Genotypic and Phenotypic Characterization of Escherichia coli ...

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Jul 19, 1995 - Thirteen Escherichia coli isolates from dogs manifesting attaching .... (ii) fK3 (5 TGG CGC TCA GCA AAT CCA GCA 3) and rK37 (5 TTT TAG.
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1996, p. 144–148 0095-1137/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 34, No. 1

Genotypic and Phenotypic Characterization of Escherichia coli Isolates from Dogs Manifesting Attaching and Effacing Lesions M. BEAUDRY, C. ZHU, J. M. FAIRBROTHER,

AND

J. HAREL*

Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculte´ de Me´decine Ve´te´rinaire, Universite´ de Montre´al, Saint-Hyacinthe, Que´bec, Canada J2S 7C6 Received 21 April 1995/Returned for modification 19 July 1995/Accepted 17 October 1995

Thirteen Escherichia coli isolates from dogs manifesting attaching and effacing lesions were characterized genetically with respect to the presence of the following virulence determinants associated with human enteropathogenic E. coli (EPEC): eaeA, encoding the outer membrane protein intimin; eaeB, which is necessary for inducing signal transduction; bfpA, encoding the bundle-forming pilus; and the EAF (stands for EPEC adherence factor) plasmid. These isolates were also analyzed phenotypically with respect to adherence to mammalian cells in vivo and in vitro. Nine of these 13 isolates were found to be eaeA positive by PCR; four of these nine were eaeB positive. The 5* end, but not the 3* end, of the eaeA gene was amplified by PCR when primers derived from the eaeA gene of EPEC were used. Six and eight of these 13 isolates were found to be bfpA positive and EAF positive, respectively. The bfpA gene and EAF locus were found on high-molecular-weight plasmids, whereas the eaeA and eaeB genes were chromosomally located when present. Only one canine E. coli isolate, 4221, which was positive for eaeA, eaeB, bfpA, and EAF, adhered to HEp-2 cells in a localized manner and was positive in the fluorescence actin staining test. The nine eaeA-positive isolates adhered to the mucosal surface of piglet ileal explants and induced some microvillus effacement. However, when tested in experimentally inoculated gnotobiotic piglets, isolate 4221 did not induce attaching and effacing lesions at any level of the intestinal tract. Our results indicate that canine E. coli isolates associated with attaching and effacing lesions share some properties with human EPEC but form a heterogeneous group. intimin (12). Studies have shown that the eaeA genes of EPEC and EHEC are functionally homologous (12, 23). Nucleotide sequences related to the eaeA gene, but not related to the bfpA gene, have been found in AEEC of the rabbit, pig, and calf (21, 45). The eaeB gene, located about 4.4 kb downstream from the end of the eaeA gene, is also necessary for the formation of A-E lesions and is involved in signal transduction events (4, 15). The predicted product of the eaeB gene is around 33 kDa (24). At least one additional product, mediated by the Cfm (stands for class four mutant) locus, appears to be necessary for A-E lesions and signal transduction (15, 36). Natural infections with AEEC in dogs have also been described (5, 14, 20). Colony hybridization with DNA probes indicated that canine AEEC isolates do not carry enterotoxin, verotoxin, or fimbrial genes common to enterotoxigenic E. coli and verotoxigenic E. coli, but they carry nucleotide sequences related to the EAF sequence and the bfpA and eaeA genes (14). In this study, we further characterized E. coli isolates from dogs with A-E lesions both genotypically with respect to the presence of virulence determinants related to AEEC and phenotypically with respect to adherence to mammalian cells in vitro and in vivo.

Escherichia coli is associated with a wide variety of intestinal diseases in humans and animals. Some pathogenic E. coli produce attaching and effacing (A-E) lesions, characterized by intimate bacterial adherence to enterocytes and disruption of the underlying cytoskeleton (12). Such isolates have been termed A-E E. coli (AEEC). A-E lesions have been associated with enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) isolates in humans and with isolates from dogs, pigs, rabbits, calves, horses, lambs, and cats (5, 7, 18, 20, 28, 32, 45). The development of A-E lesions comprises multiple steps, beginning with bacterial adherence and followed by intimate attachment of bacteria to the epithelial cell and effacement of cell microvilli (12, 35, 40, 41). Both plasmidic and chromosomal loci are involved in such processes of human EPEC infection (11). The initial adherence is manifested as the local formation of bacterial microcolonies, called localized adherence (LA), in HEp-2 and other mammalian cell lines (33, 37). The LA phenomenon is mediated by the bundle-forming pilus (Bfp), a member of the type IV pilin family (17). The structural gene bfpA is located on a high-molecular-weight plasmid which has been termed the EPEC adherence factor (EAF) (29, 33). High-molecular-weight plasmids have also been associated with adherence in human EHEC isolates and in the rabbit A-E isolate RDEC-1 (7, 10, 42). The intimate adherence of AEEC is associated with a chromosomal determinant, eaeA, which was first identified in human EPEC strains (11–13, 22, 23). The eaeA gene encodes an outer membrane protein of 94 kDa which has been termed

