Background

Few beta-glucosidase inhibitors have so far been reported from microorganisms due to the practical difficulties in performing the inhibition tests and subsequent interpretation of results. In an effort to investigate marine microbial extracts for β-glucosidase inhibitors, we developed a new protocol, using esculin as substrate in an agar plate based assay, to screen a large number of microbial extracts in a short span of time.

Results

With the new method, pale yellowish zones against the blackish brown background could be visually observed with more clarity in sample extracts where β-glucosidase inhibitor was present. The new method was compared with the closest existing method and established beyond doubt. This agar plate based procedure required about one hour for minimum 12 samples and the throughput increases with the size of the agar gel plate used.

Conclusions

The new protocol was simple, rapid and effective in detecting beta-glucosidase inhibitors in microbial extracts.

Background

Fluoroquinolone use has been listed as a risk factor for the emergence of virulent clinical strains of some bacteria. The aim of our study was to evaluate the effect of fluoroquinolone (gatifloxacin) resistance selection on differential gene expression, including the toxin genes involved in virulence, in two fluoroquinolone-resistant strains of Clostridium perfringens by comparison with their wild-type isogenic strains.

Results

DNA microarray analyses were used to compare the gene transcription of two wild types, NCTR and ATCC 13124, with their gatifloxacin-resistant mutants, NCTRR and 13124R. Transcription of a variety of genes involved in bacterial metabolism was either higher or lower in the mutants than in the wild types. Some genes, including genes for toxins and regulatory genes, were upregulated in NCTRR and downregulated in 13124R. Transcription analysis by quantitative real-time PCR (qRT-PCR) confirmed the altered expression of many of the genes that were affected differently in the fluoroquinolone-resistant mutants and wild types. The levels of gene expression and enzyme production for the toxins phospholipase C, perfringolysin O, collagenase and clostripain had decreased in 13124R and increased in NCTRR in comparison with the wild types. After centrifugation, the cytotoxicity of the supernatants of NCTRR and 13224R cultures for mouse peritoneal macrophages confirmed the increased cytotoxicity of NCTRR and the decreased cytotoxicity of 13124R in comparison with the respective wild types. Fluoroquinolone resistance selection also affected cell shape and colony morphology in both strains.

Conclusion

Our results indicate that gatifloxacin resistance selection was associated with altered gene expression in two C. perfringens strains and that the effect was strain-specific. This study clearly demonstrates that bacterial exposure to fluoroquinolones may affect virulence (toxin production) in addition to drug resistance.

Background

The efficiency of recovery and the detection limit of Legionella after co-culture with Acanthamoeba polyphaga are not known and so far no investigations have been carried out to determine the efficiency of the recovery of Legionella spp. by co-culture and compare it with that of conventional culturing methods. This study aimed to assess the detection limits of co-culture compared to culture for Legionella pneumophila in compost and air samples. Compost and air samples were spiked with known concentrations of L. pneumophila. Direct culturing and co-culture with amoebae were used in parallel to isolate L. pneumophila and recovery standard curves for both methods were produced for each sample.

Results

The co-culture proved to be more sensitive than the reference method, detecting 102-103 L. pneumophila cells in 1 g of spiked compost or 1 m3 of spiked air, as compared to 105-106 cells in 1 g of spiked compost and 1 m3 of spiked air.

Conclusions

Co-culture with amoebae is a useful, sensitive and reliable technique to enrich L. pneumophila in environmental samples that contain only low amounts of bacterial cells.

Background

Autologous platelet concentrates (PCs) have been extensively used in a variety of medical fields to promote soft and hard tissue regeneration. The significance behind their use lies in the abundance of growth factors in platelets α-granules that promotes wound healing. In addition, antibacterial properties of PCs against various bacteria have been recently pointed out. In this study, the antimicrobial effect of pure platelet-rich plasma (P-PRP) was evaluated against oral cavity microorganisms such as Enterococcus faecalis, Candida albicans, Streptococcus agalactiae, Streptococcus oralis and Pseudomonas aeruginosa. Blood samples were obtained from 17 patients who underwent oral surgery procedures involving the use of P-PRP. The antibacterial activity of P-PRP, evaluated as the minimum inhibitory concentration (MIC), was determined through the microdilution twofold serial method.

Results

P-PRP inhibited the growth of Enterococcus faecalis, Candida albicans, Streptococcus agalactiae and Streptococcus oralis, but not of Pseudomonas aeruginosa strains.

Conclusions

P-PRP is a potentially useful substance in the fight against postoperative infections. This might represent a valuable property in adjunct to the enhancement of tissue regeneration.

