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1

Burkholderia pseudomallei Capsular Polysaccharide Conjugates Provide Protection against Acute Melioidosis  

PubMed Central

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

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

2

Burkholderia pseudomallei capsular polysaccharide conjugates provide protection against acute melioidosis.  

PubMed

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

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

2014-08-01

3

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

PubMed

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. PMID:19121666

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

2008-12-01

4

Genomic patterns of pathogen evolution revealed by comparison of Burkholderia pseudomallei, the causative agent of melioidosis, to avirulent Burkholderia thailandensis  

E-print Network

Background: The Gram-negative bacterium Burkholderia pseudomallei (Bp) is the causative agent of the human disease melioidosis. To understand the evolutionary mechanisms contributing to Bp virulence, we performed a comparative ...

Yu, Yiting

5

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

PubMed Central

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

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

2015-01-01

6

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

PubMed

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

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

2015-03-01

7

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

PubMed Central

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

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

2014-01-01

8

A Prospective Study of Melioidosis After Environmental Exposure of Healthy Participants to Burkholderia pseudomallei During a Muddy Endurance Challenge.  

PubMed

In a prospective study of 123 healthy adults competing in a mud-exposing endurance challenge in the melioidosis-endemic tropical north of the Northern Territory of Australia, there were no asymptomatic seroconversions to Burkholderia pseudomallei using indirect hemagglutination assay. However, one competitor developed melioidosis attributable to infection acquired during the event. PMID:25624406

Grivas, Rebecca; Barklay, Sarah; Ruane, Amber; Mayo, Mark; Theobald, Vanessa; Freeman, Kevin; Norton, Robert; Baird, Robert W; Currie, Bart J

2015-04-01

9

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

PubMed Central

Burkholderia pseudomallei is a Gram-negative environmental bacterium that causes melioidosis, a potentially life-threatening infectious disease affecting mammals, including humans. Melioidosis symptoms are both protean and diverse, ranging from mild, localized skin infections to more severe and often fatal presentations including pneumonia, septic shock with multiple internal abscesses and occasionally neurological involvement. Several ubiquitous virulence determinants in B. pseudomallei have already been discovered. However, the molecular basis for differential pathogenesis has, until now, remained elusive. Using clinical data from 556 Australian melioidosis cases spanning more than 20 years, we identified a Burkholderia mallei-like actin polymerization bimABm gene that is strongly associated with neurological disease. We also report that a filamentous hemagglutinin gene, fhaB3, is associated with positive blood cultures but is negatively correlated with localized skin lesions without sepsis. We show, for the first time, that variably present virulence factors play an important role in the pathogenesis of melioidosis. Collectively, our study provides a framework for assessing other non-ubiquitous bacterial virulence factors and their association with disease, such as candidate loci identified from large-scale microbial genome-wide association studies. PMID:24618705

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

2014-01-01

10

Within-Host Evolution of Burkholderia pseudomallei in Four Cases of Acute Melioidosis  

PubMed Central

Little is currently known about bacterial pathogen evolution and adaptation within the host during acute infection. Previous studies of Burkholderia pseudomallei, the etiologic agent of melioidosis, have shown that this opportunistic pathogen mutates rapidly both in vitro and in vivo at tandemly repeated loci, making this organism a relevant model for studying short-term evolution. In the current study, B. pseudomallei isolates cultured from multiple body sites from four Thai patients with disseminated melioidosis were subjected to fine-scale genotyping using multilocus variable-number tandem repeat analysis (MLVA). In order to understand and model the in vivo variable-number tandem repeat (VNTR) mutational process, we characterized the patterns and rates of mutations in vitro through parallel serial passage experiments of B. pseudomallei. Despite the short period of infection, substantial divergence from the putative founder genotype was observed in all four melioidosis cases. This study presents a paradigm for examining bacterial evolution over the short timescale of an acute infection. Further studies are required to determine whether the mutational process leads to phenotypic alterations that impact upon bacterial fitness in vivo. Our findings have important implications for future sampling strategies, since colonies in a single clinical sample may be genetically heterogeneous, and organisms in a culture taken late in the infective process may have undergone considerable genetic change compared with the founder inoculum. PMID:20090837

Limmathurotsakul, Direk; Max, Tamara L.; Sarovich, Derek S.; Vogler, Amy J.; Dale, Julia L.; Ginther, Jennifer L.; Leadem, Benjamin; Colman, Rebecca E.; Foster, Jeffrey T.; Tuanyok, Apichai; Wagner, David M.; Peacock, Sharon J.; Pearson, Talima; Keim, Paul

2010-01-01

11

Burkholderia pseudomallei Known Siderophores and Hemin Uptake Are Dispensable for Lethal Murine Melioidosis  

PubMed Central

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 a novel ferritin-iron acquisition pathway as a means to sustain in vivo growth. PMID:22745846

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

2012-01-01

12

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

PubMed

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 a novel ferritin-iron acquisition pathway as a means to sustain in vivo growth. PMID:22745846

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

2012-01-01

13

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

PubMed Central

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

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

14

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

PubMed Central

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

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

2014-01-01

15

The Burkholderia pseudomallei ?asd Mutant Exhibits Attenuated Intracellular Infectivity and Imparts Protection against Acute Inhalation Melioidosis in Mice ?  

PubMed Central

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

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

2011-01-01

16

Potential Immunogenic Polypeptides of Burkholderia pseudomallei Identified by Shotgun Expression Library and Evaluation of Their Efficacy for Serodiagnosis of Melioidosis  

PubMed Central

The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei. PMID:23532805

Puah, Suat Moi; Puthucheary, SD; Chua, Kek Heng

2013-01-01

17

Neurotropic Threat Characterization of Burkholderia pseudomallei Strains  

PubMed Central

The death rate for neurologic melioidosis is high. Whether certain Burkholderia pseudomallei strains are more likely than other strains to cause central nervous system infection and whether route of infection influences the neurotropic threat remain unclear. Therefore, we compared the virulence and dissemination of Australian clinical isolates collected during October 1989–October 2012 from patients with neurologic and nonneurologic melioidosis after intranasal and subcutaneous infection of mice in an experimental model. We did not observe neurotropism as a unique characteristic of isolates from patients with neurologic melioidosis. Rather, a distinct subset of B. pseudomallei strains appear to have heightened pathogenic potential for rapid dissemination to multiple tissues, including the central nervous system, irrespective of the infection route. This finding has valuable public health ramifications for initiating appropriate and timely therapy after exposure to systemically invasive B. pseudomallei strains. Increasing understanding of B. pseudomallei pathology and its influencing factors will further reduce illness and death from this disease. PMID:25530166

Fane, Anne; Rush, Catherine; Govan, Brenda; Mayo, Mark; Currie, Bart J.; Ketheesan, Natkunam

2015-01-01

18

Flagella Are Virulence Determinants of Burkholderia pseudomallei  

Microsoft Academic Search

Received 11 June 2002\\/Returned for modification 8 October 2002\\/Accepted 23 December 2002 Burkholderia pseudomallei, a facultatively intracellular pathogen, is a flagellated and motile gram-negative bacterium and is the causative agent of melioidosis in humans. Flagella are commonly recognized as important virulence determinants expressed by bacterial pathogens since the motility phenotype imparted by these organelles often correlates with the ability of

K. L. Chua; Y. Y. Chan; Y. H. Gan

2003-01-01

19

Analysis of the Prevalence, Secretion and Function of a Cell Cycle-Inhibiting Factor in the Melioidosis Pathogen Burkholderia pseudomallei  

PubMed Central

Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif), that is injected into host cells via a Type III secretion system (T3SS) leading to arrest of cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385), which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7%) harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15) of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE) was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro. PMID:24809950

Pumirat, Pornpan; Broek, Charles Vander; Juntawieng, Niramol; Muangsombut, Veerachat; Kiratisin, Pattarachai; Pattanapanyasat, Kovit; Stevens, Joanne M.; Stevens, Mark P.; Korbsrisate, Sunee

2014-01-01

20

Efflux-Mediated Aminoglycoside and Macrolide Resistance in Burkholderia pseudomallei  

Microsoft Academic Search

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to a wide range of antimicrobial agents including b-lactams, aminoglycosides, macrolides, and polymyxins. We used Tn5-OT182 to mutagenize B. pseudomallei to identify the genes involved in aminoglycoside resistance. We report here on the identification of AmrAB-OprA, a multidrug efflux system in B. pseudomallei which is specific for both ami- noglycoside

RICHARD A. MOORE; DAVID DESHAZER; SHAUNA RECKSEIDLER; ANIA WEISSMAN; DONALD E. WOODS

21

Burkholderia pseudomallei traced to water treatment plant in Australia.  

PubMed Central

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

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

2000-01-01

22

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

PubMed

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. PMID:24710616

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

2014-06-01

23

Whole-Genome Sequences of 80 Environmental and Clinical Isolates of Burkholderia pseudomallei  

PubMed Central

Here, we present the draft genome sequences of 80 isolates of Burkholderia pseudomallei. The isolates represent clinical cases of melioidosis and environmental isolates from regions in Australia and Papua New Guinea where B. pseudomallei is endemic. The genomes provide further context for the diversity of the pathogen. PMID:25676747

Baker, Anthony L.; Chain, Patrick S.; Currie, Bart J.; Daligault, Hajnalka E.; Davenport, Karen W.; Davis, Christopher B.; Inglis, Timothy J. J.; Kaestli, Mirjam; Koren, Sergey; Mayo, Mark; Merritt, Adam J.; Sarovich, Derek S.; Warner, Jeffrey

2015-01-01

24

Whole-Genome Sequences of 80 Environmental and Clinical Isolates of Burkholderia pseudomallei.  

PubMed

Here, we present the draft genome sequences of 80 isolates of Burkholderia pseudomallei. The isolates represent clinical cases of melioidosis and environmental isolates from regions in Australia and Papua New Guinea where B. pseudomallei is endemic. The genomes provide further context for the diversity of the pathogen. PMID:25676747

Johnson, Shannon L; Baker, Anthony L; Chain, Patrick S; Currie, Bart J; Daligault, Hajnalka E; Davenport, Karen W; Davis, Christopher B; Inglis, Timothy J J; Kaestli, Mirjam; Koren, Sergey; Mayo, Mark; Merritt, Adam J; Price, Erin P; Sarovich, Derek S; Warner, Jeffrey; Rosovitz, M J

2015-01-01

25

Functional Characterization of Burkholderia pseudomallei Trimeric Autotransporters  

PubMed Central

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

Campos, Cristine G.; Byrd, Matthew S.

2013-01-01

26

Adaptation and Antibiotic Tolerance of Anaerobic Burkholderia pseudomallei ? †  

PubMed Central

The Gram-negative bacterium Burkholderia pseudomallei is the etiological agent of melioidosis and is remarkably resistant to most classes of antibacterials. Even after months of treatment with antibacterials that are relatively effective in vitro, there is a high rate of treatment failure, indicating that this pathogen alters its patterns of antibacterial susceptibility in response to cues encountered in the host. The pathology of melioidosis indicates that B. pseudomallei encounters host microenvironments that limit aerobic respiration, including the lack of oxygen found in abscesses and in the presence of nitric oxide produced by macrophages. We investigated whether B. pseudomallei could survive in a nonreplicating, oxygen-deprived state and determined if this physiological state was tolerant of conventional antibacterials. B. pseudomallei survived initial anaerobiosis, especially under moderately acidic conditions similar to those found in abscesses. Microarray expression profiling indicated a major shift in the physiological state of hypoxic B. pseudomallei, including induction of a variety of typical anaerobic-environment-responsive genes and genes that appear specific to anaerobic B. pseudomallei. Interestingly, anaerobic B. pseudomallei was unaffected by antibacterials typically used in therapy. However, it was exquisitely sensitive to drugs used against anaerobic pathogens. After several weeks of anaerobic culture, a significant loss of viability was observed. However, a stable subpopulation that maintained complete viability for at least 1 year was established. Thus, during the course of human infection, if a minor subpopulation of bacteria inhabited an oxygen-restricted environment, it might be indifferent to traditional therapy but susceptible to antibiotics frequently used to treat anaerobic infections. PMID:21537012

Hamad, Mohamad A.; Austin, Chad R.; Stewart, Amanda L.; Higgins, Mike; Vázquez-Torres, Andrés; Voskuil, Martin I.

2011-01-01

27

An Improved Selective Culture Medium Enhances the Isolation of Burkholderia pseudomallei from Contaminated Specimens  

PubMed Central

Burkholderia pseudomallei is a Gram-negative environmental bacterium found in tropical climates that causes melioidosis. Culture remains the diagnostic gold standard, but isolation of B. pseudomallei from heavily contaminated sites, such as fecal specimens, can be difficult. We recently reported that B. pseudomallei is capable of infecting the gastrointestinal tract of mice and suggested that the same may be true in humans. Thus, there is a strong need for new culture techniques to allow for efficient detection of B. pseudomallei in fecal and other specimens. We found that the addition of norfloxacin, ampicillin, and polymyxin B to Ashdown's medium (NAP-A) resulted in increased specificity without affecting the growth of 25 B. pseudomallei strains. Furthermore, recovery of B. pseudomallei from human clinical specimens was not affected by the three additional antibiotics. Therefore, we conclude that NAP-A medium provides a new tool for more sensitive isolation of B. pseudomallei from heavily contaminated sites. PMID:24062483

Goodyear, Andrew; Strange, Linda; Rholl, Drew A.; Silisouk, Joy; Dance, David A. B.; Schweizer, Herbert P.; Dow, Steven

2013-01-01

28

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

PubMed Central

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

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

2004-01-01

29

Systematic Review and Consensus Guidelines for Environmental Sampling of Burkholderia pseudomallei  

PubMed Central

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

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

30

Genomic islands from five strains of Burkholderia pseudomallei  

PubMed Central

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

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

31

Polar Lipids of Burkholderia pseudomallei Induce Different Host Immune Responses  

PubMed Central

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

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

2013-01-01

32

Functional reconstitution, gene isolation and topology modelling of porins from Burkholderia pseudomallei and Burkholderia thailandensis.  

PubMed Central

The sequences for Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis have been deposited in the DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases under the accession numbers AY312416 and AY312417 respectively. The intracellular pathogen Burkholderia pseudomallei is the causative agent of tropical melioidosis, and Burkholderia thailandensis is a closely-related Gram-negative bacterium that does not cause serious disease. Like other bacteria, the major outer membrane (OM) porins of Burkholderia strains, Bps Omp38 and Bth Omp38 may have roles in antibiotic resistance and immunity. We purified both proteins and found them to be immunologically related, SDS-resistant, heat-sensitive trimers with M (r) of approx. 110000. In functional liposome-swelling assays, both proteins showed similar permeabilities for small sugar molecules, compatible with a pore diameter of between 1.2 and 1.6 nm. Secondary structure analysis by FTIR (Fourier-transform infrared) spectroscopy revealed almost identical spectra with predominantly beta-sheet structures, typical of bacterial porins. MALDI-TOF (matrix-assisted laser-desorption ionization-time of flight) MS and ESI/MS (electrospray ionization MS) analysis of each protein showed extensive sequence similarities to the OpcP1 porin from Burkholderia cepacia (later found to be 76.5% identical). Based on information from the incomplete B. pseudomallei genome-sequencing project, the genes encoding Omp38 were identified and amplified by PCR from B. pseudomallei and B. thailandensis genomic DNA. The nucleotide sequences are 99.7% identical, and the predicted processed proteins are 100% identical. Topology prediction and molecular modelling suggest that this newly-isolated and cloned porin is a 16-stranded beta-barrel and the external loops of the protein could be important determinants of the immune response to infection. PMID:14567756

Siritapetawee, Jaruwan; Prinz, Heino; Samosornsuk, Worada; Ashley, Richard H; Suginta, Wipa

2004-01-01

33

Post-exposure therapeutic efficacy of COX-2 inhibition against Burkholderia pseudomallei.  

PubMed

Burkholderia pseudomallei is a Gram-negative, facultative intracellular bacillus and the etiologic agent of melioidosis, a severe disease in Southeast Asia and Northern Australia. Like other multidrug-resistant pathogens, the inherent antibiotic resistance of B. pseudomallei impedes treatment and highlights the need for alternative therapeutic strategies that can circumvent antimicrobial resistance mechanisms. In this work, we demonstrate that host prostaglandin E2 (PGE2) production plays a regulatory role in the pathogenesis of B. pseudomallei. PGE2 promotes B. pseudomallei intracellular survival within macrophages and bacterial virulence in a mouse model of pneumonic melioidosis. PGE2-mediated immunosuppression of macrophage bactericidal effector functions is associated with increased arginase 2 (Arg2) expression and decreased nitric oxide (NO) production. Treatment with a commercially-available COX-2 inhibitor suppresses the growth of B. pseudomallei in macrophages and affords significant protection against rapidly lethal pneumonic melioidosis when administered post-exposure to B. pseudomallei-infected mice. COX-2 inhibition may represent a novel immunotherapeutic strategy to control infection with B. pseudomallei and other intracellular pathogens. PMID:23675544

Asakrah, Saja; Nieves, Wildaliz; Mahdi, Zaid; Agard, Mallory; Zea, Arnold H; Roy, Chad J; Morici, Lisa A

2013-01-01

34

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

PubMed Central

Infection by the Gram-negative pathogen Burkholderia pseudomallei results in the disease melioidosis, acquired from the environment in parts of southeast Asia and northern Australia. Clinical symptoms of melioidosis range from acute (fever, pneumonia, septicemia, and localized infection) to chronic (abscesses in various organs and tissues, most commonly occurring in the lungs, liver, spleen, kidney, prostate and skeletal muscle), and persistent infections in humans are difficult to cure. Understanding the basic biology and genomics of B. pseudomallei is imperative for the development of new vaccines and therapeutic interventions. This formidable task is becoming more tractable due to the increasing number of B. pseudomallei genomes that are being sequenced and compared. Here, we compared three B. pseudomallei genomes, from strains MSHR668, K96243 and 1106a, to identify features that might explain why MSHR668 is more virulent than K96243 and 1106a in a mouse model of B. pseudomallei infection. Our analyses focused on metabolic, virulence and regulatory genes that were present in MSHR668 but absent from both K96243 and 1106a. We also noted features present in K96243 and 1106a but absent from MSHR668, and identified genomic differences that may contribute to variations in virulence noted among the three B. pseudomallei isolates. While this work contributes to our understanding of B. pseudomallei genomics, more detailed experiments are necessary to characterize the relevance of specific genomic features to B. pseudomallei metabolism and virulence. Functional analyses of metabolic networks, virulence and regulation shows promise for examining the effects of B. pseudomallei on host cell metabolism and will lay a foundation for future prediction of the virulence of emerging strains. Continued emphasis in this area will be critical for protection against melioidosis, as a better understanding of what constitutes a fully virulent Burkholderia isolate may provide for better diagnostic and medical countermeasure strategies. PMID:25536074

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

2014-01-01

35

Molecular Characterization of Putative Virulence Determinants in Burkholderia pseudomallei  

PubMed Central

The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei. PMID:25215325

Puah, Suat Moi; Puthucheary, S. D.; Wang, Jin Town; Pan, Yi Jiun; Chua, Kek Heng

2014-01-01

36

Accurate and Rapid Identification of the Burkholderia pseudomallei Near-Neighbour, Burkholderia ubonensis, Using Real-Time PCR  

PubMed Central

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. PMID:23967229

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

37

Use of 16S rRNA Gene Sequencing for Rapid Identification and Differentiation of Burkholderia pseudomallei and B. mallei  

Microsoft Academic Search

Burkholderia pseudomallei and B. mallei, the causative agents of melioidosis and glanders, respectively, are designated category B biothreat agents. Current methods for identifying these organisms rely on their phe- notypic characteristics and an extensive set of biochemical reactions. We evaluated the use of 16S rRNA gene sequencing to rapidly identify these two species and differentiate them from each other as

Jay E. Gee; Claudio T. Sacchi; Mindy B. Glass; Barun K. De; Robbin S. Weyant; Paul N. Levett; Anne M. Whitney; Alex R. Hoffmaster; Tanja Popovic

2003-01-01

38

Melioidosis in Traveler from Africa to Spain  

PubMed Central

The worldwide epidemiology of melioidosis is changing. We describe a case of acute melioidosis in Spain in a patient who had traveled to Africa. A novel sequence type of Burkholderia pseudomallei was identified in this patient. Clinicians should be aware of the possibility of melioidosis in travelers returning from melioidosis-nonendemic regions. PMID:24047798

Quereda, Carmen; Gil, Horacio; Anda, Pedro; Núñez-Murga, María; Cantón, Rafael; López-Vélez, Rogelio

2013-01-01

39

Burkholderia pseudomallei in cystic fibrosis and treatment complications  

PubMed Central

A healthy 29-year-old Australian man with cystic fibrosis (CF) grew Burkholderia pseudomallei on a routine sputum culture 1 month after returning from holiday in Thailand. He underwent a 12-month treatment regime with multiple antibiotics resulting in a number of adverse events. Sputum cultures were cleared of the pathogen and remain negative 8 years post-treatment. There were no clinical sequelae and no deterioration in lung function. Few reports have been published to date on melioidosis in CF patients. The proposed management for this infection includes multiple antibiotics regimens for prolonged periods of time, which may result in adverse events. Optimal treatment and length of treatment are currently determined on an individual basis.

Radhakrishna, Naghmeh; Morton, Judith

2015-01-01

40

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

PubMed

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

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

41

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

PubMed Central

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

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

42

Characterization of the Burkholderia pseudomallei K96243 Capsular Polysaccharide I Coding Region  

PubMed Central

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

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

43

Transcriptome analysis of Burkholderia pseudomallei T6SS identifies Hcp1 as a potential serodiagnostic marker.  

PubMed

Burkholderia pseudomallei, the causative agent of melioidosis, is able to survive extreme environments and utilizes various virulence factors for survival and pathogenicity. To compete and survive within these different ecological niches, B. pseudomallei has evolved specialized pathways, including the Type VI secretion systems (T6SSs), that have a role in pathogenesis as well as interbacterial interactions. We examined the expression profile of B. pseudomallei T6SS six gene clusters during infection of U937 macrophage cells. T6SS-5 was robustly transcribed while the other five clusters were not significantly regulated proposing the utility of T6SS-5 as a potential biomarker of exposure to B. pseudomallei. Transcription of T6SS regulators VirAG and BprB was also not significant during infection when compared to bacteria grown in culture. Guided by these findings, three highly expressed T6SS genes, tssJ-4, hcp1 and tssE-5, were expressed as recombinant proteins and screened against melioidosis patient sera by western analysis and ELISA. Only Hcp1 was reactive by both types of analysis. The recombinant Hcp1 protein was further evaluated against a cohort of melioidosis patients (n = 32) and non-melioidosis individuals (n = 20) sera and the data clearly indicates a higher sensitivity (93.7%) and specificity (100%) for Hcp1 compared to bacterial lysate. The detection of anti-Hcp1 antibodies in patients' sera indicating the presence of B. pseudomallei highlights the potential of Hcp1 to be further developed as a serodiagnostic marker for melioidosis. PMID:25616255

Chieng, Sylvia; Mohamed, Rahmah; Nathan, Sheila

2015-02-01

44

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

PubMed

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

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

2014-05-01

45

A Burkholderia pseudomallei Outer Membrane Vesicle Vaccine Provides Protection against Lethal Sepsis  

PubMed Central

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

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

2014-01-01

46

Correlates of Immune Protection following Cutaneous Immunization with an Attenuated Burkholderia pseudomallei Vaccine  

PubMed Central

Infections with the Gram-negative bacterium Burkholderia pseudomallei (melioidosis) are associated with high mortality, and there is currently no approved vaccine to prevent the development of melioidosis in humans. Infected patients also do not develop protective immunity to reinfection, and some individuals will develop chronic, subclinical infections with B. pseudomallei. At present, our understanding of what constitutes effective protective immunity against B. pseudomallei infection remains incomplete. Therefore, we conducted a study to elucidate immune correlates of vaccine-induced protective immunity against acute B. pseudomallei infection. BALB/c and C57BL/6 mice were immunized subcutaneously with a highly attenuated, Select Agent-excluded purM deletion mutant of B. pseudomallei (strain Bp82) and then subjected to intranasal challenge with virulent B. pseudomallei strain 1026b. Immunization with Bp82 generated significant protection from challenge with B. pseudomallei, and protection was associated with a significant reduction in bacterial burden in lungs, liver, and spleen of immunized mice. Humoral immunity was critically important for vaccine-induced protection, as mice lacking B cells were not protected by immunization and serum from Bp82-vaccinated mice could transfer partial protection to nonvaccinated animals. In contrast, vaccine-induced protective immunity was found to be independent of both CD4 and CD8 T cells. Tracking studies demonstrated uptake of the Bp82 vaccine strain predominately by neutrophils in vaccine-draining lymph nodes and by smaller numbers of dendritic cells (DC) and monocytes. We concluded that protection following cutaneous immunization with a live attenuated Burkholderia vaccine strain was dependent primarily on generation of effective humoral immune responses. PMID:24101688

Silva, Ediane B.; Goodyear, Andrew; Sutherland, Marjorie D.; Podnecky, Nicole L.; Gonzalez-Juarrero, Mercedes; Schweizer, Herbert P.

2013-01-01

47

Raman spectroscopic detection and identification of Burkholderia mallei and Burkholderia pseudomallei in feedstuff.  

PubMed

Burkholderia mallei (the etiologic agent of glanders in equines and rarely humans) and Burkholderia pseudomallei, causing melioidosis in humans and animals, are designated category B biothreat agents. The intrinsically high resistance of both agents to many antibiotics, their potential use as bioweapons, and their low infectious dose, necessitate the need for rapid and accurate detection methods. Current methods to identify these organisms may require up to 1 week, as they rely on phenotypic characteristics and an extensive set of biochemical reactions. In this study, Raman microspectroscopy, a cultivation-independent typing technique for single bacterial cells with the potential for being a rapid point-of-care analysis system, is evaluated to identify and differentiate B. mallei and B. pseudomallei within hours. Here, not only broth-cultured microbes but also bacteria isolated out of pelleted animal feedstuff were taken into account. A database of Raman spectra allowed a calculation of classification functions, which were trained to differentiate Raman spectra of not only both pathogens but also of five further Burkholderia spp. and four species of the closely related genus Pseudomonas. The developed two-stage classification system comprising two support vector machine (SVM) classifiers was then challenged by a test set of 11 samples to simulate the case of a real-world-scenario, when "unknown samples" are to be identified. In the end, all test set samples were identified correctly, even if the contained bacterial strains were not incorporated in the database before or were isolated out of animal feedstuff. Specifically, the five test samples bearing B. mallei and B. pseudomallei were correctly identified on species level with accuracies between 93.9 and 98.7 %. The sample analysis itself requires no biomass enrichment step prior to the analysis and can be performed under biosafety level 1 (BSL 1) conditions after inactivating the bacteria with formaldehyde. PMID:24880875

Stöckel, Stephan; Meisel, Susann; Elschner, Mandy; Melzer, Falk; Rösch, Petra; Popp, Jürgen

2015-01-01

48

Computational discovery and RT-PCR validation of novel Burkholderia conserved and Burkholderia pseudomallei unique sRNAs  

PubMed Central

Background The sRNAs of bacterial pathogens are known to be involved in various cellular roles including environmental adaptation as well as regulation of virulence and pathogenicity. It is expected that sRNAs may also have similar functions for Burkholderia pseudomallei, a soil bacterium that can adapt to diverse environmental conditions, which causes the disease melioidosis and is also able to infect a wide variety of hosts. Results By integrating several proven sRNA prediction programs into a computational pipeline, available Burkholderia spp. genomes were screened to identify sRNA gene candidates. Orthologous sRNA candidates were then identified via comparative analysis. From the total prediction, 21 candidates were found to have Rfam homologs. RT-PCR and sequencing of candidate sRNA genes of unknown functions revealed six putative sRNAs which were highly conserved in Burkholderia spp. and two that were unique to B. pseudomallei present in a normal culture conditions transcriptome. The validated sRNAs include potential cis-acting elements associated with the modulation of methionine metabolism and one B. pseudomallei-specific sRNA that is expected to bind to the Hfq protein. Conclusions The use of the pipeline developed in this study and subsequent comparative analysis have successfully aided in the discovery and shortlisting of sRNA gene candidates for validation. This integrated approach identified 29 B. pseudomallei sRNA genes - of which 21 have Rfam homologs and 8 are novel. PMID:23282220

2012-01-01

49

Substituted Diphenyl Ethers as a Novel Chemotherapeutic Platform against Burkholderia pseudomallei  

PubMed Central

Identification of a novel class of anti-Burkholderia compounds is key in addressing antimicrobial resistance to current therapies as well as naturally occurring resistance. The FabI enoyl-ACP reductase in Burkholderia is an underexploited target that presents an opportunity for development of a new class of inhibitors. A library of substituted diphenyl ethers was used to identify FabI1-specific inhibitors for assessment in Burkholderia pseudomallei ex vivo and murine efficacy models. Active FabI1 inhibitors were identified in a two-stage format consisting of percent inhibition screening and MIC determination by the broth microdilution method. Each compound was evaluated against the B. pseudomallei 1026b (efflux-proficient) and Bp400 (efflux-compromised) strains. In vitro screening identified candidate substituted diphenyl ethers that exhibited MICs of less than 1 ?g/ml, and enzyme kinetic assays were used to assess potency and specificity against the FabI1 enzyme. These compounds demonstrated activity in a Burkholderia ex vivo efficacy model, and two demonstrated efficacy in an acute B. pseudomallei mouse infection model. This work establishes substituted diphenyl ethers as a suitable platform for development of novel anti-Burkholderia compounds that can be used for treatment of melioidosis. PMID:24379198

Cummings, Jason E.; Beaupre, Adam J.; Knudson, Susan E.; Liu, Nina; Yu, Weixuan; Neckles, Carla; Wang, Hui; Khanna, Avinash; Bommineni, Gopal R.; Trunck, Lily A.; Schweizer, Herbert P.

2014-01-01

50

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

PubMed Central

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

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

2014-01-01

51

Randomized Soil Survey of the Distribution of Burkholderia pseudomallei in Rice Fields in Laos ? †  

PubMed Central

Melioidosis is a major cause of morbidity and mortality in Southeast Asia, where the causative organism (Burkholderia pseudomallei) is present in the soil. In the Lao People's Democratic Republic (Laos), B. pseudomallei is a significant cause of sepsis around the capital, Vientiane, and has been isolated in soil near the city, adjacent to the Mekong River. We explored whether B. pseudomallei occurs in Lao soil distant from the Mekong River, drawing three axes across northwest, northeast, and southern Laos to create nine sampling areas in six provinces. Within each sampling area, a random rice field site containing a grid of 100 sampling points each 5 m apart was selected. Soil was obtained from a depth of 30 cm and cultured for B. pseudomallei. Four of nine sites (44%) were positive for B. pseudomallei, including all three sites in Saravane Province, southern Laos. The highest isolation frequency was in east Saravane, where 94% of soil samples were B. pseudomallei positive with a geometric mean concentration of 464 CFU/g soil (95% confidence interval, 372 to 579 CFU/g soil; range, 25 to 10,850 CFU/g soil). At one site in northwest Laos (Luangnamtha), only one sample (1%) was positive for B. pseudomallei, at a concentration of 80 CFU/g soil. Therefore, B. pseudomallei occurs in Lao soils beyond the immediate vicinity of the Mekong River, alerting physicians to the likelihood of melioidosis in these areas. Further studies are needed to investigate potential climatic, soil, and biological determinants of this heterogeneity. PMID:21075883

Rattanavong, Sayaphet; Wuthiekanun, Vanaporn; Langla, Sayan; Amornchai, Premjit; Sirisouk, Joy; Phetsouvanh, Rattanaphone; Moore, Catrin E.; Peacock, Sharon J.; Buisson, Yves; Newton, Paul N.

2011-01-01

52

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

Microsoft Academic Search

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

MARY N. BURTNICK; DONALD E. WOODS

1999-01-01

53

Programmed Death Ligand 1 on Burkholderia pseudomallei-Infected Human Polymorphonuclear Neutrophils Impairs T Cell Functions.  

PubMed

Polymorphonuclear neutrophils (PMNs) are terminally differentiated cells that are involved in innate immune responses and form an early line of defense against pathogens. More recently, it has been shown that PMNs have immunosuppressive abilities on other immune cells. However, the effect of PMNs on T cell responses during bacterial infection remains to be determined. In this report, we examined the interaction of PMNs and T cells in response to infection with Burkholderia pseudomallei, the causative agent of human melioidosis. We observed that CD4(+) T cell proliferation and IFN-? production in response to polyclonal activators is significantly inhibited by uninfected PMNs, and to a greater extent B. pseudomallei-infected PMNs. Programmed death ligand 1 (PD-L1), a known regulator of T cell activation, is increased in mRNA expression in the blood of patients and upon infection of PMNs in vitro. The increased expression of PD-L1 was correlated with the degree of T cell inhibition in individuals with type 2 diabetes, a major risk factor of melioidosis. In vitro, addition of anti-PD-L1 Abs blocked this inhibitory activity and restored proliferation of CD4(+) T cells and IFN-? production, suggesting that PD-L1 on B. pseudomallei-infected PMNs is a regulatory molecule for the functions of T cells and may be involved in pathogenesis versus control of melioidosis. PMID:25801435

Buddhisa, Surachat; Rinchai, Darawan; Ato, Manabu; Bancroft, Gregory J; Lertmemongkolchai, Ganjana

2015-05-01

54

Liver abscess caused by Burkholderia pseudomallei in a young man: A case report and review of literature  

PubMed Central

Pyogenic liver abscess is a common entity in Indian subcontinent and is mostly caused by gram negative bacteria. Melioidosis is not commonly seen in India and only a few cases are reported. It can give rise to multiple abscesses at different sites including liver. We report a case of isolated liver abscess caused by Burkholderia pseudomallei (B. pseudomallei) in a 29-year-old recently diagnosed diabetic, immunocompetent male. Diagnosis was made by imaging and culture of pus aspirated from the abscess and he was treated with percutaneous pigtail catheter drainage followed by antibiotics (meropenem and trimethoprim-sulphmethoxazole). Melioidosis is an emerging infection in India and has high mortality rate, so early diagnosis and prompt management is warranted which requires clinical vigilance and an intensive microbiological workup. Clinicians should be aware of isolated liver abscess caused by B. pseudomallei in appropriate clinical settings. PMID:25325075

Pal, Partha; Ray, Sayantan; Moulick, Avijit; Dey, Subhasis; Jana, Anirban; Banerjee, Kokila

2014-01-01

55

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

PubMed Central

Melioidosis infection, caused by Burkholderia pseudomallei, is increasingly reported in Cambodia. We hypothesized that implementation of an enhanced sputum testing protocol in a provincial hospital diagnostic microbiology laboratory would increase detection of B. pseudomallei. We tested 241 sputum specimens that were deemed acceptable for culture, comparing culture in selective enrichment broth followed by sub-culture on Ashdown’s medium to standard culture methods. Two specimens (0.8%) were positive for B. pseudomallei using the enhanced protocol whereas one specimen (0.4%) was positive using standard methods. These findings demonstrate that B. pseudomallei is rarely detected in sputum at this hospital. The low frequency of B. pseudomallei in sputum specimens precludes drawing any conclusions about the relative benefits of an enhanced sputum testing protocol at this site. Promoting clinician awareness of the infection and encouraging utilization of diagnostic microbiology services are likely to be important factors in facilitating identification of melioidosis. PMID:25717370

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

2014-01-01

56

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

PubMed Central

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

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

2012-01-01

57

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

PubMed Central

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics. Ceftazidime (CAZ), the synthetic ?-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). PMID:25379514

Khosravi, Yalda; Mariappan, Vanitha; Ng, Shet-Lee

2014-01-01

58

The Condition-Dependent Transcriptional Landscape of Burkholderia pseudomallei  

PubMed Central

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

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

2013-01-01

59

Evidence for the presence in Burkholderia pseudomallei of a type III secretion system-associated gene cluster.  

PubMed

Burkholderia pseudomallei, the causative agent of melioidosis, contains a cluster of putative genes homologous to those encoding HpaP, HrcQ, HrcR, HrcS and HrpV in the plant pathogen Ralstonia solanacearum. In R. solanacearum, these genes form part of a type III secretion-associated pathogenicity island. The order of the genes in B. pseudomallei is directly equivalent to that found in R. solanacearum. The B. pseudomallei proteins share 49.5% (HpaP), 52.6% (HrcQ), 80.0% (HrcR), 72.1% (HrcS) and 46.7% (HrpV) similarity, respectively, with their equivalent R. solanacearum proteins. The presence of type III secretion-associated genes in B. pseudomallei pathogens suggests a possible role for type III secretion systems in the pathogenicity of this organism. PMID:10403415

Winstanley, C; Hales, B A; Hart, C A

1999-07-01

60

Isolation of the highly pathogenic and zoonotic agent Burkholderia pseudomallei from a pet green Iguana in Prague, Czech Republic.  

