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Sample records for burkholderia pseudomallei melioidosis

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. Clinical Definitions of Melioidosis

    PubMed Central

    Cheng, Allen C.; Currie, Bart J.; Dance, David A. B.; Funnell, Simon G. P.; Limmathurotsakul, Direk; Simpson, Andrew J. H.; Peacock, Sharon J.

    2013-01-01

    Clinical definitions of melioidosis and inhalation-acquired melioidosis (Burkholderia pseudomallei infection) are described together with the evidence used to develop these definitions. Such definitions support accurate public health reporting, preparedness planning for deliberate B. pseudomallei release, design of experimental models, and categorization of naturally acquired melioidosis. PMID:23468355

  14. 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

  15. 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

  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. 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. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  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. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  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. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. Imported Melioidosis, Israel, 2008

    PubMed Central

    Cahn, Avivit; Koslowsky, Benjamin; Nir-Paz, Ran; Temper, Violeta; Hiller, Nurit; Karlinsky, Alla; Gur, Itzhak; Hidalgo-Grass, Carlos; Heyman, Samuel N.; Moses, Allon E.

    2009-01-01

    In 2008, melioidosis was diagnosed in an agricultural worker from Thailand in the southern Jordan Valley in Israel. He had newly diagnosed diabetes mellitus, fever, multiple abscesses, and osteomyelitis. Burkholderia pseudomallei was isolated from urine and blood. Four of 10 laboratory staff members exposed to the organism received chemoprophylaxis, 3 of whom had adverse events. PMID:19891871

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. Emergence of Melioidosis in Indonesia.

    PubMed

    Tauran, Patricia M; Sennang, Nurhayana; Rusli, Benny; Wiersinga, W Joost; Dance, David; Arif, Mansyur; Limmathurotsakul, Direk

    2015-12-01

    Melioidosis is known to be highly endemic in parts of southeast Asia and northern Australia; however, cases are rarely reported in Indonesia. Here we report three cases of melioidosis in Makassar, South Sulawesi, Indonesia occurring between 2013 and 2014. Two patients died and the other was lost to follow-up. Burkholderia pseudomallei isolates from all three cases were identified by the VITEK2 Compact installed in the hospital in 2012. None of the three patients reported received antimicrobials recommended for melioidosis because of the delayed recognition of the organism. We reviewed the literature and found only seven reports of melioidosis in Indonesia. Five were reported before 1960. We suggest that melioidosis is endemic throughout Indonesia but currently under-recognized. Training on how to identify B. pseudomallei accurately and safely in all available microbiological facilities should be provided, and consideration should be given to making melioidosis a notifiable disease in Indonesia. PMID:26458777

  20. Emergence of Melioidosis in Indonesia

    PubMed Central

    Tauran, Patricia M.; Sennang, Nurhayana; Rusli, Benny; Wiersinga, W. Joost; Dance, David; Arif, Mansyur; Limmathurotsakul, Direk

    2015-01-01

    Melioidosis is known to be highly endemic in parts of southeast Asia and northern Australia; however, cases are rarely reported in Indonesia. Here we report three cases of melioidosis in Makassar, South Sulawesi, Indonesia occurring between 2013 and 2014. Two patients died and the other was lost to follow-up. Burkholderia pseudomallei isolates from all three cases were identified by the VITEK2 Compact installed in the hospital in 2012. None of the three patients reported received antimicrobials recommended for melioidosis because of the delayed recognition of the organism. We reviewed the literature and found only seven reports of melioidosis in Indonesia. Five were reported before 1960. We suggest that melioidosis is endemic throughout Indonesia but currently under-recognized. Training on how to identify B. pseudomallei accurately and safely in all available microbiological facilities should be provided, and consideration should be given to making melioidosis a notifiable disease in Indonesia. PMID:26458777

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. Cutaneous melioidosis with unusual histological features.

    PubMed

    Yeo, B; Lee, J; Alagappan, U; Pan, J Y

    2016-04-01

    Melioidosis is caused by the saprophytic gram-negative bacillus Burkholderia pseudomallei, and has varied presentations, with cutaneous manifestations occurring in about 13% of cases. The usual histopathological features of melioidosis are suppurative to chronic granulomatous inflammation. Recommended treatment of melioidosis is sequential use of intravenous followed by oral antibiotics for a few months, although oral antibiotics alone can be used in primary cutaneous melioidosis. We report a case of cutaneous melioidosis in a healthy young man, with the unusual histopathological feature of lymphoplasmacytic inflammation, and failing an initial trial of oral antibiotics alone. PMID:26299451

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  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. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. A rare cause of septic arthritis: melioidosis.

