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Molecular subtyping of bacterial isolates by characteriza- activities and transfer of standardized molecular subtyping
tion of proteins or nucleic acids has been successfully applied methodology to public health laboratories should enable more
to aid epidemiologic investigations of foodborne disease timely subtyping of clinical and food isolates. One result
outbreaks since the initial use of plasmid fingerprinting would be information useful to epidemiologists while they
nearly 20 years ago (1,2). Since that time, several methods for were investigating outbreaks. In addition, routine subtyping
identifying restriction fragment length polymorphisms on of isolates of foodborne pathogenic bacteria received by public
chromosomal DNA have been developed, and molecular health laboratories should lead to identification of outbreaks
subtyping has become an essential component of epidemiologic not readily recognizable by other means. Use of standardized
investigations of infectious diseases (3-10). subtyping methods would allow isolates to be compared from
This widespread use of molecular typing has resulted in a different parts of the country, enabling recognition of
plethora of techniques and protocols for subtyping even the nationwide outbreaks attributable to a common source of
same species of bacteria (11). Because each laboratory uses its infection, particularly those in which cases are geographically
own protocols for molecular typing and designations of separated.
patterns, the results cannot be compared with those of another In 1995, the Centers for Disease Control and Prevention
laboratory, even if both laboratories have used essentially the (CDC), with the assistance of the Association of Public Health
same methods. This lack of comparability has greatly Laboratories (APHL), selected the state public health
diminished the power of molecular subtyping methods. laboratories in Massachusetts, Minnesota, Washington, and
In 1993, during the investigation of an Escherichia coli Texas as area laboratories for a national molecular subtyping
O157:H7 outbreak caused by contaminated hamburgers network for foodborne bacterial disease surveillance. This
served in a fast-food restaurant chain in the western United network later became known as PulseNet (13). Standardized
States, Barrett et al. in our laboratory applied pulsed-field gel PFGE typing and pattern analysis technology would be
electrophoresis (PFGE) to characterize clinical and food transferred to the area laboratories, which would assume
isolates of E. coli O157:H7 and demonstrated its utility in responsibility for subtyping foodborne pathogenic bacteria
outbreak investigations (12). Subsequently, our laboratory from their states and providing subtyping service to
received numerous requests from state health departments neighboring states that requested assistance. At about the
for subtyping E. coli O157:H7. The demand soon overwhelmed same time, CDC and five state health departments, as part of
our testing capacity, and delays in subtyping isolates meant a response to emerging infectious disease threats (14),
that results were mostly useful only for laboratory implemented an active foodborne disease surveillance
confirmation of conclusions from epidemiologic investiga- program called FoodNet (15). The objectives of FoodNet were
tions. We reasoned that decentralization of subtyping to accurately estimate the burden of foodborne disease in the
United States, investigate the sources of infection in
Address for correspondence: Bala Swaminathan, Foodborne and
outbreaks and sporadic cases, and build public health
Diarrheal Diseases Branch, Centers for Disease Control and
Prevention, 1600 Clifton Road, Mailstop C03, Atlanta, GA 30333, infrastructure for dealing with emerging foodborne disease
USA; fax: 404-639-3333; e-mail: bas5@cdc.gov issues. In 1996, FoodNet included Minnesota, Oregon,
1The CDC PulseNet Task Force includes John Allan, Michelle Bird, Daniel Cameron, Wallis DeWitt, Thomas Donkar, Collette Fitzgerald, Judy Gaither, Lewis
Graves, Peggy Hayes, Jared Kerr, Kristy Kubota, Mary Ann Lambert-Fair, Susan Maslanka, Loretta McCroskey, Jeremy Miller, Heather Noll, Efrain Ribot, Nicole
Tucker, Susan Van Duyne, and the authors.
