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Genotypic characterization of enteropathogenic Escherichia coli (EPEC)isolated in Belgium from dogs and cats

Frédéric Goffaux*, Bernard China, Laurence Janssen, Jacques Mainil

Laboratory of Bacteriology, Faculty of Veterinary Medicine, University of Liège, Bd de Colonster 20/B43,4000 Liège, Belgium

Received 17 September 1999; accepted 6 July 2000

Abstract – Enteropathogenic Escherichia coli (EPEC) are isolated from man and farm animals but also fromdogs and cats. They produce typical histological lesions called ‘attaching and effacing’ lesions. Both plasmidand chromosomal elements are involved in the pathogenesis of EPEC infection. The presence of these geneticelements 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, espAand espB genes were analyzed by multiplex PCR. The results indicated the presence of those genes in thetested isolates with heterogeneity in the gene subtypes present: eaeγ-tirα-espAα-espBα (65%), eae-tir-espA-espB (29%), eaeα-tirα-espAα-espBα (6%). Moreover, the espD gene was also present in dog and cat EPEC. TheDEPEC and CEPEC form a heterogeneous group and five of them are closely related to human EPEC. © 2000Éditions scientifiques et médicales Elsevier SAS

Escheri chia coli / EPEC / genotypic characterization / dogs and cats / Belgium

1. Introduction

Attaching and effacing Escherichia coli (AEEC)are characterized by their capacity to infectintestinal epithelial cells, resulting in so-calledattaching and effacing (AE) lesions. AEEC infec-tion begins with the initial adherence, mani-fested by the local formation of bacterial micro-colonies at the cell surface, called localizedadherence (LA) [39] and is followed by theformation of AE lesion characterized by bacte-rial intimate attachment to enterocytes and dis-ruption of brush border microvilli [34]. The AElesion is associated with the accumulation of

highly organized cytoskeletal components (actin,α-actinin, myosin light chain, ezrin and talin) inepithelial cells immediately beneath the adher-

ent bacteria [14], leading to the formation of apedestal-like structure [25]. AEEC groups

together enteropathogenic E. coli (EPEC) andenterohemorrhagic E. coli (EHEC), which inaddition produce verotoxins or shigatoxins(reviewed in [30]).

Both plasmid and chromosomal loci areinvolved in the pathogenic process of humanEPEC infection. The LA is mediated by the bundle forming pilus (BFP), a member of thetype IV fimbriae family [17]. The bfpA gene islocated on a high molecular weight plasmidtermed EPEC adherence factor (EAF) [16]. A

1-kb probe was derived from the EAF plasmidand used as an epidemiological tool [35], butthis probe does not correspond to any codingregion of the plasmid [15]. In human EPECstrain E2348/69, most of the genes necessary forthe formation of the AE lesion are clustered ona 35.5-kb chromosomal pathogenicity islandcalled LEE (locus of enterocyte effacement),

* Correspondence and reprints.E-mail address: [email protected] (F. Goffaux).

Res. Microbiol. 151 (2000) 865–871 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reservedS0923250800011530/FLA

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which is inserted into an selC gene encoding thetRNA for selenocysteine [32]. The LEE is suffi-cient to confer AE activity in vitro when intro-duced 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 secretionsystem responsible for the secretion of proteinsEspA, EspB, EspD [21] and Tir [23]. The middlepart of the LEE contains two genes (eae, tir), theproducts of which promote intimate attachmentof bacteria to host cells and reorganization of cytoskeletal actin beneath adherent bacteria.The eae gene encodes a 94-kDa outer membraneprotein, the intimin, required for intimate adher-ence [22]; the tir gene encodes the intimin

receptor which is translocated into the eucary-otic cytoplasm where it becomes phosphory-lated and then incorporated into the host cellmembrane [23]. The right part of the LEE con-tains three genes: espA, espD and espB, encodingthe proteins that are essential for EPEC-mediatedsignal transduction events within the host cell,including tyrosine phosphorylation of Tir andthe increase of inositol triphosphate and cal-cium levels, leading to the AE lesion formation[10, 24, 27].

