Detection of Shiga Toxin-producing Escherichia coli on Fresh Produce Utilizing a Metagenomic Approach

Introduction: Shiga Toxin-producing Escherichia coli (STEC) is a major cause of foodborne illness and has been associated with contaminated fresh produce. Assessing the detection sensitivity of STEC on fresh produce using a metagenomic approach will aid in developing a next-generation sequencing method to screen for contamination. Additionally, metagenomic analysis provides information about the overall microbial diversity on fresh produce and how the enrichment broth shifts the indigenous microbial population with the possible outcome of optimizing the broth to enhance detection of STEC with a reduction in enrichment time.

Purpose: The objective of this study was to evaluate a metagenomic approach to the detection and strain level identification of STEC on produce in both pre-enriched samples and enriched cultures of contaminated produce.

Methods: STEC-spiked bagged spinach samples and cultures from samples processed according to the BAM method were used to prepare sequencing libraries. Sequencing data from an Illumina MiSeq platform was analyzed using an in-house k-mer signature database to determine the microbial population. The sequencing reads were examined for STEC-specific molecular markers and, when STEC reads were present in sufficient abundance, the contaminating STEC genome was assembled from the complete sequencing data set.

Results: The indigenous bacterial species on unspiked spinach was determined as well as the shifts in the microbial population at several time points during enrichment for both spiked and unspiked samples. Even for low levels of STEC contamination (10 CFU/100 g spinach), it was not necessary to complete the overnight enrichment to determine the serotype, detect STEC-specific molecular markers, and assemble the STEC genome.

Significance: Utilizing a metagenomic approach may provide a more rapid method to detect and identify STEC contamination of fresh produce, thereby enhancing food safety and expediting response time during disease outbreaks.