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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.

  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. 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

  4. Burkholderia pseudomallei Capsular Polysaccharide Conjugates Provide Protection against Acute Melioidosis

    PubMed Central

    Burtnick, Mary N.; Stokes, Margaret G. M.; Whelan, Adam O.; Williamson, E. Diane; Atkins, Timothy P.; Prior, Joann L.; Brett, Paul J.

    2014-01-01

    Burkholderia pseudomallei, the etiologic agent of melioidosis, is a CDC tier 1 select agent that causes severe disease in both humans and animals. Diagnosis and treatment of melioidosis can be challenging, and in the absence of optimal chemotherapeutic intervention, acute disease is frequently fatal. Melioidosis is an emerging infectious disease for which there are currently no licensed vaccines. Due to the potential malicious use of B. pseudomallei as well as its impact on public health in regions where the disease is endemic, there is significant interest in developing vaccines for immunization against this disease. In the present study, type A O-polysaccharide (OPS) and manno-heptose capsular polysaccharide (CPS) antigens were isolated from nonpathogenic, select-agent-excluded strains of B. pseudomallei and covalently linked to carrier proteins. By using these conjugates (OPS2B1 and CPS2B1, respectively), it was shown that although high-titer IgG responses against the OPS or CPS component of the glycoconjugates could be raised in BALB/c mice, only those animals immunized with CPS2B1 were protected against intraperitoneal challenge with B. pseudomallei. Extending upon these studies, it was also demonstrated that when the mice were immunized with a combination of CPS2B1 and recombinant B. pseudomallei LolC, rather than with CPS2B1 or LolC individually, they exhibited higher survival rates when challenged with a lethal dose of B. pseudomallei. Collectively, these results suggest that CPS-based glycoconjugates are promising candidates for the development of subunit vaccines for immunization against melioidosis. PMID:24866807

  5. Less is more: Burkholderia pseudomallei and chronic melioidosis.

    PubMed

    Nandi, Tannistha; Tan, Patrick

    2013-09-24

    The Gram-negative bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. Once considered an esoteric tropical disease confined to Southeast Asia and northern Australia, research on B. pseudomallei has recently gained global prominence due to its classification as a potential bioterrorism agent by countries such as the United States and also by increasing numbers of case reports from regions where it is not endemic. An environmental bacterium typically found in soil and water, assessing the true global prevalence of melioidosis is challenged by the fact that clinical symptoms associated with B. pseudomallei infection are extremely varied and may be confused with diverse conditions such as lung cancer, tuberculosis, or Staphyloccocus aureus infection. These diagnostic challenges, coupled with lack of awareness among clinicians, have likely contributed to underdiagnosis and the high mortality rate of melioidosis, as initial treatment is often either inappropriate or delayed. Even after antibiotic treatment, relapses are frequent, and after resolution of acute symptoms, chronic melioidosis can also occur, and the symptoms can persist for months to years. In a recent article, Price et al. [mBio 4(4):e00388-13, 2013, doi:10.1128/mBio.00388-13] demonstrate how comparative genomic sequencing can reveal the repertoire of genetic changes incurred by B. pseudomallei during chronic human infection. Their results have significant clinical ramifications and highlight B. pseudomallei's ability to survive in a wide range of potential niches within hosts, through the acquisition of genetic adaptations that optimize fitness and resource utilization.

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

    PubMed

    Gilad, Jacob

    2007-11-01

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

  7. The global distribution of Burkholderia pseudomallei and melioidosis: an update.

    PubMed

    Currie, Bart J; Dance, David A B; Cheng, Allen C

    2008-12-01

    While Southeast Asia and northern Australia are well recognized as the major endemic regions for melioidosis, recent reports have expanded the endemic zone. Severe weather events and environmental disasters such as the 2004 Asian tsunami have unmasked locations of sporadic cases and have reconfirmed endemicity in Indonesia. The endemic region now includes the majority of the Indian subcontinent, southern China, Hong Kong and Taiwan. Sporadic cases have occurred in Brazil and elsewhere in the Americas and in island communities such as New Caledonia, in the Pacific Ocean, and Mauritius in the Indian Ocean. Some of the factors that are critical to further elucidating the global distribution of Burkholderia pseudomallei and melioidosis include improved access to diagnostic laboratory facilities and formal confirmation of the identity of bacterial isolates from suspected cases.

  8. 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

  9. 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

  10. Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei.

    PubMed

    Holden, Matthew T G; Titball, Richard W; Peacock, Sharon J; Cerdeño-Tárraga, Ana M; Atkins, Timothy; Crossman, Lisa C; Pitt, Tyrone; Churcher, Carol; Mungall, Karen; Bentley, Stephen D; Sebaihia, Mohammed; Thomson, Nicholas R; Bason, Nathalie; Beacham, Ifor R; Brooks, Karen; Brown, Katherine A; Brown, Nat F; Challis, Greg L; Cherevach, Inna; Chillingworth, Tracy; Cronin, Ann; Crossett, Ben; Davis, Paul; DeShazer, David; Feltwell, Theresa; Fraser, Audrey; Hance, Zahra; Hauser, Heidi; Holroyd, Simon; Jagels, Kay; Keith, Karen E; Maddison, Mark; Moule, Sharon; Price, Claire; Quail, Michael A; Rabbinowitsch, Ester; Rutherford, Kim; Sanders, Mandy; Simmonds, Mark; Songsivilai, Sirirurg; Stevens, Kim; Tumapa, Sarinna; Vesaratchavest, Monkgol; Whitehead, Sally; Yeats, Corin; Barrell, Bart G; Oyston, Petra C F; Parkhill, Julian

    2004-09-28

    Burkholderia pseudomallei is a recognized biothreat agent and the causative agent of melioidosis. This Gram-negative bacterium exists as a soil saprophyte in melioidosis-endemic areas of the world and accounts for 20% of community-acquired septicaemias in northeastern Thailand where half of those affected die. Here we report the complete genome of B. pseudomallei, which is composed of two chromosomes of 4.07 megabase pairs and 3.17 megabase pairs, showing significant functional partitioning of genes between them. The large chromosome encodes many of the core functions associated with central metabolism and cell growth, whereas the small chromosome carries more accessory functions associated with adaptation and survival in different niches. Genomic comparisons with closely and more distantly related bacteria revealed a greater level of gene order conservation and a greater number of orthologous genes on the large chromosome, suggesting that the two replicons have distinct evolutionary origins. A striking feature of the genome was the presence of 16 genomic islands (GIs) that together made up 6.1% of the genome. Further analysis revealed these islands to be variably present in a collection of invasive and soil isolates but entirely absent from the clonally related organism B. mallei. We propose that variable horizontal gene acquisition by B. pseudomallei is an important feature of recent genetic evolution and that this has resulted in a genetically diverse pathogenic species.

  11. Advances and remaining uncertainties in the epidemiology of Burkholderia pseudomallei and melioidosis.

    PubMed

    Currie, Bart J

    2008-03-01

    Major advances have been made in molecular studies of Burkholderia pseudomallei and the immunology of melioidosis. However, there remain large gaps in understanding of the epidemiology of this enigmatic disease. Identified global distribution boundaries of melioidosis continue to expand. Recent data suggest Australian strains of B. pseudomallei may be ancestral to those from Southeast Asia, but the ecology of this environmental bacterium remains elusive. Despite the potential for rapidly progressive septicaemia, the critical virulence factors in B. pseudomallei remain to be clarified. Inhalation following aerosolization of B. pseudomallei may account for the high mortality when melioidosis occurs after severe weather events.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. Association of the melioidosis agent Burkholderia pseudomallei with water parameters in rural water supplies in Northern Australia.

    PubMed

    Draper, A D K; Mayo, M; Harrington, G; Karp, D; Yinfoo, D; Ward, L; Haslem, A; Currie, B J; Kaestli, M

    2010-08-01

    We analyzed water parameters and the occurrence of the melioidosis agent Burkholderia pseudomallei in 47 water bores in Northern Australia. B. pseudomallei was associated with soft, acidic bore water of low salinity but high iron levels. This finding aids in identifying water supplies at risk of contamination with this pathogenic bacterium.

  18. 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.

  19. 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

  20. Multilocus sequence typing and evolutionary relationships among the causative agents of melioidosis and glanders, Burkholderia pseudomallei and Burkholderia mallei.

    PubMed

    Godoy, Daniel; Randle, Gaynor; Simpson, Andrew J; Aanensen, David M; Pitt, Tyrone L; Kinoshita, Reimi; Spratt, Brian G

    2003-05-01

    A collection of 147 isolates of Burkholderia pseudomallei, B. mallei, and B. thailandensis was characterized by multilocus sequence typing (MLST). The 128 isolates of B. pseudomallei, the causative agent of melioidosis, were obtained from diverse geographic locations, from humans and animals with disease, and from the environment and were resolved into 71 sequence types. The utility of the MLST scheme for epidemiological investigations was established by analyzing isolates from captive marine mammals and birds and from humans in Hong Kong with melioidosis. MLST gave a level of resolution similar to that given by pulsed-field gel electrophoresis and identified the same three clones causing disease in animals, each of which was also associated with disease in humans. The average divergence between the alleles of B. thailandensis and B. pseudomallei was 3.2%, and there was no sharing of alleles between these species. Trees constructed from differences in the allelic profiles of the isolates and from the concatenated sequences of the seven loci showed that the B. pseudomallei isolates formed a cluster of closely related lineages that were fully resolved from the cluster of B. thailandensis isolates, confirming their separate species status. However, isolates of B. mallei, the causative agent of glanders, recovered from three continents over a 30-year period had identical allelic profiles, and the B. mallei isolates clustered within the B. pseudomallei group of isolates. Alleles at six of the seven loci in B. mallei were also present within B. pseudomallei isolates, and B. mallei is a clone of B. pseudomallei that, on population genetics grounds, should not be given separate species status.

  1. Short report: application of a polymerase chain reaction to detect Burkholderia pseudomallei in clinical specimens from patients with suspected melioidosis.

    PubMed

    Gal, Daniel; Mayo, Mark; Spencer, Emma; Cheng, Allen C; Currie, Bart J

    2005-12-01

    The diagnostic potential of a Burkholderia pseudomallei type three secretion system (TTS1) polymerase chain reaction (PCR) was examined on clinical specimens from 27 patients with sepsis in the Northern Territory of Australia, a region endemic for melioidosis. The TTS1 PCR was conducted on DNA extracted from a range of clinical specimens (blood, sputum, urine, joint, pericardial and pleural fluid, and swabs from skin lesions, throat, nose, and rectum). The PCR sensitivity in culture-positive clinical specimens from the nine confirmed patients with melioidosis was 65% and the specificity was 100%, with no PCR-positive results in specimens from 18 patients without melioidosis. The PCR based on the B. pseudomallei TTS1 has the potential to substantially improve the timeliness of diagnosis of melioidosis.

  2. Comparison of TaqMan PCR assays for detection of the melioidosis agent Burkholderia pseudomallei in clinical specimens.

    PubMed

    Kaestli, Mirjam; Richardson, Leisha J; Colman, Rebecca E; Tuanyok, Apichai; Price, Erin P; Bowers, Jolene R; Mayo, Mark; Kelley, Erin; Seymour, Meagan L; Sarovich, Derek S; Pearson, Talima; Engelthaler, David M; Wagner, David M; Keim, Paul S; Schupp, James M; Currie, Bart J

    2012-06-01

    Melioidosis is an emerging infectious disease caused by the soil bacterium Burkholderia pseudomallei. In diagnostic and forensic settings, molecular detection assays need not only high sensitivity with low limits of detection but also high specificity. In a direct comparison of published and newly developed TaqMan PCR assays, we found the TTS1-orf2 assay to be superior in detecting B. pseudomallei directly from clinical specimens. The YLF/BTFC multiplex assay (targeting the Yersinia-like fimbrial/Burkholderia thailandensis-like flagellum and chemotaxis region) also showed high diagnostic sensitivity and provides additional information on possible geographic origin.

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. Sensitive and specific molecular detection of Burkholderia pseudomallei, the causative agent of melioidosis, in the soil of tropical northern Australia.

    PubMed

    Kaestli, Mirjam; Mayo, Mark; Harrington, Glenda; Watt, Felicity; Hill, Jason; Gal, Daniel; Currie, Bart J

    2007-11-01

    Burkholderia pseudomallei, the cause of the severe disease melioidosis in humans and animals, is a gram-negative saprophyte living in soil and water of areas of endemicity such as tropical northern Australia and Southeast Asia. Infection occurs mainly by contact with wet contaminated soil. The environmental distribution of B. pseudomallei in northern Australia is still unclear. We developed and evaluated a direct soil B. pseudomallei DNA detection method based on the recently published real-time PCR targeting the B. pseudomallei type III secretion system. The method was evaluated by inoculating different soil types with B. pseudomallei dilution series and by comparing B. pseudomallei detection rate with culture-based detection rate for 104 randomly collected soil samples from the Darwin rural area in northern Australia. We found that direct soil B. pseudomallei DNA detection not only was substantially faster than culture but also proved to be more sensitive with no evident false-positive results. This assay provides a new tool to detect B. pseudomallei in soil samples in a fast and highly sensitive and specific manner and is applicable for large-scale B. pseudomallei environmental screening studies or in outbreak situations. Furthermore, analysis of the 104 collected soil samples revealed a significant association between B. pseudomallei-positive sites and the presence of animals at these locations and also with moist, reddish brown-to-reddish gray soils.

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

    PubMed Central

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

    2016-01-01

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

  10. 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

  11. Burkholderia pseudomallei penetrates the brain via destruction of the olfactory and trigeminal nerves: implications for the pathogenesis of neurological melioidosis.

    PubMed

    St John, James A; Ekberg, Jenny A K; Dando, Samantha J; Meedeniya, Adrian C B; Horton, Rachel E; Batzloff, Michael; Owen, Suzzanne J; Holt, Stephanie; Peak, Ian R; Ulett, Glen C; Mackay-Sim, Alan; Beacham, Ifor R

    2014-04-15

    ABSTRACT Melioidosis is a potentially fatal disease that is endemic to tropical northern Australia and Southeast Asia, with a mortality rate of 14 to 50%. The bacterium Burkholderia pseudomallei is the causative agent which infects numerous parts of the human body, including the brain, which results in the neurological manifestation of melioidosis. The olfactory nerve constitutes a direct conduit from the nasal cavity into the brain, and we have previously reported that B. pseudomallei can colonize this nerve in mice. We have now investigated in detail the mechanism by which the bacteria penetrate the olfactory and trigeminal nerves within the nasal cavity and infect the brain. We found that the olfactory epithelium responded to intranasal B. pseudomallei infection by widespread crenellation followed by disintegration of the neuronal layer to expose the underlying basal layer, which the bacteria then colonized. With the loss of the neuronal cell bodies, olfactory axons also degenerated, and the bacteria then migrated through the now-open conduit of the olfactory nerves. Using immunohistochemistry, we demonstrated that B. pseudomallei migrated through the cribriform plate via the olfactory nerves to enter the outer layer of the olfactory bulb in the brain within 24 h. We also found that the bacteria colonized the thin respiratory epithelium in the nasal cavity and then rapidly migrated along the underlying trigeminal nerve to penetrate the cranial cavity. These results demonstrate that B. pseudomallei invasion of the nerves of the nasal cavity leads to direct infection of the brain and bypasses the blood-brain barrier. IMPORTANCE Melioidosis is a potentially fatal tropical disease that is endemic to northern Australia and Southeast Asia. It is caused by the bacterium Burkholderia pseudomallei, which can infect many organs of the body, including the brain, and results in neurological symptoms. The pathway by which the bacteria can penetrate the brain is unknown, and

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

    PubMed

    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; Boyton, Rosemary

    2015-05-15

    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.

  13. Development of antibodies to Burkholderia pseudomallei during childhood in melioidosis-endemic northeast Thailand.

    PubMed

    Wuthiekanun, Vanaporn; Chierakul, Wirongrong; Langa, Sayan; Chaowagul, Wipada; Panpitpat, Chanathip; Saipan, Penchan; Thoujaikong, Thaksinaporn; Day, Nicholas P; Peacock, Sharon J

    2006-06-01

    A cross-sectional serological survey of 2,214 children living in northeast Thailand was conducted to define the antibody response to Burkholderia pseudomallei from birth to 14 years. There was a sharp rise in detectable antibodies from birth to 4 years followed by reactivity in approximately 60-70% of children thereafter.

  14. Burkholderia pseudomallei Penetrates the Brain via Destruction of the Olfactory and Trigeminal Nerves: Implications for the Pathogenesis of Neurological Melioidosis

    PubMed Central

    St. John, James A.; Ekberg, Jenny A. K.; Dando, Samantha J.; Meedeniya, Adrian C. B.; Horton, Rachel E.; Batzloff, Michael; Owen, Suzzanne J.; Holt, Stephanie; Peak, Ian R.; Ulett, Glen C.; Mackay-Sim, Alan; Beacham, Ifor R.

    2014-01-01

    ABSTRACT Melioidosis is a potentially fatal disease that is endemic to tropical northern Australia and Southeast Asia, with a mortality rate of 14 to 50%. The bacterium Burkholderia pseudomallei is the causative agent which infects numerous parts of the human body, including the brain, which results in the neurological manifestation of melioidosis. The olfactory nerve constitutes a direct conduit from the nasal cavity into the brain, and we have previously reported that B. pseudomallei can colonize this nerve in mice. We have now investigated in detail the mechanism by which the bacteria penetrate the olfactory and trigeminal nerves within the nasal cavity and infect the brain. We found that the olfactory epithelium responded to intranasal B. pseudomallei infection by widespread crenellation followed by disintegration of the neuronal layer to expose the underlying basal layer, which the bacteria then colonized. With the loss of the neuronal cell bodies, olfactory axons also degenerated, and the bacteria then migrated through the now-open conduit of the olfactory nerves. Using immunohistochemistry, we demonstrated that B. pseudomallei migrated through the cribriform plate via the olfactory nerves to enter the outer layer of the olfactory bulb in the brain within 24 h. We also found that the bacteria colonized the thin respiratory epithelium in the nasal cavity and then rapidly migrated along the underlying trigeminal nerve to penetrate the cranial cavity. These results demonstrate that B. pseudomallei invasion of the nerves of the nasal cavity leads to direct infection of the brain and bypasses the blood-brain barrier. PMID:24736221

  15. Burkholderia pseudomallei strain type, based on pulsed-field gel electrophoresis, does not determine disease presentation in melioidosis.

    PubMed

    Cheng, Allen C; Day, Nicholas P J; Mayo, Mark J; Gal, Daniel; Currie, Bart J

    2005-01-01

    Melioidosis, the infection due to Burkholderia pseudomallei, may present with a spectrum of severity and may affect any site in the body. Differential strain virulence and tropism suggested by previous studies would have implications for virulence and vaccine work. We explored clinical correlations using pulsed-field gel electrophoresis (PFGE) typing in a well-characterised clinical collection. Two methods of analysis were used based on band-based similarity values: first, a conventional cluster analysis formed by the unweighted paired group mean analysis, and second, an analysis of the distribution of the "within-group" and "between-group" Dice coefficient. Clinical isolates from 114 cases of melioidosis occurring in the Northern Territory, Australia were studied; 71 strain types were defined with a Simpson's index of 0.91. No correlation was found between strain type and disease severity or site of melioidosis on presentation, with no differences in similarity values found when comparing within and between-groups. In particular, isolates from patients with neurological melioidosis were not clustered. There was evidence of geographical localisation. This study suggests that the variation in strain type may not be as important as host and environmental factors in determining the pattern of disease.

  16. 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.

  17. 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

  18. 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.

  19. 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

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

    PubMed

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

    2009-07-16

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Development of hydrolysis probe-based real-time PCR for identification of virulent gene targets of Burkholderia pseudomallei and B. mallei--a retrospective study on archival cases of service members with melioidosis and glanders.

    PubMed

    Zhang, Binxue; Wear, Douglas J; Kim, H S; Weina, Peter; Stojadinovic, Alexander; Izadjoo, Mina

    2012-02-01

    Burkholderia pseudomallei and B. mallei are two highly pathogenic bacteria responsible for melioidosis and glanders, respectively. Our laboratory developed hydrolysis probe-based real-time polymerase chain reaction assays targeting type three secretion system (TTS) and transposase family protein (TFP) of B. pseudomallei and B. malli, respectively. The assays were validated for target specificity, amplification sensitivity, and reproducibility. A bacterial DNA panel, composed of B. pseudomallei (13 strains), B. mallei (11 strains), Burkholderia species close neighbors (5 strains), and other bacterial species (17 strains), was prepared for specificity testing. Reference DNAs from B. pseudomallei and B. mallei bacterial cultures were used as controls for amplification, limit of detection, and reproducibility testing. The two TaqMan assays, Bp-TTS 1 and Bm-TFP, were optimized and applied in a retrospective study of archived cases from the Armed Forces Institute of Pathology. We tested 10 formalin-fixed paraffin-embedded blocks originally from autopsy specimens of patients who died of melioidosis or glanders during or after overseas tours in 1960s. Polymerase chain reaction results confirmed that DNA samples from formalin-fixed paraffin-embedded blocks of eight patients with melioidosis were positive for Bp-TTS 1 target and two patients with glanders were positive for Bm-TFP target.

  3. 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

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

    PubMed

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

    2014-06-01

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

  5. 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.

  6. Polysaccharides and virulence of Burkholderia pseudomallei.

    PubMed

    Sarkar-Tyson, M; Thwaite, J E; Harding, S V; Smither, S J; Oyston, P C F; Atkins, T P; Titball, R W

    2007-08-01

    Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease of humans and animals. Gene clusters which encode capsular polysaccharide (type I O-PS) and LPS (type II O-PS), both of which play roles in virulence, have previously been identified. Here, the identification of two further putative clusters, type III O-PS and type IV O-PS, is reported. Mice challenged with type III O-PS or type IV O-PS mutants showed increased mean times to death (7.8 and 11.6 days) compared to those challenged with wild-type B. pseudomallei (3 days). To investigate the possible roles of polysaccharides in protection, mice were immunized with killed cells of wild-type B. pseudomallei or killed cells of B. pseudomallei with mutations in the O antigen, capsular polysaccharide, type III O-PS or type IV O-PS gene clusters. Immunization with all polysaccharide mutant strains resulted in delayed time to death compared to the naïve controls, following challenge with wild-type B. pseudomallei strain K96243. However, immunization with killed polysaccharide mutant strains conferred different degrees of protection, demonstrating the immunological importance of the polysaccharide clusters on the surface of B. pseudomallei.

  7. Groundwater seeps facilitate exposure to Burkholderia pseudomallei.

    PubMed

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

    2011-10-01

    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.

  8. Exploitation of host cells by Burkholderia pseudomallei.

    PubMed

    Stevens, Mark P; Galyov, Edouard E

    2004-04-01

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

  9. Strategies for Intracellular Survival of Burkholderia pseudomallei

    PubMed Central

    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

  10. 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

  11. Strategies toward vaccines against Burkholderia mallei and Burkholderia pseudomallei

    PubMed Central

    Bondi, Sara K; Goldberg, Joanna B

    2009-01-01

    Burkholderia mallei and Burkholderia pseudomallei are Gram-negative, rod-shaped bacteria, and are the causative agents of the diseases glanders and melioidosis, respectively. These bacteria have been recognized as important pathogens for over 100 years, yet a relative dearth of available information exists regarding their virulence determinants and immunopathology. Infection with either of these bacteria presents with nonspecific symptoms and can be either acute or chronic, impeding rapid diagnosis. The lack of a vaccine for either bacterium also makes them potential candidates for bioweaponization. Together with their high rate of infectivity via aerosols and resistance to many common antibiotics, both bacteria have been classified as category B priority pathogens by the US NIH and US CDC, which has spurred a dramatic increase in interest in these microorganisms. Attempts have been made to develop vaccines for these infections, which would not only benefit military personnel, a group most likely to be targeted in an intentional release, but also individuals who may come in contact with glanders-infected animals or live in areas where melioidosis is endemic. This review highlights some recent attempts of vaccine development for these infections and the strategies used to improve the efficacy of vaccine approaches. PMID:18980539

  12. 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

  13. 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.

  14. Development of Burkholderia mallei and pseudomallei vaccines.

    PubMed

    Silva, Ediane B; Dow, Steven W

    2013-01-01

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

  15. Comparison of diagnostic laboratory methods for identification of Burkholderia pseudomallei.

    PubMed

    Inglis, Timothy J J; Merritt, Adam; Chidlow, Glenys; Aravena-Roman, Max; Harnett, Gerry

    2005-05-01

    Limited experience and a lack of validated diagnostic reagents make Burkholderia pseudomallei, the cause of melioidosis, difficult to recognize in the diagnostic microbiology laboratory. We compared three methods of confirming the identity of presumptive B. pseudomallei strains using a collection of Burkholderia species drawn from diverse geographic, clinical, and environmental sources. The 95 isolates studied included 71 B. pseudomallei and 3 B. thailandensis isolates. The API 20NE method identified only 37% of the B. pseudomallei isolates. The agglutinating antibody test identified 82% at first the attempt and 90% including results of a repeat test with previously negative isolates. Gas-liquid chromatography analysis of bacterial fatty acid methyl esters (GLC-FAME) identified 98% of the B. pseudomallei isolates. The agglutination test produced four false positive results, one B. cepacia, one B. multivorans, and two B. thailandensis. API produced three false positive results, one positive B. cepacia and two positive B. thailandensis. GLC-FAME analysis was positive for one B. cepacia isolate. On the basis of these results, the most robust B. pseudomallei discovery pathway combines the previously recommended isolate screening tests (Gram stain, oxidase test, gentamicin and polymyxin susceptibility) with monoclonal antibody agglutination on primary culture, followed by a repeat after 24 h incubation on agglutination-negative isolates and GLC-FAME analysis. Incorporation of PCR-based identification within this schema may improve percentages of recognition further but requires more detailed evaluation. PMID:15872242

  16. 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

  17. 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

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

    DOE PAGES

    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.

  19. 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

  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. Pleuropulmonary melioidosis with osteomyelitis rib

    PubMed Central

    Neliyathodi, Suhail; Thazhathethil, Abdul Nazar; Pallivalappil, Lisha; Balakrishnan, Deepu

    2015-01-01

    Melioidosis is a multiorgan infectious disease caused by Burkholderia pseudomallei. Few cases have been reported from south India. This is a case report of pleuropulmonary melioidosis with rib osteomyelitis. PMID:25624602

  2. The Identification and Differentiation between Burkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing

    PubMed Central

    Gilling, Damian H.; Luna, Vicki Ann; Pflugradt, Cori

    2014-01-01

    The etiologic agents for melioidosis and glanders, Burkholderia mallei and Burkholderia pseudomallei respectively, are genetically similar making identification and differentiation from other Burkholderia species and each other challenging. We used pyrosequencing to determine the presence or absence of an insertion sequence IS407A within the flagellin P (fliP) gene and to exploit the difference in orientation of this gene in the two species. Oligonucleotide primers were designed to selectively target the IS407A-fliP interface in B. mallei and the fliP gene specifically at the insertion point in B. pseudomallei. We then examined DNA from ten B. mallei, ten B. pseudomallei, 14 B. cepacia, eight other Burkholderia spp., and 17 other bacteria. Resultant pyrograms encompassed the target sequence that contained either the fliP gene with the IS407A interruption or the fully intact fliP gene with 100% sensitivity and 100% specificity. These pyrosequencing assays based upon a single gene enable investigators to reliably identify the two species. The information obtained by these assays provides more knowledge of the genomic reduction that created the new species B. mallei from B. pseudomallei and may point to new targets that can be exploited in the future. PMID:27350960

  3. Contribution of gene loss to the pathogenic evolution of Burkholderia pseudomallei and Burkholderia mallei.

    PubMed

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

    2004-07-01

    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.

  4. 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

  5. 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

  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. Genomic islands from five strains of Burkholderia pseudomallei

    PubMed Central

    Tuanyok, Apichai; Leadem, Benjamin R; Auerbach, Raymond K; Beckstrom-Sternberg, Stephen M; Beckstrom-Sternberg, James S; Mayo, Mark; Wuthiekanun, Vanaporn; Brettin, Thomas S; Nierman, William C; Peacock, Sharon J; Currie, Bart J; Wagner, David M; Keim, Paul

    2008-01-01

    Background Burkholderia pseudomallei is the etiologic agent of melioidosis, a significant cause of morbidity and mortality where this infection is endemic. Genomic differences among strains of B. pseudomallei are predicted to be one of the major causes of the diverse clinical manifestations observed among patients with melioidosis. The purpose of this study was to examine the role of genomic islands (GIs) as sources of genomic diversity in this species. Results We found that genomic islands (GIs) vary greatly among B. pseudomallei strains. We identified 71 distinct GIs from the genome sequences of five reference strains of B. pseudomallei: K96243, 1710b, 1106a, MSHR668, and MSHR305. The genomic positions of these GIs are not random, as many of them are associated with tRNA gene loci. In particular, the 3' end sequences of tRNA genes are predicted to be involved in the integration of GIs. We propose the term "tRNA-mediated site-specific recombination" (tRNA-SSR) for this mechanism. In addition, we provide a GI nomenclature that is based upon integration hotspots identified here or previously described. Conclusion Our data suggest that acquisition of GIs is one of the major sources of genomic diversity within B. pseudomallei and the molecular mechanisms that facilitate horizontally-acquired GIs are common across multiple strains of B. pseudomallei. The differential presence of the 71 GIs across multiple strains demonstrates the importance of these mobile elements for shaping the genetic composition of individual strains and populations within this bacterial species. PMID:19038032

  8. Genetic diversity of Burkholderia pseudomallei isolates in Australia.

    PubMed

    Cheng, Allen C; Ward, Linda; Godoy, Daniel; Norton, Robert; Mayo, Mark; Gal, Daniel; Spratt, Brian G; Currie, Bart J

    2008-01-01

    Melioidosis is caused by the gram-negative saprophytic bacterium Burkholderia pseudomallei, which is endemic to southeast Asia and northern Australia. We have previously found evidence of geographic localization of strains based on multilocus sequence typing (MLST). In this study, we examined the diversity of 277 isolates from northern Australia, which were resolved into 159 different sequence types. No sequence types were common to both Queensland and the Northern Territory, and there was significant differentiation between the alleles present in the two regions. The considerable diversity in sequence types contrasts with the limited diversity of alleles at MLST loci, supporting previous work suggesting a high rate of recombination relative to mutation in B. pseudomallei, where new sequence types are primarily generated by reassortment of existing alleles.

  9. Ribotype differences between clinical and environmental isolates of Burkholderia pseudomallei.

    PubMed

    Trakulsomboon, S; Dance, D A; Smith, M D; White, N J; Pitt, T L

    1997-07-01

    Burkholderia pseudomallei is isolated frequently from the soil in regions where the disease melioidosis occurs. However, recent surveys in Thailand have shown that the frequency of isolation of the organism from soil samples is not directly related to the incidence of melioidosis in an area. To determine whether strain populations of B. pseudomallei prevalent in soil are gentypically related to strains causing clinical disease, rRNA BamHI restriction fragment length polymorphisms (RFLP) of 139 soil environmental isolates and 228 human isolates were compared. Two groups of ribotype patterns were found. Group I comprised 37 different ribotype patterns which were characterised by five to eight hybridisation bands of 2.8- > 23 kb. All of these ribotypes were identified among the clinical isolates, and 18 of them were also found in 59 environmental isolates. Group II was represented by 12 ribotypes found only in environmental strains. These ribotype patterns comprised one to five bands in the size range 9- > 23 kb. All but one of the 73 isolates in this group grew on a minimal medium supplemented with L-arabinose. In contrast, only 3% of the 66 isolates from the environment with group I ribotype patterns could utilise this sugar as their sole energy source. These findings suggest that B. pseudomallei strains that utilise arabinose constitute a population that is genetically distinct from other environmental and clinical strains.

  10. Comparison of Ashdown's medium, Burkholderia cepacia medium, and Burkholderia pseudomallei selective agar for clinical isolation of Burkholderia pseudomallei.

    PubMed

    Peacock, Sharon J; Chieng, Grace; Cheng, Allen C; Dance, David A B; Amornchai, Premjit; Wongsuvan, Gumphol; Teerawattanasook, Nittaya; Chierakul, Wirongrong; Day, Nicholas P J; Wuthiekanun, Vanaporn

    2005-10-01

    Ashdown's medium, Burkholderia pseudomallei selective agar (BPSA), and a commercial Burkholderia cepacia medium were compared for their abilities to grow B. pseudomallei from 155 clinical specimens that proved positive for this organism. The sensitivity of each was equivalent; the selectivity of BPSA was lower than that of Ashdown's or B. cepacia medium.

  11. 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

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

    DOE PAGES

    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

  13. 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

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

    PubMed

    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.

  15. Genome Sequence of Burkholderia pseudomallei NCTC 13392

    PubMed Central

    Sahl, Jason W.; Stone, Joshua K.; Gelhaus, H. Carl; Warren, Richard L.; Cruttwell, Caroline J.; Funnell, Simon G.; Keim, Paul

    2013-01-01

    Here, we describe the draft genome sequence of Burkholderia pseudomallei NCTC 13392. This isolate has been distributed as K96243, but distinct genomic differences have been identified. The genomic sequence of this isolate will provide the genomic context for previously conducted functional studies. PMID:23704173

  16. Melioidosis in New Caledonia.

    PubMed

    Le Hello, Simon; Currie, Bart J; Godoy, Daniel; Spratt, Brian G; Mikulski, Marc; Lacassin, Flore; Garin, Benoit

    2005-10-01

    Recognized melioidosis-endemic areas are widening. In the South Pacific, melioidosis is endemic in New Caledonia, northern Australia, and Papua New Guinea. We report the first 4 documented cases of human melioidosis from New Caledonia. Molecular typing of 2 Burkholderia pseudomallei isolates suggests a link to Australian strains.

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

    PubMed

    Price, Erin P; Sarovich, Derek S; Webb, Jessica R; Ginther, Jennifer L; Mayo, Mark; Cook, James M; Seymour, Meagan L; Kaestli, Mirjam; Theobald, Vanessa; Hall, Carina M; Busch, Joseph D; Foster, Jeffrey T; Keim, Paul; Wagner, David M; Tuanyok, Apichai; Pearson, Talima; Currie, Bart J

    2013-01-01

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

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

    PubMed

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

    2012-12-01

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

  19. 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.

  20. 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

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

    PubMed Central

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

    2016-01-01

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

  2. 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

  3. 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

  4. Experimental Phage Therapy for Burkholderia pseudomallei Infection.

    PubMed

    Guang-Han, Ong; 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

  5. Rapid Detection of Burkholderia pseudomallei in Blood Cultures Using a Monoclonal Antibody-Based Immunofluorescent Assay

    PubMed Central

    Chantratita, Narisara; Tandhavanant, Sarunporn; Wongsuvan, Gumphol; Wuthiekanun, Vanaporn; Teerawattanasook, Nittaya; Day, Nicholas P. J.; Limmathurotsakul, Direk; Peacock, Sharon J.

    2013-01-01

    Melioidosis is a severe bacterial infection caused by Burkholderia pseudomallei. Rapid antimicrobial therapy is necessary to improve patient outcome, which is aided by direct detection of B. pseudomallei in clinical samples. A drawback for all antigen assays is that the number of B. pseudomallei in blood usually falls below the achievable level of detection. We performed a prospective cohort study of 461 patients with 541 blood cultures to evaluate the utility of a pre-incubation step prior to detection of B. pseudomallei using a monoclonal antibody-based immunofluorescent assay (Mab-IFA). The Mab-IFA was positive in 74 of 76 patients with melioidosis (sensitivity = 97.4%), and negative in 385 patients who did not have blood cultures containing B. pseudomallei (specificity = 100%). The Mab-IFA could be a valuable supplementary tool for rapid detection. We recommend the use of the Mab-IFA to test blood cultures that flag positive in regions where melioidosis is endemic. PMID:24019434

  6. 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

  7. Survival of Burkholderia pseudomallei on Environmental Surfaces.

    PubMed

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

    2007-12-01

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

  8. 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

  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. Epidemiology of arabinose assimilation in burkholderia pseudomallei isolated from patients and soil in Thailand.

    PubMed

    Trakulsomboon, S; Vuddhakul, V; Tharavichitkul, P; Na-Gnam, N; Suputtamongkol, Y; Thamlikitkul, V

    1999-12-01

    Burkholderia pseudomallei is an environmental saprophyte that has been isolated widely from soil in Southeast Asia and the relationship between environmental contamination and clinical melioidosis has been established. It has been shown that the arabinose assimilation property of B. pseudonrallei is probably one of the determinants indicating virulence of this organism. Therefore, the distribution of arabinose assimilation biotypes of B. pseudomallei collected from four geographic regions of Thailand was studied in order to determine an association between arabinose assimilation of B. pseudomallei and the uneven distribution of melioidosis found among these four areas. A total of 830 isolates of B. pseudomallei (412 patient isolates and 418 soil isolates) collected from the patients and soil in four regions of Thailand in 1997 were tested for an ability to grow on a minimal agar medium supplemented with L-arabinose. All patient isolates except one could not utilise arabinose (Ara-). For 418 soil isolates, 232 (55.5%) isolates were identified as Ara type. They comprised 180 (62.5%), 36 (46.8%), 6 (35.3%) and 10 (27.8%) isolates derived from northeastern, southern, northern and central regions respectively. The ratios of Ara- to Ara, were 1.7, 0.9. 0.5 and 0.4 among isolates collected from northeastern, southern, northern and central regions respectively. The prevalence of Ara- in soil isolates in northeast is significantly higher than those in other regions. This observation suggests that in addition to the presence of B. pseudomallei in soil which is one of the factors contributing to a burden of melioidosis in northeastern Thailand, the distribution of more virulent biotype (Ara-) soil isolates is a factor contributing to a high prevalence of melioidosis in northeastern Thailand as well.

