Genetic profiles and invasion ability of Listeria monocytogenes isolated from bovine carcasses in Southern Brazil

The goals of this study were to evaluate the persistence and the virulence potential of Listeria monocytogenes isolated from beef carcasses obtained in processing facilities in the Southern region of Rio Grande do Sul, Brazil, based on pulsed field gel electrophoresis (PFGE), invasion ability in human colorectal carcinoma cells (HCT-116), InlA expression by western blot (WB) and identification of mutation points in the inlA . PFGE profiles demonstrated that L. monocytogenes isolates were grouped based on their previously identified lineages and serogroups (lineage I: serogroups IIb, n = 2, and IVb, n = 5; lineage II, serogroup IIc, n = 5), isolates with indistinguishable genetic profiles by this method were obtained from different slaughterhouses and sampling steps, with up to 3-year interval. Seven isolates showed high invasion ability (2.4 to 7.4%, lineage I, n = 6, lineage II, n = 1) in HCT and expressed InlA. Five isolates showed low cell invasion ability (0.6 to 1.4%, lineage I, n = 1, lineage II, n = 4) and did not express InlA, and two of them (lineage II, serogroup IIc) presented mutations in inlA leading to a premature stop codon (PMSC) type 19, at position 326 (GAA → TAA). The results demonstrated that most of L. monocytogenes isolates from Lineage I expressed InlA and were the most invasive in HCT indicating their high virulence potential, while most isolates from Lineage II showed attenuated invasion due to non-expression of InlA and the presence of PMSC type 19 in inlA . The obtained results demonstrated that L. monocytogenes with indistinguishable PFGE profiles can be persisting or being reintroduced in beef processing facilities in the studied region and differences on their virulence potential based on their lineages and serogroups.

Download Full-text

Listeria monocytogenes strains encoding premature stop codons in inlA invade mice and guinea pig fetuses in orally dosed dams

Journal of Medical Microbiology ◽

10.1099/jmm.0.057505-0 ◽

2013 ◽

Vol 62 (12) ◽

pp. 1799-1806 ◽

Cited By ~ 14

Author(s):

Anne Holch ◽

Hanne Ingmer ◽

Tine Rask Licht ◽

Lone Gram

Keyword(s):

Listeria Monocytogenes ◽

Guinea Pig ◽

Food Processing ◽

Guinea Pigs ◽

Stop Codon ◽

Fetal Liver ◽

Fatal Case ◽

Premature Stop Codon ◽

Tissue Samples ◽

Pregnant Mice

Listeria monocytogenes is an important food-borne bacterial pathogen and listeriosis can result in abortions in pregnant women. The bacterium can colonize food-processing environments, where specific molecular subtypes can persist for years. The purpose of this study was to determine the virulence potential of a group of food-processing persistent L. monocytogenes strains encoding a premature stop codon in inlA (encoding internalin A) by using two orally dosed models, pregnant mice and pregnant guinea pigs. A food-processing persistent strain of L. monocytogenes invaded placentas (n = 58; 10 % positive) and fetuses (3 % positive) of pregnant mice (n = 9 animals per strain), similar to a genetically manipulated murinized strain, EGD-e InlA m* (n = 61; 3 and 2 %, respectively). In pregnant guinea pigs (n = 9 animals per bacterial strain), a maternofetal strain (from a human fetal clinical fatal case) was isolated from 34 % of placenta samples (n = 50), whereas both food-processing persistent strains were found in 5 % of placenta samples (n = 36 or 37). One of the food-processing persistent strains, N53-1, was found in up to 8 % of guinea pig fetal liver and brain samples, whereas the maternofetal control was found in 6 % of fetal tissue samples. As the food-processing persistent strains carry a premature stop codon in inlA but are invasive in orally dosed pregnant mice and guinea pigs, we hypothesize that listerial crossing of the placental barrier can occur by a mechanism that is independent of an interaction between E-cadherin and InlA.

Download Full-text

Poor Invasion of Trophoblastic Cells but Normal Plaque Formation in Fibroblastic Cells despite actA Deletion in a Group of Listeria monocytogenes Strains Persisting in Some Food Processing Environments

Applied and Environmental Microbiology ◽

10.1128/aem.02862-09 ◽

2010 ◽

Vol 76 (10) ◽

pp. 3391-3397 ◽

Cited By ~ 13

Author(s):

Anne Holch ◽

Caroline Trebbien Gottlieb ◽

Marianne Halberg Larsen ◽

Hanne Ingmer ◽

Lone Gram

Keyword(s):

Listeria Monocytogenes ◽

Molecular Subtype ◽

Stop Codon ◽

Premature Stop Codon ◽

Virulence Gene ◽

Fish Processing ◽

Trophoblastic Cells ◽

Processing Plants ◽

Fibroblastic Cells ◽

Cell Spread

ABSTRACT We determined mammalian cell invasion and virulence gene (inlA, inlB, and actA) sequences of Listeria monocytogenes strains belonging to a molecular subtype (RAPD 9) that often persists in Danish fish-processing plants. These strains invaded human placental trophoblasts less efficiently than other L. monocytogenes strains, including clinical strains, and they carry a premature stop codon in inlA. Eight of 15 strains, including the RAPD 9 and maternofetal strains, had a 105-nucleotide deletion in actA that did not affect cell-to-cell spread in mouse fibroblasts. The RAPD 9 strains may still be regarded as of low virulence with respect to human listeriosis.

