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Sample records for agent burkholderia pseudomallei

  1. Pathogenesis of Burkholderia pseudomallei and Burkholderia mallei.

    PubMed

    Larsen, Joseph C; Johnson, Nathan H

    2009-06-01

    Burkholderia pseudomallei and mallei are biological agents of military significance. There has been significant research in recent years to develop medical countermeasures for these organisms. This review summarizes work which details aspects of the pathogenesis of B. pseudomallei and mallei and discusses key scientific questions and directions for future research. PMID:19585782

  2. Attenuation of a select agent-excluded Burkholderia pseudomallei capsule mutant in hamsters.

    PubMed

    Gutierrez, Maria G; Warawa, Jonathan M

    2016-05-01

    Burkholderia pseudomallei is a Tier 1 select agent and potential bioweapon. Given it is potential to cause a lethal respiratory disease, research with fully virulent B. pseudomallei is conducted in Biosafety Level 3 (BSL-3) laboratory spaces. The logistical, financial, and administrative burden of Tier 1 select agent BSL-3 research has created an interest in mitigating such burdens through the use of either attenuated B. pseudomallei strains at BSL-2, or research with surrogate species, such as Burkholderia thailandensis. Previously, attenuated B. pseudomallei auxotroph mutants (asd and purM) have been approved for exclusion from select agent requirements, allowing for in vitro studies to be conducted at BSL-2. Acapsular B. pseudomallei mutants are known to be strongly attenuated in a variety of animal models, and yet acapsular B. pseudomallei mutants do not require nutritional supplementation, and can be studied within cultured macrophages, performing phenotypically similarly to parent strains. We demonstrate that the loss of a 30.8 kb region of the wcb capsule operon allows for a dramatic >4.46 log attenuation in a hamster intraperitoneal infection model, and report that this strain, JW270, has met criteria for exclusion from select agent requirements. PMID:26836271

  3. Clinical Features and Laboratory Diagnosis of Infection with the Potential Bioterrorism Agents Burkholderia Mallei and Burkholderia Pseudomallei

    PubMed Central

    Gilad, Jacob; Schwartz, David; Amsalem, Yoram

    2007-01-01

    Burkholderia mallei and Burkholderia pseudomallei are the causative organisms of Glanders and Melioidosis, respectively. Although now rare in Western countries, both organisms have recently gained much interest because of their unique potential as bioterrorism agents. These organisms are less familiar to medical and laboratory personnel than other select bioterrorism bacterial agents and thus heightened awareness of Glanders and Melioidosis is crucial in order to enable adequate emergency preparedness and response to deliberate release of B. mallei and B. pseudomallei. The microbiological diagnosis of both species in the clinical laboratory is complicated. This paper reviews the various challenges and pitfalls associated with the diagnosis of Melioidosis and Glanders in the clinical setting, with emphasis on the role of sentinel laboratories. PMID:23675037

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

    NASA Astrophysics Data System (ADS)

    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.

  5. The melioidosis agent Burkholderia pseudomallei and related opportunistic pathogens detected in faecal matter of wildlife and livestock in northern Australia.

    PubMed

    Höger, A C R; Mayo, M; Price, E P; Theobald, V; Harrington, G; Machunter, B; Choy, J Low; Currie, B J; Kaestli, M

    2016-07-01

    The Darwin region in northern Australia has experienced rapid population growth in recent years, and with it, an increased incidence of melioidosis. Previous studies in Darwin have associated the environmental presence of Burkholderia pseudomallei, the causative agent of melioidosis, with anthropogenic land usage and proximity to animals. In our study, we estimated the occurrence of B. pseudomallei and Burkholderia spp. relatives in faecal matter of wildlife, livestock and domestic animals in the Darwin region. A total of 357 faecal samples were collected and bacteria isolated through culture and direct DNA extraction after enrichment in selective media. Identification of B. pseudomallei, B. ubonensis, and other Burkholderia spp. was carried out using TTS1, Bu550, and recA BUR3-BUR4 quantitative PCR assays, respectively. B. pseudomallei was detected in seven faecal samples from wallabies and a chicken. B. cepacia complex spp. and Pandoraea spp. were cultured from wallaby faecal samples, and B. cenocepacia and B. cepacia were also isolated from livestock animals. Various bacteria isolated in this study represent opportunistic human pathogens, raising the possibility that faecal shedding contributes to the expanding geographical distribution of not just B. pseudomallei but other Burkholderiaceae that can cause human disease. PMID:26935879

  6. Experimental Phage Therapy for Burkholderia pseudomallei Infection

    PubMed Central

    Leang-Chung, Choh; Vellasamy, Kumutha Malar; Mariappan, Vanitha; Li-Yen, Chang; Vadivelu, Jamuna

    2016-01-01

    Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen intrinsically resistant to a variety of antibiotics. Phages have been developed for use as an alternative treatment therapy, particularly for bacterial infections that do not respond to conventional antibiotics. In this study, we investigated the use of phages to treat cells infected with B. pseudomallei. Phage C34 isolated from seawater was purified and characterised on the basis of its host range and morphology using transmission electron microscopy (TEM). Phage C34 was able to lyse 39.5% of B. pseudomallei clinical strains. Due to the presence of contractile tail, phage C34 is classified as a member of the family Myoviridae, a tailed double-stranded DNA virus. When 2 × 105 A549 cells were exposed to 2 × 107 PFU of phage C34, 24 hours prior to infection with 2 × 106 CFU of B. pseudomallei, it was found that the survivability of the cells increased to 41.6 ± 6.8% as compared to 22.8 ± 6.0% in untreated control. Additionally, application of phage successfully rescued 33.3% of mice infected with B. pseudomallei and significantly reduced the bacterial load in the spleen of the phage-treated mice. These findings indicate that phage can be a potential antimicrobial agent for B. pseudomallei infections. PMID:27387381

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

    PubMed Central

    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

  8. 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

    PubMed Central

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

    2014-01-01

    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. PMID:25044501

  9. Development of Vaccines Against Burkholderia Pseudomallei

    PubMed Central

    Patel, Natasha; Conejero, Laura; De Reynal, Melanie; Easton, Anna; Bancroft, Gregory J.; Titball, Richard W.

    2011-01-01

    Burkholderia pseudomallei is a Gram-negative bacterium which is the causative agent of melioidosis, a disease which carries a high mortality and morbidity rate in endemic areas of South East Asia and Northern Australia. At present there is no available human vaccine that protects against B. pseudomallei, and with the current limitations of antibiotic treatment, the development of new preventative and therapeutic interventions is crucial. This review considers the multiple elements of melioidosis vaccine research including: (i) the immune responses required for protective immunity, (ii) animal models available for preclinical testing of potential candidates, (iii) the different experimental vaccine strategies which are being pursued, and (iv) the obstacles and opportunities for eventual registration of a licensed vaccine in humans. PMID:21991263

  10. Flagellar Glycosylation in Burkholderia pseudomallei and Burkholderia thailandensis▿

    PubMed Central

    Scott, Andrew E.; Twine, Susan M.; Fulton, Kelly M.; Titball, Richard W.; Essex-Lopresti, Angela E.; Atkins, Timothy P.; Prior, Joann L.

    2011-01-01

    Glycosylation of proteins is known to impart novel physical properties and biological roles to proteins from both eukaryotes and prokaryotes. In this study, gel-based glycoproteomics were used to identify glycoproteins of the potential biothreat agent Burkholderia pseudomallei and the closely related but nonpathogenic B. thailandensis. Top-down and bottom-up mass spectrometry (MS) analyses identified that the flagellin proteins of both species were posttranslationally modified by novel glycans. Analysis of proteins from two strains of each species demonstrated that B. pseudomallei flagellin proteins were modified with a glycan with a mass of 291 Da, while B. thailandensis flagellin protein was modified with related glycans with a mass of 300 or 342 Da. Structural characterization of the B. thailandensis carbohydrate moiety suggests that it is an acetylated hexuronic acid. In addition, we have identified through mutagenesis a gene from the lipopolysaccharide (LPS) O-antigen biosynthetic cluster which is involved in flagellar glycosylation, and inactivation of this gene eliminates flagellar glycosylation and motility in B. pseudomallei. This is the first report to conclusively demonstrate the presence of a carbohydrate covalently linked to a protein in B. pseudomallei and B. thailandensis, and it suggests new avenues to explore in order to examine the marked differences in virulence between these two species. PMID:21602339

  11. Ultrastructural effects and antibiofilm activity of LFchimera against Burkholderia pseudomallei.

    PubMed

    Puknun, Aekkalak; Kanthawong, Sakawrat; Anutrakunchai, Chitchanok; Nazmi, Kamran; Tigchelaar, Wikky; Hoeben, Kees A; Veerman, Enno C I; Bolscher, Jan G M; Taweechaisupapong, Suwimol

    2016-02-01

    Lactoferrin chimera (LFchimera), a hybrid peptide containing the two antimicrobial stretches of the innate immunity factor bovine lactoferrin, viz. LFampin265-284 and LFcin17-30, has strikingly high antimicrobial activity against the category B pathogen Burkholderia pseudomallei. The action mechanisms of LFchimera against B. pseudomallei is not fully understood. The aim of this study was to further investigate the effect of treated B. pseudomallei with LFchimera using (immune) electron microscopy. The effects of LFchimera on biofilm formation and against preformed biofilm of B. pseudomallei were also determined. After exposure to LFchimera, transmission electron microscopy revealed swelling of the periplasmic space of B. pseudomallei and a highly inhomogeneous electron density in the intracellular DNA region. Localization of LFchimera in B. pseudomallei using immunoelectron microscopy showed gold particles in intracellular structures without accumulation on the membranes. LFchimera also possessed stronger bactericidal activity than ceftazidime against B. pseudomallei grown in biofilm. Moreover, limited exposure of B. pseudomallei to LFchimera at subcidal concentration could reduce biofilm formation. Altogether, the results indicate that LFchimera possesses antibacterial and antibiofilm activities and can modulate B. pseudomallei colonization. Therefore, the efficacy of LFchimera merits further development of this agent for the therapy of melioidosis. PMID:26754671

  12. Burkholderia pseudomallei induces IL-23 production in primary human monocytes.

    PubMed

    Kulsantiwong, Panthong; Pudla, Matsayapan; Boondit, Jitrada; Wikraiphat, Chanthiwa; Dunachie, Susanna J; Chantratita, Narisara; Utaisincharoen, Pongsak

    2016-06-01

    Burkholderia pseudomallei, a gram-negative intracellular bacterium, is a causative agent of melioidosis. The bacterium has been shown to induce the innate immune response, particularly pro-inflammatory cytokine production in several of both mouse and human cell types. In the present study, we investigate host immune response in B. pseudomallei-infected primary human monocytes. We discover that wild-type B. pseudomallei is able to survive and multiply inside the primary human monocytes. In contrast, B. pseudomallei LPS mutant, a less virulent strain, is susceptible to host killing during bacterial infection. Moreover, microarray result showed that wild-type B. pseudomallei but not B. pseudomallei LPS mutant is able to activate gene expression of IL-23 as demonstrated by the up-regulation of p19 and p40 subunit expression. Consistent with gene expression analysis, the secretion of IL-23 analyzed by ELISA also showed that wild-type B. pseudomallei induces a significantly higher level of IL-23 secretion than that of B. pseudomallei LPS mutant. These results implied that IL-23 may be an important cytokine for the innate immune response during B. pseudomallei infection. The regulation of IL-23 production may drive the different host innate immune responses between patients and may relate to the severity of melioidosis. PMID:26563410

  13. Burkholderia pseudomallei Isolates in 2 Pet Iguanas, California, USA

    PubMed Central

    Zehnder, Ashley M.; Hawkins, Michelle G.; Koski, Marilyn A.; Lifland, Barry; Byrne, Barbara A.; Swanson, Alexandra A.; Rood, Michael P.; Elrod, Mindy Glass; Beesley, Cari A.; Blaney, David D.; Ventura, Jean; Hoffmaster, Alex R.; Beeler, Emily S.

    2014-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, was isolated from abscesses of 2 pet green iguanas in California, USA. The international trade in iguanas may contribute to importation of this pathogen into countries where it is not endemic and put persons exposed to these animals at risk for infection. PMID:24447394

  14. Burkholderia pseudomallei isolates in 2 pet iguanas, California, USA.

    PubMed

    Zehnder, Ashley M; Hawkins, Michelle G; Koski, Marilyn A; Lifland, Barry; Byrne, Barbara A; Swanson, Alexandra A; Rood, Michael P; Gee, Jay E; Elrod, Mindy Glass; Beesley, Cari A; Blaney, David D; Ventura, Jean; Hoffmaster, Alex R; Beeler, Emily S

    2014-02-01

    Burkholderia pseudomallei, the causative agent of melioidosis, was isolated from abscesses of 2 pet green iguanas in California, USA. The international trade in iguanas may contribute to importation of this pathogen into countries where it is not endemic and put persons exposed to these animals at risk for infection. PMID:24447394

  15. Functional Characterization of Burkholderia pseudomallei Trimeric Autotransporters

    PubMed Central

    Campos, Cristine G.; Byrd, Matthew S.

    2013-01-01

    Burkholderia pseudomallei is a tier 1 select agent and the causative agent of melioidosis, a severe and often fatal disease with symptoms ranging from acute pneumonia and septic shock to a chronic infection characterized by abscess formation in the lungs, liver, and spleen. Autotransporters (ATs) are exoproteins belonging to the type V secretion system family, with many playing roles in pathogenesis. The genome of B. pseudomallei strain 1026b encodes nine putative trimeric AT proteins, of which only four have been described. Using a bioinformatic approach, we annotated putative domains within each trimeric AT protein, excluding the well-studied BimA protein, and found short repeated sequences unique to Burkholderia species, as well as an unexpectedly large proportion of ATs with extended signal peptide regions (ESPRs). To characterize the role of trimeric ATs in pathogenesis, we constructed disruption or deletion mutations in each of eight AT-encoding genes and evaluated the resulting strains for adherence to, invasion of, and plaque formation in A549 cells. The majority of the ATs (and/or the proteins encoded downstream) contributed to adherence to and efficient invasion of A549 cells. Using a BALB/c mouse model of infection, we determined the contributions of each AT to bacterial burdens in the lungs, liver, and spleen. At 48 h postinoculation, only one strain, Bp340::pDbpaC, demonstrated a defect in dissemination and/or survival in the liver, indicating that BpaC is required for wild-type virulence in this model. PMID:23716608

  16. Strategies for Intracellular Survival of Burkholderia pseudomallei.

    PubMed

    Allwood, Elizabeth M; Devenish, Rodney J; Prescott, Mark; Adler, Ben; Boyce, John D

    2011-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high mortality that is prevalent in tropical regions of the world. A key component of the pathogenesis of melioidosis is the ability of B. pseudomallei to enter, survive, and replicate within mammalian host cells. For non-phagocytic cells, bacterial adhesins have been identified both on the bacterial surface and associated with Type 4 pili. Cell invasion involves components of one or more of the three Type 3 Secretion System clusters, which also mediate, at least in part, the escape of bacteria from the endosome into the cytoplasm, where bacteria move by actin-based motility. The mechanism of actin-based motility is not clearly understood, but appears to differ from characterized mechanisms in other bacterial species. A small proportion of intracellular bacteria is targeted by host cell autophagy, involving direct recruitment of LC3 to endosomes rather than through uptake by canonical autophagosomes. However, the majority of bacterial cells are able to circumvent autophagy and other intracellular defense mechanisms such as the induction of inducible nitric oxide synthase, and then replicate in the cytoplasm and spread to adjacent cells via membrane fusion, resulting in the formation of multi-nucleated giant cells. A potential role for host cell ubiquitin in the autophagic response to bacterial infection has recently been proposed. PMID:22007185

  17. Morphological Alteration and Survival of Burkholderia pseudomallei in Soil Microcosms.

    PubMed

    Kamjumphol, Watcharaporn; Chareonsudjai, Pisit; Taweechaisupapong, Suwimol; Chareonsudjai, Sorujsiri

    2015-11-01

    The resilience of Burkholderia pseudomallei, the causative agent of melioidosis, was evaluated in control soil microcosms and in soil microcosms containing NaCl or FeSO4 at 30°C. Iron (Fe(II)) promoted the growth of B. pseudomallei during the 30-day observation, contrary to the presence of 1.5% and 3% NaCl. Scanning electron micrographs of B. pseudomallei in soil revealed their morphological alteration from rod to coccoid and the formation of microcolonies. The smallest B. pseudomallei cells were found in soil with 100 μM FeSO4 compared with in the control soil or soil with 0.6% NaCl (P < 0.05). The colony count on Ashdown's agar and bacterial viability assay using the LIVE/DEAD(®) BacLight(™) stain combined with flow cytometry showed that B. pseudomallei remained culturable and viable in the control soil microcosms for at least 120 days. In contrast, soil with 1.5% NaCl affected their culturability at day 90 and their viability at day 120. Our results suggested that a low salinity and iron may influence the survival of B. pseudomallei and its ability to change from a rod-like to coccoid form. The morphological changes of B. pseudomallei cells may be advantageous for their persistence in the environment and may increase the risk of their transmission to humans. PMID:26324731

  18. Prevalence of Burkholderia pseudomallei in Guangxi, China.

    PubMed

    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. PMID:19538822

  19. Contribution of Gene Loss to the Pathogenic Evolution of Burkholderia pseudomallei and Burkholderia mallei

    PubMed Central

    Moore, Richard A.; Reckseidler-Zenteno, Shauna; Kim, Heenam; Nierman, William; Yu, Yan; Tuanyok, Apichai; Warawa, Jonathan; DeShazer, David; Woods, Donald E.

    2004-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis. Burkholderia thailandensis is a closely related species that can readily utilize l-arabinose as a sole carbon source, whereas B. pseudomallei cannot. We used Tn5-OT182 mutagenesis to isolate an arabinose-negative mutant of B. thailandensis. Sequence analysis of regions flanking the transposon insertion revealed the presence of an arabinose assimilation operon consisting of nine genes. Analysis of the B. pseudomallei chromosome showed a deletion of the operon from this organism. This deletion was detected in all B. pseudomallei and Burkholderia mallei strains investigated. We cloned the B. thailandensis E264 arabinose assimilation operon and introduced the entire operon into the chromosome of B. pseudomallei 406e via homologous recombination. The resultant strain, B. pseudomallei SZ5028, was able to utilize l-arabinose as a sole carbon source. Strain SZ5028 had a significantly higher 50% lethal dose for Syrian hamsters compared to the parent strain 406e. Microarray analysis revealed that a number of genes in a type III secretion system were down-regulated in strain SZ5028 when cells were grown in l-arabinose, suggesting a regulatory role for l-arabinose or a metabolite of l-arabinose. These results suggest that the ability to metabolize l-arabinose reduces the virulence of B. pseudomallei and that the genes encoding arabinose assimilation may be considered antivirulence genes. The increase in virulence associated with the loss of these genes may have provided a selective advantage for B. pseudomallei as these organisms adapted to survival in animal hosts. PMID:15213162

  20. Genomic Sequence of Burkholderia multivorans NKI379, a Soil Bacterium That Inhibits the Growth of Burkholderia pseudomallei

    PubMed Central

    Hsueh, Pei-Tan; Liu, Jong-Kang; Chen, Ya-Lei; Liu, Pei-Ju; Ni, Wen-Fan; Chen, Yao-Shen; Wu, Keh-Ming

    2015-01-01

    Burkholderia multivorans NKI379 is a soil bacterium that exhibits an antagonistic effect against the growth of Burkholderia pseudomallei, the causative agent of the infectious disease melioidosis. We report the draft genomic sequence of B. multivorans NKI379, which has a G+C content of 67% and 5,203 candidate protein-encoding genes. PMID:26586873

  1. Genomic Sequence of Burkholderia multivorans NKI379, a Soil Bacterium That Inhibits the Growth of Burkholderia pseudomallei.

    PubMed

    Hsueh, Pei-Tan; Liu, Jong-Kang; Chen, Ya-Lei; Liu, Pei-Ju; Ni, Wen-Fan; Chen, Yao-Shen; Wu, Keh-Ming; Lin, Hsi-Hsun

    2015-01-01

    Burkholderia multivorans NKI379 is a soil bacterium that exhibits an antagonistic effect against the growth of Burkholderia pseudomallei, the causative agent of the infectious disease melioidosis. We report the draft genomic sequence of B. multivorans NKI379, which has a G+C content of 67% and 5,203 candidate protein-encoding genes. PMID:26586873

  2. Draft Genome Sequence of Burkholderia pseudomallei Strain 350105, Isolated in Hainan, China, in 1976.

    PubMed

    Song, Lihua; Yu, Yonghui; Feng, Le; He, Jun; Wang, Tao; Zhu, Hong; Duan, Qing

    2015-01-01

    Burkholderia pseudomallei is the etiological agent of the potentially fatal disease melioidosis. Here, we report the draft genome sequence of a virulent water isolate obtained from the Hainan Province of China in 1976, B. pseudomallei strain 350105. PMID:26472827

  3. Draft Genome Sequence of Burkholderia pseudomallei Strain 350105, Isolated in Hainan, China, in 1976

    PubMed Central

    Yu, Yonghui; Feng, Le; He, Jun; Wang, Tao; Zhu, Hong; Duan, Qing

    2015-01-01

    Burkholderia pseudomallei is the etiological agent of the potentially fatal disease melioidosis. Here, we report the draft genome sequence of a virulent water isolate obtained from the Hainan Province of China in 1976, B. pseudomallei strain 350105. PMID:26472827

  4. Heme Oxygenase-1 and Carbon Monoxide Promote Burkholderia pseudomallei Infection.

    PubMed

    Stolt, Claudia; Schmidt, Imke H E; Sayfart, Yana; Steinmetz, Ivo; Bast, Antje

    2016-08-01

    The environmental bacterium and potential biothreat agent Burkholderia pseudomallei causes melioidosis, an often fatal infectious disease. Increased serum bilirubin has been shown to be a negative predictive factor in melioidosis patients. We therefore investigated the role of heme oxygenase-1 (HO-1), which catalyzes the degradation of heme into the bilirubin precursor biliverdin, ferrous iron, and CO during B. pseudomallei infection. We found that infection of murine macrophages induces HO-1 expression, involving activation of several protein kinases and the transcription factor nuclear erythroid-related factor 2 (Nrf2). Deficiency of Nrf2 improved B. pseudomallei clearance by macrophages, whereas Nrf2 activation by sulforaphane and tert-butylhydroquinone with subsequent HO-1 induction enhanced intracellular bacterial growth. The HO-1 inducer cobalt protoporphyrin IX diminished proinflammatory cytokine levels, leading to an increased bacterial burden in macrophages. In contrast, HO-1 gene knockdown reduced the survival of intramacrophage B. pseudomallei Pharmacological administration of cobalt protoporphyrin IX to mice resulted in an enhanced bacterial load in various organs and was associated with higher mortality of intranasally infected mice. The unfavorable outcome of B. pseudomallei infection after HO-1 induction was associated with higher serum IL-6, TNF-α, and MCP-1 levels but decreased secretion of IFN-γ. Finally, we demonstrate that the CO-releasing molecule CORM-2 increases the B. pseudomallei load in macrophages and mice. Thus, our data suggest that the B. pseudomallei-mediated induction of HO-1 and the release of its metabolite CO impair bacterial clearance in macrophages and during murine melioidosis. PMID:27316684

  5. Volatile-sulfur-compound profile distinguishes Burkholderia pseudomallei from Burkholderia thailandensis.

    PubMed

    Inglis, Timothy J J; Hahne, Dorothee R; Merritt, Adam J; Clarke, Michael W

    2015-03-01

    Solid-phase microextraction gas chromatography-mass spectrometry (SPME-GCMS) was used to show that dimethyl sulfide produced by Burkholderia pseudomallei is responsible for its unusual truffle-like smell and distinguishes the species from Burkholderia thailandensis. SPME-GCMS can be safely used to detect dimethyl sulfide produced by agar-grown B. pseudomallei. PMID:25568444

  6. Volatile-Sulfur-Compound Profile Distinguishes Burkholderia pseudomallei from Burkholderia thailandensis

    PubMed Central

    Hahne, Dorothee R.; Merritt, Adam J.; Clarke, Michael W.

    2015-01-01

    Solid-phase microextraction gas chromatography-mass spectrometry (SPME-GCMS) was used to show that dimethyl sulfide produced by Burkholderia pseudomallei is responsible for its unusual truffle-like smell and distinguishes the species from Burkholderia thailandensis. SPME-GCMS can be safely used to detect dimethyl sulfide produced by agar-grown B. pseudomallei. PMID:25568444

  7. In vitro activity of BAL30072 against Burkholderia pseudomallei.

    PubMed

    Mima, Takehiko; Kvitko, Brian H; Rholl, Drew A; Page, Malcolm G P; Desarbre, Eric; Schweizer, Herbert P

    2011-08-01

    Burkholderia pseudomallei is an intrinsically antibiotic-resistant Category B priority pathogen and the aetiological agent of melioidosis. Treatment of B. pseudomallei infection is biphasic and lengthy in order to combat the acute and chronic phases of the disease. Acute-phase treatment preferably involves an intravenous cephalosporin (ceftazidime) or a carbapenem (imipenem or meropenem). In this study, the anti-B. pseudomallei efficacy of a new monosulfactam, BAL30072, was tested against laboratory strains 1026b and 1710b and several isogenic mutant derivatives as well as a collection of clinical and environmental B. pseudomallei strains from Thailand. More than 93% of the isolates had minimal inhibitory concentrations (MICs) in the range 0.004-0.016 μg/mL. For the laboratory strain 1026b, the MIC of BAL30072 was 0.008 μg/mL, comparable with the MICs of 1.5 μg/mL for ceftazidime, 0.5 μg/mL for imipenem and 1 μg/mL for meropenem. Time-kill curves revealed that BAL30072 was rapidly bactericidal, killing >99% of bacteria in 2 h. BAL30072 activity was not significantly affected by efflux, it was only a marginal substrate of PenA β-lactamase, and activity was independent of malleobactin production and transport and the ability to transport pyochelin. In summary, BAL30072 has superior in vitro activity against B. pseudomallei compared with ceftazidime, meropenem or imipenem and it is rapidly bactericidal. PMID:21596528

  8. Groundwater Seeps Facilitate Exposure to Burkholderia pseudomallei

    PubMed Central

    Baker, Anthony; Tahani, Donald; Gardiner, Christopher; Bristow, Keith L.; Greenhill, Andrew R.; Warner, Jeffrey

    2011-01-01

    Burkholderia pseudomallei is a saprophytic bacterium which is the causative agent of melioidosis, a common cause of fatal bacterial pneumonia and sepsis in the tropics. The incidence of melioidosis is clustered spatially and temporally and is heavily linked to rainfall and extreme weather events. Clinical case clustering has recently been reported in Townsville, Australia, and has implicated Castle Hill, a granite monolith in the city center, as a potential reservoir of infection. Topsoil and water from seasonal groundwater seeps were collected around the base of Castle Hill and analyzed by quantitative real-time PCR targeting the type III secretion system genes for the presence of B. pseudomallei. The organism was identified in 65% (95% confidence interval [CI], 49.5 to 80.4) of soil samples (n = 40) and 92.5% (95% CI, 83.9 to 100) of seasonal groundwater samples (n = 40). Further sampling of water collected from roads and gutters in nearby residential areas after an intense rainfall event found that 88.2% (95% CI, 72.9 to 100) of samples (n = 16) contained viable B. pseudomallei at concentrations up to 113 CFU/ml. Comparison of isolates using multilocus sequence typing demonstrated clinical matches and close associations between environmental isolates and isolates derived from clinical samples from patients in Townsville. This study demonstrated that waterborne B. pseudomallei from groundwater seeps around Castle Hill may facilitate exposure to B. pseudomallei and contribute to the clinical clustering at this site. Access to this type of information will advise the development and implementation of public health measures to reduce the incidence of melioidosis. PMID:21873480

  9. Reliability of automated biochemical identification of Burkholderia pseudomallei is regionally dependent.

    PubMed

    Podin, Yuwana; Kaestli, Mirjam; McMahon, Nicole; Hennessy, Jann; Ngian, Hie Ung; Wong, Jin Shyan; Mohana, Anand; Wong, See Chang; William, Timothy; Mayo, Mark; Baird, Robert W; Currie, Bart J

    2013-09-01

    Misidentifications of Burkholderia pseudomallei as Burkholderia cepacia by Vitek 2 have occurred. Multidimensional scaling ordination of biochemical profiles of 217 Malaysian and Australian B. pseudomallei isolates found clustering of misidentified B. pseudomallei isolates from Malaysian Borneo. Specificity of B. pseudomallei identification in Vitek 2 and potentially other automated identification systems is regionally dependent. PMID:23784129

  10. Burkholderia pseudomallei: First case of melioidosis in Portugal.

    PubMed

    Pelerito, Ana; Nunes, Alexandra; Coelho, Susana; Piedade, Cátia; Paixão, Paulo; Cordeiro, Rita; Sampaio, Daniel; Vieira, Luís; Gomes, João Paulo; Núncio, Sofia

    2016-01-01

    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. PMID:26962474

  11. Burkholderia pseudomallei: First case of melioidosis in Portugal

    PubMed Central

    Pelerito, Ana; Nunes, Alexandra; Coelho, Susana; Piedade, Cátia; Paixão, Paulo; Cordeiro, Rita; Sampaio, Daniel; Vieira, Luís; Gomes, João Paulo; Núncio, Sofia

    2016-01-01

    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. PMID:26962474

  12. Differential Toll-Like Receptor-Signalling of Burkholderia pseudomallei Lipopolysaccharide in Murine and Human Models

    PubMed Central

    Weehuizen, Tassili A. F.; Prior, Joann L.; van der Vaart, Thomas W.; Ngugi, Sarah A.; Nepogodiev, Sergey A.; Field, Robert A.; Kager, Liesbeth M.; van ‘t Veer, Cornelis; de Vos, Alex F.; Wiersinga, W. Joost

    2015-01-01

    The Gram-negative bacterium Burkholderia pseudomallei causes melioidosis and is a CDC category B bioterrorism agent. Toll-like receptor (TLR)-2 impairs host defense during pulmonary B.pseudomallei infection while TLR4 only has limited impact. We investigated the role of TLRs in B.pseudomallei-lipopolysaccharide (LPS) induced inflammation. Purified B.pseudomallei-LPS activated only TLR2-transfected-HEK-cells during short stimulation but both HEK-TLR2 and HEK-TLR4-cells after 24 h. In human blood, an additive effect of TLR2 on TLR4-mediated signalling induced by B.pseudomallei-LPS was observed. In contrast, murine peritoneal macrophages recognized B.pseudomallei-LPS solely through TLR4. Intranasal inoculation of B.pseudomallei-LPS showed that both TLR4-knockout(-/-) and TLR2x4-/-, but not TLR2-/- mice, displayed diminished cytokine responses and neutrophil influx compared to wild-type controls. These data suggest that B.pseudomallei-LPS signalling occurs solely through murine TLR4, while in human models TLR2 plays an additional role, highlighting important differences between specificity of human and murine models that may have important consequences for B.pseudomallei-LPS sensing by TLRs and subsequent susceptibility to melioidosis. PMID:26689559

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

    PubMed Central

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

    2000-01-01

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

  14. Novel lytic bacteriophages from soil that lyse Burkholderia pseudomallei.

    PubMed

    Yordpratum, Umaporn; Tattawasart, Unchalee; Wongratanacheewin, Surasakdi; Sermswan, Rasana W

    2011-01-01

    Burkholderia pseudomallei is a Gram-negative saprophytic bacterium that causes severe sepsis with a high mortality rate in humans and a vaccine is not available. Bacteriophages are viruses of bacteria that are ubiquitous in nature. Several lysogenic phages of Burkholderia spp. have been found but information is scarce for lytic phages. Six phages, ST2, ST7, ST70, ST79, ST88 and ST96, which lyse B. pseudomallei, were isolated from soil in an endemic area. The phages belong to the Myoviridae family. The range of estimated genome sizes is 24.0-54.6 kb. Phages ST79 and ST96 lysed 71% and 67% of tested B. pseudomallei isolates and formed plaques on Burkholderia mallei but not other tested bacteria, with the exception of closely related Burkholderia thailandensis which was lysed by ST2 and ST96 only. ST79 and ST96 were observed to clear a mid-log culture by lysis within 6 h when infected at a multiplicity of infection of 0.1. As ST79 and ST96 phages effectively lysed B. pseudomallei, their potential use as a biocontrol of B. pseudomallei in the environment or alternative treatment in infected hosts could lead to benefits from phages that are available in nature. PMID:21091532

  15. Variability of Burkholderia pseudomallei strain sensitivities to chlorine disinfection.

    PubMed

    O'Connell, Heather A; Rose, Laura J; Shams, Alicia; Bradley, Meranda; Arduino, Matthew J; Rice, Eugene W

    2009-08-01

    Burkholderia pseudomallei is a select agent and the causative agent of melioidosis. Variations in previously reported chlorine and monochloramine concentration time (Ct) values for disinfection of this organism make decisions regarding the appropriate levels of chlorine in water treatment systems difficult. This study identified the variation in Ct values for 2-, 3-, and 4-log(10) reductions of eight environmental and clinical isolates of B. pseudomallei in phosphate-buffered water. The greatest calculated Ct values for a 4-log(10) inactivation were 7.8 mg.min/liter for free available chlorine (FAC) at pH 8 and 5 degrees C and 550 mg.min/liter for monochloramine at pH 8 and 5 degrees C. Ionic strength of test solutions, culture hold times in water, and cell washing were ruled out as sources of the differences in prior observations. Tolerance to FAC was correlated with the relative amount of extracellular material produced by each isolate. Solid-phase cytometry analysis using an esterase-cleaved fluorochrome assay detected a 2-log(10)-higher level of organisms based upon metabolic activity than did culture, which in some cases increased Ct values by fivefold. Despite strain-to-strain variations in Ct values of 17-fold for FAC and 2.5-fold for monochloramine, standard FAC disinfection practices utilized in the United States should disinfect planktonic populations of these B. pseudomallei strains by 4 orders of magnitude in less than 10 min at the tested temperatures and pH levels. PMID:19542324

  16. Misidentification of Burkholderia pseudomallei as Burkholderia cepacia by the VITEK 2 system.

    PubMed

    Zong, Zhiyong; Wang, Xiaohui; Deng, Yiyun; Zhou, Taoyou

    2012-10-01

    A previously healthy Chinese male returned from working in the Malaysian jungle with a fever. A blood culture grew Gram-negative bacilli that were initially identified as Burkholderia cepacia by the VITEK 2 system but were subsequently found to be Burkholderia pseudomallei by partial sequencing of the 16S rRNA gene. The identification of B. pseudomallei using commercially available automated systems is problematic and clinicians in non-endemic areas should be aware of the possibility of melioidosis in patients with a relevant travel history and blood cultures growing Burkholderia spp. PMID:22820689

  17. Burkholderia Pseudomallei Causing Bone and Joint Infections: A Clinical Update.

    PubMed

    Raja, Nadeem Sajjad; Scarsbrook, Christine

    2016-03-01

    Burkholderia pseudomallei (B. pseudomallei), a causative agent of an emerging infectious disease melioidosis, is endemic in the tropical regions of the world. Due to increased international travel, the infection is now also seen outside of the tropics. The majority of patients with identified risk factors such as diabetes mellitus, heavy alcohol use, malignancy, chronic lung and kidney disease, corticosteroid use, thalassemia, rheumatic heart disease, systemic lupus erythematosus and cardiac failure acquire this organism through percutaneous inoculation or inhalation. The clinical manifestations are variable, ranging from localized abscess formation to septicemia. Melioidotic bone and joint infections are rarely reported but are an established entity. The knee joint is the most commonly affected joint in melioidosis, followed by the ankle, hip and shoulder joints. Melioidosis should be in the differential diagnosis of bone and joint infections in residents or returning travelers from the endemic area. Melioidosis diagnosis is missed in many parts of the world due to the lack of awareness of this infection and limited laboratory training and diagnostic techniques. It also mimics other diseases such as tuberculosis. Delay in the diagnosis, or the initiation of appropriate and effective treatment against melioidosis, could worsen the outcome. Initial therapy with ceftazidime, or carbapenem with or without cotrimoxazole is recommended, followed by the oral eradication therapy (based on the antimicrobial susceptibility) with amoxicillin/clavulanic acid or cotrimoxazole. Surgical intervention remains important. This paper reviews current literature on the epidemiology, clinical features, diagnosis, and management of melioidotic bone and joint infections. PMID:26728713

  18. Outer Membrane Proteome of Burkholderia pseudomallei and Burkholderia mallei From Diverse Growth Conditions

    PubMed Central

    Schell, Mark A.; Zhao, Peng; Wells, Lance

    2016-01-01

    Burkholderia mallei and Burkholderia pseudomallei are closely related, aerosol-infective human pathogens that cause life-threatening diseases. Biochemical analyses requiring large-scale growth and manipulation at biosafety level 3 under select agent regulations are cumbersome and hazardous. We developed a simple, safe, and rapid method to prepare highly purified outer membrane (OM) fragments from these pathogens. Shotgun proteomic analyses of OMs by trypsin shaving and mass spectrometry identified >155 proteins, the majority of which are clearly outer membrane proteins (OMPs). These included: 13 porins, 4 secretins for virulence factor export, 11 efflux pumps, multiple components of a Type VI secreton, metal transport receptors, polysaccharide exporters, and hypothetical OMPs of unknown function. We also identified 20 OMPs in each pathogen that are abundant under a wide variety of conditions, including in serum and with macrophages, suggesting these are fundamental for growth and survival and may represent prime drug or vaccine targets. Comparison of the OM proteomes of B. mallei and B. pseudomallei showed many similarities but also revealed a few differences, perhaps reflecting evolution of B. mallei away from environmental survival toward host-adaptation. PMID:21391724

  19. Mycotic aneurysm caused by Burkholderia pseudomallei with negative blood cultures.

    PubMed

    Tanyaowalak, Wiriya; Sunthornyothin, Sarat; Luengtaviboon, Kittichai; Suankratay, Chusana; Kulwichit, Wanla

    2004-01-01

    We describe a case of bacterial aortitis caused by Burkholderia pseudomallei. This patient presented with prolonged fever and hoarseness of voice. Aneurysm removal with Dacron graft replacement was performed, followed by a prolonged course of antibiotics. The patient has progressed satisfactorily without recurrence of symptoms. Previous case reports are summarized. PMID:15000566

  20. Burkholderia pseudomallei Genotype Distribution in the Northern Territory, Australia.

    PubMed

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

    2016-01-01

    Melioidosis is a tropical disease of high mortality caused by the environmental bacterium, Burkholderia pseudomallei. We have collected clinical isolates from the highly endemic Northern Territory of Australia routinely since 1989, and animal and environmental B. pseudomallei isolates since 1991. Here we provide a complete record of all B. pseudomallei multilocus sequence types (STs) found in the Northern Territory to date, and distribution maps of the eight most common environmental STs. We observed surprisingly restricted geographic distributions of STs, which is contrary to previous reports suggesting widespread environmental dissemination of this bacterium. Our data suggest that B. pseudomallei from soil and water does not frequently disperse long distances following severe weather events or by migration of infected animals. PMID:26526925

  1. Burkholderia pseudomallei Differentially Regulates Host Innate Immune Response Genes for Intracellular Survival in Lung Epithelial Cells

    PubMed Central

    Vellasamy, Kumutha Malar; Mariappan, Vanitha; Shankar, Esaki M.; Vadivelu, Jamuna

    2016-01-01

    Background Burkholderia pseudomallei, the causative agent of melioidosis poses a serious threat to humankind. B. pseudomallei secretes numerous virulence proteins that alter host cell functions to escape from intracellular immune sensors. However, the events underlying disease pathogenesis are poorly understood. Methods We determined the ability of B. pseudomallei to invade and survive intracellularly in A549 human lung epithelial cells, and also investigated the early transcriptional responses using an Illumina HumanHT-12 v4 microarray platform, after three hours of exposure to live B. pseudomallei (BCMS) and its secreted proteins (CCMS). Results We found that the ability of B. pseudomallei to invade and survive intracellularly correlated with increase of multiplicity of infection and duration of contact. Activation of host carbohydrate metabolism and apoptosis as well as suppression of amino acid metabolism and innate immune responses both by live bacteria and its secreted proteins were evident. These early events might be linked to initial activation of host genes directed towards bacterial dissemination from lungs to target organs (via proposed in vivo mechanisms) or to escape potential sensing by macrophages. Conclusion Understanding the early responses of A549 cells toward B. pseudomallei infection provide preliminary insights into the likely pathogenesis mechanisms underlying melioidosis, and could contribute to development of novel intervention strategies to combat B. pseudomallei infections. PMID:27367858

  2. Systematic Review and Consensus Guidelines for Environmental Sampling of Burkholderia pseudomallei

    PubMed Central

    Limmathurotsakul, Direk; Dance, David A. B.; Wuthiekanun, Vanaporn; Kaestli, Mirjam; Mayo, Mark; Warner, Jeffrey; Wagner, David M.; Tuanyok, Apichai; Wertheim, Heiman; Yoke Cheng, Tan; Mukhopadhyay, Chiranjay; Puthucheary, Savithiri; Day, Nicholas P. J.; Steinmetz, Ivo; Currie, Bart J.; Peacock, Sharon J.

    2013-01-01

    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. PMID:23556010

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

    PubMed Central

    2012-01-01

    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. PMID:23126230

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

    PubMed

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

    2014-05-01

    The environmental Gram-negative encapsulated bacillus Burkholderia pseudomallei is the causative agent of melioidosis, a disease associated with high morbidity and mortality rates in areas of Southeast Asia and northern Australia in which the disease is endemic. B. pseudomallei is also classified as a tier I select agent due to the high level of lethality of the bacterium and its innate resistance to antibiotics, as well as the lack of an effective vaccine. Gram-negative bacteria, including B. pseudomallei, secrete outer membrane vesicles (OMVs) which are enriched with multiple protein, lipid, and polysaccharide antigens. Previously, we demonstrated that immunization with multivalent B. pseudomallei-derived OMVs protects highly susceptible BALB/c mice against an otherwise lethal aerosol challenge. In this work, we evaluated the protective efficacy of OMV immunization against intraperitoneal challenge with a heterologous strain because systemic infection with phenotypically diverse environmental B. pseudomallei strains poses another hazard and a challenge to vaccine development. We demonstrated that B. pseudomallei OMVs derived from strain 1026b afforded significant protection against septicemic infection with B. pseudomallei strain K96243. OMV immunization induced robust OMV-, lipopolysaccharide-, and capsular polysaccharide-specific serum IgG (IgG1, IgG2a, and IgG3) and IgM antibody responses. OMV-immune serum promoted bacterial killing in vitro, and passive transfer of B. pseudomallei OMV immune sera protected naive mice against a subsequent challenge. These results indicate that OMV immunization provides antibody-mediated protection against acute, rapidly lethal sepsis in mice. B. pseudomallei-derived OMVs may represent an efficacious multivalent vaccine strategy against melioidosis. PMID:24671550

  5. Comparative Burkholderia pseudomallei natural history virulence studies using an aerosol murine model of infection

    PubMed Central

    Massey, Shane; Yeager, Linsey A.; Blumentritt, Carla A.; Vijayakumar, Sudhamathi; Sbrana, Elena; Peterson, Johnny W.; Brasel, Trevor; LeDuc, James W.; Endsley, Janice J.; Torres, Alfredo G.

    2014-01-01

    Melioidosis is an endemic disease caused by the bacterium Burkholderia pseudomallei. Concerns exist regarding B. pseudomallei use as a potential bio-threat agent causing persistent infections and typically manifesting as severe pneumonia capable of causing fatal bacteremia. Development of suitable therapeutics against melioidosis is complicated due to high degree of genetic and phenotypic variability among B. pseudomallei isolates and lack of data establishing commonly accepted strains for comparative studies. Further, the impact of strain variation on virulence, disease presentation, and mortality is not well understood. Therefore, this study evaluate and compare the virulence and disease progression of B. pseudomallei strains K96243 and HBPUB10303a, following aerosol challenge in a standardized BALB/c mouse model of infection. The natural history analysis of disease progression monitored conditions such as weight, body temperature, appearance, activity, bacteremia, organ and tissue colonization (pathological and histological analysis) and immunological responses. This study provides a detailed, direct comparison of infection with different B. pseudomallei strains and set up the basis for a standardized model useful to test different medical countermeasures against Burkholderia species. Further, this protocol serves as a guideline to standardize other bacterial aerosol models of infection or to define biomarkers of infectious processes caused by other intracellular pathogens. PMID:24603493

  6. Identification of Burkholderia pseudomallei Near-Neighbor Species in the Northern Territory of Australia

    PubMed Central

    Ginther, Jennifer L.; Mayo, Mark; Warrington, Stephanie D.; Kaestli, Mirjam; Mullins, Travis; Wagner, David M.; Currie, Bart J.; Tuanyok, Apichai; Keim, Paul

    2015-01-01

    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. PMID:26121041

  7. Backbone and side-chain (1)H, (15)N, (13)C assignment and secondary structure of BPSL1445 from Burkholderia pseudomallei.

    PubMed

    Quilici, Giacomo; Berardi, Andrea; Gaudesi, Davide; Gourlay, Louise J; Bolognesi, Martino; Musco, Giovanna

    2015-10-01

    BPSL1445 is a lipoprotein produced by the Gram-negative bacterium Burkholderia pseudomallei (B. pseudomallei), the etiological agent of melioidosis. Immunodetection assays against sera patients using protein microarray suggest BPSL1445 involvement in melioidosis. Herein we report backbone, side chain NMR assignment and secondary structure for the recombinant protein. PMID:25893672

  8. Burkholderia pseudomallei rpoS mediates iNOS suppression in human hepatocyte (HC04) cells

    PubMed Central

    Sanongkiet, Sucharat; Ponnikorn, Saranyoo; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee

    2016-01-01

    Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen and the causative agent of melioidosis, a widespread disease in Southeast Asia. Reactive nitrogen, in an intermediate form of nitric oxide (NO), is one of the first lines of defense used by host cells to eliminate intracellular pathogens, through the stimulation of inducible nitric oxide synthase (iNOS). Studies in phagocytotic cells have shown that the iNOS response is muted in B. pseudomallei infection, and implicated the rpoS sigma factor as a key regulatory factor mediating suppression. The liver is a main visceral organ affected by B. pseudomallei, and there is little knowledge about the interaction of liver cells and B. pseudomallei. This study investigated the induction of iNOS, as well as autophagic flux and light-chain 3 (LC3) localization in human liver (HC04) cells in response to infection with B. pseudomallei and its rpoS deficient mutant. Results showed that the rpoS mutant was unable to suppress iNOS induction and that the mutant showed less induction of autophagy and lower co-localization with LC3, and this was coupled with a lower intracellular growth rate. Combining these results suggest that B. pseudomallei rpoS is an important factor in establishing infection in liver cells. PMID:27324398

  9. The Genetic and Molecular Basis of O-Antigenic Diversity in Burkholderia pseudomallei Lipopolysaccharide

    PubMed Central

    Tuanyok, Apichai; Stone, Joshua K.; Mayo, Mark; Kaestli, Mirjam; Gruendike, Jeffrey; Georgia, Shalamar; Warrington, Stephanie; Mullins, Travis; Allender, Christopher J.; Wagner, David M.; Chantratita, Narisara; Peacock, Sharon J.; Currie, Bart J.; Keim, Paul

    2012-01-01

    Lipopolysaccharide (LPS) is one of the most important virulence and antigenic components of Burkholderia pseudomallei, the causative agent of melioidosis. LPS diversity in B. pseudomallei has been described as typical, atypical or rough, based upon banding patterns on SDS-PAGE. Here, we studied the genetic and molecular basis of these phenotypic differences. Bioinformatics was used to determine the diversity of genes known or predicted to be involved in biosynthesis of the O-antigenic moiety of LPS in B. pseudomallei and its near-relative species. Multiplex-PCR assays were developed to target diversity of the O-antigen biosynthesis gene patterns or LPS genotypes in B. pseudomallei populations. We found that the typical LPS genotype (LPS genotype A) was highly prevalent in strains from Thailand and other countries in Southeast Asia, whereas the atypical LPS genotype (LPS genotype B) was most often detected in Australian strains (∼13.8%). In addition, we report a novel LPS ladder pattern, a derivative of the atypical LPS phenotype, associated with an uncommon O-antigen biosynthesis gene cluster that is found in only a small B. pseudomallei sub-population. This new LPS group was designated as genotype B2. We also report natural mutations in the O-antigen biosynthesis genes that potentially cause the rough LPS phenotype. We postulate that the diversity of LPS may correlate with differential immunopathogenicity and virulence among B. pseudomallei strains. PMID:22235357

  10. Burkholderia pseudomallei rpoS mediates iNOS suppression in human hepatocyte (HC04) cells.

    PubMed

    Sanongkiet, Sucharat; Ponnikorn, Saranyoo; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee

    2016-08-01

    Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen and the causative agent of melioidosis, a widespread disease in Southeast Asia. Reactive nitrogen, in an intermediate form of nitric oxide (NO), is one of the first lines of defense used by host cells to eliminate intracellular pathogens, through the stimulation of inducible nitric oxide synthase (iNOS). Studies in phagocytotic cells have shown that the iNOS response is muted in B. pseudomallei infection, and implicated the rpoS sigma factor as a key regulatory factor mediating suppression. The liver is a main visceral organ affected by B. pseudomallei, and there is little knowledge about the interaction of liver cells and B. pseudomallei This study investigated the induction of iNOS, as well as autophagic flux and light-chain 3 (LC3) localization in human liver (HC04) cells in response to infection with B. pseudomallei and its rpoS deficient mutant. Results showed that the rpoS mutant was unable to suppress iNOS induction and that the mutant showed less induction of autophagy and lower co-localization with LC3, and this was coupled with a lower intracellular growth rate. Combining these results suggest that B. pseudomallei rpoS is an important factor in establishing infection in liver cells. PMID:27324398

  11. Characterization of the Burkholderia pseudomallei K96243 Capsular Polysaccharide I Coding Region

    PubMed Central

    Cuccui, Jon; Milne, Timothy S.; Harmer, Nicholas; George, Alison J.; Harding, Sarah V.; Dean, Rachel E.; Scott, Andrew E.; Sarkar-Tyson, Mitali; Wren, Brendan W.; Prior, Joann L.

    2012-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to regions of Southeast Asia and Northern Australia. Both humans and a range of other animal species are susceptible to melioidosis, and the production of a group 3 polysaccharide capsule in B. pseudomallei is essential for virulence. B. pseudomallei capsular polysaccharide (CPS) I comprises unbranched manno-heptopyranose residues and is encoded by a 34.5-kb locus on chromosome 1. Despite the importance of this locus, the role of all of the genes within this region is unclear. We inactivated 18 of these genes and analyzed their phenotype using Western blotting and immunofluorescence staining. Furthermore, by combining this approach with bioinformatic analysis, we were able to develop a model for CPS I biosynthesis and export. We report that inactivating gmhA, wcbJ, and wcbN in B. pseudomallei K96243 retains the immunogenic integrity of the polysaccharide despite causing attenuation in the BALB/c murine infection model. Mice immunized with the B. pseudomallei K96243 mutants lacking a functional copy of either gmhA or wcbJ were afforded significant levels of protection against a wild-type B. pseudomallei K96243 challenge. PMID:22252864

  12. Distribution of Burkholderia pseudomallei in Northern Australia, a Land of Diversity

    PubMed Central

    McRobb, Evan; Kaestli, Mirjam; Price, Erin P.; Sarovich, Derek S.; Mayo, Mark; Warner, Jeffrey; Spratt, Brian G.

    2014-01-01

    Burkholderia pseudomallei is a Gram-negative soil bacillus that is the etiological agent of melioidosis and a biothreat agent. Little is known about the biogeography of this bacterium in Australia, despite its hyperendemicity in the northern region of this continent. The population structure of 953 Australian B. pseudomallei strains representing 779 and 174 isolates of clinical and environmental origins, respectively, was analyzed using multilocus sequence typing (MLST). Bayesian population structure and network SplitsTree analyses were performed on concatenated MLST loci, and sequence type (ST) diversity and evenness were examined using Simpson's and Pielou's indices and a multivariate dissimilarity matrix. Bayesian analysis found two B. pseudomallei populations in Australia that were geographically distinct; isolates from the Northern Territory were grouped mainly into the first population, whereas the majority of isolates from Queensland were grouped in a second population. Differences in ST evenness were observed between sampling areas, confirming that B. pseudomallei is widespread and established across northern Australia, with a large number of fragmented habitats. ST analysis showed that B. pseudomallei populations diversified as the sampling area increased. This observation was in contrast to smaller sampling areas where a few STs predominated, suggesting that B. pseudomallei populations are ecologically established and not frequently dispersed. Interestingly, there was no identifiable ST bias between clinical and environmental isolates, suggesting the potential for all culturable B. pseudomallei isolates to cause disease. Our findings have important implications for understanding the ecology of B. pseudomallei in Australia and for potential source attribution of this bacterium in the event of unexpected cases of melioidosis. PMID:24657869

  13. Distribution of Burkholderia pseudomallei in northern Australia, a land of diversity.

    PubMed

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

    2014-06-01

    Burkholderia pseudomallei is a Gram-negative soil bacillus that is the etiological agent of melioidosis and a biothreat agent. Little is known about the biogeography of this bacterium in Australia, despite its hyperendemicity in the northern region of this continent. The population structure of 953 Australian B. pseudomallei strains representing 779 and 174 isolates of clinical and environmental origins, respectively, was analyzed using multilocus sequence typing (MLST). Bayesian population structure and network SplitsTree analyses were performed on concatenated MLST loci, and sequence type (ST) diversity and evenness were examined using Simpson's and Pielou's indices and a multivariate dissimilarity matrix. Bayesian analysis found two B. pseudomallei populations in Australia that were geographically distinct; isolates from the Northern Territory were grouped mainly into the first population, whereas the majority of isolates from Queensland were grouped in a second population. Differences in ST evenness were observed between sampling areas, confirming that B. pseudomallei is widespread and established across northern Australia, with a large number of fragmented habitats. ST analysis showed that B. pseudomallei populations diversified as the sampling area increased. This observation was in contrast to smaller sampling areas where a few STs predominated, suggesting that B. pseudomallei populations are ecologically established and not frequently dispersed. Interestingly, there was no identifiable ST bias between clinical and environmental isolates, suggesting the potential for all culturable B. pseudomallei isolates to cause disease. Our findings have important implications for understanding the ecology of B. pseudomallei in Australia and for potential source attribution of this bacterium in the event of unexpected cases of melioidosis. PMID:24657869

  14. Genetic Control of Weight Loss During Pneumonic Burkholderia pseudomallei Infection

    PubMed Central

    Emery, Felicia D.; Parvathareddy, Jyothi; Pandey, Ashutosh K.; Cui, Yan; Williams, Robert W.; Miller, Mark A.

    2014-01-01

    Burkholderia pseudomallei (Bp) is the causal agent of a high morbidity/mortality disease syndrome known as melioidosis. This syndrome can range from acute fulminate disease to chronic, local, and disseminated infections that are often difficult to treat because Bp exhibits resistance to many antibiotics. Bp is a prime candidate for use in biological warfare/terrorism and is classified as a Tier-1 Select Agent by HHS and APHIS. It is known that inbred mouse strains display a range of susceptibility to Bp and that the murine infection model is ideal for studying acute melioidosis. Here we exploit a powerful mouse genetics resource that consists of a large family of BXD type recombinant inbred strains, to perform genome-wide linkage analysis of the weight loss phenotype following pneumonic infection with Bp. We infected parental mice and 32 BXD strains with 50-100 CFU of Bp (strain 1026b) and monitored weight retention each day over an eleven-day time course. Using the computational tools in GeneNetwork, we performed genome-wide linkage analysis to identify an interval on chromosome 12 that appears to control the weight retention trait. We then analysed and ranked positional candidate genes in this interval, several of which have intriguing connections with innate immunity, calcium homeostasis, lipid transport, host cell growth and development, and autophagy. PMID:24687986

  15. Porin Involvement in Cephalosporin and Carbapenem Resistance of Burkholderia pseudomallei

    PubMed Central

    Aunkham, Anuwat; Schulte, Albert; Winterhalter, Mathias; Suginta, Wipa

    2014-01-01

    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 Tyr119 affects

  16. The Survival of Burkholderia pseudomallei in Liquid Media

    PubMed Central

    Robertson, Jeannie; Levy, Avram; Sagripanti, Jose-Luis; Inglis, Timothy J. J.

    2010-01-01

    We studied the effect of environmental parameters on the survival of Burkholderia pseudomallei. There was a small increase in bacterial count for up to 28 days in sterilized distilled water or rain water, in water at 20°C or 40°C, and in buffered solutions of pH 4 or higher. Counts of culturable B. pseudomallei declined at pH 3, in the presence of seawater or water with concentrations of 4% salt or higher, and under refrigeration. The morphological appearances of B. pseudomallei changed under conditions that maintained culturable numbers from bacilli to coccoid cells and spiral forms under pH or salt stress. These observations indicate that B. pseudomallei can endure nutrient-depleted environments as well as a wide range of pH, salt concentrations, and temperatures for periods of up to 28 days. The relative stability of B. pseudomallei under these conditions underlines the tenacity of this species and its potential for natural dispersal in water: in surface water collections, in managed water distribution systems, and through rainfall. These survival properties help explain the recent expansion of the known melioidosis endemic zone in Australia and may have played a part in recent melioidosis outbreaks. PMID:20065001

  17. Identification of an OmpW homologue in Burkholderia pseudomallei, a protective vaccine antigen against melioidosis.

    PubMed

    Casey, William T; Spink, Natasha; Cia, Felipe; Collins, Cassandra; Romano, Maria; Berisio, Rita; Bancroft, Gregory J; McClean, Siobhán

    2016-05-17

    Burkholderia pseudomallei is the causative agent of melioidosis, which is associated with a range of clinical manifestations, including sepsis and fatal pneumonia and is endemic in Southeast Asia and Northern Australia. Treatment can be challenging and control of infection involves prolonged antibiotic therapy, yet there are no approved vaccines available to prevent infection. Our aim was to develop and assess the potential of a prophylactic vaccine candidate targeted against melioidosis. The identified candidate is the 22kDa outer membrane protein, OmpW. We previously demonstrated that this protein was immunoprotective in mouse models of Burkholderia cepacia complex (Bcc) infections. We cloned Bp_ompW in Escherichia coli, expressed and purified the protein. Endotoxin free protein administered with SAS adjuvant protected Balb/C mice (75% survival) relative to controls (25% survival) (p<0.05). A potent serological response was observed with IgG2a to IgG1 ratio of 6.0. Furthermore C57BL/6 mice were protected for up to 80 days against a lethal dose of B. pseudomallei and surpassed the efficacy of the live attenuated 2D2 positive control. BpompW is homologous across thirteen sequenced B. pseudomallei strains, indicating that it should be broadly protective against B. pseudomallei. In conclusion, we have demonstrated that BpOmpW is able to induce protective immunity against melioidosis and is likely to be an effective vaccine antigen, possibly in combination with other subunit antigens. PMID:27091689

  18. Draft Genome Sequences of Burkholderia pseudomallei and Staphylococcus aureus, Isolated from a Patient with Chronic Rhinosinusitis

    PubMed Central

    Cottrell, Kyra; Cervin, Anders

    2015-01-01

    Here, we report the draft genome sequences of Burkholderia pseudomallei and Staphylococcus aureus causing chronic rhinosinusitis. Whole-genome sequencing determined the B. pseudomallei as sequence type (ST) 1381 and the S. aureus as ST8. B. pseudomallei possessed the blaOXA-59 gene. This study illustrates the potential emergence of B. pseudomallei in cases of chronic rhinosinusitis. PMID:26430027

  19. Detection of Bacterial Virulence Genes by Subtractive Hybridization: Identification of Capsular Polysaccharide of Burkholderia pseudomallei as a Major Virulence Determinant

    PubMed Central

    Reckseidler, Shauna L.; DeShazer, David; Sokol, Pamela A.; Woods, Donald E.

