Detection and Characterization of Shiga Toxigenic <i>Escherichia coli</i> by Using Multiplex PCR Assays for <i>stx</i> ₁ , <i>stx</i> ₂ , <i>eaeA</i> , Enterohemorrhagic <i>E. coli hlyA</i> , <i>rfb</i> _O111 , and <i>rfb</i> _O157

TLDR

Shiga toxin–producing Escherichia coli (STEC) are diverse pathogens that can cause severe gastrointestinal disease, with serogroups O111 and O157 and specific virulence traits conferring higher human virulence. The study aimed to develop two multiplex PCR assays for detecting and genetically characterizing STEC in fecal or food cultures. Assay 1 employed four primer pairs to amplify stx1, stx2 variants, eaeA, and hlyA, while Assay 2 used two primer pairs targeting rfb regions of serotypes O157 and O111, yielding distinct PCR products. Validation on 52 characterized STEC strains showed 100 % concordance, assay 2 avoided false‑positive O157 in EPEC O55, and both assays identified STEC in fecal samples from five HUS patients and three bloody‑diarrhea patients while remaining negative in 31 non‑STEC cultures.

Abstract

ABSTRACT Shiga toxigenic Escherichia coli (STEC) comprises a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, certain strains appear to be of greater virulence for humans, for example, those belonging to serogroups O111 and O157 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 foodstuffs. Assay 1 utilizes four PCR primer pairs and detects the presence of stx 1 , stx 2 (including variants of stx 2 ), eaeA , and enterohemorrhagic 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. Moreover, assay 2 did not give a false-positive O157 reaction with enteropathogenic E. coli strains belonging to clonally related serogroup O55. Assays 1 and 2 detected STEC of the appropriate genotype in primary fecal cultures from five patients with hemolytic-uremic syndrome and three with bloody diarrhea. Thirty-one other primary fecal cultures from patients without evidence of STEC infection were negative.