Multilocus sequence typing breathes life into a microbial metagenome.
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Shot-gun sequencing of DNA isolated from the environment and the assembly of metagenomes from the resulting data has considerably advanced the study of microbial diversity. However, the subsequent matching of these hypothetical metagenomes to cultivable microorganisms is a limitation of such cultivation-independent methods of population analysis. Using a nucleotide sequence-based genetic typing method, multilocus sequence typing, we were able for the first time to match clonal cultivable isolates to a published and controversial bacterial metagenome, Burkholderia SAR-1, which derived from analysis of the Sargasso Sea. The matching cultivable isolates were all associated with infection and geographically widely distributed; taxonomic analysis demonstrated they were members of Burkholderia cepacia complex Group K. Comparison of the Burkholderia SAR-1 metagenome to closely related B. cepacia complex genomes indicated that it was greater than 98% intact in terms of conserved genes, and it also shared complete sequence identity with the cultivable isolates at random loci beyond the genes sampled by the multilocus sequence typing. Two features of the extant cultivable clones support the argument that the Burkholderia SAR-1 sequence may have been a contaminant in the original metagenomic survey: (i) their growth in conditions reflective of sea water was poor, suggesting the ocean was not their preferred habitat, and (ii) several of the matching isolates were epidemiologically linked to outbreaks of infection that resulted from contaminated medical devices or products, indicating an adaptive fitness of this bacterial strain towards contamination-associated environments. The ability to match identical cultivable strains of bacteria to a hypothetical metagenome is a unique feature of nucleotide sequence-based microbial typing methods; such matching would not have been possible with more traditional methods of genetic typing, such as those based on pattern matching of genomic restriction fragments or amplified DNA fragments. Overall, we have taken the first steps in moving the status of the Burkholderia SAR-1 metagenome from a hypothetical entity towards the basis for life of cultivable strains that may now be analysed in conjunction with the assembled metagenomic sequence data by the wider scientific community. (+info)
Proteomic identification and characterization of bacterial factors associated with Burkholderia cenocepacia survival in a murine host.
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Burkholderia cenocepacia is a member of the Burkholderia cepacia complex, a diverse family of Gram-negative bacteria that are serious respiratory pathogens in immunocompromised patients and individuals with cystic fibrosis. To identify putative bacterial virulence determinants, proteomic profiles were compared between two B. cenocepacia isolates that demonstrated differential persistence in a mouse model of pulmonary infection; clinical isolate C1394 is rapidly cleared from the murine lung whereas the strain variant, C1394mp2, persists. Two-dimensional (2D) gel electrophoresis was used to identify candidate proteins involved in B. cenocepacia survival in a susceptible host. The 2D proteome of the persistent isolate (C1394mp2) revealed loss of an alkyl hydroperoxide reductase subunit C (AhpC) protein spot and increased production of flagellin proteins. Loss of AhpC expression in C1394mp2 correlated with enhanced susceptibility to oxidative stress. C1394mp2 expressed increased flagellin production and enhanced swimming motility, traits that were subject to regulation by heat and low pH. Together, these results revealed differential expression and stress regulation of putative virulence determinants associated with B. cenocepacia persistence in a susceptible host. (+info)
Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo.
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Burkholderia cenocepacia, a member of the B. cepacia complex, is an opportunistic pathogen that causes serious infections in patients with cystic fibrosis. We identified a six-gene cluster in chromosome 1 encoding a two-component regulatory system (BCAL2831 and BCAL2830) and an HtrA protease (BCAL2829) hypothesized to play a role in the B. cenocepacia stress response. Reverse transcriptase PCR analysis of these six genes confirmed they are cotranscribed and comprise an operon. Genes in this operon, including htrA, were insertionally inactivated by recombination with a newly created suicide plasmid, pGPOmegaTp. Genetic analyses and complementation studies revealed that HtrA(BCAL2829) was required for growth of B. cenocepacia upon exposure to osmotic stress (NaCl or KCl) and thermal stress (44 degrees C). In addition, replacement of the serine residue in the active site with alanine (S245A) and deletion of the HtrA(BCAL2829) PDZ domains demonstrated that these areas are required for protein function. HtrA(BCAL2829) also localizes to the periplasmic compartment, as shown by Western blot analysis and a colicin V reporter assay. Using the rat agar bead model of chronic lung infection, we also demonstrated that inactivation of the htrA gene is associated with a bacterial survival defect in vivo. Together, our data demonstrate that HtrA(BCAL2829) is a virulence factor in B. cenocepacia. (+info)
Nasal immunization with Burkholderia multivorans outer membrane proteins and the mucosal adjuvant adamantylamide dipeptide confers efficient protection against experimental lung infections with B. multivorans and B. cenocepacia.
