Atomic force microscopy investigations of heterogeneities in the adhesion energies measured between pathogenic and non-pathogenic Listeria species and silicon nitride as they correlate to virulence and adherence

Atomic force microscopy (AFM) was used to probe heterogeneities in adhesion energies measured between pathogenic and non-pathogenic species of Listeria and silicon nitride in water at four levels. Adhesion energies were quantified on individual bacterial cells (cell level), bacterial cells that belonged to an individual Listeria strain but varied in their cultures (strain level), bacterial cells that belonged to an individual Listeria species but varied in their strain type (species level) and on bacterial cells that belonged to the Listeria genus but varied in their species type (genus level). To quantify heterogeneities in the adhesion energies, a heterogeneity index (HI) was defined based on quantified standard errors of mean. At the cell level, spatial variations in the adhesion energies were not observed. For the strain, species, and genus levels, the HI increased with increased adhesion energies. At the species level, the HI increased with strain virulence.     

Inactivation of adhesion and invasion of food-borne Listeria monocytogenes by bacteriocin-producing Bifidobacterium strains of human origin

Three bacteriocin-producing bifidobacterial isolates from newborns were identified as Bifidobacterium thermacidophilum (two strains) and B. thermophilum (one strain). This study was undertaken to evaluate the ability of these strains to compete with food-borne Listeria monocytogenes for adhesion and invasion sites on Caco-2 and HT-29 cells. The bifidobacteria adhered at levels ranging from 4% to 10% of the CFU added, but none of the bifidobacteria were able to invade cells. The abilities of Listeria to adhere to and to invade cells varied widely depending on the strain tested. Three groups of Listeria were identified based on invasiveness: weakly invasive, moderately invasive, and highly invasive strains. One strain from each group was tested in competition with bifidobacteria. B. thermacidophilum RBL70 was the most effective in blocking invasion of Listeria, and the decreases in invasion ranged from 38% to 90%. For all three bifidobacterial strains, contact between the cell monolayer and the bifidobacteria for 1 h before exposure to Listeria increased the degree of inhibition. Finally, visualization of competition for adhesion sites on cells by fluorescent in situ hybridization suggested that the two bacteria tended to adhere in close proximity.     

Production of thiol-dependent haemolysins by Listeria monocytogenes and related species

Twenty-six strains belonging to the five main species of the genus Listeria were examined for production of thiol-dependent exotoxins. All strains of L. monocytogenes cultured in charcoal-treated broth secreted a haemolytic factor at a level ranging from 200 to 800 haemolytic units (HU) ml-1, except for the strain EGD (1500 HU ml-1) and the type strain CIP 82110T (10 HU ml-1). The haemolytic activity reached a maximum level by 8-10 h and then rapidly declined as soon as bacterial exponential growth ceased. The titres of haemolytic activity were markedly reduced when bacteria were grown in charcoal-untreated broth. The haemolytic factor produced by L. monocytogenes strains was characterized as listeriolysin O (Mr about 60,000), a member of the group of thiol-dependent exotoxins. Strains of Listeria ivanovii also produced high levels of thiol-dependent exotoxin (about 2500 HU ml-1), in both charcoal-treated and untreated broth. Small amounts of haemolytic factor (about 9-30 HU ml-1) were also produced by Listeria seeligeri in charcoal-treated broth. The haemolysin produced by L. seeligeri was identified for the first time as a thiol-dependent exotoxin of Mr about 60,000, antigenically related to listeriolysin O. As expected, we failed to detect thiol-dependent exotoxin in the two nonhaemolytic species, Listeria innocua and Listeria welshimeri.     