    2001-01-01

    Burkholderia pseudomallei, the etiologic agent of melioidosis, is responsible for a broad spectrum of illnesses in humans and animals particularly in Southeast Asia and northern Australia, where it is endemic. Burkholderia thailandensis is a nonpathogenic environmental organism closely related to B. pseudomallei. Subtractive hybridization was carried out between these two species to identify genes encoding virulence determinants in B. pseudomallei. Screening of the subtraction library revealed A-T-rich DNA sequences unique to B. pseudomallei, suggesting they may have been acquired by horizontal transfer. One of the subtraction clones, pDD1015, encoded a protein with homology to a glycosyltransferase from Pseudomonas aeruginosa. This gene was insertionally inactivated in wild-type B. pseudomallei to create SR1015. It was determined by enzyme-linked immunosorbent assay and immunoelectron microscopy that the inactivated gene was involved in the production of a major surface polysaccharide. The 50% lethal dose (LD50) for wild-type B. pseudomallei is <10 CFU; the LD50 for SR1015 was determined to be 3.5 × 105 CFU, similar to that of B. thailandensis (6.8 × 105 CFU). DNA sequencing of the region flanking the glycosyltransferase gene revealed open reading frames similar to capsular polysaccharide genes in Haemophilus influenzae, Escherichia coli, and Neisseria meningitidis. In addition, DNA from Burkholderia mallei and Burkholderia stabilis hybridized to a glycosyltransferase fragment probe, and a capsular structure was identified on the surface of B. stabilis via immunoelectron microscopy. Thus, the combination of PCR-based subtractive hybridization, insertional inactivation, and animal virulence studies has facilitated the identification of an important virulence determinant in B. pseudomallei. PMID:11119486

  20. Curcumin rescues Caenorhabditis elegans from a Burkholderia pseudomallei infection

    PubMed Central

    Eng, Su-Anne; Nathan, Sheila

    2015-01-01

    The tropical pathogen Burkholderia pseudomallei requires long-term parenteral antimicrobial treatment to eradicate the pathogen from an infected patient. However, the development of antibiotic resistance is emerging as a threat to this form of treatment. To meet the need for alternative therapeutics, we proposed a screen of natural products for compounds that do not kill the pathogen, but in turn, abrogate bacterial virulence. We suggest that the use of molecules or compounds that are non-bactericidal (bacteriostatic) will reduce or abolish the development of resistance by the pathogen. In this study, we adopted the established Caenorhabditis elegans-B. pseudomallei infection model to screen a collection of natural products for any that are able to extend the survival of B. pseudomallei infected worms. Of the 42 natural products screened, only curcumin significantly improved worm survival following infection whilst not affecting bacterial growth. This suggested that curcumin promoted B. pseudomallei-infected worm survival independent of pathogen killing. To validate that the protective effect of curcumin was directed toward the pathogen, bacteria were treated with curcumin prior to infection. Worms fed with curcumin-treated bacteria survived with a significantly extended mean-time-to-death (p < 0.0001) compared to the untreated control. In in vitro assays, curcumin reduced the activity of known virulence factors (lipase and protease) and biofilm formation. To determine if other bacterial genes were also regulated in the presence of curcumin, a genome-wide transcriptome analysis was performed on curcumin-treated pathogen. A number of genes involved in iron acquisition and transport as well as genes encoding hypothetical proteins were induced in the presence of curcumin. Thus, we propose that curcumin may attenuate B. pseudomallei by modulating the expression of a number of bacterial proteins including lipase and protease as well as biofilm formation whilst

  1. Polar Lipids of Burkholderia pseudomallei Induce Different Host Immune Responses

    PubMed Central

    Gonzalez-Juarrero, Mercedes; Mima, Naoko; Trunck, Lily A.; Schweizer, Herbert P.; Bowen, Richard A.; Dascher, Kyle; Mwangi, Waithaka; Eckstein, Torsten M.

    2013-01-01

    Melioidosis is a disease in tropical and subtropical regions of the world that is caused by Burkholderia pseudomallei. In endemic regions the disease occurs primarily in humans and goats. In the present study, we used the goat as a model to dissect the polar lipids of B. pseudomallei to identify lipid molecules that could be used for adjuvants/vaccines or as diagnostic tools. We showed that the lipidome of B. pseudomallei and its fractions contain several polar lipids with the capacity to elicit different immune responses in goats, namely rhamnolipids and ornithine lipids which induced IFN-γ, whereas phospholipids and an undefined polar lipid induced strong IL-10 secretion in CD4+ T cells. Autologous T cells co-cultured with caprine dendritic cells (cDCs) and polar lipids of B. pseudomallei proliferated and up-regulated the expression of CD25 (IL-2 receptor) molecules. Furthermore, we demonstrated that polar lipids were able to up-regulate CD1w2 antigen expression in cDCs derived from peripheral blood monocytes. Interestingly, the same polar lipids had only little effect on the expression of MHC class II DR antigens in the same caprine dendritic cells. Finally, antibody blocking of the CD1w2 molecules on cDCs resulted in decreased expression for IFN-γ by CD4+ T cells. Altogether, these results showed that polar lipids of B. pseudomallei are recognized by the caprine immune system and that their recognition is primarily mediated by the CD1 antigen cluster. PMID:24260378

  2. The Condition-Dependent Transcriptional Landscape of Burkholderia pseudomallei

    PubMed Central

    Nandi, Tannistha; Kreisberg, Jason F.; Chua, Hui Hoon; Sun, Guangwen; Chen, Yahua; Mueller, Claudia; Conejero, Laura; Eshaghi, Majid; Ang, Roy Moh Lik; Liu, Jianhua; Sobral, Bruno W.; Korbsrisate, Sunee; Gan, Yunn Hwen; Titball, Richard W.; Bancroft, Gregory J.; Valade, Eric; Tan, Patrick

    2013-01-01

    Burkholderia pseudomallei (Bp), the causative agent of the often-deadly infectious disease melioidosis, contains one of the largest prokaryotic genomes sequenced to date, at 7.2 Mb with two large circular chromosomes (1 and 2). To comprehensively delineate the Bp transcriptome, we integrated whole-genome tiling array expression data of Bp exposed to >80 diverse physical, chemical, and biological conditions. Our results provide direct experimental support for the strand-specific expression of 5,467 Sanger protein-coding genes, 1,041 operons, and 766 non-coding RNAs. A large proportion of these transcripts displayed condition-dependent expression, consistent with them playing functional roles. The two Bp chromosomes exhibited dramatically different transcriptional landscapes — Chr 1 genes were highly and constitutively expressed, while Chr 2 genes exhibited mosaic expression where distinct subsets were expressed in a strongly condition-dependent manner. We identified dozens of cis-regulatory motifs associated with specific condition-dependent expression programs, and used the condition compendium to elucidate key biological processes associated with two complex pathogen phenotypes — quorum sensing and in vivo infection. Our results demonstrate the utility of a Bp condition-compendium as a community resource for biological discovery. Moreover, the observation that significant portions of the Bp virulence machinery can be activated by specific in vitro cues provides insights into Bp's capacity as an “accidental pathogen”, where genetic pathways used by the bacterium to survive in environmental niches may have also facilitated its ability to colonize human hosts. PMID:24068961

  3. Diverse Burkholderia Species Isolated from Soils in the Southern United States with No Evidence of B. pseudomallei

    PubMed Central

    Hall, Carina M.; Busch, Joseph D.; Shippy, Kenzie; Allender, Christopher J.; Kaestli, Mirjam; Mayo, Mark; Sahl, Jason W.; Schupp, James M.; Colman, Rebecca E.; Keim, Paul; Currie, Bart J.; Wagner, David M.

    2015-01-01

    The global distribution of the soil-dwelling bacterium Burkholderia pseudomallei, causative agent of melioidosis, is poorly understood. We used established culturing methods developed for B. pseudomallei to isolate Burkholderia species from soil collected at 18 sampling sites in three states in the southern United States (Arizona (n = 4), Florida (n = 7), and Louisiana (n = 7)). Using multi-locus sequence typing (MLST) of seven genes, we identified 35 Burkholderia isolates from these soil samples. All species belonged to the B. cepacia complex (Bcc), including B. cenocepacia, B. cepacia, B. contaminans, B. diffusa, B. metallica, B. seminalis, B. vietnamiensis and two unnamed members of the Bcc. The MLST analysis provided a high level of resolution among and within these species. Despite previous clinical cases within the U.S. involving B. pseudomallei and its close phylogenetic relatives, we did not isolate any of these taxa. The Bcc contains a number of opportunistic pathogens that cause infections in cystic fibrosis patients. Interestingly, we found that B. vietnamiensis was present in soil from all three states, suggesting it may be a common component in southern U.S. soils. Most of the Burkholderia isolates collected in this study were from Florida (30/35; 86%), which may be due to the combination of relatively moist, sandy, and acidic soils found there compared to the other two states. We also investigated one MLST gene, recA, for its ability to identify species within Burkholderia. A 365bp fragment of recA recovered nearly the same species-level identification as MLST, thus demonstrating its cost effective utility when conducting environmental surveys for Burkholderia. Although we did not find B. pseudomallei, our findings document that other diverse Burkholderia species are present in soils in the southern United States. PMID:26600238

  4. Burkholderia pseudomallei: Its Detection in Soil and Seroprevalence in Bangladesh

    PubMed Central

    Robayet, Jamshedul Alam Mohammad; Mohiuddin, Md.; Hasan, Md. Rokib

    2016-01-01

    Background Melioidosis, caused by Burkholderia pseudomallei, is an endemic disease in Bangladesh. No systematic study has yet been done to detect the environmental source of the organism and its true extent in Bangladesh. The present study attempted to isolate B. pseudomallei in soil samples and to determine its seroprevalence in several districts in Bangladesh. Methodology and Results Soil samples were collected from rural areas of four districts of Bangladesh from where culture confirmed melioidosis cases were detected earlier. Multiple soil samples, collected from 5–7 sampling points of 3–5 sites of each district, were cultured in Ashdown selective media. Suspected colonies of B. pseudomallei were identified by biochemical and serological test, and by polymerase chain reaction (PCR) using 16s rRNA specific primers. Blood samples were collected from 940 healthy individuals of four districts to determine anti- B. pseudomallei IgG antibody levels by indirect enzyme linked immunosorbent assay (ELISA) using sonicated crude antigen. Out of 179 soil samples, B. pseudomallei was isolated from two samples of Gazipur district which is located 58 km north of capital Dhaka city. Both the isolates were phenotypically identical, arabinose negative and showed specific 550bp band in PCR. Out of 940 blood samples, anti- B. pseudomallei IgG antibody, higher than the cut-off value (>0.8), was detected in 21.5% individuals. Seropositivity rate was 22.6%-30.8% in three districts from where melioidosis cases were detected earlier, compared to 9.8% in a district where no melioidosis case was either detected or reported (p<0.01). Seropositivity increased with the advancement of age from 5.3% to 30.4% among individuals aged 1–10 years and > 50 years respectively. The seropositivity rates were 26.0% and 20.6% in male and female respectively, while it was 20–27% among different occupational groups. No significant association was observed with gender (χ2 = 3.441, p = 0.064) or any

  5. Molecular evidence of Burkholderia pseudomallei genotypes based on geographical distribution

    PubMed Central

    Zulkefli, Noorfatin Jihan; Mariappan, Vanitha; Vellasamy, Kumutha Malar; Chong, Chun Wie; Thong, Kwai Lin; Ponnampalavanar, Sasheela; Vadivelu, Jamuna

    2016-01-01

    Background. Central intermediary metabolism (CIM) in bacteria is defined as a set of metabolic biochemical reactions within a cell, which is essential for the cell to survive in response to environmental perturbations. The genes associated with CIM are commonly found in both pathogenic and non-pathogenic strains. As these genes are involved in vital metabolic processes of bacteria, we explored the efficiency of the genes in genotypic characterization of Burkholderia pseudomallei isolates, compared with the established pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) schemes. Methods. Nine previously sequenced B. pseudomallei isolates from Malaysia were characterized by PFGE, MLST and CIM genes. The isolates were later compared to the other 39 B. pseudomallei strains, retrieved from GenBank using both MLST and sequence analysis of CIM genes. UniFrac and hierachical clustering analyses were performed using the results generated by both MLST and sequence analysis of CIM genes. Results. Genetic relatedness of nine Malaysian B. pseudomallei isolates and the other 39 strains was investigated. The nine Malaysian isolates were subtyped into six PFGE profiles, four MLST profiles and five sequence types based on CIM genes alignment. All methods demonstrated the clonality of OB and CB as well as CMS and THE. However, PFGE showed less than 70% similarity between a pair of morphology variants, OS and OB. In contrast, OS was identical to the soil isolate, MARAN. To have a better understanding of the genetic diversity of B. pseudomallei worldwide, we further aligned the sequences of genes used in MLST and genes associated with CIM for the nine Malaysian isolates and 39 B. pseudomallei strains from NCBI database. Overall, based on the CIM genes, the strains were subtyped into 33 profiles where majority of the strains from Asian countries were clustered together. On the other hand, MLST resolved the isolates into 31 profiles which formed three clusters

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

    PubMed

    Van Zandt, Kristopher E; Tuanyok, Apichai; Keim, Paul S; Warren, Richard L; Gelhaus, H Carl

    2012-01-01

    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 six strains as candidate for a B. pseudomallei strain panel. PMID:23057010

  7. Colony morphology variation of Burkholderia pseudomallei is associated with antigenic variation and O-polysaccharide modification.

    PubMed

    Wikraiphat, Chanthiwa; Saiprom, Natnaree; Tandhavanant, Sarunporn; Heiss, Christian; Azadi, Parastoo; Wongsuvan, Gumphol; Tuanyok, Apichai; Holden, Matthew T G; Burtnick, Mary N; Brett, Paul J; Peacock, Sharon J; Chantratita, Narisara

    2015-05-01

    Burkholderia pseudomallei is a CDC tier 1 select agent that causes melioidosis, a severe disease in humans and animals. Persistent infections are common, and there is currently no vaccine available. Lipopolysaccharide (LPS) is a potential vaccine candidate. B. pseudomallei expresses three serologically distinct LPS types. The predominant O-polysaccharide (OPS) is an unbranched heteropolymer with repeating d-glucose and 6-deoxy-l-talose residues in which the 6-deoxy-l-talose residues are variably replaced with O-acetyl and O-methyl modifications. We observed that primary clinical B. pseudomallei isolates with mucoid and nonmucoid colony morphologies from the same sample expressed different antigenic types distinguishable using an LPS-specific monoclonal antibody (MAb). MAb-reactive (nonmucoid) and nonreactive (mucoid) strains from the same patient exhibited identical LPS banding patterns by silver staining and indistinguishable genotypes. We hypothesized that LPS antigenic variation reflected modification of the OPS moieties. Mutagenesis of three genes involved in LPS synthesis was performed in B. pseudomallei K96243. Loss of MAb reactivity was observed in both wbiA (encoding a 2-O-acetyltransferase) and wbiD (putative methyl transferase) mutants. The structural characteristics of the OPS moieties from isogenic nonmucoid strain 4095a and mucoid strain 4095c were further investigated. Utilizing nuclear magnetic resonance (NMR) spectroscopy, we found that B. pseudomallei 4095a and 4095c OPS antigens exhibited substitution patterns that differed from the prototypic OPS structure. Specifically, 4095a lacked 4-O-acetylation, while 4095c lacked both 4-O-acetylation and 2-O-methylation. Our studies indicate that B. pseudomallei OPS undergoes antigenic variation and suggest that the 9D5 MAb recognizes a conformational epitope that is influenced by both O-acetyl and O-methyl substitution patterns. PMID:25776750

  8. Burkholderia pseudomallei Colony Morphotypes Show a Synchronized Metabolic Pattern after Acute Infection

    PubMed Central

    Steinmetz, Ivo; Lalk, Michael

    2016-01-01

    Background 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. Methods and Principal Findings 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. Conclusion 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. PMID:26943908

  9. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis

    PubMed Central

    Limmathurotsakul, Direk; Golding, Nick; Dance, David AB; Messina, Jane P; Pigott, David M; Moyes, Catherine L; Rolim, Dionne B; Bertherat, Eric; Day, Nicholas PJ; Peacock, Sharon J; Hay, Simon I

    2016-01-01

    Burkholderia pseudomallei, a highly pathogenic bacterium that causes melioidosis, is commonly found in soil in Southeast Asia and Northern Australia1,2. Melioidosis can be difficult to diagnose due to its diverse clinical manifestations and the inadequacy of conventional bacterial identification methods3. The bacterium is intrinsically resistant to a wide range of antimicrobials, and treatment with ineffective antimicrobials may result in case fatality rates (CFRs) exceeding 70%4,5. The importation of infected animals has, in the past, spread melioidosis to non-endemic areas6,7. The global distribution of B. pseudomallei and burden of melioidosis, however, remain poorly understood. Here, we map documented human and animal cases, and the presence of environmental B. pseudomallei, and combine this in a formal modelling framework8-10 to estimate the global burden of melioidosis. We estimate there to be 165,000 (95% credible interval 68,000-412,000) human melioidosis cases per year worldwide, of which 89,000 (36,000-227,000) die. Our estimates suggest that melioidosis is severely underreported in the 45 countries in which it is known to be endemic and that melioidosis is likely endemic in a further 34 countries which have never reported the disease. The large numbers of estimated cases and fatalities emphasise that the disease warrants renewed attention from public health officials and policy makers. PMID:26877885

  10. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis.

    PubMed

    Limmathurotsakul, Direk; Golding, Nick; Dance, David A B; Messina, Jane P; Pigott, David M; Moyes, Catherine L; Rolim, Dionne B; Bertherat, Eric; Day, Nicholas P J; Peacock, Sharon J; Hay, Simon I

    2016-01-01

    Burkholderia pseudomallei, a highly pathogenic bacterium that causes melioidosis, is commonly found in soil in Southeast Asia and Northern Australia(1,2). Melioidosis can be difficult to diagnose due to its diverse clinical manifestations and the inadequacy of conventional bacterial identification methods(3). The bacterium is intrinsically resistant to a wide range of antimicrobials, and treatment with ineffective antimicrobials may result in case fatality rates (CFRs) exceeding 70%(4,5). The importation of infected animals has, in the past, spread melioidosis to non-endemic areas(6,7). The global distribution of B. pseudomallei and the burden of melioidosis, however, remain poorly understood. Here, we map documented human and animal cases and the presence of environmental B. pseudomallei and combine this in a formal modelling framework(8-10) to estimate the global burden of melioidosis. We estimate there to be 165,000 (95% credible interval 68,000-412,000) human melioidosis cases per year worldwide, from which 89,000 (36,000-227,000) people die. Our estimates suggest that melioidosis is severely underreported in the 45 countries in which it is known to be endemic and that melioidosis is probably endemic in a further 34 countries that have never reported the disease. The large numbers of estimated cases and fatalities emphasize that the disease warrants renewed attention from public health officials and policy makers. PMID:27571754

  11. Whole-Genome Sequences of Five Burkholderia pseudomallei Isolates from Australian Cystic Fibrosis Patients

    PubMed Central

    Kidd, Timothy J.; Bell, Scott C.; Currie, Bart J.

    2015-01-01

    We report here five improved high-quality draft genomes of Burkholderia pseudomallei isolated from Australian cystic fibrosis (CF) patients. This pathogen is rarely seen in CF patients. These genomes will be used to better understand chronic carriage of B. pseudomallei in the CF lung and the within-host evolution of longitudinal isolates from these patients. PMID:25883282

  12. Characterization of Burkholderia pseudomallei Strains Using a Murine Intraperitoneal Infection Model and In Vitro Macrophage Assays

    PubMed Central

    Welkos, Susan L.; Klimko, Christopher P.; Kern, Steven J.; Bearss, Jeremy J.; Bozue, Joel A.; Bernhards, Robert C.; Trevino, Sylvia R.; Waag, David M.; Amemiya, Kei; Worsham, Patricia L.; Cote, Christopher K.

    2015-01-01

    Burkholderia pseudomallei, the etiologic agent of melioidosis, is a gram-negative facultative intracellular bacterium. This bacterium is endemic in Southeast Asia and Northern Australia and can infect humans and animals by several routes. It has also been estimated to present a considerable risk as a potential biothreat agent. There are currently no effective vaccines for B. pseudomallei, and antibiotic treatment can be hampered by nonspecific symptomology, the high incidence of naturally occurring antibiotic resistant strains, and disease chronicity. Accordingly, there is a concerted effort to better characterize B. pseudomallei and its associated disease. Before novel vaccines and therapeutics can be tested in vivo, a well characterized animal model is essential. Previous work has indicated that mice may be a useful animal model. In order to develop standardized animal models of melioidosis, different strains of bacteria must be isolated, propagated, and characterized. Using a murine intraperitoneal (IP) infection model, we tested the virulence of 11 B. pseudomallei strains. The IP route offers a reproducible way to rank virulence that can be readily reproduced by other laboratories. This infection route is also useful in distinguishing significant differences in strain virulence that may be masked by the exquisite susceptibility associated with other routes of infection (e.g., inhalational). Additionally, there were several pathologic lesions observed in mice following IP infection. These included varisized abscesses in the spleen, liver, and haired skin. This model indicated that commonly used laboratory strains of B. pseudomallei (i.e., K96243 and 1026b) were significantly less virulent as compared to more recently acquired clinical isolates. Additionally, we characterized in vitro strain-associated differences in virulence for macrophages and described a potential inverse relationship between virulence in the IP mouse model of some strains and in the

  13. Quantitative Proteomic Analysis of Burkholderia pseudomallei Bsa Type III Secretion System Effectors Using Hypersecreting Mutants

    PubMed Central

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

    2015-01-01

    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. PMID:25635268

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

    PubMed Central

    2012-01-01

    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. PMID:23217012

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

    PubMed Central

    2010-01-01

    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. PMID:20540813

  16. Genome Sequence of a Burkholderia pseudomallei Clinical Isolate from a Patient with Community-Acquired Pneumonia and Septicemia

    PubMed Central

    Vandana, K. E.; Chaitanya, T. A. K.; Shaw, Tushar; Bhat, H. Vinod; Chakrabarty, Sanjiban; Paul, Bobby; Mallya, Sandeep; Murali, T. S.; Satyamoorthy, Kapaettu

    2015-01-01

    Here, we report the draft genome sequence of Burkholderia pseudomallei CM_Manipal, the causative agent of melioidosis isolated from a diabetic patient in Manipal, southern India. The draft genome consists of 107 contigs and is 7,209,157 bp long. A total of 5,600 coding sequences (CDSs), 60 tRNAs, 12 rRNAs, and one noncoding RNA (ncRNA) were predicted from this assembly. PMID:26294629

  17. Genome Sequence of a Burkholderia pseudomallei Clinical Isolate from a Patient with Community-Acquired Pneumonia and Septicemia.

    PubMed

    Mukhopadhyay, Chiranjay; Vandana, K E; Chaitanya, T A K; Shaw, Tushar; Bhat, H Vinod; Chakrabarty, Sanjiban; Paul, Bobby; Mallya, Sandeep; Murali, T S; Satyamoorthy, Kapaettu

    2015-01-01

    Here, we report the draft genome sequence of Burkholderia pseudomallei CM_Manipal, the causative agent of melioidosis isolated from a diabetic patient in Manipal, southern India. The draft genome consists of 107 contigs and is 7,209,157 bp long. A total of 5,600 coding sequences (CDSs), 60 tRNAs, 12 rRNAs, and one noncoding RNA (ncRNA) were predicted from this assembly. PMID:26294629

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

    PubMed Central

    2012-01-01

    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 <10 colony-forming units (cfu) for all three species. In comparison, the LD50 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

  19. The chemical arsenal of Burkholderia pseudomallei is essential for pathogenicity.

    PubMed

    Biggins, John B; Kang, Hahk-Soo; Ternei, Melinda A; DeShazer, David; Brady, Sean F

    2014-07-01

    Increasing evidence has shown that small-molecule chemistry in microbes (i.e., secondary metabolism) can modulate the microbe-host response in infection and pathogenicity. The bacterial disease melioidosis is conferred by the highly virulent, antibiotic-resistant pathogen Burkholderia pseudomallei (BP). Whereas some macromolecular structures have been shown to influence BP virulence (e.g., secretion systems, cellular capsule, pili), the role of the large cryptic secondary metabolome encoded within its genome has been largely unexplored for its importance to virulence. Herein we demonstrate that BP-encoded small-molecule biosynthesis is indispensible for in vivo BP pathogenicity. Promoter exchange experiments were used to induce high-level molecule production from two gene clusters (MPN and SYR) found to be essential for in vivo virulence. NMR structural characterization of these metabolites identified a new class of lipopeptide biosurfactants/biofilm modulators (the malleipeptins) and syrbactin-type proteasome inhibitors, both of which represent overlooked small-molecule virulence factors for BP. Disruption of Burkholderia virulence by inhibiting the biosynthesis of these small-molecule biosynthetic pathways may prove to be an effective strategy for developing novel melioidosis-specific therapeutics. PMID:24884988

  20. Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head

    PubMed Central

    Edwards, Thomas E.; Phan, Isabelle; Abendroth, Jan; Dieterich, Shellie H.; Masoudi, Amir; Guo, Wenjin; Hewitt, Stephen N.; Kelley, Angela; Leibly, David; Brittnacher, Mitch J.; Staker, Bart L.; Miller, Samuel I.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.

    2010-01-01

    Background Pathogenic bacteria adhere to the host cell surface using a family of outer membrane proteins called Trimeric Autotransporter Adhesins (TAAs). Although TAAs are highly divergent in sequence and domain structure, they are all conceptually comprised of a C-terminal membrane anchoring domain and an N-terminal passenger domain. Passenger domains consist of a secretion sequence, a head region that facilitates binding to the host cell surface, and a stalk region. Methodology/Principal Findings Pathogenic species of Burkholderia contain an overabundance of TAAs, some of which have been shown to elicit an immune response in the host. To understand the structural basis for host cell adhesion, we solved a 1.35 Å resolution crystal structure of a BpaA TAA head domain from Burkholderia pseudomallei, the pathogen that causes melioidosis. The structure reveals a novel fold of an intricately intertwined trimer. The BpaA head is composed of structural elements that have been observed in other TAA head structures as well as several elements of previously unknown structure predicted from low sequence homology between TAAs. These elements are typically up to 40 amino acids long and are not domains, but rather modular structural elements that may be duplicated or omitted through evolution, creating molecular diversity among TAAs. Conclusions/Significance The modular nature of BpaA, as demonstrated by its head domain crystal structure, and of TAAs in general provides insights into evolution of pathogen-host adhesion and may provide an avenue for diagnostics. PMID:20862217

  1. Superoxide dismutase C is required for intracellular survival and virulence of Burkholderia pseudomallei.

    PubMed

    Vanaporn, Muthita; Wand, Matthew; Michell, Stephen L; Sarkar-Tyson, Mitali; Ireland, Philip; Goldman, Stan; Kewcharoenwong, Chidchamai; Rinchai, Darawan; Lertmemongkolchai, Ganjana; Titball, Richard W

    2011-08-01

    Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a life-threatening disease of humans. Within host cells, superoxide is an important mediator of pathogen killing. In this study, we have identified the B. pseudomallei K96243 sodC gene, shown that it has superoxide dismutase activity, and constructed an allelic deletion mutant of this gene. Compared with the wild-type, the mutant was more sensitive to killing by extracellular superoxide, but not to superoxide generated intracellularly. The sodC mutant showed a markedly decreased survival in J774A.1 mouse macrophages, and reduced numbers of bacteria were recovered from human polymorphonuclear neutrophils (PMNs) when compared with the wild-type. The numbers of wild-type or mutant bacteria recovered from human diabetic neutrophils were significantly lower than from normal human neutrophils. The sodC mutant was attenuated in BALB/c mice. Our results indicate that SodC plays a key role in the virulence of B. pseudomallei, but that diabetics are not more susceptible to infection because of a reduced ability of PMNs to kill by superoxide. PMID:21659326

  2. Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei.

    PubMed

    Moule, Madeleine G; Spink, Natasha; Willcocks, Sam; Lim, Jiali; Guerra-Assunção, José Afonso; Cia, Felipe; Champion, Olivia L; Senior, Nicola J; Atkins, Helen S; Clark, Taane; Bancroft, Gregory J; Cuccui, Jon; Wren, Brendan W

    2015-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survival in vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuation in vivo were identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarked bpsl2248, tex, rpiR, bpsl1728, and bpss1528 deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was tested in vitro and in vivo to confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important to in vivo virulence with roles in different stages of B. pseudomallei pathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and the tex mutant was capable of providing protective immunity against challenge with wild-type B. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates. PMID:26712202

  3. The Concentrations of Ambient Burkholderia Pseudomallei during Typhoon Season in Endemic Area of Melioidosis in Taiwan

    PubMed Central

    Yang, Chun-Yuh; Lee, Min Sheng; Ho, Chi-Kung; Mena, Kristina D.; Wang, Peng-Yau; Chen, Pei-Shih

    2014-01-01

    Background Melioidosis is a severe bacterial infection caused by Burkholderia pseudomallei with a high case-fatality rate. Epidemiological and animal studies show the possibility of inhalation transmission. However, no B. pseudomallei concentrations in ambient air have been researched. Here, we developed a method to quantify ambient B. pseudomallei and then measured concentrations of ambient B. pseudomallei during the typhoon season and the non-typhoon season to determine the factors influencing ambient B. pseudomallei levels. Methods We quantified ambient B. pseudomallei by using a filter/real-time qPCR method in the Zoynan Region in Kaohsiung, southern Taiwan. Twenty-four hour samples were collected at a sampling rate of 20 L/min every day from June 11 to December 21, 2012 including during the typhoon season (June to September) and reference season (October to December). Results We successfully developed a filtration/real-time qPCR method to quantify ambient B. pseudomallei. To our knowledge, this is the first report describing concentrations of ambient B. pseudomallei. Ambient B. pseudomallei were only detected during the typhoon season when compared to the reference season. For the typhoons affecting the Zoynan Region, the positive rates of ambient B. pseudomallei were very high at 80% to 100%. During June to December, rainfall was positively correlated with ambient B. pseudomallei with a statistical significance. Sediment at a nearby pond significantly influenced the concentration of ambient B. pseudomallei. During the typhoon month, the typhoon was positively correlated with ambient B. pseudomallei whereas wind speed was reversely correlated with ambient B. pseudomallei. Conclusions Our data suggest the possibility of transmission of B. pseudomallei via inhalation during the typhoon season. PMID:24874950

  4. Identification of a Predicted Trimeric Autotransporter Adhesin Required for Biofilm Formation of Burkholderia pseudomallei

    PubMed Central

    Lazar Adler, Natalie R.; Dean, Rachel E.; Saint, Richard J.; Stevens, Mark P.; Prior, Joann L.; Atkins, Timothy P.; Galyov, Edouard E.

    2013-01-01

    The autotransporters are a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play a role in numerous environmental and virulence-associated interactions. As part of a larger systematic study on the autotransporters of Burkholderia pseudomallei, the causative agent of the severe tropical disease melioidosis, we have constructed an insertion mutant in the bpss1439 gene encoding an unstudied predicted trimeric autotransporter adhesin. The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain. This phenotype was complemented to wild-type levels by the introduction of a full-length copy of the bpss1439 gene in trans. Examination of the wild-type and bpss1439 mutant strains under biofilm-inducing conditions by microscopy after 48 hours confirmed that the bpss1439 mutant produced less biofilm compared to wild-type. Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation. In a murine melioidosis model, the bpss1439 mutant strain demonstrated a moderate attenuation for virulence compared to the wild-type strain. This attenuation was abrogated by in trans complementation, suggesting that bpss1439 plays a subtle role in the pathogenesis of B. pseudomallei. Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A. PMID:24223950

  5. The Multiple Roles of Hypothetical Gene BPSS1356 in Burkholderia pseudomallei

    PubMed Central

    Yam, Hokchai; Abdul Rahim, Ainihayati; Mohamad, Suriani; Mahadi, Nor Muhammad; Abdul Manaf, Uyub; Shu-Chien, Alexander Chong; Najimudin, Nazalan

    2014-01-01

    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. PMID:24927285

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

    PubMed

    Yam, Hokchai; Rahim, Ainihayati Abdul; Mohamad, Suriani; Mahadi, Nor Muhammad; Manaf, Uyub Abdul; Shu-Chien, Alexander Chong; Najimudin, Nazalan

    2014-01-01

    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. PMID:24927285

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

    PubMed

    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

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

    PubMed Central

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

    2013-01-01

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

  9. What Drives the Occurrence of the Melioidosis Bacterium Burkholderia pseudomallei in Domestic Gardens?

    PubMed Central

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

    2015-01-01

    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. PMID:25803046

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

    PubMed

    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. PMID:25803046

  11. Landscape Changes Influence the Occurrence of the Melioidosis Bacterium Burkholderia pseudomallei in Soil in Northern Australia

    PubMed Central

    Kaestli, Mirjam; Mayo, Mark; Harrington, Glenda; Ward, Linda; Watt, Felicity; Hill, Jason V.; Cheng, Allen C.; Currie, Bart J.

    2009-01-01

    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. PMID:19156200

  12. Macroautophagy is essential for killing of intracellular Burkholderia pseudomallei in human neutrophils

    PubMed Central

    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. PMID:25996656

  13. Characterization of an autotransporter adhesin protein shared by Burkholderia mallei and Burkholderia pseudomallei

    PubMed Central

    2014-01-01

    Background Autotransporters form a large family of outer membrane proteins specifying diverse biological traits of Gram-negative bacteria. In this study, we report the identification and characterization of a novel autotransporter gene product of Burkholderia mallei (locus tag BMA1027 in strain ATCC 23344). Results Database searches identified the gene in at least seven B. mallei isolates and the encoded proteins were found to be 84% identical. Inactivation of the gene encoding the autotransporter in the genome of strain ATCC 23344 substantially reduces adherence to monolayers of HEp-2 laryngeal cells and A549 type II pneumocytes, as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, expression of the autotransporter on the surface of recombinant E. coli bacteria increases adherence to these cell types by 5–7 fold. The gene specifying the autotransporter was identified in the genome of 29 B. pseudomallei isolates and disruption of the gene in strain DD503 reduced adherence to NHBE cultures by 61%. Unlike B. mallei, the mutation did not impair binding of B. pseudomallei to A549 or HEp-2 cells. Analysis of sera from mice infected via the aerosol route with B. mallei and B. pseudomallei revealed that animals inoculated with as few as 10 organisms produce antibodies against the autotransporter, therefore indicating expression in vivo. Conclusions Our data demonstrate that we have identified an autotransporter protein common to the pathogenic species B. mallei and B. pseudomallei which mediates adherence to respiratory epithelial cells and is expressed in vivo during the course of aerosol infection. PMID:24731253

  14. Evaluation of a Burkholderia pseudomallei Outer Membrane Vesicle Vaccine in Nonhuman Primates.

    PubMed

    Petersen, Hailey; Nieves, Wildaliz; Russell-Lodrigue, Kasi; Roy, Chad J; Morici, Lisa A

    2014-01-01

    Burkholderia pseudomallei (Bps)is the causative agent of melioidosis and is endemic in regions of northern Australia and Southeast Asia. Bps is inherently resistant to multiple antibiotics and is considered a potential biological warfare agent by the U.S. DHHS. Therefore, effective vaccines are necessary to prevent natural infection and to safeguard against biological attack with this organism. In our previous work we have shown that immunization with naturally derived outer membrane vesicles (OMVs) from Bps provides significant protection against lethal aerosol and systemic infection in BALB/c mice. In this work, we evaluated the safety and immunogenicity of escalating doses of OMV vaccine in rhesus macaques. We show that immunization of rhesus macaques with Bps OMVs generates humoral immuneresponses to protective protein and polysaccharide antigens without any associated toxicity or reactogenicity. These results lay the groundwork for evaluation of protective efficacy of the OMV vaccine in the nonhuman primate model of melioidosis. PMID:25165491

  15. Role for the Burkholderia pseudomallei type three secretion system cluster 1 bpscN gene in virulence.

    PubMed

    D'Cruze, Tanya; Gong, Lan; Treerat, Puthayalai; Ramm, Georg; Boyce, John D; Prescott, Mark; Adler, Ben; Devenish, Rodney J

    2011-09-01

    Burkholderia pseudomallei, the causal agent of melioidosis, employs a number of virulence factors during its infection of mammalian cells. One such factor is the type three secretion system (TTSS), which is proposed to mediate the transport and secretion of bacterial effector molecules directly into host cells. The B. pseudomallei genome contains three TTSS gene clusters (designated TTSS1, TTSS2, and TTSS3). Previous research has indicated that neither TTSS1 nor TTSS2 is involved in B. pseudomallei virulence in a hamster infection model. We have characterized a B. pseudomallei mutant lacking expression of the predicted TTSS1 ATPase encoded by bpscN. This mutant was significantly attenuated for virulence in a respiratory melioidosis mouse model of infection. In addition, analyses in vitro showed diminished survival and replication in RAW264.7 cells and an increased level of colocalization with the autophagy marker protein LC3 but an unhindered ability to escape from phagosomes. Taken together, these data provide evidence that the TTSS1 bpscN gene product plays an important role in the intracellular survival of B. pseudomallei and the pathogenesis of murine infection. PMID:21768285

  16. Identification of the conserved hypothetical protein BPSL0317 in Burkholderia pseudomallei K96243

    NASA Astrophysics Data System (ADS)

    Yusoff, Nur Syamimi; Damiri, Nadzirah; Firdaus-Raih, Mohd

    2014-09-01

    Burkholderia pseudomallei K96243 is the causative agent of melioidosis, a disease which is endemic in Northern Australia and Southeastern Asia. The genome encodes several essential proteins including those currently annotated as hypothetical proteins. We studied the conservation and the essentiality of expressed hypothetical proteins in normal and different stress conditions. Based on the comparative genomics, we identified a hypothetical protein, BPSL0317, a potential essential gene that is being expressed in all normal and stress conditions. BPSL0317 is also phylogenetically conserved in the Burkholderiales order suggesting that this protein is crucial for survival among the order's members. BPSL0317 therefore has a potential to be a candidate antimicrobial drug target for this group of bacteria.

  17. Characterization of BcaA, a Putative Classical Autotransporter Protein in Burkholderia pseudomallei

    PubMed Central

    Campos, Cristine G.; Borst, Luke

    2013-01-01

    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. PMID:23340315

  18. The In vitro Antibiotic Tolerant Persister Population in Burkholderia pseudomallei is Altered by Environmental Factors

    PubMed Central

    Nierman, William C.; Yu, Yan; Losada, Liliana

    2015-01-01

    Bacterial persistence due to antibiotic tolerance is a critical aspect of antibiotic treatment failure, disease latency, and chronic or reemergent infections. The levels of persisters is especially notable for the opportunistic Gram-negative pathogens from the Burkholderia and Pseudomonas genera. We examined the rate of drug tolerant persisters in Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia cepacia complex organisms, and Pseudomonas aeruginosa at mid-log growth in LB broth culture. We found that a fraction of the antibiotic-sensitive cells from every species were tolerant to a 24 h high-dose antibiotic challenge. All tested Burkholderia strains demonstrated a drug tolerant persister population at a rate that was at least 100–500 times higher than P. aeruginosa. When challenged with at least a 10X minimum inhibitory concentration (MIC) 24 h exposure to three different antibiotics with different modes of action we found that in B. pseudomallei Bp82 each of the tree antibiotics revealed different persister fractions at each of two different growth states. This observation suggests that our assay is detecting heterogeneous persister subpopulations. Persistence in B. pseudomallei Bp82 was highly dependent on growth stage, with a surprisingly high persister fraction of >64% of the late stationary phase cells being antibiotic tolerant to 100XMIC cefotaxime. Adaptation of B. pseudomallei to distilled water storage resulted in a population of drug tolerant cells up to 100% of the non-drug-challenged viable cell count in the same cefotaxime assay. Cultivation of B. pseudomallei with a sub-inhibitory concentration of several antibiotics resulted in altered persister fractions within the population relative to cultures lacking the antibiotic. Our study provides insight into the sensitivity of the persister fraction within the population of B. pseudomallei due to environmental variables and suggests diversity within the persister population revealed by

  19. The In vitro Antibiotic Tolerant Persister Population in Burkholderia pseudomallei is Altered by Environmental Factors.

    PubMed

    Nierman, William C; Yu, Yan; Losada, Liliana

    2015-01-01

    Bacterial persistence due to antibiotic tolerance is a critical aspect of antibiotic treatment failure, disease latency, and chronic or reemergent infections. The levels of persisters is especially notable for the opportunistic Gram-negative pathogens from the Burkholderia and Pseudomonas genera. We examined the rate of drug tolerant persisters in Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia cepacia complex organisms, and Pseudomonas aeruginosa at mid-log growth in LB broth culture. We found that a fraction of the antibiotic-sensitive cells from every species were tolerant to a 24 h high-dose antibiotic challenge. All tested Burkholderia strains demonstrated a drug tolerant persister population at a rate that was at least 100-500 times higher than P. aeruginosa. When challenged with at least a 10X minimum inhibitory concentration (MIC) 24 h exposure to three different antibiotics with different modes of action we found that in B. pseudomallei Bp82 each of the tree antibiotics revealed different persister fractions at each of two different growth states. This observation suggests that our assay is detecting heterogeneous persister subpopulations. Persistence in B. pseudomallei Bp82 was highly dependent on growth stage, with a surprisingly high persister fraction of >64% of the late stationary phase cells being antibiotic tolerant to 100XMIC cefotaxime. Adaptation of B. pseudomallei to distilled water storage resulted in a population of drug tolerant cells up to 100% of the non-drug-challenged viable cell count in the same cefotaxime assay. Cultivation of B. pseudomallei with a sub-inhibitory concentration of several antibiotics resulted in altered persister fractions within the population relative to cultures lacking the antibiotic. Our study provides insight into the sensitivity of the persister fraction within the population of B. pseudomallei due to environmental variables and suggests diversity within the persister population revealed by

  20. Genomic Characterization of Burkholderia pseudomallei Isolates Selected for Medical Countermeasures Testing: Comparative Genomics Associated with Differential Virulence

    PubMed Central

    Sahl, Jason W.; Allender, Christopher J.; Colman, Rebecca E.; Califf, Katy J.; Schupp, James M.; Currie, Bart J.; Van Zandt, Kristopher E.; Gelhaus, H. Carl; Keim, Paul; Tuanyok, Apichai

    2015-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis and a potential bioterrorism agent. In the development of medical countermeasures against B. pseudomallei infection, the US Food and Drug Administration (FDA) animal Rule recommends using well-characterized strains in animal challenge studies. In this study, whole genome sequence data were generated for 6 B. pseudomallei isolates previously identified as candidates for animal challenge studies; an additional 5 isolates were sequenced that were associated with human inhalational melioidosis. A core genome single nucleotide polymorphism (SNP) phylogeny inferred from a concatenated SNP alignment from the 11 isolates sequenced in this study and a diverse global collection of isolates demonstrated the diversity of the proposed Animal Rule isolates. To understand the genomic composition of each isolate, a large-scale blast score ratio (LS-BSR) analysis was performed on the entire pan-genome; this demonstrated the variable composition of genes across the panel and also helped to identify genes unique to individual isolates. In addition, a set of ~550 genes associated with pathogenesis in B. pseudomallei were screened against the 11 sequenced genomes with LS-BSR. Differential gene distribution for 54 virulence-associated genes was observed between genomes and three of these genes were correlated with differential virulence observed in animal challenge studies using BALB/c mice. Differentially conserved genes and SNPs associated with disease severity were identified and could be the basis for future studies investigating the pathogenesis of B. pseudomallei. Overall, the genetic characterization of the 11 proposed Animal Rule isolates provides context for future studies involving B. pseudomallei pathogenesis, differential virulence, and efficacy to therapeutics. PMID:25803742

  1. Rapid Antimicrobial Susceptibility Testing of Bacillus anthracis, Yersinia pestis, and Burkholderia pseudomallei by Use of Laser Light Scattering Technology.

    PubMed

    Bugrysheva, Julia V; Lascols, Christine; Sue, David; Weigel, Linda M

    2016-06-01

    Rapid methods to determine antimicrobial susceptibility would assist in the timely distribution of effective treatment or postexposure prophylaxis in the aftermath of the release of bacterial biothreat agents such as Bacillus anthracis, Yersinia pestis, or Burkholderia pseudomallei Conventional susceptibility tests require 16 to 48 h of incubation, depending on the bacterial species. We evaluated a method that is based on laser light scattering technology that measures cell density in real time. We determined that it has the ability to rapidly differentiate between growth (resistant) and no growth (susceptible) of several bacterial threat agents in the presence of clinically relevant antimicrobials. Results were available in <4 h for B. anthracis and <6 h for Y. pestis and B. pseudomallei One exception was B. pseudomallei in the presence of ceftazidime, which required >10 h of incubation. Use of laser scattering technology decreased the time required to determine antimicrobial susceptibility by 50% to 75% for B. anthracis, Y. pestis, and B. pseudomallei compared to conventional methods. PMID:26984973

  2. Interrogation of the Burkholderia pseudomallei genome to address differential virulence among isolates

    DOE PAGESBeta

    Challacombe, Jean F.; Stubben, Chris J.; Klimko, Christopher P.; Welkos, Susan L.; Kern, Steven J.; Bozue, Joel A.; Worsham, Patricia L.; Cote, Christopher K.; Wolfe, Daniel N.; Badger, Jonathan H.

    2014-12-23

    Infection by the Gram-negative pathogen Burkholderia pseudomallei results in the disease melioidosis, acquired from the environment in parts of southeast Asia and northern Australia. Clinical symptoms of melioidosis range from acute (fever, pneumonia, septicemia, and localized infection) to chronic (abscesses in various organs and tissues, most commonly occurring in the lungs, liver, spleen, kidney, prostate and skeletal muscle), and persistent infections in humans are difficult to cure. Understanding the basic biology and genomics of B. pseudomallei is imperative for the development of new vaccines and therapeutic interventions. This formidable task is becoming more tractable due to the increasing number ofmore » B. pseudomallei genomes that are being sequenced and compared. Here, we compared three B. pseudomallei genomes, from strains MSHR668, K96243 and 1106a, to identify features that might explain why MSHR668 is more virulent than K96243 and 1106a in a mouse model of B. pseudomallei infection. Our analyses focused on metabolic, virulence and regulatory genes that were present in MSHR668 but absent from both K96243 and 1106a. We also noted features present in K96243 and 1106a but absent from MSHR668, and identified genomic differences that may contribute to variations in virulence noted among the three B. pseudomallei isolates. While this work contributes to our understanding of B. pseudomallei genomics, more detailed experiments are necessary to characterize the relevance of specific genomic features to B. pseudomallei metabolism and virulence. Functional analyses of metabolic networks, virulence and regulation shows promise for examining the effects of B. pseudomallei on host cell metabolism and will lay a foundation for future prediction of the virulence of emerging strains. Continued emphasis in this area will be critical for protection against melioidosis, as a better understanding of what constitutes a fully virulent Burkholderia isolate may provide for

  3. Interrogation of the Burkholderia pseudomallei Genome to Address Differential Virulence among Isolates

    PubMed Central

    Challacombe, Jean F.; Stubben, Chris J.; Klimko, Christopher P.; Welkos, Susan L.; Kern, Steven J.; Bozue, Joel A.; Worsham, Patricia L.; Cote, Christopher K.; Wolfe, Daniel N.

    2014-01-01

    Infection by the Gram-negative pathogen Burkholderia pseudomallei results in the disease melioidosis, acquired from the environment in parts of southeast Asia and northern Australia. Clinical symptoms of melioidosis range from acute (fever, pneumonia, septicemia, and localized infection) to chronic (abscesses in various organs and tissues, most commonly occurring in the lungs, liver, spleen, kidney, prostate and skeletal muscle), and persistent infections in humans are difficult to cure. Understanding the basic biology and genomics of B. pseudomallei is imperative for the development of new vaccines and therapeutic interventions. This formidable task is becoming more tractable due to the increasing number of B. pseudomallei genomes that are being sequenced and compared. Here, we compared three B. pseudomallei genomes, from strains MSHR668, K96243 and 1106a, to identify features that might explain why MSHR668 is more virulent than K96243 and 1106a in a mouse model of B. pseudomallei infection. Our analyses focused on metabolic, virulence and regulatory genes that were present in MSHR668 but absent from both K96243 and 1106a. We also noted features present in K96243 and 1106a but absent from MSHR668, and identified genomic differences that may contribute to variations in virulence noted among the three B. pseudomallei isolates. While this work contributes to our understanding of B. pseudomallei genomics, more detailed experiments are necessary to characterize the relevance of specific genomic features to B. pseudomallei metabolism and virulence. Functional analyses of metabolic networks, virulence and regulation shows promise for examining the effects of B. pseudomallei on host cell metabolism and will lay a foundation for future prediction of the virulence of emerging strains. Continued emphasis in this area will be critical for protection against melioidosis, as a better understanding of what constitutes a fully virulent Burkholderia isolate may provide for better

  4. Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei.

    PubMed

    Randall, Linnell B; Dobos, Karen; Papp-Wallace, Krisztina M; Bonomo, Robert A; Schweizer, Herbert P

    2015-01-01

    Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. β-Lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime-resistant clinical isolates have been described, and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded class A PenA β-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying β-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of β-lactamase activity and absence of detectable PenAC23S protein. A concomitant isoleucine-to-alanine change at position 20 in the signal peptide processing site in the PenAC23S mutant results in a nonlipidated protein (PenAI20A C23S) that is processed by signal peptidase I and exhibits β-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the profile of the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose. PMID:26711764

  5. Genome Sequence of the Historical Clinical Isolate Burkholderia pseudomallei PHLS 6

    DOE PAGESBeta

    D’haeseleer, Patrik; Johnson, Shannon L.; Davenport, Karen W.; Chain, Patrick S.; Schoeniger, Joe; Ray, Debjit; Sinha, Anupama; Williams, Kelly P.; Peña, José; Branda, Steven S.; et al

    2016-06-30

    We present the draft genome sequence ofBurkholderia pseudomalleiPHLS 6, a virulent clinical strain isolated from a melioidosis patient in Bangladesh in 1960. This draft genome consists of 39 contigs and is 7,322,181 bp long.

  6. Finished Annotated Genome Sequence of Burkholderia pseudomallei Strain Bp1651, a Multidrug-Resistant Clinical Isolate

    PubMed Central

    Sue, David; Hakovirta, Janetta; Loparev, Vladimir N.; Knipe, Kristen; Sammons, Scott A.; Ranganathan-Ganakammal, Satishkumar; Changayil, Shankar; Srinivasamoorthy, Ganesh; Weil, Michael R.; Tatusov, Roman L.; Gee, Jay E.; Elrod, Mindy G.; Hoffmaster, Alex R.; Weigel, Linda M.

    2015-01-01

    Burkholderia pseudomallei strain Bp1651, a human isolate, is resistant to all clinically relevant antibiotics. We report here on the finished genome sequence assembly and annotation of the two chromosomes of this strain. This genome sequence may assist in understanding the mechanisms of antimicrobial resistance for this pathogenic species. PMID:26634765

  7. Comparison of Whole-Genome Sequences from Two Colony Morphovars of Burkholderia pseudomallei

    PubMed Central

    Hsueh, Pei-Tan; Chen, Yao-Shen; Lin, Hsi-Hsu; Liu, Pei-Ju; Ni, Wen-Fan; Liu, Mei-Chun

    2015-01-01

    The entire genomes of two isogenic morphovars (vgh16W and vgh16R) of Burkholderia pseudomallei were sequenced. A comparison of the sequences from both strains indicates that they show 99.99% identity, are composed of 22 tandem repeated sequences with <100 bp of indels, and have 199 single-base variants. PMID:26472836

  8. Genome Sequence of the Historical Clinical Isolate Burkholderia pseudomallei PHLS 6.

    PubMed

    D'haeseleer, Patrik; Johnson, Shannon L; Davenport, Karen W; Chain, Patrick S; Schoeniger, Joe; Ray, Debjit; Sinha, Anupama; Williams, Kelly P; Peña, José; Branda, Steven S; El-Etr, Sahar

    2016-01-01

    Here, we present the draft genome sequence of Burkholderia pseudomallei PHLS 6, a virulent clinical strain isolated from a melioidosis patient in Bangladesh in 1960. The draft genome consists of 39 contigs and is 7,322,181 bp long. PMID:27365360

  9. Genome Sequence of the Historical Clinical Isolate Burkholderia pseudomallei PHLS 6

    PubMed Central

    Davenport, Karen W.; Chain, Patrick S.; Schoeniger, Joe; Ray, Debjit; Sinha, Anupama; Williams, Kelly P.; Peña, José; El-Etr, Sahar

    2016-01-01

    Here, we present the draft genome sequence of Burkholderia pseudomallei PHLS 6, a virulent clinical strain isolated from a melioidosis patient in Bangladesh in 1960. The draft genome consists of 39 contigs and is 7,322,181 bp long. PMID:27365360

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

    PubMed

    Gong, Lan; Cullinane, Meabh; Treerat, Puthayalai; Ramm, Georg; Prescott, Mark; Adler, Ben; Boyce, John D; Devenish, Rodney J

    2011-01-01

    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. PMID:21412437

  11. Burkholderia pseudomallei kynB plays a role in AQ production, biofilm formation, bacterial swarming and persistence.

    PubMed

    Butt, Aaron; Halliday, Nigel; Williams, Paul; Atkins, Helen S; Bancroft, Gregory J; Titball, Richard W

    2016-04-01

    Kynurenine formamidase (KynB) forms part of the kynurenine pathway which metabolises tryptophan to anthranilate. This metabolite can be used for downstream production of 2-alkyl-4-quinolone (AQ) signalling molecules that control virulence in Pseudomonas aeruginosa. Here we investigate the role of kynB in the production of AQs and virulence-associated phenotypes of Burkholderia pseudomallei K96243, the causative agent of melioidosis. Deletion of kynB resulted in reduced AQ production, increased biofilm formation, decreased swarming and increased tolerance to ciprofloxacin. Addition of exogenous anthranilic acid restored the biofilm phenotype, but not the persister phenotype. This study suggests the kynurenine pathway is a critical source of anthranilate and signalling molecules that may regulate B. pseudomallei virulence. PMID:26654915

  12. Burkholderia pseudomallei Capsular Polysaccharide Recognition by a Monoclonal Antibody Reveals Key Details toward a Biodefense Vaccine and Diagnostics against Melioidosis.

    PubMed

    Marchetti, Roberta; Dillon, Michael J; Burtnick, Mary N; Hubbard, Mark A; Kenfack, Marielle Tamigney; Blériot, Yves; Gauthier, Charles; Brett, Paul J; AuCoin, David P; Lanzetta, Rosa; Silipo, Alba; Molinaro, Antonio

    2015-10-16

    Burkholderia pseudomallei is the bacterium responsible for melioidosis, an infectious disease with high mortality rates. Since melioidosis is a significant public health concern in endemic regions and the organism is currently classified as a potential biothreat agent, the development of effective vaccines and rapid diagnostics is a priority. The capsular polysaccharide (CPS) expressed by B. pseudomallei is a highly conserved virulence factor and a protective antigen. Because of this, CPS is considered an attractive antigen for use in the development of both vaccines and diagnostics. In the present study, we describe the interactions of CPS with the murine monoclonal antibody (mAb) 4C4 using a multidisciplinary approach including organic synthesis, molecular biology techniques, surface plasmon resonance, and nuclear magnetic spectroscopy. Using these methods, we determined the mode of binding between mAb 4C4 and native CPS or ad hoc synthesized capsular polysaccharide fragments. Interestingly, we demonstrated that the O-acetyl moiety of CPS is essential for the interaction of the CPS epitope with mAb 4C4. Collectively, our results provide important insights into the structural features of B. pseudomallei CPS that enable antibody recognition that may help the rational design of CPS-based vaccine candidates. In addition, our findings confirm that the mAb 4C4 is suitable for use in an antibody-based detection assay for diagnosis of B. pseudomallei infections. PMID:26198038

  13. Comparison of the early host immune response to two widely diverse virulent strains of Burkholderia pseudomallei that cause acute or chronic infections in BALB/c mice.

    PubMed

    Amemiya, Kei; Dankmeyer, Jennifer L; Fetterer, David P; Worsham, Patricia L; Welkos, Susan L; Cote, Christopher K

    2015-09-01

    Burkholderia pseudomallei is the etiologic agent of melioidosis, which is endemic in Southeast Asia and Northern Australia. We previously found by the intraperitoneal (IP) route that we could discern differences in virulence in mice amongst different strains of B. pseudomallei. We report an early immune response study comparing two strains in our collection which represent the least, B. pseudomallei 1106a, and one of the most, HBPUB10134a, virulent strains in BALB/c mice. B. pseudomallei HBPUB10134a infected mouse spleens contained a 2-3 log higher bacterial burden than mice infected with B. pseudomallei 1106a 3 days post-infection (PI). More and higher amounts of inflammatory cytokines/chemokines were detected in sera and spleen extracts from B. pseudomallei HBPUB10134a than B. pseudomallei 1106a infected mice. The most prominent were IFNγ, IL-1α, IL-1β, IL-6, IL-10, IP-10, and MIG. After 7 days PI, there was a decrease in bacterial burden in spleens from 1106a infected mice and a decrease in cytokines/chemokines in sera and spleen extracts from both sets of mice. By day 14 PI we saw an increase in monocytes/macrophages, NK cells, and granulocytes in spleens from both sets of mice. No B. pseudomallei HBPUB10134a infected mice survived after this time. In summary, B. pseudomallei HBPUB10134a was more virulent and induced host innate immune responses typical of a more acute-type infection than did B. pseudomallei 1106a which produced a more chronic infection in mice. PMID:26162294

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

    PubMed Central

    2012-01-01

    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 analysis being faster than

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

    PubMed

    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. PMID:26758304

  16. Use of Whole-Genome Sequencing to Link Burkholderia pseudomallei from Air Sampling to Mediastinal Melioidosis, Australia

    PubMed Central

    Price, Erin P.; Mayo, Mark; Kaestli, Mirjam; Theobald, Vanessa; Harrington, Ian; Harrington, Glenda; Sarovich, Derek S.

    2015-01-01

    The frequency with which melioidosis results from inhalation rather than percutaneous inoculation or ingestion is unknown. We recovered Burkholderia pseudomallei from air samples at the residence of a patient with presumptive inhalational melioidosis and used whole-genome sequencing to link the environmental bacteria to B. pseudomallei recovered from the patient. PMID:26488732

  17. Use of Whole-Genome Sequencing to Link Burkholderia pseudomallei from Air Sampling to Mediastinal Melioidosis, Australia.

    PubMed

    Currie, Bart J; Price, Erin P; Mayo, Mark; Kaestli, Mirjam; Theobald, Vanessa; Harrington, Ian; Harrington, Glenda; Sarovich, Derek S

    2015-11-01

    The frequency with which melioidosis results from inhalation rather than percutaneous inoculation or ingestion is unknown. We recovered Burkholderia pseudomallei from air samples at the residence of a patient with presumptive inhalational melioidosis and used whole-genome sequencing to link the environmental bacteria to B. pseudomallei recovered from the patient. PMID:26488732

  18. Functional Characterization and Evaluation of In Vitro Protective Efficacy of Murine Monoclonal Antibodies BURK24 and BURK37 against Burkholderia pseudomallei

    PubMed Central

    Peddayelachagiri, Bhavani V.; Paul, Soumya; Makam, Shivakiran S.; Urs, Radhika M.; Kingston, Joseph J.; Tuteja, Urmil; Sripathy, Murali H.; Batra, Harsh V.

    2014-01-01

    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 bacterium to induce

  19. Highly Sensitive Direct Detection and Quantification of Burkholderia pseudomallei Bacteria in Environmental Soil Samples by Using Real-Time PCR ▿ †

    PubMed Central

    Trung, Trinh Thanh; Hetzer, Adrian; Göhler, André; Topfstedt, Eylin; Wuthiekanun, Vanaporn; Limmathurotsakul, Direk; Peacock, Sharon J.; Steinmetz, Ivo

    2011-01-01

    The soil bacterium and potential biothreat agent Burkholderia pseudomallei causes the infectious disease melioidosis, which is naturally acquired through environmental contact with the bacterium. Environmental detection of B. pseudomallei represents the basis for the development of a geographical risk map for humans and livestock. The aim of the present study was to develop a highly sensitive, culture-independent, DNA-based method that allows direct quantification of B. pseudomallei from soil. We established a protocol for B. pseudomallei soil DNA isolation, purification, and quantification by quantitative PCR (qPCR) targeting a type three secretion system 1 single-copy gene. This assay was validated using 40 soil samples from Northeast Thailand that underwent parallel bacteriological culture. All 26 samples that were B. pseudomallei positive by direct culture were B. pseudomallei qPCR positive, with a median of 1.84 × 104 genome equivalents (range, 3.65 × 102 to 7.85 × 105) per gram of soil, assuming complete recovery of DNA. This was 10.6-fold (geometric mean; range, 1.1- to 151.3-fold) higher than the bacterial count defined by direct culture. Moreover, the qPCR detected B. pseudomallei in seven samples (median, 36.9 genome equivalents per g of soil; range, 9.4 to 47.3) which were negative by direct culture. These seven positive results were reproduced using a nested PCR targeting a second, independent B. pseudomallei-specific sequence. Two samples were direct culture and qPCR negative but nested PCR positive. Five samples were negative by both PCR methods and culture. In conclusion, our PCR-based system provides a highly specific and sensitive tool for the quantitative environmental surveillance of B. pseudomallei. PMID:21803915

  20. Comparison of DNA Extraction Kits for Detection of Burkholderia pseudomallei in Spiked Human Whole Blood Using Real-Time PCR

    PubMed Central

    Podnecky, Nicole L.; Elrod, Mindy G.; Newton, Bruce R.; Dauphin, Leslie A.; Shi, Jianrong; Chawalchitiporn, Sutthinan; Baggett, Henry C.; Hoffmaster, Alex R.; Gee, Jay E.

    2013-01-01

    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 (CT) 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 5.5×104 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. PMID:23460920

  1. Evaluation of a biodegradable microparticulate polymer as a carrier for Burkholderia pseudomallei subunit vaccines in a mouse model of melioidosis.

