FOODBORNE PATHOGENS AND DISEASE

Volume 3, Number 1, 2006

© Mary Ann Liebert, Inc.

Interpretation of Pulsed-Field Gel Electrophoresis

Patterns in Foodborne Disease Investigations
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and Surveillance

TIMOTHY J. BARRETT, PETER GERNER-SMIDT, and BALA SWAMINATHAN

ABSTRACT

Since the establishment of the well-known Tenover criteria in 1995 (Tenover et al., 1995), relatively few papers
have been published about the interpretation of subtyping data generated by pulsed-field gel electrophoresis
Foodborne Pathogens and Disease 2006.3:20-31.

(PFGE). This paper describes the approach that has been used in the PulseNet network during the past 10 years.
PFGE data must always be interpreted in the proper epidemiological context and PFGE data can not alone prove
an epidemiological connection. The Tenover criteria are not generally applicable to the interpretation of PFGE
subtyping data of foodborne pathogens. The reproducibility of the method with a particular organism, the qual-
ity of the PFGE gel, the variability of the organism being subtyped, and the prevalence of the pattern in question
must always be considered. Only isolates displaying indistinguishable patterns should be included in the detec-
tion of clusters of infections or the initial case definition in a point-source outbreak. More variability (patterns
differing from each other in two to three band positions) may be accepted if the outbreak has been going on for
some time or if person-person spread is a prominent feature. If epidemiological information is sufficiently strong,
isolates with markedly different PFGE patterns may be included in an outbreak.

INTRODUCTION of the genes responsible for their virulence,

vaccine-related antigens, and drug resistance.

S UBTYPING is the characterization of bacteria

below the species and subspecies level. It
may be performed by a multitude of phenotypic
and genotypic techniques. Subtyping may be
done for taxonomic purposes, study of pop-
ulation structure of a species, phylogenetic
analyses directed at determining relatedness
of the target organism to other organisms or
delineating steps in the evolution of the tar-
get organism, or for molecular epidemiology.
Herein, we focus our attention solely on molec-
ular epidemiologic application of subtyping.
Levin et al. (1999) defined molecular epidemi-
ology as the identification of microparasites re-
sponsible for infectious disease and determining
their physical sources, their biological relation-
ships, and their route of transmission and those
From a public health standpoint, the determi-
nation of the “physical source of the micropar-
asite” that caused human illness, particularly
in an outbreak setting, and understanding its
route of transmission from the source to the af-
fected person are of paramount importance.
However, another aspect of the statement of
Levin et al. (1999) needs to be emphasized. In
considering the subtype of an organism, one
must consider all available data such as bio-
chemical reaction profiles, serotype, bacterio-
phage type, antimicrobial susceptibility profiles,
presence and/or type of surface appendages
and virulence factors; reliance on a single para-
meter for characterization should be avoided
wherever possible.
An infectious disease outbreak is defined as

Foodborne and Diarrheal Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia.

