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Applied and Environmental Microbiology, April 2001, p. 1619-1627, Vol. 67, No. 4
Food Animal Health and Management Center,
College of Veterinary Medicine, Kansas State University, Manhattan,
Kansas 66506,1 and Division of
Infectious Diseases, New England Medical Center, Boston,
Massachusetts 021112
Received 14 September 2000/Accepted 16 January 2001
Prevalence, antibiotic susceptibility, and genetic diversity were
determined for Escherichia coli O157:H7 isolated over 11 months from four beef cattle feedlots in southwest Kansas. From the
fecal pat (17,050) and environmental (7,134) samples collected, 57 isolates of E. coli O157:H7 were identified by use of
bacterial culture and latex agglutination (C/LA). PCR showed that 26 isolates were eaeA gene positive. Escherichia
coli O157:H7 was identified in at least one of the four
feedlots in 14 of the 16 collections by C/LA and in 9 of 16 collections
by PCR, but consecutive positive collections at a single feedlot were
rare. Overall prevalence in fecal pat samples was low (0.26% by C/LA,
and 0.08% by PCR). No detectable differences in prevalence or
antibiotic resistance were found between isolates collected from home
pens and those from hospital pens, where antibiotic use is high.
Resistant isolates were found for six of the eight antibiotics that
could be used to treat E. coli infections in food
animals, but few isolates were multidrug resistant. The high diversity
of isolates as measured by random amplification of polymorphic DNA and
other characteristics indicates that the majority of isolates were
unique and did not persist at a feedlot, but probably originated from
incoming cattle. The most surprising finding was the low frequency of
virulence markers among E. coli isolates identified
initially by C/LA as E. coli O157:H7. These results
demonstrate that better ways of screening and confirming E.
coli O157:H7 isolates are required for accurate determination
of prevalence.
The low infectious dose and high
virulence of Escherichia coli O157:H7 make human infections
particularly severe and life threatening (7, 28). Shiga
toxins produced by E. coli O157:H7 are the principal
virulence factors responsible for hemorrhagic colitis and hemolytic
uremic syndrome in humans (14, 15). Antibiotic treatment
is contraindicated for human E. coli O157:H7 infections,
because certain antibiotics, such as fluoroquinolones, induce Shiga
toxin-encoding bacteriophages in vivo and lead to increased expression
of Shiga toxin genes (35). Antibiotics also may cause
bacterial lysis, which could increase free Shiga toxin in the
intestinal tract (13, 32, 33; L. E. Wolf, D. W. Acheson, L. L. Lincicome, and G. T. Keusch, VTEC '97: 3rd
Int. Symp. Workshop Shiga Toxin (Verocytotoxin)-Producing E. coli Infect., abstr. V145/III, p. 60, 1997). Even though
antibiotics should not be used therapeutically for E. coli
O157:H7 infections, some evidence indicates that antibiotic resistance
in isolates is increasing (17). Because many human
E. coli O157:H7 infections are acquired from eating
undercooked contaminated beef, it is important to determine if the
organism develops resistance to antibiotics during food animal
production. In addition, it is important to determine if resistant
E. coli O157:H7 is a possible reservoir for spread of
resistance factors to other microorganisms. The Food and Drug Administration, Centers for Disease Control and Prevention, and others
believe that agricultural use of antibiotics accounts for the majority
of increases in antibiotic-resistant human isolates (30).
Antibiotics typically are used at cattle feedlots therapeutically, for
disease prophylaxes, and for growth promotion. Antibiotics also may be
used to treat horses, pets, other livestock, and crops at feedlots. If
an animal becomes sick at a feedlot, usually it is removed from its
resident (home) pen and relocated to a hospital pen, where it is
treated with antibiotics. When the animal is healthy, usually from 3 to
60 days later, it is returned to its home pen. While in the hospital
pen, animals commingle with other animals receiving antibiotics for a
variety of conditions. Therefore, one would expect to find a greater
number of resistant bacterial isolates in hospital pens than in home pens.
