Characterization of Shiga Toxin–Producing Escherichia coli Strains Isolated from Swine Feces

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FOODBORNE PATHOGENS AND DISEASE
Volume 5, Number 6. 2008
Mary Ann Liebert, Inc.
DO!: 10.1089/fpd.2008.0147
Characterization of Shiga Toxin—Producing
Escherichia co/i
Strains Isolated from Swine Feces
Pina M. Fratamico,
1
Arvind A. Bhagwat,
2
Lisa Injaian,
1
and Paula J. Fedorka-Cray3
Abstract
The virulence gene and antibiotic resistance profiles of Shiga toxin—producing
Eschericliia co/i (STEC)
strains
belonging to 58 different O:H sei-otypes (219 strains) isolated from swine feces were determined. Of the 219
isolates, 29 (13%) carried the
stx
1
gene, 14 (6%) stx
2
, 176 (80)"O) stx
2
, 46 (21%)
c'stla,
14 (6.4%)
t'stl/i,
10 (4.6%)
fedA,
94 (42.9
0
%)
astA,
25 (11.4%)
Iily,
and one (0.46%)
cdt-Ill.
None of the strains possessed the
ell, bfp,
foeG,
fanfl, fasA, finiF
41
,, cnf-1, cnf-2,
ene, cdt-I,
or
cdt-/V
genes. The strains were also tested for antibiotic
susceptibility using 16 antibiotics. The STEC isolates displayed resistance most often to tetracycline (95.4%),
sulfa methoxazole (53.4%), kanamycin (38.4%), streptomycin (34.7%), and chloramphenicol (22.4%). An
E. co/i serotype 020:H42 strain, which was positive for
stx
2
,
and astA, was resistant to all of the antibiotics
tested except for amikacin. In addition, 52 of the swine isolates, representing 16 serogroups and 30 different
scrotypes, were examined for their ability to withstand acid challenge by three types of acid resistance (AR)
pathways, AR1
(rpoS
dependent), AR2 (glutamate dependent), and AR3 (arginine dependent). None of the
strains was defective in the ARI resistancepathway, while one strain was defective in the AR2 pathway
under aerobic growth conditions but fully functional under anaerobic growth conditions. Among the three
AR pathways, the AR3 pathway offered the least protection, and 8 out of 52 strains were defective in this
pathway. The strain that was defective in AR2 was fully functional in the AR3 pathway. Since AR plays a
vital role in the survival and virulence of these strains, differences among the isolates to induce AR
pathways may play a significant role in determining their infective dose. This study demonstrates that
swine STEC comprise a heterogeneous group of organisms, and the possession of different combinations of
E. co/i
virulence genes indicate that some swine STEC can potentially cause human illness.
Introduction
E
ScHERJCH1A COLI
STRAINS
that produce one
or more types of cytotoxins known as Shiga
toxins or verotoxins are referred to as Shiga
toxin—producing
E.
coli
(STEC) or verotoxigenic
E. co/i. STEC 0157:H7,
also known as an en-
terohemorrhagic
E. co/i is
the most important
STEC in the United States. Other STEC sero-
groups, including
026, 091, 0103, 0111, 0121,
and 0145, are emerging foodborne pathogens
(Bettelheim,
2007;
Gyles,
2007).
STEC have been
classified into pathogroups A to
E
based on the
severity of diseases they cause and their asso-
ciation with outbreaks (Karmali
et al., 2003).
The
major STEC virulence factors are the Shiga tox-
ins; however, other important virulence genes
include
Ithi
(plasmid-encoded hemolysin) and
eae (intimin). STEC may also possess genes that
encode fimbrial and nonfimbrial adhesins, pro-
teases, and other toxins, including
astA
(en-
teroaggregative
E. co/i
heat-stable enterotoxin,
'Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Wvndtnoor, I'ennsvlvania.
:
Beltsvill
e
Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland.
'Richard B. Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia.
'Current address: Universit
y
of Mar
y
land, Department of Cell Biolog
y
and Molecular Genetics, College Park, Maryland.
827
'Iii

