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1

Evolution of Burkholderia pseudomallei in Recurrent Melioidosis  

PubMed Central

Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted whole-genome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis.

Hayden, Hillary S.; Lim, Regina; Brittnacher, Mitchell J.; Sims, Elizabeth H.; Ramage, Elizabeth R.; Fong, Christine; Wu, Zaining; Crist, Eva; Chang, Jean; Zhou, Yang; Radey, Matthew; Rohmer, Laurence; Haugen, Eric; Gillett, Will; Wuthiekanun, Vanaporn; Peacock, Sharon J.; Kaul, Rajinder; Miller, Samuel I.; Manoil, Colin; Jacobs, Michael A.

2012-01-01

2

Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei  

Microsoft Academic Search

Burkholderia pseudomallei is a potential bioterror agent and the causative agent of melioidosis, a severe disease that is endemic in areas of Southeast Asia and Northern Australia. Infection is often associated with bacterial dissemination to distant sites, and there are many possible disease manifestations, with melioidosis septic shock being the most severe. Eradication of the organism following infection is difficult,

Tom van der Poll; Nicholas J. White; Nicholas P. Day; Sharon J. Peacock; W. Joost Wiersinga

2006-01-01

3

Detection of Burkholderia pseudomallei DNA in Patients with Septicemic Melioidosis  

Microsoft Academic Search

Septicemic melioidosis is the most severe form of melioidosis, which is caused byBurkholderia pseudomallei. It is endemic in Southeast Asia and is the leading cause of death from community-acquired septicemia in northeast Thailand. A major factor that contributes to the high mortality is the delay in isolation and identificationofthecausativeorganism.Morethanhalfofthepatientsdiewithinthefirst2daysafterhospital admission, before bacterial cultures become positive. The present study was undertaken

TARARAJ DHARAKUL; SIRIRURG SONGSIVILAI; SIRITHIP VIRIYACHITRA; VORAVICH LUANGWEDCHAKARN; BOONRAT TASSANEETRITAP; ANDWIPADA CHAOWAGUL

1996-01-01

4

Less Is More: Burkholderia pseudomallei and Chronic Melioidosis  

PubMed Central

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

Nandi, Tannistha; Tan, Patrick

2013-01-01

5

Less is more: Burkholderia pseudomallei and chronic melioidosis.  

PubMed

The Gram-negative bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. Once considered an esoteric tropical disease confined to Southeast Asia and northern Australia, research on B. pseudomallei has recently gained global prominence due to its classification as a potential bioterrorism agent by countries such as the United States and also by increasing numbers of case reports from regions where it is not endemic. An environmental bacterium typically found in soil and water, assessing the true global prevalence of melioidosis is challenged by the fact that clinical symptoms associated with B. pseudomallei infection are extremely varied and may be confused with diverse conditions such as lung cancer, tuberculosis, or Staphyloccocus aureus infection. These diagnostic challenges, coupled with lack of awareness among clinicians, have likely contributed to underdiagnosis and the high mortality rate of melioidosis, as initial treatment is often either inappropriate or delayed. Even after antibiotic treatment, relapses are frequent, and after resolution of acute symptoms, chronic melioidosis can also occur, and the symptoms can persist for months to years. In a recent article, Price et al. [mBio 4(4):e00388-13, 2013, doi:10.1128/mBio.00388-13] demonstrate how comparative genomic sequencing can reveal the repertoire of genetic changes incurred by B. pseudomallei during chronic human infection. Their results have significant clinical ramifications and highlight B. pseudomallei's ability to survive in a wide range of potential niches within hosts, through the acquisition of genetic adaptations that optimize fitness and resource utilization. PMID:24065633

Nandi, Tannistha; Tan, Patrick

2013-09-24

6

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

PubMed

Burkholderia mallei and Burkholderia pseudomallei are the causative micro-organisms of Glanders and Melioidosis, respectively. Although now rare in Western countries, both micro-organisms have recently gained much interest because of their unique potential as bioterrorism agents. This paper reviews the epidemiology, pathogenesis, diagnosis and treatment of Melioidosis and Glanders. Recent patents relating to these micro-organisms, especially potential vaccines, are presented. Continued research and development is urgently needed, especially in regard to rapid and accurate diagnosis of melioidosis and glanders, efficacious therapy and primary and secondary prevention. PMID:18221181

Gilad, Jacob

2007-11-01

7

Genomic Patterns of Pathogen Evolution Revealed by Comparison of Burkholderia pseudomallei, the Causative Agent of Melioidosis, to Avirulent Burkholderia thailandensis.  

National Technical Information Service (NTIS)

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 genomic analysis of Bp K96243 and B. tha...

C. H. Lin H. H. Chua H. S. Kim S. H. Sim Y. Yu

2006-01-01

8

Preparation of Burkholderia pseudomallei Polysaccharide-CRM197 Conjugate, a Potential Vaccine Candidate for Glanders and Melioidosis.  

National Technical Information Service (NTIS)

Current ststus treatment of melioidosis and glanders: (1) Burkholderia mallei and B. pseudomallei are the causative agents for glanders and melioidosis, respectively; (2) Both of these organisms have been considered as potential agents for biological warf...

N. Parthasarathy D. DeShazer R. Barrais M. England D. Waag

2005-01-01

9

Highly resistant Burkholderia pseudomallei small colony variants isolated in vitro and in experimental melioidosis  

Microsoft Academic Search

Burkholderia pseudomallei is the causative agent of melioidosis, a disease in which treatment failures and relapses are common. This study reports\\u000a on slow growing B. pseudomallei `small colony variants' (SCVs), isolated either in vitro after exposure to ceftazidime, ciprofloxacin or gentamicin or from\\u000a the spleen and liver in a mouse model of melioidosis after treatment with ceftazidime. Interestingly, SCVs isolated

Susanne Häußler; Manfred Rohde; Ivo Steinmetz

1999-01-01

10

Multilocus Sequence Typing and Evolutionary Relationships among the Causative Agents of Melioidosis and Glanders, Burkholderia pseudomallei and Burkholderia mallei  

Microsoft Academic Search

A collection of 147 isolates of Burkholderia pseudomallei, B. mallei, and B. thailandensis was characterized by multilocus sequence typing (MLST). The 128 isolates of B. pseudomallei, the causative agent of melioidosis, were obtained from diverse geographic locations, from humans and animals with disease, and from the environment and were resolved into 71 sequence types. The utility of the MLST scheme

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

2003-01-01

11

Ribotyping and DNA macrorestriction analysis of isolates of Burkholderia pseudomallei from cases of melioidosis in Malaysia  

Microsoft Academic Search

Forty-nine isolates of Burkholderia pseudomallei from sporadic cases of melioidosis in Malaysia over the past 18 years were examined by BamHI ribotyping and pulsed-field gel electrophoresis (PFGE) of XbaI digests of total deoxyribonucleic acid (DNA). Twenty-four patients had septicaemic melioidosis with a mortality of 70%; mortality in the non-septicaemic disease was 16%. Five ribotype patterns were identified, 2 of which

Jamuna Vadivelu; S. D. Puthucheary; A. Mifsud; B. S. Drasar; D. A. B. Dance; T. L. Pitt

1997-01-01

12

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

Microsoft Academic Search

Natural isolates of Burkholderia pseudomallei (Bp), the causative agent of melioidosis, can exhibit significant ecological flexibility that is likely reflective of a dynamic genome. Using whole-genome Bp microarrays, we examined patterns of gene presence and absence across 94 South East Asian strains isolated from a variety of clinical, environmental, or animal sources. 86% of the Bp K96243 reference genome was

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

2008-01-01

13

Characterisation and molecular typing of Burkholderia pseudomallei: Are disease presentations of melioidosis clonally related?  

Microsoft Academic Search

Eighteen cases of culture positive melioidosis caused by Burkholderia pseudomallei, were seen in four geographically separate communities in North Queensland, Australia. The genetic inter-relatedness of the clinical isolates were compared utilising random amplification of polymorphic DNA (RAPD) and multilocus enzyme electrophoresis (MEE). The isolates segregated into two groups that correlated with clinical presentation rather than geographical location. This is the

R Norton; B Roberts; M Freeman; M Wilson; C Ashhurst-Smith; W Lock; D Brookes; J La Brooy

1998-01-01

14

Dose-response model for Burkholderia pseudomallei (melioidosis)  

Microsoft Academic Search

Aims: The objective of this study was development of a dose-response model for exposure to Burkholderia pseudomallei in different animal hosts and analysis of the results. The data sets with which the model was developed were taken from the open literature. Methods and Results: All data sets were initially tested for a trend between dose and outcome using the Cochran-Armitage

S. B. Tamrakar; C. N. Haas

2008-01-01

15

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

Microsoft Academic Search

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 genomic analysis of Bp K96243 and B. thailandensis (Bt) E264, a closely related but avirulent relative. RESULTS: We found the Bp and Bt genomes to be broadly similar, comprising two highly

Yiting Yu; H Stanley Kim; Hui Hoon Chua; Chi Ho Lin; Siew Hoon Sim; Daoxun Lin; Alan Derr; Reinhard Engels; David DeShazer; Bruce Birren; William C Nierman; Patrick Tan

2006-01-01

16

Antibiotic susceptibility of Burkholderia pseudomallei from tropical northern Australia and implications for therapy of melioidosis  

Microsoft Academic Search

From a prospective melioidosis study commencing in 1989 at Royal Darwin Hospital, 170 initial isolates of Burkholderia pseudomallei were available for susceptibility testing. Of these 163 (96%) were susceptible to meropenem\\/imipenem, ceftazidime, trimethoprim-sulphamethoxazole (SMX\\/TMP) and doxycycline. Seven (4%) showed primary resistance; three had low-level resistance to SMX\\/TMP, one to ceftriaxone and amoxycillin\\/clavulanate (AMOX\\/CA) and three to doxycycline. Of 167 patients

Adam W. J. Jenney; Gary Lum; Dale A. Fisher; Bart J. Currie

2001-01-01

17

Toxin production by Burkholderia pseudomallei strains and correlation with severity of melioidosis  

Microsoft Academic Search

An exotoxin lethal to cells in culture (cytolethal toxin, CLT) was identified in culture filtrates of Burkholderia pseudomallei, the causative organism of melioidosis. CLT could pass through a 10-kDa cut-off ultrafilter and its properties suggest that it is a peptide. Isolates from soil, animals and man showed differential cytolethality in vitro. The isolates were divided into low, medium and high

ANTJE HAASE; JULIA JANZEN; SIOBHAN BARRETT; B. CURRIE

1997-01-01

18

Recombinant Truncated Flagellin of Burkholderia pseudomallei as a Molecular Probe for Diagnosis of Melioidosis  

PubMed Central

Current serological tests for melioidosis, using impure or uncharacterized cell antigens from Burkholderia pseudomallei, have problems in detection sensitivity and specificity. Therefore, we designed and expressed the recombinant flagellin (truncated at both the N- and C-terminal ends), and used the antigen to develop an indirect enzyme-linked immunosorbent assay (ELISA) to diagnose melioidosis. Comparison of the immunoreactivities of the full-length and truncated flagellins reveals that the truncated flagellin performed much better in detection specificity and sensitivity. Only the full-length flagellin was recognized by other bacterial causing septicemia and gave a false-positive result in Western analysis, indicating that the cross-reactive epitopes were located on the more highly conserved N- and C-terminal regions of flagellin. The indirect ELISA using recombinant truncated flagellin as the antigen achieved 93.8% sensitivity and 96.3% specificity and offered a more efficient serodiagnosis of melioidosis.

Chen, Yao-Shen; Shiuan, David; Chen, Ssu-Ching; Chye, Soi-Moi; Chen, Ya-Lei

2003-01-01

19

Mechanisms of antibiotic resistance in Burkholderia pseudomallei: implications for treatment of melioidosis  

PubMed Central

Burkholderia pseudomallei is the etiologic agent of melioidosis. This multifaceted disease is difficult to treat, resulting in high morbidity and mortality. Treatment of B. pseudomallei infections is lengthy and necessitates an intensive phase (parenteral ceftazidime, amoxicillin–clavulanic acid or meropenem) and an eradication phase (oral trimethoprim–sulfamethoxazole). The main resistance mechanisms affecting these antibiotics include enzymatic inactivation, target deletion and efflux from the cell, and are mediated by chromosomally encoded genes. Overproduction and mutations in the class A PenA ?-lactamase cause ceftazidime and amoxicillin–clavulanic acid resistance. Deletion of the penicillin binding protein 3 results in ceftazidime resistance. BpeEF–OprC efflux pump expression causes trimethoprim and trimethoprim–sulfamethoxazole resistance. Although resistance is still relatively rare, therapeutic efficacies may be compromised by resistance emergence due to increased use of antibiotics in endemic regions. Novel agents and therapeutic strategies are being tested and, in some instances, show promise as anti-B. pseudomallei infectives.

Schweizer, Herbert P

2013-01-01

20

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

2012-06-26

21

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.

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

2012-01-01

22

A Proteome Reference Map of the Causative Agent of Melioidosis Burkholderia pseudomallei  

PubMed Central

Burkholderia pseudomallei is the etiologic agent of melioidosis. Using 2DE and MALDI-TOF MS, we report here a proteome reference map constructed from early stationary phase, a bacterial adaptation process. We identified 282 protein spots representing 220 ORFs; many of them have been implicated in bacterial pathogenesis. Up to 20% of identified ORFs belong to post-translational modification and stress responses. The proteome reference map will support future analysis of the bacterial gene and environmental regulation and facilitate comparative proteomics with its sibling species.

Wongtrakoongate, Patompon; Roytrakul, Sittiruk; Yasothornsrikul, Sukkid; Tungpradabkul, Sumalee

2011-01-01

23

Multilocus Sequence Typing and Evolutionary Relationships among the Causative Agents of Melioidosis and Glanders, Burkholderia pseudomallei and Burkholderia mallei  

PubMed Central

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

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

2003-01-01

24

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

PubMed

Melioidosis is an emerging infectious disease caused by the soil bacterium Burkholderia pseudomallei. In diagnostic and forensic settings, molecular detection assays need not only high sensitivity with low limits of detection but also high specificity. In a direct comparison of published and newly developed TaqMan PCR assays, we found the TTS1-orf2 assay to be superior in detecting B. pseudomallei directly from clinical specimens. The YLF/BTFC multiplex assay (targeting the Yersinia-like fimbrial/Burkholderia thailandensis-like flagellum and chemotaxis region) also showed high diagnostic sensitivity and provides additional information on possible geographic origin. PMID:22442327

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

2012-03-21

25

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.

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

26

Surprisingly low seroprevalence of Burkholderia pseudomallei in exposed healthy adults in the Darwin region of tropical Australia where melioidosis is highly endemic.  

PubMed

In the Darwin region of Australia where melioidosis is highly endemic, only 11/354 (3%) healthy residents were seropositive by indirect hemagglutination assay, despite extensive exposure to Burkholderia pseudomallei. None developed melioidosis, but some described a prior self-limiting illness. This seropositivity rate is much lower than that seen in northeast Thailand, where melioidosis is similarly highly endemic, potentially reflecting important differences between these two locations in the epidemiology of melioidosis. PMID:23536689

James, Gemma L; Delaney, Ben; Ward, Linda; Freeman, Kevin; Mayo, Mark; Currie, Bart J

2013-03-27

27

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

PubMed

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

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

2010-12-03

28

Burkholderia pseudomallei triggers altered inflammatory profiles in a whole-blood model of type 2 diabetes-melioidosis comorbidity.  

PubMed

Melioidosis is a potentially fatal disease caused by the bacterium Burkholderia pseudomallei. Type 2 diabetes (T2D) is the most common comorbidity associated with melioidosis. B. pseudomallei isolates from melioidosis patients with T2D are less virulent in animal models than those from patients with melioidosis and no identifiable risk factors. We developed an ex vivo whole-blood assay as a tool for comparison of early inflammatory profiles generated by T2D and nondiabetic (ND) individuals in response to a B. pseudomallei strain of low virulence. Peripheral blood from individuals with T2D, with either poorly controlled glycemia (PC-T2D [n = 6]) or well-controlled glycemia (WC-T2D [n = 8]), and healthy ND (n = 13) individuals was stimulated with B. pseudomallei. Oxidative burst, myeloperoxidase (MPO) release, expression of pathogen recognition receptors (TLR2, TLR4, and CD14), and activation markers (CD11b and HLA-DR) were measured on polymorphonuclear (PMN) leukocytes and monocytes. Concentrations of plasma inflammatory cytokine (interleukin-6 [IL-6], IL-12p70, tumor necrosis factor alpha [TNF-?], monocyte chemoattractant protein 1 [MCP-1], IL-8, IL-1?, and IL-10) were also determined. Following stimulation, oxidative burst and MPO levels were significantly elevated in blood from PC-T2D subjects compared to controls. Differences were also observed in expression of Toll-like receptor 2 (TLR2), CD14, and CD11b on phagocytes from T2D and ND individuals. Levels of IL-12p70, MCP-1, and IL-8 were significantly elevated in blood from PC-T2D subjects compared to ND individuals. Notably, differential inflammatory responses of PC-T2D, WC-T2D, and ND individuals to B. pseudomallei occur independently of bacterial load and confirm the efficacy of this model of T2D-melioidosis comorbidity as a tool for investigation of dysregulated PMN and monocyte responses to B. pseudomallei underlying susceptibility of T2D individuals to melioidosis. PMID:22473609

Morris, Jodie; Williams, Natasha; Rush, Catherine; Govan, Brenda; Sangla, Kunwarjit; Norton, Robert; Ketheesan, Natkunam

2012-04-02

29

Potential immunogenic polypeptides of Burkholderia pseudomallei identified by shotgun expression library and evaluation of their efficacy for serodiagnosis of melioidosis.  

PubMed

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, S D; Chua, Kek Heng

2013-03-13

30

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.

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

2013-01-01

31

Modified Virulence of Antibiotic-Induced Burkholderia pseudomallei Filaments  

Microsoft Academic Search

Melioidosis is a life-threatening bacterial infection caused by Burkholderia pseudomallei. Some antibiotics used to treat melioidosis can induce filamentation in B. pseudomallei. Despite studies on the mechanism of virulence of the bacteria, the properties of B. pseudomallei filaments and their impact on virulence have not been investigated before. To understand the characteristics of antibiotic-induced filaments, we performed in vitro assays

Kang Chen; Guang Wen Sun; Kim Lee Chua; Yunn-Hwen Gan

2005-01-01

32

Burkholderia pseudomallei Isocitrate Lyase Is a Persistence Factor in Pulmonary Melioidosis: Implications for the Development of Isocitrate Lyase Inhibitors as Novel Antimicrobials ? †  

PubMed Central

Burkholderia pseudomallei, the causative agent of melioidosis, has often been called the great “mimicker,” and clinical disease due to this organism may include acute, chronic, and latent pulmonary infections. Interestingly, chronic pulmonary melioidosis is often mistaken for tuberculosis, and this can have significant consequences, as the treatments for these two infections are radically different. The recurrent misdiagnosis of melioidosis for tuberculosis has caused many to speculate that these two bacterial pathogens use similar pathways to produce latent infections. Here we show that isocitrate lyase is a persistence factor for B. pseudomallei, and inhibiting the activity of this enzyme during experimental chronic B. pseudomallei lung infection forces the infection into an acute state, which can then be treated with antibiotics. We found that if antibiotics are not provided in combination with isocitrate lyase inhibitors, the resulting B. pseudomallei infection overwhelms the host, resulting in death. These results suggest that the inhibition of isocitrate lyase activity does not necessarily attenuate virulence as previously observed for Mycobacterium tuberculosis infections but does force the bacteria into a replicating state where antibiotics are effective. Therefore, isocitrate lyase inhibitors could be developed for chronic B. pseudomallei infections but only for use in combination with effective antibiotics.

van Schaik, Erin J.; Tom, Marina; Woods, Donald E.

2009-01-01

33

Burkholderia pseudomallei virulence: definition, stability and association with clonality  

Microsoft Academic Search

Clinical presentations of melioidosis, caused by Burkholderia pseudomallei are protean, but the mechanisms underlying development of the different forms of disease remain poorly understood. In murine melioidosis, the level of virulence of B. pseudomallei is important in disease pathogenesis and progression. In this study, we used B. pseudomallei-susceptible BALB\\/c mice to determine the virulence of a library of clinical and environmental B. pseudomallei

Glen C. Ulett; Bart J. Currie; Timothy W. Clair; Mark Mayo; Natkunam Ketheesan; Justin Labrooy; Daniel Gal; Robert Norton; Chris Ashhurst Smith; Jodie Barnes; Jeffrey Warner; Robert G. Hirst

2001-01-01

34

Comparative proteomic profiles and the potential markers between Burkholderia pseudomallei and Burkholderia thailandensis  

Microsoft Academic Search

Burkholderia pseudomallei is a bacterial pathogen causing the melioidosis disease, which is predominantly found in tropical areas of Southeast Asia and Northern Australia. Burkholderia thailandensis is a closely related species to B. pseudomallei but it is non-pathogenic species. In this study, we have constructed a proteome reference map of B. pseudomallei at the stationary phase of growth by using two-dimensional

Patompon Wongtrakoongate; Napachanok Mongkoldhumrongkul; Suthidarak Chaijan; Sumalee Kamchonwongpaisan; Sumalee Tungpradabkul

2007-01-01

35

Assessment of a DNA Vaccine Encoding Burkholderia pseudomallei Bacterioferritin.  

National Technical Information Service (NTIS)

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic in Southeast Asia and Northern Australia. The bacteria cause infection via subcutaneous or inhaled routes, resulting in either acute lethal sepsis or chronic and eventually...

J. Barnes J. McAllister N. Ketheesan P. Gauci S. Lazzaroni

2007-01-01

36

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

37

Molecular Procedure for Rapid Detection of Burkholderia mallei and Burkholderia pseudomallei  

Microsoft Academic Search

Both Burkholderia mallei and Burkholderia pseudomallei cause severe infectious diseases in humans, namely, glanders or me- lioidosis. B. pseudomallei is found in soil and water (e.g., rice paddies). Humans can be infected by soil contamination of skin abrasions, ingestion, or inhalation (5). Melioidosis is en- demic in Southeast Asia and northern Australia (14). Cases in humans or animals occur sporadically

ADOLF BAUERNFEIND; CARSTEN ROLLER; DETLEF MEYER; RENATE JUNGWIRTH; INES SCHNEIDER

1998-01-01

38

Virulent Burkholderia pseudomallei is more efficient than avirulent Burkholderia thailandensis in invasion of and adherence to cultured human epithelial cells  

Microsoft Academic Search

Burkholderia pseudomallei, a causative agent of melioidosis, is a facultative intracellular gram-negative bacillus that is closely related to its avirulent counterpart, Burkholderia thailandensis. However, pathogenic mechanisms and virulence factors of B. pseudomallei remain elusive. In the present study, we compared the invasiveness, adherence, and replication of B. pseudomallei and B. thailandensis in human respiratory epithelial cells A549. Invasion was determined

Wannapa Kespichayawattana; Pakamas Intachote; Pongsak Utaisincharoen; Stitaya Sirisinha

2004-01-01

39

Molecular insights into Burkholderia pseudomallei and Burkholderia mallei pathogenesis.  

