Quasi-metagenomics and realtime sequencing aided detection and subtyping of Salmonella enterica from food samples

Metagenomics analysis of food samples promises isolation-independent detection and subtyping of foodborne bacterial pathogens in a single workflow. Selective concentration of Salmonella genomic DNA through immunomagnetic separation (IMS) and multiple displacement amplification (MDA) were shown to shorten culture enrichment of Salmonella-spiked raw chicken breast samples by over 12 hours while permitting serotyping and high-fidelity single nucleotide polymorphisms (SNP) typing of the pathogen using short shotgun sequencing reads. The herein termed quasi-metagenomics approach was evaluated on Salmonella-spiked lettuce and black peppercorn samples as well as retail chicken parts naturally contaminated with different serotypes of Salmonella. Between 8 and 24 h culture enrichment was required for detecting and subtyping naturally occurring Salmonella from unspiked chicken parts compared with 4 to 12 h culture enrichment when Salmonella-spiked food samples were analyzed, indicating the likely need for longer culture enrichment to revive low levels of stressed or injured Salmonella cells in food. Further acceleration of the workflow was achieved by real-time nanopore sequencing. After 1.5 hours of analysis on a potable sequencer, sufficient data were generated from sequencing IMS-MDA product of a cultured-enriched lettuce sample to allow serotyping and robust phylogenetic placement of the inoculated isolate.