828

FRATAMICO ET AL.
EAST1) and
cdt
(cytolethal distending toxin)
(Gyles, 2007).
Food is the major vehicle for human trans-
mission of 0157 and non-0157 STEC, and out-
breaks of hemorrhagic colitis and hemolytic
uremic syndrome (HUS) have been linked to
contaminated food of bovine origin as well as
contaminated produce and salads (Smith and
Fratamico, 2005). STEC have been isolated from
a wide variety of farm and wild animals and,
generally, the organisms do not cause disease in
these animals (Fairbrother and Nadeau, 2006).
Ruminants, such as cattle and sheep are im-
portant reservoirs for 0157 and non-0157
STEC, and a number of studies have also shown
that swine are potential reservoirs of STEC,
including
E.
coil
0157:H7 (Feder
et cii,
2003;
Schierack
et al.,
2006; Vu-Khac et
al.,
2007).
Moreover, strains of enterotoxigenic
E. coil
(ETEC), which elaborate labile toxin (LT) and/
or stable toxins (STs); ETEC and STEC strains,
which elaborate LT, STs, and Shiga toxins; and
STEC strains can cause enteric infections in pigs
(Blanco et
al.,
1997; Vu-Khac
et al.,
2007). Pigs
may also carry the organisms and be asymp-
tomatic (Schierack
et al.,
2006; Zweifel et
al.,
2006). Fimbrial adhesins, K99, F41, K88, and
987P, encoded by fanA, filflF
41a
, fcieG,
and
fcisA,
respectively, mediate adhesion and promote
colonization of ETEC in the small intestine of
neonatal pigs. The F18 fimbriae mediate colo-
nization of both ETEC and STEC in weaned
pigs. The
astA
gene encoding the EAST1 toxin is
commonly found in swine ETEC and STEC;
however, the contribution of
astA
to coliba-
cillosis in swine caused by these organisms has
yet to be elucidated (Vu-Khac
et al.,
2007). Genes
that encode for cytolethal distending toxin
(cdt)
and cytotoxic necrotizing factor
(cuJ)
have also
been found in
E.
coil
strains from pigs with di-
arrhea or pigs that had abortions (Pohl et
al.,
1993; da Silva and da Silva Leite, 2002). STEC
strains that harbor the stX2, variant gene are
commonly associated with edema disease in
pigs but have also been isolated from healthy
pigs (Zweifel
et al.,
2006; Vu-Khac
et al.,
2007).
Furthermore, Stx2e-producing STEC have
caused diarrhea and HUS in humans (Thomas
et
al.,
1994; Friedrich
et al.,
2002).
Since the 1950s, antibiotics have been used in
agriculture to treat infections and improve
growth and feed efficiency in livestock and
poultry. Among the antibiotics approved for
nontherapeutic use in the swine industry are
aminoglycosides, such as gentamicin; fI-lactam
antibiotics, such as ampicillin and lincosamine;
and macrolides, such as erythromycin, strepto-
gramin, tetracycline, and spectinomycin. Some
of these antibiotics are important in human
clinical medicine as well (Mathew et
cii.,
2007).
Overuse of antibiotics in animal production and
human medicine may facilitate the dissemina-
tion of antibiotic resistance genes among bac-
terial populations. Under conditions simulating
the porcine ileum, Blake
et cii.
(2003) demon-
strated the transfer of antibiotic resistance
among commensal
F. coil, E.
coil
0157:H7, and
SaimoneiIci
strains. It was reported in one study
that there was a higher prevalence of antimi-
crobial resistance in bacteria from nasal, pha-
ryngeal, and fecal flora of healthy pig farmers
compared to nonfarmers, possibly due to con-
tact with antimicrobial-resistant bacteria from
pigs and the farm environment (Aubry-Damon
et
al.,
2004).
Tolerance to low pH environments encoun-
tered in certain foods and in the gastrointestinal
tract is an important virulence characteristic of
foodborne pathogens. The infectious dose cor-
responds to the pathogen's ability to withstand
gastric acid challenge (Foster, 2000).
E. coil
0157:H7 and other STEC possess multiple
mechanisms to survive in acidic environments.
Lin
et cii.
(1996) characterized three AR mecha-
nisms in
E. coil
0157:H7, which include acid re-
sistance (AR) pathways 1, 2, and 3 (AR1, AR2, and
AR3, respectively). AR] is a glucose catabolite-
repressed system apparent when the cells are
grown to stationary phase in Luria-Bertani
(LB) medium and requires RpoS (encoding the
stationary phase alternative sigma factor) and
CRP (cAMP receptor protein)-dependent genes.
AR2 and AR3 are acid-induced glutamate-
dependent and arginine-dependent systems,
respectively. Studies have shown that there is
significant interstrain variability in acid toler-
ance in STEC, and
F. coil
0157:H7 strains are
often less tolerant to acid environments com-
pared to other STEC (Molina
et al.,
2003; Large
et al.,
2005). Previously we reported the isolation
of 219 STEC strains from fecal samples from
clinically healthy pigs as part of the National