Knowing the genetic diversity of E. coli O157:H7 collected
over time could help determine if contamination at a feedlot is due to
bacteria that are transient or indigenous (resident). Transient bacteria can be introduced into the feedlot on arriving cattle; in
ingredients for cattle rations; from contaminated water sources; or by other means, such as other animals (wild or domestic), vehicles, and employees. Escherichia coli O157:H7 shed by animals
might persist for long durations in contaminated soil, water, manure, and feed and be spread to other uninfected animals (9).
Manure management, feed bunk and water trough sanitation, and feed
management all contribute to preventing transient bacteria from
becoming resident. Therefore, if bacteria collected over time are
genetically similar, the feedlot could be contaminated with a resident
strain, whereas a greater genetic diversity could indicate that strains are arriving from a variety of sources. However, LeClerc et al. (21) reported that E. coli O157:H7 strains may
become hypermutable, thereby making them more adaptable to changing
environments and perhaps increasing the organism's genetic diversity.
Genetic fingerprinting is a means by which epidemiologists have traced
back infections to their probable sources. It also has been used to
understand the ecology of E. coli O157:H7 and might be used
as part of hazard analysis and critical control point (HACCP) programs
for producers to reduce on-farm pathogens. Using genetic fingerprinting
techniques, Kudva et al. (20) found multiple strains of
E. coli O157:H7 in a single flock of sheep and showed that a
single animal shed multiple strains simultaneously and that strains
shed by individuals changed over time. Escherichia coli
O157:H7 has been isolated from animal drinking water, animal feed,
flies, and a pigeon at dairy farms in Wisconsin (27). The
majority of isolates collected at these farms had the same genetic
fingerprint. Although various methods are available for genetic
characterization of bacterial isolates, random amplification of
polymorphic DNA (RAPD) has been used successfully in the past for
E. coli O157:H7 (4, 24, 31) and is less costly
and time-consuming than other methods.
Escherichia coli O157:H7 strains typically contain genes
that encode Shiga toxins (stx1 and/or stx2),
attaching-and-effacing proteins such as intimin (eaeA), and
hemolysins (hly) (2, 6, 12, 34). The presence
of a plasmid-encoded enterohemolysin (E-Hly) in E. coli
O157:H7 was first demonstrated in 1989 by Beutin et al.
(3). Despite the presence of common virulence factors, strains of E. coli O157 may differ in their degree of
pathogenicity. Baker et al. (1) found that strains of
human origin were more virulent in gnotobiotic pigs than strains of
bovine origin.
Cattle infected with E. coli O157:H7 show no signs of
disease, and shedding of the organism in feces is sporadic and
difficult to detect. One approach to assess prevalence is by sampling
many feedlots once (USDA:APHIS:VS:Centers for Epidemiology and Animal Health: Escherichia coli O157:H7 shedding by feedlot cattle,
1995; Factors associated with Escherichia coli O157:H7 in
feces of feedlot cattle, 1997 [http: //www.aphis.usda.gov / vs / ceah / cahm / Beef_Feedlot / bffeed.htm]).
Because other studies have found E. coli O157:H7
prevalence to be low at livestock operations, many samples must be
collected to obtain a sufficient number of isolates for analysis.
The major objectives of this study were to determine the prevalence of
E. coli O157:H7 at four beef cattle feedlots by sampling repeatedly and intensively over an 11-month period and to determine if
a difference in antibiotic susceptibility could be detected between
isolates from home pens and those from hospital pens, where antibiotic
use is high. An additional objective was to determine, through use of
RAPD genetic fingerprinting, if the E. coli O157:H7 isolates
were primarily resident or transient.
Sample collection.
