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

Abstract

Shiga toxin-producing Escherichia coli (STEC) have been linked to food-borne disease outbreaks. As PCR is routinely used to screen foods for STEC, it is important that factors leading to inconsistent detection of STEC by PCR are understood. This study used whole genome sequencing (WGS) to investigate causes of inconsistent PCR detection of stx1, stx2, and serogroup-specific genes. Fifty strains isolated from Alberta feedlot cattle from three different studies were selected with inconsistent or consistent detection of stx and serogroup by PCR. All isolates were initially classified as STEC by PCR. Sequencing was performed using Illumina MiSeq® with sample library by Nextera XT. Virtual PCRs were performed using Geneious and bacteriophage content was determined using PHASTER. Sequencing coverage ranged from 47 to 102x, averaging 74x, with sequences deposited in the NCBI database. Eleven strains were confirmed by WGS as STEC having complete stxA and stxB subunits. However, truncated stx fragments occurred in twenty-two other isolates, some having multiple stx fragments in the genome. Isolates with complete stx by WGS had consistent stx1 and stx2 detection by PCR, although one also having a stx2 fragment had inconsistent stx2 PCR. For all STEC and 18/39 non-STEC, serogroups determined by PCR agreed with those determined by WGS. An additional three WGS serotypes were inconclusive and two isolates were Citrobacter spp. Results demonstrate that stx fragments associated with stx-carrying bacteriophages in the E. coli genome may contribute to inconsistent detection of stx1 and stx2 by PCR. Fourteen isolates had integrated stx bacteriophage but lacked complete or fragmentary stx possibly due to partial bacteriophage excision after sub-cultivation or other unclear mechanisms. The majority of STEC isolates (7/11) did not have identifiable bacteriophage DNA in the contig(s) where stx was located, likely increasing the stability of stx in the bacterial genome and its detection by PCR.

Phenotypic and genotypic characterization of Shiga toxin-producing Escherichia coli strains recovered from bovine carcasses in Uruguay

“…At the time of serotyping, we found some discordant results between both techniques used and some discrepancies could also be observed in the results obtained when assigning the serogroup by serology or when doing so using the Serotype Finder. The same occurred when Castro et al compared PCR and WGS results to define E. coli serogroups ( Castro et al, 2021 ). Some hypotheses to explain these discrepancies in our study are that we are dealing with new variants or new serogroups not yet described, and therefore not updated in the database used, or strains that cross-react with sera from other serogroups.…”

Isolation of Escherichia coli and Staphylococcus aureusin raw milk from refrigeration tanks: identification and antimicrobial resistance profiles

“…Resistance to several antibiotics may be attributed to the presence of certain genes, such as mcr-mediated colistin resistance and/ or plasmids from the incF and incX groups, among other factors (SAIDENBERG et al, 2020;ZAMPARETTE et al, 2020;CASTRO et al, 2021). These genes and plasmids have been previously detected in E. coli isolated from clinical and food samples in Brazil (SAIDENBERG et al, 2020;ZAMPARETTE et al, 2020) as well as in bovine faeces in Canada (CASTRO et al, 2021).…”

Characterization of Atypical Shiga Toxin Gene Sequences and Description of Stx2j, a New Subtype

“…This highlights the importance of using validated pipelines for stx gene identification to mitigate the risk of misidentification when multiple copies of the gene are present (55,60). Complementing short-read data with long-read sequencing or the use of read-mapping strategies should be considered whenever the presence of multiple gene copies, or fragments of the gene sequence (61), in a chromosome is a possibility. Two STEC strains were found to possess previously unreported variants of established stx2 subtypes (BMH-17-0027 O8:H28 stx2a; BMH-17-0036 O146:H21 stx2b).…”