PubMed

Background Melioidosis caused by Burkholderia (B.) pseudomallei is an endemic zoonotic disease mainly reported from northern Australia and Southeast Asia. In Europe, cases of human melioidosis have been reported only from patients travelling to endemic regions. Besides humans, B. pseudomallei has a very broad host range in domestic and wild animals. There are some reports about importation of B. pseudomallei-infected animals from endemic areas into Europe. The present report describes the first case of B. pseudomallei infection of a pet iguana in Europe.Case presentationIn a 5-year-old pet Iguana iguana living in a private household in Prague, Czech Republic, B. pseudomallei was isolated from pus of an abscess. The isolate VB976100 was identified by Vitek®2, MALDI-TOF mass spectrometry and polymerase chain reaction as B. pseudomallei. The molecular typing resulted in multi-locus sequence type 436 hitherto, which has been found only once worldwide in a B. pseudomallei strain isolated in the USA and originating from Guatemala. The identification as internal transcribed spacer type G indicates a close relatedness to strains mainly isolated in the Western Hemisphere. These findings support the hypothesis that the iguana became infected in this region or in a breeding facility through contact to other infected animals.ConclusionsThe present case highlights the risk of importation of the highly pathogenic and zoonotic B. pseudomallei into non-endemic regions through animal trade. Therefore, veterinarians treating animals from these areas and physicians examining patients owning such animals should include melioidosis in differential diagnosis whenever specific symptoms appear. Furthermore, veterinary authorities responsible for supervision of traders and pet shops should be aware of this risk of zoonotic transmission. PMID:25430942

Elschner, Mandy C; Hnizdo, Jan; Stamm, Ivonne; El-Adawy, Hosny; Mertens, Katja; Melzer, Falk

2014-11-28

61

Burkholderia pseudomallei Detection in Surface Water in Southern Laos Using Moore's Swabs  

PubMed Central

The causal agent of melioidosis, Burkholderia pseudomallei, has been cultured from paddy fields in the Lao PDR. We carried out a pilot study to examine the relationship between bacterial soil contamination and that of nearby surface waters in Saravane Province. Soil sampling was conducted at a depth of 30 cm (100 holes in a 45 × 45 m grid) at two sites, East and West Saravane. Moore's swabs were used for water sampling of paddy fields, lakes, rivers, boreholes, and storage tanks within 2 km of the two soil sampling sites. B. pseudomallei from soil and water were cultured on Ashdown's agar. Thirty-six percent and 6% of water samples collected around East and West Saravane, respectively, were culture positive for B. pseudomallei. Low pH and high turbidity were independently associated with culture of B. pseudomallei. Most positive water samples were from the Sedone River, downstream of the East Saravane site. Moore's swabs are simple and inexpensive tools for detecting B. pseudomallei in surface waters. PMID:22556090

Vongphayloth, Khamsing; Rattanavong, Sayaphet; Moore, Catrin E.; Phetsouvanh, Rattanaphone; Wuthiekanun, Vanaporn; Sengdouangphachanh, Amphonesavanh; Phouminh, Phonlavanh; Newton, Paul N.; Buisson, Yves

2012-01-01

62

Crystallization and preliminary crystallographic studies of the hypothetical protein BPSL1038 from Burkholderia pseudomallei.  

PubMed

Melioidosis is an infectious disease caused by the pathogenic bacterium Burkholderia pseudomallei. Whole-genome sequencing revealed that the B. pseudomallei genome includes 5855 coding DNA sequences (CDSs), of which ?25% encode hypothetical proteins. A pathogen-associated hypothetical protein, BPSL1038, was overexpressed in Escherichia coli, purified and crystallized using vapour-diffusion methods. A BPSL1038 protein crystal that grew using sodium formate as precipitant diffracted to 1.55?Å resolution. It belonged to space group C2221, with unit-cell parameters a = 85.36, b = 115.63, c = 46.73?Å. The calculated Matthews coefficient (VM) suggests that there are two molecules per asymmetric unit, with a solvent content of 48.8%. PMID:25484229

Shaibullah, Sofiyah; Mohd-Sharif, Nurhikmah; Ho, Kok Lian; Firdaus-Raih, Mohd; Nathan, Sheila; Mohamed, Rahmah; Ng, Chyan Leong

2014-12-01

63

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

PubMed Central

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

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

2012-01-01

64

Neurologic Melioidosis  

PubMed Central

Melioidosis is an important cause of morbidity and mortality in northern Australia and Southeast Asia. Diagnosis is best made by isolation of Burkholderia pseudomallei from clinical specimens. A variety of clinical presentations are described, including neurologic disease. The aim of this study was to review admissions with confirmed neurologic melioidosis to a regional hospital in a region to which melioidosis is endemic during 1995–2011. There were 12 culture-confirmed cases of neurologic melioidosis, of which two were detected by analysis of cerebrospinal fluid. Four of these cases were in children. Significant clinical features were fever, headache, and ataxia. Common changes on magnetic resonance imaging T2-weighted scans included ring-enhancing lesions and leptomeningeal enhancement. There were four deaths and an additional four patients had significant long-term neurologic sequelae. When considering the etiology of undifferentiated neurologic disease, an awareness of the possibility of neurologic melioidosis is important in disease-endemic regions. PMID:23836574

Deuble, Martin; Aquilina, Chloe; Norton, Robert

2013-01-01

65

Screening for potential anti-infective agents towards Burkholderia pseudomallei infection  

NASA Astrophysics Data System (ADS)

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.

Eng, Su Anne; Nathan, Sheila

2014-09-01

66

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

PubMed Central

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

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

2013-01-01

67

Burkholderia thailandensis as a Model System for the Study of the Virulence-Associated Type III Secretion System of Burkholderia pseudomallei?  

PubMed Central

Burkholderia pseudomallei is a bacterial pathogen that causes a broad spectrum of clinical symptoms collectively known as melioidosis. Since it can be acquired by inhalation and is difficult to eradicate due to its resistance to a wide group of antibiotics and capacity for latency, work with B. pseudomallei requires a biosafety level 3 (BSL-3) containment facility. The bsa (Burkholderia secretion apparatus)-encoded type III secretion system (TTSS) has been shown to be required for its full virulence in a number of animal models. TTSSs are export devices found in a variety of gram-negative bacteria that translocate bacterial effector proteins across host cell membranes into the cytoplasm of host cells. Although the Bsa TTSS has been shown to play an important role in the ability of B. pseudomallei to survive and replicate in mammalian cells, escape from the endocytic vacuole, and spread from cell to cell, little is known about its effectors mediating these functions. Using bioinformatics, we identified homologs of several known TTSS effectors from other bacteria in the B. pseudomallei genome. In addition, we show that orthologs of these putative effectors exist in the genome of B. thailandensis, a closely related bacterium that is rarely pathogenic to humans. By generating a Bsa TTSS mutant B. thailandensis strain, we also demonstrated that the Bsa TTSS has similar functions in the two species. Therefore, we propose B. thailandensis as a useful BSL-1 model system to study the role of the Bsa TTSS during Burkholderia infection of mammalian cells and animals. PMID:18779342

Haraga, Andrea; West, T. Eoin; Brittnacher, Mitchell J.; Skerrett, Shawn J.; Miller, Samuel I.

2008-01-01

68

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

PubMed

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. PMID:25392525

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

69

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

PubMed Central

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 extra-intestinal sites. PMID:22624016

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

2012-01-01

70

Porin Involvement in Cephalosporin and Carbapenem Resistance of Burkholderia pseudomallei  

PubMed Central

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 the permeability of the BpsOmp38 channel to neutral sugars and antimicrobial agents. PMID:24788109

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

2014-01-01

71

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

PubMed

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

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

72

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

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

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

1999-01-01

73

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

PubMed Central

Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. We constructed a mutant lacking bsaP, a homolog of the T3SS “gatekeeper” family of proteins that exert control over the timing and magnitude of effector protein secretion. Mutants lacking BsaP, or the T3SS translocon protein BipD, were observed to hypersecrete the known Bsa effector protein BopE, providing evidence of their role in post-translational control of the Bsa T3SS and representing key reagents for the identification of its secreted substrates. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hypersecreting mutants of B. pseudomallei with the isogenic parent strain and a bsaZ mutant incapable of effector protein secretion. Our study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei. PMID:25635268

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

2015-01-01

74

Evolutionary Analysis of Burkholderia pseudomallei Identifies Putative Novel Virulence Genes, Including a Microbial Regulator of Host Cell Autophagy  

PubMed Central

Burkholderia pseudomallei, the causative agent of melioidosis, contains a large pathogen genome (7.2 Mb) with ?2,000 genes of putative or unknown function. Interactions with potential hosts and environmental factors may induce rapid adaptations in these B. pseudomallei genes, which can be discerned through evolutionary analysis of multiple B. pseudomallei genomes. Here we show that several previously uncharacterized B. pseudomallei genes bearing genetic signatures of rapid adaptation (positive selection) can induce diverse cellular phenotypes when expressed in mammalian cells. Notably, several of these phenotypes are plausibly related to virulence, including multinuclear giant cell formation, apoptosis, and autophagy induction. Specifically, we show that BPSS0180, a type VI cluster-associated gene, is capable of inducing autophagy in both phagocytic and nonphagocytic mammalian cells. Following infection of macrophages, a B. pseudomallei mutant disrupted in BPSS0180 exhibited significantly decreased colocalization with LC3 and impaired intracellular survival; these phenotypes were rescued by introduction of an intact BPSS0180 gene. The results suggest that BPSS0180 may be a novel inducer of host cell autophagy that contributes to B. pseudomallei intracellular growth. More generally, our study highlights the utility of applying evolutionary principles to microbial genomes to identify novel virulence genes. PMID:24097950

Singh, Arvind Pratap; Lai, Shu-chin; Nandi, Tannistha; Chua, Hui Hoon; Ooi, Wen Fong; Ong, Catherine; Boyce, John D.; Adler, Ben

2013-01-01

75

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

PubMed Central

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 seen in human infections. PMID:24098563

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

2013-01-01

76

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

PubMed Central

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.

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

77

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

PubMed

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. PMID:21803915

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

2011-09-01

78

Physicochemical Factors Affecting the Growth of Burkholderia pseudomallei in Soil Microcosm  

PubMed Central

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

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

2014-01-01

79

Identification of the conserved hypothetical protein BPSL0317 in Burkholderia pseudomallei K96243  

NASA Astrophysics Data System (ADS)

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.

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

2014-09-01

80

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

PubMed Central

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 Bp340?bcaA and Bp340?bcaB 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 Bp340?bcaA, Bp340?bcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340?bcaA-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

Campos, Cristine G.; Borst, Luke

2013-01-01

81

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

PubMed

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 Bp340?bcaA and Bp340?bcaB 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 Bp340?bcaA, Bp340?bcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340?bcaA-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

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

2013-04-01

82

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

PubMed

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

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

83

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

PubMed Central

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

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

84

Genome-Wide Saturation Mutagenesis of Burkholderia pseudomallei K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development  

PubMed Central

ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 106 transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. PMID:24520057

Moule, Madeleine G.; Hemsley, Claudia M.; Seet, Qihui; Guerra-Assunção, José Afonso; Lim, Jiali; Sarkar-Tyson, Mitali; Clark, Taane G.; Tan, Patrick B. O.; Titball, Richard W.; Cuccui, Jon; Wren, Brendan W.

2014-01-01

85

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

PubMed Central

Background Burkholderia pseudomallei and Burkholderia mallei are gram-negative pathogens responsible for the diseases melioidosis and glanders, respectively. Both species cause disease in humans and animals and have been designated as category B select agents by the Centers for Disease Control and Prevention (CDC). Burkholderia thailandensis is a closely related bacterium that is generally considered avirulent for humans. While it can cause disease in rodents, the B. thailandensis 50% lethal dose (LD50) is typically???104-fold higher than the B. pseudomallei and B. mallei LD50 in mammalian models of infection. Here we describe an alternative to mammalian hosts in the study of virulence and host-pathogen interactions of these Burkholderia species. Results Madagascar hissing cockroaches (MH cockroaches) possess a number of qualities that make them desirable for use as a surrogate host, including ease of breeding, ease of handling, a competent innate immune system, and the ability to survive at 37°C. MH cockroaches were highly susceptible to infection with B. pseudomallei, B. mallei and B. thailandensis and the LD50 was <10 colony-forming units (cfu) for all three species. In comparison, the LD50 for Escherichia coli in MH cockroaches was >105?cfu. B. pseudomallei, B. mallei, and B. thailandensis cluster 1 type VI secretion system (T6SS-1) mutants were all attenuated in MH cockroaches, which is consistent with previous virulence studies conducted in rodents. B. pseudomallei mutants deficient in the other five T6SS gene clusters, T6SS-2 through T6SS-6, were virulent in both MH cockroaches and hamsters. Hemocytes obtained from MH cockroaches infected with B. pseudomallei harbored numerous intracellular bacteria, suggesting that this facultative intracellular pathogen can survive and replicate inside of MH cockroach phagocytic cells. The hemolymph extracted from these MH cockroaches also contained multinuclear giant cells (MNGCs) with intracellular B. pseudomallei, which indicates that infected hemocytes can fuse while flowing through the insect’s open circulatory system in vivo. Conclusions The results demonstrate that MH cockroaches are an attractive alternative to mammals to study host-pathogen interactions and may allow the identification of new Burkholderia virulence determinants. The importance of T6SS-1 as a virulence factor in MH cockroaches and rodents suggests that the primary role of this secretion system is to target evasion of the innate immune system. PMID:22892068

2012-01-01

86

The multiple roles of hypothetical gene BPSS1356 in Burkholderia pseudomallei.  

PubMed

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 ?' 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 ?BPSS1356 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 ?BPSS1356 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

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

2014-01-01

87

The Multiple Roles of Hypothetical Gene BPSS1356 in Burkholderia pseudomallei  

PubMed Central

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 ?? 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 ?BPSS1356 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 ?BPSS1356 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

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

2014-01-01

88

Two Norwegian patients with melioidosis presenting with bacteraemia and splenic and prostatic abscesses.  

PubMed

Infections caused by Burkholderia pseudomallei are rare in nonendemic areas, such as Scandinavia. We report the first two cases of melioidosis in Norway presenting with bacteraemia and splenic and prostatic abscesses, respectively. PMID:22017720

Hesstvedt, Liv; Wilhelmsen, Marianne; Mengshoel, Anne Torunn; Dyrhol-Riise, Anne Ma

2011-01-01

89

Musculoskeletal melioidosis.  

PubMed

Melioidosis is an infectious disease caused by Burkholderia pseudomallei, mostly affecting patients in Southeast Asia and northern Australia. The disease has been increasingly recognized around the world due to the increased levels of travel and population movement. Clinical manifestations of melioidosis range from fulminant septicemic illness to an indolent local infection. The disease often involves multiple organs, including the lung, spleen, liver, and other visceral organs. Musculoskeletal infection is usually seen as a part of multiorgan involvement, but localized musculoskeletal involvement may occur. The most common manifestation of musculoskeletal melioidosis is septic arthritis, followed by osteomyelitis, pyomyositis, and soft tissue abscesses. The clinical and radiological manifestations of musculoskeletal melioidosis are nonspecific, and the diagnosis needs a high level of suspicion. Associated infection of lungs and visceral organs is suggestive of melioidosis. The disease requires special laboratory facilities and treatment. Inappropriate or inadequate treatment leads to high mortality rate or long-term relapse of the disease. The causative organism of melioidosis, clinical manifestations, and imaging features of musculoskeletal melioidosis are reviewed. PMID:22081283

Pattamapaspong, Nuttaya; Muttarak, Malai

2011-11-01

90

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

PubMed Central

Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (CT) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×104 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen. PMID:23460920

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

2013-01-01

91

Dural Sinus Thrombosis in Melioidosis: The First Case Report  

Microsoft Academic Search

Melioidosis which is infection with Burkholderia pseudomallei, is an important cause of sepsis in India, southeast Asia and northern Australia. Mortality is high and treatment is problematic. Neurological melioidosis is unusual but meningoencephalitis, encephalomyelitis and brain microabscess can occur. Dural sinus thrombosis is not an uncommon cerebrovascular disorder with various etiologies. Hypercoagulable state, pregnancy, dehydration, certain blood dyscrasia and contraceptive

Suchada Niyasom; Pasiri Sithinamsuwan; Chesda Udommongkol; Jithanorm Suwantamee

92

Autochthonous Melioidosis in Humans, Madagascar, 2012 and 2013  

PubMed Central

Melioidosis is an often fatal infectious disease affecting humans and animals in the tropics. Only sporadic cases have been reported from Africa and the Indian Ocean region. We describe 2 confirmed autochthonous cases of human melioidosis in Madagascar, both from novel genotypes of Burkholderia pseudomallei. PMID:25272365

Djaomazala, Innocente; Dubois-Cauwelaert, Natasha; Raharimanga, Vaomalala; Ralison, Fidiarivony; Herindrainy, Perlinot; Andriamalala, Nivosoa C.; Sarovich, Derek S.; Mayo, Mark; Kaestli, Mirjam; Currie, Bart J.

2014-01-01

93

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

PubMed Central

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

2013-01-01

94

A Burkholderia pseudomallei Colony Variant Necessary for Gastric Colonization  

PubMed Central

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

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

95

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

PubMed Central

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

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

2014-01-01

96

Characterization and analysis of the Burkholderia pseudomallei BsaN virulence regulon  

PubMed Central

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

2014-01-01

97

BPSS1504, a Cluster 1 Type VI Secretion Gene, Is Involved in Intracellular Survival and Virulence of Burkholderia pseudomallei  

PubMed Central

Burkholderia pseudomallei is a Gram-negative rod and the causative agent of melioidosis, an emerging infectious disease of tropical and subtropical areas worldwide. B. pseudomallei harbors a remarkable number of virulence factors, including six type VI secretion systems (T6SS). Using our previously described plaque assay screening system, we identified a B. pseudomallei transposon mutant defective in the BPSS1504 gene that showed reduced plaque formation. The BPSS1504 locus is encoded within T6SS cluster 1 (T6SS1), which is known to be involved in the pathogenesis of B. pseudomallei in mammalian hosts. For further analysis, a B. pseudomallei BPSS1504 deletion (Bp?BPSS1504) mutant and complemented mutant strain were constructed. B. pseudomallei lacking the BPSS1504 gene was highly attenuated in BALB/c mice, whereas the in vivo virulence of the complemented mutant strain was fully restored to the wild-type level. The Bp?BPSS1504 mutant showed impaired intracellular replication and formation of multinucleated giant cells in macrophages compared with wild-type bacteria, whereas the induction of actin tail formation within host cells was not affected. These observations resembled the phenotype of a mutant lacking hcp1, which is an integral component of the T6SS1 apparatus and is associated with full functionality of the T6SS1. Transcriptional expression of the T6SS components vgrG, tssA, and hcp1, as well as the T6SS regulators virAG, bprC, and bsaN, was not dependent on BPSS1504 expression. However, secretion of Hcp1 was not detectable in the absence of BPSS1504. Thus, BPSS1504 seems to serve as a T6SS component that affects Hcp1 secretion and is therefore involved in the integrity of the T6SS1 apparatus. PMID:24595140

Hopf, Verena; Göhler, André; Eske-Pogodda, Kristin; Bast, Antje; Steinmetz, Ivo

2014-01-01

98

BPSS1504, a cluster 1 type VI secretion gene, is involved in intracellular survival and virulence of Burkholderia pseudomallei.  

PubMed

Burkholderia pseudomallei is a Gram-negative rod and the causative agent of melioidosis, an emerging infectious disease of tropical and subtropical areas worldwide. B. pseudomallei harbors a remarkable number of virulence factors, including six type VI secretion systems (T6SS). Using our previously described plaque assay screening system, we identified a B. pseudomallei transposon mutant defective in the BPSS1504 gene that showed reduced plaque formation. The BPSS1504 locus is encoded within T6SS cluster 1 (T6SS1), which is known to be involved in the pathogenesis of B. pseudomallei in mammalian hosts. For further analysis, a B. pseudomallei BPSS1504 deletion (Bp?BPSS1504) mutant and complemented mutant strain were constructed. B. pseudomallei lacking the BPSS1504 gene was highly attenuated in BALB/c mice, whereas the in vivo virulence of the complemented mutant strain was fully restored to the wild-type level. The Bp?BPSS1504 mutant showed impaired intracellular replication and formation of multinucleated giant cells in macrophages compared with wild-type bacteria, whereas the induction of actin tail formation within host cells was not affected. These observations resembled the phenotype of a mutant lacking hcp1, which is an integral component of the T6SS1 apparatus and is associated with full functionality of the T6SS1. Transcriptional expression of the T6SS components vgrG, tssA, and hcp1, as well as the T6SS regulators virAG, bprC, and bsaN, was not dependent on BPSS1504 expression. However, secretion of Hcp1 was not detectable in the absence of BPSS1504. Thus, BPSS1504 seems to serve as a T6SS component that affects Hcp1 secretion and is therefore involved in the integrity of the T6SS1 apparatus. PMID:24595140

Hopf, Verena; Göhler, André; Eske-Pogodda, Kristin; Bast, Antje; Steinmetz, Ivo; Breitbach, Katrin

2014-05-01

99

Proteomic analysis of the Burkholderia pseudomallei type II secretome reveals hydrolytic enzymes, novel proteins, and the deubiquitinase TssM.  

PubMed

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 ?gspD grown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent or substantially reduced in the supernatants of ?gspD 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

Burtnick, Mary N; Brett, Paul J; DeShazer, David

2014-08-01

100

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

PubMed Central

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 ?gspD grown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent or substantially reduced in the supernatants of ?gspD 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

Burtnick, Mary N.; Brett, Paul J.

2014-01-01

101

Curcumin rescues Caenorhabditis elegans from a Burkholderia pseudomallei infection  

PubMed Central

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 concomitantly regulating iron transport and other proteins of unknown function.

Eng, Su-Anne; Nathan, Sheila

2015-01-01

102

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

PubMed Central

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

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

2014-01-01

103

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

PubMed Central

Burkholderia pseudomallei, the causative agent of melioidosis has been recognized by CDC as a category B select agent. Although substantial efforts have been made for development of vaccine molecules against the pathogen, significant hurdles still remain. With no licensed vaccines available and high relapse rate of the disease, there is a pressing need for development of alternate protection strategies. Antibody-mediated passive protection is promising in this regard and our primary interest was to unravel this frontier of specific mAbs against Burkholderia pseudomallei infections, as functional characterization of antibodies is a pre-requisite to demonstrate them as protective molecules. To achieve this, we designed our study on in vitro-based approach and assessed two mAbs, namely BURK24 and BURK37, reactive with outer membrane proteins and lipopolysaccharide of the pathogen respectively, for their ability to manifest inhibitory effects on the pathogenesis mechanisms of B. pseudomallei including biofilm formation, invasion and induction of apoptosis. The experiments were performed using B. pseudomallei standard strain NCTC 10274 and a clinical isolate, B. pseudomallei 621 recovered from a septicemia patient with diabetic ailment. The growth kinetic studies of the pathogen in presence of various concentrations of each individual mAb revealed their anti-bacterial properties. Minimal inhibitory concentration and minimal bactericidal concentration of both the mAbs were determined by using standards of Clinical and Laboratory Standards Institute (CLSI) and experiments were performed using individual mAbs at their respective bacteriostatic concentration. As an outcome, both mAbs exhibited significant anti-Burkholderia pseudomallei properties. They limited the formation of biofilm by the bacterium and completely crippled its invasion into human alveolar adenocarcinoma epithelial cells. Also, the mAbs were appreciably successful in preventing the bacterium to induce apoptosis in A549 cells. The present study design revealed the protection attributes possessed by BURK24 and BURK37 that has to be further substantiated by additional in vivo studies. PMID:24614539

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

2014-01-01

104

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

PubMed Central

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

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

2014-01-01

105

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

PubMed Central

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

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

106

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

PubMed

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 2.1A 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(bd)) and LID subdomains. The two molecules in the asymmetric unit have significantly different conformations, with a backbone RMSD of 1.46 A. These two BpAdk conformations may represent 'open' Adk sub-states along the preferential pathway to the 'closed' substrate-bound state. PMID:20331978

Buchko, Garry W; Robinson, Howard; Abendroth, Jan; Staker, Bart L; Myler, Peter J

2010-04-16

107

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

SciTech Connect

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.

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

2010-04-16

108

Association of Melioidosis Incidence with Rainfall and Humidity, Singapore, 2003–2012  

PubMed Central

Soil has been considered the natural reservoir for the bacterium Burkholderia pseudomallei, which causes melioidosis. We examined 550 melioidosis cases that occurred during a 10-year period in the highly urbanized city of Singapore, where soil exposure is rare, and found that rainfall and humidity levels were associated with disease incidence. PMID:25531547

Liu, Xiang; Pang, Long; Sim, Siew Hoon; Goh, Kee Tai; Ravikumar, Sharada; Win, Mar Soe; Tan, Gladys; Cook, Alex Richard; Fisher, Dale

2015-01-01

109

Melioidosis as a Consequence of Sporting Activity  

PubMed Central

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. PMID:23732257

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

2013-01-01

110

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

SciTech Connect

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.

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. (UWASH); (Emerald)

2011-09-28

111

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

PubMed

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

Nandi, Tannistha; 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

112

A genetic programming approach for Burkholderia Pseudomallei diagnostic pattern discovery  

PubMed Central

Motivation: Finding diagnostic patterns for fighting diseases like Burkholderia pseudomallei using biomarkers involves two key issues. First, exhausting all subsets of testable biomarkers (antigens in this context) to find a best one is computationally infeasible. Therefore, a proper optimization approach like evolutionary computation should be investigated. Second, a properly selected function of the antigens as the diagnostic pattern which is commonly unknown is a key to the diagnostic accuracy and the diagnostic effectiveness in clinical use. Results: A conversion function is proposed to convert serum tests of antigens on patients to binary values based on which Boolean functions as the diagnostic patterns are developed. A genetic programming approach is designed for optimizing the diagnostic patterns in terms of their accuracy and effectiveness. During optimization, it is aimed to maximize the coverage (the rate of positive response to antigens) in the infected patients and minimize the coverage in the non-infected patients while maintaining the fewest number of testable antigens used in the Boolean functions as possible. The final coverage in the infected patients is 96.55% using 17 of 215 (7.4%) antigens with zero coverage in the non-infected patients. Among these 17 antigens, BPSL2697 is the most frequently selected one for the diagnosis of Burkholderia Pseudomallei. The approach has been evaluated using both the cross-validation and the Jack–knife simulation methods with the prediction accuracy as 93% and 92%, respectively. A novel approach is also proposed in this study to evaluate a model with binary data using ROC analysis. Contact: z.r.yang@ex.ac.uk PMID:19561021

Yang, Zheng Rong; Lertmemongkolchai, Ganjana; Tan, Gladys; Felgner, Philip L.; Titball, Richard

2009-01-01

113

Two fatal cases of melioidosis on the Thai-Myanmar border.  

PubMed

Melioidosis is endemic in areas of Southeast Asia, however, there are no published reports from the Thai-Myanmar border. We report the first two documented cases of fatal melioidosis in this region. This is of great public health importance and highlights the need to both increase clinical awareness of melioidosis on the Thai-Myanmar border, and to assess the true burden of disease in the area through improved case detection and Burkholderia pseudomallei prevalence studies. PMID:24715973

Chu, Cindy S; Winearls, Stuart; Ling, Clare; Torchinsky, Miriam Beer; Phyo, Aung Phae; Haohankunnathum, Warat; Turner, Paul; Wuthiekanun, Vanaporn; Nosten, François

2014-01-01

114

Two fatal cases of melioidosis on the Thai-Myanmar border  

PubMed Central

Melioidosis is endemic in areas of Southeast Asia, however, there are no published reports from the Thai-Myanmar border. We report the first two documented cases of fatal melioidosis in this region. This is of great public health importance and highlights the need to both increase clinical awareness of melioidosis on the Thai-Myanmar border, and to assess the true burden of disease in the area through improved case detection and Burkholderia pseudomallei prevalence studies. PMID:24715973

Nosten, François

2014-01-01

115

Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion porin  

Microsoft Academic Search

In the present paper, we describe cloning and expression of two outer membrane proteins, BpsOmp38 (from Burkholderia pseudomallei )a ndBthOmp38 (from Burkholderia thailandensis) lacking signal peptide sequences, using the pET23d(+) expres- sion vector and Escherichia coli host strain Origami(DE3). The 38 kDa proteins, expressed as insoluble inclusion bodies, were purified, solubilized in 8 M urea, and then subjected to refold-

Jaruwan SIRITAPETAWEE; Heino PRINZ; Chartchai KRITTANAI; Wipa SUGINTA

2004-01-01

116

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

PubMed Central

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

2014-01-01

117

Discovery of Inhibitors of Burkholderia pseudomallei Methionine Aminopeptidase with Antibacterial Activity.  

PubMed

Evaluation of a series of MetAP inhibitors in an in vitro enzyme activity assay led to the first identification of potent molecules that show significant growth inhibition against Burkholderia pseudomallei. Nitroxoline analogs show excellent inhibition potency in the BpMetAP1 enzyme activity assay with the lowest IC50 of 30 nM, and inhibit the growth of B. pseudomallei and B. thailandensis at concentrations ? 31 ?M. PMID:24376907

Wangtrakuldee, Phumvadee; Byrd, Matthew S; Campos, Cristine G; Henderson, Michael W; Zhang, Zheng; Clare, Michael; Masoudi, Ali; Myler, Peter J; Horn, James R; Cotter, Peggy A; Hagen, Timothy J

2013-07-01

118

Discovery of Inhibitors of Burkholderia pseudomallei Methionine Aminopeptidase with Antibacterial Activity  

PubMed Central

Evaluation of a series of MetAP inhibitors in an in vitro enzyme activity assay led to the first identification of potent molecules that show significant growth inhibition against Burkholderia pseudomallei. Nitroxoline analogues show excellent inhibition potency in the BpMetAP1 enzyme activity assay with the lowest IC50 of 30 nM and inhibit the growth of B. pseudomallei and B. thailandensis at concentrations ?31 ?M. PMID:24376907

2013-01-01

119

Radiological manifestations of melioidosis.  

PubMed

Melioidosis is a serious infection that is associated with high mortality. It is due to a Gram-negative bacterium, Burkholderia pseudomallei which is an environmental saprophyte found in wet soils. Melioidosis is endemic to northern Australia and the Southeast Asia. However, there is now increasing number of reports of imported cases to regions where this infection has not been previously encountered. Almost any organ can be affected. Like many other conditions, radiological imaging is an integral part of the diagnostic workup of melioidosis. Awareness of the various radiological manifestations can help direct appropriate investigations to achieve early diagnosis and the initiation of appropriate treatment. Generally, there are no known characteristic features on imaging that can specifically differentiate melioidosis from other infections. However, the "honeycomb" appearance has been described to be characteristic for large melioidosis liver abscesses. Simultaneous involvement of various organs is also characteristics. To date, there are few data available on the radiological manifestations of melioidosis. The present pictorial essay describes melioidosis affecting the various organs. PMID:20103424

Lim, K S; Chong, V H

2010-01-01

120

A naturally-derived outer-membrane vesicle vaccine protects against lethal pulmonary Burkholderia pseudomallei infection  

PubMed Central

B. pseudomallei, and other members of the Burkholderia, are among the most antibiotic-resistant bacterial species encountered in human infection. Mortality rates associated with severe B. pseudomallei infection approach 50% despite therapeutic treatment. A protective vaccine against B. pseudomallei would dramatically reduce morbidity and mortality in endemic areas and provide a safeguard for the U.S. and other countries against biological attack with this organism. In this study, we investigated the immunogenicity and protective efficacy of B. pseudomallei-derived outer membrane vesicles (OMVs). Vesicles are produced by Gram-negative and Gram-positive bacteria and contain many of the bacterial products recognized by the host immune system during infection. We demonstrate that subcutaneous (SC) immunization with OMVs provides significant protection against an otherwise lethal B. pseudomallei aerosol challenge in BALB/c mice. Mice immunized with B. pseudomallei OMVs displayed OMV-specific serum antibody and T-cell memory responses. Furthermore, OMV-mediated immunity appears species-specific as cross-reactive antibody and T cells were not generated in mice immunized with E. coli-derived OMVs. These results provide the first compelling evidence that OMVs represent a non-living vaccine formulation that is able to produce protective humoral and cellular immunity against an aerosolized intracellular bacterium. This vaccine platform constitutes a safe and inexpensive immunization strategy against B. pseudomallei that can be exploited for other intracellular respiratory pathogens, including other Burkholderia and bacteria capable of establishing persistent infection. PMID:21871517

Nieves, Wildaliz; Asakrah, Saja; Qazi, Omar; Brown, Katherine A.; Kurtz, Jonathan; AuCoin, David P.; McLachlan, James B.; Roy, Chad J.; Morici, Lisa A.

2011-01-01

121

Melioidosis  

MedlinePLUS

... tropical climates, especially in Southeast Asia and northern Australia where it is widespread. The bacteria causing melioidosis are found in contaminated water and soil. It is spread to humans and animals through direct contact with the contaminated source. Melioidosis ...

122

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

PubMed

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

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

2015-04-01

123

Extended loop region of Hcp1 is critical for the assembly and function of type VI secretion system in Burkholderia pseudomallei.  

PubMed

The Type VI Secretion System cluster 1 (T6SS1) is essential for the pathogenesis of Burkholderia pseudomallei, the causative agent of melioidosis, a disease endemic in the tropics. Inside host cells, B. pseudomallei escapes into the cytosol and through T6SS1, induces multinucleated giant cell (MNGC) formation that is thought to be important for bacterial cell to cell spread. The hemolysin-coregulated protein (Hcp) is both a T6SS substrate, as well as postulated to form part of the T6SS secretion tube. Our structural study reveals that Hcp1 forms hexameric rings similar to the other Hcp homologs but has an extended loop (Asp40-Arg56) that deviates significantly in position compared to other Hcp structures and may act as a key contact point between adjacent hexameric rings. When two residues within the loop were mutated, the mutant proteins were unable to stack as dodecamers, suggesting defective tube assembly. Moreover, infection with a bacterial mutant containing in situ substitution of these hcp1 residues abolishes Hcp1 secretion inside infected cells and MNGC formation. We further show that Hcp has the ability to preferentially bind to the surface of antigen-presenting cells, which may contribute to its immunogenicity in inducing high titers of antibodies seen in melioidosis patients. PMID:25648885

Lim, Yan Ting; Jobichen, Chacko; Wong, Jocelyn; Limmathurotsakul, Direk; Li, Shaowei; Chen, Yahua; Raida, Manfred; Srinivasan, Nalini; MacAry, Paul Anthony; Sivaraman, J; Gan, Yunn-Hwen

2015-01-01

124

Comparative Genomics and an Insect Model Rapidly Identify Novel Virulence Genes of Burkholderia mallei  

Microsoft Academic Search

Burkholderia pseudomallei and its host-adapted deletion clone Burkholderia mallei cause the potentially fatal human diseases melioidosis and glanders, respectively. The antibiotic resistance profile and ability to infect via aerosol of these organisms and the absence of protective vaccines have led to their classification as major biothreats and select agents. Although documented infections by these bacteria date back over 100 years,

Mark A. Schell; Lyla Lipscomb; David DeShazer

2008-01-01

125

Melioidosis Presenting as Fever and Jaundice: A Rare Presentation  

PubMed Central

Melioidosis caused by the environmental Gram-negative bacillus Burkholderia pseudomallei is endemic in northern Australia and Southeast Asia and is being described increasingly from south and west coastal regions of India. Melioidosis is known to have high mortality (14–50%) and the risk factors associated with it are diabetes mellitus and heavy alcohol abuse. Melioidosis primarily presents as pneumonia, genitourinary infection and bacteremia. We present a case of Melioidosis from North India, a 56-year-old diabetic male, presenting with fever and jaundice. His blood culture was positive for the B. pseudomallei. The hepatic involvement was in the form of jaundice with serum bilirubin value of more than 12 mg/dL, hepatic enzymes more than ten times high and without hepatic abscess. He improved with intravenous antibiotics with complete normalization of liver function tests. PMID:25755553

Tyagi, Pankaj; Shah, Vinit; Sharma, Praveen; Bansal, Naresh; Singla, Vikas; Kumar, Ashish; Arora, Anil

2013-01-01

126

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

SciTech Connect

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.

Gardner, S; Jaing, C

2012-03-27

127

Nasal Acai Polysaccharides Potentiate Innate Immunity to Protect against Pulmonary Francisella tularensis and Burkholderia pseudomallei Infections  

PubMed Central

Pulmonary Francisella tularensis and Burkholderia pseudomallei infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Activating the innate immune system provides an alternative means of treating infection and can also complement antibiotic therapies. Several natural agonists were screened for their ability to enhance host resistance to infection, and polysaccharides derived from the Acai berry (Acai PS) were found to have potent abilities as an immunotherapeutic to treat F. tularensis and B. pseudomallei infections. In vitro, Acai PS impaired replication of Francisella in primary human macrophages co-cultured with autologous NK cells via augmentation of NK cell IFN-?. Furthermore, Acai PS administered nasally before or after infection protected mice against type A F. tularensis aerosol challenge with survival rates up to 80%, and protection was still observed, albeit reduced, when mice were treated two days post-infection. Nasal Acai PS administration augmented intracellular expression of IFN-? by NK cells in the lungs of F. tularensis-infected mice, and neutralization of IFN-? ablated the protective effect of Acai PS. Likewise, nasal Acai PS treatment conferred protection against pulmonary infection with B. pseudomallei strain 1026b. Acai PS dramatically reduced the replication of B. pseudomallei in the lung and blocked bacterial dissemination to the spleen and liver. Nasal administration of Acai PS enhanced IFN-? responses by NK and ?? T cells in the lungs, while neutralization of IFN-? totally abrogated the protective effect of Acai PS against pulmonary B. pseudomallei infection. Collectively, these results demonstrate Acai PS is a potent innate immune agonist that can resolve F. tularensis and B. pseudomallei infections, suggesting this innate immune agonist has broad-spectrum activity against virulent intracellular pathogens. PMID:22438809

Skyberg, Jerod A.; Rollins, MaryClare F.; Holderness, Jeff S.; Marlenee, Nicole L.; Schepetkin, Igor A.; Goodyear, Andrew; Dow, Steven W.; Jutila, Mark A.; Pascual, David W.

