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Sample records for burkholderia cenocepacia strain

  1. Draft Genome Sequence of Burkholderia cenocepacia Strain CEIB S5-2, a Methyl Parathion- and p-Nitrophenol-Degrading Bacterium, Isolated from Agricultural Soils in Morelos, Mexico.

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    Martínez-Ocampo, Fernando; Fernández López, Maikel Gilberto; Lozano-Aguirre Beltrán, Luis Fernando; Popoca-Ursino, Elida Carolina; Ortiz-Hernández, M Laura; Sánchez-Salinas, Enrique; Ramos Quintana, Fernando; Villalobos-López, Miguel A; Dantán-González, Edgar

    2016-04-28

    Burkholderia cenocepacia is an opportunistic pathogen that belongs to Burkholderia cepacia complex (BCC). Burkholderia cenocepacia strain CEIB S5-2 was isolated from agricultural soils in Morelos, Mexico, and previously has shown its abilities for bioremediation. In this study, we report the draft genome sequence of Burkholderia cenocepacia strain CEIB S5-2.

  2. Burkholderia cenocepacia Strain CEIB S5-1, a Rhizosphere-Inhabiting Bacterium with Potential in Bioremediation

    OpenAIRE

    Martínez-Ocampo, Fernando; Lozano-Aguirre Beltrán, Luis Fernando; Hernández-Mendoza, Armando; Rojas-Espinoza, Luis Enrique; Popoca-Ursino, Elida Carolina; Ortiz-Hernández, María Laura; Sánchez-Salinas, Enrique; Ramos Quintana, Fernando; Dantán-González, Edgar

    2015-01-01

    Burkholderia cenocepacia is considered an opportunistic pathogen from humans and may cause disease in plants. A bioprospection from a plaguicide-contaminated agricultural field in Mexico identified several methyl parathion-degrading bacteria. Here, we report the draft genome sequence of B. cenocepacia strain CEIB S5-1, which gave us clues into ecological biodiversity.

  3. Burkholderia cenocepacia Strain CEIB S5-1, a Rhizosphere-Inhabiting Bacterium with Potential in Bioremediation.

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    Martínez-Ocampo, Fernando; Lozano-Aguirre Beltrán, Luis Fernando; Hernández-Mendoza, Armando; Rojas-Espinoza, Luis Enrique; Popoca-Ursino, Elida Carolina; Ortiz-Hernández, María Laura; Sánchez-Salinas, Enrique; Ramos Quintana, Fernando; Dantán-González, Edgar

    2015-03-05

    Burkholderia cenocepacia is considered an opportunistic pathogen from humans and may cause disease in plants. A bioprospection from a plaguicide-contaminated agricultural field in Mexico identified several methyl parathion-degrading bacteria. Here, we report the draft genome sequence of B. cenocepacia strain CEIB S5-1, which gave us clues into ecological biodiversity.

  4. Host evasion by Burkholderia cenocepacia

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    Shyamala eGanesan

    2012-01-01

    Full Text Available Burkholderia cenocepacia is an opportunistic respiratory pathogen of individuals with cystic fibrosis (CF. It is one of the highly transmissible species of Burkholderia cepacia complex and very resistant to almost all the antibiotics. Approximately 1/3rd of B. cenocepacia infected CF patients go on to develop fatal ‘cepacia syndrome’. During the last two decades, substantial progress has been made with regards to evasion of host innate defense mechanisms by B. cenocepacia. Almost all strains of B. cenocepacia has capacity to survive and replicate intracellularly in both airway epithelial cells and macrophages, which are primary centennials of the lung and play a pivotal role in clearance of infecting bacteria. Some strains of B. cenocepaica, which express cable pili and the associated 22kDa adhesin are also capable of transmigrating across airway epithelium and persist in mouse models of infection. In this review, we will discuss how this type of interaction between B. cenocepacia and host may lead to persistence of bacteria and contribute to lung inflammation in CF patients.

  5. Identification of Burkholderia cenocepacia strain H111 virulence factors using nonmammalian infection hosts

    DEFF Research Database (Denmark)

    Schwager, Stephan; Agnoli, Kirsty; Köthe, Manuela;

    2013-01-01

    Burkholderia cenocepacia H111, a strain isolated from a cystic fibrosis patient, has been shown to effectively kill the nematode Caenorhabditis elegans. We used the C. elegans model of infection to screen a mini-Tn5 mutant library of B. cenocepacia H111 for attenuated virulence....... Of the approximately 5,500 B. cenocepacia H111 random mini-Tn5 insertion mutants that were screened, 22 showed attenuated virulence in C. elegans. Except for the quorum-sensing regulator cepR, none of the mutated genes coded for the biosynthesis of classical virulence factors such as extracellular proteases...... or siderophores. Instead, the mutants contained insertions in metabolic and regulatory genes. Mutants attenuated in virulence in the C. elegans infection model were also tested in the Drosophila melanogaster pricking model, and those also attenuated in this model were further tested in Galleria mellonella. Six...

  6. Strains of Burkholderia cenocepacia genomovar IIIA possessing the cblA gene that are distinct from ET12.

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    Turton, Jane F; O'Brien, Emily; Megson, Brian; Kaufmann, Mary E; Pitt, Tyrone L

    2009-05-01

    Three strains of Burkholderia cenocepacia genomovar IIIA that were polymerase chain reaction positive for cblA, bcrA, and the epidemic strain marker, but were distinct from representatives of ET12 by pulsed-field gel electrophoresis, are described. One of these strains was shown to express cable pili by electron microscopy.

  7. Draft Genome Sequence of Burkholderia cenocepacia Strain 869T2, a Plant-Beneficial Endophytic Bacterium

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    Ho, Ying-Ning

    2015-01-01

    An endophytic bacterium, Burkholderia cenocepacia 869T2, isolated from vetiver grass, has shown its abilities for both in planta biocontrol and plant growth promotion. Its draft genome sequence was determined to provide insights into those metabolic pathways involved in plant-beneficial activity. This is the first genome report for endophytic B. cenocepacia. PMID:26564046

  8. Draft Genome Sequence of Burkholderia cenocepacia Strain 869T2, a Plant-Beneficial Endophytic Bacterium.

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    Ho, Ying-Ning; Huang, Chieh-Chen

    2015-11-12

    An endophytic bacterium, Burkholderia cenocepacia 869T2, isolated from vetiver grass, has shown its abilities for both in planta biocontrol and plant growth promotion. Its draft genome sequence was determined to provide insights into those metabolic pathways involved in plant-beneficial activity. This is the first genome report for endophytic B. cenocepacia.

  9. Deciphering the role of RND efflux transporters in Burkholderia cenocepacia.

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    Silvia Bazzini

    Full Text Available Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division efflux pumps are known to be among the mediators of multidrug resistance in gram-negative bacteria. Since the significance of the 16 RND efflux systems present in B. cenocepacia (named RND-1 to -16 has been only partially determined, the aim of this work was to analyze mutants of B. cenocepacia strain J2315 impaired in RND-4 and RND-9 efflux systems, and assess their role in the efflux of toxic compounds. The transcriptomes of mutants deleted individually in RND-4 and RND-9 (named D4 and D9, and a double-mutant in both efflux pumps (named D4-D9, were compared to that of the wild-type B. cenocepacia using microarray analysis. Microarray data were confirmed by qRT-PCR, phenotypic experiments, and by Phenotype MicroArray analysis. The data revealed that RND-4 made a significant contribution to the antibiotic resistance of B. cenocepacia, whereas RND-9 was only marginally involved in this process. Moreover, the double mutant D4-D9 showed a phenotype and an expression profile similar to D4. The microarray data showed that motility and chemotaxis-related genes appeared to be up-regulated in both D4 and D4-D9 strains. In contrast, these gene sets were down-regulated or expressed at levels similar to J2315 in the D9 mutant. Biofilm production was enhanced in all mutants. Overall, these results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing additional phenotypic traits important for pathogenesis.

  10. Burkholderia cenocepacia zinc metalloproteases influence resistance to antimicrobial peptides.

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    Kooi, Cora; Sokol, Pamela A

    2009-09-01

    Burkholderia cenocepacia secretes two zinc-dependent metalloproteases, designated ZmpA and ZmpB. Previously, ZmpA and ZmpB have been shown to cleave several proteins important in host defence. In this study, the ability of ZmpA and ZmpB to digest and inactivate antimicrobial peptides involved in innate immunity was examined. ZmpB but not ZmpA cleaved beta-defensin-1. ZmpA but not ZmpB cleaved the cathelicidin LL-37. Both enzymes cleaved elafin and secretory leukocyte inhibitor, which are antimicrobial peptides as well as neutrophil elastase inhibitors. Both ZmpA and ZmpB cleaved protamine, a fish antimicrobial peptide, and a zmpA zmpB mutant was more sensitive to protamine killing than the parental strain. ZmpA or ZmpB cleavage of elafin inactivated its anti-protease activity. The effect of ZmpA and ZmpB on the neutrophil proteases elastase and cathepsin G was also examined but neither enzyme was active against these host proteases. These studies suggest that ZmpA and ZmpB may influence the resistance of B. cenocepacia to host antimicrobial peptides as well as alter the host protease/anti-protease balance in chronic respiratory infections.

  11. The genome of Burkholderia cenocepacia J2315, an epidemic pathogen of cystic fibrosis patients

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    Holden, Matthew T G; Seth-Smith, Helena M B; Crossman, Lisa C

    2009-01-01

    Bacterial infections of the lungs of cystic fibrosis (CF) patients cause major complications in the treatment of this common genetic disease. Burkholderia cenocepacia infection is particularly problematic since this organism has high levels of antibiotic resistance, making it difficult to eradicate...... of this highly transmissible pathogen comprises three circular chromosomes and a plasmid and encodes a broad array of functions typical of this metabolically versatile genus, as well as numerous virulence and drug resistance functions. Although B. cenocepacia strains can be isolated from soil and can...

  12. The Genome of Burkholderia cenocepacia J2315, an Epidemic Pathogen of Cystic Fibrosis Patients ▿ †

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    Holden, Matthew T. G.; Seth-Smith, Helena M. B.; Crossman, Lisa C.; Sebaihia, Mohammed; Bentley, Stephen D.; Cerdeño-Tárraga, Ana M.; Thomson, Nicholas R.; Bason, Nathalie; Quail, Michael A.; Sharp, Sarah; Cherevach, Inna; Churcher, Carol; Goodhead, Ian; Hauser, Heidi; Holroyd, Nancy; Mungall, Karen; Scott, Paul; Walker, Danielle; White, Brian; Rose, Helen; Iversen, Pernille; Mil-Homens, Dalila; Rocha, Eduardo P. C.; Fialho, Arsenio M.; Baldwin, Adam; Dowson, Christopher; Barrell, Bart G.; Govan, John R.; Vandamme, Peter; Hart, C. Anthony; Mahenthiralingam, Eshwar; Parkhill, Julian

    2009-01-01

    Bacterial infections of the lungs of cystic fibrosis (CF) patients cause major complications in the treatment of this common genetic disease. Burkholderia cenocepacia infection is particularly problematic since this organism has high levels of antibiotic resistance, making it difficult to eradicate; the resulting chronic infections are associated with severe declines in lung function and increased mortality rates. B. cenocepacia strain J2315 was isolated from a CF patient and is a member of the epidemic ET12 lineage that originated in Canada or the United Kingdom and spread to Europe. The 8.06-Mb genome of this highly transmissible pathogen comprises three circular chromosomes and a plasmid and encodes a broad array of functions typical of this metabolically versatile genus, as well as numerous virulence and drug resistance functions. Although B. cenocepacia strains can be isolated from soil and can be pathogenic to both plants and man, J2315 is representative of a lineage of B. cenocepacia rarely isolated from the environment and which spreads between CF patients. Comparative analysis revealed that ca. 21% of the genome is unique in comparison to other strains of B. cenocepacia, highlighting the genomic plasticity of this species. Pseudogenes in virulence determinants suggest that the pathogenic response of J2315 may have been recently selected to promote persistence in the CF lung. The J2315 genome contains evidence that its unique and highly adapted genetic content has played a significant role in its success as an epidemic CF pathogen. PMID:18931103

  13. Investigation of the multifaceted iron acquisition strategies of Burkholderia cenocepacia.

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    Tyrrell, J; Whelan, N; Wright, C; Sá-Correia, I; McClean, S; Thomas, M; Callaghan, Máire

    2015-04-01

    Burkholderia cenocepacia is a bacterial pathogen which causes severe respiratory infections in cystic fibrosis (CF). These studies were aimed at gaining an insight into the iron acquisition strategies of B. cenocepacia. In iron restricted conditions, genes associated with the synthesis and utilisation of ornibactin (pvdA, orbA, orb F) were significantly upregulated compared to the expression of pyochelin associated genes (pchD, fptA). In the absence of alternative iron sources, B. cenocepacia J2315 and 715j utilised ferritin and haemin, but not transferrin or lactoferrin for growth. Significantly, mutants unable to produce ornibactin, (715j-orbI) or ornibactin and pyochelin, (715j-pobA), utilised haemin and ferritin more efficiently than the wild-type. Moreover, both mutants were also able to utilise lactoferrin for growth (P ≤ 0.01) and additionally 715j-pobA utilised transferrin (P ≤ 0.01), potentially facilitating adaptation to the host environment. Furthermore, B. cenocepacia increased ornibactin gene expression in response to pyoverdine from Pseudomonas aeruginosa (P ≤ 0.01), demonstrating the capacity to compete for iron in co-colonised niches. Pyoverdine also significantly diminished the growth of B. cenocepacia (P < 0.001) which was related to its iron chelating activity. In a study of three B. cenocepacia sequential clonal isolates obtained from a CF patient over a 3.5 year period, ornibactin upregulation in response to pyoverdine was less pronounced in the last isolate compared to the earlier isolates, as was growth in the presence of haemin and ferritin, indicating alternative iron acquisition mechanism(s) may dominate as chronic infection progresses. These data demonstrate the multifaceted iron acquisition strategies of B. cenocepacia and their capacity to be differentially activated in the presence of P. aeruginosa and during chronic infection.

  14. Construction of a large-scale Burkholderia cenocepacia J2315 transposon mutant library

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    Wong, Yee-Chin; Pain, Arnab; Nathan, Sheila

    2014-09-01

    Burkholderia cenocepacia, a pathogenic member of the Burkholderia cepacia complex (Bcc), has emerged as a significant threat towards cystic fibrosis patients, where infection often leads to the fatal clinical manifestation known as cepacia syndrome. Many studies have investigated the pathogenicity of B. cenocepacia as well as its ability to become highly resistant towards many of the antibiotics currently in use. In addition, studies have also been undertaken to understand the pathogen's capacity to adapt and survive in a broad range of environments. Transposon based mutagenesis has been widely used in creating insertional knock-out mutants and coupled with recent advances in sequencing technology, robust tools to study gene function in a genome-wide manner have been developed based on the assembly of saturated transposon mutant libraries. In this study, we describe the construction of a large-scale library of B. cenocepacia transposon mutants. To create transposon mutants of B. cenocepacia strain J2315, electrocompetent bacteria were electrotransformed with the EZ-Tn5 transposome. Tetracyline resistant colonies were harvested off selective agar and pooled. Mutants were generated in multiple batches with each batch consisting of ˜20,000 to 40,000 mutants. Transposon insertion was validated by PCR amplification of the transposon region. In conclusion, a saturated B. cenocepacia J2315 transposon mutant library with an estimated total number of 500,000 mutants was successfully constructed. This mutant library can now be further exploited as a genetic tool to assess the function of every gene in the genome, facilitating the discovery of genes important for bacterial survival and adaptation, as well as virulence.

  15. Efflux pump genes of the resistance-nodulation-division family in Burkholderia cenocepacia genome

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    Manina Giulia

    2006-07-01

    Full Text Available Abstract Background Burkholderia cenocepacia is recognized as opportunistic pathogen that can cause lung infections in cystic fibrosis patients. A hallmark of B. cenocepacia infections is the inability to eradicate the organism because of multiple intrinsic antibiotic resistance. As Resistance-Nodulation-Division (RND efflux systems are responsible for much of the intrinsic multidrug resistance in Gram-negative bacteria, this study aims to identify RND genes in the B. cenocepacia genome and start to investigate their involvement into antimicrobial resistance. Results Genome analysis and homology searches revealed 14 open reading frames encoding putative drug efflux pumps belonging to RND family in B. cenocepacia J2315 strain. By reverse transcription (RT-PCR analysis, it was found that orf3, orf9, orf11, and orf13 were expressed at detectable levels, while orf10 appeared to be weakly expressed in B. cenocepacia. Futhermore, orf3 was strongly induced by chloramphenicol. The orf2 conferred resistance to fluoroquinolones, tetraphenylphosphonium, streptomycin, and ethidium bromide when cloned and expressed in Escherichia coli KAM3, a strain lacking the multidrug efflux pump AcrAB. The orf2-overexpressing E. coli also accumulate low concentrations of ethidium bromide, which was restored to wild type level in the presence of CCCP, an energy uncoupler altering the energy of the drug efflux pump. Conclusion The 14 RND pumps gene we have identified in the genome of B. cenocepacia suggest that active efflux could be a major mechanism underlying antimicrobial resistance in this microorganism. We have characterized the ORF2 pump, one of these 14 potential RND efflux systems. Its overexpression in E. coli conferred resistance to several antibiotics and to ethidium bromide but it remains to be determined if this pump play a significant role in the antimicrobial intrinsic resistance of B. cenocepacia. The characterization of antibiotic efflux pumps in B

  16. Exploring the metabolic network of the epidemic pathogen Burkholderia cenocepacia J2315 via genome-scale reconstruction

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    Panda Gurudutta

    2011-05-01

    Full Text Available Abstract Background Burkholderia cenocepacia is a threatening nosocomial epidemic pathogen in patients with cystic fibrosis (CF or a compromised immune system. Its high level of antibiotic resistance is an increasing concern in treatments against its infection. Strain B. cenocepacia J2315 is the most infectious isolate from CF patients. There is a strong demand to reconstruct a genome-scale metabolic network of B. cenocepacia J2315 to systematically analyze its metabolic capabilities and its virulence traits, and to search for potential clinical therapy targets. Results We reconstructed the genome-scale metabolic network of B. cenocepacia J2315. An iterative reconstruction process led to the establishment of a robust model, iKF1028, which accounts for 1,028 genes, 859 internal reactions, and 834 metabolites. The model iKF1028 captures important metabolic capabilities of B. cenocepacia J2315 with a particular focus on the biosyntheses of key metabolic virulence factors to assist in understanding the mechanism of disease infection and identifying potential drug targets. The model was tested through BIOLOG assays. Based on the model, the genome annotation of B. cenocepacia J2315 was refined and 24 genes were properly re-annotated. Gene and enzyme essentiality were analyzed to provide further insights into the genome function and architecture. A total of 45 essential enzymes were identified as potential therapeutic targets. Conclusions As the first genome-scale metabolic network of B. cenocepacia J2315, iKF1028 allows a systematic study of the metabolic properties of B. cenocepacia and its key metabolic virulence factors affecting the CF community. The model can be used as a discovery tool to design novel drugs against diseases caused by this notorious pathogen.

  17. Differential roles of RND efflux pumps in antimicrobial drug resistance of sessile and planktonic Burkholderia cenocepacia cells.

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    Buroni, Silvia; Matthijs, Nele; Spadaro, Francesca; Van Acker, Heleen; Scoffone, Viola C; Pasca, Maria Rosalia; Riccardi, Giovanna; Coenye, Tom

    2014-12-01

    Burkholderia cenocepacia is notorious for causing respiratory tract infections in people with cystic fibrosis. Infections with this organism are particularly difficult to treat due to its high level of intrinsic resistance to most antibiotics. Multidrug resistance in B. cenocepacia can be ascribed to different mechanisms, including the activity of efflux pumps and biofilm formation. In the present study, the effects of deletion of the 16 operons encoding resistance-nodulation-cell division (RND)-type efflux pumps in B. cenocepacia strain J2315 were investigated by determining the MICs of various antibiotics and by investigating the antibiofilm effect of these antibiotics. Finally, the expression levels of selected RND genes in treated and untreated cultures were investigated using reverse transcriptase quantitative PCR (RT-qPCR). Our data indicate that the RND-3 and RND-4 efflux pumps are important for resistance to various antimicrobial drugs (including tobramycin and ciprofloxacin) in planktonic B. cenocepacia J2315 populations, while the RND-3, RND-8, and RND-9 efflux systems protect biofilm-grown cells against tobramycin. The RND-8 and RND-9 efflux pumps are not involved in ciprofloxacin resistance. Results from the RT-qPCR experiments on the wild-type strain B. cenocepacia J2315 suggest that there is little regulation at the level of mRNA expression for these efflux pumps under the conditions tested.

  18. Biofilms produced by Burkholderia cenocepacia: influence of media and solid supports on composition of matrix exopolysaccharides.

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    Pellizzoni, Elena; Ravalico, Fabio; Scaini, Denis; Delneri, Ambra; Rizzo, Roberto; Cescutti, Paola

    2016-02-01

    Bacteria usually grow forming biofilms, which are communities of cells embedded in a self-produced dynamic polymeric matrix, characterized by a complex three-dimensional structure. The matrix holds cells together and above a surface, and eventually releases them, resulting in colonization of other surfaces. Although exopolysaccharides (EPOLs) are important components of the matrix, determination of their structure is usually performed on samples produced in non-biofilm conditions, or indirectly through genetic studies. Among the Burkholderia cepacia complex species, Burkholderia cenocepacia is an important pathogen in cystic fibrosis (CF) patients and is generally more aggressive than other species. In the present investigation, B. cenocepacia strain BTS2, a CF isolate, was grown in biofilm mode on glass slides and cellulose membranes, using five growth media, one of which mimics the nutritional content of CF sputum. The structure of the matrix EPOLs was determined by 1H-NMR spectroscopy, while visualization of the biofilms on glass slides was obtained by means of confocal laser microscopy, phase-contrast microscopy and atomic force microscopy. The results confirmed that the type of EPOLs biosynthesized depends both on the medium used and on the type of support, and showed that mucoid conditions do not always lead to significant biofilm production, while bacteria in a non-mucoid state can still form biofilm containing EPOLs.

  19. Comparative analysis of two phenotypically-similar but genomically-distinct Burkholderia cenocepacia-specific bacteriophages

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    Lynch Karlene H

    2012-06-01

    Full Text Available Abstract Background Genomic analysis of bacteriophages infecting the Burkholderia cepacia complex (BCC is an important preliminary step in the development of a phage therapy protocol for these opportunistic pathogens. The objective of this study was to characterize KL1 (vB_BceS_KL1 and AH2 (vB_BceS_AH2, two novel Burkholderia cenocepacia-specific siphoviruses isolated from environmental samples. Results KL1 and AH2 exhibit several unique phenotypic similarities: they infect the same B. cenocepacia strains, they require prolonged incubation at 30°C for the formation of plaques at low titres, and they do not form plaques at similar titres following incubation at 37°C. However, despite these similarities, we have determined using whole-genome pyrosequencing that these phages show minimal relatedness to one another. The KL1 genome is 42,832 base pairs (bp in length and is most closely related to Pseudomonas phage 73 (PA73. In contrast, the AH2 genome is 58,065 bp in length and is most closely related to Burkholderia phage BcepNazgul. Using both BLASTP and HHpred analysis, we have identified and analyzed the putative virion morphogenesis, lysis, DNA binding, and MazG proteins of these two phages. Notably, MazG homologs identified in cyanophages have been predicted to facilitate infection of stationary phase cells and may contribute to the unique plaque phenotype of KL1 and AH2. Conclusions The nearly indistinguishable phenotypes but distinct genomes of KL1 and AH2 provide further evidence of both vast diversity and convergent evolution in the BCC-specific phage population.

  20. Reciprocal regulation by the CepIR and CciIR quorum sensing systems in Burkholderia cenocepacia

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    Malott Rebecca J

    2009-09-01

    Full Text Available Abstract Background Burkholderia cenocepacia belongs to a group of closely related organisms called the B. cepacia complex (Bcc which are important opportunistic human pathogens. B. cenocepacia utilizes a mechanism of cell-cell communication called quorum sensing to control gene expression including genes involved in virulence. The B. cenocepacia quorum sensing network includes the CepIR and CciIR regulatory systems. Results Global gene expression profiles during growth in stationary phase were generated using microarrays of B. cenocepacia cepR, cciR and cepRcciIR mutants. This is the first time CciR was shown to be a global regulator of quorum sensing gene expression. CepR was primarily responsible for positive regulation of gene expression while CciR generally exerted negative gene regulation. Many of the genes that were regulated by both quorum sensing systems were reciprocally regulated by CepR and CciR. Microarray analysis of the cepRcciIR mutant suggested that CepR is positioned upstream of CciR in the quorum sensing hierarchy in B. cenocepacia. A comparison of CepIR-regulated genes identified in previous studies and in the current study showed a substantial amount of overlap validating the microarray approach. Several novel quorum sensing-controlled genes were confirmed using qRT-PCR or promoter::lux fusions. CepR and CciR inversely regulated flagellar-associated genes, the nematocidal protein AidA and a large gene cluster on Chromosome 3. CepR and CciR also regulated genes required for iron transport, synthesis of extracellular enzymes and surface appendages, resistance to oxidative stress, and phage-related genes. Conclusion For the first time, the influence of CciIR on global gene regulation in B. cenocepacia has been elucidated. Novel genes under the control of the CepIR and CciIR quorum sensing systems in B. cenocepacia have been identified. The two quorum sensing systems exert reciprocal regulation of many genes likely enabling fine

  1. Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

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    Venturi Vittorio

    2009-09-01

    Full Text Available Abstract Background Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear. Results To investigate the contribution of efflux pumps to intrinsic drug resistance of B. cenocepacia J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes BCAS0591-BCAS0593, BCAL1674-BCAL1676, and BCAL2822-BCAL2820. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of rnd-3 also included BCAL1672, encoding a putative TetR regulator. The B. cenocepacia rnd-3 and rnd-4 mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the rnd-3 and rnd-4 mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the rnd-1 operon had no detectable phenotypes under the conditions assayed. Conclusion Two of the three inactivated RND efflux pumps in B. cenocepacia J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in B. cenocepacia is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic

  2. Identification of putative noncoding RNA genes in the Burkholderia cenocepacia J2315 genome

    DEFF Research Database (Denmark)

    Coenye, T.; Drevinek, P.; Mahenthiralingam, E.

    2007-01-01

    Noncoding RNA (ncRNA) genes are not involved in the production of mRNA and proteins, but produce transcripts that function directly as structural or regulatory RNAs. In the present study, the presence of ncRNA genes in the genome of Burkholderia cenocepacia J2315 was evaluated by combining compar...

  3. The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage.

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    Roszniowski, Bartosz; Latka, Agnieszka; Maciejewska, Barbara; Vandenheuvel, Dieter; Olszak, Tomasz; Briers, Yves; Holt, Giles S; Valvano, Miguel A; Lavigne, Rob; Smith, Darren L; Drulis-Kawa, Zuzanna

    2017-02-01

    Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7 %) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7-49.5 % identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin, and spanins) and shows 29-98 % homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4-10 and are stable at 60 °C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P < 0.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest that the AP3 phage is a promising potent agent against bacteria belonging to the most common B. cenocepacia IIIA lineage strains.

  4. Development of a multiple-locus variable-number tandem-repeat typing scheme for genetic fingerprinting of Burkholderia cenocepacia and application to nationwide epidemiological analysis.

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    Segonds, Christine; Thouverez, Michelle; Barthe, Antoine; Bossuet-Greif, Nadège; Tisseyre, Lenka; Plésiat, Patrick; Vergnaud, Gilles; Chabanon, Gérard; Pourcel, Christine

    2015-02-01

    Organisms of the Burkholderia cepacia complex are especially important pathogens in cystic fibrosis (CF), with a propensity for patient-to-patient spread and long-term respiratory colonization. B. cenocepacia and Burkholderia multivorans account for the majority of infections in CF, and major epidemic clones have been recognized throughout the world. The aim of the present study was to develop and evaluate a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) scheme for B. cenocepacia. Potential VNTR loci were identified upon analysis of the annotated genome sequences of B. cenocepacia strains AU1054, J2315, and MCO-3, and 10 of them were selected on the basis of polymorphisms and size. A collection of 100 B. cenocepacia strains, including epidemiologically related and unrelated strains, as well as representatives of the major epidemic lineages, was used to evaluate typeability, epidemiological concordance, and the discriminatory power of MLVA-10 compared with those of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Longitudinal stability was assessed by testing 39 successive isolates from 14 patients. Typeability ranged from 0.91 to 1, except for that of one marker, which was not amplified in 53% of the B. cenocepacia IIIA strains. The MLVA types were shown to be stable in chronically colonized patients and within outbreak-related strains, with excellent epidemiological concordance. Epidemic and/or globally distributed lineages (epidemic Edinburgh-Toronto electrophoretic type 12 [ET-12], sequence type 32 [ST-32], ST-122, ST-234, and ST-241) were successfully identified. Conversely, the discriminatory power of MLVA was lower than that of PFGE or MLST, although PFGE variations within the epidemic lineages sometimes masked their genetic relatedness. In conclusion, MLVA represents a promising cost-effective first-line tool in B. cenocepacia surveillance.

  5. Clinafloxacin for Treatment of Burkholderia cenocepacia Infection in a Cystic Fibrosis Patient.

    Science.gov (United States)

    Balwan, Akshu; Nicolau, David P; Wungwattana, Minkey; Zuckerman, Jonathan B; Waters, Valerie

    2016-01-01

    Respiratory infection with Burkholderia cenocepacia is associated with accelerated decline in lung function and increased mortality in cystic fibrosis (CF) patients (A. M. Jones, M. E. Dodd, J. R. W. Govan, V. Barcus, C. J. Doherty, J. Morris, and A. K. Webb, Thorax 59:948-951, 2004, http://dx.doi.org/10.1136/thx.2003.017210). B. cenocepacia often possesses innate resistance to multiple antimicrobial classes, making eradication uncommon in established infection (P. B. Davis, Am J Respir Crit Care Med 173:475-482, 2006, http://dx.doi.org/10.1164/rccm.200505-840OE). We report the use of clinafloxacin in a CF patient with advanced B. cenocepacia infection, present pharmacokinetic (PK) data, and discuss the potential therapeutic role of clinafloxacin in patients with this condition.

  6. Candidate essential genes in Burkholderia cenocepacia J2315 identified by genome-wide TraDIS

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    Yee-Chin Wong

    2016-08-01

    Full Text Available Burkholderia cenocepacia infection often leads to fatal cepacia syndrome in cystic fibrosis patients. However, antibiotic therapy rarely results in complete eradication of the pathogen due to its intrinsic resistance to many clinically available antibiotics. Recent attention has turned to the identification of essential genes as the proteins encoded by these genes may serve as potential targets for development of novel antimicrobials. In this study, we utilized TraDIS (Transposon Directed Insertion-site Sequencing as a genome-wide screening tool to facilitate the identification of B. cenocepacia genes essential for its growth and viability. A transposon mutant pool consisting of approximately 500,000 mutants was successfully constructed, with more than 400,000 unique transposon insertion sites identified by computational analysis of TraDIS datasets. The saturated library allowed for the identification of 383 genes that were predicted to be essential in B. cenocepacia. We extended the application of TraDIS to identify conditionally essential genes required for in vitro growth and revealed an additional repertoire of 439 genes to be crucial for B. cenocepacia growth under nutrient-depleted conditions. The library of B. cenocepacia mutants can subsequently be subjected to various biologically related conditions to facilitate the discovery of genes involved in niche adaptation as well as pathogenicity and virulence.

  7. Candidate Essential Genes in Burkholderia cenocepacia J2315 Identified by Genome-Wide TraDIS

    KAUST Repository

    Wong, Yee-Chin

    2016-08-22

    Burkholderia cenocepacia infection often leads to fatal cepacia syndrome in cystic fibrosis patients. However, antibiotic therapy rarely results in complete eradication of the pathogen due to its intrinsic resistance to many clinically available antibiotics. Recent attention has turned to the identification of essential genes as the proteins encoded by these genes may serve as potential targets for development of novel antimicrobials. In this study, we utilized TraDIS (Transposon Directed Insertion-site Sequencing) as a genome-wide screening tool to facilitate the identification of B. cenocepacia genes essential for its growth and viability. A transposon mutant pool consisting of approximately 500,000 mutants was successfully constructed, with more than 400,000 unique transposon insertion sites identified by computational analysis of TraDIS datasets. The saturated library allowed for the identification of 383 genes that were predicted to be essential in B. cenocepacia. We extended the application of TraDIS to identify conditionally essential genes required for in vitro growth and revealed an additional repertoire of 439 genes to be crucial for B. cenocepacia growth under nutrient-depleted conditions. The library of B. cenocepacia mutants can subsequently be subjected to various biologically related conditions to facilitate the discovery of genes involved in niche adaptation as well as pathogenicity and virulence.

  8. Interactions of Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria with epithelial and phagocytic cells.

    Science.gov (United States)

    Saldías, M Soledad; Valvano, Miguel A

    2009-09-01

    Burkholderia cenocepacia is a member of the B. cepacia complex (Bcc), a group of opportunistic bacteria that infect the airways of patients with cystic fibrosis (CF) and are extraordinarily resistant to almost all clinically useful antibiotics. Infections in CF patients with Bcc bacteria generally lead to a more rapid decline in lung function, and in some cases to the 'cepacia syndrome', a virtually deadly exacerbation of the lung infection with systemic manifestations. These characteristics of Bcc bacteria contribute to higher morbidity and mortality in infected CF patients. In the last 10 years considerable progress has been made in understanding the interactions between Bcc bacteria and mammalian host cells. Bcc isolates can survive either intracellularly within eukaryotic cells or extracellularly in host tissues. They survive within phagocytes and respiratory epithelial cells, and they have the ability to breach the respiratory epithelium layer. Survival and persistence of Bcc bacteria within host cells and tissues are believed to play a key role in pulmonary infection and to contribute to the persistent inflammation observed in patients with CF. This review summarizes recent findings concerning the interaction between Bcc bacteria and epithelial and phagocytic cells.

  9. A Pipeline for Screening Small Molecules with Growth Inhibitory Activity against Burkholderia cenocepacia.

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    Carrie Selin

    Full Text Available Infections with the bacteria Burkholderia cepacia complex (Bcc are very difficult to eradicate in cystic fibrosis patients due the intrinsic resistance of Bcc to most available antibiotics and the emergence of multiple antibiotic resistant strains during antibiotic treatment. In this work, we used a whole-cell based assay to screen a diverse collection of small molecules for growth inhibitors of a relevant strain of Bcc, B. cenocepacia K56-2. The primary screen used bacterial growth in 96-well plate format and identified 206 primary actives among 30,259 compounds. From 100 compounds with no previous record of antibacterial activity secondary screening and data mining selected a total of Bce bioactives that were further analyzed. An experimental pipeline, evaluating in vitro antibacterial and antibiofilm activity, toxicity and in vivo antibacterial activity using C. elegans was used for prioritizing compounds with better chances to be further investigated as potential Bcc antibacterial drugs. This high throughput screen, along with the in vitro and in vivo analysis highlights the utility of this experimental method to quickly identify bioactives as a starting point of antibacterial drug discovery.

  10. Burkholderia cenocepacia K56-2 trimeric autotransporter adhesin BcaA binds TNFR1 and contributes to induce airway inflammation.

    Science.gov (United States)

    Mil-Homens, Dalila; Pinto, Sandra N; Matos, Rute G; Arraiano, Cecília; Fialho, Arsenio M

    2017-04-01

    Chronic lung disease caused by persistent bacterial infections is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). CF pathogens acquire antibiotic resistance, overcome host defenses, and impose uncontrolled inflammation that ultimately may cause permanent damage of lungs' airways. Among the multiple CF-associated pathogens, Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria have become prominent contributors of disease progression. Here, we demonstrate that BcaA, a trimeric autotransporter adhesin (TAA) from the epidemic strain B. cenocepacia K56-2, is a tumor necrosis factor receptor 1-interacting protein able to regulate components of the tumor necrosis factor signaling pathway and ultimately leading to a significant production of the proinflammatory cytokine IL-8. Notably, this study is the first to demonstrate that a protein belonging to the TAA family is involved in the induction of the inflammatory response during B. cenocepacia infections, contributing to the success of the pathogen. Moreover, our results reinforce the relevance of the TAA BcaA as a multifunctional protein with a major role in B. cenocepacia virulence.

  11. Cyanide toxicity to Burkholderia cenocepacia is modulated by polymicrobial communities and environmental factors

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    Steve P. Bernier

    2016-05-01

    Full Text Available Microbes within polymicrobial communities can establish positive and negative interactions that have the potential to influence the overall behaviour of the community. Pseudomonas aeruginosa and species of the Burkholderia cepacia complex (Bcc can co-exist in the lower airways, however several studies have shown that P. aeruginosa can effectively kill the Bcc in vitro, for which hydrogen cyanide was recently proposed to play a critical role. Here we show that modification of the environment (i.e. culture medium, long-term genetic adaptation of P. aeruginosa to the cystic fibrosis (CF lung, or the addition of another bacterial species to the community can alter the sensitivity of Burkholderia cenocepacia to P. aeruginosa toxins. We specifically demonstrate that undefined rich media leads to higher susceptibility of B. cenocepacia to P. aeruginosa toxins like cyanide as compared to a synthetic medium (SCFM, that mimics the CF lung nutritional content. Overall, our study shows that the polymicrobial environment can have profound effects on negative interactions mediated by P. aeruginosa against B. cenocepacia. In fact, evolved P. aeruginosa or the presence of other species such as Staphylococcus aureus can directly abolish the direct competition mediated by cyanide and consequently maintaining a higher level of species diversity within the community.

  12. Putrescine reduces antibiotic-induced oxidative stress as a mechanism of modulation of antibiotic resistance in Burkholderia cenocepacia.

    Science.gov (United States)

    El-Halfawy, Omar M; Valvano, Miguel A

    2014-07-01

    Communication of antibiotic resistance among bacteria via small molecules is implicated in transient reduction of bacterial susceptibility to antibiotics, which could lead to therapeutic failures aggravating the problem of antibiotic resistance. Released putrescine from the extremely antibiotic-resistant bacterium Burkholderia cenocepacia protects less-resistant cells from different species against the antimicrobial peptide polymyxin B (PmB). Exposure of B. cenocepacia to sublethal concentrations of PmB and other bactericidal antibiotics induces reactive oxygen species (ROS) production and expression of the oxidative stress response regulator OxyR. We evaluated whether putrescine alleviates antibiotic-induced oxidative stress. The accumulation of intracellular ROS, such as superoxide ion and hydrogen peroxide, was assessed fluorometrically with dichlorofluorescein diacetate, while the expression of OxyR and putrescine synthesis enzymes was determined in luciferase assays using chromosomal promoter-lux reporter system fusions. We evaluated wild-type and isogenic deletion mutant strains with defects in putrescine biosynthesis after exposure to sublethal concentrations of PmB and other bactericidal antibiotics. Exogenous putrescine protected against oxidative stress induced by PmB and other antibiotics, whereas reduced putrescine synthesis resulted in increased ROS generation and a parallel increased sensitivity to PmB. Of the 3 B. cenocepacia putrescine-synthesizing enzymes, PmB induced only BCAL2641, an ornithine decarboxylase. This study reveals BCAL2641 as a critical component of the putrescine-mediated communication of antibiotic resistance and as a plausible target for designing inhibitors that would block the communication of such resistance among different bacteria, ultimately reducing the window of therapeutic failure in treating bacterial infections.

  13. Discovery of new diketopiperazines inhibiting Burkholderia cenocepacia quorum sensing in vitro and in vivo

    Science.gov (United States)

    Scoffone, Viola C.; Chiarelli, Laurent R.; Makarov, Vadim; Brackman, Gilles; Israyilova, Aygun; Azzalin, Alberto; Forneris, Federico; Riabova, Olga; Savina, Svetlana; Coenye, Tom; Riccardi, Giovanna; Buroni, Silvia

    2016-01-01

    Burkholderia cenocepacia, an opportunistic respiratory pathogen particularly relevant for cystic fibrosis patients, is difficult to eradicate due to its high level of resistance to most clinically relevant antimicrobials. Consequently, the discovery of new antimicrobials as well as molecules capable of inhibiting its virulence is mandatory. In this regard quorum sensing (QS) represents a good target for anti-virulence therapies, as it has been linked to biofilm formation and is important for the production of several virulence factors, including proteases and siderophores. Here, we report the discovery of new diketopiperazine inhibitors of the B. cenocepacia acyl homoserine lactone synthase CepI, and report their anti-virulence properties. Out of ten different compounds assayed against recombinant CepI, four were effective inhibitors, with IC50 values in the micromolar range. The best compounds interfered with protease and siderophore production, as well as with biofilm formation, and showed good in vivo activity in a Caenorhabditis elegans infection model. These molecules were also tested in human cells and showed very low toxicity. Therefore, they could be considered for in vivo combined treatments with established or novel antimicrobials, to improve the current therapeutic strategies against B. cenocepacia. PMID:27580679

  14. Evaluation of combination therapy for Burkholderia cenocepacia lung infection in different in vitro and in vivo models

    Science.gov (United States)

    Brackman, Gilles; Crabbé, Aurélie; Rigole, Petra; Vercruysse, Jurgen; Verstraete, Glenn; Cappoen, Davie; Vervaet, Chris; Cos, Paul

    2017-01-01

    Burkholderia cenocepacia is an opportunistic pathogen responsible for life-threatening infections in cystic fibrosis patients. B. cenocepacia is extremely resistant towards antibiotics and therapy is complicated by its ability to form biofilms. We investigated the efficacy of an alternative antimicrobial strategy for B. cenocepacia lung infections using in vitro and in vivo models. A screening of the NIH Clinical Collection 1&2 was performed against B. cenocepacia biofilms formed in 96-well microtiter plates in the presence of tobramycin to identify repurposing candidates with potentiator activity. The efficacy of selected hits was evaluated in a three-dimensional (3D) organotypic human lung epithelial cell culture model. The in vivo effect was evaluated in the invertebrate Galleria mellonella and in a murine B. cenocepacia lung infection model. The screening resulted in 60 hits that potentiated the activity of tobramycin against B. cenocepacia biofilms, including four imidazoles of which econazole and miconazole were selected for further investigation. However, a potentiator effect was not observed in the 3D organotypic human lung epithelial cell culture model. Combination treatment was also not able to increase survival of infected G. mellonella. Also in mice, there was no added value for the combination treatment. Although potentiators of tobramycin with activity against biofilms of B. cenocepacia were identified in a repurposing screen, the in vitro activity could not be confirmed nor in a more sophisticated in vitro model, neither in vivo. This stresses the importance of validating hits resulting from in vitro studies in physiologically relevant model systems. PMID:28248999

  15. Potential mechanisms underlying the acute lung dysfunction and bacterial extrapulmonary dissemination during Burkholderia cenocepacia respiratory infection.

    Science.gov (United States)

    Cunha, Luiz G; Assis, Maria-Cristina; Machado, Gloria-Beatriz; Assef, Ana P; Marques, Elizabeth A; Leão, Robson S; Saliba, Alessandra M; Plotkowski, Maria-Cristina

    2010-01-18

    Burkholderia cenocepacia, an opportunistic pathogen that causes lung infections in cystic fibrosis (CF) patients, is associated with rapid and usually fatal lung deterioration due to necrotizing pneumonia and sepsis, a condition known as cepacia syndrome. The key bacterial determinants associated with this poor clinical outcome in CF patients are not clear. In this study, the cytotoxicity and procoagulant activity of B. cenocepacia from the ET-12 lineage, that has been linked to the cepacia syndrome, and four clinical isolates recovered from CF patients with mild clinical courses were analysed in both in vitro and in vivo assays. B. cenocepacia-infected BEAS-2B epithelial respiratory cells were used to investigate the bacterial cytotoxicity assessed by the flow cytometric detection of cell staining with propidium iodide. Bacteria-induced procoagulant activity in cell cultures was assessed by a colorimetric assay and by the flow cytometric detection of tissue factor (TF)-bearing microparticles in cell culture supernatants. Bronchoalveolar lavage fluids (BALF) from intratracheally infected mice were assessed for bacterial proinflammatory and procoagulant activities as well as for bacterial cytotoxicity, by the detection of released lactate dehydrogenase. ET-12 was significantly more cytotoxic to cell cultures but clinical isolates Cl-2, Cl-3 and Cl-4 exhibited also a cytotoxic profile. ET-12 and CI-2 were similarly able to generate a TF-dependent procoagulant environment in cell culture supernatant and to enhance the release of TF-bearing microparticles from infected cells. In the in vivo assay, all bacterial isolates disseminated from the mice lungs, but Cl-2 and Cl-4 exhibited the highest rates of recovery from mice livers. Interestingly, Cl-2 and Cl-4, together with ET-12, exhibited the highest cytotoxicity. All bacteria were similarly capable of generating a procoagulant and inflammatory environment in animal lungs. B. cenocepacia were shown to exhibit cytotoxic

  16. Kinetic characterization of an oxidative, cooperative HMG-CoA reductase from Burkholderia cenocepacia.

    Science.gov (United States)

    Schwarz, Benjamin H; Driver, Joseph; Peacock, Riley B; Dembinski, Holly E; Corson, Melissa H; Gordon, Samuel S; Watson, Jeffrey M

    2014-02-01

    3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme in endogenous cholesterol biosynthesis in mammals and isoprenoid biosynthesis via the mevalonate pathway in other eukaryotes, archaea and some eubacteria. In most organisms that express this enzyme, it catalyzes the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have cloned and characterized the 6x-His-tagged HMGR from the opportunistic lung pathogen Burkholderia cenocepacia. Kinetic characterization shows that the enzyme prefers NAD(H) over NADP(H) as a cofactor, suggesting an oxidative physiological role for the enzyme. This hypothesis is supported by the fact that the Burkholderia cenocepacia genome lacks the genes for the downstream enzymes of the mevalonate pathway. The enzyme exhibits positive cooperativity toward the substrates of the reductive reaction, but the oxidative reaction exhibits unusual double-saturation kinetics, distinctive among characterized HMG-CoA reductases. The unusual kinetics may arise from the presence of multiple active oligomeric states, each with different Vmax values.

  17. Phenylalanine induces Burkholderia cenocepacia phenylacetic acid catabolism through degradation to phenylacetyl-CoA in synthetic cystic fibrosis sputum medium.

    Science.gov (United States)

    Yudistira, Harry; McClarty, Leigh; Bloodworth, Ruhi A M; Hammond, Sydney A; Butcher, Haley; Mark, Brian L; Cardona, Silvia T

    2011-09-01

    Synthetic cystic fibrosis sputum medium (SCFM) is rich in amino acids and supports robust growth of Burkholderia cenocepacia, a member of the Burkholderia cepacia complex (Bcc). Previous work demonstrated that B. cenocepacia phenylacetic acid (PA) catabolic genes are up-regulated during growth in SCFM and are required for full virulence in a Caenorhabditis elegans host model. In this work, we investigated the role of phenylalanine, one of the aromatic amino acids present in SCFM, as an inducer of the PA catabolic pathway. Phenylalanine degradation intermediates were used as sole carbon sources for growth and gene reporter experiments. In addition to phenylalanine and PA, phenylethylamine, phenylpyruvate, and 2-phenylacetamide were usable as sole carbon sources by wild type B. cenocepacia K56-2, but not by a PA catabolism-defective mutant. EMSA analysis showed that the binding of PaaR, the negative regulator protein of B. cenocepacia PA catabolism, to PA regulatory DNA could only be relieved by phenylacetyl-Coenzyme A (PA-CoA), but not by any of the putative phenylalanine degradation intermediates. Taken together, our results show that in B. cenocepacia, phenylalanine is catabolized to PA and induces PA catabolism through PA activation to PA-CoA. Thus, PaaR shares the same inducer with PaaX, the regulator of PA catabolism in Escherichia coli, despite belonging to a different protein family.

  18. Orderly Replication and Segregation of the Four Replicons of Burkholderia cenocepacia J2315.

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    Wen-Li Du

    2016-07-01

    Full Text Available Bacterial genomes typically consist of a single chromosome and, optionally, one or more plasmids. But whole-genome sequencing reveals about ten per-cent of them to be multipartite, with additional replicons which by size and indispensability are considered secondary chromosomes. This raises the questions of how their replication and partition is managed without compromising genome stability and of how such genomes arose. Vibrio cholerae, with a 1 Mb replicon in addition to its 3 Mb chromosome, is the only species for which maintenance of a multipartite genome has been investigated. In this study we have explored the more complex genome of Burkholderia cenocepacia (strain J2315. It comprises an extra replicon (c2 of 3.21 Mb, comparable in size to the3.87Mb main chromosome (c1, another extra replicon(c3 of 0.87 Mb and a plasmid of 0.09 Mb. The replication origin of c1 is typically chromosomal and those of c2 and c3 are plasmid-like; all are replicated bidirectionally. Fluorescence microscopy of tagged origins indicates that all initiate replication at mid-cell and segregate towards the cell quarter positions sequentially, c1-c2-p1/c3. c2 segregation is as well-phased with the cell cycle as c1, implying that this plasmid-like origin has become subject to regulation not typical of plasmids; in contrast, c3 segregates more randomly through the cycle. Disruption of individual Par systems by deletion of parAB or by addition of parS sites showed each Par system to govern the positioning of its own replicon only. Inactivation of c1, c2 and c3 Par systems not only reduced growth rate, generated anucleate cells and compromised viability but influenced processes beyond replicon partition, notably regulation of replication, chromosome condensation and cell size determination. In particular, the absence of the c1 ParA protein altered replication of all three chromosomes, suggesting that the partition system of the main chromosome is a major participant in the

  19. Burkholderia cenocepacia Differential Gene Expression during Host–Pathogen Interactions and Adaptation to the Host Environment

    Science.gov (United States)

    O’Grady, Eoin P.; Sokol, Pamela A.

    2011-01-01

    Members of the Burkholderia cepacia complex (Bcc) are important in medical, biotechnological, and agricultural disciplines. These bacteria naturally occur in soil and water environments and have adapted to survive in association with plants and animals including humans. All Bcc species are opportunistic pathogens including Burkholderia cenocepacia that causes infections in cystic fibrosis and chronic granulomatous disease patients. The adaptation of B. cenocepacia to the host environment was assessed in a rat chronic respiratory infection model and compared to that of high cell-density in vitro grown cultures using transcriptomics. The distribution of genes differentially expressed on chromosomes 1, 2, and 3 was relatively proportional to the size of each genomic element, whereas the proportion of plasmid-encoded genes differentially expressed was much higher relative to its size and most genes were induced in vivo. The majority of genes encoding known virulence factors, components of types II and III secretion systems and chromosome 2-encoded type IV secretion system were similarly expressed between in vitro and in vivo environments. Lower expression in vivo was detected for genes encoding N-acyl-homoserine lactone synthase CepI, orphan LuxR homolog CepR2, zinc metalloproteases ZmpA and ZmpB, LysR-type transcriptional regulator ShvR, nematocidal protein AidA, and genes associated with flagellar motility, Flp type pilus formation, and type VI secretion. Plasmid-encoded type IV secretion genes were markedly induced in vivo. Additional genes induced in vivo included genes predicted to be involved in osmotic stress adaptation or intracellular survival, metal ion, and nutrient transport, as well as those encoding outer membrane proteins. Genes identified in this study are potentially important for virulence during host–pathogen interactions and may be associated with survival and adaptation to the host environment during chronic lung infections. PMID:22919581

  20. Genetic relationships among Italian and Mexican maize-rhizosphere Burkholderia cepacia complex (BCC) populations belonging to Burkholderia cenocepacia IIIB and BCC6 group

    Science.gov (United States)

    2011-01-01

    Background A close association between maize roots and Burkholderia cepacia complex (BCC) bacteria has been observed in different locations globally. In this study we investigated by MultiLocus Restriction Typing (MLRT) the genetic diversity and relationships among Burkholderia cenocepacia IIIB and BCC6 populations associated with roots of maize plants cultivated in geographically distant countries (Italy and Mexico), in order to provide new insights into their population structure, evolution and ecology. Results The 31 B. cenocepacia IIIB and 65 BCC6 isolates gave rise to 29 and 39 different restriction types (RTs), respectively. Two pairs of isolates of B. cenocepacia IIIB and BCC6, recovered from both Italian and Mexican maize rhizospheres, were found to share the same RT. The eBURST (Based Upon Related Sequence Types) analysis of MLRT data grouped all the B. cenocepacia IIIB isolates into four clonal complexes, with the RT-4-complex including the 42% of them, while the majority of the BCC6 isolates (94%) were grouped into the RT-104-complex. These two main clonal complexes included RTs shared by both Italian and Mexican maize rhizospheres and a clear relationship between grouping and maize variety was also found. Grouping established by eBURST correlated well with the assessment using unweighted-pair group method with arithmetic mean (UPGMA). The standardized index of association values obtained in both B. cenocepacia IIIB and BCC6 suggests an epidemic population structure in which occasional clones emerge and spread. Conclusions Taken together our data demonstrate a wide dispersal of certain B. cenocepacia IIIB and BCC6 isolates in Mexican and Italian maize rhizospheres. Despite the clear relationship found between the geographic origin of isolates and grouping, identical RTs and closely related isolates were observed in geographically distant regions. Ecological factors and selective pressure may preferably promote some genotypes within each local microbial

  1. Genetic relationships among Italian and Mexican maize-rhizosphere Burkholderia cepacia complex (BCC populations belonging to Burkholderia cenocepacia IIIB and BCC6 group

    Directory of Open Access Journals (Sweden)

    Bevivino Annamaria

    2011-10-01

    Full Text Available Abstract Background A close association between maize roots and Burkholderia cepacia complex (BCC bacteria has been observed in different locations globally. In this study we investigated by MultiLocus Restriction Typing (MLRT the genetic diversity and relationships among Burkholderia cenocepacia IIIB and BCC6 populations associated with roots of maize plants cultivated in geographically distant countries (Italy and Mexico, in order to provide new insights into their population structure, evolution and ecology. Results The 31 B. cenocepacia IIIB and 65 BCC6 isolates gave rise to 29 and 39 different restriction types (RTs, respectively. Two pairs of isolates of B. cenocepacia IIIB and BCC6, recovered from both Italian and Mexican maize rhizospheres, were found to share the same RT. The eBURST (Based Upon Related Sequence Types analysis of MLRT data grouped all the B. cenocepacia IIIB isolates into four clonal complexes, with the RT-4-complex including the 42% of them, while the majority of the BCC6 isolates (94% were grouped into the RT-104-complex. These two main clonal complexes included RTs shared by both Italian and Mexican maize rhizospheres and a clear relationship between grouping and maize variety was also found. Grouping established by eBURST correlated well with the assessment using unweighted-pair group method with arithmetic mean (UPGMA. The standardized index of association values obtained in both B. cenocepacia IIIB and BCC6 suggests an epidemic population structure in which occasional clones emerge and spread. Conclusions Taken together our data demonstrate a wide dispersal of certain B. cenocepacia IIIB and BCC6 isolates in Mexican and Italian maize rhizospheres. Despite the clear relationship found between the geographic origin of isolates and grouping, identical RTs and closely related isolates were observed in geographically distant regions. Ecological factors and selective pressure may preferably promote some genotypes within

  2. A sensor kinase recognizing the cell-cell signal BDSF (cis-2-dodecenoic acid) regulates virulence in Burkholderia cenocepacia

    DEFF Research Database (Denmark)

    McCarthy, Y.; Yang, Liang; Twomey, K.B.

    2010-01-01

    P>Burkholderia cenocepacia is an opportunistic human pathogen that uses cis-2-dodecenoic acid (BDSF) as a quorum-sensing signal to control expression of virulence factors. BDSF is a signal molecule of the diffusible signal factor (DSF) family that was first described in the plant pathogen...... the input domain of RpfC was active in BDSF signal perception when expressed in X. campestris. Mutation of BCAM0227 gave rise to reduced cytotoxicity to Chinese hamster ovary cells and reduced virulence to Wax moth larvae and in the agar-bead mouse model of pulmonary infection. The findings identify BCAM...

  3. Survival dynamics of cystic fibrosis-related Gram-negative bacterial pathogens (Pseudomonas aeruginosa and Burkholderia cenocepacia) in Dead Sea and Atlantic Ocean waters.

    Science.gov (United States)

    Shteinberg, Michal; Kis-Papo, Tamar; Millar, Beverley C; Rendall, Jacqueline C; Downey, Damian G; Elborn, J Stuart; Moore, John E

    2015-09-01

    Clinical cystic fibrosis (CF) Pseudomonas aeruginosa (n=6) and Burkholderia cenocepacia (n=4) were inoculated in marine brines from the Dead Sea and the Atlantic Ocean and their survival was monitored over a 1 month duration. In Dead Sea samples, all P. aeruginosa and B. cenocepacia isolates were non-detectable by culture following 24 h incubation, including the non-selective enrichment samples. In the Atlantic Ocean brine, over a 1 month period, mean P. aeruginosa counts decreased by only 0.25 log10 units and mean B. cenocepacia counts decreased by approximately 4 log10 units (10,000 cfu/ml). This study demonstrated that Dead Sea brine exerted a lethal effect within 24 h on planktonic P. aeruginosa and B. cenocepacia. Thus, the Dead Sea effectively purges these organisms from its environment on a daily basis.

  4. Burkholderia cenocepacia type VI secretion system mediates escape of type II secreted proteins into the cytoplasm of infected macrophages.

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    Roberto Rosales-Reyes

    Full Text Available Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1β secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1β secretion and pyroptosis. Moreover, IL-1β secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS. We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1β secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.

  5. The exopolysaccharide gene cluster Bcam1330-Bcam1341 is involved in Burkholderia cenocepacia biofilm formation, and its expression is regulated by c-di-GMP and Bcam1349

    DEFF Research Database (Denmark)

    Fazli, Mustafa; McCarthy, Yvonne; Givskov, Michael

    2013-01-01

    In Burkholderia cenocepacia, the second messenger cyclic diguanosine monophosphate (c-di-GMP) has previously been shown to positively regulate biofilm formation and the expression of cellulose and type-I fimbriae genes through binding to the transcriptional regulator Bcam1349. Here, we provide...... evidence that cellulose and type-I fimbriae are not involved in B. cenocepacia biofilm formation in flow chambers, and we identify a novel Bcam1349/c-di-GMP-regulated exopolysaccharide gene cluster which is essential for B. cenocepacia biofilm formation. Overproduction of Bcam1349 in trans promotes wrinkly...... matrix exopolysaccharide and to be essential for flow-chamber biofilm formation. We demonstrate that Bcam1349 binds to the promoter region of genes in the Bcam1330-Bcam1341 cluster and that this binding is enhanced by the presence of c-di-GMP. Furthermore, we demonstrate that overproduction of both c...

  6. High-resolution structure of the M14-type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB-REDO strategies

    Energy Technology Data Exchange (ETDEWEB)

    Rimsa, Vadim; Eadsforth, Thomas C. [University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Joosten, Robbie P. [Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam (Netherlands); Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom)

    2014-02-01

    The structure of a bacterial M14-family carboxypeptidase determined exploiting microfocus synchrotron radiation and highly automated refinement protocols reveals its potential to act as a polyglutamylase. A potential cytosolic metallocarboxypeptidase from Burkholderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB-REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn{sup 2+}-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn{sup 2+}, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate.

  7. Burkholderia cenocepacia BC2L-C is a super lectin with dual specificity and proinflammatory activity.

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    Ondřej Sulák

    2011-09-01

    Full Text Available Lectins and adhesins are involved in bacterial adhesion to host tissues and mucus during early steps of infection. We report the characterization of BC2L-C, a soluble lectin from the opportunistic pathogen Burkholderia cenocepacia, which has two distinct domains with unique specificities and biological activities. The N-terminal domain is a novel TNF-α-like fucose-binding lectin, while the C-terminal part is similar to a superfamily of calcium-dependent bacterial lectins. The C-terminal domain displays specificity for mannose and l-glycero-d-manno-heptose. BC2L-C is therefore a superlectin that binds independently to mannose/heptose glycoconjugates and fucosylated human histo-blood group epitopes. The apo form of the C-terminal domain crystallized as a dimer, and calcium and mannose could be docked in the binding site. The whole lectin is hexameric and the overall structure, determined by electron microscopy and small angle X-ray scattering, reveals a flexible arrangement of three mannose/heptose-specific dimers flanked by two fucose-specific TNF-α-like trimers. We propose that BC2L-C binds to the bacterial surface in a mannose/heptose-dependent manner via the C-terminal domain. The TNF-α-like domain triggers IL-8 production in cultured airway epithelial cells in a carbohydrate-independent manner, and is therefore proposed to play a role in the dysregulated proinflammatory response observed in B. cenocepacia lung infections. The unique architecture of this newly recognized superlectin correlates with multiple functions including bacterial cell cross-linking, adhesion to human epithelia, and stimulation of inflammation.

  8. 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.

    Science.gov (United States)

    Bertot, Gustavo M; Restelli, Marcela A; Galanternik, Laura; Aranibar Urey, Rene C; Valvano, Miguel A; Grinstein, Saúl

    2007-06-01

    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.

  9. Phenotypic and genotypic characterisation of Burkholderia cenocepacia J2315 mutants affected in homoserine lactone and diffusible signal factor-based quorum sensing systems suggests interplay between both types of systems.

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    Claudia Udine

    Full Text Available Many putative virulence factors of Burkholderia cenocepacia are controlled by various quorum sensing (QS circuits. These QS systems either use N-acyl homoserine lactones (AHL or cis-2-dodecenoic acid ("Burkholderia diffusible signal factor", BDSF as signalling molecules. Previous work suggested that there is little cross-talk between both types of systems. We constructed mutants in B. cenocepacia strain J2315, in which genes encoding CepI (BCAM1870, CciI (BCAM0239a and the BDSF synthase (BCAM0581 were inactivated, and also constructed double (ΔcepIΔBCAM0581, ΔcciIΔBCAM0581 and ΔcepIΔcciI mutants and a triple (ΔcepIΔcciIΔBCAM0581 mutant. Subsequently we investigated phenotypic properties (antibiotic susceptibility, biofilm formation, production of AHL and BDSF, protease activity and virulence in Caenorhabditis elegans and measured gene expression in these mutants, and this in the presence and absence of added BDSF, AHL or both. The triple mutant was significantly more affected in biofilm formation, antimicrobial susceptibility, virulence in C. elegans, and protease production than either the single or double mutants. The ΔBCAM0581 mutant and the ΔcepIΔBCAM0581 and ΔcciIΔBCAM0581 double mutants produced significantly less AHL compared to the WT strain and the ΔcepI and ΔcciI single mutant, respectively. The expression of cepI and cciI in ΔBCAM0581, was approximately 3-fold and 7-fold (p<0.05 lower than in the WT, respectively. The observed differences in AHL production, expression of cepI and cciI and QS-controlled phenotypes in the ΔBCAM0581 mutant could (at least partially be restored by addition of BDSF. Our data suggest that, in B. cenocepacia J2315, AHL and BDSF-based QS systems co-regulate the same set of genes, regulate different sets of genes that are involved in the same phenotypes and/or that the BDSF system controls the AHL-based QS system. As the expression of the gene encoding the C6-HSL synthase CciI (and to a

  10. Induction of immune response to the 17 kDa OMPA Burkholderia cenocepacia polypeptide and protection against pulmonary infection in mice after nasal vaccination with an OMP nanoemulsion-based vaccine.

    Science.gov (United States)

    Makidon, P E; Knowlton, J; Groom, J V; Blanco, L P; LiPuma, J J; Bielinska, A U; Baker, J R

    2010-05-01

    Burkholderia cepacia complex (Bcc) are opportunistic bacteria associated with life-threatening illness in persons with cystic fibrosis. Once Bcc colonization is established, these antimicrobial-resistant and biofilm-forming bacteria are difficult to eradicate and are associated with increased rates of morbidity and mortality. At present, no vaccines are available to prevent the Bcc infection. There is currently a paucity of published information regarding the development of vaccines designed to prevent Burkholderia colonization. This work expands on the recent studies published by Bertot et al. [Infect Immun 75(6):2740-2752, 2007], where successful protective immune responses were generated in mice using a B. multivorans OMP-based vaccine. Here, we evaluate an experimental mucosal vaccine against Bcc using a novel mucosal adjuvant (nanoemulsion) and a novel B. cenocepacia-based OMP antigen. The OMP antigen derived from B. cenocepacia was mixed with either nanoemulsion or with PBS and delivered intranasally to CD-1 mice. Serum analysis showed robust IgG and mucosal secretory IgA immune responses in vaccinated versus control mice. The antibodies had cross-neutralizing activity against both B. cenocepacia and B. multivorans species. We found that immunized mice were protected against pulmonary colonization with B. cenocepacia. We have also identified that a 17 kDa OmpA-like protein highly conserved between Burkholderia and Ralstonia species as a new immunodominant epitope in mucosal immunization.

  11. The ornibactin biosynthesis and transport genes of Burkholderia cenocepacia are regulated by an extracytoplasmic function sigma factor which is a part of the Fur regulon.

    Science.gov (United States)

    Agnoli, Kirsty; Lowe, Carolyn A; Farmer, Kate L; Husnain, Seyyed I; Thomas, Mark S

    2006-05-01

    Burkholderia cenocepacia mutants that fail to produce the siderophore ornibactin were obtained following mutagenesis with mini-Tn5Tp. These mutants were shown to be growth restricted under conditions of iron depletion. In eight of the mutants, the transposon had integrated into one of two genes, orbI and orbJ, encoding nonribosomal peptide synthetases. In the other mutant, the transposon had inserted into an open reading frame, orbS, located upstream from orbI. The polypeptide product of orbS exhibits a high degree of similarity to the Pseudomonas aeruginosa extracytoplasmic function (ECF) sigma factor PvdS but possesses an N-terminal extension of approximately 29 amino acids that is not present in PvdS. Three predicted OrbS-dependent promoters were identified within the ornibactin gene cluster, based on their similarity to PvdS-dependent promoters. The iron-regulated activity of these promoters was shown to require OrbS. Transcription of the orbS gene was found to be under the control of an iron-regulated sigma(70)-dependent promoter. This promoter, but not the OrbS-dependent promoters, was shown to be a target for repression by the global regulator Fur. Our results demonstrate that production of ornibactin by B. cenocepacia in response to iron starvation requires transcription of an operon that is dependent on the Fur-regulated ECF sigma factor gene orbS. A mechanism is also proposed for the biosynthesis of ornibactin.

  12. Regulation of Burkholderia cenocepacia biofilm formation by RpoN and the c-di-GMP effector BerB.

    Science.gov (United States)

    Fazli, Mustafa; Rybtke, Morten; Steiner, Elisabeth; Weidel, Elisabeth; Berthelsen, Jens; Groizeleau, Julie; Bin, Wu; Zhi, Boo Zhao; Yaming, Zhang; Kaever, Volkhard; Givskov, Michael; Hartmann, Rolf W; Eberl, Leo; Tolker-Nielsen, Tim

    2017-08-01

    Knowledge about the molecular mechanisms that are involved in the regulation of biofilm formation is essential for the development of biofilm-control measures. It is well established that the nucleotide second messenger cyclic diguanosine monophosphate (c-di-GMP) is a positive regulator of biofilm formation in many bacteria, but more knowledge about c-di-GMP effectors is needed. We provide evidence that c-di-GMP, the alternative sigma factor RpoN (σ54), and the enhancer-binding protein BerB play a role in biofilm formation of Burkholderia cenocepacia by regulating the production of a biofilm-stabilizing exopolysaccharide. Our findings suggest that BerB binds c-di-GMP, and activates RpoN-dependent transcription of the berA gene coding for a c-di-GMP-responsive transcriptional regulator. An increased level of the BerA protein in turn induces the production of biofilm-stabilizing exopolysaccharide in response to high c-di-GMP levels. Our findings imply that the production of biofilm exopolysaccharide in B. cenocepacia is regulated through a cascade involving two consecutive transcription events that are both activated by c-di-GMP. This type of regulation may allow tight control of the expenditure of cellular resources. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  13. The AHL- and BDSF-dependent quorum sensing systems control specific and overlapping sets of genes in Burkholderia cenocepacia H111.

    Directory of Open Access Journals (Sweden)

    Nadine Schmid

    Full Text Available Quorum sensing in Burkholderia cenocepacia H111 involves two signalling systems that depend on different signal molecules, namely N-acyl homoserine lactones (AHLs and the diffusible signal factor cis-2-dodecenoic acid (BDSF. Previous studies have shown that AHLs and BDSF control similar phenotypic traits, including biofilm formation, proteolytic activity and pathogenicity. In this study we mapped the BDSF stimulon by RNA-Seq and shotgun proteomics analysis. We demonstrate that a set of the identified BDSF-regulated genes or proteins are also controlled by AHLs, suggesting that the two regulons partially overlap. The detailed analysis of two mutually regulated operons, one encoding three lectins and the other one encoding the large surface protein BapA and its type I secretion machinery, revealed that both AHLs and BDSF are required for full expression, suggesting that the two signalling systems operate in parallel. In accordance with this, we show that both AHLs and BDSF are required for biofilm formation and protease production.

  14. Complete Genome Sequence of the Fenitrothion-Degrading Burkholderia sp. Strain YI23

    OpenAIRE

    Lim, Jong Sung; Choi, Beom Soon; Choi, Ah Young; Kim, Kyung Duk; Kim, Dong In; Choi, Ik Young; Ka, Jong-Ok

    2012-01-01

    Burkholderia species are ubiquitous in soil environments. Many Burkholderia species isolated from various environments have the potential to biodegrade man-made chemicals. Burkholderia sp. strain YI23 was isolated from a golf course soil and identified as a fenitrothion-degrading bacterium. In this study, we report the complete genome sequence of Burkholderia sp. strain YI23.

  15. The CRP/FNR family protein Bcam1349 is a c-di-GMP effector that regulates biofilm formation in the respiratory pathogen Burkholderia cenocepacia

    DEFF Research Database (Denmark)

    Fazli, Mustafa; O'Connell, Aileen; Nilsson, Martin

    2011-01-01

    to control a wide range of functions in bacteria, but little is known about these regulatory mechanisms in B. cenocepacia. Here we investigated the role that c-di-GMP plays in the regulation of biofilm formation and virulence in B. cenocepacia. Elevated intracellular levels of c-di-GMP promoted wrinkly...... colony, pellicle and biofilm formation in B. cenocepacia. A screen for transposon mutants unable to respond to elevated levels of c-di-GMP led to the identification of the mutant bcam1349 that did not display increased biofilm and pellicle formation with excessive c-di-GMP levels, and displayed a biofilm...... larvae infection model. Taken together, these findings suggest that the Bcam1349 protein is a transcriptional regulator that binds c-di-GMP and regulates biofilm formation and virulence in B. cenocepacia in response to the level of c-di-GMP....

  16. Draft Genome Sequence of Burkholderia cordobensis Type Strain LMG 27620, Isolated from Agricultural Soils in Argentina

    Science.gov (United States)

    Draghi, Walter Omar; Mancini Villagra, Ulises M.; Wall, Luis Gabriel

    2015-01-01

    Bacteria of the genus Burkholderia are commonly found in diverse ecological niches in nature. We report here the draft genome sequence of Burkholderia cordobensis type strain LMG 27620, isolated from agricultural soil in Córdoba, Argentina. This strain harbors several genes involved in chitin utilization and phenol degradation, which make it an interesting candidate for biocontrol purposes and xenobiotic degradation in polluted environments. PMID:26494680

  17. Genomic islands from five strains of Burkholderia pseudomallei

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    Nierman William C

    2008-11-01

    Full Text Available Abstract Background Burkholderia pseudomallei is the etiologic agent of melioidosis, a significant cause of morbidity and mortality where this infection is endemic. Genomic differences among strains of B. pseudomallei are predicted to be one of the major causes of the diverse clinical manifestations observed among patients with melioidosis. The purpose of this study was to examine the role of genomic islands (GIs as sources of genomic diversity in this species. Results We found that genomic islands (GIs vary greatly among B. pseudomallei strains. We identified 71 distinct GIs from the genome sequences of five reference strains of B. pseudomallei: K96243, 1710b, 1106a, MSHR668, and MSHR305. The genomic positions of these GIs are not random, as many of them are associated with tRNA gene loci. In particular, the 3' end sequences of tRNA genes are predicted to be involved in the integration of GIs. We propose the term "tRNA-mediated site-specific recombination" (tRNA-SSR for this mechanism. In addition, we provide a GI nomenclature that is based upon integration hotspots identified here or previously described. Conclusion Our data suggest that acquisition of GIs is one of the major sources of genomic diversity within B. pseudomallei and the molecular mechanisms that facilitate horizontally-acquired GIs are common across multiple strains of B. pseudomallei. The differential presence of the 71 GIs across multiple strains demonstrates the importance of these mobile elements for shaping the genetic composition of individual strains and populations within this bacterial species.

  18. Polyphasic characterisation of Burkholderia cepaciacomplex species isolated from children with cystic fibrosis

    Directory of Open Access Journals (Sweden)

    Fernando José Vicenzi

    2016-01-01

    Full Text Available Cystic fibrosis (CF patients with Burkholderia cepacia complex (Bcc pulmonary infections have high morbidity and mortality. The aim of this study was to compare different methods for identification of Bcc species isolated from paediatric CF patients. Oropharyngeal swabs from children with CF were used to obtain isolates of Bcc samples to evaluate six different tests for strain identification. Conventional (CPT and automatised (APT phenotypic tests, polymerase chain reaction (PCR-recA, restriction fragment length polymorphism-recA, recAsequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF were applied. Bacterial isolates were also tested for antimicrobial susceptibility. PCR-recA analysis showed that 36 out of the 54 isolates were Bcc. Kappa index data indicated almost perfect agreement between CPT and APT, CPT and PCR-recA, and APT and PCR-recA to identify Bcc, and MALDI-TOF and recAsequencing to identify Bcc species. The recAsequencing data and the MALDI-TOF data agreed in 97.2% of the isolates. Based on recA sequencing, the most common species identified were Burkholderia cenocepacia IIIA (33.4%,Burkholderia vietnamiensis (30.6%, B. cenocepaciaIIIB (27.8%, Burkholderia multivorans (5.5%, and B. cepacia (2.7%. MALDI-TOF proved to be a useful tool for identification of Bcc species obtained from CF patients, although it was not able to identify B. cenocepacia subtypes.