Natural infections with EPEC and/or intesti-nal AE lesions have been described in dogs [5,20] and cats [38]. EPEC are considered as animportant cause of diarrhea in dogs, particu-larly in puppies [12]. In addition to the presenceof an eae gene, some EPEC strains isolated fromdogs harbor the espB gene and possess a highmolecular weight EAF-related plasmid carryingthe bfpA gene [4]. Moreover, eae, tir, espA, espBand espD genes have been cloned from the dog

EPEC strain 4221 and sequenced [2, 3].Canine and feline EPEC have also been iden-

tified in Belgium [28] using a colony hybridiza-tion assay with the Eae, EAF and BfpA probes.In this study, we further characterized genotypi-cally, E. coli isolates from dogs and cats for thepresence of virulence determinants related toEPEC.

2. Material and methods

2.1. Bacteria

Fourteen Eae probe-positive isolates from

dogs (DEPEC) and three Eae-positive isolatesfrom cats (CEPEC) were included in this study(refer to table I below). All were isolated fromanimals with diarrhea and/or enteritis. Thehuman 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 non-pathogenic E. coli HB101 as a negative control.

2.2. Colony hybridization

The colony blot hybridization assay was per-formed as described by Mainil et al. [29]. Theprobes (table II ) were labeled with α32P-dCTP byrandom priming using the dCTP-labeling beads(Ready to go, Pharmacia, Uppsala, Sweden)according to the instructions of the manufac-turer. LEEA corresponds to the 5’ end of theLEE, LEEB includes escV and escN genes, LEECincludes the eae gene and LEED includes espAand espB genes.

2.3. Plasmid DNA isolation and hybridization

Plasmid DNA was extracted using the modi-fied method of Kado and Liu [6]. Plasmids wereseparated by 0.5% agarose gel electrophoresisand the gel was hybridized with the BfpA probeas described by Mainil et al. [31]. The BfpAprobe was obtained by purification of PCRproducts with the QIAquick-spin PCR purifica-tion kit (Qiagen, Hilden, Germany). The probewas labeled as described in colony hybridiza-tion.

2.4. PCR reactions

The amplification of the bfpA fragment(326 bp) was performed with primers EP1 andEP2 [29]. Insertion of the LEE downstream of selC was studied using primers K255 and K260for the right junction, and K260 and K261 for theintact selC gene [32]. If the LEE is inserted

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downstream of selC, primers K255 and K260amplify a 418-bp fragment. In the absence of theLEE, primers K260 and K261 gave a 527-bpfragment since selC was intact. Four sets of fourprimers (one constant and three variable) wereused 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 amplifya 419-bp fragment of the espD gene. Upper andlower primers were chosen both in constantregions from the published sequences of theespD genes [3, 13, 36, 37].

The sequences of primers used in this studyare 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 electrophoresisin 2% agarose gels (Life Sciences International,Zellik, Belgium).

3. Results

3.1. Detection of initial adherence determinant

The presence of EPEC initial adherence deter-minant (bfpA-related gene) was investigated in14 DEPEC and three CEPEC isolates. Four iso-lates (25211-2A, 25314, 43748-1 and 43750-1)

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

Isolate and strain bfpA Multiplex PCR espD SelC disrupted Species

eae tir espA espB by LEE

25211-2A + α α α α + yes dog25314 + γ α α α + no dog26881-3 – + no dog32106-2 – γ α α α + no dog41735-2 – + no dog41927-1 – + no dog41989-3 – γ α α α + no dog42430-1 – γ α α α + no dog42481-2 – + no dog43401-2 – γ α α α + no dog43769-2 – γ α α α + no dog44053-1 – + no dog44318-3 – γ α α α + yes dog45337-2 –* γ α α α + no dog35314-1 – γ α α α + no cat43748-1 + γ α α α + no cat

43750-1 + γ α α α + no catE2348/69 + α α α α + yes humanRDEC-1 ND + ND rabbitATCC43888 ND γ γ γ γ + ND humanHB101 NA NA NA NA NA NA no

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 pCVD453 MluI / EcoRI 2 870 [32]LEEB pCVD461 EcoRI / SalI / PvuII 2 948 [32]LEEC pCVD443 SalI / StuI 1 050 [32]LEED pCVD460 SmaI / XbaI 2 300 [32]

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were BfpA-positive by PCR (table I ). The plas-mids of the isolates were extracted and hybrid-ized with the BfpA probe. High-molecular-weight plasmids were observed in all isolates( gure 1A) and five were positive by hybridiza-tion with the BfpA probe ( gure 1B). In the fiveisolates, the BfpA probe hybridized with a plas-mid 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 locationof a pathogenicity island related to the LEE

First, we investigated the presence of LEE-related sequences in 14 DEPEC and threeCEPEC. We tested the capacity of DEPEC and

CEPEC to hybridize with the LEE probes A, B,C, D in a colony blot hybridization assay. Allisolates were positive with the four LEE probes(data not shown), indicating the presence of sequences homologous to the LEE of humanEPEC.

