Investigation of domestic animals and pets as a reservoir for intimin- (eae) gene positive Escherichia coli types

Domestic animals belonging to seven different species (cattle, sheep, dogs, cats, pigs, chicken and goats) were investigated as natural reservoirs for attaching and effacing Escherichia coli (AEEC). For this, 2165 E. coli strains from faeces of 803 animals were examined for the presence of the intimin -(eae) gene as a characteristic of AEEC strains. Ten percent of the animals were found to excrete AEEC, most frequently found in sheep (19.2%) and pigs (17.6), followed by cattle (10.4%), dogs (7.2%), cats (6.5%) and poultry (2.3%). The 97 AEEC strains from animals were grouped into 44 serotypes. Only four E. coli serotypes (O2:H8, O26:[H11], O109:[H25] and O145:[H28] were found in more than one animal host species. AEEC O26:[H11] strains were most frequently isolated (13.4%) being present in cattle, poultry, pigs and sheep. A search for virulence markers associated with enterohemorrhagic E. coli (EHEC) revealed Shiga-toxin genes in three (3.1%) AEEC strains from sheep. Bundle forming pili genes as a trait of typical enteropathogenic E. coli (EPEC) were detected in four (4.1%) strains from dogs and cats. The remaining 90 AEEC strains were classified as atypical EPEC. Typing of intimin genes revealed intimin beta being present in 51.5% of the strains, followed by intimins theta (23.7%), epsilon (6.2%), kappa (5.2%), zeta (5.2%), alpha, eta and iota (each 1.0%). Our data indicate that domestic animals and pets constitute an important natural reservoir of AEEC strains, and some of these (O26:[H11], O103:H2, O128:H2, O145:[H28] and O177:[H11]) are known to occur as pathogens in humans.     

Prevalence of the lpfO113 gene cluster among Escherichia coli O157 isolates from different sources

Domestic farm animals represent an important reservoir of infection for Shiga toxin-producing Escherichia coli (STEC). Nevertheless the bacterial factors required to colonise these hosts are poorly defined. In this study, the prevalence of a recently described fimbrial gene cluster, lpfO113, among human and animal isolates of STEC was investigated. lpfO113 has been shown to play a role in the adherence of STEC O113:H21 to epithelial cells. Here the presence of the lpfAO113 gene (predicted to encode a major fimbrial subunit) was examined by PCR in E. coli of serogroups O157 and O26 isolated from pigs (n=38), cattle (n=10), and humans (n=9). In addition, we tested for several other genetic virulence markers including Shiga toxin (stx), intimin (eae), the translocated intimin receptor (tir), EHEC-hemolysin (ehx) and F18 fimbriae (fedA). Overall 45 of the 57 strains (79%) possessed the lpfAO113 gene as determined by the presence of a 573 bp PCR product. Moreover, there was a close correlation between the presence of the lpfAO113 marker and the absence of the eae gene. lpfAO113 was found in all of pig isolates, suggesting a possible role in colonisation of the porcine host. In addition, several E. coli strains isolated from pigs had two fimbrial gene markers, fedA and lpfAO113. lpfAO113 was not present in strains of E. coli O157:H7 as described previously. Overall these results show that lpfAO113 is widely distributed among eae-negative E. coli isolates and thus may represent an important adherence factor in this group of pathogens.     

Comparison of ruminant and human attaching and effacing Escherichia coli (AEEC) strains

The presence of 12 genes associated with virulence in human attaching and effacing Escherichia coli (AEEC) was studied within a collection of 20 enterohemorrhagic E. coli (EHEC) and 206 atypical enteropathogenic E. coli (EPEC) isolated from ruminants. In addition, virulence genes and the clonal relationship of 49 atypical EPEC O26 strains isolated from humans and ruminants were compared to clarify whether ruminants serve as a reservoir of atypical EPEC for humans. A great diversity in the content of virulence gene was found. Thus, the espH, espG and map genes were detected in more than 85% of ruminant AEEC strains; the tccP2, espI, efa1/lifA, ehxA and paa genes were present in 50-70% of strains; and other genes such as tccP, espP, katP and toxB were detected in <25% of strains. EHEC strains contained more virulence genes than atypical EPEC strains. Our results suggest for the first time that the efa1/lifA gene is associated with diarrhea in newborn ruminants and that the AEEC strains with the H11 flagellar antigen are potentially more virulent than the non-H11 AEEC strains. Importantly, we identified a new intimin variant gene, eaeρ, in three ruminant atypical EPEC strains. The comparison of ruminant and human EPEC O26 strains showed that some ruminant strains possess virulence gene profiles and pulse-field gel electrophoresis pulsotypes similar to those of human strains. In conclusion, our data suggest that atypical EPEC is a heterogeneous group with different pathogenic potential and that ruminants could serve as a reservoir of atypical EPEC for humans.     