Background

The ability to use erythritol as a sole carbon source is not universal among the Rhizobiaceae. Based on the relatedness to the catabolic genes in Brucella it has been suggested that the eryABCD operon may have been horizontally transferred into Rhizobium. During work characterizing a locus necessary for the transport and catabolism of erythritol, adonitol and L-arabitol in Sinorhizobium meliloti, we became interested in the differences between the erythritol loci of S. meliloti and R. leguminosarum. Utilizing the Ortholog Neighborhood Viewer from the DOE Joint Genome Institute database it appeared that loci for erythritol and polyol utilization had distinct arrangements that suggested these loci may have undergone genetic rearrangements.

Results

A data set was established of genetic loci containing erythritol/polyol orthologs for 19 different proteobacterial species. These loci were analyzed for genetic content and arrangement of genes associated with erythritol, adonitol and L-arabitol catabolism. Phylogenetic trees were constructed for core erythritol catabolic genes and contrasted with the species phylogeny. Additionally, phylogenetic trees were constructed for genes that showed differences in arrangement among the putative erythritol loci in these species.

Conclusions

Three distinct erythritol/polyol loci arrangements have been identified that reflect metabolic need or specialization. Comparison of the phylogenetic trees of core erythritol catabolic genes with species phylogeny provides evidence that is consistent with these loci having been horizontally transferred from the alpha-proteobacteria into both the beta and gamma-proteobacteria. ABC transporters within these loci adopt 2 unique genetic arrangements, and although biological data suggests they are functional erythritol transporters, phylogenetic analysis suggests they may not be orthologs and probably should be considered analogs. Finally, evidence for the presence of paralogs, and xenologs of erythritol catabolic genes in some of the genomes included in the analysis is provided.

Background

Bacterial toxin-antitoxin (TA) systems are formed by potent regulatory or suicide factors (toxins) and their short-lived inhibitors (antitoxins). Antitoxins are DNA-binding proteins and auto-repress transcription of TA operons. Transcription of multiple TA operons is activated in temporarily non-growing persister cells that can resist killing by antibiotics. Consequently, the antitoxin levels of persisters must have been dropped and toxins are released of inhibition.

Results

Here, we describe transcriptional cross-activation between different TA systems of Escherichia coli. We find that the chromosomal relBEF operon is activated in response to production of the toxins MazF, MqsR, HicA, and HipA. Expression of the RelE toxin in turn induces transcription of several TA operons. We show that induction of mazEF during amino acid starvation depends on relBE and does not occur in a relBEF deletion mutant. Induction of TA operons has been previously shown to depend on Lon protease which is activated by polyphospate accumulation. We show that transcriptional cross-activation occurs also in strains deficient for Lon, ClpP, and HslV proteases and polyphosphate kinase. Furthermore, we find that toxins cleave the TA mRNA in vivo, which is followed by degradation of the antitoxin-encoding fragments and selective accumulation of the toxin-encoding regions. We show that these accumulating fragments can be translated to produce more toxin.

Conclusion

Transcriptional activation followed by cleavage of the mRNA and disproportionate production of the toxin constitutes a possible positive feedback loop, which can fire other TA systems and cause bistable growth heterogeneity. Cross-interacting TA systems have a potential to form a complex network of mutually activating regulators in bacteria.

Background

A growing body of evidence suggests that Mycobacterium tuberculosis (Mtb) uses the host’s cholesterol as a source of carbon and energy during infection. Strains defective in cholesterol transport or degradation exhibit attenuated growth in activated macrophages and diminished infectivity in animal models. The aim of this study was to evaluate intracellular replication of a cholesterol degradation-deficient Mtb mutant in human macrophages (MØ) in vitro and assess the functional responses of Mtb mutant-infected MØ.

Results

A mutant Mtb H37Rv strain containing an inactivated kstD gene (∆kstD), which encodes 3-ketosteroid 1(2)-dehydrogenase (KstD), was previously prepared using the homologous recombination-based gene-replacement technique. A control strain carrying the kstD gene complemented with an intact kstD was also previously constructed. In this study, human resting MØ were obtained after overnight differentiation of the human monocyte-macrophage cell line THP-1. Resting MØ were further activated with interferon-γ (IFN-γ). The ability of the kstD-defective Mtb mutant strain to replicate intracellularly in human MØ was evaluated using a colony-forming assay. Nitric oxide (NO) and reactive oxygen species (ROS) production by MØ infected with wild-type or ∆kstD strains was detected using Griess reagent and chemiluminescence methods, respectively. The production of tumor necrosis factor-α and interleukin-10 by MØ after infection with wild-type or mutant Mtb was examined using enzyme-linked immunosorbent assays.

