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. The selective concentration of Salmonella genomic DNA by immunomagnetic separation (IMS) and multiple displacement amplification (MDA) shortened the time for culture enrichment of Salmonella-spiked raw chicken breast samples by over 12 h while permitting serotyping and high-fidelity single nucleotide polymorphism (SNP) typing of the pathogen using short shotgun sequencing reads. The herein-termed quasimetagenomics approach was evaluated on Salmonella-spiked lettuce and black peppercorn samples as well as retail chicken parts naturally contaminated with different serotypes of Salmonella Culture enrichment of between 8 and 24 h 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. A further acceleration of the workflow was achieved by real-time nanopore sequencing. After 1.5 h of analysis on a potable sequencer, sufficient data were generated from sequencing the IMS-MDA products of a cultured-enriched lettuce sample to enable serotyping and robust phylogenetic placement of the inoculated isolate.IMPORTANCE Both culture enrichment and next-generation sequencing remain time-consuming processes for food testing, whereas rapid methods for pathogen detection are widely available. Our study demonstrated a substantial acceleration of these processes by the use of immunomagnetic separation (IMS) with multiple displacement amplification (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 the collection of a Salmonella-contaminated lettuce sample to the phylogenetic identification of the pathogen. An improved efficiency such as this is important for further expanding the use of whole-genome and metagenomics sequencing in the microbial analysis of food. Our results suggest the potential of the quasimetagenomics approach in areas where rapid detection and subtyping of foodborne pathogens are important, such as for foodborne outbreak response and the precision tracking and monitoring of foodborne pathogens in production environments and supply chains.

Keywords: MinION; Salmonella; detection; metagenomics; subtyping.

FIG 1

Comparison of quasimetagenomics sequencing performance using different sample preparation methods. (A) Percentages of sequences from Salmonella and other individual genera among all microbial sequences generated by sequencing. Results from various sample preparation methods were evaluated. RV_8h, enrichment by RV broth for 8 h; RV-IMS-MDA_4h, enrichment by RV broth for 4 h followed by IMS and MDA. (B) Percentages of the S. Enteritidis (SE) reference genome that were sequenced by the quasimetagenomics approach using DNA samples prepared from RV broth enrichment alone and RV broth enrichment followed by IMS-MDA. (C) Percentages of Salmonella reads among all sequencing reads, including host/chicken DNA. DNA samples were prepared from RV broth enrichment alone and RV broth enrichment followed by IMS-MDA. In all samples, ∼1 CFU/g of S. Enteritidis was inoculated in each 25-g aliquot of raw chicken breast.

FIG 2

Phylogenetic clustering of the spiked chicken breast samples sequenced by the quasimetagenomics method (target) on an Illumina MiSeq platform and the genomes of recent outbreak and sporadic strains. The WGS of the spiked strain (reference) is also included. Each of the 9 dotted-line boxes shows the highlighted part of the respective tree that includes the target and reference and corresponds to a specific combination of inoculum level (0.1, 1, or 10 CFU/g) and RV broth enrichment time (4, 8, or 12 h). Bars represent unit distances of 2, 5, or 50 SNPs.

FIG 3

Phylogenetic clustering of a spiked lettuce sample sequenced by the quasimetagenomics method on a MinION device after 1.5 h (target_1.5 h) and 48 h (target_48.5 h) of sequencing. The WGS of the spiked strain (reference) is also included. Bars represent a unit distance of 100 SNPs on the tree.

FIG 4

Positive correlation between percentages of the target genome sequenced by the quasimetagenomics approach (x axis) and the C_T values from real-time PCR analysis of the IMS-MDA products (y axis). A total of 28 samples were analyzed.