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 coenriched with background microbiota, the population dynamics of the different STs was more consistent over time. To evaluate the earliest possible time point 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 cooccurring 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 cooccurring L. monocytogenes strains. Our results suggest quasimetagenomic sequencing is 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.

Keywords: IMS; ISO 11290-1; Illumina; L. monocytogenes; Listeria; MDA; Nanopore; background microbiota; immunomagnetic separation; multiple displacement amplification; population dynamics; qPCR; qualitative detection; quasimetagenomics; selective enrichment; shotgun sequencing.

FIG 1

Development of the microbiota in the enrichment cultures containing seven L. monocytogenes strains. The proportion of each L. monocytogenes ST was determined by sequencing the dapE gene either using PCR and subsequent Sanger sequencing of individual colonies (A) or amplicon sequencing of DNA isolated from culture pellets (B). Presented results are averages from four (A) or three (B) independent experiments. Results for individual experiments are shown in Fig. S1 in the supplemental material. (C) Results from sampling points analyzed with both methods were compared using PCA.

FIG 2

Growth curves for enrichment cultures containing L. monocytogenes and background microbiota. L. monocytogenes concentrations were evaluated by counting colonies on Listeria-selective agar (RLM), while total cell concentrations were evaluated by counting colonies on nonselective agar (TSA). Average values for three experiments are shown. Error bars denote the standard deviations.

FIG 3

Population dynamics in the enrichment cultures containing L. monocytogenes and background microbiota. The cultures labeled Lm5, Lm50, and Lm500 contained 1%, 9%, and 50% of L. monocytogenes, respectively, at the time of inoculation. (A) The proportion of each L. monocytogenes ST was determined using dapE amplicon sequencing. The Lm cocktail sample constituted a mixture of all seven L. monocytogenes cultures in equal proportions. The 4-h, 8-h, and 12-h samples were subjected to whole-genome MDA prior to dapE amplicon sequencing. Samples labeled with an asterisk did not yield results due to insufficient DNA concentrations. Presented results are averages from three independent experiments, except the Lm50 8-h sample, for which results were obtained for two replicates. Results for individual experiments are shown in Fig. S4. (B) The proportion of each species was determined using 16S rRNA amplicon sequencing. The microbiota cocktail sample constituted a mixture of all L. monocytogenes and background microbiota cultures in equal proportions. Presented results are averages from three independent experiments. Results for individual experiments are shown in Fig. S5. (C and D) Taxonomic assignment of reads from Nanopore Flongle sequencing, showing genus-level classification (C) and species-level classification (D) within the Listeria genus. The tables at the bottom of panels C and D show the total number of classified reads in each sample.

FIG 4

Effect of IMS. Samples were collected from Half Fraser enrichment cultures and the experiment was performed twice (replicates 2 and 3). (A) Comparison of total cell concentrations and L. monocytogenes concentrations without and with IMS. Mean values are shown, and error bars show the standard deviations. (B and C) Average relative abundances of the microbiota in 12-h samples determined by amplicon sequencing targeting the 16S rRNA (B) and the dapE gene (C). (D and E) Taxonomic assignment of reads from Illumina MiSeq quasimetagenomic shotgun sequencing of 4-h samples, showing genus-level classification (D) and species-level classification (E) within the Listeria genus. The table in panel D shows the percentage of classified reads in each sample, and the numbers printed on the columns indicate the actual number of reads classified as Listeria spp. (D) or L. monocytogenes (E).