    PubMed

    Schully, K L; Bell, M G; Prouty, A M; Gallovic, M D; Gautam, S; Peine, K J; Sharma, S; Bachelder, E M; Pesce, J T; Elberson, M A; Ainslie, K M; Keane-Myers, A

    2015-11-30

    Melioidosis, a potentially lethal disease of humans and animals, is caused by the soil-dwelling bacterium Burkholderia pseudomallei. Due to B. pseudomallei's classification as a Tier 1 Select Agent, there is substantial interest in the development of an effective vaccine. Yet, despite decades of research, no effective target, adjuvant or delivery vehicle capable of inducing protective immunity against B. pseudomallei infection has been identified. We propose a microparticulate delivery vehicle comprised of the novel polymer acetalated dextran (Ac-DEX). Ac-DEX is an acid-sensitive biodegradable carrier that can be fabricated into microparticles (MPs) that are relatively stable at pH 7.4, but rapidly degrade after phagocytosis by antigen presenting cells where the pH can drop to 5.0. As compared to other biomaterials, this acid sensitivity has been shown to enhance cross presentation of subunit antigens. To evaluate this platform as a delivery system for a melioidosis vaccine, BALB/c mice were vaccinated with Ac-DEX MPs separately encapsulating B. pseudomallei whole cell lysate and the toll-like receptor (TLR) 7/8 agonist resiquimod. This vaccine elicited a robust antibody response that included both Th1 and Th2 immunity. Following lethal intraperitoneal challenge with B. pseudomallei 1026b, vaccinated mice demonstrated a significant delay to time of death compared to untreated mice. The formulation, however, demonstrated incomplete protection indicating that lysate protein offers limited value as an antigen. Nevertheless, our Ac-DEX MPs may offer an effective delivery vehicle for a subunit B. psuedomallei vaccine. PMID:26428631

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

    PubMed

    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. PMID:26620184

  3. Discrimination of Burkholderia mallei/pseudomallei from Burkholderia thailandensis by sequence comparison of a fragment of the ribosomal protein S21 (rpsU) gene

    PubMed Central

    Frickmann, H.; Chantratita, N.; Gauthier, Y. P.; Neubauer, H.; Hagen, R. M.

    2012-01-01

    Discrimination of Burkholderia (B.) pseudomallei and B. mallei from environmental B. thailandensis is challenging. We describe a discrimination method based on sequence comparison of the ribosomal protein S21 (rpsU) gene. The rpsU gene was sequenced in ten B. pseudomallei, six B. mallei, one B. thailandensis reference strains, six isolates of B. pseudomallei, and 37 of B. thailandensis. Further rpsU sequences of six B. pseudomallei, three B. mallei, and one B. thailandensis were identified via NCBI GenBank. Three to four variable base-positions were identified within a 120-base-pair fragment, allowing discrimination of the B. pseudomallei/mallei-cluster from B. thailandensis, whose sequences clustered identically. All B. mallei and three B. pseudomallei sequences were identical, while 17/22 B. pseudomallei strains differed in one nucleotide (78A>C). Sequences of the rpsU fragment of ‘out-stander’ reference strains of B. cepacia, B. gladioli, B. plantarii, and B. vietnamensis clustered differently. Sequence comparison of the described rpsU gene fragment can be used as a supplementary diagnostic procedure for the discrimination of B. mallei/pseudomallei from B. thailandensis as well as from other species of the genus Burkholderia, keeping in mind that it does not allow for a differentiation between B. mallei and B. pseudomallei. PMID:23227305

  4. Interim report on updated microarray probes for the LLNL Burkholderia pseudomallei SNP array

    SciTech Connect

    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.

  5. Molecular Investigations of PenA-mediated β-lactam Resistance in Burkholderia pseudomallei

    PubMed Central

    Rholl, Drew A.; Papp-Wallace, Krisztina M.; Tomaras, Andrew P.; Vasil, Michael L.; Bonomo, Robert A.; Schweizer, Herbert P.

    2011-01-01

    Burkholderia pseudomallei is the etiological agent of melioidosis. Because of the bacterium’s intrinsic resistance and propensity to establish latent infections, melioidosis therapy is complicated and prolonged. Newer generation β-lactams, specifically ceftazidime, are used for acute phase therapy, but resistance to this cephalosporin has been observed. The chromosomally encoded penA gene encodes a putative twin arginine translocase (TAT)-secreted β-lactamase, and penA mutations have been implicated in ceftazidime resistance in clinical isolates. However, the role of PenA in resistance has not yet been systematically studied in isogenetic B. pseudomallei mutant backgrounds. We investigated the effects of penA deletion, point mutations, and up-regulation, as well as tat operon deletion and PenA TAT-signal sequence mutations. These experiments were made possible by employing a B. pseudomallei strain that is excluded from Select Agent regulations. Deletion of penA significantly (>4-fold) reduced the susceptibility to six of the nine β-lactams tested and ≥16-fold for ampicillin, amoxicillin, and carbenicillin. Overexpression of penA by single-copy, chromosomal expression of the gene under control of the inducible Ptac promoter, increased resistance levels for all β-lactams tested 2- to 10-fold. Recreation of the C69Y and P167S PenA amino acid substitutions previously observed in resistant clinical isolates increased resistance to ceftazidime by ≥85- and 5- to 8-fold, respectively. Similarly, a S72F substitution resulted in a 4-fold increase in resistance to amoxicillin and clavulanic acid. Susceptibility assays with PenA TAT-signal sequence and ΔtatABC mutants, as well as Western blot analysis, confirmed that PenA is a TAT secreted enzyme and not periplasmic but associated with the spheroplastic cell fraction. Lastly, we determined that two LysR-family regulators encoded by genes adjacent to penA do not play a role in transcriptional regulation of pen

  6. Development of Rapid Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Burkholderia pseudomallei

    PubMed Central

    Suttisunhakul, Vichaya; Wuthiekanun, Vanaporn; Brett, Paul J.; Khusmith, Srisin; Day, Nicholas P. J.; Burtnick, Mary N.; Limmathurotsakul, Direk

    2016-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is an environmental bacillus found in northeast Thailand. The mortality rate of melioidosis is ∼40%. An indirect hemagglutination assay (IHA) is used as a reference serodiagnostic test; however, it has low specificity in areas where the background seropositivity of healthy people is high. To improve assay specificity and reduce the time for diagnosis, four rapid enzyme-linked immunosorbent assays (ELISAs) were developed using two purified polysaccharide antigens (O-polysaccharide [OPS] and 6-deoxyheptan capsular polysaccharide [CPS]) and two crude antigens (whole-cell [WC] antigen and culture filtrate [CF] antigen) of B. pseudomallei. The ELISAs were evaluated using serum samples from 141 culture-confirmed melioidosis patients from Thailand along with 188 healthy donors from Thailand and 90 healthy donors from the United States as controls. The areas under receiver operator characteristic curves (AUROCC) using Thai controls were high for the OPS-ELISA (0.91), CF-ELISA (0.91), and WC-ELISA (0.90), while those of CPS-ELISA (0.84) and IHA (0.72) were lower. AUROCC values using U.S. controls were comparable to those of the Thai controls for all ELISAs except IHA (0.93). Using a cutoff optical density (OD) of 0.87, the OPS-ELISA had a sensitivity of 71.6% and a specificity of 95.7% for Thai controls; for U.S. controls, specificity was 96.7%. An additional 120 serum samples from tuberculosis, scrub typhus, or leptospirosis patients were evaluated in all ELISAs and resulted in comparable or higher specificities than using Thai healthy donors. Our findings suggest that antigen-specific ELISAs, particularly the OPS-ELISA, may be useful for serodiagnosis of melioidosis in areas where it is endemic and nonendemic. PMID:26912754

  7. Development of Rapid Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Burkholderia pseudomallei.

    PubMed

    Suttisunhakul, Vichaya; Wuthiekanun, Vanaporn; Brett, Paul J; Khusmith, Srisin; Day, Nicholas P J; Burtnick, Mary N; Limmathurotsakul, Direk; Chantratita, Narisara

    2016-05-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is an environmental bacillus found in northeast Thailand. The mortality rate of melioidosis is ∼40%. An indirect hemagglutination assay (IHA) is used as a reference serodiagnostic test; however, it has low specificity in areas where the background seropositivity of healthy people is high. To improve assay specificity and reduce the time for diagnosis, four rapid enzyme-linked immunosorbent assays (ELISAs) were developed using two purified polysaccharide antigens (O-polysaccharide [OPS] and 6-deoxyheptan capsular polysaccharide [CPS]) and two crude antigens (whole-cell [WC] antigen and culture filtrate [CF] antigen) of B. pseudomallei The ELISAs were evaluated using serum samples from 141 culture-confirmed melioidosis patients from Thailand along with 188 healthy donors from Thailand and 90 healthy donors from the United States as controls. The areas under receiver operator characteristic curves (AUROCC) using Thai controls were high for the OPS-ELISA (0.91), CF-ELISA (0.91), and WC-ELISA (0.90), while those of CPS-ELISA (0.84) and IHA (0.72) were lower. AUROCC values using U.S. controls were comparable to those of the Thai controls for all ELISAs except IHA (0.93). Using a cutoff optical density (OD) of 0.87, the OPS-ELISA had a sensitivity of 71.6% and a specificity of 95.7% for Thai controls; for U.S. controls, specificity was 96.7%. An additional 120 serum samples from tuberculosis, scrub typhus, or leptospirosis patients were evaluated in all ELISAs and resulted in comparable or higher specificities than using Thai healthy donors. Our findings suggest that antigen-specific ELISAs, particularly the OPS-ELISA, may be useful for serodiagnosis of melioidosis in areas where it is endemic and nonendemic. PMID:26912754

  8. Burkholderia pseudomallei identification: a comparison between the API 20NE and VITEK2GN systems.

    PubMed

    Deepak, Rama Narayana; Crawley, Brett; Phang, Elaine

    2008-12-01

    Sixty unique clinical isolates previously reported as Burkholderia pseudomallei were run in parallel through the API 20NE and VITEK2GN identification systems after 24h growth on Columbia agar with 5% sheep blood. The identifications and confidence levels determined by each test modality were compared. The VITEK2GN identified 47 (78.3%) and the API20NE identified 52 (86.7%) of the isolates as B. pseudomallei. The modal octal profile for the latter was 1156577. The alternative identification most commonly made by both systems was B. cepacia. Comparison of the two modalities gave a Cohen's kappa of 0.6008, suggesting good overall inter-test agreement. Both are good test modalities capable of identifying B. pseudomallei reliably, with the API20NE having the advantage of correctly identifying a slightly greater number of isolates, and the VITEK2 having the advantage of a shorter test turnaround time. PMID:19121685

  9. A PCR-BASED DETECTION OF BURKHOLDERIA PSEUDOMALLEI DIVERSITY USING MYOVIRIDAE PROPHAGE TYPING.

    PubMed

    Nakornpakdee, Yaowarin; Sermswan, Rasana W; Tattawasart, Unchalee; Yordpratum, Umaporn; Wongratanacheewin, Surasakdi

    2015-01-01

    PCR-based detection of Myoviridae lysogenic phages in Burkholderia pseudomallei was developed using primers targeting K96243 prophage GI2, phiE12-2 and phi52237/phiX216. Investigation of 50 clinical and 50 environmental (soil) isolates revealed that K96243 prophage GI2 was the most common (48%) among the isolates, followed by phiE12-2 (38%) and phi52237/phiX216 (35%), with K96243 prophage GI2 being significantly more frequent in soil (64%) than clinical (32%) samples. Twenty-four percent of soil isolates contained all three prophage types, while clinical isolates harbored no more than two types. Although B. pseudomallei isolates from soil were found to be more diverse based on prophage typing, all isolates were equally susceptible to a battery of lytic phages (although to different extents), suggesting the possibility of using lytic phages to control environmental B. pseudomallei. PMID:26513903

  10. Natural Infection of Burkholderia pseudomallei in an Imported Pigtail Macaque (Macaca nemestrina) and Management of the Exposed Colony

    PubMed Central

    Johnson, Crystal H; Skinner, Brianna L; Dietz, Sharon M; Blaney, David; Engel, Robyn M; Lathrop, George W; Hoffmaster, Alex R; Gee, Jay E; Elrod, Mindy G; Powell, Nathaniel; Walke, Henry

    2013-01-01

    Identification of the select agent Burkholderia pseudomallei in macaques imported into the United States is rare. A purpose-bred, 4.5-y-old pigtail macaque (Macaca nemestrina) imported from Southeast Asia was received from a commercial vendor at our facility in March 2012. After the initial acclimation period of 5 to 7 d, physical examination of the macaque revealed a subcutaneous abscess that surrounded the right stifle joint. The wound was treated and resolved over 3 mo. In August 2012, 2 mo after the stifle joint wound resolved, the macaque exhibited neurologic clinical signs. Postmortem microbiologic analysis revealed that the macaque was infected with B. pseudomallei. This case report describes the clinical evaluation of a B. pseudomallei-infected macaque, management and care of the potentially exposed colony of animals, and protocols established for the animal care staff that worked with the infected macaque and potentially exposed colony. This article also provides relevant information on addressing matters related to regulatory issues and risk management of potentially exposed animals and animal care staff. PMID:24326230

  11. T Cell Immunity to the Alkyl Hydroperoxide Reductase of Burkholderia pseudomallei: A Correlate of Disease Outcome in Acute Melioidosis

    PubMed Central

    Reynolds, Catherine; Goudet, Amélie; Jenjaroen, Kemajittra; Sumonwiriya, Manutsanun; Rinchai, Darawan; Musson, Julie; Overbeek, Saskia; Makinde, Julia; Quigley, Kathryn; Manji, Jiten; Spink, Natasha; Yos, Pagnarith; Wuthiekanun, Vanaporn; Bancroft, Gregory; Robinson, John; Lertmemongkolchai, Ganjana; Dunachie, Susanna; Maillere, Bernard; Holden, Matthew; Altmann, Daniel

    2015-01-01

    There is an urgent need for a better understanding of adaptive immunity to Burkholderia pseudomallei, the causative agent of melioidosis that is frequently associated with sepsis or death in patients in Southeast Asia and Northern Australia. The imperative to identify vaccine targets is driven both by the public health agenda in these regions and biological threat concerns. In several intracellular bacterial pathogens, alkyl hydroperoxidase reductases are upregulated as part of the response to host oxidative stress, and they can stimulate strong adaptive immunity. We show that alkyl hydroperoxidase reductase (AhpC) of B. pseudomallei is strongly immunogenic for T cells of ‘humanized’ HLA transgenic mice and seropositive human donors. Some T cell epitopes, such as p6, are able to bind diverse HLA class II heterodimers and stimulate strong T cell immunity in mice and humans. Importantly, patients with acute melioidosis who survive infection show stronger T cell responses to AhpC relative to those who do not. Although the sequence of AhpC is virtually invariant among global B. pseudomallei clinical isolates, a Cambodian isolate varies only in C-terminal truncation of the p6 T cell epitope, raising the possibility of selection by host immunity. This variant peptide is virtually unable to stimulate T cell immunity. For an infection in which there has been debate about centrality of T cell immunity in defense, these observations support a role for T cell immunity to AhpC in disease protection. PMID:25862821

  12. The Burkholderia pseudomallei Δasd Mutant Exhibits Attenuated Intracellular Infectivity and Imparts Protection against Acute Inhalation Melioidosis in Mice ▿

    PubMed Central

    Norris, Michael H.; Propst, Katie L.; Kang, Yun; Dow, Steven W.; Schweizer, Herbert P.; Hoang, Tung T.

    2011-01-01

    Burkholderia pseudomallei, the cause of serious and life-threatening diseases in humans, is of national biodefense concern because of its potential use as a bioterrorism agent. This microbe is listed as a select agent by the CDC; therefore, development of vaccines is of significant importance. Here, we further investigated the growth characteristics of a recently created B. pseudomallei 1026b Δasd mutant in vitro, in a cell model, and in an animal model of infection. The mutant was typified by an inability to grow in the absence of exogenous diaminopimelate (DAP); upon single-copy complementation with a wild-type copy of the asd gene, growth was restored to wild-type levels. Further characterization of the B. pseudomallei Δasd mutant revealed a marked decrease in RAW264.7 murine macrophage cytotoxicity compared to the wild type and the complemented Δasd mutant. RAW264.7 cells infected by the Δasd mutant did not exhibit signs of cytopathology or multinucleated giant cell (MNGC) formation, which were observed in wild-type B. pseudomallei cell infections. The Δasd mutant was found to be avirulent in BALB/c mice, and mice vaccinated with the mutant were protected against acute inhalation melioidosis. Thus, the B. pseudomallei Δasd mutant may be a promising live attenuated vaccine strain and a biosafe strain for consideration of exclusion from the select agent list. PMID:21807903

  13. Interrogation of the Burkholderia pseudomallei genome to address differential virulence among isolates

    SciTech Connect

    Challacombe, Jean F.; Stubben, Chris J.; Klimko, Christopher P.; Welkos, Susan L.; Kern, Steven J.; Bozue, Joel A.; Worsham, Patricia L.; Cote, Christopher K.; Wolfe, Daniel N.; Badger, Jonathan H.

    2014-12-23

    Infection by the Gram-negative pathogen Burkholderia pseudomallei results in the disease melioidosis, acquired from the environment in parts of southeast Asia and northern Australia. Clinical symptoms of melioidosis range from acute (fever, pneumonia, septicemia, and localized infection) to chronic (abscesses in various organs and tissues, most commonly occurring in the lungs, liver, spleen, kidney, prostate and skeletal muscle), and persistent infections in humans are difficult to cure. Understanding the basic biology and genomics of B. pseudomallei is imperative for the development of new vaccines and therapeutic interventions. This formidable task is becoming more tractable due to the increasing number of B. pseudomallei genomes that are being sequenced and compared. Here, we compared three B. pseudomallei genomes, from strains MSHR668, K96243 and 1106a, to identify features that might explain why MSHR668 is more virulent than K96243 and 1106a in a mouse model of B. pseudomallei infection. Our analyses focused on metabolic, virulence and regulatory genes that were present in MSHR668 but absent from both K96243 and 1106a. We also noted features present in K96243 and 1106a but absent from MSHR668, and identified genomic differences that may contribute to variations in virulence noted among the three B. pseudomallei isolates. While this work contributes to our understanding of B. pseudomallei genomics, more detailed experiments are necessary to characterize the relevance of specific genomic features to B. pseudomallei metabolism and virulence. Functional analyses of metabolic networks, virulence and regulation shows promise for examining the effects of B. pseudomallei on host cell metabolism and will lay a foundation for future prediction of the virulence of emerging strains. Continued emphasis in this area will be critical for protection against melioidosis, as a better understanding of what

  14. Innate Immune Responses of Pulmonary Epithelial Cells to Burkholderia pseudomallei Infection

    PubMed Central

    Sim, Siew Hoon; Liu, Yichun; Wang, Dongling; Novem, Vidhya; Sivalingam, Suppiah Paramalingam; Thong, Tuck Weng; Ooi, Eng Eong; Tan, Gladys

    2009-01-01

    Background Burkholderia pseudomallei, a facultative intracellular pathogen, causes systemic infection in humans with high mortality especially when infection occurs through an infectious aerosol. Previous studies indicated that the epithelial cells in the lung are an active participant in host immunity. In this study, we aimed to investigate the innate immune responses of lung epithelial cells against B. pseudomallei. Methodology and Principal Findings Using a murine lung epithelial cell line, primary lung epithelial cells and an inhalational murine infection model, we characterized the types of innate immunity proteins and peptides produced upon B. pseudomallei infection. Among a wide panel of immune components studied, increased levels of major pro-inflammatory cytokines IL-6 and TNFα, chemokine MCP-1, and up-regulation of secretory leukocyte protease inhibitor (SLPI) and chemokine (C-C motif) ligand 20 (CCL20) were observed. Inhibition assays using specific inhibitors suggested that NF-κB and p38 MAPK pathways were responsible for these B. pseudomallei-induced antimicrobial peptides. Conclusions Our findings indicate that the respiratory epithelial cells, which form the majority of the cells lining the epithelial tract and the lung, have important roles in the innate immune response against B. pseudomallei infection. PMID:19806192

  15. Burkholderia pseudomallei Biofilm Promotes Adhesion, Internalization and Stimulates Proinflammatory Cytokines in Human Epithelial A549 Cells

    PubMed Central

    Kunyanee, Chanikarn; Kamjumphol, Watcharaporn; Taweechaisupapong, Suwimol; Kanthawong, Sakawrat; Wongwajana, Suwin; Wongratanacheewin, Surasak; Hahnvajanawong, Chariya

    2016-01-01

    Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis. Inhalational exposure leading to pulmonary melioidosis is the most common clinical manifestation with significant mortality. However, the role of B. pseudomallei biofilm phenotype during bacterial-host interaction remains unclear. We hypothesize that biofilm phenotype may play a role in such interactions. In this study, B. pseudomallei H777 (biofilm wild type), B. pseudomallei M10 (biofilm mutant) and B. pseudomallei C17 (biofilm-complemented) strains were used to assess the contribution of biofilm to adhesion to human lung epithelial cells (A549), intracellular interactions, apoptosis/necrosis and impact on proinflammatory responses. Confocal laser scanning microscopy demonstrated that B. pseudomallei H777 and C17 produced biofilm, whereas M10 did not. To determine the role of biofilm in host interaction, we assessed the ability of each of the three strains to interact with the A549 cells at MOI 10. Strain H777 exhibited higher levels of attachment and invasion compared to strain M10 (p < 0.05). In addition, the biofilm-complemented strain, C17 exhibited restored bacterial invasion ability. Flow cytometry combined with a double-staining assay using annexin V and propidium iodide revealed significantly higher numbers of early apoptotic and late apoptotic A549 cells when these were infected with strain H777 (1.52%) and C17 (1.43%) compared to strain M10 (0.85%) (p < 0.05). Strains H777 and C17 were able to stimulate significant secretion of IL-6 and IL-8 compared with the biofilm mutant (p < 0.05). Together, these findings demonstrated the role of biofilm-associated phenotypes of B. pseudomallei in cellular pathogenesis of human lung epithelial cells with respect to initial attachment and invasion, apoptosis and proinflammatory responses. PMID:27529172

  16. Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles.

    PubMed

    Nandi, Tannistha; Holden, Matthew T G; Holden, Mathew T G; Didelot, Xavier; Mehershahi, Kurosh; Boddey, Justin A; Beacham, Ifor; Peak, Ian; Harting, John; Baybayan, Primo; Guo, Yan; Wang, Susana; How, Lee Chee; Sim, Bernice; Essex-Lopresti, Angela; Sarkar-Tyson, Mitali; Nelson, Michelle; Smither, Sophie; Ong, Catherine; Aw, Lay Tin; Hoon, Chua Hui; Michell, Stephen; Studholme, David J; Titball, Richard; Chen, Swaine L; Parkhill, Julian; Tan, Patrick

    2015-01-01

    Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity. PMID:25236617

  17. Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles

    PubMed Central

    Nandi, Tannistha; Holden, Matthew T.G.; Didelot, Xavier; Mehershahi, Kurosh; Boddey, Justin A.; Beacham, Ifor; Peak, Ian; Harting, John; Baybayan, Primo; Guo, Yan; Wang, Susana; How, Lee Chee; Sim, Bernice; Essex-Lopresti, Angela; Sarkar-Tyson, Mitali; Nelson, Michelle; Smither, Sophie; Ong, Catherine; Aw, Lay Tin; Hoon, Chua Hui; Michell, Stephen; Studholme, David J.; Titball, Richard; Chen, Swaine L.; Parkhill, Julian

    2015-01-01

    Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity. PMID:25236617

  18. The Core and Accessory Genomes of Burkholderia pseudomallei: Implications for Human Melioidosis

    PubMed Central

    Lin, Chi Ho; Karuturi, R. Krishna M.; Wuthiekanun, Vanaporn; Tuanyok, Apichai; Chua, Hui Hoon; Ong, Catherine; Paramalingam, Sivalingam Suppiah; Tan, Gladys; Tang, Lynn; Lau, Gary; Ooi, Eng Eong; Woods, Donald; Feil, Edward; Peacock, Sharon J.; Tan, Patrick

    2008-01-01

    Natural isolates of Burkholderia pseudomallei (Bp), the causative agent of melioidosis, can exhibit significant ecological flexibility that is likely reflective of a dynamic genome. Using whole-genome Bp microarrays, we examined patterns of gene presence and absence across 94 South East Asian strains isolated from a variety of clinical, environmental, or animal sources. 86% of the Bp K96243 reference genome was common to all the strains representing the Bp “core genome”, comprising genes largely involved in essential functions (eg amino acid metabolism, protein translation). In contrast, 14% of the K96243 genome was variably present across the isolates. This Bp accessory genome encompassed multiple genomic islands (GIs), paralogous genes, and insertions/deletions, including three distinct lipopolysaccharide (LPS)-related gene clusters. Strikingly, strains recovered from cases of human melioidosis clustered on a tree based on accessory gene content, and were significantly more likely to harbor certain GIs compared to animal and environmental isolates. Consistent with the inference that the GIs may contribute to pathogenesis, experimental mutation of BPSS2053, a GI gene, reduced microbial adherence to human epithelial cells. Our results suggest that the Bp accessory genome is likely to play an important role in microbial adaptation and virulence. PMID:18927621

  19. An ensemble of structures of Burkholderia pseudomallei 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase

    SciTech Connect

    Davies, Douglas R.; Staker, Bart L.; Abendroth, Jan A.; Edwards, Thomas E.; Hartley, Robert; Leonard, Jess; Kim, Hidong; Rychel, Amanda L.; Hewitt, Stephen N.; Myler, Peter J.; Stewart, Lance J.

    2011-12-07

    Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and Northern Australia. Burkholderia is responsible for melioidosis, a serious infection of the skin. The enzyme 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (PGAM) catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate, a key step in the glycolytic pathway. As such it is an extensively studied enzyme and X-ray crystal structures of PGAM enzymes from multiple species have been elucidated. Vanadate is a phosphate mimic that is a powerful tool for studying enzymatic mechanisms in phosphoryl-transfer enzymes such as phosphoglycerate mutase. However, to date no X-ray crystal structures of phosphoglycerate mutase have been solved with vanadate acting as a substrate mimic. Here, two vanadate complexes together with an ensemble of substrate and fragment-bound structures that provide a comprehensive picture of the function of the Burkholderia enzyme are reported.

  20. Gene Microarray Analyses of Daboia russelli russelli Daboiatoxin Treatment of THP-1 Human Macrophages Infected with Burkholderia pseudomallei.

    PubMed

    Perumal Samy, R; Manikandan, J; Pachiappan, A; Ooi, E E; Aw, L T; Stiles, B G; Franco, O L; Kandasamy, M; Mathi, K M; Rane, G; Siveen, K S; Arunachalam, C; Zayed, M E; Alharbi, S A; Kumar, A P; Sethi, G; Lim, L H K; Chow, V T

    2015-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In this study, the transcriptomic responses of B. pseudomallei infection of a human macrophage cell model were investigated using whole-genome microarrays. Gene expression profiles were compared between infected THP-1 human monocytic leukemia cells with or without treatment with Daboia russelli russelli daboiatoxin (DRRDbTx) or ceftazidime (antibiotic control). Microarray analyses of infected and treated cells revealed differential upregulation of various inflammatory genes such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α), cyclooxygenase (COX-2), vascular endothelial growth factor (VEGF), chemokine C-X-C motif ligand 4 (CXCL4), transcription factor p65 (NF-kB); and several genes involved in immune and stress responses, cell cycle, and lipid metabolism. Moreover, following DRR-DbTx treatment of infected cells, there was enhanced expression of the tolllike receptor 2 (TLR-2) mediated signaling pathway involved in recognition and initiation of acute inflammatory responses. Importantly, we observed that highly inflammatory cytokine gene responses were similar in infected cells exposed to DRR-DbTx or ceftazidime after 24 h. Additionally, there were increased transcripts associated with cell death by caspase activation that can promote host tissue injury. In summary, the transcriptional responses during B. pseudomallei infection of macrophages highlight a broad range of innate immune mechanisms that are activated within 24 h post-infection. These data provide insights into the transcriptomic kinetics following DRR-DbTx treatment of human macrophages infected with B. pseudomallei. PMID:26592245

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

    SciTech Connect

    Knight, M. J.; Ruaux, A.; Mikolajek, H.; Erskine, P. T.; Gill, R.; Wood, S. P.; Wood, M.; Cooper, J. B.

    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.

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

    SciTech Connect

    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.

  3. Role of Burkholderia pseudomallei Sigma N2 in Amino Acids Utilization and in Regulation of Catalase E Expression at the Transcriptional Level

    PubMed Central

    Diep, Duong Thi Hong; Phuong, Nguyen Thi Thanh; Hlaing, Mya Myintzu; Srimanote, Potjanee; Tungpradabkul, Sumalee

    2015-01-01

    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. PMID:26904748

  4. Incidental Splenic Granuloma Due to Burkholderia pseudomallei: A Case of Asymptomatic Latent Melioidosis?

    PubMed

    Chow, Tak Kuan; Eu, Lin Chuan; Chin, Kin Fah; Ong, Kien Chai; Pailoor, Jayalakshmi; Vadivelu, Jamunarani; Wong, Kum Thong

    2016-03-01

    We report a rare case of an asymptomatic latent melioidosis lesion in a posttraumatic splenectomy specimen from a diabetic patient. The 2-cm yellowish, lobulated lesion was found in the splenic parenchyma well away from the traumatized areas. Microscopically, it consisted of a central area of necrosis and exudate surrounded by macrophages, epithelioid cells, lymphocytes, and occasional multinucleated giant cells. Burkholderia bacilli were detected by a novel in situ hybridization (ISH) assay, and confirmed by polymerase chain reaction and sequencing to be Burkholderia pseudomallei. As melioidosis was not suspected initially, bacterial culture was not done but electron microscopy showed morphologically viable and dividing bacilli in the lesion. Moreover, the surgical wound became infected with B. pseudomallei several days post-surgery. After treatment with ceftazidime and trimethoprim/sulfamethoxazole, the wound infection cleared. We believe this could be a unique case of asymptomatic latent melioidosis in the spleen. In endemic countries, chronic granulomas should be investigated for B. pseudomallei infection, and if available, ISH may be helpful for diagnosis. PMID:26787155

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

    PubMed

    Baker, Anthony L; Ezzahir, Jessica; Gardiner, Christopher; Shipton, Warren; Warner, Jeffrey M

    2015-01-01

    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. PMID:26398904

  6. Characterization of lesion formation in marmosets following inhalational challenge with different strains of Burkholderia pseudomallei.

    PubMed

    Nelson, Michelle; Nunez, Alejandro; Ngugi, Sarah A; Sinclair, Adam; Atkins, Timothy P

    2015-12-01

    The marmoset model of melioidosis was used to explore whether there was any difference in the disease presentation and/or the lesion formation following inhalational challenge with one of four strains of Burkholderia pseudomallei (K96243, 1026b, HBPUB10303a and HBPUB10134a). Marmosets were challenged with a range of bacterial doses and bacterial load, histological and physiological features were determined temporally following lethal disease. Melioidosis presented as an acute, febrile disease with bacteraemia, bacterial dissemination, necrotizing hepatitis, splenitis and pneumonia which was independent of the challenge strain. Generally, there were no major differences in the manifestation of melioidosis following challenge by the different strains of B. pseudomallei; however, there were some differences in the time to death and the severity of the pathological features. The pathological features observed in the liver and spleen of animals challenged with B. pseudomallei strain 1026b were statistically less severe (P < 0.05) and less frequent. However, more severe foci of disease were evident in the lungs of animals challenged with strain 1026b. In all cases, the lesions developed from small areas of bacteria-infected macrophages surrounded by non-infected neutrophils into large lesions with both immune cell types infected. The marmoset model was a useful tool enabling the distinction of subtle difference in the pathological response to B. pseudomallei. PMID:26852689

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

    PubMed Central

    Baker, Anthony L.; Ezzahir, Jessica; Gardiner, Christopher; Shipton, Warren; Warner, Jeffrey M.

    2015-01-01

    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. PMID:26398904

  8. Out of the Ground: Aerial and Exotic Habitats of the Melioidosis Bacterium Burkholderia pseudomallei in Grasses in Australia

    PubMed Central

    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.

    2011-01-01

    Summary 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. B. 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. PMID:22176696

  9. Fatal Burkholderia pseudomallei Infection Initially Reported as a Bacillus Species, Ohio, 2013

    PubMed Central

    Doker, Thomas J.; Quinn, Celia L.; Salehi, Ellen D.; Sherwood, Joshua J.; Benoit, Tina J.; Elrod, Mindy Glass; Gee, Jay E.; Shadomy, Sean V.; Bower, William A.; Hoffmaster, Alex R.; Walke, Henry T.; Blaney, David D.; DiOrio, Mary S.

    2014-01-01

    A fatal case of melioidosis was diagnosed in Ohio one month after culture results were initially reported as a Bacillus species. To identify a source of infection and assess risk in patient contacts, we abstracted patient charts; interviewed physicians and contacts; genetically characterized the isolate; performed a Burkholderia pseudomallei antibody indirect hemagglutination assay on household contacts and pets to assess seropositivity; and collected household plant, soil, liquid, and insect samples for culturing and real-time polymerase chain reaction testing. Family members and pets tested were seronegative for B. pseudomallei. Environmental samples were negative by real-time polymerase chain reaction and culture. Although the patient never traveled internationally, the isolate genotype was consistent with an isolate that originated in Southeast Asia. This investigation identified the fifth reported locally acquired non-laboratory melioidosis case in the contiguous United States. Physicians and laboratories should be aware of this potentially emerging disease and refer positive cultures to a Laboratory Response Network laboratory. PMID:25092821

  10. Alteration of the Phenotypic and Pathogenic Patterns of Burkholderia pseudomallei that Persist in a Soil Environment

    PubMed Central

    Chen, Yao-Shen; Shieh, Wun-Ju; Goldsmith, Cynthia S.; Metcalfe, Maureen G.; Greer, Patricia W.; Zaki, Sherif R.; Chang, Hsin-Hou; Chan, Hao; Chen, Ya-Lei

    2014-01-01

    Melioidosis is caused by the soil-borne pathogen Burkholderia pseudomallei. To investigate whether the distinct phenotypic and virulent characteristics result from environmental adaptations in the soil or from the host body, two pairs of isogenic strains were generated by passages in soil or mice. After cultivation in soil, the levels of 3-hydroxytetradecanoic acid, biofilm formation, flagellar expression, and ultrastructure were altered in the bacteria. Uniformly fatal melioidosis developed as a result of infection with mouse-derived strains; however, the survival rates of mice infected with soil-derived strains prolonged. After primary infection or reinfection with soil-derived strains, the mice developed a low degree of bacterial hepatitis and bacterial colonization in the liver and bone marrow compared with mice that were infected with isogenic or heterogenic mouse-derived strains. We suggest that specific phenotypic and pathogenic patterns can be induced through infection with B. pseudomallei that has been cultured in different (soil versus mouse) environments. PMID:24445207

  11. Fatal Burkholderia pseudomallei infection initially reported as a Bacillus species, Ohio, 2013.

    PubMed

    Doker, Thomas J; Quinn, Celia L; Salehi, Ellen D; Sherwood, Joshua J; Benoit, Tina J; Glass Elrod, Mindy; Gee, Jay E; Shadomy, Sean V; Bower, William A; Hoffmaster, Alex R; Walke, Henry T; Blaney, David D; DiOrio, Mary S

    2014-10-01

    A fatal case of melioidosis was diagnosed in Ohio one month after culture results were initially reported as a Bacillus species. To identify a source of infection and assess risk in patient contacts, we abstracted patient charts; interviewed physicians and contacts; genetically characterized the isolate; performed a Burkholderia pseudomallei antibody indirect hemagglutination assay on household contacts and pets to assess seropositivity; and collected household plant, soil, liquid, and insect samples for culturing and real-time polymerase chain reaction testing. Family members and pets tested were seronegative for B. pseudomallei. Environmental samples were negative by real-time polymerase chain reaction and culture. Although the patient never traveled internationally, the isolate genotype was consistent with an isolate that originated in Southeast Asia. This investigation identified the fifth reported locally acquired non-laboratory melioidosis case in the contiguous United States. Physicians and laboratories should be aware of this potentially emerging disease and refer positive cultures to a Laboratory Response Network laboratory. PMID:25092821

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

    PubMed

    Kvitko, Brian H; Goodyear, Andrew; Propst, Katie L; Dow, Steven W; Schweizer, Herbert P

    2012-01-01

    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. pseudomallei may employ

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

    NASA Astrophysics Data System (ADS)

    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.

  14. AN IMPORTED CASE OF ACUTE MELIOIDOSIS CAUSED BY ST881 BURKHOLDERIA PSEUDOMALLEI.

    PubMed

    Zong, Zhiyong; Wang, Xiaohui; Deng, Yiyun

    2016-03-01

    A previously healthy Chinese male working in Malaysia returned to China with high fever. A blood culture showed Burkholderia pseudomallei strain WCBP1. This isolate was sequenced, showing type, ST881, which appears to be present in Malaysia. WCP1 had unusual susceptibility to aminoglycosides and habored the Yersinia-like fimbrial gene cluster for virulence. The patient's condition deteriorated rapidly but he recovered after receiving meropenem and intensive care support. Melioidosis is a potential problem among Chinese imigrant workers with strains new to China being identified. PMID:27244959

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

    PubMed

    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. PMID:16518004

  16. Effect of gamma irradiation on Burkholderia thailandensis ( Burkholderia pseudomallei surrogate) survival under combinations of pH and NaCl

    NASA Astrophysics Data System (ADS)

    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 ( 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. 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.

  17. Sequence- and Structure-Based Immunoreactive Epitope Discovery for Burkholderia pseudomallei Flagellin

    PubMed Central

    Nithichanon, Arnone; Rinchai, Darawan; Gori, Alessandro; Lassaux, Patricia; Peri, Claudio; Conchillio-Solé, Oscar; Ferrer-Navarro, Mario; Gourlay, Louise J.; Nardini, Marco; Vila, Jordi; Daura, Xavier; Colombo, Giorgio; Bolognesi, Martino; Lertmemonkolchai, Ganjana

    2015-01-01

    Burkholderia pseudomallei is a Gram-negative bacterium responsible for melioidosis, a serious and often fatal infectious disease that is poorly controlled by existing treatments. Due to its inherent resistance to the major antibiotic classes and its facultative intracellular pathogenicity, an effective vaccine would be extremely desirable, along with appropriate prevention and therapeutic management. One of the main subunit vaccine candidates is flagellin of Burkholderia pseudomallei (FliCBp). Here, we present the high resolution crystal structure of FliCBp and report the synthesis and characterization of three peptides predicted to be both B and T cell FliCBp epitopes, by both structure-based in silico methods, and sequence-based epitope prediction tools. All three epitopes were shown to be immunoreactive against human IgG antibodies and to elicit cytokine production from human peripheral blood mononuclear cells. Furthermore, two of the peptides (F51-69 and F270-288) were found to be dominant immunoreactive epitopes, and their antibodies enhanced the bactericidal activities of purified human neutrophils. The epitopes derived from this study may represent potential melioidosis vaccine components. PMID:26222657

  18. Variable Virulence Factors in Burkholderia pseudomallei (Melioidosis) Associated with Human Disease

    PubMed Central

    Webb, Jessica R.; Ward, Linda M.; Voutsinos, Marcos Y.; Tuanyok, Apichai; Mayo, Mark; Kaestli, Mirjam; Currie, Bart J.

    2014-01-01

    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 bimABm 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. PMID:24618705

  19. Production of a recombinant vaccine candidate against Burkholderia pseudomallei exploiting the bacterial N-glycosylation machinery.

    PubMed

    Garcia-Quintanilla, Fatima; Iwashkiw, Jeremy A; Price, Nancy L; Stratilo, Chad; Feldman, Mario F

    2014-01-01

    Vaccines developing immune responses toward 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 Burkholderia pseudomallei O polysaccharide (OPS II), the Campylobacter 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. PMID:25120536

  20. Production of a recombinant vaccine candidate against Burkholderia pseudomallei exploiting the bacterial N-glycosylation machinery

    PubMed Central

    Garcia-Quintanilla, Fatima; Iwashkiw, Jeremy A.; Price, Nancy L.; Stratilo, Chad; Feldman, Mario F.

    2014-01-01

    Vaccines developing immune responses toward 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 Burkholderia pseudomallei O polysaccharide (OPS II), the Campylobacter 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. PMID:25120536

  1. Physicochemical Factors Affecting the Growth of Burkholderia pseudomallei in Soil Microcosm

    PubMed Central

    Wang-ngarm, Supunnipa; Chareonsudjai, Sorujsiri; Chareonsudjai, Pisit

    2014-01-01

    Burkholderia pseudomallei causes melioidosis, the third most common cause of death from infectious diseases in northeast Thailand. Four physicochemical factors were set so that their values covered the range of the northeast, which is an endemic area. The soil pH was set at pH 4–10, soil salinity was 0.0–5.0% NaCl, total iron was 50–150 mg/kg soil, and carbon to nitrogen ratio (C/N) was 10:1 to 40:1. The experiments were carried out at 37°C, and soil moisture was maintained for 7 days. The number of viable bacterial cells was counted daily. Soil pH, salinity, Fe, and C/N ratio affected the bacterial growth. The bacterial colony was significantly (P < 0.05) reduced at soil pH > 8, soil salinity > 1% NaCl, and C/N ratio > 40:1. However, the growth of B. pseudomallei was enhanced by increasing the concentrations of iron significantly (P < 0.05). We propose using these findings to control B. pseudomallei in situ. PMID:24445210

  2. Multilocus sequence typing of 102 Burkholderia pseudomallei strains isolated from China.

    PubMed

    Fang, Y; Zhu, P; Li, Q; Chen, H; Li, Y; Ren, C; Hu, Y; Tan, Z; Gu, J; Mao, X

    2016-07-01

    The phylogenetic and epidemiological relationships of 102 Burkholderia pseudomallei clinical isolates from different geographical and population sources in China were investigated by multilocus sequence typing (MLST). The MLST data were analysed using the e-BURST algorithm, and an unweighted pair-group method with arithmetic mean dendrogram was constructed based on the pair-wise differences in the allelic profiles of the strains. Forty-one sequence types (STs) were identified, of which eight were novel (ST1341, ST1345, ST1346, ST1347, ST1348, ST1349, ST1350, ST1351). No geographical-specific or host population-specific phylogenetic lineages were identified. ST46, ST50, ST55, ST58, ST70 and ST1095 predominated, but ~44% of isolates were assigned to 45 STs illustrating high genetic diversity in the strain collection. Additionally, the phylogenetic relationships of the dominant STs in China showed significant linkeage with B. pseudomallei isolates from Thailand. Analysis of the gmhD allele suggests high genetic variation in B. pseudomallei in China. PMID:26744829

  3. [Immunotropic and immunogenic properties of Burkholderia pseudomallei surface and membrane antigens].

    PubMed

    Piven', N N; Rybkin, V S; Plekhanova, N G; Zhukova, S I; Viktorov, D V; Avrorova, I V; Demediuk, E A; Drefs, N M; Popov, S F

    2001-01-01

    The immunotropic and immunogenic properties of some chromatographic fractions of B. pseudomallei surface antigenic complex, as well as the preparations of B. pseudomallei outer and cytoplasmic membranes, were studied. The difference between the biopolymers under study in cytotoxicity, humoral and cell-mediated immunity characteristics, phagocytic activity were established. Some antigenic fractions (B, C, C1, H) showed perceptible protective activity (25-60%) in experiments on mice infected with B. pseudomallei virulent strain. One of the preparations of cytoplasmic membrane (CM-1) was also found to have protective properties (30%). Complex immunization with the antigenic complexes under study, introduced in combination with the immunomodulating agent Bromantan, was shown to enhance the protective effect. PMID:11236497

  4. The cluster 1 type VI secretion system is a major virulence determinant in Burkholderia pseudomallei.

    PubMed

    Burtnick, Mary N; Brett, Paul J; Harding, Sarah V; Ngugi, Sarah A; Ribot, Wilson J; Chantratita, Narisara; Scorpio, Angelo; Milne, Timothy S; Dean, Rachel E; Fritz, David L; Peacock, Sharon J; Prior, Joanne L; Atkins, Timothy P; Deshazer, David

    2011-04-01

    The Burkholderia pseudomallei K96243 genome encodes six type VI secretion systems (T6SSs), but little is known about the role of these systems in the biology of B. pseudomallei. In this study, we purified recombinant Hcp proteins from each T6SS and tested them as vaccine candidates in the BALB/c mouse model of melioidosis. Recombinant Hcp2 protected 80% of mice against a lethal challenge with K96243, while recombinant Hcp1, Hcp3, and Hcp6 protected 50% of mice against challenge. Hcp6 was the only Hcp constitutively produced by B. pseudomallei in vitro; however, it was not exported to the extracellular milieu. Hcp1, on the other hand, was produced and exported in vitro when the VirAG two-component regulatory system was overexpressed in trans. We also constructed six hcp deletion mutants (Δhcp1 through Δhcp6) and tested them for virulence in the Syrian hamster model of infection. The 50% lethal doses (LD(50)s) for the Δhcp2 through Δhcp6 mutants were indistinguishable from K96243 (<10 bacteria), but the LD(50) for the Δhcp1 mutant was >10(3) bacteria. The hcp1 deletion mutant also exhibited a growth defect in RAW 264.7 macrophages and was unable to form multinucleated giant cells in this cell line. Unlike K96243, the Δhcp1 mutant was only weakly cytotoxic to RAW 264.7 macrophages 18 h after infection. The results suggest that the cluster 1 T6SS is essential for virulence and plays an important role in the intracellular lifestyle of B. pseudomallei. PMID:21300775

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

    PubMed Central

    Al-Maleki, Anis Rageh; Mariappan, Vanitha; Vellasamy, Kumutha Malar; Tay, Sun Tee; Vadivelu, Jamuna

    2015-01-01

    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. PMID:25996927

  6. Whole-Genome Assemblies of 56 Burkholderia Species

    PubMed Central

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

    2014-01-01

    Burkholderia is a genus of betaproteobacteria that includes three notable human pathogens: B. cepacia, B. pseudomallei, and B. mallei. While B. pseudomallei and B. mallei are considered potential biowarfare agents, B. cepacia infections are largely limited to cystic fibrosis patients. Here, we present 56 Burkholderia genomes from 8 distinct species. PMID:25414490

  7. Whole-genome assemblies of 56 burkholderia species.

    PubMed

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

    2014-01-01

    Burkholderia is a genus of betaproteobacteria that includes three notable human pathogens: B. cepacia, B. pseudomallei, and B. mallei. While B. pseudomallei and B. mallei are considered potential biowarfare agents, B. cepacia infections are largely limited to cystic fibrosis patients. Here, we present 56 Burkholderia genomes from 8 distinct species. PMID:25414490

  8. Phylogenomic Analysis Reveals an Asian Origin for African Burkholderia pseudomallei and Further Supports Melioidosis Endemicity in Africa

    PubMed Central

    Garin, Benoit; De Smet, Birgit; Kaestli, Mirjam; Mayo, Mark; Vandamme, Peter; Jacobs, Jan; Lompo, Palpouguini; Tahita, Marc C.; Tinto, Halidou; Djaomalaza, Innocente; Currie, Bart J.

    2016-01-01

    ABSTRACT Burkholderia pseudomallei, an environmental bacterium that causes the deadly disease melioidosis, is endemic in northern Australia and Southeast Asia. An increasing number of melioidosis cases are being reported in other tropical regions, including Africa and the Indian Ocean islands. B. pseudomallei first emerged in Australia, with subsequent rare dissemination event(s) to Southeast Asia; however, its dispersal to other regions is not yet well understood. We used large-scale comparative genomics to investigate the origins of three B. pseudomallei isolates from Madagascar and two from Burkina Faso. Phylogenomic reconstruction demonstrates that these African B. pseudomallei isolates group into a single novel clade that resides within the more ancestral Asian clade. Intriguingly, South American strains reside within the African clade, suggesting more recent dissemination from West Africa to the Americas. Anthropogenic factors likely assisted in B. pseudomallei dissemination to Africa, possibly during migration of the Austronesian peoples from Indonesian Borneo to Madagascar ~2,000 years ago, with subsequent genetic diversity driven by mutation and recombination. Our study provides new insights into global patterns of B. pseudomallei dissemination and adds to the growing body of evidence of melioidosis endemicity in Africa. Our findings have important implications for melioidosis diagnosis and management in Africa. IMPORTANCE Sporadic melioidosis cases have been reported in the African mainland and Indian Ocean islands, but until recently, these regions were not considered areas where B. pseudomallei is endemic. Given the high mortality rate of melioidosis, it is crucial that this disease be recognized and suspected in all regions of endemicity. Previous work has shown that B. pseudomallei originated in Australia, with subsequent introduction into Asia; however, the precise origin of B. pseudomallei in other tropical regions remains poorly understood

  9. Phylogenomic Analysis Reveals an Asian Origin for African Burkholderia pseudomallei and Further Supports Melioidosis Endemicity in Africa.

    PubMed

    Sarovich, Derek S; Garin, Benoit; De Smet, Birgit; Kaestli, Mirjam; Mayo, Mark; Vandamme, Peter; Jacobs, Jan; Lompo, Palpouguini; Tahita, Marc C; Tinto, Halidou; Djaomalaza, Innocente; Currie, Bart J; Price, Erin P

    2016-01-01

    Burkholderia pseudomallei, an environmental bacterium that causes the deadly disease melioidosis, is endemic in northern Australia and Southeast Asia. An increasing number of melioidosis cases are being reported in other tropical regions, including Africa and the Indian Ocean islands. B. pseudomallei first emerged in Australia, with subsequent rare dissemination event(s) to Southeast Asia; however, its dispersal to other regions is not yet well understood. We used large-scale comparative genomics to investigate the origins of three B. pseudomallei isolates from Madagascar and two from Burkina Faso. Phylogenomic reconstruction demonstrates that these African B. pseudomallei isolates group into a single novel clade that resides within the more ancestral Asian clade. Intriguingly, South American strains reside within the African clade, suggesting more recent dissemination from West Africa to the Americas. Anthropogenic factors likely assisted in B. pseudomallei dissemination to Africa, possibly during migration of the Austronesian peoples from Indonesian Borneo to Madagascar ~2,000 years ago, with subsequent genetic diversity driven by mutation and recombination. Our study provides new insights into global patterns of B. pseudomallei dissemination and adds to the growing body of evidence of melioidosis endemicity in Africa. Our findings have important implications for melioidosis diagnosis and management in Africa. IMPORTANCE Sporadic melioidosis cases have been reported in the African mainland and Indian Ocean islands, but until recently, these regions were not considered areas where B. pseudomallei is endemic. Given the high mortality rate of melioidosis, it is crucial that this disease be recognized and suspected in all regions of endemicity. Previous work has shown that B. pseudomallei originated in Australia, with subsequent introduction into Asia; however, the precise origin of B. pseudomallei in other tropical regions remains poorly understood. Using

  10. Burkholderia mallei and Burkholderia pseudomallei Cluster 1 Type VI Secretion System Gene Expression Is Negatively Regulated by Iron and Zinc

    PubMed Central

    Burtnick, Mary N.; Brett, Paul J.

    2013-01-01

    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. PMID:24146925

  11. In silico analysis of Burkholderia pseudomallei genome sequence for potential drug targets.

    PubMed

    Chong, Chan-Eng; Lim, Boon-San; Nathan, Sheila; Mohamed, Rahmah

    2006-01-01

    Recent advances in DNA sequencing technology have enabled elucidation of whole genome information from a plethora of organisms. In parallel with this technology, various bioinformatics tools have driven the comparative analysis of the genome sequences between species and within isolates. While drawing meaningful conclusions from a large amount of raw material, computer-aided identification of suitable targets for further experimental analysis and characterization, has also led to the prediction of non-human homologous essential genes in bacteria as promising candidates for novel drug discovery. Here, we present a comparative genomic analysis to identify essential genes in Burkholderia pseudomallei. Our in silico prediction has identified 312 essential genes which could also be potential drug candidates. These genes encode essential proteins to support the survival of B. pseudomallei including outer-inner membrane and surface structures, regulators, proteins involved in pathogenenicity, adaptation, chaperones as well as degradation of small and macromolecules, energy metabolism, information transfer, central/intermediate/miscellaneous metabolism pathways and some conserved hypothetical proteins of unknown function. Therefore, our in silico approach has enabled rapid screening and identification of potential drug targets for further characterization in the laboratory. PMID:16922696

  12. The HicA toxin from Burkholderia pseudomallei has a role in persister cell formation

    PubMed Central

    Butt, Aaron; Higman, Victoria A.; Williams, Christopher; Crump, Matthew P.; Hemsley, Claudia M.; Harmer, Nicholas; Titball, Richard W.

    2014-01-01

    TA (toxin–antitoxin) systems are widely distributed amongst bacteria and are associated with the formation of antibiotic tolerant (persister) cells that may have involvement in chronic and recurrent disease. We show that overexpression of the Burkholderia pseudomallei HicA toxin causes growth arrest and increases the number of persister cells tolerant to ciprofloxacin or ceftazidime. Furthermore, our data show that persistence towards ciprofloxacin or ceftazidime can be differentially modulated depending on the level of induction of HicA expression. Deleting the hicAB locus from B. pseudomallei K96243 significantly reduced persister cell frequencies following exposure to ciprofloxacin, but not ceftazidime. The structure of HicA(H24A) was solved by NMR and forms a dsRBD-like (dsRNA-binding domain-like) fold, composed of a triple-stranded β-sheet, with two helices packed against one face. The surface of the protein is highly positively charged indicative of an RNA-binding protein and His24 and Gly22 were functionality important residues. This is the first study demonstrating a role for the HicAB system in bacterial persistence and the first structure of a HicA protein that has been experimentally characterized. PMID:24502667

  13. CD4+ T cell immunity to the Burkholderia pseudomallei ABC transporter LolC in melioidosis

    PubMed Central

    Chu, Karen K.; Tippayawat, Patcharaporn; Walker, Nicola J.; Harding, Sarah V.; Atkins, Helen S.; Maillere, Bernard; Bancroft, Gregory J.; Lertmemongkolchai, Ganjana; Altmann, Daniel M.

    2011-01-01

    Burkholderia pseudomallei (Bp) causes melioidosis, a disease with a wide range of possible outcomes, from seroconversion and dormancy to sepsis and death. This spectrum of host-pathogen interactions poses challenging questions about heterogeneity in immunity to Bp. Models show protection to be dependent on CD4+ cells and IFNγ, but little is known about specific target antigens. Having previously implicated the ABC transporter, LolC, in protective immunity, we here use epitope prediction, HLA binding studies, HLA-transgenic models and studies of T cells from seropositive individuals to characterize HLA-restricted LolC responses. Immunized mice showed long-lasting memory to the protein, while predictive algorithms identified epitopes within LolC that subsequently demonstrated strong HLA class II binding. Immunization of HLA-DR transgenics with LolC stimulated T cell responses to four of these epitopes. Furthermore, responsiveness of HLA-transgenics to LolC revealed a hierarchy supportive of HLA polymorphism-determined differential susceptibility. Seropositive human donors of diverse HLA class II types showed T cell responses to LolC epitopes which are conserved among Burkholderia species including B. cenocepacia, associated with life-threatening cepacia complex in cystic fibrosis patients and B. mallei, which causes glanders. These findings suggest a role for LolC epitopes in multiepitope vaccine design for melioidosis and related diseases. PMID:21182082

  14. Genetic Analysis of the CDI Pathway from Burkholderia pseudomallei 1026b

    PubMed Central

    Edman, Natasha; Chaudhuri, Swarnava; Poole, Stephen J.; Manoil, Colin; Hayes, Christopher S.; Low, David A.

    2015-01-01

    Contact-dependent growth inhibition (CDI) is a mode of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion systems. CdiA binds to receptors on susceptible target bacteria, then delivers a toxin domain derived from its C-terminus. Studies with Escherichia coli suggest the existence of multiple CDI growth-inhibition pathways, whereby different systems exploit distinct target-cell proteins to deliver and activate toxins. Here, we explore the CDI pathway in Burkholderia using the CDIIIBp1026b system encoded on chromosome II of Burkholderia pseudomallei 1026b as a model. We took a genetic approach and selected Burkholderia thailandensis E264 mutants that are resistant to growth inhibition by CDIIIBp1026b. We identified mutations in three genes, BTH_I0359, BTH_II0599, and BTH_I0986, each of which confers resistance to CDIIIBp1026b. BTH_I0359 encodes a small peptide of unknown function, whereas BTH_II0599 encodes a predicted inner membrane transport protein of the major facilitator superfamily. The inner membrane localization of BTH_II0599 suggests that it may facilitate translocation of CdiA-CTIIBp1026b toxin from the periplasm into the cytoplasm of target cells. BTH_I0986 encodes a putative transglycosylase involved in lipopolysaccharide (LPS) synthesis. ∆BTH_I0986 mutants have altered LPS structure and do not interact with CDI+ inhibitor cells to the same extent as BTH_I0986+ cells, suggesting that LPS could function as a receptor for CdiAIIBp1026b. Although ∆BTH_I0359, ∆BTH_II0599, and ∆BTH_I0986 mutations confer resistance to CDIIIBp1026b, they provide no protection against the CDIE264 system deployed by B. thailandensis E264. Together, these findings demonstrate that CDI growth-inhibition pathways are distinct and can differ significantly even between closely related species. PMID:25786241

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

    PubMed

    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

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

    PubMed Central

    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

  17. Phylogenetic Analysis of Ara+ and Ara− Burkholderia pseudomallei Isolates and Development of a Multiplex PCR Procedure for Rapid Discrimination between the Two Biotypes

    PubMed Central

    Dharakul, Tararaj; Tassaneetrithep, Boonratn; Trakulsomboon, Suwanna; Songsivilai, Sirirurg

    1999-01-01

    A Burkholderia pseudomallei-like organism has recently been identified among some soil isolates of B. pseudomallei in an area with endemic melioidosis. This organism is almost identical to B. pseudomallei in terms of morphological and biochemical profiles, except that it differs in ability to assimilate l-arabinose. These Ara+ isolates are also less virulent than the Ara− isolates in animal models. In addition, clinical isolates of B. pseudomallei available to date are almost exclusively Ara−. These features suggested that these two organisms may belong to distinctive species. In this study, the 16S rRNA-encoding genes from five clinical (four Ara− and one Ara+) and nine soil isolates (five Ara− and four Ara+) of B. pseudomallei were sequenced. The nucleotide sequences and phylogenetic analysis indicated that the 16S rRNA-encoding gene of the Ara+ biotype was similar to but distinctively different from that of the Ara− soil isolates, which were identical to the classical clinical isolates of B. pseudomallei. The nucleotide sequence differences in the 16S rRNA-encoding gene appeared to be specific for the Ara+ or Ara− biotypes. The differences were, however, not sufficient for classification into a new species within the genus Burkholderia. A simple and rapid multiplex PCR procedure was developed to discriminate between Ara− and Ara+ B. pseudomallei isolates. This new method could also be incorporated into our previously reported nested PCR system for detecting B. pseudomallei in clinical specimens. PMID:10325345

  18. Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates

    PubMed Central

    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. PMID:26484663

  19. Burkholderia pseudomallei Rapidly Infects the Brain Stem and Spinal Cord via the Trigeminal Nerve after Intranasal Inoculation.

    PubMed

    St John, James A; Walkden, Heidi; Nazareth, Lynn; Beagley, Kenneth W; Ulett, Glen C; Batzloff, Michael R; Beacham, Ifor R; Ekberg, Jenny A K

    2016-09-01

    Infection with Burkholderia pseudomallei causes melioidosis, a disease with a high mortality rate (20% in Australia and 40% in Southeast Asia). Neurological melioidosis is particularly prevalent in northern Australian patients and involves brain stem infection, which can progress to the spinal cord; however, the route by which the bacteria invade the central nervous system (CNS) is unknown. We have previously demonstrated that B. pseudomallei can infect the olfactory and trigeminal nerves within the nasal cavity following intranasal inoculation. As the trigeminal nerve projects into the brain stem, we investigated whether the bacteria could continue along this nerve to penetrate the CNS. After intranasal inoculation of mice, B. pseudomallei caused low-level localized infection within the nasal cavity epithelium, prior to invasion of the trigeminal nerve in small numbers. B. pseudomallei rapidly invaded the trigeminal nerve and crossed the astrocytic barrier to enter the brain stem within 24 h and then rapidly progressed over 2,000 μm into the spinal cord. To rule out that the bacteria used a hematogenous route, we used a capsule-deficient mutant of B. pseudomallei that does not survive in the blood and found that it also entered the CNS via the trigeminal nerve. This suggests that the primary route of entry is via the nerves that innervate the nasal cavity. We found that actin-mediated motility could facilitate initial infection of the olfactory epithelium. Thus, we have demonstrated that B. pseudomallei can rapidly infect the brain and spinal cord via the trigeminal nerve branches that innervate the nasal cavity. PMID:27382023

  20. Evaluation of Molecular Methods To Improve the Detection of Burkholderia pseudomallei in Soil and Water Samples from Laos

    PubMed Central

    Knappik, Michael; Dance, David A. B.; Rattanavong, Sayaphet; Pierret, Alain; Ribolzi, Olivier; Davong, Viengmon; Silisouk, Joy; Vongsouvath, Manivanh; Newton, Paul N.