20
 INTERPRETATION OF PFGE IN FOODBORNE DISEASE INVESTIGATIONS
a cluster of acute illnesses caused by a pathogen
that are geographically and temporally associ-
ated, and occur in excess of what is usually ex-
pected for that time and place. The smallest
outbreak consists of two cases. In a few in-
stances, where a pathogen or its toxin causes
extremely severe disease that may result in
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death (e.g., botulism), even a single case is con-
sidered and investigated as an outbreak. Only
a small proportion of enteric illnesses manifest
as common source outbreaks; nevertheless,
these outbreaks contribute greatly to the un-
derstanding of the transmission of the patho-
gens causing foodborne and diarrheal diseases,
because they offer the best opportunities to
identify the source of the outbreak by provid-
ing multiple case histories to compare for com-
mon exposure to a specific source or vehicle
(Keene, 1999). Further, the identification of the
Foodborne Pathogens and Disease 2006.3:20-31.
food, water or environmental source of infec-
tion and the institution of appropriate public
health remedial measures (e.g., recall of food,
closing of the water source, closure of an im-
plicated petting zoo) will result in prevention
of additional exposure and disease. When the
source of contamination of an outbreak is de-
termined to be a widely distributed food, the
remedial measures may have long-term impli-
cations for an entire segment of the food in-
dustry and may lead to significant changes in
specific food production and processing proto-
cols for that segment of the food industry (e.g.,
requirements for more thorough cooking of
hamburger patties in fast-food restaurants af-
ter the 1993 Western states Escherichia coli
O157:H7 outbreak). Findings from some out-
break investigations also stimulate further re-
search on the prevalence and persistence of the
pathogen in the vehicle or source of the out-
break that may lead to the development of pre-
vention strategies that may reduce or eliminate
the recurrence of similar contamination events
in the future.
BENEFITS TO PUBLIC HEALTH
Strain identification by subtyping has many
public health benefits. While patient diagnosis
and treatment are seldom impacted by the ad-
ditional information provided by subtyping,
21
subtyping has a profound impact on public
health surveillance, disease cluster identifica-
tion, and outbreak investigations. For example,
the identification of a diarrheal pathogen as Sal-
monella and determining its antimicrobial sus-
ceptibility profile provide adequate informa-
tion to a physician for the treatment of the
patient. However, further characterization of
the clinical Salmonella isolate by determination
of its serotype, bacteriophage type, its resis-
tance to antimicrobial agents, and the determi-
nation of its DNA “fingerprint” are necessary
and essential for public health officials to study
the emergence and disappearance of specific
serotypes, and for timely recognition of newly
evolving strains, particularly those that may be
resistant to treatment by multiple antimicrobial
therapies. Further, serotype and DNA “finger-
print” data enable the detection of disease clus-
ters, particularly those that are geographically
dispersed. Subtyping also assists epidemiolo-
gists in separating outbreak-associated cases
from temporally associated sporadic cases of
disease caused by the same pathogen, thereby
increasing the power of epidemiologic analy-
ses. Once the epidemiologists complete their
analysis of an outbreak and implicate a specific
food as the cause of the outbreak, isolation of
the pathogen from the implicated food and its
characterization by subtyping provide inde-
pendent confirmation of epidemiologic find-
ings.
Subtyping methods may be phenotypic or
genotypic and may vary in discriminating
power from low to very high. Examples of the
phenotyping methods are biotyping, serotyp-
ing, antimicrobial susceptibility profiles, and
bacteriophage typing. Except for bacteriophage
typing, the other phenotyping methods gener-
ally have low discriminating power and are
used primarily as first level subtyping meth-
ods. Among the plethora of DNA-based sub-
typing methods, macrorestriction analysis by
pulsed-field gel electrophoresis (PFGE) has be-
come the gold standard for bacterial pathogen
subtyping during the past ten years because
of its broad applicability, high discriminating
power, and epidemiologic concordance. The
PulseNet network has demonstrated that PFGE
patterns generated using highly standardized
PFGE protocols and their analysis by com-
 22
puter-assisted pattern normalization techniques
results in very high levels of reproducibility of
PFGE patterns within and between laboratories
(Swaminathan et al., 2001).
Despite its many advantages, the interpreta-
tion of PFGE patterns in the context of foodborne
disease epidemiology is not simple and there is
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critical need for guidelines for interpretation of
these data. The widely cited Tenover criteria
(Tenover et al., 1995) were originally developed
as guidelines for the investigation of nosocomial
outbreaks but have been universally applied.
These criteria may not be appropriate for appli-
cation to highly clonal enteric pathogens such as
Salmonella and E. coli. Further, point mutations
were thought to be one of the major contribut-
ing factors to PFGE pattern diversity at the time
the Tenover criteria were developed. However,
Kudva et al. (2002) have demonstrated that the
Foodborne Pathogens and Disease 2006.3:20-31.
PFGE patterns diversity in E. coli O157:H7 is pri-
marily attributable to insertions and deletions
and not to point mutations. Based on our ex-
tensive experience with PFGE pattern analysis
of E. coli O157:H7, Salmonella, Shigella and Liste-
ria monocytogenes, we have developed guidelines
for the interpretation of PFGE patterns of these
enteric pathogens in the context of foodborne
disease surveillance, disease cluster recognition
and outbreak investigations. We hope that these
guidelines will serve to stimulate discussion of
this topic.
INTERPRETATION OF PFGE RESULTS
First consideration: are observed differences real?
Even when a carefully standardized proce-
dure for PFGE is followed, artifacts may occur
which could lead to erroneous conclusions
about the relationship between profiles. It is
therefore important to know the nature of these
artifacts in order to recognize and correct them.
Artifacts in this context are banding patterns
that can not be reproduced on subsequent test-
ing in the same or in a different laboratory.
When the results of the subtyping of strains gen-
erated by a particular method are not repro-
ducible, there are two general explanations, one
relating to the subtyping procedure and the
other relating to the strain in question. The lat-
BARRETT ET AL.
ter depends on the stability of the typing
method, and is simply a reflection of the fact
that bacterial strains change over time. If strains