STEC STRAINS FROM SWINE FECES

829
Animal Health Swine 2000 Study (Fratamico
et al.,
2004). The objective of the present study
was to further characterize the swine STEC
strains by examining their (1) virulence gene
profiles, (2) susceptibility to 16 different antibi-
otics, and (3) ability to withstand acid challenge
by three AR pathways.
Materials and Methods
Bacterial strains and growth conditions
The STEC strains were isolated from swine
feces as described previously (Fratamico et
al.,
2004) and were serotyped at The Pennsylvania
State University,
E.
cciii
Reference Center, Uni-
versity Park, Pennsylvania. The bacteria were
kept as frozen stocks in tryptic soy broth (Becton
Dickinson, Sparks, MD) containing 20% glyc-
erol, and they were routinely grown in tryptic
soy broth or on tryptic soy agar (Becton Dick-
inson) plates at 37'C.
Detection of virulence-related genes by PCR
A colony from tryptic soy agar was resus-
pended in 200 tL of PrepMan Ultra reagent
(Applied Biosystems, Foster City, CA), and
DNA extraction was performed according to the
manufacturer's instructions. The PCR assays
were performed using the PCR Reagent System
Kit (Invitrogen, Carlsbad, CA) and a GeneAmp
PCR System 9600 thermal cycler (Applied Bio-
systems). The PCR amplification consisted of
20mM Tris-HC1 (pH 8.4), 50 m KC1, 3.0mM
M
gC
l
2,
400 1.iM of each of the four dNTPs, 2.5 U
of
Tciq
DNA polymerase, and 5 iL of template
DNA. The primers used in the PCR assays and
the sizes of the expected PCR products are listed
in Table 1. The concentrations of the primers
and the PCR cycling conditions were as de-
scribed in the original references also listed in
Table 1.
Antibiotic susceptibility testing
The isolates were tested for resistance to
16 antibiotics using the broth microdilution
method and the Sensititre automated antibiotic
susceptibility system (Trek Diagnostics Systems
Limited, Westlake, OH) according to the man-
ufacturer's instructions. The antibiotics tested
included amikacin, amoxicillin/clavulanic acid,
ampicillin, cefoxitin, ceftiofur, ceftriaxone,
cepha lothin, chloramphenicol, ciprofloxacin,
gentamicin, kanamycin, nalidixic acid, strep-
tomycin, su lfamethoxazole, tetracycline, and
trirnethoprim/ sulfarnethoxazole. Results were
interpreted using the Clinical and Laboratory
Standards Institute (CLSI, 2006) breakpoints,
when available. National Antimicrobial Re-
sistance Monitoring System (NARMS) inter-
pretive criteria, as established by the NARMS
working group, were used for antimicrobial
agents without CLSI-approved breakpoints.
Quality control strains
E.
cciii
ATCC 25922,
En-
terococcus foecauis
ATCC 29212, and Sta
phylo-
coccus aureus
ATCC 29213 were used.
Determination of acid tolerance
Growth conditions.
The strains were streaked
onto LB agar (Becton Dickinson) from frozen
stocks, and after growth for 18 hours at 37C, a
single colony was inoculated into LB growth
medium buffered with 100 mM morpholine-
ethanesulfonic acid (MES, pH 5.5) and grown
with aeration (220 rpm, 37C) (Lin
et al.,
1996).
For fermentative growth under semi-aerobic
conditions, cultures were inoculated into 3 mL
of brain heart infusion broth (Becton Dickinson)
containing 0.4% glucose (BHIG) in 13x 100 test
tubes, which were placed at a 45 inclination
angle and incubated for 18 to 20 hours at 37-C
with aeration (220 rpm) (Cui et
al.,
2001).
Acid-challenge assays.
The amino acid-
independent AR system (AR1) was analyzed
as described previously (Bhagwat
et al.,
2005).
Briefly, cells were diluted from LB-MES medium
(1:200) into minimal E medium containing 0.4°A)
glucose (EG medium) (Small
et cii.,
1994), ad-
justed to pH 3.0 with 5 N HC1. The cells were
challenged for 2 hours at 37'C, and then diluted
in 50 mM phosphate-buffered saline (pH 7.2)
and plated onto LB agar medium to determine
the viable cell count. For testing of the amino
acid-dependent AR systems, the cells were di-
luted directly from the growth medium (1:200)
into prew armed (37
-
C) EG medium adjusted to
pH 2.0 and supplemented with either gluta-
mate (1.5mM, for GDAR or AR2) or arginine
(1.5mM, for ADAR or AR3) (CLSI, 2006). The
cells were challenged at 37-
-
C for 2 hours. The

Fin gineti
t
Size
(lip)
147
172
696
333
510
431
500
450
890
552
839
597
111
411
555
350
166
210
484
230
Reference
Blanco ci aL, 1997
Blanco ci (it., 1997
Blanco ci al., 1997
Kwon et al., 2002
Tmberechts ci al.,
1992
Kwon ci al., 2002
Kwon
ci (it.,
2002
Kwon
Ci
al., 2002
Cannon at al., 1997
Pass
Ci (Ii.,
2000
Pass ci al., 2000
Wider ci tit., 1996
Osek, 2003
Tóth ci al., 2003
Tóth et al., 2003
Tóth ci al., 2003
Fratamico ci al., 1995
Fratarnico ci al., 2004
Fratamico et (it., 2004
Blanco ci al., 1997
830

FRATAMICO ET AL.
TABLE 1. VIRULENCE GENE TARGETS, PCR PRIMERS, AND PRODUCT SIZE
Gene/gene product

Printer
esila (STa)

STA-1
STA-2
es'rlh (STh)

STB-1
STB-2
eli (LT)

LTA-1
LTA-2
fasA (F6, P987)

F6-F
F6-R
fedA (1`18, F107)

FedA-1
FedA-2
titflF
41a
(F41)

F41F
F41 -R
faeG (F4, K88)

F4-F
F4ac-R
lanA (P5, K99)

F5-F
F5-R
eaeA

EAE-F
EAE-R
cnJ-I

CNFI-F
CNFI-R
cnf-2

CNF2-F
CNF2-R
bjp

bfpl-F
bfp2-R
astA (EASTI)