    2015-01-01

    Burkholderia pseudomallei is the cause of melioidosis, a severe and potentially fatal disease of humans and animals. It is endemic in northern Australia and Southeast Asia and is found in soil and surface water. The environmental distribution of B. pseudomallei worldwide and within countries where it is endemic, such as the Lao People's Democratic Republic (Laos), remains unclear. However, this knowledge is important to our understanding of the ecology and epidemiology of B. pseudomallei and to facilitate public health interventions. Sensitive and specific methods to detect B. pseudomallei in environmental samples are therefore needed. The aim of this study was to compare molecular and culture-based methods for the detection of B. pseudomallei in soil and surface water in order to identify the optimal approach for future environmental studies in Laos. Molecular detection by quantitative real-time PCR (qPCR) was attempted after DNA extraction directly from soil or water samples or after an overnight enrichment step. The positivity rates obtained by qPCR were compared to those obtained by different culture techniques. The rate of detection from soil samples by qPCR following culture enrichment was significantly higher (84/100) than that by individual culture methods and all culture methods combined (44/100; P < 0.001). Similarly, qPCR following enrichment was the most sensitive method for filtered river water compared with the sensitivity of the individual methods and all individual methods combined. In conclusion, molecular detection following an enrichment step has proven to be a sensitive and reliable approach for B. pseudomallei detection in Lao environmental samples and is recommended as the preferred method for future surveys. PMID:25819969

  1. Burkholderia pseudomallei type III secretion system mutants exhibit delayed vacuolar escape phenotypes in RAW 264.7 murine macrophages.

    PubMed

    Burtnick, Mary N; Brett, Paul J; Nair, Vinod; Warawa, Jonathan M; Woods, Donald E; Gherardini, Frank C

    2008-07-01

    Burkholderia pseudomallei is a facultative intracellular pathogen capable of surviving and replicating within eukaryotic cells. Recent studies have shown that B. pseudomallei Bsa type III secretion system 3 (T3SS-3) mutants exhibit vacuolar escape and replication defects in J774.2 murine macrophages. In the present study, we characterized the interactions of a B. pseudomallei bsaZ mutant with RAW 264.7 murine macrophages. Following uptake, the mutant was found to survive and replicate within infected RAW 264.7 cells over an 18-h period. In addition, high levels of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES, but not IL-1alpha and IL-1beta, were detected in culture supernatants harvested from infected monolayers. The subcellular location of B. pseudomallei within infected RAW 264.7 cells was determined, and as expected, the bsaZ mutant demonstrated early-vacuolar-escape defects. Interestingly, however, experiments also indicated that this mutant was capable of delayed vacuolar escape. Consistent with this finding, evidence of actin-based motility and multinucleated giant cell formation were observed between 12 and 18 h postinfection. Further studies demonstrated that a triple mutant defective in all three B. pseudomallei T3SSs exhibited the same phenotype as the bsaZ mutant, indicating that functional T3SS-1 and T3SS-2 did not appear to be responsible for the delayed escape phenotype in RAW 264.7 cells. Based upon these findings, it appears that B. pseudomallei may not require T3SS-1, -2, and -3 to facilitate survival, delayed vacuolar escape, and actin-based motility in activated RAW 264.7 macrophages. PMID:18443088

  2. Persistent Gastric Colonization with Burkholderia pseudomallei and Dissemination from the Gastrointestinal Tract following Mucosal Inoculation of Mice

    PubMed Central

    Goodyear, Andrew; Bielefeldt-Ohmann, Helle; Schweizer, Herbert; Dow, Steven

    2012-01-01

    Melioidosis is a disease of humans caused by opportunistic infection with the soil and water bacterium Burkholderia pseudomallei. Melioidosis can manifest as an acute, overwhelming infection or as a chronic, recurrent infection. At present, it is not clear where B. pseudomallei resides in the mammalian host during the chronic, recurrent phase of infection. To address this question, we developed a mouse low-dose mucosal challenge model of chronic B. pseudomallei infection and investigated sites of bacterial persistence over 60 days. Sensitive culture techniques and selective media were used to quantitate bacterial burden in major organs, including the gastrointestinal (GI) tract. We found that the GI tract was the primary site of bacterial persistence during the chronic infection phase, and was the only site from which the organism could be consistently cultured during a 60-day infection period. The organism could be repeatedly recovered from all levels of the GI tract, and chronic infection was accompanied by sustained low-level fecal shedding. The stomach was identified as the primary site of GI colonization as determined by fluorescent in situ hybridization. Organisms in the stomach were associated with the gastric mucosal surface, and the propensity to colonize the gastric mucosa was observed with 4 different B. pseudomallei isolates. In contrast, B. pseudomallei organisms were present at low numbers within luminal contents in the small and large intestine and cecum relative to the stomach. Notably, inflammatory lesions were not detected in any GI tissue examined in chronically-infected mice. Only low-dose oral or intranasal inoculation led to GI colonization and development of chronic infection of the spleen and liver. Thus, we concluded that in a mouse model of melioidosis B. pseudomallei preferentially colonizes the stomach following oral inoculation, and that the chronically colonized GI tract likely serves as a reservoir for dissemination of infection to

  3. Detection of immunoglobulins M and G using culture filtrate antigen of Burkholderia pseudomallei.

    PubMed

    Chenthamarakshan, V; Vadivelu, J; Puthucheary, S D

    2001-01-01

    IgM and IgG based ELISA systems were developed using the culture filtrate antigen (CFA) of Burkholderia pseudomallei. The assays were evaluated using 95 sera from 66 septicemic cases and 47 sera from 20 cases with localized melioidosis. In addition 65 sera from culture negative cases that were also serologically negative for other endemic infections clinically suspected of melioidosis were included. These were compared with sera from 260 non-melioidosis cases, 169 sera from individuals with high risk of acquiring the infection and 48 sera from healthy controls. The IgG-ELISA was 96% sensitive and 94% specific. All sera from cases with septicemic and localized infections and 61 of 63 sera from clinically suspected melioidosis cases were positive for IgG antibody. The geometric mean titre index (GMTI) values of IgG antibody in melioidosis cases were significantly higher (p < 0.0005) compared to that of healthy subjects, high risk group and subjects with non-melioidosis infections. The sensitivity and specificity of IgM ELISA was 74 and 99% respectively. The GMTI value of IgM antibody in the sera of melioidosis cases was significantly higher as compared to that of non-melioidosis disease controls (p < or = 0.001). These results demonstrate that the detection of IgG is a better indicator of the disease in the diagnosis of melioidosis. PMID:11173184

  4. Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei

    SciTech Connect

    Begley, Darren W.; Hartley, Robert C.; Davies, Douglas R.; Edwards, Thomas E.; Leonard, Jess T.; Abendroth, Jan; Burris, Courtney A.; Bhandari, Janhavi; Myler, Peter J.; Staker, Bart L.; Stewart, Lance J.

    2011-09-28

    As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

  5. A family history of deoxyribonuclease II: surprises from Trichinella spiralis and Burkholderia pseudomallei.

    PubMed

    MacLea, Kyle S; Krieser, Ronald J; Eastman, Alan

    2003-02-13

    Deoxyribonuclease IIalpha (DNase IIalpha) is an acidic endonuclease found in lysosomes and nuclei, and it is also secreted. Though its Caenorhabditis elegans homolog, NUC-1, is required for digesting DNA of apoptotic cell corpses and dietary DNA, it is not required for viability. However, DNase IIalpha is required in mice for correct development and viability, because undigested cell corpses lead to lesions throughout the body. Recently, we showed that, in contrast to previous reports, active DNase IIalpha consists of one contiguous polypeptide. To better analyze DNase II protein structure and determine residues important for activity, extensive database searches were conducted to find distantly related family members. We report 29 new partial or complete homologs from 21 species. Four homologs with differences at the purported active site histidine residue were detected in the parasitic nematodes Trichinella spiralis and Trichinella pseudospiralis. When these mutations were reconstructed in human DNase IIalpha, the expressed proteins were inactive. DNase II homologs were also identified in non-metazoan species. In particular, the slime-mold Dictyostelium, the protozoan Trichomonas vaginalis, and the bacterium Burkholderia pseudomallei all contain sequences with significant similarity and identity to previously cloned DNase II family members. We report an analysis of their sequences and implications for DNase II protein structure and evolution. PMID:12594037

  6. Burkholderia pseudomallei Antibodies in Individuals Living in Endemic Regions in Northeastern Brazil

    PubMed Central

    Rolim, Dionne Bezerra; Vilar, Dina Cortez F. L.; de Góes Cavalcanti, Luciano Pamplona; Freitas, Liara B. N.; Inglis, Timothy J. J.; Nobre Rodrigues, Jorge Luiz; Nagao-Dias, Aparecida Tiemi

    2011-01-01

    A seroepidemiological investigation was conducted among the population of two municipalities in Northeastern Brazil. Immunoglobulin M (IgM) and IgG antibodies to Burkholderia pseudomallei were positive in 51.27% (161 in 317 samples) and 58.49% (186), respectively. IgM titers were higher in children than in adults. On the contrary, IgG increased progressively with age. We observed a significant association between agricultural occupation and raised IgM titers (P < 0.005) and IgG titers (P < 0.001), and between construction workers and raised IgG titers (P = 0.005). Antibody IgG avidities did not correlate with age. The highest titers of antibodies (1/800) showed the highest antibody avidity indexes (P < 0.01). Most of the serum samples recognized 45-kDa and 200-kDa bands by IgG1 and IgG2 subclasses. Our study showed a high seropositivity among individuals living in endemic regions of the state of Ceará, and highlights the need for further surveillance close to water courses such as dams and rivers in Northeastern Brazil. PMID:21292903

  7. Detection of Burkholderia pseudomallei in Sputum using Selective Enrichment Broth and Ashdown's Medium at Kampong Cham Provincial Hospital, Cambodia.

    PubMed

    Nhem, Somary; Letchford, Joanne; Meas, Chea; Thann, Sovanndeth; McLaughlin, James C; Baron, Ellen Jo; West, T Eoin

    2014-01-01

    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. Given the low numbers of positive specimens, we could not conclusively determine the utility of the enhanced sputum testing protocol. However, the ramifications of identification of  B. pseudomallei are substantial, and the benefit of the enhanced testing protocol may be more apparent in patients selected based on risk factors and clinical presentation. Promoting clinician awareness of the infection and encouraging utilization of diagnostic microbiology services are also likely to be important factors in facilitating identification of melioidosis. PMID:25717370

  8. Brief communication genotyping of Burkholderia pseudomallei revealed high genetic variability among isolates from a single population group

    PubMed Central

    Zueter, Abdelrahman Mohammad; Rahman, Zaidah Abdul; Yean, Chan Yean; Harun, Azian

    2015-01-01

    Burkholderia pseudomallei is a soil dwelling Gram-negative bacteria predominates in Southeast Asia zone and the tropical part of Australia. Genetic diversity has been explored among various populations and environments worldwide. To date, little data is available on MLST profiling of clinical B. pseudomallei isolates in peninsular Malaysia. In this brief report, thirteen culture positive B. pseudomallei cases collected from a single population of Terengganu state in the Western Peninsular Malaysia and were confirmed by In-house TTS1-PCR. Isolates were subjected for multi-locus sequence typing (MLST) to explore their genotypic diversity and to investigate for possible clonal clustering of a certain sequence type. Patient’s clinical information was examined to investigate for clinical correlation among the different genotypes. In spite of small sample set, MLST results indicated predictive results; considerable genotypic diversity, predominance and novelty among B. pseudomallei collected over a single geographically-located population in Malaysia. Massive genotypic heterogeneity was observed; 8 different sequence types with predominance of sequence type 54 and discovery of two novel sequence types. However, no clear pathogenomic or organ tropism clonal relationships were predicted. PMID:26417404

  9. Effects of Colonization of the Roots of Domestic Rice (Oryza sativa L. cv. Amaroo) by Burkholderia pseudomallei

    PubMed Central

    Constantinoiu, Constantin; Gardiner, Christopher; Warner, Jeffrey

    2015-01-01

    Burkholderia pseudomallei is a saprophytic bacterium that causes melioidosis and is often isolated from rice fields in Southeast Asia, where the infection incidence is high among rice field workers. The aim of this study was to investigate the relationship between this bacterium and rice through growth experiments where the effect of colonization of domestic rice (Oryza sativa L. cv Amaroo) roots by B. pseudomallei could be observed. When B. pseudomallei was exposed to surface-sterilized seeds, the growth of both the root and the aerosphere was retarded compared to that in controls. The organism was found to localize in the root hairs and endodermis of the plant. A biofilm formed around the root and root structures that were colonized. Growth experiments with a wild rice species (Oryza meridionalis) produced similar retardation of growth, while another domestic cultivar (O. sativa L. cv Koshihikari) did not show retarded growth. Here we report B. pseudomallei infection and inhibition of O. sativa L. cv Amaroo, which might provide insights into plant interactions with this important human pathogen. PMID:25911477

  10. Diversity of 16S-23S rDNA Internal Transcribed Spacer (ITS) Reveals Phylogenetic Relationships in Burkholderia pseudomallei and Its Near-Neighbors

    PubMed Central

    Liguori, Andrew P.; Warrington, Stephanie D.; Ginther, Jennifer L.; Pearson, Talima; Bowers, Jolene; Glass, Mindy B.; Mayo, Mark; Wuthiekanun, Vanaporn; Engelthaler, David; Peacock, Sharon J.; Currie, Bart J.; Wagner, David M.; Keim, Paul; Tuanyok, Apichai

    2011-01-01

    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. PMID:22195045

  11. Exploiting the Burkholderia pseudomallei acute phase antigen BPSL2765 for structure-based epitope discovery/design in structural vaccinology.

    PubMed

    Gourlay, Louise J; Peri, Claudio; Ferrer-Navarro, Mario; Conchillo-Solé, Oscar; Gori, Alessandro; Rinchai, Darawan; Thomas, Rachael J; Champion, Olivia L; Michell, Stephen L; Kewcharoenwong, Chidchamai; Nithichanon, Arnone; Lassaux, Patricia; Perletti, Lucia; Longhi, Renato; Lertmemongkolchai, Ganjana; Titball, Richard W; Daura, Xavier; Colombo, Giorgio; Bolognesi, Martino

    2013-09-19

    We solved the crystal structure of Burkholderia pseudomallei acute phase antigen BPSL2765 in the context of a structural vaccinology study, in the area of melioidosis vaccine development. Based on the structure, we applied a recently developed method for epitope design that combines computational epitope predictions with in vitro mapping experiments and successfully identified a consensus sequence within the antigen that, when engineered as a synthetic peptide, was selectively immunorecognized to the same extent as the recombinant protein in sera from melioidosis-affected subjects. Antibodies raised against the consensus peptide were successfully tested in opsonization bacterial killing experiments and antibody-dependent agglutination tests of B. pseudomallei. Our strategy represents a step in the development of immunodiagnostics, in the production of specific antibodies and in the optimization of antigens for vaccine development, starting from structural and physicochemical principles. PMID:23993463

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

    EPA Science Inventory

    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...

  13. Disarming Burkholderia pseudomallei: Structural and Functional Characterization of a Disulfide Oxidoreductase (DsbA) Required for Virulence In Vivo

    PubMed Central

    McMahon, Róisín M.; Marshall, Laura E.; Halili, Maria; Furlong, Emily; Tay, Stephanie; Sarkar-Tyson, Mitali

    2014-01-01

    Abstract Aims: The intracellular pathogen Burkholderia pseudomallei causes the disease melioidosis, a major source of morbidity and mortality in southeast Asia and northern Australia. The need to develop novel antimicrobials is compounded by the absence of a licensed vaccine and the bacterium's resistance to multiple antibiotics. In a number of clinically relevant Gram-negative pathogens, DsbA is the primary disulfide oxidoreductase responsible for catalyzing the formation of disulfide bonds in secreted and membrane-associated proteins. In this study, a putative B. pseudomallei dsbA gene was evaluated functionally and structurally and its contribution to infection assessed. Results: Biochemical studies confirmed the dsbA gene encodes a protein disulfide oxidoreductase. A dsbA deletion strain of B. pseudomallei was attenuated in both macrophages and a BALB/c mouse model of infection and displayed pleiotropic phenotypes that included defects in both secretion and motility. The 1.9 Å resolution crystal structure of BpsDsbA revealed differences from the classic member of this family Escherichia coli DsbA, in particular within the region surrounding the active site disulfide where EcDsbA engages with its partner protein E. coli DsbB, indicating that the interaction of BpsDsbA with its proposed partner BpsDsbB may be distinct from that of EcDsbA-EcDsbB. Innovation: This study has characterized BpsDsbA biochemically and structurally and determined that it is required for virulence of B. pseudomallei. Conclusion: These data establish a critical role for BpsDsbA in B. pseudomallei infection, which in combination with our structural characterization of BpsDsbA will facilitate the future development of rationally designed inhibitors against this drug-resistant organism. Antioxid. Redox Signal. 20, 606–617. PMID:23901809

  14. Burkholderia pseudomallei in soil samples from an oceanarium in Hong Kong detected using a sensitive PCR assay

    PubMed Central

    Lau, Susanna KP; Chan, San-Yuen; Curreem, Shirly OT; Hui, Suk-Wai; Lau, Candy CY; Lee, Paul; Ho, Chi-Chun; Martelli, Paolo; Woo, Patrick CY

    2014-01-01

    Melioidosis, caused by Burkholderia pseudomallei, is an emerging infectious disease with an expanding geographical distribution. Although assessment of the environmental load of B. pseudomallei is important for risk assessment in humans or animals in endemic areas, traditional methods of bacterial culture for isolation have low sensitivities and are labor-intensive. Using a specific polymerase chain reaction (PCR) assay targeting a Tat domain protein in comparison with a bacterial culture method, we examined the prevalence of B. pseudomallei in soil samples from an oceanarium in Hong Kong where captive marine mammals and birds have contracted melioidosis. Among 1420 soil samples collected from various sites in the oceanarium over a 15-month period, B. pseudomallei was detected in nine (0.6%) soil samples using bacterial culture, whereas it was detected in 96 (6.8%) soil samples using the specific PCR assay confirmed by sequencing. The PCR-positive samples were detected during various months, with higher detection rates observed during summer months. Positive PCR detection was significantly correlated with ambient temperature (P<0.0001) and relative humidity (P=0.011) but not with daily rainfall (P=0.241) or a recent typhoon (P=0.787). PCR-positive samples were obtained from all sampling locations, with the highest detection rate in the valley. Our results suggest that B. pseudomallei is prevalent and endemic in the oceanarium. The present PCR assay is more sensitive than the bacterial culture method, and it may be used to help better assess the transmission of melioidosis and to design infection control measures for captive animals in this unique and understudied environment. PMID:26038496

  15. Seroprevalence of Burkholderia pseudomallei among Adults in Coastal Areas in Southwestern India

    PubMed Central

    Vandana, Kalwaje Eshwara; Mukhopadhyay, Chiranjay; Tellapragada, Chaitanya; Kamath, Asha; Tipre, Meghan; Bhat, Vinod; Sathiakumar, Nalini

    2016-01-01

    Background Although melioidosis, is an important disease in many Southeast Asian countries and Australia, there is limited data on its prevalence and disease burden in India. However, an increase in case reports of melioidosis in recent years indicates its endemicity in India. Aims and methods A population-based cross-sectional seroprevalence study was undertaken to determine the seroprevalence of B. pseudomallei by indirect haemagglutination assay and to investigate the associated risk determinants. Subjects were 711 adults aged 18 to 65 years residing in Udupi district, located in south-western coast of India. Key results Overall, 29% of the study subjects were seropositive (titer ≥20). Females were twice as likely to be seropositive compared to males. Rates of seroprevalence were similar in farmers and non-farmers. Besides gardening, other factors including socio-demographic, occupational and environmental factors did not show any relationship with seropositive status. Major conclusions There is a serological evidence of exposure to B. pseudomallei among adults in India. While the bacterium inhabits soil, exposure to the agent is not limited to farmers. Non-occupational exposure might play an important role in eliciting antibody response to the bacterium and may also be an important factor in disease causation. PMID:27078156

  16. Type three secretion system-mediated escape of Burkholderia pseudomallei into the host cytosol is critical for the activation of NFκB

    PubMed Central

    2014-01-01

    Background Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and Northern Australia. This Gram-negative pathogen possesses numerous virulence factors including three “injection type” type three secretion systems (T3SSs). B. pseudomallei has been shown to activate NFκB in HEK293T cells in a Toll-like receptor and MyD88 independent manner that requires T3SS gene cluster 3 (T3SS3 or T3SSBsa). However, the mechanism of how T3SS3 contributes to NFκB activation is unknown. Results Known T3SS3 effectors are not responsible for NFκB activation. Furthermore, T3SS3-null mutants are able to activate NFκB almost to the same extent as wildtype bacteria at late time points of infection, corresponding to delayed escape into the cytosol. NFκB activation also occurs when bacteria are delivered directly into the cytosol by photothermal nanoblade injection. Conclusions T3SS3 does not directly activate NFκB but facilitates bacterial escape into the cytosol where the host is able to sense the presence of the pathogen through cytosolic sensors leading to NFκB activation. PMID:24884837

  17. The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE.

    PubMed

    Treerat, Puthayalai; Alwis, Priyangi; D'Cruze, Tanya; Cullinane, Meabh; Vadivelu, Jamunarani; Devenish, Rodney J; Prescott, Mark; Adler, Ben; Boyce, John D

    2015-01-01

    Many Gram-negative pathogens use a type III secretion system (TTSS) for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC)-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness. PMID:26624293

  18. Burkholderia pseudomallei Type III Secretion System Cluster 3 ATPase BsaS, a Chemotherapeutic Target for Small-Molecule ATPase Inhibitors

    PubMed Central

    Gong, Lan; Lai, Shu-Chin; Treerat, Puthayalai; Prescott, Mark; Adler, Ben

    2015-01-01

    Melioidosis is an infectious disease of high mortality for humans and other animal species; it is prevalent in tropical regions worldwide. The pathogenesis of melioidosis depends on the ability of its causative agent, the Gram-negative bacterium Burkholderia pseudomallei, to enter and survive in host cells. B. pseudomallei can escape from the phagosome into the cytosol of phagocytic cells where it replicates and acquires actin-mediated motility, avoiding killing by the autophagy-dependent process, LC3 (microtubule-associated protein light chain 3)-associated phagocytosis (LAP). The type III secretion system cluster 3 (TTSS3) facilitates bacterial escape from phagosomes, although the mechanism has not been fully elucidated. Given the recent identification of small-molecule inhibitors of the TTSS ATPase, we sought to determine the potential of the predicted TTSS3 ATPase, encoded by bsaS, as a target for chemotherapeutic treatment of infection. A B. pseudomallei bsaS deletion mutant was generated and used as a control against which to assess the effect of inhibitor treatment. Infection of RAW 264.7 cells with wild-type bacteria and subsequent treatment with the ATPase inhibitor compound 939 resulted in reduced intracellular bacterial survival, reduced escape from phagosomes, and increased colocalization with both LC3 and the lysosomal marker LAMP1 (lysosome-associated membrane protein 1). These changes were similar to those observed for infection of RAW 264.7 cells with the bsaS deletion mutant. We propose that treatment with the ATPase inhibitor compound 939 decreased intracellular bacterial survival through a reduced ability of bacteria to escape from phagosomes and increased killing via LAP. Therefore, small-molecule inhibitors of the TTSS3 ATPase have potential as therapeutic treatments against melioidosis. PMID:25605762

  19. Burkholderia pseudomallei type III secretion system cluster 3 ATPase BsaS, a chemotherapeutic target for small-molecule ATPase inhibitors.

    PubMed

    Gong, Lan; Lai, Shu-Chin; Treerat, Puthayalai; Prescott, Mark; Adler, Ben; Boyce, John D; Devenish, Rodney J

    2015-04-01

    Melioidosis is an infectious disease of high mortality for humans and other animal species; it is prevalent in tropical regions worldwide. The pathogenesis of melioidosis depends on the ability of its causative agent, the Gram-negative bacterium Burkholderia pseudomallei, to enter and survive in host cells. B. pseudomallei can escape from the phagosome into the cytosol of phagocytic cells where it replicates and acquires actin-mediated motility, avoiding killing by the autophagy-dependent process, LC3 (microtubule-associated protein light chain 3)-associated phagocytosis (LAP). The type III secretion system cluster 3 (TTSS3) facilitates bacterial escape from phagosomes, although the mechanism has not been fully elucidated. Given the recent identification of small-molecule inhibitors of the TTSS ATPase, we sought to determine the potential of the predicted TTSS3 ATPase, encoded by bsaS, as a target for chemotherapeutic treatment of infection. A B. pseudomallei bsaS deletion mutant was generated and used as a control against which to assess the effect of inhibitor treatment. Infection of RAW 264.7 cells with wild-type bacteria and subsequent treatment with the ATPase inhibitor compound 939 resulted in reduced intracellular bacterial survival, reduced escape from phagosomes, and increased colocalization with both LC3 and the lysosomal marker LAMP1 (lysosome-associated membrane protein 1). These changes were similar to those observed for infection of RAW 264.7 cells with the bsaS deletion mutant. We propose that treatment with the ATPase inhibitor compound 939 decreased intracellular bacterial survival through a reduced ability of bacteria to escape from phagosomes and increased killing via LAP. Therefore, small-molecule inhibitors of the TTSS3 ATPase have potential as therapeutic treatments against melioidosis. PMID:25605762

  20. The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE

    PubMed Central

    Treerat, Puthayalai; Alwis, Priyangi; D’Cruze, Tanya; Cullinane, Meabh; Vadivelu, Jamunarani; Devenish, Rodney J.; Prescott, Mark; Adler, Ben; Boyce, John D.

    2015-01-01

    Many Gram-negative pathogens use a type III secretion system (TTSS) for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC)-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness. PMID:26624293

  1. New molecular interaction of IIA(Ntr) and HPr from Burkholderia pseudomallei identified by X-ray crystallography and docking studies.

    PubMed

    Kim, Mi-Sun; Lee, Hasup; Heo, Lim; Lim, Areum; Seok, Chaok; Shin, Dong Hae

    2013-09-01

    The nitrogen-related phosphoenolpyruvate phosphotransferase system (PTS(Ntr) ) is involved in controlling ammonia assimilation and nitrogen fixation. The additional role of PTS(Ntr) as a regulatory link between nitrogen and carbon utilization in Escherichia coli is assumed to be closely related to molecular functions of IIA(Ntr) in potassium homeostasis. We have determined the crystal structure of IIA(Ntr) from Burkholderia pseudomallei (BpIIA(Ntr) ), which is a causative agent of melioidosis. The crystal structure of dimeric BpIIA(Ntr) determined at 3.0 Å revealed that its active sites are mutually blocked. This dimeric state is stabilized by charge and weak hydrophobic interactions. Overall monomeric structure and the active site residues, Arg51 and His67, of BpIIA(Ntr) are well conserved with those of IIA(Ntr) enzymes from E. coli and Neisseria meningitides. Interestingly, His113 of BpIIA(Ntr) , which corresponds to a key residue in another phosphoryl group relay in the mannitol-specific enzyme EIIA family (EIIA(Mtl) ), is located away from the active site due to the loop connecting β5 and α3. Combined with other differences in molecular surface properties, these structural signatures distinguish the IIA(Ntr) family from the EIIA(Mtl) family. Since, there is no gene for NPr in the chromosome of B. pseudomallei, modeling and docking studies of the BpIIA(Ntr) -BpHPr complex has been performed to support the proposal on the NPr-like activity of BpHPr. A potential dual role of BpHPr as a nonspecific phosphocarrier protein interacting with both sugar EIIAs and IIA(Ntr) in B. pseudomallei has been discussed. PMID:23483653

  2. Solution structure of monomeric BsaL, the type III secretion needle protein of Burkholderia pseudomallei.

    PubMed

    Zhang, Lingling; Wang, Yu; Picking, Wendy L; Picking, William D; De Guzman, Roberto N

    2006-06-01

    Many gram-negative bacteria that are important human pathogens possess type III secretion systems as part of their required virulence factor repertoire. During the establishment of infection, these pathogens coordinately assemble greater than 20 different proteins into a macromolecular structure that spans the bacterial inner and outer membranes and, in many respects, resembles and functions like a syringe. This type III secretion apparatus (TTSA) is used to inject proteins into a host cell's membrane and cytoplasm to subvert normal cellular processes. The external portion of the TTSA is a needle that is composed of a single type of protein that is polymerized in a helical fashion to form an elongated tube with a central channel of 2-3 nm in diameter. TTSA needle proteins from a variety of bacterial pathogens share sequence conservation; however, no atomic structure for any TTSA needle protein is yet available. Here, we report the structure of a TTSA needle protein called BsaL from Burkholderia pseudomallei determined by nuclear magnetic resonance (NMR) spectroscopy. The central part of the protein assumes a helix-turn-helix core domain with two well-defined alpha-helices that are joined by an ordered, four-residue linker. This forms a two-helix bundle that is stabilized by interhelix hydrophobic contacts. Residues that flank this presumably exposed core region are not completely disordered, but adopt a partial helical conformation. The atomic structure of BsaL and its sequence homology with other TTSA needle proteins suggest potentially unique structural dynamics that could be linked with a universal mechanism for control of type III secretion in diverse gram-negative bacterial pathogens. PMID:16631790

  3. Whole-Genome Sequencing of Burkholderia pseudomallei Isolates from an Unusual Melioidosis Case Identifies a Polyclonal Infection with the Same Multilocus Sequence Type

    PubMed Central

    Sarovich, Derek S.; Viberg, Linda; Mayo, Mark; Kaestli, Mirjam; Tuanyok, Apichai; Foster, Jeffrey T.; Keim, Paul; Pearson, Talima; Currie, Bart J.

    2014-01-01

    Twelve Burkholderia pseudomallei isolates collected over a 32-month period from a patient with chronic melioidosis demonstrated identical multilocus sequence types (STs). However, whole-genome sequencing suggests a polyclonal infection. This study is the first to report a mixed infection with the same ST. PMID:25339397

  4. Evaluation of Polysaccharide-Based Latex Agglutination Assays for the Rapid Detection of Antibodies to Burkholderia pseudomallei.

    PubMed

    Suttisunhakul, Vichaya; Chantratita, Narisara; Wikraiphat, Chanthiwa; Wuthiekanun, Vanaporn; Douglas, Zakiya; Day, Nicholas P J; Limmathurotsakul, Direk; Brett, Paul J; Burtnick, Mary N

    2015-09-01

    Melioidosis is a severe disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Diagnosis of melioidosis currently relies on the isolation of B. pseudomallei from clinical samples, which can take several days. An indirect hemagglutination assay (IHA) is widely used for serodiagnosis, but it has a short shelf life, is poorly standardized, and requires a viable bacteria culture performed in a biosafety level 3 (BSL-3) laboratory. To improve the diagnostic methods, we have developed two rapid latex agglutination tests based on purified B. pseudomallei O-polysaccharide (OPS) and capsular polysaccharide (CPS) antigens. The immunodiagnostic potential of these tests was evaluated using serum from culture-confirmed melioidosis patients (N = 143) and healthy donors from either endemic (N = 199) or non-endemic areas (N = 90). The sensitivity of the OPS-based latex agglutination assay (OPS-latex; 84.4%) was significantly higher than both the CPS-latex (69.5%) (P < 0.001) and IHA (69.5%) (P = 0.001). When evaluated with Thai donor serum, the OPS-latex had comparable specificity (56.9%) to the CPS-latex (63.8%) (P = 0.053), but was significantly lower than the IHA (67.6%) (P = 0.002). In contrast, all tests with U.S. donor serum were highly specific (≥ 97.8%). These results suggest that polysaccharide-based latex agglutination assays may be useful for serodiagnosis of melioidosis in non-endemic areas. PMID:26123956

  5. Evaluation of Polysaccharide-Based Latex Agglutination Assays for the Rapid Detection of Antibodies to Burkholderia pseudomallei

    PubMed Central

    Suttisunhakul, Vichaya; Chantratita, Narisara; Wikraiphat, Chanthiwa; Wuthiekanun, Vanaporn; Douglas, Zakiya; Day, Nicholas P. J.; Limmathurotsakul, Direk; Brett, Paul J.; Burtnick, Mary N.

    2015-01-01

    Melioidosis is a severe disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Diagnosis of melioidosis currently relies on the isolation of B. pseudomallei from clinical samples, which can take several days. An indirect hemagglutination assay (IHA) is widely used for serodiagnosis, but it has a short shelf life, is poorly standardized, and requires a viable bacteria culture performed in a biosafety level 3 (BSL-3) laboratory. To improve the diagnostic methods, we have developed two rapid latex agglutination tests based on purified B. pseudomallei O-polysaccharide (OPS) and capsular polysaccharide (CPS) antigens. The immunodiagnostic potential of these tests was evaluated using serum from culture-confirmed melioidosis patients (N = 143) and healthy donors from either endemic (N = 199) or non-endemic areas (N = 90). The sensitivity of the OPS-based latex agglutination assay (OPS-latex; 84.4%) was significantly higher than both the CPS-latex (69.5%) (P < 0.001) and IHA (69.5%) (P = 0.001). When evaluated with Thai donor serum, the OPS-latex had comparable specificity (56.9%) to the CPS-latex (63.8%) (P = 0.053), but was significantly lower than the IHA (67.6%) (P = 0.002). In contrast, all tests with U.S. donor serum were highly specific (≥ 97.8%). These results suggest that polysaccharide-based latex agglutination assays may be useful for serodiagnosis of melioidosis in non-endemic areas. PMID:26123956

  6. Utilization of Whole-Cell MALDI-TOF Mass Spectrometry to Differentiate Burkholderia pseudomallei Wild-Type and Constructed Mutants

    PubMed Central

    Jaresitthikunchai, Janthima; Roytrakul, Sittiruk; Tungpradabkul, Sumalee

    2015-01-01

    Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has been widely adopted as a useful technology in the identification and typing of microorganisms. This study employed the whole-cell MALDI-TOF MS to identify and differentiate wild-type and mutants containing constructed single gene mutations of Burkholderia pseudomallei, a pathogenic bacterium causing melioidosis disease in both humans and animals. Candidate biomarkers for the B. pseudomallei mutants, including rpoS, ppk, and bpsI isolates, were determined. Taxon-specific and clinical isolate-specific biomarkers of B. pseudomallei were consistently found and conserved across all average mass spectra. Cluster analysis of MALDI spectra of all isolates exhibited separate distribution. A total of twelve potential mass peaks discriminating between wild-type and mutant isolates were identified using ClinProTools analysis. Two peaks (m/z 2721 and 2748 Da) were specific for the rpoS isolate, three (m/z 3150, 3378, and 7994 Da) for ppk, and seven (m/z 3420, 3520, 3587, 3688, 4623, 4708, and 5450 Da) for bpsI. Our findings demonstrated that the rapid, accurate, and reproducible mass profiling technology could have new implications in laboratory-based rapid differentiation of extensive libraries of genetically altered bacteria. PMID:26656930

  7. Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis

    PubMed Central

    Jakupciak, John P.; Wells, Jeffrey M.; Karalus, Richard J.; Pawlowski, David R.; Lin, Jeffrey S.; Feldman, Andrew B.

    2013-01-01

    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. PMID:24455204

  8. Experimental Persistent Infection of BALB/c Mice with Small-Colony Variants of Burkholderia pseudomallei Leads to Concurrent Upregulation of PD-1 on T Cells and Skewed Th1 and Th17 Responses

    PubMed Central

    See, Jia-Xiang; Samudi, Chandramathi; Saeidi, Alireza; Menon, Nivedita; Choh, Leang-Chung; Vadivelu, Jamuna; Shankar, Esaki M.

    2016-01-01

    Background Burkholderia pseudomallei (B. pseudomallei), the causative agent of melioidosis, is a deadly pathogen endemic across parts of tropical South East Asia and Northern Australia. B. pseudomallei can remain latent within the intracellular compartment of the host cell over prolonged periods of time, and cause persistent disease leading to treatment difficulties. Understanding the immunological mechanisms behind persistent infection can result in improved treatment strategies in clinical melioidosis. Methods Ten-day LD50 was determined for the small-colony variant (SCV) and its parental wild-type (WT) via intranasal route in experimental BALB/c mice. Persistent B. pseudomallei infection was generated by administrating sub-lethal dose of the two strains based on previously determined LD50. After two months, peripheral blood mononuclear cells (PBMCs) and plasma were obtained to investigate host immune responses against persistent B. pseudomallei infection. Lungs, livers, and spleens were harvested and bacterial loads in these organs were determined. Results Based on the ten-day LD50, the SCV was ~20-fold less virulent than the WT. The SCV caused higher bacterial loads in spleens compared to its WT counterparts with persistent B. pseudomallei infection. We found that the CD4+ T-cell frequencies were decreased, and the expressions of PD-1, but not CTLA-4 were significantly increased on the CD4+ and CD8+ T cells of these mice. Notably, persistent infection with the SCV led to significantly higher levels of PD-1 than the WT B. pseudomallei. Plasma IFN-γ, IL-6, and IL-17A levels were elevated only in SCV-infected mice. In addition, skewed plasma Th1 and Th17 responses were observed in SCV-infected mice relative to WT-infected and uninfected mice. Conclusion B. pseudomallei appears to upregulate the expression of PD-1 on T cells to evade host immune responses, which likely facilitates bacterial persistence in the host. SCVs cause distinct pathology and immune

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

    PubMed Central

    Kohler, Christian; Dunachie, Susanna J.; Müller, Elke; Kohler, Anne; Jenjaroen, Kemajittra; Teparrukkul, Prapit; Baier, Vico; Ehricht, Ralf; Steinmetz, Ivo

    2016-01-01

    Background 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. Methods and Principal Findings 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. Conclusions 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

  10. Sterile-α- and armadillo motif-containing protein inhibits the TRIF-dependent downregulation of signal regulatory protein α to interfere with intracellular bacterial elimination in Burkholderia pseudomallei-infected mouse macrophages.

    PubMed

    Baral, Pankaj; Utaisincharoen, Pongsak

    2013-09-01

    Burkholderia pseudomallei, the causative agent of melioidosis, evades macrophage killing by suppressing the TRIF-dependent pathway, leading to inhibition of inducible nitric oxide synthase (iNOS) expression. We previously demonstrated that virulent wild-type B. pseudomallei inhibits the TRIF-dependent pathway by upregulating sterile-α- and armadillo motif-containing protein (SARM) and by inhibiting downregulation of signal regulatory protein α (SIRPα); both molecules are negative regulators of Toll-like receptor signaling. In contrast, the less virulent lipopolysaccharide (LPS) mutant of B. pseudomallei is unable to exhibit these features and is susceptible to macrophage killing. However, the functional relationship of these two negative regulators in the evasion of macrophage defense has not been elucidated. We demonstrated here that SIRPα downregulation was observed after inhibition of SARM expression by small interfering RNA in wild-type-infected macrophages, indicating that SIRPα downregulation is regulated by SARM. Furthermore, this downregulation requires activation of the TRIF signaling pathway, as we observed abrogation of SIRPα downregulation as well as restricted bacterial growth in LPS mutant-infected TRIF-depleted macrophages. Although inhibition of SARM expression is correlated to SIRPα downregulation and iNOS upregulation in gamma interferon-activated wild-type-infected macrophages, these phenomena appear to bypass the TRIF-dependent pathway. Similar to live bacteria, the wild-type LPS is able to upregulate SARM and to prevent SIRPα downregulation, implying that the LPS of B. pseudomallei may play a crucial role in regulating the expression of these two negative regulators. Altogether, our findings show a previously unrecognized role of B. pseudomallei-induced SARM in inhibiting SIRPα downregulation-mediated iNOS upregulation, facilitating the ability of the bacterium to multiply in macrophages. PMID:23836818

  11. Malleilactone, a polyketide synthase-derived virulence factor encoded by the cryptic secondary metabolome of Burkholderia pseudomallei group pathogens

    PubMed Central

    Biggins, John B.; Ternei, Melinda A.; Brady, Sean F.

    2012-01-01

    Sequenced bacterial genomes are routinely found to contain gene clusters that are predicted to encode metabolites not seen in fermentation based studies. Pseudomallei group Burkholderia are emerging pathogens whose genomes are particularly rich in cryptic natural product biosynthetic gene clusters. We systemically probed the influence of the cryptic secondary metabolome on the virulence of these bacteria and found that the disruption of the MAL gene cluster, which is natively silent in laboratory fermentation experiments and conserved across this group of pathogens, attenuates virulence in animal models. Using a promoter exchange strategy to activate the MAL cluster we identified malleilactone, a polyketide synthase-derived cytotoxic siderophore encoded by this gene cluster. Small molecules targeting malleilactone biosynthesis either alone, or in conjunction with antibiotics, could prove useful as next-generation therapeutics for combating melioidosis and glanders. PMID:22765305

  12. Pathogenesis of percutaneous infection of goats with Burkholderia pseudomallei: clinical, pathologic, and immunological responses in chronic melioidosis

    PubMed Central

    Soffler, Carl; Bosco-Lauth, Angela M; Aboellail, Tawfik A; Marolf, Angela J; Bowen, Richard A

    2014-01-01

    Melioidosis is a severe suppurative to granulomatous infection caused by Burkholderia pseudomallei. The disease is endemic to South-East Asia and Northern Australasia and is also of interest as a potential biological weapon. Natural infection can occur by percutaneous inoculation, inhalation or ingestion, but the relative importance of each route is unknown. Experimental infection models using mice have shown inhalation to be the most lethal route of exposure, but few studies have examined the pathogenesis of percutaneous infection despite its presumptive importance in natural disease. Caprine models are useful in the study of melioidosis because goats are susceptible to natural infection by B. pseudomallei, display similar epizootiology/epidemiology to that of humans within the endemic range and develop similar pathologic lesions. Percutaneous inoculation with 104 CFU of B. pseudomallei produced disease in all experimental animals with rapid dissemination to the lungs, spleen and kidneys. Initial fever was brief, but temperatures did not return to pre-infection levels until day 18, concurrent with a dramatic lymphocytosis and the transition to chronic disease. Distribution and appearance of gross pathologic and radiographic lesions in goats were similar to caprine aerosol infection and to reported human disease. The similarities seen despite different routes of infection suggest that host or bacterial factors may be more important than the route of infection in disease pathogenesis. The nature of melioidosis in goats makes it amenable for modelling additional risk factors to produce acute clinical disease, which is important to the study of human melioidosis. PMID:24571408

  13. Novel engineered cationic antimicrobial peptides display broad-spectrum activity against Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei.

    PubMed

    Abdelbaqi, Suha; Deslouches, Berthony; Steckbeck, Jonathan; Montelaro, Ronald; Reed, Douglas S

    2016-02-01

    Broad-spectrum antimicrobials are needed to effectively treat patients infected in the event of a pandemic or intentional release of a pathogen prior to confirmation of the pathogen's identity. Engineered cationic antimicrobial peptides (eCAPs) display activity against a number of bacterial pathogens including multi-drug-resistant strains. Two lead eCAPs, WLBU2 and WR12, were compared with human cathelicidin (LL-37) against three highly pathogenic bacteria: Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. Both WLBU2 and WR12 demonstrated bactericidal activity greater than that of LL-37, particularly against F. tularensis and Y. pestis. Only WLBU2 had bactericidal activity against B. pseudomallei. WLBU2, WR12 and LL-37 were all able to inhibit the growth of the three bacteria in vitro. Because these bacteria can be facultative intracellular pathogens, preferentially infecting macrophages and dendritic cells, we evaluated the activity of WLBU2 against F. tularensis in an ex vivo infection model with J774 cells, a mouse macrophage cell line. In that model WLBU2 was able to achieve greater than 50% killing of F. tularensis at a concentration of 12.5 μM. These data show the therapeutic potential of eCAPs, particularly WLBU2, as a broad-spectrum antimicrobial for treating highly pathogenic bacterial infections. PMID:26673248

  14. A Burkholderia pseudomallei Macrophage Infectivity Potentiator-Like Protein Has Rapamycin-Inhibitable Peptidylprolyl Isomerase Activity and Pleiotropic Effects on Virulence ▿

    PubMed Central

    Norville, Isobel H.; Harmer, Nicholas J.; Harding, Sarah V.; Fischer, Gunter; Keith, Karen E.; Brown, Katherine A.; Sarkar-Tyson, Mitali; Titball, Richard W.

    2011-01-01

    Macrophage infectivity potentiators (Mips) are a group of virulence factors encoded by pathogenic bacteria such as Legionella, Chlamydia, and Neisseria species. Mips are part of the FK506-binding protein (FKBP) family, whose members typically exhibit peptidylprolyl cis-trans isomerase (PPIase) activity which is inhibitable by the immunosuppressants FK506 and rapamycin. Here we describe the identification and characterization of BPSS1823, a Mip-like protein in the intracellular pathogen Burkholderia pseudomallei. Recombinant BPSS1823 protein has rapamycin-inhibitable PPIase activity, indicating that it is a functional FKBP. A mutant strain generated by deletion of BPSS1823 in B. pseudomallei exhibited a reduced ability to survive within cells and significant attenuation in vivo, suggesting that BPSS1823 is important for B. pseudomallei virulence. In addition, pleiotropic effects were observed with a reduction in virulence mechanisms, including resistance to host killing mechanisms, swarming motility, and protease production. PMID:21859853

  15. [Immune response in experimental animals immunized with Burkholderia pseudomallei surface antigens].

    PubMed

    Avrorova, I V; Piven', N N; Zhukova, S I; Viktorov, D V; Khrapova, N P; Popov, S F

    2004-01-01

    The influence of the chromatographic fractions of B. pseudomallei surface antigenic complex (C, C1, D, H) on immune response in white rats and white mice was under study. These antigenic complexes were noted to produce perceptible stimulating effect on the immune system of white rats, in contrast to that of white mice. The immunization of the mice the above-mentioned fractions suppressed the phagocytic activity of peritoneal macrophages (PM) and slightly enhanced cell-mediated immunity. In experiments on white rats, fraction C induced the growth of specific antibody titers and stimulated the phagocytic activity of PM, as well as the indices of delayed hypersensitivity (DH). Fraction D showed a lower level of the induction of the phagocytic activity of PM and was inactive in the manifestation of cell-mediated immunity, but induced a high level of humoral immunity. Antigenic complexes C1 and H increased the phagocytic activity of PM and DH characteristics with a low level of antibody production. The studied fractions of the causative agent of melioidosis decreased the content of bactericidal cationic proteins (BCP) in rat blood neutrophils, and in mice a decreased content of BCP in phagocytes was registered. The fractions increased the activity of myeloperoxidase in blood neutrophils in mice and rats. As revealed with the use of immunoelectrophoresis, SDS PAAG electrophoresis and immunoblotting, the surface antigenic complex contained proteins of 18, 22, 39 kD and glycoproteins 42, 55, 90 kD. The latter glycoprotein was found in all the fractions under study, having protective properties. PMID:15554321

  16. 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.

    PubMed

    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

  17. 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

    PubMed Central

    Adler, Natalie R. Lazar; 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

  18. Correlation between biofilm production, antibiotic susceptibility and exopolysaccharide composition in Burkholderia pseudomallei bpsI, ppk, and rpoS mutant strains.

    PubMed

    Mongkolrob, Rungrawee; Taweechaisupapong, Suwimol; Tungpradabkul, Sumalee

    2015-11-01

    Burkholderia pseudomallei is the cause of melioidosis, a fatal tropical infectious disease, which has been reported to have a high rate of recurrence, even when an intensive dose of antibiotics is used. Biofilm formation is believed to be one of the possible causes of relapse because of its ability to increase drug resistance. EPS in biofilms have been reported to be related to the limitation of antibiotic penetration in B. pseudomallei. However, the mechanisms by which biofilms restrict the diffusion of antibiotics remain unclear. The present study presents a correlation between exopolysaccharide production in biofilm matrix and antibiotic resistance in B. pseudomallei using bpsI, ppk, and rpoS mutant strains. CLSM revealed a reduction in exopolysaccharide production and disabled micro-colony formation in B. pseudomallei mutants, which paralleled the antibiotic resistance. Different ratios of carbohydrate contents in the exopolysaccharides of the mutants were detected, although they have the same components, including glucose, galactose, mannose, and rhamnose, with the exception being that no detectable rhamnose peak was observed in the bpsI mutant. These results indicate that the correlation between these phenomena in the B. pseudomallei biofilm at least results from the exopolysaccharide, which may be under the regulation of bpsI, ppk, or rpoS genes. PMID:26486518

  19. Mapping epigenetic changes to the host cell genome induced by Burkholderia pseudomallei reveals pathogen-specific and pathogen-generic signatures of infection.

    PubMed

    Cizmeci, Deniz; Dempster, Emma L; Champion, Olivia L; Wagley, Sariqa; Akman, Ozgur E; Prior, Joann L; Soyer, Orkun S; Mill, Jonathan; Titball, Richard W

    2016-01-01

    The potential for epigenetic changes in host cells following microbial infection has been widely suggested, but few examples have been reported. We assessed genome-wide patterns of DNA methylation in human macrophage-like U937 cells following infection with Burkholderia pseudomallei, an intracellular bacterial pathogen and the causative agent of human melioidosis. Our analyses revealed significant changes in host cell DNA methylation, at multiple CpG sites in the host cell genome, following infection. Infection induced differentially methylated probes (iDMPs) showing the greatest changes in DNA methylation were found to be in the vicinity of genes involved in inflammatory responses, intracellular signalling, apoptosis and pathogen-induced signalling. A comparison of our data with reported methylome changes in cells infected with M. tuberculosis revealed commonality of differentially methylated genes, including genes involved in T cell responses (BCL11B, FOXO1, KIF13B, PAWR, SOX4, SYK), actin cytoskeleton organisation (ACTR3, CDC42BPA, DTNBP1, FERMT2, PRKCZ, RAC1), and cytokine production (FOXP1, IRF8, MR1). Overall our findings show that pathogenic-specific and pathogen-common changes in the methylome occur following infection. PMID:27484700

  20. Flexible vs Rigid Epitope Conformations for Diagnostic- and Vaccine-Oriented Applications: Novel Insights from the Burkholderia pseudomallei BPSL2765 Pal3 Epitope.

    PubMed

    Gori, Alessandro; Peri, Claudio; Quilici, Giacomo; Nithichanon, Arnone; Gaudesi, Davide; Longhi, Renato; Gourlay, Louise; Bolognesi, Martino; Lertmemongkolchai, Ganjana; Musco, Giovanna; Colombo, Giorgio

    2016-03-11

    Peptides seldom retain stable conformations if separated from their native protein structure. In an immunological context, this potentially affects the development of selective peptide-based bioprobes and, from a vaccine perspective, poses inherent limits in the elicitation of cross-reactive antibodies by candidate epitopes. Here, a 1,4-disubstituted-1,2,3-triazole-mediated stapling strategy was used to stabilize the native α-helical fold of the Pal3 peptidic epitope from the protein antigen PalBp (BPSL2765) from Burkholderia pseudomallei, the etiological agent of melioidosis. Whereas Pal3 shows no propensity to fold outside its native protein context, the engineered peptide (Pal3H) forms a stable α-helix, as assessed by MD, NMR, and CD structural analyses. Importantly, Pal3H shows an enhanced ability to discriminate between melioidosis patient subclasses in immune sera reactivity tests, demonstrating the potential of the stapled peptide for diagnostic purposes. With regard to antibody elicitation and related bactericidal activities, the linear peptide is shown to elicit a higher response. On these bases, we critically discuss the implications of epitope structure engineering for diagnostic- and vaccine-oriented applications. PMID:27623032

  1. Mapping epigenetic changes to the host cell genome induced by Burkholderia pseudomallei reveals pathogen-specific and pathogen-generic signatures of infection

    PubMed Central

    Cizmeci, Deniz; Dempster, Emma L.; Champion, Olivia L.; Wagley, Sariqa; Akman, Ozgur E.; Prior, Joann L.; Soyer, Orkun S.; Mill, Jonathan; Titball, Richard W.

    2016-01-01

    The potential for epigenetic changes in host cells following microbial infection has been widely suggested, but few examples have been reported. We assessed genome-wide patterns of DNA methylation in human macrophage-like U937 cells following infection with Burkholderia pseudomallei, an intracellular bacterial pathogen and the causative agent of human melioidosis. Our analyses revealed significant changes in host cell DNA methylation, at multiple CpG sites in the host cell genome, following infection. Infection induced differentially methylated probes (iDMPs) showing the greatest changes in DNA methylation were found to be in the vicinity of genes involved in inflammatory responses, intracellular signalling, apoptosis and pathogen-induced signalling. A comparison of our data with reported methylome changes in cells infected with M. tuberculosis revealed commonality of differentially methylated genes, including genes involved in T cell responses (BCL11B, FOXO1, KIF13B, PAWR, SOX4, SYK), actin cytoskeleton organisation (ACTR3, CDC42BPA, DTNBP1, FERMT2, PRKCZ, RAC1), and cytokine production (FOXP1, IRF8, MR1). Overall our findings show that pathogenic-specific and pathogen-common changes in the methylome occur following infection. PMID:27484700

  2. Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: isolation of motility mutants and molecular characterization of the flagellin structural gene.

    PubMed Central

    DeShazer, D; Brett, P J; Carlyon, R; Woods, D E

    1997-01-01

    Burkholderia pseudomallei is a human and animal pathogen in tropical regions, especially Southeast Asia and northern Australia. Currently little is known about the genetics and molecular biology of this organism. In this report, we describe the mutagenesis of B. pseudomallei with the transposon Tn5-OT182. B. pseudomallei 1026b transposon mutants were obtained at a frequency of 4.6 x 10(-4) per initial donor cell, and the transposon inserted randomly into the chromosome. We used Tn5-OT182 to identify the flagellin structural gene, fliC. We screened 3,500 transposon mutants and identified 28 motility mutants. Tn5-OT182 integrated into 19 unique genetic loci encoding proteins with homology to Escherichia coli and Salmonella typhimurium flagellar and chemotaxis proteins. Two mutants, MM35 and MM36, contained Tn5-OT182 integrations in fliC. We cloned and sequenced fliC and used it to complement MM35 and MM36 in trans. The fliC transcriptional start site and a sigmaF-like promoter were identified by primer extension analysis. We observed a significant difference in the expression of two distinct fliC-lacZ transcriptional fusions during bacterial growth, suggesting the presence of a latent intragenic transcriptional terminator in fliC. There was no significant difference in the virulence of 1026b compared to that of MM36 in diabetic rats or Syrian hamsters, suggesting that flagella and/or motility are probably not virulence determinants in these animal models of B. pseudomallei infection. A phylogenetic analysis based on the flagellins from a variety of bacterial species supported the recent transfer of B. pseudomallei from the genus Pseudomonas to Burkholderia. PMID:9079894

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

    PubMed

    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. PMID:27091931

  4. Structural characterization of the Ser324Thr variant of the catalase-peroxidase (KatG) from Burkholderia pseudomallei.

    PubMed

    Deemagarn, Taweewat; Carpena, Xavier; Singh, Rahul; Wiseman, Ben; Fita, Ignacio; Loewen, Peter C

    2005-01-01

    The Ser315Thr variant of the catalase-peroxidase KatG from Mycobacterium tuberculosis imparts resistance to the pro-drug isonicotinic acid hydrazide (isoniazid) through a failure to convert it to the active drug, isonicotinoyl-NAD. The equivalent variant in KatG from Burkholderia pseudomallei, Ser324Thr, has been constructed, revealing catalase and peroxidase activities that are similar to those of the native enzyme. The other activities of the variant protein, including the NADH oxidase, the isoniazid hydrazinolysis and isonicotinoyl-NAD synthase activities are reduced by 60-70%. The crystal structure of the variant differs from that of the native enzyme in having the methyl group of Thr324 situated in the entrance channel to the heme cavity, in a modified water matrix in the entrance channel and heme cavity, in lacking the putative perhydroxy modification on the heme, in the multiple locations of a few side-chains, and in the presence of an apparent perhydroxy modification on the indole nitrogen atom of the active-site Trp111. The position of the methyl group of Thr324 creates a constriction or narrowing of the channel leading to the heme cavity, providing an explanation for the lower reactivity towards isoniazid and the slower rate of isonicotinoyl-NAD synthesis. PMID:15567407

  5. Distribution of immunoglobulin classes and IgG subclasses against a culture filtrate antigen of Burkholderia pseudomallei in melioidosis patients.

    PubMed

    Chenthamarakshan, V; Kumutha, M V; Vadivelu, J; Puthucheary, S D

    2001-01-01

    The class and subclass distribution of antibody response to the culture filtrate antigen (CFA) of Burkholderia pseudomallei was examined in the sera of 45 septicaemic and 17 localised melioidosis cases and 40 cases clinically suspected of melioidosis and the results were compared with those from high-risk and healthy control groups. The geometric mean titre index (GMTI) values for all classes and subclasses of immunoglobulins examined were higher for sera from the proven and clinically suspected melioidosis cases than for the control groups. However, the highest response in the three patient groups was that of IgG with GMTIs ranging from 219.4 to 291.6 and the lowest was for IgM with GMTIs of 22.5, 24.3 and 28.7. The IgA response was intermediate with GMTIs ranging from 119.2 to 170. The GMTIs were highest for IgG in septicaemic and localised infections and for IgA and IgM in localised infections. As regards IgG subclass distribution, IgG1 and IgG2 were the predominant subclasses produced against the CFA in contrast to IgG3 and IgG4, which were produced in low amounts. None of the sera from the control groups had any significant titres of antibodies. PMID:11192506

  6. Redefining the PF06864 Pfam Family Based on Burkholderia pseudomallei PilO2Bp S-SAD Crystal Structure

    PubMed Central

    Manjasetty, Babu A.; Yero, Daniel; Perletti, Lucia; Belrhali, Hassan; Daura, Xavier; Gourlay, Louise J.; Bolognesi, Martino

    2014-01-01

    Type IV pili are surface-exposed filaments and bacterial virulence factors, represented by the Tfpa and Tfpb types, which assemble via specific machineries. The Tfpb group is further divided into seven variants, linked to heterogeneity in the assembly machineries. Here we focus on PilO2Bp, a protein component of the Tfpb R64 thin pilus variant assembly machinery from the pathogen Burkholderia pseudomallei. PilO2Bp belongs to the PF06864 Pfam family, for which an improved definition is presented based on newly derived Hidden Markov Model (HMM) profiles. The 3D structure of the N-terminal domain of PilO2Bp (N-PilO2Bp), here reported, is the first structural representative of the PF06864 family. N-PilO2Bp presents an actin-like ATPase fold that is shown to be present in BfpC, a different variant assembly protein; the new HMM profiles classify BfpC as a PF06864 member. Our results provide structural insight into the PF06864 family and on the Type IV pili assembly machinery. PMID:24728008

  7. Genome-wide prediction and annotation of Burkholderia pseudomallei AraC/XylS family transcription regulator.

    PubMed

    Lim, Boon-San; Chong, Chan-Eng; Zamrod, Zulkeflie; Nathan, Sheila; Mohamed, Rahmah

    2007-01-01

    Many members of the AraC/XylS family transcription regulator have been proven to play a critical role in regulating bacterial virulence factors in response to environmental stress. By using the Hidden Markov Model (HMM) profile built from the alignment of a 99 amino acid conserved domain sequence of 273 AraC/XylS family transcription regulators, we detected a total of 45 AraC/XylS family transcription regulators in the genome of the Gram-negative pathogen, Burkholderia pseudomallei. Further in silico analysis of each detected AraC/XylS family transcription regulatory protein and its neighboring genes allowed us to make a first-order guess on the role of some of these transcription regulators in regulating important virulence factors such as those involved in three type III secretion systems and biosynthesis of pyochelin, exopolysaccharide (EPS) and phospholipase C. This paper has demonstrated an efficient and systematic genome-wide scale prediction of the AraC/XylS family that can be applied to other protein families. PMID:18391231

  8. Detection of Burkholderia pseudomallei in Sputum using Selective Enrichment Broth and Ashdown’s Medium at Kampong Cham Provincial Hospital, Cambodia

    PubMed Central

    Nhem, Somary; Letchford, Joanne; Meas, Chea; Thann, Sovanndeth; McLaughlin, James C.; Baron, Ellen Jo; West, T. Eoin

    2015-01-01

    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. Given the low numbers of positive specimens, we could not conclusively determine the utility of the enhanced sputum testing protocol. However, the ramifications of identification of  B. pseudomallei are substantial, and the benefit of the enhanced testing protocol may be more apparent in patients selected based on risk factors and clinical presentation. Promoting clinician awareness of the infection and encouraging utilization of diagnostic microbiology services are also likely to be important factors in facilitating identification of melioidosis. PMID:25717370

  9. Glyphosate Resistance as a Novel Select-Agent-Compliant, Non-Antibiotic-Selectable Marker in Chromosomal Mutagenesis of the Essential Genes asd and dapB of Burkholderia pseudomallei▿

    PubMed Central

    Norris, Michael H.; Kang, Yun; Lu, Diana; Wilcox, Bruce A.; Hoang, Tung T.

