APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2009, p. 5927–5937
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 18
Escherichia coli O157:H7 Strains That Persist in Feedlot Cattle Are
Genetically Related and Demonstrate an Enhanced Ability To
Adhere to Intestinal Epithelial Cells?
Brandon A. Carlson,1Kendra K. Nightingale,1Gary L. Mason,2John R. Ruby,3W. Travis Choat,4
Guy H. Loneragan,5Gary C. Smith,1John N. Sofos,1and Keith E. Belk1*
Center for Meat Safety & Quality, Department of Animal Sciences, Colorado State University, Fort Collins, Colorado 80523-11711;
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523-16192;
JBS Packerland Inc., Green Bay, Wisconsin 543113; Elanco Animal Health, Greenfield, Indiana 461404; and
Feedlot Research Group, West Texas A&M University, Canyon, Texas 79016-00015
Received 28 April 2009/Accepted 10 July 2009
A longitudinal study was conducted to investigate the nature of Escherichia coli O157:H7 colonization of
feedlot cattle over the final 100 to 110 days of finishing. Rectal fecal grab samples were collected from an initial
sample population of 788 steers every 20 to 22 days and microbiologically analyzed to detect E. coli O157:H7.
The identities of presumptive colonies were confirmed using a multiplex PCR assay that screened for gene
fragments unique to E. coli O157:H7 (rfbE and fliCh7) and other key virulence genes (eae, stx1, and stx2).
Animals were classified as having persistent shedding (PS), transient shedding (TS), or nonshedding (NS)
status if they consecutively shed the same E. coli O157:H7 genotype (based on the multiplex PCR profile),
exhibited variable E. coli O157 shedding, or never shed morphologically typical E. coli O157, respectively.
Overall, 1.0% and 1.4% of steers were classified as PS and NS animals, respectively. Characterization of 132
E. coli O157:H7 isolates from PS and TS animals by pulsed-field gel electrophoresis (PFGE) typing yielded 32
unique PFGE types. One predominant PFGE type accounted for 53% of all isolates characterized and persisted
in cattle throughout the study. Isolates belonging to this predominant and persistent PFGE type demonstrated
an enhanced (P < 0.0001) ability to adhere to Caco-2 human intestinal epithelial cells compared to isolates
belonging to less common PFGE types but exhibited equal virulence expression. Interestingly, the attachment
efficacy decreased as the genetic divergence from the predominant and persistent subtype increased. Our data
support the hypothesis that certain E. coli O157:H7 strains persist in feedlot cattle, which may be partially
explained by an enhanced ability to colonize the intestinal epithelium.
Escherichia coli serotype O157:H7 was first linked to human
illness in the early 1980s, when it was determined to cause
severe abdominal pain with initially watery diarrhea that pro-
gressed to grossly bloody diarrhea accompanied by little or no
fever (42). Initially, E. coli O157:H7 can cause nonbloody di-
arrhea through attachment to, and subsequent destruction of,
intestinal microvilli (24). In addition to microvillus damage,
serious health complications can arise due to the ability of E.
coli O157:H7 to produce Shiga toxins (Stx1 and Stx2). Shiga
toxins are very potent cytotoxins that are absorbed into the
intestinal microvasculature and initiate apoptosis of vascular
epithelium, resulting in hemorrhagic colitis (41). Persistent
uptake of these toxins may lead to more severe manifestations
of disease, such as hemolytic-uremic syndrome, which may
ultimately result in kidney failure (24). Most recent estimates
have identified E. coli O157:H7 as the cause of at least 70,000
cases of food-borne illness annually in the United States, and
in 4% of cases life-threatening hemolytic-uremic syndrome
develops (37). Epidemiological studies have implicated the
consumption of meat, dairy products, produce, and water con-
taminated by animal feces, as well as person-to-person contact
and direct contact with farm animals or their environment, as
routes of E. coli O157:H7 transmission leading to human ill-
It is generally accepted that cattle and other animals are the
major reservoir of E. coli O157:H7, but it is still not clear if
animals are colonized for prolonged periods with E. coli
O157:H7 or if they transiently shed this organism following
repeated exposure to it through ingestion of contaminated
feedstuffs or water or through exposure to other contaminated
environmental sources. Based on results of numerous epide-
miological studies (4, 6, 21, 30, 32), the prevalence of E. coli
O157:H7 in feedlot cattle is highly variable and can range from
less than 1% to 80%. Several other studies (7, 8, 23) have
found evidence of persistent E. coli O157:H7 colonization in
individual cattle, supporting the hypothesis that at least some
animals are susceptible to persistent E. coli O157:H7 coloni-
zation. Multiple experimental inoculation studies (15, 23, 39,
46) showed that E. coli O157:H7 persists in the bovine gastro-
intestinal (GI) tract for at least 14 days up to 140 days postin-
fection. Studies have implicated the lower GI tract and specif-
ically the recto-anal junction (RAJ) as the major location of E.
coli O157:H7 colonization and proliferation (9, 12, 23, 39);
however, this organism also can be found throughout the bo-
vine GI tract (7, 8, 31, 40, 54).
It stands to reason that if the E. coli O157:H7 prevalence in
* Corresponding author. Mailing address: Center for Meat Safety &
Quality, Department of Animal Sciences, Colorado State University, 7C
(970) 491-0278. E-mail: Keith.Belk@ColoState.edu.
?Published ahead of print on 17 July 2009.
cattle presented for harvest were reduced, there would be a
decrease in the probability of beef product contamination, if
good manufacturing procedures were used. Although there is
consensus concerning the importance of preharvest pathogen
mitigation and its role in minimizing entry of E. coli O157:H7
into harvest facilities, there is disagreement about the signifi-
cance of “supershedders” (animals that excrete large quantities
of a pathogen for various amounts of time) for E. coli O157:H7
transmission dynamics at the preharvest level (12, 34, 35, 39).
Utilizing statistical modeling, researchers have estimated that,
on average, the prevalence of “supershedders” in a population
is 4% and that these animals excrete 50 times more E. coli
O157:H7 than other animals colonized by this organism (34).
Additionally, the same researchers suggested that approxi-
mately 80% of E. coli O157:H7 transmission is generated by a
few “supershedders” (35).
Research by our group discovered a unique association be-
tween E. coli O157:H7 prevalence in pen floor fecal pats and
carcass contamination by this pathogen (57). When the prev-
alence in fecal pats from a pen floor exceeded 20%, carcasses
of animals from the pen had E. coli O157:H7 prevalence values
of 14.3, 2.9, and 0.7% before evisceration, after evisceration,
and after final intervention, respectively. However, when the
prevalence in pen floor fecal pats was less than 20%, the
preeviscerated carcass prevalence value was 6.3%, and there
was no detectable E. coli O157:H7 contamination of carcass
samples after evisceration and after final intervention (57).
