESIS, ECOLOGY, AND STRESS RESPONSES
FURTHER CONSIDERED
A tremendous amount of information has been obtained to date on the mechanisms by which
L. monocytogenes interacts with mammalian host cells [2]. The next few years, nonetheless, promise
to further expand the frontiers and to help us understand how pathogenesis actually evolved in the
history of this organism.
Does L. monocytogenes interact with nonmammalian animal systems in ways that may have set
the stage for its evolving as a pathogen of mammals, including humans? The terrestrial ecosystem
which Listeria often inhabits in its “nonpathogenic” mode abounds in many animal forms that may
interact with the bacteria; protozoa, nematodes, insects and their larvae; all these may serve as
amplification reservoirs, and they may themselves be actually infected by the pathogen. In this context,
recent reports of Drosophila melanogaster as a potential model system for listerial pathogenesis [1,7]
remind us of the hidden surprises that the pathogenesis-related study of this organism holds.
Future studies will address whether virulence of Listeria to mammals is perhaps increased
following the microorganism’s passage through invertebrate hosts, as is known for other pathogens
[4]. If virulence is indeed enhanced following such passages, a subsequent question is whether this
contributes to the observed differences in the prevalence of certain serotypes, or clonal groups, in
human illness. These are questions that this author anticipates will be productively explored in the
next few years and, similar to many others, eagerly awaits the findings.
Further studies along these lines also hold potential for development of a new generation of
models for pathogenesis of the organism. Potential hosts with highly characterized genomes and
with available libraries of mutants of key developmental genes, such as Drosophila and the nematode
Caenorhabditis elegans, hold further promise of understanding the genetic give-and-take operating
in the evolution of pathogenesis, through implementation of a molecular version of Koch’s postu-
lates that encompasses both host and pathogen.
How the genome is expressed under selected circumstances, such as during infection (or coloni-
zation) of animals, or as the pathogen becomes established and persists in the processing plant, will
undoubtedly continue to be explored with heavy reliance on the accumulating genomic and proteomic
knowledge. In addition to currently pursued lines of research such as the expression arrays, new tools
will undoubtedly be developed to get glimpses of gene expression of the organism in situ.
There is little doubt that the currently developing appreciation of Listeria’s existence as a
component of biofilms will be strengthened and that gene and protein expression profiles of the
organism will be investigated in the actual surface associations that are of relevance both to its
ecology and its pathogenesis, and to the food industry. Currently our understanding along these
lines remains primitive.
In this context, a key component of interest is the associations of Listeria with other microbes.
Characterization of the microbial communities with which Listeria is associated in nature, and in
the processing plants and foods, will occupy the Listeria community in the next few years, with
hopefully intriguing results. Community-based perspectives will also reach out to other areas of
keen interest, such as the study of stress response. The stress response of the microorganism has
been extensively investigated in selected strains, but in the admittedly artificial, isolated system of
pure monocultures. Although such studies will still be needed to provide the database of how
individual strains respond to various stresses, the biological and ecological relevance of the findings
would be greatly complemented by investigations of how the organism responds and copes with
the stresses encountered in the actual microbial communities of which it is a denizen.
This necessarily includes further understanding of not only how Listeria may interact with
other bacterial species, but also how different strains of L. monocytogenes may interact with each
other. In ecosystems commonly serving as harborage sites in food processing plants, such as floor
drains, this author’s laboratory and others have found that multiple strains of L. monocytogenes
typically do coexist, although their population dynamics, physical associations, and interstrain
interactions remain completely unknown at this time.
The establishment and augmentation of a database of microbial consortia or communities that
might harbor the organism ought to be accompanied by bio-mathematical modeling of the com-
plexity and dynamics of community establishment and compositional shifts. Such modeling would
need to be developed to take into adequate account the distinct variables (community composition
and diversity, environmental conditions) that shape the quite different ecosystems that are of
relevance to the organism. Clearly, modeling of community shifts and strain composition, and
prevalence in the food processing plant may involve variables very different from those in ecosys-
tems such as soil and vegetation, or the gastrointestinal tract of animals.
CONCLUDING THOUGHTS
An expansion in the genomic and proteomic characterization of L. monocytogenes will continue
to occupy scientists in the next few years, and will also provide tools for targeted functional analysis
of the organism. Expected deliverable outcomes will include a new generation of detection, mon-
itoring, and subtyping strategies, not only with unprecedented potential but also with significant
challenges at the experimental and interpretational levels. These challenges would need to be
carefully addressed if the new tools and findings are to fully realize their potential for public health,
epidemiology, and the food industry. Other challenges are of nonscientific nature, and they involve
costs and accessibility of the technology.
Genomics- and proteomics-based tools are expensive, and their costs will likely remain high,
especially if the technology becomes proprietary and relies on automated equipment and standardized
reagents. It will be the task of an enlightened Listeria community to develop programs and venues
to ensure that high-resolution tools of the expected high potential will be accessible to all members
of the community for the purposes of further understanding the organism in the natural environment,
in the processing plant, and as a human and animal pathogen.
At the basic science level, the projected shifts from contained laboratory monocultures to
communities and surface-associated systems to be analyzed in situ will constitute novel venues for
the study of the organism and will result in findings that are currently nonexistent, enriching our
views of how the organism survives in relevant ecosystems and responds to environmental stresses.
Projected shifts from pathogenesis studies focusing on mammalian models (animals and cell lines)
not only may result in potentially novel models for pathogenesis studies, but will also elucidate
aspects of Listeria’s lifestyle that have remained largely hidden, including a whole spectrum of the
organism’s interactions with nonmammalian biota in its various habitats.
This author has little doubt that, with the proper direction of resources and encouragement of
young scientists, the next few years will bring to fruition a new generation of studies that will
continue to justify this organism’s reputation as both intriguing and interesting, and to clarify its
evolution as a pathogen of concern to public health, as well as to the food industry. In so many
ways, for students of Listeria, the best is still to come.
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in the life cycle of intracellular bacterial pathogens. Environ. Microbiol. 2: 251–265.
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RESEARCH NEEDS: A FOOD SAFETY PERSPECTIVE

EWEN C.D. TODD
In the United States, data for the passive foodborne outbreak reporting system at the state level
submitted to CDC may be unrepresentative of the real illness situation, and the information gathered
is typically incomplete. However, outbreaks that are investigated provide the best process to identify
the food vehicles associated with illnesses. Obtaining better data depends on the commitment by
the different public health authorities from local to national levels to provide the necessary resources
for extensive case interviews, along with the use of molecular typing methods such as PFGE to
match cases with implicated food products. This will allow policy making and inspection services
to better monitor the effectiveness of regulations and other programs to reduce listeriosis. At present,
it is unclear how much of a correlation exists between outbreak reports, FoodNet data on listeriosis,
and prevalence and concentration of L. monocytogenes in ready-to-eat (RTE) foods. For instance,
FSIS regulatory testing for L. monocytogenes showed a 25% reduction of positive samples collected
in 2003 over 2002, recalls dropped from 40 in 2002 to 14 in 2003, and there were no large outbreaks
involving meat and poultry products since 2003. The 2003 CDC FoodNet preliminary data showed
a slight increase in listeriosis incidence after a 4-year downward trend [2]; there were 3.3 cases of
listeriosis for every 1 million people in 2003, compared with 2.7 cases per million in 2002. This
translates into 951 cases in the United States, but this is different from the 629 cases of listeriosis
provisionally reported by the CDC Electronic Foodborne Outbreak Reporting System (EFORS)
[13] for the same year. The assumption is that the active reporting system (FoodNet) is more
accurate than the passive system (EFORS), and thus, while it would appear that L. monocytogenes
contamination is down, cases are probably up slightly.
How does a regulatory agency interpret this collection of information? Although it is recognized
that disease patterns have to be measured over several years in order to see long-term trends, these
types of challenges make policy setting difficult for food control. Regulators will view the con-
tamination reduction positively, but maybe some key foods are not being tested. For instance, they
may be sampling commercially packaged products but not those products that are supplied from
“under the table.” This issue has been raised for noncommercial Latino soft cheeses and some kinds
of ethnic sausages that have been linked to illness. Options for federally inspected establishments
in preventing product contamination and outgrowth in retail operations appear to be limited.
Regulatory strategies that attempt to project the responsibility onto inspected processing establish-
ments may not be effective in significantly reducing the likelihood of foodborne listeriosis from
the deli counter and other retail-produced items. As the quality of EFORS data improves, with the
use of PFGE to match cases and food products and with the use of an extensive case interview
form for all lab-confirmed cases, regulatory agencies like FSIS will be better able to correlate
changes in regulations with changes in illness. In time, it is hoped that active and passive systems
will be more coordinated, and data therefore better correlated.
In summary, long-term trends in the incidence of listeriosis reflect changes in regulation,
changing industry practices, consumption patterns, educational approaches, demographics, diag-
nostic methods, and health-care-seeking behavior. Consequently, the relative impact of regulations
will be difficult to measure precisely.
REGULATORY TOLERANCE
The United States and some other countries do not have any regulatory tolerance for L. monocy-
togenes in RTE foods. This can be interpreted as the most restrictive approach for limiting L.
monocytogenes contamination in products available for consumption. Yet, outbreaks have occurred,
and it is assumed that many sporadic cases have arisen since this rule was established. In fact,
based on disease surveillance data, rates do not appear substantially different in countries with or
without such a policy, even allowing for variability with different national surveillance systems
(0.3–7.8 cases per million population). A petition by industry groups is now with the FDA seeking
change in its policy to 100 CFU/g, similar to that in other countries. One argument presented by
USDA is that it is not in a position to set a regulatory tolerance partly because it cannot identify
the end users of the products (presumably some would be high-risk individuals with compromised
immune systems). However, the FAO/WHO risk assessment of L. monocytogenes in RTE foods
[4] indicates that nearly all cases of listeriosis result from the consumption of pathogen levels
exceeding 100 CFU/g by several orders of magnitude, even in immunocompromised subpopula-
tions. The FDA/FSIS/CDC [8] risk assessment demonstrates that the probability of mortality at the
5th percentile for the neonatal population (worst-case scenario) is 1 in 1.7 × 108. The model also
predicts that consumption of low numbers of L. monocytogenes has a low probability of causing
illness; for example, when the level of L. monocytogenes was assumed not to exceed 1000 CFU/g,
the number of cases in the United States would be less than 26 per year versus the estimated 2500
[4]. The report states that the vast majority of listeriosis cases are associated with foods that do
not meet current standards for L. monocytogenes in foods, whether the standard is “zero tolerance”
(e.g., <0.04 CFU/g) or 100 CFU/g. In other words, dealing with compliance is just as important
as setting a standard. For instance, the FAO/WHO model predicts that if a limit of 0.04 CFU/g
with a 0.018% defect rate (2133 cases) was replaced by a 100 CFU/g limit and a 0.001% defect
rate (124 cases), the predicted result based on the scenario would be an approximate 95% reduction
in foodborne listeriosis. Thus, risk assessments give government potential tools to alter policies so
that the public would be at even lower risk.
It can be argued that the zero-tolerance approach to dealing with L. monocytogenes was a cautious
enforcement policy based on the state of the science during the 1980s—a time when there was no
understanding of the ubiquity of the microorganism, nor effective methods for finding it in foods and
food processing environments. Since then, knowledge has increased sufficiently to be able to set a
regulatory limit. Regulatory agencies and the industry will then be able to focus their resources in
proportion to the level of public health significance on measures to provide a strong incentive for
development of products that do not support growth of L. monocytogenes, encourage aggressive
sampling programs, and facilitate collection of better quantitative data on L. monocytogenes.
CONTROL OPTIONS
Because problems still exist with RTE meats and poultry, FSIS has proposed three possible options
for industry to adopt to reduce the risk of L. monocytogenes contamination in RTE meat and poultry
products [10–12]. These include the use of (1) a postlethality treatment and addition of antimicrobial
agents, (2) a postlethality treatment or addition of antimicrobial agent, and (3) neither of these with
increased sanitation used as the only Listeria control strategy. Coupled with these alternatives are
differing degrees of verification testing, with alternative (3) requiring the most. Some details of the
testing were revealed in the January 2005 FSIS RTE001 program for sampling (postlethality-
exposed RTE meat and poultry products only) [13]. A survey of 2900 establishments found that
27% have started using an antimicrobial agent, 17% have started using postlethality treatments,
and 59% have started enhanced testing for L. monocytogenes or Listeria-like organisms. If done
properly, postlethality treatments will be effective in destroying the pathogen, and antimicrobial
agents will eliminate or at least prevent growth of the organism (presumably low numbers from
sporadic environmental contamination) to potentially hazardous levels. However, the concentrations
of these antimicrobials will likely show wide variability, which would compromise their effective-
ness. Establishments that produce certain high-risk products may be able to use levels of lactate
that are acceptable for incorporation into their product, but likely ineffective. Attempting to inhibit
L. monocytogenes outgrowth with greater lactate/diacetate combinations, and at higher levels, alters
the palatability and marketability of the product. However, some low-risk deli products containing
antimicrobial agent formulations may also represent a significant hazard in retail deli operations if
refrigeration is not properly controlled, especially where these are sliced, handled, and packaged.
These agents become increasingly less effective in inhibiting outgrowth as the storage temperature
rises. The only truly effective way to verify and validate the effectiveness of an antimicrobial agent
in a product is to conduct challenge tests using inoculated product held throughout the shelf life.
Even if these controls are followed, problems may remain. Any L. monocytogenes-positive
products produced under alternative (2) (use of antimicrobials to prevent growth) would still be
subject to recall. The degree of reduction of Listeria contamination is uncertain because the new
testing procedures will be the only control measure under alternative (3), which may represent over
50% of all RTE operations. Alternative (3) is statistically based and depends on development and
implementation of a regular and adequate testing regimen for food contact surfaces in the particular
facility. However, unlike the use of postlethality treatments and antimicrobial agents, verification
testing is very much dependent on worker expertise and commitment. The noncompliance (NC)
records are well documented from very small plants (56%), and most establishments with NC
records had chosen alternative (3) (the least protective alternative) to control L. monocytogenes. In
addition, most L. monocytogenes-related NCs concern fully cooked, perishable products. Managers
and employees in very small plants will most likely have the least knowledge and expertise of all
producers of RTE meat and poultry products. Because companies typically look for Listeria spp.
rather than L. monocytogenes, due to the cost of testing and the adverse consequences of finding
a positive test result, a minimum number of samples are likely to be tested with a possible cover
up of any undesirable findings. In other words, the human element is much more vulnerable to
lack of compliance than a measurable heat treatment or addition of a specified amount of an
antimicrobial agent, both of which would be carefully monitored at critical control points in the
HACCP plan.
Retail delicatessens present a potential public health concern but are not currently under the
active purview of a federal agency. Most states have adopted the Food Code as their regulatory
food safety framework. Unfortunately, retail delis complied with all Food Code controls only some
of the time [7].
RISKS ASSOCIATED WITH PROCESSED FOOD

Deli Meats
The FDA/FSIS/CDC [8] risk assessment indicated that deli meats were the products associated
with the highest number of cases. Outbreaks involving deli meats have been reported over several
years and recalls occur on a regular basis. In one outbreak in 2002, 54 people were diagnosed with
listeriosis associated with consumption of sliced meat, which resulted in over 27 million pounds
of product at risk. Although much of the product had been consumed or discarded, the company
experienced more than $100 million in lost sales. Scenarios to evaluate the impact of changing the
maximum storage time were run for deli meats, with the simulation end point being the predicted
annual mortality for the elderly subpopulation. The simulations showed that reducing storage time
by half (28-day baseline to 14 days) reduced the median number of listeriosis cases in the elderly
population from 228 to 197 (13.6%), whereas shortening storage time to 10 days further reduced
the cases to 154 (32.5%). The exercise concluded that achieving a 50% reduction in listeriosis
cases from consumption of deli meats would require eliminating storage above approximately 8°C
or all storage times longer than 8 days. The interaction of modifying both storage time and
temperature was also simulated. An example of a combination that would reduce cases of listeriosis
by 50% was 10°C for no more than 11 days. This assessment was based on generic deli meat that
supported Listeria growth.
The ILSI report on Achieving Continuous Improvement in Reductions in Foodborne Listeriosis—
A Risk-Based Approach [9] outlines the five most effective strategies to control L. monocytogenes in
high-risk foods:
1. Good manufacturing practices, standard sanitation operating procedures and HACCP

programs to minimize environmental L. monocytogenes contamination and to prevent
cross-contamination in processing plants and at retail
2. An intensive environmental sampling program in processing plants along with an effec-
tive corrective action plan to reduce the likelihood of contamination of high-risk foods
3. Time and temperature controls throughout the entire distribution and storage period including
establishing acceptable storage times for foods that support growth of L. monocytogenes
to high numbers
4. Reformulating foods to prevent or retard L. monocytogenes growth, and using postpackaging
treatments to destroy L. monocytogenes on products
5. Using postpackaging treatments to destroy L. monocytogenes on products
FSIS is incorporating four of these approaches into the Interim Rule (2004) [9], but much less
emphasis is placed on the third strategy to have effective time and temperature controls.
More Focus on Retail Product
The entire Interim Rule [12] has focused on minimizing Listeria contamination of RTE meat
and poultry products at the producer level. Only in the FSIS December 2004 Docket are potential
problems at retail, such as Listeria transfer and cross-contamination during slicing and pack-
aging, mentioned. Although some research is being done to estimate the contribution of these
operations to the overall caseload, more work is clearly needed. This is important because the
risk of illness from delicatessen-sliced meats is approximately seven times higher than pre-
packaged sliced products coming directly from the manufacturer. In the survey by Gombas
et al. [6], 0.4% of prepackaged, compared to 2.7% of delicatessen-sliced luncheon meats, were
contaminated with L. monocytogenes. Even more problematic is the fact that four of every five
consumers purchase deli-sliced meats as opposed to products sliced and packaged at the manu-
facturing facility. Given an estimated 320 fatal listeriosis cases each year from RTE meat products,
242 of these deaths could be considered associated with the consumption of delicatessen-sliced
luncheon meats alone. Problems with retail delis have been documented. Only 73% of them
complied with all Food Code controls [7]. Proper holding time and temperature were observed
only 43.3% of the time, and opportunity for equipment to become contaminated was seen over
79.4% of the time. Both of these are critical controls for RTE foods. The Docket [12] indicates
that small and very small establishments need better guidance, and a similar strategy will be
needed for retail establishments. At present, the incidence of L. monocytogenes on food contact
surfaces within delicatessen environments remains unknown along with the most likely routes
of contamination. These two areas, along with more vigilant training of delicatessen workers,
are in need of further study.
Other Foods of Concern
Deli meats and pasteurized milk are widely consumed by the population at large. However, there is
some risk for RTE foods less frequently eaten by the general population being contaminated and causing
listeriosis. These include smoked fish, crustaceans, pâtés, and some cheeses. Certain aficionados, who
are often in the higher-risk category such as the aged, may consume these frequently. Cheeses of
concern are soft unripened varieties (usually Hispanic or Latino in North America) and fresh soft
cheeses made from unpasteurized milk. Smoked fish and raw milk cheeses may be sold by small family
operations “under the counter” or at the farm, and are not identified during routine retail store inspec-
tions. These products are sought after by gourmands or those with strong cultural links to the product,
and their purchase is difficult to monitor, let alone control. In countries or states where dairy regulations
prevent consumers from buying raw milk in stores or directly from farmers, some individuals are
entering into cow share or farm share agreements with the farmer. In a cow share agreement, consumers
pay a farmer a fee corresponding to the share owned for boarding, caring for, and milking the cow [5].
The cow shareowner then is given milk from the cow without any formal purchase being conducted.
This arrangement is similar to arrangements of owning a share in a racehorse or a bull. Where the
regulations specifically forbid cow share agreements, consumers and farmers may set up corporations
in which consumers hold nonvoting shares, although these are more difficult and expensive to set up
than a cow share program. The same approach can be made for any products made from raw milk.
Other would-be consumers tell the farmer that they are using the milk for their pets, but they drink
most of it themselves. Arguments given for seeking out raw milk products are that milk from the cow
is a highly health-promoting fresh food, and pasteurization damages its quality (by destroying enzymes,
vitamins, and beneficial bacteria, denaturing milk proteins, and promoting pathogens through lack of
competition by the beneficial lactic acid organisms) [1]. There is also a cultural element in the
consumption of raw milk products and other traditionally produced RTE foods for centuries without
any large-scale concern. Thus, eliminating the small proportion of any population consuming such
RTE foods with increased risks of listeriosis and other enteric diseases will be very difficult.
LABELING AND CONSUMER EDUCATION

