An extracellular serine protease produced by Vibrio vulnificus NCIMB 2137, a metalloprotease-gene negative strain isolated from a diseased eel

Vibrio vulnificus is a ubiquitous estuarine microorganism but causes fatal systemic infections in immunocompromised humans, cultured eels or shrimps. An extracellular metalloprotease VVP/VvpE has been reported to be a potential virulence factor of the bacterium; however, a few strains isolated from a diseased eel or shrimp were recently found to produce a serine protease termed VvsA, but not VVP/VvpE. In the present study, we found that these strains had lost the 80 kb genomic region including the gene encoding VVP/VvpE. We also purified VvsA from the culture supernatant through ammonium sulfate fractionation, gel filtration and ion-exchange column chromatography, and the enzyme was demonstrated to be a chymotrypsin-like protease, as well as those from some vibrios. The gene vvsA was shown to constitute an operon with a downstream gene vvsB, and several Vibrio species were found to have orthologues of vvsAB. These findings indicate that the genes vvp/vvpE and vvsAB might be mobile genetic elements

Genetic analysis of the capsule polysaccharide (K antigen) and exopolysaccharide genes in pandemic Vibrio parahaemolyticus O3:K6

<p>Abstract</p> <p>Background</p> <p>Pandemic <it>Vibrio parahaemolyticus </it>has undergone rapid changes in both K- and O-antigens, making detection of outbreaks more difficult. In order to understand these rapid changes, the genetic regions encoding these antigens must be examined. In <it>Vibrio cholerae </it>and <it>Vibrio vulnificus</it>, both O-antigen and capsular polysaccharides are encoded in a single region on the large chromosome; a similar arrangement in pandemic <it>V. parahaemolyticus </it>would help explain the rapid serotype changes. However, previous reports on "capsule" genes are controversial. Therefore, we set out to clarify and characterize these regions in pandemic <it>V. parahaemolyticus </it>O3:K6 by gene deletion using a chitin based transformation strategy.</p> <p>Results</p> <p>We generated different deletion mutants of putative polysaccharide genes and examined the mutants by immuno-blots with O and K specific antisera. Our results showed that O- and K-antigen genes are separated in <it>V. parahaemolyticus </it>O3:K6; the region encoding both O-antigen and capsule biosynthesis in other vibrios, i.e. genes between <it>gmhD </it>and <it>rjg</it>, determines the K6-antigen but not the O3-antigen in <it>V. parahaemolyticus</it>. The previously identified "capsule genes" on the smaller chromosome were related to exopolysaccharide synthesis, not K-antigen.</p> <p>Conclusion</p> <p>Understanding of the genetic basis of O- and K-antigens is critical to understanding the rapid changes in these polysaccharides seen in pandemic <it>V. parahaemolyticus. </it>This report confirms the genetic location of K-antigen synthesis in <it>V. parahaemolyticus </it>O3:K6 allowing us to focus future studies of the evolution of serotypes to this region.</p

Comparative efficacies of different antibiotic treatments to eradicate nontypeable Haemophilus influenzae infection

<p>Abstract</p> <p>Background</p> <p>Nonencapsulated and nontypeable <it>Haemophilus influenzae </it>(NTHi) is a major cause of human respiratory tract infections. Some strains of NTHi can cause invasive diseases such as septicemia and meningitis, even if <it>H. influenzae </it>is not generally considered to be an intracellular pathogen. There have been very few reports about the therapeutic efficacy of antibiotics against respiratory tract infection caused by NTHi in mice because it is difficult for <it>H. influenzae </it>to infect mice. Therefore, we evaluated the efficacy of antibiotics against NTHi in both a cell culture model and a mouse model of infection.</p> <p>Methods</p> <p>We used six strains of NTHi isolated from adult patients with chronic otitis media, namely three β-lactamase-negative ampicillin (AMP)-resistant (BLNAR) strains and three β-lactamase-negative AMP-susceptible (BLNAS) strains, to evaluate the efficacy of AMP, cefcapene (CFPN), levofloxacin (LVX), clarithromycin (CLR), and azithromycin (AZM) in both a cell culture infection model and a mouse infection model. In the cell culture infection model, strains that invade A549 human alveolar epithelial cells were treated with each antibiotic (1 μg/ml). In the mouse infection model, female C57BL/6 mice were intraperitoneally injected with cyclophosphamide (200 mg/kg) three days before intranasal infection with 1 × 10⁹colony-forming units (CFU) of NTHi and on the day of infection. After infection, the mice were orally administered each antibiotic three times daily for three days, except for AZM, which was administered once daily for three days, at a dose of 100 mg/kg/day.</p> <p>Results</p> <p>In the cell culture infection model, it was found that two BLNAR strains were able to enter the cell monolayers by the process of macropinocytosis, and treatment with LVX yielded good bactericidal activity against both strains inside the cells. In the mouse infection model, no bacteria were detected by means of plating the lung homogenates of LVX-treated mice at day 4 after infection, while more than 10⁵CFU of bacteria per tissue sample were detected in nontreated mice.</p> <p>Conclusion</p> <p>Our findings show the outcome and rich benefits of fluoroquinolone treatment of respiratory infections caused by either invasive or noninvasive BLNAR strains of NTHi.</p

Retrospective application of transposon-directed insertion-site sequencing to investigate niche-specific virulence of Salmonella Typhimurium in cattle.