  19. Polyphasic characterisation of Burkholderia cepacia complex species isolated from children with cystic fibrosis

    Science.gov (United States)

    Vicenzi, Fernando José; Pillonetto, Marcelo; de Souza, Helena Aguilar Peres Homem de Mello; Palmeiro, Jussara Kasuko; Riedi, Carlos Antônio; Rosario-Filho, Nelson Augusto; Dalla-Costa, Libera Maria

    2016-01-01

    Cystic fibrosis (CF) patients with Burkholderia cepacia complex (Bcc) pulmonary infections have high morbidity and mortality. The aim of this study was to compare different methods for identification of Bcc species isolated from paediatric CF patients. Oropharyngeal swabs from children with CF were used to obtain isolates of Bcc samples to evaluate six different tests for strain identification. Conventional (CPT) and automatised (APT) phenotypic tests, polymerase chain reaction (PCR)-recA, restriction fragment length polymorphism-recA, recAsequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) were applied. Bacterial isolates were also tested for antimicrobial susceptibility. PCR-recA analysis showed that 36 out of the 54 isolates were Bcc. Kappa index data indicated almost perfect agreement between CPT and APT, CPT and PCR-recA, and APT and PCR-recA to identify Bcc, and MALDI-TOF and recAsequencing to identify Bcc species. The recAsequencing data and the MALDI-TOF data agreed in 97.2% of the isolates. Based on recA sequencing, the most common species identified were Burkholderia cenocepacia IIIA (33.4%),Burkholderia vietnamiensis (30.6%), B. cenocepaciaIIIB (27.8%), Burkholderia multivorans (5.5%), and B. cepacia (2.7%). MALDI-TOF proved to be a useful tool for identification of Bcc species obtained from CF patients, although it was not able to identify B. cenocepacia subtypes. PMID:26814642

  20. Biocontrol of Burkholderia cepacia complex bacteria and bacterial phytopathogens by Bdellovibrio bacteriovorus.

    Science.gov (United States)

    McNeely, Damian; Chanyi, Ryan M; Dooley, James S; Moore, John E; Koval, Susan F

    2017-04-01

    Bdellovibrio and like organisms are predatory bacteria that have the unusual property of using the cytoplasmic constituents of other Gram-negative bacteria as nutrients. These predators may thus provide an alternative approach to the biocontrol of human and plant pathogens. Predators were isolated on Burkholderia cenocepacia K56-2 and J2315 as prey cells, in enrichment cultures with soil and sewage. Three isolates (DM7C, DM8A, and DM11A) were identified as Bdellovibrio bacteriovorus on the basis of morphology, a periplasmic life cycle, and 16S rRNA gene sequencing. The prey range of these isolates was tested on Burkholderia cepacia complex bacteria and several phytopathogenic bacteria of agricultural importance. Of 31 strains of the Burkholderia cepacia complex tested, only 4 were resistant to predation by strain DM7C. A subset of 9 of the prey tested were also susceptible to strains DM8A and DM11A. Of 12 phytopathogens tested, 4 were resistant to strains DM7C and DM8A, and only 2 were resistant to strain DM11A. Thus, Bdellovibrio bacteriovorus strains retrieved from environmental samples on 2 Burkholderia cenocepacia isolates from cystic fibrosis patients did not distinguish in their prey range between other isolates of that pathogen or phytopathogens. Such strains hold promise as potential wide-spectrum biocontrol agents.

  1. Genomic sequence and activity of KS10, a transposable phage of the Burkholderia cepacia complex

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    Shrivastava Savita

    2008-12-01

    Full Text Available Abstract Background The Burkholderia cepacia complex (BCC is a versatile group of Gram negative organisms that can be found throughout the environment in sources such as soil, water, and plants. While BCC bacteria can be involved in beneficial interactions with plants, they are also considered opportunistic pathogens, specifically in patients with cystic fibrosis and chronic granulomatous disease. These organisms also exhibit resistance to many antibiotics, making conventional treatment often unsuccessful. KS10 was isolated as a prophage of B. cenocepacia K56-2, a clinically relevant strain of the BCC. Our objective was to sequence the genome of this phage and also determine if this prophage encoded any virulence determinants. Results KS10 is a 37,635 base pairs (bp transposable phage of the opportunistic pathogen Burkholderia cenocepacia. Genome sequence analysis and annotation of this phage reveals that KS10 shows the closest sequence homology to Mu and BcepMu. KS10 was found to be a prophage in three different strains of B. cenocepacia, including strains K56-2, J2315, and C5424, and seven tested clinical isolates of B. cenocepacia, but no other BCC species. A survey of 23 strains and 20 clinical isolates of the BCC revealed that KS10 is able to form plaques on lawns of B. ambifaria LMG 19467, B. cenocepacia PC184, and B. stabilis LMG 18870. Conclusion KS10 is a novel phage with a genomic organization that differs from most phages in that its capsid genes are not aligned into one module but rather separated by approximately 11 kb, giving evidence of one or more prior genetic rearrangements. There were no potential virulence factors identified in KS10, though many hypothetical proteins were identified with no known function.

  2. Draft Genome Sequence of Burkholderia gladioli Strain UCD-UG_CHAPALOTE (Phylum Proteobacteria)

    Science.gov (United States)

    Ettinger, Cassandra L.; Shehata, Hanan R.; Johnston-Monje, David; Raizada, Manish N.

    2015-01-01

    Here, we present the draft genome of Burkholderia gladioli strain UCD-UG_CHAPALOTE. This strain is an endophyte isolated from surface sterilized seeds of an ancient Mexican landrace of corn, Chapalote. The genome contains 8,527,129 bp in 109 scaffolds. PMID:25614570

  3. Proof that Burkholderia strains form effective symbioses with legumes: a study of novel Mimosa-nodulating strains from South America.

    Science.gov (United States)

    Chen, Wen-Ming; de Faria, Sergio M; Straliotto, Rosângela; Pitard, Rosa M; Simões-Araùjo, Jean L; Chou, Jui-Hsing; Chou, Yi-Ju; Barrios, Edmundo; Prescott, Alan R; Elliott, Geoffrey N; Sprent, Janet I; Young, J Peter W; James, Euan K

    2005-11-01

    Twenty Mimosa-nodulating bacterial strains from Brazil and Venezuela, together with eight reference Mimosa-nodulating rhizobial strains and two other beta-rhizobial strains, were examined by amplified rRNA gene restriction analysis. They fell into 16 patterns and formed a single cluster together with the known beta-rhizobia, Burkholderia caribensis, Burkholderia phymatum, and Burkholderia tuberum. The 16S rRNA gene sequences of 15 of the 20 strains were determined, and all were shown to belong to the genus Burkholderia; four distinct clusters could be discerned, with strains isolated from the same host species usually clustering very closely. Five of the strains (MAP3-5, Br3407, Br3454, Br3461, and Br3469) were selected for further studies of the symbiosis-related genes nodA, the NodD-dependent regulatory consensus sequences (nod box), and nifH. The nodA and nifH sequences were very close to each other and to those of B. phymatum STM815, B. caribensis TJ182, and Cupriavidus taiwanensis LMG19424 but were relatively distant from those of B. tuberum STM678. In addition to nodulating their original hosts, all five strains could also nodulate other Mimosa spp., and all produced nodules on Mimosa pudica that had nitrogenase (acetylene reduction) activities and structures typical of effective N2-fixing symbioses. Finally, both wild-type and green fluorescent protein-expressing transconjugant strains of Br3461 and MAP3-5 produced N2-fixing nodules on their original hosts, Mimosa bimucronata (Br3461) and Mimosa pigra (MAP3-5), and hence this confirms strongly that Burkholderia strains can form effective symbioses with legumes.

  4. Susceptibility of Caenorhabditis elegans to Burkholderia infection depends on prior diet and secreted bacterial attractants.

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    Vaughn S Cooper

    Full Text Available The nematode Caenorhabditis elegans may be killed by certain pathogenic bacteria and thus is a model organism for studying interactions between bacteria and animal hosts. However, growing nematodes on prey bacteria may influence their susceptibility to potential pathogens. A method of axenic nematode culture was developed to isolate and quantify interactions between C. elegans and potentially pathogenic strains of the Burkholderia cepacia complex. Studying these dynamics in liquid solution rather than on agar surfaces minimized nematode avoidance behavior and resolved more differences among isolates. Most isolates of B. cenocepacia, B. ambifaria and B. cepacia caused 60-80% mortality of nematodes after 7 days, whereas isolates of B. multivorans caused less mortality (<25% and supported nematode reproduction. However, some B. cenocepacia isolates recovered from chronic infections were much less virulent (5-28% mortality. As predicted, prior diet altered the outcome of interactions between nematodes and bacteria. When given the choice between Burkholderia and E. coli as prey on agar, axenically raised nematodes initially preferred most lethal Burkholderia isolates to E. coli as a food source, but this was not the case for nematodes fed E. coli, which avoided toxic Burkholderia. This food preference was associated with the cell-free supernatant and thus secreted compounds likely mediated bacterial-nematode interactions. This model, which isolates interactions between bacteria and nematodes from the effects of prior feeding, demonstrates that bacteria can influence nematode behavior and their susceptibility to pathogens.

  5. Genome sequence of the Lebeckia ambigua-nodulating "Burkholderia sprentiae" strain WSM5005(T.).

    Science.gov (United States)

    Reeve, Wayne; De Meyer, Sofie; Terpolilli, Jason; Melino, Vanessa; Ardley, Julie; Rui, Tian; Tiwari, Ravi; Howieson, John; Yates, Ron; O'Hara, Graham; Lu, Megan; Bruce, David; Detter, Chris; Tapia, Roxanne; Han, Cliff; Wei, Chia-Lin; Huntemann, Marcel; Han, James; Chen, I-Min; Mavromatis, Konstantinos; Markowitz, Victor; Szeto, Ernest; Ivanova, Natalia; Mikhailova, Natalia; Ovchinnikova, Galina; Pagani, Ioanna; Pati, Amrita; Goodwin, Lynne; Peters, Lin; Pitluck, Sam; Woyke, Tanja; Kyrpides, Nikos

    2013-12-20

    "Burkholderia sprentiae" strain WSM5005(T) is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated in Australia from an effective N2-fixing root nodule of Lebeckia ambigua collected in Klawer, Western Cape of South Africa, in October 2007. Here we describe the features of "Burkholderia sprentiae" strain WSM5005(T), together with the genome sequence and its annotation. The 7,761,063 bp high-quality-draft genome is arranged in 8 scaffolds of 236 contigs, contains 7,147 protein-coding genes and 76 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.

  6. Genome sequence of the Lebeckia ambigua-nodulating “Burkholderia sprentiae” strain WSM5005T

    Science.gov (United States)

    Reeve, Wayne; De Meyer, Sofie; Terpolilli, Jason; Melino, Vanessa; Ardley, Julie; Rui, Tian; Tiwari, Ravi; Howieson, John; Yates, Ron; O’Hara, Graham; Lu, Megan; Bruce, David; Detter, Chris; Tapia, Roxanne; Han, Cliff; Wei, Chia-Lin; Huntemann, Marcel; Han, James; Chen, I-Min; Mavromatis, Konstantinos; Markowitz, Victor; Szeto, Ernest; Ivanova, Natalia; Mikhailova, Natalia; Ovchinnikova, Galina; Pagani, Ioanna; Pati, Amrita; Goodwin, Lynne; Peters, Lin; Pitluck, Sam; Woyke, Tanja; Kyrpides, Nikos

    2013-01-01

    Burkholderia sprentiae” strain WSM5005T is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated in Australia from an effective N2-fixing root nodule of Lebeckia ambigua collected in Klawer, Western Cape of South Africa, in October 2007. Here we describe the features of “Burkholderia sprentiae” strain WSM5005T, together with the genome sequence and its annotation. The 7,761,063 bp high-quality-draft genome is arranged in 8 scaffolds of 236 contigs, contains 7,147 protein-coding genes and 76 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program. PMID:24976894

  7. Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT.

    OpenAIRE

    Haigler, B E; Suen, W C; Spain, J C

    1996-01-01

    4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene by Burkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenas...

  8. Role of lipase in Burkholderia cepacia complex (Bcc) invasion of lung epithelial cells.

    Science.gov (United States)

    Mullen, T; Markey, K; Murphy, P; McClean, S; Callaghan, M

    2007-12-01

    The Burkholderia cepacia complex (Bcc) is a group of ten closely related species associated with life-threatening infection in cystic fibrosis (CF). These bacteria are highly antibiotic resistant, with some strains transmissible, and in a subgroup of patients, they can cause a rapid and fatal necrotising pneumonia. The Bcc organisms produce a range of exoproducts with virulence potential, including exopolysaccharide, proteases and lipases. Many members of the Bcc are also capable of epithelial cell invasion, although the mechanism(s) involved are poorly understood. This study investigates a role for Bcc lipase in epithelial cell invasion by Bcc strains. Lipase activity was measured in eight species of the Bcc. Strains that produced high levels of lipase were predominantly from the B. multivorans and B. cenocepacia species. Pre-treatment of two epithelial cell lines with Bcc lipase significantly increased invasion by two B. multivorans strains and one B. cenocepacia strain and did not affect either plasma membrane or tight junction integrity. Inhibition of Bcc lipase production by the lipase inhibitor Orlistat significantly decreased invasion by both B. multivorans and B. cenocepacia strains in a concentration-dependent manner. This study demonstrates the extent of lipase production across the Bcc and establishes a potential role for lipase in Bcc epithelial cell invasion.

  9. A comparison of virulence of intraperitoneal infection of Burkholderia mallei strains in guinea-pigs

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    Eslampanah, M.

    2015-12-01

    Full Text Available Male guinea pigs show high susceptibility to Burkholderia mallei and have been used as animal models in glanders studies. The purpose of our study was to elucidate glanders comparative pathogenesis in guinea pigs. We present here the histological changes and bacterial isolation that develop over time in guinea pigs inoculated intraperitoneally (IP with two strain of B. mallei. Ten male guinea pigs were inoculated intraperitoneally with either the standard strain of Burkholderia mallei or B. mallei strain from Siberian tiger at the Tehran zoo individually, then euthanized at multiple time points post inoculation. Histopathologic changes were similar in both groups and consisted of pyogranulomatous inflammation. In the standard strain study guinea pigs, changes were first seen at 48 hours in liver and heart then in spleen, lung, and kidney at day 3. These changes generally reached maximal incidence and severity by day 3 but decreased by comparison in all tissues except the liver, lung and kidney. Changes were first seen in Siberian tiger strain study guinea pigs also at 48 hours in lung, liver and spleen. At day 3, changes were present in liver, spleen and mediastinal lymph nodes. These changes were maximal at day 4 and 5. In contrast there are differences in incidence and severity between the two strain study guinea pigs. Our findings based on histopathological study indicate that Siberian tiger strain has more severity in gross and necropsy examination but in pathologic lesion was qualitatively similar generally. Additionally, by bacterial isolation, we confirmed the presence of B. mallei.

  10. Identification and cloning of four riboswitches from Burkholderia pseudomallei strain K96243

    Science.gov (United States)

    Munyati-Othman, Noor; Fatah, Ahmad Luqman Abdul; Piji, Mohd Al Akmarul Fizree Bin Md; Ramlan, Effirul Ikhwan; Raih, Mohd Firdaus

    2015-09-01

    Structured RNAs referred as riboswitches have been predicted to be present in the genome sequence of Burkholderia pseudomallei strain K96243. Four of the riboswitches were identified and analyzed through BLASTN, Rfam search and multiple sequence alignment. The RNA aptamers belong to the following riboswitch classifications: glycine riboswitch, cobalamin riboswitch, S-adenosyl-(L)-homocysteine (SAH) riboswitch and flavin mononucleotide (FMN) riboswitch. The conserved nucleotides for each aptamer were identified and were marked on the secondary structure generated by RNAfold. These riboswitches were successfully amplified and cloned for further study.

  11. Burkholderia mallei CLH001 Attenuated Vaccine Strain Is Immunogenic and Protects against Acute Respiratory Glanders.

    Science.gov (United States)

    Hatcher, Christopher L; Mott, Tiffany M; Muruato, Laura A; Sbrana, Elena; Torres, Alfredo G

    2016-08-01

    Burkholderia mallei is the causative agent of glanders, an incapacitating disease with high mortality rates in respiratory cases. Its endemicity and ineffective treatment options emphasize its public health threat and highlight the need for a vaccine. Live attenuated vaccines are considered the most viable vaccine strategy for Burkholderia, but single-gene-deletion mutants have not provided complete protection. In this study, we constructed the select-agent-excluded B. mallei ΔtonB Δhcp1 (CLH001) vaccine strain and investigated its ability to protect against acute respiratory glanders. Here we show that CLH001 is attenuated, safe, and effective at protecting against lethal B. mallei challenge. Intranasal administration of CLH001 to BALB/c and NOD SCID gamma (NSG) mice resulted in complete survival without detectable colonization or abnormal organ histopathology. Additionally, BALB/c mice intranasally immunized with CLH001 in a prime/boost regimen were fully protected against lethal challenge with the B. mallei lux (CSM001) wild-type strain.

  12. Diversities in virulence, antifungal activity, pigmentation and DNA fingerprint among strains of Burkholderia glumae.

    Science.gov (United States)

    Karki, Hari S; Shrestha, Bishnu K; Han, Jae Woo; Groth, Donald E; Barphagha, Inderjit K; Rush, Milton C; Melanson, Rebecca A; Kim, Beom Seok; Ham, Jong Hyun

    2012-01-01

    Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice. In this study, 11 naturally avirulent and nine virulent strains of B. glumae native to the southern United States were characterized in terms of virulence in rice and onion, toxofalvin production, antifungal activity, pigmentation and genomic structure. Virulence of B. glumae strains on rice panicles was highly correlated to virulence on onion bulb scales, suggesting that onion bulb can be a convenient alternative host system to efficiently determine the virulence of B. glumae strains. Production of toxoflavin, the phytotoxin that functions as a major virulence factor, was closely associated with the virulence phenotypes of B. glumae strains in rice. Some strains of B. glumae showed various levels of antifungal activity against Rhizoctonia solani, the causal agent of sheath blight, and pigmentation phenotypes on casamino acid-peptone-glucose (CPG) agar plates regardless of their virulence traits. Purple and yellow-green pigments were partially purified from a pigmenting strain of B. glumae, 411gr-6, and the purple pigment fraction showed a strong antifungal activity against Collectotrichum orbiculare. Genetic variations were detected among the B. glumae strains from DNA fingerprinting analyses by repetitive element sequence-based PCR (rep-PCR) for BOX-A1R-based repetitive extragenic palindromic (BOX) or enterobacterial repetitive intergenic consensus (ERIC) sequences of bacteria; and close genetic relatedness among virulent but pigment-deficient strains were revealed by clustering analyses of DNA fingerprints from BOX-and ERIC-PCR.

  13. Diversities in virulence, antifungal activity, pigmentation and DNA fingerprint among strains of Burkholderia glumae.

    Directory of Open Access Journals (Sweden)

    Hari S Karki

    Full Text Available Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice. In this study, 11 naturally avirulent and nine virulent strains of B. glumae native to the southern United States were characterized in terms of virulence in rice and onion, toxofalvin production, antifungal activity, pigmentation and genomic structure. Virulence of B. glumae strains on rice panicles was highly correlated to virulence on onion bulb scales, suggesting that onion bulb can be a convenient alternative host system to efficiently determine the virulence of B. glumae strains. Production of toxoflavin, the phytotoxin that functions as a major virulence factor, was closely associated with the virulence phenotypes of B. glumae strains in rice. Some strains of B. glumae showed various levels of antifungal activity against Rhizoctonia solani, the causal agent of sheath blight, and pigmentation phenotypes on casamino acid-peptone-glucose (CPG agar plates regardless of their virulence traits. Purple and yellow-green pigments were partially purified from a pigmenting strain of B. glumae, 411gr-6, and the purple pigment fraction showed a strong antifungal activity against Collectotrichum orbiculare. Genetic variations were detected among the B. glumae strains from DNA fingerprinting analyses by repetitive element sequence-based PCR (rep-PCR for BOX-A1R-based repetitive extragenic palindromic (BOX or enterobacterial repetitive intergenic consensus (ERIC sequences of bacteria; and close genetic relatedness among virulent but pigment-deficient strains were revealed by clustering analyses of DNA fingerprints from BOX-and ERIC-PCR.

  14. Diversities in Virulence, Antifungal Activity, Pigmentation and DNA Fingerprint among Strains of Burkholderia glumae

    Science.gov (United States)

    Karki, Hari S.; Shrestha, Bishnu K.; Han, Jae Woo; Groth, Donald E.; Barphagha, Inderjit K.; Rush, Milton C.; Melanson, Rebecca A.; Kim, Beom Seok; Ham, Jong Hyun

    2012-01-01

    Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice. In this study, 11 naturally avirulent and nine virulent strains of B. glumae native to the southern United States were characterized in terms of virulence in rice and onion, toxofalvin production, antifungal activity, pigmentation and genomic structure. Virulence of B. glumae strains on rice panicles was highly correlated to virulence on onion bulb scales, suggesting that onion bulb can be a convenient alternative host system to efficiently determine the virulence of B. glumae strains. Production of toxoflavin, the phytotoxin that functions as a major virulence factor, was closely associated with the virulence phenotypes of B. glumae strains in rice. Some strains of B. glumae showed various levels of antifungal activity against Rhizoctonia solani, the causal agent of sheath blight, and pigmentation phenotypes on casamino acid-peptone-glucose (CPG) agar plates regardless of their virulence traits. Purple and yellow-green pigments were partially purified from a pigmenting strain of B. glumae, 411gr-6, and the purple pigment fraction showed a strong antifungal activity against Collectotrichum orbiculare. Genetic variations were detected among the B. glumae strains from DNA fingerprinting analyses by repetitive element sequence-based PCR (rep-PCR) for BOX-A1R-based repetitive extragenic palindromic (BOX) or enterobacterial repetitive intergenic consensus (ERIC) sequences of bacteria; and close genetic relatedness among virulent but pigment-deficient strains were revealed by clustering analyses of DNA fingerprints from BOX-and ERIC-PCR. PMID:23028972

  15. Genome sequence of Burkholderia mimosarum strain LMG 23256(T), a Mimosa pigra microsymbiont from Anso, Taiwan.

    Science.gov (United States)

    Willems, Anne; Tian, Rui; Bräu, Lambert; Goodwin, Lynne; Han, James; Liolios, Konstantinos; Huntemann, Marcel; Pati, Amrita; Woyke, Tanja; Mavrommatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Reeve, Wayne

    2014-06-15

    Burkholderia mimosarum strain LMG 23256(T) is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Mimosa pigra (giant sensitive plant). LMG 23256(T) was isolated from a nodule recovered from the roots of the M. pigra growing in Anso, Taiwan. LMG 23256(T) is highly effective at fixing nitrogen with M. pigra. Here we describe the features of B. mimosarum strain LMG 23256(T), together with genome sequence information and its annotation. The 8,410,967 bp high-quality-draft genome is arranged into 268 scaffolds of 270 contigs containing 7,800 protein-coding genes and 85 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

  16. Genome sequence of Burkholderia mimosarum strain LMG 23256T, a Mimosa pigra microsymbiont from Anso, Taiwan

    Science.gov (United States)

    Willems, Anne; Tian, Rui; Bräu, Lambert; Goodwin, Lynne; Han, James; Liolios, Konstantinos; Huntemann, Marcel; Pati, Amrita; Woyke, Tanja; Mavrommatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Reeve, Wayne

    2013-01-01

    Burkholderia mimosarum strain LMG 23256T is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Mimosa pigra (giant sensitive plant). LMG 23256T was isolated from a nodule recovered from the roots of the M. pigra growing in Anso, Taiwan. LMG 23256T is highly effective at fixing nitrogen with M. pigra. Here we describe the features of B. mimosarum strain LMG 23256T, together with genome sequence information and its annotation. The 8,410,967 bp high-quality-draft genome is arranged into 268 scaffolds of 270 contigs containing 7,800 protein-coding genes and 85 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project. PMID:25197434

  17. [Homology analysis and historical tracing for inter-continental Burkholderia pseudomallei strains of sequence type 562].

    Science.gov (United States)

    Zheng, X; Wang, L X; Wu, H; Chen, H; Zhu, X; He, J R; Xia, L X; Li, W

    2017-05-10

    Objective: To understand the homology of sequence type 562 (ST562) strains of Burkholderia pseudomallei which circulated in two separate continents (Asia and Australia) at different times. Methods:SpeⅠrestriction fragments and 4-locus multiple locus variable number tandem repeat analysis (MLVA-4) profiles were extracted from MSHR5858 (ST562 Australia strain) and 350105 (ST562 historical strain of Hainan) genomes respectively by in silico analysis and then compared with the PFGE and MLVA-4 results of five ST562 clinical isolates from Hainan to test their homology. Synteny and homology between MSHR5858 and 350105 genomes were evaluated with bioinformatics methods. Results: Five ST562 clinical strains from Hainan shared same PFGE pattern (similarity>97%) and this pattern coincided to the map of SpeⅠrestriction fragments of Australian strain MSHR5858. The amounts of genomic restriction fragments (SpeⅠ) for MSHR5858 and 350105 were 31 and 34 respectively, with 31 of them matched by each other. Five ST562 clinical strains of Hainan were distinct by MLVA-4 profiles, among which HPPH43 (MLVA-4 profile: 10, 8, 10, 8) was close to Australia strain MSHR5858 (10, 8, 8, 6), containing identical repeat numbers at VNTR loci 2341k and 1788k; while HK003 (11, 8, 15, 7) and HK061 (11, 8, 17, 7) similar to Hainan historical strain 350105 (11, 8, 11, 8), with same repeat numbers at loci 2341k and 1788k also. High-degree synteny and consistency on genomic contents were observed between 350105 and MSHR5858, indicating a similar origin for the 2 strains. Conclusion: All inter-continental and historical ST562 strains of B. pseudomallei had similar genomic characteristics, supporting the assumption that they had a common origin. Also, it is possible that Hainan historical strain 350105 is the ancestor of all circulating ST562 strains.

  18. An objective approach for Burkholderia pseudomallei strain selection as challenge material for medical countermeasures efficacy testing

    Directory of Open Access Journals (Sweden)

    Kristopher E. Van Zandt

    2012-09-01

    Full Text Available Burkholderia pseudomallei is the causative agent of melioidosis, a rare disease of biodefense concern with high mortality and extreme difficulty in treatment. No human vaccines are available that protect against B. pseudomallei infection, and with the current limitations of antibiotic treatment, the development of new preventative and therapeutic interventions is crucial. Although clinical trials could be used to test the efficacy of new medical countermeasures (MCMs, the high mortality rates associated with melioidosis raises significant ethical issues concerning treating individuals with new compounds with unknown efficacies. The US Food and Drug Administration (FDA has formulated a set of guidelines for the licensure of new MCMs to treat diseases in which it would be unethical to test the efficacy of these drugs in humans. The FDA Animal Rule 21 CFR 314 calls for consistent, well-characterized B. pseudomallei strains to be used as challenge material in animal models. In order to facilitate the efficacy testing of new MCMs for melioidosis using animal models, we intend to develop a well-characterized panel of strains for use. This panel will comprise of strains that were isolated from human cases, have a low passage history, are virulent in animal models, and are well characterized phenotypically and genotypically. We have reviewed published and unpublished data on various B. pseudomallei strains to establish an objective method for selecting the strains to be included in the panel of B. pseudomallei strains with attention to five categories: animal infection models, genetic characterization, clinical and passage history, and availability of the strain to the research community. We identified 109 strains with data in at least one of the five categories, scored each strain based on the gathered data and identified 6 strains as candidate for a B. pseudomallei strain panel.

  19. [GENOTYPING OF THE BURKHOLDERIA MALLEI STRAINS BASED ON DIFFERENT REGION ANALYSIS].

    Science.gov (United States)

    Bondareva, O S; Savchenko, S S; Tkachenko, G A; Ledeneva, M L; Lemasova, L V; Antonov, V A

    2016-01-01

    Development of the genotyping methods of glanders agent is urgent due to its high pathogenicity, lack of effective preventive measures and threat of the use of Burkholderia mallei as a biological weapon. In this work we proposed a scheme for the typing of the B. mallei strains based on different region analysis (DFR). The choice of variable loci differentially presented in various strains of glanders agents was performed by analyzing annotated whole-genome sequences of the B. mallei strains. Primers and fluorescence probes were designed for 9 selected loci. The amplification conditions for different regions were optimized in two variants: with electrophoretic detection and hybridization-fluorescence detection in the strip format. The possibility of applying the DFR analysis to genetic characterization of strains was assessed in 14 B. mallei strains. The genetic profiles of the studied B. mallei strains revealed that the developed DFR-typing scheme was characterized by high discrimination power (Hunter-Gaston index value was 0.92), reproducibility, rapidity, easy interpretation, and applicability for epidemiological surveillance of glanders.

  20. Rapid emergence of a ceftazidime-resistant Burkholderia multivorans strain in a cystic fibrosis patient.

    Science.gov (United States)

    Stokell, Joshua R; Gharaibeh, Raad Z; Steck, Todd R

    2013-12-01

    Burkholderia multivorans poses a serious health threat to cystic fibrosis patients due to innate resistance to multiple antibiotics and acquisition of resistance to a range of antibiotics due to the frequent use of antibiotics to treat chronic infections. Monitoring antibiotic susceptibility is crucial to managing patient care. We identified the rapid emergence of a ceftazidime-resistant strain in a single patient within four days during a hospitalization for treatment of an exacerbation. B. multivorans was isolated from expectorated sputum samples using Burkholderia cepacia selective agar. A macrodilution assay was performed on all isolates to determine the minimum inhibitory concentration of ceftazidime. Approximately 4000 colonies were scored to identify the percent of ceftazidime-resistant colonies. Extracted DNA was used to determine the total bacterial counts and abundance of B. multivorans using quantitative PCR. An increase from no detectable B. multivorans ceftazidime-resistant colonies to over 75% of all colonies tested occurred within a four-day period. The resistant population remained dominant in 6 of the 8 samples in the following 17 months of the study. qPCR revealed an association between change in the percent of resistant colonies and abundance of B. multivorans, but not of total bacteria. No association was found between the acquisition of resistance to ceftazidime and other antibiotics commonly used to treat B. multivorans infections. The rapid emergence of a ceftazidime-resistant by B. multivorans strain occurred during a hospitalization while under selective pressure of antibiotics. The resistant strain maintained dominance in the B. multivorans population which resulted in an overall decline in a patient health and treatment efficacy. Copyright © 2013 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  1. Comparing in vitro and in vivo virulence phenotypes of Burkholderia pseudomallei type G strains.

    Science.gov (United States)

    Lewis, Eric R G; Kilgore, Paul B; Mott, Tiffany M; Pradenas, Gonzalo A; Torres, Alfredo G

    2017-01-01

    Burkholderia pseudomallei (Bpm) is a saprophytic rod-shaped gram-negative bacterium and the causative agent of melioidosis. This disease has previously been described as endemic in areas such as northern Australia and Southeast Asia, but, more recently, a better understanding of the epidemiology of melioidosis indicated that the disease is distributed worldwide, including regions of the Americas and Africa. A 16S-23S rDNA internal transcribed spacer (ITS) typing system has been developed for Bpm and has revealed that ITS types C, E, and hybrid CE are mainly associated with Australia and Southeast Asia while type G strains are more associated with cases of melioidosis in the Western Hemisphere. The purpose of the current study was to determine the in vitro and in vivo virulence profiles of the understudied Bpm type G strains Ca2009, Ca2013a, Mx2013, and 724644 and compared such phenotypes to the commonly studied Bpm type C strain K96243. We evaluated virulence by measuring invasion/uptake and survival of these Bpm strains in murine respiratory epithelial LA-4 cells and alveolar macrophage MH-S cells using different multiplicity of infections (MOIs of 1 and 10). We also calculated the lethal dose 50 values (LD50) in BALB/c mice that were inoculated intranasally with either Ca2009, Ca2013a, or Mx2013. Overall, the virulence and lethality phenotypes of Bpm type G strains were similar to the Bpm type C strain K96243. Additional comparative analyses between the Bpm ITS types may lead to a better understanding of the contribution of the ITS type to the epidemiology and ecology of Bpm strains.

  2. Identification and onion pathogenicity of Burkholderia cepacia complex isolates from the onion rhizosphere and onion field soil.

    Science.gov (United States)

    Jacobs, Janette L; Fasi, Anthony C; Ramette, Alban; Smith, James J; Hammerschmidt, Raymond; Sundin, George W

    2008-05-01

    Burkholderia cepacia complex strains are genetically related but phenotypically diverse organisms that are important opportunistic pathogens in patients with cystic fibrosis (CF,) as well as pathogens of onion and banana, colonizers of the rhizospheres of many plant species, and common inhabitants of bulk soil. Genotypic identification and pathogenicity characterization were performed on B. cepacia complex isolates from the rhizosphere of onion and organic soils in Michigan. A total of 3,798 putative B. cepacia complex isolates were recovered on Pseudomonas cepacia azelaic acid tryptamine and trypan blue tetracycline semiselective media during the 2004 growing season from six commercial onion fields located in two counties in Michigan. Putative B. cepacia complex isolates were identified by hybridization to a 16S rRNA gene probe, followed by duplex PCR using primers targeted to the 16S rRNA gene and recA sequences and restriction fragment length polymorphism analysis of the recA sequence. A total of 1,290 isolates, 980 rhizosphere and 310 soil isolates, were assigned to the species B. cepacia (160), B. cenocepacia (480), B. ambifaria (623), and B. pyrrocinia (27). The majority of isolates identified as B. cepacia (85%), B. cenocepacia (90%), and B. ambifaria (76%) were pathogenic in a detached onion bulb scale assay and caused symptoms of water soaking, maceration, and/or necrosis. A phylogenetic analysis of recA sequences from representative B. cepacia complex type and panel strains, along with isolates collected in this study, revealed that the B. cenocepacia isolates associated with onion grouped within the III-B lineage and that some strains were closely related to strain AU1054, which was isolated from a CF patient. This study revealed that multiple B. cepacia complex species colonize the onion rhizosphere and have the potential to cause sour skin rot disease of onion. In addition, the onion rhizosphere is a natural habitat and a potential environmental source

  3. Bacteria of the Burkholderia cepacia complex are cyanogenic under biofilm and colonial growth conditions

    Directory of Open Access Journals (Sweden)

    Hoshino Saiko

    2008-06-01

    Full Text Available Abstract Background The Burkholderia cepacia complex (Bcc is a collection of nine genotypically distinct but phenotypically similar species. They show wide ecological diversity and include species that are used for promoting plant growth and bio-control as well species that are opportunistic pathogens of vulnerable patients. Over recent years the Bcc have emerged as problematic pathogens of the CF lung. Pseudomonas aeruginosa is another important CF pathogen. It is able to synthesise hydrogen cyanide (HCN, a potent inhibitor of cellular respiration. We have recently shown that HCN production by P. aeruginosa may have a role in CF pathogenesis. This paper describes an investigation of the ability of bacteria of the Bcc to make HCN. Results The genome of Burkholderia cenocepacia has 3 putative HCN synthase encoding (hcnABC gene clusters. B. cenocepacia and all 9 species of the Bcc complex tested were able to make cyanide at comparable levels to P. aeruginosa, but only when grown surface attached as colonies or during biofilm growth on glass beads. In contrast to P. aeruginosa and other cyanogenic bacteria, cyanide was not detected during planktonic growth of Bcc strains. Conclusion All species in the Bcc are cyanogenic when grown as surface attached colonies or as biofilms.

  4. Strain Identification of Burkholderia cepacia Palleroni and Holmes and Pseudomonas fluorescens Migula Associated to Maize Crop by Polyphasic Taxonomy

    OpenAIRE

    Annia Hernández Rodríguez; María Esther González Vega; Alberto Caballero Núñez; Ariel Medina Concepción; Madelaine Quiñónez Pantoja; Mayra Heydrich Pérez; Ana Niurka Hernández Lauzardo; Monica Höfte

    2004-01-01

    A polyphasic taxonomic study, which included phenotypic characterization, the indirect immunofluorescence (IIF), and the polymerase chain reaction (PCR) was conducted for the identification of Burkholderia cepacia and Pseudomonas fluorescens strains previously isolated from maize rhizosphere. Conventional methods were used (API 20 NE, BioMeuriux), specific hyper-immune antiserum against the species under study, and specific primers were designed out of subunits 16S of the ribosomal RNA of B....

  5. Genetic Diversity of Burkholderia contaminans Isolates from Cystic Fibrosis Patients in Argentina

    OpenAIRE

    Martina, Pablo F.; Bettiol, Marisa; Vescina, Cecilia; Montanaro, Patricia; Mannino, Maria Constanza; Prieto, Claudia Inés; Vay, Carlos Alberto; Naumann, Dieter; Schmitt, Juergen; Yantorno, Osvaldo Miguel; Lagares, Antonio; Bosch, María Alejandra

    2013-01-01

    A total of 120 Burkholderia cepacia complex isolates collected during 2004?2010 from 66 patients in two cystic fibrosis reference centers in Argentina were analyzed. Burkholderia contaminans was the species most frequently recovered (57.6%), followed by Burkholderia cenocepacia (15%), a species distribution not reported so far. The recA-PCR-based techniques applied to the B. contaminans isolates revealed that 85% of the population carried the recA-ST-71 allele. Our results showed the utili...

  6. Purification, biochemical characterization, and genetic cloning of the phytase produced by Burkholderia sp. strain a13.

    Science.gov (United States)

    Graminho, Eduardo Rezende; Takaya, Naoki; Nakamura, Akira; Hoshino, Takayuki

    2015-01-01

    A phytase-producing bacterium, Burkholderia sp. a13 (JCM 30421), was isolated from Lake Kasumigaura by enrichment cultivation using minimum medium containing phytic acid as the sole phosphorus source. The phytase production by strain a13 was induced by the presence of phytic acid and repressed by the addition of glucose. The purified enzyme had a molecular weight of 44 kDa and a phytase activity of 174 μmol min(-1) mg(-1). The enzyme showed broad substrate specificity, but the highest activity was observed with phytic acid. The enzyme activity was strongly inhibited by Cu(2+), Zn(2+), Hg(2+), and iodoacetic acid, indicating the requirement of a thiol group for the activity. Genetic cloning reveals that the mature portion of this enzyme consists of 428 amino acids with a calculated molecular weight of 46 kDa. The amino acid sequence showed the highest similarity to the phytase produced by Hafnia alvei with 48% identity; it also contained histidine acid phosphatase (HAP) motifs (RHGXRXP and HD), indicating the classification of this enzyme in the HAP phytase family. We have successfully expressed the cloned gene in Escherichia coli from its putative initiation codon, showing that the gene actually encodes the phytase.

  7. Biochemical Characterization of 3-Methyl-4-nitrophenol degradation in Burkholderia sp. Strain SJ98

    Directory of Open Access Journals (Sweden)

    Jun eMin

    2016-05-01

    Full Text Available Several strains have been reported to grow on 3-methyl-4-nitrophenol (3M4NP, the primary breakdown product of the excessively used insecticide fenitrothion. However, the microbial degradation of 3M4NP at molecular and biochemical levels remains unknown. Here, methyl-1,4-benzoquinone (MBQ and methylhydroquinone (MHQ,rather than catechol proposed previously, were identified as the intermediates before ring cleavage during 3M4NP degradation by Burkholderia sp. strain SJ98. Real-time quantitative PCR analysis indicated that the pnpABA1CDEF cluster involved in para-nitrophenol (PNP and 2-chloro-4-nitrophenol (2C4NP catabolism was also likely responsible for 3M4NP degradation in this strain. Purified PNP 4-monooxygenase (PnpA is able to catalyze the monooxygenation of 3M4NP to MBQ and exhibited an apparent Km value of 20.3±2.54 μM for 3M4NP, and pnpA is absolutely necessary for the catabolism of 3M4NP by gene knock-out and complementation. PnpB, a 1,4-benzoquinone reductase catalyzes the reduction of MBQ to MHQ, and also found to enhance PnpA activity in vitro in the conversion of 3M4NP to MBQ. By sequential catalysis assays, PnpCD, PnpE and PnpF were likely involved in the lower pathway of 3M4NP catabolism. Although NpcCD, NpcE and NpcF are able to catalyze the sequential conversion of MHQ in vitro, these enzymes are unlikely involved in 3M4NP catabolism because their coding genes were not upregulated by 3M4NP induction in vivo. These results revealed that the enzymes involved in PNP and 2C4NP catabolism were also responsible for 3M4NP degradation in strain SJ98. This fills a gap in our understanding of the microbial degradation of 3M4NP at molecular and biochemical levels and also provides another example to illustrate the adaptive flexibility in microbial catabolism for structurally similar compounds.

  8. Burkholderia cepacia complex infection in an Adult Cystic Fibrosis unit in Madrid.

    Science.gov (United States)

    Correa-Ruiz, Ana; Girón, Rosa; Buendía, Buenaventura; Medina-Pascual, M José; Valenzuela, Claudia; López-Brea, Manuel; Sáez-Nieto, Juan Antonio

    2013-12-01

    Burkholderia cepacia complex have emerged as significant pathogens in cystic fibrosis (CF) patients due to the risk of cepacia syndrome and the innate multi-resistance of the microorganisms to antibiotics. The aim of this study was to describe the antimicrobial susceptibility profiles, the genotypes and subtypes of BCC, and the clinical evolution of CF patients with BCC. The lung function and Brasfield and Shwachman score were assessed in 12 patients. BCC were identified and susceptibility was studied by MicroScan (Siemens). Species and genospecies of BCC were confirmed by molecular methods in a Reference Centre (Majadahonda). BCC were identified in 12 of 70 patients (17.1%) over a ten year period. The mean age to colonization by BCC was 24.4 years (SD: 7.71). B. cenocepacia was isolated in 4 patients (33.3%), B. contaminans was isolated in 3 patients (25%), both B. vietnamiensis and B. stabilis were isolated in 2 patients (16.7%), and B. cepacia, B. multivorans and B. late were isolated in one patient (8.3%). Among the B. cenocepacia, subtype IIIa was identified in two strains, and subtype IIIb was identified in the other two strains. There was susceptibility to meropenem in 90% of BCC, 80% to cotrimoxazole, 60% to minocycline, 50% to ceftazidime, and 40% to levofloxacin. B. cenocepacia was the most prevalent species among the BCC isolated in CF adult patients, and subtypes IIIa and IIIb were identified in the 50% of the strains. Meropenem and cotrimoxazole showed the best activity. Copyright © 2012 Elsevier España, S.L. All rights reserved.

  9. Genome sequence of the acid-tolerant Burkholderia sp. strain WSM2230 from Karijini National Park, Australia.

    Science.gov (United States)

    Walker, Robert; Watkin, Elizabeth; Tian, Rui; Bräu, Lambert; O'Hara, Graham; Goodwin, Lynne; Han, James; Lobos, Elizabeth; Huntemann, Marcel; Pati, Amrita; Woyke, Tanja; Mavromatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Reeve, Wayne

    2014-06-15

    Burkholderia sp. strain WSM2230 is an aerobic, motile, Gram-negative, non-spore-forming acid-tolerant rod isolated from acidic soil collected in 2001 from Karijini National Park, Western Australia, using Kennedia coccinea (Coral Vine) as a host. WSM2230 was initially effective in nitrogen-fixation with K. coccinea, but subsequently lost symbiotic competence. Here we describe the features of Burkholderia sp. strain WSM2230, together with genome sequence information and its annotation. The 6,309,801 bp high-quality-draft genome is arranged into 33 scaffolds of 33 contigs containing 5,590 protein-coding genes and 63 RNA-only encoding genes. The genome sequence of WSM2230 failed to identify nodulation genes and provides an explanation for the observed failure of the laboratory grown strain to nodulate. The genome of this strain is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

  10. φX216, a P2-like bacteriophage with broad Burkholderia pseudomallei and B. mallei strain infectivity

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    Kvitko Brian H

    2012-12-01

    Full Text Available Abstract Background Burkholderia pseudomallei and B. mallei are closely related Category B Select Agents of bioterrorism and the causative agents of the diseases melioidosis and glanders, respectively. Rapid phage-based diagnostic tools would greatly benefit early recognition and treatment of these diseases. There is extensive strain-to-strain variation in B. pseudomallei genome content due in part to the presence or absence of integrated prophages. Several phages have previously been isolated from B. pseudomallei lysogens, for example φK96243, φ1026b and φ52237. Results We have isolated a P2-like bacteriophage, φX216, which infects 78% of all B. pseudomallei strains tested. φX216 also infects B. mallei, but not other Burkholderia species, including the closely related B. thailandensis and B. oklahomensis. The nature of the φX216 host receptor remains unclear but evidence indicates that in B. mallei φX216 uses lipopolysaccharide O-antigen but a different receptor in B. pseudomallei. The 37,637 bp genome of φX216 encodes 47 predicted open reading frames and shares 99.8% pairwise identity and an identical strain host range with bacteriophage φ52237. Closely related P2-like prophages appear to be widely distributed among B. pseudomallei strains but both φX216 and φ52237 readily infect prophage carrying strains. Conclusions The broad strain infectivity and high specificity for B. pseudomallei and B. mallei indicate that φX216 will provide a good platform for the development of phage-based diagnostics for these bacteria.

  11. Genome sequence of the acid-tolerant Burkholderia sp. strain WSM2232 from Karijini National Park, Australia.

    Science.gov (United States)

    Walker, Robert; Watkin, Elizabeth; Tian, Rui; Bräu, Lambert; O'Hara, Graham; Goodwin, Lynne; Han, James; Reddy, Tatiparthi; Huntemann, Marcel; Pati, Amrita; Woyke, Tanja; Mavromatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Reeve, Wayne

    2014-06-15

    Burkholderia sp. strain WSM2232 is an aerobic, motile, Gram-negative, non-spore-forming acid-tolerant rod that was trapped in 2001 from acidic soil collected from Karijini National Park (Australia) using Gastrolobium capitatum as a host. WSM2232 was effective in nitrogen fixation with G. capitatum but subsequently lost symbiotic competence during long-term storage. Here we describe the features of Burkholderia sp. strain WSM2232, together with genome sequence information and its annotation. The 7,208,311 bp standard-draft genome is arranged into 72 scaffolds of 72 contigs containing 6,322 protein-coding genes and 61 RNA-only encoding genes. The loss of symbiotic capability can now be attributed to the loss of nodulation and nitrogen fixation genes from the genome. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

  12. Study of class I integron in a Burkholderia cepacia complex strain isolated from blood colture

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    Linda Furlanis

    2011-06-01

    Full Text Available The Burkholderia cepacia complex (Bcc consists of several species that cause lung infections in patients with cystic fibrosis but are also capable to colonize immunocompromised patients. Once established, the infection is usually difficult to eradicate, as Bcc is intrinsically resistant to many antibiotics. Besides, the acquisition of additional resistance determinants by horizontal gene transfer makes very difficult the therapeutic approach to these infections. Among horizontally acquired DNAs, integrons have been frequently reported in many Gramnegative bacteria that affect human health, but they have not been found frequently in Burkholderia isolates until now. In the present work we report on a Bcc isolate, recovered from the blood of an immunocompromised patient, that carries a 2.3 kb class I integron already described in a Salmonella enterica isolate eight years ago, coding for aacA4, aadA1 and catB2 in its cassette array.

  13. Transcriptional responses of the bacterium Burkholderia terrae BS001 to the fungal host Lyophyllum sp strain Karsten under soil-mimicking conditions

    NARCIS (Netherlands)

    Ul Haq, Irshad; Dini-Andreote, Francisco; van Elsas, Jan Dirk

    2017-01-01

    In this study, the mycosphere isolate Burkholderia terrae BS001 was confronted with the soil fungus Lyophyllum sp. strain Karsten on soil extract agar plates in order to examine its transcriptional responses over time. At the initial stages of the experiment (T1-day 3; T2-day 5), contact between bot

  14. The role of hydrophobicity and surface receptors at hyphae of Lyophyllum sp. strain Karsten in the interaction with Burkholderia terrae BS001 : Implications for interactions in soil

    NARCIS (Netherlands)

    Vila, Taissa; Nazir, Rashid; Rozental, Sonia; dos Santos, Giulia M. P.; Calixto, Renata O.R.; Barreto-Bergter, Eliana; Wick, Lukas Y.; van Elsas, Jan Dirk

    2016-01-01

    The soil bacterium Burkholderia terrae strain BS001 can interact with varying soil fungi, using mechanisms that range from the utilization of carbon/energy sources such as glycerol to the ability to reach novel territories in soil via co-migration with growing fungal mycelia. Here, we investigate th

  15. Garlic revisited: antimicrobial activity of allicin-containing garlic extracts against Burkholderia cepacia complex.

    Science.gov (United States)

    Wallock-Richards, Daynea; Doherty, Catherine J; Doherty, Lynsey; Clarke, David J; Place, Marc; Govan, John R W; Campopiano, Dominic J

    2014-01-01

    The antimicrobial activities of garlic and other plant alliums are primarily based on allicin, a thiosulphinate present in crushed garlic bulbs. We set out to determine if pure allicin and aqueous garlic extracts (AGE) exhibit antimicrobial properties against the Burkholderia cepacia complex (Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. We prepared an AGE from commercial garlic bulbs and used HPLC to quantify the amount of allicin therein using an aqueous allicin standard (AAS). Initially we determined the minimum inhibitory concentrations (MICs) of the AGE against 38 Bcc isolates; these MICs ranged from 0.5 to 3% (v/v). The antimicrobial activity of pure allicin (AAS) was confirmed by MIC and minimum bactericidal concentration (MBC) assays against a smaller panel of five Bcc isolates; these included three representative strains of the most clinically important species, B. cenocepacia. Time kill assays, in the presence of ten times MIC, showed that the bactericidal activity of AGE and AAS against B. cenocepacia C6433 correlated with the concentration of allicin. We also used protein mass spectrometry analysis to begin to investigate the possible molecular mechanisms of allicin with a recombinant form of a thiol-dependent peroxiredoxin (BCP, Prx) from B. cenocepacia. This revealed that AAS and AGE modifies an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. To our knowledge, we report the first evidence that allicin and allicin-containing garlic extracts possess inhibitory and bactericidal activities against the Bcc. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit investigation as adjuncts to existing antibiotics.

  16. Garlic revisited: antimicrobial activity of allicin-containing garlic extracts against Burkholderia cepacia complex.

    Directory of Open Access Journals (Sweden)

    Daynea Wallock-Richards

    Full Text Available The antimicrobial activities of garlic and other plant alliums are primarily based on allicin, a thiosulphinate present in crushed garlic bulbs. We set out to determine if pure allicin and aqueous garlic extracts (AGE exhibit antimicrobial properties against the Burkholderia cepacia complex (Bcc, the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. We prepared an AGE from commercial garlic bulbs and used HPLC to quantify the amount of allicin therein using an aqueous allicin standard (AAS. Initially we determined the minimum inhibitory concentrations (MICs of the AGE against 38 Bcc isolates; these MICs ranged from 0.5 to 3% (v/v. The antimicrobial activity of pure allicin (AAS was confirmed by MIC and minimum bactericidal concentration (MBC assays against a smaller panel of five Bcc isolates; these included three representative strains of the most clinically important species, B. cenocepacia. Time kill assays, in the presence of ten times MIC, showed that the bactericidal activity of AGE and AAS against B. cenocepacia C6433 correlated with the concentration of allicin. We also used protein mass spectrometry analysis to begin to investigate the possible molecular mechanisms of allicin with a recombinant form of a thiol-dependent peroxiredoxin (BCP, Prx from B. cenocepacia. This revealed that AAS and AGE modifies an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. To our knowledge, we report the first evidence that allicin and allicin-containing garlic extracts possess inhibitory and bactericidal activities against the Bcc. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit investigation as adjuncts to existing antibiotics.

  17. Disinfection of Burkholderia cepacia complex from non-touch taps in a neonatal nursery.

    Science.gov (United States)

    Kotsanas, Despina; Brett, Judith; Kidd, Tim J; Stuart, Rhonda L; Korman, Tony M

    2008-01-01

    Burkholderia cepacia complex (Bcc) comprises nine closely related species or genomovars. It is an important causative agent of opportunistic infections and waterborne nosocomial infections. B. cepacia (formerly genomovar I) was identified from the blood culture of a baby in our neonatal unit (NU) in March 2005. B. cepacia was isolated four times from clinical specimens since the introduction of non-touch taps in the NU from 2000 to 2005 and only once from 1994 to 2000. Environmental samples were collected from the NU, including tap water from non-touch taps. Clinical and environmental isolates of Bcc were characterized using molecular identification and strain typing. A literature review was undertaken to delineate a method for eradication of Bcc. Several variations for hot water eradication of the organism from the taps were attempted. Genotyping and molecular analysis revealed that tap water isolates were B. cenocepacia which was a different species from the B. cepacia isolated from blood cultures of the neonate. However, B. cenocepacia has been known to cause nosocomial outbreaks and it was eventually eradicated from the NU by using repeated thermal shock (hot water at 65 degrees C for 10 min), changing taps and decolonizing sinks with hypochlorite. Molecular typing is useful in assisting the investigation of Bcc nosocomial infections.

  18. Burkholderia cepacia complex Phage-Antibiotic Synergy (PAS): antibiotics stimulate lytic phage activity.

    Science.gov (United States)

    Kamal, Fatima; Dennis, Jonathan J

    2015-02-01

    The Burkholderia cepacia complex (Bcc) is a group of at least 18 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. Bcc organisms possess high levels of innate antimicrobial resistance, and alternative therapeutic strategies are urgently needed. One proposed alternative treatment is phage therapy, the therapeutic application of bacterial viruses (or bacteriophages). Recently, some phages have been observed to form larger plaques in the presence of sublethal concentrations of certain antibiotics; this effect has been termed phage-antibiotic synergy (PAS). Those reports suggest that some antibiotics stimulate increased production of phages under certain conditions. The aim of this study is to examine PAS in phages that infect Burkholderia cenocepacia strains C6433 and K56-2. Bcc phages KS12 and KS14 were tested for PAS, using 6 antibiotics representing 4 different drug classes. Of the antibiotics tested, the most pronounced effects were observed for meropenem, ciprofloxacin, and tetracycline. When grown with subinhibitory concentrations of these three antibiotics, cells developed a chain-like arrangement, an elongated morphology, and a clustered arrangement, respectively. When treated with progressively higher antibiotic concentrations, both the sizes of plaques and phage titers increased, up to a maximum. B. cenocepacia K56-2-infected Galleria mellonella larvae treated with phage KS12 and low-dose meropenem demonstrated increased survival over controls treated with KS12 or antibiotic alone. These results suggest that antibiotics can be combined with phages to stimulate increased phage production and/or activity and thus improve the efficacy of bacterial killing.

  19. Trimeric autotransporter adhesins in members of the Burkholderia cepacia complex: a multifunctional family of proteins implicated in virulence

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    Arsénio Mendes Fialho

    2011-12-01

    Full Text Available Trimeric autotransporter adhesins (TAAs are multimeric surface proteins, involved in various biological traits of pathogenic Gram-negative bacteria including adherence, biofilm formation, invasion, survival within eukaryotic cells, serum resistance and cytotoxicity. TAAs have a modular architecture composed by a conserved membrane-anchored C-terminal domain and a variable number of stalk and head domains. In this study, a bioinformatic approach has been used to analyze the distribution and architecture of TAAs among Burkholderia cepacia complex (Bcc genomes. Fifteen genomes were probed revealing a total of 74 encoding sequences. Compared with other bacterial species, the Bcc genomes contain a disproportionately large number of TAAs (two genes to up to 8 genes, such as in B.cenocepacia. Phylogenetic analysis showed that the TAAs grouped into at least eight distinct clusters. TAAs with serine-rich repeats are clearly well separated from others, thereby representing a different evolutionary lineage. Comparative gene mapping across Bcc genomes reveals that TAA genes are inserted within conserved synteny blocks. We further focused our analysis on the epidemic strain B. cenocepacia J2315 in which 7 TAAs were annotated. Among these, 3 TAA-encoding genes (BCAM019, BCAM0223 and BCAM0224 are organized into a cluster and are candidates for multifunctional virulence factors. Here we review the current insights into the functional role of BCAM0224 as a model locus.

  20. Burkholderia cepacia complex isolates survive intracellularly without replication within acidic vacuoles of Acanthamoeba polyphaga.

    Science.gov (United States)

    Lamothe, Julie; Thyssen, Sandra; Valvano, Miguel A

    2004-12-01

    We have previously demonstrated that isolates of the Burkholderia cepacia complex can survive intracellularly in murine macrophages and in free-living Acanthamoeba. In this work, we show that the clinical isolates B. vietnamiensis strain CEP040 and B. cenocepacia H111 survived but did not replicate within vacuoles of A. polyphaga. B. cepacia-containing vacuoles accumulated the fluid phase marker Lysosensor Blue and displayed strong blue fluorescence, indicating that they had low pH. In contrast, the majority of intracellular bacteria within amoebae treated with the V-ATPse inhibitor bafilomycin A1 localized in vacuoles that did not fluoresce with Lysosensor Blue. Experiments using bacteria fluorescently labelled with chloromethylfluorescein diacetate demonstrated that intracellular bacteria remained viable for at least 24 h. In contrast, Escherichia coli did not survive within amoebae after 2 h post infection. Furthermore, intracellular B. vietnamiensis CEP040 retained green fluorescent protein within the bacterial cytoplasm, while this protein rapidly escaped from the cytosol of phagocytized heat-killed bacteria into the vacuolar lumen. Transmission electron microscopy analysis confirmed that intracellular Burkholderia cells were structurally intact. In addition, both Legionella pneumophila- and B. vietnamiensis-containing vacuoles did not accumulate cationized ferritin, a compound that localizes within the lysosome. Thus, our observations support the notion that B. cepacia complex isolates can use amoebae as a reservoir in the environment by surviving without intracellular replication within an acidic vacuole that is distinct from the lysosomal compartment.

  1. Investigating Burkholderia cepacia complex populations recovered from Italian maize rhizosphere by multilocus sequence typing.

    Science.gov (United States)

    Dalmastri, Claudia; Baldwin, Adam; Tabacchioni, Silvia; Bevivino, Annamaria; Mahenthiralingam, Eshwar; Chiarini, Luigi; Dowson, Christopher

    2007-07-01

    The Burkholderia cepacia complex (BCC) comprises at least nine closely related species of abundant environmental microorganisms. Some of these species are highly spread in the rhizosphere of several crop plants, particularly of maize; additionally, as opportunistic pathogens, strains of the BCC are capable of colonizing humans. We have developed and validated a multilocus sequence typing (MLST) scheme for the BCC. Although widely applied to understand the epidemiology of bacterial pathogens, MLST has seen limited application to the population analysis of species residing in the natural environment; we describe its novel application to BCC populations within maize rhizospheres. 115 BCC isolates were recovered from the roots of different maize cultivars from three different Italian regions over a 9-year period (1994-2002). A total of 44 sequence types (STs) were found of which 41 were novel when compared with existing MLST data which encompassed a global database of 1000 clinical and environmental strains representing nearly 400 STs. In this study of rhizosphere isolates approximately 2.5 isolates per ST was found, comparable to that found for the whole BCC population. Multilocus sequence typing also resolved inaccuracies associated with previous identification of the maize isolates based on recA gene restriction fragment length polymorphims and species-specific polymerase chain reaction. The 115 maize isolates comprised the following BCC species groups, B. ambifaria (39%), BCC6 (29%), BCC5 (10%), B. pyrrocinia (8%), B. cenocepacia IIIB (7%) and B. cepacia (6%), with BCC5 and BCC6 potentially constituting novel species groups within the complex. Closely related clonal complexes of strains were identified within B. cepacia, B. cenocepacia IIIB, BCC5 and BCC6, with one of the BCC5 clonal complexes being distributed across all three sampling sites. Overall, our analysis demonstrates that the maize rhizosphere harbours a massive diversity of novel BCC STs, so that their

  2. Characterization of clinically-attenuated Burkholderia mallei by whole genome sequencing: candidate strain for exclusion from Select Agent lists.

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    Steven E Schutzer

    Full Text Available BACKGROUND: Burkholderia mallei is an understudied biothreat agent responsible for glanders which can be lethal in humans and animals. Research with this pathogen has been hampered in part by constraints of Select Agent regulations for safety reasons. Whole genomic sequencing (WGS is an apt approach to characterize newly discovered or poorly understood microbial pathogens. METHODOLOGY/PRINCIPAL FINDINGS: We performed WGS on a strain of B. mallei, SAVP1, previously pathogenic, that was experimentally infected in 6 equids (4 ponies, 1 mule, 1 donkey, natural hosts, for purposes of producing antibodies. Multiple high inocula were used in some cases. Unexpectedly SAVP1 appeared to be avirulent in the ponies and mule, and attenuated in the donkey, but induced antibodies. We determined the genome sequence of SAVP1 and compared it to a strain that was virulent in horses and a human. In comparison, this phenotypic avirulent SAVP1 strain was missing multiple genes including all the animal type III secretory system (T3SS complex of genes demonstrated to be essential for virulence in mice and hamster models. The loss of these genes in the SAVP1 strain appears to be the consequence of a multiple gene deletion across insertion sequence (IS elements in the B. mallei genome. Therefore, the strain by itself is unlikely to revert naturally to its virulent phenotype. There were other genes present in one strain and not the other and vice-versa. CONCLUSION/SIGNIFICANCE: The discovery that this strain of B. mallei was both avirulent in the natural host ponies, and did not possess T3SS associated genes may be fortuitous to advance biodefense research. The deleted virulence-essential T3SS is not likely to be re-acquired naturally. These findings may provide a basis for exclusion of SAVP1 from the Select Agent regulation or at least discussion of what else would be required for exclusion. This exclusion could accelerate research by investigators not possessing BSL-3

  3. Two stable variants of Burkholderia pseudomallei strain MSHR5848 express broadly divergent in vitro phenotypes associated with their virulence differences

    Science.gov (United States)

    Cote, C. K.; Chase, C. J.; Koehler, J. W.; Klimko, C. P.; Ladner, J. T.; Rozak, D. A.; Wolcott, M. J.; Fetterer, D. P.; Kern, S. J.; Koroleva, G. I.; Lovett, S. P.; Palacios, G. F.; Toothman, R. G.; Bozue, J. A.; Worsham, P. L.; Welkos, S. L.

    2017-01-01

    Burkholderia pseudomallei (Bp), the agent of melioidosis, causes disease ranging from acute and rapidly fatal to protracted and chronic. Bp is highly infectious by aerosol, can cause severe disease with nonspecific symptoms, and is naturally resistant to multiple antibiotics. However, no vaccine exists. Unlike many Bp strains, which exhibit random variability in traits such as colony morphology, Bp strain MSHR5848 exhibited two distinct and relatively stable colony morphologies on sheep blood agar plates: a smooth, glossy, pale yellow colony and a flat, rough, white colony. Passage of the two variants, designated “Smooth” and “Rough”, under standard laboratory conditions produced cultures composed of > 99.9% of the single corresponding type; however, both could switch to the other type at different frequencies when incubated in certain nutritionally stringent or stressful growth conditions. These MSHR5848 derivatives were extensively characterized to identify variant-associated differences. Microscopic and colony morphology differences on six differential media were observed and only the Rough variant metabolized sugars in selective agar. Antimicrobial susceptibilities and lipopolysaccharide (LPS) features were characterized and phenotype microarray profiles revealed distinct metabolic and susceptibility disparities between the variants. Results using the phenotype microarray system narrowed the 1,920 substrates to a subset which differentiated the two variants. Smooth grew more rapidly in vitro than Rough, yet the latter exhibited a nearly 10-fold lower lethal dose for mice than Smooth. Finally, the Smooth variant was phagocytosed and replicated to a greater extent and was more cytotoxic than Rough in macrophages. In contrast, multiple locus sequence type (MLST) analysis, ribotyping, and whole genome sequence analysis demonstrated the variants’ genetic conservation; only a single consistent genetic difference between the two was identified for further

  4. Characterization of the Burkholderia mallei tonB Mutant and Its Potential as a Backbone Strain for Vaccine Development.

    Science.gov (United States)

    Mott, Tiffany M; Vijayakumar, Sudhamathi; Sbrana, Elena; Endsley, Janice J; Torres, Alfredo G

    2015-01-01

    In this study, a Burkholderia mallei tonB mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis. Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 10(4) CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001. Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis.

  5. Characterization of the Burkholderia mallei tonB Mutant and Its Potential as a Backbone Strain for Vaccine Development.

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    Tiffany M Mott

    Full Text Available In this study, a Burkholderia mallei tonB mutant (TMM001 deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis.Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 10(4 CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001.Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis.

  6. Multiphasic characterization of a plant growth promoting bacterial strain, Burkholderia sp, 7016 and its effect on tomato growth in the field

    Institute of Scientific and Technical Information of China (English)

    GAO Miao[1; ZHOU Jian-jiao[1; WANG En-tao[2; CHEN Qian[1; XU Jing[1; SUN Jian-guana[1

    2015-01-01

    Aiming at searching for plant growth promoting rhizobacteria (PGPR), a bacterium strain coded as 7016 was isolated from soybean rhizosphere and was characterized in the present study. It was identified as Burkholderia sp. based on 16S rDNA sequence analysis, as well as phenotypic and biochemical characterizations. This bacterium presented nitrogenase activity, 1-aminocyclopropane-l-carboxylic acid (ACC) deaminase activity and phosphate solubilizing ability; inhibited the growth of Sclerotinia sclerotiorum, Gibberella zeae and Verticillium dahliae; and produced small quantities of indole acetic acid (IAA). In green house experiments, significant increases in shoot height and weight, root length and weight, and stem diameter were observed on tomato plants in 30 d after inoculation with strain 7016. Result of 16S rDNA PCR-DGGE showed that 7016 survived in the rhizosphere of tomato seedlings. In the field experiments, Burkholderia sp. 7016 enhanced the tomato yield and significantly promoted activities of soil urease, phosphatase, sucrase, and catalase. All these results demonstrated Burkholderia sp. 7016 as a valuable PGPR and a candidate of biofertilizer.

  7. Multiphasic characterization of a plant growth promoting bacterial strain, Burkholderia sp. 7016 and its effect on tomato growth in the ifeld

    Institute of Scientific and Technical Information of China (English)

    GAO Miao; ZHOU Jian-jiao; WANG En-tao; CHEN Qian; XU Jing; SUN Jian-guang

    2015-01-01

    Aiming at searching for plant growth promoting rhizobacteria (PGPR), a bacterium strain coded as 7016 was isolated from soybean rhizosphere and was characterized in the present study. It was identiifed as Burkholderia sp. based on 16S rDNA sequence analysis, as wel as phenotypic and biochemical characterizations. This bacterium presented nitrogenase activity, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity and phosphate solubilizing ability;inhibited the growth of Sclerotinia sclerotiorum, Gibberel a zeae and Verticil ium dahliae;and produced smal quantities of indole acetic acid (IAA). In green house experiments, signiifcant increases in shoot height and weight, root length and weight, and stem diameter were observed on tomato plants in 30 d after inoculation with strain 7016. Result of 16S rDNA PCR-DGGE showed that 7016 survived in the rhizosphere of tomato seedlings. In the ifeld experiments, Burkholderia sp. 7016 enhanced the tomato yield and signiifcantly promoted activities of soil urease, phosphatase, sucrase, and catalase. Al these results demonstrated Burkholderia sp. 7016 as a valuable PGPR and a candidate of biofertilizer.

  8. Characterization of in vitro phenotypes of pathogenic Burkholderia strains isolated from infected mice

    Science.gov (United States)

    2017-06-13

    These media were SBA, GTA, AA, OF- PBL , BCA, and BCSA. In addition, 297 skim milk agar was included to assay for protease production. Because Bm...Strain Characteristic:* 1- SBAP 2- GTA 3- Ashdown/+gent 4- OF- PBL variant: 406e parent 1- size 2.5–3 mm - 3 mm 1.5–2 mm 1 mm 2- color of colony

  9. Aerosol phage therapy efficacy in Burkholderia cepacia complex respiratory infections.

    Science.gov (United States)

    Semler, Diana D; Goudie, Amanda D; Finlay, Warren H; Dennis, Jonathan J

    2014-07-01

    Phage therapy has been suggested as a potential treatment for highly antibiotic-resistant bacteria, such as the species of the Burkholderia cepacia complex (BCC). To address this hypothesis, experimental B. cenocepacia respiratory infections were established in mice using a nebulizer and a nose-only inhalation device. Following infection, the mice were treated with one of five B. cenocepacia-specific phages delivered as either an aerosol or intraperitoneal injection. The bacterial and phage titers within the lungs were assayed 2 days after treatment, and mice that received the aerosolized phage therapy demonstrated significant decreases in bacterial loads. Differences in phage activity were observed in vivo. Mice that received phage treatment by intraperitoneal injection did not demonstrate significantly reduced bacterial loads, although phage particles were isolated from their lung tissue. Based on these data, aerosol phage therapy appears to be an effective method for treating highly antibiotic-resistant bacterial respiratory infections, including those caused by BCC bacteria.

  10. Growth promotion and colonization of switchgrass (Panicum virgatum cv. Alamo by bacterial endophyte Burkholderia phytofirmans strain PsJN

    Directory of Open Access Journals (Sweden)

    Kim Seonhwa

    2012-05-01

    Full Text Available Abstract Background Switchgrass is one of the most promising bioenergy crop candidates for the US. It gives relatively high biomass yield and can grow on marginal lands. However, its yields vary from year to year and from location to location. Thus it is imperative to develop a low input and sustainable switchgrass feedstock production system. One of the most feasible ways to increase biomass yields is to harness benefits of microbial endophytes. Results We demonstrate that one of the most studied plant growth promoting bacterial endophytes, Burkholderia phytofirmans strain PsJN, is able to colonize and significantly promote growth of switchgrass cv. Alamo under in vitro, growth chamber, and greenhouse conditions. In several in vitro experiments, the average fresh weight of PsJN-inoculated plants was approximately 50% higher than non-inoculated plants. When one-month-old seedlings were grown in a growth chamber for 30 days, the PsJN-inoculated Alamo plants had significantly higher shoot and root biomass compared to controls. Biomass yield (dry weight averaged from five experiments was 54.1% higher in the inoculated treatment compared to non-inoculated control. Similar results were obtained in greenhouse experiments with transplants grown in 4-gallon pots for two months. The inoculated plants exhibited more early tillers and persistent growth vigor with 48.6% higher biomass than controls. We also found that PsJN could significantly promote growth of switchgrass cv. Alamo under sub-optimal conditions. However, PsJN-mediated growth promotion in switchgrass is genotype specific. Conclusions Our results show B. phytofirmans strain PsJN significantly promotes growth of switchgrass cv. Alamo under different conditions, especially in the early growth stages leading to enhanced production of tillers. This phenomenon may benefit switchgrass establishment in the first year. Moreover, PsJN significantly stimulated growth of switchgrass cv. Alamo under sub

  11. The third replicon of members of the Burkholderia cepacia Complex, plasmid pC3, plays a role in stress tolerance.

    Science.gov (United States)

    Agnoli, Kirsty; Frauenknecht, Carmen; Freitag, Roman; Schwager, Stephan; Jenul, Christian; Vergunst, Annette; Carlier, Aurelien; Eberl, Leo

    2014-02-01

    The metabolically versatile Burkholderia cepacia complex (Bcc) occupies a variety of niches, including the plant rhizosphere and the cystic fibrosis lung (where it is often fatal to the patient). Bcc members have multipartite genomes, of which the third replicon, pC3 (previously chromosome 3), has been shown to be a nonessential megaplasmid which confers virulence and both antifungal and proteolytic activity on several strains. In this study, pC3 curing was extended to cover strains of 16 of the 17 members of the Bcc, and the phenotypes conferred by pC3 were determined. B. cenocepacia strains H111, MCO-3, and HI2424 were previously cured of pC3; however, this had not proved possible in the epidemic strain K56-2. Here, we investigated the mechanism of this unexpected stability and found that efficient toxin-antitoxin systems are responsible for maintaining pC3 of strain K56-2. Identification of these systems allowed neutralization of the toxins and the subsequent deletion of K56-2pC3. The cured strain was found to exhibit reduced antifungal activity and was attenuated in both the zebrafish and the Caenorhabditis elegans model of infection. We used a PCR screening method to examine the prevalence of pC3 within 110 Bcc isolates and found that this replicon was absent in only four cases, suggesting evolutionary fixation. It is shown that plasmid pC3 increases the resistance of B. cenocepacia H111 to various stresses (oxidative, osmotic, high-temperature, and chlorhexidine-induced stresses), explaining the prevalence of this replicon within the Bcc.

  12. Draft Genome Sequence of the Soil Bacterium Burkholderia terrae Strain BS001, Which Interacts with Fungal Surface Structures

    DEFF Research Database (Denmark)

    Nazir, Rashid; Hansen, Martin A.; Sorensen, Soren

    2012-01-01

    Burkholderia terrae BS001 is a soil bacterium which was originally isolated from the mycosphere of the ectomycorrhizal fungus Laccaria proxima. It exhibits a range of fungus-interacting traits which reveal its propensity to actively interact at fungal interfaces. Here, we present the approximatel...

  13. Selection of nitrogen-fixing deficient Burkholderia vietnamiensis strains by cystic fibrosis patients: involvement of nif gene deletions and auxotrophic mutations.