Thus, we hypothesize that animals which persistently excrete
normal levels of E. coli O157:H7 over prolonged periods (per-
sistent shedders [PS]) rather than animals that periodically
shed abnormally high levels (supershedders) are the most sig-
nificant source of E. coli O157:H7 contamination in the food
continuum. Although previous studies suggested that cattle
may be persistently colonized by E. coli O157:H7 and shed this
organism in their feces for prolonged periods, molecular sub-
typing data are required to further investigate whether cattle
are persistently colonized by the same strain (i.e., molecular
subtype) or if they are repeatedly exposed to different strains
through contaminated feedstuffs, water, or other environmen-
tal sources. Thus, the objectives of this study were to determine
if naturally colonized feedlot cattle persistently shed E. coli
O157:H7, using combined cultural microbiological analyses,
molecular subtyping approaches, and in vitro virulence pheno-
type assays to probe the factors (agent, host, environment, or a
combination of these factors) that contribute to the complex
ecology of E. coli O157:H7 persistence at the preharvest level.
MATERIALS AND METHODS
Study design. Holstein steers from different calf farms in Wisconsin (n ? 788)
consuming a high-concentrate finishing ration at a commercial feedlot in eastern
Kansas (all research protocols were reviewed and approved by the Colorado
State University Animal Care and Use Committee [approval 05-233A-01]) that
had never been exposed to directly fed antimicrobials (e.g., Bovamine) were
enrolled in the study. The steers were housed in five pens; three of the pens
shared fence lines, and the two remaining pens were independently located in
other areas of the feedlot. All animals were fed the same finishing diet during the
sampling period. Animals were permanently removed from the study population
following treatment for any clinical illness.
Rectal fecal grab samples were collected every 20 to 22 days during the final
100 to 110 days (June to October) of the finishing period. Every animal was
sampled during the first two collection periods to establish its E. coli O157:H7
shedding status along with the prevalence of shedding in the population of
feedlot cattle studied (Table 1). Animals whose E. coli O157:H7 shedding status
varied during the first two sample collections were excluded from further sample
collection as the focus of this study was to further investigate the PS status. The
four remaining sample collections focused on identification of animals that were
either consecutively E. coli O157:H7 positive or consecutively E. coli O157
negative as determined using the results for each previous sample collection.
Additionally, a random subset of animals whose E. coli O157:H7 shedding status
varied was included during each of the final four sample collections to determine
if these animals reverted back to their original shedding patterns.
At every predetermined collection period, steers were processed through con-
ventional processing facilities, where rectal fecal grab samples were collected
from each targeted animal. Feces were transferred to a sterile Whirl-Pak bag
(Nasco, Modesto, CA) and subsequently placed in a cooler with ice packs.
Following sampling, the fecal samples were transported to the Pathogen Reduc-
tion Laboratory of the Center for Meat Safety & Quality at Colorado State
University (Fort Collins, CO), where they were stored at 4°C until a microbio-
logical analysis was performed (within 48 h after collection).
Analysis of fecal E. coli O157:H7. Fecal samples (10 g) were enriched using
procedures outlined by Barkocy-Gallagher et al. (5). Following incubation, fecal
slurries were stored at 4°C until they were subjected to immunomagnetic bead
separation. Immunomagnetic bead separation was performed as described by
Barkocy-Gallagher et al. (3), and ultimately 50 ?l of each sample was plated onto
Rainbow agar (Biolog Inc., Hayward, CA) supplemented with 10 mg/liter of
novobiocin (Sigma-Aldrich, St. Louis, MO) and 0.8 mg/liter of potassium tellu-
rite (Sigma), as well as sorbitol MacConkey agar (Becton, Dickinson and Com-
pany, Sparks, MD) supplemented with 20 mg/liter of novobiocin and 2.5 mg/liter
of potassium tellurite (mSMAC). Rainbow plates were incubated for 24 ? 2 h at
37°C, and mSMAC plates were incubated for 36 ? 2 h at 37°C. After incubation,
up to three colonies displaying E. coli O157:H7 morphology were selected for
each medium and initially screened for the O157 antigen using the RIM E. coli
O157:H7 latex agglutination test (Remel, Lenexa, KS). All agglutination-positive
colonies were cultured in 5 ml of tryptic soy broth for 24 ? 2 h at 37°C, streaked
onto mSMAC, and incubated for 36 ? 2 h at 37°C to determine purity.
Presumptive colonies were confirmed to be E. coli O157:H7 colonies by a
multiplex PCR assay performed using a 96-well plate format and 25-?l reaction
mixtures that included a primer master mixture containing forward and reverse
primers at concentrations specified previously (25) to amplify rfbE (which en-
codes the O157 antigen), fliCh7(which encodes the H7 antigen), eae (which
encodes intimin), stx1(which encodes Shiga toxin 1), and stx2(which encodes
Shiga toxin 2), GoTaq Green master mixture (Promega, Madison, WI), nuclease-
free water, and a DNA template. The PCR cycling conditions outlined by Hu et
al. (25) were used. PCR products were separated by electrophoresis in 2%
agarose gels, stained with ethidium bromide, and visualized with UV illumina-
tion. Multiplex PCR profiles were assigned based on the presence of each
targeted gene. Isolates in which amplicons corresponding to rfbE, fliCh7, and at
least one stx gene were detected were designated E. coli O157:H7 isolates. For
the purposes of this study, we chose to follow up with animals that consecutively
shed the most common E. coli O157:H7 genotype (indicated by the presence of
all five genes, as determined by multiplex PCR [Table 1]). Up to three E. coli
O157:H7 isolates from each positive fecal sample were stored in 15% glycerol at
?80°C for subsequent further characterization.
Enumeration of E. coli O157:H7 PS animals. A five-tube most-probable-
number MPN assay (19) was used to enumerate E. coli O157:H7 in fecal samples
TABLE 1. Distribution of E. coli O157 virulence genes among
animals during the first two antemortem collection periods
rfb fliCh7eae stx1stx2
rfb eae stx1stx2
arfb encodes the O157 antigen, fliCh7encodes the H7 antigen, eae encodes
intimin, stx1encodes Shiga toxin 1, and stx2encodes Shiga toxin 2.
bAll 788 animals were sampled during collections 1 and 2.
5928 CARLSON ET AL.APPL. ENVIRON. MICROBIOL.
collected from PS animals just before slaughter to determine whether these
animals excreted elevated quantities of E. coli O157:H7 in their feces. A 10-g
aliquot of each fecal sample was combined with 90 ml of Butterfield’s phosphate
buffer (BPB) (Becton) and pummeled in a stomacher for 2 min. Three 1:10 serial
dilutions were prepared from each BPB sample. From each of a sample’s three
serial BPB dilutions, 1 ml was removed and added to five different tubes of lauryl
tryptose broth (LTB) (Becton), resulting in a total of 15 tubes for each sample.
