Listeria monocytogenes Triggers the Cell Surface Expression of Gp96 Protein and Interacts with Its N Terminus to Support Cellular Infection

Listeria monocytogenes is an intracellular food-borne pathogen causing listeriosis in humans. This bacterium deploys an arsenal of virulence factors that act in concert to promote cellular infection. Bacterial surface proteins are of primary importance in the process of host cell invasion. They interact with host cellular receptors, inducing/modulating specific cellular responses. We previously identified Vip, a Listeria surface protein covalently attached to the bacterial cell wall acting as a key virulence factor. We have shown that Vip interacts with Gp96 localized at the surface of host cells during invasion and that this interaction is critical for a successful infection in vivo. To better understand the importance of Vip-Gp96 interaction during infection, we aimed to characterize this interaction at the molecular level. Here we demonstrate that, during infection, L. monocytogenes triggers the cellular redistribution of Gp96, inducing its exposure at the cell surface. Upon infection, Gp96 N-terminal domain is exposed to the extracellular milieu in L2071 fibroblasts and interacts with Vip expressed by Listeria. We identified Gp96 (Asp¹–Leu¹⁷⁰) as sufficient to interact with Vip; however, we also showed that the region Tyr¹⁷⁹–Leu³⁹⁰ of Gp96 is important for the interaction. Our findings unravel the Listeria-induced surface expression of Gp96 and the topology of its insertion on the plasma membrane and improve our knowledge on the Vip-Gp96 interaction during Listeria infection.     

Cell-mediated killing of Listeria monocytogenes by leucocin C producing Escherichia coli

Listeria monocytogenes is a foodborne pathogen causing listeriosis. Listeria in foods can be inhibited with bacteriocins or bacteriocin producing cultures. The aim of this study was to enhance the killing of L. monocytogenes by binding bacteriocin producing Escherichia coli cells to Listeria cells. Antilisterial E. coli was obtained by transferring leucocin C production from Leuconostoc carnosum 4010. For binding of E. coli cells to Listeria cells, the Listeria phage endolysin PlyP35 cell wall binding domain (CBD) was displayed on E. coli cell surface as FliC::CBD chimeric protein in flagella. CBD insertion in flagella was confirmed by Western analysis and enterokinase cleavage. By mixing isolated flagella with L. monocytogenes WSLC 1019 cells, the FliC::CBD flagella was shown to bind to Listeria cells. However, the wild type flagella also attached to Listeria cells masking putative additional binding mediated by the CBD. Yet, the cell-mediated leucocin C killing resulted in two-log reduction of Listeria, whereas the corresponding amount of leucocin C in spent culture medium could only inhibit growth without bacteriocidal effect. Cells binding Listeria and secreting antilisterial peptides may have applications in protection against listeriosis as they kill Listeria better than free antilisterial peptides.     

Deletion of the membrane protein Lmo0412 increases the virulence of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that causes gastroenteritis, meningitis, encephalitis and maternofetal infections. 20–30% of eubacterial ORFs are predicted to encode membrane proteins. The bacterial cytoplasmic membrane is a macromolecular structure, which plays a key role for the pathogenesis. Despite this, little knowledge exists regarding the function of cytoplasmic membrane proteins of Listeria during infection. Here, we investigated a predicted membrane protein of the pathogen L. monocytogenes, Lmo0412, of unknown function. Lmo0412 is only present in the Listeria genus and low conserved in the non-pathogenic species L. innocua. Bacterial fractionation and western blot analyses showed that Lmo0412 was only detectable in the membrane of L. monocytogenes EGDe during logarithmic growth phase. lmo0412 expression in L. monocytogenes was down-regulated during in vitro infection of JEG-3 epithelial cells. An L. monocytogenes mutant deficient in this membrane protein showed increased invasion of Caco-2 and NRK-49F host cells using in vitro infection models. Moreover, the lack of Lmo0412 in this deletion mutant increased the viable bacteria counts in the spleen and liver of mice compared to the wild type strain. Taken together, these data suggest a selective advantage conferred by the absence of Lmo0412 for the virulence of L. monocytogenes.     