EASTI-1
EASTI -2
Cdt-I

CDT-IS
CDT-lAS
cdt-111

CDT-IllS
CDT-Ill
AS
cdt-/V

CDT-IVS
CDT-IVAS
h/v
933
MFS1-F
MFSI-R
six
1
SLTI-F
SLTI-R
six2

SLT2-F
SLT2-R
StS2
e

VT2e-A
VT2e-B
Oligonucleotide sequence (5'-3')
TTAATAGCACCCGGTACAAGCAGG
CTTGACTCTTCAAAACAGAAAAT[AC
ATCGCATTTCTTCTFGC ATC
GGGCGCCAA AGCATGCTCC
GGCGACAG ATTATACCGTGC
CCGAATTCTGUATATATGTC
TCTGCTCTTAAAGCTACTGG
AACTCCACCGTTTGTATCAG
GTGAAAAGACTAGTGTITATTTC
CTTGTAAGTAACCGCGTAAGC
GAGGGACTTTCATCT1TfAG
AGTCCATFCCATFATAGGC
GGTGATTTCAATGGTTCGGTC
CCCAGCCGACGATTCAGAACCCCT
TGCGACTACCAATGCrFCTG
TATCCACCATTAGACGGAGC
GTGGCGAATACTGGCGAGACT
CCCCATTCTTmCACCGTCG
GGCGACAAATGCAGTA1TGCTTGG
GACGTTGGTTGCGGTAATTTTGGC
GTGAGGCTCAACGAGATTATGCACTG
CCACGCTTCTTCTTCAGTTGTI7CCTC
GATTGAATCTGCAATGGTGC
GGAflACTCTCCTCACATAT
CCATCAACACAGTATATC
GTCGCGAGTGACGGCTTTGT
CAATAGTCGCCCACAGGA
ATAATCAAGAACACCACCAC
GAAAGTAAATGGAATATAAATGTCCG
TTTGTGTCGGTGCAGCAGGGAAAA
CCTGATGGUCAGGAGGCTGGTTC
TTGCTCCAGAATCTATACCT
ACGATGTGCTTFATTCTGGA
CTTCACGTCACCATACATAT
TGTAACTGGAAAGGTGGAGTATAC
GCTATTCTG ACTCAACGAAAAATAAC
GTTTTTC1TCGGTATCCTATTCC
GATGCATCTCTGGTCATTCTATTAC
CCTTAACTAAAAGGAATATA
CTGGTGGTGTATGATTAATA
viable cell counts were determined after acid
challenge by diluting the cells in phosphate-
buffered saline (50 mM, pH 7.2) and plating
immediately onto LB agar. As per the previous
classification scheme (Bhagwat et al.,
2005), if
the surviving population after the acid chal-
lenge was _-1.0%, the isolate was considered
resistant for the particular pathway. The AR
phenotype was tested at least three times and
each experiment had two replicates. Control
experiments were performed for each strain as
described b
y
Lin
et al.
(1996), which involved
either growing the cells under fermentative (in
BHIG) growth conditions (AR1 assays) or sub-
jecting the cells to pH 2 in the absence of glu-
tamate or arginine in the challenge medium
(AR2 and AR3 assays, respectively). Less than
0.001% of the cells survived acid challenge at
pH 2.0 without addition of either glutamate or
arginine. For statistical analyses, SigmaStat 3.0
software (Ashburn, VA) was used. Cell survival
data were analyzed by one-way analysis of
variance (ANOVA) to determine statistical dif-
ferences between means of treatments.

