Revelation by Single-Nucleotide Polymorphism Genotyping That Mutations Leading to a Premature Stop Codon in inlA Are Common among Listeria monocytogenes Isolates from Ready-To-Eat Foods but Not Human Listeriosis Cases

Abstract

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...

Diverse Geno- and Phenotypes of Persistent Listeria monocytogenes Isolates from Fermented Meat Sausage Production Facilities in Portugal

“…These reduced invasion phenotypes appear to be due to different mechanisms, including the following: (i) different premature stop codon (PMSC) mutations in the inlA gene, which encodes a protein (internalin A) that promotes L. monocytogenes internalization into human epithelial cells (36); (ii) reduced transcription of inlA; and (iii) reduced swarming. While PMSC mutations in inlA have been described extensively in both clinical and food L. monocytogenes isolates collected worldwide, with at least 18 different naturally occurring inlA mutations identified so far (17,25,27,29,46,48,52,56,59,67,68), only limited previous evidence (58) exists for attenuated invasion associated with reduced inlA transcript levels or reduced swarming. While swarming motility has previously been shown to contribute to the ability of L. monocytogenes to adhere to and invade Caco-2 cells, as well as its ability to cause disease in a mouse infection model (2,11,50), further characterization of isogenic mutants will be necessary to confirm that specific mechanisms (e.g., reduced swarming) are responsible for reduced invasion efficiency.…”

“…b nt, nucleotide; aa, amino acids. c Premature stop codon (PMSC) mutations were classified by the method of Van Stelten et al (67). d Strain FSL F8-151 showed reduced swarming compared to strain 10403S at 37°C; swarming at this temperature was similar to that for a ⌬flaA control strain (Fig.…”

Listeria monocytogenes Associated with New Zealand Seafood Production and Clinical Cases: Unique Sequence Types, Truncated InlA, and Attenuated Invasiveness

“…A truncated InlA was not evident in any of the 61 L. monocytogenes isolates from abortion cases in France (43). Furthermore, Van Stelten et al (23) found that only 5.1% of human isolates have a PMSC in inlA, while two other studies in the United States provided further evidence that an inlA PMSC is uncommon in strains that cause listeriosis (22,42). Nightingale et al (24), however, revealed that 36.5% of the 52 human clinical isolates selected from ribotypes commonly associated with PMSCs carried an inlA PMSC, placing in doubt previous assumptions that a functional InlA protein is required for effective invasion.…”

“…Partial inlA DNA sequences spanning regions associated with previously defined PMSCs (mutation types 1 to 18) (23,24) were obtained for the New Zealand clinical and environmental isolates. PCRs were performed as previously described using primers inlA-F and inlA-R (24).…”

“…Screening of L. monocytogenes isolated from human clinical cases, ready-to-eat (RTE) foods, and foodprocessing environments has revealed mutations in inlA leading to premature stop codons (PMSCs) (21)(22)(23), particularly among lineage II isolates of serotypes 1/2a and 1/2c (6). To date, at least 18 naturally occurring single nucleotide polymorphisms leading to a PMSC in inlA have been identified (23). Strains containing these PMSCs produce a truncated InlA protein and are generally less invasive than those without a PMSC (24).…”