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Sample records for burkholderia pseudomallei clinical

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

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    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.

  2. Development and validation of Burkholderia pseudomallei-specific real-time PCR assays for clinical, environmental or forensic detection applications.

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    Erin P Price

    Full Text Available The bacterium Burkholderia pseudomallei causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating B. pseudomallei from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all B. pseudomallei from other Burkholderiaceae. Currently available molecular assays for B. pseudomallei detection lack rigorous validation across large in silico datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ, limit of detection (LoD, linearity, ruggedness and robustness to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel B. pseudomallei specific assays, 122018 and 266152, using a dual-probe approach to differentiate B. pseudomallei from B. thailandensis, B. oklahomensis and B. thailandensis-like species; other species failed to amplify. Species specificity was validated across a large DNA panel (>2,300 samples comprising Burkholderia spp. and non-Burkholderia bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published B. pseudomallei-specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100% specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using B. pseudomallei as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated B. pseudomallei assays for the scientific research community.

  3. Genomic islands as a marker to differentiate between clinical and environmental Burkholderia pseudomallei.

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    Thanatchaporn Bartpho

    Full Text Available Burkholderia pseudomallei, as a saprophytic bacterium that can cause a severe sepsis disease named melioidosis, has preserved several extra genes in its genome for survival. The sequenced genome of the organism showed high diversity contributed mainly from genomic islands (GIs. Comparative genome hybridization (CGH of 3 clinical and 2 environmental isolates, using whole genome microarrays based on B. pseudomallei K96243 genes, revealed a difference in the presence of genomic islands between clinical and environmental isolates. The largest GI, GI8, of B. pseudomallei was observed as a 2 sub-GI named GIs8.1 and 8.2 with distinguishable %GC content and unequal presence in the genome. GIs8.1, 8.2 and 15 were found to be more common in clinical isolates. A new GI, GI16c, was detected on chromosome 2. Presences of GIs8.1, 8.2, 15 and 16c were evaluated in 70 environmental and 64 clinical isolates using PCR assays. A combination of GIs8.1 and 16c (positivity of either GI was detected in 70% of clinical isolates and 11.4% of environmental isolates (P0.05. Some virulence genes located in the absent GIs and the difference of GIs seems to contribute less to bacterial virulence. The PCR detection of 2 GIs could be used as a cost effective and rapid tool to detect potentially virulent isolates that were contaminated in soil.

  4. Characterization of ceftazidime resistance mechanisms in clinical isolates of Burkholderia pseudomallei from Australia.

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    Derek S Sarovich

    Full Text Available Burkholderia pseudomallei is a gram-negative bacterium that causes the serious human disease, melioidosis. There is no vaccine against melioidosis and it can be fatal if not treated with a specific antibiotic regimen, which typically includes the third-generation cephalosporin, ceftazidime (CAZ. There have been several resistance mechanisms described for B. pseudomallei, of which the best described are amino acid changes that alter substrate specificity in the highly conserved class A β-lactamase, PenA. In the current study, we sequenced penA from isolates sequentially derived from two melioidosis patients with wild-type (1.5 µg/mL and, subsequently, resistant (16 or ≥256 µg/mL CAZ phenotypes. We identified two single-nucleotide polymorphisms (SNPs that directly increased CAZ hydrolysis. One SNP caused an amino acid substitution (C69Y near the active site of PenA, whereas a second novel SNP was found within the penA promoter region. In both instances, the CAZ resistance phenotype corresponded directly with the SNP genotype. Interestingly, these SNPs appeared after infection and under selection from CAZ chemotherapy. Through heterologous cloning and expression, and subsequent allelic exchange in the native bacterium, we confirmed the role of penA in generating both low-level and high-level CAZ resistance in these clinical isolates. Similar to previous studies, the amino acid substitution altered substrate specificity to other β-lactams, suggesting a potential fitness cost associated with this mutation, a finding that could be exploited to improve therapeutic outcomes in patients harboring CAZ resistant B. pseudomallei. Our study is the first to functionally characterize CAZ resistance in clinical isolates of B. pseudomallei and to provide proven and clinically relevant signatures for monitoring the development of antibiotic resistance in this important pathogen.

  5. 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.

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

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    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.

  7. Burkholderia pseudomallei Data Gap Analysis

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    2015-11-01

    conducting this review is to identify the major biodefense relevant data gaps critical to better understanding B. pseudomallei infections and how they are...of patients (Table 2-1). Much of the data has excellent demographic and clinical resolution including age, sex, symptoms, duration of illness, risk...inhalation and enteral. Presentations include pneumonia , genitourinary, cutaneous, general bacteremia, septic arthritis and neurological

  8. The effect of methanolic extract of Tamarindus indica Linn. on the growth of clinical isolates of Burkholderia pseudomallei.

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    Muthu, Shankar Esaki; Nandakumar, Subhadra; Rao, Usha Anand

    2005-12-01

    Burkholderia pseudomallei (Pseudomonas pseudomallei) causes melioidosis, a life-threatening infection common among paddy cultivators in Southeast Asian countries. No plant materials have been investigated for its activity against B. pseudomallei. Therefore, a preliminary study was carried out using disc diffusion and minimum inhibitory concentration (MIC) methods to evaluate the anti-B. pseudomallei activity of five Indian medicinal plants documented to have been used for several ailments in the ancient Indian scriptures. The leaf extracts of Tamarindus indica, Lawsonia inermis, and Hibiscus rosa-sinensis, the rhizome extracts of Curcuma longa and the seeds of Vigna radiata were prepared using methanol as solvent. The disc diffusion and MIC methods were used to assess the anti-B. pseudomallei activity of the plants tested. Only methanol leaf extracts of Tamarindus indica exhibited anti-B. pseudomallei activity starting from disc concentrations of 150 mug by the disc diffusion method. The other plants failed to show any zone of inhibition. MIC assay revealed that the MIC and minimum bactericidal concentration (MBC) for B. pseudomallei were 125 mug/ml. Our preliminary finding showed that methanolic extracts of Tamarindus indica has anti-B. pseudomallei inhibitory potentials under in vitro conditions. Extensive animal studies may be required before investigating the role of Tamarindus indica for treating melioidosis.

  9. Prevalence of Burkholderia pseudomallei in Guangxi, China.

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    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.

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

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    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.

  11. Survival of Burkholderia pseudomallei on Environmental Surfaces.

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    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.

  12. Development of Burkholderia mallei and pseudomallei vaccines

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    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.

  13. Exploitation of host cells by Burkholderia pseudomallei.

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    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.

  14. 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.

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

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    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.

  16. Development of Burkholderia mallei and pseudomallei vaccines.

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    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

  17. SDS-PAGE analysis of whole cell protein and outer memr-bane protein patterns of clinical isolates of Burkholderia pseud-omallei

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    Apichart Nontprasert; Cheeraratana Cheeramakara; Sasithon Pukrittayakamee; David AB Dance; Ty L Pitt; Michael D Smith; Sirivan Vanijanonta; Nicholas J White

    2009-01-01

    Objective:To investigate the banding patterns of whole cell protein (WCP)and outer membrane protein (OMP)of Burkholderia pseudomallei (B.pseudomallei)in clinical isolates from patients with melioidosis. Methods:WCP and OMP of of B.pseudomallei in 50 clinical isolates,from 47 patients with melioidosis were prepared and separated by polyacrylamide gel electrophoresis (SDS-PAGE)using 10% gels and stained with Coomassie brilliant blue.The banding patterns were compared by using a laser densitometer and dendrogram. Results:There were 6 different banding patterns of WCP and 2 types of OMP.Type 1-5 WCP had 8 common protein bands at 19.0 -45.0 kDa with identical OMP pattern.The banding patterns of WCP in type 6 were distinct from the others and also its OMP profile.The majority of clinical isolates (37 /50,74%)were in type 1 WCP.Of the remaining isolates,8 were in type 2,2 in type 3,and one each was in type 4 to 6.There was no significant association between the WCP typing and the demographic or clinical features of the investigated patients.Conclusion:Despite the wide variation of clinical features of melioidosis,the results of this study show that B.pseudomallei had a few differences in the WCP and OMP profiles.Therefore typing of WCP and OMP,using SDS-PAGE analysis,could be an alternative method for phenotypic differentiation in clinical iso-lates of B.pseudomallei.

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

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    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...

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

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    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

  20. 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

  1. PCR-based identification of Burkholderia pseudomallei Identificação de Burkholderia pseudomallei baseada em PCR

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    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

  2. Colonization or spontaneous resolution:Expanding the role for Burkholderia pseudomallei

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    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.

  3. 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.

  4. 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.

  5. Survival and Intra-Nuclear Trafficking of Burkholderia pseudomallei: Strategies of Evasion from Immune Surveillance?

    Science.gov (United States)

    Vadivelu, Jamuna; Vellasamy, Kumutha Malar; Thimma, Jaikumar; Mariappan, Vanitha; Kang, Wen-Tyng; Choh, Leang-Chung; Wong, Kum Thong

    2017-01-01

    Background During infection, successful bacterial clearance is achieved via the host immune system acting in conjunction with appropriate antibiotic therapy. However, it still remains a tip of the iceberg as to where persistent pathogens namely, Burkholderia pseudomallei (B. pseudomallei) reside/hide to escape from host immune sensors and antimicrobial pressure. Methods We used transmission electron microscopy (TEM) to investigate post-mortem tissue sections of patients with clinical melioidosis to identify the localisation of a recently identified gut microbiome, B. pseudomallei within host cells. The intranuclear presence of B. pseudomallei was confirmed using transmission electron microscopy (TEM) of experimentally infected guinea pig spleen tissues and Live Z-stack, and ImageJ analysis of fluorescence microscopy analysis of in vitro infection of A549 human lung epithelial cells. Results TEM investigations revealed intranuclear localization of B. pseudomallei in cells of infected human lung and guinea pig spleen tissues. We also found that B. pseudomallei induced actin polymerization following infection of A549 human lung epithelial cells. Infected A549 lung epithelial cells using 3D-Laser scanning confocal microscopy (LSCM) and immunofluorescence microscopy confirmed the intranuclear localization of B. pseudomallei. Conclusion B. pseudomallei was found within the nuclear compartment of host cells. The nucleus may play a role as an occult or transient niche for persistence of intracellular pathogens, potentially leading to recurrrent episodes or recrudescence of infection. PMID:28045926

  6. Burkholderia pseudomallei enhances maturation of bone marrow-derived dendritic cells.

    Science.gov (United States)

    Williams, Natasha L; Kloeze, Eveline; Govan, Brenda L; Körner, Heinrich; Ketheesan, Natkunam

    2008-12-01

    T-cell activation is essential for protection against Burkholderia pseudomallei infection. Using bone marrow-derived dendritic cells (BMDC) isolated from partially resistant C57BL/6 and susceptible BALB/c mice, the degree of BMDC activation in the presence of B. pseudomallei was investigated. Maturation, cytokine production and internalization of B. pseudomallei by BMDC was assessed in response to infection with a highly virulent and a low-virulent clinical isolate. Maturation was determined by identifying the up-regulation of cell-surface markers CD11c and CD86. IL-1beta and IL-12p40 expression were assessed by reverse-transcriptase PCR. The uptake of B. pseudomallei by BMDC was measured using an internalization assay. This study demonstrated that B. pseudomallei isolates stimulate the maturation of BMDC to the same degree regardless of virulence. However, maturation of BMDC was significantly increased in BALB/c mice compared with C57BL/6 mice. Additionally, the uptake of B. pseudomallei by BMDC was significantly greater with the highly virulent isolate compared with the low-virulent isolate. Expression of IL-12 and IL-1beta following infection with B. pseudomallei was up-regulated. The differences observed may have implications in the development of an effective immune response to B. pseudomallei.

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

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    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.

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

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    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

  9. Burkholderia pseudomallei is frequently detected in groundwater that discharges to major watercourses in northern Australia.

    Science.gov (United States)

    Baker, Anthony L; Warner, Jeffrey M

    2016-07-01

    Burkholderia pseudomallei is the environmental bacterium that causes the serious disease melioidosis. Recently, a high prevalence of viable B. pseudomallei was reported from natural groundwater seeps around Castle Hill, a clinical focus of melioidosis in Townsville, Australia. This study sought to expand previous findings to determine the extent of B. pseudomallei in more diverse natural groundwater seeps in northern Queensland to ascertain if the presence of the organism in groundwater on Castle Hill was an isolated occurrence. Analysis of water samples (n = 26) obtained from natural groundwater seeps following an intensive rainfall event in the Townsville region determined the presence of B. pseudomallei DNA in duplicates of 18 samples (69.2 % [95 % CI, 51.5 to 87.0]). From 26 water samples, a single isolate of B. pseudomallei was recovered despite plating of both pre-enriched samples and original water samples onto selective media, indicating that the sensitivity of these molecular techniques far exceeds culture-based methods. Furthermore, the identification of new environments endemic for melioidosis may be more effectively determined by analysing surface groundwater seeps than by the analysis of random soil samples. This study suggests that a higher incidence of melioidosis following monsoonal rains may be partially the result of exposure to groundwater sources carrying B. pseudomallei, and that modifications to public health messages in endemic regions may be warranted. Moreover, these findings have implications for predictive models of melioidosis, effective models requiring consideration of topographical and surface hydrological data.

  10. 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.

  11. 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.

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

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    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.

  13. 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.

  14. 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

  15. 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.

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

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    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

  17. 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.

  18. 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.

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

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    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.

  20. 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.

  1. Interleukin 10 inhibits pro-inflammatory cytokine responses and killing of Burkholderia pseudomallei

    Science.gov (United States)

    Kessler, Bianca; Rinchai, Darawan; Kewcharoenwong, Chidchamai; Nithichanon, Arnone; Biggart, Rachael; Hawrylowicz, Catherine M.; Bancroft, Gregory J.; Lertmemongkolchai, Ganjana

    2017-01-01

    Melioidosis, caused by Burkholderia pseudomallei, is endemic in northeastern Thailand and Northern Australia. Severe septicemic melioidosis is associated with high levels of pro-inflammatory cytokines and is correlated with poor clinical outcomes. IL-10 is an immunoregulatory cytokine, which in other infections can control the expression of pro-inflammatory cytokines, but its role in melioidosis has not been addressed. Here, whole blood of healthy seropositive individuals (n = 75), living in N. E. Thailand was co-cultured with B. pseudomallei and production of IL-10 and IFN-γ detected and the cellular sources identified. CD3− CD14+ monocytes were the main source of IL-10. Neutralization of IL-10 increased IFN-γ, IL-6 and TNF-α production and improved bacteria killing. IFN-γ production and microbicidal activity were impaired in individuals with diabetes mellitus (DM). In contrast, IL-10 production was unimpaired in individuals with DM, resulting in an IL-10 dominant cytokine balance. Neutralization of IL-10 restored the IFN-γ response of individuals with DM to similar levels observed in healthy individuals and improved killing of B. pseudomallei in vitro. These results demonstrate that monocyte derived IL-10 acts to inhibit potentially protective cell mediated immune responses against B. pseudomallei, but may also moderate the pathological effects of excessive cytokine production during sepsis. PMID:28216665

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

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    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

  3. Source-identifying biomarker ions between environmental and clinical Burkholderia pseudomallei using whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS.

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    Suthamat Niyompanich

    Full Text Available Burkholderia pseudomallei is the causative agent of melioidosis, which is an endemic disease in Northeast Thailand and Northern Australia. Environmental reservoirs, including wet soils and muddy water, serve as the major sources for contributing bacterial infection to both humans and animals. The whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS has recently been applied as a rapid, accurate, and high-throughput tool for clinical diagnosis and microbiological research. In this present study, we employed a whole-cell MALDI-TOF MS approach for assessing its potency in clustering a total of 11 different B. pseudomallei isolates (consisting of 5 environmental and 6 clinical isolates with respect to their origins and to further investigate the source-identifying biomarker ions belonging to each bacterial group. The cluster analysis demonstrated that six out of eleven isolates were grouped correctly to their sources. Our results revealed a total of ten source-identifying biomarker ions, which exhibited statistically significant differences in peak intensity between average environmental and clinical mass spectra using ClinProTools software. Six out of ten mass ions were assigned as environmental-identifying biomarker ions (EIBIs, including, m/z 4,056, 4,214, 5,814, 7,545, 7,895, and 8,112, whereas the remaining four mass ions were defined as clinical-identifying biomarker ions (CIBIs consisting of m/z 3,658, 6,322, 7,035, and 7,984. Hence, our findings represented, for the first time, the source-specific biomarkers of environmental and clinical B. pseudomallei.

  4. Landscape changes influence the occurrence of the melioidosis bacterium Burkholderia pseudomallei in soil in northern Australia.

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    Mirjam Kaestli

    Full Text Available BACKGROUND: The soil-dwelling saprophyte bacterium Burkholderia pseudomallei is the cause of melioidosis, a severe disease of humans and animals in southeast Asia and northern Australia. Despite the detection of B. pseudomallei in various soil and water samples from endemic areas, the environmental habitat of B. pseudomallei remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: We performed a large survey in the Darwin area in tropical Australia and screened 809 soil samples for the presence of these bacteria. B. pseudomallei were detected by using a recently developed and validated protocol involving soil DNA extraction and real-time PCR targeting the B. pseudomallei-specific Type III Secretion System TTS1 gene cluster. Statistical analyses such as multivariable cluster logistic regression and principal component analysis were performed to assess the association of B. pseudomallei with environmental factors. The combination of factors describing the habitat of B. pseudomallei differed between undisturbed sites and environmentally manipulated areas. At undisturbed sites, the occurrence of B. pseudomallei was found to be significantly associated with areas rich in grasses, whereas at environmentally disturbed sites, B. pseudomallei was associated with the presence of livestock animals, lower soil pH and different combinations of soil texture and colour. CONCLUSIONS/SIGNIFICANCE: This study contributes to the elucidation of environmental factors influencing the occurrence of B. pseudomallei and raises concerns that B. pseudomallei may spread due to changes in land use.

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

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    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.

  6. Molecular phylogeny of Burkholderia pseudomallei from a remote region of Papua New Guinea.

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    Anthony Baker

    Full Text Available BACKGROUND: The island of New Guinea is located midway between the world's two major melioidosis endemic regions of Australia and Southeast Asia. Previous studies in Papua New Guinea have demonstrated autochthonous melioidosis in Balimo, Western province. In contrast to other regions of endemicity, isolates recovered from both environmental and clinical sources demonstrate narrow genetic diversity over large spatial and temporal scales. METHODOLOGY/PRINCIPAL FINDINGS: We employed molecular typing techniques to determine the phylogenetic relationships of these isolates to each other and to others worldwide to aid in understanding the origins of the Papua New Guinean isolates. Multi-locus sequence typing of the 39 isolates resolved three unique sequence types. Phylogenetic reconstruction and Structure analysis determined that all isolates were genetically closer to those from Australia than those from Southeast Asia. Gene cluster analysis however, identified a Yersinia-like fimbrial gene cluster predominantly found among Burkholderia pseudomallei derived from Southeast Asia. Higher resolution VNTR typing and phylogenetic reconstruction of the Balimo isolates resolved 24 genotypes with long branch lengths. These findings are congruent with long term persistence in the region and a high level of environmental stability. CONCLUSIONS/SIGNIFICANCE: Given that anthropogenic influence has been hypothesized as a mechanism for the dispersal of B. pseudomallei, these findings correlate with limited movement of the indigenous people in the region. The palaeogeographical and anthropogenic history of Australasia and the results from this study indicate that New Guinea is an important region for the further study of B. pseudomallei origins and dissemination.

  7. What drives the occurrence of the melioidosis bacterium Burkholderia pseudomallei in domestic gardens?

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    Mirjam Kaestli

    2015-03-01

    Full Text Available Melioidosis is an often fatal infectious disease affecting humans and animals in tropical regions and is caused by the saprophytic environmental bacterium Burkholderia pseudomallei. Domestic gardens are not only a common source of exposure to soil and thus to B. pseudomallei, but they also have been found to contain more B. pseudomallei than other environments. In this study we addressed whether anthropogenic manipulations common to gardens such as irrigation or fertilizers change the occurrence of B. pseudomallei. We conducted a soil microcosm experiment with a range of fertilizers and soil types as well as a longitudinal interventional study over three years on an experimental fertilized field site in an area naturally positive for B. pseudomallei. Irrigation was the only consistent treatment to increase B. pseudomallei occurrence over time. The effects of fertilizers upon these bacteria depended on soil texture, physicochemical soil properties and biotic factors. Nitrates and urea increased B. pseudomallei load in sand while phosphates had a positive effect in clay. The high buffering and cation exchange capacities of organic material found in a commercial potting mix led to a marked increase in soil salinity with no survival of B. pseudomallei after four weeks in the potting mix sampled. Imported grasses were also associated with B. pseudomallei occurrence in a multivariate model. With increasing population density in endemic areas these findings inform the identification of areas in the anthropogenic environment with increased risk of exposure to B. pseudomallei.

  8. What drives the occurrence of the melioidosis bacterium Burkholderia pseudomallei in domestic gardens?

    Science.gov (United States)

    Kaestli, Mirjam; Harrington, Glenda; Mayo, Mark; Chatfield, Mark D; Harrington, Ian; Hill, Audrey; Munksgaard, Niels; Gibb, Karen; Currie, Bart J

    2015-03-01

    Melioidosis is an often fatal infectious disease affecting humans and animals in tropical regions and is caused by the saprophytic environmental bacterium Burkholderia pseudomallei. Domestic gardens are not only a common source of exposure to soil and thus to B. pseudomallei, but they also have been found to contain more B. pseudomallei than other environments. In this study we addressed whether anthropogenic manipulations common to gardens such as irrigation or fertilizers change the occurrence of B. pseudomallei. We conducted a soil microcosm experiment with a range of fertilizers and soil types as well as a longitudinal interventional study over three years on an experimental fertilized field site in an area naturally positive for B. pseudomallei. Irrigation was the only consistent treatment to increase B. pseudomallei occurrence over time. The effects of fertilizers upon these bacteria depended on soil texture, physicochemical soil properties and biotic factors. Nitrates and urea increased B. pseudomallei load in sand while phosphates had a positive effect in clay. The high buffering and cation exchange capacities of organic material found in a commercial potting mix led to a marked increase in soil salinity with no survival of B. pseudomallei after four weeks in the potting mix sampled. Imported grasses were also associated with B. pseudomallei occurrence in a multivariate model. With increasing population density in endemic areas these findings inform the identification of areas in the anthropogenic environment with increased risk of exposure to B. pseudomallei.

  9. 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.

  10. 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.

  11. 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

  12. Analysis of the prevalence, secretion and function of a cell cycle-inhibiting factor in the melioidosis pathogen Burkholderia pseudomallei.

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    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.

  13. Analysis of the prevalence, secretion and function of a cell cycle-inhibiting factor in the melioidosis pathogen Burkholderia pseudomallei.

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    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.

  14. Environmental Free-Living Amoebae Isolated from Soil in Khon Kaen, Thailand, Antagonize Burkholderia pseudomallei

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    Noinarin, Parumon; Chareonsudjai, Pisit; Wangsomnuk, Pinich; Wongratanacheewin, Surasak

    2016-01-01

    Presence of Burkholderia pseudomallei in soil and water is correlated with endemicity of melioidosis in Southeast Asia and northern Australia. Several biological and physico-chemical factors have been shown to influence persistence of B. pseudomallei in the environment of endemic areas. This study was the first to evaluate the interaction of B. pseudomallei with soil amoebae isolated from B. pseudomallei-positive soil site in Khon Kaen, Thailand. Four species of amoebae, Paravahlkampfia ustiana, Acanthamoeba sp., Naegleria pagei, and isolate A-ST39-E1, were isolated, cultured and identified based on morphology, movement and 18S rRNA gene sequence. Co-cultivation combined with a kanamycin-protection assay of B. pseudomallei with these amoebae at MOI 20 at 30°C were evaluated during 0–6 h using the plate count technique on Ashdown’s agar. The fate of intracellular B. pseudomallei in these amoebae was also monitored by confocal laser scanning microscopy (CLSM) observation of the CellTracker™ Orange-B. pseudomallei stained cells. The results demonstrated the ability of P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 to graze B. pseudomallei. However, the number of internalized B. pseudomallei substantially decreased and the bacterial cells disappeared during the observation period, suggesting they had been digested. We found that B. pseudomallei promoted the growth of Acanthamoeba sp. and isolate A-ST39-E1 in co-cultures at MOI 100 at 30°C, 24 h. These findings indicated that P. ustiana, Acanthamoeba sp. and isolate A-ST39-E1 may prey upon B. pseudomallei rather than representing potential environmental reservoirs in which the bacteria can persist. PMID:27898739

  15. 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.

  16. 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

  17. 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.

  18. Systematic review and consensus guidelines for environmental sampling of Burkholderia pseudomallei.

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    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.

  19. Burkholderia Pseudomallei is genetically diverse in agricultural land in Northeast Thailand.

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    Vanaporn Wuthiekanun

    Full Text Available BACKGROUND: The soil-dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis. Extreme structuring of genotype and genotypic frequency has been demonstrated for B. pseudomallei in uncultivated land, but its distribution and genetic diversity in agricultural land where most human infections are probably acquired is not well defined. METHODS: Fixed-interval soil sampling was performed in a rice paddy in northeast Thailand in which 100 grams of soil was sampled at a depth of 30 cm from 10x10 sampling points each measuring 2.5 m by 2.5 m. Soil was cultured for the presence of B. pseudomallei and genotyping of colonies present on primary culture plates was performed using a combination of pulsed-field gel electrophoresis (PFGE and multilocus sequence typing (MLST. PRINCIPAL FINDINGS: B. pseudomallei was cultured from 28/100 samples. Genotyping of 630 primary colonies drawn from 11 sampling points demonstrated 10 PFGE banding pattern types, which on MLST were resolved into 7 sequence types (ST. Overlap of genotypes was observed more often between sampling points that were closely positioned. Two sampling points contained mixed B. pseudomallei genotypes, each with a numerically dominant genotype and one or more additional genotypes present as minority populations. CONCLUSIONS: Genetic diversity and structuring of B. pseudomallei exists despite the effects of flooding and the physical and chemical processes associated with farming. These findings form an important baseline for future studies of environmental B. pseudomallei.

  20. Land use and soil type determine the presence of the pathogen Burkholderia pseudomallei in tropical rivers.

    Science.gov (United States)

    Ribolzi, Olivier; Rochelle-Newall, Emma; Dittrich, Sabine; Auda, Yves; Newton, Paul N; Rattanavong, Sayaphet; Knappik, Michael; Soulileuth, Bounsamai; Sengtaheuanghoung, Oloth; Dance, David A B; Pierret, Alain

    2016-04-01

    Burkholderia pseudomallei is the bacterium that causes melioidosis in humans. While B. pseudomallei is known to be endemic in South East Asia (SEA), the occurrence of the disease in other parts of the tropics points towards a potentially large global distribution. We investigated the environmental factors that influence the presence (and absence) of B. pseudomallei in a tropical watershed in SEA. Our main objective was to determine whether there is a link between the presence of the organism in the hydrographic network and the upstream soil and land-use type. The presence of B. pseudomallei was determined using a specific quantitative real-time PCR assay following enrichment culture. Land use, soil, geomorphology, and environmental data were then analyzed using partial least squares discriminant analysis (PLSDA) to compare the B. pseudomallei positive and negative sites. Soil type in the surrounding catchment and turbidity had a strong positive influence on the presence (acrisols and luvisols) or absence (ferralsols) of B. pseudomallei. Given the strong apparent links between soil characteristics, water turbidity, and the presence/absence of B. pseudomallei, actions to raise public awareness about factors increasing the risk of exposure should be undertaken in order to reduce the incidence of melioidosis in regions of endemicity.