PubMed

Burkholderia pseudomallei and Burkholderia mallei are closely related gram-negative bacteria that can cause serious diseases in humans and animals. This review summarizes the current and rapidly expanding knowledge on the specific virulence factors employed by these pathogens and their roles in the pathogenesis of melioidosis and glanders. In particular, the contributions of recently identified virulence factors are described in the context of the intracellular lifestyle of these pathogens. Throughout this review, unique and shared virulence features of B. pseudomallei and B. mallei are discussed. PMID:20528691

Galyov, Edouard E; Brett, Paul J; DeShazer, David

2010-01-01

40

Quantitative recovery of Burkholderia pseudomallei from soil in Thailand  

Microsoft Academic Search

Melioidosis is common in north-eastern Thailand, but is reported rarely from the adjacent areas of central Thailand, although rice farming is common to both regions. Quantitative soil cultures for Burkholderia pseudomallei were therefore prepared on 12 rice farms in both regions. B. pseudomallei was isolated from a similar proportion of rice fields in the central region (612) and in the

Michael D. Smith; Vanaporn Wuthiekanun; Amanda L. Walsh; Nicholas J. White

1995-01-01

41

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

PubMed

Burkholderia pseudomallei and Burkholderia mallei are gram-negative bacilli that are the causative agents of melioidosis and glanders, respectively. Both humans and animals are susceptible to both diseases. There is currently no vaccine available for the prevention of disease. We report the protective efficacy of heat-inactivated Burkholderia thailandensis, B. mallei or B. pseudomallei cells as vaccines against murine melioidosis and glanders. Immunisation with heat-inactivated B. pseudomallei cells provided the highest levels of protection against either melioidosis or glanders. These studies indicate the longer term potential for heat-inactivated bacteria to be developed as vaccines against melioidosis and glanders. PMID:19490962

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

2009-05-31

42

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

PubMed

Burkholderia pseudomallei and B. mallei are two highly pathogenic bacteria responsible for melioidosis and glanders, respectively. Our laboratory developed hydrolysis probe-based real-time polymerase chain reaction assays targeting type three secretion system (TTS) and transposase family protein (TFP) of B. pseudomallei and B. malli, respectively. The assays were validated for target specificity, amplification sensitivity, and reproducibility. A bacterial DNA panel, composed of B. pseudomallei (13 strains), B. mallei (11 strains), Burkholderia species close neighbors (5 strains), and other bacterial species (17 strains), was prepared for specificity testing. Reference DNAs from B. pseudomallei and B. mallei bacterial cultures were used as controls for amplification, limit of detection, and reproducibility testing. The two TaqMan assays, Bp-TTS 1 and Bm-TFP, were optimized and applied in a retrospective study of archived cases from the Armed Forces Institute of Pathology. We tested 10 formalin-fixed paraffin-embedded blocks originally from autopsy specimens of patients who died of melioidosis or glanders during or after overseas tours in 1960s. Polymerase chain reaction results confirmed that DNA samples from formalin-fixed paraffin-embedded blocks of eight patients with melioidosis were positive for Bp-TTS 1 target and two patients with glanders were positive for Bm-TFP target. PMID:22360070

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

2012-02-01

43

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

44

Role of quorum sensing in the pathogenicity of Burkholderia pseudomallei  

Microsoft Academic Search

Burkholderia pseudomallei is the causative agent of human and animal melioidosis. The role of quorum sensing (QS) in the in vivo pathogenicity of B. pseudomallei via inhalational exposure of BALB\\/c mice and intraperitoneal challenge of Syrian hamsters has not been reported. This investigation demonstrates that B. pseudomallei encodes a minimum of three luxI and five luxR homologues that are involved

Ricky L. Ulrich; David DeShazer; Ernst E. Brueggemann; Harry B. Hines; Petra C. Oyston; Jeffrey A. Jeddeloh

2004-01-01

45

Genomic Diversity of Burkholderia pseudomallei Clinical Isolates: Subtractive Hybridization Reveals a Burkholderia mallei-Specific Prophage in B. pseudomallei 1026b  

Microsoft Academic Search

Burkholderia pseudomallei is the etiologic agent of the disease melioidosis and is a category B biological threat agent. The genomic sequence of B. pseudomallei K96243 was recently determined, but little is known about the overall genetic diversity of this species. Suppression subtractive hybridization was employed to assess the genetic variability between two distinct clinical isolates of B. pseudomallei, 1026b and

David DeShazer

2004-01-01

46

Development of a Polymerase Chain Reaction Assay for the Specific Identification of Burkholderia mallei and Differentiation from Burkholderia pseudomallei and Other Closely Related Burkholderiaceae.  

National Technical Information Service (NTIS)

Burkholderia mallei and Burkholderia pseudomallei, the etiologic agents responsible for glanders and melioidosis, respectively, are genetically and phenotypically similar and are category B biothreat agents. We used an in silico approach to compare the B....

R. L. Ulrich M. P. Ulrich M. A. Schell H. S. Kim D. DeShazer

2005-01-01

47

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

Microsoft Academic Search

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

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

2004-01-01

48

Multilocus Sequence Typing of Historical Burkholderia pseudomallei Isolates Collected in Southeast Asia from 1964 to 1967 Provides Insight into the Epidemiology of Melioidosis  

Microsoft Academic Search

A collection of 207 historically relevant Burkholderia pseudomallei isolates was analyzed by multilocus sequence typing (MLST). The strain collection contains environmental isolates obtained from a geographical distribution survey of B. pseudomallei isolates in Thailand (1964 to 1967), as well as stock cultures and colony variants from the U.S. Army Medical Research Unit (Malaysia), the Walter Reed Army Institute for Research,

Roberta L. McCombie; Richard A. Finkelstein; Donald E. Woods

2006-01-01

49

Brain abscesses caused by Burkholderia pseudomallei.  

PubMed

Burkholderia pseudomallei is an important human pathogen in tropical areas, particularly South East Asia and Northern Australia. A fatal case of meliodosis presenting as brain abscesses is described. The patient deteriorated despite treatment and died 21 days after admission. Burkholderia pseudomallei was only isolated after administration of corticosteroids, whilst on treatment with antibiotics to which the organism later showed in vitro sensitivity. Magnetic resonance imaging was more sensitive than computed tomography in diagnosing early brainstem infection in this patient. Physicians working outside the endemic areas must be attuned to the possibility of melioidosis in any patient with an appropriate history of travel to endemic areas. The combination of striking early, extensive, confluent T2 hyperintensity with disproportionately small enhancing lesions may be characteristic of meliodosis. PMID:9661950

Padiglione, A; Ferris, N; Fuller, A; Spelman, D

1998-05-01

50

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

Microsoft Academic Search

BACKGROUND: The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across

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

2007-01-01

51

Adhesion and Invasion of Human Lung Epithelial Cells by Burkholderia Pseudomallei.  

National Technical Information Service (NTIS)

Melioidosis is a potentially lethal infection that is endemic in Northern Australia and Southeast Asia. The causative bacterium, Burkholderia pseudomallei, is capable of adhering to and invading a number of mammalian cells. Lung epithelial cells are parti...

S. Shahin D. Proll

2004-01-01

52

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

National Technical Information Service (NTIS)

Background:The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized large tandem repeat arrays and their distribution throug...

C. L. Friedman H. Hornstra J. M. Schupp J. M. U'Ren T. Pearson

2007-01-01

53

Pathogenesis of Burkholderia pseudomallei and Burkholderia mallei.  

PubMed

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

Larsen, Joseph C; Johnson, Nathan H

2009-06-01

54

Biological activities of lipopolysaccharide of Burkholderia (Pseudomonas) pseudomallei  

Microsoft Academic Search

Endotoxic activities of lipopolysaccharide (LPS) isolated from Burkholderia (Pseudomonas) pseudomallei, a causative agent of melioidosis, were investigated. Compared to an enterobacterial LPS (SAE-LPS), B. pseudomallei LPS (BP-LPS) exhibited weaker pyrogenic activity in rabbits, lethal toxicity in galactosamine-sensitized mice and murine macrophage activation, i.e. production of tumor necrosis factor, interleukin-6 and nitric oxide. BP-LPS, on the other hand, exhibited stronger mitogenic

Motohiro Matsuura; Kazuyoshi Kawahara; Takayuki Ezaki; masayasu Nakano

1996-01-01

55

Model of Differential Susceptibility to Mucosal Burkholderia pseudomallei Infection  

Microsoft Academic Search

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with protean clinical manifestations. The major route of infection is thought to be through subcutaneous inoculation of contami- nated soil and water, although ingestion and inhalation of contaminated aerosols are also possible. This study examines infection through the intranasal route in a murine model to mimic infection through inhalation.

Boping Liu; Ghee Chong Koo; Eu Hian Yap; Kim Lee Chua; Yunn-Hwen Gan

2002-01-01

56

Biochemical characteristics of clinical and environmental isolates of Burkholderia pseudomallei  

Microsoft Academic Search

The biochemical characteristics of 213 isolates of Burkholderia pseudomallei from patients with melioidosis and 140 isolates from the soil in central and northeastern Thailand were compared. Whereas the biochemical profiles of all the clinical isolates were similar, all soil isolates from the central area and 25% of isolates from northeastern Thailand comprised a different phenotype. This was characterised by the

VANAPORN WUTHIEKANUN; M. D. Smith; D. A. B. Dance; AMANDA L. WALSH; T. L. PITTT; N. J. White

1996-01-01

57

DNA microarray-based detection and identification of Burkholderia mallei, Burkholderia pseudomallei and Burkholderia spp.  

PubMed

We developed a rapid oligonucleotide microarray assay based on genetic markers for the accurate identification and differentiation of Burkholderia (B.) mallei and Burkholderia pseudomallei, the agents of glanders and melioidosis, respectively. These two agents were clearly identified using at least 4 independent genetic markers including 16S rRNA gene, fliC, motB and also by novel species-specific target genes, identified by in silico sequence analysis. Specific hybridization signal profiles allowed the detection and differentiation of up to 10 further Burkholderia spp., including the closely related species Burkholderia thailandensis and Burkholderia-like agents, such as Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia vietnamiensis, Burkholderia ambifaria, and Burkholderia gladioli, which are often associated with cystic fibrosis (CF) lung disease. The assay was developed using the easy-to-handle and economical ArrayTube (AT) platform. A representative strain panel comprising 44 B. mallei, 32 B. pseudomallei isolates, and various Burkholderia type strains were examined to validate the test. Assay specificity was determined by examination of 40 non-Burkholderia strains. PMID:19366627

Schmoock, Gernot; Ehricht, Ralf; Melzer, Falk; Rassbach, Astrid; Scholz, Holger C; Neubauer, Heinrich; Sachse, Konrad; Mota, Rinaldo Aparecido; Saqib, Muhammad; Elschner, Mandy

2009-04-12

58

BALB\\/c and C57Bl\\/6 mice infected with virulent Burkholderia pseudomallei provide contrasting animal models for the acute and chronic forms of human melioidosis  

Microsoft Academic Search

Burkholderia pseudomalleiis the aetiological agent of melioidosis, a life-threatening bacterial disease occurring in many species of animals, including man. Infection in humans commonly manifests as one of three clinical presentations: acute, subacute or chronic disease. Investigations were undertaken to assess the suitability of BALB\\/c and C57Bl\\/6 mice as animal models for the different forms of human melioidosis. The course of

Alison K Leakey; Glen C Ulett; Robert G Hirst

1998-01-01

59

Groundwater Seeps Facilitate Exposure to Burkholderia pseudomallei ?  

PubMed Central

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

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

2011-01-01

60

Environmental factors affecting Burkholderia pseudomallei biofilm formation.  

PubMed

Melioidosis is highly prevalent in Northeast Thailand which is associated with high incidence of Burkholderia pseudomallei present in the soil of this region. B. pseudomallei when present in biofilm becomes resistant to numerous environmental factors and also to certain antibiotics. In this study, we examined the effects of several environmentally relevant factors (salinity, iron, manganese and temperature) on biofilm formation of four clinical ribotypes of B. pseudomallei commonly found in Northeast Thailand. The results showed that biofilm formation increased when B. pseudomallei were grown in modified Vogel and Bonner's medium containing 0.85-1.7 M NaCl or 100-500 microM iron (FeSO4). Low temperature (20 degrees C) also induced more biofilm formation than 30 degrees C or 37 degrees C. On the other hand, protease production and bacterial motility were adversely affected but not in the case of low temperature. Results from this study should be useful in the development of prevention measures or controlling B. pseudomallei biofilm formation in the environment. PMID:23682440

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

2013-01-01

61

Development of ceftazidime resistance in an acute Burkholderia pseudomallei infection  

PubMed Central

Burkholderia pseudomallei, a bacterium that causes the disease melioidosis, is intrinsically resistant to many antibiotics. First-line antibiotic therapy for treating melioidosis is usually the synthetic ?-lactam, ceftazidime (CAZ), as almost all B. pseudomallei strains are susceptible to this drug. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, which can lead to mortality if therapy is not switched to a different drug in a timely manner. Serial B. pseudomallei isolates obtained from an acute Thai melioidosis patient infected by a CAZ susceptible strain, who ultimately succumbed to infection despite being on CAZ therapy for the duration of their infection, were analyzed. Isolates that developed CAZ resistance due to a proline to serine change at position 167 in the ?-lactamase PenA were identified. Importantly, these CAZ resistant isolates remained sensitive to the alternative melioidosis treatments; namely, amoxicillin-clavulanate, imipenem, and meropenem. Lastly, real-time polymerase chain reaction-based assays capable of rapidly identifying CAZ resistance in B. pseudomallei isolates at the position 167 mutation site were developed. The ability to rapidly identify the emergence of CAZ resistant B. pseudomallei populations in melioidosis patients will allow timely alterations in treatment strategies, thereby improving patient outcomes for this serious disease.

Sarovich, Derek S; Price, Erin P; Limmathurotsakul, Direk; Cook, James M; Von Schulze, Alex T; Wolken, Spenser R; Keim, Paul; Peacock, Sharon J; Pearson, Talima

2012-01-01

62

Ecology of Burkholderia pseudomallei and the interactions between environmental Burkholderia spp. and human–animal hosts  

Microsoft Academic Search

Early workers thought that melioidosis was a zoonosis with a reservoir in rodents, but we now know that Burkholderia pseudomallei is a widely distributed environmental saprophyte. In northeast Thailand, two thirds of paddy fields yield the organism, and 80% of children have antibodies by the time they are 4 years old. However, interpretation of these results has been complicated by

David A. B Dance

2000-01-01

63

A Mutant of Burkholderia pseudomallei, Auxotrophic in the Branched Chain Amino Acid Biosynthetic Pathway, Is Attenuated and Protective in a Murine Model of Melioidosis  

Microsoft Academic Search

Using a transposon mutagenesis approach, we have identified a mutant of Burkholderia pseudomallei that is auxotrophic for branched chain amino acids. The transposon was shown to have interrupted the ilvI gene encoding the large subunit of the acetolactate synthase enzyme. Compared to the wild type, this mutant was significantly attenuated in a murine model of disease. Mice inoculated intraperitoneally with

T. Atkins; R. G. Prior; K. Mack; P. Russell; M. Nelson; P. C. F. Oyston; G. Dougan; R. W. Titball

2002-01-01

64

A simple method to detect and differentiate Burkholderia pseudomallei and Burkholderia thailandensis using specific flagellin gene primers  

Microsoft Academic Search

We have previously shown that Burkholderia pseudomallei, the causative pathogen of melioidosis, may be discriminated from the closely related non-pathogenic species Burkholderia thailandensis by the presence of a 15 base pair deletion in the flagellin gene of B. thailandensis. Using specific flagellin gene primers flanking the distinctive region, PCR products of 191 and 176bp in size were detected for B.

Piengchan Sonthayanon; Piamnukul Krasao; Vannaporn Wuthiekanun; Sakol Panyim; Sumalee Tungpradabkul

2002-01-01

65

Nonrandom Distribution of Burkholderia pseudomallei Clones in Relation to Geographical Location and Virulence  

Microsoft Academic Search

Burkholderia pseudomallei is a soil-dwelling saprophyte and the causative agent of melioidosis, a life- threatening human infection. Most cases are reported from northeast Thailand and northern Australia. Using multilocus sequence typing (MLST), we have compared (i) soil and invasive isolates from northeast Thailand and (ii) invasive isolates from Thailand and Australia. A total of 266 Thai B. pseudomallei isolates were

Mongkol Vesaratchavest; Sarinna Tumapa; Nicholas P. J. Day; Vanaporn Wuthiekanun; Wirongrong Chierakul; Matthew T. G. Holden; Nicholas J. White; Bart J. Currie; Brian G. Spratt; Edward J. Feil; Sharon J. Peacock

2006-01-01

66

Biological Relevance of Colony Morphology and Phenotypic Switching by Burkholderia pseudomallei  

Microsoft Academic Search

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes

Narisara Chantratita; Vanaporn Wuthiekanun; Khaemaporn Boonbumrung; R. Tiyawisutsri; M. Vesaratchavest; D. Limmathurotsakul; W. Chierakul; S. Wongratanacheewin; S. Pukritiyakamee; N. J. White; N. P. J. Day; S. J. Peacock

2007-01-01

67

Interaction between Burkholderia pseudomallei and Acanthamoeba Species Results in Coiling Phagocytosis, Endamebic Bacterial Survival, and Escape  

Microsoft Academic Search

Burkholderia pseudomallei causes melioidosis, a potentially fatal disease whose clinical outcomes include rapid- onset septicemia and relapsing and delayed-onset infections. Like other facultative intracellular bacterial patho- gens, B. pseudomallei is capable of survival in human phagocytic cells, but unlike mycobacteria, Listeria monocytogenes, and Salmonella serovar Typhimurium, the species has not been reported to survive as an endosymbiont in free-living amebae.

TIMOTHY J. J. INGLIS; PAUL RIGBY; TERRY A. ROBERTSON; NICHOLE S. DUTTON; MANDY HENDERSON; BARBARA J. CHANG

2000-01-01

68

Rapid presumptive identification of Burkholderia pseudomallei with real-time PCR assays using fluorescent hybridization probes  

Microsoft Academic Search

Burkholderia pseudomallei (the etiologic agent of melioidosis) can cause pyogenic or granulomatous lesions in almost any organ. Septicemia has a case fatality rate of >40%. Early diagnosis and appropriate antibiotic therapy are crucial for survival, but cultivation, biochemical identification, and conventional PCR of B. pseudomallei are time consuming. We established real-time PCR assays using fluorescent hybridization probes targeting the 16S

Herbert Tomaso; Tyrone L. Pitt; Olfert Landt; Sascha Al Dahouk; Holger C. Scholz; Emil C. Reisinger; Lisa D. Sprague; Ilka Rathmann; Heinrich Neubauer

2005-01-01

69

Distribution of type III secretion gene clusters in Burkholderia pseudomallei, B. thailandensis and B. mallei  

Microsoft Academic Search

Burkholderia pseudomallei, the causative agent of melioidosis, carries a cluster of genes closely related in organisation to the type III secretion (TTS) system gene clusters of the plant pathogens Ralstonia solanacearum and Xanthomonas spp. The TTS gene cluster (TTS1) is present only in B. pseudomallei and not in avirulent B. thailandensis. Adjacent to the gene cluster encoding putative secreton structural

LUCILLE RAINBOW; C. ANTHONY HART; CRAIG WINSTANLEY

70

Effectiveness of a Simplified Method for Isolation of Burkholderia pseudomallei from Soil  

PubMed Central

Detection of environmental Burkholderia pseudomallei indicates a risk for melioidosis and is important for the development of a global risk map. We describe a simple method for detecting B. pseudomallei using direct culture of soil in enrichment broth. This gives a rate of positivity comparable to that obtained with a standard method but is cheaper and labor saving.

Wuthiekanun, Vanaporn; Amornchai, Premjit; Wongsuwan, Gumphol; Day, Nicholas P. J.; Peacock, Sharon J.

2012-01-01

71

Burkholderia pseudomallei Infection of T Cells Leads to T-Cell Costimulation Partially Provided by Flagellin  

Microsoft Academic Search

Burkholderia pseudomallei is the causative agent of melioidosis. While adaptive immunity has been shown to be important for host resistance to B. pseudomallei, the direct interaction of the bacteria with adaptive immune cells such as T and B cells is not well known. To address this question, we infected Jurkat T cells, as well as human primary CD4 and CD8

Zhiyong Ye; C. M. L. Lee; G. W. Sun; Y.-H. Gan

2008-01-01

72

A CLUSTER OF MELIOIDOSIS CASES FROM AN ENDEMIC REGION IS CLONAL AND IS LINKED TO THE WATER SUPPLY USING MOLECULAR TYPING OF BURKHOLDERIA PSEUDOMALLEI ISOLATES  

Microsoft Academic Search

Abstract. Nine cases of melioidosis with four deaths occurred over a 28-month period in members,of a small remote Aboriginal community,in the top end of the Northern Territory of Australia. Typing by pulsed-field gel electrophoresis showed,isolates of Burkholderia pseudomalleifrom six of the cases to be clonal and also identical to an isolate from the community water supply, but not to soil

Bart J. Currie; Mark Mayo; Nicholas M. Anstey; Phillip Donohoe; Antje Haase; David J. Kemp

73

Strategies for Intracellular Survival of Burkholderia pseudomallei  

PubMed Central

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

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

2011-01-01

74

Molecular Characterization of Genetic Loci Required for Secretion of Exoproducts in Burkholderia pseudomallei  

Microsoft Academic Search

Previous studies have demonstrated that Burkholderia pseudomallei secretes protease, lipase, and phospho- lipase C (PLC) into the extracellular milieu, but their mechanisms of secretion and roles in pathogenesis have not been elucidated. In this study, we isolated and characterized 29 transposon mutants unable to secrete protease, lipase, and PLC. Melioidosis is an infection caused by the gram-negative ba- cillus Burkholderia

DAVID DESHAZER; PAUL J. BRETT; MARY N. BURTNICK; DONALD E. WOODS

1999-01-01

75

Macrophage-lymphocyte interactions mediate anti- Burkholderia pseudomallei activity  

Microsoft Academic Search

The mechanisms involved in the pathogenesis of melioidosis, caused by the intracellular bacterium Burkholderia pseudomallei, are unclear. C57BL\\/6 mice are resistant to infection, while BALB\\/c mice are highly susceptible. Previous studies have demonstrated that peritoneal exudate cell preparations enriched for macrophages are capable of effectively eliminating intracellular pathogens. In this study we present evidence showing that interaction of macrophages with

Glen C Ulett; Natkunam Ketheesan; Robert G Hirst

1998-01-01

76

Modified Virulence of Antibiotic-Induced Burkholderia pseudomallei Filaments  

PubMed Central

Melioidosis is a life-threatening bacterial infection caused by Burkholderia pseudomallei. Some antibiotics used to treat melioidosis can induce filamentation in B. pseudomallei. Despite studies on the mechanism of virulence of the bacteria, the properties of B. pseudomallei filaments and their impact on virulence have not been investigated before. To understand the characteristics of antibiotic-induced filaments, we performed in vitro assays to compare several aspects of virulence between normal, nonfilamentous and filamentous B. pseudomallei. Normal, nonfilamentous B. pseudomallei could cause the lysis of monocytic cells, while filaments induced by sublethal concentrations of ceftazidime, ofloxacin, or trimethoprim show decreased lysis of monocytic cells, especially after prolonged antibiotic exposure. The motility of the filamentous bacteria was reduced compared to that of nonfilamentous bacteria. However, the filamentation was reversible when the antibiotics were removed, and the revertant bacteria recovered their motility and ability to lyse monocytic cells. Meanwhile, antibiotic resistance developed in revertant bacteria exposed to ceftazidime at the MIC. Our study highlights the danger of letting antibiotic concentration drop to the MIC or sub-MICs during antibiotic treatment of melioidosis. This could potentially give rise to a temporary reduction of bacterial virulence, only to result in bacteria that are equally virulent but more resistant to antibiotics, should the antibiotics be reduced or removed.

Chen, Kang; Sun, Guang Wen; Chua, Kim Lee; Gan, Yunn-Hwen

2005-01-01

77

Strategies toward vaccines against Burkholderia mallei and Burkholderia pseudomallei.  

PubMed

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

Bondi, Sara K; Goldberg, Joanna B

2008-11-01

78

Molecular phylogeny of Burkholderia pseudomallei  

Microsoft Academic Search

In terms of population structure, the species Burkholderia pseudomallei contains both clonal and non-clonal elements. By indexing variation in rRNA loci using the restriction endonuclease BamHI, we found that two ribotypes (types 1 and 3) are predominant in nature. Ribotype 3 is prevalent in Asian countries while ribotype 1 is more widespread. Some disease association was suggested for 4 ribotypes

Tyrone L Pitt; Suwanna Trakulsomboon; David A. B Dance

2000-01-01

79

Development of Burkholderia mallei and pseudomallei vaccines.  

PubMed

Burkholderia mallei and Burkholderia pseudomallei are Gram-negative bacteria that cause glanders and melioidosis, respectively. Inhalational infection with either organism can result in severe and rapidly fatal pneumonia. Inoculation by the oral and cutaneous routes can also produce infection. Chronic infection may develop after recovery from acute infection with both agents, and control of infection with antibiotics requires prolonged treatment. Symptoms for both meliodosis and glanders are non-specific, making diagnosis difficult. B. pseudomallei can be located in the environment, but in the host, B. mallei and B. psedomallei are intracellular organisms, and infection results in similar immune responses to both agents. Effective early innate immune responses are critical to controlling the early phase of the infection. Innate immune signaling molecules such as TLR, NOD, MyD88, and pro-inflammatory cytokines such as IFN-? and TNF-? play key roles in regulating control of infection. Neutrophils and monocytes are critical cells in the early infection for both microorganisms. Both monocytes and macrophages are necessary for limiting dissemination of B. pseudomallei. In contrast, the role of adaptive immune responses in controlling Burkholderia infection is less well understood. However, T cell responses are critical for vaccine protection from Burkholderia infection. At present, effective vaccines for prevention of glanders or meliodosis have not been developed, although recently development of Burkholderia vaccines has received renewed attention. This review will summarize current and past approaches to develop B. mallei and B. pseudomalllei vaccines, with emphasis on immune mechanisms of protection and the challenges facing the field. At present, immunization with live attenuated bacteria provides the most effective and durable immunity, and it is important therefore to understand the immune correlates of protection induced by live attenuated vaccines. Subunit vaccines have typically provided less robust immunity, but are safer to administer to a wider variety of people, including immune compromised individuals because they do not reactivate or cause disease. The challenges facing B. mallei and B. pseudomalllei vaccine development include identification of broadly protective antigens, design of efficient vaccine delivery and adjuvant systems, and a better understanding of the correlates of protection from both acute and chronic infection. PMID:23508691

Silva, Ediane B; Dow, Steven W

2013-03-11

80

Autotransporters and Their Role in the Virulence of Burkholderia pseudomallei and Burkholderia mallei.  

