Variability in Characterizing Escherichia coli from Cattle Feces: A Cautionary Tale

Shiga toxin-producing Escherichia coli (STEC) are diverse bacteria, with seven serogroups (O26, O45, O103, O111, O121, O145, O157; "Top 7") of interest due to their predominance in human disease. Confirmation of STEC relies on a combination of culturing, immunological and molecular assays, but no single gold standard for identification exists. In this study, we compared analysis of STEC between three independent laboratories (LAB) using different methodologies. In LAB A, colonies of Top 7 were picked after serogroup-specific immunomagnetic separation of feces from western-Canadian slaughter cattle. A fraction of each colony was tested by PCR (stx1, stx2, eae, O group), and Top 7 isolates were saved as glycerol stocks (n = 689). In LAB B, a subsample of isolates (n = 171) were evaluated for stx1 and stx2 using different primer sets. For this, approximately half of the PCR were performed using original DNA template provided by LAB A and half using DNA extracted from sub-cultured isolates. All Top 7 isolates were sub-cultured by LAB A and shipped to LAB C for traditional serotyping (TS) to determine O and H groups, with PCR-confirmation of virulence genes using a third set of primers. By TS, 76% of O groups (525/689) matched PCR-determined O groups. Lowest proportions (p < 0.05) of O group matches between PCR and TS (62.6% and 69.8%) occurred for O26 and O45 serogroups, respectively. PCR-detection of stx differed most between LAB A and LAB C. Excluding isolates where O groups by PCR and TS did not match, detection of stx1 was most consistent (p < 0.01) for O111 and O157:H7/NM. In contrast, for O45 and O103, stx1 was detected in >65% of isolates by LAB A and <5% by LAB C. Stx2 was only detected by LAB C in isolates of serogroups O121, O145, and O157:H7/NM. LAB B also detected stx2 in O26 and O157:H12/H29, while LAB A detected stx2 in all serogroups. Excluding O111 and O157:H7/NM, marked changes in stx detection were observed between initial isolation and sub-cultures of the same isolate. While multiple explanations exist for discordant O-typing between PCR and TS and for differences in stx detection across labs, these data suggest that assays for STEC classification may require re-evaluation and/or standardization.