We found that replication of mutant Mtb was attenuated in resting MØ compared to the wild-type or complemented strains. Moreover, the mutant was unable to inhibit the NO and ROS production induced through Toll-like receptor 2 (TLR2) signaling in infected resting MØ. In contrast, mutant and wild-type Mtb behaved similarly in MØ activated with IFN-γ before and during infection.

Conclusions

The Mtb mutant ∆kstD strain, which is unable to use cholesterol as a source of carbon and energy, has a limited ability to multiply in resting MØ following infection, reflecting a failure of the ∆kstD strain to inhibit the TLR2-dependent bactericidal activity of resting MØ.

Contributing reviewers

The editors of BMC Microbiology would like to thank all our reviewers who have contributed to the journal in Volume 12 (2012).

Background

Bacteriocins are protein antimicrobial agents that are produced by all prokaryotic lineages. Escherichia coli strains frequently produce the bacteriocins known as colicins. One of the most prevalent colicins, colicin M, can kill susceptible cells by hydrolyzing the peptidoglycan lipid II intermediate, which arrests peptidoglycan polymerization steps and provokes cell lysis. Due to the alarming rise in antibiotic resistance and the lack of novel antimicrobial agents, colicin M has recently received renewed attention as a promising antimicrobial candidate. Here the effects of subinhibitory concentrations of colicin M on whole genome transcription in E. coli were investigated, to gain insight into its ecological role and for purposes related to antimicrobial therapy.

Results

Transcriptome analysis revealed that exposure to subinhibitory concentrations of colicin M altered expression of genes involved in envelope, osmotic and other stresses, including genes of the CreBC two-component system, exopolysaccharide production and cell motility. Nonetheless, there was no induction of biofilm formation or genes involved in mutagenesis.

Conclusion

At subinhibitory concentrations colicin M induces an adaptive response primarily to protect the bacterial cells against envelope stress provoked by peptidoglycan damage. Among the first induced were genes of the CreBC two-component system known to promote increased resistance against colicins M and E2, providing novel insight into the ecology of colicin M production in natural environments. While an adaptive response was induced nevertheless, colicin M application did not increase biofilm formation, nor induce SOS genes, adverse effects that can be provoked by a number of traditional antibiotics, providing support for colicin M as a promising antimicrobial agent.

Background

In humans, Streptococcus agalactiae or group B streptococcus (GBS) is a frequent coloniser of the rectovaginal tract, a major cause of neonatal infectious disease and an emerging cause of disease in non-pregnant adults. In addition, Streptococcus agalactiae causes invasive disease in fish, compromising food security and posing a zoonotic hazard. We studied the molecular epidemiology of S. agalactiae in fish and other aquatic species to assess potential for pathogen transmission between aquatic species and humans.

Methods

Isolates from fish (n = 26), seals (n = 6), a dolphin and a frog were characterized by pulsed-field gel electrophoresis, multilocus sequence typing and standardized 3-set genotyping, i.e. molecular serotyping and profiling of surface protein genes and mobile genetic elements.

Results

Four subpopulations of S. agalactiae were identified among aquatic isolates. Sequence type (ST) 283 serotype III-4 and its novel single locus variant ST491 were detected in fish from Southeast Asia and shared a 3-set genotype identical to that of an emerging ST283 clone associated with invasive disease of adult humans in Asia. The human pathogenic strain ST7 serotype Ia was also detected in fish from Asia. ST23 serotype Ia, a subpopulation that is normally associated with human carriage, was found in all grey seals, suggesting that human effluent may contribute to microbial pollution of surface water and exposure of sea mammals to human pathogens. The final subpopulation consisted of non-haemolytic ST260 and ST261 serotype Ib isolates, which belong to a fish-associated clonal complex that has never been reported from humans.

Conclusions

The apparent association of the four subpopulations of S. agalactiae with specific groups of host species suggests that some strains of aquatic S. agalactiae may present a zoonotic or anthroponotic hazard. Furthermore, it provides a rational framework for exploration of pathogenesis and host-associated genome content of S. agalactiae strains.

Background

Dengue virus (DENV) infection represents a significant public health problem in many subtropical and tropical countries. Although genetically closely related, the four serotypes of DENV differ in antigenicity for which cross protection among serotypes is limited. It is also believed that both multi-serotype infection as well as the evolution of viral antigenicity may have confounding effects in increased dengue epidemics. Numerous studies have been performed that investigated genetic diversity of DENV, but the precise mechanism(s) of dengue virus evolution are not well understood.