The inoculated LTB tubes were incubated for 24 h at 37°C. After incubation, the
contents of all turbid LTB tubes were streaked on mSMAC and appropriately
incubated, and the identities of morphologically typical colonies were confirmed
by performing multiplex PCR as previously described.
GI tissue and content samples. All animals identified as PS (n ? 8) and
nonshedders (NS) (n ? 11), as well as a subsample of animals identified as
transient shedders (TS) (n ? 18), were harvested at a commercial facility in the
upper Midwest. The entire GI tract (esophagus, reticulum, rumen, omasum,
abomasum, gall bladder, small intestine, large intestine, colon, and bung) was
collected from each animal and transported to a vacant area of the facility to
allow sample collection. Additionally, the liver of each animal was examined for
the presence of abscesses. Samples of tissue and contents were collected asep-
tically from the reticulum, rumen, omasum, abomasum, duodenum (proximal to
the anterior side of the first loop), ileocecal valve, distal colon (?60 cm proximal
to the anus), RAJ, and two mesenteric lymph nodes (at a position ?30 cm
proximal to the anterior root of the mesentery and the ileal-cecal colic node)
(only tissue samples were collected from lymph nodes) for microbiological
analysis. Tissue samples were washed with sterile phosphate-buffered saline
(PBS) with 0.05% Tween 20 (Sigma) to remove visible organic matter before
they were placed into a Whirl-Pak bag (Nasco). All microbial samples were
placed in ice pack-filled coolers and shipped to the Pathogen Reduction
Laboratory of the Center for Meat Safety & Quality at Colorado State
University (Fort Collins, CO).
GI tissue and content samples were analyzed microbiologically as described
above. Ten-gram aliquots of the epithelial layer of each tissue were aseptically
removed and placed in a Whirl-Pak bag containing 90 ml of phosphate-buffered
tryptic soy broth (5). Lymph nodes were first aseptically trimmed to remove
excess adipose tissue, and a 10-g aliquot of each tissue sample was placed in a
Whirl-Pak bag containing 90 ml of phosphate-buffered tryptic soy broth. All
tissue samples were pummeled (IUL Instruments, Barcelona, Spain) for 2 min
and processed as described above. Additionally, pH measurements were ob-
tained by preparing an additional 1:10 dilution of each GI content sample with
distilled water, pummeling (IUL Instruments) the preparation for 2 min, and
submerging a glass pH electrode (Denver Instruments, Arvada, CO) into the
GI tissue samples from selected PS (n ? 3) and NS (n ? 3) animals were fixed
in 4% paraformaldehyde and shipped to the Colorado State University Pathol-
ogy Diagnostic Laboratory, where they were embedded in paraffin using an
automated tissue processor, 5-?m sections were cut with a microtome, and the
sections were stained with hematoxylin and eosin. Histopathological evaluation
and characterization were performed by a trained pathologist who was blinded
with respect to sample identification information.
PFGE. Pulsed-field gel electrophoresis (PFGE) typing of E. coli O157:H7
isolates was performed using the standardized Centers for Disease Control and
Prevention PulseNet protocol (10). At least one isolate from all six sample
collections for each PS animal and representative isolates from TS animals were
selected for characterization by PFGE. All isolates were previously confirmed to
be E. coli O157:H7 isolates using the eae, stx1, and stx2genes before PFGE
analysis. Briefly, isolates were grown on tryptic soy agar (Becton) plates and
incubated at 37°C for 18 h. Bacterial cultures were imbedded in 1% agarose
(SeaKem Gold Agarose; Cambrex BioScience Rockland, Inc., Rockland, ME),
lysed, washed, and digested with XbaI overnight at 37°C. Restricted agarose
plugs were then placed into 1% agarose gels and electrophoresed with a CHEF
Mapper XA (Bio-Rad Laboratories, Hercules, CA) for 21 h with switch times of
2.16 s to 54.17 s. XbaI-digested Salmonella enterica serovar Braenderup (H9812)
DNA was used as a reference size standard (26). Agarose gels were stained with
ethidium bromide, and the resulting images were captured with a FOTO/Analyst
Investigator system (FOTODYNE, Inc., Hartland, WI). PFGE patterns then
were analyzed and compared using the Applied Maths Bionumerics software
package (v3.5; Applied Maths, Saint-Matins-Latem, Belgium). Similarity clus-
tering analyses were performed with Bionumerics software using the un-
weighted-pair group matching algorithm and the Dice correlation coefficient
Cell attachment assay. The attachment efficiencies of E. coli O157:H7 isolates
representing the predominant PFGE subtype were compared to those of the E.
coli O157:H7 isolates representing the more genetically diverse PFGE subtypes
by performing attachment assays using the Caco-2 human intestinal epithelial
cell line. Up to 10 antemortem fecal isolates representing each of the following
categories were selected for characterization by this Caco-2 attachment assay: (i)
the dominant PFGE subtype, (ii) subtypes closely related to the dominant sub-
type, (iii) subtypes possibly related to the dominant subtype, and (iv) subtypes
distantly related to the dominant subtype (based on the criteria defined previ-
ously ). Caco-2 cells were seeded into 24-well flat-bottom plates (Corning
Inc., Corning, NY) at a density of 1 ? 105cells/well in Dulbecco’s modified
Eagle’s medium (Gibco, Grand Island, NY) containing 20% heat-inactivated
fetal bovine serum (Gibco) without antibiotics and grown to confluence (approx-
imately 72 h). E. coli O157:H7 overnight cultures were prepared by inoculating
a colony isolated in a single well into a 10-ml tube containing brain heart infusion
(BHI) (Becton) broth and incubating the tube at 37°C for 12 to 18 h without
shaking. Overnight E. coli O157:H7 cultures (1 ml) were pelleted by centrifuga-
tion (11,337 ? g, 5 min) and reconstituted in 1 ml of PBS. Confluent Caco-2
monolayers were infected with approximately 2 ? 107E. coli O157:H7 cells/well.
After infection for 3 h at 37°C, nonadherent bacteria were removed by washing
preparations three times with PBS. Caco-2 cells were lysed by adding 0.5 ml of
ice-cold sterile ultrapure water and vigorous pipetting, followed by vortexing of
the cell suspension. Adherent E. coli O157:H7 cells, along with the cells in
overnight E. coli O157:H7 cultures, were enumerated by spread plating appro-
priate serial dilutions onto BHI medium plates, in duplicate. The BHI medium
plates were incubated at 37°C for 24 h, and the resultant CFU were enumerated.