Four beef cattle feedlots (designated M,
S, R, and V) in southwest Kansas were studied. Each feedlot was chosen
for inclusion because it was large-scale (>35,000 animal capacity) and
well managed and because pens in the feedlot were used continuously throughout the year. The cattle in the feedlots came from a variety of
sources. Animals at each feedlot are kept in a pen with up to 250 animals for approximately 145 days until they are shipped to slaughter
and another lot of cattle is placed in the pen. During the period of
study, these feedlots reported using antibiotics therapeutically, but
not prophylactically or for growth promotion. Samples were collected at
each feedlot every 3 weeks throughout an 11-month period (16 collections). Typically, 30 home pens and all hospital and buller pens
(pens with steers removed from their home pen because they were mounted
repeatedly by other steers) were sampled at each feedlot during each of
the 16 collections. One teaspoon of feces was collected from each of
five different fresh fecal pats on the pen floor and placed together in
a new zip-lock plastic bag. Six bags were collected from each study pen. Drinking water (50 ml) and rations (50 g) from each pen were sampled at each collection. Standing lagoons at each feedlot and individual ration components at each feedlot's feed mill also were
sampled. Samples were placed in a cooler with frozen cool packs. At the
end of a sampling day, new frozen cool packs were placed in the cooler,
and the cooler was sealed and shipped by next-day delivery to the laboratory.
Selective enrichment and isolation of E. coli
O157:H7.
On arrival at the laboratory, each composite fecal sample
was mixed, and 1 g of feces or feed or 1 ml of water was removed and put into 10 ml of universal pre-enrichment broth (Difco, Inc., Detroit, Mich.) with a 15-µg/ml final concentration of novobiocin (Sigma-Aldrich, St. Louis, Mo.) and incubated at 42°C for 18 to 24 h. Then, the tube was vortexed, and a swab sample was plated onto sorbitol-MacConkey agar. The plate was streaked for isolation and
incubated at 42°C for 18 h.
Identification by C/LA.
Identification by the combination of
bacterial culture and latex agglutination (C/LA) was performed as
follows. After incubation, up to five suspected E. coli
O157:H7 colonies (gray-white) were picked and inoculated into
triple-sugar iron agar slants and incubated at 42°C for 18 to 24 h. After incubation, suspected E. coli isolates (yellow
slant and butt) were plated onto O157 Rainbow agar (Biolog, Inc., Hayward, Calif.) and incubated at 42°C for 18 to 24 h.
Suspected O157 isolates (gray-black) were picked and streaked onto
blood agar plates (BAP), which were incubated at 42°C for 18 to
24 h. Then, isolated colonies were checked for O157 LA (Remel,
Lenexa, Kans.). Any isolates that agglutinated with the O157 latex were checked for H7 agglutination (Remel). If the isolate did not
agglutinate with the H7 latex, the isolate was transferred to another
BAP, incubated at 42°C for 18 to 24 h, and then rechecked for H7
agglutination. If the isolate again did not agglutinate with the H7
latex, the isolate was cultured and checked a third time.
Biochemical confirmation.
Isolates that agglutinated with
the O157 latex were inoculated into an API20E strip (bioMerieux Vitek,
Hazelwood, Mo.) for identification according to the manufacturer's specifications.
Storage.
If the API strip identified the isolate as E. coli, then the isolate was inoculated onto BAP and incubated at
42°C for 18 to 24 h. The isolate was inoculated onto Protect
beads (Key Scientific Products, Round Rock, Tex.) according to the
manufacturer's specifications and frozen at Antibiotic susceptibility testing.
Isolates were streaked
for isolation onto BAP and incubated overnight at 37°C. Following
incubation, an autoinoculator (Sensititre/Alamar AccuMed, Westlake,
Ohio) was used to inoculate the wells of a 96-well plate with bacteria
to a standard cell density. Isolates were inoculated into both a
breakpoint panel with 17 antibiotics and a broth microdilution panel
with 8 antibiotics. The antibiotics selected for the broth
microdilution panel are used commonly at feedlots and are included on
panels used by the National Antimicrobial Resistance Monitoring System
(29). The breakpoint panel was a standard panel
distributed by the manufacturer to test human isolates. The
concentration of each antibiotic was selected by the manufacturer
(Sensititre/Alamar) on the basis of National Committee for Clinical
Laboratory Standards recommendations (23). After
inoculation, the plates were incubated overnight at 37°C. Following
incubation, the plates were read with an automated reader (Sensititre/Alamar AccuMed), and results were generated by use of SAMS
software (Sensititre/Alamar AccuMed).
