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PubMed1999

A predictive model to determine the effects of pH, milkfat, and temperature on thermal inactivation of Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen of significance because of its comparatively high heat resistance, zero tolerance in ready-to-eat foods, and growth at refrigeration temperatures. A 3 x 3 x 3 factorial study was done to determine the effects of milkfat (0%, 2.5%, 5.0%), pH (5.0, 6.0, 7.0), and processing temperature (55 degrees C, 60 degrees C, 65 degrees C) on the thermal resistance of L. monocytogenes in a formulated and homogenized milk system. Data were fit to a modified Gompertz equation where parameter estimates characterized three regions of a survival curve: the shoulder, maximum slope, and tail. Statistical analysis was done for each of the 27 individual treatment sets to visualize individual effects on parameter estimates and to evaluate how well the Gompertz equation represented the data. A regression model for the Gompertz equation was generated to predict the logarithmic surviving fraction of L. monocytogenes based on all 27 treatments and their single and interactive effects. The shoulder region of the survival curve was affected by pH; however, the maximum slope was affected by temperature, milkfat, and the interaction of temperature x milkfat. Validation of the model suggests that the predictions are best suited for processing above 62 degrees C. Trends over time for a 4-log reduction in cells (4D values) were evaluated using results from the 27 individual treatment sets, the regression model for the Gompertz equation, and a linear equation. At lower temperatures, 4D values by the three methods varied by twofold. At higher temperatures, all methods gave similar 4D values, suggesting that death became more linear. Based on this study all three factors affect heat resistance for specific regions of a survival curve, and a predictive model was developed that can be used as a preliminary estimate for L. monocytogenes inactivation.

Animals↗

PubMed1991

Early effects of lead on bone marrow cell responsiveness in mice challenged with Listeria monocytogenes.

Listeria monocytogenes challenge of lead-treated mice results in increased mortality. Since macrophage development constitutes the initial phase of the immune response to L. monocytogenes, bone marrow and spleens from Pb-treated mice that were infected with L. monocytogenes were analyzed for their ability to form colonies when exposed to the macrophage growth factor CSF-1. Serum colony-stimulating activity also was evaluated. Data obtained indicate the Pb exposure results in decreased responsiveness of bone marrow and spleen cells to CSF-1 while colony-stimulating activity in serum rises. This lack of bone marrow-derived macrophage development may contribute to the increased mortality observed with L. monocytogenes challenged. Pb-treated mice.

Animals↗

PubMedDate not supplied

Quantitative assessment of hard surface disinfectant activity against the foodborne pathogen Listeria monocytogenes.

Listeria monocytogenes is an important foodborne pathogen that must be controlled to ensure food safety. For the years 2003 and 2004, L. monocytogenes caused 20 deaths per 100 listeriosis cases and was responsible for most food recalls for pathogen contamination. The objective of this work was to develop a quantitative method to assess disinfectant activity against L. monocytogenes. Standard procedures for testing disinfectants against 3 bacteria are described in the AOAC Official Methods of Analysis as use-dilution methods. No standard methods are provided for L. monocytogenes. In this study, preliminary efficacy of a quaternary ammonium compound with hydroperoxide ion was determined for 25 bacterial strains. The zones of inhibition ranged from 7.0 to 12.5 mm, and the minimum inhibitory concentration ranged from 5 to 250 ppm. For final efficacy, stainless steel carriers were contaminated with L. monocytogenes and tested separately for 5, 10, and 15 min in disinfectant or phenol. After exposure, the carriers were placed into 2 series of D/E neutralization broth. For 3 replications with duplicate samples, the phenol coefficient was 3.3. This research presents a technique-sensitive method that provides quantitative data for comparison and analysis of disinfectant activity against L. monocytogenes.

Bacteria↗

PubMed2002

Passive immunization with convalescent serum, or oral immunization with formalin-killed organisms, does not protect mice against gastrointestinal challenge with Listeria monocytogenes.

Listeria monocytogenes is an important foodborne pathogen in both humans and animals. In addition, murine listeriosis is a widely used model for studying the molecular pathogenesis of an intracellular pathogen, and the regulation of protective cellular immunity. Little attention has been paid to protective immunity against L. monocytogenes in the gastrointestinal tract, where a secretory immune response might prevent attachment of the bacteria to the intestinal epithelium. In this study we found that neither opsonization of L. monocytogenes with immune serum, nor repeated oral administration of killed L. monocytogenes, protected mice against gastrointestinal challenge with L. monocytogenes.

