Surveillance of Listeria monocytogenes: Early Detection, Population Dynamics, and Quasimetagenomic Sequencing during Selective Enrichment

Abstract

In this study we addressed different aspects regarding the implementation of quasimetagenomic sequencing as a hybrid surveillance method in combination with enrichment for early detection of Listeria monocytogenes in the food industry. Different experimental enrichment cultures were used, comprising seven L. monocytogenes strains of different sequence types (STs), with and without a background microbiota community. To assess whether the proportions of the different STs changed over time during enrichment, the growth and population dynamics were assessed using dapE colony sequencing and dapE and 16S rRNA amplicon sequencing. There was a tendency of some STs to have a higher relative abundance during the late stage of enrichment when L. monocytogenes was enriched without background microbiota. When co-enriched with background microbiota, the population dynamics of the different STs was more consistent over time. To evaluate the earliest possible timepoint during enrichment that allows the detection of L. monocytogenes and at the same time the generation of genetic information that enables an estimation regarding the strain diversity in a sample, quasimetagenomic sequencing was performed early during enrichment in the presence of the background microbiota using Oxford Nanopore Technologies Flongle and Illumina MiSeq sequencing. The application of multiple displacement amplification (MDA) enabled detection of L. monocytogenes (and the background microbiota) after only 4 h of enrichment using both applied sequencing approaches. The MiSeq sequencing data additionally enabled the prediction of co-occurring L. monocytogenes strains in the samples. Importance We showed that a combination of a short primary enrichment combined with MDA and Nanopore sequencing can accelerate the traditional process of cultivation and identification of L. monocytogenes . The use of Illumina MiSeq sequencing additionally allowed us to predict the presence of co-occurring L. monocytogenes strains. Our results suggest quasimetagenomic sequencing to be a valuable and promising hybrid surveillance tool for the food industry that enables faster identification of L. monocytogenes during early enrichment. Routine application of this approach could lead to more efficient and proactive actions in the food industry that prevent contamination and subsequent product recalls and food destruction, economic and reputational losses and human listeriosis cases.

Research progress on detection techniques for point-of-care testing of foodborne pathogens

“…The POCT technology provides simple, fast, and sensitive platforms for the detection of foodborne pathogens and will become a powerful multi-functional tool for food safety, biological threat detection, and environmental monitoring. However, there are still shortcomings that require researchers to continuously improve the existing detection technologies ( He et al, 2021b ; Wagner et al, 2021 ). The sensitivity, specificity, ease of operation and detection speed, cost, and application of the techniques for detecting foodborne pathogens are given in Table 1 .…”

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.…”

The Saprophytic Lifestyle of Listeria monocytogenes and Entry Into the Food-Processing Environment

“…New approaches combining quasimetagenomic approaches with microbiome analyses based on 16 S RNA sequencing provide good insights into the co-selection of L. monocytogenes in the presence of a diverse background flora (e.g., Serratia , Pseudomonas , and Enterococcus , Bacillus ) during selective enrichment. Wagner et al (2021) tended to use a shortened enrichment time of 4 h for molecular detection to fully capture L. monocytogenes genotype diversity based on molecular methods.…”