PubMed

Burkholderia pseudomallei and Burkholderia mallei are closely related Gram-negative bacteria responsible for the infectious diseases melioidosis and glanders, respectively. Autotransporters (ATs) comprise a large and diverse family of secreted and outer membrane proteins that includes virulence-associated invasins, adhesins, proteases, and actin-nucleating factors. The B. pseudomallei K96243 genome contains 11 predicted ATs, eight of which share homologs in the B. mallei ATCC 23344 genome. This review distils key findings from in silico, in vitro, and in vivo studies on the ATs of B. pseudomallei and B. mallei. To date, the best characterized of the predicted ATs of B. pseudomallei and B. mallei is BimA, a predicted trimeric AT mediating actin-based motility which varies in sequence and mode of action between Burkholderia species. Of the remaining eight predicted B. pseudomallei trimeric autotransporters, five of which are also present in B. mallei, two (BoaA and BoaB), have been implicated in bacterial adhesion to epithelial cells. Several predicted Burkholderia ATs are recognized by human humoral and cell-mediated immunity, indicating that they are expressed during infection and may be useful for diagnosis and vaccine-mediated protection. Further studies on the mode of secretion and functions of Burkholderia ATs will facilitate the rational design of control strategies. PMID:21811486

Lazar Adler, Natalie R; Stevens, Joanne M; Stevens, Mark P; Galyov, Edouard E

2011-07-15

81

Autotransporters and Their Role in the Virulence of Burkholderia pseudomallei and Burkholderia mallei  

PubMed Central

Burkholderia pseudomallei and Burkholderia mallei are closely related Gram-negative bacteria responsible for the infectious diseases melioidosis and glanders, respectively. Autotransporters (ATs) comprise a large and diverse family of secreted and outer membrane proteins that includes virulence-associated invasins, adhesins, proteases, and actin-nucleating factors. The B. pseudomallei K96243 genome contains 11 predicted ATs, eight of which share homologs in the B. mallei ATCC 23344 genome. This review distils key findings from in silico, in vitro, and in vivo studies on the ATs of B. pseudomallei and B. mallei. To date, the best characterized of the predicted ATs of B. pseudomallei and B. mallei is BimA, a predicted trimeric AT mediating actin-based motility which varies in sequence and mode of action between Burkholderia species. Of the remaining eight predicted B. pseudomallei trimeric autotransporters, five of which are also present in B. mallei, two (BoaA and BoaB), have been implicated in bacterial adhesion to epithelial cells. Several predicted Burkholderia ATs are recognized by human humoral and cell-mediated immunity, indicating that they are expressed during infection and may be useful for diagnosis and vaccine-mediated protection. Further studies on the mode of secretion and functions of Burkholderia ATs will facilitate the rational design of control strategies.

Adler, Natalie R. Lazar; Stevens, Joanne M.; Stevens, Mark P.; Galyov, Edouard E.

2011-01-01

82

Survival of Burkholderia pseudomallei in Water  

Microsoft Academic Search

BACKGROUND: The ability of Burkholderia pseudomallei to survive in water likely contributes to its environmental persistence in endemic regions. To determine the physiological adaptations which allow B. pseudomallei to survive in aqueous environments, we performed microarray analyses of B. pseudomallei cultures transferred from Luria broth (LB) to distilled water. FINDINGS: Increased expression of a gene encoding for a putative membrane

Richard A Moore; Apichai Tuanyok; Donald E Woods

2008-01-01

83

Structures of phosphopantetheine adenylyltransferase from Burkholderia pseudomallei  

PubMed Central

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the fourth of five steps in the coenzyme A biosynthetic pathway, reversibly transferring an adenylyl group from ATP onto 4?-phosphopantetheine to yield dephospho-coenzyme A and pyrophosphate. Burkholderia pseudomallei is a soil- and water-borne pathogenic bacterium and the etiologic agent of melioidosis, a potentially fatal systemic disease present in southeast Asia. Two crystal structures are presented of the PPAT from B. pseudomallei with the expectation that, because of the importance of the enzyme in coenzyme A biosynthesis, they will aid in the search for defenses against this pathogen. A crystal grown in ammonium sulfate yielded a 2.1?Å resolution structure that contained dephospho-coenzyme A with partial occupancy. The overall structure and ligand-binding interactions are quite similar to other bacterial PPAT crystal structures. A crystal grown at low pH in the presence of coenzyme A yielded a 1.6?Å resolution structure in the same crystal form. However, the experimental electron density was not reflective of fully ordered coenzyme A, but rather was only reflective of an ordered 4?-diphosphopantetheine moiety.

Edwards, Thomas E.; Leibly, David J.; Bhandari, Janhavi; Statnekov, Jacob B.; Phan, Isabelle; Dieterich, Shellie H.; Abendroth, Jan; Staker, Bart L.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.

2011-01-01

84

Clinical definitions of melioidosis.  

PubMed

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

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

2013-03-01

85

(1)H, (13)C, (15)N backbone and side chain NMR resonance assignments of BPSL1050 from Burkholderia pseudomallei.  

PubMed

BPSL1050 is a 13.9 kDa protein produced by the Gram-negative bacterium Burkholderia pseudomallei, the etiological agent of melioidosis. Immunodetection assays against sera patients using protein microarray suggest BPSL1050 involvement in melioidosis. Herein we report its backbone and side chains NMR assignment. PMID:23616103

Gaudesi, Davide; Quilici, Giacomo; Musco, Giovanna

2013-04-25

86

Diagnostic use of Burkholderia pseudomallei selective media in a low prevalence setting.  

PubMed

Routine use of selective media improves diagnosis of Burkholderia pseudomallei, but resources may be limited in endemic developing countries. To maximise yield in the relatively low-prevalence setting of Kuala Lumpur, Malaysia, B. pseudomallei selective agar and broth were compared with routine media for 154 respiratory specimens from patients with community-acquired disease. Selective media detected three additional culture-positive specimens and one additional melioidosis patient, at a consumables cost of US$75. Burkholderia pseudomallei was not isolated from 74 diabetic foot ulcer samples. Following careful local evaluation, focused use of selective media may be cost-effective. PMID:22112687

Roesnita, Baharudin; Tay, Sun Tee; Puthucheary, Savithri D; Sam, I-Ching

2011-11-21

87

Type III secretion system cluster 3 is required for maximal virulence of Burkholderia pseudomallei in a hamster infection model  

Microsoft Academic Search

Burkholderia pseudomallei, the etiological agent of melioidosis, is an animal pathogen capable of inducing a highly fatal septicemia. B. pseudomallei possesses three type III secretion system (TTSS) clusters, two of which (TTSS1 and TTSS2) are homologous to the TTSS of the plant pathogen Ralstonia solanacearum, and one (TTSS3) is homologous to the Salmonella SPI-1 mammalian pathogenicity island. We have demonstrated

Jonathan Warawa; Donald E. Woods

2005-01-01

88

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

Microsoft Academic Search

Burkholderia pseudomallei, the etiologic agent of melioidosis, is responsible for a broad spectrum of illnesses in humans and animals particularly in Southeast Asia and northern Australia, where it is endemic. Burkhold- eria thailandensis is a nonpathogenic environmental organism closely related to B. pseudomallei. Subtractive hybridization was carried out between these two species to identify genes encoding virulence determinants in B.

SHAUNA L. RECKSEIDLER; DAVID DESHAZER; PAMELA A. SOKOL; DONALD E. WOODS

2001-01-01

89

Novel Selective Medium for Isolation of Burkholderia pseudomallei  

Microsoft Academic Search

Isolation of Burkholderia pseudomallei currently relies on the use of Ashdown's selective agar (ASA). We designed a new selective agar (Burkholderia pseudomallei selective agar (BPSA)) to improve recovery of the more easily inhibited strains of B. pseudomallei. B. pseudomallei, Burkholderia cepacia, and Pseudomonas aerugi- nosa were used to determine the selectivity and sensitivity of BPSA. BPSA was more inhibitory to

K. Howard; T. J. J. Inglis

2003-01-01

90

Functional characterization of Burkholderia pseudomallei trimeric autotransporters.  

PubMed

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; Cotter, Peggy A

2013-05-28

91

Induction of multiple chemokine and colony-stimulating factor genes in experimental Burkholderia pseudomallei infection  

Microsoft Academic Search

Melioidosis is a disease of the tropics caused by the facultative intracellular bacterium Burkholderia pseudomallei. In human infection, increased levels of IFN-? in addition to the chemokines interferon-?-inducible protein 10 (IP-10) and monocyte interferon-?-inducible protein (Mig) have been demonstrated. However, the role of these and other chemokines in the pathogenesis of melioidosis remains unknown. Using BALB\\/c and C57BL\\/6 mice as

Jodie L Barnes; Glen C Ulett; Natkunam Ketheesan; Timothy Clair; Phillip M Summers; Robert G Hirst

2001-01-01

92

BpeAB-OprB, a Multidrug Efflux Pump in Burkholderia pseudomallei  

Microsoft Academic Search

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to a wide range of antimicrobial agents, including -lactams, aminoglycosides, macrolides, and polymyxins. An operon, bpeR- bpeA-bpeB-oprB, which encodes a putative repressor, a membrane fusion protein, an inner membrane protein, and an outer membrane protein, respectively, of a multidrug efflux pump of the resistance-nodulation-division family was identified in B. pseudomallei.

Y. Y. Chan; T. M. C. Tan; Y. M. Ong; K. L. Chua

2004-01-01

93

Sequencing and characterization of a novel serine metalloprotease from Burkholderia pseudomallei  

Microsoft Academic Search

Burkholderia pseudomallei, a Gram-negative bacterium is found in the soil and water, mainly in Southeast Asia and Northern Australia. It is responsible for melioidosis in human and animals. The bacteria produce several potential virulent factors such as extracellular protease, hemolysin, lipase and lecithinase. The isolation of virulence genes and the study of their functions will contribute to our understanding of

May-Ann Lee; Yichun Liu

2000-01-01

94

Characterisation of an acapsular mutant of Burkholderia pseudomallei identified by signature tagged mutagenesis  

Microsoft Academic Search

A Burkholderia pseudomallei mutant which was attenuated in a mouse model of melioidosis was identified by a signature tagged mutagenesis approach. The transposon was shown to be inserted into a gene within the capsular biosynthetic operon. Compared with the wild-type bacteria this mutant demonstrated a 105-fold increase in the median lethal dose in a mouse model and it did not

TIMOTHY ATKINS; RICHARD PRIOR; KERRI MACK; PAUL RUSSELL; MICHELLE NELSON; JILL ELLIS; PETRA C. F. OYSTON; GORDON DOUGAN; RICHARD W. TITBALL

2002-01-01

95

Complete Genome Sequence of the Encephalomyelitic Burkholderia pseudomallei Strain MSHR305.  

PubMed

We describe the complete genome sequence of Burkholderia pseudomallei MSHR305, a clinical isolate taken from a fatal encephalomyelitis case, a rare form of melioidosis. This sequence will be used for comparisons to identify the genes that are involved in neurological cases. PMID:23969058

Stone, Joshua K; Johnson, Shannon L; Bruce, David C; Detter, J Chris; Mayo, Mark; Currie, Bart J; Gelhaus, H Carl; Keim, Paul; Tuanyok, Apichai

2013-08-22

96

Clinical features and laboratory diagnosis of infection with the potential bioterrorism agents burkholderia mallei and burkholderia pseudomallei.  

PubMed

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

Gilad, Jacob; Schwartz, David; Amsalem, Yoram

2007-09-01

97

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

PubMed Central

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

Gilad, Jacob; Schwartz, David; Amsalem, Yoram

2007-01-01

98

EPIDEMIOLOGY OF BURKHOLDERIA PSEUDOMALLEI IN THAILAND  

Microsoft Academic Search

The distribution of Burkholderia pseudomalleiin soil collected from four regions of Thailand and the frequency of B. pseudomallei infections in patients attending government hospitals throughout Thailand in 1997 were surveyed. A total of 3,585 soil samples collected from 896 sites in four regions of Thailand were cultured for B. pseudomallei using selective enrichment broth and modified Ashdown's agar. The organism

VARAPORN VUDDHAKUL; PRASIT THARAVICHITKUL; NARISORN NA-NGAM; SIROJ JITSURONG; BANYONG KUNTHAWA; PITAK NOIMAY; PANPETH NOIMAY; ANUCHIT BINLA; VISANU THAMLIKITKUL

1999-01-01

99

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.

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

100

Nitric oxide-dependent killing of aerobic, anaerobic and persistent Burkholderia pseudomallei  

PubMed Central

Burkholderia pseudomallei infections are fastidious to treat with conventional antibiotic therapy, often involving a combination of drugs and long-term regimes. Bacterial genetic determinants contribute to the resistance of B. pseudomallei to many classes of antibiotics. In addition, anaerobiosis and hypoxia in abscesses typical of melioidosis select for persistent populations of B. pseudomallei refractory to a broad spectrum of antibacterials. We tested the susceptibility of B. pseudomallei to the drugs hydroxyurea, spermine NONOate and DETA NONOate that release nitric oxide (NO). Our investigations indicate that B. pseudomallei are killed by NO in a concentration and time-dependent fashion. The cytoxicity of this diatomic radical against B. pseudomallei depends on both the culture medium and growth phase of the bacteria. Rapidly growing, but not stationary phase, B. pseudomallei are readily killed upon exposure to the NO donor spermine NONOate. NO also has excellent antimicrobial activity against anaerobic B. pseudomallei. In addition, persistent bacteria highly resistant to most conventional antibiotics are remarkably susceptible to NO. Sublethal concentrations of NO inhibited the enzymatic activity of [4Fe-4S]-cofactored aconitase of aerobic and anaerobic B. pseudomallei. The strong anti-B. pseudomallei activity of NO described herein merits further studies on the application of NO-based antibiotics for the treatment of melioidosis.

Jones-Carson, Jessica; Laughlin, James R.; Stewart, Amanda L.; Voskuil, Martin I.; Vazquez-Torres, Andres

2012-01-01

101

Comparative proteomic profiles and the potential markers between Burkholderia pseudomallei and Burkholderia thailandensis.  

PubMed

Burkholderia pseudomallei is a bacterial pathogen causing the melioidosis disease, which is predominantly found in tropical areas of Southeast Asia and Northern Australia. Burkholderia thailandensis is a closely related species to B. pseudomallei but it is non-pathogenic species. In this study, we have constructed a proteome reference map of B. pseudomallei at the stationary phase of growth by using two-dimensional gel electrophoresis with a pH 4-7 immobilized pH gradient combined with matrix-assisted laser desorption ionization time of flight mass spectrometry. Approximately 550 spots could be detected by Coomassie brilliant blue G-250 staining, and 88 spots representing 77 unique proteins were identified. Eleven of the gene products were found in multiple spots indicating as isoforms. In attempt to detect distinctive expressed proteins between a virulent and a non-virulent species, the use of comparative proteomic profiles under the same condition were performed. We could identify more than 20 different spots. Twelve out of 14 spots are detected in B. pseudomallei and six proteins have been identified and indicated that they are involved in virulent characters of bacteria. Two hypothetical proteins were expressed and found only in B. pseudomallei. These proteins are potential markers to distinguish between these two species. Our study also provides a useful information of global intracellular protein expression and is a valuable starting point for analyzing a proteomic pathogenicity of the bacterial pathogen. PMID:17030112

Wongtrakoongate, Patompon; Mongkoldhumrongkul, Napachanok; Chaijan, Suthidarak; Kamchonwongpaisan, Sumalee; Tungpradabkul, Sumalee

2006-09-03

102

Bioluminescent Diagnostic Imaging to Characterize Altered Respiratory Tract Colonization by the Burkholderia Pseudomallei Capsule Mutant  

PubMed Central

Pneumonia is a common manifestation of the potentially fatal disease melioidosis, caused by the select agent bacteria Burkholderia pseudomallei. In this study we describe a new model system to investigate pulmonary melioidosis in vivo using bioluminescent-engineered bacteria in a murine respiratory disease model. Studies were performed to validate that the stable, light producing B. pseudomallei strain JW280 constitutively produced light in biologically relevant host–pathogen interactions. Hairless outbred SKH1 mice were used to enhance the ability to monitor B. pseudomallei respiratory disease, and were found to be similarly susceptible to respiratory melioidosis as BALB/c mice. This represents the first demonstration of in vivo diagnostic imaging of pulmonary melioidosis permitting the detection of B. pseudomallei less than 24?h post-infection. Diagnostic imaging of pulmonary melioidosis revealed distinct temporal patterns of bacterial colonization unique to both BALB/c and SKH1 mice. Validation of these model systems included the use of the previously characterized capsule mutant, which was found to colonize the upper respiratory tract at significantly higher levels than the wild type strain. These model systems allow for high resolution detection of bacterial pulmonary disease which will facilitate studies of therapeutics and basic science evaluation of melioidosis.

Warawa, Jonathan M.; Long, Dan; Rosenke, Rebecca; Gardner, Don; Gherardini, Frank C.

2011-01-01

103

Burkholderia pseudomallei genome plasticity associated with genomic island variation  

PubMed Central

Background Burkholderia pseudomallei is a soil-dwelling saprophyte and the cause of melioidosis. Horizontal gene transfer contributes to the genetic diversity of this pathogen and may be an important determinant of virulence potential. The genome contains genomic island (GI) regions that encode a broad array of functions. Although there is some evidence for the variable distribution of genomic islands in B. pseudomallei isolates, little is known about the extent of variation between related strains or their association with disease or environmental survival. Results Five islands from B. pseudomallei strain K96243 were chosen as representatives of different types of genomic islands present in this strain, and their presence investigated in other B. pseudomallei. In silico analysis of 10 B. pseudomallei genome sequences provided evidence for the variable presence of these regions, together with micro-evolutionary changes that generate GI diversity. The diversity of GIs in 186 isolates from NE Thailand (83 environmental and 103 clinical isolates) was investigated using multiplex PCR screening. The proportion of all isolates positive by PCR ranged from 12% for a prophage-like island (GI 9), to 76% for a metabolic island (GI 16). The presence of each of the five GIs did not differ between environmental and disease-associated isolates (p > 0.05 for all five islands). The cumulative number of GIs per isolate for the 186 isolates ranged from 0 to 5 (median 2, IQR 1 to 3). The distribution of cumulative GI number did not differ between environmental and disease-associated isolates (p = 0.27). The presence of GIs was defined for the three largest clones in this collection (each defined as a single sequence type, ST, by multilocus sequence typing); these were ST 70 (n = 15 isolates), ST 54 (n = 11), and ST 167 (n = 9). The rapid loss and/or acquisition of gene islands was observed within individual clones. Comparisons were drawn between isolates obtained from the environment and from patients with melioidosis in order to examine the role of genomic islands in virulence and clinical associations. There was no reproducible association between the individual or cumulative presence of five GIs and a range of clinical features in 103 patients with melioidosis. Conclusion Horizontal gene transfer of mobile genetic elements can rapidly alter the gene repertoire of B. pseudomallei. This study confirms the utility of a range of approaches in defining the presence and significance of genomic variation in natural populations of B. pseudomallei.

Tumapa, Sarinna; Holden, Matthew TG; Vesaratchavest, Mongkol; Wuthiekanun, Vanaporn; Limmathurotsakul, Direk; Chierakul, Wirongrong; Feil, Edward J; Currie, Bart J; Day, Nicholas PJ; Nierman, William C; Peacock, Sharon J

2008-01-01

104

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

2013-05-09

105

Polysaccharide Microarray Technology for the Detection of Burkholderia Pseudomallei and Burkholderia Mallei Antibodies.  

National Technical Information Service (NTIS)

A polysaccharide microarray platform was prepared by immobilizing Burkholderia pseudomallei and Burkholderia mallei polysaccharides. This polysaccharide array was tested with success for detecting B. pseudomallei and B. mallei serum (human and animal) ant...

D. DeShazer D. M. Waag M. England N. Parthasarathy

2006-01-01

106

Neurological melioidosis  

Microsoft Academic Search

Neurological abnormalities have long been recognised in animals with melioidosis, including laboratory rodents and sheep in the first Australian outbreak in 1949. Autopsies in animals have shown microabscesses and lymphocytic infiltration to be present on occasion in the same animal, but Burkholderia pseudomallei is usually able to be grown from central nervous system (CNS) tissue. In humans CNS melioidosis is

Bart J Currie; Dale A Fisher; Diane M Howard; James N. C Burrow

2000-01-01

107

Genetic Diversity and Microevolution of Burkholderia pseudomallei in the Environment  

PubMed Central

Background The soil dwelling Gram-negative pathogen Burkholderia pseudomallei is the cause of melioidosis. The diversity and population structure of this organism in the environment is poorly defined. Methods and Findings We undertook a study of B. pseudomallei in soil sampled from 100 equally spaced points within 237.5 m2 of disused land in northeast Thailand. B. pseudomallei was present on direct culture of 77/100 sampling points. Genotyping of 200 primary plate colonies from three independent sampling points was performed using a combination of pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Twelve PFGE types and nine sequence types (STs) were identified, the majority of which were present at only a single sampling point. Two sampling points contained four STs and the third point contained three STs. Although the distance between the three sampling points was low (7.6, 7.9, and 13.3 meters, respectively), only two STs were present in more than one sampling point. Each of the three samples was characterized by the localized expansion of a single B. pseudomallei clone (corresponding to STs 185, 163, and 93). Comparison of PFGE and MLST results demonstrated that two STs contained strains with variable PFGE banding pattern types, indicating geographic structuring even within a single MLST-defined clone. Conclusions We discuss the implications of this extreme structuring of genotype and genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies.

Limmathurotsakul, Direk; Vesaratchavest, Mongkol; Thanwisai, Aunchalee; Amornchai, Premjit; Tumapa, Sarinna; Feil, Edward J.; Day, Nicholas P.; Peacock, Sharon J.

2008-01-01

108

Evaluating Burkholderia pseudomallei Bip proteins as vaccines and Bip antibodies as detection agents.  

PubMed

Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS-3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS-3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti-BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme-linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei. PMID:17995960

Druar, Chris; Yu, Fei; Barnes, Jodie L; Okinaka, Richard T; Chantratita, Narisara; Beg, Steve; Stratilo, Chad W; Olive, Andrew J; Soltes, Glenn; Russell, Michelle L; Limmathurotsakul, Direk; Norton, Robert E; Ni, Sally X; Picking, William D; Jackson, Paul J; Stewart, Don I H; Tsvetnitsky, Vadim; Picking, Wendy L; Cherwonogrodzky, John W; Ketheesan, Natkunam; Peacock, Sharon J; Wiersma, Erik J

2007-11-11

109

Molecular identification and typing of Burkholderia pseudomallei and Burkholderia mallei: when is enough enough?  

PubMed

Burkholderia mallei and B. pseudomallei are highly pathogenic microorganisms for both humans and animals. Moreover, they are regarded as potential agents of bioterrorism. Thus, rapid and unequivocal detection and identification of these dangerous pathogens is critical. In the present study, we describe the use of an optimized protocol for the early diagnosis of experimental glanders and melioidosis and for the rapid differentiation and typing of Burkholderia strains. This experience with PCR-based identification methods indicates that single PCR targets (23S and 16S rRNA genes, 16S-23S intergenic region, fliC and type III secretion gene cluster) should be used with caution for identification of B. mallei and B. pseudomallei, and need to be used alongside molecular methods such as gene sequencing. Several molecular typing procedures have been used to identify genetically related B. pseudomallei and B. mallei isolates, including ribotyping, pulsed-field gel electrophoresis and multilocus sequence typing. However, these methods are time consuming and technically challenging for many laboratories. RAPD, variable amplicon typing scheme, Rep-PCR, BOX-PCR and multiple-locus variable-number tandem repeat analysis have been recommended by us for the rapid differentiation of B. mallei and B. pseudomallei strains. PMID:19121675

Antonov, Valery A; Tkachenko, Galina A; Altukhova, Viktoriya V; Savchenko, Sergey S; Zinchenko, Olga V; Viktorov, Dmitry V; Zamaraev, Valery S; Ilyukhin, Vladimir I; Alekseev, Vladimir V

2008-12-01

110

An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei.  

PubMed

Burkholderia pseudomallei and B. mallei are Gram-negative bacterial pathogens that cause melioidosis in humans and glanders in horses, respectively. Both bacteria are classified as category B select agents in the United States. Due to strict select-agent regulations, the number of antibiotic selection markers approved for use in these bacteria is greatly limited. Approved markers for B. pseudomallei include genes encoding resistance to kanamycin (Km), gentamicin (Gm), and zeocin (Zeo); however, wild type B. pseudomallei is intrinsically resistant to these antibiotics. Selection markers for B. mallei are limited to Km and Zeo resistance genes. Additionally, there are few well developed counter-selection markers for use in Burkholderia. The use of SacB as a counter-selection method has been of limited success due to the presence of endogenous sacBC genes in the genomes of B. pseudomallei and B. mallei. These impediments have greatly hampered the genetic manipulation of B. pseudomallei and B. mallei and currently few reliable tools for the genetic manipulation of Burkholderia exist. To expand the repertoire of genetic tools for use in Burkholderia, we developed the suicide plasmid pMo130, which allows for the compliant genetic manipulation of the select agents B. pseudomallei and B. mallei using allelic exchange. pMo130 harbors an aphA gene which allows for Km selection, the reporter gene xylE, which allows for reliable visual detection of Burkholderia transformants, and carries a modified sacB gene that allows for the resolution of co-integrants. We employed this system to generate multiple unmarked and in-frame mutants in B. pseudomallei, and one mutant in B. mallei. This vector significantly expands the number of available tools that are select-agent compliant for the genetic manipulation of B. pseudomallei and B. mallei. PMID:19010402

Hamad, Mohamad A; Zajdowicz, Sheryl L; Holmes, Randall K; Voskuil, Martin I

2008-10-28

111

An Allelic Exchange System for Compliant Genetic Manipulation of the Select Agents Burkholderia pseudomallei and Burkholderia mallei  

PubMed Central

Burkholderia pseudomallei and B. mallei are Gram-negative bacterial pathogens that cause melioidosis in humans and glanders in horses, respectively. Both bacteria are classified as category B select agents in the United States. Due to strict select-agent regulations, the number of antibiotic selection markers approved for use in these bacteria is greatly limited. Approved markers for B. pseudomallei include genes encoding resistance to kanamycin (Km), gentamicin (Gm), and zeocin (Zeo); however, wild type B. pseudomallei is intrinsically resistant to these antibiotics. Selection markers for B. mallei are limited to Km and Zeo resistance genes. Additionally, there are few well developed counter-selection markers for use in Burkholderia. The use of SacB as a counter-selection method has been of limited success due to the presence of endogenous sacBC genes in the genomes of B. pseudomallei and B. mallei. These impediments have greatly hampered the genetic manipulation of B. pseudomallei and B. mallei and currently few reliable tools for the genetic manipulation of Burkholderia exist. To expand the repertoire of genetic tools for use in Burkholderia, we developed the suicide plasmid pMo130, which allows for the compliant genetic manipulation of the select agents B. pseudomallei and B. mallei using allelic exchange. pMo130 harbors an aphA gene which allows for Km selection, the reporter gene xylE, which allows for reliable visual detection of Burkholderia transformants, and carries a modified sacB gene that allows for the resolution of co-integrants. We employed this system to generate multiple unmarked and in-frame mutants in B. pseudomallei, and one mutant in B. mallei. This vector significantly expands the number of available tools that are select-agent compliant for the genetic manipulation of B. pseudomallei and B. mallei.

