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Res. Microbiol.

151 (2000) 865871


2000 ditions scientifiques et mdicales Elsevier SAS. All rights reserved
S0923250800011530/FLA

Genotypic characterization of enteropathogenic Escherichia coli (EPEC)


isolated in Belgium from dogs and cats
Frdric Goffaux*, Bernard China, Laurence Janssen, Jacques Mainil
Laboratory of Bacteriology, Faculty of Veterinary Medicine, University of Lige, Bd de Colonster 20/B43,
4000 Lige, Belgium
Received 17 September 1999; accepted 6 July 2000

Abstract Enteropathogenic Escherichia coli (EPEC) are isolated from man and farm animals but also from
dogs and cats. They produce typical histological lesions called attaching and effacing lesions. Both plasmid
and chromosomal elements are involved in the pathogenesis of EPEC infection. The presence of these genetic
elements was investigated in 14 dog and three cat EPEC isolates. A bfpA-related gene was detected in five of
the 17 isolates in association with high molecular weight plasmids, and a locus of enterocyte effacement (LEE)
was present in all isolates. The LEE was inserted in the selC region in only 12% of the isolates. The eae, tir, espA
and espB genes were analyzed by multiplex PCR. The results indicated the presence of those genes in the
tested isolates with heterogeneity in the gene subtypes present: eae-tir-espA-espB (65%), eae-tir-espAespB (29%), eae-tir-espA-espB (6%). Moreover, the espD gene was also present in dog and cat EPEC. The
DEPEC and CEPEC form a heterogeneous group and five of them are closely related to human EPEC. 2000
ditions scientifiques et mdicales Elsevier SAS
Escherichia coli / EPEC / genotypic characterization / dogs and cats / Belgium

1. Introduction
Attaching and effacing Escherichia coli (AEEC)
are characterized by their capacity to infect
intestinal epithelial cells, resulting in so-called
attaching and effacing (AE) lesions. AEEC infection begins with the initial adherence, manifested by the local formation of bacterial microcolonies at the cell surface, called localized
adherence (LA) [39] and is followed by the
formation of AE lesion characterized by bacterial intimate attachment to enterocytes and disruption of brush border microvilli [34]. The AE
lesion is associated with the accumulation of
highly organized cytoskeletal components (actin,
-actinin, myosin light chain, ezrin and talin) in
epithelial cells immediately beneath the adher-

* Correspondence and reprints.


E-mail address: fgoffaux@ulg.ac.be (F. Goffaux).

ent bacteria [14], leading to the formation of a


pedestal-like structure [25]. AEEC groups
together enteropathogenic E. coli (EPEC) and
enterohemorrhagic E. coli (EHEC), which in
addition produce verotoxins or shigatoxins
(reviewed in [30]).
Both plasmid and chromosomal loci are
involved in the pathogenic process of human
EPEC infection. The LA is mediated by the
bundle forming pilus (BFP), a member of the
type IV fimbriae family [17]. The bfpA gene is
located on a high molecular weight plasmid
termed EPEC adherence factor (EAF) [16]. A
1-kb probe was derived from the EAF plasmid
and used as an epidemiological tool [35], but
this probe does not correspond to any coding
region of the plasmid [15]. In human EPEC
strain E2348/69, most of the genes necessary for
the formation of the AE lesion are clustered on
a 35.5-kb chromosomal pathogenicity island
called LEE (locus of enterocyte effacement),

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F. Goffaux et al. / Res. Microbiol. 151 (2000) 865871