2012-01-01

128

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

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-? response in a restimulation assay using whole blood from seropositive donors and were recognised by seropositive human sera from the endemic area. To conclude, several predicted autotransporters contribute to B. pseudomallei virulence and BpaC may do so by conferring resistance against complement-mediated killing. PMID:25830295

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

129

Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion porin  

PubMed Central

In the present paper, we describe cloning and expression of two outer membrane proteins, BpsOmp38 (from Burkholderia pseudomallei) and BthOmp38 (from Burkholderia thailandensis) lacking signal peptide sequences, using the pET23d(+) expression vector and Escherichia coli host strain Origami(DE3). The 38 kDa proteins, expressed as insoluble inclusion bodies, were purified, solubilized in 8 M urea, and then subjected to refolding experiments. As seen on SDS/PAGE, the 38 kDa band completely migrated to ?110 kDa when the purified monomeric proteins were refolded in a buffer system containing 10% (w/v) Zwittergent® 3-14, together with a subsequent heating to 95 °C for 5 min. CD spectroscopy revealed that the 110 kDa proteins contained a predominant ?-sheet structure, which corresponded completely to the structure of the Omp38 proteins isolated from B. pseudomallei and B. thailandensis. Immunoblot analysis using anti-BpsOmp38 polyclonal antibodies and peptide mass analysis by MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS confirmed that the expressed proteins were BpsOmp38 and BthOmp38. The anti-BpsOmp38 antibodies considerably exhibited the inhibitory effects on the permeation of small sugars through the Omp38-reconstituted liposomes. A linear relation between relative permeability rates and Mr of neutral sugars and charged antibiotics suggested strongly that the in vitro re-assembled Omp38 functioned fully as a diffusion porin. PMID:15329048

2004-01-01

130

Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis  

PubMed Central

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

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

2014-01-01

131

Sterile-?- and Armadillo Motif-Containing Protein Inhibits the TRIF-Dependent Downregulation of Signal Regulatory Protein ? To Interfere with Intracellular Bacterial Elimination in Burkholderia pseudomallei-Infected Mouse Macrophages  

PubMed Central

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

Baral, Pankaj

2013-01-01

132

Involvement of Signal Regulatory Protein ?, a Negative Regulator of Toll-Like Receptor Signaling, in Impairing the MyD88-Independent Pathway and Intracellular Killing of Burkholderia pseudomallei-Infected Mouse Macrophages  

PubMed Central

The facultative intracellular Gram-negative bacterium Burkholderia pseudomallei is the causative agent of melioidosis and is known for its ability to evade the Toll-like receptor (TLR)-mediated innate immune response. Previously it has been demonstrated that this bacterium was able to suppress the MyD88-independent pathway and can survive macrophage intracellular killing. However, the underlying mechanisms responsible for the suppression of this pathway are not fully understood. In the present study, we showed that both living and heat-killed B. pseudomallei bacteria restrict the TLR signaling response, particularly macrophage inducible nitric oxide synthase (iNOS) expression, by preventing downregulation of constitutively expressed signal regulatory protein ? (SIRP?) molecule, a known negative regulator of TLR signaling. In contrast, a lipopolysaccharide (LPS) mutant of B. pseudomallei, a less virulent strain, was able to downregulate SIRP? expression in mouse macrophages. However, depletion of constitutively expressed SIRP? was able to induce the gene expression downstream of TLR signaling pathways (particularly the MyD88-independent pathway), such as that of the iNOS gene, leading to enhanced macrophage intracellular killing of B. pseudomallei. Induction of gene expression was consistent with the enhanced degradation pattern of I?B? with SIRP? depletion. Additionally, the downregulation of SIRP? expression with upregulation of iNOS was observed when the macrophages were pretreated with gamma interferon (IFN-?) prior to the infection, suggesting that the enhanced intracellular killing of bacteria by IFN-? is associated with the decreased SIRP? expression. Altogether our findings demonstrate that B. pseudomallei evades macrophage intracellular killing by preventing the downregulation of SIRP? that results in the inhibition of gene expression downstream of the MyD88-independent pathway. PMID:22988019

Baral, Pankaj

2012-01-01

133

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

PubMed Central

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

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

2013-01-01

134

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

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 IFN?, 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 IFN?. This suggests that effective immune therapies would need to elicit both antibodies and Th1-adaptive responses for successful prevention/eradication of melioidosis. PMID:25144195

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

2014-01-01

135

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

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 IFN?, 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 IFN?. This suggests that effective immune therapies would need to elicit both antibodies and Th1-adaptive responses for successful prevention/eradication of melioidosis. PMID:25144195

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

2014-08-01

136

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

PubMed

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

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

2004-05-01

137

Regulatory role of GSK3? in the activation of NF-?B and modulation of cytokine levels in Burkholderia pseudomallei-infected PBMC isolated from streptozotocin-induced diabetic animals.  

PubMed

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-3? (GSK3?) plays an important role in the innate inflammatory response caused by bacterial pathogens. The present study was conducted to investigate the effects of GSK3? inhibition by LiCl on levels of pro- and anti-inflammatory cytokines; and the activity of transcription factor NF-?B 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-?, IL-12 and IL-10) and phosphorylation of NF-?B 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 GSK3? in normal PBMC, an effect absent in infected diabetic PBMC. Inhibition of GSK3? by LiCl lowered the levels of pro-inflammatory cytokines (TNF-? and IL-12) in both normal and diabetic PBMC. Similarly, phosphorylated NF- ?B (pNF-?B) 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 GSK3? inhibition. More importantly, GSK3? 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? in diabetic PBMC resulted in the inactivation of NF-?B and modulation of inflammatory cytokine levels. This is evidence that dysregulation of GSK3? is a contributing factor in the molecular basis of innate dysfunction and susceptibility of diabetic host to melioidosis infection. PMID:25801253

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

2015-03-01

138

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

PubMed Central

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

Burtnick, Mary N.; Brett, Paul J.

2013-01-01

139

Genetic Analysis of the CDI Pathway from Burkholderia pseudomallei 1026b  

PubMed Central

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

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

2015-01-01

140

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

PubMed

A pre-requisite for bacterial pathogenesis is the successful interaction of a pathogen with a host. One mechanism used by a broad range of Gram negative bacterial pathogens is to deliver effector proteins directly into host cells through a dedicated type III secretion system where they modulate host cell function. The cycle inhibiting factor (Cif) family of effector proteins, identified in a growing number of pathogens that harbour functional type III secretion systems and have a wide host range, arrest the eukaryotic cell cycle. Here, the crystal structures of Cifs from the insect pathogen/nematode symbiont Photorhabdus luminescens (a gamma-proteobacterium) and human pathogen Burkholderia pseudomallei (a beta-proteobacterium) are presented. Both of these proteins adopt an overall fold similar to the papain sub-family of cysteine proteases, as originally identified in the structure of a truncated form of Cif from Enteropathogenic E. coli (EPEC), despite sharing only limited sequence identity. The structure of an N-terminal region, referred to here as the 'tail-domain' (absent in the EPEC Cif structure), suggests a surface likely to be involved in host-cell substrate recognition. The conformation of the Cys-His-Gln catalytic triad is retained, and the essential cysteine is exposed to solvent and addressable by small molecule reagents. These structures and biochemical work contribute to the rapidly expanding literature on Cifs, and direct further studies to better understand the molecular details of the activity of these proteins. PMID:19440549

Crow, Allister; Race, Paul R; Jubelin, Grégory; Varela Chavez, Carolina; Escoubas, Jean-Michel; Oswald, Eric; Banfield, Mark J

2009-01-01

141

Acute melioidosis outbreak in Western Australia.  

PubMed Central

A cluster of acute melioidosis cases occurred in a remote, coastal community in tropical Western Australia. Molecular typing of Burkholderia pseudomallei isolates from culture-confirmed cases and suspected environmental sources by pulsed-field gel electrophoresis (PFGE) of XbaI chromosomal DNA digests showed that a single PFGE type was responsible for five cases of acute infection in a community of around 300 during a 5 week period. This temporal and geographical clustering of acute melioidosis cases provided a unique opportunity to investigate the environmental factors contributing to this disease. B. pseudomallei isolated from a domestic tap at the home of an asymptomatic seroconverter was indistinguishable by PFGE. Possible contributing environmental factors included an unusually acid communal water supply, unrecordable chlorine levels during the probable exposure period, a nearby earth tremor, and gusting winds during the installation of new water and electricity supplies. The possible role of the potable water supply as a source of B. pseudomallei was investigated further. PMID:10694154

Inglis, T. J.; Garrow, S. C.; Adams, C.; Henderson, M.; Mayo, M.; Currie, B. J.

1999-01-01

142

Source-Identifying Biomarker Ions between Environmental and Clinical Burkholderia pseudomallei Using Whole-Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)  

PubMed Central

Burkholderia pseudomallei is the causative agent of melioidosis, which is an endemic disease in Northeast Thailand and Northern Australia. Environmental reservoirs, including wet soils and muddy water, serve as the major sources for contributing bacterial infection to both humans and animals. The whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has recently been applied as a rapid, accurate, and high-throughput tool for clinical diagnosis and microbiological research. In this present study, we employed a whole-cell MALDI-TOF MS approach for assessing its potency in clustering a total of 11 different B. pseudomallei isolates (consisting of 5 environmental and 6 clinical isolates) with respect to their origins and to further investigate the source-identifying biomarker ions belonging to each bacterial group. The cluster analysis demonstrated that six out of eleven isolates were grouped correctly to their sources. Our results revealed a total of ten source-identifying biomarker ions, which exhibited statistically significant differences in peak intensity between average environmental and clinical mass spectra using ClinProTools software. Six out of ten mass ions were assigned as environmental-identifying biomarker ions (EIBIs), including, m/z 4,056, 4,214, 5,814, 7,545, 7,895, and 8,112, whereas the remaining four mass ions were defined as clinical-identifying biomarker ions (CIBIs) consisting of m/z 3,658, 6,322, 7,035, and 7,984. Hence, our findings represented, for the first time, the source-specific biomarkers of environmental and clinical B. pseudomallei. PMID:24914956

Srisanga, Kitima; Roytrakul, Sittiruk; Tungpradabkul, Sumalee

2014-01-01

143

Melioidosis: Epidemiology, Pathophysiology, and Management  

PubMed Central

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

Cheng, Allen C.; Currie, Bart J.

2005-01-01

144

Intramolecular aglycon delivery enables the synthesis of 6-deoxy-?-D-manno-heptosides as fragments of Burkholderia pseudomallei and Burkholderia mallei capsular polysaccharide.  

PubMed

Burkholderia pseudomallei and Burkholderia mallei are potential bioterrorism agents. They express the same capsular polysaccharide (CPS), a homopolymer featuring an unusual [?3)-2-O-acetyl-6-deoxy-?-D-manno-heptopyranosyl-(1?] as the repeating unit. This CPS is known to be one of the main targets of the adaptive immune response in humans and therefore represents a crucial subunit candidate for vaccine development. Herein, the stereoselective synthesis of mono- and disaccharidic fragments of the B. pseudomallei and B. mallei CPS repeating unit is reported. The synthesis of 6-deoxy-?-D-manno-heptosides was investigated using both inter- and intramolecular glycosylation strategies from thio-manno-heptose that was modified with 2-naphthylmethyl (NAP) at C2. We show here that NAP-mediated intramolecular aglycon delivery (IAD) represents a suitable approach for the stereocontrolled synthesis of 6-deoxy-?-D-manno-heptosides without the need for rigid 4,6-O-cyclic protection of the sugar skeleton. The IAD strategy is highly modular, as it can be applied to structurally diverse acceptors with complete control of stereoselectivity. Problematic hydrogenation of the acetylated disaccharides was overcome by using a microfluidic continuous flow reactor. PMID:24786555

Tamigney Kenfack, Marielle; Blériot, Yves; Gauthier, Charles

2014-05-16

145

Burkholderia vaccines: are we moving forward?  

PubMed Central

The genus Burkholderia consists of diverse species which includes both “friends” and “foes.” Some of the “friendly” Burkholderia spp. are extensively used in the biotechnological and agricultural industry for bioremediation and biocontrol. However, several members of the genus including B. pseudomallei, B. mallei, and B. cepacia, are known to cause fatal disease in both humans and animals. B. pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively, while B. cepacia infection is lethal to cystic fibrosis (CF) patients. Due to the high rate of infectivity and intrinsic resistance to many commonly used antibiotics, together with high mortality rate, B. mallei and B. pseudomallei are considered to be potential biological warfare agents. Treatments of the infections caused by these bacteria are often unsuccessful with frequent relapse of the infection. Thus, we are at a crucial stage of the need for Burkholderia vaccines. Although the search for a prophylactic therapy candidate continues, to date development of vaccines has not advanced beyond research to human clinical trials. In this article, we review the current research on development of safe vaccines with high efficacy against B. pseudomallei, B. mallei, and B. cepacia. It can be concluded that further research will enable elucidation of the potential benefits and risks of Burkholderia vaccines. PMID:23386999

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

146

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

PubMed Central

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

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

147

Transverse myelitis secondary to Melioidosis; A case report  

PubMed Central

Background Melioidosis has become an emerging infection in Sri Lanka; a country which is considered non endemic for it. Paraplegia due to Burkholderia pseudomallei is a very rare entity encountered even in countries where the disease is endemic. There are no reported cases of transverse myelitis due to melioidosis in Sri Lankan population thus we report the first case. Case presentation A 21?year old farmer presented with sudden onset bi lateral lower limb weakness, numbness and urine retention. Examination revealed flaccid areflexic lower limbs with a sensory loss of all modalities and a sensory level at T10 together with sphincter involvement. MRI of the thoracolumbar spine showed extensive myelitis of the thoracic spine complicating left psoas abscess without definite extension to the spinal cord or cord compression. Burkholderia pseudomallei was isolated from the psoas abscess pus cultures and the diagnosis of melioidosis was confirmed with high titers of Burkholderia pseudomallei antibodies and positive PCR. He was treated with high doses of IV ceftazidime and oral cotrimoxazole for one month with a plan to continue cotrimoxazole and doxycycline till one year. Patient’s general condition improved but the residual neurological problems persisted. Conclusion The exact pathogenesis of spinal cord melioidosis is not quite certain except in the cases where there is direct microbial invasion, which does not appear to be the case in our patient. We postulate our patient’s presentation could be due to ischemia of the spinal cord following septic embolisation or thrombosis of spinal artery due to the abscess nearby. A neurotrophic exotoxin causing myelitis or post infectious immunological demyelination is yet another possibility. This emphasizes the necessity of further studies to elucidate the exact pathogenesis in this type of presentations. Health care professionals in Sri Lanka, where this is an emerging infection, need to improve their knowledge regarding this disease and should have high degree of suspicion to make a correct and a timely diagnosis to reduce the morbidity and mortality due to Burkholderia pseudomallei infection. It is highly likely that this infection is under diagnosed in developing countries where diagnostic facilities are minimal. Therefore strategies to improve the awareness and upgrade the diagnostic facilities need to be implemented in near future. PMID:23020820

2012-01-01

148

Pulmonary melioidosis in Cambodia: A prospective study  

PubMed Central

Background Melioidosis is a disease caused by Burkholderia pseudomallei and considered endemic in South-East Asia but remains poorly documented in Cambodia. We report the first series of hospitalized pulmonary melioidosis cases identified in Cambodia describing clinical characteristics and outcomes. Methods We characterized cases of acute lower respiratory infections (ALRI) that were identified through surveillance in two provincial hospitals. Severity was defined by systolic blood pressure, cardiac frequency, respiratory rate, oxygen saturation and body temperature. B. pseudomallei was detected in sputum or blood cultures and confirmed by API20NE gallery. We followed up these cases between 6 months and 2 years after hospital discharge to assess the cost-of-illness and long-term outcome. Results During April 2007 - January 2010, 39 ALRI cases had melioidosis, of which three aged ?2 years; the median age was 46 years and 56.4% were males. A close contact with soil and water was identified in 30 patients (76.9%). Pneumonia was the main radiological feature (82.3%). Eleven patients were severe cases. Twenty-four (61.5%) patients died including 13 who died within 61 days after discharge. Of the deceased, 23 did not receive any antibiotics effective against B. pseudomallei. Effective drugs that were available did not include ceftazidime. Mean total illness-related costs was of US$65 (range $25-$5000). Almost two-thirds (61.5%) incurred debt and 28.2% sold land or other belongings to pay illness-related costs. Conclusions The observed high fatality rate is likely explained by the lack or limited access to efficient antibiotics and under-recognition of the disease among clinicians, which led to inappropriate therapy. PMID:21569563

2011-01-01

149

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

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

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

2014-01-01

150

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

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

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

2014-10-01

151

rpsU-based discrimination within the genus Burkholderia.  

PubMed

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

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

2014-06-01

152

rpsU-based discrimination within the genus Burkholderia  

PubMed Central

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

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

2014-01-01

153

The epidemiology of melioidosis in Australia and Papua New Guinea.  

PubMed

Melioidosis was first described in Australia in an outbreak in sheep in 1949 in north Queensland (22 degrees S). Human melioidosis was first described from Townsville (19 degrees S) in 1950. Melioidosis is hyperendemic in the Top End of the Northern Territory (NT) and as in parts of northeastern Thailand it is the commonest cause of fatal community-acquired septicemic pneumonia. In the 9 years since 1989 the prospective NT melioidosis study at Royal Darwin Hospital (12 degrees S) has documented 206 culture confirmed cases of melioidosis, with an average annual incidence of 16.5/100,000. Melioidosis is also seen in the north of Western Australia and north Queensland, including the Torres Strait Islands, but is uncommon in adjacent Papua New Guinea. Serological studies suggest that infection is rare in the Port Moresby region, but there is emerging evidence of melioidosis from Western Province. The NT study has documented inoculating events in 52 (25%) of cases, with an incubation period of 1-21 days (mean 9 days); 84% of cases had acute disease from presumed recent acquisition and 13% had chronic disease (sick, > 2 months). In 4% there was evidence of possible reactivation from a latent focus; 28 of 153 (18%) males had prostatic abscesses. The overall mortality was 21% (43 cases), with a mortality rate in septicemic cases (95) of 39% and in non-septicemic cases (103) of 4%. Pneumonia was the commonest presentation in both groups and, in addition, eight patients (two deaths) presented with melioidosis encephalomyelitis. Melioidosis clusters in temperate Australia are attributed to animals imported from the north. Molecular typing of Burkholderia pseudomallei isolates from temperate southwest Western Australia showed clonality over 25 years. In this outbreak and in studies from the NT, some soil isolates are molecularly identical to epidemiologically related animal and human isolates. Molecular typing has implicated the water supply in two clonal outbreaks in remote aboriginal communities in northern Australia. Further prospective collaborative studies are required to evaluate whether there are truly regional differences in clinical features of melioidosis and to better understand how B. pseudomallei is acquired from the environment. PMID:10674639

Currie, B J; Fisher, D A; Howard, D M; Burrow, J N; Selvanayagam, S; Snelling, P L; Anstey, N M; Mayo, M J

2000-02-01

154

Natural history of inhalation melioidosis in rhesus macaques (Macaca mulatta) and African green monkeys (Chlorocebus aethiops).  

PubMed

Burkholderia pseudomallei, the causative agent of melioidosis, is recognized as a serious health threat due to its involvement in septic and pulmonary infections in areas of endemicity and is recognized by the Centers for Disease Control and Prevention as a category B biothreat agent. An animal model is desirable to evaluate the pathogenesis of melioidosis and medical countermeasures. A model system that represents human melioidosis infections is essential in this process. A group of 10 rhesus macaques (RMs) and 10 African green monkeys (AGMs) was exposed to aerosolized B. pseudomallei 1026b. The first clinical signs were fever developing 24 to 40 h postexposure followed by leukocytosis resulting from a high percentage of neutrophils. Dyspnea manifested 2 to 4 days postexposure. In the AGMs, an increase in interleukin 1? (IL-1?), IL-6, IL-8, gamma interferon (IFN-?), and tumor necrosis factor alpha (TNF-?) was observed. In the RMs, IL-1?, IL-6, and TNF-? increased. All the RMs and AGMs had various degrees of bronchopneumonia, with inflammation consisting of numerous neutrophils and a moderate number of macrophages. Both the RMs and the AGMs appear to develop a melioidosis infection that closely resembles that seen in acute human melioidosis. However, for an evaluation of medical countermeasures, AGMs appear to be a more appropriate model. PMID:22778104

Yeager, John J; Facemire, Paul; Dabisch, Paul A; Robinson, Camenzind G; Nyakiti, David; Beck, Katie; Baker, Reese; Pitt, M Louise M

2012-09-01

155

Overexpression of the Endothelial Protein C Receptor Is Detrimental during Pneumonia-Derived Gram-negative Sepsis (Melioidosis)  

PubMed Central

Background The endothelial protein C receptor (EPCR) enhances anticoagulation by accelerating activation of protein C to activated protein C (APC) and mediates anti-inflammatory effects by facilitating APC-mediated signaling via protease activated receptor-1. We studied the role of EPCR in the host response during pneumonia-derived sepsis instigated by Burkholderia (B.) pseudomallei, the causative agent of melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia. Methodology/Principal Findings Soluble EPCR was measured in plasma of patients with septic culture-proven melioidosis and healthy controls. Experimental melioidosis was induced by intranasal inoculation of B. pseudomallei in wild-type (WT) mice and mice with either EPCR-overexpression (Tie2-EPCR) or EPCR-deficiency (EPCR?/?). Mice were sacrificed after 24, 48 or 72 hours. Organs and plasma were harvested to measure colony forming units, cellular influxes, cytokine levels and coagulation parameters. Plasma EPCR-levels were higher in melioidosis patients than in healthy controls and associated with an increased mortality. Tie2-EPCR mice demonstrated enhanced bacterial growth and dissemination to distant organs during experimental melioidosis, accompanied by increased lung damage, neutrophil influx and cytokine production, and attenuated coagulation activation. EPCR?/? mice had an unremarkable response to B. pseudomallei infection as compared to WT mice, except for a difference in coagulation activation in plasma. Conclusion/Significance Increased EPCR-levels correlate with accelerated mortality in patients with melioidosis. In mice, transgenic overexpression of EPCR aggravates outcome during Gram-negative pneumonia-derived sepsis caused by B. pseudomallei, while endogenous EPCR does not impact on the host response. These results add to a better understanding of the regulation of coagulation during severe (pneumo)sepsis. PMID:23875041

Kager, Liesbeth M.; Schouten, Marcel; Wiersinga, W. Joost; de Boer, J. Daan; Lattenist, Lionel C. W.; Roelofs, Joris J. T. H.; Meijers, Joost C. M.; Levi, Marcel; Dondorp, Arjen M.; Esmon, Charles T.; van 't Veer, Cornelis; van der Poll, Tom

2013-01-01

156

Interaction of Interferon gamma-induced reactive oxygen species with ceftazidime leads to synergistic killing of intracellular Burkholderia pseudomallei.  

PubMed

Burkholderia pseudomallei, a facultative intracellular pathogen, causes severe infections and is inherently refractory to many antibiotics. Previous studies from our group have shown that interferon gamma (IFN-?) interacts synergistically with the antibiotic ceftazidime to kill bacteria in infected macrophages. The present study aimed to identify the underlying mechanism of that interaction. We first showed that blocking reactive oxygen species (ROS) pathways reversed IFN-?- and ceftazidime-mediated killing, which led to our hypothesis that IFN-?-induced ROS interacted with ceftazidime to synergistically kill Burkholderia bacteria. Consistent with this hypothesis, we also observed that buthionine sulfoximine (BSO), another inducer of ROS, could substitute for IFN-? to similarly potentiate the effect of ceftazidime on intracellular killing. Next, we observed that IFN-? induced ROS-mediated killing of intracellular but not extracellular bacteria. On the other hand, ceftazidime effectively reduced extracellular bacteria but was not capable of intracellular killing when applied at 10 ?g/ml. We investigated the exact role of IFN-?-induced ROS responses on intracellular bacteria and notably observed a lack of actin polymerization associated with Burkholderia bacteria in IFN-?-treated macrophages, which led to our finding that IFN-?-induced ROS blocks vacuolar escape. Based on these results, we propose a model in which synergistically reduced bacterial burden is achieved primarily through separate and compartmentalized killing: intracellular killing by IFN-?-induced ROS responses and extracellular killing by ceftazidime. Our findings suggest a means of enhancing antibiotic activity against Burkholderia bacteria through combination with drugs that induce ROS pathways or otherwise target intracellular spread and/or replication of bacteria. PMID:25070108

Mosovsky, Kara; Silva, Ediane; Troyer, Ryan; Propst-Graham, Katie; Dow, Steven

2014-10-01

157

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

NASA Astrophysics Data System (ADS)

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.

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

2010-04-01

158

The role of short-chain dehydrogenase/oxidoreductase, induced by salt stress, on host interaction of B. pseudomallei  

PubMed Central

Background Burkholderia pseudomallei is the causative agent of melioidosis, a frequently occurring disease in northeastern Thailand, where soil and water high in salt content are common. Using microarray analysis, we previously showed that B. pseudomallei up-regulated a short-chain dehydrogenase/oxidoreductase (SDO) under salt stress. However, the importance of SDO in B. pseudomallei infection is unknown. This study aimed to explore the function of B. pseudomallei SDO, and to investigate its role in interactions between B. pseudomallei and host cells. Results Bioinformatics analysis of B. pseudomallei SDO structure, based on homology modeling, revealed a NAD+ cofactor domain and a catalytic triad containing Ser149, Tyr162, and Lys166. This is similar to Bacillus megaterium glucose 1-dehydrogenase. To investigate the role of this protein, we constructed a B. pseudomallei SDO defective mutant, measured glucose dehydrogenase (GDH) activity, and tested the interactions with host cells. The B. pseudomallei K96243 wild type exhibited potent GDH activity under condition containing 300 mM NaCl, while the mutant showed activity levels 15 times lower. Both invasion into the A549 cell line and early intracellular survival within the J774A.1 macrophage cell were impaired in the mutant. Complementation of SDO was able to restore the mutant ability to produce GDH activity, invade epithelial cells, and survive in macrophages. Conclusions Our data suggest that induced SDO activity during salt stress may facilitate B. pseudomallei invasion and affect initiation of successful intracellular infection. Identifying the role of B. pseudomallei SDO provides a better understanding of the association between bacterial adaptation and pathogenesis in melioidosis. PMID:24382268

2014-01-01

159

Galleria mellonella as a model system to test the pharmacokinetics and efficacy of antibiotics against Burkholderia pseudomallei.  

PubMed

Mammalian models of infection are paramount to elucidating the mechanisms of bacterial pathogenesis and are also used for evaluating the efficacy of novel antimicrobials before the commencement of human trials. In this study, Galleria mellonella was used to determine the efficacy of antibiotics towards a Burkholderia thailandensis infection in G. mellonella larvae. Kanamycin, imipenem, ceftazidime, doxycycline and ciprofloxacin could all provide some protection when given 1 h before challenge with B. thailandensis; however, at 2 h or 6 h post challenge, imipenem and kanamycin were unable to rescue larvae. The most effective antibiotic for the prevention or treatment of disease was ceftazidime. Pharmacokinetic properties of a single dose of these antibiotics in G. mellonella larvae were also determined, and it was demonstrated that this model is useful for approximating the antibiotic response in humans. The G. mellonella model was used to screen a panel of novel antimicrobials for activity towards B. thailandensis and Burkholderia pseudomallei, and three novel compounds with antibiotic activity were identified. These results support the hypothesis that G. mellonella can be used to screen antimicrobial efficacy. This is the first study to determine the pharmacokinetic parameters of clinically relevant antibiotics in this model system. PMID:23402703

Thomas, Rachael J; Hamblin, Karleigh A; Armstrong, Stuart J; Müller, Claudia M; Bokori-Brown, Monika; Goldman, Stan; Atkins, Helen S; Titball, Richard W

2013-04-01

160

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

PubMed Central

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

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

1997-01-01

161

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

PubMed Central

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 uniquely capable to characterize the fate of lung-delivered pathogen. PMID:25569630

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

2015-01-01

162

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

PubMed

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. PMID:25477002

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

2014-12-01

163

Nitric Oxide from IFN?-Primed Macrophages Modulates the Antimicrobial Activity of ?-Lactams against the Intracellular Pathogens Burkholderia pseudomallei and Nontyphoidal Salmonella  

PubMed Central

Our investigations show that nonlethal concentrations of nitric oxide (NO) abrogate the antibiotic activity of ?-lactam antibiotics against Burkholderia pseudomallei, Escherichia coli and nontyphoidal Salmonella enterica serovar Typhimurium. NO protects B. pseudomallei already exposed to ?-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 ?-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 ?-lactams. The degree of NO-induced ?-lactam antibiotic tolerance seems to be inversely proportional to the proton motive force (PMF), and thus the dissipation of ?H+ and ?? electrochemical gradients of the PMF prevents ?-lactam-mediated killing. According to this model, NO generated by IFN?-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 IFN?-primed macrophages. Our investigations indicate that NO modulates the antimicrobial activity of ?-lactam antibiotics. PMID:25121731

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

164

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

PubMed

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

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

2005-01-01

165

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

PubMed Central

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

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

2014-01-01

166

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

PubMed

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

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

2015-03-01

167

A non-invasive intratracheal inoculation method for the study of pulmonary melioidosis  

PubMed Central

Pulmonary melioidosis, a disease manifestation caused by the bacterium Burkholderia pseudomallei, has been studied using aerosols or intranasal (IN) inoculation in small animal models. Both have inherent disadvantages which may not accurately model primary pulmonary melioidosis in humans. Intratracheal inoculation (IT) by direct visualization of the tracheal opening offers an alternative technique for infection that overcomes the disadvantages of aerosol and IN challenge. In this study, we describe a method which requires relatively inexpensive equipment, little training, and is compliant with the operational constraints of a BSL3 laboratory. Results obtained using trypan blue demonstrated that an inoculum can be accurately delivered into the lungs of mice within a biosafety cabinet (BSC). Whole body imaging and histopathology confirmed that mice inoculated intratracheally with B. pseudomallei develop the primary focus of infection in the lungs, and not the nasal passages which can lead to invasion of the central nervous system and potential neurologic complications. Further, based on colony counts and bioluminescent imaging, dissemination to secondary organs occurred as expected. Taken together, this intratracheal method of inoculation fulfills four goals: (1) to accurately deliver B. pseudomallei into the lungs of the animal model, (2) to avoid potentially confounding complications due to primary infections at sites other than the lung, (3) to maintain normal organ dissemination, and (4) to be BSL3 compliant. PMID:23267442

Revelli, David A.; Boylan, Julie A.; Gherardini, Frank C.

2012-01-01

168

Protection against Experimental Melioidosis following Immunisation with a Lipopolysaccharide-Protein Conjugate  

PubMed Central

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. It is refractory to antibiotic treatment and there is currently no licensed vaccine. In this report we detail the construction and protective efficacy of a polysaccharide-protein conjugate composed of B. pseudomallei lipopolysaccharide and the Hc fragment of tetanus toxin. Immunisation of mice with the lipopolysaccharide-conjugate led to significantly reduced bacterial burdens in the spleen 48 hours after challenge and afforded significant protection against a lethal challenge with B. pseudomallei. The conjugate generated significantly higher levels of antigen-specific IgG1 and IgG2a than in lipopolysaccharide-immunised mice. Immunisation with the conjugate also demonstrated a bias towards Th1 type responses, evidenced by high levels of IgG2a. In contrast, immunisation with unconjugated lipopolysaccharide evoked almost no IgG2a demonstrating a bias towards Th2 type responses. This study demonstrates the effectiveness of this approach in the development of an efficacious and protective vaccine against melioidosis. PMID:24892035

Scott, Andrew E.; Ngugi, Sarah A.; Laws, Thomas R.; Corser, David; Lonsdale, Claire L.; D'Elia, Riccardo V.; Titball, Richard W.; Williamson, E. Diane; Atkins, Timothy P.; Prior, Joann L.

2014-01-01

169

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

E-print Network

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

Bart J. Currie

170

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

PubMed

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

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

2015-04-01

171

Screen of whole blood responses to flagellin identifies TLR5 variation associated with outcome in melioidosis.  

PubMed

Melioidosis is a severe infection caused by the flagellated bacterium Burkholderia pseudomallei. The nonsense polymorphism TLR51174C>T is associated with improved outcome in Thais with melioidosis. We hypothesized that other TLR5 variants may modulate the host response and determine outcome in melioidosis. We genotyped 12 TLR5 variants selected de novo from the HapMap database and examined the association of each with cytokines induced by flagellin stimulation of whole blood from healthy Thai subjects. We found a blunted cytokine response for three related markers that were in linkage disequilibrium (LD) with a non-synonymous variant, TLR51846T>C. Carriers of TLR51846T>C had broadly impaired cytokine responses induced by flagellin. TLR51846T>C was associated with protection against death in melioidosis patients (odds ratio: 0.62, 95% confidence interval: 0.42-0.93, P=0.021). We observed no impairment in TLR51846C-dependent nuclear factor ?B activation, however, suggesting an alternative mechanism for the effect. We found that TLR51846T>C was in strong LD with TLR51174C>T. Many of the blunted cytokine responses observed and the association of TLR51846T>C with survival in melioidosis patients may be attributable to TLR51174C>T, but we could not exclude an independent effect of TLR51846T>C. These data identify novel associations for TLR51846T>C, enhance our understanding of TLR5 genetic architecture in Thais and highlight the role of TLR5 in melioidosis. PMID:24285178

Chantratita, N; Tandhavanant, S; Myers, N D; Chierakul, W; Robertson, J D; Mahavanakul, W; Singhasivanon, P; Emond, M J; Peacock, S J; West, T E

2014-03-01

172

Cervical abscesses due to co-infection with Burkholderia pseudomallei, Salmonella enterica serovar Stanley and Mycobacterium tuberculosis in a patient with diabetes mellitus  

PubMed Central

Background Infections due to Mycobacterium tuberculosis, Burkholderia pseudomallei and non-typhoidal Salmonella cause significant morbidity and mortality throughout the world. These intracellular pathogens share some common predisposing factors and clinical features. Co-infection with two of these organisms has been reported previously but, to our knowledge, this is the first time that infection with all three has been reported in one person. Case presentation In September 2010, a 58-year-old diabetic Malaysian male presented with fever and a fluctuant mass on the right side of his neck. B. pseudomallei was isolated from an aspirate of this lesion and there was radiological evidence of disseminated infection in the liver and spleen. The recurrence of clinical symptoms over ensuing months prompted further aspiration and biopsy of a cervical abscess and underlying lymph nodes. Salmonella enterica serovar Stanley and then M. tuberculosis were identified from these specimens by culture and molecular methods. The patient responded to targeted medical management of each of these infections. Conclusion In endemic settings, a high index of suspicion and adequate tissue sampling are imperative in identifying these pathogenic organisms. Diabetes was identified as a predisposing factor in this case while our understanding of other potential risk factors is evolving. PMID:24209898

2013-01-01

173

Imaging patterns in melioidosis.  

PubMed

Melioidosis is an infectious disease caused by Pseudomonas pseudomallei. It is seldom diagnosed promptly and, if untreated, can lead to an 80-100% mortality rate. Twenty-eight patients with melioidosis were identified over a 6 year period, and their imaging patterns were analysed. Respiratory infections were the commonest form of presentation, frequently shown as diffuse airspace consolidation, and accounted for the highest mortality. Visceral and musculoskeletal infections were associated with chronicity and a high relapse rate. Multifocal splenic abscesses were a common occurrence. Septic arthritis of the knee was frequently seen. The majority of patients had diabetes mellitus and chronic ill-health. An increased awareness of the disease can contribute to its early detection and appropriate treatment. PMID:7487762

Tan, A P; Pui, M H; Tan, L K

1995-08-01

174

Use of the phytopathogenic effect for studies of burkholderia virulence.  