    Science.gov (United States)

    Menard, Aymeric; Monnez, Claire; Estrada de Los Santos, Paulina; Segonds, Christine; Caballero-Mellado, Jesus; Lipuma, John J; Chabanon, Gerard; Cournoyer, Benoit

    2007-05-01

    Burkholderia vietnamiensis is the third most prevalent species of the Burkholderia cepacia complex (Bcc) found in cystic fibrosis (CF) patients. Its ability at fixing nitrogen makes it one of the main Bcc species showing strong filiations with environmental reservoirs. In this study, 83% (29 over 35) of the B. vietnamiensis CF isolates and 100% of the environmental ones (over 29) were found expressing the dinitrogenase complex (encoded by the nif cluster) which is essential in N(2) fixation. Among the deficient strains, two were found growing with ammonium chloride suggesting that they were defective in N(2) fixation, and four with amino acids supplements suggesting that they were harbouring auxotrophic mutations. To get insights about the genetic events that led to the emergence of the N(2)-fixing defective strains, a genetic analysis of B. vietnamiensis nitrogen-fixing property was undertaken. A 40-kb-long nif cluster and nif regulatory genes were identified within the B. vietnamiensis strain G4 genome sequence, and analysed. Transposon mutagenesis and nifH genetic marker exchanges showed the nif cluster and several other genes like gltB (encoding a subunit of the glutamate synthase) to play a key role in B. vietnamiensis ability at growing in nitrogen-free media. nif cluster DNA probings of restricted genomic DNA blots showed a full deletion of the nif cluster for one of the N(2)-fixing defective strain while the other one showed a genetic organization similar to the one of the G4 strain. For 17% of B. vietnamiensis clinical strains, CF lungs appeared to have favoured the selection of mutations or deletions leading to N(2)-fixing deficiencies.

  14. Unusual Multiple Production of N-Acylhomoserine Lactones a by Burkholderia sp. Strain C10B Isolated from Dentine Caries

    Directory of Open Access Journals (Sweden)

    Share Yuan Goh

    2014-05-01

    Full Text Available Bacteria realize the ability to communicate by production of quorum sensing (QS molecules called autoinducers, which regulate the physiological activities in their ecological niches. The oral cavity could be a potential area for the presence of QS bacteria. In this study, we report the isolation of a QS bacterial isolate C10B from dentine caries. Preliminary screening using Chromobacterium violaceum CV026 biosensor showed that isolate C10B was able to produce N-acylhomoserine lactones (AHLs. This bacterium was further identified as a member of Burkholderia, an opportunistic pathogen. The isolated Burkholderia sp. was confirmed to produce N-hexanoyl-L-homoserine lactone (C6-HSL, N-octanoyl-L-homoserine lactone (C8-HSL, N-decanoyl-L-homoserine lactone (C10-HSL and N-dodecanoyl-L-homoserine lactone (C12-HSL.

  15. In vitro study of biological activity of four strains of Burkholderia gladioli pv. agaricicola and identification of their bioactive metabolites using

    Directory of Open Access Journals (Sweden)

    Hazem S. Elshafie

    2017-02-01

    Full Text Available This research was carried out to study in vitro antibacterial activity of 4 strains of Burkholderia gladioli pv. agaricicola (Bga against G+ve Bacillus megaterium and G−ve Escherichia coli, haemolytic activity against the cell membrane of erythrocytes, the production of extracellular hydrolytic enzymes and finally, the pathogenicity against Agaricus bisporus flesh blocks. Chemical structure of bioactive substances of the most bioactive strain (ICMP 11096 was established using gas chromatography–mass spectrometry (GC–MS. All the studied Bga strains inhibited the growth of the two tested bacteria although some growing substrates negatively influenced the antimicrobial substance production. The same Bga strains showed highly haemolytic activity and were able to produce 3 hydrolytic enzymes, i.e. chitinase, glucanase and protease. In pathogenicity assays, the considered Bga strains resulted virulent for A. bisporus. The GC–MS for compounds from Bga ICMP 11096 were compatible with the structure of two bioactive fatty acids identified as methyl stearate and ethanol 2-butoxy phosphate with mass spectrum m/e 298 and 398, respectively.

  16. Preliminary data on antibacterial activity of Echinacea purpurea-associated bacterial communities against Burkholderia cepacia complex strains, opportunistic pathogens of Cystic Fibrosis patients.

    Science.gov (United States)

    Chiellini, Carolina; Maida, Isabel; Maggini, Valentina; Bosi, Emanuele; Mocali, Stefano; Emiliani, Giovanni; Perrin, Elena; Firenzuoli, Fabio; Mengoni, Alessio; Fani, Renato

    2017-03-01

    Burkholderia cepacia complex bacteria (Bcc) represent a serious threat for immune-compromised patient affected by Cystic Fibrosis (CF) since they are resistant to many substances and to most antibiotics. For this reason, the research of new natural compounds able to inhibit the growth of Bcc strains has raised new interest during the last years. A source of such natural compounds is represented by medicinal plants and, in particular, by bacterial communities associated with these plants able to produce molecules with antimicrobial activity. In this work, a panel of 151 (endophytic) bacteria isolated from three different compartments (rhizospheric soil, roots, and stem/leaves) of the medicinal plant Echinacea purpurea were tested (using the cross-streak method) for their ability to inhibit the growth of 10 Bcc strains. Data obtained revealed that bacteria isolated from the roots of E. purpurea are the most active in the inhibition of Bcc strains, followed by bacteria isolated from the rhizospheric soil, and endophytes from stem/leaf compartment. At the same time, Bcc strains of environmental origin showed a higher resistance toward inhibition than the Bcc strains with clinical (i.e. CF patients) origin. Differences in the inhibition activity of E. purpurea-associated bacteria are mainly linked to the environment -the plant compartment- rather than to their taxonomical position. Copyright © 2016 Elsevier GmbH. All rights reserved.

  17. CHROMOSOMAL MULTIPLICITY IN BURKHOLDERIA CEPACIA

    Science.gov (United States)

    We have used CHEF gel electrophoresis to screen preparations of large DNA from different Burkholderia cepacia isolates for the presence of DNA species corresponding to the linearized forms of the three chromosomes of 3.4,2.5, and 0.9 Mb identified in B. cepacia strain 17616. DNA ...

  18. Metabolism of 2-hydroxy-1-naphthoic acid and naphthalene via gentisic acid by distinctly different sets of enzymes in Burkholderia sp. strain BC1.

    Science.gov (United States)

    Chowdhury, Piyali Pal; Sarkar, Jayita; Basu, Soumik; Dutta, Tapan K

    2014-05-01

    Burkholderia sp. strain BC1, a soil bacterium, isolated from a naphthalene balls manufacturing waste disposal site, is capable of utilizing 2-hydroxy-1-naphthoic acid (2H1NA) and naphthalene individually as the sole source of carbon and energy. To deduce the pathway for degradation of 2H1NA, metabolites isolated from resting cell culture were identified by a combination of chromatographic and spectrometric analyses. Characterization of metabolic intermediates, oxygen uptake studies and enzyme activities revealed that strain BC1 degrades 2H1NA via 2-naphthol, 1,2,6-trihydroxy-1,2-dihydronaphthalene and gentisic acid. In addition, naphthalene was found to be degraded via 1,2-dihydroxy-1,2-dihydronaphthalene, salicylic acid and gentisic acid, with the putative involvement of the classical nag pathway. Unlike most other Gram-negative bacteria, metabolism of salicylic acid in strain BC1 involves a dual pathway, via gentisic acid and catechol, with the latter being metabolized by catechol 1,2-dioxygenase. Involvement of a non-oxidative decarboxylase in the enzymic transformation of 2H1NA to 2-naphthol indicates an alternative catabolic pathway for the bacterial degradation of hydroxynaphthoic acid. Furthermore, the biochemical observations on the metabolism of structurally similar compounds, naphthalene and 2-naphthol, by similar but different sets of enzymes in strain BC1 were validated by real-time PCR analyses.

  19. Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4

    Directory of Open Access Journals (Sweden)

    Kah Yan How

    2015-08-01

    Full Text Available Quorum sensing is a mechanism for regulating proteobacterial gene expression in response to changes in cell population. In proteobacteria, N-acyl homoserine lactone (AHL appears to be the most widely used signalling molecules in mediating, among others, the production of extracellular virulence factors for survival. In this work, the genome of B. cepacia strain GG4, a plasmid-free strain capable of AHL synthesis was explored. In silico analysis of the 6.6 Mb complete genome revealed the presence of a LuxI homologue which correspond to Type I quorum sensing. Here, we report the molecular cloning and characterization of this LuxI homologue, designated as BurI. This 609 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3. The purified protein was approximately 25 kDa and is highly similar to several autoinducer proteins of the LuxI family among Burkholderia species. To verify the AHL synthesis activity of this protein, high resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-hexanoylhomoserine lactone, N-octanoylhomoserine lactone and 3-hydroxy-octanoylhomoserine lactone from induced E. coli BL21 harboring the recombinant BurI. Our data show, for the first time, the cloning and characterization of the LuxI homologue from B. cepacia strain GG4 and confirmation of its AHL synthesis activity.

  20. Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4.

    Science.gov (United States)

    How, Kah Yan; Hong, Kar Wai; Chan, Kok-Gan

    2015-01-01

    Quorum sensing is a mechanism for regulating proteobacterial gene expression in response to changes in cell population. In proteobacteria, N-acyl homoserine lactone (AHL) appears to be the most widely used signalling molecules in mediating, among others, the production of extracellular virulence factors for survival. In this work, the genome of B. cepacia strain GG4, a plasmid-free strain capable of AHL synthesis was explored. In silico analysis of the 6.6 Mb complete genome revealed the presence of a LuxI homologue which correspond to Type I quorum sensing. Here, we report the molecular cloning and characterization of this LuxI homologue, designated as BurI. This 609 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was approximately 25 kDa and is highly similar to several autoinducer proteins of the LuxI family among Burkholderia species. To verify the AHL synthesis activity of this protein, high resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-hexanoylhomoserine lactone, N-octanoylhomoserine lactone and 3-hydroxy-octanoylhomoserine lactone from induced E. coli BL21 harboring the recombinant BurI. Our data show, for the first time, the cloning and characterization of the LuxI homologue from B. cepacia strain GG4 and confirmation of its AHL synthesis activity.

  1. Interplay between 4-Hydroxy-3-Methyl-2-Alkylquinoline and N-Acyl-Homoserine Lactone Signaling in a Burkholderia cepacia Complex Clinical Strain

    Directory of Open Access Journals (Sweden)

    Annelise Chapalain

    2017-06-01

    Full Text Available Species from the Burkholderia cepacia complex (Bcc share a canonical LuxI/LuxR quorum sensing (QS regulation system named CepI/CepR, which mainly relies on the acyl-homoserine lactone (AHL, octanoyl-homoserine lactone (C8-HSL as signaling molecule. Burkholderia ambifaria is one of the least virulent Bcc species, more often isolated from rhizospheres where it exerts a plant growth-promoting activity. However, clinical strains of B. ambifaria display distinct features, such as phase variation and higher virulence properties. Notably, we previously reported that under laboratory conditions, only clinical strains of the B. ambifaria species produced 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs via expression of the hmqABCDEFG operon. HMAQs are the methylated counterparts of the 4-hydroxy-2-alkylquinolines (HAQs produced by the opportunistic human pathogen Pseudomonas aeruginosa, in which they globally contribute to the bacterial virulence and survival. We have found that unlike P. aeruginosa’s HAQs, HMAQs do not induce their own production. However, they indirectly regulate the expression of the hmqABCDEFG operon. In B. ambifaria, a strong link between CepI/CepR-based QS and HMAQs is proposed, as we have previously reported an increased production of C8-HSL in HMAQ-negative mutants. Here, we report the identification of all AHLs produced by the clinical B. ambifaria strain HSJ1, namely C6-HSL, C8-HSL, C10-HSL, 3OHC8-HSL, 3OHC10-HSL, and 3OHC12-HSL. Production of significant levels of hydroxylated AHLs prompted the identification of a second complete LuxI/LuxR-type QS system relying on 3OHC10-HSL and 3OHC12-HSL, that we have named CepI2/CepR2. The connection between these two QS systems and the hmqABCDEFG operon, responsible for HMAQs biosynthesis, was investigated. The CepI/CepR system strongly induced the operon, while the second system appears moderately involved. On the other hand, a HMAQ-negative mutant overproduces AHLs from both QS systems

  2. A comparison of the response of two Burkholderia fungorum strains grown as planktonic cells versus biofilm to dibenzothiophene and select polycyclic aromatic hydrocarbons.

    Science.gov (United States)

    Khoei, Nazanin Seyed; Andreolli, Marco; Lampis, Silvia; Vallini, Giovanni; Turner, Raymond J

    2016-10-01

    In natural environments, bacteria often exist in close association with surfaces and interfaces by establishing biofilms. Here, we report on the ability of Burkholderia fungorum strains DBT1 and 95 to survive in high concentrations of hydrocarbons, and we compare their growth as a biofilm vs. planktonic cells. The 2 compounds tested were dibenzothiophene (DBT) and a mixture of naphthalene, phenanthrene, and pyrene (5:2:1) as representative compounds of thiophenes and polycyclic aromatic hydrocarbons (PAHs), respectively. The results showed that both strains were able to degrade DBT and to survive in the presence of up to a 2000 mg·L(-1) concentration of this compound both as a biofilm and as free-living cells. Moreover, B. fungorum DBT1 showed reduced tolerance towards the mixed PAHs (2000 mg·L(-1) naphthalene, 800 mg·L(-1) phenanthrene, and 400 mg·L(-1) pyrene) both as a biofilm and as free-living cells. Conversely, biofilms of B. fungorum 95 enhanced resistance against these toxic compounds compared with planktonic cells (P hydrocarbon-contaminated sites.

  3. The Role of Hydrophobicity and Surface Receptors at Hyphae of Lyophyllum sp. Strain Karsten in the Interaction with Burkholderia terrae BS001 – Implications for Interactions in Soil

    Science.gov (United States)

    Vila, Taissa; Nazir, Rashid; Rozental, Sonia; dos Santos, Giulia M. P.; Calixto, Renata O. R.; Barreto-Bergter, Eliana; Wick, Lukas Y.; van Elsas, Jan Dirk

    2016-01-01

    The soil bacterium Burkholderia terrae strain BS001 can interact with varying soil fungi, using mechanisms that range from the utilization of carbon/energy sources such as glycerol to the ability to reach novel territories in soil via co-migration with growing fungal mycelia. Here, we investigate the intrinsic properties of the B. terrae BS001 interaction with the basidiomycetous soil fungus Lyophyllum sp. strain Karsten. In some experiments, the ascomycetous Trichoderma asperellum 302 was also used. The hyphae of Lyophyllum sp. strain Karsten were largely hydrophilic on water-containing media versus hydrophobic when aerial, as evidenced by contact angle analyses (CA). Co-migration of B. terrae strain BS001 cells with the hyphae of the two fungi occurred preferentially along the - presumably hydrophilic - soil-dwelling hyphae, whereas aerial hyphae did not allow efficient migration, due to reduced thickness of their surrounding mucous films. Moreover, the cell numbers over the length of the hyphae in soil showed an uneven distribution, i.e., the CFU numbers increased from minima at the inoculation point to maximal numbers in the middle of the extended hyphae, then decreasing toward the terminal side. Microscopic analyses of the strain BS001 associations with the Lyophyllum sp. strain Karsten hyphae in the microcosms confirmed the presence of B. terrae BS001 cells on the mucous matter that was present at the hyphal surfaces of the fungi used. Cell agglomerates were found to accumulate at defined sites on the hyphal surfaces, which were coined ‘fungal-interactive’ hot spots. Evidence was further obtained for the contention that receptors for a physical bacterium-fungus interaction occur at the Lyophyllum sp. strain Karsten hyphal surface, in which the specific glycosphingolipid ceramide monohexoside (CMH) plays an important role. Thus, bacterial adherence may be mediated by heterogeneously distributed fungal-specific receptors, implying the CMH moieties. This

  4. The role of hydrophobicity and surface receptors at hyphae of Lyophyllum sp strain Karsten in the interaction with Burkholderia terrae BS001 – implications for interactions in soil.

    Directory of Open Access Journals (Sweden)

    Taissa Vieira Machado Vila

    2016-10-01

    Full Text Available The soil bacterium Burkholderia terrae strain BS001 can interact with varying soil fungi, using mechanisms that range from the utilization of carbon/energy sources such as glycerol to the ability to reach novel territories in soil via co-migration with growing fungal mycelia. Here, we investigate the intrinsic properties of the B. terrae BS001 interaction with the basidiomycetous soil fungus Lyophyllum sp. strain Karsten. In some experiments, the ascomycetous Trichoderma asperellum 302 was also used. The hyphae of Lyophyllum sp. strain Karsten were largely hydrophilic on water-containing media versus hydrophobic when aerial, as evidenced by contact angle analyses (CA. Co-migration of B. terrae strain BS001 cells with the hyphae of the two fungi occurred preferentially along the - presumably hydrophilic - soil-dwelling hyphae, whereas aerial hyphae did not allow efficient migration, due to reduced thickness of their surrounding mucous films. Moreover, the cell numbers over the length of the hyphae in soil showed an uneven distribution, i.e. the CFU numbers increased from minima at the inoculation point to maximal numbers in the middle of the extended hyphae, then decreasing towards the terminal side. Microscopic analyses of the strain BS001 associations with the Lyophyllum sp. strain Karsten hyphae in the microcosms confirmed the presence of B. terrae BS001 cells on the mucous matter that was present at the hyphal surfaces of the fungi used. Cell agglomerates were found to accumulate at defined sites on the hyphal surfaces, which were coined ‘fungal-interactive’ hot spots. Evidence was further obtained for the contention that receptors for a physical bacterium-fungus interaction occur at the Lyophyllum sp. strain Karsten hyphal surface, in which the specific glycosphingolipid ceramide mono hexoside (CMH plays an important role. Thus, bacterial adherence may be mediated by heterogeneously-distributed fungal-specific receptors, implying the CMH

  5. The three-species consortium of genetically improved strains Cupriavidus necator RW112, Burkholderia xenovorans RW118, and Pseudomonas pseudoalcaligenes RW120, grows with technical polychlorobiphenyl, Aroclor 1242

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    Regina-Michaela eWittich

    2013-04-01

    Full Text Available Burkholderia xenovorans LB400, Cupriavidus necator H850, and Pseudomonas pseudoalcaligenes KF707 are bacterial strains able to mineralize biphenyl and to co-oxidize many of its halogenated derivatives (PCBs. Only strain LB400 also mineralizes a few mono- and dichlorobiphenyls, due to the presence of a functioning chlorocatechol pathway. Here, we used a Tn5-based minitransposon shuttle system to chromosomically introduce genes tcbRCDEF, encoding the chlorocatechol pathway into KF707, and genes cbdABC encoding a 2-chlorobenzoate 1,2-dioxygenase into KF707 and LB400, as well as transposon Tn4653 from the TOL plasmid providing genes xylXYZL, encoding a broad-range toluate (methylbenzoate dioxygenase and its dihydrodiol dehydrogenase, to extend the range for the mineralization of halogenated benzoates in LB400 and in KF707. The engineered derivatives of LB400, and KF707 thus gained the ability for the mineralization of all isomeric monochloro- and bromobenzoates of the so-called lower pathway which, consequently, also allowed the mineralization of all monochlorobiphenyls and a number of di- and trichlorobiphenyls, thus preventing the accumulation of halobenzoates and of catabolites thereof. LB400 and KF707 also grow with the two commercial PCB formulations, Aroclor 1221 and Aroclor 1232, as the sole carbon and energy sources, but not with higher halogenated PCB mixtures, similar to the already published strain RW112. Repeated exposition of the modified LB400 to short pulses of UV light, over a prolonged period of time, allowed the isolation of a derivative of LB400, termed RW118, capable of growth with Aroclor 1016 still containing only traces of biphenyl, and in co-culture with modified KF707 termed RW120, and modified H850 (RW112 with Aroclor 1242, the commercial mixture already void of biphenyl and monochlorobiphenyls.

  6. The Three-Species Consortium of Genetically Improved Strains Cupriavidus necator RW112, Burkholderia xenovorans RW118, and Pseudomonas pseudoalcaligenes RW120 Grows with Technical Polychlorobiphenyl, Aroclor 1242

    Science.gov (United States)

    Hernández-Sánchez, Verónica; Lang, Elke; Wittich, Regina-Michaela

    2013-01-01

    Burkholderia xenovorans LB400, Cupriavidus necator H850, and Pseudomonas pseudoalcaligenes KF707 are bacterial strains able to mineralize biphenyl and to co-oxidize many of its halogenated derivatives (PCBs). Only strain LB400 also mineralizes a few mono- and dichlorobiphenyls, due to the presence of a functioning chlorocatechol pathway. Here, we used a Tn5-based minitransposon shuttle system to chromosomically introduce genes tcbRCDEF, encoding the chlorocatechol pathway into KF707, and genes cbdABC encoding a 2-chlorobenzoate 1,2-dioxygenase into KF707 and LB400, as well as transposon Tn4653 from the TOL plasmid, providing genes xylXYZL, encoding a broad-range toluate (methylbenzoate) dioxygenase and its dihydrodiol dehydrogenase, to extend the range for the mineralization of halogenated benzoates in LB400 and in KF707 through co-oxidation of halobenzoates into chlorocatechols. The engineered derivatives of LB400 and KF707 thus gained the ability for the mineralization of all isomeric monochloro- and bromobenzoates of the so-called lower pathway which, consequently, also allowed the mineralization of all monochlorobiphenyls and a number of di- and trichlorobiphenyls, thus preventing the accumulation of halobenzoates and of catabolites thereof. LB400 and KF707 also grow with the two commercial PCB formulations, Aroclor 1221 and Aroclor 1232, as the sole carbon and energy sources, but not with higher halogenated PCB mixtures, similar to the already published strain RW112. Repeated exposition of the modified LB400 to short pulses of UV light, over a prolonged period of time, allowed the isolation of a derivative of LB400, termed RW118, capable of growth with Aroclor 1016 still containing only traces of biphenyl, and in co-culture with modified KF707 termed RW120, and modified H850 (RW112) with Aroclor 1242, the commercial mixture already void of biphenyl and monochlorobiphenyls. PMID:23658554

  7. Genes involved in degradation of para-nitrophenol are differentially arranged in form of non-contiguous gene clusters in Burkholderia sp. strain SJ98.

    Directory of Open Access Journals (Sweden)

    Surendra Vikram

    Full Text Available Biodegradation of para-Nitrophenol (PNP proceeds via two distinct pathways, having 1,2,3-benzenetriol (BT and hydroquinone (HQ as their respective terminal aromatic intermediates. Genes involved in these pathways have already been studied in different PNP degrading bacteria. Burkholderia sp. strain SJ98 degrades PNP via both the pathways. Earlier, we have sequenced and analyzed a ~41 kb fragment from the genomic library of strain SJ98. This DNA fragment was found to harbor all the lower pathway genes; however, genes responsible for the initial transformation of PNP could not be identified within this fragment. Now, we have sequenced and annotated the whole genome of strain SJ98 and found two ORFs (viz., pnpA and pnpB showing maximum identity at amino acid level with p-nitrophenol 4-monooxygenase (PnpM and p-benzoquinone reductase (BqR. Unlike the other PNP gene clusters reported earlier in different bacteria, these two ORFs in SJ98 genome are physically separated from the other genes of PNP degradation pathway. In order to ascertain the identity of ORFs pnpA and pnpB, we have performed in-vitro assays using recombinant proteins heterologously expressed and purified to homogeneity. Purified PnpA was found to be a functional PnpM and transformed PNP into benzoquinone (BQ, while PnpB was found to be a functional BqR which catalyzed the transformation of BQ into hydroquinone (HQ. Noticeably, PnpM from strain SJ98 could also transform a number of PNP analogues. Based on the above observations, we propose that the genes for PNP degradation in strain SJ98 are arranged differentially in form of non-contiguous gene clusters. This is the first report for such arrangement for gene clusters involved in PNP degradation. Therefore, we propose that PNP degradation in strain SJ98 could be an important model system for further studies on differential evolution of PNP degradation functions.

  8. Draft Genomes for Eight Burkholderia mallei Isolates from Turkey

    Science.gov (United States)

    Daligault, H. E.; Davenport, K. W.; Minogue, T. D.; Bishop-Lilly, K. A.; Broomall, S. M.; Bruce, D. C.; Coyne, S. R.; Frey, K. G.; Gibbons, H. S.; Jaissle, J.; Koroleva, G. I.; Ladner, J. T.; Lo, C.-C.; Munk, C.; Wolcott, M. J.; Palacios, G. F.; Redden, C. L.; Rosenzweig, C. N.; Scholz, M. B.; Chain, P. S.

    2016-01-01

    Burkholderia mallei, the etiologic agent of glanders, is a Gram-negative, nonmotile, facultative intracellular pathogen. Although glanders has been eradicated from many parts of the world, the threat of B. mallei being used as a weapon is very real. Here we present draft genome assemblies of 8 Burkholderia mallei strains that were isolated in Turkey. PMID:26744368

  9. Natural Burkholderia mallei infection in Dromedary, Bahrain.

    Science.gov (United States)

    Wernery, Ulrich; Wernery, Renate; Joseph, Marina; Al-Salloom, Fajer; Johnson, Bobby; Kinne, Joerg; Jose, Shanti; Jose, Sherry; Tappendorf, Britta; Hornstra, Heidie; Scholz, Holger C

    2011-07-01

    We confirm a natural infection of dromedaries with glanders. Multilocus variable number tandem repeat analysis of a Burkholderia mallei strain isolated from a diseased dromedary in Bahrain revealed close genetic proximity to strain Dubai 7, which caused an outbreak of glanders in horses in the United Arab Emirates in 2004.

  10. Phenotypic characterization of a novel virulence-factor deletion strain of Burkholderia mallei that provides partial protection against inhalational glanders in mice

    Directory of Open Access Journals (Sweden)

    Joel A. Bozue

    2016-02-01

    Full Text Available Burkholderia mallei (Bm is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN, and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders.

  11. Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei That Provides Partial Protection against Inhalational Glanders in Mice

    Science.gov (United States)

    Bozue, Joel A.; Chaudhury, Sidhartha; Amemiya, Kei; Chua, Jennifer; Cote, Christopher K.; Toothman, Ronald G.; Dankmeyer, Jennifer L.; Klimko, Christopher P.; Wilhelmsen, Catherine L.; Raymond, Jolynn W.; Zavaljevski, Nela; Reifman, Jaques; Wallqvist, Anders

    2016-01-01

    Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders. PMID:26955620

  12. Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei That Provides Partial Protection against Inhalational Glanders in Mice.

    Science.gov (United States)

    Bozue, Joel A; Chaudhury, Sidhartha; Amemiya, Kei; Chua, Jennifer; Cote, Christopher K; Toothman, Ronald G; Dankmeyer, Jennifer L; Klimko, Christopher P; Wilhelmsen, Catherine L; Raymond, Jolynn W; Zavaljevski, Nela; Reifman, Jaques; Wallqvist, Anders

    2016-01-01

    Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders.

  13. Genome-centric evaluation of Burkholderia sp. strain SRS-W-2-2016 resistant to high concentrations of uranium and nickel isolated from the Savannah River Site (SRS, USA

    Directory of Open Access Journals (Sweden)

    Ashish Pathak

    2017-06-01

    Full Text Available Savannah River Site (SRS, an approximately 800-km2 former nuclear weapons production facility located near Aiken, SC remains co-contaminated by heavy metals and radionuclides. To gain a better understanding on microbially-mediated bioremediation mechanisms, several bacterial strains resistant to high concentrations of Uranium (U and Nickel (Ni were isolated from the Steeds Pond soils located within the SRS site. One of the isolated strains, designated as strain SRS-W-2-2016, grew robustly on both U and Ni. To fully understand the arsenal of metabolic functions possessed by this strain, a draft whole genome sequence (WGS was obtained, assembled, annotated and analyzed. Genome-centric evaluation revealed the isolate to belong to the Burkholderia genus with close affiliation to B. xenovorans LB400, an aggressive polychlorinated biphenyl-degrader. At a coverage of 90×, the genome of strain SRS-W-2-2016 consisted of 8,035,584 bases with a total number of 7071 putative genes assembling into 191 contigs with an N50 contig length of 134,675 bases. Several gene homologues coding for resistance to heavy metals/radionuclides were identified in strain SRS-W-2-2016, such as a suite of outer membrane efflux pump proteins similar to nickel/cobalt transporter regulators, peptide/nickel transport substrate and ATP-binding proteins, permease proteins, and a high-affinity nickel-transport protein. Also noteworthy were two separate gene fragments in strain SRS-W-2-2016 homologous to the spoT gene; recently correlated with bacterial tolerance to U. Additionally, a plethora of oxygenase genes were also identified in the isolate, potentially involved in the breakdown of organic compounds facilitating the strain's successful colonization and survival in the SRS co-contaminated soils. The WGS project of Burkholderia sp. strain SRS-W-2-2016 is available at DDBJ/ENA/GenBank under the accession #MSDV00000000.

  14. Test of aerobic TCE degradation by willows (Salix viminalis) and willows inoculated with TCE-cometabolizing strains of Burkholderia cepacia

    DEFF Research Database (Denmark)

    Clausen, Lauge Peter Westergaard; Broholm, Mette Martina; Gosewinkel, Ulrich Bay

    2017-01-01

    Trichloroethylene (TCE) is a widespread soil and groundwater pollutant and clean-up is often problematic and expensive. Phytoremediation may be a cost-effective solution at some sites. This study investigates TCE degradation by willows (S. viminalis) and willows inoculated with three strains of B...

  15. Use of a Burkholderia cenocepacia ABTS Oxidizer in a Microbial Fuel Cell

    Science.gov (United States)

    Microbial fuel cells (MFCs) often use biological processes to generate electrons from organic material contained in the anode chamber and abiotic processes employing atmospheric oxygen as the oxidant in the cathode chamber. This study investigated the accumulation of an oxidant in bacterial cultures...

  16. Draft genome sequence of Burkholderia sordidicola S170, a potential plant growth promoter isolated from coniferous forest soil in the Czech Republic

    DEFF Research Database (Denmark)

    Lladó, Salvador; Xu, Zhuofei; Sørensen, Søren Johannes

    2014-01-01

    Burkholderia species are key players in the accumulation of carbon from cellulose decomposition in coniferous forest ecosystems. We report here the draft genome of Burkholderia sordidicola strain S170, containing features associated with known genes involved in plant growth promotion, the biologi......Burkholderia species are key players in the accumulation of carbon from cellulose decomposition in coniferous forest ecosystems. We report here the draft genome of Burkholderia sordidicola strain S170, containing features associated with known genes involved in plant growth promotion...

  17. Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei

    Directory of Open Access Journals (Sweden)

    Janse Ingmar

    2013-02-01

    Full Text Available Abstract Background Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance. Methods Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated. Results A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction. Conclusions The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.

  18. Selection and evaluation of reference genes for RT-qPCR expression studies on Burkholderia tropica strain Ppe8, a sugarcane-associated diazotrophic bacterium grown with different carbon sources or sugarcane juice.

    Science.gov (United States)

    da Silva, Paula Renata Alves; Vidal, Marcia Soares; de Paula Soares, Cleiton; Polese, Valéria; Simões-Araújo, Jean Luís; Baldani, José Ivo

    2016-11-01

    Among the members of the genus Burkholderia, Burkholderia tropica has the ability to fix nitrogen and promote sugarcane plant growth as well as act as a biological control agent. There is little information about how this bacterium metabolizes carbohydrates as well as those carbon sources found in the sugarcane juice that accumulates in stems during plant growth. Reverse transcription quantitative PCR (RT-qPCR) can be used to evaluate changes in gene expression during bacterial growth on different carbon sources. Here we tested the expression of six reference genes, lpxC, gyrB, recA, rpoA, rpoB, and rpoD, when cells were grown with glucose, fructose, sucrose, mannitol, aconitic acid, and sugarcane juice as carbon sources. The lpxC, gyrB, and recA were selected as the most stable reference genes based on geNorm and NormFinder software analyses. Validation of these three reference genes during strain Ppe8 growth on the same carbon sources showed that genes involved in glycogen biosynthesis (glgA, glgB, glgC) and trehalose biosynthesis (treY and treZ) were highly expressed when Ppe8 was grown in aconitic acid relative to other carbon sources, while otsA expression (trehalose biosynthesis) was reduced with all carbon sources. In addition, the expression level of the ORF_6066 (gluconolactonase) gene was reduced on sugarcane juice. The results confirmed the stability of the three selected reference genes (lpxC, gyrB, and recA) during the RT-qPCR and also their robustness by evaluating the relative expression of genes involved in glycogen and trehalose biosynthesis when strain Ppe8 was grown on different carbon sources and sugarcane juice.

  19. Characterization of plant-growth-promoting effects and concurrent promotion of heavy metal accumulation in the tissues of the plants grown in the polluted soil by Burkholderia strain LD-11.

    Science.gov (United States)

    Huang, Gui-Hai; Tian, Hui-Hui; Liu, Hai-Ying; Fan, Xian-Wei; Liang, Yu; Li, You-Zhi

    2013-01-01

    Plant-growth-promoting (PGP) bacteria especially with the resistance to multiple heavy metals are helpful to phytoremediation. Further development of PGP bacteria is very necessary because of the extreme diversity of plants, soils, and heavy metal pollution. A Burkholderia sp. strain, numbered LD-11, was isolated, which showed resistances to multiple heavy metals and antibiotics. It can produce indole-3-acetic acid, 1-aminocyclopropane-1-carboxylic acid deaminase and siderophores. Inoculation with the LD-11 improved germination of seeds of the investigated vegetable plants in the presence of Cu, promoted elongation of roots and hypocotyledonary axes, enhanced the dry weights of the plants grown in the soils polluted with Cu and/or Pb, and increased activity of the soil urease and the rhizobacteria diversity. Inoculation with the LD-11 significantly enhanced Cu and/or Pb accumulation especially in the roots of the plants grown in the polluted soils. Notably, LD-11 could produce siderophores in the presence of Cu. Conclusively, the PGP effects and concurrent heavy metal accumulation in the plant tissues results from combined effects of the above-mentioned multiple factors. Cu is an important element that represses production of the siderophore by the bacteria. Phytoremediation by synergistic use of the investigated plants and the bacterial strain LD-11 is a phytoextraction process.

  20. BIOAUGMENTATION WITH BURKHOLDERIA CEPACIA PR1301 FOR IN SITU BIOREMEDIATION OF TRICHLOROETHYLENE CONTAMINATED GROUNDWATER (RESEARCH BRIEF)

    Science.gov (United States)

    A pilot field study was conducted at the Moffett Federal Airfield, Mountain View, California, to determine whether effective in-situ aerobic cometabolic biodegradation of TCE could be accomplished through bioaugmentation with a genetically modified strain of Burkholderia cepacia ...

  1. Molecular characterization of a novel family VIII esterase from burkholderia multivorans UWC10

    CSIR Research Space (South Africa)

    Rashamuse, KJ

    2007-02-01

    Full Text Available An esterase producing Burkholderia multivorans UWC10 strain was isolated by culture enrichment. A shotgun library of B. multivorans UWC10 genomic DNA was screened for esterase activity and a recombinant clone conferring an esterolytic phenotype...

  2. Common duckweed (Lemna minor is a versatile high-throughput infection model for the Burkholderia cepacia complex and other pathogenic bacteria.

    Directory of Open Access Journals (Sweden)

    Euan L S Thomson

    Full Text Available Members of the Burkholderia cepacia complex (Bcc have emerged in recent decades as problematic pulmonary pathogens of cystic fibrosis (CF patients, with severe infections progressing to acute necrotizing pneumonia and sepsis. This study presents evidence that Lemna minor (Common duckweed is useful as a plant model for the Bcc infectious process, and has potential as a model system for bacterial pathogenesis in general. To investigate the relationship between Bcc virulence in duckweed and Galleria mellonella (Greater wax moth larvae, a previously established Bcc infection model, a duckweed survival assay was developed and used to determine LD50 values. A strong correlation (R(2 = 0.81 was found between the strains' virulence ranks in the two infection models, suggesting conserved pathways in these vastly different hosts. To broaden the application of the duckweed model, enteropathogenic Escherichia coli (EPEC and five isogenic mutants with previously established LD50 values in the larval model were tested against duckweed, and a strong correlation (R(2 = 0.93 was found between their raw LD50 values. Potential virulence factors in B. cenocepacia K56-2 were identified using a high-throughput screen against single duckweed plants. In addition to the previously characterized antifungal compound (AFC cluster genes, several uncharacterized genes were discovered including a novel lysR regulator, a histidine biosynthesis gene hisG, and a gene located near the gene encoding the recently characterized virulence factor SuhB(Bc. Finally, to demonstrate the utility of this model in therapeutic applications, duckweed was rescued from Bcc infection by treating with bacteriophage at 6-h intervals. It was observed that phage application became ineffective at a timepoint that coincided with a sharp increase in bacterial invasion of plant tissue. These results indicate that common duckweed can serve as an effective infection model for the investigation of bacterial

  3. Structural relationship of the lipid A acyl groups to activation of murine Toll-like receptor 4 by lipopolysaccharides from pathogenic strains of Burkholderia mallei, Acinetobacter baumannii and Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Kirill V Korneev

    2015-11-01

    Full Text Available Toll-like receptor 4 (TLR4 is required for activation of innate immunity upon recognition of lipopolysaccharide (LPS of Gram-negative bacteria. The ability of TLR4 to respond to a particular LPS species is important since insufficient activation may not prevent bacterial growth while excessive immune reaction may lead to immunopathology associated with sepsis. Here we investigated the biological activity of LPS from Burkholderia mallei that causes glanders, and from the two well-known opportunistic pathogens Acinetobacter baumannii and Pseudomonas aeruginosa (causative agents of nosocomial infections. For each bacterial strain, R-form LPS preparations were purified by hydrophobic chromatography and the chemical structure of lipid A, an LPS structural component, was elucidated by HR-MALDI-TOF mass spectrometry. The biological activity of LPS samples was evaluated by their ability to induce production of proinflammatory cytokines, such as IL-6 and TNF, by bone marrow-derived macrophages (BMDM. Our results demonstrate direct correlation between the biological activity of LPS from these pathogenic bacteria and the extent of their lipid A acylation.

  4. Structural Relationship of the Lipid A Acyl Groups to Activation of Murine Toll-Like Receptor 4 by Lipopolysaccharides from Pathogenic Strains of Burkholderia mallei, Acinetobacter baumannii, and Pseudomonas aeruginosa

    Science.gov (United States)

    Korneev, Kirill V.; Arbatsky, Nikolay P.; Molinaro, Antonio; Palmigiano, Angelo; Shaikhutdinova, Rima Z.; Shneider, Mikhail M.; Pier, Gerald B.; Kondakova, Anna N.; Sviriaeva, Ekaterina N.; Sturiale, Luisa; Garozzo, Domenico; Kruglov, Andrey A.; Nedospasov, Sergei A.; Drutskaya, Marina S.; Knirel, Yuriy A.; Kuprash, Dmitry V.

    2015-01-01

    Toll-like receptor 4 (TLR4) is required for activation of innate immunity upon recognition of lipopolysaccharide (LPS) of Gram-negative bacteria. The ability of TLR4 to respond to a particular LPS species is important since insufficient activation may not prevent bacterial growth while excessive immune reaction may lead to immunopathology associated with sepsis. Here, we investigated the biological activity of LPS from Burkholderia mallei that causes glanders, and from the two well-known opportunistic pathogens Acinetobacter baumannii and Pseudomonas aeruginosa (causative agents of nosocomial infections). For each bacterial strain, R-form LPS preparations were purified by hydrophobic chromatography and the chemical structure of lipid A, an LPS structural component, was elucidated by HR-MALDI-TOF mass spectrometry. The biological activity of LPS samples was evaluated by their ability to induce production of proinflammatory cytokines, such as IL-6 and TNF, by bone marrow-derived macrophages. Our results demonstrate direct correlation between the biological activity of LPS from these pathogenic bacteria and the extent of their lipid A acylation. PMID:26635809

  5. 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▿

    Science.gov (United States)

    Bertot, Gustavo M.; Restelli, Marcela A.; Galanternik, Laura; Aranibar Urey, Rene C.; Valvano, Miguel A.; Grinstein, Saúl

    2007-01-01

    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. PMID:17296759

  6. Burkholderia caballeronis sp. nov., a nitrogen fixing species isolated from tomato (Lycopersicon esculentum) with the ability to effectively nodulate Phaseolus vulgaris.

    Science.gov (United States)

    Martínez-Aguilar, Lourdes; Salazar-Salazar, Corelly; Méndez, Rafael Díaz; Caballero-Mellado, Jesús; Hirsch, Ann M; Vásquez-Murrieta, María Soledad; Estrada-de los Santos, Paulina

    2013-12-01

    During a survey of Burkholderia species with potential use in agrobiotechnology, a group of 12 strains was isolated from the rhizosphere and rhizoplane of tomato plants growing in Mexico (Nepantla, Mexico State). A phylogenetic analysis of 16S rRNA gene sequences showed that the strains are related to Burkholderia kururiensis and Burkholderia mimosarum (97.4 and 97.1 %, respectively). However, they induced effective nitrogen-fixing nodules on roots of Phaseolus vulgaris. Based on polyphasic taxonomy, the group of strains represents a novel species for which the name Burkholderia caballeronis sp. nov. is proposed. The type species is TNe-841(T) (= LMG 26416(T) = CIP 110324(T)).

  7. Unusual distribution of Burkholderia cepacia complex species in Danish cystic fibrosis clinics may stem from restricted transmission between patients

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, Niels; Johansen, Helle Krogh; Fenger, Mette G;

    2010-01-01

    Forty-four of 48 Burkholderia cepacia complex strains cultured from Danish cystic fibrosis patients were Burkholderia multivorans, a distribution of species that has not been reported before. Although cases of cross infections were demonstrated, no major epidemic clone was found. The species...... distribution may represent the sporadic acquisition of bacteria from the environment....

  8. Draft Genome Sequence of Flavobacterium sp. Strain TAB 87, Able To Inhibit the Growth of Cystic Fibrosis Bacterial Pathogens Belonging to the Burkholderia cepacia Complex

    Science.gov (United States)

    Presta, Luana; Inzucchi, Ilaria; Bosi, Emanuele; Fondi, Marco; Perrin, Elena; Miceli, Elisangela; Tutino, Maria Luisa; Lo Giudice, Angelina

    2016-01-01

    We report here the draft genome sequence of the Flavobacterium sp. TAB 87 strain, isolated from Antarctic seawater during a summer campaign near the French Antarctic station Dumont d’Urville (60°40′S, 40°01′E). It will allow for comparative genomics and the fulfillment of both fundamental and application-oriented investigations. It allowed the recognition of genes associated with the production of bioactive compounds and antibiotic resistance. PMID:27198032

  9. Exploring the HME and HAE1 efflux systems in the genus Burkholderia

    Directory of Open Access Journals (Sweden)

    Pasca Maria

    2010-06-01

    Full Text Available Abstract Background The genus Burkholderia includes a variety of species with opportunistic human pathogenic strains, whose increasing global resistance to antibiotics has become a public health problem. In this context a major role could be played by multidrug efflux pumps belonging to Resistance Nodulation Cell-Division (RND family, which allow bacterial cells to extrude a wide range of different substrates, including antibiotics. This study aims to i identify rnd genes in the 21 available completely sequenced Burkholderia genomes, ii analyze their phylogenetic distribution, iii define the putative function(s that RND proteins perform within the Burkholderia genus and iv try tracing the evolutionary history of some of these genes in Burkholderia. Results BLAST analysis of the 21 Burkholderia sequenced genomes, using experimentally characterized ceoB sequence (one of the RND family counterpart in the genus Burkholderia as probe, allowed the assembly of a dataset comprising 254 putative RND proteins. An extensive phylogenetic analysis revealed the occurrence of several independent events of gene loss and duplication across the different lineages of the genus Burkholderia, leading to notable differences in the number of paralogs between different genomes. A putative substrate [antibiotics (HAE1 proteins/heavy-metal (HME proteins] was also assigned to the majority of these proteins. No correlation was found between the ecological niche and the lifestyle of Burkholderia strains and the number/type of efflux pumps they possessed, while a relation can be found with genome size and taxonomy. Remarkably, we observed that only HAE1 proteins are mainly responsible for the different number of proteins observed in strains of the same species. Data concerning both the distribution and the phylogenetic analysis of the HAE1 and HME in the Burkholderia genus allowed depicting a likely evolutionary model accounting for the evolution and spreading of HME and HAE

  10. Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. nodule bacteria on two Mimosa spp. in Costa Rica.

    Science.gov (United States)

    Barrett, Craig F; Parker, Matthew A

    2006-02-01

    rRNA gene sequencing and PCR assays indicated that 215 isolates of root nodule bacteria from two Mimosa species at three sites in Costa Rica belonged to the genera Burkholderia, Cupriavidus, and Rhizobium. This is the first report of Cupriavidus sp. nodule symbionts for Mimosa populations within their native geographic range in the neotropics. Burkholderia spp. predominated among samples from Mimosa pigra (86% of isolates), while there was a more even distribution of Cupriavidus, Burkholderia, and Rhizobium spp. on Mimosa pudica (38, 37, and 25% of isolates, respectively). All Cupriavidus and Burkholderia genotypes tested formed root nodules and fixed nitrogen on both M. pigra and M. pudica, and sequencing of rRNA genes in strains reisolated from nodules verified identity with inoculant strains. Inoculation tests further indicated that both Cupriavidus and Burkholderia spp. resulted in significantly higher plant growth and nodule nitrogenase activity (as measured by acetylene reduction assays) relative to plant performance with strains of Rhizobium. Given the prevalence of Burkholderia and Cupriavidus spp. on these Mimosa legumes and the widespread distribution of these plants both within and outside the neotropics, it is likely that both beta-proteobacterial genera are more ubiquitous as root nodule symbionts than previously believed.

  11. Prevalence and Identification of Burkholderia pseudomallei and Near-Neighbor Species in the Malabar Coastal Region of India.

    Science.gov (United States)

    Peddayelachagiri, Bhavani V; Paul, Soumya; Nagaraj, Sowmya; Gogoi, Madhurjya; Sripathy, Murali H; Batra, Harsh V

    2016-09-01

    Accurate identification of pathogens with biowarfare importance requires detection tools that specifically differentiate them from near-neighbor species. Burkholderia pseudomallei, the causative agent of a fatal disease melioidosis, is one such biothreat agent whose differentiation from its near-neighbor species is always a challenge. This is because of its phenotypic similarity with other Burkholderia species which have a wide spread geographical distribution with shared environmental niches. Melioidosis is a major public health concern in endemic regions including Southeast Asia and northern Australia. In India, the disease is still considered to be emerging. Prevalence surveys of this saprophytic bacterium in environment are under-reported in the country. A major challenge in this case is the specific identification and differentiation of B. pseudomallei from the growing list of species of Burkholderia genus. The objectives of this study included examining the prevalence of B. pseudomallei and near-neighbor species in coastal region of South India and development of a novel detection tool for specific identification and differentiation of Burkholderia species. Briefly, we analyzed soil and water samples collected from Malabar coastal region of Kerala, South India for prevalence of B. pseudomallei. The presumptive Burkholderia isolates were identified using recA PCR assay. The recA PCR assay identified 22 of the total 40 presumptive isolates as Burkholderia strains (22.72% and 77.27% B. pseudomallei and non-pseudomallei Burkholderia respectively). In order to identify each isolate screened, we performed recA and 16S rDNA sequencing. This two genes sequencing revealed that the presumptive isolates included B. pseudomallei, non-pseudomallei Burkholderia as well as non-Burkholderia strains. Furthermore, a gene termed D-beta hydroxybutyrate dehydrogenase (bdha) was studied both in silico and in vitro for accurate detection of Burkholderia genus. The optimized bdha

  12. Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species.

    Directory of Open Access Journals (Sweden)

    Heleen Van Acker

    Full Text Available The presence of persister cells has been proposed as a factor in biofilm resilience. In the present study we investigated whether persister cells are present in Burkholderia cepacia complex (Bcc biofilms, what the molecular basis of antimicrobial tolerance in Bcc persisters is, and how persisters can be eradicated from Bcc biofilms. After treatment of Bcc biofilms with high concentrations of various antibiotics often a small subpopulation survived. To investigate the molecular mechanism of tolerance in this subpopulation, Burkholderia cenocepacia biofilms were treated with 1024 µg/ml of tobramycin. Using ROS-specific staining and flow cytometry, we showed that tobramycin increased ROS production in treated sessile cells. However, approximately 0.1% of all sessile cells survived the treatment. A transcriptome analysis showed that several genes from the tricarboxylic acid cycle and genes involved in the electron transport chain were downregulated. In contrast, genes from the glyoxylate shunt were upregulated. These data indicate that protection against ROS is important for the survival of persisters. To confirm this, we determined the number of persisters in biofilms formed by catalase mutants. The persister fraction in ΔkatA and ΔkatB biofilms was significantly reduced, confirming the role of ROS detoxification in persister survival. Pretreatment of B. cenocepacia biofilms with itaconate, an inhibitor of isocitrate lyase (ICL, the first enzyme in the glyoxylate shunt, reduced the persister fraction approx. 10-fold when the biofilms were subsequently treated with tobramycin. In conclusion, most Bcc biofilms contain a significant fraction of persisters that survive treatment with high doses of tobramycin. The surviving persister cells downregulate the TCA cycle to avoid production of ROS and at the same time activate an alternative pathway, the glyoxylate shunt. This pathway may present a novel target for combination therapy.

  13. Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex.

    Science.gov (United States)

    Bartholdson, S Josefin; Brown, Alan R; Mewburn, Ben R; Clarke, David J; Fry, Stephen C; Campopiano, Dominic J; Govan, John R W

    2008-08-01

    The species that presently constitute the Burkholderia cepacia complex (Bcc) have multiple roles; they include soil and water saprophytes, bioremediators, and plant, animal and human pathogens. Since the first description of pathogenicity in the Bcc was based on sour skin rot of onion bulbs, this study returned to this plant host to investigate the onion-associated phenotype of the Bcc. Many Bcc isolates, which were previously considered to be non-mucoid, produced copious amounts of exopolysaccharide (EPS) when onion tissue was provided as the sole nutrient. EPS production was not species-specific, was observed in isolates from both clinical and environmental sources, and did not correlate with the ability to cause maceration of onion tissue. Chemical analysis suggested that the onion components responsible for EPS induction were primarily the carbohydrates sucrose, fructose and fructans. Additional sugars were investigated, and all alcohol sugars tested were able to induce EPS production, in particular mannitol and glucitol. To investigate the molecular basis for EPS biosynthesis, we focused on the highly conserved bce gene cluster thought to be involved in cepacian biosynthesis. We demonstrated induction of the bce gene cluster by mannitol, and found a clear correlation between the inability of representatives of the Burkholderia cenocepacia ET12 lineage to produce EPS and the presence of an 11 bp deletion within the bceB gene, which encodes a glycosyltransferase. Insertional inactivation of bceB in Burkholderia ambifaria AMMD results in loss of EPS production on sugar alcohol media. These novel and surprising insights into EPS biosynthesis highlight the metabolic potential of the Bcc and show that a potential virulence factor may not be detected by routine laboratory culture. Our results also highlight a potential hazard in the use of inhaled mannitol as an osmolyte to improve mucociliary clearance in individuals with cystic fibrosis.

  14. Plant-associated symbiotic Burkholderia species lack hallmark strategies required in mammalian pathogenesis.

    Directory of Open Access Journals (Sweden)

    Annette A Angus

    Full Text Available Burkholderia is a diverse and dynamic genus, containing pathogenic species as well as species that form complex interactions with plants. Pathogenic strains, such as B. pseudomallei and B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic pathogens, infecting humans or animals with a compromised immune system. Although some of the opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the risk of infection to infants, the elderly, and people who are immunocompromised has not only resulted in a restriction on their use, but has also limited the application of non-pathogenic, symbiotic species, several of which nodulate legume roots or have positive effects on plant growth. However, recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct lineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts. A number of environmental strains that promote plant growth or degrade xenobiotics are also included in the symbiotic lineage. Many of these species have the potential to enhance agriculture in areas where fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted by climate change. Here we address the pathogenic potential of several of the symbiotic Burkholderia strains using bioinformatics and functional tests. A series of infection experiments using Caenorhabditis elegans and HeLa cells, as well as genomic characterization of pathogenic loci, show that the risk of opportunistic infection by symbiotic strains such as B. tuberum is extremely low.

  15. Plant-associated symbiotic Burkholderia species lack hallmark strategies required in mammalian pathogenesis.

    Science.gov (United States)

    Angus, Annette A; Agapakis, Christina M; Fong, Stephanie; Yerrapragada, Shailaja; Estrada-de los Santos, Paulina; Yang, Paul; Song, Nannie; Kano, Stephanie; Caballero-Mellado, Jésus; de Faria, Sergio M; Dakora, Felix D; Weinstock, George; Hirsch, Ann M

    2014-01-01

    Burkholderia is a diverse and dynamic genus, containing pathogenic species as well as species that form complex interactions with plants. Pathogenic strains, such as B. pseudomallei and B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic pathogens, infecting humans or animals with a compromised immune system. Although some of the opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the risk of infection to infants, the elderly, and people who are immunocompromised has not only resulted in a restriction on their use, but has also limited the application of non-pathogenic, symbiotic species, several of which nodulate legume roots or have positive effects on plant growth. However, recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct lineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts. A number of environmental strains that promote plant growth or degrade xenobiotics are also included in the symbiotic lineage. Many of these species have the potential to enhance agriculture in areas where fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted by climate change. Here we address the pathogenic potential of several of the symbiotic Burkholderia strains using bioinformatics and functional tests. A series of infection experiments using Caenorhabditis elegans and HeLa cells, as well as genomic characterization of pathogenic loci, show that the risk of opportunistic infection by symbiotic strains such as B. tuberum is extremely low.

  16. Burkholderia pseudomallei traced to water treatment plant in Australia.

    Science.gov (United States)

    Inglis, T. J.; Garrow, S. C.; Henderson, M.; Clair, A.; Sampson, J.; O'Reilly, L.; Cameron, B.

    2000-01-01

    Burkholderia pseudomallei was isolated from environmental specimens 1 year after an outbreak of acute melioidosis in a remote coastal community in northwestern Australia. B. pseudomallei was isolated from a water storage tank and from spray formed in a pH-raising aerator unit. Pulsed-field gel electrophoresis confirmed the aerator and storage tank isolates were identical to the outbreak strain, WKo97. PMID:10653571

  17. Draft genome sequence of Burkholderia sordidicola S170, a potential plant growth promoter isolated from coniferous forest soil in the Czech Republic

    DEFF Research Database (Denmark)

    Lladó, Salvador; Xu, Zhuofei; Sørensen, Søren Johannes;

    2014-01-01

    Burkholderia species are key players in the accumulation of carbon from cellulose decomposition in coniferous forest ecosystems. We report here the draft genome of Burkholderia sordidicola strain S170, containing features associated with known genes involved in plant growth promotion, the biologi...

  18. Draft Genome Sequence of Burkholderia sordidicola S170, a Potential Plant Growth Promoter Isolated from Coniferous Forest Soil in the Czech Republic.

    Science.gov (United States)

    Lladó, Salvador; Xu, Zhuofei; Sørensen, Søren J; Baldrian, Petr

    2014-08-14

    Burkholderia species are key players in the accumulation of carbon from cellulose decomposition in coniferous forest ecosystems. We report here the draft genome of Burkholderia sordidicola strain S170, containing features associated with known genes involved in plant growth promotion, the biological control of plant diseases, and green remediation technologies. Copyright © 2014 Lladó et al.

  19. Architecture of Burkholderia cepacia complex σ70 gene family: evidence of alternative primary and clade-specific factors, and genomic instability

    Directory of Open Access Journals (Sweden)

    Menard Aymeric

    2007-09-01

    Full Text Available Abstract Background The Burkholderia cepacia complex (Bcc groups bacterial species with beneficial properties that can improve crop yields or remediate polluted sites but can also lead to dramatic human clinical outcomes among cystic fibrosis (CF or immuno-compromised individuals. Genome-wide regulatory processes of gene expression could explain parts of this bacterial duality. Transcriptional σ70 factors are components of these processes. They allow the reversible binding of the DNA-dependent RNA polymerase to form the holoenzyme that will lead to mRNA synthesis from a DNA promoter region. Bcc genome-wide analyses were performed to investigate the major evolutionary trends taking place in the σ70 family of these bacteria. Results Twenty σ70 paralogous genes were detected in the Burkholderia cenocepacia strain J2315 (Bcen-J2315 genome, of which 14 were of the ECF (extracytoplasmic function group. Non-ECF paralogs were related to primary (rpoD, alternative primary, stationary phase (rpoS, flagellin biosynthesis (fliA, and heat shock (rpoH factors. The number of σ70 genetic determinants among this genome was of 2,86 per Mb. This number is lower than the one of Pseudomonas aeruginosa, a species found in similar habitats including CF lungs. These two bacterial groups showed strikingly different σ70 family architectures, with only three ECF paralogs in common (fecI-like, pvdS and algU. Bcen-J2315 σ70 paralogs showed clade-specific distributions. Some paralogs appeared limited to the ET12 epidemic clone (ecfA2, particular Bcc species (sigI, the Burkholderia genus (ecfJ, ecfF, and sigJ, certain proteobacterial groups (ecfA1, ecfC, ecfD, ecfE, ecfG, ecfL, ecfM and rpoS, or were broadly distributed in the eubacteria (ecfI, ecfK, ecfH, ecfB, and rpoD-, rpoH-, fliA-like genes. Genomic instability of this gene family was driven by chromosomal inversion (ecfA2, recent duplication events (ecfA and RpoD, localized (ecfG and large scale deletions (sig

  20. Screening and fermentation conditions of lipase-producing strain NJY-I-7%脂肪酶产生菌NJY-1-7的选育及发酵条件的研究

    Institute of Scientific and Technical Information of China (English)

    贺胜英; 陈爱军; 杜华明; 黄遵锡

    2011-01-01

    从云南省福贡县的玉米地土壤样品中筛选到一株耐高温产碱性脂肪酶的菌株NJY-1-7,通过16SrDNA鉴定该菌株为伯克氏菌(Burkholderia cenocepacia).对其发酵条件进行研究结果表明:该菌株产酶的最适培养时间为48h,酶促反应的最适作用pH值为9.0,最适作用温度为50℃,摇瓶发酵最适产酶条件为橄榄油1%,MgSO4·7H20.05%,可溶性淀粉03%,酵母膏0.5%,单蒸水70mL.在此条件下发酵脂肪酶酶活力为42.00U/mL.%A thermostable alkaline lipase-producing strain NJY-1-7 was isolated from soil of corn field in Fugong county of Yunnan. The strain was identified as Burkholderia pseudomallei the methods of 16S rDNA. The optimum pH value and temperature for lipase activity was 9.0 and 50℃ respectively. The optimum conditions for enzyme production were as follows: cultivation with shaking flask for 48h with the medium composed of olive oil 1%, MgSO4·7H2O 0.05%, soluble starch 0.3%, yeast extract 0.5%, distilled H2O 70ml. Under the optimum conditions, lipase activity could reach 42.00U/ml.

  1. 吡咯伯克霍尔德氏菌A12发酵培养基的优化及抑菌物质理化性质研究%Fermentation Medium of Antagonistic Strain Burkholderia pyrrocinia A12 and Characterisics of Its Antibacterial Substance

    Institute of Scientific and Technical Information of China (English)

    韩超; 刘爱新

    2012-01-01

    Optimized fermentation medium affecting production of antagonistic substance produced by Burkholderia pyrrocinia A12 and the characterisics of its antibacterial substance extracted with ammonium sulfate method were studied.The results showed that beef extract and peptone were optimum nutrientsubstance for production of antagonistic substance produced by B.pyrrocinia strain A12.Antifungal substances were stable to the heat treatment and not sensitive to trypsin,pepsin,proteinase K and UV radiation,which is suitable for playing a role on antibacterial activity in neutral conditions.%对拮抗细菌Burkholderia pyrrocinia菌株A12的发酵培养基进行了优化,并利用硫酸铵盐析法得到抑菌物质粗提物,对其理化性质进行了初步探索.结果表明,牛肉膏、蛋白胨是发酵培养基中最佳营养物质,有利于菌株A12抗菌物质的产生;胞外抑菌活性物质具有一定的热稳定性,对胃蛋白酶、胰蛋白酶、蛋白酶K及紫外线照射均不敏感,适于在中性条件下发挥作用.

  2. The phylogenetic distribution and ecological role of carbon monoxide oxidation in the genus Burkholderia.

    Science.gov (United States)

    Weber, Carolyn F; King, Gary M

    2012-01-01

    Burkholderia is a physiologically and ecologically diverse genus that occurs commonly in assemblages of soil and rhizosphere bacteria. Although Burkholderia is known for its heterotrophic versatility, we demonstrate that 14 distinct environmental isolates oxidized carbon monoxide (CO) and possessed the gene encoding the catalytic subunit of form I CO dehydrogenase (coxL). DNA from a Burkholderia isolate obtained from a passalid beetle also contained coxL as do the genomic sequences of species H160 and Ch1-1. Isolates were able to consume CO at concentrations ranging from 100 ppm (vol/vol) to sub-ambient ( 2.5 mM), but mixotrophic consumption of CO and pyruvate occurred when initial pyruvate concentrations were lower (c. 400 lM). With the exception of an isolate most closely related to Burkholderia cepacia, all CO-oxidizing isolates examined were members of a nonpathogenic clade and were most closely related to Burkholderia species, B. caledonica, B. fungorum, B. oxiphila, B. mimosarum, B. nodosa, B. sacchari, B. bryophila, B. ferrariae, B. ginsengesoli, and B. unamae. However, none of these type strains oxidized CO or contained coxL based on results from PCR analyses. Collectively, these results demonstrate that the presence of CO oxidation within members of the Burkholderia genus is variable but it is most commonly found among rhizosphere inhabitants that are not closely related to B. cepacia.

  3. Comparative Genomics of Burkholderia singularis sp. nov., a Low G+C Content, Free-Living Bacterium That Defies Taxonomic Dissection of the Genus Burkholderia

    Directory of Open Access Journals (Sweden)

    Peter Vandamme

    2017-09-01

    Full Text Available Four Burkholderia pseudomallei-like isolates of human clinical origin were examined by a polyphasic taxonomic approach that included comparative whole genome analyses. The results demonstrated that these isolates represent a rare and unusual, novel Burkholderia species for which we propose the name B. singularis. The type strain is LMG 28154T (=CCUG 65685T. Its genome sequence has an average mol% G+C content of 64.34%, which is considerably lower than that of other Burkholderia species. The reduced G+C content of strain LMG 28154T was characterized by a genome wide AT bias that was not due to reduced GC-biased gene conversion or reductive genome evolution, but might have been caused by an altered DNA base excision repair pathway. B. singularis can be differentiated from other Burkholderia species by multilocus sequence analysis, MALDI-TOF mass spectrometry and a distinctive biochemical profile that includes the absence of nitrate reduction, a mucoid appearance on Columbia sheep blood agar, and a slowly positive oxidase reaction. Comparisons with publicly available whole genome sequences demonstrated that strain TSV85, an Australian water isolate, also represents the same species and therefore, to date, B. singularis has been recovered from human or environmental samples on three continents.

  4. Biochemical and functional studies on the Burkholderia cepacia complex bceN gene, encoding a GDP-D-mannose 4,6-dehydratase.

    Directory of Open Access Journals (Sweden)

    Sílvia A Sousa

    Full Text Available This work reports the biochemical and functional analysis of the Burkholderia cenocepacia J2315 bceN gene, encoding a protein with GDP-D-mannose 4,6-dehydratase enzyme activity (E.C.4.2.1.47. Data presented indicate that the protein is active when in the tetrameric form, catalyzing the conversion of GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. This sugar nucleotide is the intermediary necessary for the biosynthesis of GDP-D-rhamnose, one of the sugar residues of cepacian, the major exopolysaccharide produced by environmental and human, animal and plant pathogenic isolates of the Burkholderia cepacia complex species. Vmax and Km values of 1.5±0.2 µmol.min(-1.mg(-1 and 1024±123 µM, respectively, were obtained from the kinetic characterization of the B. cenocepacia J2315 BceN protein by NMR spectroscopy, at 25°C and in the presence of 1 mol MgCl2 per mol of protein. The enzyme activity was strongly inhibited by the substrate, with an estimated Ki of 2913±350 µM. The lack of a functional bceN gene in a mutant derived from B. cepacia IST408 slightly reduced cepacian production. However, in the B. multivorans ATCC17616 with bceN as the single gene in its genome with predicted GMD activity, a bceN mutant did not produce cepacian, indicating that this gene product is required for cepacian biosynthesis.

  5. Biochemical and Functional Studies on the Burkholderia cepacia Complex bceN Gene, Encoding a GDP-D-Mannose 4,6-Dehydratase

    Science.gov (United States)

    Pinheiro, Pedro F.; Leitão, Jorge H.

    2013-01-01

    This work reports the biochemical and functional analysis of the Burkholderia cenocepacia J2315 bceN gene, encoding a protein with GDP-D-mannose 4,6-dehydratase enzyme activity (E.C.4.2.1.47). Data presented indicate that the protein is active when in the tetrameric form, catalyzing the conversion of GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. This sugar nucleotide is the intermediary necessary for the biosynthesis of GDP-D-rhamnose, one of the sugar residues of cepacian, the major exopolysaccharide produced by environmental and human, animal and plant pathogenic isolates of the Burkholderia cepacia complex species. Vmax and Km values of 1.5±0.2 µmol.min−1.mg−1 and 1024±123 µM, respectively, were obtained from the kinetic characterization of the B. cenocepacia J2315 BceN protein by NMR spectroscopy, at 25°C and in the presence of 1 mol MgCl2 per mol of protein. The enzyme activity was strongly inhibited by the substrate, with an estimated Ki of 2913±350 µM. The lack of a functional bceN gene in a mutant derived from B. cepacia IST408 slightly reduced cepacian production. However, in the B. multivorans ATCC17616 with bceN as the single gene in its genome with predicted GMD activity, a bceN mutant did not produce cepacian, indicating that this gene product is required for cepacian biosynthesis. PMID:23460819

  6. Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

    Science.gov (United States)

    Su, Shengchang; Bangar, Hansraj; Saldanha, Roland; Pemberton, Adin; Aronow, Bruce; Dean, Gary E; Lamkin, Thomas J; Hassett, Daniel J

    2014-10-01

    Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages.

  7. Innate Immune Response to Burkholderia mallei

    Science.gov (United States)

    2017-02-16

    PREVENTIVE STRATEGY TO GLANDERS: Antibiotic resistance associated with Burkholderia infection is on the rise (27). Even with optimal antibiotic treatment...27. Rhodes KA, Schweizer HP. Antibiotic resistance in Burkholderia species. Drug resistance updates : reviews and commentaries in antimicrobial and...highly contagious and often fatal disease, glanders. With its high rate of infectivity via aerosol and recalcitrance towards antibiotics , this

  8. Accurate and rapid identification of the Burkholderia pseudomallei near-neighbour, Burkholderia ubonensis, using real-time PCR.

    Directory of Open Access Journals (Sweden)

    Erin P Price

    Full Text Available Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc, a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdown's medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdown's agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdown's-positive colonies that are not B. pseudomallei.

  9. The role of siderophores in metal homeostasis of members of the genus Burkholderia.

    Science.gov (United States)

    Mathew, Anugraha; Jenul, Christian; Carlier, Aurelien L; Eberl, Leo

    2016-02-01

    Although members of the genus Burkholderia can utilize a high-affinity iron uptake system to sustain growth under iron-limiting conditions, many strains also produce siderophores, suggesting that they may serve alternative functions. Here we demonstrate that the two Burkholderia siderophores pyochelin and ornibactin can protect the cells from metal toxicity and thus play an alternative role in metal homeostasis. We also demonstrate that metals such as copper and zinc induce the production of ornibactin. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  10. Burkholderia and Cupriavidus spp. are the preferred symbionts of Mimosa spp. in southern China.

    Science.gov (United States)

    Liu, XiaoYun; Wei, Shuang; Wang, Fang; James, Euan K; Guo, XiaoYe; Zagar, Catherine; Xia, Liu Gui; Dong, Xin; Wang, Yi Peng

    2012-05-01

    Rhizobia were isolated from invasive Mimosa spp. (M. diplotricha and M. pudica) in Dehong district of the province of Yunnan in subtropical southern China. Almost all of the 98 isolates were β-rhizobia in the genera Burkholderia and Cupriavidus. These strains were analysed for their distribution characteristics together with strains from a previous study from Sishuangbanna. The proportion of nodules containing each β-rhizobial genus varied between Mimosa species, with Cupriavidus being predominant in M. diplotricha nodules (63.3% compared to 36.7% occupation with Burkholderia), but with M. pudica showing a slight preference for Burkholderia over Cupriavidus, with them occupying 56.5% and 43.5% of nodules, respectively. The symbiosis-essential genes nodA and nifH were present in all the Burkholderia and Cupriavidus strains tested, and their phylogenies indicated that these Mimosa symbionts share symbiotic genes with native South American rhizobia. The evolutionary discrepancies among 16S rRNA genes, nodA and nifH of Mimosa spp. symbionts, suggests that the nod and nif genes of β-rhizobia evolved independently. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  11. Quorum Sensing Influences Burkholderia thailandensis Biofilm Development and Matrix Production.

    Science.gov (United States)

    Tseng, Boo Shan; Majerczyk, Charlotte D; Passos da Silva, Daniel; Chandler, Josephine R; Greenberg, E Peter; Parsek, Matthew R

    2016-10-01

    Members of the genus Burkholderia are known to be adept at biofilm formation, which presumably assists in the survival of these organisms in the environment and the host. Biofilm formation has been linked to quorum sensing (QS) in several bacterial species. In this study, we characterized Burkholderia thailandensis biofilm development under flow conditions and sought to determine whether QS contributes to this process. B. thailandensis biofilm formation exhibited an unusual pattern: the cells formed small aggregates and then proceeded to produce mature biofilms characterized by "dome" structures filled with biofilm matrix material. We showed that this process was dependent on QS. B. thailandensis has three acyl-homoserine lactone (AHL) QS systems (QS-1, QS-2, and QS-3). An AHL-negative strain produced biofilms consisting of cell aggregates but lacking the matrix-filled dome structures. This phenotype was rescued via exogenous addition of the three AHL signals. Of the three B. thailandensis QS systems, we show that QS-1 is required for proper biofilm development, since a btaR1 mutant, which is defective in QS-1 regulation, forms biofilms without these dome structures. Furthermore, our data show that the wild-type biofilm biomass, as well as the material inside the domes, stains with a fucose-binding lectin. The btaR1 mutant biofilms, however, are negative for fucose staining. This suggests that the QS-1 system regulates the production of a fucose-containing exopolysaccharide in wild-type biofilms. Finally, we present data showing that QS ability during biofilm development produces a biofilm that is resistant to dispersion under stress conditions. The saprophyte Burkholderia thailandensis is a close relative of the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis, which is contracted from its environmental reservoir. Since most bacteria in the environment reside in biofilms, B. thailandensis is an ideal model organism for

  12. Identification of Burkholderia mallei and Burkholderia pseudomallei adhesins for human respiratory epithelial cells

    Directory of Open Access Journals (Sweden)

    Hogan Robert J

    2010-09-01

    Full Text Available Abstract Background Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. A well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. In contrast, the means by which B. pseudomallei and B. mallei adhere to cells are poorly defined. The purpose of this study was to identify adherence factors expressed by these organisms. Results Comparative sequence analyses identified a gene product in the published genome of B. mallei strain ATCC23344 (locus # BMAA0649 that resembles the well-characterized Yersinia enterocolitica autotransporter adhesin YadA. The gene encoding this B. mallei protein, designated boaA, was expressed in Escherichia coli and shown to significantly increase adherence to human epithelial cell lines, specifically HEp2 (laryngeal cells and A549 (type II pneumocytes, as well as to cultures of normal human bronchial epithelium (NHBE. Consistent with these findings, disruption of the boaA gene in B. mallei ATCC23344 reduced adherence to all three cell types by ~50%. The genomes of the B. pseudomallei strains K96243 and DD503 were also found to contain boaA and inactivation of the gene in DD503 considerably decreased binding to monolayers of HEp2 and A549 cells and to NHBE cultures. A second YadA-like gene product highly similar to BoaA (65% identity was identified in the published genomic sequence of B. pseudomallei strain K96243 (locus # BPSL1705. The gene specifying this protein, termed boaB, appears to be B. pseudomallei-specific. Quantitative attachment assays demonstrated that recombinant E. coli expressing BoaB displayed greater binding to A549 pneumocytes, HEp2 cells and NHBE cultures. Moreover, a boaB mutant of B. pseudomallei DD503 showed decreased adherence to these respiratory cells. Additionally, a B. pseudomallei strain lacking expression of both boaA and boaB was impaired in its ability to

  13. Insecticide applications to soil contribute to the development of Burkholderia mediating insecticide resistance in stinkbugs.

    Science.gov (United States)

    Tago, Kanako; Kikuchi, Yoshitomo; Nakaoka, Sinji; Katsuyama, Chie; Hayatsu, Masahito

    2015-07-01

    Some soil Burkholderia strains are capable of degrading the organophosphorus insecticide, fenitrothion, and establish symbiosis with stinkbugs, making the host insects fenitrothion-resistant. However, the ecology of the symbiotic degrading Burkholderia adapting to fenitrothion in the free-living environment is unknown. We hypothesized that fenitrothion applications affect the dynamics of fenitrothion-degrading Burkholderia, thereby controlling the transmission of symbiotic degrading Burkholderia from the soil to stinkbugs. We investigated changes in the density and diversity of culturable Burkholderia (i.e. symbiotic and nonsymbiotic fenitrothion degraders and nondegraders) in fenitrothion-treated soil using microcosms. During the incubation with five applications of pesticide, the density of the degraders increased from less than the detection limit to around 10(6)/g of soil. The number of dominant species among the degraders declined with the increasing density of degraders; eventually, one species predominated. This process can be explained according to the competitive exclusion principle using V(max) and K(m) values for fenitrothion metabolism by the degraders. We performed a phylogenetic analysis of representative strains isolated from the microcosms and evaluated their ability to establish symbiosis with the stinkbug Riptortus pedestris. The strains that established symbiosis with R. pedestris were assigned to a cluster including symbionts commonly isolated from stinkbugs. The strains outside the cluster could not necessarily associate with the host. The degraders in the cluster predominated during the initial phase of degrader dynamics in the soil. Therefore, only a few applications of fenitrothion could allow symbiotic degraders to associate with their hosts and may cause the emergence of symbiont-mediated insecticide resistance.

