Application of a strain-level shotgun metagenomics approach on food samples: resolution of the source of a Salmonella food-borne outbreak

Abstract

Food-borne outbreak investigation currently relies on the time-consuming and challenging bacterial isolation from food, to be able to link food-derived strains to more easily obtained isolates from infected people. When no food isolate can be obtained, the source of the outbreak cannot be unambiguously determined. Shotgun metagenomics approaches applied to the food samples could circumvent this need for isolation from the suspected source, but require downstream strain-level data analysis to be able to accurately link to the human isolate. Until now, this approach has not yet been applied outside research settings to analyse real food-borne outbreak samples. In September 2019, a Salmonella outbreak occurred in a hotel school in Bruges, Belgium, affecting over 200 students and teachers. Following standard procedures, the Belgian National Reference Center for human salmonellosis and the National Reference Laboratory for Salmonella in food and feed used conventional analysis based on isolation, serotyping and MLVA (multilocus variable number tandem repeat analysis) comparison, followed by whole-genome sequencing, to confirm the source of the contamination over 2 weeks after receipt of the sample, which was freshly prepared tartar sauce in a meal cooked at the school. Our team used this outbreak as a case study to deliver a proof of concept for a short-read strain-level shotgun metagenomics approach for source tracking. We received two suspect food samples: the full meal and some freshly made tartar sauce served with this meal, requiring the use of raw eggs. After analysis, we could prove, without isolation, that Salmonella was present in both samples, and we obtained an inferred genome of a Salmonella enterica subsp. enterica serovar Enteritidis that could be linked back to the human isolates of the outbreak in a phylogenetic tree. These metagenomics-derived outbreak strains were separated from sporadic cases as well as from another outbreak circulating in Europe at the same time period. This is, to our knowledge, the first Salmonella food-borne outbreak investigation uniquely linking the food source using a metagenomics approach and this in a fast time frame.

Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies

“…Therefore, a metagenomics approach has been proposed as an alternative to the currently performed microbiological analyses requiring a not-always-straightforward isolation of the pathogenic strain. As previously described (Buytaers et al, 2020), Illumina sequencing may be used to obtain the full information necessary for outbreak investigation from metagenomics samples, without the need for isolation, and this to the strain level, after about one full week of lab work (Buytaers et al, 2021b). However, not only the need for more proofs of concept but also the high cost of such an analysis impact its potential implementation as a routine practice.…”

“…However, not only the need for more proofs of concept but also the high cost of such an analysis impact its potential implementation as a routine practice. To render the analysis somehow more cost-effective, while still taking the required coverage into account, 8-12 samples were pooled into one Illumina MiSeq run in previous studies (Leonard et al, 2016;Buytaers et al, 2020Buytaers et al, , 2021b. However, it might not always be possible to analyze this number of samples as the number of available food samples during outbreak investigation varies and is not gathered at a single time point.…”

“…In our previous work, we have presented a metagenomics approach to obtain the same level of precision as the conventional bacterial detection methods and isolate's WGS, through direct sequencing of all DNA in the sample after enrichment in a nonselective medium following the ISO standard ISO 13136:2012 (ISO: International Organization for standardization, 2012; Buytaers et al, 2020Buytaers et al, , 2021b. After short-read sequencing of 12 DNA extracts with or without removal of host DNA in a 48-h Illumina MiSeq run, we were able to link the pathogenic strains derived from metagenomics sequencing of samples containing multiple strains of the same species (Escherichia coli) to human isolates from the same outbreak (Buytaers et al, 2020).…”

Use of Whole Genome Sequencing by the Federal Interagency Collaboration for Genomics for Food and Feed Safety in the United States

“…Limited shotgun metagenomic and targeted 16S rRNA amplicon sequencing methods are routinely used for food safety and clinical applications to test the specificity and sensitivity of pathogen detection methods in specific environments (9,23,37,48). However, current foodborne pathogen detection workflows rely on culture-based methods that can take weeks to complete.…”

“…Although most infections that occur each year in the United States are sporadic events (i.e., without a known connection with other infections or outbreaks), public health officials investigate and report an average of ca. 900 foodborne outbreaks (defined as two or more people with the same illness linked to consumption of the same contaminated food or drink) (9). A focus in public health investigations on those illnesses that are most likely to be part of a foodborne outbreak provides investigators more food exposure and laboratory information and enables those investigators to better identify, control, and prevent food safety problems.…”