which is inserted into an selC gene encoding the


tRNA for selenocysteine [32]. The LEE is sufficient to confer AE activity in vitro when introduced into a nonpathogenic E. coli strain [33].
The LEE can be divided into three functional
regions. The left part of the LEE contains genes
(esc and sep) that encode for a type III secretion
system responsible for the secretion of proteins
EspA, EspB, EspD [21] and Tir [23]. The middle
part of the LEE contains two genes (eae, tir), the
products of which promote intimate attachment
of bacteria to host cells and reorganization of
cytoskeletal actin beneath adherent bacteria.
The eae gene encodes a 94-kDa outer membrane
protein, the intimin, required for intimate adherence [22]; the tir gene encodes the intimin
receptor which is translocated into the eucaryotic cytoplasm where it becomes phosphorylated and then incorporated into the host cell
membrane [23]. The right part of the LEE contains three genes: espA, espD and espB, encoding
the proteins that are essential for EPEC-mediated
signal transduction events within the host cell,
including tyrosine phosphorylation of Tir and
the increase of inositol triphosphate and calcium levels, leading to the AE lesion formation
[10, 24, 27].
Natural infections with EPEC and/or intestinal AE lesions have been described in dogs [5,
20] and cats [38]. EPEC are considered as an
important cause of diarrhea in dogs, particularly in puppies [12]. In addition to the presence
of an eae gene, some EPEC strains isolated from
dogs harbor the espB gene and possess a high
molecular weight EAF-related plasmid carrying
the bfpA gene [4]. Moreover, eae, tir, espA, espB
and espD genes have been cloned from the dog
EPEC strain 4221 and sequenced [2, 3].
Canine and feline EPEC have also been identified in Belgium [28] using a colony hybridization assay with the Eae, EAF and BfpA probes.
In this study, we further characterized genotypically, E. coli isolates from dogs and cats for the
presence of virulence determinants related to
EPEC.

2. Material and methods


2.1. Bacteria

Fourteen Eae probe-positive isolates from


dogs (DEPEC) and three Eae-positive isolates
from cats (CEPEC) were included in this study
(refer to table I below). All were isolated from
animals with diarrhea and/or enteritis. The
human EPEC strain E2348/69 (O127:H6) [11],
the human EHEC strain ATCC43888 (O157:H7),
and the rabbit EPEC strain RDEC-1 (O15) [7]
were included as positive controls and the nonpathogenic E. coli HB101 as a negative control.
2.2. Colony hybridization

The colony blot hybridization assay was performed as described by Mainil et al. [29]. The
probes (table II) were labeled with 32P-dCTP by
random priming using the dCTP-labeling beads
(Ready to go, Pharmacia, Uppsala, Sweden)
according to the instructions of the manufacturer. LEEA corresponds to the 5 end of the
LEE, LEEB includes escV and escN genes, LEEC
includes the eae gene and LEED includes espA
and espB genes.
2.3. Plasmid DNA isolation and hybridization

Plasmid DNA was extracted using the modified method of Kado and Liu [6]. Plasmids were
separated by 0.5% agarose gel electrophoresis
and the gel was hybridized with the BfpA probe
as described by Mainil et al. [31]. The BfpA
probe was obtained by purification of PCR
products with the QIAquick-spin PCR purification kit (Qiagen, Hilden, Germany). The probe
was labeled as described in colony hybridization.
2.4. PCR reactions

The amplification of the bfpA fragment


(326 bp) was performed with primers EP1 and
EP2 [29]. Insertion of the LEE downstream of
selC was studied using primers K255 and K260
for the right junction, and K260 and K261 for the
intact selC gene [32]. If the LEE is inserted

F. Goffaux et al. / Res. Microbiol. 151 (2000) 865871

867

Table I. Results of PCR analysis of 14 DEPEC and three CEPEC isolates.


Isolate and strain
25211-2A
25314
26881-3
32106-2
41735-2
41927-1
41989-3
42430-1
42481-2
43401-2
43769-2
44053-1
44318-3
45337-2
35314-1
43748-1
43750-1
E2348/69
RDEC-1
ATCC43888
HB101

bfpA
+
+

+
+
+
ND
ND
NA

Multiplex PCR

espD

eae

tir

espA

espB

NA

NA

NA

NA

SelC disrupted

Species

by LEE
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
NA

yes
no
no
no
no
no
no
no
no
no
no
no
yes
no
no
no
no
yes
ND
ND
no

dog
dog
dog
dog
dog
dog
dog
dog
dog
dog
dog
dog
dog
dog
cat
cat
cat
human
rabbit
human

NA, no amplification. ND, not done. * Positive by plasmid hybridization.

