Salmonella enterica and Escherichia coli in Wheat Flour: Detection and Serotyping by a Quasimetagenomic Approach Assisted by Magnetic Capture, Multiple-Displacement Amplification, and Real-Time Sequencing

Abstract

Food safety is a new area for novel applications of metagenomics analysis, which not only can detect and subtype foodborne pathogens in a single workflow but may also produce additional information with in-depth analysis capabilities. In this study, we applied a quasimetagenomic approach by combining short-term enrichment, immunomagnetic separation (IMS), multiple-displacement amplification (MDA), and nanopore sequencing real-time analysis for simultaneous detection of Salmonella and Escherichia coli in wheat flour. Tryptic soy broth was selected for the 12-h enrichment of samples at 42°C. Enrichments were subjected to IMS using beads capable of capturing both Salmonella and E. coli. MDA was performed on harvested beads, and amplified DNA fragments were subjected to DNA library preparation for sequencing. Sequencing was performed on a portable device with real-time basecalling adaptability, and resulting sequences were subjected to two parallel pipelines for further analysis. After 1 h of sequencing, the quasimetagenomic approach could detect all targets inoculated at approximately 1 CFU/g flour to the species level. Discriminatory power was determined by simultaneous detection of dual inoculums of Salmonella and E. coli, absence of detection in control samples, and consistency in microbial flora composition of the same flour samples over several rounds of experiments. The total turnaround time for detection was approximately 20 h. Longer sequencing for up to 15 h enabled serotyping for many of the samples with more than 99% genome coverage, which could be subjected to other appropriate genetic analysis pipelines in less than a total of 36 h. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) and Salmonella are of serious concern in low-moisture foods, including wheat flour and its related products, causing illnesses, outbreaks, and recalls. The development of advanced detection methods based on molecular principles of analysis is essential to incorporate into interventions intended to reduce the risk from these pathogens. In this work, a quasimetagenomic method based on real-time sequencing analysis and assisted by magnetic capture and DNA amplification was developed. This protocol is capable of detecting multiple Salmonella and/or E. coli organisms in the sample within less than a day, and it can also generate sufficient whole-genome sequences of the target organisms suitable for subsequent bioinformatics analysis. Multiplex detection and identification were accomplished in less than 20 h and additional whole-genome analyses of different nature were attained within 36 h, in contrast to the several days required in previous sequencing pipelines.

Application of MinION sequencing as a tool for the rapid detection and characterization of Listeria monocytogenes in smoked salmon

“…Up to date, very few studies have been published with regard to the implementation of DNA sequencing as a simultaneous detection and characterization tool for food samples, and even less have performed a detailed evaluation of its analytical performance (Hyeon et al, 2018 ; Forghani et al, 2020 ; Ottesen et al, 2020 ; Azinheiro et al, 2021 ; Commichaux et al, 2021 ; Wagner et al, 2021 ). This may be in part due to the complexity involved in the recovery of the microorganisms to reach an acceptable concentration for the sequencing analysis.…”

“…These techniques are typically applied to pure microbial cultures in order to typify them and in metagenomic studies to characterize the populations present in the sample (González-Escalona et al, 2019 ; Jagadeesan et al, 2019 ; Chen et al, 2020 ). However, if a laboratory is interested in the direct detection of the microbial pathogens present in a food sample, the workflows must be adapted for this particular application, and it is in this context that major gaps exist, as only a handful of studies have been reported (Katz et al, 2017 ; Forghani et al, 2020 ; Ottesen et al, 2020 ; Townsend et al, 2020 ; Commichaux et al, 2021 ). Most of these studies take advantage of the so-called “quasimetagenomics” approach described by Hyeon et al ( 2018 ), which, in brief, is based on a primary sample enrichment followed by immunomagnetic separation (IMS) to concentrate on one specific pathogen, whole-genome amplification (WGA) to increase its DNA concentration, and finally identification and characterization of the pathogen by DNA sequencing.…”

Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies

“…The MinION sequencing resulted in a larger output, allowing the potential applicability to other samples, regardless of the quality of the flow cell (number of pores). The work of Forghani et al (2020) showed that a quasimetagenomics method to the strain level with MinION sequencing can be extended to other strains of STEC and other bacterial pathogens without a problem. However, our study, although only including one serotype, showed the potential of the metagenomics approach for samples presenting a population of several different E. coli strains (including nonpathogenic strains).…”

“…They obtained 65 and 70 SNP difference to the WGS isolate reference of the spiked bacteria after 1.5 and 48.5 h of sequencing, respectively (Hyeon et al, 2018). A similar quasimetagenomics method was used to target Shiga toxin-producing E. coli (STEC) and Salmonella in contaminated flour samples (Forghani et al, 2020). The method proved successful to cluster (without specifying the SNP differences) the metagenomics-obtained strain to the spiked isolate, for multiple single-spiked strains of each pathogen and also on samples cospiked with one strain of each of the two pathogen species.…”

A Review of Isothermal Amplification Methods and Food-Origin Inhibitors against Detecting Food-Borne Pathogens

“…MDA randomly and massively amplifies single-cell genomic DNA and is compatible with whole genome amplification (WGA) [51,52]. MDA is conducted using modified random hexamer primers, phi29 DNA polymerases (strand-displacing DNA polymerase from bacteriophage Ø29), denatured template DNA, and dNTPs [53].…”