    PubMed

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

    2013-10-01

    Melioidosis is a pyogenic infection with high mortality caused by the bacterium Burkholderia pseudomallei. As the clinical presentation is not distinctive, a high index of clinical suspicion is required for diagnosis. We present a case of a 50-year-old farmer who was diabetic and a chronic alcoholic, who presented to us with pneumonia, followed by septic arthritis. He was ultimately diagnosed as having melioidosis. PMID:24067292

  12. 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

  13. 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

  14. 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

  15. Neurologic Melioidosis: Case Report of a Rare Cause of Acute Flaccid Paralysis.

    PubMed

    Andersen, Erik W; Mackay, Mark T; Ryan, Monique M

    2016-03-01

    Acute flaccid paralysis is associated with inflammation, infection, or tumors in the spinal cord or peripheral nerves. Melioidosis (Burkholderia pseudomallei infection) can rarely cause this presentation. We describe a case of spinal melioidosis in a 4-year-old boy presenting with flaccid paralysis, and review the literature on this rare disease. PMID:26778096

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. [Pulmonary melioidosis].

    PubMed

    Perret, J L; Vidal, D; Thibault, F

    1998-12-01

    Melioidosis is most frequently encountered in pulmonary localization. Melioidosis is an infectious disease caused by Burkholderia pseudomallei first described by Whitmore in 1912 in Burma. B. pseudomallei is a Gram negative rod belonging to the Pseudomonadaceae family. Soil and water are the natural reservoirs for the germ which is a specific pathogen for several mammal species. Long endemic in Southeast Asia and several tropical zones, B. pseudomallei has recently been found in temperate zones, including France. Human contamination occurs via the transcutaneous route and often leads to dormant inapparent infection. Many conditions, such as diabetes, renal lithiasis, various circumstances of immunodepression or stress, facilitate clinical manifestations which vary greatly. Pulmonary manifestations may be acute and extensive, producing a torpid pseudo-tuberculous condition or a variety of clinical and radiological features mimicking other diseases. Bacteriological and serological tests may be negative. Exposure in an endemic zone, the notion of a favorable context, weight loss, cavitary images on successive chest x-rays and the presence of extra-pulmonary localizations may be suggestive. Ceftazidime or the amoxicillin-clavulanic acid combination are indicated, but mortality in acute forms still reaches 40%. Relapse can be expected if the treatment duration is too short. PMID:10100350

  2. Cutaneous Melioidosis Cluster Caused by Contaminated Wound Irrigation Fluid

    PubMed Central

    Merritt, Adam J.; Peck, Mariani; Gayle, Dionne; Levy, Avram; Ler, Yi-Horng; Raby, Edward; Gibbs, Tristan M.

    2016-01-01

    Melioidosis usually occurs after environmental exposure to Burkholderia pseudomallei in the tropics. A cluster of 5 cutaneous melioidosis cases occurred in suburban southwest Australia after an earlier case in January 2012. We collected environmental samples at the first patient’s home in January 2012 and from a nearby health center in December 2013 after 2 new cases occurred in the same postal district. We isolated genotypically identical B. pseudomallei from the first patient and 5 other patients in the district. Environmental sampling implicated an opened bottle of saline wound irrigation fluid containing >106 B. pseudomallei/mL. The bottle included instructions to discard within 24 hours of opening. No further cases of B. pseudomallei infection occurred after removing the contaminated bottle. This cutaneous melioidosis cluster demonstrates that B. pseudomallei can survive and disseminate in widely used medical fluids beyond its known geographic distribution, highlighting a need to use these products according to manufacturers’ instructions. PMID:27438887

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. MALDI-TOF MS contribution to diagnosis of melioidosis in a nonendemic country in three French travellers.