Download Full-text

Spontaneous Loss of Virulence in Natural Populations of Listeria monocytogenes

Infection and Immunity ◽

10.1128/iai.00541-17 ◽

2017 ◽

Vol 85 (11) ◽

Cited By ~ 41

Author(s):

Mylène M. Maury ◽

Viviane Chenal-Francisque ◽

Hélène Bracq-Dieye ◽

Lei Han ◽

Alexandre Leclercq ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Stop Codon ◽

Premature Stop Codon ◽

Host Cells ◽

Single Amino Acid ◽

Listeriolysin O ◽

Loss Of Function ◽

Phenotypic Marker ◽

Content Type ◽

Virulence Attenuation

ABSTRACT The pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene (hly) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes. Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA. Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA−/LLO−) mutants belonged to phylogenetically diverse clades of L. monocytogenes, and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA−/LLO− mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen.

Download Full-text

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

Applied and Environmental Microbiology ◽

10.1128/aem.03305-13 ◽

2013 ◽

Vol 80 (4) ◽

pp. 1489-1497 ◽

Cited By ~ 16

Author(s):

Cristina D. Cruz ◽

Andrew R. Pitman ◽

Sally A. Harrow ◽

Graham C. Fletcher

Keyword(s):

New Zealand ◽

Listeria Monocytogenes ◽

Stop Codon ◽

Premature Stop Codon ◽

Potential Health ◽

Content Type ◽

Processing Plants ◽

Seafood Processing ◽

Sequence Types ◽

Clinical Cases

ABSTRACTListeriosis is caused by the food-borne pathogenListeria monocytogenes, which can be found in seafood and processing plants. To evaluate the risk to human health associated with seafood production in New Zealand, multi-virulence-locus sequence typing (MVLST) was used to define the sequence types (STs) of 31L. monocytogenesisolates collected from seafood-processing plants, 15 from processed foods, and 6 from human listeriosis cases. The STs of these isolates were then compared with those from a collection of seafood isolates and epidemic strains from overseas. A total of 17 STs from New Zealand clustered into two lineages: seafood-related isolates in lineages I and II and all human isolates in lineage II. None of the New Zealand STs matched previously described STs from other countries. Isolates (belonging to ST01-N and ST03-N) from mussels and their processing environments, however, were identical to those of sporadic listeriosis cases in New Zealand. ST03-N isolates (16 from mussel-processing environments, 2 from humans, and 1 from a mussel) contained aninlApremature stop codon (PMSC) mutation. Therefore, the levels of invasiveness of 22 isolates from ST03-N and the three other common STs were compared using human intestinal epithelial Caco-2 cell lines. STs carryinginlAPMSCs, including ST03-N isolates associated with clinical cases, had a low invasion phenotype. The close relatedness of some clinical and environmental strains, as revealed by identical MVLST profiles, suggests that local and persistent environmental strains in seafood-processing environments pose a potential health risk. Furthermore, a PMSC ininlAdoes not appear to giveL. monocytogenesa noninvasive profile.

Download Full-text

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

Applied and Environmental Microbiology ◽

10.1128/aem.02651-09 ◽

2010 ◽

Vol 76 (9) ◽

pp. 2783-2790 ◽

Cited By ~ 83

Author(s):

A. Van Stelten ◽

J. M. Simpson ◽

T. J. Ward ◽

K. K. Nightingale

Keyword(s):

United States ◽

Single Nucleotide Polymorphism ◽

Listeria Monocytogenes ◽

Stop Codon ◽

Human Health Risk ◽

Premature Stop Codon ◽

The United States ◽

Nucleotide Polymorphism ◽

Pathogenic Potential ◽

Single Nucleotide

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.

Download Full-text

Development and Implementation of a Multiplex Single-Nucleotide Polymorphism Genotyping Assay for Detection of Virulence-Attenuating Mutations in the Listeria monocytogenes Virulence-Associated Gene inlA

Applied and Environmental Microbiology ◽

10.1128/aem.01138-08 ◽

2008 ◽

Vol 74 (23) ◽

pp. 7365-7375 ◽

Cited By ~ 38

Author(s):

A. Van Stelten ◽

K. K. Nightingale

Keyword(s):

Single Nucleotide Polymorphism ◽

Listeria Monocytogenes ◽

Stop Codon ◽

Premature Stop Codon ◽

Snp Genotyping ◽

Regulatory Agencies ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Genotyping Assay ◽

Virulence Attenuation

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.