STEC STRAINS FROM SWINE FECES
Results and Discussion
Serotypes of the STEC isolates
and virulence profiles
The 219 swine STEC isolates consisted of 27
different 0 serogroups, including 18 0 (non-
typeable) strains and one strain that gave auto-
agglutination, and 58 0:H serotypes (Table 2).
Thirty of the strains (14%) were serogroup 0
nontypeable (0), and of these, three possessed
the stx
1
gene, one had
stx7,
and 26 had
StX2e, in
combination with different virulence genes.
Thirty-four percent (75/219) of the strains be-
longed to serogroup 08 associated with seven
different H types. Only four of the strains har-
bored stx
2
, and the rest had StX
7
e. A review of the
literature by Bettelheim (2007) on the isolation
of STEC serogroups from food, humans, and
healthy and diseased animals showed that
E.
coil
serogroup 08 has been isolated over 100
times from food, humans, and animals, includ-
ing pigs. The most common serotypes were
08:H, 08:H9, and 08:H19, and in the present
study these serotypes accounted for 88% (66/75)
of the 08 STEC strains. There were 22
E. co/i
0100 strains that were H nontypeable (H
-
) or
H30. All of the strains possessed stx, except
one that had stx
2
. Serogroups 0 nontypeablc,
08, and 0100 accounted for 58% (127/219) of
the swine STEC strains. This is in agreement
with results of Kaufmann
et al.
(2006) who also
found that
E.
coil
belonging to serogroups 0
nontypeable, 08, and 0100 represented 73%
(33/45) of the STEC strains isolated from heal-
thy pigs at slaughter in Switzerland.
Characterization of 266 STEC strains associ-
ated with human infections, including severe
enteritis, HUS, and mild diarrhea, showed that
the strains belonged to 81 serotypes and had 40
different combinations of virulence markers
(Prager et
at.,
2005). There was no clear link
between various virulence gene combinations
and the different clinical syndromes. In the pres-
ent study, the swine STEC strains possessed
22 different combinations of virulence genes.
Forty-three percent (94/219) of the STEC strains
harbored
nstA
that encodes for the EAST1 toxin.
Other investigators also found that the
astA
gene was common in swine STEC (Kaufmann
et at.,
2006; Zweifel
et at.,
2006; Vu-Khac et at.,
2007). In the present study, 29/219 isolates
831
had stx
1
, 14 had stx
2
, and 176 possessed StX2e A
number of serogroups, including 0, 07, 08,
096, 0100, 0120, 0121, and 0159 possessed the
stx
2
gene, in addition to either
estlb
and
astA,
fc'dA and astA, or
astfl.
Of the 29 strains that
harbored stx
1
, seven belonged to serogroup
091. stx
l
was the only virulence gene in four
091 strains, 14 strains harbored
stx
1
and
h1y933,
and one strain had stx
1
,
astA,
and
111Y133.
Bettel-
heim (2007) found that 091 STEC serotypes
091:H , 091:H10, 091:H14, and 091:H21 pre-
dominated among strains isolated from food
and from healthy and diseased humans and
animals. One third of the reports on 091 de-
scribed cases of human illness. In the current
study, 16 strains belonged to serogroup 091; 10
strains were serotype 091:H and two were
091:1-114, and these strains had
stx
1
and
111,'933.
Two 091:H12 strains possessed stx
1
and
h1y933,
and two 091:H44 strains possessed
stx
2
.
only.
Genomic subtractive hybridization was used
to identify 42 sequences in a strain of 091:H21
that caused HUS that were absent from STEC
strains of serotype 06:1-110 (Pradel
et at.,
2002).
Nineteen corresponded to unique DNA se-
quences in F. co/i 0157:H7 EDL933, including
prophage sequences and putative virulence
genes, while other sequences were related to
virulence plasmids found in
Shigelia
and path-
ogenic
E. coil,
Hybridization with a stx
2
DNA
probe showed that the 091:H2 strain had at
least three copies of the
stx
2
gene. The investi-
gators suggested that the transfer of virulence-
linked mobile genetic elements contributes to
the evolution of pathogenic STEC strains. Dif-
ferent types of fimbriae, including F4 (encoded
by
faeG),
F5
(fanA),
F41
(flfliF
41a
),
and 987P
(fasA)
mediate colonization of ETEC and STEC
in the porcine small intestine. In the current
study, PCR assays were positive only for the
fedA
gene that encodes for the major subunit of
F18 fimbriae, and
fedA
was found only in strains
belonging to serogroup 0121 (Table 2). PCR
assays were negative for the
elf, fasA, firnF41a,
faeG, fanA, bfp,
cuif-1,
cnf-2,
cdt-I, cdt-IV,
and
eaL'A GEN
in all of the swine STEC strains.
Many of the STEC serogroups that were iso-
lated, including serogroups 020, 091, 0101,
and 0121 have been associated with human
illness. As an example, E.
coil
serotypes 091:H,
091:1-110, 091:H21, and 091:H40 have been

832

FRATAMICO ET AL.
TABLE 2. SEROTYPES AND VIRULENCE GENES OF THE SWINE SHIGA ToxiN-PRoDucING
EScIIERIcIIIA COLI
(STEC)
ISOLATES (N_219)a
Serotype

Total

stx1

stx2

stx2,

estla

estlb

fedA

astA

-
-_
cdt-III

111y33
A:H

I

+

+
O,H

2

+

+

+
O:H

I

+

+

+

+
Q:H

5

+

+

+
I

+

+
O:H

I

+
0:H20

I

+

+
0:H30

3

+

+

+
0:H30

I

+

+
0:H30

2

+

+
O:H30

5

+
O:H30

I

+
0:H34

1

+

+
O:H4

1

+
0:H42

I

-

+

+
O:H42

I

+
O:H51

I

+
o :H51
and
24

I

+
0-:H9

I
02:H44

I

+
05:H17

I

-r
07:H"

I

+

+

+
08:H

2

+

+

+

+
08:H

4

+

+

+
08:H

I

+

+

+
08:H

5

+

+
08:H

6

+

+
08:H

4

+

+
08:H

13

+
08:H

2

+
08:H3

I

+

+
08:H9

2

+

+

+
08:1-19

6

+

+
08:1-19

8

+

+
08:H9

4

+
08:H9

I

+
08:H17

2

+
08:H19

4

+

+
08:H19

4

+
08:H20

I

H-

+
08:1-120

2

+

+
08:1-120
and
37

1

+
08:H20
and
37

2

+
09:1-117

1

-

+

+
09:1-117

1

-

+
09:H19

1

-
09:H9

1
011:H25

1

±
015:H

1

+
020:H

3

+

+
020:1-119

I

+
020:H30

5

+
020:H42

3

+

+
057:H

1

+

+
057:1-114

1

+
065:H

1

+
068:H

1

+

+
069:H26

2

+

+
078:H

1

+
091:H

1

+

+

+
(continued)

STEC STRAINS FROM SWINE FECES

833
TABLE
2.
(CONTINUED)
Scrotipe

Total

stx1

stx2

StX2e

estla

estib

fedA

astA

cdt-Ill

hlys
091:1-1

9

+

+
091:1
-
112

2

+

±
091:1
-
114

2

+

+
091:F144

2

+
096:H5

I

+
OiOO:H

I

+

+
0100:H3()