  1. Drug susceptibility and biofilm formation of Burkholderia pseudomallei in nutrient-limited condition.

    Science.gov (United States)

    Anutrakunchai, C; Sermswan, R W; Wongratanacheewin, S; Puknun, A; Taweechaisupapong, S

    2015-06-01

    Burkholderia pseudomallei is the causative agent of melioidosis, which can form biofilms and microcolonies in vivo and in vitro. One of the hallmark characteristics of the biofilm-forming bacteria is that they can be up to 1,000 times more resistant to antibiotics than their free-living counterpart. Bacteria also become highly tolerant to antibiotics when nutrients are limited. One of the most important causes of starvation induced tolerance in vivo is biofilm growth. However, the effect of nutritional stress on biofilm formation and drug tolerance of B. pseudomallei has never been reported. Therefore, this study aims to determine the effect of nutrient-limited and enriched conditions on drug susceptibility of B. pseudomallei in both planktonic and biofilm forms in vitro using broth microdilution method and Calgary biofilm device, respectively. The biofilm formation of B. pseudomallei in nutrient-limited and enriched conditions was also evaluated by a modified microtiter-plate test. Six isolates of ceftazidime (CAZ)-susceptible and four isolates of CAZ-resistant B. pseudomallei were used. The results showed that the minimum bactericidal concentrations of CAZ against B. pseudomallei in nutrient-limited condition were higher than those in enriched condition. The drug susceptibilities of B. pseudomallei biofilm in both enriched and nutrient-limited conditions were more tolerant than those of planktonic cells. Moreover, the quantification of biofilm formation by B. pseudomallei in nutrient-limited condition was significantly higher than that in enriched condition. These data indicate that nutrient-limited condition could induce biofilm formation and drug tolerance of B. pseudomallei.

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

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    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. 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.

  4. 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

  5. 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

  6. 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.

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

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    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.

  8. A predicted structure of the cytochrome c oxidase from Burkholderia pseudomallei

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    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...

  9. Evaluation of six commercial DNA extraction kits for recovery of Burkholderia pseudomallei DNA.

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    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.

  10. Out of the ground: aerial and exotic habitats of the melioidosis bacterium Burkholderia pseudomallei in grasses in Australia.

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    Kaestli, Mirjam; Schmid, Michael; Mayo, Mark; Rothballer, Michael; Harrington, Glenda; Richardson, Leisha; Hill, Audrey; Hill, Jason; Tuanyok, Apichai; Keim, Paul; Hartmann, Anton; Currie, Bart J

    2012-08-01

    Melioidosis is an emerging infectious disease of humans and animals in the tropics caused by the soil bacterium Burkholderia pseudomallei. Despite high fatality rates, the ecology of B.pseudomallei remains unclear. We used a combination of field and laboratory studies to investigate B.pseudomallei colonization of native and exotic grasses in northern Australia. Multivariable and spatial analyses were performed to determine significant predictors for B.pseudomallei occurrence in plants and soil collected longitudinally from field sites. In plant inoculation experiments, the impact of B.pseudomallei upon these grasses was studied and the bacterial load semi-quantified. Fluorescence in situ hybridization and confocal laser scanning microscopy were performed to localize the bacteria in plants. Burkholderia pseudomallei was found to inhabit not only the rhizosphere and roots but also aerial parts of specific grasses. This raises questions about the potential spread of B.pseudomallei by grazing animals whose droppings were found to be positive for these bacteria. In particular, B.pseudomallei readily colonized exotic grasses introduced to Australia for pasture. The ongoing spread of these introduced grasses creates new habitats suitable for B.pseudomallei survival and may be an important factor in the evolving epidemiology of melioidosis seen both in northern Australia and elsewhere globally.

  11. 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

  12. Rapid and Sensitive Multiplex Detection of Burkholderia pseudomallei-Specific Antibodies in Melioidosis Patients Based on a Protein Microarray Approach.

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    Christian Kohler

    2016-07-01

    Full Text Available The environmental bacterium Burkholderia pseudomallei causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against B. pseudomallei, including the reference indirect haemagglutination assay (IHA, lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against B. pseudomallei is still lacking.In this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified B. pseudomallei proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001. Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response.Our protein array provides a standardized, rapid, easy-to-perform test for the detection of B. pseudomallei-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our high-throughput assay might be useful

  13. Phenotypic and functional characterization of human memory T cell responses to Burkholderia pseudomallei.

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    Patcharaporn Tippayawat

    Full Text Available BACKGROUND: Infection with the Gram-negative bacterium Burkholderia pseudomallei is an important cause of community-acquired lethal sepsis in endemic regions in southeast Asia and northern Australia and is increasingly reported in other tropical areas. In animal models, production of interferon-gamma (IFN-gamma is critical for resistance, but in humans the characteristics of IFN-gamma production and the bacterial antigens that are recognized by the cell-mediated immune response have not been defined. METHODS: Peripheral blood from 133 healthy individuals who lived in the endemic area and had no history of melioidosis, 60 patients who had recovered from melioidosis, and 31 other patient control subjects were stimulated by whole bacteria or purified bacterial proteins in vitro, and IFN-gamma responses were analyzed by ELISPOT and flow cytometry. FINDINGS: B. pseudomallei was a potent activator of human peripheral blood NK cells for innate production of IFN-gamma. In addition, healthy individuals with serological evidence of exposure to B. pseudomallei and patients recovered from active melioidosis developed CD4(+ (and CD8(+ T cells that recognized whole bacteria and purified proteins LolC, OppA, and PotF, members of the B. pseudomallei ABC transporter family. This response was primarily mediated by terminally differentiated T cells of the effector-memory (T(EMRA phenotype and correlated with the titer of anti-B. pseudomallei antibodies in the serum. CONCLUSIONS: Individuals living in a melioidosis-endemic region show clear evidence of T cell priming for the ability to make IFN-gamma that correlates with their serological status. The ability to detect T cell responses to defined B. pseudomallei proteins in large numbers of individuals now provides the opportunity to screen candidate antigens for inclusion in protein or polysaccharide-conjugate subunit vaccines against this important but neglected disease.

  14. Antimicrobial Susceptibility and Genetic Characterisation of Burkholderia pseudomallei Isolated from Malaysian Patients

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    Yalda Khosravi

    2014-01-01

    Full Text Available Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics. Ceftazidime (CAZ, the synthetic β-lactam, is normally used as the first-line antibiotic therapy for treatment of melioidosis. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, leading to mortality if therapy is not switched to a different antibiotic(s in a timely manner. In this study, susceptibilities of 81 B. pseudomallei isolates to nine different antimicrobial agents were determined using the disk diffusion method, broth microdilution test and Etest. Highest percentage of susceptibility was demonstrated to CAZ, amoxicillin/clavulanic acid, meropenem, imipenem, and trimethoprim/sulfamethoxazole. Although these drugs demonstrated the highest percentage of susceptibility in B. pseudomallei, the overall results underline the importance of the emergence of resistance in this organism. PCR results showed that, of the 81 B. pseudomallei, six multidrug resistant (MDR isolates carried bpeB, amrB, and BPSS1119 and penA genes. Genotyping of the isolates using random amplified polymorphic DNA analysis showed six different PCR fingerprinting patterns generated from the six MDR isolates clusters (A and eight PCR fingerprinting patterns generated for the remaining 75 non-MDR isolates clusters (B.

  15. Environmental Attributes Influencing the Distribution of Burkholderia pseudomallei in Northern Australia.

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    Anthony L Baker

    Full Text Available Factors responsible for the spatial and temporal clustering of Burkholderia pseudomallei in the environment remain to be elucidated. Whilst laboratory based experiments have been performed to analyse survival of the organism in various soil types, such approaches are strongly influenced by alterations to the soil micro ecology during soil sanitisation and translocation. During the monsoonal season in Townsville, Australia, B. pseudomallei is discharged from Castle Hill (an area with a very high soil prevalence of the organism by groundwater seeps and is washed through a nearby area where intensive sampling in the dry season has been unable to detect the organism. We undertook environmental sampling and soil and plant characterisation in both areas to ascertain physiochemical and macro-floral differences between the two sites that may affect the prevalence of B. pseudomallei. In contrast to previous studies, the presence of B. pseudomallei was correlated with a low gravimetric water content and low nutrient availability (nitrogen and sulphur and higher exchangeable potassium in soils favouring recovery. Relatively low levels of copper, iron and zinc favoured survival. The prevalence of the organism was found to be highest under the grasses Aristida sp. and Heteropogon contortus and to a lesser extent under Melinis repens. The findings of this study indicate that a greater variety of factors influence the endemicity of melioidosis than has previously been reported, and suggest that biogeographical boundaries to the organisms' distribution involve complex interactions.

  16. Screening for potential anti-infective agents towards Burkholderia pseudomallei infection

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    Eng, Su Anne; Nathan, Sheila

    2014-09-01

    The established treatment for melioidosis is antibiotic therapy. However, a constant threat to this form of treatment is resistance development of the causative agent, Burkholderia pseudomallei, towards antibiotics. One option to circumvent this threat of antibiotic resistance is to search for new alternative anti-infectives which target the host innate immune system and/or bacterial virulence. In this study, 29 synthetic compounds were evaluated for their potential to increase the lifespan of an infected host. The nematode Caenorhabditis elegans was adopted as the infection model as its innate immune pathways are homologous to humans. Screens were performed in a liquid-based survival assay containing infected worms exposed to individual compounds and survival of untreated and compound-treated worms were compared. A primary screen identified nine synthetic compounds that extended the lifespan of B. pseudomallei-infected worms. Subsequently, a disc diffusion test was performed on these selected compounds to delineate compounds into those that enhanced the survival of worms via antimicrobial activity i.e. reducing the number of infecting bacteria, or into those that did not target pathogen viability. Out of the nine hits selected, two demonstrated antimicrobial effects on B. pseudomallei. Therefore, the findings from this study suggest that the other seven identified compounds are potential anti-infectives which could protect a host against B. pseudomallei infection without developing the risk of drug resistance.

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

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    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.

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

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    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.

  19. 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.

  20. Altered Proteome of Burkholderia pseudomallei Colony Variants Induced by Exposure to Human Lung Epithelial Cells.

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    Anis Rageh Al-Maleki

    Full Text Available Burkholderia pseudomallei primary diagnostic cultures demonstrate colony morphology variation associated with expression of virulence and adaptation proteins. This study aims to examine the ability of B. pseudomallei colony variants (wild type [WT] and small colony variant [SCV] to survive and replicate intracellularly in A549 cells and to identify the alterations in the protein expression of these variants, post-exposure to the A549 cells. Intracellular survival and cytotoxicity assays were performed followed by proteomics analysis using two-dimensional gel electrophoresis. B. pseudomallei SCV survive longer than the WT. During post-exposure, among 259 and 260 protein spots of SCV and WT, respectively, 19 were differentially expressed. Among SCV post-exposure up-regulated proteins, glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase (CbbA and betaine aldehyde dehydrogenase were associated with adhesion and virulence. Among the down-regulated proteins, enolase (Eno is implicated in adhesion and virulence. Additionally, post-exposure expression profiles of both variants were compared with pre-exposure. In WT pre- vs post-exposure, 36 proteins were differentially expressed. Of the up-regulated proteins, translocator protein, Eno, nucleoside diphosphate kinase (Ndk, ferritin Dps-family DNA binding protein and peptidyl-prolyl cis-trans isomerase B were implicated in invasion and virulence. In SCV pre- vs post-exposure, 27 proteins were differentially expressed. Among the up-regulated proteins, flagellin, Eno, CbbA, Ndk and phenylacetate-coenzyme A ligase have similarly been implicated in adhesion, invasion. Protein profiles differences post-exposure provide insights into association between morphotypic and phenotypic characteristics of colony variants, strengthening the role of B. pseudomallei morphotypes in pathogenesis of melioidosis.

  1. Nematode Peptides with Host-Directed Anti-inflammatory Activity Rescue Caenorhabditis elegans from a Burkholderia pseudomallei Infection

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    Lim, Mei-Perng; Firdaus-Raih, Mohd; Nathan, Sheila

    2016-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is among a growing number of bacterial pathogens that are increasingly antibiotic resistant. Antimicrobial peptides (AMPs) have been investigated as an alternative approach to treat microbial infections, as generally, there is a lower likelihood that a pathogen will develop resistance to AMPs. In this study, 36 candidate Caenorhabditis elegans genes that encode secreted peptides of nematodes. RNA interference (RNAi)-based knockdown of 12/34 peptide-encoding genes resulted in enhanced nematode susceptibility to B. pseudomallei without affecting worm fitness. A microdilution test demonstrated that two peptides, NLP-31 and Y43C5A.3, exhibited anti-B. pseudomallei activity in a dose dependent manner on different pathogens. Time kill analysis proposed that these peptides were bacteriostatic against B. pseudomallei at concentrations up to 8× MIC90. The SYTOX green assay demonstrated that NLP-31 and Y43C5A.3 did not disrupt the B. pseudomallei membrane. Instead, gel retardation assays revealed that both peptides were able to bind to DNA and interfere with bacterial viability. In parallel, microscopic examination showed induction of cellular filamentation, a hallmark of DNA synthesis inhibition, of NLP-31 and Y43C5A.3 treated cells. In addition, the peptides also regulated the expression of inflammatory cytokines in B. pseudomallei infected macrophage cells. Collectively, these findings demonstrate the potential of NLP-31 and Y43C5A.3 as anti-B. pseudomallei peptides based on their function as immune modulators. PMID:27672387

  2. Global transcriptional profiling of Burkholderia pseudomallei under salt stress reveals differential effects on the Bsa type III secretion system

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    Singsuksawat Ekapot

    2010-06-01

    Full Text Available Abstract Background Burkholderia pseudomallei is the causative agent of melioidosis where the highest reported incidence world wide is in the Northeast of Thailand, where saline soil and water are prevalent. Moreover, recent reports indicate a potential pathogenic role for B. pseudomallei in cystic fibrosis lung disease, where an increased sodium chloride (NaCl concentration in airway surface liquid has been proposed. These observations raise the possibility that high salinity may represent a favorable niche for B. pseudomallei. We therefore investigated the global transcriptional response of B. pseudomallei to increased salinity using microarray analysis. Results Transcriptome analysis of B. pseudomallei under salt stress revealed several genes significantly up-regulated in the presence of 320 mM NaCl including genes associated with the bsa-derived Type III secretion system (T3SS. Microarray data were verified by reverse transcriptase-polymerase chain reactions (RT-PCR. Western blot analysis confirmed the increased expression and secretion of the invasion-associated type III secreted proteins BipD and BopE in B. pseudomallei cultures at 170 and 320 mM NaCl relative to salt-free medium. Furthermore, salt-treated B. pseudomallei exhibited greater invasion efficiency into the lung epithelial cell line A549 in a manner partly dependent on a functional Bsa system. Conclusions B. pseudomallei responds to salt stress by modulating the transcription of a relatively small set of genes, among which is the bsa locus associated with invasion and virulence. Expression and secretion of Bsa-secreted proteins was elevated in the presence of exogenous salt and the invasion efficiency was enhanced. Our data indicate that salinity has the potential to influence the virulence of B. pseudomallei.

  3. 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.

  4. The Madagascar hissing cockroach as a novel surrogate host for Burkholderia pseudomallei, B. mallei and B. thailandensis

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    Fisher Nathan A

    2012-06-01

    Full Text Available Abstract Background Burkholderia pseudomallei and Burkholderia mallei are gram-negative pathogens responsible for the diseases melioidosis and glanders, respectively. Both species cause disease in humans and animals and have been designated as category B select agents by the Centers for Disease Control and Prevention (CDC. Burkholderia thailandensis is a closely related bacterium that is generally considered avirulent for humans. While it can cause disease in rodents, the B. thailandensis 50% lethal dose (LD50 is typically ≥ 104-fold higher than the B. pseudomallei and B. mallei LD50 in mammalian models of infection. Here we describe an alternative to mammalian hosts in the study of virulence and host-pathogen interactions of these Burkholderia species. Results Madagascar hissing cockroaches (MH cockroaches possess a number of qualities that make them desirable for use as a surrogate host, including ease of breeding, ease of handling, a competent innate immune system, and the ability to survive at 37°C. MH cockroaches were highly susceptible to infection with B. pseudomallei, B. mallei and B. thailandensis and the LD50 was 50 for Escherichia coli in MH cockroaches was >105 cfu. B. pseudomallei, B. mallei, and B. thailandensis cluster 1 type VI secretion system (T6SS-1 mutants were all attenuated in MH cockroaches, which is consistent with previous virulence studies conducted in rodents. B. pseudomallei mutants deficient in the other five T6SS gene clusters, T6SS-2 through T6SS-6, were virulent in both MH cockroaches and hamsters. Hemocytes obtained from MH cockroaches infected with B. pseudomallei harbored numerous intracellular bacteria, suggesting that this facultative intracellular pathogen can survive and replicate inside of MH cockroach phagocytic cells. The hemolymph extracted from these MH cockroaches also contained multinuclear giant cells (MNGCs with intracellular B. pseudomallei, which indicates that infected hemocytes can

  5. Osteopontin impairs host defense during established gram-negative sepsis caused by Burkholderia pseudomallei (melioidosis.

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    Gerritje J W van der Windt

    Full Text Available BACKGROUND: Melioidosis, caused by infection with Burkholderia (B. pseudomallei, is a severe illness that is endemic in Southeast Asia. Osteopontin (OPN is a phosphorylated glycoprotein that is involved in several immune responses including induction of T-helper 1 cytokines and recruitment of inflammatory cells. METHODOLOGY AND PRINCIPAL FINDINGS: OPN levels were determined in plasma from 33 melioidosis patients and 31 healthy controls, and in wild-type (WT mice intranasally infected with B. pseudomallei. OPN function was studied in experimental murine melioidosis using WT and OPN knockout (KO mice. Plasma OPN levels were elevated in patients with severe melioidosis, even more so in patients who went on to die. In patients who recovered plasma OPN concentrations had decreased after treatment. In experimental melioidosis in mice plasma and pulmonary OPN levels were also increased. Whereas WT and OPN KO mice were indistinguishable during the first 24 hours after infection, after 72 hours OPN KO mice demonstrated reduced bacterial numbers in their lungs, diminished pulmonary tissue injury, especially due to less necrosis, and decreased neutrophil infiltration. Moreover, OPN KO mice displayed a delayed mortality as compared to WT mice. OPN deficiency did not influence the induction of proinflammatory cytokines. CONCLUSIONS: These data suggest that sustained production of OPN impairs host defense during established septic melioidosis.

  6. 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.

  7. Environmental Survival, Military Relevance, and Persistence of Burkholderia Pseudomallei

    Science.gov (United States)

    2007-04-01

    septicaemic melioidosis (White el al., 1989). More recently, carbapenems have been found to be at least as effective and may have superior intracellular...whether the carbapenems are superior to ceftazidime in terms of clinical outcomes. Investigators think that a combination of ceftazidime and co

  8. 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.

  9. Detection of Burkholderia pseudomallei toxin-mediated inhibition of protein synthesis using a Caenorhabditis elegans ugt–29 biosensor

    Science.gov (United States)

    Wong, Rui-Rui; Kong, Cin; Lee, Song-Hua; Nathan, Sheila

    2016-01-01

    Toxins are believed to play a crucial role in Burkholderia pseudomallei pathogenicity, however to date, only a few have been identified. The discovery of additional toxic molecules is limited by the lack of a sensitive indicator of B. pseudomallei toxicity. Previously, from a whole genome transcriptome analysis of B. pseudomallei-infected Caenorhabditis elegans, we noted significant overexpression of a number of worm genes encoding detoxification enzymes, indicating the host’s attempt to clear bacterial toxic molecules. One of these genes, ugt–29, a family member of UDP-glucuronosyltransferases, was the most robustly induced phase II detoxification gene. In this study, we show that strong induction of ugt–29 is restricted to infections by the most virulent species among the pathogens tested. We also noted that ugt–29 is activated upon disruption of host protein synthesis. Hence, we propose that UGT–29 could be a promising biosensor to detect B. pseudomallei toxins that compromise host protein synthesis. The identification of bactobolin, a polyketide-peptide hybrid molecule, as a toxic molecule of B. pseudomallei further verifies the utilization of this surveillance system to search for bacterial toxins. Hence, a ugt–29 based reporter should be useful in screening for other molecules that inhibit host protein synthesis. PMID:27273550

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

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    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

  11. 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

  12. Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif)

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    Ng, Mei Ying; Wang, Mei; Casey, Patrick J.; Gan, Yunn-Hwen; Hagen, Thilo

    2017-01-01

    Cycle inhibiting factors (Cifs) are virulence proteins secreted by the type III secretion system of some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Cif is known to function to deamidate Nedd8, leading to inhibition of Cullin E3 ubiquitin ligases (CRL) and consequently induction of cell cycle arrest. Here we show that Cif can function as a potent activator of MAPK/ERK signaling without significant activation of other signaling pathways downstream of receptor tyrosine kinases. Importantly, we found that the ability of Cif to activate ERK is dependent on its deamidase activity, but independent of Cullin E3 ligase inhibition. This suggests that apart from Nedd8, other cellular targets of Cif-dependent deamidation exist. We provide evidence that the mechanism involved in Cif-mediated ERK activation is dependent on recruitment of the Grb2-SOS1 complex to the plasma membrane. Further investigation revealed that Cif appears to modify the phosphorylation status of SOS1 in a region containing the CDC25-H and proline-rich domains. It is known that prolonged Cullin E3 ligase inhibition leads to cellular apoptosis. Therefore, we hypothesize that ERK activation is an important mechanism to counter the pro-apoptotic effects of Cif. Indeed, we show that Cif dependent ERK activation promotes phosphorylation of the proapoptotic protein Bim, thereby potentially conferring a pro-survival signal. In summary, we identified a novel deamidation-dependent mechanism of action of the B. pseudomallei virulence factor Cif/CHBP to activate MAPK/ERK signaling. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian signaling pathways. PMID:28166272

  13. 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.

  14. 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.

  15. BurkDiff: a real-time PCR allelic discrimination assay for Burkholderia pseudomallei and B. mallei.

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    Jolene R Bowers

    Full Text Available A real-time PCR assay, BurkDiff, was designed to target a unique conserved region in the B. pseudomallei and B. mallei genomes containing a SNP that differentiates the two species. Sensitivity and specificity were assessed by screening BurkDiff across 469 isolates of B. pseudomallei, 49 isolates of B. mallei, and 390 isolates of clinically relevant non-target species. Concordance of results with traditional speciation methods and no cross-reactivity to non-target species show BurkDiff is a robust, highly validated assay for the detection and differentiation of B. pseudomallei and B. mallei.

  16. The Burkholderia pseudomallei type III secretion system and BopA are required for evasion of LC3-associated phagocytosis.

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    Lan Gong

    Full Text Available Burkholderia pseudomallei is the causative agent of melioidosis, a fatal infectious disease endemic in tropical regions worldwide, and especially prevalent in southeast Asia and northern Australia. This intracellular pathogen can escape from phagosomes into the host cytoplasm, where it replicates and infects adjacent cells. We previously demonstrated that, in response to B. pseudomallei infection of macrophage cell line RAW 264.7, a subset of bacteria co-localized with the autophagy marker protein, microtubule-associated protein light chain 3 (LC3, implicating autophagy in host cell defence against infection. Recent reports have suggested that LC3 can be recruited to both phagosomes and autophagosomes, thereby raising questions regarding the identity of the LC3-positive compartments in which invading bacteria reside and the mechanism of the autophagic response to B. pseudomallei infection. Electron microscopy analysis of infected cells demonstrated that the invading bacteria were either free in the cytosol, or sequestered in single-membrane phagosomes rather than double-membrane autophagosomes, suggesting that LC3 is recruited to B. pseudomallei-containing phagosomes. Partial or complete loss of function of type III secretion system cluster 3 (TTSS3 in mutants lacking the BopA (effector or BipD (translocator proteins respectively, resulted in delayed or no escape from phagosomes. Consistent with these observations, bopA and bipD mutants both showed a higher level of co-localization with LC3 and the lysosomal marker LAMP1, and impaired survival in RAW264.7 cells, suggesting enhanced killing in phagolysosomes. We conclude that LC3 recruitment to phagosomes stimulates killing of B. pseudomallei trapped in phagosomes. Furthermore, BopA plays an important role in efficient escape of B. pseudomallei from phagosomes.

  17. 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.

  18. Crystal structures of Cif from bacterial pathogens Photorhabdus luminescens and Burkholderia pseudomallei.

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    Allister Crow

    Full Text Available A pre-requisite for bacterial pathogenesis is the successful interaction of a pathogen with a host. One mechanism used by a broad range of Gram negative bacterial pathogens is to deliver effector proteins directly into host cells through a dedicated type III secretion system where they modulate host cell function. The cycle inhibiting factor (Cif family of effector proteins, identified in a growing number of pathogens that harbour functional type III secretion systems and have a wide host range, arrest the eukaryotic cell cycle. Here, the crystal structures of Cifs from the insect pathogen/nematode symbiont Photorhabdus luminescens (a gamma-proteobacterium and human pathogen Burkholderia pseudomallei (a beta-proteobacterium are presented. Both of these proteins adopt an overall fold similar to the papain sub-family of cysteine proteases, as originally identified in the structure of a truncated form of Cif from Enteropathogenic E. coli (EPEC, despite sharing only limited sequence identity. The structure of an N-terminal region, referred to here as the 'tail-domain' (absent in the EPEC Cif structure, suggests a surface likely to be involved in host-cell substrate recognition. The conformation of the Cys-His-Gln catalytic triad is retained, and the essential cysteine is exposed to solvent and addressable by small molecule reagents. These structures and biochemical work contribute to the rapidly expanding literature on Cifs, and direct further studies to better understand the molecular details of the activity of these proteins.

  19. Diversity of 16S-23S rDNA internal transcribed spacer (ITS reveals phylogenetic relationships in Burkholderia pseudomallei and its near-neighbors.

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    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.

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

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    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

  1. Comparison of DNA extraction kits for detection of Burkholderia pseudomallei in spiked human whole blood using real-time PCR.

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    Nicole L Podnecky

    Full Text Available Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1 assay. Crossing threshold (C T values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10(4 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.

  2. Genome-wide analysis reveals loci encoding anti-macrophage factors in the human pathogen Burkholderia pseudomallei K96243.

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    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.

  3. Capsule influences the deposition of critical complement C3 levels required for the killing of Burkholderia pseudomallei via NADPH-oxidase induction by human neutrophils.

    Science.gov (United States)

    Woodman, Michael E; Worth, Randall G; Wooten, R Mark

    2012-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD(50) via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo.

  4. Capsule influences the deposition of critical complement C3 levels required for the killing of Burkholderia pseudomallei via NADPH-oxidase induction by human neutrophils.

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    Michael E Woodman

    Full Text Available Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD(50 via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo.

  5. Crystallization and preliminary X-ray diffraction analysis of BipD, a virulence factor from Burkholderia pseudomallei

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    Knight, M. J.; Ruaux, A.; Mikolajek, H.; Erskine, P. T.; Gill, R.; Wood, S. P. [School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX (United Kingdom); Wood, M. [Institute of Animal Health, Division of Environmental Microbiology, Institute for Animal Health, Compton Laboratory, Berkshire RG20 7NN (United Kingdom); Cooper, J. B., E-mail: j.b.cooper@soton.ac.uk [School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX (United Kingdom)

    2006-08-01

    BipD is likely to be a component of a type-III protein secretion system (TTSS) in B. pseudomallei. Native and selenomethionyl-BipD proteins have been expressed and crystals have been obtained which diffract to 2.1 Å. Burkholderia pseudomallei, the causative agent of melioidosis, possesses a protein-secretion apparatus that is similar to those found in Salmonella and Shigella. A major function of these secretion systems is to secrete virulence-associated proteins into target cells of the host organism. The BipD gene of B. pseudomallei encodes a secreted virulence factor that is similar in sequence and most likely functionally analogous to IpaD from Shigella and SipD from Salmonella. Thus, the BipD protein is likely to be a component of a type III protein-secretion system (TTSS) in B. pseudomallei. Proteins in the same class as BipD, such as IpaD and SipD, are thought to act as extracellular chaperones to help the hydrophobic translocator proteins enter the target cell membrane, where they form a pore and might even link the translocon pore with the secretion needle. There is evidence that the translocator proteins also bind an integrin which stimulates actin-mediated insertion of the bacterium into the host-cell membrane. Native BipD has been crystallized in a monoclinic crystal form that diffracts X-rays to 2.5 Å resolution. BipD protein which incorporates selenomethionine (SeMet-BipD) has also been expressed and forms crystals which diffract to a higher resolution of 2.1 Å.

  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. BurkDiff: A Real-Time PCR Allelic Discrimination Assay for Burkholderia Pseudomallei and B. mallei

    OpenAIRE

    Jolene R Bowers; Engelthaler, David M.; Jennifer L Ginther; Talima Pearson; Peacock, Sharon J; Apichai Tuanyok; Wagner, David M.; Currie, Bart J.; Keim, Paul S.