Labeling to advise consumers that products have received some type of treatment to reduce contam-
ination (such as postlethality treatment or added antimicrobial agents) needs to be considered along with
safety-based dates and advice on proper storage conditions. The interim final rule states that establish-
ments can declare any processing methodology on their label that they use to address L. monocytogenes.
However, at present, no company is using such incentive labeling on a voluntary basis. Although such
information could be perceived by industries as an advantageous marketing strategy, it is not certain
how the public would respond. At present, it is probable that industry perceives that labeling statements
would be too confusing or misinterpreted by consumers. More research is needed on labeling as an
effective means of educating consumers about proper food storage and handling practices, including
the use of focus groups, to help develop labeling statements. However, growth studies are also required
to determine how long and under what conditions RTE foods can be stored before L. monocytogenes
will reach unacceptable levels at the time of consumption. The FSIS Interim Rule [12] suggests no
more than a 2-log increase, which could be interpreted as 100–1000 CFU/g, assuming that most
processed packages contain no, or very low levels of, L. monocytogenes, e.g., < 0.04 CFU/g. However,
any “best consumed by” date will depend on the product, storage temperature and length of storage.
Different labels also may be required for different types of consumers (high vs. low risk). Science-
based education and risk communication strategies aimed at susceptible populations and focused on
high-risk foods should be delivered through health-care providers or other credible sources. Exquisitely
sensitive consumers may become ill when exposed to low numbers of L. monocytogenes or other
opportunistic pathogens. So reducing the risk to this population could be achieved by maintaining
them on restricted low microbial diets during those periods when they are most severely immuno-
compromised. The ILSI report [9] indicates the critical importance of education and communication.
High-risk individuals, i.e., the elderly, pregnant women, and most immunocompromised individuals,
should receive guidance on healthy eating habits, including specific information on high-risk foods
that should be avoided, and strategies to reduce their risk, such as thorough cooking, avoidance of
cross-contamination, and short-term refrigerated storage of cooked perishable foods. Those at low
risk for listeriosis should receive information on safe food handling practices, preferably starting at
a preschool age. L. monocytogenes labeling statements should be further developed by conducting
focus group research studies to produce statements that would provide flexibility to the industry while
still remaining truthful and not misleading. Consumer education messages can also be delivered using
multimedia channels, e.g., radio and television networks for Hispanic or Arab-American communities,
Web sites, and mobile exhibits and community health fairs. Apart from consumer education, there
are education and training needs for regulatory agencies, especially for inspecting personnel when
new approaches are being taken for risk-based policies.
REFERENCES
1. A Campaign for Real Milk. 2005. Cow Share and Management Agreement. http://www.realm-
ilk.com/cowshare-austr.doc. Accessed March 21, 2005.
2. CDC. 2004. Centers for Disease Control and Prevention. Preliminary FoodNet data on the incidence
of infection with pathogens transmitted commonly through food—selected sites, United States, 2003.
Morb. Mort. Wkly. Rep. 53: 338–343.
3. CDC. 2004. Centers for Disease Control and Prevention. Food Outbreak Response and Surveillance
Unit Electronic Foodborne Outbreak Investigation and Reporting System (EFORS) http:
//www.cdc.gov/foodborneoutbreaks/reporting_outbreak.htm. Accessed March 21, 2005.
4. FAO/WHO, Buchanan, R., R. Lindqvist, T. Ross, M. Smith, E. Todd, and R. Whiting. 2004. Risk
Assessment of Listeria monocytogenes in Ready-to-Eat Foods—Technical report. Microbiological
Risk Assessment Series 5, 304 pp.
5. Fausett, M.R. and K.C. Dhuyvetter. 1995. Beef Cow Leasing Arrangements MF-2163. Kansas State
University Agricultural Experiment Station and Cooperative Extension Service. http: //www.oznet.ksu.edu/
library/agec2/mf2163.pdf. Accessed March 21, 2005.
6. Gombas, D.E., Y. Chen, R.S. Claver, and V.N. Scott. 2003. Survey of Listeria monocytogenes in ready-
to-eat foods. J. Food Prot. 66: 559–569.
7. FDA. 2000. Report of the FDA Retail Food Program Database of Foodborne Illness Risk Factors,
prepared by the FDA Retail Food Program Steering Committee. http://www.cfsan.fda.gov/acrobat/
retrsk.pdf. Accessed August 7, 2006.
8. FDA/FSIS/CDC. 2003. Center for Food Safety and Applied Nutrition, Food and Drug Administra-
tion/Food Safety and Inspection Service, USDA and Centers for Disease Control and Prevention.
Quantitative assessment of the relative risk to public health from foodborne Listeria monocytogenes
among selected categories of ready-to-eat foods. http://www.foodsafety.gov/~dms/lmr2-toc.html.
9. ILSI. 2004. Achieving continuous improvement in reductions in foodborne listeriosis. reduction in
foodborne listeriosis, draft report by an expert panel. International Life Sciences Institute, Washington,
DC. http: //www.ilsi.org/publications/pubslist.cfm?publicationid=541. Accessed March 21, 2005.
10. USDA. 2003. Control of Listeria monocytogenes in Ready-to-Eat Meat and Poultry Products; Final Rule,
9 CFR Part 430. Federal Register 68 (109): 34207–34254. http://a257.g.akamaitech.net/7/257/2422/
14mar20010800/edocket.access.gpo.gov/2003/pdf/03–14173.pdf. Accessed March 21, 2005.
11. USDA. 2003. Verification Procedures for the Listeria monocytogenes Regulation and Microbial Sam-
pling of Ready-to-Eat (RTE) Products for the FSIS Verification Testing Program. FSIS Directive
10,240.4, October 2, pp. 1–25. http://www.fsis.usda.gov/oppde/rdad/FSISDirectives/10240-4.pdf.
Accessed March 21, 2005.
12. USDA. 2004. Control of Listeria monocytogenes in Ready-to-Eat Meat and Poultry Products. 9 CFR Part
430. Federal Register 69(231): 70051. http://www.fsis.usda.gov/oppde/rdad/frpubs/97-013FE.pdf.
Accessed March 21, 2005.
13. USDA. 2004. FSIS Notice Listeria monocytogenes Risk-Based Verification Testing Program–Phase
1: Introduction of a New Sampling Project—RTE001 61-04. December 23, pp. 1–5.
Index
A Animal listeriosis
animals, transmission to, 57–72
Aarnisalo studies, 291 cattle, 62–66
Abachin studies, 118 crustaceans, 71–72
Abdalla studies, 467 ecology, 40–42
Abdel-Gawad studies, 443 fish, 71–72
Abou-Donia and Al-Medhagi studies, 467 fowl, 67–69
Abou-Eleinin studies, 366 fundamentals, 55
Academic perspective goats, 60–62
food safety perspective, 835–840 humans, transmission to, 57
fundamentals, 830–831, 834–835 incidence, 56
gene expression, 833–834 minor species, 69–71
genomic endowment, 831–833 predisposing factors, 56–57
proteomic endowment, 831–833 sheep, 57–60
Accessory virulence factors swine, 66–67
catalase, 131 transmission, 40–42, 57–72
fundamentals, 130 treatment, 72
iron uptake systems, 132 Anjaria, Brahmbhatt and, studies, 522
protein p60, 130–131 Anjou, France, 37
stress response mediators, 131–132 Anne (Queen of England), 2
superoxide dismutase, 131 Antelope, 70
Acidity, 169, 169–171 Antibody-based methods, 264–267
Acid sanitizers, 194 Antimicrobial agents and additives, 589, 598–599
Acriflavine, 221–222 Antimicrobial components, foods
Active packaging, 196–197 antioxidants, 180–181
Adak studies, 98 benzoic acid derivatives, 175, 178
Addison, Farber and, studies, 292–293 extracts, plant, 181–182, 182
Adesiyun studies, 522 fatty acids, 178–180
AFLP, see Amplified fragment length polymorphism food processing importance, 174–184
(AFLP) free fatty acids, 178–179, 179
Agents, selective, 219–223, 220 herbs, 181–182, 182
Aguado studies, 661, 745 hydrogen peroxide, 183
Ahmed studies, 467 lactate, 175, 176
Al-Azawi, Al-Ghazali and, studies, 30 lactoferrin, 184
Al-Ghazali and al-Azawi studies, 30 lactoperoxidase system, 183–184
Al-Medhagi, Abou-Donia and, studies, 467 lysozyme, 182–183
Alpas and Bozoglu studies, 673 monoesters, 179–180
Al-Sheddy and Richter studies, 579 organic acids, 175, 175–178
Alternative processing technologies parabens, 175, 178
fundamentals, 187 plant extracts, 181–182, 182
high-intensity pulsed light, 188–189 potassium sorbate, 175, 177–178
high-pressure processing, 189, 189 salt, 174–175
ionizing radiations, 187–188, 188 smoke, 181
irradiation, 187–189 sodium benzoate, 175, 178
pulsed electric field processing, 189–190, 190 sodium diacetate, 175, 176–177
ultraviolet radiation, 188–189 sodium nitrite, 180
Amelang and Doores studies, 391 sodium propionate, 175, 177
Amezquita and Brashears studies, 558 spices, 181–182, 182
Amoril and Bhunia studies, 516 Antimicrobial susceptibility testing, 286
Amplified fragment length polymorphism (AFLP), Antioxidants, 180–181
294–295 Arias studies, 364
Amtsberg studies, 528 Arimi studies, 36, 39
Animal feeds, 32–34 ARS-MMLA media, 229–230
843
Arumugaswamy studies, 662 modified-atmosphere packaging, 548–549, 549

Ashenafi studies, 446 transmission pathways, 43
Ashton, Wesley and, studies, 37 Beerens and Tahon-Castel studies, 221
Asia, 662 Behavior, FDA perspective, 817
Asperger studies, 452 Behavior, fermented products
Aspic, 328–329 bacterial surface-ripened cheeses, 454–456
Asymptomatic carriage, 91, 92–93, 93 blue cheese, 453, 454
Asymptomatic carriers, 67 brick cheese, 455–456
Attachment to surfaces, 190–192, 191 brine solutions, 483–486, 484–486
Aureli, Gianfranceschi and, studies, 666 Bulgarian white-pickled cheese, 467
Australia Camembert cheese, 449–453, 450–451
foodborne infection, 332 cheddar cheese, 459, 460, 461–462
food processing, facilities, 747–748, 748 Chihuahua cheese, 465
listeriosis outbreak, 332 coagulants, 447–448, 448
risk assessment, 773–774 colby cheese, 458–459, 459
standards and criteria, 791–793 cold-pack cheese food, 476–477, 476–477
Austria, 796 coloring agents, 448–449
Autio studies, 631, 638, 744 composition of cheese, 478–481, 479–480
Autoclaved milk, 380–387 cottage cheese, 471–474, 472
Automation, 259 cream cheese, 474
Avery studies, 548 Domiati cheese, 467
Ayre studies, 444 ewe’s milk cheese, 468–470
Aytac and Gorris studies, 667 feta cheese, 465–467, 466
fundamentals, 446
B goat’s milk cheese, 468–470
Gouda cheese, 458
Babic studies, 670
hard cheeses, 457–463
Bachmann and Spahr studies, 456
hard Italian-type cheese, 463
Bacillus relationship, 4
Hispanic cheeses, 464–465
Back studies, 480
Kachkaval cheese, 468–469
Bacteria identification, Listeria fundamentals
Maasdam cheese, 458
biochemical characteristics, 10
Manchego cheese, 464–465, 469
culture, 9
Mexican Manchego cheese, 464–465
genus characteristics, 9–10
mold-ripened cheeses, 449–453
metabolism, 10
mozzarella cheese, 457
morphology, 9
Parmesan cheese, 462–463, 463
nutritional requirements, 10
pasteurized process cheese, 477
species identification, 10–12
pickled cheeses, 465–468
Bacterial surface-ripened cheeses, 454–456
Queso blanco cheese, 464
Bacteriocins
Queso de los Ibores cheese, 464
biocontrol, 185–186
raw milk, 481
meat products, 557–559
semisoft cheeses, 457–463
Bacteriophage typing, 285–286
soft Italian cheese, 457
Baigent, Donnelly and, studies, 225–226
soft unripened cheese, 471–474
Bailey studies, 68, 229, 232, 577
starter distillates, 448–449
Bakery and pastry items, 177, 243
Sudanese white-pickled cheese, 467–468
Ball studies, 605–606
Swiss cheese, 462
Baloga and Harlander studies, 288, 297
Taleggio cheese, 456
Banks studies, 452, 461
Tilsiter cheese, 456
Bannerman and Bille studies, 218, 223–224, 431
Trappist cheese, 456
Bannerman studies, 286
Turkish white-brined cheese, 467
Barbalho studies, 586
whey, 482, 482–483
Barbosa studies, 532
whey cheeses, 474–477
Barrier establishment, 715
Yugoslavian white-pickled cheese, 468
Bats, 41
Behavior, fish and seafood products
Batt, Norton and, studies, 262
fundamentals, 636
Baxter studies, 34
growth, 638–640
Bearns and Girard studies, 227, 229
inactivation, 643–646, 644
Beckers studies, 227, 363, 428
inhibition, 640–643, 641
Beef, see also Cattle; Cow’s milk
survival, 638–640
cooked roast beef, 535
transmission modes, 636–638
growth and survival, 530–533
Index 845
Behavior, meat products sweetened condensed milk, 387

bacteriocins, 557–559 ultrafiltered milk, 388
beef, 530–533, 535, 548–549, 549 Behrsing studies, 673
cooked products, 534–538 Belgium, 31
cooked roast beef, 535 Bemrah studies, 775–776
cooked smoked sausage, 540–542, 542 Benagozzi studies, 32
cured ham, 534–535 Bendig and Strangeways studies, 661
domestic livestock, 528 Benech studies, 461
dry fermented sausage, 545–547 Benkerroum and Sandine studies, 474
fermented sausage, 544, 558–559 Benzoic acid derivatives, 175, 178
fresh sausage, 538–540, 539–540 Berang studies, 391
fundamentals, 527–528 Berche studies, 60
growth, 530–533 Berrang studies, 666, 731
ham, cured, 534–535 Berry studies, 558
lamb, 533–534, 549–550, 550 Beuchat, Brackett and, studies, 600, 604, 639
luncheon meats, 535–538, 536–537 Beuchat, Holliday and, studies, 393
modified-atmosphere packaging, Beuchat, Hwang and, studies, 589
547–557 Beuchat, Taormina and, studies, 556
pork, 533–534, 550–552, 551 Beuchat and Brackett studies, 665, 667–668, 670, 675
raw ground meat, 557–558 Beuchat and Doyle studies, 670
raw meat, 530–533 Beuchat studies, 663, 667, 670, 814
ready-to-eat products, 534–538 Bhunia, Amoril and, studies, 516
roast beef, cooked, 535 Bierne and Cossart studies, 116
sausage, 538–547, 549 Biester and Schwarte studies, 66, 217
semidry fermented sausage, 544–545 Bile salt hydrolase, 129–130
smoked sausage, 540–543, 542 Bille, Bannerman and, studies, 218, 223–224, 431
specialty items, 543–544 Bille and Rocourt studies, 284, 297
survival, 530–533 Biochemical characteristics, 10
thermal inactivation, 552–557, 553–556 Biocontrol, 185–186
tissue localization, 528–530 Biofilms
uncooked smoked sausage, 543 factory and equipment design, 696–697, 697–704, 699,
unfermented sausage, 538–547 702–704
Behavior, plant origin products formation, 190–192, 191
chemical sanitizers, 668–669 fundamentals, 685–692, 686–692
fruits and fruit juices, 672–673, 674 maintenance and repair practices, 693, 693–697
fundamentals, 663, 672–676, 674 Biosensors, rapid method detection, 269–271, 270
growth, 663–666, 664 Blanco studies, 234
heat, 667–668 Blenden studies, 67
inactivation, 667–671 Blood donations, 100
modified atmosphere storage, 666–667 Blood-free plating media, 229
plant components, 669–670 Blue-mold or hard cheese, 314
processing techniques, 671, 671 Blue mussels, 32
survival, 663–666 Blue-veined cheese, 453–454
Behavior, poultry products Bockemühl studies, 221
chicken, raw, 587–590, 588 Boerlin studies, 287, 293, 637
cooked products, 590–592, 591–593 Bohner and Bradley studies, 708–709
fundamentals, 587 Bojsen-Møller studies, 217–218
growth, 587–592, 588, 591–593 Bolton and Frank studies, 481
ready-to-eat products, 590–592, 591–593 Borucki studies, 296
thermal inactivation, 593–597, 594–596 Boston (United States), 37
Behavior, unfermented dairy products Botzler studies, 30
autoclaved milk, 380–387 Bourry studies, 64
chocolate milk, 380–387 Bovine, see Cattle
cream, 380–387 Bower, Betsy, 647
evaporated milk, 387 Boyle studies, 552, 755
fundamentals, 376 Bozoglu, Alpas and, studies, 673
intensively pasteurized milk, 379–380 Brackett, Beuchat and, studies, 665, 667–668, 670, 675
mixed cultures, 388–389 Brackett, Cassiday and, studies, 218
other products, 367–376 Brackett, Hao and, studies, 669
pasteurized milk, 367–376, 379–380 Brackett and Beuchat studies, 600, 604, 639
raw milk, 377–379, 378–380 Brackett studies, 71, 655–676, 814–819
Braden studies, 305–349 CAMP tests, 10–11

