DETECTION AND CHARACTERIZATION OF SHIGA TOXIGENIC ESCHERICHIA-COLI BYUSING MULTIPLEX PCR ASSAYS FOR STX(1), STX(2), EAEA, ENTEROHEMORRHAGIC ESCHERICHIA-COLI HLYA, RFB(O111), AND RFB(O157)

Shiga toxigenic Escherichia coli (STEC) comprises a diverse group of o rganisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, certain strains appear to be of greater virul ence for humans, for example, those belonging to serogroups O111 and O 157 and those with particular combinations of other putative virulence traits. We have developed two multiplex PCR assays for the detection and genetic characterization of STEC in cultures of feces or foodstuff s. Assay 1 utilizes four PCR primer pairs and detects the presence of stx(1), stx(2) (including variants of stx(2)), eaeA, and enterohemorrh agic E. coli hlyA, generating amplification products of 180, 255, 384, and 534 bp, respectively. Assay 2 uses two primer pairs specific for portions of the rfb (O-antigen-encoding) regions of E. coli serotypes O157 and O111, generating PCR products of 259 and 406 bp, respectively . The two assays were validated by testing 52 previously characterized STEC strains and observing 100% agreement with previous results. More over, assay 2 did not give a false-positive O157 reaction with enterop athogenic E. coli strains belonging to clonally related serogroup O55. Assays 1 and 2 detected STEC of the appropriate genotype in primary f ecal cultures from five patients with hemolytic-uremic syndrome and th ree with bloody diarrhea. Thirty-one other primary fecal cultures from patients without evidence of STEC infection were negative.