In view of these results, the insertion site of

the LEE was determined by PCR. A 418-bpamplicon was obtained with two DEPEC iso-lates (25211-2A and 44318-3), indicating that theLEE is inserted downstream of selC, a n d a527-bp amplicon with 15 isolates, indicatingthat the LEE is inserted elsewhere on the chro-mosome, since in these isolates the selC gene isintact (table I ).

Table III. Primers used in this study.

Gene Primers and sequences Length of the PCRproduct (bp)

Reference

bfpA EP1: 5’-AATGGTGCTTGCGCTTGCTGC-3 ’ 326 [19]EP2: 5’-GCCGCTTTATCCAACCTGGTA-3’

Disrupted K260: 5’-GAGCGAATATTCCGATATACTGGTT-3’ 418 [32]selC K255: 5’-GGTTGAGTCGATTGATCTCTGG-3’Intact selC K260: 5’-GAGCGAATATTCCGATATACTGGTT-3’ 527 [32]

K261: 5’-CCTGCAAATAAACACGGCGCAT-3’eaeα B73: 5’-TACTGAGATTAAGGCTGATAA-3 ’ 452 [8]

B138: 5’-GACCAGAAGAAGATCCA-3’eaeγ B73: 5’-TACTGAGATTAAGGCTGATAA-3 ’ 778 [8]

B74: 5’-AGGAAGAGGGTTTTGTGTT-3 ’eae B73: 5’-TACTGAGATTAAGGCTGATAA-3 ’ 520 [8]

B137: 5’-TGTATGTCGCACTCTGATT-3’tirα B139: 5’-CRCCKCCAYTACCTTCACA-3’ 342 [8]

B152: 5’-CGCTAACCTCCAAACCATT-3’tirγ B139: 5’-CRCCKCCAYTACCTTCACA-3’’ 781 [8]

B141: 5’-GTCGGCAGTTTCAGTTTCAC-3

tir B139: 5’-CRCCKCCAYTACCTTCACA-3’ 560 [8]B140: 5’-GATTTTTCCCTCGCCACTA-3’espBα B148: 5’-GCCGTTTTTGAGAGCCA-3’-3’ 94 [8]

B151: 5’-TCCCCAGGACAGATGAGATespB γ B148: 5’-GCCGTTTTTGAGAGCCA-3’ 188 [8]

B150: 5’-GCACCAGCAGCCTTTGA-3’espB B148: 5’-GCCGTTTTTGAGAGCCA-3’ 233 [8]

B149: 5’-CTTTCCGTTGCCTTAGT-3’espAα B163: 5’-TGAGGCATCTAARGMGTC-3’ 98 [8]

B165: 5’-GCTGGCTATTATTGACCG-3’espAγ B163: 5’-TGAGGCATCTAARGMGTC-3’ 180 [8]

B164: 5’-ATCACGAATACCAGTTACA-3’espA B163: 5’-TGAGGCATCTAARGMGTC-3’ 269 [8]

B166: 5’-TGCCTTTCTTATTCTTGTCA-3’espD B213: 5’-GCTGGATTTACAACTGG-3’ 419 this study

B214: 5’-NTTTCTCTTCGGCTTTY-3 ’


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3.3. Presence and typingof eae , t i r , espA , espB and espD genes

We used four multiplex PCRs on our isolatesto test the presence of and to type eae, tir, espAand espB genes. An amplicon was obtained forall four genes in all isolates (table I ). The eae genemainly observed (65%) was of the eaeγ type. Forthe tir gene, the tirα type was dominant (71%).For the espA gene, the espAα type was mainlyobserved (71%). The same result was obtainedfor the espB gene. No tirγ, espAγ, or espBγ typewas detected. When associating the results of the four multiplex PCRs we observed threedifferent associations: 11/17 were eaeγ-tirα-espAα-espBα, 5/17 were eae-tir-espA-espB,and 1/17 was eaeα-tirα-espAα-espBα. To com-

plete the pathotype we also tested isolates forthe presence of an espD-related gene. PCR resultsshowed that all isolates were espD-positive(table I ).