Bradley, Bohner and, studies, 708–709 Canada
Brahmbhatt and Anjaria studies, 522 animal feeds, 33
Brashears, Amezquita and, studies, 558 foodborne infection, 314–315
Bréand studies, 555 monitoring programs, 419–421, 420, 508–514
Breer and Schöpfer studies, 518 plant origin products, 660
Breer studies, 431, 483, 584 risk assessment, 775
Brehm-Stecher studies, 257–274 seafood products, 333
Brehm studies, 136 standards and criteria, 794–796, 795
Bremer and Osborne studies, 644 surveillance programs, 419–421, 420
Brick cheese transmission to animals, 64–65, 67
cold enrichment, 217 vegetables, 335
fermented dairy products, 455–456 Canaries, 41
Brie cheese, 231 Canillac and Mourney studies, 747
Brie de Meaux, 97, 313–314 Cantoni, Comi and, studies, 584
Briggs, Donnelly and, studies, 383 Cantoni studies, 430, 518
Brine solutions, 483–486, 484–486, see also specific solution Cantor, Schwartz and, studies, 291
Brosch studies, 291–292 Capita studies, 599
Buazzi studies, 457, 462 Carlier studies, 552
Buchanan and Klawitter studies, 557 Carlin and Nguyen-the studies, 665
Buchanan studies, 220, 229–230, 603, 659, 777 Carlin studies, 665
Buchrieser studies, 1–12, 291 Carminati, Ribeiro and, studies, 444
Budu-Amoako studies, 643 Carpenter, Harrison and, studies, 597
Buffalo, 41 Carpenter and Harrison studies, 594–595
Bulgaria, 56 Carpentier, Midelet and, studies, 538
Bulgarian white-pickled cheese, 467 Carvacrol, 196
Buni studies, 527 Casolari studies, 526
Bunning, Peeler and, studies, 778 Cassiday and Brackett studies, 218
Bunning studies, 167 Cassiday studies, 231
Burden estimates, foodborne infection, 348–349 Cassin studies, 778
Busani studies, 599 Catalase, 131
Busch and Donnelly studies, 245 Cats, 41
Busse, Sulzer and, studies, 452 Cattle, see also Beef; Cow’s milk
Butter, see also Dairy products animal feeds, 32–33
foodborne infection, 318 animal listeriosis, 62–66
human outbreaks, 37 fecal material, 41
potassium sorbate, 177 predisposing factors, 56–57
unfermented dairy products, 392–393 soil contamination, 30
Buyong studies, 462 Caugant studies, 287
Cauliflower, 337
C Ceftazidime, 223
Cell separation and concentration, 266–267
Cabbage Cellular adhesion, 118
antioxidants, 181 Cereals, 177
direct plating media, 231 Channel catfish, 71
epidemic listeriosis, 94 Characteristics, food processing importance
fecal and sewage contamination, 31 acid damage and tolerance, 170–171
soil contamination, 30 acidity, 169–171
CAC, see Codex Alimentarius Commission (CAC) acid sanitizers, 194
Cagri studies, 599 active packaging, 196–197
Cai studies, 296 alternative processing technologies, 187–190
Calcinated calcium solution, 196 antimicrobial components, 174–184
California (United States) antioxidants, 180–181
listeriosis outbreak, 329 attachment to surfaces, 190–192, 191
Mexican-style cheese, 315 bacteriocins, 185–186
transmission to animals, 63, 71–72 benzoic acid derivatives, 175, 178
Camembert cheese biocontrol, 185–186
cold enrichment, 217 biofilm formation, 190–192, 191
fatty acid monoesters, 178 chlorine and chlorinated compounds, 193–194
fermented dairy products, 449–453, 450–451 cold tolerance, 161–163, 162
shrimp, 337 elevated sublethal temperature, 163
Index 847
enhanced acid tolerance, 171 Chasseignaux studies, 522, 743

extrinsic factors, heat resistance, 167 Cheddar cheese
fatty acid monoesters, 179–180 cold enrichment, 217
fatty acids and related compounds, 178–180 fermented dairy products, 459–460, 461–462
free fatty acids, 178–179 Cheese and fermented products, see also specific type
freezing temperature, 163–164 bacterial surface-ripened cheeses, 454–456
fundamentals, 158–159 behavior, 446–486
growth, water activity, 171–172, 172 blue cheese, 453, 454
growth kinetics, 160–161, 161 brick cheese, 455–456
growth temperature, 159–163 brine solutions, 483–486, 484–486
heat resistance, 167–168 Bulgarian white-pickled cheese, 467
herbs, 181–182, 182 Camembert cheese, 449–453, 450–451
high-intensity pulsed light, 188–189 Canadian monitoring programs, 419–421, 420
high-pressure processing, 189, 189 cheddar cheese, 459, 460, 461–462
high salt concentration, 174 Chihuahua cheese, 465
hydrogen peroxide, 183 coagulants, 447–448, 448
inactivation, low pH, 169–170 colby cheese, 458–459, 459
intrinsic factors, heat resistance, 167–168 cold-pack cheese food, 476–477, 476–477
ionizing radiations, 187–188, 188 coloring agents, 448–449
irradiation, 187–189 composition of cheese, 478–481, 479–480
kinetics, thermal inactivation, 165–167 cottage cheese, 471–474, 472
lactate, 175, 176 cream, 436–445
lactoferrin, 184 cream cheese, 474
lactoperoxidase system, 183–184 cultured buttermilk, 439–441, 440
lethal temperature, 164–168 cultured cream, 441
live fermentate, 185 cultured milks, 436–445
low pH, 169–170 domestic cheese recalls, 407–414
lysozyme, 182–183 Domiati cheese, 467
miscellaneous sanitizing agents, 195–196 ergo, 445–446
modified atmosphere, 186–187 European countries, 418–419, 421–435
multiple antimicrobial treatments, 197, 197–198 ewe’s milk cheese, 468–470
optimum temperature, 159–160 febrile gastroenteritis, 342–343
organic acids and salts, 175, 175–178 fermented milks, 436–446
osmotolerance factors, 172–173, 172–173 feta cheese, 465–467, 466
ozone, 194–195, 195 France, 414–418, 421
parabens, 175, 178 fraser broth, 227
physiology, high salt concentration, 174–175 fundamentals, 313–317, 342–343, 406–407
plant extracts, 181–182, 182 Germany monitoring programs, 428–430, 429
potassium sorbate, 175, 177–178 goat’s milk cheese, 468–470
pulsed electric field processing, 189–190, 190 Gouda cheese, 458
quaternary ammonium compounds, 194 hard cheeses, 457–463
range, temperature, 159–160 hard Italian-type cheese, 463
salt concentration, 174 Hispanic cheeses, 464–465
salts, 174–178 hydrogen peroxide, 183
sanitizers, 192–196 imported cheese, 414–419
smoke, 181 Italy monitoring programs, 430
sodium benzoate, 175, 178 Kachkaval cheese, 468–469
sodium diacetate, 175, 176–177 kefir, 445
sodium nitrite, 180 labneh, 445–446
sodium propionate, 175, 177 Maasdam cheese, 458
spices, 181–182, 182 Manchego cheese, 464–465, 469
stress adaptation, 163 mesophilic starter cultures, 436–439, 437–438
surrogate microorganisms, 168 Mexican Manchego cheese, 464–465
survival, 171–172, 172, 174 mold-ripened cheeses, 449–453
temperature, 159–168 monitoring programs, 421
thermal inactivation, 164–167 mozzarella cheese, 457
tolerance, acid, 170–171 non-European countries, 435
ultraviolet radiation, 188–189 Parmesan cheese, 462–463, 463
virulence variations, 163 pasteurized process cheese, 477
water activity, 171–173 pickled cheeses, 465–468
Charpentier and Courvalin studies, 286 potassium sorbate, 177
Queso blanco cheese, 464 Cold-pack cheese food

Queso de los Ibores cheese, 464 fermented dairy products, 476–477, 476–477
raw milk, 481 sodium propionate, 177
recalls, 407–419 Cold-smoked fish products
semisoft cheeses, 457–463 direct plating media, 230
soft Italian cheese, 457 foodborne infection, 334–335
soft unripened cheese, 471–474 smoking, 181
starter cultures, 436–445 Cold-smoked rainbow trout, 340–341
starter distillates, 448–449 Cold tolerance, 161–163, 162
Sudanese white-pickled cheese, 467–468 Coleslaw
surveillance programs, 407–435 fecal and sewage contamination, 31
Swiss cheese, 462 human outbreaks, 37
Switzerland monitoring programs, 430–432, soil contamination, 30
431 transmission, 36
Taleggio cheese, 456 Collins studies, 4
thermophilic starter cultures, 441–442, 441–443 Collins-Thompson, Slade and, studies, 222, 224–225, 362
Tilsiter cheese, 456 Coloring agents, 448–449
Trappist cheese, 456 Comi and Cantoni studies, 584
Turkish white-brined cheese, 467 Comi and Valenti studies, 463
U.S. surveillance programs, 407–419 Commercial availability, 258–259
whey, 482, 482–483 Comparison of methods, 297, 298
whey cheeses, 474–477 Composition of cheese, 478–481, 479–480
yogurt, 443–444 Connecticut (United States)
Yugoslavian white-pickled cheese, 468 deli meat and frankfurters, 321
Cheese production facilities, 743 foodborne infection, 328
Chemical sanitizers, 668–669 Conner studies, 663
Chemotaxonomy, 3–4 Consequences, enhanced tolerance, 170–171
Chen and Hotchkiss studies, 474 Consumer education, 839–840
Chicken, raw, 577–579, 587–590, 588 Consumer exposure
Chicken products, 332–333, see also Poultry products FDA perspective, research needs, 816–817
Chickens, 41, 67 industry perspective, research needs, 821–827
Chihuahua cheese, 465 specific measures for reducing, 102
China, 775 Consumption, risk assessment, 784
Chinchillas, 41 Contamination prevention, 821–823
Chip-based microanalytical systems, 271, 272 Continental Europe, 582–583, 584–585, see also Europe
Chitosan, 196 Control, febrile gastroenteritis, 343–344
Chlorine and chlorinated compounds, 193–194 Control guidelines, poultry products, 597–599
Chlorohexidine, 196 Control options, 837–838
Chocolate milk, see also Milk; Raw milk Conventional methods, detection and isolation
febrile gastroenteritis, 338–339 acriflavine, 221–222
gastrointestinal illness, 90 agents, selective, 219–223, 220
unfermented dairy products, 380–387 ARS-MMLA media, 229–230
Chocolate production facilities blood-free plating media, 229
England, 742 ceftazidime, 223
Europe, 741–742 cold enrichment, 217–219
Choi studies, 439, 443–444, 523 direct plating media comparisons, 230–232
Chromosomal DNA restriction endonuclease analysis, Food and Drug Administration method, 234–238, 236–237
287–288 Fraser broth, 226–227
Chung studies, 529 fundamentals, 216–217, 248
Cirigliano and Keller studies, 393 gum base nalidixic acid, 229
Cirigliano studies, 544 β-hemolysis, 233–234
Claire studies, 604 incubation conditions, 232
Clarifiers, 750 injured Listeria recovery, 244–248
Cleaning, control method, 707–710 International Dairy Federation method, 238–239, 239
Clinical manifestations, humans, 87–88 isolation media, 227–232
Coagulants, 447–448, 448 lithium chloride, 220–221
Codex Alimentarius Commission (CAC), lithium chloride-phenylethanol-moxalactam agar, 228
783–790 McBride Listeria agar, 227–228
Colburn studies, 23, 32 modified Oxford agar, 228
Colby cheese, 458–459 moxalactam, 223
Cold enrichment, 217–219 nalidixic acid, 221
Index 849
Netherlands Government Food Inspection Service, 243 Cultured buttermilk, 439–441, 440
oblique illumination, 232–234, 233 Cultured cream, 441
official methods, 234–243 Cultured milks, 436–445
Oxford agar, 228 Cured ham, 534–535
PALCAM agar, 228–229 Curtis and Mitchell studies, 286
phenylethanol, 220–221 Curtis studies, 228
plating, 219–223 Cutter and Siragusa studies, 559
polymyxin B, 222–223 Cytoplasm growth, see Host cell cytoplasm growth
potassium tellurite, 219–220
potassium thiocyanate, 222 D
recovering injured Listeria, 244–248
research advances, 244 Dairy processing facilities
selective enrichment, 219–223 control methods, 750–754
selective media, 223–243 Europe, 739–741, 740–741
thallous acetate, 222 Sweden, 746–747
trypaflavine, 221–222 United States, 720–723, 721–724
USDA-FSIS method, 239–243, 240 Dairy products, see also Fermented milks and dairy
UVM broth, 225–226 products; Unfermented dairy products
Conventional methods, subtyping, 284–286 foodborne infection, 310–320, 338–339, 342–343
Cooked products isolation methods for, 234
meat products, 505, 506–508, 507–508, 513, 534–538 McBride Listeria agar, 228
poultry products, 586–587, 590–592, 591–593 USDA-FSIS method, 243
Cooked roast beef, 535 Daley, Farber and, studies, 537
Cooked smoked sausage, 540–542, 542 Dalgliesh, Stephanie, 647
Corn and tuna salad, 340 Dalu and Feresu studies, 445
Corned beef, 755 Damage mechanisms, 170–171
Corrective actions, HACCP, 718 Dancz studies, 122
Corry, Lewis and, studies, 218 DaSilva studies, 435
Cortesi studies, 640 Dauphin studies, 743
Cossart, Bierne and, studies, 116 Davidson studies, 362
Cossart, Johansson and, studies, 135 Davies studies, 475, 550
Cossart studies, 116 Dean and Zottola studies, 391
Costa Rica Debevere, El-Ziny and, studies, 473–474
neonatal disease, 88 Debevere, Zeitoun and, studies, 588
transmission pathways, 43 De Boer and van Netten studies, 543
Costs, 801–807, 802–803, 805–806 Dedie studies, 377
Cottage cheese Deer, 41
cold enrichment, 217 Degnan studies, 642
fermented dairy products, 471–474, 472 Deibel, Griffith and, studies, 444
Cottin studies, 474, 528 Delaquis studies, 668
Courvalin, Charpentier and, studies, 286 Deli meats, see also Luncheon meats; Ready-to-eat meat
Cows, water contamination, 32 and poultry products
Cow’s milk, 358–366, see also Beef; Cattle epidemic listeriosis, 96–97
Cox studies, 27, 578, 739, 741, 745, 748 foodborne infection, 321–327
Crabmeat, see also Fish and seafood products food safety perspective, 838
fatty acid monoesters, 178 Dendritic cells uptake, 118–119
human outbreaks, 37 Denmark
Cream asymptomatic carriage, 91, 93
fermented milks, 436–445 foodborne infection, 314
unfermented dairy products, 380–387, 381–382, listeriosis outbreak, 329
384–386 sporadic incidence, 98
Cream cheese, 474 standards and criteria, 796–797
Criteria and standards, 790–807 transmission to animals, 63, 65, 69
Critical control point, 717 De Roin studies, 690
Critical limits, 717 Desmarchelier studies, 601
Cross-connections, 752 Despierres studies, 222
Crustaceans Destro studies, 32, 292, 733
animal listeriosis, 71–72 Destruction and detection, FDA perspective, 818–819
fecal material, 41 Detection, rapid methods
fish and seafood products, 626, 630 advances, 265–266
Culture, 9 antibody-based methods, 264–267
automation, 259 Devlieghere studies, 551

