Microbiology 17 GPB Aerobic and Facultative 496-555

MICROBIOLOGY
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AEROBIC AND FACULTATIVE
GRAM-POSITIVE RODS
THE MICHENER INSTITUTE for Applied Health Sciences
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MICROBIOLOGY
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AEROBIC AND FACULTATIVE GRAM-POSITIVE RODS
Acknowledgements
Author
John TarBush, B Sc., A.R.T .
Contributors And Reviewers

Christine Moore, B Sc., A.R.T.
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MICROBIOLOGY
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TABLE OF CONTENTS
OBJECTIVES 1
REFERENCES 5
INTRODUCTION 10
A. Regular/Sporeforming/Aerobic Or Facultative Anaerobic 10

Bacilli
1. BACILLUS 11
Taxonomy 11
Natural Habitat 11
Clinical Significance 11
ANTHRAX - B. ANTHRACIS 12
FOOD POISONING - B. CERUS 13
OPPORTUNISTIC INFECTIONS 13
Laboratory Diagnosis 14
SPECIMENS - COLLECTION AND HANDLING 14
DIRECT DETECTION 15
ISOLATION/IDENTIFICATION 15
Susceptibility Testing And Treatment 18
B. Regular/Non-Sporeforming/Facultative Anaerobic Bacilli 19

1. LISTERIA 19
Taxonomy 19
Natural Habitat 19
2. ERYSIPELOTHRIX 25
3. LACTOBACILLUS 27
4. KURTHIA 28
C. Irregular/Non-Sporeforming/Aerobic-Facultative 29
Anaerobic Bacilli
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1. CORYNEBACTERIUM 32
Taxonomy 32
Natural Habitat 32
DIPHTHERIA - C.diphtheriae 34
Opportunistic Infections 35
SPECIMENS-COLLECTION AND HANDLING 36
2. ARCANOBACTERIUM 40
3. MISCELLANEOUS GENERA 41
D. Branching/Filamentous/Aerobic Bacilli (Aerobic 42

Actinomycetes)
1. NOCARDIA 46
2. MISCELLANEOUS GENERA 48
SELF-ASSESSMENT QUESTIONS 50
SELF-ASSESSMENT ANSWERS 54
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OBJECTIVES
1. Describe how to separate aerobic gram-positive bacilli based upon cellular shape and presence of
endospores. Discuss the meaning and significance of "regular" versus "irregular" cellular morphology. Give
the titles for the 4 classifications as described in this module.
Regular / Sporeforming / Aerobic or Facultative Anaerobic Bacilli:
Bacillus species
1. State the basic Gram stain cellular characteristics of members of the genus, Bacillus.
2. Discuss the natural habitat of members of the genus, Bacillus.
3. Name the two most recognized Bacillus species and their clinical significance.
4. Give the 3 clinical manifestations of anthrax. State which form is the most common in humans.
5. Discuss the major clinical features of food-poisoning due to B. cereus.
6. Recognize common underlying conditions which lead to opportunistic infections caused by Bacillus
species. Name 3 opportunistic infections caused by Bacillus species.
7. State the safety concerns and practices involved when working with specimens and/or cultures
suspected to contain Bacillus anthracis.
8. Give the circumstances when speciation of Bacillus-like organisms is warranted.
9. Name differential tests and results used to distinguish Bacillus from Clostridium species.
10. State key colonial and physiological/ biochemical characteristics of B. anthracis.
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Listeria species
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11. Name the Listeria species associated with human disease.
12. Discuss L. monocytogenes with regard to:

 Natural habitat
 The common name for the various diseases caused by this species
 Disease transmission
 Specific populations who get infections
 Common clinical manifestations
13. Pertaining to the diagnosis of listeriosis, Discuss:

 Appropriate clinical specimens
 Typical cellular and colonial characteristics
 Tests and results needed to establish a genus-level identification
 Tests and results needed to differentiate L. monocytogenes from other beta-hemolytic Listeria
 Name the antibiotic(s) of choice for treatment
Erysipelothrix species
14. Name the most clinically significant species in the genus Erysipelothrix.
15. Describe the natural habitat and clinical significance of E. rhusiopathiae.
16. Recognize the typical cellular and colonial characteristics of E. rhusiopathiae.
17. State the tests and results for differentiating E. rhusiopathiae from Listeria.
Lactobacillus species
18. Discuss the clinical significance and the key characteristics of Lactobacilli.
Kurthia species
19. Discuss the clinical significance and the key characteristics of members of the genus Kurthia.
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Irregular / Non-sporeforming / Aerobic -Facultative Anaerobic Bacilli:
20. Recognize the facultative anaerobic genera found in this category
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21. Discuss the terms, "diphtheroid" and "coryneform"
22. Recognize the key tests or characteristics used to differentiate genera in this category
23. Give the name of the genus that accounts for most coryneforms found in clinical specimens
Corynebacterium species
24. Name 2 of the most clinically significant species in the genus, Corynebacterium.
25. Discuss the organism C. diphtheriae in terms of:

 Natural habitat
 Clinical significance and manifestations
 Incidence of infection
26. Describe the host which is prone to infections caused by other Corynebacterium species. Recognize
the clinical significance of non-diphtheriae Corynebacterium species. Discuss the unique clinical
significance of C. jeikeium.
27. Discuss issues around the culturing for C. diphtheriae.
28. Recognize situations when isolates of Corynebacterium species other than C. diphtheriae are
identified.
29. Discuss the key characteristics of the genus Corynebacterium.
30. State basic identification tests used to screen Corynebacterium-like species for C. diphtheriae.
31. Discuss the clinical significance and the key characteristics of Arcanobacterium haemolyticum.
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Branching / Filamentous / Aerobic Bacilli (Aerobic Actinomycetes):
32. Describe the basic characteristics of members of this category of gram-positive bacilli. Recognize
the medically important genera found in aerobic Actinomycetes group.
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33. State the importance of direct microscopy in the diagnosis and management of aerobic Actinomycete
infections.
34. Recognize the tests required to achieve genus-level identification of these organisms.
35. State two characteristics that are useful for preliminary grouping of medically important aerobic
Actinomycetes.
36. Discuss the principle of acid-fast staining and of the modified acid-fast stain.
37. Discuss the following areas, with regard to the genus Nocardiae:
 Natural habitat
 Clinical significance and manifestations
 Unique specimen requirements for laboratory diagnosis
 Cellular morphology
 Acid-fast staining characteristics
 Colonial characteristics
38. Recognize important features of aerobic Actinomycete genera other than Nocardiae.
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REFERENCES
1. Balows, A. (Editor), Laboratory Diagnosis of Infectious Diseases, Principles and Practices,

Springer-Verlag, New York, 1988.
2. Baron, E. J., Medical Microbiology, A Short Course, Wiley-Liss, New York City, N.Y., 1994.
3. Canada Communicable Disease Report, Notifiable Diseases Annual Summary, Supplement, Health
Canada, Vol. 25S6, August 1999.
4. Committee Comments, Survey B-9806, Laboratory Proficiency Testing Program (LPTP), Section
2.2, Page 406-407, August 11, 1998.
5. Coyle, M. B. et al, Coryneform Bacteria in Infectious Diseases: Clinical and Laboratory Aspects,
Clinical Microbiology Reviews, Vol. 3, No.3, page 227 - 246, July 1990.
6. Forbes B.A., Bailey and Scott's Diagnostic Microbiology, 10th Edition, Mosby Inc., St. Louis,
Missouri, 1998.
7. Isada, C. M., Infectious Diseases Handbook, 3rd Edition, Lexi-Comp Inc., Hudson, Ohio, 1999.
8. Isenberg H. (Editor), Clinical Microbiology Procedures Handbook, Vol. 1, ASM Publications, 1992.
9. Janda, W. M., Corynebacterium Species and the Coryneform Bacteria Part I: New and Emerging
Species In the Genus Corynebacterium, Clinical Microbiology Newsletter, Vol. 20, No.6, March 15,
1998.
10. Janda, W. M., Corynebacterium Species and the Coryneform Bacteria Part II: Current Status of the
CDC Coryneform Groups, Clinical Microbiology Newsletter, Vol. 20, No.7, April 1, 1998.
11. Koneman E.W., Color Atlas and Textbook of Diagnostic Microbiology, 5th Edition, Lippincott,
Philadelphia, 1997.
12. Lennette, E. H., Editor in Chief, Manual of Clinical Microbiology, 4th Edition, ASM Press,
Washington D.C., 1985.
13. Mandell, G. L., Editor, Principles and Practice of Infectious Diseases, 4th Edition, Churchill
Livingstone, New York, 1995.
14. McNeil, M. M., The Medically Important Aerobic Actinomycetes: Epidemiology and Microbiology,
Clinical Microbiology Reviews, Vol. 7, No.3, pages 357-417, July, 1994.
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15. Morbidity and Mortality Weekly Report, Supplement, Summary of Notifiable Diseases, United
States, Vol. 47, No. 53, 1998.
16. Murray, P. R., Editor in Chief, Manual of Clinical Microbiology, 7th Edition, ASM Press, Washington
D.C., 1999.
17. NCCLS M7-A5, Vol. 20, No.2, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
that Grow Aerobically; Approved Standard -Fifth Edition, January 2000.
18. Spiegel, C.A., Bacterial Vaginosis: Changes in Laboratory Practice, Clinical Microbiology
Newsletter, Vol. 21, No.5, March 1, 1999 .
19. The Sanford Guide to Antimicrobial Therapy, 28th Edition, Antimicrobial Therapy Inc., Vienna,
Virginia, 1998.
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MICROBIOLOGY
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INTRODUCTION
There are many genera and a multitude of species that can be characterized as aerobic, gram-positive
bacilli. Their clinical significance varies. B. anthracis, for example, is one of the most highly pathogenic
bacteria known to mankind. However, a great number of other members of this broad group commonly
recovered from clinical specimens, belonging to the genera Bacillus,Corynebacterium, and Lactobacillus,
are usually considered to be laboratory contaminants or a part of the normal microflora of humans.
The technologist should take note that many of these bacteria, which appear to lack virulence, can cause
opportunistic infections in immunocompromised hosts. The opportunistic potential of these organisms will
be discussed throughout this module.
Taxonomists have developed a taxonomic framework to help microbiologists deal with this heterogeneous
group of organisms. Gram-positive bacilli can be placed into one of four subcategories based upon the
following characteristics:
 Presence of endospores (spores)
 Cellular shape
Some members produce endospores (endo = inside) or spores in the presence of oxygen, when growth
conditions are unfavourable (i.e. when nutrients are lacking). The vegetative cell of these species is capable
of producing a single spore. When growth conditions deteriorate, vegetative cells disintegrate releasing their
spores into the environment. Spores can persist in the environment for many years. Each spore will
germinate into a single vegetative cell when favourable growth conditions return.
Based upon the shape of the bacterial cell, members of this broad group can be categorized as "regular",
"irregular" or "branched." If the longitudinal sides of the rod-shaped cell are generally straight and parallel
to each they are classified as "regular." The term "irregular" is used to describe rod-shaped cells with
longitudinal sides that are curved and not parallel. A third group of organisms exhibit "tree-like" branching.
Cells of these organisms are usually filamentous (elongated).
It should be understood that organisms in this group are either strict aerobes (i.e. they do not grow at all
under anaerobic conditions) or they are facultative anaerobes (i.e. they can grow anaerobically but prefer
to grow under aerobic conditions).
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According to these attributes, aerobic gram-positive bacilli can be divided into 4 groups:
1. Regular, sporeforming, aerobic or facultative anaerobic bacilli
2. Regular, non-sporeforming, facultative anaerobic bacilli
3. Irregular, non-sporeforming, facultative anaerobic bacilli
4. Branching, filamentous, aerobic bacilli (aerobic Actinomycetes)
The inter-relationships of these groups are depicted in Figure 1.
The technologist should understand that "absolute" allocation of organisms to categories (as above) is not
possible. Sometimes there is significant variability amongst species within the same genus. For instance,
most Clostridium species are strict anaerobes and are associated with other anaerobic gram-positive bacilli.
However, some Clostridium species (e.g. C. tertium and C. histolyticum) are aerotolerant (able to grow in
the presence of oxygen) and could be included with the aerobic, gram-positive bacilli. Likewise, many
Lactobacillus species are isolated from clinical specimens under aerobic conditions. However, some species
in this genus are strict anaerobes.
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Figure 1. Differentiation of common aerobic gram-positive bacilli