change quickly enough, different subcultures of
the same strain may show different subtypes
(Iguchi et al., 2002). On the other hand, differ-
ences due to the subtyping procedure usually
manifest as artifacts introduced by small, often
unrecognized differences in the subtyping pro-
cedure. Sometimes these small differences in
procedure manifest as subtle differences in band
separation (e.g., one thick band versus two thin-
ner bands), but with PFGE the most common ar-
tifact is probably incomplete restriction.
When DNA is restricted, the restriction en-
zyme will ideally cleave the DNA every time
it meets its recognition sequence, cutting the
DNA in a well-defined number of pieces of
equally well defined size. If the restriction is in-
complete, some DNA molecules will be com-
pletely restricted whereas others will not be
cleaved at all the recognition sequences. This
will result in the generation of more fragments,
some with the expected size, and some larger
fragments containing one or more restriction
sites that were not cleaved. In a PFGE gel, this
will show up as additional bands, usually at
the top of the gel. However, if the incomplete
restriction is little pronounced, as may be the
case if a restriction site is methylated (McClel-
land et al., 1994), this may result in the addi-
tion of a single or a few extra bands. Bands pre-
sent due to incomplete restriction are typically
of a weaker intensity than their neighbors
(“ghost bands”). The presence of such ghost
bands should always trigger a repeated re-
striction and analysis of the isolate in question.
Another common error causing lack of re-
peatability is working with cultures that are not
pure. If a culture contains more than one strain,
there is obviously a risk of picking different
strains when selecting single colonies for re-
peating subtyping of the same culture. In case
of swarming bacteria such as Campylobacter, it
may be difficult to obtain a pure culture or pick
a single colony. In this case, the subtyping results
will be completely unpredictable and repeated
subtyping of such cultures will almost invariably
lead to different results. Media or culture condi-
tions preventing swarming should be used for
obtaining a pure culture.
 INTERPRETATION OF PFGE IN FOODBORNE DISEASE INVESTIGATIONS
If an organism causing an outbreak is part of
the normal flora of humans, animals, or food,
there is always a risk of overlooking the out-
break strain in a particular specimen if multi-
ple strains are present but only one colony is
investigated. This may be the case with Listeria
monocytogenes, which is widely distributed in
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the environment and in food (Tham et al., 2000,
2002); the solution to this problem is to pick
multiple colonies from the primary cultures for
subtyping. Organisms such as Clostridium per-
fringens, which may be either a normal com-
mensal or cause disease if the strain produces
enterotoxin, present another problem. For such
organisms, only colonies producing entero-
toxin should be selected for further subtyping
in an outbreak setting (Lukinmaa et al., 2002).
Foodborne Pathogens and Disease 2006.3:20-31.
Tenover criteria and sources of variability in
PFGE patterns
In 1995, Tenover et al. published a paper on
the interpretation of PFGE profile differences.
According to the authors, profiles differing
from each other in the position of up to three
bands should be considered closely related and
profiles differing by up to six bands should be
considered possibly related. The reasoning be-
hind this recommendation is that a single ge-
netic event (a point mutation in a restriction
site, a deletion or an insertion) would result in
up to three-band differences. Thus, a three-
band difference would be the result of one ge-
netic event and a six-band difference the result
of two genetic events. A point mutation in a re-
striction site will lead to the loss of that site,
and the two original fragments with the re-
striction site will merge to one larger fragment,
leading to a three-band difference (loss of two
fragments, gain of one). Conversely, if a re-
striction site is gained by a point mutation, a
large fragment is split into two smaller frag-
ments, also resulting in a three-band difference.
Deletions and insertions will show as a change
in the size of one band leading to a two-band
difference (loss of a band of one size, gain of a
band of another). If the insertion/deletion con-
tains a restriction site, additional fragments
may be gained or lost relative to the pattern of
the strain without the insertion/deletion.
Over the past 10 years, the Tenover criteria
23
have proven to be useful, especially when com-
paring profiles of nosocomial pathogens and in
suspected settings of ongoing transmission. For
example, Aucken et al. (2000) compared PFGE
patterns obtained with XbaI digestion of ge-
nomic DNA from 28 unrelated isolates of Ser-
ratia marcescens and 29 isolates from two out-
breaks. Twenty-six of the 28 unrelated isolates
had unique profiles with more than 10-band
differences. Twenty-seven of the 29 outbreak
isolates could be assigned to the correct out-
break based on PFGE results, with most iso-
lates differing by less than three bands. Three
isolates differed by five to seven bands.
As useful as the Tenover criteria have been
in many circumstances, they are clearly not ap-
plicable to all situations. The criteria are very
general and assume that all genomic fragments
are visible in the gel and that the plasmid con-
tent in the strains is stable, at least for the plas-
mids visible in the gel. If these assumptions
were always correct, all profiles would either
be identical or differ from each other in the po-
sition of at least two bands (a change in band
size being interpreted as loss of one band and
gain of another). It is clear that the plasmid con-
tent of bacterial cells is not stable; plasmids are
often gained or lost. PFGE profiles differing
from each other by a single band are common,
at least in foodborne pathogens. Thus, these as-
sumptions are not always sound.
One potential cause of a one band difference
is the presence of an extra plasmid in one of
the strains. Plasmids may be visible in a PFGE
gel, often as intense bands. This intensity may
be due to the presence of multiple copies of the
plasmid versus the single copy of the chromo-
some. If plasmids are digested by the restric-
tion enzyme used, the resulting fragments will
migrate as a function of their size in a PFGE
gel, just like the chromosomal fragments. For
example, in XbaI digested preparations of Sal-
monella Enteritidis, the 57-kb virulence plasmid
is visible in the gel and strains lacking this plas-
mid may differ from strains harboring this
plasmid by just this single band (Buchrieser et
al., 1994; Powell et al., 1994). However, if the
plasmids are not in linear conformation, their
migration is unpredictable. Large undigested
plasmids will typically move very slowly and
may not be visible in a PFGE gel, but they may
 24 BARRETT ET AL.