Serotypes, virulence genes and intimin types of verotoxin-producing Escherichia coli and enteropathogenic E. coli isolated from healthy dairy goats in Spain

Faecal samples from 222 healthy dairy goats on 12 farms in Spain, as well as bulk tank milk samples of these farms, were screened for the presence of verotoxin-producing Escherichia coli (VTEC) and enteropathogenic E. coli (EPEC). VTEC and EPEC were isolated in 47.7 and 7.7% of the animals, respectively. VTEC were isolated more frequently from adults and replacement animals than from goat kids. In contrast, EPEC were detected more frequently from goat kids than from replacement animals and adults. VTEC or EPEC strains were not detected in the bulk tank milk samples. Although a selective enrichment protocol was used, the serotype O157:H7 was not detected. The most frequent serotypes among the 106 VTEC strains isolated from goats were O5:H-, O76:H19, O126:H8, O146:H21, ONT:H- and ONT:H21. None VTEC strain was eae-positive. The absence of the eae gene in the VTEC strains could indicate that these strains are less virulent for humans that the classical eae-positive enterohaemorrhagic E. coli types. However, 16% of VTEC strains isolated from healthy goats belonged to serotypes associated with haemolytic uraemic syndrome in humans. The ehxA gene was detected in 84.9 and 52.9% of the VTEC and EPEC from goats, respectively. The beta1, theta/gamma2 and zeta were the most frequent intimin types among the 17 EPEC strains studied and the most prevalent serotypes of these strains were O156:H25 and O177:H11. Our data show that in Spain healthy goats are an important reservoir of VTEC and EPEC, and a potential source of infection for humans.     

Domestic Cats Constitute a Natural Reservoir of Human Enteropathogenic Escherichia coli Types

Feces of 70 diarrhoeic and 230 non‐diarrhoeic domestic cats from São Paulo, Brazil were investigated for enteropathogenic (EPEC), enterohaemorrhagic (EHEC) and enterotoxigenic (ETEC) Escherichia coli types. While ETEC and EHEC strains were not found, 15 EPEC strains were isolated from 14 cats, of which 13 were non‐diarrhoeic, and one diarrhoeic. None of 15 EPEC strains carried the bfpA gene or the EPEC adherence factor plasmid, indicating atypical EPEC types. The EPEC strains were heterogeneous with regard to intimin types, such as eae‐θ (three strains), eae‐κ (n = 3), eae‐α1 (n = 2), eae‐ι (n = 2), one eae‐α2, eae‐β1 and eae‐η each, and two were not typeable. The majority of the EPEC isolates adhered to HEp‐2 cells in a localized adherence‐like pattern and were positive for fluorescence actin staining. The EPEC strains belonged to 12 different serotypes, including O111:H25 and O125:H6, which are known to be pathogens in humans. Multi locus sequence typing revealed a close genetic similarity between the O111:H25 and O125:H6 strains from cats, dogs and humans. Our results show that domestic cats are colonized by EPEC, including serotypes previously described as human pathogens. As these EPEC strains are also isolated from humans, a cycle of mutual infection by EPEC between cats and its households cannot be ruled out, though the transmission dynamics among the reservoirs are not yet understood clearly.     