Administration, Oral↗

PubMed2003

Sigma(B)-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen that can persist and grow under a wide variety of environmental conditions including low pH and high osmolarity. The alternative sigma factor sigma(B) contributes to L. monocytogenes survival under extreme conditions. The purpose of this study was to identify and confirm specific sigma(B)-dependent genes in L. monocytogenes and to characterize their expression patterns under various stress conditions. opuCA, lmo1421 and bsh were identified as putative sigma(B)-dependent genes based on the presence of a predicted sigma(B)-dependent promoter sequence upstream of each gene. opuCA and lmo1421 encode known and putative compatible solute transporter proteins, respectively, and bsh encodes a conjugated bile salt hydrolase (BSH). Reporter fusions and semi-quantitative RT-PCR techniques were used to confirm sigma(B)-dependent regulation of these stress-response genes and to determine their expression patterns in response to environmental stresses. RT-PCR demonstrated that opuCA, lmo1421 and bsh transcript levels are reduced in stationary-phase L. monocytogenes deltasigB cells relative to levels present in wild-type cells. Furthermore, BSH activity is abolished in a L. monocytogenes deltasigB strain. RT-PCR confirmed growth-phase-dependent expression of opuCA, with highest levels of expression in stationary-phase cells. The L. monocytogenes wild-type strain exhibited two- and threefold induction of opuCA expression and seven- and fivefold induction of lmo1421 expression following 10 and 15 min exposure to 0.5 M KCl, respectively, as determined by RT-PCR, suggesting rapid induction of sigma(B) activity in exponential-phase L. monocytogenes upon exposure to salt stress. Single-copy chromosomal opuCA-gus reporter fusions also showed significant induction of opuCA expression following exposure of exponential-phase cells to increased salt concentrations (0.5 M NaCl or 0.5 M KCl). In conjunction with recent findings that indicate a role for opuCA and bsh in L. monocytogenes virulence, the data presented here provide further evidence of specific sigma(B)-mediated contributions to both environmental stress resistance and intra-host survival in L. monocytogenes.

ATP-Binding Cassette Transporters↗

PubMed1997

Host-pathogen interactions during entry and actin-based movement of Listeria monocytogenes.

Listeria monocytogenes is a pathogenic bacterium that induces its own uptake into mammalian cells, and spreads from one cell to another by an actin-based motility process. Entry into host cells involves the bacterial surface proteins InlA (internalin) and InlB. The receptor for InlA is the cell adhesion molecule E-cadherin. InlB-mediated entry requires activation of the host protein phosphoinositide (PI) 3-kinase, probably in response to engagement of a receptor. Actin-based movement of L. monocytogenes is mediated by the bacterial surface protein ActA. The N-terminal region of this protein is necessary and sufficient for polymerization of host cell actin. Other host proteins involved in bacterial motility include profilin, Vasodilator-Stimulated Phosphoprotein (VASP), the Arp2/Arp3 complex, and cofilin. Studies of entry and intracellular movement of L. monocytogenes could lead to a better understanding of receptor-ligand signaling and dynamics of actin polymerization in mammalian cells.

Actins↗

PubMed1986

Synthesis and secretion of interferon by murine fibroblasts in response to intracellular Listeria monocytogenes.

Listeria monocytogenes, a gram-positive facultative intracellular bacterium, was shown to be capable of infecting and proliferating in murine embryo fibroblasts. During exponential proliferation, the doubling time of the bacterium was determined to be 2.5 h intracellularly, compared with 25 min extracellularly. Progressive intracellular growth of listeriae ultimately resulted in the destruction of initially infected cells and the spread of infection to neighboring cells. Listeria infection induced fibroblasts to synthesize considerable quantities of an acid-stable interferon that proved to be antigenically indistinguishable from both polyinosinic-polycytidylic acid-induced and virus-induced interferon.

Animals↗

PubMed1995

Utilization of exogenous siderophores and natural catechols by Listeria monocytogenes.

Listeria monocytogenes does not produce siderophores for iron acquisition. We demonstrate that a number of microbial siderophores and natural iron-binding compounds are able to promote the growth of iron-starved L. monocytogenes. We suggest that the ability of L. monocytogenes to use a variety of exogenous siderophores and natural catechols accounts for its ubiquitous character.