    2009-01-01

    Genetic manipulation of the category B select agents Burkholderia pseudomallei and Burkholderia mallei has been stifled due to the lack of compliant selectable markers. Hence, there is a need for additional select-agent-compliant selectable markers. We engineered a selectable marker based on the gat gene (encoding glyphosate acetyltransferase), which confers resistance to the common herbicide glyphosate (GS). To show the ability of GS to inhibit bacterial growth, we determined the effective concentrations of GS against Escherichia coli and several Burkholderia species. Plasmids based on gat, flanked by unique flip recombination target (FRT) sequences, were constructed for allelic-replacement. Both allelic-replacement approaches, one using the counterselectable marker pheS and the gat-FRT cassette and one using the DNA incubation method with the gat-FRT cassette, were successfully utilized to create deletions in the asd and dapB genes of wild-type B. pseudomallei strains. The asd and dapB genes encode an aspartate-semialdehyde dehydrogenase (BPSS1704, chromosome 2) and dihydrodipicolinate reductase (BPSL2941, chromosome 1), respectively. Mutants unable to grow on media without diaminopimelate (DAP) and other amino acids of this pathway were PCR verified. These mutants displayed cellular morphologies consistent with the inability to cross-link peptidoglycan in the absence of DAP. The B. pseudomallei 1026b Δasd::gat-FRT mutant was complemented with the B. pseudomallei asd gene on a site-specific transposon, mini-Tn7-bar, by selecting for the bar gene (encoding bialaphos/PPT resistance) with PPT. We conclude that the gat gene is one of very few appropriate, effective, and beneficial compliant markers available for Burkholderia select-agent species. Together with the bar gene, the gat cassette will facilitate various genetic manipulations of Burkholderia select-agent species. PMID:19648360

  10. Backbone chemical shift assignments for the sensor domain of the Burkholderia pseudomallei histidine kinase RisS: "missing" resonances at the dimer interface.

    PubMed

    Buchko, Garry W; Edwards, Thomas E; Hewitt, Stephen N; Phan, Isabelle Q H; Van Voorhis, Wesley C; Miller, Samuel I; Myler, Peter J

    2015-10-01

    Using a deuterated sample, all the observable backbone (1)H(N), (15)N, (13)C(a), and (13)C' chemical shifts for the dimeric, periplasmic sensor domain of the Burkholderia pseudomallei histidine kinase RisS were assigned. Approximately one-fifth of the amide resonances are "missing" in the (1)H-(15)N HSQC spectrum and map primarily onto α-helices at the dimer interface observed in a crystal structure suggesting this region either undergoes intermediate timescale motion (μs-ms) and/or is heterogeneous. PMID:25957069

  11. Unprecedented Melioidosis Cases in Northern Australia Caused by an Asian Burkholderia pseudomallei Strain Identified by Using Large-Scale Comparative Genomics

    PubMed Central

    Smith, Emma J.; MacHunter, Barbara; Harrington, Glenda; Theobald, Vanessa; Hall, Carina M.; Hornstra, Heidie M.; McRobb, Evan; Podin, Yuwana; Mayo, Mark; Sahl, Jason W.; Wagner, David M.; Keim, Paul; Kaestli, Mirjam; Currie, Bart J.

    2015-01-01

    Melioidosis is a disease of humans and animals that is caused by the saprophytic bacterium Burkholderia pseudomallei. Once thought to be confined to certain locations, the known presence of B. pseudomallei is expanding as more regions of endemicity are uncovered. There is no vaccine for melioidosis, and even with antibiotic administration, the mortality rate is as high as 40% in some regions that are endemic for the infection. Despite high levels of recombination, phylogenetic reconstruction of B. pseudomallei populations using whole-genome sequencing (WGS) has revealed surprisingly robust biogeographic separation between isolates from Australia and Asia. To date, there have been no confirmed autochthonous melioidosis cases in Australia caused by an Asian isolate; likewise, no autochthonous cases in Asia have been identified as Australian in origin. Here, we used comparative genomic analysis of 455 B. pseudomallei genomes to confirm the unprecedented presence of an Asian clone, sequence type 562 (ST-562), in Darwin, northern Australia. First observed in Darwin in 2005, the incidence of melioidosis cases attributable to ST-562 infection has steadily risen, and it is now a common strain in Darwin. Intriguingly, the Australian ST-562 appears to be geographically restricted to a single locale and is genetically less diverse than other common STs from this region, indicating a recent introduction of this clone into northern Australia. Detailed genomic and epidemiological investigations of new clinical and environmental B. pseudomallei isolates in the Darwin region and ST-562 isolates from Asia will be critical for understanding the origin, distribution, and dissemination of this emerging clone in northern Australia. PMID:26607593

  12. AFN-1252 is a potent inhibitor of enoyl-ACP reductase from Burkholderia pseudomallei--Crystal structure, mode of action, and biological activity.

    PubMed

    Rao, Krishnamurthy Narasimha; Lakshminarasimhan, Anirudha; Joseph, Sarah; Lekshmi, Swathi U; Lau, Ming-Seong; Takhi, Mohammed; Sreenivas, Kandepu; Nathan, Sheila; Yusof, Rohana; Abd Rahman, Noorsaadah; Ramachandra, Murali; Antony, Thomas; Subramanya, Hosahalli

    2015-05-01

    Melioidosis is a tropical bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei; Bpm), a Gram-negative bacterium. Current therapeutic options are largely limited to trimethoprim-sulfamethoxazole and β-lactam drugs, and the treatment duration is about 4 months. Moreover, resistance has been reported to these drugs. Hence, there is a pressing need to develop new antibiotics for Melioidosis. Inhibition of enoyl-ACP reducatase (FabI), a key enzyme in the fatty acid biosynthesis pathway has shown significant promise for antibacterial drug development. FabI has been identified as the major enoyl-ACP reductase present in B. pseudomallei. In this study, we evaluated AFN-1252, a Staphylococcus aureus FabI inhibitor currently in clinical development, for its potential to bind to BpmFabI enzyme and inhibit B. pseudomallei bacterial growth. AFN-1252 stabilized BpmFabI and inhibited the enzyme activity with an IC50 of 9.6 nM. It showed good antibacterial activity against B. pseudomallei R15 strain, isolated from a melioidosis patient (MIC of 2.35 mg/L). X-ray structure of BpmFabI with AFN-1252 was determined at a resolution of 2.3 Å. Complex of BpmFabI with AFN-1252 formed a symmetrical tetrameric structure with one molecule of AFN-1252 bound to each monomeric subunit. The kinetic and thermal melting studies supported the finding that AFN-1252 can bind to BpmFabI independent of cofactor. The structural and mechanistic insights from these studies might help the rational design and development of new FabI inhibitors. PMID:25644789

  13. Unprecedented Melioidosis Cases in Northern Australia Caused by an Asian Burkholderia pseudomallei Strain Identified by Using Large-Scale Comparative Genomics.

    PubMed

    Price, Erin P; Sarovich, Derek S; Smith, Emma J; MacHunter, Barbara; Harrington, Glenda; Theobald, Vanessa; Hall, Carina M; Hornstra, Heidie M; McRobb, Evan; Podin, Yuwana; Mayo, Mark; Sahl, Jason W; Wagner, David M; Keim, Paul; Kaestli, Mirjam; Currie, Bart J

    2016-02-01

    Melioidosis is a disease of humans and animals that is caused by the saprophytic bacterium Burkholderia pseudomallei. Once thought to be confined to certain locations, the known presence of B. pseudomallei is expanding as more regions of endemicity are uncovered. There is no vaccine for melioidosis, and even with antibiotic administration, the mortality rate is as high as 40% in some regions that are endemic for the infection. Despite high levels of recombination, phylogenetic reconstruction of B. pseudomallei populations using whole-genome sequencing (WGS) has revealed surprisingly robust biogeographic separation between isolates from Australia and Asia. To date, there have been no confirmed autochthonous melioidosis cases in Australia caused by an Asian isolate; likewise, no autochthonous cases in Asia have been identified as Australian in origin. Here, we used comparative genomic analysis of 455 B. pseudomallei genomes to confirm the unprecedented presence of an Asian clone, sequence type 562 (ST-562), in Darwin, northern Australia. First observed in Darwin in 2005, the incidence of melioidosis cases attributable to ST-562 infection has steadily risen, and it is now a common strain in Darwin. Intriguingly, the Australian ST-562 appears to be geographically restricted to a single locale and is genetically less diverse than other common STs from this region, indicating a recent introduction of this clone into northern Australia. Detailed genomic and epidemiological investigations of new clinical and environmental B. pseudomallei isolates in the Darwin region and ST-562 isolates from Asia will be critical for understanding the origin, distribution, and dissemination of this emerging clone in northern Australia. PMID:26607593

  14. A high-content imaging assay for the quantification of the Burkholderia pseudomallei induced multinucleated giant cell (MNGC) phenotype in murine macrophages

    PubMed Central

    2014-01-01

    Background Burkholderia pseudomallei (Bp), a Gram-negative, motile, facultative intracellular bacterium is the causative agent of melioidosis in humans and animals. The Bp genome encodes a repertoire of virulence factors, including the cluster 3 type III secretion system (T3SS-3), the cluster 1 type VI secretion system (T6SS-1), and the intracellular motility protein BimA, that enable the pathogen to invade both phagocytic and non-phagocytic cells. A unique hallmark of Bp infection both in vitro and in vivo is its ability to induce cell-to-cell fusion of macrophages to form multinucleated giant cells (MNGCs), which to date are semi-quantitatively reported following visual inspection. Results In this study we report the development of an automated high-content image acquisition and analysis assay to quantitate the Bp induced MNGC phenotype. Validation of the assay was performed using T6SS-1 (∆hcp1) and T3SS-3 (∆bsaZ) mutants of Bp that have been previously reported to exhibit defects in their ability to induce MNGCs. Finally, screening of a focused small molecule library identified several Histone Deacetylase (HDAC) inhibitors that inhibited Bp-induced MNGC formation of macrophages. Conclusions We have successfully developed an automated HCI assay to quantitate MNGCs induced by Bp in macrophages. This assay was then used to characterize the phenotype of the Bp mutants for their ability to induce MNGC formation and identify small molecules that interfere with this process. Successful application of chemical genetics and functional reverse genetics siRNA approaches in the MNGC assay will help gain a better understanding of the molecular targets and cellular mechanisms responsible for the MNGC phenotype induced by Bp, by other bacteria such as Mycobacterium tuberculosis, or by exogenously added cytokines. PMID:24750902

  15. 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)

    PubMed Central

    Srisanga, Kitima; Roytrakul, Sittiruk; Tungpradabkul, Sumalee

    2014-01-01

    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. PMID:24914956

  16. Contribution of the BacT/Alert MB Mycobacterium Bottle to Bloodstream Infection Surveillance in Thailand: Added Yield for Burkholderia pseudomallei

    PubMed Central

    Higdon, Melissa; Kaewpan, Anek; Makprasert, Sirirat; Yuenprakhon, Somkhit; Tawisaid, Kittisak; Dejsirilert, Surang; Whistler, Toni; Baggett, Henry C.

    2015-01-01

    Community-acquired bloodstream infections cause substantial morbidity and mortality worldwide, but microbiology capacity and surveillance limitations have challenged good descriptions of pathogen distribution in many regions, including Southeast Asia. Active surveillance for bloodstream infections has been conducted in two rural Thailand provinces for >7 years. Blood specimens were divided into two culture bottles, one optimized for aerobic growth (F bottle) and a second for enhanced growth of mycobacteria (MB bottle), and processed with the BactT/Alert 3D system. Because the routine use of MB culture bottles is resource intensive (expensive and requires prolonged incubation), we assessed the added yield of MB bottles by comparing the proportion of pathogens detected by MB versus that by F bottles from 2005 to 2012. Of 63,066 blood cultures, 7,296 (12%) were positive for at least one pathogen; the most common pathogens were Escherichia coli (28%), Burkholderia pseudomallei (11%), Klebsiella pneumoniae (9%), and Staphylococcus aureus (6%). Two bottles improved the yield overall, but the added yield attributable to the MB bottles was limited to a few pathogens. In addition to the detection of mycobacteria and some fungi, MB bottles improved the detection of B. pseudomallei (27% [MB] versus 8% [F]; P < 0.0001), with added benefit if therapy was initiated prior to the blood culture. The targeted use of MB bottles is warranted for patients at risk for mycobacterial and fungal infections and for infection with B. pseudomallei, a common cause of septicemia in Thailand. PMID:25588650

  17. Burkholderia vaccines: are we moving forward?

    PubMed Central

    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. PMID:23386999

  18. Evaluation of the Bruker Biotyper Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry System for Identification of Clinical and Environmental Isolates of Burkholderia pseudomallei

    PubMed Central

    Wang, He; Chen, Ya-Lei; Teng, Shih-Hua; Xu, Zhi-Peng; Xu, Ying-Chun; Hsueh, Po-Ren

    2016-01-01

    Burkholderia pseudomallei is not represented in the current version of Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) system. A total of 66 isolates of B. pseudomallei, including 30 clinical isolates collected from National Taiwan University Hospital (NTUH, n = 27) and Peking Union Medical College Hospital (PUMCH, n = 3), and 36 isolates of genetically confirmed strains, including 13 from clinical samples and 23 from environmental samples, collected from southern Taiwan were included in this study. All these isolates were identified by partial 16S rDNA gene sequencing analysis and the Bruker Biotyper MALDI-TOF MS system. Among the 30 isolates initially identified as B. pseudomallei by conventional identification methods, one was identified as B. cepacia complex (NTUH) and three were identified as B. putida (PUMCH) by partial 16S rDNA gene sequencing analysis and Bruker Biotyper MALDI-TOF MS system. The Bruker Biotyper MALDI-TOF MS system misidentified 62 genetically confirmed B. pseudomallei isolates as B. thailandensis or Burkholderia species (score values, 1.803–2.063) when the currently available database (DB 5627) was used. However, using a newly created MALDI-TOF MS database (including B. pseudomallei NTUH-3 strain), all isolates were correctly identified as B. pseudomallei (score values >2.000, 100%). An additional 60 isolates of genetically confirmed B. cepacia complex and B. putida were also evaluated by the Bruker Biotyper MALDI-TOF MS system using the newly created database and none of these isolates were identified as B. pseudomallei. MALDI-TOF MS is a versatile and robust tool for the rapid identification of B. pseudomallei using the enhanced database. PMID:27092108

  19. Spectroscopic and kinetic investigation of the reactions of peroxyacetic acid with Burkholderia pseudomallei catalase-peroxidase, KatG.

    PubMed

    Ivancich, Anabella; Donald, Lynda J; Villanueva, Jacylyn; Wiseman, Ben; Fita, Ignacio; Loewen, Peter C

    2013-10-15

    Catalase-peroxidases or KatGs can utilize organic peroxyacids and peroxides instead of hydrogen peroxide to generate the high-valent ferryl-oxo intermediates involved in the catalase and peroxidase reactions. In the absence of peroxidatic one-electron donors, the ferryl intermediates generated with a low excess (10-fold) of peroxyacetic acid (PAA) slowly decay to the ferric resting state after several minutes, a reaction that is demonstrated in this work by both stopped-flow UV-vis absorption measurements and EPR spectroscopic characterization of Burkholderia pseudomallei KatG (BpKatG). EPR spectroscopy showed that the [Fe(IV)═O Trp330(•+)], [Fe(IV)═O Trp139(•)], and [Fe(IV)═O Trp153(•)] intermediates of the peroxidase-like cycle of BpKatG ( Colin, J. Wiseman, B. Switala, J. Loewen, P. C. Ivancich, A. ( 2009 ) J. Am. Chem. Soc. 131 , 8557 - 8563 ), formed with a low excess of PAA at low temperature, are also generated with a high excess (1000-fold) of PAA at room temperature. However, under high excess conditions, there is a rapid conversion to a persistent [Fe(IV)═O] intermediate. Analysis of tryptic peptides of BpKatG by mass spectrometry before and after treatment with PAA showed that specific tryptophan (including W330, W139, and W153), methionine (including Met264 of the M-Y-W adduct), and cysteine residues are either modified with one, two, or three oxygen atoms or could not be identified in the spectrum because of other undetermined modifications. It was concluded that these oxidized residues were the source of electrons used to reduce the excess of PAA to acetic acid and return the enzyme to the ferric state. Treatment of BpKatG with PAA also caused a loss of catalase activity towards certain substrates, consistent with oxidative disruption of the M-Y-W adduct, and a loss of peroxidase activity, consistent with accumulation of the [Fe(IV)═O] intermediate and the oxidative modification of the W330, W139, and W153. PAA, but not H2O2 or tert

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

    PubMed Central

    Bowers, Jolene R.; Engelthaler, David M.; Ginther, Jennifer L.; Pearson, Talima; Peacock, Sharon J.; Tuanyok, Apichai; 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 robust, highly validated assay for the detection and differentiation of B. pseudomallei and B. mallei. PMID:21103048

  1. Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063.

    PubMed

    Gourlay, Louise J; Peano, Clelia; Deantonio, Cecilia; Perletti, Lucia; Pietrelli, Alessandro; Villa, Riccardo; Matterazzo, Elena; Lassaux, Patricia; Santoro, Claudio; Puccio, Simone; Sblattero, Daniele; Bolognesi, Martino

    2015-11-01

    The 1.8 Å resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using β-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography. PMID:26527140

  2. Use of the phytopathogenic effect for studies of Burkholderia virulence.

    PubMed

    Molchanova, E V; Ageeva, N P

    2015-02-01

    The phytopathogenic effect of the pseudomallei group Burkholderia is demonstrated on the Peireskia aculeata model. A method for evaluation of the effect is suggested. The effect correlates with the levels of Burkholderia pseudomallei, Burkholderia mallei, and Burkholderia thailandensis virulence for laboratory animals. P. aculeata can be used as a model for preliminary studies of the virulence of the above species. PMID:25705037

  3. The Burkholderia pseudomallei Enoyl-Acyl Carrier Protein Reductase FabI1 Is Essential for In Vivo Growth and Is the Target of a Novel Chemotherapeutic with Efficacy

    PubMed Central

    Cummings, Jason E.; Kingry, Luke C.; Rholl, Drew A.; Schweizer, Herbert P.

    2014-01-01

    The bacterial fatty acid biosynthesis pathway is a validated target for the development of novel chemotherapeutics. However, since Burkholderia pseudomallei carries genes that encode both FabI and FabV enoyl-acyl carrier protein (ACP) reductase homologues, the enoyl-ACP reductase that is essential for in vivo growth needs to be defined so that the correct drug target can be chosen for development. Accordingly, ΔfabI1, ΔfabI2, and ΔfabV knockout strains were constructed and tested in a mouse model of infection. Mice infected with a ΔfabI1 strain did not show signs of morbidity, mortality, or dissemination after 30 days of infection compared to the wild-type and ΔfabI2 and ΔfabV mutant strains that had times to mortality of 60 to 84 h. Although signs of morbidity and mortality of ΔfabI2 and ΔfabV strains were not significantly different from those of the wild-type strain, a slight delay was observed. A FabI1-specific inhibitor was used to confirm that inhibition of FabI1 results in reduced bacterial burden and efficacy in an acute B. pseudomallei murine model of infection. This work establishes that FabI1 is required for growth of Burkholderia pseudomallei in vivo and is a potential molecular target for drug development. PMID:24277048

  4. Expression, purification, crystallization and preliminary crystallographic analysis of BipD, a component of the Burkholderia pseudomallei type III secretion system

    SciTech Connect

    Roversi, Pietro; Johnson, Steven; Field, Terry; Deane, Janet E.; Galyov, Edouard E.; Lea, Susan M.

    2006-09-01

    A construct consisting of residues 10–310 of mature BipD, a component of the B. pseudomallei type III secretion system, has been crystallized. Native BipD crystals and SeMet and K{sub 2}PtCl{sub 4} derivative crystals have undergone preliminary crystallographic analysis. A construct consisting of residues 10–310 of BipD, a component of the Burkholderia pseudomallei type III secretion system (T3SS), has been overexpressed as a GST fusion, cleaved from the GST tag and purified. Crystals were grown of native and selenomethionine-labelled BipD. The crystals grow in two different polymorphs from the same condition. The first polymorph belongs to space group C222, with unit-cell parameters a = 103.98, b = 122.79, c = 49.17 Å, a calculated Matthews coefficient of 2.4 Å{sup 3} Da{sup −1} (47% solvent content) and one molecule per asymmetric unit. The second polymorph belongs to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 136.47, b = 89.84, c = 50.15 Å, and a calculated Matthews coefficient of 2.3 Å{sup 3} Da{sup −1} (45% solvent content) for two molecules per asymmetric unit (analysis of the self-rotation function indicates the presence of a weak twofold non-crystallographic symmetry axis in this P2{sub 1}2{sub 1}2 form). The native crystals of both forms give diffraction data to 2.7 Å resolution, while the SeMet-labelled P2{sub 1}2{sub 1}2 crystals diffract to 3.3 Å resolution. A K{sub 2}PtCl{sub 4} derivative of the P2{sub 1}2{sub 1}2 form was also obtained and data were collected to 2.7 Å with radiation of wavelength λ = 0.933 Å. The Pt-derivative anomalous difference Patterson map revealed two self-peaks on the Harker sections.

  5. Recombinant Salmonella Expressing Burkholderia mallei LPS O Antigen Provides Protection in a Murine Model of Melioidosis and Glanders

    PubMed Central

    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. PMID:26148026

  6. Expression, purification, crystallization and preliminary crystallographic analysis of BipD, a component of the Burkholderia pseudomallei type III secretion system

    PubMed Central

    Roversi, Pietro; Johnson, Steven; Field, Terry; Deane, Janet E.; Galyov, Edouard E.; Lea, Susan M.

    2006-01-01

    A construct consisting of residues 10–310 of BipD, a component of the Burkholderia pseudomallei type III secretion system (T3SS), has been overexpressed as a GST fusion, cleaved from the GST tag and purified. Crystals were grown of native and selenomethionine-labelled BipD. The crystals grow in two different polymorphs from the same condition. The first polymorph belongs to space group C222, with unit-cell parameters a = 103.98, b = 122.79, c = 49.17 Å, a calculated Matthews coefficient of 2.4 Å3 Da−1 (47% solvent content) and one molecule per asymmetric unit. The second polymorph belongs to space group P21212, with unit-cell parameters a = 136.47, b = 89.84, c = 50.15 Å, and a calculated Matthews coefficient of 2.3 Å3 Da−1 (45% solvent content) for two molecules per asymmetric unit (analysis of the self-rotation function indicates the presence of a weak twofold non-crystallographic symmetry axis in this P21212 form). The native crystals of both forms give diffraction data to 2.7 Å resolution, while the SeMet-labelled P21212 crystals diffract to 3.3 Å resolution. A K2PtCl4 derivative of the P21212 form was also obtained and data were collected to 2.7 Å with radiation of wavelength λ = 0.933 Å. The Pt-derivative anomalous difference Patterson map revealed two self-peaks on the Harker sections. PMID:16946464

  7. Identification of CD4+ T-cell epitope and investigation of HLA distribution for the immunogenic proteins of Burkholderia pseudomallei using in silico approaches - A key vaccine development strategy for melioidosis.

    PubMed

    Swetha, Rayapadi G; Sandhya, Madangopal; Ramaiah, Sudha; Anbarasu, Anand

    2016-07-01

    Melioidosis is a serious infectious diseases affecting multi-organ system in humans with high mortality rate. The disease is caused by the bacterium, Burkholderia pseudomallei and it is intrinsically resistant to many antibiotics. Thus, there is an urgent need for protective vaccine against B. pseudomallei; which may reduce morbidity and mortality in endemic areas. The identification of peptides that bind to major histocompatibility complex II class helps in understanding the nature of immune response and identifying T-cell epitopes for the design of new vaccines. Previous studies indicate that, ompA, bipB, fliC and groEL proteins of B. pseudomallei stimulate CD4+ T-cell immune response and act as protective immunogens. However, the data for CD4+ T-cell epitopes of these immunogenic proteins are very limited. Hence, in this present study we attempted to identify CD4+ T-cell epitopes in B. pseudomallei immunogenic proteins using in silico approaches. We did population coverage analysis for these identified epitopic core sequences to identify individuals in endemic areas expected to respond to a given set of these epitopes on the basis of HLA genotype frequencies. We observed that eight epitopic core sequences, two from each immunogenic protein, were associated with the maximum number of HLA-DR binding alleles. These eight peptides are found to be immunogenic in more than 90% of population in endemic areas considered. Thus, these eight peptides containing epitopic core sequences may act as probable vaccine candidates and they may be considered for the development of epitope-based vaccines for melioidosis. PMID:27086038

  8. Antibacterial activity of a lectin-like Burkholderia cenocepacia protein.

    PubMed

    Ghequire, Maarten G K; De Canck, Evelien; Wattiau, Pierre; Van Winge, Iris; Loris, Remy; Coenye, Tom; De Mot, René

    2013-08-01

    Bacteriocins of the LlpA family have previously been characterized in the γ-proteobacteria Pseudomonas and Xanthomonas. These proteins are composed of two MMBL (monocot mannose-binding lectin) domains, a module predominantly and abundantly found in lectins from monocot plants. Genes encoding four different types of LlpA-like proteins were identified in genomes from strains belonging to the Burkholderia cepacia complex (Bcc) and the Burkholderia pseudomallei group. A selected recombinant LlpA-like protein from the human isolate Burkholderia cenocepacia AU1054 displayed narrow-spectrum genus-specific antibacterial activity, thus representing the first functionally characterized bacteriocin within this β-proteobacterial genus. Strain-specific killing was confined to other members of the Bcc, with mostly Burkholderia ambifaria strains being susceptible. In addition to killing planktonic cells, this bacteriocin also acted as an antibiofilm agent. PMID:23737242

  9. 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

    PubMed Central

    Becka, Scott A.; Taracila, Magdalena A.; Winkler, Marisa L.; Gatta, Julian A.; Rholl, Drew A.; Schweizer, Herbert P.

    2015-01-01

    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

  10. Regulatory role of GSK3β in the activation of NF-κB and modulation of cytokine levels in Burkholderia pseudomallei-infected PBMC isolated from streptozotocin-induced diabetic animals.

    PubMed

    Maniam, P; Nurul Aiezzah, Z; Mohamed, R; Embi, N; Hasidah, M S

    2015-03-01

    Increased susceptibility of diabetics to melioidosis, a disease caused by the Burkholderia pseudomallei bacterium is believed to be attributed to dysfunction of the innate immune system. However, the underlying mechanism of the innate susceptibility is not well-understood. Glycogen synthase kinase-3β (GSK3β) plays an important role in the innate inflammatory response caused by bacterial pathogens. The present study was conducted to investigate the effects of GSK3β inhibition by LiCl on levels of pro- and anti-inflammatory cytokines; and the activity of transcription factor NF-κB in B. pseudomallei-infected peripheral blood mononuclear cells (PBMC) derived from diabetic-induced and normal Sprague Dawley rats. In addition, the effects of LiCl on intracellular bacterial counts were also investigated. Infection of PBMC from diabetic and normal rats with B. pseudomallei resulted in elevated levels of cytokines (TNF-α, IL-12 and IL-10) and phosphorylation of NF-κB in both cell types. Intracellular bacterial counts decreased with time in both cell types during infection. However bacterial clearance was less prominent in diabetic PBMC. Burkholderia pseudomallei infection also caused inactivation (Ser9 phosphorylation) of GSK3β in normal PBMC, an effect absent in infected diabetic PBMC. Inhibition of GSK3β by LiCl lowered the levels of pro-inflammatory cytokines (TNF-α and IL-12) in both normal and diabetic PBMC. Similarly, phosphorylated NF- κB (pNF-κB) levels in both cell types were decreased with LiCl treatment. Also, LiCl was able to significantly decrease the intracellular bacterial count in normal as well as diabetic PBMC. Interestingly, the levels of anti-inflammatory cytokine IL-10 in both normal and diabetic PBMC were further elevated with GSK3β inhibition. More importantly, GSK3β in infected diabetic PBMC was inactivated as in their non-diabetic counterparts upon LiCl treatment. Taken together, our results suggest that inhibition of dysregulated GSK3

  11. Antibiotic resistance in Burkholderia species.

    PubMed

    Rhodes, Katherine A; Schweizer, Herbert P

    2016-09-01

    The genus Burkholderia comprises metabolically diverse and adaptable Gram-negative bacteria, which thrive in often adversarial environments. A few members of the genus are prominent opportunistic pathogens. These include Burkholderia mallei and Burkholderia pseudomallei of the B. pseudomallei complex, which cause glanders and melioidosis, respectively. Burkholderia cenocepacia, Burkholderia multivorans, and Burkholderia vietnamiensis belong to the Burkholderia cepacia complex and affect mostly cystic fibrosis patients. Infections caused by these bacteria are difficult to treat because of significant antibiotic resistance. The first line of defense against antimicrobials in Burkholderia species is the outer membrane penetration barrier. Most Burkholderia contain a modified lipopolysaccharide that causes intrinsic polymyxin resistance. Contributing to reduced drug penetration are restrictive porin proteins. Efflux pumps of the resistance nodulation cell division family are major players in Burkholderia multidrug resistance. Third and fourth generation β-lactam antibiotics are seminal for treatment of Burkholderia infections, but therapeutic efficacy is compromised by expression of several β-lactamases and ceftazidime target mutations. Altered DNA gyrase and dihydrofolate reductase targets cause fluoroquinolone and trimethoprim resistance, respectively. Although antibiotic resistance hampers therapy of Burkholderia infections, the characterization of resistance mechanisms lags behind other non-enteric Gram-negative pathogens, especially ESKAPE bacteria such as Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. PMID:27620956

  12. SpeI restriction enzyme displays greater discriminatory power than XbaI enzyme does in a pulsed-field gel electrophoresis study on 146 clinical Burkholderia pseudomallei isolates.

    PubMed

    Chua, Kek Heng; See, Kah Heng; Thong, Kwai Lin; Puthucheary, S D

    2011-01-01

    Restriction enzymes SpeI and XbaI were used in a pulsed-field gel electrophoresis (PFGE) study for molecular characterization of 146 clinical Burkholderia pseudomallei isolates. The PFGE parameters were optimized to enable comparable, reproducible, and robust results. The optimized parameters for both SpeI and XbaI restriction enzymes used in this study were 200 V and a pulse time of 5 to 65 s for a 28-h runtime. Using SpeI, 9 different clusters were identified, whereas 6 clusters were identified by XbaI digestion, which exhibited 85% similarity to SpeI. SpeI (discrimination index [D]=0.854) showed higher discriminatory power than XbaI did (D=0.464). PMID:21617308

  13. A Unique Set of the Burkholderia Collagen-Like Proteins Provides Insight into Pathogenesis, Genome Evolution and Niche Adaptation, and Infection Detection

    PubMed Central

    Bachert, Beth A.; Choi, Soo J.; Snyder, Anna K.; Rio, Rita V. M.; Durney, Brandon C.; Holland, Lisa A.; Amemiya, Kei; Welkos, Susan L.; Bozue, Joel A.; Cote, Christopher K.; Berisio, Rita; Lukomski, Slawomir

    2015-01-01

    Burkholderia pseudomallei and Burkholderia mallei, classified as category B priority pathogens, are significant human and animal pathogens that are highly infectious and broad-spectrum antibiotic resistant. Currently, the pathogenicity mechanisms utilized by Burkholderia are not fully understood, and correct diagnosis of B. pseudomallei and B. mallei infection remains a challenge due to limited detection methods. Here, we provide a comprehensive analysis of a set of 13 novel Burkholderia collagen-like proteins (Bucl) that were identified among B. pseudomallei and B. mallei select agents. We infer that several Bucl proteins participate in pathogenesis based on their noncollagenous domains that are associated with the components of a type III secretion apparatus and membrane transport systems. Homology modeling of the outer membrane efflux domain of Bucl8 points to a role in multi-drug resistance. We determined that bucl genes are widespread in B. pseudomallei and B. mallei; Fischer’s exact test and Cramer’s V2 values indicate that the majority of bucl genes are highly associated with these pathogenic species versus nonpathogenic B. thailandensis. We designed a bucl-based quantitative PCR assay which was able to detect B. pseudomallei infection in a mouse with a detection limit of 50 CFU. Finally, chromosomal mapping and phylogenetic analysis of bucl loci revealed considerable genomic plasticity and adaptation of Burkholderia spp. to host and environmental niches. In this study, we identified a large set of phylogenetically unrelated bucl genes commonly found in Burkholderia select agents, encoding predicted pathogenicity factors, detection targets, and vaccine candidates. PMID:26356298

  14. Complete genome sequences for 59 burkholderia isolates, both pathogenic and near neighbor.

    PubMed

    Johnson, Shannon L; Bishop-Lilly, Kimberly A; Ladner, Jason T; Daligault, Hajnalka E; Davenport, Karen W; Jaissle, James; Frey, Kenneth G; Koroleva, Galina I; Bruce, David C; Coyne, Susan R; Broomall, Stacey M; Li, Po-E; Teshima, Hazuki; Gibbons, Henry S; Palacios, Gustavo F; Rosenzweig, C Nicole; Redden, Cassie L; Xu, Yan; Minogue, Timothy D; Chain, Patrick S

    2015-01-01

    The genus Burkholderia encompasses both pathogenic (including Burkholderia mallei and Burkholderia pseudomallei, U.S. Centers for Disease Control and Prevention Category B listed), and nonpathogenic Gram-negative bacilli. Here we present full genome sequences for a panel of 59 Burkholderia strains, selected to aid in detection assay development. PMID:25931592

  15. Complete Genome Sequences for 59 Burkholderia Isolates, Both Pathogenic and Near Neighbor

    PubMed Central

    Bishop-Lilly, Kimberly A.; Ladner, Jason T.; Daligault, Hajnalka E.; Davenport, Karen W.; Jaissle, James; Frey, Kenneth G.; Koroleva, Galina I.; Bruce, David C.; Coyne, Susan R.; Broomall, Stacey M.; Li, Po-E; Teshima, Hazuki; Gibbons, Henry S.; Palacios, Gustavo F.; Rosenzweig, C. Nicole; Redden, Cassie L.; Xu, Yan; Minogue, Timothy D.; Chain, Patrick S.

    2015-01-01

    The genus Burkholderia encompasses both pathogenic (including Burkholderia mallei and Burkholderia pseudomallei, U.S. Centers for Disease Control and Prevention Category B listed), and nonpathogenic Gram-negative bacilli. Here we present full genome sequences for a panel of 59 Burkholderia strains, selected to aid in detection assay development. PMID:25931592

  16. Complete Genome Sequences for 59 Burkholderia Isolates, Both Pathogenic and Near Neighbor

    SciTech Connect

    Johnson, Shannon L.; Bishop-Lilly, Kimberly A.; Ladner, Jason T.; Daligault, Hajnalka E.; Davenport, Karen W.; Jaissle, James; Frey, Kenneth G.; Koroleva, Galina I.; Bruce, David C.; Coyne, Susan R.; Broomall, Stacey M.; Li, Po-E; Teshima, Hazuki; Gibbons, Henry S.; Palacios, Gustavo F.; Rosenzweig, C. Nicole; Redden, Cassie L.; Xu, Yan; Minogue, Timothy D.; Chain, Patrick S.

    2015-04-30

    The genus Burkholderia encompasses both pathogenic (including Burkholderia mallei and Burkholderia pseudomallei, U.S. Centers for Disease Control and Prevention Category B listed), and nonpathogenic Gram-negative bacilli. Presented in this document are full genome sequences for a panel of 59 Burkholderia strains, selected to aid in detection assay development.

  17. Burkholderia stagnalis sp. nov. and Burkholderia territorii sp. nov., two novel Burkholderia cepacia complex species from environmental and human sources.

    PubMed

    De Smet, Birgit; Mayo, Mark; Peeters, Charlotte; Zlosnik, James E A; Spilker, Theodore; Hird, Trevor J; LiPuma, John J; Kidd, Timothy J; Kaestli, Mirjam; Ginther, Jennifer L; Wagner, David M; Keim, Paul; Bell, Scott C; Jacobs, Jan A; Currie, Bart J; Vandamme, Peter

    2015-07-01

    Nine Burkholderia cepacia complex (Bcc) bacteria were isolated during environmental surveys for the ecological niche of Burkholderia pseudomallei, the aetiological agent of melioidosis, in the Northern Territory of Australia. They represented two multi-locus sequence analysis-based clusters, referred to as Bcc B and Bcc L. Three additional environmental and clinical Bcc B isolates were identified upon deposition of the sequences in the PubMLST database. Analysis of the concatenated nucleotide sequence divergence levels within both groups (1.4 and 1.9%, respectively) and towards established Bcc species (4.0 and 3.9%, respectively) demonstrated that the two taxa represented novel Bcc species. All 12 isolates were further characterized using 16S rRNA and recA gene sequence analysis, RAPD analysis, DNA base content determination, fatty acid methyl ester analysis and biochemical profiling. Analysis of recA gene sequences revealed a remarkable diversity within each of these taxa, but, together, the results supported the affiliation of the two taxa to the Bcc. Bcc B strains can be differentiated from most other Bcc members by the assimilation of maltose. Bcc L strains can be differentiated from other Bcc members by the absence of assimilation of N-acetylglucosamine. The names Burkholderia stagnalis sp. nov. with type strain LMG 28156(T) ( = CCUG 65686(T)) and Burkholderia territorii sp. nov. with type strain LMG 28158(T) ( = CCUG 65687(T)) are proposed for Bcc B and Bcc L bacteria, respectively. PMID:25872960

  18. Burkholderia Hep_Hap autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

    PubMed Central

    Tiyawisutsri, Rachaneeporn; Holden, Matthew TG; Tumapa, Sarinna; Rengpipat, Sirirat; Clarke, Simon R; Foster, Simon J; Nierman, William C; Day, Nicholas PJ; Peacock, Sharon J

    2007-01-01

    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. PMID:17362501

  19. Burkholderia rhizoxinica sp. nov. and Burkholderia endofungorum sp. nov., bacterial endosymbionts of the plant-pathogenic fungus Rhizopus microsporus.

    PubMed

    Partida-Martinez, Laila P; Groth, Ingrid; Schmitt, Imke; Richter, Walter; Roth, Martin; Hertweck, Christian

    2007-11-01

    Several strains of the fungus Rhizopus microsporus harbour endosymbiotic bacteria for the production of the causal agent of rice seedling blight, rhizoxin, and the toxic cyclopeptide rhizonin. R. microsporus and isolated endobacteria were selected for freeze-fracture electron microscopy, which allowed visualization of bacterial cells within the fungal cytosol by their two parallel-running envelope membranes and by the fine structure of the lipopolysaccharide layer of the outer membrane. Two representatives of bacterial endosymbionts were chosen for phylogenetic analyses on the basis of full 16S rRNA gene sequences, which revealed that the novel fungal endosymbionts formed a monophyletic group within the genus Burkholderia. Inter-sequence similarities ranged from 98.94 to 100%, and sequence similarities to members of the Burkholderia pseudomallei group, the closest neighbours, were 96.74-97.38%. In addition, the bacterial strains were distinguished from their phylogenetic neighbours by their fatty acid profiles and other biochemical characteristics. The phylogenetic studies based on 16S rRNA gene sequence data, together with conclusive DNA-DNA reassociation experiments, strongly support the proposal that these strains represent two novel species within the genus Burkholderia, for which the names Burkholderia rhizoxinica sp. nov. (type strain, HKI 454T=DSM 19002T=CIP 109453T) and Burkholderia endofungorum sp. nov. (type strain, HKI 456T=DSM 19003T=CIP 109454T) are proposed. PMID:17978222

  20. The art of persistence-the secrets to Burkholderia chronic infections.

    PubMed

    Lewis, Eric R G; Torres, Alfredo G

    2016-08-01

    The Gram-negative proteobacteria genus Burkholderia encompasses multiple bacterial species that are pathogenic to humans and other vertebrates. Two pathogenic species of interest within this genus are Burkholderia pseudomallei (Bpm) and the B. cepacia complex (Bcc); the former is the causative agent of melioidosis in humans and other mammals, and the latter is associated with pneumonia in immunocompromised patients. One understudied and shared characteristic of these two pathogenic groups is their ability to persist and establish chronic infection within the host. In this review, we will explore the depth of knowledge about chronic infections caused by persistent Bpm and Bcc. We examine the host risk factors and immune responses associated with more severe chronic infections. We also discuss host adaptation and phenotypes associated with persistent Burkholderia species. Lastly, we survey how other intracellular bacteria associated with chronic infections are combatted and explore possible future applications to target Burkholderia Our goal is to highlight understudied areas that should be addressed for a more thorough understanding of chronic Burkholderia infections and how to combat them. PMID:27440810

  1. Exploiting molecular virulence determinants in Burkholderia to develop vaccine antigens.

    PubMed

    Casey, William Thomas; McClean, Siobhán

    2015-01-01

    The Burkholderia genus is a highly diverse group of species that are distributed throughout a wide range of environments and habitats. Among this group, which is remarkable for its adaptability to a wider range of environmental conditions including disinfectants and organic solvents, are a subgroup that represents some of the most difficult to treat infections. This subgroup includes Burkholderia pseudomallei, the causative agent of melioidosis; B. mallei, the causative agent of glanders and B. cepacia complex (Bcc) which causes opportunistic infections in people with cystic fibrosis and immunocompromised patients. The latter pathogen is itself a group of 18 distinct, but, closely related species. The adaptability of this group allows the expression of a rich selection of molecular virulence determinants to facilitate its survival in the diverse habitats that it colonises. This review will describe a selection of these associated with human infection; comparing them across the three pathogens and highlighting their potential roles as vaccine candidates. Better integration of the knowledge on the pathogenesis and molecular determinants of virulence for these Burkholderia spp may allow the development of more efficacious vaccines. PMID:25850766

  2. Pulsed-field gel electrophoresis as a discriminatory typing technique for the biothreat agent burkholderia mallei.

    PubMed

    Chantratita, Narisara; Vesaratchavest, Mongkol; Wuthiekanun, Vanaporn; Tiyawisutsri, Rachaneeporn; Ulziitogtokh, Tsedev; Akcay, Erhan; Day, Nicholas P J; Peacock, Sharon J

    2006-03-01

    Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) was used to type 21 laboratory strains of Burkholderia mallei. We demonstrated good resolution by PFGE together with clustering of some geographically related isolates, and confirmed previous observations that B. mallei is clonal as defined by MLST. PMID:16525089

  3. rpsU-based discrimination within the genus Burkholderia.

    PubMed

    Frickmann, H; Neubauer, H; Loderstaedt, U; Derschum, H; Hagen, R M

    2014-06-01

    Sequencing of the gene rpsU reliably delineates saprophytic Burkholderia (B.) thailandensis from highly pathogenic B. mallei and B. pseudomallei. We analyzed the suitability of this technique for the delineation of the B. pseudomallei complex from other Burkholderia species. Both newly recorded and previously deposited sequences of well-characterized or reference strains (n = 84) of Azoarcus spp., B. ambifaria, B. anthina, B. caledonica, B. caribensis, B. caryophylli, B. cenocepacia, B. cepacia, B. cocovenenans, B. dolosa, B. fungorum, B. gladioli, B. glathei, B. glumae, B. graminis, B. hospita, B. kururensis, B. mallei, B. multivorans, B. phenazinium, B. phenoliruptrix, B. phymatum, B. phytofirmans, B. plantarii, B. pseudomallei, B. pyrrocinia, B. stabilis, B. thailandensis, B. ubonensis, B. vietnamiensis, B. xenovorans, not further defined Burkholderia spp., and the outliers Cupriavidus metallidurans, Laribacter hongkongensis, Pandorea norimbergensis, and Ralstonia pickettii were included in a multiple sequence analysis. Multiple sequence alignments led to the delineation of four major clusters, rpsU-I to rpsU-IV, with a sequence homology >92%. The B. pseudomallei complex formed the complex rpsU-II. Several Burkholderia species showed 100% sequence homology. This procedure is useful for the molecular confirmation or exclusion of glanders or melioidosis from primary patient material. Further discrimination within the Burkholderia genus requires other molecular approaches. PMID:24883196

  4. rpsU-based discrimination within the genus Burkholderia

    PubMed Central

    Neubauer, H.; Loderstaedt, U.; Derschum, H.; Hagen, R. M.

    2014-01-01

    Sequencing of the gene rpsU reliably delineates saprophytic Burkholderia (B.) thailandensis from highly pathogenic B. mallei and B. pseudomallei. We analyzed the suitability of this technique for the delineation of the B. pseudomallei complex from other Burkholderia species. Both newly recorded and previously deposited sequences of well-characterized or reference strains (n = 84) of Azoarcus spp., B. ambifaria, B. anthina, B. caledonica, B. caribensis, B. caryophylli, B. cenocepacia, B. cepacia, B. cocovenenans, B. dolosa, B. fungorum, B. gladioli, B. glathei, B. glumae, B. graminis, B. hospita, B. kururensis, B. mallei, B. multivorans, B. phenazinium, B. phenoliruptrix, B. phymatum, B. phytofirmans, B. plantarii, B. pseudomallei, B. pyrrocinia, B. stabilis, B. thailandensis, B. ubonensis, B. vietnamiensis, B. xenovorans, not further defined Burkholderia spp., and the outliers Cupriavidus metallidurans, Laribacter hongkongensis, Pandorea norimbergensis, and Ralstonia pickettii were included in a multiple sequence analysis. Multiple sequence alignments led to the delineation of four major clusters, rpsU-I to rpsU-IV, with a sequence homology >92%. The B. pseudomallei complex formed the complex rpsU-II. Several Burkholderia species showed 100% sequence homology. This procedure is useful for the molecular confirmation or exclusion of glanders or melioidosis from primary patient material. Further discrimination within the Burkholderia genus requires other molecular approaches. PMID:24883196

  5. Burkholderia terrae BS001 migrates proficiently with diverse fungal hosts through soil and provides protection from antifungal agents

    PubMed Central

    Nazir, Rashid; Tazetdinova, Diana I.; van Elsas, Jan Dirk

    2014-01-01

    Soil bacteria can benefit from co-occurring soil fungi in respect of the acquisition of carbonaceous nutrients released by fungal hyphae and the access to novel territories in soil. Here, we investigated the capacity of the mycosphere-isolated bacterium Burkholderia terrae BS001 to comigrate through soil along with hyphae of the soil fungi Trichoderma asperellum, Rhizoctonia solani, Fusarium oxysporum, F. oxysporum pv lini, Coniochaeta ligniaria, Phanerochaete velutina, and Phallus impudicus. We used Lyophyllum sp. strain Karsten as the reference migration-inciting fungus. Bacterial migration through presterilized soil on the extending fungal hyphae was detected with six of the seven test fungi, with only Phallus impudicus not showing any bacterial transport. Much like with Lyophyllum sp. strain Karsten, intermediate (106–108 CFU g-1 dry soil) to high (>108 CFU g-1 dry soil) strain BS001 cell population sizes were found at the hyphal migration fronts of four fungi, i.e., T. asperellum, Rhizoctonia solani, F. oxysporum and F. oxysporum pv lini, whereas for two fungi, Coniochaeta ligniaria and Phanerochaete velutina, the migration responses were retarded and population sizes were lower (103–106 CFU g-1 dry soil). Consistent with previous data obtained with the reference fungus, migration with the migration-inciting fungi occurred only in the direction of the hyphal growth front. Remarkably, Burkholderia terrae BS001 provided protection from several antifungal agents to the canonical host Lyophyllum sp. strain Karsten. Specifically, this host was protected from Pseudomonas fluorescens strain CHA0 metabolites, as well as from the anti-fungal agent cycloheximide. Similar protection by strain BS001was observed for T. asperellum, and, to a lower extent, F. oxysporum and Rhizoctonia solani. The protective effect may be related to the consistent occurrence of biofilm-like cell layers or agglomerates at the surfaces of the protected fungi. The current study represents

  6. Efflux pump-mediated drug resistance in Burkholderia

    PubMed Central

    Podnecky, Nicole L.; Rhodes, Katherine A.; Schweizer, Herbert P.

    2015-01-01

    Several members of the genus Burkholderia are prominent pathogens. Infections caused by these bacteria are difficult to treat because of significant antibiotic resistance. Virtually all Burkholderia species are also resistant to polymyxin, prohibiting use of drugs like colistin that are available for treatment of infections caused by most other drug resistant Gram-negative bacteria. Despite clinical significance and antibiotic resistance of Burkholderia species, characterization of efflux pumps lags behind other non-enteric Gram-negative pathogens such as Acinetobacter baumannii and Pseudomonas aeruginosa. Although efflux pumps have been described in several Burkholderia species, they have been best studied in Burkholderia cenocepacia and B. pseudomallei. As in other non-enteric Gram-negatives, efflux pumps of the resistance nodulation cell division (RND) family are the clinically most significant efflux systems in these two species. Several efflux pumps were described in B. cenocepacia, which when expressed confer resistance to clinically significant antibiotics, including aminoglycosides, chloramphenicol, fluoroquinolones, and tetracyclines. Three RND pumps have been characterized in B. pseudomallei, two of which confer either intrinsic or acquired resistance to aminoglycosides, macrolides, chloramphenicol, fluoroquinolones, tetracyclines, trimethoprim, and in some instances trimethoprim+sulfamethoxazole. Several strains of the host-adapted B. mallei, a clone of B. pseudomallei, lack AmrAB-OprA, and are therefore aminoglycoside and macrolide susceptible. B. thailandensis is closely related to B. pseudomallei, but non-pathogenic to humans. Its pump repertoire and ensuing drug resistance profile parallels that of B. pseudomallei. An efflux pump in B. vietnamiensis plays a significant role in acquired aminoglycoside resistance. Summarily, efflux pumps are significant players in Burkholderia drug resistance. PMID:25926825

  7. Insights into β-Lactamases from Burkholderia Species, Two Phylogenetically Related yet Distinct Resistance Determinants*

    PubMed Central

    Papp-Wallace, Krisztina M.; Taracila, Magdalena A.; Gatta, Julian A.; Ohuchi, Nozomi; Bonomo, Robert A.; Nukaga, Michiyoshi

    2013-01-01

    Burkholderia cepacia complex and Burkholderia pseudomallei are opportunistic human pathogens. Resistance to β-lactams among Burkholderia spp. is attributable to expression of β-lactamases (e.g. PenA in B. cepacia complex and PenI in B. pseudomallei). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor-resistant carbapenemase, most similar to KPC-2 (the most clinically significant serine carbapenemase), whereas PenI is an extended spectrum β-lactamase. PenA hydrolyzes β-lactams with kcat values ranging from 0.38 ± 0.04 to 460 ± 46 s−1 and possesses high kcat/kinact values of 2000, 1500, and 75 for β-lactamase inhibitors. PenI demonstrates the highest kcat value for cefotaxime of 9.0 ± 0.9 s−1. Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explain the lower kcat values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. Conversely, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of β-lactamases, which can lead to the rational design of novel agents against these pathogens. PMID:23658015

  8. Versatile Dual-Technology System for Markerless Allele Replacement in Burkholderia pseudomallei▿ †

    PubMed Central

    López, Carolina M.; Rholl, Drew A.; Trunck, Lily A.; Schweizer, Herbert P.

    2009-01-01

    Burkholderia pseudomallei is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a lacZα gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed gusA indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or sacB-based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an araC-PBAD-I-sceI expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved Δasd Escherichia coli mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI- and sacB-based methods afforded by pEXKm5 in conjunction with E. coli RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory B. pseudomallei strains. Finally, sacB-based counterselection was employed to isolate a defined chromosomal fabD(Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme. PMID:19700544

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

    PubMed Central

    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

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

    PubMed Central

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

    2014-01-01

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

  11. Divergent homologs of the predicted small RNA BpCand697 in Burkholderia spp.

    NASA Astrophysics Data System (ADS)

    Damiri, Nadzirah; Mohd-Padil, Hirzahida; Firdaus-Raih, Mohd

    2015-09-01

    The small RNA (sRNA) gene candidate, BpCand697 was previously reported to be unique to Burkholderia spp. and is encoded at 3' non-coding region of a putative AraC family transcription regulator gene. This study demonstrates the conservation of BpCand697 sequence across 32 Burkholderia spp. including B. pseudomallei, B. mallei, B. thailandensis and Burkholderia sp. by integrating both sequence homology and secondary structural analyses of BpCand697 within the dataset. The divergent sequence of BpCand697 was also used as a discriminatory power in clustering the dataset according to the potential virulence of Burkholderia spp., showing that B. thailandensis was clearly secluded from the virulent cluster of B. pseudomallei and B. mallei. Finally, the differential co-transcript expression of BpCand697 and its flanking gene, bpsl2391 was detected in Burkholderia pseudomallei D286 after grown under two different culture conditions using nutrient-rich and minimal media. It is hypothesized that the differential expression of BpCand697-bpsl2391 co-transcript between the two standard prepared media might correlate with nutrient availability in the culture media, suggesting that the physical co-localization of BpCand697 in B. pseudomallei D286 might be directly or indirectly involved with the transcript regulation of bpsl2391 under the selected in vitro culture conditions.

  12. Combining Functional and Structural Genomics to Sample the Essential Burkholderia Structome

    PubMed Central

    Baugh, Loren; Gallagher, Larry A.; Patrapuvich, Rapatbhorn; Clifton, Matthew C.; Gardberg, Anna S.; Edwards, Thomas E.; Armour, Brianna; Begley, Darren W.; Dieterich, Shellie H.; Dranow, David M.; Abendroth, Jan; Fairman, James W.; Fox, David; Staker, Bart L.; Phan, Isabelle; Gillespie, Angela; Choi, Ryan; Nakazawa-Hewitt, Steve; Nguyen, Mary Trang; Napuli, Alberto; Barrett, Lynn; Buchko, Garry W.; Stacy, Robin; Myler, Peter J.; Stewart, Lance J.; Manoil, Colin; Van Voorhis, Wesley C.

    2013-01-01

    Background 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. Methodology/Principal Findings 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. Conclusions/Significance This collection of structures, solubility and experimental essentiality data

  13. Protection against Experimental Melioidosis following Immunization with Live Burkholderia thailandensis Expressing a manno-Heptose Capsule

    PubMed Central

    Laws, Thomas R.; D'Elia, Riccardo V.; Stokes, Margaret G. M.; Nandi, Tannistha; Williamson, E. Diane; Tan, Patrick; Prior, Joann L.; Atkins, Timothy P.

    2013-01-01

    Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. It is highly resistant to antibiotic treatment, and there is currently no licensed vaccine. Burkholderia thailandensis is a close relative of Burkholderia pseudomallei but is essentially avirulent in mammals. In this report, we detail the protective efficacy of immunization with live B. thailandensis E555, a strain which has been shown to express an antigenic capsule similar to that of B. pseudomallei. Immunization with E555 induced significant protection against a lethal intraperitoneal B. pseudomallei challenge in a mouse model of infection, with no mice succumbing to infection over the course of the study, even with challenges of up to 6,000 median lethal doses. By comparison, mice immunized with B. thailandensis not expressing a B. pseudomallei-like capsule had significantly decreased levels of protection. E555-immunized mice had significantly higher levels of IgG than mice immunized with noncapsulated B. thailandensis, and these antibody responses were primarily directed against the capsule. PMID:23677322

  14. Mechanisms of Disease: Host-Pathogen Interactions between Burkholderia Species and Lung Epithelial Cells

    PubMed Central

    David, Jonathan; Bell, Rachel E.; Clark, Graeme C.

    2015-01-01

    Members of the Burkholderia species can cause a range of severe, often fatal, respiratory diseases. A variety of in vitro models of infection have been developed in an attempt to elucidate the mechanism by which Burkholderia spp. gain entry to and interact with the body. The majority of studies have tended to focus on the interaction of bacteria with phagocytic cells with a paucity of information available with regard to the lung epithelium. However, the lung epithelium is becoming more widely recognized as an important player in innate immunity and the early response to infections. Here we review the complex relationship between Burkholderia species and epithelial cells with an emphasis on the most pathogenic species, Burkholderia pseudomallei and Burkholderia mallei. The current gaps in knowledge in our understanding are highlighted along with the epithelial host-pathogen interactions that offer potential opportunities for therapeutic intervention. PMID:26636042

  15. [Comparative analysis of total cell protein electrophoregram of pathogenic Burkholderia].

    PubMed

    Budchenko, A A; Iliukhin, V I; Viktorov, D V

    2005-01-01

    Whole-cell proteins of 22 strain of Burkhoderia pseudomallei, including 13 B. mallei, 5 B. cepacia strains and 14 strains of opportunistically pathogenic Pseudomonas defined by 1D SDC-PAAG electrophoresis. Electrophoregrams contained 35 to 45 protein fractions sized 19 to 130 kDa, which were highly reproductive. On the basis of computer-aided comparative analysis of protein patterns the interspecies and intraspecies grouping of studied microorganisms was made. The cluster analysis of the similarity matrix of protein spectra made it possible to allocate two groups of strains at the level of similarity of 78%. Group I was formed by Burkholderia species that previously belonged to the II RNA-DNA homology group of Pseudomonas: B. pseudomallei, B. mallei, B. cepacia. All Pseudomonas species were added to the 2nd Group: P. aeruginosa, P. stutzeri, P. testosterone, P. fluorescens, P. putida, P. mendocina. Four phenons were isolated among the strains of B. pseudomallei and 2 phenons--among the strains of B. mallei at the threshold similarity level (89%). The authors conclude that the comparative analysis of electrophoregrams of whole-cell proteins can be useful in the identification and typing of pathogenic Burkholderia. PMID:15954473

  16. Snake Cathelicidin NA-CATH and Smaller Helical Antimicrobial Peptides Are Effective against Burkholderia thailandensis

    PubMed Central

    Blower, Ryan J.; Barksdale, Stephanie M.; van Hoek, Monique L.

    2015-01-01

    Burkholderia thailandensis is a Gram-negative soil bacterium used as a model organism for B. pseudomallei, the causative agent of melioidosis and an organism classified category B priority pathogen and a Tier 1 select agent for its potential use as a biological weapon. Burkholderia species are reportedly “highly resistant” to antimicrobial agents, including cyclic peptide antibiotics, due to multiple resistance systems, a hypothesis we decided to test using antimicrobial (host defense) peptides. In this study, a number of cationic antimicrobial peptides (CAMPs) were tested in vitro against B. thailandensis for both antimicrobial activity and inhibition of biofilm formation. Here, we report that the Chinese cobra (Naja atra) cathelicidin NA-CATH was significantly antimicrobial against B. thailandensis. Additional cathelicidins, including the human cathelicidin LL-37, a sheep cathelicidin SMAP-29, and some smaller ATRA peptide derivatives of NA-CATH were also effective. The D-enantiomer of one small peptide (ATRA-1A) was found to be antimicrobial as well, with EC50 in the range of the L-enantiomer. Our results also demonstrate that human alpha-defensins (HNP-1 & -2) and a short beta-defensin-derived peptide (Peptide 4 of hBD-3) were not bactericidal against B. thailandensis. We also found that the cathelicidin peptides, including LL-37, NA-CATH, and SMAP-29, possessed significant ability to prevent biofilm formation of B. thailandensis. Additionally, we show that LL-37 and its D-enantiomer D-LL-37 can disperse pre-formed biofilms. These results demonstrate that although B. thailandensis is highly resistant to many antibiotics, cyclic peptide antibiotics such as polymyxin B, and defensing peptides, some antimicrobial peptides including the elapid snake cathelicidin NA-CATH exert significant antimicrobial and antibiofilm activity towards B. thailandensis. PMID:26196513

  17. Snake Cathelicidin NA-CATH and Smaller Helical Antimicrobial Peptides Are Effective against Burkholderia thailandensis.