FSL J1-208, a virulent uncommon phylogenetic lineage IV Listeria monocytogenes strain with a small chromosome size and a putative virulence plasmid carrying internalin-like genes

The bacterial genus Listeria contains both saprotrophic and facultative pathogenic species. A small genome size has been suggested to be associated with the loss of pathogenic potential of L. welshimeri and L. seeligeri. In this paper we present data on the genome of L. monocytogenes strain FSL J1-208, a representative of phylogenetic lineage IV. Although this strain was isolated from a clinical case in a caprine host and has no decreased invasiveness in human intestinal epithelial cells, our analyses show that this strain has one of the smallest Listeria chromosomes reported to date (2.78 Mb). The chromosome contains 2,772 protein-coding genes, including well-characterized virulence-associated genes, such as inlA, inlB, and inlC and the full prfA gene cluster. The small genome size is mainly caused by the absence of prophages in the genome of L. monocytogenes FSL J1-208, and further analyses showed that the total size of prophage-related regions is highly correlated to chromosome size in the genus Listeria. L. monocytogenes FSL J1-208 carries a unique type of plasmid of approximately 80 kbp that does not carry genes annotated as being involved in resistance to antibiotics or heavy metals. The accessory genes in this plasmid belong to the internalin family, a family of virulence-associated proteins, and therefore this is the first report of a potential virulence plasmid in the genus Listeria.     

Polyacrylamide Gel Identification of Bacterial L-Forms and Mycoplasma Species of Human Origin

Polyacrylamide gel electrophoretic patterns of acidified phenol extracts prepared from whole cells can be used for the identification of bacterial L-forms and Mycoplasma species of human origin. Ten human Mycoplasma serotypes and eight L-forms belonging to five different genera were studied. The gel patterns were sufficiently distinct and reproducible that it was possible not only to identify L-forms at the genus level (group with streptococci) and different Mycoplasma serotypes but also to differentiate between the two of them. The parentage of L-forms of Streptobacillus moniliformis L1, Listeria monocytogenes, Streptococcus MG, and Staphylococcus aureus Smith strain was established by relating their gel patterns directly to parent bacteria. It was found that an L-form designated S. moniliformis An (ATCC 14220) was actually an L-form of Proteus. In addition, it was shown electrophoretically that no relationship existed between the Streptococcus MG L-form and M. pneumoniae. The applicability of this method as a diagnostic and taxonomic tool for the differentiation of L-forms and mycoplasmas is discussed.     

Bacterial adhesion at synthetic surfaces.

A systematic investigation into the effect of surface chemistry on bacterial adhesion was carried out. In particular, a number of physicochemical factors important in defining the surface at the molecular level were assessed for their effect on the adhesion of Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli. The primary experiments involved the grafting of groups varying in hydrophilicity, hydrophobicity, chain length, and chemical functionality onto glass substrates such that the surfaces were homogeneous and densely packed with functional groups. All of the surfaces were found to be chemically well defined, and their measured surface energies varied from 15 to 41 mJ. m(-2). Protein adsorption experiments were performed with (3)H-labelled bovine serum albumin and cytochrome c prior to bacterial attachment studies. Hydrophilic uncharged surfaces showed the greatest resistance to protein adsorption; however, our studies also showed that the effectiveness of poly(ethyleneoxide) (PEO) polymers was not simply a result of its hydrophilicity and molecular weight alone. The adsorption of the two proteins approximately correlated with short-term cell adhesion, and bacterial attachment for L. monocytogenes and E. coli also correlated with the chemistry of the underlying substrate. However, for S. aureus and S. typhimurium a different pattern of attachment occurred, suggesting a dissimilar mechanism of cell attachment, although high-molecular-weight PEO was still the least-cell-adsorbing surface. The implications of this for in vivo attachment of cells suggest that hydrophilic passivating groups may be the best method for preventing cell adsorption to synthetic substrates provided they can be grafted uniformly and in sufficient density at the surface.     

The attachment to, and invasion of HeLa cells by Salmonella typhimurium: the contribution of mannose-sensitive and mannose-resistant haemagglutinating activities

The association of the haemagglutinating activities of Salmonella typhimurium cultures with bacterial adhesion to HeLa cells, and the internalization of the bacteria by HeLa cells, was studied. Adhesion was not inhibited by alpha-methyl-D-mannoside (i.e. adhesion was mannose-resistant), and only four of the six strains tested produced type 1 fimbriae and the associated mannose-sensitive haemagglutinin (MSHA). The other two strains belonged to the non-fimbriate FIRN biogroup. Cultures of all six strains contained a mannose-resistant haemagglutinating (MRHA) activity when grown at 37 degrees C, but cultures of only one fimbriate and one non-fimbriate strain did so when grown at 18 degrees C. From the comparison of cultures grown at 18 degrees C and 37 degrees C, and of mutant strains with the phenotypes MRHA-negative/MSHA-positive, or MRHA-positive/MSHA-negative, it was concluded that the MRHA activity was responsible for the attachment of salmonellae to HeLa cells. Only bacterial adhesion that was resistant to mannose resulted in the internalization of the bacteria by the HeLa cells.     

