Purpose: The objective of this study was to evaluate the comprehensiveness of a metagenomics approach for detection and strain level identification of STEC on bagged spinach using pathogenic STEC strains of a variety of serotypes and Shiga toxin subtypes.
Methods: Bagged spinach was spiked with one of 12 STEC strains at a level of 0.1 CFU/g spinach and processed according to the U.S. FDA BAM protocol. Sequencing data generated from each sample was used to determine molecular serotype and STEC-specific virulence genes by BLAST analysis, identify the microbial communities present in the enriched sample using a discriminative k-mer method, and perform E. coli core gene SNP analysis on de novo assemblies of the metagenomic sequencing data.
Results: Bacterial community analysis determined that E. coli was a major component of the population in most samples, but molecular serotyping using the metagenomic data revealed the presence of indigenous E. coli in some samples. Despite the presence of additional E. coli strains, the serotype and virulence genes of the spiked STEC, including correct Shiga toxin subtype, were detected in 92% of the samples. E. coli core gene SNP analysis of the metagenomic sequencing data correctly placed the spiked STEC in a phylogeny of related strains in cases where the indigenous E. coli did not predominate.
Significance: Utilizing a shotgun metagenomics approach to characterize STEC contaminating bagged spinach may expedite the time necessary to ascertain the risk level to public health and response time during outbreaks.