also be visible almost anywhere in the gel Interpreting differences in the context of
(Buchrieser et al., 1994; Barton et al. 1995). organism diversity
Because of the variability in how plasmids
Optimal interpretation of the differences (or
may migrate, it has been suggested that frag-
lack thereof) between the PFGE patterns of two
ments of a size less than 125 kb should be ex-
isolates depends largely on the variability of
cluded from analysis (Olsen et al., 1994). Even
the organism being typed. Some organisms are
this drastic measure may not solve the prob-
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highly clonal while others demonstrate extreme

lem, however, as plasmids may appear in a
variability. Helicobacter pylori, for example, shows
higher size range than 125 kb, either because
so much diversity that molecular subtyping is
they actually are that large (“megaplasmids”)
used primarily to determine if an infection is new
or because they have not been digested by the
or recurring, and contributes little to the under-
restriction enzyme used and are not in a linear
standing of H. pylori epidemiology (Taylor et al.,
conformation (Barton et al., 1995). It is well-
1995). Even within a genus, species or serotypes
known that plasmids, including megaplas-
mids, are common in foodborne pathogens
(Bradbury et al., 1983; Wachsmuth et al., 1991;
Olsen et al., 1994; Barton et al., 1995; Iteman et
al., 1996; McLauchlin et al., 1997), but the in-
fluence of the plasmid content on the PFGE
Foodborne Pathogens and Disease 2006.3:20-31.

