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.     

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.     

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.     

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.     

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.     

Phylogenetic Profiles of In-House Microflora in Drains at a Food Production Facility: Comparison and Biocontrol Implications of Listeria-Positive and -Negative Bacterial Populations

Listeria species experience complex interactions with other microorganisms, which may promote growth and colonization of the organism in local environments or negatively affect them. This study investigated the microbial community at a food production facility, examining interactions between Listeria and the associated microbiome. Listeria species can be transferred between zones in the production environment by individuals or equipment, and drains may act as a reservoir for the organism, reflecting the microbial flora potentially in the production environment. Drains that were colonized by Listeria species and those determined to be free of Listeria were examined. In each case, 16S rRNA gene analysis was performed using the PhyloChip platform. Some general similarities in bacterial population structure were observed when Listeria-negative and -positive drain communities were compared, with some distinct differences also noted. These included increased populations of the genera Prevotella and Janthinobacterium associated with the absence of Listeria species, whereas Enterococcus and Rhodococcus were in higher abundance in drains colonized by Listeria species. Based on these results, a selection of bacterial species were grown in coculture biofilm with a Listeria monocytogenes strain identified as having colonized a drain at the facility. Mixed-species biofilm experiments showed that Janthinobacterium inhibited attachment and subsequent biofilm formation of L. monocytogenes; however, Enterococcus gallinarum significantly increased it. The results of this study suggest the microbial community in food processing facilities can impact the colonization of Listeria species and that influencing the microbiome in favor of antilisterial species may reduce the colonization of Listeria species and limit the likelihood of product/process contamination.     

Domain shuffling and module engineering of Listeria phage endolysins for enhanced lytic activity and binding affinity

Bacteriophage endolysins are peptidoglycan hydrolases employed by the virus to lyse the host at the end of its multiplication phase. They have found many uses in biotechnology; not only as antimicrobials, but also for the development of novel diagnostic tools for rapid detection of pathogenic bacteria. These enzymes generally show a modular organization, consisting of N‐terminal enzymatically active domains (EADs) and C‐terminal cell wall‐binding domains (CBDs) which specifically target the enzymes to their substrate in the bacterial cell envelope. In this work, we used individual functional modules of Listeria phage endolysins to create fusion proteins with novel and optimized properties for labelling and lysis of Listeria cells. Chimaeras consisting of individual EAD and CBD modules from PlyPSA and Ply118 endolysins with different binding specificity and catalytic activity showed swapped properties. EAD118–CBDPSA fusion proteins exhibited up to threefold higher lytic activity than the parental endolysins. Recombineering different CBD domains targeting various Listeria cell surfaces into novel heterologous tandem proteins provided them with extended recognition and binding properties, as demonstrated by fluorescent GFP‐tagged CBD fusions. It was also possible to combine the binding specificities of different single CBDs in heterologous tandem CBD constructs such as CBD500‐P35 and CBDP35‐500, which were then able to recognize the majority of Listeria strains. Duplication of CBD500 increased the equilibrium cell wall binding affinity by approximately 50‐fold, and the enzyme featuring tandem CBD modules showed increased activity at higher ionic strength. Our results demonstrate that modular engineering of endolysins is a powerful approach for the rational design and optimization of desired functional properties of these proteins.     

Isolation of bacteria of the genus <Emphasis Type="Italic">Variovorax</Emphasis> from the <Emphasis Type="Italic">Thioploca</Emphasis> mats of Lake Baikal

