Shiga Toxigenic and Atypical Enteropathogenic
Escherichia coli in the Feces and Carcasses

of Slaughtered Pigs

Clarissa Araújo Borges,1 Lı́via Gerbasi Beraldo,1 Renato Pariz Maluta,2 Marita Vedovelli Cardozo,1

Beatriz Ernestina Cabilio Guth,3 Everlon Cid Rigobelo,4 and Fernando Antônio de Ávila1

Abstract

Escherichia coli is a pathogen of major importance in swine and public health. To determine the prevalence of
Shiga toxigenic E. coli (STEC) and enteropathogenic E. coli (EPEC), samples were collected from the feces and
carcasses of swines. In total, 441 samples were collected in four samplings, of which 141 samples tested positive
for either the stx1, stx2, and/or eae genes. From the positive samples, one STEC and 15 atypical EPEC (aEPEC)
isolates were obtained, and all originated from the same sampling. In addition to eae, lpfAO157/OI-141, ehxA, toxB,
and lpfAO113 were present in the aEPEC isolates. The only stx2-containing isolate carried stx2e and belonged to
serotype O103:HNT. Resistance to four or more antimicrobials was found in almost half of the isolates, and some
isolates shared the same fingerprint patterns by enterobacterial repetitive intergenic consensus–polymerase
chain reaction (ERIC-PCR). The presence of certain virulence genes and the high level of resistance to antimi-
crobials, as well as the possible fecal contamination of carcasses showed that some of the isolates are of public
health concern.

Introduction

Escherichia coli is an important pathogen in swine
medicine and public health (Bertschinger and Fairbrother,

1999; Kaper et al., 2004). There are several pathotypes of E. coli,
which are characterized by the presence of different virulence
factors. The genes responsible for these factors are located on
chromosomes, plasmids, or phages (Teng et al., 2004; Trabulsi,
1999). Shiga toxin–producing (STEC) and enteropathogenic
Escherichia coli (EPEC) represent two of the six different cate-
gories of diarrheagenic E. coli that can cause disease in hu-
mans (Kaper et al., 2004).

STEC, which is defined by the production of two Shiga
toxins, Stx1 and/or Stx2, is a zoonotic pathogen that is a major
cause of diarrhea worldwide. In humans, STEC can cause
hemorrhagic colitis (HC), which can progress and cause se-
vere extraintestinal complications, such as hemolytic-uremic
syndrome (HUS) (Paton and Paton, 1998). Stx2 is more closely
related to these diseases than Stx1 (Miceli et al., 1999; Siegler
et al., 2003). Although E. coli O157:H7 is the most widely
recognized pathogenic STEC serotype, non-O157 STEC ser-
ogroups, including O26, O103, O111, and O113, are also

commonly associated with this disease (Nataro and Kaper,
1998; Acheson et al., 2000).

There are three Stx1 subtypes (Stx1a, Stx1c, and Stx1d) and
seven Stx2 subtypes (Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f,
and Stx2g) according to the subtyping nomenclature proposal
put forth at the 7th International Symposium on Shiga Toxin
(Verocytotoxin)–Producing Escherichia coli Infection, held in
Buenos Aires, in 2009.

EPEC, which is characterized by the presence of the eae
gene and the absence of the toxin Stx, can be typical (tEPEC) or
atypical (aEPEC). The former, tEPEC, contains both the
plasmid EAF and the gene cluster bfp, whereas the latter,
aEPEC, lacks both EAF and bfp (Trabulsi et al., 2002). A pre-
vious report demonstrated that aEPEC is the second major
cause of E. coli–related infantile diarrhea in Brazil (Araujo
et al., 2007).

Other virulence factors that may contribute to the patho-
genicity of E. coli have been found. These include the ehxA
gene, which encodes a hemolysin that is responsible for the
formation of pores in host cells (Schmidt et al., 1996), and
putative adhesins encoded outside of the loci of enterocyte
effacement (LEE), such as iha, efa1, toxB, lpfAO157/OI-141,

1Departamento de Patologia Veterinária, Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, and 4Faculdade de Zootecnia de
Dracena, Universidade Estadual Paulista, São Paulo, Brasil.