Table II. Probes used in the colony hybridization assay.


Probe

Plasmid

Enzymes

Size (bp)

Reference

LEEA
LEEB
LEEC
LEED

pCVD453
pCVD461
pCVD443
pCVD460

MluI / EcoRI
EcoRI / SalI / PvuII
SalI / StuI
SmaI / XbaI

2 870
2 948
1 050
2 300

[32]
[32]
[32]
[32]

downstream of selC, primers K255 and K260


amplify a 418-bp fragment. In the absence of the
LEE, primers K260 and K261 gave a 527-bp
fragment since selC was intact. Four sets of four
primers (one constant and three variable) were
used for amplification and typing of eae, tir,
espA and espB genes by multiplex PCR [8]
following the classification of Adu-Bobie et al.
[1]. Primers B213 and B214 were used to amplify
a 419-bp fragment of the espD gene. Upper and
lower primers were chosen both in constant
regions from the published sequences of the
espD genes [3, 13, 36, 37].
The sequences of primers used in this study
are presented in table III. DNA to be amplified

was released from whole organisms by boiling.


PCR were performed in a Gene Cycler (Bio-Rad,
Hercules, CA, USA) as described previously [8].
PCR products were analyzed by electrophoresis
in 2% agarose gels (Life Sciences International,
Zellik, Belgium).

3. Results
3.1. Detection of initial adherence determinant

The presence of EPEC initial adherence determinant (bfpA-related gene) was investigated in
14 DEPEC and three CEPEC isolates. Four isolates (25211-2A, 25314, 43748-1 and 43750-1)

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F. Goffaux et al. / Res. Microbiol. 151 (2000) 865871

Table III. Primers used in this study.


Gene

Primers and sequences

Length of the PCR


product (bp)

Reference

bfpA

EP1: 5-AATGGTGCTTGCGCTTGCTGC-3
EP2: 5-GCCGCTTTATCCAACCTGGTA-3
K260: 5-GAGCGAATATTCCGATATACTGGTT-3
K255: 5-GGTTGAGTCGATTGATCTCTGG-3
K260: 5-GAGCGAATATTCCGATATACTGGTT-3
K261: 5-CCTGCAAATAAACACGGCGCAT-3
B73: 5-TACTGAGATTAAGGCTGATAA-3
B138: 5-GACCAGAAGAAGATCCA-3
B73: 5-TACTGAGATTAAGGCTGATAA-3
B74: 5-AGGAAGAGGGTTTTGTGTT-3
B73: 5-TACTGAGATTAAGGCTGATAA-3
B137: 5-TGTATGTCGCACTCTGATT-3
B139: 5-CRCCKCCAYTACCTTCACA-3
B152: 5-CGCTAACCTCCAAACCATT-3
B139: 5-CRCCKCCAYTACCTTCACA-3
B141: 5-GTCGGCAGTTTCAGTTTCAC-3
B139: 5-CRCCKCCAYTACCTTCACA-3
B140: 5-GATTTTTCCCTCGCCACTA-3
B148: 5-GCCGTTTTTGAGAGCCA-3-3
B151: 5-TCCCCAGGACAGATGAGAT
B148: 5-GCCGTTTTTGAGAGCCA-3
B150: 5-GCACCAGCAGCCTTTGA-3
B148: 5-GCCGTTTTTGAGAGCCA-3
B149: 5-CTTTCCGTTGCCTTAGT-3
B163: 5-TGAGGCATCTAARGMGTC-3
B165: 5-GCTGGCTATTATTGACCG-3
B163: 5-TGAGGCATCTAARGMGTC-3
B164: 5-ATCACGAATACCAGTTACA-3
B163: 5-TGAGGCATCTAARGMGTC-3
B166: 5-TGCCTTTCTTATTCTTGTCA-3
B213: 5-GCTGGATTTACAACTGG-3
B214: 5-NTTTCTCTTCGGCTTTY-3

326

[19]

418

[32]