profiles of foodborne pathogens has not been

well characterized.
A second cause of one-band differences in
PFGE patterns is the possibility that one or
more of the fragments generated by deletion,
insertion, or point mutation are so small that
they migrate off the gel. On the other hand, it
should also be kept in mind that even changes
affecting larger fragments will not always be
readily apparent. Only comparatively large
deletions or insertions will be detectable by
PFGE. Band size differences of 1–2% (0.5–1 kb
at the bottom and
10–15 kb at the top of the
gel) are not detectable under most conditions.
It has been shown that changes in PFGE pro-
files of Shiga toxin–producing E. coli O157 are
caused primarily by deletions and insertions
and not by point mutations in the restriction
sites (Kudva et al., 2002). Little is known about
the cause of changes in the PFGE profiles of
other foodborne pathogens. Even if point mu-
tations are common, the rareness of restriction
sites for enzymes used in PFGE would dictate
that only a tiny fraction of mutations would be
expected to occur within a specific restriction
site and thus change the PFGE pattern. It seems
likely that in many, if not most cases, several
genetic events have likely occurred before the
PFGE pattern is visibly altered, and the Ten-
over criteria represent a conservative estimate
FIG. 1. Pulsed-field gel electrophoresis (PFGE) patterns
of the number of genetic events behind changes of S. Baildon (A) and S. Bareilly (B) in the PulseNet USA
in PFGE profiles. until 2000.
 INTERPRETATION OF PFGE IN FOODBORNE DISEASE INVESTIGATIONS
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FIG. 2. Pulsed-field gel electrophoresis (PFGE) patterns
of S. Baildon in the PulseNet database as of 2005.
may vary greatly in genetic diversity. Dendro-
grams of PFGE patterns of two Salmonella
serotypes from the PulseNet database are shown
in Figure 1. As of 2000, S. Baildon isolates were
all highly similar (Fig. 1a), while S. Bareilly iso-
lates were highly diverse (Fig. 1b). Matching
PFGE patterns for isolates of S. Bareilly would
Foodborne Pathogens and Disease 2006.3:20-31.
thus be more likely to indicate epidemiologic re-
latedness than matching patterns for S. Baildon,
while minor differences between S. Baildon pat-
terns would be more significant than the same
differences in S. Bareilly patterns. Recent S. Bail-
don isolates have begun to show more diversity
(Fig. 2), but the primary pattern still constitutes
most of the database. S. Bareilly patterns con-
tinue to be highly diverse.
In addition to the diversity of subtypes of the
organism being tested, one must also consider
the prevalence of the particular subtype or pat-
tern. A recent study in our laboratory found
considerable diversity in S. Heidelberg PFGE
patterns, but 33/60 (55%) apparently unrelated
isolates had the same PFGE pattern (Kubota et
al., 2000). This pattern represented about half
of all S. Heidelberg isolates in our collection
from each time period sampled dating back to
1985. The remaining 27 isolates were divided
among 21 PFGE patterns. In the absence of epi-
demiologic information, one would have to
conclude that isolates sharing one of the less
common patterns are more likely have come
from a common source than isolates sharing the
predominant pattern.
Ongoing transmission versus a point source
outbreak: interpreting differences in
different settings
Time is also a critical factor in interpreting
typing results. The shorter the duration of an
25
outbreak, the less time the outbreak strain has
to undergo mutations that change the PFGE
pattern. Foodborne disease outbreaks often re-
sult from a single contamination event where
all of the patients are exposed to the same con-
taminated food at the same time, and variabil-
ity among isolates from patients is expected to
be minimal. Hospital or community outbreaks,
on the other hand, are frequently prolonged,
with strains being passed from person to per-
son with much more opportunity for muta-
tional changes in PFGE patterns. An outbreak
of Shigella flexneri serotype 2a in Taiwan town-
ship in 1996 illustrates both situations (Chen et
al., 2003).
During the month of August, when the out-
break was identified, all outbreak-related iso-
lates had the same PFGE pattern. By the end of
December, eight variants with differences of
three or fewer bands were detected. By the end
of 2000, a total of 50 variations of the original
pattern had been seen. A similar situation was
reported during a large person-to-person out-
break of S. sonnei in Massachusetts in 1999
(Hackbarth et al., 2000). The outbreak was rec-
ognized in June, when isolates from three cases
were found to have indistinguishable PFGE
patterns. A second pattern had emerged by
July, and by December of that year, 21 differ-
ent PFGE patterns had been seen in isolates
from persons who could be epidemiologically
connected.
In a multistate outbreak of listeriosis in the
United States in 1998, outbreak-associated iso-
lates included variants of those with the major
PFGE pattern isolated from patients and the
epidemiologically implicated food product. Of
108 cases identified as part of this outbreak, 77
