Development and Implementation of a Multiplex Single-Nucleotide Polymorphism Genotyping Assay for Detection of Virulence-Attenuating Mutations in theListeria monocytogenesVirulence-Associated GeneinlA

Abstract

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.

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

“…Three isolates with PFGE type 356 (two from processor D and one from processor I) carried inlA PMSC type 4, which has been previously observed in isolates from the United States (52,68) and Portugal, namely, in one isolate with the same PFGE type collected from processor D on another sampling date (17). Three isolates representing 3 different PFGE profiles (profiles 365, 342, and 376) carried inlA PMSC mutation type 6 ( tation was also previously identified by Felício et al (17) in two isolates with PFGE profile 361 obtained from the same processor J samples that yielded one of the two isolates with this PMSC here.…”

“…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.…”

Presencia de Listeria monocytogenes en alimentos listos para el consumo, procedentes de plazas de mercado y delicatessen de supermercados de cadena, Bogotá, D.C, 2002-2008

“…Listeria monocytogenes es un patógeno, facultativo intracelular y oportunista (1,2,4,5,9,10,24,32,36). Sus manifestaciones clínicas se pueden agrupar en dos categorías: listeriosis invasiva y listeriosis no invasiva (1,16,17,28).…”

“…Sus manifestaciones clínicas se pueden agrupar en dos categorías: listeriosis invasiva y listeriosis no invasiva (1,16,17,28). La listeriosis invasiva (2,24,28,37) se produce cuando la infección ocasionada por las células bacterianas atraviesa la barrera intestinal y alcanza sistemas y órganos que son blanco de infección, como son el sistema nervioso central y el útero en la mujer embarazada (1,2,11,13,37), ocasionando meningitis (2,9,11,12,15,19,21,24,31,36), bacteriemias (2,3,9,12,19,31), septicemias (2,9,11,13,15,19,21,24,31,36), meningoencefalitis (2,9,21,24,31), absceso cerebral o espinal, listeriosis pontobulbar y romboencefalitis (2,3,13,21,30,38,39).…”

“…En los países industrializados, como Estados Unidos, en donde la listeriosis es de notificación obligatoria en todos los estados desde el 2001 (23), se reportan 2.500 casos anuales de listeriosis ocasionados por alimentos y muere una de cada cinco personas afectadas (5,6,10,(23)(24)(25).…”