Introduction

Traditionally, the investigation of suspect food samples during an outbreak investigation requires the samples to be enriched in order to reach detectable levels. However, a bias can occur during the enrichment of the food due to competition between the various living organisms or the cultivation parameters. Moreover, the current methods require a subsequent but not always successful isolation. In part due to these two drawbacks, not all outbreaks can be resolved. Shotgun metagenomics is a sequencing method involving the analysis of the genetic material directly extracted from a sample, without the need for an isolation of the bacteria. By sequencing and analysing the DNA of the sample, it can identify the presence of various pathogens to the strain level. Moreover, adaptive sampling is a new tool proposed during nanopore sequencing to target certain DNA strands to be sequenced preferably. The combination of metagenomics and adaptive sampling could allow to circumvent the isolation but also the enrichment of the food sample.

Materials and Methods

As a proof-of-concept, we explored the use of adaptive sampling using various databases (depletion of the food matrix i.e. potato, enrichment of a selection of foodborne pathogens, enrichment of a database of Staphylococcus aureus) compared to shotgun metagenomics without adaptive sampling on DNA of mashed potatoes spiked with DNA of S. aureus at a level of 0.5%. The strain was previously associated with a foodborne outbreak. Additionally, the living S. aureus strain was spiked into mashed potatoes at a level of 10⁵ CFU per 25 grams and three DNA extraction kits were tested, in combination with two databases for adaptive sampling, following whole genome amplification to increase the amount of genetic material to meet sequencing standards. The strain-level data analysis was performed as previously described (Buytaers et al. 2021).

Discussion

Our results showed that the combination of whole genome amplification, metagenomics and adaptive sampling using a database of S. aureus genomes outperformed shotgun metagenomics and adaptive sampling using other databases. It allowed strain-level analysis of foodborne outbreaks without the need for culture enrichment, thereby enabling faster investigations and facilitating precise pathogen characterization, contributing to improved food safety and public health.