had L. monocytogenes with the major outbreak
pattern, 29 were infected with a variant that dif-
fered from the major outbreak strain by the ab-
sence of an 80-kb ApaI restriction fragment, and
two were infected with a variant that differed
by the absence of the 80-kb fragment and the
presence of a 190-kb fragment. Epidemiologic
data strongly associated all three types with the
outbreak. All three types and an additional
variant (containing the 190-kb fragment) were
found in foods obtained from the implicated
food processing facility. The investigators hy-
pothesized that the major strain may have been
 26
a resident of the processing plant for a dura-
tion long enough to produce the genetic vari-
ants (Graves, 2005).
In contrast to these descriptions of ongoing
transmission, outbreaks with a point source
and little secondary transmission are typically
yield isolates with a more limited range of pat-
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terns. Two other Shigella outbreaks illustrate
this point. The first outbreak occurred in Janu-
ary, 2000 and involved a contaminated food
product (bean dip) (CDC, 2000). The subtyping
results were typical of a foodborne (point
source) outbreak. S. sonnei isolates from 25 per-
sons and two foods were available for subtyp-
ing. Isolates from 21 patients and both foods
were indistinguishable by PFGE, and the other
isolates had highly similar PFGE patterns. A
second outbreak occurred in July and August
of 1998, and was due to consumption of cont-
Foodborne Pathogens and Disease 2006.3:20-31.
aminated parsley at restaurants in the United
States and Canada (Naimi et al., 2003). Seventy-
seven percent of the isolates had one of two
closely related PFGE patterns. These outbreaks
illustrate the impact of time and transmission
on PFGE patterns, and emphasize the impor-
tance of the outbreak setting in interpreting
PFGE results.
Lab results do not tell the whole story
Perhaps the most important factor to con-
sider in interpreting PFGE results is context:
how the laboratory results fit together with epi-
demiologic and environmental investigations.
The simple fact that common PFGE patterns ex-
ist for many organisms demonstrates that in-
distinguishable PFGE patterns alone do not
unequivocally demonstrate an epidemiologic
connection. Even if PFGE patterns could prove
that two isolates were the same strain, it is al-
ways possible that two people could have been
infected with the same strain by different
routes.
It is perhaps less obvious, but equally true
that clearly differing patterns do not prove that
isolates are epidemiologically unrelated. At
least four distinct PFGE patterns (differing by
three or more bands) were found among E. coli
O157:H7 isolates from persons who ate at the
same restaurant in Seattle over the course of a
BARRETT ET AL.
few days in 1993 (Jackson et al., 2000). A
case/control study implicated salad bar items,
apparently due to cross-contamination from
raw beef in the restaurant kitchen. If epidemi-
ologists had relied solely on molecular subtyp-
ing data rather than patient interviews and en-
vironmental observations of the restaurant, the
source of this outbreak would likely have been
unidentified.
WATERBORNE OUTBREAKS
OF PATHOGENS USUALLY
ASSOCIATED WITH FOOD
Contaminated drinking water has the
propensity to cause large outbreaks of diar-
rheal disease (Hrudy et al., 2003). Molecular
subtyping may have less utility in the early de-
tection of some outbreaks caused by contami-
nation of potable water supplies with diarrheal
bacterial pathogens if the outbreaks have a
very sharp epidemic curve resulting from large
numbers of people in a geographically local-
ized area being exposed to the infecting
pathogens over a very short window of time.
These outbreaks are easily detected by sharp
increases in the number of patients seeking
medical attention or even by the rapid deple-
tion of over-the-counter anti-diarrheal medica-
tions in stores in a specific area (Hrudy et al.,
2003; CDC, 1999; Mac Kenzie et al., 1994). Nev-
ertheless, molecular subtyping has provided
critical information for the investigation of
waterborne outbreaks and for tracking their
sources.
In an outbreak in Walkerton, Canada, a sin-
gle E. coli O157:H7 strain was isolated from the
case-patients and from the farm that was iden-
tified as the source of contamination. In con-
trast, four SmaI PFGE types of C. jejuni were
isolated from outbreak-associated case-patients
(Clark et al., 2003). The four outbreak-associ-
ated PFGE patterns shared a common back-
bone and differed in the presence or absence of
four DNA fragments in the middle portion of
the DNA “fingerprint.” Another large dual-
pathogen outbreak of waterborne disease oc-
curred in upstate New York following a county
fair in 1999. Once again, E. coli O157 and C. je-
 INTERPRETATION OF PFGE IN FOODBORNE DISEASE INVESTIGATIONS
juni were the two pathogens that were in-
volved. However, in this outbreak, PFGE
analysis of E. coli O157:H7 showed that 65% of
the patient isolates, and 63% of the isolates
from the implicated water source shared the
same PFGE profile. An additional 27% of the
