Evaluation of known and putative Escherichia coli O157:H7 virulence genes

The research aimed to establish a pig model for the pathogenesis of E. coli O157:H7 and to evaluate the roles of verotoxin (VT) and putative virulence genes in the pig model and in HEp-2 and IPEC-J2 cells. Oral infection of pigs with the E. coli O157:H7 strain 86-24 was unsuitable but ligated pig ileal loops were successfully used to evaluate intestinal adherence of O157:H7 strain 86-24 and its mutants. There was more extensive attaching and effacing (AE) lesion formation in the ileum compared with the spiral colon. In both the in-vitro and the in-vivo systems culture conditions affected adherence; some effects applied to both systems but others varied between the two systems. Growth in MacConkey broth resulted in failure to adhere to the cultured cells but had no effect on adherence in-vivo. Patterns of adherence to the pig intestinal IPEC-J2 cells were similar to those with HEp-2 cells but adherence tended to be more extensive with IPEC-J2 cells. Mutants of O157:H7 strain 86-24 defective in vt2 and putative virulence genes from O-islands (OI) 7, 15 and 48, including the urease genes, the tellurite-resistance genes, aidA and the entire OI-7, were generated by allele exchange and the lambda-red recombination system. The mutants that lacked vt2 had decreased adherence to both cells and pig ileal epithelium. Knockout of the tellurite-resistance genes caused a reduction in adherence to cultured cells. Complemented vt2 and tellurite resistance mutants were restored to wild type levels of adherence in in-vitro assays. Mutants lacking urease, Iha or the AIDA-I-like adhesin of OI-15 adhered like the wild type to the cultured cells but had reduced adherence in the ileum. The complementing plasmid-encoded genes ureC and iha failed to restore adherence of the mutants, suggesting that secondary mutations or regulatory factors may have been involved. Mutants of OI-7 and aidA of OI-48 had no effect on adherence to cultured cells or the pig ileum. The data support a role for VT in colonization of the host intestine and identify tellurite-resistance as another factor involved in adherence of E. coli O157:H7.