  11. PCR-based Methodologies Used to Detect and Differentiate the Burkholderia pseudomallei complex: B. pseudomallei, B. mallei, and B. thailandensis.

    PubMed

    Lowe, Woan; March, Jordon K; Bunnell, Annette J; O'Neill, Kim L; Robison, Richard A

    2014-01-01

    Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. The high mortality rates of glanders and melioidosis, their potential use as bioweapons, and their low infectious dose, necessitate the need for rapid and accurate detection methods. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, due to the difficulty in developing a sensitive, selective, and accurate assay. The development of PCR technologies has revolutionized diagnostic testing and these detection methods have become popular due to their speed, sensitivity, and accuracy. The purpose of this review is to provide a comprehensive overview and evaluation of the advancements in PCR-based detection and differentiation methodologies for the B. pseudomallei complex, and examine their potential uses in diagnostic and environmental testing.

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

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

    2014-12-23

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

  14. 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

  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. 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.

  17. Protective response to subunit vaccination against intranasal Burkholderia mallei and B. pseudomallei challenge

    PubMed Central

    Whitlock, Gregory C.; Deeraksa, Arpaporn; Qazi, Omar; Judy, Barbara M.; Taylor, Katherine; Propst, Katie L.; Duffy, Angie J.; Johnson, Kate; Kitto, G. Barrie; Brown, Katherine A.; Dow, Steven W.; Torres, Alfredo G.; Estes, D. Mark

    2013-01-01

    Burkholderia mallei and B. pseudomallei are Gram-negative pathogenic bacteria, responsible for the diseases glanders and melioidosis, respectively. Furthermore, there is currently no vaccine available against these Burkholderia species. In this study, we aimed to identify protective proteins against these pathogens. Immunization with recombinant B. mallei Hcp1 (type VI secreted/structural protein), BimA (autotransporter protein), BopA (type III secreted protein), and B. pseudomallei LolC (ABC transporter protein) generated significant protection against lethal inhaled B. mallei ATCC23344 and B. pseudomallei 1026b challenge. Immunization with BopA elicited the greatest protective activity, resulting in 100% and 60% survival against B. mallei and B. pseudomallei challenge, respectively. Moreover, sera from recovered mice demonstrated reactivity with the recombinant proteins. Dendritic cells stimulated with each of the different recombinant proteins showed distinct cytokine patterns. In addition, T cells from immunized mice produced IFN-γ following in vitro re-stimulation. These results indicated therefore that it was possible to elicit cross-protective immunity against both B. mallei and B. pseudomallei by vaccinating animals with one or more novel recombinant proteins identified in B. mallei. PMID:24379895

  18. Protective response to subunit vaccination against intranasal Burkholderia mallei and B. pseudomallei challenge.

    PubMed

    Whitlock, Gregory C; Deeraksa, Arpaporn; Qazi, Omar; Judy, Barbara M; Taylor, Katherine; Propst, Katie L; Duffy, Angie J; Johnson, Kate; Kitto, G Barrie; Brown, Katherine A; Dow, Steven W; Torres, Alfredo G; Estes, D Mark

    2010-01-01

    Burkholderia mallei and B. pseudomallei are Gram-negative pathogenic bacteria, responsible for the diseases glanders and melioidosis, respectively. Furthermore, there is currently no vaccine available against these Burkholderia species. In this study, we aimed to identify protective proteins against these pathogens. Immunization with recombinant B. mallei Hcp1 (type VI secreted/structural protein), BimA (autotransporter protein), BopA (type III secreted protein), and B. pseudomallei LolC (ABC transporter protein) generated significant protection against lethal inhaled B. mallei ATCC23344 and B. pseudomallei 1026b challenge. Immunization with BopA elicited the greatest protective activity, resulting in 100% and 60% survival against B. mallei and B. pseudomallei challenge, respectively. Moreover, sera from recovered mice demonstrated reactivity with the recombinant proteins. Dendritic cells stimulated with each of the different recombinant proteins showed distinct cytokine patterns. In addition, T cells from immunized mice produced IFN-γ following in vitro re-stimulation. These results indicated therefore that it was possible to elicit cross-protective immunity against both B. mallei and B. pseudomallei by vaccinating animals with one or more novel recombinant proteins identified in B. mallei.

  19. Burkholderia pseudomallei Class A β-Lactamase Mutations That Confer Selective Resistance against Ceftazidime or Clavulanic Acid Inhibition

    PubMed Central

    Tribuddharat, Chanwit; Moore, Richard A.; Baker, Patricia; Woods, Donald E.

    2003-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is inherently resistant to a variety of antibiotics including aminoglycosides, macrolides, polymyxins, and β-lactam antibiotics. Despite resistance to many β-lactams, ceftazidime and β-lactamase inhibitor-β-lactam combinations are commonly used for treatment of melioidosis. Here, we examine the enzyme kinetics of β-lactamase isolated from mutants resistant to ceftazidime and clavulanic acid inhibition and describe specific mutations within conserved motifs of the β-lactamase enzyme which account for these resistance patterns. Sequence analysis of regions flanking the B. pseudomallei penA gene revealed a putative regulator gene located downstream of penA. We have cloned and sequenced the penA gene from B. mallei and found it to be identical to penA from B. pseudomallei. PMID:12821450

  20. 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.

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

    PubMed

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

    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

  2. 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

  3. 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

  4. 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

  5. Antimicrobial susceptibility and genetic characterisation of Burkholderia pseudomallei isolated from Malaysian patients.

    PubMed

    Khosravi, Yalda; Vellasamy, Kumutha Malar; Mariappan, Vanitha; Ng, Shet-Lee; Vadivelu, Jamuna

    2014-01-01

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

  6. Antimicrobial activity of Tachyplesin 1 against Burkholderia pseudomallei: an in vitro and in silico approach

    PubMed Central

    Lee, Lyn-Fay; Mariappan, Vanitha; Vellasamy, Kumutha Malar

    2016-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many conventional antibiotics. Therefore, alternative antimicrobial agents such as antimicrobial peptides (AMPs) are extensively studied to combat this issue. Our study aims to identify and understand the mode of action of the potential AMP(s) that are effective against B. pseudomallei in both planktonic and biofilm state as well as to predict the possible binding targets on using in vitro and in silico approaches. In the in vitro study, 11 AMPs were tested against 100 B. pseudomallei isolates for planktonic cell susceptibility, where LL-37, and PG1, demonstrated 100.0% susceptibility and TP1 demonstrated 83% susceptibility. Since the B. pseudomallei activity was reported on LL-37 and PG1, TP1 was selected for further investigation. TP1 inhibited B. pseudomallei cells at 61.69 μM, and membrane blebbing was observed using scanning electron microscopy. Moreover, TP1 inhibited B. pseudomallei cell growth, reaching bactericidal endpoint within 2 h post exposure as compared to ceftazidime (CAZ) (8 h). Furthermore, TP1 was shown to suppress the growth of B. pseudomallei cells in biofilm state at concentrations above 221 μM. However, TP1 was cytotoxic to the mammalian cell lines tested. In the in silico study, molecular docking revealed that TP1 demonstrated a strong interaction to the common peptide or inhibitor binding targets for lipopolysaccharide of Escherichia coli, as well as autolysin, pneumolysin, and pneumococcal surface protein A (PspA) of Streptococcus pneumoniae. Homology modelled B. pseudomallei PspA protein (YDP) also showed a favourable binding with a strong electrostatic contribution and nine hydrogen bonds. In conclusion, TP1 demonstrated a good potential as an anti-B. pseudomallei agent. PMID:27812400

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

    PubMed

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

    2015-06-01

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

  8. Biogeography of Burkholderia pseudomallei in the Torres Strait Islands of Northern Australia.

    PubMed

    Baker, Anthony; Mayo, Mark; Owens, Leigh; Burgess, Graham; Norton, Robert; McBride, William John Hannan; Currie, Bart J; Warner, Jeffrey

    2013-08-01

    It has been hypothesized that biogeographical boundaries are a feature of Burkholderia pseudomallei ecology, and they impact the epidemiology of melioidosis on a global scale. This study examined the relatedness of B. pseudomallei sourced from islands in the Torres Strait of Northern Australia to determine if the geography of isolated island communities is a determinant of the organisms' dispersal. Environmental sampling on Badu Island in the Near Western Island cluster recovered a single clone. An additional 32 clinical isolates from the region were sourced. Isolates were characterized using multilocus sequence typing and a multiplex PCR targeting the flagellum gene cluster. Gene cluster analysis determined that 69% of the isolates from the region encoded the ancestral Burkholderia thailandensis-like flagellum and chemotaxis gene cluster, a proportion significantly lower than that reported from mainland Australia and consistent with observations of isolates from southern Papua New Guinea. A goodness-of-fit test indicated that there was geographic localization of sequence types throughout the archipelago, with the exception of Thursday Island, the economic and cultural hub of the region. Sequence types common to mainland Australia and Papua New Guinea were identified. These findings demonstrate for the first time an environmental reservoir for B. pseudomallei in the Torres Strait, and multilocus sequence typing suggests that the organism is not randomly distributed throughout this region and that seawater may provide a barrier to dispersal of the organism. Moreover, these findings support an anthropogenic dispersal hypothesis for the spread of B. pseudomallei throughout this region.

  9. Comparison of four selective media for the isolation of Burkholderia mallei and Burkholderia pseudomallei.

    PubMed

    Glass, Mindy B; Beesley, Cari A; Wilkins, Patricia P; Hoffmaster, Alex R

    2009-06-01

    Currently there are no commercially available selective media indicated for the isolation of Burkholderia mallei and Burkholderia pseudomallei. Ashdown's agar, a custom selective medium for isolation of B. pseudomallei, is well described in the literature but unavailable commercially. Three commercially available media, Burkholderia cepacia selective agar (BCSA), oxidative-fermentative-polymyxin B-bacitracin-lactose (OFPBL) agar, and Pseudomonas cepacia (PC) agar are recommended for isolation of B. cepacia from respiratory secretions of cystic fibrosis patients. We evaluated the sensitivity and selectivity of these four media using 20 B. mallei, 20 B. pseudomallei, 20 Burkholderia spp., and 15 diagnostically challenging organisms. Ashdown's agar was the most sensitive medium for the isolation of B. pseudomallei, but it was unable to support growth of B. mallei. Pseudomonas cepacia agar was highly sensitive and selective for both organisms. In non-endemic areas, we suggest the use of the commercially available PC agar for the isolation of B. mallei and B. pseudomallei.

  10. 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.

  11. 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.

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

    PubMed Central

    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

  13. Short report: Failure of Burkholderia pseudomallei to grow in an automated blood culture system.

    PubMed

    Teerawattanasook, Nittaya; Limmathurotsakul, Direk; Day, Nicholas P J; Wuthiekanun, Vanaporn

    2014-12-01

    We compared the organisms isolated from 30,210 pairs of blood culture bottles by using BacT/Alert system and the conventional system. Overall, 2,575 (8.5%) specimens were culture positive for pathogenic organisms. The sensitivity for detection of pathogenic organisms with the BACT/Alert system (85.6%, 2,203 of 2,575) was significantly higher than that with the conventional method (74.1%, 1,908 of 2,575; P < 0.0001). However, Burkholderia pseudomallei was isolated less often with the BacT/ALERT system (73.5%, 328 of 446) than with the conventional system (90.3%, 403 of 446; P < 0.0001). This finding suggests that use of the conventional culture method in conjunction with the BacT/Alert system may improve the isolation rate for B. pseudomallei in melioidosis-endemic areas.

  14. Diffusion and activity of antibiotics against Burkholderia pseudomallei biofilms.

    PubMed

    Pibalpakdee, Phannarai; Wongratanacheewin, Surasakdi; Taweechaisupapong, Suwimol; Niumsup, Pannika R

    2012-04-01

    The diffusion and activity of ceftazidime (CAZ), imipenem (IPM) and trimethoprim/sulfamethoxazole (TMP/SMX) against Burkholderia pseudomallei biofilms were comparatively tested using the high biofilm-producing strain B. pseudomallei 377 and the biofilm-defective mutant B. pseudomallei M6. Biofilms were generated by inoculation of bacteria on polycarbonate membranes placed on the surface of tryptic soy agar plates. The results showed that diffusion of TMP/SMX through B. pseudomallei biofilms was similar for both strains. However, diffusion of CAZ and IPM was significantly faster through strain M6 biofilm in comparison with strain 377 biofilm. The viabilities of strain 377 biofilm were significantly higher than those observed with strain M6 for all antibiotics challenged at 4 h, suggesting that the biofilm-forming capacity may be involved in antibiotic susceptibilities in B. pseudomallei. These results re-emphasise the importance of biofilm for antibiotic resistance in B. pseudomallei.

  15. Immunogenic recombinant Burkholderia pseudomallei MprA serine protease elicits protective immunity in mice

    PubMed Central

    Chin, Chui-Yoke; Tan, Swee-Chen; Nathan, Sheila

    2012-01-01

    Burkholderia pseudomallei is resistant to a diverse group of antimicrobials including third generation cephalosporins whilst quinolones and aminoglycosides have no reliable effect. As therapeutic options are limited, development of more effective forms of immunotherapy is vital to avoid a fatal outcome. In an earlier study, we reported on the B. pseudomallei serine MprA protease, which is relatively stable over a wide pH and temperature range and digests physiological proteins. The present study was carried out to evaluate the immunogenicity and protective efficacy of the MprA as a potential vaccine candidate. In BALB/c mice immunized with recombinant MprA protease (smBpF4), a significantly high IgG titer was detectable. Isotyping studies revealed that the smBpF4-specific antibodies produced were predominantly IgG1, proposing that immunization with smBpF4 triggered a Th2 immune response. Mice were immunized with smBpF4 and subsequently challenged with B. pseudomallei via the intraperitoneal route. Whilst control mice succumbed to the infection by day 9, smBpF4-immunized mice were protected against the lethal challenge and survived beyond 25 days post-infection. In conclusion, MprA is immunogenic in melioidosis patients whilst also eliciting a strong immune response upon bacterial challenge in mice and presents itself as a potential vaccine candidate for the treatment of melioidosis. PMID:22919676

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

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

    PubMed

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

    2016-09-01

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

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

    PubMed Central

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

    2016-01-01

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

  3. 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

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

    PubMed

    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.

  5. 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

  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. 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

  8. Colony Morphology Variation of Burkholderia pseudomallei Is Associated with Antigenic Variation and O-Polysaccharide Modification

    PubMed Central

    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.

    2015-01-01

    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

  9. 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

  10. A model of immunity to Burkholderia pseudomallei: unique responses following immunization and acute lethal infection.

    PubMed

    Ulett, Glen C; Labrooy, Justin T; Currie, Bart J; Barnes, Jodie L; Ketheesan, Natkunam

    2005-01-01

    Burkholderia pseudomallei, the etiological agent of melioidosis, causes significant mortality in endemic regions, but little is known regarding the immune mechanisms required for successful protective immunity. To establish a model of immunization that could be used to study this we screened a library of B. pseudomallei strains for immunogenicity in mice. BALB/c mice were immunized with test strains, and 2 weeks later were given a lethal challenge (LC) of virulent B. pseudomallei. Among 49 strains tested, a single strain, CL04, exhibited strong immunoprotective capacity. Interestingly, CL04 had been cultured from a patient with chronic colonization of B. pseudomallei, which is a rare phenomenon. Mice immunized with 0.1 x LD50 (5 x 10(3) CFU) of CL04 had significantly better survival and lower bacterial loads after LC compared to naïve controls. Dose-response analysis demonstrated more robust immunity after higher immunizing doses, and bacterial inactivation by gamma irradiation diminished the protective effect, indicating a requirement for viable organism for immunity. CL04-induced immunity was demonstrated both in B. pseudomallei-susceptible BALB/c and -resistant C57BL/6 mice. We investigated the gene profile of CL04-induced immunity by analyzing responses to immunization using cDNA microarray. Unique responses involving granulocyte macrophage colony stimulating factor (GM-CSF), the proapoptotic regulator Bad and cyclin-dependent kinase (CDK5) were detected in immunized mice, but these responses were absent in naïve-LC mice. Further, responses differed between mouse strains, indicating dependence on host genetic background. This model will be useful in identifying elements of the immune response required for successful adaptive immunity against B. pseudomallei.

  11. 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

  12. Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping

    PubMed Central

    U'Ren, Jana M; Schupp, James M; Pearson, Talima; Hornstra, Heidie; Friedman, Christine L Clark; Smith, Kimothy L; Daugherty, Rebecca R Leadem; Rhoton, Shane D; Leadem, Ben; Georgia, Shalamar; Cardon, Michelle; Huynh, Lynn Y; DeShazer, David; Harvey, Steven P; Robison, Richard; Gal, Daniel; Mayo, Mark J; Wagner, David; Currie, Bart J; Keim, Paul

    2007-01-01

    Background The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing ~18,000 generations. Results B. pseudomallei contains a preponderance of tandem repeat loci throughout its genome, many of which are duplicated elsewhere in the genome. The majority of these loci are composed of repeat motif lengths of 6 to 9 bp with 4 to 10 repeat units and are predominately located in intergenic regions of the genome. Across geographically diverse B. pseudomallei and B.mallei isolates, the 32 VNTR loci displayed between 7 and 28 alleles, with Nei's diversity values ranging from 0.47 and 0.94. Mutation rates for these loci are comparable (>10-5 per locus per generation) to that of the most diverse tandemly repeated regions found in other less diverse bacteria. Conclusion The frequency, location and duplicate nature of tandemly repeated regions within the B. pseudomallei genome indicate that these tandem repeat regions may play a role in generating and maintaining adaptive genomic variation. Multiple-locus VNTR analysis revealed extensive diversity within the global isolate set containing B. pseudomallei and B. mallei, and it detected genotypic differences within clonal lineages of both species that were identical using previous

  13. Within-Host Evolution of Burkholderia pseudomallei over a Twelve-Year Chronic Carriage Infection

    PubMed Central

    Price, Erin P.; Sarovich, Derek S.; Mayo, Mark; Tuanyok, Apichai; Drees, Kevin P.; Kaestli, Mirjam; Beckstrom-Sternberg, Stephen M.; Babic-Sternberg, James S.; Kidd, Timothy J.; Bell, Scott C.; Keim, Paul; Pearson, Talima; Currie, Bart J.

    2013-01-01

    ABSTRACT Burkholderia pseudomallei causes the potentially fatal disease melioidosis. It is generally accepted that B. pseudomallei is a noncommensal bacterium and that any culture-positive clinical specimen denotes disease requiring treatment. Over a 23-year study of melioidosis cases in Darwin, Australia, just one patient from 707 survivors has developed persistent asymptomatic B. pseudomallei carriage. To better understand the mechanisms behind this unique scenario, we performed whole-genome analysis of two strains isolated 139 months apart. During this period, B. pseudomallei underwent several adaptive changes. Of 23 point mutations, 78% were nonsynonymous and 43% were predicted to be deleterious to gene function, demonstrating a strong propensity for positive selection. Notably, a nonsense mutation inactivated the universal stress response sigma factor RpoS, with pleiotropic implications. The genome underwent substantial reduction, with four deletions in chromosome 2 resulting in the loss of 221 genes. The deleted loci included genes involved in secondary metabolism, environmental survival, and pathogenesis. Of 14 indels, 11 occurred in coding regions and 9 resulted in frameshift mutations that dramatically affected predicted gene products. Disproportionately, four indels affected lipopolysaccharide biosynthesis and modification. Finally, we identified a frameshift mutation in both P314 isolates within wcbR, an important component of the capsular polysaccharide I locus, suggesting virulence attenuation early in infection. Our study illustrates a unique clinical case that contrasts a high-consequence infectious agent with a long-term commensal infection and provides further insights into bacterial evolution within the human host. PMID:23860767

  14. CD4+ T cell epitopes of FliC conserved between strains of Burkholderia: implications for vaccines against melioidosis and cepacia complex in cystic fibrosis.

    PubMed

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

    2014-12-15

    Burkholderia pseudomallei is the causative agent of melioidosis characterized by pneumonia and fatal septicemia and prevalent in Southeast Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The B. pseudomallei flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed B. pseudomallei FliC peptide binding affinity to multiple HLA class II alleles and 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 B. pseudomallei and the related Burkholderia species associated with Cepacia Complex CF. B. pseudomallei 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 B. pseudomallei-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 B. pseudomallei FliC epitope also cross-reacted with orthologous FliC sequences from Burkholderia multivorans and Burkholderia 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 Ag 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 B. pseudomallei endemic regions as well as CF patients.

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

    PubMed

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

    2015-04-01

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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

    2016-03-01

    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

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

    PubMed Central

    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

    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. 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

  3. Highly sensitive direct detection and quantification of Burkholderia pseudomallei bacteria in environmental soil samples by using real-time PCR.

    PubMed

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

    2011-09-01

    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 × 10(4) genome equivalents (range, 3.65 × 10(2) to 7.85 × 10(5)) 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.

  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 Central

    Randall, Linnell B.; Dobos, Karen; Papp-Wallace, Krisztina M.; Bonomo, Robert A.

    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. 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

  10. 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

  11. Genomic characterization of Burkholderia pseudomallei isolates selected for medical countermeasures testing: comparative genomics associated with differential virulence.

    PubMed

    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.

  12. Clinical evaluation of a type III secretion system real-time PCR assay for diagnosing melioidosis.

    PubMed

    Meumann, Ella M; Novak, Ryan T; Gal, Daniel; Kaestli, Mirjam E; Mayo, Mark; Hanson, Joshua P; Spencer, Emma; Glass, Mindy B; Gee, Jay E; Wilkins, Patricia P; Currie, Bart J

    2006-08-01

    A Burkholderia pseudomallei type III secretion system real-time PCR assay was evaluated on clinical specimens in a region where melioidosis is endemic. The PCR was positive in 30/33 (91%) patients with culture-confirmed melioidosis. All six patients with melioidosis septic shock were blood PCR positive, suggesting potential for rapid diagnosis and commencement of appropriate therapy.

  13. 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

  14. 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.

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

    PubMed

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

    2016-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is among a growing number of bacterial pathogens that are increasingly antibiotic resistant. Antimicrobial peptides (AMPs) have been investigated as an alternative approach to treat microbial infections, as generally, there is a lower likelihood that a pathogen will develop resistance to AMPs. In this study, 36 candidate Caenorhabditis elegans genes that encode secreted peptides of <150 amino acids and previously shown to be overexpressed during infection by B. pseudomallei were identified from the expression profile of infected nematodes. RNA interference (RNAi)-based knockdown of 12/34 peptide-encoding genes resulted in enhanced nematode susceptibility to B. pseudomallei without affecting worm fitness. A microdilution test demonstrated that two peptides, NLP-31 and Y43C5A.3, exhibited anti-B. pseudomallei activity in a dose dependent manner on different pathogens. Time kill analysis proposed that these peptides were bacteriostatic against B. pseudomallei at concentrations up to 8× MIC90. The SYTOX green assay demonstrated that NLP-31 and Y43C5A.3 did not disrupt the B. pseudomallei membrane. Instead, gel retardation assays revealed that both peptides were able to bind to DNA and interfere with bacterial viability. In parallel, microscopic examination showed induction of cellular filamentation, a hallmark of DNA synthesis inhibition, of NLP-31 and Y43C5A.3 treated cells. In addition, the peptides also regulated the expression of inflammatory cytokines in B. pseudomallei infected macrophage cells. Collectively, these findings demonstrate the potential of NLP-31 and Y43C5A.3 as anti-B. pseudomallei peptides based on their function as immune modulators. PMID:27672387

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

    PubMed Central

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

    2016-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is among a growing number of bacterial pathogens that are increasingly antibiotic resistant. Antimicrobial peptides (AMPs) have been investigated as an alternative approach to treat microbial infections, as generally, there is a lower likelihood that a pathogen will develop resistance to AMPs. In this study, 36 candidate Caenorhabditis elegans genes that encode secreted peptides of <150 amino acids and previously shown to be overexpressed during infection by B. pseudomallei were identified from the expression profile of infected nematodes. RNA interference (RNAi)-based knockdown of 12/34 peptide-encoding genes resulted in enhanced nematode susceptibility to B. pseudomallei without affecting worm fitness. A microdilution test demonstrated that two peptides, NLP-31 and Y43C5A.3, exhibited anti-B. pseudomallei activity in a dose dependent manner on different pathogens. Time kill analysis proposed that these peptides were bacteriostatic against B. pseudomallei at concentrations up to 8× MIC90. The SYTOX green assay demonstrated that NLP-31 and Y43C5A.3 did not disrupt the B. pseudomallei membrane. Instead, gel retardation assays revealed that both peptides were able to bind to DNA and interfere with bacterial viability. In parallel, microscopic examination showed induction of cellular filamentation, a hallmark of DNA synthesis inhibition, of NLP-31 and Y43C5A.3 treated cells. In addition, the peptides also regulated the expression of inflammatory cytokines in B. pseudomallei infected macrophage cells. Collectively, these findings demonstrate the potential of NLP-31 and Y43C5A.3 as anti-B. pseudomallei peptides based on their function as immune modulators.

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

    PubMed

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

    2016-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is among a growing number of bacterial pathogens that are increasingly antibiotic resistant. Antimicrobial peptides (AMPs) have been investigated as an alternative approach to treat microbial infections, as generally, there is a lower likelihood that a pathogen will develop resistance to AMPs. In this study, 36 candidate Caenorhabditis elegans genes that encode secreted peptides of <150 amino acids and previously shown to be overexpressed during infection by B. pseudomallei were identified from the expression profile of infected nematodes. RNA interference (RNAi)-based knockdown of 12/34 peptide-encoding genes resulted in enhanced nematode susceptibility to B. pseudomallei without affecting worm fitness. A microdilution test demonstrated that two peptides, NLP-31 and Y43C5A.3, exhibited anti-B. pseudomallei activity in a dose dependent manner on different pathogens. Time kill analysis proposed that these peptides were bacteriostatic against B. pseudomallei at concentrations up to 8× MIC90. The SYTOX green assay demonstrated that NLP-31 and Y43C5A.3 did not disrupt the B. pseudomallei membrane. Instead, gel retardation assays revealed that both peptides were able to bind to DNA and interfere with bacterial viability. In parallel, microscopic examination showed induction of cellular filamentation, a hallmark of DNA synthesis inhibition, of NLP-31 and Y43C5A.3 treated cells. In addition, the peptides also regulated the expression of inflammatory cytokines in B. pseudomallei infected macrophage cells. Collectively, these findings demonstrate the potential of NLP-31 and Y43C5A.3 as anti-B. pseudomallei peptides based on their function as immune modulators.

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

    PubMed Central

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

    2016-01-01

    Burkholderia pseudomallei, the causative agent of melioidosis, is among a growing number of bacterial pathogens that are increasingly antibiotic resistant. Antimicrobial peptides (AMPs) have been investigated as an alternative approach to treat microbial infections, as generally, there is a lower likelihood that a pathogen will develop resistance to AMPs. In this study, 36 candidate Caenorhabditis elegans genes that encode secreted peptides of <150 amino acids and previously shown to be overexpressed during infection by B. pseudomallei were identified from the expression profile of infected nematodes. RNA interference (RNAi)-based knockdown of 12/34 peptide-encoding genes resulted in enhanced nematode susceptibility to B. pseudomallei without affecting worm fitness. A microdilution test demonstrated that two peptides, NLP-31 and Y43C5A.3, exhibited anti-B. pseudomallei activity in a dose dependent manner on different pathogens. Time kill analysis proposed that these peptides were bacteriostatic against B. pseudomallei at concentrations up to 8× MIC90. The SYTOX green assay demonstrated that NLP-31 and Y43C5A.3 did not disrupt the B. pseudomallei membrane. Instead, gel retardation assays revealed that both peptides were able to bind to DNA and interfere with bacterial viability. In parallel, microscopic examination showed induction of cellular filamentation, a hallmark of DNA synthesis inhibition, of NLP-31 and Y43C5A.3 treated cells. In addition, the peptides also regulated the expression of inflammatory cytokines in B. pseudomallei infected macrophage cells. Collectively, these findings demonstrate the potential of NLP-31 and Y43C5A.3 as anti-B. pseudomallei peptides based on their function as immune modulators. PMID:27672387

  19. 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

  20. 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

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

    PubMed

    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

  2. The Effect of Environmental Conditions on Biofilm Formation of Burkholderia pseudomallei Clinical Isolates

    PubMed Central

    Ramli, Nur Siti K.; Eng Guan, Chua; Nathan, Sheila; Vadivelu, Jamuna

    2012-01-01

    Burkholderia pseudomallei, a Gram-negative saprophytic bacterium, is the causative agent of the potentially fatal melioidosis disease in humans. In this study, environmental parameters including temperature, nutrient content, pH and the presence of glucose were shown to play a role in in vitro biofilm formation by 28 B. pseudomallei clinical isolates, including four isolates with large colony variants (LCVs) and small colony variants (SCVs) morphotypes. Enhanced biofilm formation was observed when the isolates were tested in LB medium, at 30°C, at pH 7.2, and in the presence of as little as 2 mM glucose respectively. It was also shown that all SVCs displayed significantly greater capacity to form biofilms than the corresponding LCVs when cultured in LB at 37°C. In addition, octanoyl-homoserine lactone (C8-HSL), a quorum sensing molecule, was identified by mass spectrometry analysis in bacterial isolates referred to as LCV CTH, LCV VIT, SCV TOM, SCV CTH, 1 and 3, and the presence of other AHL's with higher masses; decanoyl-homoserine lactone (C10-HSL) and dodecanoyl-homoserine lactone (C12-HSL) were also found in all tested strain in this study. Last but not least, we had successfully acquired two Bacillus sp. soil isolates, termed KW and SA respectively, which possessed strong AHLs degradation activity. Biofilm formation of B. pseudomallei isolates was significantly decreased after treated with culture supernatants of KW and SA strains, demonstrating that AHLs may play a role in B. pseudomallei biofilm formation. PMID:22970167

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

    PubMed Central

    2013-01-01

    Background Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance. Methods Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated. Results A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction. Conclusions The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification. PMID:23409683

  4. A Burkholderia pseudomallei Colony Variant Necessary for Gastric Colonization

    PubMed Central

    Austin, C. R.; Goodyear, A. W.; Bartek, I. L.; Stewart, A.; Sutherland, M. D.; Silva, E. B.; Zweifel, A.; Vitko, N. P.; Tuanyok, A.; Highnam, G.; Mittelman, D.; Keim, P.; Schweizer, H. P.; Vázquez-Torres, A.; Dow, S. W. C.

    2015-01-01

    ABSTRACT  Diverse colony morphologies are a hallmark of Burkholderia pseudomallei recovered from infected patients. We observed that stresses that inhibit aerobic respiration shifted populations of B. pseudomallei from the canonical white colony morphotype toward two distinct, reversible, yet relatively stable yellow colony variants (YA and YB). As accumulating evidence supports the importance of B. pseudomallei enteric infection and gastric colonization, we tested the response of yellow variants to hypoxia, acidity, and stomach colonization. Yellow variants exhibited a competitive advantage under hypoxic and acidic conditions and alkalized culture media. The YB variant, although highly attenuated in acute virulence, was the only form capable of colonization and persistence in the murine stomach. The accumulation of extracellular DNA (eDNA) was a characteristic of YB as observed by 4′,6-diamidino-2-phenylindole (DAPI) staining of gastric tissues, as well as in an in vitro stomach model where large amounts of eDNA were produced without cell lysis. Transposon mutagenesis identified a transcriptional regulator (BPSL1887, designated YelR) that when overexpressed produced the yellow phenotype. Deletion of yelR blocked a shift from white to the yellow forms. These data demonstrate that YB is a unique B. pseudomallei pathovariant controlled by YelR that is specifically adapted to the harsh gastric environment and necessary for persistent stomach colonization. PMID:25650400

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

    PubMed

    Prasertsincharoen, Noppadol; Constantinoiu, Constantin; Gardiner, Christopher; Warner, Jeffrey; Elliman, Jennifer

    2015-07-01

    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.

  6. 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

  7. Characterization of BPSS1521 (bprD), a Regulator of Burkholderia pseudomallei Virulence Gene Expression in the Mouse Model

    PubMed Central

    Wongsurawat, Thidathip; Taweechaisupapong, Suwimol; Karoonutaisiri, Nitsara; Talaat, Adel M.; Wongratanacheewin, Surasakdi; Ernst, Robert K.; Sermswan, Rasana W.

    2014-01-01

    The Gram-negative saprophytic bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe infectious disease of both humans and animals. Severity of the disease is thought to be dependent on both the health status of the host, including diabetes mellitus and kidney disease, and bacterial-derived factors. To identify the bacterial factors important during an acute infection, gene expression profiles in the spleen, lung, and liver of BALB/c (Th2 prototype) and C57BL/6 mice (Th1 prototype) were determined using DNA microarrays. This analysis identified BPSS1521 (bprD), a predicted transcriptional regulator located in the type III secretion system (T3SS-3) operon, to be up regulated, specifically in C57BL/6 mice. BALB/c mice infected with a bprD mutant showed a shorter time to death and increased inflammation, as determined by histopathological analysis and enumeration of bacteria in the spleen. Elevated numbers of multinucleated giant cells (MNGCs), which is the hallmark of melioidosis, were detected in both the wild-type and the bprD mutants; a similar elevation occurs in melioidosis patients. One striking observation was the increased expression of BPSS1520 (bprC), located downstream of bprD, in the bprD mutant. BprC is a regulator of T6SS-1 that is required for the virulence of B. pseudomallei in murine infection models. Deletion of bprD led to the overexpression of bprC and a decreased time to death. bprD expression was elevated in C57BL/6 —as compared to BALB/c—mice, suggesting a role for BprD in the natural resistance of C57BL/6 mice to B. pseudomallei. Ultimately, this analysis using mice with different immune backgrounds may enhance our understanding of the outcomes of infection in a variety of models. PMID:25111708

  8. Glibenclamide impairs responses of neutrophils against Burkholderia pseudomallei by reduction of intracellular glutathione

    PubMed Central

    Kewcharoenwong, Chidchamai; Rinchai, Darawan; Nithichanon, Arnone; Bancroft, Gregory J.; Ato, Manabu; Lertmemongkolchai, Ganjana

    2016-01-01

    The major risk factor for melioidosis, an infectious disease caused by B. pseudomallei, is diabetes mellitus. More than half of diabetic melioidosis patients in Thailand were prescribed glibenclamide. Recent evidence demonstrates that glibenclamide reduces pro-inflammatory cytokine production by polymorphonuclear neutrophils (PMNs) of diabetic individuals in response to this bacterial infection. However, the mechanisms by which glibenclamide affects cytokine production are unknown. We found that PMNs from glibenclamide-treated diabetic individuals infected with live B. pseudomallei in vitro showed lower free glutathione (GSH) levels compared with those of healthy individuals. Glibenclamide decreased GSH levels and glutathione peroxidase (GPx) of PMNs after exposed to live B. pseudomallei. Moreover, glibenclamide reduced cytokine production and migration capacity of infected PMNs, whereas GSH could restore these functions. Taken together, our data show a link between the effect of glibenclamide on GSH and PMN functions in response to B. pseudomallei that may contribute to the susceptibility of diabetic individuals to B. pseudomallei infection. PMID:27713554

  9. Characterization and analysis of the Burkholderia pseudomallei BsaN virulence regulon

    PubMed Central

    2014-01-01

    Background Burkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis. A conserved type III secretion system (T3SS3) and type VI secretion system (T6SS1) are critical for intracellular survival and growth. The T3SS3 and T6SS1 genes are coordinately and hierarchically regulated by a TetR-type regulator, BspR. A central transcriptional regulator of the BspR regulatory cascade, BsaN, activates a subset of T3SS3 and T6SS1 loci. Results To elucidate the scope of the BsaN regulon, we used RNAseq analysis to compare the transcriptomes of wild-type B. pseudomallei KHW and a bsaN deletion mutant. The 60 genes positively-regulated by BsaN include those that we had previously identified in addition to a polyketide biosynthesis locus and genes involved in amino acid biosynthesis. BsaN was also found to repress the transcription of 51 genes including flagellar motility loci and those encoding components of the T3SS3 apparatus. Using a promoter-lacZ fusion assay in E. coli, we show that BsaN together with the chaperone BicA directly control the expression of the T3SS3 translocon, effector and associated regulatory genes that are organized into at least five operons (BPSS1516-BPSS1552). Using a mutagenesis approach, a consensus regulatory motif in the promoter regions of BsaN-regulated genes was shown to be essential for transcriptional activation. Conclusions BsaN/BicA functions as a central regulator of key virulence clusters in B. pseudomallei within a more extensive network of genetic regulation. We propose that BsaN/BicA controls a gene expression program that facilitates the adaption and intracellular survival of the pathogen within eukaryotic hosts. PMID:25085508

  10. 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

  11. Cross-Species Comparison of the Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei Quorum-Sensing Regulons

    PubMed Central

    Majerczyk, Charlotte D.; Brittnacher, Mitchell J.; Jacobs, Michael A.; Armour, Christopher D.; Radey, Matthew C.; Bunt, Richard; Hayden, Hillary S.; Bydalek, Ryland

    2014-01-01

    Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei (the Bptm group) are close relatives with very different lifestyles: B. pseudomallei is an opportunistic pathogen, B. thailandensis is a nonpathogenic saprophyte, and B. mallei is a host-restricted pathogen. The acyl-homoserine lactone quorum-sensing (QS) systems of these three species show a high level of conservation. We used transcriptome sequencing (RNA-seq) to define the quorum-sensing regulon in each species, and we performed a cross-species analysis of the QS-controlled orthologs. Our analysis revealed a core set of QS-regulated genes in all three species, as well as QS-controlled factors shared by only two species or unique to a given species. This global survey of the QS regulons of B. pseudomallei, B. thailandensis, and B. mallei serves as a platform for predicting which QS-controlled processes might be important in different bacterial niches and contribute to the pathogenesis of B. pseudomallei and B. mallei. PMID:25182491

  12. Cross-species comparison of the Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei quorum-sensing regulons.

    PubMed

    Majerczyk, Charlotte D; Brittnacher, Mitchell J; Jacobs, Michael A; Armour, Christopher D; Radey, Matthew C; Bunt, Richard; Hayden, Hillary S; Bydalek, Ryland; Greenberg, E Peter

    2014-11-01

    Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei (the Bptm group) are close relatives with very different lifestyles: B. pseudomallei is an opportunistic pathogen, B. thailandensis is a nonpathogenic saprophyte, and B. mallei is a host-restricted pathogen. The acyl-homoserine lactone quorum-sensing (QS) systems of these three species show a high level of conservation. We used transcriptome sequencing (RNA-seq) to define the quorum-sensing regulon in each species, and we performed a cross-species analysis of the QS-controlled orthologs. Our analysis revealed a core set of QS-regulated genes in all three species, as well as QS-controlled factors shared by only two species or unique to a given species. This global survey of the QS regulons of B. pseudomallei, B. thailandensis, and B. mallei serves as a platform for predicting which QS-controlled processes might be important in different bacterial niches and contribute to the pathogenesis of B. pseudomallei and B. mallei.