Coselection of Cadmium and Benzalkonium Chloride Resistance in Conjugative Transfers from Nonpathogenic Listeria spp. to Other Listeriae

Resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) may be an important contributor to the ability of Listeria spp. to persist in the processing plant environment. Although a plasmid-borne disinfectant resistance cassette (bcrABC) has been identified in Listeria monocytogenes, horizontal transfer of these genes has not been characterized. Nonpathogenic Listeria spp. such as L. innocua and L. welshimeri are more common than L. monocytogenes in food processing environments and may contribute to the dissemination of disinfectant resistance genes in listeriae, including L. monocytogenes. In this study, we investigated conjugative transfer of resistance to BC and to cadmium from nonpathogenic Listeria spp. to other nonpathogenic listeriae, as well as to L. monocytogenes. BC-resistant L. welshimeri and L. innocua harboring bcrABC, along with the cadmium resistance determinant cadA2, were able to transfer resistance to other nonpathogenic listeriae as well as to L. monocytogenes of diverse serotypes, including strains from the 2011 cantaloupe outbreak. Transfer among nonpathogenic Listeria spp. was noticeably higher at 25°C than at 37°C, whereas acquisition of resistance by L. monocytogenes was equally efficient at 25 and 37°C. When the nonpathogenic donors were resistant to both BC and cadmium, acquisition of cadmium resistance was an effective surrogate for transfer of resistance to BC, suggesting coselection between these resistance attributes. The results suggest that nonpathogenic Listeria spp. may behave as reservoirs for disinfectant and heavy metal resistance genes for other listeriae, including the pathogenic species L. monocytogenes.     

In Vivo Transcriptional Profiling of Listeria monocytogenes and Mutagenesis Identify New Virulence Factors Involved in Infection

Listeria monocytogenes is a human intracellular pathogen able to colonize host tissues after ingestion of contaminated food, causing severe invasive infections. In order to gain a better understanding of the nature of host–pathogen interactions, we studied the L. monocytogenes genome expression during mouse infection. In the spleen of infected mice, ≈20% of the Listeria genome is differentially expressed, essentially through gene activation, as compared to exponential growth in rich broth medium. Data presented here show that, during infection, Listeria is in an active multiplication phase, as revealed by the high expression of genes involved in replication, cell division and multiplication. In vivo bacterial growth requires increased expression of genes involved in adaptation of the bacterial metabolism and stress responses, in particular to oxidative stress. Listeria interaction with its host induces cell wall metabolism and surface expression of virulence factors. During infection, L. monocytogenes also activates subversion mechanisms of host defenses, including resistance to cationic peptides, peptidoglycan modifications and release of muramyl peptides. We show that the in vivo differential expression of the Listeria genome is coordinated by a complex regulatory network, with a central role for the PrfA-SigB interplay. In particular, L. monocytogenes up regulates in vivo the two major virulence regulators, PrfA and VirR, and their downstream effectors. Mutagenesis of in vivo induced genes allowed the identification of novel L. monocytogenes virulence factors, including an LPXTG surface protein, suggesting a role for S-layer glycoproteins and for cadmium efflux system in Listeria virulence.     

Isolation and characterization of Listeria monocytogenes from tropical seafood of Kerala, India

Listeria monocytogenes, which is an intracellular pathogen, causes various illnesses in human as well as in animals. The pathogenicity of this organism depends upon the presence of different virulence genes. A total of 324 tropical seafood and fishery environmental samples were screened for L. monocytogenes. The incidence of the human pathogenic species L. monocytogenes was 1.2 % of the samples. Listeria spp. was detected in 32.3, 27.1, and 5 % of fresh, frozen, and dry fish samples, respectively. Listeria innocua was found to be the most prevalent species of Listeria in the tropical seafood and environmental samples of Kerala. Listeria monocytogenes and L. innocua isolates were confirmed by multiplex PCR. L. monocytogenes isolates from the four positive samples showed phosphatidylinositol-specific phospholipase C reaction on Chromocult® Listeria selective agar. Molecular characterization of L. monocytogenes isolates for virulence genes revealed the presence of β-hemolysin (hly), plcA, actA, metalloprotease (mpl), iap and prfA genes in all the isolates recovered from the positive samples.     