Hamad, Mohamad A.; Zajdowicz, Sheryl L.; Holmes, Randall K.; Voskuil, Martin I.

2009-01-01

112

Comparative assessment of the intracellular survival of the Burkholderia pseudomallei bopC mutant.  

PubMed

Burkholderia pseudomallei, the causative agent of melioidosis, is a Gram-negative saprophytic bacterium capable of surviving within phagocytic cells. To assess the role of BopC (a type III secreted effector protein) in the pathogenesis of B. pseudomallei, a B. pseudomallei bopC mutant was used to infect J774A.1 macrophage-like cells. The bopC mutant showed significantly reduced intracellular survival in infected macrophages compared to wild-type B. pseudomallei. In addition, the bopC mutant displayed delayed escape from endocytic vesicles compared with the wild-type strain. This indicates that BopC is important, and at least in part, needed for intracellular survival of B. pseudomallei. PMID:23990305

Srinon, Varintip; Muangman, Sunsiree; Imyaem, Nithima; Muangsombut, Veerachat; Lazar Adler, Natalie R; Galyov, Edouard E; Korbsrisate, Sunee

2013-08-30

113

Genome Sequence of Burkholderia pseudomallei NCTC 13392.  

PubMed

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

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

2013-05-23

114

Genome Sequence of Burkholderia pseudomallei NCTC 13392  

PubMed Central

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

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

2013-01-01

115

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

Microsoft Academic Search

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 I11 secretion-associated pathogenicity island. The order of the genes in B. pseudomallei is directly equivalent to that found in R. solanacearum.

C. WINSTANLEY; B. A. HALES; C. A. HART

1999-01-01

116

The Capsular Polysaccharide of Burkholderia pseudomallei Contributes to Survival in Serum by Reducing Complement Factor C3b Deposition  

Microsoft Academic Search

Burkholderia pseudomallei produces an extracellular polysaccharide capsule -3)-2-O-acetyl-6-deoxy--D- manno-heptopyranose-(1- which has been shown to be an essential virulence determinant. The addition of purified capsule was shown to increase the virulence of a capsule mutant strain in the Syrian hamster model of acute melioidosis. An increase in the number of wild-type B. pseudomallei cells in the blood was seen by 48

Shauna L. Reckseidler-Zenteno; Rebekah DeVinney; Donald E. Woods

2005-01-01

117

Lipopolysaccharide from Nonvirulent Ara1 Burkholderia pseudomallei Isolates Is Immunologically Indistinguishable from Lipopolysaccharide from Virulent Ara2 Clinical Isolates  

Microsoft Academic Search

Different lines of evidence suggest that a discrepancy between the distribution of Burkholderia (Pseudomonas) pseudomallei in the environment and the distribution of the disease melioidosis is attributable, at least in part, to phenotypic differences between clinical and some environmental isolates. Two antigenically and biochemi- cally distinct biotypes have been described, only one of which is virulent. In this study, lipopolysaccharides

NARISARA ANUNTAGOOL; PAKAMAS INTACHOTE; VANAPORN WUTHIEKANUN; NICHOLAS J. WHITE; STITAYA SIRISINHA

118

Passive protection against Burkholderia pseudomallei infection in mice by monoclonal antibodies against capsular polysaccharide, lipopolysaccharide or proteins  

Microsoft Academic Search

Burkholderia pseudomallei, the aetiological agent of melioidosis, is endemic in south-east Asia and northern Australia, where it is an important cause of human disease. There is no vaccine available and antibiotic therapy is associated with high relapse rates. A panel of seven monoclonal antibodies (MAbs) that recognise capsular polysaccharide, lipopolysaccharide or proteins was produced and their ability to protect mice

S. M. JONES; J. F. ELLIS; P. RUSSELL; K. F. GRIFFIN; P. C. F. OYSTON

119

Burkholderia pseudomallei Induces Cell Fusion and Actin-Associated Membrane Protrusion: a Possible Mechanism for Cell-to-Cell Spreading  

Microsoft Academic Search

Burkholderia pseudomallei, a facultative intracellular bacterium, is the causative agent of a broad spectrum of diseases collectively known as melioidosis. Its ability to survive inside phagocytic and nonphagocytic cells and to induce multinucleated giant cell (MNGC) formation has been demonstrated. This study was designed to assess a possible mechanism(s) leading to this cellular change, using virulent and nonvirulent strains of

W. Kespichayawattana; S. Rattanachetkul; T. Wanun; P. Utaisincharoen; S. Sirisinha

2000-01-01

120

Obligatory Role of Gamma Interferon for Host Survival in a Murine Model of Infection with Burkholderia pseudomallei  

Microsoft Academic Search

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium capable of causing either acute lethal sepsis or chronic but eventually fatal disease in infected individuals. However, despite the clinical importance of this infection in areas where it is endemic, there is essentially no information on the mechanisms of protective immunity to the bacterium. We describe here a murine

P. SANTANIRAND; V. S. HARLEY; D. A. B. DANCE; B. S. DRASAR; G. J. BANCROFT

1999-01-01

121

Specificity and Functional Activity of Anti-Burkholderia pseudomallei Polysaccharide Antibodies  

Microsoft Academic Search

The lipopolysaccharide (LPS) of Burkholderia pseudomallei, the causative agent of melioidosis, consists of two O-antigenic polysaccharides designated O-PS I and O-PS II. In this study, the O-PS specificity and functional activity of a protective polyclonal antiserum and an immunoglobulin M (IgM) monoclonal antibody were determined. The polyclonal antiserum recognized both O-PS I and O-PS II, while the monoclonal antibody was

MAY HO; TINEKE SCHOLLAARDT; MICHAEL D. SMITH; MALCOLM B. PERRY; PAUL J. BRETT; WIPADA CHAOWAGUL; LARRY E. BRYAN; Ubol Ratchatani

1997-01-01

122

groEL Encodes a Highly Antigenic Protein in Burkholderia pseudomallei  

Microsoft Academic Search

No recombinant protein is available for serodiagnosis of melioidosis. In this study, we report the cloning of the groEL gene, which encodes an immunogenic protein of Burkholderia pseudomallei. Bidirectional DNA sequencing of groEL revealed that the gene contained a single open reading frame encoding 546 amino acid residues with a predicted molecular mass of 57.1 kDa. Basic Local Alignment Search

PATRICK C. Y. WOO; PATRICIA K. L. LEUNG; SAMSON S. Y. WONG; PAK-LEUNG HO; KWOK-YUNG YUEN

2001-01-01

123

Human melioidosis, Malawi, 2011.  

PubMed

A case of human melioidosis caused by a novel sequence type of Burkholderia pseudomallei occurred in a child in Malawi, southern Africa. A literature review showed that human cases reported from the continent have been increasing. PMID:23735189

Katangwe, Thembi; Purcell, Janet; Bar-Zeev, Naor; Denis, Brigitte; Montgomery, Jacqui; Alaerts, Maaike; Heyderman, Robert Simon; Dance, David A B; Kennedy, Neil; Feasey, Nicholas; Moxon, Christopher Alan

2013-06-01

124

Novel Selective Medium for Isolation of Burkholderia pseudomallei  

PubMed Central

Isolation of Burkholderia pseudomallei currently relies on the use of Ashdown's selective agar (ASA). We designed a new selective agar (Burkholderia pseudomallei selective agar [BPSA]) to improve recovery of the more easily inhibited strains of B. pseudomallei. B. pseudomallei, Burkholderia cepacia, and Pseudomonas aeruginosa were used to determine the selectivity and sensitivity of BPSA. BPSA was more inhibitory to P. aeruginosa and B. cepacia and should make recognition of Burkholderia species easier due to distinctive colony morphology. BPSA also inhibited Enterococcus, Escherichia, Staphylococcus, and Streptococcus. These results indicate that BPSA is a potential replacement for ASA.

Howard, K.; Inglis, T. J. J.

2003-01-01

125

Protection against experimental melioidosis following immunization with live Burkholderia thailandensis expressing a manno-heptose capsule.  

PubMed

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

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

2013-05-15

126

Role of RelA and SpoT in Burkholderia pseudomallei virulence and immunity.  

PubMed

Burkholderia pseudomallei is a Gram-negative soil bacterium and the causative agent of melioidosis, a disease of humans and animals. It is also listed as a category B bioterrorism threat agent by the U.S. Centers for Disease Control and Prevention, and there is currently no melioidosis vaccine available. Small modified nucleotides such as the hyperphosphorylated guanosine molecules ppGpp and pppGpp play an important role as signaling molecules in prokaryotes. They mediate a global stress response under starvation conditions and have been implicated in the regulation of virulence and survival factors in many bacterial species. In this study, we created a relA spoT double mutant in B. pseudomallei strain K96243, which lacks (p)ppGpp-synthesizing enzymes, and investigated its phenotype in vitro and in vivo. The B. pseudomallei ?relA ?spoT mutant displayed a defect in stationary-phase survival and intracellular replication in murine macrophages. Moreover, the mutant was attenuated in the Galleria mellonella insect model and in both acute and chronic mouse models of melioidosis. Vaccination of mice with the ?relA ?spoT mutant resulted in partial protection against infection with wild-type B. pseudomallei. In summary, (p)ppGpp signaling appears to represent an essential component of the regulatory network governing virulence gene expression and stress adaptation in B. pseudomallei, and the ?relA ?spoT mutant may be a promising live-attenuated vaccine candidate. PMID:22778096

Müller, Claudia M; Conejero, Laura; Spink, Natasha; Wand, Matthew E; Bancroft, Gregory J; Titball, Richard W

2012-07-09

127

Burkholderia pseudomallei transcriptional adaptation in macrophages  

PubMed Central

Background Burkholderia pseudomallei is a facultative intracellular pathogen of phagocytic and non-phagocytic cells. How the bacterium interacts with host macrophage cells is still not well understood and is critical to appreciate the strategies used by this bacterium to survive and how intracellular survival leads to disease manifestation. Results Here we report the expression profile of intracellular B. pseudomallei following infection of human macrophage-like U937 cells. During intracellular growth over the 6?h infection period, approximately 22?% of the B. pseudomallei genome showed significant transcriptional adaptation. B. pseudomallei adapted rapidly to the intracellular environment by down-regulating numerous genes involved in metabolism, cell envelope, motility, replication, amino acid and ion transport system and regulatory function pathways. Reduced expression in catabolic and housekeeping genes suggested lower energy requirement and growth arrest during macrophage infection, while expression of genes encoding anaerobic metabolism functions were up regulated. However, whilst the type VI secretion system was up regulated, expression of many known virulence factors was not significantly modulated over the 6hours of infection. Conclusions The transcriptome profile described here provides the first comprehensive view of how B. pseudomallei survives within host cells and will help identify potential virulence factors and proteins that are important for the survival and growth of B. pseudomallei within human cells.

2012-01-01

128

Melioidosis in traveler from Africa to Spain.  

PubMed

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

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

2013-10-01

129

Survival of Burkholderia pseudomallei on Environmental Surfaces?  

PubMed Central

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

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

2007-01-01

130

Human Immune Responses to Burkholderia pseudomallei Characterized by Protein Microarray Analysis  

PubMed Central

Background.?We aimed to determine the antibody and T cell responses to Burkholderia pseudomallei of humans to select candidate vaccine antigens. Methods.?For antibody profiling, a protein microarray of 154 B. pseudomallei proteins was probed with plasma from 108 healthy individuals and 72 recovered patients. Blood from 20 of the healthy and 30 of the recovered individuals was also obtained for T cell assays. Results.?Twenty-seven proteins distinctively reacted with human plasma following environmental exposure or clinical melioidosis. We compared the responses according to the patient’s history of subsequent relapse, and antibody response to BPSL2765 was higher in plasma from individuals who had only 1 episode of disease than in those with recurrent melioidosis. A comparison of antibody and T cell responses to 5 B. pseudomallei proteins revealed that BimA and flagellin-induced responses were similar but that BPSS0530 could induce T cell responses in healthy controls more than in recovered patients. Conclusions.?By combining large-scale antibody microarrays and assays of T cell–mediated immunity, we identified a panel of novel B. pseudomallei proteins that show distinct patterns of reactivity in different stages of human melioidosis. These proteins may be useful candidates for development of subunit-based vaccines and in monitoring the risks of treatment failure and relapse.

Suwannasaen, Duangchan; Mahawantung, Jirawan; Chaowagul, Wipada; Felgner, Philip L.; Davies, Huw; Bancroft, Gregory J.; Titball, Richard W.

2011-01-01

131

Experimental acute respiratory Burkholderia pseudomallei infection in BALB/c mice  

PubMed Central

Burkholderia pseudomallei is the causative agent of melioidosis, which is considered a potential deliberate release agent. The objective of this study was to establish and characterise a relevant, acute respiratory Burkholderia pseudomallei infection in BALB/c mice. Mice were infected with 100 B. pseudomallei strain BRI bacteria by the aerosol route (approximately 20 median lethal doses). Bacterial counts within lung, liver, spleen, brain, kidney and blood over 5 days were determined and histopathological and immunocytochemical profiles were assessed. Bacterial numbers in the lungs reached approximately 108 cfu/ml at day 5 post-infection. Bacterial numbers in other tissues were lower, reaching between 103 and 105 cfu/ml at day 4. Blood counts remained relatively constant at approximately 1.0 × 102 cfu/ml. Foci of acute inflammation and necrosis were seen within lungs, liver and spleen. These results suggest that the BALB/c mouse is highly susceptible to B. pseudomallei by the aerosol route and represents a relevant model system of acute human melioidosis.

Lever, Mark S; Nelson, Michelle; Stagg, Anthony J; Beedham, Richard J; Simpson, Andrew J H

2009-01-01

132

Development and Evaluation of a Real-Time PCR Assay Targeting the Type III Secretion System of Burkholderia pseudomallei  

PubMed Central

Here we report on the development of a discriminatory real-time assay for the rapid identification of Burkholderia pseudomallei isolates and the evaluation of this assay for sensitivity against related species and detection in spiked human blood samples. The assay targets a 115-base-pair region within orf2 of the B. pseudomallei type III secretion system gene cluster and distinguishes B. pseudomallei from other microbial species. Assay performance was evaluated with 224 geographically, temporally, and clinically diverse B. pseudomallei isolates from the Centers for Disease Control and Prevention strain collection. This represents the first real-time PCR for rapid and sensitive identification of B. pseudomallei that has been tested for cross-reactivity with 23 Burkholderia mallei, 5 Burkholderia thailandensis, and 35 Burkholderia and 76 non-Burkholderia organisms which have historically presented diagnostic challenges. The assay performed with 100% specificity. The limit of detection was found to be 76 femtograms of DNA (equivalent to 5.2 × 103 genome equivalents per ml) in a single PCR. In spiked human blood, the assay could detect as few as 8.4 × 103 CFU per ml. This rapid assay is a valuable tool for identification of B. pseudomallei and may improve diagnosis in regions endemic for melioidosis.

Novak, Ryan T.; Glass, Mindy B.; Gee, Jay E.; Gal, Daniel; Mayo, Mark J.; Currie, Bart J.; Wilkins, Patricia P.

2006-01-01

133

Dictyostelium discoideum as a Model System for Identification of Burkholderia pseudomallei Virulence Factors?§  

PubMed Central

Burkholderia pseudomallei is an emerging bacterial pathogen and category B biothreat. Human infections with B. pseudomallei (called melioidosis) present as a range of manifestations, including acute septicemia and pneumonia. Although melioidosis can be fatal, little is known about the molecular basis of B. pseudomallei pathogenicity, in part because of the lack of simple, genetically tractable eukaryotic models to facilitate en masse identification of virulence determinants or explore host-pathogen interactions. Two assays, one high-throughput and one quantitative, were developed to monitor levels of resistance of B. pseudomallei and the closely related nearly avirulent species Burkholderia thailandensis to predation by the phagocytic amoeba Dictyostelium discoideum. The quantitative assay showed that levels of resistance to, and survival within, amoeba by these bacteria and their known virulence mutants correlate well with their published levels of virulence in animals. Using the high-throughput assay, we screened a 1,500-member B. thailandensis transposon mutant library and identified 13 genes involved in resistance to predation by D. discoideum. Orthologs of these genes were disrupted in B. pseudomallei, and nearly all mutants had similarly decreased resistance to predation by D. discoideum. For some mutants, decreased resistance also correlated with reduced survival in and cytotoxicity toward macrophages, as well as attenuated virulence in mice. These observations suggest that some factors required by B. pseudomallei for resistance to environmental phagocytes also aid in resistance to phagocytic immune cells and contribute to disease in animals. Thus, D. discoideum provides a novel, high-throughput model system for facilitating inquiry into B. pseudomallei virulence.

Hasselbring, Benjamin M.; Patel, Maharsh K.; Schell, Mark A.

2011-01-01

134

Enzymatic and molecular characterisation of leucine aminopeptidase of Burkholderia pseudomallei.  

PubMed

BACKGROUND: Leucine aminopeptidase (LAP) has been known to be a housekeeping protease, DNA-binding protein and repressor or activator in the operon regulation of virulence-associated genes in several bacterial species. LAP activity was consistently detected in overnight cultures of Burkholderia pseudomallei, the causative agent of melioidosis and this enzyme was partially purified and characterised in this study. The intra- and inter-species nucleotide and deduced amino acid sequence variation of LAP encoding gene (pepA) was determined. A pepA/PCR-RFLP assay was designed to facilitate the identification of major LAP sequence types amongst clinical and environmental isolates of B. pseudomallei. RESULTS: LAP activity was detected in B. pseudomallei culture supernantants by zymographic analysis. Optimum activity was at pH 9 and stable at 50[degree sign]C. Enhanced enzymatic activity was observed in the presence of metallic ions Mg2+, Ca2+, Na+ and K+. LAP activity was inhibited by EDTA, 1,10-phenanthroline, amastatin, Mn2+ and Zn2+. Sequence analysis of the complete nucleotide and deduced amino acid sequences of LAP-encoding (pepA) gene showed close genetic relatedness to B. mallei (similarity 99.7%/99.6%), but not with B. thailandensis (96.4%/96.4%). Eight pepA sequence types were identified by comparison with a 596 bp DNA fragment encompassing central regions of the pepA gene. A pepA/PCR-RFLP was designed to differentiate pepA sequence types. Based on restriction analysis with StuI and HincII enzymes of the amplified pepA gene, clinical and environmental isolates showed different predominant RFLP types. Type I was the most predominant type amongst 71.4% (65/91) of the clinical isolates, while Type II was predominant in 55.6% (5/9) of the environmental isolates. CONCLUSIONS: This study showed that LAP is a secretory product of B. pseudomallei with features similar to LAP of other organisms. Identification of major LAP sequence types of B. pseudomallei was made possible based on RFLP analysis of the pepA gene. The high LAP activity detected in both B. pseudomallei and B. thailandensis, suggests that LAP is probably a housekeeping enzyme rather than a virulence determinant. PMID:23682954

Mun, Liew Siew; Tee, Tay Sun; Puthucheary, Savithiri D

2013-05-17

135

Structure of a Burkholderia pseudomallei Trimeric Autotransporter Adhesin Head  

PubMed Central

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

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

2010-01-01

136

Novel gain of function approaches for vaccine candidate identification in Burkholderia pseudomallei  

PubMed Central

The Gram-negative bacterium Burkholderia pseudomallei is a serious environmental pathogen and the causative agent of the often fatal melioidosis. Disease occurs following exposure to contaminated water or soil, usually through cuts in the skin or via inhalation. However, the underlying mechanisms of pathogenicity remain poorly understood. B. pseudomallei is endemic to South East Asia and Northern Australia where infections are associated with antibiotic resistance and high mortality rates. Categorization of the pathogen as a potential biowarfare agent has also made research into vaccine development a high priority. Recent genome-scale screening has produced a large number of putative gene candidates from B. pseudomallei with the potential for development into vaccines. This mini-review will discuss the advantages and limitations of this novel approach, how these new techniques can complement existing strategies, and outline aims for future research.

Dowling, Andrea J.

2013-01-01

137

Flagellar Glycosylation in Burkholderia pseudomallei and Burkholderia thailandensis?  

PubMed Central

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

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

2011-01-01

138

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

PubMed

Burkholderia pseudomallei (Pseudomonas pseudomallei) causes melioidosis, a life-threatening infection common among paddy cultivators in Southeast Asian countries. No plant materials have been investigated for its activity against B. pseudomallei. Therefore, a preliminary study was carried out using disc diffusion and minimum inhibitory concentration (MIC) methods to evaluate the anti-B. pseudomallei activity of five Indian medicinal plants documented to have been used for several ailments in the ancient Indian scriptures. The leaf extracts of Tamarindus indica, Lawsonia inermis, and Hibiscus rosa-sinensis, the rhizome extracts of Curcuma longa and the seeds of Vigna radiata were prepared using methanol as solvent. The disc diffusion and MIC methods were used to assess the anti-B. pseudomallei activity of the plants tested. Only methanol leaf extracts of Tamarindus indica exhibited anti-B. pseudomallei activity starting from disc concentrations of 150 mug by the disc diffusion method. The other plants failed to show any zone of inhibition. MIC assay revealed that the MIC and minimum bactericidal concentration (MBC) for B. pseudomallei were 125 mug/ml. Our preliminary finding showed that methanolic extracts of Tamarindus indica has anti-B. pseudomallei inhibitory potentials under in vitro conditions. Extensive animal studies may be required before investigating the role of Tamarindus indica for treating melioidosis. PMID:16518004

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

2005-12-01

139

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

140

Isolates of Burkholderia pseudomallei from Northern Australia Are Distinct by Multilocus Sequence Typing, but Strain Types Do Not Correlate with Clinical Presentation  

Microsoft Academic Search

Melioidosis is the disease caused by the saprophytic organism Burkholderia pseudomallei. Previous studies have suggested some strain tropism and differential virulence. In this study, we defined strains by multilocus sequence typing (MLST) of isolates taken from the Top End of Australia's Northern Territory and compared the results with those of other strains typed worldwide. We specifically sought clinical and geographical

Allen C. Cheng; Daniel Godoy; Mark Mayo; Daniel Gal; Brian G. Spratt; Bart J. Currie

2004-01-01

141

The PmlI-PmlR Quorum-Sensing System in Burkholderia pseudomallei Plays a Key Role in Virulence and Modulates Production of the MprA Protease  

Microsoft Academic Search

Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infection of humans and animals. The virulence of this pathogen is thought to depend on a number of secreted proteins, including the MprA metalloprotease. We observed that MprA is produced upon entry into the stationary phase, when the cell density is high, and this prompted us to study cell

E. Valade; F. M. Thibault; Y. P. Gauthier; M. Palencia; M. Y. Popoff; D. R. Vidal

2004-01-01

142

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.

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

143

Characterization of the capsular polysaccharide of Burkholderia (Pseudomonas) pseudomallei 304b.  

PubMed Central

Burkholderia (Pseudomonas) pseudomallei is the causative agent of melioidosis, a bacterial infection of considerable morbidity in areas of endemicity of Southeast Asia and northern Australia. Clinical isolates of B. pseudomallei have been demonstrated to produce a lipopolysaccharide (LPS) containing two separate and chemically distinct antigenic O polysaccharides against which infected patients produced antibodies. A putative capsular polysaccharide (CPS) has also been reported and is thought to be antigenically conserved based on results of serological studies with clinical B. pseudomallei isolates. In the present study, the CPS isolated from B. pseudomallei 304b from northeastern Thailand was found to have an [alpha]D of +99 degrees (water), was composed of D-galactose (D-Gal), 3-deoxy-D-manno-2-octulosonic acid (KDO), and O-acetyl 3:1:1), and was a linear unbranched polymer of repeating tetrasaccharide units having the following structure: -3)-2-O-Ac-beta-D-Galp-(1-4)-alpha-D-Galp-(1-3)-beta-D -Galp-(1-5)-beta-D-KDOp-(2-. Sera from 13 of 15 patients with different clinical manifestations of melioidosis but not normal controls recognize the CPS, which suggests that it is immunogenic and raises the possibility that it may have a role as a vaccine candidate and/or diagnostic agent.