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A. Regular / Sporeforming / Aerobic Or Facultative Anaerobic Bacilli
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Organisms in this group have a "regular" cellular morphology. Bacterial cells typically range from 0.5 to
1.2 µm in width by 2.5 to 10 µm in length; generally wider and longer than members of the group, Regular /
Non-Sporeforming / Facultative Anaerobes. Group members produce endospores (or spores), that appear as
unstained areas within the bacterial cell in Gram-stained preparations because spores do not retain the
crystal violet or safranin. They are either strict aerobes or facultative anaerobes.
Genera that fit the above criteria are grouped in the family Bacillaceae. The most familiar genera in this
family are Bacillus and Clostridium. This section of the module will focus on the genus Bacillus. Since
most Clostridium species prefer anaerobic conditions for growth, they are usually described in context with
other anaerobes. Students should consult module 070-1-MI, "Anaerobes", for details about Clostridium
species.
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1. BACILLUS
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Taxonomy
A number of taxonomic changes to the Bacillus genus have been-made in the past few years that
should be acknowledged here. Five new genera -Alicyclobacillus,Paenibacillus, Brevibacillus,
Aneurinibacillus, and Virgibacillus - were created from realigned Bacillus species. Unfortunately,
differentiation of the new genera and Bacillus is extremely challenging at the genus level. This will
likely remain so until genus characteristics are established.
The genus Bacillus comprises 50 species. It contains the well known species (prior to
taxonomic changes), such as: B. subtilis, B. anthracis, B. cereus, B. licheniformis. B.megaterium. B.
pumilus. B. sphaericus, and B. thuringiensis.
Natural Habitat
Bacillus species are widespread in nature. They exist in a variety of soil and aquatic ecosystems.
Some species prefer elevated temperatures (as high as 75°C) and are labeled as thermophiles. Others
exist at only cold temperatures (psychrophiles). Bacillus species are also found at extremes of
acidity and alkalinity. Their spores move from one habitat to another with the movement of soils,
dusts, and aerosols. Spores also can be found in dried foods, such as spices and powdered milk.
Some Bacillus species are part of the normal intestinal flora of humans and other animals. B.
anthracis is considered to be an obligate pathogen of animals and humans. Outside of the animal or
human host (i.e. in the soil) it exists most often as a spore.
Clinical Significance
Two species, B. anthracis and B. cereus, are capable of causing serious illnesses. Other Bacillus
species appear to be less pathogenic. Generally, infections caused by Bacillus species can be
grouped as follows:
 Anthrax - caused by B. anthracis
 Food-poisoning - caused by B. cereus
 Opportunistic infections - caused by a number of Bacillus species
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ANTHRAX - B. anthracis
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Anthrax is the most recognized clinical condition caused by a Bacillus species. Before the introduction of a
protective vaccine, anthrax was a leading cause of death in herbivores (e.g. cattle). Anthrax is also a life-
threatening infection in humans. Although the disease is uncommon in Canada and other developed
countries, it is still endemic in certain parts of the world.
The environmental reservoir of B. anthracis is the soil. Spores of B. anthracis are seeded into the soil from
animals (e.g. sheep, goats, horses, and cattle) that die of anthrax. Also, the organism can exist in the
gastrointestinal tract of animals that do not develop the disease.
Human anthrax cases can be classified as either (i), industrial or (ii), non-industrial. Industrial cases occur in
workers employed in the processing of wool, hair, hides, bones or other animal products. Non-industrial
cases originate from close contact with infected animals or their carcasses - after death from anthrax.
Three clinical forms of anthrax have been described: cutaneous, intestinal, and pulmonary. Cutaneous
anthrax occurs after spores gain access through breaks in the skin during the handling of infected materials.
Over a period of 2 to 5 days, a small pimple at the point of entry evolves into a blackened, necrotic ulcer.
Approximately 95 to 99% of human anthrax cases are of this type. Fewer than 20% of untreated cases of
cutaneous anthrax are fatal. Intestinal anthrax -marked by bloody diarrhea, vomiting, fever, and abdominal
pain - develops as a result of the ingestion of infected meat. Pulmonary anthrax stems from the inhalation of
infectious spores. Macrophages in the lungs carry the spores to the lymphatic system. Here they germinate
and multiply. If left untreated, organisms typically spill into the bloodstream leading to fatal septicemia and
toxemia. The case mortality rate for untreated pulmonary anthrax is close to 100%.
A rapid presumptive diagnosis is essential. A history of animal exposure and the presence of black, necrotic
ulcerations are grounds for suspicion of anthrax.
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FOOD-POISONING - B. cereus
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The Bacillus species next in importance to B. anthracis as a human pathogen is B. cereus. It is the causative
agent of two distinct food-poisoning syndromes:
 Diarrheal - long incubation form
 Emetic - short incubation form
The diarrheal form appears 8 to 12 hours (long incubation) after eating foods contaminated with the heat-
labile enterotoxin of B. cereus. This form of illness, characterized by abdominal pain and watery diarrhea,
usually resolves after 24 to 36 hours. The emetic form (short incubation) begins only 1 to 5 hours after
ingesting foods laced with a heat-stable enterotoxin. Symptoms include vomiting and abdominal cramps.
This type of food-poisoning runs its course within 10 hours of onset. Both syndromes arise from B. cereus
spores that have survived cooking. During food storage, the spores are able to germinate producing
vegetative cells. These cells multiply and release enterotoxins into the food. B. cereus food-poisoning is
regarded as an "intoxication" rather than an infection.
OPPORTUNISTIC INFECTIONS
Many Bacillus species, other than B. anthracis and B. cereus, can behave as opportunistic pathogens,
targeting the immunocompromised host. The number of reports linking Bacillus species to serious
infections is on the increase. Operative procedures, immunosuppression, traumatic wounds, burns,
hemodialysis and parenteral drug abuse are the usual conditions or events leading to opportunistic
infections.
The full magnitude of these types of infections is not fully appreciated. Many infections go undiagnosed in
the laboratory because Bacillus species are often quickly dismissed as laboratory contaminants or normal
flora. Technologists should be aware that a predominant or pure growth of a Bacillus species in culture may
be of significance. The patient's clinical presentation may determine the significance of positive culture
results. For this reason, it is important that the laboratory relay potentially significant culture findings to the
patient's physician.
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Opportunistic infections caused by Bacillus species are as follows:
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Bacteremia: Most cases are associated with intravenous drug users, patients with indwelling catheters or
patients immunosuppressed due to cancer or chemotherapy.
Endophthalmitis: Endophthalmitis, an infection of the internal chambers of the eye, occurs following
penetrating trauma to the eye or as a consequence of bacterial seeding from a bacteremic episode within the
same host. Bacillus species (in particular B. cereus) may be the second most commonly isolated organism
after Staphylococcus epidermidis in post-traumatic cases. This type of infection has a rapid course and can
cause permanent loss of sight. Direct Gram stains and cultures of eye aspirates must be examined carefully
with Bacillus species in mind.
Miscellaneous infections: Bacillus species can cause a number of infections in the immunosuppressed host
such as osteomyelitis, necrotizing fasciitis, and endocarditis; infections of surgical and burn sites; and
infections of the central nervous system, lower respiratory tract and deep wounds.
Laboratory Diagnosis
SPECIMENS - COLLECTION AND HANDLING
The exudate from cutaneous anthrax lesions can be collected onto a swab for culture and stained smears. In
severely ill patients (advanced pulmonary anthrax), blood cultures should be collected. If the patient is
treated before specimens can be collected, paired serum specimens - one obtained upon initial examination
and the second > 10 days later may yield confirmatory results.
Diagnosis of suspected Bacillus cereus food poisoning is achieved by recovering the organism from cultures
of implicated foods. There is little benefit in culturing the patient's stool since gastrointestinal tract
colonization is common in healthy individuals.
No special collection and handling procedures are required to recover Bacillus species from specimens.
Aerobic spore-formers are hardy organisms that survive transport to the laboratory either in freshly
collected specimens or in a standard transport system.
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DIRECT DETECTION
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The endospores of Bacillus species may not be evident in direct smears of clinical specimens. Typically,
these organisms Gram stain as gram-positive bacilli but occasional strains will appear gram-negative. In
tissue, B. anthracis often appears encapsulated. This feature is lost when grown on routine artificial media.
ISOLATION / IDENTIFICATION
Most Bacillus species and related genera grow well on 5% sheep blood agar, chocolate agar, routine blood
culture media and in commonly used nutrient broths. They grow poorly or not at all on MacConkey agar.
Phenylethyl alcohol agar (PEA) is useful for the isolation of Bacillus species from contaminated specimens.
Most species will produce visible growth on blood agar within 18 to 24 hours of incubation at 35°C, in
ambient air or 5% CO2.
After 24 hours of incubation on blood agar, colonies of B. anthracis are gray, flat, 4 to 5 mm in diameter,
non-hemolytic and have an irregular margin. The surface of the colony may have a "Medusa head"
appearance due to the projection of numerous filamentous chains of bacilli.
Colonies of B. cereus grown on blood agar after overnight incubation are 3 to 8 mm in diameter, raised,
with a gray to greenish frosted glass appearance and irregular margins. Most strains produce a large zone of
beta hemolysis. The colonies of many other Bacillus species encountered in clinical specimens are typically
large, beta-hemolytic, flat, and tend to have a frosted glass appearance and irregular edges.
Various cellular characteristics are useful in the identification of aerobic endosporeforming bacteria.
Consequently, a Gram stain should be performed on Bacillus-like colonies. Bacillus species may be
coccobacillary (0.5 µm wide by 1.2 µm long) or large and "box-shaped" (2.5 µm wide by 10 µm in length).
Spores are larger, more regular in shape, size and position than other types of cellular inclusions. They may
be elliptical, spherical, or egg-shaped. Spores are located centrally within the cell, between the end and the
centre of the cell (sub-terminal) or at one end of the cell (terminal). Sometimes the diameter of the spore
exceeds the width of the bacterial cell making cells look "swollen."
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Spore characteristics are enhanced when preparations are stained with a spore stain. The Wirtz-Conklin
spore stain uses malachite green as the primary stain and safranin as the counterstain. Spores stain green and
the rest of the cell stains red.
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On Gram-stained smears from culture growth, the bacterial cells of B. anthracis occur in chains and contain
elliptical-shaped, subterminal spores that do not cause the cell to swell. Cells of B. cereus are not swollen
and contain elliptical spores that are located centrally or subterminally.
Occasionally cells in Gram stained smears are Bacillus-like but lack endospores or stain as gram-negative
bacilli. Endospore production can be induced in cultures by i) incubating cultures at higher temperatures
(i.e. 45°C); ii) growing the organism in the presence of a 30 µg vancomycin disc (disc diffusion test); or iii)
growing the bacteria on esculin or urea agar.
Some strains of Bacillus species will not retain the crystal violet upon Gram staining and will stain as
"gram-negatives." The 3% KOH string test may be helpful in differentiating gram-negative staining
Bacillus species from "true" gram-negative bacilli. Most gram-negative bacilli will denature and become
viscous or sticky when colonies are mixed with a solution of 3% KOH, whereas Bacillus species will
remain in a smooth suspension.
Many technologists think that all Bacillus species are strict aerobes. This genus, however, contains species
that are capable of growing under anaerobic conditions (facultative anaerobes). Also, aerotolerant
Clostridium species (another sporeformer) may grow on aerobically incubated plates. Using a few simple
tests, the technologist can readily distinguish Bacillus species from Clostridium species (Table 1). Bacillus-
like colonies should be subcultured and incubated under both aerobic and anaerobic conditions. Bacillus
species produce larger colonies under aerobic conditions, produce endospores only under aerobic conditions
and are catalase-positive.
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Table 1. Differentiation of Bacillus species from Clostridium species.
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Each laboratory should develop an in-house protocol for the identification of Bacillus-like species. The
level of identification of these organisms should take into account the type of medical service (i.e. in-patient
versus out-patient), the level of immunosuppression in the patient population being served, and the
resources available to the laboratory.
Technologists can use the following as a guide for when to identify Bacillus-like species found in routine
cultures:
 Ideally, all isolates from normally sterile body sites (i.e. blood, CSF, and vitreous humor) should be
identified to genus level; species level identification may be appropriate if the appearance of the
organism is consistent with the clinical history
 A pure or predominate growth of a Bacillus-like species from a wound should be identified to the
genus level; species level identification may be appropriate if the same organism is recovered in
serial cultures
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All laboratories should be able to identify B. anthracis, even though the organism is rarely recovered in
clinical specimens in North America. B. anthracis should be suspected if typical non-hemolytic "Medusa
head" colonies are observed on sheep blood agar and the organism is non-motile (many of the Bacillus
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species recovered in clinical specimens are motile). These findings should be discussed with senior
laboratory personnel. Key tests and results for differentiating suspect isolates of B. anthracis from other
Bacillus species are shown in Table 2. B. anthracis infections must be reported to the Ministry of Health.
Table 2. Key characteristics for distinguishing B. anthracis from other Bacillus species (excerpt from
Koneman, 5th edition).
A detailed account of Bacillus speciation is beyond the scope of this module. Readers seeking an in-depth
discussion of this subject should consult any of the textbooks listed in the REFERENCES section of this
module.
Susceptibility Testing &Treatment
There are no standard susceptibility testing methods recognized by North American laboratories for
Bacillus species.
B. anthracis is almost always susceptible to penicillin, gentamicin, erythromycin, ciprofloxacin, and