2Departamento de Genética e Evolução, Universidade Estadual de Campinas, São Paulo, Brasil.
3Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, São Paulo, Brasil.

FOODBORNE PATHOGENS AND DISEASE
Volume 9, Number 12, 2012
ª Mary Ann Liebert, Inc.
DOI: 10.1089/fpd.2012.1206

1119



lpfAO157/OI-154, lpfAO113, and saa (Toma et al., 2004). Several
recent studies demonstrated that some genes, such as efa1,
lpfA, paa, and ehxA (Afset et al., 2006; Narimatsu et al., 2010),
are associated with EPEC isolated from humans with diarrhea
and could therefore act as zoonotic potential predictors.

Previous studies reported the presence of STEC and EPEC
in the feces of pigs (Fratamico et al., 2004; Vu-Khac et al., 2007;
Oporto et al., 2008) and STEC in their carcasses (Botteldoorn
et al., 2003; Leung et al., 2001). However, these studies did not
focus on EPEC from carcasses. A number of studies report the
prevalence of putative adhesins in STEC and EPEC from
humans and several animal species (Afset et al., 2006; Leotta
et al., 2006; Toma et al., 2006; Aidar-Ugrinovich et al., 2007;
Cergole-Novella et al., 2007; Islam et al., 2008; Narimatsu et al.,
2010). It would therefore be of great interest to investigate the
prevalence of E. coli in pigs.

Due to the importance of E. coli–related foodborne diseases,
this study focused on pig feces and carcasses, which were
examined for the presence of both STEC and EPEC. We also
characterized the isolates for virulence genes, enterobacterial
repetitive intergenic consensus (ERIC)–polymerase chain re-
action (PCR) fingerprint patterns, and antimicrobial resis-
tance levels.

Materials and Methods

Sampling and initial procedures

A total of 441 samples were collected from pig feces and
carcasses between March and August 2010 at three slaugh-
terhouses in São Paulo State, Brazil. In slaughterhouse 1, two
samplings were performed on two different days (S1a and
S1b), while at the other two slaughterhouses, a single sam-
pling (S2 and S3) was performed. In total, 96 samples (52 from
feces and 44 from carcasses), 139 samples (70 from feces and
69 from carcasses), 122 samples (62 from feces and 60 from
carcasses), and 84 samples (42 from feces and 42 from car-
casses) were collected from S1a, S1b, S2, and S3, respectively.
Feces were collected directly from the rectum of each animal
using sterile swabs. Carcass samples from slaughtered pigs
were collected with a sterile sponge rubbed over the leg, ribs,
shoulder, and neck, for a total of 100 cm2. All swabs were
deposited in tubes containing peptone water (5 mL), and all
sponges were placed in bags containing 15 mL of the same
medium. All samples were transported to the laboratory in
thermal boxes with ice.

Detection of STEC and EPEC by PCR

For the detection of STEC- and EPEC-positive samples,
1 mL of peptone water from the tubes and bags containing
feces and carcass samples, respectively, were transferred to
tubes containing 5 mL of Brilliant Green broth, incubated at
37�C for 24 h under static conditions. After incubation, 100 lL
of each culture were streaked onto MacConkey agar (MA)
plates and incubated at 37�C for 24 h. The DNA template was
prepared from confluent MA cultures by thermal lyses
(available at www.apzec.ca/en/APZEC/Protocols/pdfs/
ECL_PCR_Protocol.pdf ). Multiplex PCR was performed on
the samples to detect the presence of the stx1, stx2, and eae
genes (China et al., 1996). The following parameters were used
in the PCR: 4 lL of DNA template was added to a mixture
containing 0.4 lL of 10 mM dNTPs, 2 lL of 10 · buffer

