Listeria monocytogenes utilizes internalin A (InlA; encoded by inlA) to cross the intestinal barrier to establish a systemic infection. Multiple naturally occurring mutations leading to a premature stop codon (PMSC) in inlA have been reported worldwide, and these mutations are causally associated with attenuated virulence. Five inlA PMSC mutations recently discovered among isolates from France and the United States were included as additional markers in our previously described inlA single-nucleotide polymorphism (SNP) genotyping assay. This assay was used to screen >1,000 L. monocytogenes isolates from ready-to-eat (RTE) foods (n ‫؍‬ 502) and human listeriosis cases (n ‫؍‬ 507) for 18 inlA PMSC mutations. A significantly (P < 0.0001) greater proportion of RTE food isolates (45.0%) carried a PMSC mutation in inlA compared to human clinical isolates (5.1%). The proportion of L. monocytogenes with or without PMSC mutations in inlA was similar among isolates from different RTE food categories except for deli meats, which included a marginally higher proportion (P ‫؍‬ 0.12) of isolates carrying a PMSC in inlA. We also analyzed the distribution of epidemic clone (EC) strains, which have been linked to the majority of listeriosis outbreaks worldwide and are overrepresented among sporadic cases in the United States. We observed a significant (P < 0.05) overrepresentation of EC strains in deli and seafood salads and a significant (P < 0.05) underrepresentation of EC strains in smoked seafood. These results provide important data to predict the human health risk of exposure to L. monocytogenes strains that differ in pathogenic potential through consumption of contaminated RTE foods.Listeria monocytogenes is the etiological agent of listeriosis, a potentially life-threatening food-borne disease that primarily affects individuals with underlying immune-compromising circumstances. Listeriosis is associated with an exceptionally high hospitalization rate of 85 to 90% and 20 to 30% of cases are fatal. L. monocytogenes infections account for nearly 30% of all fatalities attributed to known food-borne pathogens each year in the United States (18). Although L. monocytogenes is readily inactivated by cooking and pasteurization, subsequent crosscontamination of ready-to-eat (RTE) food products exposed to the food processing plant environment following a lethality treatment represents the route through which RTE foods become contaminated (31). L. monocytogenes is a hardy pathogen; it is relatively resistant to acid, is able to grow in high salt concentrations, and is capable of growing at refrigeration temperatures (2, 4). Because L. monocytogenes tolerates intrinsic and extrinsic properties of food typically used to control the growth of pathogens, consumption of RTE foods contaminated by L. monocytogenes represents a significant health risk for immunocompromised individuals. The combined foodborne route of L. monocytogenes transmission and severity of listeriosis prompted establishment of strict regulations regarding the presence...

The virulence factor internalin A (InlA) facilitates the uptake of Listeria monocytogenes by epithelial cells that express the human isoform of E-cadherin. Previous studies identified naturally occurring premature stop codon (PMSC) mutations in inlA and demonstrated that these mutations are responsible for virulence attenuation. We assembled >1,700 L. monocytogenes isolates from diverse sources representing 90 EcoRI ribotypes. A subset of this isolate collection was selected based on ribotype frequency and characterized by a Caco-2 cell invasion assay. The sequencing of inlA genes from isolates with attenuated invasion capacities revealed three novel inlA PMSCs which had not been identified previously among U.S. isolates. Since ribotypes include isolates with and without inlA PMSCs, we developed a multiplex single-nucleotide polymorphism (SNP) genotyping assay to detect isolates with virulence-attenuating PMSC mutations in inlA. The SNP genotyping assay detects all inlA PMSC mutations that have been reported worldwide and verified in this study to date by the extension of unlabeled primers with fluorescently labeled dideoxynucleoside triphosphates. We implemented the SNP genotyping assay to characterize human clinical and food isolates representing common ribotypes associated with novel inlA PMSC mutations. PMSCs in inlA were significantly (ribotypes DUP-1039C and DUP-1045B; P < 0.001) or marginally (ribotype DUP-1062D; P ‫؍‬ 0.11) more common among food isolates than human clinical isolates. SNP genotyping revealed a fourth novel PMSC mutation among U.S. L. monocytogenes isolates, which was observed previously among isolates from France and Portugal. This SNP genotyping assay may be implemented by regulatory agencies and the food industry to differentiate L. monocytogenes isolates carrying virulence-attenuating PMSC mutations in inlA from strains representing the most significant health risk.

