Influence of preadsorbed milk proteins on adhesion of Listeria monocytogenes to hydrophobic and hydrophilic silica surfaces.

The adsorption of beta-lactoglobulin, bovine serum albumin, alpha-lactalbumin, and beta-casein for 8 h and beta-lactoglobulin and bovine serum albumin for 1 h at silanized silica surfaces of low and high hydrophobicity, followed by incubation in buffer and contact with Listeria monocytogenes, resulted in different numbers of cells adhered per unit of surface area. Adhesion to both surfaces was greatest when beta-lactoglobulin was present and was lowest when bovine serum albumin was present. Preadsorption of alpha-lactalbumin and beta-casein showed an intermediate effect on cell adhesion. Adsorption of beta-lactoglobulin for 1 h resulted in a generally lower number of cells adhered compared with the 8-h adsorption time, while the opposite result was observed with respect to bovine serum albumin. The adhesion data were explainable in terms of the relative rates of arrival to the surface and postadsorptive conformational change among the proteins, in addition to the extent of surface coverage in each case.     

Postadaptational resistance to benzalkonium chloride and subsequent physicochemical modifications of Listeria monocytogenes

Many studies have demonstrated that bacteria, including Listeria monocytogenes, are capable of adapting to disinfectants used in industrial settings after prolonged exposure to sublethal concentrations. However, the consequent alterations of the cell surface due to sanitizer adaptation of this pathogen are not fully understood. Two resistant and four sensitive L. monocytogenes strains from different sources were progressively subcultured with increasing sublethal concentrations of a surfactant, benzalkonium chloride (BC). To evaluate the effects of acquired tolerance to BC, parent and adapted strains were compared by using several morphological and physiological tests. Sensitive strains showed at least a fivefold increase in the MIC, while the MIC doubled for resistant strains after the adaptation period. The hydrophobicities of cells of parent and adapted strains were similar. Serological testing indicated that antigen types 1 and 4 were both present on the cell surface of adapted cells. The data suggest that efflux pumps are the major mechanism of adaptation in sensitive strains and are less important in originally resistant isolates. A different, unknown mechanism was responsible for the original tolerance of resistant isolates. In an originally resistant strain, there was a slight shift in the fatty acid profile after adaptation, whereas sensitive strains had similar profiles. Electron micrographs revealed morphological differences after adaptation. The changes in cell surface antigens, efflux pump utilization, and fatty acid profiles suggest that different mechanisms are used by resistant and sensitive strains for adaptation to BC.     

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.     

Limitation of adhesion and growth of Listeria monocytogenes on stainless steel surfaces by Staphylococcus sciuri biofilms

The adhesion and subsequent development of Listeria monocytogenes on stainless steel was studied in the absence and in the presence of a Staphylococcus sciuri biofilm. In the three growth media studied, the percentage of adherent cells was reduced to nearly the same extent by the presence of 1-day biofilms of Staph. sciuri for the two strains of L. monocytogenes studied. One-day biofilms of Staph. sciuri exhibited the same exopolysaccharide content per square centimetre, although they colonized from 3.5 to 35% of the stainless steel depending on the growth media. This suggests that extracellular substances rather than cell-to-cell interactions were involved in the decreased adhesion. After 3 days of culture, Staphylococcus biofilms prevented the adherent L. monocytogenes population from increasing within the biofilm, leading to an average logarithmic cfu difference of 0.9-2.7 between the pure and mixed culture. A competition for nutrients by Staph. sciuri was observed in one of the three media. A role for extracellular polysaccharides produced by the Staphylococcus biofilm in preventing the adhesion of L. monocytogenes and in modifying the balance existing between its planktonic and biofilm phase is hypothesized. A higher proportion of L. monocytogenes cells was observed in the planktonic phase in mixed cultures, suggesting that the extracellular substances produced by Staph sciuri biofilms and involved in the decreased adhesion of L. monocytogenes could modify the balance existing between planktonic and biofilm populations. In addition, co-cultures of L. monocytogenes and Staph. sciuri in broth showed competition for nutrients for Staph. sciuri in one of the three media.     

Characterization of Listeria monocytogenes strains involved in invasive and noninvasive listeriosis outbreaks by PCR-based fingerprinting techniques

A total of 32 Listeria monocytogenes strains (16 from a recent outbreak of invasive listeriosis and 16 from two outbreaks of noninvasive listeriosis, all three occurring in Italy) were characterized by PCR-ribotyping, arbitrarily primed PCR (AP-PCR), and the recently developed infrequent-restriction-site PCR (IRS-PCR). The discriminatory ability of the techniques, first evaluated on 29 unrelated L. monocytogenes food isolates using Simpson's index of diversity, was 0.714 for PCR-ribotyping, 0.690 for AP-PCR, and 0.919 for IRS-PCR. IRS-PCR was also more capable of distinguishing among strains from the invasive listeriosis outbreak: three different clusters were identified by IRS-PCR compared to two clusters identified by both PCR-ribotyping and AP-PCR. Within each of the two outbreaks of noninvasive listeriosis, the patterns were practically identical, as demonstrated by all three techniques. Only IRS-PCR succeeded in clearly discriminating the strains related to noninvasive listeriosis from all of the other strains included in this study, including those from the outbreak of invasive listeriosis. This finding may suggest the presence of unique differences in their DNA sequences.     