  13. 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

  14. Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM

    PubMed Central

    Burtnick, Mary N.; Brett, Paul J.

    2014-01-01

    Burkholderia pseudomallei, the etiologic agent of melioidosis, is an opportunistic pathogen that harbors a wide array of secretion systems, including a type II secretion system (T2SS), three type III secretion systems (T3SS), and six type VI secretion systems (T6SS). The proteins exported by these systems provide B. pseudomallei with a growth advantage in vitro and in vivo, but relatively little is known about the full repertoire of exoproducts associated with each system. In this study, we constructed deletion mutations in gspD and gspE, T2SS genes encoding an outer membrane secretin and a cytoplasmic ATPase, respectively. The secretion profiles of B. pseudomallei MSHR668 and its T2SS mutants were noticeably different when analyzed by SDS-PAGE. We utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins present in the supernatants of B. pseudomallei MSHR668 and B. pseudomallei ΔgspD grown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent or substantially reduced in the supernatants of ΔgspD strains. Many of these proteins were putative hydrolytic enzymes, including 12 proteases, two phospholipases, and a chitinase. Biochemical assays validated the LC-MS/MS results and demonstrated that the export of protease, phospholipase C, and chitinase activities is T2SS dependent. Previous studies had failed to identify the mechanism of secretion of TssM, a deubiquitinase that plays an integral role in regulating the innate immune response. Here we present evidence that TssM harbors an atypical signal sequence and that its secretion is mediated by the T2SS. This study provides the first in-depth characterization of the B. pseudomallei T2SS secretome. PMID:24866793

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

    PubMed

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

    2015-06-01

    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.

  16. 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

  17. Oropharyngeal Aspiration of Burkholderia mallei and Burkholderia pseudomallei in BALB/c Mice

    PubMed Central

    Schully, Kevin L.; Bell, Matthew G.; Ward, Jerrold M.; Keane-Myers, Andrea M.

    2014-01-01

    Burkholderia mallei and Burkholderia pseudomallei are potentially lethal pathogens categorized as biothreat agents due, in part, to their ability to be disseminated via aerosol. There are no protective vaccines against these pathogens and treatment options are limited and cumbersome. Since disease severity is greatest when these agents are inhaled, efforts to develop pre- or post-exposure prophylaxis focus largely on inhalation models of infection. Here, we demonstrate a non-invasive and technically simple method for affecting the inhalational challenge of BALB/c mice with B. pseudomallei and B. mallei. In this model, two investigators utilized common laboratory tools such as forceps and a micropipette to conduct and characterize an effective and reproducible inhalational challenge of BALB/c mice with B. mallei and B. pseudomallei. Challenge by oropharyngeal aspiration resulted in acute disease. Additionally, 50% endpoints for B. pseudomallei K96243 and B. mallei ATCC 23344 were nearly identical to published aerosol challenge methods. Furthermore, the pathogens disseminated to all major organs typically targeted by these agents where they proliferated. The pro-inflammatory cytokine production in the proximal and peripheral fluids demonstrated a rapid and robust immune response comparable to previously described murine and human studies. These observations demonstrate that OA is a viable alternative to aerosol exposure. PMID:25503969

  18. Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis.

    PubMed

    Kanthawong, Sakawrat; Puknun, Aekkalak; Bolscher, Jan G M; Nazmi, Kamran; van Marle, Jan; de Soet, Johannes J; Veerman, Enno C I; Wongratanacheewin, Surasakdi; Taweechaisupapong, Suwimol

    2014-10-01

    LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265-284 and lactoferricin17-30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.

  19. Melioidosis from Contaminated Bore Water and Successful UV Sterilization

    PubMed Central

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

    2013-01-01

    Two cases of melioidosis at a residence in rural northern Australia were linked to the unchlorinated domestic bore (automated well) water supply, which was found to have a high concentration of Burkholderia pseudomallei. Using multilocus sequence typing, clinical B. pseudomallei isolates from both cases were identical to an isolate from the bore water supply. A simple UV sterilizer reduced B. pseudomallei from the domestic water supply to undetectable levels. We have shown that UV treatment is highly effective for remediation of water contaminated with B. pseudomallei and recommend its consideration in households where individuals may be at heightened risk of contracting melioidosis. PMID:23751401

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

    PubMed

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

    2013-08-01

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

  1. Septicaemic Melioidosis: Case Report from a Non-Endemic Area

    PubMed Central

    Khedar, Raghuvir Singh; Joad, Shabbar HK; Gupta, Rajeev

    2014-01-01

    Melioidosis is a clinically diverse disease caused by the facultative intracellular Gram-negative bacterium, Burkholderia pseudomallei. In recent times melioidosis has been increasingly reported in India especially from the southern and coastal states. We report a fatal case of septicaemic melioidosis from the state of Rajasthan with a view to increase awareness about the existence of this disease in an area yet unrecognized. PMID:25653950

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

    PubMed

    Niyompanich, Suthamat; Srisanga, Kitima; 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

  3. 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.

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

    PubMed

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

    2002-12-01

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

  5. 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.

  6. 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

  7. Analyses of the Distribution Patterns of Burkholderia pseudomallei and Associated Phages in Soil Samples in Thailand Suggest That Phage Presence Reduces the Frequency of Bacterial Isolation

    PubMed Central

    Withatanung, Patoo; Chantratita, Narisara; Muangsombut, Veerachat; Saiprom, Natnaree; Lertmemongkolchai, Ganjana; Klumpp, Jochen; Clokie, Martha R. J.; Galyov, Edouard E.

    2016-01-01

    Background Burkholderia pseudomallei is a soil saprophytic bacterium that causes melioidosis. The infection occurs through cutaneous inoculation, inhalation or ingestion. Bacteriophages (phages) in the same ecosystem may significantly impact the biology of this bacterium in the environment, and in their culturability in the laboratory. Methods/Principal Findings The soil samples were analysed for the presence of bacteria using culture methods, and for phages using plaque assays on B. pseudomallei strain 1106a lawns. Of the 86 soil samples collected from northeastern Thailand, B. pseudomallei was cultured from 23 (26.7%) samples; no phage capable of infecting B. pseudomallei was detected in these samples. In contrast, phages capable of infecting B. pseudomallei, but no bacteria, were present in 10 (11.6%) samples. B. pseudomallei and their phages were co-isolated from only 3 (3.5%) of soil samples. Since phage capable of infecting B. pseudomallei could not have appeared in the samples without the prior presence of bacteria, or exposure to bacteria nearby, our data suggest that all phage-positive/bacteria-negative samples have had B. pseudomallei in or in a close proximity to them. Taken together, these findings indicate that the presence of phages may influence the success of B. pseudomallei isolation. Transmission electron microscopy revealed that the isolated phages are podoviruses. The temperate phages residing in soil-isolated strains of B. pseudomallei that were resistant to the dominant soil borne phages could be induced by mitomycin C. These induced-temperate phages were closely related, but not identical, to the more dominant soil-isolated phage type. Conclusion/Significance The presence of podoviruses capable of infecting B. pseudomallei may affect the success of the pathogen isolation from the soil. The currently used culture-based methods of B. pseudomallei isolation appear to under-estimate the bacterial abundance. The detection of phage capable of

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

    PubMed

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

    2015-01-01

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

  9. Clinical, Environmental, and Serologic Surveillance Studies of Melioidosis in Gabon, 2012–2013

    PubMed Central

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

    2015-01-01

    Burkholderia pseudomallei, an environmental gram-negative bacillus, is the causative agent of melioidosis and a bio-threat agent. Reports of B. pseudomallei isolation from soil and animals in East and West Africa suggest that melioidosis might be more widely distributed than previously thought. Because it has been found in equatorial areas with tropical climates, we hypothesized that B. pseudomallei could exist in Gabon. During 2012–2013, we conducted a seroprevalance study in which we set up microbiology facilities at a large clinical referral center and prospectively screened all febrile patients by conducting blood cultures and testing for B. pseudomallei and related species; we also determined whether B. pseudomallei could be isolated from soil. We discovered a novel B. pseudomallei sequence type that caused lethal septic shock and identified B. pseudomallei and B. thailandensis in the environment. Our data suggest that melioidosis is emerging in Central Africa but is unrecognized because of the lack of diagnostic microbiology facilities. PMID:25530077

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

    PubMed

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

    2015-01-01

    Burkholderia pseudomallei, an environmental gram-negative bacillus, is the causative agent of melioidosis and a bio-threat agent. Reports of B. pseudomallei isolation from soil and animals in East and West Africa suggest that melioidosis might be more widely distributed than previously thought. Because it has been found in equatorial areas with tropical climates, we hypothesized that B. pseudomallei could exist in Gabon. During 2012-2013, we conducted a seroprevalance study in which we set up microbiology facilities at a large clinical referral center and prospectively screened all febrile patients by conducting blood cultures and testing for B. pseudomallei and related species; we also determined whether B. pseudomallei could be isolated from soil. We discovered a novel B. pseudomallei sequence type that caused lethal septic shock and identified B. pseudomallei and B. thailandensis in the environment. Our data suggest that melioidosis is emerging in Central Africa but is unrecognized because of the lack of diagnostic microbiology facilities. PMID:25530077

  11. 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

  12. 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

  13. 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.

  14. Melioidosis as a consequence of sporting activity.

    PubMed

    Hill, Audrey A; Mayo, Mark; Kaestli, Mirjam; Price, Erin P; Richardson, Leisha J; Godoy, Daniel; Spratt, Brian G; Currie, Bart J

    2013-08-01

    In the tropical city of Darwin, Northern Territory, Australia, dry season soil sampling cultured Burkholderia pseudomallei from 7 (70%) of 10 sports fields. However, during the 23 years of the Darwin Prospective Melioidosis Study, only 5 (0.6%) of 785 melioidosis cases have been attributed to infection from sports fields. In one soccer player with cutaneous melioidosis, B. pseudomallei cultured from the player was identical by multilocus sequence typing and multilocus variable-number tandem repeat analysis with an isolate recovered from soil at the location on the sports field where he was injured. Melioidosis is uncommon in otherwise healthy sports persons in melioidosis-endemic regions but still needs consideration in persons with abrasion injuries that involve contact with soil.

  15. 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

  16. 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.

  17. 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

  18. [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

  19. Melioidosis of Chest Wall Masquerading as a Tubercular Cold Abscess

    PubMed Central

    Vishnu Prasad, Nelamangala Ramakrishnaiah; Balasubramaniam, Gopal; Karthikeyan, Vilvapathy Senguttuvan; Ramesh, Chakravarti Kapil; Srinivasan, Krishnamachari

    2012-01-01

    Melioidosis caused by Burkholderia pseudomallei, an important human pathogen in the tropical regions causes protean and multisystem clinical manifestations. A 50-year-old man on treatment for pulmonary tuberculosis developed a chest wall abscess. With a suspicion of tuberculous cold abscess, pus culture was done and it revealed Burkholderia pseudomallei. He was treated with 10 days of ceftazidime and incision and drainage was done. Wound healed well and he has now completed three months of oral cotrimoxazole eradication therapy and is on follow-up without recurrence. We report this case for the unusual presentation of melioidosis and the diagnostic challenge posed due to clinical similarity with tuberculosis. PMID:23741590

  20. Neonatal melioidosis: a case report from India.

    PubMed

    Noyal, M J C; Harish, B N; Bhat, V; Parija, S C

    2009-01-01

    Melioidosis, caused by Burkholderia pseudomallei , is an infectious disease of major public health importance in Southeast Asia and Australia. We report, for the first time from the Indian subcontinent, a case of melioidosis in a neonate, its clinical presentation, microbiological diagnosis, possible mode of transmission and outcome. A pre-term female baby developed respiratory distress soon after birth. The child was febrile, had tachypnea, grunting, normal heart rate with a low pulse volume and poor peripheral perfusion. Chest X-ray revealed right-sided bronchopneumonia. B. pseudomallei was isolated from the blood culture of the neonate collected aseptically. The neonate was successfully treated with meropenem. PMID:19584512

  1. Structural and immunological characterization of Burkholderia pseudomallei O-polysaccharide-flagellin protein conjugates.

    PubMed Central

    Brett, P J; Woods, D E

    1996-01-01

    The O-polysaccharide moiety of Burkholderia pseudomallei 319a lipopolysaccharide was covalently linked to flagellin protein isolated from the same strain. A glycoconjugate incorporating adipic acid dihydrazide as a spacer molecule elicited high-titer immunoglobulin G responses to both the protein and carbohydrate components of the construct. This immunoglobulin G was capable of protecting diabetic rats from challenge with a heterologous B. pseudomallei strain. PMID:8698517

  2. Septicemic melioidosis in a transplant recipient causing graft dysfunction

    PubMed Central

    Sathiavageesan, S.

    2016-01-01

    Melioidosis, an infectious disease caused by Burkholderia pseudomallei, is endemic in the Northern Australia and South-East Asia. It is an emerging disease in the Indian subcontinent. Melioidosis tends to run a potentially lethal course in immunocompromised individuals and data in renal transplantation are scarce. The clinical presentation of melioidosis is diverse, mimicking several other infectious diseases. Diagnosis could be delayed in transplant recipients. Choice and duration of antimicrobial therapy, management of immunosuppression, and patient and graft outcomes are other issues to be addressed. We report septicemic melioidosis with pulmonary involvement in a 32-year old renal transplant recipient that caused acute allograft dysfunction.

  3. [Melioidosis: an emerging tropical disease].

    PubMed

    Valade, E; Thibault, F M; Biot, F V; Vidal, D R

    2009-10-01

    Melioidosis is an infection affecting both human and animal health. The causative agent is Burkholderia pseudomallei, a Gram-negative soil bacterium. Melioidosis is endemic in tropical areas of Southeast Asia and Northern Australia, and sporadic in many other countries. Clinical presentation is variable ranging from acute septicemia, isolated pulmonary infection, or chronic granulomatous lesions to asymptomatic forms with positive serology. There is no vaccine and treatment is difficult because B. pseudomallei is resistant to a wide range of antibiotics. Relapses are common. B. pseudomallei is listed as a biological risk class 3 and considered as a potential bioterrorism agent due to its high virulence by inhalation, to the difficulty of treatment, and to the lack of vaccine. PMID:20025169

  4. 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

  5. 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

  6. 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

  7. Eukaryotic pathways targeted by the type III secretion system effector protein, BipC, involved in the intracellular lifecycle of Burkholderia pseudomallei

    PubMed Central

    Kang, Wen-Tyng; Vellasamy, Kumutha Malar; Vadivelu, Jamuna

    2016-01-01

    Burkholderia pseudomallei, the etiological agent for melioidosis, is known to secrete a type III secretion system (TTSS) protein into the host’s internal milieu. One of the TTSS effector protein, BipC, has been shown to play an important role in the B. pseudomallei pathogenesis. To identify the host response profile that was directly or indirectly regulated by this protein, genome-wide transcriptome approach was used to examine the gene expression profiles of infected mice. The transcriptome analysis of the liver and spleen revealed that a total of approximately 1,000 genes were transcriptionally affected by BipC. Genes involved in bacterial invasion, regulation of actin cytoskeleton, and MAPK signalling pathway were over-expressed and may be specifically regulated by BipC in vivo. These results suggest that BipC mainly targets pathways related to the cellular processes which could modulate the cellular trafficking processes. The host transcriptional response exhibited remarkable differences with and without the presence of the BipC protein. Overall, the detailed picture of this study provides new insights that BipC may have evolved to efficiently manipulate host-cell pathways which is crucial in the intracellular lifecycle of B. pseudomallei. PMID:27634329

  8. Eukaryotic pathways targeted by the type III secretion system effector protein, BipC, involved in the intracellular lifecycle of Burkholderia pseudomallei.

    PubMed

    Kang, Wen-Tyng; Vellasamy, Kumutha Malar; Vadivelu, Jamuna

    2016-01-01

    Burkholderia pseudomallei, the etiological agent for melioidosis, is known to secrete a type III secretion system (TTSS) protein into the host's internal milieu. One of the TTSS effector protein, BipC, has been shown to play an important role in the B. pseudomallei pathogenesis. To identify the host response profile that was directly or indirectly regulated by this protein, genome-wide transcriptome approach was used to examine the gene expression profiles of infected mice. The transcriptome analysis of the liver and spleen revealed that a total of approximately 1,000 genes were transcriptionally affected by BipC. Genes involved in bacterial invasion, regulation of actin cytoskeleton, and MAPK signalling pathway were over-expressed and may be specifically regulated by BipC in vivo. These results suggest that BipC mainly targets pathways related to the cellular processes which could modulate the cellular trafficking processes. The host transcriptional response exhibited remarkable differences with and without the presence of the BipC protein. Overall, the detailed picture of this study provides new insights that BipC may have evolved to efficiently manipulate host-cell pathways which is crucial in the intracellular lifecycle of B. pseudomallei. PMID:27634329

  9. 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

  10. 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.

  11. 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

  12. C-reactive protein in the diagnosis of melioidosis.

    PubMed

    Cheng, Allen C; Obrien, Matthew; Jacups, Susan P; Anstey, Nicholas M; Currie, Bart J

    2004-05-01

    Previous work suggested that C-reactive protein (CRP) may be a useful test in the diagnosis of melioidosis, the infection caused by Burkholderia pseudomallei. We reviewed patients with culture-confirmed melioidosis to define the role of this inflammatory marker in the diagnosis of melioidosis. In 175 patients, we found that the admission CRP level may be normal or only mildly elevated, including patients with severe sepsis, fatal cases, and in relapsed melioidosis. In a multivariate analysis, sepsis and bacteremia were more strongly associated with mortality than CRP. Admission levels of CRP are not a sensitive marker for the presence of melioidosis and a normal level cannot be used to exclude acute, chronic, or relapsed melioidosis in febrile patients in or from endemic regions.

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

    PubMed

    Marques, Maria Angela de Mello; Zimmermann, Pia; Messelhäußer, Ute; Sing, Andreas

    2012-12-01

    Six commercially available DNA extraction kits, as well as thermal lysis and proteinase K DNA extraction were evaluated regarding bacterial inactivation, DNA yield and purity, and their use in a Burkholderia pseudomallei real-time PCR. While all methods successfully inactivated the bacteria, by measuring DNA purity and the level of detection by real-time PCR, the proteinase K method was the most sensitive.

  14. Melioidosis risk in a tropical industrial environment.

    PubMed

    Inglis, Timothy J J; Levy, Avram; Merritt, Adam J; Hodge, Meredith; McDonald, Robert; Woods, Donald E

    2009-01-01

    An investigation into the risk of occupationally acquired melioidosis at a mine site in northern Australia found that 45 (13%) of 345 staff had serologic evidence of exposure and 14 (4%) had recent exposure to Burkholderia pseudomallei or closely related bacteria. There was only one culture-confirmed case of melioidosis in mine staff during the study period. The lack of overt infection directly attributable to work activities despite detectable B. pseudomallei on site, the absence of an association between positive serology and occupational activity on site, or duration of employment in the mining industry stand against a significant occupationally acquired infection risk on this industrial site. Workplace exposure to a dust-generating tropical environment in the melioidosis-endemic north of Australia did not appear to pose a measurable increase in infection risk. The effect of long-term climatic trends on this potential biologic threat requires further study.

  15. Physicochemical Properties Influencing Presence of Burkholderia pseudomallei in Soil from Small Ruminant Farms in Peninsular Malaysia.

    PubMed

    Musa, Hassan Ismail; Hassan, Latiffah; Shamsuddin, Zulkifli Hj; Panchadcharam, Chandrawathani; Zakaria, Zunita; Abdul Aziz, Saleha

    2016-01-01

    Soil is considered to be a major reservoir of Burkholderia pseudomallei in the environment. This paper investigates soil physicochemical properties that may influence presence of B. pseudomallei in soil samples from small ruminant farms in Peninsular Malaysia. Soil samples were collected from the farms and cultured for B. pseudomallei. The texture, organic matter and water contents, pH, elemental contents, cation exchange capacities, carbon, sulfur and nitrogen contents were determined. Analysis of soil samples that were positive and negative for B. pseudomallei using multivariable logistic regression found that the odds of bacterial isolation from soil was significantly higher for samples with higher contents of iron (OR = 1.01, 95%CI = 1.00-1.02, p = 0.03), water (OR = 1.28, 95%CI = 1.05-1.55, p = 0.01) and clay (OR = 1.54, 95%CI = 1.15-2.06, p = 0.004) compared to the odds of isolation in samples with lower contents of the above variables. These three factors may have favored the survival of B. pseudomallei because iron regulates expression of respiratory enzymes, while water is essential for soil ecology and agent's biological processes and clay retains water and nutrients. PMID:27635652

  16. Physicochemical Properties Influencing Presence of Burkholderia pseudomallei in Soil from Small Ruminant Farms in Peninsular Malaysia

    PubMed Central

    Panchadcharam, Chandrawathani; Zakaria, Zunita; Abdul Aziz, Saleha

    2016-01-01

    Soil is considered to be a major reservoir of Burkholderia pseudomallei in the environment. This paper investigates soil physicochemical properties that may influence presence of B. pseudomallei in soil samples from small ruminant farms in Peninsular Malaysia. Soil samples were collected from the farms and cultured for B. pseudomallei. The texture, organic matter and water contents, pH, elemental contents, cation exchange capacities, carbon, sulfur and nitrogen contents were determined. Analysis of soil samples that were positive and negative for B. pseudomallei using multivariable logistic regression found that the odds of bacterial isolation from soil was significantly higher for samples with higher contents of iron (OR = 1.01, 95%CI = 1.00–1.02, p = 0.03), water (OR = 1.28, 95%CI = 1.05–1.55, p = 0.01) and clay (OR = 1.54, 95%CI = 1.15–2.06, p = 0.004) compared to the odds of isolation in samples with lower contents of the above variables. These three factors may have favored the survival of B. pseudomallei because iron regulates expression of respiratory enzymes, while water is essential for soil ecology and agent’s biological processes and clay retains water and nutrients. PMID:27635652

  17. Isolation of Polymyxin B-Susceptible Mutants of Burkholderia pseudomallei and Molecular Characterization of Genetic Loci Involved in Polymyxin B Resistance

    PubMed Central

    Burtnick, Mary N.; Woods, Donald E.

    1999-01-01

    Burkholderia pseudomallei is a gram-negative bacterium that causes the disease known as melioidosis. This pathogen is endemic to Southeast Asia and northern Australia and is particularly problematic in northeastern Thailand. It has been previously reported that B. pseudomallei is resistant to the killing action of cationic antimicrobial peptides, including human neutrophil peptide, protamine sulfate, poly-l-lysine, magainins, and polymyxins. Recently, we have also found that the virulent clinical isolate B. pseudomallei 1026b is capable of replicating in media containing polymyxin B at concentrations of >100 mg/ml. In order to identify genetic loci that are associated with this particular resistance phenotype, we employed a Tn5-OT182 mutagenesis system in coordination with a replica plating screen to isolate polymyxin B-susceptible mutants. Of the 17,000 Tn5-OT182 mutants screened via this approach, five polymyxin B-susceptible mutants were obtained. Three of these mutants harbored Tn5-OT182 insertions within a genetic locus demonstrating strong homology to the lytB gene present in other gram-negative bacteria. Of the remaining two mutants, one contained a transposon insertion in a locus involved in lipopolysaccharide core biosynthesis (waaF), while the other contained an insertion in an open reading frame homologous to UDP-glucose dehydrogenase genes. Isogenic mutants were also constructed via allelic exchange and used in complementation analysis studies to further characterize the relative importance of each of the various genetic loci with respect to the polymyxin B resistance phenotype exhibited by B. pseudomallei 1026b. PMID:10543742

  18. 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

  19. 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

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

    PubMed

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

    2016-03-11

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

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

    PubMed

    Antonov, V A; Iliukhin, V I

    2005-01-01

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

  2. Structure-based design of a B cell antigen from B. pseudomallei.

    PubMed

    Gaudesi, Davide; Peri, Claudio; Quilici, Giacomo; Gori, Alessandro; Ferrer-Navarro, Mario; Conchillo-Solé, Oscar; Thomas, Rachael; Nithichanon, Arnone; Lertmemongkolchai, Ganjana; Titball, Richard; Daura, Xavier; Colombo, Giorgio; Musco, Giovanna

    2015-03-20

    Burkholderia pseudomallei is the etiological agent of melioidosis, a severe endemic disease in South-East Asia, causing septicemia and organ failure with high mortality rates. Current treatments and diagnostic approaches are largely ineffective. The development of new diagnostic tools and vaccines toward effective therapeutic opportunities against B. pseudomallei is therefore an urgent priority. In the framework of a multidisciplinary project tackling melioidosis through reverse and structural vaccinology, BPSL1050 was identified as a candidate for immunodiagnostic and vaccine development based on its reactivity against the sera of melioidosis patients. We determined its NMR solution structure and dynamics, and by novel computational methods we predicted immunogenic epitopes that once synthesized were able to elicit the production of antibodies inducing the agglutination of the bacterium and recognizing both BPSL1050 and B. pseudomallei crude extracts. Overall, these results hold promise for novel chemical biology approaches in the discovery of new diagnostic and prophylactic tools against melioidosis.

  3. Endemic Melioidosis in Residents of Desert Region after Atypically Intense Rainfall in Central Australia, 2011

    PubMed Central

    Yip, Teem-Wing; Hewagama, Saliya; Mayo, Mark; Price, Erin P.; Sarovich, Derek S.; Bastian, Ivan; Baird, Robert W.; Spratt, Brian G.

    2015-01-01

    After heavy rains and flooding during early 2011 in the normally arid interior of Australia, melioidosis was diagnosed in 6 persons over a 4-month period. Although the precise global distribution of the causal bacterium Burkholderia pseudomallei remains to be determined, this organism can clearly survive in harsh and even desert environments outside the wet tropics. PMID:25988301

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

    PubMed

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

    2012-01-01

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

  5. Mycotic aneurysm caused by Burkholderia pseudomallei in a previously healthy returning traveller

    PubMed Central

    Bodilsen, Jacob; Vammen, Sten; Fuursted, Kurt; Hjort, Ulla

    2014-01-01

    Burkholderia pseudomallei is a common cause of serious, difficult to treat infections in South-East Asia and Northern Australia, but is a rare imported pathogen in the USA and Europe. We report a case of a patient with a mycotic aneurysm caused by B. pseudomallei in a previously healthy returning traveller. The patient presented with 4 weeks of abdominal pain and intermittent fever after a brief vacation in Thailand. The aneurysm was excised and replaced by an autologous deep vein graft, and the patient was treated for 6 months with antibiotics adjusted according to postoperative renal impairment. Twenty-four months after surgery the patient is well and without relapse. PMID:25246454

  6. 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

  7. Laboratory diagnosis of melioidosis: Past, present and future

    PubMed Central

    Lau, Susanna KP; Sridhar, Siddharth; Ho, Chi-Chun; Chow, Wang-Ngai; Lee, Kim-Chung; Lam, Ching-Wan; Yuen, Kwok-Yung

    2015-01-01

    Melioidosis is an emerging, potentially fatal disease caused by Burkholderia pseudomallei, which requires prolonged antibiotic treatment to prevent disease relapse. However, difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes. Isolation of B. pseudomallei from clinical specimens has been improved with the use of selective media. However, even with positive cultures, identification of B. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. Commercial identification systems may fail to distinguish between B. pseudomallei and closely related species such as Burkholderia thailandensis. Genotypic identification of suspected isolates can be achieved by sequencing of gene targets such as groEL which offer higher discriminative power than 16S rRNA. Specific PCR-based identification of B. pseudomallei has also been developed using B. pseudomallei-specific gene targets such as Type III secretion system and Tat-domain protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolutionary technique for pathogen identification, has been shown to be potentially useful for rapid identification of B. pseudomallei, although existing databases require optimization by adding reference spectra for B. pseudomallei. Despite these advances in bacterial identification, diagnostic problems encountered in culture-negative cases remain largely unresolved. Although various serological tests have been developed, they are generally unstandardized “in house” assays and have low sensitivities and specificities. Although specific PCR assays have been applied to direct clinical and environmental specimens, the sensitivities for diagnosis remain to be evaluated. Metabolomics is an uprising tool for studying infectious diseases and may offer a novel approach for exploring potential diagnostic biomarkers. The metabolomics profiles of B

  8. Laboratory diagnosis of melioidosis: past, present and future.

    PubMed

    Lau, Susanna K P; Sridhar, Siddharth; Ho, Chi-Chun; Chow, Wang-Ngai; Lee, Kim-Chung; Lam, Ching-Wan; Yuen, Kwok-Yung; Woo, Patrick C Y

    2015-06-01

    Melioidosis is an emerging, potentially fatal disease caused by Burkholderia pseudomallei, which requires prolonged antibiotic treatment to prevent disease relapse. However, difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes. Isolation of B. pseudomallei from clinical specimens has been improved with the use of selective media. However, even with positive cultures, identification of B. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. Commercial identification systems may fail to distinguish between B. pseudomallei and closely related species such as Burkholderia thailandensis. Genotypic identification of suspected isolates can be achieved by sequencing of gene targets such as groEL which offer higher discriminative power than 16S rRNA. Specific PCR-based identification of B. pseudomallei has also been developed using B. pseudomallei-specific gene targets such as Type III secretion system and Tat-domain protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolutionary technique for pathogen identification, has been shown to be potentially useful for rapid identification of B. pseudomallei, although existing databases require optimization by adding reference spectra for B. pseudomallei. Despite these advances in bacterial identification, diagnostic problems encountered in culture-negative cases remain largely unresolved. Although various serological tests have been developed, they are generally unstandardized "in house" assays and have low sensitivities and specificities. Although specific PCR assays have been applied to direct clinical and environmental specimens, the sensitivities for diagnosis remain to be evaluated. Metabolomics is an uprising tool for studying infectious diseases and may offer a novel approach for exploring potential diagnostic biomarkers. The metabolomics profiles of B

  9. 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.

  10. [Identification of the causative agents of glanders and melioidosis by polymerase chain reaction].

    PubMed

    Tkachenko, G A; Antonov, V A; Zamaraev, V S; Iliukhin, V I

    2003-01-01

    Burkholderia mallei and B. pseudomallei are causative agents of glanders and melioidosis, respectively, i.e. severe and fatal infection diseases of man and animal. The computer-based analysis of the 23S rRNA gene sites was used for selecting the primers. Two pairs of primers were chosen for the identification of B. mallei and Bpseudomallei. DNAs from 48 B. pseudomallei and 15 strains of B. mallei, unlike from other geterological bacteria, were positively amplified. Therefore, the method of polymerase chain reaction can be used in laboratory diagnosis of glanders and melioidosis.

  11. 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 ...

  12. 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

  13. 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

  14. 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.

  15. Cellular Reporter Screens for Inhibitors of Burkholderia pseudomallei Targets in Pseudomonas aeruginosa

    PubMed Central

    Moir, D. T.; Di, M.; Moore, R. A.; Schweizer, H. P.; Woods, D. E.

    2009-01-01

    Summary To facilitate the discovery of new therapeutics for Burkholderia pseudomallei infections, we have developed cellular reporter screens for inhibitors of B. pseudomallei targets in the surrogate host Pseudomonas aeruginosa. P. aeruginosa strains carrying deletions of essential genes were engineered to be dependent on the IPTG-regulated expression of their B. pseudomallei orthologs on a broad-host-range plasmid. P. aeruginosa genes which are upregulated in response to depletion of each target gene product were fused to the Photorhabdus luminescens luxCDABE operon via pGSV3-lux-SpR to generate reporter strains with increased bioluminescence upon target inhibition. A total of 11 of 19 B. pseudomallei genes complemented deletions of their orthologs in P. aeruginosa. The dependence of growth on IPTG levels varied from complete dependence (ftsQ, gyrA, glmU, secA), to slower growth in the absence of IPTG (coaD, efp, mesJ), to apparently normal growth in the absence of IPTG (ligA, lpxA, folA, ipk). Reporter screening strains have been constructed for three gene targets (gyrA, glmU, secA), and one (gyrA) has been applied to 68,000 compounds resulting in a primary hit rate of 0.5% and a confirmed hit rate of 0.06% including several fluoroquinolones. These results provide proof of principle for surrogate cellular reporter screens as a useful approach to identify inhibitors of essential gene products. PMID:19121678

  16. Cellular reporter screens for inhibitors of Burkholderia pseudomallei targets in Pseudomonas aeruginosa.

    PubMed

    Moir, Donald T; Di, Ming; Moore, Richard A; Schweizer, Herbert P; Woods, Donald E

    2008-12-01

    To facilitate the discovery of new therapeutics for Burkholderia pseudomallei infections, we have developed cellular reporter screens for inhibitors of B. pseudomallei targets in the surrogate host Pseudomonas aeruginosa. Pseudomonas aeruginosa strains carrying deletions of essential genes were engineered to be dependent on the isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated expression of their B. pseudomallei orthologues on a broad-host-range plasmid. Pseudomonas aeruginosa genes which are upregulated in response to depletion of each target gene product, were fused to the Photorhabdus luminescens luxCDABE operon via pGSV3-lux-Sp(R) to generate reporter strains with increased bioluminescence upon target inhibition. A total of 11 of 19 B. pseudomallei genes complemented deletions of their orthologues in P. aeruginosa. The dependence of growth on IPTG levels varied from complete dependence (ftsQ, gyrA, glmU, secA) to slower growth in the absence of IPTG (coaD, efp, mesJ), to apparently normal growth in the absence of IPTG (ligA, lpxA, folA, ipk). Reporter screening strains have been constructed for three gene targets (gyrA, glmU, secA), and one (gyrA) has been applied to 68,000 compounds resulting in a primary hit rate of 0.5% and a confirmed hit rate of 0.06%, including several fluoroquinolones. These results provide proof of principle for surrogate cellular reporter screens as a useful approach to identify inhibitors of essential gene products. PMID:19121678

  17. Knock-out and pull-out recombineering protocols for naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei

    PubMed Central

    Kang, Yun; Norris, Michael H.; Wilcox, Bruce A.; Tuanyok, Apichai; Keim, Paul S.; Hoang, Tung T.