Human heat-shock protein 60 receptor-coated paramagnetic beads show improved capture of Listeria monocytogenes in the presence of other Listeria in food

Aims: To investigate the suitability of human Hsp60, a receptor for Listeria adhesion protein (LAP), on paramagnetic beads (PMB) to capture Listeria monocytogenes from food in the presence of other Listeria to facilitate rapid and specific detection of this pathogen. Methods and Results: Commercially available streptavidin‐coated PMBs were linked with biotinylated Hsp60 (PMB‐Hsp60), and the bacterial capture efficiency from pure culture and meat samples was determined. Capture rate was also compared with the monoclonal antibody (MAb)‐C11E9‐coated beads (PMB‐C11E9) and the commercial Dynabeads anti‐Listeria. Captured cells were detected and quantified by plating on selective medium, quantitative real‐time PCR (qPCR) and a light‐scattering sensor. Overall, all ligand‐coated beads had similar capture efficiency (varied from 1·8 to 9·2%) for L. monocytogenes under the conditions employed, and the minimum cell number required to achieve such capture was 10³ CFU ml^?1. PMB‐Hsp60 had significantly greater capture efficiency for pathogenic Listeria (P < 0·0001) than the nonpathogenic Listeria. In contrast, PMB‐C11E9 and Dynabeads anti‐Listeria had similar capture efficiency for both. The efficacy of all PMBs to capture L. monocytogenes in the presence of Listeria innocua from food matrices was compared. Although Dynabeads anti‐Listeria had the overall best capture efficiency, PMB‐Hsp60 was able to selectively capture L. monocytogenes even in the presence of 10–100‐fold more L. innocua cells from enriched meat samples. Conclusions: Data show that the human cell receptor, Hsp60, is suitable for the capture of pathogenic Listeria on PMB in the presence of other Listeria in food. Significance and Impact of the Study: As pathogen interaction with host cells is highly specific, host cell receptors could be used as alternate capture molecules on PMB to aid in specific detection of pathogens.     

Development of a Novel Selective and Differential Medium for the Isolation of Listeria monocytogenes

A new medium (lecithin and levofloxacin [LL] medium) is described for the isolation of Listeria monocytogenes from food samples. LL medium includes lecithin from soybeans for the detection of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) produced by L. monocytogenes. Levofloxacin is incorporated to inhibit the growth of microorganisms other than L. monocytogenes, especially Bacillus cereus, shown to possess PI-PLC and PC-PLC activities. L. monocyogenes produced white colonies with a halo on LL medium, whereas Listeria innocua appeared as white colonies without a halo. Levofloxacin at 0.20 mg/liter completely inhibited the growth of B. cereus, while the growth of L. monocytogenes was unaffected. In the second phase of the study, the sensitivity and the specificity of LL medium were compared to those of modified Oxford agar (MOX) and two chromogenic media (Brilliance Listeria agar and CHROMagar Listeria), using a total of 250 food samples. From 200 unspiked food samples, the specificity of LL medium (96.0%) was superior to that of MOX (72.0%) and similar to the specificities of Brilliance Listeria agar (96.5%) and CHROMagar Listeria (94.5%). From 50 spiked food samples, LL medium and CHROMagar Listeria represented the highest sensitivities (96.0%), followed by Brilliance Listeria agar (92.0%) and MOX (54.0%). Also, LL medium showed the highest confirmation rate (98.8%), followed by Brilliance Listeria agar (98.7%), CHROMagar Listeria (98.3%), and MOX (52.0%). On the basis of its good specificity and cost effectiveness, LL medium is useful for the isolation of L. monocytogenes from food samples.     

Microsporidia Are Natural Intracellular Parasites of the Nematode Caenorhabditis elegans

For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.     

Modeling of Spatially Referenced Environmental and Meteorological Factors Influencing the Probability of Listeria Species Isolation from Natural Environments

