Molecular Detection of Non-O157 Shiga Toxin-Producing Escherichia coli (STEC) Directly from Stool Using Multiplex qPCR Assays

Abstract

Non-O157 Shiga toxin-producing E. coli (STEC) can cause outbreaks that have great economic and health impact. Since the implementation of STEC screening in Alberta in 2018, it is also essential to have a molecular serotyping method with faster turnaround time for cluster identification and surveillance purposes. This study sought to perform molecular serotyping of the top six non-O157 (O26, O45, O103, O111, O121 and O145) STEC serotypes directly from stools and enrichment broths compared to conventional methods on isolates. Multiplex, serotyping qPCR assays were used to determine sensitivity and specificity of the top six non-O157 STEC serotypes. Sensitivity and specificity were assessed for both singleplex and multiplex qPCR assays for comparison of the top six serotypes. Blinded stool specimens (n = 116) or broth samples (n = 482) submitted from frontline microbiology laboratories for STEC investigation were analyzed by qPCR. Both singleplex and multiplex assays were comparable, and we observed 100% specificity with a limit of detection of 100 colony-forming units per mL. Direct molecular serotyping from stool specimens mostly correlated (88%) with conventional serotyping of the cultured isolate. In cases of discordant serotypes, the top six non-O157 STEC mixed infections were identified and confirmed by culture and conventional serotyping. Detection of non-O157 STEC can be done directly from stool specimens using multiplex PCR assays with the ability to identify mixed infections, which would otherwise remain undetected by conventional serotyping of a single colony. This method can be easily implemented into a frontline diagnostic laboratory to enhance surveillance of non-O157 STEC, as more frontline microbiology laboratories move to culture independent assays.

Keywords: STEC; broth 5; culture; qPCR; serotyping.

Figure 1

STEC serotyping workflow comparing enriched broth culture and stool direct serotyping to conventional serotyping provided by the Public Health Agency of Canada—National Microbiology Laboratory (PHAC-NML), Winnipeg, Manitoba, Canada. Positive stool (n = 116) and broth (n = 482) samples were directly molecularly serotyped using qPCR. Additionally, broth cultures were plated onto CHROMagar™ STEC (Micronostyx. Ottawa, ON, Canada) where 3 colonies were picked and characterized for stx using qPCR. One of the isolates was then sent to the PHAC-NML for conventional serotyping and result generated was then compared to the in-house direct serotyping. Any discordant result between direct and conventional serotyping was treated as a mixed infection and an attempt was made to isolate the additional serotype for confirmation.