    PubMed

    Walewski, V; Méchaï, F; Billard-Pomares, T; Juguet, W; Jauréguy, F; Picard, B; Tandjaoui-Lambiotte, Y; Carbonnelle, E; Bouchaud, O

    2016-07-01

    Melioidosis is an endemic disease in Southeast Asia and northern Australia. An increasing number of cases are being reported in nonendemic countries, making the diagnosis less obvious. We discuss the identification of Burkholderia pseudomallei using matrix-assisted desorption ionization-time of flight mass spectrometry on the occasion of recent cases of imported melioidosis in French travellers. PMID:27222715

  9. 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

  10. 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

  11. 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

  12. 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

  13. The epidemiology of melioidosis in the Balimo region of Papua New Guinea.

    PubMed

    Warner, J M; Pelowa, D B; Gal, D; Rai, G; Mayo, M; Currie, B J; Govan, B; Skerratt, L F; Hirst, R G

    2008-07-01

    The distribution of Burkholderia pseudomallei was determined in soil collected from a rural district in Papua New Guinea (PNG) where melioidosis had recently been described, predominately affecting children. In 274 samples, 2.6% tested culture-positive for B. pseudomallei. Pulsed-field gel electrophoresis using SpeI digests and rapid polymorphic DNA PCR with five primers demonstrated a single clone amongst clinical isolates and isolates cultured from the environment that was commonly used by children from whom the clinical isolates were derived. We concluded that individuals in this region most probably acquired the organism through close contact with the environment at these sites. Burkholderia thailandensis, a closely related Burkholderia sp. was isolated from 5.5% of samples tested, an observation similar to that of melioidosis-endemic areas in Thailand. This is the first report of an environmental reservoir for melioidosis in PNG and confirms the Balimo district in PNG as melioidosis endemic. PMID:17714600

  14. 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

  15. 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

  16. The man with the boggy head: cranial melioidosis.

    PubMed

    Kuan, Y C; How, S H; Ng, T H; Fauzi, A R

    2010-02-01

    Melioidosis is known to cause abscesses in various organs, including the cranium, though less commonly. We present a patient with scalp abscess and subdural empyema that was visible on computed tomography of the brain. The neurosurgical drainage grew Burkholderia pseudomallei. Despite our best effort to treat the patient using parenteral antibiotics and surgical drainage, the patient did not survive. PMID:20358143

  17. 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

  18. 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

  19. 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

  20. Evaluation of Surrogate Animal Models of Melioidosis

    PubMed Central

    Warawa, Jonathan Mark

    2010-01-01

    Burkholderia pseudomallei is the Gram-negative bacterial pathogen responsible for the disease melioidosis. B. pseudomallei establishes disease in susceptible individuals through multiple routes of infection, all of which may proceed to a septicemic disease associated with a high mortality rate. B. pseudomallei opportunistically infects humans and a wide range of animals directly from the environment, and modeling of experimental melioidosis has been conducted in numerous biologically relevant models including mammalian and invertebrate hosts. This review seeks to summarize published findings related to established animal models of melioidosis, with an aim to compare and contrast the virulence of B. pseudomallei in these models. The effect of the route of delivery on disease is also discussed for intravenous, intraperitoneal, subcutaneous, intranasal, aerosol, oral, and intratracheal infection methodologies, with a particular focus on how they relate to modeling clinical melioidosis. The importance of the translational validity of the animal models used in B. pseudomallei research is highlighted as these studies have become increasingly therapeutic in nature. PMID:21772830

  1. Melioidosis: Molecular Aspects of Pathogenesis

    PubMed Central

    Stone, Joshua K.; DeShazer, David; Brett, Paul J.; Burtnick, Mary N.

    2015-01-01

    SUMMARY Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, a multifaceted disease that is highly endemic in Southeast Asia and northern Australia. This facultative intracellular pathogen possesses a large genome that encodes a wide array of virulence factors that promote survival in vivo by manipulating host cell processes and disarming elements of the host immune system. Antigens and systems that play key roles in B. pseudomallei virulence include capsular polysaccharide, lipopolysaccharide, adhesins, specialized secretion systems, actin-based motility and various secreted factors. This review provides an overview of the current and steadily expanding knowledge regarding the molecular mechanisms used by this organism to survive within a host and their contribution to the pathogenesis of melioidosis. PMID:25312349

  2. 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

  3. 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

  4. 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

  5. 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.