Exploitation of host cell cytoskeleton and signalling during Listeria monocytogenes entry into mammalian cells

Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells during infection isa key issue for the understanding how this food-borne pathogen causes a pleiotropic disease ranging from gastro-enteritis to meningitis and abortions. Using multidisciplinary approaches, essentially combining bacterial genetics and cell biology, we have identified two bacterial proteins critical for entry into target cells, InlA and InlB. Their cellular ligands have been also identified: InlA interacts with the adhesion molecule E-cadherin, while InlB interacts with the receptor for the globular head of the complement factor Clq (gClq-R), with the hepatocyte growth factor receptor (c-Met) and with glycosaminoglycans(including heparan sulphate). The dynamic interaction between these cellular receptors and the actin cytoskeleton is currently under investigation. Several intracellular molecules have been recognized as key effectors for Listeria entry into target cells,including catenins (implicated in the connection of E-cadherin to actin) and the actin depolymerising factor/cofilin (involved in the rearrangement of the cytoskeleton in the InlB-dependent internalisation pathway). At the organism level, species specificity has been discovered concerning the interaction between InlA and E-cadherin, leading to the generation of transgenic mice expressing the human E-cadherin, in which the critical role of InlA in the crossing of the intestinal barrier has been clearly determined. Listeria appears as an instrumental model for addressing critical questions concerning both the complex process of bacterial pathogenesis and also fundamental molecular processes, such as phagocytosis.     

Natural atypical Listeria innocua strains with Listeria monocytogenes pathogenicity island 1 genes

Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentation-negative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.     

Listeria monocytogenes LO28: surface physicochemical properties and ability to form biofilms at different temperatures and growth phases

The surface physicochemical properties of Listeria monocytogenes LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents. The effect of these parameters on adhesion and biofilm formation by L. monocytogenes LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene [PTFE]) surfaces was assessed. The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature. The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature. However, biofilm formation was faster on the hydrophilic substratum. After 5 days at 37 or 20 degrees C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37 degrees C. At 8 degrees C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE. The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation. The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.     

Exploitation of host cell cytoskeleton and signalling during Listeria monocytogenes entry into mammalian cells

Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells during infection is a key issue for the understanding how this food-borne pathogen causes a pleiotropic disease ranging from gastro-enteritis to meningitis and abortions. Using multidisciplinary approaches, essentially combining bacterial genetics and cell biology, we have identified two bacterial proteins critical for entry into target cells, InlA and InlB. Their cellular ligands have been also identified: InlA interacts with the adhesion molecule E-cadherin, while InlB interacts with the receptor for the globular head of the complement factor C1q (gC1q-R), with the hepatocyte growth factor receptor (c-Met) and with glycosaminoglycans (including heparan sulphate). The dynamic interaction between these cellular receptors and the actin cytoskeleton is currently under investigation. Several intracellular molecules have been recognized as key effectors for Listeria entry into target cells, including catenins (implicated in the connection of E-cadherin to actin) and the actin depolymerising factor/cofilin (involved in the rearrangement of the cytoskeleton in the InlB-dependent internalisation pathway). At the organism level, species specificity has been discovered concerning the interaction between InlA and E-cadherin, leading to the generation of transgenic mice expressing the human E-cadherin, in which the critical role of InlA in the crossing of the intestinal barrier has been clearly determined. Listeria appears as an instrumental model for addressing critical questions concerning both the complex process of bacterial pathogenesis and also fundamental molecular processes, such as phagocytosis.     