    2010-01-01

    A real-time PCR assay, BurkDiff, was designed to target a unique conserved region in the B. pseudomallei and B. mallei genomes containing a SNP that differentiates the two species. Sensitivity and specificity were assessed by screening BurkDiff across 469 isolates of B. pseudomallei, 49 isolates of B. mallei, and 390 isolates of clinically relevant non-target species. Concordance of results with traditional speciation methods and no cross-reactivity to non-target species show BurkDiff is a ro...

  8. Toll-like receptor 2 impairs host defense in gram-negative sepsis caused by Burkholderia pseudomallei (Melioidosis.

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    W Joost Wiersinga

    2007-07-01

    Full Text Available BACKGROUND: Toll-like receptors (TLRs are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis. METHODS AND FINDINGS: Patient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2, TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury. CONCLUSIONS: Patients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in

  9. 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.

  10. 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.

  11. Production and characterization of chimeric monoclonal antibodies against Burkholderia pseudomallei and B. mallei using the DHFR expression system.

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    Hyung-Yong Kim

    Full Text Available Burkholderia pseudomallei (BP and B. mallei (BM are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7 were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65°C/90 min, sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1 reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM. The cMAb BP7 2C6 (cMAb CK2 recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14∼28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3 reacted with lipopolysaccharides (38∼52 kDa in BP; 38∼60 kDa in B. thailandensis. Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection.

  12. Characterization of pathogenesis of and immune response to Burkholderia pseudomallei K96243 using both inhalational and intraperitoneal infection models in BALB/c and C57BL/6 mice

    Science.gov (United States)

    Bearss, Jeremy J.; Hunter, Melissa; Dankmeyer, Jennifer L.; Fritts, Kristen A.; Klimko, Christopher P.; Weaver, Chris H.; Shoe, Jennifer L.; Quirk, Avery V.; Toothman, Ronald G.; Webster, Wendy M.; Fetterer, David P.; Bozue, Joel A.; Worsham, Patricia L.; Welkos, Susan L.; Amemiya, Kei; Cote, Christopher K.

    2017-01-01

    Burkholderia pseudomallei, the etiologic agent of melioidosis, is a Gram negative bacterium designated as a Tier 1 threat. This bacterium is known to be endemic in Southeast Asia and Northern Australia and can infect humans and animals by several routes. Inhalational melioidosis has been associated with monsoonal rains in endemic areas and is also a significant concern in the biodefense community. There are currently no effective vaccines for B. pseudomallei and antibiotic treatment can be hampered by non-specific symptomology and also the high rate of naturally occurring antibiotic resistant strains. Well-characterized animal models will be essential when selecting novel medical countermeasures for evaluation prior to human clinical trials. Here, we further characterize differences between the responses of BALB/c and C57BL/6 mice when challenged with low doses of a low-passage and well-defined stock of B. pseudomallei K96243 via either intraperitoneal or aerosol routes of exposure. Before challenge, mice were implanted with a transponder to collect body temperature readings, and daily body weights were also recorded. Mice were euthanized on select days for pathological analyses and determination of the bacterial burden in selected tissues (blood, lungs, liver, and spleen). Additionally, spleen homogenate and sera samples were analyzed to better characterize the host immune response after infection with aerosolized bacteria. These clinical, pathological, and immunological data highlighted and confirmed important similarities and differences between these murine models and exposure routes. PMID:28235018

  13. 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.

  14. Production of a Recombinant Vaccine Candidate against Burkholderia pseudomallei Exploiting the Bacterial N-Glycosylation Machinery

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    Fatima eGarcia-Quintanilla

    2014-07-01

    Full Text Available Vaccines developing immune responses towards surface carbohydrates conjugated to proteins are effective in preventing infection and death by bacterial pathogens. Traditional production of these vaccines utilizes complex synthetic chemistry to acquire and conjugate the glycan to a protein. However, glycoproteins produced by bacterial protein glycosylation systems are significantly easier to produce, and could possible be used as vaccine candidates. In this work we functionally expressed the B. pseudomallei O polysaccharide (OPS II, the C. jejuni oligosaccharyltransferase (OTase, and a suitable glycoprotein (AcrA in a designer E. coli strain with a higher efficiency for production of glycoconjugates. We were able to produce and purify the OPS II-AcrA glycoconjugate, and MS analysis confirmed correct glycan was produced and attached. We observed the attachment of the O-acetylated deoxyhexose directly to the acceptor protein, which expands the range of substrates utilized by the OTase PglB. Injection of the glycoprotein into mice generated an IgG immune response against B. pseudomallei, and this response was partially protective against an intranasal challenge. Our experiments show that bacterial engineered glycoconjugates can be utilized as vaccine candidates against B. pseudomallei. Additionally, our new E. coli strain SDB1 is more efficient in glycoprotein production, and could have additional applications in the future.

  15. Burkholderia pseudomallei Capsule Exacerbates Respiratory Melioidosis but Does Not Afford Protection against Antimicrobial Signaling or Bacterial Killing in Human Olfactory Ensheathing Cells.

    Science.gov (United States)

    Dando, Samantha J; Ipe, Deepak S; Batzloff, Michael; Sullivan, Matthew J; Crossman, David K; Crowley, Michael; Strong, Emily; Kyan, Stephanie; Leclercq, Sophie Y; Ekberg, Jenny A K; St John, James; Beacham, Ifor R; Ulett, Glen C

    2016-07-01

    Melioidosis, caused by the bacterium Burkholderia pseudomallei, is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery of B. pseudomallei to establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improved B. pseudomallei survival in vivo and triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response to B. pseudomallei infection, we infected human olfactory ensheathing cells (OECs) in vitro and measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of the B. pseudomallei in a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I in B. pseudomallei survival in vivo following inhalation infection and the antibacterial signaling network that exists in human OM and OECs.

  16. 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.

  17. A genomic survey of positive selection in Burkholderia pseudomallei provides insights into the evolution of accidental virulence.

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    Tannistha Nandi

    2010-04-01

    Full Text Available Certain environmental microorganisms can cause severe human infections, even in the absence of an obvious requirement for transition through an animal host for replication ("accidental virulence". To understand this process, we compared eleven isolate genomes of Burkholderia pseudomallei (Bp, a tropical soil microbe and causative agent of the human and animal disease melioidosis. We found evidence for the existence of several new genes in the Bp reference genome, identifying 282 novel genes supported by at least two independent lines of supporting evidence (mRNA transcripts, database homologs, and presence of ribosomal binding sites and 81 novel genes supported by all three lines. Within the Bp core genome, 211 genes exhibited significant levels of positive selection (4.5%, distributed across many cellular pathways including carbohydrate and secondary metabolism. Functional experiments revealed that certain positively selected genes might enhance mammalian virulence by interacting with host cellular pathways or utilizing host nutrients. Evolutionary modifications improving Bp environmental fitness may thus have indirectly facilitated the ability of Bp to colonize and survive in mammalian hosts. These findings improve our understanding of the pathogenesis of melioidosis, and establish Bp as a model system for studying the genetics of accidental virulence.

  18. Detection of Burkholderia pseudomallei in Sputum using Selective Enrichment Broth and Ashdown’s Medium at Kampong Cham Provincial Hospital, Cambodia [v1; ref status: indexed, http://f1000r.es/4w7

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    Somary Nhem

    2014-12-01

    Full Text Available Melioidosis infection, caused by Burkholderia pseudomallei, is increasingly reported in Cambodia. We hypothesized that implementation of an enhanced sputum testing protocol in a provincial hospital diagnostic microbiology laboratory would increase detection of B. pseudomallei. We tested 241 sputum specimens that were deemed acceptable for culture, comparing culture in selective enrichment broth followed by sub-culture on Ashdown’s medium to standard culture methods. Two specimens (0.8% were positive for B. pseudomallei using the enhanced protocol whereas one specimen (0.4% was positive using standard methods. These findings demonstrate that B. pseudomallei is rarely detected in sputum at this hospital. The low frequency of B. pseudomallei in sputum specimens precludes drawing any conclusions about the relative benefits of an enhanced sputum testing protocol at this site. Promoting clinician awareness of the infection and encouraging utilization of diagnostic microbiology services are likely to be important factors in facilitating identification of melioidosis.

  19. Characterization of the mrgRS locus of the opportunistic pathogen Burkholderia pseudomallei: temperature regulates the expression of a two-component signal transduction system

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    Dance David AB

    2006-08-01

    Full Text Available Abstract Background Burkholderia pseudomallei is a saprophyte in tropical environments and an opportunistic human pathogen. This versatility requires a sensing mechanism that allows the bacterium to respond rapidly to altered environmental conditions. We characterized a two-component signal transduction locus from B. pseudomallei 204, mrgR and mrgS, encoding products with extensive homology with response regulators and histidine protein kinases of Escherichia coli, Bordetella pertussis, and Vibrio cholerae. Results The locus was present and expressed in a variety of B. pseudomallei human and environmental isolates but was absent from other Burkholderia species, B. cepacia, B. cocovenenans, B. plantarii, B. thailandensis, B. vandii, and B. vietnamiensis. A 2128 bp sequence, including the full response regulator mrgR, but not the sensor kinase mrgS, was present in the B. mallei genome. Restriction fragment length polymorphism downstream from mrgRS showed two distinct groups were present among B. pseudomallei isolates. Our analysis of the open reading frames in this region of the genome revealed that transposase and bacteriophage activity may help explain this variation. MrgR and MrgS proteins were expressed in B. pseudomallei 204 cultured at different pH, salinity and temperatures and the expression was substantially reduced at 25°C compared with 37°C or 42°C but was mostly unaffected by pH or salinity, although at 25°C and 0.15% NaCl a small increase in MrgR expression was observed at pH 5. MrgR was recognized by antibodies in convalescent sera pooled from melioidosis patients. Conclusion The results suggest that mrgRS regulates an adaptive response to temperature that may be essential for pathogenesis, particularly during the initial phases of infection. B. pseudomallei and B. mallei are very closely related species that differ in their capacity to adapt to changing environmental conditions. Modifications in this region of the genome may

  20. Exposing a β-Lactamase "Twist": the Mechanistic Basis for the High Level of Ceftazidime Resistance in the C69F Variant of the Burkholderia pseudomallei PenI β-Lactamase.

    Science.gov (United States)

    Papp-Wallace, Krisztina M; Becka, Scott A; Taracila, Magdalena A; Winkler, Marisa L; Gatta, Julian A; Rholl, Drew A; Schweizer, Herbert P; Bonomo, Robert A

    2016-02-01

    Around the world, Burkholderia spp. are emerging as pathogens highly resistant to β-lactam antibiotics, especially ceftazidime. Clinical variants of Burkholderia pseudomallei possessing the class A β-lactamase PenI with substitutions at positions C69 and P167 are known to demonstrate ceftazidime resistance. However, the biochemical basis for ceftazidime resistance in class A β-lactamases in B. pseudomallei is largely undefined. Here, we performed site saturation mutagenesis of the C69 position and investigated the kinetic properties of the C69F variant of PenI from B. pseudomallei that results in a high level of ceftazidime resistance (2 to 64 mg/liter) when expressed in Escherichia coli. Surprisingly, quantitative immunoblotting showed that the steady-state protein levels of the C69F variant β-lactamase were ∼4-fold lower than those of wild-type PenI (0.76 fg of protein/cell versus 4.1 fg of protein/cell, respectively). However, growth in the presence of ceftazidime increases the relative amount of the C69F variant to greater than wild-type PenI levels. The C69F variant exhibits a branched kinetic mechanism for ceftazidime hydrolysis, suggesting there are two different conformations of the enzyme. When incubated with an anti-PenI antibody, one conformation of the C69F variant rapidly hydrolyzes ceftazidime and most likely contributes to the higher levels of ceftazidime resistance observed in cell-based assays. Molecular dynamics simulations suggest that the electrostatic characteristics of the oxyanion hole are altered in the C69F variant. When ceftazidime was positioned in the active site, the C69F variant is predicted to form a greater number of hydrogen-bonding interactions than PenI with ceftazidime. In conclusion, we propose "a new twist" for enhanced ceftazidime resistance mediated by the C69F variant of the PenI β-lactamase based on conformational changes in the C69F variant. Our findings explain the biochemical basis of ceftazidime resistance in B

  1. 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

  2. Survey of innate immune responses to Burkholderia pseudomallei in human blood identifies a central role for lipopolysaccharide.

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    Narisara Chantratita

    Full Text Available B. pseudomallei is a gram-negative bacterium that causes the tropical infection melioidosis. In northeast Thailand, mortality from melioidosis approaches 40%. As exemplified by the lipopolysaccharide-Toll-like receptor 4 interaction, innate immune responses to invading bacteria are precipitated by activation of host pathogen recognition receptors by pathogen associated molecular patterns. Human melioidosis is characterized by up-regulation of pathogen recognition receptors and pro-inflammatory cytokine release. In contrast to many gram-negative pathogens, however, the lipopolysaccharide of B. pseudomallei is considered only weakly inflammatory. We conducted a study in 300 healthy Thai subjects to investigate the ex vivo human blood response to various bacterial pathogen associated molecular patterns, including lipopolysaccharide from several bacteria, and to two heat-killed B. pseudomallei isolates. We measured cytokine levels after stimulation of fresh whole blood with a panel of stimuli. We found that age, sex, and white blood cell count modulate the innate immune response to B. pseudomallei. We further observed that, in comparison to other stimuli, the innate immune response to B. pseudomallei is most highly correlated with the response to lipopolysaccharide. The magnitude of cytokine responses induced by B. pseudomallei lipopolysaccharide was significantly greater than those induced by lipopolysaccharide from Escherichia coli and comparable to many responses induced by lipopolysaccharide from Salmonella minnesota despite lower amounts of lipid A in the B. pseudomallei lipopolysaccharide preparation. In human monocytes stimulated with B. pseudomallei, addition of polymyxin B or a TLR4/MD-2 neutralizing antibody inhibited the majority of TNF-α production. Challenging existing views, our data indicate that the innate immune response to B. pseudomallei in human blood is largely driven by lipopolysaccharide, and that the response to B

  3. Nitric oxide from IFNγ-primed macrophages modulates the antimicrobial activity of β-lactams against the intracellular pathogens Burkholderia pseudomallei and Nontyphoidal Salmonella.

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    Jessica Jones-Carson

    2014-08-01

    Full Text Available Our investigations show that nonlethal concentrations of nitric oxide (NO abrogate the antibiotic activity of β-lactam antibiotics against Burkholderia pseudomallei, Escherichia coli and nontyphoidal Salmonella enterica serovar Typhimurium. NO protects B. pseudomallei already exposed to β-lactams, suggesting that this diatomic radical tolerizes bacteria against the antimicrobial activity of this important class of antibiotics. The concentrations of NO that elicit antibiotic tolerance repress consumption of oxygen (O2, while stimulating hydrogen peroxide (H2O2 synthesis. Transposon insertions in genes encoding cytochrome c oxidase-related functions and molybdenum assimilation confer B. pseudomallei a selective advantage against the antimicrobial activity of the β-lactam antibiotic imipenem. Cumulatively, these data support a model by which NO induces antibiotic tolerance through the inhibition of the electron transport chain, rather than by potentiating antioxidant defenses as previously proposed. Accordingly, pharmacological inhibition of terminal oxidases and nitrate reductases tolerizes aerobic and anaerobic bacteria to β-lactams. The degree of NO-induced β-lactam antibiotic tolerance seems to be inversely proportional to the proton motive force (PMF, and thus the dissipation of ΔH+ and ΔΨ electrochemical gradients of the PMF prevents β-lactam-mediated killing. According to this model, NO generated by IFNγ-primed macrophages protects intracellular Salmonella against imipenem. On the other hand, sublethal concentrations of imipenem potentiate the killing of B. pseudomallei by NO generated enzymatically from IFNγ-primed macrophages. Our investigations indicate that NO modulates the antimicrobial activity of β-lactam antibiotics.

  4. Structural characterization of Burkholderia pseudomallei adenylate kinase (Adk): Profound asymmetry in the crystal structure of the 'open' state

    Energy Technology Data Exchange (ETDEWEB)

    Buchko, G.W.; Robinson, H.; Abendroth, J.; Staker, B. L.; Myler, P. J.

    2010-04-16

    In all organisms adenylate kinases (Adks) play a vital role in cellular energy metabolism and nucleic acid synthesis. Due to differences in catalytic properties between the Adks found in prokaryotes and in the cytoplasm of eukaryotes, there is interest in targeting this enzyme for new drug therapies against infectious bacterial agents. Here we report the 2.1 {angstrom} resolution crystal structure for the 220-residue Adk from Burkholderia pseudomallei (BpAdk), the etiological agent responsible for the infectious disease melioidosis. The general structure of apo BpAdk is similar to other Adk structures, composed of a CORE subdomain with peripheral ATP-binding (ATP{sub bd}) and LID subdomains. The two molecules in the asymmetric unit have significantly different conformations, with a backbone RMSD of 1.46 {angstrom}. These two BpAdk conformations may represent 'open' Adk sub-states along the preferential pathway to the 'closed' substrate-bound state.

  5. 广西地区类鼻疽伯克霍尔德氏菌的分布调查%Prevalence of Burkholderia pseudomallei in Guangxi province

    Institute of Scientific and Technical Information of China (English)

    马广强; 王倩; 朱金华; 叶荷平; 万红娇; 汪建民

    2013-01-01

    目的 调查类鼻疽伯克霍尔德氏菌在广西地区的详细分布情况.方法 于2007年底对广西地区的14个地级市进行了大规模调查,共采集了77个地点的154个土壤样品和130个水样.采样通过细菌分离培养方法、16S rDNA基因测序和鞭毛基因检测的方法进行鉴定分析,对类鼻疽伯克霍尔德氏菌检出情况的地理分布进行绘图记录.结果 154份土壤样品中分离纯化出13株类鼻疽伯克霍尔德氏菌,阳性率为8.4%,没有在水样中发现该菌;分离纯化结果与16S rDNA基因测序和鞭毛基因检测的结果完全一致.分离纯化类鼻疽伯克霍尔德氏菌阳性的采样点分布在中东、南宁、钦州、放城、北海5个地区.结论 广西地区的类鼻疽伯克霍尔德氏菌主要分布在北回归线以南,南海附近地区.%Objective To detect the prevalence and distribution of Burkholderia pseudomallei (B. pseudomallei) in Guangxi province, China, Methods Samples were harvested from several locations in Guangxi region in the year of 2007. A total of 154 soil samples and 130 water samples were screened for B. pseudomallei by bacterial culture, and identification by PCR for species-specific 16S rDNA and flagellin genes. Results B. pseudomallei was detected in 8. 4% of the soil samples but in none of the water samples. These results were identical with those from species-specific 16S rDNA and flagellin genes. All positive samples were confined to 5 locations including Zhongdong, Nanning, Qinzhou, Fangcheng and Beihai. Conclusion Almost all positive samples are isolated from places in the south of the Tropic of Cancer and nearby the South China Sea.

  6. 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.

  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. Genomic overview and progression of Burkholderia pseudomallei%类鼻疽杆菌基因组学概述及进展

    Institute of Scientific and Technical Information of China (English)

    方瑶; 顾江; 李倩; 于波; 王海光; 毛旭虎

    2012-01-01

    类鼻疽伯克霍尔德杆菌是一种人体机会致病菌,导致的疾病称类鼻疽,是一种典型的热带疾病,能感染机体几乎任何器官.类鼻疽杆菌分布广泛,主要存在于水及土壤中,其耐受性极强,能在土壤中存活几十年之久,被美国CDC列为B类生物恐怖剂.随着病原菌的扩散和人类活动的影响,全球范围内关于类鼻疽病例报道的增加,越来越多的证据表明类鼻疽是一种正在扩散的人兽共患病.海南、广东等地正是类鼻疽感染的重灾区,而国内类鼻疽杆菌的研究尚少,也没有类鼻疽杆菌研究的标准株和基因组学理论基础,获取国际标准菌株资源受到西方国家的限制,鉴于类鼻疽杆菌的特殊性、类鼻疽流行的严峻形势及科研壁垒,加强类鼻疽的研究有着重要的社会意义和军事价值.本文总结了类鼻疽杆菌(K96243标准株)的全基因组的特点,结合当今的DNA测序的发展现状,指出开展国内类鼻疽杆菌全基因测序的必要性和紧迫性.%Burkholderia pseudomallei is an opportunistic pathogen which can cause melioidosis, a typical tropical dis ease, referring to almost all organs. Burkholderia pseudomallei is found in widespread water and soil and can survive as long as decades, also classified as B category pathogen by the CDC of USA. With the diffusion of pathogen and the increasing nuni ber of victims, it is believed that melioidosis is unfolding. In China, Hainan and Guangdong is disaster area of melioidosis. However, study on melioidosis still could not receive enough attention with no international standard for bacteria or whole ge nome sequencing data. In view of the specificity of the pathogen, draconic prevalence of melioidosis and research barrier, it has significant benefit for public health and military use to further the research of Burkholderia pseudomallei in China. This review will point out the necessity of whole genome sequencing of Chinese standard bacteria

  10. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Science.gov (United States)

    Lazar Adler, Natalie R; Stevens, Mark P; Dean, Rachel E; Saint, Richard J; Pankhania, Depesh; Prior, Joann L; Atkins, Timothy P; Kessler, Bianca; Nithichanon, Arnone; Lertmemongkolchai, Ganjana; Galyov, Edouard E

    2015-01-01

    Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were

  11. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Directory of Open Access Journals (Sweden)

    Natalie R Lazar Adler

    Full Text Available Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA. Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE. A single mutant (bpaC was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA, those attenuated for virulence and net intracellular replication (BpaE, the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA. Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors

  12. Molecular and Cellular Basis of Pathogenesis of Burkholderia pseudomallei%类鼻疽伯克霍尔德菌致病机制的分子与细胞基础

    Institute of Scientific and Technical Information of China (English)

    曹旺斌; 贺英

    2012-01-01

    类鼻疽伯克霍尔德菌(Burkholderia pseudomallei)是引起主要发生在东南亚和澳大利亚北部的一种人兽共患病类鼻疽的潜在病原。明确该疾病致病机理及其宿主与病原的互作将加强对该条件致病菌的发病机制的理解。本研究从影响细菌致病性的毒力因子和病原与宿主互作两个方面阐述类鼻疽伯克霍德尔菌的致病机理,为该菌的有效控制提供可能的机制。%Burkholderia pseudomallei is a potential bioterror agent and the causative agent of melioidosis, a se- vere disease that is endemic in areas of Southeast Asia and Northern Australia. Studying how the disease is ac- quired and the host-pathogen interactions involved will underpin understanding of the bacteria, This review presents an overview focused on current knowledge of putative virulence factors and the host-pathogen interac- tions,led to understanding of the infection process of Burkholderia pseudomallei and effective control strategy.

  13. Delineating the importance of serum opsonins and the bacterial capsule in affecting the uptake and killing of Burkholderia pseudomallei by murine neutrophils and macrophages.

    Science.gov (United States)

    Mulye, Minal; Bechill, Michael P; Grose, William; Ferreira, Viviana P; Lafontaine, Eric R; Wooten, R Mark

    2014-08-01

    Infection of susceptible hosts by the encapsulated Gram-negative bacterium Burkholderia pseudomallei (Bp) causes melioidosis, with septic patients attaining mortality rates ≥ 40%. Due to its high infectivity through inhalation and limited effective therapies, Bp is considered a potential bioweapon. Thus, there is great interest in identifying immune effectors that effectively kill Bp. Our goal is to compare the relative abilities of murine macrophages and neutrophils to clear Bp, as well as determine the importance of serum opsonins and bacterial capsule. Our findings indicate that murine macrophages and neutrophils are inherently unable to clear either unopsonized Bp or the relatively-avirulent acapsular bacterium B. thailandensis (Bt). Opsonization of Bp and Bt with complement or pathogen-specific antibodies increases macrophage-uptake, but does not promote clearance, although antibody-binding enhances complement deposition. In contrast, complement opsonization of Bp and Bt causes enhanced uptake and killing by neutrophils, which is linked with rapid ROS induction against bacteria exhibiting a threshold level of complement deposition. Addition of bacteria-specific antibodies enhances complement deposition, but antibody-binding alone cannot elicit neutrophil clearance. Bp capsule provides some resistance to complement deposition, but is not anti-phagocytic or protective against reactive oxygen species (ROS)-killing. Macrophages were observed to efficiently clear Bp only after pre-activation with IFNγ, which is independent of serum- and/or antibody-opsonization. These studies indicate that antibody-enhanced complement activation is sufficient for neutrophil-clearance of Bp, whereas macrophages are ineffective at clearing serum-opsonized Bp unless pre-activated with IFNγ. This suggests that effective immune therapies would need to elicit both antibodies and Th1-adaptive responses for successful prevention/eradication of melioidosis.

  14. Delineating the importance of serum opsonins and the bacterial capsule in affecting the uptake and killing of Burkholderia pseudomallei by murine neutrophils and macrophages.

    Directory of Open Access Journals (Sweden)

    Minal Mulye

    2014-08-01

    Full Text Available Infection of susceptible hosts by the encapsulated Gram-negative bacterium Burkholderia pseudomallei (Bp causes melioidosis, with septic patients attaining mortality rates ≥ 40%. Due to its high infectivity through inhalation and limited effective therapies, Bp is considered a potential bioweapon. Thus, there is great interest in identifying immune effectors that effectively kill Bp. Our goal is to compare the relative abilities of murine macrophages and neutrophils to clear Bp, as well as determine the importance of serum opsonins and bacterial capsule. Our findings indicate that murine macrophages and neutrophils are inherently unable to clear either unopsonized Bp or the relatively-avirulent acapsular bacterium B. thailandensis (Bt. Opsonization of Bp and Bt with complement or pathogen-specific antibodies increases macrophage-uptake, but does not promote clearance, although antibody-binding enhances complement deposition. In contrast, complement opsonization of Bp and Bt causes enhanced uptake and killing by neutrophils, which is linked with rapid ROS induction against bacteria exhibiting a threshold level of complement deposition. Addition of bacteria-specific antibodies enhances complement deposition, but antibody-binding alone cannot elicit neutrophil clearance. Bp capsule provides some resistance to complement deposition, but is not anti-phagocytic or protective against reactive oxygen species (ROS-killing. Macrophages were observed to efficiently clear Bp only after pre-activation with IFNγ, which is independent of serum- and/or antibody-opsonization. These studies indicate that antibody-enhanced complement activation is sufficient for neutrophil-clearance of Bp, whereas macrophages are ineffective at clearing serum-opsonized Bp unless pre-activated with IFNγ. This suggests that effective immune therapies would need to elicit both antibodies and Th1-adaptive responses for successful prevention/eradication of melioidosis.

  15. Clinical, environmental, and serologic surveillance studies of melioidosis in Gabon, 2012-2013.

    Science.gov (United States)

    Wiersinga, W Joost; Birnie, Emma; Weehuizen, Tassili A F; Alabi, Abraham S; Huson, Michaëla A M; Huis in 't Veld, Robert A G; Mabala, Harry K; Adzoda, Gregoire K; Raczynski-Henk, Yannick; Esen, Meral; Lell, Bertrand; Kremsner, Peter G; Visser, Caroline E; Wuthiekanun, Vanaporn; Peacock, Sharon J; van der Ende, Arie; Limmathurotsakul, Direk; Grobusch, Martin P

    2015-01-01

    Burkholderia pseudomallei, an environmental gram-negative bacillus, is the causative agent of melioidosis and a bio-threat agent. Reports of B. pseudomallei isolation from soil and animals in East and West Africa suggest that melioidosis might be more widely distributed than previously thought. Because it has been found in equatorial areas with tropical climates, we hypothesized that B. pseudomallei could exist in Gabon. During 2012-2013, we conducted a seroprevalance study in which we set up microbiology facilities at a large clinical referral center and prospectively screened all febrile patients by conducting blood cultures and testing for B. pseudomallei and related species; we also determined whether B. pseudomallei could be isolated from soil. We discovered a novel B. pseudomallei sequence type that caused lethal septic shock and identified B. pseudomallei and B. thailandensis in the environment. Our data suggest that melioidosis is emerging in Central Africa but is unrecognized because of the lack of diagnostic microbiology facilities.

  16. Characterization of integrons in Burkholderia cepacia clinical isolates

    Directory of Open Access Journals (Sweden)

    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.

  17. Survey of antimicrobial resistance in clinical Burkholderia pseudomallei isolates over two decades in Northeast Thailand.