Many pathogens have the ability to survive and multiply in abiotic environments, representing a possible reservoir and source of human and animal exposure. Our objective was to develop a methodological framework to study spatially explicit environmental and meteorological factors affecting the probability of pathogen isolation from a location. Isolation of Listeria spp. from the natural environment was used as a model system. Logistic regression and classification tree methods were applied, and their predictive performances were compared. Analyses revealed that precipitation and occurrence of alternating freezing and thawing temperatures prior to sample collection, loam soil, water storage to a soil depth of 50 cm, slope gradient, and cardinal direction to the north are key predictors for isolation of Listeria spp. from a spatial location. Different combinations of factors affected the probability of isolation of Listeria spp. from the soil, vegetation, and water layers of a location, indicating that the three layers represent different ecological niches for Listeria spp. The predictive power of classification trees was comparable to that of logistic regression. However, the former were easier to interpret, making them more appealing for field applications. Our study demonstrates how the analysis of a pathogen''s spatial distribution improves understanding of the predictors of the pathogen''s presence in a particular location and could be used to propose novel control strategies to reduce human and animal environmental exposure.The transmission cycle of many pathogens involves biotic hosts and abiotic environments. After infection of a host with a pathogen like Listeria monocytogenes, Bacillus anthracis, enterohemorrhagic Escherichia coli, Salmonella spp., or Toxoplasma gondii, large numbers of the pathogen may be shed into the environment where, under favorable conditions, they may survive, multiply, and infect new hosts, including humans (6, 11, 13, 30, 37). It is important to identify spatially explicit environmental and meteorological factors that favor a pathogen''s presence in a particular environmental location. That information could be used to design novel measures to reduce the presence of the pathogen in the environment and prevent exposure and infection of animal and human hosts. For analysis of pathogens'' spatial distribution in the environment, geographic information systems (GIS) integrated with standard statistical and epidemiological methods provide tremendous opportunities (5).Detection of pathogens in environmental samples is usually based on culturing methods without enumeration, resulting in presence/absence data. For such data, a standard statistical approach to predict microbial presence as influenced by covariates would be logistic regression (LR). However, classification trees (CT) have recently been suggested as a powerful yet simple alternative to LR in ecological studies (7, 48). It is therefore of interest to contrast the performance of the CT with that of the standard LR approach in predicting pathogen isolation from a spatial location.The objective of this study was to develop a methodological framework to study spatially explicit determinants affecting the local probability of pathogen isolation by using Listeria spp. as a model system. Specifically, our goals were (i) to examine the effect of environmental and meteorological factors on isolation of Listeria spp. from a spatial location and from soil, vegetation, and water layers of a location and (ii) to compare the predictive performance of LR and CT models. The genus Listeria was chosen as a model system because of the convenience of gathering data (Listeria bacteria are relatively prevalent in the environment; they have been isolated from 28% of sampled locations in the natural environment [38]) and because the genus Listeria includes the human-pathogenic species L. monocytogenes. There are currently six species in the genus Listeria, including two known pathogens (L. monocytogenes and Listeria ivanovii) and four nonpathogens (Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi). L. ivanovii is predominantly an animal pathogen, infecting ruminants, while L. monocytogenes can cause disease (listeriosis) in a wide range of animal species, including humans. In addition, a new Listeria species, “Listeria marthii” sp. nov., has recently been identified (L. M. Graves, L. O. Helsel, A. G. Steigerwalt, R. E. Morey, M. I. Daneshvar, S. E. Roof, R. H. Orsi, E. D. Fortes, S. R. Millilo, H. C. den Bakker, M. Wiedmann, B. Swaminathan, and B. D. Sauders, submitted for publication).     

How the interaction of Listeria monocytogenes and Acanthamoeba spp. affects growth and distribution of the food borne pathogen

Listeria monocytogenes is a foodborne opportunistic pathogen capable to switch from an environmental saprophyte to a potentially fatal human pathogen. The fact that the pathogen maintains the genes suitable for an elaborate infectious process indicates that these genes are required to survive in the environment. However, no environmental host reservoir for L. monocytogenes has been identified so far. The similarity of free-living, bacteria-scavenging amoebae to macrophages led to the hypothesis that protozoa may represent the missing link in the ecology and pathology of L. monocytogenes. Consequently, numerous studies have been published reporting on the potential of Acanthamoeba spp. to serve as host for a variety of pathogenic bacteria. However, the data on the interaction of L. monocytogenes with Acanthamoeba spp. are inconsistent and relatively little information on the impact of this interaction on growth and distribution of the foodborne pathogen is currently available. Hence, this review focuses on the interaction of L. monocytogenes and Acanthamoeba spp. affecting survival and growth of the foodborne pathogen in natural and man-made environments, in order to highlight the potential impact of this interplay on food safety and human health.     

A Novel Therapy for Melanoma Developed in Mice: Transformation of Melanoma into Dendritic Cells with Listeria monocytogenes

Listeria monocytogenes is a gram-positive bacteria and human pathogen widely used in cancer immunotherapy because of its capacity to induce a specific cytotoxic T cell response in tumours. This bacterial pathogen strongly induces innate and specific immunity with the potential to overcome tumour induced tolerance and weak immunogenicity. Here, we propose a Listeria based vaccination for melanoma based in its tropism for these tumour cells and its ability to transform in vitro and in vivo melanoma cells into matured and activated dendritic cells with competent microbicidal and antigen processing abilities. This Listeria based vaccination using low doses of the pathogen caused melanoma regression by apoptosis as well as bacterial clearance. Vaccination efficacy is LLO dependent and implies the reduction of LLO-specific CD4⁺ T cell responses, strong stimulation of innate pro-inflammatory immune cells and a prevalence of LLO-specific CD8⁺ T cells involved in tumour regression and Listeria elimination. These results support the use of low doses of pathogenic Listeria as safe melanoma therapeutic vaccines that do not require antibiotics for bacterial removal.     