The attachment efficiency of each E. coli O157:H7 isolate was expressed as a
percentage based on the initial inoculum that was recovered as adherent E. coli
O157:H7 cells. The attachment efficiency of each isolate was measured in dupli-
cate wells in at least three independent experiments.
Statistical analysis. The GI content pH data were analyzed with PROC
MIXED of SAS (version 9.3; SAS Institute, Cary, NC). Analysis of variance
techniques were employed to determine if there were differences (P ? 0.05)
among main effects, including GI tissue location and shedding status, as well as
all appropriate interactions.
The proportion of the study population expected to be classified as PS or NS
animals by chance alone was calculated by multiplying the prevalence estimates
of animals shedding and not shedding E. coli O157:H7 with the five-gene mul-
tiplex PCR profile at each of the six time points. These joint probabilities
represented the expected frequency in each category, which was compared to the
observed number in each category by using chi-square goodness-of-fit tests.
Within-table independence (i.e., independence of the expected and observed
outcomes) was determined using a P value of ?0.05.
Chi-square analysis was utilized to detect differences in subtype frequency
between shedding status groups using the PROC FREQ procedure of SAS.
Initially, data were collapsed to form three subtype categories: the dominant
subtype, subtypes that differed from the dominant subtype by 1 to 3 bands, and
subtypes that differed from the dominant subtype by 4 to ?7 bands before
analysis. The estimated probability of shedding a given PFGE subtype during
antemortem collection was analyzed using a repeated-measures generalized es-
timating equations marginal logistic model with PROC GLIMMIX of SAS with
the empirical difference in standard errors. Differences between predicted prob-
abilities were considered significant at P values of ?0.05.
The difference between the log-transformed value for cells inoculated into a
well and the log-transformed counts for adherent cells recovered in the well was
used as the dependent variable to compare Caco-2 attachment data. Analysis of
variance techniques were employed to determine if there was a difference be-
tween the attachment abilities of different PFGE subtype categories, as previ-
ously described. Differences in attachment efficiency were analyzed using PROC
MIXED of SAS with least-squares means generated for each PFGE subtype
category. Ultimately, least-squares means were separated using pairwise t tests
incorporating a Tukey adjustment, and significant inferences were noted when
differences between means were detected at the P ? 0.05 level.
E. coli O157:H7 carriage in feedlot steers. All animals en-
rolled in this study were sampled during the first two sample
collections, which allowed us to determine the overall preva-
lence of E. coli O157:H7 in the study population. Presumptive
E. coli O157:H7 isolates were characterized by using a five-
gene multiplex PCR that detects gene fragments unique to
serotype O157:H7 along with genes encoding three key viru-
lence determinants (eae, stx1, and stx2). Overall, 45.5 and
VOL. 75, 2009 PERSISTENCE OF E. COLI O157:H7 IN FEEDLOT CATTLE5929
58.7% of the study population shed E. coli isolates belonging to
serotype O157 and carrying at least one stx gene during col-
lections 1 and 2, respectively (Table 1). Over the first two
collection periods, animals shed E. coli O157 isolates with five
virulence genotypes as determined by their multiplex PCR
profiles (Table 1). During the first two collection periods, E.
coli O157:H7 isolates possessing the eae, stx1, and stx2genes
(five-gene multiplex PCR profile) were shed most frequently,
as 39.8% and 33.6% of animals shed isolates with this genotype
during the first and second collections, respectively (Table 1).
Since the purpose of this study was to determine if cattle
become persistently colonized by E. coli O157:H7, which would
imply that the same E. coli O157:H7 strain is able to persist in
the GI tract of a given animal over time, our strategy for the
remaining four sample collections was to target animals that
consistently shed the predominant genotype (isolates with the
five-gene multiplex PCR profile). As a result, the prevalence of
less common genotypes could not be determined after the first
two sample collections, and we focused on monitoring the
persistence of the dominant genotype throughout the study
(Table 2). Specifically, PS animals were defined as animals that
shed an E. coli O157:H7 isolate carrying eae, stx1, and stx2
(five-gene-positive multiplex genotype) over the six collection
periods. Animals that intermittently shed an E. coli O157:H7
isolate with a genotype that included all three virulence factors
were classified as TS animals, while animals that never shed
morphologically typical E. coli O157 (i.e., colonies that were
not able to rapidly ferment sorbitol) during the 110-day sample
collection period were classified as NS animals. Overall, based
on these criteria, 8 of 788 animals (1.0%) were classified as PS
animals, while 11 of 788 animals (1.4%) never shed a detect-
able amount of morphologically typical E. coli O157 and were
thus classified as NS animals. Chi-square goodness-of-fit tests
revealed the independence of the observed and expected fre-
quency of PS status (P ? 0.001), while the number of animals
classified as having NS status was similar to that expected by
chance alone (P ? 0.23). The remaining 769 animals were
classified as TS animals since they shed an E. coli O157 isolate
carrying at least one stx gene at least once during this study.
The distribution of both PS and NS animals was balanced for
the five pens, and each pen contained at least one animal with
each E. coli O157:H7 shedding status. These results demon-
strated that small subpopulations of cattle in a feedlot popu-
lation appear to be persistently colonized by E. coli O157:H7
for extended periods of time during the final 100 to 110 days of
Feces from the eight animals classified as PS animals that
were collected during the final antemortem sampling were
examined using the five-tube MPN methodology. Only one PS
animal (PS-7) shed E. coli O157:H7 at levels (46 MPN/g)
detectable by our method. The remaining seven PS animals
shed E. coli O157:H7 at levels below the detectable limit, 1.8
MPN/g (data not shown). Our results indicate that an animal
that becomes persistently colonized by E. coli O157:H7 does
not necessarily shed high levels of the organism in its feces.
GI tissue and GI content analysis. E. coli O157:H7 was
detected in tissue and content samples collected from both
upper and lower sites in the GI tracts of PS and TS animals
(Table 3). Although the differences were not significant, more
lower GI tissue and content samples from PS animals than
from TS animals tested positive for E. coli O157:H7. An upper
GI tissue sample (omasum) and an anterior root lymph node
tissue sample from only one PS animal tested positive for E.
coli O157:H7. Two upper GI tissue samples (reticulum and
omasum) from two different TS animals tested positive for E.
coli O157:H7. No gall bladder samples were positive for E. coli
O157:H7 regardless of the previous shedding status (Table 3).