Animals↗

PubMed2000

Listeriolysin O as a reporter to identify constitutive and in vivo-inducible promoters in the pathogen Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular gram-positive bacterium capable of growing in the cytoplasm of infected host cells. Bacterial escape from the phagosomal vacuole of infected cells is mainly mediated by the pore-forming hemolysin listeriolysin O (LLO) encoded by hly. LLO-negative mutants of L. monocytogenes are avirulent in the mouse model. We have developed a genetic system with hly as a reporter gene allowing the identification of both constitutive and in vivo-inducible promoters of this pathogen. Genomic libraries were created by randomly inserting L. monocytogenes chromosomal fragments upstream of the promoterless hly gene cloned into gram-positive and gram-negative shuttle vectors and expressed in an LLO-negative mutant strain. With this hly-based promoter trap system, combined with access to the L. monocytogenes genome database, we identified 20 in vitro-transcribed genes, including genes encoding (i) p60, a previously known virulence gene, (ii) a putative new hemolysin, and (iii) two proteins of the general protein secretion pathway. By using the hly-based system as an in vivo expression technology tool, nine in vivo-induced loci of L. monocytogenes were identified, including genes encoding (i) the previously known in vivo-inducible phosphatidylinositol phospholipase C and (ii) a putative N-acetylglucosamine epimerase, possibly involved in teichoic acid biosynthesis. The use of hly as a reporter is a simple and powerful alternative to classical methods for transcriptional analysis to monitor promoter activity in L. monocytogenes.

Amino Acid Sequence↗

PubMed2000

Electron paramagnetic resonance studies of the membrane fluidity of the foodborne pathogenic psychrotroph Listeria monocytogenes.

Listeria monocytogenes is a foodborne psychrotrophic pathogen that grows at refrigeration temperatures. Previous studies of fatty acid profiles of wild-type and cold-sensitive, branched-chain fatty acid deficient mutants of L. monocytogenes suggest that the fatty acid 12-methyltetradecanoic (anteiso-C(15:0)) plays a critical role in low-temperature growth of L. monocytogenes, presumably by maintaining membrane fluidity. The fluidity of isolated cytoplasmic membranes of wild-type (SLCC53 and 10403S), and a cold-sensitive mutant (cld-1) of L. monocytogenes, grown with and without the supplementation of 2-methylbutyric acid, has been studied using a panel of hydrocarbon-based nitroxides (2N10, 3N10, 4N10, and 5N10) and spectral deconvolution and simulation methods to obtain directly the Lorentzian line widths and hence rotational correlation times (tau(c)) and motional anisotropies of the nitroxides in the fast motional region. tau(c) values over the temperature range of -7 degrees C to 50 degrees C were similar for the membranes of strains SLCC53 and 10403S grown at 10 degrees C and 30 degrees C, and for strain cld-1 grown with 2-methylbutyric acid supplementation (which restores branched-chain fatty acids) at 30 degrees C. However, strain cld-1 exhibited a threefold higher tau(c) when grown without 2-methylbutyric acid supplementation (deficient in branched-chain fatty acids) compared to strains SLCC53, 10403S, and supplemented cld-1. No evidence was seen for a clear lipid phase transition in any sample. We conclude that the fatty acid anteiso-C(15:0) imparts an essential fluidity to the L. monocytogenes membrane that permits growth at refrigeration temperatures.

Anisotropy↗

PubMed2006

The response regulator ResD modulates virulence gene expression in response to carbohydrates in Listeria monocytogenes.

Listeria monocytogenes is a versatile bacterial pathogen that is able to accommodate to diverse environmental and host conditions. Presently, we have identified a L. monocytogenes two-component response regulator, ResD that is required for the repression of virulence gene expression known to occur in the presence of easily fermentable carbohydrates not found inside host organisms. Structurally and functionally, ResD resembles the respiration regulator ResD in Bacillus subtilis as deletion of the L. monocytogenes resD reduces respiration and expression of cydA, encoding a subunit of cytochrome bd. The resD mutation also reduces expression of mptA encoding the EIIABman component of a mannose/glucose-specific PTS system, indicating that ResD controls sugar uptake. This notion was supported by the poor growth of resD mutant cells that was alleviated by excess of selected carbohydrates. Despite the growth deficient phenotype of the mutant in vitro the mutation did not affect intracellular multiplication in epithelial or macrophage cell lines. When examining virulence gene expression we observed traditional induction by charcoal in both mutant and wild-type cells whereas the repression observed in wild-type cells by fermentable carbohydrates did not occur in resD mutant cells. Thus, ResD is a central regulator of L. monocytogenes when present in the external environment.