PubMed

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

Molchanova, E V; Ageeva, N P

2015-02-01

175

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

PubMed

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. PMID:23805570

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

176

In Vitro Susceptibilities of Burkholderia mallei in Comparison to Those of Other Pathogenic Burkholderia spp  

Microsoft Academic Search

The in vitro antimicrobial susceptibilities of isolates of Burkholderia mallei to 16 antibiotics were assessed and compared with the susceptibilities of Burkholderia pseudomallei and Burkholderia cepacia. The antibiotic susceptibility profile of B. mallei resembled that of B. pseudomallei more closely than that of B. cepacia, which corresponds to their similarities in terms of biochemistry, antigenicity, and pathogenicity. Ceftazidime, imi- penem,

D. J. KENNY; P. RUSSELL; D. ROGERS; S. M. ELEY; R. W. TITBALL

1999-01-01

177

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

PubMed Central

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

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

2010-01-01

178

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

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, ?fabI1, ?fabI2, and ?fabV knockout strains were constructed and tested in a mouse model of infection. Mice infected with a ?fabI1 strain did not show signs of morbidity, mortality, or dissemination after 30 days of infection compared to the wild-type and ?fabI2 and ?fabV mutant strains that had times to mortality of 60 to 84 h. Although signs of morbidity and mortality of ?fabI2 and ?fabV 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

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

2014-01-01

179

Sulphonylurea Usage in Melioidosis Is Associated with Severe Disease and Suppressed Immune Response  

PubMed Central

Background Melioidosis is a problem in the developing tropical regions of Southeast Asia and Northern Australia where the the Gram negative saprophytic bacillus Burkholderia pseudomallei is endemic with the risk of fulminant septicaemia. While diabetes mellitus is a well-established risk factor for melioidiosis, little is known if specific hypoglycemic agents may differentially influence the susceptibility and clinical course of infection with B. pseudomallei (Bp). Methodology/Principal Findings In this cohort study, patients with pre-existing diabetes and melioidosis were retrospectively studied. Outcome measures: mortality, length of stay and development of complications (namely hypotension, intubation, renal failure and septicaemia) were studied in relation to prior diabetic treatment regimen. Peripheral blood mononuclear cells (PBMC) from diabetic patients and healthy PBMC primed with metformin, glyburide and insulin were stimulated with purified Bp antigens in vitro. Immune response and specific immune pathway mediators were studied to relate to the clinical findings mechanistically. Of 74 subjects, 44 (57.9%) had sulphonylurea-containing diabetic regimens. Patient receiving sulphonylureas had more severe septic complications (47.7% versus 16.7% p?=?0.006), in particular, hypotension requiring intropes (p?=?0.005). There was also a trend towards increased mortality in sulphonylurea-users (15.9% versus 3.3% p?=?0.08). In-vitro, glyburide suppressed inflammatory cytokine production in a dose-dependent manner. An effect of the drug was the induction of IL-1R-associated kinase-M at the level of mRNA transcription. Conclusion/Significance Sulphonylurea treatment results in suppression of host inflammatory response and may put patients at higher risk for adverse outcomes in melioidosis. PMID:24762472

Liu, Xiang; Foo, Geraldine; Lim, Wan Peng; Ravikumar, Sharada; Sim, Siew Hoon; Win, Mar Soe; Goh, Jessamine Geraldine; Lim, Joan Hui Juan; Ng, Ying Hui; Fisher, Dale; Khoo, Chin Meng

2014-01-01

180

In Vivo Manipulation of ?9+ T Cells in the Common Marmoset (Callithrix Jacchus) with Phosphoantigen and Effect on the Progression of Respiratory Melioidosis  

PubMed Central

Burkholderia pseudomallei is a dangerous human pathogen. Phosphoantigens specifically the target primate specific ?9+?2+ T cells subset and some have been developed as potential immunotherapeutics. Previously, we demonstrated that, when stimulated with the phosphoantigen CHDMAPP, ?9+?2+ T cells aid in the killing of intracellular B. pseudomallei bacteria. Moreover, we found that common marmoset (Callithrix Jacchus) ?9+ T cells increase in frequency and respond to the phosphoantigen CHDMAPP and/or B. pseudomallei, in combination with IL-2, in a similar manner to human ?9+?2+ T cells. Here we evaluate the efficacy of the phosphoantigen CHDMAPP, in combination with IL-2, as a therapy against B. pseudomallei infection, in vivo. We found that the previous studies predicted the in vivo responsiveness of ?9+ T cells to the CHDMAPP+IL-2 treatment and significant expansion of the numbers of peripheral and splenic ?9+ T cells were observed. This effect was similar to those reported in other primate species treated with phosphoantigen. Furthermore, splenocytes were retrieved 7 days post onset of treatment, restimulated with CHDMAPP or heat-killed B. pseudomallei and the cultured ?9+ T cells demonstrated no reduction in IFN-? response when CHDMAPP+IL-2 animals were compared to IL-2 only treated animals. Using an established model of B. pseudomallei infection in the marmoset, we assessed the potential for using phosphoantigen as a novel immunotherapy. The CHDMAPP treatment regime had no effect on the progression of respiratory melioidosis and this was despite the presence of elevated numbers of ?9+ T cells in the spleen, liver and lung and an increased proportion of IFN-?+ cells in response to infection. We therefore report that the common marmoset has proven a good model for studying the effect in vivo of ?9+ T cell stimulation; however, ?9+ T cells have little or no effect on the progression of lethal, respiratory B. pseudomallei infection. PMID:24098670

Laws, Thomas R.; Nelson, Michelle; Bonnafous, Cecile; Sicard, Helene; Taylor, Christopher; Salguero, Francisco Javier; Atkins, Timothy P.; Oyston, Petra C. F.; Rowland, Caroline A.

2013-01-01

181

Elucidation of the Regulon and cis-Acting Regulatory Element of HrpB, the AraC-Type Regulator of a Plant Pathogen-Like Type III Secretion System in Burkholderia pseudomallei?†  

PubMed Central

The human pathogen Burkholderia pseudomallei possesses multiple type III secretion system (T3SS) gene clusters. One of these, the B. pseudomallei T3SS2 (T3SS2bp) gene cluster, which apparently plays no role in animal virulence, is also found in six additional Burkholderia spp. and is very similar to T3SSs found in phytopathogenic Xanthomonas spp. and Ralstonia solanacearum. The T3SS2bp gene cluster also encodes an AraC-type regulatory protein (HrpBbp) that is an ortholog of HrpB, the master regulator of the R. solanacearum T3SS (T3SSrso) and its secreted effectors. Transcriptome analysis showed that HrpBbp activates the expression of T3SS2bp genes, as well as their orthologs in R. solanacearum. In addition to activating T3SS2bp, HrpBbp also upregulates the expression of ?30 additional B. pseudomallei genes, including some that may confer production of adhesive pili, a polyketide toxin, several putative T3SS2bp-secreted effectors, and components of a regulatory cascade. T3SS2bp promoter regions were found to contain a conserved DNA motif (p2bp box) identical in sequence and position to the hrpII box required for HrpB-dependent T3SSrso transcription activation. The p2bp box is also present in the promoter regions of the essentially identical T3SS found in the very closely related species Burkholderia thailandensis (T3SS2bt). Analysis of p2bp box mutants showed that it is essential for HrpBbp-mediated transcription activation in both species. Although it has been suggested that T3SS2bp and T3SS2bt may function in phytopathogenicity, we were unable to demonstrate a phytopathogenic phenotype for B. thailandensis in three different plant hosts. PMID:21335458

Lipscomb, Lyla; Schell, Mark A.

2011-01-01

182

Development of an acute model of inhalational melioidosis in the common marmoset (Callithrix jacchus)  

PubMed Central

Studies of inhalational melioidosis were undertaken in the common marmoset (Callithrix jacchus). Following exposure to an inhaled challenge with aerosolized Burkholderia pseudomallei, lethal infection was observed in marmosets challenged with doses below 10 cfu; a precise LD50 determination was not possible. The model was further characterized using a target challenge dose of approximately 102 cfu. A separate pathogenesis time-course experiment was also conducted. All animals succumbed, between 27 and 78 h postchallenge. The challenge dose received and the time to the humane endpoint (1 °C below normal body temperature postfever) were correlated. The first indicator of disease was an increased core body temperature (Tc), at 22 h postchallenge. This coincided with bacteraemia and bacterial dissemination. Overt clinical signs were first observed 3–5 h later. A sharp decrease (typically within 3–6 h) in the Tc was observed prior to humanely culling the animals in the lethality study. Pathology was noted in the lung, liver and spleen. Disease progression in the common marmoset appears to be consistent with human infection in terms of bacterial spread, pathology and physiology. The common marmoset can therefore be considered a suitable animal model for further studies of inhalational melioidosis. PMID:22122591

Nelson, Michelle; Dean, Rachel E; Salguero, Francisco J; Taylor, Christopher; Pearce, Peter C; Simpson, Andrew J H; Lever, Mark S

2011-01-01

183

Burkholderia thailandensis Is Virulent in Drosophila melanogaster  

PubMed Central

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

Pilátová, Martina; Dionne, Marc S.

2012-01-01

184

Whole-genome assemblies of 56 burkholderia species.  

PubMed

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

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

2014-01-01

185

Whole-Genome Assemblies of 56 Burkholderia Species  

PubMed Central

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

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

186

Ribotype analysis of Pseudomonas pseudomallei isolates.  

PubMed Central

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

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

1993-01-01

187

Siderophore production by Pseudomonas pseudomallei.  

PubMed Central

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

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

1991-01-01

188

Sequence-Defined Transposon Mutant Library of Burkholderia thailandensis  

PubMed Central

ABSTRACT We constructed a near-saturation transposon mutant library for Burkholderia thailandensis, a low-virulence surrogate for the causative agent of melioidosis (Burkholderia pseudomallei). A primary set of nearly 42,000 unique mutants (~7.5 mutants/gene) was generated using transposon Tn5 derivatives. The strains carry insertions in 87% of the predicted protein-coding genes of the organism, corresponding to nearly all of those nonessential for growth on nutrient agar. To achieve high genome coverage, we developed procedures for efficient sequence identification of insertions in extremely GC-rich regions of DNA. To facilitate strain distribution, we created a secondary library with two mutants per gene for which most transposon locations had been confirmed by resequencing. A map of mutations in the two-allele library and procedures for obtaining strains can be found at http://tools.nwrce.org/tn_mutants/ and http://www.gs.washington.edu/labs/manoil/. The library should facilitate comprehensive mutant screens and serve as a source of strains to test predicted genotype-phenotype associations. PMID:24194535

Gallagher, Larry A.; Ramage, Elizabeth; Patrapuvich, Rapatbhorn; Weiss, Eli; Brittnacher, Mitch; Manoil, Colin

2013-01-01

189

Incidence, risk factors and clinical epidemiology of melioidosis: a complex socio-ecological emerging infectious disease in the Alor Setar region of Kedah, Malaysia  

PubMed Central

Background Melioidosis, a severe and fatal infectious disease caused by Burkholderia pseudomallei, is believed to an emerging global threat. However, data on the natural history, risk factors, and geographic epidemiology of the disease are still limited. Methods We undertook a retrospective analysis of 145 confirmed cases extracted from a hospital-based Melioidosis Registry set up from 2005 in Hospital Sultanah Bahiyah, Alor Setar, Kedah state, Malaysia, in order to provide a first description of the contemporary incidence, risk factors, and clinical epidemiology of the disease in this putatively high risk region of the country. Results The incidence of melioidosis in Alor Setar is remarkably high at 16.35 per 100,000 population per year. The mean age of patients was 50.40 years, with infection varying nonlinearly with age. Males (75.2%; P < 0.0001) predominated and the majority of cases were Malays (88.9%). The overall, crude mortality rate among the study patients was 33.8%. The proportions of cases and deaths were significantly greater among patients involved in farming, forestry and fishing and the unemployed (?2 = 30.57, P < 0.0001). A majority of cases (62.75%) were culture positive, with mortality in these patients being 45.05%. A large proportion (83.0%) of culture positives was also bacteremic. Pneumonia accounted for 42.06% of primary diagnoses followed in importance by soft tissue abscess. In patients with pneumonia and who were culture positive, the mortality rate was as high as 65.00%. Diabetes mellitus constituted the major underlying risk factor for developing and dying from melioidosis, occurring in 57% of all diagnosed cases. The age distribution of diabetes paralleled that of melioidosis cases. There were linear associations between cases and deaths with monthly rainfall. Conclusions Melioidosis represents a complex socio-ecological public health problem in Kedah, being strongly related with age, occupation, rainfall and predisposing chronic diseases, such as diabetes mellitus. Among cases, bacteremic patients were associated with significantly high mortality despite provision of the recommended antibacterial therapy. The burden of this disease is likely to grow in this region unless better informed interventions targeted at high-risk groups and associated diseases are urgently implemented. PMID:20964837

2010-01-01

190

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

PubMed

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-?, TNF-?, IL-1?, and IL-6) equivalent to the levels of B. pseudomallei in primary PBMC cultures of NHPs and humans. When we examined IL-1? 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. PMID:25450887

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

2015-01-01

191

Melioidosis diagnostic workshop, 2013.  

PubMed

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

Hoffmaster, Alex R; 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

2015-02-01

192

Melioidosis Diagnostic Workshop, 20131  

PubMed Central

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

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

193

Animal melioidosis in Australia  

Microsoft Academic Search

Melioidosis was first diagnosed in Australia in sheep in 1949. While it has been considered endemic in tropical Australia, there have been animal outbreaks in southwest Western Australia and southern Queensland. Infection occurs in many species, with both latency and a wide range of clinical manifestations. Some species may develop melioidosis only if immunocompromised. Sheep and goats are particularly susceptible,

Jodie Low Choy; Mark Mayo; Anton Janmaat; Bart J Currie

2000-01-01

194

Stable, site-specific fluorescent tagging constructs optimized for burkholderia species.  

PubMed

Several vectors that facilitate stable fluorescent labeling of Burkholderia pseudomallei and Burkholderia thailandensis were constructed. These vectors combined the effectiveness of the mini-Tn7 site-specific transposition system with fluorescent proteins optimized for Burkholderia spp., enabling bacterial tracking during cellular infection. PMID:20851961

Norris, Michael H; Kang, Yun; Wilcox, Bruce; Hoang, Tung T

2010-11-01

195

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

PubMed Central

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

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

196

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

PubMed Central

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 Melioidosis Congress 2013, Bangkok, Thailand, 18–20 September 2013 (abstract OS VII-04). PMID:25803048

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

197

Melioidosis in a returned traveller  

PubMed Central

Melioidosis is an infection endemic to Southeast Asia and Northern Australia, and is associated with significant morbidity and mortality. The present report describes a case of chronic melioidosis in a returning traveller from the Philippines. Clinical suspicion of this illness is warranted in individuals with a history of travel to endemic regions. Safety in handling clinical specimens is paramount because laboratory transmission has been described. PMID:25285129

Chagla, Zain; Aleksova, Natasha; Quirt, Jaclyn; Emery, Joel; Kraeker, Christian; Haider, Shariq

2014-01-01

198

Cutaneous melioidosis in a healthy Danish man after travelling to South-East Asia.  

PubMed

A healthy Danish man presented with infected prepatellar bursitis 8 months after being involved in a car accident in Malaysia resulting in exposure of a laceration of his knee to stagnant water. Tissue samples grew Burkholderia pseudomallei and diagnostic work up revealed no secondary foci. The patient was successfully treated with surgical debridement and 3 months of oral trimethoprim-sulfamethoxazole. At 6 months follow-up the patient was without relapse. PMID:25596295

Bodilsen, Jacob; Langgaard, Henrik; Nielsen, Hans Linde

2015-01-01

199

Fatal Melioidosis in Goats in Bangkok, Thailand  

PubMed Central

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. PMID:24891468

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

2014-01-01

200

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

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 the demarcation of different groups of Burkholderia spp. and they also offer novel and useful targets for the development of diagnostic assays for the clinically important members of the BCC or the pseudomallei groups. Based upon the results of phylogenetic analyses, the identified CSIs and the pathogenicity profile of Burkholderia species, we are proposing a division of the genus Burkholderia into two genera. In this new proposal, the emended genus Burkholderia will correspond to the Clade I and it will contain only the clinically relevant and phytopathogenic Burkholderia species. All other Burkholderia spp., which are primarily environmental, will be transferred to a new genus Paraburkholderia gen. nov. PMID:25566316

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

2014-01-01

201

Antibacterial activity of a lectin-like Burkholderia cenocepacia protein.  

PubMed

Bacteriocins of the LlpA family have previously been characterized in the ?-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 ?-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

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

2013-08-01

202

High-Redundancy Draft Sequencing of 15 Clinical and Environmental Burkholderia Strains?  

PubMed Central

The Gram-negative Burkholderia genus includes several species of intracellular bacterial pathogens that pose substantial risk to humans. In this study, we have generated draft genome sequences of 15 strains of B. oklahomensis, B. pseudomallei, B. thailandensis, and B. ubonensis to an average sequence read coverage of 25- to 40-fold. PMID:20870763

Mukhopadhyay, Sanghamitra; Thomason, Maureen K.; Lentz, Shannon; Nolan, Nichole; Willner, Kristin; Gee, Jay E.; Glass, Mindy B.; Inglis, Timothy J. J.; Merritt, Adam; Levy, Avram; Sozhamannan, Shanmuga; Mateczun, Al; Read, Timothy D.

2010-01-01

203

Melioidosis Acquired by Traveler to Nigeria  

PubMed Central

We describe melioidosis associated with travel to Nigeria in a woman with diabetes, a major predisposing factor for this infection. With the prevalence of diabetes projected to increase dramatically in many developing countries, the global reach of melioidosis may expand. PMID:21762592

Salam, Alex P.; Khan, Nisa; Malnick, Henry; Kenna, Dervla T.D.; Dance, David A.B.

2011-01-01

204

Genetic Tools for Allelic Replacement in Burkholderia Species? †  

PubMed Central

Allelic replacement in the Burkholderia genus has been problematic due to the lack of appropriate counter-selectable and selectable markers. The counter-selectable marker sacB, commonly used in gram-negative bacteria, is nonselective on sucrose in many Burkholderia species. In addition, the use of antibiotic resistance markers of clinical importance for the selection of desirable genetic traits is prohibited in the United States for two potential bioterrorism agents, Burkholderia mallei and Burkholderia pseudomallei. Here, we engineered a mutated counter-selectable marker based on the B. pseudomallei PheS (the ?-subunit of phenylalanyl tRNA synthase) protein and tested its effectiveness in three different Burkholderia species. The mutant PheS protein effectively killed 100% of the bacteria in the presence of 0.1% p-chlorophenylalanine. We assembled the mutant pheS on several allelic replacement vectors, in addition to constructing selectable markers based on tellurite (Telr) and trimethoprim (Tpr) resistance that are excisable by flanking unique FLP recombination target (FRT) sequences. As a proof of concept, we utilized one of these gene replacement vectors (pBAKA) and the Telr-FRT cassette to produce a chromosomal mutation in the Burkholderia thailandensis betBA operon, which codes for betaine aldehyde dehydrogenase and choline dehydrogenase. Chromosomal resistance markers could be excised by the introduction of pFLP-AB5 (Tpr), which is one of two constructed flp-containing plasmids, pFLP-AB4 (Telr) and pFLP-AB5 (Tpr). These flp-containing plasmids harbor the mutant pheS gene and allow self curing on media that contain p-chlorophenylalanine after Flp-FRT excision. The characterization of the ?betBA::Telr-FRT and ?betBA::FRT mutants indicated a defect in growth with choline as a sole carbon source, while these mutants grew as well as the wild type with succinate and glucose as alternative carbon sources. PMID:18502918

Barrett, Ashley R.; Kang, Yun; Inamasu, Ken S.; Son, Mike S.; Vukovich, Joseph M.; Hoang, Tung T.

2008-01-01

205

The epidemiology of melioidosis in Australia and Papua New Guinea  

Microsoft Academic Search

Melioidosis was first described in Australia in an outbreak in sheep in 1949 in north Queensland (22°S). Human melioidosis was first described from Townsville (19°S) in 1950. Melioidosis is hyperendemic in the Top End of the Northern Territory (NT) and as in parts of northeastern Thailand it is the commonest cause of fatal community-acquired septicemic pneumonia. In the 9 years

Bart J Currie; Dale A Fisher; Diane M Howard; James N. C Burrow; Sudarshan Selvanayagam; Paul L Snelling; Nicholas M Anstey; Mark J Mayo

2000-01-01

206

DNA-Based Diagnostic Approaches for Identification of Burkholderia cepacia Complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia Genomovars I and III  

Microsoft Academic Search

Bacteria of the Burkholderia cepacia complex consist of five discrete genomic species, including genomovars I and III and three new species: Burkholderia multivorans (formerly genomovar II), Burkholderia stabilis (formerly genomovar IV), and Burkholderia vietnamiensis (formerly genomovar V). Strains of all five genomo- vars are capable of causing opportunistic human infection, and microbiological identification of these closely related species is difficult.

ESHWAR MAHENTHIRALINGAM; JOCELYN BISCHOF; SEAN K. BYRNE; CHRISTOPHER RADOMSKI; JULIAN E. DAVIES; YOSSEF AV-GAY

207

The effect of glibenclamide on the pathogenesis of melioidosis  

E-print Network

........................................................................ 2!1.2! Epidemiology ........................................................................................... 12!1.3! Clinical features of melioidosis ............................................................... 20!1.4! Diagnosis... .................................................................................... 70!2.1! Introduction ............................................................................................ 70!2.2! Methods ................................................................................................... 82!2.3! Results...

Koh, Gavin Christian Kia Wee

2012-03-06

208

Management of melioidosis osteomyelitis and septic arthritis.  

PubMed

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. PMID:25628295

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

2015-02-01

209

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

PubMed Central

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

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

2013-01-01

210

Global Analysis of the Burkholderia thailandensis Quorum Sensing-Controlled Regulon  

PubMed Central

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

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

2014-01-01

211

Crystal Structures of Cif from Bacterial Pathogens Photorhabdus luminescens and Burkholderia pseudomallei  

Microsoft Academic Search

A pre-requisite for bacterial pathogenesis is the successful interaction of a pathogen with a host. One mechanism used by a broad range of Gram negative bacterial pathogens is to deliver effector proteins directly into host cells through a dedicated type III secretion system where they modulate host cell function. The cycle inhibiting factor (Cif) family of effector proteins, identified in

Allister Crow; Paul R. Race; Grégory Jubelin; Carolina Varela Chavez; Jean-Michel Escoubas; Eric Oswald; Mark J. Banfield; Bostjan Kobe

2009-01-01

212

Detection of Pseudomonas pseudomallei by PCR and hybridization.  

PubMed Central

A molecular method for the detection of Pseudomonas pseudomallei was developed on the basis of the differences in the 23S rRNA sequences of related species of the genus Pseudomonas. An 18-base oligonucleotide probe, designed following partial sequencing of 23s ribosomal DNA (rDNA), was used for the identification and detection of P. pseudomallei either by hybridization or by direct PCR. Optimal detection was obtained by hybridization of the probe with PCR-amplified rDNA rather than with total genomic DNA or colony blots. One nanogram of template DNA amplified in a PCR mixture containing 14% glycerol could be detected in slot blots hybridized with the digoxigenin-labelled probe and the lumigen PPD detection system. Amplified rDNA sequences from 41 P. pseudomallei strains of various origins hybridized with the probe. The probe also hybridized with three Pseudomonas mallei reference strains under conditions of high stringency but failed to hybridize with amplified rDNA sequences from other closely related Pseudomonas spp. PCR with a conserved primer and the 18-base oligonucleotide probe (direct PCR) specifically amplified P. pseudomallei and P. mallei. By using these methods, approximately 10(4) P. pseudomallei cells per ml could be detected in artificially inoculated blood samples and in blood dried on filter paper following Chelex extraction. The detection limit in blood was increased to 10(2) cells per ml by concentration of bacteria from 0.5 ml of blood or by a 24-h blood culture enrichment prior to PCR. Approximately 10(3) cells per ml were detected in seeded sputum samples. The detection times by direct PCR and indirect PCR and then probe hybridization were approximately 5 h and 24 h, respectively. These results indicate that amplification of conserved rDNA sequences by PCR directly or by hybridization with a probe to PCR fragments offers promise for the detection of P. pseudomallei and P. mallei. Images PMID:7519629

Lew, A E; Desmarchelier, P M

1994-01-01

213

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 ...

214

Whitmore's disease: an uncommon urological presentation.  

PubMed

The incidence of prostatic abscesses has much decreased in the antibiotic era. We present an uncommon cause of prostatic abscess secondary to melioidosis, also known as Whitmore's disease or pseudoglanders. The disease is endemic in South East Asia and Australia. Although India is considered endemic for Burkholderia pseudomallei, the causative organism of melioidosis, not many cases have been reported. Most of the reported cases from India are from the South-West coastal regions of Kerala and Karnataka, Vellore, West Bengal and Bihar. Our index patient was successfully treated with parenteral antibiotics and endoscopic deroofing of the abscess. PMID:24518392

Naganathan, Karthickeyan; Pillai, Sunil Bhaskara; Kumar, Praveen; Hegde, Padmaraj

2014-01-01

215

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

Federal Register 2010, 2011, 2012, 2013, 2014

...For example, a 20-year prospective study of melioidosis in northern Australia...shock (Ref. 1). A 9-year prospective study of melioidosis in northeast...Cases From the 20 Year Darwin Prospective Study,'' Public Library of...

2012-11-07

216

DNA-Based Diagnostic Approaches for Identification of Burkholderia cepacia Complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia Genomovars I and III  

PubMed Central

Bacteria of the Burkholderia cepacia complex consist of five discrete genomic species, including genomovars I and III and three new species: Burkholderia multivorans (formerly genomovar II), Burkholderia stabilis (formerly genomovar IV), and Burkholderia vietnamiensis (formerly genomovar V). Strains of all five genomovars are capable of causing opportunistic human infection, and microbiological identification of these closely related species is difficult. The 16S rRNA gene (16S rDNA) and recA gene of these bacteria were examined in order to develop rapid tests for genomovar identification. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA revealed sequence polymorphisms capable of identifying B. multivorans and B. vietnamiensis but insufficient to discriminate strains of B. cepacia genomovars I and III and B. stabilis. RFLP analysis of PCR-amplified recA demonstrated sufficient nucleotide sequence variation to enable separation of strains of all five B. cepacia complex genomovars. Complete recA nucleotide sequences were obtained for 20 strains representative of the diversity of the B. cepacia complex. Construction of a recA phylogenetic tree identified six distinct clusters (recA groups): B. multivorans, B. vietnamiensis, B. stabilis, genomovar I, and the subdivision of genomovar III isolates into two recA groups, III-A and III-B. Alignment of recA sequences enabled the design of PCR primers for the specific detection of each of the six latter recA groups. The recA gene was found on the largest chromosome within the genome of B. cepacia complex strains and, in contrast to the findings of a previous study, only a single copy of the gene was present. In conclusion, analysis of the recA gene of the B. cepacia complex provides a rapid and robust nucleotide sequence-based approach to identify and classify this taxonomically complex group of opportunistic pathogens. PMID:10970351

Mahenthiralingam, Eshwar; Bischof, Jocelyn; Byrne, Sean K.; Radomski, Christopher; Davies, Julian E.; Av-Gay, Yossef; Vandamme, Peter

2000-01-01

217

Evaluation of Recombinant Proteins of Burkholderia mallei for Serodiagnosis of Glanders  

PubMed Central

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. PMID:22695165

Kumar, Subodh; Malik, Praveen

2012-01-01

218

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

PubMed Central

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

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

219

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

PubMed Central

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 ?-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

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

220

Intravenous Therapy Duration and Outcomes in Melioidosis: A New Treatment Paradigm  

PubMed Central

Background International melioidosis treatment guidelines recommend a minimum 10 to 14 days’ intravenous antibiotic therapy (intensive phase), followed by 3 to 6 months’ oral therapy (eradication phase). This approach is associated with rates of relapse, defined as recurrence following the eradication phase, that can exceed 5%. Rates of recrudescence, defined as recurrence during the eradication phase, have not previously been reported. In response to low eradication phase completion rates in Australia, a local guideline has evolved over the last ten years recommending a longer minimum intensive phase duration for many cases of melioidosis. Methodology/ Principal Findings This retrospective cohort study reviews antibiotic duration for the first episode of care for all patients diagnosed with melioidosis and surviving the intensive phase during a recent three year period in the tropical north of Australia’s Northern Territory; we also review adherence to the current local guideline and treatment outcomes. Of 215 first episodes of melioidosis surviving the intensive phase, the median (interquartile range) intensive phase duration was 26 (14-34) days. One hundred and eight (50.2%) patients completed eradication therapy; 58 (27.0%) patients took no eradication therapy. At 28 months’ follow-up, one (0.5%) relapse and eleven (5.1%) recrudescences had occurred. On exact logistic regression analysis, the only independent risk factors for recrudescence were self-discharge during the intensive phase (odds ratio 6.2 [95% confidence interval 1.2-30.0]) and septic shock (odds ratio 5.3 [95% confidence interval 1.1-25.7]). Conclusions/ Significance Relapsed melioidosis is rare in patients who receive a minimum intensive phase duration specified by our guideline and extended according to clinical progress. Recrudescence rates may improve with reductions in rates of self-discharge. Given the low relapse rate despite a high rate of eradication therapy non-adherence, the duration and necessity of eradication therapy for different patients after guideline-concordant intensive therapy should be evaluated further. PMID:25811783

Pitman, Matthew C.; Luck, Tara; Marshall, Catherine S.; Anstey, Nicholas M.; Ward, Linda; Currie, Bart J.

2015-01-01

221

Structural characterization of Burkholderia pseudomallei adenylate kinase (Adk): Profound asymmetry in the crystal structure of the ‘open’ state  

Microsoft Academic Search

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 2.1Å resolution crystal structure for

Garry W. Buchko; Howard Robinson; Jan Abendroth; Bart L. Staker; Peter J. Myler

2010-01-01

222

Occurrence of Multiple Genomovars of Burkholderia cepacia in Cystic Fibrosis Patients and Proposal of Burkholderia multivorans sp. nov  

Microsoft Academic Search

We performed an integrated genotypic and phenotypic analysis of 128 strains of the genera Burkholderia, Rabtonia, and Pseudomonas in order to study the taxonomic structure of Burkholderia cepacia and its relation- ships with other Burkholderia species. Our data show that presumed B. cepacia strains isolated from cystic fibrosis patients belong to at least five distinct genomic species, one of which

P. VANDAMME; B. HOLMES; M. VANCANNEYT; T. COENYE; B. HOSTE; R. COOPMAN; H. REVETS; S. LAUWERS; M. GILLIS; K. KERSTERS; J. R. W. GOVAN

223

Host Evasion by Burkholderia cenocepacia  

PubMed Central

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

Ganesan, Shyamala; Sajjan, Umadevi S.

2012-01-01

224

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

PubMed

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. PMID:25472981

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

2015-02-01

225

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...

226

Splenic Abscesses in a Returning Traveler  

PubMed Central

Burkholderia, an aerobic gram-negative rod, is the causative organism behind melioidosis and is a common soil and water organism found predominantly in South-East Asia. We report the case of a 68 year-old man returning from an extended trip to the Philippines, with splenic hypodense lesions on abdominal computer tomography scan, later confirmed to be culture-positive for Burkholderia pseudomallei. The patient was treated with a course of intravenous ceftazidime followed by eradication therapy with oral doxycycline and trimethoprim-sulfamethoxazole. He recovered with complete resolution of symptoms at follow up. In a returning traveler from an endemic area, melioidosis should be considered as part of the differential for any febrile illness with abscesses.

Guo, Richard F.; Wong, Frances L.; Perez, Mario L.

2015-01-01

227

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

NASA Astrophysics Data System (ADS)

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)-1? 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-1? production by PMNs.

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

2013-11-01

228

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

PubMed

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?:?1?7c, summed feature 3 (C16?:?1?7c and/or C16?:?1?6c) 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)). PMID:25563916

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

2015-03-01

229

Role of phages in the pathogenesis of Burkholderia or “Where are the toxin genes in Burkholderia phages?”  

PubMed Central

Summary Most bacteria of the genus Burkholderia are soil- and rhizosphere- associated, noted for their metabolic plasticity in the utilization of a wide range of organic compounds as carbon sources. Many Burkholderia species are also opportunistic human and plant pathogens and the distinction between environmental, plant, and human pathogens is not always clear. Burkholderia phages are not uncommon and multiple cryptic prophages are identifiable in the sequenced Burkholderia genomes. Phages have played a crucial role in the transmission of virulence factors among many important pathogens, however, the data does not yet support a significant correlation between phages and pathogenicity in the Burkholderia. This may be due to the role of Burkholderia as a “versaphile” such that selection is occurring in several niches, including roles as a pathogen and in the context of environmental survival. PMID:17719265

Summer, Elizabeth J.; Gill, Jason J.; Upton, Chris; Gonzalez, Carlos F.; Young, Ry

2007-01-01

230

Comparative Metabolic Systems Analysis of Pathogenic Burkholderia  

PubMed Central

Burkholderia cenocepacia and Burkholderia multivorans are opportunistic drug-resistant pathogens that account for the majority of Burkholderia cepacia complex infections in cystic fibrosis patients and also infect other immunocompromised individuals. While they share similar genetic compositions, B. cenocepacia and B. multivorans exhibit important differences in pathogenesis. We have developed reconciled genome-scale metabolic network reconstructions of B. cenocepacia J2315 and B. multivorans ATCC 17616 in parallel (designated iPY1537 and iJB1411, respectively) to compare metabolic abilities and contextualize genetic differences between species. The reconstructions capture the metabolic functions of the two species and give insight into similarities and differences in their virulence and growth capabilities. The two reconstructions have 1,437 reactions in common, and iPY1537 and iJB1411 have 67 and 36 metabolic reactions unique to each, respectively. After curating the extensive reservoir of metabolic genes in Burkholderia, we identified 6 genes essential to growth that are unique to iPY1513 and 13 genes uniquely essential to iJB1411. The reconstructions were refined and validated by comparing in silico growth predictions to in vitro growth capabilities of B. cenocepacia J2315, B. cenocepacia K56-2, and B. multivorans ATCC 17616 on 104 carbon sources. Overall, we identified functional pathways that indicate B. cenocepacia can produce a wider array of virulence factors compared to B. multivorans, which supports the clinical observation that B. cenocepacia is more virulent than B. multivorans. The reconciled reconstructions provide a framework for generating and testing hypotheses on the metabolic and virulence capabilities of these two related emerging pathogens. PMID:24163337

Bartell, Jennifer A.; Yen, Phillip; Varga, John J.; Goldberg, Joanna B.

2014-01-01

231

An ancient but promiscuous host–symbiont association between Burkholderia gut symbionts and their heteropteran hosts  

Microsoft Academic Search

Here, we investigated 124 stinkbug species representing 20 families and 5 superfamilies for their Burkholderia gut symbionts, of which 39 species representing 6 families of the superfamilies Lygaeoidea and Coreoidea were Burkholderia-positive. Diagnostic PCR surveys revealed high frequencies of Burkholderia infection in natural populations of the stinkbugs, and substantial absence of vertical transmission of Burkholderia infection to their eggs. In

Yoshitomo Kikuchi; Takahiro Hosokawa; Takema Fukatsu

2011-01-01

232

VgrG-5 Is a Burkholderia Type VI Secretion System-Exported Protein Required for Multinucleated Giant Cell Formation and Virulence  

PubMed Central

The type VI secretion system (T6SS) has emerged as a critical virulence factor for the group of closely related Burkholderia spp. that includes Burkholderia pseudomallei, B. mallei, and B. thailandensis. While the genomes of these bacteria, referred to as the Bptm group, appear to encode several T6SSs, we and others have shown that one of these, type VI secretion system 5 (T6SS-5), is required for virulence in mammalian infection models. Despite its pivotal role in the pathogenesis of the Bptm group, the effector repertoire of T6SS-5 has remained elusive. Here we used quantitative mass spectrometry to compare the secretome of wild-type B. thailandensis to that of a mutant harboring a nonfunctional T6SS-5. This analysis identified VgrG-5 as a novel secreted protein whose export depends on T6SS-5 function. Bioinformatics analysis revealed that VgrG-5 is a specialized VgrG protein that harbors a C-terminal domain (CTD) conserved among Bptm group species. We found that a vgrG-5 ?CTD mutant is avirulent in mice and is unable to stimulate the fusion of host cells, a hallmark of the Bptm group previously shown to require T6SS-5 function. The singularity of VgrG-5 as a detected T6SS-5 substrate, taken together with the essentiality of its CTD for virulence, suggests that the protein is critical for the effector activity of T6SS-5. Intriguingly, we show that unlike the bacterial-cell-targeting T6SSs characterized so far, T6SS-5 localizes to the bacterial cell pole. We propose a model whereby the CTD of VgrG-5—, propelled by T6SS-5—, plays a key role in inducing membrane fusion, either by the recruitment of other factors or by direct participation. PMID:24452686

Singh, Pragya; Robertson, Johanna D.; LeRoux, Michele; Skerrett, Shawn J.; Goodlett, David R.; West, T. Eoin; Mougous, Joseph D.

2014-01-01

233

VgrG-5 is a Burkholderia type VI secretion system-exported protein required for multinucleated giant cell formation and virulence.  

PubMed

The type VI secretion system (T6SS) has emerged as a critical virulence factor for the group of closely related Burkholderia spp. that includes Burkholderia pseudomallei, B. mallei, and B. thailandensis. While the genomes of these bacteria, referred to as the Bptm group, appear to encode several T6SSs, we and others have shown that one of these, type VI secretion system 5 (T6SS-5), is required for virulence in mammalian infection models. Despite its pivotal role in the pathogenesis of the Bptm group, the effector repertoire of T6SS-5 has remained elusive. Here we used quantitative mass spectrometry to compare the secretome of wild-type B. thailandensis to that of a mutant harboring a nonfunctional T6SS-5. This analysis identified VgrG-5 as a novel secreted protein whose export depends on T6SS-5 function. Bioinformatics analysis revealed that VgrG-5 is a specialized VgrG protein that harbors a C-terminal domain (CTD) conserved among Bptm group species. We found that a vgrG-5 ?CTD mutant is avirulent in mice and is unable to stimulate the fusion of host cells, a hallmark of the Bptm group previously shown to require T6SS-5 function. The singularity of VgrG-5 as a detected T6SS-5 substrate, taken together with the essentiality of its CTD for virulence, suggests that the protein is critical for the effector activity of T6SS-5. Intriguingly, we show that unlike the bacterial-cell-targeting T6SSs characterized so far, T6SS-5 localizes to the bacterial cell pole. We propose a model whereby the CTD of VgrG-5-, propelled by T6SS-5-, plays a key role in inducing membrane fusion, either by the recruitment of other factors or by direct participation. PMID:24452686

Schwarz, Sandra; Singh, Pragya; Robertson, Johanna D; LeRoux, Michele; Skerrett, Shawn J; Goodlett, David R; West, T Eoin; Mougous, Joseph D

2014-04-01

234

9 CFR 121.4 - Overlap select agents and toxins.  

Code of Federal Regulations, 2013 CFR

...HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS...Burkholderia mallei; *Burkholderia pseudomallei; Hendra virus; Nipah virus; Rift Valley fever virus; Venezuelan equine...

2013-01-01

235

9 CFR 121.4 - Overlap select agents and toxins.  

Code of Federal Regulations, 2014 CFR

...HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS...Burkholderia mallei; *Burkholderia pseudomallei; Hendra virus; Nipah virus; Rift Valley fever virus; Venezuelan equine...

2014-01-01

236

9 CFR 121.4 - Overlap select agents and toxins.  

Code of Federal Regulations, 2012 CFR

...HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS...Burkholderia mallei; Burkholderia pseudomallei; Hendra virus; Nipah virus; Rift Valley fever virus; Venezuelan...

2012-01-01

237

42 CFR 73.9 - Responsible Official.  

Code of Federal Regulations, 2013 CFR

...neurotoxin producing species of Clostridium, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Ebola viruses, Marburg virus, Variola major virus (Smallpox virus), Variola minor (Alastrim), or Yersinia pestis....

2013-10-01

238

42 CFR 73.9 - Responsible Official.  

Code of Federal Regulations, 2014 CFR

...neurotoxin producing species of Clostridium, Burkholderia mallei, Burkholderia pseudomallei Francisella tularensis, Ebola viruses, , Marburg virus, Variola major virus (Smallpox virus), Variola minor (Alastrim), or Yersinia pestis....