Three strains of gram-negative bacteria were isolated from the mats of colorless sulfur bacteria Thioploca (Lake Baikal). The cells of new strains are motile with peritrichous flagella. Bacteria are aerobic, obligate chemoorganoheterotrophs growing within the pH range of 3.0–8.8 with the optimum at 8.3 and within the temperature range of 5–42°C with the optimum at 28°C. The cells contained menaquinones MK-8 H2 as the major component, as well as MK-7 H2 (less than 15%), while the content of ubiquinone Q8 was at least an order of magnitude lower. The G+C content of DNA in the new strains varied from 67.4 to 69.9 mol %. The level of DNA-DNA hybridization between the strains ranged from 80 to 94%, indicating that all the isolates belonged to one species. Analysis of the 16S rRNA gene nucleotide sequences of the type strain (Gen-Bank HQ400611) revealed close homologues among the known species of the genus Variovorax: 98% resemblance with the type strains of the species V. paradoxus, V. soli, V. ginsengisoli, and V. boronicumulans and 96% similarity with the type strain of V. dokdonensis. However, since the isolates differed significantly in the composition of fatty acids and isoprenoid quinones from the nearest neighbors in the phylogenetic tree, they cannot be related implicitly to the known species.     

Differentiation of Pectobacterium and Dickeya spp. phytopathogens using infrared spectroscopy and machine learning analysis

Pectobacterium and Dickeya spp. are soft rot Pectobacteriaceae that cause aggressive diseases on agricultural crops leading to substantial economic losses. The accurate, rapid and low‐cost detection of these pathogenic bacteria are very important for controlling their spread, reducing the consequent financial loss and for producing uninfected potato seed tubers for future generations. Currently used methods for the identification of these bacterial pathogens at the strain level are based mainly on molecular techniques, which are expensive. We used an alternative method, infrared spectroscopy, to measure 24 strains of five species of Pectobacterium and Dickeya. Measurements were then analyzed using machine learning methods to differentiate among them at the genus, species and strain levels. Our results show that it is possible to differentiate among different bacterial pathogens with a success rate of ~99% at the genus and species levels and with a success rate of over 94% at the strain level.     

Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity

The synthesis of extracellular molecules such as biosurfactants should have major consequences on bacterial adhesion. These molecules may be adsorbed on surfaces and modify their hydrophobicities. Certain strains of Bacillus subtilis synthesize the lipopeptides, which exhibit antibiotic and surface active properties. In this study the high-performance liquid chromatography (HPLC) analysis of the culture supernatants of the seven B. subtilis strains, showed that the lipopeptide profile varied greatly according to the strain. Among the three lipopeptide types, only iturin A was produced by all B. subtilis strains. Bacterial hydrophobicity, evaluated by the water contact angle measurements and the hydrophobic interaction chromatography, varied according to the strain. Two strains (ATCC 15476 and ATCC 15811) showing extreme behaviors in term of hydrophobicity were selected to study surfactin and iturin A effects on bacterial hydrophobicity. The two lipopeptides modified the B. subtilis surface hydrophobicity. Their effects varied according to the bacterial surface hydrophobic character, the lipopeptide type and the concentration. Lipopeptide adsorption increased the hydrophobicity of the hydrophilic strain but decreased that of the hydrophobic. Comparison of lipopeptide effects on B. subtilis surface hydrophobicity showed that surfactin was more effective than iturin A for the two strains tested.     

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.     

Isolation of Paenibacillus sp. and Assessment of its Potential for Enhancing Mineral Weathering

One mineral-solubilizing bacterial strain designated KT was isolated from a soil in Henan Province, China. The full-length 16S rRNA gene sequence showed 95.2 to 96.7% similarity to the bacteria of the genus Paenibacillus, indicating that the strain KT belonged to the genus Paenibacillus. The potential of this strain to release potassium from silicate minerals was investigated using a potassium-bearing rock as the sole source of potassium to support its growth. After inoculation for 7 days, the concentrations of water-soluble Al, Ca and Fe released from the potassium-bearing rock in active bacterial culture were higher than those from the control with autoclaved inoculum, but the concentration of water-soluble K in active bacterial culture was similar to that in the control. The concentrations of HNO₃-extractable Al, Ca, Fe and K from the bacterial culture were also higher than those in the control. These results showed strain KT was able to release potassium from potassium-bearing rock. These results have important implications for extraction of potassium from rocks to support plant growth.     