    PubMed

    Blower, Ryan J; Barksdale, Stephanie M; van Hoek, Monique L

    2015-01-01

    Burkholderia thailandensis is a Gram-negative soil bacterium used as a model organism for B. pseudomallei, the causative agent of melioidosis and an organism classified category B priority pathogen and a Tier 1 select agent for its potential use as a biological weapon. Burkholderia species are reportedly "highly resistant" to antimicrobial agents, including cyclic peptide antibiotics, due to multiple resistance systems, a hypothesis we decided to test using antimicrobial (host defense) peptides. In this study, a number of cationic antimicrobial peptides (CAMPs) were tested in vitro against B. thailandensis for both antimicrobial activity and inhibition of biofilm formation. Here, we report that the Chinese cobra (Naja atra) cathelicidin NA-CATH was significantly antimicrobial against B. thailandensis. Additional cathelicidins, including the human cathelicidin LL-37, a sheep cathelicidin SMAP-29, and some smaller ATRA peptide derivatives of NA-CATH were also effective. The D-enantiomer of one small peptide (ATRA-1A) was found to be antimicrobial as well, with EC50 in the range of the L-enantiomer. Our results also demonstrate that human alpha-defensins (HNP-1 & -2) and a short beta-defensin-derived peptide (Peptide 4 of hBD-3) were not bactericidal against B. thailandensis. We also found that the cathelicidin peptides, including LL-37, NA-CATH, and SMAP-29, possessed significant ability to prevent biofilm formation of B. thailandensis. Additionally, we show that LL-37 and its D-enantiomer D-LL-37 can disperse pre-formed biofilms. These results demonstrate that although B. thailandensis is highly resistant to many antibiotics, cyclic peptide antibiotics such as polymyxin B, and defensing peptides, some antimicrobial peptides including the elapid snake cathelicidin NA-CATH exert significant antimicrobial and antibiofilm activity towards B. thailandensis. PMID:26196513

  18. Airborne Transmission of Melioidosis to Humans from Environmental Aerosols Contaminated with B. pseudomallei

    PubMed Central

    Lin, Hsi-Hsun; Liu, Pei-Ju; Ni, Wei-Fan; Hsueh, Pei-Tan; Liang, Shih-Hsiung; Chen, Chialin; Chen, Ya-Lei

    2015-01-01

    Melioidosis results from an infection with the soil-borne pathogen Burkholderia pseudomallei, and cases of melioidosis usually cluster after rains or a typhoon. In an endemic area of Taiwan, B. pseudomallei is primarily geographically distributed in cropped fields in the northwest of this area, whereas melioidosis cases are distributed in a densely populated district in the southeast. We hypothesized that contaminated cropped fields generated aerosols contaminated with B. pseudomallei, which were carried by a northwesterly wind to the densely populated southeastern district. We collected soil and aerosol samples from a 72 km2 area of land, including the melioidosis-clustered area and its surroundings. Aerosols that contained B. pseudomallei-specific TTSS (type III secretion system) ORF2 DNA were well distributed in the endemic area but were rare in the surrounding areas during the rainy season. The concentration of this specific DNA in aerosols was positively correlated with the incidence of melioidosis and the appearance of a northwesterly wind. Moreover, the isolation rate in the superficial layers of the contaminated cropped field in the northwest was correlated with PCR positivity for aerosols collected from the southeast over a 2-year period. According to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analyses, PFGE Type Ia (ST58) was the predominant pattern linking the molecular association among soil, aerosol and human isolates. Thus, the airborne transmission of melioidosis moves from the contaminated soil to aerosols and/or to humans in this endemic area. PMID:26061639

  19. Airborne Transmission of Melioidosis to Humans from Environmental Aerosols Contaminated with B. pseudomallei.

    PubMed

    Chen, Pei-Shih; Chen, Yao-Shen; Lin, Hsi-Hsun; Liu, Pei-Ju; Ni, Wei-Fan; Hsueh, Pei-Tan; Liang, Shih-Hsiung; Chen, Chialin; Chen, Ya-Lei

    2015-06-01

    Melioidosis results from an infection with the soil-borne pathogen Burkholderia pseudomallei, and cases of melioidosis usually cluster after rains or a typhoon. In an endemic area of Taiwan, B. pseudomallei is primarily geographically distributed in cropped fields in the northwest of this area, whereas melioidosis cases are distributed in a densely populated district in the southeast. We hypothesized that contaminated cropped fields generated aerosols contaminated with B. pseudomallei, which were carried by a northwesterly wind to the densely populated southeastern district. We collected soil and aerosol samples from a 72 km2 area of land, including the melioidosis-clustered area and its surroundings. Aerosols that contained B. pseudomallei-specific TTSS (type III secretion system) ORF2 DNA were well distributed in the endemic area but were rare in the surrounding areas during the rainy season. The concentration of this specific DNA in aerosols was positively correlated with the incidence of melioidosis and the appearance of a northwesterly wind. Moreover, the isolation rate in the superficial layers of the contaminated cropped field in the northwest was correlated with PCR positivity for aerosols collected from the southeast over a 2-year period. According to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analyses, PFGE Type Ia (ST58) was the predominant pattern linking the molecular association among soil, aerosol and human isolates. Thus, the airborne transmission of melioidosis moves from the contaminated soil to aerosols and/or to humans in this endemic area. PMID:26061639

  20. Members of the genus Burkholderia: good and bad guys

    PubMed Central

    Eberl, Leo; Vandamme, Peter

    2016-01-01

    In the 1990s several biocontrol agents on that contained Burkholderia strains were registered by the United States Environmental Protection Agency (EPA). After risk assessment these products were withdrawn from the market and a moratorium was placed on the registration of Burkholderia-containing products, as these strains may pose a risk to human health. However, over the past few years the number of novel Burkholderia species that exhibit plant-beneficial properties and are normally not isolated from infected patients has increased tremendously. In this commentary we wish to summarize recent efforts that aim at discerning pathogenic from beneficial Burkholderia strains. PMID:27303639

  1. Members of the genus Burkholderia: good and bad guys.

    PubMed

    Eberl, Leo; Vandamme, Peter

    2016-01-01

    In the 1990s several biocontrol agents on that contained Burkholderia strains were registered by the United States Environmental Protection Agency (EPA). After risk assessment these products were withdrawn from the market and a moratorium was placed on the registration of Burkholderia-containing products, as these strains may pose a risk to human health. However, over the past few years the number of novel Burkholderia species that exhibit plant-beneficial properties and are normally not isolated from infected patients has increased tremendously. In this commentary we wish to summarize recent efforts that aim at discerning pathogenic from beneficial Burkholderia strains. PMID:27303639

  2. Draft Genomes for Eight Burkholderia mallei Isolates from Turkey

    PubMed Central

    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

  3. Draft Genomes for Eight Burkholderia mallei Isolates from Turkey.

    PubMed

    Daligault, H E; Johnson, S L; 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

  4. Draft Genomes for Eight Burkholderia mallei Isolates from Turkey

    DOE PAGESBeta

    Daligault, H. E.; Johnson, Shannon L.; 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.; et al

    2016-01-07

    Burkholderia mallei, the etiologic agent of glanders, is a Gram-negative, nonmotile, facultative intracellular pathogen. Though glanders have been eradicated from many parts of the world, the threat ofB. malleibeing used as a weapon is very real. We, then, present draft genome assemblies of 8Burkholderia malleistrains that were isolated in Turkey.

  5. Burkholderia thailandensis Is Virulent in Drosophila melanogaster

    PubMed Central

    Pilátová, Martina; Dionne, Marc S.

    2012-01-01

    Melioidosis is a serious infectious disease endemic to Southeast Asia and Northern Australia. This disease is caused by the Gram-negative bacterium Burkholderia pseudomallei; Burkholderia thailandensis is a closely-related organism known to be avirulent in humans. B. thailandensis has not previously been used to infect Drosophila melanogaster. We examined the effect of B. thailandensis infection on fly survival, on antimicrobial peptide expression, and on phagocytic cells. In the fruit fly, which possesses only an innate immune system, B. thailandensis is highly virulent, causing rapid death when injected or fed. One intriguing aspect of this infection is its temperature dependence: infected flies maintained at 25°C exhibit rapid bacterial proliferation and death in a few days, while infected animals maintained at 18°C exhibit very slow bacterial proliferation and take weeks to die; this effect is due in part to differences in immune activity of the host. Death in this infection is likely due at least in part to a secreted toxin, as injection of flies with sterile B. thailandensis-conditioned medium is able to kill. B. thailandensis infection strongly induces the expression of antimicrobial peptides, but this is insufficient to inhibit bacterial proliferation in infected flies. Finally, the function of fly phagocytes is not affected by B. thailandensis infection. The high virulence of B. thailandensis in the fly suggests the possibility that this organism is a natural pathogen of one or more invertebrates. PMID:23209596

  6. Siderophore production by Pseudomonas pseudomallei.

    PubMed Central

    Yang, H M; Chaowagul, W; Sokol, P A

    1991-01-01

    Eighty-four strains of Pseudomonas pseudomallei isolated from patients with melioidosis were examined for siderophore production. All the strains were shown to produce siderophore both on chrome azurol S agar plates and in liquid medium under iron-deficient conditions. Chemical assays indicated that the siderophore belongs to the hydroxamate class. Addition of iron to the culture medium resulted in increased culture growth with markedly decreased yield of siderophore. Siderophore produced by strain U7 was purified by gel filtration chromatography, and the molecular weight was estimated to be 1,000. When this partially purified siderophore was added to culture medium, it promoted iron uptake by P. pseudomallei in the presence of EDTA and enhanced growth of the organism in the presence of transferrin. We have given this siderophore the trivial name malleobactin. PMID:1825486

  7. CD4+ T cell epitopes of FliC conserved between strains of Burkholderia - implications for vaccines against melioidosis and Cepacia Complex in Cystic Fibrosis

    PubMed Central

    Musson, Julie A.; Reynolds, Catherine J; Rinchai, Darawan; Nithichanon, Arnone; Khaenam, Prasong; Favry, Emmanuel; Spink, Natasha; Chu, Karen KY; De Soyza, Anthony; Bancroft, Gregory J; Lertmemongkolchai, Ganjana; Maillere, Bernard; Boyton, Rosemary J; Altmann, Daniel M.; Robinson, John H.

    2014-01-01

    Burkholderia pseudomallei (Bp), is the causative agent of melioidosis, characterized by pneumonia and fatal septicemia and prevalent in SE Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The Bp flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed Bp FliC peptide binding affinity to multiple HLA class II alleles, then assessed CD4 T cell immunity in HLA class II transgenic mice and in seropositive individuals in Thailand. T cell hybridomas were generated to investigate cross-reactivity between Bp and the related Burkholderia species associated with Cepacia Complex CF. Bp FliC contained several peptide sequences with ability to bind multiple HLA class II alleles. Several peptides were shown to encompass strong CD4 T cell epitopes in Bp-exposed individuals and in HLA transgenic mice. In particular, the p38 epitope is robustly recognized by CD4 T cells of seropositive donors across diverse HLA haplotypes. T cell hybridomas against an immunogenic Bp FliC epitope also cross-reacted with orthologous FliC sequences from B. multivorans and B. cenocepacia, important pathogens in CF. Epitopes within FliC were accessible for processing and presentation from live or heat-killed bacteria, demonstrating that flagellin enters the HLA class II antigen presentation pathway during infection of macrophages with B. cenocepacia. Collectively, the data support the possibility of incorporating FliC T cell epitopes into vaccination programs targeting both at-risk individuals in Bp endemic regions as well as CF patients. PMID:25392525

  8. Developing Peptide Mimotopes of Capsular Polysaccharides and Lipopolysaccharides Protective Antigens of Pathogenic Burkholderia Bacteria.

    PubMed

    Guo, Pengfei; Zhang, Jing; Tsai, Shien; Li, Bingjie; Lo, Shyh-Ching

    2016-06-01

    Burkholderia pseudomallei (BP) and Burkholderia mallei (BM) are two species of pathogenic Burkholderia bacteria. Our laboratory previously identified four monoclonal antibodies (MAbs) that reacted against Burkholderia capsular polysaccharides (PS) and lipopolysaccharides (LPS) and effectively protected against a lethal dose of BP/BM infections in mice. In this study, we used phage display panning against three different phage peptide libraries to select phage clones specifically recognized by each of the four protective MAbs. After sequencing a total of 179 candidate phage clones, we examined in detail six selected phage clones carrying different peptide inserts for the specificity of binding by the respective target MAbs. Chemically synthesized peptides corresponding to those displayed by the six phage clones were conjugated to keyhole limpet hemocyanin carrier protein and tested for their binding specificity to the respective protective MAbs. The study revealed that four of the six peptides, all derived from the library displaying dodecapeptides, functioned well as "mimotopes" of Burkholderia PS and LPS as demonstrated by a high degree of specific competition against the binding of three protective MAbs to BP and BM. Our results suggest that the four selected peptide mimics corresponding to PS/LPS protective antigens of BP and BM could potentially be developed into peptide vaccines against pathogenic Burkholderia bacteria. PMID:27328059

  9. Ribotype analysis of Pseudomonas pseudomallei isolates.

    PubMed Central

    Sexton, M M; Goebel, L A; Godfrey, A J; Choawagul, W; White, N J; Woods, D E

    1993-01-01

    No epidemiological typing system to differentiate among Pseudomonas pseudomallei isolates has been available. Ribotype analysis was developed and used to examine 74 clinical and 10 environmental isolates of P. pseudomallei from Thailand. Six P. pseudomallei ribotypes were identified from restriction fragment polymorphisms of EcoRI chromosomal digests. The predominant ribotype, A, was found in 59 of the isolates examined. By using patterns from hybridizations with SalI, HindIII, and PstI restriction digests, isolates of ribotype A were subdivided into a further five subtypes, giving a total of 10 differentiable P. pseudomallei types. In 23 of 34 melioidosis patients studied, multiple P. pseudomallei isolates were present. In all but one of these patients, a single ribotype of the organism was present. Isolation of two different ribotypes of P. pseudomallei from one patient, one each in sputum and urine, suggests that superinfection may have occurred. The ribotype was shown to be conserved during the course of antibiotic treatments in seven patients studied, although the antibiotic sensitivity patterns in the isolates from these patients varied. The prevalence of subtype A1 in clinical and environmental specimens suggests that this strain may be predominant in this geographical location. These results demonstrate the usefulness of the ribotyping method for epidemiological studies of P. pseudomallei. Images PMID:7679401

  10. Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

    PubMed Central

    Zhang, Zheng; Jakkaraju, Sriram; Blain, Joy; Gogol, Kenneth; Zhao, Lei; Hartley, Robert C.; Karlsson, Courtney A.; Staker, Bart L.; Stewart, Lance J.; Myler, Peter J.; Clare, Michael; Begley, Darren W.; Horn, James R.; Hagen, Timothy J

    2013-01-01

    Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series. PMID:24157367

  11. Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei.

    PubMed

    Zhang, Zheng; Jakkaraju, Sriram; Blain, Joy; Gogol, Kenneth; Zhao, Lei; Hartley, Robert C; Karlsson, Courtney A; Staker, Bart L; Edwards, Thomas E; Stewart, Lance J; Myler, Peter J; Clare, Michael; Begley, Darren W; Horn, James R; Hagen, Timothy J

    2013-12-15

    Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series. PMID:24157367

  12. Plant-Associated Symbiotic Burkholderia Species Lack Hallmark Strategies Required in Mammalian Pathogenesis

    PubMed Central

    Fong, Stephanie; Yerrapragada, Shailaja; Estrada-de los Santos, Paulina; Yang, Paul; Song, Nannie; Kano, Stephanie; de Faria, Sergio M.; Dakora, Felix D.; Weinstock, George; Hirsch, Ann M.

    2014-01-01

    Burkholderia is a diverse and dynamic genus, containing pathogenic species as well as species that form complex interactions with plants. Pathogenic strains, such as B. pseudomallei and B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic pathogens, infecting humans or animals with a compromised immune system. Although some of the opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the risk of infection to infants, the elderly, and people who are immunocompromised has not only resulted in a restriction on their use, but has also limited the application of non-pathogenic, symbiotic species, several of which nodulate legume roots or have positive effects on plant growth. However, recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct lineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts. A number of environmental strains that promote plant growth or degrade xenobiotics are also included in the symbiotic lineage. Many of these species have the potential to enhance agriculture in areas where fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted by climate change. Here we address the pathogenic potential of several of the symbiotic Burkholderia strains using bioinformatics and functional tests. A series of infection experiments using Caenorhabditis elegans and HeLa cells, as well as genomic characterization of pathogenic loci, show that the risk of opportunistic infection by symbiotic strains such as B. tuberum is extremely low. PMID:24416172

  13. Distinct colicin M-like bacteriocin-immunity pairs in Burkholderia

    PubMed Central

    Ghequire, Maarten G. K.; De Mot, René

    2015-01-01

    The Escherichia coli bacteriocin colicin M (ColM) acts via degradation of the cell wall precursor lipid II in target cells. ColM producers avoid self-inhibition by a periplasmic immunity protein anchored in the inner membrane. In this study, we identified colM-like bacteriocin genes in genomes of several β-proteobacterial strains belonging to the Burkholderia cepacia complex (Bcc) and the Burkholderia pseudomallei group. Two selected Burkholderia ambifaria proteins, designated burkhocins M1 and M2, were produced recombinantly and showed antagonistic activity against Bcc strains. In their considerably sequence-diverged catalytic domain, a conserved aspartate residue equally proved pivotal for cytotoxicity. Immunity to M-type burkhocins is conferred upon susceptible strains by heterologous expression of a cognate gene located either upstream or downstream of the toxin gene. These genes lack homology with currently known ColM immunity genes and encode inner membrane-associated proteins of two distinct types, differing in predicted transmembrane topology and moiety exposed to the periplasm. The addition of burkhocins to the bacteriocin complement of Burkholderia reveals a wider phylogenetic distribution of ColM-like bacteriotoxins, beyond the γ-proteobacterial genera Escherichia, Pectobacterium and Pseudomonas, and illuminates the diversified nature of immunity-providing proteins. PMID:26610609

  14. Phylogenomic Study of Burkholderia glathei-like Organisms, Proposal of 13 Novel Burkholderia Species and Emended Descriptions of Burkholderia sordidicola, Burkholderia zhejiangensis, and Burkholderia grimmiae

    PubMed Central

    Peeters, Charlotte; Meier-Kolthoff, Jan P.; Verheyde, Bart; De Brandt, Evie; Cooper, Vaughn S.; Vandamme, Peter

    2016-01-01

    Partial gyrB gene sequence analysis of 17 isolates from human and environmental sources revealed 13 clusters of strains and identified them as Burkholderia glathei clade (BGC) bacteria. The taxonomic status of these clusters was examined by whole-genome sequence analysis, determination of the G+C content, whole-cell fatty acid analysis and biochemical characterization. The whole-genome sequence-based phylogeny was assessed using the Genome Blast Distance Phylogeny (GBDP) method and an extended multilocus sequence analysis (MLSA) approach. The results demonstrated that these 17 BGC isolates represented 13 novel Burkholderia species that could be distinguished by both genotypic and phenotypic characteristics. BGC strains exhibited a broad metabolic versatility and developed beneficial, symbiotic, and pathogenic interactions with different hosts. Our data also confirmed that there is no phylogenetic subdivision in the genus Burkholderia that distinguishes beneficial from pathogenic strains. We therefore propose to formally classify the 13 novel BGC Burkholderia species as Burkholderia arvi sp. nov. (type strain LMG 29317T = CCUG 68412T), Burkholderia hypogeia sp. nov. (type strain LMG 29322T = CCUG 68407T), Burkholderia ptereochthonis sp. nov. (type strain LMG 29326T = CCUG 68403T), Burkholderia glebae sp. nov. (type strain LMG 29325T = CCUG 68404T), Burkholderia pedi sp. nov. (type strain LMG 29323T = CCUG 68406T), Burkholderia arationis sp. nov. (type strain LMG 29324T = CCUG 68405T), Burkholderia fortuita sp. nov. (type strain LMG 29320T = CCUG 68409T), Burkholderia temeraria sp. nov. (type strain LMG 29319T = CCUG 68410T), Burkholderia calidae sp. nov. (type strain LMG 29321T = CCUG 68408T), Burkholderia concitans sp. nov. (type strain LMG 29315T = CCUG 68414T), Burkholderia turbans sp. nov. (type strain LMG 29316T = CCUG 68413T), Burkholderia catudaia sp. nov. (type strain LMG 29318T = CCUG 68411T) and Burkholderia peredens sp. nov. (type strain LMG 29314T = CCUG

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

    PubMed

    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. PMID:26305954

  16. Burkholderia genome mining for nonribosomal peptide synthetases reveals a great potential for novel siderophores and lipopeptides synthesis.

    PubMed

    Esmaeel, Qassim; Pupin, Maude; Kieu, Nam Phuong; Chataigné, Gabrielle; Béchet, Max; Deravel, Jovana; Krier, François; Höfte, Monica; Jacques, Philippe; Leclère, Valérie

    2016-06-01

    Burkholderia is an important genus encompassing a variety of species, including pathogenic strains as well as strains that promote plant growth. We have carried out a global strategy, which combined two complementary approaches. The first one is genome guided with deep analysis of genome sequences and the second one is assay guided with experiments to support the predictions obtained in silico. This efficient screening for new secondary metabolites, performed on 48 gapless genomes of Burkholderia species, revealed a total of 161 clusters containing nonribosomal peptide synthetases (NRPSs), with the potential to synthesize at least 11 novel products. Most of them are siderophores or lipopeptides, two classes of products with potential application in biocontrol. The strategy led to the identification, for the first time, of the cluster for cepaciachelin biosynthesis in the genome of Burkholderia ambifaria AMMD and a cluster corresponding to a new malleobactin-like siderophore, called phymabactin, was identified in Burkholderia phymatum STM815 genome. In both cases, the siderophore was produced when the strain was grown in iron-limited conditions. Elsewhere, the cluster for the antifungal burkholdin was detected in the genome of B. ambifaria AMMD and also Burkholderia sp. KJ006. Burkholderia pseudomallei strains harbor the genetic potential to produce a novel lipopeptide called burkhomycin, containing a peptidyl moiety of 12 monomers. A mixture of lipopeptides produced by Burkholderia rhizoxinica lowered the surface tension of the supernatant from 70 to 27 mN·m(-1) . The production of nonribosomal secondary metabolites seems related to the three phylogenetic groups obtained from 16S rRNA sequences. Moreover, the genome-mining approach gave new insights into the nonribosomal synthesis exemplified by the identification of dual C/E domains in lipopeptide NRPSs, up to now essentially found in Pseudomonas strains. PMID:27060604

  17. Virulence of Burkholderia mallei Quorum-Sensing Mutants

    PubMed Central

    Majerczyk, Charlotte; Kinman, Loren; Han, Tony; Bunt, Richard

    2013-01-01

    Many Proteobacteria use acyl-homoserine lactone-mediated quorum-sensing (QS) to activate specific sets of genes as a function of cell density. QS often controls the virulence of pathogenic species, and in fact a previous study indicated that QS was important for Burkholderia mallei mouse lung infections. To gain in-depth information on the role of QS in B. mallei virulence, we constructed and characterized a mutant of B. mallei strain GB8 that was unable to make acyl-homoserine lactones. The QS mutant showed virulence equal to that of its wild-type parent in an aerosol mouse infection model, and growth in macrophages was indistinguishable from that of the parent strain. Furthermore, we assessed the role of QS in B. mallei ATCC 23344 by constructing and characterizing a mutant strain producing AiiA, a lactonase enzyme that degrades acyl-homoserine lactones. Although acyl-homoserine lactone levels in cultures of this strain are very low, it showed full virulence. Contrary to the previous report, we conclude that QS is not required for acute B. mallei infections of mice. QS may be involved in some stage of chronic infections in the natural host of horses, or the QS genes may be remnants of the QS network in B. pseudomallei from which this host-adapted pathogen evolved. PMID:23429539

  18. Unraveling the B. pseudomallei Heptokinase WcbL: From Structure to Drug Discovery

    PubMed Central

    Vivoli, Mirella; Isupov, Michail N.; Nicholas, Rebecca; Hill, Andrew; Scott, Andrew E.; Kosma, Paul; Prior, Joann L.; Harmer, Nicholas J.

    2015-01-01

    Summary Gram-negative bacteria utilize heptoses as part of their repertoire of extracellular polysaccharide virulence determinants. Disruption of heptose biosynthesis offers an attractive target for novel antimicrobials. A critical step in the synthesis of heptoses is their 1-O phosphorylation, mediated by kinases such as HldE or WcbL. Here, we present the structure of WcbL from Burkholderia pseudomallei. We report that WcbL operates through a sequential ordered Bi-Bi mechanism, loading the heptose first and then ATP. We show that dimeric WcbL binds ATP anti-cooperatively in the absence of heptose, and cooperatively in its presence. Modeling of WcbL suggests that heptose binding causes an elegant switch in the hydrogen-bonding network, facilitating the binding of a second ATP molecule. Finally, we screened a library of drug-like fragments, identifying hits that potently inhibit WcbL. Our results provide a novel mechanism for control of substrate binding and emphasize WcbL as an attractive anti-microbial target for Gram-negative bacteria. PMID:26687481

  19. An ERp57-mediated disulphide exchange promotes the interaction between Burkholderia cenocepacia and epithelial respiratory cells

    PubMed Central

    Pacello, Francesca; D’Orazio, Melania; Battistoni, Andrea

    2016-01-01

    Previous studies have demonstrated that extracellular glutathione reduces the ability of the Cystic Fibrosis pathogen Burkholderia cenocepacia to infect primary or immortalized epithelial respiratory cells. We report here that the adhesion and invasion ability of B. cenocepacia is limited also by thiol-oxidizing and disulphide-reducing agents and by protein disulfide isomerase (PDI) inhibitors. PDI inhibitors also reduce the proinflammatory response elicited by cells in response to Burkholderia. These findings indicate that a membrane-associated PDI catalyzes thiol/disulphide exchange reactions which favor bacterial infection. The combined use of selective PDI inhibitors, RNA silencing and specific antibodies identified ERp57 as a major PDI involved in the interaction between B. cenocepacia and epithelial cells. This study contributes to the elucidation of the Burkholderia pathogenic mechanisms by showing that this microorganism exploits a membrane-associated host protein to infect epithelial cells and identifies ERp57 as a putative pharmacological target for the treatment of Burkholderia lung infections. PMID:26879174

  20. Viperatoxin-II: A novel viper venom protein as an effective bactericidal agent.

    PubMed

    Samy, Ramar Perumal; Stiles, Bradley G; Chinnathambi, Arunachalam; Zayed, M E; Alharbi, Sulaiman Ali; Franco, Octavio Luiz; Rowan, Edward G; Kumar, Alan Prem; Lim, Lina H K; Sethi, Gautam

    2015-01-01

    Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have become a rising threat to public health. There is an urgent need for development of promising new therapeutic agents against drug resistant bacteria like S. aureus. This report discusses purification and characterization of proteins from Indian Russell's viper snake venom. Novel 15-kDa proteins called "Viperatoxin" (VipTx-I and VipTx-II) were extracted from the whole venom and evaluated using in vitro antimicrobial experiments. The N-terminal amino acid sequence of "Viperatoxin" showed high sequence homology to daboiatoxin isolated from the same venom and also matched phospholipase A2 (PLA2) enzymes isolated from other snake venoms. In an in vitro plate assay, VipTx-II but not VipTx-I showed strong antimicrobial effects against S. aureus and Burkholderia pseudomallei (KHW & TES), Proteus vulgaris and P. mirabilis. The VipTx-II was further tested by a broth-dilution assay at 100-3.1 μg/ml concentrations. The most potent bactericidal effect was found at the lowest dilutions (MICs of 6.25 μg/ml) against B. pseudomallei, S. aureus and P. vulgaris (MICs of 12.25 μg/ml). Electron microscopic investigation revealed that the protein-induced bactericidal potency was closely associated with pore formation and membrane damage, even at the lowest concentrations (<20 μg/ml). The toxin caused a low level of cytotoxic effects as observed in human (THP-1) cells at higher concentrations. Molecular weight determinations of VipTx-II by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one major, along with a few minor bands. The results indicate that VipTx-II plays a significant role in bactericidal and membrane damaging effects in vitro. Non-cytotoxic properties on human cells highlight it as a promising candidate for further evaluation of antimicrobial potential in vivo. PMID:26793432

  1. Biological Relevance of Colony Morphology and Phenotypic Switching by Burkholderia pseudomallei▿

    PubMed Central

    Chantratita, Narisara; Wuthiekanun, Vanaporn; Boonbumrung, Khaemaporn; Tiyawisutsri, Rachaneeporn; Vesaratchavest, Mongkol; Limmathurotsakul, Direk; Chierakul, Wirongrong; Wongratanacheewin, Surasakdi; Pukritiyakamee, Sasithorn; White, Nicholas J.; Day, Nicholas P. J.; Peacock, Sharon J.

    2007-01-01

    Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42°C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development. PMID:17114252

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

    PubMed Central

    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

  3. AFN-1252 is a potent inhibitor of enoyl-ACP reductase from Burkholderia pseudomallei—Crystal structure, mode of action, and biological activity

    PubMed Central

    Narasimha Rao, Krishnamurthy; Lakshminarasimhan, Anirudha; Joseph, Sarah; Lekshmi, Swathi U; Lau, Ming-Seong; Takhi, Mohammed; Sreenivas, Kandepu; Nathan, Sheila; Yusof, Rohana; Abd Rahman, Noorsaadah; Ramachandra, Murali; Antony, Thomas; Subramanya, Hosahalli

    2015-01-01

    Melioidosis is a tropical bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei; Bpm), a Gram-negative bacterium. Current therapeutic options are largely limited to trimethoprim-sulfamethoxazole and β-lactam drugs, and the treatment duration is about 4 months. Moreover, resistance has been reported to these drugs. Hence, there is a pressing need to develop new antibiotics for Melioidosis. Inhibition of enoyl-ACP reducatase (FabI), a key enzyme in the fatty acid biosynthesis pathway has shown significant promise for antibacterial drug development. FabI has been identified as the major enoyl-ACP reductase present in B. pseudomallei. In this study, we evaluated AFN-1252, a Staphylococcus aureus FabI inhibitor currently in clinical development, for its potential to bind to BpmFabI enzyme and inhibit B. pseudomallei bacterial growth. AFN-1252 stabilized BpmFabI and inhibited the enzyme activity with an IC50 of 9.6 nM. It showed good antibacterial activity against B. pseudomallei R15 strain, isolated from a melioidosis patient (MIC of 2.35 mg/L). X-ray structure of BpmFabI with AFN-1252 was determined at a resolution of 2.3 Å. Complex of BpmFabI with AFN-1252 formed a symmetrical tetrameric structure with one molecule of AFN-1252 bound to each monomeric subunit. The kinetic and thermal melting studies supported the finding that AFN-1252 can bind to BpmFabI independent of cofactor. The structural and mechanistic insights from these studies might help the rational design and development of new FabI inhibitors. PMID:25644789

  4. [Classify species of Pseudomonas pseudomallei into serotypes].

    PubMed

    Han, O; Li, L; Zhao, Z

    1990-10-01

    Species of P. pseudomallei can be classified into two serotypes, serotype I and serotype II, based on whether or not it contains a thermolabile antigen beside a thermostable one. Under the condition of lack of typing serum, by means of serum absorption test, we recognized a strain which contains a major thermolabile antigen. The antigen was purified by Sephadex G-200, and it was used to inoculate rabbits. With the immunoserum at hand, we identified 68 of the domestic chinese strains and 6 of alien strains for serotyping by bi-directional agar diffusion test. The results showed that 68 strains were identified serotype I, 3 strains serotype II, and the remaining 3 comparable with those reported indicating that strains of serotype I were found mostly in Asia, and that the serotype are unrelated to their existing environments, nor that of animal bodies, but connected with their existence in geographic distribution. PMID:2251832

  5. CHROMOSOMAL MULTIPLICITY IN BURKHOLDERIA CEPACIA

    EPA Science Inventory

    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 ...

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

    PubMed Central

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

    2015-01-01

    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. PMID:26305954

  7. A gold nanoparticle-linked glycoconjugate vaccine against Burkholderia mallei

    PubMed Central

    Gregory, Anthony E.; Judy, Barbara M.; Qazi, Omar; Blumentritt, Carla A.; Brown, Katherine A.; Shaw, Andrew M.; Torres, Alfredo G.; Titball, Richard W.

    2014-01-01

    Burkholderia mallei are Gram-negative bacteria, responsible for the disease glanders. B. mallei has recently been classified as a Tier 1 agent owing to the fact that this bacterial species can be weaponised for aerosol release, has a high mortality rate and demonstrates multi-drug resistance. Furthermore, there is no licensed vaccine available against this pathogen. Lipopolysaccharide (LPS) has previously been identified as playing an important role in generating host protection against Burkholderia infection. In this study, we present gold nanoparticles (AuNPs) functionalised with a glycoconjugate vaccine against glanders. AuNPs were covalently coupled with one of three different protein carriers (TetHc, Hcp1 and FliC) followed by conjugation to LPS purified from a non-virulent clonal relative, B. thailandensis. Glycoconjugated LPS generated significantly higher antibody titres and compared with LPS alone. Further, they improved protection against a lethal inhalation challenge of B. mallei in the murine model of infection. PMID:25194998

  8. Corneal ulcer caused by Pseudomonas pseudomallei: report of three cases.

    PubMed

    Siripanthong, S; Teerapantuwat, S; Prugsanusak, W; Suputtamongkol, Y; Viriyasithavat, P; Chaowagul, W; Dance, D A; White, N J

    1991-01-01

    We report three cases of corneal ulcer caused by Pseudomonas pseudomallei. In all cases corneal trauma preceded the development of extensive ulcers, subconjunctival abscesses, and hypopyon. Treatment for a total of 8 weeks with topical and/or parenteral ceftazidime followed by amoxicillin-clavulanic acid produced resolution of infection in each case. PMID:2041966

  9. The relationship of biofilm production to biocontrol activity of Burkholderia pyrrocinia FP62

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foliar biocontrol agent (BCA) efficacy is often inconsistent due to poor colonization and survival on plant surfaces. Burkholderia pyrrocinia FP62, a superior leaf colonist and BCA of Botrytis cinerea, forms unsaturated biofilms on plant surfaces. To determine the relationship between biocontrol act...

  10. Biocidal and Sporicidal Efficacy of Pathoster(®) 0.35% and Pathoster(®) 0.50% Against Bacterial Agents in Potential Bioterrorism Use.

    PubMed

    Candeliere, Antonio; Campese, Emanuele; Donatiello, Adelia; Pagano, Stefania; Iatarola, Michela; Tolve, Francesco; Antonino, Leonardo; Fasanella, Antonio

    2016-01-01

    The use of products that can neutralize or significantly reduce the microbial load and that are not harmful to human health and the environment represents a milestone in the fight against the spread of infectious diseases. Peracetic acid, besides being an excellent sterilizing and sporicidal agent, is harmless to humans and the environment when it is used in a common dosage. However, the high costs and loss of efficacy of the product very quickly after its reconstitution limit its use. We evaluated the efficacy and stability of 2 commercial products, based on stabilized peracetic acid (Pathoster(®) 0.35% and Pathoster(®) 0.50%) used against spores of Bacillus anthracis and spores of Bacillus cereus and vegetative forms of Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Brucella abortus, and Brucella melitensis. The efficacy tests were based on the direct contact of the products with a standard suspension of the bacteria. The stability of the products was defined as the period of time during which the biocidal and sporicidal properties remained unchanged. The limit of effectiveness was the period after which the product was unable to exert a complete sterilization after a contact of 5 minutes with at least 1 of the 8 bacteria used in this work. Both formulations showed good efficacy against the microorganisms used in the study, confirming the utility of peracetic acid as a sterilizing product. After the reconstitution, Pathoster(®) 0.35% was stable until 16±1 days, while Pathoster(®) 0.50% was stable until 24±1 days. The formulations used in this study showed good performance and a significant stability of peracetic acid. PMID:27482880

  11. Biocidal and Sporicidal Efficacy of Pathoster® 0.35% and Pathoster® 0.50% Against Bacterial Agents in Potential Bioterrorism Use

    PubMed Central

    Candeliere, Antonio; Donatiello, Adelia; Pagano, Stefania; Iatarola, Michela; Tolve, Francesco; Antonino, Leonardo; Fasanella, Antonio

    2016-01-01

    The use of products that can neutralize or significantly reduce the microbial load and that are not harmful to human health and the environment represents a milestone in the fight against the spread of infectious diseases. Peracetic acid, besides being an excellent sterilizing and sporicidal agent, is harmless to humans and the environment when it is used in a common dosage. However, the high costs and loss of efficacy of the product very quickly after its reconstitution limit its use. We evaluated the efficacy and stability of 2 commercial products, based on stabilized peracetic acid (Pathoster® 0.35% and Pathoster® 0.50%) used against spores of Bacillus anthracis and spores of Bacillus cereus and vegetative forms of Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Brucella abortus, and Brucella melitensis. The efficacy tests were based on the direct contact of the products with a standard suspension of the bacteria. The stability of the products was defined as the period of time during which the biocidal and sporicidal properties remained unchanged. The limit of effectiveness was the period after which the product was unable to exert a complete sterilization after a contact of 5 minutes with at least 1 of the 8 bacteria used in this work. Both formulations showed good efficacy against the microorganisms used in the study, confirming the utility of peracetic acid as a sterilizing product. After the reconstitution, Pathoster® 0.35% was stable until 16±1 days, while Pathoster® 0.50% was stable until 24±1 days. The formulations used in this study showed good performance and a significant stability of peracetic acid. PMID:27482880

  12. Implications of amino acid substitutions in GyrA at position 83 in terms of oxolinic acid resistance in field isolates of Burkholderia glumae, a causal agent of bacterial seedling rot and grain rot of rice.

    PubMed

    Maeda, Yukiko; Kiba, Akinori; Ohnishi, Kouhei; Hikichi, Yasufumi

    2004-09-01

    Oxolinic acid (OA), a quinolone, inhibits the activity of DNA gyrase composed of GyrA and GyrB and shows antibacterial activity against Burkholderia glumae. Since B. glumae causes bacterial seedling rot and grain rot of rice, both of which are devastating diseases, the emergence of OA-resistant bacteria has important implications on rice cultivation in Japan. Based on the MIC of OA, 35 B. glumae field isolates isolated from rice seedlings grown from OA-treated seeds in Japan were divided into sensitive isolates (OSs; 0.5 microg/ml), moderately resistant isolates (MRs; 50 microg/ml), and highly resistant isolates (HRs; > or =100 microg/ml). Recombination with gyrA of an OS, Pg-10, led MRs and HRs to become OA susceptible, suggesting that gyrA mutations are involved in the OA resistance of field isolates. The amino acid at position 83 in the GyrA of all OSs was Ser, but in all MRs and HRs it was Arg and Ile, respectively. Ser83Arg and Ser83Ile substitutions in the GyrA of an OS, Pg-10, resulted in moderate and high OA resistance, respectively. Moreover, Arg83Ser and Ile83Ser substitutions in the GyrA of MRs and HRs, respectively, resulted in susceptibility to OA. These results suggest that Ser83Arg and Ser83Ile substitutions in GyrA are commonly responsible for resistance to OA in B. glumae field isolates. PMID:15345450

  13. DIFFERENTIATION OF EXOTOXIN AND OTHER BIOLOGICALLY ACTIVE SUBSTANCES IN PSEUDOMONAS PSEUDOMALLEI FILTRATES

    PubMed Central

    Heckly, Robert J.

    1964-01-01

    Heckly, Robert J. (University of California, Berkeley). Differentiation of exotoxin and other biologically active substances in Pseudomonas pseudomallei filtrates. J. Bacteriol. 88:1730–1736. 1964.—Denaturing agents such as phenol, formaldehyde, and urea reduced lethal toxicity and proteolytic activity of partially purified preparations from Pseudomonas pseudomallei at about the same rate. Neither toxin nor enzyme was stable at pH 11, when the solution was adjusted with sodium hydroxide, but there was a slight difference in their rates of inactivation. However, under certain conditions, ammonium hydroxide destroyed most of the enzymatic activity with only a slight effect on lethality. Conversely, toxin was less stable in acid solutions than was the enzyme. Thus, treatment with ammonium hydroxide or acetic acid yielded preparations with either a low or a high enzyme-to-toxin ratio, indicating that lethality was not dependent on enzyme activity. Although proteolysis of any one of the essential factors in the blood coagulation system can inhibit clotting of blood, the potent anticoagulant activity of culture filtrates was not associated with its proteolytic activity, but was directly correlated with lethal toxicity. It is of considerable interest that the necrotoxicity was, however, associated with enzymatic activity and not with lethality. Serological reactivity of the enzyme, as well as its proteolytic activity, was altered by ammonium hydroxide. Similarly, antigenicity and toxicity of the lethal toxin were reduced by acidification. Each acid- or alkali-treated preparation produced a single precipitin line in double diffusion in agar when reacted with antisera produced by injection of crude filtrate. Partially purified preparations, having both lethal and enzymatic activity, produced two lines, one identifiable with the enzyme preparation, and one with the toxin. Furthermore, specific precipitation with the respective antisera removed either enzyme or toxin from

  14. Comparative genome-wide analysis reveals that Burkholderia contaminans MS14 possesses multiple antimicrobial biosynthesis genes but not major genetic loci required for pathogenesis.

    PubMed

    Deng, Peng; Wang, Xiaoqiang; Baird, Sonya M; Showmaker, Kurt C; Smith, Leif; Peterson, Daniel G; Lu, Shien

    2016-06-01

    Burkholderia contaminans MS14 shows significant antimicrobial activities against plant and animal pathogenic fungi and bacteria. The antifungal agent occidiofungin produced by MS14 has great potential for development of biopesticides and pharmaceutical drugs. However, the use of Burkholderia species as biocontrol agent in agriculture is restricted due to the difficulties in distinguishing between plant growth-promoting bacteria and the pathogenic bacteria. The complete MS14 genome was sequenced and analyzed to find what beneficial and virulence-related genes it harbors. The phylogenetic relatedness of B. contaminans MS14 and other 17 Burkholderia species was also analyzed. To research MS14's potential virulence, the gene regions related to the antibiotic production, antibiotic resistance, and virulence were compared between MS14 and other Burkholderia genomes. The genome of B. contaminans MS14 was sequenced and annotated. The genomic analyses reveal the presence of multiple gene sets for antimicrobial biosynthesis, which contribute to its antimicrobial activities. BLAST results indicate that the MS14 genome harbors a large number of unique regions. MS14 is closely related to another plant growth-promoting Burkholderia strain B. lata 383 according to the average nucleotide identity data. Moreover, according to the phylogenetic analysis, plant growth-promoting species isolated from soils and mammalian pathogenic species are clustered together, respectively. MS14 has multiple antimicrobial activity-related genes identified from the genome, but it lacks key virulence-related gene loci found in the pathogenic strains. Additionally, plant growth-promoting Burkholderia species have one or more antimicrobial biosynthesis genes in their genomes as compared with nonplant growth-promoting soil-isolated Burkholderia species. On the other hand, pathogenic species harbor multiple virulence-associated gene loci that are not present in nonpathogenic Burkholderia species. The MS14

  15. Burkholderia humisilvae sp. nov., Burkholderia solisilvae sp. nov. and Burkholderia rhizosphaerae sp. nov., isolated from forest soil and rhizosphere soil.

    PubMed

    Lee, Jae-Chan; Whang, Kyung-Sook

    2015-09-01

    Strains Y-12(T) and Y-47(T) were isolated from mountain forest soil and strain WR43(T) was isolated from rhizosphere soil, at Daejeon, Korea. The three strains grew at 10-55 °C (optimal growth at 28-30 °C), at pH 3.0-8.0 (optimal growth at pH 6.0) and in the presence of 0-4.0% (w/v) NaCl, growing optimally in the absence of added NaCl. On the basis of 16S rRNA gene sequence analysis, the three strains were found to belong to the genus Burkholderia, showing the closest phylogenetic similarity to Burkholderia diazotrophica JPY461(T) (97.2-97.7%); the similarity between the three sequences ranged from 98.3 to 98.7%. Additionally, the three strains formed a distinct group in phylogenetic trees based on the housekeeping genes recA and gyrB. The predominant ubiquinone was Q-8, the major fatty acids were C16 : 0 and C17  : 0 cyclo and the DNA G+C content of the novel isolates was 61.6-64.4 mol%. DNA-DNA relatedness among the three strains and the type strains of the closest species of the genus Burkholderia was less than 50%. On the basis of 16S rRNA, recA and gyrB gene sequence similarities, chemotaxonomic and phenotypic data, the three strains represent three novel species within the genus Burkholderia, for which the names Burkholderia humisilvae sp. nov. (type strain Y-12(T)= KACC 17601(T) = NBRC 109933(T) = NCAIM B 02543(T)), Burkholderia solisilvae sp. nov. (type strain Y-47(T) = KACC 17602(T)= NBRC 109934(T) = NCAIM B 02539(T)) and Burkholderia rhizosphaerae sp. nov. (type strain WR43(T) = KACC 17603(T) = NBRC 109935(T) = NCAIM B 02541(T)) are proposed. PMID:26031294

  16. 40 CFR 725.1075 - Burkholderia cepacia complex.

    Code of Federal Regulations, 2010 CFR

    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...

  17. Gene and protein expression in response to different growth temperatures and oxygen availability in Burkholderia thailandensis.

    PubMed

    Peano, Clelia; Chiaramonte, Fabrizio; Motta, Sara; Pietrelli, Alessandro; Jaillon, Sebastien; Rossi, Elio; Consolandi, Clarissa; Champion, Olivia L; Michell, Stephen L; Freddi, Luca; Falciola, Luigi; Basilico, Fabrizio; Garlanda, Cecilia; Mauri, Pierluigi; De Bellis, Gianluca; Landini, Paolo

    2014-01-01

    Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors. PMID:24671187

  18. Gene and Protein Expression in Response to Different Growth Temperatures and Oxygen Availability in Burkholderia thailandensis

    PubMed Central

    Peano, Clelia; Chiaramonte, Fabrizio; Motta, Sara; Pietrelli, Alessandro; Jaillon, Sebastien; Rossi, Elio; Consolandi, Clarissa; Champion, Olivia L.; Michell, Stephen L.; Freddi, Luca; Falciola, Luigi; Basilico, Fabrizio; Garlanda, Cecilia; Mauri, Pierluigi; De Bellis, Gianluca; Landini, Paolo

    2014-01-01

    Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors. PMID:24671187

  19. Viperatoxin-II: A novel viper venom protein as an effective bactericidal agent

    PubMed Central

    Samy, Ramar Perumal; Stiles, Bradley G.; Chinnathambi, Arunachalam; Zayed, M.E.; Alharbi, Sulaiman Ali; Franco, Octavio Luiz; Rowan, Edward G.; Kumar, Alan Prem; Lim, Lina H.K.; Sethi, Gautam

    2015-01-01

    Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have become a rising threat to public health. There is an urgent need for development of promising new therapeutic agents against drug resistant bacteria like S. aureus. This report discusses purification and characterization of proteins from Indian Russell’s viper snake venom. Novel 15-kDa proteins called “Viperatoxin” (VipTx-I and VipTx-II) were extracted from the whole venom and evaluated using in vitro antimicrobial experiments. The N-terminal amino acid sequence of “Viperatoxin” showed high sequence homology to daboiatoxin isolated from the same venom and also matched phospholipase A2 (PLA2) enzymes isolated from other snake venoms. In an in vitro plate assay, VipTx-II but not VipTx-I showed strong antimicrobial effects against S. aureus and Burkholderia pseudomallei (KHW & TES), Proteus vulgaris and P. mirabilis. The VipTx-II was further tested by a broth-dilution assay at 100–3.1 μg/ml concentrations. The most potent bactericidal effect was found at the lowest dilutions (MICs of 6.25 μg/ml) against B. pseudomallei, S. aureus and P. vulgaris (MICs of 12.25 μg/ml). Electron microscopic investigation revealed that the protein-induced bactericidal potency was closely associated with pore formation and membrane damage, even at the lowest concentrations (<20 μg/ml). The toxin caused a low level of cytotoxic effects as observed in human (THP-1) cells at higher concentrations. Molecular weight determinations of VipTx-II by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one major, along with a few minor bands. The results indicate that VipTx-II plays a significant role in bactericidal and membrane damaging effects in vitro. Non-cytotoxic properties on human cells highlight it as a promising candidate for further evaluation of antimicrobial potential in vivo. PMID:26793432

  20. Host Evasion by Burkholderia cenocepacia

    PubMed Central

    Ganesan, Shyamala; Sajjan, Umadevi S.

    2012-01-01

    Burkholderia cenocepacia is an opportunistic respiratory pathogen of individuals with cystic fibrosis (CF). Some strains of B. cenocepacia are highly transmissible and resistant to almost all antibiotics. Approximately one-third of B. cenocepacia infected CF patients go on to develop fatal “cepacia syndrome.” During the last two decades, substantial progress has been made with regards to evasion of host innate defense mechanisms by B. cenocepacia. Almost all strains of B. cenocepacia have the capacity to survive and replicate intracellularly in both airway epithelial cells and macrophages, which are primary sentinels of the lung and play a pivotal role in clearance of infecting bacteria. Those strains of B. cenocepacia, which express both cable pili and the associated 22 kDa adhesin are also capable of transmigrating across airway epithelium and persist in mouse models of infection. In this review, we will discuss how this type of interaction between B. cenocepacia and host may lead to persistence of bacteria as well as lung inflammation in CF patients. PMID:22919590

  1. Host evasion by Burkholderia cenocepacia.

    PubMed

    Ganesan, Shyamala; Sajjan, Umadevi S

    2011-01-01

    Burkholderia cenocepacia is an opportunistic respiratory pathogen of individuals with cystic fibrosis (CF). Some strains of B. cenocepacia are highly transmissible and resistant to almost all antibiotics. Approximately one-third of B. cenocepacia infected CF patients go on to develop fatal "cepacia syndrome." During the last two decades, substantial progress has been made with regards to evasion of host innate defense mechanisms by B. cenocepacia. Almost all strains of B. cenocepacia have the capacity to survive and replicate intracellularly in both airway epithelial cells and macrophages, which are primary sentinels of the lung and play a pivotal role in clearance of infecting bacteria. Those strains of B. cenocepacia, which express both cable pili and the associated 22 kDa adhesin are also capable of transmigrating across airway epithelium and persist in mouse models of infection. In this review, we will discuss how this type of interaction between B. cenocepacia and host may lead to persistence of bacteria as well as lung inflammation in CF patients. PMID:22919590

  2. Strains from the Burkholderia cepacia Complex: Relationship to Opportunistic Pathogens

    PubMed Central

    Vandamme, Peter; Mahenthiralingam, Eshwar

    2003-01-01

    Burkholderia cepacia-like organisms attract much interest from the agricultural industry as natural promoters of plant growth and biological control agents, and for bioremediation. Some of these organisms, however, cause life-threatening infections, particularly in cystic fibrosis patients for whom this multi-resistant bacterium is a major pathogen. The biodiversity of this group of bacteria is severely underestimated, and current identification procedures are inadequate. Presumed B. cepacia isolates belong to at least nine distinct genomic species (genomovars), referred to collectively as the B. cepacia complex. All these B. cepacia complex genomovars have been isolated from clinical and environmental sources. There are no phenotypic, genomic, or taxonomic grounds to differentiate environmental and clinical strains of the B. cepacia complex or to use the source of isolation to assess the safety of biopesticides containing members of the B. cepacia complex. PMID:19265996

  3. Use of the Common Marmoset to Study Burkholderia mallei Infection

    PubMed Central

    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 104 to 2.5 X 105 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 103 bacteria exhibited mild non-specific signs of illness and were culture negative. One marmoset inoculated with 2.5 X 103 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. PMID

  4. Natural Burkholderia mallei Infection in Dromedary, Bahrain

    PubMed Central

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

    2011-01-01

    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. PMID:21762586

  5. GENOME ANALYSIS OF BURKHOLDERIA CEPACIA AC1100

    EPA Science Inventory

    Burkholderia cepacia is an important organism in bioremediation of environmental pollutants and it is also of increasing interest as a human pathogen. The genomic organization of B. cepacia is being studied in order to better understand its unusual adaptive capacity and genome pl...

  6. A polar-localized iron-binding protein determines the polar targeting of Burkholderia BimA autotransporter and actin tail formation.

    PubMed

    Lu, Qiuhe; Xu, Yue; Yao, Qing; Niu, Miao; Shao, Feng

    2015-03-01

    Intracellular bacterial pathogens including Shigella, Listeria, Mycobacteria, Rickettsia and Burkholderia spp. deploy a specialized surface protein onto one pole of the bacteria to induce filamentous actin tail formation for directional movement within host cytosol. The mechanism underlying polar targeting of the actin tail proteins is unknown. Here we perform a transposon screen in Burkholderia thailandensis and identify a conserved bimC that is required for actin tail formation mediated by BimA from B. thailandensis and its closely related pathogenic species B. pseudomallei and B. mallei. bimC is located upstream of bimA in the same operon. Loss of bimC results in even distribution of BimA on the outer membrane surface, where actin polymerization still occurs. BimC is targeted to the same bacterial pole independently of BimA. BimC confers polar targeting of BimA prior to BimA translocation across bacterial inner membrane. BimC is an iron-binding protein, requiring a four-cysteine cluster at the carboxyl terminus. Mutation of the cysteine cluster disrupts BimC polar localization. Truncation analyses identify the transmembrane domain in BimA being responsible for its polar targeting. Consistently, BimC can interact with BimA transmembrane domain in an iron binding-dependent manner. Our study uncovers a new mechanism that determines the polar distribution of bacteria-induced actin tail in infected host cells. PMID:25293534

  7. Comparative metabolic systems analysis of pathogenic Burkholderia.

    PubMed

    Bartell, Jennifer A; Yen, Phillip; Varga, John J; Goldberg, Joanna B; Papin, Jason A

    2014-01-01

    Burkholderia cenocepacia and Burkholderia multivorans are opportunistic drug-resistant pathogens that account for the majority of Burkholderia cepacia complex infections in cystic fibrosis patients and also infect other immunocompromised individuals. While they share similar genetic compositions, B. cenocepacia and B. multivorans exhibit important differences in pathogenesis. We have developed reconciled genome-scale metabolic network reconstructions of B. cenocepacia J2315 and B. multivorans ATCC 17616 in parallel (designated iPY1537 and iJB1411, respectively) to compare metabolic abilities and contextualize genetic differences between species. The reconstructions capture the metabolic functions of the two species and give insight into similarities and differences in their virulence and growth capabilities. The two reconstructions have 1,437 reactions in common, and iPY1537 and iJB1411 have 67 and 36 metabolic reactions unique to each, respectively. After curating the extensive reservoir of metabolic genes in Burkholderia, we identified 6 genes essential to growth that are unique to iPY1513 and 13 genes uniquely essential to iJB1411. The reconstructions were refined and validated by comparing in silico growth predictions to in vitro growth capabilities of B. cenocepacia J2315, B. cenocepacia K56-2, and B. multivorans ATCC 17616 on 104 carbon sources. Overall, we identified functional pathways that indicate B. cenocepacia can produce a wider array of virulence factors compared to B. multivorans, which supports the clinical observation that B. cenocepacia is more virulent than B. multivorans. The reconciled reconstructions provide a framework for generating and testing hypotheses on the metabolic and virulence capabilities of these two related emerging pathogens. PMID:24163337

  8. Comparative Metabolic Systems Analysis of Pathogenic Burkholderia

    PubMed Central

    Bartell, Jennifer A.; Yen, Phillip; Varga, John J.; Goldberg, Joanna B.

    2014-01-01

    Burkholderia cenocepacia and Burkholderia multivorans are opportunistic drug-resistant pathogens that account for the majority of Burkholderia cepacia complex infections in cystic fibrosis patients and also infect other immunocompromised individuals. While they share similar genetic compositions, B. cenocepacia and B. multivorans exhibit important differences in pathogenesis. We have developed reconciled genome-scale metabolic network reconstructions of B. cenocepacia J2315 and B. multivorans ATCC 17616 in parallel (designated iPY1537 and iJB1411, respectively) to compare metabolic abilities and contextualize genetic differences between species. The reconstructions capture the metabolic functions of the two species and give insight into similarities and differences in their virulence and growth capabilities. The two reconstructions have 1,437 reactions in common, and iPY1537 and iJB1411 have 67 and 36 metabolic reactions unique to each, respectively. After curating the extensive reservoir of metabolic genes in Burkholderia, we identified 6 genes essential to growth that are unique to iPY1513 and 13 genes uniquely essential to iJB1411. The reconstructions were refined and validated by comparing in silico growth predictions to in vitro growth capabilities of B. cenocepacia J2315, B. cenocepacia K56-2, and B. multivorans ATCC 17616 on 104 carbon sources. Overall, we identified functional pathways that indicate B. cenocepacia can produce a wider array of virulence factors compared to B. multivorans, which supports the clinical observation that B. cenocepacia is more virulent than B. multivorans. The reconciled reconstructions provide a framework for generating and testing hypotheses on the metabolic and virulence capabilities of these two related emerging pathogens. PMID:24163337

  9. Functional and genomic insights into the pathogenesis of Burkholderia species to rice.

    PubMed

    Naughton, Lynn M; An, Shi-Qi; Hwang, Ingyu; Chou, Shan-Ho; He, Yong-Qiang; Tang, Ji-Liang; Ryan, Robert P; Dow, J Maxwell

    2016-03-01

    A number of species of bacteria from the genus Burkholderia have been shown to be causal agents of diseases of rice. These diseases, caused by Burkholderia glumae, B. gladioli and B. plantarii, are becoming increasingly common across the globe. This is particularly so for B. glumae, whose ability to grow at elevated temperatures suggests that it may become a prevalent problem in an era of global warming. Despite the increasing threat to rice, relatively little is known about the virulence mechanisms employed by these pathogens. Work over the last 5 years has provided an increasing insight into these factors and their control by environmental and other cues. In addition, the determination of a number of genome sequences has allowed bioinformatic predictions of further possible mechanisms, which can now be investigated experimentally. Here, we review recent advances in the understanding of virulence of Burkholderia to rice, to include discussion of the roles of toxins, type II secreted enzymes, type III secreted effectors and motility as well as their regulation by quorum sensing, two-component systems and cyclic di-GMP signalling. Finally, we consider a number of approaches for the control of bacterial virulence through the modulation of quorum sensing and toxin degradation. PMID:26690879

  10. 40 CFR 725.1075 - Burkholderia cepacia complex.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS Significant New Uses for 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...

  11. Development of a loop-mediated isothermal amplification assay for rapid detection of Burkholderia mallei.

    PubMed

    Mirzai, S; Safi, S; Mossavari, N; Afshar, D; Bolourchian, M

    2016-01-01

    The present study was conducted to establish a Loop-mediated isothermal amplification (LAMP) technique for the rapid detection of B. mallei the etiologic agent of glanders, a highly contagious disease of equines. A set of six specific primers targeting integrase gene cluster were designed for the LAMP test. The reaction was optimized using different temperatures and time intervals. The specificity of the assay was evaluated using DNA from B.pseudomallei and Pseudomonas aeruginosa. The LAMP products were analyzed both visually and under UV light after electrophoresis. The optimized conditions were found to be at 63ºC for 60 min. The assay showed high specificity and sensitivity. It was concluded that the established LAMP assay is a rapid, sensitive and practical tool for detection of B. mallei and early diagnosis of glanders. PMID:27609471

  12. The promise of bacteriophage therapy for Burkholderia cepacia complex respiratory infections.

    PubMed

    Semler, Diana D; Lynch, Karlene H; Dennis, Jonathan J

    2011-01-01

    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 investigation is not only comprised of phage isolation, in vitro phage characterization and assessment of in vivo infection model efficacy, but also adapting aerosol drug delivery techniques to aerosol phage formulation delivery and storage. PMID:22919592

  13. The Promise of Bacteriophage Therapy for Burkholderia cepacia Complex Respiratory Infections

    PubMed Central

    Semler, Diana D.; Lynch, Karlene H.; Dennis, Jonathan J.

    2012-01-01

    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 investigation is not only comprised of phage isolation, in vitro phage characterization and assessment of in vivo infection model efficacy, but also adapting aerosol drug delivery techniques to aerosol phage formulation delivery and storage. PMID:22919592

  14. Incidence of Burkholderia mallei infection among indigenous equines in India

    PubMed Central

    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

  15. Indole-3-Acetic Acid Produced by Burkholderia heleia Acts as a Phenylacetic Acid Antagonist to Disrupt Tropolone Biosynthesis in Burkholderia plantarii

    PubMed Central

    Wang, Mengcen; Tachibana, Seiji; Murai, Yuta; Li, Li; Lau, Sharon Yu Ling; Cao, Mengchao; Zhu, Guonian; Hashimoto, Makoto; Hashidoko, Yasuyuki

    2016-01-01

    Burkholderia heleia PAK1-2 is a potent biocontrol agent isolated from rice rhizosphere, as it prevents bacterial rice seedling blight disease caused by Burkholderia plantarii. Here, we isolated a non-antibacterial metabolite from the culture fluid of B. heleia PAK1-2 that was able to suppress B. plantarii virulence and subsequently identified as indole-3-acetic acid (IAA). IAA suppressed the production of tropolone in B. plantarii in a dose-dependent manner without any antibacterial and quorum quenching activity, suggesting that IAA inhibited steps of tropolone biosynthesis. Consistent with this, supplementing cultures of B. plantarii with either L-[ring-2H5]phenylalanine or [ring-2H2~5]phenylacetic acid revealed that phenylacetic acid (PAA), which is the dominant metabolite during the early growth stage, is a direct precursor of tropolone. Exposure of B. plantarii to IAA suppressed production of both PAA and tropolone. These data particularly showed that IAA produced by B. heleia PAK1-2 disrupts tropolone production during bioconversion of PAA to tropolone via the ring-rearrangement on the phenyl group of the precursor to attenuate the virulence of B. plantarii. B. heleia PAK1-2 is thus likely a microbial community coordinating bacterium in rhizosphere ecosystems, which never eliminates phytopathogens but only represses production of phytotoxins or bacteriocidal substances. PMID:26935539

  16. Indole-3-Acetic Acid Produced by Burkholderia heleia Acts as a Phenylacetic Acid Antagonist to Disrupt Tropolone Biosynthesis in Burkholderia plantarii.