2014-10-01

239

42 CFR 73.4 - Overlap select agents and toxins.  

Code of Federal Regulations, 2014 CFR

...Brucella suis; Burkholderia mallei *; Burkholderia pseudomallei *; Hendra virus; Nipah virus; Rift Valley fever virus; Venezuelan equine encephalitis virus (c) Genetic Elements, Recombinant and/or Synthetic Nucleic Acids,...

2014-10-01

240

Clonally related Burkholderia contaminans among ventilated patients without cystic fibrosis.  

PubMed

We investigated a cluster of 10 Burkholderia cepacia complex-positive cultures among ventilated patients and those with a tracheostomy in an acute care hospital. Isolates from 5 patients had outbreak-strain-related Burkholderia contaminans. Isolates of B. cepacia complex unrelated to the outbreak strain were cultured from a sink drain. The investigation identified practices that might have led to contamination of patient respiratory care supplies with tap water, which might have contributed to the cluster. PMID:23973426

Peterson, Amy E; Chitnis, Amit S; Xiang, Nan; Scaletta, Joseph M; Geist, Robert; Schwartz, Jennifer; Dement, Jamie; Lawlor, Elizabeth; Lipuma, John J; O'Connell, Heather; Noble-Wang, Judith; Kallen, Alexander J; Hunt, D Charles

2013-12-01

241

Burkholderia Species Are Major Inhabitants of White Lupin Cluster Roots?†  

PubMed Central

The formation of cluster roots by plants represents a highly efficient strategy for acquisition of sparingly available phosphate. This particular root type is characterized by a densely branched structure and high exudation of organic acids and protons, which are likely to influence the resident bacterial community. Until now, the identity of the bacterial populations living in cluster roots has not been investigated. We applied cultivation-dependent and cultivation-independent methods to characterize the dominant bacterial genera inhabiting the growing cluster roots of white lupin. We observed a high relative abundance of Burkholderia species (up to 58% of all isolated strains and 44% of all retrieved 16S rRNA sequences) and a significant enrichment with increasing cluster root age. Most of the sequences retrieved clustered together with known plant- or fungus-associated Burkholderia species, while only one of 98 sequences was affiliated with the Burkholderia cepacia complex. In vitro assays revealed that Burkholderia strains were much more tolerant to low pH than non-Burkholderia strains. Moreover, many strains produced large amounts of siderophores and were able to utilize citrate and oxalate as carbon sources. These features seem to represent important traits for the successful colonization and maintenance of Burkholderia species in white lupin cluster roots. PMID:21908626

Weisskopf, Laure; Heller, Stefanie; Eberl, Leo

2011-01-01

242

[Electron microscopic study of Burkholderia cepacia biofilms].  

PubMed

Using the methods of transmission electron microscopy, the structure of the biofilms formed by the bacterium Burkholderia cepacia (clinical isolate and mutants with an increased and decreased ability to produce biofilms) were investigated. The biofilms were obtained on a liquid nutrient medium or on an abiotic surface (polystyrene). It has been demonstrated that the cultures of the studied strains differ in some morphological and functional characteristics. In biofilms, changes in the size and submicroscopic organization of all the components of bacterial cells occur. Staining biofilms with ruthenium red revealed the presence of exopolysaccharides in the intercellular space. The differences in the ultrastructure of bacterial films formed on nutrient medium and abiotic surfaces were demonstrated. PMID:18365723

Smirnova, T A; Didenko, L V; Andreev, A L; Aklekseeva, N V; Stepanova, T V; Romanova, Iu M

2008-01-01

243

A physical genome map of the Burkholderia cepacia type strain.  

PubMed

Burkholderia cepacia (basonym Pseudomonas cepacia), the type species of the new genus Burkholderia, is of interest, not only because of its broad catabolic capacity and its ability to antagonize soil-borne plant pathogens, but also because of its causative role in infections in man, which are particularly evident in patients with cystic fibrosis. A physical map of the 8.1 Mb genome of the B. cepacia type-strain ATCC 25416 was constructed by applying two-dimensional pulsed-field gel electrophoresis techniques. Placed onto the macrorestriction map were 38 SpeI, 11 SwaI, 11 PacI, 11 PmeI and six I-CeuI sites, resulting in an average resolution of 105 kbp. Random single-hit linearization by irradiation and restriction mapping uncovered the presence of four circular replicons of 3.65 Mb, 3.17 Mb, 1.07 Mb and 200 kbp in size. The largest replicon harbours four rrn operons while the other two Megabase-size replicons each contain a single rrn operon, suggesting that the genome has three chromosomes and a large plasmid. Within the beta subdivision of proteobacteria, the existence of multiple replicons is not confined to B. cepacia. The phylogenetically related species Burkholderia glumae, Burkholderia pickettii, Burkholderia solanacearum, Alcaligenes eutrophus and the so far unassigned Pseudomonas glathei were also found to harbour more than one Megabase-size replicon. PMID:7476209

Rodley, P D; Römling, U; Tümmler, B

1995-07-01

244

Effect of colony morphology variation of Burkholderia pseudomallei on intracellular survival and resistance to antimicrobial environments in human macrophages in vitro  

E-print Network

of bacterial survival during the estab- lishment of persistent infection in the host is adaptation to hypoxia in the host microenvironment [14]. This study demonstrated that all 3 isogenic morphotypes were able to tolerate a low oxygen concentration... ml of TSB and incubated at 37°C in air with shaking at 200 rpm for 28 h. At 2 h intervals, 100 ?l of bacterial culture was removed, serially diluted 10-fold in PBS, and the bacter- ial count determined by plating on Ashdown agar in duplicate...

Tandhavanant, Sarunporn; Thanwisai, Aunchalee; Limmathurotsakul, Direk; Korbsrisate, Sunee; Day, Nicholas P J; Peacock, Sharon J; Chantratita, Narisara

2010-11-30

245

Genomic acquisition of a capsular polysaccharide virulence cluster by non-pathogenic Burkholderia isolates  

E-print Network

genomic islands, while 26 regions were novel. Variant B. thailandensis isolates exhibited isolated acquisition of a capsular polysaccharide biosynthesis gene cluster (B. pseudomallei-like capsular polysaccharide) closely resembling a similar cluster in B...

Sim, Bernice Meng Qi; Chantratita, Narisara; Ooi, Wen Fong; Nandi, Tannistha; Tewhey, Ryan; Wuthiekanun, Vanaporn; Thaipadungpanit, Janjira; Tumapa, Sarinna; Ariyaratne, Pramila; Sung, Wing-Kin; Sem, Xiao Hui; Chua, Hui Hoon; Ramnarayanan, Kalpana; Lin, Chi Ho; Liu, Yichun; Feil, Edward J; Glass, Mindy B; Tan, Gladys; Peacock, Sharon J; Tan, Patrick

2010-08-27

246

The genus burkholderia: analysis of 56 genomic sequences.  

PubMed

The genus Burkholderia consists of a number of very diverse species, both in terms of lifestyle (which varies from category B pathogens to apathogenic soil bacteria and plant colonizers) and their genetic contents. We have used 56 publicly available genomes to explore the genomic diversity within this genus, including genome sequences that are not completely finished, but are available from the NCBI database. Defining the pan- and core genomes of species results in insights in the conserved and variable fraction of genomes, and can verify (or question) historic, taxonomic groupings. We find only several hundred genes that are conserved across all Burkholderia genomes, whilst there are more than 40,000 gene families in the Burkholderia pan-genome. A BLAST matrix visualizes the fraction of conserved genes in pairwise comparisons. A BLAST atlas shows which genes are actually conserved in a number of genomes, located and visualized with reference to a chosen genome. Genomic islands are common in many Burkholderia genomes, and most of these can be readily visualized by DNA structural properties of the chromosome. Trees that are based on relatedness of gene family content yield different results depending on what genes are analyzed. Some of the differences can be explained by errors in incomplete genome sequences, but, as our data illustrate, the outcome of phylogenetic trees depends on the type of genes that are analyzed. PMID:19696499

Ussery, D W; Kiil, K; Lagesen, K; Sicheritz-Pontén, T; Bohlin, J; Wassenaar, T M

2009-01-01

247

Burkholderia bryophila sp. nov. and Burkholderia megapolitana sp. nov., moss-associated species with antifungal and plant-growth-promoting properties.  

PubMed

A polyphasic taxonomic study including DNA-DNA reassociation experiments and an extensive biochemical characterization was performed on 14 Burkholderia isolates from moss gametophytes of nutrient-poor plant communities on the southern Baltic Sea coast in northern Germany. The strains were classified within two novel species, for which the names Burkholderia bryophila sp. nov. and Burkholderia megapolitana sp. nov. are proposed. The former species also includes isolates from grassland and agricultural soil collected in previous studies. Strains Burkholderia bryophila 1S18(T) (=LMG 23644(T) =CCUG 52993(T)) and Burkholderia megapolitana A3(T) (=LMG 23650(T) =CCUG 53006(T)) are the proposed type strains. They were isolated from Sphagnum rubellum and Aulacomnium palustre, respectively, growing in the 'Ribnitzer Grosses Moor' nature reserve (Mecklenburg-Pommern, Germany). All moss isolates of both novel species showed antifungal activity against phytopathogens as well as plant-growth-promoting properties. PMID:17911288

Vandamme, Peter; Opelt, Katja; Knöchel, Nadine; Berg, Christian; Schönmann, Susan; De Brandt, Evie; Eberl, Leo; Falsen, Enevold; Berg, Gabriele

2007-10-01

248

Removal of Burkholderia cepacia biofilms with oxidants  

NASA Technical Reports Server (NTRS)

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. cepacia associated with both primary and secondary biofilms. The amount of biocide needed to inactivate 50% of planktonic B. cepacia in 10 min at 25 degrees C was 8.4, 0.5, and 0.2 mg l-1 for iodine, chlorine, and ozone, respectively. The data suggest that iodine maynot be the best chemical for treating of biofilms when removal of cellular material is required.

Koenig, D. W.; Mishra, S. K.; Pierson, D. L.

1995-01-01

249

An ancient but promiscuous host-symbiont association between Burkholderia gut symbionts and their heteropteran hosts.  

PubMed

Here, we investigated 124 stinkbug species representing 20 families and 5 superfamilies for their Burkholderia gut symbionts, of which 39 species representing 6 families of the superfamilies Lygaeoidea and Coreoidea were Burkholderia-positive. Diagnostic PCR surveys revealed high frequencies of Burkholderia infection in natural populations of the stinkbugs, and substantial absence of vertical transmission of Burkholderia infection to their eggs. In situ hybridization confirmed localization of the Burkholderia in their midgut crypts. In the lygaeoid and coreoid stinkbugs, development of midgut crypts in their alimentary tract was coincident with the Burkholderia infection, suggesting that the specialized morphological configuration is pivotal for establishment and maintenance of the symbiotic association. The Burkholderia symbionts were easily isolated as pure culture on standard microbiological media, indicating the ability of the gut symbionts to survive outside the host insects. Molecular phylogenetic analysis showed that the gut symbionts of the lygaeoid and coreoid stinkbugs belong to a ?-proteobacterial clade together with Burkholderia isolates from soil environments and Burkholderia species that induce plant galls. On the phylogeny, the stinkbug-associated, environmental and gall-forming Burkholderia strains did not form coherent groups, indicating host-symbiont promiscuity among these stinkbugs. Symbiont culturing revealed that slightly different Burkholderia genotypes often coexist in the same insects, which is also suggestive of host-symbiont promiscuity. All these results strongly suggest an ancient but promiscuous host-symbiont relationship between the lygaeoid/coreoid stinkbugs and the Burkholderia gut symbionts. Possible mechanisms as to how the environmentally transmitted promiscuous symbiotic association has been stably maintained in the evolutionary course are discussed. PMID:20882057

Kikuchi, Yoshitomo; Hosokawa, Takahiro; Fukatsu, Takema

2011-03-01

250

An ancient but promiscuous host–symbiont association between Burkholderia gut symbionts and their heteropteran hosts  

PubMed Central

Here, we investigated 124 stinkbug species representing 20 families and 5 superfamilies for their Burkholderia gut symbionts, of which 39 species representing 6 families of the superfamilies Lygaeoidea and Coreoidea were Burkholderia-positive. Diagnostic PCR surveys revealed high frequencies of Burkholderia infection in natural populations of the stinkbugs, and substantial absence of vertical transmission of Burkholderia infection to their eggs. In situ hybridization confirmed localization of the Burkholderia in their midgut crypts. In the lygaeoid and coreoid stinkbugs, development of midgut crypts in their alimentary tract was coincident with the Burkholderia infection, suggesting that the specialized morphological configuration is pivotal for establishment and maintenance of the symbiotic association. The Burkholderia symbionts were easily isolated as pure culture on standard microbiological media, indicating the ability of the gut symbionts to survive outside the host insects. Molecular phylogenetic analysis showed that the gut symbionts of the lygaeoid and coreoid stinkbugs belong to a ?-proteobacterial clade together with Burkholderia isolates from soil environments and Burkholderia species that induce plant galls. On the phylogeny, the stinkbug-associated, environmental and gall-forming Burkholderia strains did not form coherent groups, indicating host–symbiont promiscuity among these stinkbugs. Symbiont culturing revealed that slightly different Burkholderia genotypes often coexist in the same insects, which is also suggestive of host–symbiont promiscuity. All these results strongly suggest an ancient but promiscuous host–symbiont relationship between the lygaeoid/coreoid stinkbugs and the Burkholderia gut symbionts. Possible mechanisms as to how the environmentally transmitted promiscuous symbiotic association has been stably maintained in the evolutionary course are discussed. PMID:20882057

Kikuchi, Yoshitomo; Hosokawa, Takahiro; Fukatsu, Takema

2011-01-01

251

Molecular epidemiology of Burkholderia cepacia, Stenotrophomonas maltophilia , and Alcaligenes xylosoxidans in a cystic fibrosis center  

Microsoft Academic Search

Burkholderia cepacia, Stenotrophomonas maltophilia, andAlcaligenes xylosoxidans have been isolated with increasing frequency from the sputum of patients with cystic fibrosis in a pediatric hospital. In 1994–95, 27 of 120 patients were persistently colonized, 17 withBurkholderia cepacia, eight withAlcaligenes xylosoxidans, and five withStenotrophomonas maltophilia. Genotyping of 220 clinical isolates revealed that most of theBurkholderia cepacia strains were clonally related, suggesting either

H. Vu-Thien; D. Moissenet; M. Valcin; C. Dulot; G. Tournier; A. Garbarg-Chenon

1996-01-01

252

Iron acquisition mechanisms of the Burkholderia cepacia complex  

Microsoft Academic Search

The Burkholderia cepacia complex (Bcc) is comprised of at least 10 closely related species of Gram-negative proteobacteria that are associated with\\u000a infections in certain groups of immunocompromised individuals, particularly those with cystic fibrosis. Infections in humans\\u000a tend to occur in the lungs, which present an iron-restricted environment to a prospective pathogen, and accordingly members\\u000a of the Bcc appear to possess

Mark S. Thomas

2007-01-01

253

Nodulation of Cyclopia spp. (Leguminosae, Papilionoideae) by Burkholderia tuberum  

PubMed Central

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 ?-rhizobial strain can effectively nodulate papilioinoid legumes. Conclusions Papilionoid legumes from widely different tribes can be nodulated by ?-rhizobia, forming both indeterminate (Cyclopia) and determinate (Macroptilium) nodules. PMID:17881339

Elliott, Geoffrey N.; Chen, Wen-Ming; Bontemps, Cyril; Chou, Jui-Hsing; Young, J. Peter W.; Sprent, Janet I.; James, Euan K.

2007-01-01

254

Identification and enzymatic characterization of acid phosphatase from Burkholderia gladioli  

PubMed Central

Background The genus Burkholderia is widespread in diverse ecological niches, the majority of known species are soil bacteria that exhibit different types of non-pathogenic interactions with plants. Burkholderia species are versatile organisms that solubilize insoluble minerals through the production of organic acids, which increase the availability of nutrients for the plant. Therefore these bacteria are promising candidates for biotechnological applications. Results Burkholderia sp. (R 3.25 isolate) was isolated from agricultural soil in Ponta Grossa-PR-Brazil and identified through analysis of the 16S rDNA as a strain classified as Burkholderia gladioli. The expression of membrane-bound acid phosphatase (MBAcP) was strictly regulated with optimal expression at a concentration of phosphorus 5 mM. The apparent optimum pH for the hydrolysis of p-nitrophenylphosphate (PNPP) was 6.0. The hydrolysis of PNPP by the enzyme exhibited a hyperbolic relationship with increasing concentration of substrate and no inhibition by excess of substrate was observed. Kinetic data revealed that the hydrolysis of PNPP exhibited cooperative kinetics with n?=?1.3, Vm?=?113.5 U/mg and K0.5?=?65 ?M. The PNPPase activity was inhibited by vanadate, p-hydroxymercuribenzoate, arsenate and phosphate, however the activity was not inhibited by calcium, levamisole, sodium tartrate, EDTA, zinc, magnesium, cobalt, ouabain, oligomycin or pantoprazol. Conclusion The synthesis of membrane-bound non-specific acid phosphatase, strictly regulated by phosphate, and its properties suggest that this bacterium has a potential biotechnological application to solubilize phosphate in soils with low levels of this element, for specific crops. PMID:24713147

2014-01-01

255

Degradation of parabens by Pseudomonas beteli and Burkholderia latens  

Microsoft Academic Search

p-Hydroxybenzoic acid esters (parabens) are commonly used antimicrobial preservatives in pharmaceutical formulations. Two microorganisms, isolated from non-sterile methyl paraben (MP) and propyl paraben (PP) solutions, were found to degrade the respective parabens. Identification by 16S rRNA partial gene sequencing revealed them to be Pseudomonas beteli and Burkholderia latens, respectively. The present work describes a previously unreported interaction of the parabens

Aeshna Amin; Sateesh Chauhan; Manish Dare; Arvind Kumar Bansal

2010-01-01

256

Deciphering the Role of RND Efflux Transporters in Burkholderia cenocepacia  

Microsoft Academic Search

Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF) pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division) efflux pumps are known to be among the mediators of multidrug resistance in Gram-negative bacteria. Since the

Silvia Bazzini; Claudia Udine; Andrea Sass; Maria Rosalia Pasca; Francesca Longo; Giovanni Emiliani; Marco Fondi; Elena Perrin; Francesca Decorosi; Carlo Viti; Luciana Giovannetti; Livia Leoni; Renato Fani; Giovanna Riccardi; Eshwar Mahenthiralingam; Silvia Buroni; Mark Alexander Webber

2011-01-01

257

Draft Genome Sequence of the Lignin-Degrading Burkholderia sp. Strain LIG30, Isolated from Wet Tropical Forest Soil  

PubMed Central

Burkholderia species are common soil Betaproteobacteria capable of degrading recalcitrant aromatic compounds and xenobiotics. Burkholderia sp. strain LIG30 was isolated from wet tropical forest soil and is capable of utilizing lignin as a sole carbon source. Here we report the draft genome sequence of Burkholderia sp. strain LIG30. PMID:24948777

Woo, Hannah L.; Utturkar, Sagar; Klingeman, Dawn; Simmons, Blake A.; DeAngelis, Kristen M.; Brown, Steven D.

2014-01-01

258

Draft Genome Sequence of the Organophosphorus Compound-Degrading Burkholderia zhejiangensis Strain CEIB S4-3  

PubMed Central

Burkholderia species are widely distributed in the environment. A Burkholderia zhejiangensis strain was isolated from pesticide-contaminated soil from an agricultural field in Mexico and identified as an organophosphorus compound-degrading bacterium. In this study, we report the draft genome sequence of Burkholderia zhejiangensis strain CEIB S4-3. PMID:25523778

Hernández-Mendoza, Armando; Martínez-Ocampo, Fernando; Lozano-Aguirre Beltrán, Luis Fernando; Popoca-Ursino, Elida Carolina; Ortiz-Hernández, Laura; Sánchez-Salinas, Enrique

2014-01-01

259

Whole-Genome Analysis of Quorum-Sensing Burkholderia sp. Strain A9.  

PubMed

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

Chan, Kok-Gan; Chen, Jian Woon; Tee, Kok Keng; Chang, Chien-Yi; Yin, Wai-Fong; Chan, Xin-Yue

2015-01-01

260

Whole-Genome Analysis of Quorum-Sensing Burkholderia sp. Strain A9  

PubMed Central

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

Chen, Jian Woon; Tee, Kok Keng; Chang, Chien-Yi; Yin, Wai-Fong; Chan, Xin-Yue

2015-01-01

261

Immunoproteomic Analysis of Proteins Expressed by Two Related Pathogens, Burkholderia multivorans and Burkholderia cenocepacia, during Human Infection  

PubMed Central

Burkholderia cepacia complex (Bcc) is an opportunistic bacterial pathogen that causes chronic infections in people with cystic fibrosis (CF). It is a highly antibiotic resistant organism and Bcc infections are rarely cleared from patients, once they are colonized. The two most clinically relevant species within Bcc are Burkholderia cenocepacia and Burkholderia multivorans. The virulence of these pathogens has not been fully elucidated and the virulence proteins expressed during human infection have not been identified to date. Furthermore, given its antibiotic resistance, prevention of infection with a prophylactic vaccine may represent a better alternative than eradication of an existing infection. We have compared the immunoproteome of two strains each from these two species of Bcc, with the aim of identifying immunogenic proteins which are common to both species. Fourteen immunoreactive proteins were exclusive to both B. cenocepacia strains, while 15 were exclusive to B. multivorans. A total of 15 proteins were immunogenic across both species. DNA-directed RNA polymerase, GroEL, 38kDa porin and elongation factor-Tu were immunoreactive proteins expressed by all four strains examined. Many proteins which were immunoreactive in both species, warrant further investigations in order to aid in the elucidation of the mechanisms of pathogenesis of this difficult organism. In addition, identification of some of these could also allow the development of protective vaccines which may prevent colonisation. PMID:24260482

Shinoy, Minu; Dennehy, Ruth; Coleman, Lorraine; Carberry, Stephen; Schaffer, Kirsten; Callaghan, Máire; Doyle, Sean; McClean, Siobhán

2013-01-01

262

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...

263

A Fine-Scale Phylogenetic Analysis of Free-Living Burkholderia Species in Sugarcane Field Soil  

PubMed Central

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 (107 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.

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

264

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 ...

265

A Fine-Scale Phylogenetic Analysis of Free-Living Burkholderia Species in Sugarcane Field Soil.  

PubMed

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. PMID:25410730

Tago, Kanako; Itoh, Hideomi; Kikuchi, Yoshitomo; Hori, Tomoyuki; Sato, Yuya; Nagayama, Atsushi; Okubo, Takashi; Navarro, Ronald; Aoyagi, Tomo; Hayashi, Kentaro; Hayatsu, Masahito

2014-11-20

266

Expression of Caenorhabditis elegans antimicrobial peptide NLP-31 in Escherichia coli  

NASA Astrophysics Data System (ADS)

Burkholderia pseudomallei is the causative agent of melioidosis, a fulminant disease endemic in Southeast Asia and Northern Australia. The standardized form of therapy is antibiotics treatment; however, the bacterium has become increasingly resistant to these antibiotics. This has spurred the need to search for alternative therapeutic agents. Antimicrobial peptides (AMPs) are small proteins that possess broad-spectrum antimicrobial activity. In a previous study, the nematode Caenorhabditis elegans was infected by B. pseudomallei and a whole animal transcriptome analysis identified a number of AMP-encoded genes which were induced significantly in the infected worms. One of the AMPs identified is NLP-31 and to date, there are no reports of anti-B. pseudomallei activity demonstrated by NLP-31. To produce NLP-31 protein for future studies, the gene encoding for NLP-31 was cloned into the pET32b expression vector and transformed into Escherichia coli BL21(DE3). Protein expression was induced with 1 mM IPTG for 20 hours at 20°C and recombinant NLP-31 was detected in the soluble fraction. Taken together, a simple optimized heterologous production of AMPs in an E. coli expression system has been successfully developed.

Lim, Mei-Perng; Nathan, Sheila

2014-09-01

267

A Structural Biology Approach Enables the Development of Antimicrobials Targeting Bacterial Immunophilins  

PubMed Central

Macrophage infectivity potentiators (Mips) are immunophilin proteins and essential virulence factors for a range of pathogenic organisms. We applied a structural biology approach to characterize a Mip from Burkholderia pseudomallei (BpML1), the causative agent of melioidosis. Crystal structure and nuclear magnetic resonance analyses of BpML1 in complex with known macrocyclics and other derivatives led to the identification of a key chemical scaffold. This scaffold possesses inhibitory potency for BpML1 without the immunosuppressive components of related macrocyclic agents. Biophysical characterization of a compound series with this scaffold allowed binding site specificity in solution and potency determinations for rank ordering the set. The best compounds in this series possessed a low-micromolar affinity for BpML1, bound at the site of enzymatic activity, and inhibited a panel of homologous Mip proteins from other pathogenic bacteria, without demonstrating toxicity in human macrophages. Importantly, the in vitro activity of BpML1 was reduced by these compounds, leading to decreased macrophage infectivity and intracellular growth of Burkholderia pseudomallei. These compounds offer the potential for activity against a new class of antimicrobial targets and present the utility of a structure-based approach for novel antimicrobial drug discovery. PMID:24366729

Fox, David; Jenner, Dominic; Juli, Christina; Pierce, Phillip G.; Abendroth, Jan; Muruthi, Muigai; Safford, Kris; Anderson, Vanessa; Atkins, Kateri; Barnes, Steve R.; Moen, Spencer O.; Raymond, Amy C.; Stacy, Robin; Myler, Peter J.; Staker, Bart L.; Harmer, Nicholas J.; Norville, Isobel H.; Holzgrabe, Ulrike; Sarkar-Tyson, Mitali; Edwards, Thomas E.; Lorimer, Donald D.

2014-01-01

268

Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. Nodule Bacteria on two Mimosa spp. in Costa Rica  

PubMed Central

rRNA gene sequencing and PCR assays indicated that 215 isolates of root nodule bacteria from two Mimosa species at three sites in Costa Rica belonged to the genera Burkholderia, Cupriavidus, and Rhizobium. This is the first report of Cupriavidus sp. nodule symbionts for Mimosa populations within their native geographic range in the neotropics. Burkholderia spp. predominated among samples from Mimosa pigra (86% of isolates), while there was a more even distribution of Cupriavidus, Burkholderia, and Rhizobium spp. on Mimosa pudica (38, 37, and 25% of isolates, respectively). All Cupriavidus and Burkholderia genotypes tested formed root nodules and fixed nitrogen on both M. pigra and M. pudica, and sequencing of rRNA genes in strains reisolated from nodules verified identity with inoculant strains. Inoculation tests further indicated that both Cupriavidus and Burkholderia spp. resulted in significantly higher plant growth and nodule nitrogenase activity (as measured by acetylene reduction assays) relative to plant performance with strains of Rhizobium. Given the prevalence of Burkholderia and Cupriavidus spp. on these Mimosa legumes and the widespread distribution of these plants both within and outside the neotropics, it is likely that both ?-proteobacterial genera are more ubiquitous as root nodule symbionts than previously believed. PMID:16461667

Barrett, Craig F.; Parker, Matthew A.

2006-01-01

269

Burkholderia species are the most common and preferred nodulating symbionts of the Piptadenia group (tribe Mimoseae).  

PubMed

Burkholderia legume symbionts (also called ?-rhizobia) are ancient in origin and are the main nitrogen-fixing symbionts of species belonging to the large genus Mimosa in Brazil. We investigated the extent of the affinity between Burkholderia and species in the tribe Mimoseae by studying symbionts of the genera Piptadenia (P.), Parapiptadenia (Pp.), Pseudopiptadenia (Ps.), Pityrocarpa (Py.), Anadenanthera (A.) and Microlobius (Mi.), all of which are native to Brazil and are phylogenetically close to Mimosa, and which together with Mimosa comprise the "Piptadenia group". We characterized 196 strains sampled from 18 species from 17 locations in Brazil using two neutral markers and two symbiotic genes in order to assess their species affiliations and the evolution of their symbiosis genes. We found that Burkholderia are common and highly diversified symbionts of species in the Piptadenia group, comprising nine Burkholderia species, of which three are new ones and one was never reported as symbiotic (B. phenoliruptrix). However, ?-rhizobia were also detected and were occasionally dominant on a few species. A strong sampling site effect on the rhizobial nature of symbionts was detected, with the symbiont pattern of the same legume species changing drastically from location to location, even switching from ? to ?-rhizobia. Coinoculation assays showed a strong affinity of all the Piptadenia group species towards Burkholderia genotypes, with the exception of Mi. foetidus. Phylogenetic analyses of neutral and symbiotic markers showed that symbiosis genes in Burkholderia from the Piptadenia group have evolved mainly through vertical transfer, but also by horizontal transfer in two species. PMID:23691052

Bournaud, Caroline; de Faria, Sergio Miana; dos Santos, José Miguel Ferreira; Tisseyre, Pierre; Silva, Michele; Chaintreuil, Clémence; Gross, Eduardo; James, Euan K; Prin, Yves; Moulin, Lionel

2013-01-01

270

Characterization and Inference of Gene Gain/Loss Along Burkholderia Evolutionary History  

PubMed Central

A comparative analysis of 60 complete Burkholderia genomes was conducted to obtain insight in the evolutionary history behind the diversity and pathogenicity at species level. A concatenated multiprotein phyletic pattern and a dataset with Burkholderia clusters of orthologous genes (BuCOGs) were constructed. The extent of horizontal gene transfer (HGT) was assessed using a Markov based probabilistic method. A reconstruction of the gene gains and losses history shows that more than half of the Burkholderia genes families are inferred to have experienced HGT at least once during their evolution. Further analysis revealed that the number of gene gain and loss was correlated with the branch length. Genomic islands (GEIs) analysis based on evolutionary history reconstruction not only revealed that most genes in ancient GEIs were gained but also suggested that the fraction of the genome located in GEIs in the small chromosomes is higher than in the large chromosomes in Burkholderia. The mapping of coexpressed genes onto biological pathway schemes revealed that pathogenicity of Burkholderia strains is probably mainly determined by the gained genes in its ancestor. Taken together, our results strongly support that gene gain and loss especially in ancient evolutionary history play an important role in strain divergence, pathogenicity determinants of Burkholderia and GEIs formation. PMID:22084562

Zhu, Bo; Zhou, Shengli; Lou, Miaomiao; Zhu, Jun; Li, Bin; Xie, Guanlin; Jin, GuLei; De Mot, René

2011-01-01

271

Aerosol Phage Therapy Efficacy in Burkholderia cepacia Complex Respiratory Infections  

PubMed Central

Phage therapy has been suggested as a potential treatment for highly antibiotic-resistant bacteria, such as the species of the Burkholderia cepacia complex (BCC). To address this hypothesis, experimental B. cenocepacia respiratory infections were established in mice using a nebulizer and a nose-only inhalation device. Following infection, the mice were treated with one of five B. cenocepacia-specific phages delivered as either an aerosol or intraperitoneal injection. The bacterial and phage titers within the lungs were assayed 2 days after treatment, and mice that received the aerosolized phage therapy demonstrated significant decreases in bacterial loads. Differences in phage activity were observed in vivo. Mice that received phage treatment by intraperitoneal injection did not demonstrate significantly reduced bacterial loads, although phage particles were isolated from their lung tissue. Based on these data, aerosol phage therapy appears to be an effective method for treating highly antibiotic-resistant bacterial respiratory infections, including those caused by BCC bacteria. PMID:24798268

Semler, Diana D.; Goudie, Amanda D.; Finlay, Warren H.

2014-01-01

272

Use of the Common Marmoset to Study Burkholderia mallei Infection  

PubMed Central

Burkholderia mallei is a host-adapted bacterium that does not persist outside of its equine reservoir. The organism causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by B. mallei typically occurs via the respiratory or percutaneous route, and the most common manifestations are life-threatening pneumonia and bacteremia. Glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. There is no vaccine to protect against B. mallei and there is concern regarding its use as a biothreat agent. Thus, experiments were performed to establish a non-human primate model of intranasal infection to study the organism and develop countermeasures. Groups of marmosets (Callithrix jacchus) were inoculated intranasally with B. mallei strain ATCC 23344 and monitored for clinical signs of illness for up to 13 days. We discovered that 83% of marmosets inoculated with doses of 2.5 X 104 to 2.5 X 105 bacteria developed acute lethal infection within 3–4 days. Signs of disease were severe and included lethargy, inappetence, conjunctivitis, mucopurulent and hemorrhagic nasal discharges, and increased respiratory effort with abdominal lifts. Burkholderia mallei was cultured from the lungs, spleen and liver of these animals, and pathologic examination of tissues revealed lesions characteristic of glanders. Challenge experiments also revealed that 91% of animals infected with doses ranging from 25 to 2.5 X 103 bacteria exhibited mild non-specific signs of illness and were culture negative. One marmoset inoculated with 2.5 X 103 organisms developed moderate signs of disease and reached humane end-points 8 days post-infection. The liver and spleen of this animal were colonized with the agent and pathological analysis of tissues showed nasal, splenic and hepatic lesions. Taken together, these data indicate that the marmoset is a suitable model to study respiratory infection by B. mallei. PMID:25860021

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

2015-01-01

273

40 CFR 180.1325 - Heat-killed Burkholderia spp. strain A396 cells and spent fermentation media exemption from the...  

Code of Federal Regulations, 2014 CFR

...Heat-killed Burkholderia spp. strain A396 cells and spent fermentation media exemption...Heat-killed Burkholderia spp. strain A396 cells and spent fermentation media exemption...heat-killed Burkholderia spp. strain A396 cells and spent fermentation media in or...

2014-07-01

274

Diazotrophic Burkholderia Species Associated with Field-Grown Maize and Sugarcane  

PubMed Central

Until recently, diazotrophy was known in only one of the 30 formally described species of Burkholderia. Novel N2-fixing plant-associated Burkholderia species such as B. unamae, B. tropica, and B. xenovorans have been described, but their environmental distribution is scarcely known. In the present study, the occurrence of N2-fixing Burkholderia species associated with different varieties of sugarcane and maize growing in regions of Mexico and Brazil was analyzed. Only 111 out of more than 900 isolates recovered had N2-fixing ability as demonstrated by the acetylene reduction assay. All 111 isolates also yielded a PCR product with primers targeting the nifH gene, which encodes a key enzyme in the process of nitrogen fixation. These 111 isolates were confirmed as belonging to the genus Burkholderia by using a new 16S rRNA-specific primer pair for diazotrophic species (except B. vietnamiensis) and closely related nondiazotrophic Burkholderia. In Mexico, many isolates of B. unamae (predominantly associated with sugarcane) and B. tropica (more often associated with maize) were recovered. However, in Brazil B. tropica was not identified among the isolates analyzed, and only a few B. unamae isolates were recovered from one sugarcane variety. Most Brazilian diazotrophic Burkholderia isolates (associated with both sugarcane and maize plants) belonged to a novel species, as revealed by amplified 16S rRNA gene restriction profiles, 16S rRNA gene sequencing, and protein electrophoresis. In addition, transmissibility factors such as the cblA and esmR genes, identified among clinical and environmental isolates of opportunistic pathogens of B. cenocepacia and other species of the B. cepacia complex, were not detected in any of the plant-associated diazotrophic Burkholderia isolates analyzed. PMID:16672447

Perin, L.; Martínez-Aguilar, L.; Castro-González, R.; Estrada-de los Santos, P.; Cabellos-Avelar, T.; Guedes, H. V.; Reis, V. M.; Caballero-Mellado, J.

2006-01-01

275

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

PubMed Central

Plant roots and shoots harbor complex bacterial communities. Early seed and plantlet colonization plays a key role in determining which bacterial populations will successfully invade plant tissues, yet the mechanisms enabling plants to select for beneficial rather than harmful populations are largely unknown. In this study, we demonstrate a role of oxalate as a determinant in this selection process, using members of the genus Burkholderia as model organisms. Oxalotrophy, i.e., the ability to use oxalate as a carbon source, was found to be a property strictly associated with plant-beneficial species of the Burkholderia genus, while plant pathogenic (B. glumae, B. plantarii) or human opportunistic pathogens (Burkholderia cepacia complex strains) were unable to degrade oxalate. We further show that oxalotrophy is required for successful plant colonization by the broad host endophyte Burkholderia phytofirmans PsJN: an engineered ?oxc mutant, which lost the ability to grow on oxalate, was significantly impaired in early colonization of both lupin and maize compared with the wild-type. This work suggests that in addition to the role of oxalate in heavy metal tolerance of plants and in virulence of phytopathogenic fungi, it is also involved in specifically recruiting plant-beneficial members from complex bacterial communities. PMID:24409174

Kost, Thomas; Stopnisek, Nejc; Agnoli, Kirsty; Eberl, Leo

2014-01-01

276

Diversity of the parB and repA genes of the Burkholderia cepacia complex and their utility for rapid identification of Burkholderia cenocepacia  

Microsoft Academic Search

BACKGROUND: Burkholderia cenocepacia is the most prominent species of the B. cepacia complex (Bcc), a group of nine closely related and difficult to identify bacteria that cause serious infections in patients with cystic fibrosis. Despite its clinical relevance, identification of B. cenocepacia as a single species is unavailable, as it splits by a widely used recA gene-based PCR identification method

Pavel Drevinek; Adam Baldwin; Christopher G Dowson; Eshwar Mahenthiralingam

2008-01-01

277

9 CFR 121.9 - Responsible official.  

Code of Federal Regulations, 2013 CFR

...facsimile, or email: African horse sickness virus, African swine fever virus, avian influenza virus (highly pathogenic), Bacillus...Burkholderia mallei, Burkholderia pseudomallei, classical swine fever virus, foot-and-mouth disease virus, virulent...

2013-01-01

278

9 CFR 121.9 - Responsible official.  

Code of Federal Regulations, 2014 CFR

...facsimile, or email: African horse sickness virus, African swine fever virus, avian influenza virus (highly pathogenic), Bacillus...Burkholderia mallei, Burkholderia pseudomallei, classical swine fever virus, foot-and-mouth disease virus, virulent...