    2013-01-01

    Summary Phage λ Red proteins are powerful tools for pulling- and knocking-out chromosomal fragments but have been limited to the γ-proteobacteria. Procedures are described here to easily knock-out (KO) and pull-out (PO) chromosomal DNA fragments from naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei. This system takes advantage of published compliant counter-selectable and selectable markers (sacB, pheS, gat, and the arabinose utilization operon) and λ Red mutant proteins. pheS-gat (KO) or oriT-ColE1ori-gat-ori1600-rep (PO) PCR fragments are generated with flanking 40–45 bp homologies to targeted regions incorporated on PCR primers. One-step recombination is achieved by incubating the PCR product with cells expressing λ Red proteins and subsequent selection on glyphosate-containing medium. This procedure takes approximately 10 days and is advantageous over previously published protocols: i) smaller PCR products reduce primer numbers and amplification steps, ii) PO fragments for downstream manipulation in E. coli, and iii) chromosomal KO increases flexibility for downstream processing. PMID:21738123

  18. 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

  19. 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

  20. 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

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

    PubMed

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

    2014-08-01

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

  2. Delineating the Importance of Serum Opsonins and the Bacterial Capsule in Affecting the Uptake and Killing of Burkholderia pseudomallei by Murine Neutrophils and Macrophages

    PubMed Central

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

    2014-01-01

    Infection of susceptible hosts by the encapsulated Gram-negative bacterium Burkholderia pseudomallei (Bp) causes melioidosis, with septic patients attaining mortality rates ≥40%. Due to its high infectivity through inhalation and limited effective therapies, Bp is considered a potential bioweapon. Thus, there is great interest in identifying immune effectors that effectively kill Bp. Our goal is to compare the relative abilities of murine macrophages and neutrophils to clear Bp, as well as determine the importance of serum opsonins and bacterial capsule. Our findings indicate that murine macrophages and neutrophils are inherently unable to clear either unopsonized Bp or the relatively-avirulent acapsular bacterium B. thailandensis (Bt). Opsonization of Bp and Bt with complement or pathogen-specific antibodies increases macrophage-uptake, but does not promote clearance, although antibody-binding enhances complement deposition. In contrast, complement opsonization of Bp and Bt causes enhanced uptake and killing by neutrophils, which is linked with rapid ROS induction against bacteria exhibiting a threshold level of complement deposition. Addition of bacteria-specific antibodies enhances complement deposition, but antibody-binding alone cannot elicit neutrophil clearance. Bp capsule provides some resistance to complement deposition, but is not anti-phagocytic or protective against reactive oxygen species (ROS)-killing. Macrophages were observed to efficiently clear Bp only after pre-activation with IFNγ, which is independent of serum- and/or antibody-opsonization. These studies indicate that antibody-enhanced complement activation is sufficient for neutrophil-clearance of Bp, whereas macrophages are ineffective at clearing serum-opsonized Bp unless pre-activated with IFNγ. This suggests that effective immune therapies would need to elicit both antibodies and Th1-adaptive responses for successful prevention/eradication of melioidosis. PMID:25144195

  3. Therapeutic Administration of a Monoclonal Anti-Il-1β Antibody Protects Against Experimental Melioidosis

    PubMed Central

    Weehuizen, Tassili A. F.; Lankelma, Jacqueline M.; De Jong, Hanna K.; De Boer, Onno J.; Roelofs, Joris J. T. H.; Day, Nicholas P.; Gram, Hermann; De Vos, Alex F.; Wiersinga, W. Joost

    2016-01-01

    ABSTRACT Background: Melioidosis, caused by the gram-negative bacterium Burkholderia pseudomallei, is a common cause of community-acquired sepsis in Southeast Asia and Northern Australia. The NLRP3 inflammasome and its downstream product interleukin-1 beta (IL-1β) have been proposed to play crucial roles in melioidosis. In this study, we characterized the role of IL-1β more closely and we assessed its therapeutic potential. Methods: mRNA expression of inflammasome components was determined in isolated leukocytes of 32 healthy controls and 34 patients with sepsis caused by B pseudomallei. Wild-type (WT), NLRP3-deficient (Nlrp3−/−), and Asc−/− mice were infected with B pseudomallei. In additional experiments, infected WT mice were treated with an anti-IL-1β antibody. After 24, 48, and 72 hours (h) mice were sacrificed and organs were harvested. Furthermore, survival studies were performed. Results: Patients with melioidosis exhibited lower mRNA levels of caspase-1, NLRP3, and ASC. Bacterial dissemination and organ damage were increased in B pseudomallei-infected Nlrp3−/− and Asc−/− mice, together with a reduced pulmonary cell influx. Anti-IL-1β treatment of B pseudomallei challenged mice resulted in strongly reduced bacterial counts, organ damage, and pulmonary granulocyte influx together with reduced mortality. Postponement of anti-IL-1β treatment for 24 h postinfection still protected mice during melioidosis. Conclusion: Expression of caspase-1, NLRP3, and ASC is altered in melioidosis patients. In mice, both NLRP3 and ASC contribute to the host defense against melioidosis. Anti-IL-1β treatment protects mice against B pseudomallei infection and might be a novel treatment strategy in melioidosis. PMID:27219859

  4. Outcomes of patients with melioidosis treated with meropenem.

    PubMed

    Cheng, Allen C; Fisher, Dale A; Anstey, Nicholas M; Stephens, Dianne P; Jacups, Susan P; Currie, Bart J

    2004-05-01

    Melioidosis, an infection due to Burkholderia pseudomallei, is endemic in southeast Asia and northern Australia. We reviewed our experience with meropenem in the treatment of severe melioidosis in 63 patients over a 6-year period. Outcomes were similar to those of ceftazidime-treated patients (n = 153) despite a deliberate selection bias to more-unwell patients receiving meropenem. The mortality among meropenem-treated patients was 19%. One patient had a possible drug fever associated with the use of meropenem. We conclude that meropenem (1 g or 25 mg/kg every 8 h intravenously for >/=14 days) is an alternative to ceftazidime and imipenem in the treatment of melioidosis. The use of meropenem may be associated with improved outcomes in patients with severe sepsis associated with melioidosis.

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. Further evaluation of a rapid diagnostic test for melioidosis in an area of endemicity.

    PubMed

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

    2004-05-01

    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.

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

    PubMed

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

    2013-06-01

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

  12. 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

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

    PubMed Central

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

    2016-01-01

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

  14. 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

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

    PubMed

    Spring-Pearson, Senanu M; Stone, Joshua K; Doyle, Adina; Allender, Christopher J; Okinaka, Richard T; Mayo, Mark; Broomall, Stacey M; Hill, Jessica M; Karavis, Mark A; Hubbard, Kyle S; Insalaco, Joseph M; McNew, Lauren A; Rosenzweig, C Nicole; Gibbons, Henry S; Currie, Bart J; Wagner, David M; Keim, Paul; Tuanyok, Apichai

    2015-01-01

    The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

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

    PubMed Central

    Spring-Pearson, Senanu M.; Stone, Joshua K.; Doyle, Adina; Allender, Christopher J.; Okinaka, Richard T.; Mayo, Mark; Broomall, Stacey M.; Hill, Jessica M.; Karavis, Mark A.; Hubbard, Kyle S.; Insalaco, Joseph M.; McNew, Lauren A.; Rosenzweig, C. Nicole; Gibbons, Henry S.; Currie, Bart J.; Wagner, David M.; Keim, Paul; Tuanyok, Apichai

    2015-01-01

    The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is ‘open’, with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order. PMID:26484663

  17. Clinical guideline for diagnosis and management of melioidosis.

    PubMed

    Inglis, Timothy J J; Rolim, Dionne B; Rodriguez, Jorge L N

    2006-01-01

    Melioidosis is an emerging infection in Brazil and neighbouring South American countries. The wide range of clinical presentations include severe community-acquired pneumonia, septicaemia, central nervous system infection and less severe soft tissue infection. Diagnosis depends heavily on the clinical microbiology laboratory for culture. Burkholderia pseudomallei, the bacterial cause of melioidosis, is easily cultured from blood, sputum and other clinical samples. However, B. pseudomallei can be difficult to identify reliably, and can be confused with closely related bacteria, some of which may be dismissed as insignificant culture contaminants. Serological tests can help to support a diagnosis of melioidosis, but by themselves do not provide a definitive diagnosis. The use of a laboratory discovery pathway can help reduce the risk of missing atypical B. pseudomallei isolates. Recommended antibiotic treatment for severe infection is either intravenous Ceftazidime or Meropenem for several weeks, followed by up to 20 weeks oral treatment with a combination of trimethoprim-sulphamethoxazole and doxycycline. Consistent use of diagnostic microbiology to confirm the diagnosis, and rigorous treatment of severe infection with the correct antibiotics in two stages; acute and eradication, will contribute to a reduction in mortality from melioidosis. PMID:16547571

  18. 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

  19. A Quadruplex Real-Time PCR Assay for the Rapid Detection and Differentiation of the Most Relevant Members of the B. pseudomallei Complex: B. mallei, B. pseudomallei, and B. thailandensis

    PubMed Central

    Lowe, Chinn-Woan; Thiriot, Joseph D.; Heder, Michael J.; March, Jordon K.; Drake, David S.; Lew, Cynthia S.; Bunnell, Annette J.; Moore, Emily S.; O'Neill, Kim L.; Robison, Richard A.

    2016-01-01

    The Burkholderia pseudomallei complex classically consisted of B. mallei, B. pseudomallei, and B. thailandensis, but has now expanded to include B. oklahomensis, B. humptydooensis, and three unassigned Burkholderia clades. Methods for detecting and differentiating the B. pseudomallei complex has been the topic of recent research due to phenotypic and genotypic similarities of these species. B. mallei and B. pseudomallei are recognized as CDC Tier 1 select agents, and are the causative agents of glanders and melioidosis, respectively. Although B. thailandensis and B. oklahomensis are generally avirulent, both display similar phenotypic characteristics to that of B. pseudomallei. B. humptydooensis and the Burkholderia clades are genetically similar to the B. pseudomallei complex, and are not associated with disease. Optimal identification of these species remains problematic, and PCR-based methods can resolve issues with B. pseudomallei complex detection and differentiation. Currently, no PCR assay is available that detects the major species of the B. pseudomallei complex. A real-time PCR assay in a multiplex single-tube format was developed to simultaneously detect and differentiate B. mallei, B. pseudomallei, and B. thailandensis, and a common sequence found in B. pseudomallei, B. mallei, B. thailandensis, and B. oklahomensis. A total of 309 Burkholderia isolates and 5 other bacterial species were evaluated. The assay was 100% sensitive and specific, demonstrated sensitivity beyond culture and GC methods for the isolates tested, and is completed in about an hour with a detection limit between 2.6pg and 48.9pg of gDNA. Bioinformatic analyses also showed the assay is likely 100% specific and sensitive for all 84 fully sequenced B. pseudomallei, B. mallei, B. thailandensis, and B. oklahomensis strains currently available in GenBank. For these reasons, this assay could be a rapid and sensitive tool in the detection and differentiation for those species of the B

  20. 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

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

    PubMed

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

    2003-02-13

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

  2. Distinct human antibody response to the biological warfare agent Burkholderia mallei.

    PubMed

    Varga, John J; Vigil, Adam; DeShazer, David; Waag, David M; Felgner, Philip; Goldberg, Joanna B

    2012-10-01

    The genetic similarity between Burkholderia mallei (glanders) and Burkholderia pseudomallei (melioidosis) had led to the general assumption that pathogenesis of each bacterium would be similar. In 2000, the first human case of glanders in North America since 1945 was reported in a microbiology laboratory worker. Leveraging the availability of pre-exposure sera for this individual and employing the same well-characterized protein array platform that has been previously used to study a large cohort of melioidosis patients in southeast Asia, we describe the antibody response in a human with glanders. Analysis of 156 peptides present on the array revealed antibodies against 17 peptides with a > 2-fold increase in this infection. Unexpectedly, when the glanders data were compared with a previous data set from B. pseudomallei infections, there were only two highly increased antibodies shared between these two infections. These findings have implications in the diagnosis and treatment of B. mallei and B. pseudomallei infections.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-06-01

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

  5. Efficacy of post exposure administration of doxycycline in a murine model of inhalational melioidosis.

    PubMed

    Gelhaus, H Carl; Anderson, Michael S; Fisher, David A; Flavin, Michael T; Xu, Ze-Qi; Sanford, Daniel C

    2013-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis. Treatment of melioidosis is suboptimal and developing improved melioidosis therapies requires animal models. In this report, we exposed male BALB/c mice to various amounts of aerosolized B. pseudomallei 1026b to determine lethality. After establishing a median lethal dose (LD(50)) of 2,772 colony forming units (cfu)/animal, we tested the ability of doxycycline administered 6 hours after exposure to a uniformly lethal dose of ~20 LD(50) to prevent death and eliminate bacteria from the lung and spleens. Tissue bacterial burdens were examined by PCR analysis. We found that 100% of mice treated with doxycycline survived and B. pseudomallei DNA was not amplified from the lungs or spleens of most surviving mice. We conclude the BALB/c mouse is a useful model of melioidosis. Furthermore, the data generated in this mouse model indicate that doxycycline is likely to be effective in post-exposure prophylaxis of melioidosis. PMID:23359492

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

    PubMed

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

    2004-12-15

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

  7. Melioidosis: an emerging infection in Taiwan?

    PubMed Central

    Hsueh, P. R.; Teng, L. J.; Lee, L. N.; Yu, C. J.; Yang, P. C.; Ho, S. W.; Luh, K. T.

    2001-01-01

    From January 1982 to May 2000, 17 infections caused by Burkholderia pseudomallei were diagnosed in 15 patients in Taiwan; almost all the infections were diagnosed from 1994 to May 2000. Of the 15 patients, 9 (60%) had underlying diseases, and 10 (67%) had bacteremic pneumonia. Thirteen (76%) episodes of infection were considered indigenous. Four patients died of melioidosis. Seventeen B. pseudomallei isolates, recovered from eight patients from November 1996 to May 2000, were analyzed to determine their in vitro susceptibilities to 14 antimicrobial agents, cellular fatty acid and biochemical reaction profiles, and randomly amplified polymorphic DNA patterns. Eight strains (highly related isolates) were identified. All isolates were arabinose non-assimilators and were susceptible to amoxicillin-clavulanate, piperacillin-tazobactam, imipenem, and meropenem. No spread of the strain was documented. PMID:11384520

  8. Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79.

    PubMed

    Khakhum, Nittaya; Yordpratum, Umaporn; Boonmee, Atcha; Tattawasart, Unchalee; Rodrigues, Jorge L M; Sermswan, Rasana W

    2016-12-01

    The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn(2+). When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5-9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however, the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases. PMID:27637947

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

    PubMed

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

    2006-06-01

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

  10. Melioidosis: A Rare Cause of Liver Abscess.

    PubMed

    Martin, Peter Franz M San; Teh, Catherine S C; Casupang, Ma Amornetta J

    2016-01-01

    Case Presentation. This is a case of a 44-year-old male, farmer, known to be diabetic, presenting with two-week history of vague abdominal pain associated with high grade fever. Abdominal CT scan showed localized liver abscess at segment 8 measuring 7.5 × 6.8 × 6.1 cm. Patient subsequently underwent laparoscopic ultrasound guided pigtail insertion for drainage of abscess. Culture studies showed moderate growth of Burkholderia pseudomallei in which the patient completed seven days of IV Meropenem. On follow-up after 12 weeks of oral Sulfamethoxazole/Trimethoprim, taken twice a day, the patient remained asymptomatic with no residual findings based on the abdominal ultrasound. Discussion. Diagnosis of melioidosis, a known "great masquerader," relies heavily on culture studies. Consensus with regard to the management of liver abscess caused by Burkholderia pseudomallei has not yet been established due to the rarity of cases. Surgical intervention through either a percutaneous or open drainage has shown good outcomes compared to IV antibiotics alone. In Philippines, the possibility of underreporting is highly plausible. This write-up serves not only to report a rare presentation of melioidosis but also to add to the number of cases reported in the country, possibly indicative of disease emergence. PMID:27529039

  11. Melioidosis: A Rare Cause of Liver Abscess

    PubMed Central

    Teh, Catherine S. C.; Casupang, Ma. Amornetta J.

    2016-01-01

    Case Presentation. This is a case of a 44-year-old male, farmer, known to be diabetic, presenting with two-week history of vague abdominal pain associated with high grade fever. Abdominal CT scan showed localized liver abscess at segment 8 measuring 7.5 × 6.8 × 6.1 cm. Patient subsequently underwent laparoscopic ultrasound guided pigtail insertion for drainage of abscess. Culture studies showed moderate growth of Burkholderia pseudomallei in which the patient completed seven days of IV Meropenem. On follow-up after 12 weeks of oral Sulfamethoxazole/Trimethoprim, taken twice a day, the patient remained asymptomatic with no residual findings based on the abdominal ultrasound. Discussion. Diagnosis of melioidosis, a known “great masquerader,” relies heavily on culture studies. Consensus with regard to the management of liver abscess caused by Burkholderia pseudomallei has not yet been established due to the rarity of cases. Surgical intervention through either a percutaneous or open drainage has shown good outcomes compared to IV antibiotics alone. In Philippines, the possibility of underreporting is highly plausible. This write-up serves not only to report a rare presentation of melioidosis but also to add to the number of cases reported in the country, possibly indicative of disease emergence. PMID:27529039

  12. Whole-Genome Sequencing Confirms that Burkholderia pseudomallei Multilocus Sequence Types Common to Both Cambodia and Australia Are Due to Homoplasy

    PubMed Central

    De Smet, Birgit; Mayo, Mark; Theobald, Vanessa; Kham, Chun; Heng, Seiha; Thong, Phe; Holden, Matthew T. G.; Peacock, Sharon J.; Spratt, Brian G.; Jacobs, Jan A.; Vandamme, Peter; Currie, Bart J.

    2014-01-01

    Burkholderia pseudomallei isolates with shared multilocus sequence types (STs) have not been isolated from different continents. We identified two STs shared between Australia and Cambodia. Whole-genome analysis revealed substantial diversity within STs, correctly identified the Asian or Australian origin, and confirmed that these shared STs were due to homoplasy. PMID:25392354

  13. NLRC4 and TLR5 each contribute to host defense in respiratory melioidosis.

    PubMed

    West, T Eoin; Myers, Nicolle D; Chantratita, Narisara; Chierakul, Wirongrong; Limmathurotsakul, Direk; Wuthiekanun, Vanaporn; Miao, Edward A; Hajjar, Adeline M; Peacock, Sharon J; Liggitt, H Denny; Skerrett, Shawn J

    2014-09-01

    Burkholderia pseudomallei causes the tropical infection melioidosis. Pneumonia is a common manifestation of melioidosis and is associated with high mortality. Understanding the key elements of host defense is essential to developing new therapeutics for melioidosis. As a flagellated bacterium encoding type III secretion systems, B. pseudomallei may trigger numerous host pathogen recognition receptors. TLR5 is a flagellin sensor located on the plasma membrane. NLRC4, along with NAIP proteins, assembles a canonical caspase-1-dependent inflammasome in the cytoplasm that responds to flagellin (in mice) and type III secretion system components (in mice and humans). In a murine model of respiratory melioidosis, Tlr5 and Nlrc4 each contributed to survival. Mice deficient in both Tlr5 and Nlrc4 were not more susceptible than single knockout animals. Deficiency of Casp1/Casp11 resulted in impaired bacterial control in the lung and spleen; in the lung much of this effect was attributable to Nlrc4, despite relative preservation of pulmonary IL-1β production in Nlrc4(-/-) mice. Histologically, deficiency of Casp1/Casp11 imparted more severe pulmonary inflammation than deficiency of Nlrc4. The human NLRC4 region polymorphism rs6757121 was associated with survival in melioidosis patients with pulmonary involvement. Co-inheritance of rs6757121 and a functional TLR5 polymorphism had an additive effect on survival. Our results show that NLRC4 and TLR5, key components of two flagellin sensing pathways, each contribute to host defense in respiratory melioidosis. PMID:25232720

  14. NLRC4 and TLR5 Each Contribute to Host Defense in Respiratory Melioidosis

    PubMed Central

    West, T. Eoin; Myers, Nicolle D.; Chantratita, Narisara; Chierakul, Wirongrong; Limmathurotsakul, Direk; Wuthiekanun, Vanaporn; Miao, Edward A.; Hajjar, Adeline M.; Peacock, Sharon J.; Liggitt, H. Denny; Skerrett, Shawn J.

    2014-01-01

    Burkholderia pseudomallei causes the tropical infection melioidosis. Pneumonia is a common manifestation of melioidosis and is associated with high mortality. Understanding the key elements of host defense is essential to developing new therapeutics for melioidosis. As a flagellated bacterium encoding type III secretion systems, B. pseudomallei may trigger numerous host pathogen recognition receptors. TLR5 is a flagellin sensor located on the plasma membrane. NLRC4, along with NAIP proteins, assembles a canonical caspase-1-dependent inflammasome in the cytoplasm that responds to flagellin (in mice) and type III secretion system components (in mice and humans). In a murine model of respiratory melioidosis, Tlr5 and Nlrc4 each contributed to survival. Mice deficient in both Tlr5 and Nlrc4 were not more susceptible than single knockout animals. Deficiency of Casp1/Casp11 resulted in impaired bacterial control in the lung and spleen; in the lung much of this effect was attributable to Nlrc4, despite relative preservation of pulmonary IL-1β production in Nlrc4−/− mice. Histologically, deficiency of Casp1/Casp11 imparted more severe pulmonary inflammation than deficiency of Nlrc4. The human NLRC4 region polymorphism rs6757121 was associated with survival in melioidosis patients with pulmonary involvement. Co-inheritance of rs6757121 and a functional TLR5 polymorphism had an additive effect on survival. Our results show that NLRC4 and TLR5, key components of two flagellin sensing pathways, each contribute to host defense in respiratory melioidosis. PMID:25232720

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

    PubMed Central

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

    2013-01-01

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

  16. Natural infection of Burkholderia pseudomallei in an imported pigtail macaque (Macaca nemestrina) and management of the exposed colony.

    PubMed

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

    2013-01-01

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

  17. Burkholderia vaccines: are we moving forward?

    PubMed

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

    2013-01-01

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

  18. Melioidosis: epidemiology, pathophysiology, and management.

    PubMed

    Cheng, Allen C; Currie, Bart J

    2005-04-01

    Melioidosis, caused by the gram-negative saprophyte Burkholderia pseudomallei, is a disease of public health importance in southeast Asia and northern Australia that is associated with high case-fatality rates in animals and humans. It has the potential for epidemic spread to areas where it is not endemic, and sporadic case reports elsewhere in the world suggest that as-yet-unrecognized foci of infection may exist. Environmental determinants of this infection, apart from a close association with rainfall, are yet to be elucidated. The sequencing of the genome of a strain of B. pseudomallei has recently been completed and will help in the further identification of virulence factors. The presence of specific risk factors for infection, such as diabetes, suggests that functional neutrophil defects are important in the pathogenesis of melioidosis; other studies have defined virulence factors (including a type III secretion system) that allow evasion of killing mechanisms by phagocytes. There is a possible role for cell-mediated immunity, but repeated environmental exposure does not elicit protective humoral or cellular immunity. A vaccine is under development, but economic constraints may make vaccination an unrealistic option for many regions of endemicity. Disease manifestations are protean, and no inexpensive, practical, and accurate rapid diagnostic tests are commercially available; diagnosis relies on culture of the organism. Despite the introduction of ceftazidime- and carbapenem-based intravenous treatments, melioidosis is still associated with a significant mortality attributable to severe sepsis and its complications. A long course of oral eradication therapy is required to prevent relapse. Studies exploring the role of preventative measures, earlier clinical identification, and better management of severe sepsis are required to reduce the burden of this disease.

  19. Melioidosis: Epidemiology, Pathophysiology, and Management

    PubMed Central

    Cheng, Allen C.; Currie, Bart J.

    2005-01-01

    Melioidosis, caused by the gram-negative saprophyte Burkholderia pseudomallei, is a disease of public health importance in southeast Asia and northern Australia that is associated with high case-fatality rates in animals and humans. It has the potential for epidemic spread to areas where it is not endemic, and sporadic case reports elsewhere in the world suggest that as-yet-unrecognized foci of infection may exist. Environmental determinants of this infection, apart from a close association with rainfall, are yet to be elucidated. The sequencing of the genome of a strain of B. pseudomallei has recently been completed and will help in the further identification of virulence factors. The presence of specific risk factors for infection, such as diabetes, suggests that functional neutrophil defects are important in the pathogenesis of melioidosis; other studies have defined virulence factors (including a type III secretion system) that allow evasion of killing mechanisms by phagocytes. There is a possible role for cell-mediated immunity, but repeated environmental exposure does not elicit protective humoral or cellular immunity. A vaccine is under development, but economic constraints may make vaccination an unrealistic option for many regions of endemicity. Disease manifestations are protean, and no inexpensive, practical, and accurate rapid diagnostic tests are commercially available; diagnosis relies on culture of the organism. Despite the introduction of ceftazidime- and carbapenem-based intravenous treatments, melioidosis is still associated with a significant mortality attributable to severe sepsis and its complications. A long course of oral eradication therapy is required to prevent relapse. Studies exploring the role of preventative measures, earlier clinical identification, and better management of severe sepsis are required to reduce the burden of this disease. PMID:15831829

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

    PubMed Central

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

    2014-01-01

    Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253-, P2cysZK-, and P1-driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT-mini-Tn7T-Km, pmini-Tn7-gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP- and RFP-tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild-type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high-throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages. PMID:25044501

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

    PubMed

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

    2014-10-01

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

  2. The public health implications of melioidosis.

    PubMed

    Inglis, Timothy J J; Sousa, Anastácio Q

    2009-02-01

    Melioidosis, which is caused by the bacterium Burkholderia pseudomallei, is a potentially fatal tropical infection, little known outside its main endemic zone of Southeast Asia and northern Australia. Though it has received more attention in recent years on account of its claimed suitability as a biological weapon agent, the principal threat from melioidosis is a result of naturally occurring events. Occasional case clusters, sporadic cases outside the known endemic zone and infections in unusual demographic groups highlight a changing epidemiology. As melioidosis is the result of an environmental encounter and not person-to-person transmission, subtle changes in its epidemiology indicate a role environmental factors, such as man-made disturbances of soil and surface water. These have implications for travel, occupational and tropical medicine and in particular for risk assessment and prevention. Practical problems with definitive laboratory diagnosis, antibiotic treatment and the current lack of a vaccine underline the need for prevention through exposure avoidance and other environmental health measures. It is likely that the increasing population burden of the tropical zone and extraction of resources from the humid tropics will increase the prevalence of melioidosis. Climate change-driven extreme weather events will both increase the prevalence of infection and gradually extend its main endemic zone. PMID:19578632

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

    NASA Astrophysics Data System (ADS)

    Yoon, Yohan; Kim, Jae-Hun; Byun, Myung-Woo; Choi, Kyoung-Hee; Lee, Ju-Woon

    2010-04-01

    This study evaluated the effect of gamma irradiation on Burkholderia thailandensis ( Burkholderia pseudomallei surrogate; potential bioterrorism agent) survival under different levels of NaCl and pH. B. thailandensis in Luria Bertani broth supplemented with NaCl (0-3%), and pH-adjusted to 4-7 was treated with gamma irradiation (0-0.5 kGy). Surviving cell counts of bacteria were then enumerated on tryptic soy agar. Data for the cell counts were also used to calculate D10 values (the dose required to reduce 1 log CFU/mL of B. thailandensis). Cell counts of B. thailandensis were decreased ( P<0.05) as irradiation dose increased, and no differences ( P≥0.05) in cell counts of the bacteria were observed among different levels of NaCl and pH. D10 values ranged from 0.04 to 0.07 kGy, regardless of NaCl and pH level. These results indicate that low doses of gamma irradiation should be a useful treatment in decreasing the potential bioterrorism bacteria, which may possibly infect humans through foods.

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

    PubMed

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

    2016-02-01

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

  5. Melioidosis in animals: a review on epizootiology, diagnosis and clinical presentation.

    PubMed

    Sprague, L D; Neubauer, H

    2004-09-01

    Melioidosis, an infectious disease caused by Burkholderia pseudomallei is an emerging disease with high impact on animals and man. In different animal species, the clinical course varies and delayed diagnosis poses risks for the dissemination of the agent in non-endemic areas. Not only migration and transport of animals around the world but also tourism increases the risk that melioidosis can leave its endemic boundaries and establish itself elsewhere. Detection of the agent is a major challenge, as the agent has to be handled in laboratories of biosafety level 3 and test kits are not yet commercially available. Veterinarians and doctors should be aware of melioidosis not only as an agent of public interest but also in terms of a bioterrorist attack. The aim of this review is to describe the agent, its aetiology, the manifestation in a variety of animal species as well as to describe diagnostic procedures, typing techniques and countermeasures.

  6. A case of systemic melioidosis: unravelling the etiology of chronic unexplained fever with multiple presentations.

    PubMed

    Mohanty, Srujana; Pradhan, Gourahari; Panigrahi, Manoj Kumar; Mohapatra, Prasanta Raghab; Mishra, Baijayantimala

    2016-01-01

    Melioidosis, caused by the environmental saprophyte, Burkholderia pseudomallei, is an important public health problem in Southeast Asia and Northern Australia. It is being increasingly reported from other parts, including India, China, and North and South America expanding the endemic zone of the disease. We report a case of systemic melioidosis in a 58-year-old diabetic, occupationally-unexposed male patient, who presented with chronic fever, sepsis, pneumonia, pleural effusion and subcutaneous abscess, was undiagnosed for long, misidentified as Pseudomonas aeruginosa infection elsewhere, but was saved due to correct identification of the etiologic agent and timely institution of appropriate therapy at our institute. A strong clinical and microbiological suspicion for melioidosis should be considered in the differential diagnosis of acute pyrexia of unknown origin, acute respiratory distress syndrome and acute onset of sepsis, especially in the tropics. PMID:27238172

  7. Melioidosis in animals: a review on epizootiology, diagnosis and clinical presentation.

    PubMed

    Sprague, L D; Neubauer, H

    2004-09-01

    Melioidosis, an infectious disease caused by Burkholderia pseudomallei is an emerging disease with high impact on animals and man. In different animal species, the clinical course varies and delayed diagnosis poses risks for the dissemination of the agent in non-endemic areas. Not only migration and transport of animals around the world but also tourism increases the risk that melioidosis can leave its endemic boundaries and establish itself elsewhere. Detection of the agent is a major challenge, as the agent has to be handled in laboratories of biosafety level 3 and test kits are not yet commercially available. Veterinarians and doctors should be aware of melioidosis not only as an agent of public interest but also in terms of a bioterrorist attack. The aim of this review is to describe the agent, its aetiology, the manifestation in a variety of animal species as well as to describe diagnostic procedures, typing techniques and countermeasures. PMID:15525357

  8. Consensus on the Development of Vaccines against Naturally Acquired Melioidosis

    PubMed Central

    Funnell, Simon G.P.; Torres, Alfredo G.; Morici, Lisa A.; Brett, Paul J.; Dunachie, Susanna; Atkins, Timothy; Altmann, Daniel M.; Bancroft, Gregory; Peacock, Sharon J.

    2015-01-01

    Several candidates for a vaccine against Burkholderia pseudomallei, the causal bacterium of melioidosis, have been developed, and a rational approach is now needed to select and advance candidates for testing in relevant nonhuman primate models and in human clinical trials. Development of such a vaccine was the topic of a meeting in the United Kingdom in March 2014 attended by international candidate vaccine developers, researchers, and government health officials. The focus of the meeting was advancement of vaccines for prevention of natural infection, rather than for protection from the organism’s known potential for use as a biological weapon. A direct comparison of candidate vaccines in well-characterized mouse models was proposed. Knowledge gaps requiring further research were identified. Recommendations were made to accelerate the development of an effective vaccine against melioidosis. PMID:25992835

  9. An intriguing case of locked jaw secondary to melioidosis.

    PubMed

    Vaid, Tejasvini; Rao, Karthik; Hande, Handattu Manjunath

    2015-01-01

    A 56-year-old woman presented with fever, pain and restriction of movement of the right temporomandibular joint. She was premorbidly diagnosed to have type 2 diabetes mellitus and rheumatoid arthritis. Local examination revealed a poorly demarcated severely tender, erythematous swelling in the right preauricular region. All haematological and biochemical investigations were within normal limits. MRI of the neck revealed the presence of a masticator space infection with intramuscular abscess involving the masseter and the temporalis muscles along with intracranial extension. Osteomyelitic changes were detected in the right mandibular condyle, temporal bone and in the temporomandibular joint. Melioidosis was suspected due to this unique clinical presentation of an abscess at an unusual and atypical site. Blood cultures identified the Gram-negative bacilli Burkholderia pseudomallei, which established the diagnosis of Melioidosis. Remarkable improvement was attained with antibiotics meropenem and cotrimoxazole, deferring the need for any surgical intervention. PMID:26628312

  10. Indirect hemagglutination assay in patients with melioidosis in northern Australia.

    PubMed

    Cheng, Allen C; O'brien, Mathew; Freeman, Kevin; Lum, Gary; Currie, Bart J

    2006-02-01

    Melioidosis is caused by the saprophytic organism Burkholderia pseudomallei. The use of the indirect hemagglutination assay (IHA) has found widespread use in areas endemic for this disease. Using this assay, we explored the serologic profile of 275 patients with culture-confirmed melioidosis in the Northern Territory of Australia. Based on a threshold titer of 1:40, the sensitivity of the IHA on admission was 56%. Female patients, those with positive blood cultures, and those with pneumonia independently predicted a negative IHA result. Most patients (68%) with negative admission IHA titers subsequently seroconverted. Most patients (92%) with positive admission IHA titers had persistently positive IHA titers. Relapses were not observed in 36 patients who had a negative IHA at least 1 month after admission, irrespective of initial admission IHA. The IHA has limited utility as a diagnostic test for acute disease, and most patients subsequently have persistently positive titers after recovery from illness.

  11. Neurologic melioidosis in an imported pigtail macaque (Macaca nemestrina).

    PubMed

    Ritter, J M; Sanchez, S; Jones, T L; Zaki, S R; Drew, C P

    2013-11-01

    Burkholderia pseudomallei is the cause of melioidosis in humans and other animals. Disease occurs predominately in Asia and Australia. It is rare in North America, and affected people and animals typically have a history of travel to (in human cases) or importation from (in animal cases) endemic areas. We describe the gross and histopathologic features and the microbiologic, molecular, and immunohistochemical diagnoses of a case of acute meningoencephalomyelitis and focal pneumonia caused by B. pseudomallei infection in a pigtail macaque that was imported from Indonesia to the United States for research purposes. This bacterium has been classified as a Tier 1 overlap select agent and toxin; therefore, recognition of pathologic features, along with accurate and timely confirmatory diagnostic testing, in naturally infected research animals is imperative to protect animals and personnel in the laboratory animal setting.

  12. Treatment and prophylaxis of melioidosis

    PubMed Central

    Dance, David

    2014-01-01

    Melioidosis, infection with Burkholderia pseudomallei, is being recognised with increasing frequency and is probably more common than currently appreciated. Treatment recommendations are based on a series of clinical trials conducted in Thailand over the past 25 years. Treatment is usually divided into two phases: in the first, or acute phase, parenteral drugs are given for ≥10 days with the aim of preventing death from overwhelming sepsis; in the second, or eradication phase, oral drugs are given, usually to complete a total of 20 weeks, with the aim of preventing relapse. Specific treatment for individual patients needs to be tailored according to clinical manifestations and response, and there remain many unanswered questions. Some patients with very mild infections can probably be cured by oral agents alone. Ceftazidime is the mainstay of acute-phase treatment, with carbapenems reserved for severe infections or treatment failures and amoxicillin/clavulanic acid (co-amoxiclav) as second-line therapy. Trimethoprim/sulfamethoxazole (co-trimoxazole) is preferred for the eradication phase, with the alternative of co-amoxiclav. In addition, the best available supportive care is needed, along with drainage of abscesses whenever possible. Treatment for melioidosis is unaffordable for many in endemic areas of the developing world, but the relative costs have reduced over the past decade. Unfortunately there is no likelihood of any new or cheaper options becoming available in the immediate future. Recommendations for prophylaxis following exposure to B. pseudomallei have been made, but the evidence suggests that they would probably only delay rather than prevent the development of infection. PMID:24613038

  13. Comprehensive identification of virulence factors required for respiratory melioidosis using Tn-seq mutagenesis

    PubMed Central

    Gutierrez, Maria G.; Yoder-Himes, Deborah R.; Warawa, Jonathan M.

