Quasimetagenomics-Based and Real-Time-Sequencing-Aided Detection and Subtyping of Salmonella enterica from Food Samples

Abstract

Metagenomics analysis of food samples promises isolation-independent detection and subtyping of foodborne bacterial pathogens in a single workflow. Selective concentration of genomic DNA through immunomagnetic separation (IMS) and multiple displacement amplification (MDA) were shown to shorten culture enrichment of-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 -spiked lettuce and black peppercorn samples as well as retail chicken parts naturally contaminated with different serotypes of Between 8 and 24 h culture enrichment was required for detecting and subtyping naturally occurring from unspiked chicken parts compared with 4 to 12 h culture enrichment when-spiked food samples were analyzed, indicating the likely need for longer culture enrichment to revive low levels of stressed or injured 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. Both culture enrichment and next-generation sequencing remain to be time-consuming processes for food testing where rapid methods for pathogen detection are widely available. Our study demonstrated substantial acceleration of the respective process through IMS-MDA and real-time nanopore sequencing. In one example, the combined use of the two methods delivered a less than 24 h turnaround time from a -contaminated lettuce sample to phylogenetic identification of the pathogen. Improved efficiency like this is important for further expanding the use of whole genome and metagenomics sequencing in microbial analysis of food. Our results suggest the potential of the quasi-metagenomics approach in areas where rapid detection and subtyping of foodborne pathogens is important, such as foodborne outbreak response and precision tracking and monitoring of foodborne pathogens in production environments and supply chains.

The Present and Future of Whole Genome Sequencing (WGS) and Whole Metagenome Sequencing (WMS) for Surveillance of Antimicrobial Resistant Microorganisms and Antimicrobial Resistance Genes across the Food Chain

“…Despite the demonstrated potential of WGS and WMS tools within surveillance programs, their routine use does require its transformation into cheaper, user-friendly approaches that could be used on-site by personnel not specialized in big data management [ 146 ]. In this regard, prototypes of some miniaturized sequencers have recently become available and have demonstrated potential to be used on-site, generating results in real time [ 147 ].…”

A New Whole Genome Culture-Independent Diagnostic Test (WG-CIDT) for Rapid Detection of Salmonella in Lettuce

“…The time period from the first handling of contaminated lettuce to completing all bioinformatic analysis was 28 h and was shorter or similar to time reported by other genome-based assays (Leonard et al, 2016;Hyeon et al, 2018). We are currently employing the use of nanopore sequencing to accelerate the speed of testing and reduce assay period to at least half the time and to develop a mobile model that can be applied to unintentional and intentional contamination of food.…”

“…Other published procedures are able to achieve a detection limit of 10-70 cfu/g, i.e., 250-1,750/25 g (Pérez-Rodríguez et al, 2014;Rodriguez-Lazaro et al, 2014) which makes our assay more sensitive. Recently, Hyeon et al (2018) combined selective immunomagnetic separation of Salmonella with a multiple displacement amplification procedure to prepare DNA template that were sequenced with the Illumina MiSeq sequencing procedure followed by extensive bioinformatic procedures including bacterial genome assembly. The procedure led to the identification of Salmonella in chicken and serotype designation of Salmonella Enteritidis except at low level contamination of a theoretical 2.5 cfu/25 g of chicken.…”

NanoARG: A web service for identification of antimicrobial resistance elements from nanopore-derived environmental metagenomes

“…The heavily infected urine sample (HIU) shows Escherichia coli as the dominant species, which is expected given that this particular metagenome consists of a spiked MDR E. coli strain with urine from a healthy patient (34) (see Figure 10D ). Similarly, the metagenome from food sample (LSS) consists of a Salmonella enterica contaminated sample (69). As a result, Salmonella enterica is the most abundant followed closely by a synthetic construct that refers to artificial samples containing a mixture of different strains (see Figure 10C ).…”