(100 mM Tris-HCl, pH 8.8 at 25�C, 500 mM KCl, 0.8% [v/v]
Nonidet P40), 1.6 lL of 25 mM MgCl2, 0.8 lL of each 10 pM
primer, and 1 unit of Taq DNA polymerase (Fermentas, Eur-
ope). MilliQ water was added for a total volume of 20 lL. The
amplification cycles were performed in an Eppendorf Mas-
tercycler Gradient thermocycler under the following condi-
tions: t1, 5 min at 94�C; t2, 30 sec at 94�C; t3, 45 sec at 50�C; t4,
1 min at 72�C; t2–t4, 25 repeated cycles; and t5, 7 min at 72�C.
Ten individual colonies from each positive sample, which was
defined by the presence of at least one of the three studied
genes, were also tested by PCR to isolate STEC and EPEC
strains (a methodological approach from the Reference La-
boratory for Escherichia coli [EcL] Université de Montréal).
From these isolates, the remaining tests were performed.

Detection of other virulence genes

Other virulence genes were also detected by PCR. The
conditions were the same as above except that the annealing
temperatures were 53�C, 52�C, 39�C, 49�C, 59�C, 55�C, 47�C,
and 56�C, respectively, for duplex PCR of iha, toxB genes,
and simple PCR of saa, lpfAO113, lpfAO157/OI-141, lpfAO157/OI-154,
ehxA, efa1, and bfpA genes. The primers used have been
described previously (Schmidt et al., 1995, 2001; Nicholls
et al., 2000; Doughty et al., 2002; Szalo et al., 2002; Paton and
Paton 2002; Tarr et al., 2002; Toma et al., 2004; Vidal et al.,
2004). For the differentiation of stx2 variants, RFLP-PCR
method was carried out as previously described (Cergole-
Novella et al., 2006). Identification of stx2e subtype was per-
formed with the primers and PCR conditions described by
Pièrard et al. (1998).

ERIC–PCR

E. coli isolates were subjected to Enterobacterial Repetitive
Intergenic Consensus (ERIC-PCR) reactions with the primers
ERIC1R (5¢ ATGTAAGCTCCTGGGGATTCAC-3¢) and
ERIC2 (5¢ AAGTAAGTGACTGGGGTGAGCG-3¢) (Versa-
lovic et al., 1991). Each ERIC-PCR reaction was performed in a
total volume of 20 lL, as described previously. The PCR am-
plification thermal profile was based on a previous report
(Silveira et al., 2002) with minor modifications: t1, 5 min at
94�C; t2, 30 sec at 94�C; t3, 45 sec at 52�C; t4, 1 min at 72�C; t2–
t4, 30 repeated cycles; and t5, 7 min at 72�C. The similarities in
fragments were compared using a Dice coefficient at 1% tol-
erance and 0.5% optimization, and a dendrogram was con-
structed with the Unweighted Pair Group with Arithmetic
Mean clustering method using the software BioNumerics.

Biochemical characteristics and antimicrobial
susceptibility testing

All STEC and EPEC isolates were tested for lactose fer-
mentation, indole production, methyl red reactions, Voges-
Proskauer, and citrate utilization, as well as for the production
of urease and hydrogen sulfide (H2S) (Mac Faddin, 1976).
Antimicrobial disk susceptibility tests were performed using
the disk diffusion method (CLSI, 2009). The antimicrobials
tested were ampicillin (10 lg), cephalothin (30 lg), strepto-
mycin (10 lg), gentamicin (10 lg), nalidixic acid (30 lg), ci-
profloxacin (5 lg), chloramphenicol (30 lg), tetracycline
(30 lg), nitrofurantoin (300 lg), and sulfamethoxazole + tri-
methoprim (25 lg).

1120 BORGES ET AL.



Serotyping

The determination of the EPEC serogroup was performed
with the slide agglutination technique, using anti-O sera
against the classical EPEC somatic antigens O26, O55, O111,
O119, O114, O125, O142, O158, O86, O126, O127, and O128
(Probac, São Paulo, Brazil). STEC serotyping with all somatic
and flagellar antigens was performed at a reference laboratory
(Instituto Adolfo Lutz, São Paulo, Brazil).