2014-01-01

279

Draft Genome Sequence of Burkholderia pyrrocinia Lyc2, a Biological Control Strain That Can Suppress Multiple Plant Microbial Pathogens  

PubMed Central

Burkholderia pyrrocinia strain Lyc2 was isolated from the tobacco rhizosphere in China. This bacterium exhibits a remarkable capacity to inhibit the growth of multiple pathogens and shows strong suppression of cotton seedling damping-off. Here, we present the draft genome sequence of Burkholderia pyrrocinia strain Lyc2. PMID:25278535

Wang, Xiao-Qiang; Showmaker, Kurt C.; Yu, Xiao-Qing; Bi, Tao; Hsu, Chuan-Yu; Baird, Sonya M.; Peterson, Daniel G.

2014-01-01

280

Whole-Genome Sequence of Burkholderia sp. Strain RPE67, a Bacterial Gut Symbiont of the Bean Bug Riptortus pedestris  

PubMed Central

Burkholderia sp. strain RPE67 is a bacterial symbiont isolated from a field-collected bean bug, Riptortus pedestris. To understand the genetic basis of the insect-microbe symbiosis, we performed whole-genome sequencing of the Burkholderia strain, revealing an 8.69-Mb genome consisting of three chromosomes and three plasmids. PMID:24948758

Takeshita, Kazutaka; Shibata, Tomoko F.; Nikoh, Naruo; Nishiyama, Tomoaki; Hasebe, Mitsuyasu; Fukatsu, Takema; Shigenobu, Shuji

2014-01-01

281

Genome Annotation of Burkholderia sp. SJ98 with Special Focus on Chemotaxis Genes  

PubMed Central

Burkholderia sp. strain SJ98 has the chemotactic activity towards nitroaromatic and chloronitroaromatic compounds. Recently our group published draft genome of strain SJ98. In this study, we further sequence and annotate the genome of stain SJ98 to exploit the potential of this bacterium. We specifically annotate its chemotaxis genes and methyl accepting chemotaxis proteins. Genome of Burkholderia sp. SJ98 was annotated using PGAAP pipeline that predicts 7,268 CDSs, 52 tRNAs and 3 rRNAs. Our analysis based on phylogenetic and comparative genomics suggest that Burkholderia sp. YI23 is closest neighbor of the strain SJ98. The genes involved in the chemotaxis of strain SJ98 were compared with genes of closely related Burkholderia strains (i.e. YI23, CCGE 1001, CCGE 1002, CCGE 1003) and with well characterized bacterium E. coli K12. It was found that strain SJ98 has 37 che genes including 19 methyl accepting chemotaxis proteins that involved in sensing of different attractants. Chemotaxis genes have been found in a cluster along with the flagellar motor proteins. We also developed a web resource that provides comprehensive information on strain SJ98 that includes all analysis data (http://crdd.osdd.net/raghava/genomesrs/burkholderia/). PMID:23940608

Kumar, Shailesh; Vikram, Surendra; Raghava, Gajendra Pal Singh

2013-01-01

282

The Organization of the Quorum Sensing luxI/R Family Genes in Burkholderia  

PubMed Central

Members of the Burkholderia genus of Proteobacteria are capable of living freely in the environment and can also colonize human, animal and plant hosts. Certain members are considered to be clinically important from both medical and veterinary perspectives and furthermore may be important modulators of the rhizosphere. Quorum sensing via N-acyl homoserine lactone signals (AHL QS) is present in almost all Burkholderia species and is thought to play important roles in lifestyle changes such as colonization and niche invasion. Here we present a census of AHL QS genes retrieved from public databases and indicate that the local arrangement (topology) of QS genes, their location within chromosomes and their gene neighborhoods show characteristic patterns that differ between the known Burkholderia clades. In sequence phylogenies, AHL QS genes seem to cluster according to the local gene topology rather than according to the species, which suggests that the basic topology types were present prior to the appearance of current Burkholderia species. The data are available at http://net.icgeb.org/burkholderia/. PMID:23820583

Choudhary, Kumari Sonal; Hudaiberdiev, Sanjarbek; Gelencsér, Zsolt; Coutinho, Bruna Gonçalves; Venturi, Vittorio; Pongor, Sándor

2013-01-01

283

The Tomato Rhizosphere, an Environment Rich in Nitrogen-Fixing Burkholderia Species with Capabilities of Interest for Agriculture and Bioremediation?  

PubMed Central

Burkholderia strains are promising candidates for biotechnological applications. Unfortunately, most of these strains belong to species of the Burkholderia cepacia complex (Bcc) involved in human infections, hampering potential applications. Novel diazotrophic Burkholderia species, phylogenetically distant from the Bcc species, have been discovered recently, but their environmental distribution and relevant features for agro-biotechnological applications are little known. In this work, the occurrence of N2-fixing Burkholderia species in the rhizospheres and rhizoplanes of tomato plants field grown in Mexico was assessed. The results revealed a high level of diversity of diazotrophic Burkholderia species, including B. unamae, B. xenovorans, B. tropica, and two other unknown species, one of them phylogenetically closely related to B. kururiensis. These N2-fixing Burkholderia species exhibited activities involved in bioremediation, plant growth promotion, or biological control in vitro. Remarkably, B. unamae and B. kururiensis grew with aromatic compounds (phenol and benzene) as carbon sources, and the presence of aromatic oxygenase genes was confirmed in both species. The rhizospheric and endophyte nature of B. unamae and its ability to degrade aromatic compounds suggest that it could be used in rhizoremediation and for improvement of phytoremediation. B. kururiensis and other Burkholderia sp. strains grew with toluene. B. unamae and B. xenovorans exhibited ACC (1-aminocyclopropane-1-carboxylic acid) deaminase activity, and the occurrence of acdS genes encoding ACC deaminase was confirmed. Mineral phosphate solubilization through organic acid production appears to be the mechanism used by most diazotrophic Burkholderia species, but in B. tropica, there presumably exists an additional unknown mechanism. Most of the diazotrophic Burkholderia species produced hydroxamate-type siderophores. Certainly, the N2-fixing Burkholderia species associated with plants have great potential for agro-biotechnological applications. PMID:17601817

Caballero-Mellado, Jesús; Onofre-Lemus, Janette; Estrada-de los Santos, Paulina; Martínez-Aguilar, Lourdes

2007-01-01

284

Genome-guided discovery of diverse natural products from Burkholderia sp  

PubMed Central

Burkholderia species have emerged as a new source of diverse natural products. This mini-review covers all natural products discovered in recent years from Burkholderia sp. by genome-guided approaches – these refer to the use of bacterial genome sequence as an entry point for in silico structural prediction, wet lab experimental design and execution. While reliable structural prediction based on cryptic biosynthetic gene cluster sequence was not always possible due to noncanonical domains and/or module organization of a deduced biosynthetic pathway, a molecular genetic method was often employed to detect or alter the expression level of the gene cluster to achieve an observable phenotype, which facilitated downstream natural product purification and identification. Those examples of natural product discovery from Burkholderia sp. provide a practical guidance for future exploration of Gram-negative bacteria as a new source of natural products. PMID:24212473

Liu, Xiangyang; Cheng, Yi-Qiang

2013-01-01

285

Monitoring Therapeutic Treatments against Burkholderia Infections Using Imaging Techniques  

PubMed Central

Burkholderia mallei, the etiologic agent of glanders, are Category B select agents with biothreat potential, and yet effective therapeutic treatments are lacking. In this study, we showed that CpG administration increased survival, demonstrating protection in the murine glanders model. Bacterial recovery from infected lungs, liver and spleen was significantly reduced in CpG-treated animals as compared with non-treated mice. Reciprocally, lungs of CpG-treated infected animals were infiltrated with higher levels of neutrophils and inflammatory monocytes, as compared to control animals. Employing the B. mallei bioluminescent strain CSM001 and the Neutrophil-Specific Fluorescent Imaging Agent, bacterial dissemination and neutrophil trafficking were monitored in real-time using multimodal in vivo whole body imaging techniques. CpG-treatment increased recruitment of neutrophils to the lungs and reduced bioluminescent bacteria, correlating with decreased bacterial burden and increased protection against acute murine glanders. Our results indicate that protection of CpG-treated animals was associated with recruitment of neutrophils prior to infection and demonstrated, for the first time, simultaneous real time in vivo imaging of neutrophils and bacteria. This study provides experimental evidence supporting the importance of incorporating optimized in vivo imaging methods to monitor disease progression and to evaluate the efficacy of therapeutic treatment during bacterial infections. PMID:24349761

Mott, Tiffany M.; Johnston, R. Katie; Vijayakumar, Sudhamathi; Estes, D. Mark; Motamedi, Massoud; Sbrana, Elena; Endsley, Janice J.; Torres, Alfredo G.

2013-01-01

286

Monitoring Therapeutic Treatments against Burkholderia Infections Using Imaging Techniques.  

PubMed

Burkholderia mallei, the etiologic agent of glanders, are Category B select agents with biothreat potential, and yet effective therapeutic treatments are lacking. In this study, we showed that CpG administration increased survival, demonstrating protection in the murine glanders model. Bacterial recovery from infected lungs, liver and spleen was significantly reduced in CpG-treated animals as compared with non-treated mice. Reciprocally, lungs of CpG-treated infected animals were infiltrated with higher levels of neutrophils and inflammatory monocytes, as compared to control animals. Employing the B. mallei bioluminescent strain CSM001 and the Neutrophil-Specific Fluorescent Imaging Agent, bacterial dissemination and neutrophil trafficking were monitored in real-time using multimodal in vivo whole body imaging techniques. CpG-treatment increased recruitment of neutrophils to the lungs and reduced bioluminescent bacteria, correlating with decreased bacterial burden and increased protection against acute murine glanders. Our results indicate that protection of CpG-treated animals was associated with recruitment of neutrophils prior to infection and demonstrated, for the first time, simultaneous real time in vivo imaging of neutrophils and bacteria. This study provides experimental evidence supporting the importance of incorporating optimized in vivo imaging methods to monitor disease progression and to evaluate the efficacy of therapeutic treatment during bacterial infections. PMID:24349761

Mott, Tiffany M; Johnston, R Katie; Vijayakumar, Sudhamathi; Estes, D Mark; Motamedi, Massoud; Sbrana, Elena; Endsley, Janice J; Torres, Alfredo G

2013-06-01

287

Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia.  

PubMed

The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms. Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P. aeruginosa alone. The interactions between P. aeruginosa biofilms and B. cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship. Using the Calgary Biofilm Device (CBD), members of all genomovars of the B. cepacia complex were shown to form biofilms, including those isolated from CF lungs. Mixed species biofilm formation between CF isolates of P. aeruginosa and B. cepacia was readily achieved using the CBD. Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition. Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure. Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B. cepacia complex. Characterization of the soluble inhibitor suggested pyocyanin as the active compound. PMID:11718549

Tomlin, K L; Coll, O P; Ceri, H

2001-10-01

288

Binding of protegrin-1 to Pseudomonas aeruginosa and Burkholderia cepacia  

PubMed Central

Background Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia. Methods The PG-1 sensitivity/resistance and PG-1 binding properties of P. aeruginosa and B. cepacia were assessed using radial diffusion assays, radioiodinated PG-1, and surface plasmon resonance (BiaCore). Results The six P. aeruginosa strains examined were very sensitive to PG-1, exhibiting minimal active concentrations from 0.0625–0.5 ?g/ml in radial diffusion assays. In contrast, all five B. cepacia strains examined were greater than 10-fold to 100-fold more resistant, with minimal active concentrations ranging from 6–10 ?g/ml. When incubated with a radioiodinated variant of PG-1, a sensitive P. aeruginosa strain bound considerably more protegrin molecules per cell than a resistant B. cepacia strain. Binding/diffusion and surface plasmon resonance assays revealed that isolated lipopolysaccharide (LPS) and lipid A from the sensitive P. aeruginosa strains bound PG-1 more effectively than LPS and lipid A from resistant B. cepacia strains. Conclusion These findings support the hypothesis that the relative resistance of B. cepacia to protegrin is due to a reduced number of PG-1 binding sites on the lipid A moiety of its LPS. PMID:11980587

Albrecht, Mark T; Wang, Wei; Shamova, Olga; Lehrer, Robert I; Schiller, Neal L

2002-01-01

289

Identification of quorum sensing-controlled genes in Burkholderia ambifaria.  

PubMed

The Burkholderia cepacia complex (Bcc) comprises strains with a virulence potential toward immunocompromised patients as well as plant growth-promoting rhizobacteria (PGPR). Owing to the link between quorum sensing (QS) and virulence, most studies among Bcc species have been directed toward QS of pathogenic bacteria. We have investigated the QS of B. ambifaria, a PGPR only infrequently recovered from patients. The cepI gene, responsible for the synthesis of the main signaling molecule N-octanoylhomoserine lactone (C8 -HSL), was inactivated. Phenotypes of the B. ambifaria cepI mutant we observed, such as increased production of siderophores and decreased proteolytic and antifungal activities, are in agreement with those of other Bcc cepI mutants. The cepI mutant was then used as background strain for a whole-genome transposon-insertion mutagenesis strategy, allowing the identification of 20 QS-controlled genes, corresponding to 17 loci. The main functions identified are linked to antifungal and antimicrobial properties, as we have identified QS-controlled genes implicated in the production of pyrrolnitrin, burkholdines (occidiofungin-like molecules), and enacyloxins. This study provides insights in the QS-regulated functions of a PGPR, which could lead to beneficial potential biotechnological applications. PMID:23382083

Chapalain, Annelise; Vial, Ludovic; Laprade, Natacha; Dekimpe, Valérie; Perreault, Jonathan; Déziel, Eric

2013-04-01

290

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

PubMed Central

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

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

2001-01-01

291

Mixotrophic metabolism in Burkholderia kururiensis subsp. thiooxydans subsp. nov., a facultative chemolithoautotrophic thiosulfate oxidizing bacterium isolated from rhizosphere soil and proposal for classification of the type strain of Burkholderia kururiensis as Burkholderia kururiensis subsp. kururiensis subsp. nov  

Microsoft Academic Search

A thiosulfate-oxidizing facultative chemolithoautotrophic Burkholderia sp. strain ATSB13T was previously isolated from rhizosphere soil of tobacco plant. Strain ATSB13T was aerobic, Gram-staining-negative, rod shaped and motile by means of sub-terminal flagellum. Strain ATSB13T exhibited mixotrophic growth in a medium containing thiosulfate plus acetate. A phylogenetic study based on 16S rRNA gene\\u000a sequence analysis indicated that strain ATSB13T was most closely

Rangasamy Anandham; Pandiyan Indira Gandhi; Soon Wo Kwon; Tong Min Sa; Yong Ki Kim; Hyeong Jin Jee

2009-01-01

292

Burkholderia ginsengiterrae sp. nov. and Burkholderia panaciterrae sp. nov., antagonistic bacteria against root rot pathogen Cylindrocarpon destructans, isolated from ginseng soil.  

PubMed

Strain DCY85(T) and DCY85-1(T), isolated from rhizosphere of ginseng, were rod-shaped, Gram-reaction-negative, strictly aerobic, catalase positive and oxidase negative. 16S rRNA gene sequence analysis revealed that strain DCY85(T) as well as DCY85-1(T) belonged to the genus Burkholderia and were closely related to Burkholderia fungorum KACC 12023(T) (98.1 and 98.0 % similarity, respectively). The major polar lipids of strain DCY85(T) and DCY85-1(T) were phosphatidylethanolamine, one unidentified aminolipid and two unidentified phospholipids. The major fatty acids of both strains are C16:0, C18:1 ?7c and summed feature 3 (C16:1 ?6c and/or C16:1 ?7c). The predominant isoprenoid quinone of each strain DCY85(T) and DCY85-1(T) was ubiquinone (Q-8) and the G+C content of their genomic DNA was 66.0 and 59.4 mol%, respectively, which fulfill the characteristic range of the genus Burkholderia. The polyamine content of both DCY85(T) and DCY85-1(T) was putrescine. Although both DCY85(T) and DCY85-1(T) have highly similar 16S rRNA and identical RecA and gyrB sequences, they show differences in phenotypic and chemotaxonomic characteristics. DNA-DNA hybridization results proved the consideration of both strains as two different species. Based on the results from our polyphasic characterization, strain DCY85(T) and DCY85-1(T) are considered novel Burkholderia species for which the name Burkholderia ginsengiterrae sp. nov and Burkholderia panaciterrae sp. nov are, respectively, proposed. An emended description of those strains is also proposed. DCY85(T) and DCY85-1(T) showed antagonistic activity against the common root rot pathogen of ginseng, Cylindrocarpon destructans. The proposed type strains are DCY85(T) (KCTC 42054(T) = JCM 19888(T)) and DCY85-1(T) (KCTC 42055(T) = JCM 19889(T)). PMID:25537097

Farh, Mohamed El-Agamy; Kim, Yeon-Ju; Van An, Hoang; Sukweenadhi, Johan; Singh, Priyanka; Huq, Md Amdadul; Yang, Deok-Chun

2015-04-01

293

Quorum Sensing Controls Flagellar Morphogenesis in Burkholderia glumae  

PubMed Central

Burkholderia glumae is a motile plant pathogenic bacterium that has multiple polar flagella and one LuxR/LuxI-type quorum sensing (QS) system, TofR/TofI. A QS-dependent transcriptional regulator, QsmR, activates flagellar master regulator flhDC genes. FlhDC subsequently activates flagellar gene expression in B. glumae at 37°C. Here, we confirm that the interplay between QS and temperature is critical for normal polar flagellar morphogenesis in B. glumae. In the wild-type bacterium, flagellar gene expression and flagellar number were greater at 28°C compared to 37°C. The QS-dependent flhC gene was significantly expressed at 28°C in two QS-defective (tofI::? and qsmR::?) mutants. Thus, flagella were present in both tofI::? and qsmR::? mutants at 28°C, but were absent at 37°C. Most tofI::? and qsmR::? mutant cells possessed polar or nonpolar flagella at 28°C. Nonpolarly flagellated cells processing flagella around cell surface of both tofI::? and qsmR::? mutants exhibited tumbling and spinning movements. The flhF gene encoding GTPase involved in regulating the correct placement of flagella in other bacteria was expressed in QS mutants in a FlhDC-dependent manner at 28°C. However, FlhF was mislocalized in QS mutants, and was associated with nonpolar flagellar formation in QS mutants at 28°C. These results indicate that QS-independent expression of flagellar genes at 28°C allows flagellar biogenesis, but is not sufficient for normal polar flagellar morphogenesis in B. glumae. Our findings demonstrate that QS functions together with temperature to control flagellar morphogenesis in B. glumae. PMID:24416296

Jang, Moon Sun; Goo, Eunhye; An, Jae Hyung; Kim, Jinwoo; Hwang, Ingyu

2014-01-01

294

Investigation of the multifaceted iron acquisition strategies of Burkholderia cenocepacia.  

PubMed

Burkholderia cenocepacia is a bacterial pathogen which causes severe respiratory infections in cystic fibrosis (CF). These studies were aimed at gaining an insight into the iron acquisition strategies of B. cenocepacia. In iron restricted conditions, genes associated with the synthesis and utilisation of ornibactin (pvdA, orbA, orb F) were significantly upregulated compared to the expression of pyochelin associated genes (pchD, fptA). In the absence of alternative iron sources, B. cenocepacia J2315 and 715j utilised ferritin and haemin, but not transferrin or lactoferrin for growth. Significantly, mutants unable to produce ornibactin, (715j-orbI) or ornibactin and pyochelin, (715j-pobA), utilised haemin and ferritin more efficiently than the wild-type. Moreover, both mutants were also able to utilise lactoferrin for growth (P ? 0.01) and additionally 715j-pobA utilised transferrin (P ? 0.01), potentially facilitating adaptation to the host environment. Furthermore, B. cenocepacia increased ornibactin gene expression in response to pyoverdine from Pseudomonas aeruginosa (P ? 0.01), demonstrating the capacity to compete for iron in co-colonised niches. Pyoverdine also significantly diminished the growth of B. cenocepacia (P < 0.001) which was related to its iron chelating activity. In a study of three B. cenocepacia sequential clonal isolates obtained from a CF patient over a 3.5 year period, ornibactin upregulation in response to pyoverdine was less pronounced in the last isolate compared to the earlier isolates, as was growth in the presence of haemin and ferritin, indicating alternative iron acquisition mechanism(s) may dominate as chronic infection progresses. These data demonstrate the multifaceted iron acquisition strategies of B. cenocepacia and their capacity to be differentially activated in the presence of P. aeruginosa and during chronic infection. PMID:25725797

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

2015-04-01

295

Cytotoxicity Associated with Trichloroethylene Oxidation in Burkholderia cepacia G4  

PubMed Central

The effects of trichloroethylene (TCE) oxidation on toluene 2-monooxygenase activity, general respiratory activity, and cell culturability were examined in the toluene-oxidizing bacterium Burkholderia cepacia G4. Nonspecific damage outpaced inactivation of toluene 2-monooxygenase in B. cepacia G4 cells. Cells that had degraded approximately 0.5 ?mol of TCE (mg of cells?1) lost 95% of their acetate-dependent O2 uptake activity (a measure of general respiratory activity), yet toluene-dependent O2 uptake activity decreased only 35%. Cell culturability also decreased upon TCE oxidation; however, the extent of loss varied greatly (up to 3 orders of magnitude) with the method of assessment. Addition of catalase or sodium pyruvate to the surfaces of agar plates increased enumeration of TCE-injured cells by as much as 100-fold, indicating that the TCE-injured cells were ultrasensitive to oxidative stress. Cell suspensions that had oxidized TCE recovered the ability to grow in liquid minimal medium containing lactate or phenol, but recovery was delayed substantially when TCE degradation approached 0.5 ?mol (mg of cells?1) or 66% of the cells' transformation capacity for TCE at the cell density utilized. Furthermore, among B. cepacia G4 cells isolated on Luria-Bertani agar plates from cultures that had degraded approximately 0.5 ?mol of TCE (mg of cells?1), up to 90% were Tol? variants, no longer capable of TCE degradation. These results indicate that a toxicity threshold for TCE oxidation exists in B. cepacia G4 and that once a cell suspension has exceeded this toxicity threshold, the likelihood of reestablishing an active, TCE-degrading biomass from the cells will decrease significantly. PMID:11319088

Yeager, Chris M.; Bottomley, Peter J.; Arp, Daniel J.

2001-01-01

296

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

Microsoft Academic Search

BACKGROUND: Burkholderia cenocepacia, an opportunistic pathogen that causes lung infections in cystic fibrosis (CF) patients, is associated with rapid and usually fatal lung deterioration due to necrotizing pneumonia and sepsis, a condition known as cepacia syndrome. The key bacterial determinants associated with this poor clinical outcome in CF patients are not clear. In this study, the cytotoxicity and procoagulant activity

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

2010-01-01

297

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

PubMed

Symbiotic bacteria are common in insects and intimately affect the various aspects of insect host biology. In a number of insect symbiosis models, it has been possible to elucidate the effects of the symbiont on host biology, whereas there is a limited understanding of the impact of the association on the bacterial symbiont, mainly due to the difficulty of cultivating insect symbionts in vitro. Furthermore, the molecular features that determine the establishment and persistence of the symbionts in their host (i.e., symbiotic factors) have remained elusive. However, the recently established model, the bean bug Riptortus pedestris, provides a good opportunity to study bacterial symbiotic factors at a molecular level through their cultivable symbionts. Bean bugs acquire genus Burkholderia cells from the environment and harbor them as gut symbionts in the specialized posterior midgut. The genome of the Burkholderia symbiont was sequenced, and the genomic information was used to generate genetically manipulated Burkholderia symbiont strains. Using mutant symbionts, we identified several novel symbiotic factors necessary for establishing a successful association with the host gut. In this review, these symbiotic factors are classified into three categories based on the colonization dynamics of the mutant symbiont strains: initiation, accommodation, and persistence factors. In addition, the molecular characteristics of the symbiotic factors are described. These newly identified symbiotic factors and on-going studies of the Riptortus-Burkholderia symbiosis are expected to contribute to the understanding of the molecular cross-talk between insects and bacterial symbionts that are of ecological and evolutionary importance. PMID:25521625

Kim, Jiyeun Kate; Lee, Bok Luel

2015-01-01

298

CHARACTERIZATION OF THE BIOFILM PHENOTYPE OF BURKHOLDERIA SP., FP62 AND ITS ROLE IN BIOCONTROL  

Technology Transfer Automated Retrieval System (TEKTRAN)

Biofilm formation of Burkholderia sp. (FP62) on plant leaves and its role in the biocontrol of Botrytis cinerea(Bc) was examined on geranium. A library of mini-Tn5 lacZ1 transposon mutants was screened for biofilm formation in a polystyrene microtiter plate assay. Mutants deficient in biofilm form...

299

Species Abundance and Diversity of Burkholderia cepacia Complex in the Environment  

Microsoft Academic Search

Despite considerable interest in studying Burkholderia cepacia complex in the environment, we still do not have efficient methods to detect, isolate, and screen large numbers of B. cepacia isolates. To better describe the ecology and diversity of B. cepacia complex, a colony hybridization assay was developed to detect specifically all species of the complex based on polymorphism of the variable

Alban Ramette; John J. LiPuma; James M. Tiedje

2005-01-01

300

Susceptibility of Caenorhabditis elegans to Burkholderia Infection Depends on Prior Diet and Secreted Bacterial Attractants  

PubMed Central

The nematode Caenorhabditis elegans may be killed by certain pathogenic bacteria and thus is a model organism for studying interactions between bacteria and animal hosts. However, growing nematodes on prey bacteria may influence their susceptibility to potential pathogens. A method of axenic nematode culture was developed to isolate and quantify interactions between C. elegans and potentially pathogenic strains of the Burkholderia cepacia complex. Studying these dynamics in liquid solution rather than on agar surfaces minimized nematode avoidance behavior and resolved more differences among isolates. Most isolates of B. cenocepacia, B. ambifaria and B. cepacia caused 60–80% mortality of nematodes after 7 days, whereas isolates of B. multivorans caused less mortality (<25%) and supported nematode reproduction. However, some B. cenocepacia isolates recovered from chronic infections were much less virulent (5–28% mortality). As predicted, prior diet altered the outcome of interactions between nematodes and bacteria. When given the choice between Burkholderia and E. coli as prey on agar, axenically raised nematodes initially preferred most lethal Burkholderia isolates to E. coli as a food source, but this was not the case for nematodes fed E. coli, which avoided toxic Burkholderia. This food preference was associated with the cell-free supernatant and thus secreted compounds likely mediated bacterial-nematode interactions. This model, which isolates interactions between bacteria and nematodes from the effects of prior feeding, demonstrates that bacteria can influence nematode behavior and their susceptibility to pathogens. PMID:19956737

Cooper, Vaughn S.; Carlson, Wendy A.; LiPuma, John J.

2009-01-01

301

Burkholderia phymatum Strains Capable of Nodulating Phaseolus vulgaris Are Present in Moroccan Soils ?  

PubMed Central

Phylogenetic analysis of 16S rRNA, nodC, and nifH genes of four bacterial strains isolated from root nodules of Phaseolus vulgaris grown in Morocco soils were identified as Burkholderia phymatum. All four strains formed N2-fixing nodules on P. vulgaris and Mimosa, Acacia, and Prosopis species and reduced acetylene to ethylene when cultured ex planta. PMID:20472732

Talbi, C.; Delgado, M. J.; Girard, L.; Ramírez-Trujillo, A.; Caballero-Mellado, J.; Bedmar, E. J.

2010-01-01

302

Drosophila melanogaster as a Model Host for the Burkholderia cepacia Complex  

E-print Network

Drosophila melanogaster as a Model Host for the Burkholderia cepacia Complex Jose´e Castonguay with vertebrate organisms. Methodology/Principal Findings: The aim of this study was to establish Drosophila, validity of the Drosophila infection model was confirmed with B. cenocepacia K56-2 mutants known to be less

Paris-Sud XI, Université de

303

Nickel and cadmium ions inhibit quorum sensing and biofilm formation without affecting viability in Burkholderia multivorans  

E-print Network

Nickel and cadmium ions inhibit quorum sensing and biofilm formation without affecting viability transcriptase polymerase chain reaction (q-PCR) assays, we show that sub-millimolar concentrations of nickel (Ni2þ ) and cadmium (Cd2þ ) inhibit biofilm formation by the bacterium Burkholderia multivorans through

Alvarez, Pedro J.

304

Characterization of a Novel Two-Component System in Burkholderia cenocepacia.  

PubMed

Two-component systems are important regulatory systems that allow bacteria to adjust to environmental conditions, and in some bacteria are used in pathogenesis. We identified a novel two-component system in Burkholderia cenocepacia, an opportunistic pathogen that causes pneumonia in cystic fibrosis (CF) patients. The putative operon encodes BceS, a sensor kinase, and BceR, a response regulator. Our studies indicated that the bceR mutant showed a statistically significant decrease in protease, swimming motility, and quorum sensing when compared to the wild-type, but there was no significant difference in phospholipase C activity, swarming, and biofilm formation. In addition, the mutant showed a statistically significant reduction in virulence compared to the wild-type using the alfalfa plant model. Examination of the Burkholderia cepacia complex (a group of organisms that are phenotypically similar, but genotypically distinct) revealed that this system is prevalent in B. ambifaria, B. multivorans, B. vietnamiensis and B. dolosa. Interestingly, all these organisms have been associated with CF patients. The collective results indicate that BceSR influences various activities important in Burkholderia physiology and possibly pathogenesis. This information could be important in the design of novel therapeutics for Burkholderia infections. PMID:25519693

Merry, Callie R; Perkins, Michael; Mu, Lin; Peterson, Bridget K; Knackstedt, Rebecca W; Weingart, Christine L

2015-04-01

305

NOVEL ORGANIZATION OF THE GENES FOR PHTHALATE DEGRADATION FROM BURKHOLDERIA CEPACIA DBO1  

EPA Science Inventory

Burkholderia cepacia DBO1 is able to utilize phthalate as the sole source of carbon and energy for growth. Two overlapping cosmid clones containing the genes for phthalate degradation were isolated from this strain. Subcloning and activity analysis localized the genes for phthala...

306

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

307

Drosophila melanogaster as a Model Host for the Burkholderia cepacia Complex  

Microsoft Academic Search

BackgroundColonization with bacterial species from the Burkholderia cepacia complex (Bcc) is associated with fast health decline among individuals with cystic fibrosis. In order to investigate the virulence of the Bcc, several alternative infection models have been developed. To this end, the fruit fly is increasingly used as surrogate host, and its validity to enhance our understanding of host-pathogen relationships has

Josée Castonguay-Vanier; Ludovic Vial; Julien Tremblay; Eric Déziel; François Leulier

2010-01-01

308

Draft Genome Sequence of an Aniline-Degrading Bacterium, Burkholderia sp. K24  

PubMed Central

Burkholderia sp. K24 is an aniline-degrading soil bacterium that utilizes aniline and its analogues as sole carbon and nitrogen sources. Here, we report the draft genome sequence of this strain that consists of 8,344,181 bp, with a G+C content of 61.7%. PMID:25477408

Lee, Sang-Yeop; Yun, Sung Ho; Choi, Chi-Won; Lee, Dong-Gi; Choi, Jong Soon; Kahng, Hyung-Yeel

2014-01-01

309

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

E-print Network

with an extensive and varied set of adapted mechanisms to cope with and modulate host-cell environments. One clone of B. pseu- domallei, with 1000 genes lost in an adaptation to equine hosts. Thus, the genesNovel Burkholderia mallei Virulence Factors Linked to Specific Host-Pathogen Protein Interactions

310

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

PubMed

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

Ettinger, Cassandra L; Shehata, Hanan R; Johnston-Monje, David; Raizada, Manish N; Eisen, Jonathan A

2015-01-01

311

Burkholderia diazotrophica sp. nov., isolated from root nodules of Mimosa spp.  

PubMed

Five strains, JPY461(T), JPY359, JPY389, DPU-3 and STM4206 were isolated from nitrogen-fixing nodules on the roots of Mimosa spp. and their taxonomic positions were investigated using a polyphasic approach. All five strains grew at 15-40 °C (optimum, 30-37 °C), at pH 4.0-8.0 (optimum, pH 6.0-7.0) and with 0-1?% (w/v) NaCl [optimum, 0?% (w/v)]. On the basis of 16S rRNA gene sequence analysis, a representative strain (JPY461(T)) showed 97.2?% sequence similarity to the closest related species Burkholderia acidipaludis SA33(T), a similarity of 97.2?% to Burkholderia terrae KMY02(T), 97.1?% to Burkholderia phymatum STM815(T) and 97.1?% to Burkholderia hospita LMG 20598(T). The predominant fatty acids of the five novel strains were summed feature 2 (comprising C(16?:?1) iso I and/or C(14?:?0) 3-OH), summed feature 3 (comprising C(16?:?1)?7c and/or C(16?:?1)?6c), C(16?:?0) , C(16?:?0) 3-OH, C(17?:?0) cyclo, C(18?:?1)?7c and C(19?:?0) cyclo ?8c. The major isoprenoid quinone was Q-8 and the DNA G+C content of the strains was 63.0-65.0 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid, an unidentified aminolipid and several unidentified phospholipids. The DNA-DNA relatedness of the novel strain with respect to recognized species of the genus Burkholderia was less than 54?%. On the basis of 16S rRNA and recA gene sequence similarities, chemotaxonomic and phenotypic data, the five strains represent a novel species in the genus Burkholderia, for which the name Burkholderia diazotrophica sp. nov. is proposed with the type strain, JPY461(T) (?=?LMG 26031(T)?=?BCRC 80259(T)?=?KCTC 23308(T)). PMID:22467155

Sheu, Shih-Yi; Chou, Jui-Hsing; Bontemps, Cyril; Elliott, Geoffrey N; Gross, Eduardo; dos Reis Junior, Fabio Bueno; Melkonian, Rémy; Moulin, Lionel; James, Euan K; Sprent, Janet I; Young, J Peter W; Chen, Wen-Ming

2013-02-01

312

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

PubMed

The genus Burkholderia (?-Proteobacteria) currently comprises more than 60 species, including parasites, symbionts and free-living organisms. Several new species of Burkholderia have recently been described showing a great diversity of phenotypes. We examined the diversity of Burkholderia spp in environmental samples collected from Caatinga and Atlantic rainforest biomes of Bahia, Brazil. Legume nodules were collected from five locations, and 16S rDNA and recA genes of the isolated microorganisms were analyzed. Thirty-three contigs of 16S rRNA genes and four contigs of the recA gene related to the genus Burkholderia were obtained. The genetic dissimilarity of the strains ranged from 0 to 2.5% based on 16S rDNA analysis, indicating two main branches: one distinct branch of the dendrogram for the B. cepacia complex and another branch that rendered three major groups, partially reflecting host plants and locations. A dendrogram designed with sequences of this research and those designed with sequences of Burkholderia-type strains and the first hit BLAST had similar topologies. A dendrogram similar to that constructed by analysis of 16S rDNA was obtained using sequences of the fragment of the recA gene. The 16S rDNA sequences enabled sufficient identification of relevant similarities and groupings amongst isolates and the sequences that we obtained. Only 6 of the 33 isolates analyzed via 16S rDNA sequencing showed high similarity with the B. cepacia complex. Thus, over 3/4 of the isolates have potential for biotechnological applications. PMID:23546947

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

2013-01-01

313

Quantification of burkholderia coxL genes in Hawaiian volcanic deposits.  

PubMed

Isolation of multiple carbon monoxide (CO)-oxidizing Burkholderia strains and detection by culture-independent approaches suggest that Burkholderia may be an important component of CO-oxidizing communities in Hawaiian volcanic deposits. The absolute and relative abundance of the bacteria in these communities remains unknown, however. In this study, a quantitative PCR (Q-PCR) approach has been developed to enumerate Burkholderia coxL genes (large subunit of carbon monoxide dehydrogenase). This represents the first attempt to enumerate coxL genes from CO oxidizers in environmental samples. coxL copy numbers have been determined for samples from three sites representing a vegetation gradient on a 1959 volcanic deposit that included unvegetated cinders (bare), edges of vegetated sites (edge), and sites within tree stands (canopy). Q-PCR has also been used to estimate copy numbers of Betaproteobacteria 16S rRNA gene copy numbers and total Bacteria 16S rRNA. coxL genes could not be detected in the bare site (detection limit, > or = 4.7 x 10(3) copies per reaction) but average 1.0 x 10(8) + or - 2.4 x 10(7) and 8.6 x 10(8) + or - 7.6 x 10(7) copies g(-1) (dry weight) in edge and canopy sites, respectively, which differ statistically (P = 0.0007). Average Burkholderia coxL gene copy numbers, expressed as a percentage of total Bacteria 16S rRNA gene copy numbers, are 6.2 and 0.7% for the edge and canopy sites, respectively. Although the percentage of Burkholderia coxL is lower in the canopy site, significantly greater gene copy numbers demonstrate that absolute abundance of coxL increases in vegetated sites and contributes to the expansion of CO oxidizer communities during biological succession on volcanic deposits. PMID:20139318

Weber, C F; King, G M

2010-04-01

314

Genome sequences of Burkholderia sp. strains CCGE1002 and H160, isolated from legume nodules in Mexico and Brazil.  

PubMed

The genome sequences of Burkholderia sp. strains CCGE1002 from Mexico and H160 from Brazil, isolated from legume nodules, are reported. Their gene contents in relation to plant-microbe interactions and xenobiotic degradation are discussed. PMID:23209196

Ormeño-Orrillo, Ernesto; Rogel, Marco A; Chueire, Ligia Maria Oliveira; Tiedje, James M; Martínez-Romero, Esperanza; Hungria, Mariangela

2012-12-01

315

Draft Genome Sequences of Two Burkholderia multivorans Sequential Isolates from a Chronic Lung Infection of a Cystic Fibrosis Patient.  