MATERIALS AND METHODS Bacterial strains. Thirteen canine E. coli strains isolated from the intestinal contents of dogs with diarrhea demonstrating typical A-E lesions were examined in this study. Ten of them were initially characterized in a previous study (14). Strains 2680-3, 3549-4-1, and 4097-2 were isolated from the same dogs as isolates 2680-1, 3549-1, and 4097-3, respectively. Bacteria were stored on Dorset medium at 48C and grown on blood agar or in Trypticase soy broth (Difco) at 378C (45). The E. coli reference strain, E2348/69 (eaeA1 eaeB1 bfpA1 EAF1), was used for the preparation of probes. Strains 862 (negative for eaeA eaeB bfpA EAF) and H-7290 (eaeA1 eaeB1 bfpA1 EAF1) were used as negative and positive controls, respectively (11, 13, 45). The porcine E. coli O45 strain, 86-1390 (eaeA1), which induced A-E lesions in vitro and in vivo, was used as a positive control in ileal

* Corresponding author. Mailing address: Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculte´ de Me´decine Ve´te´rinaire, Universite´ de Montre´al, C. P. 5000, Saint-Hyacinthe, Que ´bec, Canada J2S 7C6. Phone: (514) 773-8521, ext. 8233. Fax: (514) 778-8108. Electronic mail address: [email protected]. 144