  14. Oxalotrophy, a widespread trait of plant-associated Burkholderia species, is involved in successful root colonization of lupin and maize by Burkholderia phytofirmans.

    Directory of Open Access Journals (Sweden)

    Thomas eKost

    2014-01-01

    Full Text Available Plant roots and shoots harbour complex bacterial communities. Early seed and plantlet colonization plays a key role in determining which bacterial populations will successfully invade plant tissues, yet the mechanisms enabling plants to select for beneficial rather than harmful populations are largely unknown. In this study, we demonstrate a role of oxalate as a determinant in this selection process, using members of the genus Burkholderia as model organisms. Oxalotrophy, i.e. the ability to use oxalate as a carbon source, was found to be a property strictly associated with plant-beneficial species of the Burkholderia genus, while plant pathogenic (B. glumae, B. plantarii or human opportunistic pathogens (Burkholderia cepacia complex strains were unable to degrade oxalate. We further show that oxalotrophy is required for successful plant colonization by the broad host endophyte Burkholderia phytofirmans PsJN: an engineered Δoxc mutant, which lost the ability to grow on oxalate, was significantly impaired in early colonization of both lupin and maize compared with the wild-type. This work suggests that in addition to the role of oxalate in heavy metal tolerance of plants and in virulence of phytopathogenic fungi, it is also involved in specifically recruiting plant-beneficial members from complex bacterial communities.

  15. NCBI nr-aa BLAST: CBRC-CREM-01-1310 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1310 ref|YP_624641.1| acyltransferase 3 [Burkholderia cenocepacia AU 1...054] ref|YP_837010.1| acyltransferase 3 [Burkholderia cenocepacia HI2424] ref|ZP_01565190.1| acyltransferase 3 [Burkholder...ia cenocepacia MC0-3] gb|ABF79668.1| acyltransferase 3 [Burkholderia cenocepacia AU 1054] gb|A...BK10117.1| acyltransferase 3 [Burkholderia cenocepacia HI2424] gb|EAV56867.1| acyltransferase 3 [Burkholderia cenocepacia MC0-3] YP_624641.1 1e-65 56% ...

  16. The symbiotic role of O-antigen of Burkholderia symbiont in association with host Riptortus pedestris.

    Science.gov (United States)

    Kim, Jiyeun Kate; Park, Ha Young; Lee, Bok Luel

    2016-07-01

    Riptortus pedestris harboring Burkholderia symbiont is a useful symbiosis model to study the molecular interactions between insects and bacteria. We recently reported that the lipopolysaccharide O-antigen is absent in the Burkholderia symbionts isolated from Riptortus guts. Here, we investigated the symbiotic role of O-antigen comprehensively in the Riptortus-Burkholderia model. Firstly, Burkholderia mutant strains deficient of O-antigen biosynthesis genes were generated and confirmed for their different patterns of the lipopolysaccharide by electrophoretic analysis. The O-antigen-deficient mutant strains initially exhibited a reduction of infectivity, having significantly lower level of symbiont population at the second-instar stage. However, both the wild-type and O-antigen mutant symbionts exhibited a similar level of symbiont population from the third-instar stage, indicating that the O-antigen deficiency did not affect the bacterial persistence in the host midgut. Taken together, we showed that the lipopolysaccharide O-antigen of gut symbiont plays an exclusive role in the initial symbiotic association.

  17. Burkholderia, a Genus Rich in Plant-Associated Nitrogen Fixers with Wide Environmental and Geographic Distribution

    Science.gov (United States)

    Estrada-De Los Santos, Paulina; Bustillos-Cristales, Rocío; Caballero-Mellado, Jesús

    2001-01-01

    The genus Burkholderia comprises 19 species, including Burkholderia vietnamiensis which is the only known N2-fixing species of this bacterial genus. The first isolates of B. vietnamiensis were recovered from the rhizosphere of rice plants grown in a phytotron, but its existence in natural environments and its geographic distribution were not reported. In the present study, most N2-fixing isolates recovered from the environment of field-grown maize and coffee plants cultivated in widely separated regions of Mexico were phenotypically identified as B. cepacia using the API 20NE system. Nevertheless, a number of these isolates recovered from inside of maize roots, as well as from the rhizosphere and rhizoplane of maize and coffee plants, showed similar or identical features to those of B. vietnamiensis TVV75T. These features include nitrogenase activity with 10 different carbon sources, identical or very similar nifHDK hybridization patterns, very similar protein electrophoregrams, identical amplified 16S rDNA restriction (ARDRA) profiles, and levels of DNA-DNA reassociation higher than 70% with total DNA from strain TVV75T. Although the ability to fix N2 is not reported to be a common feature among the known species of the genus Burkholderia, the results obtained show that many diazotrophic Burkholderia isolates analyzed showed phenotypic and genotypic features different from those of the known N2-fixing species B. vietnamiensis as well as from those of B. kururiensis, a bacterium identified in the present study as a diazotrophic species. DNA-DNA reassociation assays confirmed the existence of N2-fixing Burkholderia species different from B. vietnamiensis. In addition, this study shows the wide geographic distribution and substantial capability of N2-fixing Burkholderia spp. for colonizing diverse host plants in distantly separated environments. PMID:11375196

  18. Properties of Polyhydroxyalkanoate Granules and Bioemulsifiers from Pseudomonas sp. and Burkholderia sp. Isolates Growing on Glucose.

    Science.gov (United States)

    Sacco, Laís Postai; Castellane, Tereza Cristina Luque; Lopes, Erica Mendes; de Macedo Lemos, Eliana Gertrudes; Alves, Lúcia Maria Carareto

    2016-03-01

    A Burkholderia and Pseudomonas species designated as AB4 and AS1, respectively, were isolated from soil containing decomposing straw or sugar cane bagasse collected from Brazil. This study sought to evaluate the capacities of culture media, cell-free medium, and crude lysate preparations (containing PHB inclusion bodies) from bacterial cell cultures to stabilize emulsions with several hydrophobic compounds. Four conditions showed good production of bioemulsifiers (E24 ≥ 50 %), headed by substantially cell-free media from bacterial cell cultures in which bacterial isolates from Burkholderia sp. strain AB4 and Pseudomonas sp. strain AS1 were grown. Our results revealed that the both isolates (AB4 and AS1 strains) exhibited high emulsification indices (indicating usefulness in bioremediation) and good stabilities.

  19. Biodegradation of PAHs by Burkholderia sp. VITRSB1 Isolated from Marine Sediments

    Directory of Open Access Journals (Sweden)

    T. Revathy

    2015-01-01

    Full Text Available The polycyclic aromatic hydrocarbons (PAHs pollution to the environment is a major threat to the living organisms, and hence the degradation of these PAHs is necessary. Studies on PAHs degrading bacteria have focussed on terrestrial microbes and the potential of marine derived microbes is undermined. Herein we report the isolation and characterization of PAHs degrading Burkholderia sp. from lagoon sediments collected at the Southern coast of India. The strain was Gram negative, rod-shaped, motile, and ∼2–5 μm in length. Based on the phylogenetic data the strain was identified as Burkholderia and designated as VITRSB1. Initial PAHs degradation ability of the strain was assessed using basal salt medium supplemented with diesel, kerosene, toluene, aniline, naphthalene, and phenol. The strain was found to be effectively degrading kerosene, diesel, toluene, and aniline even at higher concentration (1%. However, naphthalene and aniline were degraded only at lower concentration (0.1% and phenol, camphor, and DAP inhibited the growth of the strain. Furthermore, the degraded end products of the PAHs were determined using FTIR. Notably, none of the end products were found to be toxic to the biosphere. Our results indicate that the isolated Burkholderia sp. could be a prospective candidate for the effective degradation of selective PAHs.

  20. The Role of Reactive Oxygen Species in Antibiotic-Induced Cell Death in Burkholderia cepacia Complex Bacteria.

    Directory of Open Access Journals (Sweden)

    Heleen Van Acker

    Full Text Available It was recently proposed that bactericidal antibiotics, besides through specific drug-target interactions, kill bacteria by a common mechanism involving the production of reactive oxygen species (ROS. However, this mechanism involving the production of hydroxyl radicals has become the subject of a lot of debate. Since the contribution of ROS to antibiotic mediated killing most likely depends on the conditions, differences in experimental procedures are expected to be at the basis of the conflicting results. In the present study different methods (ROS specific stainings, gene-expression analyses, electron paramagnetic resonance, genetic and phenotypic experiments, detection of protein carbonylation and DNA oxidation to measure the production of ROS upon antibiotic treatment in Burkholderia cepacia complex (Bcc bacteria were compared. Different classes of antibiotics (tobramycin, ciprofloxacin, meropenem were included, and both planktonic and biofilm cultures were studied. Our results indicate that some of the methods investigated were not sensitive enough to measure antibiotic induced production of ROS, including the spectrophotometric detection of protein carbonylation. Secondly, other methods were found to be useful only in specific conditions. For example, an increase in the expression of OxyR was measured in Burkholderia cenocepacia K56-2 after treatment with ciprofloxacin or meropenem (both in biofilms and planktonic cultures but not after treatment with tobramycin. In addition results vary with the experimental conditions and the species tested. Nevertheless our data strongly suggest that ROS contribute to antibiotic mediated killing in Bcc species and that enhancing ROS production or interfering with the protection against ROS may form a novel strategy to improve antibiotic treatment.

  1. Isolation and Identification of Burkholderia glumae from Symptomless Rice Seeds

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A survey on isolation and detection of the casual organism of bacterial grain rot of rice was conducted during 1997-2006.In 2006,six pathogenic bacterial strains were isolated from two symptomiess seed samples of rice (Oryza sativa L.) originally produced in Hainan Province and then planted in Zhejiang Province,China.They were identified as Burkholderia glumae which is the causal organism of bacterial grain rot of rice by physiological characteristics,colony morphology,pathogenicity test,Biolog,fatty acid methyl ester (FAME) analysis and RAPD-PCR compared with the four standard reference strains.It is confirmed that there is the infection of B.glumae in so-called 'health looking seeds'.

  2. 40 CFR 725.1075 - Burkholderia cepacia complex.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Burkholderia cepacia complex. 725.1075... Specific Microorganisms § 725.1075 Burkholderia cepacia complex. (a) Microorganism and significant new uses subject to reporting. (1) The microorganisms identified as the Burkholderia cepacia complex defined...

  3. In vitro antibiotic susceptibilities of Burkholderia mallei (causative agent of glanders) determined by broth microdilution and E-test.

    Science.gov (United States)

    Heine, H S; England, M J; Waag, D M; Byrne, W R

    2001-07-01

    In vitro susceptibilities to 28 antibiotics were determined for 11 strains of Burkholderia mallei by the broth microdilution method. The B. mallei strains demonstrated susceptibility to aminoglycosides, macrolides, quinolones, doxycycline, piperacillin, ceftazidime, and imipenem. For comparison and evaluation, 17 antibiotic susceptibilities were also determined by the E-test. E-test values were always lower than the broth dilution values. Establishing and comparing antibiotic susceptibilities of specific B. mallei strains will provide reference information for assessing new antibiotic agents.

  4. Genetic diversity of Burkholderia (Proteobacteria) species from the Caatinga and Atlantic rainforest biomes in Bahia, Brazil.

    Science.gov (United States)

    Santini, A C; Santos, H R M; Gross, E; Corrêa, R X

    2013-03-11

    The genus Burkholderia (β-Proteobacteria) currently comprises more than 60 species, including parasites, symbionts and free-living organisms. Several new species of Burkholderia have recently been described showing a great diversity of phenotypes. We examined the diversity of Burkholderia spp in environmental samples collected from Caatinga and Atlantic rainforest biomes of Bahia, Brazil. Legume nodules were collected from five locations, and 16S rDNA and recA genes of the isolated microorganisms were analyzed. Thirty-three contigs of 16S rRNA genes and four contigs of the recA gene related to the genus Burkholderia were obtained. The genetic dissimilarity of the strains ranged from 0 to 2.5% based on 16S rDNA analysis, indicating two main branches: one distinct branch of the dendrogram for the B. cepacia complex and another branch that rendered three major groups, partially reflecting host plants and locations. A dendrogram designed with sequences of this research and those designed with sequences of Burkholderia-type strains and the first hit BLAST had similar topologies. A dendrogram similar to that constructed by analysis of 16S rDNA was obtained using sequences of the fragment of the recA gene. The 16S rDNA sequences enabled sufficient identification of relevant similarities and groupings amongst isolates and the sequences that we obtained. Only 6 of the 33 isolates analyzed via 16S rDNA sequencing showed high similarity with the B. cepacia complex. Thus, over 3/4 of the isolates have potential for biotechnological applications.

  5. Evaluation of a latex agglutination assay for the identification of Burkholderia pseudomallei and Burkholderia mallei.

    Science.gov (United States)

    Duval, Brea D; Elrod, Mindy G; Gee, Jay E; Chantratita, Narisara; Tandhavanant, Sarunporn; Limmathurotsakul, Direk; Hoffmaster, Alex R

    2014-06-01

    Cases of melioidosis and glanders are rare in the United States, but the etiologic agents of each disease (Burkholderia pseudomallei and Burkholderia mallei, respectively) are classified as Tier 1 select agents because of concerns about their potential use as bioterrorism agents. A rapid, highly sensitive, and portable assay for clinical laboratories and field use is required. Our laboratory has further evaluated a latex agglutination assay for its ability to identify B. pseudomallei and B. mallei isolates. This assay uses a monoclonal antibody that specifically recognizes the capsular polysaccharide produced by B. pseudomallei and B. mallei, but is absent in closely related Burkholderia species. A total of 110 B. pseudomallei and B. mallei were tested, and 36 closely related Burkholderia species. The latex agglutination assay was positive for 109 of 110 (99.1% sensitivity) B. pseudomallei and B. mallei isolates tested.

  6. Development of ceftazidime resistance in an acute Burkholderia pseudomallei infection

    Directory of Open Access Journals (Sweden)

    Sarovich DS

    2012-08-01

    Full Text Available Derek S Sarovich,1,2,* Erin P Price,1,2,* Direk Limmathurotsakul,3 James M Cook,1 Alex T Von Schulze,1 Spenser R Wolken,1 Paul Keim,1 Sharon J Peacock,3,4 Talima Pearson1 1Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ, USA; 2Tropical and Emerging Infectious Diseases Division, Menzies School of Health Research, Darwin, Australia; 3Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 4Department of Medicine, University of Cambridge, Cambridge, United Kingdom*These authors contributed equally to this workAbstract: Burkholderia pseudomallei, a bacterium that causes the disease melioidosis, is intrinsically resistant to many antibiotics. First-line antibiotic therapy for treating melioidosis is usually the synthetic β-lactam, ceftazidime (CAZ, as almost all B. pseudomallei strains are susceptible to this drug. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, which can lead to mortality if therapy is not switched to a different drug in a timely manner. Serial B. pseudomallei isolates obtained from an acute Thai melioidosis patient infected by a CAZ susceptible strain, who ultimately succumbed to infection despite being on CAZ therapy for the duration of their infection, were analyzed. Isolates that developed CAZ resistance due to a proline to serine change at position 167 in the β-lactamase PenA were identified. Importantly, these CAZ resistant isolates remained sensitive to the alternative melioidosis treatments; namely, amoxicillin-clavulanate, imipenem, and meropenem. Lastly, real-time polymerase chain reaction-based assays capable of rapidly identifying CAZ resistance in B. pseudomallei isolates at the position 167 mutation site were developed. The ability to rapidly identify the emergence of CAZ resistant B. pseudomallei populations in melioidosis patients will allow timely alterations in treatment strategies

  7. Burkholderia Vaccines: Are We Moving Forward?

    Directory of Open Access Journals (Sweden)

    Leang-Chung eChoh

    2013-02-01

    Full Text Available The genus Burkholderia consists of diverse species which includes both ‘friends’ and ‘foes’. Some of the ‘friendly’ Burkholderia spp. are extensively used in the biotechnological and agricultural industry for bioremediation and biocontrol. However, several members of the genus including B. pseudomallei, B. mallei and B. cepacia, are known to cause fatal disease in both humans and animals. B. pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively, while B. cepacia infection is lethal to cystic fibrosis patients. Due to the high rate of infectivity and intrinsic resistance to many commonly used antibiotics, together with high mortality rate, B. mallei and B. pseudomallei are considered to be potential biological warfare agents. Treatments of the infections caused by these bacteria are often unsuccessful with frequent relapse of the infection. Thus, we are at a crucial stage of the need for Burkholderia vaccines. Although the search for a prophylactic therapy candidate continues, to date development of vaccines has not advanced beyond research to human clinical trials. In this article, we review the current research on development of safe vaccines with high efficacy against B. pseudomallei, B. mallei and B. cepacia. It can be concluded that further research will enable elucidation of the potential benefits and risks of Burkholderia vaccines.

  8. Burkholderia vaccines: are we moving forward?

    Science.gov (United States)

    Choh, Leang-Chung; Ong, Guang-Han; Vellasamy, Kumutha M; Kalaiselvam, Kaveena; Kang, Wen-Tyng; Al-Maleki, Anis R; Mariappan, Vanitha; Vadivelu, Jamuna

    2013-01-01

    The genus Burkholderia consists of diverse species which includes both "friends" and "foes." Some of the "friendly" Burkholderia spp. are extensively used in the biotechnological and agricultural industry for bioremediation and biocontrol. However, several members of the genus including B. pseudomallei, B. mallei, and B. cepacia, are known to cause fatal disease in both humans and animals. B. pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively, while B. cepacia infection is lethal to cystic fibrosis (CF) patients. Due to the high rate of infectivity and intrinsic resistance to many commonly used antibiotics, together with high mortality rate, B. mallei and B. pseudomallei are considered to be potential biological warfare agents. Treatments of the infections caused by these bacteria are often unsuccessful with frequent relapse of the infection. Thus, we are at a crucial stage of the need for Burkholderia vaccines. Although the search for a prophylactic therapy candidate continues, to date development of vaccines has not advanced beyond research to human clinical trials. In this article, we review the current research on development of safe vaccines with high efficacy against B. pseudomallei, B. mallei, and B. cepacia. It can be concluded that further research will enable elucidation of the potential benefits and risks of Burkholderia vaccines.

  9. Volcanic Soils as Sources of Novel CO-Oxidizing Paraburkholderia and Burkholderia: Paraburkholderia hiiakae sp. nov., Paraburkholderia metrosideri sp. nov., Paraburkholderia paradisi sp. nov., Paraburkholderia peleae sp. nov., and Burkholderia alpina sp. nov. a Member of the Burkholderia cepacia Complex

    Science.gov (United States)

    Weber, Carolyn F.; King, Gary M.

    2017-01-01

    Previous studies showed that members of the Burkholderiales were important in the succession of aerobic, molybdenum-dependent CO oxidizing-bacteria on volcanic soils. During these studies, four isolates were obtained from Kilauea Volcano (Hawai‘i, USA); one strain was isolated from Pico de Orizaba (Mexico) during a separate study. Based on 16S rRNA gene sequence similarities, the Pico de Orizaba isolate and the isolates from Kilauea Volcano were provisionally assigned to the genera Burkholderia and Paraburkholderia, respectively. Each of the isolates possessed a form I coxL gene that encoded the catalytic subunit of carbon monoxide dehydrogenase (CODH); none of the most closely related type strains possessed coxL or oxidized CO. Genome sequences for Paraburkholderia type strains facilitated an analysis of 16S rRNA gene sequence similarities and average nucleotide identities (ANI). ANI did not exceed 95% (the recommended cutoff for species differentiation) for any of the pairwise comparisons among 27 reference strains related to the new isolates. However, since the highest 16S rRNA gene sequence similarity among this set of reference strains was 98.93%, DNA-DNA hybridizations (DDH) were performed for two isolates whose 16S rRNA gene sequence similarities with their nearest phylogenetic neighbors were 98.96 and 99.11%. In both cases DDH values were <16%. Based on multiple variables, four of the isolates represent novel species within the Paraburkholderia: Paraburkholderia hiiakae sp. nov. (type strain I2T = DSM 28029T = LMG 27952T); Paraburkholderia paradisi sp. nov. (type strain WAT = DSM 28027T = LMG 27949T); Paraburkholderia peleae sp. nov. (type strain PP52-1T = DSM 28028T = LMG 27950T); and Paraburkholderia metrosideri sp. nov. (type strain DNBP6-1T = DSM 28030T = LMG 28140T). The remaining isolate represents the first CO-oxidizing member of the Burkholderia cepacia complex: Burkholderia alpina sp. nov. (type strain PO-04-17-38T = DSM 28031T = LMG 28138T

  10. Workshop on treatment of and postexposure prophylaxis for Burkholderia pseudomallei and B. mallei Infection, 2010.

    Science.gov (United States)

    Lipsitz, Rebecca; Garges, Susan; Aurigemma, Rosemarie; Baccam, Prasith; Blaney, David D; Cheng, Allen C; Currie, Bart J; Dance, David; Gee, Jay E; Larsen, Joseph; Limmathurotsakul, Direk; Morrow, Meredith G; Norton, Robert; O'Mara, Elizabeth; Peacock, Sharon J; Pesik, Nicki; Rogers, L Paige; Schweizer, Herbert P; Steinmetz, Ivo; Tan, Gladys; Tan, Patrick; Wiersinga, W Joost; Wuthiekanun, Vanaporn; Smith, Theresa L

    2012-12-01

    The US Public Health Emergency Medical Countermeasures Enterprise convened subject matter experts at the 2010 HHS Burkholderia Workshop to develop consensus recommendations for postexposure prophylaxis against and treatment for Burkholderia pseudomallei and B. mallei infections, which cause melioidosis and glanders, respectively. Drugs recommended by consensus of the participants are ceftazidime or meropenem for initial intensive therapy, and trimethoprim/sulfamethoxazole or amoxicillin/clavulanic acid for eradication therapy. For postexposure prophylaxis, recommended drugs are trimethoprim/sulfamethoxazole or co-amoxiclav. To improve the timely diagnosis of melioidosis and glanders, further development and wide distribution of rapid diagnostic assays were also recommended. Standardized animal models and B. pseudomallei strains are needed for further development of therapeutic options. Training for laboratory technicians and physicians would facilitate better diagnosis and treatment options.

  11. Burkholderia glumae en el cultivo de arroz en Costa Rica.

    OpenAIRE

    Andrea Quesada-Gonz\\u00E1lez; Fernando Garc\\u00EDa-Santamar\\u00EDa

    2014-01-01

    Burkholderia glumae en el cultivo de arroz en Costa Rica. El objetivo de este trabajo fue determinar la presencia de Burkholderia glumae en arroz en Costa Rica. La bacteria Burkholderia glumae está asociada al cultivo del arroz en el que provoca la enfermedad llamada añublo bacterial. Bajo condiciones ambientales favorables, la densidad bacteriana aumenta, lo que provoca que, bajo un sistema de regulación denominado quorum sensing, se expresen sus mecanismos de virulencia mediante la ...

  12. Symbiotic factors in Burkholderia essential for establishing an association with the bean bug, Riptortus pedestris.

    Science.gov (United States)

    Kim, Jiyeun Kate; Lee, Bok Luel

    2015-01-01

    Symbiotic bacteria are common in insects and intimately affect the various aspects of insect host biology. In a number of insect symbiosis models, it has been possible to elucidate the effects of the symbiont on host biology, whereas there is a limited understanding of the impact of the association on the bacterial symbiont, mainly due to the difficulty of cultivating insect symbionts in vitro. Furthermore, the molecular features that determine the establishment and persistence of the symbionts in their host (i.e., symbiotic factors) have remained elusive. However, the recently established model, the bean bug Riptortus pedestris, provides a good opportunity to study bacterial symbiotic factors at a molecular level through their cultivable symbionts. Bean bugs acquire genus Burkholderia cells from the environment and harbor them as gut symbionts in the specialized posterior midgut. The genome of the Burkholderia symbiont was sequenced, and the genomic information was used to generate genetically manipulated Burkholderia symbiont strains. Using mutant symbionts, we identified several novel symbiotic factors necessary for establishing a successful association with the host gut. In this review, these symbiotic factors are classified into three categories based on the colonization dynamics of the mutant symbiont strains: initiation, accommodation, and persistence factors. In addition, the molecular characteristics of the symbiotic factors are described. These newly identified symbiotic factors and on-going studies of the Riptortus-Burkholderia symbiosis are expected to contribute to the understanding of the molecular cross-talk between insects and bacterial symbionts that are of ecological and evolutionary importance. © 2014 Wiley Periodicals, Inc.

  13. Autotransporters and their role in the virulence of Burkholderia pseudomallei and Burkholderia mallei.

    Directory of Open Access Journals (Sweden)

    Natalie eLazar Adler

    2011-07-01

    Full Text Available Burkholderia pseudomallei and Burkholderia mallei are closely related Gram-negative bacteria responsible for the infectious diseases melioidosis and glanders, respectively. Autotransporters (ATs comprise a large and diverse family of secreted and outer membrane proteins that includes virulence-associated invasins, adhesins, proteases and actin-nucleating factors. The B. pseudomallei K96243 genome contains eleven predicted ATs, eight of which share homologues in the B. mallei ATCC 23344 genome. This review distils key findings from in silico, in vitro and in vivo studies on the ATs of B. pseudomallei and B. mallei. To date, the best characterized of the predicted ATs of B. pseudomallei and B. mallei is BimA, a predicted trimeric AT mediating actin-based motility which varies in sequence and mode of action between Burkholderia species. Of the remaining eight predicted B. pseudomallei trimeric autotransporters, five of which are also present in B. mallei, two (BoaA and BoaB, have been implicated in bacterial adhesion to epithelial cells. Several predicted Burkholderia ATs are recognized by human humoral and cell-mediated immunity, indicating that they are expressed during infection and may be useful for diagnosis and vaccine-mediated protection. Further studies on the mode of secretion and functions of Burkholderia ATs will facilitate the rational design of control strategies.

  14. Use of the common marmoset to study Burkholderia mallei infection.

    Directory of Open Access Journals (Sweden)

    Tomislav Jelesijevic

    Full Text Available Burkholderia mallei is a host-adapted bacterium that does not persist outside of its equine reservoir. The organism causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by B. mallei typically occurs via the respiratory or percutaneous route, and the most common manifestations are life-threatening pneumonia and bacteremia. Glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. There is no vaccine to protect against B. mallei and there is concern regarding its use as a biothreat agent. Thus, experiments were performed to establish a non-human primate model of intranasal infection to study the organism and develop countermeasures. Groups of marmosets (Callithrix jacchus were inoculated intranasally with B. mallei strain ATCC 23344 and monitored for clinical signs of illness for up to 13 days. We discovered that 83% of marmosets inoculated with doses of 2.5 X 10(4 to 2.5 X 10(5 bacteria developed acute lethal infection within 3-4 days. Signs of disease were severe and included lethargy, inappetence, conjunctivitis, mucopurulent and hemorrhagic nasal discharges, and increased respiratory effort with abdominal lifts. Burkholderia mallei was cultured from the lungs, spleen and liver of these animals, and pathologic examination of tissues revealed lesions characteristic of glanders. Challenge experiments also revealed that 91% of animals infected with doses ranging from 25 to 2.5 X 10(3 bacteria exhibited mild non-specific signs of illness and were culture negative. One marmoset inoculated with 2.5 X 10(3 organisms developed moderate signs of disease and reached humane end-points 8 days post-infection. The liver and spleen of this animal were colonized with the agent and pathological analysis of tissues showed nasal, splenic and hepatic lesions. Taken together, these data indicate that the marmoset is a suitable model to study respiratory infection by B

  15. Use of the common marmoset to study Burkholderia mallei infection.

    Science.gov (United States)

    Jelesijevic, Tomislav; Zimmerman, Shawn M; Harvey, Stephen B; Mead, Daniel G; Shaffer, Teresa L; Estes, D Mark; Michel, Frank; Quinn, Frederick D; Hogan, Robert J; Lafontaine, Eric R

    2015-01-01

    Burkholderia mallei is a host-adapted bacterium that does not persist outside of its equine reservoir. The organism causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by B. mallei typically occurs via the respiratory or percutaneous route, and the most common manifestations are life-threatening pneumonia and bacteremia. Glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. There is no vaccine to protect against B. mallei and there is concern regarding its use as a biothreat agent. Thus, experiments were performed to establish a non-human primate model of intranasal infection to study the organism and develop countermeasures. Groups of marmosets (Callithrix jacchus) were inoculated intranasally with B. mallei strain ATCC 23344 and monitored for clinical signs of illness for up to 13 days. We discovered that 83% of marmosets inoculated with doses of 2.5 X 10(4) to 2.5 X 10(5) bacteria developed acute lethal infection within 3-4 days. Signs of disease were severe and included lethargy, inappetence, conjunctivitis, mucopurulent and hemorrhagic nasal discharges, and increased respiratory effort with abdominal lifts. Burkholderia mallei was cultured from the lungs, spleen and liver of these animals, and pathologic examination of tissues revealed lesions characteristic of glanders. Challenge experiments also revealed that 91% of animals infected with doses ranging from 25 to 2.5 X 10(3) bacteria exhibited mild non-specific signs of illness and were culture negative. One marmoset inoculated with 2.5 X 10(3) organisms developed moderate signs of disease and reached humane end-points 8 days post-infection. The liver and spleen of this animal were colonized with the agent and pathological analysis of tissues showed nasal, splenic and hepatic lesions. Taken together, these data indicate that the marmoset is a suitable model to study respiratory infection by B. mallei.

  16. Burkholderia contaminans Biofilm Regulating Operon and Its Distribution in Bacterial Genomes

    Science.gov (United States)

    Semenov, Andrey N.; Gintsburg, Alexandr L.

    2016-01-01

    Biofilm formation by Burkholderia spp. is a principal cause of lung chronic infections in cystic fibrosis patients. A “lacking biofilm production” (LBP) strain B. contaminans GIMC4587:Bct370-19 has been obtained by insertion modification of clinical strain with plasposon mutagenesis. It has an interrupted transcriptional response regulator (RR) gene. The focus of our investigation was a two-component signal transduction system determination, including this RR. B. contaminans clinical and LBP strains were analyzed by whole genome sequencing and bioinformatics resources. A four-component operon (BiofilmReg) has a key role in biofilm formation. The relative location (i.e., by being separated by another gene) of RR and histidine kinase genes is unique in BiofilmReg. Orthologs were found in other members of the Burkholderiales order. Phylogenetic analysis of strains containing BiofilmReg operons demonstrated evidence for earlier inheritance of a three-component operon. During further evolution one lineage acquired a fourth gene, whereas others lost the third component of the operon. Mutations in sensor domains have created biodiversity which is advantageous for adaptation to various ecological niches. Different species Burkholderia and Achromobacter strains all demonstrated similar BiofilmReg operon structure. Therefore, there may be an opportunity to develop a common drug which is effective for treating all these causative agents. PMID:28070515

  17. Burkholderia contaminans Biofilm Regulating Operon and Its Distribution in Bacterial Genomes

    Directory of Open Access Journals (Sweden)

    Olga L. Voronina

    2016-01-01

    Full Text Available Biofilm formation by Burkholderia spp. is a principal cause of lung chronic infections in cystic fibrosis patients. A “lacking biofilm production” (LBP strain B. contaminans GIMC4587:Bct370-19 has been obtained by insertion modification of clinical strain with plasposon mutagenesis. It has an interrupted transcriptional response regulator (RR gene. The focus of our investigation was a two-component signal transduction system determination, including this RR. B. contaminans clinical and LBP strains were analyzed by whole genome sequencing and bioinformatics resources. A four-component operon (BiofilmReg has a key role in biofilm formation. The relative location (i.e., by being separated by another gene of RR and histidine kinase genes is unique in BiofilmReg. Orthologs were found in other members of the Burkholderiales order. Phylogenetic analysis of strains containing BiofilmReg operons demonstrated evidence for earlier inheritance of a three-component operon. During further evolution one lineage acquired a fourth gene, whereas others lost the third component of the operon. Mutations in sensor domains have created biodiversity which is advantageous for adaptation to various ecological niches. Different species Burkholderia and Achromobacter strains all demonstrated similar BiofilmReg operon structure. Therefore, there may be an opportunity to develop a common drug which is effective for treating all these causative agents.

  18. Burkholderia contaminans Biofilm Regulating Operon and Its Distribution in Bacterial Genomes.

    Science.gov (United States)

    Voronina, Olga L; Kunda, Marina S; Ryzhova, Natalia N; Aksenova, Ekaterina I; Semenov, Andrey N; Romanova, Yulia M; Gintsburg, Alexandr L

    2016-01-01

    Biofilm formation by Burkholderia spp. is a principal cause of lung chronic infections in cystic fibrosis patients. A "lacking biofilm production" (LBP) strain B. contaminans GIMC4587:Bct370-19 has been obtained by insertion modification of clinical strain with plasposon mutagenesis. It has an interrupted transcriptional response regulator (RR) gene. The focus of our investigation was a two-component signal transduction system determination, including this RR. B. contaminans clinical and LBP strains were analyzed by whole genome sequencing and bioinformatics resources. A four-component operon (BiofilmReg) has a key role in biofilm formation. The relative location (i.e., by being separated by another gene) of RR and histidine kinase genes is unique in BiofilmReg. Orthologs were found in other members of the Burkholderiales order. Phylogenetic analysis of strains containing BiofilmReg operons demonstrated evidence for earlier inheritance of a three-component operon. During further evolution one lineage acquired a fourth gene, whereas others lost the third component of the operon. Mutations in sensor domains have created biodiversity which is advantageous for adaptation to various ecological niches. Different species Burkholderia and Achromobacter strains all demonstrated similar BiofilmReg operon structure. Therefore, there may be an opportunity to develop a common drug which is effective for treating all these causative agents.

  19. Development of Burkholderia mallei and pseudomallei vaccines

    Directory of Open Access Journals (Sweden)

    Ediane Batista Silva

    2013-03-01

    Full Text Available B. mallei and B. pseudomallei are Gram-negative bacteria that cause glanders and melioidosis, respectively. Inhalational infection with either organism can result in severe and rapidly fatal pneumonia. Inoculation by the oral and cutaneous routes can also produce infection. chronic infection develops after recovery from acute infection with both agents, and control of infection with antibiotics requires prolonged treatment. Symptoms for both meliodosis and glanders are non-specific, making diagnosis difficult.B. pseudomallei can be located in the environment, but in the host, B. mallei and B. psedomallei are intracellular organisms. Thefection results in similar immune responses to both agents. Effective early innate immune responses are critical to controlling the early phase of the infection. Innate immune signaling molecules such as TLR, NOD, MyD88 and pro-inflammatory cytokines such as IFN- and TNF-α play key roles in regulating control of infection. Neutrophils and monocytes are critical cells in the early infection for these microorganisms. Both monocytes and macrophages are necessary for limiting dissemination of B. pseudomallei. In contrast, the role of adaptive immune responses in controlling Burkholderia infection is less well understood. However, T cell responses are critical for vaccine protection from Burkholderia infection. At present, effective vaccines for prevention of glanders or meliodosis have not been developed, although recently progress of Burkholderia vaccines has received renewed attention.This review will summarize current and past approaches to develop Burkholderia mallei and pseudomalllei vaccines, with emphasis on immune mechanisms of protection and the challenges facing the field. At present, immunization with live attenuated bacteria provides the most effective and durable immunity, and it is important therefore to understand the immune correlates of protection induced by live attenuated vaccines.

  20. Survival of Burkholderia pseudomallei on Environmental Surfaces.

    Science.gov (United States)

    Shams, Alicia M; Rose, Laura J; Hodges, Lisa; Arduino, Matthew J

    2007-12-01

    The survival of the biothreat agent Burkholderia pseudomallei on the surfaces of four materials was measured by culture and esterase activity analyses. The culture results demonstrated that this organism persisted for <24 h to <7 days depending on the material, bacterial isolate, and suspension medium. The persistence determined by analysis of esterase activity, as measured with a ScanRDI solid-phase cytometer, was always longer than the persistence determined by culture analysis.

  1. Burkholderia mallei and Burkholderia pseudomallei: the causative micro-organisms of glanders and melioidosis.

    Science.gov (United States)

    Gilad, Jacob

    2007-11-01

    Burkholderia mallei and Burkholderia pseudomallei are the causative micro-organisms of Glanders and Melioidosis, respectively. Although now rare in Western countries, both micro-organisms have recently gained much interest because of their unique potential as bioterrorism agents. This paper reviews the epidemiology, pathogenesis, diagnosis and treatment of Melioidosis and Glanders. Recent patents relating to these micro-organisms, especially potential vaccines, are presented. Continued research and development is urgently needed, especially in regard to rapid and accurate diagnosis of melioidosis and glanders, efficacious therapy and primary and secondary prevention.

  2. Detection of Burkholderia pseudomallei O-antigen serotypes in near-neighbor species

    Directory of Open Access Journals (Sweden)

    Stone Joshua K

    2012-11-01

    Full Text Available Abstract Background Burkholderia pseudomallei is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS as a potential vaccine target because it is known as one of the most important antigenic epitopes in B. pseudomallei. Complicating this strategy are the four different B. pseudomallei LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of Burkholderia species. Here, we identified the presence of multiple B. pseudomallei O-antigen types and sero-crossreactivity in its near-neighbor species. Results PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of B. mallei and B. thailandensis strains contained the typical O-antigen type A. In contrast, most of B. ubonensis and B. thailandensis-like strains expressed the atypical O-antigen types B and B2, respectively. Most B. oklahomensis strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in B. thailandensis 82172, B. ubonensis MSMB108, and Burkholderia sp. MSMB175. Interestingly, B. thailandensis-like MSMB43 contained a novel serotype B positive O-antigen. Conclusions This study expands the number of species which express B. pseudomallei O-antigen types. Further work is required to elucidate the full structures and how closely these are to the B. pseudomallei O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development.

  3. Development of Burkholderia mallei and pseudomallei vaccines.

    Science.gov (United States)

    Silva, Ediane B; Dow, Steven W

    2013-01-01

    Burkholderia mallei and Burkholderia pseudomallei are Gram-negative bacteria that cause glanders and melioidosis, respectively. Inhalational infection with either organism can result in severe and rapidly fatal pneumonia. Inoculation by the oral and cutaneous routes can also produce infection. Chronic infection may develop after recovery from acute infection with both agents, and control of infection with antibiotics requires prolonged treatment. Symptoms for both meliodosis and glanders are non-specific, making diagnosis difficult. B. pseudomallei can be located in the environment, but in the host, B. mallei and B. psedomallei are intracellular organisms, and infection results in similar immune responses to both agents. Effective early innate immune responses are critical to controlling the early phase of the infection. Innate immune signaling molecules such as TLR, NOD, MyD88, and pro-inflammatory cytokines such as IFN-γ and TNF-α play key roles in regulating control of infection. Neutrophils and monocytes are critical cells in the early infection for both microorganisms. Both monocytes and macrophages are necessary for limiting dissemination of B. pseudomallei. In contrast, the role of adaptive immune responses in controlling Burkholderia infection is less well understood. However, T cell responses are critical for vaccine protection from Burkholderia infection. At present, effective vaccines for prevention of glanders or meliodosis have not been developed, although recently development of Burkholderia vaccines has received renewed attention. This review will summarize current and past approaches to develop B. mallei and B. pseudomalllei vaccines, with emphasis on immune mechanisms of protection and the challenges facing the field. At present, immunization with live attenuated bacteria provides the most effective and durable immunity, and it is important therefore to understand the immune correlates of protection induced by live attenuated vaccines. Subunit

  4. NCBI nr-aa BLAST: CBRC-BTAU-01-3033 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-3033 ref|ZP_00977438.1| COG2207: AraC-type DNA-binding domain-containing proteins [Burkholder...ia cenocepacia PC184] ref|YP_623398.1| transcriptional regulator, AraC family [Burkholder...ia cenocepacia AU 1054] ref|YP_838462.1| transcriptional regulator, AraC family [Burkholderia cenocepaci...a HI2424] gb|ABF78425.1| transcriptional regulator, AraC family [Burkholderia cen...ocepacia AU 1054] gb|ABK11569.1| transcriptional regulator, AraC family [Burkholderia cenocepacia HI2424] gb

  5. Mining host-pathogen protein interactions to characterize Burkholderia mallei infectivity mechanisms.

    Directory of Open Access Journals (Sweden)

    Vesna Memišević

    2015-03-01

    Full Text Available Burkholderia pathogenicity relies on protein virulence factors to control and promote bacterial internalization, survival, and replication within eukaryotic host cells. We recently used yeast two-hybrid (Y2H screening to identify a small set of novel Burkholderia proteins that were shown to attenuate disease progression in an aerosol infection animal model using the virulent Burkholderia mallei ATCC 23344 strain. Here, we performed an extended analysis of primarily nine B. mallei virulence factors and their interactions with human proteins to map out how the bacteria can influence and alter host processes and pathways. Specifically, we employed topological analyses to assess the connectivity patterns of targeted host proteins, identify modules of pathogen-interacting host proteins linked to processes promoting infectivity, and evaluate the effect of crosstalk among the identified host protein modules. Overall, our analysis showed that the targeted host proteins generally had a large number of interacting partners and interacted with other host proteins that were also targeted by B. mallei proteins. We also introduced a novel Host-Pathogen Interaction Alignment (HPIA algorithm and used it to explore similarities between host-pathogen interactions of B. mallei, Yersinia pestis, and Salmonella enterica. We inferred putative roles of B. mallei proteins based on the roles of their aligned Y. pestis and S. enterica partners and showed that up to 73% of the predicted roles matched existing annotations. A key insight into Burkholderia pathogenicity derived from these analyses of Y2H host-pathogen interactions is the identification of eukaryotic-specific targeted cellular mechanisms, including the ubiquitination degradation system and the use of the focal adhesion pathway as a fulcrum for transmitting mechanical forces and regulatory signals. This provides the mechanisms to modulate and adapt the host-cell environment for the successful establishment of

  6. Mining Host-Pathogen Protein Interactions to Characterize Burkholderia mallei Infectivity Mechanisms

    Science.gov (United States)

    Memišević, Vesna; Zavaljevski, Nela; Rajagopala, Seesandra V.; Kwon, Keehwan; Pieper, Rembert; DeShazer, David; Reifman, Jaques; Wallqvist, Anders

    2015-01-01

    Burkholderia pathogenicity relies on protein virulence factors to control and promote bacterial internalization, survival, and replication within eukaryotic host cells. We recently used yeast two-hybrid (Y2H) screening to identify a small set of novel Burkholderia proteins that were shown to attenuate disease progression in an aerosol infection animal model using the virulent Burkholderia mallei ATCC 23344 strain. Here, we performed an extended analysis of primarily nine B. mallei virulence factors and their interactions with human proteins to map out how the bacteria can influence and alter host processes and pathways. Specifically, we employed topological analyses to assess the connectivity patterns of targeted host proteins, identify modules of pathogen-interacting host proteins linked to processes promoting infectivity, and evaluate the effect of crosstalk among the identified host protein modules. Overall, our analysis showed that the targeted host proteins generally had a large number of interacting partners and interacted with other host proteins that were also targeted by B. mallei proteins. We also introduced a novel Host-Pathogen Interaction Alignment (HPIA) algorithm and used it to explore similarities between host-pathogen interactions of B. mallei, Yersinia pestis, and Salmonella enterica. We inferred putative roles of B. mallei proteins based on the roles of their aligned Y. pestis and S. enterica partners and showed that up to 73% of the predicted roles matched existing annotations. A key insight into Burkholderia pathogenicity derived from these analyses of Y2H host-pathogen interactions is the identification of eukaryotic-specific targeted cellular mechanisms, including the ubiquitination degradation system and the use of the focal adhesion pathway as a fulcrum for transmitting mechanical forces and regulatory signals. This provides the mechanisms to modulate and adapt the host-cell environment for the successful establishment of host infections

  7. Burkholderia kirstenboschensis sp. nov. nodulates papilionoid legumes indigenous to South Africa.

    Science.gov (United States)

    Steenkamp, Emma T; van Zyl, Elritha; Beukes, Chrizelle W; Avontuur, Juanita R; Chan, Wai Yin; Palmer, Marike; Mthombeni, Lunghile S; Phalane, Francina L; Sereme, T Karabo; Venter, Stephanus N

    2015-12-01

    Despite the diversity of Burkholderia species known to nodulate legumes in introduced and native regions, relatively few taxa have been formally described. For example, the Cape Floristic Region of South Africa is thought to represent one of the major centres of diversity for the rhizobial members of Burkholderia, yet only five species have been described from legumes occurring in this region and numerous are still awaiting taxonomic treatment. Here, we investigated the taxonomic status of 12 South African root-nodulating Burkholderia isolates from native papilionoid legumes (Hypocalyptus coluteoides, H. oxalidifolius, H. sophoroides and Virgilia oroboides). Analysis of four gene regions (16S rRNA, recA, atpD and rpoB) revealed that the isolates represent a genealogically unique and exclusive assemblage within the genus. Its distinctness was supported by all other aspects of the polyphasic approach utilized, including the genome-based criteria DNA-DNA hybridization (≥70.9%) and average nucleotide identities (≥96%). We accordingly propose the name B. kirstenboschensis sp. nov. for this taxon with isolate Kb15(T) (=LMG 28727(T); =SARC 695(T)) as its type strain. Our data showed that intraspecific genome size differences (≥0.81 Mb) and the occurrence of large DNA regions that are apparently unique to single individuals (16-23% of an isolate's genome) can significantly limit the value of data obtained from DNA-DNA hybridization experiments. Substitution of DNA-DNA hybridization with whole genome sequencing as a prerequisite for the description of Burkholderia species will undoubtedly speed up the pace at which their diversity are documented, especially in hyperdiverse regions such as the Cape Floristic Region.

  8. Intrinsic Resistance of Burkholderia cepacia Complex to Benzalkonium Chloride.

    Science.gov (United States)

    Ahn, Youngbeom; Kim, Jeong Myeong; Kweon, Ohgew; Kim, Seong-Jae; Jones, Richard C; Woodling, Kellie; Gamboa da Costa, Gonçalo; LiPuma, John J; Hussong, David; Marasa, Bernard S; Cerniglia, Carl E

    2016-11-22

    Pharmaceutical products that are contaminated with Burkholderia cepacia complex (BCC) bacteria may pose serious consequences to vulnerable patients. Benzyldimethylalkylammonium chloride (BZK) cationic surfactants are extensively used in medical applications and have been implicated in the coselection of antimicrobial resistance. The ability of BCC to degrade BZK, tetradecyldimethylbenzylammonium chloride (C14BDMA-Cl), dodecyldimethylbenzylammonium chloride (C12BDMA-Cl), decyldimethylbenzylammonium chloride (C10BDMA-Cl), hexyldimethylbenzylammonium chloride, and benzyltrimethylammonium chloride was determined by incubation in 1/10-diluted tryptic soy broth (TSB) to determine if BCC bacteria have the ability to survive and inactivate these disinfectants. With BZK, C14BDMA-Cl, and C12BDMA-Cl, inhibition of the growth of 20 BCC strains was observed in disinfectant solutions that ranged from 64 to 256 µg/ml. The efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone increased the sensitivity of bacteria to 64 µg/ml BZK. The 20 BCC strains grew well in 1/10-diluted TSB medium with BZK, C12BDMA-Cl, and C10BDMA-Cl; they absorbed and degraded the compounds in 7 days. Formation of benzyldimethylamine and benzylmethylamine as the initial metabolites suggested that the cleavage of the C alkyl-N bond occurred as the first step of BZK degradation by BCC bacteria. Proteomic data confirmed the observed efflux activity and metabolic inactivation via biodegradation in terms of BZK resistance of BCC bacteria, which suggests that the two main resistance mechanisms are intrinsic and widespread.

  9. Polymorphisms within the prnD and pltC genes from pyrrolnitrin and pyoluteorin-producing Pseudomonas and Burkholderia spp

    NARCIS (Netherlands)

    Souza, J.T.; Raaijmakers, J.M.

    2003-01-01

    Pyrrolnitrin (PRN) and pyoluteorin (PLT) are broad-spectrum antibiotics produced by several strains of Pseudomonas and Burkholderia species. Both antibiotics play an important role in the suppression of multiple plant pathogenic fungi. Primers were developed from conserved sequences and amplified pr

  10. Competition Experiments for Legume Infection Identify Burkholderia phymatum as a Highly Competitive β-Rhizobium

    Science.gov (United States)

    Lardi, Martina; de Campos, Samanta Bolzan; Purtschert, Gabriela; Eberl, Leo; Pessi, Gabriella

    2017-01-01

    Members of the genus Burkholderia (β-proteobacteria) have only recently been shown to be able to establish a nitrogen-fixing symbiosis with several legumes, which is why they are also referred to as β-rhizobia. Therefore, very little is known about the competitiveness of these species to nodulate different legume host plants. In this study, we tested the competitiveness of several Burkholderia type strains (B. diazotrophica, B. mimosarum, B. phymatum, B. sabiae, B. symbiotica and B. tuberum) to nodulate four legumes (Phaseolus vulgaris, Macroptilium atropurpureum, Vigna unguiculata and Mimosa pudica) under our closely defined growth conditions. The assessment of nodule occupancy of these species on different legume host plants revealed that B. phymatum was the most competitive strain in the three papilionoid legumes (bean, cowpea and siratro), while B. mimosarum outcompeted the other strains in mimosa. The analysis of phenotypes known to play a role in nodulation competitiveness (motility, exopolysaccharide production) and additional in vitro competition assays among β-rhizobial strains suggested that B. phymatum has the potential to be a very competitive legume symbiont. PMID:28861050

  11. Competition Experiments for Legume Infection Identify Burkholderia phymatum as a Highly Competitive β-Rhizobium

    Directory of Open Access Journals (Sweden)

    Martina Lardi

    2017-08-01

    Full Text Available Members of the genus Burkholderia (β-proteobacteria have only recently been shown to be able to establish a nitrogen-fixing symbiosis with several legumes, which is why they are also referred to as β-rhizobia. Therefore, very little is known about the competitiveness of these species to nodulate different legume host plants. In this study, we tested the competitiveness of several Burkholderia type strains (B. diazotrophica, B. mimosarum, B. phymatum, B. sabiae, B. symbiotica and B. tuberum to nodulate four legumes (Phaseolus vulgaris, Macroptilium atropurpureum, Vigna unguiculata and Mimosa pudica under our closely defined growth conditions. The assessment of nodule occupancy of these species on different legume host plants revealed that B. phymatum was the most competitive strain in the three papilionoid legumes (bean, cowpea and siratro, while B. mimosarum outcompeted the other strains in mimosa. The analysis of phenotypes known to play a role in nodulation competitiveness (motility, exopolysaccharide production and additional in vitro competition assays among β-rhizobial strains suggested that B. phymatum has the potential to be a very competitive legume symbiont.

  12. Identification of volatile compounds produced by the bacterium Burkholderia tropica that inhibit the growth of fungal pathogens

    Science.gov (United States)

    Tenorio-Salgado, Silvia; Tinoco, Raunel; Vazquez-Duhalt, Rafael; Caballero-Mellado, Jesus; Perez-Rueda, Ernesto

    2013-01-01

    It has been documented that bacteria from the Burkholderia genera produce different kinds of compounds that inhibit plant pathogens, however in Burkholderia tropica, an endophytic diazotrophic and phosphate-solubilizing bacterium isolated from a wide diversity of plants, the capacity to produce antifungal compounds has not been evaluated. In order to expand our knowledge about Burkholderia tropica as a potential biological control agent, we analyzed 15 different strains of this bacterium to evaluate their capacities to inhibit the growth of four phytopathogenic fungi, Colletotrichum gloeosporioides, Fusarium culmorum, Fusarium oxysporum and Sclerotium rolffsi. Diverse analytical techniques, including plant root protection and dish plate growth assays and gas chromatography-mass spectroscopy showed that the fungal growth inhibition was intimately associated with the volatile compounds produced by B. tropica and, in particular, two bacterial strains (MTo293 and TTe203) exhibited the highest radial mycelial growth inhibition. Morphological changes associated with these compounds, such as disruption of fungal hyphae, were identified by using photomicrographic analysis. By using gas chromatography-mass spectroscopy technique, 18 volatile compounds involved in the growth inhibition mechanism were identified, including α-pinene and limonene. In addition, we found a high proportion of bacterial strains that produced siderophores during growth with different carbon sources, such as alanine and glutamic acid; however, their roles in the antagonism mechanism remain unclear. PMID:23680857

  13. Solubilization of insoluble inorganic phosphate by Burkholderia cepacia DA23 isolated from cultivated soil Solubilização de fosfato inorgânico insolúvel por Burkholderia cepacea DA23 isolada de solo cultivado

    Directory of Open Access Journals (Sweden)

    Ok-Ryul Song

    2008-03-01

    Full Text Available A mineral phosphate solubilizing bacterium, Burkholderia cepacia DA23 has been isolated from cultivated soils. Phosphate-solubilizing activities of the strain against three types of insoluble phosphate were quantitatively determined. When 3% of glucose concentration was used for carbon source, the strain had a marked mineral phosphate-solubilizing activity. Mineral phosphate solubilization was directly related to the pH drop by the strain. Analysis of the culture medium by high pressure liquid chromatography identified gluconic acid as the main organic acid released by Burkholderia cepacia DA23. Gluconic acid production was apparently the result of the glucose dehydrogenase activity and glucose dehydrogenase was affected by phosphate regulation.Uma bactéria capaz de solubilizar fosfato mineral, Burkholderia cepacea DA23, foi isolada de solo cultivado. A capacidade dessa bactéria solubilizar o fosfato de três tipos de fosfato insolúvel foi quantificada. Quando foi utilizada glicose a 3% como fonte de carbono, a bactéria apresentou uma intensa atividade solubilizante de fosfato, sendo a solubilização diretamente relacionada com a queda de pH causada pela bactéria. A análise do meio de cultura por cromatografia líquida de alta pressão indicou o ácido glicônico como principal ácido produzido por Burkholderia cepacea DA23. Aparentemente, a produção de ácido glicônico foi causada pela atividade da glicose desidrogenase. A enzima foi afetada pela regulação do fosfato.

  14. Use of a Safe, Reproducible, and Rapid Aerosol Delivery Method to Study Infection by Burkholderia pseudomallei and Burkholderia mallei in Mice

    Science.gov (United States)

    Lafontaine, Eric R.; Zimmerman, Shawn M.; Shaffer, Teresa L.; Michel, Frank; Gao, Xiudan; Hogan, Robert J.

    2013-01-01

    Burkholderia pseudomallei, the etiologic agent of melioidosis, is a saprophytic bacterium readily isolated from wet soils of countries bordering the equator. Burkholderia mallei is a host-adapted clone of B. pseudomallei that does not persist outside of its equine reservoir and causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by these organisms typically occurs via percutaneous inoculation or inhalation of aerosols, and the most common manifestation is severe pneumonia leading to fatal bacteremia. Glanders and melioidosis are difficult to diagnose and require prolonged antibiotic therapy with low success rates. There are no vaccines available to protect against either Burkholderia species, and there is concern regarding their use as biological warfare agents given that B. mallei has previously been utilized in this manner. Hence, experiments were performed to establish a mouse model of aerosol infection to study the organisms and develop countermeasures. Using a hand-held aerosolizer, BALB/c mice were inoculated intratracheally with strains B. pseudomallei 1026b and B. mallei ATCC23344 and growth of the agents in the lungs, as well as dissemination to the spleen, were examined. Mice infected with 102, 103 and 104 organisms were unable to control growth of B. mallei in the lungs and bacteria rapidly disseminated to the spleen. Though similar results were observed in mice inoculated with 103 and 104 B. pseudomallei cells, animals infected with 102 organisms controlled bacterial replication in the lungs, dissemination to the spleen, and the extent of bacteremia. Analysis of sera from mice surviving acute infection revealed that animals produced antibodies against antigens known to be targets of the immune response in humans. Taken together, these data show that small volume aerosol inoculation of mice results in acute disease, dose-dependent chronic infection, and immune responses that correlate with those

  15. Use of a safe, reproducible, and rapid aerosol delivery method to study infection by Burkholderia pseudomallei and Burkholderia mallei in mice.

    Directory of Open Access Journals (Sweden)

    Eric R Lafontaine

    Full Text Available Burkholderia pseudomallei, the etiologic agent of melioidosis, is a saprophytic bacterium readily isolated from wet soils of countries bordering the equator. Burkholderia mallei is a host-adapted clone of B. pseudomallei that does not persist outside of its equine reservoir and causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by these organisms typically occurs via percutaneous inoculation or inhalation of aerosols, and the most common manifestation is severe pneumonia leading to fatal bacteremia. Glanders and melioidosis are difficult to diagnose and require prolonged antibiotic therapy with low success rates. There are no vaccines available to protect against either Burkholderia species, and there is concern regarding their use as biological warfare agents given that B. mallei has previously been utilized in this manner. Hence, experiments were performed to establish a mouse model of aerosol infection to study the organisms and develop countermeasures. Using a hand-held aerosolizer, BALB/c mice were inoculated intratracheally with strains B. pseudomallei 1026b and B. mallei ATCC23344 and growth of the agents in the lungs, as well as dissemination to the spleen, were examined. Mice infected with 10(2, 10(3 and 10(4 organisms were unable to control growth of B. mallei in the lungs and bacteria rapidly disseminated to the spleen. Though similar results were observed in mice inoculated with 10(3 and 10(4 B. pseudomallei cells, animals infected with 10(2 organisms controlled bacterial replication in the lungs, dissemination to the spleen, and the extent of bacteremia. Analysis of sera from mice surviving acute infection revealed that animals produced antibodies against antigens known to be targets of the immune response in humans. Taken together, these data show that small volume aerosol inoculation of mice results in acute disease, dose-dependent chronic infection, and immune responses

  16. Use of a safe, reproducible, and rapid aerosol delivery method to study infection by Burkholderia pseudomallei and Burkholderia mallei in mice.

    Science.gov (United States)

    Lafontaine, Eric R; Zimmerman, Shawn M; Shaffer, Teresa L; Michel, Frank; Gao, Xiudan; Hogan, Robert J

    2013-01-01

    Burkholderia pseudomallei, the etiologic agent of melioidosis, is a saprophytic bacterium readily isolated from wet soils of countries bordering the equator. Burkholderia mallei is a host-adapted clone of B. pseudomallei that does not persist outside of its equine reservoir and causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by these organisms typically occurs via percutaneous inoculation or inhalation of aerosols, and the most common manifestation is severe pneumonia leading to fatal bacteremia. Glanders and melioidosis are difficult to diagnose and require prolonged antibiotic therapy with low success rates. There are no vaccines available to protect against either Burkholderia species, and there is concern regarding their use as biological warfare agents given that B. mallei has previously been utilized in this manner. Hence, experiments were performed to establish a mouse model of aerosol infection to study the organisms and develop countermeasures. Using a hand-held aerosolizer, BALB/c mice were inoculated intratracheally with strains B. pseudomallei 1026b and B. mallei ATCC23344 and growth of the agents in the lungs, as well as dissemination to the spleen, were examined. Mice infected with 10(2), 10(3) and 10(4) organisms were unable to control growth of B. mallei in the lungs and bacteria rapidly disseminated to the spleen. Though similar results were observed in mice inoculated with 10(3) and 10(4) B. pseudomallei cells, animals infected with 10(2) organisms controlled bacterial replication in the lungs, dissemination to the spleen, and the extent of bacteremia. Analysis of sera from mice surviving acute infection revealed that animals produced antibodies against antigens known to be targets of the immune response in humans. Taken together, these data show that small volume aerosol inoculation of mice results in acute disease, dose-dependent chronic infection, and immune responses that correlate

  17. 应用聚合酶链式反应快速特异鉴定假单胞菌和伯克霍尔德氏菌(R)-3-羟基酯酰载酯蛋白-辅酶A转酰基酶基因%Specific Identification of ( R )-3-hydroxyacyl-ACP: CoA Transacylase Gene from Pseudomonas and Burkholderia Strains by Polymerase Chain Reaction

    Institute of Scientific and Technical Information of China (English)

    郑重; 陈金春; 田鸿磊; 贝锋锋; 陈国强

    2005-01-01

    聚羟基脂肪酸酯(PHA)是一类具有广泛应用前景的可降解生物塑料.因其可以以葡萄糖等廉价底物直接发酵生产PHA而日益受到重视.目前的研究表明在积累中长链PHA的假单胞菌中,由phaG基因编码的(R)-3-羟基酯酰载酯蛋白-辅酶A转酰基酶(PhaG)起关键作用,但目前为止对该蛋白还知之甚少.通过聚合酶链式反应(PCR)建立了一种快速、特异鉴定phaG基因的方法,应用该方法成功地从两株积累不同PHA的假单胞菌Pseudomonas stutzeri 1317和Pseudomonas nitroreducens0802中分别克隆得到phaG基因,并在phaG基因突变株Pseudomonasputida PHAGN-21中表达成功.同时,还首次报道了从非假单胞菌菌株Burkholderia caryophylli AS 1.2741中鉴定得到phaG基因,提示PhaG介导的中长链PHA合成途径作为一种通用的代谢模式在细菌中广泛存在,为进一步实现从廉价的非相关底物合成中长链PHA提供了必要的分子生物学基础.%Polyhydroxyalkanoates (PHA) were biodegradable thermoplastics. Due to their broad applications, direct biosynthesis of PHA from inexpensive substrates, such as carbohydrates, is actively pursued. It has been recently revealed that (R)-3-hydroxyacyl-ACP: CoA transacylase (PhaG) played an important role in this pathway. In this study, a polymerase chain reaction(PCR) protocol was developed for the rapid and specific identification of phaG gene from various bacteria. Using the PCR strategy, the complete open reading frames of two phaG genes from Pseudomonas stutzeri 1317 and Pseudomonas nitroreducens 0802were cloned from the genomic DNA and functionally expressed in Pseudomonas putida PHAGN-21. Furthermore, this strategy was successful applied in non-Pseudomonas strains, such as Burkholderia. These results suggest that PhaG-mediated pathway of medium-chain-length polyhydroxyalkanoates was widespread among bacteria.

  18. Mouse model of sublethal and lethal intraperitoneal glanders (Burkholderia mallei).

    Science.gov (United States)

    Fritz, D L; Vogel, P; Brown, D R; Deshazer, D; Waag, D M

    2000-11-01

    Sixty male BALB/c mice were inoculated intraperitoneally with either a sublethal or a lethal dose of Burkholderia mallei China 7 strain, then killed at multiple time points postinoculation. Histopathologic changes were qualitatively similar in both groups and consisted of pyogranulomatous inflammation. In sublethal study mice, changes were first seen at 6 hours in mediastinal lymph nodes, then in spleen, liver, peripheral lymph nodes, and bone marrow at day 3. These changes generally reached maximal incidence and severity by day 4 but decreased by comparison in all tissues except the liver. Changes were first seen in lethal study mice also at 6 hours in mediastinal lymph nodes and in spleens. At day 1, changes were present in liver, peripheral lymph nodes, and bone marrow. The incidence and severity of these changes were maximal at day 2. In contrast to sublethal study mice, the incidence and severity of the changes did not decrease through the remainder of the study. The most significant difference between the two groups was the rapid involvement of the spleen in the lethal study mice. Changes indicative of impaired vascular perfusion were more frequently seen in the sublethal study mice. Our findings indicate that mice are susceptible to B. mallei infection and may serve as an appropriate model for glanders infection in a resistant host such as human beings. Additionally, by immunoelectron microscopy, we showed the presence of type I O-antigenic polysaccharide (capsular) antigen surrounding B. mallei.

  19. Monitoring Therapeutic Treatments against Burkholderia Infections Using Imaging Techniques

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    Tiffany M. Mott

    2013-05-01

    Full Text Available Burkholderia mallei, the etiologic agent of glanders, are Category B select agents with biothreat potential, and yet effective therapeutic treatments are lacking. In this study, we showed that CpG administration increased survival, demonstrating protection in the murine glanders model. Bacterial recovery from infected lungs, liver and spleen was significantly reduced in CpG-treated animals as compared with non-treated mice. Reciprocally, lungs of CpG-treated infected animals were infiltrated with higher levels of neutrophils and inflammatory monocytes, as compared to control animals. Employing the B. mallei bioluminescent strain CSM001 and the Neutrophil-Specific Fluorescent Imaging Agent, bacterial dissemination and neutrophil trafficking were monitored in real-time using multimodal in vivo whole body imaging techniques. CpG-treatment increased recruitment of neutrophils to the lungs and reduced bioluminescent bacteria, correlating with decreased bacterial burden and increased protection against acute murine glanders. Our results indicate that protection of CpG-treated animals was associated with recruitment of neutrophils prior to infection and demonstrated, for the first time, simultaneous real time in vivo imaging of neutrophils and bacteria. This study provides experimental evidence supporting the importance of incorporating optimized in vivo imaging methods to monitor disease progression and to evaluate the efficacy of therapeutic treatment during bacterial infections.

  20. Burkholderia pseudomallei: First case of melioidosis in Portugal

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    Ana Pelerito

    2016-01-01

    Full Text Available Burkholderia pseudomallei is a Gram-negative bacillus and the causative agent of melioidosis, a serious infection associated with high mortality rate in humans. It can be naturally found as an environmental saprophyte in soil or stagnant water, and rice paddies that predominate in regions of endemicity such as Northeast Thailand. B. pseudomallei is a Biosafety Level 3 organism due to risks of aerosolization and severe disease and is now included in formal emergency preparedness plans and guidelines issued by various authorities in the United States and Europe. Here, we report the first case of imported melioidosis in Portugal. B. pseudomallei was isolated from the patient's blood as well as from a left gluteal abscess pus. The isolate strain showed the unusual resistance profile to first-line eradication therapy trimethroprim/sulfamethoxazole. Whole genome sequencing revealed its similarity with isolates from Southeast Asia, suggesting the Thai origin of this Portuguese isolate, which is in agreement with a recent patient's travel to Thailand.

  1. Genetic diversity and microevolution of Burkholderia pseudomallei in the environment.

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    Narisara Chantratita

    Full Text Available BACKGROUND: The soil dwelling Gram-negative pathogen Burkholderia pseudomallei is the cause of melioidosis. The diversity and population structure of this organism in the environment is poorly defined. METHODS AND FINDINGS: We undertook a study of B. pseudomallei in soil sampled from 100 equally spaced points within 237.5 m(2 of disused land in northeast Thailand. B. pseudomallei was present on direct culture of 77/100 sampling points. Genotyping of 200 primary plate colonies from three independent sampling points was performed using a combination of pulsed field gel electrophoresis (PFGE and multilocus sequence typing (MLST. Twelve PFGE types and nine sequence types (STs were identified, the majority of which were present at only a single sampling point. Two sampling points contained four STs and the third point contained three STs. Although the distance between the three sampling points was low (7.6, 7.9, and 13.3 meters, respectively, only two STs were present in more than one sampling point. Each of the three samples was characterized by the localized expansion of a single B. pseudomallei clone (corresponding to STs 185, 163, and 93. Comparison of PFGE and MLST results demonstrated that two STs contained strains with variable PFGE banding pattern types, indicating geographic structuring even within a single MLST-defined clone. CONCLUSIONS: We discuss the implications of this extreme structuring of genotype and genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies.

  2. Phylogenetic and degradation characterization of Burkholderia cepacia WZ1 degrading herbicide quinclorac.

    Science.gov (United States)

    Lü, Zhenmei; Min, Hang; Wu, Shuwen; Ruan, Aidong

    2003-11-01

    Strain WZI capable of degrading quinclorac was isolated from a pesticide manufactory soil and considered to be Burkholderia cepacia, belonged to bacteria, Proteobacteria, beta-Proteobacteria, based on morphology, physio-biochemical properties, whole cell fatty acid analysis and a partial sequencing of 16S rDNA. Strain WZ1 decomposed 90% of quinclorac at original concentration of 1000 mg L(-1) within 11 days. GC/MS analysis showed that the strain degraded quinclorac to 3,7-dichloro-8-quinoline and the cracked residue 2-chloro, 1,4-benzenedicarboxylic acid, indicating that the metabolic pathway was initiated by process of decarboxylation followed by cleavage of the aromatic ring. Stain WZ1 was also able to degrade some other herbicides and aromatic compounds, including 2,4,5-T, phenol, naphthalene and hydrochinone etc. This paper describes for the first time Phylogenetic and degradation characterization of a pure bacterium which, is able to mineralize quinclorac.

  3. Genomovars of Burkholderia cepacia Complex from Rice Rhizosphere and Clinic in China

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Burkholderia cepacia is regarded as a genetically distinct but phenotypically similar bacteria group referring to Burkholderia cepacia complex (Bcc), which is found not only in clinic but also in rice growing environment. It is very important in microbial safety of rice for us to understand the genomovar status of Bcc. Genomovar analysis was performed among 87 Bcc isolates by means of Hae Ⅲ-recA RFLP assays and species-specific PCR tests. Three genomovars were found from the rice rhizosphere including Ⅰ, ⅢB and Ⅴ, and genomovar Ⅴ was predominant. Genomovars Ⅰ, ⅢA and ⅢB existed in the clinical samples, and genomovar ⅢA was the most popular. It showed that genomovar composition was different between the Bcc strains from the rice rhizosphere and clinical environment. Simultaneously, the results revealed the genetic diversity of Bcc strains from the rice rhizosphere, and genomovar Ⅲ referred as virulent species in clinic also existed in the rice rhizosphere.

  4. Nonribosomal peptides and polyketides of Burkholderia: new compounds potentially implicated in biocontrol and pharmaceuticals.

    Science.gov (United States)

    Esmaeel, Qassim; Pupin, Maude; Jacques, Philippe; Leclère, Valérie

    2017-05-25

    Bacteria belonging to the genus Burkholderia live in various ecological niches and present a significant role in the environments through the excretion of a wide variety of secondary metabolites including modular nonribosomal peptides (NRPs) and polyketides (PKs). These metabolites represent a widely distributed biomedically and biocontrol important class of natural products including antibiotics, siderophores, and anticancers as well as biopesticides that are considered as a novel source that can be used to defend ecological niche from competitors and to promote plant growth. The aim of this review is to present all NRPs produced or potentially produced by strains of Burkholderia, as NRPs represent a major source of active compounds implicated in biocontrol. The review is a compilation of results from a large screening we have performed on 48 complete sequenced genomes available in NCBI to identify NRPS gene clusters, and data found in the literature mainly because some interesting compounds are produced by strains not yet sequenced. In addition to NRPs, hybrids NRPs/PKs are also included. Specific features about biosynthetic gene clusters and structures of the modular enzymes responsible for the synthesis, the biological activities, and the potential uses in agriculture and pharmaceutical of NRPs and hybrids NRPs/PKs will also be discussed.

  5. Solution conformation and dynamics of exopolysaccharides from Burkholderia species.

    Science.gov (United States)

    Pol-Fachin, Laercio; Serrato, Rodrigo V; Verli, Hugo

    2010-09-03

    Exopolysaccharides (EPSs) from the Burkholderia genus are proposed to be involved in pathological conditions in humans, such as cystic fibrosis and septicemia, as well as in the stability of soil aggregates. Hence, considering that the conformational and dynamic aspects of such EPSs may influence their biological activity, the current work employs a series of molecular dynamics simulations on di-, oligo-, and polysaccharide fragments of three EPSs, from Burkholderia caribensis, Burkholderia cepacia, and Burkholderia pseudomallei, with previously determined NOE data, to obtain a conformational description of such EPSs at the atomic level. As the obtained results show good agreement with the experimental data, pointing to the adequacy of the employed methodology to accurately describe the dynamics of polysaccharides, the strategy was also employed to predict the conformational behavior of an additional compound, from Burkholderia tropica, for which NOE signals are not available. Taking into account the potential importance of EPSs on the interaction of Burkholderia bacteria with distinct environments, it may be expected that a greater understanding of their structural aspects may contribute to controlling their pathological roles and potential agricultural applications.

  6. Intrinsic Resistance of Burkholderia cepacia Complex to Benzalkonium Chloride

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    Youngbeom Ahn

    2016-11-01

    Full Text Available Pharmaceutical products that are contaminated with Burkholderia cepacia complex (BCC bacteria may pose serious consequences to vulnerable patients. Benzyldimethylalkylammonium chloride (BZK cationic surfactants are extensively used in medical applications and have been implicated in the coselection of antimicrobial resistance. The ability of BCC to degrade BZK, tetradecyldimethylbenzylammonium chloride (C14BDMA-Cl, dodecyldimethylbenzylammonium chloride (C12BDMA-Cl, decyldimethylbenzylammonium chloride (C10BDMA-Cl, hexyldimethylbenzylammonium chloride, and benzyltrimethylammonium chloride was determined by incubation in 1/10-diluted tryptic soy broth (TSB to determine if BCC bacteria have the ability to survive and inactivate these disinfectants. With BZK, C14BDMA-Cl, and C12BDMA-Cl, inhibition of the growth of 20 BCC strains was observed in disinfectant solutions that ranged from 64 to 256 µg/ml. The efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone increased the sensitivity of bacteria to 64 µg/ml BZK. The 20 BCC strains grew well in 1/10-diluted TSB medium with BZK, C12BDMA-Cl, and C10BDMA-Cl; they absorbed and degraded the compounds in 7 days. Formation of benzyldimethylamine and benzylmethylamine as the initial metabolites suggested that the cleavage of the C alkyl-N bond occurred as the first step of BZK degradation by BCC bacteria. Proteomic data confirmed the observed efflux activity and metabolic inactivation via biodegradation in terms of BZK resistance of BCC bacteria, which suggests that the two main resistance mechanisms are intrinsic and widespread.

