Application of quasimetagenomics methods to define microbial diversity and subtype Listeria monocytogenes in dairy and seafood production facilities

Abstract

In developed countries, the human diet is predominated by food commodities, which have been manufactured, processed, and stored in a food production facility. Little is known about the application of metagenomic sequencing approaches for detecting foodborne pathogens, such as L. monocytogenes, and characterizing microbial diversity in food production ecosystems. In this work, we investigated the utility of 16S rRNA amplicon and quasimetagenomic sequencing for the taxonomic and phylogenetic classification of Listeria culture enrichments of environmental swabs collected from dairy and seafood production facilities. We demonstrated that single-nucleotide polymorphism (SNP) analyses of L. monocytogenes metagenome-assembled genomes (MAGs) from quasimetagenomic data sets can achieve similar resolution as culture isolate whole-genome sequencing. To further understand the impact of genome coverage on MAG SNP cluster resolution, an in silico downsampling approach was employed to reduce the percentage of target pathogen sequence reads, providing an initial estimate of required MAG coverage for subtyping resolution of L. monocytogenes.

Keywords: Listeria monocytogenes; environmental microbiology; food-borne pathogens; genomics; metagenomics; microbial ecology; microbiome; seafood and dairy production facilities.

Fig 1

Shannon diversity of enrichments for each sample collected from the seven dairy firms and five seafood firms. Color of the dot indicates the regulatory culture result (blue = Listeria culture negative, red = Listeria monocytogenes culture positive, green = culture positive for non-monocytogenes Listeria). Percentage of L. monocytogenes culture-positive samples is indicated at the bottom.

Fig 2

Shannon diversity of 16S rRNA results from 355 environmental swab culture enrichments via box plots. These samples are segregated by the food production facility type (dairy or seafood) and culture results for L. monocytogenes: negative dairy (n = 181), positive dairy (n = 64), negative seafood (n = 79), positive seafood (n = 31).

Fig 3

PCA of Bray Curtis dissimilarity distances between the 355 UVM culture enrichments. (A) Beta-diversity between samples collected from the 12 food manufacturing firms (AC, AM, G, I, J, O, and V = dairy firms; A, AS, F, L, and W = seafood firms). (B) Beta-diversity of samples collected from dairy manufacturing firms (n = 245) and seafood manufacturing firms (n = 110).

Fig 4

Chord diagrams of shared taxa found in environmental swab culture enrichments taken from seven dairy (A) and five seafood (B) facilities. All genera less than 3% relative abundance after 16S rRNA amplicon MAPseq analysis have been incorporated into “other_genera <3%.”

Fig 5

Hypothetical floorplan of dairy production facility FirmAM. Room legend indicates separate rooms of the facility where a specific step in food production occurs. The relative areas sampled (dots) are marked and color coded to reflect the regulatory culture result (blue = negative, red = Listeria monocytogenes positive, green = Listeria innocua positive). The swab collection sites (n = 68) were grouped into 10 sampling areas: Area A (Zone 3), Area B (Zone 2, 3), Area C (Zone 1, 3), Area D (Zone 1, 2, 3), Area E (Zone 3), Area F (Zone 1, 2, 3), Area G (Zone 1, 2), Area H (Zone 1, 2), Area I (Zone 3), and Area J (Zone 3). This floorplan is not drawn to scale.

Fig 6

PCA of Bray Curtis dissimilarity distances from culture enrichments from dairy firm AM cleaning and processing rooms. (A) Beta-diversity between all samples taken from the 10 areas. (B) Areas A (n = 4), B (n = 9), C (n = 8), and G (n = 12) had significant differences in beta diversities when compared to the other 6 areas (D, E, F, H, I, J).

Fig 7

Phylogeny of Listeria pure isolates (WGS) and MAG determined by SNP analysis. WGS and MAG are designated by name and color (Listeria monocytogenes MAG = red, Listeria monocytogenes WGS = black, Listeria innocua MAG = green). Samples were named according to the blinded firm name followed by the designation of dairy or seafood manufacturer, whether the sample was sequencing using shotgun metagenomics (MAG) or pure isolate WGS and the environmental swab number. Samples that resolved to Listeria monocytogenes lineage I, lineage II, and Listeria innocua are included in the tree.

Fig 8

In silico fastq dilutions of three metagenomic assembled Listeria genomes (A–C). The first sample, FirmI_Dairy_MAG_S105 (A), had both a high relative abundance of L. monocytogenes and a significant number of L. monocytogenes lineage I reads. The second sample, FirmG_Dairy_MAG_S170 (B), had a high relative abundance and number of L. monocytogenes lineage I reads but also exhibited reads for L. monocytogenes lineage II. The third and final sample, FirmI_Dairy_MAG_S064 (C), was culture positive for both L. moncytogenes and L. innocua, but shotgun metagenomic sequencing and analysis only identified reads and relative abundance for L. innocua. Panel D indicates the sequencing reads and relative abundance data mapped to Listeria for all three samples.