527

[32]

452

[8]

778

[8]

520

[8]

342

[8]

781

[8]

560

[8]

94

[8]

188

[8]

233

[8]

98

[8]

180

[8]

269

[8]

419

this study

Disrupted
selC
Intact selC
eae
eae
eae
tir
tir
tir
espB
espB
espB
espA
espA
espA
espD

were BfpA-positive by PCR (table I). The plasmids of the isolates were extracted and hybridized with the BfpA probe. High-molecularweight plasmids were observed in all isolates
(figure 1A) and five were positive by hybridization with the BfpA probe (figure 1B). In the five
isolates, the BfpA probe hybridized with a plasmid ranging from 60 to 70 MDa. Therefore, one
isolate (45337-2) was BfpA-negative by PCR,
but positive by plasmid hybridization.
3.2. Presence and location
of a pathogenicity island related to the LEE

First, we investigated the presence of LEErelated sequences in 14 DEPEC and three


CEPEC. We tested the capacity of DEPEC and

CEPEC to hybridize with the LEE probes A, B,


C, D in a colony blot hybridization assay. All
isolates were positive with the four LEE probes
(data not shown), indicating the presence of
sequences homologous to the LEE of human
EPEC.
In view of these results, the insertion site of
the LEE was determined by PCR. A 418-bp
amplicon was obtained with two DEPEC isolates (25211-2A and 44318-3), indicating that the
LEE is inserted downstream of selC, and a
527-bp amplicon with 15 isolates, indicating
that the LEE is inserted elsewhere on the chromosome, since in these isolates the selC gene is
intact (table I).

F. Goffaux et al. / Res. Microbiol. 151 (2000) 865871

Figure 1. Plasmid profiles (A) and hybridization with the BfpA


probe (B) from DEPEC and CEPEC isolates. Lane 1: 25211-A2;
lane 2: 25314; lane: 3: 26881-3; lane 4: 32106-2; lane 5: 41735-2;
lane 6: 41927-1; lane 7: 41989-3; lane 8: 42430-1; lane 9: 42481-2;
lane 10: 43401-2; lane 11: 43769-2; lane 12: 44053-1; lane 13:
44318-3; lane 14: 45337-2; lane 15: 35314-1; lane 16: 43748-1;
lane 17: 43750-1; lane 18: E2348/69; and lane 19: HB101.

3.3. Presence and typing


of eae, tir, espA, espB and espD genes

We used four multiplex PCRs on our isolates


to test the presence of and to type eae, tir, espA
and espB genes. An amplicon was obtained for
all four genes in all isolates (table I). The eae gene
mainly observed (65%) was of the eae type. For
the tir gene, the tir type was dominant (71%).
For the espA gene, the espA type was mainly
observed (71%). The same result was obtained
for the espB gene. No tir, espA, or espB type
was detected. When associating the results of
the four multiplex PCRs we observed three
different associations: 11/17 were eae-tirespA-espB, 5/17 were eae-tir-espA-espB,
and 1/17 was eae-tir-espA-espB. To complete the pathotype we also tested isolates for
the presence of an espD-related gene. PCR results
showed that all isolates were espD-positive
(table I).

