Comparative Evaluation of Genomic and Laboratory Approaches for Determination of Shiga Toxin Subtypes in Escherichia coli

Abstract

The determination of Shiga toxin (ST) subtypes can be an important element in the risk characterization of foodborne ST-producing Escherichia coli (STEC) isolates for making risk management decisions. ST subtyping methods include PCR techniques based on electrophoretic or pyrosequencing analysis of amplicons and in silico techniques based on whole genome sequence analysis using algorithms that can be readily incorporated into bioinformatics analysis pipelines for characterization of isolates by their genetic composition. The choice of technique will depend on the performance characteristics of the method and an individual laboratory's access to specialized equipment or personnel. We developed two whole genome sequence-based ST subtyping tools: (i) an in silico PCR algorithm requiring genome assembly to replicate a reference PCR-based method developed by the Statens Serum Institut (SSI) and (ii) an assembly-independent routine in which raw sequencing results are mapped to a database of known ST subtype sequence variants (V-Typer). These tools were evaluated alongside the SSI reference PCR method and a recently described PCR-based pyrosequencing technique. The V-Typer method results corresponded closely with the reference method in the analysis of 67 STEC cultures obtained from a World Health Organization National Reference Laboratory. In contrast, the in silico PCR method failed to detect ST subtypes in several cases, a result which we attribute to assembly-induced errors typically encountered with repetitive gene sequences. The V-Typer can be readily integrated into bioinformatics protocols used in the identification and characterization of foodborne STEC isolates.

Systematic Evaluation of Whole Genome Sequence-Based Predictions of Salmonella Serotype and Antimicrobial Resistance

“…We considered the possibility that ARG detection may be more sensitive using raw-read sequences as this would alleviate errors arising from repeat regions and assembly of contaminating agents impacting genome assembly as has been observed in other studies (Carrillo et al, 2016;Clausen et al, 2016;Low et al, 2019). Assembly tools were found to have an impact on the ability to detect ARGs, particularly at low genome coverage where percentages of correctly identified genes were significantly lower in SKESA-assemblies.…”

Applications of Genomics in Regulatory Food Safety Testing in Canada

“…This real-time WGS approach produces high-resolution characterization of bacterial pathogens at a cost and within a timeframe that are similar to standard microbiological techniques and has the potential to replace lengthy biochemical characterization and molecular and serological typing procedures widely used in food testing laboratories. Our laboratory is currently studying strategies for broad implementation of WGS technology in support of regulatory food inspection objectives through the detection, identification, and characterization of priority bacterial pathogens such as STEC, Salmonella enterica, and Listeria monocytogenes [5,[23][24][25]. We have developed guidelines and implemented validated methods and bioinformatics tools for automated analyses of sequence data to ensure reliability and reproducibility of WGS-based analyses [24,26].…”

“…These properties are readily discoverable through the analysis of WGS data. For instance, the Shiga toxin subtype can be reliably determined using the V-typer tool, which is an automated assembly-independent subtyping module that can be integrated in a bioinformatics pipeline for the analysis of foodborne STEC isolates [23]. Yet another possibility would be to define priority STEC on the basis of contemporary public health data (reviewed periodically) identifying STEC serogroups most frequently associated with illness in a given jurisdiction.…”

The Integrated Rapid Infectious Disease Analysis (IRIDA) Platform

“…In contrast, WGS can inform investigators about numerous infectious pathogen traits in a single "assay", often with reduced turnaround time and with improved analytical power. As such, public health and food safety authorities are undergoing a historic transition, as WGS-based applications increasingly replace many molecular and phenotypic assays, ranging from serotyping and other molecular level microbial characterization for pathogen identification and surveillance as well as outbreak response, to antimicrobial resistance (AMR) and virulence prediction for risk assessment (9)(10)(11)(12)(13)(14)(15)(16)(17).…”