Download Full-text

Potential Ad Hoc Markers of Persistence and Virulence in Canadian Listeria monocytogenes Food and Clinical Isolates

Journal of Food Protection ◽

10.4315/0362-028x.jfp-19-028 ◽

2019 ◽

Vol 82 (11) ◽

pp. 1909-1921 ◽

Cited By ~ 2

Author(s):

JACQUELINE UPHAM ◽

STEPHEN CHEN ◽

ELIZABETH BOUTILIER ◽

LISA HODGES ◽

MIKAELA EISEBRAUN ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Ad Hoc ◽

Stop Codon ◽

Premature Stop Codon ◽

In Silico Analysis ◽

Clinical Isolates ◽

Intestinal Epithelial ◽

Virulence Protein ◽

Islet 1 ◽

Experimental Findings

ABSTRACT The Listeria monocytogenes gene inlA, encoding a surface virulence protein, was examined for the presence of premature stop codon (PMSC) mutations in 82 isolates obtained by the Canadian Food Inspection Agency (CFIA) from foods and food contact surfaces. These mutations were coanalyzed for the presence of stress survival islet 1 (SSI-1) and for the abilities of the isolates to invade Caco-2 intestinal epithelial cells and form biofilms on polystyrene. PMSC mutations were present in one-third of the isolates (predominantly those of serogroup 1/2a), and their presence was correlated with a noninvasive phenotype. The presence of SSI-1 and the ability to form biofilms were also linked to the 1/2a serogroup. Serogroup 4b isolates lacked inlA PMSC mutations and were invasive, but neither formed biofilms nor carried SSI-1. To expand upon these experimental findings, an in silico analysis was performed on L. monocytogenes genomes from Canadian databases of 278 food isolates and 607 clinical isolates. The prevalence of inlA PMSC mutations in genomes of food isolates was significantly higher (P < 0.0001) than that in clinical isolates. Also, a three-codon deletion in inlA associated with a hyperinvasive phenotype was more prevalent in genomes from clinical isolates (primarily of clonal complex 6, serogroup 4b) than in those from food isolates (P < 0.001). In contrast, SSI-1 was significantly overrepresented (P < 0.001) in genomes from food isolates. We propose the hypothesis that SSI-1 and inlA play a role in the evolution of Canadian L. monocytogenes strains into either a virulent (represented by serogroup 4b clinical isolates) or an environmentally persistent (represented by serogroup 1/2a food isolates) phenotype. The combined presence of SSI-1 and inlA PMSC mutations have potential for use as genetic markers for risk assessment when L. monocytogenes is recovered from foods, indicating low potential for pathogenesis.

Download Full-text

Genomic Diversity of Listeria monocytogenes Isolated from Clinical and Non-Clinical Samples in Chile

Genes ◽

10.3390/genes9080396 ◽

2018 ◽

Vol 9 (8) ◽

pp. 396 ◽

Cited By ~ 11

Author(s):

Viviana Toledo ◽

Henk den Bakker ◽

Juan Hormazábal ◽

Gerardo González-Rocha ◽

Helia Bello-Toledo ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Stop Codon ◽

Premature Stop Codon ◽

Severe Infection ◽

Risk Groups ◽

Genomic Diversity ◽

Clinical Samples ◽

Pathogenic Potential ◽

Stress Survival ◽

Sequence Types

Listeria monocytogenes is the causative agent of listeriosis, which is an uncommon but severe infection associated with high mortality rates in humans especially in high-risk groups. This bacterium survives a variety of stress conditions (e.g., high osmolality, low pH), which allows it to colonize different niches especially niches found in food processing environments. Additionally, a considerable heterogeneity in pathogenic potential has been observed in different strains. In this study, 38 isolates of L. monocytogenes collected in Chile from clinical samples (n = 22) and non-clinical samples (n = 16) were analyzed using whole genome sequencing (WGS) to determine their genomic diversity. A core genome Single Nucleotide Polymorphism (SNP) tree using 55 additional L. monocytogenes accessions classified the Chilean isolates in lineages I (n = 25) and II (n = 13). In silico, Multi-locus sequence typing (MLST) differentiated the isolates into 13 sequence types (ST) in which the most common were ST1 (15 isolates) and ST9 (6 isolates) and represented 55% of the isolates. Genomic elements associated with virulence (i.e., LIPI-1, LIPI-3, inlA, inlB, inlC, inlG, inlH, inlD, inlE, inlK, inlF, and inlJ) and stress survival (i.e., stress survival islet 1 and stress survival islet 2) were unevenly distributed among clinical and non-clinical isolates. In addition, one novel inlA premature stop codon (PMSC) was detected. Comparative analysis of L. monocytogenes circulating in Chile revealed the presence of globally distributed sequence types along with differences among the isolates analyzed at a genomic level specifically associated with virulence and stress survival.