Results

We investigated genome-wide genetic diversity and nucleotide substitution patterns in the four serotypes among samples collected from different countries in Asia and Central and South America and sequenced as part of the Genome Sequencing Center for Infectious Diseases at the Broad Institute. We applied bioinformatics, statistical and coalescent simulation methods to investigate diversity of codon sequences of DENV samples representing the four serotypes. We show that fixation of nucleotide substitutions is more prominent among the inter-continental isolates (Asian and American) of serotypes 1, 2 and 3 compared to serotype 4 isolates (South and Central America) and are distributed in a non-random manner among the genes encoded by the virus. Nearly one third of the negatively selected sites are associated with fixed mutation sites within serotypes. Our results further show that of all the sites showing evidence of recombination, the majority (~84%) correspond to sites under purifying selection in the four serotypes. The analysis further shows that genetic recombination occurs within specific codons, albeit with low frequency (< 5% of all recombination sites) throughout the DENV genome of the four serotypes and reveals significant enrichment (p < 0.05) among sites under purifying selection in the virus.

Conclusion

The study provides the first evidence for intracodon recombination in DENV and suggests that within codons, genetic recombination has a significant role in maintaining extensive purifying selection of DENV in natural populations. Our study also suggests that fixation of beneficial mutations may lead to virus evolution via translational selection of specific sites in the DENV genome.

Background

Glucose is a signaling molecule which regulates multiple events in eukaryotic organisms and the most preferred carbon source in the fission yeast Schizosaccharomyces pombe. The ability of this yeast to grow in the absence of glucose becomes strongly limited due to lack of enzymes of the glyoxylate cycle that support diauxic growth. The stress-activated protein kinase (SAPK) pathway and its effectors, Sty1 MAPK and transcription factor Atf1, play a critical role in the adaptation of fission yeast to grow on alternative non-fermentable carbon sources by inducing the expression of fbp1+ gene, coding for the gluconeogenic enzyme fructose-1,6-bisphosphatase. The cell integrity Pmk1 pathway is another MAPK cascade that regulates various processes in fission yeast, including cell wall construction, cytokinesis, and ionic homeostasis. Pmk1 pathway also becomes strongly activated in response to glucose deprivation but its role during glucose exhaustion and ensuing adaptation to respiratory metabolism is currently unknown.

Results

We found that Pmk1 activation in the absence of glucose takes place only after complete depletion of this carbon source and that such activation is not related to an endogenous oxidative stress. Notably, Pmk1 MAPK activation relies on de novo protein synthesis, is independent on known upstream activators of the pathway like Rho2 GTPase, and involves PKC ortholog Pck2. Also, the Glucose/cAMP pathway is required operative for full activation of the Pmk1 signaling cascade. Mutants lacking Pmk1 displayed a partial growth defect in respiratory media which was not observed in the presence of glucose. This phenotype was accompanied by a decreased and delayed expression of transcription factor Atf1 and target genes fbp1+ and pyp2+. Intriguingly, the kinetics of Sty1 activation in Pmk1-less cells was clearly altered during growth adaptation to non-fermentable carbon sources.

Conclusions

Unknown upstream elements mediate Pck2-dependent signal transduction of glucose withdrawal to the cell integrity MAPK pathway. This signaling cascade reinforces the adaptive response of fission yeast to such nutritional stress by enhancing the activity of the SAPK pathway.

Background

Hfq is an RNA chaperone protein that has been broadly implicated in sRNA function in bacteria. Here we describe the construction and characterization of a null allele of the gene that encodes the RNA chaperone Hfq in Shewanella oneidensis strain MR-1, a dissimilatory metal reducing bacterium.

Results

Loss of hfq in S. oneidensis results in a variety of mutant phenotypes, all of which are fully complemented by addition of a plasmid-borne copy of the wild type hfq gene. Aerobic cultures of the hfq∆ mutant grow more slowly through exponential phase than wild type cultures, and hfq∆ cultures reach a terminal cell density in stationary phase that is ~2/3 of that observed in wild type cultures. We have observed a similar growth phenotype when the hfq∆ mutant is cultured under anaerobic conditions with fumarate as the terminal electron acceptor, and we have found that the hfq∆ mutant is defective in Cr(VI) reduction. Finally, the hfq∆ mutant exhibits a striking loss of colony forming units in extended stationary phase and is highly sensitive to oxidative stress induced by H2O2 or methyl viologen (paraquat).

Conclusions

The hfq mutant in S. oneidensis exhibits pleiotropic phenotypes, including a defect in metal reduction. Our results also suggest that hfq mutant phenotypes in S. oneidensis may be at least partially due to increased sensitivity to oxidative stress.