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Chronic lung infection by opportunistic pathogens, such as Pseudomonas aeruginosa and members of the Burkholderia cepacia complex, is a major cause of morbidity and mortality in patients with cystic fibrosis. Outer membrane proteins (OMPs) of gram-negative bacteria are promising vaccine antigen candidates. In this study, we evaluated the immunogenicity, protection, and cross-protection conferred by intranasal vaccination of mice with OMPs from B. multivorans plus the mucosal adjuvant adamantylamide dipeptide (AdDP). Robust mucosal and systemic immune responses were stimulated by vaccination of naive animals with OMPs from B. multivorans and B. cenocepacia plus AdDP. Using a mouse model of chronic pulmonary infection, we observed enhanced clearance of B. multivorans from the lungs of vaccinated animals, which correlated with OMP-specific secretory immunoglobulin A responses. Furthermore, OMP-immunized mice showed rapid resolution of the pulmonary infection with virtually no lung pathology after bacterial challenge with B. multivorans. In addition, we demonstrated that administration of B. multivorans OMP vaccine conferred protection against B. cenocepacia challenge in this mouse infection model, suggesting that OMPs provide cross-protection against the B. cepacia complex. Therefore, we concluded that mucosal immunity to B. multivorans elicited by intranasal vaccination with OMPs plus AdDP could prevent early steps of colonization and infection with B. multivorans and also ameliorate lung tissue damage, while eliciting cross-protection against B. cenocepacia. These results support the notion that therapies leading to increased mucosal immunity in the airways may help patients with cystic fibrosis. (+info)
Multiscale responses of microbial life to spatial distance and environmental heterogeneity in a patchy ecosystem.
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Spatial distance (SD) and environmental heterogeneity (EH) are currently thought to represent major factors shaping genetic variation and population abundance, but their relative importance is still poorly understood. Because EH varies at multiple spatial scales, so too are microbial variables expected to vary. The determination of SD x EH interactions at multiple scales is, however, not a trivial exercise, especially when one examines their effects on microbial abundance and genomic similarities. Here we assessed those interactions at all scales perceptible in a patchy environment composed of known plant species and of heterogeneous soil physical and chemical parameters. For free-living, soil-borne Burkholderia ambifaria, genomic similarities responded to most of the spatial scales that the experimental sampling scheme could reveal, despite limited dispersal of the individuals. Species abundance and community composition were, however, responding to much smaller scales more consistent with local responses to EH. Our results suggest that whole-genome similarities may reflect the simultaneous effects of both SD and EH in microbial populations, but the pure effects of each factor only contributed to < 2% of the total genetic variation. The large amount of unexplained variation that remains after considering most environmental, spatial, and biological interactions is then posited to be the result of noise introduced by unmeasured environmental and spatial variability, sampling effects, and neutral ecological drift. (+info)
Emerging cystic fibrosis pathogens: incidence and antimicrobial resistance.
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We examined the frequency of isolation and the antimicrobial resistance of Burkholderia cepacia complex, Stenotrophomonas maltophilia and Achromobacter xylosoxidans in cystic fibrosis patients from 2000 to 2004. Strains susceptibility to tobramycin, piperacillin/tazobactam, imipenem, gentamicin, ciprofloxacin and ceftazidime was determined by disc diffusion assay. B. cepacia complex showed a very high resistance also to ciprofloxacin reaching 100% in 2004. S. maltophilia and A. xvylosoxidans showed high rates of antimicrobial resistance both aminoglycoside and ciprofloxacin. It is very important to monitor the percentage of isolation of these species over time to verify strains resistance to antibiotics and also to test new combinations of antimicrobial agents. (+info)
Characterization of SodC, a periplasmic superoxide dismutase from Burkholderia cenocepacia.
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Burkholderia cenocepacia is a gram-negative, non-spore-forming bacillus and a member of the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly in phagocytic cells and can produce at least one superoxide dismutase (SOD). The inability of O2- to cross the cytoplasmic membrane, coupled with the periplasmic location of Cu,ZnSODs, suggests that periplasmic SODs protect bacteria from superoxide that has an exogenous origin (for example, when cells are faced with reactive oxygen intermediates generated by host cells in response to infection). In this study, we identified the sodC gene encoding a Cu,ZnSOD in B. cenocepacia and demonstrated that a sodC null mutant was not sensitive to a H2O2, 3-morpholinosydnonimine, or paraquat challenge but was killed by exogenous superoxide generated by the xanthine/xanthine oxidase method. The sodC mutant also exhibited a growth defect in liquid medium compared to the parental strain, which could be complemented in trans. The mutant was killed more rapidly than the parental strain was killed in murine macrophage-like cell line RAW 264.7, but killing was eliminated when macrophages were treated with an NADPH oxidase inhibitor. We also confirmed that SodC is periplasmic and identified the metal cofactor. B. cenocepacia SodC was resistant to inhibition by H2O2 and was unusually resistant to KCN for a Cu,ZnSOD. Together, these observations establish that B. cenocepacia produces a periplasmic Cu,ZnSOD that protects this bacterium from exogenously generated O2- and contributes to intracellular survival of this bacterium in macrophages. (+info)
A putative gene cluster for aminoarabinose biosynthesis is essential for Burkholderia cenocepacia viability.
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Using a conditional mutagenesis strategy we demonstrate here that a gene cluster encoding putative aminoarabinose (Ara4N) biosynthesis enzymes is essential for the viability of Burkholderia cenocepacia. Loss of viability is associated with dramatic changes in bacterial cell morphology and ultrastructure, increased permeability to propidium iodide, and sensitivity to sodium dodecyl sulfate, suggesting a general cell envelope defect caused by the lack of Ara4N. (+info)