  6. A Review of Melioidosis Cases in the Americas.

    PubMed

    Benoit, Tina J; Blaney, David D; Doker, Thomas J; Gee, Jay E; Elrod, Mindy G; Rolim, Dionne B; Inglis, Timothy J J; Hoffmaster, Alex R; Bower, William A; Walke, Henry T

    2015-12-01

    Melioidosis is a bacterial infection caused by Burkholderia pseudomallei, a gram-negative saprophytic bacillus. Cases occur sporadically in the Americas with an increasing number of cases observed among people with no travel history to endemic countries. To better understand the incidence of the disease in the Americas, we reviewed the literature, including unpublished cases reported to the Centers for Disease Control and Prevention. Of 120 identified human cases, occurring between 1947 and June 2015, 95 cases (79%) were likely acquired in the Americas; the mortality rate was 39%. Burkholderia pseudomallei appears to be widespread in South, Central, and North America. PMID:26458779

  7. 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

  8. 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

  9. Association between activities related to routes of infection and clinical manifestations of melioidosis.

    PubMed

    Lim, C; Peacock, S J; Limmathurotsakul, D

    2016-01-01

    We sought associations between route of infection by Burkholderia pseudomallei and clinical manifestations in 330 cases of melioidosis in northeast Thailand using bivariate multivariable logistic regression models. Activities related to skin inoculation were negatively associated with bacteraemia, activities related to ingestion were associated with bacteraemia, and activities related to inhalation were associated with pneumonia. Our study suggests that route of infection is one of the factors related to clinical manifestations of melioidosis. PMID:26417852

  10. Association between activities related to routes of infection and clinical manifestations of melioidosis

    PubMed Central

    Lim, C.; Peacock, S.J.; Limmathurotsakul, D.

    2016-01-01

    We sought associations between route of infection by Burkholderia pseudomallei and clinical manifestations in 330 cases of melioidosis in northeast Thailand using bivariate multivariable logistic regression models. Activities related to skin inoculation were negatively associated with bacteraemia, activities related to ingestion were associated with bacteraemia, and activities related to inhalation were associated with pneumonia. Our study suggests that route of infection is one of the factors related to clinical manifestations of melioidosis. PMID:26417852

  11. 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

  12. 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

  13. 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

  14. Melioidosis

    MedlinePlus

    ... disease, is an infectious disease that can infect humans or animals. The disease is caused by the bacterium Burkholderia ... contaminated water and soil. It is spread to humans and animals through direct contact with the contaminated source. Specimens ...

  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. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. Melioidosis in New Caledonia: a dominant strain in a transmission hotspot.

    PubMed

    Melot, B; Colot, J; Lacassin, F; Tardieu, S; Lapisardi, E; Mayo, M; Price, E P; Sarovich, D S; Currie, B J; Goarant, C

    2016-04-01

    Melioidosis is an infectious disease caused by Burkholderia pseudomallei, a bacterium endemic in Southeast Asia and northern Australia. In New Caledonia, sporadic cases were first described in 2005; since then, more cases have been identified. To improve our understanding of melioidosis epidemiology in New Caledonia, we compared the local cases and B. pseudomallei isolates with those from endemic areas. Nineteen melioidosis cases have been diagnosed in New Caledonia since 1999, mostly severe and with frequent bacteraemia, leading to three (16%) fatalities. All but one occurred in the North Province. Besides sporadic cases caused by non-clonal strains, we also identified a hotspot of transmission related to a clonal group of B. pseudomallei that is phylogenetically related to Australian strains. PMID:26542622

  3. Melioidosis in a patient on monoclonal antibody therapy for psoriatic arthritis.

    PubMed

    Commons, R J; Grivas, R; Currie, B J

    2014-12-01

    Melioidosis is caused by the environmental bacterium Burkholderia pseudomallei and can present with severe sepsis. Predisposing risk factors are present in 80% of cases. Monoclonal antibodies are increasingly prescribed for varied medical conditions. This report describes the first known case of melioidosis in a patient whose only risk factor for disease is treatment with a monoclonal antibody. Prescribers of monoclonal antibodies and other immunosuppressants should ensure that their patients are aware of the potential risk of melioidosis prior to travel and the precautions that should be taken. PMID:25442759

