A mariner-based transposition system for Listeria monocytogenes

In this study, we developed a new mariner-based transposition system for Listeria monocytogenes. The mariner-based system has a high rate of transposition and a low rate of plasmid retention, and transposition is very random, making it an ideal tool for high-throughput transposon mutagenesis in L. monocytogenes.     

Listeriolysin O: a key protein of Listeria monocytogenes with multiple functions

Cholesterol-dependent cytolysins (CDCs) are produced by a large number of pathogenic Gram-positive bacteria. Most of these single-chain proteins are secreted in the extracellular medium. Among the species producing CDCs, only two species belonging to the genus Listeria (Listeria monocytogenes and Listeria ivanovii) are able to multiply intracellularly and release their toxins in the phagosomal compartment of the infected host cell. This review provides an updated overview on the importance of listeriolysin O (LLO) in the pathogenicity of L. monocytogenes, focusing mainly on two aspects: (1) the structure-function relationship of LLO and (2) its role in intra- and extracellular signalling. We first examine the specific sequence determinants, or protein domains, that make this cytolysin so well adapted to the intracellular lifestyle of L. monocytogenes. The roles that LLO has in cellular signalling events in the context of relations to pathogenesis are also discussed.     

Physiological and biochemical studies on psychrotolerance in Listeria monocytogenes

Studies were undertaken to explain the ability of Listeria monocytogenes to grow at low temperatures in a chemostat. It was found that when grown in continuous culture at a dilution rate of 0·02 h⁻¹ L. monocytogenes had a lower proportion of anteiso -17:0, and a higher proportion of anteiso -15:0, and smaller chain fatty acids when grown at 10°C compared to 30°C. A previously unreported glycolipid was only seen after growth at low temperature. Growth temperature had no effect on the rate of glucose uptake.     

Construction,characterization, and use of two Listeria monocytogenes site-specific phage integration vectors

Two site-specific shuttle integration vectors were developed with two different chromosomal bacteriophage integration sites to facilitate strain construction in Listeria monocytogenes. The first vector, pPL1, utilizes the listeriophage U153 integrase and attachment site within the comK gene for chromosomal insertion. pPL1 contains a useful polylinker, can be directly conjugated from Escherichia coli into L. monocytogenes, forms stable, single-copy integrants at a frequency of approximately 10(-4) per donor cell, and can be used in the L. monocytogenes 1/2 and 4b serogroups. Methods for curing endogenous prophages from the comK attachment site in 10403S-derived strains were developed. pPL1 was used to introduce the hly and actA genes at comK-attBB' in deletion strains derived from 10403S and SLCC-5764. These strains were tested for second-site complementation in hemolysin assays, plaquing assays, and cell extract motility assays. Unlike plasmid-complemented strains, integrated pPL1-complemented strains were fully virulent in the mouse 50% lethal dose assay. Additionally, the PSA phage attachment site on the L. monocytogenes chromosome was characterized, and pPL1 was modified to integrate at this site. The listeriophage PSA integrates in the 3' end of an arginine tRNA gene. There are 17 bp of DNA identity between the bacterial and phage attachment sites. The PSA prophage DNA sequence reconstitutes a complete tRNA(Arg) gene. The modified vector, pPL2, was integration proficient at the same frequency as pPL1 in common laboratory serotype 1/2 strains as well as serotype 4b strains.     

The use of Listeria monocytogenes as a DNA delivery vector for cancer gene therapy

Listeria monocytogenes is an intracellular pathogen that lyses the phagosomal vacuole of infected cells, proliferates in the host cell cytoplasm and can actively enter adjacent cells. The pathogen is therefore well suited to exploitation as a vector for the delivery of DNA to target cells as the lifecycle favors cellular targeting with vector amplification and the potential for cell-to-cell spread. We have recently demonstrated DNA transfer by L. monocytogenes in growing tumors in murine models. Our approach exploited an ampicillin sensitive stain of L. monocytogenes which can be lysed through systemic administration of ampicillin to facilitate release of plasmid DNA for expression by infected mammalian cells. Here, we discuss the implications of this technology and the potential for future improvements of the system.     