Histology and pathology. Histological analyses of tissue sam-
ples collected from PS and TS animals in this study did not
reveal any notable differences between tissues of PS and NS
animals. As expected, all tissue samples from PS and NS ani-
mals were normal or had minor lesions commonly found in GI
tissues characteristic of fed cattle. In addition, the livers exam-
ined from animals representing the PS, TS, and NS groups had
no visible surface lesions (data not shown). Thus, there do not
appear to be significant physiological differences between an-
imals that become persistently colonized by E. coli O157:H7
and animals that are not colonized by this organism.
Molecular characterization. At least one fecal isolate was
selected to represent each antemortem collection period for
each of the eight PS animals along with all postmortem isolates
(for a total of 82 isolates), and a random representative set of
fecal isolates was selected for 16 TS animals along with all
postmortem isolates (for a total of 50 isolates), resulting in a
set of 132 isolates that were characterized by PFGE typing.
TABLE 2. Prevalence of E. coli O157:H7 in feedlot cattle during
the final phase of finishing
No. of animals
No. of positive
aPositive samples were samples that contained E. coli O157:H7 with the eae,
stx1, and stx2virulence genes. This E. coli O157:H7 genotype was targeted as it
was the most common genotype identified during the first two sample collections
and the purpose of this study was to gain insight into persistent shedding of E.
TABLE 3. Distribution of postmortem GI tissues and contents
positive for E. coli O157:H7 among PS and TS animals
No. of positive samples
PS (n ? 8) TS (n ? 18)
Ileal-cecal colic node
Anterior root node
5930 CARLSON ET AL.APPL. ENVIRON. MICROBIOL.
The 132 E. coli O157:H7 isolates analyzed by PFGE typing
were classified into 32 different subtypes (Fig. 1; Table 4). A
single, predominant PFGE subtype (subtype F) (Fig. 1; Table
4) accounted for 53% of the 132 isolates characterized. A
chi-square test of independence showed that subtype F was
distributed similarly in the PS and TS animal populations (P ?
0.05), and this subtype persisted throughout the study (Fig. 2).
Twenty-two of the 32 unique PFGE subtypes were found in PS
animals, and 17 of the 32 unique PFGE subtypes were found
exclusively in PS animals. Interestingly, only five PFGE sub-
types (subtypes E, F, H, I, and N) overlapped in the PS and TS
animal populations (Table 4). All eight PS animals shed sub-
type F during the first sample collection, at least twice over the
entire collection period, and in at least two consecutive sample
collection periods (Table 5). One PS animal (PS-4) shed sub-
type F over the entire collection period, while two other PS
animals (PS-1 and PS-7) shed subtype F consecutively over the
first four sample collections (Table 5). During fecal sample
collection, PS animals shed 15 PFGE subtypes in addition to
subtype F, and 10 of these subtypes differed from subtype F by
three or fewer bands (Table 4). Tenover et al. (53) concluded
that strains with differences of three or fewer bands compared
to a reference strain (subtype F was used as the reference
strain) were closely related. The remaining five subtypes dif-
fered from subtype F by four or more bands and thus were not
considered closely related to PFGE subtype F according to the
criteria of Tenover et al. (53). The postmortem tissue and GI
content sample collection yielded an additional 10 unique
PFGE types for PS and TS animals that were not present
during antemortem fecal sample collections, and 4 of these
postmortem PFGE subtypes were closely related to the dom-
inant antemortem subtype, subtype F.
The ability of the predominant E. coli O157:H7 subtype to
persist in the GI tracts of feedlot cattle was indicated by the
percentage of animals that shed this subtype in their feces
TABLE 4. PFGE characterization of E. coli O157:H7 isolates from
PS and TS animals collected ante- and postmortem
No. of band
No. of isolates
No. of isolates
Total66 (16) 39 (11)
FIG. 1. Partial dendrogram and representative banding patterns
for the 32 unique PFGE banding patterns, including the banding
pattern for the E. coli O157:H7 isolate (Kansas) obtained in a previous
study (11) conducted 2 years before the current study. The Kansas
isolate was determined to have the same virulence genotype as the
other E. coli O157:H7 isolates utilizing multiplex PCR before the
PFGE analysis. The letters on the right correspond to the PFGE
subtypes shown in Table 4.
VOL. 75, 2009PERSISTENCE OF E. COLI O157:H7 IN FEEDLOT CATTLE 5931
throughout the study. More specifically, the estimated proba-
bilities of finding subtype F were 89.1, 77.0, 78.5, 59.3, 36.0, and
30.2% for sample collections 1, 2, 3, 4, 5, and 6, respectively
(Fig. 2). Although there was a decrease (P ? 0.05) in the
probability of shedding the dominant subtype with time, the
dominant subtype was still detected and actually accounted for
31.6% (6/19) of the isolates collected during the final antemor-
tem collection. Interestingly, our results also suggest that the
dominant E. coli O157:H7 molecular subtype (subtype F) un-
derwent microevolutionary changes during the study, as shown
by the emergence of molecular subtypes that were closely re-
lated to the dominant subtype and the decline in the presence
of the dominant subtype as the study progressed (Fig. 2).
These results indicate that a dominant E. coli O157:H7 strain
(subtype F) and other closely related strains persisted in the
population of feedlot cattle over a 100- to 110-day period.
In an effort to further investigate the ability of E. coli
O157:H7 to persist in the feedlot environment, we molecularly
characterized an additional E. coli O157:H7 isolate from a
previous study performed by Childs et al. (11) that was col-
lected from the environment of the same feedlot that was used
in the current study more than 2 years before our cattle ar-
rived. This E. coli O157:H7 isolate (designated the Kansas
isolate) was analyzed first with multiplex PCR to determine its
genotype. After it was determined that the Kansas isolate had
the same genotype as the persistent E. coli O157:H7 strain
obtained in the current study (based on a five-gene multiplex
PCR profile), the Kansas isolate was characterized by PFGE
typing and compared to isolates obtained in the current study
(Fig. 1). The Kansas isolate differed by only a single band from
the dominant subtype and exhibited 86% similarity (Fig. 1),
supporting the hypothesis that there is long-term persistence of
closely related E. coli O157:H7 strains in the feedlot environ-
Cell attachment. The abilities of E. coli O157:H7 isolates
belonging to the dominant PFGE subtype (subtype F), closely
related PFGE subtypes (?3-band difference from subtype F),
possibly related subtypes (between 4- and 6-band difference
from subtype F), and divergent subtypes (?7-band difference
from subtype F) to adhere to the Caco-2 human intestinal
epithelial cell line were compared. The attachment efficiency
was expressed as the percentage of the initial inoculum that
FIG. 2. Estimated probabilities of encountering the three PFGE subtype categories during antemortem sampling. The probability of encoun-
tering an E. coli O157:H7 isolate belonging to the dominant subtype, a subtype differing from the dominant subtype by 1 to 3 bands, or a subtype
differing from the dominant subtype by 4 to ?7 bands is indicated on the y axis, while the collections are indicated on the x axis. Isolates possibly
related (4- to 6-band difference) to the dominant subtype and isolates divergent (?7-band difference) from the dominant subtype were collapsed
to form the 4- to ?7-band difference subtype category due to low numbers. The low frequency of the 4- to ?7-band difference subtype category
prevented inclusion of this category in the analysis, which prevented calculation of errors. The error bars indicate the standard error calculated
for each estimated probability. For each independent collection, the estimated probability of encountering the dominant subtype was greater (P ?