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

    PubMed

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

    2016-02-01

    The epidemiologic status of melioidosis in Sri Lanka was unclear from the few previous case reports. We established laboratory support for a case definition and started a nationwide case-finding study. Suspected Burkholderia pseudomallei isolates were collated, identified by polymerase chain reaction assay, referred for Matrix Assisted Laser Desorption Ionization-Time of Flight analysis and multilocus sequence typing (MLST), and named according to the international MLST database. Between 2006 and early 2014, there were 32 patients with culture-confirmed melioidosis with an increasing annual total and a falling fatality rate. Patients were predominantly from rural communities, diabetic, and male. The major clinical presentations were sepsis, pneumonia, soft tissue and joint infections, and other focal infection. Burkholderia pseudomallei isolates came from all parts of Sri Lanka except the Sabaragamuwa Province, the south central hill country, and parts of northern Sri Lanka. Bacterial isolates belonged to 18 multilocus sequence types, one of which (ST 1137) was associated with septicemia and a single-organ focus (Fisher's exact, P = 0.004). Melioidosis is an established endemic infection throughout Sri Lanka, and is caused by multiple genotypes of B. pseudomallei, which form a distinct geographic group based upon related sequence types (BURST) cluster at the junction of the southeast Asian and Australasian clades. PMID:26621560

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. Fatal Melioidosis in a Newborn from Hainan, China.

    PubMed

    Fang, Yao; Chen, Hai; Zhu, Xiong; Mao, Xuhu

    2016-08-01

    Herein, we report a case of fatal melioidosis in a newborn. The newborn died of serious melioidosis with respiratory and multiorgan failure at 16 days of age despite extensive treatment with antibiotics and methylprednisolone. Burkholderia pseudomallei was isolated from the infant's blood and cerebrospinal fluid and identified as a novel sequence type (ST-1341). His father had cough and fever when the newborn was born, and a localized patchy infiltration on the right upper lung was seen in chest radiography, but B. pseudomallei was not isolated. Two years later, the father developed cough and fever again, and the same novel sequence type of B. pseudomallei was isolated from the blood of the father. It is postulated that transmission of B. pseudomallei from the father to the newborn might have occurred during close contact in the first couple of days after birth. Given the high mortality of neonatal melioidosis, particular attention must be paid when the caretakers of the newborn develop fever of unknown origin in a melioidosis-endemic region. PMID:27162267

  11. Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis.

    PubMed

    Houghton, Raymond L; Reed, Dana E; Hubbard, Mark A; Dillon, Michael J; Chen, Hongjing; Currie, Bart J; Mayo, Mark; Sarovich, Derek S; Theobald, Vanessa; Limmathurotsakul, Direk; Wongsuvan, Gumphol; Chantratita, Narisara; Peacock, Sharon J; Hoffmaster, Alex R; Duval, Brea; Brett, Paul J; Burtnick, Mary N; Aucoin, David P

    2014-03-01

    Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation. PMID:24651568

  12. 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

  13. 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

  14. 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

  15. Further Evaluation of a Rapid Diagnostic Test for Melioidosis in an Area of Endemicity

    PubMed Central

    O'Brien, Mathew; Freeman, Kevin; Lum, Gary; Cheng, Allen C.; Jacups, Susan P.; Currie, Bart J.

    2004-01-01

    Immunochromatographic test (ICT) kits for the rapid detection of immunoglobulin G (IgG) and IgM antibodies to Burkholderia pseudomallei were compared to the indirect hemagglutination (IHA) assay. In 138 culture-confirmed melioidosis cases, sensitivities were 80, 77, and 88% for IHA, ICT IgG, and ICT IgM, respectively. In a prospective study of 160 consecutive sera samples sent for melioidosis serology, respective specificities were 91, 90, and 69, positive predictive values were 41, 32, and 18, and negative predictive values were 99, 98, and 100%. ICT IgM kits are unreliable for diagnosis of melioidosis, but ICT IgG kits may be useful for diagnosing travelers presenting with possible melioidosis who return from regions where melioidosis is endemic. PMID:15131200

  16. 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

  17. 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

  18. 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

  19. Impaired TLR5 Functionality Is Associated with Survival in Melioidosis

    PubMed Central

    Chantratita, Narisara; Chierakul, Wirongrong; Limmathurotsakul, Direk; Wuthiekanun, Vanaporn; Myers, Nicolle D.; Emond, Mary J.; Wurfel, Mark M.; Hawn, Thomas R.; Peacock, Sharon J.; Skerrett, Shawn J.

