Listeria monocytogenes strains are known to harbour plasmids that confer resistance to sanitizers, heavy metals, and antibiotics; however, very little research has been conducted into how plasmids may influence L. monocytogenes’ ability to tolerate food-related stresses. To investigate this, a library (n = 93) of L. monocytogenes plasmid sequences were compared. Plasmid sequences were divided into two groups (G1 and G2) based on a repA phylogeny. Twenty-six unique plasmid types were observed, with 13 belonging to each of the two repA-based groups. G1 plasmids were significantly (p < 0.05) smaller than G2 plasmids but contained a larger diversity of genes. The most prevalent G1 plasmid (57,083 bp) was observed in 26 strains from both Switzerland and Canada and a variety of serotypes. Quantitative PCR (qPCR) revealed a >2-fold induction of plasmid-contained genes encoding an NADH peroxidase, cadmium ATPase, multicopper oxidase, and a ClpL chaperone protein during growth under salt (6% NaCl) and acid conditions (pH 5) and ProW, an osmolyte transporter, under salt stress conditions. No differences in salt and acid tolerance were observed between plasmid-cured and wildtype strains. This work highlights the abundance of specific plasmid types among food-related L. monocytogenes strains, the unique characteristics of G1 and G2 plasmids, and the possible contributions of plasmids to L. monocytogenes tolerance to food-related stresses.

Prophages have long been regarded as an important contributor to the evolution of Salmonella and Verotoxin-producing E. coli (VTEC), members of the Enterobacteriaceae that cause millions of cases of foodborne illness in North America. In S. Typhimurium, prophages provide many of the genes required for invasion; similarly, in VTEC, the Verotoxin-encoding genes are located in cryptic prophages. The ability of prophages to quickly acquire and lose genes have driven their rapid evolution, leading to highly diversified populations of phages that can infect distantly-related bacterial hosts. To defend against foreign genetic materials (i.e., phages), bacteria have evolved Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) immunity, consisting of variable spacer regions that match short nucleic acid sequences of invaders previously encountered. The number of spacer regions varies widely amongst Enterobacteriaceae, and there is currently no clear consensus if the accumulation of spacers is linked to genomic prophage abundance. Given the immense prophage diversity and contribution to bacterial host phenotypes, we analyzed the prophage sequences within 118 strains of Salmonella and VTEC, 117 of which are of agricultural origin. Overall, 130 unique prophage sequences were identified and they were found to be remarkably diverse with &lt;50% nucleotide similarity, particularly with the Gifsy-1 group which was identified in several Salmonella serovars and interestingly, a strain of VTEC. Additionally, we identified a novel plasmid-like phage that carried antibiotic resistance and bacteriocin resistance genes. The strains analyzed carried at least six distinct spacers which did not possess homology to prophages identified in the same genome. In fact, only a fraction of all identified spacers (14%) possessed significant homology to known prophages. Regression models did not discern a correlation between spacer and prophage abundance in our strains, although the relatively high number of spacers in our strains (an average of 27 in Salmonella and 19 in VTEC) suggest that high rates of infection may occur in agricultural niches and be a contributing driver in bacterial evolution. Cumulatively, these results shed insight into prophage diversity of Salmonella and VTEC, which will have further implications when informing development of phage therapies against these foodborne pathogens.

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