2

±

+
0100:H30

2

±

+

+
0100:H30

4

+

+
0100:H30

1

+

+
0100:1130

12

+
0101:H

3

+

+
O101:H

1

+
0120:1-1

15

+

+
0120:H30

I

+
0121:H

2

-1-

+

+
0121:H

1

-i-

+

+
0121:H10

5

+

+

+
0121:1-110

2

+

+

+
0152:H

1

±
0159:H

1

+

+
0159:H

I

+

+

+

+
0159:H34

1

+
0159:1-14

1

+

+

+

+
0159:H4

I

+

+

+
O16O:H

1
0163:H

4

-
0163:H41 and 51

1
OXIO:H

1

+

+

+

+
0X18:H

I

-
OXI8:1-123

1

-
0X18:H24 and 56

1

-
Total

219

29

14

176

46

14

10

94

1

25
The following virulence genes were not detected by the PCR in any tested serotvpes: c//,
fioA,f
zn
i
F
41,,f
a
c
G
,f
anA
,
bfp,
cnf-1, cnf-2,
oft-1, cdt-/V.
and
'Ot'fl(;FN.
b
The strain carries the
fliC
gene; however, the restriction fragment length polymorphism pattern does not match that of
any of the
known 53 F.
co/i
H types.
A, autoagglutination; 0 and H 0 and 1-1 nontypeable (no reaction with any of the standard antisera).
associated with cases of bloody diarrhea and
HUS (Bonnet
et al.,
1998; Beutin
et al., 2004;
Betteiheim,
2007). E. co/i
0121:1-119 was associ-
ated with an outbreak of hemorrhagic colitis
with three cases of HUS at a lake in Connecticut
in 1999 (McCarthy
et al.,
2001). In the current
study, four
stx
2
-producing
E. coli
0101:H
strains were isolated from swine feces. Stx2e-
producing strains of
E. co/i
0101 have been
isolated from patients with diarrhea and HUS
(Thomas
et al.,
1994; Franke
ct al.,
1995). Franke
et al.
(1995) determined the relatedness of
E. coli
0101 strains from humans and pigs. The DNA
sequence of the
stx
2
gene of the human isolate
was identical to that of a classical swine STEC
0139 strain that causes edema disease, and the
porcine 0101 strains showed greater than 99%
similarity to 0139. There was a high degree of
genetic relatedness among the human and por-
cine 0101 strains based on DNA fingerprinting.
However, virulence factors typically found in
porcine STEC, such as ST (encoded by
est), LT
(elf),
and
F107 (fedA)
fimbriae, and in human
STEC, such as EaeA and Hly, were not found
in the
E. co/i
0101 strains, thus suggesting that
the human Stx2e-producing
E. co/i 0101
strain
caused illness via a different pathogenic mech-
anism.
Antibiotic resistance in the swine STEC strains
The utilization of antimicrobial compounds
in swine production may contribute to the
increased prevalence of antibiotic-resistant

834

FRATAMICO ET AL.
TABLE 3. NUMBER AND PERCENTAGE OF SWINE
Siiic
TOXIN-PRODUCING
EscIfEu!cI!M COLt
(STEC) STRAINS
SHOWING SPECJFJC ANTIBIOTIC RESISTANCE PATTERNS
(tc= 219)
Resistance
pattern