Download Full-text

Intensive Environmental Surveillance Plan for Listeria monocytogenes in Food Producing Plants and Retail Stores of Central Italy: Prevalence and Genetic Diversity

Foods ◽

10.3390/foods10081944 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1944

Author(s):

Gabriella Centorotola ◽

Fabrizia Guidi ◽

Guglielmo D’Aurizio ◽

Romolo Salini ◽

Marco Di Domenico ◽

...

Keyword(s):

Genetic Diversity ◽

Listeria Monocytogenes ◽

Core Genome ◽

Stop Codon ◽

Central Italy ◽

Premature Stop Codon ◽

Grocery Stores ◽

Environmental Surveillance ◽

Retail Grocery

Listeria monocytogenes (Lm) can persist in food processing environments (FPEs), surviving environmental stresses and disinfectants. We described an intensive environmental monitoring plan performed in Central Italy and involving food producing plants (FPPs) and retail grocery stores (RSs). The aim of the study was to provide a snapshot of the Lm circulation in different FPEs during a severe listeriosis outbreak, using whole genome sequencing (WGS) to investigate the genetic diversity of the Lm isolated, evaluating their virulence and stress resistance profiles. A total of 1217 samples were collected in 86 FPEs with 12.0% of positive surfaces at FPPs level and 7.5% at RSs level; 133 Lm isolates were typed by multilocus sequencing typing (MLST) and core genome MLST (cgMLST). Clonal complex (CC) 121 (25.6%), CC9 (22.6%), CC1 (11.3%), CC3 (10.5%), CC191 (4.5%), CC7 (4.5%) and CC31 (3.8%) were the most frequent MLST clones. Among the 26 cgMLST clusters obtained, 5 of them persisted after sanitization and were re-isolated during the follow-up sampling. All the CC121 harboured the Tn6188_qac gene for tolerance to benzalkonium chloride and the stress survival islet SSI-2. The CC3, CC7, CC9, CC31 and CC191 carried the SSI-1. All the CC9 and CC121 strains presented a premature stop codon in the inlA gene. In addition to the Lm Pathogenicity Island 1 (LIPI-1), CC1, CC3 and CC191 harboured the LIPI-3. The application of intensive environmental sampling plans for the detection and WGS analysis of Lm isolates could improve surveillance and early detection of outbreaks.

Download Full-text

Hypo- and Hyper-Virulent Listeria monocytogenes Clones Persisting in Two Different Food Processing Plants of Central Italy

Microorganisms ◽

10.3390/microorganisms9020376 ◽

2021 ◽

Vol 9 (2) ◽

pp. 376

Author(s):

Fabrizia Guidi ◽

Massimiliano Orsini ◽

Alexandra Chiaverini ◽

Marina Torresi ◽

Patrizia Centorame ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Food Processing ◽

Stop Codon ◽

Genetic Relationships ◽

Central Italy ◽

Premature Stop Codon ◽

Small Scale ◽

Processing Plant ◽

Cadmium Resistance

A total of 66 Listeria monocytogenes (Lm) isolated from 2013 to 2018 in a small-scale meat processing plant and a dairy facility of Central Italy were studied. Whole Genome Sequencing and bioinformatics analysis were used to assess the genetic relationships between the strains and investigate persistence and virulence abilities. The biofilm forming-ability was assessed in vitro. Cluster analysis grouped the Lm from the meat plant into three main clusters: two of them, both belonging to CC9, persisted for years in the plant and one (CC121) was isolated in the last year of sampling. In the dairy facility, all the strains grouped in a CC2 four-year persistent cluster. All the studied strains carried multidrug efflux-pumps genetic determinants (sugE, mdrl, lde, norM, mepA). CC121 also harbored the Tn6188 specific for tolerance to Benzalkonium Chloride. Only CC9 and CC121 carried a Stress Survival Islet and presented high-level cadmium resistance genes (cadA1C1) carried by different plasmids. They showed a greater biofilm production when compared with CC2. All the CC2 carried a full-length inlA while CC9 and CC121 presented a Premature Stop Codon mutation correlated with less virulence. The hypo-virulent clones CC9 and CC121 appeared the most adapted to food-processing environments; however, even the hyper-virulent clone CC2 warningly persisted for a long time. The identification of the main mechanisms promoting Lm persistence in a specific food processing plant is important to provide recommendations to Food Business Operators (FBOs) in order to remove or reduce resident Lm.

Download Full-text