biosensors, 269–271, 270 de Vries and Strikwerda studies, 63
cell separation and concentration, 266–267 Diagnosis, humans, 100–101
chip-based microanalytical systems, 271, 272 Diagnostic targets, 259
commercial availability, 258–259 Dickson, Niebuhr and, studies, 530
diagnostic targets, 259 Dickson studies, 529
DNA microarrays, 262–263 Dietary risk factors, 98–100
flow cytometry, 267–269, 268–269 Dieuleveux and Gueguen studies, 470
fluorescence in situ hybridization, 263–264 Dijkstra studies, 31, 33, 41, 377
fundamentals, 257–259, 274 Direct plating media comparisons, 230–232
generic species vs. specific species, 259 DNA
ideal detection, 258 macrorestriction analysis, 291–292, 293
nucleic-acid-based methods, 259–264 microarrays, 262–263
polymerase chain reaction, 260–262 relatedness, 6–7
potential pitfalls, 265 sequence-based strategies, 25, 295–297
recombinant bateriophage, 264 Documentation procedures, 718–719
spectroscopic methods, 271–274, 273 Dogan and Erkmen studies, 673
traditional methods, 258 Dogs, 41, 70–71
Detection and isolation, conventional methods Doherty studies, 556
acriflavine, 221–222 Domestic cheeses, 407–414, see also Fermented products
agents, selective, 219–223, 220 and cheese
ARS-MMLA media, 229–230 Domestic livestock, see also specific type
blood-free plating media, 229 incidence, 56
ceftazidime, 223 meat products, 528
cold enrichment, 217–219 Domestic seafood, 618–626, see also Fish and seafood
direct plating media comparisons, 230–232 products
Food and Drug Administration method, 234–238, Domiati cheese, 467
236–237 Dominguez-Rodriguez studies, 64
Fraser broth, 226–227 Dominguez studies, 469
fundamentals, 216–217, 248 Donachie studies, 37
gum base nalidixic acid, 229 Donald studies, 33
β-hemolysis, 233–234 Donker-Voet, Welshimer and, studies, 28
incubation conditions, 232 Donker-Voet and Seeliger studies, 6
injured Listeria recovery, 244–248 Donnelly, Busch and, studies, 245
International Dairy Federation method, 238–239, Donnelly, Klausner and, studies, 722
239 Donnelly, Ngutter and, studies, 244
isolation media, 227–232 Donnelly, Roth and, studies, 248
lithium chloride, 220–221 Donnelly, Sallam and, studies, 245
lithium chloride-phenylethanol-moxalactam agar, 228 Donnelly and Baigent studies, 225–226
McBride Listeria agar, 227–228 Donnelly and Briggs studies, 383
modified Oxford agar, 228 Donnelly studies, 215–248, 359, 364
moxalactam, 223 Doores, Amelang and, studies, 391
nalidixic acid, 221 Dorsa studies, 555, 644
Netherlands Government Food Inspection Service, Dose-response assessment, 784–785
243 Doyle, Beuchat and, studies, 670
oblique illumination, 232–234, 233 Doyle, Glass and, studies, 534–546, 558, 604
official methods, 234–243 Doyle, Lammerding and, studies, 243
Oxford agar, 228 Doyle and Schoeni studies, 223–224, 226,
PALCAM agar, 228–229 229, 238
phenylethanol, 220–221 Doyle studies, 167, 238, 369, 378, 394, 556, 814
plating, 219–223 Drevets studies, 119
polymyxin B, 222–223 Dry areas, cleaning, 707
potassium tellurite, 219–220 Dry fermented sausage, 545–547
potassium thiocyanate, 222 Dry milk, see Nonfat dry milk
recovering injured Listeria, 244–248 Duarte studies, 230
research advances, 244 Ducks, see also Poultry products
selective enrichment, 219–223 asymptomatic carriers, 67
selective media, 223–243 fecal material, 41
thallous acetate, 222 thermal inactivation, 597
trypaflavine, 221–222 Duffy studies, 534
USDA-FSIS method, 239–243, 240 Durst studies, 163
UVM broth, 225–226 D-value, 165–167
Index 851
E neonatal disease, 88
predisposing factors, 56
Eagles, 41 sporadic incidence, 98
Early onset, neonatal disease, 87–88 transmission to animals, 59, 67
Ecology Engulfment, 167
animal feeds, 32–34 Enhanced acid tolerance, 171
animal listeriosis, 40–42 Enrichment method, 22–23
enumeration methods, 23–24 Enumeration, 23–24, 234–235, 818
environments, various, 27 Environmental signals affect, 136
food-processing environment, 36 Environments, 27
fundamentals, 21–22 Epidemic listeriosis, humans, 94, 95, 96–97
gene expression regulation, 34–35 Epidemiological patterns, 94–100, 95
globals pathways and reservoirs, 42–44 Equines, see Horses
human listeriosis, 35–40 Equipment design, 697–704
isolation method, 22–23 Ergo, 445–446
limitations, current methods, 24–25 Erickson and Jenkins studies, 604
molecular detection, 25–27, 26 Ericsson studies, 294
multiplication, stress conditions, 27–35 Erkmen, Dogan and, studies, 673
natural environment, 35–42 Erkmen studies, 467
outbreaks, 37–38 EU, see European Union (EU)
pitfalls, current methods, 24–25 Europe
sewage, 30–31 cheese and fermented products, 418–419, 421–435
soil, 28, 29, 30 epidemic listeriosis, 96
sporadic cases, 38–40 food processing, facilities, 739–742
stress conditions, 27–35 meat products, 523–527, 524–525
study methods, 22–27 plant origin products, 660–661
subtyping methods, 25–27, 26 poultry products, 580–587
survival, stress conditions, 27–35 standards and criteria, 796–799
traditional enrichment method, 22–23 transmission to animals, 67
transmission dynamics, 35–44 European Union (EU), 799–800, 800
various environments, 27 Evans studies, 742
vegetation, 28, 29, 30 Evaporated milk, 387
water, 31–32 Evolutionary aspects, 138
Economic impact, see Risk assessment, regulatory control, Ewe’s milk, see also Lambs; Sheep
and economic impact cheese and fermented dairy products, 468–470
Egg processing facilities unfermented dairy products, 366, 366–367
control methods, 757 Exposure assessment, 635
United States, 732 Extracts, plant, 181–182, 182
Eggs and egg products, see also Poultry products Extrinsic factors, 167
fundamentals, 599–600
growth, 601–603, 601–604 F
incidence, 600–601
isolation methods for, 234 Factors, food processing facilities, 710–711
thermal inactivation, 165, 604–606 Factory design, 697–704
USDA-FSIS method, 241 Fairchild and Foegeding studies, 168
Eilertz studies, 469 Fanelli, Zaika and, studies, 160
Eklund studies, 617–646, 733 FAO/WHO risk assessment significance, 819–820
Elevated sublethal temperature, 163 Farber, Sontakke and, studies, 291
El-Gazzar and Marth studies, 447–449, 675 Farber, Zhang and, studies, 668
El-Gazzar studies, 388, 439 Farber and Addison studies, 292–293
Elimination, FDA perspective, 818–819 Farber and Daley studies, 537
El-Khateib studies, 559 Farber studies
Elliot and Kvenberg studies, 634 Camembert cheese, 503–559
El-Shenawy and Marth studies, 471 Canadian cheeses sold, 453
El-Ziny and Debevere studies, 473–474 cheeses exported to Canada, 420
Embarek studies, 622, 642 fermented sausages, 523, 546
Encephalitis, animal feeds, 34 generation times, 383
England growth and survival, 639
epidemic listeriosis, 97 incidence, 600
foodborne infection, 327 nalidixic acid, 221
food processing, facilities, 742–743 raw cow’s milk isolation, 362
listeriosis outbreak, 327, 330 risk assessment, 775–777
selective enrichment media, 224 blue cheese, 453, 454

thermal inactivation, 552, 595 brick cheese, 455–456
vegetable incidence, 660 brine solutions, 483–486, 484–486
Farm environment, 750 Bulgarian white-pickled cheese, 467
Farrag and Marth studies, 388–389 Camembert cheese, 449–453, 450–451
Farrag studies, 387 Canadian monitoring programs, 419–421, 420
Fatty acid monoesters, 179–180 cheddar cheese, 459, 460, 461–462
Fatty acids, 178–180 Chihuahua cheese, 465
FDA/FSIS risk assessment significance, 819–820 coagulants, 447–448, 448
FDA perspective, research needs colby cheese, 458–459, 459
behavior in foods, 817 cold-pack cheese food, 476–477, 476–477
consumer exposure, 816–817 coloring agents, 448–449
destruction, 818–819 composition of cheese, 478–481, 479–480
detection, 818 cottage cheese, 471–474, 472
elimination, 818–819 cream, 436–445
enumeration, 818 cream cheese, 474
FAO/WHO risk assessment significance, 819–820 cultured buttermilk, 439–441, 440
FDA/FSIS risk assessment significance, 819–820 cultured cream, 441
fundamentals, 815 cultured milks, 436–445
pathogenicity, 817–818 domestic cheese recalls, 407–414
risk assessment significance, 819–820 Domiati cheese, 467
FDA surveys, 618–626, 619–625 ergo, 445–446
Febrile gastroenteritis European countries, 418–419, 421–435
cheese, 342–343 ewe’s milk cheese, 468–470
chocolate milk, 338–339 fermented milks, 436–446
cold-smoked rainbow trout, 340–341 feta cheese, 465–467, 466
control, 343–344 France, 414–418, 421
corn and tuna salad, 340 fundamentals, 313–317, 342–343, 406–407
fresh, raw-milk cheese, 342–343 Germany monitoring programs, 428–430, 429
fundamentals, 336–337, 337, 343–344 goat’s milk cheese, 468–470
ready-to-eat meat and poultry products, 341–342 Gouda cheese, 458
rice salad, 338 hard cheeses, 457–463
shrimp, 337–338 hard Italian-type cheese, 463
Fecal carriage, see Asymptomatic carriage Hispanic cheeses, 464–465
Fecal materials, transmission, 41 imported cheese, 414–419
Fedio studies, 474 Italy monitoring programs, 430
Fenlon, David, 44 Kachkaval cheese, 468–469
Fenlon studies, 28, 30, 33, 41, 637 kefir, 445
Feresu, Dalu and, studies, 445 labneh, 445–446
Feresu and Jones studies, 3, 10 Maasdam cheese, 458
Ferguson and Shelef studies, 675 Manchego cheese, 464–465, 469
Fermented milks and dairy products, see also Dairy products mesophilic starter cultures, 436–439, 437–438
control methods, 754 Mexican Manchego cheese, 464–465
cream, 436–445 mold-ripened cheeses, 449–453
cultured buttermilk, 439–441, 440 monitoring programs, 421
cultured cream, 441 mozzarella cheese, 457
cultured milks, 436–445 non-European countries, 435
ergo, 445–446 Parmesan cheese, 462–463, 463
food processing, facilities, 754 pasteurized process cheese, 477
fundamentals, 436 pickled cheeses, 465–468
kefir, 445 Queso blanco cheese, 464
labneh, 445–446 Queso de los Ibores cheese, 464
mesophilic starter cultures, 436–439, 437–438 raw milk, 481
polymyxin B, 223 recalls, 407–419
starter cultures, 436–445 semisoft cheeses, 457–463
thermophilic starter cultures, 441–442, 441–443 soft Italian cheese, 457
traditional products, 445–446 soft unripened cheese, 471–474
yogurt, 443–444 starter cultures, 436–445
Fermented products and cheese starter distillates, 448–449
bacterial surface-ripened cheeses, 454–456 Sudanese white-pickled cheese, 467–468
behavior, 446–486 surveillance programs, 407–435
Index 853
Swiss cheese, 462 Fluid milk, 317–318, see also Milk

Switzerland monitoring programs, 430–432, 431 Fluorescence in situ hybridization, 263–264
Taleggio cheese, 456 Foegeding, Fairchild and, studies, 168
thermophilic starter cultures, 441–442, 441–443 Foegeding, Leasor and, studies, 600
Tilsiter cheese, 456 Foegeding and Leasor studies, 603, 605
Trappist cheese, 456 Foegeding and Stanley studies, 606
Turkish white-brined cheese, 467 Foegeding studies, 558
U.S. surveillance programs, 407–419 Food and Drug Administration method, 234–238, 236–237
whey, 482, 482–483 Foodborne infection
whey cheeses, 474–477 aspic, 328–329
yogurt, 443–444 Australia, 332
Yugoslavian white-pickled cheese, 468 blue-mold or hard cheese, 314
Fermented sausage, 544, 558–559, 560 Brie de Meaux, 313–314
Ferreira and Lund studies, 474 burden estimates, 348–349
Ferron and Michard studies, 243 butter, 318
Feta cheese, 465–467 Canada, 314–315
Fett, Ukuku and, studies, 673 cheese, 310–317, 342–343
Filling, control methods, 752 chicken products, 332–333
Finfish, 630 chocolate milk, 338–339
Finland cold-smoked fish products, 334–335
foodborne infection, 340–341 cold-smoked rainbow trout, 340–341
food processing, facilities, 744 Connecticut (United States), 328
human outbreaks, 37 dairy products, 310–320, 338–339, 342–343
Fish and seafood processing facilities deli meat, 321–327
control methods, 757–758 Denmark, 314
Europe, 741 England, 327
Finland, 744 febrile gastroenteritis, 336–344, 337, 343–344
United States, 732–738, 733, 735–737 Finland, 340–341
Fish and seafood products fluid milk, 317–318
animal listeriosis, 71–72 France, 313–314, 330–332
behavior, 636–646 frankfurters, 321–327
crustaceans, 626, 630 fresh, raw-milk cheese, 342–343
domestic seafood, 618–626 fundamentals, 306, 307
exposure assessment, 635 identification as, 308, 308–309
FDA surveys, 618–626, 619–625 illicitly produced/distributed cheese, 315–317
fecal material, 41 Ireland, 327
finfish, 630 Italy, 338
foodborne infection, 333–335, 337–338, 340–341 Japan, 343
fundamentals, 617–618, 646 Los Angeles County, 310–312, 311
growth, 638–640 Maryland (United States), 328
hazard identification and characterization, 635 meat products, 320–333
human listeriosis, 632–633 Mexican-style cheese, 310–312
imported seafood, 618–626 molecular subtyping role, 309–310
incidence, 627–629 mussels, smoked, 334
inhibition, 640–643, 641 New York (United States), 328
isolation methods for, 234 New Zealand, 341–342
lightly processed fish products, 631–632 outbreak detection and investigation, 309–310, 310
McBride Listeria agar, 228 paté, 327–328
regulatory aspects, 634–635 pork tongue, 328–329
risk assessment, 635–636 poultry products, 320–333
shellfish, 630 Quebec, Canada, 314–315
smoked fish products, 630–631 raw-milk fresh, unripened cheese, 315–317
surveys, 618–632 raw-milk soft cheese, 314–315
survival, 638–640 ready-to-eat meat and poultry products, 321–327,
transmission modes, 636–638 341–342
USDA-FSIS method, 242 reduction, 333
Fisher studies, 668 rice salad, 338
Flanders studies, 245 rillettes, 330–331
Fleming, Kyung and, studies, 670 seafood products, 333–335, 337–338, 340–341
Flies, fecal material, 41 semihard soft cheese, 314–315
Flow cytometry, 267–269, 268–269 shrimp, 337–338
smoked mussels, 334 miscellaneous production facilities, 742–743, 745–746

soft, unripened Mexican-style cheese, 310–312 monitoring, 711, 717
sporadic listeriosis, 344–348 Netherlands, 745–746
surveillance, 344–349, 346–347 packaging, 752
Sweden, 342–343 pasteurization, 750–752, 751
Switzerland, 312–313 pipeline connections, 752
United States, 321–328, 337–339, 341–342, 344–349 poultry processing facilities, 730–731, 731, 741–742,
Vacherin Mont d’Or, 312–313 742, 743, 756–757
vegetables, 335–336 rebagged products, 755
Wales, 327 reclaimed and reworked products, 753
Food composition, 167 record-keeping procedures, 718–719
FoodNet repair practices, 693, 693–697
clusters of listeriosis, 345 roast beef, 755
historical development, 98 sampling plans, 713–715, 714
surveillance, 347–348 sanitation, control method, 707–710
Food processing, facilities seafood processing facilities, 732–738, 733, 735–737,
Australia, 747–748, 748 741, 744, 757–758
barrier establishment, 715 separators, 750
biofilms, 685–704 smoked-salmon processing facilities, 743
cheese production facilities, 743 Spain, 745
chocolate production facilities, 741–742 Sweden, 746–747
clarifiers, 750 Switzerland, 747
cleaning, control method, 707–710 traditional control approaches, 704–710
corned beef, 755 traffic patterns, 710
corrective actions, 718 United Kingdom, 742–743
critical control point, 717 United States, 719–738
critical limits, 717 vegetable processing facilities, 738, 745, 758–759
cross-connections, 752 verification procedures, 718
dairy processing facilities, 720–723, 721–724, 739–741, wet processing areas, cleaning, 707–709
740–741, 746–747, 750–754 Food processing, importance of characteristics
documentation procedures, 718–719 acid damage and tolerance, 170–171
dry areas, cleaning, 707 acidity, 169–171
egg processing facilities, 732, 757 acid sanitizers, 194
England, 742–743 active packaging, 196–197
equipment design, 697–704 alternative processing technologies, 187–190
Europe, 739–742 antimicrobial components, 174–184
factors to consider, 710–711 antimicrobial components, foods, 174–184
factory design, 697–704 antioxidants, 180–181
farm environment, 750 attachment to surfaces, 190–192, 191
fermented dairy products, 754 bacteriocins, 185–186
filling, 752 benzoic acid derivatives, 175, 178
Finland, 744 biocontrol, 185–186
France, 743 biofilm formation, 190–192, 191
frankfurters, 755–756 chlorine and chlorinated compounds, 193–194
frozen dairy products, 753 cold tolerance, 161–163, 162
fruit processing facilities, 738, 758–759 elevated sublethal temperature, 163
fundamentals, 683–684, 683–685 enhanced acid tolerance, 171
growth, 685–704 extrinsic factors, heat resistance, 167
hazard analysis critical control point, 711–713, 712, fatty acid monoesters, 179–180
716–719 fatty acids and related compounds, 178–180
household kitchens, 748–749 free fatty acids, 178–179
hygiene, 38 freezing temperature, 163–164
incidence, 719–748 fundamentals, 158–159
industry-specific control approaches, 749–759 growth, water activity, 171–172, 172
Italy, 744 growth kinetics, 160–161, 161
kitchens, household, 748–749 growth temperature, 159–163
link-type products, 755–756 heat resistance, 167–168
luncheon meats, 756 herbs, 181–182, 182
maintenance practices, 693, 693–697 high-intensity pulsed light, 188–189
meat processing facilities, 724–730, 725–726, 728–730, high-pressure processing, 189, 189
741, 744, 754–756 high salt concentration, 174
Index 855
hydrogen peroxide, 183 fluid milk, 317