0.05) than the probability of encountering strains that differed by one to three bands (indicated by an asterisk). Also, for an individual collection,
a dagger indicates that the probability of encountering the dominant subtype and subtypes with 1 to 3 band differences was greater (P ? 0.05) than
the probability of encountering subtypes with 4- to ?7-band differences. Dif., differences.
TABLE 5. Distribution of PFGE subtypes for each PS animal
during the antemortem collection period
5932CARLSON ET AL.APPL. ENVIRON. MICROBIOL.
adhered to host cells, and the mean attachment efficiencies for
the strain categories described above ranged from 10.7 to
53.9% (Fig. 3). E. coli O157:H7 isolates belonging to the per-
sistent PFGE subtype (subtype F) demonstrated an enhanced
(P ? 0.05) ability to adhere to human intestinal epithelial cells
compared to isolates belonging to closely related, possibly re-
lated, and genetically divergent subtypes. A relationship was
observed between genetic diversity and attachment efficacy; as
the genetic difference from subtype F increased (based on the
number of band differences), the ability to attach to Caco-2
cells decreased (Fig. 3). As a reference, an E. coli O157:H7
isolate obtained from a food sample associated with an out-
break of human illness (ATCC 43895) with the same genotype
(five-gene multiplex PCR profile) was included in all cell at-
tachment assays. The attachment efficiencies of the E. coli
O157:H7 isolates representing the dominant subtype were
more than threefold greater than that of the outbreak-associ-
ated isolate (Fig. 3). The results of the Caco-2 attachment
assays suggest that E. coli O157:H7 subtypes that persist in
cattle have an enhanced ability to adhere to human intestinal
To date, there has not been an extensive investigation of the
molecular ecology of E. coli O157:H7 persistence and shedding
in naturally colonized feedlot cattle. Our results demonstrate
that closely related E. coli O157:H7 strains may persist in the
feedlot ecosystem (i.e., cattle and the feedlot environment) for
extended periods, which may be explained in part by an en-
hanced ability of these persistent strains to adhere to intestinal
epithelial cells. Additionally, we demonstrated that most
(97.6%) feedlot steers shed Shiga toxin-encoding E. coli O157
during the final 100 to 110 days of the feeding period. Molec-
ular characterization of E. coli O157:H7 isolates revealed that
a predominant E. coli O157:H7 strain persisted throughout the
study and that this persistent strain diversified during the study.
Further phenotypic characterization of isolates belonging to
the persistent subtype and closely related and more genetically
divergent PFGE subtypes using a cell culture attachment assay
revealed an increased attachment efficiency of the persistent E.
coli O157:H7 strain found in the feedlot population. Our re-
sults illustrate that certain E. coli O157:H7 strains may persist
in cattle populations and that, ultimately, these strains may
represent an increased risk to human health due to the in-
creased likelihood that they could enter the human food supply
and due to their subsequent enhanced ability to attach to
human intestinal epithelial cells.
Most feedlot cattle appear to shed Shiga toxin-encoding E.
coli O157 at some point during the final phase of finishing.
During collections 1 and 2, we detected an E. coli O157 isolate
carrying at least one Shiga toxin-encoding gene in 45.5 and
58.7% of all animals, respectively. Before now, the point prev-
alence of Shiga toxin-encoding E. coli O157, a more indicative
estimate of true risk (e.g., a Shiga toxin-encoding E. coli O157
would elicit a regulatory response if it was transferred to a
carcass ), was reported to range from 0.3 to 19.7% in
feedlot cattle (28). More importantly, we observed that 97.6%
of steers shed a Shiga toxin-encoding E. coli O157 isolate at
least once during the final 100 to 110 days of the feeding
period, a level that had been reported previously. During the
first two sample collection periods, 39.8 and 33.6% of the
animals shed an E. coli O157:H7 isolate with the same geno-
type (i.e., carrying the eae, stx1, and stx2genes), which are
higher percentages than those previously reported (12, 18, 27,
45, 50, 55, 56).
Small subpopulations of feedlot cattle appear to become
persistently colonized by E. coli O157:H7. Few studies have
evaluated the persistence of E. coli O157:H7 fecal shedding in
FIG. 3. Caco-2 attachment efficacies for E. coli O157:H7 isolates representing genetically diverse PFGE subtypes and an E. coli O157:H7 isolate
known to have caused an outbreak of food-borne illness, which was included as a reference. The data are expressed as the percentage of adherent
E. coli O157:H7 cells recovered from each well. The PFGE subtype or subtype category is indicated on the x axis, while the percent attachment
is indicated on the y axis. The bars indicate the least-squares means for attachment efficacies obtained for at least two strains for each subtype
category (except for the 6- and ?7-band bars, where only one E. coli O157:H7 strain was assayed for each category) for three independent
experiments. The error bars indicate the standard errors calculated for each least-squares mean. Bars that do not contain the same letter are
significantly different (P ? 0.05). The outbreak strain was not included in the analysis, but data for this strain are included for reference. Dif.,
VOL. 75, 2009 PERSISTENCE OF E. COLI O157:H7 IN FEEDLOT CATTLE 5933
large populations of feedlot cattle naturally colonized by this
human pathogen. We found that 1% of feedlot steers persis-
tently shed an E. coli O157:H7 isolate with the same genotype
(i.e., a strain carrying eae, stx1, and stx2) during the final 100
days of the feeding period, a level that is significantly greater
than expected by chance. Intensive fecal sampling (multiple
samples collected each day) that was conducted with two dif-
ferent cohorts of 6- to 11-month-old dairy calves revealed two
animals in each cohort (14 and 12.5% of the animals) that
persistently shed E. coli O157:H7 for 4 and 15 days (43).