    Science.gov (United States)

    Wuthiekanun, Vanaporn; Amornchai, Premjit; Saiprom, Natnaree; Chantratita, Narisara; Chierakul, Wirongrong; Koh, Gavin C K W; Chaowagul, Wipada; Day, Nicholas P J; Limmathurotsakul, Direk; Peacock, Sharon J

    2011-11-01

    A 21-year survey conducted in northeast Thailand of antimicrobial resistance to parenteral antimicrobial drugs used to treat melioidosis identified 24/4,021 (0.6%) patients with one or more isolates resistant to ceftazidime (n = 8), amoxicillin-clavulanic acid (n = 4), or both drugs (n = 12). Two cases were identified at admission, and the remainder were detected a median of 15 days after starting antimicrobial therapy. Resistance to carbapenem drugs was not detected. These findings support the current prescribing recommendations for melioidosis.

  18. 抗类鼻疽伯克霍尔德菌多抗血清的制备及评价%Preparation and evaluation of multi-antibody serum of Burkholderia pseudomallei

    Institute of Scientific and Technical Information of China (English)

    方瑶; 顾江; 王海光; 于波; 毛旭虎

    2012-01-01

    目的制备抗类鼻疽伯克霍尔德菌(Burkholderia pseudoamllei,其抗原简写为BP)多抗血清,评价不同处理方式对抗原免疫原性的影响.方法 采用超声破碎、甲醛灭活、热灭活3种方式处理细菌抗原接种新西兰兔和BALB/C小鼠,检测不同抗原免疫动物抗血清的ELISA效价及凝集试验效价.结果 (1)ELISA方案最佳条件:二抗稀释度为1/8 000,抗原包被浓度为4 μg/mL或8 μg/mL;凝集反应最佳条件:细菌浓度为6×109 CFU/mL、凝集温度为37 ℃、观察时刻为4 h.(2)超声破碎抗原(U-BP)免疫新西兰兔和小鼠的抗血清ELISA效价高达1/64 000,最高血清凝集效价分别为1/32和1/64,甲醛灭活抗原(F-BP)抗血清ELISA效价高达1/16 000,凝集效价为1/128和1/64;热灭活抗原(H-BP)抗血清ELISA效价达1/8 000,凝集效价为1/16和1/64.(3)F-BP和H-BP具有较强的免疫原性,U-BP在小鼠中的免疫原性较弱(P<0.05).结论 建立了抗类鼻疽伯克霍尔德杆菌多克隆抗体的检测方法,制备了高效价的抗类鼻疽伯克霍尔德杆菌多抗血清;甲醛灭活和热灭活细菌具有较好的免疫原性,为下一步类鼻疽伯克霍尔德杆菌的致病机制研究和疫苗研发打下了基础.%Objective To prepare multi-antibody of Burkholderia pseudomallei(B. Pseudomallei) and evaluate the immunogenici-ty of antigens under different inactivation treatments. Methods Bacterial antigen was inactivated by three different methods, including ultrasound cracking, formaldehyde inactivation and heat inactivation, and inoculated into New-Zealand rabbits and BALB/C mice, followed by quality and specificity evaluation by ELISA and agglutination test. Results 1. The optimized condition of ELISA was 1:8000 of the dilution of secondary antibodies, 4 or 8fig/mL of the concentration of coating antigen. The optimized condition of agglutination test was 6×109 cfu/mL of bacteria density, 37 ℃ of reaction temperature, and 4 h for observation. 2. ELISA titer

  19. CpG oligodeoxyribonucleotides protect mice from Burholderia pseudomallei but not Francisella tularensis Schu 54 aersols

    Science.gov (United States)

    2010-01-01

    CpG ODN 10103 performs comparably in mice to CpG ODN 7909 (5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’), which was previously reported to protect the BALB/c mice...SHORT REPORT Open Access CpG oligodeoxyribonucleotides protect mice from Burkholderia pseudomallei but not Francisella tularensis Schu S4 aerosols...that CpG oligodeoxyribonucleotides (ODN) protect mice from various bacterial pathogens, including Burkholderia pseudomallei and Francisella tularensis

  20. Protective efficacy of heat-inactivated B. thailandensis, B. mallei or B. pseudomallei against experimental melioidosis and glanders.

    Science.gov (United States)

    Sarkar-Tyson, Mitali; Smither, Sophie J; Harding, S V; Atkins, Timothy P; Titball, Richard W

    2009-07-16

    Burkholderia pseudomallei and Burkholderia mallei are gram-negative bacilli that are the causative agents of melioidosis and glanders, respectively. Both humans and animals are susceptible to both diseases. There is currently no vaccine available for the prevention of disease. We report the protective efficacy of heat-inactivated Burkholderia thailandensis, B. mallei or B. pseudomallei cells as vaccines against murine melioidosis and glanders. Immunisation with heat-inactivated B. pseudomallei cells provided the highest levels of protection against either melioidosis or glanders. These studies indicate the longer term potential for heat-inactivated bacteria to be developed as vaccines against melioidosis and glanders.

  1. Towards a rapid molecular diagnostic for melioidosis: comparison of DNA extraction methods from clinical specimens

    Science.gov (United States)

    Richardson, Leisha J; Kaestli, Mirjam; Mayo, Mark; Bowers, Jolene R; Tuanyok, Apichai; Schupp, Jim; Engelthaler, David; Wagner, David M; Keim, Paul S; Currie, Bart J

    2011-01-01

    Optimising DNA extraction from clinical samples for Burkholderia pseudomallei Type III secretion system real-time PCR in suspected melioidosis patients confirmed that urine and sputum are useful diagnostic samples. Direct testing on blood remains problematic; testing DNA extracted from plasma was superior to DNA from whole blood or buffy coat. PMID:22108495

  2. 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.

  3. 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

  4. Heat stable antimicrobial activity of Burkholderia gladioli OR1 against clinical drug resistant isolates

    Science.gov (United States)

    Bharti, Pratibha; Anand, Vivek; Chander, Jagdish; Singh, Inder Pal; Singh, Tej Vir; Tewari, Rupinder

    2012-01-01

    Background & objectives: Drug resistant microbes are a serious challenge to human health. During the search for novel antibiotics/inhibitors from the agricultural soil, a bacterial colony was found to inhibit the growth of clinical isolates including Staphylococcus (resistant to amikacin, ciprofloxacin, clindamycin, clinafloxacin, erythromycin, gentamicin and methicillin) and Candida (resistant to fluconazole and itraconazole). The culture was identified as Burkholderia gladioli and produced at least five different antimicrobial compounds which were highly stable at high temperature (121°C) and in the broad pH range (3.0-11.0). We report here the antimicrobial activity of B. gladioli against drug resistant bacterial pathogens. Methods: The bacterial culture was identified using morphological, biochemical and 16S rRNA gene sequencing techniques. The antimicrobial activity of the identified organism against a range of microbial pathogens was checked by Kirby-Bauer's disc diffusion method. The antimicrobial compounds in the cell free supernatant were chloroform-extracted and separated by thin layer chromatography (TLC). Results: B. gladioli OR1 exhibited broad spectrum antimicrobial activity against drug resistant clinical isolates belonging to various genera of bacteria (Staphylococcus, Enterobacter, Enterococcus, Acinetobacter and Citrobacter) and a fungus (Candida). Based on TLC profile and bioautography studies, the chloroform extract of B. gladioli OR1 consisted of at least three anti-staphylococcal and two anti-Candida metabolites. The antimicrobial activity was heat stable (121°C/20 min) as well as pH stable (3.0-11.0). Interpretation & conclusions: The bacterial soil isolate, B. gladioli OR1 possessed the ability to kill various drug resistant bacteria and a fungus. This organism produced many antimicrobial metabolites which might have the potential to be used as antibiotics in future. PMID:22771597

  5. 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

  6. Development of a Polymerase Chain Reaction Assay for Detection of Burkholderia mallei, a Potent Biological Warfare Agent

    OpenAIRE

    2016-01-01

    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 ...

  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. 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.

  9. 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.

  10. 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....

  11. 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.

  12. Assessment of a DNA Vaccine Encoding Burkholderia pseudomallei Bacterioferritin

    Science.gov (United States)

    2007-08-01

    bacterioferritin gene from Brucella abortus, when delivered to mice as a DNA vaccine, evokes a potent Th1 immune response, including strong IFN-γ...blocking buffer containing goat anti-mouse IgG alkaline phosphatase conjugate (Sigma) at a dilution of 1:30000 for 1hr at room temperature. Following...Walravens, and J. J. Letesson. 2001. Induction of immune response in BALB/c mice with a DNA vaccine encoding bacterioferritin or P39 of Brucella

  13. 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.

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

    Science.gov (United States)

    2013-06-23

    Wallqvist‡ Burkholderia mallei is an infectious intracellular pathogen whose virulence and resistance to antibiotics makes it a potential bioterrorism agent...ingestion, inhalation, or skin abrasion. Given their considerable antibiotic resistance, ability to infect via aerosol, and absence of vaccines, these... equine hosts. Thus, the genes retained in B. mallei share a high sequence similarity to genes common to B. pseudomallei (3), and many virulence

  15. 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...

  16. Clinically lesser known entity in India: A Report of two cases of Melioidosis

    Directory of Open Access Journals (Sweden)

    Purabi Barman

    2013-01-01

    Full Text Available Melioidosis is endemic in the South Asian regions, like Thailand, Singapore Malaysia and Australia. The disease is more pronounced in the southern part of the country. It is caused by Burkholderia pseudomallei which causes systemic involvement, morbidity and mortality associated with the disease is high. Due to highly varied clinical presentation, and low general awareness this infection is largely underdiagnosed and under reported in our country. Most laboratories in the country still rely on conventional culturing methods with their low sensitivity, adding to the under reporting. To enhance physician awareness we describe here two cases who presented to our institute after months of misdiagnosis.

  17. Clinically lesser known entity in India: A Report of two cases of Melioidosis.

    Science.gov (United States)

    Barman, Purabi; Kaur, Ravneet; Kumar, Kamlesh

    2013-01-01

    Melioidosis is endemic in the South Asian regions, like Thailand, Singapore Malaysia and Australia. The disease is more pronounced in the southern part of the country. It is caused by Burkholderia pseudomallei which causes systemic involvement, morbidity and mortality associated with the disease is high. Due to highly varied clinical presentation, and low general awareness this infection is largely underdiagnosed and under reported in our country. Most laboratories in the country still rely on conventional culturing methods with their low sensitivity, adding to the under reporting. To enhance physician awareness we describe here two cases who presented to our institute after months of misdiagnosis.

  18. Clinical guideline for diagnosis and management of melioidosis

    Directory of Open Access Journals (Sweden)

    Inglis Timothy J.J.

    2006-01-01

    Full Text Available Melioidosis is an emerging infection in Brazil and neighbouring South American countries. The wide range of clinical presentations include severe community-acquired pneumonia, septicaemia, central nervous system infection and less severe soft tissue infection. Diagnosis depends heavily on the clinical microbiology laboratory for culture. Burkholderia pseudomallei, the bacterial cause of melioidosis, is easily cultured from blood, sputum and other clinical samples. However, B. pseudomallei can be difficult to identify reliably, and can be confused with closely related bacteria, some of which may be dismissed as insignificant culture contaminants. Serological tests can help to support a diagnosis of melioidosis, but by themselves do not provide a definitive diagnosis. The use of a laboratory discovery pathway can help reduce the risk of missing atypical B. pseudomallei isolates. Recommended antibiotic treatment for severe infection is either intravenous Ceftazidime or Meropenem for several weeks, followed by up to 20 weeks oral treatment with a combination of trimethoprim-sulphamethoxazole and doxycycline. Consistent use of diagnostic microbiology to confirm the diagnosis, and rigorous treatment of severe infection with the correct antibiotics in two stages; acute and eradication, will contribute to a reduction in mortality from melioidosis.

  19. Bichat guidelines for the clinical management of glanders and melioidosis and bioterrorism-related glanders and melioidosis.

    Science.gov (United States)

    Bossi, Philippe; Tegnell, Anders; Baka, Agoritsa; Van Loock, Frank; Hendriks, Jan; Werner, Albrecht; Maidhof, Heinrich; Gouvras, Georgios

    2004-12-15

    Glanders and melioidosis are two infectious diseases that are caused by Burkholderia mallei and Burkholderia pseudomallei respectively. Infection may be acquired through direct skin contact with contaminated soil or water. Ingestion of such contaminated water or dust is another way of contamination. Glanders and melioidosis have both been studied for weaponisation in several countries in the past. They produce similar clinical syndromes. The symptoms depend upon the route of infection but one form of the disease may progress to another, or the disease might run a chronic relapsing course. Four clinical forms are generally described: localised infection, pulmonary infection, septicaemia and chronic suppurative infections of the skin. All treatment recommendations should be adapted according to the susceptibility reports from any isolates obtained. Post-exposure prophylaxis with trimethoprim-sulfamethoxazole is recommended in case of a biological attack. There is no vaccine available for humans.

  20. Burkholderia Hep_Hag autotransporter (BuHA proteins elicit a strong antibody response during experimental glanders but not human melioidosis

    Directory of Open Access Journals (Sweden)

    Foster Simon J

    2007-03-01

    Full Text Available Abstract Background The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei. Results Using bacteriophage-mediated immunoscreening we identified genes expressed in vivo during experimental equine glanders infection. A family of immunodominant antigens were identified that share protein domain architectures with hemagglutinins and invasins. These have been designated Burkholderia Hep_Hag autotransporter (BuHA proteins. A total of 110/207 positive clones (53% of a B. mallei expression library screened with sera from two infected horses belonged to this family. This contrasted with 6/189 positive clones (3% of a B. pseudomallei expression library screened with serum from 21 patients with culture-proven melioidosis. Conclusion Members of the BuHA proteins are found in other Gram-negative bacteria and have been shown to have important roles related to virulence. Compared with other bacterial species, the genomes of both B. mallei and B. pseudomallei contain a relative abundance of this family of proteins. The domain structures of these proteins suggest that they function as multimeric surface proteins that modulate interactions of the cell with the host and environment. Their effect on the cellular immune response to B. mallei and their potential as diagnostics for glanders requires further study.

  1. 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.

  2. Recovery efficiencies for Burkholderia thailandensis from various aerosol sampling media

    Directory of Open Access Journals (Sweden)

    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.

  3. Macrophage and Galleria mellonella infection models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis

    Directory of Open Access Journals (Sweden)

    Michell Stephen L

    2011-01-01

    Full Text Available Abstract Background Burkholderia pseudomallei is the causative agent of melioidosis, a tropical disease of humans with a variable and often fatal outcome. In murine models of infection, different strains exhibit varying degrees of virulence. In contrast, two related species, B. thailandensis and B. oklahomensis, are highly attenuated in mice. Our aim was to determine whether virulence in mice is reflected in macrophage or wax moth larvae (Galleria mellonella infection models. Results B. pseudomallei strains 576 and K96243, which have low median lethal dose (MLD values in mice, were able to replicate and induce cellular damage in macrophages and caused rapid death of G. mellonella. In contrast, B. pseudomallei strain 708a, which is attenuated in mice, showed reduced replication in macrophages, negligible cellular damage and was avirulent in G. mellonella larvae. B. thailandensis isolates were less virulent than B. pseudomallei in all of the models tested. However, we did record strain dependent differences. B. oklahomensis isolates were the least virulent isolates. They showed minimal ability to replicate in macrophages, were unable to evoke actin-based motility or to form multinucleated giant cells and were markedly attenuated in G. mellonella compared to B. thailandensis. Conclusions We have shown that the alternative infection models tested here, namely macrophages and Galleria mellonella, are able to distinguish between strains of B. pseudomallei, B. thailandensis and B. oklahomensis and that these differences reflect the observed virulence in murine infection models. Our results indicate that B. oklahomensis is the least pathogenic of the species investigated. They also show a correlation between isolates of B. thailandensis associated with human infection and virulence in macrophage and Galleria infection models.

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

    Directory of Open Access Journals (Sweden)

    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

  5. Neutrophil elastase causes tissue damage that decreases host tolerance to lung infection with burkholderia species.

    Directory of Open Access Journals (Sweden)

    Manoranjan Sahoo

    2014-08-01

    Full Text Available Two distinct defense strategies can protect the host from infection: resistance is the ability to destroy the infectious agent, and tolerance is the ability to withstand infection by minimizing the negative impact it has on the host's health without directly affecting pathogen burden. Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and causes melioidosis. We have recently shown that inflammasome-triggered pyroptosis and IL-18 are equally important for resistance to B. pseudomallei, whereas IL-1β is deleterious. Here we show that the detrimental role of IL-1β during infection with B. pseudomallei (and closely related B. thailandensis is due to excessive recruitment of neutrophils to the lung and consequent tissue damage. Mice deficient in the potentially damaging enzyme neutrophil elastase were less susceptible than the wild type C57BL/6J mice to infection, although the bacterial burdens in organs and the extent of inflammation were comparable between C57BL/6J and elastase-deficient mice. In contrast, lung tissue damage and vascular leakage were drastically reduced in elastase-deficient mice compared to controls. Bradykinin levels were higher in C57BL/6 than in elastase-deficient mice; administration of a bradykinin antagonist protected mice from infection, suggesting that increased vascular permeability mediated by bradykinin is one of the mechanisms through which elastase decreases host tolerance to melioidosis. Collectively, these results demonstrate that absence of neutrophil elastase increases host tolerance, rather than resistance, to infection by minimizing host tissue damage.

  6. 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.

  7. Tandem Repeat Regions within the Burkholderia pseudomallei Genome and their Application for High-Resolution Genotyping

    Science.gov (United States)

    2007-03-30

    multilocus sequence typing (MLST) [6]. RAPD detects differences in genomes by amplifying segments of unknown DNA. Drawbacks to this technique include the...Australian isolates using MLST exhibited no overlap between sequence types for the two countries [50]. However, phylogenetic analysis of these data...Aanensen DM, Pitt TL, Kinoshita R, Spratt BG: Multilocus sequence typing and evolutionary rela- tionships among the causative agents of melioidosis

  8. Genetic analysis of the CDI pathway from Burkholderia pseudomallei 1026b

    OpenAIRE

    Koskiniemi, S; Garza-Sánchez, F; Edman, N; Chaudhuri, S.; Poole, SJ; Manoil, C; Hayes, CS; Low, DA

    2015-01-01

    © 2015 Koskiniemi et al. 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...

  9. Discrimination of Pathogenic vs. Nonpathogenic Francisella tularensis and Burkholderia pseudomallei Using Proteomics Mass Spectrometry

    Science.gov (United States)

    2011-03-01

    assignments. Validated peptide sequences, differentially present or absent in various strains (STB matrices), were visualized as assignment bitmaps...PERL, MATLAB, and Microsoft Visual Basic. 3. RESULTS AND DISCUSSION The current project characterized and identified pathogenic and nonpathogenic...ECa_O157H7EDt9|ECOt.UTie ABC«.51!2| AEt «.MlHEllBA(41_AP5|BAPH_BP|6V»PH_5G|r»O_RBS0|ECEN_AU1054|BaC_rK;|BMAl. SO.O_MOR5ITAN5 ECCl.536|ECOt_536|ECOl._CFT073

  10. 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.

  11. Clinical and economic burden of community-acquired pneumonia amongst adults in the Asia-Pacific region.

    Science.gov (United States)

    Song, Jae-Hoon; Thamlikitkul, Visanu; Hsueh, Po-Ren

    2011-08-01

    Community-acquired pneumonia (CAP) is an important cause of mortality and morbidity amongst adults in the Asia-Pacific region. Literature published between 1990 and May 2010 on the clinical and economic burden of CAP amongst adults in this region was reviewed. CAP is a significant health burden with significant economic impact in this region. Chronic obstructive pulmonary disease, cardiovascular disease, diabetes mellitus and advanced age were risk factors for CAP. Aetiological agents included Streptococcus pneumoniae, Klebsiella pneumoniae, Gram-negative bacteria, Mycobacterium tuberculosis, Burkholderia pseudomallei, Staphylococcus aureus and atypical pathogens (Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella spp.), with important differences in the prevalence of these pathogens within the region. Antibiotic resistance was significant but was not linked to excess mortality. Aetiological pathogens remained susceptible to newer antimicrobial agents. Rational antibiotic use is essential for preventing resistance, and increased surveillance is required to identify future trends in incidence and aetiology and to drive treatment and prevention strategies.

  12. 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

  13. Virulent Burkholderia species mimic host actin polymerases to drive actin-based motility

    Science.gov (United States)

    Benanti, Erin L.; Nguyen, Catherine M.; Welch, Matthew D.

    2015-01-01

    Summary Burkholderia pseudomallei and B. mallei are bacterial pathogens that cause melioidosis and glanders, while their close relative B. thailandensis is nonpathogenic. All use the trimeric autotransporter BimA to facilitate actin-based motility, host cell fusion and dissemination. Here, we show that BimA orthologs mimic different host actin-polymerizing proteins. B. thailandensis BimA activates the host Arp2/3 complex. In contrast, B. pseudomallei and B. mallei BimA mimic host Ena/VASP actin polymerases in their ability to nucleate, elongate and bundle filaments by associating with barbed ends, as well as in their use of WH2 motifs and oligomerization for activity. Mechanistic differences among BimA orthologs resulted in distinct actin filament organization and motility parameters, which affected the efficiency of cell fusion during infection. Our results identify bacterial Ena/VASP mimics and reveal that pathogens imitate the full spectrum of host actin-polymerizing pathways, suggesting that mimicry of different polymerization mechanisms influences key parameters of infection. PMID:25860613

  14. Synthesis of di- and tetrasaccharide containing 6-deoxytalose from the O-antigenic polysaccharide of B.pseudomallei strain 304b

    Institute of Scientific and Technical Information of China (English)

    Shi Qiang Yan; Xue Min Wu; Xiao Mei Liang; Jian Jun Zhang; Dao Quan Wang

    2009-01-01

    Synthesis of the disaccharide β-D-Glup-(1→3)-6-deoxy-α-L-Talp Ⅱ,and its dimer Ⅲ from the O-antigenic polysaccharide of Burkholderia pseudomallei strain 304b,were achieved through assembly of suitably synthesized building blocks,4-methoxylphenyl 6-deoxy-2,4-di-O-benzoyl-α-L-talopyranoside (7),3-O-allyl-2,4,6-tri-O-benzoyl-α,β-D-glucopyranosyl trichloroacetimidate (8) and 2,3,4,6-tetra-O-benzoyi-α,β-D-glucopyranosyl trichloroacetimidate (Ⅱ).The total yield of Ⅲ from 4-methoxyiphenyl 2,3-O-isopropylidene-α-L-rhamnopyranoside (1) was 18%.

  15. 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 ...

  16. 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

  17. Killing of Pseudomonas pseudomallei by polymorphonuclear leukocytes and peritoneal macrophages from chicken, sheep, swine and rabbits.

    Science.gov (United States)

    Markova, N; Kussovski, V; Radoucheva, T

    1998-07-01

    Differences in the kinetics of Pseudomonas pseudomallei killing by peritoneal macrophages (PM) and polymorphonuclear leucocytes (PMNL) from chickens, sheep, swine and rabbits were found. P. pseudomallei was rapidly killed by porcine PM and PMNL. However the bacterial killing by ovine and lapine PM and PMNL proceeded at a slower rate. In contrast, chicken PM and PMNL ingested and killed the lowest number of P. pseudomallei bacteria. The differences in the bactericidal activity of PM and PMNL from different animal species correlated with the level of their acid phosphatase and glycolytic activity.

  18. NCBI nr-aa BLAST: CBRC-ACAR-01-0265 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-0265 ref|YP_001062117.1| cytosine permease [Burkholderia pseudomallei ...668] ref|ZP_01765971.1| cytosine permease [Burkholderia pseudomallei 305] gb|ABN88339.1| cytosine permease [Burkholder...ia pseudomallei 668] gb|EBA49758.1| cytosine permease [Burkholderia pseudomallei 305] YP_001062117.1 0.008 27% ...

  19. 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...

  20. A case-control study of community-acquired Acinetobacter baumannii pneumonia and melioidosis pneumonia in northeast Thailand: an emerging fatal disease with unique clinical features.

    Science.gov (United States)

    Patamatamkul, Samadhi; Klungboonkrong, Voravan; Praisarnti, Pakawas; Jirakiat, Kittitouch

    2017-01-01

    Acinetobacter baumannii is the emerging cause of severe and often fatal gram-negative, community-acquired pneumonia (CAP-AB) in Thailand. Due to its rarity, its specific clinical features are ill defined. In this retrospective study, we compared the demographic data, risk factors, clinical characteristics, radiographic pattern, and microbiological data between CAP-AB and Burkholderia pseudomallei CAP (CAP-BP) to identify the clinical features and risk factors of CAP-AB. CAP-AB was associated with a more productive cough and a shorter duration of symptoms, while CAP-BP was associated with more musculoskeletal symptoms. The white blood cell and neutrophil counts were significantly lower in the CAP-AB group. Gram staining of the sputum revealed a significantly higher amount of bacteria in the CAP-AB group. Lobar infiltration and unilateral right lung involvement were the most common radiographic patterns in the CAP-AB group. CAP-AB is associated with severe pneumonia and has unique clinical features that distinguish it from CAP-BP.

  1. Glanders in animals: a review on epidemiology, clinical presentation, diagnosis and countermeasures.

    Science.gov (United States)

    Khan, I; Wieler, L H; Melzer, F; Elschner, M C; Muhammad, G; Ali, S; Sprague, L D; Neubauer, H; Saqib, M

    2013-06-01

    Glanders or farcy, caused by Burkholderia mallei, is an infectious and zoonotic disease of solipeds. Horses, donkeys and mules are the only known natural reservoir of B. mallei. Although glanders has been eradicated from most countries, it has regained the status of a re-emerging disease because of the numerous recent outbreaks. Pre-symptomatic or carrier animals are the potential source of infection for the healthy equine population and play a crucial role in the spreading of the infectious agent. Glanders is characterized by ulcerating nodular lesions of the skin and mucous membrane. Generalized symptoms include fever, malaise, depression, cough, anorexia and weight loss. Burkholderia mallei can invade its host through mucous membranes, gastrointestinal tract and the integument. Its virulence mechanisms and pathogenesis are not yet completely understood. A major problem when using serological tests for diagnosing glanders is the occurrence of false-positive and false-negative results leading to difficulties in international trade with equids and to the spread of glanders to disease-free regions. Moreover, poor tests critically result in poor control of disease. These tests are not only incapable of discriminating between B. mallei and B. pseudomallei antibodies, they are also unable to differentiate between malleinized and naturally infected animals. Combined use of both serological and molecular detection methods increases the detection rate of glanders. Countermeasures against glanders include early detection of disease in susceptible animals, stringent quarantine measures, testing and safe destruction of infected carcasses, adequate compensation to the animal owners, disinfection of infected premises and awareness about glanders and the zoonotic implications through veterinary extension services. An account of the clinical picture and successful experimental therapy of spontaneous equine glanders is also given.

  2. 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.

  3. 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.

  4. 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 ...

  5. Burkholderia cepacia Complex Regulation of Virulence Gene Expression: A Review

    Science.gov (United States)

    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

  6. 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.

  7. 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

  8. Melioidosis from contaminated bore water and successful UV sterilization.

    Science.gov (United States)

    McRobb, Evan; Kaestli, Mirjam; Mayo, Mark; Price, Erin P; Sarovich, Derek S; Godoy, Daniel; Spratt, Brian G; Currie, Bart J

    2013-08-01

    Two cases of melioidosis at a residence in rural northern Australia were linked to the unchlorinated domestic bore (automated well) water supply, which was found to have a high concentration of Burkholderia pseudomallei. Using multilocus sequence typing, clinical B. pseudomallei isolates from both cases were identical to an isolate from the bore water supply. A simple UV sterilizer reduced B. pseudomallei from the domestic water supply to undetectable levels. We have shown that UV treatment is highly effective for remediation of water contaminated with B. pseudomallei and recommend its consideration in households where individuals may be at heightened risk of contracting melioidosis.

  9. 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.

  10. 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

  11. NCBI nr-aa BLAST: CBRC-PCAP-01-1647 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PCAP-01-1647 ref|YP_337232.1| Rhs element Vgr protein [Burkholderia pseudomall...ei 1710b] ref|YP_001061795.1| Rhs element Vgr protein [Burkholderia pseudomallei 668] ref|ZP_01769751.1| Rhs... element Vgr protein [Burkholderia pseudomallei 305] ref|ZP_04520653.1| Rhs element Vgr protein [Burkholderi...a pseudomallei MSHR346] ref|ZP_04892045.1| Rhs element Vgr protein [Burkholderia ...pseudomallei 1655] ref|ZP_04893856.1| Rhs element Vgr protein [Burkholderia pseudomallei Pasteur 52237] ref|

  12. NCBI nr-aa BLAST: CBRC-MEUG-01-0169 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available subunit [Burkholderia pseudomallei Pakistan 9] gb|EEH26464.1| putative DNA-directed RNA polymerase II, large subunit [Burkholderia pseudomallei Pakistan 9] ZP_03792652.1 2e-14 37% ...