Inhibitory activity of surfactin,produced by different Bacillus subtilis subsp. subtilis strains,against Listeria monocytogenes sensitive and bacteriocin-resistant strains

Three surfactin-producing Bacillus subtilis strains, C4, M1 and G2III, previously isolated from honey and intestines from the Apis mellifera L. bee, were phylogenetically characterized at sub-species level as B. subtilis subsp. subtilis using gyrA gene sequencing. The antagonistic effect of surfactin was studied against seven different Listeria monocytogenes strains, 6 of which were resistant to bacteriocins. Surfactin showed anti-Listeria activity against all 7 strains and a dose of 0.125 mg/mL of surfactin was enough to inhibit this pathogen. Surfactin sintetized by B. subtilis subsp. subtilis C4 inhibited the pathogen in lower concentrations, 0.125 mg/mL, followed by G2III and M1 with 0.25 and 1 mg/mL, respectively. In particular, a dose of 0.125 mg/mL reduced the viability of L. monocytogenes 99/287 RB6, a bacteriocin-resistant strain, to 5 log orders. Surfactin assayed maintained anti-Listeria activity within a pH range of between 2 and 10, after heat treatment (boiling for 10 min and autoclaving at 121 °C for 15 min) and after treatment with proteolytic enzymes. These results suggest that surfactin can be used as a new tool for prevention and the control of L. monocytogenes in different environments, for example, in the food industry.     

Comparison of the Prevalences and Diversities of Listeria Species and Listeria monocytogenes in an Urban and a Rural Agricultural Watershed

Foods and related processing environments are commonly contaminated with the pathogenic Listeria monocytogenes. To investigate potential environmental reservoirs of Listeria spp. and L. monocytogenes, surface water and point source pollution samples from an urban and a rural municipal water supply watershed in Nova Scotia, Canada, were examined over 18 months. Presumptive Listeria spp. were cultured from 72 and 35% of rural and urban water samples, respectively, with 24% of the positive samples containing two or three different Listeria spp. The L. innocua (56%) and L. welshimeri (43%) groups were predominant in the rural and urban watersheds, respectively. Analysis by the TaqMan assay showed a significantly (P < 0.05) higher prevalence of L. monocytogenes of 62% versus 17% by the culture-based method. Both methods revealed higher prevalences in the rural watershed and during the fall and winter seasons. Elevated Escherichia coli (≥100 CFU/100 ml) levels were not associated with the pathogen regardless of the detection method. Isolation of Listeria spp. were associated with 70 times higher odds of isolating L. monocytogenes (odds ratio = 70; P < 0.001). Serogroup IIa was predominant (67.7%) among the 285 L. monocytogenes isolates, followed by IVb (16.1%), IIb (15.8%), and IIc (0.4%). L. monocytogenes was detected in cow feces and raw sewage but not in septic tank samples. Pulsotyping of representative water (n = 54) and local human (n = 19) isolates suggested genetic similarities among some environmental and human L. monocytogenes isolates. In conclusion, temperate surface waters contain a diverse Listeria species population and could be a potential reservoir for L. monocytogenes, especially in rural agricultural watersheds.     

Plasmids in Listeria

Thirty-two Listeria strains, including L. monocytogenes of the different Seeliger serovars, L. grayi and L. murrayi, have been analyzed for the presence of plasmids. A cryptic plasmid of 38.5 Md was found in seven strains: in four L. monocytogenes (serovars $\text{1}\text{2}\text{c}$, 4b, 4d, and Ivanov 1), and in both the L. grayi and the single L. murrayi strains. Restriction enzyme cleavage analysis with two restriction enzymes suggests that the plasmid is a single molecular species, pRYC16. Conjugation experiments between Streptococcus agalactiae BM6101 containing the resistance plasmid pIP501 and L. monocytogenes, L. grayi, or L. Murrayi demonstrated that pIP501 is transferred to Listeria at a mean frequency of 10^?6. pIP501 is fully expressed in Listeria and promotes its own transfer between strains of Listeria and from Listeria back to Streptococcus. It is suggested that a circulation of plasmids can take place among members of the genera Listeria and Streptococcus.     