doxycycline. B. cereus endophthalmitis requires aggressive and timely antibiotic treatment. Intravitreal
(within the globe of the eye) administration of vancomycin plus amikacin and systemic administration of
clindamycin or vancomycin is effective.
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Regular / Non-Sporeforming / Facultative Anaerobic Bacilli
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Organisms in this group have a "regular" cellular morphology. The longitudinal sides of the rod-shaped
cell are usually straight and parallel to each other. No endospores are produced. They are facultative
anaerobes in that they can grow anaerobically but prefer to grow under aerobic conditions.
This group is comprised of the following genera:

 Listeria
 Erysipelothrix
 Lactobacillus
 Kurthia
The key tests used to differentiate these genera are shown in Table 3.
1. LISTERIA
Taxonomy
The genus Listeria contains the following species: L. monocytogenes, L.ivanovii (including subspecies
londoniensis), L. seeligeri, L. welshimeri, L. innocua, L. grayi, and L. denitrificans. Only L. monocytogenes
is associated with human disease. The other species are non-pathogenic or have uncertain pathogenicity.
The rest of this section focuses on L. monocytogenes.
Natural Habitat
Soil and decaying vegetable matter are considered to be the primary natural habitats for L.
monocytogenes. It has been isolated from a wide variety of sources including freshwater,
saltwater, and sewage; animal feed, fresh and frozen poultry, red meat, and meat products; fish;
raw dairy products, including milk, cheese, and ice cream; raw fruits and vegetables; and from
human and animal feces. More than 40 different species of mammals (including cattle and
sheep) and 17 species of birds harbour this organism.
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Table 3 Differentiation of the genera belonging to the Regular / Non-Sporeforming / Facultative
Anaerobic Bacilli group. Key test results are shaded (modified from 5th Edition of Diagnostic
Microbiology)
^ "Diphtheroid" is a term used to describe any gram-positive bacillus that resembles Corynebacterium
diphtheriae on Gram stain (i.e. pleomorphic cells that tend to form "picket fence" and "Chinese letters"
arrangements on Gram-stained smears)
Abbreviations and symbols:

BETA HEMOLYSIS, Beta hemolysis on sheep blood agar, MOTILITY, by wet mount or semi-solid
medium, H2S in TSI, H2S production in triple sugar iron (TSI) agar slants, ESCULIN, Esculin
hydrolysis, +, positive reaction, -, negative reaction, V, variable reaction
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Listeriosis is the term used to describe the different types of infections caused by L. monocytogenes. Most
cases of listeriosis result from the ingestion of contaminated foods. Sporadic cases or outbreaks of listeriosis
have been linked to the consumption of contaminated coleslaw, cabbage, cheese and ice cream. The ability
of the organism to survive and even mulltiply under refrigeration is an attribute that promotes its food-borne
transmission. Direct handling of infected animal tissues can lead to cutaneous listeriosis, an infection
reported among veterinarians and abattoir workers. Some nosocomial outbreaks of listeriosis have occurred
in newborn nursenes as a result of contaminated delivery rooms. In most cases of listeriosis, the exact
source of the organism is never pinpointed.
L. monocytogenes appears to target certain populations. Those at highest risk include patients with organ
transplants, malignancies, AIDS and patients receiving corticosteroids. Pregnant women, neonates and
alcoholics are also at risk for infection. Approximately 34% of all cases of listeriosis occur in pregnant
women, 50% occur in non-pregnant adults with severe underlying diseases, and 14% occur in previously
healthy adults.
Acquisition of the organism via the oral route usually results in an asymptomatic carrier state. Carriers often
shed the organism in their feces; usually for a month or less. This species can be a transient member of the
intestinal flora of healthy humans.
In some individuals, however, the organism can penetrate the intestinal mucosa, circulate in the bloodstream
causing septicemia and infections in other areas of the body (e.g. meningitis, encephalitis, and intra-
abdominal abscesses). The organism is capable of persisting inside the cells of the monocyte-macrophage
system of the body making infections difficult to treat.
Septicemia and meningitis are the most common clinical manifestations of listeriosis in older children and
adults. Listeria septicemia, with or without bacteremia, can mimic gram-negative sepsis (i.e., high fever and
hypotension). Many of these infections are clinically labeled as "sepsis of unknown origin" until diagnosed
by a positive blood culture. L. monocytogenes is the most common cause of community-acquired meningitis
in immunocompromised people. Listeria meningitis is known for its variable clinical presentation. CSF
analysis results are highly variable. For instance, differential cell counts can yield nearly 100%
polymorphonuclear cells in one case and 100% mononuclear cells in the next. Approximately 20 to 50% of
Listeria meningitis infections end in death.
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Cerebritis (inflammation of the brain stem, cerebellum and cerebral cortex) is a clinical illness that is
increasingly recognized and reported. Patients with this infection often complain only of headache and fever
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or manifest varying degrees of paralysis. The CSF may not yield many leucocytes. In addition, protein and
sugar readings may be normal or slightly abnormal.
In pregnant women, L. monocytogenes can cause a ''flu-like'' illness, which may present with diarrhea and
flank pain. Diagnosis is usually based upon a positive blood culture. If the infection is left untreated, the
organism may cross the placenta leading to intrauterine infection of the fetus. Some in utero infections
result in abortion, stillbirth, or premature birth.
One severe and often lethal infection arising from transplacental spread is granulomatosis infantiseptica.
It is an "early onset" neonatal infection that is apparent within hours of birth. Abscesses or granulomas are
usually observed in a number of organs throughout the body, such as the liver and brain. Observation of
gram-positive bacilli in direct gram stains of amniotic fluid, conjunctival exudates, CSF, blood, or skin
lesions can be life-saving - guiding early antibiotic therapy.
Some neonates present with ''late onset" disease in the first week to several weeks postpartum. These
neonates often acquire the organism as they pass through a colonized or infected birth canal. After an initial
bacteremic phase, the brain and meninges often become infected.
L. monocytogenes can cause a number of focal infections such as skin-ulcers, purulent conjunctivitis,
subacute bacterial endocarditis, osteomyelitis, arthritis, peritonitis, and acute hepatitis.
L. monocytogenes can be recovered in culture from a number of specimens including blood, CSF, amniotic
fluid, placenta, and fetal tissue. At least 10 ml of CSF should be collected because the organism may be
present in low numbers in this specimen type.
Specimens form normally sterile body sites can be inoculated directly onto tryptic soy agar (TSA)
containing 5% sheep blood. Both manual and automated blood culture techniques are reliable for the
isolation of the organism from blood samples.
Specimens obtained from non-sterile body sites should be selectively enriched for Listeria before being
setup on culture plates. Cold enrichment is the traditional method used. At lower temperatures (i.e., 4ºC), L.
monocytogenes actually multiplies while other contaminating flora do not. The method involves mixing one
part of specimen with nine parts of nutrient broth. The mixture is then held at 4ºC for several days to 2
months and is subcultured onto solid media at regular intervals until the organism is recovered.
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Refer to the most current Edition of the Manual of Clinical Microbiology for details surrounding other
selective enrichment methods.
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In direct Gram stains of clinical specimens, L. monocytogenes typically appears as a nonbranching, non-
sporeforming, regular, short (0.5 to 2 µm long by 0.4 to 0.5 µm wide), gram-positive bacillus that occurs
singly or in short chains. The organism may be found inside polymorphonuclear leucocytes and monocytes.
Pleomorphic strains are not uncommon. Coccobacillary forms in pairs are often mistaken for Streptococcus
pneumoniae. If Gram smears are over-decolourized, rods may appear gram-negative and be confused for
Haemophilus influenzae. Similarly, L. monocytogenes may occur in the "palisade" cellular arrangement
characteristic of coryneforms or diphtheroids.
After 24 hours of incubation on sheep blood agar - in ambient air, in increased CO2 or under anaerobic
conditions - colonies of L. monocytogenes are typically small, gray-white and weakly beta-hemolytic. Often
beta hemolysis is most pronounced underneath the colonies and can be readily seen by moving the colony
aside with an inoculating wire. Colonies of Group B streptococcus, which usually are weakly beta-
hemolytic, are easily mistaken for colonies of L. monocytogenes. A Gram stain of the colonial growth is an
invaluable tool to rule-out Group B streptococcus, which occurs as a gram-positive coccus in pairs and
chains.
Genus-level identification is achievable with the following combination of test results:

 Regular, gram-positive bacilli without spores
 Catalase-positive
 "Tumbling" motility-positive
 Glucose fermentation-positive
 Esculin hydrolysis-positive
 Voges-Proskauer (VP) - positive
 Methyl red-positive
Streptococcus species (notably, Group B) are easily differentiated from Listeria species based upon their
cellular morphology and arrangement, lack of motility and a negative catalase test. Erysipelothrix species
(see next section), winch can resemble Listeria in a Gram smear, are catalase-negative and non-motile.
Lactobacillus species, found along with Listeria in clinical specimens, can be differentiated based upon a
negative catalase test.
Motility is probably the most recognized characteristic for the genus Lisreria. It can be determined in either
a wet mount (hanging drop) or in a semisolid motility medium. Using the wet mount method, a pure growth
of the test organism is grown in a broth medium (e.g. nutrient broth) for 6 hours at 22 to 25°C. A drop of
this suspension is viewed under the microscope with reduced light. Cells of Listeria species spin around in
circles, flip-flop, or tumble end-over-end; often described as "tumbling motility."
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Detection of beta hemolysis is a good beginning point for differentiating L. monocytogenes from the other
Listeria species (Table 4). Only two Listeria species, other than L. monocytogenes, are beta hemolyoc -L.
ivanovii and L. seeligeri. Both L. monocytogenes and L. seeligeri produce a narrow zone of beta hemolysis,
whereas the zone produced by L. ivanovii is much wider.
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The CAMP test, used to differentiate Group B streptococcus from other streptococcus species, can be used
to differentiate L.ivanovii from L. monocytogenes. A beta-hemolytic strain of Staphylococcus aureus is
streaked in a straight line through the middle of a sheep blood agar plate. The test isolate of Listeria is
streaked at a right angle to (but not touching) the S. aureus line (see Figure 3 in Module 063-1-MI). L.
monocytogenes produces a rectangular-shaped area of beta hemolysis (enhanced hemolysis) near the line of
S. aureus; as opposed to L. ivanovii, which shows no area of enhanced beta hemolysis. Acid production
from xylose can be used to differentiate L. monocytogenes (xylose-negative) from L. seeligeri (xylose-
positive).
Multi-test identification systems are commercially available for the identification of Listeria species (e.g.
the API-Listeria test from bioMerieux Vitek Inc. and Micro-ID Listeria from Organon Teknika). Also, Gen-
Probe markets a DNA probe assay for the rapid identification of L. monocytogenes from primary culture
plates.
Table 4. Key biochemical and physiologic characteristics of beta hemolytic Listeria species
(modified from Manual of Clinical Microbiology, 7th Edition)
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Susceptibility Testing & Treatment
The National Committee for Clinical Laboratory Standards (NCCLS) has approved a modified broth
dilution method for determining the susceptibility of Listeria species.
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The susceptibility and resistance pattern of L. monocytogenes to various antimicrobial agents has been
relatively stable for many years. The organism has remained susceptible to penicillin, ampicillin,
gentamicin, erythromycin, and tetracycline in laboratory susceptibility tests. Penicillin or ampicillin with or
without an aminoglycoside (e.g. gentamicin) is the most widely used regimen for treatment of listeriosis.
2. ERYSIPELOTHRIX
There are two species in the genus Erysipelothrix, E. rhusiopathiae and E. tonsillarum. Because E.
tonsillarum has not yet been isolated from human specimens, it will not be discussed here.
E. rhusiopathiae is carried by a variety of animals but is most frequently associated with pigs. The organism
can be recovered from food, water and soil that has been contaminated with the feces and urine of infected
animals.
This species has been recognized as the causative agent of swine erysipelas, characterized by septicemia
and arthritis, in a number of farm animals such as swine, calves,lambs, and turkeys. Occasionally, E.
rhusiopathiae can cause a zoonotic infection in humans known as erysipeloid (not to be confused with
human erysipelas - a skin infection caused by Streptococcus pyogenes).
Erysipeloid, a localized cellulitis of the hands and fingers, is largely an occupational disease of workers who
handle meat, poultry, fish, and fertilizer. The organism gains entry through breaks in the skin. After a 2 to 7
day incubation period, patients develop purple-coloured, non-suppurative (i.e. non pus-producing) lesions,
which are raised with well-defined edges. Untreated infections may spread to nearby lymph nodes and
joints. There are rare reports of severe systemic infections involving septicemia and endocarditis, often in
immunocompromised patients.
A biopsy for culture and Gram stain, collected from the advancing edge of the lesion, is the method of
choice for diagnosis of erysipeloid. Because the organism typically resides deep in the subcutaneous layer,
the biopsy should sample the entire thickness of the lesion. Blood cultures are usually negative for
uncomplicated infections but can be helpful in the diagnosis of systemic disease.
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In Gram stains of biopsy specimens, E. rhusiopathiae may appear as regular, long, slender, gram-positive
bacilli.
Macerated tissue can be planted onto either blood or chocolate agar. A selective medium is usually not
indicated if the infected site was properly cleansed before biopsy. Tissue should also be placed into an
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infusion broth. Inoculated media can be incubated at 35ºC aerobically or in 5% CO2. Over a 7 day
incubation period, the broth is subcultured to blood agar and primary plates are examined for growth.
Colonies of E. rhusiopathiae are usually pinpoint in size after 24 hours incubation (<0.1 to 0.5mm in
diameter). By 48 hours, two distinct colony types can be seen. One morphotype is described as being small
(0.3 to 1.5 mm in diameter), smooth, transparent, convex, and circular with a regular edge. The other
morphotype is larger, rougher (with matte surface), flatter and has an irregular edge. Colonies are either
alpha or nonhemolytic.
Cells from smooth colonies Gram stain as short, non-branching, non-sporeforming, gram-positive bacilli or
coccobacilli (0.2 to 0.5 µm wide by 0.8 to 2.0 µm in length) with rounded ends. Cells from rough colonies
appear as long filaments, ranging from 4 to 60 µm in length.
E. rhusiopathiae is the only gram-positive, non-sporeforming bacillus, other than rare strains of Kurthia
species, capable of producing H2S in triple sugar iron (TSI) agar (i.e. blackennig of the medium along the
stab line or throughout the butt of the slant). The organism is also catalase-negative, non-motile, esculin-
negative, and alpha or non-hemolytic on blood agar; characteristics that separate it from Listeria species
(Table 3).
Penicillin is the antibiotic of choice for treatment of both localized infections (erysipeloid) and systemic
infections.
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3. LACTOBACILLUS
Lactobacillus species are normal inhabitants of the human mouth, intestinal tract, and vagina. Lactobacilli
are an important component of the normal vaginal flora of reproductive women. They produce acid, which
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serves to keep unwanted organisms from colonizing and infecting the vagina. Also, they reside in a number
of other animals and are recovered from a variety of food products.
Lactobacillus species are not virulent. Usually, these organisms garner little attention when recovered in
culture and are readily dismissed as "normal flora." However, Lactobacillus species can cause opportunistic
infections such as bacteremia, pneumonia, and meningitis in immunocompromised patients. Also,
technologists must be able to differentiate lactobacilli from other more clinically significant organisms such
as viridans streptococci.
Although most species of Lactobacillus are facultative anaerobes, approximately 20% of human isolates are
obligately (strictly) anaerobic.
There are currently 45 or more documented species, making speciation of lactobacilli challenging. Current
identification tests used for this purpose lack both accuracy and reproducibility. Identification to the genus
level is usually sufficient (Table 3). This level of identification is usually built upon cellular morphology
(under Gram stain), colonial characteristics, and the catalase reaction.
Lactobacilli are gram-positive, non-sporeforming bacilli that vary from long and slender forms to short
coccoid forms. Spiral and other pleomorphic forms are also common. The observation of bacilli in chains is
highly suggestive of a Lactobacillus species. This chain formation is best seen when a Gram stain is made
from a thioglycollate broth culture.
Lactobacillus species grow well on blood and chocolate agars. Because many species are capable of
producing small alpha-or non-hemolytic colonies at 24 hours of aerobic incubation, they are easily confused
for other more significant organisms with a similar colonial picture; namely, Erysipelothrix, Leuconostoc,
Aerococcus, Pediococcus and viridans streptococci. Other species may produce larger colonies with a rough
surface.
All Lactobacilli are negative for catalase, motility, H2S production, and esculin hydrolysis. They exhibit
vancomycin resistance on disk testing. They produce a major lactic acid peak from glucose in gas
chromatography.
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4. KURTHIA
Kurthia species are frequently isolated from the environment (soil, water and wood), from animals,
(chickens, pigs and dogs) and from meat and meat products. A number of strains of Kurthia species have
been recovered from a variety of human samples. To date, there IS no evidence to indicate that these species
cause infection in humans.
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These organisms form large, non-hemolytic, cream to yellow colonies on blood agar under aerobic
conditions. Bacterial cells are large (0.8 to 1.2 µm wide and 2 to 4 µm long), regular, non-sporeforming and
often occur in chains (Table 3). Kurthia species can be confused for Lactobacilli in Gram stains. Unlike
lactobacilli, Kurthia species are strictly aerobic and do not ferment carbohydrates. They are catalase-
positive and motile like Listeria monocytogenes. They differ from L. monocytogenes in their strict aerobic
growth, non-hemolytic quality on blood agar, and failure to hydrolyze esculin.
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C. Irregular / Non-Sporeforming / Aerobic-Facultative Anaerobic Bacilli
Members of this large group have the following general characteristics:

 "Irregular" cellular morphology - the longitudinal sides of the rod-shaped cell are usually curved
and not parallel to each other
 No endospores are produced (see introduction of module for discussion)
 Facultatively anaerobic
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Genera in this group, such as Arthrobacter, Aureobacterium, Brevibacterium, Caseobacter, Clavibacter,
Curtobacterium, and Microbacterium, are "aerobes." Because they are found primarily in the environment
and are not usually associated with human infection, they will not be discussed further.
Generally, the genera in this group that cause infections in humans are facultatively anaerobic. This
subgroup includes Arcanobacterium, Corynebacterium, Dermabacter, Gardnerella, Propionibacterium,
Rothia, and Turicella species. These organisms will be the focus of this module (Table 5).
The terms "diphtheroid" and "coryneform" are used extensively to describe these organisms. These terms
have been applied to species belonging to one or more of the genera mentioned above (see Figure 2).
Clinical microbiologists will often call a gram-positive bacillus that resembles C. diphtheriae a
"diphtheroid."
Originally, "coryneform" was a descriptive term used to identify the club-shaped cellular morphology of
Corynebacterium species ("coryne", meaning club in Greek). Even though the term was intended to
describe only Corynebacterium species, many microbiologists use the term to describe any aerobically
growing, non-sporeforming, irregularly shaped, non-partially acid fast, gram-positive bacillus - or all of the
genera discussed above.
Basic tests that are available to most laboratories can be used to identify most facultatively anaerobic
coryneform bacteria grown in culture. The following tests can be used to get reliable identifications (refer to
Janda - Clinical Microbiology Newsletter - for details):
 Cellular morphology (shape, size, arrangement)
 Colonial morphology (size, pigmentation, odour, hemolysis)
 Catalase (using 3% hydrogen peroxide)
 Motility (preferably hanging drop from 18 to 24 hour broth culture)
 Relation to oxygen (aerobic, facultatively anaerobic, anaerobic)
 Fermentation or oxidation of sugars (preferably in semisolid cystine Trypticase agar [CTA] medium)
 Nitrate reduction (preferably read after 48 to 72 hours of incubation)
 Urea hydrolysis (Christenson's medium; read for up to 72 hours)
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 Esculin hydrolysis (read for up to 72 hours)