PubMed

Burkholderia multivorans belongs to the Burkholderia cepacia complex, which comprises opportunistic pathogens infecting cystic fibrosis (CF) patients. Here, we report the genome sequences and annotations of two sequential B. multivorans clinical isolates (D2095 and D2214) displaying different traits. The differences in the genomic contents of these isolates may provide clues regarding the evolution of B. multivorans within the airways of a CF patient. PMID:25676757

Silva, Inês N; Santos, Pedro M; Moreira, Leonilde M

2015-01-01

316

Evidence of Environmental and Vertical Transmission of Burkholderia Symbionts in the Oriental Chinch Bug, Cavelerius saccharivorus (Heteroptera: Blissidae)  

PubMed Central

The vertical transmission of symbiotic microorganisms is omnipresent in insects, while the evolutionary process remains totally unclear. The oriental chinch bug, Cavelerius saccharivorus (Heteroptera: Blissidae), is a serious sugarcane pest, in which symbiotic bacteria densely populate the lumen of the numerous tubule-like midgut crypts that the chinch bug develops. Cloning and sequence analyses of the 16S rRNA genes revealed that the crypts were dominated by a specific group of bacteria belonging to the genus Burkholderia of the Betaproteobacteria. The Burkholderia sequences were distributed into three distinct clades: the Burkholderia cepacia complex (BCC), the plant-associated beneficial and environmental (PBE) group, and the stinkbug-associated beneficial and environmental group (SBE). Diagnostic PCR revealed that only one of the three groups of Burkholderia was present in ?89% of the chinch bug field populations tested, while infections with multiple Burkholderia groups within one insect were observed in only ?10%. Deep sequencing of the 16S rRNA gene confirmed that the Burkholderia bacteria specifically colonized the crypts and were dominated by one of three Burkholderia groups. The lack of phylogenetic congruence between the symbiont and the host population strongly suggested host-symbiont promiscuity, which is probably caused by environmental acquisition of the symbionts by some hosts. Meanwhile, inspections of eggs and hatchlings by diagnostic PCR and egg surface sterilization demonstrated that almost 30% of the hatchlings vertically acquire symbiotic Burkholderia via symbiont-contaminated egg surfaces. The mixed strategy of symbiont transmission found in the oriental chinch bug might be an intermediate stage in evolution from environmental acquisition to strict vertical transmission in insects. PMID:25038101

Itoh, Hideomi; Aita, Manabu; Nagayama, Atsushi; Meng, Xian-Ying; Kamagata, Yoichi; Navarro, Ronald; Hori, Tomoyuki; Ohgiya, Satoru

2014-01-01

317

Draft Genome Sequences of Two Burkholderia multivorans Sequential Isolates from a Chronic Lung Infection of a Cystic Fibrosis Patient  

PubMed Central

Burkholderia multivorans belongs to the Burkholderia cepacia complex, which comprises opportunistic pathogens infecting cystic fibrosis (CF) patients. Here, we report the genome sequences and annotations of two sequential B. multivorans clinical isolates (D2095 and D2214) displaying different traits. The differences in the genomic contents of these isolates may provide clues regarding the evolution of B. multivorans within the airways of a CF patient. PMID:25676757

Silva, Inês N.; Santos, Pedro M.

2015-01-01

318

Studies on Endophytic Colonization Ability of Two Upland Rice Endophytes, Rhizobium sp. and Burkholderia sp., Using Green Fluorescent Protein Reporter  

Microsoft Academic Search

Colonization ability of the two endophytic bacteria, isolated from surface sterilized roots of upland cultivated rice viz.,\\u000a Rhizobium sp. and Burkholderia sp., was compared after genetically tagging them with a constitutively expressing green fluorescent protein gene (gfp\\/gusA). Confocal laser scanning microscopy (CLSM) of gnotobiotically grown seedlings of Narendradhan 97, inoculated with gfp\\/gusA-tagged endophytes, revealed that both Rhizobium sp. and Burkholderia

Manoj Kumar Singh; Chanda Kushwaha; Ramesh Kumar Singh

2009-01-01

319

Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis  

Microsoft Academic Search

Background  \\u000a Burkholderia cenocepacia is a member of the Burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. B. cenocepacia isolate J2315 has been genome sequenced and is representative of a virulent, epidemic CF strain (ET12). Its genome encodes\\u000a multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or

Andrea Sass; Angela Marchbank; Elizabeth Tullis; John J LiPuma; Eshwar Mahenthiralingam

2011-01-01

320

Burkholderia (basonym Pseudomonas) cepacia binding to lipid receptors.  

PubMed Central

Piliated Burkholderia (formerly Pseudomonas) cepacia from sputa of cys tic fibrosis patients in Toronto, Canada, were shown earlier to bind to purified mucins and to a protein receptor on epithelial cells via a 22-kDa adhesin located on unique cable pili. However, a second receptor, thought to be lipid in nature, was also identified on cells and appeared to serve as the major cell receptor for poorly piliated or nonpiliated isolates. In the present study in vitro approaches were used to identify putative lipid receptors for B. cepacia and to explore the nature of the binding interaction. As judged by thin-layer chromatography overlay assays, the best receptors were digalactosylceramide and globotriosylceramide (Gb(3)). Both contain and unsubstituted terminal Gal alpha 1-4Gal sequence. B cepacia also bound moderately to galactosylceramide, gangliotriosylceramide, and gangliotetraosylceramide. Binding to glycolipids was not affected by tetramethylurea, a hydrophobic-bond-breaking adhesin for GB(3). Binding to glycolipids was not affected by tetramethylurea, a hydrophobic-bond-breaking agent. Binding was influenced by the structure of the ceramide, which probably affects the presentation of the agent. Binding was influenced by the structure of the ceramide, which probably affects the presentation of the carbohydrate epitope to the bacteria. Gb(3) was also the major receptor in lipid extracts of human erythrocytes, human buccal epithelial cells and HEp-2 laryngeal epithelial cells. In a receptor-based enzyme-linked immunosorbent assay, binding to Gb(3) within a phospholipid-cholesterol mixture (a membrane-like environment) increased and then approached saturation as a direct function of increasing bacterial concentration. The calculated value of K(a) (3.06 X 10(-8) ml/CFU), the affinity constant, was almost identical to the K(a) calculated earlier for B. cepacia binding to a set of lipid receptors in buccal epithelial cells (1.5 X 10(-8) to 2.0 X 10(-8) ml/CFU). Our findings suggest that within cell membranes, galactose-containing glycolipids, particularly Gb(3) are good candidates for receptors for B. cepacia, particularly for isolates in which cable pili are poorly expressed. PMID:8606110

Sylvester, F A; Sajjan, U S; Forstner, J F

1996-01-01

321

Revelation of the ability of Burkholderia sp. USM (JCM 15050) PHA synthase to polymerize 4-hydroxybutyrate monomer  

PubMed Central

The nutrition-versatility of Burkholderia sp. strain USM (JCM 15050) has initiated the studies on the use of this bacterium for polyhydroxyalkanoate (PHA) production. To date, the Burkholderia sp. has been reported to synthesize 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxy-4-methylvalerate monomers. In this study, the PHA biosynthetic genes of this strain were successfully cloned and characterized. The PHA biosynthetic cluster of this strain consisted of a PHA synthase (phaC), ?-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthesis regulator (phaR). The translated products of these genes revealed identities to corresponding proteins of Burkholderia vietnamiensis (99–100?%) and Cupriavidus necator H16 (63–89%). Heterologous expression of phaCBs conferred PHA synthesis to the PHA-negative Cupriavidus necator PHB¯4, confirming that phaCBs encoded functionally active protein. PHA synthase activity measurements revealed that the crude extracts of C. necator PHB¯4 transformant showed higher synthase activity (243 U/g) compared to that of wild-types Burkholderia sp. (151 U/g) and C. necator H16 (180 U/g). Interestingly, the transformant C. necator PHB¯4 harbouring Burkholderia sp. PHA synthase gene accumulated poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with 4-hydroxybutyrate monomer as high as up to 87?mol% from sodium 4-hydroxybutyrate. The wild type Burkholderia sp. did not have the ability to produce this copolymer. PMID:22877240

2012-01-01

322

Bioassay-guided isolation of a low molecular weight PHB from Burkholderia sp. with phytotoxic activity.  

PubMed

This work reports on the bioassay-guided isolation and identification of the macrocyclic pentolide 1, a cyclic polyhydroxybutyrate (PHB) with low molecular weight. This metabolite is produced by Burkholderia sp. and it exhibited phytotoxic activity in a Lemna minor bioassay. Its structure was determined by (1)H and (13)C NMR, heteronuclear multiple quantum correlation, heteronuclear multiple bond correlation, IR, and electrospray ionization tandem mass spectrometry analyses. The period for maximum production of the pentolide was optimized and determined on the basis of multiple reaction monitoring experiments at 15 days. The potential of Burkholderia sp. as a producer of higher biopolymers of PHB was also investigated. The methodology employed here accelerated the isolation and characterization of a phytotoxic metabolite whose structure can serve as a model for the synthesis of new classes of herbicides. PMID:23722946

Petta, Tânia; Raichardt, Leandro; Melo, Itamar S; Moraes, Luiz A B

2013-08-01

323

Origin, duplication and reshuffling of plasmid genes: Insights from Burkholderia vietnamiensis G4 genome.  

PubMed

Using a computational pipeline based on similarity networks reconstruction we analysed the 1133 genes of the Burkholderia vietnamiensis (Bv) G4 five plasmids, showing that gene and operon duplication played an important role in shaping the plasmid architecture. Several single/multiple duplications occurring at intra- and/or interplasmids level involving 253 paralogous genes (stand-alone, clustered or operons) were detected. An extensive gene/operon exchange between plasmids and chromosomes was also disclosed. The larger the plasmid, the higher the number and size of paralogous fragments. Many paralogs encoded mobile genetic elements and duplicated very recently, suggesting that the rearrangement of the Bv plastic genome is ongoing. Concerning the "molecular habitat" and the "taxonomical status" (the Preferential Organismal Sharing) of Bv plasmid genes, most of them have been exchanged with other plasmids of bacteria belonging (or phylogenetically very close) to Burkholderia, suggesting that taxonomical proximity of bacterial strains is a crucial issue in plasmid-mediated gene exchange. PMID:24576463

Maida, Isabel; Fondi, Marco; Orlandini, Valerio; Emiliani, Giovanni; Papaleo, Maria Cristiana; Perrin, Elena; Fani, Renato

2014-01-01

324

Evolution of an endofungal Lifestyle: Deductions from the Burkholderia rhizoxinica Genome  

Microsoft Academic Search

Background  \\u000a Burkholderia rhizoxinica is an intracellular symbiont of the phytopathogenic zygomycete Rhizopus microsporus, the causative agent of rice seedling blight. The endosymbiont produces the antimitotic macrolide rhizoxin for its host.\\u000a It is vertically transmitted within vegetative spores and is essential for spore formation of the fungus. To shed light on\\u000a the evolution and genetic potential of this model organism, we

Gerald Lackner; Nadine Moebius; Laila P Partida-Martinez; Sebastian Boland; Christian Hertweck

2011-01-01

325

Purification and properties of a highly enantioselective l-menthyl acetate hydrolase from Burkholderia cepacia  

Microsoft Academic Search

A highly enantioselective l-menthyl acetate esterase was purified to homogeneity from Burkholderia cepacia ATCC 25416, with a recovery of 4.8% and a fold purification of 22.7. The molecular weight of the esterase was found to be 37kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was “MGARTDA”, and there was no homology in contrast to other

Lijuan Yu; Yan Xu; Xiaowei Yu

2009-01-01

326

Burkholderia grimmiae sp. nov., isolated from a xerophilous moss (Grimmia montana).  

PubMed

A Gram-staining-negative, rod-shaped, non-spore-forming bacterium, designated strain R27(T), was isolated from the moss Grimmia montana, collected from Beijing Songshan National Nature Reserve, China, and characterized by using a polyphasic taxonomic approach. The predominant fatty acids of strain R27(T) were C18:1?7c (33.6%), C16:0 (16.3%), summed feature 3 (C16:1?7c and/or C16:1?6c; 15.8%) and C17:0 cyclo (8.7%) and its major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, three uncharacterized aminolipids and an unknown phospholipid. Strain R27(T) contained Q-8 as the dominant isoprenoid quinone and the G+C content of its genomic DNA was 64.6 mol%. On the basis of 16S rRNA gene sequence comparison, strain R27(T) showed 99.1% similarity to the closest related type strain, Burkholderia zhejiangensis OP-1(T), and 97.6% similarity to Burkholderia glathei ATCC 29195(T). However, the DNA-DNA relatedness between strain R27(T) and B. zhejiangensis CCTCC AB 2010354(T) and B. glathei ATCC 29195(T) was 10.2 and 14.9%, respectively. Based on 16S rRNA and rpoB gene sequence similarities and phenotypic and chemotaxonomic data, strain R27(T) is considered to represent a novel species of the genus Burkholderia, for which the name Burkholderia grimmiae sp. nov. is proposed. The type strain is R27(T) (=CGMCC 1.11013(T) =DSM 25160(T)). PMID:23087167

Tian, Yang; Kong, Bi He; Liu, Su Lin; Li, Chun Li; Yu, Rong; Liu, Lei; Li, Yan Hong

2013-06-01

327

Identification of Burkholderia spp. in the clinical microbiology laboratory: comparison of conventional and molecular methods  

Microsoft Academic Search

Cystic fibrosis (CF) predisposes patients to bacterial colonization and\\u000a infection of the lower airways. Several species belonging to the genus\\u000a Burkholderia are potential CF-related pathogens, but microbiological\\u000a identification may be complicated. This situation is not in the least due\\u000a to the poorly defined taxonomic status of these bacteria, and further\\u000a validation of the available diagnostic assays is required. A total

Pelt van C; C. M. Verduin; W. H. F. Goessens; M. C. Vos; B. Tummler; C. Segonds; F. Reubsaet; Belkum van A. F; H. A. Verbrugh

1999-01-01

328

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

PubMed

Burkholderia pathogenicity relies on protein virulence factors to control and promote bacterial internalization, survival, and replication within eukaryotic host cells. We recently used yeast two-hybrid (Y2H) screening to identify a small set of novel Burkholderia proteins that were shown to attenuate disease progression in an aerosol infection animal model using the virulent Burkholderia mallei ATCC 23344 strain. Here, we performed an extended analysis of primarily nine B. mallei virulence factors and their interactions with human proteins to map out how the bacteria can influence and alter host processes and pathways. Specifically, we employed topological analyses to assess the connectivity patterns of targeted host proteins, identify modules of pathogen-interacting host proteins linked to processes promoting infectivity, and evaluate the effect of crosstalk among the identified host protein modules. Overall, our analysis showed that the targeted host proteins generally had a large number of interacting partners and interacted with other host proteins that were also targeted by B. mallei proteins. We also introduced a novel Host-Pathogen Interaction Alignment (HPIA) algorithm and used it to explore similarities between host-pathogen interactions of B. mallei, Yersinia pestis, and Salmonella enterica. We inferred putative roles of B. mallei proteins based on the roles of their aligned Y. pestis and S. enterica partners and showed that up to 73% of the predicted roles matched existing annotations. A key insight into Burkholderia pathogenicity derived from these analyses of Y2H host-pathogen interactions is the identification of eukaryotic-specific targeted cellular mechanisms, including the ubiquitination degradation system and the use of the focal adhesion pathway as a fulcrum for transmitting mechanical forces and regulatory signals. This provides the mechanisms to modulate and adapt the host-cell environment for the successful establishment of host infections and intracellular spread. PMID:25738731

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

2015-03-01

329

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

PubMed Central

Burkholderia pathogenicity relies on protein virulence factors to control and promote bacterial internalization, survival, and replication within eukaryotic host cells. We recently used yeast two-hybrid (Y2H) screening to identify a small set of novel Burkholderia proteins that were shown to attenuate disease progression in an aerosol infection animal model using the virulent Burkholderia mallei ATCC 23344 strain. Here, we performed an extended analysis of primarily nine B. mallei virulence factors and their interactions with human proteins to map out how the bacteria can influence and alter host processes and pathways. Specifically, we employed topological analyses to assess the connectivity patterns of targeted host proteins, identify modules of pathogen-interacting host proteins linked to processes promoting infectivity, and evaluate the effect of crosstalk among the identified host protein modules. Overall, our analysis showed that the targeted host proteins generally had a large number of interacting partners and interacted with other host proteins that were also targeted by B. mallei proteins. We also introduced a novel Host-Pathogen Interaction Alignment (HPIA) algorithm and used it to explore similarities between host-pathogen interactions of B. mallei, Yersinia pestis, and Salmonella enterica. We inferred putative roles of B. mallei proteins based on the roles of their aligned Y. pestis and S. enterica partners and showed that up to 73% of the predicted roles matched existing annotations. A key insight into Burkholderia pathogenicity derived from these analyses of Y2H host-pathogen interactions is the identification of eukaryotic-specific targeted cellular mechanisms, including the ubiquitination degradation system and the use of the focal adhesion pathway as a fulcrum for transmitting mechanical forces and regulatory signals. This provides the mechanisms to modulate and adapt the host-cell environment for the successful establishment of host infections and intracellular spread. PMID:25738731

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

2015-01-01

330

Insights into the Role of Extracellular Polysaccharides in Burkholderia Adaptation to Different Environments  

PubMed Central

The genus Burkholderia comprises more than 60 species able to adapt to a wide range of environments such as soil and water, and also colonize and infect plants and animals. They have large genomes with multiple replicons and high gene number, allowing these bacteria to thrive in very different niches. Among the properties of bacteria from the genus Burkholderia is the ability to produce several types of exopolysaccharides (EPSs). The most common one, cepacian, is produced by the majority of the strains examined irrespective of whether or not they belong to the Burkholderia cepacia complex (Bcc). Cepacian biosynthesis proceeds by a Wzy-dependent mechanism, and some of the B. cepacia exopolysaccharide (Bce) proteins have been functionally characterized. In vitro studies showed that cepacian protects bacterial cells challenged with external stresses. Regarding virulence, bacterial cells with the ability to produce EPS are more virulent in several animal models of infection than their isogenic non-producing mutants. Although the production of EPS within the lungs of cystic fibrosis (CF) patients has not been demonstrated, the in vitro assessment of the mucoid phenotype in serial Bcc isolates from CF patients colonized for several years showed that mucoid to non-mucoid transitions are relatively frequent. This morphotype variation can be induced under laboratory conditions by exposing cells to stress such as high antibiotic concentration. Clonal isolates where mucoid to non-mucoid transition had occurred showed that during lung infection, genomic rearrangements, and mutations had taken place. Other phenotypic changes include variations in motility, chemotaxis, biofilm formation, bacterial survival rate under nutrient starvation and virulence. In this review, we summarize major findings related to EPS biosynthesis by Burkholderia and the implications in broader regulatory mechanisms important for cell adaptation to the different niches colonized by these bacteria. PMID:22919582

Ferreira, Ana S.; Silva, Inês N.; Oliveira, Vítor H.; Cunha, Raquel; Moreira, Leonilde M.

2011-01-01

331

Mechanism of resistance to an antitubercular 2-thiopyridine derivative that is also active against Burkholderia cenocepacia.  

PubMed

The discovery of new compounds that are able to inhibit the growth of Burkholderia cenocepacia is of primary importance for cystic fibrosis patients. Here, the mechanism of resistance to a new pyridine derivative already shown to be effective against Mycobacterium tuberculosis and to have good activity toward B. cenocepacia was investigated. Increased expression of a resistance-nodulation-cell division (RND) efflux system was detected in the resistant mutants, thus confirming their important roles in B. cenocepacia antibiotic resistance. PMID:24395233

Scoffone, Viola C; Spadaro, Francesca; Udine, Claudia; Makarov, Vadim; Fondi, Marco; Fani, Renato; De Rossi, Edda; Riccardi, Giovanna; Buroni, Silvia

2014-01-01

332

Extreme Antimicrobial Peptide and Polymyxin B Resistance in the Genus Burkholderia  

PubMed Central

Cationic antimicrobial peptides and polymyxins are a group of naturally occurring antibiotics that can also possess immunomodulatory activities. They are considered a new source of antibiotics for treating infections by bacteria that are resistant to conventional antibiotics. Members of the genus Burkholderia, which includes various human pathogens, are inherently resistant to antimicrobial peptides. The resistance is several orders of magnitude higher than that of other Gram-negative bacteria such as Escherichia coli, Salmonella enterica, or Pseudomonas aeruginosa. This review summarizes our current understanding of antimicrobial peptide and polymyxin B resistance in the genus Burkholderia. These bacteria possess major and minor resistance mechanisms that will be described in detail. Recent studies have revealed that many other emerging Gram-negative opportunistic pathogens may also be inherently resistant to antimicrobial peptides and polymyxins and we propose that Burkholderia sp. are a model system to investigate the molecular basis of the resistance in extremely resistant bacteria. Understanding resistance in these types of bacteria will be important if antimicrobial peptides come to be used regularly for the treatment of infections by susceptible bacteria because this may lead to increased resistance in the species that are currently susceptible and may also open up new niches for opportunistic pathogens with high inherent resistance. PMID:21811491

Loutet, Slade A.; Valvano, Miguel A.

2011-01-01

333

Extreme Antimicrobial Peptide and Polymyxin B Resistance in the Genus Burkholderia  

PubMed Central

Cationic antimicrobial peptides and polymyxins are a group of naturally occurring antibiotics that can also possess immunomodulatory activities. They are considered a new source of antibiotics for treating infections by bacteria that are resistant to conventional antibiotics. Members of the genus Burkholderia, which includes various human pathogens, are inherently resistant to antimicrobial peptides. The resistance is several orders of magnitude higher than that of other Gram-negative bacteria such as Escherichia coli, Salmonella enterica, or Pseudomonas aeruginosa. This review summarizes our current understanding of antimicrobial peptide and polymyxin B resistance in the genus Burkholderia. These bacteria possess major and minor resistance mechanisms that will be described in detail. Recent studies have revealed that many other emerging Gram-negative opportunistic pathogens may also be inherently resistant to antimicrobial peptides and polymyxins and we propose that Burkholderia sp. are a model system to investigate the molecular basis of the resistance in extremely resistant bacteria. Understanding resistance in these types of bacteria will be important if antimicrobial peptides come to be used regularly for the treatment of infections by susceptible bacteria because this may lead to increased resistance in the species that are currently susceptible and may also open up new niches for opportunistic pathogens with high inherent resistance. PMID:22919572

Loutet, Slade A.; Valvano, Miguel A.

2011-01-01

334

Identification of Specific and Universal Virulence Factors in Burkholderia cenocepacia Strains by Using Multiple Infection Hosts? †  

PubMed Central

Over the past few decades, strains of the Burkholderia cepacia complex have emerged as important pathogens for patients suffering from cystic fibrosis. Identification of virulence factors and assessment of the pathogenic potential of Burkholderia strains have increased the need for appropriate infection models. In previous studies, different infection hosts, including mammals, nematodes, insects, and plants, have been used. At present, however, the extent to which the virulence factors required to infect different hosts overlap is not known. The aim of this study was to analyze the roles of various virulence factors of two closely related Burkholderia cenocepacia strains, H111 and the epidemic strain K56-2, in a multihost pathogenesis system using four different model organisms, namely, Caenorhabditis elegans, Galleria mellonella, the alfalfa plant, and mice or rats. We demonstrate that most of the identified virulence factors are specific for one of the infection models, and only three factors were found to be essential for full pathogenicity in several hosts: mutants defective in (i) quorum sensing, (ii) siderophore production, and (iii) lipopolysaccharide biosynthesis were attenuated in at least three of the infection models and thus may represent promising targets for the development of novel anti-infectives. PMID:19528212

Uehlinger, Susanne; Schwager, Stephan; Bernier, Steve P.; Riedel, Kathrin; Nguyen, David T.; Sokol, Pamela A.; Eberl, Leo

2009-01-01

335

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

PubMed Central

Burkholderia species are key players in the accumulation of carbon from cellulose decomposition in coniferous forest ecosystems. We report here the draft genome of Burkholderia sordidicola strain S170, containing features associated with known genes involved in plant growth promotion, the biological control of plant diseases, and green remediation technologies. PMID:25125648

Xu, Zhuofei; Sørensen, Søren J.; Baldrian, Petr

2014-01-01

336

Burkholderia symbiotica sp. nov., isolated from root nodules of Mimosa spp. native to north-east Brazil.  

PubMed

Four strains, designated JPY-345(T), JPY-347, JPY-366 and JPY-581, were isolated from nitrogen-fixing nodules on the roots of two species of Mimosa, Mimosa cordistipula and Mimosa misera, that are native to North East Brazil, and their taxonomic positions were investigated by using a polyphasic approach. All four strains grew at 15-43 °C (optimum 35 °C), at pH 4-7 (optimum pH 5) and with 0-2 % (w/v) NaCl (optimum 0 % NaCl). On the basis of 16S rRNA gene sequence analysis, strain JPY-345(T) showed 97.3 % sequence similarity to the closest related species Burkholderia soli GP25-8(T), 97.3 % sequence similarity to Burkholderia caryophylli ATCC25418(T) and 97.1 % sequence similarity to Burkholderia kururiensis KP23(T). The predominant fatty acids of the strains were C(18 : 1)?7c (36.1 %), C(16 : 0) (19.8 %) and summed feature 3, comprising C(16 : 1)?7c and/or C(16 : 1)?6c (11.5 %). The major isoprenoid quinone was Q-8 and the DNA G+C content of the strains was 64.2-65.7 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized aminophospholipids and phospholipids. DNA-DNA hybridizations between the novel strain and recognized species of the genus Burkholderia yielded relatedness values of <51.8 %. On the basis of 16S rRNA and recA gene sequence similarities and chemotaxonomic and phenotypic data, the four strains represent a novel species in the genus Burkholderia, for which the name Burkholderia symbiotica sp. nov. is proposed. The type strain is JPY-345(T) (= LMG 26032(T) = BCRC 80258(T) = KCTC 23309(T)). PMID:22081715

Sheu, Shih-Yi; Chou, Jui-Hsing; Bontemps, Cyril; Elliott, Geoffrey N; Gross, Eduardo; James, Euan K; Sprent, Janet I; Young, J Peter W; Chen, Wen-Ming

2012-09-01

337

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

PubMed Central

Background Genomic analysis of bacteriophages infecting the Burkholderia cepacia complex (BCC) is an important preliminary step in the development of a phage therapy protocol for these opportunistic pathogens. The objective of this study was to characterize KL1 (vB_BceS_KL1) and AH2 (vB_BceS_AH2), two novel Burkholderia cenocepacia-specific siphoviruses isolated from environmental samples. Results KL1 and AH2 exhibit several unique phenotypic similarities: they infect the same B. cenocepacia strains, they require prolonged incubation at 30°C for the formation of plaques at low titres, and they do not form plaques at similar titres following incubation at 37°C. However, despite these similarities, we have determined using whole-genome pyrosequencing that these phages show minimal relatedness to one another. The KL1 genome is 42,832 base pairs (bp) in length and is most closely related to Pseudomonas phage 73 (PA73). In contrast, the AH2 genome is 58,065 bp in length and is most closely related to Burkholderia phage BcepNazgul. Using both BLASTP and HHpred analysis, we have identified and analyzed the putative virion morphogenesis, lysis, DNA binding, and MazG proteins of these two phages. Notably, MazG homologs identified in cyanophages have been predicted to facilitate infection of stationary phase cells and may contribute to the unique plaque phenotype of KL1 and AH2. Conclusions The nearly indistinguishable phenotypes but distinct genomes of KL1 and AH2 provide further evidence of both vast diversity and convergent evolution in the BCC-specific phage population. PMID:22676492

2012-01-01

338

Evolution of an endofungal Lifestyle: Deductions from the Burkholderia rhizoxinica Genome  

PubMed Central

Background Burkholderia rhizoxinica is an intracellular symbiont of the phytopathogenic zygomycete Rhizopus microsporus, the causative agent of rice seedling blight. The endosymbiont produces the antimitotic macrolide rhizoxin for its host. It is vertically transmitted within vegetative spores and is essential for spore formation of the fungus. To shed light on the evolution and genetic potential of this model organism, we analysed the whole genome of B. rhizoxinica HKI 0454 - a type strain of endofungal Burkholderia species. Results The genome consists of a structurally conserved chromosome and two plasmids. Compared to free-living Burkholderia species, the genome is smaller in size and harbors less transcriptional regulator genes. Instead, we observed accumulation of transposons over the genome. Prediction of primary metabolic pathways and transporters suggests that endosymbionts consume host metabolites like citrate, but might deliver some amino acids and cofactors to the host. The rhizoxin biosynthesis gene cluster shows evolutionary traces of horizontal gene transfer. Furthermore, we analysed gene clusters coding for nonribosomal peptide synthetases (NRPS). Notably, B. rhizoxinica lacks common genes which are dedicated to quorum sensing systems, but is equipped with a large number of virulence-related factors and putative type III effectors. Conclusions B. rhizoxinica is the first endofungal bacterium, whose genome has been sequenced. Here, we present models of evolution, metabolism and tools for host-symbiont interaction of the endofungal bacterium deduced from whole genome analyses. Genome size and structure suggest that B. rhizoxinica is in an early phase of adaptation to the intracellular lifestyle (genome in transition). By analysis of tranporters and metabolic pathways we predict how metabolites might be exchanged between the symbiont and its host. Gene clusters for biosynthesis of secondary metabolites represent novel targets for genomic mining of cryptic natural products. In silico analyses of virulence-associated genes, secreted proteins and effectors might inspire future studies on molecular mechanisms underlying bacterial-fungal interaction. PMID:21539752

2011-01-01

339

Purine biosynthesis-deficient Burkholderia mutants are incapable of symbiotic accommodation in the stinkbug  

PubMed Central

The Riptortus–Burkholderia symbiotic system represents a promising experimental model to study the molecular mechanisms involved in insect–bacterium symbiosis due to the availability of genetically manipulated Burkholderia symbiont. Using transposon mutagenesis screening, we found a symbiosis-deficient mutant that was able to colonize the host insect but failed to induce normal development of host's symbiotic organ. The disrupted gene was identified as purL involved in purine biosynthesis. In vitro growth impairment of the purL mutant and its growth dependency on adenine and adenosine confirmed the functional disruption of the purine synthesis gene. The purL mutant also showed defects in biofilm formation, and this defect was not rescued by supplementation of purine derivatives. When inoculated to host insects, the purL mutant was initially able to colonize the symbiotic organ but failed to attain a normal infection density. The low level of infection density of the purL mutant attenuated the development of the host's symbiotic organ at early instar stages and reduced the host's fitness throughout the nymphal stages. Another symbiont mutant-deficient in a purine biosynthesis gene, purM, showed phenotypes similar to those of the purL mutant both in vitro and in vivo, confirming that the purL phenotypes are due to disrupted purine biosynthesis. These results demonstrate that the purine biosynthesis genes of the Burkholderia symbiont are critical for the successful accommodation of symbiont within the host, thereby facilitating the development of the host's symbiotic organ and enhancing the host's fitness values. PMID:24088627

Kim, Jiyeun Kate; Jang, Ho Am; Won, Yeo Jin; Kikuchi, Yoshitomo; Heum Han, Sang; Kim, Chan-Hee; Nikoh, Naruo; Fukatsu, Takema; Lee, Bok Luel

2014-01-01

340

Isolation and Characterization of Burkholderia rinojensis sp. nov., a Non-Burkholderia cepacia Complex Soil Bacterium with Insecticidal and Miticidal Activities  

PubMed Central

Isolate A396, a bacterium isolated from a Japanese soil sample demonstrated strong insecticidal and miticidal activities in laboratory bioassays. The isolate was characterized through biochemical methods, fatty acid methyl ester (FAME) analysis, sequencing of 16S rRNA, multilocus sequence typing and analysis, and DNA-DNA hybridization. FAME analysis matched A396 to Burkholderia cenocepacia, but this result was not confirmed by 16S rRNA or DNA-DNA hybridization. 16S rRNA sequencing indicated closest matches with B. glumae and B. plantarii. DNA-DNA hybridization experiments with B. plantarii, B. glumae, B. multivorans, and B. cenocepacia confirmed the low genetic similarity (11.5 to 37.4%) with known members of the genus. PCR-based screening showed that A396 lacks markers associated with members of the B. cepacia complex. Bioassay results indicated two mechanisms of action: through ingestion and contact. The isolate effectively controlled beet armyworms (Spodoptera exigua; BAW) and two-spotted spider mites (Tetranychus urticae; TSSM). In diet overlay bioassays with BAW, 1% to 4% (vol/vol) dilution of the whole-cell broth caused 97% to 100% mortality 4 days postexposure, and leaf disc treatment bioassays attained 75% ± 22% mortality 3 days postexposure. Contact bioassays led to 50% larval mortality, as well as discoloration, stunting, and failure to molt. TSSM mortality reached 93% in treated leaf discs. Activity was maintained in cell-free supernatants and after heat treatment (60°C for 2 h), indicating that a secondary metabolite or excreted thermostable enzyme might be responsible for the activity. Based on these results, we describe the novel species Burkholderia rinojensis, a good candidate for the development of a biocontrol product against insect and mite pests. PMID:24096416

Fernandez, Lorena E.; Koivunen, Marja; Yang, April; Flor-Weiler, Lina; Marrone, Pamela G.

2013-01-01

341

Isolation and characterization of Burkholderia rinojensis sp. nov., a non-Burkholderia cepacia complex soil bacterium with insecticidal and miticidal activities.  

PubMed

Isolate A396, a bacterium isolated from a Japanese soil sample demonstrated strong insecticidal and miticidal activities in laboratory bioassays. The isolate was characterized through biochemical methods, fatty acid methyl ester (FAME) analysis, sequencing of 16S rRNA, multilocus sequence typing and analysis, and DNA-DNA hybridization. FAME analysis matched A396 to Burkholderia cenocepacia, but this result was not confirmed by 16S rRNA or DNA-DNA hybridization. 16S rRNA sequencing indicated closest matches with B. glumae and B. plantarii. DNA-DNA hybridization experiments with B. plantarii, B. glumae, B. multivorans, and B. cenocepacia confirmed the low genetic similarity (11.5 to 37.4%) with known members of the genus. PCR-based screening showed that A396 lacks markers associated with members of the B. cepacia complex. Bioassay results indicated two mechanisms of action: through ingestion and contact. The isolate effectively controlled beet armyworms (Spodoptera exigua; BAW) and two-spotted spider mites (Tetranychus urticae; TSSM). In diet overlay bioassays with BAW, 1% to 4% (vol/vol) dilution of the whole-cell broth caused 97% to 100% mortality 4 days postexposure, and leaf disc treatment bioassays attained 75% ± 22% mortality 3 days postexposure. Contact bioassays led to 50% larval mortality, as well as discoloration, stunting, and failure to molt. TSSM mortality reached 93% in treated leaf discs. Activity was maintained in cell-free supernatants and after heat treatment (60°C for 2 h), indicating that a secondary metabolite or excreted thermostable enzyme might be responsible for the activity. Based on these results, we describe the novel species Burkholderia rinojensis, a good candidate for the development of a biocontrol product against insect and mite pests. PMID:24096416

Cordova-Kreylos, Ana Lucia; Fernandez, Lorena E; Koivunen, Marja; Yang, April; Flor-Weiler, Lina; Marrone, Pamela G

2013-12-01

342

Involvement of Two Plasmids in Fenitrothion Degradation by Burkholderia sp. Strain NF100  

PubMed Central

A bacterium capable of utilizing fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate) as a sole carbon source was isolated from fenitrothion-treated soil. This bacterium was characterized taxonomically as being a member of the genus Burkholderia and was designated strain NF100. NF100 first hydrolyzed an organophosphate bond of fenitrothion, forming 3-methyl-4-nitrophenol, which was further metabolized to methylhydroquinone. The ability to degrade fenitrothion was found to be encoded on two plasmids, pNF1 and pNF2. PMID:10742273

Hayatsu, Masahito; Hirano, Motoko; Tokuda, Shinichi

2000-01-01

343

Fatal Burkholderia gladioli infection misidentified as Empedobacter brevis in a lung transplant recipient with cystic fibrosis.  

PubMed

Data describing the risk of lung transplantation (LT), clinical features, and outcomes of patients with cystic fibrosis (CF) infected with Burkholderia gladioli are limited. Herein, we report a case of disseminated B. gladioli infection characterized by bacteremia, necrotizing pneumonia, lung abscess, and empyema in a lung transplant recipient with CF, highlight the importance of accurate microbiological identification, and review published outcomes of LT in CF patients infected with B. gladioli, which include cases of pneumonia, tracheobronchitis, bacteremia, and abscesses, and demonstrate an all-cause 1-year mortality of approximately 23%, often after combined medical and surgical treatment. PMID:22429703

Brizendine, K D; Baddley, J W; Pappas, P G; Leon, K J; Rodriguez, J M

2012-08-01

344

The Promise of Bacteriophage Therapy for Burkholderia cepacia Complex Respiratory Infections  

PubMed Central

In recent times, increased attention has been given to evaluating the efficacy of phage therapy, especially in scenarios where the bacterial infectious agent of interest is highly antibiotic resistant. In this regard, phage therapy is especially applicable to infections caused by the Burkholderia cepacia complex (BCC) since members of the BCC are antibiotic pan-resistant. Current studies in BCC phage therapy are unique from many other avenues of phage therapy research in that the investigation is not only comprised of phage isolation, in vitro phage characterization and assessment of in vivo infection model efficacy, but also adapting aerosol drug delivery techniques to aerosol phage formulation delivery and storage. PMID:22919592

Semler, Diana D.; Lynch, Karlene H.; Dennis, Jonathan J.