Background: Salmonella enterica subspecies enterica is an animal and zoonotic pathogen of global importance. Cattle are a significant reservoir of human non-typhoidal salmonellosis and can suffer enteric and systemic disease owing to the ability of Salmonella to survive within the bovine lymphatic system and intestines. Contamination of food can occur due to the incorporation of contaminated peripheral lymph nodes or by direct contamination of carcasses with gut contents. It is essential to understand the mechanisms used by Salmonella to enter and persist within the bovine lymphatic system and how they differ from those required for intestinal colonization to minimize zoonotic infections. Results: Transposon-directed insertion site sequencing (TraDIS) was applied to pools of mutants recovered from mesenteric lymph nodes (MLNs) draining the distal ileum of calves after oral inoculation with a library of 8550 random S. Typhimurium mini-Tn5Km2 mutants in pools of 475 mutants per calf. A total of 8315 mutants representing 2852 different genes were detected in MLNs and their in vivo fitness was calculated. Using the same improved algorithm for analysis of transposon-flanking sequences, the identity and phenotype of mutants recovered from the distal ileal mucosa of the same calves was also defined, enabling comparison with previously published data and of mutant phenotypes across the tissues. Phenotypes observed for the majority of mutants were highly significantly correlated in the two tissues. However, 32 genes were identified in which transposon insertions consistently resulted in differential fitness in the ileal wall and MLNs, suggesting niche-specific roles for these genes in pathogenesis. Defined null mutations affecting ptsN and spvC were confirmed to result in tissue-specific phenotypes in calves, thus validating the TraDIS dataset. Conclusions: This validation of the role of thousands of Salmonella genes and identification of genes with niche-specific roles in a key target species will inform the design of control strategies for bovine salmonellosis and zoonotic infections, for which efficacious and cross-protective vaccines are currently lacking

Metagenomics - a guide from sampling to data analysis

Metagenomics applies a suite of genomic technologies and bioinformatics tools to directly access the genetic content of entire communities of organisms. The field of metagenomics has been responsible for substantial advances in microbial ecology, evolution, and diversity over the past 5 to 10 years, and many research laboratories are actively engaged in it now. With the growing numbers of activities also comes a plethora of methodological knowledge and expertise that should guide future developments in the field. This review summarizes the current opinions in metagenomics, and provides practical guidance and advice on sample processing, sequencing technology, assembly, binning, annotation, experimental design, statistical analysis, data storage, and data sharing. As more metagenomic datasets are generated, the availability of standardized procedures and shared data storage and analysis becomes increasingly important to ensure that output of individual projects can be assessed and compared

Salmonella paratyphi C: Genetic Divergence from Salmonella choleraesuis and Pathogenic Convergence with Salmonella typhi

BACKGROUND: Although over 1400 Salmonella serovars cause usually self-limited gastroenteritis in humans, a few, e.g., Salmonella typhi and S. paratyphi C, cause typhoid, a potentially fatal systemic infection. It is not known whether the typhoid agents have evolved from a common ancestor (by divergent processes) or acquired similar pathogenic traits independently (by convergent processes). Comparison of different typhoid agents with non-typhoidal Salmonella lineages will provide excellent models for studies on how similar pathogens might have evolved. METHODOLOGIES/PRINCIPAL FINDINGS: We sequenced a strain of S. paratyphi C, RKS4594, and compared it with previously sequenced Salmonella strains. RKS4594 contains a chromosome of 4,833,080 bp and a plasmid of 55,414 bp. We predicted 4,640 intact coding sequences (4,578 in the chromosome and 62 in the plasmid) and 152 pseudogenes (149 in the chromosome and 3 in the plasmid). RKS4594 shares as many as 4346 of the 4,640 genes with a strain of S. choleraesuis, which is primarily a swine pathogen, but only 4008 genes with another human-adapted typhoid agent, S. typhi. Comparison of 3691 genes shared by all six sequenced Salmonella strains placed S. paratyphi C and S. choleraesuis together at one end, and S. typhi at the opposite end, of the phylogenetic tree, demonstrating separate ancestries of the human-adapted typhoid agents. S. paratyphi C seemed to have suffered enormous selection pressures during its adaptation to man as suggested by the differential nucleotide substitutions and different sets of pseudogenes, between S. paratyphi C and S. choleraesuis. CONCLUSIONS: S. paratyphi C does not share a common ancestor with other human-adapted typhoid agents, supporting the convergent evolution model of the typhoid agents. S. paratyphi C has diverged from a common ancestor with S. choleraesuis by accumulating genomic novelty during adaptation to man