    2015-01-01

    Respiratory melioidosis is a disease presentation of the biodefense pathogen, Burkholderia pseudomallei, which is frequently associated with a lethal septicemic spread of the bacteria. We have recently developed an improved respiratory melioidosis model to study the pathogenesis of Burkholderia pseudomallei in the lung (intubation-mediated intratracheal [IMIT] inoculation), which more closely models descriptions of human melioidosis, including prominent septicemic spread from the lung and reduced involvement of the upper respiratory tract. We previously demonstrated that the Type 3 Secretion System cluster 3 (T3SS3) is a critical virulence determinant for B. pseudomallei when delivered directly into the lung. We decided to comprehensively identify all virulence determinants required for respiratory melioidosis using the Tn-seq phenotypic screen, as well as to investigate which virulence determinants are required for dissemination to the liver and spleen. While previous studies have used Tn-seq to identify essential genes for in vitro cultured B. pseudomallei, this represents the first study to use Tn-seq to identify genes required for in vivo fitness. Consistent with our previous findings, we identified T3SS3 as the largest genetic cluster required for fitness in the lung. Furthermore, we identified capsular polysaccharide and Type 6 Secretion System cluster 5 (T6SS5) as the two additional major genetic clusters facilitating respiratory melioidosis. Importantly, Tn-seq did not identify additional, novel large genetic systems supporting respiratory melioidosis, although these studies identified additional small gene clusters that may also play crucial roles in lung fitness. Interestingly, other previously identified virulence determinants do not appear to be required for lung fitness, such as lipopolysaccharide. The role of T3SS3, capsule, and T6SS5 in lung fitness was validated by competition studies, but only T3SS3 was found to be important for respiratory

  14. Comprehensive identification of virulence factors required for respiratory melioidosis using Tn-seq mutagenesis.

    PubMed

    Gutierrez, Maria G; Yoder-Himes, Deborah R; Warawa, Jonathan M

    2015-01-01

    Respiratory melioidosis is a disease presentation of the biodefense pathogen, Burkholderia pseudomallei, which is frequently associated with a lethal septicemic spread of the bacteria. We have recently developed an improved respiratory melioidosis model to study the pathogenesis of Burkholderia pseudomallei in the lung (intubation-mediated intratracheal [IMIT] inoculation), which more closely models descriptions of human melioidosis, including prominent septicemic spread from the lung and reduced involvement of the upper respiratory tract. We previously demonstrated that the Type 3 Secretion System cluster 3 (T3SS3) is a critical virulence determinant for B. pseudomallei when delivered directly into the lung. We decided to comprehensively identify all virulence determinants required for respiratory melioidosis using the Tn-seq phenotypic screen, as well as to investigate which virulence determinants are required for dissemination to the liver and spleen. While previous studies have used Tn-seq to identify essential genes for in vitro cultured B. pseudomallei, this represents the first study to use Tn-seq to identify genes required for in vivo fitness. Consistent with our previous findings, we identified T3SS3 as the largest genetic cluster required for fitness in the lung. Furthermore, we identified capsular polysaccharide and Type 6 Secretion System cluster 5 (T6SS5) as the two additional major genetic clusters facilitating respiratory melioidosis. Importantly, Tn-seq did not identify additional, novel large genetic systems supporting respiratory melioidosis, although these studies identified additional small gene clusters that may also play crucial roles in lung fitness. Interestingly, other previously identified virulence determinants do not appear to be required for lung fitness, such as lipopolysaccharide. The role of T3SS3, capsule, and T6SS5 in lung fitness was validated by competition studies, but only T3SS3 was found to be important for respiratory

  15. Development of capsular polysaccharide-based glycoconjugates for immunization against melioidosis and glanders.

    PubMed

    Burtnick, Mary N; Heiss, Christian; Roberts, Rosemary A; Schweizer, Herbert P; Azadi, Parastoo; Brett, Paul J

    2012-01-01

    Burkholderia pseudomallei and Burkholderia mallei, the etiologic agents of melioidosis and glanders, respectively, cause severe disease in humans and animals and are considered potential agents of biological warfare and terrorism. Diagnosis and treatment of infections caused by these pathogens can be challenging and, in the absence of chemotherapeutic intervention, acute disease is frequently fatal. At present, there are no human or veterinary vaccines available for immunization against these emerging/re-emerging infectious diseases. One of the long term objectives of our research, therefore, is to identify and characterize protective antigens expressed by B. pseudomallei and B. mallei and use them to develop efficacious vaccine candidates. Previous studies have demonstrated that the 6-deoxy-heptan capsular polysaccharide (CPS) expressed by these bacterial pathogens is both a virulence determinant and a protective antigen. Consequently, this carbohydrate moiety has become an important component of the various subunit vaccines that we are currently developing in our laboratory. In the present study, we describe a reliable method for isolating CPS antigens from O-polysaccharide (OPS) deficient strains of B. pseudomallei; including a derivative of the select agent excluded strain Bp82. Utilizing these purified CPS samples, we also describe a simple procedure for covalently linking these T-cell independent antigens to carrier proteins. In addition, we demonstrate that high titer IgG responses can be raised against the CPS component of such constructs. Collectively, these approaches provide a tangible starting point for the development of novel CPS-based glycoconjugates for immunization against melioidosis and glanders.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2007-01-01

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

  18. Present and future therapeutic strategies for melioidosis and glanders.

    PubMed

    Estes, D Mark; Dow, Steven W; Schweizer, Herbert P; Torres, Alfredo G

    2010-03-01

    Burkholderia pseudomallei and Burkholderia mallei are the causative agents of melioidosis and glanders, respectively. Both Gram-negative pathogens are endemic in many parts of the world. Although natural acquisition of these pathogens is rare in the majority of countries, these bacteria have recently gained much interest because of their potential as bioterrorism agents. In modern times, their potential destructive impact on public health has escalated owing to the ability of these pathogens to cause opportunistic infections in diabetic and perhaps otherwise immunocompromised people, two growing populations worldwide. For both pathogens, severe infection in humans carries a high mortality rate, both species are recalcitrant to antibiotic therapy - B. pseudomallei more so than B. mallei - and no licensed vaccine exists for either prophylactic or therapeutic use. The potential malicious use of these organisms has accelerated the investigation of new ways to prevent and to treat the diseases. The availability of several B. pseudomallei and B. mallei genome sequences has greatly facilitated target identification and development of new therapeutics. This review provides a compilation of literature covering studies in antimelioidosis and antiglanders antimicrobial drug discovery, with a particular focus on potential novel therapeutic approaches to combat these diseases.

  19. Type 3 Secretion System Cluster 3 Is a Critical Virulence Determinant for Lung-Specific Melioidosis

    PubMed Central

    Gutierrez, Maria G.; Pfeffer, Tia L.; Warawa, Jonathan M.

    2015-01-01

    Burkholderia pseudomallei, the bacterial agent of melioidosis, causes disease through inhalation of infectious particles, and is classified as a Tier 1 Select Agent. Optical diagnostic imaging has demonstrated that murine respiratory disease models are subject to significant upper respiratory tract (URT) colonization. Because human melioidosis is not associated with URT colonization as a prominent presentation, we hypothesized that lung-specific delivery of B. pseudomallei may enhance our ability to study respiratory melioidosis in mice. We compared intranasal and intubation-mediated intratracheal (IMIT) instillation of bacteria and found that the absence of URT colonization correlates with an increased bacterial pneumonia and systemic disease progression. Comparison of the LD50 of luminescent B. pseudomallei strain, JW280, in intranasal and IMIT challenges of albino C57BL/6J mice identified a significant decrease in the LD50 using IMIT. We subsequently examined the LD50 of both capsular polysaccharide and Type 3 Secretion System cluster 3 (T3SS3) mutants by IMIT challenge of mice and found that the capsule mutant was attenuated 6.8 fold, while the T3SS3 mutant was attenuated 290 fold, demonstrating that T3SS3 is critical to respiratory melioidosis. Our previously reported intranasal challenge studies, which involve significant URT colonization, did not identify a dissemination defect for capsule mutants; however, we now report that capsule mutants exhibit significantly reduced dissemination from the lung following lung-specific instillation, suggesting that capsule mutants are competent to spread from the URT, but not the lung. We also report that a T3SS3 mutant is defective for dissemination following lung-specific delivery, and also exhibits in vivo growth defects in the lung. These findings highlight the T3SS3 as a critical virulence system for respiratory melioidosis, not only in the lung, but also for subsequent spread beyond the lung using a model system

  20. Validation of ten new polymorphic tandem repeat loci and application to the MLVA typing of Burkholderia pseudomallei isolates collected in Singapore from 1988 to 2004.

    PubMed

    Michelle Wong Su Yen; Lisanti, Olivier; Thibault, François; Toh Su San; Loh Gek Kee; Hilaire, Valérie; Jiali, Lim; Neubauer, Heinrich; Vergnaud, Gilles; Ramisse, Vincent

    2009-06-01

    Multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) has been shown to be very promising for the typing of Burkholderia pseudomallei and mallei. The currently available set of loci requires high resolution allele size measurement due to short repeat units. The present work was aimed at expanding the available set of VNTR loci, and generating data from a collection of 102 B. pseudomallei strains isolated in Singapore between 1988 and 2004 including few additional strains of various origins as references. Ten new VNTRs with a longer array size have been identified compatible with standard agarose gel separation, and a reference database of 72 genotypes was created which can be queried on the Internet.

  1. Comparative experimental subcutaneous glanders and melioidosis in the common marmoset (Callithrix jacchus)

    PubMed Central

    Nelson, Michelle; Salguero, Francisco J; Dean, Rachel E; Ngugi, Sarah A; Smither, Sophie J; Atkins, Timothy P; Lever, Mark S

    2014-01-01

    Glanders and melioidosis are caused by two distinct Burkholderia species and have generally been considered to have similar disease progression. While both of these pathogens are HHS/CDC Tier 1 agents, natural infection with both these pathogens is primarily through skin inoculation. The common marmoset (Callithrix jacchus) was used to compare disease following experimental subcutaneous challenge. Acute, lethal disease was observed in marmosets following challenge with between 26 and 1.2 × 108 cfu Burkholderia pseudomallei within 22–85 h. The reproducibility and progression of the disease were assessed following a challenge of 1 × 102 cfu of B. pseudomallei. Melioidosis was characterised by high levels of bacteraemia, focal microgranuloma progressing to non-necrotic multifocal solid lesions in the livers and spleens and multi-organ failure. Lethal disease was observed in 93% of animals challenged with Burkholderia mallei, occurring between 5 and 10.6 days. Following challenge with 1 × 102 cfu of B. mallei, glanders was characterised with lymphatic spread of the bacteria and non-necrotic, multifocal solid lesions progressing to a multifocal lesion with severe necrosis and pneumonia. The experimental results confirmed that the disease pathology and presentation is strikingly different between the two pathogens. The marmoset provides a model of the human syndrome for both diseases facilitating the development of medical countermeasures. PMID:25477002

  2. Comparative experimental subcutaneous glanders and melioidosis in the common marmoset (Callithrix jacchus).

    PubMed

    Nelson, Michelle; Salguero, Francisco J; Dean, Rachel E; Ngugi, Sarah A; Smither, Sophie J; Atkins, Timothy P; Lever, Mark S

    2014-12-01

    Glanders and melioidosis are caused by two distinct Burkholderia species and have generally been considered to have similar disease progression. While both of these pathogens are HHS/CDC Tier 1 agents, natural infection with both these pathogens is primarily through skin inoculation. The common marmoset (Callithrix jacchus) was used to compare disease following experimental subcutaneous challenge. Acute, lethal disease was observed in marmosets following challenge with between 26 and 1.2 × 10(8) cfu Burkholderia pseudomallei within 22-85 h. The reproducibility and progression of the disease were assessed following a challenge of 1 × 10(2) cfu of B. pseudomallei. Melioidosis was characterised by high levels of bacteraemia, focal microgranuloma progressing to non-necrotic multifocal solid lesions in the livers and spleens and multi-organ failure. Lethal disease was observed in 93% of animals challenged with Burkholderia mallei, occurring between 5 and 10.6 days. Following challenge with 1 × 10(2) cfu of B. mallei, glanders was characterised with lymphatic spread of the bacteria and non-necrotic, multifocal solid lesions progressing to a multifocal lesion with severe necrosis and pneumonia. The experimental results confirmed that the disease pathology and presentation is strikingly different between the two pathogens. The marmoset provides a model of the human syndrome for both diseases facilitating the development of medical countermeasures.

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

    PubMed

    Barman, Purabi; Kaur, Ravneet; Kumar, Kamlesh

    2013-01-01

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

  4. Triggering Receptor Expressed on Myeloid Cells (TREM)-2 Impairs Host Defense in Experimental Melioidosis

    PubMed Central

    Weehuizen, Tassili A. F.; Hommes, Tijmen J.; Lankelma, Jacqueline M.; de Jong, Hanna K.; Roelofs, Joris. J.T.H.; de Vos, Alex F.; Colonna, Marco; van der Poll, Tom; Wiersinga, W. Joost

    2016-01-01

    Background Triggering receptor expressed on myeloid cells (TREM) -1 and TREM-2 are key regulators of the inflammatory response that are involved in the clearance of invading pathogens. Melioidosis, caused by the "Tier 1" biothreat agent Burkholderia pseudomallei, is a common form of community-acquired sepsis in Southeast-Asia. TREM-1 has been suggested as a biomarker for sepsis and melioidosis. We aimed to characterize the expression and function of TREM-1 and TREM-2 in melioidosis. Methodology/Principal Findings Wild-type, TREM-1/3 (Trem-1/3-/-) and TREM-2 (Trem-2-/-) deficient mice were intranasally infected with live B. pseudomallei and killed after 24, and/or 72 h for the harvesting of lungs, liver, spleen, and blood. Additionally, survival studies were performed. Cellular functions were further analyzed by stimulation and/or infection of isolated cells. TREM-1 and TREM-2 expression was increased both in the lung and liver of B. pseudomallei-infected mice. Strikingly, Trem-2-/-, but not Trem-1/3-/-, mice displayed a markedly improved host defense as reflected by a strong survival advantage together with decreased bacterial loads, less inflammation and reduced organ injury. Cellular responsiveness of TREM-2, but not TREM-1, deficient blood and bone-marrow derived macrophages (BMDM) was diminished upon exposure to B. pseudomallei. Phagocytosis and intracellular killing of B. pseudomallei by BMDM and alveolar macrophages were TREM-1 and TREM-2-independent. Conclusions/Significance We found that TREM-2, and to a lesser extent TREM-1, plays a remarkable detrimental role in the host defense against a clinically relevant Gram-negative pathogen in mice: TREM-2 deficiency restricts the inflammatory response, thereby decreasing organ damage and mortality. PMID:27253382

  5. Comparison of efficacy of ciprofloxacin and doxycycline against experimental melioidosis and glanders.

    PubMed

    Russell, P; Eley, S M; Ellis, J; Green, M; Bell, D L; Kenny, D J; Titball, R W

    2000-06-01

    Melioidosis and glanders are caused by the closely related species Burkholderia pseudomallei and Burkholderia mallei, respectively. Whereas melioidosis is a significant cause of morbidity in south-east Asia, glanders is extremely rare. The efficacies of ciprofloxacin and doxycycline were assessed against a strain of B. pseudomallei and a strain of B. mallei which were susceptible to both antimicrobials in vitro. Porton outbred mice and Syrian hamsters were given 40 mg/kg of either doxycycline or ciprofloxacin twice daily by sc injection according to one of three regimens: dosing starting 48 h before challenge and continuing for 5 days postchallenge; 5 days' therapy starting immediately after challenge; 5 days' therapy starting 24 h after challenge. Mice were challenged ip with B. pseudomallei 4845 and hamsters were challenged ip with B. mallei 23344. Antimicrobial efficacy was determined by the shift in the median lethal dose (MLD). Ciprofloxacin prophylaxis and immediate therapy both raised the MLD of B. pseudomallei to 4 x 10(6) cfu from 19 cfu in untreated animals, but therapeutic ciprofloxacin only raised the MLD to 180 cfu. The results for doxycycline were similar. Ciprofloxacin prophylaxis raised the MLD of B. mallei 23344 to 4.6 x 10(5) cfu compared with 4 cfu in untreated controls. Immediate therapy raised the MLD to 7.0 x 10(4) cfu and therapy raised the MLD to 1.6 x 10(3) cfu. All regimens of doxycycline protected hamsters against challenges of up to 2 x 10(7) cfu. Despite using a susceptible strain of B. pseudomallei, neither antimicrobial was effective when used therapeutically. The timely administration of either antimicrobial, however, was effective in preventing symptomatic infection. Doxycycline was the superior of the two antimicrobials against experimental glanders although relapse did occur in treated animals approximately 4-5 weeks after challenge.

  6. Backbone chemical shift assignments for the sensor domain of the Burkholderia pseudomallei histidine kinase RisS – “missing” resonances at the dimer interface

    PubMed Central

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

    2015-01-01

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

  7. Melioidosis: reactivation during radiation therapy

    SciTech Connect

    Jegasothy, B.V.; Goslen, J.B.; Salvatore, M.A.

    1980-05-01

    Melioidosis is caused by Pseudomonas pseudomallei, a gram-negative, motile bacillus which is a naturally occurring soil saprophyte. The organism is endemic in Southeast Asia, the Philippines, Australia, and parts of Central and South America. Most human disease occurs from infection acquired in these countries. Infection with P pseudomallei may produce no apparent clinical disease. Acute pneumonitis or septicemia may result from inhalation of the organism, and inoculation into sites of trauma may cause localized skin abscesses, or the disease may remain latent and be reactivated months or years later by trauma, burns, or pneumococcal pneumonia, diabetic ketoacidosis, influenza, or bronchogenic carcinoma. The last is probably the commonest form of melioidosis seen in the United States. We present the first case of reactivation of melioidosis after radiation therapy for carcinoma of the lung, again emphasizing the need to consider melioidosis in a septic patient with a history of travel, especially to Southeast Asia.

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

    PubMed Central

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

    2015-01-01

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

  9. Beclin 1 is required for starvation-enhanced, but not rapamycin-enhanced, LC3-associated phagocytosis of Burkholderia pseudomallei in RAW 264.7 cells.

    PubMed

    Li, Xuelei; Prescott, Mark; Adler, Ben; Boyce, John D; Devenish, Rodney J

    2013-01-01

    LC3-associated phagocytosis (LAP) of Burkholderia pseudomallei by murine macrophage (RAW 264.7) cells is an intracellular innate defense mechanism. Beclin 1, a protein with several roles in autophagic processes, is known to be recruited to phagosomal membranes as a very early event in LAP. We sought to determine whether knockdown of Beclin 1 by small interfering RNA (siRNA) would affect recruitment of LC3 and subsequent LAP of infecting B. pseudomallei. Both starvation and rapamycin treatment can induce Beclin 1-dependent autophagy. Therefore, we analyzed the consequences of Beclin 1 knockdown for LAP in infected cells that had been either starved or treated with rapamycin by determining the levels of bacterial colocalization with LC3 and intracellular survival. Concurrently, we confirmed the location of bacteria as either contained in phagosomes or free in the cytoplasm. We found that both rapamycin and starvation treatment enhanced LAP of B. pseudomallei but that the rapamycin response is Beclin 1 independent whereas the starvation response is Beclin 1 dependent.

  10. Nitric Oxide from IFNγ-Primed Macrophages Modulates the Antimicrobial Activity of β-Lactams against the Intracellular Pathogens Burkholderia pseudomallei and Nontyphoidal Salmonella

    PubMed Central

    Jones-Carson, Jessica; Zweifel, Adrienne E.; Tapscott, Timothy; Austin, Chad; Brown, Joseph M.; Jones, Kenneth L.; Voskuil, Martin I.; Vázquez-Torres, Andrés

    2014-01-01

    Our investigations show that nonlethal concentrations of nitric oxide (NO) abrogate the antibiotic activity of β-lactam antibiotics against Burkholderia pseudomallei, Escherichia coli and nontyphoidal Salmonella enterica serovar Typhimurium. NO protects B. pseudomallei already exposed to β-lactams, suggesting that this diatomic radical tolerizes bacteria against the antimicrobial activity of this important class of antibiotics. The concentrations of NO that elicit antibiotic tolerance repress consumption of oxygen (O2), while stimulating hydrogen peroxide (H2O2) synthesis. Transposon insertions in genes encoding cytochrome c oxidase-related functions and molybdenum assimilation confer B. pseudomallei a selective advantage against the antimicrobial activity of the β-lactam antibiotic imipenem. Cumulatively, these data support a model by which NO induces antibiotic tolerance through the inhibition of the electron transport chain, rather than by potentiating antioxidant defenses as previously proposed. Accordingly, pharmacological inhibition of terminal oxidases and nitrate reductases tolerizes aerobic and anaerobic bacteria to β-lactams. The degree of NO-induced β-lactam antibiotic tolerance seems to be inversely proportional to the proton motive force (PMF), and thus the dissipation of ΔH+ and ΔΨ electrochemical gradients of the PMF prevents β-lactam-mediated killing. According to this model, NO generated by IFNγ-primed macrophages protects intracellular Salmonella against imipenem. On the other hand, sublethal concentrations of imipenem potentiate the killing of B. pseudomallei by NO generated enzymatically from IFNγ-primed macrophages. Our investigations indicate that NO modulates the antimicrobial activity of β-lactam antibiotics. PMID:25121731

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

    PubMed Central

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

    2016-01-01

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

  12. Imported melioidosis with an isolated cutaneous presentation in a 90-year-old traveller from Bangladesh.

    PubMed

    Ezzedine, K; Malvy, D; Steels, E; De Dobbeeler, G; Struelens, M; Jacobs, F; Heenen, M

    2007-02-01

    Melioidosis is a tropical disease caused by infection with the bacterium Burkholderia pseudomallei. Most cases present as an acute febrile illness with severe pneumonia and sepsis. Sub-acute and late onset disease can also occur Melioidosis has been diagnosed among travellers who contracted the disease while staying in endemic areas during the rainy season. We report a case of travel-associated B. pseudomallei cutaneous infection in a febrile 90-year-old woman with diabetes mellitus, with early stage manifestations of an isolated inoculation lesion. A 32 weeks' treatment with oral amoxicillin-clavulanate and doxycycline combination regimen led to resolution of the lesion and lack of relapse over fifteen months of follow-up. Melioidosis should be considered in the differential diagnosis of unusual subacute cutaneous lesions in a febrile patients returning from endemic areas, as successful management largely depends on early diagnosis and specific long-term suppressive antimicrobial therapy at an early stage of the course of the disease. PMID:17402688

  13. Protection against Experimental Melioidosis with a Synthetic manno-Heptopyranose Hexasaccharide Glycoconjugate

    PubMed Central

    2016-01-01

    Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei and is associated with high morbidity and mortality rates in endemic areas. Antibiotic treatment is protracted and not always successful; even with appropriate therapy, up to 40% of individuals presenting with melioidosis in Thailand succumb to infection. In these circumstances, an effective vaccine has the potential to have a dramatic impact on both the scale and the severity of disease. Currently, no vaccines are licensed for human use. A leading vaccine candidate is the capsular polysaccharide consisting of a homopolymer of unbranched 1→3 linked 2-O-acetyl-6-deoxy-β-d-manno-heptopyranose. Here, we present the chemical synthesis of this challenging antigen using a novel modular disaccharide assembly approach. The resulting hexasaccharide was coupled to the nontoxic Hc domain of tetanus toxin as a carrier protein to promote recruitment of T-cell help and provide a scaffold for antigen display. Mice immunized with the glycoconjugate developed IgM and IgG responses capable of recognizing native capsule, and were protected against infection with over 120 × LD50 of B. pseudomallei strain K96243. This is the first report of the chemical synthesis of an immunologically relevant and protective hexasaccharide fragment of the capsular polysaccharide of B. pseudomallei and serves as the rational starting point for the development of an effective licensed vaccine for this emerging infectious disease. PMID:27124182

  14. Comparison of Five Commercial Nucleic Acid Extraction Kits for the PCR-based Detection of Burkholderia Pseudomallei DNA in Formalin-Fixed, Paraffin-Embedded Tissues

    PubMed Central

    Obersteller, Sonja; Neubauer, Heinrich; Hagen, Ralf Matthias; Frickmann, Hagen

    2016-01-01

    The extraction and further processing of nucleic acids (NA) from formalin-fixed paraffin-embedded (FFPE) tissues for microbiological diagnostic polymerase chain reaction (PCR) approaches is challenging. Here, we assessed the effects of five different commercially available nucleic acid extraction kits on the results of real-time PCR. FFPE samples from organs of Burkholderia pseudomallei-infected Swiss mice were subjected to processing with five different extraction kits from QIAGEN (FFPE DNA Tissue Kit, EZ1 DNA Tissue Kit, DNA Mini Kit, DNA Blood Mini Kit, and FlexiGene DNA Kit) in combination with three different real-time PCRs targeting B. pseudomallei-specific sequences of varying length after 16 years of storage. The EZ1 DNA Tissue Kit and the DNA Mini Kit scored best regarding the numbers of successful PCR reactions. In case of positive PCR, differences regarding the cycle-threshold (Ct) values were marginal. The impact of the applied extraction kits on the reliability of PCR from FFPE material seems to be low. Interfering factors like the quality of the dewaxing procedure or the sample age appear more important than the selection of specialized FFPE kits. PMID:27766174

  15. Tracing melioidosis back to the source: using whole-genome sequencing to investigate an outbreak originating from a contaminated domestic water supply.

    PubMed

    McRobb, Evan; Sarovich, Derek S; Price, Erin P; Kaestli, Mirjam; Mayo, Mark; Keim, Paul; Currie, Bart J

    2015-04-01

    Melioidosis, a disease of public health importance in Southeast Asia and northern Australia, is caused by the Gram-negative soil bacillus Burkholderia pseudomallei. Melioidosis is typically acquired through environmental exposure, and case clusters are rare, even in regions where the disease is endemic. B. pseudomallei is classed as a tier 1 select agent by the Centers for Disease Control and Prevention; from a biodefense perspective, source attribution is vital in an outbreak scenario to rule out a deliberate release. Two cases of melioidosis within a 3-month period at a residence in rural northern Australia prompted an investigation to determine the source of exposure. B. pseudomallei isolates from the property's groundwater supply matched the multilocus sequence type of the clinical isolates. Whole-genome sequencing confirmed the water supply as the probable source of infection in both cases, with the clinical isolates differing from the likely infecting environmental strain by just one single nucleotide polymorphism (SNP) each. For the first time, we report a phylogenetic analysis of genomewide insertion/deletion (indel) data, an approach conventionally viewed as problematic due to high mutation rates and homoplasy. Our whole-genome indel analysis was concordant with the SNP phylogeny, and these two combined data sets provided greater resolution and a better fit with our epidemiological chronology of events. Collectively, this investigation represents a highly accurate account of source attribution in a melioidosis outbreak and gives further insight into a frequently overlooked reservoir of B. pseudomallei. Our methods and findings have important implications for outbreak source tracing of this bacterium and other highly recombinogenic pathogens. PMID:25631791

  16. Tracing Melioidosis Back to the Source: Using Whole-Genome Sequencing To Investigate an Outbreak Originating from a Contaminated Domestic Water Supply

    PubMed Central

    McRobb, Evan; Kaestli, Mirjam; Mayo, Mark; Keim, Paul

    2015-01-01

    Melioidosis, a disease of public health importance in Southeast Asia and northern Australia, is caused by the Gram-negative soil bacillus Burkholderia pseudomallei. Melioidosis is typically acquired through environmental exposure, and case clusters are rare, even in regions where the disease is endemic. B. pseudomallei is classed as a tier 1 select agent by the Centers for Disease Control and Prevention; from a biodefense perspective, source attribution is vital in an outbreak scenario to rule out a deliberate release. Two cases of melioidosis within a 3-month period at a residence in rural northern Australia prompted an investigation to determine the source of exposure. B. pseudomallei isolates from the property's groundwater supply matched the multilocus sequence type of the clinical isolates. Whole-genome sequencing confirmed the water supply as the probable source of infection in both cases, with the clinical isolates differing from the likely infecting environmental strain by just one single nucleotide polymorphism (SNP) each. For the first time, we report a phylogenetic analysis of genomewide insertion/deletion (indel) data, an approach conventionally viewed as problematic due to high mutation rates and homoplasy. Our whole-genome indel analysis was concordant with the SNP phylogeny, and these two combined data sets provided greater resolution and a better fit with our epidemiological chronology of events. Collectively, this investigation represents a highly accurate account of source attribution in a melioidosis outbreak and gives further insight into a frequently overlooked reservoir of B. pseudomallei. Our methods and findings have important implications for outbreak source tracing of this bacterium and other highly recombinogenic pathogens. PMID:25631791

  17. Tracing melioidosis back to the source: using whole-genome sequencing to investigate an outbreak originating from a contaminated domestic water supply.

    PubMed

    McRobb, Evan; Sarovich, Derek S; Price, Erin P; Kaestli, Mirjam; Mayo, Mark; Keim, Paul; Currie, Bart J

    2015-04-01

    Melioidosis, a disease of public health importance in Southeast Asia and northern Australia, is caused by the Gram-negative soil bacillus Burkholderia pseudomallei. Melioidosis is typically acquired through environmental exposure, and case clusters are rare, even in regions where the disease is endemic. B. pseudomallei is classed as a tier 1 select agent by the Centers for Disease Control and Prevention; from a biodefense perspective, source attribution is vital in an outbreak scenario to rule out a deliberate release. Two cases of melioidosis within a 3-month period at a residence in rural northern Australia prompted an investigation to determine the source of exposure. B. pseudomallei isolates from the property's groundwater supply matched the multilocus sequence type of the clinical isolates. Whole-genome sequencing confirmed the water supply as the probable source of infection in both cases, with the clinical isolates differing from the likely infecting environmental strain by just one single nucleotide polymorphism (SNP) each. For the first time, we report a phylogenetic analysis of genomewide insertion/deletion (indel) data, an approach conventionally viewed as problematic due to high mutation rates and homoplasy. Our whole-genome indel analysis was concordant with the SNP phylogeny, and these two combined data sets provided greater resolution and a better fit with our epidemiological chronology of events. Collectively, this investigation represents a highly accurate account of source attribution in a melioidosis outbreak and gives further insight into a frequently overlooked reservoir of B. pseudomallei. Our methods and findings have important implications for outbreak source tracing of this bacterium and other highly recombinogenic pathogens.

  18. Melioidosis Vaccines: A Systematic Review and Appraisal of the Potential to Exploit Biodefense Vaccines for Public Health Purposes

    PubMed Central

    Lubell, Yoel; Koh, Gavin C. K. W.; White, Lisa J.; Day, Nicholas P. J.; Titball, Richard W.

    2012-01-01

    Background Burkholderia pseudomallei is a Category B select agent and the cause of melioidosis. Research funding for vaccine development has largely considered protection within the biothreat context, but the resulting vaccines could be applicable to populations who are at risk of naturally acquired melioidosis. Here, we discuss target populations for vaccination, consider the cost-benefit of different vaccination strategies and review potential vaccine candidates. Methods and Findings Melioidosis is highly endemic in Thailand and northern Australia, where a biodefense vaccine might be adopted for public health purposes. A cost-effectiveness analysis model was developed, which showed that a vaccine could be a cost-effective intervention in Thailand, particularly if used in high-risk populations such as diabetics. Cost-effectiveness was observed in a model in which only partial immunity was assumed. The review systematically summarized all melioidosis vaccine candidates and studies in animal models that had evaluated their protectiveness. Possible candidates included live attenuated, whole cell killed, sub-unit, plasmid DNA and dendritic cell vaccines. Live attenuated vaccines were not considered favorably because of possible reversion to virulence and hypothetical risk of latent infection, while the other candidates need further development and evaluation. Melioidosis is acquired by skin inoculation, inhalation and ingestion, but routes of animal inoculation in most published studies to date do not reflect all of this. We found a lack of studies using diabetic models, which will be central to any evaluation of a melioidosis vaccine for natural infection since diabetes is the most important risk factor. Conclusion Vaccines could represent one strand of a public health initiative to reduce the global incidence of melioidosis. PMID:22303489

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

    PubMed

    Molchanova, E V; Ageeva, N P

    2015-02-01

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

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

    PubMed

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

    2015-07-01

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

  1. Environmental management procedures following fatal melioidosis in a captive chimpanzee (Pan troglodytes).

    PubMed

    Sommanustweechai, Angkana; Kasantikul, Tanit; Somsa, Wachirawit; Wongratanacheewin, Surasakdi; Sermswan, Rasana W; Kongmakee, Piyaporn; Thomas, Warissara; Kamolnorranath, Sumate; Siriaroonrat, Boripat; Bush, Mitchell; Banlunara, Wijit

    2013-06-01

    A 40-yr-old male captive chimpanzee (Pan troglodytes) presented with depression and anorexia for 7 days. The tentative diagnosis, following a physical examination under anesthesia, was pneumonia with sepsis. Despite antibiotic treatment and supportive care the chimpanzee died a week following presentation. Gross pathology confirmed severe purulent pneumonia and diffuse hepatosplenic abscesses. Detected in serum at the time of the initial examination, the melioidosis serum antibody titer was elevated (> 1:512). Soil samples were collected from three sites in the exhibit at three depths of 5, 15, and 30 cm. By direct and enrichment culture, positive cultures for Burkholderia pseudomallei were found at 5 and 15 cm in one site. The other two sites were positive by enrichment culture at the depth of 5 cm. To prevent disease in the remaining seven troop members, they were relocated to permit a soil treatment with calcium oxide. The exhibit remained empty for approximately 1 yr before the chimpanzees were returned. During that period, the soil in the exhibit area was again cultured as before and all samples were negative for B. pseudomallei. Following the soil treatment in the exhibit, all chimpanzees have remained free of clinical signs consistent with melioidosis.

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

    PubMed Central

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

    2014-01-01

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

  3. The Epidemiology and Clinical Spectrum of Melioidosis: 540 Cases from the 20 Year Darwin Prospective Study

    PubMed Central

    Currie, Bart J.; Ward, Linda; Cheng, Allen C.

    2010-01-01

    Background Over 20 years, from October 1989, the Darwin prospective melioidosis study has documented 540 cases from tropical Australia, providing new insights into epidemiology and the clinical spectrum. Principal Findings The principal presentation was pneumonia in 278 (51%), genitourinary infection in 76 (14%), skin infection in 68 (13%), bacteremia without evident focus in 59 (11%), septic arthritis/osteomyelitis in 20 (4%) and neurological melioidosis in 14 (3%). 298 (55%) were bacteremic and 116 (21%) developed septic shock (58 fatal). Internal organ abscesses and secondary foci in lungs and/or joints were common. Prostatic abscesses occurred in 76 (20% of 372 males). 96 (18%) had occupational exposure to Burkholderia pseudomallei. 118 (22%) had a specific recreational or occupational incident considered the likely infecting event. 436 (81%) presented during the monsoonal wet season. The higher proportion with pneumonia in December to February supports the hypothesis of infection by inhalation during severe weather events. Recurrent melioidosis occurred in 29, mostly attributed to poor adherence to therapy. Mortality decreased from 30% in the first 5 years to 9% in the last five years (p<0.001). Risk factors for melioidosis included diabetes (39%), hazardous alcohol use (39%), chronic lung disease (26%) and chronic renal disease (12%). There was no identifiable risk factor in 20%. Of the 77 fatal cases (14%), 75 had at least one risk factor; the other 2 were elderly. On multivariate analysis of risk factors, age, location and season, the only independent predictors of mortality were the presence of at least one risk factor (OR 9.4; 95% CI 2.3–39) and age ≥50 years (OR 2.0; 95% CI 1.2–2.3). Conclusions Melioidosis should be seen as an opportunistic infection that is unlikely to kill a healthy person, provided infection is diagnosed early and resources are available to provide appropriate antibiotics and critical care. PMID:21152057

  4. Retrospective Study on Fatal Melioidosis in Captive Zoo Animals in Thailand.

    PubMed

    Kasantikul, T; Sommanustweechai, A; Polsrila, K; Kongkham, W; Chaisongkram, C; Sanannu, S; Kongmakee, P; Narongwanichgarn, W; Bush, M; Sermswan, R W; Banlunara, W

    2016-10-01

    Melioidosis is caused by Burkholderia pseudomallei and is an important zoonotic infectious disease causing high mortality from fulminant septicaemia in humans and a wide variety of animal species. The incidence of fatal melioidosis in zoo animals has been significant in many Thai zoos. A total number of 32 cases were evaluated throughout the Thai zoo animal populations. The highest prevalence of disease has been reported from the north-eastern region followed by the zoos in the southern part of the country, approximately 47% and 38%, respectively, while the other zoos reported sporadic infections. Herbivores and non-human primates were the most commonly affected animals with incidences of 59% and 28%, respectively. This appears to be a seasonal correlation with the highest incidence of melioidosis in zoo animals reported in the rainy season (44%) or subdivided monthly in June (19%) followed by September and November (16% and 12%, respectively). The route of infection and the incubation period still remain unclear. This retrospective study examined the clinical presentation in various zoo species, pathological findings and epidemiological data as well as conducting an in depth literature review.

  5. [In vitro antibiotic susceptibility compliance with efficacy of chemotherapy in infections due to pathogenic Burkholderias].

    PubMed

    Iliukhin, V I; Senina, T V; Trushkina, M N; Shubnikova, E V; Antonov, Iu V; Andropova, N V

    2009-01-01

    Among the known species of Burkholderia only two are obligate pathogens, i.e., B. mallei and B. pseudomallei, causative agents of glanders and melioidosis respectively. The other species are saprophytes as natural inhabitants of water reservoirs and soil, still capable of causing opportunistic infections in humans and animals under definite conditions. All the species of Burkholderia are characterized by high resistance to antibacterials, including antibiotics. By the MICs, the most efficient chemotherapeutics against pathogenic burkholderias are tetracyclines, fluoroquinolones, penems and combined sulfanilamides. In the treatment of experimental glanders and melioidosis the set of the effective drugs had the inverse variation dependence on the infection severity and the desease process rate. Co-trimoxasole showed the best results, then followed doxicycline, ciprofioxacin and ceftazidime in the diminishing succession. The modification of the method for determination of antibiotic susceptibility with addition of native blood to the medium and the subculture under the atmosphere of 5% CO2 was shown useful in estimation of the prospects of the use of chemotherapeutics for the treatment of Burkholderia infections. PMID:20201399

  6. Influence of the molybdenum cofactor biosynthesis on anaerobic respiration, biofilm formation and motility in Burkholderia thailandensis.

    PubMed

    Andreae, Clio A; Titball, Richard W; Butler, Clive S

    2014-01-01

    Burkholderia thailandensis is closely related to Burkholderia pseudomallei, a bacterial pathogen and the causative agent of melioidosis. B. pseudomallei can survive and persist within a hypoxic environment for up to one year and has been shown to grow anaerobically in the presence of nitrate. Currently, little is known about the role of anaerobic respiration in pathogenesis of melioidosis. Using B. thailandensis as a model, a library of 1344 transposon mutants was created to identify genes required for anaerobic nitrate respiration. One transposon mutant (CA01) was identified with an insertion in BTH_I1704 (moeA), a gene required for the molybdopterin biosynthetic pathway. This pathway is involved in the synthesis of a molybdopterin cofactor required for a variety of molybdoenzymes, including nitrate reductase. Disruption of molybdopterin biosynthesis prevented growth under anaerobic conditions, when using nitrate as the sole terminal electron acceptor. Defects in anaerobic respiration, nitrate reduction, motility and biofilm formation were observed for CA01. Mutant complementation with pDA-17:BTH_I1704 was able to restore anaerobic growth on nitrate, nitrate reductase activity and biofilm formation, but did not restore motility. This study highlights the potential importance of molybdoenzyme-dependent anaerobic respiration in the survival and virulence of B. thailandensis.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2006-01-01

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

  9. Barriers and Recommended Interventions to Prevent Melioidosis in Northeast Thailand: A Focus Group Study Using the Behaviour Change Wheel

    PubMed Central

    Suntornsut, Pornpan; Wongsuwan, Nittayasee; Malasit, Mayura; Kitphati, Rungreung; Michie, Susan; Peacock, Sharon J.