 Acid production from glucose, maltose, sucrose, mannitol, and xylose (read for up to 72 hours)
 CAMP reaction (enhanced beta hemolysis in proximity to beta hemolysin producing strain of S.
aureus -resulting in an arrowhead"configuration)
 Reverse CAMP reaction (beta hemolysis in proximity to the beta hemolysin producing strain of S.
aureus is inhibited by certain coryneform bacteria resulting in a triangular zone devoid of beta
hemolysis)
 Test for lipophilia (growth stimulation in presence of lipid)
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Commercial identification systems are popular alternatives to the conventional identification scheme shown
above. API (RAPID) Coryne system (bioMerieux), RapID CB Plus (Remel), and Biolog GP plate (Biolog)
are three systems that have been evaluated in the literature.
Off-line conventional tests may still be required with these systems. Although prominent Corynebacterium
species are usually reliably identified, it is always important to correlate each identification with basic
colonial and cellular characteristics.
Identification of less frequently encountered coryneform bacteria or coryneform strains that are not
ildentified by traditional identification algorithms may require more sophisticated identification tools (i.e.,
detection of mycolic acid chains using gas chromatography) available only in reference laboratories.
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1. CORYNEBACTERIUM
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Most of the coryneform-like bacteria encountered in clinical cultures belong to the genus Corynebacterium.
Taxonomy
Taxonomic changes have been made to the genus Corynebacterium based upon genetic analyses,
chemotaxonomic methods (i.e., detection of mycolic acids and cellular fatty acid studies) and phenotypic
techniques (i.e., carbohydrates assimilation tests and detection of preformed enzymes). New
Corynebacterium species have been described and several old Corynebacterium species have been placed
into other genera.
The genus Corynebacterium is presently composed of 46 species; some of which are found in plants,
animals, foods, or the environment.
Recent genetic studies indicate that the genus Corynebacterium is more closely related to Mycobacterium,
Nocardia, and Rhodococcus species (bacteria that are partially or fully acid-fast) than to other coryneforms
covered in this module.
Natural Habitat
Corynebacteria are found on the skin and mucous membranes of humans and other animals. C. diphtheriae
is found only in humans. This organism is recovered in cultures of the nasopharynx and skin of healthy
individuals (carriers). Skin colonization (with no infection) is usually associated with poor hygiene (i.e.,
drug users). In general, C. diphtheriae is not commonly isolated from healthy humans. C. jeikeium inhabits
the skin and can be isolated from inanimate hospital surfaces. Many other noteworthy species occupy site-
specific niches of the human body (Table 6).
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Thirty-one species have been implicated in human infections. The mode of transmission and the clinical
significance of the species most frequently associated with infection are presented in Table 6. The most
recognized species is C. diphtheriae, the causative agent of diphtheria. The other species act as
opportunistic pathogens.
DIPHTHERIA - C. diphtheriae
Before the advent of protective vaccines, diphtheria was a leading cause of death worldwide. Today, the
disease is very rare. Only 1 case was reported to Health Canada for the entire nation m 1997. No significant
outbreaks of disease have been reported in North America in the past 10 years. However, sporadic cases do
occur, especially amongst native or indigenous North Americans. A fatal case has not been reported in
Canada since 1983.
The main manifestation of diphtheria is an upper respiratory tract illness marked by a sore throat, local
lymphadenitis, low-grade fever, malaise and headache. Aerosols produced by convalescent and healthy
carriers of C. diphtheriae serve as the primary mode of transmission. In classic diphtheria, inflammation in
a variety of areas of the upper respiratory tract (especially the pharynx) ensues due to the action of a potent
exotoxin. In approximately 50 % of cases of respiratory diphtheria a thick "pseudomembrane" forms over
the mucosa of the tonsils, palate, pharynx and/or nasopharynx. In untreated cases the "pseudomembrane"
can obstruct the airway causing suffocation.
The exotoxin produced by C. diphtheriae in the upper respiratory tract may enter the bloodstream and
damage a number of organs, especially the heart and the peripheral nervous system. A mortality rate of 10 to
30% is associated with systemic complications of diphtheria; primarily congestive heart failure and cardiac
arrhythmia.
Systemic diphtheria is caused only by strains of C. diphtheriae that have the tox gene that encodes for
exotoxin production. The tox gene is acquired from a specific bacteriophage (a virus that infects bacteria).
Non-toxigenic strains of C. diphtheriae (strains lacking the toxin gene) are isolated from carriers more often
than toxigenic strains. Although they are capable of causmg pharyngitis, they do not cause systemic disease.
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C. diphtheriae can also cause cutaneous diphtheria. Usually the infection results when organisms
colonizing the skin gain entry into deeper skin layers through a wound. Exotoxin production can lead to the
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development of a non-healing, ulcerative lesion with a dirty-gray pseudomembrane and one or more
systemic complications as seen in respiratory diphtheria.
The diagnosis of diphtheria may be a challenge in Canada due to rarity of the disease and the overlap of
symptoms with other illnesses. The pharyngitis of diphtheria must be differentiated from the more common
pharyngeal conditions, namely streptococcal pharyngitis ("strep throat"), adenovirus infection, infectious
mononucleosis, and Vincent's disease. Detection of the pseudomembrane upon patient examination is
presumptive evidence of diphtheria. Other clues include signs of toxin-mediated, systemic complications.
However, laboratory tests are required to confirm the clinical diagnosis.
Opportunistic Infections
Most infections caused by Corynebacterium species other than C. diphtheriae occur in the
immunocompromised host.
C. jeikeium (formerly CDC group JK): C. jeikeium is the most commonly isolated Corynebacterium
species from human infections. It has been implicated as the infectious agent in a number of diseases; such
as bacterial endocarditis (usually involving a prosthetic valve), pneumonia, peritonitis, and infections of
skin and surgical wounds. A large percentage of hospitalized pattents become colonized with the organism,
in the rectum, axilla, and inguinal areas. Factors that predispose patients to infections with C. jeikeium
include prolonged hospitalization, neutropenia (shortage of neutrophils), use of multiple antibiotics, and
disruption of the skin(e.g., implantation of medical devices or surgery). The organism is characteristically
resistant to a wide range of antimicrobial agents, which may make treatment more difficult.
Other opportunistic pathogens: C. glucuronolyticum has been isolated from male patients with
genitourinary tract infections, particularly from semen specimens collected from patients with prostatitis. C.
macginleyi has been implicated in eye infections. C. minutissimum causes erythrasma - a superficial skin
infection characterized by scaly, brownish-red lesions (that fluoresce a coral red colour under a Wood's
light). C. pseudodiphtheriticum is an important emerging pathogen. It can cause respiratory tract infections
such as tracheobronchitis, necrotizing tracheitis, pneumonia, and lung abscesses. C. pseudotuberculosis
causes rare cases of acute or chronic lymphadenitis in animal handlers. C. riegelii is associated with urinary
tract infections in females. C. striatum is an emerging pathogen; reported in cases of bacteremia,
pneumonia, lung abscesses, and wound infections. C. ulcerans infections occur most often among persons
exposed to cattle. This species can cause a diphtheria-like pharyngitis and skin ulcers. Some toxigenic
species are capable of causing pseudomembrane formation and systemic complications seen in classic
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diphtheria. C. urealyticum (formerly CDC group D2) is a well-documented cause of alkaline-encrusted

cystitis (chronic inflammatory condition involving the bladder wall) and other urinary tract infections.
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These infections are difficult to treat because of the organism's resistance to commonly used antimicrobial
agents.
SPECIMENS - COLLECTION AND HANDLING
There are no special specimen collection, transport and processing considerations or requirements for
Corynebacterium species other than C. diphtheriae.
The specimens appropriate for the diagnosis of respiratory diphtheria include a throat swab (sampling of
inflamed areas and underneath the pseudomembrane), a portion of a pseudomembrane (if observed), and a
nasopharyngeal swab. Sampling of the throat and the nasopharynx increases the recovery rate. Swabs can be
sent to the laboratory in a semisolid transport media (e.g., Amies). If the transport time is expected to
exceed 24 hours, the use of silica gel transport media is recommended.
Since most clinical laboratories do not routinely screen throat specimens for C. diphtheriae it is imperative
that the clinician notify the laboratory when an investigation for C. diphtheriae is required.
Laboratories must develop their own protocol for dealing with C. diphtheriae investigations. Specific
testing options have been outlined in Laboratory Proficiency Testing Program (LPTP) Commitee
Comments (see References).
Requests for C. diphtheriae culture are exceedingly rare. Many laboratories no longer have the required
media and the expertise needed to isolate and identify C. diphtheriae and have opted to refer specimens
directly to a reference laboratory. The reader should refer to a reputable textbook for details around
specimen processing, isolation procedures, and toxin srudies specific for C. diphtheriae.
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It is difficult to determine the significance of Corynebacterium species other than C. diphtheriae in
cultures when (1) many species are part of the normal flora of humans and (2), they are not highly
pathogenic. Technologists should consider identification of corynebacteria to the species level in the
following circumstances:
 Isolates from normally sterile body sites that appear to correlate with clinical symptoms
 Isolates from severely debilitated patients or patients whose defense mechanisms are altered by
trauma, surgery or long-term chemotherapy, antibiotic treatment or intravenous therapy
 Isolates (recovered from adequately collected specimens) that occur as the predominant organism
and other organisms (if present) are of low pathogenicity; especially if coryneform bacteria are
seen in the direct gram stain and leucocytes are present
 Isolates recovered from multiple samples from the same body site
 Isolates recovered from urine cultures with a clinically significant colony count
Corynebacterium species have the following characteristics:

 In Gram-stained smears (Figure 2):
- bacterial cells are rod-shaped, gram-positive, and non-sporeforming
- cells are pleomorphic or irregular - usually "club-shaped"
- cells tend to occur in "picket fence" and "Chinese letter" arrangements
 Catalase-positive
 Non-motile
 Non-acid fast staining
 Most species are facultative anaerobes and ferment carbohydrates
 Cell walls contain mycolic acid chains (only genus in coryneform group)
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All Corynebacterium species grow readily on 5% sheep blood agar. Some lipophilic or lipid-requiring
species produce much larger colonies on blood agar supplemented with 1% Tween 80 (a lipid source). The
latter species will not grow well on commercially available chocolate agar that contains hemin powder
instead of lysed red blood cells (a source of lipid). Corynebacterium species also grow well in most blood
culture broth formulations and in nutrient and thioglycollate broths. They do not grow on MacConkey
agar.
C. diphtheriae is commonly divided into 4 biotypes - gravis, mitis, belfanti, and intermedius - based upon
colonial morphology and biochemical reactions. C. diphtheriae biotype intermedius produces a small, gray
or translucent colony which prefers growth on lipid-containing media. The other 3 biotypes produce larger,
white or opaque colonies that are indistinguishable from each other.
Colonies of C. jeikeium are tiny, dry, low, entire and grayish-white. Refer to Table 7 for the colonial
morphology of other clinically significant Corynebacterium species.
Detection of cystinase and pyrazinamidase enzymes can be used to separate C. diphtheriae group organisms
(C. diphtheriae , C. ulcerans and C. pseudotuberculosis) from other Corynebacterium species. The former
group is cystinase-positive and pyrazinamidase-negative while the latter group is cystinase-negative and
pyrazinamidase-variable (usually positive). Cystinase production can be detected using diagnostic tablets
(Rosco, Taastrup, Denmark) or by noting a brown halo around colonies on Tinsdale medium, a selective
medium for the isolation of C. diphtheriae. Diagnostic tablets are available for detection of pyrazinamidase.
The urease test can be used to distinguish C. diphtheriae (urease-negative) from C. ulcerans and C.
pseudotuberculosis (urease-positive).
C. jeikeium is a strict aerobe. It is usually resistant to 2 or more classes of antibiotics, pyrazinamidase-