4. Discussion

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

isolated from animals with diarrhea, enteritis orsepticemia, 5.5% were Eae probe-positive indogs and 5% in cats. In this work, 14 canine andthree feline EPEC isolates, associated with diar-rhea and/or enteritis, were further character-

ized for the presence of virulence determinantsrelated to human EPEC.

The canine and feline isolates tested in thisstudy carry high-molecular-weight plasmidswith approximately the same size as the 60-MDaEAF plasmid of human EPEC strain E2348/69.But only 29% of our isolates possess a bfpA-related gene located on a high-molecular-weightplasmid. This may indicate that most DEPECproduce their own specific adhesin, if any. More-over, the fact that one isolate was BfpA-negative by PCR but positive by plasmid hybridizationmay indicate the presence of a variant of thebfpA gene in this isolate. We did not furtherinvestigate the isolates for the presence of EAFfactor on a high molecular weight plasmid sinceEAF 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 felineisolates were EAF probe-positive but Eae probe-negative.

The LEE is a pathogenicity island conserved

among all intestinal pathogens which producethe AE lesion, including E. coli (EPEC andEHEC), Hafnia alvei and Citrobacter rodentium[32]. Results of colony blot hybridizations withfour LEE probes scattered along the LEE showedthat a LEE-related structure is probably alsopresent in 14 DEPEC and three CEPEC isolates.Interestingly, in most isolates (88%) the LEE wasnot 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 toshow the presence of different types of geneslocated in the LEE (eae, tir, espA and espB genes).Surprisingly, the associations between theintimin gene and the intimin receptor gene weremainly heterologous (65%). Indeed, an eaeγ geneis associated with a tirα gene in 11 isolates.These results were not expected, since the eaegene encodes intimin and the tir gene encodes

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


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the intimin receptor. Nevertheless, homologouscombinations are also present: eae with tir infive isolates and eaeα with tirα in one isolate.These results raise the question of the comple-mentary regions between the intimin and its

receptor. On the other hand, the associations between espA and espB genes are more conclu-sive since there are only homologous associa-tions: espAα with espBα, espA with espB. Theseresults are consistent with the fact that the EspAprotein is required for EspB translocation intohost cells [26]. The espB multiplex PCR seems to be very sensitive to detect this gene. Indeed, aprevious study showed that only 44% of DEPECisolates were positive with the EspB probe [4], but all our isolates were positive by PCR. Thiscould be explained by the fact that the EspBprobe is derived from the espB gene of thehuman EPEC strain E2348/69 [10]. Therefore,this probe would only be able to detect theespBa gene. We also show that all DEPEC andCEPEC isolates possessed an espD-related gene.

The pathotypes of DEPEC and CEPEC iso-lates determined by multiplex PCR are hetero-geneous (three different types for 16 isolates).This is in agreement with the results obtainedfor bovine AEEC isolates (two pathotypes for 71isolates) [8]. Interestingly, the three pathotypes

present in dogs and cats are also found inhuman AEEC isolates [8]. We can observe thatthe bfpA gene is mainly associated with theeaeγ-tirα-espAα-espBα pathotype. As previouslydescribed [4], the DEPEC and CEPEC form aheterogeneous group, and five of them areclosely related to human EPEC as they present apathotype found in human EPEC and alsopossess the bfpA gene detected in human EPEC but not in that of calf AEEC [9, 19]. We evenobserved that one DEPEC isolate (25211-A2),having a LEE inserted into selC, presented thesame pathotype as human EPEC strain E2348/69(eaeα-tirα-espAα-espBα). The question of domes-tic carnivores as an animal reservoir for humanEPEC is worth asking.

In a previous description of EPEC infection indogs, some animals suffered from concomitantenteric infections [5, 12]. Unfortunately, here, nofurther laboratory examinations were per-

formed. It is therefore difficult to determine theexact role of the EPEC in disease. But in thepresent work we show that DEPEC and CEPECpossess virulence factors related to EPEC; there-fore they could be potential pathogens in dogs

and cats. Moreover, in vivo experiments showedthat the LEE of four DEPEC isolates (25211-A2,41735-2, 43769-2 and 44318-3) were functionalsince these isolates were able to produce AElesions in a rabbit intestinal loop assay (China etal., personal communication).

Acknowledgments

The authors thank Vinciane Pirson and Eti-enne Jacquemin for their technical assistance.Frédéric Goffaux is a fellow of the FRIA (Fondsde la Recherche appliquée à l’Industrie et àl’Agriculture).

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