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explant culture and pig infections (46). Positive controls for detection of adhesin pap, sfa, and afa genes were described previously (9, 27). Plasmid DNA isolation and hybridization. Plasmid DNA was extracted from cells by using the modified method of Kado and Liu (8). Plasmids separated by 0.5% agarose gel electrophoresis were hybridized with the probes for different virulence determinants as described previously (19). Appropriate positive control strains were included on each filter. Preparation of DNA probes. Gene probes for various virulence determinants were derived from recombinant plasmids or generated by PCR as described previously (8, 10). A probe for the eaeA gene was derived from plasmid pCVD434 (23) by digestion with SalI and KpnI and electroelution of the resulting 1-kb fragment. The BfpA probe was an 816-bp EcoRI fragment from plasmid pMSD207 (10). The EAF probe was a 1-kb BamHI-SalI fragment derived from plasmid pMAR22 (31, 33). DNA fragments were labeled with [a-32P]dCTP by using an oligolabelling kit (Pharmacia LKB Biotechnology Inc., Baie d’Urfe´, Que´bec, Canada) according to the instructions of the manufacturer. Preparation of bacterial DNA for PCR and enzymatic amplification. DNA to be amplified was released from whole organisms by boiling as described previously (9). For PCR, 10 ml of DNA (1 ml of purified plasmid control) was mixed with 2 U of Taq polymerase, 200 mM deoxynucleoside triphosphate, 0.5 mM primers, and 13 buffer (103 buffer is 50 mM KCl, 10 mM Tris-HCl [pH 8.3], and 1.5 mM MgCl2 to a final concentration of 3 mM) in a final volume of 50 ml (26). The reaction mixtures were overlaid with oil. The samples were heated at 808C for 5 min (hot start) after the Taq polymerase (GIBCO BRL) was added, and the samples were subjected to PCR in a thermal cycler. The program used was 948C for 1 min, 558C for 1 min, and 728C for 2 min for 25 cycles, followed by a final 7-min extension at 728C. A volume of 10 ml of each reaction mixture was subjected to electrophoresis on a 2% agarose gel; this was followed by staining with ethidium bromide. For amplification of the eaeA gene (45), two sets of primers were used: (i) fM1 (59 CAT TAT GGA ACG GCA GAG GT 39) and rYu4 (59 ATC TTC TGC GTA CTG CGT TCA 39) amplifying the 59 region and (ii) fK3 (59 TGG CGC TCA GCA AAT CCA GCA 39) and rK37 (59 TTT TAG ACA AGT GGC CAT AAG C 39) amplifying the 39 end. An amplified fragment of 790 bp corresponded to a positive result for the 59 end, and a 434-bp fragment corresponded to a positive result for the 39 end. The amplification of the eaeB fragment (930 bp) was performed with primers feaeB (59 TAT CGA TAA TAA CAA TGC GG 39) and reaeB (59 CAT GCG ATT AAT AAG GTC AG 39) derived from the eaeB sequence (13). Three sets of 25-mer primers were used for amplification of genes in the pap, afa, and sfa fimbrial operons as described previously (9, 27). HEp-2 cell adherence and FAS. The ability of canine E. coli isolates to adhere to HEp-2 cells in the presence of D-mannose was determined as previously described (45). Fluorescence actin staining (FAS) was performed by the method of Knutton et al. (25). Fixed and washed cells were treated with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 5 min. After three washes in PBS, the slides were stained with a 5-mg/ml solution of fluorescein isothiocyanatephalloidin (Sigma Chemical Co., St. Louis, Mo.) in PBS for 20 min. The slides were then washed as described above and mounted in FA mounting fluid (Difco Laboratories, Detroit, Mich.). Fluorescence and phase-contrast micrographs of the same field were recorded. To test the effect of cytochalasin-D (CD) on bacterial adherence to HEp-2 cells, increasing volumes (0.5 to 5 ml) of CD (Sigma) in dimethyl sulfoxide were added to the HEp-2 cell supernatants 15 min before changing the medium and adding bacteria to the HEp-2 cell monolayer, as described above, to give a final CD concentration ranging between 1 and 5 mg ml21. The HEp-2 cell adherence and FAS experiments were then performed as mentioned above. Examination of A-E capacity in vitro and in vivo. Pig ileal explant culture was used as an in vitro model for the study of A-E activity of canine E. coli isolates as previously described (46). Overnight bacterial cultures were placed on the villus surface of ileal fragments and incubated at 378C for 8 h with 5% CO2 on a rocking platform. Each isolate was tested in at least three independent experiments. Bacterial adherence was recorded as follows: 2, no bacterial attachment; 1, small scattered areas of attachment; 11, large areas of attachment; and 111, extensive attachment, as previously described (46). The A-E capacity of isolate 4221 was further examined by experimental inoculation of two gnotobiotic piglets according to the previously described method (45). Two gnotobiotic piglets were also inoculated with pig A-E strain 86-1390 as a positive control. The gnotobiotic piglets were given 10 ml of an overnight culture of E. coli and kept for 120 h postinoculation. Intestinal tissues were collected and processed for light microscopy and transmission electron microscopy as previously described (45).

RESULTS Detection of virulence determinants by PCR. Thirteen E. coli isolates from dogs showing A-E lesions were tested for the presence of EPEC virulence determinants by PCR. Nine of these isolates were positive when the EaeA primers fM1 and rYu4 were used for amplification of the 59 end of the eaeA gene (Table 1). However, none of these canine isolates was

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TABLE 1. Distribution of eaeA, eaeB, bfpA, and EAF-related nucleotide sequences and adherence to mammalian cells by canine E. coli isolates

bfpA

EAF

LA on HEp-2 cells

1 1 2 2 1 1 2 2 2 1 1 2 2 2 1 2

1 1 1 2 1 1 2 1 2 1 1 2 2 2 1 2

1 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2

Presence of: Isolate

a

eaeA

4221 4225 2680-1 5289 4097-3 4083 1171 3549-1 3403 2507 4097-2 2680-3 3549-4 862e E2348/69e 86-1390e

1 1 1 1 1 1 1 1 1 2 2 2 2 2 1 1

a,b

eaeB

1 1 1 1 2 2 2 2 2 2 2 2 2 2 1 2

c

c

Adherence to ileal explantsd

111 111 111 111 111 111 111 111 111 2 2 2 2 2 111 111

a

As detected by PCR. As detected by colony hybridization. c As detected by plasmid hybridization. d As observed by light microscopy. 2, no bacterial attachment; 111, extensive attachment. e Strain 862 is a negative control and strains E2348/69 and 86-1390 are positive controls for A-E activity. b