positive, alkaline phosphatase-positive and produces acid for glucose and sometimes maltose, oxidatively.
Consult Table 7 for differentiating characteristics for other frequently reported Corynebacterium species
(key characteristics are shaded).
The commonest Corynebacterium species appear to identify reliably using commercial identification
systems with or without supplementary tests.
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Susceptibility Testing & Treatment
The National Committee for Clinical Laboratory Standards (NCCLS) has not published guidelines for the
susceptibility testing of coryneform bacteria. A method is described in the 7th edition of the Manual of
Clinical Microbiology that some laboratories use. It is a modified disc diffusion method involving: 1) the
use of Mueller-Hinton agar supplemented with 5% sheep blood, 2) incubation at 35°C in ambient air for 24
to 48 hours, and 3) the use of interpretive standards used for staphylococci or streptococci.
Diphtheria antitoxin is recommended for the treatment of pharyngeal diphtheria. Antibiotic therapy against
C. diphtheriae is also indicated to reduce the bacterial burden and thus decrease the production of exotoxin.
The organism is susceptible to a number of agents but in most cases erythromycin or penicillin is
recommended.
C. jeikeium is highly resistant to a number of antibiotics. Strains are susceptible to vancomycin, variably
susceptible to tetracycline and/or erythromycin, and resistant to most other agents (e.g., penicillins,
cephalosporins, aminoglycosides, clindamycin, and rifampin). Vancomycin is the drug of choice.
Other Corynebacterium species (with exception of C. urealyticum) are generally susceptible to

erythromycin, most beta-lactam antibiotics (penicillins and cephalosporins), and vancomycin. C.
urealyticum strains may be almost as resistant to antibiotics as C. jeikeium.
2. ARCANOBACTERIUM
The genus Arcanobacterium comprises 4 species. A. haemolyticum, the most recognized species, is
associated with pharyngitis and a number of other infections in humans. Formation of a "pseudomembrane"
resembling the type produced in diphtheria may occur in some cases of pharyngitis. The natural habitat of
arcanobacteria has not been established.
All four species are beta hemolytic on sheep blood agar; although the hemolysis is less pronounced than that
produced by beta-hemolytic streptococci. Colonies of A.haemolyticum are small (< 0.5 mm in diameter after
48 hours incubation at 37°C). Isolates from the respiratory tract usually produce rough colonies whereas
isolates from wounds are usually smooth. Pitting can be seen when colonies are removed from the
agar. Gram-stained smears reveal thin, irregular, club-shaped, curved rods in "V" formations. Occasionally,
cells exhibit rudimentary branching. The catalase test is the easiest method for differentiating
Arcanobacterium species (catalase-negative) from Corynebacterium species (catalase-positive). Refer to
Table 5 for other genus characteristics.
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3. MISCELLANEOUS GENERA
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Tests and characteristics used in the differentiation of other facultative anaerobic coryneforms are shown in
Table 5.
The genus Dermabacter comprises only one species - D. hominis. This organism is part of the normal skin
flora of humans but it can cause opportunistic wound infections and bacteremia. Colonies are 1 to 1.5 mm
in diameter after 48 hours of incubation, whitish, convex and give off a pungent odour.
The genus Rothia contains only one species - R. dentocariosa. This organism is a normal inhabitant of the
human oropharynx. It can cause endocarditis, septicemia, and pneumonia in the immunocompromised host.
Colonies of R. dentocariosa are whitish, raised, and can present with a smooth, rough or "spoked"
appearance (lines radiating from a central point).
The genus Turicella has only one species – T. otitidis. This organism is recovered from middle-ear fluids
from patients with otitis media. Colonies are 1 to 2 mm in diameter on blood agar after 48 hours of
incubation, whitish, creamy, and become yellowish with age.
The genus Gardnerella is comprised of only one species - G. vaginalis. Although a normal inhabitant of the
vagina of 40% of healthy reproductive females, this species can be part of the altered vaginal flora
associated with a non-inflammatory infection known as bacterial vaginosis. It has also been linked to
endometritis and post-partum sepsis. The "gold standard" for diagnosis of bacterial vaginosis is direct
microscopic examination of the vaginal discharge. Culture for G. vaginalis is not recommended in this
setting. In Gram-stained smears, G. vaginalis appears as thin, gram-variable bacilli (gram variable means
that some cells stain gram-positive and some stain gram-negative).
Five species are found in the genus - Propionibacterium. They are part of the normal oral cavity and skin
flora of humans. Acne, endocarditis, infections seeded from foreign bodies (e.g. central nervous system
shunts), and the SAPHO syndrome (synovitis, acne, pustulosis, hypertostosis, and osteomyelitis) have been
attributed to propionibacteria. P. acnes is the most common gram-positive, non-sporeforming, anaerobic
bacillus encountered in clinical specimens. Although the species is considered to be an obligate anaerobe,
some strains will grow aerobically under increased CO2. Because the organism can exhibit coryneform
Gram stain characteristics (i.e. pleomorphic cells in "diphtheroid" arrangements), the species deserves
mention here. P. acnes forms whitish, smooth, entire, convex colonies that range from 1 to 2 mm in
diameter on anaerobic blood agar after 48 hours of incubation. An organism with the colonial and cellular
characteristics described above that is both indole and catalase-positive is usually P. acnes.
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D. Branching / Filamentous / Aerobic Bacilli (Aerobic Actinomycetes)
284
The actinomycetes are a large and diverse group of gram-positive bacilli. Actinomycetes are classically
defined as organisms that produce branched, filamentous cells (hyphae) that either form spores or reproduce
by fragmentation. The rate and extent of elongation and branching is dependent on the strain of
actinomycetes, the growth medium, and the temperature of incubation. The hyphae produced by
actinomycetes may penetrate the surface of agar media, lay on the surface (substrate hyphae), or project into
the air (aerial hyphae).
Actinomycetes can be categorized as aerobic, facultatively anaerobic, or obligately anaerobic. There are
more than 40 genera that are described as aerobic actinomycetes. The medically important aerobic genera
include Actinomadura, Corynebacterium, Dermatophilus, Gordona, Mycobacterium, Nocardia,
Nocardiopsis, Oerskovia, Rhodococcus, Streptomyces, and Tsukamurella.
Only medically important aerobic actinomycetes that exhibit filamentous, branching cells are covered in
this section. Corynebacterium and Mycobacterium species are excluded from this discussion. This is
because Corynebacterium and Mycobacterium species do not exhibit branching (exception M. fortuitum,
which may produce branching at acute angles).
Aerobic actinomycetes are not frequently isolated in the clinical laboratory. However, these organisms can
cause serious human infection, especially in the immunocompromised host. The diagnosis of these
infections has been hindered by a combination of clinical and microbiologic difficulties, including their
often non-specific clinical presentation, a need for invasive biopsy procedures, difficulty in isolation, and
imperfect taxonomic classification.
Since Nocardia species and other aerobic actinomycetes are ubiquitous in nature, the isolation of these
organisms from non-sterile clinical specimens (e.g., sputum) does not provide conclusive evidence of
infection. Their presence could be a result of laboratory contamination or colonization. Culture findings
must be correlated with the direct microscopic examination of stained preparations from the same sample.
Besides qualifying culture findings, direct microscopic studies may yield results that may assist the clinician
in patient management. For instance, an effective antibiotic regimen may be initiated prior to the availability
of culture results.
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Aerobic actinomycetes grow well on most of the non-selective media used for the isolation of bacteria
(including mycobacteria) and fungi; such as blood agar and Sabouraud dextrose agar. Although most species
285
grow at 35ºC, more isolates can be recovered at 30°C (Nocardia asteroides, grows best at 30°C). Because
these organisms are slow growers, incubation of primary culture media must be extended from 2 to 4 weeks.
Inoculated plates should be sealed to prevent dehydration. Bacterial overgrowth is also a problem when
attempting to isolate aerobic actinomycetes from specimens collected from non-sterile body sites (e.g.,
lower respiratory tract samples). Thayer-Martin agar (for isolation of Neisseria gonorrhoeae) and buffered-
charcoal-yeast extract or BCYE agar (for isolation of Legionella species) have been used successfully for
the recovery of nocardia from these specimens.
Blood specimens can be collected into conventional blood culture bottles, biphasic (liquid and solid media
in the same bottle) culture bottles, and automated blood culture systems. For optimal detection of these
organisms, some experts recommend that blind subcultures of blood culture bottles be performed at regular
intervals and at the end of the incubation period.
Aerobic actinomycetes can be identified to the genus level using the following battery of tests:
 cellular morphology (i.e., branching, filament thickness),
 acid-fastness (using a modified acid fast stain),
 colonial characteristics (i.e. substrate filaments versus aerial filaments),
 growth at 50°C,
 metabolism of glucose (i.e., oxidative or fermentative utilization),
 arylsulfatase production,
 growth in lysozyme, and
 analysis of cell wall constituents (i.e., presence or absence of meso-diaminopimelic acid [meso-
DAP], mycolic acids, and sugars)
Most laboratories are unable to perform some of these tests making genus level identifications unrealistic.
Laboratories may opt to presumptively identify only the more frequently isolated members relying upon a
reputable reference laboratory for confirmation. Others may choose to refer out all clinically significant
isolates. For information around speciation of Nocardia and other aerobic actinomycetes, the reader should
refer to an excellent review written by McNeil and Brown (see References).
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Key characteristics for differentiating medically important aerobic actinomycetes at the genus level are
shown in Table 8. The observation of aerial hyphae and the acid-fastness of culture growth (partially acid-
fast versus non acid-fast) are two useful identification tools for the preliminary breakdown of these
organisms.
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Since Nocardia species account for the majority of infections caused by aerobic actinomycetes, this genus
will be discussed in detail in the following section.
ACID-FASTNESS
Determining acid-fastness is a valuable test for the identification of aerobic actinomycetes.