Identification of Listeria monocytogenes genes involved in salt and alkaline-pH tolerance

The capacity of Listeria monocytogenes to tolerate salt and alkaline stresses is of particular importance, as this pathogen is often exposed to such environments during food processing and food preservation. We screened a library of Tn917-lacZ insertional mutants in order to identify genes involved in salt and/or alkaline tolerance. We isolated six mutants sensitive to salt stress and 12 mutants sensitive to salt and alkaline stresses. The position of the insertion of the transposon was located in 15 of these mutants. In six mutants the transposon was inserted in intergenic regions, and in nine mutants it was inserted in genes. Most of the genes have unknown functions, but sequence comparisons indicated that they encode putative transporters.     

单核细胞增生李斯特菌单克隆抗体的制备与鉴定

以单核细胞增生李斯特菌细胞碎片免疫BALB/c小鼠,间接ELISA法成功筛选获得2株稳定分泌抗LM的单克隆杂交瘤细胞株4A7、4H11.抗体效价为1∶160 000以及1∶20 000,亚型为IgG1、IgG2a,Dot-ELISA结果表明4A7和4H11单克隆抗体具有很好的属特异性,Western blot分析表明4A7、4H11抗体分别与单核细胞增生李斯特菌62 kDa以及32 kDa外膜蛋白抗原表位结合,胶体金免疫电镜实验进一步确证以上抗体可有效识别单核细胞增生李斯特菌细胞表面抗原.     

Characterization of the calcium-binding sites of Listeria monocytogenes InlB

The Listeria monocytogenes protein InlB promotes invasion of mammalian cells through activation of the receptor tyrosine kinase Met. The InlB N-cap, a approximately 40 residue part of the domain that binds Met, was previously observed to bind two calcium ions in a novel and unusually exposed manner. Because subsequent work raised questions about the existence of these calcium-binding sites, we assayed calcium binding in solution to the InlB N-cap. We show that calcium ions are bound with dissociation constants in the low micromolar range at the two identified sites, and that the sites interact with one another. We demonstrate that the calcium ions are not required for structure, and also find that they have no appreciable effect on Met activation or intracellular invasion. Therefore, our results indicate that the sites are fortuitous in InlB, but also suggest that the simple architecture of the sites may be adaptable for protein engineering purposes.     

Characterization of Listeria monocytogenes isolates by esterase electrophoresis.

Multiple bands of alpha-naphthyl-propionyl esterase (alpha NPE) activity observed following starch gel electrophoresis of cell extracts allowed 219 Listeria monocytogenes isolates from milk, nondairy foods, and clinical and veterinary sources to be assigned to 17 different alpha NPE types. Multilocus enzyme electrophoresis (MEE) analysis was used to obtain electrophoretic mobility data for alpha NPEs and nine other metabolic enzymes; on the basis of these data, the 219 strains were assigned to 59 electrophoretic types. Each of the methods separated the strains into two main groups, and there was extensive agreement on assignment of the strains to the groups. Although the method is less discriminatory than MEE, the usefulness of alpha NPE electrophoresis alone as a rapid, simple typing method for L. monocytogenes for certain purposes is indicated by agreement among alpha NPE, MEE, and restriction fragment length polymorphism types as molecular markers for persistent and transient L. monocytogenes strains found in sequential raw-milk and nondairy food samples obtained from different processors.     

Morphological and Physiological Characterization of Listeria monocytogenes Subjected to High Hydrostatic Pressure

High hydrostatic pressure is a new food preservation technology known for its capacity to inactivate spoilage and pathogenic microorganisms. That inactivation is usually assessed by the number of colonies growing on solid media after treatment. Under normal conditions the method does not permit recovery of damaged cells and may underestimate the number of cells that will remain viable and grow after a few days in high-pressure-processed foodstuffs. This study investigated the damage inflicted on Listeria monocytogenes cells treated by high pressure for 10 min at 400 MPa in pH 5.6 citrate buffer. Under these conditions, no cell growth occurred after 48 h on plate count agar. Scanning electron microscopy, light scattering by flow cytometry, and cell volume measurements were compared to evaluate the morphological changes in cells after pressurization. All these methods revealed that cellular morphology was not really affected. Esterase activity, as assessed either by enzymatic activity assays or by carboxy fluorescein diacetate fluorescence monitored by flow cytometry, was dramatically lowered, but not totally obliterated, under the effects of treatment. The measurement of propidium iodide uptake followed by flow cytometry demonstrated that membrane integrity was preserved in a small part of the population, although the membrane potential measured by analytical methods or evaluated by oxonol uptake was reduced from −86 to −5 mV. These results showed that such combined methods as fluorescent dyes monitored by flow cytometry and physiological activity measurements provide valuable indications of cellular viability.     