Characterization of root-nodulating bacteria associated to <Emphasis Type="Italic">Prosopis farcta</Emphasis> growing in the arid regions of Tunisia

Diversity of 50 bacterial isolates recovered from root nodules of Prosopis farcta grown in different arid soils in Tunisia, was investigated. Characterization of isolates was assessed using a polyphasic approach including phenotypic characteristics, 16S rRNA gene PCR–RFLP and sequencing, nodA gene sequencing and MLSA. It was found that most of isolates are tolerant to high temperature (40°C) and salinity (3%). Genetic characterization emphasizes that isolates were assigned to the genus Ensifer (80%), Mesorhizobium (4%) and non-nodulating endophytic bacteria (16%). Forty isolates belonging to the genus Ensifer were affiliated to Ensifer meliloti, Ensifer xinjiangense/Ensifer fredii and Ensifer numidicus species. Two isolates belonged to the genus Mesorhizobium. Eight isolates failing to renodulate their host plant were endophytic bacteria and belonged to Bacillus, Paenibacillus and Acinetobacter genera. Symbiotic properties of nodulating isolates showed a diversity in their capacity to infect their host plant and fix atmospheric nitrogen. Isolate PG29 identified as Ensifer meliloti was the most effective one. Ability of Prosopis farcta to establish symbiosis with rhizobial species confers an important advantage for this species to be used in reforestation programs. This study offered the first systematic information about the diversity of microsymbionts nodulating Prosopis farcta in the arid regions of Tunisia.     

Simultaneous hydrocarbon biodegradation and biosurfactant production by oilfield-selected bacteria

Aims: To study the bacterial diversity associated with hydrocarbon biodegradation potentiality and biosurfactant production of Tunisian oilfields bacteria. Methods and Results: Eight Tunisian hydrocarbonoclastic oilfields bacteria have been isolated and selected for further characterization studies. Phylogenetic analysis revealed that three thermophilic strains belonged to the genera Geobacillus, Bacillus and Brevibacillus, and that five mesophilic strains belonged to the genera Pseudomonas, Lysinibacillus, Achromobacter and Halomonas. The bacterial strains were cultivated on crude oil as sole carbon and energy sources, in the presence of different NaCl concentrations (1, 5 and 10%, w/v), and at 37 or 55°C. The hydrocarbon biodegradation potential of each strain was quantified by GC–MS. Strain C450R, phylogenetically related to the species Pseudomonas aeruginosa, showed the maximum crude oil degradation potentiality. During the growth of strain C450R on crude oil (2%, v/v), the emulsifying activity (E24) and glycoside content increased and reached values of 77 and 1·33 g l^?1, respectively. In addition, the surface tension (ST) decreased from 68 to 35·1 mN m^?1, suggesting the production of a rhamnolipid biosurfactant. Crude biosurfactant had been partially purified and characterized. It showed interest stability against temperature and salinity increasing and important emulsifying activity against oils and hydrocarbons. Conclusions: The results of this study showed the presence of diverse aerobic bacteria in Tunisian oilfields including mesophilic, thermophilic and halotolerant strains with interesting aliphatic hydrocarbon degradation potentiality, mainly for the most biosurfactant produced strains. Significance and Impact of the Study: It may be suggested that the bacterial isolates are suitable candidates for practical field application for effective in situ bioremediation of hydrocarbon‐contaminated sites.     

Role of hydrophobicity in adhesion of wild yeast isolated from the ultrafiltration membranes of an apple juice processing plant

The role of cell surface hydrophobicity in the adhesion to stainless steel (SS) of 11 wild yeast strains isolated from the ultrafiltration membranes of an apple juice processing plant was investigated. The isolated yeasts belonged to four species: Candida krusei (5 isolates), Candida tropicalis (2 isolates), Kluyveromyces marxianus (3 isolates) and Rhodotorula mucilaginosa (1 isolate). Surface hydrophobicity was measured by the microbial adhesion to solvents method. Yeast cells and surfaces were incubated in apple juice and temporal measurements of the numbers of adherent cells were made. Ten isolates showed moderate to high hydrophobicity and 1 strain was hydrophilic. The hydrophobicity expressed by the yeast surfaces correlated positively with the rate of adhesion of each strain. These results indicated that cell surface hydrophobicity governs the initial attachment of the studied yeast strains to SS surfaces common to apple juice processing plants.     