Masoud, H; Ho, M; Schollaardt, T; Perry, M B

1997-01-01

144

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

PubMed Central

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

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

2013-01-01

145

Cystic Fibrosis Patient with Burkholderia pseudomallei Infection Acquired in Brazil?  

PubMed Central

Burkholderia pseudomallei is rarely isolated from cystic fibrosis patients outside known areas of endemicity. We report the recovery of B. pseudomallei from the sputum of a cystic fibrosis patient who lives in Brazil. We highlight the importance of careful attention to unusual nonfermentative gram-negative rods in cystic fibrosis patients.

Barth, Afonso Luis; de Abreu e Silva, Fernando Antonio; Hoffmann, Anneliese; Vieira, Maria Izolete; Zavascki, Alexandre Prehn; Ferreira, Alex Guerra; da Cunha, Luiz Gonzaga; Albano, Rodolpho Mattos; de Andrade Marques, Elizabeth

2007-01-01

146

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

PubMed Central

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

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

2011-01-01

147

Isolation of Burkholderia pseudomallei from a case of septicaemia--a case report.  

PubMed

Meliodosis, an infectious disease caused by Burkholderia pseudomallei has been recognized as an emerging infectious disease in India. The infection is under diagnosed and underreported, and hence considered a rare disease. Majority of the cases have been documented from the states with heavy rainfall. The present case being reported is a middle-aged woman who had developed a fulminant infection following exposure to stagnant floodwater in the city of Hyderabad. To the best of our knowledge, this is the first case of Melioidosis being reported from this part of the country. PMID:17642999

Anuradha, K; Meena, A K; Lakshmi, V

148

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.

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

149

The Condition-Dependent Transcriptional Landscape of Burkholderia pseudomallei.  

PubMed

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

Ooi, Wen Fong; Ong, Catherine; 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

2013-09-12

150

Current studies on the pathogenesis of melioidosis  

Microsoft Academic Search

Burkholderia pseudomallei is a major cause of bacterial septicemias in many parts of the world, particularly Thailand; the known geographic range of the organism appears to be enlarging as awareness of the organism and the disease it causes – melioidosis – increases. B. pseudomallei is intrinsically resistant to most antibiotics, and our knowledge of B. pseudomallei pathogenesis is lacking. Thus,

Donald E Woods; David DeShazer; Richard A Moore; Paul J Brett; Mary N Burtnick; Shauna L Reckseidler; Michelle D Senkiw

1999-01-01

151

Neutrophil Extracellular Traps Exhibit Antibacterial Activity against Burkholderia pseudomallei and Are Influenced by Bacterial and Host Factors  

PubMed Central

Burkholderia pseudomallei is the causative pathogen of melioidosis, of which a major predisposing factor is diabetes mellitus. Polymorphonuclear neutrophils (PMNs) kill microbes extracellularly by the release of neutrophil extracellular traps (NETs). PMNs play a key role in the control of melioidosis, but the involvement of NETs in killing of B. pseudomallei remains obscure. Here, we showed that bactericidal NETs were released from human PMNs in response to B. pseudomallei in a dose- and time-dependent manner. B. pseudomallei-induced NET formation required NADPH oxidase activation but not phosphatidylinositol-3 kinase, mitogen-activated protein kinases, or Src family kinase signaling pathways. B. pseudomallei mutants defective in the virulence-associated Bsa type III protein secretion system (T3SS) or capsular polysaccharide I (CPS-I) induced elevated levels of NETs. NET induction by such mutants was associated with increased bacterial killing, phagocytosis, and oxidative burst by PMNs. Taken together the data imply that T3SS and the capsule may play a role in evading the induction of NETs. Importantly, PMNs from diabetic subjects released NETs at a lower level than PMNs from healthy subjects. Modulation of NET formation may therefore be associated with the pathogenesis and control of melioidosis.

Riyapa, Donporn; Buddhisa, Surachat; Korbsrisate, Sunee; Cuccui, Jon; Wren, Brendan W.; Stevens, Mark P.; Ato, Manabu

2012-01-01

152

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

PubMed

It has been hypothesized that biogeographical boundaries are a feature of Burkholderia pseudomallei ecology, and they impact the epidemiology of melioidosis on a global scale. This study examined the relatedness of B. pseudomallei sourced from islands in the Torres Strait of Northern Australia to determine if the geography of isolated island communities is a determinant of the organisms' dispersal. Environmental sampling on Badu Island in the Near Western Island cluster recovered a single clone. An additional 32 clinical isolates from the region were sourced. Isolates were characterized using multilocus sequence typing and a multiplex PCR targeting the flagellum gene cluster. Gene cluster analysis determined that 69% of the isolates from the region encoded the ancestral Burkholderia thailandensis-like flagellum and chemotaxis gene cluster, a proportion significantly lower than that reported from mainland Australia and consistent with observations of isolates from southern Papua New Guinea. A goodness-of-fit test indicated that there was geographic localization of sequence types throughout the archipelago, with the exception of Thursday Island, the economic and cultural hub of the region. Sequence types common to mainland Australia and Papua New Guinea were identified. These findings demonstrate for the first time an environmental reservoir for B. pseudomallei in the Torres Strait, and multilocus sequence typing suggests that the organism is not randomly distributed throughout this region and that seawater may provide a barrier to dispersal of the organism. Moreover, these findings support an anthropogenic dispersal hypothesis for the spread of B. pseudomallei throughout this region. PMID:23698533

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

2013-05-22

153

Latex agglutination for rapid detection of Pseudomonas pseudomallei antigen in urine of patients with melioidosis  

Microsoft Academic Search

A latex agglutination test for the detection of Pseudomonas pseudomallei antigen in urine was evaluated for the rapid diagnosis of melioidosis. With unconcentrated urine, antigen was detected in only 18% of patients with melioidosis overall. However, when urine was concentrated 100-fold, antigen was detected in 47% overall and in 67% of patients with septicaemia or disseminated infection, in whom a

M D Smith; V Wuthiekanun; A L Walsh; N Teerawattanasook; V Desakorn; Y Suputtamongkol; T L Pitt; N J White

1995-01-01

154

Revised structures for the predominant O-polysaccharides expressed by Burkholderia pseudomallei and Burkholderia mallei.  

PubMed

O-Polysaccharides (OPS) were isolated from purified Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharides by mild-acid hydrolysis and gel-permeation chromatography. 1-D and 2-D (1)H and (13)C NMR spectroscopy experiments revealed that the OPS antigens were unbranched heteropolymers with the following structures: Collectively, our results demonstrate that the predominant OPS antigens expressed by B. pseudomallei and B. mallei isolates are structurally more complex than previously described and provide evidence that different capping residues are used by these closely related pathogens to terminate chain elongation. Additionally, they confirm that Burkholderia thailandensis and B. pseudomallei express OPS antigens that are essentially identical to one another. PMID:24056008

Heiss, Christian; Burtnick, Mary N; Roberts, Rosemary A; Black, Ian; Azadi, Parastoo; Brett, Paul J

2013-08-24

155

Comparison of Rapid, Automated Ribotyping and DNA Macrorestriction Analysis of Burkholderia pseudomallei  

PubMed Central

An automated ribotyping device (RiboPrinter) was used to determine the ribotypes of a collection of Burkholderia pseudomallei isolates. In a preliminary evaluation with the restriction enzymes BamHI and EcoRI, the protocol with EcoRI was more discriminating. The reproducibilities of the ribotypes obtained with EcoRI (EcoRI ribotypes) were determined by testing three levels of bacterial loads. The performance of the manufacturer's software was assessed by comparing the machine-optimized ribotypes with the type determined from the original gel image analyzed with Bionumerics software. The library of B. pseudomallei EcoRI ribotypes was then compared with the ribotypes obtained by DNA macrorestriction analysis of XbaI digests by pulsed-field gel electrophoresis. The typeability of B. pseudomallei by EcoRI ribotyping was 100%, and the discrimination index was 0.94. The slightly greater discrimination provided by DNA macrorestriction analysis (0.96) was achieved at the expense of a significantly longer processing time of 6 days, although the method was only half the cost of automated ribotyping. Typeability by macrorestriction analysis was lower (97%) unless a thiourea step was added to neutralize the action of Tris-dependent endonucleases. The digital record of B. pseudomallei isolates analyzed thus far provides a useful resource for future epidemiological studies and will help shorten the response time in the event of a further melioidosis outbreak or the deliberate release of B. pseudomallei as a biohazard.

Inglis, Timothy J. J.; O'Reilly, Lyn; Foster, Niki; Clair, Adele; Sampson, Judy

2002-01-01

156

Burkholderia pseudomallei detection in surface water in southern Laos using Moore's swabs.  

PubMed

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

2012-05-01

157

A heterodimer comprised of two bovine lactoferrin antimicrobial peptides exhibits powerful bactericidal activity against Burkholderia pseudomallei.  

PubMed

Melioidosis is a severe infectious disease that is endemic in Southeast Asia and Northern Australia. Burkholderia pseudomallei, the causative agent of this disease, has developed resistance to an increasing list of antibiotics, demanding a search for novel agents. Lactoferricin and lactoferrampin are two antimicrobial domains of lactoferrin with a broad spectrum of antimicrobial activity. A hybrid peptide (LFchimera) containing lactoferrampin (LFampin265-284) and a part of lactoferricin (LFcin17-30) has strikingly higher antimicrobial activities compared to the individual peptides. In this study, the antimicrobial activities of this chimeric construct (LFchimera1), as well as of another one containing LFcin17-30 and LFampin268-284, a shorter fragment of LFampin265-284 (LFchimera2), and the constituent peptides were tested against 7 isolates of B. pseudomallei and compared to the preferential antibiotic ceftazidime (CAZ). All isolates including B. pseudomallei 979b shown to be resistant to CAZ, at a density of 10(5) CFU/ml, could be killed by 5-10 ?M of LFchimera1 within 2 h, while the other peptides as well as the antibiotic CAZ only inhibited the B. pseudomallei strains resulting in an overgrowth in 24 h. These data indicate that LFchimera1 could be considered for development of therapeutic agents against B. pseudomallei. PMID:23404819

Puknun, Aekkalak; Bolscher, Jan G M; Nazmi, Kamran; Veerman, Enno C I; Tungpradabkul, Sumalee; Wongratanacheewin, Surasakdi; Kanthawong, Sakawrat; Taweechaisupapong, Suwimol

2013-02-13

158

Development and Validation of Burkholderia pseudomallei-Specific Real-Time PCR Assays for Clinical, Environmental or Forensic Detection Applications  

PubMed Central

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

Price, Erin P.; Dale, Julia L.; Cook, James M.; Sarovich, Derek S.; Seymour, Meagan L.; Ginther, Jennifer L.; Kaufman, Emily L.; Beckstrom-Sternberg, Stephen M.; Mayo, Mark; Kaestli, Mirjam; Glass, Mindy B.; Gee, Jay E.; Wuthiekanun, Vanaporn; Warner, Jeffrey M.; Baker, Anthony; Foster, Jeffrey T.; Tan, Patrick; Tuanyok, Apichai; Limmathurotsakul, Direk; Peacock, Sharon J.; Currie, Bart J.; Wagner, David M.; Keim, Paul; Pearson, Talima

2012-01-01

159

Characterization of Ceftazidime Resistance Mechanisms in Clinical Isolates of Burkholderia pseudomallei from Australia  

PubMed Central

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

Sarovich, Derek S.; Price, Erin P.; Von Schulze, Alex T.; Cook, James M.; Mayo, Mark; Watson, Lindsey M.; Richardson, Leisha; Seymour, Meagan L.; Tuanyok, Apichai; Engelthaler, David M.; Pearson, Talima; Peacock, Sharon J.; Currie, Bart J.; Keim, Paul; Wagner, David M.

2012-01-01

160

Diffusion and activity of antibiotics against Burkholderia pseudomallei biofilms.  

PubMed

The diffusion and activity of ceftazidime (CAZ), imipenem (IPM) and trimethoprim/sulfamethoxazole (TMP/SMX) against Burkholderia pseudomallei biofilms were comparatively tested using the high biofilm-producing strain B. pseudomallei 377 and the biofilm-defective mutant B. pseudomallei M6. Biofilms were generated by inoculation of bacteria on polycarbonate membranes placed on the surface of tryptic soy agar plates. The results showed that diffusion of TMP/SMX through B. pseudomallei biofilms was similar for both strains. However, diffusion of CAZ and IPM was significantly faster through strain M6 biofilm in comparison with strain 377 biofilm. The viabilities of strain 377 biofilm were significantly higher than those observed with strain M6 for all antibiotics challenged at 4 h, suggesting that the biofilm-forming capacity may be involved in antibiotic susceptibilities in B. pseudomallei. These results re-emphasise the importance of biofilm for antibiotic resistance in B. pseudomallei. PMID:22364716

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

2012-02-23

161

Bacteraemic melioidosis pneumonia: impact on outcome, clinical and radiological features  

Microsoft Academic Search

Objectives. Melioidosis is an endemic disease in South-east (SE) Asia and bacteraemia in melioidosis is associated with high mortality. We describe some clinical and radiological features of bacteraemic pneumonia due to Burkholderia pseudomallei as well as a comparison with bacteraemic patients without pneumonia.Methods. Patients with positive blood cultures for B. pseudomallei from October 1997 to November 2001 were included. Patients

Amartya Mukhopadhyay; Kang Hoe Lee; Paul Ananth Tambyah

2004-01-01

162

Polysaccharide Specific Monoclonal Antibodies Provide Passive Protection against Intranasal Challenge with Burkholderia pseudomallei  

PubMed Central

Burkholderia pseudomallei is a Gram-negative bacillus that is the causative agent of melioidosis. The bacterium is inherently resistant to many antibiotics and mortality rates remain high in endemic areas. The lipopolysaccharide (LPS) and capsular polysaccharide (CPS) are two surface-associated antigens that contribute to pathogenesis. We previously developed two monoclonal antibodies (mAbs) specific to the CPS and LPS; the CPS mAb was shown to identify antigen in serum and urine from melioidosis patients. The goal of this study was to determine if passive immunization with CPS and LPS mAbs alone and in combination would protect mice from a lethal challenge with B. pseudomallei. Intranasal (i.n.) challenge experiments were performed with B. pseudomallei strains 1026b and K96423. Both mAbs provided significant protection when administered alone. A combination of mAbs was protective when low doses were administered. In addition, combination therapy provided a significant reduction in spleen colony forming units (cfu) compared to results when either the CPS or LPS mAbs were administered alone.

AuCoin, David P.; Reed, Dana E.; Marlenee, Nicole L.; Bowen, Richard A.; Thorkildson, Peter; Judy, Barbara M.; Torres, Alfredo G.; Kozel, Thomas R.

2012-01-01

163

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.

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

2012-01-01

164

Monoclonal antibody-based immunofluorescence microscopy for the rapid identification of Burkholderia pseudomallei in clinical specimens.  

PubMed

The diagnosis of melioidosis depends on the culture of Burkholderia pseudomallei, which takes at least 48 hours. We used a polyclonal-FITC-based immunofluorescence microscopic assay (Pab-IFA) on clinical samples to provide a rapid presumptive diagnosis. This has limitations including photobleaching and batch-to-batch variability. This study evaluated an IFA based on a monoclonal antibody specific to B. pseudomallei (Mab-IFA) and Alexa Fluor 488. A diagnostic evaluation was performed on a prospective cohort of 951 consecutive patients with suspected melioidosis. A total of 1,407 samples were tested. Test accuracy was defined against culture as the gold standard, and was also compared against Pab-IFA. A total of 88 samples from 64 patients were culture positive for B. pseudomallei. The diagnostic sensitivity and specificity of the Mab-IFA was comparable to the Pab-IFA (48.4% versus 45.3% for sensitivity, and 99.8% versus 98.8% for specificity). We have incorporated the Mab-IFA into our routine practice. PMID:23716405

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

2013-05-28

165

Exploiting the Burkholderia pseudomallei Acute Phase Antigen BPSL2765 for Structure-Based Epitope Discovery/Design in Structural Vaccinology.  

PubMed

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

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

2013-08-29

166

SHORT REPORT: A RAPID METHOD FOR THE DIFFERENTIATION OF BURKHOLDERIA PSEUDOMALLEI AND BURKHOLDERIA THAILANDENSIS  

Microsoft Academic Search

A rapid method for the identification and differentiation of Burkholderia pseudomallei and Burkholderia thailandensis colonies is described. It consists of simultaneous use of 2 monoclonal antibody-based latex agglutination test systems. The anti-lipopolysaccharide test reacts with both species, whereas the anti-exopolysaccharide reacts only with B. pseudomallei. Compared with classical biochemical tests, the method is highly reproducibleand accurate . It is particularly

VANAPORN WUTHIEKANUN; NARISARA ANUNTAGOOL; NICHOLAS J. WHITE; STITAYA SIRISINHA

2002-01-01

167

Protease production by Burkholderia pseudomallei and virulence in mice.  

PubMed

The aim of this study was to assess protease production and virulence of various Burkholderia pseudomallei strains. Protease activity was evaluated in filtrates from cultures grown for 50 h in TSB Dialysate by azocasein hydrolysis, and expressed as absorbancy at 405 nm. Virulence was assessed in 8 weeks old SWISS mice, by intraperitoneal injection of 6-6 x 10(5) CFU, and the LD50 was calculated after 30 days by the method of Reed and Muench. The lethal activity was studied for five strains of B. pseudomallei and the type strains of Burkholderia pseudomallei, Burkholderia mallei, and Burkholderia cepacia. The three type strains appeared to be low protease producers (A405 = 0.11, 0.09 and 0.00, respectively) and avirulent. The two more virulent B. pseudomallei strains exhibited significantly different LD50, 3.5 x 10(2) (IPP 6068 VIR) versus 2.1 x 10(5) CFU/mouse (40/97), and protease activities (A405 = 0.046 and 0.79, respectively). Moreover, the avirulent parent of IPP 6068 (AG), was a better protease producer than the 6068 VIR strain, A405 = 0.26 versus 0.046. These results suggest that there is no correlation between virulence and level of exoproteolytic activity, when B. pseudomallei is injected to mice via the intraperitoneal route. PMID:10674652

Gauthier, Y P; Thibault, F M; Paucod, J C; Vidal, D R

2000-02-01

168

Role for the Burkholderia pseudomallei Type Three Secretion System Cluster 1 bpscN Gene in Virulence ?  

PubMed Central

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

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

2011-01-01

169

Clinically practical seminested PCR for Burkholderia pseudomallei quantitated by enzyme immunoassay with and without solution hybridization.  

PubMed Central

Diagnosis of melioidosis, an infectious disease caused by Burkholderia pseudomallei (formerly Pseudomonas pseudomallei), is made initially by antibody testing, which is not always sensitive or specific. We have developed two seminested PCR protocols combined with enzyme immunoassay (EIA) to detect the conserved ribosomal regulatory region of B. pseudomallei. Both PCRs used one biotinylated primer for capturing PCR products on EIA plates. One system, termed solution hybridization EIA (SHEIA), hybridized PCR products with a digoxigenin-labeled probe in solution. Another system, termed primer-labeled EIA (PLEIA), used a digoxigenin-labeled nested primer to generate products that were directly detected without hybridization. To prevent amplicon contamination, pre-PCR uracil DNA glycosylase treatment or post-PCR UV irradiation was incorporated into each system. By a rapid method of blood sample preparation for PCR, these systems had sensitivities of 75 bacteria per ml for SHEIA and 300 bacteria per ml for PLEIA. No nonspecific amplification of other bacterial DNAs was detected. This seminested PCR coupled with SHEIA or PLEIA fulfills all the requirements for a diagnostic test to be used in developing countries where B. pseudomallei is endemic.

Kunakorn, M; Markham, R B

1995-01-01

170

Neurologic melioidosis.  

PubMed

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

2013-07-08

171

Antibiotic susceptibility of 65 isolates of Burkholderia pseudomallei and Burkholderia mallei to 35 antimicrobial agents  

Microsoft Academic Search

Objectives: Fifty isolates of Burkholderia pseudomallei and 15 isolates of Burkholderia mallei were tested for their susceptibilities to 35 antimicrobial agents, including agents not previously tested against these bacteria. Methods: MICs were determined by agar dilution in Mueller-Hinton medium. Results: Among the antibiotics tested, lower MICs were obtained with imipenem, ceftazidime, piperacil- lin, piperacillin\\/tazobactam, doxycycline and minocycline. Fluoroquinolones and aminoglycosides

F. M. Thibault; E. Hernandez; D. R. Vidal; M. Girardet; D. Cavallo

2004-01-01

172

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

Microsoft Academic Search

Burkholderia 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

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

2011-01-01

173

Potential of recombinant flagellin fragment from Burkholderia thailandensis as an antigen for melioidosis antibody detection by indirect ELISA.  

PubMed

Non-pathogenic Burkholderia thailandensis may be used as a model for Burkholderia pseudomallei due to the genetic similarity of these species. Moreover, the experimental manipulation of B. thailandensis is safer. In this study, we constructed recombinant flagellin protein fragments of B. thailandensis E264 (FLAG300, FLAG600, FLAG900, and FLAGFL fragments) and used fragments as the antigen to detect melioidosis antibodies by an indirect enzyme-linked immunosorbent assay (indirect ELISA). The serum samples consisted of serodiagnostic melioidosis sera (N = 52), septicemic sera caused by other bacteria (disease control) (N = 16) and healthy donor sera (N = 40). The area under the receiver operating characteristic (ROC) curve at optimal value (mean plus standard deviation) of all constructed fragments to melioidosis antibodies detection ranged from 0.654 to 0.953. The highest area under the ROC curve (AUROCC, 0.953) for FLAG300 fragment at 1600 serum titer revealed the best performance of melioidosis diagnosis. The indirect ELISA using this fragment as the antigen possessed 82.7% sensitivity and 94.6% specificity. PMID:23159530

Wajanarogana, Sumet; Nimnuch, Phitchapat; Thongmee, Acharawan; Kritsiriwuthinan, Kanyanan

2012-11-15

174

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.

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

2012-01-01

175

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

PubMed

Burkholderia pseudomallei, the etiological agent of melioidosis, causes significant mortality in endemic regions, but little is known regarding the immune mechanisms required for successful protective immunity. To establish a model of immunization that could be used to study this we screened a library of B. pseudomallei strains for immunogenicity in mice. BALB/c mice were immunized with test strains, and 2 weeks later were given a lethal challenge (LC) of virulent B. pseudomallei. Among 49 strains tested, a single strain, CL04, exhibited strong immunoprotective capacity. Interestingly, CL04 had been cultured from a patient with chronic colonization of B. pseudomallei, which is a rare phenomenon. Mice immunized with 0.1 x LD50 (5 x 10(3) CFU) of CL04 had significantly better survival and lower bacterial loads after LC compared to naïve controls. Dose-response analysis demonstrated more robust immunity after higher immunizing doses, and bacterial inactivation by gamma irradiation diminished the protective effect, indicating a requirement for viable organism for immunity. CL04-induced immunity was demonstrated both in B. pseudomallei-susceptible BALB/c and -resistant C57BL/6 mice. We investigated the gene profile of CL04-induced immunity by analyzing responses to immunization using cDNA microarray. Unique responses involving granulocyte macrophage colony stimulating factor (GM-CSF), the proapoptotic regulator Bad and cyclin-dependent kinase (CDK5) were detected in immunized mice, but these responses were absent in naïve-LC mice. Further, responses differed between mouse strains, indicating dependence on host genetic background. This model will be useful in identifying elements of the immune response required for successful adaptive immunity against B. pseudomallei. PMID:16027024

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

2005-06-08

176

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

PubMed Central

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

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

2007-01-01

177

Protease production by Burkholderia pseudomallei and virulence in mice  

Microsoft Academic Search

The aim of this study was to assess protease production and virulence of various Burkholderia pseudomallei strains. Protease activity was evaluated in filtrates from cultures grown for 50 h in TSB Dialysate by azocasein hydrolysis, and expressed as absorbancy at 405 nm. Virulence was assessed in 8 weeks old SWISS mice, by intraperitoneal injection of 6–6×105 CFU, and the LD50

Y. P Gauthier; F. M Thibault; J. C Paucod; D. R Vidal

2000-01-01

178

Mycotic aneurysm caused by Burkholderia pseudomallei with negative blood cultures.  

PubMed

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

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

2004-01-01

179

Antimicrobial resistance to ceftazidime involving loss of penicillin-binding protein 3 in Burkholderia pseudomallei  

PubMed Central

Known mechanisms of resistance to ?-lactam antibiotics include ?-lactamase expression, altered drug target, decreased bacterial permeability, and increased drug efflux. Here, we describe a unique mechanism of ?-lactam resistance in the biothreat organism Burkholderia pseudomallei (the cause of melioidosis), associated with treatment failure during prolonged ceftazidime therapy of natural infection. Detailed comparisons of the initial ceftazidime-susceptible infecting isolate and subsequent ceftazidime-resistant variants from six patients led us to identify a common, large-scale genomic loss involving a minimum of 49 genes in all six resistant strains. Mutational analysis of wild-type B. pseudomallei demonstrated that ceftazidime resistance was due to deletion of a gene encoding a penicillin-binding protein 3 (BPSS1219) present within the region of genomic loss. The clinical ceftazidime-resistant variants failed to grow using commonly used laboratory culture media, including commercial blood cultures, rendering the variants almost undetectable in the diagnostic laboratory. Melioidosis is notoriously difficult to cure and clinical treatment failure is common in patients treated with ceftazidime, the drug of first choice across most of Southeast Asia where the majority of cases are reported. The mechanism described here represents an explanation for ceftazidime treatment failure, and may be a frequent but undetected resistance event.