    2016-01-01

    Background Melioidosis, an often fatal infectious disease in Northeast Thailand, is caused by skin inoculation, inhalation or ingestion of the environmental bacterium, Burkholderia pseudomallei. The major underlying risk factor for melioidosis is diabetes mellitus. Recommendations for melioidosis prevention include using protective gear such as rubber boots and gloves when in direct contact with soil and environmental water, and consuming bottled or boiled water. Only a small proportion of people follow such recommendations. Methods Nine focus group discussions were conducted to evaluate barriers to adopting recommended preventive behaviours. A total of 76 diabetic patients from northeast Thailand participated in focus group sessions. Barriers to adopting the recommended preventive behaviours and future intervention strategies were identified using two frameworks: the Theoretical Domains Framework and the Behaviour Change Wheel. Results Barriers were identified in the following five domains: (i) knowledge, (ii) beliefs about consequences, (iii) intention and goals, (iv) environmental context and resources, and (v) social influence. Of 76 participants, 72 (95%) had never heard of melioidosis. Most participants saw no harm in not adopting recommended preventive behaviours, and perceived rubber boots and gloves to be hot and uncomfortable while working in muddy rice fields. Participants reported that they normally followed the behaviour of friends, family and their community, the majority of whom did not wear boots while working in rice fields and did not boil water before drinking. Eight intervention functions were identified as relevant for the intervention: (i) education, (ii) persuasion, (iii) incentivisation, (iv) coercion, (v) modeling, (vi) environmental restructuring, (vii) training, and (viii) enablement. Participants noted that input from role models in the form of physicians, diabetic clinics, friends and families, and from the government via mass media

  10. A Thrombomodulin Mutation that Impairs Active Protein C Generation Is Detrimental in Severe Pneumonia-Derived Gram-Negative Sepsis (Melioidosis)

    PubMed Central

    Kager, Liesbeth M.; Wiersinga, W. Joost; Roelofs, Joris J. T. H.; de Boer, Onno J.; Weiler, Hartmut; van 't Veer, Cornelis; van der Poll, Tom

    2014-01-01

    Background During severe (pneumo)sepsis inflammatory and coagulation pathways become activated as part of the host immune response. Thrombomodulin (TM) is involved in a range of host defense mechanisms during infection and plays a pivotal role in activation of protein C (PC) into active protein C (APC). APC has both anticoagulant and anti-inflammatory properties. In this study we investigated the effects of impaired TM-mediated APC generation during melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia caused by Burkholderia (B.) pseudomallei. Methodology/Principal Findings (WT) mice and mice with an impaired capacity to activate protein C due to a point mutation in their Thbd gene (TMpro/pro mice) were intranasally infected with B. pseudomallei and sacrificed after 24, 48 or 72 hours for analyses. Additionally, survival studies were performed. When compared to WT mice, TMpro/pro mice displayed a worse survival upon infection with B. pseudomallei, accompanied by increased coagulation activation, enhanced lung neutrophil influx and bronchoalveolar inflammation at late time points, together with increased hepatocellular injury. The TMpro/pro mutation had limited if any impact on bacterial growth and dissemination. Conclusion/Significance TM-mediated protein C activation contributes to protective immunity after infection with B. pseudomallei. These results add to a better understanding of the regulation of the inflammatory and procoagulant response during severe Gram-negative (pneumo)sepsis. PMID:24762740

  11. Outbreak of melioidosis and leptospirosis co-infection following a rescue operation.

    PubMed

    Sapian, M; Khair, M T; How, S H; Rajalingam, R; Sahhir, K; Norazah, A; Khebir, V; Jamalludin, A R

    2012-06-01

    We analyzed the epidemiological data of all people who were involved in the search and rescue operation in Lubuk Yu, a natural recreational forest with waterfall and stream. The hospital admission records of the cases who fulfilled the case definition and the environmental samples result taken at Lubuk Yu recreational area were studied. 153 people were exposed to this outbreak, 85 (55.5%) were professional rescuers from various government agencies and 68 (44.5%) were villagers. 21 fulfilled the case definition. Ten cases were confirmed melioidosis, six melioidosis alone and four coinfected with leptospirosis. There were eight deaths in this outbreak, seven were villagers and one professional rescuer. Overall case fatality was 70%. All confirmed melioidosis cases and seven who died had diabetes mellitus. The morbidity rate were higher among the villagers, 23.5% compared to professional rescuers, 5.9%. The case fatality rate were also higher in this group which was 100% compared to 33.3% in professional rescuers. The soil and water samples in Lubuk Yu recreational area were positive for leptospira and Burkholderia pseudomallei. The presence of co-infection and co-morbidities especially diabetes mellitus among the exposed led to the high mortality in this outbreak hence a high index of suspicion is important among the healthcare professionals in the management of melioidosis cases. To avoid similar incident in future, search and rescue operation should be only conducted by professional rescuers with appropriate personal protective equipment. A register of rescuers should be maintained for surveillance and follow up if necessary. PMID:23082420

  12. Immune Modulation as an Effective Adjunct Post-exposure Therapeutic for B. pseudomallei

    PubMed Central

    Afzali, Maryam F.; Cummings, Jason E.; Legare, Marie E.; Tjalkens, Ronald B.; Allen, Christopher P.; Slayden, Richard A.; Hanneman, William H.

    2016-01-01

    Melioidosis is caused by the facultative intracellular bacterium Burkholderia pseudomallei and is potentially fatal. Despite a growing global burden and high fatality rate, little is known about the disease. Recent studies demonstrate that cyclooxygenase-2 (COX-2) inhibition is an effective post-exposure therapeutic for pulmonary melioidosis, which works by inhibiting the production of prostaglandin E2 (PGE2). This treatment, while effective, was conducted using an experimental COX-2 inhibitor that is not approved for human or animal use. Therefore, an alternative COX-2 inhibitor needs to be identified for further studies. Tolfenamic acid (TA) is a non-steroidal anti-inflammatory drug (NSAID) COX-2 inhibitor marketed outside of the United States for the treatment of migraines. While this drug was developed for COX-2 inhibition, it has been found to modulate other aspects of inflammation as well. In this study, we used RAW 264.7 cells infected with B pseudomallei to analyze the effect of TA on cell survival, PGE2 production and regulation of COX-2 and nuclear factor- kappaB (NF-ĸB) protein expression. To evaluate the effectiveness of post-exposure treatment with TA, results were compared to Ceftazidime (CZ) treatments alone and the co-treatment of TA with a sub-therapeutic treatment of CZ determined in a study of BALB/c mice. Results revealed an increase in cell viability in vitro with TA and were able to reduce both COX-2 expression and PGE2 production while also decreasing NF-ĸB activation during infection. Co-treatment of orally administered TA and a sub-therapeutic treatment of CZ significantly increased survival outcome and cleared the bacterial load within organ tissue. Additionally, we demonstrated that post-exposure TA treatment with sub-therapeutic CZ is effective to treat melioidosis in BALB/c mice. PMID:27792775

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

    PubMed

    Lewis, Eric R G; Torres, Alfredo G

    2016-08-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Casey, William Thomas; McClean, Siobhán

    2015-01-01

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

  16. Whole-Genome Assemblies of 56 Burkholderia Species

    PubMed Central

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

    2014-01-01

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

  17. [Glanders, melioidosis and biowarfare].

    PubMed

    Guilhot, Amélie; Bricaire, François; Bossi, Philippe

    2005-01-29

    MANY COMMON FACTORS: Glanders and melioidosis are infectious diseases that are caused by the bacteria of the Burkholderia species. These infections are endemic in tropical regions and can lead to la broad spectrum of common clinical manifestations. TWO PRINCIPLE CLINICAL FORMS: The most frequent clinical presentation is the pulmonary form, which can mimic pulmonary tuberculosis. The septicemic form is the most severe form, and lethal in nearly 50% of cases. WEAPONS FOR BIOTERRORISM AND WAR: Very few organisms are required to cause disease by aerosolisation, which could be the main route of contamination for humans after a deliberate release. This property has permitted yet the use of these bacteria as biological warfare weapon during the past century. We have to consider these agents as possible biological warfare agents. Europeans guidelines for treatment and post-exposure prophylaxis are detailed.

  18. Ribotype analysis of Pseudomonas pseudomallei isolates.

    PubMed Central

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

    1993-01-01

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

  19. Siderophore production by Pseudomonas pseudomallei.

    PubMed Central

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

    1991-01-01

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

  20. Melioidosis: the tip of the iceberg?

    PubMed Central

    Dance, D A

    1991-01-01

    For nearly 80 years clinical melioidosis has been considered a rare disease. This bacterial infection is caused by Pseudomonas pseudomallei, a saprophyte found in soil and surface water of endemic areas. Consequently, those who have most contact with soil, the rural poor, are likely to be at greatest risk of infection. Since the diversity of clinical manifestations necessitates the isolation and identification of the causative organism for a definitive diagnosis of melioidosis and the population at greatest risk within endemic areas rarely have access to an appropriate level of health care, the disease has probably been underrecognized. Melioidosis is now known to be an important cause of human morbidity and mortality in Thailand, and this may be true throughout Southeast Asia, which is usually regarded as the main endemic area for the disease. In Australia, melioidosis causes a smaller number of human infections, while disease among livestock has important economic and possible public health implications. Sporadic reports of the infection indicate its presence in several other tropical regions: in the Indian subcontinent, Africa, and Central and South America. Clinical melioidosis may be highly prevalent in these areas, but underdiagnosed as a result of a lack of awareness of the clinical and microbiological features of the disease, or simply because of a lack of health care facilities. Furthermore, during the last two decades the importation and transmission of melioidosis within nontropical zones have been documented. The causative organism is not difficult to grow, and modern antibiotics have improved disease prognosis. Further studies are needed to determine the true worldwide distribution and prevalence of melioidosis so that improved therapeutic and preventive measures can be developed and applied. PMID:2004347

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

    SciTech Connect

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

    2015-04-30

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

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

    PubMed Central

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

    2015-01-01

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

  3. Characterization of cellular immune response and innate immune signaling in human and nonhuman primate primary mononuclear cells exposed to Burkholderia mallei.

    PubMed

    Alam, Shahabuddin; Amemiya, Kei; Bernhards, Robert C; Ulrich, Robert G; Waag, David M; Saikh, Kamal U

    2015-01-01

    Burkholderia pseudomallei infection causes melioidosis and is often characterized by severe sepsis. Although rare in humans, Burkholderia mallei has caused infections in laboratory workers, and the early innate cellular response to B. mallei in human and nonhuman primates has not been characterized. In this study, we examined the primary cellular immune response to B. mallei in PBMC cultures of non-human primates (NHPs), Chlorocebus aethiops (African Green Monkeys), Macaca fascicularis (Cynomolgus macaque), and Macaca mulatta (Rhesus macaque) and humans. Our results demonstrated that B. mallei elicited strong primary pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β, and IL-6) equivalent to the levels of B. pseudomallei in primary PBMC cultures of NHPs and humans. When we examined IL-1β and other cytokine responses by comparison to Escherichia coli LPS, African Green Monkeys appears to be most responsive to B. mallei than Cynomolgus or Rhesus. Characterization of the immune signaling mechanism for cellular response was conducted by using a ligand induced cell-based reporter assay, and our results demonstrated that MyD88 mediated signaling contributed to the B. mallei and B. pseudomallei induced pro-inflammatory responses. Notably, the induced reporter activity with B. mallei, B. pseudomallei, or purified LPS from these pathogens was inhibited and cytokine production was attenuated by a MyD88 inhibitor. Together, these results show that in the scenario of severe hyper-inflammatory responses to B. mallei infection, MyD88 targeted therapeutic intervention may be a successful strategy for therapy.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-04-01

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

  6. Reactivation of latent melioidosis.

    PubMed

    Johnson, A B; Ali, N

    1990-09-01

    Reports of melioidosis in residents of European countries are rare. We describe a case of reactivation of latent melioidosis in a United Kingdom resident. The case demonstrates the lack of clinical response to chemotherapy despite proven in vitro sensitivity of the organism to the drugs used. It is important to consider melioidosis as a cause of septicaemic illness in patients who have travelled to, or been resident in South-East Asia.

  7. Reactivation of latent melioidosis.

    PubMed Central

    Johnson, A. B.; Ali, N.

    1990-01-01

    Reports of melioidosis in residents of European countries are rare. We describe a case of reactivation of latent melioidosis in a United Kingdom resident. The case demonstrates the lack of clinical response to chemotherapy despite proven in vitro sensitivity of the organism to the drugs used. It is important to consider melioidosis as a cause of septicaemic illness in patients who have travelled to, or been resident in South-East Asia. PMID:2235805

  8. Melioidosis Diagnostic Workshop, 20131

    PubMed Central

    AuCoin, David; Baccam, Prasith; Baggett, Henry C.; Baird, Rob; Bhengsri, Saithip; Blaney, David D.; Brett, Paul J.; Brooks, Timothy J.G.; Brown, Katherine A.; Chantratita, Narisara; Cheng, Allen C.; Dance, David A.B.; Decuypere, Saskia; Defenbaugh, Dawn; Gee, Jay E.; Houghton, Raymond; Jorakate, Possawat; Lertmemongkolchai, Ganjana; Limmathurotsakul, Direk; Merlin, Toby L.; Mukhopadhyay, Chiranjay; Norton, Robert; Peacock, Sharon J.; Rolim, Dionne B.; Simpson, Andrew J.; Steinmetz, Ivo; Stoddard, Robyn A.; Stokes, Martha M.; Sue, David; Tuanyok, Apichai; Whistler, Toni; Wuthiekanun, Vanaporn; Walke, Henry T.

    2015-01-01

    Melioidosis is a severe disease that can be difficult to diagnose because of its diverse clinical manifestations and a lack of adequate diagnostic capabilities for suspected cases. There is broad interest in improving detection and diagnosis of this disease not only in melioidosis-endemic regions but also outside these regions because melioidosis may be underreported and poses a potential bioterrorism challenge for public health authorities. Therefore, a workshop of academic, government, and private sector personnel from around the world was convened to discuss the current state of melioidosis diagnostics, diagnostic needs, and future directions. PMID:25626057

  9. [The use of polymerase chain reaction for detection of the agents of glanders and melioidosis using experimental infection].

    PubMed

    Altukhova, V V; Antonov, V A; Tkachenko, G A; Zinchenko, O V; Zamaraev, V S; Plekhanova, N G; Iliukhin, V I; Trofimov, D Iu

    2007-01-01

    Glanders and melioidosis are severe infectious diseases of people and animals. The causative agents of these infections refer to the potential agents of bioterrorism of group B. In this work the possibility of use of flagellin-based primers for the identification of B. mallei and B. pseudomallei and for diagnosis of experimental glanders and melioidosis was studied. The obtained results permit to make a conclusion that PCR using the developed primers may be recommended for the incorporation in the scheme of laboratory diagnosis of glanders and melioidosis both for the identification of clean cultures and in experimental clinical material.

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

    PubMed Central

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

    2015-01-01

    Background In this study, a Burkholderia mallei tonB mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis. Methodology/Principal Findings Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 104 CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001. Conclusions/Significance Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis. PMID:26114445

  11. Melioidosis: a case report.

    PubMed

    Barman, Purabi; Sidhwa, Harish; Shirkhande, Pinak A

    2011-04-01

    Burkhloderia pseudomallei has recently gained importance as an emerging pathogen in India. It causes various clinical manifestations like pneumoniae, septicaemia, arthritis, abscess etc. Cases have been reported from Southeast Asia mainly Thailand, Malaysia, Vietnam, etc. In India, few cases have been reported mainly from the southern part of the country. Patient was a 65-year-old male and presented with fever 1 month back, cough and breathlessness for same period, swelling on both ankles from 7 days. B. pseudomallei was isolated from endotracheal secretions, blood cultures, leg wound. He was successfully treated with Imipenem and Doxycycline and put on maintenance therapy now, and is currently doing well.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  14. Sudden unexplained death syndrome--a new manifestation in melioidosis?

    PubMed Central

    Yap, E. H.; Chan, Y. C.; Goh, K. T.; Chao, T. C.; Heng, B. H.; Thong, T. W.; Tan, H. C.; Thong, K. T.; Jacob, E.; Singh, M.

    1991-01-01

    The indirect haemagglutination (IHA) test using sensitized turkey erythrocytes and the indirect immunofluorescence assay (IgM-IFA) was confirmed to be sensitive in the detection of a recent or current Pseudomonas pseudomallei infection in 19 culture-confirmed Singapore melioidosis patients. All were found to have antibody titres from 4 to 32768 in the IHA test and 10 to 320 in the IgM-IFA test. When these tests were employed on sera from 16 immigrant Thai construction workers who died of sudden unexplained death syndrome (SUDS) and 73 healthy Thai fellow workers, 93.8% and 68.8% of SUDS cases had IHA titre of greater than or equal to 4 and IgM-IFA titre of greater than or equal to 10 respectively, in contrast to 39.7% and 12.3% found among healthy Thai workers. These data indicate that at the time of death, most of the SUDS patients had an active infection with P. pseudomallei, possibly resulting from reactivation of a latent infection. The aetiological role of P. pseudomallei as the major cause of SUDS is discussed. PMID:1721589

  15. Efflux pump-mediated drug resistance in Burkholderia.

    PubMed

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

    2015-01-01

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

  16. Efflux pump-mediated drug resistance in Burkholderia

    PubMed Central

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

    2015-01-01

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

  17. Public Awareness of Melioidosis in Thailand and Potential Use of Video Clips as Educational Tools

    PubMed Central

    Chansrichavala, Praveen; Wongsuwan, Nittayasee; Suddee, Suthee; Malasit, Mayura; Hongsuwan, Maliwan; Wannapinij, Prapass; Kitphati, Rungreung; Day, Nicholas P. J.; Michie, Susan; Peacock, Sharon J.; Limmathurotsakul, Direk

    2015-01-01

    Background Melioidosis causes more than 1,000 deaths in Thailand each year. Infection occurs via inoculation, ingestion or inhalation of the causative organism (Burkholderia pseuodmallei) present in soil and water. Here, we evaluated public awareness of melioidosis using a combination of population-based questionnaire, a public engagement campaign to obtain video clips made by the public, and viewpoints on these video clips as potential educational tools about the disease and its prevention. Methods A questionnaire was developed to evaluate public awareness of melioidosis, and knowledge about its prevention. From 1 March to 31 April 2012, the questionnaire was delivered to five randomly selected adults in each of 928 districts in Thailand. A video clip contest entitled “Melioidosis, an infectious disease that Thais must know” was run between May and October 2012. The best 12 video clips judged by a contest committee were shown to 71 people at risk from melioidosis (diabetics). Focus group interviews were used to evaluate their perceptions of the video clips. Results Of 4,203 Thais who completed our study questionnaire, 74% had never heard of melioidosis, and 19% had heard of the disease but had no further knowledge. Most participants in all focus group sessions felt that video clips were beneficial and could positively influence them to increase adherence to recommended preventive behaviours, including drinking boiled water and wearing protective gear if in contact with soil or environmental water. Participants suggested that video clips should be presented in the local dialect with simple words rather than medical terms, in a serious manner, with a doctor as the one presenting the facts, and having detailed pictures of each recommended prevention method. Conclusions In summary, public awareness of melioidosis in Thailand is very low, and video clips could serve as a useful medium to educate people and promote disease prevention. Presented in Part World

  18. [Some properties of plasmocoagulase of the causative agents of glanders and melioidosis].

    PubMed

    Narbutovich, N I; Lomova, L V; Ageeva, N P; Kucheriaeva, V T; Seimova, I K

    2005-01-01

    Criteria for the evaluation of the plasmocoagulase activity of natural isolates and mutant strains of the causative agents of glanders and melioidosis were worked out, which made it possible to subdivide them by this sign into pathogens with high, moderate and low activity. Plasmocoagulase produced by pathogenic Burkholderia was shown to be a thermolabile enzyme, comparatively stable with respect to the action of such chemico-biological agents as hydrogen peroxide and chloramine.

  19. Chronic melioidosis: a report of the first case in Japan.

    PubMed

    Arakawa, M; Mitsui, T; Miki, R; Yabuuchi, E

    1993-02-01

    A 41-year-old Japanese male with uncontrolled diabetes mellitus and alcoholic liver dysfunction developed melioidosis after his business trip to Indonesia and Singapore in 1988. His disease started with spiked fever on the following day after extraction of a tooth, and a liver abscess developed, followed by abscesses in the spleen and in the subphrenic space. In spite of splenectomy and intensive antimicrobial treatments for three months, he developed parotitis, prostatitis, and abscess of the right submandibular gland at 5 to 16-month interval. Pseudomonas pseudomallei was isolated from the blood and pus from each abscess. The lung was not involved. At present, he has returned to work, with continued intravenous instillation of imipenem/cilastatin.

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

    PubMed Central

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

    2014-01-01

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

  1. The Treatment of Melioidosis

    PubMed Central

    Inglis, Timothy J.J.

    2010-01-01

    Melioidosis is a complex bacterial infection, treatment of which combines the urgency of treating rapidly fatal Gram negative septicaemia with the need for eradication of long-term persistent disease in pulmonary, soft tissue, skeletal and other organ systems. Incremental improvements in treatment have been made as a result of multicentre collaboration across the main endemic region of Southeast Asia and northern Australia. There is an emerging consensus on the three main patterns of antimicrobial chemotherapy; initial (Phase 1) treatment, subsequent eradication (Phase 2) therapy and most recently post-exposure (Phase 0) prophylaxis. The combination of agents used, duration of therapy and need for adjunct modalities depends on the type, severity and antimicrobial susceptibility of infection. New antibiotic and adjunct therapies are at an investigational stage but on currently available data are unlikely to make a significant impact on this potentially fatal infection. PMID:27713302

  2. Fatal melioidosis in goats in Bangkok, Thailand.

    PubMed

    Tonpitak, Walaiporn; Sornklien, Chulabha; Chawanit, Mongkol; Pavasutthipaisit, Suvarin; Wuthiekanun, Vanaporn; Hantrakun, Viriya; Amornchai, Premjit; Thaipadungpanit, Janjira; Day, Nicholas P J; Yingst, Samuel; Peacock, Sharon J; Limmathurotsakul, Direk

    2014-08-01

    Bangkok, Thailand, is a city considered to be at low risk for melioidosis. We describe 10 goats that died of melioidosis in Bangkok. Half of them were born and reared in the city. Multilocus sequence typing ruled out an outbreak. This finding challenges the assumption that melioidosis is rarely acquired in central Thailand.

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2013-08-01

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

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

    PubMed

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

    2013-08-01

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

  7. [The comparative evalution of informativeness of immunologic and molecular genetic methods and means during stages of specific indication of melioidosis agent].

    PubMed

    Prokhvatilova, E V; Antonov, V A; Viktorov, D V; Khrapova, N P; Tkachenko, G A; Iliukhin, V I; Zakharova, I B; Grishina, M A; Plekhanova, N G; Novickaia, I V; Kulakov, M Ia; Bulatova, T V; Korsakova, I I; Savchenko, S S; Bondareva, O S; Teteriatnikova, N N; Senina, T V; Lopasteĭskaia, Ia A; Baturin, A A; Kulikova, A S

    2014-12-01

    The reference-center of monitoring of agents of glanders and melioidosis carried out testing of reagents kits for diagnostic of agent of melioidosis and other close-related species of Burkholderiae in vitro. At the stage of specific identification of pathogenic Burkholderiae the diagnostic possibilities of commercial and experimental kits of reagents for express- and rapid analysis were evaluated. The criteria of evaluation of diagnostic value of kits of reagents were sensitivity, specificity and time of implementation of studies. The analysis with application of mono- and multi-locus amplification systems, including real-time polymerase chain reaction permitted during 5-6 hours to implement identification and differentiation of Burkholderia pseufomallei, B. thailandensis and B. cepacia.

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

    PubMed Central

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

    2013-01-01

    Abstract Bacteriocins of the LlpA family have previously been characterized in the γ-proteobacteria Pseudomonas and Xanthomonas. These proteins are composed of two MMBL (monocot mannose-binding lectin) domains, a module predominantly and abundantly found in lectins from monocot plants. Genes encoding four different types of LlpA-like proteins were identified in genomes from strains belonging to the Burkholderia cepacia complex (Bcc) and the Burkholderia pseudomallei group. A selected recombinant LlpA-like protein from the human isolate Burkholderia cenocepacia AU1054 displayed narrow-spectrum genus-specific antibacterial activity, thus representing the first functionally characterized bacteriocin within this β-proteobacterial genus. Strain-specific killing was confined to other members of the Bcc, with mostly Burkholderia ambifaria strains being susceptible. In addition to killing planktonic cells, this bacteriocin also acted as an antibiofilm agent. Bacteriocins mediate highly selective antagonism among closely related bacteria but such antimicrobial proteins have not yet been reported in Burkholderia. We identified a lectin-like protein of the LlpA family in a Burkholderia cenocepacia human isolate that strain-specifically and selectively kills planktonic and biofilm cells of other Burkholderia cepacia complex members. PMID:23737242

  9. Intensity of exposure and incidence of melioidosis in Thai children.

    PubMed

    Cheng, Allen C; Wuthiekanun, Vanaporn; Limmathurotsakul, Direk; Chierakul, Wirongrong; Peacock, Sharon J

    2008-12-01

    There is a high background seroprevalence of antibodies to Burkholderia pseudomallei in Thailand that limits its use as a diagnostic tool. It is believed that this results from childhood exposure to the bacterium in mud and surface water. The increasing prevalence of antibodies with age is a marker of the intensity of exposure. A susceptible-infected-susceptible (SIS) model was calibrated with data on seroprevalence in children (<15 years) in Udon Thani and Ubon Ratchathani (n=2214). In this mathematical model, children were assumed to gain antibodies at a constant rate related to exposure events, and waning antibody response occurred at a constant rate. The intensity of exposure appeared to be higher in Udon Thani than in Ubon Ratchathani, with 24% vs. 11% of patients becoming seropositive each year. In Udon Thani children, antibodies appeared to be long-lasting, compared with those in Ubon Ratchathani, where the mean duration was 5.2 years. Based on an estimated paediatric disease incidence in Ubon Ratchathani of 4.15 per 100,000 population, it is estimated that approximately 1 in 4600 antibody-producing exposures results in clinical infection. Childhood seroprevalence can be used as a marker of intensity of exposure. Further work to separate the effect of exposure to B. thailandensis and cross-reactivity to B. pseudomallei is proposed.

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

    PubMed Central

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

    2015-01-01

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

  11. The prevalence of human melioidosis in Northern Queensland.

    PubMed

    Ashdown, L R; Guard, R W

    1984-05-01

    Sera from 9,047 individuals from Northern Queensland were examined for the presence of hemagglutinating antibodies to Pseudomonas pseudomallei, the causative agent of melioidosis, and 512 (5.7%) were found to have titers of 1:40 or greater. The distribution of positive reactors in various groups was uneven, and significantly higher prevalences of positive antibody titers were found in the sera from Aborigines (7.9-10.6%), Torres Strait Islanders (7.8%), Vietnamese refugees (29%) and from persons with certain medical conditions including chronic alcoholism (15%), chronic infections (14.8%), diabetes mellitus (8.6%) and liver disease (12.9%). There were significantly fewer positive reactors (1.4%) amongst the armed forces stationed in Northern Queensland. At present, the boundaries of the major endemic region of Australia extend north from Rockhampton along the coast to Darwin and inland, west from Rockhampton to Tennant Creek in central Australia. Townsville was found to have the highest prevalence (5.2%) of positive reactors of all urban populations of Northern Queensland. The extent of the disease is such that it can no longer be considered a rare infection in Northern Queensland.

  12. Phylogenetic analysis of burkholderia species by multilocus sequence analysis.

    PubMed

    Estrada-de los Santos, Paulina; Vinuesa, Pablo; Martínez-Aguilar, Lourdes; Hirsch, Ann M; Caballero-Mellado, Jesús

    2013-07-01

    Burkholderia comprises more than 60 species of environmental, clinical, and agro-biotechnological relevance. Previous phylogenetic analyses of 16S rRNA, recA, gyrB, rpoB, and acdS gene sequences as well as genome sequence comparisons of different Burkholderia species have revealed two major species clusters. In this study, we undertook a multilocus sequence analysis of 77 type and reference strains of Burkholderia using atpD, gltB, lepA, and recA genes in combination with the 16S rRNA gene sequence and employed maximum likelihood and neighbor-joining criteria to test this further. The phylogenetic analysis revealed, with high supporting values, distinct lineages within the genus Burkholderia. The two large groups were named A and B, whereas the B. rhizoxinica/B. endofungorum, and B. andropogonis groups consisted of two and one species, respectively. The group A encompasses several plant-associated and saprophytic bacterial species. The group B comprises the B. cepacia complex (opportunistic human pathogens), the B. pseudomallei subgroup, which includes both human and animal pathogens, and an assemblage of plant pathogenic species. The distinct lineages present in Burkholderia suggest that each group might represent a different genus. However, it will be necessary to analyze the full set of Burkholderia species and explore whether enough phenotypic features exist among the different clusters to propose that these groups should be considered separate genera.

  13. Melioidosis and glanders modulation of the innate immune system: barriers to current and future vaccine approaches.

    PubMed

    Aschenbroich, Sophie A; Lafontaine, Eric R; Hogan, Robert J

    2016-09-01

    Burkholderia pseudomallei and Burkholderia mallei are pathogenic bacteria causing fatal infections in animals and humans. Both organisms are classified as Tier 1 Select Agents owing to their highly fatal nature, potential/prior use as bioweapons, severity of disease via respiratory exposure, intrinsic resistance to antibiotics, and lack of a current vaccine. Disease manifestations range from acute septicemia to chronic infection, wherein the facultative intracellular lifestyle of these organisms promotes persistence within a broad range of hosts. This ability to thrive intracellularly is thought to be related to exploitation of host immune response signaling pathways. There are currently considerable gaps in our understanding of the molecular strategies employed by these pathogens to modulate these pathways and evade intracellular killing. A better understanding of the specific molecular basis for dysregulation of host immune responses by these organisms will provide a stronger platform to identify novel vaccine targets and develop effective countermeasures.

  14. Melioidosis and glanders modulation of the innate immune system: barriers to current and future vaccine approaches.

    PubMed

    Aschenbroich, Sophie A; Lafontaine, Eric R; Hogan, Robert J

    2016-09-01

    Burkholderia pseudomallei and Burkholderia mallei are pathogenic bacteria causing fatal infections in animals and humans. Both organisms are classified as Tier 1 Select Agents owing to their highly fatal nature, potential/prior use as bioweapons, severity of disease via respiratory exposure, intrinsic resistance to antibiotics, and lack of a current vaccine. Disease manifestations range from acute septicemia to chronic infection, wherein the facultative intracellular lifestyle of these organisms promotes persistence within a broad range of hosts. This ability to thrive intracellularly is thought to be related to exploitation of host immune response signaling pathways. There are currently considerable gaps in our understanding of the molecular strategies employed by these pathogens to modulate these pathways and evade intracellular killing. A better understanding of the specific molecular basis for dysregulation of host immune responses by these organisms will provide a stronger platform to identify novel vaccine targets and develop effective countermeasures. PMID:27010618

  15. [Comparative assessment of DNA extraction methods for identification of glanders and melioidosis etiological agents by PCR].

    PubMed

    Zinchenko, O V; Antonov, V A; Tkachenko, G A; Altukhova, V V; Zamaraev, V S; Piven', N N; Goloseev, Iu A; Vasil'ev, V P; Lomova, L V; Alekseev, V V

    2008-01-01

    Pathogenic Burkholderia are considered as a cause of dangerous infections and potential agents of bioterrorism. Comparative assessment of different methods of extraction and purification of DNA for PCR analysis of pure cultures and samples contaminated by etiological agents of glanders and melioidosis was performed. Samples of soil and food artificially contaminated by pathogenic Burkholderia as well as organs of infected animals were tested. DNA was extracted by methods of boiling, nucleosorption with presence of guanidine thiocyanate, guanidine thiocyanatephenol extraction, guanidine thiocyanate-phenol extraction with additional purification of DNA by nucleosorption. Amplification was performed by "Flash" technique and detector of fluorescence was used for analysis of PCR products. Utilization of the recommended methods of preparation depending on the nature of sample let to detect by the "Flash" technique the etiological agents of glanders and melioidosis in concentration =10(3) microbial cells per ml. Choice of DNA extraction and purification methods is determined by type of a sample and presence in it of admixtures inhibiting PCR.

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

    PubMed Central

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

    2015-01-01

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

  17. Genetic and phenotypic diversity in Burkholderia: contributions by prophage and phage-like elements

    PubMed Central

    2010-01-01

    Background Burkholderia species exhibit enormous phenotypic diversity, ranging from the nonpathogenic, soil- and water-inhabiting Burkholderia thailandensis to the virulent, host-adapted mammalian pathogen B. mallei. Genomic diversity is evident within Burkholderia species as well. Individual isolates of Burkholderia pseudomallei and B. thailandensis, for example, carry a variety of strain-specific genomic islands (GIs), including putative pathogenicity and metabolic islands, prophage-like islands, and prophages. These GIs may provide some strains with a competitive advantage in the environment and/or in the host relative to other strains. Results Here we present the results of analysis of 37 prophages, putative prophages, and prophage-like elements from six different Burkholderia species. Five of these were spontaneously induced to form bacteriophage particles from B. pseudomallei and B. thailandensis strains and were isolated and fully sequenced; 24 were computationally predicted in sequenced Burkholderia genomes; and eight are previously characterized prophages or prophage-like elements. The results reveal numerous differences in both genome structure and gene content among elements derived from different species as well as from strains within species, due in part to the incorporation of additional DNA, or 'morons' into the prophage genomes. Implications for pathogenicity are also discussed. Lastly, RNAseq analysis of gene expression showed that many of the genes in ϕ1026b that appear to contribute to phage and lysogen fitness were expressed independently of the phage structural and replication genes. Conclusions This study provides the first estimate of the relative contribution of prophages to the vast phenotypic diversity found among the Burkholderiae. PMID:20667135

  18. Genus-wide acid tolerance accounts for the biogeographical distribution of soil Burkholderia populations.

    PubMed

    Stopnisek, Nejc; Bodenhausen, Natacha; Frey, Beat; Fierer, Noah; Eberl, Leo; Weisskopf, Laure

    2014-06-01

    Bacteria belonging to the genus Burkholderia are highly versatile with respect to their ecological niches and lifestyles, ranging from nodulating tropical plants to causing melioidosis and fatal infections in cystic fibrosis patients. Despite the clinical importance and agronomical relevance of Burkholderia species, information about the factors influencing their occurrence, abundance and diversity in the environment is scarce. Recent findings have demonstrated that pH is the main predictor of soil bacterial diversity and community structure, with the highest diversity observed in neutral pH soils. As many Burkholderia species have been isolated from low pH environments, we hypothesized that acid tolerance may be a general feature of this genus, and pH a good predictor of their occurrence in soils. Using a combination of environmental surveys at trans-continental and local scales, as well as in vitro assays, we show that, unlike most bacteria, Burkholderia species have a competitive advantage in acidic soils, but are outcompeted in alkaline soils. Physiological assays and diversity analysis based on 16S rRNA clone libraries demonstrate that pH tolerance is a general phenotypic trait of the genus Burkholderia. Our results provide a basis for building a predictive understanding of the biogeographical patterns exhibited by Burkholderia sp.

  19. Particle-size dependent effects in the Balb/c murine model of inhalational melioidosis

    PubMed Central

    Thomas, Richard J.; Davies, C.; Nunez, A.; Hibbs, S.; Eastaugh, L.; Harding, S.; Jordan, J.; Barnes, K.; Oyston, P.; Eley, S.

    2012-01-01

    Deposition of Burkholderia pseudomallei within either the lungs or nasal passages of the Balb/c murine model resulted in different infection kinetics. The infection resulting from the inhalation of B. pseudomallei within a 12 μm particle aerosol was prolonged compared to a 1 μm particle aerosol with a mean time-to-death (MTD) of 174.7 ± 14.9 h and 73.8 ± 11.3 h, respectively. Inhalation of B. pseudomallei within 1 μm or 12 μm particle aerosols resulted in a median lethal dose (MLD) of 4 and 12 cfu, respectively. The 12 μm particle inhalational infection was characterized by a marked involvement of the nasal mucosa and extension of bacterial colonization and inflammatory lesions from the olfactory epithelium through the olfactory nerves (or tracts) to the olfactory bulb (100%), culminating in abscessation of the brain (33%). Initial involvement of the upper respiratory tract lymphoid tissues (nasal-associated lymphoid tissue (NALT) and cervical lymph nodes) was observed in both the 1 and 12 μm particle inhalational infections (80–85%). Necrotising alveolitis and bronchiolitis were evident in both inhalational infections, however, lung pathology was greater after inhalation of the 1 μm particle aerosol with pronounced involvement of the mediastinal lymph node (50%). Terminal disease was characterized by bacteraemia in both inhalational infections with dissemination to the spleen, liver, kidneys, and thymus. Treatment with co-trimoxazole was more effective than treatment with doxycycline irrespective of the size of the particles inhaled. Doxycycline was more effective against the 12 μm particle inhalational infection as evidenced by increased time to death. However, both treatment regimes exhibited significant relapse when therapy was discontinued with massive enlargement and abscessation of the lungs, spleen, and cervical lymph nodes observed. PMID:22919690

  20. Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

    PubMed Central

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

    2013-01-01

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

  1. Human melioidosis reported by ProMED

    PubMed Central

    Nasner-Posso, Katherinn Melissa; Cruz-Calderón, Stefania; Montúfar-Andrade, Franco E.; Dance, David A.B.; Rodriguez-Morales, Alfonso J.