Observation of E. coli O157:H7 fecal shedding in feedlot cattle
determined that a small number of animals (n ? 8) shed the
organism for a maximum of 4.5 weeks, about three-quarters of
the time that we found E. coli O157:H7 persistence to last, and
the remaining animals in the sample population shed this bac-
terium for 2.5 weeks on average (30). For dairy cattle, the
duration of E. coli O157:H7 shedding was estimated to be
approximately 1 month (6). These previous studies of naturally
colonized populations were limited by the lack of molecular
subtyping to characterize isolates in order to determine if an-
imals were persistently colonized by the same E. coli O157:H7
strain or if they were continuously exposed to, and subse-
quently shed, genetically diverse strains. Experimental inocu-
lation of calves with E. coli O157:H7 established that shedding
occurred for periods between 14 and 140 days long (7, 15, 39,
44). Alternatively, experimental inoculation of cattle that were
?0.5 year old increased the minimum number of days of per-
sistent fecal shedding to 29 days but reduced the maximum
shedding time to only 98 days (15, 23, 46). Assessment of
previously published data supported the hypothesis that there
is a general trend of increased E. coli O157:H7 shedding du-
ration for neonates and calves less than 1 year old. As the
animal age increases, persistent shedding decreases and is in-
It is plausible that the eight animals that we found to be
colonized by the same E. coli O157:H7 strain throughout this
study continued to be reexposed to the organism via animal-
to-animal transmission or a contaminated pen environment,
which resulted in the persistent E. coli O157:H7 shedding
status. Animal-to-animal transmission was previously observed
when uninoculated young calves (10 weeks old) that commin-
gled with calves inoculated with E. coli O157:H7 began shed-
ding the organism (7). However, animal-to-animal transmis-
sion of E. coli O157:H7 was found to be very inefficient and
unlikely for a group of 5- to 8-month-old calves (46), further
substantiating our conclusion that the animals were persis-
tently colonized with E. coli O157:H7, particularly since the PS
animals were fed and maintained in different pens. Addition-
ally, it appears that observations from one time point to the
next are not independent with regard to positive animals. Fur-
thermore, previous literature suggests that while the microbi-
ological assays are highly sensitive, the sampling methodology
is quite insensitive (17a). Thus, the true PS population was
likely underestimated and the true NS population overesti-
mated; if so, there is even more true dependency from time
point to time point than that observed herein.
E. coli O157:H7 may show specificity for colonization of the
lower GI tract, and there do not appear to be notable his-
topathological differences between PS and NS animals. We did
not obtain conclusive evidence regarding the preferential site
of E. coli O157:H7 colonization due to the limited prevalence
of this organism in the GI tract tissue and content samples
collected postmortem, which was presumably a result of trans-
portation stress (2) and extended lairage (38) at the plant. A
comparable situation was reported for a population of sheep
inoculated with E. coli O157:H7 that shed detectable amounts
of the organism in their feces, but the organism could not be
detected in any GI tract tissue or content samples following
necropsy (23). Despite the limited E. coli O157:H7 prevalence,
we found that at least one ileal-cecal sample and one RAJ
tissue sample were positive for E. coli O157:H7 both in animals
identified as PS animals and in animals identified as TS ani-
mals. We also obtained three positive tissue samples from the
fore-stomach, the earliest GI site believed to be a site of E. coli
O157:H7 propagation (8). Recent research has concluded that
the colon (23, 52), specifically an area designated the RAJ, is
the principal site of E. coli O157:H7 colonization (33, 39, 46),
and this colonization site is particularly important for E. coli
O157:H7 excretion in feces (12). In cattle naturally colonized
by E. coli O157, tissue samples obtained 1 cm from the RAJ
contained larger quantities of E. coli O157 than samples ob-
tained 15 cm from the RAJ (33). A relationship between in-
creased E. coli O157:H7 concentration and areas closest to the
RAJ was observed with tissue samples obtained from experi-
mentally infected animals, naturally colonized animals, and
calves exposed to infected animals (39). It was concluded that
animals are more likely to become consistent long-term shed-
ders when they are infected with E. coli O157:H7 directly at the
RAJ rather than through oral inoculation (46). While our
findings do not confirm the preferential site of E. coli O157:H7
colonization in cattle, they do corroborate the conclusion that
the lower GI tract is the preferred site of colonization.
Histopathological evaluation of the all of the GI tracts ob-
tained from PS, TS, and NS animals did not reveal any gross
abnormalities or discernible lesions. More specifically, no at-
taching and effacing (A/E) lesions, which are characteristic of
E. coli O157:H7 colonization, were identified in PS animal GI
tissue samples. All animals enrolled in the study remained
healthy throughout the entire time of sample collection, and
cattle colonized with E. coli O157:H7 do not generally have
clinical symptoms (15). There does, however, appear to be an
association between animal age and susceptibility to clinical
symptoms for animals exposed to very high levels of E. coli
O157:H7. Experimental infection of young calves (?12 h old
or 30 to 36 h old) with 10 logs of E. coli O157:H7 resulted in
severe diarrhea and A/E lesions throughout the lower GI tract
(16). In contrast, 1-day-old calves remained clinically normal
after inoculation with 8 logs of E. coli O157:H7 (44), an inoc-
ulation level believed to encourage A/E lesion development
(17). Once animals reach 3 weeks of age, their susceptibility to
E. coli O157:H7 infection (10 logs) appears to diminish; E. coli
O157:H7 infection results in slight increases in the body tem-
perature and watery diarrhea, but these symptoms last for only
a couple of days following inoculation and no A/E lesions are
formed (8, 15). Still, infection of 3- to 4-month-old calves with
10 logs of E. coli O157:H7 caused watery diarrhea (17) and
A/E lesion formation (17, 52) and in one case resulted in
translocation of the bacteria to the gall bladder, where they
were able to produce A/E lesions (52). The general consensus
that E. coli O157:H7 colonization is not associated with clinical
5934CARLSON ET AL.APPL. ENVIRON. MICROBIOL.
symptoms in animals that are at least 1 year old (15, 23) is
further validated by the lack of pathological symptoms ob-
served for the animals enrolled in our study. Furthermore,
histopathological comparisons between PS and NS animals
support the conclusion that host-associated factors do not ap-
pear to be as significant as E. coli O157:H7’s ability to orches-
trate persistent colonization in cattle more than 1 year old.