Background

Mycoplasmas-contamination of Orientia tsutsugamushi, one of the obligated intracellular bacteria, is a very serious problem in in vitro studies using cell cultures because mycoplasmas have significant influence on the results of scientific studies. Only a recommended decontamination method is to passage the contaminated O. tsutsugamushi strains through mice to eliminate only mycoplasmas under influence of their immunity. However, this method sometimes does not work especially for low virulent strains of O. tsutsugamushi which are difficult to propagate in mice. In this study, we tried to eliminate mycoplasmas contaminants from both high virulent and low virulent strains of the contaminated O. tsutsugamushi by repeating passage through cell cultures with antibiotics in vitro.

Results

We cultured a contaminated, high virulent strain of O. tsutsugamushi using a mouse lung fibroblasts cell line, L-929 cell in the culture medium containing lincomycin at various concentrations and repeated passages about every seven days. At the passage 5 only with 10 μg/ml of lincomycin, we did not detect mycoplasmas by two PCR based methods whereas O. tsutsugamushi continued good growth. During following four passages without lincomycin, mycoplasmas did not recover. These results suggested that mycoplasmas were completely eliminated from the high virulent strain of O. tsutsugamushi. Furthermore, by the same procedures with 10 μg/ml of lincomycin, we also eliminated mycoplasmas from a contaminated, low virulent strain of O. tsutsugamushi. Our additional assay showed that 50 μg/ml of lyncomycin did not inhibit the growth of O. tsutsugamushi, although MICs of many mycoplasmas contaminants were less than 6 μg/ml as shown previously.

Conclusion

Our results showed an alternative method to eliminate mycoplasmas from the contaminated O. tsutsugamushi strains in place of in vivo passage through mice. Especially this notable method works for the decontamination not only from the high virulent strain also from the low virulent strain of O. tsutsugamushi. For further elimination, lincomycin at the limit concentration, which does not inhibit the growth of O. tsutsugamushi, can possibly eliminate most mycoplasmas from contaminated O. tsutsugamushi strains.

Background

Heavy-metals exert considerable stress on the environment worldwide. This study assessed the resistance to and bioremediation of heavy-metals by selected protozoan and bacterial species in highly polluted industrial-wastewater. Specific variables (i.e. chemical oxygen demand, pH, dissolved oxygen) and the growth/die-off-rates of test organisms were measured using standard methods. Heavy-metal removals were determined in biomass and supernatant by the Inductively Couple Plasma Optical Emission Spectrometer. A parallel experiment was performed with dead microbial cells to assess the biosorption ability of test isolates.

Results

The results revealed that the industrial-wastewater samples were highly polluted with heavy-metal concentrations exceeding by far the maximum limits (in mg/l) of 0.05-Co, 0.2-Ni, 0.1-Mn, 0.1-V, 0.01-Pb, 0.01-Cu, 0.1-Zn and 0.005-Cd, prescribed by the UN-FAO. Industrial-wastewater had no major effects on Pseudomonas putida, Bacillus licheniformis and Peranema sp. (growth rates up to 1.81, 1.45 and 1.43 d-1, respectively) compared to other test isolates. This was also revealed with significant COD increases (p < 0.05) in culture media inoculated with living bacterial isolates (over 100%) compared to protozoan isolates (up to 24% increase). Living Pseudomonas putida demonstrated the highest removal rates of heavy metals (Co-71%, Ni-51%, Mn-45%, V-83%, Pb-96%, Ti-100% and Cu-49%) followed by Bacillus licheniformis (Al-23% and Zn-53%) and Peranema sp. (Cd-42%). None of the dead cells were able to remove more than 25% of the heavy metals. Bacterial isolates contained the genes copC, chrB, cnrA3 and nccA encoding the resistance to Cu, Cr, Co-Ni and Cd-Ni-Co, respectively. Protozoan isolates contained only the genes encoding Cu and Cr resistance (copC and chrB genes). Peranema sp. was the only protozoan isolate which had an additional resistant gene cnrA3 encoding Co-Ni resistance.

Conclusion

Significant differences (p < 0.05) observed between dead and living microbial cells for metal-removal and the presence of certain metal-resistant genes indicated that the selected microbial isolates used both passive (biosorptive) and active (bioaccumulation) mechanisms to remove heavy metals from industrial wastewater. This study advocates the use of Peranema sp. as a potential candidate for the bioremediation of heavy-metals in wastewater treatment, in addition to Pseudomonas putida and Bacillus licheniformis.