  7. Burkholderia xernovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility

    Energy Technology Data Exchange (ETDEWEB)

    Chain, Patrick S. G. [Lawrence Livermore National Laboratory (LLNL); Denef, Vincent [University of California, Berkeley; Konstantinidis, Konstantinos T [Michigan State University, East Lansing; Vergez, Lisa [Lawrence Livermore National Laboratory (LLNL); Agullo, Loreine [Universidad Tecnica Federico Santa Maria, Casilla 110-V; Reyes, Valeria Latorre [Universidad Tecnica Federico Santa Maria, Casilla 110-V; Hauser, Loren John [ORNL; Cordova, Macarena [Universidad Tecnica Federico Santa Maria, Casilla 110-V; Gomez, Luis [Universidad Tecnica Federico Santa Maria, Casilla 110-V; Gonzalez, Myriam [Universidad Tecnica Federico Santa Maria, Casilla 110-V; Land, Miriam L [ORNL; Lao, Victoria [Lawrence Livermore National Laboratory (LLNL); Larimer, Frank W [ORNL; LiPuma, John J [University of Michigan; Mahenthiralingam, Eshwar [Cardiff University, Wales; Malfatti, Stephanie [Lawrence Livermore National Laboratory (LLNL); Marx, Christopher J [Harvard University; Parnell, J Jacob [Michigan State University, East Lansing; Ramette, Alban [Michigan State University, East Lansing; Richardson, P M [U.S. Department of Energy, Joint Genome Institute; Seeger, Michael [Universidad Tecnica Federico Santa Maria, Casilla 110-V; Smith, Daryl [University of British Columbia, Vancouver; Spilker, Theodore [University of Michigan; Sul, Woo Jun [Michigan State University, East Lansing; Tsoi, Tamara V [Michigan State University, East Lansing; Zhulin, Igor B [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Tiedje, James M. [Michigan State University, East Lansing

    2006-01-01

    Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven 'central aromatic' and twenty 'peripheral aromatic' pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.

  8. Differential intracellular fate of Burkholderia pseudomallei 844 and Burkholderia thailandensis UE5 in human monocyte-derived dendritic cells and macrophages

    Directory of Open Access Journals (Sweden)

    Engering Anneke

    2009-04-01

    Full Text Available Abstract Background Burkholderia pseudomallei (Bp is a category B biothreat organism that causes a potentially fatal disease in humans and animals, namely melioidosis. Burkholderia thailandensis (Bt is another naturally occurring species that is very closely related to Bp. However, despite this closely related genotype, Bt is considered avirulent as it does not cause the disease. In the present study, we compared the growth kinetics of B. pseudomallei strain 844 (Bp-844 in human monocyte-derived dendritic cells (MoDCs and macrophages (Mφs, as well as its ability to stimulate host cell responses with those of B. thailandensis strain UE5 (Bt-UE5. Results Primary human MoDCs and Mφs were infected with Bp-844 and its intracellular growth kinetics and ability to induce host cell responses were evaluated. The results were compared with those obtained using the Bt-UE5. In human MoDCs, both bacteria were similar in respect to their ability to survive and replicate intracellularly, induce upregulation of costimulatory molecules and cytokines and bias T helper cell differentiation toward a Th1 phenotype. By contrast, the two bacteria exhibited different growth kinetics in human Mφs, where the intracellular growth of Bt-UE5, but not Bp-844, was significantly suppressed. Moreover, the ability of Mφs to kill Bp-844 was markedly enhanced following stimulation with IFN-γ. Conclusion The data presented showed that while both strains were similar in their ability to survive and replicate in human MoDCs, only Bp-844 could readily replicate in human Mφs. Both bacteria induced similar host cellular responses, particularly with regard to their ability to bias T cell differentiation toward a Th1 phenotype.

  9. Exploitation of host cells by Burkholderia pseudomallei.

    Science.gov (United States)

    Stevens, Mark P; Galyov, Edouard E

    2004-04-01

    Intracellular bacterial pathogens have evolved mechanisms to enter and exit eukaryotic cells using the power of actin polymerisation and to subvert the activity of cellular enzymes and signal transduction pathways. The proteins deployed by bacteria to subvert cellular processes often mimic eukaryotic proteins in their structure or function. Studies on the exploitation of host cells by the facultative intracellular pathogen Burkholderia pseudomallei are providing novel insights into the pathogenesis of melioidosis, a serious invasive disease of animals and humans that is endemic in tropical and subtropical areas. B. pseudomallei can invade epithelial cells, survive and proliferate inside phagocytes, escape from endocytic vesicles, form actin-based membrane protrusions and induce host cell fusion. Here we review current understanding of the molecular mechanisms underlying these processes.

  10. Novel Burkholderia mallei Virulence Factors Linked to Specific Host-Pathogen Protein Interactions*

    Science.gov (United States)

    Memišević, Vesna; Zavaljevski, Nela; Pieper, Rembert; Rajagopala, Seesandra V.; Kwon, Keehwan; Townsend, Katherine; Yu, Chenggang; Yu, Xueping; DeShazer, David; Reifman, Jaques; Wallqvist, Anders

    2013-01-01

    Burkholderia mallei is an infectious intracellular pathogen whose virulence and resistance to antibiotics makes it a potential bioterrorism agent. Given its genetic origin as a commensal soil organism, it is equipped with an extensive and varied set of adapted mechanisms to cope with and modulate host-cell environments. One essential virulence mechanism constitutes the specialized secretion systems that are designed to penetrate host-cell membranes and insert pathogen proteins directly into the host cell's cytosol. However, the secretion systems' proteins and, in particular, their host targets are largely uncharacterized. Here, we used a combined in silico, in vitro, and in vivo approach to identify B. mallei proteins required for pathogenicity. We used bioinformatics tools, including orthology detection and ab initio predictions of secretion system proteins, as well as published experimental Burkholderia data to initially select a small number of proteins as putative virulence factors. We then used yeast two-hybrid assays against normalized whole human and whole murine proteome libraries to detect and identify interactions among each of these bacterial proteins and host proteins. Analysis of such interactions provided both verification of known virulence factors and identification of three new putative virulence proteins. We successfully created insertion mutants for each of these three proteins using the virulent B. mallei ATCC 23344 strain. We exposed BALB/c mice to mutant strains and the wild-type strain in an aerosol challenge model using lethal B. mallei doses. In each set of experiments, mice exposed to mutant strains survived for the 21-day duration of the experiment, whereas mice exposed to the wild-type strain rapidly died. Given their in vivo role in pathogenicity, and based on the yeast two-hybrid interaction data, these results point to the importance of these pathogen proteins in modulating host ubiquitination pathways, phagosomal escape, and actin

  11. Alteration in cell surface properties of Burkholderia spp. during surfactant-aided biodegradation of petroleum hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Mohanty, Sagarika; Mukherji, Suparna [Indian Institute of Technology Bombay, Mumbai (India). Centre for Environmental Science and Engineering (CESE)

    2012-04-15

    Chemical surfactants may impact microbial cell surface properties, i.e., cell surface hydrophobicity (CSH) and cell surface charge, and may thus affect the uptake of components from non-aqueous phase liquids (NAPLs). This work explored the impact of Triton X-100, Igepal CA 630, and Tween 80 (at twice the critical micelle concentration, CMC) on the cell surface characteristics of Burkholderia cultures, Burkholderia cepacia (ES1, aliphatic degrader) and Burkholderia multivorans (NG1, aromatic degrader), when grown on a six-component model NAPL. In the presence of Triton X-100, NAPL biodegradation was enhanced from 21% to 60% in B. cepacia and from 18% to 53% in B. multivorans. CSH based on water contact angle (50-52 ) was in the same range for both strains while zeta potential at neutral pH was -38 and -31 mV for B. cepacia and B. multivorans, respectively. In the presence of Triton X-100, their CSH increased to greater than 75 and the zeta potential decreased. This induced a change in the mode of uptake and initiated aliphatic hydrocarbon degradation by B. multivorans and increased the rate of aliphatic hydrocarbon degradation in B. cepacia. Igepal CA 630 and Tween 80 also altered the cell surface properties. For B. cepacia grown in the presence of Triton X-100 at two and five times its CMC, CSH increased significantly in the log growth phase. Growth in the presence of the chemical surfactants also affected the abundance of chemical functional groups on the cell surface. Cell surface changes had maximum impact on NAPL degradation in the presence of emulsifying surfactants, Triton X-100 and Igepal CA630.

  12. Biosurfactant Production by Pseudomonas aeruginosa and Burkholderia gladioli Isolated from Mangrove Sediments Using Alternative Substrates

    Directory of Open Access Journals (Sweden)

    Karla Maria Catter

    2016-10-01

    Full Text Available Biosurfactants are surface-active agents produced by a variety of microorganisms. To make biosurfactant production economically feasible, several alternative carbon sources have been proposed. This study describes biosurfactant production by strains of Pseudomonas aeruginosa and Burkholderia gladioli isolated from mangrove sediments in Northeastern Brazil and cultured in mineral media enriched with waste cooking oil. The biosurfactants were tested for drop collapse, emulsion formation and stability and surface tension. P. aeruginosa performed better both at lowering the surface tension (from 69 to 28 mN/m and at forming stable emulsions (approximately 80% at 48 hours of culture. The strains tested in this study were found to be efficient biosurfactant producers when cultured on substrates enriched with vegetable oil. DOI: http://dx.doi.org/10.17807/orbital.v8i5.771

  13. Cometabolic degradation of trichloroethylene by Burkholderia cepacia G4 with poplar leaf homogenate.

    Science.gov (United States)

    Kang, Jun Won; Doty, Sharon Lafferty

    2014-07-01

    Trichloroethylene (TCE), a chlorinated organic solvent, is one of the most common and widespread groundwater contaminants worldwide. Among the group of TCE-degrading aerobic bacteria, Burkholderia cepacia G4 is the best-known representative. This strain requires the addition of specific substrates, including toluene, phenol, and benzene, to induce the enzymes to degrade TCE. However, the substrates are toxic and introducing them into the soil can result in secondary contamination. In this study, poplar leaf homogenate containing natural phenolic compounds was tested for the ability to induce the growth of and TCE degradation by B. cepacia G4. The results showed that the G4 strain could grow and degrade TCE well with the addition of phytochemicals. The poplar leaf homogenate also functioned as an inducer of the toluene-ortho-monooxygenase (TOM) gene in B. cepacia G4.

  14. Burkholderia pseudomallei virulence: definition, stability and association with clonality.

    Science.gov (United States)

    Ulett, G C; Currie, B J; Clair, T W; Mayo, M; Ketheesan, N; Labrooy, J; Gal, D; Norton, R; Smith, C A; Barnes, J; Warner, J; Hirst, R G

    2001-07-01

    Clinical presentations of melioidosis, caused by Burkholderia pseudomallei are protean, but the mechanisms underlying development of the different forms of disease remain poorly understood. In murine melioidosis, the level of virulence of B. pseudomallei is important in disease pathogenesis and progression. In this study, we used B. pseudomallei-susceptible BALB/c mice to determine the virulence of a library of clinical and environmental B. pseudomallei isolates from Australia and Papua New Guinea. Among 42 non-arabinose-assimilating (ara(-)) isolates, LD(50) ranged from 10 to > 10(6) CFU. There were numerous correlations between virulence and disease presentation in patients; however, this was not a consistent observation. Virulence did not correlate with isolate origin (i.e. clinical vs environmental), since numerous ara(-) environmental isolates were highly virulent. The least virulent isolate was a soil isolate from Papua New Guinea, which was arabinose assimilating (ara(+)). Stability of B. pseudomallei virulence was investigated by in vivo passage of isolates through mice and repetitive in vitro subculture. Virulence increased following in vivo exposure in only one of eight isolates tested. In vitro subculture on ferric citrate-containing medium caused attenuation of virulence, and this correlated with changes in colony morphology. Pulsed-field gel electrophoresis and randomly amplified polymorphic DNA typing demonstrated that selected epidemiologically related isolates that had variable clinical outcomes and different in vivo virulence were clonal strains. No molecular changes were observed in isolates after in vivo or in vitro exposure despite changes in virulence. These results indicate that virulence of selected B. pseudomallei isolates is variable, being dependent on factors such as iron bioavailability. They also support the importance of other variables such as inoculum size and host risk factors in determining the clinical severity of melioidosis.

  15. Burkholderia pseudomallei genome plasticity associated with genomic island variation

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    Currie Bart J

    2008-04-01

    Full Text Available Abstract Background Burkholderia pseudomallei is a soil-dwelling saprophyte and the cause of melioidosis. Horizontal gene transfer contributes to the genetic diversity of this pathogen and may be an important determinant of virulence potential. The genome contains genomic island (GI regions that encode a broad array of functions. Although there is some evidence for the variable distribution of genomic islands in B. pseudomallei isolates, little is known about the extent of variation between related strains or their association with disease or environmental survival. Results Five islands from B. pseudomallei strain K96243 were chosen as representatives of different types of genomic islands present in this strain, and their presence investigated in other B. pseudomallei. In silico analysis of 10 B. pseudomallei genome sequences provided evidence for the variable presence of these regions, together with micro-evolutionary changes that generate GI diversity. The diversity of GIs in 186 isolates from NE Thailand (83 environmental and 103 clinical isolates was investigated using multiplex PCR screening. The proportion of all isolates positive by PCR ranged from 12% for a prophage-like island (GI 9, to 76% for a metabolic island (GI 16. The presence of each of the five GIs did not differ between environmental and disease-associated isolates (p > 0.05 for all five islands. The cumulative number of GIs per isolate for the 186 isolates ranged from 0 to 5 (median 2, IQR 1 to 3. The distribution of cumulative GI number did not differ between environmental and disease-associated isolates (p = 0.27. The presence of GIs was defined for the three largest clones in this collection (each defined as a single sequence type, ST, by multilocus sequence typing; these were ST 70 (n = 15 isolates, ST 54 (n = 11, and ST 167 (n = 9. The rapid loss and/or acquisition of gene islands was observed within individual clones. Comparisons were drawn between isolates obtained

  16. Porin involvement in cephalosporin and carbapenem resistance of Burkholderia pseudomallei.

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    Anuwat Aunkham

    Full Text Available BACKGROUND: Burkholderia pseudomallei (Bps is a Gram-negative bacterium that causes frequently lethal melioidosis, with a particularly high prevalence in the north and northeast of Thailand. Bps is highly resistant to many antimicrobial agents and this resistance may result from the low drug permeability of outer membrane proteins, known as porins. PRINCIPAL FINDINGS: Microbiological assays showed that the clinical Bps strain was resistant to most antimicrobial agents and sensitive only to ceftazidime and meropenem. An E. coli strain defective in most porins, but expressing BpsOmp38, exhibited considerably lower antimicrobial susceptibility than the control strain. In addition, mutation of Tyr119, the most prominent pore-lining residue in BpsOmp38, markedly altered membrane permeability, substitution with Ala (mutant BpsOmp38Y119A enhanced uptake of the antimicrobial agents, while substitution with Phe (mutant BpsOmp38Y119F inhibited uptake. Channel recordings of BpsOmp38 reconstituted in a planar black lipid membrane (BLM suggested that the higher permeability of BpsOmp38Y119A was caused by widening of the pore interior through removal of the bulky side chain. In contrast, the lower permeability of BpsOmp38Y119F was caused by introduction of the hydrophobic side chain (Phe, increasing the 'greasiness' of the pore lumen. Significantly, liposome swelling assays showed no permeation through the BpsOmp38 channel by antimicrobial agents to which Bps is resistant (cefoxitin, cefepime, and doripenem. In contrast, high permeability to ceftazidime and meropenem was observed, these being agents to which Bps is sensitive. CONCLUSION/SIGNIFICANCE: Our results, from both in vivo and in vitro studies, demonstrate that membrane permeability associated with BpsOmp38 expression correlates well with the antimicrobial susceptibility of the virulent bacterium B. pseudomallei, especially to carbapenems and cephalosporins. In addition, substitution of the residue

  17. Characterization of the papilionoid-Burkholderia interaction in the Fynbos biome: The diversity and distribution of beta-rhizobia nodulating Podalyria calyptrata (Fabaceae, Podalyrieae).

    Science.gov (United States)

    Lemaire, Benny; Van Cauwenberghe, Jannick; Verstraete, Brecht; Chimphango, Samson; Stirton, Charles; Honnay, Olivier; Smets, Erik; Sprent, Janet; James, Euan K; Muasya, A Muthama

    2016-02-01

    The South African Fynbos soils are renowned for nitrogen-fixing Burkholderia associated with diverse papilionoid legumes of the tribes Crotalarieae, Hypocalypteae, Indigofereae, Phaseoleae and Podalyrieae. However, despite numerous rhizobial studies in the region, the symbiotic diversity of Burkholderia has not been investigated in relation to a specific host legume and its geographical provenance. This study analyzed the diversity of nodulating strains of Burkholderia from the legume species Podalyria calyptrata. Diverse lineages were detected that proved to be closely related to Burkholderia taxa, originating from hosts in other legume tribes. By analyzing the genetic variation of chromosomal (recA) and nodulation (nodA) sequence data in relation to the sampling sites we assessed the geographical distribution patterns of the P. calyptrata symbionts. Although we found a degree of genetically differentiated rhizobial populations, a correlation between genetic (recA and nodA) and geographic distances among populations was not observed, suggesting high rates of dispersal and rhizobial colonization within Fynbos soils.

  18. Inhibition of Burkholderia multivorans Adhesion to Lung Epithelial Cells by Bivalent Lactosides

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    Trinidad Velasco-Torrijos

    2012-08-01

    Full Text Available Burkholderia cepacia complex (Bcc is an opportunistic pathogen in cystic fibrosis patients which is inherently resistant to antimicrobial agents. The mechanisms of attachment and pathogenesis of Bcc, a group of 17 species, are poorly understood. The most commonly identified Bcc species in newly colonised patients, Burkholderia multivorans, continues to be acquired from the environment. Development of therapies which can prevent or reduce the risk of colonization on exposure to Bcc in the environment would be a better alternative to antimicrobial agents. Previously, it has been shown that Bcc strains bound to many glycolipid receptors on lung epithelia. Using a real-time PCR method to quantify the levels of binding of B. multivorans to the lung epithelial cells, we have examined glycoconjugate derivatives for their potential to inhibit host cell attachment. Bivalent lactosides previously shown to inhibit galectin binding significantly reduced the attachment of B. multivorans to CF lung epithelial cells at micromolar concentrations. This was in contrast to monosaccharides and lactose, which were only effective in the millimolar range. Development of glycoconjugate therapies such as these, which inhibit attachment to lung epithelial cells, represent an alternative means of preventing infection with inherently antimicrobially resistant pathogens such as B. multivorans.

  19. Characterization Of Pathogenesis Of And Immune Response To Burkholderia Pseudomallei K9243 Using Both Inhalational And Intraperitoneal Infection Models In BALB/c and C57BL/6 Mice

    Science.gov (United States)

    2017-02-24

    ecological emerging infectious disease in the Alor Setar region of Kedah, Malaysia . BMC 1098 Infect Dis, 2010. 10: p. 302. 1099 12. Limmathurotsakul, D...1181 50. Amemiya, K., et al., Comparison of the early host immune response to two widely diverse virulent 1182 strains of Burkholderia pseudomallei

  20. Burkholderia mallei and Burkholderia pseudomallei cluster 1 type VI secretion system gene expression is negatively regulated by iron and zinc.

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    Mary N Burtnick

    Full Text Available Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1 expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G or minimal media plus casamino acids (M9CG facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.

  1. Transfer of 13 species of the genus Burkholderia to the genus Caballeronia and reclassification of Burkholderia jirisanensis as Paraburkholderia jirisanensis comb. nov.

    Science.gov (United States)

    Dobritsa, Anatoly P; Linardopoulou, Elena V; Samadpour, Mansour

    2017-09-07

    A recent study of a group of Burkholderia glathei-like bacteria resulted in the description of 13 novel species of the genus Burkholderia. However, our analysis of phylogenetic positions of these species and their molecular signatures (conserved protein sequence indels) showed that they belong to the genus Caballeronia, and we propose to transfer them to this genus. The reclassified species names are proposed as Caballeroniaarationis comb. nov., Caballeroniaarvi comb. nov., Caballeroniacalidae comb. nov., Caballeroniacatudaia comb. nov., Caballeroniaconcitans comb. nov., Caballeroniafortuita comb. nov., Caballeroniaglebae comb. nov., Caballeroniahypogeia comb. nov., Caballeroniapedi comb. nov., Caballeroniaperedens comb. nov., Caballeroniaptereochthonis comb. nov., Caballeroniatemeraria comb. nov. and Caballeronia turbans comb. nov. It is also proposed to reclassify Burkholderia jirisanensis as Paraburkholderiajirisanensis comb. nov. Based on the results of the polyphasic study, B. jirisanensis had been described as a member of the A-group of the genus Burkholderiaand the most closely related to Burkholderia rhizosphaerae, Burkholderia humisilvae and Burkholderia solisilvae currently classified as belonging to the genus Paraburkholderia.

  2. Burkholderia pseudomallei musculoskeletal infections (melioidosis in India

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    Pandey Vivek

    2010-01-01

    Full Text Available Melioidosis, an infection due to gram negative Burkholderia pseudomallei, is an important cause of sepsis in east Asia especially Thailand and northern Australia. It usually causes abscesses in lung, liver, spleen, skeletal muscle and parotids especially in patients with diabetes, chronic renal failure and thalassemia. Musculoskeletal melioidosis is not common in India even though sporadic cases have been reported mostly involving soft tissues. During a two-year-period, we had five patients with musculoskeletal melioidosis. All patients presented with multifocal osteomyelitis, recurrent osteomyelitis or septic arthritis. One patient died early because of septicemia and multi-organ failure. All patients were diagnosed on the basis of positive pus culture. All patients were treated by surgical debridement followed by a combination of antibiotics; (ceftazidime, amoxy-clavulanic acid, co-trimoxazole and doxycycline for six months except for one who died due to fulminant septicemia. All other patients recovered completely with no recurrences. With increasing awareness and better diagnostic facilities, probably musculoskeletal melioidosis will be increasingly diagnosed in future.

  3. Prevalence of Burkholderia pseudomallei in Guangxi, China.

    Science.gov (United States)

    Ma, G; Zheng, D; Cai, Q; Yuan, Z

    2010-01-01

    Melioidosis, an infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei, is now recognized as an important public health problem in Southeast Asia and tropical northern Australia. Although B. pseudomallei has been detected in various water and soil samples in southeast China, the enviromental distribution of B. pseudomallei in China is unclear. In the winter months of 2007, 154 and 130 soil and water samples, respectively, were collected from several locations in Guangxi, China. The samples were screened for B. pseudomallei by bacterial culture and identification and confirmed by PCR for species-specific 16S rDNA and flagellin genes. B. pseudomallei was detected in 8.4% of the soil samples but in none of the water samples. All positive samples were confined to a single low-lying region from rice paddy fields. Counts of B. pseudomallei ranged from 23 to 521 c.f.u./g soil. This is the first geographical distribution survey of B. pseudomallei in soil in Guangxi, China, and the data are of importance for further evaluating the impact of this pathogen on melioidosis in this region.

  4. Revised approach for identification of isolates within the Burkholderia cepacia complex and description of clinical isolates not assigned to any of the known genomovars.

    Science.gov (United States)

    Turton, Jane F; Arif, Nazia; Hennessy, Daneeta; Kaufmann, Mary E; Pitt, Tyrone L

    2007-09-01

    One hundred thirty-eight clinical isolates of the Burkholderia cepacia complex (Bcc) were identified using a modified strategy that involved PCR detection of the cblA gene for the ET12 lineage simultaneously with detection of the Bcc recA PCR product; recA sequence cluster analysis also was part of the strategy. Four strains could not be assigned to any of the known genomovars.

  5. Recent Advances in Burkholderia mallei and B. pseudomallei Research

    Science.gov (United States)

    Hatcher, Christopher L.; Muruato, Laura A.

    2015-01-01

    Burkholderia mallei and Burkholderia pseudomallei are Gram-negative organisms, which are etiological agents of glanders and melioidosis, respectively. Although only B. pseudomallei is responsible for a significant number of human cases, both organisms are classified as Tier 1 Select Agents and their diseases lack effective diagnosis and treatment. Despite a recent resurgence in research pertaining to these organisms, there are still a number of knowledge gaps. This article summarizes the latest research progress in the fields of B. mallei and B. pseudomallei pathogenesis, vaccines, and diagnostics. PMID:25932379

  6. A conserved two-component regulatory system, PidS/PidR, globally regulates pigmentation and virulence-related phenotypes of Burkholderia glumae.

    Science.gov (United States)

    Karki, Hari Sharan; Barphagha, Inderjit Kaur; Ham, Jong Hyun

    2012-09-01

    Burkholderia glumae is a rice pathogenic bacterium that causes bacterial panicle blight. Some strains of this pathogen produce dark brown pigments when grown on casamino-acid peptone glucose (CPG) agar medium. A pigment-positive and highly virulent strain of B. glumae, 411gr-6, was randomly mutagenized with mini-Tn5gus, and the resulting mini-Tn5gus derivatives showing altered pigmentation phenotypes were screened on CPG agar plates to identify the genetic elements governing the pigmentation of B. glumae. In this study, a novel two-component regulatory system (TCRS) composed of the PidS sensor histidine kinase and the PidR response regulator was identified as an essential regulatory factor for pigmentation. Notably, the PidS/PidR TCRS was also required for the elicitation of the hypersensitive response on tobacco leaves, indicating the dependence of the hypersensitive response and pathogenicity (Hrp) type III secretion system of B. glumae on this regulatory factor. In addition, B. glumae mutants defective in the PidS/PidR TCRS showed less production of the phytotoxin, toxoflavin, and less virulence on rice panicles and onion bulbs relative to the parental strain, 411gr-6. The presence of highly homologous PidS and PidR orthologues in other Burkholderia species suggests that PidS/PidR-family TCRSs may exert the same or similar functions in different Burkholderia species, including both plant and animal pathogens.

  7. Burkholderia cepacia XXVI siderophore with biocontrol capacity against Colletotrichum gloeosporioides.

    Science.gov (United States)

    de Los Santos-Villalobos, Sergio; Barrera-Galicia, Guadalupe Coyolxauhqui; Miranda-Salcedo, Mario Alberto; Peña-Cabriales, Juan José

    2012-08-01

    Colletotrichum gloeosporioides is the causal agent of anthracnose in mango. Burkholderia cepacia XXVI, isolated from mango rhizosphere and identified by 16S rDNA sequencing as a member of B. cepacia complex, was more effective than 6 other mango rhizosphere bacteria in inhibiting the model mango pathogen, C. gloeosporioides ATCC MYA 456. Biocontrol of this pathogen was demonstrated on Petri-dishes containing PDA by > 90 % reduction of surface colonization. The nature of the biocontrol metabolite(s) was characterized via a variety of tests. The inhibition was almost exclusively due to production of agar-diffusible, not volatile, metabolite(s). The diffusible metabolite(s) underwent thermal degradation at 70 and 121 °C (1 atm). Tests for indole acetic acid production and lytic enzyme activities (cellulase, glucanase and chitinase) by B. cepacia XXVI were negative, indicating that these metabolites were not involved in the biocontrol effect. Based on halo formation and growth inhibition of the pathogen on the diagnostic medium, CAS-agar, as well as colorimetric tests we surmised that strain XXVI produced a hydroxamate siderophore involved in the biocontrol effect observed. The minimal inhibitory concentration test showed that 0.64 μg ml(-1) of siderophore (Deferoxamine mesylate salt-equivalent) was sufficient to achieve 91.1 % inhibition of the pathogen growth on Petri-dishes containing PDA. The biocontrol capacity against C. gloeosporioides ATCC MYA 456 correlated directly with the siderophore production by B. cepacia XXVI: the highest concentration of siderophore production in PDB on day 7, 1.7 μg ml(-1) (Deferoxamine mesylate salt-equivalent), promoted a pathogen growth inhibition of 94.9 %. The growth of 5 additional strains of C. gloeosporioides (isolated from mango "Ataulfo" orchards located in the municipality of Chahuites, State of Oaxaca in Mexico) was also inhibited when confronted with B. cepacia XXVI. Results indicate that B. cepacia XXVI or its

  8. In Vitro Antifungal Activity of Burkholderia gladioli pv. agaricicola against Some Phytopathogenic Fungi

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    Hazem S. Elshafie

    2012-12-01

    Full Text Available The trend to search novel microbial natural biocides has recently been increasing in order to avoid the environmental pollution from use of synthetic pesticides. Among these novel natural biocides are the bioactive secondary metabolites of Burkholderia gladioli pv. agaricicola (Bga. The aim of this study is to determine antifungal activity of Bga strains against some phytopathogenic fungi. The fungicidal tests were carried out using cultures and cell-free culture filtrates against Botrytis cinerea, Aspergillus flavus, Aspergillus niger, Penicillium digitatum, Penicillium expansum, Sclerotinia sclerotiorum and Phytophthora cactorum. Results demonstrated that all tested strains exert antifungal activity against all studied fungi by producing diffusible metabolites which are correlated with their ability to produce extracellular hydrolytic enzymes. All strains significantly reduced the growth of studied fungi and the bacterial cells were more bioactive than bacterial filtrates. All tested Bulkholderia strains produced volatile organic compounds (VOCs, which inhibited the fungal growth and reduced the growth rate of Fusarium oxysporum and Rhizoctonia solani. GC/MS analysis of VOCs emitted by strain Bga 11096 indicated the presence of a compound that was identified as 1-methyl-4-(1-methylethenyl-cyclohexene, a liquid hydrocarbon classified as cyclic terpene. This compound could be responsible for the antifungal activity, which is also in agreement with the work of other authors.

  9. Survival of Burkholderia pseudomallei in Water

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    Woods Donald E

    2008-05-01

    Full Text Available Abstract Background The ability of Burkholderia pseudomallei to survive in water likely contributes to its environmental persistence in endemic regions. To determine the physiological adaptations which allow B. pseudomallei to survive in aqueous environments, we performed microarray analyses of B. pseudomallei cultures transferred from Luria broth (LB to distilled water. Findings Increased expression of a gene encoding for a putative membrane protein (BPSL0721 was confirmed using a lux-based transcriptional reporter system, and maximal expression was noted at approximately 6 hrs after shifting cells from LB to water. A BPSL0721 deficient mutant of B. pseudomallei was able to survive in water for at least 90 days indicating that although involved, BPSL0721 was not essential for survival. BPSL2961, a gene encoding a putative phosphatidylglycerol phosphatase (PGP, was also induced when cells were shifted to water. This gene is likely involved in cell membrane biosynthesis. We were unable to construct a PGP mutant suggesting that the gene is not only involved in survival in water but is essential for cell viability. We also examined mutants of polyhydroxybutyrate synthase (phbC, lipopolysaccharide (LPS oligosaccharide and capsule synthesis, and these mutations did not affect survival in water. LPS mutants lacking outer core were found to lose viability in water by 200 days indicating that an intact LPS core provides an outer membrane architecture which allows prolonged survival in water. Conclusion The results from these studies suggest that B. pseudomallei survival in water is a complex process that requires an LPS molecule which contains an intact core region.

  10. NCBI nr-aa BLAST: CBRC-AGAM-04-0109 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-04-0109 ref|ZP_01563785.1| NADH:flavin oxidoreductase/NADH oxidase [Burkholderia... cenocepacia MC0-3] gb|EAV58290.1| NADH:flavin oxidoreductase/NADH oxidase [Burkholderia cenocepacia ...MC0-3] gb|EAY65940.1| NADH:flavin oxidoreductase/NADH oxidase [Burkholderia cenocepacia PC184] ZP_01563785.1 4.6 26% ...

  11. The multiple roles of hypothetical gene BPSS1356 in Burkholderia pseudomallei.

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    Hokchai Yam

    Full Text Available Burkholderia pseudomallei is an opportunistic pathogen and the causative agent of melioidosis. It is able to adapt to harsh environments and can live intracellularly in its infected hosts. In this study, identification of transcriptional factors that associate with the β' subunit (RpoC of RNA polymerase was performed. The N-terminal region of this subunit is known to trigger promoter melting when associated with a sigma factor. A pull-down assay using histidine-tagged B. pseudomallei RpoC N-terminal region as bait showed that a hypothetical protein BPSS1356 was one of the proteins bound. This hypothetical protein is conserved in all B. pseudomallei strains and present only in the Burkholderia genus. A BPSS1356 deletion mutant was generated to investigate its biological function. The mutant strain exhibited reduced biofilm formation and a lower cell density during the stationary phase of growth in LB medium. Electron microscopic analysis revealed that the ΔBPSS1356 mutant cells had a shrunken cytoplasm indicative of cell plasmolysis and a rougher surface when compared to the wild type. An RNA microarray result showed that a total of 63 genes were transcriptionally affected by the BPSS1356 deletion with fold change values of higher than 4. The expression of a group of genes encoding membrane located transporters was concurrently down-regulated in ΔBPSS1356 mutant. Amongst the affected genes, the putative ion transportation genes were the most severely suppressed. Deprivation of BPSS1356 also down-regulated the transcriptions of genes for the arginine deiminase system, glycerol metabolism, type III secretion system cluster 2, cytochrome bd oxidase and arsenic resistance. It is therefore obvious that BPSS1356 plays a multiple regulatory roles on many genes.

  12. Burkholderia spp. are the most competitive symbionts of Mimosa, particularly under N-limited conditions.

    Science.gov (United States)

    Elliott, Geoffrey N; Chou, Jui-Hsing; Chen, Wen-Ming; Bloemberg, Guido V; Bontemps, Cyril; Martínez-Romero, Esperanza; Velázquez, Encarna; Young, J Peter W; Sprent, Janet I; James, Euan K

    2009-04-01

    Bacteria isolated from Mimosa nodules in Taiwan, Papua New Guinea, Mexico and Puerto Rico were identified as belonging to either the alpha- or beta-proteobacteria. The beta-proteobacterial Burkholderia and Cupriavidus strains formed effective symbioses with the common invasive species Mimosa diplotricha, M. pigra and M. pudica, but the alpha-proteobacterial Rhizobium etli and R. tropici strains produced a range of symbiotic phenotypes from no nodulation through ineffective to effective nodulation, depending on Mimosa species. Competition studies were performed between three of the alpha-proteobacteria (R. etli TJ167, R. tropici NGR181 and UPRM8021) and two of the beta-rhizobial symbionts (Burkholderia mimosarum PAS44 and Cupriavidus taiwanensis LMG19424) for nodulation of these invasive Mimosa species. Under flooded conditions, B. mimosarum PAS44 out-competed LMG19424 and all three alpha-proteobacteria to the point of exclusion. This advantage was not explained by initial inoculum levels, rates of bacterial growth, rhizobia-rhizobia growth inhibition or individual nodulation rate. However, the competitive domination of PAS44 over LMG19424 was reduced in the presence of nitrate for all three plant hosts. The largest significant effect was for M. pudica, in which LMG19424 formed 57% of the nodules in the presence of 0.5 mM potassium nitrate. In this host, ammonium also had a similar, but lesser, effect. Comparable results were also found using an N-containing soil mixture, and environmental N levels are therefore suggested as a factor in the competitive success of the bacterial symbiont in vivo.

  13. Burkholderia pseudomallei: A potential zoonosis in the southeastern United States

    Science.gov (United States)

    Burkholderia pseudomallei, the causative agent of melioidosis, is an underreported zoonosis in many countries where environmental conditions may be favorable for B. pseudomallei. This soil saprophyte is most often detected in tropical areas such as Southeast Asia and Northern Australia where the cas...

  14. Novel recombinant ethyl ferulate esterase from Burkholderia multivorans

    CSIR Research Space (South Africa)

    Rashamuse, KJ

    2007-11-01

    Full Text Available techniques. Detailed molecular identification based on species-specific primers and two conserved genes (16S rRNA and recA) led to the identification of the isolate as Burkholderia multivorans UWC10. A gene (designated estEFH5) encoding an EFH enzyme...

  15. Intrinsic Resistance of Burkholderia cepacia Complex to Benzalkonium Chloride

    OpenAIRE

    Youngbeom Ahn; Jeong Myeong Kim; Ohgew Kweon; Seong-Jae Kim; Jones, Richard C.; Kellie Woodling; Goncalo Gamboa da Costa; LiPuma, John J.; David Hussong; Marasa, Bernard S.; Cerniglia, Carl E.

    2016-01-01

    ABSTRACT Pharmaceutical products that are contaminated with Burkholderia cepacia complex (BCC) bacteria may pose serious consequences to vulnerable patients. Benzyldimethylalkylammonium chloride (BZK) cationic surfactants are extensively used in medical applications and have been implicated in the coselection of antimicrobial resistance. The ability of BCC to degrade BZK, tetradecyldimethylbenzylammonium chloride (C14BDMA-Cl), dodecyldimethylbenzylammonium chloride (C12BDMA-Cl), decyldimethyl...

  16. Symbiotic ß-proteobacteria beyond legumes: Burkholderia in Rubiaceae.

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    Brecht Verstraete

    Full Text Available Symbiotic ß-proteobacteria not only occur in root nodules of legumes but are also found in leaves of certain Rubiaceae. The discovery of bacteria in plants formerly not implicated in endosymbiosis suggests a wider occurrence of plant-microbe interactions. Several ß-proteobacteria of the genus Burkholderia are detected in close association with tropical plants. This interaction has occurred three times independently, which suggest a recent and open plant-bacteria association. The presence or absence of Burkholderia endophytes is consistent on genus level and therefore implies a predictive value for the discovery of bacteria. Only a single Burkholderia species is found in association with a given plant species. However, the endophyte species are promiscuous and can be found in association with several plant species. Most of the endophytes are part of the plant-associated beneficial and environmental group, but others are closely related to B. glathei. This soil bacteria, together with related nodulating and non-nodulating endophytes, is therefore transferred to a newly defined and larger PBE group within the genus Burkholderia.

  17. Melioidosis caused by Burkholderia pseudomallei in drinking water, Thailand, 2012.

    Science.gov (United States)

    Limmathurotsakul, Direk; Wongsuvan, Gumphol; Aanensen, David; Ngamwilai, Sujittra; Saiprom, Natnaree; Rongkard, Patpong; Thaipadungpanit, Janjira; Kanoksil, Manas; Chantratita, Narisara; Day, Nicholas P J; Peacock, Sharon J

    2014-02-01

    We identified 10 patients in Thailand with culture-confirmed melioidosis who had Burkholderia pseudomallei isolated from their drinking water. The multilocus sequence type of B. pseudomallei from clinical specimens and water samples were identical for 2 patients. This finding suggests that drinking water is a preventable source of B. pseudomallei infection.

  18. Burkholderia phymatum is a highly effective nitrogen-fixing symbiont of Mimosa spp. and fixes nitrogen ex planta.

    Science.gov (United States)

    Elliott, Geoffrey N; Chen, Wen-Ming; Chou, Jui-Hsing; Wang, Hui-Chun; Sheu, Shih-Yi; Perin, Liamara; Reis, Veronica M; Moulin, Lionel; Simon, Marcelo F; Bontemps, Cyril; Sutherland, Joan M; Bessi, Rosana; de Faria, Sergio M; Trinick, Michael J; Prescott, Alan R; Sprent, Janet I; James, Euan K

    2007-01-01

    * The ability of Burkholderia phymatum STM815 to effectively nodulate Mimosa spp., and to fix nitrogen ex planta, was compared with that of the known Mimosa symbiont Cupriavidus taiwanensis LMG19424. * Both strains were equally effective symbionts of M. pudica, but nodules formed by STM815 had greater nitrogenase activity. STM815 was shown to have a broader host range across the genus Mimosa than LMG19424, nodulating 30 out of 31 species, 21 of these effectively. LMG19424 effectively nodulated only nine species. GFP-marked variants were used to visualise symbiont presence within nodules. * STM815 gave significant acetylene reduction assay (ARA) activity in semisolid JMV medium ex planta, but no ARA activity was detected with LMG19424. 16S rDNA sequences of two isolates originally from Mimosa nodules in Papua New Guinea (NGR114 and NGR195A) identified them as Burkholderia phymatum also, with nodA, nodC and nifH genes of NGR195A identical to those of STM815. * B. phymatum is therefore an effective Mimosa symbiont with a broad host range, and is the first reported beta-rhizobial strain to fix nitrogen in free-living culture.

  19. Role of phosphate solubilizing Burkholderia spp. for successful colonization and growth promotion of Lycopodium cernuum L. (Lycopodiaceae) in lateritic belt of Birbhum district of West Bengal, India.

    Science.gov (United States)

    Ghosh, Ranjan; Barman, Soma; Mukherjee, Rajib; Mandal, Narayan C

    2016-02-01

    Profuse growth of Lycpodium cernuum L. was found in phosphate deficient red lateritic soil of West Bengal, India. Interaction of vesicular-arbuscular mycorrhiza (VAM) with Lycopodium rhizoids were described earlier but association of PGPR with their rhizoids were not studied. Three potent phosphate solubilizing bacterial strains (P4, P9 and P10) associated with L. cernuum rhizoids were isolated and identified by 16S rDNA homologies on Ez-Taxon database as Burkholderia tropica, Burkholderia unamae and Burkholderia cepacia respectively. Day wise kinetics of phosphate solubilization against Ca3(PO4)2 suggested P4 (580.56±13.38 μg ml(-1)) as maximum mineral phosphate solubilizer followed by P9 (517.12±17.15 μg ml(-1)) and P10 (485.18±14.23 μg ml(-1)) at 28 °C. Release of bound phosphates by isolated strains from ferric phosphate (FePO4), aluminum phosphate (AlPO4) and four different complex rock phosphates indicated their very good phosphate solubilizng efficacy. Nitrogen independent solubilizition also supports their nitrogen fixing capabilities. Inhibition of P solubilization by calcium salts and induction by EDTA suggested pH dependent chelation of metal cations by all of the isolates. Rhizoidal colonization potentials of Burkholderia spp. were confirmed by in planta experiment and also using scanning electron microscope (SEM). Increases of total phosphate content in Lycopodium plants upon soil treatment with these isolates were also recorded. In addition siderophore production on CAS agar medium, tryptophan dependent IAA production and antifungal activities against pathogenic fungi by rhizospheric isolates deep-rooted that they have definite role in nutrient mobilization for successful colonization of L. cernuum in nutrient deficient lateritic soil.

  20. Screening a mushroom extract library for activity against Acinetobacter baumannii and Burkholderia cepacia and the identification of a compound with anti-Burkholderia activity

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    Rott Marc

    2010-01-01

    Full Text Available Abstract Background Acinetobacter baumannii and species within the Burkholderia cepacia complex (BCC are significant opportunistic bacterial pathogens of humans. These species exhibit a high degree of antibiotic resistance, and some clinical isolates are resistant to all currently available antimicrobial drugs used for treatment. Thus, new drugs are needed to treat infections by these species. Mushrooms could be a potential source for new drugs to treat A. baumannii and BCC infections. Methods The aim of this study was to screen a library of crude extracts from 330 wild mushrooms by disk diffusion assays for antibacterial activity against A. baumannii and Burkholderia cepacia in the hope of identifying a novel natural drug that could be used to treat infections caused by these species. Once positive hits were identified, the extracts were subjected to bioassay-guided separations to isolate and identify the active drug molecules. MICs were performed to gauge the in vitro activity of the purified compounds. Results Only three crude extracts (0.9% had activity against A. baumannii and B. cepacia. Compounds from two of these extracts had MICs greater than 128 μg/ml, and further analyses were not performed. From the third extract, prepared from Leucopaxillus albissimus, 2-aminoquinoline (2-AQ was isolated. This compound exhibited a modest MIC in vitro against strains from nine different BCC species, including multi-drug resistant clinical isolates (MIC = 8-64 μg/ml, and a weak MIC (128 μg/ml against A baumannii. The IC50 against a murine monocyte line was 1.5 mg/ml. Conclusion The small number of positive hits in this study suggests that finding a new drug from mushrooms to treat Gram-negative bacterial infections may be difficult. Although 2-AQ was identified in one mushroom, and it was shown to inhibit the growth of multi-drug resistant BCC isolates, the relatively high MICs (8-128 μg/ml for both A. baumannii and BCC strains suggests that 2-AQ

  1. Exploring the Anti-Burkholderia cepacia Complex Activity of Essential Oils: A Preliminary Analysis

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    Isabel Maida

    2014-01-01

    Full Text Available In this work we have checked the ability of the essential oils extracted from six different medicinal plants (Eugenia caryophyllata, Origanum vulgare, Rosmarinus officinalis, Lavandula officinalis, Melaleuca alternifolia, and Thymus vulgaris to inhibit the growth of 18 bacterial type strains belonging to the 18 known species of the Burkholderia cepacia complex (Bcc. These bacteria are opportunistic human pathogens that can cause severe infection in immunocompromised patients, especially those affected by cystic fibrosis (CF, and are often resistant to multiple antibiotics. The analysis of the aromatograms produced by the six oils revealed that, in spite of their different chemical composition, all of them were able to contrast the growth of Bcc members. However, three of them (i.e., Eugenia caryophyllata, Origanum vulgare, and Thymus vulgaris were particularly active versus the Bcc strains, including those exhibiting a high degree or resistance to ciprofloxacin, one of the most used antibiotics to treat Bcc infections. These three oils are also active toward both environmental and clinical strains (isolated from CF patients, suggesting that they might be used in the future to fight B. cepacia complex infections.

  2. Bioleaching remediation of heavy metal-contaminated soils using Burkholderia sp. Z-90.

    Science.gov (United States)

    Yang, Zhihui; Zhang, Zhi; Chai, Liyuan; Wang, Yong; Liu, Yi; Xiao, Ruiyang

    2016-01-15

    Bioleaching is an environment-friendly and economical technology to remove heavy metals from contaminated soils. In this study, a biosurfactant-producing strain with capacity of alkaline production was isolated from cafeteria sewer sludge and its capability for removing Zn, Pb, Mn, Cd, Cu, and As was investigated. Phylogenetic analysis using 16S rDNA gene sequences confirmed that the strain belonged to Burkholderia sp. and named as Z-90. The biosurfactant was glycolipid confirmed by thin layer chromatography and Fourier-transform infrared spectroscopy. Z-90 broth was then used for bioleaching remediation of heavy metal-contaminated soils. The removal efficiency was 44.0% for Zn, 32.5% for Pb, 52.2% for Mn, 37.7% for Cd, 24.1% for Cu and 31.6% for As, respectively. Mn, Zn and Cd were more easily removed from soil than Cu, Pb and As, which was attributed to the presence of high acid-soluble fraction of Mn, Zn and Cd and high residual fraction of Cu, Pb and As. The heavy metal removal in soils was contributed to the adhesion of heavy metal-contaminated soil minerals with strain Z-90 and the formation of a metal complex with biosurfactant.

  3. Pyrrolnitrin from Burkholderia cepacia: antibiotic activity against fungi and novel activities against streptomycetes.

    Science.gov (United States)

    el-Banna, N; Winkelmann, G

    1998-07-01

    A bacterial strain identified as Burkholderia cepacia NB-1 was isolated from water ponds in the botanical garden in Tübingen, Germany, and was found to produce a broad spectrum phenylpyrrole antimicrobial substance active against filamentous fungi, yeasts and Gram-positive bacteria. In batch culture containing glycerol and L-glutamic acid, the isolate NB-1 produced the antibiotic optimally late in the growth phase and accumulated a main portion in their cells. Isolation and purification of the antibiotic from Burkholderia (Pseudomonas) cepacia NB-1 by acetone extraction, gel filtration on Sephadex LH-20 and preparative HPLC yielded 0.54 mg l-1 of a pure substance. Spectroscopic data (HPLC, MS and NMR) confirmed that the compound was pyrrolnitrin [3-chloro-4-(2'-nitro-3'-chloro-phenyl) pyrrole]. Pyrrolnitrin has an inhibitory effect on the electron transport system, as demonstrated by isolated mitochondria from Neurospora crassa 74 A. This inhibition was relieved by N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD), indicating that pyrrolnitrin blocked the electron transfer between the dehydrogenases and the cytochrome components of the respiratory chain. Among Gram-positive bacteria, pyrrolnitrin was most active against certain Streptomyces species, especially S. antibioticus, which has not previously been described in the literature. In the presence of pyrrolnitrin, aerial mycelium and spore formation of Strep. antibioticus was suppressed, although growth continued via substrate mycelium. The new findings of inhibition of streptomycetes and their secondary metabolism by pyrrolnitrin may contribute to the fact that Pseudomonas species predominate in soil and compete even with antibiotic-producing Streptomyces.

  4. Quantitative proteomic analysis of Burkholderia pseudomallei Bsa type III secretion system effectors using hypersecreting mutants.

    Science.gov (United States)

    Vander Broek, Charles W; Chalmers, Kevin J; Stevens, Mark P; Stevens, Joanne M

    2015-04-01

    Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. We constructed a mutant lacking bsaP, a homolog of the T3SS "gatekeeper" family of proteins that exert control over the timing and magnitude of effector protein secretion. Mutants lacking BsaP, or the T3SS translocon protein BipD, were observed to hypersecrete the known Bsa effector protein BopE, providing evidence of their role in post-translational control of the Bsa T3SS and representing key reagents for the identification of its secreted substrates. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hypersecreting mutants of B. pseudomallei with the isogenic parent strain and a bsaZ mutant incapable of effector protein secretion. Our study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei.

  5. Immobilization of Burkholderia sp. lipase on a ferric silica nanocomposite for biodiesel production.

    Science.gov (United States)

    Tran, Dang-Thuan; Chen, Ching-Lung; Chang, Jo-Shu

    2012-04-15

    In this work, lipase produced from an isolated strain Burkholderia sp. C20 was immobilized on magnetic nanoparticles to catalyze biodiesel synthesis. Core-shell nanoparticles were synthesized by coating Fe(3)O(4) core with silica shell. The nanoparticles treated with dimethyl octadecyl [3-(trimethoxysilyl) propyl] ammonium chloride were used as immobilization supporters. The Burkholderia lipase was then bound to the synthesized nanoparticles for immobilization. The protein binding efficiency on alkyl-functionalized Fe(3)O(4)-SiO(2) was estimated as 97%, while the efficiency was only 76% on non-modified Fe(3)O(4)-SiO(2). Maximum adsorption capacity of lipase on alkyl-functionalized Fe(3)O(4)-SiO(2) was estimated as 29.45 mg g(-1) based on Langmuir isotherm. The hydrolytic kinetics (using olive oil as substrate) of the lipase immobilized on alkyl-grafted Fe(3)O(4)-SiO(2) followed Michaelis-Menten model with a maximum reaction rate and a Michaelis constant of 6251 Ug(-1) and 3.65 mM, respectively. Physical and chemical properties of the nanoparticles and the immobilized lipase were characterized by Brunauer-Emmett-Teller (BET) analysis, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FT-IR). Moreover, the immobilized lipase was used to catalyze the transesterification of olive oil with methanol to produce fatty acid methyl esters (FAMEs), attaining a FAMEs conversion of over 90% within 30 h in batch operation when 11 wt% immobilized lipase was employed. The immobilized lipase could be used for ten cycles without significant loss in its transesterification activity.

  6. Comparison of the in vitro and in vivo susceptibilities of Burkholderia mallei to Ceftazidime and Levofloxacin

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    Torres Alfredo G

    2009-05-01

    Full Text Available Abstract Background Burkholderia mallei is a zoonotic Gram negative bacterium which primarily infects solipeds but can cause lethal disease in humans if left untreated. The effect of two antibiotics with different modes of action on Burkholderia mallei strain ATCC23344 was investigated by using in vitro and in vivo studies. Results Determination of minimal inhibitory concentrations (MICs in vitro was done by the agar diffusion method and the dilution method. The MICs of levofloxacin and ceftazidime were in the similar range, 2.5 and 5.0 μg/ml, respectively. Intracellular susceptibility of the bacterium to these two antibiotics in J774A.1 mouse macrophages in vitro was also investigated. Macrophages treated with antibiotics demonstrated uptake of the drugs and reduced bacterial loads in vitro. The efficacy of ceftazidime and levofloxacin were studied in BALB/c mice as post-exposure treatment following intranasal B. mallei infection. Intranasal infection with 5 × 105 CFUs of B. mallei resulted in 90% death in non-treated control mice. Antibiotic treatments 10 days post-infection proved to be effective in vivo with all antibiotic treated mice surviving to day 34 post-infection. The antibiotics did not result in complete clearance of the bacterial infection and presence of the bacteria was found in lungs and spleens of the survivors, although bacterial burden recovered from levofloxacin treated animals appeared reduced compared to ceftazidime. Conclusion Both antibiotics demonstrated utility for the treatment of glanders, including the ability for intracellular penetration and clearance of organisms in vitro.

  7. Characterization of the Burkholderia thailandensis SOS response by using whole-transcriptome shotgun sequencing.

    Science.gov (United States)

    Ulrich, Ricky L; Deshazer, David; Kenny, Tara A; Ulrich, Melanie P; Moravusova, Anna; Opperman, Timothy; Bavari, Sina; Bowlin, Terry L; Moir, Donald T; Panchal, Rekha G

    2013-10-01

    The bacterial SOS response is a well-characterized regulatory network encoded by most prokaryotic bacterial species and is involved in DNA repair. In addition to nucleic acid repair, the SOS response is involved in pathogenicity, stress-induced mutagenesis, and the emergence and dissemination of antibiotic resistance. Using high-throughput sequencing technology (SOLiD RNA-Seq), we analyzed the Burkholderia thailandensis global SOS response to the fluoroquinolone antibiotic, ciprofloxacin (CIP), and the DNA-damaging chemical, mitomycin C (MMC). We demonstrate that a B. thailandensis recA mutant (RU0643) is ∼4-fold more sensitive to CIP in contrast to the parental strain B. thailandensis DW503. Our RNA-Seq results show that CIP and MMC treatment (P SOS response were induced and include lexA, uvrA, dnaE, dinB, recX, and recA. At the genome-wide level, we found an overall decrease in gene expression, especially for genes involved in amino acid and carbohydrate transport and metabolism, following both CIP and MMC exposure. Interestingly, we observed the upregulation of several genes involved in bacterial motility and enhanced transcription of a B. thailandensis genomic island encoding a Siphoviridae bacteriophage designated E264. Using B. thailandensis plaque assays and PCR with B. mallei ATCC 23344 as the host, we demonstrate that CIP and MMC exposure in B. thailandensis DW503 induces the transcription and translation of viable bacteriophage in a RecA-dependent manner. This is the first report of the SOS response in Burkholderia spp. to DNA-damaging agents. We have identified both common and unique adaptive responses of B. thailandensis to chemical stress and DNA damage.

  8. Burkholderia pseudomallei known siderophores and hemin uptake are dispensable for lethal murine melioidosis.

    Directory of Open Access Journals (Sweden)

    Brian H Kvitko

    Full Text Available Burkholderia pseudomallei is a mostly saprophytic bacterium, but can infect humans where it causes the difficult-to-manage disease melioidosis. Even with proper diagnosis and prompt therapeutic interventions mortality rates still range from >20% in Northern Australia to over 40% in Thailand. Surprisingly little is yet known about how B. pseudomallei infects, invades and survives within its hosts, and virtually nothing is known about the contribution of critical nutrients such as iron to the bacterium's pathogenesis. It was previously assumed that B. pseudomallei used iron-acquisition systems commonly found in other bacteria, for example siderophores. However, our previous discovery of a clinical isolate carrying a large chromosomal deletion missing the entire malleobactin gene cluster encoding the bacterium's major high-affinity siderophore while still being fully virulent in a murine melioidosis model suggested that other iron-acquisition systems might make contributions to virulence. Here, we deleted the major siderophore malleobactin (mba and pyochelin (pch gene clusters in strain 1710b and revealed a residual siderophore activity which was unrelated to other known Burkholderia siderophores such as cepabactin and cepaciachelin, and not due to increased secretion of chelators such as citrate. Deletion of the two hemin uptake loci, hmu and hem, showed that Hmu is required for utilization of hemin and hemoglobin and that Hem cannot complement a Hmu deficiency. Prolonged incubation of a hmu hem mutant in hemoglobin-containing minimal medium yielded variants able to utilize hemoglobin and hemin suggesting alternate pathways for utilization of these two host iron sources. Lactoferrin utilization was dependent on malleobactin, but not pyochelin synthesis and/or uptake. A mba pch hmu hem quadruple mutant could use ferritin as an iron source and upon intranasal infection was lethal in an acute murine melioidosis model. These data suggest that B

  9. Bacterial Cell Wall Synthesis Gene uppP Is Required for Burkholderia Colonization of the Stinkbug Gut

    Science.gov (United States)

    Kim, Jiyeun Kate; Lee, Ho Jin; Kikuchi, Yoshitomo; Kitagawa, Wataru; Nikoh, Naruo

    2013-01-01

    To establish a host-bacterium symbiotic association, a number of factors involved in symbiosis must operate in a coordinated manner. In insects, bacterial factors for symbiosis have been poorly characterized at the molecular and biochemical levels, since many symbionts have not yet been cultured or are as yet genetically intractable. Recently, the symbiotic association between a stinkbug, Riptortus pedestris, and its beneficial gut bacterium, Burkholderia sp., has emerged as a promising experimental model system, providing opportunities to study insect symbiosis using genetically manipulated symbiotic bacteria. Here, in search of bacterial symbiotic factors, we targeted cell wall components of the Burkholderia symbiont by disruption of uppP gene, which encodes undecaprenyl pyrophosphate phosphatase involved in biosynthesis of various bacterial cell wall components. Under culture conditions, the ΔuppP mutant showed higher susceptibility to lysozyme than the wild-type strain, indicating impaired integrity of peptidoglycan of the mutant. When administered to the host insect, the ΔuppP mutant failed to establish normal symbiotic association: the bacterial cells reached to the symbiotic midgut but neither proliferated nor persisted there. Transformation of the ΔuppP mutant with uppP-encoding plasmid complemented these phenotypic defects: lysozyme susceptibility in vitro was restored, and normal infection and proliferation in the midgut symbiotic organ were observed in vivo. The ΔuppP mutant also exhibited susceptibility to hypotonic, hypertonic, and centrifugal stresses. These results suggest that peptidoglycan cell wall integrity is a stress resistance factor relevant to the successful colonization of the stinkbug midgut by Burkholderia symbiont. PMID:23747704

  10. Identification and Antagonism Study of a Novel Chitinase-producing Bacterium Burkholderia Sp.C3 against Phytopathogenic Fungi

    Institute of Scientific and Technical Information of China (English)

    金虹; TAO Yong

    2006-01-01

    Through a modified agar well diffusion assay, antagonism of a novel chitinase-producing strain C3 against the phytopathogenic fungi including Phoma wasabiae Yokogi,Heterostrophus, Exserohilum Turcicum, Curwularia (Walk) Boed, Thantephorus cucumris, Fusarium graminearum was tested. The data showed that the crude cxtracts of strain C3 had stable antifungal activity in the range of pH 5.0 to pH 8.0. The active components were heat labile and sensitive to proteinase K. A series of experiments supported that the compound responsible for inhibitory activity appeared to be ehitinase. The 16s rDNA analysis indicated that C3 was subject to genus Burkholderia. Pbenotypic characterization of C3 was also consisted with the result of molecular identification.

  11. Characterization of integrons in Burkholderia cepacia clinical isolates

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    Linda Furlanis

    2010-03-01

    Full Text Available Burkholderia cepacia is an opportunistic pathogen able to colonize the airways of Cystic Fibrosis (CF patients, frequently developing chronic infections. In 20% of cases these infections cause severe and poorly controlled pathological situations because of the intrinsic antibiotic resistance expressed by the microorganism. CF patients are often subjected to antibiotic therapy: this facilitates the acquisition of antibiotic resistance determinants by the infecting bacteria. Integrons are mobile genetic elements that are widespread in bacterial populations and favor the acquisition of gene cassettes coding for these determinants.The presence of class 1 integrons was investigated by PCR with primers specific for the 5’ and 3’ ends in Burkholderia isolates recovered from patients in treatment at the CF center of Friuli Venezia Giulia. The same integron, carrying an uncommon allelic form (Ib of the aacA4 gene in its cassette array and conferring resistance to some aminoglycosides, was found in two independent isolates (different RAPD profiles infecting two different patients. In both isolates the integron was carried by plasmids and was still present 3 and 6 years later the first finding. Despite the exchange of integrons between bacterial pathogens is fully described, these items were not frequently found in Burkholderia isolates. Although the clinical relevance of the integron we identified is low (a single gene cassette encoding a widespread resistance,we feel concerned that these genetic elements begin to circulate in this bacterial species, as this could make more and more troublesome the treatment of infections notoriously difficult to eradicate.

  12. Stress conditions triggering mucoid morphotype variation in Burkholderia species and effect on virulence in Galleria mellonella and biofilm formation in vitro.

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    Inês N Silva

    Full Text Available Burkholderia cepacia complex (Bcc bacteria are opportunistic pathogens causing chronic respiratory infections particularly among cystic fibrosis patients. During these chronic infections, mucoid-to-nonmucoid morphotype variation occurs, with the two morphotypes exhibiting different phenotypic properties. Here we show that in vitro, the mucoid clinical isolate Burkholderia multivorans D2095 gives rise to stable nonmucoid variants in response to prolonged stationary phase, presence of antibiotics, and osmotic and oxidative stresses. Furthermore, in vitro colony morphotype variation within other members of the Burkholderia genus occurred in Bcc and non-Bcc strains, irrespectively of their clinical or environmental origin. Survival to starvation and iron limitation was comparable for the mucoid parental isolate and the respective nonmucoid variant, while susceptibility to antibiotics and to oxidative stress was increased in the nonmucoid variants. Acute infection of Galleria mellonella larvae showed that, in general, the nonmucoid variants were less virulent than the respective parental mucoid isolate, suggesting a role for the exopolysaccharide in virulence. In addition, most of the tested nonmucoid variants produced more biofilm biomass than their respective mucoid parental isolate. As biofilms are often associated with increased persistence of pathogens in the CF lungs and are an indicative of different cell-to-cell interactions, it is possible that the nonmucoid variants are better adapted to persist in this host environment.

  13. Stress conditions triggering mucoid morphotype variation in Burkholderia species and effect on virulence in Galleria mellonella and biofilm formation in vitro.

    Science.gov (United States)

    Silva, Inês N; Tavares, Andreia C; Ferreira, Ana S; Moreira, Leonilde M

    2013-01-01

    Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens causing chronic respiratory infections particularly among cystic fibrosis patients. During these chronic infections, mucoid-to-nonmucoid morphotype variation occurs, with the two morphotypes exhibiting different phenotypic properties. Here we show that in vitro, the mucoid clinical isolate Burkholderia multivorans D2095 gives rise to stable nonmucoid variants in response to prolonged stationary phase, presence of antibiotics, and osmotic and oxidative stresses. Furthermore, in vitro colony morphotype variation within other members of the Burkholderia genus occurred in Bcc and non-Bcc strains, irrespectively of their clinical or environmental origin. Survival to starvation and iron limitation was comparable for the mucoid parental isolate and the respective nonmucoid variant, while susceptibility to antibiotics and to oxidative stress was increased in the nonmucoid variants. Acute infection of Galleria mellonella larvae showed that, in general, the nonmucoid variants were less virulent than the respective parental mucoid isolate, suggesting a role for the exopolysaccharide in virulence. In addition, most of the tested nonmucoid variants produced more biofilm biomass than their respective mucoid parental isolate. As biofilms are often associated with increased persistence of pathogens in the CF lungs and are an indicative of different cell-to-cell interactions, it is possible that the nonmucoid variants are better adapted to persist in this host environment.

  14. Burkholderia bacteria infectiously induce the proto-farming symbiosis of Dictyostelium amoebae and food bacteria.

    Science.gov (United States)

    DiSalvo, Susanne; Haselkorn, Tamara S; Bashir, Usman; Jimenez, Daniela; Brock, Debra A; Queller, David C; Strassmann, Joan E

    2015-09-01

    Symbiotic associations can allow an organism to acquire novel traits by accessing the genetic repertoire of its partner. In the Dictyostelium discoideum farming symbiosis, certain amoebas (termed "farmers") stably associate with bacterial partners. Farmers can suffer a reproductive cost but also gain beneficial capabilities, such as carriage of bacterial food (proto-farming) and defense against competitors. Farming status previously has been attributed to amoeba genotype, but the role of bacterial partners in its induction has not been examined. Here, we explore the role of bacterial associates in the initiation, maintenance, and phenotypic effects of the farming symbiosis. We demonstrate that two clades of farmer-associated Burkholderia isolates colonize D. discoideum nonfarmers and infectiously endow them with farmer-like characteristics, indicating that Burkholderia symbionts are a major driver of the farming phenomenon. Under food-rich conditions, Burkholderia-colonized amoebas produce fewer spores than uncolonized counterparts, with the severity of this reduction being dependent on the Burkholderia colonizer. However, the induction of food carriage by Burkholderia colonization may be considered a conditionally adaptive trait because it can confer an advantage to the amoeba host when grown in food-limiting conditions. We observed Burkholderia inside and outside colonized D. discoideum spores after fruiting body formation; this observation, together with the ability of Burkholderia to colonize new amoebas, suggests a mixed mode of symbiont transmission. These results change our understanding of the D. discoideum farming symbiosis by establishing that the bacterial partner, Burkholderia, is an important causative agent of the farming phenomenon.

  15. Functional Characterization of OXA-57, a Class D β-Lactamase from Burkholderia pseudomallei

    OpenAIRE

    Keith, Karen E.; Oyston, Petra C.; Crossett, Ben; Fairweather, Neil F.; Titball, Richard W.; Walsh, Timothy R.; Brown, Katherine A.

    2005-01-01

    Class D β-lactamase OXA-57 was identified in a range of isolates of Burkholderia pseudomallei and Burkholderia thailandensis. Comparative kinetic analyses of wild-type and mutant forms of B. pseudomallei OXA-57 are reported. Implications of these data for β-lactam resistance and the proposed role of Ser-104 in β-lactam hydrolysis are discussed.

  16. Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes.

    Science.gov (United States)

    De Meyer, Sofie E; Briscoe, Leah; Martínez-Hidalgo, Pilar; Agapakis, Christina M; de-Los Santos, Paulina Estrada; Seshadri, Rekha; Reeve, Wayne; Weinstock, George; O'Hara, Graham; Howieson, John G; Hirsch, Ann M

    2016-08-01

    Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.

  17. Genetic diversity of Mimosa pudica rhizobial symbionts in soils of French Guiana: investigating the origin and diversity of Burkholderia phymatum and other beta-rhizobia.

    Science.gov (United States)

    Mishra, Ravi P N; Tisseyre, Pierre; Melkonian, Rémy; Chaintreuil, Clémence; Miché, Lucie; Klonowska, Agnieszka; Gonzalez, Sophie; Bena, Gilles; Laguerre, Gisèle; Moulin, Lionel

    2012-02-01

    The genetic diversity of 221 Mimosa pudica bacterial symbionts trapped from eight soils from diverse environments in French Guiana was assessed by 16S rRNA PCR-RFLP, REP-PCR fingerprints, as well as by phylogenies of their 16S rRNA and recA housekeeping genes, and by their nifH, nodA and nodC symbiotic genes. Interestingly, we found a large diversity of beta-rhizobia, with Burkholderia phymatum and Burkholderia tuberum being the most frequent and diverse symbiotic species. Other species were also found, such as Burkholderia mimosarum, an unnamed Burkholderia species and, for the first time in South America, Cupriavidus taiwanensis. The sampling site had a strong influence on the diversity of the symbionts sampled, and the specific distributions of symbiotic populations between the soils were related to soil composition in some cases. Some alpha-rhizobial strains taxonomically close to Rhizobium endophyticum were also trapped in one soil, and these carried two copies of the nodA gene, a feature not previously reported. Phylogenies of nodA, nodC and nifH genes showed a monophyly of symbiotic genes for beta-rhizobia isolated from Mimosa spp., indicative of a long history of interaction between beta-rhizobia and Mimosa species. Based on their symbiotic gene phylogenies and legume hosts, B. tuberum was shown to contain two large biovars: one specific to the mimosoid genus Mimosa and one to South African papilionoid legumes. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  18. A Burkholderia sacchari cell factory: production of poly-3-hydroxybutyrate, xylitol and xylonic acid from xylose-rich sugar mixtures.

    Science.gov (United States)

    Raposo, Rodrigo S; de Almeida, M Catarina M D; de Oliveira, M da Conceição M A; da Fonseca, M Manuela; Cesário, M Teresa

    2017-01-25

    Efficient production of poly-3-hydroxybutyrate (P(3HB)) based on glucose-xylose mixtures simulating different types of lignocellulosic hydrolysate (LCH) was addressed using Burkholderia sacchari, a wild strain capable of metabolizing both sugars and producing P(3HB). Carbon catabolite repression was avoided by maintaining glucose concentration below 10g/L. Xylose concentrations above 30g/L were inhibitory for growth and production. In fed-batch cultivations, pulse size and feed addition rate were controlled in order to reach high productivities and efficient sugar consumptions. High xylose uptake and P(3HB) productivity were attained with glucose-rich mixtures (glucose/xylose ratio in the feed=1.5w/w) using high feeding rates, while with xylose-richer feeds (glucose/xylose=0.8w/w), a lower feeding rate is a robust strategy to avoid xylose build-up in the medium. Xylitol production was observed with xylose concentrations in the medium above 30-40g/L. With sugar mixtures featuring even lower glucose/xylose ratios, i.e. xylose-richer feeds (glucose/xylose=0.5), xylonic acid (a second byproduct) was produced. This is the first report of the ability of Burkholderia sacchari to produce both xylitol and xylonic acid. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Aerogenic vaccination with a Burkholderia mallei auxotroph protects against aerosol-initiated glanders in mice.

    Science.gov (United States)

    Ulrich, Ricky L; Amemiya, Kei; Waag, David M; Roy, Chad J; DeShazer, David

    2005-03-14

    Burkholderia mallei is an obligate mammalian pathogen that causes the zoonotic disease glanders. Two live attenuated B. mallei strains, a capsule mutant and a branched-chain amino acid auxotroph, were evaluated for use as vaccines against aerosol-initiated glanders in mice. Animals were aerogenically vaccinated and serum samples were obtained before aerosol challenge with a high-dose (>300 times the LD50) of B. mallei ATCC 23344. Mice vaccinated with the capsule mutant developed a Th2-like Ig subclass antibody response and none survived beyond 5 days. In comparison, the auxotrophic mutant elicited a Th1-like Ig subclass antibody response and 25% of the animals survived for 1 month postchallenge. After a low-dose (5 times the LD50) aerosol challenge, the survival rates of auxotroph-vaccinated and unvaccinated animals were 50 and 0%, respectively. Thus, live attenuated strains that promote a Th1-like Ig response may serve as promising vaccine candidates against aerosol infection with B. mallei.

  20. Strategies for PCR based detection of Burkholderia pseudomallei DNA in paraffin wax embedded tissues.

    Science.gov (United States)

    Hagen, R M; Gauthier, Y P; Sprague, L D; Vidal, D R; Zysk, G; Finke, E-J; Neubauer, H

    2002-12-01

    Recently, several cases of melioidosis imported to Europe have been reported. The diagnosis of the acute or chronic infection remains challenging. This report describes an optimised protocol for fast and reliable DNA preparation for use in two different polymerase chain reaction (PCR) assays, namely: (1) a seminested PCR assay targeting a genus specific sequence of the ribosomal protein subunit 21 (rpsU) gene and (2) a nested PCR assay targeting the gene encoding the filament forming flagellin (fliC). Various strains of Burkholderia spp, strains of closely related genera, and spleen tissue samples of experimentally infected mice were investigated. The combination of PCR and sequencing of the amplicons resulted in high sensitivity and specificity. These procedures may allow rapid, sensitive, and reliable detection of B pseudomallei DNA in routinely formalin fixed and paraffin wax embedded samples, thus providing a safe diagnostic tool and avoiding the cultivation of a risk group 3 agent. In addition, this method could be useful for retrospective histopathological investigations.

  1. Survival and susceptibility of Burkholderia cepacia complex in chlorhexidine gluconate and benzalkonium chloride.

    Science.gov (United States)

    Kim, Jeong Myeong; Ahn, Youngbeom; LiPuma, John J; Hussong, David; Cerniglia, Carl E

    2015-06-01

    The Burkholderia cepacia complex (BCC) includes opportunistic pathogenic bacteria that have occasionally been recovered from various pharmaceutical products, including antiseptics and disinfectants. Plausible reasons for the contamination include intrinsic sources, such as inadequate process controls, especially for water or equipment used during product manufacture, or extrinsic sources, such as improper handling and dilution or distribution in contaminated containers. Because the survival of BCC in antiseptics is a concern to the public health and pharmaceutical industry, we determined minimum inhibitory concentrations (MICs) of 36 BCC strains against the antiseptics, following exposure to chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions (1-500 µg/ml for each chemical). Susceptibility to CHX and BZK varied across the BCC strains and was recorded as mean 90.3 and 111.1 µg/ml, respectively, at initial inoculation, which was significantly higher than the 46.4 and 61.1 µg/ml levels measured for BCC incubated in water for 40 days. After determining antiseptic MICs of individual BCC strains, BCC recovery was measured on Tryptic Soy Agar (TSA), Reasoner's Second Agar (R2A) and diluted preparations of these media under their sub-MICs. The survival of BCC was monitored for 14 days (336 h) in sub-MICs diluted to less than their antiseptic susceptible concentration value. Diluted TSA and R2A media exhibited greater efficiency of recovery for most BCC strains from the CHX and BZK solutions than full strength TSA or R2A. For BCC survival in antiseptic solutions, the cell number of BCC decreased rapidly within the first 20 min in both antiseptics, but after this, recovery remained constant in CHX and increased in BZK over the 14 day incubation period. The results indicate that BCC in water can remain viable with low susceptibility to antiseptics for 14 days, which suggests the necessity for improved detection methods and control measures to monitor

  2. A Burkholderia pseudomallei outer membrane vesicle vaccine provides protection against lethal sepsis.

    Science.gov (United States)

    Nieves, Wildaliz; Petersen, Hailey; Judy, Barbara M; Blumentritt, Carla A; Russell-Lodrigue, Kasi; Roy, Chad J; Torres, Alfredo G; Morici, Lisa A

    2014-05-01

    The environmental Gram-negative encapsulated bacillus Burkholderia pseudomallei is the causative agent of melioidosis, a disease associated with high morbidity and mortality rates in areas of Southeast Asia and northern Australia in which the disease is endemic. B. pseudomallei is also classified as a tier I select agent due to the high level of lethality of the bacterium and its innate resistance to antibiotics, as well as the lack of an effective vaccine. Gram-negative bacteria, including B. pseudomallei, secrete outer membrane vesicles (OMVs) which are enriched with multiple protein, lipid, and polysaccharide antigens. Previously, we demonstrated that immunization with multivalent B. pseudomallei-derived OMVs protects highly susceptible BALB/c mice against an otherwise lethal aerosol challenge. In this work, we evaluated the protective efficacy of OMV immunization against intraperitoneal challenge with a heterologous strain because systemic infection with phenotypically diverse environmental B. pseudomallei strains poses another hazard and a challenge to vaccine development. We demonstrated that B. pseudomallei OMVs derived from strain 1026b afforded significant protection against septicemic infection with B. pseudomallei strain K96243. OMV immunization induced robust OMV-, lipopolysaccharide-, and capsular polysaccharide-specific serum IgG (IgG1, IgG2a, and IgG3) and IgM antibody responses. OMV-immune serum promoted bacterial killing in vitro, and passive transfer of B. pseudomallei OMV immune sera protected naive mice against a subsequent challenge. These results indicate that OMV immunization provides antibody-mediated protection against acute, rapidly lethal sepsis in mice. B. pseudomallei-derived OMVs may represent an efficacious multivalent vaccine strategy against melioidosis.