Immune responses to Listeria monocytogenes

Listeria monocytogenes is a Gram-positive bacterium that is often used to study the mammalian immune response to infection because it is easy to culture, is relatively safe to work with and causes a highly predictable infection in laboratory mice. The broad application of this mouse model has resulted in a torrent of studies characterizing the contributions of different cytokines, receptors, adaptors and effector molecules to resistance against infection with Listeria monocytogenes. These studies, which are yielding one of the most comprehensive pictures of the 'battle' between host and microorganism, are reviewed here.     

Interaction of Listeria monocytogenes autolysin amidase with glycosaminoglycans promotes listerial adhesion to mouse hepatocytes

Adherence to the cell surface is a key event during infection of pathogenic microorganisms. We have previously reported that autolysin amidase (Ami) of Listeria monocytogenes promotes an efficient listerial adherence to mouse hepatocytes and requires for listerial pathogenicity. Cell wall anchoring (CWA) domain of Ami has been shown to bind lipoteichoic acid on listerial cell wall but the binding of Ami to host cell surface molecules remains to be determined. In this study, we present evidence here that Ami promotes efficient adherence of L. monocytogenes to mouse hepatocytes mediated by glycosaminoglycans (GAGs). The adhesion of L. monocytogenes wild type but not Ami-deficient mutant to the hepatocytes was dramatically attenuated by 4-nitrophenyl-β-D-xylopyranoside, a specific inhibitor of GAG association to cell surface. Full-length and truncated Ami were used to investigate the binding of Ami to GAGs and we found that four-repeated CWA of Ami is sufficient to bind GAGs on the host cell surface. Competitive assay and surface plasmon resonance demonstrated that Ami interacts with sulfated GAGs but not non-sulfated GAGs. The results suggest that Ami acts as an adhesin of L. monocytogenes to hepatocytes by interaction via its four-repeated CWA domain and sulfated GAGs.     

Neuroinfections due to Listeria monocytogenes

Listeria monocytogenes is not a rare pathogen causing meningitis, mainly in small children and in close contacts to livestock. The pathogen is naturally resistant to cephalosporins and some glycopeptides as well, therefore despite of syndromologic diagnosis of meningitis and initial therapy with 3rd generation cephalosporins according to the guidelines therapeutic failures with clinical consequences may occur.     

Identification of the Listeria monocytogenes virulence factors involved in the CAMP reaction

There is a need to identify the virulence factors involved in the synergistic lysis of erythrocytes (CAMP reaction) by Listeria monocytogenes and either Staphylococcus aureus or Corynebacterium equi , in order to assess the relationship between the CAMP reaction and virulence of L. monocytogenes . The ability of various L. monocytogenes mutants to secrete listeriolysin O and phospholipases, and to produce lysis of sheep blood agar was determined. The results suggest that the CAMP reaction with Coryne. equi involves listeriolysin O and Coryne. equi cholesterol oxidase, and that the reaction with Staph. aureus involves either of the phospholipases C produced by L. monocytogenes . A modified CAMP test, which incorporates cholesterol oxidase into sheep blood agar, is proposed for the rapid (4–6 h) identification of L. monocytogenes.     

Characterization of iap gene in Listeria monocytogenes strains isolated in Japan

Variation of the iap gene region (407bp) encoding an invasion-associated protein p60 was studied on 12 strains of Listeria monocytogenes of different origin in Japan. These 12 strains are known to have 2 types of serotype (1/2a and 4b) and have a diversity among the strains (Saito et al., 1998). The dye-primer cycle sequencing method was employed to determine the genomic structure, and the nucleotide sequences obtained were compared with those of reference strain SV 1/2a EGD. Differences found in the nucleotides were as follows; point mutations of 33 variations in 32 places; an insertion and 3 deletions of 3 bases; AAT position (po.) 1282-1283, and GCA po. 1307-1309, ACA po. 1412-1414, AAT po. 1439-1444, respectively. Different repeating numbers by 6 base unit, ACA AAT, were also found in the tandem repeat region (po. 1394-1423). Classification of 12 strains was attempted, then 8, 4 and 5 types were obtained from the point mutations, the insertions and deletions, and the repeating numbers, respectively. Consequently, 8 patterns were profiled regardless of each serotype. From these results, genomic structures were partially clarified in the iap gene 407bp of L. monocytogenes isolated in Japan. Then, the possibility of detailed epidemiology for L. monocytogenes infection using a combination of serotype and genome structure was suggested because of the previous polymorphism thought to be due to the nucleotide differences in the region.     

Participation of DnaK in expression of genes involved in virulence of Listeria monocytogenes

The 16S-23S rDNA internal transcribed spacer regions of the acetic acid bacteria were sequenced and evaluated for molecular identification of these bacteria. All the sequenced spacers contained genes for tRNA(Ile) and tRNA(Ala), and the antitermination element. The sequences revealed 56.8-78.3% similarity. By PCR amplification of the spacers from 57 strains of acetic acid bacteria, single products of similar sizes were produced. Digestion of the spacers by HaeIII and HpaII restriction enzymes resulted in 12 distinct groups of restriction types. All the restriction profiles obtained after analysis of microbial populations from vinegar matched one of the 12 groups.