RAPD primers.
To narrow the search for arbitrary primers
that would be suitable for this study, six primers that had been
discriminatory in E. coli studies by others (22,
31) were used for RAPD. From these, the two primers that had
shown the greatest ability to discriminate among E. coli
O157:H7 isolates in preliminary testing were selected. All primers were
purchased from Integrated DNA, Inc., Coralville, Iowa. The 10-mer
primers used were 1254 (5'CCG CAG CCA A 3') and 1283 (5' GCG ATC CCC A
3'). The four primers that were not discriminatory were HLWL85, OPB17,
1247, and 1290. The E. coli O157:H7 isolates were analyzed
with each of the two primers in at least three independent reactions,
and bands that were clearly and consistently detected were considered to establish a fingerprint for that isolate.
RAPD procedure.
Isolates were grown for 18 to 24 h at
42°C in tubes with 5 ml of brain heart infusion broth. After
incubation, the broth suspension was checked for optical density.
Isolates were diluted with broth to standardize optical density and
centrifuged. Total DNA (chromosomal and plasmid) was extracted by
resuspending the cell pellet in 100 µl of Tris EDTA containing 20%
Chelex. The suspension was incubated at 55°C for 15 min and then
boiled for 7 min. Cell debris and Chelex were removed by
centrifugation. For RAPD profiles of plasmid DNA, isolates were grown
for 18 to 24 h at 42°C in brain heart infusion broth, and
plasmids were extracted with a Qiagen (Valencia, Calif.) plasmid
isolation kit. According to the manufacturer's specifications, this
kit can isolate plasmids that range from 1 to 100 MDa in size. The DNA
concentrations were about 5 ng/µl, as determined by the DNA dipstick
method (Invitrogen, Carlsbad, Calif.). Each PCR mixture consisted of
1× reaction buffer, 1.5 mM MgCl2, 2 µM primer,
200 µM each deoxynucleoside triphosphates (dNTPs), 2.5 U of
Taq polymerase (Fisher, St. Louis, Mo.); and 5 µl of DNA
in a total reaction volume of 50 µl. Amplification was done with a
thermal cycler (model PTC-100; MJ Research,Watertown, Mass.), which was
set for four cycles at 94°C for 4 min, 37°C for 4 min, and 72°C
for 4 min; followed by 30 cycles at 94°C for 1 min, 37°C for 1 min,
and 72°C for 1 min; followed by 1 cycle at 72°C for 10 min.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1619-1627.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Prevalence, Antibiotic Susceptibility, and Diversity of
Escherichia coli O157:H7 Isolates from a
Longitudinal Study of Beef Cattle Feedlots
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C for long-term storage.
Virulence genes. The following primers (New England Medical Center Hospital, Boston, Mass.) were used to analyze the isolates for the presence of virulence genes: stx1 forward primer (AAA TCG CCA TTC GTT GAC TAC TTC T), stx1 reverse primer (TGT CCA TTC TGG CAA CTC GCG ATG CA), stx2 forward primer (CAG TCG TCA CTC ACT GGT TTC ATC A), stx2 reverse primer (GGA TAT TCT CCC CAC TCT GAC ACC), eaeA forward primer (CAG GTC GTC GTG TCT GCT AAA), eaeA reverse primer (TCA GCG TGG TTG GAT CAA CCT), hlyA forward primer (GCA TCA TCA AGC GTA CGT TCC), and hlyA reverse primer (AAT GAG CCA AGC TGG TTA AGC T). The annealing temperatures were 63°C for the stx1 and stx2 primers and 59°C for the eaeA and hlyA genes. Amplification was done with a 94°C denaturation step for 1 min and an annealing step for 1 min, followed by 72°C for 1 min, for 40 cycles, followed by 1 cycle at 72°C for 10 min. The PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining.
Data analysis. All data were entered into computer spreadsheets (Microsoft Excel, Bellevue, Wash.) and then converted to SAS (26) data sets, and tables were constructed to report prevalence data. For this study, the sampling unit was the pen and the population of inference comprised pens at large-scale beef cattle feedlots. Prevalence was computed as the number of pens with E. coli O157:H7 divided by the number of pens sampled for collection and feedlot combinations.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Prevalence.