An Improved Cloning Vector for Construction of Gene Replacements in Listeria monocytogenes

Listeria monocytogenes is a gram-positive, facultative intracellular bacterium implicated in severe food-borne illness (listeriosis) in humans. The construction of well-defined gene replacements in the genome of L. monocytogenes has been instrumental to several genetic studies of the virulence and other attributes of the organism. Construction of such mutations by currently available procedures, however, tends to be labor intensive, and gene replacement mutants are sometimes difficult to recover due to lack of direct selection for the construct. In this study we describe the construction and use of plasmid vector pGF-EM, which can be conjugatively transferred from Escherichia coli S17-1 to L. monocytogenes and which provides the genetic means for direct selection of gene replacements.     

PCR detection of Listeria monocytogenes: a study of multiple factors affecting sensitivity

AIMS: To test, under comparable conditions, several parameters affecting sensitivity of PCR detection in order to establish a PCR procedure suitable for the routine detection of Listeria monocytogenes in food. METHODS AND RESULTS: Beef samples artificially inoculated were used to determine sensitivity of PCR detection under different parameters. As few as 1 CFU g(-1) were detected by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) of 1 ml aliquot and PCR amplification with primers directed to the hlyA gene. This PCR protocol was applied in 60 naturally contaminated foods, comparing two enrichment procedures with the traditional culture method. The highest number of positives was recorded by PCR following a 24-h pre-enrichment step at 30 degrees C and a 24-h enrichment step at 37 degrees C. Afterwards, it was applied in 217 naturally contaminated foods and 56 of them tested positive for L. monocytogenes in which only 17 tested positive using the culture method. CONCLUSIONS: The PCR procedure described has proved to be a rapid and sensitive method suitable for the routine analysis of different types of food. SIGNIFICANCE AND IMPACT OF THE STUDY: The method proposed for the detection of L. monocytogenes, has been validated in naturally contaminated food and is suitable to implement in the food industry.     

The potential use of nisin to control Listeria monocytogenes in poultry

Nisin (Nisaplin^TM) was added to scald water and used to treat Listeria monocytogenes which were either directly suspended in the water or attached to turkey skin. There was at least a 1 log decrease of listeria for the nisin treatments. This was followed by further reductions when the samples were stored under refrigeration. Heat exhibited a synergistic relationship with the nisin when the cells were directly suspended in the scald water. A 2 log decrease was observed immediately after this treatment and there was complete elimination of the listeria after 48 h of refrigeration. However, this relationship was not as obvious when the cells were first attached to the turkey skin. In these latter experiments, the nisin treatments resulted in reductions in the listeria levels but they were smaller than those observed when the cells were directly suspended in the scald water.     

Development of a biofilm model for Listeria monocytogenes EGD-e

The ability to form persistent biofilms makes the pathogenic bacterium Listeria monocytogenes a hazardous contaminant in food processing environments. Growth and biofilm formation of L. monocytogenes EGD-e were studied in defined medium (HTM) and in tryptic soy broth (TSB) with different supplements. TSB + 1% glucose gave optimal results. Using this medium, biofilm development on the model surface polystyrene (microtiter plate) was monitored by the standard crystal violet staining for adherent cells after bacterial cultivation for 24 and 48 h at five different temperatures (4, 18, 25, 30 and 37°C). In parallel, the matrix exopolysaccharide formed after 48 h of incubation was quantified by staining with ruthenium red. In both assays incubation at 30°C yielded the highest values. The formation of larger scale biofilms on dialysis membranes, placed on TSB agar with 1% glucose for 48 h, was studied by scanning electron microscopy. Contiguous and multilayered biofilms were observed at 18, 25, 30 and 37°C incubation temperature. The methodology is suitable for quantitative and microscopic studies and, in addition, yields sufficient cell mass for subsequent biochemical and molecular biological analyses.     