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. NCBI nr-aa BLAST: CBRC-OPRI-01-0333 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OPRI-01-0333 ref|YP_109945.1| hypothetical protein BPSL3349 [Burkholderia pseu...domallei K96243] ref|ZP_02449621.1| hypothetical protein Bpse9_22577 [Burkholderia pseudomallei 91] emb|CAH3...7362.1| putative membrane protein [Burkholderia pseudomallei K96243] YP_109945.1 7.2 42% ...

  16. NCBI nr-aa BLAST: CBRC-TBEL-01-1988 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TBEL-01-1988 ref|ZP_01331076.1| hypothetical protein BpseS_03000920 [Burkholderia... pseudomallei S13] ref|ZP_01334471.1| hypothetical protein Bpse4_03003005 [Burkholderia pseudomallei 406e...] gb|EDO83829.1| Rhs element Vgr protein [Burkholderia pseudomallei 406e] ZP_01331076.1 2e-07 26% ...

  17. NCBI nr-aa BLAST: CBRC-PTRO-01-0110 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PTRO-01-0110 ref|ZP_00896661.1| hypothetical protein Bpse110_02000926 [Burkholder...ia pseudomallei 1106b] ref|YP_001074743.1| Rhs element Vgr protein [Burkholderia pseudomallei 1106a] gb|A...BN93621.1| Rhs element Vgr protein [Burkholderia pseudomallei 1106a] ZP_00896661.1 8e-07 27% ...

  18. Prevalence of melioidosis in patients with suspected pulmonary tuberculosis and sputum smear negative for acid-fast bacilli in northeast Thailand.

    Science.gov (United States)

    Suntornsut, Pornpan; Kasemsupat, Kriangsak; Silairatana, Santi; Wongsuvan, Gumphol; Jutrakul, Yaowaruk; Wuthiekanun, Vanaporn; Day, Nicholas P J; Peacock, Sharon J; Limmathurotsakul, Direk

    2013-11-01

    The clinical and radiological features of pulmonary melioidosis can mimic tuberculosis. We prospectively evaluated 118 patients with suspected pulmonary tuberculosis who were acid-fast bacilli (AFB) smear negative at Udon Thani Hospital, northeast Thailand. Culture of residual sputum from AFB testing was positive for Burkholderia pseudomallei in three patients (2.5%; 95% confidence interval [CI] 0.5-7.3%). We propose that in melioidosis-endemic areas, residual sputum from AFB testing should be routinely cultured for B. pseudomallei.

  19. The epidemiology and clinical spectrum of melioidosis: 540 cases from the 20 year Darwin prospective study.

    Directory of Open Access Journals (Sweden)

    Bart J Currie

    Full Text Available BACKGROUND: Over 20 years, from October 1989, the Darwin prospective melioidosis study has documented 540 cases from tropical Australia, providing new insights into epidemiology and the clinical spectrum. PRINCIPAL FINDINGS: The principal presentation was pneumonia in 278 (51%, genitourinary infection in 76 (14%, skin infection in 68 (13%, bacteremia without evident focus in 59 (11%, septic arthritis/osteomyelitis in 20 (4% and neurological melioidosis in 14 (3%. 298 (55% were bacteremic and 116 (21% developed septic shock (58 fatal. Internal organ abscesses and secondary foci in lungs and/or joints were common. Prostatic abscesses occurred in 76 (20% of 372 males. 96 (18% had occupational exposure to Burkholderia pseudomallei. 118 (22% had a specific recreational or occupational incident considered the likely infecting event. 436 (81% presented during the monsoonal wet season. The higher proportion with pneumonia in December to February supports the hypothesis of infection by inhalation during severe weather events. Recurrent melioidosis occurred in 29, mostly attributed to poor adherence to therapy. Mortality decreased from 30% in the first 5 years to 9% in the last five years (p<0.001. Risk factors for melioidosis included diabetes (39%, hazardous alcohol use (39%, chronic lung disease (26% and chronic renal disease (12%. There was no identifiable risk factor in 20%. Of the 77 fatal cases (14%, 75 had at least one risk factor; the other 2 were elderly. On multivariate analysis of risk factors, age, location and season, the only independent predictors of mortality were the presence of at least one risk factor (OR 9.4; 95% CI 2.3-39 and age ≥ 50 years (OR 2.0; 95% CI 1.2-2.3. CONCLUSIONS: Melioidosis should be seen as an opportunistic infection that is unlikely to kill a healthy person, provided infection is diagnosed early and resources are available to provide appropriate antibiotics and critical care.

  20. 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.

  1. 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.

  2. 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.

  3. 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...

  4. 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.

  5. NCBI nr-aa BLAST: CBRC-AGAM-05-0010 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-05-0010 ref|ZP_00895641.1| hypothetical protein Bpse110_02002081 [Burkholderia pse...udomallei 1106b] ref|ZP_01214082.1| hypothetical protein Bpse17_02000720 [Burkholderia pseudomallei... 1710a] ref|ZP_01318224.1| hypothetical protein Bpse1_03002406 [Burkholderia pseudomallei 1655] ref|ZP_01324...763.1| hypothetical protein BpseP_03001411 [Burkholderia pseudomallei Pasteur] re...f|ZP_01330574.1| hypothetical protein BpseS_03001271 [Burkholderia pseudomallei S13] ref|ZP_01335179.1| hypothetical protein Bpse

  6. NCBI nr-aa BLAST: CBRC-XTRO-01-0586 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0586 ref|ZP_00892508.1| hypothetical protein Bpse110_02005257 [Burkholder...ia pseudomallei 1106b] ref|ZP_01315437.1| hypothetical protein Bpse1_03005219 [Burkholderia pseudomallei ...1655] ref|ZP_01326482.1| hypothetical protein BpseS_03005487 [Burkholderia pseudomallei S13] ref|YP_00107577...5.1| hypothetical protein BURPS1106A_A1741 [Burkholderia pseudomallei 1106a] gb|A...BN93338.1| conserved hypothetical protein [Burkholderia pseudomallei 1106a] ZP_00892508.1 2.7 33% ...

  7. NCBI nr-aa BLAST: CBRC-OPRI-01-0923 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OPRI-01-0923 ref|YP_001066912.1| iron-sulfur cluster binding protein [Burkholder...ia pseudomallei 1106a] ref|ZP_02471985.1| iron-sulfur cluster binding protein [Burkholderia pseudomallei B...7210] ref|ZP_04813470.1| iron-sulfur cluster binding protein [Burkholderia pseudomallei 1106b] gb|ABN90317.1...| iron-sulfur cluster binding protein [Burkholderia pseudomallei 1106a] gb|EES240...95.1| iron-sulfur cluster binding protein [Burkholderia pseudomallei 1106b] YP_001066912.1 3.0 32% ...

  8. 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...

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

    Directory of Open Access Journals (Sweden)

    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.

  10. 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

  11. 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.

    Science.gov (United States)

    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.

  12. 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.

  13. 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.

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    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

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

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    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.

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

    Science.gov (United States)

    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.

  18. 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

  19. 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.

  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. 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...

  2. 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

  3. 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 ...

  4. 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.

  5. 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'.

  6. Glanders: off to the races with Burkholderia mallei.

    Science.gov (United States)

    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. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. NCBI nr-aa BLAST: CBRC-PTRO-07-0003 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PTRO-07-0003 ref|ZP_00892892.1| hypothetical protein Bpse110_02004856 [Burkholderia pse...udomallei 1106b] ref|ZP_01213659.1| hypothetical protein Bpse17_02001368 [Burkholderia pseudomallei... 1710a] ref|ZP_01320796.1| hypothetical protein BpseP_03005463 [Burkholderia pseudomallei Pasteur] ZP_00892892.1 1e-13 34% ...

  10. NCBI nr-aa BLAST: CBRC-PTRO-20-0065 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PTRO-20-0065 ref|ZP_00892892.1| hypothetical protein Bpse110_02004856 [Burkholderia pse...udomallei 1106b] ref|ZP_01213659.1| hypothetical protein Bpse17_02001368 [Burkholderia pseudomallei... 1710a] ref|ZP_01320796.1| hypothetical protein BpseP_03005463 [Burkholderia pseudomallei Pasteur] ZP_00892892.1 2e-06 34% ...

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  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. Structure and in silico substrate-binding mode of ADP-L-glycero-D-manno-heptose 6-epimerase from Burkholderia thailandensis.

    Science.gov (United States)

    Kim, Mi-Sun; Lim, Areum; Yang, Seung Won; Park, Jimin; Lee, Daeun; Shin, Dong Hae

    2013-04-01

    ADP-L-glycero-D-manno-heptose 6-epimerase (AGME), the product of the rfaD gene, is the last enzyme in the heptose-biosynthesis pathway; it converts ADP-D-glycero-D-manno-heptose (ADP-D,D-Hep) to ADP-L-glycero-D-manno-heptose (ADP-L,D-Hep). AGME contains a catalytic triad involved in catalyzing hydride transfer with the aid of NADP(+). Defective lipopolysaccharide is found in bacterial mutants lacking this gene. Therefore, it is an interesting target enzyme for a novel epimerase inhibitor for use as a co-therapy with antibiotics. The crystal structure of AGME from Burkholderia thailandensis (BtAGME), a surrogate organism for studying the pathogenicity of melioidosis caused by B. pseudomallei, has been determined. The crystal structure determined with co-purified NADP(+) revealed common as well as unique structural properties of the AGME family when compared with UDP-galactose 4-epimerase homologues. They form a similar architecture with conserved catalytic residues. Nevertheless, there are differences in the substrate- and cofactor-binding cavities and the oligomerization domains. Structural comparison of BtAGME with AGME from Escherichia coli indicates that they may recognize their substrate in a `lock-and-key' fashion. Unique structural features of BtAGME are found in two regions. The first region is the loop between β8 and β9, affecting the binding affinity of BtAGME for the ADP moiety of ADP-D,D-Hep. The second region is helix α8, which induces decamerization at low pH that is not found in other AGMEs. With the E210G mutant, it was observed that the resistance of the wild type to acid-induced denaturation is related to the decameric state. An in silico study was performed using the Surflex-Dock GeomX module of the SYBYL-X 1.3 software to predict the catalytic mechanism of BtAGME with its substrate, ADP-D,D-Hep. In the in silico study, the C7'' hydroxymethyl group of ADP-D,D-Hep is predicted to form hydrogen bonds to Ser116 and Gln293. With the aid of these

  15. Integrated Semiconductor-based Diagnostics System for Multiplexed Genomic Amplification and Electrochemical Detection of Biothreat Agents

    Science.gov (United States)

    2009-01-01

    contains all of the reagents and a microarray to test for Bacillus anthracis, Clostridium botulinum, Yersinia pestis, Francisella tularensis, Burkholderia pseudomallei, Brucella suis, Vibrio cholera, and Yersinia enterocolitica .

  16. 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.

  17. 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.

  18. Draft Genome Sequence of Burkholderia sp. MR1, a Methylarsenate-Reducing Bacterial Isolate from Florida Golf Course Soil

    Science.gov (United States)

    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

  19. A Possible Link between Infection with Burkholderia Bacteria and Systemic Lupus Erythematosus Based on Epitope Mimicry

    Directory of Open Access Journals (Sweden)

    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.

  20. Melioidosis:a rare cause of anterior chest wall abscess

    Institute of Scientific and Technical Information of China (English)

    Rashidi Ahmad; Azhar Amir Hamzah; Ahmad Kasfi Abdul Rahman; Phee Kheng Cheah

    2010-01-01

    Melioidosis is an unusual tropical infectious disease caused by the gram-negative bacterium Burkholderia pseudomallei, which was formerly known asPseudomonas pseudomallei. Melioidosis is characterized by abscess formation and it may manifest in any part of the human body, however, musculoskeletel melioidosis is uncommon and chest wall melioidosis is very rare. To determine the exact organism based solely on clinical presentation poses a great challenge to the physician. Yet, delay administration of antibiotic may be harmful. We describe a diabetic patient who had anterior chest wall melioidosis that mimicsStaphylococcus aureus infection. A description of his presentation and management, along with a review of literature is presented.

  1. CHLORINE INACTIVATION OF CATEGORY "A" BIO-TERRORISM AGENTS

    Science.gov (United States)

    This poster presents information on the inactivation of select bioterrorist agents. Information will be presented on chlorine disinfection of vegetative cells of Brucella suis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis and endos...

  2. 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.

  3. 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

  4. Monitoring Therapeutic Treatments against Burkholderia Infections Using Imaging Techniques

    Directory of Open Access Journals (Sweden)

    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.

  5. 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.

  6. 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.

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

    Science.gov (United States)

    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.

  8. 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.

  9. 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

  10. A rare cause of septic arthritis: melioidosis.

    Science.gov (United States)

    Caldera, Aruna Sanjeewa; Kumanan, Thirunavukarasu; Corea, Enoka

    2013-10-01

    Melioidosis is a pyogenic infection with high mortality caused by the bacterium Burkholderia pseudomallei. As the clinical presentation is not distinctive, a high index of clinical suspicion is required for diagnosis. We present a case of a 50-year-old farmer who was diabetic and a chronic alcoholic, who presented to us with pneumonia, followed by septic arthritis. He was ultimately diagnosed as having melioidosis.

  11. 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

  12. Transport of nanoparticles and tobramycin-loaded liposomes in Burkholderia cepacia complex biofilms.

    Directory of Open Access Journals (Sweden)

    Anne-Sophie Messiaen

    Full Text Available Due to the intrinsic resistance of Burkholderia cepacia complex (Bcc to many antibiotics and the production of a broad range of virulence factors, lung infections by these bacteria, primarily occurring in cystic fibrosis (CF patients, are very difficult to treat. In addition, the ability of Bcc organisms to form biofilms contributes to their persistence in the CF lung. As Bcc infections are associated with poor clinical outcome, there is an urgent need for new effective therapies to treat these infections. In the present study, we investigated whether liposomal tobramycin displayed an increased anti-biofilm effect against Bcc bacteria compared to free tobramycin. Single particle tracking (SPT was used to study the transport of positively and negatively charged nanospheres in Bcc biofilms as a model for the transport of liposomes. Negatively charged nanospheres became immobilized in close proximity of biofilm cell clusters, while positively charged nanospheres interacted with fiber-like structures, probably eDNA. Based on these data, encapsulation of tobramycin in negatively charged liposomes appeared promising for targeted drug delivery. However, the anti-biofilm effect of tobramycin encapsulated into neutral or anionic liposomes did not increase compared to that of free tobramycin. Probably, the fusion of the anionic liposomes with the negatively charged bacterial surface of Bcc bacteria was limited by electrostatic repulsive forces. The lack of a substantial anti-biofilm effect of tobramycin encapsulated in neutral liposomes could be further investigated by increasing the liposomal tobramycin concentration. However, this was hampered by the low encapsulation efficiency of tobramycin in these liposomes.

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

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    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.

  14. 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.

  15. Intrinsic Resistance of Burkholderia cepacia Complex to Benzalkonium Chloride

    Directory of Open Access Journals (Sweden)

    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.

  16. Influence of neutrophil defects on Burkholderia cepacia complex pathogenesis

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  18. 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.

  19. 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...

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

    DEFF Research Database (Denmark)

    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 eradica...

  1. 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

  2. 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.

  3. 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.

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

    Science.gov (United States)

    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.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. NCBI nr-aa BLAST: CBRC-ACAR-01-0265 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-0265 ref|ZP_00896538.1| hypothetical protein Bpse110_02001132 [Burkholderia pse...udomallei 1106b] ref|ZP_01330877.1| hypothetical protein BpseS_03001038 [Burkholderia pseudomallei S13] ZP_00896538.1 0.005 27% ...

  11. NCBI nr-aa BLAST: CBRC-MEUG-01-1926 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MEUG-01-1926 ref|ZP_02401667.1| hypothetical protein BpseD_05380 [Burkholderia pse...udomallei DM98] ref|ZP_02488822.1| hypothetical protein BpseN_05018 [Burkholderia pseudomallei NCTC 13177] ZP_02401667.1 3e-06 31% ...

  12. NCBI nr-aa BLAST: CBRC-XTRO-01-3152 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-3152 ref|ZP_01335736.1| hypothetical protein Bpse4_03001703 [Burkholderia... pseudomallei 406e] gb|EDO84333.1| ABC transporter, ATP-binding protein [Burkholderia pseudomallei 406e] ZP_01335736.1 2e-25 34% ...

  13. NCBI nr-aa BLAST: CBRC-TGUT-37-0067 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TGUT-37-0067 ref|YP_001061172.1| Rhs element Vgr protein [Burkholderia pseudom...allei 668] gb|ABN85966.1| Rhs element Vgr protein [Burkholderia pseudomallei 668] YP_001061172.1 7e-26 29% ...

  14. NCBI nr-aa BLAST: CBRC-AGAM-03-0063 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-03-0063 ref|YP_332515.1| putative lipoprotein [Burkholderia pseudomallei ...1710b] gb|ABA48419.1| putative lipoprotein [Burkholderia pseudomallei 1710b] YP_332515.1 7e-12 31% ...

  15. NCBI nr-aa BLAST: CBRC-DMEL-04-0043 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DMEL-04-0043 ref|ZP_01767027.1| putative transcriptional regulator [Burkholderia... pseudomallei 305] gb|EBA48406.1| putative transcriptional regulator [Burkholderia pseudomallei 305] ZP_01767027.1 0.11 41% ...

  16. NCBI nr-aa BLAST: CBRC-PHAM-01-0697 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PHAM-01-0697 ref|ZP_04811055.1| tash protein pest motif family [Burkholderia p...seudomallei 1106b] gb|EES21680.1| tash protein pest motif family [Burkholderia pseudomallei 1106b] ZP_04811055.1 4e-22 36% ...

  17. NCBI nr-aa BLAST: CBRC-PHAM-01-0502 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PHAM-01-0502 ref|YP_001061172.1| Rhs element Vgr protein [Burkholderia pseudom...allei 668] gb|ABN85966.1| Rhs element Vgr protein [Burkholderia pseudomallei 668] YP_001061172.1 1e-103 72% ...

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

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1310 ref|YP_001066820.1| acyltransferase family protein [Burkholderia ...pseudomallei 1106a] gb|ABN91330.1| acyltransferase family protein [Burkholderia pseudomallei 1106a] YP_001066820.1 1e-51 46% ...

  19. NCBI nr-aa BLAST: CBRC-PTRO-10-0064 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PTRO-10-0064 ref|YP_107518.1| putative lipoprotein [Burkholderia pseudomallei ...K96243] emb|CAH34885.1| putative lipoprotein [Burkholderia pseudomallei K96243] YP_107518.1 8e-37 40% ...

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

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-01-0052 ref|YP_333983.1| hypothetical protein BURPS1710b_2591 [Burkholder...ia pseudomallei 1710b] gb|ABA48937.1| hypothetical protein BURPS1710b_2591 [Burkholderia pseudomallei 1710b] YP_333983.1 5e-11 27% ...

  1. NCBI nr-aa BLAST: CBRC-PHAM-01-0697 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PHAM-01-0697 ref|ZP_04969012.1| tash protein pest motif family [Burkholderia p...seudomallei 406e] gb|EDO88606.1| tash protein pest motif family [Burkholderia pseudomallei 406e] ZP_04969012.1 7e-21 36% ...

  2. NCBI nr-aa BLAST: CBRC-CJAC-01-0777 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CJAC-01-0777 ref|YP_001061172.1| Rhs element Vgr protein [Burkholderia pseudom...allei 668] gb|ABN85966.1| Rhs element Vgr protein [Burkholderia pseudomallei 668] YP_001061172.1 9e-15 33% ...

  3. NCBI nr-aa BLAST: CBRC-AGAM-03-0021 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-03-0021 ref|YP_331887.1| Planctomycete extracellular domain protein [Burkholder...ia pseudomallei 1710b] gb|ABA51048.1| Planctomycete extracellular domain protein [Burkholderia pseudomallei 1710b] YP_331887.1 0.005 31% ...

  4. NCBI nr-aa BLAST: CBRC-PABE-01-0051 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PABE-01-0051 ref|YP_107941.1| putative lipoprotein [Burkholderia pseudomallei ...K96243] emb|CAH35314.1| putative lipoprotein [Burkholderia pseudomallei K96243] YP_107941.1 1e-06 35% ...

  5. NCBI nr-aa BLAST: CBRC-GACU-19-0032 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GACU-19-0032 ref|YP_001061379.1| proline iminopeptidase [Burkholderia pseudoma...llei 668] gb|ABN85444.1| proline iminopeptidase [Burkholderia pseudomallei 668] YP_001061379.1 8.0 31% ...

  6. NCBI nr-aa BLAST: CBRC-AGAM-02-0064 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-02-0064 ref|YP_335617.1| haemagluttinin family protein [Burkholderia pseu...domallei 1710b] gb|ABA51851.1| haemagluttinin family protein [Burkholderia pseudomallei 1710b] YP_335617.1 0.0 31% ...

  7. NCBI nr-aa BLAST: CBRC-GACU-23-0103 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GACU-23-0103 ref|YP_332516.1| hypothetical protein BURPS1710b_1104 [Burkholder...ia pseudomallei 1710b] gb|ABA49681.1| hypothetical protein BURPS1710b_1104 [Burkholderia pseudomallei 1710b] YP_332516.1 6e-32 37% ...

  8. NCBI nr-aa BLAST: CBRC-CJAC-01-0102 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CJAC-01-0102 ref|YP_111980.1| lipoprotein [Burkholderia pseudomallei K96243] e...mb|CAH39452.1| putative lipoprotein [Burkholderia pseudomallei K96243] YP_111980.1 6e-35 47% ...

  9. NCBI nr-aa BLAST: CBRC-ATHA-05-0020 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ATHA-05-0020 ref|YP_001061443.1| hypothetical protein BURPS668_A0437 [Burkholder...ia pseudomallei 668] gb|ABN88237.1| hypothetical protein BURPS668_A0437 [Burkholderia pseudomallei 668] YP_001061443.1 0.22 50% ...

  10. NCBI nr-aa BLAST: CBRC-PCAP-01-1647 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PCAP-01-1647 ref|ZP_03792095.1| Rhs element Vgr protein [Burkholderia pseudomallei Pakistan... 9] gb|EEH27396.1| Rhs element Vgr protein [Burkholderia pseudomallei Pakistan 9] ZP_03792095.1 1e-12 26% ...

  11. Two fatal cases of melioidosis on the Thai-Myanmar border [v1; ref status: indexed, http://f1000r.es/2os

    Directory of Open Access Journals (Sweden)

    Cindy S. Chu

    2014-01-01

    Full Text Available Melioidosis is endemic in areas of Southeast Asia, however, there are no published reports from the Thai-Myanmar border.  We report the first two cases of fatal melioidosis in this region. This is of great public health importance and highlights the need to increase clinical awareness of melioidosis on the Thai-Myanmar border and to assess the true burden of disease in the area through improved case detection and Burkholderia pseudomallei prevalence studies.

  12. Two fatal cases of melioidosis on the Thai-Myanmar border [v2; ref status: indexed, http://f1000r.es/373

    Directory of Open Access Journals (Sweden)

    Cindy S. Chu

    2014-03-01

    Full Text Available Melioidosis is endemic in areas of Southeast Asia, however, there are no published reports from the Thai-Myanmar border. We report the first two documented cases of fatal melioidosis in this region. This is of great public health importance and highlights the need to both increase clinical awareness of melioidosis on the Thai-Myanmar border, and to assess the true burden of disease in the area through improved case detection and Burkholderia pseudomallei prevalence studies.

  13. 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

  14. 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

    Directory of Open Access Journals (Sweden)

    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

  15. 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)).

  16. 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.

  17. POLYCLONAL OUTBREAK OF BLOODSTREAM INFECTIONS CAUSED BY Burkholderia cepacia COMPLEX IN HEMATOLOGY AND BONE MARROW TRANSPLANT OUTPATIENT UNITS

    Directory of Open Access Journals (Sweden)

    Icaro Boszczowski

    2014-01-01

    Full Text Available Aim: The objective was to describe an outbreak of bloodstream infections by Burkholderia cepacia complex (Bcc in bone marrow transplant and hematology outpatients. Methods: On February 15, 2008 a Bcc outbreak was suspected. 24 cases were identified. Demographic and clinical data were evaluated. Environment and healthcare workers' (HCW hands were cultured. Species were determined and typed. Reinforcement of hand hygiene, central venous catheter (CVC care, infusion therapy, and maintenance of laminar flow cabinet were undertaken. 16 different HCWs had cared for the CVCs. Multi-dose heparin and saline were prepared on counter common to both units. Findings: 14 patients had B. multivorans (one patient had also B. cenopacia, six non-multivorans Bcc and one did not belong to Bcc. Clone A B. multivorans occurred in 12 patients (from Hematology; in 10 their CVC had been used on February 11/12. Environmental and HCW cultures were negative. All patients were treated with meropenem, and ceftazidime lock-therapy. Eight patients (30% were hospitalized. No deaths occurred. After control measures (multidose vial for single patient; CVC lock with ceftazidime; cleaning of laminar flow cabinet; hand hygiene improvement; use of cabinet to store prepared medication, no new cases occurred. Conclusions: This polyclonal outbreak may be explained by a common source containing multiple species of Bcc, maybe the laminar flow cabinet common to both units. There may have been contamination by B. multivorans (clone A of multi-dose vials.

  18. 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

  19. 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.

  20. 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.

  1. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  3. 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.

  4. 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.

  5. 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...

  6. 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...

  7. 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...

  8. 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

  9. 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.

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

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    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

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

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    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.

  12. Extreme antimicrobial peptide and polymyxin B resistance in the genus Burkholderia

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    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.

  13. 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

  14. 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

  15. 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...

  16. 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.

  17. Pathological findings and diagnostic implications of a rhesus macaque (Macacca mulatta) model of aerosol exposure to Burkholderia mallei (glanders).

    Science.gov (United States)

    Yingst, Samuel L; Facemire, Paul; Chuvala, Lara; Norwood, David; Wolcott, Mark; Huzella, Louis

    2015-06-01

    Burkholderia mallei is a Gram-negative bacillus that causes a pneumonic disease known as glanders in equids and humans, and a lymphatic infection known as farcy, primarily in equids. With the potential to infect humans by the respiratory route, aerosol exposure can result in severe, occasionally fatal, pneumonia. Today, glanders infections in humans are rare, likely due to less frequent contact with infected equids than in the past. Acutely ill humans often have non-specific clinical signs and in order to diagnose cases, especially in scenarios of multiple cases in an unexpected setting, rapid diagnostics for B. mallei may be critical. The pathogenesis of acute glanders in the rhesus macaque (Macaca mulatta) was studied as an initial effort to improve diagnostic methods. In the study described here, the diagnostic techniques of PCR, culture and histopathology were compared. The results indicated that PCR may provide rapid, non-invasive diagnosis of glanders in some cases. As expected, PCR results were positive in lung tissue in 11/12 acutely infected rhesus macaques, but more importantly in terms of diagnostic algorithm development, PCR results were frequently positive in non-invasive samples such as broncho-alveolar lavage or nasal swabs (7/12) and occasionally in blood (3/12). However, conventional bacterial culture failed to recover bacteria in many of these samples. The study showed that the clinical presentation of aerosol-exposed rhesus macaques is similar to descriptions of human glanders and that PCR has potential for rapid diagnosis of outbreaks, if not individual cases.

  18. Chronic infection of cystic fibrosis patient airways by a single clone of Burkholderia cepacia: replacement of non-mucoid to mucoid morphotype Infecção pulmonar crônica por um único clone de Burkholderia cepacia: substituição do morfotipo não mucóide por mucóide

    Directory of Open Access Journals (Sweden)

    Ana Paula D'Alincourt Carvalho

    2003-11-01

    Full Text Available Mucoid Burkholderia cepacia morphotype emerged within a nine year follow-up of a cystic fibrosis patient. Clinical data suggested a linkage between the mucoid phenotype isolation and the deterioration of the patient's condition. Despite of the phenotypic variation, molecular typing showed that the patient was chronically infected with B. cepacia complex isolates belonging to a same genetic clone.O presente trabalho descreve a emergência de cepas mucoides do complexo B. cepacia em um paciente com Fibrose Cística dentro de um acompanhamento bacteriológico prospectivo de nove anos. Os dados clínicos sugerem a associação entre o isolamento do morfotipo mucoide e a deterioração clínica do paciente. Apesar da variação fenotípica, os testes moleculares mostraram que o paciente manteve-se cronicamente infectado por cepas de mesma origem clonal.