Maths, Kortrijk, Belgium); the change-over will be completed Developing Standardized Protocols
in 2001. Each PulseNet laboratory has all the above Standardized protocols for foodborne bacterial pathogens
equipment and has the capability to normalize the patterns, were developed in priority order based on the ability of PFGE
compare them with other patterns, and maintain local to discriminate among strains of the organism and the
databases of PFGE patterns for each bacterial pathogen of epidemiologic utility of the resulting data. Standardized
interest. PFGE protocols have been developed for E. coli O157:H7,
Salmonella enterica serotype Typhimurium, L. monocytoge-
National Database of PFGE Patterns and nes, and Shigella species. The S. Typhimurium protocol is
Associated Epidemiologic Information applicable to most other nontyphoidal Salmonella serotypes,
A national database of PFGE patterns is being assembled including S. Enteritidis. However, neither PFGE nor other
for foodborne bacterial pathogens. These databases reside on molecular subtyping methods provide acceptable discrimina-
a PulseNet server at CDC. For each bacterial pathogen, the tion among strains of this highly clonal serotype. Standard
normalized PFGE pattern is associated with a pattern PFGE protocols for Campylobacter jejuni, C. coli, and
database and a database of epidemiologic and clinical Clostridium perfringens (7) are being developed and
information for isolates. One isolate may be associated with validated. Although C. jejuni and C. coli infections are
more than one PFGE pattern in the database because common, developing a standardized PFGE protocol for these
PulseNet protocols may call for the use of more than one organisms was not a high priority because they infrequently
restriction enzyme to achieve appropriate discrimination cause outbreaks. On the other hand, although outbreaks of C.
between epidemiologically unrelated isolates. The E. coli perfringens infections are seldom widespread, state and local
O157:H7 database is functional; databases for nontyphoidal public health laboratories requested a standardized
Salmonella serotypes and Listeria monocytogenes are under subtyping protocol to assist with local outbreak investiga-
construction. tions. All PulseNet protocols are 1-day procedures based on
Seven PulseNet laboratories (four state public health the PFGE protocol developed by the Washington State Public
laboratories, FDA-CFSAN, USDA-FSIS, and CDC) have Health Laboratory in response to the need for more rapid
direct access to the PulseNet database server through the techniques (16). All new protocols and modifications of
Internet, enabling them to submit normalized PFGE patterns existing protocols are evaluated initially at the developing
and associated epidemiologic information. (The DST version laboratory, followed by a second evaluation at CDC, alpha-
of the Molecular Analyst software creates a special “bundle” testing at one or two PulseNet laboratories, and beta-testing
file for comparison with the national database.) Laboratories at several PulseNet laboratories before they are adopted as
query the national database for identical matches or closely official PulseNet protocols. Evaluation criteria include
related patterns (>95% related under specified conditions). If reproducibility of patterns, appropriateness of the strain used
identical or close matches to the submitted patterns are as the reference standard, and robustness of the procedure.
found, the submitting laboratory can access epidemiologic Once a protocol is officially adopted, no changes can be made
information associated with those patterns from the text except by a petition to CDC’s PulseNet Task Force, discussion
database. When a PulseNet participating laboratory logs on of the proposed changes, and adoption of the proposal by
to the PulseNet server, it will display a “recent match” PulseNet laboratories. The PulseNet Task Force at CDC is
message if two or more laboratories submit identical or closely composed of personnel who carry out PulseNet-related
related patterns within a specified time. This alert provides activities. The Task Force members develop and evaluate
an early warning to PulseNet laboratories about possible protocols, provide technical support for participating
multisite foodborne disease outbreaks. laboratories, organize and conduct training workshops,
PulseNet laboratories that do not yet have direct online administer the certification program and proficiency testing
access to the PulseNet server may still electronically submit program, and maintain the national databases of PFGE
raw TIFF images and normalized PFGE patterns (bundle patterns for the bacteria under surveillance in PulseNet.