    2013-01-01

    Melioidosis is infection caused by the flagellated saprophyte Burkholderia pseudomallei. TLR5 is a pathogen recognition receptor activated by bacterial flagellin. We studied a genetic variant that encodes a defective TLR5 protein, TLR51174C>T, to elucidate the role of TLR5 in melioidosis. We measured NF-κB activation induced by B. pseudomallei in human embryonic kidney–293 cells transfected with TLR5 and found that B. pseudomallei induced TLR51174C- but not TLR51174T-dependent activation of NF-κB. We tested the association of TLR51174C>T with outcome in 600 Thai subjects with melioidosis. In a dominant model, TLR51174C>T was associated with protection against in-hospital death (adjusted odds ratio: 0.20; 95% confidence interval: 0.08–0.50; p = 0.001) and organ failure (adjusted odds ratio: 0.37; 95% confidence interval: 0.19–0.71; p = 0.003). We analyzed blood cytokine production induced by flagellin or heat-killed B. pseudomallei by TLR51174C>T genotype in healthy subjects. Flagellin induced lower monocyte-normalized levels of IL-6, IL-8, TNF-α, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1β in carriers of TLR51174T compared with carriers of TLR51174C. B. pseudomallei induced lower monocyte-normalized levels of IL-10 in carriers of TLR51174T. We conclude that the hypofunctional genetic variant TLR51174C>T is associated with reduced organ failure and improved survival in melioidosis. This conclusion suggests a deleterious immunoregulatory effect of TLR5 that may be mediated by IL-10 and identifies this receptor as a potential therapeutic target in melioidosis. PMID:23447684

  20. Impaired TLR5 functionality is associated with survival in melioidosis.

    PubMed

    West, T Eoin; Chantratita, Narisara; Chierakul, Wirongrong; Limmathurotsakul, Direk; Wuthiekanun, Vanaporn; Myers, Nicolle D; Emond, Mary J; Wurfel, Mark M; Hawn, Thomas R; Peacock, Sharon J; Skerrett, Shawn J

    2013-04-01

    Melioidosis is infection caused by the flagellated saprophyte Burkholderia pseudomallei. TLR5 is a pathogen recognition receptor activated by bacterial flagellin. We studied a genetic variant that encodes a defective TLR5 protein, TLR5(1174C)>T, to elucidate the role of TLR5 in melioidosis. We measured NF-κB activation induced by B. pseudomallei in human embryonic kidney-293 cells transfected with TLR5 and found that B. pseudomallei induced TLR5(1174C)- but not TLR5(1174T)-dependent activation of NF-κB. We tested the association of TLR5(1174C)>T with outcome in 600 Thai subjects with melioidosis. In a dominant model, TLR5(1174C)>T was associated with protection against in-hospital death (adjusted odds ratio: 0.20; 95% confidence interval: 0.08-0.50; p = 0.001) and organ failure (adjusted odds ratio: 0.37; 95% confidence interval: 0.19-0.71; p = 0.003). We analyzed blood cytokine production induced by flagellin or heat-killed B. pseudomallei by TLR5(1174C)>T genotype in healthy subjects. Flagellin induced lower monocyte-normalized levels of IL-6, IL-8, TNF-α, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1β in carriers of TLR5(1174T) compared with carriers of TLR5(1174C). B. pseudomallei induced lower monocyte-normalized levels of IL-10 in carriers of TLR5(1174T). We conclude that the hypofunctional genetic variant TLR5(1174C)>T is associated with reduced organ failure and improved survival in melioidosis. This conclusion suggests a deleterious immunoregulatory effect of TLR5 that may be mediated by IL-10 and identifies this receptor as a potential therapeutic target in melioidosis. PMID:23447684

  1. 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

  2. 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

  3. 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

  4. Host Responses to Melioidosis and Tuberculosis Are Both Dominated by Interferon-Mediated Signaling

    PubMed Central

    Koh, Gavin C. K. W.; Schreiber, M. Fernanda; Bautista, Ruben; Maude, Rapeephan R.; Dunachie, Susanna; Limmathurotsakul, Direk; Day, Nicholas P. J.; Dougan, Gordon; Peacock, Sharon J.