  19. 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

  20. 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.

  1. 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.

  2. NCBI nr-aa BLAST: CBRC-SARA-01-0144 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-SARA-01-0144 ref|YP_104995.1| alcohol dehydrogenase, zinc-containing [Burkholder...ia mallei ATCC 23344] ref|YP_111924.1| alcohol dehydrogenase [Burkholderia pseudomallei K96243] ref|ZP_004...40487.1| COG1064: Zn-dependent alcohol dehydrogenases [Burkholderia mallei GB8 horse 4] ref|YP_336176.1| alc...ohol dehydrogenase, zinc-containing [Burkholderia pseudomallei 1710b] ref|ZP_0093...0750.1| COG1064: Zn-dependent alcohol dehydrogenases [Burkholderia mallei FMH] ref|ZP_00934487.1| COG1064: Z

  3. 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

    were used to visualize AHL-mediated communication in mixed biofilms, which were cultivated either in artificial flow chambers or in alginate beads in mouse lung tissue. In both model systems B. cepacia was capable of perceiving the AHL signals produced by P. aeruginosa, while the latter strain did......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...

  4. 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.

  5. 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.

  6. 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.

  7. 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...

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  10. 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...

  11. 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...

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

    Science.gov (United States)

    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.

  13. 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.

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

    Science.gov (United States)

    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.

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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.

  18. 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.

  19. 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.

  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. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

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

    Science.gov (United States)

    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.

  8. 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.

  9. 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) ...

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  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. 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...

  18. 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.

  19. 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.

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. Sri Lankan National Melioidosis Surveillance Program Uncovers a Nationwide Distribution of Invasive Melioidosis.

    Science.gov (United States)

    Corea, Enoka M; Merritt, Adam J; Ler, Yi-Horng; Thevanesam, Vasanthi; Inglis, Timothy J J

    2016-02-01

    The epidemiologic status of melioidosis in Sri Lanka was unclear from the few previous case reports. We established laboratory support for a case definition and started a nationwide case-finding study. Suspected Burkholderia pseudomallei isolates were collated, identified by polymerase chain reaction assay, referred for Matrix Assisted Laser Desorption Ionization-Time of Flight analysis and multilocus sequence typing (MLST), and named according to the international MLST database. Between 2006 and early 2014, there were 32 patients with culture-confirmed melioidosis with an increasing annual total and a falling fatality rate. Patients were predominantly from rural communities, diabetic, and male. The major clinical presentations were sepsis, pneumonia, soft tissue and joint infections, and other focal infection. Burkholderia pseudomallei isolates came from all parts of Sri Lanka except the Sabaragamuwa Province, the south central hill country, and parts of northern Sri Lanka. Bacterial isolates belonged to 18 multilocus sequence types, one of which (ST 1137) was associated with septicemia and a single-organ focus (Fisher's exact, P = 0.004). Melioidosis is an established endemic infection throughout Sri Lanka, and is caused by multiple genotypes of B. pseudomallei, which form a distinct geographic group based upon related sequence types (BURST) cluster at the junction of the southeast Asian and Australasian clades.

  10. 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.

  11. T-Cell Responses Are Associated with Survival in Acute Melioidosis Patients.

    Directory of Open Access Journals (Sweden)

    Kemajittra Jenjaroen

    2015-10-01

    Full Text Available Melioidosis is an increasingly recognised cause of sepsis and death across South East Asia and Northern Australia, caused by the bacterium Burkholderia pseudomallei. Risk factors include diabetes, alcoholism and renal disease, and a vaccine targeting at-risk populations is urgently required. A better understanding of the protective immune response in naturally infected patients is essential for vaccine design.We conducted a longitudinal clinical and immunological study of 200 patients with melioidosis on admission, 12 weeks (n = 113 and 52 weeks (n = 65 later. Responses to whole killed B. pseudomallei were measured in peripheral blood mononuclear cells (PBMC by interferon-gamma (IFN-γ ELIspot assay and flow cytometry and compared to those of control subjects in the region with diabetes (n = 45 and without diabetes (n = 43.We demonstrated strong CD4+ and CD8+ responses to B. pseudomallei during acute disease, 12 weeks and 52 weeks later. 28-day mortality was 26% for melioidosis patients, and B. pseudomallei-specific cellular responses in fatal cases (mean 98 IFN-γ cells per million PBMC were significantly lower than those in the survivors (mean 142 IFN-γ cells per million PBMC in a multivariable logistic regression model (P = 0.01. A J-shaped curve association between circulating neutrophil count and mortality was seen with an optimal count of 4000 to 8000 neutrophils/μl. Melioidosis patients with known diabetes had poor diabetic control (median glycated haemoglobin HbA1c 10.2%, interquartile range 9.2-13.1 and showed a stunted B. pseudomallei-specific cellular response during acute illness compared to those without diabetes.The results demonstrate the role of both CD4+ and CD8+ T-cells in protection against melioidosis, and an interaction between diabetes and cellular responses. This supports development of vaccine strategies that induce strong T-cell responses for the control of intracellular pathogens such as B. pseudomallei.

  12. Disease: H00317 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available lling bacillus Burkholderia pseudomallei. It predominantly affects people in regular contact with soil and w...ater and is endemic in areas of Southeast Asia and Northern Australia. There are many possible disease manif

  13. 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.

  14. A CpG oligonucleotide can protect mice from a low aerosol challenge dose of Burkholderia mallei.

    Science.gov (United States)

    Waag, David M; McCluskie, Michael J; Zhang, Ningli; Krieg, Arthur M

    2006-03-01

    Treatment with an oligodeoxynucleotide (ODN) containing CPG motifs (CpG ODN 7909) was found to protect BALB/c mice from lung infection or death after aerosol challenge with Burkholderia mallei. Protection was associated with enhanced levels of gamma interferon (IFN-gamma)-inducible protein 10, interleukin-12 (IL-12), IFN-gamma, and IL-6. Preexposure therapy with CpG ODNs may protect victims of a biological attack from glanders.

  15. A CpG Oligonucleotide Can Protect Mice from a Low Aerosol Challenge Dose of Burkholderia mallei

    OpenAIRE

    Waag, David M.; Michael J. McCluskie; Zhang, Ningli; Krieg, Arthur M.

    2006-01-01

    Treatment with an oligodeoxynucleotide (ODN) containing CPG motifs (CpG ODN 7909) was found to protect BALB/c mice from lung infection or death after aerosol challenge with Burkholderia mallei. Protection was associated with enhanced levels of gamma interferon (IFN-γ)-inducible protein 10, interleukin-12 (IL-12), IFN-γ, and IL-6. Preexposure therapy with CpG ODNs may protect victims of a biological attack from glanders.

  16. 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.

  17. Evaluation of PCR, DNA hybridization and immunomagnetic separation — PCR for detection of Burkholderia mallei in artificially inoculated environmental samples

    OpenAIRE

    2010-01-01

    Glanders is highly contagious disease of equines, caused by Burkholderia mallei. The disease though rare, can be transmitted to humans. Here, we report a strategy for rapid detection of B. mallei from environmental samples. Different bacteriological media were evaluated and brain heart infusion broth medium with selective supplements (BHIB-SS) of penicillin (200 U/ml) and crystal violet (1:10,00000) was found to support the maximum growth of B. mallei even in the presence of other bacteria li...

  18. Endophytic colonization of rice (Oryza sativa L.) by the diazotrophic bacterium Burkholderia kururiensis and its ability to enhance plant growth

    OpenAIRE

    Mattos, Katherine A; Vania L.M. Pádua; Alexandre Romeiro; Hallack,Leticia F.; Bianca C. Neves; Tecia M.U. Ulisses; Claudia F. Barros; Adriane R Todeschini; Previato, José O.; Lucia Mendonça-Previato

    2008-01-01

    Burkholderia kururiensis is a diazotrophic bacterium originally isolated from a polluted aquifer environment and presents a high level of similarity with the rice endophyte "B. brasilensis" species. This work assessed the ability of B. kururiensis to endophytically colonize rice plantlets by monitoring different tissues of root-inoculated plants for the presence of bacterial growth in different media, electron microscopy and by 16S rDNA analysis. Observations of roots, stems and leaves of ino...

  19. 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....

  20. Genome Sequence Alterations Detected upon Passage of Burkholderia mallei ATCC 23344 in Culture and in Mammalian Hosts

    Science.gov (United States)

    2006-09-05

    Bacillus anthracis and Bacillus cereus . Antimicrob Agents Chemother 2002, 46(2):511-513. 39. Jones AL, Needham RH, Clancy A, Knoll KM, Rubens CE...Background Burkholderia mallei is a nonmotile, Gram-negative bacillus and the causative agent of a severe disease known as glan- ders. Humans are accidental...isolate, three in the human liver isolate, and five in the human blood isolate Table 1: Perfect simple sequence repeats (SSRs) identified in the B

  1. Exploration of the transcription factors that regulate the expression of the haloacid operon in Burkholderia caribensis MBA4

    OpenAIRE

    Deng, Liyu; 鄧麗瑜

    2014-01-01

    Bacterial dehalogenase is a key enzyme involved in bioremediation of halogenated organic compounds. A dehalogenase, Deh4a, was isolated from the Gram-negative bacterium Burkholderia caribensis MBA4, which can utilize haloacetic acids as carbon source. The haloacid operon in MBA4 was identified and characterized. It is composed of the structural genes forDeh4a and a transporter Deh4p. Transcription of this operon is negatively regulated, but the mechanism and the relevant regulator are still p...

  2. 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

  3. Changes in the repertoire of natural antibodies caused by immunization with bacterial antigens

    DEFF Research Database (Denmark)

    Shilova, N V; Navakouski, M J; Huflejt, M

    2011-01-01

    The repertoire of natural anti-glycan antibodies in naïve chickens and in chickens immunized with bacteria Burkholderia mallei, Burkholderia pseudomallei, and Francisella tularensis as well as with peptides from an outer membrane protein of B. pseudomallei was studied. A relatively restricted...... pattern of natural antibodies (first of all IgY against bacterial cell wall peptidoglycan fragments, L-Rha, and core N-acetyllactosamine) shrank and, moreover, the level of detectable antibodies decreased as a result of immunization....

  4. A new bacterial disease of carnation in Portugal caused by Burkholderia andropogonis

    Directory of Open Access Journals (Sweden)

    Madalena Eloy

    2008-12-01

    Full Text Available 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. Pathogenicity tests on china pink plants, re-isolation of the pathogen from inoculated plants and further PCR testing confirmed the identification of the bacterium. Infected plants came from an open air nursery and the whole production was destroyed to avoid dissemination of the pathogen.A ocorrência da mancha bacteriana do craveiro causada por Burkholderia andropogonis é pela primeira vez assinalada em Portugal. Os sintomas observados consistiam em manchas em forma de olho-de-perdiz em todos os órgãos aéreos das plantas afectadas, frequentemente circundadas por halos hidrópicos nas folhas. À medida que a doença progredia, as lesões adquiriam uma coloração castanha escura, acabando os órgãos afectados por secar. A identificação do agente causal da doença baseou-se no estudo dos seus caracteres fenotípicos e na Reacção em Cadeia da Polimerase (PCR, utilizando os iniciadores específicos Pf/Pr dirigidos à região 16S rDNA de B. andropogonis. A identificação foi confirmada por ensaios de patogenicidade em cravinas, reisolamento do agente causal da doença a partir das plantas inoculadas e novos ensaios PCR. As plantas infectadas provinham de um viveiro ao ar livre e toda a produção foi destruída a fim de evitar a disseminação do patogéneo.

  5. Decoding the folding of Burkholderia glumae lipase: folding intermediates en route to kinetic stability.

    Directory of Open Access Journals (Sweden)

    Kris Pauwels

    Full Text Available The lipase produced by Burkholderia glumae folds spontaneously into an inactive near-native state and requires a periplasmic chaperone to reach its final active and secretion-competent fold. The B. glumae lipase-specific foldase (Lif is classified as a member of the steric-chaperone family of which the propeptides of α-lytic protease and subtilisin are the best known representatives. Steric chaperones play a key role in conferring kinetic stability to proteins. However, until present there was no solid experimental evidence that Lif-dependent lipases are kinetically trapped enzymes. By combining thermal denaturation studies with proteolytic resistance experiments and the description of distinct folding intermediates, we demonstrate that the native lipase has a kinetically stable conformation. We show that a newly discovered molten globule-like conformation has distinct properties that clearly differ from those of the near-native intermediate state. The folding fingerprint of Lif-dependent lipases is put in the context of the protease-prodomain system and the comparison reveals clear differences that render the lipase-Lif systems unique. Limited proteolysis unveils structural differences between the near-native intermediate and the native conformation and sets the stage to shed light onto the nature of the kinetic barrier.

  6. lux-Marking and application of carbofuran degrader Burkholderia cepacia PCL3.

    Science.gov (United States)

    Plangklang, Pensri; Reungsang, Alissara

    2011-10-01

    A luxAB-mutant of the carbofuran degrading bacterium Burkholderia cepacia PCL3 was successfully constructed with the capability to emit a luminescence signal of 1.6×10(-3)RLUcfu(-1). The mutant has a growth pattern and carbofuran degradation ability similar to PCL3 wild-type. The luminescent emission by PCL3:luxAB1 directly correlated with the metabolic activity of the cells. The optimal pH, temperature and n-decanal concentration for luminescence emission are 7.0, 35°C and 0.01%, respectively. PCL3:luxAB1 was used to assess the toxicity of carbofuran and carbofuran phenol in basal salt medium (BSM) in which the different sensitivity of the cells is dependent on the biomass concentration. With the luciferase system, the degradative fraction of the augmented PCL3:luxAB1 and the difference between the active augmented PCL3:luxAB1 and indigenous microorganisms at the contaminated site could be indicated.

  7. 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.

  8. Bioremediation of refinery wastewater using immobilised Burkholderia cepacia and Corynebacterium sp and their transconjugants

    Directory of Open Access Journals (Sweden)

    Abdullahi T. Ajao

    2013-07-01

    Full Text Available When oil spill occurs, it poses serious toxic hazards to all forms of life. Mixed culture of Burkholderia cepacia and Corynebacterium sp isolated from refinery sludge using selective enrichment technique was used for bioremediation of refinery wastewater in a laboratoryscale bioreactor. Physicochemical parameters of both raw and treated water were as determined and compared with Federal Environ - mental Protection Agency (FEPA-limit, Abuja, Nigeria to asses the efficiency of the bioremediation process. Each of the bacterium was screened for the presence of plasmid DNA and for the involvement or otherwise of plasmid in the bioremediation of wastewater. The immobilised cells showed percentage decrease in chemical oxygen demand (97%, biochemical oxygen demand (94%, phenol (98%, total petroleum hydrocarbon (79%, oil and grease (90% of the refinery waste water after 20 days of treatment while their transconjugants showed the multiplicative effect by achieving the same percentage after 10 days of treatment. Therefore, the findings revealed that bioaugmentation of wastewater using transmissible catabolic plasmid will enhance efficiency of the bioremediation by spreading the plasmid among indigenous microbial community either through horizontal gene transfer or transformation.

  9. [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.

  10. 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.

  11. Evaluation of recombinant proteins of Burkholderia mallei for serodiagnosis of glanders.

    Science.gov (United States)

    Pal, Vijai; Kumar, Subodh; Malik, Praveen; Rai, Ganga Prasad

    2012-08-01

    Glanders is a contagious disease caused by the Gram-negative bacillus Burkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e., crude preparations of whole cells. False-positive results obtained from other diagnostic tests utilizing crude antigens lead to financial losses to animal owners, and false-negative results can turn a risk into a possible threat. In this study, we report on the identification of diagnostic targets using bioinformatics tools for serodiagnosis of glanders. The identified gene sequences were cloned and expressed as recombinant proteins. The purified recombinant proteins of B. mallei were used in an indirect ELISA format for serodiagnosis of glanders. Two recombinant proteins, 0375H and 0375TH, exhibited 100% sensitivity and specificity for glanders diagnosis. The proteins also did not cross-react with sera from patients with the closely related disease melioidosis. The results of this investigation highlight the potential of recombinant 0375H and 0375TH proteins in specific and sensitive diagnosis of glanders.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Degradation of 4-nitrocatechol by Burkholderia cepacia: a plasmid-encoded novel pathway.

    Science.gov (United States)

    Chauhan, A; Samanta, S K; Jain, R K

    2000-05-01

    Pseudomonas cepacia RKJ200 (now described as Burkholderia cepacia) has been shown to utilize p-nitrophenol (PNP) as sole carbon and energy source. The present work demonstrates that RKJ200 utilizes 4-nitrocatechol (NC) as the sole source of carbon, nitrogen and energy, and is degraded with concomitant release of nitrite ions. Several lines of evidence, including thin layer chromatography, gas chromatography, 1H-nuclear magnetic resonance, gas chromatography-mass spectrometry, spectral analyses and quantification of intermediates by high performance liquid chromatography, have shown that NC is degraded via 1,2, 4-benzenetriol (BT) and hydroquinone (HQ) formation. Studies carried out on a PNP- derivative and a PNP+ transconjugant also demonstrate that the genes for the NC degradative pathway reside on the plasmid present in RKJ200; the same plasmid had earlier been shown to encode genes for PNP degradation, which is also degraded via HQ formation. It is likely, therefore, that the same sets of genes encode the further metabolism of HQ in NC and PNP degradation.

  14. Bacterial-Feeding Nematode Growth and Preference for Biocontrol Isolates of the Bacterium Burkholderia cepacia.

    Science.gov (United States)

    Carta, L K

    2000-12-01

    The potential of different bacterial-feeding Rhabditida to consume isolates of Burkholderia cepacia with known agricultural biocontrol ability was examined. Caenorhabditis elegans, Diploscapter sp., Oscheius myriophila, Pelodera strongyloides, Pristionchus pacificus, Zeldia punctata, Panagrellus redivivus, and Distolabrellus veechi were tested for growth on and preference for Escherichia coli OP50 or B. cepacia maize soil isolates J82, BcF, M36, Bc2, and PHQM100. Considerable growth and preference variations occurred between nematode taxa on individual bacterial isolates, and between different bacterial isolates on a given nematode. Populations of Diploscapter sp. and P. redivivus were most strongly suppressed. Only Z. punctata and P. pacificus grew well on all isolates, though Z. punctata preferentially accumulated on all isolates and P. pacificus had no preference. Oscheius myriophila preferentially accumulated on growth-supportive Bc2 and M36, and avoided less supportive J82 and PHQM100. Isolates with plant-parasitic nematicidal properties and poor fungicidal properties supported the best growth of three members of the Rhabditidae, C. elegans, O. myriophila, and P. strongyloides. Distolabrellus veechi avoided commercial nematicide M36 more strongly than fungicide J82.

  15. Susceptibility of Opportunistic Burkholderia glumae to Copper Surfaces Following Wet or Dry Surface Contact

    Directory of Open Access Journals (Sweden)

    Zhouqi Cui

    2014-07-01

    Full Text Available Burkholderia glumae has been proposed to have a potential risk to vulnerable communities. In this work, we investigated the antibacterial activity and mechanism of copper surfaces against multi-drug resistant B. glumae from both patients and rice plants. The susceptibility of B. glumae to copper surfaces was noted by a significant decline in viable bacterial counts, relative to the slight reduction of stainless steel and polyvinylchloride, both of which were used as control surfaces. The mode of action of bacterial killing was determined by examing the mutagenicity, DNA damage, copper ions accumulation, and membrane damage in bacterial cells. The results indicated that the cells exposed to copper surfaces did not cause severe DNA lesions or increase the mutation frequencies, but resulted in a loss of cell membrane integrity within minutes. Furthermore, bacterial cells exposed to copper surfaces accumulated significantly higher amounts of copper compared to control surfaces. Overall, this study showed that metallic copper had strong antibacterial effect against B. glumae by causing DNA and membrane damage, cellular accumulation of copper, and cell death following DNA degradation, which could be utilized to reduce the risk of bacterial contamination and infection.

  16. Susceptibility of opportunistic Burkholderia glumae to copper surfaces following wet or dry surface contact.

    Science.gov (United States)

    Cui, Zhouqi; Ibrahim, Muhammad; Yang, Chunlan; Fang, Yuan; Annam, Hussain; Li, Bin; Wang, Yanli; Xie, Guan-Lin; Sun, Guochang

    2014-07-09

    Burkholderia glumae has been proposed to have a potential risk to vulnerable communities. In this work, we investigated the antibacterial activity and mechanism of copper surfaces against multi-drug resistant B. glumae from both patients and rice plants. The susceptibility of B. glumae to copper surfaces was noted by a significant decline in viable bacterial counts, relative to the slight reduction of stainless steel and polyvinylchloride, both of which were used as control surfaces. The mode of action of bacterial killing was determined by examing the mutagenicity, DNA damage, copper ions accumulation, and membrane damage in bacterial cells. The results indicated that the cells exposed to copper surfaces did not cause severe DNA lesions or increase the mutation frequencies, but resulted in a loss of cell membrane integrity within minutes. Furthermore, bacterial cells exposed to copper surfaces accumulated significantly higher amounts of copper compared to control surfaces. Overall, this study showed that metallic copper had strong antibacterial effect against B. glumae by causing DNA and membrane damage, cellular accumulation of copper, and cell death following DNA degradation, which could be utilized to reduce the risk of bacterial contamination and infection.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. Enhanced Polychlorinated Biphenyl Removal in a Switchgrass Rhizosphere by Bioaugmentation with Burkholderia xenovorans LB400.

    Science.gov (United States)

    Liang, Yi; Meggo, Richard; Hu, Dingfei; Schnoor, Jerald L; Mattes, Timothy E

    2014-10-01

    Phytoremediation makes use of plants and associated microorganisms to clean up soils and sediments contaminated with inorganic and organic pollutants. In this study, switchgrass (Panicum virgatum) was used to test for its efficiency in improving the removal of three specific polychlorinated biphenyl (PCB) congeners (PCB 52, 77 and 153) in soil microcosms. The congeners were chosen for their ubiquity, toxicity, and recalcitrance. After 24 weeks of incubation, loss of 39.9 ± 0.41% of total PCB molar mass was observed in switchgrass treated soil, significantly higher than in unplanted soil (29.5 ± 3.4%) (p<0.05). The improved PCB removal in switchgrass treated soils could be explained by phytoextraction processes and enhanced microbial activity in the rhizosphere. Bioaugmentation with Burkholderia xenovorans LB400 was performed to further enhance aerobic PCB degradation. The presence of LB400 was associated with improved degradation of PCB 52, but not PCB 77 or PCB 153. Increased abundances of bphA (a functional gene that codes for a subunit of PCB-degrading biphenyl dioxygenase in bacteria) and its transcript were observed after bioaugmentation. The highest total PCB removal was observed in switchgrass treated soil with LB400 bioaugmentation (47.3 ± 1.22 %), and the presence of switchgrass facilitated LB400 survival in the soil. Overall, our results suggest the combined use of phytoremediation and bioaugmentation could be an efficient and sustainable strategy to eliminate recalcitrant PCB congeners and remediate PCB-contaminated soil.

  2. 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.

  3. [Bacteria of the Burkholderia cepacia complex: specific features of diagnostics, genome organization and metabolism].

    Science.gov (United States)

    Shaginian, I A; Chernukha, M Iu

    2003-01-01

    Modern data, related with the identification and typing of the complex B. cepacia bacteria, are analyzed in the article by using the poly-phase taxonomic approach. An optimal scheme for identifying and typing the complex B. cepacia bacteria, involving the microbiological and molecular-biological methods of laboratory diagnostics, is presented. The key and assumed factors of pathogenicity of the discussed bacteria are described. The possible phylogenetic relations of the complex B. cepacia bacteria with phytopathgens as well as with pathogenic bacteria of species Burkholderia, Pseudomonas, Escherichia, B. mallei, B. pdeudomallei, P. seruginosa and E. coli are described. A possible role of genome alterations and mutations in the genome of the complex B. cepacia bacteria (with the latter genome having unusual properties, i.e. a big size, and a considerable quantity of insertion sequences) in creating the conditions for the "pulsing" evolution "jerks", i.e. for a rapid change-over from saprophytism in the soil to a pathogenic causative agent of a viral-and-bacteriological infection. Such mechanism can be regarded as a rapid and radical adaptation of a microorganism under the conditions of changing ecological niches.

  4. Biosynthesis of antifungal and antibacterial polyketides by Burkholderia gladioli in coculture with Rhizopus microsporus.

    Science.gov (United States)

    Ross, Claudia; Opel, Viktoria; Scherlach, Kirstin; Hertweck, Christian

    2014-12-01

    Fungi-bacteria interactions can impact the course of fungal infection and biotechnological use. The mucoralean fungus Rhizopus microsporus, traditionally used in food fermentations (tempe and sufu), is frequently accompanied by Burkholderia gladioli pv. cocovenenans. When producing tempe bongkrek, the bacterial contamination can lead to lethal food-related intoxications caused by the respiratory toxin bongkrekic acid. To unveil the metabolic potential of the fungus-associated bacterium, we sequenced its genome, assigned secondary metabolite biosynthesis gene clusters and monitored the metabolic profile under various growth conditions. In addition to the bongkrekic acid biosynthesis gene cluster we found gene clusters coding for the biosynthesis of toxoflavin and a complex polyketide. The orphan polyketide synthase gene cluster was activated under conditions that emulate tempe production, which enabled isolation and structure elucidation of four members of the enacyloxin family of antibiotics, out of which one is new. Moreover, we found that the fungus positively influences the growth of the bacteria and dramatically increases bongkrekic acid production in stationary culture, which inhibits the growth of the fungus. These results showcase the context-dependent formation of antifungal and antibacterial agents at the fungal-bacterial interface, which may also serve as a model for scenarios observed in mixed infections.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. [Melioidosis in a Danish tourist returning from North-eastern Thailand].

    Science.gov (United States)

    Leth, Steffen; Wang, Mikala; Deutch, Susanna

    2014-06-09

    Melioidosis, an infectious disease caused by Burkholderia pseudomallei, is endemic in South East Asia and Northern Australia. It has a wide clinical diversity, spanning from asymptomatic cases to rapid septic shock and death. We present a case of pulmonary melioidosis in a Danish tourist returning from North-eastern Thailand. The patient was treated with intravenous ceftazidime followed by oral therapy with trimethoprim/sulfamethoxazole and subsequently switched to doxycycline due to abnormal liver function tests and eosinophilia, with no sign of relapse two months after antibiotic cessation.

  7. Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01 bio-converts (+)-catechin and (-)-epicatechin to (+)-taxifolin.

    Science.gov (United States)

    Otsuka, Yuichiro; Matsuda, Motoki; Sonoki, Tomonori; Sato-Izawa, Kanna; Goodell, Barry; Jelison, Jody; Navarro, Ronald R; Murata, Hitoshi; Nakamura, Masaya

    2016-12-01

    This study characterized the enzymatic ability of a cell-free extract from an acidophilic (+)-catechin degrader Burkholderia oxyphila (OX-01). The crude OX-01 extracts were able to transform (+)-catechin and (-)-epicatechin into (+)-taxifolin via a leucocyanidin intermediate in a two-step oxidation. Enzymatic oxidation at the C-4 position was carried out anaerobically using H2O as an oxygen donor. The C-4 oxidation occurred only in the presence of the 2R-catechin stereoisomer, with the C-3 stereoisomer not affecting the reaction. These results suggest that the OX-01 may have evolved to target both (+)-catechin and (-)-epicatechin, which are major structural units in plants.

  8. Complex of Burkholderia cepacia lipase with transition state analogue of 1-phenoxy-2-acetoxybutane: biocatalytic, structural and modelling study.