Attaching and effacing Escherichia coli isolated from dogs in Brazil: characteristics and serotypic relationship to human enteropathogenic E. coli (EPEC)

Escherichia coli isolates recovered from 182 fecal specimens from dogs up to five months old from the cities of S?o Paulo and Campinas, SP, Brazil, were examined by polymerase chain reaction (PCR) for several virulence factors and properties. The eae gene was found in 23 isolates of E. coli from 22 dogs, 19 of 146 (13%) from dogs with diarrhea and 3 of 36 (8.3%) from dogs with no diarrhea. Two different eae+ isolates were recovered from one dog with diarrhea. Isolates from two dogs with diarrhea harbored the bfpA gene, and none of the isolates possessed genes for enterotoxins, the EAF plasmid or Shiga toxins. PCR showed that, among the 23 isolates, eight were positive for beta intimin, six for gamma, two for, one for alpha, one for kappa, and five showed no amplification with any of the nine pairs of specific intimin primers used. PCR also showed that the LEE (locus of enterocyte effacement) was inserted in selC in four isolates, likely in pheU in seven isolates, and in undetermined sites in twelve isolates. Fifteen isolates adhered to HEp-2 cells and were fluorescence actin staining (FAS) positive. The predominant adherence pattern was the localized adherence-like (LAL) pattern. The eae-positive isolates belonged to a wide diversity of serotypes, including O111:H25, O119:H2 and O142:H6, which are serotypes that are common among human EPEC. These results confirmed the presence of EPEC in dogs (DEPEC) with and without diarrhea. The virulence factors found in these strains were similar to those in human EPEC, leading to the possibility that EPEC may move back and forth among human and canine populations.     

Serobiotypes and virulence genes of Escherichia coli strains isolated from diarrheic and healthy rabbits in Brazil

A total of 178 Escherichia coli isolates from diarrheic and healthy rabbits in the S?o Paulo State (Brazil) were serobiotyped and investigated by PCR for the presence of virulence genes. Among the 90 (50.6%) isolates which possessed the eae gene, 74 were from diarrheic animals and all but one encoded intimin beta. Sixty five (72.2%) of the eae+ isolates had insertion of the locus of enterocyte effacement locus in the pheU locus, 11 (12.2%) in the selC and 14 (15.6%) did not insert in either of these loci. All isolates were negative for genes of the E. coli enterotoxins, Stx1, Stx2, CNF1, CNF2 and EHEC hemolysin. The O132:H2 serotype was dominant, being present in 63 isolates (70%) of the 90 eae+ isolates, and 57 of the 63 isolates of this serotype belonged to biotype 30. PCR detected the gene for AF/R2 fimbriae in 75 (83.3%) of the 90 eae+ isolates. Adherence to HeLa cells was best detected following 6h incubation and a positive fluorescence actin staining (FAS) test was given by 52 isolates. These data show that isolates of E. coli associated with diarrhea in rabbits in Brazil possess the genotype and phenotype typically associated with rabbit enteropathogenic E. coli (EPEC). We conclude that EPEC that possess the eae gene are a common cause of diarrhea in Brazilian rabbit farms and that the pathogenic eae+ AF/R2+ isolates of O132:H2:B30 serobiotype are especially predominant.     

Subtypes of intimin among non-toxigenic Escherichia coli from diarrheic calves in Brazil.

One hundred and five strains of Escherichia coli that were isolated from calves with diarrhea in the state of São Paulo, Brazil, and were negative for enterotoxins and cytotoxins, were examined for the eae gene. Four (3.8%) strains were positive by polymerase chain reaction (PCR) and were shown to produce intimin by using Western blot with specific antiserum against the conserved N-terminal region of intimin. Subtyping of the intimins was done by PCR with specific primers and by Western blot with specific antisera against the C-terminal variable region of the protein. Three of these isolates (O?:H11, O26:H-, O123:H1) produced the beta subtype of intimin, and the 4th (0103:H2) produced intimin that was not typable. The 0103:H2 and the O26:H-isolates adhered to HEp-2 cells with diffuse adherence and localized-like adherence patterns, respectively. The other strains did not adhere to HEp-2 cells. To our knowledge, this is the first report of the occurrence of a subtype of intimin described for human enteropathogenic E. coli among bovine diarrheogenic E. coli. It is also the first report from Brazil demonstrating the presence of bovine E. coli harboring the eae gene.     

