Molecular characterisation of the recA locus in clinical isolates of verocytotoxigenic E. coli O157:H7

Molecular epidemiology of verocytoxigenic Escherichia coli O157:H7 is important to help elucidate reservoirs and modes of transmission, particularly between animals and humans. As the recA gene locus is now beginning to gain application in bacterial genotyping schemes, and as it has not been examined previously in E. coli O157 isolates, this study aims to examine potential polymorphic variation as a possible epidemiological marker for the subspecies characterisation of clinically significant verocytotoxigenic E. coli O157:H7. A novel polymerase chain reaction (PCR) assay was designed to target a 638 bp region of the recA gene in E. coli O157 isolates. The PCR amplification of genomic DNA from extracted organisms was able to generate an amplicon of the expected size (approximately 638 bp) for all E. coli O157:H7 examined (n=80), as well as for other non-O157 F. coli and other members of the Enterobacteriaeceae including Citrobacter, Hafnia, Shigella, Enterobacter and Providencia. Subsequent restriction fragment length polymorphism (RFLP) and single-stranded conformational polymorphism (SSCP) analyses of these recA amplicons were able to differentiate E. coli O157 from the organisms examined, but were unable to distinguish between 79 isolates of wild-type E. coli O157, suggesting a highly conserved recA gene structure within the local population of organisms examined.