Characterization of Salmonella Type III Secretion Hyper-Activity Which Results in Biofilm-Like Cell Aggregation

We have previously reported the cloning of the Salmonella enterica serovar Typhimurium SPI-1 secretion system and the use of this clone to functionally complement a ΔSPI-1 strain for type III secretion activity. In the current study, we discovered that S. Typhimurium cultures containing cloned SPI-1 display an adherent biofilm and cell clumps in the media. This phenotype was associated with hyper-expression of SPI-1 type III secretion functions. The biofilm and cell clumps were associated with copious amounts of secreted SPI-1 protein substrates SipA, SipB, SipC, SopB, SopE, and SptP. We used a C-terminally FLAG-tagged SipA protein to further demonstrate SPI-1 substrate association with the cell aggregates using fluorescence microscopy and immunogold electron microscopy. Different S. Typhimurium backgrounds and both flagellated and nonflagellated strains displayed the biofilm phenotype. Mutations in genes essential for known bacterial biofilm pathways (bcsA, csgBA, bapA) did not affect the biofilms formed here indicating that this phenomenon is independent of established biofilm mechanisms. The SPI-1-mediated biofilm was able to massively recruit heterologous non-biofilm forming bacteria into the adherent cell community. The results indicate a bacterial aggregation phenotype mediated by elevated SPI-1 type III secretion activity with applications for engineered biofilm formation, protein purification strategies, and antigen display

Molecular Basis of Increased Serum Resistance among Pulmonary Isolates of Non-typeable Haemophilus influenzae

Non-typeable Haemophilus influenzae (NTHi), a common commensal of the human pharynx, is also an opportunistic pathogen if it becomes established in the lower respiratory tract (LRT). In comparison to colonizing isolates from the upper airway, LRT isolates, especially those associated with exacerbations of chronic obstructive pulmonary disease, have increased resistance to the complement- and antibody-dependent, bactericidal effect of serum. To define the molecular basis of this resistance, mutants constructed in a serum resistant strain using the mariner transposon were screened for loss of survival in normal human serum. The loci required for serum resistance contribute to the structure of the exposed surface of the bacterial outer membrane. These included loci involved in biosynthesis of the oligosaccharide component of lipooligosaccharide (LOS), and vacJ, which functions with an ABC transporter encoded by yrb genes in retrograde trafficking of phospholipids from the outer to inner leaflet of the cell envelope. Mutations in vacJ and yrb genes reduced the stability of the outer membrane and were associated with increased cell surface hyrophobicity and phospholipid content. Loss of serum resistance in vacJ and yrb mutants correlated with increased binding of natural immunoglobulin M in serum as well as anti-oligosaccharide mAbs. Expression of vacJ and the yrb genes was positively correlated with serum resistance among clinical isolates. Our findings suggest that NTHi adapts to inflammation encountered during infection of the LRT by modulation of its outer leaflet through increased expression of vacJ and yrb genes to minimize recognition by bactericidal anti-oligosaccharide antibodies

Analysis of Pools of Targeted Salmonella Deletion Mutants Identifies Novel Genes Affecting Fitness during Competitive Infection in Mice

Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS)

The Regulatory Network of Natural Competence and Transformation of Vibrio cholerae

The human pathogen Vibrio cholerae is an aquatic bacterium frequently encountered in rivers, lakes, estuaries, and coastal regions. Within these environmental reservoirs, the bacterium is often found associated with zooplankton and more specifically with their chitinous exoskeleton. Upon growth on such chitinous surfaces, V. cholerae initiates a developmental program termed “natural competence for genetic transformation.” Natural competence for transformation is a mode of horizontal gene transfer in bacteria and contributes to the maintenance and evolution of bacterial genomes. In this study, we investigated competence gene expression within this organism at the single cell level. We provide evidence that under homogeneous inducing conditions the majority of the cells express competence genes. A more heterogeneous expression pattern was observable on chitin surfaces. We hypothesize that this was the case due to the heterogeneity around the chitin surface, which might vary extensively with respect to chitin degradation products and autoinducers; these molecules contribute to competence induction based on carbon catabolite repression and quorum-sensing pathways, respectively. Therefore, we investigated the contribution of these two signaling pathways to natural competence in detail using natural transformation assays, transcriptional reporter fusions, quantitative RT–PCR, and immunological detection of protein levels using Western blot analysis. The results illustrate that all tested competence genes are dependent on the transformation regulator TfoX. Furthermore, intracellular cAMP levels play a major role in natural transformation. Finally, we demonstrate that only a minority of genes involved in natural transformation are regulated in a quorum-sensing-dependent manner and that these genes determine the fate of the surrounding DNA. We conclude with a model of the regulatory circuit of chitin-induced natural competence in V. cholerae