Phenotypic and genotypic characteristics of non-O157 Shiga toxin-producing Escherichia coli (STEC) from Swiss cattle

A total of 42 Shiga toxin-producing (STEC) strains from slaughtered healthy cattle in Switzerland were characterized by phenotypic and genotypic traits. The 42 sorbitol-positive, non-O157 STEC strains belonged to 26 O:H serotypes (including eight new serotypes) with four serotypes (O103:H2, O113:H4, O116:H-, ONT:H-) accounting for 38.1% of strains. Out of 16 serotypes previously found in human STEC (71% of strains), nine serotypes (38% of strains) were serotypes that have been associated with hemolytic-uremic syndrome (HUS). Polymerase chain reaction (PCR) analysis showed that 18 (43%) strains carried the stx1 gene, 20 strains (48%) had the stx2 gene, and four (9%) strains had both stx1 and stx2 genes. Of strains encoding for stx2 variants, 63% were positive for stx2 subtype. Enterohemolysin (ehxA), intimin (eae), STEC autoagglutinating adhesin (saa) were detected in 17%, 21%, and 19% of the strains, respectively. Amongst the seven intimin-positive strains, one possessed intimin type beta1 (O5:H-), one intimin gamma1 (O145:H), one intimin gamma2/theta, (O111:H21), and four intimin epsilon (O103:H2). The strains belonged to 29 serovirotypes (association between serotypes and virulence factors). O103:H2 stx1eae-epsilon ehxA, O116:H- stx2, and ONT:H- stx2c were the most common accounting for 29% of the strains. Only one strain (2.4%) of serovirotype O145:H- stx1stx2eae-gamma1ehxA showed a pattern of highly virulent human strains. This is the first study providing characterization data of bovine non-O157 STEC in Switzerland, and underlining the importance of the determination of virulence factors (including intimin types) in addition to serotypes to assess the potential pathogenicity of these strains for humans.     

Phylogenetic background of attaching and effacing <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from animals

Detection and distribution of eae gene in forty-four attaching and effacing Escherichia coli (AEEC) strains of animal origin were investigated. Association of distinct intimin alleles with phylogenetic background were assessed among strains in comparison with different serogroups. Phylogenetic analysis showed that 31 EHEC/eae+ STEC strains belong to groups A, B1 and E, 13 EPEC strains segregated in B1 and B2. Moreover, group A possessed the eae gamma2/theta type, group B1 the eae beta1, eae kappa, eae zeta, and eae epsilon types, group B2 the eae alpha1, eae alpha2 and eae iota types, while the group E possessed the eae gamma1 type. The presence of numerous eae-types show that EPEC and EHEC/eae+ STEC tested have a high genetic homology within each phylogenetic group.     

Phenotypic and genetic characterization of enteropathogenic Escherichia coli (EPEC) and entero-aggregative E. coli (EAEC) from diarrhoeal and non-diarrhoeal children in Libya

A total of 50 Escherichia coli strains isolated in a Libyan hospital (20 from children with diarrhoea and 30 from healthy children) were investigated for their pathotypes and virulence traits. Altogether nine eae-positive (enteropathogenic E. coli, EPEC) and nine aggR-positive (entero-aggregative E. coli, EAEC) strains were identified. Significantly (P=0.001) more EPEC strains were identified from diarrhoeal patients (n=8) than from healthy controls (n=1), while six EAEC strains were identified from diarrhoeal and three from healthy children. Typical (eae(+), EAF(+), bfp(+)) EPEC strains (n=6) belonged to classical EPEC serogroups O55, O114, O127 and showed localized adherence on Hela cells. EAEC strains revealed genetic heterogeneity but uniformly adhered to HeLa cultures in an entero-aggregative adherence pattern. Antibiotic resistance frequently, characterized the strains. Sixty-eight percentage of the strains were resistant against at least one antibiotic and 30% harbored a class 1 integron independently of their clinical background. This is the first report from North Africa demonstrating the significance of EPEC and EAEC.     