Na
Tet

57

26.0
SulTet

32

14.6
KanSulTet

17

7.8
KanStrSu ITet

15

6.8
ChlKanStrSulTet

11

5.0
KanTet

8

3.7
AmpChlKanStrSulTet

5

2.3
AmpTet

5

2.3
ChlStrSulTet

5

2.3
KanStrTet

5

2.3
StrTet

5

2.3
Pan-susceptible

5

2.3
AmpCepChlKanStrSulTet

4

1.8
ChiSulTet

4

1.8
ChiTet

4

1.8
StrSulTet

4

1.8
AmpSullet

3

1.4
ChlKanStrTet

3

1.4
AmpCepKanStrSullet

2

0.9
Am pCepKanStrTet

2

0.9
AmpGenKanStrSulTet

2

0.9
ArnpKanStrSullet

2

0.9
ChiKanSulTet

2

0.9
ChlStrlet

2

0.9
Str

2

0.9
AmoAmpFoxCefAxoCep

0.5
ChlCipGenKanNalStrSulTetTri
ArnoCefSulTet

0.5
AmpCepChlKanStrTet

I

0.5
AmpCepChlStrlet

1

(3.5
A mpChlGenKanStrSulTetTri

I

0.5
AmpChlKanStrTet

I

0.5
ArnpChlStrTet

I

0.5
AmpChlSulTet

I

0.5
ArnpSul

I

0.5
CcfSul

0.5
ChlGenKanStrSulTet

I

0.5
ChiSul

1

0.5
KanSulTetlri

1

0.5
Total

219

100.0
Arno,
amoxici!!in
,
/clavulanic acid; Amp, ampicillin; Fox,
cefoxitin;
Cef, cettiour; Axo, ceftriaxone; Cep, cephalothin; Chi,
chloramphenicol; Cip, ciprofloxacin; Gen, gentanticin; Kan, kana-
mycin; Nal, nalidixic acid; Sir, streptomycin; So!, sulfamethox-
azole;
let, tetrac
y
cline; Tni, trimethoprim/sulfamethoxazole.
foodborne pathogens. Stephan and Shumacher
(2001) reported that increases in resistance to
streptomycin, sulphonamide, and tetracycline
in non-0157 STEC isolated from asymptomatic
meat industry workers, healthy slaughter pigs,
ground beef, beef carcasses, and cattle may be
due to widespread use of these antibiotics in the
early finishing phase of fattened pigs and also
in cattle. Eight Stx2e-producing STEC 0100:H
strains from healthy pigs were resistant to eight
antibiotics.
The spread of resistance among bacteria is
associated with mobile DNA elements such as
plasmids, transposons, and integrons. In addi-
tion, multiple resistances to antibiotics in
E. coli
and
Salmonella
have been linked to the
inarRAB
operon induced by a variety of chemical and
antimicrobial agents (Sulavik et
al.,
1997). Sul-
fonamides, f3-lactams, tetracyclines, and ami-
noglycosides, such as amikacin, gentamicin,
kanamycin, streptomycin, and apramycin, are
widely used in swine production for growth
promotion, as well as for treatment and pre-
vention of disease. Thus, it is not unexpected
that resistance to some of these antibiotics was
observed in the swine STEC examined in the
present study. Only 2.3% (5/219) of the STEC
strains were susceptible to all 16 antibiotics
tested, 27% (59/219) were resistant to one anti-
biotic, 26% (57/219) were resistant to two, 16%
(35/219) were resistant to three, 13% (29/219)
were resistant to four, 8% (17/219) were resis-
tant to five, 5
0
%
(
11/219) were resistant to six,
2% (4/219) were resistant to seven, one strain
was resistant to eight, and one was resistant to
15 antibiotics (serotype 020:1-142, possesses stx2(,
and
astA)
(Table 3). The STEC isolates displayed
resistance most often to tetracycline (95.4%),
sulfamethoxazole (53.4%), kanamycin (38.4%),
streptomycin (34.
7%), chlora mphenicol (22.4%),
and ampicillin (15.1%) (Table 4).
Acid tolerance
Fifty-two out of 219 swine STEC strains were
examined for acid tolerance through the AR1,
AR2, and AR3 pathways. The 52 strains con-
sisted of 16 serogroups and 30 different 0:H
serotypes. The strains that were tested included
the 29 strains that carried the
stx
1
gene, 13 of the
14 strains that carried stx
2
, and 10 strains that
carried stx
2
.. One factor critical to the AR1 sys-
tem is the stationary phase-associated sigma
factor aS,
the product of the
rpoS
gene (Cui
et al.,
2001; Price
et al.
2004). None of the strains was
defective in the rpoS-mediated acid-tolerance
pathway (AR1), which is tested at pH 3 for 2
hours in minimal E medium with glucose and
with no exogenous amino acids (Table 5). Under
aerobic growth conditions the a' factor is also

"t-2c'
et/a, asiA,
611933
six
Sf52,., edit,,
aSIA,
lily933
,Vt52e, estici,
asiA
SIX,
St-VIII
six1
.5137,
estlb, asiA, cdt-111
'•
KanSulTetTri
Tet
ChlKanStrSulTet
AmpChlGenKanStrSulTetTri
Tet
Tet
Pan-susceptible
ArnpChl KanStrSu]Tet
AmpChl KanStrTet
AR-3
AR-2 aerobic pathway
AR-3
AR-3
AR-3
AR-3
AR-3
AR-3
AR-3
STEC STRAINS FROM SWINE FECES

835
TABLE 4. PERCENTAGES
OF SWINE
SHIGA TOXIN-
PRODUCING Esci-milcElA
COLI
(STEC) STRAINS (N=219)
SUSCEPTIBLE, RESISTANT, OR SHOWING INTERMEDIATE
RESISTANCE
TO
THE ANTIBIOTIcS TESTED
Ant itnicro b/al Susceptible Intermediate Resistance
Amikacin