Results

After testing the confluent growth from fecal and carcass
samples, we found differences in the prevalence of genes re-
lated to STEC (stx) and EPEC (eae) in the different slaughter-
houses. The samples from S1b had the greatest percentage of
eae, whereas the samples from S3 showed the highest per-
centage of stx2. In addition, eae was more prevalent than stx2
in all samplings, except in S3. In all samplings, stx1 was not
found. Furthermore, samples that were positive for at least
one gene were found more often in the fecal rather than the
carcass samples. The prevalence of these genes is shown in
Table 1. After testing ten colonies from each of the 141 positive
samples for the presence of the stx1, stx2, and eae gene, we
found only one STEC isolate (stx2 + stx1- eae-) in a carcass
sample and none in the feces. Therefore, the prevalence of
STEC isolates was 0.4% of the total carcasses sampled (215),
and no STEC isolates were detected in the feces. Moreover,
eight fecal and seven carcass samples each yielded a single
isolate of aEPEC (stx1-, stx2-, eae + ). The prevalence of aEPEC
was 3.5% and 3.2% in the fecal and carcasses, respectively,
relative to the total number of feces (226) and carcasses (215)
samples. All sixteen isolates described in this work originated
from the second sampling of slaughterhouse 1 (S1b).

The lpfAO113 (46.6%) gene was the most prevalent in the
aEPEC isolates. The lpfAO157/OI-141 (13.3%), ehxA (13.3%), and
toxB (13.3%) genes were also detected in the aEPEC isolates.
There was no aEPEC isolate that contained the lpfAO157/OI-154,
saa, efa1, iha, or bfp gene. The absence of bfp indicated that all of
the EPECs isolated in this study were considered atypical
EPEC (aEPEC). The only stx2-containing isolate presented
stx2e subtype and was serotyped as O103:HNT. None of the

aEPEC isolates belonged to the classical EPEC serogroups
tested.

All of the isolates in this study were tested against ten
antimicrobial agents. The most common resistances were
to tetracycline (93.7%), nalidixic acid (81.3%), and ampicillin
(50.0%). Less frequent resistances were to chlorampheni-
col (33.3%), streptomycin (31.2%), cephalothin (25.0%), gen-
tamicin (18.7%), sulfamethoxazole/trimethoprim (12.5%),
nitrofurantoin (0%), and ciprofloxacin (0%). Multidrug resis-
tance was found in 43.8% of the isolates, which was defined as
resistance against four or more antimicrobials. Some isolates
showed the same fingerprint pattern by ERIC-PCR, and two
of these samples, one from carcass (46C) and one from feces
(25R), also shared the same virulence genes and antimicrobial
resistance profile (Fig. 1). All isolates showed typical charac-
teristics of Escherichia coli in biochemical tests.

Discussion

In the present study, we identified STEC and aEPEC iso-
lates from swine feces and carcasses. Some of these isolates
showed similar genetic profiles by ERIC-PCR and virulence
genes, including putative adhesins.

According to Table 1, the percentage of samples containing
stx1, eae, or stx2/eae varied between slaughterhouses, which
could possibly be attributed to the batch of animals that each
slaughterhouse had received. The animals were likely to have
been exposed to different sanitary managements at the farms of
origin. The PCR sampling yielded more positive results when
compared to the PCR from the isolates. These findings may be
attributed to the PCR being performed with confluent cultures
from MacConkey Agar. These results would represent all
Gram-negative microbiota from each sample, thereby enhanc-
ing the detection levels of STEC and EPEC. Because only a few
PCR-positive isolates were detected from these samples, it is
apparent that STEC and EPEC isolates occur at a much lower
level in swine compared to E. coli of different pathotypes.