inactivation, low pH, 169–170 foodborne infection, 313–314, 330–332
intrinsic factors, heat resistance, 167–168 food processing, facilities, 743
ionizing radiations, 187–188, 188 human outbreaks, 37
irradiation, 187–189 listeriosis outbreak, 330, 332
kinetics, thermal inactivation, 165–167 predisposing factors, 56
lactate, 175, 176 risk assessment, 775–776
lactoferrin, 184 sporadic incidence, 98
lactoperoxidase system, 183–184 standards and criteria, 797
lethal temperature, 164–168 surveillance programs, 414–418, 421, 428
live fermentate, 185 transmission to animals, 67
low pH, 169–170 USDA-FSIS method, 243
lysozyme, 182–183 Franco studies, 585
miscellaneous sanitizing agents, 195–196 Frank, Bolton and, studies, 481
modified atmosphere, 186–187 Frankfurters, see also Hot dogs
multiple antimicrobial treatments, 197, 197–198 foodborne infection, 321–327
optimum temperature, 159–160 food processing, facilities, 755–756
organic acids and salts, 175, 175–178 organic acids and salts, 176–177
osmotolerance factors, 172–173, 172–173 potassium sorbate, 177–178
ozone, 194–195, 195 sodium benzoate, 178
parabens, 175, 178 Fraser broth, 226–227
physiology, high salt concentration, 174–175 Free fatty acids, 178–179, 179
plant extracts, 181–182, 182 Freezing, temperature, 163–164
potassium sorbate, 175, 177–178 Fresh, raw-milk cheese, 342–343
pulsed electric field processing, 189–190, 190 Fresh sausage, 538–540, 539–540, see also Sausages
quaternary ammonium compounds, 194 Frogs, 41
range, temperature, 159–160 Frozen dairy products, 753
salts, 174–178 Fruit processing facilities
sanitizers, 192–196 control methods, 758–759
smoke, 181 United States, 738
sodium benzoate, 175, 178 Fruits and fruit juices, 671–674, 672
sodium diacetate, 175, 176–177 Fuad studies, 636
sodium nitrite, 180 Fuchs and Surendran studies, 630
sodium propionate, 175, 177 Fuerst, Hugenholtz and, studies, 704
spices, 181–182, 182 Fundamentals
stress adaptation, 163 bacteria identification, 9–12
surrogate microorganisms, 168 biochemical characteristics, 10
survival, 171–172, 172, 174 chemotaxonomy, 3–4
temperature, 159–168 culture, 9
thermal inactivation, 164–167 fundamentals, 4–5, 12
tolerance, acid, 170–171 genus characteristics, 9–10
ultraviolet radiation, 188–189 historical background, 1–2
virulence variations, 163 metabolism, 10
water activity, 171–173 morphology, 9
Food-processing environments, 36, see also Food numerical taxonomy, 3
processing, facilities nutritional requirements, 10
Food safety perspective phylogenetic position, 3–5
consumer education, 839–840 rRNA sequencing, 4
control options, 837–838 species identification, 10–12
deli meats, 838 taxonomy, 5–9
foods of concern, 839 whole genome sequencing, 4, 5
labeling, 839–840 Future issues, 139
processed food risks, 838–839 Fuzi and Pillis studies, 222
regulatory tolerance, 836–837
retail product focus, 838–839 G
Foods of concern, 839
Fowl, 67–69 Gabis, Silliker and, studies, 721
Foxes, 41 Gabis studies, 483, 723
France, see also Rillettes Gahan studies, 461
cheese and fermented products, 414–418 Galindo-Cuspinera studies, 449
epidemic listeriosis, 97 Gallagher, Sumner and, studies, 773
Gamma radiation, 589–590 animal feeds, 34

Garayzabal and Genigeorgis studies, 232 animal listeriosis, 60–62
Garayzabal studies, 374 cheese and fermented dairy products, 468–470
Garcia-Gimeno studies, 667 unfermented dairy products, 366, 366–367
Garland, Soonthoranant and, studies, 32 water contamination, 32
Garrec studies, 31 Godreuil studies, 286
Gastrointestinal illness, 90–93 Goebel studies, 111–139
Gaya studies, 61 Goetz studies, 125
Gazelles, 70 Gohil studies, 522, 662
Geese, 67 Golan, Kuchler and, studies, 803
Geisen studies, 451 Golden studies, 231
Gendel studies, 291 Goldfine and Wadsworth studies, 119
Gene expression Goldfine studies, 119
academic perspective, 833–834 Gombas studies, 413, 804
molecular virulence determinants, 132–137 Gómez studies, 271
regulation in stress conditions, 34–35 Gorris, Aytac and, studies, 667
Generic species vs. specific species, 259 Gouda cheese, 458
Genigeorgis, Garayzabal and, studies, 232 Gouet studies, 530–531
Genigeorgis studies, 478, 577–579, 742 Goulet studies, 98
Genome sequencing Graham, Kerlin and, studies, 66
fundamentals, 4, 5, 27–28 Grant’s gazelle, 70
groups, 6 Grau and Vanderlinde studies, 527, 532, 535
molecular virulence determinants, 137–138 Graves studies, 283–297, 324
Genomic endowment, 831–833 Gray, M.L., 5
Genotypic markers, 11–12 Gray and Killinger studies, 41, 218
Genus characteristics, 9–10 Gray studies, 9, 32, 216–219, 258, 814
Georgia (United States) Great Britain
deli meat and frankfurters, 325 animal feeds, 33
Mexican-style cheese, 316 neonatal disease, 88
Germany transmission to animals, 59, 62
asymptomatic carriage, 91 Greece, 31
fluid milk, 317 Gregorio studies, 221
monitoring programs, 428–430, 429 Griffith and Deibel studies, 444
predisposing factors, 56 Grif studies, 39, 91
risk assessment, 777 Grøntsol studies, 33
standards and criteria, 797 Ground meat, raw, see also specific type
surveillance programs, 429 fraser broth, 226–227
Gerner-Smidt, Norrung and, studies, 287–288, 297 meat products, 557–558
Gerner-Smidt studies, 288 Groves and Welshimer studies, 6
Gianfranceschi and Aureli studies, 666 Grow studies, 274
Gibbons studies, 2 Growth
Gilbert, McLauchlin and, studies, 526 egg products, 601–603, 601–604
Gilbert, Pini and, studies, 433, 581, 583 enrichment and isolation methods, 22
Gilbert studies, 516 factors, 10
Gill and Reichel studies, 548 fish and seafood products, 638–640
Gillespie studies, 526 food processing, facilities, 685–704
Gill studies, 2 kinetics, 160–161, 161
Gilmour, Harvey and, studies, 36, 363, 660, 747 meat products, 530–533
Gilmour, Kells and, studies, 230 nutritional requirements, 10
Gilmour, Lawrence and, studies, 36, 586 plant origin products, 663–666, 664
Giovanacci studies, 522 poultry products, 587–592
Giraffes, 41 prevention, 824–826
Girard, Bearns and, studies, 227, 229 risk assessment, regulatory control, and economic
Girard, McBride and, studies, 220, 227 impact, 784
Gitter studies, 33, 581 temperature, 159–163
Glass and Doyle studies, 534–546, 558, 604 water activity, 171–172, 172
Glass studies, 464, 477, 539 Gudbjornsdottir studies, 741
Global pathways and reservoirs, 42–44 Gueguen, Dieuleveux and, studies, 470
Globals pathways and reservoirs, 42–44 Guerra studies, 294
Glutaraldehyde, 196 Guinea pigs, 41
Goats and goat’s milk Gulf Coast, United States, 32, 72
Index 857
Gulls, 41 Hewitt studies, 804

Gulmez and Guven studies, 445 Hicks and Lund studies, 473
Gum base nalidixic acid, 229 Higgins, Parish and, studies, 672
Gurtler studies, 681–759 Higgins and Robinson studies, 10
Guven, Gulmez and, studies, 445 High-intensity pulsed light, 188–189
Guyer and Jemmi studies, 639 High-pressure processing, 189, 189
Hill, Nancy, 647
H Hill, Walter E., 647
Hispanic cheeses, see Mexican-style cheese
Halle, Germany, 317 Historical background, 1–2
Ham Hitchins studies, 778
direct plating media, 231–232 Hobson studies, 527
meat products, 534–535 Hofer studies, 221
thermal inactivation, 165 Hoffman and Wiedmann studies, 242
Hamon and Peron studies, 286 Hoffman studies, 631, 638
Hao and Brackett studies, 669 Höhne studies, 528
Hao studies, 226, 231, 663, 669 Holliday and Beuchat studies, 393
Hard cheeses, 457–463 Holt studies, 273
Hard Italian-type cheese, 463 Hong Kong, 775
Hard salami, 172 Hooper-Kinder studies, 515
Harlander, Baloga and, studies, 288, 297 Horses
Harrison, Carpenter and, studies, 594–595 fecal material, 41
Harrison, Shineman and, studies, 639 transmission to, 69–70
Harrison and Carpenter studies, 597 Host cell cytoplasm growth, 125–126
Harrison and Huang studies, 643 Hotchkiss, Chen and, studies, 474
Harrison studies, 639 Hot dogs, see also Frankfurters
Hart studies, 587 dietary risk factors, 99–100
Harvey and Gilmour studies, 36, 363, 660, 747 epidemic listeriosis, 96
Haydon studies, 43 human outbreaks, 37
Hayes studies, 64, 223, 225, 243, 358 Household kitchens, 748–749
Hazard analysis critical control point (HACCP) Huang, Harrison and, studies, 643
corrective actions, 718 Huang studies, 592
critical control points, 717 Hudson and Mead studies, 742
critical limits, 717 Hudson and Mott studies, 640
fundamentals, 711–713, 712 Huffman, Randy, 827
hazard analysis, 716–717 Hugenholtz and Fuerst studies, 704
monitoring procedures, 717 Hughey studies, 453, 482, 486, 538, 669
need for methods, 216 Huhtanen studies, 589
plan development, 331 Hulphers studies, 1
record-keeping and validation, 718–719 Humans, listeriosis
required by USDA-FSIS, 333 asymptomatic carriage, 91, 92–93, 93
team, 716 blood donations, 100
verification procedures, 718 clinical manifestations, 87–88
Hazard identification and characterization, fish, 635 diagnosis, 100–101
Heat dietary risk factors, 98–100
food processing importance, 167–168 early onset, neonatal disease, 87–88
plant origin products, 667–668 epidemic listeriosis, 94, 95, 96–97
temperature, 167 epidemiological patterns, 94–100, 95
Heinitz, Maxine, 647 fish and seafood products, 632–633
Heisick studies, 659–660, 662 fundamentals, 85–86, 86
Helloin studies, 452 gastrointestinal illness, 90–93
Hemolysin, 110 incidence, 97–98, 99
β-hemolysis, 233–234 infection, 87–88
Henderson-Hasselbalch equation, 176 invasive disease, 88–90
Henry studies, 232–233 late onset, neonatal disease, 88
Herald and Zottola studies, 529 neonatal disease, 87–88
Herbs, 181–182, 182 noninvasive disease, 90–93
Heredia studies, 516 nonpregnant adults, 88–90
Herman, Rijpens and, studies, 230 nosocomial transmission, 100
Hernandez-Sanchez, Solano-Lopez and, studies, 464–465 outbreaks, 37–38
Hessen studies, 66 pregnancy, 87
prevention, 101–102 other products, 367–376

sexual transmission, 100 pasteurized milk, 367–376
sporadic disease, 97–100, 99 raw cow’s milk, 358–366
transmission dynamics, 35–40 raw ewe’s milk, 366, 366–367
treatment, 101 raw goats milk, 366, 366–367
water contamination, 32 Incubation conditions, 232
Humans, transmission to, 57 Indiana (United States), 65
Humboldt-Arcata Bay, 72 Industry perspective, research needs, 819–827
Hungary, 56 consumer exposure, 821–827
Hungerford, Jim, 647 contamination prevention, 821–823
Hunter studies, 283–297 FAO/WHO risk assessment significance, 819–820
Husu studies, 41, 578 FDA/FSIS risk assessment significance, 819–820
Hwang and Beuchat studies, 589 fundamentals, 819, 827
Hydrogen peroxide, 183, 196 growth prevention, 824–826
Hyraxes, 41 industry perspective, 819–827
outbreaks vs. sporadic cases, 820–821
I postpasteurization, 823–824
risk assessment significance, 819–820
Ice cream, see also Dairy products Industry-specific control approaches, 749–759
direct plating media, 231 Infection, humans, 87–88, see also Humans, listeriosis
unfermented dairy products, 391–392 Ingham studies, 590
Ideal detection, 258 Inhibition, fish and seafood products, 640–643, 641
Identification, 308, 308–309 Initial contamination, 783–784
Ikonomov and Todorov studies, 467–468 Injured Listeria recovery, 244–248
Illicitly produced/distributed cheese, 315–317, see also Intensively pasteurized milk, 379–380, see also Pasteurized
Mexican-style cheese milk
Imitation crabmeat, see also Fish and seafood products International, risk assessment, 781–790, 800–801
fatty acid monoesters, 178 International Dairy Federation method, 238–239, 239
human outbreaks, 37 Intracellular motility, 126–129, 127
Imported cheese, 414–419, see also specific type Intrinsic factors, 167–168
Imported seafood, 618–626, see also Fish and seafood Invasive disease, 88–90, see also Humans, listeriosis
products Iodophors, 195–196
Inactivation Ionizing radiations, 187–188, 188
low pH, 169–170 Iowa (United States), 62, 66–67
plant origin products, 667–673 Ireland, 97, 327, 330
Incidence Iron uptake systems, 132
animal listeriosis, 56 Irradiation, 187–189
egg products, 600–601 Isaac, Kwantes and, studies, 580–581
fish and seafood products, 627–629 Islam studies, 599
food processing, facilities, 719–748 Isolation and detection, conventional methods
fruits, 671–672 acriflavine, 221–222
humans, listeriosis in, 97–98, 99 agents, selective, 219–223, 220
raw vegetables, 657–663 ARS-MMLA media, 229–230
Incidence, meat products blood-free plating media, 229
Canadian monitoring program results, 508–514, ceftazidime, 223
514–518 cold enrichment, 217–219
cooked meat, 505 direct plating media comparisons, 230–232
cooked products, 505, 506–508, 507–508, 513 Food and Drug Administration method, 234–238,
Europe, 519–521, 523–527, 524–525 236–237
non-European countries, 527 Fraser broth, 226–227
non-North American countries, 516, 518–523, 519–521 fundamentals, 216–217, 248
North America, 515–516, 517–518, 523 gum base nalidixic acid, 229
raw meat, 505, 514–523 β-hemolysis, 233–234
ready-to-eat products, 508, 513, 514–515, 523–527 incubation conditions, 232
recalls and regulatory actions, 507–508, 509–513, injured Listeria recovery, 244–248
514, 516 International Dairy Federation method, 238–239, 239
sausage, 523–527 isolation media, 227–232
USDA-FSIS monitoring program, 504–508, 506–508 lithium chloride, 220–221
Incidence, unfermented dairy products lithium chloride-phenylethanol-moxalactam agar, 228
fundamentals, 358 McBride Listeria agar, 227–228
incidence, 358–376 modified Oxford agar, 228
Index 859
moxalactam, 223 K
nalidixic acid, 221
Netherlands Government Food Inspection Kabuki studies, 723
Service, 243 Kachkaval cheese, 468–469
oblique illumination, 232–234, 233 Kallipolitis studies, 137
official methods, 234–243 Kamat studies, 671
Oxford agar, 228 Kampelmacher studies, 64, 93
PALCAM agar, 228–229 Kaneko studies, 662
phenylethanol, 220–221 Kanuganti studies, 534
plating, 219–223 Karaioannoglou and Xenos studies, 552
polymyxin B, 222–223 Katic studies, 468
potassium tellurite, 219–220 Kaya and Schmidt studies, 531
potassium thiocyanate, 222 Kaymaz, Sarumehmetoglu and, studies, 467
recovering injured Listeria, 244–248 Kaysner studies, 639
research advances, 244 Kefir, 445
selective enrichment, 219–223 Keller, Cirigliano and, studies, 393
selective media, 223–243 Kells and Gilmour studies, 230
thallous acetate, 222 Kentucky (United States), 65
trypaflavine, 221–222 Kerlin and Graham studies, 66
USDA-FSIS method, 239–243, 240 Kerr studies, 196
UVM broth, 225–226 Keusch, Jemmi and, studies, 646
Isolation media, 227–232 Khan studies, 222, 533–534
Isolation method, 22–23 Khattab studies, 444
Issa and Ryser studies, 446 Killinger, Gray and, studies, 41, 218
Italy Kim studies, 457, 669
foodborne infection, 338 Kinetics
food processing, facilities, 744 growth, temperature, 160–161, 161
standards and criteria, 798 thermal inactivation, food processing importance,
surveillance programs, 430 165–167
Ivanov studies, 5–6 Kitchens, household, 748–749
Kitts, Wong and, studies, 600
Klausner and Donnelly studies, 722
J Klawitter, Buchanan and, studies, 557
Japan Klein and Juneja studies, 262
foodborne infection, 343 Knabel studies, 245
sporadic incidence, 98 Kornacki studies, 240, 681–759
Jayaro studies, 64 Kovácsné Domjan studies, 527
Jeffers studies, 38 Kovincic studies, 456
Jemmi, Guyer and, studies, 639 Kramer and Jones studies, 222
Jemmi, Trüssel and, studies, 543, 545 Kreft, J., 139
Jemmi and Keusch studies, 646 Kreft and Vazquez-Boland studies, 136
Jemmi studies, 639 Kuchler and Golan studies, 803
Jenkins, Erickson and, studies, 604 Kuhn studies, 111–139
Jinneman studies, 617–646 Kvenberg, Elliot and, studies, 634
Johannessen studies, 661 Kvenburg studies, 645
Johannson and Cossart studies, 135 Kwantes and Isaac studies, 580–581
Johannson studies, 135 Kyung and Fleming studies, 670
Johansson studies, 230
Johnson, Jan, 647 L
Johnson, Lungu and, studies, 599
Johnson, Siragusa and, studies, 223 Labeling, 839–840
Johnson studies, 257–274, 528, 530–531, 533, Labneh, 445–446
545–546 Lachica studies, 232
Jones, Feresu and, studies, 3, 10 Lactate, 175, 176
Jones, Kramer and, studies, 222 Lactoferrin, 184
Jones, Wilkinson and, studies, 3 Lactoperoxidase system, 183–184
Jonesia denitrificans, 8 Lado studies, 157–198
Jones studies, 3 Lahti studies, 559
Juneja, Klein and, studies, 262 Lambs, 533–534, 549–550, 550, see also Ewe’s milk;
Juneja studies, 535 Sheep
Junttila studies, 547 Lammerding and Doyle studies, 243
Lamont and Postlethwaite studies, 93 Listeria murrayi, 7–8

