AUTHOR=Buytaers Florence E. , Verhaegen Bavo , Van Nieuwenhuysen Tom , Roosens Nancy H. C. , Vanneste Kevin , Marchal Kathleen , De Keersmaecker Sigrid C. J. 

TITLE=Strain-level characterization of foodborne pathogens without culture enrichment for outbreak investigation using shotgun metagenomics facilitated with nanopore adaptive sampling

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 15 - 2024

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1330814

DOI=10.3389/fmicb.2024.1330814

ISSN=1664-302X

ABSTRACT=Shotgun metagenomics has previously proven effective in the investigation of foodborne outbreaks by providing rapid and comprehensive insights into the microbial contaminant. However, culture enrichment of the sample has remained a prerequisite, despite the potential impact on pathogen detection resulting from the growth competition. To circumvent the need for culture enrichment, we explored the use of adaptive sampling using various databases for a targeted nanopore sequencing, compared to shotgun metagenomics alone. This was done first on DNA of mashed potatoes mixed with DNA of a Staphylococcus aureus strain previously associated with a foodborne outbreak, by either depleting the potato sequencing reads or enriching for the pathogen sequencing reads through adaptive sampling. Additionally, living S. aureus were spiked at 10 5 CFU into 25 g of mashed potatoes. Three  DNA extraction kits were tested, in combination with enrichment using adaptive sampling, following whole genome amplification. While the use of a host removal DNA extraction kit and a database of foodborne pathogens allowed rapid detection of the pathogen, the most complete characterization was achieved when using solely a database of S. aureus combined with a conventional DNA extraction kit, enabling accurate placement of the strain on a phylogenetic tree alongside outbreak cases. Overall, this method shows great potential for strain-level analysis of foodborne outbreaks without the need for culture enrichment, thereby enabling faster investigations and facilitating precise pathogen characterization. The integration of adaptive sampling with shotgun metagenomics presents a valuable strategy for more efficient and targeted analysis of microbial communities in foodborne outbreaks, contributing to improved food safety and public health.