In this study, the prevalence of STEC isolates, both in car-
casses and feces, was similar to those reported in other studies
conducted in pigs, which found frequencies ranging from 0%
to 1% (Leung et al., 2001; Lindblad 2007; Oporto et al., 2008).
Using the same methodology described in this study, we

Table 1. Prevalence of Stx1, Stx2, and eae in Samples of Feces and Carcasses

of Pig from Different Slaughterhouses

S1a1 S1b2 S23 S34

(n = 96) (n = 139) (n = 122) (n = 84)

Feces Carcass Feces Carcass Feces Carcass Feces Carcass

52 44 70 69 62 60 42 42

n % n % n % n % n % n % n % n %

stx2 0 0 13 18.6 4 5.8 3 4.8 2 3.3 15 35.7 4 9.5
eae 17 32.7 3 6.8 25 35.7 25 36.2 13 20.9 3 5.0 2 4.7 1 2.4
stx2/eae 5 9.6 0 2 2.8 1 1.4 0 0 2 4.7 1 2.4

1Slaughterhouse 1, sampling a.
2Slaughterhouse 1, sampling b.
3Slaughterhouse 2.
4Slaughterhouse 3.
stx1 was not found in any slaughterhouse.

STEC AND EPEC IN PIGS 1121



found that the prevalence of STEC isolates from sheep and
buffalo samples were greater than the prevalence in pigs
(unpublished data). These findings suggest that pigs might
not be an important reservoir for STEC.

The STEC isolate identified in this study was positive for
Stx2e, a subtype commonly associated with edema disease in
pigs but is rarely detected in human infections (Friedrich et al.,
2002; Zweifel et al., 2006; Vu-Khac et al., 2007). We also found
that this strain belonged to the serogroup O103, which is not
common in swine but is considered a serious public health
threat because it has been recovered from infected patients.
The serotype usually related to human infection is O103:H2
and possess another stx subtype (Mariani-Kurkdjian et al.,
1993; Guth et al., 2005). However, these findings suggest that
healthy pigs cannot be excluded as a potential source of
human infection.

We did not find any O157:H7 isolates in our study. These
results were similar to the findings from studies conducted in
France, Belgium, and Ireland using swine carcasses (Bouvet
et al., 2001; Botteldoorn et al., 2003; Lenahan et al., 2009).
However, the use of a more specific technique for O157:H7,
such as immunomagnetic separation (IMS), may improve
detection of this microorganism.

Previous reports demonstrated a prevalence range between
1.4% and 18.5% of aEPEC in pigs with diarrhea (Frydendahl
et al., 2002; Malik et al., 2006; Vu-Khac et al., 2007). We found
that the prevalence of aEPEC isolated from the feces was
much lower compared to another study that was conducted in
healthy pigs. This study found that 14% of the samples were
positive for aEPEC (Malik et al., 2006), suggesting that the
occurrence of aEPEC varies with the batch rather than health
status. However, these studies used different methods and
approaches to detect aEPEC, and it is therefore possible that
the differences in methods could impact the results.

In Korea, previous studies demonstrated that STEC and
EPEC strains were identified in 2.0% and 2.5%, respectively,
of the isolates collected from pork meat. A similar study
conducted in New Zealand found that 4.0% were STEC iso-
lates, whereas in the United States, the prevalence of this
pathotype was 18% (Samadpour et al., 1994; Brooks et al., 2001;
Lee et al., 2009). These data demonstrated that meat, including
pork, could potentially act as a reservoir for pathogenic E. coli
in humans. A study conducted in cows concluded that E. coli
that contaminates meat can originate from feces (Aslam et al.,
2003). It is likely that E. coli from feces have contaminated the
meat in this study. The isolates that showed 100% similarity
by ERIC-PCR and shared the same genetic profile suggest that
this ocurred.