Recombinant probiotic expressing Listeria adhesion protein attenuates Listeria monocytogenes virulence in vitro

Background

Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection.

Methodology/Principal Findings

The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (Lbp^LAP) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to Lbp^LAP for 1, 4, 15, or 24 h significantly (P<0.05) reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, Lbp^LAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. Lbp^LAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, Lbp^LAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h.

Conclusions/Significance

Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, Lbp^LAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant probiotics in preventing L. monocytogenes infection in high-risk populations.     

Analysis of the Role of OpuC, an Osmolyte Transport System, in Salt Tolerance and Virulence Potential of Listeria monocytogenes

The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the external environment prior to ingestion and subsequently within the animal host. Growth at high salt concentrations and low temperatures is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We utilized a novel system for generating chromosomal mutations (based on a lactococcal pWVO1-derived Ori⁺ RepA⁻ vector, pORI19) to identify a listerial OpuC homologue. Mutating the operon in two strains of L. monocytogenes revealed significant strain variation in the observed activity of OpuC. Radiolabeled osmolyte uptake studies, together with growth experiments in defined media, linked OpuC to carnitine and glycine betaine uptake in Listeria. We also investigated the role of OpuC in contributing to the growth and survival of Listeria in an animal (murine) model of infection. Altering OpuC resulted in a significant reduction in the ability of Listeria to colonize the upper small intestine and cause subsequent systemic infection following peroral inoculation.     

Isolation and Confirmation of <Emphasis Type="Italic">Listeria</Emphasis> Species from Seafood off Goa Region by Polymerase Chain Reaction

Several food borne outbreaks have highlighted the importance of Listeria monocytogenes to the public health and have been recognized as an emerging, important food borne pathogen, and a causative agent of listerioses. A number of genes are involved in the manifestation of Listeria virulence, hlyA is one among them. In the present study, 111 marine fish samples including prawns, finfishes and bivalves were screened for the presence of Listeria species. The isolates were characterized biochemically and further L. monocytogenes were confirmed by polymerase chain reaction (PCR) technique using the hlyA gene as a tool to differentiate between L. monocytogenes and other non-pathogenic Listeria species. Out of 111 samples 5 (4.5%) samples were positive for L. monocytogenes. Among the three different types of samples bivalves were found to have maximum percent (12.5) of L. monocytogenes followed by prawns (3.84) and finfishes (2.9). Among all the 111 samples, 15 (13.51%) samples were positive for other Listeria species. It was observed that Listeria occurrence is more in shellfishes than in fin fishes. All the isolates were sensitive towards five different antibiotics in sequence ciprofloxacin > sulphafurazole > norfloxacin > ampicillin and gentamicin.     

The Presence of the Internalin Gene in Natural Atypically Hemolytic Listeria innocua Strains Suggests Descent from L. monocytogenes

The atypical hemolytic Listeria innocua strains PRL/NW 15B95 and J1-023 were previously shown to contain gene clusters analogous to the pathogenicity island (LIPI-1) present in the related foodborne gram-positive facultative intracellular pathogen Listeria monocytogenes, which causes listeriosis. LIPI-1 includes the hemolysin gene, thus explaining the hemolytic activity of the atypical L. innocua strains. No other L. monocytogenes-specific virulence genes were found to be present. In order to investigate whether any other specific L. monocytogenes genes could be identified, a global approach using a Listeria biodiversity DNA array was applied. According to the hybridization results, the isolates were defined as L. innocua strains containing LIPI-1. Surprisingly, evidence for the presence of the L. monocytogenes-specific inlA gene, previously thought to be absent, was obtained. The inlA gene codes for the InlA protein which enables bacterial entry into some nonprofessional phagocytic cells. PCR and sequence analysis of this region revealed that the flanking genes of the inlA gene at the upstream, 5′-end region were similar to genes found in L. monocytogenes serotype 4b isolates, whereas the organization of the downstream, 3′-end region was similar to that typical of L. innocua. Sequencing of the inlA region identified a small stretch reminiscent of the inlB gene of L. monocytogenes. The presence of two clusters of L. monocytogenes-specific genes makes it unlikely that PRL/NW 15B95 and J1-023 are L. innocua strains altered by horizontal transfer. It is more likely that they are distinct relics of the evolution of L. innocua from an ancestral L. monocytogenes, as postulated by others.