    2013-01-01

    Melioidosis (Burkholderia pseudomallei infection) is a common cause of community-acquired sepsis in Northeast Thailand and northern Australia. B. pseudomallei is a soil saprophyte endemic to Southeast Asia and northern Australia. The clinical presentation of melioidosis may mimic tuberculosis (both cause chronic suppurative lesions unresponsive to conventional antibiotics and both commonly affect the lungs). The two diseases have overlapping risk profiles (e.g., diabetes, corticosteroid use), and both B. pseudomallei and Mycobacterium tuberculosis are intracellular pathogens. There are however important differences: the majority of melioidosis cases are acute, not chronic, and present with severe sepsis and a mortality rate that approaches 50% despite appropriate antimicrobial therapy. By contrast, tuberculosis is characteristically a chronic illness with mortality <2% with appropriate antimicrobial chemotherapy. We examined the gene expression profiles of total peripheral leukocytes in two cohorts of patients, one with acute melioidosis (30 patients and 30 controls) and another with tuberculosis (20 patients and 24 controls). Interferon-mediated responses dominate the host response to both infections, and both type 1 and type 2 interferon responses are important. An 86-gene signature previously thought to be specific for tuberculosis is also found in melioidosis. We conclude that the host responses to melioidosis and to tuberculosis are similar: both are dominated by interferon-signalling pathways and this similarity means gene expression signatures from whole blood do not distinguish between these two diseases. PMID:23383015

  5. Investigation of Recurrent Melioidosis in Lao People's Democratic Republic by Multilocus Sequence Typing

    PubMed Central

    Rachlin, Audrey; Dittrich, Sabine; Phommasone, Koukeo; Douangnouvong, Anousone; Phetsouvanh, Rattanaphone; Newton, Paul N.; Dance, David A. B.

    2016-01-01

    Melioidosis is an infectious disease caused by the saprophytic bacterium Burkholderia pseudomallei. In northeast Thailand and northern Australia, where the disease is highly endemic, a range of molecular tools have been used to study its epidemiology and pathogenesis. In the Lao People's Democratic Republic (Laos) where melioidosis has been recognized as endemic since 1999, no such studies have been undertaken. We used a multilocus sequence typing scheme specific for B. pseudomallei to investigate nine cases of culture-positive recurrence occurring in 514 patients with melioidosis between 2010 and 2015: four were suspected to be relapses while the other five represented reinfections. In addition, two novel sequence types of the bacterium were identified. The low overall recurrence rates (2.4%) and proportions of relapse and reinfection in the Laos are consistent with those described in the recent literature, reflecting the effective use of appropriate antimicrobial therapy. PMID:27001759

  6. Investigation of Recurrent Melioidosis in Lao People's Democratic Republic by Multilocus Sequence Typing.

    PubMed

    Rachlin, Audrey; Dittrich, Sabine; Phommasone, Koukeo; Douangnouvong, Anousone; Phetsouvanh, Rattanaphone; Newton, Paul N; Dance, David A B

    2016-06-01

    Melioidosis is an infectious disease caused by the saprophytic bacterium Burkholderia pseudomallei In northeast Thailand and northern Australia, where the disease is highly endemic, a range of molecular tools have been used to study its epidemiology and pathogenesis. In the Lao People's Democratic Republic (Laos) where melioidosis has been recognized as endemic since 1999, no such studies have been undertaken. We used a multilocus sequence typing scheme specific for B. pseudomallei to investigate nine cases of culture-positive recurrence occurring in 514 patients with melioidosis between 2010 and 2015: four were suspected to be relapses while the other five represented reinfections. In addition, two novel sequence types of the bacterium were identified. The low overall recurrence rates (2.4%) and proportions of relapse and reinfection in the Laos are consistent with those described in the recent literature, reflecting the effective use of appropriate antimicrobial therapy. PMID:27001759

  7. Melioidosis and idiopathic pulmonary hemosiderosis: a cast-iron case.

    PubMed

    Gerhardy, Benjamin; Simpson, Graham

    2013-12-01

    Melioidosis is an infection with clinical importance in northern Australia due to the high associated mortality despite appropriate therapy. This report presents a case of acute pulmonary melioidosis on a background remarkable for the absence of typical risk factors for infection, but the presence of a high iron pulmonary microenvironment consequent to idiopathic pulmonary hemosiderosis. In light of recent genetic analysis of Burkholderia pseudomallei, we postulate that the patient inadvertently provided a high-substrate environment for the iron-scavenging ability of B. pseudomallei's siderophore associated virulence factors, giving her a unique major risk factor for infection. This highlights the importance of considering individual patient factors in addition to population-wide risk factors in the differential diagnosis of a serious illness, and the value of genetic analysis of clinically significant pathogens. PMID:25473541

  8. 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

  9. 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

  10. 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