Identification of thermo-acidophilic bacteria isolated from the soil of several Japanese fruit orchards

Aims: To investigate the occurrence and distribution of thermo‐acidophilic bacteria (TAB) associated with various commercial fruit crop soils in Japan and to assess their ability to produce the odorous phenolic compound, guaiacol. Methods and Results: Phylogenetic analysis based on the 5′ end of the 16S rRNA gene (approximately 500 bp), was performed on 62 TAB isolated from the soil of several Japanese fruit orchards. The results suggested that 60 of the bacterial strains analysed belonged to the genus Alicyclobacillus, while the remaining two belonged to the genus Bacillus. The majority of strains (58%) were identified as Alicyclobacillus acidoterrestris. This group partitioned into three phylogenetically distinct subgroups (A–C). Isolates identified as A. acidiphilus (two strains), A. acidoterrestris (36 strains), and A. hesperidum subsp. aigle (one strain), produced guaiacol from vanillic acid. Levels of guaiacol production varied significantly among strains. The guaiacol producing phenotype was conserved among certain species, however no correlation was observed between levels of guaiacol production and 16S rRNA gene‐based phylogenetic relatedness. Conclusions: Alicyclobacillus acidoterrestris and Alicyclobacillus contaminans were widely distributed among various fruit orchards in Japan. Guaiacol production was common at the species/subspecies level; however the amount of guaiacol produced by each strain varied significantly. Significance and Impact of the Study: This study provides a comprehensive phylogenetic survey of Alicyclobacillus species in Japanese fruit orchards. Quality control standards for guaiacol producing Alicyclobacillus have also been described.     

李斯特菌毒力因子及其进化

李斯特菌属包含6个种,毒力各有差异。在细菌耐受外界环境、黏附侵袭及细胞内感染过程中,毒力因子各司其职又相互协作。毒力基因常聚集为毒力岛,其中PrfA依赖型毒力基因簇(LIPI-1)与内化素岛(LIPI-2)是致病种最重要的两个毒力岛。李斯特菌各个种可能来源于同一个携带有完整毒力岛的祖先,在长期进化过程中,通过基因水平转移或重组、整合等事件,演化为目前流行的6个种。噬菌体、转座子、质粒等可能扮演着毒力进化执行者的角色。一些天然非典型菌株是目前研究的热点,如含有LIPI-1的无害李斯特菌和缺失LIPI-1的塞氏李斯特菌,其演化进程可能尚未达到或已超越目前流行的状态,为李斯特菌毒力进化的研究提供了重要线索。     

Wolbachia infection and incompatibility dynamics in experimental selection lines

High and low levels of Wolbachia-induced cytoplasmic incompatibility (CI) were selected for in the parasitic wasp Nasonia vitripennis, in the single-infected strain Ti277. After nine generations of selection, males from lines selected for high incompatibility level (HI lines) were significantly more incompatible with uninfected females (AsymC) than the maternal strain. The reverse response, a full compatibility with AsymC, was observed in eight out of 12 lines selected for low incompatibility (LO lines), correlated with loss of Wolbachia infection. Bacterial density estimates in the eggs of some HI lines increased significantly. The procedure for line maintenance resulted in introgression of AsymC nuclear genome into the Ti277 background. Significant changes of CI level and bacterial density due to the introgression were also observed in the control lines, possibly reflecting an effect of host genotype on bacterial density and CI. After selection had been relaxed for six generations, bacterial density in the five high-infected HI lines declined back to a level comparable to the other lines. The data are consistent with the ‘bacterial dosage’ model, but with an upper threshold of bacterial infection above which there is no correlation between infection level and CI level. We further investigate the maternal transmission of bacterial density by a mother–daughter regression on bacterial density. The pattern observed is consistent with a density dependent regulation of bacterial numbers around an ‘equilibrium’ density, independent of any effects of CI. The equilibrium value is likely to be determined by both bacterial strain and host genotype.     

Phenotypic characterization of the Xenorhabdus bacterial symbiont of a Texas strain of the entomopathogenic nematode Steinernema riobrave, and characterization of the Xenorhabdus bovienii bacterial symbiont of a Newfoundland strain of Steinernema feltiae

Two bacterial symbionts of entomopathogenic nematodes, one of which originated from Texas, U.S.A., and the other from Newfoundland, Canada, were characterized phenotypically. These strains belonged to the genus Xenorhabdus. The Newfoundland (NF) strain was shown to be X. bovienii but the Texas (TX) strain was not identified at the species level. Four additional cultures of Xenorhabdus were included in the study. These were a strain of X. bovienii (Ume?), which was from a nematode of European origin, and strains of X. nematophilus, X. beddingii, and X. poinarii. The tests used in this study indicated identical properties for the NF (North American) and Ume? (European) strains of X. bovienii. These could be differentiated from the other strains studied by their failure to grow at 34 degrees C and resistance to low concentrations of a mixture of amoxilline and clavulanic acid. The Xenorhabdus TX strain could be differentiated from the other strains studied by its failure to grow at 10 degrees C. Of the tests done, approximately 30 were useful in distinguishing between the strains and species studied.     