Animals↗

PubMed2003

Antibodies present in normal human serum inhibit invasion of human brain microvascular endothelial cells by Listeria monocytogenes.

Listeria monocytogenes causes meningitis and encephalitis in humans and crosses the blood-brain barrier by yet unknown mechanisms. The interaction of the bacteria with different types of endothelial cells was recently analyzed, and it was shown that invasion into, but not adhesion to, human brain microvascular endothelial cells (HBMEC) depends on the product of the inlB gene, the surface molecule InlB, which is a member of the internalin multigene family. In the present study we analyzed the role of the medium composition in the interaction of L. monocytogenes with HBMEC, and we show that invasion of HBMEC is strongly inhibited in the presence of adult human serum. The strong inhibitory activity, which is not present in fetal calf serum, does not inhibit uptake by macrophage-like J774 cells but does also inhibit invasion of Caco-2 epithelial cells. The inhibitory component of human serum was identified as being associated with L. monocytogenes-specific antibodies present in the human serum. Human newborn serum (cord serum) shows only a weak inhibitory activity on the invasion of HBMEC by L. monocytogenes.

Animals↗

PubMed2000

Osmoprotectants and cryoprotectants for Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen that can grow in high osmotic strength environments and at refrigeration temperatures. Glycine betaine, proline betaine, acetylcarnitine, carnitine, gamma-butyrobetaine and 3-dimethylsulphoniopropionate all acted as osmoprotectants, as evidenced by an increase in growth rate of L. monocytogenes 10403S and Scott A when provided with these compounds, while being stressed in defined medium containing 0.7 M NaCl. These same compounds exhibited cryoprotective activity, as evidenced by increasing the growth rate of L. monocytogenes at 5 degrees C. Ectoine, hydroxy ectoine, pipecolic acid and proline were ineffective as osmoprotectants or cryoprotectants under these conditions. The presence of osmoprotectants and cryoprotectants in foods may provide compounds assisting L. monocytogenes to overcome the barriers of high osmotic strength and low temperature that otherwise control microbial growth.

Acetylcarnitine↗

PubMed2004

Use of monoclonal antibodies that recognize p60 for identification of Listeria monocytogenes.

Listeria monocytogenes causes major food-borne outbreaks of disease worldwide. Specific identification of this microorganism is of utmost importance to public health and industry. Listeria species are known to secrete a 60-kDa protein collectively termed p60, which is encoded by the iap (invasion-associated protein) gene and secreted in large quantities into the growth media. p60 is a highly immunogenic murein hydrolase that is essential for cell division. Due to these properties, p60 is an ideal diagnostic target for the development of immunological detection systems for L. monocytogenes. We report here two independent lines of monoclonal antibody (MAb): p6007, which specifically recognizes L. monocytogenes p60, and p6017, which reacts with a wide range of Listeria p60 proteins. By combining these antibodies with a polyclonal antibody, we developed efficient sandwich enzyme-linked immunosorbent assay (ELISA) systems which can specifically identify L. monocytogenes or generally detect Listeria species. Since an excess amount of the peptide corresponding to PepA or PepD did not interfere with the ELISA, and direct ELISAs were unable to detect both peptides, we concluded that the epitope presumed to be recognized by p6007 or p6017 could be distinguished from PepA and PepD as described by Bubert et al. (Appl. Environ. Microbiol. 60:3120-3127, 1997). To our best knowledge, this is the first example of an immunological identification system that uses p60-recognizing MAbs.

Animals↗

PubMed1996

Effect of medium-chain fatty acids in mould ripened cheese on the growth of Listeria monocytogenes.

Listeria monocytogenes and List. innocua were isolated from commercial soft ripened and blue-veined cheeses manufactured in France, mainly from Brie cheese made from unpasteurized milk. Five isolates were List. monocytogenes serotype 1/2 and two were List. innocua. Examination of Bleu d'Auvergne cheese with the cryoscanning electron microscope showed that many conidia spores were present in the blue veins in close contact with the cheese surface. There were few conidia spores in the Brie, mostly on the outside of the cheese but not in contact with the surface. High concentrations of free dodecanoic (lauric) acid (1.77-2.50 g/kg cheese) and tetradecanoic (myristic) acid (2.54-6.38 g/kg cheese) were found in the veins of the blue cheese, but concentrations in the white regions were much lower. Free lauric and myristic acids were not detected in the Brie cheeses. There was no difference in the overall fatty acid composition of the fat in the surface ripened and blue-veined cheeses, although higher concentrations of free medium-chain fatty acids were found in a blue cheese compared with a surface ripened cheese. The pH and fat content were higher in regions with obvious fungal growth, the blue veins of Fourme d'Ambert and the rind of Brie. Free lauric acid dissolved in butteroil inhibited multiplication in broth at pH 7.0 of a test strain of List. monocytogenes isolated from Bleu d'Auvergne. Some inhibition was seen with hexanoic, octanoic, decanoic and tetradecanoic acids. We suggest that the presence of localized concentrations of free medium-chain fatty acids (dissolved in the fat) in the blue veins of blue mould ripened cheese could act as natural preservatives and inhibit the growth of listerias in conditions where (if present), one would otherwise expect them to grow.

