Predominance of L. monocytogenes Lineage I Clones in Wastewater, Ruminants, and Natural Environments

Abstract

Listeria monocytogenes is a saprophytic bacterium and a foodborne pathogen of humans and animals. Little is known about its distribution and genetic diversity across different environments within the same geographical region. We conducted a large-scale longitudinal study in southeastern Spain monitoring Listeria spp. in untreated wastewater, ruminant farms, and natural environments over four seasons (N = 1490 samples, N = 545 isolates) and in food and food-processing environments (N = 7395 samples, N = 255 isolates). Listeria spp. were more abundant in host-associated than natural environments, and non-pathogenic Listeria were more prevalent than L. monocytogenes in both niches. L. monocytogenes was detected in 42.7%, 11.4%, 4.2%, and 3.4% of wastewater, ruminant farms, natural environments, and food-related samples, respectively. Hypervirulent lineage I accounted for 82.9% of L. monocytogenes isolates from wastewater, ruminant farms, and natural environments, while lineage II represented 74.1% in food-related samples. Among 255 L. monocytogenes cgMLST types, 5% were shared across environments, demonstrating circulation between different environments. Persistent L. monocytogenes clones were detected in food processing environments and ruminant farms. Our data suggest anthropogenic activities and livestock drive Listeria spp. dissemination. These results provide insights into the interactions of Listeria spp. in the environment, improving surveillance strategies to reduce pathogen transmission, food contamination, and clinical cases.

Keywords: cgMLST; ecology; foodborne; pathogens; persistence; virulence; whole genome sequencing.

FIGURE 1

Number of Listeria spp. isolates obtained in this study from wastewater (n = 234 samples from 4 sites), ruminant faeces (n = 512 from 4 farms), ruminant farm environments (n = 288 from 4 farms), natural environments (n = 456 from 3 sites), and food processing environments (FPE) (N = 7395 from regional food surveillance). Listeria spp. isolated from wastewater, ruminant faeces, ruminant farms, and natural environments were sampled during four consecutive seasons (spring 2021 to winter 2022). L. monocytogenes isolated from food and food processing environments were sampled during four consecutive years (spring 2019 to winter 2023). In the food and food processing environments category, the prevalence of other species than L. monocytogenes could not be assessed, since these isolates were obtained from public health authorities who exclusively surveyed L. monocytogenes , disregarding the possible presence of other Listeria spp. Circle size is proportional to the number of isolates. † Listeria sensu stricto; ∆ Listeria sensu lato.

FIGURE 2

Prevalence of L. monocytogenes positive samples among wastewater, ruminant faeces, farm environment, natural environment, and food processing environments (FPE). L. monocytogenes prevalence in wastewater, ruminant farms, and natural environment was assessed from spring 2021 to winter 2022; whereas L. monocytogenes prevalence among food and food processing environments was assessed from spring 2019 to winter 2023. N indicates the number of analysed samples.

FIGURE 3

(a) Diversity of L. monocytogenes isolates collected in this study (N = 465) based on cgMLST (1748 loci) analyses. Distribution of sublineages (SLs) in wastewater (n = 100), ruminant faeces (n = 75), farm environment (n = 16), natural environment (n = 19), and food processing environments (FPE) (n = 255) L. monocytogenes isolates. Corresponding clonal complexes (CCs), defined on the basis of seven‐locus MLST, are shown in brackets. (b) Distribution of CCs from the five major food‐product categories of this study. (c) Minimum spanning tree based on the cgMLST profiles L. monocytogenes observed in each sampled ecological niche in this study. Circle sizes are proportional to the number of isolates and are coloured by source type, as in panel (a). CCs, defined on the basis of seven‐locus MLST, are shown in each circle. Dashed lines delimitate lineages and are coloured according to the phylogenetic lineage (red, lineage I; blue, lineage II). Values shown in connecting lines denote the number of allelic differences between profiles.

FIGURE 4

Genetic diversity of 465  L. monocytogenes isolates sequenced in this study. cgMLST single linkage dendrogram. Branches are coloured by phylogenetic lineage (LI, red; LII, blue). SLs with more than 10 isolates are labelled in the tree. The inner and outer rings show the isolate's source and season, respectively, according to the colour codes shown on the left. Colour‐filled dark boxes indicate the presence of selected genetic traits: Listeria pathogenic islands (LIPI‐1, LIPI‐3, and LIPI‐4), internalin (inlB), benzalkonium chloride (bcrABC, emrC), pH or oxidative stress (SSI‐1, SSI‐2). Brown‐filled stars represent genes with truncations leading to Internalin A (inlA) premature stop codons (PMSCs).

FIGURE 5

Prevalence of genetic traits involved in virulence and acquired resistance towards disinfectants. L. monocytogenes isolates were obtained from wastewater (n = 100), ruminant faeces (n = 75), farm environment (n = 16), natural environment (n = 19), and food processing environments (FPE) (n = 255). Listeria pathogenic islands (LIPI‐3 and LIPI‐4), acquired premature stop codons (PMSCs) in inlA, SSI‐1, SSI‐2, and acquired resistance loci towards benzalkonium chloride (bcrABC and emrC) are shown. Statistically significant associations (Fisher's exact test) are indicated with asterisks. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

FIGURE 6

(a) Prevalence of L. monocytogenes (Lm) and non‐pathogenic (NP) Listeria spp. positive samples among natural environment (n = 81) and host‐related Listeria spp. isolates (n = 405). Host‐related category (N = 746) contained samples from wastewater (n = 234) and ruminant faeces (n = 512). Listeria spp. prevalence in natural environmental samples (N = 456) and in host‐related samples (N = 746) was assessed from spring 2021 to winter 2022. Statistically significant associations (Fisher's exact test) of Listeria spp. with origin ‘Natural environments’ or ‘Host‐related’) are indicated with asterisks. **p ≤ 0.01, ****p ≤ 0.0001. (b) Ratio of non‐pathogenic (NP) Listeria spp. versus L. monocytogenes (Lm) isolates in natural environment and host‐related environments.