    PubMed

    Wang, Mengcen; Tachibana, Seiji; Murai, Yuta; Li, Li; Lau, Sharon Yu Ling; Cao, Mengchao; Zhu, Guonian; Hashimoto, Makoto; Hashidoko, Yasuyuki

    2016-01-01

    Burkholderia heleia PAK1-2 is a potent biocontrol agent isolated from rice rhizosphere, as it prevents bacterial rice seedling blight disease caused by Burkholderia plantarii. Here, we isolated a non-antibacterial metabolite from the culture fluid of B. heleia PAK1-2 that was able to suppress B. plantarii virulence and subsequently identified as indole-3-acetic acid (IAA). IAA suppressed the production of tropolone in B. plantarii in a dose-dependent manner without any antibacterial and quorum quenching activity, suggesting that IAA inhibited steps of tropolone biosynthesis. Consistent with this, supplementing cultures of B. plantarii with either L-[ring-(2)H5]phenylalanine or [ring-(2)H2~5]phenylacetic acid revealed that phenylacetic acid (PAA), which is the dominant metabolite during the early growth stage, is a direct precursor of tropolone. Exposure of B. plantarii to IAA suppressed production of both PAA and tropolone. These data particularly showed that IAA produced by B. heleia PAK1-2 disrupts tropolone production during bioconversion of PAA to tropolone via the ring-rearrangement on the phenyl group of the precursor to attenuate the virulence of B. plantarii. B. heleia PAK1-2 is thus likely a microbial community coordinating bacterium in rhizosphere ecosystems, which never eliminates phytopathogens but only represses production of phytotoxins or bacteriocidal substances. PMID:26935539

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

    PubMed

    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. PMID:27271739

  18. Environmental Transmission of the Gut Symbiont Burkholderia to Phloem-Feeding Blissus insularis.

    PubMed

    Xu, Yao; Buss, Eileen A; Boucias, Drion G

    2016-01-01

    The plant-phloem-feeding Blissus insularis possesses specialized midgut crypts, which harbor a dense population of the exocellular bacterial symbiont Burkholderia. Most individual B. insularis harbor a single Burkholderia ribotype in their midgut crypts; however, a diverse Burkholderia community exists within a host population. To understand the mechanism underlying the consistent occurrence of various Burkholderia in B. insularis and their specific association, we investigated potential gut symbiont transmission routes. PCR amplification detected a low titer of Burkholderia in adult reproductive tracts; however, fluorescence in situ hybridization assays failed to produce detectable signals in these tracts. Furthermore, no Burkholderia-specific PCR signals were detected in eggs and neonates, suggesting that it is unlikely that B. insularis prenatally transmits gut symbionts via ovarioles. In rearing experiments, most nymphs reared on St. Augustinegrass treated with cultured Burkholderia harbored the cultured Burkholderia strains. Burkholderia was detected in the untreated host grass of B. insularis, and most nymphs reared on untreated grass harbored a Burkholderia ribotype that was closely related to a plant-associated Burkholderia strain. These findings revealed that B. insularis neonates acquired Burkholderia primarily from the environment (i.e., plants and soils), even though the possibility of acquisition via egg surface cannot be excluded. In addition, our study explains how the diverse Burkholderia symbiont community in B. insularis populations can be maintained. PMID:27548682

  19. Environmental Transmission of the Gut Symbiont Burkholderia to Phloem-Feeding Blissus insularis

    PubMed Central

    Xu, Yao; Buss, Eileen A.; Boucias, Drion G.

    2016-01-01

    The plant-phloem-feeding Blissus insularis possesses specialized midgut crypts, which harbor a dense population of the exocellular bacterial symbiont Burkholderia. Most individual B. insularis harbor a single Burkholderia ribotype in their midgut crypts; however, a diverse Burkholderia community exists within a host population. To understand the mechanism underlying the consistent occurrence of various Burkholderia in B. insularis and their specific association, we investigated potential gut symbiont transmission routes. PCR amplification detected a low titer of Burkholderia in adult reproductive tracts; however, fluorescence in situ hybridization assays failed to produce detectable signals in these tracts. Furthermore, no Burkholderia-specific PCR signals were detected in eggs and neonates, suggesting that it is unlikely that B. insularis prenatally transmits gut symbionts via ovarioles. In rearing experiments, most nymphs reared on St. Augustinegrass treated with cultured Burkholderia harbored the cultured Burkholderia strains. Burkholderia was detected in the untreated host grass of B. insularis, and most nymphs reared on untreated grass harbored a Burkholderia ribotype that was closely related to a plant-associated Burkholderia strain. These findings revealed that B. insularis neonates acquired Burkholderia primarily from the environment (i.e., plants and soils), even though the possibility of acquisition via egg surface cannot be excluded. In addition, our study explains how the diverse Burkholderia symbiont community in B. insularis populations can be maintained. PMID:27548682

  20. Burkholderia cepacia Complex as Human Pathogens

    PubMed Central

    LiPuma, John J.

    2003-01-01

    Although sporadic human infection due to Burkholderia cepacia has been reported for many years, it has been only during the past few decades that species within the B. cepacia complex have emerged as significant opportunistic human pathogens. Individuals with cystic fibrosis, the most common inherited genetic disease in Caucasian populations, or chronic granulomatous disease, a primary immunodeficiency, are particularly at risk of life-threatening infection. Despite advances in our understanding of the taxonomy, microbiology, and epidemiology of B. cepacia complex, much remains unknown regarding specific human virulence factors. The broad-spectrum antimicrobial resistance demonstrated by most strains limits current therapy of infection. Recent research efforts are aimed at a better appreciation of the pathogenesis of human infection and the development of novel therapeutic and prophylactic strategies. PMID:19265997

  1. 42 CFR 73.9 - Responsible Official.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Clostridium, Burkholderia mallei, Burkholderia pseudomallei Francisella tularensis, Ebola viruses, , Marburg virus, Variola major virus (Smallpox virus), Variola minor (Alastrim), or Yersinia pestis. The...

  2. 42 CFR 73.9 - Responsible Official.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Clostridium, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Ebola viruses, Marburg virus, Variola major virus (Smallpox virus), Variola minor (Alastrim), or Yersinia pestis. The...

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

    PubMed Central

    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

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

    PubMed Central

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

    2015-01-01

    Background 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. Methodology/Principal Findings 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 104 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. Conclusions/Significance 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. PMID:26114445

  5. Distinguishing species of the Burkholderia cepacia complex and Burkholderia gladioli by automated ribotyping.

    PubMed

    Brisse, S; Verduin, C M; Milatovic, D; Fluit, A; Verhoef, J; Laevens, S; Vandamme, P; Tümmler, B; Verbrugh, H A; van Belkum, A

    2000-05-01

    Several species belonging to the genus Burkholderia are clinically relevant, opportunistic pathogens that inhabit major environmental reservoirs. Consequently, the availability of means for adequate identification and epidemiological characterization of individual environmental or clinical isolates is mandatory. In the present communication we describe the use of the Riboprinter microbial characterization system (Qualicon, Warwick, United Kingdom) for automated ribotyping of 104 strains of Burkholderia species from diverse sources, including several publicly accessible collections. The main outcome of this analysis was that all strains were typeable and that strains of Burkholderia gladioli and of each species of the B. cepacia complex, including B. multivorans, B. stabilis, and B. vietnamiensis, were effectively discriminated. Furthermore, different ribotypes were discerned within each species. Ribotyping results were in general agreement with strain classification based on restriction fragment analysis of 16S ribosomal amplicons, but the resolution of ribotyping was much higher. This enabled automated molecular typing below the species level. Cluster analysis of the patterns obtained by ribotyping (riboprints) showed that within B. gladioli, B. multivorans, and B. cepacia genomovar VI, the different riboprints identified always clustered together. Riboprints of B. cepacia genomovars I and III, B. stabilis, and B. vietnamiensis did not show distinct clustering but rather exhibited the formation of loose assemblages within which several smaller, genomovar-specific clusters were delineated. Therefore, ribotyping proved useful for genomovar identification. Analysis of serial isolates from individual patients demonstrated that infection with a single ribotype had occurred, despite minor genetic differences that were detected by pulsed-field gel electrophoresis of DNA macrorestriction fragments. The automated approach allows very rapid and reliable identification and

  6. Isolation and characterization of an abamectin-degrading Burkholderia cepacia-like GB-01 strain.

    PubMed

    Ali, Shinawar Waseem; Li, Rong; Zhou, Wei-You; Sun, Ji-Quan; Guo, Peng; Ma, Ji-Ping; Li, Shun-Peng

    2010-06-01

    Abamectin is widely used in agriculture as an insecticide and in veterinary as an anti-parasitic agent, and has caused great environmental pollution by posing potential risk to non-target soil invertebrates and nearby aquatic systems. A bacterium designated GB-01, which was capable of degrading abamectin, was isolated from soil by enrichment culture method. On the basis of morphological, physiological and biochemical characteristics, combined with phylogenetic analysis of 16S rRNA gene, the bacterium GB-01 was identified as Burkholderia cepacia-like species. The bacterium GB-01 was able to utilize abamectin as its sole carbon source for growth, and could degrade more than 90% of abamectin at initial concentrations of 50 and 100 mg l(-1) in mineral salt medium in 30 and 36 h, respectively. The longer degradation cycle was observed with abamectin concentrations higher than 100 mg l(-1). Optimal growth temperatures and pH values with highest degradation rate were 30-35 degrees C and 7-8, respectively. Two new degradation products were identified and characterized by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based mass spectral data and a plausible partial degradation pathway of abamectin was proposed. This is the first report in which an abamectin-degrading Burkholderia species isolated from soil was identified and characterized. PMID:19937266

  7. Outbreak of Subclinical Mastitis in a Flock of Dairy Sheep Associated with Burkholderia cepacia Complex Infection

    PubMed Central

    Berriatua, E.; Ziluaga, I.; Miguel-Virto, C.; Uribarren, P.; Juste, R.; Laevens, S.; Vandamme, P.; Govan, J. R. W.

    2001-01-01

    An outbreak of subclinical mastitis in a flock of 620 milking sheep was investigated. Microbiological and epidemiological analyses identified the causative agent as belonging to the Burkholderia cepacia complex (formerly Pseudomonas cepacia). Every ewe in the milking flock was individually tested for subclinical mastitis on two separate occasions, 6 weeks apart, by the California (rapid) mastitis test (CMT). The proportion of CMT-positive ewes was 69 of 393 (17.6%) on the first sampling and 27 of 490 (5.5%) on the second sampling. Pure B. cepacia cultures identified with the API 20 NE system were grown from 64 of 96 (66.7%) CMT-positive ewes and from 1 of 33 (3.0%) CMT-negative ewes. Statistical analysis confirmed the significant association between a positive CMT result and a positive culture result for B. cepacia complex. Additional polyphasic taxonomic analyses of eight isolates showed that seven belonged to B. cepacia genomovar III; the remaining isolate was identified as Burkholderia vietnamiensis (formerly B. cepacia genomovar V). Bacteriological investigation of samples from milking equipment and other environmental sites failed to identify “B. cepacia” in any of the samples taken. To our knowledge, this is the first report of an outbreak of natural infection in animals caused by B. cepacia complex and the first description of B. cepacia complex infection in sheep. PMID:11230416

  8. Resistance of the Burkholderia cepacia complex to fosmidomycin and fosmidomycin derivatives.

    PubMed

    Messiaen, Anne-Sophie; Verbrugghen, Thomas; Declerck, Charlotte; Ortmann, Regina; Schlitzer, Martin; Nelis, Hans; Van Calenbergh, Serge; Coenye, Tom

    2011-09-01

    The Burkholderia cepacia complex (BCC) is a group of 17 closely related opportunistic pathogens that are able to infect the respiratory tract of cystic fibrosis patients. BCC bacteria are intrinsically resistant to many antibiotics and are therefore difficult to eradicate. Fosmidomycin could be a new therapeutic agent to treat BCC infections as it inhibits 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in the non-mevalonate pathway essential in BCC bacteria for isoprenoid synthesis. In this study, the antimicrobial activity of fosmidomycin and eight fosmidomycin derivatives towards 40 BCC strains was investigated. All BCC strains were resistant to fosmidomycin, although addition of glucose-6-phosphate reduced the minimum inhibitory concentration values of FR900098, the fosmidomycin acetyl derivative, from 512 mg/L to 64 mg/L for Burkholderia multivorans and B. cepacia. This enhanced activity was linked to increased expression of the genes involved in glycerol-3-phosphate transport, which appears to be the only route for fosmidomycin import in BCC bacteria. Furthermore, upregulation of a fosmidomycin resistance gene (fsr) encoding an efflux pump was observed during fosmidomycin and FR900098 treatment. These results strongly suggest that the observed resistance in BCC bacteria is due to insufficient uptake accompanied by fosmidomycin and FR900098 efflux. PMID:21724375

  9. Use of a recombinant burkholderia intracellular motility a protein for immunodiagnosis of glanders.

    PubMed

    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. PMID:21752949

  10. Burkholderia cepacia Complex Vaccines: Where Do We Go from here?

    PubMed Central

    Pradenas, Gonzalo A.; Ross, Brittany N.; Torres, Alfredo G.

    2016-01-01

    Burkholderia comprises a wide variety of environmental Gram-negative bacteria. Burkholderia cepacia complex (Bcc) includes several Burkholderia species that pose a health hazard as they are able to cause respiratory infections in patients with chronic granulomatous disease and cystic fibrosis. Due to the intrinsic resistance to a wide array of antibiotics and naturally occurring immune evasion strategies, treatment of Bcc infections often proves to be unsuccessful. To date, limited work related to vaccine development has been performed for Bcc pathogens. In this review, we have gathered key aspects of Bcc research that have been reported in recent years related to vaccine efforts, virulence, immune responses, and animal models, and use this information to inform the research community of areas of opportunity toward development of a viable Bcc vaccine. PMID:27092530

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

    PubMed

    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

  12. Agricultural use of Burkholderia (Pseudomonas) cepacia: a threat to human health?

    PubMed Central

    Holmes, A.; Govan, J.; Goldstein, R.

    1998-01-01

    In the past 2 decades, Burkholderia cepacia has emerged as a human pathogen causing numerous outbreaks, particularly among cystic fibrosis (CF) patients. One highly transmissible strain has spread across North America and Britain, and another between hospitalized CF and non-CF patients. Meanwhile, the organism has been developed as a biopesticide for protecting crops against fungal diseases and has potential as a bioremediation agent for breaking down recalcitrant herbicides and pesticides. However, B. cepacia is inherently resistant to multiple antibiotics; selection of strains "safe" for environmental application is not at present possible phenotypically or genotypically; molecular epidemiology and phylogenetic studies demonstrate that highly transmissible strains emerge randomly; and the organism has a capacity for rapid mutation and adaptation (facilitated by numerous insertion sequences), and a large, complex genome divided into separate chromosomes. Therefore, the widespread agricultural use of B. cepacia should be approached with caution. PMID:9621192

  13. Burkholderia thailandensis oacA Mutants Facilitate the Expression of Burkholderia mallei-Like O Polysaccharides▿

    PubMed Central

    Brett, Paul J.; Burtnick, Mary N.; Heiss, Christian; Azadi, Parastoo; DeShazer, David; Woods, Donald E.; Gherardini, Frank C.

    2011-01-01

    Previous studies have shown that the O polysaccharides (OPS) expressed by Burkholderia mallei are similar to those produced by Burkholderia thailandensis except that they lack the 4-O-acetyl modifications on their 6-deoxy-α-l-talopyranosyl residues. In the present study, we describe the identification and characterization of an open reading frame, designated oacA, expressed by B. thailandensis that accounts for this phenomenon. Utilizing the B. thailandensis and B. mallei lipopolysaccharide (LPS)-specific monoclonal antibodies Pp-PS-W and 3D11, Western immunoblot analyses demonstrated that the LPS antigens expressed by the oacA mutant, B. thailandensis ZT0715, were antigenically similar to those produced by B. mallei ATCC 23344. In addition, immunoblot analyses demonstrated that when B. mallei ATCC 23344 was complemented in trans with oacA, it synthesized B. thailandensis-like LPS antigens. To elucidate the structure of the OPS moieties expressed by ZT0715, purified samples were analyzed via nuclear magnetic resonance spectroscopy. As predicted, these studies demonstrated that the loss of OacA activity influenced the O acetylation phenotype of the OPS moieties. Unexpectedly, however, the results indicated that the O methylation status of the OPS antigens was also affected by the loss of OacA activity. Nonetheless, it was revealed that the LPS moieties expressed by the oacA mutant reacted strongly with the B. mallei LPS-specific protective monoclonal antibody 9C1-2. Based on these findings, it appears that OacA is required for the 4-O acetylation and 2-O methylation of B. thailandensis OPS antigens and that ZT0715 may provide a safe and cost-effective source of B. mallei-like OPS to facilitate the synthesis of glanders subunit vaccine candidates. PMID:21115721

  14. Burkholderia: an update on taxonomy and biotechnological potential as antibiotic producers.

    PubMed

    Depoorter, Eliza; Bull, Matt J; Peeters, Charlotte; Coenye, Tom; Vandamme, Peter; Mahenthiralingam, Eshwar

    2016-06-01

    Burkholderia is an incredibly diverse and versatile Gram-negative genus, within which over 80 species have been formally named and multiple other genotypic groups likely represent new species. Phylogenetic analysis based on the 16S rRNA gene sequence and core genome ribosomal multilocus sequence typing analysis indicates the presence of at least three major clades within the genus. Biotechnologically, Burkholderia are well-known for their bioremediation and biopesticidal properties. Within this review, we explore the ability of Burkholderia to synthesise a wide range of antimicrobial compounds ranging from historically characterised antifungals to recently described antibacterial antibiotics with activity against multiresistant clinical pathogens. The production of multiple Burkholderia antibiotics is controlled by quorum sensing and examples of quorum sensing pathways found across the genus are discussed. The capacity for antibiotic biosynthesis and secondary metabolism encoded within Burkholderia genomes is also evaluated. Overall, Burkholderia demonstrate significant biotechnological potential as a source of novel antibiotics and bioactive secondary metabolites. PMID:27115756

  15. Removal of Burkholderia cepacia biofilms with oxidants

    NASA Technical Reports Server (NTRS)

    Koenig, D. W.; Mishra, S. K.; Pierson, D. L.

    1995-01-01

    Iodine is used to disinfect the water system aboard US space shuttles and is the anticipated biocide for the international space station. Water quality on spacecraft must be maintained at the highest possible levels for the safety of the crew. Furthermore, the treatment process used to maintain the quality of water on research must be robust and operate for long periods with minimal crew intervention. Biofilms are recalcitrant and pose a major threat with regard to chronic contamination of spacecraft water systems. We measured the effectiveness of oxidizing biocides on the removal and regrowth of Burkholderia (Pseudomonas) cepacia biofilms. B. cepacia, isolated from the water distribution system of the space shuttle Discovery, was grown in continuous culture to produce a bacterial contamination source for biofilm formation and removal studies. A 10(7) CFU ml-1 B. cepacia suspension, in distilled water, was used to form biofilms on 3000 micrometers2 glass surfaces. Rates of attachment were measured directly with image analysis and were found to be 7.8, 15.2, and 22.8 attachment events h-1 for flow rates of 20.7, 15.2, and 9.8 ml min-1, respectively. After 18 h of formation, the B. cepacia biofilms were challenged with oxidants (ozone, chlorine, and iodine) and the rates of biofilm removal determined by image analysis. Fifty percent of the biofilm material was removed in the first hour of continous treatment with 24 mg l-1 chlorine or 2 mg l-1 ozone. Iodine (48 mg l-1) did not remove any measurable cellular material after 6 h continuous contact. After this first removal of biofilms by the oxidants, the surface was allowed to refoul and was again treated with the biocide. Iodine was the only compound that was unable to remove cellular debris from either primary or secondary biofilms. Moreover, treating primary biofilms with iodine increased the rate of formation of secondary biofilms, from 4.4 to 5.8 attachment events h-1. All the oxidants tested inactivated the B

  16. Species Distribution and Ribotype Diversity of Burkholderia cepacia Complex Isolates from French Patients with Cystic Fibrosis

    PubMed Central

    Brisse, Sylvain; Cordevant, Christophe; Vandamme, Peter; Bidet, Philippe; Loukil, Chawki; Chabanon, Gérard; Lange, Marc; Bingen, Edouard

    2004-01-01

    A total of 153 Burkholderia cepacia strains obtained from 153 French patients with cystic fibrosis were identified as Burkholderia multivorans (51.6%) or Burkholderia cenocepacia (45.1%). Eighty-two genotypes were identified using PvuII and EcoRI ribotyping. B. multivorans genotype A (found in 32 French patients) and two other genotypes were also identified among isolates from Austrian, German, Italian, and Canadian patients. PMID:15472352

  17. An ancient but promiscuous host-symbiont association between Burkholderia gut symbionts and their heteropteran hosts.

    PubMed

    Kikuchi, Yoshitomo; Hosokawa, Takahiro; Fukatsu, Takema

    2011-03-01

    Here, we investigated 124 stinkbug species representing 20 families and 5 superfamilies for their Burkholderia gut symbionts, of which 39 species representing 6 families of the superfamilies Lygaeoidea and Coreoidea were Burkholderia-positive. Diagnostic PCR surveys revealed high frequencies of Burkholderia infection in natural populations of the stinkbugs, and substantial absence of vertical transmission of Burkholderia infection to their eggs. In situ hybridization confirmed localization of the Burkholderia in their midgut crypts. In the lygaeoid and coreoid stinkbugs, development of midgut crypts in their alimentary tract was coincident with the Burkholderia infection, suggesting that the specialized morphological configuration is pivotal for establishment and maintenance of the symbiotic association. The Burkholderia symbionts were easily isolated as pure culture on standard microbiological media, indicating the ability of the gut symbionts to survive outside the host insects. Molecular phylogenetic analysis showed that the gut symbionts of the lygaeoid and coreoid stinkbugs belong to a β-proteobacterial clade together with Burkholderia isolates from soil environments and Burkholderia species that induce plant galls. On the phylogeny, the stinkbug-associated, environmental and gall-forming Burkholderia strains did not form coherent groups, indicating host-symbiont promiscuity among these stinkbugs. Symbiont culturing revealed that slightly different Burkholderia genotypes often coexist in the same insects, which is also suggestive of host-symbiont promiscuity. All these results strongly suggest an ancient but promiscuous host-symbiont relationship between the lygaeoid/coreoid stinkbugs and the Burkholderia gut symbionts. Possible mechanisms as to how the environmentally transmitted promiscuous symbiotic association has been stably maintained in the evolutionary course are discussed. PMID:20882057

  18. Performance of Traditional and Molecular Methods for Detecting Biological Agents in Drinking Water

    USGS Publications Warehouse

    Francy, Donna S.; Bushon, Rebecca N.; Brady, Amie M.G.; Bertke, Erin E.; Kephart, Christopher M.; Likirdopulos, Christina A.; Mailot, Brian E.; Schaefer, Frank W., III; Lindquist, H.D. Alan

    2009-01-01

    To reduce the impact from a possible bioterrorist attack on drinking-water supplies, analytical methods are needed to rapidly detect the presence of biological agents in water. To this end, 13 drinking-water samples were collected at 9 water-treatment plants in Ohio to assess the performance of a molecular method in comparison to traditional analytical methods that take longer to perform. Two 100-liter samples were collected at each site during each sampling event; one was seeded in the laboratory with six biological agents - Bacillus anthracis (B. anthracis), Burkholderia cepacia (as a surrogate for Bu. pseudomallei), Francisella tularensis (F. tularensis), Salmonella Typhi (S. Typhi), Vibrio cholerae (V. cholerae), and Cryptospordium parvum (C. parvum). The seeded and unseeded samples were processed by ultrafiltration and analyzed by use of quantiative polymerase chain reaction (qPCR), a molecular method, and culture methods for bacterial agents or the immunomagnetic separation/fluorescent antibody (IMS/FA) method for C. parvum as traditional methods. Six replicate seeded samples were also processed and analyzed. For traditional methods, recoveries were highly variable between samples and even between some replicate samples, ranging from below detection to greater than 100 percent. Recoveries were significantly related to water pH, specific conductance, and dissolved organic carbon (DOC) for all bacteria combined by culture methods, but none of the water-quality characteristics tested were related to recoveries of C. parvum by IMS/FA. Recoveries were not determined by qPCR because of problems in quantifying organisms by qPCR in the composite seed. Instead, qPCR results were reported as detected, not detected (no qPCR signal), or +/- detected (Cycle Threshold or 'Ct' values were greater than 40). Several sample results by qPCR were omitted from the dataset because of possible problems with qPCR reagents, primers, and probes. For the remaining 14 qPCR results

  19. Vertical transmission explains the specific Burkholderia pattern in Sphagnum mosses at multi-geographic scale

    PubMed Central

    Bragina, Anastasia; Cardinale, Massimiliano; Berg, Christian; Berg, Gabriele

    2013-01-01

    The betaproteobacterial genus Burkholderia is known for its versatile interactions with its hosts that can range from beneficial to pathogenic. A plant-beneficial-environmental (PBE) Burkholderia cluster was recently separated from the pathogen cluster, yet still little is known about burkholderial diversity, distribution, colonization, and transmission patterns on plants. In our study, we applied a combination of high-throughput molecular and microscopic methods to examine the aforementioned factors for Burkholderia communities associated with Sphagnum mosses – model plants for long-term associations – in Austrian and Russian bogs. Analysis of 16S rRNA gene amplicons libraries revealed that most of the Burkholderia are part of the PBE group, but a minor fraction was closely related to B. glathei and B. andropogonis from the pathogen cluster. Notably, Burkholderia showed highly similar composition patterns for each moss species independent of the geographic region, and Burkholderia-specific fluorescent in situ hybridization of Sphagnum gametophytes exhibited similar colonization patterns in different Sphagnum species at multi-geographic scales. To explain these patterns, we compared the compositions of the surrounding water, gametophyte-, and sporophyte-associated microbiome at genus level and discovered that Burkholderia were present in the Sphagnum sporophyte and gametophyte, but were absent in the flark water. Therefore, Burkholderia is a part of the core microbiome transmitted from the moss sporophyte to the gametophyte. This suggests a vertical transmission of Burkholderia strains, and thus underlines their importance for the plants themselves. PMID:24391630

  20. Characterization of the Burkholderia thailandensis SOS Response by Using Whole-Transcriptome Shotgun Sequencing

    PubMed Central

    Ulrich, Ricky L.; DeShazer, David; Kenny, Tara A.; Ulrich, Melanie P.; Moravusova, Anna; Opperman, Timothy; Bavari, Sina; Bowlin, Terry L.; Moir, Donald T.

    2013-01-01

    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 < 0.01) resulted in the differential expression of 344 genes in B. thailandensis and 210 genes in RU0643. Several genes associated with the 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 ϕE264. 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. PMID:23872555

  1. Postinfection Biological Control of Oomycete Pathogens of Pea by Burkholderia cepacia AMMDR1.

    PubMed

    Heungens, K; Parke, J L

    2001-04-01

    ABSTRACT Burkholderia cepacia AMMDR1 is a biocontrol agent that reduces Pythium damping-off and Aphanomyces root rot severity on peas in the field. We studied the effect of B. cepacia AMMDR1 on post-infection stages in the life cycles of these pathogens, including mycelial colonization of the host, production of oogonia, and production of secondary zoospore inoculum. We used Burkholderia cepacia 1324, a seed and rootcolonizing but antibiosis-deficient Tn5 mutant of B. cepacia AMMDR1, to study mechanisms of biological control other than antibiosis. B. cepacia AMMDR1 significantly reduced Pythium aphanidermatum postinfection colonization and damping-off of pea seeds, even when the bacteria were applied 12 h after zoospore inoculation. B. cepacia AMMDR1 also significantly reduced colonization of taproots by Aphanomyces euteiches mycelium, but only when the bacteria were applied at high population densities at the site of zoospore inoculation. The antibiosisdeficient mutant, B. cepacia 1324, had no effect on mycelial colonization of seeds or roots by Pythium aphanidermatum nor A. euteiches, suggesting that antibiosis is the primary mechanism of biological control. B. cepacia AMMDR1, but not B. cepacia 1324, reduced production of A. euteiches oogonia. This effect occurred even when the population size of B. cepacia AMMDR1 was too small to cause a reduction in lesion length early on in the infection process and may result from in situ antibiotic production. B. cepacia AMMDR1 had no effect on the production of secondary zoospores of A. euteiches from infected roots. The main effects of B. cepacia AMMDR1 on postinfection stages in the life cycles of these pathogens therefore were reductions in mycelial colonization by Pythium aphanidermatum and in formation of oogonia by A. euteiches. No mechanism other than antibiosis could be identified. PMID:18943851

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

    PubMed Central

    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

  3. Nucleic Acid Similarities Among Pseudomonas pseudomallei, Pseudomonas multivorans, and Actinobacillus mallei1

    PubMed Central

    Rogul, M.; Brendle, J. J.; Haapala, D. K.; Alexander, A. D.

    1970-01-01

    Annealing experiments on membrane filters were carried out with deoxyribonucleic acids (DNA) from selected strains of the nomen-species of Pseudomonas, Actinobacillus, Chromobacterium, and Micrococcus, with the use of DNA of Pseudomonas pseudomallei and Actinobacillus mallei as reference materials. Under the usual conditions employed in these experiments, the results were not quantitatively reproducible. Incorporation of dimethylsulfoxide (DMSO) into the incubation medium greatly increased differences in comparative binding. DNA binding in agar matrices was examined in the presence and absence of DMSO at various incubation temperatures. It was found that the greatest specificity, stability, and total binding for DNA containing high amounts of guanine and cytosine occurred in the presence of DMSO. Under the most stringent annealing conditions permitted in agar, DNA species from P. pseudomallei and A. mallei in the presence of DMSO demonstrated interspecific relative bindings of 76 to 86% when compared to the homologous reactions. The thermal elution midpoints (Em) of these duplexed interspecific DNA species were quite close to the homologous Em values. The relative bindings of P. multivorans DNA types to either reference DNA ranged between 6 to 27%, and the Em values were 4 to 7 C less than those for the homologous reactions. PMID:5438051

  4. More than skin deep: moisturizing body milk and Burkholderia cepacia

    PubMed Central

    Irwin, Amy E; Price, Connie Savor

    2008-01-01

    Alvarez-Lerma and colleagues observed over an 18-day period that five critically ill patients admitted to a multidisciplinary 18-bed intensive care unit contracted Burkholderia cepacia from unopened containers of moisturizing body milk, calling into question the use in critical care settings of cosmetic products that do not guarantee sterilization during the manufacturing process. Is this the answer to the problem, however, or should the use of lotions in such settings be re-examined? PMID:18304377

  5. Atypical presentation of chronic granulomatous disease with Burkholderia cepacia.

    PubMed

    Vining, Mac; Sharma, Nirupma; Guill, Margaret

    2014-01-01

    Chronic granulomatous disease (CGD) is a rare inherited disorder of neutrophil oxidative burst. In patients with CGD, phagocyte destruction of catalase-producing organisms is impaired, resulting in recurrent and potentially fatal infections. Burkholderia cepacia, a catalase-producing organism, is known to infect patients with dysfunctional immune systems. We report a case of a 3-year-old boy with this rare infection that unravelled the diagnosis of CGD. PMID:25103315

  6. Atypical presentation of chronic granulomatous disease with Burkholderia cepacia

    PubMed Central

    Vining, Mac; Sharma, Nirupma; Guill, Margaret

    2014-01-01

    Chronic granulomatous disease (CGD) is a rare inherited disorder of neutrophil oxidative burst. In patients with CGD, phagocyte destruction of catalase-producing organisms is impaired, resulting in recurrent and potentially fatal infections. Burkholderia cepacia, a catalase-producing organism, is known to infect patients with dysfunctional immune systems. We report a case of a 3-year-old boy with this rare infection that unravelled the diagnosis of CGD. PMID:25103315

  7. Nodulation of Cyclopia spp. (Leguminosae, Papilionoideae) by Burkholderia tuberum

    PubMed Central

    Elliott, Geoffrey N.; Chen, Wen-Ming; Bontemps, Cyril; Chou, Jui-Hsing; Young, J. Peter W.; Sprent, Janet I.; James, Euan K.

    2007-01-01

    Background and Aims Species of the genus Burkholderia, from the Betaproteobacteria, have been isolated from legume nodules, but so far they have only been shown to form symbioses with species of Mimosa, sub-family Mimosoideae. This work investigates whether Burkholderia tuberum strains STM678 (isolated from Aspalathus carnosa) and DUS833 (from Aspalathus callosa) can nodulate species of the South African endemic papilionoid genera Cyclopia (tribe Podalyrieae) and Aspalathus (Crotalarieae) as well as the promiscuous legume Macroptilium atropurpureum (Phaseoleae). Method Bacterial strains and the phylogeny of their symbiosis-related (nod) genes were examined via 16S rRNA gene sequencing. Seedlings were grown in liquid culture and inoculated with one of the two strains of B. tuberum or with Sinorhizobium strain NGR 234 (from Lablab purpureus), Mesorhizobium strain DUS835 (from Aspalathus linearis) or Methylobacterium nodulans (from Crotalaria podocarpa). Some nodules, inoculated with green fluorescence protein (GFP)-tagged strains, were examined by light and electron microscopy coupled with immunogold labelling with a Burkholderia-specific antibody. The presence of active nitrogenase was checked by immunolabelling of nitrogenase and by the acetylene reduction assay. B. tuberum STM678 was also tested on a wide range of legumes from all three sub-families. Key Results Nodules were not formed on any of the Aspalathus spp. Only B. tuberum nodulated Cyclopia falcata, C. galioides, C. genistoides, C. intermedia and C. pubescens. It also effectively nodulated M. atropurpureum but no other species tested. GFP-expressing inoculant strains were located inside infected cells of C. genistoides, and bacteroids in both Cyclopia spp. and M. atropurpureum were immunogold-labelled with antibodies against Burkholderia and nitrogenase. Nitrogenase activity was also shown using the acetylene reduction assay. This is the first demonstration that a β-rhizobial strain can effectively

  8. Identification and enzymatic characterization of acid phosphatase from Burkholderia gladioli

    PubMed Central

    2014-01-01

    Background The genus Burkholderia is widespread in diverse ecological niches, the majority of known species are soil bacteria that exhibit different types of non-pathogenic interactions with plants. Burkholderia species are versatile organisms that solubilize insoluble minerals through the production of organic acids, which increase the availability of nutrients for the plant. Therefore these bacteria are promising candidates for biotechnological applications. Results Burkholderia sp. (R 3.25 isolate) was isolated from agricultural soil in Ponta Grossa-PR-Brazil and identified through analysis of the 16S rDNA as a strain classified as Burkholderia gladioli. The expression of membrane-bound acid phosphatase (MBAcP) was strictly regulated with optimal expression at a concentration of phosphorus 5 mM. The apparent optimum pH for the hydrolysis of p-nitrophenylphosphate (PNPP) was 6.0. The hydrolysis of PNPP by the enzyme exhibited a hyperbolic relationship with increasing concentration of substrate and no inhibition by excess of substrate was observed. Kinetic data revealed that the hydrolysis of PNPP exhibited cooperative kinetics with n = 1.3, Vm = 113.5 U/mg and K0.5 = 65 μM. The PNPPase activity was inhibited by vanadate, p-hydroxymercuribenzoate, arsenate and phosphate, however the activity was not inhibited by calcium, levamisole, sodium tartrate, EDTA, zinc, magnesium, cobalt, ouabain, oligomycin or pantoprazol. Conclusion The synthesis of membrane-bound non-specific acid phosphatase, strictly regulated by phosphate, and its properties suggest that this bacterium has a potential biotechnological application to solubilize phosphate in soils with low levels of this element, for specific crops. PMID:24713147

  9. 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

    SciTech Connect

    Fischer, N. O.

    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.

  10. Epidemiology and molecular characterization of a clone of Burkholderia cenocepacia responsible for nosocomial pulmonary tract infections in a French intensive care unit.

    PubMed

    Graindorge, Arnault; Menard, Aymeric; Neto, Manuelle; Bouvet, Claude; Miollan, Roger; Gaillard, Sandrine; de Montclos, Henri; Laurent, Frédéric; Cournoyer, Benoit

    2010-01-01

    Clustered cases of nosocomial pulmonary infections were observed in a French intensive care unit. Biochemical tests showed the etiologic agents to be part of the Bcc (Bcc). recA polymerase chain reaction-restriction fragment length polymorphism analysis and molecular phylogeny positioned the isolates into Burkholderia cenocepacia. Their recA sequences were found identical to those of ET12 strains responsible of necrotic pneumonia in cystic fibrosis patients. Analyses of a multi locus sequence typing genes set confirmed this proximity and suggested a wide distribution among occidental countries but could not resolve their phylogenetic position unambiguously. A novel marker, ecfB, indicated a significant phylogenetic divergence from ET12 strains. Pulse field gel electrophoresis analysis of SpeI-restricted total genomic DNA of the strains showed a unique profile indicative of a clonal outbreak. Environmental hospital screenings indicated cross-contamination between staff and patients. Bcc strains from outdoor environments were not related to this clone but indicated the presence of Burkholderia multivorans and Burkholderia vietnamiensis. PMID:19716254

  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.

    PubMed

    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

    2016-01-01

    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. PMID:27125479

  12. 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

    PubMed Central

    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.

    2016-01-01

    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. PMID:27125479

  13. Whole-Genome Analysis of Quorum-Sensing Burkholderia sp. Strain A9

    PubMed Central

    Chen, Jian Woon; Tee, Kok Keng; Chang, Chien-Yi; Yin, Wai-Fong; Chan, Xin-Yue

    2015-01-01

    Burkholderia spp. rely on N-acyl homoserine lactone as quorum-sensing signal molecules which coordinate their phenotype at the population level. In this work, we present the whole genome of Burkholderia sp. strain A9, which enables the discovery of its N-acyl homoserine lactone synthase gene. PMID:25745000

  14. Diversity and distribution of Burkholderia cepacia complex in the rhizosphere of rice and maize.

    PubMed

    Zhang, Lixin; Xie, Guanlin

    2007-01-01

    A survey of Burkholderia cepacia complex (Bcc) species was conducted in agricultural fields within Hangzhou, China. Out of the 251 bacterial isolates recovered on the selective media from the rhizosphere of rice and maize, 112 of them were assigned to Bcc by PCR assays. The species composition of the Bcc isolates was analyzed by a combination of recA-restriction fragment length polymorphism assays, species-specific PCR tests and recA gene sequencing. The results revealed that the majority belong to B. cepacia, Burkholderia cenocepacia recA lineage IIIB, Burkholderia vietnamiensis and Burkholderia pyrrocinia. Burkholderia cenocepacia and B. vietnamiensis dominated the rhizosphere of maize and rice, respectively, indicating that species composition and abundance of Bcc may vary dramatically in different crop rhizospheres. In addition, one isolate (R456) formed a single discrete cluster within the phylogenetic analysis of the Bcc recA gene, and it may belong to a new genomovar. PMID:17233735

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

    PubMed

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

    2012-01-01

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

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

    PubMed

    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. PMID:27183720

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

    PubMed Central

    2010-01-01

    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 HAE1 systems in the

  18. Characterization of the Poly-β-1,6-N-Acetylglucosamine Polysaccharide Component of Burkholderia Biofilms ▿

    PubMed Central

    Yakandawala, Nandadeva; Gawande, Purushottam V.; LoVetri, Karen; Cardona, Silvia T.; Romeo, Tony; Nitz, Mark; Madhyastha, Srinivasa

    2011-01-01

    We demonstrated the production of poly-β-1,6-N-acetylglucosamine (PNAG) polysaccharide in the biofilms of Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia cepacia, and Burkholderia cenocepacia using an immunoblot assay for PNAG. These results were confirmed by further studies, which showed that the PNAG hydrolase, dispersin B, eliminated immunoreactivity of extracts from the species that were tested (B. cenocepacia and B. multivorans). Dispersin B also inhibited biofilm formation and dispersed preformed biofilms of Burkholderia species. These results imply a role for PNAG in the maintenance of Burkholderia biofilm integrity. While PNAG was present in biofilms of all of the wild-type test organisms, a ΔpgaBC mutant of B. multivorans (Mu5) produced no detectable PNAG, indicating that these genes are needed for Burkholderia PNAG formation. Furthermore, restoration of PNAG production in PNAG negative E. coli TRXWMGΔC (ΔpgaC) by complementation with B. multivorans pgaBCD confirmed the involvement of these genes in Burkholderia PNAG production. While the confocal scanning laser microscopy of untreated wild-type B. multivorans showed thick, multilayered biofilm, Mu5 and dispersin B-treated wild-type biofilms were thin, poorly developed, and disrupted, confirming the involvement of PNAG in B. multivorans biofilm formation. Thus, PNAG appears to be an important component of Burkholderia biofilms, potentially contributing to its resistance to multiple antibiotics and persistence during chronic infections, including cystic fibrosis-associated infection. PMID:21984237

  19. Microbial degradation of quinoline by immobilized cells of Burkholderia pickettii.

    PubMed

    Jianlong, Wang; Xiangchun, Quan; Liping, Han; Yi, Qian; Hegemann, Werner

    2002-05-01

    A quinoline-biodegrading microorganism was isolated from activated sludge of coke-oven wastewater treatment plant using quinoline as sole carbon and nitrogen source. It is a gram negative, rod-shaped and aerobic strain, which was identified as Burkholderia pickettii. The biodegradation of quinoline was carried out with this isolated strain. Analysis by high performance liquid chromatography and gas chromatography/mass spectrum (GC/MS) revealed that 2-hydroxyquinoline (2-OH-Q) was the first intermediate in the course of quinoline biodegradation. A novel immobilization carrier, that is, polyvinyl alcohol (PVA)-gauze hybrid carrier, was developed. The isolated strain was immobilized by two different immobilizing techniques and used for the quinolinerdegradation. It was found that biodegradation rate of quinoline by the microorganisms immobilized on PVA-gauze hybrid carrier was faster than that by the microorganisms immobilized in PVA gel beads. Kinetics of quinoline biodegradation by cells of Burkholderia pickettii immobilized on PVA-gauze hybrid carrier was investigated. The results demonstrate that quinoline degradation could be described by zero-order reaction rate equation when the initial quinoline concentration was in the range of 50-500 mg l(-1). PMID:12108721

  20. Burkholderia cepacia lipase is a promising biocatalyst for biofuel production.

    PubMed

    Sasso, Francesco; Natalello, Antonino; Castoldi, Simone; Lotti, Marina; Santambrogio, Carlo; Grandori, Rita

    2016-07-01

    Lipases resistant to inhibition and denaturation by methanol are valuable tools for biotechnological applications, in particular for biofuel production. Microbial lipases have attracted a great deal of interest because of their stability at high concentrations of organic solvents. Burkholderia cepacia lipase (BCL) is tested here for robustness towards methanol in terms of conformational stability and catalytic activity in transesterification assays. This lipase turns out to be even more tolerant than the homologous and better characterized enzyme from Burkholderia glumae. BCL unfolding transition, as monitored by far-UV circular dichroism (CD) and intrinsic fluorescence, displays a Tm above 60°C in the presence of 50% methanol. The protein unfolds at low pH, and the organic solvent affects the nature of the denatured state under acidic conditions. The protein performs well in transesterification assays upon prolonged incubations at high methanol concentrations. BCL is highly tolerant to methanol and displays particularly high conformational stability under conditions employed for transesterification reactions. These features depict BCL as a promising enzyme for biofuel industry. PMID:27067648

  1. Bovine lactoferrin interacts with cable pili of Burkholderia cenocepacia.

    PubMed

    Ammendolia, Maria Grazia; Bertuccini, Lucia; Iosi, Francesca; Minelli, Fabio; Berlutti, Francesca; Valenti, Piera; Superti, Fabiana

    2010-06-01

    In this study we evaluated the ability of lactoferrin, the most abundant antimicrobial protein in airway secretions, to bind the surface structures of a Burkholderia strain cystic fibrosis-isolated. Burkholderia cenocepacia is a gram-negative bacterium involved as respiratory pathogen in cystic fibrosis patient infections. This bacterium possesses filamentous structures, named cable pili that have been proposed as virulence factors because of their ability to bind to respiratory epithelia and mucin. Previously, we demonstrated that bovine lactoferrin was able to influence the efficiency of invasion of different iron-regulated morphological forms of B. cenocepacia. Bovine lactoferrin showed to efficiently inhibit invasion of alveolar epithelial cells by free-living bacteria or iron-induced aggregates or biofilm. Results of the present study demonstrate that bovine lactoferrin is also able to specifically bind to B. cenocepacia cells and show that cable pili are involved in this interaction. The attachment of bovine lactoferrin to pili led to a reduced binding of bacterial cells to mucin. Since cable pili are implicated in mediating the bacterial interactions with mucin and epithelial cells, lactoferrin binding to these structures could play an important role in neutralizing bacterial infection in cystic fibrosis patients. PMID:20364433

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

    PubMed

    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. PMID:25250879

  3. Importance of topology for glycocluster binding to Pseudomonas aeruginosa and Burkholderia ambifaria bacterial lectins.

    PubMed

    Ligeour, Caroline; Dupin, Lucie; Angeli, Anthony; Vergoten, Gérard; Vidal, Sébastien; Meyer, Albert; Souteyrand, Eliane; Vasseur, Jean-Jacques; Chevolot, Yann; Morvan, François

    2015-12-14

    Pseudomonas aeruginosa (PA) and Burkholderia ambifaria (BA) are two opportunistic Gram negative bacteria and major infectious agents involved in lung infection of cystic fibrosis patients. Both bacteria can develop resistance to conventional antibiotherapies. An alternative strategy consists of targeting virulence factors in particular lectins with high affinity ligands such as multivalent glycoclusters. LecA (PA-IL) and LecB (PA-IIL) are two tetravalent lectins from PA that recognise galactose and fucose respectively. BambL lectin from BA is trimeric with 2 binding sites per monomer and is also specific for fucose. These three lectins are potential therapeutic targets in an anti-adhesive anti-bacterial approach. Herein, we report the synthesis of 18 oligonucleotide pentofuranose-centered or mannitol-centered glycoclusters leading to tri-, penta- or decavalent clusters with different topologies. The linker arm length between the core and the carbohydrate epitope was also varied leading to 9 galactoclusters targeting LecA and 9 fucoclusters targeting both LecB and BambL. Their dissociation constants (Kd) were determined using a DNA-based carbohydrate microarray technology. The trivalent xylo-centered galactocluster and the ribo-centered fucocluster exhibited the best affinity for LecA and LecB respectively while the mannitol-centered decafucocluster displayed the best affinity to BambL. These data demonstrated that the topology and nature of linkers were the predominant factors for achieving high affinity rather than valency. PMID:26412676

  4. Characterizing uncommon Burkholderia cepacia complex isolates from an outbreak in a haemodialysis unit.

    PubMed

    Souza, Andrea V; Moreira, Cláudia R; Pasternak, Jacyr; Hirata, Maria de Lurdes; Saltini, Denise Alves; Caetano, Viviane Cristina; Ciosak, Suely; Azevedo, Fátima M; Severino, Patricia; Vandamme, Peter; Magalhães, Vanda D

    2004-10-01

    An outbreak of bacteraemia in a haemodialysis unit where 65 episodes of infection involved 35 outpatients is reported. Burkholderia cepacia complex was the agent most frequently recovered from blood. Thirty-three environmental and clinical isolates of B. cepacia complex were characterized by whole-cell protein electrophoresis and recA-RFLP profile. Fourteen isolates were genomovar I and 16 isolates were not classifiable by their recA-RFLP pattern. Ribotyping, random amplification of polymorphic DNA (RAPD) and integron profile were used to explore the clonality of the isolates, and revealed multiple strain genotypes. Four ribotypes and RAPD types and three integron patterns were identified. The water supply was identified as the source of the outbreak, and inappropriate cleaning and a leak in the reverse osmosis tubing connection were the probable causes of contamination. B. cepacia complex was still recovered from blood of patients even after apparently adequate measures were taken and water quality standards were met, suggesting that higher standards for water quality should be adopted in haemodialysis units. The genomovars recovered here were distinct from those commonly reported for cystic fibrosis isolates. PMID:15358822

  5. TRACKING THE RESPONSE OF BURKHOLDERIA CEPACIA G4 5223-PR1 IN AQUIFER MICROCOSMS

    EPA Science Inventory

    The introduction of bacteria into the environment for bioremediation purposes (bioaugmentation) requires analysis and monitoring of microbial population dynamics to define persistence and activity from both efficacy and risk assessment perspectives, Burkholderia cepacia G4 5223-P...

  6. AQUIFER PROTIST RESPONSE AND THE POTENTIAL FOR TCE BIOREMEDIATION WITH BURKHOLDERIA CEPACIA G4 PR1

    EPA Science Inventory

    The introduction of bacteria into the environment for bioremediation purposes (bioaugmentation) requires analysis and monitoring of the persistence and activity of microbial population for efficacy and risk assessment purposes. Burkholderia cepacia G4 PR123 and PR131 constitutive...

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

    EPA Science Inventory

    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 ...

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

    PubMed

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

    2016-02-01

    Although members of the genus Burkholderia can utilize a high-affinity iron uptake system to sustain growth under iron-limiting conditions, many strains also produce siderophores, suggesting that they may serve alternative functions. Here we demonstrate that the two Burkholderia siderophores pyochelin and ornibactin can protect the cells from metal toxicity and thus play an alternative role in metal homeostasis. We also demonstrate that metals such as copper and zinc induce the production of ornibactin. PMID:26621188

  9. Burkholderia Species Are Major Inhabitants of White Lupin Cluster Roots▿†

    PubMed Central

    Weisskopf, Laure; Heller, Stefanie; Eberl, Leo

    2011-01-01

    The formation of cluster roots by plants represents a highly efficient strategy for acquisition of sparingly available phosphate. This particular root type is characterized by a densely branched structure and high exudation of organic acids and protons, which are likely to influence the resident bacterial community. Until now, the identity of the bacterial populations living in cluster roots has not been investigated. We applied cultivation-dependent and cultivation-independent methods to characterize the dominant bacterial genera inhabiting the growing cluster roots of white lupin. We observed a high relative abundance of Burkholderia species (up to 58% of all isolated strains and 44% of all retrieved 16S rRNA sequences) and a significant enrichment with increasing cluster root age. Most of the sequences retrieved clustered together with known plant- or fungus-associated Burkholderia species, while only one of 98 sequences was affiliated with the Burkholderia cepacia complex. In vitro assays revealed that Burkholderia strains were much more tolerant to low pH than non-Burkholderia strains. Moreover, many strains produced large amounts of siderophores and were able to utilize citrate and oxalate as carbon sources. These features seem to represent important traits for the successful colonization and maintenance of Burkholderia species in white lupin cluster roots. PMID:21908626

  10. Detection of cultured and uncultured Burkholderia cepacia complex bacteria naturally occurring in the maize rhizosphere.

    PubMed

    Pirone, Luisa; Chiarini, Luigi; Dalmastri, Claudia; Bevivino, Annamaria; Tabacchioni, Silvia

    2005-11-01

    The species composition of a Burkholderia cepacia complex population naturally occurring in the maize rhizosphere was investigated by using both culture-dependent and culture-independent methods. B. cepacia complex isolates were recovered from maize root slurry on the two selective media Pseudomonas cepacia azelaic acid tryptamine (PCAT) and trypan blue tetracycline (TB-T) and subjected to identification by a combination of restriction fragment length polymorphism (RFLP) analysis and species-specific polymerase chain reaction (PCR) tests of the recA gene. DNA extracted directly from root slurry was examined by means of nested PCR to amplify recA gene with species-specific B. cepacia complex primers and to obtain a library of PCR amplified recA genes. Using the culture-dependent method the species Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia ambifaria and Burkholderia pyrrocinia were identified, whereas using the culture-independent method also the species Burkholderia vietnamiensis was detected. The latter method also allowed us to highlight a higher diversity within the B. cenocepacia species. In fact, by using the culture-independent method the species B. cenocepacia recA lineages IIIA and IIID besides B. cenocepacia recA lineage IIIB were detected. Moreover, higher heterogeneity of recA RFLP patterns was observed among clones assigned to the species B. cenocepacia than among B. cenocepacia isolates from selective media. PMID:16232288

  11. Molecular mechanisms underlying the close association between soil Burkholderia and fungi.

    PubMed

    Stopnisek, Nejc; Zühlke, Daniela; Carlier, Aurélien; Barberán, Albert; Fierer, Noah; Becher, Dörte; Riedel, Katharina; Eberl, Leo; Weisskopf, Laure

    2016-01-01

    Bacterial species belonging to the genus Burkholderia have been repeatedly reported to be associated with fungi but the extent and specificity of these associations in soils remain undetermined. To assess whether associations between Burkholderia and fungi are widespread in soils, we performed a co-occurrence analysis in an intercontinental soil sample collection. This revealed that Burkholderia significantly co-occurred with a wide range of fungi. To analyse the molecular basis of the interaction, we selected two model fungi frequently co-occurring with Burkholderia, Alternaria alternata and Fusarium solani, and analysed the proteome changes caused by cultivation with either fungus in the widespread soil inhabitant B. glathei, whose genome we sequenced. Co-cultivation with both fungi led to very similar changes in the B. glathei proteome. Our results indicate that B. glathei significantly benefits from the interaction, which is exemplified by a lower abundance of several starvation factors that were highly expressed in pure culture. However, co-cultivation also gave rise to stress factors, as indicated by the increased expression of multidrug efflux pumps and proteins involved in oxidative stress response. Our data suggest that the ability of Burkholderia to establish a close association with fungi mainly lies in the capacities to utilize fungal-secreted metabolites and to overcome fungal defense mechanisms. This work indicates that beneficial interactions with fungi might contribute to the survival strategy of Burkholderia species in environments with sub-optimal conditions, including acidic soils. PMID:25989372

  12. Genus-wide acid tolerance accounts for the biogeographical distribution of soil Burkholderia populations.

    PubMed

    Stopnisek, Nejc; Bodenhausen, Natacha; Frey, Beat; Fierer, Noah; Eberl, Leo; Weisskopf, Laure

    2014-06-01

    Bacteria belonging to the genus Burkholderia are highly versatile with respect to their ecological niches and lifestyles, ranging from nodulating tropical plants to causing melioidosis and fatal infections in cystic fibrosis patients. Despite the clinical importance and agronomical relevance of Burkholderia species, information about the factors influencing their occurrence, abundance and diversity in the environment is scarce. Recent findings have demonstrated that pH is the main predictor of soil bacterial diversity and community structure, with the highest diversity observed in neutral pH soils. As many Burkholderia species have been isolated from low pH environments, we hypothesized that acid tolerance may be a general feature of this genus, and pH a good predictor of their occurrence in soils. Using a combination of environmental surveys at trans-continental and local scales, as well as in vitro assays, we show that, unlike most bacteria, Burkholderia species have a competitive advantage in acidic soils, but are outcompeted in alkaline soils. Physiological assays and diversity analysis based on 16S rRNA clone libraries demonstrate that pH tolerance is a general phenotypic trait of the genus Burkholderia. Our results provide a basis for building a predictive understanding of the biogeographical patterns exhibited by Burkholderia sp. PMID:23945027

  13. Burkholderia gladioli infection isolated from the blood cultures of newborns in the neonatal intensive care unit.

    PubMed

    Zhou, F; Ning, H; Chen, F; Wu, W; Chen, A; Zhang, J

    2015-08-01

    Burkholderia gladioli was described as a plant pathogen, and it is a rare cause of infection in humans that is primarily associated with human pulmonary infections, such as chronic granulomatous disease and cystic fibrosis. The neonatal respiratory system is not fully developed and cannot expel bacterial aerosol properly. A total of 2,676 newborns in the neonatal intensive care unit were retrospectively analysed in Putian City, Fujian Province, China, from 2011 to 2014. All of the blood samples were tested for C-reactive protein (CRP), procalcitonin (PCT) and white blood cell (WBC). B. gladioli infections were determined and analysed using a blood culture system. Antibiotic susceptibility testing was performed using the K-B method. Of the 2,676 participants, 87 (3.25 %) had a positive B. gladioli blood culture that occurred >72 h after birth, including a premature group (54.0 %, asphyxia [vs. 9.20 %], fever [vs. 13.80 %], pneumonia [vs. 6.90 %] and hyperbilirubinaemia [vs. 8.05 %]) and newborns with necrotising enterocolitis (NEC) (vs. 5.75 %). The mean ± standard deviation (SD) of the CRP level was 12.31 ± 0.26 mg/L and that of the PCT level was 1.53 ± 0.21 ng/ml in the 87 B. gladioli-infected newborns. Most of the B. gladioli isolates were sensitive to many antimicrobial agents and did not lead to serious consequences. All of the B. gladioli-infected newborns were unhealthy, especially the premature infants. B. gladioli might be a causative bacteraemia agent in neonates, it appears to have pathogenic potential in newborns and its sensitivity to antibiotics may be a beneficial factor. PMID:25926303

  14. Fosmidomycin Decreases Membrane Hopanoids and Potentiates the Effects of Colistin on Burkholderia multivorans Clinical Isolates

    PubMed Central

    Malott, Rebecca J.; Wu, Chia-Hung; Lee, Tracy D.; Hird, Trevor J.; Dalleska, Nathan F.; Zlosnik, James E. A.; Newman, Dianne K.

    2014-01-01

    Burkholderia cepacia complex (Bcc) pulmonary infections in people living with cystic fibrosis (CF) are difficult to treat because of the extreme intrinsic resistance of most isolates to a broad range of antimicrobials. Fosmidomycin is an antibacterial and antiparasitic agent that disrupts the isoprenoid biosynthesis pathway, a precursor to hopanoid biosynthesis. Hopanoids are involved in membrane stability and contribute to polymyxin resistance in Bcc bacteria. Checkerboard MIC assays determined that although isolates of the Bcc species B. multivorans were highly resistant to treatment with fosmidomycin or colistin (polymyxin E), antimicrobial synergy was observed in certain isolates when the antimicrobials were used in combination. Treatment with fosmidomycin decreased the MIC of colistin for isolates as much as 64-fold to as low as 8 μg/ml, a concentration achievable with colistin inhalation therapy. A liquid chromatography-tandem mass spectrometry technique was developed for the accurate quantitative determination of underivatized hopanoids in total lipid extracts, and bacteriohopanetetrol cyclitol ether (BHT-CE) was found to be the dominant hopanoid made by B. multivorans. The amount of BHT-CE made was significantly reduced upon fosmidomycin treatment of the bacteria. Uptake assays with 1-N-phenylnaphthylamine were used to determine that dual treatment with fosmidomycin and colistin increases membrane permeability, while binding assays with boron-dipyrromethene-conjugated polymyxin B illustrated that the addition of fosmidomycin had no impact on polymyxin binding. This work indicates that pharmacological suppression of membrane hopanoids with fosmidomycin treatment can increase the susceptibility of certain clinical B. multivorans isolates to colistin, an agent currently in use to treat pulmonary infections in CF patients. PMID:24957830

  15. In vivo Bioluminescence Imaging of Burkholderia mallei Respiratory Infection and Treatment in the Mouse Model

    PubMed Central

    Massey, Shane; Johnston, Katie; Mott, Tiffany M.; Judy, Barbara M.; Kvitko, Brian H.; Schweizer, Herbert P.; Estes, D. Mark; Torres, Alfredo G.

    2011-01-01

    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 5 × 103 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. PMID:21904535

  16. Identification, molecular characterisation and antimicrobial susceptibility of genomovars of the Burkholderia cepacia complex in Spain.

    PubMed

    Medina-Pascual, M J; Valdezate, S; Villalón, P; Garrido, N; Rubio, V; Saéz-Nieto, J A

    2012-12-01

    Burkholderia spp. strains collected in Spain over a 13-year period from patients with cystic fibrosis (CF) (n = 148), non-CF patients (n = 103) and from environmental sources (n = 64) were characterised. One hundred and forty-one of the examined strains were involved in seven suspected nosocomial disease outbreaks. Strains were identified by their 16s rRNA and recA genes. Their genetic relatedness, the possession of cable pili and the B. cepacia epidemic strain marker (BCESM), and their susceptibility to antimicrobial agents were studied using pulsed-field gel electrophoresis (PFGE), cblA and esmR genes analysis, and by the E-test, respectively. The genomovar distribution for the 315 strains was as follows: B. stabilis 29.5 %, B. cepacia 14.9 %, B. multivorans 11.1 %, B. cenocepacia IIIA 9.5 %, B. vietnamiensis 3.8 %, B. cenocepacia IIIB 3.5 %, and B. ambifaria and B. pyrrocinia 0.3 % each. The genetic diversity of the B. cepacia complex (Bcc) was ample, with 57 different SpeI types, showing a genetic similarity of 36.4-96.6 %. No strain carried cblA, whereas 25 B. cenocepacia genotypes harboured BCESM (23 from patients with CF). Antimicrobial resistance rates to tobramycin (TOB; 86 %) and imipenem (IPM; 67 %) were high. The strains from patients with CF showed significantly greater resistance to piperacillin (PIP), levofloxacin (LVX) and co-trimoxazole (SXT) than those isolated from non-CF patients (p < 0.05). In conclusion, B. cenocepacia was the most prevalent genomovar found in patients with CF (19.1 %), whereas B. cepacia was the most common among non-CF patients (20.7 %). B. stabilis (47.6 %) was the most common environmental genomovar. Susceptibility to antimicrobial agents depended on genomovar status and strain origin. PMID:22855365

  17. Aerosol Phage Therapy Efficacy in Burkholderia cepacia Complex Respiratory Infections

    PubMed Central

    Semler, Diana D.; Goudie, Amanda D.; Finlay, Warren H.