Background

The oral spirochete bacterium Treponema denticola is associated with both the incidence and severity of periodontal disease. Although the biological or phenotypic properties of a significant number of T. denticola isolates have been reported in the literature, their genetic diversity or phylogeny has never been systematically investigated. Here, we describe a multilocus sequence analysis (MLSA) of 20 of the most highly studied reference strains and clinical isolates of T. denticola; which were originally isolated from subgingival plaque samples taken from subjects from China, Japan, the Netherlands, Canada and the USA.

Results

The sequences of the 16S ribosomal RNA gene, and 7 conserved protein-encoding genes (flaA, recA, pyrH, ppnK, dnaN, era and radC) were successfully determined for each strain. Sequence data was analyzed using a variety of bioinformatic and phylogenetic software tools. We found no evidence of positive selection or DNA recombination within the protein-encoding genes, where levels of intraspecific sequence polymorphism varied from 18.8% (flaA) to 8.9% (dnaN). Phylogenetic analysis of the concatenated protein-encoding gene sequence data (ca. 6,513 nucleotides for each strain) using Bayesian and maximum likelihood approaches indicated that the T. denticola strains were monophyletic, and formed 6 well-defined clades. All analyzed T. denticola strains appeared to have a genetic origin distinct from that of ‘Treponema vincentii’ or Treponema pallidum. No specific geographical relationships could be established; but several strains isolated from different continents appear to be closely related at the genetic level.

Conclusions

Our analyses indicate that previous biological and biophysical investigations have predominantly focused on a subset of T. denticola strains with a relatively narrow range of genetic diversity. Our methodology and results establish a genetic framework for the discrimination and phylogenetic analysis of T. denticola isolates, which will greatly assist future biological and epidemiological investigations involving this putative ‘periodontopathogen’.

Background

Diffusely adherent Escherichia coli (DAEC) have been considered a diarrheagenic category of E. coli for which several potential virulence factors have been described in the last few years. Despite this, epidemiological studies involving DAEC have shown inconsistent results. In this work, two different collections of DAEC possessing Afa/Dr genes, from children and adults, were studied regarding characteristics potentially associated to virulence.

Results

DAEC strains were recovered in similar frequencies from diarrheic and asymptomatic children, and more frequently from adults with diarrhea (P < 0.01) than from asymptomatic adults. Association with diarrhea (P < 0.05) was found for SAT-positive strains recovered from children and for curli-positive strains recovered from adults. Mixed biofilms involving DAEC and a Citrobacter freundii strain have shown an improved ability to form biofilms in relation to the monocultures. Control strains have shown a greater diversity of Afa/Dr adhesins and higher frequencies of cellulose, TTSS, biofilm formation and induction of IL-8 secretion than strains from cases of diarrhea in children.

Conclusions

DAEC strains possessing Afa/Dr genes isolated from children and adults represent two different bacterial populations. DAEC strains carrying genes associated to virulence can be found as part of the normal microbiota present in asymptomatic children.

Background

A prerequisite for the virulence of the facultative intracellular bacterium Francisella tularensis is effective intramacrophage proliferation, which is preceded by phagosomal escape into the cytosol, and ultimately leads to host cell death. Many components essential for the intracellular life cycle are encoded by a gene cluster, the Francisella pathogenicity island (FPI), constituting a type VI secretion system.

Results

We characterized the FPI mutant ΔpdpC of the live vaccine strain (LVS) of F. tularensis and found that it exhibited lack of intracellular replication, incomplete phagosomal escape, and marked attenuation in the mouse model, however, unlike a phagosomally contained FPI mutant, it triggered secretion of IL-1β, albeit lower than LVS, and markedly induced LDH release.

Conclusions

The phenotype of the ΔpdpC mutant appears to be unique compared to previously described F. tularensis FPI mutants.

Background

Uterine infections in dairy cows lower profitability of dairy operations. Infections of the reproductive tract are related to the overgrowth of pathogenic bacteria during the first three weeks after parturition. However, alterations in the vaginal microbiota composition in the first weeks after parturition remain poorly documented.

Results

In this study, bacteria isolated from the vagina of healthy pregnant, and infected postpartum cows were characterised by random amplification of polymorphic DNA (RAPD) analysis and partial 16S ribosomal RNA (rDNA) gene sequencing. Populations of bacilli and lactic acid bacteria of the genera Enterococcus, Lactobacillus, and Pediococcus were present in both healthy and infected cows. Infected cows had a significant increase in the vaginal enteric bacteria population which consisted mainly of Escherichia coli. Three E. coli isolates harboured the gene coding for Shiga-like-toxin (SLT) I or II. Several isolates of the Pediococcus acidilactici were found to produce the bacteriocin pediocin AcH/PA-1. Quantitative PCR analyses of vaginal mucus samples collected from ten metritic cows before and after parturition confirmed the presence of the Lactobacillus group (Lactobacillus spp., Pediococcus spp., Leuconostoc spp., and Weissella spp.); Enterobacteriaceae, E. coli, and bacilli. The presence of the pediocin AcH/PA-1 structural gene and SLT genes were also confirmed with qPCR.