  3. Molecular signatures and phylogenomic analysis of the genus Burkholderia: proposal for division of this genus into the emended genus Burkholderia containing pathogenic organisms and a new genus Paraburkholderia gen. nov. harboring environmental species.

    Science.gov (United States)

    Sawana, Amandeep; Adeolu, Mobolaji; Gupta, Radhey S

    2014-01-01

    The genus Burkholderia contains large number of diverse species which include many clinically important organisms, phytopathogens, as well as environmental species. However, currently, there is a paucity of biochemical or molecular characteristics which can reliably distinguish different groups of Burkholderia species. We report here the results of detailed phylogenetic and comparative genomic analyses of 45 sequenced species of the genus Burkholderia. In phylogenetic trees based upon concatenated sequences for 21 conserved proteins as well as 16S rRNA gene sequence based trees, members of the genus Burkholderia grouped into two major clades. Within these main clades a number of smaller clades including those corresponding to the clinically important Burkholderia cepacia complex (BCC) and the Burkholderia pseudomallei groups were also clearly distinguished. Our comparative analysis of protein sequences from Burkholderia spp. has identified 42 highly specific molecular markers in the form of conserved sequence indels (CSIs) that are uniquely found in a number of well-defined groups of Burkholderia spp. Six of these CSIs are specific for a group of Burkholderia spp. (referred to as Clade I in this work) which contains all clinically relevant members of the genus (viz. the BCC and the B. pseudomallei group) as well as the phytopathogenic Burkholderia spp. The second main clade (Clade II), which is composed of environmental Burkholderia species, is also distinguished by 2 identified CSIs that are specific for this group. Additionally, our work has also identified multiple CSIs that serve to clearly demarcate a number of smaller groups of Burkholderia spp. including 3 CSIs that are specific for the B. cepacia complex, 4 CSIs that are uniquely found in the B. pseudomallei group, 5 CSIs that are specific for the phytopathogenic Burkholderia spp. and 22 other CSI that distinguish two groups within Clade II. The described molecular markers provide highly specific means for

  4. Burkholderia glumae EN EL CULTIVO DE ARROZ EN COSTA RICA

    Directory of Open Access Journals (Sweden)

    Andrea Quesada-Gonz\\u00E1lez

    2014-01-01

    Full Text Available Burkholderia glumae en el cultivo de arroz en Costa Rica. El objetivo de este trabajo fue determinar la presencia de Burkholderia glumae en arroz en Costa Rica. La bacteria Burkholderia glumae está asociada al cultivo del arroz en el que provoca la enfermedad llamada añublo bacterial. Bajo condiciones ambientales favorables, la densidad bacteriana aumenta, lo que provoca que, bajo un sistema de regulación denominado quorum sensing, se expresen sus mecanismos de virulencia mediante la activación de genes responsables para la síntesis de la toxoflavina, que bloquea el flujo de nutrientes, para la biogénesis de flagelos y la respuesta quimiotáctica, y la producción de la enzima catalasa. Las plantas desarrollan la sintomatología que finalmente conlleva a un vaneamiento del grano provocando pérdidas económicas importantes. Se investigó la situación referente a la contaminación del grano de arroz causado por esta bacteria en Costa Rica durante los años 2009 y 2010, mediante un convenio entre la Corporación Nacional Arrocera y el Laboratorio de Fitopatología del Centro de Investigación en Protección de Cultivos de la Universidad de Costa Rica. Se usó la metodología de PCR de punto final recomendada por investigadores del Centro Internacional de Agricultura Tropical en Colombia y se reforzó la identificación, por medio de técnicas de microbiología convencional. Se obtuvieron resultados que indican la presencia de la bacteria en Costa Rica, la primera información sobre la prevalencia de un fitopatógeno bacteriano de gran importancia para el sector arrocero.

  5. NCBI nr-aa BLAST: CBRC-PMAR-01-0179 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PMAR-01-0179 ref|ZP_01560690.1| hypothetical protein Bcenmc03DRAFT_2689 [Burkh...olderia cenocepacia MC0-3] gb|EAV61335.1| hypothetical protein Bcenmc03DRAFT_2689 [Burkholderia cenocepacia MC0-3] ZP_01560690.1 3e-07 28% ...

  6. NCBI nr-aa BLAST: CBRC-BTAU-01-2512 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-2512 ref|ZP_01560879.1| hypothetical protein Bcenmc03DRAFT_2878 [Burkh...olderia cenocepacia MC0-3] gb|EAV61524.1| hypothetical protein Bcenmc03DRAFT_2878 [Burkholderia cenocepacia MC0-3] ZP_01560879.1 0.005 27% ...

  7. NCBI nr-aa BLAST: CBRC-XTRO-01-2967 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-2967 ref|ZP_01566576.1| hypothetical protein Bcenmc03DRAFT_1196 [Burkh...olderia cenocepacia MC0-3] gb|EAV55453.1| hypothetical protein Bcenmc03DRAFT_1196 [Burkholderia cenocepacia MC0-3] ZP_01566576.1 4e-25 22% ...

  8. NCBI nr-aa BLAST: CBRC-FCAT-01-0851 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-FCAT-01-0851 ref|ZP_01560690.1| hypothetical protein Bcenmc03DRAFT_2689 [Burkh...olderia cenocepacia MC0-3] gb|EAV61335.1| hypothetical protein Bcenmc03DRAFT_2689 [Burkholderia cenocepacia MC0-3] ZP_01560690.1 0.039 33% ...

  9. NCBI nr-aa BLAST: CBRC-AGAM-03-0017 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-03-0017 ref|ZP_01562088.1| hypothetical protein Bcenmc03DRAFT_2302 [Burkh...olderia cenocepacia MC0-3] gb|EAV60091.1| hypothetical protein Bcenmc03DRAFT_2302 [Burkholderia cenocepacia MC0-3] ZP_01562088.1 4e-04 26% ...

  10. NCBI nr-aa BLAST: CBRC-AGAM-01-0052 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-01-0052 ref|ZP_01564777.1| hypothetical protein Bcenmc03DRAFT_4887 [Burkh...olderia cenocepacia MC0-3] gb|EAV57364.1| hypothetical protein Bcenmc03DRAFT_4887 [Burkholderia cenocepacia MC0-3] ZP_01564777.1 5e-05 28% ...

  11. NCBI nr-aa BLAST: CBRC-FCAT-01-0154 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-FCAT-01-0154 ref|ZP_01560690.1| hypothetical protein Bcenmc03DRAFT_2689 [Burkh...olderia cenocepacia MC0-3] gb|EAV61335.1| hypothetical protein Bcenmc03DRAFT_2689 [Burkholderia cenocepacia MC0-3] ZP_01560690.1 0.039 33% ...

  12. NCBI nr-aa BLAST: CBRC-OCUN-01-1280 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OCUN-01-1280 ref|ZP_01566981.1| hypothetical protein Bcenmc03DRAFT_7238 [Burkh...olderia cenocepacia MC0-3] gb|EAV54955.1| hypothetical protein Bcenmc03DRAFT_7238 [Burkholderia cenocepacia MC0-3] ZP_01566981.1 1.1 38% ...

  13. NCBI nr-aa BLAST: CBRC-XTRO-01-3129 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-3129 ref|ZP_01566576.1| hypothetical protein Bcenmc03DRAFT_1196 [Burkh...olderia cenocepacia MC0-3] gb|EAV55453.1| hypothetical protein Bcenmc03DRAFT_1196 [Burkholderia cenocepacia MC0-3] ZP_01566576.1 2e-37 47% ...

  14. NCBI nr-aa BLAST: CBRC-XTRO-01-0105 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0105 ref|ZP_01566576.1| hypothetical protein Bcenmc03DRAFT_1196 [Burkh...olderia cenocepacia MC0-3] gb|EAV55453.1| hypothetical protein Bcenmc03DRAFT_1196 [Burkholderia cenocepacia MC0-3] ZP_01566576.1 4e-12 21% ...

  15. NCBI nr-aa BLAST: CBRC-CBRE-01-0787 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CBRE-01-0787 ref|YP_840388.1| PE-PGRS family protein [Burkholderia cenocepacia... HI2424] gb|ABK13495.1| PE-PGRS family protein [Burkholderia cenocepacia HI2424] YP_840388.1 7e-04 28% ...

  16. NCBI nr-aa BLAST: CBRC-CJAC-01-0283 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CJAC-01-0283 ref|YP_840388.1| PE-PGRS family protein [Burkholderia cenocepacia... HI2424] gb|ABK13495.1| PE-PGRS family protein [Burkholderia cenocepacia HI2424] YP_840388.1 2e-09 26% ...

  17. NCBI nr-aa BLAST: CBRC-XTRO-01-0143 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0143 ref|ZP_01564992.1| hypothetical protein Bcenmc03DRAFT_7449 [Burkholder...ia cenocepacia MC0-3] gb|EAV57107.1| hypothetical protein Bcenmc03DRAFT_7449 [Burkholderia cenocepacia MC0-3] ZP_01564992.1 0.002 23% ...

  18. NCBI nr-aa BLAST: CBRC-CJAC-01-1085 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CJAC-01-1085 ref|ZP_01559985.1| conserved hypothetical protein [Burkholderia c...enocepacia MC0-3] gb|EAV61841.1| conserved hypothetical protein [Burkholderia cenocepacia MC0-3] ZP_01559985.1 2e-58 63% ...

  19. NCBI nr-aa BLAST: CBRC-PVAM-01-0739 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PVAM-01-0739 ref|YP_626364.1| PE-PGRS family protein [Burkholderia cenocepacia... AU 1054] gb|ABF81391.1| PE-PGRS family protein [Burkholderia cenocepacia AU 1054] YP_626364.1 5e-10 26% ...

  20. NCBI nr-aa BLAST: CBRC-FRUB-02-0738 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-FRUB-02-0738 ref|ZP_01567160.1| Haemagluttinin motif [Burkholderia cenocepacia... MC0-3] gb|EAV54748.1| Haemagluttinin motif [Burkholderia cenocepacia MC0-3] ZP_01567160.1 4e-24 29% ...

  1. NCBI nr-aa BLAST: CBRC-DMEL-08-0031 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DMEL-08-0031 ref|YP_838849.1| Haemagluttinin domain protein [Burkholderia ceno...cepacia HI2424] gb|ABK11956.1| Haemagluttinin domain protein [Burkholderia cenocepacia HI2424] YP_838849.1 3e-09 31% ...

  2. NCBI nr-aa BLAST: CBRC-CREM-01-1362 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1362 ref|ZP_01564035.1| 200 kDa antigen p200, putative [Burkholderia c...enocepacia MC0-3] gb|EAV58079.1| 200 kDa antigen p200, putative [Burkholderia cenocepacia MC0-3] ZP_01564035.1 7e-36 33% ...

  3. NCBI nr-aa BLAST: CBRC-DSIM-01-0065 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DSIM-01-0065 ref|ZP_01559985.1| conserved hypothetical protein [Burkholderia c...enocepacia MC0-3] gb|EAV61841.1| conserved hypothetical protein [Burkholderia cenocepacia MC0-3] ZP_01559985.1 8e-50 46% ...

  4. NCBI nr-aa BLAST: CBRC-RNOR-23-0023 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-RNOR-23-0023 ref|YP_840446.1| YadA C-terminal domain protein [Burkholderia cen...ocepacia HI2424] gb|ABK13553.1| YadA C-terminal domain protein [Burkholderia cenocepacia HI2424] YP_840446.1 1.0 31% ...

  5. NCBI nr-aa BLAST: CBRC-AGAM-03-0072 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-03-0072 ref|ZP_01560354.1| hypothetical protein Bcenmc03DRAFT_5869 [Burkholderia... cenocepacia MC0-3] gb|EAV62210.1| hypothetical protein Bcenmc03DRAFT_5869 [Burkholderia cenocepacia MC0-3] ZP_01560354.1 0.002 26% ...

  6. NCBI nr-aa BLAST: CBRC-PHAM-01-1691 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PHAM-01-1691 ref|YP_001773964.1| hypothetical protein Bcenmc03_6354 [Burkholderia... cenocepacia MC0-3] gb|ACA95469.1| conserved hypothetical protein [Burkholderia cenocepacia MC0-3] YP_001773964.1 2e-67 45% ...

  7. NCBI nr-aa BLAST: CBRC-FCAT-01-1259 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-FCAT-01-1259 ref|ZP_01559985.1| conserved hypothetical protein [Burkholderia c...enocepacia MC0-3] gb|EAV61841.1| conserved hypothetical protein [Burkholderia cenocepacia MC0-3] ZP_01559985.1 1e-19 42% ...

  8. NCBI nr-aa BLAST: CBRC-RNOR-09-0007 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-RNOR-09-0007 ref|YP_621794.1| hypothetical protein Bcen_1918 [Burkholderia cen...ocepacia AU 1054] gb|ABF76821.1| conserved hypothetical protein [Burkholderia cenocepacia AU 1054] YP_621794.1 1e-53 70% ...

  9. NCBI nr-aa BLAST: CBRC-BTAU-01-1684 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-1684 ref|YP_838849.1| Haemagluttinin domain protein [Burkholderia ceno...cepacia HI2424] gb|ABK11956.1| Haemagluttinin domain protein [Burkholderia cenocepacia HI2424] YP_838849.1 0.001 27% ...

  10. NCBI nr-aa BLAST: CBRC-AGAM-03-0017 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-03-0017 ref|ZP_01561792.1| conserved hypothetical protein [Burkholderia c...enocepacia MC0-3] gb|EAV60539.1| conserved hypothetical protein [Burkholderia cenocepacia MC0-3] ZP_01561792.1 0.004 27% ...

  11. NCBI nr-aa BLAST: CBRC-CREM-01-1290 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1290 ref|ZP_01562587.1| uracil-xanthine permease [Burkholderia cenocep...acia MC0-3] gb|EAV59325.1| uracil-xanthine permease [Burkholderia cenocepacia MC0-3] ZP_01562587.1 2e-94 68% ...

  12. NCBI nr-aa BLAST: CBRC-AGAM-03-0072 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-03-0072 ref|ZP_01567096.1| conserved hypothetical protein [Burkholderia c...enocepacia MC0-3] gb|EAV54827.1| conserved hypothetical protein [Burkholderia cenocepacia MC0-3] ZP_01567096.1 3e-05 26% ...

  13. NCBI nr-aa BLAST: CBRC-PVAM-01-0284 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PVAM-01-0284 ref|YP_002233319.1| putative lipoprotein [Burkholderia cenocepaci...a J2315] emb|CAR54555.1| putative lipoprotein [Burkholderia cenocepacia J2315] YP_002233319.1 8e-96 64% ...

  14. NCBI nr-aa BLAST: CBRC-FCAT-01-0154 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-FCAT-01-0154 ref|ZP_01567157.1| conserved hypothetical protein [Burkholderia c...enocepacia MC0-3] gb|EAV54750.1| conserved hypothetical protein [Burkholderia cenocepacia MC0-3] ZP_01567157.1 0.030 31% ...

  15. NCBI nr-aa BLAST: CBRC-XTRO-01-0143 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0143 ref|ZP_01560289.1| conserved hypothetical protein [Burkholderia c...enocepacia MC0-3] gb|EAV62145.1| conserved hypothetical protein [Burkholderia cenocepacia MC0-3] ZP_01560289.1 0.001 27% ...

  16. NCBI nr-aa BLAST: CBRC-PMAR-01-0763 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PMAR-01-0763 ref|YP_623013.1| Haemagluttinin motif protein [Burkholderia cenoc...epacia AU 1054] gb|ABF78040.1| Haemagluttinin motif protein [Burkholderia cenocepacia AU 1054] YP_623013.1 3e-71 28% ...

  17. NCBI nr-aa BLAST: CBRC-XTRO-01-2343 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-2343 ref|YP_626365.1| Outer membrane autotransporter barrel [Burkholderia... cenocepacia AU 1054] gb|ABF81392.1| Outer membrane autotransporter barrel [Burkholderia cenocepacia AU 1054] YP_626365.1 6e-04 29% ...

  18. NCBI nr-aa BLAST: CBRC-PMAR-01-0718 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PMAR-01-0718 ref|YP_838849.1| Haemagluttinin domain protein [Burkholderia ceno...cepacia HI2424] gb|ABK11956.1| Haemagluttinin domain protein [Burkholderia cenocepacia HI2424] YP_838849.1 4e-85 26% ...

  19. NCBI nr-aa BLAST: CBRC-CJAC-01-0227 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CJAC-01-0227 ref|ZP_01566546.1| Collagen triple helix repeat [Burkholderia cen...ocepacia MC0-3] gb|EAV55423.1| Collagen triple helix repeat [Burkholderia cenocepacia MC0-3] ZP_01566546.1 2e-18 52% ...

  20. NCBI nr-aa BLAST: CBRC-GACU-01-0025 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GACU-01-0025 ref|ZP_01566546.1| Collagen triple helix repeat [Burkholderia cen...ocepacia MC0-3] gb|EAV55423.1| Collagen triple helix repeat [Burkholderia cenocepacia MC0-3] ZP_01566546.1 8e-15 33% ...

  1. Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing

    Directory of Open Access Journals (Sweden)

    Karger Axel

    2012-10-01

    Full Text Available Abstract Background Burkholderia (B. pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms. Results A collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343 was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain. Conclusions Handling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS

  2. Development of a Selective Medium for the Fungal Pathogen Fusarium graminearum Using Toxoflavin Produced by the Bacterial Pathogen Burkholderia glumae

    Directory of Open Access Journals (Sweden)

    Boknam Jung

    2013-12-01

    Full Text Available The ascomycete fungus Fusarium graminearum is a major causal agent for Fusarium head blight in cereals and produces mycotoxins such as trichothecenes and zearalenone. Isolation of the fungal strains from air or cereals can be hampered by various other airborne fungal pathogens and saprophytic fungi. In this study, we developed a selective medium specific to F. graminearum using toxoflavin produced by the bacterial pathogen Burkholderia glumae. F. graminearum was resistant to toxoflavin, while other fungi were sensitive to this toxin. Supplementing toxoflavin into medium enhanced the isolation of F. graminearum from rice grains by suppressing the growth of saprophytic fungal species. In addition, a medium with or without toxoflavin exposed to wheat fields for 1 h had 84% or 25%, respectively, of colonies identified as F. graminearum. This selection medium provides an efficient tool for isolating F. graminearum, and can be adopted by research groups working on genetics and disease forecasting.

  3. Phosphorus uptake of an arbuscular mycorrhizal fungus is not effected by the biocontrol bacterium ¤Burkholderia cepacia¤

    DEFF Research Database (Denmark)

    Ravnskov, S.; Larsen, J.; Jakobsen, I.

    2002-01-01

    The biocontrol bacterium Burkholderia cepacia is known to suppress a broad range of root pathogenic fungi, while its impact on other beneficial non-target organisms such as arbuscular mycorrhizal (AM) fungi is unknown. Direct interactions between five B. cepacia strains and the AM fungus, Glomus...... the amount of branched PLFAs suggesting a reduction in the population of Gram-positive bacteria in these cases. In conclusion, the B. cepacia seems to have no impact on neither mycorrhiza formation nor on the functioning of the AM fungus G. intraradices in terms of P transport, whereas our results suggest...... that mycorrhiza might have adverse effects on B. cepacia. (C) 2002 Elsevier Science Ltd. All rights reserved....

  4. Characterization of BcaA, a putative classical autotransporter protein in Burkholderia pseudomallei.

    Science.gov (United States)

    Campos, Cristine G; Borst, Luke; Cotter, Peggy A

    2013-04-01

    Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3' to bcaA is Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB mutants to wild-type B. pseudomallei in vitro demonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen.

  5. Molecular basis of rare aminoglycoside susceptibility and pathogenesis of Burkholderia pseudomallei clinical isolates from Thailand.

    Directory of Open Access Journals (Sweden)

    Lily A Trunck

    Full Text Available BACKGROUND: Burkholderia pseudomallei is intrinsically resistant to aminoglycosides and macrolides, mostly due to AmrAB-OprA efflux pump expression. We investigated the molecular mechanisms of aminoglycoside susceptibility exhibited by Thai strains 708a, 2188a, and 3799a. METHODOLOGY/PRINCIPAL FINDINGS: qRT-PCR revealed absence of amrB transcripts in 708a and greatly reduced levels in 2188a and 3799a. Serial passage on increasing gentamicin concentrations yielded 2188a and 3799a mutants that became simultaneously resistant to other aminoglycosides and macrolides, whereas such mutants could not be obtained with 708a. Transcript analysis showed that the resistance of the 2188a and 3799a mutants was due to upregulation of amrAB-oprA expression by unknown mechanism(s. Use of a PCR walking strategy revealed that the amrAB-oprA operon was missing in 708a and that this loss was associated with deletion of more than 70 kb of genetic material. Rescue of the amrAB-oprB region from a 708a fosmid library and sequencing showed the presence of a large chromosome 1 deletion (131 kb and 141 kb compared to strains K96243 and 1710b, respectively. This deletion not only removed the amrAB-oprA operon, but also the entire gene clusters for malleobactin and cobalamin synthesis. Other genes deleted included the anaerobic arginine deiminase pathway, putative type 1 fimbriae and secreted chitinase. Whole genome sequencing and PCR analysis confirmed absence of these genes from 708a. Despite missing several putative virulence genes, 708a was fully virulent in a murine melioidosis model. CONCLUSIONS/SIGNIFICANCE: Strain 708a may be a natural candidate for genetic manipulation experiments that use Select Agent compliant antibiotics for selection and validates the use of laboratory-constructed Delta(amrAB-oprA mutants in such experiments.

  6. An Oxygen-Sensing Two-Component System in the Burkholderia cepacia Complex Regulates Biofilm, Intracellular Invasion, and Pathogenicity

    Science.gov (United States)

    Liao, Tiffany L.; Boisvert, Nicole M.; Priebe, Gregory P.

    2017-01-01

    Burkholderia dolosa is a member of the Burkholderia cepacia complex (BCC), which is a group of bacteria that cause chronic lung infection in patients with cystic fibrosis (CF) and can be associated with outbreaks carrying high morbidity and mortality. While investigating the genomic diversity of B. dolosa strains collected from an outbreak among CF patients, we previously identified fixL as a gene showing signs of strong positive selection. This gene has homology to fixL of the rhizobial FixL/FixJ two-component system. The goals of this study were to determine the functions of FixLJ and their role in virulence in B. dolosa. We generated a fixLJ deletion mutant and complemented controls in B. dolosa strain AU0158. Using a fixK-lacZ reporter we found that FixLJ was activated in low oxygen in multiple BCC species. In a murine pneumonia model, the B. dolosa fixLJ deletion mutant was cleared faster from the lungs and spleen than wild-type B. dolosa strain AU0158 at 7 days post infection. Interestingly, the fixLJ deletion mutant made more biofilm, albeit with altered structure, but was less motile than strain AU0158. Using RNA-seq with in vitro grown bacteria, we found ~11% of the genome was differentially expressed in the fixLJ deletion mutant relative to strain AU0158. Multiple flagella-associated genes were down-regulated in the fixLJ deletion mutant, so we also evaluated virulence of a fliC deletion mutant, which lacks a flagellum. We saw no difference in the ability of the fliC deletion mutant to persist in the murine model relative to strain AU0158, suggesting factors other than flagella caused the phenotype of decreased persistence. We found the fixLJ deletion mutant to be less invasive in human lung epithelial and macrophage-like cells. In conclusion, B. dolosa fixLJ is a global regulator that controls biofilm formation, motility, intracellular invasion/persistence, and virulence. PMID:28046077

  7. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions

    DEFF Research Database (Denmark)

    Schwarz, Sandra; West, T Eoin; Boyer, Frédéric

    2010-01-01

    lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ¿T6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions......Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial...... cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain...

  8. Real-time Fluorescence PCR Method for Detection of Burkholderia glumae from Rice

    Institute of Scientific and Technical Information of China (English)

    FANG Yuan; XU Li-hui; TIAN Wen-xiao; HUAI Yan; YU Shan-hong; LOU Miao-miao; XIE Guan-lin

    2009-01-01

    Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It's quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×104 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1:100.

  9. Degradation of toluene by ortho cleavage enzymes in Burkholderia fungorum FLU100

    Science.gov (United States)

    Dobslaw, Daniel; Engesser, Karl-Heinrich

    2015-01-01

    Burkholderia fungorum FLU100 simultaneously oxidized any mixture of toluene, benzene and mono-halogen benzenes to (3-substituted) catechols with a selectivity of nearly 100%. Further metabolism occurred via enzymes of ortho cleavage pathways with complete mineralization. During the transformation of 3-methylcatechol, 4-carboxymethyl-2-methylbut-2-en-4-olide (2-methyl-2-enelactone, 2-ML) accumulated transiently, being further mineralized only after a lag phase of 2 h in case of cells pre-grown on benzene or mono-halogen benzenes. No lag phase, however, occurred after growth on toluene. Cultures inhibited by chloramphenicol after growth on benzene or mono-halogen benzenes were unable to metabolize 2-ML supplied externally, even after prolonged incubation. A control culture grown with toluene did not show any lag phase and used 2-ML as a substrate. This means that 2-ML is an intermediate of toluene degradation and converted by specific enzymes. The conversion of 4-methylcatechol as a very minor by-product of toluene degradation in strain FLU100 resulted in the accumulation of 4-carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone, 4-ML) as a dead-end product, excluding its nature as a possible intermediate. Thus, 3-methylcyclohexa-3,5-diene-1,2-diol, 3-methylcatechol, 2-methyl muconate and 2-ML were identified as central intermediates of productive ortho cleavage pathways for toluene metabolism in B. fungorum FLU100. PMID:25130674

  10. Bioactive and Structural Metabolites of Pseudomonas and Burkholderia Species Causal Agents of Cultivated Mushrooms Diseases1

    Science.gov (United States)

    Andolfi, Anna; Cimmino, Alessio; Cantore, Pietro Lo; Iacobellis, Nicola Sante; Evidente, Antonio

    2008-01-01

    Pseudomonas tolaasii, P. reactans and Burkholderia gladioli pv. agaricicola, are responsible of diseases on some species of cultivated mushrooms. The main bioactive metabolites produced by both Pseudomonas strains are the lipodepsipeptides (LDPs) tolaasin I and II and the so called White Line Inducing Principle (WLIP), respectively, LDPs which have been extensively studied for their role in the disease process and for their biological properties. In particular, their antimicrobial activity and the alteration of biological and model membranes (red blood cell and liposomes) was established. In the case of tolaasin I interaction with membranes was also related to the tridimensional structure in solution as determined by NMR combined with molecular dynamic calculation techniques. Recently, five news minor tolaasins, tolaasins A–E, were isolated from the culture filtrates of P. tolaasii and their chemical structure was determined by extensive use of NMR and MS spectroscopy. Furthermore, their antimicrobial activity was evaluated on target micro-organisms (fungi—including the cultivated mushrooms Agaricus bisporus, Lentinus edodes, and Pleurotus spp.—chromista, yeast and bacteria). The Gram positive bacteria resulted the most sensible and a significant structure-activity relationships was apparent. The isolation and structure determination of bioactive metabolites produced by B. gladioli pv. agaricicola are still in progress but preliminary results indicate their peptide nature. Furthermore, the exopolysaccharide (EPS) from the culture filtrates of B. gladioli pv. agaricicola, as well as the O-chain and lipid A, from the lipopolysaccharide (LPS) of the three bacteria, were isolated and the structures determined. PMID:19787100

  11. Bioactive and structural metabolites of pseudomonas and burkholderia species causal agents of cultivated mushrooms diseases.

    Science.gov (United States)

    Andolfi, Anna; Cimmino, Alessio; Cantore, Pietro Lo; Iacobellis, Nicola Sante; Evidente, Antonio

    2008-05-09

    Pseudomonas tolaasii, P. reactans and Burkholderia gladioli pv. agaricicola, are responsible of diseases on some species of cultivated mushrooms. The main bioactive metabolites produced by both Pseudomonas strains are the lipodepsipeptides (LDPs) tolaasin I and II and the so called White Line Inducing Principle (WLIP), respectively, LDPs which have been extensively studied for their role in the disease process and for their biological properties. In particular, their antimicrobial activity and the alteration of biological and model membranes (red blood cell and liposomes) was established. In the case of tolaasin I interaction with membranes was also related to the tridimensional structure in solution as determined by NMR combined with molecular dynamic calculation techniques. Recently, five news minor tolaasins, tolaasins A-E, were isolated from the culture filtrates of P. tolaasii and their chemical structure was determined by extensive use of NMR and MS spectroscopy. Furthermore, their antimicrobial activity was evaluated on target micro-organisms (fungi-including the cultivated mushrooms Agaricus bisporus, Lentinus edodes, and Pleurotus spp.-chromista, yeast and bacteria). The Gram positive bacteria resulted the most sensible and a significant structure-activity relationships was apparent. The isolation and structure determination of bioactive metabolites produced by B. gladioli pv. agaricicola are still in progress but preliminary results indicate their peptide nature. Furthermore, the exopolysaccharide (EPS) from the culture filtrates of B. gladioli pv. agaricicola, as well as the O-chain and lipid A, from the lipopolysaccharide (LPS) of the three bacteria, were isolated and the structures determined.

  12. Complete Genome sequence of Burkholderia phymatum STM815T, a broad host range and efficient nitrogen-fixing symbiont of Mimosa species

    Science.gov (United States)

    Moulin, Lionel; Klonowska, Agnieszka; Caroline, Bournaud; Booth, Kristina; Vriezen, Jan A.C.; Melkonian, Rémy; James, Euan K.; Young, J. Peter W.; Bena, Gilles; Hauser, Loren; Land, Miriam; Kyrpides, Nikos; Bruce, David; Chain, Patrick; Copeland, Alex; Pitluck, Sam; Woyke, Tanja; Lizotte-Waniewski, Michelle; Bristow, Jim; Riley, Margaret

    2014-01-01

    Burkholderia phymatum is a soil bacterium able to develop a nitrogen-fixing symbiosis with species of the legume genus Mimosa, and is frequently found associated specifically with Mimosa pudica. The type strain of the species, STM 815T, was isolated from a root nodule in French Guiana in 2000. The strain is an aerobic, motile, non-spore forming, Gram-negative rod, and is a highly competitive strain for nodulation compared to other Mimosa symbionts, as it also nodulates a broad range of other legume genera and species. The 8,676,562 bp genome is composed of two chromosomes (3,479,187 and 2,697,374 bp), a megaplasmid (1,904,893 bp) and a plasmid hosting the symbiotic functions (595,108 bp). PMID:25197461

  13. Complete Genome sequence of Burkholderia phymatum STM815, a broad host range and efficient nitrogen-fixing symbiont of Mimosa species

    Energy Technology Data Exchange (ETDEWEB)

    Moulin, Lionel [UMR, France; Klonowska, Agnieszka [UMR, France; Caroline, Bournaud [UMR, France; Booth, Kristina [University of Massachusetts; Vriezen, Jan A.C. [University of Massachusetts; Melkonian, Remy [UMR, France; James, Euan [James Hutton Institute, Dundee, United Kingdom; Young, Peter W. [University of York, United Kingdom; Bena, Gilles [UMR, France; Hauser, Loren John [ORNL; Land, Miriam L [ORNL; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Bruce, David [Los Alamos National Laboratory (LANL); Chain, Patrick S. G. [Lawrence Livermore National Laboratory (LLNL); Copeland, A [U.S. Department of Energy, Joint Genome Institute; Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Woyke, Tanja [U.S. Department of Energy, Joint Genome Institute; Lizotte-Waniewski, Michelle [University of Massachusetts; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Riley, Monica [Woods Hole Oceanographic Institution (WHOI), Woods Hole

    2014-01-01

    Burkholderia phymatum is a soil bacterium able to develop a nitrogen-fixing symbiosis with species of the legume genus Mimosa, and is frequently found associated specifically with Mimosa pudica. The type strain of the species, STM 815T, was isolated from a root nodule in French Guiana in 2000. The strain is an aerobic, motile, non-spore forming, Gram-negative rod, and is a highly competitive strain for nodulation compared to other Mimosa symbionts, as it also nodulates a broad range of other legume genera and species. The 8,676,562 bp genome is composed of two chromosomes (3,479,187 and 2,697,374 bp), a megaplasmid (1,904,893 bp) and a plasmid hosting the symbiotic functions (595,108 bp).

  14. Complete Genome sequence of Burkholderia phymatum STM815(T), a broad host range and efficient nitrogen-fixing symbiont of Mimosa species.

    Science.gov (United States)

    Moulin, Lionel; Klonowska, Agnieszka; Caroline, Bournaud; Booth, Kristina; Vriezen, Jan A C; Melkonian, Rémy; James, Euan K; Young, J Peter W; Bena, Gilles; Hauser, Loren; Land, Miriam; Kyrpides, Nikos; Bruce, David; Chain, Patrick; Copeland, Alex; Pitluck, Sam; Woyke, Tanja; Lizotte-Waniewski, Michelle; Bristow, Jim; Riley, Margaret

    2014-06-15

    Burkholderia phymatum is a soil bacterium able to develop a nitrogen-fixing symbiosis with species of the legume genus Mimosa, and is frequently found associated specifically with Mimosa pudica. The type strain of the species, STM 815(T), was isolated from a root nodule in French Guiana in 2000. The strain is an aerobic, motile, non-spore forming, Gram-negative rod, and is a highly competitive strain for nodulation compared to other Mimosa symbionts, as it also nodulates a broad range of other legume genera and species. The 8,676,562 bp genome is composed of two chromosomes (3,479,187 and 2,697,374 bp), a megaplasmid (1,904,893 bp) and a plasmid hosting the symbiotic functions (595,108 bp).

  15. Interim report on updated microarray probes for the LLNL Burkholderia pseudomallei SNP array

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, S; Jaing, C

    2012-03-27

    The overall goal of this project is to forensically characterize 100 unknown Burkholderia isolates in the US-Australia collaboration. We will identify genome-wide single nucleotide polymorphisms (SNPs) from B. pseudomallei and near neighbor species including B. mallei, B. thailandensis and B. oklahomensis. We will design microarray probes to detect these SNP markers and analyze 100 Burkholderia genomic DNAs extracted from environmental, clinical and near neighbor isolates from Australian collaborators on the Burkholderia SNP microarray. We will analyze the microarray genotyping results to characterize the genetic diversity of these new isolates and triage the samples for whole genome sequencing. In this interim report, we described the SNP analysis and the microarray probe design for the Burkholderia SNP microarray.

  16. Identification of Burkholderia spp. in the clinical microbiology laboratory: comparison of conventional and molecular methods

    NARCIS (Netherlands)

    C. van Pelt (Cindy); C.M. Verduin (Cees); W.H.F. Goessens (Wil); M.C. Vos (Margreet); B. Tummler; C. Segonds; F. Reubsaet; A.F. van Belkum (Alex); H.A. Verbrugh (Henri)

    1999-01-01

    textabstractCystic fibrosis (CF) predisposes patients to bacterial colonization and infection of the lower airways. Several species belonging to the genus Burkholderia are potential CF-related pathogens, but microbiological identification may be complicated. This situat

  17. Determining the Biochemical Properties of the Oxalate Biosynthetic Component (Obc)1 from Burkholderia mallei

    OpenAIRE

    Lambert, Peter M.; Nakata, Paul A.

    2016-01-01

    Oxalic acid is produced by a variety of organisms ranging from simple microbes to complex animals. This acid has been proposed to fulfill various physiological and pathological functions which vary between organisms. In bacteria from the Burkholderia genus, oxalate secretion has been shown to be quorum sensing dependent and to support pathogenicity and cell viability. In light of the critical roles of oxalate in Burkholderia as well as other organisms, it is surprising that our understanding ...

  18. Development of Galleria mellonella as an Alternative Infection Model for the Burkholderia cepacia Complex▿

    OpenAIRE

    Seed, Kimberley D.; Dennis, Jonathan J.

    2008-01-01

    Burkholderia is an important bacterial genus with a complex taxonomy that contains species of both ecological and pathogenic importance, including nine closely related species collectively termed the Burkholderia cepacia complex (BCC). In order to more thoroughly investigate the virulence of this bacterial complex of microorganisms, alternative infection models would be useful. To this end, we have adapted and developed the use of the Galleria mellonella wax moth larvae as a host for examinin...

  19. Type VI Secretion is a Major Virulence Determinant in Burkholderia Mallei

    Science.gov (United States)

    2007-06-01

    infections can also occur in felines , camels and goats. Humans are accidental hosts of B. mallei and the majority of cases have been the result of...occupational contact with infected horses. Whereas equines are generally infected orally , the primary route of infection in humans is contamination of skin...Effects of Burkholderia pseudomallei and other Burkholderia species on eukarotic cells in tissue culture. Microbios 96: 71–93. Holm, M.M., Vanlerberg, S.L

  20. Potential of Burkholderia seminalis TC3.4.2R3 as Biocontrol Agent Against Fusarium oxysporum Evaluated by Mass Spectrometry Imaging

    Science.gov (United States)

    Araújo, Francisca Diana da Silva; Araújo, Welington Luiz; Eberlin, Marcos Nogueira

    2017-02-01

    Species of genus Burkholderia display different interaction profiles in the environment, causing either several diseases in plants and animals or being beneficial to some plants, promoting their growth, and suppressing phytopathogens. Burkholderia spp. also produce many types of biomolecules with antimicrobial activity, which may be commercially used to protect crops of economic interest, mainly against fungal diseases. Herein we have applied matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to investigate secondary metabolites produced by B. seminalis TC3.4.2R3 in monoculture and coculture with plant pathogen Fusarium oxysporum. The siderophore pyochelin and the rhamnolipid Rha-Rha-C15-C14 were detected in wild-type B. seminalis strain, and their productions were found to vary in mutant strains carrying disruptions in gene clusters associated with antimicrobial compounds. Two mycotoxins were detected in F. oxysporum. During coculture with B. seminalis, metabolites probably related to defense mechanisms of these microorganisms were observed in the interspecies interaction zone. Our findings demonstrate the effective application of MALDI-MSI in the detection of bioactive molecules involved in the defense mechanism of B. seminalis, and these findings suggest the potential use of this bacterium in the biocontrol of plant diseases caused by F. oxysporum.

  1. Potential of Burkholderia seminalis TC3.4.2R3 as Biocontrol Agent Against Fusarium oxysporum Evaluated by Mass Spectrometry Imaging

    Science.gov (United States)

    Araújo, Francisca Diana da Silva; Araújo, Welington Luiz; Eberlin, Marcos Nogueira

    2017-05-01

    Species of genus Burkholderia display different interaction profiles in the environment, causing either several diseases in plants and animals or being beneficial to some plants, promoting their growth, and suppressing phytopathogens. Burkholderia spp. also produce many types of biomolecules with antimicrobial activity, which may be commercially used to protect crops of economic interest, mainly against fungal diseases. Herein we have applied matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to investigate secondary metabolites produced by B. seminalis TC3.4.2R3 in monoculture and coculture with plant pathogen Fusarium oxysporum. The siderophore pyochelin and the rhamnolipid Rha-Rha-C15-C14 were detected in wild-type B. seminalis strain, and their productions were found to vary in mutant strains carrying disruptions in gene clusters associated with antimicrobial compounds. Two mycotoxins were detected in F. oxysporum. During coculture with B. seminalis, metabolites probably related to defense mechanisms of these microorganisms were observed in the interspecies interaction zone. Our findings demonstrate the effective application of MALDI-MSI in the detection of bioactive molecules involved in the defense mechanism of B. seminalis, and these findings suggest the potential use of this bacterium in the biocontrol of plant diseases caused by F. oxysporum.

  2. Molecular mechanisms underlying the close association between soil Burkholderia and fungi

    Science.gov (United States)

    Stopnisek, Nejc; Zühlke, Daniela; Carlier, Aurélien; Barberán, Albert; Fierer, Noah; Becher, Dörte; Riedel, Katharina; Eberl, Leo; Weisskopf, Laure

    2016-01-01

    Bacterial species belonging to the genus Burkholderia have been repeatedly reported to be associated with fungi but the extent and specificity of these associations in soils remain undetermined. To assess whether associations between Burkholderia and fungi are widespread in soils, we performed a co-occurrence analysis in an intercontinental soil sample collection. This revealed that Burkholderia significantly co-occurred with a wide range of fungi. To analyse the molecular basis of the interaction, we selected two model fungi frequently co-occurring with Burkholderia, Alternaria alternata and Fusarium solani, and analysed the proteome changes caused by cultivation with either fungus in the widespread soil inhabitant B. glathei, whose genome we sequenced. Co-cultivation with both fungi led to very similar changes in the B. glathei proteome. Our results indicate that B. glathei significantly benefits from the interaction, which is exemplified by a lower abundance of several starvation factors that were highly expressed in pure culture. However, co-cultivation also gave rise to stress factors, as indicated by the increased expression of multidrug efflux pumps and proteins involved in oxidative stress response. Our data suggest that the ability of Burkholderia to establish a close association with fungi mainly lies in the capacities to utilize fungal-secreted metabolites and to overcome fungal defense mechanisms. This work indicates that beneficial interactions with fungi might contribute to the survival strategy of Burkholderia species in environments with sub-optimal conditions, including acidic soils. PMID:25989372

  3. Molecular mechanisms underlying the close association between soil Burkholderia and fungi.

    Science.gov (United States)

    Stopnisek, Nejc; Zühlke, Daniela; Carlier, Aurélien; Barberán, Albert; Fierer, Noah; Becher, Dörte; Riedel, Katharina; Eberl, Leo; Weisskopf, Laure

    2016-01-01

    Bacterial species belonging to the genus Burkholderia have been repeatedly reported to be associated with fungi but the extent and specificity of these associations in soils remain undetermined. To assess whether associations between Burkholderia and fungi are widespread in soils, we performed a co-occurrence analysis in an intercontinental soil sample collection. This revealed that Burkholderia significantly co-occurred with a wide range of fungi. To analyse the molecular basis of the interaction, we selected two model fungi frequently co-occurring with Burkholderia, Alternaria alternata and Fusarium solani, and analysed the proteome changes caused by cultivation with either fungus in the widespread soil inhabitant B. glathei, whose genome we sequenced. Co-cultivation with both fungi led to very similar changes in the B. glathei proteome. Our results indicate that B. glathei significantly benefits from the interaction, which is exemplified by a lower abundance of several starvation factors that were highly expressed in pure culture. However, co-cultivation also gave rise to stress factors, as indicated by the increased expression of multidrug efflux pumps and proteins involved in oxidative stress response. Our data suggest that the ability of Burkholderia to establish a close association with fungi mainly lies in the capacities to utilize fungal-secreted metabolites and to overcome fungal defense mechanisms. This work indicates that beneficial interactions with fungi might contribute to the survival strategy of Burkholderia species in environments with sub-optimal conditions, including acidic soils.

  4. The effect of environmental conditions on biofilm formation of Burkholderia pseudomallei clinical isolates.

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    Nur Siti K Ramli

    Full Text Available Burkholderia pseudomallei, a Gram-negative saprophytic bacterium, is the causative agent of the potentially fatal melioidosis disease in humans. In this study, environmental parameters including temperature, nutrient content, pH and the presence of glucose were shown to play a role in in vitro biofilm formation by 28 B. pseudomallei clinical isolates, including four isolates with large colony variants (LCVs and small colony variants (SCVs morphotypes. Enhanced biofilm formation was observed when the isolates were tested in LB medium, at 30 °C, at pH 7.2, and in the presence of as little as 2 mM glucose respectively. It was also shown that all SVCs displayed significantly greater capacity to form biofilms than the corresponding LCVs when cultured in LB at 37 °C. In addition, octanoyl-homoserine lactone (C(8-HSL, a quorum sensing molecule, was identified by mass spectrometry analysis in bacterial isolates referred to as LCV CTH, LCV VIT, SCV TOM, SCV CTH, 1 and 3, and the presence of other AHL's with higher masses; decanoyl-homoserine lactone (C(10-HSL and dodecanoyl-homoserine lactone (C(12-HSL were also found in all tested strain in this study. Last but not least, we had successfully acquired two Bacillus sp. soil isolates, termed KW and SA respectively, which possessed strong AHLs degradation activity. Biofilm formation of B. pseudomallei isolates was significantly decreased after treated with culture supernatants of KW and SA strains, demonstrating that AHLs may play a role in B. pseudomallei biofilm formation.

  5. Genotyping of Burkholderia mallei from an outbreak of glanders in Bahrain suggests multiple introduction events.

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    Holger C Scholz

    2014-09-01

    Full Text Available Glanders, caused by the gram-negative bacterium Burkholderia mallei, is a highly infectious zoonotic disease of solipeds causing severe disease in animals and men. Although eradicated from many Western countries, it recently emerged in Asia, the Middle-East, Africa, and South America. Due to its rareness, little is known about outbreak dynamics of the disease and its epidemiology.We investigated a recent outbreak of glanders in Bahrain by applying high resolution genotyping (multiple locus variable number of tandem repeats, MLVA and comparative whole genome sequencing to B. mallei isolated from infected horses and a camel. These results were compared to samples obtained from an outbreak in the United Arab Emirates in 2004, and further placed into a broader phylogeographic context based on previously published B. mallei data. The samples from the outbreak in Bahrain separated into two distinct clusters, suggesting a complex epidemiological background and evidence for the involvement of multiple B. mallei strains. Additionally, the samples from Bahrain were more closely related to B. mallei isolated from horses in the United Arab Emirates in 2004 than other B. mallei which is suggestive of repeated importation to the region from similar geographic sources.High-resolution genotyping and comparative whole genome analysis revealed the same phylogenetic patterns among our samples. The close relationship of the Dubai/UAE B. mallei populations to each other may be indicative of a similar geographic origin that has yet to be identified for the infecting strains. The recent emergence of glanders in combination with worldwide horse trading might pose a new risk for human infections.

  6. Glanders: off to the races with Burkholderia mallei.

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    Whitlock, Gregory C; Estes, D Mark; Torres, Alfredo G

    2007-12-01

    Burkholderia mallei, the etiologic agent of the disease known as glanders, is primarily a disease affecting horses and is transmitted to humans by direct contact with infected animals. The use of B. mallei as a biological weapon has been reported and currently, there is no vaccine available for either humans or animals. Despite the history and highly infective nature of B. mallei, as well as its potential use as a bio-weapon, B. mallei research to understand the pathogenesis and the host responses to infection remains limited. Therefore, this minireview will focus on current efforts to elucidate B. mallei virulence, the associated host immune responses elicited during infection and discuss the feasibility of vaccine development.

  7. Burkholderia cepacia Complex Regulation of Virulence Gene Expression: A Review

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    Sousa, Sílvia A.; Feliciano, Joana R.; Pita, Tiago; Guerreiro, Soraia I.; Leitão, Jorge H.

    2017-01-01

    Burkholderia cepacia complex (Bcc) bacteria emerged as opportunistic pathogens in cystic fibrosis and immunocompromised patients. Their eradication is very difficult due to the high level of intrinsic resistance to clinically relevant antibiotics. Bcc bacteria have large and complex genomes, composed of two to four replicons, with variable numbers of insertion sequences. The complexity of Bcc genomes confers a high genomic plasticity to these bacteria, allowing their adaptation and survival to diverse habitats, including the human host. In this work, we review results from recent studies using omics approaches to elucidate in vivo adaptive strategies and virulence gene regulation expression of Bcc bacteria when infecting the human host or subject to conditions mimicking the stressful environment of the cystic fibrosis lung. PMID:28106859

  8. Global and regional dissemination and evolution of Burkholderia pseudomallei

    Science.gov (United States)

    Chewapreecha, Claire; Holden, Matthew T. G.; Vehkala, Minna; Välimäki, Niko; Yang, Zhirong; Harris, Simon R; Mather, Alison E.; Tuanyok, Apichai; De Smet, Birgit; Le Hello, Simon; Bizet, Chantal; Mayo, Mark; Wuthiekanun, Vanaporn; Limmathurotsakul, Direk; Phetsouvanh, Rattanaphone; Spratt, Brian G; Corander, Jukka; Keim, Paul; Dougan, Gordon; Dance, David A. B.; Currie, Bart J; Parkhill, Julian; Peacock, Sharon J.

    2017-01-01

    The environmental bacterium Burkholderia pseudomallei causes an estimated 165,000 cases of human melioidosis per year worldwide, and is also classified as a biothreat agent. We used whole genome sequences of 469 B. pseudomallei isolates from 30 countries collected over 79 years to explore its geographic transmission. Our data point to Australia as an early reservoir, with transmission to Southeast Asia followed by onward transmission to South Asia, and East Asia. Repeated reintroduction was observed within the Malay Peninsula, and between countries bordered by the Mekong river. Our data support an African origin of the Central and South American isolates with introduction of B. pseudomallei into the Americas between 1650 and 1850, providing a temporal link with the slave trade. We also identified geographically distinct genes/variants in Australasian or Southeast Asian isolates alone, with virulence-associated genes being among those overrepresented. This provides a potential explanation for clinical manifestations of melioidosis that are geographically restricted. PMID:28112723

  9. Burkholderia glumae en el cultivo de arroz en Costa Rica.

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    Andrea Quesada-González

    2014-07-01

    Full Text Available El objetivo de este trabajo fue determinar la presencia de Burkholderia glumae en arroz en Costa Rica. La bacteria Burkholderia glumae está asociada al cultivo del arroz en el que provoca la enfermedad llamada añublo bacterial. Bajo condiciones ambientales favorables, la densidad bacteriana aumenta, lo que provoca que, bajo un sistema de regulación denominado quorum sensing, se expresen sus mecanismos de virulencia mediante la activación de genes responsables para la síntesis de la toxoflavina, que bloquea el flujo de nutrientes, para la biogénesis de flagelos y la respuesta quimiotáctica, y la producción de la enzima catalasa. Las plantas desarrollan la sintomatología que finalmente conlleva a un vaneamiento del grano provocando pérdidas económicas importantes. Se investigó la situación referente a la contaminación del grano de arroz causado por esta bacteria en Costa Rica durante los años 2009 y 2010, mediante un convenio entre la Corporación Nacional Arrocera y el Laboratorio de Fitopatología del Centro de Investigación en Protección de Cultivos de la Universidad de Costa Rica. Se usó la metodología de PCR de punto final recomendada por investigadores del Centro Internacional de Agricultura Tropical en Colombia y se reforzó la identificación, por medio de técnicas de microbiología convencional. Se obtuvieron resultados que indican la presencia de la bacteria en Costa Rica, la primera información sobre la prevalencia de un fitopatógeno bacteriano de gran importancia para el sector arrocero.

  10. Effects of the inoculation of Burkholderia vietnamensis and related endophytic diazotrophic bacteria on grain yield of rice.

    Science.gov (United States)

    Govindarajan, Munusamy; Balandreau, Jacques; Kwon, Soon-Wo; Weon, Hang-Yeon; Lakshminarasimhan, Cunthipuram

    2008-01-01

    During a survey of endophytic diazotrophic bacteria associated with different rice varieties in Tamilnadu, some "endophytes" were obtained. Thirteen bacterial isolates from surface-sterilized roots and shoots were obtained in pure culture, which produced indole acetic acid (IAA) and reduced acetylene to ethylene. Polymerase chain reaction (PCR) amplification confirmed the presence of nif-H gene in all the isolates. Morphological, biochemical, and molecular characteristics indicated that all of them belonged to the genus Burkholderia One of them, MGK3, was consistently more active in reducing acetylene, and 16S rDNA sequences of isolate MGK3 confirmed its identification as Burkholderia vietnamiensis. Colonization of rice root was confirmed by strain MGK3 marked with gusA gene. The inoculated roots showed a blue color, which was most intense at the points of lateral root emergence and at the root tip. Transverse sections of roots, 15 days after inoculation, revealed beta-glucuronidase (GUS) activity within many of the cortical intercellular spaces next to the stele and within the aerenchyma. Nitrogen fixation was quantified by using (15)N isotope dilution method with two different cultivars grown in pot and field experiments. Higher nitrogen fixation was observed in variety Ponni than in ADT-43, where nearly 42% (field) and 40% (pot) of the nitrogen was derived from the atmosphere (% Ndfa). Isolate MGK3 was used to inoculate rice seedlings in a comparison with four other diazotrophs, viz., Gluconacetobacter diazotrophicus LMG7603, Herbaspirillum seropedicae LMG6513, Azospirillum lipoferum 4B LMG4348, and B. vietnamiensis LMG10929. They were used to conduct two pot and four field inoculation experiments. MGK3 alone, and combined with other diazotrophs, performed best under both pot and field conditions: combined inoculation produced yield increases between 9.5 and 23.6%, while MGK3 alone increased yield by 5.6 to 12.16% over the uninoculated control treatment.

  11. Functional characterization and evaluation of in vitro protective efficacy of murine monoclonal antibodies BURK24 and BURK37 against Burkholderia pseudomallei.

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    Bhavani V Peddayelachagiri

    Full Text Available Burkholderia pseudomallei, the causative agent of melioidosis has been recognized by CDC as a category B select agent. Although substantial efforts have been made for development of vaccine molecules against the pathogen, significant hurdles still remain. With no licensed vaccines available and high relapse rate of the disease, there is a pressing need for development of alternate protection strategies. Antibody-mediated passive protection is promising in this regard and our primary interest was to unravel this frontier of specific mAbs against Burkholderia pseudomallei infections, as functional characterization of antibodies is a pre-requisite to demonstrate them as protective molecules. To achieve this, we designed our study on in vitro-based approach and assessed two mAbs, namely BURK24 and BURK37, reactive with outer membrane proteins and lipopolysaccharide of the pathogen respectively, for their ability to manifest inhibitory effects on the pathogenesis mechanisms of B. pseudomallei including biofilm formation, invasion and induction of apoptosis. The experiments were performed using B. pseudomallei standard strain NCTC 10274 and a clinical isolate, B. pseudomallei 621 recovered from a septicemia patient with diabetic ailment. The growth kinetic studies of the pathogen in presence of various concentrations of each individual mAb revealed their anti-bacterial properties. Minimal inhibitory concentration and minimal bactericidal concentration of both the mAbs were determined by using standards of Clinical and Laboratory Standards Institute (CLSI and experiments were performed using individual mAbs at their respective bacteriostatic concentration. As an outcome, both mAbs exhibited significant anti-Burkholderia pseudomallei properties. They limited the formation of biofilm by the bacterium and completely crippled its invasion into human alveolar adenocarcinoma epithelial cells. Also, the mAbs were appreciably successful in preventing the

  12. Burkholderia mallei and Burkholderia pseudomallei stimulate differential inflammatory responses from human alveolar type II cells (ATII and macrophages.

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    Richard eLu

    2012-12-01

    Full Text Available Alveolar type II pneumocytes (ATII and alveolar macrophages (AM play a crucial role in the lung’s innate immune response. Burkholderia pseudomallei (BP and Burkholderia mallei (BM are facultative Gram-negative bacilli that cause melioidosis and glanders, respectively. The inhalation of these pathogens can cause lethal disease and death in humans. We sought to compare the pathogenesis of and host responses to BP and BM through contact with human primary ATII cells and monocytes-derived macrophages (MDM. We hypothesized that because BP and BM induce different disease outcomes, each pathogen would induce distinct, unique host immune responses from resident pulmonary cells. Our findings showed that BP adhered readily to ATII cells compared to BM. BP, but not BM, was rapidly internalized by macrophages where it replicated to high numbers. Further, BP induced significantly higher levels of pro-inflammatory cytokine secretion from ATII cells (IL-6, IL-8 and macrophages (IL-6, TNFα at 6h post-infection compared to BM (p<0.05. Interestingly, BM induced the anti-inflammatory cytokine, IL-10, in ATII cells and macrophages at 6h post-infection, with delayed induction of inflammatory cytokines at 24h post-infection. Because BP is flagellated and produces LPS, we confirmed that it stimulated both Toll-like receptor (TLR 4 and TLR5 via NF-κb activation while the non-flagellated BM stimulated only TLR4. These data show the differences in BP and BM pathogenicity in the lung when infecting human ATII cells and macrophages and demonstrate the ability of these pathogens to elicit distinct immune responses from resident lung cells which may open new targets for therapeutic intervention to fight against these pathogens.

  13. Isolation rates of Burkholderia pseudomallei among the four regions in Thailand.

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    Leelarasamee, A; Trakulsomboon, S; Kusum, M; Dejsirilert, S

    1997-03-01

    This study aimed to compare the isolation rates of Burkholderia pseudomallei among community-based hospitals located in the central, north, northeast, and south of Thailand. A questionnaire inquiring about the number of isolation of B. pseudomallei from various clinical specimens during 1994-95 were mailed to 141 community-based hospitals. Of these, 125 hospitals (88.6%) responded to the questionnaire. Microbiological laboratory was not available in thirty hospitals. Data from 95 remaining hospitals with capability to do bacterial culture showed that B. pseudomallei was never isolated in 49 hospitals. Eleven, 9, 19 and 7 hospitals where B. pseudomallei has been isolated, are located in the central, north, northeast and south of Thailand respectively. From these 46 hospitals, a total of 1,131 strains of B. pseudomallei were isolated from 407,263 specimens in 1994 and 1,165 strains from 440,541 specimens in 1995. However, the isolation was most frequent in northeastern hospitals, which accounted for 890 and 964 strains in 1994 and 1995 respectively while only 94, 76, 71 and 83, 75, 43 strains were simultaneously isolated during the 2-year period in those located in central, north and south respectively. The isolation rates of B. speudomallei in 1994 and 1995 were 4.2 and 4.1 per 1,000 clinical specimens in northeastern hospitals as compared to 1.1, 1.8, 1.1 and 1.1, 1.2, 0.7 in those located in central, north and south respectively. Ubon Ratchathani, Nakhon Ratchasima, Buri Ram, Khon Kaen and Udon Thani were the five provinces which exhibited the highest isolation rates as follows; 244, 150, 147, 127, 100 and 218, 128, 114, 119, 58, in 1994 and 1995, respectively. It was concluded that B.pseudomallei was most commonly isolated in the northeast of Thailand. Under-recognition of B. pseudomallei may prevail not only in other parts of Thailand but in some areas of the northeast as well.

  14. Draft Genome Sequence of Burkholderia sp. MR1, a Methylarsenate-Reducing Bacterial Isolate from Florida Golf Course Soil

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    Pawitwar, Shashank S.; Utturkar, Sagar M.; Brown, Steven D.; Yoshinaga, Masafumi

    2015-01-01

    To elucidate the environmental organoarsenical biocycle, we isolated a soil organism, Burkholderia sp. MR1, which reduces relatively nontoxic pentavalent methylarsenate to the more toxic trivalent methylarsenite, with the goal of identifying the gene for the reductase. Here, we report the draft genome sequence of Burkholderia sp. MR1. PMID:26044439

  15. Draft Genome Sequence of Burkholderia sp. MR1, a Methylarsenate-Reducing Bacterial Isolate from Florida Golf Course Soil

    OpenAIRE

    Pawitwar, Shashank S.; Utturkar, Sagar M.; Brown, Steven D.; Yoshinaga, Masafumi; Rosen, Barry P.

    2015-01-01

    To elucidate the environmental organoarsenical biocycle, we isolated a soil organism, Burkholderia sp. MR1, which reduces relatively nontoxic pentavalent methylarsenate to the more toxic trivalent methylarsenite, with the goal of identifying the gene for the reductase. Here, we report the draft genome sequence of Burkholderia sp. MR1.

  16. Burkholderia phytofirmans PsJN reduces damages to freezing temperature in Arabidopsis thaliana

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    Fan eSU

    2015-10-01

    Full Text Available Several plant growth-promoting rhizobacteria (PGPR are known to improve plant tolerance to multiple stresses, including low temperatures. However, mechanisms underlying this protection are still poorly understood. The aim of this study was to evaluate the role of the endophytic PGPR, Burkholderia phytofirmans strain PsJN (Bp PsJN, on Arabidopsis thaliana cold tolerance using photosynthesis parameters as physiological markers.Under standard conditions, our results indicated that Bp PsJN inoculation led to growth promotion of Arabidopsis plants without significant modification on photosynthesis parameters and chloroplast organization. However, bacterial colonization induced a cell wall strengthening in the mesophyllImpact of inoculation modes (either on seeds or by soil irrigation and their effects overnight at 0, -1 or -3°C, were investigated by following photosystem II (PSII activity and gas exchanges. Following low temperatures stress, a decrease of photosynthesis parameters was observed. In addition, during three consecutive nights or days at -1°C, PSII activity was monitored. Pigment contents, RuBisCO protein abundance, expression of several genes including RbcS, RbcL, CBF1, CBF2, CBF3, ICE1, COR15a, and COR78 were evaluated at the end of exposure. To assess the impact of the bacteria on cell ultrastructure under low temperatures, microscopic observations were achieved. Results indicated that freezing treatment induced significant changes in PSII activity as early as the first cold day, whereas the same impact on PSII activity was observed only during the third cold night. The significant effects conferred by PsJN were differential accumulation of pigments, and reduced expression of RbcL and COR78. Microscopical observations showed an alteration/disorganization in A. thaliana leaf mesophyll cells independently of the freezing treatments. The presence of bacteria during the three successive nights or days did not significantly improved A

  17. Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

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    Senanu M Spring-Pearson

    Full Text Available The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

  18. Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

    Science.gov (United States)

    Spring-Pearson, Senanu M; Stone, Joshua K; Doyle, Adina; Allender, Christopher J; Okinaka, Richard T; Mayo, Mark; Broomall, Stacey M; Hill, Jessica M; Karavis, Mark A; Hubbard, Kyle S; Insalaco, Joseph M; McNew, Lauren A; Rosenzweig, C Nicole; Gibbons, Henry S; Currie, Bart J; Wagner, David M; Keim, Paul; Tuanyok, Apichai

    2015-01-01

    The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

  19. Burkholderia pseudomallei Colony Morphotypes Show a Synchronized Metabolic Pattern after Acute Infection.

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    Philipp Gierok

    2016-03-01

    Full Text Available Burkholderia pseudomallei is a water and soil bacterium and the causative agent of melioidosis. A characteristic feature of this bacterium is the formation of different colony morphologies which can be isolated from environmental samples as well as from clinical samples, but can also be induced in vitro. Previous studies indicate that morphotypes can differ in a number of characteristics such as resistance to oxidative stress, cellular adhesion and intracellular replication. Yet the metabolic features of B. pseudomallei and its different morphotypes have not been examined in detail so far. Therefore, this study aimed to characterize the exometabolome of B. pseudomallei morphotypes and the impact of acute infection on their metabolic characteristics.We applied nuclear magnetic resonance spectroscopy (1H-NMR in a metabolic footprint approach to compare nutrition uptake and metabolite secretion of starvation induced morphotypes of the B. pseudomallei strains K96243 and E8. We observed gluconate production and uptake in all morphotype cultures. Our study also revealed that among all morphotypes amino acids could be classified with regard to their fast and slow consumption. In addition to these shared metabolic features, the morphotypes varied highly in amino acid uptake profiles, secretion of branched chain amino acid metabolites and carbon utilization. After intracellular passage in vitro or murine acute infection in vivo, we observed a switch of the various morphotypes towards a single morphotype and a synchronization of nutrient uptake and metabolite secretion.To our knowledge, this study provides first insights into the basic metabolism of B. pseudomallei and its colony morphotypes. Furthermore, our data suggest, that acute infection leads to the synchronization of B. pseudomallei colony morphology and metabolism through yet unknown host signals and bacterial mechanisms.

  20. Extraction of lipase from Burkholderia cepacia by PEG/Phosphate ATPS and its biochemical characterization

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    Giovana da Silva Padilha

    2012-02-01

    Full Text Available This work aimed to study the partitioning of a lipase produced by Burkholderia cepacia in PEG/Phosphate aqueous two phase system (ATPS and its characterization. Lipase was produced by B. cepacia strains in a fermenter. Enzyme partitioning occurred at pH 6.0 and 8.0, using PEG 1500 and 6000 on two tie lines. Metal ions, pH and temperature effects on enzyme activity were evaluated. Five milliliter of 7.5% olive oil emulsion with 2.5% gumarabic in 0.1M sodium phosphate buffer at pH 8.0 and 37ºC were used for the activity determinations. Results showed that crude stratum from B. cepacia was partitioned by PEG1500/phosphate ATPS at pH 6.0 or 8.0 for, which the partitioning coefficients were 108-and 209-folds. Lipase presented optimal activity conditions at 37ºC and pH 8.0; it showed pH-stability for 4 h of incubation at different pH values at 37ºC. Metal ions such as Mn2+ , Co2+, I-and Ca2+ sustained enzymatic activities; however, it was inhibited by the presence of Fe2+, Hg2+ and Al3+ . Km and Vmax values were 0.258 U/mg and 43.90 g/L, respectively. A molecular weight of 33 kDa and an isoelectric point at pH 5.0 were determined by SDS-PAGE and IFS electrophoresis, respectively.

  1. In vivo bioluminescence imaging of Burkholderia mallei respiratory infection and treatment in the mouse model

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    Shane eMassey

    2011-08-01

    Full Text Available Bioluminescent imaging (BLI technology is a powerful tool for monitoring infectious disease progression and treatment approaches. BLI is particularly useful for tracking fastidious intracellular pathogens that might be difficult to recover from certain organs. Burkholderia mallei, the causative agent of glanders, is a facultative intracellular pathogen and has been classified by the CDC as a Category B select agent due to its highly infectious nature and potential use as a biological weapon. Very little is known regarding pathogenesis or treatment of glanders. We investigated the use of bioluminescent reporter constructs to monitor the dynamics of infection as well as the efficacy of therapeutics for B. mallei in real time. A stable luminescent reporter B. mallei strain was created using the pUTmini-Tn5::luxKm2 plasmid and used to monitor glanders in the BALB/c murine model. Mice were infected via the intranasal route with 5x103 bacteria and monitored by BLI at 24, 48 and 72 h. We verified that our reporter construct maintained similar virulence and growth kinetics compared to wild-type B. mallei and confirmed that it maintains luminescent stability in the presence or absence of antibiotic selection. The luminescent signal was initially seen in the lungs, and progressed to the liver and spleen over the course of infection. We demonstrated that antibiotic treatment 24 h post-infection resulted in reduction of bioluminescence that can be attributed to decreased bacterial burden in target organs. These findings suggest that BLI can be used to monitor disease progression and efficacy of therapeutics during glanders infections. Finally, we report an alternative method to mini-Tn5::luxKm2 transposon using mini-Tn7-lux elements that insert site-specifically at known genomic attachment sites and that can also be used to tag bacteria.

  2. Degradation of toluene by ortho cleavage enzymes in Burkholderia fungorum FLU100.

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    Dobslaw, Daniel; Engesser, Karl-Heinrich

    2015-01-01

    Burkholderia fungorum FLU100 simultaneously oxidized any mixture of toluene, benzene and mono-halogen benzenes to (3-substituted) catechols with a selectivity of nearly 100%. Further metabolism occurred via enzymes of ortho cleavage pathways with complete mineralization. During the transformation of 3-methylcatechol, 4-carboxymethyl-2-methylbut-2-en-4-olide (2-methyl-2-enelactone, 2-ML) accumulated transiently, being further mineralized only after a lag phase of 2 h in case of cells pre-grown on benzene or mono-halogen benzenes. No lag phase, however, occurred after growth on toluene. Cultures inhibited by chloramphenicol after growth on benzene or mono-halogen benzenes were unable to metabolize 2-ML supplied externally, even after prolonged incubation. A control culture grown with toluene did not show any lag phase and used 2-ML as a substrate. This means that 2-ML is an intermediate of toluene degradation and converted by specific enzymes. The conversion of 4-methylcatechol as a very minor by-product of toluene degradation in strain FLU100 resulted in the accumulation of 4-carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone, 4-ML) as a dead-end product, excluding its nature as a possible intermediate. Thus, 3-methylcyclohexa-3,5-diene-1,2-diol, 3-methylcatechol, 2-methyl muconate and 2-ML were identified as central intermediates of productive ortho cleavage pathways for toluene metabolism in B. fungorum FLU100. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  3. Copper as an antibacterial agent for human pathogenic multidrug resistant Burkholderia cepacia complex bacteria.

    Science.gov (United States)

    Ibrahim, Muhammad; Wang, Fang; Lou, Miao-miao; Xie, Guan-lin; Li, Bin; Bo, Zhu; Zhang, Gou-qing; Liu, He; Wareth, Abdul

    2011-12-01

    The Burkholderia cepacia complex (Bcc) consists of 17 closely related multidrug resistant bacterial species that are difficult to eradicate. Copper has recently gained attention as an antimicrobial agent because of its inhibitory effects on bacteria, yeast, and viruses. The objective of this study was to evaluate the antibacterial activity of copper surfaces and copper powder against members of the B. cepacia complex. The antibacterial activity of different copper surfaces was evaluated by incubating them with Bcc strain suspensions (5×10(7)cfu/ml). The bacterial survival counts were calculated and the data for various copper surfaces were compared to the data for stainless steel and polyvinylchloride, which were used as control surfaces. The antibacterial activity of copper powder was determined with the diffusimetrical technique and the zone of inhibition was evaluated with paper disks. A single cell gel electrophoresis assay, staining assays, and inductively coupled plasma mass spectroscopy were performed to determine the mechanism responsible for the bactericidal activity. The results showed a significant decrease in the viable bacterial count after exposure to copper surfaces. Moreover, the copper powder produced a large zone of inhibition and there was a significantly higher influx of copper ions into the bacterial cells that were exposed to copper surfaces compared to the controls. The present study demonstrates that metallic copper has an antibacterial effect against Bcc bacteria and that copper adversely affects the bacterial cellular structure, thus resulting in cell death. These findings suggest that copper could be utilized in health care facilities to reduce the bioburden of Bcc species, which may protect susceptible members of the community from bacterial infection.

  4. Structural characterization of an acidic exoheteropolysaccharide produced by the nitrogen-fixing bacterium Burkholderia tropica.

    Science.gov (United States)

    Serrato, Rodrigo V; Sassaki, Guilherme L; Gorin, Philip A J; Cruz, Leonardo M; Pedrosa, Fábio O; Choudhury, Biswa; Carlson, Russell W; Iacomini, Marcello

    2008-09-05

    An acidic exopolysaccharide (EPS) produced by the diazotrophic bacterium Burkholderia tropica, strain Ppe8, was isolated from the culture supernatant of bacteria grown in a synthetic liquid medium containing mannitol and glutamate. Monosaccharide composition showed Rha, Glc and GlcA in a 2.0:2.0:1.0 molar ratio, respectively. Further structural characterization was performed by a combination of NMR, mass spectrometry and chemical methods. Partial acid hydrolysis of EPS provided a mixture of acidic oligosaccharides that were characterized by ESI-MS, giving rise to ions with m/z 193 (GlcA-H)(-), 339 (GlcA,Rha-H)(-), 501 (GlcA,Rha,Glc-H)(-), 647 (GlcA,Rha2,Glc,-H)(-), 809 (GlcA,Rha2,Glc2,-H)(-) and 851 (GlcA,Rha2,Glc2,OAc-H)(-). Carboxyreduced EPS (EPS-CR) had Glc and Rha in a 3:2 ratio, present as d- and l-enantiomers, respectively. Methylation and NMR analysis of EPS and EPS-CR showed a main chain containing 2,4-di-O-Rhap, 3-O-Rhap and 4-O-Glcp. A GlcA side chain unit was found in the acidic EPS, substituting O-4 of α-l-Rhap units. This was observed as a non-reducing end unit of glucopyranose in the EPS-CR. Acetyl esters occured at O-2 of β-l-Rhap units. From the combined results herein, we determined the structure of the exocellular polysaccharide produced by B. tropica, Ppe8, as being a pentasaccharide repeating unit as shown.

  5. A Possible Link between Infection with Burkholderia Bacteria and Systemic Lupus Erythematosus Based on Epitope Mimicry

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    Wei Zhang

    2008-01-01

    Full Text Available We previously demonstrated that purified polyclonal and monoclonal anti-dsDNA antibodies bind a 15-mer peptide ASPVTARVLWKASHV in ELISA and Dot blot. This 15-mer peptide partial sequence ARVLWKASH shares similarity with burkholderia bacterial cytochrome B 561 partial sequence ARVLWRATH. In this study, we show that purified anti-dsDNA antibodies react with burkholderia fungorum bacterial cell lysates in Western blot. We used anti-dsDNA antibodies to make an anti-dsDNA antibodies affinity column and used this column to purify the burkholderia fungorum bacterial protein. Purified anti-dsDNA antibodies bind specifically to purified bacterial antigen and purified bacterial antigen blocked the anti-dsDNA antibodies binding to dsDNA antigen. Sera with anti-dsDNA antibodies bind specifically to purified bacterial antigen. We obtained protein partial sequence of RAGTDEGFG which is shared with burkholderia bacterial transcription regulator protein sequence. Sera with anti-dsDNA antibodies bind to RAGTDEGFG peptide better than control groups. These data support our hypothesis that the origin of anti-dsDNA antibodies in SLE may be associated with burkholderia bacterial infection.