100

0

0
Arnoxicillin/

98.2

0.9

0.9
clavulanic acid
Ampicillin

84.9

0

15.1
Cefoxltin

99.5

0

0.5
Ceftiofur

98.6

0

1.4
Ccftriaxone

99.5

0

0.5
Cephalothin

72.1

22.8

5
Chloramphenicol

75.3

2.3

22.4
Ciprotloxacin

99.5

0

0.5
Gentarnicin

96.8

0.9

2.3
Kanarnycin

61.6

0

38.4
Nalidixic acid

99.5

0

0.5
Streptomycin

65.3

0

34.7
Sulfamethoxazole

46.6

0

53.4
Tetracycline

3.2

1.4

95.4
Irirnethoprirn/

98.6

0

1.4
sul Ia methoxazole
necessary to induce a second AR system in-
volving a glutamate d eca rboxyla tion-an ti porter
system (GDAR, AR2), which protects the cell
under extreme acid conditions (pH 2.0) pro-
vided glutamate is available in the external
medium (Price et
al.,
2004). One STEC strain,
serogroup 0 :H51, that harbored stx
2
was de-
fective in the AR2 pathway (<1.0% survival)
under aerobic induction. The rpoS-mediated AR
system is not operative under anaerobic or fer:
mentatively metabolizing cells, nor is the &'
factor necessary to induce GDAR (Foster, 2000;
Castanie-Coronet and Foster, 2001). In the cur-
rent study, the glutamate-dependent and
rpoS-
mediated AR systems provided a significantly
higher level of AR (88 + 18% cell survival for
ARI and AR2) compared to that observed for
arginme-dependent acid resistance (39±11%
cell survival) (AR3;
p
<0.05). Unlike two previ-
ous comprehensive studies, in which a large
proportion of clinical and foodborne pathogenic
strains of
E.
coli
with dysfunctional RpoS were
detected (Waterman and Small, 1996; Bhagwat
et
al.,
2005), most isolates in this study did not
appear to have a mutation in
rpoS.
This con-
clusion is based on the fact that 52 out of 52
strains had functional AR1, while 51 out of 52
strains were able to induce AR2 under aerobic
growth conditions. However, several strains
(8 out of 52) were defective in the AR3 pathway
(<1.0% survival), which is arginine dependent
and is induced under anaerobic conditions in
the presence of glucose. In one previous study,
E. coli
0157:H7 strains lacking the AR3 pathway
showed comparable survival in apple cider and
during passage through the gastrointestinal
tract of calves (Price et (i!., 2004). It was thus
proposed that the ARI and/or AR2 systems
may be required for survival of
E.
co/i
in the
bovine gastrointestinal tract and in apple cider
and that different AR systems are used based
on exposure to different acidic environments
(Price et
al.,
2004). In the current study, the
strain defective in AR2 had a fully functional
AR3 pathway. All of the eight strains that were
defective in the AR3 pathway had a functional
T\RI.F
5. SEROTYPES AND VIRULENCE ANI) ANTIBIOTIC RESISTANCE PROFILES
OF
STRAINS Di;rcrivr
IN ACID
RESISTANCE (AR) MECHANISMS
Sero/iipe of

Virulence

Antibiotic resistance

Defect in
sa
OIL
STECstrain'

Siniploj6lc

profile

AR nurhanini
0 :H
0 :H51
08tH
08:11
08:H
08tH 17
015tH
0159:H4
0160:H
'Out of the 52 swine STEC strains examined for the AR1, AR2, and AR3 pathways, the nine strains shown were defective in one of
the acid resistance pathways. STEC, Shiga toxin-producing
Esc/u'ridiia colt.
Amp, ampicillin; Chi, chloraniphenicol; Gen, gentamicin; Kan, kanamycin; Str, streptomycin; Sul, sulfamethoxazole; Tet,
tetracycline; Tn, trimethoprim,/sulfamethoxazole.

836

FRATAMICO ET AL.
AR2 pathway. There were no strains that were
defective in two AR pathways. The data high-
light the biological significance of having mul-
tiple acid resistance pathways in STEC strains.
Comparing survival of
E. coli
0157:H7 strains to
non-0157 STEC strains by the AR1, AR2, and
AR3 pathways, Large et
al.
(2005) did not find
that E. coli
0157:H7 strains had higher survival
rates compared to non-0157 strains. They also
found that there was considerable variation in
acid tolerance with the three AR mechanisms
among the strains examined. Furthermore,
Molina
et al.
(2003) found that a STEC 091:H21
strain survived longer when challenged at pH
2.5 compared to other STEC strains, including
0157:H7. In the present study, swine STEC
strains defective in AR3 or AR2 aerobic path-
ways and possessing stx
1
,
stx
2
,
or stx
2
, and none
of the other virulence genes were resistant only
to tetracycline (Table 5), A STEC strain serotype
08:H defective in AR3 possessing stx7,
estla,
and
astA
was resistant to eight antibiotics, and
an
E. coli
serotype 0159:H4 strain also defective
in AR3 and possessing stx
2
,
estib, astA,
and
cdt-
ill was resistant to six antibiotics.
Conclusion
This study demonstrated that STEC isolated
from clinically healthy swine belong to many
different serotypes and have numerous viru-
lence gene profiles. Although it is unknown if
these strains can cause disease in humans, the
presence of Shiga toxin genes and other impor-
tant STEC and ETEC virulence genes indicate
that these strains may have human pathogenic
potential. However, none of the strains pos-
sessed the
eae
gene, which encodes intimin, an
important virulence factor. Further studies are
needed to understand the intricate interplay
between virulence factors and to identify addi-
tional virulence genes that may play a role in the
pathogenesis of STEC strains. Among the three
acid resistance pathways, AR1 and AR2 pro-
vided significantly higher protection, and no
strain was defective in more than one acid re-
sistance pathway. The ability of STEC to tolerate
acid environments and the ease at which F.
coli
strains are known to acquire virulence factors
and antibiotic resistance genes via mobile ge-
netic elements likely leads to the evolution of
pathogenic STEC clones. STEC are found in
many animal species; however, the extent to
which animals, including pigs play a role in the
epidemiology of human STEC infection war-
rants further investigation.
Acknowledgments
We are grateful to Lori Bagi at the Eastern
Regional Research Center (ERRC) and Jovita
Haro at the Richard B. Russell Research Center
for their technical assistance. We also thank Dr.
James Smith (ERRC) for critical reading of the
manuscript. Mention of trade names or com-
mercial products is solely for the purpose of
providing specific information and does not
imply recommendation or endorsement by the
U.S. Department of Agriculture.
Disclosure Statement
No competing financial interests exist.
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Anti-
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Esc/,crichia co/i;
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Eschericlua
co/i
strains isolated
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al.
Characterization of
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Address reprint requests to:
Pina M. Fratamico, Ph.D.
Eastern Regional Research Center
Agricultural Research Service
U. S. Department of Agriculture
600 East Mermaid Lane
Wyndmoor, PA 19038
E-mail:
pina.fratamico@ars.usda.gov
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