    2015-01-01

    Summary Objective There are limited sources describing the global burden of emerging diseases. A review of human melioidosis reported by ProMED was performed and the reliability of the data retrieved assessed in comparison to published reports. The effectiveness of ProMED was evaluated as a source of epidemiological data by focusing on melioidosis. Methods Using the keyword ‘melioidosis’ in the ProMED search engine, all of the information from the reports and collected data was reviewed using a structured form, including the year, country, gender, occupation, number of infected individuals, and number of fatal cases. Results One hundred and twenty-four entries reported between January 1995 and October 2014 were identified. A total of 4630 cases were reported, with death reported in 505 cases, suggesting a misleadingly low overall case fatality rate (CFR) of 11%. Of 20 cases for which the gender was reported, 12 (60%) were male. Most of the cases were reported from Australia, Thailand, Singapore, Vietnam, and Malaysia, with sporadic reports from other countries. Conclusions Internet-based reporting systems such as ProMED are useful to gather information and synthesize knowledge on emerging infections. Although certain areas need to be improved, ProMED provided good information about melioidosis. PMID:25975651

  2. Guillain Barre syndrome as a manifestation of neurological melioidosis

    PubMed Central

    Krovvidi, Rajesh; Mridula, Rukmini K.; Jabeen, S. A.; Meena, A. K.

    2013-01-01

    Neurological melioidosis is a very rare and very few cases have been reported from India. Presentation is an extremely varied and as this disease is associated with high mortality, high index of suspicion is needed to diagnose and treat. In this context, we report a patient presenting as Guillain Barre syndrome evaluated as melioidosis. PMID:24339608

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

    PubMed

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

    2014-01-01

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

  4. Management of melioidosis osteomyelitis and septic arthritis.

    PubMed

    Shetty, R P; Mathew, M; Smith, J; Morse, L P; Mehta, J A; Currie, B J

    2015-02-01

    Little information is available about several important aspects of the treatment of melioidosis osteomyelitis and septic arthritis. We undertook a retrospective review of 50 patients with these conditions in an attempt to determine the effect of location of the disease, type of surgical intervention and duration of antibiotic treatment on outcome, particularly complications and relapse. We found that there was a 27.5% risk of osteomyelitis of the adjacent bone in patients with septic arthritis in the lower limb. Patients with septic arthritis and osteomyelitis of an adjacent bone were in hospital significantly longer (p = 0.001), needed more operations (p = 0.031) and had a significantly higher rate of complications and re-presentation (p = 0.048). More than half the patients (61%), most particularly those with multifocal bone and joint involvement, and those with septic arthritis and osteomyelitis of an adjacent bone who were treated operatively, needed more visits to theatre.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Ghequire, Maarten G K; De Mot, René

    2015-11-27

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

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

    PubMed Central

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

    2016-01-01

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

  8. Clinical-Epidemiological Features of 13 Cases of Melioidosis in Brazil

    PubMed Central

    Bandeira, Tereza J. P. G.; Cordeiro, Rossana A.; Grangeiro, Thalles B.; Lima, Rita A. C.; Ribeiro, Joyce F.; Castelo-Branco, Débora S. C. M.; Rodrigues, Jorge L. N.; Coelho, Ivo C. B.; Magalhães, Francisco G.; Rocha, Marcos F. G.; Sidrim, José J. C.

    2012-01-01

    The aim of this work was to catalog the clinical and ecoepidemiological characteristics of melioidosis in Brazil. The clinical-epidemiological features of melioidosis in Ceará are similar to those in other regions where the disease is endemic. These findings support the inclusion of this Brazilian state as part of the zone of endemicity for melioidosis. PMID:22814457

  9. Cerebral melioidosis for the first time in the western hemisphere

    PubMed Central

    Vestal, Matthew L.; Wong, Emily B.; Milner, Dan A.; Gormley, William B.; Dunn, Ian F.

    2015-01-01

    This report is the first published case of cerebral melioidosis in the western hemisphere. In this paper the authors review the literature on neurological melioidosis and its presentation and treatment in endemic areas, describe the clinical course of this unique case of a presentation of the disease with cranial abscess in the US, review the pathological and radiological findings associated with this seminal case, and put forth recommendations for recognizing and treating possible future instances of the disease within the western hemisphere. PMID:23767895

  10. Global Analysis of the Burkholderia thailandensis Quorum Sensing-Controlled Regulon

    PubMed Central

    Majerczyk, Charlotte; Brittnacher, Mitchell; Jacobs, Michael; Armour, Christopher D.; Radey, Mathew; Schneider, Emily; Phattarasokul, Somsak; Bunt, Richard

    2014-01-01

    Burkholderia thailandensis contains three acyl-homoserine lactone quorum sensing circuits and has two additional LuxR homologs. To identify B. thailandensis quorum sensing-controlled genes, we carried out transcriptome sequencing (RNA-seq) analyses of quorum sensing mutants and their parent. The analyses were grounded in the fact that we identified genes coding for factors shown previously to be regulated by quorum sensing among a larger set of quorum-controlled genes. We also found that genes coding for contact-dependent inhibition were induced by quorum sensing and confirmed that specific quorum sensing mutants had a contact-dependent inhibition defect. Additional quorum-controlled genes included those for the production of numerous secondary metabolites, an uncharacterized exopolysaccharide, and a predicted chitin-binding protein. This study provides insights into the roles of the three quorum sensing circuits in the saprophytic lifestyle of B. thailandensis, and it provides a foundation on which to build an understanding of the roles of quorum sensing in the biology of B. thailandensis and the closely related pathogenic Burkholderia pseudomallei and Burkholderia mallei. PMID:24464461

  11. Inactivation of Burkholderia cepacia Complex Phage KS9 gp41 Identifies the Phage Repressor and Generates Lytic Virions▿ †

    PubMed Central

    Lynch, Karlene H.; Seed, Kimberley D.; Stothard, Paul; Dennis, Jonathan J.

    2010-01-01

    The Burkholderia cepacia complex (BCC) is made up of at least 17 species of Gram-negative opportunistic bacterial pathogens that cause fatal infections in patients with cystic fibrosis and chronic granulomatous disease. KS9 (vB_BcenS_KS9), one of a number of temperate phages isolated from BCC species, is a prophage of Burkholderia pyrrocinia LMG 21824. Transmission electron micrographs indicate that KS9 belongs to the family Siphoviridae and exhibits the B1 morphotype. The 39,896-bp KS9 genome, comprised of 50 predicted genes, integrates into the 3′ end of the LMG 21824 GTP cyclohydrolase II open reading frame. The KS9 genome is most similar to uncharacterized prophage elements in the genome of B. cenocepacia PC184 (vB_BcenZ_ PC184), as well as Burkholderia thailandensis phage φE125 and Burkholderia pseudomallei phage φ1026b. Using molecular techniques, we have disrupted KS9 gene 41, which exhibits similarity to genes encoding phage repressors, producing a lytic mutant named KS9c. This phage is incapable of stable lysogeny in either LMG 21824 or B. cenocepacia strain K56-2 and rescues a Galleria mellonella infection model from experimental B. cenocepacia K56-2 infections at relatively low multiplicities of infection. These results readily demonstrate that temperate phages can be genetically engineered to lytic form and that these modified phages can be used to treat bacterial infections in vivo. PMID:19939932

  12. Screening and expression of selected taxonomically conserved and unique hypothetical proteins in Burkholderia pseudomallei K96243

    NASA Astrophysics Data System (ADS)

    Akhir, Nor Azurah Mat; Nadzirin, Nurul; Mohamed, Rahmah; Firdaus-Raih, Mohd

    2015-09-01

    Hypothetical proteins of bacterial pathogens represent a large numbers of novel biological mechanisms which could belong to essential pathways in the bacteria. They lack functional characterizations mainly due to the inability of sequence homology based methods to detect functional relationships in the absence of detectable sequence similarity. The dataset derived from this study showed 550 candidates conserved in genomes that has pathogenicity information and only present in the Burkholderiales order. The dataset has been narrowed down to taxonomic clusters. Ten proteins were selected for ORF amplification, seven of them were successfully amplified, and only four proteins were successfully expressed. These proteins will be great candidates in determining the true function via structural biology.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2000-06-01

    Four species of synanthropic flies were trapped in downtown Kuala Lumpur: Chrysomya megacephala, Chrysomya rufifacies, Musca domestica, and Musca sorbens. Burkholderia pseudomallei, the organism causing melioidosis, was the dominant bacteria isolated from Chrysomya megacephala. Klebsiella oxytoca, commonly associated with nosocomial infections, was commonly isolated from Chrysomya megacephala, Musca domestica, and Musca sorbens. Aeromonas hydrophila, the bacteria causing gastroenteritis, was predominantly isolated from Chrysomya megacephala and also from Musca domestica and Musca sorbens. A total of 18 bacterial species was isolated from the synanthropic flies trapped. Burkholderia pseudomallei had been reported for the first time.

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

    PubMed

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

    2016-06-01

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

  16. Genomic characterization of JG068, a novel virulent podovirus active against Burkholderia cenocepacia

    PubMed Central

    2013-01-01

    Background As is true for many other antibiotic-resistant Gram-negative pathogens, members of the Burkholderia cepacia complex (BCC) are currently being assessed for their susceptibility to phage therapy as an antimicrobial treatment. The objective of this study was to perform genomic and limited functional characterization of the novel BCC phage JG068 (vB_BceP_JG068). Results JG068 is a podovirus that forms large, clear plaques on Burkholderia cenocepacia K56-2. Host range analysis indicates that this phage can infect environmental, clinical, and epidemic isolates of Burkholderia multivorans, B. cenocepacia, Burkholderia stabilis, and Burkholderia dolosa, likely through interaction with the host lipopolysaccharide as a receptor. The JG068 chromosome is 41,604 base pairs (bp) in length and is flanked by 216 bp short direct terminal repeats. Gene expression originates from both host and phage promoters and is in the forward direction for all 49 open reading frames. The genome sequence shows similarity to Ralstonia phage ϕRSB1, Caulobacter phage Cd1, and uncharacterized genetic loci of blood disease bacterium R229 and Burkholderia pseudomallei 1710b. CoreGenesUniqueGenes analysis indicates that JG068 belongs to the Autographivirinae subfamily and ϕKMV-like phages genus. Modules within the genome encode proteins involved in DNA-binding, morphogenesis, and lysis, but none associated with pathogenicity or lysogeny. Similar to the signal-arrest-release (SAR) endolysin of ϕKMV, inducible expression of the JG068 SAR endolysin causes lysis of Escherichia coli that is dependent on the presence of an N-terminal signal sequence. In an in vivo assay using the Galleria mellonella infection model, treatment of B. cenocepacia K56-2-infected larvae with JG068 results in a significant increase in larval survival. Conclusions As JG068 has a broad host range, does not encode virulence factors, is obligately lytic, and has activity against an epidemic B. cenocepacia strain in vivo

  17. Use of a recombinant burkholderia intracellular motility a protein for immunodiagnosis of glanders.

    PubMed

    Kumar, Subodh; Malik, Praveen; Verma, Shailendra Kumar; Pal, Vijai; Gautam, Vandana; Mukhopadhyay, Chiranjay; Rai, Ganga Prasad

    2011-09-01

    Glanders, caused by the Gram-negative, nonmotile bacterium Burkholderia mallei, is a contagious and highly fatal disease of equines. During the last decade, the number of glanders outbreaks has increased steadily. The disease also has high zoonotic significance and B. mallei is listed biological warfare agent. The complement fixation test (CFT) is a routinely used and internationally recognized test to screen equine sera for the glanders. However, discrepant results have been observed using the CFT. The low sensitivity and specificity of the CFT and enzyme-linked immunosorbent assay (ELISA) have been linked to the use of crude test antigens. We expressed a novel recombinant Burkholderia intracellular motility A (rBimA) protein in Escherichia coli for the diagnosis of equine glanders. Purified rBimA was used in an indirect ELISA format. All of the 21 true-positive serum samples used in the study tested positive, whereas only 17 of the 1,524 potentially negative sera tested positive by indirect ELISA, thus exhibiting 100% sensitivity and 98.88% specificity. Also, rBimA protein did not react with melioidosis patient and normal healthy human serum samples, showing its high specificity. The developed assay can be used as a simple and rapid tool for diagnosis of glanders in equine serum samples. An Indian patent (1328/DEL/2010) has been filed for the reagent.

  18. CHROMOSOMAL MULTIPLICITY IN BURKHOLDERIA CEPACIA

    EPA Science Inventory

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

  19. Metabolic profiling of Burkholderia cenocepacia, Burkholderia ambifaria, and Burkholderia pyrrocinia isolates from maize rhizosphere.

    PubMed

    Alisi, Chiara; Lasinio, Giovanna Jona; Dalmastri, Claudia; Sprocati, AnnaRosa; Tabacchioni, Silvia; Bevivino, Annamaria; Chiarini, Luigi

    2005-10-01

    Burkholderia cenocepacia, Burkholderia ambifaria, and Burkholderia pyrrocinia are the Burkholderia cepacia complex (Bcc) species most frequently associated with roots of crop plants. To investigate the ecophysiological diversity of these species, metabolic profiling of maize rhizosphere isolates was carried out by means of the Biolog system, using GN2 and SFN2 plates and different parameters related to optical density (OD). The metabolic profiles produced by the SFN2 and GN2 plates were identical, but the SFN2's narrower range of OD values and significantly longer reaction times made these plates less suitable for differentiation of isolates. Principal component analysis of maximum OD (ODM) and maximum substrate oxidation rate (muM) data generated by GN2 plates allowed the selection of a reduced number of carbon sources. Statistical analysis of ODM values highlighted marked differences between the metabolic profiles of B. cenocepacia and B. ambifaria, whereas metabolic profiles of B. pyrrocinia clustered very often with those of B. cenocepacia. Analysis of the mu(M) parameter resulted in a slightly lower differentiation among the three Bcc species and a higher metabolic diversity within the single species, in particular within B. cenocepacia. Finally, B. cenocepacia and B. pyrrocinia showed generally higher oxidation rates than B. ambifaria on those GN2 substrates that commonly occur in maize root exudates. PMID:16328653

  20. Melioidosis as a Cause of Acute Abdomen in Immuno-Competent Male from Eastern India

    PubMed Central

    Karuna, Tadepalli; Khadanga, Sagar; Dugar, Dharmendra; Sau, Biyanka; Bhoi, Priyadarshini

    2015-01-01

    Though melioidosis is rare in India, it has gained importance as one of the most potent emerging infections. In India, the cases have been under-reported because of the lack of awareness. The majority of cases present with multifocal pyogenic infections with septicemia. We present an unusual case of melioidosis presenting as acute intestinal perforation. The organism was ceftazidime resistant, and we successfully treated the case with imipenem and doxycyclin. This case highlights ruling out the possibility of melioidosis in acute abdomen and existence of ceftazidime resistant cases in India. PMID:25949062

  1. A case of melioidosis of the breast present at screening mammography.

    PubMed

    Lengren, J; Withey, G; Hack, J

    2002-02-01

    A previously well 54-year-old woman presented for screening mammography with a 3-day history of an inflamed lump in her right breast. She was subsequently admitted to hospital with acute melioidosis where right breast abscesses were drained. Following recovery and discharge, she was reviewed at the mammographic screening unit where her previous abnormal mammogram was found to be due to melioidosis. To our knowledge this is the first reported case of melioidosis of the breast and its appearance on mammography and ultrasound are described.

  2. Melioidosis as a cause of acute abdomen in immuno-competent male from eastern India.

    PubMed

    Karuna, Tadepalli; Khadanga, Sagar; Dugar, Dharmendra; Sau, Biyanka; Bhoi, Priyadarshini

    2015-01-01

    Though melioidosis is rare in India, it has gained importance as one of the most potent emerging infections. In India, the cases have been under-reported because of the lack of awareness. The majority of cases present with multifocal pyogenic infections with septicemia. We present an unusual case of melioidosis presenting as acute intestinal perforation. The organism was ceftazidime resistant, and we successfully treated the case with imipenem and doxycyclin. This case highlights ruling out the possibility of melioidosis in acute abdomen and existence of ceftazidime resistant cases in India. PMID:25949062

  3. Metabolomic Profiling of Plasma from Melioidosis Patients Using UHPLC-QTOF MS Reveals Novel Biomarkers for Diagnosis

    PubMed Central

    Lau, Susanna K. P.; Lee, Kim-Chung; Lo, George C. S.; Ding, Vanessa S. Y.; Chow, Wang-Ngai; Ke, Tony Y. H.; Curreem, Shirly O. T.; To, Kelvin K. W.; Ho, Deborah T. Y.; Sridhar, Siddharth; Wong, Sally C. Y.; Chan, Jasper F. W.; Hung, Ivan F. N.; Sze, Kong-Hung; Lam, Ching-Wan; Yuen, Kwok-Yung; Woo, Patrick C. Y.

    2016-01-01

    To identify potential biomarkers for improving diagnosis of melioidosis, we compared plasma metabolome profiles of melioidosis patients compared to patients with other bacteremia and controls without active infection, using ultra-high-performance liquid chromatography-electrospray ionization-quadruple time-of-flight mass spectrometry. Principal component analysis (PCA) showed that the metabolomic profiles of melioidosis patients are distinguishable from bacteremia patients and controls. Using multivariate and univariate analysis, 12 significant metabolites from four lipid classes, acylcarnitine (n = 6), lysophosphatidylethanolamine (LysoPE) (n = 3), sphingomyelins (SM) (n = 2) and phosphatidylcholine (PC) (n = 1), with significantly higher levels in melioidosis patients than bacteremia patients and controls, were identified. Ten of the 12 metabolites showed area-under-receiver operating characteristic curve (AUC) >0.80 when compared both between melioidosis and bacteremia patients, and between melioidosis patients and controls. SM(d18:2/16:0) possessed the largest AUC when compared, both between melioidosis and bacteremia patients (AUC 0.998, sensitivity 100% and specificity 91.7%), and between melioidosis patients and controls (AUC 1.000, sensitivity 96.7% and specificity 100%). Our results indicate that metabolome profiling might serve as a promising approach for diagnosis of melioidosis using patient plasma, with SM(d18:2/16:0) representing a potential biomarker. Since the 12 metabolites were related to various pathways for energy and lipid metabolism, further studies may reveal their possible role in the pathogenesis and host response in melioidosis. PMID:26927094

  4. Burkholderia humi sp. nov., Burkholderia choica sp. nov., Burkholderia telluris sp. nov., Burkholderia terrestris sp. nov. and Burkholderia udeis sp. nov.: Burkholderia glathei-like bacteria from soil and rhizosphere soil.

    PubMed

    Vandamme, Peter; De Brandt, Evie; Houf, Kurt; Salles, Joana Falcão; Dirk van Elsas, Jan; Spilker, Theodore; Lipuma, John J

    2013-12-01

    Analysis of partial gyrB gene sequences revealed six taxa in a group of 17 Burkholderia glathei-like isolates which were further examined by (GTG)5-PCR fingerprinting, 16S rRNA gene sequence analysis, DNA-DNA hybridizations, determination of the DNA G+C content, whole-cell fatty acid analysis and an analysis of cell and colony morphology and more than 180 biochemical characteristics. The results demonstrated that one taxon consisting of three human clinical isolates represented Burkholderia zhejiangensis, a recently described methyl-parathion-degrading bacterium isolated from a wastewater-treatment system in China. The remaining taxa represented five novel species isolated from soil or rhizosphere soil samples, and could be distinguished by both genotypic and phenotypic characteristics. We therefore propose to formally classify these bacteria as Burkholderia humi sp. nov. (type strain, LMG 22934(T) = CCUG 63059(T)), Burkholderia choica sp. nov. (type strain, LMG 22940(T) = CCUG 63063(T)), Burkholderia telluris sp. nov. (type strain, LMG 22936(T) = CCUG 63060(T)), Burkholderia udeis sp. nov. (type strain, LMG 27134(T) = CCUG 63061(T)) and Burkholderia terrestris sp. nov. (type strain, LMG 22937(T) = CCUG 63062(T)).

  5. Dosing regimens of cotrimoxazole (trimethoprim-sulfamethoxazole) for melioidosis.

    PubMed

    Cheng, Allen C; McBryde, Emma S; Wuthiekanun, Vanaporn; Chierakul, Wirongrong; Amornchai, Premjit; Day, Nicholas P J; White, Nicholas J; Peacock, Sharon J

    2009-10-01

    Melioidosis is an infectious disease with a propensity for relapse, despite prolonged antibiotic eradication therapy for 12 to 20 weeks. A pharmacokinetic (PK) simulation study was performed to determine the optimal dosing of cotrimoxazole (trimethoprim-sulfamethoxazole [TMP-SMX]) used in current eradication regimens in Thailand and Australia. Data for bioavailability, protein binding, and coefficients of absorption and elimination were taken from published literature. Apparent volumes of distribution were correlated with body mass and were estimated separately for Thai and Australian populations. In vitro experiments demonstrated concentration-dependent killing. In Australia, the currently used eradication regimen (320 [TMP]/1,600 [SMX] mg every 12 h [q12h]) was predicted to achieve the PK-pharmacodynamic (PD) target (an area under the concentration-time curve from 0 to 24 h/MIC ratio of >25 for both TMP and SMX) for strains with the MIC90 of Australian strains (< or = 1/19 mg/liter). In Thailand, the former regimen of 160/800 mg q12h would not be expected to attain the target for strains with an MIC of > or = 1/19 mg/liter, but the recently implemented weight-based regimen (<40 kg [body weight], 160/800 mg q12h; 40 to 60 kg, 240/1,200 mg q12h; >60 kg, 320/1,600 mg q12h) would be expected to achieve adequate concentrations for strains with an MIC of < or = 1/19 mg/liter. The results were sensitive to the variance of the PK parameters. Prospective PK-PD studies of Asian populations are needed to optimize TMP-SMX dosing in melioidosis.

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

    PubMed

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

    2012-08-01

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

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

    PubMed

    Lee, Jae-Chan; Whang, Kyung-Sook

    2015-09-01

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

  8. [The use of fluorescent liposomal immunoanalysis for the diagnostics and indication of glanders and melioidosis].

    PubMed

    Skopinskaia, S N; Iarkov, S P; Khramov, E N; Khrapova, N P; Piven', N N; Prokhvatilova, E V

    2007-01-01

    The authors have developed a homogenous method for the detection of the cells, surface antigen, and Ag8 of glanders and melioidosis pathogens and antibodies to them, based on the compliment-dependent lysis of Ag8-sensitized liposomes. The method is highly specific; its sensitivity to antibodies to glanders and melioidosis pathogens is 24 to 440 times higher than that of RID: its sensitivity to Ag8 is 100 ng/ml corresponding to 1.25x10-10 M, and its sensitivity to cells is 10(5) to 10(6) cells/ml).

  9. 40 CFR 725.1075 - Burkholderia cepacia complex.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

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

  10. The Twin Arginine Translocation System Is Essential for Aerobic Growth and Full Virulence of Burkholderia thailandensis

    PubMed Central

    Wagley, Sariqa; Hemsley, Claudia; Thomas, Rachael; Moule, Madeleine G.; Vanaporn, Muthita; Andreae, Clio; Robinson, Matthew; Goldman, Stan; Wren, Brendan W.; Butler, Clive S.

    2014-01-01

    The twin arginine translocation (Tat) system in bacteria is responsible for transporting folded proteins across the cytoplasmic membrane, and in some bacteria, Tat-exported substrates have been linked to virulence. We report here that the Tat machinery is present in Burkholderia pseudomallei, B. mallei, and B. thailandensis, and we show that the system is essential for aerobic but not anaerobic growth. Switching off of the Tat system in B. thailandensis grown anaerobically resulted in filamentous bacteria, and bacteria showed increased sensitivity to some β-lactam antibiotics. In Galleria mellonella and zebrafish infection models, the Tat conditional mutant was attenuated. The aerobic growth-restricted phenotype indicates that Tat substrates may play a functional role in oxygen-dependent energy conservation. In other bacteria, aerobic growth restriction in Tat mutants has been attributed to the inability to translocate PetA, the Rieske iron-sulfur protein which forms part of the quinol-cytochrome c oxidoreductase complex. Here, we show that PetA is not responsible for aerobic growth restriction in B. thailandensis. However, we have identified an operon encoding 2 proteins of unknown function (BTH_I2176 and BTH_I2175) that play a role in aerobic growth restriction, and we present evidence that BTH_I2176 is Tat translocated. PMID:24214943

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

    PubMed Central

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

    2014-01-01

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

  12. Burkholderia cordobensis sp. nov., from agricultural soils.

    PubMed

    Draghi, Walter O; Peeters, Charlotte; Cnockaert, Margo; Snauwaert, Cindy; Wall, Luis G; Zorreguieta, Angeles; Vandamme, Peter

    2014-06-01

    Two Gram-negative, rod-shaped bacteria were isolated from agricultural soils in Córdoba province in central Argentina. Their 16S rRNA gene sequences demonstrated that they belong to the genus Burkholderia, with Burkholderia zhejiangensis as most closely related formally named species; this relationship was confirmed through comparative gyrB sequence analysis. Whole-cell fatty acid analysis supported their assignment to the genus Burkholderia. Burkholderia sp. strain YI23, for which a whole-genome sequence is available, represents the same taxon, as demonstrated by its highly similar 16S rRNA (100% similarity) and gyrB (99.1-99.7%) gene sequences. The results of DNA-DNA hybridization experiments and physiological and biochemical characterization further substantiated the genotypic and phenotypic distinctiveness of the Argentinian soil isolates, for which the name Burkholderia cordobensis sp. nov. is proposed, with strain MMP81(T) ( = LMG 27620(T) = CCUG 64368(T)) as the type strain.

  13. Burkholderia pyrrocinia in cystic fibrosis lung transplantation: a case report.

    PubMed

    Savi, D; De Biase, R Valerio; Amaddeo, A; Anile, M; Venuta, F; Ruberto, F; Simmonds, N; Cimino, G; Quattrucci, S

    2014-01-01

    Infection with Burkholderia species is typically considered a contraindication leading to transplantation in cystic fibrosis (CF). However, the risks posed by different Burkholderia species on transplantation outcomes are poorly defined. We present the case of a patient with CF who underwent lung transplantation due to a severe respiratory failure from chronic airways infection with Burkholderia pyrrocinia (B. cepacia genomovar IX) and pan-resistant Pseudomonas aeruginosa. The postoperative course was complicated by recurrent B. pyrrocinia infections, ultimately lea ding to uncontrollable sepsis and death. This is the first case report in CF of Burkholderia pyrrocinia infection and lung transplantation, providing further evidence of the high risk nature of the Burkholderia species.

  14. Host Evasion by Burkholderia cenocepacia

    PubMed Central

    Ganesan, Shyamala; Sajjan, Umadevi S.

    2012-01-01

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

  15. The use of animal infection models to study the pathogenesis of melioidosis and glanders.

    PubMed

    Woods, Donald E

    2002-11-01

    The use of animal infection models is central to the study of microbial pathogenesis. In combination with genetic, immunological and antigen purification techniques, much can be learned regarding the pathogenesis of diseases caused by microorganisms. This update focuses on the recent use of animal infection models to study the pathogenesis of melioidosis and glanders.

  16. Natural Burkholderia mallei infection in Dromedary, Bahrain.

    PubMed

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

    2011-07-01

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

  17. GENOME ANALYSIS OF BURKHOLDERIA CEPACIA AC1100

    EPA Science Inventory

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

  18. Burkholderia monticola sp. nov., isolated from mountain soil.

    PubMed

    Baek, Inwoo; Seo, Boram; Lee, Imchang; Yi, Hana; Chun, Jongsik

    2015-02-01

    An ivory/yellow, Gram-stain-negative, short-rod-shaped, aerobic bacterial strain, designated JC2948(T), was isolated from a soil sample taken from Gwanak Mountain, Republic of Korea. 16S rRNA gene sequence analysis indicated that strain JC2948(T) belongs to the genus Burkholderia. The test strain showed highest sequence similarities to Burkholderia tropica LMG 22274(T) (97.6 %), Burkholderia acidipaludis NBRC 101816(T) (97.5 %), Burkholderia tuberum LMG 21444(T) (97.5 %), Burkholderia sprentiae LMG 27175(T) (97.4 %), Burkholderia terricola LMG 20594(T) (97.3 %) and Burkholderia diazotrophica LMG 26031(T) (97.1 %). Based on average nucleotide identity (ANI) values, the new isolate represents a novel genomic species as it shows less than 90 % ANI values with other closely related species. Also, other phylosiological and biochemical comparisons allowed the phenotypic differentiation of strain JC2948(T) from other members of the genus Burkholderia. Therefore, we suggest that this strain should be classified as the type strain of a novel species of the genus Burkholderia. The name Burkholderia monticola sp. nov. (type strain, JC2948(T) = JCM 19904(T) = KACC 17924(T)) is proposed.

  19. Burkholderia megalochromosomata sp. nov., isolated from grassland soil.

    PubMed

    Baek, Inwoo; Seo, Boram; Lee, Imchang; Lee, Kihyun; Park, Sang-Cheol; Yi, Hana; Chun, Jongsik

    2015-03-01

    A Gram-stain negative, rod-shaped, non-spore-forming, obligate aerobic bacterial strain, JC2949(T), was isolated from grassland soil in Gwanak Mountain, Seoul, Republic of Korea. Phylogenetic analysis, based on 16S rRNA sequences, indicated that strain JC2949(T) belongs to the genus Burkholderia, showing highest sequence similarities with Burkholderia grimmiae R27(T) (98.8 %), Burkholderia cordobensis LMG 27620(T) (98.6 %), Burkholderia jiangsuensis MP-1T(T) (98.6 %), Burkholderia zhejiangensis OP-1(T) (98.5 %), Burkholderia humi LMG 22934(T) (97.5 %), Burkholderia terrestris LMG 22937(T) (97.3 %), Burkholderia telluris LMG 22936(T) (97.2 %) and Burkholderia glathei ATCC 29195(T) (97.0 %). The major fatty acids of strain JC2949(T) were C18 : 1ω7c, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. Its predominant polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and an unknown amino phospholipid. The dominant isoprenoid quinone was ubiquinone Q-8. The pairwise average nucleotide identity values between strain JC2949(T) and the genomes of 30 other species of the genus Burkholderia ranged from 73.4-90.4 %, indicating that the isolate is a novel genomic species within this genus. Based on phenotypic and chemotaxonomic comparisons, it is clear that strain JC2949(T) represents a novel species of the genus Burkholderia. We propose the name for this novel species to be Burkholderia megalochromosomata sp. nov. The type strain is JC2949(T) ( = KACC 17925(T) = JCM 19905(T)).

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

    PubMed Central

    Eberl, Leo; Vandamme, Peter

    2016-01-01

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

  1. Discrimination of Burkholderia multivorans and Burkholderia vietnamiensis from Burkholderia cepacia genomovars I, III, and IV by PCR.

    PubMed

    Bauernfeind, A; Schneider, I; Jungwirth, R; Roller, C

    1999-05-01

    We present a PCR procedure for identification of Burkholderia cepacia, Burkholderia multivorans, and Burkholderia vietnamiensis. 16S and 23S ribosomal DNAs (rDNAs) of B. multivorans and B. vietnamiensis were sequenced and aligned with published sequences for definition of species-specific 18-mer oligonucleotide primers. Specific antisense 16S rDNA primers (for B. cepacia, 5'-AGC ACT CCC RCC TCT CAG-3'; for B. multivorans, 5'-AGC ACT CCC GAA TCT CTT-3') and 23S rDNA primers (for B. vietnamiensis, 5'-TCC TAC CAT GCG TGC AA-3') were paired with a general sense primer of 16S rDNAs (5'-AGR GTT YGA TYM TGG CTC AG-3') or with a sense primer of 23S rDNA (5'-CCT TTG GGT CAT CCT GGA-3'). PCR with these primers under optimized conditions is appropriate to specifically and rapidly identify B. multivorans, B. vietnamiensis, and B. cepacia (genomovars I, III, and IV are not discriminated). In comparison with the polyphasic taxonomic analyses presently necessary for species and genomovar identification within the B. cepacia complex, our procedure is more rapid and easier to perform and may contribute to clarifying the clinical significance of individual members of the complex in cystic fibrosis.

  2. Short report: Predictors of severe disease in melioidosis patients in Kuala Lumpur, Malaysia.

    PubMed

    Mohd Roslani, Ardita Dewi Roslani; Tay, Sun Tee; Puthucheary, Savithri D; Rukumani, Devi V; Sam, I-Ching

    2014-12-01

    The predictors of severe disease or death were determined for 85 melioidosis patients in Kuala Lumpur, Malaysia. Most of the patients were male, > 40 years old, and diabetic. Severe disease or death occurred in 28 (32.9%) cases. Lower lymphocyte counts and positive blood cultures were significant independent predictors of severe disease, but age, presentations with pneumonia, inappropriate empirical antibiotics, or flagellin types of the infecting isolates were not. Knowledge of local predictors of severe disease is useful for clinical management.

  3. Glibenclamide reduces pro-inflammatory cytokine production by neutrophils of diabetes patients in response to bacterial infection

    NASA Astrophysics Data System (ADS)

    Kewcharoenwong, Chidchamai; Rinchai, Darawan; Utispan, Kusumawadee; Suwannasaen, Duangchan; Bancroft, Gregory J.; Ato, Manabu; Lertmemongkolchai, Ganjana

    2013-11-01

    Type 2 diabetes mellitus is a major risk factor for melioidosis, which is caused by Burkholderia pseudomallei. Our previous study has shown that polymorphonuclear neutrophils (PMNs) from diabetic subjects exhibited decreased functions in response to B. pseudomallei. Here we investigated the mechanisms regulating cytokine secretion of PMNs from diabetic patients which might contribute to patient susceptibility to bacterial infections. Purified PMNs from diabetic patients who had been treated with glibenclamide (an ATP-sensitive potassium channel blocker for anti-diabetes therapy), showed reduction of interleukin (IL)-1β and IL-8 secretion when exposed to B. pseudomallei. Additionally, reduction of these pro-inflammatory cytokines occurred when PMNs from diabetic patients were treated in vitro with glibenclamide. These findings suggest that glibenclamide might be responsible for the increased susceptibility of diabetic patients, with poor glycemic control, to bacterial infections as a result of its effect on reducing IL-1β production by PMNs.

  4. Draft Genomes for Eight Burkholderia mallei Isolates from Turkey

    PubMed Central

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

    2016-01-01

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

  5. 40 CFR 725.1075 - Burkholderia cepacia complex.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS Significant New Uses for Specific Microorganisms § 725.1075 Burkholderia cepacia complex. (a) Microorganism and significant new uses subject to reporting. (1) The microorganisms identified as the Burkholderia cepacia complex defined...

  6. Draft Genomes for Eight Burkholderia mallei Isolates from Turkey

    DOE PAGES

    Daligault, H. E.; Johnson, Shannon L.; Davenport, K. W.; Minogue, T. D.; Bishop-Lilly, K. A.; Broomall, S. M.; Bruce, D. C.; Coyne, S. R.; Frey, K. G.; Gibbons, H. S.; et al

    2016-01-07

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

  7. 40 CFR 725.1075 - Burkholderia cepacia complex.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... CONTROL ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS Significant New Uses for Specific Microorganisms § 725.1075 Burkholderia cepacia complex. (a) Microorganism and significant new uses subject to reporting. (1) The microorganisms identified as the Burkholderia cepacia complex defined...

  8. A preliminary survey for human immunodeficient virus (HIV) infections in tuberculosis and melioidosis patients in Ubon Ratchathani, Thailand.

    PubMed

    Kanai, K; Kurata, T; Akksilp, S; Auwanit, W; Chaowagul, V; Naigowit, P

    1992-01-01

    A preliminary survey was conducted for the prevalence of HIV infections in pulmonary tuberculosis and melioidosis patients in Ubon Ratchathani province, in Thailand, the second largest province in population which supplies labors to Bangkok metropolis. In this province, tuberculosis is prevalent in a higher rate than in most other provinces and melioidosis is endemic. Four HIV-seropositives were found in a total of 551 suspected and culture-positive cases of pulmonary tuberculosis, while no HIV-seropositive was found in 121 melioidosis patients. In view of the rapidly expanding HIV-infections in Thailand, a strict watch will be needed on the future epidemiological status of HIV-infection in tuberculous patient.

  9. Histone Deacetylase Inhibitors from Burkholderia Thailandensis

    PubMed Central

    Klausmeyer, Paul; Shipley, Suzanne; Zuck, Karina M.; McCloud, Thomas G.

    2011-01-01

    Bioactivity guided fractionation of an extract of Burkholderia thailandensis led to the isolation and identification of a new cytotoxic depsipeptide and its dimer. Both compounds potently inhibited the function of histone deacetylases 1 and 4. The monomer, spiruchostatin C (2), was tested side-by-side with the clinical depsipeptide FK228 (1, Istodax®, romidepsin) in a murine hollow fiber assay consisting of 12 implanted tumor cell lines. Spiruchostatin C (2) showed good activity towards LOX IMVI melanoma cells and NCI-H522 non small cell lung cancer cells. Overall, however, FK228 (1) showed a superior in vivo antitumor profile compared to the new compound. PMID:21967146

  10. Burkholderia rhynchosiae sp. nov., isolated from Rhynchosia ferulifolia root nodules.

    PubMed

    De Meyer, Sofie E; Cnockaert, Margo; Ardley, Julie K; Trengove, Robert D; Garau, Giovanni; Howieson, John G; Vandamme, Peter

    2013-11-01

    Two strains of Gram-stain-negative, rod-shaped bacteria were isolated from root nodules of the South African legume Rhynchosia ferulifolia and authenticated on this host. Based on phylogenetic analysis of the 16S rRNA gene, strains WSM3930 and WSM3937(T) belonged to the genus Burkholderia, with the highest degree of sequence similarity to Burkholderia terricola (98.84 %). Additionally, the housekeeping genes gyrB and recA were analysed since 16S rRNA gene sequences are highly similar between closely related species of the genus Burkholderia. The results obtained for both housekeeping genes, gyrB and recA, showed the highest degree of sequence similarity of the novel strains towards Burkholderia caledonica LMG 19076(T) (94.2 % and 94.5 %, respectively). Chemotaxonomic data, including fatty acid profiles and respiratory quinone data supported the assignment of strains WSM3930 and WSM3937(T) to the genus Burkholderia. DNA-DNA hybridizations, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains WSM3930 and WSM3937(T) from the most closely related species of the genus Burkholderia with validly published names. We conclude, therefore, that these strains represent a novel species for which the name Burkholderia rhynchosiae sp. nov. is proposed, with strain WSM3937(T) ( = LMG 27174(T) = HAMBI 3354(T)) as the type strain.