  6. The Organization of the Quorum Sensing luxI/R Family Genes in Burkholderia

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    Sándor Pongor

    2013-07-01

    Full Text Available Members of the Burkholderia genus of Proteobacteria are capable of living freely in the environment and can also colonize human, animal and plant hosts. Certain members are considered to be clinically important from both medical and veterinary perspectives and furthermore may be important modulators of the rhizosphere. Quorum sensing via N-acyl homoserine lactone signals (AHL QS is present in almost all Burkholderia species and is thought to play important roles in lifestyle changes such as colonization and niche invasion. Here we present a census of AHL QS genes retrieved from public databases and indicate that the local arrangement (topology of QS genes, their location within chromosomes and their gene neighborhoods show characteristic patterns that differ between the known Burkholderia clades. In sequence phylogenies, AHL QS genes seem to cluster according to the local gene topology rather than according to the species, which suggests that the basic topology types were present prior to the appearance of current Burkholderia species. The data are available at http://net.icgeb.org/burkholderia/.

  7. Modified sublimation to isolate phenanthrene-degrading bacteria of the genera Sphingomonas and Burkholderia from Xiamen oil port.

    Science.gov (United States)

    Huang, X; Tian, Y; Luo, Y R; Liu, H J; Zheng, Wei; Zheng, T L

    2008-01-01

    Sublimation was developed by Alley and Brown (2000) in order to isolate bacterial strains that were capable of degrading water insoluble compounds. In this study, sublimation was modified by the use of nutritional agar plates, instead of mineral salt agar, to isolate phenanthrene-degrading bacteria from a mixed culture that had been enriched under the selective pressure of high phenanthrene content. Five strains were obtained with different morphology and degradation ability. Based on the 16S rDNA sequence, two of them were classified as species of the genus Sphingomonas; the others as species of the genus Burkholderia. Denaturing gradient gel electrophoresis (DGGE) was introduced to detect dynamic changes in the bacterial community during enrichment batch culture, and to determine any correlation between the five isolates and the phenanthrene-degrading consortium. The DGGE profile indicated that these five isolates corresponded to four dominant bands of the consortium. Compared to traditional means of isolation, we concluded that modified sublimation is effective and more convenient.

  8. Identification of new regulatory genes involved in the pathogenic functions of the rice-pathogenic bacterium Burkholderia glumae.

    Science.gov (United States)

    Melanson, Rebecca A; Barphagha, Inderjit; Osti, Surendra; Lelis, Tiago P; Karki, Hari S; Chen, Ruoxi; Shrestha, Bishnu K; Ham, Jong Hyun

    2017-02-01

    Burkholderia glumae is an emerging plant-pathogenic bacterium that causes disease in rice in several of the major rice-producing areas throughout the world. In the southern United States, B. glumae is the major causal agent of bacterial panicle blight of rice and has caused severe yield losses in recent decades. Despite its importance, few management options are available for diseases caused by B. glumae, and knowledge of how this pathogen causes disease is limited. In an effort to identify novel factors that contribute to the pathogenicity of B. glumae, random mutagenesis using the miniTn5gus transposon was performed on two strains of B. glumae. Resultant mutants were screened in the laboratory for altered phenotypes in various known or putative virulence factors, including toxoflavin, lipase and extracellular polysaccharides. Mutants that exhibited altered phenotypes compared to their parent strain were selected and subsequently characterized using a PCR-based method to identify the approximate location of the transposon insertion. Altogether, approximately 20 000 random mutants were screened and 51 different genes were identified as having potential involvement in the production of toxoflavin, lipase and/or extracellular polysaccharide. Especially, two regulatory genes, ntpR and tepR, encoding a LysR-type transcriptional regulator and a σ54-dependent response regulator, respectively, were discovered in this study as new negative regulatory factors for the production of toxoflavin, the major phytotoxin synthesized by B. glumae and involved in bacterial pathogenesis.

  9. Development of hydrolysis probe-based real-time PCR for identification of virulent gene targets of Burkholderia pseudomallei and B. mallei--a retrospective study on archival cases of service members with melioidosis and glanders.

    Science.gov (United States)

    Zhang, Binxue; Wear, Douglas J; Kim, H S; Weina, Peter; Stojadinovic, Alexander; Izadjoo, Mina

    2012-02-01

    Burkholderia pseudomallei and B. mallei are two highly pathogenic bacteria responsible for melioidosis and glanders, respectively. Our laboratory developed hydrolysis probe-based real-time polymerase chain reaction assays targeting type three secretion system (TTS) and transposase family protein (TFP) of B. pseudomallei and B. malli, respectively. The assays were validated for target specificity, amplification sensitivity, and reproducibility. A bacterial DNA panel, composed of B. pseudomallei (13 strains), B. mallei (11 strains), Burkholderia species close neighbors (5 strains), and other bacterial species (17 strains), was prepared for specificity testing. Reference DNAs from B. pseudomallei and B. mallei bacterial cultures were used as controls for amplification, limit of detection, and reproducibility testing. The two TaqMan assays, Bp-TTS 1 and Bm-TFP, were optimized and applied in a retrospective study of archived cases from the Armed Forces Institute of Pathology. We tested 10 formalin-fixed paraffin-embedded blocks originally from autopsy specimens of patients who died of melioidosis or glanders during or after overseas tours in 1960s. Polymerase chain reaction results confirmed that DNA samples from formalin-fixed paraffin-embedded blocks of eight patients with melioidosis were positive for Bp-TTS 1 target and two patients with glanders were positive for Bm-TFP target.

  10. Caracterização fenotípica e molecular de amostras de Burkholderia mallei isoladas na Região Nordeste do Brasil Phenotypic and molecular characterization of Burkholderia mallei isolated in northeastern Brazil

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    Karla P.C. Silva

    2009-05-01

    Full Text Available Objetivou-se com este trabalho realizar o estudo bioquímico e molecular de amostras de Burkholderia mallei isoladas de eqüídeos com diagnóstico clínico e sorológico para o mormo e provenientes da Região Metropolitana do Recife-PE e Zona da Mata dos Estados de Alagoas e Pernambuco. Foram realizadas as técnicas microbiológicas para o isolamento e identificação fenotípica de B. mallei e as técnicas moleculares de ribotipagem-PCR e RAPD-PCR. Das oito amostras estudadas, quatro apresentaram pequenas variações fenotípicas. Nas técnicas moleculares, as amostras formaram quatro grupos de diferentes perfis ribotípicos, demonstrando também quatro perfis genotípicos. Houve associação nos resultados da Ribotipagem-PCR e RAPD-PCR. As variações nos perfis ribotípicos e genotípicos foram associadas às diferentes regiões estudadas. De acordo com os resultados obtidos, conclui-se que as pequenas variações bioquímicas não estão associadas aos diferentes perfis moleculares e que essas diferenças demonstram uma heterogeneidade que está associada à procedência das amostras, indicando que a infecção nos animais ocorre por clones diferentes das amostras analisadas.The objective of this paper was to study the molecular performance and phenotypic characterization of Burkholderia mallei isolated from horses with clinical and serological diagnosis of glanders, originating from the Metropolitan District of Recife and Zona da Mata of Pernambuco and Alagoas. The isolation and biochemical identification of B. mallei was carried out by microbiological and molecular techniques of PCR-fingerprinting and RAPD-PCR. From the eight samples studied, four showed little phenotype variations. In the molecular tests, the samples formed 4 groups of different ribotype profiles and 4 genotype profiles. There was some association of PCR-fingerprinting with RAPD-PCR results. It was concluded that the slight biochemical variations were not associated with

  11. Degradation of parabens by Pseudomonas beteli and Burkholderia latens.

    Science.gov (United States)

    Amin, Aeshna; Chauhan, Sateesh; Dare, Manish; Bansal, Arvind Kumar

    2010-06-01

    p-Hydroxybenzoic acid esters (parabens) are commonly used antimicrobial preservatives in pharmaceutical formulations. Two microorganisms, isolated from non-sterile methyl paraben (MP) and propyl paraben (PP) solutions, were found to degrade the respective parabens. Identification by 16S rRNA partial gene sequencing revealed them to be Pseudomonas beteli and Burkholderia latens, respectively. The present work describes a previously unreported interaction of the parabens with P. beteli and B. latens. Degradation of MP at various concentrations by P. beteli, followed a logarithmic pattern, while that of PP by B. latens was found to be linear. It was subsequently observed that P. beteli could degrade only MP, while B. latens could degrade both the parabens. Absence of HPLC chromatogram peaks of expected degradation products indicated that the parabens were used up as a carbon source. The behaviour of pathogens (Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus niger) of the pharmacopoeial preservative effectiveness test (PET), towards MP, showed that none had the ability to degrade the paraben. It was concluded that, for a paraben-preserved multi-dose ophthalmic formulation, the sole use of the four pathogens that are recommended by the pharmacopoeia for PET can falsely indicate the formulation to be effective against 'in-use' contamination.

  12. RNAseq-based Transcriptome Analysis of Burkholderia glumae Quorum Sensing.

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    Kim, Sunyoung; Park, Jungwook; Kim, Ji Hyeon; Lee, Jongyun; Bang, Bongjun; Hwang, Ingyu; Seo, Young-Su

    2013-09-01

    Burkholderia glumae causes rice grain rot and sheath rot by producing toxoflavin, the expression of which is regulated by quorum sensing (QS). The QS systems of B. glumae rely on N-octanoyl homoserine lactone, synthesized by TofI and its cognate receptor TofR, to activate the genes for toxoflavin biosynthesis and an IclR-type transcriptional regulator gene, qsmR. To understand genome-wide transcriptional profiling of QS signaling, we employed RNAseq of the wild-type B. glumae BGR1 with QS-defective mutant, BGS2 (BGR1 tofI::Ω) and QS-dependent transcriptional regulator mutant, BGS9 (BGR1 qsmR::Ω). A comparison of gene expression profiling among the wild-type BGR1 and the two mutants before and after QS onset as well as gene ontology (GO) enrichment analysis from differential expressed genes (DEGs) revealed that genes involved in motility were highly enriched in TofI-dependent DEGs, whereas genes for transport and DNA polymerase were highly enriched in QsmR-dependent DEGs. Further, a combination of pathways with these DEGs and phenotype analysis of mutants pointed to a couple of metabolic processes, which are dependent on QS in B. glumae, that were directly or indirectly related with bacterial motility. The consistency of observed bacterial phenotypes with GOs or metabolic pathways in QS-regulated genes implied that integration RNAseq with GO enrichment or pathways would be useful to study bacterial physiology and phenotypes.

  13. Incidence of Burkholderia mallei infection among indigenous equines in India

    Science.gov (United States)

    Malik, Praveen; Singha, Harisankar; Goyal, Sachin K; Khurana, Sandip K; Tripathi, Badri Naryan; Dutt, Abha; Singh, Dabal; Sharma, Neeraj; Jain, Sanjay

    2015-01-01

    Burkholderia mallei is the causative agent of glanders which is a highly contagious and fatal disease of equines. Considering the nature and severity of the disease in equines, and potential of transmission to human beings, glanders is recognised as a ‘notifiable’ disease in many countries. An increasing number of glanders outbreaks throughout the Asian continents, including India, have been noticed recently. In view of the recent re-emergence of the disease, the present study was undertaken to estimate the prevalence of glanders among indigenous equines from different parts of India. Serum samples were analysed by complement fixation test (CFT) and ELISA for the detection of B mallei specific antibodies. A total of 7794 equines, which included 4720 horses, 1881 donkeys and 1193 mules were sampled from April 2011 to December 2014 from 10 states of India. Serologically, 36 equines (pony=7, mules=10, horses=19) were found to be positive for glanders by CFT and indirect-ELISA. The highest number of cases were detected in Uttar Pradesh (n=31) followed by Himachal Pradesh (n=4) and Chhattisgarh (n=1). Isolation of B mallei was attempted from nasal and abscess swabs collected from seropositive equines. Four isolates of B mallei were cultured from nasal swabs of two mules and two ponies. Identity of the isolates was confirmed by PCR and sequencing of fliP gene fragment. The study revealed circulation of B mallei in northern India and the need for continued surveillance to support the eradication. PMID:26457190

  14. Evolving serodiagnostics by rationally designed peptide arrays: the Burkholderia paradigm in Cystic Fibrosis

    Science.gov (United States)

    Peri, Claudio; Gori, Alessandro; Gagni, Paola; Sola, Laura; Girelli, Daniela; Sottotetti, Samantha; Cariani, Lisa; Chiari, Marcella; Cretich, Marina; Colombo, Giorgio

    2016-09-01

    Efficient diagnosis of emerging and novel bacterial infections is fundamental to guide decisions on therapeutic treatments. Here, we engineered a novel rational strategy to design peptide microarray platforms, which combines structural and genomic analyses to predict the binding interfaces between diverse protein antigens and antibodies against Burkholderia cepacia complex infections present in the sera of Cystic Fibrosis (CF) patients. The predicted binding interfaces on the antigens are synthesized in the form of isolated peptides and chemically optimized for controlled orientation on the surface. Our platform displays multiple Burkholderia-related epitopes and is shown to diagnose infected individuals even in presence of superinfections caused by other prevalent CF pathogens, with limited cost and time requirements. Moreover, our data point out that the specific patterns determined by combined probe responses might provide a characterization of Burkholderia infections even at the subtype level (genomovars). The method is general and immediately applicable to other bacteria.

  15. Identification of Burkholderia pseudomallei Near-Neighbor Species in the Northern Territory of Australia.

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    Jennifer L Ginther

    Full Text Available Identification and characterization of near-neighbor species are critical to the development of robust molecular diagnostic tools for biothreat agents. One such agent, Burkholderia pseudomallei, a soil bacterium and the causative agent of melioidosis, is lacking in this area because of its genomic diversity and widespread geographic distribution. The Burkholderia genus contains over 60 species and occupies a large range of environments including soil, plants, rhizospheres, water, animals and humans. The identification of novel species in new locations necessitates the need to identify the true global distribution of Burkholderia species, especially the members that are closely related to B. pseudomallei. In our current study, we used the Burkholderia-specific recA sequencing assay to analyze environmental samples from the Darwin region in the Northern Territory of Australia where melioidosis is endemic. Burkholderia recA PCR negative samples were further characterized using 16s rRNA sequencing for species identification. Phylogenetic analysis demonstrated that over 70% of the bacterial isolates were identified as B. ubonensis indicating that this species is common in the soil where B. pseudomallei is endemic. Bayesian phylogenetic analysis reveals many novel branches within the B. cepacia complex, one novel B. oklahomensis-like species, and one novel branch containing one isolate that is distinct from all other samples on the phylogenetic tree. During the analysis with recA sequencing, we discovered 2 single nucleotide polymorphisms in the reverse priming region of B. oklahomensis. A degenerate primer was developed and is proposed for future use. We conclude that the recA sequencing technique is an effective tool to classify Burkholderia and identify soil organisms in a melioidosis endemic area.

  16. Real-Time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei

    Science.gov (United States)

    2005-10-01

    1 Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei Vipin K. Rastogi1, Tu-chen Cheng1, Lisa Collins1 and Jennifer Bagley2 1...A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B.pseudomallei 5a. CONTRACT NUMBER 5b...risk. There is currently no real - time PCR assay for detection of both of these pathogens. Primers and probes corresponding to specific genomic regions

  17. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions.

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    Sandra Schwarz

    Full Text Available Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs of Burkholderia thailandensis (B. thai in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.

  18. Biodegradation of 3-Nitrotyrosine by Burkholderia sp. Strain JS165 and Variovorax paradoxus JS171

    Science.gov (United States)

    2006-02-01

    and B. Minambres. 2004. The homogentisate pathway: a central catabolic pathway involved in the degradation of L- phenylalanine , L-tyrosine, and 3...acetate-3- hydroxylase . A two-protein component enzyme. J. Biol. Chem. 267:25848–25855. 4. Bruhn, C., H. Lenke, and H.-J. Knackmuss. 1987. Nitrosubstituted

  19. Recovery efficiencies for Burkholderia thailandensis from various aerosol sampling media

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    Paul eDabisch

    2012-06-01

    Full Text Available Burkholderia thailandensis is used in the laboratory as a surrogate of the more virulent B. pseudomallei. Since inhalation is believed to be a natural route of infection for B. pseudomallei, many animal studies with B. pseudomallei and B. thailandensis utilize the inhalation route of exposure. The aim of the present study was to quantify the recovery efficiency of culturable B. thailandensis from several common aerosol sampling devices to ensure that collected microorganisms could be reliably recovered post-collection. The sampling devices tested included 25-mm gelatin filters, 25-mm stainless steel disks used in Mercer cascade impactors, and two types of glass impingers. The results demonstrate that while several processing methods tested resulted in significantly lower physical recovery efficiencies than other methods, it was possible to obtain culturable recovery efficiencies for B. thailandensis and physical recovery efficiencies for 1 μm fluorescent spheres of at least 0.95 from all of the sampling media tested given an appropriate sample processing procedure. The results of the present study also demonstrated that the bubbling action of liquid media in all-glass impingers (AGIs can result in physical loss of material from the collection medium, although additional studies are needed to verify the exact mechanisms involved. Overall, the results of this study demonstrate that the collection mechanism as well as the post-collection processing method can significantly affect the recovery from and retention of culturable microorganisms in sampling media, potentially affecting the calculated airborne concentration and any subsequent estimations of risk or dose derived from such data.

  20. Influence of neutrophil defects on Burkholderia cepacia complex pathogenesis

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    Laura A. Porter

    2011-11-01

    Full Text Available The Burkholderia cepacia complex (Bcc is a group of Gram-negative bacteria that are ubiquitous in the environment and have emerged as opportunistic pathogens in immunocompromised patients. The primary patient populations infected with Bcc include individuals with cystic fibrosis (CF, as well as those with chronic granulomatous disease (CGD. While Bcc infection in CF is better characterized than in CGD, these two genetic diseases are not obviously similar and it is currently unknown if there is any commonality in host immune defects that is responsible for the susceptibility to Bcc. CF is caused by mutations in the CF transmembrane conductance regulator, resulting in manifestations in various organ systems, however the major cause of morbidity and mortality is currently due to bacterial respiratory infections. CGD, on the other hand, is a genetic disorder that is caused by defects in phagocyte NADPH oxidase. Because of the defect in CGD, phagocytes in these patients are unable to produce reactive oxygen species, which results in increased susceptibility to bacterial and fungal infections. Despite this significant defect in microbial clearance, the spectrum of pathogens frequently implicated in infections in CGD is relatively narrow and includes some bacterial species that are considered almost pathognomonic for this disorder. Very little is known about the cause of the specific susceptibility to Bcc over other potential pathogens more prevalent in the environment, and a better understanding of specific mechanisms required for bacterial virulence has become a high priority. This review will summarize both the current knowledge and future directions related to Bcc virulence in immunocompromised individuals with a focus on the roles of bacterial factors and neutrophil defects in pathogenesis.

  1. Burkholderia pseudomallei is spatially distributed in soil in northeast Thailand.

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    Direk Limmathurotsakul

    Full Text Available BACKGROUND: Melioidosis is a frequently fatal infectious disease caused by the soil dwelling Gram-negative bacterium Burkholderia pseudomallei. Environmental sampling is important to identify geographical distribution of the organism and related risk of infection to humans and livestock. The aim of this study was to evaluate spatial distribution of B. pseudomallei in soil and consider the implications of this for soil sampling strategies. METHODS AND FINDINGS: A fixed-interval sampling strategy was used as the basis for detection and quantitation by culture of B. pseudomallei in soil in two environmental sites (disused land covered with low-lying scrub and rice field in northeast Thailand. Semivariogram and indicator semivariogram were used to evaluate the distribution of B. pseudomallei and its relationship with range between sampling points. B. pseudomallei was present on culture of 80/100 sampling points taken from the disused land and 28/100 sampling points from the rice field. The median B. pseudomallei cfu/gram from positive sampling points was 378 and 700 for the disused land and the rice field, respectively (p = 0.17. Spatial autocorrelation of B. pseudomallei was present, in that samples taken from areas adjacent to sampling points that were culture positive (negative for B. pseudomallei were also likely to be culture positive (negative, and samples taken from areas adjacent to sampling points with a high (low B. pseudomallei count were also likely to yield a high (low count. Ranges of spatial autocorrelation in quantitative B. pseudomallei count were 11.4 meters in the disused land and 7.6 meters in the rice field. CONCLUSIONS: We discuss the implications of the uneven distribution of B. pseudomallei in soil for future environmental studies, and describe a range of established geostatistical sampling approaches that would be suitable for the study of B. pseudomallei that take account of our findings.

  2. Polar Lipids of Burkholderia pseudomallei Induce Different Host Immune Responses

    Science.gov (United States)

    Gonzalez-Juarrero, Mercedes; Mima, Naoko; Trunck, Lily A.; Schweizer, Herbert P.; Bowen, Richard A.; Dascher, Kyle; Mwangi, Waithaka; Eckstein, Torsten M.

    2013-01-01

    Melioidosis is a disease in tropical and subtropical regions of the world that is caused by Burkholderia pseudomallei. In endemic regions the disease occurs primarily in humans and goats. In the present study, we used the goat as a model to dissect the polar lipids of B. pseudomallei to identify lipid molecules that could be used for adjuvants/vaccines or as diagnostic tools. We showed that the lipidome of B. pseudomallei and its fractions contain several polar lipids with the capacity to elicit different immune responses in goats, namely rhamnolipids and ornithine lipids which induced IFN-γ, whereas phospholipids and an undefined polar lipid induced strong IL-10 secretion in CD4+ T cells. Autologous T cells co-cultured with caprine dendritic cells (cDCs) and polar lipids of B. pseudomallei proliferated and up-regulated the expression of CD25 (IL-2 receptor) molecules. Furthermore, we demonstrated that polar lipids were able to up-regulate CD1w2 antigen expression in cDCs derived from peripheral blood monocytes. Interestingly, the same polar lipids had only little effect on the expression of MHC class II DR antigens in the same caprine dendritic cells. Finally, antibody blocking of the CD1w2 molecules on cDCs resulted in decreased expression for IFN-γ by CD4+ T cells. Altogether, these results showed that polar lipids of B. pseudomallei are recognized by the caprine immune system and that their recognition is primarily mediated by the CD1 antigen cluster. PMID:24260378

  3. Growth on mannitol-rich media elicits a genome-wide transcriptional response in Burkholderia multivorans that impacts on multiple virulence traits in an exopolysaccharide-independent manner.

    Science.gov (United States)

    Denman, Carmen C; Robinson, Matthew T; Sass, Andrea M; Mahenthiralingam, Eshwar; Brown, Alan R

    2014-01-01

    In common with other members of the Burkholderia cepacia complex (BCC), Burkholderia multivorans is capable of producing exopolysaccharide (EPS) when grown on certain mannitol-rich media. The significance of the resulting mucoid phenotype and the genome-wide response to mannitol has never been characterized despite its clinical relevance following the approval of a dried-powder preparation of mannitol as an inhaled osmolyte therapy for cystic fibrosis (CF) patients. In the present study we defined the transcriptional response of B. multivorans ATCC 17616, a model genome-sequenced strain of environmental origin, to growth on mannitol-rich yeast extract media (MYEM). EPS-dependent and -independent impact of MYEM on virulence-associated traits was assessed in both strain ATCC 17616 and the CF isolate B. multivorans C1576. Our studies revealed a significant transcriptional response to MYEM encompassing approximately 23 % of predicted genes within the genome. Strikingly, this transcriptional response identified that EPS induction occurs in ATCC 17616 without the upregulation of the bce-I and bce-II EPS gene clusters, despite their pivotal role in EPS biosynthesis. Of approximately 20 differentially expressed putative virulence factors, 16 exhibited upregulation including flagella, ornibactin, oxidative stress proteins and phospholipases. MYEM-grown B. multivorans also exhibited enhanced motility, biofilm formation and epithelial cell invasion. In contrast to these potential virulence enhancements, MYEM-grown B. multivorans C1576 showed attenuated virulence in the Galleria mellonella infection model. All of the observed phenotypic responses occurred independently of EPS production, highlighting the profound impact that mannitol-based growth has on the physiology and virulence of B. multivorans.

  4. Burkholderia ambifaria and B. caribensis promote growth and increase yield in grain amaranth (Amaranthus cruentus and A. hypochondriacus) by improving plant nitrogen uptake.

    Science.gov (United States)

    Parra-Cota, Fannie I; Peña-Cabriales, Juan J; de Los Santos-Villalobos, Sergio; Martínez-Gallardo, Norma A; Délano-Frier, John P

    2014-01-01

    Grain amaranth is an emerging crop that produces seeds having high quality protein with balanced amino-acid content. However, production is restricted by agronomic limitations that result in yields that are lower than those normally produced by cereals. In this work, the use of five different rhizobacteria were explored as a strategy to promote growth and yields in Amaranthus hypochondriacus cv. Nutrisol and A. cruentus cv. Candil, two commercially important grain amaranth cultivars. The plants were grown in a rich substrate, high in organic matter, nitrogen (N), and phosphorus (P) and under greenhouse conditions. Burkholderia ambifaria Mex-5 and B. caribensis XV proved to be the most efficient strains and significantly promoted growth in both grain amaranth species tested. Increased grain yield and harvest index occurred in combination with chemical fertilization when tested in A. cruentus. Growth-promotion and improved yields correlated with increased N content in all tissues examined. Positive effects on growth also occurred in A. cruentus plants grown in a poor soil, even after N and P fertilization. No correlation between non-structural carbohydrate levels in roots of inoculated plants and growth promotion was observed. Conversely, gene expression assays performed at 3-, 5- and 7-weeks after seed inoculation in plants inoculated with B. caribensis XV identified a tissue-specific induction of several genes involved in photosynthesis, sugar- and N- metabolism and transport. It is concluded that strains of Burkholderia effectively promote growth and increase seed yields in grain amaranth. Growth promotion was particularly noticeable in plants grown in an infertile soil but also occurred in a well fertilized rich substrate. The positive effects observed may be attributed to a bio-fertilization effect that led to increased N levels in roots and shoots. The latter effect correlated with the differential induction of several genes involved in carbon and N metabolism

  5. A heterodimer comprised of two bovine lactoferrin antimicrobial peptides exhibits powerful bactericidal activity against Burkholderia pseudomallei

    NARCIS (Netherlands)

    Puknun, A.; Bolscher, J.G.M.; Nazmi, K.; Veerman, E.C.I.; Tungpradabkul, S.; Wongratanacheewin, S.; Kanthawong, S.; Taweechaisupapong, S.

    2013-01-01

    Melioidosis is a severe infectious disease that is endemic in Southeast Asia and Northern Australia. Burkholderia pseudomallei, the causative agent of this disease, has developed resistance to an increasing list of antibiotics, demanding a search for novel agents. Lactoferricin and lactoferrampin ar

  6. N-acylhomoserine-lactone-mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms

    DEFF Research Database (Denmark)

    Riedel, K; Hentzer, Morten; Geisenberger, O

    2001-01-01

    Pseudomonas aeruginosa and Burkholderia cepacia are capable of forming mixed biofilms in the lungs of cystic fibrosis patients. Both bacteria employ quorum-sensing systems, which rely on N-acylhomoserine lactone (AHL) signal molecules, to co-ordinate expression of virulence factors with the forma...

  7. Enhanced biodegradation of anthracene in acidic soil by inoculated Burkholderia sp. VUN10013.

    Science.gov (United States)

    Somtrakoon, Khanitta; Suanjit, Sudarat; Pokethitiyook, Prayad; Kruatrachue, Maleeya; Lee, Hung; Upatham, Suchart

    2008-08-01

    The ability of Burkholderia sp. VUN10013 to degrade anthracene in microcosms of two acidic Thai soils was studied. The addition of Burkholderia sp. VUN10013 (initial concentration of 10(5) cells g(-1) dry soil) to autoclaved soil collected from the Plew District, Chanthaburi Province, Thailand, supplemented with anthracene (50 mg kg(-1) dry soil) resulted in complete degradation of the added anthracene within 20 days. In contrast, under the same test conditions but using autoclaved soil collected from the Kitchagude District, Chanthaburi Province, Thailand, only approximately 46.3% of the added anthracene was degraded after 60 days of incubation. In nonautoclaved soils, without adding the VUN10013 inocula, 22.8 and 19.1% of the anthracene in Plew and Kitchagude soils, respectively, were degraded by indigenous bacteria after 60 days. In nonautoclaved soil inoculated with Burkholderia sp. VUN10013, the rate and extent of anthracene degradation were considerably better than those seen in autoclaved soils or in uninoculated nonautoclaved soils in that only 8.2 and 9.1% of anthracene remained in nonautoclaved Plew and Kitchagude soils, respectively, after 10 days of incubation. The results showed that the indigenous microorganisms in the pristine acidic soils have limited ability to degrade anthracene. Inoculation with the anthracene-degrading Burkholderia sp. VUN10013 significantly enhanced anthracene degradation in such acidic soils. The indigenous microorganisms greatly assisted the VUN10013 inoculum in anthracene degradation, especially in the more acidic Kitchagude soil.

  8. N-acylhomoserine-lactone-mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms

    DEFF Research Database (Denmark)

    Riedel, K.; Hentzer, Morten; Geisenberger, O.;

    2001-01-01

    Pseudomonas aeruginosa and Burkholderia cepacia are capable of forming mixed biofilms in the lungs of cystic fibrosis patients. Both bacteria employ quorum-sensing systems, which rely on N-acylhomoserine lactone (AHL) signal molecules, to co- ordinate expression of virulence factors with the form...

  9. Molecular Characterization of Genetic Loci Required for Secretion of Exoproducts in Burkholderia pseudomallei

    OpenAIRE

    DeShazer, David; Brett, Paul J.; Mary N Burtnick; Woods, Donald E.

    1999-01-01

    Previous studies have demonstrated that Burkholderia pseudomallei secretes protease, lipase, and phospholipase C (PLC) into the extracellular milieu, but their mechanisms of secretion and roles in pathogenesis have not been elucidated. In this study, we isolated and characterized 29 transposon mutants unable to secrete protease, lipase, and PLC.

  10. Changes in agricultural management drive the diversity of Burkholderia species isolated from soil on PLAT medium

    NARCIS (Netherlands)

    Salles, JF; Samyn, E; Vandamme, P; van Veen, JA; van Elsas, JD

    2006-01-01

    In order to assess the diversity of culturable Burkholderia populations in rhizosphere and bulk soil and to evaluate how different agricultural management regimes and land use history affect this diversity, four treatments were evaluated: permanent grassland; grassland converted into maize monocultu

  11. Changes in agricultural management drive the diversity of Burkholderia species isolated from soil on PCAT medium

    NARCIS (Netherlands)

    Salles, J.F.; Samyn, E.; Vandamme, P.; Van Veen, J.A.; van Elsas, J.D.

    2006-01-01

    Abstract In order to assess the diversity of culturable Burkholderia populations in rhizosphere and bulk soil and to evaluate how different agricultural management regimes and land use history affect this diversity, four treatments were evaluated: permanent grassland; grassland converted into maize

  12. A disruption of ctpA encoding carboxy-terminal protease attenuates Burkholderia mallei and induces partial protection in CD1 mice.

    Science.gov (United States)

    Bandara, Aloka B; DeShazer, David; Inzana, Thomas J; Sriranganathan, Nammalwar; Schurig, Gerhardt G; Boyle, Stephen M

    2008-09-01

    Burkholderia mallei is the etiologic agent of glanders in solipeds (horses, mules and donkeys), and incidentally in carnivores and humans. Little is known about the molecular mechanisms of B. mallei pathogenesis. The putative carboxy-terminal processing protease (CtpA) of B. mallei is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. All species and isolates of Burkholderia carry a highly conserved copy of ctpA. We studied the involvement of CtpA on growth, cell morphology, persistence, and pathogenicity of B. mallei. A sucrose-resistant strain of B. mallei was constructed by deleting a major portion of the sacB gene of the wild type strain ATCC 23344 by gene replacement, and designated as strain 23344DeltasacB. A portion of the ctpA gene (encoding CtpA) of strain 23344DeltasacB was deleted by gene replacement to generate strain 23344DeltasacBDeltactpA. In contrast to the wild type ATCC 23344 or the sacB mutant 23344DeltasacB, the ctpA mutant 23344DeltasacBDeltactpA displayed altered cell morphologies with partially or fully disintegrated cell envelopes. Furthermore, relative to the wild type, the ctpA mutant displayed slower growth in vitro and less ability to survive in J774.2 murine macrophages. The expression of mRNA of adtA, the gene downstream of ctpA was similar among the three strains suggesting that disruption of ctpA did not induce any polar effects. As with the wild type or the sacB mutant, the ctpA mutant exhibited a dose-dependent lethality when inoculated intraperitoneally into CD1 mice. The CD1 mice inoculated with a non-lethal dose of the ctpA mutant produced specific serum immunoglobulins IgG1 and IgG2a and were partially protected against challenge with wild type B. mallei ATCC 23344. These findings suggest that CtpA regulates in vitro growth, cell morphology and intracellular survival of B. mallei, and a ctpA mutant protects CD1 mice against glanders.

  13. Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei.

    Science.gov (United States)

    Godoy, Daniel; Randle, Gaynor; Simpson, Andrew J; Aanensen, David M; Pitt, Tyrone L; Kinoshita, Reimi; Spratt, Brian G

    2003-05-01

    A collection of 147 isolates of Burkholderia pseudomallei, B. mallei, and B. thailandensis was characterized by multilocus sequence typing (MLST). The 128 isolates of B. pseudomallei, the causative agent of melioidosis, were obtained from diverse geographic locations, from humans and animals with disease, and from the environment and were resolved into 71 sequence types. The utility of the MLST scheme for epidemiological investigations was established by analyzing isolates from captive marine mammals and birds and from humans in Hong Kong with melioidosis. MLST gave a level of resolution similar to that given by pulsed-field gel electrophoresis and identified the same three clones causing disease in animals, each of which was also associated with disease in humans. The average divergence between the alleles of B. thailandensis and B. pseudomallei was 3.2%, and there was no sharing of alleles between these species. Trees constructed from differences in the allelic profiles of the isolates and from the concatenated sequences of the seven loci showed that the B. pseudomallei isolates formed a cluster of closely related lineages that were fully resolved from the cluster of B. thailandensis isolates, confirming their separate species status. However, isolates of B. mallei, the causative agent of glanders, recovered from three continents over a 30-year period had identical allelic profiles, and the B. mallei isolates clustered within the B. pseudomallei group of isolates. Alleles at six of the seven loci in B. mallei were also present within B. pseudomallei isolates, and B. mallei is a clone of B. pseudomallei that, on population genetics grounds, should not be given separate species status.

  14. Genome-wide analysis reveals loci encoding anti-macrophage factors in the human pathogen Burkholderia pseudomallei K96243.

    Directory of Open Access Journals (Sweden)

    Andrea J Dowling

    Full Text Available Burkholderia pseudomallei is an important human pathogen whose infection biology is still poorly understood. The bacterium is endemic to tropical regions, including South East Asia and Northern Australia, where it causes melioidosis, a serious disease associated with both high mortality and antibiotic resistance. B. pseudomallei is a Gram-negative facultative intracellular pathogen that is able to replicate in macrophages. However despite the critical nature of its interaction with macrophages, few anti-macrophage factors have been characterized to date. Here we perform a genome-wide gain of function screen of B. pseudomallei strain K96243 to identify loci encoding factors with anti-macrophage activity. We identify a total of 113 such loci scattered across both chromosomes, with positive gene clusters encoding transporters and secretion systems, enzymes/toxins, secondary metabolite, biofilm, adhesion and signal response related factors. Further phenotypic analysis of four of these regions shows that the encoded factors cause striking cellular phenotypes relevant to infection biology, including apoptosis, formation of actin 'tails' and multi-nucleation within treated macrophages. The detailed analysis of the remaining host of loci will facilitate genetic dissection of the interaction of this important pathogen with host macrophages and thus further elucidate this critical part of its infection cycle.

  15. Biodegradation of crystal violet using Burkholderia vietnamiensis C09V immobilized on PVA-sodium alginate-kaolin gel beads.

    Science.gov (United States)

    Cheng, Ying; Lin, HongYan; Chen, Zuliang; Megharaj, Mallavarapu; Naidu, Ravi

    2012-09-01

    The strain, Burkholderia vietnamiensis C09V was immobilized on PVA-alginate-kaolin gel beads as a biomaterial to improve the degradation of crystal violet from aqueous solution. The results show that 98.6% (30 mg L(-1)) crystal violet was removed from aqueous solution using immobilized cells on PVA-alginate-kaolin gel beads, while 94.0% crystal violet was removed by free cells after degradation at the pH 5 and 30°C for 30 h. Kinetics studies show that the pseudo-second-order kinetics well described the adsorption of crystal violet on the PVA-alginate-kaolin beads. Biodegradation of crystal violet on immobilized cells was fitted well by first-order reaction kinetics, indicating that CV was adsorbed onto kaolin and followed their degradation by immobilized cells onto the the PVA-alginate-kaolin beads. Characterization with SEM shows that cells attached well to the surface of PVA-alginate-kaolin beads, leading to improved crystal violet transfer from aqueous solution to immobilized cells. In addition, UV-vis show that the absorption peak at 588 nm was reduced by the degraded N-bond linkages, as well as the formation of degrading products were observed by Fourier transform infrared (FTIR). These results suggest that crystal violet was biodegraded to N,N-dimethylaminophenol and Michler's Ketone prior to these intermediates being further degraded. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Development of a Polymerase Chain Reaction Assay for Detection of Burkholderia mallei, a Potent Biological Warfare Agent

    Directory of Open Access Journals (Sweden)

    Vijai Pal

    2016-09-01

    Full Text Available Burkholderia mallei is the etiological agent of glanders, primarily a disease of equines. B. mallei is closely related to B. pseudomallei, the causative agent of melioidosis. Therefore, detection of B. mallei and its differentiation from B. pseudomallei, has always been troublesome. In present investigation, a B. mallei specific DNA sequence was identified by performing BLASTn search using ~3000 ORFs of B. mallei NCTC 10229. A polymerase chain reaction (PCR assay with internal amplification control (IAC was developed for detection of B. mallei and its differentiation from B. pseudomallei. The PCR assay could amplify a specific 224-bp fragment from all the six B. mallei strains used in the study, whereas other closely related organisms were tested negative. The detection limit of the assay was found to be 10 pg of purified DNA of B. mallei. Incorporation of IAC in the assay makes the results reliable as false negative results which may arise due to presence of PCR inhibitors, can be avoided. For validation, the assay was tested on tap water, Bengal gram and grass artificially spiked with B. mallei. The developed assay can be used as a simple and rapid tool for detection of B. mallei.

  17. Biocontrol of Late Blight (Phytophthora capsici Disease and Growth Promotion of Pepper by Burkholderia cepacia MPC-7

    Directory of Open Access Journals (Sweden)

    Mao Sopheareth

    2013-03-01

    Full Text Available A chitinolytic bacterial strain having strong antifungal activity was isolated and identified as Burkholderia cepacia MPC-7 based on 16S rRNA gene analysis. MPC-7 solubilized insoluble phosphorous in hydroxyapatite agar media. It produced gluconic acid and 2-ketogluconic acid related to the decrease in pH of broth culture. The antagonist produced benzoic acid (BA and phenylacetic acid (PA. The authentic compounds, BA and PA, showed a broad spectrum of antimicrobial activity against yeast, several bacterial and fungal pathogens in vitro. To demonstrate the biocontrol efficiency of MPC-7 on late blight disease caused by Phytophthora capsici, pepper plants in pot trials were treated with modified medium only (M, M plus zoospore inoculation (MP, MPC-7 cultured broth (B and B plus zoospore inoculation (BP. With the sudden increase in root mortality, plants in MP wilted as early as five days after pathogen inoculation. However, plant in BP did not show any symptom of wilting until five days. Root mortality in BP was markedly reduced for as much as 50%. Plants in B had higher dry weight, P concentration in root, and larger leaf area compared to those in M and MP. These results suggested that B. cepacia MPC-7 should be considered as a candidate for the biological fertilizer as well as antimicrobial agent for pepper plants.

  18. Novel engineered cationic antimicrobial peptides display broad-spectrum activity against Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei.

    Science.gov (United States)

    Abdelbaqi, Suha; Deslouches, Berthony; Steckbeck, Jonathan; Montelaro, Ronald; Reed, Douglas S

    2016-02-01

    Broad-spectrum antimicrobials are needed to effectively treat patients infected in the event of a pandemic or intentional release of a pathogen prior to confirmation of the pathogen's identity. Engineered cationic antimicrobial peptides (eCAPs) display activity against a number of bacterial pathogens including multi-drug-resistant strains. Two lead eCAPs, WLBU2 and WR12, were compared with human cathelicidin (LL-37) against three highly pathogenic bacteria: Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. Both WLBU2 and WR12 demonstrated bactericidal activity greater than that of LL-37, particularly against F. tularensis and Y. pestis. Only WLBU2 had bactericidal activity against B. pseudomallei. WLBU2, WR12 and LL-37 were all able to inhibit the growth of the three bacteria in vitro. Because these bacteria can be facultative intracellular pathogens, preferentially infecting macrophages and dendritic cells, we evaluated the activity of WLBU2 against F. tularensis in an ex vivo infection model with J774 cells, a mouse macrophage cell line. In that model WLBU2 was able to achieve greater than 50% killing of F. tularensis at a concentration of 12.5 μM. These data show the therapeutic potential of eCAPs, particularly WLBU2, as a broad-spectrum antimicrobial for treating highly pathogenic bacterial infections.

  19. Culture conditions for the production of an acidic exopolysaccharide by the nitrogen-fixing bacterium Burkholderia tropica.

    Science.gov (United States)

    Serrato, Rodrigo V; Sassaki, Guilherme L; Cruz, Leonardo M; Pedrosa, Fábio O; Gorin, Philip A J; Iacomini, Marcello

    2006-05-01

    The endophytic diazotrophic bacterium Burkholderia tropica, strain Ppe8, produced copious amounts of exopolysaccharide (EPS) on batch growth in liquid synthetic media containing mannitol and glutamate as carbon and nitrogen sources. The effect of various aeration regimes and carbon source concentrations on EPS production was determined, as well as the effects of temperature and time of growth. The degree of aeration had a great influence on the yield of EPS, in contrast with the C:N ratio of the medium. Growth temperature also affected the EPS yield after the first 24 h of culture but seemed to be irrelevant after that. After isolation and purification, the EPS was analyzed by high-performance size exclusion chromatography and multiangle laser light scattering (HPSEC-MALLS), revealing a molecular mass of 300 kDa. The acid hydrolyzate of EPS was examined by HPLC and found to contain Glc, Rha, GlcA, and an aldobiouronic acid. The latter was found to have a GlcA and a Rha unit. Carboxy-reduced EPS contained Glc and Rha (3:2). The monosaccharide composition of the native acidic EPS was calculated as GlcA, Glc, and Rha in a molar ratio of 1:2:2.

  20. Diversity of 16S-23S rDNA internal transcribed spacer (ITS reveals phylogenetic relationships in Burkholderia pseudomallei and its near-neighbors.

    Directory of Open Access Journals (Sweden)

    Andrew P Liguori

    Full Text Available Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species.

  1. Molecular investigations of PenA-mediated beta-lactam resistance inBurkholderia pseudomallei

    Directory of Open Access Journals (Sweden)

    Drew A Rholl

    2011-07-01

    Full Text Available Burkholderia pseudomallei is the etiological agent of melioidosis. Because of the bacterium’s intrinsic resistance and propensity to establish latent infections, melioidosis therapy is complicated and prolonged. Newer generation b-lactams, specifically ceftazidime, are used for acute phase therapy, but resistance to the drugs has been observed. The chromosomally-encoded penA gene encodes a putative twin arginine translocase (TAT-secreted b-lactamase, and penA mutations have been implicated in ceftazidime resistance in clinical isolates. However, the role of PenA in resistance has not yet been systematically studied in isogenetic B. pseudomallei mutant backgrounds. We investigated the effects of penA deletion, point mutations, and up-regulation, as well as tat operon deletion and PenA TAT signal sequence mutations. These experiments were made possible by employing a B. pseudomallei strain that is excluded from Select Agent regulations. Deletion of penA significantly (>4-fold reduced the susceptibility to six of the nine b-lactams tested and >16-fold for ampicillin, amoxicillin and carbenicillin. Over-expression of penA by single-copy, chromosomal expression of the gene under control of the inducible Ptac promoter, increased resistance levels for all b-lactams tested 2- to 10-fold. Recreation of the C69Y and P167S PenA amino acid substitutions previously observed in resistant clinical isolates increased resistance to ceftazidime by >85 and 5 to 8-fold, respectively. Similarly, a S72F substitution resulted in a 4-fold increase in resistance to amoxicillin + clavulanic acid. Susceptibility assays with PenA TAT signal sequence and tatABC mutants, as well as Western blot analysis, confirmed that PenA is a TAT secreted enzyme and not periplasmic but associated with the spheroplastic cell fraction. Lastly, we determined that two LysR-family regulators encoded by genes adjacent to penA do not play a role in transcriptional regulation of pen

  2. Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping

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    Harvey Steven P

    2007-03-01

    Full Text Available Abstract Background The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing ~18,000 generations. Results B. pseudomallei contains a preponderance of tandem repeat loci throughout its genome, many of which are duplicated elsewhere in the genome. The majority of these loci are composed of repeat motif lengths of 6 to 9 bp with 4 to 10 repeat units and are predominately located in intergenic regions of the genome. Across geographically diverse B. pseudomallei and B.mallei isolates, the 32 VNTR loci displayed between 7 and 28 alleles, with Nei's diversity values ranging from 0.47 and 0.94. Mutation rates for these loci are comparable (>10-5 per locus per generation to that of the most diverse tandemly repeated regions found in other less diverse bacteria. Conclusion The frequency, location and duplicate nature of tandemly repeated regions within the B. pseudomallei genome indicate that these tandem repeat regions may play a role in generating and maintaining adaptive genomic variation. Multiple-locus VNTR analysis revealed extensive diversity within the global isolate set containing B. pseudomallei and B. mallei, and it detected genotypic differences within clonal lineages of both species that were

  3. Activation of Toll-like receptors by Burkholderia pseudomallei

    Directory of Open Access Journals (Sweden)

    Jansson-Hutson Malinka J

    2008-08-01

    Full Text Available Abstract Background Melioidosis, a lethal tropical infection that is endemic in southeast Asia and northern Australia, is caused by the saprophytic Gram-negative bacterium Burkholderia pseudomallei. Overall mortality approaches 40% yet little is known about mechanisms of host defense. Toll-like receptors (TLRs are host transmembrane receptors that recognize conserved pathogen molecular patterns and induce an inflammatory response. The lipopolysaccharide (LPS of Gram-negative bacteria is a potent inducer of the host innate immune system. TLR4, in association with MD-2, is the archetype receptor for LPS although B. pseudomallei LPS has been previously identified as a TLR2 agonist. We examined TLR signaling induced by B. pseudomallei, B. pseudomallei LPS, and B. pseudomallei lipid A using gain-of-function transfection assays of NF-κB activation and studies of TLR-deficient macrophages. Results In HEK293 cells transfected with murine or human TLRs, CD14, and MD-2, heat-killed B. pseudomallei activated TLR2 (in combination with TLR1 or TLR6 and TLR4. B. pseudomallei LPS and lipid A activated TLR4 and this TLR4-mediated signaling required MD-2. In TLR2-/- macrophages, stimulation with heat-killed B. pseudomallei augmented TNF-α and MIP-2 production whereas in TLR4-/- cells, TNF-α, MIP-2, and IL-10 production was reduced. Cytokine production by macrophages stimulated with B. pseudomallei LPS or lipid A was entirely dependent on TLR4 but was increased in the absence of TLR2. TLR adaptor molecule MyD88 strongly regulated TNF-α production in response to heat-killed B. pseudomallei. Conclusion B. pseudomallei activates TLR2 and TLR4. In the presence of MD-2, B. pseudomallei LPS and lipid A are TLR4 ligands. Although the macrophage cytokine response to B. pseudomallei LPS or lipid A is completely dependent on TLR4, in TLR2-/- macrophages stimulated with B. pseudomallei, B. pseudomallei LPS or lipid A, cytokine production is augmented. Other MyD88

  4. PCR-based identification of Burkholderia pseudomallei Identificação de Burkholderia pseudomallei baseada em PCR

    Directory of Open Access Journals (Sweden)

    Adam Merritt

    2006-10-01

    Full Text Available DNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99, but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.As técnicas de amplificação de DNA estão sendo cada vez mais utilizadas em laboratórios clínicos para a confirmação da identificação de bact

  5. Biodegradation of s-triazine herbicide atrazine by Enterobacter cloacae and Burkholderia cepacia sp. from long-term treated sugarcane-cultivated soils in Kenya.

    Science.gov (United States)

    Ngigi, Anastasiah N; Getenga, Zachary M; Boga, Hamadi I; Ndalut, Paul K

    2012-09-01

    In this study soils from sugarcane-cultivated fields were screened for bacterial species capable of atrazine (6-chloro-N²-ethyl-N⁴-isopropyl-1,3,5-triazine-2,4-diamine) degradation due to long exposure of the soils to this herbicide. To enrich for atrazine degraders, Minimal Salt Medium containing atrazine as the sole N source and glucose as the C source was inoculated with soils impacted with this herbicide and incubated. Bacterial growth was monitored by measuring optical density. The degradation of atrazine was followed by measuring residual atrazine in liquid cultures over a given time period by high performance liquid chromatography. Bacterial strains isolated from the enrichment cultures were characterized by biochemical tests and identified by 16S rRNA gene sequencing. Two bacterial strains coded ISL 8 and ISL 15 isolated from two different fields were shown to have 94 and 96% 16S rRNA gene sequence similarity to Burkholderia cepacia respectively. Another bacterial sp., ISL 14 was closely related to Enterobacter cloacae with a 96% 16S rRNA gene sequence similarity. There was not much difference between the extents of atrazine degradation by the enrichment cultures with communities (79-82% applied amount) from which pure strains were isolated and the pure strains themselves in liquid cultures that showed a degradation of 53-83% of applied amount. The study showed existence of bacterial strains in different sugarcane-cultivated fields which can use atrazine as a nitrogen source. The bacterial strains isolated can be used to enhance the degradation of atrazine in contaminated soils where atrazine is still considered to be recalcitrant.

  6. Total protein extraction and 2-D gel electrophoresis methods for Burkholderia species.

    Science.gov (United States)

    Velapatiño, Billie; Zlosnik, James E A; Hird, Trevor J; Speert, David P

    2013-10-15

    The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

  7. Distinct human antibody response to the biological warfare agent Burkholderia mallei.

    Science.gov (United States)

    Varga, John J; Vigil, Adam; DeShazer, David; Waag, David M; Felgner, Philip; Goldberg, Joanna B

    2012-10-01

    The genetic similarity between Burkholderia mallei (glanders) and Burkholderia pseudomallei (melioidosis) had led to the general assumption that pathogenesis of each bacterium would be similar. In 2000, the first human case of glanders in North America since 1945 was reported in a microbiology laboratory worker. Leveraging the availability of pre-exposure sera for this individual and employing the same well-characterized protein array platform that has been previously used to study a large cohort of melioidosis patients in southeast Asia, we describe the antibody response in a human with glanders. Analysis of 156 peptides present on the array revealed antibodies against 17 peptides with a > 2-fold increase in this infection. Unexpectedly, when the glanders data were compared with a previous data set from B. pseudomallei infections, there were only two highly increased antibodies shared between these two infections. These findings have implications in the diagnosis and treatment of B. mallei and B. pseudomallei infections.

  8. Enhanced bioconversion of ethylene glycol to glycolic acid by a newly isolated Burkholderia sp. EG13.

    Science.gov (United States)

    Gao, Xiaoxin; Ma, Zhengfei; Yang, Limin; Ma, Jiangquan

    2014-10-01

    Burkholderia sp. EG13 with high ethylene glycol-oxidizing activity was isolated from soil, which could be used for the synthesis of glycolic acid from the oxidation of ethylene glycol. Using the resting cells of Burkholderia sp. EG13 as biocatalysts, the optimum reaction temperature and pH were 30 °C and 6.0, respectively. After 24 h of biotransformation, the yield of glycolic acid from 200 mM ethylene glycol was 98.8 %. Furthermore, an integrated bioprocess for the production of glycolic acid which involved in situ product removal (ISPR) was investigated. Using fed-batch method with ISPR, a total of 793 mM glycolic acid has been accumulated in the reaction mixture after the 4th feed.

  9. Novel Burkholderia mallei Virulence Factors Linked to Specific Host-Pathogen Protein Interactions

    Science.gov (United States)

    2013-06-23

    fundamental molecular host-pathogen interac- tions that mediate these activities have gathered less atten- tion. Although the transcriptional immune response...Hines, H. B., and Jeddeloh, J. A. (2004) Quorum sensing: A transcriptional regulatory system involved in the pathogenicity of Burkholderia mallei...orthology database for prokaryotes and eukaryotes inferred by evolutionary evidence. BMC Bioinformatics 13, 143 25. Yu, C., Zavaljevski, N., Desai, V

  10. Combining functional and structural genomics to sample the essential Burkholderia structome.

    Directory of Open Access Journals (Sweden)

    Loren Baugh

    Full Text Available The genus Burkholderia includes pathogenic gram-negative bacteria that cause melioidosis, glanders, and pulmonary infections of patients with cancer and cystic fibrosis. Drug resistance has made development of new antimicrobials critical. Many approaches to discovering new antimicrobials, such as structure-based drug design and whole cell phenotypic screens followed by lead refinement, require high-resolution structures of proteins essential to the parasite.We experimentally identified 406 putative essential genes in B. thailandensis, a low-virulence species phylogenetically similar to B. pseudomallei, the causative agent of melioidosis, using saturation-level transposon mutagenesis and next-generation sequencing (Tn-seq. We selected 315 protein products of these genes based on structure-determination criteria, such as excluding very large and/or integral membrane proteins, and entered them into the Seattle Structural Genomics Center for Infection Disease (SSGCID structure determination pipeline. To maximize structural coverage of these targets, we applied an "ortholog rescue" strategy for those producing insoluble or difficult to crystallize proteins, resulting in the addition of 387 orthologs (or paralogs from seven other Burkholderia species into the SSGCID pipeline. This structural genomics approach yielded structures from 31 putative essential targets from B. thailandensis, and 25 orthologs from other Burkholderia species, yielding an overall structural coverage for 49 of the 406 essential gene families, with a total of 88 depositions into the Protein Data Bank. Of these, 25 proteins have properties of a potential antimicrobial drug target i.e., no close human homolog, part of an essential metabolic pathway, and a deep binding pocket. We describe the structures of several potential drug targets in detail.This collection of structures, solubility and experimental essentiality data provides a resource for development of drugs against

  11. Evaluation of six commercial DNA extraction kits for recovery of Burkholderia pseudomallei DNA.

    Science.gov (United States)

    Marques, Maria Angela de Mello; Zimmermann, Pia; Messelhäußer, Ute; Sing, Andreas

    2012-12-01

    Six commercially available DNA extraction kits, as well as thermal lysis and proteinase K DNA extraction were evaluated regarding bacterial inactivation, DNA yield and purity, and their use in a Burkholderia pseudomallei real-time PCR. While all methods successfully inactivated the bacteria, by measuring DNA purity and the level of detection by real-time PCR, the proteinase K method was the most sensitive.

  12. A predicted structure of the cytochrome c oxidase from Burkholderia pseudomallei

    OpenAIRE

    Mohd. Raih,Mohd. Firdaus; Sailan,Ahmad Tarmidi; Zamrod,Zulkeflie; Embi,Mohd. Noor; Mohamed, Rahmah

    2003-01-01

    Cytochrome c oxidase, the terminal enzyme of the respiratory chains of mitochondria and aerobic bacteria, catalyzes electron transfer from cytochrome c to molecular oxygen. The enzyme belongs to the haem-copper-containing oxidases superfamily. A recombinant plasmid carrying a 2.0 kb insert from a Burkholderia pseudomallei genomic library was subjected to automated DNA sequencing utilizing a primer walking strategy. Analysis of the 2002 bp insert revealed a 1536 bp open reading frame predicted...

  13. The Twin Arginine Translocation System Is Essential for Aerobic Growth and Full Virulence of Burkholderia thailandensis

    OpenAIRE

    Wagley, Sariqa; Hemsley, Claudia; Thomas, Rachael; Madeleine G Moule; Vanaporn, Muthita; Andreae, Clio; Robinson, Matthew; Goldman, Stan; Brendan W. Wren; Butler, Clive S.; Richard W Titball

    2014-01-01

    The twin arginine translocation (Tat) system in bacteria is responsible for transporting folded proteins across the cytoplasmic membrane, and in some bacteria, Tat-exported substrates have been linked to virulence. We report here that the Tat machinery is present in Burkholderia pseudomallei, B. mallei, and B. thailandensis, and we show that the system is essential for aerobic but not anaerobic growth. Switching off of the Tat system in B. thailandensis grown anaerobically resulted in filamen...

  14. Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex

    OpenAIRE

    Bartholdson, S. Josefin; Brown, Alan R.; Mewburn, Ben R.; Clarke, David J.; Fry, Stephen C.; Campopiano, Dominic J.; John R W Govan

    2008-01-01

    The species that presently constitute the Burkholderia cepacia complex (Bcc) have multiple roles; they include soil and water saprophytes, bioremediators, and plant, animal and human pathogens. Since the first description of pathogenicity in the Bcc was based on sour skin rot of onion bulbs, this study returned to this plant host to investigate the onion-associated phenotype of the Bcc. Many Bcc isolates, which were previously considered to be non-mucoid, produced copious amounts of exopolysa...

  15. Type III Secretion: a Virulence Factor Delivery System Essential for the Pathogenicity of Burkholderia mallei

    OpenAIRE

    Ulrich, Ricky L; DeShazer, David

    2004-01-01

    By creating mutations in the Burkholderia mallei ATCC 23344 animal pathogen-like type III secretion system (TTSS), this study analyzes the correlation between type III secretion and the pathogenicity of ATCC 23344 in vivo. Mutagenesis demonstrated that a functional TTSS was required for the full pathogenicity of ATCC 23344 in the BALB/c mouse and Syrian hamster models of infection. However, vaccination with each mutant failed to elicit a protective immunity against challenge with wild-type AT...

  16. In Vitro Activity of Fusidic Acid (CEM-102, Sodium Fusidate) against Staphylococcus aureus Isolates from Cystic Fibrosis Patients and Its Effect on the Activities of Tobramycin and Amikacin against Pseudomonas aeruginosa and Burkholderia cepacia▿

    Science.gov (United States)

    McGhee, Pamela; Clark, Catherine; Credito, Kim; Beachel, Linda; Pankuch, Glenn A.; Appelbaum, Peter C.; Kosowska-Shick, Klaudia

    2011-01-01

    We tested the MICs of fusidic acid (CEM-102) plus other agents against 40 methicillin-resistant Staphylococcus aureus (MRSA) isolates from cystic fibrosis patients and the activities of fusidic acid with or without tobramycin or amikacin against Pseudomonas aeruginosa, MRSA, and Burkholderia cepacia isolates from cystic fibrosis patients in a 24-h time-kill study. Fusidic acid was potent (MICs, 0.125 to 0.5 μg/ml; a single 500-mg dose of fusidic acid at 8 h averaged 8 to 12. 5 μg/ml with 91 to 97% protein binding) against all MRSA strains. No antagonism was observed; synergy occurred for one MRSA strain treated with fusidic acid plus tobramycin. PMID:21343445

  17. In vitro activity of fusidic acid (CEM-102, sodium fusidate) against Staphylococcus aureus isolates from cystic fibrosis patients and its effect on the activities of tobramycin and amikacin against Pseudomonas aeruginosa and Burkholderia cepacia.

    Science.gov (United States)

    McGhee, Pamela; Clark, Catherine; Credito, Kim; Beachel, Linda; Pankuch, Glenn A; Appelbaum, Peter C; Kosowska-Shick, Klaudia

    2011-05-01

    We tested the MICs of fusidic acid (CEM-102) plus other agents against 40 methicillin-resistant Staphylococcus aureus (MRSA) isolates from cystic fibrosis patients and the activities of fusidic acid with or without tobramycin or amikacin against Pseudomonas aeruginosa, MRSA, and Burkholderia cepacia isolates from cystic fibrosis patients in a 24-h time-kill study. Fusidic acid was potent (MICs, 0.125 to 0.5 μg/ml; a single 500-mg dose of fusidic acid at 8 h averaged 8 to 12. 5 μg/ml with 91 to 97% protein binding) against all MRSA strains. No antagonism was observed; synergy occurred for one MRSA strain treated with fusidic acid plus tobramycin.

  18. Global analysis of the Burkholderia thailandensis quorum sensing-controlled regulon.

    Science.gov (United States)

    Majerczyk, Charlotte; Brittnacher, Mitchell; Jacobs, Michael; Armour, Christopher D; Radey, Mathew; Schneider, Emily; Phattarasokul, Somsak; Bunt, Richard; Greenberg, E Peter

    2014-04-01

    Burkholderia thailandensis contains three acyl-homoserine lactone quorum sensing circuits and has two additional LuxR homologs. To identify B. thailandensis quorum sensing-controlled genes, we carried out transcriptome sequencing (RNA-seq) analyses of quorum sensing mutants and their parent. The analyses were grounded in the fact that we identified genes coding for factors shown previously to be regulated by quorum sensing among a larger set of quorum-controlled genes. We also found that genes coding for contact-dependent inhibition were induced by quorum sensing and confirmed that specific quorum sensing mutants had a contact-dependent inhibition defect. Additional quorum-controlled genes included those for the production of numerous secondary metabolites, an uncharacterized exopolysaccharide, and a predicted chitin-binding protein. This study provides insights into the roles of the three quorum sensing circuits in the saprophytic lifestyle of B. thailandensis, and it provides a foundation on which to build an understanding of the roles of quorum sensing in the biology of B. thailandensis and the closely related pathogenic Burkholderia pseudomallei and Burkholderia mallei.

  19. Extreme antimicrobial peptide and polymyxin B resistance in the genus Burkholderia

    Directory of Open Access Journals (Sweden)

    Slade A. Loutet

    2011-07-01

    Full Text Available Cationic antimicrobial peptides and polymyxins are a group of naturally occurring antibiotics that can also possess immunomodulatory activities. They are considered a new source of antibiotics for treating infections by bacteria that are resistant to conventional antibiotics. Members of the genus Burkholderia, which includes various human pathogens, are inherently resistant to antimicrobial peptides. The resistance is several orders of magnitude higher than that of other Gram-negative bacteria such as Escherichia coli, Salmonella enterica, or Pseudomonas aeruginosa. This review summarizes our current understanding of antimicrobial peptide and polymyxin B resistance in the genus Burkholderia. These bacteria possess major and minor resistance mechanisms that will be described in detail. Recent studies have revealed that many other emerging Gram-negative opportunistic pathogens may also be inherently resistant to antimicrobial peptides and polymyxins and we propose that Burkholderia species are a model system to investigate the molecular basis of the resistance in extremely resistant bacteria. Understanding resistance in these types of bacteria will be important if antimicrobial peptides come to be used regularly for the treatment of infections by susceptible bacteria because this may lead to increased resistance in the species that are currently susceptible and may also open up new niches for opportunistic pathogens with high inherent resistance.

  20. Oxyfunctionalization of pyridine derivatives using whole cells of Burkholderia sp. MAK1

    Science.gov (United States)

    Stankevičiūtė, Jonita; Vaitekūnas, Justas; Petkevičius, Vytautas; Gasparavičiūtė, Renata; Tauraitė, Daiva; Meškys, Rolandas

    2016-01-01

    Pyridinols and pyridinamines are important intermediates with many applications in chemical industry. The pyridine derivatives are in great demand as synthons for pharmaceutical products. Moreover, pyridines are used either as biologically active substances or as building blocks for polymers with unique physical properties. Application of enzymes or whole cells is an attractive strategy for preparation of hydroxylated pyridines since the methods for chemical synthesis of pyridinols, particularly aminopyridinols, are usually limited or inefficient. Burkholderia sp. MAK1 (DSM102049), capable of using pyridin-2-ol as the sole carbon and energy source, was isolated from soil. Whole cells of Burkholderia sp. MAK1 were confirmed to possess a good ability to convert different pyridin-2-amines and pyridin-2-ones into their 5-hydroxy derivatives. Moreover, several methylpyridines as well as methylated pyrazines were converted to appropriate N-oxides. In conclusion, regioselective oxyfunctionalization of pyridine derivatives using whole cells of Burkholderia sp. MAK1 is a promising method for the preparation of various pyridin-5-ols and pyridin-N-oxides. PMID:27982075

  1. Effect of gamma irradiation on Burkholderia thailandensis ( Burkholderia pseudomallei surrogate) survival under combinations of pH and NaCl

    Science.gov (United States)

    Yoon, Yohan; Kim, Jae-Hun; Byun, Myung-Woo; Choi, Kyoung-Hee; Lee, Ju-Woon

    2010-04-01

    This study evaluated the effect of gamma irradiation on Burkholderia thailandensis ( Burkholderia pseudomallei surrogate; potential bioterrorism agent) survival under different levels of NaCl and pH. B. thailandensis in Luria Bertani broth supplemented with NaCl (0-3%), and pH-adjusted to 4-7 was treated with gamma irradiation (0-0.5 kGy). Surviving cell counts of bacteria were then enumerated on tryptic soy agar. Data for the cell counts were also used to calculate D10 values (the dose required to reduce 1 log CFU/mL of B. thailandensis). Cell counts of B. thailandensis were decreased ( Pbacteria were observed among different levels of NaCl and pH. D10 values ranged from 0.04 to 0.07 kGy, regardless of NaCl and pH level. These results indicate that low doses of gamma irradiation should be a useful treatment in decreasing the potential bioterrorism bacteria, which may possibly infect humans through foods.

  2. Effect of gamma irradiation on Burkholderia thailandensis (Burkholderia pseudomallei surrogate) survival under combinations of pH and NaCl

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Yohan; Kim, Jae-Hun; Byun, Myung-Woo [Team for Radiation Food Science and Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup, Jeollabuk 580-185 (Korea, Republic of); Choi, Kyoung-Hee [Department of Oral Microbiology, College of Dentistry, Wonkwang University, Iksan, Jeollabuk 570-749 (Korea, Republic of); Lee, Ju-Woon, E-mail: sjwlee@kaeri.re.k [Team for Radiation Food Science and Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup, Jeollabuk 580-185 (Korea, Republic of)

    2010-04-15

    This study evaluated the effect of gamma irradiation on Burkholderia thailandensis (Burkholderia pseudomallei surrogate; potential bioterrorism agent) survival under different levels of NaCl and pH. B. thailandensis in Luria Bertani broth supplemented with NaCl (0-3%), and pH-adjusted to 4-7 was treated with gamma irradiation (0-0.5 kGy). Surviving cell counts of bacteria were then enumerated on tryptic soy agar. Data for the cell counts were also used to calculate D{sub 10} values (the dose required to reduce 1 log CFU/mL of B. thailandensis). Cell counts of B. thailandensis were decreased (P<0.05) as irradiation dose increased, and no differences (P>=0.05) in cell counts of the bacteria were observed among different levels of NaCl and pH. D{sub 10} values ranged from 0.04 to 0.07 kGy, regardless of NaCl and pH level. These results indicate that low doses of gamma irradiation should be a useful treatment in decreasing the potential bioterrorism bacteria, which may possibly infect humans through foods.

  3. Transfer of eleven species of the genus Burkholderia to the genus Paraburkholderia and proposal of Caballeronia gen. nov. to accommodate twelve species of the genera Burkholderia and Paraburkholderia.

    Science.gov (United States)

    Dobritsa, Anatoly P; Samadpour, Mansour

    2016-08-01

    It has been proposed to split the genus Burkholderia into two genera according to phylogenetic clustering: (1) a genus retaining this name and consisting mainly of animal and plant pathogens and (2) the genus Paraburkholderia including so-called environmental bacteria. The latter genus name has been validly published recently. During the period between the effective and valid publications of the genus name Paraburkholderia, 16 novel species of the genus Burkholderiawere described, but only two of them can be classified as members of this genus based on the emended genus description. Analysis of traits and phylogenetic positions of the other 11 species shows that they belong to the genus Paraburkholderia, and we propose to transfer them to this genus. The reclassified species names are proposed as Paraburkholderia dipogonis comb. nov., Paraburkholderia ginsengiterrae comb. nov., Paraburkholderia humisilvae comb. nov., Paraburkholderia insulsa comb. nov., Paraburkholderia kirstenboschensis comb. nov., Paraburkholderia metalliresistens comb. nov., Paraburkholderia monticola comb. nov., Paraburkholderia panaciterrae comb. nov., Paraburkholderia rhizosphaerae comb. nov., Paraburkholderia solisilvae comb. nov. and Paraburkholderia susongensis comb. nov. The remaining three species are transferred to the new genus Caballeronia gen. nov. proposed to accommodate twelve species of the genera Burkholderia and Paraburkholderia forming a distinctive clade in phylogenetic trees. The new genus members are Caballeronia choica comb. nov., Caballeronia cordobensis comb. nov., Caballeronia glathei comb. nov., Caballeronia grimmiae comb. nov., Caballeronia humi comb. nov., Caballeronia megalochromosomata comb. nov., Caballeronia jiangsuensis comb. nov., Caballeronia sordidicola comb. nov., Caballeronia telluris comb. nov., Caballeronia terrestris comb. nov., Caballeronia udeis comb. nov., and Caballeronia zhejiangensis comb. nov.

  4. Investigating the Role of the Host Multidrug Resistance Associated Protein Transporter Family in Burkholderia cepacia Complex Pathogenicity Using a Caenorhabditis elegans Infection Model.

    Directory of Open Access Journals (Sweden)

    Pietro Tedesco

    Full Text Available This study investigated the relationship between host efflux system of the non-vertebrate nematode Caenorhabditis elegans and Burkholderia cepacia complex (Bcc strain virulence. This is the first comprehensive effort to profile host-transporters within the context of Bcc infection. With this aim, two different toxicity tests were performed: a slow killing assay that monitors mortality of the host by intestinal colonization and a fast killing assay that assesses production of toxins. A Virulence Ranking scheme was defined, that expressed the toxicity of the Bcc panel members, based on the percentage of surviving worms. According to this ranking the 18 Bcc strains were divided in 4 distinct groups. Only the Cystic Fibrosis isolated strains possessed profound nematode killing ability to accumulate in worms' intestines. For the transporter analysis a complete set of isogenic nematode single Multidrug Resistance associated Protein (MRP efflux mutants and a number of efflux inhibitors were interrogated in the host toxicity assays. The Bcc pathogenicity profile of the 7 isogenic C. elegans MRP knock-out strains functionality was classified in two distinct groups. Disabling host transporters enhanced nematode mortality more than 50% in 5 out of 7 mutants when compared to wild type. In particular mrp-2 was the most susceptible phenotype with increased mortality for 13 out 18 Bcc strains, whereas mrp-3 and mrp-4 knock-outs had lower mortality rates, suggesting a different role in toxin-substrate recognition. The use of MRP efflux inhibitors in the assays resulted in substantially increased (>40% on average mortality of wild-type worms.

  5. Investigating the Role of the Host Multidrug Resistance Associated Protein Transporter Family in Burkholderia cepacia Complex Pathogenicity Using a Caenorhabditis elegans Infection Model

    Science.gov (United States)

    Tedesco, Pietro; Visone, Marco; Parrilli, Ermenegilda; Tutino, Maria Luisa; Perrin, Elena; Maida, Isabel; Fani, Renato; Ballestriero, Francesco; Santos, Radleigh; Pinilla, Clemencia; Di Schiavi, Elia; Tegos, George; de Pascale, Donatella

    2015-01-01

    This study investigated the relationship between host efflux system of the non-vertebrate nematode Caenorhabditis elegans and Burkholderia cepacia complex (Bcc) strain virulence. This is the first comprehensive effort to profile host-transporters within the context of Bcc infection. With this aim, two different toxicity tests were performed: a slow killing assay that monitors mortality of the host by intestinal colonization and a fast killing assay that assesses production of toxins. A Virulence Ranking scheme was defined, that expressed the toxicity of the Bcc panel members, based on the percentage of surviving worms. According to this ranking the 18 Bcc strains were divided in 4 distinct groups. Only the Cystic Fibrosis isolated strains possessed profound nematode killing ability to accumulate in worms’ intestines. For the transporter analysis a complete set of isogenic nematode single Multidrug Resistance associated Protein (MRP) efflux mutants and a number of efflux inhibitors were interrogated in the host toxicity assays. The Bcc pathogenicity profile of the 7 isogenic C. elegans MRP knock-out strains functionality was classified in two distinct groups. Disabling host transporters enhanced nematode mortality more than 50% in 5 out of 7 mutants when compared to wild type. In particular mrp-2 was the most susceptible phenotype with increased mortality for 13 out 18 Bcc strains, whereas mrp-3 and mrp-4 knock-outs had lower mortality rates, suggesting a different role in toxin-substrate recognition. The use of MRP efflux inhibitors in the assays resulted in substantially increased (>40% on average) mortality of wild-type worms. PMID:26587842

  6. Burkholderia thailandensis harbors two identical rhl gene clusters responsible for the biosynthesis of rhamnolipids

    Directory of Open Access Journals (Sweden)

    Woods Donald E

    2009-12-01

    Full Text Available Abstract Background Rhamnolipids are surface active molecules composed of rhamnose and β-hydroxydecanoic acid. These biosurfactants are produced mainly by Pseudomonas aeruginosa and have been thoroughly investigated since their early discovery. Recently, they have attracted renewed attention because of their involvement in various multicellular behaviors. Despite this high interest, only very few studies have focused on the production of rhamnolipids by Burkholderia species. Results Orthologs of rhlA, rhlB and rhlC, which are responsible for the biosynthesis of rhamnolipids in P. aeruginosa, have been found in the non-infectious Burkholderia thailandensis, as well as in the genetically similar important pathogen B. pseudomallei. In contrast to P. aeruginosa, both Burkholderia species contain these three genes necessary for rhamnolipid production within a single gene cluster. Furthermore, two identical, paralogous copies of this gene cluster are found on the second chromosome of these bacteria. Both Burkholderia spp. produce rhamnolipids containing 3-hydroxy fatty acid moieties with longer side chains than those described for P. aeruginosa. Additionally, the rhamnolipids produced by B. thailandensis contain a much larger proportion of dirhamnolipids versus monorhamnolipids when compared to P. aeruginosa. The rhamnolipids produced by B. thailandensis reduce the surface tension of water to 42 mN/m while displaying a critical micelle concentration value of 225 mg/L. Separate mutations in both rhlA alleles, which are responsible for the synthesis of the rhamnolipid precursor 3-(3-hydroxyalkanoyloxyalkanoic acid, prove that both copies of the rhl gene cluster are functional, but one contributes more to the total production than the other. Finally, a double ΔrhlA mutant that is completely devoid of rhamnolipid production is incapable of swarming motility, showing that both gene clusters contribute to this phenotype. Conclusions Collectively, these

  7. The Autotransporter BpaB Contributes to the Virulence of Burkholderia mallei in an Aerosol Model of Infection.

    Directory of Open Access Journals (Sweden)

    Shawn M Zimmerman

    Full Text Available Burkholderia mallei is a highly pathogenic bacterium that causes the zoonosis glanders. Previous studies indicated that the genome of the organism contains eight genes specifying autotransporter proteins, which are important virulence factors of Gram-negative bacteria. In the present study, we report the characterization of one of these autotransporters, BpaB. Database searches identified the bpaB gene in ten B. mallei isolates and the predicted proteins were 99-100% identical. Comparative sequence analyses indicate that the gene product is a trimeric autotransporter of 1,090 amino acids with a predicted molecular weight of 105-kDa. Consistent with this finding, we discovered that recombinant bacteria expressing bpaB produce a protein of ≥ 300-kDa on their surface that is reactive with a BpaB-specific monoclonal antibody. Analysis of sera from mice infected with B. mallei indicated that animals produce antibodies against BpaB during the course of disease, thus establishing production of the autotransporter in vivo. To gain insight on its role in virulence, we inactivated the bpaB gene of B. mallei strain ATCC 23344 and determined the median lethal dose of the mutant in a mouse model of aerosol infection. These experiments revealed that the bpaB mutation attenuates virulence 8-14 fold. Using a crystal violet-based assay, we also discovered that constitutive production of BpaB on the surface of B. mallei promotes biofilm formation. To our knowledge, this is the first report of a biofilm factor for this organism.