A single E. coli O157:H7 strain may persist in a population
of feedlot cattle. We observed that a single predominant PFGE
subtype accounted for 53% of all isolates characterized; fur-
thermore, 87% of the isolates belonged to the predominant
PFGE subtype or to PFGE subtypes that were closely related
to the predominant subtype (only one to three bands were
different ). Our data contribute to the growing body of
evidence indicating that E. coli O157:H7 persists in cattle pop-
ulations. More specifically, along with several previous studies
(6, 43, 45, 48, 51), our study supports the conclusion that a
given population of feedlot cattle appears to be colonized by a
single predominant strain and a few closely related strains. The
predominant PFGE subtype in our study was disseminated into
each animal pen, was found on each sample collection date,
and was shed by each PS animal on at least two consecutive
sample collection dates. Similarly, in another study, all 54 E.
coli O157 isolates obtained from two different dairies belonged
to the same PFGE subtype (43). Analysis of the genetic diver-
sity of E. coli O157:H7 collected from four dairies located in a
30-km area in southern Alberta also demonstrated that there
was a highly clonal E. coli O157:H7 population, as three dom-
inant subtypes, which were detected at every dairy, accounted
for a majority of the isolates characterized (50). It can be
argued that dairy farm environments have an increased likeli-
hood of sustaining highly clonal E. coli O157:H7 populations
because of reduced animal turnover compared to commercial
feedlots, where new animals presumably are the main source of
new E. coli O157:H7 subtypes. However, feedlots also appear
to maintain highly related populations of E. coli O157:H7, even
with their increased animal turnover rates. For example, PFGE
analyses of 103 and 230 E. coli O157:H7 isolates obtained from
two different commercial feedlots revealed that isolates clus-
tered with 80% similarity (45) and that 60% of the isolates
belonged to four closely related subtypes (32), respectively.
Additionally, indistinguishable E. coli O157:H7 subtypes were
recovered from two feedlots that were approximately 100 km
apart and did not share any common source of animals that
entered them (56). Previously, we demonstrated that transit to,
or holding at, the processing plant can introduce E. coli
O157:H7 isolates with diverse PFGE subtypes (11). We ob-
tained similar results, as 10 unique E. coli O157:H7 subtypes
that were never observed during antemortem sampling were
obtained from either GI tract tissue or GI content samples.
The persistence of predominant E. coli O157:H7 subtypes in
beef cattle feedlots was characterized and was determined to
last for several years (32). Strains of E. coli O157:H7 persist in
the environment (1) and can potentially be rapidly dissemi-
nated throughout a cattle population (45). We compared the
PFGE banding pattern of the dominant PFGE subtype ob-
served in the current study to that of an E. coli O157:H7 strain
(with the same genotype) obtained from an environmental
sample (11) from the same feedlot 2 years before the arrival of
our cattle and determined that the two subtypes were highly
related, with only one band difference and only 14% diver-
gence from the dominant subtype (Fig. 1). Our findings pro-
vide further evidence that certain E. coli O157:H7 strains likely
persist in the feedlot environment and subsequently colonize
E. coli O157:H7 strains that persist in cattle populations
demonstrate an enhanced ability to adhere to intestinal epi-
thelial cells. Based on our observations that a predominant
PFGE subtype and other closely related PFGE subtypes ac-
counted for the majority of E. coli O157:H7 isolates obtained
from the population of feedlot cattle studied here and that
these subtypes persisted in these cattle throughout the study,
we hypothesized that these strains may represent an “ecotype”
that adapted to colonize and persist in the GI tract. We ex-
plored this hypothesis by characterization of E. coli O157:H7
isolates representing the predominant and persistent PFGE
subtype (subtype F), along with isolates representing PFGE
subtypes that were closely related, possibly related, and dis-
tantly related to subtype F. Our investigation showed that E.
coli O157:H7 isolates that represent the predominant PFGE
subtype demonstrate an enhanced (P ? 0.05) ability to adhere
to the Caco-2 human intestinal epithelial cell line. Interest-
ingly, as genetic diversity of the predominant PFGE subtype
increases, attachment efficacy decreases. E. coli O157:H7 iso-
lates representing the predominant subtype demonstrated an
ability to attach to Caco-2 cells that was greater than that of a
reference E. coli O157:H7 isolate from an outbreak of human
illness, substantiating the human-pathogenic potential of E.
coli O157:H7 strains that persist in feedlot cattle.
We characterized the attachment efficacies of E. coli
O157:H7 isolates using a human intestinal epithelial cell line
because of the lack of an immortal bovine intestinal epithelial
cell line. Although our initial objectives were to elucidate the
nature of E. coli O157:H7 colonization and persistence in the
bovine GI tract, we discovered that E. coli O157:H7 strains that
persist in feedlot cattle appear to have an accentuated ability to
adhere to human intestinal epithelial cells, which is essential
for disease manifestation. E. coli O157:H7 depends on intimin
and the translocated intimin receptor (Tir) for intimate adher-
ence to a host cell (29). The role of intimin and its importance
in bacterial adherence have been investigated and validated
using bovine models (14, 22, 47) and appear to be no different
in human cell lines (13). We do not discount the significance of
intimin in bacterial attachment, but taking into consideration
the fact that all of the E. coli O157:H7 isolates screened during
the attachment assay contained the gene responsible for in-
timin production, our results provide evidence that there are
other influential mechanisms that are responsible for attach-
ment efficacy. Further work is required to elucidate the mo-
lecular mechanisms responsible for the disparate attachment
efficacies of diverse E. coli O157:H7 strains that encode the
same virulence determinants.
Conclusions. We provide compelling evidence that in a pop-
ulation of healthy feedlot cattle, a small subpopulation of an-
imals appears to become persistently colonized by closely re-
lated E. coli O157:H7 strains. We found no physiological
differences between animals that we classified as PS, TS, and
NS based on our observations of animal health status and
postmortem histopathology. In addition, PS and TS animals
appeared to become colonized by a single predominant E. coli
VOL. 75, 2009PERSISTENCE OF E. COLI O157:H7 IN FEEDLOT CATTLE 5935
O157:H7 molecular subtype along with other closely related
molecular types, supporting the hypothesis that new genotypes
emerged. Finally, our findings provide evidence that cattle may
be more likely to be colonized by E. coli O157:H7 molecular
subtypes that demonstrate accentuated human-pathogenic po-
tential, as shown by the enhanced ability of persistent strains to
adhere to human intestinal epithelial cells. Additionally, it
stands to reason that there is an increased likelihood that these
E. coli O157:H7 subtypes are transferred through the produc-
tion continuum and subsequently into the human population
because of their increased prevalence in feedlot cattle. Our
results highlight the importance of preharvest food safety in-
terventions to reduce the load of E. coli O157:H7 that enters
the human food supply and support the conclusion that such
efforts should be targeted at strains that persist in cattle pop-
ulations which seem to represent the greatest risk to human
health. Further research is needed to elucidate the underlying
pathogen factors associated with persistent colonization of
healthy cattle by E. coli O157:H7, including work to further
probe molecular mechanisms associated with enhanced adhe-
sion to intestinal cells and to develop mitigation strategies to
control E. coli O157:H7 in feedlot populations with the ulti-
mate goal of reducing the risk of human infection.
This work was supported by the Beef Checkoff and by the Colorado
State University Agricultural Experiment Station.
We are indebted to M. Wiedmann and Y. Soyer of Cornell Univer-
sity for their expertise and assistance with PFGE typing and PFGE
pattern cluster analysis.
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