From 24,184 samples collected, 45 samples were
positive (0.19%). Multiple colonies were taken from each sample, and
57 total isolates of E. coli O157:H7 were identified by use
of C/LA (Table 1). Forty-four of the
positive samples were fecal pat samples (Table
2), and one was a water trough sample
(3,186 collected). Of 2,871 ration samples, 581 ingredient samples, 312 lagoon water samples, 24 horse fecal samples, and 160 fecal pat samples
from temporary holding pens at feedlot R, none tested positive for E. coli O157:H7.
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Antibiotic susceptibility.
Some isolates were resistant to
antibiotics not approved for feedlot animals (Table
3). Resistant isolates were found for six
of the eight (86%) antibiotics that could be used to treat E. coli infections in feedlot animals. All 57 isolates had
intermediate resistance to the other two approved antibiotics. All
isolates were resistant to tilmicosin, an antibiotic that is used in
feed at many cattle feedlots. All but one isolate was susceptible to gentamicin that has been used for cattle. However, its use in feedlot
cattle is now prohibited in the United States, and there is a voluntary
moratorium on its extra-label use in cattle. Most isolates were
susceptible to other antibiotics used at feedlots, such as
trimethoprim-sulfamethoxazole and ceftiofur. All isolates were
susceptible to ciprofloxacin, an antibiotic used widely in human
medicine and similar to enrofloxacin, which recently has been approved
for use in cattle. All isolates were resistant to antibiotics that do
not target E. coli, except tylosin, to which all 57 isolates
had intermediate susceptibility.
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Isolates with multiple resistance.
Most isolates were not
multiresistant (Table 4). Some isolates
with multiple resistance were resistant to antibiotics that are not
approved for use in feedlot animals. Of the isolates that were
resistant to five or more antibiotics, four were from hospital pens,
three were from home pens, and one was from a buller pen; none were
from the same sample. Three of the eight multiply-resistant isolates
were from one collection time, but from two feedlots. All were
collected in spring from three feedlots.
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RAPD profiles of total DNA.
Of the 57 isolates, 54 were
characterized by RAPD profiles of total DNA, and 16 unique patterns
were identified (Fig. 1 and Table 1).
Most isolates were either RAPD pattern 1 or RAPD pattern 2 (Table
5) and were found at all four feedlots.
The remaining 14 RAPD patterns were identified only once. Clusters of
unique RAPD patterns distinguished the feedlots from one another.
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RAPD profiles of plasmid DNA. Similar diversity (16 patterns) was found in the RAPD profiles of plasmid DNA isolated from the 54 E. coli O157:H7 isolates tested (Table 1).
Virulence genes. The PCR showed that more than half the isolates did not have the virulence genes stx1, stx2, eaeA, and hlyA (Table 1). Of the 26 isolates that had at least one of the stx genes, 38.5% had both genes, and 57.7% had only stx2. One isolate (3.8%) had only stx1. All 26 isolates contained both the eaeA gene and the hlyA gene.
Overall diversity. When all combinations of virulence factors, DNA patterns, and antibiotic resistance were considered, even greater diversity was found among the isolates. From samples in which two isolates were picked, neither isolate had the same RAPD patterns of total DNA and plasmid DNA. One sample had five isolates that were characterized. Two of these had the same RAPD patterns of total DNA and plasmid DNA and the same virulence genes, but were different in their antibiotic profiles. When all measures that were used to characterize the diversity of the isolates were considered, all but 8 of the 57 isolates were unique.
Comparison of all isolates with PCR-confirmed isolates. We found that 31 of the 57 isolates did not have either the eaeA gene or one or both of the stx genes. This afforded the opportunity to compare non-type-specific E. coli with E. coli O157:H7. The salient difference between the two confirmation methods was a general halving of the culture prevalence rates.
