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

Abstract

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.

Inconsistent PCR detection of Shiga toxin-producing Escherichia coli: Insights from whole genome sequence analyses

“…A significant finding of the present study was that intermittent false stx positives could in twenty-two cases be possibly related to presence of fragments of stx-encoding phages (Table 2), especially as genomes of the majority of strains possessed multiple fragments of identifiable stx-encoding phages (Table 3). The Conrad et al [10] primers used at initial isolation have had positive amplification of stx even with one or two base-pair mismatches [33], but the possible intermittent binding of primers to stx fragments has not been previously reported, likely as only a subset of stx fragments may have influenced PCR results. Larger fragments with highest stx sequence integrity would be the most likely to intermittently bind to PCR primers, although it was not possible in the present study to verify which if any of the stx fragments led to false-positive PCRs.…”

“…In summary, generic E. coli strains showed false positive amplification for serogroups: O26 (n = 6), O45 (n = 2), O103 (n = 6), O145 (n = 2), and O157 (n = 5). The exceptions were O121 which had stable serogroup detection (Table 4) and O111 which was not included in this study due to previously noted stable serogroup and stx 1 detection [33].…”

“…Although there may be difficulties in isolation of STEC, and it has been established that PCR assays across laboratories can produce variable results for detection of Shiga toxin genes due PLOS ONE use of different equipment and methods [22,33], it was expected that the use of the same assay by the same laboratory staff with the same equipment and conditions would produce consistent results. However, on re-growth of isolates collected in previous studies, and repeated PCR, detection of stx 1 and/or stx 2 showed variation for some isolates.…”

The Characterisation of Diarrhoeagenic Verotoxin Producing Non-O157 Escherichia coli among Young Children in Kuantan, Malaysia

“…Similar results of O26 serogroup have been reported from Malaysia (25%), Asia-Pacific (7.2%) ( 40 ), Europe (3.5%) ( 41 ), Africa (7%) ( 42 ) and America (17%), ( 43 ) which are in agreement with our findings. However, a higher frequency of O26 serotype was observed in Iran (43.75%) ( 44 ), Malaysia (18.4%) ( 12 ) and Canada (62.6%) ( 45 ). Epidemiological surveillance indicates that the persistence of non-O157 serotypes, E. coli O26 is the one of the major serotypes of concern.…”

Role of Recent Therapeutic Applications and the Infection Strategies of Shiga Toxin-Producing Escherichia coli

“…The HUS-associated STEC list and their non-motile derivatives were therefore expanded in Table 1A . These are the seven STEC priority serotypes most often associated with HC and HUS infections and sporadic cases worldwide ( Stanford et al., 2018 ); USDA study on the classification of non-O157 STEC from meat products are reported in Table 1B .…”