Chantratita, Narisara; Rholl, Drew A.; Sim, Bernice; Wuthiekanun, Vanaporn; Limmathurotsakul, Direk; Amornchai, Premjit; Thanwisai, Aunchalee; Chua, Hui Hoon; Ooi, Wen Fong; Holden, Matthew T. G.; Day, Nicholas P.; Tan, Patrick; Schweizer, Herbert P.; Peacock, Sharon J.

2011-01-01

180

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

PubMed Central

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

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

2007-01-01

181

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.

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

2013-01-01

182

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

PubMed

Burkholderia pseudomallei, the etiologic agent of melioidosis, is a saprophytic bacterium readily isolated from wet soils of countries bordering the equator. Burkholderia mallei is a host-adapted clone of B. pseudomallei that does not persist outside of its equine reservoir and causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by these organisms typically occurs via percutaneous inoculation or inhalation of aerosols, and the most common manifestation is severe pneumonia leading to fatal bacteremia. Glanders and melioidosis are difficult to diagnose and require prolonged antibiotic therapy with low success rates. There are no vaccines available to protect against either Burkholderia species, and there is concern regarding their use as biological warfare agents given that B. mallei has previously been utilized in this manner. Hence, experiments were performed to establish a mouse model of aerosol infection to study the organisms and develop countermeasures. Using a hand-held aerosolizer, BALB/c mice were inoculated intratracheally with strains B. pseudomallei 1026b and B. mallei ATCC23344 and growth of the agents in the lungs, as well as dissemination to the spleen, were examined. Mice infected with 10(2), 10(3) and 10(4) organisms were unable to control growth of B. mallei in the lungs and bacteria rapidly disseminated to the spleen. Though similar results were observed in mice inoculated with 10(3) and 10(4) B. pseudomallei cells, animals infected with 10(2) organisms controlled bacterial replication in the lungs, dissemination to the spleen, and the extent of bacteremia. Analysis of sera from mice surviving acute infection revealed that animals produced antibodies against antigens known to be targets of the immune response in humans. Taken together, these data show that small volume aerosol inoculation of mice results in acute disease, dose-dependent chronic infection, and immune responses that correlate with those seen in human infections. PMID:24098563

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

2013-10-02

183

Burkholderia pseudomallei Proteins Presented by Monocyte-Derived Dendritic Cells Stimulate Human Memory T Cells In Vitro?  

PubMed Central

Melioidosis is a severe infectious disease caused by the saprophytic facultative intracellular pathogen Burkholderia pseudomallei. The disease is endemic in Southeast Asia and Northern Australia, and no effective vaccine exists. To describe human cell-mediated immune responses to B. pseudomallei and to identify candidate antigens for vaccine development, the ability of antigen-pulsed monocyte-derived dendritic cells (moDCs) to trigger autologous T-cell responses to B. pseudomallei and its products was tested. moDCs were prepared from healthy individuals exposed or not exposed to B. pseudomallei, based on serological evidence. These were pulsed with heat-killed B. pseudomallei or purified antigens, including ABC transporters (LolC, OppA, and PotF), Bsa type III secreted proteins (BipD and BopE), tandem repeat sequence-containing proteins (Rp1 and Rp2), flagellin, and heat shock proteins (Hsp60 and Hsp70), prior to being mixed with autologous T-cell populations. After pulsing of cells with either heat-killed B. pseudomallei, LolC, or Rp2, coculturing the antigen-pulsed moDCs with T cells elicited gamma interferon production from CD4+ T cells from seropositive donors at levels greater than those for seronegative donors. These antigens also induced granzyme B (cytotoxic) responses from CD8+ T cells. Activation of antigen-specific CD4+ T cells required direct contact with moDCs and was therefore not dependent on soluble mediators. Rp peptide epitopes recognized by T cells in healthy individuals were identified. Our study provides valuable novel data on the induction of human cell-mediated immune responses to B. pseudomallei and its protein antigens that may be exploited in the rational development of vaccines to combat melioidosis.

Tippayawat, Patcharaporn; Pinsiri, Maneerat; Rinchai, Darawan; Riyapa, Donporn; Romphruk, Amornrat; Gan, Yunn-Hwen; Houghton, Raymond L.; Felgner, Philip L.; Titball, Richard W.; Stevens, Mark P.; Galyov, Edouard E.; Bancroft, Gregory J.; Lertmemongkolchai, Ganjana

2011-01-01

184

Latex agglutination for rapid detection of Pseudomonas pseudomallei antigen in urine of patients with melioidosis.  

PubMed Central

A latex agglutination test for the detection of Pseudomonas pseudomallei antigen in urine was evaluated for the rapid diagnosis of melioidosis. With unconcentrated urine, antigen was detected in only 18% of patients with melioidosis overall. However, when urine was concentrated 100-fold, antigen was detected in 47% overall and in 67% of patients with septicaemia or disseminated infection, in whom a rapid diagnosis is most important. The specificity of the test was 100%. These results compared favourably with an enzyme immunoassay. This latex agglutination test is a simple, rapid and highly specific method of diagnosing melioidosis, and will be particularly useful in areas with limited laboratory facilities.

Smith, M D; Wuthiekanun, V; Walsh, A L; Teerawattanasook, N; Desakorn, V; Suputtamongkol, Y; Pitt, T L; White, N J

1995-01-01

185

Expression and characterization of an iron-containing superoxide dismutase from Burkholderia pseudomallei.  

PubMed

A superoxide dismutase (SOD) gene from Burkholderia pseudomallei, the causative agent of melioidosis, was cloned and expressed in Escherichia coli, and its product was functionally and physically characterized. The gene has an open-reading frame of 579 bp. The deduced amino acid sequence has 192 residues with a calculated molecular mass of ~22 kDa. Sequence comparison with other bacterial SODs showed that the protein contains typical metal-binding motifs and other Fe-SOD-conserved residues. The sequence has substantial similarity with other bacterial Fe-SOD sequences. The enzymatic activity of the expressed protein was inhibited by hydrogen peroxide but not by sodium azide or potassium cyanide, attributes that indeed are characteristic of typical bacterial Fe-SODs. Western blotting with antiserum against the recombinant Fe-SOD revealed that it is expressed in B. pseudomallei. Transformed E. coli that expressed the Fe-SOD had significantly increased SOD activity and was highly tolerant to paraquat-mediated replication inhibition, compared to transformed cells carrying an empty vector. Our results provide a basis for further biochemical characterization of the enzyme and elucidation of its role in the pathogenesis of B. pseudomallei. PMID:23274991

Cho, Min-Hee; Shin, Yong-Woo; Chun, Jeong-Hoon; Hong, Kee-Jong; Na, Byoung-Kuk; Rhie, Gi-Eun; Seong, Baik-Lin; Yoo, Cheon-Kwon

2012-12-30

186

The detection of insertion sequences within the human pathogen Burkholderia pseudomallei which have been identified previously in Burkholderia cepacia  

Microsoft Academic Search

Using primers designed from the nucleotide sequences of five insertion elements identified previously in Burkholderia cepacia, the presence of two insertion sequences (IS406 and IS407) was detected in chromosomal DNA isolated from strains of the human pathogen Burkholderia pseudomallei. The IS407 homologue was cloned from B. pseudomallei NCTC 4845 and nucleotide sequenced to confirm its identity and degree of homology

Kerri Mack; Richard W Titball

1998-01-01

187

Specificity and functional activity of anti-Burkholderia pseudomallei polysaccharide antibodies.  

PubMed Central

The lipopolysaccharide (LPS) of Burkholderia pseudomallei, the causative agent of melioidosis, consists of two O-antigenic polysaccharides designated O-PS I and O-PS II. In this study, the O-PS specificity and functional activity of a protective polyclonal antiserum and an immunoglobulin M (IgM) monoclonal antibody were determined. The polyclonal antiserum recognized both O-PS I and O-PS II, while the monoclonal antibody was O-PS II specific. Both mediated phagocytic killing of B. pseudomallei by polymorphonuclear leukocytes. Patients acutely infected with B. pseudomallei also produced antibodies to the two O-PSs, but these antibodies were not produced by asymptomatic individuals from an area of endemicity who were seropositive by an indirect hemagglutination test using sonicated heat-killed whole organisms as antigen. IgM antibodies were detected only in patients with localized infection. IgG antibodies were detected in all acutely infected patients, but there was no significant difference in antibody levels among patients with localized infection, patients who survived septicemic illness, and patients who died from septicemic illness. Further analysis of the IgG response revealed production of IgG1 and IgG2 antibodies by all patient groups, while an IgG3 response was seen only in survivors of septicemic infection. IgG4 was not detectable even when a fivefold-lower serum dilution was used. Patient sera also mediated phagocytic killing by polymorphonuclear leukocytes, and the killing effect was enhanced by complement. These results suggest that antibodies to the LPS O-polysaccharides of B. pseudomallei are protective by promoting phagocytic killing. The antibodies develop during human infection and may facilitate clearance of the organisms, as seen in a diabetic rat model of B. pseudomallei infection.

Ho, M; Schollaardt, T; Smith, M D; Perry, M B; Brett, P J; Chaowagul, W; Bryan, L E

1997-01-01

188

Pathogenesis of and immunity to melioidosis  

Microsoft Academic Search

While Burkholderia pseudomallei, the causative agent of melioidosis, is becoming increasingly recognized as a significant cause of morbidity and mortality in regions to which it is endemic, no licensed vaccine preparation currently exists for immunization against the disease. Therefore, one of the primary goals of our research has been to identify and characterize antigens expressed by B. pseudomallei isolates for

Paul J Brett; Donald E Woods

2000-01-01

189

Evaluation of PCR for Diagnosis of Melioidosis  

Microsoft Academic Search

Melioidosis is a potentially fatal infectious disease caused by an organism of the soil, Burkholderia pseudomallei (formerly Pseudomonas pseudomallei). It is endemic in Southeast Asia and northern Australia. Most cases occur during the mon- soonal wet season. The fatality rate of septicemic cases is high, up to 70% in northern Thailand (6, 24) and 47% in the tropical top end

ANTJE HAASE; MAREE BRENNAN; SIOBHAN BARRETT; YVONNE WOOD; SARAH HUFFAM; BART CURRIE

1998-01-01

190

Imported Melioidosis, Israel, 2008  

PubMed Central

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

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

2009-01-01

191

Molecular Investigations of PenA-mediated ?-lactam Resistance in Burkholderia pseudomallei  

PubMed Central

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

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

2011-01-01

192

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

2013-01-22

193

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.

Campos, Cristine G.; Borst, Luke

2013-01-01

194

Quorum sensing negatively regulates multinucleate cell formation during intracellular growth of Burkholderia pseudomallei in macrophage-like cells.  

PubMed

Burkholderia pseudomallei is a Gram-negative environmental bacterium and the causative agent of melioidosis, a potentially fatal, acute or chronic disease endemic in the tropics. Acyl homoserine lactone (AHL)-mediated quorum sensing and signalling have been associated with virulence and biofilm formation in numerous bacterial pathogens. In the canonical acyl-homoserine lactone signalling paradigm, AHLs are detected by a response regulator. B. pseudomallei encodes three AHL synthases, encoded by bpsI1, bpsI2 and bpsI3, and five regulator genes. In this study, we mutated the B. pseudomallei AHL synthases individually and in double and triple combination. Five AHLs were detected and quantified by tandem liquid chromatography-mass spectroscopy. The major AHLs produced were N-octanoylhomoserine lactone and N-(3-hydroxy-decanoyl)homoserine lactone, the expression of which depended on bpsI1 and bpsI2, respectively. B. pseudomallei infection of macrophage cells causes cell fusion, leading to multinucleated cells (3 or more nuclei per cell). A triple mutant defective in production of all three AHL synthases was associated with a striking phenotype of massively enhanced host cellular fusion in macrophages. However, neither abrogation of host cell fusion, achieved by mutation of bimA or hcp1, nor enhancement of fusion altered intracellular replication of B. pseudomallei. Furthermore, when tested in murine models of acute melioidosis the AHL synthase mutants were not attenuated for virulence. Collectively, this study identifies important new aspects of the genetic basis of AHL synthesis in B. pseudomallei and the roles of these AHLs in systemic infection and in cell fusion in macrophages for this important human pathogen. PMID:23704903

Horton, Rachel E; Grant, Gary D; Matthews, Ben; Batzloff, Michael; Owen, Suzzanne J; Kyan, Stephanie; Flegg, Cameron P; Clark, Amanda M; Ulett, Glen C; Morrison, Nigel; Peak, Ian R; Beacham, Ifor R

2013-05-21

195

Quorum Sensing Negatively Regulates Multinucleate Cell Formation during Intracellular Growth of Burkholderia pseudomallei in Macrophage-Like Cells  

PubMed Central

Burkholderia pseudomallei is a Gram-negative environmental bacterium and the causative agent of melioidosis, a potentially fatal, acute or chronic disease endemic in the tropics. Acyl homoserine lactone (AHL)-mediated quorum sensing and signalling have been associated with virulence and biofilm formation in numerous bacterial pathogens. In the canonical acyl-homoserine lactone signalling paradigm, AHLs are detected by a response regulator. B. pseudomallei encodes three AHL synthases, encoded by bpsI1, bpsI2 and bpsI3, and five regulator genes. In this study, we mutated the B. pseudomallei AHL synthases individually and in double and triple combination. Five AHLs were detected and quantified by tandem liquid chromatography-mass spectroscopy. The major AHLs produced were N-octanoylhomoserine lactone and N-(3-hydroxy-decanoyl)homoserine lactone, the expression of which depended on bpsI1 and bpsI2, respectively. B. pseudomallei infection of macrophage cells causes cell fusion, leading to multinucleated cells (3 or more nuclei per cell). A triple mutant defective in production of all three AHL synthases was associated with a striking phenotype of massively enhanced host cellular fusion in macrophages. However, neither abrogation of host cell fusion, achieved by mutation of bimA or hcp1, nor enhancement of fusion altered intracellular replication of B. pseudomallei. Furthermore, when tested in murine models of acute melioidosis the AHL synthase mutants were not attenuated for virulence. Collectively, this study identifies important new aspects of the genetic basis of AHL synthesis in B. pseudomallei and the roles of these AHLs in systemic infection and in cell fusion in macrophages for this important human pathogen.

Horton, Rachel E.; Grant, Gary D.; Matthews, Ben; Batzloff, Michael; Owen, Suzzanne J.; Kyan, Stephanie; Flegg, Cameron P.; Clark, Amanda M.; Ulett, Glen C.; Morrison, Nigel; Peak, Ian R.; Beacham, Ifor R.

2013-01-01

196

Burkholderia pseudomallei: another emerging pathogen in cystic fibrosis  

PubMed Central

Background: Burkholderia pseudomallei is an important cause of acute fulminant pneumonia and septicaemia in tropical regions of northern Australia and south east Asia. Subacute and chronic forms of the disease also occur. There have been three recent reports of adults with cystic fibrosis (CF) who presumably acquired B pseudomallei infection during extended vacations or residence in either Thailand or northern Australia. Methods: The clinical course, molecular characteristics, serology and response to treatment are described in four adult CF patients infected with B pseudomallei. Polymerase chain reaction (PCR) based methods were used to confirm B pseudomallei and exclude B cepacia complex. Genotyping was performed using randomly amplified polymorphic DNA (RAPD) PCR and pulsed field gel electrophoresis (PFGE). Results: Four patients are described with a mean duration of infection of 32 months. All but one patient lived in tropical Queensland. Two patients (with the longest duration of infection) deteriorated clinically and one subsequently died of respiratory failure. Both responded to intravenous treatment specifically targeting B pseudomallei. Another patient suffered two severe episodes of acute bronchopneumonia following acquisition of B pseudomallei. Eradication of the organism was not possible in any of the cases. PFGE of a sample isolate from each patient revealed the strains to be unique and RAPD analysis showed retention of the same strain within an individual over time. Conclusions: These findings support a potential pathogenic role for B pseudomallei in CF lung disease, producing both chronic infection and possibly acute bronchopneumonia. Identical isolates are retained over time and are unique, consistent with likely environmental acquisition and not person to person spread. B pseudomallei is emerging as a significant pathogen for patients with CF residing and holidaying in the tropics.

O'Carroll, M; Kidd, T; Coulter, C; Smith, H; Rose, B; Harbour, C; Bell, S

2003-01-01

197

Characterization and mutagenesis of fur gene from Burkholderia pseudomallei  

Microsoft Academic Search

A homolog of the ferric uptake regulator gene (fur) was isolated from Burkholderia pseudomallei (Bp) by a reverse genetic technique. Sequencing of a 2.2kb DNA fragment revealed an open reading frame with extensive homology to bacterial Fur proteins. The cloned gene encodes a 16kDa protein that cross-reacts with a polyclonal anti-Escherichia coli Fur serum. The transcription start site was determined

Suvit Loprasert; Ratiboot Sallabhan; Wirongrong Whangsuk; Skorn Mongkolsuk

2000-01-01

198

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.

2012-01-01

199

Melioidosis  

MedlinePLUS

... disease, is an infectious disease that can infect humans or animals. The disease is caused by the bacterium Burkholderia pseudomallei . It is predominately a disease of tropical climates, especially in Southeast ... spread to humans and animals through direct contact with the contaminated ...

200

Burkholderia pseudomallei osteomyelitis of the metatarsal in an infant.  

PubMed

Burkoholderia pseudomallei is an emerging cause of localized musculoskeletal infections. We report the case of a 9-month-old infant with isolated primary chronic osteomyelitis of the fifth metatarsal. Radiographs showed expansion and thickening of the cortex. The metatarsal had lytic lesions with scalloped margins; no periosteal reaction or sequestration was seen. Surgical debridement provided removal of infected material and adequate drainage by saucerization. B. pseudomallei was isolated from purulent material, and histologic examination revealed granulomatous inflammation. The child responded rapidly to a 2-week intravenous course of ceftazidime. The present case highlights the need for an awareness of melioidosis as a new differential diagnosis for a nontuberculous, granulomatous inflammation in those living in or visiting tropical regions. PMID:23395629

James, Deeptiman; Madhuri, Vrisha; Gahukamble, Abhay Deodas; Choudhrie, Lisa; Pancharatnam, Padmaja

2013-02-08

201

Human single-chain Fv antibodies against Burkholderia mallei and Burkholderia pseudomallei.  

PubMed

Much effort has been devoted to the development of mouse monoclonal antibodies that react specifically with Burkholderia mallei and Burkholderia pseudomallei for diagnostic and/or therapeutic purposes. Our present study focused on the screening of a phage-displayed nonimmune human single-chain Fv (scFv) antibody library against heat-killed B. mallei and B. pseudomallei for the generation of human scFv antibodies specific to the two pathogenic species of bacteria. Using two different panning procedures, we obtained seven different scFv phage antibodies that interacted with the heat-killed whole bacterial cells of B. mallei and B. pseudomallei. Our results demonstrate that panning of a human scFv antibody library against heat-killed whole bacterial cells may provide a valuable strategy for developing human monoclonal antibodies against the highly pathogenic bacteria. PMID:17392491

Zou, Nianxiang; Newsome, Tamara; Li, Bingjie; Tsai, Shien; Lo, Shyh-Ching

2007-04-01

202

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.

2013-01-01

203

Use of a Real-Time PCR TaqMan Assay for Rapid Identification and Differentiation of Burkholderia pseudomallei and Burkholderia mallei  

Microsoft Academic Search

A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype Burkholderia pseudomallei and Burkholderia mallei isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neigh- bors, such as Burkholderia thailandensis and Burkholderia cepacia. The intracellular, pathogenic bacteria Burkholderia pseudomallei and Burkholderia mallei are the causative

Jana M. U'Ren; Matthew N. Van Ert; James M. Schupp; W. Ryan Easterday; Tatum S. Simonson; Richard T. Okinaka; Talima Pearson; Paul Keim

2005-01-01

204

A structure-based strategy for epitope discovery in Burkholderia pseudomallei OppA antigen.  

PubMed

We present an approach integrating structural and computational biology with immunological tests to identify epitopes in the OppA antigen from the Gram-negative pathogen Burkholderia pseudomallei, the etiological agent of melioidosis. The crystal structure of OppA(Bp), reported here at 2.1 Å resolution, was the basis for a computational analysis that identified three potential epitopes. In parallel, antigen proteolysis and immunocapturing allowed us to identify three additional peptides. All six potential epitopes were synthesized as free peptides and tested for their immunoreactivity against sera from healthy seronegative, healthy seropositive, and recovered melioidosis patients. Three synthetic peptides allowed the different patient groups to be distinguished, underlining the potential of this approach. Extension of the computational analysis, including energy-based decomposition methods, allowed rationalizing results of the predictive analyses and the immunocapture epitope mapping. Our results illustrate a structure-based epitope discovery process, whose application may expand our perspectives in the diagnostic and vaccine design fields. PMID:23159127

Lassaux, Patricia; Peri, Claudio; Ferrer-Navarro, Mario; Gourlay, Louise J; Gori, Alessandro; Conchillo-Solé, Oscar; Rinchai, Darawan; Lertmemongkolchai, Ganjana; Longhi, Renato; Daura, Xavier; Colombo, Giorgio; Bolognesi, Martino

2012-11-15

205

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

PubMed

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

2013-02-27

206

Application of Polymerase Chain Reaction to Detect Burkholderia Pseudomallei and Brucella Species in Buffy Coat from Patients with Febrile Illness Among Rural and Peri-Urban Population  

PubMed Central

Context: Melioidosis and Brucellosis are important endemic infections among people in India, especially in rural settings. Conventional detection techniques have several limitations. Only a few studies exist on the prevalence of Melioidosis and Brucellosis in rural area especially in India. Aim: We sought to evaluate detection of Burkholderia pseudomallei and Brucella spp. among patients presenting febrile illness. Material and Methods: Previously described polymerase chain reaction (PCR) assays for both pathogens were evaluated with Deoxyribonucleic acid extracts of buffy coat samples collected from 301 patients recruited prospectively. Data was not amenable to statistical analysis. Results: The PCR showed specific amplification and no non-specific amplification with heterologous Gram-negative bacilli. The lower limit of detection of the assay for B. pseudomallei was determined to be 1 colony-forming unit /mL and for Brucella it was 1.95 × 103 plasmids per microliter. Blood culture in automated blood culture system was negative for all the samples. This prospective study carried out in southern India for the first time. PCR for Brucella was positive in 1% of the patient samples whereas 0.3% was positive for B. pseudomallei. Conclusion: The finding of Brucella and Burkholderia infections in our populations leads us to suggest that tests for Brucella and B. pseudomallei should also form part of a diagnostic platform for patients with Pyrexia of unknown origin in tropical developing countries.

Nandagopal, Balaji; Sankar, Sathish; Lingesan, Karthikeyan; Appu, KC; Sridharan, Gopalan; Gopinathan, Anilkumar

2012-01-01

207

INDIRECT HEMAGGLUTINATION ASSAY IN PATIENTS WITH MELIOIDOSIS IN NORTHERN AUSTRALIA  

Microsoft Academic Search

Melioidosis is caused by the saprophytic organism Burkholderia pseudomallei. The use of the indirect hemagglutination assay (IHA) has found widespread use in areas endemic for this disease. Using this assay, we explored the serologic profile of 275 patients with culture-confirmed melioidosis in the Northern Territory of Australia. Based on a threshold titer of 1:40, the sensitivity of the IHA on

ALLEN C. CHENG; KEVIN FREEMAN; GARY LUM; BART J. CURRIE

2006-01-01

208

Demonstration of a Cell?Mediated Immune Response in Melioidosis  

Microsoft Academic Search

+ ( ) and ac- P ! .004 tivated CD8 + T cells ( ) in cell cultures from patients. The development of such a cell- P ! .035 mediated immune response in patients may be essential for their survival. Melioidosis is a bacterial infection caused by Burkholderia pseudomallei. It occurs primarily in tropical and subtropical regions (1). B. pseudomallei

Natkunam Ketheesan

2002-01-01

209

Genomic Islands as a Marker to Differentiate between Clinical and Environmental Burkholderia pseudomallei  

PubMed Central

Burkholderia pseudomallei, as a saprophytic bacterium that can cause a severe sepsis disease named melioidosis, has preserved several extra genes in its genome for survival. The sequenced genome of the organism showed high diversity contributed mainly from genomic islands (GIs). Comparative genome hybridization (CGH) of 3 clinical and 2 environmental isolates, using whole genome microarrays based on B. pseudomallei K96243 genes, revealed a difference in the presence of genomic islands between clinical and environmental isolates. The largest GI, GI8, of B. pseudomallei was observed as a 2 sub-GI named GIs8.1 and 8.2 with distinguishable %GC content and unequal presence in the genome. GIs8.1, 8.2 and 15 were found to be more common in clinical isolates. A new GI, GI16c, was detected on chromosome 2. Presences of GIs8.1, 8.2, 15 and 16c were evaluated in 70 environmental and 64 clinical isolates using PCR assays. A combination of GIs8.1 and 16c (positivity of either GI) was detected in 70% of clinical isolates and 11.4% of environmental isolates (P<0.001). Using BALB/c mice model, no significant difference of time to mortality was observed between K96243 isolate and three isolates without GIs under evaluation (P>0.05). Some virulence genes located in the absent GIs and the difference of GIs seems to contribute less to bacterial virulence. The PCR detection of 2 GIs could be used as a cost effective and rapid tool to detect potentially virulent isolates that were contaminated in soil.