Cheese↗

PubMed2006

Regulated translation of listeriolysin O controls virulence of Listeria monocytogenes.

Listeria monocytogenes is an intracytosolic bacterial pathogen that escapes from the phagosome using a secreted cytolysin, listeriolysin O (LLO). In the host cytosol, LLO activity is minimized to prevent pore formation in the host plasma membrane; premature lysis of the infected host cell exposes the bacteria to extracellular immune defences of the host and is detrimental to infection. Here we identified nucleotide substitutions in the coding sequence of the LLO gene (hly) that did not alter the protein sequence, yet caused over-production of LLO, cytotoxicity and loss of virulence. These phenotypes were independent of the promoter and, under conditions in which the mutants produced more LLO protein than wild type, levels of hly mRNA were similar. Finally, negative regulation of LLO was maintained even when bacteria were engineered to produce elevated levels of the wild-type hly transcript. Together, our data demonstrate that translational regulation of LLO is critical for L. monocytogenes pathogenesis.

Animals↗

PubMed2004

Intraspecific phylogeny and lineage group identification based on the prfA virulence gene cluster of Listeria monocytogenes.

Listeria monocytogenes is a serious food-borne pathogen that can cause invasive disease in humans and other animals and has been the leading cause of food recalls due to microbiological concerns in recent years. In order to test hypotheses regarding L. monocytogenes lineage composition, evolution, ecology, and taxonomy, a robust intraspecific phylogeny was developed based on prfA virulence gene cluster sequences from 113 L. monocytogenes isolates. The results of the multigene phylogenetic analyses confirm that L. monocytogenes comprises at least three evolutionary lineages, demonstrate that lineages most frequently (lineage 1) and least frequently (lineage 3) associated with human listeriosis are sister-groups, and reveal for the first time that the human epidemic associated serotype 4b is prevalent among strains from lineage 1 and lineage 3. In addition, a PCR-based test for lineage identification was developed and used in a survey of food products demonstrating that the low frequency of association between lineage 3 isolates and human listeriosis cases likely reflects rarity of exposure and not reduced virulence for humans as has been previously suggested. However, prevalence data do suggest lineage 3 isolates may be better adapted to the animal production environment than the food-processing environment. Finally, analyses of haplotype diversity indicate that lineage 1 has experienced a purge of genetic variation that was not observed in the other lineages, suggesting that the three L. monocytogenes lineages may represent distinct species within the framework of the cohesion species concept.

Animals↗

PubMed2000

Role of listeriolysin O in cell-to-cell spread of Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a host vacuolar compartment and grows rapidly in the cytosol. Listeriolysin O (LLO) is a secreted pore-forming protein essential for the escape of L. monocytogenes from the vacuole formed upon initial internalization. However, its role in intracellular growth and cell-to-cell spread events has not been testable by a genetic approach. In this study, purified six-His-tagged LLO (HisLLO) was noncovalently coupled to the surface of nickel-treated LLO-negative mutants. Bound LLO mediated vacuolar escape in approximately 2% of the mutants. After 5.5 h of growth, cytosolic bacteria were indistinguishable from wild-type bacteria with regard to formation of pseudopod-like extensions, here termed listeriopods, and spread to adjacent cells. However, bacteria in adjacent cells failed to multiply and were found in double-membrane vacuoles. Addition of bound LLO to mutants lacking LLO and two distinct phospholipases C (PLCs) also resulted in spread to adjacent cells, but these triple mutants became trapped in multiple-membrane vacuoles that are reminiscent of autophagocytic vacuoles. These studies show that neither LLO nor the PLCs are necessary for listeriopod formation and uptake of bacteria into neighboring cells but that LLO is required for the escape of L. monocytogenes from the double-membrane vacuole that forms upon cell-to-cell spread.

Animals↗