Virulence gene profiles and intimin subtypes of Shiga toxin-producing Escherichia coli isolated from healthy and diarrhoeic calves

The virulence properties of Shiga toxin-producing Escherichia coli (STEC) strains isolated from diarrhoeic and non-diarrhoeic calves were compared. The strains were also tested for O157:H7, O111 and O26 serotypes, using PCR and conventional serotyping methods. E coli strains isolated from 297 faecal samples, from 200 diarrhoeic and 97 non-diarrhoeic calves, were screened by multiplex PCR assay for the stx1, stx2, eae and Ehly virulence genes. STECs were recovered from 8 per cent of diarrhoeic calves and 10.3 per cent of non-diarrhoeic calves. The predominant virulence gene profile was stx1/eae/Ehly (47.3 per cent) among isolates from diarrhoeic calves and eae/Ehly (36.8 per cent) among isolates from non-diarrhoeic calves. Among three tested serogroups, the predominant serogroup was O26 (18.4 per cent), and O157:H7 was not detected. Intimin subtyping by restriction fragment length polymorphism analysis revealed only three intimin subtypes (β, γ and ). A significant difference was observed in the distribution of Int- between two groups. Int- was present in 50 per cent of the isolates from diarrhoeic calves and in 11.1 per cent of the isolates from non-diarrhoeic calves; this difference was statistically significant (P=0.01).     

A national surveillance of Shiga toxin-producing Escherichia coli in food-producing animals in Japan

To assess the public health risk, the prevalence and anti-microbial resistance of Shiga toxin-producing Escherichia coli (STEC) among food-producing animals were studied throughout Japan. Faecal samples were collected from healthy animals of 272 cattle, 179 pigs, and 158 broilers on 596 farms in all 47 Japanese prefectures. STEC were isolated from 62 (23%) cattle and 32 (14%) pig samples but from no chicken samples. Of the bovine isolates, 19 belonged to serotypes frequently implicated in human disease (O157:H7/non-motile (NM)/H not typeable, O26:NM/H11/H21/H not typeable, O113:H21, and O145:NM). The eae genes were observed in 37% of bovine isolates; among them one O145:NM and all four O157 isolates possessed eae-gamma1, and one O145:NM, one O103:H11, and all five O26 isolates possessed eae-beta1 gene. Among the swine isolates, stx2e were dominant, and serotypes frequently implicated in human diseases or eae-positive isolates were not observed. Bovine isolates showed less anti-microbial resistance, but six isolates of 26:NM/H11 and O145:NM were multi-resistant and may need careful monitoring. Swine isolates showed various resistance patterns; chloramphenicol resistance patterns were more common than in bovine isolates. This first national study of STEC in the Japanese veterinary field should aid our understanding of Japan's STEC status.     

Serotypes, virulence genes, intimin types and PFGE profiles of Escherichia coli isolated from piglets with diarrhoea in Slovakia

Two hundred and fifty Escherichia coli isolates from diarrhoeic and healthy piglets were serotyped and tested for the presence of virulence genes for fimbriae, intimin, heat-labile (LT) and heat-stable (STa and STb) enterotoxins, Stx toxins, and enteroaggregative heat-stable 1 (EAST1) enterotoxin by polymerase chain reaction (PCR). Although 220 isolates from diarrhoeic piglets belonged to 43 O serogroups and 77 O:H serotypes, 60% were of one of the 10 serogroups O2, O8, O15, O54, O84, O101, O141, O147, O149 and O157, and 60% belonged to only 10 serotypes (O8:H-, O54:H-, O84:H7, O101:H-, O141:H-, O141:H4, O147:H-, O149:H10, O163:H-, and ONT:H-). PCR showed that 79% of 220 isolates carried genes for at least one of the virulence factors tested. The gene encoding for EAST1 was the most prevalent (65%) followed by those encoding for STb (49%), LT (42%), STa (13%), and Stx2e (4%). Eighty-three (38%) of the 220 E. coli isolates carried the gene for F4 (K88), whereas genes for F18, F5 (K99), F41, F6 (P987), F17, and intimin (eae) were detected in 9%, 3%, 3%, 3%, 1%, and 3%, respectively. Seropathotype O149:H10:F4:LT/STb/EAST1 (70 isolates) was the most common, representing 32% of isolates. Pulsed-field gel electrophoresis (PFGE) analysis with XbaI of 15 O149:H10 representative isolates from diarrhoeic piglets distinguished 14 types. The 15 isolates exhibited a wide variability of distinct restriction patterns though all belonged to the same serotype (O149:H10), and all but one showed identical virulence determinants (F4, LT, STb, and EAST1). Among 30 isolates from healthy piglets only two virulence genes were detected: EAST1 (26%) and eae (17%). In total, 12 isolates were positives for the eae gene: five isolates had intimin beta1, four possessed intimin theta and three showed intimin type xiB. This is believed to be the first study describing the presence of intimin type xiB in E. coli of porcine origin.     