  8. The Autotransporter BpaB Contributes to the Virulence of Burkholderia mallei in an Aerosol Model of Infection.

    Science.gov (United States)

    Zimmerman, Shawn M; Michel, Frank; Hogan, Robert J; Lafontaine, Eric R

    2015-01-01

    Burkholderia mallei is a highly pathogenic bacterium that causes the zoonosis glanders. Previous studies indicated that the genome of the organism contains eight genes specifying autotransporter proteins, which are important virulence factors of Gram-negative bacteria. In the present study, we report the characterization of one of these autotransporters, BpaB. Database searches identified the bpaB gene in ten B. mallei isolates and the predicted proteins were 99-100% identical. Comparative sequence analyses indicate that the gene product is a trimeric autotransporter of 1,090 amino acids with a predicted molecular weight of 105-kDa. Consistent with this finding, we discovered that recombinant bacteria expressing bpaB produce a protein of ≥ 300-kDa on their surface that is reactive with a BpaB-specific monoclonal antibody. Analysis of sera from mice infected with B. mallei indicated that animals produce antibodies against BpaB during the course of disease, thus establishing production of the autotransporter in vivo. To gain insight on its role in virulence, we inactivated the bpaB gene of B. mallei strain ATCC 23344 and determined the median lethal dose of the mutant in a mouse model of aerosol infection. These experiments revealed that the bpaB mutation attenuates virulence 8-14 fold. Using a crystal violet-based assay, we also discovered that constitutive production of BpaB on the surface of B. mallei promotes biofilm formation. To our knowledge, this is the first report of a biofilm factor for this organism.

  9. Spatio-temporal responses of Arabidopsis leaves in photosynthetic performance and metabolite contents to Burkholderia phytofirmans PsJN

    Directory of Open Access Journals (Sweden)

    Fan eSu

    2016-03-01

    Full Text Available A valuable strategy to improve crop yield consists in the use of plant growth-promoting rhizobacteria (PGPRs. However, the influence of PGPR colonization on plant physiology is largely unknown. PGPR Burkholderia phytofirmans strain PsJN (Bp PsJN colonized only Arabidopsis thaliana roots after seed or soil inoculation. Foliar bacteria were detected only after leaf infiltration. Since different bacterial times of presence and/or locations in host plant could lead to different plant physiological responses, photosynthesis and metabolite profiles in A. thaliana leaves were thus investigated following leaf, root or seed inoculation with Bp PsJN. Only Bp PsJN leaf colonization transiently decreased cyclic electron transport and effective quantum yield of photosystem I (PSI, and prevented a decrease in net photosynthesis and stomatal opening compared to the corresponding control. Metabolomic analysis revealed that soluble sugars, amino acids or their derivatives accumulated differently in all Bp PsJN-inoculated plants. Octanoic acid accumulated only in case of inoculated plants. Modifications in vitamin, organic acid such as tricarboxylic acid intermediates, and hormone amounts were dependent on bacterial time of presence and location. Additionally, a larger array of amino acids and hormones (auxin, cytokinin, abscisic acid were modified by seed inoculation with Bp PsJN. Our work thereby provides evidence that relative short-term inoculation with Bp PsJN altered physiological status of A.thaliana leaves, whereas long-term bacterization triggered modifications on a larger set of metabolites. Our data highlighted the changes displayed during this plant-microbe interaction to trigger physiological and metabolic responses that could explain the increase in plant growth or stress tolerance conferred by the presence of Bp PsJN.

  10. Analysis of the prevalence, secretion and function of a cell cycle-inhibiting factor in the melioidosis pathogen Burkholderia pseudomallei.

    Science.gov (United States)

    Pumirat, Pornpan; Broek, Charles Vander; Juntawieng, Niramol; Muangsombut, Veerachat; Kiratisin, Pattarachai; Pattanapanyasat, Kovit; Stevens, Joanne M; Stevens, Mark P; Korbsrisate, Sunee

    2014-01-01

    Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif), that is injected into host cells via a Type III secretion system (T3SS) leading to arrest of cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385), which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7%) harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15) of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE) was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro.

  11. Analysis of the prevalence, secretion and function of a cell cycle-inhibiting factor in the melioidosis pathogen Burkholderia pseudomallei.

    Directory of Open Access Journals (Sweden)

    Pornpan Pumirat

    Full Text Available Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif, that is injected into host cells via a Type III secretion system (T3SS leading to arrest of cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385, which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7% harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15 of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro.

  12. Recombinant Salmonella Expressing Burkholderia mallei LPS O Antigen Provides Protection in a Murine Model of Melioidosis and Glanders.

    Directory of Open Access Journals (Sweden)

    Dina A Moustafa

    Full Text Available Burkholderia pseudomallei and Burkholderia mallei are the etiologic agents of melioidosis and glanders, respectively. These bacteria are highly infectious via the respiratory route and can cause severe and often fatal diseases in humans and animals. Both species are considered potential agents of biological warfare; they are classified as category B priority pathogens. Currently there are no human or veterinary vaccines available against these pathogens. Consequently efforts are directed towards the development of an efficacious and safe vaccine. Lipopolysaccharide (LPS is an immunodominant antigen and potent stimulator of host immune responses. B. mallei express LPS that is structurally similar to that expressed by B. pseudomallei, suggesting the possibility of constructing a single protective vaccine against melioidosis and glanders. Previous studies of others have shown that antibodies against B. mallei or B. pseudomallei LPS partially protect mice against subsequent lethal virulent Burkholderia challenge. In this study, we evaluated the protective efficacy of recombinant Salmonella enterica serovar Typhimurium SL3261 expressing B. mallei O antigen against lethal intranasal infection with Burkholderia thailandensis, a surrogate for biothreat Burkholderia spp. in a murine model that mimics melioidosis and glanders. All vaccine-immunized mice developed a specific antibody response to B. mallei and B. pseudomallei O antigen and to B. thailandensis and were significantly protected against challenge with a lethal dose of B. thailandensis. These results suggest that live-attenuated SL3261 expressing B. mallei O antigen is a promising platform for developing a safe and effective vaccine.

  13. Recombinant Salmonella Expressing Burkholderia mallei LPS O Antigen Provides Protection in a Murine Model of Melioidosis and Glanders.

    Science.gov (United States)

    Moustafa, Dina A; Scarff, Jennifer M; Garcia, Preston P; Cassidy, Sara K B; DiGiandomenico, Antonio; Waag, David M; Inzana, Thomas J; Goldberg, Joanna B

    2015-01-01

    Burkholderia pseudomallei and Burkholderia mallei are the etiologic agents of melioidosis and glanders, respectively. These bacteria are highly infectious via the respiratory route and can cause severe and often fatal diseases in humans and animals. Both species are considered potential agents of biological warfare; they are classified as category B priority pathogens. Currently there are no human or veterinary vaccines available against these pathogens. Consequently efforts are directed towards the development of an efficacious and safe vaccine. Lipopolysaccharide (LPS) is an immunodominant antigen and potent stimulator of host immune responses. B. mallei express LPS that is structurally similar to that expressed by B. pseudomallei, suggesting the possibility of constructing a single protective vaccine against melioidosis and glanders. Previous studies of others have shown that antibodies against B. mallei or B. pseudomallei LPS partially protect mice against subsequent lethal virulent Burkholderia challenge. In this study, we evaluated the protective efficacy of recombinant Salmonella enterica serovar Typhimurium SL3261 expressing B. mallei O antigen against lethal intranasal infection with Burkholderia thailandensis, a surrogate for biothreat Burkholderia spp. in a murine model that mimics melioidosis and glanders. All vaccine-immunized mice developed a specific antibody response to B. mallei and B. pseudomallei O antigen and to B. thailandensis and were significantly protected against challenge with a lethal dose of B. thailandensis. These results suggest that live-attenuated SL3261 expressing B. mallei O antigen is a promising platform for developing a safe and effective vaccine.

  14. Draft Genome Sequences of Symbiotic and Nonsymbiotic Rhizopus microsporus Strains CBS 344.29 and ATCC 62417.

    Science.gov (United States)

    Horn, Fabian; Üzüm, Zerrin; Möbius, Nadine; Guthke, Reinhard; Linde, Jörg; Hertweck, Christian

    2015-01-22

    Specific Rhizopus microsporus pathovars harbor bacterial endosymbionts (Burkholderia rhizoxinica) for the production of a phytotoxin. Here, we present the draft genome sequences of two R. microsporus strains, one symbiotic (ATCC 62417), and one endosymbiont-free (CBS 344.29). The gene predictions were supported by RNA sequencing (RNA-seq) data. The functional annotation sets the basis for comparative analyses. Copyright © 2015 Horn et al.

  15. Isolation of Burkholderia cepacia JB12 from lead- and cadmium-contaminated soil and its potential in promoting phytoremediation with tall fescue and red clover.

    Science.gov (United States)

    Jin, Zhong Min; Sha, Wei; Zhang, Yan Fu; Zhao, Jing; Ji, Hongyang

    2013-07-01

    Phytoremediation combined with suitable microorganisms and biodegradable chelating agents can be a means of reclaiming lands contaminated by toxic heavy metals. We investigated the ability of a lead- and cadmium-resistant bacterial strain (JB12) and the biodegradable chelator ethylenediamine-N,N'-disuccinic acid (EDDS) to improve absorption of these metals from soil by tall fescue and red clover. Strain JB12 was isolated from contaminated soil samples, analysed for lead and cadmium resistance, and identified as Burkholderia cepacia. Tall fescue and red clover were grown in pots to which we added JB12, (S,S)-EDDS, combined JB12 and EDDS, or water only. Compared with untreated plants, the biomass of plants treated with JB12 was significantly increased. Concentrations of lead and cadmium in JB12-treated plants increased significantly, with few exceptions. Plants treated with EDDS responded variably, but in those treated with combined EDDS and JB12, heavy metal concentrations increased significantly in tall fescue and in the aboveground parts of red clover. We conclude that JB12 is resistant to lead and cadmium. Its application to the soil improved the net uptake of these heavy metals by experimental plants. The potential for viable phytoremediation of lead- and cadmium-polluted soils with tall fescue and red clover combined with JB12 was further enhanced by the addition of EDDS.

  16. Fermentation optimization for non-positional specificity lipase production of Burkholderia cepacia S31%Burkholderia cepacia S31细菌高产位置非特异性脂肪酶的发酵条件优化

    Institute of Scientific and Technical Information of China (English)

    卢亚萍; 汪瑾; 张充; 吕凤霞; 别小妹; 陆兆新

    2012-01-01

    从油脂污染的土壤中分离获得了1株高效产脂肪酶的细菌S31,经鉴定为Burkholderia cepacia(洋葱伯克霍尔德菌).B.cepacia S31所产脂肪酶具有活性高、耐高温、耐有机溶剂和位置非特异性水解甘油三酯等优良特性.为了进一步提高S31菌株的产酶量,对该菌产酶的发酵条件进行优化.通过单因子试验筛选出最佳碳源为麸皮,最佳氮源为蛋白胨,最佳诱导物为Tween-80.通过对培养基各组分及外部培养条件因素的正交试验,确定S31菌产脂肪酶的摇瓶发酵最优条件为:以20 g·L-1麸皮、10 g·L-1蛋白胨、40g·L-1Tween-80、0.5g·L-1MgSO4和2g·L-1K2 HPO4为培养基(pH 7.0),250 mL三角瓶装40 mL培养基,3%接种量,30℃、180 r·min-1培养66h可获得最理想的酶产量,达283.6 U·mL-1,比优化前提高2.73倍.%A lipase-producing strain named S31 from the soil of a cole plantation was isolated,which was identified as Burkholderia cepacia. S31 lipase had a variety of highly desirable characteristics, such as high activity, temperature stability, organic solvents tolerance and hydrolysis of triglyceride without positional specificity. In order to enhance the enzyme productivity, the culture conditions were improved. Initially, single-factor experiments were used to evaluate the optimal carbon source, nitrogen source and inducer, which were bran, peptone and Tween-80, respectively. According to the orthogonal tests of media components and fermentation parameters,the optimal culture conditions were determined as follows;the culture medium containing of 20 g·L-1 bran, l0g·L-1 peptone,40 g·L-1 Tween-80,0.5 g·L-1 MgSO4 and 2 g·L-1 K2HPO4 with initial pH 7. 0. The overnight culture was inoculated to 40 mL medium in 250 mL shaking flask,3% inocution amount,and fermented at 30 ℃ with 180 r·min-1 shaking for 66 h. The maximum lipase activity reached a high level(283.6 U·mL-1 ) .which was improved 2.73 folds compared with that under the original

  17. A new bacterial disease of carnation in Portugal caused by Burkholderia andropogonis

    OpenAIRE

    Madalena Eloy; Leonor Cruz

    2008-01-01

    The occurrence of a leaf spot disease of carnation caused by Burkholderia andropogonis is recorded for the first time in Portugal. Symptoms consisted of ‘eyespot’ lesions on all aerial plant parts, often bordered by water-soaked halos on the leaves. As the disease progressed lesions became dark brown and affected areas dried out. Phenotypic studies and Polymerase Chain Reaction using specific primers Pf/Pr targeted to 16S rDNA of B. andropogonis were used to identify the pathogen. Pathogenici...

  18. A rare case of community acquired Burkholderia cepacia infection presenting as pyopneumothorax in an immunocompetent individual

    Institute of Scientific and Technical Information of China (English)

    Suman S Karanth; Hariharan Regunath; Kiran Chawla; Mukhyaprana Prabhu

    2012-01-01

    Burkholderia cepacia (B. cepacia) infection is rarely reported in an immunocompetent host. It is a well known occurence in patients with cystic fibrosis and chronic granulomatous disease where it increases both morbidity and mortality. It has also been included in the list of organisms causing nosocomial infections in an immunocompetent host, most of them transmitted from the immunocompromised patient in which this organism harbors. We report a rare case of isolation of B. cepacia from the bronchoalveolar lavage fluid of an immunocompetent agriculturist who presented with productive cough and fever associated with a pyopneumothorax. This is the first case of community acquired infection reported in an immunocompetent person in India.

  19. Eradication of Burkholderia cepacia Using Inhaled Aztreonam Lysine in Two Patients with Bronchiectasis

    Directory of Open Access Journals (Sweden)

    A. Iglesias

    2014-01-01

    Full Text Available There are not many articles about the chronic bronchial infection/colonization in patients with underlying lung disease other than cystic fibrosis (CF, especially with non-CF bronchiectasis (NCFBQ. The prevalence of B. cepacia complex is not well known in NCFBQ. The vast majority of published clinical data on Burkholderia infection in individuals with CF is comprised of uncontrolled, anecdotal, and/or single center experiences, and no consensus has emerged regarding treatment. We present two cases diagnosed with bronchiectasis (BQ of different etiology, with early pulmonary infection by B. cepacia complex, which was eradicated with inhaled aztreonam lysine.

  20. A rare case of community acquired Burkholderia cepacia infection presenting as pyopneumothorax in an immunocompetent individual

    Directory of Open Access Journals (Sweden)

    Suman S Karanth

    2012-02-01

    Full Text Available Burkholderia cepacia (B. cepacia infection is rarely reported in an immunocompetent host. It is a well known occurence in patients with cystic fibrosis and chronic granulomatous disease where it increases both morbidity and mortality. It has also been included in the list of organisms causing nosocomial infections in an immunocompetent host, most of them transmitted from the immunocompromised patient in which this organism harbors. We report a rare case of isolation of B. cepacia from the bronchoalveolar lavage fluid of an immunocompetent agriculturist who presented with productive cough and fever associated with a pyopneumothorax. This is the first case of community acquired infection reported in an immunocompetent person in India.

  1. The promise of bacteriophage therapy for Burkholderia cepacia complex respiratory infections.

    Directory of Open Access Journals (Sweden)

    Diana Dawn Semler

    2012-01-01

    Full Text Available In recent times, increased attention has been given to evaluating the efficacy of phage therapy, especially in scenarios where the bacterial infectious agent of interest is highly antibiotic resistant. In this regard, phage therapy is especially applicable to infections caused by the Burkholderia cepacia complex (BCC since members of the BCC are antibiotic pan-resistant. Current studies in BCC phage therapy are unique from many other avenues of phage therapy research in that the research is not only comprised of phage isolation, in vitro phage characterization and in vivo infection model efficacy, but also adapting aerosol drug delivery techniques to aerosol phage formulation delivery and storage.

  2. Investigating early stages of biocorrosion with XPS: AISI 304 stainless steel exposed to Burkholderia species

    Science.gov (United States)

    Johansson, Leena-Sisko; Saastamoinen, Tuomas

    1999-04-01

    We have investigated the interactions of an exopolymer-producing bacteria, Burkholderia sp. with polished AISI 304 stainless steel substrates using X-ray photoelectron spectroscopy (XPS). Steel coupons were exposed to the pure bacteria culture in a specially designed flowcell for 6 h during which the experiment was monitored in situ with an optical microscope. XPS results verified the formation of biofilm containing extracellular polymer on all the samples exposed to bacteria. Sputter results indicated that some ions needed for metabolic processes were trapped within the biofilm. Changes in the relative Fe concentration and Fe 2p peak shape indicated that also iron had accumulated into the biofilm.

  3. Stereoselective lipases from Burkholderia sp., cloning and their application to preparation of methyl (R)-N-(2,6-dimethylphenyl)alaninate, a key intermediate for (R)-Metalaxyl.

    Science.gov (United States)

    Park, Oh-Jin; Lee, Sang-Hyun

    2005-11-04

    Two microbial strains (referred to as MC 16-3 and 99-2-1) that produce extracellular lipases were isolated from soil samples and identified as Burkholderia species. The lipases were partially purified by isopropyl alcohol precipitation and gave molecular weight of 33kDa. The lipases were characterized in terms of stereoselectivity with racemic methoxyethyl (R,S)-N-(2,6-dimethylphenyl)alaninate and the genes encoding the proteins have been identified by homology alignment of lipases reported belonging to I.2 subfamily and their complete DNA sequences were determined. The lipases will be useful for the preparation of methyl (R)-N-(2,6-dimethylphenyl)alaninate, a key intermediate for the synthesis of (R)-Metalaxyl, which is one of the best-selling fungicides.

  4. Detection of the reemerging agent Burkholderia mallei in a recent outbreak of glanders in the United Arab Emirates by a newly developed fliP-based polymerase chain reaction assay.

    Science.gov (United States)

    Scholz, Holger C; Joseph, Marina; Tomaso, Herbert; Al Dahouk, Sascha; Witte, Angela; Kinne, Joerg; Hagen, Ralph M; Wernery, Renate; Wernery, Ulrich; Neubauer, Heinrich

    2006-04-01

    A polymerase chain reaction (PCR) assay targeting the flagellin P (fliP)-I S407A genomic region of Burkholderia mallei was developed for the specific detection of this organism in pure cultures and clinical samples from a recent outbreak of equine glanders. Primers deduced from the known fliP-IS407A sequence of B. mallei American Type Culture Collection (ATCC) 23344(T) allowed the specific amplification of a 989-bp fragment from each of the 20 B. mallei strains investigated, whereas other closely related organisms tested negative. The detection limit of the assay was 10 fg for purified DNA of B. mallei ATCC 23344(T). B. mallei DNA was also amplified from various tissues of horses with a generalized B. mallei infection. The developed PCR assay can be used as a simple and rapid tool for the specific and sensitive detection of B. mallei in clinical samples.

  5. Genomic and functional analyses of the 2-aminophenol catabolic pathway and partial conversion of its substrate into picolinic acid in Burkholderia xenovorans LB400.

    Directory of Open Access Journals (Sweden)

    Bernardita Chirino

    Full Text Available 2-aminophenol (2-AP is a toxic nitrogen-containing aromatic pollutant. Burkholderia xenovorans LB400 possess an amn gene cluster that encodes the 2-AP catabolic pathway. In this report, the functionality of the 2-aminophenol pathway of B. xenovorans strain LB400 was analyzed. The amnRJBACDFEHG cluster located at chromosome 1 encodes the enzymes for the degradation of 2-aminophenol. The absence of habA and habB genes in LB400 genome correlates with its no growth on nitrobenzene. RT-PCR analyses in strain LB400 showed the co-expression of amnJB, amnBAC, amnACD, amnDFE and amnEHG genes, suggesting that the amn cluster is an operon. RT-qPCR showed that the amnB gene expression was highly induced by 2-AP, whereas a basal constitutive expression was observed in glucose, indicating that these amn genes are regulated. We propose that the predicted MarR-type transcriptional regulator encoded by the amnR gene acts as repressor of the amn gene cluster using a MarR-type regulatory binding sequence. This report showed that LB400 resting cells degrade completely 2-AP. The amn gene cluster from strain LB400 is highly identical to the amn gene cluster from P. knackmussi strain B13, which could not grow on 2-AP. However, we demonstrate that B. xenovorans LB400 is able to grow using 2-AP as sole nitrogen source and glucose as sole carbon source. An amnBA (- mutant of strain LB400 was unable to grow with 2-AP as nitrogen source and glucose as carbon source and to degrade 2-AP. This study showed that during LB400 growth on 2-AP this substrate was partially converted into picolinic acid (PA, a well-known antibiotic. The addition of PA at lag or mid-exponential phase inhibited LB400 growth. The MIC of PA for strain LB400 is 2 mM. Overall, these results demonstrate that B. xenovorans strain LB400 posses a functional 2-AP catabolic central pathway, which could lead to the production of picolinic acid.

  6. 对贵州发酵米粉与新玉米中椰毒伯克氏菌的调查%Investigation on Burkholderia cocoyenenans and Bongkeric acid of the fermented foodand new harvesting corn in Guizhou province

    Institute of Scientific and Technical Information of China (English)

    魏桂兰; 许成栋; 赵松华; 张莹; 陈瑛

    2001-01-01

    For understanding the polluted situation of Burkholderia cocovenenans and Bongkreic acid in the fermented food as well as new harvesting corn is Guizhou province, 104 samples of fermented rice food and 53 samples of new harvesting corn were investigated in 1986 in nine districts of Guizhou province. 3 strains of suspicious microbes and 3 samples of suspicious food were carried out experimental identification. The authors also analyzed and investigated 3 cases due to Burkholderia cocvenenans and Bongkreic acid of food poisoning. Burkholderia cocovenenans and Bongkreic acid were demonstrated in the poisoning food,and it was strongly verified that 3 poisoning samples was caused by Bongkreic acid produced from Burkholderin cocovenenans via microbe evaluation,fungus strains poisoning experiment, animal poisoning experiment,and AQUA soaking liquid of poisoning sample of animal poisoning experiment.%为了解我省发酵食品与新收玉米中椰毒伯克氏菌及米酵菌酸的污染情况,对贵州省9个地区的104份发酵米粉类食品与1986年新收未入库的53份玉米样品进行调查。对分离的3株可疑菌,3份可疑食品进行确证试验,并对3起因椰毒伯克氏菌污染食品产生未酵菌酸所致的食物中毒进行调查分析。从中毒样品中检出椰毒伯克氏菌及米酵菌酸,经细菌学鉴定、菌株产毒试验、动物毒性试验、中毒样品水浸液的动物毒性试验证实其中毒均系椰毒伯克氏菌污染食品产生米酵菌酸所致。

  7. The cep quorum-sensing system of Burkholderia cepacia H111 controls biofilm formation and swarming motility

    DEFF Research Database (Denmark)

    Huber, B.; Riedel, K.; Hentzer, Morten;

    2001-01-01

    Burkholderia cepacia and Pseudomonas aeruginosa often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, the isolation of random mini-Tn5 insertion mutants of B. cepacia H111 defective in biofilm formation on an abiotic surface is reported. It is demons...

  8. South african papilionoid legumes are nodulated by diverse burkholderia with unique nodulation and nitrogen-fixation Loci.

    Science.gov (United States)

    Beukes, Chrizelle W; Venter, Stephanus N; Law, Ian J; Phalane, Francina L; Steenkamp, Emma T

    2013-01-01

    The root-nodule bacteria of legumes endemic to the Cape Floristic Region are largely understudied, even though recent reports suggest the occurrence of nodulating Burkholderia species unique to the region. In this study, we considered the diversity and evolution of nodulating Burkholderia associated with the endemic papilionoid tribes Hypocalypteae and Podalyrieae. We identified distinct groups from verified rhizobial isolates by phylogenetic analyses of the 16S rRNA and recA housekeeping gene regions. In order to gain insight into the evolution of the nodulation and diazotrophy of these rhizobia we analysed the genes encoding NifH and NodA. The majority of these 69 isolates appeared to be unique, potentially representing novel species. Evidence of horizontal gene transfer determining the symbiotic ability of these Cape Floristic Region isolates indicate evolutionary origins distinct from those of nodulating Burkholderia from elsewhere in the world. Overall, our findings suggest that Burkholderia species associated with fynbos legumes are highly diverse and their symbiotic abilities have unique ancestries. It is therefore possible that the evolution of these bacteria is closely linked to the diversification and establishment of legumes characteristic of the Cape Floristic Region.

  9. Degradation of toluene and trichloroethylene by Burkholderia cepacia G4 in growth-limited fed-batch culture

    NARCIS (Netherlands)

    Mars, Astrid E.; Houwing, Joukje; Dolfing, Jan; Janssen, Dick B.

    1996-01-01

    Burkholderia (Pseudomonas) cepacia G4 was cultivated in a fed-batch bioreactor on either toluene or toluene plus trichloroethylene (TCE), The culture was allowed to reach a constant cell density under conditions in which the amount of toluene supplied equals the maintenance energy demand of the cult

  10. Synthesis of a selective inhibitor of a fucose binding bacterial lectin from Burkholderia ambifaria.

    Science.gov (United States)

    Richichi, Barbara; Imberty, Anne; Gillon, Emilie; Bosco, Rosa; Sutkeviciute, Ieva; Fieschi, Franck; Nativi, Cristina

    2013-06-28

    Burkholderia ambifaria is a bacterium member of the Burkholderia cepacia complex (BCC), a closely related group of Gram-negative bacteria responsible for "cepacia syndrome" in immunocompromised patients. B. ambifaria produces BambL, a fucose-binding lectin that displays fine specificity to human fucosylated epitopes. Here, we report the first example of a synthetic ligand able to selectively bind, in the micromolar range, the pathogen-lectin BambL. The synthetic routes for the preparation of the α conformationally constrained fucoside are described, focusing on a totally diastereoselective inverse electron demand [4 + 2] Diels-Alder reaction. Isothermal titration calorimetry (ITC) demonstrated that this compound binds to the pathogen-associated lectin BambL with an affinity comparable to that of natural fucose-containing oligosaccharides. No binding was observed by LecB, a fucose-binding lectin from Pseudomonas aeruginosa, and the differences in affinity between the two lectins could be rationalized by modeling. Furthermore, SPR analyses showed that this fucomimetic does not bind to the human fucose-binding lectin DC-SIGN, thus supporting the selective binding profile towards B. ambifaria lectin.

  11. Outbreak of subclinical mastitis in a flock of dairy sheep associated with Burkholderia cepacia complex infection.

    Science.gov (United States)

    Berriatua, E; Ziluaga, I; Miguel-Virto, C; Uribarren, P; Juste, R; Laevens, S; Vandamme, P; Govan, J R

    2001-03-01

    An outbreak of subclinical mastitis in a flock of 620 milking sheep was investigated. Microbiological and epidemiological analyses identified the causative agent as belonging to the Burkholderia cepacia complex (formerly Pseudomonas cepacia). Every ewe in the milking flock was individually tested for subclinical mastitis on two separate occasions, 6 weeks apart, by the California (rapid) mastitis test (CMT). The proportion of CMT-positive ewes was 69 of 393 (17.6%) on the first sampling and 27 of 490 (5.5%) on the second sampling. Pure B. cepacia cultures identified with the API 20 NE system were grown from 64 of 96 (66.7%) CMT-positive ewes and from 1 of 33 (3.0%) CMT-negative ewes. Statistical analysis confirmed the significant association between a positive CMT result and a positive culture result for B. cepacia complex. Additional polyphasic taxonomic analyses of eight isolates showed that seven belonged to B. cepacia genomovar III; the remaining isolate was identified as Burkholderia vietnamiensis (formerly B. cepacia genomovar V). Bacteriological investigation of samples from milking equipment and other environmental sites failed to identify "B. cepacia" in any of the samples taken. To our knowledge, this is the first report of an outbreak of natural infection in animals caused by B. cepacia complex and the first description of B. cepacia complex infection in sheep.

  12. Functional characterisation of Burkholderia pseudomallei biotin protein ligase: A toolkit for anti-melioidosis drug development.

    Science.gov (United States)

    Bond, Thomas E H; Sorenson, Alanna E; Schaeffer, Patrick M

    2017-06-01

    Burkholderia pseudomallei (Bp) is the causative agent of melioidosis. The bacterium is responsible for 20% of community-acquired sepsis cases and 40% of sepsis-related mortalities in northeast Thailand, and is intrinsically resistant to aminoglycosides, macrolides, rifamycins, cephalosporins, and nonureidopenicillins. There is no vaccine and its diagnosis is problematic. Biotin protein ligase (BirA) which is essential for fatty acid synthesis has been proposed as a drug target in bacteria. Very few bacterial BirA have been characterized, and a better understanding of these enzymes is necessary to further assess their value as drug targets. BirA within the Burkholderia genus have not yet been investigated. We present for the first time the cloning, expression, purification and functional characterisation of the putative Bp BirA and orthologous B. thailandensis (Bt) biotin carboxyl carrier protein (BCCP) substrate. A GFP-tagged Bp BirA was produced and applied for the development of a high-throughput (HT) assay based on our differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP) principle as well as an electrophoretic mobility shift assay. Our biochemical data in combination with the new HT DSF-GTP and biotinylation activity assay could facilitate future drug screening efforts against this drug-resistant organism. Copyright © 2017 Elsevier GmbH. All rights reserved.

  13. Burkholderia cepacia complex: Virulence characteristics, importance and relationship with cystic fibrosis

    Directory of Open Access Journals (Sweden)

    Melo Coutinho Henrique

    2007-07-01

    Full Text Available Background : Burkholderia cepacia has been described as a cause of opportunist infections in patients with immune deficiency because of the high transmission rates. Actually the B. cepacia is subdivided in nine different genomic species that show morphological similarity, called genomovars. High mortality rates have been associated with infections caused by genomovars in susceptible patients; antibiotics are not efficient because of the high resistance level and genomic mutability. Little is known about the epidemiological traits of this bacterium; therefore, their isolation remains a relevant technical problem.Aims : The objective of this review is to describe Burkholderia cepacia as a bacterial complex with high pathogenicity and variability of habitats. Materials and Methods : A systematic search was realized using the international bibliographic databanks SCIELO, HIGHWIRE, PUBMED, SCIRUS and LILACS to provide a useful and practical review for the health workers that do not know this microorganism. Conclusions : Today, B. cepacia complex is a very important problem for the acquired immunodeficiency syndrome and cystic fibrosis patients. The immunodeficiency caused by these diseases is a positive factor for this microorganism to infect and kill these patients. Therefore, this opportunistic pathogen should be pointed out as a risk to these patients, and hospitals all over the world must be prepared to detect and combat this bacterium.

  14. Use of a recombinant burkholderia intracellular motility a protein for immunodiagnosis of glanders.

    Science.gov (United States)

    Kumar, Subodh; Malik, Praveen; Verma, Shailendra Kumar; Pal, Vijai; Gautam, Vandana; Mukhopadhyay, Chiranjay; Rai, Ganga Prasad

    2011-09-01

    Glanders, caused by the Gram-negative, nonmotile bacterium Burkholderia mallei, is a contagious and highly fatal disease of equines. During the last decade, the number of glanders outbreaks has increased steadily. The disease also has high zoonotic significance and B. mallei is listed biological warfare agent. The complement fixation test (CFT) is a routinely used and internationally recognized test to screen equine sera for the glanders. However, discrepant results have been observed using the CFT. The low sensitivity and specificity of the CFT and enzyme-linked immunosorbent assay (ELISA) have been linked to the use of crude test antigens. We expressed a novel recombinant Burkholderia intracellular motility A (rBimA) protein in Escherichia coli for the diagnosis of equine glanders. Purified rBimA was used in an indirect ELISA format. All of the 21 true-positive serum samples used in the study tested positive, whereas only 17 of the 1,524 potentially negative sera tested positive by indirect ELISA, thus exhibiting 100% sensitivity and 98.88% specificity. Also, rBimA protein did not react with melioidosis patient and normal healthy human serum samples, showing its high specificity. The developed assay can be used as a simple and rapid tool for diagnosis of glanders in equine serum samples. An Indian patent (1328/DEL/2010) has been filed for the reagent.

  15. Chronic suppurative joint effusion due to burkholderia pseudomallei: A case report

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    Madhavi Deshmukh

    2013-01-01

    Full Text Available Burkholderia pseudomallei, a Gram-negative bacillus is the causative agent of Melioidosis, a glanders-like disease, primarily a disease of animals. Melioidosis has been only a rare and sporadic disease in humans outside its endemic region. Currently, diagnosis of B. pseudomallei in the clinical laboratory is very difficult, owing to low awareness of physicians to the nonspecific clinical manifestations, lack of responsiveness among microbiologists outside endemic areas, identification systems in the average sentinel laboratory, and the biosafety conditions necessary to process these organisms. We report a case of chronic left hip joint effusion in a known case of diabetes mellitus. Gram stain of computed tomography (CT-guided aspirate from the joint revealed Gram-negative bacilli along with pus cells. Culture was confirmed as Burkholderia pseudomallei on Vitek2C, which was sensitive to ceftazidime and trimethoprim/sulfmethoxazole. Unfortunately, patient could not be started on appropriate antibiotics due to delay in detection and patient succumbed to severe septicemia. This case is reported to highlight importance of automated identification and sensitivity especially in nonendemic areas and unusual antibiogram of this organism for which disc diffusion method is not standardized.

  16. Macroautophagy is essential for killing of intracellular Burkholderia pseudomallei in human neutrophils.

    Science.gov (United States)

    Rinchai, Darawan; Riyapa, Donporn; Buddhisa, Surachat; Utispan, Kusumawadee; Titball, Richard W; Stevens, Mark P; Stevens, Joanne M; Ogawa, Michinaga; Tanida, Isei; Koike, Masato; Uchiyama, Yasuo; Ato, Manabu; Lertmemongkolchai, Ganjana

    2015-01-01

    Neutrophils play a key role in the control of Burkholderia pseudomallei, the pathogen that causes melioidosis. Here, we show that survival of intracellular B. pseudomallei was significantly increased in the presence of 3-methyladenine or lysosomal cathepsin inhibitors. The LC3-flux was increased in B. pseudomallei-infected neutrophils. Concordant with this result, confocal microscopy analyses using anti-LC3 antibodies revealed that B. pseudomallei-containing phagosomes partially overlapped with LC3-positive signal at 3 and 6 h postinfection. Electron microscopic analyses of B. pseudomallei-infected neutrophils at 3 h revealed B. pseudomallei-containing phagosomes that occasionally fused with phagophores or autophagosomes. Following infection with a B. pseudomallei mutant lacking the Burkholderia secretion apparatus Bsa Type III secretion system, neither this characteristic structure nor bacterial escape into the cytosol were observed. These findings indicate that human neutrophils are able to recruit autophagic machinery adjacent to B. pseudomallei-containing phagosomes in a Type III secretion system-dependent manner.

  17. Variable virulence factors in Burkholderia pseudomallei (melioidosis associated with human disease.

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    Derek S Sarovich

    Full Text Available Burkholderia pseudomallei is a Gram-negative environmental bacterium that causes melioidosis, a potentially life-threatening infectious disease affecting mammals, including humans. Melioidosis symptoms are both protean and diverse, ranging from mild, localized skin infections to more severe and often fatal presentations including pneumonia, septic shock with multiple internal abscesses and occasionally neurological involvement. Several ubiquitous virulence determinants in B. pseudomallei have already been discovered. However, the molecular basis for differential pathogenesis has, until now, remained elusive. Using clinical data from 556 Australian melioidosis cases spanning more than 20 years, we identified a Burkholderia mallei-like actin polymerization bimA(Bm gene that is strongly associated with neurological disease. We also report that a filamentous hemagglutinin gene, fhaB3, is associated with positive blood cultures but is negatively correlated with localized skin lesions without sepsis. We show, for the first time, that variably present virulence factors play an important role in the pathogenesis of melioidosis. Collectively, our study provides a framework for assessing other non-ubiquitous bacterial virulence factors and their association with disease, such as candidate loci identified from large-scale microbial genome-wide association studies.

  18. Research Status and Prospect ofBurkholderia glumae, the Pathogen Causing Bacterial Panicle Blight

    Institute of Scientific and Technical Information of China (English)

    CUI Zhou-qi; ZHU Bo; XIE Guan-lin; LI Bin; HUANG Shi-wen

    2016-01-01

    Bacterial panicle blight caused by Burkholderia glumae is one of the most severe seed-borne bacterial diseases of rice in the world. Currently, this disease has affected many countries of Asia, Africa, South and North America. It is a typical example of the shifting from minor plant disease to major disease due to the changes of environmental conditions. Some virulent factors of B. glumae have been identified, including toxoflavins and lipases, whose productions are dependent on the TofI/TofR quorum-sensing system, and type III effectors. In spite of its economic significance, neither effective control measure for this disease nor resistant rice variety is currently available. In recent years, genomics, transcriptomics and other molecular methods have provided useful information for better understanding the molecular mechanisms underlyingB. glumaevirulence and the rice defence mechanisms against pathogens. For the prevention of this pathogen, our laboratory has developed a rapid and sensitive multiplex PCR assay for detecting and distinguishingB. glumae from otherBurkholderia species. This improved understanding ofB. glumae will shed new light on bacterial panicle blight disease management.

  19. Research Status and Prospect of Burkholderia glumae, the Pathogen Causing Bacterial Panicle Blight

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    Cui Zhou-qi

    2016-05-01

    Full Text Available Bacterial panicle blight caused by Burkholderia glumae is one of the most severe seed-borne bacterial diseases of rice in the world. Currently, this disease has affected many countries of Asia, Africa, South and North America. It is a typical example of the shifting from minor plant disease to major disease due to the changes of environmental conditions. Some virulent factors of B. glumae have been identified, including toxoflavins and lipases, whose productions are dependent on the TofI/TofR quorum-sensing system, and type III effectors. In spite of its economic significance, neither effective control measure for this disease nor resistant rice variety is currently available. In recent years, genomics, transcriptomics and other molecular methods have provided useful information for better understanding the molecular mechanisms underlying B. glumae virulence and the rice defence mechanisms against pathogens. For the prevention of this pathogen, our laboratory has developed a rapid and sensitive multiplex PCR assay for detecting and distinguishing B. glumae from other Burkholderia species. This improved understanding of B. glumae will shed new light on bacterial panicle blight disease management.

  20. Maize responds to Azotobacter sp and Burkholderia sp inoculation at reduced dose of nitrogen fertilizer

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    Juan Manuel Sánchez-Yáñez

    2014-03-01

    Full Text Available The positive maize response to inoculation with plant growth promoting bacteria (PGPB as Azotobacter sp and Burkholderia sp an endophytic type, are an alternative to reduced and optimize nitrogen fertilizer (NF dose, recommended for this plant, without adversely affect its growth. The aim of this study was to analyze maize respond to inoculation with Azotobacter sp and Burkholderia sp at the dose 50% of FN. Used an experimental design of randomized blocks. By response variables: percent germination (%, the shoot and root phenology: plant height (PH, root length (RL and biomass: shoot fresh weight (SFW and root fresh weight (RFW, the shoot dry weight (SDW and root dry weight (RDW. The results indicated a positive maize respond to PGPB inoculation at germination, seedling and flowering level, reached a RDW of 7.03 g, statistically significant value compared with 2.60 g of RDW non inoculated maize feed with NF dose recommended regard as relative control (RC. This suggests a synergistic interaction among these PGPB in synthesis of plant growth promoting substances (PGPS on maize, to optimize the reduced NF dose.

  1. Effectiveness of various Pseudomonas spp. and Burkholderia caryophylli containing ACC-deaminase for improving growth and yield of wheat (Triticum aestivum L.).

    Science.gov (United States)

    Shaharoona, B; Jamro, G M; Zahir, Z A; Arshad, M; Memon, K S

    2007-08-01

    This study assessed the possible role of different traits in selected plant growth-promoting rhizobacteria (PGPR) for improving wheat growth and yield under natural conditions. Rhizobacteria exhibiting 1-aminocyclopropane-1-carboxylate (ACC)-deaminase activity were isolated and screened for their growth-promoting activity in wheat under axenic conditions. Five isolates belonging to Pseudomonas and one Burkholderia caryophylli isolate that showed promising performances under axenic conditions were selected and characterized for in vitro ACC-deaminase activity, chitinase activity, auxin production, P solubilization, and root colonization. These isolates were then used as inocula for wheat cultivated under natural conditions in pot and/or field trials. Significant increases in root elongation, root weight, tillers per pot, 1,000-grain weight, and grain and straw yields were observed in response to inoculation with PGPR in the pot trials. Inoculation with these PGPR was also effective under field conditions and increased the wheat growth and yield significantly. However, the efficacy of the strains was inconsistent under the axenic, pot, and field conditions. Pseudomonasfluorescens (ACC50), which exhibited a relatively high in vitro ACC-deaminase activity, chitinase activity, auxin production, and P solubilization and more intensive root colonization, was the most efficient isolate under the field conditions. Therefore, these results demonstrated that ACC-deaminase activity is an efficient parameter for the selection of promising PGPR under axenic conditions. However, additional traits of PGPR, including auxin production, chitinase activity, P solubilization, and root colonization, are also important for selecting PGPR as biofertilizers.

  2. An Outbreak of Burkholderia cepacia Pseudobacteraemia caused by Intrinsically Contaminated Commercial 0.5% Chlorhexidine Solution in Neonatal Intensive Care Units.

    Science.gov (United States)

    Song, Je Eun; Kwak, Yee Gyung; Um, Tae Hyun; Cho, Chong Rae; Kim, Sollip; Park, In Sook; Hwang, Jong Hee; Kim, Namhee; Oh, Gang-Bok

    2017-09-18

    Burkholderia cepacia is intrinsically resistant to certain antiseptics. We experienced a sudden increase in the frequency of isolation of B. cepacia from blood cultures in a neonatal intensive care unit (NICU) of a university-affiliated hospital. An investigation was conducted to identify the source and to intervene in the ongoing infections. The cases were defined as patients with positive blood cultures for B. cepacia in a NICU between November 2014 and January 2015. We reviewed medical records and interviewed NICU health care workers. Samples of suspected antiseptics, blood culture bottles, cotton balls, gauze, and a needle used in the NICU were analysed microbiologically. During the outbreak period, B. cepacia was identified in 25 blood cultures obtained from 21 patients. The clinical features of the patients were suggestive of pseudobacteraemia. Regarding environmental samples, B. cepacia was cultured from 0.5% chlorhexidine gluconate (CHG) solution products that had been used as a skin antiseptic during blood drawing in the NICU. The clinical B. cepacia isolate and two strains obtained from 0.5% CHG exhibited identical pulsed-field gel electrophoresis patterns. After the CHG products were withdrawn, the outbreak was resolved. The pseudobacteraemia cases were caused by contaminated 0.5% CHG from a single company. Stricter government regulation is needed to prevent contamination of disinfectants during manufacturing. In addition, microbial contamination of antiseptics and disinfectants should be suspected when a B. cepacia outbreak occurs in hospitalised patients. Copyright © 2017. Published by Elsevier Ltd.

  3. Role of Burkholderia pseudomallei Sigma N2 in Amino Acids Utilization and in Regulation of Catalase E Expression at the Transcriptional Level

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    Duong Thi Hong Diep

    2015-01-01

    Full Text Available Burkholderia pseudomallei is the causative agent of melioidosis. The complete genome sequences of this pathogen have been revealed, which explain some pathogenic mechanisms. In various hostile conditions, for example, during nitrogen and amino acid starvation, bacteria can utilize alternative sigma factors such as RpoS and RpoN to modulate genes expression for their adaptation and survival. In this study, we demonstrate that mutagenesis of rpoN2, which lies on chromosome 2 of B. pseudomallei and encodes a homologue of the sigma factor RpoN, did not alter nitrogen and amino acid utilization of the bacterium. However, introduction of B. pseudomallei rpoN2 into E. coli strain deficient for rpoN restored the ability to utilize amino acids. Moreover, comparative partial proteomic analysis of the B. pseudomallei wild type and its rpoN2 isogenic mutant was performed to elucidate its amino acids utilization property which was comparable to its function found in the complementation assay. By contrast, the rpoN2 mutant exhibited decreased katE expression at the transcriptional and translational levels. Our finding indicates that B. pseudomallei RpoN2 is involved in a specific function in the regulation of catalase E expression.

  4. Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis

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    John P. Jakupciak

    2013-01-01

    Full Text Available Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations.

  5. IN SITU BIOREMEDIATION OF TRICHLOROETHYLENE USING BURKHOLDERIA CEPACIA G4 PR1: ANALYSIS OF MICROBIAL ECOLOGY PARAMETERS FOR RISK ASSESSMENT (RESEARCH BRIEF)

    Science.gov (United States)

    The introduction of bacteria into aquifers for bioremediation purposes requires monitoring of the persistence and activity of microbial populations for efficacy and risk assessment purposes. Burkholderia cepacia G4 PR1 constitutively expresses a toluene ortho-monooxygenase (tom) ...

  6. Genetic analysis of the CDI pathway from Burkholderia pseudomallei 1026b.

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    Sanna Koskiniemi

    Full Text Available Contact-dependent growth inhibition (CDI is a mode of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion systems. CdiA binds to receptors on susceptible target bacteria, then delivers a toxin domain derived from its C-terminus. Studies with Escherichia coli suggest the existence of multiple CDI growth-inhibition pathways, whereby different systems exploit distinct target-cell proteins to deliver and activate toxins. Here, we explore the CDI pathway in Burkholderia using the CDIIIBp1026b system encoded on chromosome II of Burkholderia pseudomallei 1026b as a model. We took a genetic approach and selected Burkholderia thailandensis E264 mutants that are resistant to growth inhibition by CDIIIBp1026b. We identified mutations in three genes, BTH_I0359, BTH_II0599, and BTH_I0986, each of which confers resistance to CDIIIBp1026b. BTH_I0359 encodes a small peptide of unknown function, whereas BTH_II0599 encodes a predicted inner membrane transport protein of the major facilitator superfamily. The inner membrane localization of BTH_II0599 suggests that it may facilitate translocation of CdiA-CTIIBp1026b toxin from the periplasm into the cytoplasm of target cells. BTH_I0986 encodes a putative transglycosylase involved in lipopolysaccharide (LPS synthesis. ∆BTH_I0986 mutants have altered LPS structure and do not interact with CDI⁺ inhibitor cells to the same extent as BTH_I0986⁺ cells, suggesting that LPS could function as a receptor for CdiAIIBp1026b. Although ∆BTH_I0359, ∆BTH_II0599, and ∆BTH_I0986 mutations confer resistance to CDIIIBp1026b, they provide no protection against the CDIE264 system deployed by B. thailandensis E264. Together, these findings demonstrate that CDI growth-inhibition pathways are distinct and can differ significantly even between closely related species.

  7. Burkholderia phytofirmans inoculation-induced changes on the shoot cell anatomy and iron accumulation reveal novel components of Arabidopsis-endophyte interaction that can benefit downstream biomass deconstruction

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    Shuai eZhao

    2016-01-01

    Full Text Available It is known that plant growth promoting bacteria (PGPB elicit positive effects on plant growth and biomass yield. However, the actual mechanism behind the plant-PGPB interaction is poorly understood, and the literature is scarce regarding the thermochemical pretreatability and enzymatic degradability of biomass derived from PGPB-inoculated plants. Most recent transcriptional analyses of PGPB strain Burkholderia phytofirmans PsJN inoculating potato in literature and Arabidopsis in our present study have revealed the expression of genes for ferritin and the biosynthesis and transport of siderophores (i.e. the molecules with high affinity for iron, respectively. The expression of such genes in the shoots of PsJN-inoculated plants prompted us to propose that PsJN-inoculation can improve the host plant’s iron uptake and accumulation, which facilitates the downstream plant biomass pretreatment and conversion to simple sugars. In this study, we employed B. phytofirmans PsJN to inoculate the Arabidopsis thaliana plants, and conducted the first investigation for its effects on the biomass yield, the anatomical organization of stems, the iron accumulation, and the pretreatment and enzymatic hydrolysis of harvested biomass. The results showed that the strain PsJN stimulated plant growth in the earlier period of plant development and enlarged the cell size of stem piths, and it also indeed enhanced the essential metals uptake and accumulation in host plants. Moreover, we found that the PsJN-inoculated plant biomass released more glucose and xylose after hot water pretreatment and subsequent co-saccharification, which provided a novel insight into development of lignocellulosic biofuels from renewable biomass resources.

  8. Perturbation of the two-component signal transduction system, BprRS, results in attenuated virulence and motility defects in Burkholderia pseudomallei.

    Science.gov (United States)

    Lazar Adler, Natalie R; Allwood, Elizabeth M; Deveson Lucas, Deanna; Harrison, Paul; Watts, Stephen; Dimitropoulos, Alexandra; Treerat, Puthayalai; Alwis, Priyangi; Devenish, Rodney J; Prescott, Mark; Govan, Brenda; Adler, Ben; Harper, Marina; Boyce, John D

    2016-05-04

    Burkholderia pseudomallei is the causative agent of melioidosis, a severe invasive disease of humans and animals. Initial screening of a B. pseudomallei signature-tagged mutagenesis library identified an attenuated mutant with a transposon insertion in a gene encoding the sensor component of an uncharacterised two-component signal transduction system (TCSTS), which we designated BprRS. Single gene inactivation of either the response regulator gene (bprR) or the sensor histidine kinase gene (bprS) resulted in mutants with reduced swarming motility and reduced virulence in mice. However, a bprRS double mutant was not attenuated for virulence and displayed wild-type levels of motility. The transcriptomes of the bprS, bprR and bprRS mutants were compared with the transcriptome of the parent strain K96243. Inactivation of the entire BprRS TCSTS (bprRS double mutant) resulted in altered expression of only nine genes, including both bprR and bprS, five phage-related genes and bpss0686, encoding a putative 5, 10-methylene tetrahydromethanopterin reductase involved in one carbon metabolism. In contrast, the transcriptomes of each of the bprR and bprS single gene mutants revealed more than 70 differentially expressed genes common to both mutants, including regulatory genes and those required for flagella assembly and for the biosynthesis of the cytotoxic polyketide, malleilactone. Inactivation of the entire BprRS TCSTS did not alter virulence or motility and very few genes were differentially expressed indicating that the definitive BprRS regulon is relatively small. However, loss of a single component, either the sensor histidine kinase BprS or its cognate response regulator BprR, resulted in significant transcriptomic and phenotypic differences from the wild-type strain. We hypothesize that the dramatically altered phenotypes of these single mutants are the result of cross-regulation with one or more other TCSTSs and concomitant dysregulation of other key regulatory genes.

  9. Dissection of quorum-sensing genes in Burkholderia glumae reveals non-canonical regulation and the new regulatory gene tofM for toxoflavin production.

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    Ruoxi Chen

    Full Text Available Burkholderia glumae causes bacterial panicle blight of rice and produces major virulence factors, including toxoflavin, under the control of the quorum-sensing (QS system mediated by the luxI homolog, tofI, and the luxR homolog, tofR. In this study, a series of markerless deletion mutants of B. glumae for tofI and tofR were generated using the suicide vector system, pKKSacB, for comprehensive characterization of the QS system of this pathogen. Consistent with the previous studies by other research groups, ΔtofI and ΔtofR strains of B. glumae did not produce toxoflavin in Luria-Bertani (LB broth. However, these mutants produced high levels of toxoflavin when grown in a highly dense bacterial inoculum (∼ 10(11 CFU/ml on solid media, including LB agar and King's B (KB agar media. The ΔtofI/ΔtofR strain of B. glumae, LSUPB201, also produced toxoflavin on LB agar medium. These results indicate the presence of previously unknown regulatory pathways for the production of toxoflavin that are independent of tofI and/or tofR. Notably, the conserved open reading frame (locus tag: bglu_2g14480 located in the intergenic region between tofI and tofR was found to be essential for the production of toxoflavin by tofI and tofR mutants on solid media. This novel regulatory factor of B. glumae was named tofM after its homolog, rsaM, which was recently identified as a novel negative regulatory gene for the QS system of another rice pathogenic bacterium, Pseudomonas fuscovaginae. The ΔtofM strain of B. glumae, LSUPB286, produced a less amount of toxoflavin and showed attenuated virulence when compared with its wild type parental strain, 336gr-1, suggesting that tofM plays a positive role in toxoflavin production and virulence. In addition, the observed growth defect of the ΔtofI strain, LSUPB145, was restored by 1 µM N-octanoyl homoserine lactone (C8-HSL.

  10. Growth of Burkholderia sacchari LFM 101 cultivated in glucose, sucrose and glycerol at different temperatures

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    Valkirea Matos Nascimento

    Full Text Available ABSTRACT Polyhydroxyalkanoates (PHAs have attracted major industrial interest as alternatives to conventional plastics. They are produced by several bacteria as cytoplasmic inclusions when nutrients are in limited supply. Among the many factors influencing bacterial growth, the effect of temperature on both specific growth rates and growth yields in terms of carbon source intake is of considerable interest. This study aimed to evaluate the influence of the bacterium Burkholderia sacchari LFM 101 on growth and PHA production, using glucose, sucrose or glycerol as a carbon source, at 30 and 35 °C. The results showed that B. sacchari cultured with glucose at 35 °C presented both higher productivity and polymer yield in dried cell mass. There were no differences in growth rates (μmax in sucrose and glucose. The growth conditions studied were not favorable to glycerol consumption due to limitations in the energy supply from glycerol.

  11. Biodiesel production from Jatropha oil catalyzed by immobilized Burkholderia cepacia lipase on modified attapulgite.

    Science.gov (United States)

    You, Qinghong; Yin, Xiulian; Zhao, Yuping; Zhang, Yan

    2013-11-01

    Lipase from Burkholderia cepacia was immobilized on modified attapulgite by cross-linking reaction for biodiesel production with jatropha oil as feedstock. Effects of various factors on biodiesel production were studied by single-factor experiment. Results indicated that the best conditions for biodiesel preparation were: 10 g jatropha oil, 2.4 g methanol (molar ratio of oil to methanol is 1:6.6) being added at 3h intervals, 7 wt% water, 10 wt% immobilized lipase, temperature 35°C, and time 24h. Under these conditions, the maximum biodiesel yield reached 94%. The immobilized lipase retained 95% of its relative activity during the ten repeated batch reactions. The half-life time of the immobilized lipase is 731 h. Kinetics was studied and the Vmax of the immobilized lipases were 6.823 mmol L(-1). This immobilized lipase catalyzed process has potential industrial use for biodiesel production to replace chemical-catalyzed method.

  12. Ceftazidime resistance inBurkholderia pseudomallei:First report from India

    Institute of Scientific and Technical Information of China (English)

    Bijayini Behera; TLVD Prasad Babu; A Kamalesh; Gangadhar Reddy

    2012-01-01

    ABSTRACT Melioidosis, a disease of public health importance in Southeast Asia and Northern Australia, of late has shown an increasing trend in India, particularly Southern India. We describe a case of a39-year-old diabetic patient with left elbow septic arthritis, multiple liver, splenic abscesses, pneumonia, pleural effusion, followed by sepsis syndrome. Blood cultures and culture of the joint aspirate yielded pure growth ofBurkholderia pseudomallei (B. pesudomallei), sensitive to carbapenem, co-trimoxazole and resistant to ceftazidime. The patient was successfully treated with imipenem- cilastin.He was discharged on co-trimoxazole to complete the24 weeks course and follow-up has continued to date. The patient continues to remain asymptomatic. The case re-emphasizes the need to monitor the trend ofB. pseudomallei in India, particularly the development of ceftazidime resistance, which incidentally is the drug of choice.

  13. Chemotaxis of Pseudomonas stutzeri OX1 and Burkholderia cepacia G4 toward chlorinated ethenes.

    Science.gov (United States)

    Vardar, Gönül; Barbieri, Paola; Wood, Thomas K

    2005-03-01

    The chemotactic responses of Pseudomonas putida F1, Burkholderia cepacia G4, and Pseudomonas stutzeri OX1 were investigated toward toluene, trichloroethylene (TCE), tetrachloroethylene (PCE), cis-1,2-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), 1,1-dichloroethylene (1,1-DCE), and vinyl chloride (VC). P. stutzeri OX1 and P. putida F1 were chemotactic toward toluene, PCE, TCE, all DCEs, and VC. B. cepacia G4 was chemotactic toward toluene, PCE, TCE, cis-DCE, 1,1-DCE, and VC. Chemotaxis of P. stutzeri OX1 grown on o-xylene vapors was much stronger than when grown on o-cresol vapors toward some chlorinated ethenes. Expression of toluene-o-xylene monooxygenase (ToMO) from touABCDEF appears to be required for positive chemotaxis attraction, and the attraction is stronger with the touR (ToMO regulatory) gene.

  14. Colonization or spontaneous resolution:Expanding the role for Burkholderia pseudomallei

    Institute of Scientific and Technical Information of China (English)

    Kushal Naha; Barkur Ananthakrishna Shastry; Kavitha Saravu

    2014-01-01

    A 19-year-old Asian Indian female presented with productive cough since the past one month and low grade fever since the past two weeks. She was diagnosed with pulmonary tuberculosis and treated with antitubercular drugs. Subsequently, delayed cultures of bronchoalveolar lavage fluid grewBurkholderia pseudomallei (B. pseudomallei). On follow up the patient reported significant subjective improvement and ESR progressively returned to normal. In summary, this case report raises two distinct and equally intriguing roles forB. pseudomallei,i.e. respiratory colonization and spontaneously resolving pulmonary infection. The pathogenic potential ofB. pseudomallei, the etiologic agent of melioidosis, is well known. Confirmation of either colonization or spontaneous resolution, would potentially spare many patients unnecessary and expensive therapy with broad-spectrum antibiotics, and contribute to more rational usage of antibiotics, especially in co-infection withMycobacterium tuberculosis andB. pseudomallei-two bacterial diseases with closely similar clinical, radiologic and histopathologic features.

  15. Optimization of Lipase Production by Burkholderia sp. Using Response Surface Methodology

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    Shiow-Ling Lee

    2012-11-01

    Full Text Available Response surface methodology (RSM was employed to optimize the extracellular lipase production by Burkholderia sp. HL-10. Preliminary tests showed that olive oil, tryptone and Tween-80 exhibited significant effects on the lipase production. The optimum concentrations of these three components were determined using a faced-centered central composite design (FCCCD. The analysis of variance revealed that the established model was significant (p < 0.01. The optimized medium containing 0.65% olive oil (v/v, 2.42% tryptone (w/v and 0.15% Tween-80 (v/v resulted in a maximum activity of 122.3 U/mL, about three fold higher than that in basal medium. Approximately 99% of validity of the predicted value was achieved.

  16. Evaluation of a Burkholderia pseudomallei Outer Membrane Vesicle Vaccine in Nonhuman Primates.

    Science.gov (United States)

    Petersen, Hailey; Nieves, Wildaliz; Russell-Lodrigue, Kasi; Roy, Chad J; Morici, Lisa A

    2014-01-01

    Burkholderia pseudomallei (Bps)is the causative agent of melioidosis and is endemic in regions of northern Australia and Southeast Asia. Bps is inherently resistant to multiple antibiotics and is considered a potential biological warfare agent by the U.S. DHHS. Therefore, effective vaccines are necessary to prevent natural infection and to safeguard against biological attack with this organism. In our previous work we have shown that immunization with naturally derived outer membrane vesicles (OMVs) from Bps provides significant protection against lethal aerosol and systemic infection in BALB/c mice. In this work, we evaluated the safety and immunogenicity of escalating doses of OMV vaccine in rhesus macaques. We show that immunization of rhesus macaques with Bps OMVs generates humoral immuneresponses to protective protein and polysaccharide antigens without any associated toxicity or reactogenicity. These results lay the groundwork for evaluation of protective efficacy of the OMV vaccine in the nonhuman primate model of melioidosis.

  17. Validation of a monoclonal antibody-based immunofluorescent assay to detect Burkholderia pseudomallei in blood cultures.

    Science.gov (United States)

    Dulsuk, Adul; Paksanont, Suporn; Sangchankoom, Adisak; Ekchariyawat, Peeraya; Phunpang, Rungnapa; Jutrakul, Yaowaruk; Chantratita, Narisara; West, T Eoin

    2017-01-22

    Identification of Burkholderia pseudomallei, the cause of melioidosis, using routine methods takes several days. Use of a monoclonal antibody-based immunofluorescent assay (IFA) on positive blood cultures may speed diagnosis. We tested the diagnostic accuracy of the IFA on 545 blood cultures positive for Gram-negative organisms at Udon Thani Hospital, Thailand, between June 2015 and August 2016. Sensitivity of the IFA was 100% and specificity was 99.6%. The median decrease in time to pathogen identification between the IFA result and routine methods was 28 h (IQR 25-51), p<0.0001. The IFA accurately expedites the diagnosis of melioidosis. © The Author 2017. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.

  18. Real-time Fluorescent Quantitative PCR Detection for Burkholderia gladioli pv.alliicola%洋葱腐烂病菌荧光定量PCR检测体系的建立

    Institute of Scientific and Technical Information of China (English)

    张仑; 高文娜; 殷幼平; 王中康

    2013-01-01

    唐菖蒲伯克霍尔德菌洋葱致病型(Burkholderia gladioli pv.alliicola),是重要的植物病原细菌检疫性有害生物.本研究的目的是建立特异、灵敏、快速的TaqMan探针荧光定量PCR方法用于检测洋葱腐烂病菌(B.gladioli pv.alliicola).利用洋葱腐烂病菌保守基因序列设计和筛选特异性引物和TaqMan探针,优化PCR反应体系和反应条件.荧光定量PCR菌液检测灵敏度达到1.02×102 CFU/mL,DNA检测灵敏度达到1.73×10-4 ng/μL,反应时间仅需1h左右,对14种伯克氏菌属Burkholderia)参比菌种的特异性检测结果显示,洋葱腐烂病菌具有明显的扩增曲线生长,表明建立的荧光定量PCR体系具有非常高的特异性.同时对洋葱(Allium cepa)种子进行了模拟带菌检测,结果表明,建立的洋葱腐烂病菌荧光定量PCR检测体系能够检出模拟样品中的洋葱腐烂病菌,验证了检测体系的实用性.本研究建立的洋葱腐烂病菌荧光定量PCR检测体系能够有效用于洋葱腐烂病菌的鉴定,为进出口口岸和基层检验检疫部门提供了一种简便、快速、准确的洋葱腐烂病菌分子检测手段.%Burkholderia gladioli pv. alliicola is an important plant pathogen. This research was to establish the specific, sensitive and fast Taqman Real-time PCR for detection of B. gladioli pv. alliicola. The specific genome region of B. gladioli pv. alliicola was used for the design of Taqman probe and specific primers. The reaction conditions was optimized. Fourteen reference strains were used for the specificity test, the sensitivity of the Real-time PCR was tested as well. The sensitivity of bacterial suspension of Real-time PCR reached 1.02×102 CFU/mL and the sensitivity of DNA reached 1.73×104 ng/μL. Total reaction time took about 1 h or so. Among 14 reference strains belong to the genus of Burkholderia, Real-time PCR showed an extremely high specificity. The simulation test was performed on the onion (Allium cepa

  19. Two Stable Variants of Burkholderia pseudomallei Strain MSHR5848 Express Broadly Divergent in vitro Phenotypes Associated with their Virulence Differences

    Science.gov (United States)

    2016-11-21

    and/or N sources (ref - Kleber, H.P. FEMS Microbiol Let 147:1-9, 1997; Shah, MolMicro 2008 ). They and other polyamines are widely distributed and...13192; Mott, PLoSNegTropDis 2015; Kvitko et al. 2012 PLoSNegTropDis; Braun V. FEMS Microbiol Rev. 1995; 16: 295–307 ). The more efficient...superoxide radicals leading to intracellular oxidative stress (ref – Chasteen, T. FEM Mic Rev 2009; Fuentes, D. JBact 2007). Maintenance of the cell

  20. Effect of two types of biosurfactants on phenanthrene availability to the bacterial bioreporter Burkholderia sartisoli strain RP037

    NARCIS (Netherlands)

    Tecon, R.; Van der Meer, J.R.

    2010-01-01

    Biosurfactants are tensio-active agents that have often been proposed as a means to enhance the aqueous solubility of hydrophobic organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs). Biosurfactant-producing bacteria such as those belonging to the genus Pseudomonas might therefore e

  1. The phn Genes of Burkholderia sp. Strain RP007 Constitute a Divergent Gene Cluster for Polycyclic Aromatic Hydrocarbon Catabolism

    OpenAIRE

    1999-01-01

    Cloning and molecular ecological studies have underestimated the diversity of polycyclic aromatic hydrocarbon (PAH) catabolic genes by emphasizing classical nah-like (nah, ndo, pah, and dox) sequences. Here we report the description of a divergent set of PAH catabolic genes, the phn genes, which although isofunctional to the classical nah-like genes, show very low homology. This phn locus, which contains nine open reading frames (ORFs), was isolated on an 11.5-kb HindIII fragment from phenant...

  2. Transports of acetate and haloacetate in Burkholderia species MBA4 are operated by distinct systems

    Directory of Open Access Journals (Sweden)

    Su Xianbin

    2012-11-01

    Full Text Available Abstract Background Acetate is a commonly used substrate for biosynthesis while monochloroacetate is a structurally similar compound but toxic and inhibits cell metabolism by blocking the citric acid cycle. In Burkholderia species MBA4 haloacetate was utilized as a carbon and energy source for growth. The degradation of haloacid was mediated by the production of an inducible dehalogenase. Recent studies have identified the presence of a concomitantly induced haloacetate-uptake activity in MBA4. This uptake activity has also been found to transport acetate. Since acetate transporters are commonly found in bacteria it is likely that haloacetate was transported by such a system in MBA4. Results The haloacetate-uptake activity of MBA4 was found to be induced by monochloroacetate (MCA and monobromoacetate (MBA. While the acetate-uptake activity was also induced by MCA and MBA, other alkanoates: acetate, propionate and 2-monochloropropionate (2MCPA were also inducers. Competing solute analysis showed that acetate and propionate interrupted the acetate- and MCA- induced acetate-uptake activities. While MCA, MBA, 2MCPA, and butyrate have no effect on acetate uptake they could significantly quenched the MCA-induced MCA-uptake activity. Transmembrane electrochemical potential was shown to be a driving force for both acetate- and MCA- transport systems. Conclusions Here we showed that acetate- and MCA- uptake in Burkholderia species MBA4 are two transport systems that have different induction patterns and substrate specificities. It is envisaged that the shapes and the three dimensional structures of the solutes determine their recognition or exclusion by the two transport systems.

  3. Systematic review and consensus guidelines for environmental sampling of Burkholderia pseudomallei.

    Directory of Open Access Journals (Sweden)

    Direk Limmathurotsakul

    Full Text Available BACKGROUND: Burkholderia pseudomallei, a Tier 1 Select Agent and the cause of melioidosis, is a Gram-negative bacillus present in the environment in many tropical countries. Defining the global pattern of B. pseudomallei distribution underpins efforts to prevent infection, and is dependent upon robust environmental sampling methodology. Our objective was to review the literature on the detection of environmental B. pseudomallei, update the risk map for melioidosis, and propose international consensus guidelines for soil sampling. METHODS/PRINCIPAL FINDINGS: An international working party (Detection of Environmental Burkholderia pseudomallei Working Party (DEBWorP was formed during the VIth World Melioidosis Congress in 2010. PubMed (January 1912 to December 2011 was searched using the following MeSH terms: pseudomallei or melioidosis. Bibliographies were hand-searched for secondary references. The reported geographical distribution of B. pseudomallei in the environment was mapped and categorized as definite, probable, or possible. The methodology used for detecting environmental B. pseudomallei was extracted and collated. We found that global coverage was patchy, with a lack of studies in many areas where melioidosis is suspected to occur. The sampling strategies and bacterial identification methods used were highly variable, and not all were robust. We developed consensus guidelines with the goals of reducing the probability of false-negative results, and the provision of affordable and 'low-tech' methodology that is applicable in both developed and developing countries. CONCLUSIONS/SIGNIFICANCE: The proposed consensus guidelines provide the basis for the development of an accurate and comprehensive global map of environmental B. pseudomallei.

  4. Understanding the Pathogenicity of Burkholderia contaminans, an Emerging Pathogen in Cystic Fibrosis

    Science.gov (United States)

    Nunvar, Jaroslav; Kalferstova, Lucie; Bloodworth, Ruhi A. M.; Kolar, Michal; Degrossi, Jose; Lubovich, Silvina; Cardona, Silvia T.; Drevinek, Pavel

    2016-01-01

    Several bacterial species from the Burkholderia cepacia complex (Bcc) are feared opportunistic pathogens that lead to debilitating lung infections with a high risk of developing fatal septicemia in cystic fibrosis (CF) patients. However, the pathogenic potential of other Bcc species is yet unknown. To elucidate clinical relevance of Burkholderia contaminans, a species frequently isolated from CF respiratory samples in Ibero-American countries, we aimed to identify its key virulence factors possibly linked with an unfavorable clinical outcome. We performed a genome-wide comparative analysis of two isolates of B. contaminans ST872 from sputum and blood culture of a female CF patient in Argentina. RNA-seq data showed significant changes in expression for quorum sensing-regulated virulence factors and motility and chemotaxis. Furthermore, we detected expression changes in a recently described low-oxygen-activated (lxa) locus which encodes stress-related proteins, and for two clusters responsible for the biosynthesis of antifungal and hemolytic compounds pyrrolnitrin and occidiofungin. Based on phenotypic assays that confirmed changes in motility and in proteolytic, hemolytic and antifungal activities, we were able to distinguish two phenotypes of B. contaminans that coexisted in the host and entered her bloodstream. Whole genome sequencing revealed that the sputum and bloodstream isolates (each representing a distinct phenotype) differed by over 1,400 mutations as a result of a mismatch repair-deficient hypermutable state of the sputum isolate. The inferred lack of purifying selection against nonsynonymous mutations and the high rate of pseudogenization in the derived isolate indicated limited evolutionary pressure during evolution in the nutrient-rich, stable CF sputum environment. The present study is the first to examine the genomic and transcriptomic differences between longitudinal isolates of B. contaminans. Detected activity of a number of putative virulence

  5. Bukholderia strains promote Mimosa spp. growth but not Macroptilium atropurpureum

    Directory of Open Access Journals (Sweden)

    Kaliane Sírio Araújo

    Full Text Available ABSTRACT The aim of this study was to evaluate the relationship and symbiotic efficiency of 14 strains of Burkholderia isolated from rupestrian grasslands, using M. atropurpureum and Mimosa tenuiflora as trap plants, with the species M. atropurpureum, Mimosa bimucronata and M. foliolosa. For the nodulation and symbiotic efficiency test in M. atropurpureum, long-neck bottles containing nutrient solution were used. The experiments with Mimosa spp. were carried out in tubes containing vermiculite (160 cm3 and sand (80 cm3 (2:1. The parameters under evaluation were number of nodules, nodules dry matter production, shoots dry matter, roots dry matter, and total dry matter production for all the species analyzed; and plant height, diameter, and the Dickson quality index for Mimosa species. Of the 14 tested strains, two nodulated M. atropurpureum; however, they were ineffective in promoting plant growth. All the tested