Lanciotti studies, 393 Listeria seeligeri, 6–7
Landefeld and Seskin studies, 803 Listeria welshimeri, 6–7
Lappi studies, 734 Listeriophages, 196
Larsen studies, 216, 485 Li studies, 665
Larson studies, 483 Lithium chloride, 220–221
Late onset, neonatal disease, 88 Lithium chloride-phenylethanol-moxalactam agar, 228
Lawrence and Gilmour studies, 36, 586 Live fermentate, 185
Lawrence studies, 292, 743 Livestock, domestic, see also specific type
Leasor, Foegeding and, studies, 603, 605 incidence, 56
Leasor and Foegeding studies, 600 meat products, 528
Lecuit studies, 117 Llamas, 70
Lee, McClain and, studies, 226, 228 Localization in tissues, 528–530
Lee and McClain studies, 223, 228, 239 Loessner studies, 232
Lee studies, 532, 666 Lorber, Ooi and, studies, 336
Lefier studies, 273 Los Angeles County
Legan studies, 825 asymptomatic carriage, 93
Lehmann and Schönberg studies, 285 epidemic listeriosis, 96
Leighton studies, 222 foodborne infection, 310–312, 311
Lemaitre studies, 285 human outbreaks, 37
Lemmings, 41 sporadic incidence, 98, 344–345
Leningham studies, 2 surveillance, 347
Lennon studies, 71 Lovett studies, 228, 234, 534, 638
Lessons learned, 137–138 Low pH, 169, 169–170, see also Acidity
Lethal temperature, 164–168 Low studies, 34, 41
Lettuce, 30 Lozniewski studies, 31
Lety studies, 122 Ludwig studies, 4
Leuchner studies, 379 Lukasova studies, 442
Levre studies, 526 Luncheon meats, see also Deli meats; Ready-to-eat meat
Lewis and Corry studies, 218 and poultry products
Lieval studies, 587 food processing, facilities, 756
Liewen, Peters and, studies, 385 meat products, 535–538, 536–537
Lightly processed fish products, 631–632, see also Fish Lund, Ferreira and, studies, 474
and seafood products Lund, Hicks and, studies, 473
Limitations, ecology study methods, 24–25 Lund, Nguyen-the and, studies, 670
Lindqvist and Westöö studies, 777–778 Lunden studies, 744
Link-type products, 755–756, see also specific type Lund studies, 243
Lin studies, 274, 659 Lungu and Johnson studies, 599
Lister, Arthur, 2 Ly and Müller studies, 42
Lister, Joseph Jackson, 2 Lysozyme
Listeria denitrificans, 8 antimicrobial components, foods, 182–183
Listeria fundamentals food processing importance, 182–183
bacteria identification, 9–12
biochemical characteristics, 10 M
chemotaxonomy, 3–4
culture, 9 MA, see Modified atmosphere (MA)
fundamentals, 4–5, 12 Maasdam cheese, 458
genus characteristics, 9–10 MacGowan studies, 28, 516
historical background, 1–2 Mackey studies, 556
metabolism, 10 Macrophages uptake, 118–119
morphology, 9 Madden, Moore and, studies, 600
numerical taxonomy, 3 Madden studies, 634
nutritional requirements, 10 Maini studies, 518
phylogenetic position, 3–5 Maintenance practices, 693, 693–697
rRNA sequencing, 4 Maisner-Patin studies, 452
species identification, 10–12 Mammalian cells
taxonomy, 5–9 cellular adhesion, 118
whole genome, 4, 5 dendritic cells uptake, 118–119
Listeria grayi, 7–8 fundamentals, 113
Listeria ivanovii, 6–7 macrophages uptake, 118–119
Listeria monocytogenes, 6–7 nonprofessional phagocytic cells, 114, 115, 116–118
Index 861
Manchego cheese, 464–465, 469 Italy, 744

Manu-Tawiah studies, 550 United States, 724–730, 725–726, 728–730
Marchisio studies, 456 Meats and meat products, see also Ready-to-eat meat and
Marine environments, 32 poultry products
Marquet-van der Mee studies, 285 bacteriocins, 557–559
Marshall and Schmidt studies, 383, 389 beef, 530–533, 535, 548–549, 549
Marth, El-Gazzar and, studies, 447–449, 675 behavior, 527–559
Marth, El-Shenawy and, studies, 471 Canadian monitoring program results, 508–514
Marth, Farrag and, studies, 388–389 cooked products, 505, 506–508, 507–508, 513, 534–538
Marth, Papageorgiou and, studies, 453, 465, 484–485 cooked roast beef, 535
Marth, Pearson and, studies, 182, 385–387 cooked smoked sausage, 540–542, 542
Marth, Rosenow and, studies, 381, 383–385, 392 cured ham, 534–535
Marth, Ryser and, studies domestic livestock, 528
brick cheese, 455–456 dry fermented sausage, 545–547
brine solutions, 484–485 Europe, 523–527, 524–525
Camembert cheese, 449–451 fermented sausage, 544, 558–559, 560
cheddar cheese, 459, 461 foodborne infection, 320–333
cold enrichment, 217–218 fresh sausage, 538–540, 539–540
cold-pack cheese food, 476 fundamentals, 504
International Dairy Federation method, 238 growth, 530–533
lithium chloride/phenylethanol, 221 ham, cured, 534–535
selective enrichment and plating, 219 incidence, 504–527
survival in cheddar cheese, 413 isolation methods for, 234
whey, 482–483 lamb, 533–534, 549–550, 550
Marth, Schaack and, studies, 436–439, 441–443, luncheon meats, 535–538, 536–537
446, 471 modified-atmosphere packaging, 547–557
Marth, Wenzel and, studies, 377 non-European countries, 527
Marth, Yousef and, studies, 221, 458, 462, 481 non-North American countries, 516, 518–523,
Marth studies, 814–815 519–521
Martin studies, 229, 232 North America, 515–516, 517–518, 523
Maryland (United States), 328 pork, 533–534, 550–552, 551
Mascola studies, 93 potassium sorbate, 177
Massachusetts (United States) raw ground meat, 557–558
epidemic listeriosis, 94 raw meat, 505, 514–523, 530–533
fluid milk, 317 ready-to-eat products, 508, 513, 514–515, 523–527,
human outbreaks, 37 534–538
listeriosis outbreak, 330 recalls and regulatory actions, 507–508, 509–513,
transmission to animals, 65 514, 516
vegetables, 336 roast beef, cooked, 535
Massa studies, 430, 433 sausage, 523–527, 538–547, 549
Mathew and Ryser studies, 437 semidry fermented sausage, 544–545
Mathew studies, 380 smoked sausage, 540–543, 542
Matthews studies, 62 specialty items, 543–544
Mazurier studies, 292 survival, 530–533
McAuliffe studies, 473 thermal inactivation, 552–557, 553–556
McBride and Girard studies, 220, 227 tissue localization, 528–530
McBride Listeria agar, 227–228 uncooked smoked sausage, 543
McClain, Lee and, studies, 223, 228, 239 unfermented sausage, 538–547
McClain and Lee studies, 226, 228 USDA-FSIS monitoring program results,
McKellar studies, 541, 591 504–508
McLauchlin and Gilbert studies, 526 Mechanisms
McLauchlin and Nichols studies, 38 acid damage and tolerance, 170–171
McLauchlin studies, 38, 285, 334 food processing importance, 164–165
McNab, Truscott and, studies, 227 positive regulatory factor A, 134–136
Mead, Hudson and, studies, 742 temperature, 164–165
Mead studies, 98, 348 thermal inactivation, 164–165
Measurement, thermal inactivation kinetics, 165 Medicated soap, 196
Meat processing facilities Mehta and Tatini studies, 461
control methods, 754–756 Mesophilic starter cultures, 436–439, 437–438
Europe, 741 Metabolism, 10
Finland, 744 Metaxopoulos, Samelis and, studies, 535
Methods Molecular subtyping role, 309–310

comparison of subtyping, 297, 298 Molecular virulence determinants
conventional subtyping methods, 284–286 accessory virulence factors, 130–132
ecology, 22–27 bile salt hydrolase, 129–130
enrichment method, 22–23 catalase, 131
enumeration methods, 23–24 cellular adhesion, 118
isolation method, 22–23 dendritic cells uptake, 118–119
limitations, ecology study methods, 24–25 environmentals signals affect, 136
molecular detection methods, 25–27, 26, 287–297 evolutionary aspects, 138
pitfalls, ecology study methods, 24–25 fundamentals, 111–113, 112–113
subtyping, 25–27, 26, 284–297, 298 future issues, 139
traditional enrichment method, 22–23 gene expression, 132–137
Mexican Manchego cheese, 464–465 genome sequence, 137–138
Mexican-style cheese host cell cytoplasm growth, 125–126
epidemic listeriosis, 96 intracellular motility, 126–129, 127
foodborne infection, 310–312 iron uptake systems, 132
human outbreaks, 37 lessons learned, 137–138
illicitly produced/distributed cheese, 315–317 macrophages uptake, 118–119
Meyer-Broseta studies, 363 mammalian cells, 113–119
Mice mechanisms, 134–136
animal feeds, 32 nonprofessional phagocytic cells, 114, 115, 116–118
fecal material, 41 phagocytic vacuole escape, 120–125, 121, 123
human outbreaks, 39 positive regulatory factor A, 132–137, 133
Michard, Ferron and, studies, 243 promoters, 134–136
Michigan (United States), 325 protein p60, 130–131
Microarrays, DNA, 262–263 regulation, 137
Middle East, 662–663 stress response mediators, 131–132
Midelet and Carpentier studies, 538 superoxide dismutase, 131
Miles, Wilson and, studies, 2 transcripts, 134–136
Milk, see also Chocolate milk; Raw milk two-component systems, 137
human outbreaks, 37 Molin, Ternstrom and, studies, 584
lactoferrin, 184 Monitoring programs, see also Surveillance
sanitizing agents, 195 Canada, 419–421, 420, 508–514
Miller studies, 556 cheese and fermented products, 419–421, 420,
Mink, 41 428–432, 429, 431
Minor species, animal listeriosis, 69–71 food processing, facilities, 711, 717
Miscellaneous production facilities France, 421
Netherlands, 745–746 Germany, 428–430, 429
United Kingdom, 742–743 Italy, 430
Miscellaneous sanitizing agents, 195–196 meats and meat products, 504–514
Missouri (United States), 61–62, 67 Switzerland, 430–432, 431
Mitchell, Curtis and, studies, 286 USDA-FSIS monitoring program results, 504–508
Mixed cultures, 388–390 Monoesters, 179–180
Modified atmosphere (MA) Monolaurin, 196
fundamentals, 186–187 Moore and Madden studies, 600
storage, plant origin products, 666–667 Morgan studies, 469
Modified-atmosphere (MA) packaging Morgen, Skovgaard and, studies, 226, 584
beef, 548–549, 549 Morphology, 9
fundamentals, 547–548 Morris and Ribeiro studies, 543, 586
lamb, 549–550, 550 Mosupye and von Holy studies, 662
pork, 550–552, 551 Motes studies, 32
sausages, 549 Mott, Hudson and, studies, 640
thermal inactivation, meats, 552–557, 553–556 Mounier studies, 126
Modified Oxford agar, 228 Mourney, Canillac and, studies, 747
Moir studies, 473 Moxalactam, 223
Mold-ripened cheeses Mozzarella cheese
fermented dairy products, 449–453 fermented dairy products, 457
PALCAM agar, 229 free fatty acids, 178
Molecular methods Müller, Ly and, studies, 42
detection, 25–27, 26 Multilocus enzyme electrophoresis, 287
subtyping, 287–297 Multiple antimicrobial treatments, 197, 197–198
Index 863
Multiplication, stress conditions, 27–35 Norrung and Gerner-Smidt studies, 287–288, 297
Muraoka studies, 362 Norrung studies, 521
Muriana, Wang and, studies, 523 North America
Muriana studies, 556, 598, 605 epidemic listeriosis, 96
Murphy studies, 590, 597–598 meat products, 515–516, 517–518, 523
Murray, E.G.D., 5 standards and criteria, 793–796
Murray, Webb and Swann studies, 1 transmission to animals, 62
Murray and Pirie studies, 2 North Carolina (United States), 315–316
Murray studies, 216–217, 306 Northolt studies, 376–377, 379, 384, 458, 482
Mushrooms, 227 Norton and Batt studies, 262
Mussels, smoked, 334 Norton studies, 36–39, 305–349, 637
Nosocomial transmission, 100
N Notermans studies, 604, 777, 783
Nova Scotia
Nalidixic acid, 221
epidemic listeriosis, 94
Natural environment, 35–42
human outbreaks, 37
Neonatal disease, 87–88
transmission, 36
Netherlands
Nucleic-acid-based methods
food processing, facilities, 745–746
DNA microarrays, 262–263
predisposing factors, 56
fluorescence in situ hybridization, 263–264
risk assessment, 777
standards and criteria, 798
polymerase chain reaction, 260–262
transmission to animals, 59, 67
recombinant bateriophage, 264
Netherlands Government Food Inspection Service, 22, 243
Numerical taxonomy, 3
Newman and Weiner studies, 135
Nutritional requirements, 10
New South Wales, 60, 806
Nyachuba studies, 215–248
New York (United States)
Nyfeldt, Schmidt and, studies, 63
deli meat and frankfurters, 321, 325
Nyfeldt studies, 2
foodborne infection, 328
Nykanen studies, 642
transmission to animals, 65
New Zealand
costs, 807
O
foodborne infection, 341–342 Oblique illumination, 232–234, 233
smoked mussels, 334 O’Donoghue studies, 293
standards and criteria, 791–793 Official methods, detection and isolation, 234–243
Ngutter and Donnelly studies, 244 Ohio (United States), 65, 321
Nguyen-the, Carlin and, studies, 665 Ojeniyi studies, 584
Nguyen-the and Lund studies, 670 Oklahoma, 98
Nichols, McLauchlin and, studies, 38 Olafson studies, 32
Niebuhr and Dickson studies, 530 Olsen studies, 392
Nilsson studies, 642 Olson studies, 219
Nisin, 185–187, 196 Omary studies, 667
Noah studies, 243 Ontario, Canada, 65
Nocera studies, 288, 297 Ooi and Lorber studies, 336
Nogva studies, 262 Optimum temperature, 159–160
Nohuman primates, 71 Organic acids, 175, 175–178
Non-European countries Ortel studies, 222
cheese and fermented products, 435 Osborne, Bremer and, studies, 644
meat products, 527 Osmotolerance factors, 172–173, 172–173
Nonfat dry milk, 393–394 Outbreaks
Nonfluid dairy products detection and investigation, 309–310, 310
butter, 392–393 ecology, 37–38
fundamentals, 391 ten or more cases, 307
ice cream, 391–392 vs. sporadic cases, 820–821
nonfat dry milk, 393–394 Oxford agar, 228
Noninvasive disease, 90–93 Oysters, 231–232
Non-North American countries, 516, 518–523, 519–521 Ozone, 194–195, 195
Nonpregnant adults, 88–90
Nonprofessional phagocytic cells, 114, 115, 116–118, P
see also Phagocytic vacuole escape
Non-United States products, 574–576, 580–587 Pace studies, 643
Non-United States programs, 419–435 Pacific Northwest, 65
Pacific oysters, 32 Pickled cheeses, 465–468