湘江干流浮游生物群落结构及水质状况分析

为了解湘江干流水质与生物群落现状, 2010-2011年对湘江的水质和浮游生物的分布进行了采样分析, 利用香农-威纳多样性指数分析湘江干流浮游生物群落特征, 并结合水质理化指标评价其水体营养状态。结果表明, 共检出浮游植物8门99属164种, 丰度变化在(1.83-51.1)104cells/L, 生物量变化在0.03-0.60 mg/L; 浮游动物80种, 密度变化在2.16-76.34个/L, 生物量变化在0.01-1.95 mg/L。各采样点的浮游植物多样性指数变化在2.47-5.43, 浮游动物多样性指数变化在1.63-3.38, 浮游生物的多样性较好, 显示出湘江干流的浮游生物群落处于较稳定的状态。对湘江m-m指示种种类组成和群落结构分析, 结果显示湘江水体属于中污带水质; 综合营养状态指数(TIL)在38.27-51.97, 均值为43.73, 属于中营养水平; 综合水质生物学和化学评价结果可知湘江干流的水质较差, 但无明显向富营养化转化的趋势。       

Biophysical parameters influence actin-based movement, trajectory, and initiation in a cell-free system

Using a biochemically complex cytoplasmic extract to reconstitute actin-based motility of Listeria monocytogenes and polystyrene beads coated with the bacterial protein ActA, we have systematically varied a series of biophysical parameters and examined their effects on initiation of motility, particle speed, speed variability, and path trajectory. Bead size had a profound effect on all aspects of motility, with increasing size causing slower, straighter movement and inhibiting symmetry-breaking. Speed also was reduced by extract dilution, by addition of methylcellulose, and paradoxically by addition of excess skeletal muscle actin, but it was enhanced by addition of nonmuscle (platelet) actin. Large, persistent individual variations in speed were observed for all conditions and their relative magnitude increased with extract dilution, indicating that persistent alterations in particle surface properties may be responsible for intrinsic speed variations. Trajectory curvature was increased for smaller beads and also for particles moving in the presence of methylcellulose or excess skeletal muscle actin. Symmetry breaking and movement initiation occurred by two distinct modes: either stochastic amplification of local variation for small beads in concentrated extracts, or gradual accumulation of strain in the actin gel for large beads in dilute extracts. Neither mode was sufficient to enable spherical particles to break symmetry in the cytoplasm of living cells.     

Novel experimental study of receptor-mediated bacterial adhesion under the influence of fluid shear

Dynamic adhesion of cells to surfaces is a vital step in a variety of biochemical and physiological phenomena. Bacterial adhesion is responsible not only for problems associated with biofouling and biofilm formation in the biochemical industry but also in the initiation of certain infectious diseases. In this study, we report the effect of critical parameters, such as receptor and ligand densities and shear rate, on receptor-mediated dynamic bacterial adhesion. Adhesion of a pathogenic strain of Staphylococcus aureus to immobilized collagen was studied. The receptor density on the cell surface was varied by harvesting cells at different growth times and was quantified using flow cytometry. Dynamic adhesion experiments were conducted over a range of physiologically relevant shear rates (50 to 1500 s(-1)) using a parallel-plate flow chamber. Video microscopy coupled with digital image processing was employed to quantify adhesion. A semiquantitative comparison between experimental results and theoretical data obtained using a previously proposed mathematical model was also performed. The results suggest that dynamic adhesion is dependent on receptor density and shear rate, but independent of ligand density. This report demonstrates the feasibility of using bacteria to study fundamental aspects of receptor-mediated dynamic adhesion.