Conclusions

In conclusion, overgrowth of pathogenic bacteria, particularly E. coli, after parturition likely contributes to the development of metritis. Our microbiota analysis extends the information related to the composition of commensal bacteria in the bovine female reproductive tract and may facilitate the development of novel intervention strategies for prevention of uterine infections in dairy cows.

Background

Several cases of legionellosis have been diagnosed in the same French thermal spa in 1986, 1994 and 1997. L. pneumophila serogroup 1 (Lp1) strains have been isolated from several patients, but the source of contamination was not identified despite the presence of different Lp1 in water samples of the three natural springs feeding the spa at this period.

Results

Our strategy was to investigate L. pneumophila (Lp) strains from natural biofilms developed in a sulphur-rich warm spring of this contaminated site. Biofilm analysis revealed the presence of three Lp serogroups (Lp1, Lp10 and Lp12). Surprisingly, Lp10 and Lp12 were not reported in the previous described studies from water samples. Besides, the new seven Lp1 we isolated exhibit a high molecular diversity and have been differentiated in five classes according to their DNA genome patterns obtained by PFGE and mip sequences. It must be noted that these DNA patterns are original and unknown in databases. Interestingly, the 27 Lp environmental strains we isolated display a higher cytotoxicity and virulence towards the amoeba Acanthamoeba castellanii than those of known Lp1 epidemic strains.

Conclusion

The characteristics of Legionella pneumophila Lp1 strains isolated from the warm spring are in agreement with their presence in biofilms and their probable long-term persistence in this ecosystem.

Background

The Staphylococcus aureus RecU protein is homologous to a Bacillus subtilis Holliday junction resolvase. Interestingly, RecU is encoded in the same operon as PBP2, a penicillin-binding protein required for cell wall synthesis and essential for the full expression of resistance in Methicillin Resistant S. aureus strains. In this work we have studied the role of RecU in the clinical pathogen S. aureus.

Results

Depletion of RecU in S. aureus results in the appearance of cells with compact nucleoids, septa formed over the DNA and anucleate cells. RecU-depleted cells also show increased septal recruitment of the DNA translocase SpoIIIE, presumably to resolve chromosome segregation defects. Additionally cells are more sensitive to DNA damaging agents such as mitomycin C or UV radiation. Expression of RecU from the ectopic chromosomal spa locus showed that co-expression of RecU and PBP2 was not necessary to ensure correct cell division, a process that requires tight coordination between chromosome segregation and septal cell wall synthesis.

Conclusions

RecU is required for correct chromosome segregation and DNA damage repair in S. aureus. Co-expression of recU and pbp2 from the same operon is not required for normal cell division.

Background

Listeriosis is a severe infection which mainly affects pregnant women, neonates and immuno-compromised adults. ANSES’s Laboratory for Food safety has been the European Union Reference Laboratory (EURL) for L. monocytogenes in the food chain since 2006. Pulsed Field Gel Electrophoresis (PFGE) is routinely used in the EURL for the surveillance of L. monocytogenes isolated from foods, animals and the environment. One of the main EURL activities is to evaluate alternative molecular subtyping methods to PFGE, and integrate their use within the National Reference Laboratories (NRL) network. Since 2008, the United Kingdom (UK)-NRL for L. monocytogenes at the Health Protection Agency (HPA), London, has used fluorescent Amplified Fragment Length Polymorphism (fAFLP) for the routine surveillance of L. monocytogenes isolated from human clinical cases, food and food processing environments in the UK. This study compares fAFLP with PFGE for subtyping L. monocytogenes.

Results

A panel of 109 L. monocytogenes isolates from either human cases of listeriosis, foods, food processing environments and animals were used for the comparative evaluation. Among these, 2 strains were tested from duplicate culture by both methods. The panel also included field isolates, isolates associated with outbreaks or sporadic cases and reference strains. The two strains tested in duplicate displayed the same fAFLP and PFGE types. Strains known to be epidemiologically associated with one another were found to have unique PFGE and fAFLP types. FAFLP and PFGE divided the strains into 76 and 82 distinct profiles, or types, respectively. The discriminatory index calculated was 0.993 and 0.996 for fAFLP and PFGE, respectively.

