Characterization of virulence-attenuated Listeria monocytogenes common among food and food-associated environments but rarely associated with disease

Studies were conducted to characterize virulence-attenuated Listeria monocytogenes isolates carrying single nucleotide polymorphisms (SNPs) leading to a premature stop codon (PMSC) in the key virulence gene, inlA. The virulence factor Internalin A (InlA; encoded by inlA), facilitates the uptake of L. monocytogenes by epithelial cells that express the human isoform of E-cadherin and allows for pathogen traversal across the intestinal barrier in order to establish a systemic infection. Previous studies have identified 18 naturally occurring inlA SNPs, to date, and have demonstrated that these mutations are responsible for attenuated mammalian virulence. L. monocytogenes contains at least two distinct subpopulations characterized by stark differences in pathogenic potential, including (i) a small subpopulation of epidemic clone strains, which have been linked to the majority of listeriosis cases and (ii) a significant subpopulation of strains commonly isolated from foods that carry virulence-attenuating (VA) SNPs in inlA. These mutations therefore represent suitable genetic markers to identify L. monocytogenes isolates with attenuated virulence. Previously, we developed a multiplex single-nucleotide polymorphism genotyping assay capable of screening for the presence of all known VA SNPs in inlA (mutation types 1-18). Briefly, the SNP genotyping assay relies on single-base-pair extension or “minisequencing”. We previously used this assay to determine the prevalence of inlA PMSC mutations among a large representative collection of L. monocytogenes isolates from ready-to-eat (RTE) foods and human listeriosis cases. Results showed that a significantly (P<0.0001) greater proportion (45.0%) of isolates from RTE foods carry a VA SNP in inlA as compared to human clinical isolates (5.1%). Herein, we wanted to specifically investigate the prevalence of VA SNPs in inlA among L. monocytogenes isolates from environments associated with RTE food production and handling (i.e. processing plant and retail environments). More than 700 L. monocytogenes isolates from RTE food processing plant and retail environments from the U.S. were screened for the presence or absence of the seven most common VA SNPs in inlA in the U.S. (mutation types 1-7). Mutation types 8-18 were not investigated due to their underrepresentation in the U.S. (e.g. from 252 food and human isolates found to carry a VA SNP in inlA, only 4 isolates carried a mutation type belonging to types 8-18). Overall, 26.4% of isolates from RTE food processing plant and 32.6% of isolates from retail environments carried a VA SNP in inlA. Food contact surfaces sampled at retail establishments were significantly (P<0.0001) more likely to be contaminated by a L. monocytogenes isolate carrying a VA SNP in inlA (56% of 55 isolates) as compared to non-food contact surfaces (28% of 264 isolates). Overall, data obtained here, coupled with our previous work, indicate a significant proportion of L. monocytogenes isolated from RTE foods and food production and handling environments carry a VA SNP in inlA, and consequently, have a decreased ability to cause disease.
The current L. monocytogenes risk assessment predicts that the average individual consumes a food serving containing low (< 1x103) or intermediate (between 1x103 and 1x106) levels of L. monocytogenes 19 and 2.4 times per year, respectively, without developing disease. This data, combined with the high prevalence of VA L. monocytogenes in foods and food-associated environments, led us to hypothesize that natural exposure to VA strains through consumption of contaminated food may confer protective population immunity against listeriosis and thus, current food safety measures to minimize exposure to any L. monocytogenes strain in food may have adverse epidemiological outcomes at the population level. This hypothesis could additionally elucidate why declines in L. monocytogenes contamination of deli meats (a food vehicle responsible for almost 90% of human listeriosis cases) and the incidence of human disease have not paralleled each other despite considerable efforts by government and industry to prevent exposure to L. monocytogenes through contaminated food. We therefore conducted studies to orally inoculate mice with a range of doses of a L. monocytogenes strain carrying a VA SNP in inlA, to assess the ability of these strains to (i) raise listeria-specific CD8+ T cell-mediated immune responses and (ii) confer protection against a subsequent challenge by a fully-virulent strain. Results provided evidence that after exposure to L. monocytogenes carrying a VA SNP in inlA, there is an expansion of Listeria-specific CD8+ T effector cells that ultimately lead to a stably maintained pool of central memory CD8+ T cells capable of providing long-term immunity. Overall, level of immune protection was dependent on dose of initial exposure. Data from the vaccine challenge study demonstrated that protection could be provided by L. monocytogenes carrying a VA SNP in inlA at higher inoculum levels pertaining to relevant doses of VA strains per food serving (e.g. 2x105 CFU/serving to 1.36x106 CFU/serving); these primary inoculum levels proved to provide protective immunity in both the vaccine challenge model and CD8+ T cell model. At lower exposure doses relevant to food contamination with fully-virulent strains (e.g. 2x103 CFU), however, protection was not provided, which could indicate more virulent strains of L. monocytogenes in the food supply might not play as great of role in priming our immune systems to subsequent infection, and VA strains may play a larger role in immune priming since we previously determined they are frequently isolated from foods, and overall, at significantly higher levels of contamination (>10,000-fold higher than fully-virulent strains). This confirms that memory CD8+ T cells generated by VA strains are capable of providing protective immunity at levels they are commonly isolated from foods and that lack of a functional InlA protein does not impair protection against a secondary L. monocytogenes infection. Ultimately, these results will provide critical data for revision of current and development of future risk assessments and regulatory initiatives regarding the presence of L. monocytogenes in RTE foods.