  11. Burkholderia humi sp. nov., isolated from peat soil.

    PubMed

    Srinivasan, Sathiyaraj; Kim, Jinsoo; Kang, Sang-Rim; Jheong, Weon-Hwa; Lee, Sang-Seob

    2013-03-01

    A Gram-negative, aerobic, short-rod-shaped, non-motile bacterium designated Rs7(T), was isolated from peat soil collected from Russia and was characterized to determine its taxonomic position. 16S rRNA gene sequence analysis revealed that the strain Rs7(T) belongs to the class Betaproteobacteria. The highest degree of sequence similarities were determined to be with Burkholderia tropica Ppe8(T) (98.4 %), Burkholderia unamae MTI-641(T) (97.8 %), Burkholderia bannensis E25(T) (97.7 %), Burkholderia heleia SA41(T) (97.0 %), and Burkholderia sacchari IPT101(T) (97.0 %). Chemotaxonomic data revealed that the strain Rs7(T) possesses ubiquinone Q-8. The polar lipid profile of strain Rs7(T) contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and an unknown amino phospholipid. The predominant fatty acids were C(16:0), C(19:0) cyclo ω8c, and C(17:0) cyclo, all of which corroborated the assignment of the strain to the genus Burkholderia. The DNA G+C content was 63.2 mol%. DNA-DNA hybridization experiments showed less than 37.8 % DNA relatedness with closely related type strains, thus confirming separate species status. The results of physiological and biochemical tests allowed phenotypic differentiation of strain Rs7(T) from the members of the genus Burkholderia. Based on these data, Rs7(T) (=KEMC 7302-068(T) = JCM 18069(T)) should be classified as the type strain for a novel Burkholderia species, for which the name Burkholderia humi sp. nov. is proposed.

  12. Melioidosis presenting as an infected intrathoracic subclavian artery pseudoaneurysm treated with femoral vein interposition graft.

    PubMed

    Schindler, Nancy; Calligaro, Keith D; Dougherty, Matthew J; Diehl, James; Modi, Ketang H; Braffman, Michael N

    2002-03-01

    We present the first case of in situ replacement of an infected subclavian artery using superficial femoral vein and the fourth reported case of an infected arterial pseudoaneurysm caused by pseudomonas pseudomallei. Sepsis and hoarseness developed in a 58-year-old man after recent travel to Borneo, Indonesia. Indirect laryngoscopy revealed a paralyzed right vocal cord. Computed tomography and arteriography revealed a 6.5-cm pseudoaneurysm of the proximal right subclavian artery. Blood cultures grew pseudomonas pseudomallei. An abnormal cardiac stress test prompted a coronary angiography, which revealed severe coronary artery disease.The patient underwent coronary artery bypass and in situ replacement of the infected subclavian artery pseudoaneurysm with a superficial femoral vein, along with placement of a pectoralis major muscle flap to cover the vein graft. Operative cultures of the pseudoaneurysm grew pseudomonas pseudomallei. The patient was treated with a 6-week course of intravenous ceftazidime and oral doxycycline and then continued on oral amoxicillin-clavulanate. One week after discontinuing intravenous antibiotics, the patient presented to the emergency department with a rapidly expanding, pulsatile mass in the right supraclavicular space. He was taken emergently to the operating room. After hypothermic circulatory arrest was accomplished, the disrupted vein graft and aneurysm cavity were resected and the subclavian artery was oversewn proximally and distally. Parenteral ceftazidime was continued for 3 months and oral amoxicillin-clavulanate (augmentin) was continued indefinitely. There was no evidence of infection clinically or by computed tomographic scan 2 years later. Although autogenous vein replacement of infected arteries and grafts may be successful in the majority of cases, this strategy should probably be avoided when particularly virulent bacteria such as the organism in this case are present.

  13. Modulation of Human Airway Barrier Functions during Burkholderia thailandensis and Francisella tularensis Infection Running Title: Airway Barrier Functions during Bacterial Infections.

    PubMed

    Blume, Cornelia; David, Jonathan; Bell, Rachel E; Laver, Jay R; Read, Robert C; Clark, Graeme C; Davies, Donna E; Swindle, Emily J

    2016-01-01

    The bronchial epithelium provides protection against pathogens from the inhaled environment through the formation of a highly-regulated barrier. In order to understand the pulmonary diseases melioidosis and tularemia caused by Burkholderia thailandensis and Fransicella tularensis, respectively, the barrier function of the human bronchial epithelium were analysed. Polarised 16HBE14o- or differentiated primary human bronchial epithelial cells (BECs) were exposed to increasing multiplicities of infection (MOI) of B. thailandensis or F. tularensis Live Vaccine Strain and barrier responses monitored over 24-72 h. Challenge of polarized BECs with either bacterial species caused an MOI- and time-dependent increase in ionic permeability, disruption of tight junctions, and bacterial passage from the apical to the basolateral compartment. B. thailandensis was found to be more invasive than F. tularensis. Both bacterial species induced an MOI-dependent increase in TNF-α release. An increase in ionic permeability and TNF-α release was induced by B. thailandensis in differentiated BECs. Pretreatment of polarised BECs with the corticosteroid fluticasone propionate reduced bacterial-dependent increases in ionic permeability, bacterial passage, and TNF-α release. TNF blocking antibody Enbrel(®) reduced bacterial passage only. BEC barrier properties are disrupted during respiratory bacterial infections and targeting with corticosteroids or anti-TNF compounds may represent a therapeutic option. PMID:27527221

  14. Modulation of Human Airway Barrier Functions during Burkholderia thailandensis and Francisella tularensis Infection Running Title: Airway Barrier Functions during Bacterial Infections

    PubMed Central

    Blume, Cornelia; David, Jonathan; Bell, Rachel E.; Laver, Jay R.; Read, Robert C.; Clark, Graeme C.; Davies, Donna E.; Swindle, Emily J.

    2016-01-01

    The bronchial epithelium provides protection against pathogens from the inhaled environment through the formation of a highly-regulated barrier. In order to understand the pulmonary diseases melioidosis and tularemia caused by Burkholderia thailandensis and Fransicella tularensis, respectively, the barrier function of the human bronchial epithelium were analysed. Polarised 16HBE14o- or differentiated primary human bronchial epithelial cells (BECs) were exposed to increasing multiplicities of infection (MOI) of B. thailandensis or F. tularensis Live Vaccine Strain and barrier responses monitored over 24–72 h. Challenge of polarized BECs with either bacterial species caused an MOI- and time-dependent increase in ionic permeability, disruption of tight junctions, and bacterial passage from the apical to the basolateral compartment. B. thailandensis was found to be more invasive than F. tularensis. Both bacterial species induced an MOI-dependent increase in TNF-α release. An increase in ionic permeability and TNF-α release was induced by B. thailandensis in differentiated BECs. Pretreatment of polarised BECs with the corticosteroid fluticasone propionate reduced bacterial-dependent increases in ionic permeability, bacterial passage, and TNF-α release. TNF blocking antibody Enbrel® reduced bacterial passage only. BEC barrier properties are disrupted during respiratory bacterial infections and targeting with corticosteroids or anti-TNF compounds may represent a therapeutic option. PMID:27527221

  15. Environmental Transmission of the Gut Symbiont Burkholderia to Phloem-Feeding Blissus insularis

    PubMed Central

    Xu, Yao; Buss, Eileen A.; Boucias, Drion G.

    2016-01-01

    The plant-phloem-feeding Blissus insularis possesses specialized midgut crypts, which harbor a dense population of the exocellular bacterial symbiont Burkholderia. Most individual B. insularis harbor a single Burkholderia ribotype in their midgut crypts; however, a diverse Burkholderia community exists within a host population. To understand the mechanism underlying the consistent occurrence of various Burkholderia in B. insularis and their specific association, we investigated potential gut symbiont transmission routes. PCR amplification detected a low titer of Burkholderia in adult reproductive tracts; however, fluorescence in situ hybridization assays failed to produce detectable signals in these tracts. Furthermore, no Burkholderia-specific PCR signals were detected in eggs and neonates, suggesting that it is unlikely that B. insularis prenatally transmits gut symbionts via ovarioles. In rearing experiments, most nymphs reared on St. Augustinegrass treated with cultured Burkholderia harbored the cultured Burkholderia strains. Burkholderia was detected in the untreated host grass of B. insularis, and most nymphs reared on untreated grass harbored a Burkholderia ribotype that was closely related to a plant-associated Burkholderia strain. These findings revealed that B. insularis neonates acquired Burkholderia primarily from the environment (i.e., plants and soils), even though the possibility of acquisition via egg surface cannot be excluded. In addition, our study explains how the diverse Burkholderia symbiont community in B. insularis populations can be maintained. PMID:27548682

  16. Environmental Transmission of the Gut Symbiont Burkholderia to Phloem-Feeding Blissus insularis.

    PubMed

    Xu, Yao; Buss, Eileen A; Boucias, Drion G

    2016-01-01

    The plant-phloem-feeding Blissus insularis possesses specialized midgut crypts, which harbor a dense population of the exocellular bacterial symbiont Burkholderia. Most individual B. insularis harbor a single Burkholderia ribotype in their midgut crypts; however, a diverse Burkholderia community exists within a host population. To understand the mechanism underlying the consistent occurrence of various Burkholderia in B. insularis and their specific association, we investigated potential gut symbiont transmission routes. PCR amplification detected a low titer of Burkholderia in adult reproductive tracts; however, fluorescence in situ hybridization assays failed to produce detectable signals in these tracts. Furthermore, no Burkholderia-specific PCR signals were detected in eggs and neonates, suggesting that it is unlikely that B. insularis prenatally transmits gut symbionts via ovarioles. In rearing experiments, most nymphs reared on St. Augustinegrass treated with cultured Burkholderia harbored the cultured Burkholderia strains. Burkholderia was detected in the untreated host grass of B. insularis, and most nymphs reared on untreated grass harbored a Burkholderia ribotype that was closely related to a plant-associated Burkholderia strain. These findings revealed that B. insularis neonates acquired Burkholderia primarily from the environment (i.e., plants and soils), even though the possibility of acquisition via egg surface cannot be excluded. In addition, our study explains how the diverse Burkholderia symbiont community in B. insularis populations can be maintained. PMID:27548682

  17. Burkholderia sprentiae sp. nov., isolated from Lebeckia ambigua root nodules.

    PubMed

    De Meyer, Sofie E; Cnockaert, Margo; Ardley, Julie K; Maker, Garth; Yates, Ron; Howieson, John G; Vandamme, Peter

    2013-11-01

    Seven Gram-stain-negative, rod-shaped bacteria were isolated from Lebeckia ambigua root nodules and authenticated on this host. Based on the 16S rRNA gene phylogeny, they were shown to belong to the genus Burkholderia, with the representative strain WSM5005(T) being most closely related to Burkholderia tuberum (98.08 % sequence similarity). Additionally, these strains formed a distinct group in phylogenetic trees based on the housekeeping genes gyrB and recA. Chemotaxonomic data including fatty acid profiles and analysis of respiratory quinones supported the assignment of the strains to the genus Burkholderia. Results of DNA-DNA hybridizations, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of our strains from the closest species of the genus Burkholderia with a validly published name. Therefore, these strains represent a novel species for which the name Burkholderia sprentiae sp. nov. (type strain WSM5005(T) = LMG 27175(T) = HAMBI 3357(T)) is proposed.

  18. Burkholderia dilworthii sp. nov., isolated from Lebeckia ambigua root nodules.

    PubMed

    De Meyer, Sofie E; Cnockaert, Margo; Ardley, Julie K; Van Wyk, Ben-Erik; Vandamme, Peter A; Howieson, John G

    2014-04-01

    Three strains of Gram-stain-negative, rod-shaped bacteria were isolated from Lebeckia ambigua root nodules and authenticated on this host. Based on the 16S rRNA gene sequence phylogeny, they were shown to belong to the genus Burkholderia, with the representative strain WSM3556(T) being most closely related to Burkholderia caledonica LMG 23644(T) (98.70 % 16S rRNA gene sequence similarity) and Burkholderia rhynchosiae WSM3937(T) (98.50 %). Additionally, these strains formed a distinct group in phylogenetic trees of the housekeeping genes gyrB and recA. Chemotaxonomic data, including fatty acid profiles and analysis of respiratory quinones, supported the assignment of our strains to the genus Burkholderia. Results of DNA-DNA hybridizations, MALDI-TOF MS analysis and physiological and biochemical tests allowed genotypic and phenotypic differentiation of our strains from their nearest neighbour species. Therefore, these strains represent a novel species, for which the name Burkholderia dilworthii sp. nov. is proposed, with the type strain WSM3556(T) ( = LMG 27173(T) = HAMBI 3353(T)).

  19. Ornibactin production and transport properties in strains of Burkholderia vietnamiensis and Burkholderia cepacia (formerly Pseudomonas cepacia).

    PubMed

    Meyer, J M; Van, V T; Stintzi, A; Berge, O; Winkelmann, G

    1995-10-01

    Several strains of Burkholderia vietnamiensis, isolated from the rhizosphere of rice plants, and four strains formerly known as Pseudomonas cepacia including two collection strains and two clinical isolates were compared for siderophore production and iron uptake. The B. vietnamiensis (TVV strains) as well as the B. cepacia strains (ATCC 25416 and ATCC 17759) and the clinical isolates K132 and LMG 6999 were all found to produce ornibactins under iron starvation. The two ATCC strains of B. cepacia additionally produced the previously described siderophores, pyochelin and cepabactin. Analysis of the ratio of isolated ornibactins (C4, C6 and C8) by HPLC revealed nearly identical profiles. Supplementation of the production medium with ornithine (20 mM) resulted in a 2.5-fold increase in ornibactin synthesis. Ornibactin-mediated iron uptake was independent of the length of the acyl side chain and was observed with all strains of B. vietnamiensis and B. cepacia, but was absent with strains of Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas stutzeri, known to produce pyoverdines or desferriferrioxamines as siderophores. These results suggest that ornibactin production is a common feature of all Burkholderia strains and that these strains develop an ornibactin-specific iron transport system which is distinct from the pyoverdine-specific transport in Pseudomonas strains. PMID:7580051

  20. Burkholderia cepacia Complex Vaccines: Where Do We Go from here?

    PubMed Central

    Pradenas, Gonzalo A.; Ross, Brittany N.; Torres, Alfredo G.

    2016-01-01

    Burkholderia comprises a wide variety of environmental Gram-negative bacteria. Burkholderia cepacia complex (Bcc) includes several Burkholderia species that pose a health hazard as they are able to cause respiratory infections in patients with chronic granulomatous disease and cystic fibrosis. Due to the intrinsic resistance to a wide array of antibiotics and naturally occurring immune evasion strategies, treatment of Bcc infections often proves to be unsuccessful. To date, limited work related to vaccine development has been performed for Bcc pathogens. In this review, we have gathered key aspects of Bcc research that have been reported in recent years related to vaccine efforts, virulence, immune responses, and animal models, and use this information to inform the research community of areas of opportunity toward development of a viable Bcc vaccine. PMID:27092530

  1. 40 CFR 725.1075 - Burkholderia cepacia complex.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Burkholderia cepacia complex. 725.1075 Section 725.1075 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES... significant new use is any use other than research and development in the degradation of chemicals...

  2. 40 CFR 725.1075 - Burkholderia cepacia complex.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Burkholderia cepacia complex. 725.1075 Section 725.1075 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES... significant new use is any use other than research and development in the degradation of chemicals...

  3. Removal of Burkholderia cepacia biofilms with oxidants

    NASA Technical Reports Server (NTRS)

    Koenig, D. W.; Mishra, S. K.; Pierson, D. L.

    1995-01-01

    Iodine is used to disinfect the water system aboard US space shuttles and is the anticipated biocide for the international space station. Water quality on spacecraft must be maintained at the highest possible levels for the safety of the crew. Furthermore, the treatment process used to maintain the quality of water on research must be robust and operate for long periods with minimal crew intervention. Biofilms are recalcitrant and pose a major threat with regard to chronic contamination of spacecraft water systems. We measured the effectiveness of oxidizing biocides on the removal and regrowth of Burkholderia (Pseudomonas) cepacia biofilms. B. cepacia, isolated from the water distribution system of the space shuttle Discovery, was grown in continuous culture to produce a bacterial contamination source for biofilm formation and removal studies. A 10(7) CFU ml-1 B. cepacia suspension, in distilled water, was used to form biofilms on 3000 micrometers2 glass surfaces. Rates of attachment were measured directly with image analysis and were found to be 7.8, 15.2, and 22.8 attachment events h-1 for flow rates of 20.7, 15.2, and 9.8 ml min-1, respectively. After 18 h of formation, the B. cepacia biofilms were challenged with oxidants (ozone, chlorine, and iodine) and the rates of biofilm removal determined by image analysis. Fifty percent of the biofilm material was removed in the first hour of continous treatment with 24 mg l-1 chlorine or 2 mg l-1 ozone. Iodine (48 mg l-1) did not remove any measurable cellular material after 6 h continuous contact. After this first removal of biofilms by the oxidants, the surface was allowed to refoul and was again treated with the biocide. Iodine was the only compound that was unable to remove cellular debris from either primary or secondary biofilms. Moreover, treating primary biofilms with iodine increased the rate of formation of secondary biofilms, from 4.4 to 5.8 attachment events h-1. All the oxidants tested inactivated the B

  4. Removal of Burkholderia cepacia biofilms with oxidants.

    PubMed

    Koenig, D W; Mishra, S K; Pierson, D L

    1995-01-01

    Iodine is used to disinfect the water system aboard US space shuttles and is the anticipated biocide for the international space station. Water quality on spacecraft must be maintained at the highest possible levels for the safety of the crew. Furthermore, the treatment process used to maintain the quality of water on research must be robust and operate for long periods with minimal crew intervention. Biofilms are recalcitrant and pose a major threat with regard to chronic contamination of spacecraft water systems. We measured the effectiveness of oxidizing biocides on the removal and regrowth of Burkholderia (Pseudomonas) cepacia biofilms. B. cepacia, isolated from the water distribution system of the space shuttle Discovery, was grown in continuous culture to produce a bacterial contamination source for biofilm formation and removal studies. A 10(7) CFU ml-1 B. cepacia suspension, in distilled water, was used to form biofilms on 3000 micrometers2 glass surfaces. Rates of attachment were measured directly with image analysis and were found to be 7.8, 15.2, and 22.8 attachment events h-1 for flow rates of 20.7, 15.2, and 9.8 ml min-1, respectively. After 18 h of formation, the B. cepacia biofilms were challenged with oxidants (ozone, chlorine, and iodine) and the rates of biofilm removal determined by image analysis. Fifty percent of the biofilm material was removed in the first hour of continous treatment with 24 mg l-1 chlorine or 2 mg l-1 ozone. Iodine (48 mg l-1) did not remove any measurable cellular material after 6 h continuous contact. After this first removal of biofilms by the oxidants, the surface was allowed to refoul and was again treated with the biocide. Iodine was the only compound that was unable to remove cellular debris from either primary or secondary biofilms. Moreover, treating primary biofilms with iodine increased the rate of formation of secondary biofilms, from 4.4 to 5.8 attachment events h-1. All the oxidants tested inactivated the B

  5. Burkholderia: an update on taxonomy and biotechnological potential as antibiotic producers.

    PubMed

    Depoorter, Eliza; Bull, Matt J; Peeters, Charlotte; Coenye, Tom; Vandamme, Peter; Mahenthiralingam, Eshwar

    2016-06-01

    Burkholderia is an incredibly diverse and versatile Gram-negative genus, within which over 80 species have been formally named and multiple other genotypic groups likely represent new species. Phylogenetic analysis based on the 16S rRNA gene sequence and core genome ribosomal multilocus sequence typing analysis indicates the presence of at least three major clades within the genus. Biotechnologically, Burkholderia are well-known for their bioremediation and biopesticidal properties. Within this review, we explore the ability of Burkholderia to synthesise a wide range of antimicrobial compounds ranging from historically characterised antifungals to recently described antibacterial antibiotics with activity against multiresistant clinical pathogens. The production of multiple Burkholderia antibiotics is controlled by quorum sensing and examples of quorum sensing pathways found across the genus are discussed. The capacity for antibiotic biosynthesis and secondary metabolism encoded within Burkholderia genomes is also evaluated. Overall, Burkholderia demonstrate significant biotechnological potential as a source of novel antibiotics and bioactive secondary metabolites. PMID:27115756

  6. Vertical transmission explains the specific Burkholderia pattern in Sphagnum mosses at multi-geographic scale

    PubMed Central

    Bragina, Anastasia; Cardinale, Massimiliano; Berg, Christian; Berg, Gabriele

    2013-01-01

    The betaproteobacterial genus Burkholderia is known for its versatile interactions with its hosts that can range from beneficial to pathogenic. A plant-beneficial-environmental (PBE) Burkholderia cluster was recently separated from the pathogen cluster, yet still little is known about burkholderial diversity, distribution, colonization, and transmission patterns on plants. In our study, we applied a combination of high-throughput molecular and microscopic methods to examine the aforementioned factors for Burkholderia communities associated with Sphagnum mosses – model plants for long-term associations – in Austrian and Russian bogs. Analysis of 16S rRNA gene amplicons libraries revealed that most of the Burkholderia are part of the PBE group, but a minor fraction was closely related to B. glathei and B. andropogonis from the pathogen cluster. Notably, Burkholderia showed highly similar composition patterns for each moss species independent of the geographic region, and Burkholderia-specific fluorescent in situ hybridization of Sphagnum gametophytes exhibited similar colonization patterns in different Sphagnum species at multi-geographic scales. To explain these patterns, we compared the compositions of the surrounding water, gametophyte-, and sporophyte-associated microbiome at genus level and discovered that Burkholderia were present in the Sphagnum sporophyte and gametophyte, but were absent in the flark water. Therefore, Burkholderia is a part of the core microbiome transmitted from the moss sporophyte to the gametophyte. This suggests a vertical transmission of Burkholderia strains, and thus underlines their importance for the plants themselves. PMID:24391630

  7. Molecular method to assess the diversity of Burkholderia species in environmental samples.

    PubMed

    Salles, Joana Falcão; De Souza, Francisco Adriano; van Elsas, Jan Dirk

    2002-04-01

    In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.

  8. Molecular Method To Assess the Diversity of Burkholderia Species in Environmental Samples

    PubMed Central

    Salles, Joana Falcão; De Souza, Francisco Adriano; van Elsas, Jan Dirk

    2002-01-01

    In spite of the importance of many members of the genus Burkholderia in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the Burkholderia diversity in soil samples. Primers specific for the genus Burkholderia were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from Burkholderia and non-Burkholderia species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus Burkholderia. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14 Burkholderia species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of Burkholderia species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the Burkholderia communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns. PMID:11916673

  9. Glutathione deficiency in type 2 diabetes impairs cytokine responses and control of intracellular bacteria

    PubMed Central

    Tan, Kai Soo; Lee, Kok Onn; Low, Kee Chung; Gamage, Akshamal Mihiranga; Liu, Yichun; Tan, Gek-Yen Gladys; Koh, Hui Qi Vanessa; Alonso, Sylvie; Gan, Yunn-Hwen

    2012-01-01

    Individuals with type 2 diabetes are at increased risk of acquiring melioidosis, a disease caused by Burkholderia pseudomallei infection. Although up to half of melioidosis patients have underlying diabetes, the mechanisms involved in this increased susceptibility are unknown. We found that B. pseudomallei–infected PBMCs from diabetic patients were impaired in IL-12p70 production, which resulted in decreased IFN-γ induction and poor bacterial killing. The defect was specific to the IL-12–IFN-γ axis. Defective IL-12 production was also observed during Mycobacterium tuberculosis infection, in which diabetes is likewise known to be a strong risk factor. In contrast, IL-12 production in diabetic cells was not affected upon Salmonella enterica infection or in response to TLR2, -3, -4, and -5 ligands. Poor IL-12 production correlated with a deficiency in intracellular reduced glutathione (GSH) concentrations in diabetic patients. Addition of GSH or N-acetylcysteine to PBMCs selectively restored IL-12 and IFN-γ production and improved bacterial killing. Furthermore, the depletion of GSH in mice led to increased susceptibility to melioidosis, reduced production of IL-12p70, and poorer disease outcome. Our data thus establish a link between GSH deficiency in diabetes and increased susceptibility to melioidosis that may open up new therapeutic avenues to protect diabetic patients against some intracellular bacterial pathogens. PMID:22546856

  10. Nodulation of Cyclopia spp. (Leguminosae, Papilionoideae) by Burkholderia tuberum

    PubMed Central

    Elliott, Geoffrey N.; Chen, Wen-Ming; Bontemps, Cyril; Chou, Jui-Hsing; Young, J. Peter W.; Sprent, Janet I.; James, Euan K.

    2007-01-01

    Background and Aims Species of the genus Burkholderia, from the Betaproteobacteria, have been isolated from legume nodules, but so far they have only been shown to form symbioses with species of Mimosa, sub-family Mimosoideae. This work investigates whether Burkholderia tuberum strains STM678 (isolated from Aspalathus carnosa) and DUS833 (from Aspalathus callosa) can nodulate species of the South African endemic papilionoid genera Cyclopia (tribe Podalyrieae) and Aspalathus (Crotalarieae) as well as the promiscuous legume Macroptilium atropurpureum (Phaseoleae). Method Bacterial strains and the phylogeny of their symbiosis-related (nod) genes were examined via 16S rRNA gene sequencing. Seedlings were grown in liquid culture and inoculated with one of the two strains of B. tuberum or with Sinorhizobium strain NGR 234 (from Lablab purpureus), Mesorhizobium strain DUS835 (from Aspalathus linearis) or Methylobacterium nodulans (from Crotalaria podocarpa). Some nodules, inoculated with green fluorescence protein (GFP)-tagged strains, were examined by light and electron microscopy coupled with immunogold labelling with a Burkholderia-specific antibody. The presence of active nitrogenase was checked by immunolabelling of nitrogenase and by the acetylene reduction assay. B. tuberum STM678 was also tested on a wide range of legumes from all three sub-families. Key Results Nodules were not formed on any of the Aspalathus spp. Only B. tuberum nodulated Cyclopia falcata, C. galioides, C. genistoides, C. intermedia and C. pubescens. It also effectively nodulated M. atropurpureum but no other species tested. GFP-expressing inoculant strains were located inside infected cells of C. genistoides, and bacteroids in both Cyclopia spp. and M. atropurpureum were immunogold-labelled with antibodies against Burkholderia and nitrogenase. Nitrogenase activity was also shown using the acetylene reduction assay. This is the first demonstration that a β-rhizobial strain can effectively

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

    PubMed

    HECKLY, R J

    1964-12-01

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

  12. Whole-Genome Analysis of Quorum-Sensing Burkholderia sp. Strain A9

    PubMed Central

    Chen, Jian Woon; Tee, Kok Keng; Chang, Chien-Yi; Yin, Wai-Fong; Chan, Xin-Yue

    2015-01-01

    Burkholderia spp. rely on N-acyl homoserine lactone as quorum-sensing signal molecules which coordinate their phenotype at the population level. In this work, we present the whole genome of Burkholderia sp. strain A9, which enables the discovery of its N-acyl homoserine lactone synthase gene. PMID:25745000

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

    PubMed

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

    2015-09-01

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

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

    PubMed Central

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

    2016-01-01

    Burkholderia cenocepacia is an opportunistic pathogen that belongs to Burkholderia cepacia complex (BCC). Burkholderia cenocepacia strain CEIB S5-2 was isolated from agricultural soils in Morelos, Mexico, and previously has shown its abilities for bioremediation. In this study, we report the draft genome sequence of Burkholderia cenocepacia strain CEIB S5-2. PMID:27125479

  15. Diversity and distribution of Burkholderia cepacia complex in the rhizosphere of rice and maize.

    PubMed

    Zhang, Lixin; Xie, Guanlin

    2007-01-01

    A survey of Burkholderia cepacia complex (Bcc) species was conducted in agricultural fields within Hangzhou, China. Out of the 251 bacterial isolates recovered on the selective media from the rhizosphere of rice and maize, 112 of them were assigned to Bcc by PCR assays. The species composition of the Bcc isolates was analyzed by a combination of recA-restriction fragment length polymorphism assays, species-specific PCR tests and recA gene sequencing. The results revealed that the majority belong to B. cepacia, Burkholderia cenocepacia recA lineage IIIB, Burkholderia vietnamiensis and Burkholderia pyrrocinia. Burkholderia cenocepacia and B. vietnamiensis dominated the rhizosphere of maize and rice, respectively, indicating that species composition and abundance of Bcc may vary dramatically in different crop rhizospheres. In addition, one isolate (R456) formed a single discrete cluster within the phylogenetic analysis of the Bcc recA gene, and it may belong to a new genomovar. PMID:17233735

  16. Burkholderia fungorum sp. nov. and Burkholderia caledonica sp. nov., two new species isolated from the environment, animals and human clinical samples.

    PubMed

    Coenye, T; Laevens, S; Willems, A; Ohlén, M; Hannant, W; Govan, J R; Gillis, M; Falsen, E; Vandamme, P

    2001-05-01

    A polyphasic taxonomic study that included DNA-DNA hybridizations, DNA base ratio determinations, 16S rDNA sequence analyses, whole-cell protein and fatty acid analyses and an extensive biochemical characterization was performed on 16 strains isolated from the environment, animals and human clinical samples. The isolates belonged to the genus Burkholderia, were phylogenetically closely related to Burkholderia graminis, Burkholderia caribensis and Burkholderia phenazinium and had G+C contents between 61.9 and 62.2 mol%. Seven strains isolated from the rhizosphere were assigned to Burkholderia caledonica sp. nov. [type strain LMG 19076T (= CCUG 42236T)]. Nine strains isolated from the environment, animals and human clinical samples were assigned to Burkholderia fungorum sp. nov. [type strain LMG 16225T (= CCUG 31961T)]. Differential tests for B. graminis, B. caribensis, B. phenazinium, B. caledonica and B. fungorum include the following: assimilation of trehalose, citrate, DL-norleucine, adipate and sucrose; nitrate reduction; growth in the presence of 0.5% NaCl; and beta-galactosidase activity. PMID:11411678

  17. Burkholderia eburnea sp. nov., isolated from peat soil.

    PubMed

    Kang, Sang Rim; Srinivasan, Sathiyaraj; Lee, Sang Seob

    2014-04-01

    A novel aerobic bacterium, designated strain RR11(T), was isolated from peat soil and was characterized by using a polyphasic taxonomic approach and identified in order to determine its taxonomic position. Strain RR11(T) is a Gram-negative, non-sporulating, motile, short-rod-shaped bacterium. 16S rRNA gene sequence analysis identified this strain as a member of the genus Burkholderia of the class Betaproteobacteria. The highest degrees of gene sequence similarity were found with Burkholderia tropica Ppe8(T) (98.0 %), B. bannensis E25(T) (97.3 %), B. ferrariae FeGI01(T) (97.1 %), B. unamae MTI-641(T) (97.1 %) and B. heleia SA41(T) (97.1 %). Strain RR11(T) had the following chemotaxonomic characteristics: the major ubiquinone was Q-8, the DNA G+C content was 60.8 mol%, the major fatty acids were C16 : 0, C19 : 0 cyclo ω8c and C17 : 0 cyclo and the polar lipid profile contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and an unknown aminophospholipid. Based on its morphological, physiological and chemotaxonomic characteristics, together with 16S rRNA gene sequence comparison results, strain RR11(T) represents a novel species, for which the name Burkholderia eburnea sp. nov. is proposed. The type strain is strain RR11(T) ( = KEMC 7302-065(T) = JCM 18070(T)).

  18. Combined use of a specific probe and PCAT medium to study Burkholderia in soil.

    PubMed

    Pallud, C; Viallard, V; Balandreau, J; Normand, P; Grundmann, G

    2001-10-01

    Due to its pathogenic traits and agricultural benefits, there is some challenge in detecting Burkholderia in the soil environment. In this perspective, an existing semi-selective medium, (PCAT), was combined with a Burkholderia specific molecular probe. Using the complete 16S rRNA sequences of all available Burkholderia species type strains, we selected the following sequence: 5'-ACCCTCTGTTCCGACCATTGTATGA-3'. The probe was validated against GenBank sequences, with dot blots and colony hybridization tests. A diversity study of all strains growing on a PCAT plate after plating a soil dilution (75 strains) was carried out with ARDRA analysis and colony hybridization tests. All the hybridizing strains belonged to genus Burkholderia. The major type of non-hybridizing isolates belonged to Pseudomonas (16S rRNA sequencing). Both tools were combined to compare the Burkholderia populations in a rhizosphere (maize) and a non-rhizosphere soil. Based on hybridizing PCAT isolates, we were able to show an increase in Burkholderia populations in the maize rhizosphere. This genus represented 2% and 16% of the total cultivable microflora in the non-rhizosphere and rhizosphere soils, respectively. Although PCAT was shown not to be appropriate to routinely enumerate Burkholderia populations in soil, it allowed environmental investigations at the genus level, when combined with a molecular specific probe. PMID:11566224

  19. Characterization of the Poly-β-1,6-N-Acetylglucosamine Polysaccharide Component of Burkholderia Biofilms ▿

    PubMed Central

    Yakandawala, Nandadeva; Gawande, Purushottam V.; LoVetri, Karen; Cardona, Silvia T.; Romeo, Tony; Nitz, Mark; Madhyastha, Srinivasa

    2011-01-01

    We demonstrated the production of poly-β-1,6-N-acetylglucosamine (PNAG) polysaccharide in the biofilms of Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia cepacia, and Burkholderia cenocepacia using an immunoblot assay for PNAG. These results were confirmed by further studies, which showed that the PNAG hydrolase, dispersin B, eliminated immunoreactivity of extracts from the species that were tested (B. cenocepacia and B. multivorans). Dispersin B also inhibited biofilm formation and dispersed preformed biofilms of Burkholderia species. These results imply a role for PNAG in the maintenance of Burkholderia biofilm integrity. While PNAG was present in biofilms of all of the wild-type test organisms, a ΔpgaBC mutant of B. multivorans (Mu5) produced no detectable PNAG, indicating that these genes are needed for Burkholderia PNAG formation. Furthermore, restoration of PNAG production in PNAG negative E. coli TRXWMGΔC (ΔpgaC) by complementation with B. multivorans pgaBCD confirmed the involvement of these genes in Burkholderia PNAG production. While the confocal scanning laser microscopy of untreated wild-type B. multivorans showed thick, multilayered biofilm, Mu5 and dispersin B-treated wild-type biofilms were thin, poorly developed, and disrupted, confirming the involvement of PNAG in B. multivorans biofilm formation. Thus, PNAG appears to be an important component of Burkholderia biofilms, potentially contributing to its resistance to multiple antibiotics and persistence during chronic infections, including cystic fibrosis-associated infection. PMID:21984237

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

    EPA Science Inventory

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

  1. AQUIFER PROTIST RESPONSE AND THE POTENTIAL FOR TCE BIOREMEDIATION WITH BURKHOLDERIA CEPACIA G4 PR1

    EPA Science Inventory

    The introduction of bacteria into the environment for bioremediation purposes (bioaugmentation) requires analysis and monitoring of the persistence and activity of microbial population for efficacy and risk assessment purposes. Burkholderia cepacia G4 PR123 and PR131 constitutive...

  2. A fine-scale phylogenetic analysis of free-living Burkholderia species in sugarcane field soil.

    PubMed

    Tago, Kanako; Itoh, Hideomi; Kikuchi, Yoshitomo; Hori, Tomoyuki; Sato, Yuya; Nagayama, Atsushi; Okubo, Takashi; Navarro, Ronald; Aoyagi, Tomo; Hayashi, Kentaro; Hayatsu, Masahito

    2014-01-01

    The diversity and abundance of Burkholderia species in sugarcane field soils were investigated by a 16S rRNA gene-based approach using genus-specific primers. A total of 365,721 sequences generated by the Illumina MiSeq platform were assigned to the genus Burkholderia. Nearly 58% of these sequences were placed in a previously defined cluster, including stinkbug symbionts. Quantitative PCR analysis revealed a consistent number of 16S rRNA gene copies for Burkholderia species (10(7) g(-1) soil) across the sampled fields. C/N, pH, and nitrate concentrations were important factors shaping the Burkholderia community structure; however, their impacts were not significant considering the overall genus size.

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

    PubMed Central

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

    2016-01-01

    Previous studies have demonstrated that extracellular glutathione reduces the ability of the Cystic Fibrosis pathogen Burkholderia cenocepacia to infect primary or immortalized epithelial respiratory cells. We report here that the adhesion and invasion ability of B. cenocepacia is limited also by thiol-oxidizing and disulphide-reducing agents and by protein disulfide is