patient isolates were represented by a PFGE
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profile that differed from the major pattern by
two bands. The remaining isolates were also
variants of the major PFGE profile with 1–3-
band differences from the major pattern (Bopp
et al., 2003). These data suggest that the prog-
enitor of the outbreak strain may have per-
sisted on the farm (from which cow manure
was suspected to have leached into the well
that served as the source of water for the county
fair) long enough to diverge into variants ob-
served by PFGE typing. Changes in E. coli
O157:H7 PFGE patterns over time have been
Foodborne Pathogens and Disease 2006.3:20-31.
demonstrated in vitro (Iguchi et al., 2002).
Interestingly, there was much less genetic di-
versity among the C. jejuni isolates (83% of the
isolates had the same PFGE profile) associated
with this outbreak when compared with the
Walkerton, Canada outbreak. It is possible that
the C. jejuni may have contaminated the water
source from a source other than the farm be-
cause no C. jejuni was recovered from any of
the samples taken from that farm.
A waterborne salmonellosis outbreak of S.
Bareilly infections in 95 persons spread across
10 states highlights another aspect of inter-
pretation of PFGE data in the context of epi-
demiologic information. PFGE subtyping of
the S. Bareilly isolates from case-patients re-
vealed numerous PFGE patterns. However,
the epidemiologic investigation revealed that
the infections were associated with drinking
water from private wells and springs in the
southeastern United States and from drinking
bottled water from a bottling operation that
used water from a spring in that area (Epi-Aid
Report 00-61; CDC, unpublished data). Be-
cause S. Bareilly is an amphibian-associated
serotype, investigators hypothesized that mul-
tiple S. Bareilly strains may have persisted in
amphibian populations in and around the dif-
ferent water sources to confound the inter-
pretation of the PFGE patterns associated with
this outbreak.
27
HOW DIFFERENT SHOULD
PFGE PATTERNS BE
CONSIDERED “DIFFERENT”?
This simplest and probably most useful way
to answer this question is to consider patterns
that show any discernable differences to be dif-
ferent patterns. This is the approach that
PulseNet has chosen. In point source outbreaks
(typical of most foodborne outbreaks) the vast
majority of outbreak related isolates display the
same profile (Mølbak et al., 1999; Naimi et al.,
2003). In investigating recognized outbreaks, it
is fairly easy and often useful to designate pat-
terns that differ slightly from the primary out-
break pattern as “subpatterns” or “related pat-
terns.” In surveillance for cluster detection,
such an approach quickly becomes unwieldy
and could easily be misleading. A recent multi-
state outbreak of E. coli O157 illustrates the ra-
tionale of this approach (Gerner-Smidt et al.,
2005).
In the summer of 2002, a cluster of cases of
E. coli O157 infections was detected in Col-
orado. It soon became evident that cases were
occurring in other states. In accordance with es-
tablished PulseNet protocol, only patients with
isolates displaying patterns indistinguishable
from the original outbreak pattern were in-
cluded in the initial case interviews. Thirty-
seven isolates of the outbreak pattern were up-
loaded to the PulseNet server during the
outbreak period. During the same time period,
forty-one isolates of a frequently encountered
pattern that differed from the outbreak pattern
by a single band were also uploaded. The case-
control study pointed to ground beef from one
particular manufacturer as the source of the
outbreak. If the interviews had included cases
infected with the related, frequently encoun-
tered strain in the case definition, the results of
the case-control study might have been less
conclusive. While some of the patients infected
with isolates having the common, non-out-
break pattern may have actually acquired their
infection from eating meat from the implicated
producer, the fact that this pattern was com-
monly seen before the outbreak argues that
most probably did not. Including these patients
in the case interviews would have made iden-
 28
tifying the source of the outbreak much more
difficult. At the same time, epidemiologic evi-
dence supported the inclusion of two siblings
as part of the outbreak, even though their iso-
lates had the frequently encountered (non-out-
break) pattern. While it has been well docu-
mented that a strain may undergo changes
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during an outbreak causing minor differences
in the number or position of the DNA frag-
ments in a PFGE gel (Proctor et al., 2002),
including variants in the case definition
may complicate the interview studies of the
outbreak, especially if one of the variants
represents a common pattern. This example
illustrates the complexities involved in inter-
pretation of outbreak investigation data and
underscores the importance of considering all
the available evidence in assessing strain relat-
edness.
Foodborne Pathogens and Disease 2006.3:20-31.
AVOIDING “FALSE ALARMS” IN