    Science.gov (United States)

    Luić, M; Tomić, S; Lescić, I; Ljubović, E; Sepac, D; Sunjić, V; Vitale, L; Saenger, W; Kojic-Prodić, B

    2001-07-01

    In a series of four racemic phenoxyalkyl-alkyl carbinols, 1-phenoxy-2-hydroxybutane (1) is enantioselectively acetylated by Burkholderia cepacia (formerly Pseudomonas cepacia) lipase with an E value > or = 200, whereas for the other three racemates E was found to be analogue with a tetrahedral P-atom, (R(P),S(P))-O-(2R)-(1-phenoxybut-2-yl)methylphosphonic acid chloride was prepared and crystallized in complex with B. cepacia lipase. The X-ray structure of the complex was determined, allowing to compare the conformation of the inhibitor with results of molecular modelling.

  9. 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 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......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...

  10. Burkholderia phytofirmans PsJN reduces damages to freezing temperature in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    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

  11. In vivo bioluminescence imaging of Burkholderia mallei respiratory infection and treatment in the mouse model

    Directory of Open Access Journals (Sweden)

    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.

  12. Genotyping of Burkholderia mallei from an outbreak of glanders in Bahrain suggests multiple introduction events.

    Directory of Open Access Journals (Sweden)

    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.

  13. 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

  14. Survey of Bartonella spp. in U.S. bed bugs detects Burkholderia multivorans but not Bartonella.

    Directory of Open Access Journals (Sweden)

    Virna L Saenz

    Full Text Available Bed bugs (Cimex lectularius L. have resurged in the United States and globally. Bed bugs are hematophagous ectoparasites of humans and other animals, including domestic pets, chickens, and bats, and their blood feeding habits contribute to their potential as disease vectors. Several species of Bartonella are re-emergent bacterial pathogens that also affect humans, domestic pets, bats and a number of other wildlife species. Because reports of both bed bugs and Bartonella have been increasing in the U.S., and because their host ranges can overlap, we investigated whether the resurgences of these medically important pathogens and their potential vector might be linked, by screening for Bartonella spp. in bed bugs collected from geographic areas where these pathogens are prevalent and from bed bugs that have been in culture in the laboratory for several years. We screened a total of 331 bed bugs: 316 bed bugs from 36 unique collections in 29 geographic locations in 13 states, 10 bed bugs from two colonies maintained in the laboratory for 3 yr, and 5 bed bugs from a colony that has been in culture since before the recent resurgence of bed bugs. Bartonella spp. DNA was screened using a polymerase chain reaction assay targeting the 16S-23S rRNA intergenic transcribed spacer region. Bartonella DNA was not amplified from any bed bug, but five bed bugs from four different apartments of an elderly housing building in North Carolina contained DNA sequences that corresponded to Burkholderia multivorans, an important pathogen in nosocomial infections that was not previously linked to an arthropod vector.

  15. 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.

  16. Homology modeling and protein engineering of alkane monooxygenase in Burkholderia thailandensis MSMB121: in silico insights.

    Science.gov (United States)

    Jain, Chakresh Kumar; Gupta, Money; Prasad, Yamuna; Wadhwa, Gulshan; Sharma, Sanjeev Kumar

    2014-07-01

    The degradation of hydrocarbons plays an important role in the eco-balancing of petroleum products, pesticides and other toxic products in the environment. The degradation of hydrocarbons by microbes such as Geobacillus thermodenitrificans, Burkhulderia, Gordonia sp. and Acinetobacter sp. has been studied intensively in the literature. The present study focused on the in silico protein engineering of alkane monooxygenase (ladA)-a protein involved in the alkane degradation pathway. We demonstrated the improvement in substrate binding energy with engineered ladA in Burkholderia thailandensis MSMB121. We identified an ortholog of ladA monooxygenase found in B. thailandensis MSMB121, and showed it to be an enzyme involved in an alkane degradation pathway studied extensively in Geobacillus thermodenitrificans. Homology modeling of the three-dimensional structure of ladA was performed with a crystal structure (protein databank ID: 3B9N) as a template in MODELLER 9v11, and further validated using PROCHECK, VERIFY-3D and WHATIF tools. Specific amino acids were substituted in the region corresponding to amino acids 305-370 of ladA protein, resulting in an enhancement of binding energy in different alkane chain molecules as compared to wild protein structures in the docking experiments. The substrate binding energy with the protein was calculated using Vina (Implemented in VEGAZZ). Molecular dynamics simulations were performed to study the dynamics of different alkane chain molecules inside the binding pockets of wild and mutated ladA. Here, we hypothesize an improvement in binding energies and accessibility of substrates towards engineered ladA enzyme, which could be further facilitated for wet laboratory-based experiments for validation of the alkane degradation pathway in this organism.

  17. Survey of Bartonella spp. in U.S. bed bugs detects Burkholderia multivorans but not Bartonella.

    Science.gov (United States)

    Saenz, Virna L; Maggi, Ricardo G; Breitschwerdt, Edward B; Kim, Jung; Vargo, Edward L; Schal, Coby

    2013-01-01

    Bed bugs (Cimex lectularius L.) have resurged in the United States and globally. Bed bugs are hematophagous ectoparasites of humans and other animals, including domestic pets, chickens, and bats, and their blood feeding habits contribute to their potential as disease vectors. Several species of Bartonella are re-emergent bacterial pathogens that also affect humans, domestic pets, bats and a number of other wildlife species. Because reports of both bed bugs and Bartonella have been increasing in the U.S., and because their host ranges can overlap, we investigated whether the resurgences of these medically important pathogens and their potential vector might be linked, by screening for Bartonella spp. in bed bugs collected from geographic areas where these pathogens are prevalent and from bed bugs that have been in culture in the laboratory for several years. We screened a total of 331 bed bugs: 316 bed bugs from 36 unique collections in 29 geographic locations in 13 states, 10 bed bugs from two colonies maintained in the laboratory for 3 yr, and 5 bed bugs from a colony that has been in culture since before the recent resurgence of bed bugs. Bartonella spp. DNA was screened using a polymerase chain reaction assay targeting the 16S-23S rRNA intergenic transcribed spacer region. Bartonella DNA was not amplified from any bed bug, but five bed bugs from four different apartments of an elderly housing building in North Carolina contained DNA sequences that corresponded to Burkholderia multivorans, an important pathogen in nosocomial infections that was not previously linked to an arthropod vector.

  18. Inactivation of [Fe-S] metalloproteins mediates nitric oxide-dependent killing of Burkholderia mallei.

    Directory of Open Access Journals (Sweden)

    Jessica Jones-Carson

    Full Text Available BACKGROUND: Much remains to be known about the mechanisms by which O(2-dependent host defenses mediate broad antimicrobial activity. METHODOLOGY/PRINCIPAL FINDINGS: We show herein that reactive nitrogen species (RNS generated by inducible nitric oxide (NO synthase (iNOS account for the anti-Burkholderia mallei activity of IFNgamma-primed macrophages. Inducible NOS-mediated intracellular killing may represent direct bactericidal activity, because B. mallei showed an exquisite sensitivity to NO generated chemically. Exposure of B. mallei to sublethal concentrations of NO upregulated transcription of [Fe-S] cluster repair genes, while damaging the enzymatic activity of the [Fe-S] protein aconitase. To test whether [Fe-S] clusters are critical targets for RNS-dependent killing of B. mallei, a mutation was constructed in the NO-induced, [Fe-S] cluster repair regulator iscR. Not only was the iscR mutant hypersusceptible to iNOS-mediated killing, but its aconitase pool was readily oxidized by NO donors as compared to wild-type controls. Although killed by authentic H(2O(2, which also oxidizes [Fe-S] clusters, B. mallei appear to be resilient to NADPH oxidase-mediated cytotoxicity. The poor respiratory burst elicited by this bacterium likely explains why the NADPH oxidase is nonessential to the killing of B. mallei while it is still confined within phagosomes. CONCLUSIONS/SIGNIFICANCE: Collectively, these findings have revealed a disparate role for NADPH oxidase and iNOS in the innate macrophage response against the strict aerobe B. mallei. To the best of our knowledge, this is the first instance in which disruption of [Fe-S] clusters is demonstrated as cause of the bactericidal activity of NO congeners.

  19. Extraction of lipase from Burkholderia cepacia by PEG/Phosphate ATPS and its biochemical characterization

    Directory of Open Access Journals (Sweden)

    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.

  20. Profiling of Burkholderia cepacia secretome at mid-logarithmic and early-stationary phases of growth.

    Directory of Open Access Journals (Sweden)

    Vanitha Mariappan

    Full Text Available BACKGROUND: Burkholderia cepacia is a Gram-negative pathogen that causes serious respiratory infections in immunocompromised patients and individuals with cystic fibrosis. This bacterium is known to release extracellular proteins that may be involved in virulence. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, B. cepacia grown to mid-logarithmic and early-stationary phases were investigated on their ability to invade and survive intracellularly in A549 lung epithelial cells in order to discern the fate of these bacteria in the pathogenesis of B. cepacia lung infections in in vitro condition. The early-stationary phase B. cepacia was demonstrated to be more invasive than mid-logarithmic phase. In addition, culture supernatants of B. cepacia obtained from these phases of growth were also demonstrated to cause different cytotoxic potency on the A549 human lung epithelial cells. Profiling of the supernatants using the gel-based proteomics approach identified 43 proteins that were commonly released in both the growth phases and 40 proteins newly-released at the early-stationary phase. The latter proteins may account for the higher cytotoxic activity of the early-stationary culture supernatant compared to that obtained at the mid-logarithmic phase. Among the newly-released proteins in the early-stationary phase supernatant were flagellar hook-associated domain protein (FliD, flagellar hook-associated protein (FlgK, TonB-dependent siderophore (Fiu, Elongation factor G (FusA, phosphoglycerate kinase (Pgk and sulfatase (AslA which are known for their virulence. CONCLUSION/SIGNIFICANCE: Differences in the ability of B. cepacia to invade and survive intracellularly inside the epithelial cells at different phases of growth may improve our understanding of the varied disease progressions associated with B. cepacia infections. In addition, the identified culture supernatant proteins may be used as targets for the development of new strategies to

  1. Manganese scavenging and oxidative stress response mediated by type VI secretion system in Burkholderia thailandensis.

    Science.gov (United States)

    Si, Meiru; Zhao, Chao; Burkinshaw, Brianne; Zhang, Bing; Wei, Dawei; Wang, Yao; Dong, Tao G; Shen, Xihui

    2017-02-27

    Type VI secretion system (T6SS) is a versatile protein export machinery widely distributed in Gram-negative bacteria. Known to translocate protein substrates to eukaryotic and prokaryotic target cells to cause cellular damage, the T6SS has been primarily recognized as a contact-dependent bacterial weapon for microbe-host and microbial interspecies competition. Here we report contact-independent functions of the T6SS for metal acquisition, bacteria competition, and resistance to oxidative stress. We demonstrate that the T6SS-4 in Burkholderia thailandensis is critical for survival under oxidative stress and is regulated by OxyR, a conserved oxidative stress regulator. The T6SS-4 is important for intracellular accumulation of manganese (Mn(2+)) under oxidative stress. Next, we identified a T6SS-4-dependent Mn(2+)-binding effector TseM, and its interacting partner MnoT, a Mn(2+)-specific TonB-dependent outer membrane transporter. Similar to the T6SS-4 genes, expression of mnoT is regulated by OxyR and is induced under oxidative stress and low Mn(2+) conditions. Both TseM and MnoT are required for efficient uptake of Mn(2+) across the outer membrane under Mn(2+)-limited and -oxidative stress conditions. The TseM-MnoT-mediated active Mn(2+) transport system is also involved in contact-independent bacteria-bacteria competition and bacterial virulence. This finding provides a perspective for understanding the mechanisms of metal ion uptake and the roles of T6SS in bacteria-bacteria competition.

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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.

  7. Bacteria-associated haemophagocytic syndrome and septic pulmonary embolism caused by Burkholderia cepacia complex in a woman with chronic granulomatous disease.

    Science.gov (United States)

    Hisano, Michi; Sugawara, Kana; Tatsuzawa, Osamu; Kitagawa, Michihiro; Murashima, Atsuko; Yamaguchi, Koushi

    2007-05-01

    Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by recurrent infections with certain types of bacteria and fungi. Presented herein is the case of a 29 year old woman with CGD who suffered from bacteria-associated haemophagocytic syndrome and a septic pulmonary embolism following a uterine infection and sepsis, caused by Burkholderia cepacia complex.

  8. 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.

  9. Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24.

    Directory of Open Access Journals (Sweden)

    Sang-Yeop Lee

    Full Text Available Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs, including benzene, toluene, and xylene (BTX, as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the β-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX.

  10. 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.

  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. Purification and Biochemical Characterization of D-hydantoinase and D-N-carbamoylase from Burkholderia cepecia, njut01

    Institute of Scientific and Technical Information of China (English)

    李家璜; 李苏平; 严明; 姚忠; 欧阳平凯

    2005-01-01

    Hydantoinase and N-carbamoylase play important roles in the production of optically pure amino acids from racemic 5-monosubstituted hydantoins. In this report, hydantoinase and the N-carbamoylase from Burkholderia cepecia, njut01 were purified to homogeneity by chromatography (Pharmacia Explorer 100 system). The substrate specificity, enantioselectivity, pH dependence of activity and temperature stability of the activity were characterized. The results show that the hydantoinase and N-carbamoylase induced from Burkholderia cepecia, njut01 are both strict D-stereo selective enzymes. They both hydrolyze substrates with side chains containing aliphatic and aromatic residues with higher activity and affinity toward aromatic than aliphatic substituted substrates. The hydantoinase is a homotetramer with subtmit molecular weight near 52,000 and is active between pH 6.5 and 10 with an optimum near pH 9.0. The enzyme is active at temperatures up to 60~C, however, it appears instable at higher temperatures. The subunit molecular weight of N-carbamoylase is about 35KD. The N-carbamoylase is active in the pH range from 6.0 to 9.5. The optira-pH is 7.2 and the optinfizing bioconversion temperature of the N-carbamyolase is 52℃.

  13. Antimicrobial Peptides Containing Unnatural Amino Acid Exhibit Potent Bactericidal Activity against ESKAPE Pathogens

    Science.gov (United States)

    2013-01-01

    bacterial strains as previously reported by Hicks and co-workers45 Bacteria Compound 23 64 61 Yersinia pestis C092 500 62.5 32.2 Brucella melitensis...16M 500 125 125 Brucella abortus 2308 500 62.5 125 Francisella tularensis 250 0.04 125 Burkholderia mallei 500 0.04 500 Burkholderia pseudomallei 500

  14. Disease: H00316 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available d to humans by direct contact with infected animals as veterinarians, grooms, and farmers. The causative age...allei. Mil Med 174:647-51 (2009) PMID:18221181 (description) Gilad J Burkholderia mallei and Burkholderia pseudomallei: the causa

  15. [Molecular-genetic approaches to diagnosis and intraspecific typing of causative agents of glanders and melioidosis].

    Science.gov (United States)

    Antonov, V A; Iliukhin, V I

    2005-01-01

    Pathogenic Burkholderia--Burkholderia mallei and Burkholderia pseudomallei--are causative agents of glanders and melioidosis, severe infectious diseases of man and animals. They are regarded as potential agents of bioterrorism. The existing bacteriological and immunological methods of identification of B. mallei and B. pseudomallei are not efficient enough for the rapid diagnosis and typing of strains. Described in the paper are molecular methods of detection of the agents by PCR, hybridization and strain typing made on the basis of bacterial total cell protein profiles, RAPD, ribotyping as well as of plasmid and DNA microrestriction analyses.

  16. Purification and characterization of toluene 2-monooxygenase from Burkholderia cepacia G4.

    Science.gov (United States)

    Newman, L M; Wackett, L P

    1995-10-31

    Recent in vivo studies indicate that ring monooxygenation is a widespread mechanism by which bacteria metabolize aromatic hydrocarbons and obtain carbon and energy. In this study, toluene 2-monooxygenase from Burkholderia (formerly Pseudomonas) cepacia G4 was purified to homogeneity and found to be a three-component enzyme system. The reconstituted enzyme system oxidized toluene to o-cresol and o-cresol to 3-methylcatechol, an important intermediate for growth of the bacterium on toluene. Steady-state kinetic parameters measured for the water-soluble substrate o-cresol were a Km of 0.8 microM and a Vmax of 131 nmol min-1 (mg of hydroxylase protein)-1. The three protein components were (1) a 40 kDa polypeptide containing one FAD and a [2Fe2S] cluster, (2) a 10.4 kDa polypeptide that contained no identifiable metals or organic cofactors, and (3) a 211 kDa alpha 2 beta 2 gamma 2 component containing five to six iron atoms. The 40 kDa flavo-iron-sulfur protein oxidized NADH and transferred electrons to cytochrome c, dyes, and the alpha 2 beta 2 gamma 2 component. It is analogous to other NADH oxidoreductase components found in a wide range of bacterial mono- and dioxygenases. The 10.4 kDa component, added to the other two components and NADH, increased toluene oxidation rates 10-fold. The alpha 2 beta 2 gamma 2 component was indicated to contain the site for toluene binding and hydroxylation by the following observations: (1) tight binding to a toluene affinity column; (2) oxidation of toluene after reduction of the protein with dithionite and adding O2; (3) H2O2-dependent toluene oxidation and catalase activity; and (4) spectroscopic studies of the iron atoms in the component. The alpha 2 beta 2 gamma 2 component had no significant absorbance in the visible region. EPR spectroscopy yielded a signal at g = 16 upon addition of > 2 equiv of electrons per 2 Fe atoms. Taken with the quantitation of five to six iron atoms, the data suggest that the alpha 2 beta 2 gamma 2

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Research progress in quorum sensing and quorum quenching of the rice pathogen Burkholderia plantarii%水稻病原菌 Burkholderia plantarii群体感应和群体淬灭研究进展

    Institute of Scientific and Technical Information of China (English)

    MATSUMOTO Haruna; 王蒙岑; 桂文君; 郭逸蓉; 朱国念

    2016-01-01

    植物伯克霍尔德菌 Burkholderia plantarii 是引起水稻秧苗细菌性立枯病的重要病原菌之一,其侵染性、繁殖力及适应性均很强,严重威胁中国水稻生产。文章围绕 B. plantarii 的发生、危害及致病机理,着重论述了细菌群体感应系统(quorum sensing,QS)的生理功能及其在B. plantarii 致病力调控方面的最新研究进展,并进一步从根际微生物互作角度,综述了种间信号分子对病原菌群体淬灭(quorum quenching)的作用机制,同时结合种间信号分子的独特性,展望了其在新型微生物杀菌剂研发中的重要性和应用潜力。%Burkholderia plantarii is a causal agent of bacterial seedling blight of rice. This pathogen is of strong infectivity, reproductive capacity and adapt ability, and thus poses a serious threat to rice production. Hereby, the occurrence, damage and pathogenic mechanism of B. plantarii are introduced. Research progress of physiological traits and the regulation of pathogenicity controlled by bacterial quorum sensing (QS) in B. plantarii are also discussed. Moreover, from the point of view of microbial interaction in rhizosphere, the mechanisms of quorum quenching by interspecific signaling molecules against bacterial pathogens are summarized. According to the characteristics of the interspecies signaling molecules, their importance and application potential in development of novel bactericides are also previewed.

  1. Humoral Immune Responses in a Human Case of Glanders

    OpenAIRE

    Waag, David M.; England, Marilyn J.; DeShazer, David

    2012-01-01

    Within 2 months of acquiring glanders, a patient developed 8-, 16-, and 4-fold increases, respectively, in specific IgA, IgG, and IgM serological titers against Burkholderia mallei. Within 14 months of infection, the titers decreased to the baseline. Serum from this patient was also highly reactive against Burkholderia pseudomallei whole cells. Burkholderia mallei whole cells did not react with sera from patients with other diseases. Therefore, an assay using a B. mallei cellular diagnostic a...

  2. 类鼻疽伯克霍尔德菌胞内运动相关蛋白BimA的生物学特性研究进展%Biological characteristics and research progress about Burkholderia pseudomallei intracellular movement associated protein (BimA)

    Institute of Scientific and Technical Information of China (English)

    胡艺; 方瑶; 毛旭虎

    2016-01-01

    类鼻疽伯克霍尔德菌是一种热带医学病原体,现被美国CDC提升为1类致病菌严加防范.类鼻疽伯克霍尔德菌所导致的疾病称为类鼻疽,致死率和复发率都很高,广泛流行于东南亚及我国南海周边地区,已成为严峻的公共卫生问题.作为一种兼性胞内菌,类鼻疽伯克霍尔德菌的胞内运动能力为其逃逸机体免疫和细胞间的扩散提供了重要基础.胞内动力相关蛋白A(BimA)是一种类鼻疽菌自分泌的、与其胞内运动相关的关键分子,它通过操控肌动蛋白实现干预细菌的胞内运动,在类鼻疽菌的感染和致病中发挥着重要作用.全面、深入地了解BimA在类鼻疽菌感染中的作用机制对寻找有效预防和控制该病原的传播等方面具有重要意义.本文即综述了BimA蛋白的生物学特性分析,结合研究报道探讨其主导细菌胞内运动、干扰宿主胞内免疫、实现细菌胞间传播的机制及意义.

  3. NCBI nr-aa BLAST: CBRC-XTRO-01-0683 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0683 ref|YP_991995.1| major facilitator family transporter [Burkholder...ia mallei SAVP1] ref|YP_001028442.1| putative 4-hydroxybenzoate transporter [Burkholderia mallei NCTC 10229]... ref|YP_001057942.1| major facilitator family transporter [Burkholderia pseudomallei 668] ref|YP_001079676.1...| major facilitator family transporter [Burkholderia mallei NCTC 10247] gb|ABM523...08.1| major facilitator family transporter [Burkholderia mallei SAVP1] gb|ABN00840.1| putative 4-hydroxybenz

  4. Effect of Azospirillum brasilense and Burkholderia unamae Bacteria on Maize Photosynthetic Activity Evaluated Using the Photoacoustic Technique

    Science.gov (United States)

    Gordillo-Delgado, F.; Marín, E.; Calderón, A.

    2016-09-01

    In this work, the photosynthetic process of maize plants ( Zea mays), which were grown using seeds inoculated with plant growth promoting bacteria Azospirillum brasilense and Burkholderia unamae, was monitored. Photothermal and photobaric signals obtained by a time-resolved photoacoustic measurement configuration were used for measuring the oxygen evolution rate in situ. A frequency-resolved configuration of the method was utilized to determine the oxygen diffusion coefficient and the thermal diffusivity of the maize leaves. The latter parameters, which can be used as indicators of the photosynthetic activity of maize, are found to vary according to the plant-microbe interaction. Treatment with plant growth promoting bacteria induced a decrease in the oxygen diffusion coefficient of about 20 %.

  5. Biodiesel synthesis and conformation of lipase from Burkholderia cepacia in room temperature ionic liquids and organic solvents.

    Science.gov (United States)

    Liu, Yun; Chen, Dawei; Yan, Yunjun; Peng, Cheng; Xu, Li

    2011-11-01

    Biodiesel synthesis and conformation of Burkholderia cepacia lipase (BCL) were studied in 19 different room temperature ionic liquids (RTLLs) with a range of cation and anion structures. Overall, anion selection had a greater influence on biodiesel conversion than cation choice. RTILs containing Tf2N- and PF6- anions were suitable reaction media, while RTIL of [OmPy][BF4] was the best reaction medium with a biodiesel yield of 82.2±1.2%. RTILs with strong water miscible properties showed very low biodiesel yields. Conformational analysis by FT-IR revealed that higher biodiesel conversion in RTILs was correlated with a low tendency in α-helix content of BCL. An ultrasound-assisted biocatalysis process in RTILs was used to improve mass transfer rate, leading to 83% reduction of the reaction time for biodiesel production.

  6. Enhancing plant disease suppression by Burkholderia vietnamiensis through chromosomal integration of Bacillus subtilis chitinase gene chi113.

    Science.gov (United States)

    Zhang, Xinjian; Huang, Yujie; Harvey, Paul R; Ren, Yan; Zhang, Guangzhi; Zhou, Hongzi; Yang, Hetong

    2012-02-01

    Burkholderia vietnamiensis P418 is a plant growth-promoting rhizobacteria. A chitinase gene from Bacillus subtilis was cloned and stably integrated into the chromosome of using the transposon delivery vector, pUTkm1. Chitinase activity was detected in recombinant P418-37 but not in wild type P418. Recombinant P418-37 retained the in vitro growth rate, N(2)-fixation and phosphate and potassium-solubilizing characteristics of the wild type. P418-37 significantly (P Bipolaris sorokiniana, Verticillium dahliae and Gaeumannomyces graminis var. tritici compared with P418. In planta disease suppression assays indicated that P418-37 significantly (P < 0.05) enhanced suppression of wheat sheath blight (R. cerealis), cotton Fusarium wilt (F. oxysporium f.sp. vasinfectum) and tomato gray mould (Botrytis cinerea), relative to the wild type.

  7. 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.

  8. Melioidosis in acute cholangitis of diabetic patient: a forgotten diagnosis

    Directory of Open Access Journals (Sweden)

    Mohamad N

    2012-08-01

    Full Text Available Nasir Mohamad,1 Suresh Ponnusamy,2 Sunita Devi,3 Rishya Manikam,4 Ilya Irinaz Idrus,1 Nor Hidayah Abu Bakar51Department of Emergency Medicine, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia; 2AIMST University, Bedong, Malaysia; 3Hospital Sultan Abdul Halim, Sungai Petani, Malaysia; 4University Malaya Medical Centre, Kuala Lumpur, Malaysia; 5Department of Pathology, Hospital Raja Perempuan Zainab II, Kota Bharu, MalaysiaAbstract: Melioidosis presents with a wide range of clinical presentations, which include severe community-acquired pneumonia, septicemia, central nervous system infection, and less severe soft tissue infection. Hence, its diagnosis depends heavily on the clinical microbiology laboratory for culture. In this case report, we describe an atypical presentation of melioidosis in a 52-year-old man who had fever, right upper-abdominal pain, and jaundice for 15 days. Melioidosis caused by Burkholderia pseudomallei was subsequently diagnosed from blood culture. As a primary care physician, high suspicion index is of great importance. High suspicion index of melioidosis in a high-risk group patient, such as the patient with diabetes mellitus and diabetic foot, is crucial in view of atypical presentations of pseudomonas sepsis. A correct combination of antibiotic administration in the early phase of therapy will determine its successful outcome.Keywords: Burkholderia pseudomallei, atypical, high suspicion, primary care

  9. Fucose-binding lectin from opportunistic pathogen Burkholderia ambifaria binds to both plant and human oligosaccharidic epitopes.

    Science.gov (United States)

    Audfray, Aymeric; Claudinon, Julie; Abounit, Saïda; Ruvoën-Clouet, Nathalie; Larson, Göran; Smith, David F; Wimmerová, Michaela; Le Pendu, Jacques; Römer, Winfried; Varrot, Annabelle; Imberty, Anne

    2012-02-03

    Burkholderia ambifaria is generally associated with the rhizosphere of plants where it has biocontrol effects on other microorganisms. It is also a member of the Burkholderia cepacia complex, a group of closely related bacteria that cause lung infections in immunocompromised patients as well as in patients with granulomatous disease or cystic fibrosis. Our previous work indicated that fucose on human epithelia is a frequent target for lectins and adhesins of lung pathogens (Sulák, O., Cioci, G., Lameignère, E., Balloy, V., Round, A., Gutsche, I., Malinovská, L., Chignard, M., Kosma, P., Aubert, D. F., Marolda, C. L., Valvano, M. A., Wimmerová, M., and Imberty, A. (2011) PLoS Pathog. 7, e1002238). Analysis of the B. ambifaria genome identified BambL as a putative fucose-binding lectin. The 87-amino acid protein was produced recombinantly and demonstrated to bind to fucosylated oligosaccharides with a preference for αFuc1-2Gal epitopes. Crystal structures revealed that it associates as a trimer with two fucose-binding sites per monomer. The overall fold is a six-bladed β-propeller formed by oligomerization as in the Ralstonia solanacearum lectin and not by sequential domains like the fungal fucose lectin from Aleuria aurantia. The affinity of BambL for small fucosylated glycans is very high as demonstrated by microcalorimetry (K(D) < 1 μM). Plant cell wall oligosaccharides and human histo-blood group oligosaccharides H-type 2 and Lewis Y are bound with equivalent efficiency. Binding to artificial glycosphingolipid-containing vesicles, human saliva, and lung tissues confirmed that BambL could recognize a wide spectrum of fucosylated epitopes, albeit with a lower affinity for biological material from nonsecretor individuals.

  10. 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.

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

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    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.

  12. 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.