2012-01-01

345

Comparative Evaluation of the BD Phoenix and VITEK 2 Automated Instruments for Identification of Isolates of the Burkholderia cepacia Complex  

Microsoft Academic Search

We evaluated two new automated identification systems, the BD Phoenix (Becton Dickinson) and the VITEK 2 (bioMérieux), for identification of isolates of the Burkholderia cepacia complex (BCC). The test sample included 42 isolates of the highly virulent and epidemic genomovar III, 45 isolates of B. multivorans, and 47 isolates of other members of the BCC. Rates of correct identification by

Sylvain Brisse; Stefania Stefani; Jan Verhoef; Alex Van Belkum; Peter Vandamme; Wil Goessens

2002-01-01

346

South African Papilionoid Legumes Are Nodulated by Diverse Burkholderia with Unique Nodulation and Nitrogen-Fixation Loci  

PubMed Central

The root-nodule bacteria of legumes endemic to the Cape Floristic Region are largely understudied, even though recent reports suggest the occurrence of nodulating Burkholderia species unique to the region. In this study, we considered the diversity and evolution of nodulating Burkholderia associated with the endemic papilionoid tribes Hypocalypteae and Podalyrieae. We identified distinct groups from verified rhizobial isolates by phylogenetic analyses of the 16S rRNA and recA housekeeping gene regions. In order to gain insight into the evolution of the nodulation and diazotrophy of these rhizobia we analysed the genes encoding NifH and NodA. The majority of these 69 isolates appeared to be unique, potentially representing novel species. Evidence of horizontal gene transfer determining the symbiotic ability of these Cape Floristic Region isolates indicate evolutionary origins distinct from those of nodulating Burkholderia from elsewhere in the world. Overall, our findings suggest that Burkholderia species associated with fynbos legumes are highly diverse and their symbiotic abilities have unique ancestries. It is therefore possible that the evolution of these bacteria is closely linked to the diversification and establishment of legumes characteristic of the Cape Floristic Region. PMID:23874611

Beukes, Chrizelle W.; Venter, Stephanus N.; Law, Ian J.; Phalane, Francina L.; Steenkamp, Emma T.

2013-01-01

347

APPLICATION OF BURKHOLDERIA CEPACIA AND TRICHODERMA VIRENS , ALONE AND IN COMBINATIONS, AGAINST MELOIDOGYNE INCOGNITA ON BELL PEPPER  

Microsoft Academic Search

Meyer, S. L. F, D. P. Roberts, D. J. Chitwood, L. K. Carta, R. D. Lumsden, and W. Mao. 2001. Applica- tion of Burkholderia cepacia and Trichoderma virens , alone and in combinations, against Meloidogyne in- cognita on bell pepper. Nematropica 31:75-86. Bell pepper ( Capsicum annuum L.) seeds and seedlings were treated with three potentially benefi- cial microbes, applied

Susan L. F. Meyer; Daniel P. Roberts; David J. Chitwood; Lynn K. Carta; Robert D. Lumsden; Weili Mao

2001-01-01

348

South african papilionoid legumes are nodulated by diverse burkholderia with unique nodulation and nitrogen-fixation Loci.  

PubMed

The root-nodule bacteria of legumes endemic to the Cape Floristic Region are largely understudied, even though recent reports suggest the occurrence of nodulating Burkholderia species unique to the region. In this study, we considered the diversity and evolution of nodulating Burkholderia associated with the endemic papilionoid tribes Hypocalypteae and Podalyrieae. We identified distinct groups from verified rhizobial isolates by phylogenetic analyses of the 16S rRNA and recA housekeeping gene regions. In order to gain insight into the evolution of the nodulation and diazotrophy of these rhizobia we analysed the genes encoding NifH and NodA. The majority of these 69 isolates appeared to be unique, potentially representing novel species. Evidence of horizontal gene transfer determining the symbiotic ability of these Cape Floristic Region isolates indicate evolutionary origins distinct from those of nodulating Burkholderia from elsewhere in the world. Overall, our findings suggest that Burkholderia species associated with fynbos legumes are highly diverse and their symbiotic abilities have unique ancestries. It is therefore possible that the evolution of these bacteria is closely linked to the diversification and establishment of legumes characteristic of the Cape Floristic Region. PMID:23874611

Beukes, Chrizelle W; Venter, Stephanus N; Law, Ian J; Phalane, Francina L; Steenkamp, Emma T

2013-01-01

349

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

PubMed Central

Burkholderia cenocepacia is considered an opportunistic pathogen from humans and may cause disease in plants. A bioprospection from a plaguicide-contaminated agricultural field in Mexico identified several methyl parathion-degrading bacteria. Here, we report the draft genome sequence of B. cenocepacia strain CEIB S5-1, which gave us clues into ecological biodiversity. PMID:25744996

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

2015-01-01

350

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

PubMed

Burkholderia cenocepacia is considered an opportunistic pathogen from humans and may cause disease in plants. A bioprospection from a plaguicide-contaminated agricultural field in Mexico identified several methyl parathion-degrading bacteria. Here, we report the draft genome sequence of B. cenocepacia strain CEIB S5-1, which gave us clues into ecological biodiversity. PMID:25744996

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

2015-01-01

351

A RAPD-derived STS marker is linked to a bacterial wilt ( Burkholderia caryophylli ) resistance gene in carnation  

Microsoft Academic Search

Bacterial wilt caused by Burkholderia caryophylli is one of the most important and damaging diseases of carnations (Dianthus caryophyllus) in Japan. We aimed to identify random amplified polymorphic DNA (RAPD) markers associated with the genes controlling bacterial wilt resistance in a resistance-segregating population of 134 progeny plants derived from a cross between ‘Carnation Nou No. 1’ (a carnation breeding line

Takashi Onozaki; Natsu Tanikawa; Mitsuyasu Taneya; Kiyofumi Kudo; Takuya Funayama; Hiroshi Ikeda; Michio Shibata

2004-01-01

352

Suppression of Bacterial Wilt and Fusarium Wilt by a Burkholderia nodosa Strain Isolated from Kalimantan Soils, Indonesia.  

PubMed

A trial was conducted to suppress bacterial wilt of tomato (BWT) caused by Ralstonia solanacearum using biocontrol agents (BCAs) isolated from soils in Kalimantan, Indonesia. Five isolates were selected from 270 isolates as better performing BCAs through screening four times using a pumice medium. The isolates selected were identified as Burkholderia nodosa, Burkholderia sacchari, Burkholderia pyrrocinia and Burkholderia terricola according to 16S rDNA sequences, fatty acid composition and carbon source utilization patterns. Among them, B. nodosa G5.2.rif1 had significant suppressive effects on Fusarium wilt of tomato (FWT) and spinach (FWS) as well as BWT. When B. nodosa G5.2rif1 was inoculated into a pumice medium in combination with sucrose, it showed even more stable disease suppression for BWT, but not for FWS. This suppression was considered to mainly occur through competition for nutrients. In two times greenhouse experiments for BWT using pots comparable in size to those used commercially, B. nodosa G5.2rif1 significantly suppressed the disease index by 33-79%, with no inhibitory effects on the growth, yield and quality of tomatoes. PMID:21558699

Nion, Yanetri Asi; Toyota, Koki

2008-01-01

353

Improvement of bioremediation by Pseudomonas and Burkholderia by mutants of the Vitreoscilla hemoglobin gene ( vgb ) integrated into their chromosomes  

Microsoft Academic Search

Using genetic engineering, the Vitreoscilla (bacterial) hemoglobin gene (vgb) was integrated stably into the chromosomes of Pseudomonas aeruginosa and Burkholderia sp. strain DNT. This was done for both wild type vgb and two site-directed mutants of vgb that produce Vitreoscilla hemoglobin (VHb) with lowered oxygen affinities; in all cases functional VHb was expressed. Similar to previous results, the wild type

Yongsoon Kim; Dale A. Webster; Benjamin C. Stark

2005-01-01

354

Polyhydroxyalkanoate biosynthesis and simultaneous remotion of organic inhibitors from sugarcane bagasse hydrolysate by Burkholderia sp.  

PubMed

Burkholderia sp. F24, originally isolated from soil, was capable of growth on xylose and removed organic inhibitors present in a hemicellulosic hydrolysate and simultaneously produced poly-3-hydroxybutyrate (P3HB). Using non-detoxified hydrolysate, Burkholderia sp. F24 reached a cell dry weight (CDW) of 6.8 g L(-1), containing 48 % of P3HB and exhibited a volumetric productivity (PP3HB) of 0.10 g L(-1) h(-1). Poly-3-hydroxybutyrate-co-3-hydroxyvalerate copolymers (P3HB-co-3HV) were produced using xylose and levulinic acid (LA) as carbon sources. In shake flask cultures, the 3HV content in the copolymer increased from 9 to 43 mol% by adding LA from 1.0 to 5.0 g L(-1). In high cell density cultivation using concentrated hemicellulosic hydrolysate F24 reached 25.04 g L(-1) of CDW containing 49 % of P3HB and PP3HB of 0.28 g L(-1 )h(-1). Based on these findings, second-generation ethanol and bioplastics from sugarcane bagasse is proposed. PMID:25059637

Lopes, Mateus Schreiner Garcez; Gomez, José Gregório Cabrera; Taciro, Marilda Keico; Mendonça, Thatiane Teixeira; Silva, Luiziana Ferreira

2014-09-01

355

Tangled bank of experimentally evolved Burkholderia biofilms reflects selection during chronic infections.  

PubMed

How diversity evolves and persists in biofilms is essential for understanding much of microbial life, including the uncertain dynamics of chronic infections. We developed a biofilm model enabling long-term selection for daily adherence to and dispersal from a plastic bead in a test tube. Focusing on a pathogen of the cystic fibrosis lung, Burkholderia cenocepacia, we sequenced clones and metagenomes to unravel the mutations and evolutionary forces responsible for adaptation and diversification of a single biofilm community during 1,050 generations of selection. The mutational patterns revealed recurrent evolution of biofilm specialists from generalist types and multiple adaptive alleles at relatively few loci. Fitness assays also demonstrated strong interference competition among contending mutants that preserved genetic diversity. Metagenomes from five other independently evolved biofilm lineages revealed extraordinary mutational parallelism that outlined common routes of adaptation, a subset of which was found, surprisingly, in a planktonic population. These mutations in turn were surprisingly well represented among mutations that evolved in cystic fibrosis isolates of both Burkholderia and Pseudomonas. These convergent pathways included altered metabolism of cyclic diguanosine monophosphate, polysaccharide production, tricarboxylic acid cycle enzymes, global transcription, and iron scavenging. Evolution in chronic infections therefore may be driven by mutations in relatively few pathways also favored during laboratory selection, creating hope that experimental evolution may illuminate the ecology and selective dynamics of chronic infections and improve treatment strategies. PMID:23271804

Traverse, Charles C; Mayo-Smith, Leslie M; Poltak, Steffen R; Cooper, Vaughn S

2013-01-15

356

Stenotrophomonas, Achromobacter, and nonmelioid Burkholderia species: antimicrobial resistance and therapeutic strategies.  

PubMed

Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and nonmelioid Burkholderia species, namely, Burkholderia cepacia complex, collectively are a group of troublesome nonfermenters. Although not inherently virulent organisms, these environmental Gram negatives can complicate treatment in those who are immunocompromised, critically ill in the intensive care unit and those patients with suppurative lung disease, such as cystic fibrosis. Through a range of intrinsic antimicrobial resistance mechanisms, virulence factors, and the ability to survive in biofilms, these opportunistic pathogens are well suited to persist, both in the environment and the host. Treatment recommendations are hindered by the difficulties in laboratory identification, the lack of reproducibility of antimicrobial susceptibility testing, the lack of clinical breakpoints, and the absence of clinical outcome data. Despite trimethoprim-sulfamethoxazole often being the mainstay of treatment, resistance is widely encountered, and alternative regimens, including combination therapy, are often used. This review will highlight the important aspects and unique challenges that these three nonfermenters pose, and, in the absence of clinical outcome data, our therapeutic recommendations will be based on reported antimicrobial susceptibility and pharmacokinetic/pharmacodynamic profiles. PMID:25643274

Abbott, Iain J; Peleg, Anton Y

2015-02-01

357

Substrate preference of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase in Burkholderia thailandensis.  

PubMed

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) plays crucial roles in the production of autoinducers and methionine metabolism. Putative genes encoding MTAN and AdoHcyase from Burkholderia thailandensis were cloned and characterized. The K(m) values of MTAN for 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) were 19 and 58 ?M, respectively. The catalytic efficiency of MTAN for SAH was only 0.004% of the value for MTA, indicating an almost complete substrate preference of MTAN for MTA. The results of autoinducer-2 assay of B. thailandensis and recombinants indicated that LuxS enzyme activity was lacking in Burkholderia species. Instead, AdoHcyase hydrolysed SAH directly to homocysteine and adenosine in the activated methyl cycle. Meanwhile, the K(m) value of AdoHcyase for SAH was determined to be 40 ?M. Sequence analysis revealed that MTAN had much higher diversity than AdoHcyase, which likely contributes to its substrate preference for MTA. Furthermore, the phylogenetic tree of MTAN sequences revealed that LuxS(+) bacteria could be discriminated from LuxS(-) bacteria. These results suggested that the substrate preference of MTAN for MTA and SAH degradation pathway evolved with the bacterial-activated methyl cycle. PMID:23198993

Gao, Qiang; Zheng, Dasheng; Yuan, Zhiming

2013-02-01

358

Application of lipase from Burkholderia cepacia in the degradation of agro-industrial effluent.  

PubMed

This study aimed to analyze the physical and chemical characteristics of Amano PS commercial lipase - Burkholderia cepacia and lipase produced by Burkholderia cepacia strain ATCC 25416, in addition to studying the hydrolysis of agro-industrial effluent collected in a fried potato industry. The optimum temperature for increasing lipase activity was 37 °C. The temperature increase caused a decrease in thermostability of lipase, and the commercial lipase was less stable, with values of 10.5, 4.6 and 4.9%, respectively, lower than those obtained by lipase from strain ATCC 25416, at temperatures of 40, 50 and 60 °C. The enzymatic activity was higher in alkaline conditions, achieving better results at pH 8.0. The pH was the variable that most influenced the hydrolysis of triacylglycerides of the agro-industrial effluent, followed by enzyme concentration, and volume of gum arabic used in the reaction medium. Thus, it can be observed that the enzymatic hydrolytic process of the studied effluent presents a premising contribution to reduction of environmental impacts of potato chip processing industries. PMID:25860696

Mello Bueno, Pabline Rafaella; de Oliveira, Tatianne Ferreira; Castiglioni, Gabriel Luis; Soares Júnior, Manoel Soares; Ulhoa, Cirano Jose

2015-01-01

359

PCR-based detection and identification of Burkholderia cepacia complex pathogens in sputum from cystic fibrosis patients.  

PubMed

PCR amplification of the recA gene followed by restriction fragment length polymorphism (RFLP) analysis was investigated for the rapid detection and identification of Burkholderia cepacia complex genomovars directly from sputum. Successful amplification of the B. cepacia complex recA gene from cystic fibrosis (CF) patient sputum samples containing B. cepacia genomovar I, Burkholderia multivorans, B. cepacia genomovar III, Burkholderia stabilis, and Burkholderia vietnamiensis was demonstrated. In addition, the genomovar identifications determined directly from sputum were the same as those obtained after selective culturing. Sensitivity experiments revealed that recA-based PCR could reliably detect B. cepacia complex organisms to concentrations of 10(6) CFU g of sputum(-1). To fully assess the diagnostic value of the method, sputum samples from 100 CF patients were screened for B. cepacia complex infection by selective culturing and recA-based PCR. Selective culturing identified 19 samples with presumptive B. cepacia complex infection, which was corroborated by phenotypic analyses. Of the culture-positive sputum samples, 17 were also detected directly by recA-based PCR, while 2 samples were negative. The isolates cultured from both recA-negative sputum samples were subsequently identified as Burkholderia gladioli. RFLP analysis of the recA amplicons revealed 2 patients (12%) infected with B. multivorans, 11 patients (65%) infected with B. cepacia genomovar III-A, and 4 patients (23%) infected with B. cepacia genomovar III-B. These results demonstrate the potential of recA-based PCR-RFLP analysis for the rapid detection and identification of B. cepacia complex genomovars directly from sputum. Where the sensitivity of the assay proves a limitation, sputum samples can be analyzed by selective culturing followed by recA-based analysis of the isolate. PMID:11724828

McDowell, A; Mahenthiralingam, E; Moore, J E; Dunbar, K E; Webb, A K; Dodd, M E; Martin, S L; Millar, B C; Scott, C J; Crowe, M; Elborn, J S

2001-12-01

360

Gut Symbiotic Bacteria of the Genus Burkholderia in the Broad-Headed Bugs Riptortus clavatus and Leptocorisa chinensis (Heteroptera: Alydidae)  

PubMed Central

The Japanese common broad-headed bugs Riptortus clavatus and Leptocorisa chinensis possess a number of crypts in the posterior region of the midgut, whose lumen contains a copious amount of bacterial cells. We characterized the gut symbiotic bacteria by using molecular phylogenetic analysis, light and electron microscopy, in situ hybridization, and PCR-based detection techniques. Restriction fragment length polymorphism analysis of 16S rRNA gene clones suggested that a single bacterium dominated the microbiota in the crypts of the both bug species. The predominant 16S rRNA gene sequences obtained from different individuals and species of the bugs were not identical but were very similar to each other. Homology searches in the DNA databases revealed that the sequences showed the highest levels of similarity (96% to 99%) to the sequences of Burkholderia spp. belonging to the ? subdivision of the class Proteobacteria. In situ hybridization with specific oligonucleotide probes confirmed the localization of the Burkholderia symbiont in the lumen of the midgut crypts. Electron microscopy showed that the lumen of the crypts was filled with rod-shaped bacteria of a single morphotype. Molecular phylogenetic analysis demonstrated that the Burkholderia symbionts of the bugs formed a well-defined monophyletic group, although the group also contained several environmental Burkholderia strains. The phylogenetic relationship of the Burkholderia symbionts did not reflect the relationship of the host bug species at all. The sequences from R. clavatus and the sequences from L. chinensis did not form clades but were intermingled in the phylogeny, suggesting that horizontal transmission of the symbiont might have occasionally occurred between populations and species of the bugs. PMID:16000818

Kikuchi, Yoshitomo; Meng, Xian-Ying; Fukatsu, Takema

2005-01-01

361

IN SITU BIOREMEDIATION OF TRICHLOROETHYLENE USING BURKHOLDERIA CEPACIA G4 PR1: ANALYSIS OF MICROBIAL ECOLOGY PARAMETERS FOR RISK ASSESSMENT (RESEARCH BRIEF)  

EPA Science Inventory

The introduction of bacteria into aquifers for bioremediation purposes requires monitoring of the persistence and activity of microbial populations for efficacy and risk assessment purposes. Burkholderia cepacia G4 PR1 constitutively expresses a toluene ortho-monooxygenase (tom) ...

362

Characterization of a multimetal resistant Burkholderia fungorum isolated from an e-waste recycling area for its potential in Cd sequestration  

Microsoft Academic Search

The Cd resistance mechanism of a Burkholderia strain isolated from paddy soil of an electronic waste recycling site was characterized for its potential application in\\u000a metal bioremediation. 16S rRNA gene analysis revealed phylogenetic relatedness of this strain to Burkholderia fungorum. When grown in broth supplemented with Cd, this strain could decrease the bio-available Cd concentration through biosorption\\u000a of Cd with

Jun-hui Zhang; Hang Min

2010-01-01

363

Burkholderia dabaoshanensis sp. nov., a Heavy-Metal-Tolerant Bacteria Isolated from Dabaoshan Mining Area Soil in China  

PubMed Central

Heavy-metal-tolerant bacteria, GIMN1.004T, was isolated from mine soils of Dabaoshan in South China, which were acidic (pH 2–4) and polluted with heavy metals. The isolation was Gram-negative, aerobic, non-spore-forming, and rod-shaped bacteria having a cellular width of 0.5?0.6 µm and a length of 1.3?1.8 µm. They showed a normal growth pattern at pH 4.0–9.0 in a temperature ranging from 5°C to 40°C.The organism contained ubiquinone Q-8 as the predominant isoprenoid quinine, and C16?0, summed feature 8 (C18?1?7c and C18?1?6c), C18?0, summed feature 3 (C16?1?7c or iso-C15?0 2-OH), C17?0 cyclo, C18?1?9c, C19?0 cyclo ?8c, C14?0 as major fatty acid. These profiles were similar to those reported for Burkholderia species. The DNA G+C % of this strain was 61.6%. Based on the similarity to 16S rRNA gene sequence, GIMN1.004T was considered to be in the genus Burkholderia. The similarities of 16S rRNA gene sequence between strain GIMN1.004T and members of the genus Burkholderia were 96?99.4%, indicating that this novel strain was phylogenetically related to members of that genus. The novel strain showed the highest sequence similarities to Burkholderia soli DSM 18235T (99.4%); Levels of DNA-DNA hybridization with DSM 18235T was 25%. Physiological and biochemical tests including cell wall composition analysis, differentiated phenotype of this strain from that closely related Burkholderia species. The isolation had great tolerance to cadmium with MIC of 22 mmol/L, and adsorbability of 144.94 mg/g cadmium,and it was found to exhibit antibiotic resistance characteristics. The adsorptive mechanism of GIMN1.004T for cadmium depended on the action of the amide,carboxy and phosphate of cell surface and producing low-molecular-weight (LMW ) organic acids to complex or chelated Cd2+.Therefore, the strain GIMN1.004T represented a new cadmium resistance species, which was tentatively named as Burkholderia dabaoshanensis sp. nov. The strain type is GIMN1.004T (?=?CCTCC M 209109T?=? NRRL B-59553T ). PMID:23226514

Zhu, Honghui; Guo, Jianhua; Chen, Meibiao; Feng, Guangda; Yao, Qing

2012-01-01

364

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

PubMed

Trichloroethylene (TCE), a chlorinated organic solvent, is one of the most common and widespread groundwater contaminants worldwide. Among the group of TCE-degrading aerobic bacteria, Burkholderia cepacia G4 is the best-known representative. This strain requires the addition of specific substrates, including toluene, phenol, and benzene, to induce the enzymes to degrade TCE. However, the substrates are toxic and introducing them into the soil can result in secondary contamination. In this study, poplar leaf homogenate containing natural phenolic compounds was tested for the ability to induce the growth of and TCE degradation by B. cepacia G4. The results showed that the G4 strain could grow and degrade TCE well with the addition of phytochemicals. The poplar leaf homogenate also functioned as an inducer of the toluene-ortho-monooxygenase (TOM) gene in B. cepacia G4. PMID:24992516

Kang, Jun Won; Doty, Sharon Lafferty

2014-07-01

365

Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.  

PubMed Central

Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF). This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P. aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B. cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism. While understanding the mechanism of conversion to mucoidy in P. aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B. cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity. PMID:8840786

Govan, J R; Deretic, V

1996-01-01

366

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

PubMed

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

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

2014-06-15

367

Clinical and in vitro evidence for the antimicrobial therapy in Burkholderia cepacia complex infections.  

PubMed

Treatment of infections caused by Burkholderia cepacia complex (Bcc) in cystic fibrosis (CF) patients poses a complex problem. Bcc is multidrug-resistant due to innate and acquired mechanisms of resistance. As CF patients receive multiple courses of antibiotics, susceptibility patterns of strains from CF patients may differ from those noted in strains from non-CF patients. Thus, there was a need for assessing in vitro and clinical data to guide antimicrobial therapy in these patients. A systematic search of literature, followed by extraction and analysis of available information from human and in vitro studies was done. The results of the analysis are used to address various aspects like use of antimicrobials for pulmonary and non-pulmonary infections, use of combination versus monotherapy, early eradication, duration of therapy, route of administration, management of biofilms, development of resistance during therapy, pharmacokinetics-pharmacodynamics correlations, therapy in post-transplant patients and newer drugs in Bcc-infected CF patients. PMID:25772031

Gautam, Vikas; Shafiq, Nusrat; Singh, Meenu; Ray, Pallab; Singhal, Lipika; Jaiswal, Nishant P; Prasad, Amber; Singh, Shaunik; Agarwal, Amit

2015-05-01

368

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

PubMed Central

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

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

2013-01-01

369

Antimicrobial properties of an oxidizer produced by Burkholderia cenocepacia P525.  

PubMed

A compound with both oxidizing properties and antibiotic properties was extracted and purified from broth cultures of Burkholderia cenocepacia strain P525. A four step purification procedure was used to increase its specific activity ~400-fold and to yield a HPLC-UV chromatogram containing a single major peak. Size exclusion chromatography suggests a molecular mass of ~1,150 and UV spectroscopy suggests the presence of a polyene structure consisting of as many as six conjugated double bonds. Biological studies indicate that the compound is bacteriostatic. Enterobacter soli and E. aerogenes cells incubated with the compound exhibit a longer lag phase of growth. The bacteriostatic activity is greater at pH 3 than at pH 5. Bacteria such as B. cenocepacia strain P525 may have value in the agricultural industry as biocontrol agents. PMID:24384626

Hunter, William J; Manter, Dan K

2014-05-01

370

Optimization of Lipase Production by Burkholderia sp. Using Response Surface Methodology  

PubMed Central

Response surface methodology (RSM) was employed to optimize the extracellular lipase production by Burkholderia sp. HL-10. Preliminary tests showed that olive oil, tryptone and Tween-80 exhibited significant effects on the lipase production. The optimum concentrations of these three components were determined using a faced-centered central composite design (FCCCD). The analysis of variance revealed that the established model was significant (p < 0.01). The optimized medium containing 0.65% olive oil (v/v), 2.42% tryptone (w/v) and 0.15% Tween-80 (v/v) resulted in a maximum activity of 122.3 U/mL, about three fold higher than that in basal medium. Approximately 99% of validity of the predicted value was achieved. PMID:23203100

Lo, Chia-Feng; Yu, Chi-Yang; Kuan, I-Ching; Lee, Shiow-Ling

2012-01-01

371

Two quorum sensing systems control biofilm formation and virulence in members of the Burkholderia cepacia complex  

PubMed Central

The Burkholderia cepacia complex (Bcc) consists of 17 closely related species that are problematic opportunistic bacterial pathogens for cystic fibrosis patients and immunocompromised individuals. These bacteria are capable of utilizing two different chemical languages: N-acyl homoserine lactones (AHLs) and cis-2-unsaturated fatty acids. Here we summarize the current knowledge of the underlying molecular architectures of these communication systems, showing how they are interlinked and discussing how they regulate overlapping as well as specific sets of genes. A particular focus is laid on the role of these signaling systems in the formation of biofilms, which are believed to be highly important for chronic infections. We review genes that have been implicated in the sessile lifestyle of this group of bacteria. The new emerging role of the intracellular second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) as a downstream regulator of the fatty acid signaling cascade and as a key factor in biofilm formation is also discussed. PMID:23799665

Suppiger, Angela; Schmid, Nadine; Aguilar, Claudio; Pessi, Gabriella; Eberl, Leo

2013-01-01

372

Identification of aldolase and ferredoxin reductase within the dbt operon of Burkholderia fungorum DBT1.  

PubMed

Burkholderia fungorum DBT1, first isolated from settling particulate matter of an oil refinery wastewater, is a bacterial strain which has been shown capable of utilizing several polycyclic aromatic hydrocarbons (PAHs) including dibenzothiophene (DBT). In particular, this microbe is able to efficiently degrade DBT through the Kodama pathway. Previous investigations have lead to the identification of six genes, on a total of eight, required for DBT degradation. In the present study, a combined experimental/computational approach was adopted to identify and in silico characterize the two missing genes, namely a ferredoxin reductase and a hydratase-aldolase. Thus, the finding of all enzymatic components of the Kodama pathway in B. fungorum DBT1 makes this bacterial strain amenable for possible exploitation in soil bioremediation protocols. PMID:23686744

Piccoli, Stefano; Andreolli, Marco; Giorgetti, Alejandro; Zordan, Fabio; Lampis, Silvia; Vallini, Giovanni

2014-05-01

373

Diazotrophic Burkholderia species isolated from the Amazon region exhibit phenotypical, functional and genetic diversity.  

PubMed

Forty-eight Burkholderia isolates from different land use systems in the Amazon region were compared to type strains of Burkholderia species for phenotypic and functional characteristics that can be used to promote plant growth. Most of these isolates (n=46) were obtained by using siratro (Macroptilium atropurpureum - 44) and common bean (Phaseolus vulgaris - 2) as the trap plant species; two isolates were obtained from nodules collected in the field from Indigofera suffruticosa and Pithecellobium sp. The evaluated characteristics were the following: colony characterisation on "79" medium, assimilation of different carbon sources, enzymatic activities, solubilisation of phosphates, nitrogenase activity and antifungal activity against Fusarium oxysporium f. sp. phaseoli. Whole cell protein profiles, 16S rRNA, gyrB, and recA gene sequencing and multilocus sequence typing were used to identify the isolates. The isolates showed different cultural and biochemical characteristics depending on the legume species from which they were obtained. Except for one isolate from I. suffruticosa, all isolates were able to solubilise calcium phosphate and present nitrogenase activity under free-living conditions. Only one isolate from common beans, showed antifungal activity. The forty four isolates from siratro nodules were identified as B. fungorum; isolates UFLA02-27 and UFLA02-28, obtained from common bean plants, were identified as B. contaminans; isolate INPA89A, isolated from Indigofera suffruticosa, was a close relative of B. caribensis but could not be assigned to an established species; isolate INPA42B, isolated from Pithecellobium sp., was identified as B. lata. This is the first report of nitrogenase activity in B. fungorum, B. lata and B. contaminans. PMID:22609342

da Silva, Krisle; Cassetari, Alice de Souza; Lima, Adriana Silva; De Brandt, Evie; Pinnock, Eleanor; Vandamme, Peter; Moreira, Fatima Maria de Souza

2012-06-01

374

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

NASA Astrophysics Data System (ADS)

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

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

2014-09-01

375

Chemotaxis of Burkholderia sp. Strain SJ98 towards chloronitroaromatic compounds that it can metabolise  

PubMed Central

Background Burkholderia sp. strain SJ98 is known for its chemotaxis towards nitroaromatic compounds (NACs) that are either utilized as sole sources of carbon and energy or co-metabolized in the presence of alternative carbon sources. Here we test for the chemotaxis of this strain towards six chloro-nitroaromatic compounds (CNACs), namely 2-chloro-4-nitrophenol (2C4NP), 2-chloro-3-nitrophenol (2C3NP), 4-chloro-2-nitrophenol (4C2NP), 2-chloro-4-nitrobenzoate (2C4NB), 4-chloro-2-nitrobenzoate (4C2NB) and 5-chloro-2-nitrobenzoate (5C2NB), and examine its relationship to the degradation of such compounds. Results Strain SJ98 could mineralize 2C4NP, 4C2NB and 5C2NB, and co-metabolically transform 2C3NP and 2C4NB in the presence of an alternative carbon source, but was unable to transform 4C2NP under these conditions. Positive chemotaxis was only observed towards the five metabolically transformed CNACs. Moreover, the chemotaxis was induced by growth in the presence of the metabolisable CNAC. It was also competitively inhibited by the presence of nitroaromatic compounds (NACs) that it could metabolise but not by succinate or aspartate. Conclusions Burkholderia sp. strain SJ98 exhibits metabolic transformation of, and inducible chemotaxis towards CNACs. Its chemotactic responses towards these compounds are related to its previously demonstrated chemotaxis towards NACs that it can metabolise, but it is independently inducible from its chemotaxis towards succinate or aspartate. PMID:22292983

2012-01-01

376

Characterization of the Burkholderia thailandensis SOS Response by Using Whole-Transcriptome Shotgun Sequencing  

PubMed Central

The bacterial SOS response is a well-characterized regulatory network encoded by most prokaryotic bacterial species and is involved in DNA repair. In addition to nucleic acid repair, the SOS response is involved in pathogenicity, stress-induced mutagenesis, and the emergence and dissemination of antibiotic resistance. Using high-throughput sequencing technology (SOLiD RNA-Seq), we analyzed the Burkholderia thailandensis global SOS response to the fluoroquinolone antibiotic, ciprofloxacin (CIP), and the DNA-damaging chemical, mitomycin C (MMC). We demonstrate that a B. thailandensis recA mutant (RU0643) is ?4-fold more sensitive to CIP in contrast to the parental strain B. thailandensis DW503. Our RNA-Seq results show that CIP and MMC treatment (P < 0.01) resulted in the differential expression of 344 genes in B. thailandensis and 210 genes in RU0643. Several genes associated with the SOS response were induced and include lexA, uvrA, dnaE, dinB, recX, and recA. At the genome-wide level, we found an overall decrease in gene expression, especially for genes involved in amino acid and carbohydrate transport and metabolism, following both CIP and MMC exposure. Interestingly, we observed the upregulation of several genes involved in bacterial motility and enhanced transcription of a B. thailandensis genomic island encoding a Siphoviridae bacteriophage designated ?E264. Using B. thailandensis plaque assays and PCR with B. mallei ATCC 23344 as the host, we demonstrate that CIP and MMC exposure in B. thailandensis DW503 induces the transcription and translation of viable bacteriophage in a RecA-dependent manner. This is the first report of the SOS response in Burkholderia spp. to DNA-damaging agents. We have identified both common and unique adaptive responses of B. thailandensis to chemical stress and DNA damage. PMID:23872555

Ulrich, Ricky L.; DeShazer, David; Kenny, Tara A.; Ulrich, Melanie P.; Moravusova, Anna; Opperman, Timothy; Bavari, Sina; Bowlin, Terry L.; Moir, Donald T.

2013-01-01

377

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

PubMed Central

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

O’Grady, Eoin P.; Sokol, Pamela A.

2011-01-01

378

SNaPBcen: a Novel and Practical Tool for Genotyping Burkholderia cenocepacia  

PubMed Central

Burkholderia cenocepacia is the most prevalent and feared member of the Burkholderia cepacia complex in lung infections of cystic fibrosis (CF). Genotyping and monitoring of long-term colonization are critical at clinical units; however, the differentiation of specific lineages performed by multilocus sequence typing (MLST) is still limited to a small number of isolates due to the high cost and time-consuming procedure. The aim of this study was to optimize a protocol (the SNaPBcen assay) for extensive bacterial population studies. The strategy used for the SNaPBcen assay is based on targeting single nucleotide polymorphisms (SNPs) located in MLST genes instead of sequencing full MLST sequences. Nonpolymorphic and redundant MLST positions were eliminated, and a set of 24 polymorphisms included in the SNaPBcen assay ensures a high-resolution genomic characterization. These polymorphisms were identified based on the comparative analysis of 137 B. cenocepacia MLST profiles available online (http://pubmlst.org/bcc/). The group of 81 clinical isolates of B. cenocepacia examined in this study using the SNaPBcen assay revealed 51 distinct profiles, and a final discriminatory power of 0.9997 compared with MLST was determined. The SNaPBcen assay was able to reveal isolates with microvariations and the presence of multiple clonal variants in patients chronically colonized with B. cenocepacia. Main phylogenetic subgroups IIIA, IIIB, and IIIC of B. cenocepacia could be separated by the Gl94R polymorphism included in the panel. The SNaPBcen assay proved to be a rapid and robust alternative to the standard MLST for B. cenocepacia, allowing the simultaneous analysis of multiple polymorphisms following amplification and mini-sequencing reactions. PMID:23761147

Eusebio, Nadia; Coutinho, Carla P.; Sá-Correia, Isabel

2013-01-01

379

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

PubMed

The Burkholderia cepacia complex (Bcc) is a group of at least 18 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. Bcc organisms possess high levels of innate antimicrobial resistance, and alternative therapeutic strategies are urgently needed. One proposed alternative treatment is phage therapy, the therapeutic application of bacterial viruses (or bacteriophages). Recently, some phages have been observed to form larger plaques in the presence of sublethal concentrations of certain antibiotics; this effect has been termed phage-antibiotic synergy (PAS). Those reports suggest that some antibiotics stimulate increased production of phages under certain conditions. The aim of this study is to examine PAS in phages that infect Burkholderia cenocepacia strains C6433 and K56-2. Bcc phages KS12 and KS14 were tested for PAS, using 6 antibiotics representing 4 different drug classes. Of the antibiotics tested, the most pronounced effects were observed for meropenem, ciprofloxacin, and tetracycline. When grown with subinhibitory concentrations of these three antibiotics, cells developed a chain-like arrangement, an elongated morphology, and a clustered arrangement, respectively. When treated with progressively higher antibiotic concentrations, both the sizes of plaques and phage titers increased, up to a maximum. B. cenocepacia K56-2-infected Galleria mellonella larvae treated with phage KS12 and low-dose meropenem demonstrated increased survival over controls treated with KS12 or antibiotic alone. These results suggest that antibiotics can be combined with phages to stimulate increased phage production and/or activity and thus improve the efficacy of bacterial killing. PMID:25452284

Kamal, Fatima; Dennis, Jonathan J

2015-02-01

380

Endophytic Colonization of Vitis vinifera L. by Plant Growth-Promoting Bacterium Burkholderia sp. Strain PsJN  

Microsoft Academic Search

Patterns of colonization of Vitis vinifera L. cv. Chardonnay plantlets by a plant growth-promoting bacterium, Burkholderia sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with gfp (PsJN::gfp2x) or gusA (PsJN::gusA11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed

Stephane Compant; Birgit Reiter; Angela Sessitsch; Jerzy Nowak; Christophe Clement; E. Ait Barka

2005-01-01

381

Burkholderia xenovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility  

PubMed Central

Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven “central aromatic” and twenty “peripheral aromatic” pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes. PMID:17030797

Chain, Patrick S. G.; Denef, Vincent J.; Konstantinidis, Konstantinos T.; Vergez, Lisa M.; Agulló, Loreine; Reyes, Valeria Latorre; Hauser, Loren; Córdova, Macarena; Gómez, Luis; González, Myriam; Land, Miriam; Lao, Victoria; Larimer, Frank; LiPuma, John J.; Mahenthiralingam, Eshwar; Malfatti, Stephanie A.; Marx, Christopher J.; Parnell, J. Jacob; Ramette, Alban; Richardson, Paul; Seeger, Michael; Smith, Daryl; Spilker, Theodore; Sul, Woo Jun; Tsoi, Tamara V.; Ulrich, Luke E.; Zhulin, Igor B.; Tiedje, James M.

2006-01-01