Shedding of food-borne pathogens and microbiological carcass contamination in rabbits at slaughter

To obtain microbiological data from rabbits at slaughter, 500 fecal samples and 500 carcasses samples were examined. All samples tested negative for Listeria and Salmonella. Campylobacter were detected in two fecal samples. Of the 500 fecal samples, 45.8% tested positive for eae (intimin), 1.2% for stx (Shiga toxin), and 1.8% for both eae and stx. By colony hybridization, 56 eae positive Escherichia coli strains were isolated. Among them, 27 strains (48.2%) were of the serotypes O178:H7 and O153:H7, whereas 15 strains (26.8%) belonged to a serogroup that has not yet been described (O(CB10681):H7). All strains possessed intimin beta1 and the translocated intimin receptor (tir) capable of being tyrosine phosphorylated. None of the strains harbored the genes for Shiga toxins, EAST1 (astA), bundlin (bfpA), or the EAF plasmid. Slaughter rabbits therefore constitute a reservoir for certain atypical enteropathogenic Escherichia coli. On rabbit carcasses, average total bacterial counts accounted for 3.3 log CFU cm(-2). Enterobacteriaceae and coagulase positive staphylococci (CPS) were detected on 118 (23.6%) and 153 (30.6%) carcasses, respectively. Enterobacteriaceae and CPS counts of positive samples were mainly <1.5 log CFU cm(-2). Among 153 selected CPS isolates, 98.7% were identified as Staphylococcus aureus. None of the 151 isolated strains harbored the gene for methicillin resistance (mecA). Genes for staphylococcal enterotoxins (SE) were detected in 102 strains. The combinations of seg and sei (53 strains) and sed, seg, sei, and sej (27 strains) dominated.     

Virulence profiles of Shiga toxin 2e-producing Escherichia coli isolated from healthy pig at slaughter

Recently, virulence patterns of Stx2e-producing Escherichia coli from pigs with edema disease and from humans were compared and strains from diseased pigs were reported to be unlikely human pathogens [Sonntag, A.K., Bielaszewska, M., Mellmann, A., Dierksen, N., Schierack, P., Wieler, L.H., Schmidt, M.A., Karch, H., 2005. Shiga toxin 2e-producing Escherichia coli isolates from humans and pigs differ in their virulence profiles and interactions with intestinal epithelial cells. Appl. Environ. Microbiol. 71, 8855-8863]. In the present study, 31 Shiga toxin-producing E. coli (STEC) strains harboring stx2e, which were previously isolated out of fecal samples from healthy pigs at slaughter [Kaufmann, M., Zweifel, C., Blanco, M., Blanco, J.E., Blanco, J., Beutin, L., Stephan, R., 2006. Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland. J. Food Prot. 69, 260-266], were characterized by phenotypic and genotypic traits. Nine of the thirty-one sorbitol-positive non-O157 STEC (stx2e) isolated from healthy pigs belonged to serotypes found in STEC isolated from humans, including two serotypes (O9:H-, O26:H-) reported in association with hemolytic-uremic syndrome. Otherwise, the serotypes were different from those isolated from cases of edema disease in pigs. The eae (intimin) gene, which is strongly correlated with severe human disease, was not detected. Moreover, all strains were lacking the genes for enterohemolysin (ehxA), porcine A/E associated protein (paa), STEC autoagglutinating adhesin (saa) and the serin protease EspI (espI). Nine strains tested positive for astA (EAST1), one O141:H17 strain for fedA (F18 fimbrial adhesin) and one O159:H- strain for terF (tellurite resistance). Similar to the Stx2e-producing E. coli isolated from humans, which are mainly lacking further virulence factors, genes of an iron uptake system on the high-pathogenicity island (irp2, fyuA) were detected in three ONT:H10 and ONT:H19 strains from healthy pigs. Consequently, although the isolated strains are unlikely to be associated with severe human diseases, healthy pigs cannot be excluded as a potential source of human infection with Stx2e-producing STEC.     