Internalin A (InlA; encoded by inlA) facilitates the crossing of the intestinal barrier by Listeria monocytogenes. Mutations leading to a premature stop codon (PMSC) in inlA and thus attenuated mammalian virulence have been reported. We recently characterized 502 L. monocytogenes food isolates from a retail survey and 507 human clinical isolates from multiple U.S. states with respect to the presence/absence of inlA mutations. The objective of this study was to investigate the hypothesis that dose responses for human listeriosis vary between L. monocytogenes strains with and those without a PMSC in inlA. Subtype-specific prevalence and concentration distributions in food, along with epidemiologic and consumption data, were input into established doseresponse models to generate an r value (probability of a cell causing illness). Under the conservative assumption that L. monocytogenes levels at retail represent levels consumed, mean log 10 r values were ؊8.1 and ؊10.7 for L. monocytogenes subtypes with genes encoding a full-length and a truncated InlA, respectively. L. monocytogenes carrying a 5 frameshift mutation in a homopolymeric tract showed a mean log 10 r value of ؊12.1. Confidence intervals for the r values and their differences varied depending on subtypes. When the increase in concentration of L. monocytogenes subtypes between retail and consumption was considered, mean log 10 r values were reduced to ؊10.4, ؊13.8, and ؊12.8 for the subtypes with genes encoding a full-length InlA, for the subtypes carrying a PMSC in inlA, and for all L. monocytogenes isolates regardless of subtype, respectively. Our study provides further quantitative evidence that L. monocytogenes subtypes vary in abilities and relative likelihoods of causing human disease, which were mechanistically related to defined genetic markers.Listeria monocytogenes continues to represent a major challenge for the ready-to-eat (RTE) food industry due to the ubiquitous presence of this pathogen along the food supply continuum, its food-borne route of transmission, and the exceptionally high hospitalization (90%) and mortality (20 to 30%) rates associated with invasive listeriosis (17,22,45). L. monocytogenes isolates can be classified into four genetic lineages, including two common ones termed lineages I and II (43); lineage I isolates have been reported to be overrepresented among isolates from human clinical cases in the United States, while lineage II isolates appear overrepresented among isolates from contaminated foods (13). Studies combining molecular subtyping and in vitro virulence phenotype assays provided initial evidence for heterogeneity in virulence among L. monocytogenes molecular subtypes (e.g., ribotypes) beyond the genetic lineage level (13,25,46). Specifically, a subpopulation of highly clonal strains (termed epidemic clones) has been linked to most listeriosis outbreaks worldwide and epidemic clone strains appear to be overrepresented among sporadic listeriosis cases in the United States (13,17,40,42). On the other hand, recent...

Listeria monocytogenes contains (i) epidemic clone (EC) strains, which have been linked to the majority of listeriosis outbreaks worldwide and are overrepresented among sporadic cases in the United States, and (ii) strains commonly isolated from ready-to-eat foods that carry a mutation leading to a premature stop codon (PMSC) in inlA, which encodes the key virulence factor internalin A (InlA). Internalin A binds certain isoforms of the cellular receptor E-cadherin to facilitate crossing the intestinal barrier during the initial stages of an L. monocytogenes infection. Juvenile guinea pigs, which express the human isoform of E-cadherin that binds InlA, were intragastrically challenged with a range of doses of (i) an EC strain associated with a listeriosis outbreak or (ii) a strain carrying a PMSC mutation in inlA. Recovery of L. monocytogenes from tissues (i.e., liver, spleen, mesenteric lymph nodes, and ileum) was used to develop strain-specific dose-response curves on the basis of individual and combined organ data. Modeling of individual and combined organ data revealed an approximate 1.2 to 1.3 log 10 increase in the median infectious dose for the strain carrying a PMSC in inlA relative to that for the EC strain. Inclusion of the strain parameter significantly improved the goodness of fit for individual and combined organ models, indicating a significant shift in median infectious dose for guinea pigs challenged with an inlA PMSC strain compared to that for guinea pigs challenged with an EC strain. Results from this work provide evidence that the L. monocytogenes dose-response relationship is strain specific and will provide critical data for enhancement of current risk assessments and development of future risk assessments.

cIn Listeria monocytogenes, 18 mutations leading to premature stop codons (PMSCs) in the virulence gene inlA have been identified to date. While most of these mutations represent nucleotide substitutions, a frameshift deletion in a 5= seven-adenine homopolymeric tract (HT) in inlA has also been reported. This HT may play a role in phase variation and was first identified among L. monocytogenes lineage II ribotype DUP-1039C isolates. In order to better understand the distribution of different inlA mutations in this ribotype, a newly developed multiplex real-time PCR assay was used to screen 368 DUP-1039C isolates from human, animal, and food-associated sources for three known 5= inlA HT alleles: (i) wild-type (WT) (A 7 ), (ii) frameshift (FS) (A 6 ), and (iii) guanine interruption (A 2 GA 4 ) alleles. Additionally, 228 DUP-1039C isolates were screened for all inlA PMSCs; data on the presence of all inlA PMSCs for the other 140 isolates were obtained from previous studies. The statistical analysis based on 191 epidemiologically unrelated strains showed that strains with inlA PMSC mutations (n ‫؍‬ 41) were overrepresented among food-associated isolates, while strains encoding full-length InlA (n ‫؍‬ 150) were overrepresented among isolates from farm animals and their environments. Furthermore, the A 6 allele was overrepresented and the A 7 allele was underrepresented among food isolates, while the A 6 allele was underrepresented among farm and animal isolates. Our results indicate that genetic variation in inlA contributes to niche adaptation within the lineage II subtype DUP-1039C. Listeria monocytogenes is a food-borne pathogen and the etiological agent of listeriosis, a severe invasive disease that can affect both humans and animals (1). More than 99% of human listeriosis cases are estimated to be transmitted through food (2). Despite its presence in a wide range of environments and foods, the majority of human listeriosis infections appear to be linked to consumption of contaminated ready-to-eat (RTE) foods (3) that support L. monocytogenes growth. Numerous studies have indicated that not all L. monocytogenes strains are equally associated with invasive disease. For example, McLauchlin reported that three (1/2a, 1/2b, and 4b) of the 13 serotypes of L. monocytogenes were responsible for 90% of 1,363 listeriosis cases from the United Kingdom (4). Additionally, multiple studies using both DNA band-based and sequence-based subtyping methods have shown that L. monocytogenes forms a structured population composed of at least four divergent lineages (I, II, III, and IV), which in a number of studies have been suggested to differ in their associations with different sources and in their pathogenic potentials (5-9). The majority of L. monocytogenes isolates belong to lineages I and II, which contain the serotypes most commonly associated with human clinical cases; serotypes 1/2b and 4b group into lineage I, while serotypes1/2a and 1/2c group into lineage II. Lineage III and IV strains are rare and usually isolated from ...