positive when primers fK3 and rK37 were used for amplification of the 39 end of the eaeA gene (data not shown). Four of the eaeA-positive isolates were also eaeB positive as detected by PCR (Table 1) and colony hybridization. None of the canine isolates was positive for amplification of afa, pap, or sfa by PCR (data not shown). Analysis of plasmid content by probe hybridization. Highmolecular-weight plasmids ranging from 30 to 70 MDa were observed in all canine isolates (Fig. 1A). High-molecularweight plasmids on Southern blots of eight and six isolates were positive by hybridization for the EAF and BfpA probes, respectively (Fig. 1B and C; Table 1). However, none of these plasmids hybridized with the EaeA probe (data not shown). Two isolates (4221 and 4225) were positive for eaeA, eaeB, bfpA, and EAF. Six isolates (2680-1, 4097-3, 4083, 3549-1, 2507, and 4097-2) were positive for bfpA and/or EAF and were either eaeA positive or eaeA negative. Three isolates (5289, 1171, and 3403) were negative for bfpA and EAF but positive for eaeA, and two isolates (2680-3 and 3549-4) were negative for all the virulence factors tested (Table 1). Adherence of canine AEEC to HEp-2 cells. Of thirteen canine E. coli isolates tested, only isolate 4221 adhered to HEp-2 cells as examined by Giemsa staining of the monolayers (Table 1). At 3 h following inoculation, the isolate attached in a localized manner to HEp-2 cells, forming clusters of 10 to 30 bacterial cells on the HEp-2 cell surface (Fig. 2). Areas of fluorescence on the HEp-2 cells beneath adhering bacteria were observed by FAS, indicating actin condensation (Fig. 2C and D). However, this reaction was inhibited in the presence of CD, although the LA pattern and the number of bacterial clusters on HEp-2 cells were not apparently affected (data not shown). The EPEC strain E2348/69 adhered to HEp-2 cells in a localized manner and induced actin accumulation, which was inhibited by the presence of CD. The adhesive capacity and induction of accumulation of filamentous actin of isolate 4221 were similar to those seen with human EPEC strain E2348/69.

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DISCUSSION

FIG. 1. Plasmid profiles (A) and hybridization with EAF (B) and with BfpA (C) of canine E. coli isolates. The arrows (B and C) indicate the high-molecularweight plasmid of positive control H-7290. Strain numbers are indicated on the top. Molecular size (in megadaltons) is indicated on the left.

Adherence to piglet enterocytes. When tested in piglet ileal explants, the nine eaeA-positive but none of the eaeA-negative E. coli isolates from dogs appeared to adhere closely to ileal enterocytes on examination by light microscopy (Table 1). A multifocal extensive bacterial colonization of enterocyte brush borders, similar to that observed for the pig and human A-E positive-control strains 86-1390 and E2348/69, respectively, was observed (data not shown) (46). Further examination by transmission electron microscopy of explants inoculated with canine isolate 4221 revealed that bacteria were closely adherent to enterocytes with occasional microvillus effacement and pedestal formation (Fig. 3). The A-E pig and human positivecontrol strains demonstrated intimate attachment to and extensive microvillus effacement of the enterocytes from ileal explants (data not shown) as previously observed (46). However, the effacement of microvilli by isolate 4221 was less extensive than that observed for the positive-control strains. The A-E capacity of isolate 4221 was further examined in experimentally inoculated gnotobiotic piglets. Both of two inoculated piglets appeared clinically normal up to 96 h postinoculation. A moderate diarrhea was observed at 120 h postinoculation. No bacterial attachment to intestinal enterocytes was observed in the duodenum, jejunum, ileum, cecum, or colon on examination by light microscopy or transmission electron microscopy at 120 h postinoculation. In contrast, strain 86-1390 induced extensive A-E lesions in experimentally inoculated pigs at the same time postinoculation (data not shown).