Bartpho, Thanatchaporn; Wongsurawat, Thidathip; Wongratanacheewin, Surasakdi; Talaat, Adel M.; Karoonuthaisiri, Nitsara; Sermswan, Rasana W.

2012-01-01

210

Antimicrobial action of the cyclic peptide bactenecin on Burkholderia pseudomallei correlates with efficient membrane permeabilization.  

PubMed

Burkholderia pseudomallei is a category B agent that causes Melioidosis, an acute and chronic disease with septicemia. The current treatment regimen is a heavy dose of antibiotics such as ceftazidime (CAZ); however, the risk of a relapse is possible. Peptide antibiotics are an alternative to classical antibiotics as they exhibit rapid action and are less likely to result in the development of resistance. The aim of this study was to determine the bactericidal activity against B. pseudomallei and examine the membrane disrupting abilities of the potent antimicrobial peptides: bactenecin, RTA3, BMAP-18 and CA-MA. All peptides exhibited >97% bactericidal activity at 20 µM, with bactenecin having slightly higher activity. Long term time-kill assays revealed a complete inhibition of cell growth at 50 µM bactenecin and CA-MA. All peptides inhibited biofilm formation comparable to CAZ, but exhibited faster kinetics (within 1 h). Bactenecin exhibited stronger binding to LPS and induced perturbation of the inner membrane of live cells. Interaction of bactenecin with model membranes resulted in changes in membrane fluidity and permeability, leading to leakage of dye across the membrane at levels two-fold greater than that of other peptides. Modeling of peptide binding on the membrane showed stable and deep insertion of bactenecin into the membrane (up to 9 Å). We propose that bactenecin is able to form dimers or large ?-sheet structures in a concentration dependent manner and subsequently rapidly permeabilize the membrane, leading to cytosolic leakage and cell death in a shorter period of time compared to CAZ. Bactenecin might be considered as a potent antimicrobial agent for use against B. pseudomallei. PMID:23785532

Madhongsa, Kanjana; Pasan, Supaluk; Phophetleb, Onanong; Nasompag, Sawinee; Thammasirirak, Sompong; Daduang, Sakda; Taweechaisupapong, Suwimol; Lomize, Andrei L; Patramanon, Rina

2013-06-13

211

Imaging spectrum of thoracic melioidosis.  

PubMed

Melioidosis is predominantly a tropical disease caused by Burkholderia pseudomallei, a soil-dwelling gram-negative, aerobic bacillus that is distributed primarily in southeast Asia and northern Australia. In this pictorial essay, we provide an illustrated review and conceptual framework of the protean imaging features of this infection, along with a brief description of its clinical manifestations and demographic features. PMID:22781630

Ko, Sheung-Fat; Kung, Chia-Te; Lee, Yi-Wei; Ng, Shu-Hang; Huang, Chung Cheng; Lee, Chen-Hsiang

2013-05-01

212

Capsule Influences the Deposition of Critical Complement C3 Levels Required for the Killing of Burkholderia pseudomallei via NADPH-Oxidase Induction by Human Neutrophils  

PubMed Central

Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD50 via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo.

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

2012-01-01

213

Temperature-Regulated Microcolony Formation by Burkholderia pseudomallei Requires pilA and Enhances Association with Cultured Human Cells  

PubMed Central

Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease that is endemic to Northern Australia and Southeast Asia and is acquired from soil or water. Adherence of B. pseudomallei 08 to cultured cells increases dramatically following prior growth at 30°C or less compared to that following prior growth at 37°C. Here, we show that this occurs almost entirely as the result of microcolony formation (bacterium-bacterium interactions) following growth at 27°C but not at 37°C, which considerably enhances bacterial association with eukaryotic cells. Further, we demonstrate that the type IVA pilin-encoding gene, pilA, is essential for microcolony development by B. pseudomallei 08, and thus optimum association with eukaryotic cells, but is not required for direct adherence (bacterium-cell interactions). In contrast, although the B. pseudomallei genome sequence strain, K96243, also contains transcriptionally active pilA, microcolony formation rarely occurs following growth at either 27°C or 37°C and cell association occurs significantly less than with strain 08. Analysis of pilA transcription in 08 identified that pilA is dramatically upregulated under microcolony-forming conditions, viz., growth at low temperature, and association with eukaryotic cells; the pattern of transcription of pilA in K96243 differed from that in 08. Our study also suggests that biofilm formation by B. pseudomallei 08 and K96243 on polyvinylchloride is not mediated by pilA. Adherence and microcolony formation, and pilA transcription, vary between strains, consistent with known genomic variation in B. pseudomallei, and these phenotypes may be relevant to colonization from the environment.

Boddey, Justin A.; Flegg, Cameron P.; Day, Chris J.; Beacham, Ifor R.; Peak, Ian R.

2006-01-01

214

Burkholderia mallei and Burkholderia pseudomallei stimulate differential inflammatory responses from human alveolar type II cells (ATII) and macrophages.  

PubMed

Alveolar type II pneumocytes (ATII) and alveolar macrophages (AM) play a crucial role in the lung's innate immune response. Burkholderia pseudomallei (BP) and Burkholderia mallei (BM) are facultative Gram-negative bacilli that cause melioidosis and glanders, respectively. The inhalation of these pathogens can cause lethal disease and death in humans. We sought to compare the pathogenesis of and host responses to BP and BM through contact with human primary ATII cells and monocytes-derived macrophages (MDM). We hypothesized that because BP and BM induce different disease outcomes, each pathogen would induce distinct, unique host immune responses from resident pulmonary cells. Our findings showed that BP adhered readily to ATII cells compared to BM. BP, but not BM, was rapidly internalized by macrophages where it replicated to high numbers. Further, BP-induced significantly higher levels of pro-inflammatory cytokine secretion from ATII cells (IL-6, IL-8) and macrophages (IL-6, TNF?) at 6 h post-infection compared to BM (p < 0.05). Interestingly, BM-induced the anti-inflammatory cytokine, IL-10, in ATII cells and macrophages at 6 h post-infection, with delayed induction of inflammatory cytokines at 24 h post-infection. Because BP is flagellated and produces LPS, we confirmed that it stimulated both Toll-like receptor (TLR) 4 and TLR5 via NF-?b activation while the non-flagellated BM stimulated only TLR4. These data show the differences in BP and BM pathogenicity in the lung when infecting human ATII cells and macrophages and demonstrate the ability of these pathogens to elicit distinct immune responses from resident lung cells which may open new targets for therapeutic intervention to fight against these pathogens. PMID:23293773

Lu, Richard; Popov, Vsevolod; Patel, Jignesh; Eaves-Pyles, Tonyia

2012-12-28

215

Burkholderia mallei and Burkholderia pseudomallei stimulate differential inflammatory responses from human alveolar type II cells (ATII) and macrophages  

PubMed Central

Alveolar type II pneumocytes (ATII) and alveolar macrophages (AM) play a crucial role in the lung's innate immune response. Burkholderia pseudomallei (BP) and Burkholderia mallei (BM) are facultative Gram-negative bacilli that cause melioidosis and glanders, respectively. The inhalation of these pathogens can cause lethal disease and death in humans. We sought to compare the pathogenesis of and host responses to BP and BM through contact with human primary ATII cells and monocytes-derived macrophages (MDM). We hypothesized that because BP and BM induce different disease outcomes, each pathogen would induce distinct, unique host immune responses from resident pulmonary cells. Our findings showed that BP adhered readily to ATII cells compared to BM. BP, but not BM, was rapidly internalized by macrophages where it replicated to high numbers. Further, BP-induced significantly higher levels of pro-inflammatory cytokine secretion from ATII cells (IL-6, IL-8) and macrophages (IL-6, TNF?) at 6 h post-infection compared to BM (p < 0.05). Interestingly, BM-induced the anti-inflammatory cytokine, IL-10, in ATII cells and macrophages at 6 h post-infection, with delayed induction of inflammatory cytokines at 24 h post-infection. Because BP is flagellated and produces LPS, we confirmed that it stimulated both Toll-like receptor (TLR) 4 and TLR5 via NF-?b activation while the non-flagellated BM stimulated only TLR4. These data show the differences in BP and BM pathogenicity in the lung when infecting human ATII cells and macrophages and demonstrate the ability of these pathogens to elicit distinct immune responses from resident lung cells which may open new targets for therapeutic intervention to fight against these pathogens.

Lu, Richard; Popov, Vsevolod; Patel, Jignesh; Eaves-Pyles, Tonyia

2012-01-01

216

Melioidosis: acute and chronic disease, relapse and re-activation  

Microsoft Academic Search

In melioidosis-endemic regions the importance of re-activation of Burkholderia pseudomallei from latent foci remains unclear. This topic was assessed in a 10-year prospective study (1989–1999) of melioidosis in the tropical north of the Northern Territory of Australia, together with other aspects of the nature of melioidosis. Incubation period from defined inoculating events was previously ascertained as 1–21 (mean 9) days.

Bart J. Currie; Dale A. Fisher; Nicholas M. Anstey; Susan P. Jacups

2000-01-01

217

Genome-Wide Analysis Reveals Loci Encoding Anti-Macrophage Factors in the Human Pathogen Burkholderia pseudomallei K96243  

PubMed Central

Burkholderia pseudomallei is an important human pathogen whose infection biology is still poorly understood. The bacterium is endemic to tropical regions, including South East Asia and Northern Australia, where it causes melioidosis, a serious disease associated with both high mortality and antibiotic resistance. B. pseudomallei is a Gram-negative facultative intracellular pathogen that is able to replicate in macrophages. However despite the critical nature of its interaction with macrophages, few anti-macrophage factors have been characterized to date. Here we perform a genome-wide gain of function screen of B. pseudomallei strain K96243 to identify loci encoding factors with anti-macrophage activity. We identify a total of 113 such loci scattered across both chromosomes, with positive gene clusters encoding transporters and secretion systems, enzymes/toxins, secondary metabolite, biofilm, adhesion and signal response related factors. Further phenotypic analysis of four of these regions shows that the encoded factors cause striking cellular phenotypes relevant to infection biology, including apoptosis, formation of actin ‘tails’ and multi-nucleation within treated macrophages. The detailed analysis of the remaining host of loci will facilitate genetic dissection of the interaction of this important pathogen with host macrophages and thus further elucidate this critical part of its infection cycle.

Dowling, Andrea J.; Wilkinson, Paul A.; Holden, Matthew T. G.; Quail, Michael A.; Bentley, Stephen D.; Reger, Julia; Waterfield, Nicholas R.; Titball, Richard W.; ffrench-Constant, Richard H.

2010-01-01

218

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

PubMed

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

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

2012-08-01

219

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

PubMed Central

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

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

2012-01-01

220

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

221

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

222

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

PubMed

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. PMID:21359640

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

2011-02-26

223

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

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

2009-01-01

224

Structural characterization of the lipopolysaccharide O antigens of Burkholderia pseudomallei.  

PubMed Central

A serologically typical strain of Burkholderia pseudomallei (strain 304b) was found to produce two S-type lipopolysaccharides (LPS) differing in the chemical structures of their O-polysaccharide (O-PS) components. Structural analysis revealed that one O-antigenic polysaccharide (O-PS I) is an unbranched high-molecular-weight polymer of 1,3-linked 2-O-acetyl-6-deoxy-beta-D-manno-heptopyranose residues. The other LPS O antigen (O-PS II) is an unbranched polymer of repeating disaccharide units having the structure -3)-beta-D-glucopyranose-(1-3)-6-deoxy-alpha-L-talopyranose-(1- in which ca. 33% of the L-6dTalp residues bear 2-O-methyl and 4-O-acetyl substituents while the other L-6dTalp residues carry only 2-O-acetyl substituents. Analysis of a serologically atypical strain of B. pseudomallei (strain 824a) produced a single LPS O-PS which was chemically identical to the 6-deoxy-D-manno-hepan O-PS I. The production of two distinct LPS raises the interesting question of their relative immunogenicities and consequently their relative importance for diagnostic serology and for the possible development of conjugate vaccines.

Perry, M B; MacLean, L L; Schollaardt, T; Bryan, L E; Ho, M

1995-01-01

225

Melioidosis: an important cause of pneumonia in residents of and travellers returned from endemic regions  

Microsoft Academic Search

Melioidosis: an important cause of pneumonia in residents of and travellers returned from endemic regions. B.J. Currie. #ERS Journals Ltd 2003. ABSTRACT: Melioidosis is endemic in South East Asia, Asia and northern Australia. Infection usually follows percutaneous inoculation or inhalation of the causative bacterium, Burkholderia pseudomallei, which is present in soil and surface water in the endemic region. While 20-36%

B. J. Currie

2003-01-01

226

Relapsing melioidosis as cause of iliac mycotic aneurysm: An indigenous case in Taiwan  

Microsoft Academic Search

Melioidosis, an infectious disease caused by Burkholderia pseudomallei, an aerobic gram-negative bacillus, is normally transmitted through skin wounds and contact with infected human beings and animals. Its primary source is rice paddy soil and stagnant water. Melioidosis manifesting as an arterial mycotic aneurysm is rare, and, to our knowledge, infected true and false aneurysms of the iliac artery have never

Chwan-yau Luo; Wen-Chien Ko; Hsin-Chun Lee; Yu-Jen Yang

2003-01-01

227

A rare cause of septic arthritis: melioidosis.  

PubMed

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

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

2013-09-25

228

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

PubMed Central

Background Burkholderia (B.) pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms. Results A collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14?Da specifically and reproducibly found in the spectra of this strain. Conclusions Handling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed.

2012-01-01

229

Identifi cation of Melioidosis Outbreak by Multilocus Variable Number Tandem Repeat Analysis  

Microsoft Academic Search

Endemic melioidosis is caused by genetically diverse Burkholderia pseudomallei strains. However, clonal out- breaks (multiple cases caused by 1 strain) have occurred, such as from contaminated potable water. B. pseudomallei is designated a group B bioterrorism agent, which neces- sitates rapidly recognizing point-source outbreaks. Pulsed- fi eld gel electrophoresis (PFGE) and multilocus sequence typing (MLST) can identify genetically related isolates,

Bart J. Currie; Asha Haslem; Talima Pearson; Heidie Hornstra; Benjamin Leadem; Mark Mayo; Daniel Gal; Linda Ward; Daniel Godoy; Brian G. Spratt; Paul Keim

230

Role and Significance of Quantitative Urine Cultures in Diagnosis of Melioidosis  

Microsoft Academic Search

Melioidosis is associated with significant mortality in countries in which it is endemic. Previous studies have demonstrated that quantitative Burkholderia pseudomallei counts in blood are predictive of mortality. Here we examine the relationship between outcomes and quantitative B. pseudomallei counts in urine. A total of 755 patients presenting to Sappasithiprasong Hospital, Ubon Ratchathani, northeast Thailand (in the northeast part of

Direk Limmathurotsakul; Vanaporn Wuthiekanun; Wirongrong Chierakul; Allen C. Cheng; Bina Maharjan; Wipada Chaowagul; Nicholas J. White; Nicholas P. J. Day; Sharon J. Peacock

2005-01-01

231

Characterization of a Murine Model of Melioidosis: Comparison of Different Strains of Mice  

Microsoft Academic Search

Melioidosis is an infectious disease caused by the saprophytic gram-negative rod Burkholderia pseudomallei. The aim of this study was to establish and characterize a murine model of melioidosis to provide a basis for further investigations on the pathogenesis of the disease. After intravenous infection with B. pseudomallei, C57BL\\/6 mice were found to be significantly more resistant than BALB\\/c mice. There

I. HOPPE; B. BRENNEKE; M. ROHDE; A. KREFT; S. HAUßLER; A. REGANZEROWSKI; I. STEINMETZ

1999-01-01

232

Role of Inducible Nitric Oxide Synthase and NADPH Oxidase in Early Control of Burkholderia pseudomallei Infection in Mice  

Microsoft Academic Search

Infection with the soil bacterium Burkholderia pseudomallei can result in a variety of clinical outcomes, including asymptomatic infection. The initial immune defense mechanisms which might contribute to the various outcomes after environmental contact with B. pseudomallei are largely unknown. We have previously shown that relatively resistant C57BL\\/6 mice can restrict bacterial B. pseudomallei growth more efficiently within 1 day after

Katrin Breitbach; Sonja Klocke; Thomas Tschernig; Nico van Rooijen; Ulrich Baumann; Ivo Steinmetz

2006-01-01

233

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

PubMed

Burkholderia 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 Escherichia 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

2011-08-24

234

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

Microsoft Academic Search

BackgroundBurkholderia pseudomallei is a Category B select agent and the cause of melioidosis. Research funding for vaccine development has largely considered protection within the biothreat context, but the resulting vaccines could be applicable to populations who are at risk of naturally acquired melioidosis. Here, we discuss target populations for vaccination, consider the cost-benefit of different vaccination strategies and review potential

Sharon J. Peacock; Direk Limmathurotsakul; Yoel Lubell; Gavin C. K. W. Koh; Lisa J. White; Nicholas P. J. Day; Richard W. Titball

2012-01-01

235

Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei.  

PubMed

BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis. Like its Salmonella homologues SopE and SopE2, BopE is a guanine nucleotide exchange factor for Rho GTPases. It is thought that, in order to be secreted by the type III system, proteins must be unfolded or only partially folded. As part of a study of B. pseudomallei virulence proteins, we have expressed, purified and characterized the catalytic domain of BopE (amino acids 78-261). Analytical ultracentrifugation experiments in conjunction with analytical size exclusion chromatography show that BopE(78-261) is monomeric in aqueous solution. CD spectroscopy indicates that the protein is predominantly alpha-helical, with predicted secondary structure composition of 59% alpha-helix and 7% beta-strand. NMR spectroscopy confirms that BopE(78-261) adopts a single, stable conformation. In differential scanning calorimetry experiments, thermal denaturation of BopE(78-261) (T(m) 52 degrees C) is reversible. Also, the secondary structure composition of BopE(78-261) changes little over a range of pH values from 3.5 to 10.5. BopE may therefore fold spontaneously to a functional form upon secretion into the host cell cytoplasm, and retains a native or native-like fold in varied environments. These properties are likely to be advantageous for a secreted bacterial effector protein. PMID:15063321

Upadhyay, Abhishek; Williams, Christopher; Gill, Andrew C; Philippe, Didier L; Davis, Kenneth; Taylor, Lowrie A; Stevens, Mark P; Galyov, Edouard E; Bagby, Stefan

2004-04-01

236

Near-atomic resolution analysis of BipD, a component of the type III secretion system of Burkholderia pseudomallei  

PubMed Central

Burkholderia pseudomallei, the causative agent of melioidosis, possesses a type III protein secretion apparatus that is similar to those found in Salmonella and Shigella. A major function of these secretion systems is to inject virulence-associated proteins into target cells of the host organism. The bipD gene of B. pseudomallei encodes a secreted virulence factor that is similar in sequence and is most likely to be functionally analogous to IpaD from Shigella and SipD from Salmonella. Proteins in this family are thought to act as extracellular chaperones at the tip of the secretion needle to help the hydrophobic translocator proteins enter the target cell membrane, where they form a pore and may also link the translocon pore with the secretion needle. BipD has been crystallized in a monoclinic crystal form that diffracted X-rays to 1.5?Å resolution and the structure was refined to an R factor of 16.1% and an R free of 19.8% at this resolution. The putative dimer interface that was observed in previous crystal structures was retained and a larger surface area was buried in the new crystal form.

Pal, M.; Erskine, P. T.; Gill, R. S.; Wood, S. P.; Cooper, J. B.

2010-01-01

237

Immunostimulatory CpG Oligodeoxynucleotide Confers Protection in a Murine Model of Infection with Burkholderia pseudomallei  

Microsoft Academic Search

Although CpG oligodeoxynucleotides (CpG ODNs) are known to enhance resistance against infection in a number of animal models, little is known about the CpG-induced protection against acute fatal sepsis such as that associated with the highly virulent bacterium Burkholderia pseudomallei. We previously demonstrated in an in vitro study that immunostimulatory CpG ODN 1826 enhances phagocytosis of B. pseudomallei and induces

Surasakdi Wongratanacheewin; Wannapa Kespichayawattana; Pakamas Intachote; Sathit Pichyangkul; Rasana W. Sermswan; Arthur M. Krieg; Stitaya Sirisinha

2004-01-01

238

Rapid Identification of Burkholderia pseudomallei by Latex Agglutination Based on an Exopolysaccharide-Specific Monoclonal Antibody  

Microsoft Academic Search

We recently identified a constitutively expressed exopolysaccharide of Burkholderia pseudomallei which is composed of a unique linear tetrasaccharide repeating unit consisting of three galactose residues and one 3-deoxy-D-manno-2-octulosonic acid residue. In this study we developed a latex agglutination test based on monoclonal antibody 3015, which is specific for this exopolysaccharide, and evaluated this test for rapid identification of B. pseudomallei

I. STEINMETZ; A. REGANZEROWSKI; B. BRENNEKE; S. HAUSSLER; A. SIMPSON; N. J. WHITE

1999-01-01

239

Innate Immune Responses of Pulmonary Epithelial Cells to Burkholderia pseudomallei Infection  

Microsoft Academic Search

BackgroundBurkholderia pseudomallei, a facultative intracellular pathogen, causes systemic infection in humans with high mortality especially when infection occurs through an infectious aerosol. Previous studies indicated that the epithelial cells in the lung are an active participant in host immunity. In this study, we aimed to investigate the innate immune responses of lung epithelial cells against B. pseudomallei.Methodology and Principal FindingsUsing

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

2009-01-01

240

Differential intracellular fate of Burkholderia pseudomallei 844 and Burkholderia thailandensis UE5 in human monocyte-derived dendritic cells and macrophages  

PubMed Central

Background Burkholderia pseudomallei (Bp) is a category B biothreat organism that causes a potentially fatal disease in humans and animals, namely melioidosis. Burkholderia thailandensis (Bt) is another naturally occurring species that is very closely related to Bp. However, despite this closely related genotype, Bt is considered avirulent as it does not cause the disease. In the present study, we compared the growth kinetics of B. pseudomallei strain 844 (Bp-844) in human monocyte-derived dendritic cells (MoDCs) and macrophages (M?s), as well as its ability to stimulate host cell responses with those of B. thailandensis strain UE5 (Bt-UE5). Results Primary human MoDCs and M?s were infected with Bp-844 and its intracellular growth kinetics and ability to induce host cell responses were evaluated. The results were compared with those obtained using the Bt-UE5. In human MoDCs, both bacteria were similar in respect to their ability to survive and replicate intracellularly, induce upregulation of costimulatory molecules and cytokines and bias T helper cell differentiation toward a Th1 phenotype. By contrast, the two bacteria exhibited different growth kinetics in human M?s, where the intracellular growth of Bt-UE5, but not Bp-844, was significantly suppressed. Moreover, the ability of M?s to kill Bp-844 was markedly enhanced following stimulation with IFN-?. Conclusion The data presented showed that while both strains were similar in their ability to survive and replicate in human MoDCs, only Bp-844 could readily replicate in human M?s. Both bacteria induced similar host cellular responses, particularly with regard to their ability to bias T cell differentiation toward a Th1 phenotype.

Charoensap, Jaruek; Utaisincharoen, Pongsak; Engering, Anneke; Sirisinha, Stitaya

2009-01-01

241

Rapid Identification of Burkholderia pseudomallei by Latex Agglutination Based on an Exopolysaccharide-Specific Monoclonal Antibody  

PubMed Central

We recently identified a constitutively expressed exopolysaccharide of Burkholderia pseudomallei which is composed of a unique linear tetrasaccharide repeating unit consisting of three galactose residues and one 3-deoxy-d-manno-2-octulosonic acid residue. In this study we developed a latex agglutination test based on monoclonal antibody 3015, which is specific for this exopolysaccharide, and evaluated this test for rapid identification of B. pseudomallei grown on agar plates. All 74 environmental and clinical B. pseudomallei strains tested, originating from different areas of Southeast Asia, northern Australia, and Africa, showed a strong and specific agglutination. B. pseudomallei-like organisms and a variety of other bacteria did not react. In conclusion this monoclonal antibody-based test is a simple, rapid, and highly specific method for identifying B. pseudomallei culture isolates from different geographic areas.

Steinmetz, I.; Reganzerowski, A.; Brenneke, B.; Haussler, S.; Simpson, A.; White, N. J.

1999-01-01

242

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.

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

243

RpoS and oxidative stress conditions regulate succinyl-CoA: 3-ketoacid-coenzyme A transferase (SCOT) expression in Burkholderia pseudomallei.  

