A Practical Method to Implement Strain-Level Metagenomics-Based Foodborne Outbreak Investigation and Source Tracking in Routine.

Florence E Buytaers,Denis Piérard,Assia Saltykova,Sarah Denayer,Sigrid C J De Keersmaecker,Nancy H C Roosens,Bavo Verhaegen,Kathleen Marchal,Kevin Vanneste

doi:10.3390/microorganisms8081191

Abstract

The management of a foodborne outbreak depends on the rapid and accurate identification of the responsible food source. Conventional methods based on isolation of the pathogen from the food matrix and target-specific real-time polymerase chain reactions (qPCRs) are used in routine. In recent years, the use of whole genome sequencing (WGS) of bacterial isolates has proven its value to collect relevant information for strain characterization as well as tracing the origin of the contamination by linking the food isolate with the patient’s isolate with high resolution. However, the isolation of a bacterial pathogen from food matrices is often time-consuming and not always successful. Therefore, we aimed to improve outbreak investigation by developing a method that can be implemented in reference laboratories to characterize the pathogen in the food vehicle without its prior isolation and link it back to human cases. We tested and validated a shotgun metagenomics approach by spiking food pathogens in specific food matrices using the Shiga toxin-producing Escherichia coli (STEC) as a case study. Different DNA extraction kits and enrichment procedures were investigated to obtain the most practical workflow. We demonstrated the feasibility of shotgun metagenomics to obtain the same information as in ISO/TS 13136:2012 and WGS of the isolate in parallel by inferring the genome of the contaminant and characterizing it in a shorter timeframe. This was achieved in food samples containing different E. coli strains, including a combination of different STEC strains. For the first time, we also managed to link individual strains from a food product to isolates from human cases, demonstrating the power of shotgun metagenomics for rapid outbreak investigation and source tracking.

Highlights

Food contaminations with pathogens are a major burden on our society, affecting an estimated 600 million people a year and impacting socioeconomic development at various levels [1]
In case of foodborne outbreak investigation, the microbiological analysis of the probable responsible food vehicle is performed at two levels and consists of the detection of the pathogen, followed by the association of the food vehicle to the human cases using typing of the food isolate
All workflows allowed a characterization of the pathogen after enrichment, comparable to the conventional method, but without prior isolation: i.e., detection of Shiga toxin-producing Escherichia coli (STEC) in the sample, determination of the serotype and virulence factors of interest, and retrieval of the reads corresponding to the STEC strain to perform phylogenetic tracing back to the Limburg outbreak

Summary

Introduction

Food contaminations with pathogens are a major burden on our society, affecting an estimated 600 million people a year and impacting socioeconomic development at various levels [1]. In the last decade, whole genome sequencing (WGS) of the isolate has been proposed as a higher resolution alternative for the full characterization of the micro-organisms [3,4] This approach allows the detection of all genes present on the bacterial genome in just one test as well as phylogenetic analysis to link cases of food and human origin at the single nucleotide level. This resulted in recommendations from the European Centre for Disease Prevention and Control (ECDC) and the European Food Safety Authority (EFSA) to implement WGS on isolates in Europe for surveillance and outbreak investigation for a short list of priority pathogens and diseases [5,6,7]

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