A comparison of Shiga-toxin negative Escherichia coli O157 aflagellate and intimin deficient mutants in porcine in vitro and in vivo models of infection

Isolation of Shiga-toxin (Stx) positive Escherichia coli O157:H7 from commercially grown pigs has been reported. Furthermore, experimental infection studies have demonstrated that Stx-positive E. coli O157:H7 can persist in 12-week-old experimentally orally inoculated conventional pigs for up to 2 months and that persistence was not dependent upon intimin. We have shown that the flagellum of Stx-negative E. coli O157:H7 does not have a role to play in pathogenesis in ruminant models whereas, in poultry, the flagellum of E. coli O157:H7 was important for long-term persistent infection. The contribution of the flagellum of Stx-negative E. coli O157 in the colonisation of pigs was investigated by adherence assays on a porcine (IPI-21) cell line, porcine in vitro organ culture (IVOC) and experimental oral inoculation of conventional 14-week-old pigs. E. coli O157:H7 NCTC12900nal(r) and isogenic aflagellate and intimin deficient mutants adhered equally well to IPI-21 cells. In porcine IVOC association assays, E. coli O157:H7 NCTC12900nal(r) was associated in significantly higher numbers to tissues from the caecum and the terminal rectum than other sites. The aflagellate and intimin deficient mutants significantly adhered in greater numbers to more IVOC gastrointestinal tissues than the parent. Groups of 14-week-old pigs were dosed orally with 10(10)CFU/10ml of either E. coli O157:H7 NCTC12900nal(r) or isogenic aflagellate and intimin deficient mutants and recovery of each test strain was similar. Histological analysis of pig tissues at post mortem examination revealed that E. coli O157 specifically stained bacteria were associated with the mucosa of the ascending and spiral colon. These data suggest that colonisation and persistence of Stx-negative E. coli O157:H7 in pigs, involves mechanisms that do not require the flagellum or intimin.     

Characterization of atypical Enteropathogenic Escherichia coli (aEPEC) isolated from dogs

Enteropathogenic Escherichia coli (EPEC), an important human pathogen has the ability to form attaching and effacing lesions on the intestinal epithelium and has been isolated from a wide range of species. Two EPEC subgroups are recognized: typical (tEPEC) and atypical (aEPEC) strains, differing by the presence of EAF plasmid and bundle-forming pilus (BFP) in typical strains and their absence in atypical strains. This study searched the presence of EPEC strains in 101 fecal samples of diarrheic (n=65) and non-diarrheic (n=36) dogs from two cities in Rio de Janeiro State, Brazil. The isolates were evaluated for the presence of eae, tir, espA, espB and bfpA genes, EAF plasmid, and for the insertion site of the LEE locus. Cell-adherence assays, fluorescent actin staining (FAS) test, hemolysin production and serotyping were also performed. Twenty eight aEPEC isolates were recovered from 48 eae-positive fecal samples, 24 from diarrheic animals and 4 from non-diarrheic ones. PCR showed that most isolates was positive for β or γ intimin, and for β or α subtypes of tir, espA and espB. Six isolates showed a selC insertion of locus LEE. Only two isolates from the same diarrheic animal harbored the bfpA gene, and none presented the EAF plasmid. Most isolates was FAS-positive and showed a localized adherence-like (LAL) in a 6h HeLa cell-adherence assay. A wide diversity of serotypes was detected including O4:H16 and O51:H40, previously described in human disease. The phenotypic and genotypic markers of aEPEC isolated from diarrheic and non-diarrheic dogs were similar to those found in isolates recovered from human disease.     