4. Discussion
In a previous study, Mainil et al. [28] found
that of 798 canine and 113 feline E. coli isolates

869

isolated from animals with diarrhea, enteritis or


septicemia, 5.5% were Eae probe-positive in
dogs and 5% in cats. In this work, 14 canine and
three feline EPEC isolates, associated with diarrhea and/or enteritis, were further characterized for the presence of virulence determinants
related to human EPEC.
The canine and feline isolates tested in this
study carry high-molecular-weight plasmids
with approximately the same size as the 60-MDa
EAF plasmid of human EPEC strain E2348/69.
But only 29% of our isolates possess a bfpArelated gene located on a high-molecular-weight
plasmid. This may indicate that most DEPEC
produce their own specific adhesin, if any. Moreover, the fact that one isolate was BfpA-negative
by PCR but positive by plasmid hybridization
may indicate the presence of a variant of the
bfpA gene in this isolate. We did not further
investigate the isolates for the presence of EAF
factor on a high molecular weight plasmid since
EAF is not associated with a virulence factor;
moreover, EAF does not seem to be specific of
EPEC in dogs and cats. Indeed, previous reports
[4, 28] showed that some canine and feline
isolates were EAF probe-positive but Eae probenegative.
The LEE is a pathogenicity island conserved
among all intestinal pathogens which produce
the AE lesion, including E. coli (EPEC and
EHEC), Hafnia alvei and Citrobacter rodentium
[32]. Results of colony blot hybridizations with
four LEE probes scattered along the LEE showed
that a LEE-related structure is probably also
present in 14 DEPEC and three CEPEC isolates.
Interestingly, in most isolates (88%) the LEE was
not located in the selC gene on the chromosome.
This is in agreement with results obtained with
bovine and human AEEC strains [18, 40].
By using multiplex PCR, we were able to
show the presence of different types of genes
located in the LEE (eae, tir, espA and espB genes).
Surprisingly, the associations between the
intimin gene and the intimin receptor gene were
mainly heterologous (65%). Indeed, an eae gene
is associated with a tir gene in 11 isolates.
These results were not expected, since the eae
gene encodes intimin and the tir gene encodes

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F. Goffaux et al. / Res. Microbiol. 151 (2000) 865871

the intimin receptor. Nevertheless, homologous


combinations are also present: eae with tir in
five isolates and eae with tir in one isolate.
These results raise the question of the complementary regions between the intimin and its
receptor. On the other hand, the associations
between espA and espB genes are more conclusive since there are only homologous associations: espA with espB, espA with espB. These
results are consistent with the fact that the EspA
protein is required for EspB translocation into
host cells [26]. The espB multiplex PCR seems to
be very sensitive to detect this gene. Indeed, a
previous study showed that only 44% of DEPEC
isolates were positive with the EspB probe [4],
but all our isolates were positive by PCR. This
could be explained by the fact that the EspB
probe is derived from the espB gene of the
human EPEC strain E2348/69 [10]. Therefore,
this probe would only be able to detect the
espBa gene. We also show that all DEPEC and
CEPEC isolates possessed an espD-related gene.
The pathotypes of DEPEC and CEPEC isolates determined by multiplex PCR are heterogeneous (three different types for 16 isolates).
This is in agreement with the results obtained
for bovine AEEC isolates (two pathotypes for 71
isolates) [8]. Interestingly, the three pathotypes
present in dogs and cats are also found in
human AEEC isolates [8]. We can observe that
the bfpA gene is mainly associated with the
eae-tir-espA-espB pathotype. As previously
described [4], the DEPEC and CEPEC form a
heterogeneous group, and five of them are
closely related to human EPEC as they present a
pathotype found in human EPEC and also
possess the bfpA gene detected in human EPEC
but not in that of calf AEEC [9, 19]. We even
observed that one DEPEC isolate (25211-A2),
having a LEE inserted into selC, presented the
same pathotype as human EPEC strain E2348/69
(eae-tir-espA-espB). The question of domestic carnivores as an animal reservoir for human
EPEC is worth asking.
In a previous description of EPEC infection in
dogs, some animals suffered from concomitant
enteric infections [5, 12]. Unfortunately, here, no
further laboratory examinations were per-

formed. It is therefore difficult to determine the


exact role of the EPEC in disease. But in the
present work we show that DEPEC and CEPEC
possess virulence factors related to EPEC; therefore they could be potential pathogens in dogs
and cats. Moreover, in vivo experiments showed
that the LEE of four DEPEC isolates (25211-A2,
41735-2, 43769-2 and 44318-3) were functional
since these isolates were able to produce AE
lesions in a rabbit intestinal loop assay (China et
al., personal communication).

Acknowledgments
The authors thank Vinciane Pirson and Etienne Jacquemin for their technical assistance.
Frdric Goffaux is a fellow of the FRIA (Fonds
de la Recherche applique lIndustrie et
lAgriculture).

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