RIG‐I detects infection with live Listeria by sensing secreted bacterial nucleic acids

Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill‐defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG‐I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG‐I‐dependent IL‐1β‐production and inflammasome activation. The signalling molecule CARD9 contributed to IL‐1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG‐I provides a mechanistic explanation for efficient induction of immunity by live bacteria.     

Dissimilatory sulphate reduction in hypersaline coastal pans: an integrated microbiological and geochemical study

Here, we report on the spatial and temporal variation in sulphate‐reducing bacterial community structure and activity in three hypersaline coastal pans. Community structure was determined using denaturing gradient gel electrophoresis (DGGE). Cluster analysis of DGGE patterns indicated that similar microbial populations were generally found in individual pans but varied from one pan to the other. Sulphate reducing bacteria (SRB) were quantified by competitive polymerase chain reaction based on the amplification of the dsrAB genes. Cell numbers and in situ sulphate reduction activities varied between seasons and pans but in general showed low variation in depth. Sulphate reduction activity was not correlated with microbial population size indicating that community composition is relevant for specific microbial processes. Principal component analysis coupled with correlation analyses suggested that salinity, sulphate concentration, C/N ratio and pH were the most important factors in explaining variations in SRB community composition. Most sequences derived from DGGE amplicons belonged to members of the Desulfobacteraceae and Desulfohalobiaceae families.     

Variance in composition of inquiline communities in leaves of Sarracenia purpurea L. on multiple spatial scales

A survey of the abundances of species that inhabit the water-bearing leaves of the pitcher plant Sarracenia purpurea was conducted at several different spatial scales in northern Florida. Individual leaves are hosts to communities of inquiline species, including mosquitoes, midges, mites, copepods, cladocerans, and a diverse bacterial assemblage. Inquiline communities were quantified from four pitchers per plant, three plants per subpopulation, two subpopulations per population, and three populations. Species varied in abundance at different spatial scales. Variation in the abundances of mosquitoes and copepods was not significantly associated with any spatial scale. Midges varied in abundance at the level of populations; one population contained significantly more midges than the other two. Cladocerans varied at the level of the subpopulation, whereas mites varied at the level of the individual plants. Bacterial communities were described by means of Biolog plates, which quantify the types of carbon media used by the bacteria in each pitcher. Bacterial communities were found to vary significantly in composition among individual plants but not among populations or subpopulations. These results suggest that independent factors determining the abundances of individual species are important in determining community patterns in pitcher-plant inquilines.     

Intestinal inflammation alters mucosal carbohydrate foraging and monosaccharide incorporation into microbial glycans

Endogenous carbohydrates released from the intestinal mucus represent a constant source of nutrients to the intestinal microbiota. Mucus‐derived carbohydrates can also be used as building blocks in the biosynthesis of bacterial cell wall components, thereby influencing host mucosal immunity. To assess the uptake of endogenous carbohydrates by gut microbes in healthy mice and during intestinal inflammation, we applied azido‐monosaccharides that can be tracked on bacterial cell walls after conjugation with fluorophores. In interleukin‐10 deficient mice, changes in the gut microbiota were accompanied by decreased carbohydrate hydrolase activities and increased lumenal concentrations of host glycan‐derived monosaccharides. Tracking of the monosaccharide N‐azidoacetylglucosamine (GlcNAz) in caecum bacteria revealed a preferential incorporation of this carbohydrate by Xanthomonadaceae in healthy mice and by Bacteroidaceae in interleukin‐10 deficient mice. These GlcNAz‐positive Bacteroidaceae fractions mainly belonged to the species B. acidifaciens and B. vulgatus. Growth of Bacteroides species in the presence of specific monosaccharides changed their stimulatory activity toward CD11c⁺ dendritic cells. Expression of activation markers and cytokine production was highest after stimulation of dendritic cells with B. vulgatus. The variable incorporation of monosaccharides by related Bacteroides species underline the necessity to investigate intestinal bacteria down to the species level when addressing microbiota‐host interactions.