Structural and immunochemical studies of teichoic acid of Listeria monocytogenes

An immunologically active teichoic acid component was isolated from the cell wall of Listeria monocytogenes strain EGD. The teichoic acid component, accounting for about 20% of the weight of cell wall, contained N-acetylglucosamine, rhamnose, ribitol, and phosphorus in a molar ratio of 0.95 : 1.0 : 0.97 : 0.98. The molecular weight of the teichoic acid chain was about 120,000 as analyzed by gel filtration. The probable structure was deduced from the results of methylation analysis, Smith degradation, and proton magnetic resonance spectrometry of the teichoic acid, together with the characterization of fragments obtained by treatment with hydrofluoric acid, as follows: (formula; see text) Inhibition testing with monosaccharide and fragments obtained from HF treatment of Listeria teichoic acid in the quantitative precipitin reaction suggested that the rhamnose residue is a major antigenic determinant.     

Listeria monocytogenes: a multifaceted model

The opportunistic intracellular pathogen Listeria monocytogenes has become a paradigm for the study of host-pathogen interactions and bacterial adaptation to mammalian hosts. Analysis of L. monocytogenes infection has provided considerable insight into how bacteria invade cells, move intracellularly, and disseminate in tissues, as well as tools to address fundamental processes in cell biology. Moreover, the vast amount of knowledge that has been gathered through in-depth comparative genomic analyses and in vivo studies makes L. monocytogenes one of the most well-studied bacterial pathogens.     

Development of a Synthetic Minimal Medium for Listeria monocytogenes

A defined solid and liquid minimal medium, HTM, which contained methionine and cysteine as the sole amino acids, was developed for Listeria monocytogenes. Complex broth-grown L. monocytogenes had to adapt to HTM by inducing amino acid biosyntheis. HTM is the simplest minimal medium available for growth of L. monocytogenes.     

Listeria monocytogenes, a Food-Borne Pathogen

[This corrects the article on p. 485 in vol. 55.].     

Caenorhabditis elegans is a model host for Listeria monocytogenes

Here we report that Caenorhabditis elegans nematodes fed Listeria monocytogenes die over the course of several days, as a consequence of an accumulation of bacteria in the worm intestine. Mutant strains previously shown to be important for virulence in mammalian models were also found to be attenuated in their virulence in C. elegans. However, ActA, which is required for actin-based intracellular motility, appears to be dispensable during infection of C. elegans, indicating that L. monocytogenes remains extracellular in C. elegans.     

Caenorhabditis elegans Is a Model Host for Listeria monocytogenes

Here we report that Caenorhabditis elegans nematodes fed Listeria monocytogenes die over the course of several days, as a consequence of an accumulation of bacteria in the worm intestine. Mutant strains previously shown to be important for virulence in mammalian models were also found to be attenuated in their virulence in C. elegans. However, ActA, which is required for actin-based intracellular motility, appears to be dispensable during infection of C. elegans, indicating that L. monocytogenes remains extracellular in C. elegans.     

Acid-tolerant Listeria monocytogenes persist in a model food system fermented with nisin-producing bacteria

AIMS: To investigate the induction of the acid tolerance response (ATR) in Listeria monocytogenes and to assess the persistence of the pathogen in broth fermented using a nisin-producing starter culture. METHODS AND RESULTS: Lactic, acetic and hydrochloric acids were used to induce the ATR in L. monocytogenes growing at early exponential phase. Cells were then challenged in medium acidified to pH 3.5 with the same acid. Only lactic acid induced a detectable ATR. ATR+ cells maintained their initial numbers after 1 h exposure while ATR- were reduced by c. 4 log10 CFU. ATR+ or ATR- cells were also inoculated in M17G broth fermented with nisin-producing (nis+) or control (nis-) Lactococcus lactis. When exposed to nisin, the numbers of ATR+ cells were c. 2 log10 CFU higher than non detectable ATR- cells at day 3. In the absence of nisin (nis- culture), L. monocytogenes was recovered from all ATR+ and ATR- samples after 30 days. In contrast, no L. monocytogenes were recovered from any nis+ATR- samples but four of five nis+ATR+ samples were positive for L. monocytogenes after 30 days. CONCLUSIONS: The ATR confers cross-resistance to nisin for at least 30 days in a system fermented by nisin-producing bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The cross-resistance induced by the ATR should be considered for the safety of foods fermented with bacteriocin-producing cultures.     

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.