Conclusions

The discriminatory ability of fAFLP was similar to that of PFGE for the subtyping of L. monocytogenes isolates. As a less labour intensive technique fAFLP may be a better method to use than PFGE in investigating outbreaks of human listeriosis and tracking the source of contamination in food processing facilities in real time.

Background

The microorganisms intended for use as probiotics in aquaculture should exert antimicrobial activity and be regarded as safe not only for the aquatic hosts but also for their surrounding environments and humans. The objective of this work was to investigate the antimicrobial/bacteriocin activity against fish pathogens, the antibiotic susceptibility, and the prevalence of virulence factors and detrimental enzymatic activities in 99 Lactic Acid Bacteria (LAB) (59 enterococci and 40 non-enterococci) isolated from aquatic animals regarded as human food.

Results

These LAB displayed a broad antimicrobial/bacteriocin activity against the main Gram-positive and Gram-negative fish pathogens. However, particular safety concerns based on antibiotic resistance and virulence factors were identified in the genus Enterococcus (86%) (Enterococcus faecalis, 100%; E. faecium, 79%). Antibiotic resistance was also found in the genera Weissella (60%), Pediococcus (44%), Lactobacillus (33%), but not in leuconostocs and lactococci. Antibiotic resistance genes were found in 7.5% of the non-enterococci, including the genera Pediococcus (12.5%) and Weissella (6.7%). One strain of both Pediococcus pentosaceus and Weissella cibaria carried the erythromycin resistance gene mef(A/E), and another two P. pentosaceus strains harboured lnu(A) conferring resistance to lincosamides. Gelatinase activity was found in E. faecalis and E. faecium (71 and 11%, respectively), while a low number of E. faecalis (5%) and none E. faecium exerted hemolytic activity. None enterococci and non-enterococci showed bile deconjugation and mucin degradation abilities, or other detrimental enzymatic activities.

Conclusions

To our knowledge, this is the first description of mef(A/E) in the genera Pediococcus and Weissella, and lnu(A) in the genus Pediococcus. The in vitro subtractive screening presented in this work constitutes a valuable strategy for the large-scale preliminary selection of putatively safe LAB intended for use as probiotics in aquaculture.

Background

Several genotyping protocols have been described to study Candida albicans strains with different sensitivity values. In this study we have analyzed the genetic relatedness and the antifungal susceptibility of several Candida albicans strains isolated from a patient who from suffered recurrent candiduria for a period of five years. Strains were genotyped using Microsatellite Length Polymorphism (MLP) with three microsatellite markers (HIS 3, EF 3 and CDC 3), and a new method based on high resolution melting (HRM) was developed to analyze the microsatellite region. This method was compared with the conventional technique that uses capillary electrophoresis.

Results

MICs of the isolates showed the existence of fluconazole susceptible and resistant strains. An inter-colony test using single concentration (8 and 16 mg/l) of fluconazole revealed the coexistence of both fluconazole susceptible and resistant strains. Both genotyping analysis methods showed that all the patient’s isolates had a clonal origin. HRM analysis method developed was able to accurately establish strain relatedness and presented a discriminatory power of 0.77.

Conclusions

Although HRM analysis method presented a lower discriminatory power compared to methods based on capillary electrophoresis, it provided a more cost-effective and suitable alternative for genotyping C. albicans in a clinical laboratory.

Background

Deviations in composition and diversity of intestinal microbiota in infancy have been associated with both the development and recurrence of atopic eczema. Thus, we decided to use a deep and global microarray-based method to characterize the diversity and temporal changes of the intestinal microbiota in infancy and to define specific bacterial signatures associated with eczema. Faecal microbiota at 6 and 18 months of age were analysed from 34 infants (15 with eczema and 19 healthy controls) selected from a prospective follow-up study based on the availability of faecal samples. The infants were originally randomized to receive either Lactobacillus rhamnosus GG or placebo.

Results

Children with eczema harboured a more diverse total microbiota than control subjects as assessed by the Simpson’s reciprocal diversity index of the microarray profiles. Composition of the microbiota did not differ between study groups at age of 6 months, but was significantly different at age of 18 months as assessed by MCPP (p=0.01). At this age healthy children harboured 3 -fold greater amount of members of the Bacteroidetes (p=0.01). Microbiota of children suffering from eczema had increased abundance of the Clostridium clusters IV and XIVa, which are typically abundant in adults. Probiotic Lactobacillus rhamnosus GG supplementation in early infancy was observed to have minor long-term effects on the microbiota composition.

Conclusion

A diverse and adult-type microbiota in early childhood is associated with eczema and it may contribute to the perpetuation of eczema.