    2014-01-01

    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. PMID:24798268

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

    PubMed

    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. PMID:24798268

  19. High Confidence Prediction of Essential Genes in Burkholderia Cenocepacia

    PubMed Central

    Juhas, Mario; Stark, Manuel; von Mering, Christian; Lumjiaktase, Puthapoom; Crook, Derrick W.; Valvano, Miguel A.; Eberl, Leo

    2012-01-01

    Background Essential genes are absolutely required for the survival of an organism. The identification of essential genes, besides being one of the most fundamental questions in biology, is also of interest for the emerging science of synthetic biology and for the development of novel antimicrobials. New antimicrobial therapies are desperately needed to treat multidrug-resistant pathogens, such as members of the Burkholderia cepacia complex. Methodology/Principal Findings We hypothesize that essential genes may be highly conserved within a group of evolutionary closely related organisms. Using a bioinformatics approach we determined that the core genome of the order Burkholderiales consists of 649 genes. All but two of these identified genes were located on chromosome 1 of Burkholderia cenocepacia. Although many of the 649 core genes of Burkholderiales have been shown to be essential in other bacteria, we were also able to identify a number of novel essential genes present mainly, or exclusively, within this order. The essentiality of some of the core genes, including the known essential genes infB, gyrB, ubiB, and valS, as well as the so far uncharacterized genes BCAL1882, BCAL2769, BCAL3142 and BCAL3369 has been confirmed experimentally in B. cenocepacia. Conclusions/Significance We report on the identification of essential genes using a novel bioinformatics strategy and provide bioinformatics and experimental evidence that the large majority of the identified genes are indeed essential. The essential genes identified here may represent valuable targets for the development of novel antimicrobials and their detailed study may shed new light on the functions required to support life. PMID:22768221

  20. Production of bioactive volatiles by different Burkholderia ambifaria strains.

    PubMed

    Groenhagen, Ulrike; Baumgartner, Rita; Bailly, Aurélien; Gardiner, Amber; Eberl, Leo; Schulz, Stefan; Weisskopf, Laure

    2013-07-01

    Increasing evidence indicates that volatile compounds emitted by bacteria can influence the growth of other organisms. In this study, the volatiles produced by three different strains of Burkholderia ambifaria were analysed and their effects on the growth of plants and fungi, as well as on the antibiotic resistance of target bacteria, were assessed. Burkholderia ambifaria emitted highly bioactive volatiles independently of the strain origin (clinical environment, rhizosphere of pea, roots of maize). These volatile blends induced significant biomass increase in the model plant Arabidopsis thaliana as well as growth inhibition of two phytopathogenic fungi (Rhizoctonia solani and Alternaria alternata). In Escherichia coli exposed to the volatiles of B. ambifaria, resistance to the aminoglycoside antibiotics gentamicin and kanamycin was found to be increased. The volatile blends of the three strains were similar, and dimethyl disulfide was the most abundant compound. Sulfur compounds, ketones, and aromatic compounds were major groups in all three volatile profiles. When applied as pure substance, dimethyl disulfide led to increased plant biomass, as did acetophenone and 3-hexanone. Significant fungal growth reduction was observed with high concentrations of dimethyl di- and trisulfide, 4-octanone, S-methyl methanethiosulphonate, 1-phenylpropan-1-one, and 2-undecanone, while dimethyl trisulfide, 1-methylthio-3-pentanone, and o-aminoacetophenone increased resistance of E. coli to aminoglycosides. Comparison of the volatile profile produced by an engineered mutant impaired in quorum-sensing (QS) signalling with the corresponding wild-type led to the conclusion that QS is not involved in the regulation of volatile production in B. ambifaria LMG strain 19182. PMID:23832658

  1. Unusual Presentation of Melioidosis in a Case of Pseudoaneurysm of Descending Thoracic Aorta: Review of Two Case Reports

    PubMed Central

    Padmaja, Kanne; Lakshmi, Vemu; Sudhaharan, Sukanya; Venkata Surya Malladi, Subbalaxmi; Gopal, Palanki; Venkata Ravinuthala, Kumar

    2015-01-01

    Introduction: Melioidosis is a rapidly fatal infectious disease caused by Burkholderia pseudomallei, an agent of potential biothreat, endemic in several parts of India. Most melioidosis-induced infected aneurysms are located in the abdominal or thoracic aorta. Case Presentation: We reported two unusual cases of melioidosis resulting in pseudoaneurysm of the descending thoracic aorta. In both cases, blood cultures yielded B. pseudomallei. The first patient was managed with resection of aneurysm and reconstruction with Dacron graft followed by medical treatment and was discharged uneventfully. The second patient died within one week of admission before the infecting etiological agent was identified and aneurysmal repair was planned. Conclusions: A high clinical index of suspicion, especially in areas of endemicity is essential for timely management of intracavitary infected pseudoaneurysms caused by B. pseudomallei and use of rapid microbiological techniques, such as bact/alert 3D system, which enables rapid and early recovery of the etiological agent. PMID:26380820

  2. Activity of Tobramycin against Cystic Fibrosis Isolates of Burkholderia cepacia Complex Grown as Biofilms

    PubMed Central

    Kennedy, Sarah; Beaudoin, Trevor; Yau, Yvonne C. W.; Caraher, Emma; Zlosnik, James E. A.; Speert, David P.; LiPuma, John J.; Tullis, Elizabeth

    2015-01-01

    Pulmonary infection with Burkholderia cepacia complex in cystic fibrosis (CF) patients is associated with more-rapid lung function decline and earlier death than in CF patients without this infection. In this study, we used confocal microscopy to visualize the effects of various concentrations of tobramycin, achievable with systemic and aerosolized drug administration, on mature B. cepacia complex biofilms, both in the presence and absence of CF sputum. After 24 h of growth, biofilm thickness was significantly reduced by exposure to 2,000 μg/ml of tobramycin for Burkholderia cepacia, Burkholderia multivorans, and Burkholderia vietnamiensis; 200 μg/ml of tobramycin was sufficient to reduce the thickness of Burkholderia dolosa biofilm. With a more mature 48-h biofilm, significant reductions in thickness were seen with tobramycin at concentrations of ≥100 μg/ml for all Burkholderia species. In addition, an increased ratio of dead to live cells was observed in comparison to control with tobramycin concentrations of ≥200 μg/ml for B. cepacia and B. dolosa (24 h) and ≥100 μg/ml for Burkholderia cenocepacia and B. dolosa (48 h). Although sputum significantly increased biofilm thickness, tobramycin concentrations of 1,000 μg/ml were still able to significantly reduce biofilm thickness of all B. cepacia complex species with the exception of B. vietnamiensis. In the presence of sputum, 1,000 μg/ml of tobramycin significantly increased the dead-to-live ratio only for B. multivorans compared to control. In summary, although killing is attenuated, high-dose tobramycin can effectively decrease the thickness of B. cepacia complex biofilms, even in the presence of sputum, suggesting a possible role as a suppressive therapy in CF. PMID:26503664

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

    PubMed Central

    Kost, Thomas; Stopnisek, Nejc; Agnoli, Kirsty; Eberl, Leo

    2014-01-01

    Plant roots and shoots harbor 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. PMID:24409174

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

    PubMed

    Kost, Thomas; Stopnisek, Nejc; Agnoli, Kirsty; Eberl, Leo; Weisskopf, Laure

    2014-01-01

    Plant roots and shoots harbor 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. PMID:24409174

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

    PubMed

    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. PMID:26044439

  6. Isolation of indigenous larvicidal microbial control agents of mosquitos: the Malaysian experience.

    PubMed

    Lee, H L; Seleena, P

    1990-06-01

    A nationwide screening program searching for microbial control agents of mosquitos was initiated in Malaysia in 1986. A total of 725 samples were collected and 2,394 bacterial colonies were isolated and screened for larvicidal activity. From such screening, 20 Bacillus thuringiensis, 6 B. sphaericus, 1 Clostridium bifermentans and 2 Pseudomonas pseudomallei larvicidal isolates were obtained. Of these, a new B. thuringiensis named as subspecies malaysianensis was found, while the C. bifermentans was also a new anaerobe individualized as serovar malaysia. It was concluded that this screening program was highly successful. PMID:2237596

  7. The Organization of the Quorum Sensing luxI/R Family Genes in Burkholderia

    PubMed Central

    Choudhary, Kumari Sonal; Hudaiberdiev, Sanjarbek; Gelencsér, Zsolt; Coutinho, Bruna Gonçalves; Venturi, Vittorio; Pongor, Sándor

    2013-01-01

    Members of the Burkholderia genus of Proteobacteria are capable of living freely in the environment and can also colonize human, animal and plant hosts. Certain members are considered to be clinically important from both medical and veterinary perspectives and furthermore may be important modulators of the rhizosphere. Quorum sensing via N-acyl homoserine lactone signals (AHL QS) is present in almost all Burkholderia species and is thought to play important roles in lifestyle changes such as colonization and niche invasion. Here we present a census of AHL QS genes retrieved from public databases and indicate that the local arrangement (topology) of QS genes, their location within chromosomes and their gene neighborhoods show characteristic patterns that differ between the known Burkholderia clades. In sequence phylogenies, AHL QS genes seem to cluster according to the local gene topology rather than according to the species, which suggests that the basic topology types were present prior to the appearance of current Burkholderia species. The data are available at http://net.icgeb.org/burkholderia/. PMID:23820583

  8. Genome Annotation of Burkholderia sp. SJ98 with Special Focus on Chemotaxis Genes

    PubMed Central

    Kumar, Shailesh; Vikram, Surendra; Raghava, Gajendra Pal Singh

    2013-01-01

    Burkholderia sp. strain SJ98 has the chemotactic activity towards nitroaromatic and chloronitroaromatic compounds. Recently our group published draft genome of strain SJ98. In this study, we further sequence and annotate the genome of stain SJ98 to exploit the potential of this bacterium. We specifically annotate its chemotaxis genes and methyl accepting chemotaxis proteins. Genome of Burkholderia sp. SJ98 was annotated using PGAAP pipeline that predicts 7,268 CDSs, 52 tRNAs and 3 rRNAs. Our analysis based on phylogenetic and comparative genomics suggest that Burkholderia sp. YI23 is closest neighbor of the strain SJ98. The genes involved in the chemotaxis of strain SJ98 were compared with genes of closely related Burkholderia strains (i.e. YI23, CCGE 1001, CCGE 1002, CCGE 1003) and with well characterized bacterium E. coli K12. It was found that strain SJ98 has 37 che genes including 19 methyl accepting chemotaxis proteins that involved in sensing of different attractants. Chemotaxis genes have been found in a cluster along with the flagellar motor proteins. We also developed a web resource that provides comprehensive information on strain SJ98 that includes all analysis data (http://crdd.osdd.net/raghava/genomesrs/burkholderia/). PMID:23940608

  9. Phylogenetically Diverse Burkholderia Associated with Midgut Crypts of Spurge Bugs, Dicranocephalus spp. (Heteroptera: Stenocephalidae).

    PubMed

    Kuechler, Stefan Martin; Matsuura, Yu; Dettner, Konrad; Kikuchi, Yoshitomo

    2016-06-25

    Diverse phytophagous heteropteran insects, commonly known as stinkbugs, are associated with specific gut symbiotic bacteria, which have been found in midgut cryptic spaces. Recent studies have revealed that members of the stinkbug families Coreidae and Alydidae of the superfamily Coreoidea are consistently associated with a specific group of the betaproteobacterial genus Burkholderia, called the "stinkbug-associated beneficial and environmental (SBE)" group, and horizontally acquire specific symbionts from the environment every generation. However, the symbiotic system of another coreoid family, Stenocephalidae remains undetermined. We herein investigated four species of the stenocephalid genus Dicranocephalus. Examinations via fluorescence in situ hybridization (FISH) and transmission electron microscopy (TEM) revealed the typical arrangement and ultrastructures of midgut crypts and gut symbionts. Cloning and molecular phylogenetic analyses of bacterial genes showed that the midgut crypts of all species are colonized by Burkholderia strains, which were further assigned to different subgroups of the genus Burkholderia. In addition to the SBE-group Burkholderia, a number of stenocephalid symbionts belonged to a novel clade containing B. sordidicola and B. udeis, suggesting a specific symbiont clade for the Stenocephalidae. The symbiotic systems of stenocephalid bugs may provide a unique opportunity to study the ongoing evolution of symbiont associations in the stinkbug-Burkholderia interaction. PMID:27265344

  10. A N2-fixing endophytic Burkholderia sp. associated with maize plants cultivated in Mexico.

    PubMed

    Estrada, Paulina; Mavingui, Patrick; Cournoyer, Benoit; Fontaine, Fanette; Balandreau, Jacques; Caballero-Mellado, Jesus

    2002-04-01

    In the frame of a survey of potentially endophytic N2-fixing Burkholderia associated with maize in Mexico, its country of origin, the soil of an indigenous maize field near Oaxaca was studied. Under laboratory conditions, plant seedlings of two ancient maize varieties were used as a trap to select endophyte candidates from the soil sample. Among the N2 fixers isolated from inside plant tissues and able to grow on PCAT medium, the most abundant isolates belonged to genus Burkholderia (API 20NE, rrs sequences). Representative isolates obtained from roots and shoots of different plants appeared identical (rrs and nifH RFLP), showing that they were closely related. In addition, their 16S rDNA sequences differed from described Burkholderia species and, phylogenetically, they constituted a separate deep-branching new lineage in genus Burkholderia. This indicated that these isolates probably constituted a new species. An inoculation experiment confirmed that these N2-fixing Burkholderia isolates could densely colonize the plant tissues of maize. More isolates of this group were subsequently obtained from field-grown maize and teosinte plants. It was hypothesized that strains of this species had developed a sort of primitive symbiosis with one of their host plants, teosinte, which persisted during the domestication of teosinte into maize. PMID:12030700

  11. Phylogenetically Diverse Burkholderia Associated with Midgut Crypts of Spurge Bugs, Dicranocephalus spp. (Heteroptera: Stenocephalidae)

    PubMed Central

    Kuechler, Stefan Martin; Matsuura, Yu; Dettner, Konrad; Kikuchi, Yoshitomo

    2016-01-01

    Diverse phytophagous heteropteran insects, commonly known as stinkbugs, are associated with specific gut symbiotic bacteria, which have been found in midgut cryptic spaces. Recent studies have revealed that members of the stinkbug families Coreidae and Alydidae of the superfamily Coreoidea are consistently associated with a specific group of the betaproteobacterial genus Burkholderia, called the “stinkbug-associated beneficial and environmental (SBE)” group, and horizontally acquire specific symbionts from the environment every generation. However, the symbiotic system of another coreoid family, Stenocephalidae remains undetermined. We herein investigated four species of the stenocephalid genus Dicranocephalus. Examinations via fluorescence in situ hybridization (FISH) and transmission electron microscopy (TEM) revealed the typical arrangement and ultrastructures of midgut crypts and gut symbionts. Cloning and molecular phylogenetic analyses of bacterial genes showed that the midgut crypts of all species are colonized by Burkholderia strains, which were further assigned to different subgroups of the genus Burkholderia. In addition to the SBE-group Burkholderia, a number of stenocephalid symbionts belonged to a novel clade containing B. sordidicola and B. udeis, suggesting a specific symbiont clade for the Stenocephalidae. The symbiotic systems of stenocephalid bugs may provide a unique opportunity to study the ongoing evolution of symbiont associations in the stinkbug-Burkholderia interaction. PMID:27265344

  12. Diversity of Cultivated Endophytic Bacteria from Sugarcane: Genetic and Biochemical Characterization of Burkholderia cepacia Complex Isolates▿

    PubMed Central

    Mendes, Rodrigo; Pizzirani-Kleiner, Aline A.; Araujo, Welington L.; Raaijmakers, Jos M.

    2007-01-01

    Bacteria were isolated from the rhizosphere and from inside the roots and stems of sugarcane plants grown in the field in Brazil. Endophytic bacteria were found in both the roots and the stems of sugarcane plants, with a significantly higher density in the roots. Many of the cultivated endophytic bacteria were shown to produce the plant growth hormone indoleacetic acid, and this trait was more frequently found among bacteria from the stem. 16S rRNA gene sequence analysis revealed that the selected isolates of the endophytic bacterial community of sugarcane belong to the genera of Burkholderia, Pantoea, Pseudomonas, and Microbacterium. Bacterial isolates belonging to the genus Burkholderia were the most predominant among the endophytic bacteria. Many of the Burkholderia isolates produced the antifungal metabolite pyrrolnitrin, and all were able to grow at 37°C. Phylogenetic analyses of the 16S rRNA gene and recA gene sequences indicated that the endophytic Burkholderia isolates from sugarcane are closely related to clinical isolates of the Burkholderia cepacia complex and clustered with B. cenocepacia (gv. III) isolates from cystic fibrosis patients. These results suggest that isolates of the B. cepacia complex are an integral part of the endophytic bacterial community of sugarcane in Brazil and reinforce the hypothesis that plant-associated environments may act as a niche for putative opportunistic human pathogenic bacteria. PMID:17905875

  13. Burkholderia of Plant-Beneficial Group are Symbiotically Associated with Bordered Plant Bugs (Heteroptera: Pyrrhocoroidea: Largidae)

    PubMed Central

    Takeshita, Kazutaka; Matsuura, Yu; Itoh, Hideomi; Navarro, Ronald; Hori, Tomoyuki; Sone, Teruo; Kamagata, Yoichi; Mergaert, Peter; Kikuchi, Yoshitomo

    2015-01-01

    A number of phytophagous stinkbugs (order Heteroptera: infraorder Pentatomomorpha) harbor symbiotic bacteria in a specific midgut region composed of numerous crypts. Among the five superfamilies of the infraorder Pentatomomorpha, most members of the Coreoidea and Lygaeoidea are associated with a specific group of the genus Burkholderia, called the “stinkbug-associated beneficial and environmental (SBE)” group, which is not vertically transmitted, but acquired from the environment every host generation. A recent study reported that, in addition to these two stinkbug groups, the family Largidae of the superfamily Pyrrhocoroidea also possesses a Burkholderia symbiont. Despite this recent finding, the phylogenetic position and biological nature of Burkholderia associated with Largidae remains unclear. Based on the combined results of fluorescence in situ hybridization, cloning analysis, Illumina deep sequencing, and egg inspections by diagnostic PCR, we herein demonstrate that the largid species are consistently associated with the “plant-associated beneficial and environmental (PBE)” group of Burkholderia, which are phylogenetically distinct from the SBE group, and that they maintain symbiosis through the environmental acquisition of the bacteria. Since the superfamilies Coreoidea, Lygaeoidea, and Pyrrhocoroidea are monophyletic in the infraorder Pentatomomorpha, it is plausible that the symbiotic association with Burkholderia evolved at the common ancestor of the three superfamilies. However, the results of this study strongly suggest that a dynamic transition from the PBE to SBE group, or vice versa, occurred in the course of stinkbug evolution. PMID:26657305

  14. Gene Expression Profiling of Burkholderia cenocepacia at the Time of Cepacia Syndrome: Loss of Motility as a Marker of Poor Prognosis?

    PubMed Central

    Kalferstova, Lucie; Kolar, Michal; Fila, Libor; Vavrova, Jolana

    2015-01-01

    Cepacia syndrome (CS) is a fatal septic condition that develops in approximately 20% of cystic fibrosis (CF) patients chronically infected with the Burkholderia cepacia complex (Bcc). The most common causative agent is Burkholderia cenocepacia, a clinically dominant Bcc species that contains the globally distributed epidemic strain sequence type 32 (ST32). Using microarrays, we compared the transcriptomes of ST32 isolates from the bloodstream at the time of CS with their sputum counterparts recovered 1 to 2 months prior to the development of CS. Global gene expression profiles of blood isolates revealed greater activities of the virulence genes involved in the type III secretion system, the bacterial exopolysaccharide cepacian, and quorum sensing, while reduced expression was demonstrated for flagellar genes. Furthermore, a nonmotile phenotype (as evaluated by a swimming motility assay) was identified in blood isolates from 6 out of 8 patients with CS; this phenotype was traceable to 24 months prior to the onset of CS. Loss of motility was not observed in any of the 89 ST32 isolates recovered over the course of chronic infection from 17 patients without CS. In conclusion, the gene expression of Bcc bacteria disseminated during CS has been elucidated for the first time. This study demonstrated marked differences at the transcriptome level between isogenic ST32 isolates that are attributable to the stage and site of infection. The finding of a nonmotile B. cenocepacia isolate may serve as a warning sign for the development of CS in the near future. PMID:25694518

  15. The antibiotics of choice for the treatment of melioidosis in Indian set up.

    PubMed

    Shaw, T; Tellapragada, C; Eshwara, V K; Bhat, H V; Mukhopadhyay, C

    2016-01-01

    Therapeutic options for the treatment of melioidosis caused by Burkholderia pseudomallei are limited due to the inherent resistance conferred by this pathogen to various groups of antibiotics. Witnessing an increase in the number of microbiological culture-confirmed cases of melioidosis at our settings in the past few years, we undertook this study to estimate the minimum inhibitory concentrations of clinical isolates of B. pseudomallei against the four commonly employed antimicrobial agents in the patient management at our settings, namely, ceftazidime, meropenem, trimethoprim-sulfamethoxazole and doxycycline. All isolates were susceptible to the antibiotics tested, except for one isolate which showed resistance to doxycycline (minimum inhibitory concentration [MIC]: 32 μg/ml). MIC50 and 90 for all the four antibiotics were estimated. From this study, we conclude that the clinical isolates of B. pseudomallei from the southern part of India are well susceptible to the commonly employed antimicrobial agents for therapy. PMID:27514960

  16. [Phylogenetic analysis of the genes for naphthalene and phenanthrene degradation in Burkholderia sp. strains].

    PubMed

    Izmalkova, T Yu; Sazonova, O I; Kosheleva, I A; Boronin, A M

    2013-06-01

    The genetic systems responsible for naphthalene and phenanthrene catabolism have been analyzed in the five strains of Burkholderia sp. isolated from soil samples (West Siberia) contaminated by heavy residual fuel oil and in the strain Burkholderia sp. BS3702 from the laboratory collection isolated from soil samples of the coke gas works (Vidnoe, Moscow oblast). The results of this work demonstrate that naphthalene and phenanthrene degradation in the above strains is encoded by the sequences not homologous to the classical nah genes of pseudomonades. In the Burkholderia sp. BS3702 strain, the initial stages of phenanthrene degradation and the subsequent stages of salicylate degradation are controlled by the sequences of different evolutionary origins (phn and nag genes). PMID:24450193

  17. Naturally Occurring Class A ß-Lactamases from the Burkholderia cepacia Complex ▿

    PubMed Central

    Poirel, Laurent; Rodriguez-Martinez, José-Manuel; Plésiat, Patrick; Nordmann, Patrice

    2009-01-01

    Chromosomally encoded ß-lactamases from the Burkholderia cepacia complex species (formerly Pseudomonas cepacia) were characterized. Cloning and sequencing identified an Ambler class A ß-lactamase (PenB) from B. cenocepacia. It shares 82% amino acid identity with the PenA ß-lactamases previously identified from B. multivorans 249. Its expression was dependent upon a LysR-type regulatory protein. Its narrow-spectrum hydrolysis activity mostly included penicillins but also included expanded-spectrum cephalosporins and aztreonam at lower levels. In that study, Pen-like ß-lactamases (PenC, PenD, PenE, PenF) that shared 63 to 92% identity with PenB from B. cenocepacia were identified from other Burkholderia species. The corresponding ß-lactamase genes might be used as genetic tools for accurate Burkholderia species identification. PMID:19075063

  18. Evolving serodiagnostics by rationally designed peptide arrays: the Burkholderia paradigm in Cystic Fibrosis.

    PubMed

    Peri, Claudio; Gori, Alessandro; Gagni, Paola; Sola, Laura; Girelli, Daniela; Sottotetti, Samantha; Cariani, Lisa; Chiari, Marcella; Cretich, Marina; Colombo, Giorgio

    2016-01-01

    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. PMID:27615705

  19. Genetic structure of a lotic population of Burkholderia (Pseudomonas) cepacia

    SciTech Connect

    Wise, M.G.; Shimkets, L.J.; McArthur, J.V.

    1995-05-01

    The genetic structure of a population of Burkholderia (Pseudomonas) cepacia isolated from a southeastern blackwater stream was investigated by using multilocus enzyme electrophoresis to examine the allelic variation in eight structural gene loci. Overall, 213 isolates were collected at transect points along the stream continuum, from both the sediments along the bank and the water column. Multilocus enzyme electrophoresis analysis revealed 164 distinct electrophoretic types, and the mean genetic diversity of the entire population was 0.574. Genetic diversity values did not vary spatially along the stream continuum. From a canonical discriminant analysis, Mahalonobis distances (measurements of genetic similarity between populations) revealed significant differences among the subpopulations at the sediment sampling points, suggesting bacterial adaptation to a heterogeneous (or patchy) microgeographical environment. Multilocus linkage disequilibrium analysis of the isolates revealed only limited association between alleles, suggesting frequent recombination, relative to binary fission, in this population. Furthermore, the dendrogram created from the data of this study and the allele mismatch distribution are typical of a population characterized by extensive genetic mixing. We suggest that B. cepacia be added to the growing list of bacteria that are not obligatorily clonal. 41 refs., 5 figs., 3 tabs.

  20. Burkholderia Sepsis in Children as a Hospital-Acquired Infection

    PubMed Central

    Kim, Kyu Yeun; Yong, Dongeun; Lee, Kyungwon; Kim, Ho-Seong

    2016-01-01

    Purpose Hospital-acquired Burkholderia cepacia (B. cepacia) infection are not commonly recorded in patients without underlying lung disease, such as cystic fibrosis and chronic granulomatous disease. However, in 2014, B. cepacia appeared more frequently in pediatric blood samples than in any other year. In order to access this situation, we analyzed the clinical characteristics of B. cepacia infections in pediatric patients at our hospital. Materials and Methods We conducted a retrospective study of blood isolates of B. cepacia taken at our hospital between January 2004 and December 2014. Patient clinical data were obtained by retrospective review of electronic medical records. We constructed a dendrogram for B. cepacia isolates from two children and five adult patients. Results A total of 14 pediatric patients and 69 adult patients were identified as having B. cepacia bacteremia. In 2014, higher rates of B. cepacia bacteremia were observed in children. Most of them required Intensive Care Unit (ICU) care (12/14). In eleven children, sputum cultures were examined, and five of these children had the same strain of B. cepacia that grew out from their blood samples. Antibiotics were administered based on antibiotic sensitivity results. Four children expired despite treatment. Compared to children, there were no demonstrative differences in adults, except for history of ICU care. Conclusion Although there were not many pediatric cases at our hospital, awareness of colonization through hospital-acquired infection and effective therapy for infection of B. cepacia is needed, as it can cause mortality and morbidity. PMID:26632388

  1. Identification of quorum sensing-controlled genes in Burkholderia ambifaria

    PubMed Central

    Chapalain, Annelise; Vial, Ludovic; Laprade, Natacha; Dekimpe, Valérie; Perreault, Jonathan; Déziel, Eric

    2013-01-01

    The Burkholderia cepacia complex (Bcc) comprises strains with a virulence potential toward immunocompromised patients as well as plant growth–promoting rhizobacteria (PGPR). Owing to the link between quorum sensing (QS) and virulence, most studies among Bcc species have been directed toward QS of pathogenic bacteria. We have investigated the QS of B. ambifaria, a PGPR only infrequently recovered from patients. The cepI gene, responsible for the synthesis of the main signaling molecule N-octanoylhomoserine lactone (C8-HSL), was inactivated. Phenotypes of the B. ambifaria cepI mutant we observed, such as increased production of siderophores and decreased proteolytic and antifungal activities, are in agreement with those of other Bcc cepI mutants. The cepI mutant was then used as background strain for a whole-genome transposon-insertion mutagenesis strategy, allowing the identification of 20 QS-controlled genes, corresponding to 17 loci. The main functions identified are linked to antifungal and antimicrobial properties, as we have identified QS-controlled genes implicated in the production of pyrrolnitrin, burkholdines (occidiofungin-like molecules), and enacyloxins. This study provides insights in the QS-regulated functions of a PGPR, which could lead to beneficial potential biotechnological applications. PMID:23382083

  2. Binding of protegrin-1 to Pseudomonas aeruginosa and Burkholderia cepacia

    PubMed Central

    Albrecht, Mark T; Wang, Wei; Shamova, Olga; Lehrer, Robert I; Schiller, Neal L

    2002-01-01

    Background Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia. Methods The PG-1 sensitivity/resistance and PG-1 binding properties of P. aeruginosa and B. cepacia were assessed using radial diffusion assays, radioiodinated PG-1, and surface plasmon resonance (BiaCore). Results The six P. aeruginosa strains examined were very sensitive to PG-1, exhibiting minimal active concentrations from 0.0625–0.5 μg/ml in radial diffusion assays. In contrast, all five B. cepacia strains examined were greater than 10-fold to 100-fold more resistant, with minimal active concentrations ranging from 6–10 μg/ml. When incubated with a radioiodinated variant of PG-1, a sensitive P. aeruginosa strain bound considerably more protegrin molecules per cell than a resistant B. cepacia strain. Binding/diffusion and surface plasmon resonance assays revealed that isolated lipopolysaccharide (LPS) and lipid A from the sensitive P. aeruginosa strains bound PG-1 more effectively than LPS and lipid A from resistant B. cepacia strains. Conclusion These findings support the hypothesis that the relative resistance of B. cepacia to protegrin is due to a reduced number of PG-1 binding sites on the lipid A moiety of its LPS. PMID:11980587

  3. Identification of sodium chloride-regulated genes in Burkholderia cenocepacia.

    PubMed

    Bhatt, Shantanu; Weingart, Christine L

    2008-05-01

    Previous studies have suggested that the airways of cystic fibrosis (CF) patients have elevated sodium chloride (NaCl) levels due to the malfunctioning of the CF transmembrane conductance regulator protein. For bacteria to survive in this high-salt environment, they must adjust by altering the regulation of gene expression. Among the different bacteria inhabiting the airways of CF patients is the opportunistic pathogen Burkholderia cenocepacia. Previous studies have indicated that B. cenocepacia produces a toxin and cable pili under high osmolar conditions. We used transposon mutagenesis to identify NaCl-regulated genes in the clinical strain B. cenocepacia K56-2. Six transconjugants were induced with increasing NaCl concentration. The DNA flanking the transposon was sequenced and five distinct open reading frames were identified encoding the following putative proteins: an integrase, an NAD-dependent deacetylase, TolB, an oxidoreductase, and a novel hypothetical protein. The collective results of this study provide important information about the physiology of B. cenocepacia when faced with osmotic stress and suggest the identity of significant virulence mechanisms in this opportunistic pathogen. PMID:18288523

  4. Burkholderia Species Are the Most Common and Preferred Nodulating Symbionts of the Piptadenia Group (Tribe Mimoseae)

    PubMed Central

    Bournaud, Caroline; de Faria, Sergio Miana; dos Santos, José Miguel Ferreira; Tisseyre, Pierre; Silva, Michele; Chaintreuil, Clémence; Gross, Eduardo; James, Euan K.; Prin, Yves; Moulin, Lionel

    2013-01-01

    Burkholderia legume symbionts (also called α-rhizobia) are ancient in origin and are the main nitrogen-fixing symbionts of species belonging to the large genus Mimosa in Brazil. We investigated the extent of the affinity between Burkholderia and species in the tribe Mimoseae by studying symbionts of the genera Piptadenia (P.), Parapiptadenia (Pp.), Pseudopiptadenia (Ps.), Pityrocarpa (Py.), Anadenanthera (A.) and Microlobius (Mi.), all of which are native to Brazil and are phylogenetically close to Mimosa, and which together with Mimosa comprise the “Piptadenia group”. We characterized 196 strains sampled from 18 species from 17 locations in Brazil using two neutral markers and two symbiotic genes in order to assess their species affiliations and the evolution of their symbiosis genes. We found that Burkholderia are common and highly diversified symbionts of species in the Piptadenia group, comprising nine Burkholderia species, of which three are new ones and one was never reported as symbiotic (B. phenoliruptrix). However, α-rhizobia were also detected and were occasionally dominant on a few species. A strong sampling site effect on the rhizobial nature of symbionts was detected, with the symbiont pattern of the same legume species changing drastically from location to location, even switching from β to α-rhizobia. Coinoculation assays showed a strong affinity of all the Piptadenia group species towards Burkholderia genotypes, with the exception of Mi. foetidus. Phylogenetic analyses of neutral and symbiotic markers showed that symbiosis genes in Burkholderia from the Piptadenia group have evolved mainly through vertical transfer, but also by horizontal transfer in two species. PMID:23691052

  5. Solubilization of insoluble inorganic phosphate by Burkholderia cepacia DA23 isolated from cultivated soil

    PubMed Central

    Song, Ok-Ryul; Lee, Seung-Jin; Lee, Yong-Seok; Lee, Sang-Cheol; Kim, Keun-Ki; Choi, Yong-Lark

    2008-01-01

    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. PMID:24031195

  6. Toluene 2-Monooxygenase-Dependent Growth of Burkholderia cepacia G4/PR1 on Diethyl Ether

    PubMed Central

    Hur, H.; Newman, L. M.; Wackett, L. P.; Sadowsky, M. J.

    1997-01-01

    Aerobic bacterial growth on aromatic hydrocarbons typically requires oxygenase enzymes, which are known to fortuitously oxidize nongrowth substrates. In this study, we found that oxidation of diethyl ether by toluene 2-monooxygenase supported more rapid growth of Burkholderia cepacia G4/PR1 than did the aromatic substrates n-propylbenzene and o-xylene. The wild-type Burkholderia cepacia G4 failed to grow on diethyl ether. Purified toluene 2-monooxygenase protein components oxidized diethyl ether stoichiometrically to ethanol and acetaldehyde. Butyl methyl ether, diethyl sulfide, and 2-chloroethyl ethyl ether were oxidized by B. cepacia G4/PR1. PMID:16535583

  7. Burkholderia cepacia septicemia in a patient with acute myeloid leukemia in postchemotherapy bone marrow aplasia

    PubMed Central

    Mihaila, Romeo-Gabriel; Blaga, Lucian

    2013-01-01

    The patients with hematologic malignancies are predisposed to develop infections with unusual bacteria, like Burkholderia cepacia, which is frequently resistant to many antibiotics and antiseptics. We present the case of a female patient with acute myeloid leukemia type 2 on the background of myelodysplastic syndrome, from whom Burkholderia cepacia was isolated in blood culture, after the 2nd cycle of induction. She was sensitive to ceftazidime, but its eradication was not easy. Five other patients were contaminated with this bacteria, but all of them had favourable evolution. The case is discussed in the context of those similar in literature. PMID:24353735

  8. Burkholderia ginsengiterrae sp. nov. and Burkholderia panaciterrae sp. nov., antagonistic bacteria against root rot pathogen Cylindrocarpon destructans, isolated from ginseng soil.

    PubMed

    Farh, Mohamed El-Agamy; Kim, Yeon-Ju; Van An, Hoang; Sukweenadhi, Johan; Singh, Priyanka; Huq, Md Amdadul; Yang, Deok-Chun

    2015-04-01

    Strain DCY85(T) and DCY85-1(T), isolated from rhizosphere of ginseng, were rod-shaped, Gram-reaction-negative, strictly aerobic, catalase positive and oxidase negative. 16S rRNA gene sequence analysis revealed that strain DCY85(T) as well as DCY85-1(T) belonged to the genus Burkholderia and were closely related to Burkholderia fungorum KACC 12023(T) (98.1 and 98.0 % similarity, respectively). The major polar lipids of strain DCY85(T) and DCY85-1(T) were phosphatidylethanolamine, one unidentified aminolipid and two unidentified phospholipids. The major fatty acids of both strains are C16:0, C18:1 ω7c and summed feature 3 (C16:1 ω6c and/or C16:1 ω7c). The predominant isoprenoid quinone of each strain DCY85(T) and DCY85-1(T) was ubiquinone (Q-8) and the G+C content of their genomic DNA was 66.0 and 59.4 mol%, respectively, which fulfill the characteristic range of the genus Burkholderia. The polyamine content of both DCY85(T) and DCY85-1(T) was putrescine. Although both DCY85(T) and DCY85-1(T) have highly similar 16S rRNA and identical RecA and gyrB sequences, they show differences in phenotypic and chemotaxonomic characteristics. DNA-DNA hybridization results proved the consideration of both strains as two different species. Based on the results from our polyphasic characterization, strain DCY85(T) and DCY85-1(T) are considered novel Burkholderia species for which the name Burkholderia ginsengiterrae sp. nov and Burkholderia panaciterrae sp. nov are, respectively, proposed. An emended description of those strains is also proposed. DCY85(T) and DCY85-1(T) showed antagonistic activity against the common root rot pathogen of ginseng, Cylindrocarpon destructans. The proposed type strains are DCY85(T) (KCTC 42054(T) = JCM 19888(T)) and DCY85-1(T) (KCTC 42055(T) = JCM 19889(T)). PMID:25537097

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

    PubMed

    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. PMID:25725797

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

    PubMed Central

    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

  11. Draft Genome Sequence of the Rice Endophyte Burkholderia kururiensis M130

    PubMed Central

    Coutinho, Bruna Gonçalves; Passos da Silva, Daniel; Previato, José Osvaldo

    2013-01-01

    Burkholderia kururiensis M130 is one of the few characterized rice endophytes and was isolated from surface-sterilized rice roots. This bacterium shows strong growth-promoting effects, being able to increase rice yields. Here we present its draft genome sequence, which contains important traits for endophytic life and plant growth promotion. PMID:23558537

  12. Repeated Burkholderia cepacia Peritonitis in a Patient Undergoing Continuous Ambulatory Peritoneal Dialysis

    PubMed Central

    Apostolovic, BL; Velickovic-Radovanovic, RM; Andjelkovic-Apostolovic, MR; Cvetkovic, TP; Dinic, MM; Radivojevic, JD

    2015-01-01

    ABSTRACT Burkholderia cepacia (B cepacia) is a rare opportunistic pathogen in continuous ambulatory peritoneal dialysis (CAPD) peritonitis. We describe the first case of repeated B cepacia CAPD peritonitis, occurring in an outpatient environment, treated with antimicrobial medication without peritoneal catheter removal. B cepacia may lead to repeat infection, therefore, we should insist on catheter removal during each peritonitis episode. PMID:26426187

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

    PubMed

    Ho, Ying-Ning; Huang, Chieh-Chen

    2015-01-01

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

  14. A bioinformatics approach to the determination of genes involved in endophytic behavior in Burkholderia spp.

    PubMed

    Ali, Shimaila; Duan, Jin; Charles, Trevor C; Glick, Bernard R

    2014-02-21

    The vast majority of plants harbor endophytic bacteria that colonize a portion of the plant's interior tissues without harming the plant. Like plant pathogens, endophytes gain entry into their plants hosts through various mechanisms. Bacterial endophytes display a broad range of symbiotic interactions with their host plants. The molecular bases of these plant-endophyte interactions are currently not fully understood. In the present study, a set of genes possibly responsible for endophytic behavior for genus Burkholderia was predicted and then compared and contrasted with a number (nine endophytes from different genera) of endophytes by comparative genome analysis. The nine endophytes included Burkholderia phytofirmans PsJN, Burkholderia spp. strain JK006, Azospirillum lipoferum 4B, Enterobacter cloacae ENHKU01, Klebsiella pneumoniae 342, Pseudomonas putida W619, Enterobacter spp. 638, Azoarcus spp. BH72, and Serratia proteamaculans 568. From the genomes of the analyzed bacterial strains, a set of bacterial genes orthologs was identified that are predicted to be involved in determining the endophytic behavior of Burkholderia spp. The genes and their possible functions were then investigated to establish a potential connection between their presence and the role they play in bacterial endophytic behavior. Nearly all of the genes identified by this bioinformatics procedure encode function previously suggested in other studies to be involved in endophytic behavior. PMID:24513137

  15. NOVEL ORGANIZATION OF THE GENES FOR PHTHALATE DEGRADATION FROM BURKHOLDERIA CEPACIA DBO1

    EPA Science Inventory

    Burkholderia cepacia DBO1 is able to utilize phthalate as the sole source of carbon and energy for growth. Two overlapping cosmid clones containing the genes for phthalate degradation were isolated from this strain. Subcloning and activity analysis localized the genes for phthala...

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

    PubMed

    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. PMID:26875632

  17. Antimicrobial Properties of an Oxidizer Produced by Burkholderia cenocepacia P525

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A compound with both oxidizing properties and antibiotic properties was extracted and purified from broth cultures of Burkholderia cenocepacia strain P525. A four step purification procedure was used to increase its specific activity ~ 400 fold and to yield a HPLC- UV chromatogram containing a sing...

  18. Complete genome sequence of the lipase producing strain Burkholderia glumae PG1.

    PubMed

    Voget, Sonja; Knapp, Andreas; Poehlein, Anja; Vollstedt, Christel; Streit, Wolfgang; Daniel, Rolf; Jaeger, Karl-Erich

    2015-06-20

    The Gram-negative proteobacterium Burkholderia glumae PG1 produces a lipase of biotechnological interest, which is used for the production of enantiopure pharmaceuticals. In order to better understand the underlying mechanisms and provide a basis for further studies, we present here the complete genome sequence of B. glumae PG1. PMID:25848987

  19. Burkholderia phymatum Strains Capable of Nodulating Phaseolus vulgaris Are Present in Moroccan Soils ▿

    PubMed Central

    Talbi, C.; Delgado, M. J.; Girard, L.; Ramírez-Trujillo, A.; Caballero-Mellado, J.; Bedmar, E. J.

    2010-01-01

    Phylogenetic analysis of 16S rRNA, nodC, and nifH genes of four bacterial strains isolated from root nodules of Phaseolus vulgaris grown in Morocco soils were identified as Burkholderia phymatum. All four strains formed N2-fixing nodules on P. vulgaris and Mimosa, Acacia, and Prosopis species and reduced acetylene to ethylene when cultured ex planta. PMID:20472732

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

    PubMed

    Vicenzi, Fernando José; Pillonetto, Marcelo; Souza, Helena Aguilar Peres Homem de Mello de; 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, recA sequencing, 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 recA sequencing to identify Bcc species. The recA sequencing 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

  1. Characterization of a novel two-component system in Burkholderia cenocepacia.

    PubMed

    Merry, Callie R; Perkins, Michael; Mu, Lin; Peterson, Bridget K; Knackstedt, Rebecca W; Weingart, Christine L

    2015-04-01

    Two-component systems are important regulatory systems that allow bacteria to adjust to environmental conditions, and in some bacteria are used in pathogenesis. We identified a novel two-component system in Burkholderia cenocepacia, an opportunistic pathogen that causes pneumonia in cystic fibrosis (CF) patients. The putative operon encodes BceS, a sensor kinase, and BceR, a response regulator. Our studies indicated that the bceR mutant showed a statistically significant decrease in protease, swimming motility, and quorum sensing when compared to the wild-type, but there was no significant difference in phospholipase C activity, swarming, and biofilm formation. In addition, the mutant showed a statistically significant reduction in virulence compared to the wild-type using the alfalfa plant model. Examination of the Burkholderia cepacia complex (a group of organisms that are phenotypically similar, but genotypically distinct) revealed that this system is prevalent in B. ambifaria, B. multivorans, B. vietnamiensis and B. dolosa. Interestingly, all these organisms have been associated with CF patients. The collective results indicate that BceSR influences various activities important in Burkholderia physiology and possibly pathogenesis. This information could be important in the design of novel therapeutics for Burkholderia infections. PMID:25519693

  2. Burkholderia tropica sp. nov., a novel nitrogen-fixing, plant-associated bacterium.

    PubMed

    Reis, V M; Estrada-de los Santos, P; Tenorio-Salgado, S; Vogel, J; Stoffels, M; Guyon, S; Mavingui, P; Baldani, V L D; Schmid, M; Baldani, J I; Balandreau, J; Hartmann, A; Caballero-Mellado, J

    2004-11-01

    In an ecological survey of nitrogen-fixing bacteria isolated from the rhizosphere and as endophytes of sugarcane, maize and teosinte plants in Brazil, Mexico and South Africa, a new phylogenetically homogeneous group of N(2)-fixing bacteria was identified within the genus Burkholderia. This polyphasic taxonomic study included microscopic and colony morphology, API 20NE tests and growth on different culture media at different pH and temperatures, as well as carbon source assimilation tests and whole-cell protein pattern analysis. Analysis of 16S rRNA gene sequences showed 99.2-99.9 % similarity within the novel species and 97.2 % similarity to the closest related species, Burkholderia sacchari. The novel species was composed of four distinct amplified 16S rDNA restriction analysis groups. The DNA-DNA reassociation values within the novel species were greater than 70 % and less than 42 % for the closest related species, B. sacchari. Based on these results and on many phenotypic characteristics, a novel N(2)-fixing species is proposed for the genus Burkholderia, Burkholderia tropica sp. nov., with the type strain Ppe8(T) (=ATCC BAA-831(T)=LMG 22274(T)=DSM 15359(T)). B. tropica was isolated from plants grown in geographical regions with climates ranging from temperate subhumid to hot humid. PMID:15545451

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

    PubMed

    Ettinger, Cassandra L; Shehata, Hanan R; Johnston-Monje, David; Raizada, Manish N; Eisen, Jonathan A

    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

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

    PubMed Central

    Ho, Ying-Ning

    2015-01-01

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

  5. Biocontrol of Late Blight (Phytophthora capsici) Disease and Growth Promotion of Pepper by Burkholderia cepacia MPC-7

    PubMed Central

    Sopheareth, Mao; Chan, Sarun; Naing, Kyaw Wai; Lee, Yong Seong; Hyun, Hae Nam; Kim, Young Cheol; Kim, Kil Yong

    2013-01-01

    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. PMID:25288930

  6. Biocontrol of Late Blight (Phytophthora capsici) Disease and Growth Promotion of Pepper by Burkholderia cepacia MPC-7.

    PubMed

    Sopheareth, Mao; Chan, Sarun; Naing, Kyaw Wai; Lee, Yong Seong; Hyun, Hae Nam; Kim, Young Cheol; Kim, Kil Yong

    2013-03-01

    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. PMID:25288930

  7. Powder formulation of Burkholderia cepacia for control of rape seed damping-off caused by Rhizoctonia solani.

    PubMed

    Sharifi-Tehrani, A; Ahmadzadeh, M; Sarani, S; Farzaneh, M

    2007-01-01

    Talc-based formulation of Burkholderia cepaci strain Bu1 was tested as seed and soil drenchs separately for its ability to control Rhizoctonia soloni the causal agent of rape seed damping-off in greenhouse and field trials. In general, the formulated bacteria was more effective to suppress the disease than the suspension of bacteria cells in carboxymethylcellulose solution (1% w/v), in both greenhouse and field trials. The formulation of strain Bul as soil and seed treatments had the greatest effect on reducing the rape seed damping-off in greenhouse and field trials (66.7, 53.3, 64.4 and 40% respectively). The formulation of strain Bu1 as soil and seed treatments were the most effective treatments to increase the root dry weights in the infected soil in greenhouse. The formulation of strain Bul as soil drench had the greatest effect on enhancement of the fresh weight of roots and stem fresh and dry weights. The formulation of strain Bu1 stored at 4 degrees C exhibited better shelf Life and efficacy in vitro than it's counterpart stored at 25 degrees C. PMID:18399433

  8. Agent Orange

    MedlinePlus

    ... Index Agent Orange Agent Orange Home Facts about Herbicides Veterans' Diseases Birth Defects Benefits Exposure Locations Provider ... millions of gallons of Agent Orange and other herbicides on trees and vegetation during the Vietnam War. ...

  9. Minocycline activity tested against Acinetobacter baumannii complex, Stenotrophomonas maltophilia, and Burkholderia cepacia species complex isolates from a global surveillance program (2013).

    PubMed

    Flamm, Robert K; Castanheira, Mariana; Streit, Jennifer M; Jones, Ronald N

    2016-07-01

    Clinical isolates of Acinetobacter baumannii complex (1312), Stenotrophomonas maltophilia (464), and Burkholderia cepacia species complex (30) were selected from medical centers in the United States (USA), Europe and the Mediterranean (EU-M) region, Latin America, and Asia Pacific. Only one isolate per infected patient episode was included and local identifications were confirmed by the monitoring laboratory. Susceptibility testing was performed at the monitoring laboratory using the reference broth microdilution method as described by Clinical and Laboratory Standards Institute (CLSI). A. baumannii complex were classified as MDR (multi-drug resistant [MDR]; nonsusceptible to ≥1 agent in ≥3 antimicrobial classes) and extensively drug-resistant (XDR; nonsusceptible to ≥1 agent in all but ≤2 antimicrobial classes). A total of 81.6% of A. baumannii complex were MDR. Colistin was the most active agent against MDR A. baumannii complex. Minocycline was the most active "tetracycline" against these organisms based on susceptibility. Against B. cepacia, trimethoprim-sulfamethoxazole (MIC90, 2 μg/mL; 100.0% susceptible) was the most active agent tested. Overall, minocycline was the most active tetracycline tested against A. baumannii complex and S. maltophilia isolates collected from patients throughout EU-M, USA, Latin America, and the Asia-Pacific. Minocycline, particularly the intravenous formulation, has activity against several ESKAPE pathogens and merits consideration in seriously ill patients where treatment options may be limited due to the presence of MDR bacteria. PMID:27112832

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

    PubMed

    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. PMID:26059639

  11. Epidemiology of Burkholderia cepacia complex species recovered from cystic fibrosis patients: issues related to patient segregation.

    PubMed

    McDowell, Andrew; Mahenthiralingam, Eshwar; Dunbar, Kerstin E A; Moore, John E; Crowe, Mary; Elborn, J Stuart

    2004-07-01

    Studies of the prevalence of Burkholderia cepacia complex species amongst cystic fibrosis (CF) patients in different geographical regions, and the association between cross-infection and putative transmissibility markers, will further our understanding of these organisms and help to address infection-control issues. In this study, B. cepacia complex isolates from CF patients in different regions of Europe were analysed. Isolates were examined for B. cepacia complex species and putative transmissibility markers [cable pilin subunit gene (cblA) and the B. cepacia epidemic strain marker (BCESM)]. Sporadic and cross-infective strains were identified by random amplification of polymorphic DNA (RAPD). In total, 79% of patients were infected with Burkholderia cenocepacia (genomovar III), 18% with Burkholderia multivorans (genomovar II) and less than 5% of patients with B. cepacia (genomovar I), Burkholderia stabilis (genomovar IV) or Burkholderia vietnamiensis (genomovar V). The cblA and BCESM transmissibility markers were only detected in strains of B. cenocepacia. The BCESM was a more sensitive marker for transmissible B. cenocepacia strains than cblA, although sporadic B. cenocepacia strains containing the BCESM, but lacking cblA, were also observed. Furthermore, clusters of cross-infection with transmissibility marker-negative strains of B. multivorans were identified. In conclusion, B. cenocepacia was the greatest cause of cross-infection, and the most widely distributed B. cepacia complex species, within these CF populations. However, cross-infection was not exclusive to B. cenocepacia and cblA and the BCESM were not absolute markers for transmissible B. cenocepacia, or other B. cepacia complex strains. It is therefore suggested that CF centres cohort patients based on the presence or absence of B. cepacia complex infection and not on the basis of transmissibility marker-positive B. cenocepacia as previously suggested. PMID:15184539

  12. Complete genome sequence of Burkholderia sp. strain PAMC28687, a potential octopine-utilizing bacterium isolated from Antarctica lichen.

    PubMed

    Han, So-Ra; Yu, Sang-Cheol; Ahn, Do-Hwan; Park, Hyun; Oh, Tae-Jin

    2016-05-20

    We report the complete genome sequence of Burkholderia sp. PAMC28687, which was isolated from the Antarctica lichen Useea sp., for better understanding of its catabolic traits in utilizing octopine as a source of carbon/nitrogen between Burkholderia and lichen. The genome consists of three circular chromosomes with five circular plasmids for the total 6,881,273bp sized genome with a G+C content of 58.14%. PMID:27034021

  13. Draft Genome Sequences of Two Burkholderia multivorans Sequential Isolates from a Chronic Lung Infection of a Cystic Fibrosis Patient

    PubMed Central

    Silva, Inês N.; Santos, Pedro M.

    2015-01-01

    Burkholderia multivorans belongs to the Burkholderia cepacia complex, which comprises opportunistic pathogens infecting cystic fibrosis (CF) patients. Here, we report the genome sequences and annotations of two sequential B. multivorans clinical isolates (D2095 and D2214) displaying different traits. The differences in the genomic contents of these isolates may provide clues regarding the evolution of B. multivorans within the airways of a CF patient. PMID:25676757

  14. Classification of the biphenyl- and polychlorinated biphenyl-degrading strain LB400T and relatives as Burkholderia xenovorans sp. nov.

    PubMed

    Goris, Johan; De Vos, Paul; Caballero-Mellado, Jesús; Park, Joonhong; Falsen, Enevold; Quensen, John F; Tiedje, James M; Vandamme, Peter

    2004-09-01

    Strain LB400T is the best-studied polychlorinated biphenyl (PCB) degrader. This organism has previously been allocated in the genus Burkholderia, since its 16S rRNA gene sequence shows 98.6 % sequence similarity to the type strains of Burkholderia graminis and Burkholderia terricola. A polyphasic study was undertaken to clarify the actual taxonomic position of this biotechnologically important organism and of two strains, one recovered from a blood culture vial and one from a coffee plant rhizosphere, both of which resembled strain LB400T in their whole-cell protein patterns. DNA-DNA hybridization experiments revealed that the three strains represented a single novel species, for which the name Burkholderia xenovorans sp. nov. is proposed. Strains of this novel species can be differentiated phenotypically from nearly all other Burkholderia species by their inability to assimilate L-arabinose. The whole-cell fatty acid profile of B. xenovorans strains is consistent with their classification in the genus Burkholderia, with 18 : 1omega7c, 16 : 1omega7c, 16 : 0, 14 : 0 3OH, 16 : 0 3OH, 17 : 0 cyclo and 14 : 0 being the most abundant fatty acids. The G + C content of the species varies between 62.4 and 62.9 mol%. The type strain of B. xenovorans is LB400T (= LMG 21463T = CCUG 46959T = NRRL B-18064T). PMID:15388727

  15. Bioconversion of AHX to AOH by resting cells of Burkholderia contaminans CH-1.

    PubMed

    Choi, Jae-Hoon; Kikuchi, Ayaka; Pumkaeo, Panyapon; Hirai, Hirofumi; Tokuyama, Shinji; Kawagishi, Hirokazu

    2016-10-01

    Fairy rings are zones of stimulated grass growth owing to the interaction between a fungus and a plant. We previously reported the discovery of two novel plant-growth regulating compounds related to forming fairy rings, 2-azahypoxanthine (AHX) and 2-aza-8-oxohypoxanthine (AOH). In this study, a bacterial strain CH-1 was isolated from an airborne-contaminated nutrient medium containing AHX. The strain converted AHX to AOH and identified as Burkholderia contaminans based on the gene sequence of its 16S rDNA. The quantitative production of AOH by resting cells of the strain was achieved. Among seven Burkholderia species, two bacteria and two yeasts tested, B. contaminans CH-1 showed the highest rate of conversion of AHX to AOH. By batch system, up to 10.6 mmol AHX was converted to AOH using the resting cells. The yield of this process reached at 91%. PMID:27387894

  16. Synthesis of the tetrasaccharide outer core fragment of Burkholderia multivorans lipooligosaccharide.

    PubMed

    Ziaco, Marcello; De Castro, Cristina; Silipo, Alba; Corsaro, Maria Michela; Molinaro, Antonio; Iadonisi, Alfonso; Lanzetta, Rosa; Parrilli, Michelangelo; Bedini, Emiliano

    2015-02-11

    The first synthesis of the outer core fragment of Burkholderia multivorans lipooligosaccharide [β-D-Glc-(1→3)-α-D-GalNAc-(1→3)-β-D-GalNAc-(1→3)-L-Rha] as α-allyl tetrasaccharide was accomplished. The glycosylations involving GalNAc units were studied in depth testing them under several conditions. This allowed the building of both the α- and the β-configured glycosidic bonds by employing the same GalNAc glycosyl donor, thus considerably shortening the total number of synthetic steps. The target tetrasaccharide was synthesized with an allyl aglycone to allow its future conjugation with an immunogenic protein en route to the development of a synthetic neoglycoconjugate vaccine against the Burkholderia cepacia pathogens. PMID:24933233

  17. Quorum Sensing Controls Swarming Motility of Burkholderia glumae through Regulation of Rhamnolipids.

    PubMed

    Nickzad, Arvin; Lépine, François; Déziel, Eric

    2015-01-01

    Burkholderia glumae is a plant pathogenic bacterium that uses an acyl-homoserine lactone-mediated quorum sensing system to regulate protein secretion, oxalate production and major virulence determinants such as toxoflavin and flagella. B. glumae also releases surface-active rhamnolipids. In Pseudomonas aeruginosa and Burkholderia thailandensis, rhamnolipids, along with flagella, are required for the social behavior called swarming motility. In the present study, we demonstrate that quorum sensing positively regulates the production of rhamnolipids in B. glumae and that rhamnolipids are necessary for swarming motility also in this species. We show that a rhlA- mutant, which is unable to produce rhamnolipids, loses its ability to swarm, and that this can be complemented by providing exogenous rhamnolipids. Impaired rhamnolipid production in a quorum sensing-deficient B. glumae mutant is the main factor responsible for its defective swarming motility behaviour. PMID:26047513

  18. σ54-Dependent Response to Nitrogen Limitation and Virulence in Burkholderia cenocepacia Strain H111

    PubMed Central

    Lardi, Martina; Aguilar, Claudio; Pedrioli, Alessandro; Omasits, Ulrich; Suppiger, Angela; Cárcamo-Oyarce, Gerardo; Schmid, Nadine; Ahrens, Christian H.

    2015-01-01

    Members of the genus Burkholderia are versatile bacteria capable of colonizing highly diverse environmental niches. In this study, we investigated the global response of the opportunistic pathogen Burkholderia cenocepacia H111 to nitrogen limitation at the transcript and protein expression levels. In addition to a classical response to nitrogen starvation, including the activation of glutamine synthetase, PII proteins, and the two-component regulatory system NtrBC, B. cenocepacia H111 also upregulated polyhydroxybutyrate (PHB) accumulation and exopolysaccharide (EPS) production in response to nitrogen shortage. A search for consensus sequences in promoter regions of nitrogen-responsive genes identified a σ54 consensus sequence. The mapping of the σ54 regulon as well as the characterization of a σ54 mutant suggests an important role of σ54 not only in control of nitrogen metabolism but also in the virulence of this organism. PMID:25841012

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

    PubMed

    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. PMID:26578147

  20. Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

    PubMed Central

    Velapatiño, Billie; Zlosnik, James E. A.; Hird, Trevor J.; Speert, David P.

    2013-01-01

    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. PMID:24192802

  1. Biological Agents

    MedlinePlus

    ... to Z Index Contact Us FAQs What's New Biological Agents This page requires that javascript be enabled ... and Health Topics A-Z Index What's New Biological agents include bacteria, viruses, fungi, other microorganisms and ...

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

    PubMed Central

    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

  3. Draft Genome Sequence of Burkholderia sp. Strain CCA53, Isolated from Leaf Soil

    PubMed Central

    Kimura, Zen-ichiro; Yusoff, Mohd Zulkhairi Mohd; Nakashima, Nobutaka; Hoshino, Tamotsu

    2016-01-01

    Burkholderia sp. strain CCA53 was isolated from leaf soil collected in Higashi-Hiroshima City in Hiroshima Prefecture, Japan. Here, we present a draft genome sequence of this strain, which consists of a total of 4 contigs containing 6,647,893 bp, with a G+C content of 67.0% and comprising 9,329 predicted coding sequences. PMID:27389268

  4. Draft Genome Sequence of Burkholderia sp. Strain CCA53, Isolated from Leaf Soil.

    PubMed

    Akita, Hironaga; Kimura, Zen-Ichiro; Yusoff, Mohd Zulkhairi Mohd; Nakashima, Nobutaka; Hoshino, Tamotsu

    2016-01-01

    Burkholderia sp. strain CCA53 was isolated from leaf soil collected in Higashi-Hiroshima City in Hiroshima Prefecture, Japan. Here, we present a draft genome sequence of this strain, which consists of a total of 4 contigs containing 6,647,893 bp, with a G+C content of 67.0% and comprising 9,329 predicted coding sequences. PMID:27389268

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

    PubMed

    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. PMID:26472229

  6. Burkholderia cepacia as a cause of ecthyma gangrenosum-like lesion.

    PubMed

    Aygencel, G; Dizbay, M; Sahin, G

    2008-06-01

    Ecthyma gangrenosum (EG) is considered to be a pathognomonic sign of Pseudomonas aeruginosa septicemia and is predominantly seen in immunosuppressed patients. Although P. aeruginosa is the most recognized pathogen of EG, numerous other organisms have been reported to cause clinically identical lesions. We report a case of an EG-like eruption caused by Burkholderia cepacia, in an intensive care unit patient with multiple myeloma. PMID:17962903

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

    PubMed Central

    Revathy, T.; Jayasri, M. A.; Suthindhiran, K.

    2015-01-01

    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