In contrast to other studies on E. coli (Paton et al., 2001;
Cergole-Novella et al., 2007; Vu-Khac and Cornick, 2008), we
did not find isolates possessing both the ehxA and saa genes.
Instead, our isolates contained only the ehxA gene, which
had already been reported from EPEC isolates obtained from
healthy cattle (Aidar-Ugrinovich et al., 2007). Other reports
(Osek et al., 2003; Tatarczak et al., 2005) identified the pres-
ence of lpfAO113 loci in 100% and 61% of STEC from pigs.
Osek et al. (2003) suggest that LPFO113 contributed to the
colonization of the porcine host. As this gene was also found
in our study, it is possible that a similar event occurs in
porcine aEPEC.

Previous studies did not find lpfAO157/OI-141 and toxB in
STEC collected from pigs (Tatarczak et al., 2005; Osek et al.,
2006). To the best of our knowledge, this is the first report of
the lpfAO157/OI-141, lpfAO113 and toxB genes being found in
aEPEC isolated from swine.

The ehxA gene has been previously found in E. coli from
humans with diarrhea, hemorrhagic colitis and hemolytic
uremic syndrome (Cookson et al., 2007; Pradel et al., 2008). The

FIG. 1. Dendrogram of enterobacterial repetitive intergenic consensus (ERIC)–polymerase chain reaction (PCR) profiles
showing virulence genes and antimicrobial resistance of the 16 isolates of Escherichia coli from pigs. R, feces; C, carcass; AMP,
ampicillin; GEN, gentamicin; STR, streptomycin; CEP, cephalothin; STX, trimethoprim/sulfamethoxazole; CHL, chloram-
phenicol; NAL, nalidixic acid; TET, tetracycline.

1122 BORGES ET AL.

http://online.liebertpub.com/action/showImage?doi=10.1089/fpd.2012.1206&iName=master.img-000.jpg&w=373&h=240


lpfAO113 gene was also associated with aEPEC that causes
diarrhea in humans (Afset et al., 2006). Thus, the isolates re-
ported in our study that had these genes (13R, 15C, 25R, 46C,
67C) may have zoonotic potential.

Multidrug resistance was found in almost half of the E. coli
isolates from pigs, which were resistant against four or more
antimicrobials. This result is in agreement with other studies
conducted in Brazil, Germany, Switzerland, and the United
States that reported multidrug resistance of E. coli isolated
from healthy pigs (Stephan and Schumacher, 2001; Fratamico
et al., 2004; Von Müffling et al., 2007; Costa et al., 2010). This
result is not surprising because antimicrobials have been used
for many years for prophylactic purposes as well as for
growth promotion. The widespread use of antibiotics has
probably contributed to high rates of resistance (Mathew
et al., 1999; Stephan and Shumacher, 2001).

Vieira et al. (2011) reported strong and significant correla-
tions between resistances to aminoglycosides (streptomycin),
aminopenicillins (ampicillin), and fluoroquinolones (nalidixic
acid) in E. coli isolates from humans and pigs in many Euro-
pean countries. Our results also demonstrated increased re-
sistance of these isolates to the same class of antimicrobials.
Because these are important antimicrobials used in human
medicine, the results from this study are of great public health
concern (Collignon et al., 2009).

In conclusion, this study showed that pig carcasses and
feces carry aEPEC and STEC, as well as some putative ad-
hesins. The presence of certain genes, together with the high
level of resistance to antimicrobials and possible fecal con-
tamination of carcasses, demonstrate that E. coli from pork
may pose a concern to public health.

Acknowledgments

We thank Instituto Adolfo Lutz for the serotyping. C.A.B.,
L.G.B., and M.V.C. received scholarships from Coordenação
de Aperfeiçoamento de Pessoal de Nı́vel Superior, and R.P.M.
(process 2008/00417-0) received a scholarship from Fundação
de Amparo à Pesquisa do Estado de São Paulo during the
development of this work.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:
Clarissa Araújo Borges, M.D.

Via de Acesso Prof. Paulo Donato Castellane s/n
Jaboticabal, CEP 14884-900

São Paulo, Brasil

E-mail: lissa_bio@yahoo.com

STEC AND EPEC IN PIGS 1125