The modified Ziehl-Neelsen or Kinyoun acid fast stains are used for this purpose.
The cell walls of acid-fast organisms contain mycolic acids that make the cell wall
impervious to fuchsin, the primary dye used in acid fast staining methods. Heat or phenol
must be used to promote the entry of the primary dye into the bacterium.
The mycolic acid molecules of acid-fast organisms retain the primary dye, withstanding the
decolourization action of acid-alcohol. This resistance to decolourization is termed "acid-
fastness."
Some organisms are classified as partially or weakly acid-fast wherein bacilli and filaments
appear both acid-fast and non acid-fast in the same preparation. Partial acid-fastness can be
detected if 1% H2SO4 is used instead of the acid-alcohol decolourizer (used for staining
Mycobacterium species).
An acid-fast organism retains the primary stain (basic fuchsin) and appears red. Non acid--
fast organisms are readily decolourized and take up the blue colour of the counterstain
(methylene blue).
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Table 8 Characteristics of medically important aerobic actinomycetes
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1. NOCARDIA
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There are currently 8 medically important Nocardia species; namely, N. asteroides complex (Types I, II, IV
and VI), N. brasiliensis, N. brevicatena, N. farcinica, N. nova, N. otitidiscaviarum (formerly, N. caviae), N.
pseudobrasiliensis, and N. transvalensis. N. asteroides accounts for most Nocardia infections.
Nocardia species are ubiquitous in the environment and are primarily responsible for the decomposition of
plant material. Humans become infected by inhaling dust contaminated with organisms or by traumatic
inoculation of Nocardiae into subcutaneous tissues. Infections occur most often in severely
immunocompromised individuals; especially in those with defective T-cell immunity, such as organ
transplant recipients, patients with malignancies, those receiving corticosteroids, and persons with AIDS.
Pulmonary nocardiosis, the most common clinical syndrome, is often a progressive, disseminated and life-
threatening infection in immunosuppressed patients. The formation of multiple abscesses in the lungs
generally leads to a necrotizing pneumonia with cavitation. Disseminated nocardiosis occurs in 50% of
patients with pulmonary infection.
Once disseminated, Nocardiae are capable of causing abscesses in virtually every organ of the body;
especially in brain tissue.
Direct inoculation into the skin may lead to actinomycetoma, an infection relatively uncommon in North
America but not uncommon in tropical or subtropical regions. Actinomycetoma is a chronic, disfiguring
skin and soft tissue infection marked by multiple draining sinuses. In North America, inoculation usually
results in cellulitis and/or lymphocutaneous syndrome (involvement of the local lymphatic system).
Since the portal of entry for most cases of nocardiosis is the respiratory tract, the commonest specimens
submitted to the laboratory include sputum, bronchoalveolar lavage fluid, transtracheal aspirates, or lung
tissue. If actinomycetoma is being queried, aspirates rather than swabs should be collected. The general
principles of specimen collection, transport, and storage are applicable to most aerobic actinomycetes.
However, if specimens are suspected of carrying Nocardiae, they should NOT be refrigerated. It appears
that some Nocardia strains lose their viability after exposure to temperatures near freezing. It should be
noted that not all Nocardia species survive the N-acetylcysteine digestion procedure used to optimize
recovery of pathogens from sputum and bronchial washings.
If possible, specimens submitted for nocardia investigations should be spread out in a petri dish and
searched for clumps of organisms that resemble granules. Granules should be extracted and crushed
between two glass microscope slides for microscopic examination. Duplicate direct smears of the clinical
material should always be prepared; one smear for Gram staining and the other for modified Kinyoun
staining.
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In direct Gram-stained preparations, Nocardiae occur as gram-positive, beaded (alternating stained and non-
stained areas), branched, filamentous hyphae that measure from 0.5 µm to 1.0 µm in width to as much as 20
µm in length. Right angle branching is diagnostic for Nocardia species. The Gomori methenamine silver
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stain and the Brown and Brenn tissue Gram staining procedure can be used to detect organisms in tissue
samples.
Direct acid-fast staining of clinical material is also informative. Nocardia species usually stain partially
acid-fast. However, strain-to-strain variation in acid-fast staning - from non acid-fast to partially acid-fast
-should be expected. Acid fast staining is difficult to perform reliably and interpretation of stain results must
be made with caution; in context with other identification tools. Variable results have been reported,
dependant upon the type of culture medium supporting the growth. Media containing high levels of lipid,
such as Lowenstein-Jensen medium, are best for demonstrating acid-fastness.
Cultures should be incubated for at least 4 weeks before reporting as "no growth" for Nocardiae. Most
Nocardia strains can be detected in 10 to 14 days. Incubation in 5 to 10% CO2 enhances growth.
The morphological features of immature nocardial colonies can be assessed under a stereoscopic
microscope. It is important to look for the presence of true branched substrate hyphae, aerial hyphae, and
sporulation. The substrate hyphae of Nocardia species appear as thin filaments that branch at right angles.
Secondary branching is also a common characteristic. Upon aging, filaments may begin to break up
(fragmentation) into individual bacilli-shaped (bacillary) and coccus-shaped (coccoid) cells. Scanning up
and down through multiple focal planes will reveal the presence of aerial hyphae; a morphological
characteristic that is not produced in Corynebacterium, Gordona, Mycobacterium, Rhodococcus, and
Tsukamurella species (Table 8). It should be noted, however, that aerial hyphae may be absent, sparse, or
very abundant. Short chains of conidia may be found on the aerial hyphae, which are rarely seen on the
substrate hyphae.
With time, colonies with aerial hyphae become "chalky" looking to the unaided eye as opposed to colonies
that lack aerial hyphae (or immature colonies) that remain smooth looking. Colonial colour is highly
variable from species to species depending upon the support medium and the incubation temperature used.
Most strains of N. asteroides vary from salmon pink to orange on Sabouraud dextrose agar and heart
infusion agar.
An organism that displays aerial hyphae, partial acid-fastness, and right-angle branched filaments is
typically a Nocardia species. Nocardiae are capable of growing in a broth containing lysozyme, do not
grow at 50°C, ferment glucose, and have mycolic acids, meso DAP, arabinose and galactose as cell wall
constituents (Table 8).
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To date, there are no NCCLS antimicrobial susceptibility testing methods available for aerobic
actinomycetes. The typically slow-growing nature of these organisms and the difficulty in making even
suspensions in saline have thwarted most attempts in applying testing formats used for other
microorganisms. Despite the lack of standardized testing, some experts recommend that susceptibility
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testing be performed on all important clinical isolates; preferably at a specialized reference laboratory.
Sulfonamides with or without trimethoprim have been the mainstay of antimicrobial treatment of
nocardiosis for many years.
2. MISCELLANEOUS GENERA
The genus, Actinomadura, comprises 26 species. A. madurae is a frequent cause of actinomycetoma (see
Nocardia). Members occur as fine, gram-positive, non-fragmenting bacilli that exhibit branched substrate
hyphae and aerial hyphae, which can each carry up to 15 arthrospores. All species are non-acid fast, unable
to grow in lysozyme, do not grow at 50°C, and metabolize glucose oxidatively. Cell walls are devoid of
mycolic acids, positive for meso-DAP and positive for the sugar - madurose.
The genus, Nocardiopsis, includes 7 species. N. dassonvillei has been linked to mycetomas and other skin
infections. Species demonstrate aerial hyphae and are non-acid fast. Typically, species produce long,
branched substrate hyphae and aerial hyphae that fragment into "zigzag" chains of arthrospores. All species
are non-acid fast, unable to grow in lysozyme, do not grow at 50°C, and utilize glucose oxidatively. Cell
walls are negative for mycolic acids but positive for meso-DAP.
The genus, Streptomyces, is a vast collection of organisms. S. somaliensis is a causative agent of

actinomycetoma. Infrequently, Streptomyces species can lead to septicemia, pulmonary infections, and brain
abscesses. Like Nocardiae, Streptomyces species produce both substrate and aerial hyphae. Both hyphal
types bear long chains of conidia. Most species produce dry, chalky, heaped, gray-white colonies that emit a
pungent "musty-basement" odour. Streptomyces species are non-acid fast, unable to grow in lysozyme, do
not grow at 50°C, and utilize glucose oxidatively. Cell walls contain the L-DAP isomer but lack mycolic
acids.
Gordona species have been implicated as the causative agent in skin infections, chronic pulmonary disease,
catheter-associated sepsis, and wound infections. These organisms appear as gram-positive, weakly acid-
fast, thin, beaded coccobacilli with minimal branching. No aerial hyphae are produced. Gordona species are
exhibit variable growth in lysozyme, do not grow at 50°C, and utilize glucose oxidatively. Cell walls
contain mycolic acids, the meso-DAP isomer and two diagnostic sugars - arabinose and galactose.
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The genus, Rhodococcus, comprises a diverse group of organisms that demonstrate variable morphology,
biochemical characteristics, growth patterns, and pathogenicity R. equi is considered to be the most
clinically significant species. It can lead to potentially life-threatening infections in immunocompromised
patients; especially in individuals infected with HIV. Infections include actinomycetoma, bacteremia,
pneumonia, lung abscesses, and catheter-related sepsis. The microscopic morphology of R. equi in cultures
291
is cyclic. Cells vary from bacillary to coccoid depending upon the incubation time and growth conditions.
Rhodococcus species do not produce aerial hyphae and are weakly acid-fast. The classic colony type of R.
equi is pale pink and slimy in 2 to 4 days on blood agar. Rhodococcus species exhibit variable growth in
lysozyme, do not grow at 50°C, and utilize glucose oxidatively. Cell walls contain mycolic acids,
the meso DAP isomer and two diagnostic sugars - arabinose and galactose.
Tsukamurella species are gram-positive, weakly acid-fast organisms that grow best at temperatures below
human body temperature. No aerial hyphae are produced. Cells are long rods that fragment into 3 pieces,
which then separate and grow independently. T.paurometabola, the most recognized species, has been
implicated in a number of cases of catheter-related sepsis. Tsukamurella species exhibit growth in lysozyme,
do not grow at 50ºC, and utilize glucose oxidatively. Cell walls contain mycolic acids, the meso DAP
isomer and two diagnostic sugars - arabinose and galactose.
Dermatophilus species have a unique life cycle that begins and ends in the production of motile spores. D.
congolensis is the primary species in the genus. It causes a relatively rare exudative skin infection (with
encrustations) known as dermatophilosis. Humans acquire the infection after handling tissues of infected
animals. The diagnosis of dermatophilosis depends upon the observation of the typical structures of D.
congolensis in clinical samples and the isolation and identification of the organism in culture. Typically,
branched filaments divide in their transverse and longitudinal planes, forming packets of eight coccoid or
cuboidal-shaped motile cells or spores. Cells are non acid-fast. Gray-white colonies measuring 0.5 to 1.0
mm in diameter form after 24 hours of incubation on brain heart infusion agar containing horse blood. D.
congolensis do not grow at 50°C. Cell walls do not contain mycolic acids. The organism is positive for
glucose fermentation, the meso DAP isomer and one diagnostic sugar - madurose.
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MICROBIOLOGY
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SELF ASSESSMENT QUESTIONS

292
[1] 1. What two characteristics are used to categorise aerobic gram-positive bacilli?
[2] 2. What is meant by the adjectives “regular” and “irregular” with regards to cellular morphology?
[1] 3. Under what conditions are endospores produced by Bacillus species?
[1] 4. Name the two most recognized Bacillus species.
[3] 5. a) What are the 3 clinical manifestations of anthrax?