CLUSTER DETECTION
If subtyping is to be useful for cluster detec-
tion and alerting public health authorities to
potential outbreaks, there must be a balance be-
tween reporting too many clusters, many of
which will prove to be “false alarms,” and fail-
ing to identify real outbreak possibilities. One
of the advantages of PulseNet is that the data-
bases are large and contain data many years of
data. These data provides a historical baseline
against which possible clustered cases may be
compared. The cluster detection level for a
common profile should be higher than for a
rare or unique profile to avoid reacting to ac-
cumulations of common profiles that are to be
expected to occur by chance only (“false
alarms”). It also helps to identify organisms
where PFGE is not sufficiently discriminatory
and where alternative subtyping methods
should be applied in case clusters are detected.
While cluster profiles should be compared
with the full database to determine the fre-
quency of the observed pattern, the time factor
should also be considered. The dynamics of
subtypes are constantly changing, and a pat-
tern that is common in the database as a whole
may not have been common for several years.
BARRETT ET AL.
Such a pattern would suggest reacting to a
lower number of reports than if the pattern oc-
curred evenly over the past several years.
RECOMMENDATIONS FOR
INTERPRETING PFGE RESULTS
When any differences in PFGE patterns are
observable, the patterns should be reported as
different. Likewise, patterns that are indistin-
guishable should be reported as such. Indistin-
guishable is a more accurate term than “identi-
cal,” since it implies only that any differences
are not observable under the conditions used.
Interpretation of PFGE results, however, should
include each of the following steps.
1. The gel should be of sufficient quality to be
properly interpreted. A gel that includes
partial digestions or other artifacts, or in
which the bands are not sharp and clear,
should be re-run without attempting to in-
terpret the results. Interpreting a poor qual-
ity gel can be misleading, and can actually
be worse than having no results at all.
2. Consider the diversity of the organism be-
ing tested. Large databases such as those
maintained by PulseNet should provide suf-
ficient data to make reasonable determina-
tions of diversity. If the organism displays
little diversity, one should be cautious in as-
suming that closely related patterns, or even
indistinguishable patterns, indicate a high
likelihood of a common source. PFGE using
a second enzyme may be useful. Other avail-
able laboratory data such as phage type, bio-
chemical profile, antimicrobial susceptibility
profile, or multilocus sequence type should
also be considered. If the organism being
tested shows substantial diversity, one must
still consider whether there are clonal pop-
ulations within a non-clonal organism, as is
the case with S. Heidelberg. If the PFGE pat-
tern in question matches the pattern of a
large clonal group, the overall diversity of
the serotype is of little relevance and addi-
tional data is needed to interpret the PFGE
results. On the other hand, when an organ-
ism demonstrates extreme variability, such
 INTERPRETATION OF PFGE IN FOODBORNE DISEASE INVESTIGATIONS
as that seen with H. pylori, any pattern
matches are probably significant.
3. After establishing the diversity of the or-
ganism and whether or not there are clonal
populations, one should then consider the
outbreak setting. If it appears to be a point
source outbreak without continued trans-
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mission, only very minor differences are
likely to be observed and isolates that differ
by several bands are probably not part of the
outbreak (unless it is a multi-strain out-
break). When there is ongoing transmission,
such as a community outbreak of shigellosis,
more variability should be expected. Isolates
that are temporally or geographically re-
lated, and have PFGE patterns that are more
similar to patterns known to be outbreak-as-
sociated than to other patterns in the data-
base, may be considered potentially out-
Foodborne Pathogens and Disease 2006.3:20-31.
break-related and worth further laboratory
study or epidemiologic investigation.
CONCLUSION
Bacterial subtyping has proven useful for
outbreak investigations and cluster detection
in so many settings over the years that its util-
ity is seldom questioned. However, the inter-
pretation of subtyping results can still prove
problematic. It can not be overemphasized
that proper interpretation can only be made in
context. The reproducibility of the subtyping
method, quality of the PFGE gel, variability of
the organism being subtyped, and prevalence
of a particular pattern are all critical factors.
Perhaps the most important factor, however,
is how the laboratory data fit with the epi-
demiologic and environmental information.
PFGE results alone can not establish an epi-
demiologic connection between isolates. All of
the available information must be considered
in order to interpret subtyping results appro-
priately.
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