files) to the PulseNet database administration team by e-mail
or through file transfer protocols (ftp). The team compares the Quality Control and Assurance Program
submitted patterns with those in the national database and e- A quality assurance program has been instituted for
mails the results to the submitting laboratory as quickly as PulseNet to ensure the integrity of results obtained with the
possible. We expect that direct access to the PulseNet server standardized PFGE techniques. This program requires strict
will be available to all participating laboratories that have adherence to the standardized PFGE protocols (17). In
satisfactorily completed certification requirements by June addition, the quality assurance program consists of standards
30, 2001. for training, analytical procedures, documentation, and
Figure 2. Distribution by
date and pulsed-field gel
electrophoresis subtype
of Salmonella ser.
Typhimurium isolates
received by the Minne-
sota Department of
Health, August 14-Sep-
tember 14, 1995. Data
provided by Jeffrey
Bender and John Besser,
Minnesota Department
of Health.
The use of molecular subtyping as part of routine PFGE patterns should not be considered proof that the
surveillance has benefits beyond outbreak detection. persons had a common exposure, merely that the isolates in
Temporal clustering of unrelated cases is not uncommon, and question share a common ancestry. Moreover, outbreaks can
without molecular subtyping, valuable public health be caused by more than one subtype, so that differences in
resources can be wasted investigating pseudo-outbreaks. In PFGE pattern alone cannot prove that isolates did not have a
June and July 1994, an outbreak of E. coli O157:H7 infections common source (24,25).
was suspected when the New Jersey Department of Health
and Senior Services received reports of 48 culture-confirmed Requirements for Effective Functioning
cases; only four were reported during the same period in 1993 Although the area laboratories are set up to assist
(23). PFGE subtyping found most isolates to have unique neighboring state public health laboratories that are not
patterns, indicating that a large outbreak was unlikely. The PulseNet participants, every state must have PFGE
probable reason for the sudden increase in case reports was subtyping capacity for optimum performance of the network.
the concomitant increase in the number of laboratories A dramatic indication of this was provided during the 1997
culturing stools for E. coli O157:H7 (Figure 3). ground beef-associated E. coli O157:H7 outbreak in Colorado.
Although PulseNet has proven invaluable in detecting When the outbreak pattern was posted on PulseNet ListServ,
foodborne disease outbreaks and facilitating their investiga- most laboratories that were network participants responded
tion, molecular subtyping is an adjunct to epidemiologic within 48 hours that they had no matching PFGE patterns
investigation and not a replacement for it. The observation from recent E. coli O157:H7 isolates. In contrast, it took more
that isolates from two or more persons have indistinguishable than 2 months to identify a case in Kentucky (not a PulseNet
participant state in 1997) that was related to the Colorado
outbreak. The Association of Public Health Laboratories has
determined that PulseNet participation is a core capacity for
all state and territorial public health departments in the
United States.
For the network to work efficiently in detecting foodborne
disease outbreaks through routine surveillance, PulseNet
laboratories must perform, at a minimum, routine PFGE
subtyping of E. coli O157:H7 and L. monocytogenes as soon as
isolates are received. In addition, they must perform PFGE
subtyping of other foodborne pathogenic bacteria (Campylo-
bacter jejuni and C. coli, Salmonella serotypes, Shigella spp.,
Bacillus cereus, Vibrio cholerae, V. parahaemolyticus,
V. vulnificus, Clostridium botulinum, C. perfringens, Yersinia
enterocolitica) rapidly when the number of isolates received
by the laboratory exceeds the expected number for that
period. Unfortunately, microbiologists at state and local
Figure 3. Reported cases of culture-confirmed Escherichia coli
public health laboratories often have responsibilities for all
O157:H7 infection and percentage of surveyed laboratories routinely pathogenic bacteria and may not be able to type incoming
testing all stool specimens for E. coli O157:H7, New Jersey, January isolates of foodborne pathogenic bacteria in a timely manner.
1991 through July 1994 (10). In addition, like public health surveillance in general, PulseNet