Application of Metagenomic Sequencing to Food Safety: Detection of Shiga Toxin-Producing Escherichia coli on Fresh Bagged Spinach

Abstract

Culture-independent diagnostics reduce the reliance on traditional (and slower) culture-based methodologies. Here we capitalize on advances in next-generation sequencing (NGS) to apply this approach to food pathogen detection utilizing NGS as an analytical tool. In this study, spiking spinach with Shiga toxin-producing Escherichia coli (STEC) following an established FDA culture-based protocol was used in conjunction with shotgun metagenomic sequencing to determine the limits of detection, sensitivity, and specificity levels and to obtain information on the microbiology of the protocol. We show that an expected level of contamination (ϳ10 CFU/100 g) could be adequately detected (including key virulence determinants and strain-level specificity) within 8 h of enrichment at a sequencing depth of 10,000,000 reads. We also rationalize the relative benefit of static versus shaking culture conditions and the addition of selected antimicrobial agents, thereby validating the long-standing culture-based parameters behind such protocols. Moreover, the shotgun metagenomic approach was informative regarding the dynamics of microbial communities during the enrichment process, including initial surveys of the microbial loads associated with bagged spinach; the microbes found included key genera such as Pseudomonas, Pantoea, and Exiguobacterium. Collectively, our metagenomic study highlights and considers various parameters required for transitioning to such sequencing-based diagnostics for food safety and the potential to develop better enrichment processes in a high-throughput manner not previously possible. Future studies will investigate new species-specific DNA signature target regimens, rational design of medium components in concert with judicious use of additives, such as antibiotics, and alterations in the sample processing protocol to enhance detection. Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that can cause severe illness such as hemorrhagic colitis and hemolytic-uremic syndrome (HUS) in humans (1). Fresh leafy vegetables have been implicated in many foodborne disease outbreaks, including an STEC O157:H7 outbreak associated with consumption of fresh ready-to-eat bagged spinach in 2006 that caused 100 hospitalizations and five deaths (2, 3). In an unrelated outbreak, bagged baby spinach was recalled in 2010 due to STEC O157:H7 contamination (4). The Centers for Disease Control and Prevention reported that between 1973 and 2012, STEC was second only to norovirus in the number of outbreaks associated with leafy vegetables in the United States, of which 46 of the 49 STEC outbreaks were caused by a serogroup O157 strain (2). Although STEC did not cause the greatest number of outbreaks, it did lead to by far the greatest number of hospitalizations for a given microorganism, with 46% of the total hospitalizations due to all leafy-vegetable-associated outbreaks attributed to STEC. Illness due to ingestion of STEC-contaminated leafy greens led to nine deaths, including six from spinach (2). With ...

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“…We validated the accuracy of each approach by processing spinach metagenome data from samples that had been spiked with the STEC O157:H7 Sakai in a previous study (11). We observed that PanPhlAn was the most sensitive approach.…”

“…Application of strain-level analysis to characterize enteric bacteria in nunu. Leonard et al previously used metagenomic sequencing to detect E. coli in spinach that had been intentionally spiked with E. coli O157:H7 strain Sakai (11). We downloaded the metagenomic reads from that study (16 samples), and we subjected them to StrainPhlAn, MetaMLST, and PanPhlAn analysis to confirm that these tools can accurately detect pathogens in food samples; MetaMLST was used for multilocus sequence typing, StrainPhlAn was used for phylogenetic identification, and PanPhlAn was used for functional characterization.…”

“…Metagenomic sequencing enables elucidation of the roles of microorganisms during food production (7)(8)(9), and it can be used to track microorganisms of interest through the food production chain, as illustrated by Yang et al (10), who used whole-metagenome shotgun sequencing to track pathogenic species in the beef production chain. Indeed, metagenomic sequencing can be used to detect pathogens in foods to monitor outbreaks of foodborne illnesses (11), but few studies have done so, because of the limited taxonomic resolution achievable using these methods. Typically, 16S rRNA gene sequencing provides genus-level taxonomic resolution (12), and although sub-genus-level classification is achievable using species classifiers (13) or oligotyping (14,15), these methods cannot accurately discriminate between strains.…”

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