  13. Toxin-Antitoxin Systems in Clinical Pathogens

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    Laura Fernández-García

    2016-07-01

    Full Text Available Toxin-antitoxin (TA systems are prevalent in bacteria and archaea. Although not essential for normal cell growth, TA systems are implicated in multiple cellular functions associated with survival under stress conditions. Clinical strains of bacteria are currently causing major human health problems as a result of their multidrug resistance, persistence and strong pathogenicity. Here, we present a review of the TA systems described to date and their biological role in human pathogens belonging to the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. and others of clinical relevance (Escherichia coli, Burkholderia spp., Streptococcus spp. and Mycobacterium tuberculosis. Better understanding of the mechanisms of action of TA systems will enable the development of new lines of treatment for infections caused by the above-mentioned pathogens.

  14. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Technical Report

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    Fischer, N. O. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-01-13

    The goal of this proposal is to demonstrate that colocalization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. In the third quarter of the third year, F344 rats vaccinated with adjuvanted NLP formulations were challenged with F. tularensis SCHU S4 at Battelle. Preliminary data indicate that up to 65% of females vaccinated intranasally with an NLP-based formulation survived this challenge, compared to only 20% survival of naïve animals. In addition, NLPs were successfully formulated with Burkholderia protein antigens. IACUC approval for immunological assessments in BALB/c mice was received and we anticipate that these assessments will begin by March 2015, pending ACURO approval.

  15. Indagine epidemiologica locale sulle infezioni sostenute da Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderia cepacia e sensibilità agli antibiotici di questi microrganismi.

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    Valeria Di Marcello

    2007-03-01

    Full Text Available Background: The aim of this local surveillance study was to determine the distribution of Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderia cepacia in our geographic area, their impact in the hospital and community acquired infections and their resistance to antimicrobial agents currently used in the treatment of infections due to these microrganisms. Materials and Methods: During the period January 2001 - June 2003, 14.200 clinical isolates were collected from urine,wounds, catheters, body fluids, blood, respiratory tract specimens. Bacterial identifications were performed according to the standard methods (Murray, 2003 and antibiotic susceptibility tests were carry out in microassay by the automated system MicroScan (Dade Behring, Milano, Italy.The following antimicrobial agents were tested: piperacillin (PIP, ticarcillin (TIC, piperacillin-tazobactam (TZP, ticarcillin-clavulanic acid (TTC, ceftazidime (CAZ, ceftriaxone (CRO, aztreonam (ATM, imipenem (IPM, trimethoprim-sulfamethoxazole (SXT, gentamicin (CN, amikacin (AK, tobramycin (TOB, ciprofloxacin (CIP. Results: A total of 994 Pseudomonadaceae were isolated from in- (67% and out-patients (33%.They were P.aeruginosa (81%, other Pseudomonas species as P.fluorescens and P.putida (8%, S.maltophilia (9% and B.cepacia (2%.The great majority of the strains were collected from respiratory tract specimens (70% and urine (15%.The divisions from which derived the greater quantity of isolates were pediatric (33.8%, intensive care (22.7% and pneumology (10% units.Antibiotics more active against P. aeruginosa were IPM, CAZ,AK and TZP. IPM was effective against B. cepacia also.The other drugs, except SXT, displayed against this microrganism high rates of resistance. Even S. maltophilia was not susceptible to much antimicrobial agents, whereas SXT was the drug more active against this germ. Conclusion: P. aeruginosa was the microrganism more frequently isolated among non-fermenting Gram

  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. 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.

  18. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Annual Technical Report

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, N. O. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-04-16

    The goal of this proposal is to demonstrate that co-localization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of recombinant subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. NLPs are are biocompatible, high-density lipoprotein mimetics that are amenable to the incorporation of multiple, chemically-disparate adjuvant and antigen molecules. We hypothesize that the ability to co-localize optimized adjuvant formulations with subunit antigens within a single particle will enhance the stimulation and activation of key immune effector cells, increasing the protective efficacy of subunit antigen-based vaccines. While Burkholderia spp. and F. tularensis subunit antigens are the focus of this proposal, we anticipate that this approach is applicable to a wide range of DOD-relevant biothreat agents. The F344 rat aerosol challenge model for F. tularensis has been successfully established at Battelle under this contract, and Year 3 efficacy studies performed at Battelle demonstrated that an NLP vaccine formulation was able to enhance survival of female F344 rats relative to naïve animals. In addition, Year 3 focused on the incorporation of multiple Burkholderia antigens (both polysaccharides and proteins) onto adjuvanted NLPs, with immunological analysis poised to begin in the next quarter.

  19. 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.

  20. 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.

  1. 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

  2. The potential of TaqMan Array Cards for detection of multiple biological agents by real-time PCR.

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    Phillip A Rachwal

    Full Text Available The TaqMan Array Card architecture, normally used for gene expression studies, was evaluated for its potential to detect multiple bacterial agents by real-time PCR. Ten PCR assays targeting five biological agents (Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis were incorporated onto Array Cards. A comparison of PCR performance of each PCR in Array Card and singleplex format was conducted using DNA extracted from pure bacterial cultures. When 100 fg of agent DNA was added to Array Card channels the following levels of agent detection (where at least one agent PCR replicate returned a positive result were observed: Y. pestis 100%, B. mallei & F. tularensis 93%; B. anthracis 71%; B. pseudomallei 43%. For B. mallei & pseudomallei detection the BPM2 PCR, which detects both species, outperformed PCR assays specific to each organism indicating identification of the respective species would not be reproducible at the 100 fg level. Near 100% levels of detection were observed when 100 fg of DNA was added to each PCR in singleplex format with singleplex PCRs also returning sporadic positives at the 10 fg per PCR level. Before evaluating the use of Array Cards for the testing of environmental and clinical sample types, with potential levels of background DNA and PCR inhibitors, users would therefore have to accept a 10-fold reduction in sensitivity of PCR assays on the Array Card format, in order to benefit for the capacity to test multiple samples for multiple agents. A two PCR per agent strategy would allow the testing of 7 samples for the presence of 11 biological agents or 3 samples for 23 biological agents per card (with negative control channels.

  3. Trimethoprim/sulfamethoxazole (co-trimoxazole) prophylaxis is effective against acute murine inhalational melioidosis and glanders.

    Science.gov (United States)

    Barnes, Kay B; Steward, Jackie; Thwaite, Joanne E; Lever, M Stephen; Davies, Carwyn H; Armstrong, Stuart J; Laws, Thomas R; Roughley, Neil; Harding, Sarah V; Atkins, Timothy P; Simpson, Andrew J H; Atkins, Helen S

    2013-06-01

    Burkholderia pseudomallei is the causative agent of the disease melioidosis, which is prevalent in tropical countries and is intractable to a number of antibiotics. In this study, the antibiotic co-trimoxazole (trimethoprim/sulfamethoxazole) was assessed for the post-exposure prophylaxis of experimental infection in mice with B. pseudomallei and its close phylogenetic relative Burkholderia mallei, the causative agent of glanders. Co-trimoxazole was effective against an inhalational infection with B. pseudomallei or B. mallei. However, oral co-trimoxazole delivered twice daily did not eradicate infection when administered from 6h post exposure for 14 days or 21 days, since infected and antibiotic-treated mice succumbed to infection following relapse or immunosuppression. These data highlight the utility of co-trimoxazole for prophylaxis both of B. pseudomallei and B. mallei and the need for new approaches for the treatment of persistent bacterial infection.

  4. Biostimulation of soil polluted by 40000 ppm of waste motor oil and phytoremediation with Cicer arietinum and Burkholderia cepacia

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    Meza-Ramírez Janitzi Yunuén

    2016-08-01

    Full Text Available Soil polluted by 40000 ppm of waste residual oil (WRO, is a relative high hydrocarbons mix concentration according to Mexican regulation related with as the well know NOM-138-SEMARNAT/SSA1-2003 (NOM-138. Due to cause lost soil´s fertility, inhibiting microbial life and reducing vegetal production. To NOM-138 the highest limit of hydrocarbons mix allowed in soil is equal to 4400 ppm/kg. Aims of this research were: i Biostimulation of soil polluted by 40000 ppm of WRO by vermicompost and/or bovine compost, ii Phytoremediation by Cicer arietinum and Burkholderia cepacia to reduce WRO at below value compared to highest according to NOM-138. Results showed that biostimulation of soil with bovine compost eliminated WRO at 24000 ppm in 49 days. Then phytoremediation by C. arietinum and B. cepacia decreased WRO at 2760 ppm value below to compare to highest concentration allowed to NOM-138. It´s concluded that biore-mediation of soil impacted by relatively high concentration of WRO, the best strategy was to apply both biostimulation/phytoremediation that separate.

  5. Critical role of quorum sensing-dependent glutamate metabolism in homeostatic osmolality and outer membrane vesiculation in Burkholderia glumae

    Science.gov (United States)

    Kang, Yongsung; Goo, Eunhye; Kim, Jinwoo; Hwang, Ingyu

    2017-01-01

    Metabolic homeostasis in cooperative bacteria is achieved by modulating primary metabolism in a quorum sensing (QS)-dependent manner. A perturbed metabolism in QS mutants causes physiological stress in the rice bacterial pathogen Burkholderia glumae. Here, we show that increased bacterial osmolality in B. glumae is caused by unusually high cellular concentrations of glutamate and betaine generated by QS deficiencies. QS negatively controls glutamate uptake and the expression of genes involved in the glutamine synthetase and glutamine oxoglutarate aminotransferase cycles. Thus, cellular glutamate levels were significantly higher in the QS mutants than in the wild type, and they caused hyperosmotic cellular conditions. Under the hypotonic conditions of the periplasm in the QS mutants, outer membrane bulging and vesiculation were observed, although these changes were rescued by knocking out the gltI gene, which encodes a glutamate transporter. Outer membrane modifications were not detected in the wild type. These results suggest that QS-dependent glutamate metabolism is critical for homeostatic osmolality. We suggest that outer membrane bulging and vesiculation might be the outcome of a physiological adaptation to relieve hypotonic osmotic stress in QS mutants. Our findings reveal how QS functions to maintain bacterial osmolality in a cooperative population. PMID:28272446

  6. Selection of support materials for immobilization of Burkholderia cepacia PCL3 in treatment of carbofuran-contaminated water.

    Science.gov (United States)

    Laocharoen, S; Plangklang, P; Reungsang, A

    2013-01-01

    This study investigated the utilization of agricultural matrices as the support materials for cell immobilization to improve the technique of bioremediation. Coir, bulrush, banana stem and water hyacinth stem in both delignified and undelignified forms were used to immobilize Burkholderia cepacia PCL3 in bioremediation of carbofuran at 5 mg l(-1) in synthetic wastewater. Undelignified coir was found to be the most suitable support material for cell immobilization, giving the short half-life of carbofuran of 3.40 d (2.8 times shorter than the treatments with free cells). In addition, it could be reused three times without a loss in ability to degrade carbofuran. The growth and degradation ability of free cells were completely inhibited at the initial carbofuran concentrations of 250 mg l(-1), while there was no inhibitory effect of carbofuran on the immobilized cells. The results indicated a great potential for using the agricultural matrices as support material for cell immobilization to improve the overall efficiency of carbofuran bioremediation in contaminated water by B. cepacia PCL3.

  7. Enzymatic transesterification of microalgal oil from Chlorella vulgaris ESP-31 for biodiesel synthesis using immobilized Burkholderia lipase.

    Science.gov (United States)

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

    2012-03-01

    An indigenous microalga Chlorella vulgaris ESP-31 grown in an outdoor tubular photobioreactor with CO(2) aeration obtained a high oil content of up to 63.2%. The microalgal oil was then converted to biodiesel by enzymatic transesterification using an immobilized lipase originating from Burkholderia sp. C20. The conversion of the microalgae oil to biodiesel was conducted by transesterification of the extracted microalgal oil (M-I) and by transesterification directly using disrupted microalgal biomass (M-II). The results show that M-II achieved higher biodiesel conversion (97.3 wt% oil) than M-I (72.1 wt% oil). The immobilized lipase worked well when using wet microalgal biomass (up to 71% water content) as the oil substrate. The immobilized lipase also tolerated a high methanol to oil molar ratio (>67.93) when using the M-II approach, and can be repeatedly used for six cycles (or 288 h) without significant loss of its original activity.

  8. Outbreak of Burkholderia cepacia complex bacteremia in a chemotherapy day care unit due to intrinsic contamination of an antiemetic drug

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    T Singhal

    2015-01-01

    Full Text Available Background: In the end of 2009, a large number of patients with cancer undergoing chemotherapy at the day care unit of a private hospital in Mumbai, India developed Burkholderia cepacia complex (BCC blood stream infection (BSI. Objective: The objectives were to identify the source of the outbreak and terminate the outbreak as rapidly as possible. Materials and Methods: All infection control protocols and processes were reviewed. Intensive training was started for all nursing staff involved in patient care. Cultures were sent from the environment (surfaces, water, air, intravenous fluids, disinfectants and antiseptics and opened/unopened medication. Results: A total of 13 patients with cancer with tunneled catheters were affected with BCC BSI. The isolates were of similar antimicrobial sensitivity. No significant breach of infection control protocols could be identified. Cultures from the prepared intravenous medication bags grew BCC. Subsequently, culture from unused vials of the antiemetic granisetron grew BCC, whereas those from the unopened IV fluid bag and chemotherapy medication were negative. On review, it was discovered that the outbreak started when a new brand of granisetron was introduced. The result was communicated to the manufacturer and the brand was withdrawn. There were no further cases. Conclusions: This outbreak was thus linked to intrinsic contamination of medication vials. We acknowledge a delay in identifying the source as we were concentrating more on human errors in medication preparation and less on intrinsic contamination. We recommend that in an event of an outbreak, unopened vials be cultured at the outset.

  9. A novel nucleoside kinase from Burkholderia thailandensis: a member of the phosphofructokinase B-type family of enzymes.

    Science.gov (United States)

    Ota, Hiroko; Sakasegawa, Shin-Ichi; Yasuda, Yuko; Imamura, Shigeyuki; Tamura, Tomohiro

    2008-12-01

    The genome of the mesophilic Gram-negative bacterium Burkholderia thailandensis contains an open reading frame (i.e. the Bth_I1158 gene) that has been annotated as a putative ribokinase and PFK-B family member. Notably, although the deduced amino acid sequence of the gene showed only 29% similarity to the recently identified nucleoside kinase from hyperthermophilic archaea Methanocaldococcus jannaschii, 15 of 17 residues reportedly involved in the catalytic activity of M. jannaschii nucleoside kinase were conserved. The gene was cloned and functionally overexpressed in Rhodococcus erythropolis, and the purified enzyme was characterized biochemically. The substrate specificity of the enzyme was unusually broad for a bacterial PFK-B protein, and the specificity extended not only to purine and purine-analog nucleosides but also to uridine. Inosine was the most effective phosphoryl acceptor, with the highest k(cat)/K(m) value (80 s(-1).mm(-1)) being achieved when ATP served as the phosphoryl donor. By contrast, this enzyme exhibited no activity toward ribose, indicating that the recombinant enzyme was a nucleoside kinase rather than a ribokinase. To our knowledge, this is the first detailed analysis of a bacterial nucleoside kinase in the PFK-B family.

  10. 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.

  11. 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.

  12. Metabolomic and proteomic insights into carbaryl catabolism by Burkholderia sp. C3 and degradation of ten N-methylcarbamates.

    Science.gov (United States)

    Seo, Jong-Su; Keum, Young-Soo; Li, Qing X

    2013-11-01

    Burkholderia sp. C3, an efficient polycyclic aromatic hydrocarbon degrader, can utilize nine of the ten N-methylcarbamate insecticides including carbaryl as a sole source of carbon. Rapid hydrolysis of carbaryl in C3 is followed by slow catabolism of the resulting 1-naphthol. This study focused on metabolomes and proteomes in C3 cells utilizing carbaryl in comparison to those using glucose or nutrient broth. Sixty of the 867 detected proteins were involved in primary metabolism, adaptive sensing and regulation, transport, stress response, and detoxification. Among the 41 proteins expressed in response to carbaryl were formate dehydrogenase, aldehyde-alcohol dehydrogenase and ethanolamine utilization protein involved in one carbon metabolism. Acetate kinase and phasin were 2 of the 19 proteins that were not detected in carbaryl-supported C3 cells, but detected in glucose-supported C3 cells. Down-production of phasin and polyhydroxyalkanoates in carbaryl-supported C3 cells suggests insufficient carbon sources and lower levels of primary metabolites to maintain an ordinary level of metabolism. Differential metabolomes (~196 identified polar metabolites) showed up-production of metabolites in pentose phosphate pathways and metabolisms of cysteine, cystine and some other amino acids, disaccharides and nicotinate, in contract to down-production of most of the other amino acids and hexoses. The proteomic and metabolomic analyses showed that carbaryl-supported C3 cells experienced strong toxic effects, oxidative stresses, DNA/RNA damages and carbon nutrient deficiency.

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

    Science.gov (United States)

    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.

  14. Rhamnolipids from non-pathogenic Burkholderia thailandensis E264: Physicochemical characterization, antimicrobial and antibiofilm efficacy against oral hygiene related pathogens.

    Science.gov (United States)

    Elshikh, Mohamed; Funston, Scott; Chebbi, Alif; Ahmed, Syed; Marchant, Roger; Banat, Ibrahim M

    2017-05-25

    Biosurfactants are naturally occurring surface active compounds that have mainly been exploited for environmental applications and consumer products, with their biomedical efficacy an emerging area of research. Rhamnolipids area major group of biosurfactants that have been reported for their antimicrobial and antibiofilm efficacy. One of the main limiting factors for scaled up production and downstream applications of rhamnolipids is the fact that they are predominantly produced from the opportunistic pathogen Pseudomonas aeruginosa. In this article, we have reported the production and characterisation of long chain rhamnolipids from non-pathogenic Burkholderia thailandensis E264 (ATCC 700388). We have also investigated the antibacterial and antibiofilm properties of these rhamnolipids against some oral pathogens (Streptococcus oralis, Actinomyces naeslundii, Neisseria mucosa and Streptococcus sanguinis), important for oral health and hygiene. Treating these bacteria with different concentrations of long chain rhamnolipids resulted in a reduction of 3-4 log of bacterial viability, placing these rhamnolipids close to being classified as biocidal. Investigating long chain rhamnolipid efficacy as antibiofilm agents for prospective oral-related applications revealed good potency against oral-bacteria biofilms in a co-incubation experiments, in a pre-coated surface format, in disrupting immature biofilms and has shown excellent combination effect with Lauryl Sodium Sulphate which resulted in a drastic decrease in its minimal inhibitory concentration against different bacteria. Investigating the rhamnolipid permeabilization effect along with their ability to induce the formation of reactive oxygen species has shed light on the mechanism through which inhibition/killing of bacteria may occur.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. 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.

  17. Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain.

    Science.gov (United States)

    de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

    2014-06-01

    The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution.

  18. 77 FR 66850 - Public Workshop on Burkholderia: Exploring Current Issues and Identifying Regulatory Science Gaps

    Science.gov (United States)

    2012-11-07

    ... patients with septic shock (Ref. 1). A 9-year prospective study of melioidosis in northeast Thailand found... Human Melioidosis in Northeast Thailand,'' American Journal of Tropical Medicine and Hygiene, vol. 82(6.... Chierakul, et al., ``Risk Factors for Recurrent Melioidosis in Northeast Thailand,'' Clinical...

  19. Agricultural Use of Burkholderia (Pseudomonas) Cepacia: A Threat to Human Health?

    Science.gov (United States)

    1998-06-01

    effects of fungi such as Fusarium , Pythium, Rhizoctonia, Cylindrocarpum, and Botrytis. These widespread fungal pathogens cause seedling loss in...and two agricultural isolates from the rhizopheres of rice and maize were examined (38). The clinical isolates had identical 16S ribosomal rDNA

  20. 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

  1. Determinación de la Infección de Burkholderia glumae en Semillas de Variedades Comerciales Colombianas de Arroz Determination of the Infeccion of Burkholderia glumae in Comercial Colombian Rice Varieties

    Directory of Open Access Journals (Sweden)

    Nathalia María Vanesa Flórez Zapata

    2011-12-01

    Full Text Available El añublo bacterial de la panícula del arroz, ocasionado por Burkholderia glumae, es una enfermedad cada vez más difundida en Colombia. Por tal motivo es importante implementar un sistema de evaluación de la infección de este agente fitopatógeno en semilla, ya que ésta es una de las posibles fuentes de la enfermedad en campo. Semillas previamente desinfectadas, fueron sumergidas en suspensiones bacterianas y colocadas en cámara húmeda por siete días, luego de los cuales fueron evaluadas. Este sistema fue validado a través del análisis de la severidad de la infección en siete variedades de arroz y tres concentraciones del patógeno. El análisis reveló que las variedades F60, Panorama y F174, presentaron la menor severidad de la infección, aunque estadísticamente similar entre sí; mientras que, las variedades F369 y F733 presentaron niveles de severidad significativamente mayores y similares entre sí. Todas las variedades, con excepción de F60, presentaron diferencias entre las concentraciones del patógeno evaluadas, encontrándose una relación positiva entre concentración del inóculo y la severidad, la cual osciló según la variedad en la que fue estimada. Se concluyó que el modelo de evaluación de la infección fue exitoso dado que permitió encontrar diferencias en la severidad de la infección entre las variedades de arroz y las concentraciones de inóculo.The bacterial panicle blight of rice caused by Burkholderia glumae, is a disease that has gained increasing distribution in Colombia. Therefore it is important to implement an assessment system of infection of this phytopathogen in seeds, since this is one of the possible disease sources on the field. Thus, previously disinfected seeds were dipped in the bacterial suspension and placed in a moist chamber for 7 days, after which they were assessed. This assessment system, was validated by analyzing the infection severity level on seven rice varieties and three

  2. QUORUM SENSING AND 3-INDOLE ACETIC ACID DEGRADATION PLAY A ROLE IN COLONIZATION AND PLANT GROWTH PROMOTION OF ARABIDOPSIS THALIANA BY BURKHOLDERIA PHYTOFIRMANS

    OpenAIRE

    ZUÑIGA SEPULVEDA, ANA ANGELICA

    2013-01-01

    La comunicación molecular en la rizósfera tiene un importante papel en la regulación de rasgos que caracterizan la asociación planta-bacteria. Burkholderia phytofirmans PsJN es una bacteria promotora del crecimiento de plantas (PGPR) bien caracterizada y capaz de establecer interacciones rizosféricas y endofiticas con diferentes especies de plantas. Para una efectiva promoción del crecimiento es necesario que las PGPR tengan una colonización competente del hospedero. Utilizando cepas...

  3. Nanolipoprotein Particles (NLPs) as Versatile Vaccine Platforms for Co-delivery of Multiple Adjuvants with Subunit Antigens from Burkholderia spp. and F. tularensis - Technical Report

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, N. O. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-01-06

    The goal of this proposal is to demonstrate that colocalization of protein subunit antigens and adjuvants on nanolipoprotein particles (NLPs) can increase the protective efficacy of subunit antigens from Burkholderia spp. and Francisella tularensis against an aerosol challenge. In the second quarter of the third year, LLNL finalized all immunological assessments of NLP vaccine formulations in the F344 model. Battelle has immunized rats with three unique NLP formulations by either intramuscular or intranasal administration. All inoculations have been completed, and protective efficacy against an aerosolized challenge will begin at the end of October, 2014.

  4. Bacteria belonging to the genus Burkholderia are obligatory symbionts of the eriococcids Acanthococcus aceris Signoret, 1875 and Gossyparia spuria (Modeer, 1778) (Insecta, Hemiptera, Coccoidea).

    Science.gov (United States)

    Michalik, Katarzyna; Szklarzewicz, Teresa; Kalandyk-Kołodziejczyk, Małgorzata; Jankowska, Władysława; Michalik, Anna

    2016-05-01

    In the fat body cells of the scale insects, Gossyparia spuria and Acanthococcus aceris, numerous rod-shaped symbiotic bacteria occur. Molecular analyses have revealed that these microorganisms are closely related to the widely distributed bacterium Burkholderia. Ultrastructural observations have revealed that the bacteria are transovarially (vertically) transmitted from the mother to offspring. The microorganisms leave the fat body cells and invade ovarioles containing vitellogenic oocytes. They pass through the follicular epithelium in the neck region of the ovariole and enter the perivitelline space. Next, the symbionts infest the anterior region of the oocyte.

  5. Endophytic colonization of rice (Oryza sativa L. by the diazotrophic bacterium Burkholderia kururiensis and its ability to enhance plant growth

    Directory of Open Access Journals (Sweden)

    Katherine A. Mattos

    2008-09-01

    Full Text Available Burkholderia kururiensis is a diazotrophic bacterium originally isolated from a polluted aquifer environment and presents a high level of similarity with the rice endophyte "B. brasilensis" species. This work assessed the ability of B. kururiensis to endophytically colonize rice plantlets by monitoring different tissues of root-inoculated plants for the presence of bacterial growth in different media, electron microscopy and by 16S rDNA analysis. Observations of roots, stems and leaves of inoculated rice plantlets by electron microscopy revealed B. kururiensis colonization predominantly on root hair zones, demonstrating endophytic colonization primarily through the endodermis, followed by spreading into xylem vessels, a possible pathway leading to aerial parts. Although indifferent for the bacterial growth itself, addition of a nitrogen source was a limiting factor for endophytic colonization. As endophytic colonization was directly associated to an enhanced plant development, production of phytohormone auxin/indole-3-acetic acid by B. kururiensis was assayed with transgenic rice plantlets containing an auxin-responsive reporter (DR5-GUS. Our findings suggest the ability of auxin production by plant-associated B. kururiensis which may have a stimulatory effect on plant development, as evidenced by activation of DR5-GUS. We hereby demonstrate, for the first time, the ability of B. kururiensis to endophytically colonize rice, promoting both plant growth and rice grain yield.Burkholderia kururiensis é uma bactéria diazotrófica, originalmente isolada de um ambiente aquático poluído e apresenta alto nível de similaridade com a espécie endofítica "B. brasilensis" encontrada na planta de arroz. Este artigo demonstrou a habilidade de B. kururiensis colonizar endofiticamente plântulas de arroz, após esta bactéria ter sido inoculada na raiz das plantas. Esta capacidade foi confirmada pelo crescimento bacteriano em diferentes tecidos da planta

  6. 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.

  7. 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.

  8. 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

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. 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.

  12. Evaluation of PCR, DNA hybridization and immunomagnetic separation - PCR for detection of Burkholderia mallei in artificially inoculated environmental samples.

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    Merwyn, S; Kumar, S; Agarwal, G S; Rai, G P

    2010-06-01

    Glanders is highly contagious disease of equines, caused by Burkholderia mallei. The disease though rare, can be transmitted to humans. Here, we report a strategy for rapid detection of B. mallei from environmental samples. Different bacteriological media were evaluated and brain heart infusion broth medium with selective supplements (BHIB-SS) of penicillin (200 U/ml) and crystal violet (1:10,00000) was found to support the maximum growth of B. mallei even in the presence of other bacteria like Escherichia coli and Staphylococcus aureus. A polymerase chain reaction (PCR) and a DNA hybridization method was standardized for 823 bp specific dNA sequence of B. mallei. To enable the quicker and direct enrichment of B. mallei bacteria from environmental samples, an immunomagnetic separation (IMS) method was also standardized. Water, husk, grass and gram samples were artificially contaminated by B. mallei bacteria and after enrichment of B. mallei in BHIB-SS, detection was carried out by PCR and DNA hybridization. PCR was found to be a better method of the two with a detection limit of 10(4)-10(6) CFU/ml (6 h enrichment in BHIB-SS) in water and other particulate matrices. Detection by PCR in the above samples without enrichment in BHIBSS was carried out following IMS where the detection limit was about 1-2 log higher than PCR following enrichment in BHIB-SS. We recommend PCR for 823 bp for detection of B. mallei from environmental samples either following enrichment in BHIB-SS or IMS. IMS-PCR method may be preferred in situations where numbers of B. mallei bacteria are expected to be high and results are required in short time.

  13. The Autotransporter BpaB Contributes to the Virulence of Burkholderia mallei in an Aerosol Model of Infection.

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    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.

  14. Spatio-temporal responses of Arabidopsis leaves in photosynthetic performance and metabolite contents to Burkholderia phytofirmans PsJN

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    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.

  15. Isolations of enteric pathogens from synanthropic flies trapped in downtown Kuala Lumpur.

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    Sulaiman, S; Othman, M Z; Aziz, A H<