When only the PCR-confirmed isolates were considered, overall prevalence was reduced to 0.08%. One isolate from the single positive (by C/LA) water sample was confirmed by PCR. The trend of hospital pens having a higher prevalence than home pens was not detectable when only PCR-confirmed isolates were analyzed (Table 2). The frequency of finding at least one positive feedlot at a collection dropped from 14 to 9 of 16 collections. Although feedlot prevalence was lower at each feedlot, the feedlot prevalence of PCR-confirmed E. coli O157:H7 was still lower at feedlot M (6.3%) than at the other feedlots (V, 18.8%; R, 18.8%; and S, 37.5%). Only at feedlot S was E. coli O157:H7 found from consecutive collections, where it was isolated from two consecutive collections 3 weeks apart. Although the number was fewer, the PCR-confirmed isolates were resistant to the same antibiotics as all isolates (Table 3). Only two of the nine colonies that were resistant to five or more antibiotics had the eaeA gene (Table 4). When only the 26 isolates with the eaeA gene were considered (Table 1), five RAPD patterns of total DNA were identified (Table 5). These PCR-confirmed isolates included multiple picks from a total of 20 samples. Of those samples with multiple isolates, none had the same combinations of RAPD patterns, virulence genes, or antimicrobial resistance pattern.| |
DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Prevalence. Prevalence of E. coli O157:H7 was low at southwest Kansas cattle feedlots sampled intensively over time. Had only one isolate instead of up to five been selected from each culture plate, the prevalence would have been considerably lower, illustrating the importance of selecting multiple isolates from a plate. Improved methodology, such as immunomagnetic separation (5), might have increased our prevalence estimates, but it is costly and time-consuming and increases sensitivity only when the organism is present in very low concentrations. However, overestimation of prevalence (false positives) can result from using current methods such as polyclonal LA.
Others have reported similar low prevalence rates in cattle (8, 10, 11, 25, 28), but higher prevalence rates have been reported recently by Keen and Elder (16). That study used a new antigen capture test to screen multiple isolates from a sample before further testing, effectively increasing the probability of selecting a positive isolate.Antibiotic susceptibility. We expected to observe a difference in the antibiograms of isolates collected from home pens versus hospital pens, where convalescent animals had been treated recently with antibiotics. Because we had so few isolates, we were not able to detect a difference statistically.
We expected that most isolates would be resistant to antibiotics that are used heavily at feedlots, but that was not the case. For example, less than half of the isolates were resistant to tetracycline, which was one of the most heavily used antibiotics on feedlots. No isolate was susceptible to newer drugs (e.g., florfenicol, spectinomycin, and rifampin) that are used at feedlots. Prudent use of these newer antibiotics will be necessary to retain their efficacy and for resistant bacterial populations to revert to a susceptible phenotype. We expected isolates from cattle feedlots to be susceptible to antibiotics not approved for feedlot animals. However, some isolates were resistant to cefazolin and cephalothin. Perhaps the organisms had acquired resistance from other organisms, were coselected through genetic linkage of resistance factors, or were resistant to antibiotics in the same class. The resistant organisms also could have been of human origin or could be naturally resistant to the antibiotics. When reporting resistance patterns, researchers need to consider whether the antibiotic targets the organism. If it does not, resistance would not be associated with use or misuse of the antibiotic. Current concern about the increase of antibiotic resistance can be mitigated partially when the antimicrobial agent-bacterium combination is considered. For example, the data reported here could be interpreted to mean that E. coli O157:H7 is a public health concern because it is highly resistant to antibiotics such as vancomycin. Because this antibiotic does not target this organism and never would be used as treatment for an E. coli O157:H7 infection, this interpretation is incorrect. Before broad statements are made about an increase in antibiotic resistance because of antibiotic use, the antibiotics considered should target the organism, and evidence should be provided that use of antibiotics has caused this increase in antibiotic resistance. The combination of resistance and whether the antibiotic targets the organism also must be addressed when reporting that organisms are multidrug resistant. Isolates in this study with multiple resistances were resistant to all but two antibiotics (cefazolin and gentamicin) that target E. coli and are approved for use in feedlot animals. Why are isolates resistant when cattle are not treated for an E. coli infection? Even though antibiotic treatment is not used for E. coli O157:H7 infections in humans or food animals, we found that a third of the isolates were resistant to one or more antibiotics. One likely explanation for this resistance is the relative ease with which resistance factors are exchanged among promiscuous bacteria (21). Another possibility is that cattle are being treated with antibiotics for other conditions, thereby selecting for resistant populations of E. coli.Genetic diversity. The surprisingly high degree of genetic diversity indicates that E. coli O157:H7 probably was brought into these feedlots on the hide or in the feces of incoming cattle. Although some RAPD patterns of total DNA were unique to a feedlot, their infrequent occurrence does not support the hypothesis that strains are resident, but rather indicates that they were unique to incoming cattle. Some patterns were present in isolates at all feedlots and occurred with higher frequency, suggesting that they were resident strains. However, because of our sampling intensity, if they were resident strains, they would have been recovered at more collections. Isolates with common RAPD patterns of total DNA did not have the same RAPD profiles of plasmid DNA or virulence markers, further indicating the diversity of the organism and that the isolates were not resident strains.
