Phenotypic and genotypic characterisation of Escherichia coli strains serogrouped as enteropathogenic E. coli (EPEC) isolated from pasteurised milk

Abstract Fifty-six Escherichia coli strains, serogrouped as EPEC, isolated from three different brands of pasteurised milk commercialised in Rio de Janeiro, Brazil, were tested for enteropathogenicity markers. Most of the strains (71.4%) were adherent to HEp-2 cells. The adherent strains were distributed among 7 EPEC serogroups (O26, O55, O111, O114, O125, O127, O128, O158). Although almost half of these strains (33.9%) presented unrecognisable adherence phenotypes, classical adherence patterns (localised-like, aggregative and diffuse adherence) described for E. coli and epidemiologically associated with diarrheagenic strains were observed. None of the strains showed typical localised adherence, usually associated with EPEC strains, but 4 of them displayed a localised-like adherence (LAL) phenotype, characterised by fewer and less compact microcolonies but that is still associated with diarrheagenic strains as well as strains of non-human origin. Indeed, 3 of these 4 strains were able to elicit the attaching–effacing lesion (FAS-positive), the central feature of EPEC pathogenesis, and hybridised with bfp A and eae DNA probes. The other LAL-positive strain hybridised with the bfp A probe but gave negative results for the eae probe and FAS assays. Interestingly, all LAL-positive strains produced amplicons of 200 bp in the PCR for bfp A, instead of the expected 326 bp fragment. PCR reactions for stx 1 and stx 2, two shiga-toxin-encoding genes, gave negative results. Typing of LEE-associated genes by PCR showed the profile eae (β), tir (β), esp A (α) and esp B (α) for one of the LAL-positive strain. The most prevalent adherence phenotype was the aggregative pattern which is observed in strains epidemiologically associated with persistent diarrhea. Additionally, one strain promoted complete detachment of the Hep-2 cell monolayer after 3 h of infection which might be related to the production of citotoxins, a feature that has been increasingly observed in clinical strains. The possession of EPEC-related O and H antigens is no longer deemed an essential characteristic of true pathogenic EPEC strains, emphasising the importance of routinely screen for virulence markers in E. coli strains isolated from foods. Our results are in accordance with data from the literature that demonstrate that environmental strains display atypical features but yet are capable of eliciting the classical A/E lesion and thus must be considered as potentially pathogenic. Further, our results demonstrate the potential of pasteurised milk as a vehicle for transmission of diarrheagenic E. coli in Brazil.