Packaging, control methods, 752 Pigeons, 41
Pagan studies, 198 Pigs, 41, see also Swine
Painter studies, 85–102 Pillis, Fuzi and, studies, 222
PALCAM agar, 228–229 Pine studies, 380
Palumbo and Williams studies, 577 Pingulkar studies, 662, 665
Paoli studies, 265 Pini and Gilbert studies, 433, 581, 583
Papageorgiou and Marth studies, 453, 465, 484–485 Pinkerton, Pat, 647
Papageorgiou studies, 466, 475 Pipeline connections, 752
Parabens, 175, 178 Pirie, Murray and, studies, 2
Parish and Higgins studies, 672 Pirie studies, 2
Park studies, 600 Pitfalls
Parmesan cheese, 462–463 ecology study methods, 24–25
Parrots, 41 rapid method detection, 265
Partridges, 41 Pitt studies, 437, 442
Pass-through phenomenon, 42 Plant components, 669–670
Pasteurization, control methods, 750–752, 751 Plant extracts, 181–182, 182
Pasteurized cheese spreads, 185–186 Plant origin products
Pasteurized milk Asia, 662
direct plating media, 231 behavior, 663–676, 674
epidemic listeriosis, 94, 96 Canada, 660
unfermented dairy products, 367–376, 379–380 chemical sanitizers, 668–669
Pasteurized process cheese, 477 Europe, 660–661
Pastry and bakery items fruits and fruit juices, 671–674, 672
baked goods, 243 fundamentals, 655–656
potassium sorbate, 177 growth, 663–666, 664
USDA-FSIS method, 243 heat, 667–668
Pâté inactivation, 667–673
epidemic listeriosis, 97, 317 incidence, 657–663, 671–672
foodborne infection, 327–330 Middle East, 662–663
human outbreaks, 37 modified atmosphere storage, 666–667
Paterson studies, 6, 601 plant components, 669–670
Pathogenicity, 817–818 processing techniques, 671, 671
Patterson studies, 589 raw vegetables, 657–663, 658
PCR, see Polymerase chain reaction (PCR) risk from, 656–657, 657
Pearson and Marth studies, 182, 385–387 survival, 663–666, 664
Peccio studies, 744 United States, 658–660
Pediocin, 186 Plant processing facilities
Peeler and Bunning studies, 778 Australia, 747–748, 748
PEF, see Pulsed electric field (PEF) processing barrier establishment, 715
Pelroy studies, 640–641 biofilms, 685–704
Peng and Sheref studies, 244 cheese production facilities, 743
Pennsylvania (United States), 319–320, 326 chocolate production facilities, 741–742
Peptone water, 196 clarifiers, 750
Peron, Hamon and, studies, 286 cleaning, control method, 707–710
Peters and Liewen studies, 385 corned beef, 755
Peterson studies, 640 corrective actions, 718
Petran studies, 658 critical control point, 717
PFGE, subtyping, 291–292, 293 critical limits, 717
Phage typing (bateriophage typing), 285–286 cross-connections, 752
Phagocytic vacuole escape, 120–125, 121, 123, see also dairy processing facilities, 720–723, 721–724, 739–741,
Nonprofessional phagocytic cells 740–741, 746–747, 750–754
Pheasants, 41, 67 documentation procedures, 718–719
Phenotypic markers, 11 dry areas, cleaning, 707
Phenylethanol, 220–221 egg processing facilities, 732, 757
Philadelphia, Pennsylvania, 319–320 England, 742–743
Phylogenetic position, 3–5 equipment design, 697–704
Physiology Europe, 739–742
high salt concentration, 174–175 factors to consider, 710–711
resistance to heat, 168 factory design, 697–704
Pickett, Sword and, studies, 286 farm environment, 750
Index 865
fermented dairy products, 754 Portnoy, Tilney and, studies, 126

filling, 752 Portnoy studies, 122, 131
Finland, 744 Positive regulatory factor A
France, 743 environmental signals, 136
frankfurters, 755–756 fundamentals, 132–134, 133
frozen dairy products, 753 mechanism, 134–136
fruit processing facilities, 738, 758–759 promoters, 134–136
fundamentals, 683–684, 683–685 regulation, 136
growth, 685–704 transcripts, 134–136
hazard analysis critical control point, 711–713, 712, two-component systems, 136
716–719 Postlethwaite, Lamont and, studies, 93
household kitchens, 748–749 Postpackaging pasteurization, 598
hygiene, 38 Postpasteurization, 823–824
incidence, 719–748 Potassium sorbate, 175, 177–178
industry-specific control approaches, 749–759 Potassium tellurite, 219–220
Italy, 744 Potassium thiocyanate, 222
kitchens, household, 748–749 Potel studies, 2, 306
link-type products, 755–756 Pothuri studies, 642
luncheon meats, 756 Poultry processing facilities
maintenance practices, 693, 693–697 control methods, 756–757
meat processing facilities, 724–730, 725–726, 728–730, England, 742, 742
741, 744, 754–756 Europe, 741
miscellaneous production facilities, 742–743, 745–746 France, 743
monitoring, 711, 717 United States, 730–731, 731
Netherlands, 745–746 Poultry products, see also Raw chicken; Raw turkey;
packaging, 752 Ready-to-eat meat and poultry products
pasteurization, 750–752, 751 antimicrobial agents and additives, 589, 598–599
pipeline connections, 752 behavior in, 587–597
poultry processing facilities, 730–731, 731, 741–742, chicken, raw, 577–579, 587–590
742, 743, 756–757 continental Europe, 582–583, 584–585
rebagged products, 755 control guidelines, 597–599
reclaimed and reworked products, 753 cooked products, 586–587, 590–592, 591–593
record-keeping procedures, 718–719 deli meat and frankfurters, 321–327
repair practices, 693, 693–697 European products, 580–587
roast beef, 755 frankfurters and deli meat, 321–327
sampling plans, 713–715, 714 fraser broth, 226
sanitation, control method, 707–710 fundamentals, 320–321, 332–333, 572
seafood processing facilities, 732–738, 733, 735–737, gamma radiation, 589–590
741, 744, 757–758 growth, 587–592
separators, 750 isolation methods for, 234
smoked-salmon processing facilities, 743 moxalactam, 223
Spain, 745 non-United States products, 574–576, 580–587
Sweden, 746–747 PALCAM agar, 229
Switzerland, 747 postpackaging pasteurization, 598
traditional control approaches, 704–710 raw chicken, 587–590, 588
traffic patterns, 710 raw poultry, 580–586
United Kingdom, 742–743 raw turkey, 578–580, 579
United States, 719–738 ready-to-eat products, 586–587, 590–592, 597–599
vegetable processing facilities, 738, 745, 758–759 recalls, 574, 575–576
verification procedures, 718 reduction of listeriosis, 333
wet processing areas, cleaning, 707–709 thermal inactivation, 593–597, 594–596
Plating, 219–223 transmission pathways, 43
Polymerase chain reaction (PCR), 260–262 turkey, raw, 578–580, 579
Polymyxin B, 222–223 United Kingdom, 580–584, 582–583
Pork, see also Swine United States products, 577–579
foodborne infection, 328–329 USDA control guidelines, 597–599
human outbreaks, 37 USDA-FSIS, 241, 572–574, 574
meat products, 533–534, 550–552, 551 Poysky studies, 646
plant extracts, 181–182 Predisposing factors, 56–57
processing facilities, 743 Pregnancy, 87
Porritt, Sutherland and, studies, 748 Prevention, 101–102
Pritchard studies, 245, 721 Rasmussen studies, 295

Process, risk assessment, 771–773 Rats, 41
Processed food risks, 838–839 Raw chicken, 587–590, 588, see also Poultry products; Raw
Processed meat, 241 poultry
Processing techniques, 671, 671 Raw cow’s milk, 358–366, see also Milk
Promoters, 134–136 Raw ewe’s milk, 366, 366–367, see also Sheep
Protein p60, 130–131 Raw fish, 242, see also Fish and seafood products
Proteomic endowment, 831–833 Raw goats milk, 366, 366–367, see also Goats and goat’s milk
Pucci studies, 473 Raw ground meat, 226, 557–558, see also Meats and meat
Pulsed electric field (PEF) processing, 189–190, 190 products
Pulsed-field gel electrophoresis (PFGE) Rawles studies, 639
DNA macrorestriction analysis, 291–292, 293 Raw meat, see also Meats and meat products
molecular subtyping role, 309 meat products, 505, 514–523, 530–533
PulseNet moxalactam, 223
clusters of listeriosis, 345 USDA-FSIS method, 241
deli meat and frankfurters, 321, 324–326 Raw milk, see also Chocolate milk; Milk
DNA macrorestriction analysis, 292 cheese and fermented products, 481
epidemiological patterns, 94 FDA method, 238
FDA method, 238 fraser broth, 226
as surveillance system, 309–310 hydrogen peroxide, 183
PurePulse system, 189 PALCAM agar, 229
selective enrichment and plating, 224–225
Q unfermented dairy products, 377–379, 378–380
Raw-milk fresh, unripened cheese, 315–317, see also
Quaternary ammonium compounds, 194 Fermented products and cheese
Quebec, Canada, 314–315 Raw-milk soft cheese, 314–315, see also Fermented
Queen Anne (of England), 2 products and cheese
Queso blanco cheese, see Mexican-style cheese Raw poultry, see also Poultry products; Raw chicken
Queso de los Ibores cheese, 464 direct plating media, 232
Queso fresco cheese, see Mexican-style cheese fraser broth, 227
Quinto studies, 383, 389 incidence, 580–586
Raw turkey, 578–580, 579, see also Poultry products;
R Turkey and turkey products
Raw vegetables, see also Vegetables
Rabbits, 41 epidemic listeriosis, 94
Rainbow trout, 71, 340–341 isolation methods for, 234
Rajkowski studies, 380, 485 live fermentate, 185
Ralovich studies, 221, 225, 228 McBride Listeria agar, 228
Random amplification of polymorphic DNA (RAPD), PALCAM agar, 229
292–294 plant origin products, 657–663, 658
Rapid method detection Razavilar studies, 478
advances, 265–266 REA, see Restriction endonuclease analysis (REA)
antibody-based methods, 264–267 Read, Ralston, 814
automation, 259 Ready-to-eat meat and poultry products, see also Deli
biosensors, 269–271, 270 meats; Luncheon meats; Meats and meat products;
cell separation and concentration, 266–267 Poultry products
chip-based microanalytical systems, 271, 272 epidemic listeriosis, 96–97
commercial availability, 258–259 febrile gastroenteritis, 341–342
diagnostic targets, 259 foodborne infection, 321–327, 341–342
DNA microarrays, 262–263 Ready-to-eat products
flow cytometry, 267–269, 268–269 meat products, 508, 513, 514–515, 523–527, 534–538
fluorescence in situ hybridization, 263–264 poultry products, 586–587, 590–592, 597–599
fundamentals, 257–259, 274 risk assessment, regulatory control, and economic
generic species vs. specific species, 259 impact, 783
ideal detection, 258 REB, see Repetitive element-based (REB) typing
nucleic-acid-based methods, 259–264 Rebagged products, 755
polymerase chain reaction, 260–262 Recalls
potential pitfalls, 265 fermented products, 407–419
recombinant bateriophage, 264 meat products, 507–508, 509–513, 514, 516
spectroscopic methods, 271–274, 273 poultry products, 574, 575–576
traditional methods, 258 unfermented products, 370, 371–373
Index 867
Reclaimed and reworked products, 753 Rijpens studies, 574, 587

Recombinant bateriophage, 264 Rillettes
Record-keeping procedures, 718–719 epidemic listeriosis, 97
Recovering injured Listeria, 244–248 fluid milk similarity, 317
Reduction, 333 foodborne infection, 330–331
Reglier-Poupet studies, 125 shelf life, 332
Regulation, gene expression, 136 Ripabelli studies, 294
Regulations, see also Risk assessment, regulatory control, Risk assessment
and economic impact fish and seafood products, 635–636
fish and seafood products, 634–635 plant origin products, 656–657, 657
tolerance, 836–837 significance, 819–820
Reichel, Gill and, studies, 548 Risk assessment, regulatory control, and economic impact
Reindeer, 70 Australia, 773–774, 791–793
Repair practices, 693, 693–697 Austria, 796
Repetitive element-based (REB) typing, 294 Canada, 775, 794–796, 795
Research advances, 244 characterization of risk, 785–789, 786–788
Research needs China, 775
academic perspective, 830–835 Codex Alimentarius Commission, 783–790
behavior in foods, 817 consumption, 784
consumer education, 839–840 costs, 801–807, 802–803, 805–806
consumer exposure, 816–817, 821–827 criteria and standards, 790–807
contamination prevention, 821–823 Denmark, 796–797
control options, 837–838 dose-response assessment, 784–785
deli meats, 838 European countries, 796–799
destruction, 818–819 European Union, 799–800, 800
detection, 818 France, 775–776, 797
elimination, 818–819 fundamentals, 768–771
enumeration, 818 Germany, 777, 797
FAO/WHO risk assessment significance, 819–820 growth, 784
FDA/FSIS risk assessment significance, 819–820 Hong Kong, 775
FDA perspective, 815–819 initial contamination, 783–784
food safety perspective, 835–840 internationally, 781–790, 800–801
foods of concern, 839 Italy, 798
fundamentals, 814–815 Netherlands, 777, 798
gene expression, 833–834 New Zealand, 791–793
genomic endowment, 831–833 North America, 793–796
growth prevention, 824–826 process, 771–773
industry perspective, 819–827 ready-to-eat foods, 783
labeling, 839–840 Slovenia, 798
outbreaks vs. sporadic cases, 820–821 standards and criteria, 790–807
pathogenicity, 817–818 summary, 790
postpasteurization, 823–824 Sweden, 777–778
processed food risks, 838–839 United Kingdom, 798–799
proteomic endowment, 831–833 United States, 778–781, 782, 793–794
regulatory tolerance, 836–837 Rivera-Betancourt studies, 724
retail product focus, 838–839 Roast beef, 535, 755, see also Beef
risk assessment significance, 819–820 Robbins studies, 129
Restriction endonuclease analysis (REA), 287–288 Robertson studies, 527, 661
Restriction fragment length polymorphism, 288–291, 289–290 Robinson, Higgins and, studies, 10
Retail product focus, 838–839 Robinson studies, 160
Rho studies, 523 Rocourt, Bille and, studies, 284, 297
Ribeiro, Morris and, studies, 543, 586 Rocourt studies, 1–12, 285–286
Ribeiro and Carminati studies, 444 Rodriguez-Saona studies, 274
Ribotyping, 288–290, 288–291 Rodriguez studies, 222–223, 225, 231, 469
Rice salad, 37, 338 Roe deer, 70
Richard, Siswanto and, studies, 382 Romania, 71
Richard studies, 452 Rooks, 41
Richter, Al-Sheddy and, studies, 579 Rørvik studies, 631, 637, 640
Ridley studies, 290 Rosenow and Marth studies, 381, 383–385, 392
Riedo studies, 337 Roth and Donnelly studies, 248
Rijpens and Herman studies, 230 rRNA sequencing, 4
Rudi studies, 296 sodium dichloroisocyanurate, 196

Russell studies, 600 Ultra-Kleen, 196
Russia, 66, 72 Sarumehmetoglu and Kaymaz studies, 467
Rusul studies, 585 Sauders studies, 21–44
Ryser, Issa and, studies, 446 Saudi Arabia, 59
Ryser, Mathew and, studies, 437 Saunders studies, 290
Ryser and Marth studies Sausages
brick cheese, 455–456 cooked smoked sausage, 540–542, 542
brine solutions, 484–485 dry fermented sausage, 545–547
Camembert cheese, 449–451 fermented sausage, 544, 558–559
cheddar cheese, 459, 461 fraser broth, 227
cold enrichment, 217–218 fresh sausage, 538–540, 539–540
cold-pack cheese food, 476 fundamentals, 538–547, 549
International Dairy Federation method, 238 incidence, 523–527
lithium chloride/phenylethanol, 221 modified-atmosphere packaging, 549
selective enrichment and plating, 219 plant extracts, 181–182
survival in cheddar cheese, 413 semidry fermented sausage, 544–545
whey, 482–483 smoked sausage, 540–543, 542
Ryser studies uncooked smoked sausage, 543
cold enrichment, 217 unfermented sausage, 538–547
current characterization methods limitations, 24 Scandinavia, 61
fermented dairy products, 405–486 Schaack and Marth studies, 436–439, 441–443, 446, 471
injured recovery, 246 Schabinski, Urbach and, studies, 601, 604
poultry and egg products, 571–606 Schaffer studies, 461
RiboPrinter, 290 Schlech studies, 671
unfermented dairy products, 357–394 Schmidt, Kaya and, studies, 531
Ryu studies, 522, 662 Schmidt, Marshall and, studies, 383, 389
Schmidt and Nyfeldt studies, 63
S Schoeni, Doyle and, studies, 223–224, 226, 229, 238
Schönberg, Lehmann and, studies, 285
Salamah studies, 662 Schönberg studies, 284, 430
Salcedo studies, 296 Schöpfer, Breer and, studies, 518
Sallam and Donnelly studies, 245 Schultz studies, 2, 64
Salmon, 165 Schuman, Sheldon and, studies, 603
Saltijeral studies, 435 Schwarte, Biester and, studies, 66, 217
Salts, 174–178 Schwartz and Cantor studies, 291
Salvat studies, 743 Scotland, 31
Samelis and Metaxopoulos studies, 535 Scott, Jenny, 827
Samelis studies, 475, 547 Scott studies, 806
Sampling plans, 713–715, 714 Seafood products, see Fish and seafood products
Sandine, Benkerroum and, studies, 474 Seeliger, Donker-Voet and, studies, 6
San Francisco, 98 Seeliger, H.P.R., 5
Sanitation, control method, 707–710 Seeliger, Weiss and, studies, 28
Sanitizers Seeliger studies, 6, 221, 407, 814
acid type, 194 Selective enrichment and plating
calcinated calcium solution, 196 acriflavine, 221–222
carvacrol, 196 agents, 219–223, 220
chitosan, 196 caftazidime, 223
chlorine and chlorinated compounds, fundamentals, 219
193–194 lithium chloride, 220–221
chlorohexidine, 196 moxalactam, 223
fundamentals, 192–193 nalidixic acid, 221
glutaraldehyde, 196 phenylethanol, 220–221
hydrogen peroxide, 196 polymyxin B, 222–223
iodophors, 195–196 potassium tellurite, 219–220
listeriophages, 196 potassium thiocyanate, 222
medicated soap, 196 thallous acetate, 222
monolaurin, 196 trypaflavine, 221–222
ozone, 194–195, 195 Selective enrichment media
peptone water, 196 ARS-MMLA, 229–230
quaternary ammonium compounds, 194 blood-free plating, 229
Index 869
comparative evaluation, 230–232 Siswanto and Richard studies, 382