The high diversity observed may make epidemiological traceback investigations of human infections to feedlots difficult. The organism's rare and sporadic occurrence makes it a poor choice as a sentinel organism for HACCP monitoring programs.Virulence genes. The presence of virulence markers has been used extensively in hybridization and PCR-based methods of detecting E. coli O157:H7. Some investigators have reported differences in virulence between E. coli O157:H7 strains recovered from humans and strains isolated from animals (18). If not all virulence markers are tested, then the molecular methods of detection might fail to identify the organism or to distinguish diversity among strains. Production of Shiga toxin may be higher in isolates derived from hospital pens in our study, and this testing is under way. Studies that do not test for virulence markers may overreport E. coli O157:H7 prevalence. For instance, commonly used polyclonal antibody tests may result in false positives (19). Conversely, some detection methods that require an isolate may underreport E. coli O157:H7 prevalence because of a lack of sensitivity or specificity.
Comparison of all isolates with PCR-confirmed isolates. Research laboratories typically have used C/LA as the means of identifying E. coli O157:H7 when an isolate is required for further characterization, such as antibiotic susceptibility testing. The results presented here show that this methodology results in more than 50% false positives. Even when the same isolates are compared by two different PCR methods, different results can occur. The results from our PCR-confirmed isolates do not agree precisely (82% agreement) with those for the same isolates tested by Kimura et al. (19). Possible reasons for this discrepancy are the different primers used for each test, the difference in test types, and the laboratory-to-laboratory variation. These results illustrate the variability that can occur in results when different methods and laboratories are used to identify and characterize E. coli O157:H7. Nevertheless, genetic confirmation of isolates is highly recommended to increase the accuracy of all future prevalence studies.
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ACKNOWLEDGMENTS |
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United States Department of Agriculture Cooperative State Research Education, and Extension Service grant 95-37201-2127, Food and Drug Administration grant FD-U-001574, and National Institutes of Health grant AI 39067 supported this work.
We thank Lori Helmle, Christy Davison, and Matthew Selee for valuable field collection support. We also thank Mike Hornback, Aaron Carman, David Stuever, Dusty Woods, Thao Ngo, Ramona Chitrakar, and Christa Irwin for skillful technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Food Animal Health and Management Center, College of Veterinary Medicine, Kansas State University, 1800 Denison Ave., Manhattan, KS 66506. Phone: (785) 532-4262. Fax: (785) 532-4288. E-mail: galland{at}vet.ksu.edu.
This is contribution no. 01-108-J from the Kansas Agricultural
Experiment Station.
Present address: Department of Microbiology, College of Veterinary
Medicine and Biomedical Sciences, Colorado State University, Fort
Collins, CO 80523.
§ Present address: Division of Biological Sciences, Emporia State University, Emporia, KS 66801.
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Top
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Discussion
References
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Applied and Environmental Microbiology, April 2001, p. 1619-1627, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1619-1627.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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