Serotypes and analysis of distribution of Shiga toxin producing Escherichia coli from cattle and sheep in the lower North Island, New Zealand

AIMS: To serotype a subset of Shiga toxin-producing Escherichia coli (STEC) isolates from cattle and sheep to determine whether any corresponding serotypes have been implicated in human diarrhoeal disease, both in New Zealand and worldwide, and to examine the distribution of STEC and enteropathogenic Escherichia coli (EPEC) amongst cattle (calves, heifers and dairy) and sheep (lambs, rams and ewes), to assess whether carriage of identified bacterial genotypes may be associated with a particular age of animal. METHODS: Recto-anal mucosal swabs (RAMS) were taken from 91 calves, 24 heifers and 72 dairy cattle, and 46 lambs, 50 ewes and 36 rams, from four sites in the Manawatu and Rangitikei regions of New Zealand. Strains of E. coli selected from primary isolation plates were subjected to a multiplex polymerase chain reaction (PCR), to determine the presence of Shiga toxin genes (stx1 and stx2) and the E. coli attaching and effacing gene (eae). RESULTS: Overall, 186/319 (58.3%) animals sampled were positive for stx1, stx2, or eae isolates. More sheep (43.9%) were stx1-positive than cattle (2.7%; p = 0.036), and amongst sheep more lambs and ewes were stx1-positive than rams (p = 0.036). Amongst cattle, more calves and heifers were eae-positive than dairy cows (p = 0.030). Two or more different STEC were isolated from at least 28 (9%) animals (three cattle and 25 sheep), based on their stx/eae genotype. Enterohaemolysin genes were found in 39/51 (76%) isolates serotyped. Twenty-one different serotypes were detected, including O5:H-, O9:H51, O26:H11, O84:H-/H2 and O149:H8 from cattle, and O26:H11, O65:H-, O75:H8, O84:H-, O91:H-, O128:H2 and O174:H8 from sheep; O84:H-, O26:H11, O5:H-, O91:H- and O128:H2 serotypes have been associated with human disease. CONCLUSIONS: If nationally representative, this study confirms that cattle and sheep in New Zealand may be a major reservoir of STEC serotypes that have been recognised as causative agents of diarrhoeal disease in humans. Distribution of STEC and EPEC in cattle and sheep indicates that direct contact with, in particular, calves or their faeces, or exposure to environments cross contaminated with ruminant faeces, may represent an increased risk factor for human disease in New Zealand.     

Serotypes,phage types and virulence genes of shiga-producing Escherichia coli isolated from sheep in Spain

PROBLEM ADDRESSED: Shiga toxin-producing Escherichia coli (STEC), have emerged as food poisoning pathogens which can cause severe diseases in humans. OBJECTIVE: The aim of this study was to determinate the serotypes and virulence genes of STEC strains isolated from sheep in Spain, with the purpose of determining whether sheep represent a potential source of STEC pathogenic for humans. METHODS AND APPROACH: Faecal swabs obtained from 697 healthy lambs on 35 flocks in Spain during the years 2000 and 2001 were examined for STEC using phenotypic (Vero cells) and genotypic (PCR) methods. RESULTS: STEC O157:H7 strains were isolated from seven (1%) animals in six flocks, whereas non-O157 STEC strains were isolated from 246 (35%) lambs in 33 flocks. A total of 253 ovine STEC strains were identified in this study. PCR showed that 110 (43%) strains carried stx(1) genes, 10 (4%) possessed stx(2) genes and 133 (53%) both stx(1) and stx(2). Enterohaemolysin (ehxA) and intimin (eae) virulence genes were detected in 120 (47%) and in 9 (4%) of the STEC strains. STEC strains belonged to 22 O serogroups and 44 O:H serotypes. However, 70% were of one of these six serogroups (O6, O91, O117, O128, O146, O166) and 71% belonged to only nine serotypes (O6:H10, O76:H19, O91:H-, O117:H-, O128:H-, O128:H2, O146:H21, O157:H7, O166:H28). A total of 10 new O:H serotypes not previously reported in STEC strains were found in this study. Seven strains of serotype O157:H7 possessed intimin type gamma1, and two strains of serotype O156:H- had the new intimin zeta. STEC O157:H7 strains were phage types 54 (four strains), 34 (two strains) and 14 (one strain). CONCLUSIONS: This study confirms that healthy sheep are a major reservoir of STEC pathogenic for humans. However, because the eae gene is present only in a very small proportion of ovine non-O157 STEC, most ovine strains may be less pathogenic.