b) What form of anthrax is most commonly seen in humans?
[2] 6. Food poisoning caused by B. cereus is regarded as an_________________ rather than an

infection – why?
[2] 7. Name 3 factors (events) that predispose patients to opportunistic infections caused by Bacillus
species other than B. anthracis and B. cereus?
[2] 8. Name 3 examples of opportunistic infections caused by Bacillus species.
[2] 9. Explain why work with specimens and/or cultures, suspected of containing B. anthracis, must be
carried out in a biological safety cabinet.
[2] 10. Describe the cellular characteristics of Bacillus species.
[4] 11. a) What spore-forming genus other than the genus Bacillus contains species that are capable of
growing under aerobic conditions?
b) What characteristics separate members of the genus in a) from Bacillus species.
[1] 12. Which of the following test result(s) do not describe B. anthracis (there could be more than one
response)?
a) Beta-hemolysis positive
b) Non-motile
c) Penicillin-susceptible
d) No growth on PEA
e) Negative for salicin fermentation
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[1] 13. Which of the following genera are not part of the “Regular / Non-sporeforming / Facultative
Anaerobic Bacilli” group?
a) Listeria
b) Kurthia
c) Lactobacillus
d) Erysipelothrix
e) Corynebacterium
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[1] 14. What is the most common bacterial cause of community-acquired meningitis in
immunocompromised people?
[2] 15. Describe the Gram stain morphology and arrangement of Listeria monocytogenes.
[1] 16. Which of the following test result(s) are not consistent with a Listeria species (there could be
more than one response)?
b) Catalase-positive
c) Esculin-hydrolysis positive
d) Non-motile at room temperature
e) VP-positive
[1] 17. What bacterium is the causative agent of swine erysipelas in animals and erysipeloid in
humans?
[1] 18. Which of the following test result(s) are not consistent with E. rhusiopathiae (there could be
more than one response)?
b) Catalase-positive
c) Esculin-hydrolysis positive
d) Positive tumbling motility
e) All of the above
[1] 19. Lactobacillus species are normal inhabitants of the _____________, _______________, and the
_________________ of humans.
[3] 20. a) Name 3 genera that belong to the category – “Irregular / Non-sporeforming / Aerobic-
Facultative Anaerobic Bacilli.
b) Which facultatively anaerobic genus is recovered most often in cultures of clinical
specimens?
[3] 21. a) Name the bacterium that is the primary cause of diphtheria.
b) What is the primary mode of transmission leading respiratory diphtheria?
c) Name the complications of diphtheria which can result in significant mortality?
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[2] 22. a) What species of Corynebacterium is recovered from clinical cultures more frequently than
any other Corynebacterium species?
b) Infections caused by this organism are difficult to treat - why?
[2] 23. Name 3 circumstances when Corynebacterium species other than C. diphtheriae are identified
to the species level?
[1] 24. Which of the following test result(s) are not consistent with Corynebacterium species
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(there could be more than one response)?
a) Catalase-positive
b) Most species are beta-hemolytic
c) Non-acid fast staining
d) Cell walls contain mycolic acids
e) Cells are pleomorphic - usually club-shaped
f) Cells are partially acid-fast
[4] 25. Match the characteristics / methods / results in Column A which correspond with the gram-
positive genus or species in Column B (there may be multiple correct characteristics for each
species).
[1] 26. Which of the following statements about C. jeikeium is false (there could be more than one
response)?
a) It is found on skin and hospital surfaces.
b) It is catalase-positive and a strict aerobe.
c) It can cause septicemia, wound infections and endocarditis in compromised hospital
patients.
d) Infections are usually spread by aerosols.
e) It is usually vancomycin-resistant.
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[1] 27. Name the species covered in this module that is beta-hemolytic, pits agar media, and is capable
of causing pharyngitis.
[2] 28. a) Name 3 genera that belong to the category – “Branching / Filamentous / Aerobic Bacilli”
b) Which genus is recovered most often in cultures of clinical specimens?
[2] 29. State the primary function of direct Gram stain of clinical specimens submitted for workup of
aerobic actinomycetes.
295
[2] 30. Name the two characteristics that are used to group and sub-group aerobic actinomycetes.
[1] 31. Which of the following statement(s) about Nocardia species is/are false (there could be more
than one response)?
a) Most forms of nocardiosis are characterized by the absence of abscesses.
b) They are ubiquitous in nature.
c) Cells are diphtheroid-like and acid-fast.
d) It can cause pulmonary nocardiosis – usually in immunocompromised patients.
e) They never produce aerial hyphae.
[2] 32. Describe the Gram stain characteristics (cellular morphology and arrangement) of “classic”
nocardiae.
TOTAL 57
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MICROBIOLOGY
840-828
SELF ASSESSMENT ANSWERS
1. Presence of endospores and cellular shape
2. Cells are considered to be "regular" in shape if the longitudinal sides of the bacillus are straight and
parallel to each other. Cells are described as "irregular" if the longitudinal sides are curved and not
parallel.
3. Aerobic conditions
4. B. anthracis and B. cereus
5. a) Cutaneous, intestinal and pulmonary; b) cutaneous

296
6. Intoxication; Gastroenteritis is caused by the action of pre-formed enterotoxins in the food. There is
no infectious process.
7. Operative procedures, traumatic wounds, and parenteral drug abuse

8. Bacteremia, endophthalmitis, and surgical wound infections
9. Spores of B. anthracis are infectious by the respiratory route. Manipulations of specimens

(submitted for anthrax investigation) and known positive cultures could generate aerosols. These
manipulations must be carried out in biological safety cabinet to protect the worker.
10. Bacillus species may be coccobacillary (0.5 µm wide by 1.2 µm long) or large and "box-shaped"
(2.5 µm wide by 10 µm in length). Spores may be elliptical, spherical, or egg-shaped. Spores vary in
their location in the cell; from a central position, between the end and the centre of the cell (sub-
terminal) or at one end of the cell (terminal). Sometimes the diameter of the spore exceeds the width
of the bacterial cell making cells look "swollen"
11. a) Clostridium; b) endospore production (under aerobic or anaerobic conditions), colony size (under
aerobic or anaerobic conditions), and catalase reaction
12. a
13. e
14. L. monocytogenes
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15. In direct Gram stains of clinical specimens, L. monocytogenes typically appears as a non-branching,
non-sporeforming, regular, short (0.5 to 2 µm long by 0.4 to 0.5 µm wide), gram-positive bacillus
that occurs singly or in short chains.
16. d
17. E. rhusiopathiae
18. a, b, c, d, or all of the above
19. Human mouth, intestinal tract and vagina
20. a) Corynebacterium, Arcanobacterium, and Rothia (see Page 37 for more examples); b)
Corynebacterium
21. a) Corynebacterium diphtheriae; b) droplet transmission; c) congestive heart failure and cardiac
arrhythmia
22. a) Corynebacterium jeikeium; b) This species is usually resistant to a number of classes of

antibiotics. As a result, there are few treatment options.
297
23. Any 3 of the following situations:
 Isolates from normally sterile body sites that appear to correlate with clinical symptoms
 Isolates from severely debilitated patients or patients whose defense mechanisms are altered by
trauma, surgery or long-term chemotherapy, antibiotic treatment or intravenous therapy
 Isolates (recovered from adequately collected specimens) that occur as the predominant
organism and other organisms (if present) are of low pathogenicity; especially if coryneform
bacteria are seen in the direct gram stain and leucocytes are present
 Isolates recovered from multiple samples from the same body site
 Isolates recovered from urine cultures with a clinically significant colony count
24. b, f
25. a. 1, 6, 10
b. 7, 9
c. 2, 5
d. 3, 4, 8
26. d, e
840-828 56 2715
27. Arcanobacterium haemolyticum
28. a) Any 3 of the following genera:

There are more than 40 genera that are described as aerobic actinomycetes. Only the following
genera exhibit branching: Actinomadura, Dermatophilus, Gordona, Nocardia, Nocardiopsis,
Oerskovia, Rhodococcus, Streptomyces, and Tsukamurella.
b) Nocardia
29. Discovery of aerobic in cultures does not imply infection. These organisms are widespread in nature
and can occur in cultures as a contaminant or as part of the normal flora in non-sterile body sites
(e.g. sputum). Direct Gram stains give the culture findings some context. If there is strong evidence
of an infectious process (branching bacilli plus WBCs) positive culture findings can be given more
diagnostic value. Also, the direct Gram stain provides rapid diagnostic information - before the
availability of culture results.
30. Presence of aerial hyphae and acid-fastness (acid-fast staining characteristics)
31. c, e
298
32. Thin, filamentous, gram-positive bacilli (some beading may be seen) that produce righ-angle
branches
299

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MICROBIOLOGY

THE MICHENER INSTITUTE for Applied Health Sciences

Contributors And Reviewers

© 2000 by THE MICHENER INSTITUTE for Applied Health Sciences

A. Regular/Sporeforming/Aerobic Or Facultative Anaerobic 10

B. Regular/Non-Sporeforming/Facultative Anaerobic Bacilli 19

D. Branching/Filamentous/Aerobic Bacilli (Aerobic 42

Regular / Sporeforming / Aerobic or Facultative Anaerobic Bacilli:

2. Discuss the natural habitat of members of the genus, Bacillus.

5. Discuss the major clinical features of food-poisoning due to B. cereus.

8. Give the circumstances when speciation of Bacillus-like organisms is warranted.

10. State key colonial and physiological/ biochemical characteristics of B. anthracis.

12. Discuss L. monocytogenes with regard to:

13. Pertaining to the diagnosis of listeriosis, Discuss:

15. Describe the natural habitat and clinical significance of E. rhusiopathiae.

16. Recognize the typical cellular and colonial characteristics of E. rhusiopathiae.

Irregular / Non-sporeforming / Aerobic -Facultative Anaerobic Bacilli:

20. Recognize the facultative anaerobic genera found in this category

25. Discuss the organism C. diphtheriae in terms of:

27. Discuss issues around the culturing for C. diphtheriae.

29. Discuss the key characteristics of the genus Corynebacterium.

Branching / Filamentous / Aerobic Bacilli (Aerobic Actinomycetes):

1. Balows, A. (Editor), Laboratory Diagnosis of Infectious Diseases, Principles and Practices,

1. Regular, sporeforming, aerobic or facultative anaerobic bacilli

2. Regular, non-sporeforming, facultative anaerobic bacilli

3. Irregular, non-sporeforming, facultative anaerobic bacilli

4. Branching, filamentous, aerobic bacilli (aerobic Actinomycetes)

The inter-relationships of these groups are depicted in Figure 1.

Figure 1. Differentiation of common aerobic gram-positive bacilli

A. Regular / Sporeforming / Aerobic Or Facultative Anaerobic Bacilli

Opportunistic infections caused by Bacillus species are as follows:

SPECIMENS - COLLECTION AND HANDLING

Table 1. Differentiation of Bacillus species from Clostridium species.

Susceptibility Testing &Treatment

B. anthracis is almost always susceptible to penicillin, gentamicin, erythromycin, ciprofloxacin, and

Regular / Non-Sporeforming / Facultative Anaerobic Bacilli

This group is comprised of the following genera:

Abbreviations and symbols:

Genus-level identification is achievable with the following combination of test results:

Susceptibility Testing & Treatment

C. Irregular / Non-Sporeforming / Aerobic-Facultative Anaerobic Bacilli

Members of this large group have the following general characteristics:

 Esculin hydrolysis (read for up to 72 hours)

diphtheria. C. urealyticum (formerly CDC group D2) is a well-documented cause of alkaline-encrusted

SPECIMENS - COLLECTION AND HANDLING

Corynebacterium species have the following characteristics:

C. jeikeium is a strict aerobe. It is usually resistant to 2 or more classes of antibiotics, pyrazinamidase-

Other Corynebacterium species (with exception of C. urealyticum) are generally susceptible to

D. Branching / Filamentous / Aerobic Bacilli (Aerobic Actinomycetes)

Determining acid-fastness is a valuable test for the identification of aerobic actinomycetes.

Table 8 Characteristics of medically important aerobic actinomycetes

The genus, Streptomyces, is a vast collection of organisms. S. somaliensis is a causative agent of

SELF ASSESSMENT QUESTIONS

[1] 3. Under what conditions are endospores produced by Bacillus species?

[1] 4. Name the two most recognized Bacillus species.

[3] 5. a) What are the 3 clinical manifestations of anthrax?

[2] 6. Food poisoning caused by B. cereus is regarded as an_________________ rather than an

[2] 8. Name 3 examples of opportunistic infections caused by Bacillus species.

[2] 10. Describe the cellular characteristics of Bacillus species.

SELF ASSESSMENT ANSWERS

1. Presence of endospores and cellular shape

4. B. anthracis and B. cereus

5. a) Cutaneous, intestinal and pulmonary; b) cutaneous

7. Operative procedures, traumatic wounds, and parenteral drug abuse