We have demonstrated the presence of virulence determinant genes, eaeA, eaeB, bfpA, and the EAF sequence of human EPEC, in canine E. coli isolates associated with A-E lesions (14). This is an indication that the canine isolates were more closely related to human EPEC isolates than other AEEC isolates obtained from pigs (45), rabbits (34), or calves (30). Nevertheless, the inability to adhere to HEp-2 cells in an LA pattern with one exception indicates that these isolates do differ from human EPEC. In this study, we further confirmed the location of the eaeA gene on the chromosome and the location of the bfpA gene on plasmids in these canine isolates. The eaeA-related sequences were detected in most of the canine isolates, indicating the importance of the eaeA gene in the canine AEEC. This is in accordance with the finding that the presence of nucleotide sequence homology to the eaeA gene is a common feature of bacteria capable of inducing A-E lesions, including human AEEC, animal AEEC, Citrobacter freundii, and Hafnia alvei (1, 2, 38). One of the striking features of E. coli strains capable of inducing A-E lesions is the presence of high-molecular-weight plasmids which may encode adhesive factors or participate in the regulation of bacterial pathogenesis (12, 17, 22, 30). We have noted that the canine E. coli isolates tested in this study carried high-molecular-weight plasmids ranging from 30 to 70 MDa and were mostly EAF positive and bfpA positive. The presence of the bfpA gene in eaeA-positive canine isolates may suggest a close relatedness of these isolates to human EPEC, in light of the fact that the bfpA gene was not detected in AEEC isolates from pigs (45), rabbits (34), and calves (30). Our results also indicated that the presence of the bfpA gene was not highly correlated to the presence of the EAF plasmid in canine isolates. Similar findings have been demonstrated with human AEEC isolates (26). The finding that one canine isolate, 4221, adhered in a localized manner to HEp-2 cells and shows a positive FAS reaction which is inhibited by CD indicates that this isolate closely resembles phenotypically human EPEC strain E2348/69. In previous studies, the presence of the bfpA gene has been highly correlated with characteristic LA to HEp-2 or HeLa cells in vitro (12, 33). The apparent inability of other canine isolates possessing the bfpA gene to adhere to HEp-2 cells might be due to lack of expression of Bfp or functional differences in the expressed BfpA protein. We are presently investigating these possibilities. When tested in piglet ileal explants, the eaeA-positive canine E. coli isolates were able to adhere closely to enterocytes but appeared to induce less severe effacement of microvilli, at least in the case of isolate 4221, than AEEC isolates from humans and pigs. This could be due to a lack of recognition of speciesspecific receptors by bacterial factors necessary for efficient effacement of microvilli. Lack of recognition of species-specific receptors could also explain the absence of bacterial attachment and microvillus effacement in gnotobiotic pigs inoculated with the canine isolate 4221 (6, 39, 45). It is likely that functional differences between the eaeA gene products of canine, porcine, and human strains resulted in differences in bacterial attachment in pig intestinal tissues in vivo and in vitro. Supporting this is the finding that the 59 extremity but not the 39 extremity of the eaeA gene of canine isolates was amplified by PCR with primers derived from the eaeA gene of the human EPEC strain. Studies have demonstrated high homology at the N-terminal ends and less homology at the C-terminal ends of the intimins of EPEC, EHEC, C. freundii, and H. alvei, suggesting different binding properties and biological activities of

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FIG. 2. HEp-2 cells infected with a canine E. coli isolate showing no bacterial attachment to the cell monolayer (A) or with isolate 4221 showing LA (B and C) and actin accumulation beneath the attached bacteria (D). (A and B) Giemsa staining; magnification, 3400. (C and D) Phase-contrast micrograph and FAS, respectively, of the same field.

these proteins (3, 16, 44). Another possibility is that the canine isolate lacks an adhesive factor additional to Bfp, which mediates the initial attachment of bacteria to porcine enterocytes in vivo. Human and rabbit AEEC isolates possess different adhesive factors, which might confer tissue specificity (7, 42, 43). Differences in the attachment of canine isolates in vitro and in vivo could also be due to the absence of additional determi-

nants necessary for a complete A-E activity in the pig intestinal epithelium, since A-E capacity is multifactorial (21). To summarize, we have demonstrated the presence of the eaeA, eaeB, and bfpA genes and high-molecular-weight plasmids in E. coli isolates associated with canine enteric colibacillosis. These isolates mostly possess the eaeA gene, a common determinant of AEEC, but are heterogeneous with respect to other known determinants of EPEC, such as EAF, bfpA, and eaeB genes. Ongoing work is aimed at identifying and further characterizing the virulence factors in A-E activity of canine isolates. ACKNOWLEDGMENTS This work was supported in part by the Medical Research Council of Canada grant MT11720, the Conseil de Recherche en Peˆche et en Agro-alimentaire du Que´bec grant 2982, and the Fonds pour la Formation des Chercheurs et l’Aide `a la Recherche du Que´bec (FCAR) grant 0214. We are grateful to M. Jacques for helpful discussions and to B. Foiry and C. Desautels for technical assistance. REFERENCES

FIG. 3. Transmission electron micrograph of the ileal explant (bar 5 1 mm) showing bacteria (isolate 4221) adherent to piglet enterocytes.

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