Food and Drug Administration method, 234–238, 236–237 Sizmur and Walker studies, 660, 666
Fraser broth, 226–227 Skovgaard and Morgen studies, 226, 584
fundamentals, 223–225 Skovgaard studies, 67
gum base nalidixic acid medium, 229 Skunks, 41
β-hemolysis, 233–234 Slabospits’ Kii studies, 66
incubation conditions, 232 Slade and Collins-Thompson studies, 222,
International Dairy Federation method, 238–239, 239 224–225, 362
isolation media, 227–232 Slade studies, 362
LPM agar, 228 Slavchev studies, 393
McBride Listeria agar, 227–228 Sleath, Watkins and, studies, 23, 30–31
modified Oxford agar, 228 Slovenia, 798
Netherlands Government Food Inspective Service, 243 Slutsker studies, 85–102
oblique illumination, 232–234, 233 Smoke, 181
official methods, 234–243 Smoked fish products
Oxford agar, 228 fish and seafood products, 630–631
PALCAM agar, 228–229 processing plants, 38–39
USDA-FSIS method, 239–243, 240 Smoked mussels, 334
UVM broth, 225–226 Smoked-salmon processing facilities, 743
Selective media, 223–243 Smoked sausage, 540–543, 542
Seman studies, 539, 825 Sodium benzoate, 175, 178
Semidry fermented sausage, 544–545 Sodium diacetate, 175, 176–177
Semihard soft cheese, 314–315 Sodium dichloroisocyanurate, 196
Semisoft cheeses, 457–463 Sodium nitrite, 180
Separators, 750 Sodium propionate, 175, 177
Serotyping Soft, unripened Mexican-style cheese, 310–312,
historical developments, 6 see also Mexican-style cheese
subtyping, 284–285 Soft cheese
Seskin, Landefeld and, studies, 803 epidemic listeriosis, 96
Sewage, 30–31 FDA method, 238
Sexual transmission, 100 human outbreaks, 37
Shamsuzzaman studies, 590 PALCAM agar, 229
Sheehan studies, 134 Soft Italian cheese, 457
Sheep, see also Ewe’s milk; Lambs Soft mold-ripened cheeses, 230
animal feeds, 33–34 Soft unripened cheese, 471–474
animal listeriosis, 57–60 Soil, 28, 29, 30
fecal material, 41 Solano-Lopez and Hernandez-Sanchez studies, 464–465
soil contamination, 30 Sontakke and Farber studies, 291
water contamination, 32 Soonthoranant and Garland studies, 32
Sheldon and Schuman studies, 603 Soriano studies, 522, 661
Shelef, Ferguson and, studies, 675 Soultos studies, 581
Shelef, Sionkowski and, studies, 602 Sows, see Swine
Shelef and Yang studies, 589 Spahr, Bachmann and, studies, 456
Shelef studies, 531 Spain, 745
Shellfish, 630, see also Fish and seafood products Specialty items, 543–544
Sheref, Peng and, studies, 244 Species identification, 10–12
Sheridan studies, 516, 549 Specific species vs. generic species, 259
Shineman and Harrison studies, 639 Spectroscopic methods, 271–274, 273
Shrimp, see also Fish and seafood products Spices, 181–182, 182
fatty acid monoesters, 178 Sporadic human cases
febrile gastroenteritis, 337–338 dietary risk factors, 98–100
water contamination, 32 foodborne infection, 344–348
Sielaff studies, 530 incidence, 97–98, 99
Sikes studies, 444 transmission dynamics, 38–40
Silk studies, 246 Stajner studies, 441, 471
Silliker and Gabis studies, 721 Standards and criteria, 790–807
Sindoni studies, 56 Stanley, Foegeding and, studies, 606
Sionkowski and Shelef studies, 602 Starlings, 41
Sipka studies, 468 Starter cultures, 436–445
Siragusa, Cutter and, studies, 559 Starter distillates, 448–449
Siragusa and Johnson studies, 223 Stecchini studies, 457
Stegeman studies, 534 plant origin products, 663–666, 664

Steinbruegge studies, 664 stress conditions, 27–35
Stilton cheese, 186 water activity, 171–172, 172
Strain, resistance to heat, 167–168 Sutherland and Porritt studies, 748
Strangeways, Bendig and, studies, 661 Swaminathan studies, 283–297
Stress Swann, Murray, Web and, studies, 1
ecology, 27–35 Sweden
elevated sublethal temperatures, 163 cold-smoked fish products, 334
response mediators, 131–132 foodborne infection, 342–343
Strikwerda, de Vries and, studies, 63 food processing, facilities, 746–747
Stuart and Welshimer studies, 6–7 risk assessment, 777–778
Study methods, ecology, 22–27 transmission to animals, 63
Subtyping Sweetened condensed milk, 387
amplified fragment length polymorphism, 294–295 Swine, 66–67, see also Pigs
antimicrobial susceptibility testing, 286 Swiss cheese, 462
bacteriocin typing, 286 Switzerland
bacteriophage typing, 285–286 epidemic listeriosis, 96
chromosomal DNA restriction endonuclease analysis, fermented dairy products, 430–432, 431
287–288 foodborne infection, 312–313
comparison of methods, 297, 298 food processing, facilities, 747
conventional methods, 284–286 listeriosis outbreak, 329
DNA macrorestriction analysis, 291–292, 293 monitoring programs, 430–432, 431
DNA-sequence-based strategies, 295–297 transmission to animals, 62, 71
ecology study methods, 25–27, 26 Sword and Pickett studies, 286
methods comparison, 297, 298 T
molecular methods, 287–297
multilocus enzyme electrophoresis, 287 Tahon-Castel, Beerens and, studies, 221
PFGE, 291–292, 293 Taleggio cheese, 456
phage typing, 285–286 Taormina and Beuchat studies, 556
random amplification of polymorphic DNA, 292–294 Tasmania, smoked mussels, 334
repetitive element-based subtyping, 294 Tatini, Mehta and, studies, 461
restriction fragment length polymorphism, 288–291, Tawfik studies, 467
289–290 Taxonomy, 5–9, see also Phylogenetic position
ribotyping, 288–290, 288–291 Temperature
serotyping, 284–285 cold tolerance, 161–163, 162
Sudanese white-pickled cheese, 467–468 culture, 9
Sulzer and Busse studies, 452 D-value, 165–167
Sumner and Gallagher studies, 773 elevated sublethal temperature, 163
Superoxide dismutase, 131 engulfment, 167
Surendran, Fuchs and, studies, 630 extrinsic factors, 167
Surrogate microorganisms, 168 food composition, 167
Surveillance, see also Monitoring programs freezing, 163–164
Canada, 419–421, 420 growth, 159–163
domestic cheese, 407–414 heat resistance, 167
FDA method, 234 intrinsic factors, 167–168
foodborne infection, 344–349, 346–347 kinetics, thermal inactivation, 165–167
France, 414–418, 421, 428 kinetics of growth, 160–161, 161
Germany, 428–430, 429 lethal temperature, 164–168
imported cheese, 414–419 measurement, thermal inactivation kinetics, 165
Italy, 430 mechanisms, thermal inactivation, 164–165
non-United States programs, 419–435 optimum, 159–160
Switzerland, 430–432, 431 physiological state, 168
Western European countries, 418–419 range, 159–160
Surveys, 618–632 strain, 167–168
Survival stress adaptation, 163
enrichment and isolation methods, 22 surrogate microorganisms, 168
fish and seafood products, 638–640 thermal inactivation, 164–167
high salt concentration, 174 virulence variations, 163
low pH, 169 z-value, 165–167
meat products, 530–533 Temperature-dependent virulence, gene expression, 134–136
Index 871
Tennessee (United States) within natural environments, 42

deli meat and frankfurters, 321 sporadic human cases, 38–40
transmission to animals, 64 Trappist cheese, 456
Ternstrom and Molin studies, 584 Treatment
Terplan studies, 363, 450, 455, 483, 743 animal listeriosis, 72
Texas (United States) human listeriosis, 101
Mexican-style cheese, 316 Truscott and McNab studies, 227
vegetables, 336 Trüssel and Jemmi studies, 543, 545
Thallous acetate, 222 Trypaflavine, 221–222
Tham studies, 469 Tsigarda studies, 548
Thermal inactivation Tsiotsias studies, 475
egg products, 604–606 Turkey and turkey products, see also Poultry products
kinetics, 165–167 asymptomatic carriers, 67
meat products, 552–557, 553–556 dietary risk factors, 99–100
mechanisms, 164–165 epidemic listeriosis, 96–97
poultry products, 593–597, 594–596 fecal material, 41
Thermophilic starter cultures, 441–442, human outbreaks, 37
441–443 organic acids and salts, 176–177
Thimothe studies, 734, 737 potassium sorbate, 177–178
Thompson, Daryl, 647 Turkish white-brined cheese, 467
Thunberg studies, 660 Turtles, 41
Ticks, 41 Two-component systems, 136
Tilney and Portnoy studies, 126
Tilsiter cheese, 456 U
Tipparaju studies, 444
Tissue localization, 528–530 Ukuku and Fett studies, 673
Todd studies, 767–807 Ultrafiltered milk, 388
Todorov, Ikonomov and, studies, 467–468 Ultra-Kleen, 196
Todorov studies, 379 Ultraviolet radiation, 188–189
Tolerance, acid, 170–171 Uncooked smoked sausage, 543
Tompkin studies, 690, 727, 819–827 Unfermented dairy products, see also Dairy products
Traditional methods autoclaved milk, 380–387
control approaches, 704–710 behavior, 376–388
enrichment, 22–23 butter, 392–393
fermented milks, 445–446 chocolate milk, 380–387
rapid method detection, 258 cream, 380–387
Traffic patterns, 710 evaporated milk, 387
Transcripts, 134–136 fundamentals, 357–358
Transmission, to animals ice cream, 391–392
cattle, 62–66 incidence, 358–376
crustaceans, 71–72 intensively pasteurized milk, 379–380
ecology, 40–42 mixed cultures, 388–389, 390
fish, 71–72 nonfat dry milk, 393–394
fowl, 67–69 nonfluid dairy products, 391–394
fundamentals, 55 other products, 367–376
goats, 60–62 pasteurized milk, 367–376, 379–380
incidence, 56 raw cow’s milk, 358–366
minor species, 69–71 raw ewe’s milk, 366, 366–367
predisposing factors, 56–57 raw goats milk, 366, 366–367
sheep, 57–60 raw milk, 377–379, 378–380
swine, 66–67 sweetened condensed milk, 387
transmission dynamics, 40–42 ultrafiltered milk, 388
treatment, 72 Unfermented sausage, 538–547
Transmission dynamics United Kingdom
animals listeriosis, 40–42 asymptomatic carriage, 93
food-processing environments, 36 fluid milk, 317
global pathways and reservoirs, 42–44 food processing, facilities, 742–743
human listeriosis, 35–40 poultry products, 580–584, 582–583
human outbreaks, 37–38 predisposing factors, 57
modes, fish and seafood products, standards and criteria, 798–799
636–638 water contamination, 31
United States Vegetables, processing facilities

alternative processing technologies, 187 control methods, 758–759
animal feeds, 33 Spain, 745
asymptomatic carriage, 91 United States, 738
cheese and fermented products, 407–419 Vegetation, 28, 29, 30
chocolate milk, 338–339 Vela studies, 41
epidemic listeriosis, 96 Venables studies, 435
foodborne infection, 321–327 Venkitanarayanan studies, 673
food processing, facilities, 719–738 Verification procedures, HACCP, 718
frankfurters and deli meat, 321–327 Viable but not culturable (VBNC) state, 24
human outbreaks, 37 Virulence variations, 163
meningitis relationship, 88–89 Vogel studies, 631
neonatal disease, 88 Voles, 41
paté, 328 von Holy, Mosupye and, studies, 662
plant origin products, 658–660 Vorster studies, 527
poultry products, 577–579 Vorst studies, 576
predisposing factors, 56 Vos studies, 294
prevention, 101
ready-to-eat meat and poultry products, 341–342 W
risk assessment, 778–781, 782
shrimp, 337–338 Waak studies, 746
sporadic incidence, 98 Wadsworth, Goldfine and, studies, 119
standards and criteria, 793–794 Wales
surveillance, 344–349, 407–419 epidemic listeriosis, 97
thermal inactivation, 165 foodborne infection, 327
transmission to animals, 63–64, 66 listeriosis outbreak, 327, 330
ultraviolet radiation, 188 neonatal disease, 88
University of Vermont (UVM) broth sporadic incidence, 98
detection and isolation, 225–226 Walker, Sizmur and, studies, 660, 666
enrichment and isolation, 22 Walker studies, 592, 722
Unnerstad studies, 297 Wang and Muriana studies, 523
Unpasteurized cheese, 96, see also specific type Wang studies, 522
Urbach and Schabinski studies, 601, 604 Wan studies, 452
USDA control guidelines, 597–599 Warburton studies, 243–244
USDA-FSIS Warriner studies, 530
detection and isolation method, 239–243, 240 Washington (United States), 315, 317
monitoring program, meat products, 504–508 Water, survival in stress conditions, 31–32
testing program, poultry products, 572–574, 574 Water activity
zero tolerance, 321 food processing importance, 171–173
UVM, see University of Vermont (UVM) broth growth, 171–172, 172
Uyttendaele studies, 522, 581, 585 osmotolerance factors, 172–173, 172–173
survival, 171–172, 172
V Watkins and Sleath studies, 23, 30–31
Weagant studies, 620
Vacherin Mont d’Or, 312–313 Webb and Swann, Murray, studies, 1
Valenti, Comi and, studies, 463 Wederquist studies, 591
Validation, HACCP, 718 Weiner, Newman and, studies, 135
Vanderlinde, Grau and, studies, 527, 532, 535 Weiss and Seeliger studies, 28
Van Laack studies, 536 Weis studies, 23
van Netten, De Boer and, studies, 543 Wekell studies, 617–646
van Netten studies, 223, 227–229, 661 Welshimer, Groves and, studies, 6
Van Nierop studies, 585 Welshimer, H.J., 5
van Renterghem studies, 23, 663 Welshimer, Stuart and, studies, 6–7
Van Schothorst studies, 777 Welshimer and Donker-Voet studies, 28
Varabioff studies, 527 Welshimer studies, 30
Various environments, ecology, 27 Wendorff studies, 542
Vasquez-Salinas studies, 364 Wenzel and Marth studies, 377
Vazquez-Boland, Kreft and, studies, 136 Wernars studies, 293
Vazquez-Boland studies, 24 Wesley and Ashton studies, 37
VBNC, see Viable but not culturable (VBNC) state Wesley studies, 55–72, 579
Vegetables, 335–336, see also Raw vegetables Western European countries, 418–419
Index 873
Westöö, Lindqvist and, studies, 777–778 Y

Wet processing areas, cleaning, 707–709
Whey Yakima County, 317
cheese and fermented dairy products, 474–477, 482, Yang, Shelef and, studies, 589
482–483 Yan studies, 688, 690
pulsed electric field processing, 190 Yen studies, 552
Whittenbury studies, 28 Yogurt, 443–444
Whole genome sequencing, 4, 5 Yousef and Marth studies, 221, 458,
Wiedmann, Hoffman and, studies, 242 462, 481
Wiedmann studies, 21–44, 290 Yousef studies, 157–198, 449, 461, 557
Wilkinson and Jones studies, 3 Yugoslavia, 67
Williams, Palumbo and, studies, 577 Yugoslavian white-pickled cheese, 468
Williams, T., 139
Wilson and Miles studies, 2
Wilson studies, 516 Z
Wimpfheimer studies, 550, 577, 587–588
Zaika and Fanelli studies, 160
Wolyniak, Cecilia, 647
Zaika studies, 541
Wong and Kitts studies, 600
Zeitoun and Debevere studies, 588
Wood and Woodbine studies, 163
Zhang and Farber studies, 668
Woodbine, Wood and, studies, 163
Zhu studies, 599
Wramby studies, 63
Zottola, Dean and, studies, 391
Zottola, Herald and, studies, 529
X Zuniga-Estrada studies, 443
Xenos, Karaioannoglou and, studies, 552 z-value, 165–167

Listeria Listeriosis and Food Safety Third Edition

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International Standard Book Number-10: 0-8247-5750-5 (Hardcover)

Library of Congress Cataloging-in-Publication Data

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Preface to the Third Edition

Preface to the Second Edition

Preface to the First Edition

L. monocytogenes as the causative agent of listeriosis

Michael Kuhn Brian D. Sauders

Beatrice H. Lado Chris Scherf

Elmer H. Marth Laurence Slutsker

2 Listeria, Listeriosis, and Food Safety

The Genus Listeria and Listeria monocytogenes 3

PHYLOGENETIC POSITION OF THE GENUS LISTERIA

4 Listeria, Listeriosis, and Food Safety

WHOLE GENOME SEQUENCING

The Genus Listeria and Listeria monocytogenes 5

a coryneform bacterium as evidenced by numerical phenetic studies, chemotaxonomic properties,

TAXONOMY OF THE GENUS LISTERIA

6 Listeria, Listeriosis, and Food Safety

L. MONOCYTOGENES, L. IVANOVII, L. WELSHIMERI, AND L. SEELIGERI

Genomic group 1 contained the type strain of L. monocytogenes, thus corresponding to L.

The Genus Listeria and Listeria monocytogenes 7

L. GRAYI (AND “L. MURRAYI”)

8 Listeria, Listeriosis, and Food Safety

slight difference in G +C percent DNA content (42 vs. 36 to 38) [43,175]

PRESENT STATE OF THE TAXONOMY OF THE GENUS LISTERIA

The Genus Listeria and Listeria monocytogenes 9

IDENTIFICATION OF BACTERIA OF THE GENUS LISTERIA

10 Listeria, Listeriosis, and Food Safety

Metabolism and Biochemical Characteristics

Listeria is aerobic, microaerophilic, facultatively anaerobic, catalase positive (rare catalase-negative

The Genus Listeria and Listeria monocytogenes 11

R. equi [15,48,65,74,75,108,109,113,144,170]. Positive CAMP tests are indicated by an enhanced

16S rRNA sequencing [30]

12 Listeria, Listeriosis, and Food Safety

multiplex PCR based on a single reaction [16]

The Genus Listeria and Listeria monocytogenes 13

14 Listeria, Listeriosis, and Food Safety

The Genus Listeria and Listeria monocytogenes 15

16 Listeria, Listeriosis, and Food Safety

The Genus Listeria and Listeria monocytogenes 17

18 Listeria, Listeriosis, and Food Safety

The Genus Listeria and Listeria monocytogenes 19

20 Listeria, Listeriosis, and Food Safety

22 Listeria, Listeriosis, and Food Safety

METHODS TO STUDY THE ECOLOGY OF LISTERIA SPECIES

TRADITIONAL ENRICHMENT AND ISOLATION METHODS

Ecology of Listeria Species and L. monocytogenes in the Natural Environment 23

24 Listeria, Listeriosis, and Food Safety

LIMITATIONS AND PITFALLS OF CURRENT ENVIRONMENTAL CHARACTERIZATION

Ecology of Listeria Species and L. monocytogenes in the Natural Environment 25

MOLECULAR DETECTION AND SUBTYPING METHODS

26 Listeria, Listeriosis, and Food Safety

FSL C1-103 human epidemic Oct 1998 Troy, NY DUP-1044A

Ecology of Listeria Species and L. monocytogenes in the Natural Environment 27

ECOLOGY OF LISTERIA SPECIES IN DIFFERENT ENVIRONMENTS

SURVIVAL AND MULTIPLICATION OF LISTERIA SPP. UNDER

28 Listeria, Listeriosis, and Food Safety

LISTERIA SPP. IN SOIL AND VEGETATION

Ecology of Listeria Species and L. monocytogenes in the Natural Environment 29

30 Listeria, Listeriosis, and Food Safety