PubMed

Burkholderia pseudomallei, a pathogenic gram-negative bacterium, causes the severe human disease melioidosis. This organism can survive in eukaryotic host cells by escaping reactive oxygen species via the regulation of stress responsive sigma factors, including RpoS. In B. pseudomallei, RpoS has been reported to play a role in the oxidative stress response through enhanced activity of OxyR and catalase. In this study, the RpoS dependent oxidative stress responsive system was further characterized using comparative proteomic analysis. The proteomic profiles of wild-type B. pseudomallei following exposure to H2 O2 and between wild-type and the rpoS mutant strains were analyzed. Using stringent criteria, 13 oxidative responsive proteins, eight of which are regulated by RpoS, were identified with high confidence. It was observed that ScoA, a subunit of the SCOT enzyme not previously shown to be involved directly in the oxidative stress response, is significantly down-regulated after hydrogen peroxide treatment. ScoA and ScoB have been predicted to be organized in a single operon using computational methods: in this study it was confirmed by RT-PCR that these genes are indeed co-transcribed as a single mRNA. The present study is the first to report a role for RpoS in the down-regulation of SCOT expression in response to oxidative stress in B. pseudomallei. PMID:23808410

Chutoam, Palatip; Charoensawan, Varodom; Wongtrakoongate, Patompon; Kum-Arth, Apiratana; Buphamalai, Pisanu; Tungpradabkul, Sumalee

2013-09-01

244

Burkholderia pseudomallei-Induced Expression of a Negative Regulator, Sterile-? and Armadillo Motif-Containing Protein, in Mouse Macrophages: a Possible Mechanism for Suppression of the MyD88-Independent Pathway ?  

PubMed Central

Burkholderia pseudomallei, a causative agent of melioidosis, is a Gram-negative facultative intracellular bacterium that can survive and multiply in macrophages. Previously, we demonstrated that B. pseudomallei failed to activate gene expression downstream of the MyD88-independent pathway, particularly the expression of beta interferon (IFN-?) and inducible nitric oxide synthase (iNOS), leading to the inability of macrophages to kill this bacterium. In the present report, we extended our study to show that B. pseudomallei was able to activate sterile-? and Armadillo motif (SARM)-containing protein, a known negative regulator of the MyD88-independent pathway. Both live B. pseudomallei and heat-killed B. pseudomallei were able to upregulate SARM expression in a time-dependent manner in mouse macrophage cell line RAW 264.7. The expression of SARM required bacterial internalization, as it could be inhibited by cytochalasin D. In addition, the intracellular survival of B. pseudomallei was suppressed in SARM-deficient macrophages. Increased expression of IFN-? and iNOS and degradation of I?B? correlated with enhanced macrophage killing capability. These results demonstrated that B. pseudomallei modulated macrophage defense mechanisms by upregulating SARM, thus leading to the suppression of IFN-? and iNOS needed for bacterial elimination.

Pudla, M.; Limposuwan, K.; Utaisincharoen, P.

2011-01-01

245

Melioidosis in an adult male.  

PubMed

Infection with Burkholderia pseudomallei has been described, albeit rarely, patients in Bangladesh. Infection usually follows percutaneous inoculation or inhalation of the causative bacterium, which is present in soil and surface water in the endemic region. A 35-year-young male farmer presented with prolonged fever and significant weight loss. Patient gradually deteriorated despite getting different antibiotics including intravenous ceftriaxone and metronidazole. Panels of investigations were done which revealed no diagnostic confirmation except uncontrolled diabetes and multiple abscesses in different organs. Melioidosis was suspected and serum samples were positive for Burkholderia pseudomallei antibody. The case illustrates the importance of non-specific nature of the clinical presentation and high index of suspicion of uncommon diseases like melioidosis where the disease has not been considered as an endemic. PMID:23715373

Majumder, M I; Haque, M M; Ahmed, M W; Alam, M N; Rahman, M W; Akter, F; Basher, A; Maude, R J; Faiz, M A

2013-04-01

246

Proteomic analysis of colony morphology variants of Burkholderia pseudomallei defines a role for the arginine deiminase system in bacterial survival  

PubMed Central

Colony morphology variation of Burkholderia pseudomallei is a notable feature of a proportion of primary clinical cultures from patients with melioidosis. Here, we examined the hypothesis that colony morphology switching results in phenotypic changes associated with enhanced survival under adverse conditions. We generated isogenic colony morphology types II and III from B. pseudomallei strain 153 type I, and compared their protein expression profiles using 2D gel electrophoresis. Numerous proteins were differentially expressed, the most prominent of which were flagellin, arginine deiminase (AD) and carbamate kinase (CK), which were over-expressed in isogenic types II and III compared with parental type I. AD and CK (encoded by arcA and arcC) are components of the arginine deiminase system (ADS) which facilitates acid tolerance. Reverse transcriptase PCR of arcA and arcC mRNA expression confirmed the proteomic results. Transcripts of parental type I strain 153 arcA and arcC were increased in the presence of arginine, in a low oxygen concentration and in acid. Comparison of wild type with arcA and arcC defective mutants demonstrated that the B. pseudomallei ADS was associated with survival in acid, but did not appear to play a role in intracellular survival or replication within the mouse macrophage cell line J774A.1. These data provide novel insights into proteomic alterations that occur during the complex process of morphotype switching, and lend support to the idea that this is associated with a fitness advantage in vivo.

Chantratita, Narisara; Tandhavanant, Sarunporn; Wikraiphat, Chanthiwa; Trunck, Lily A.; Rholl, Drew A.; Thanwisai, Aunchalee; Saiprom, Natnaree; Limmathurotsakul, Direk; Korbsrisate, Sunee; Day, Nicholas P.J.; Schweizer, Herbert P.; Peacock, Sharon J.

2012-01-01

247

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

Microsoft Academic Search

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,

Muhammad RA Hassan; Subhada P Pani; Ng P Peng; Kirtanaa Voralu; Natesan Vijayalakshmi; Ranjith Mehanderkar; Norasmidar A Aziz; Edwin Michael

2010-01-01

248

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

Microsoft Academic Search

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

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

2012-01-01

249

Genetic Tools for Select-Agent-Compliant Manipulation of Burkholderia pseudomallei  

Microsoft Academic Search

Because of Burkholderia pseudomallei's classification as a select agent in the United States, genetic manip- ulation of this bacterium is strictly regulated. Only a few antibiotic selection markers, including gentamicin, kanamycin, and zeocin, are currently approved for use with this bacterium, but wild-type strains are highly resistant to these antibiotics. To facilitate routine genetic manipulations of wild-type strains, several new

Kyoung-Hee Choi; Takehiko Mima; Yveth Casart; Drew Rholl; Ayush Kumar; Ifor R. Beacham; Herbert P. Schweizer

2008-01-01

250

Burkholderia pseudomallei infection in a cystic fibrosis patient from the Caribbean: A case report  

PubMed Central

Burkholderia pseudomallei is a pathogen identified with increasing frequency in the respiratory tracts of cystic fibrosis (CF) patients from endemic areas such as Southeast Asia and northern Australia. The following report describes the first known reported case in a CF patient from the Caribbean attending a North American CF clinic.

Corral, Dimas Mateos; Coates, Allan L; Yau, Yvonne CW; Tellier, Raymond; Glass, Mindy; Jones, Steven M; Waters, Valerie J

2008-01-01

251

Melioidosis—an unusual cause of septic arthritis  

Microsoft Academic Search

Melioidosis is an infection caused by Burkholderia pseudomallei. It is an important human pathogen in the tropical area. The clinical manifestations are protean with multisystem involvement.\\u000a Septic arthritis and prostatic abscess are rare but well-recognized forms of the disease. Herein we report a case of melioidosis\\u000a presenting with a rare combination of septic arthritis, prostatic abscess, and septicemia.

Joe Thomas; Nambiar Veettil Jayachandran; Pradeep Kumar Shenoy Chandrasekhara; V. Lakshmi; Gumdal Narsimulu

2008-01-01

252

Outcomes of Patients with Melioidosis Treated with Meropenem  

Microsoft Academic Search

Melioidosis, an infection due to Burkholderia pseudomallei, is endemic in southeast Asia and northern Australia. We reviewed our experience with meropenem in the treatment of severe melioidosis in 63 patients over a 6-year period. Outcomes were similar to those of ceftazidime-treated patients (n 153) despite a deliberate selection bias to more-unwell patients receiving meropenem. The mortality among meropenem- treated patients

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

2004-01-01

253

Efficacy of Post Exposure Administration of Doxycycline in a Murine Model of Inhalational Melioidosis  

PubMed Central

Burkholderia pseudomallei is the causative agent of melioidosis. Treatment of melioidosis is suboptimal and developing improved melioidosis therapies requires animal models. In this report, we exposed male BALB/c mice to various amounts of aerosolized B. pseudomallei 1026b to determine lethality. After establishing a median lethal dose (LD50) of 2,772 colony forming units (cfu)/animal, we tested the ability of doxycycline administered 6?hours after exposure to a uniformly lethal dose of ~20 LD50 to prevent death and eliminate bacteria from the lung and spleens. Tissue bacterial burdens were examined by PCR analysis. We found that 100% of mice treated with doxycycline survived and B. pseudomallei DNA was not amplified from the lungs or spleens of most surviving mice. We conclude the BALB/c mouse is a useful model of melioidosis. Furthermore, the data generated in this mouse model indicate that doxycycline is likely to be effective in post-exposure prophylaxis of melioidosis.

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

2013-01-01

254

CpG Oligodeoxyribonucleotides Protect Mice from Burkholderia Pseudomallei but not Francisella Tularensis Schu S4 Aerosols.  

National Technical Information Service (NTIS)

Studies have shown that CpG oligodeoxyribonucleotides (ODN) protect mice from various bacterial pathogens, including Burkholderia pseudomallei and Francisella tularensis live vaccine strain (LVS), when administered before parenteral challenge. Given the p...

D. A. Rozak H. C. Gelhaus L. Huzella M. Smith M. Zadeh

2010-01-01

255

Development and evaluation of a real-time PCR assay targeting the type III secretion system of Burkholderia pseudomallei  

Microsoft Academic Search

Here we report on the development of a discriminatory real-time assay for the rapid identification of Burkholderia pseudomallei isolates and the evaluation of this assay for sensitivity against related species and detection in spiked human blood samples. The assay targets a 115-base-pair region within orf2 of the B. pseudomallei type III secretion system gene cluster and distinguishes B. pseudomallei from

Ryan T. Novak; Mindy B. Glass; Jay E. Gee; Daniel Gal; Mark J. Mayo; Bart J. Currie; Patricia P. Wilkins

2006-01-01

256

High-level resistance to fluoroquinolones and cephalosporins in Burkholderia pseudomallei and closely related species.  

PubMed

The molecular mechanisms involved in the development of a high level of resistance to a wide range of antimicrobials in Burkholderia pseudomallei and closely related species have not been sufficiently investigated. In the present study, the properties of B. pseudomallei, B. mallei and B. thailandensis mutants with increased resistance to fluoroquinolones and cephalosporins were analysed. Resistance to pefloxacin, ofloxacin and ceftazidime in B. pseudomallei and B. thailandensis was accompanied by an increased resistance to aminoglycosides, beta-lactams, macrolides and chloramphenicol, whereas mutants of B. mallei were characterized by a narrower spectrum of resistance. With the use of the differential display technique, we demonstrated that multiple resistant variants of B. pseudomallei, B. mallei and B. thailandensis had an increased expression of putative efflux transporters belonging to the resistance-nodulation division superfamily and the major facilitator superfamily. With the application of PCR-single-stranded conformational polymorphism (PCR-SSCP) and sequencing, point mutations in gyrA quinolone-resistance determining region were detected in the part of multiple resistant B. pseudomallei and B. mallei mutants. These results indicate that various molecular mechanisms are involved in the development of multiple drug resistance in pathogenic Burkholderia and may be useful for further studying the adaptability of this microorganism and optimization of treatment. PMID:19121669

Viktorov, Dimitry V; Zakharova, Irina B; Podshivalova, Maria V; Kalinkina, Elena V; Merinova, Olga A; Ageeva, Natalya P; Antonov, Valery A; Merinova, Lyudmila K; Alekseev, Vladimir V

2008-12-01

257

Burkholderia pseudomallei kills Caenorhabditis elegans through virulence mechanisms distinct from intestinal lumen colonization  

PubMed Central

The nematode Caenorhabditis elegans is hypersusceptible to Burkholderia pseudomallei infection. However, the virulence mechanisms underlying rapid lethality of C. elegans upon B. pseudomallei infection remain poorly defined. To probe the host-pathogen interaction, we constructed GFP-tagged B. pseudomallei and followed bacterial accumulation within the C. elegans intestinal lumen. Contrary to slow-killing by most bacterial pathogens, B. pseudomallei caused fairly limited intestinal lumen colonization throughout the period of observation. Using grinder-defective mutant worms that allow the entry of intact bacteria also did not result in full intestinal lumen colonization. In addition, we observed a significant decline in C. elegans defecation and pharyngeal pumping rates upon B. pseudomallei infection. The decline in defecation rates ruled out the contribution of defecation to the limited B. pseudomallei colonization. We also demonstrated that the limited intestinal lumen colonization was not attributed to slowed host feeding as bacterial loads did not change significantly when feeding was stimulated by exogenous serotonin. Both these observations confirm that B. pseudomallei is a poor colonizer of the C. elegans intestine. To explore the possibility of toxin-mediated killing, we examined the transcription of the C. elegans ABC transporter gene, pgp-5, upon B. pseudomallei infection of the ppgp-5::gfp reporter strain. Expression of pgp-5 was highly induced, notably in the pharynx and intestine, compared with Escherichia coli-fed worms, suggesting that the host actively thwarted the pathogenic assaults during infection. Collectively, our findings propose that B. pseudomallei specifically and continuously secretes toxins to overcome C. elegans immune responses.

Ooi, Soon-Keat; Lim, Tian-Yeh; Lee, Song-Hua; Nathan, Sheila

2012-01-01

258

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

PubMed

Burkholderia pseudomallei (BP) and B. mallei (BM) are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs) against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7) were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs) against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR) amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO)-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65°C/90 min), sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1) reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM). The cMAb BP7 2C6 (cMAb CK2) recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14?28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3) reacted with lipopolysaccharides (38?52 kDa in BP; 38?60 kDa in B. thailandensis). Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection. PMID:21573027

Kim, Hyung-Yong; Tsai, Shien; Lo, Shyh-Ching; Wear, Douglas J; Izadjoo, Mina J

2011-05-09

259

Development of signature-tagged mutagenesis in Burkholderia pseudomallei to identify genes important in survival and pathogenesis.  

PubMed

Burkholderia pseudomallei, the causative agent of melioidosis, is an important human pathogen in Southeast Asia and northern Australia for which a vaccine is unavailable. A panel of 892 double signature-tagged mutants was screened for virulence using an intranasal BALB/c mouse model of infection. A novel DNA tag microarray identified 33 mutants as being attenuated in spleens, while 6 were attenuated in both lungs and spleens. The transposon insertion sites in spleen-attenuated mutants revealed genes involved in several stages of capsular polysaccharide biosynthesis and DNA replication and repair, a putative oxidoreductase, ABC transporters, and a lipoprotein that may be important in intercellular spreading. The six mutants identified as missing in both lungs and spleens were found to have insertions in recA involved in the SOS response and DNA repair; putative auxotrophs of leucine, threonine, p-aminobenzoic acid, and a mutant with an insertion in aroB causing auxotrophy for aromatic compounds were also found. Murine challenge studies revealed partial protection in BALB/c mice vaccinated with the aroB mutant. The refined signature-tagged mutagenesis approach developed in this study was used to efficiently identify attenuating mutants from this highly pathogenic species and could be applied to other organisms. PMID:17189432

Cuccui, J; Easton, A; Chu, K K; Bancroft, G J; Oyston, P C F; Titball, R W; Wren, B W

2006-12-22

260

DISCRIMINATION OF Burkholderia mallei/pseudomallei FROM Burkholderia thailandensis BY SEQUENCE COMPARISON OF A FRAGMENT OF THE RIBOSOMAL PROTEIN S21 (RPSU) GENE  

PubMed Central

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

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

2012-01-01

261

A Burkholderia pseudomallei Toxin Inhibits Helicase Activity of Translation Factor eIF4A  

PubMed Central

The structure of BPSL1549, a protein of unknown function from Burkholderia pseudomallei reveals a similarity to E. coli cytotoxic necrotizing factor 1. We found that BPSL1549 acted as a potent cytotoxin against eukaryotic cells and was lethal when administered to mice. Expression levels of bpsl1549 correlate with conditions expected to promote or suppress pathogenicity. BPSL1549 promotes deamidation of Gln339 of the translation initiation factor eIF4A, abolishing its helicase activity and inhibiting translation. We propose to name BPSL1549 Burkholderia Lethal Factor 1 (BLF1).

Cruz, Abimael; Hautbergue, Guillaume M.; Artymiuk, Peter J.; Baker, Patrick J.; Bokori-Brown, Monika; Chang, Chung-Te; Dickman, Mark J.; Essex-Lopresti, Angela; Harding, Sarah V.; Mahadi, Nor Muhammad; Marshall, Laura E.; Mobbs, George W.; Mohamed, Rahmah; Nathan, Sheila; Ngugi, Sarah A.; Ong, Catherine; Ooi, Wen Fong; Partridge, Lynda J.; Phillips, Helen L.; Raih, M. Firdaus; Ruzhenikov, Sergei; Sarkar-Tyson, Mitali; Sedelnikova, Svetlana E.; Smither, Sophie J.; Tan, Patrick; Titball, Richard W.; Wilson, Stuart A.; Rice, David W.

2012-01-01

262

Survival, sublethal injury, and recovery of environmental Burkholderia pseudomallei in soil subjected to desiccation.  

PubMed

Environmental Burkholderia pseudomallei isolated from sandy soil at Castle Hill, Townsville, in the dry tropic region of Queensland, Australia, was inoculated into sterile-soil laboratory microcosms subjected to variable soil moisture. Survival and sublethal injury of the B. pseudomallei strain were monitored by recovery using culture-based methods. Soil extraction buffer yielded higher recoveries as an extraction agent than sterile distilled water. B. pseudomallei was not recoverable when inoculated into desiccated soil but remained recoverable from moist soil subjected to 91 days' desiccation and showed a growth response to increased soil moisture over at least 113 days. Results indicate that endemic dry tropic soil may act as a reservoir during the dry season, with an increase in cell number and potential for mobilization from soil into water in the wet season. PMID:23377947

Larsen, Eloise; Smith, James J; Norton, Robert; Corkeron, Maree

2013-02-01

263

Survival, Sublethal Injury, and Recovery of Environmental Burkholderia pseudomallei in Soil Subjected to Desiccation  

PubMed Central

Environmental Burkholderia pseudomallei isolated from sandy soil at Castle Hill, Townsville, in the dry tropic region of Queensland, Australia, was inoculated into sterile-soil laboratory microcosms subjected to variable soil moisture. Survival and sublethal injury of the B. pseudomallei strain were monitored by recovery using culture-based methods. Soil extraction buffer yielded higher recoveries as an extraction agent than sterile distilled water. B. pseudomallei was not recoverable when inoculated into desiccated soil but remained recoverable from moist soil subjected to 91 days' desiccation and showed a growth response to increased soil moisture over at least 113 days. Results indicate that endemic dry tropic soil may act as a reservoir during the dry season, with an increase in cell number and potential for mobilization from soil into water in the wet season.

Larsen, Eloise; Smith, James J.; Norton, Robert

2013-01-01

264

Detection of Burkholderia pseudomallei in blood samples using polymerase chain reaction  

Microsoft Academic Search

A highly sensitive, specific, rapid and simple method to detectBurkholderia pseudomalleiin blood samples was developed. Two 22-base oligonucleotide primers, based on sequences from a specific DNA probe, were used for amplification of bacterial DNA by the polymerase chain reaction (PCR). Amplification with these primers yielded a 178-base pair product in 100 clinical isolates ofB. pseudomallei. As little as 0·5 fg

A. Rattanathongkom; R. W. Sermswan; S. Wongratanacheewin

1997-01-01

265

Attachment of Burkholderia pseudomallei to pharyngeal epithelial cells: a highly pathogenic bacteria with low attachment ability  

Microsoft Academic Search

Respiratory infections are initiated by the attachment of bacteria to pharyngeal epithelial cells. We studied the attachment of Burkholderia pseudomallei to pharyngeal epithelial cells. After one, two, three, and four washes, there were 22.6 6 8.9, 15.7 6 7.0, 6.8 6 3.1, and 4.6 6 1.1 (mean 6 SD) attached bacteria\\/cell, respectively. If the bacterial concentration was maintained at 1

Misao Tao; Akemi Omori; Prasit Tharavichikul; Tsuyoshi Nagatake

266

Effects of soil pH, temperature and water content on the growth of Burkholderia pseudomallei  

Microsoft Academic Search

Optimum conditions were determined for the growth ofBurkholderia pseudomallei in natural soils or waters. It grows better in paddy soil, crop-covered and fallow field than in fresh and salty water. Although\\u000a the optimal temperature and pH for the growth were 37 or 42 °C, and 6.5 or 7.5 in an environmental-mimicking soil medium,\\u000a this bacterium can still grow at 4

Y. S. Chen; S. C. Chen; C. M. Kao; Y. L. Chen

2003-01-01

267

Alanine Racemase Mutants of Burkholderia pseudomallei and Burkholderia mallei and Use of Alanine Racemase as a Non-Antibiotic-Based Selectable Marker  

PubMed Central

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous d-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for d-alanine. During log phase growth without d-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ?flgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages.

Zajdowicz, Sheryl L. W.; Jones-Carson, Jessica; Vazquez-Torres, Andres; Jobling, Michael G.; Gill, Ronald E.; Holmes, Randall K.

2011-01-01

268

In Vitro and In Vivo studies of monoclonal antibodies with prominent bactericidal activity against Burkholderia pseudomallei and Burkholderia mallei.  

PubMed

Our laboratory has developed more than a hundred mouse monoclonal antibodies (MAbs) against Burkholderia pseudomallei and Burkholderia mallei. These antibodies have been categorized into different groups based on their specificities and the biochemical natures of their target antigens. The current study first examined the bactericidal activities of a number of these MAbs by an in vitro opsonic assay. Then, the in vivo protective efficacy of selected MAbs was evaluated using BALB/c mice challenged intranasally with a lethal dose of the bacteria. The opsonic assay using dimethyl sulfoxide-treated human HL-60 cells as phagocytes revealed that 19 out of 47 tested MAbs (40%) have prominent bactericidal activities against B. pseudomallei and/or B. mallei. Interestingly, all MAbs with strong opsonic activities are those with specificity against either the capsular polysaccharides (PS) or the lipopolysaccharides (LPS) of the bacteria. On the other hand, none of the MAbs reacting to bacterial proteins or glycoproteins showed prominent bactericidal activity. Further study revealed that the antigenic epitopes on either the capsular PS or LPS molecules were readily available for binding in intact bacteria, while the epitopes on proteins/glycoproteins were less accessible to the MAbs. Our in vivo study showed that four MAbs reactive to either the capsular PS or LPS were highly effective in protecting mice against lethal bacterial challenge. The result is compatible with that of our in vitro study. The MAbs with the highest protective efficacy are those reactive to either the capsular PS or LPS of the Burkholderia bacteria. PMID:21450976

Zhang, Shimin; Feng, Shaw-Huey; Li, Bingjie; Kim, Hyung-Yong; Rodriguez, Joe; Tsai, Shien; Lo, Shyh-Ching

2011-03-30

269

In Vitro and In Vivo Studies of Monoclonal Antibodies with Prominent Bactericidal Activity against Burkholderia pseudomallei and Burkholderia mallei?  

PubMed Central

Our laboratory has developed more than a hundred mouse monoclonal antibodies (MAbs) against Burkholderia pseudomallei and Burkholderia mallei. These antibodies have been categorized into different groups based on their specificities and the biochemical natures of their target antigens. The current study first examined the bactericidal activities of a number of these MAbs by an in vitro opsonic assay. Then, the in vivo protective efficacy of selected MAbs was evaluated using BALB/c mice challenged intranasally with a lethal dose of the bacteria. The opsonic assay using dimethyl sulfoxide-treated human HL-60 cells as phagocytes revealed that 19 out of 47 tested MAbs (40%) have prominent bactericidal activities against B. pseudomallei and/or B. mallei. Interestingly, all MAbs with strong opsonic activities are those with specificity against either the capsular polysaccharides (PS) or the lipopolysaccharides (LPS) of the bacteria. On the other hand, none of the MAbs reacting to bacterial proteins or glycoproteins showed prominent bactericidal activity. Further study revealed that the antigenic epitopes on either the capsular PS or LPS molecules were readily available for binding in intact bacteria, while the epitopes on proteins/glycoproteins were less accessible to the MAbs. Our in vivo study showed that four MAbs reactive to either the capsular PS or LPS were highly effective in protecting mice against lethal bacterial challenge. The result is compatible with that of our in vitro study. The MAbs with the highest protective efficacy are those reactive to either the capsular PS or LPS of the Burkholderia bacteria.

Zhang, Shimin; Feng, Shaw-Huey; Li, Bingjie; Kim, Hyung-Yong; Rodriguez, Joe; Tsai, Shien; Lo, Shyh-Ching

2011-01-01

270

Epidemiological Surveillance of Melioidosis in Singapore  

Microsoft Academic Search

During the period 1989 to 1996, a total of 372 cases of melioidosis, with 147 deaths, were reported, giving a mean annual incid ence rate of 1.7 per 100 000 population and a case-fatality rate of 39.5%. Majority (89%) of the clinical cases were confirmed by culture of Burkholderia pseudomallei, while the others were presumptive cases based on a single

B H Heng; K T Goh; E H Yap; H Loh; M Yeo

271

ELISA and immuno-polymerase chain reaction assays for the sensitive detection of melioidosis.  

PubMed

Melioidosis is caused by the Gram-negative bacterium Burkholderia pseudomallei. The gold standard for diagnosis is culture, which requires at least 3-4 days to obtain a result, hindering successful treatment of acute disease. An indirect haemagglutination assay (IHA) is often used but lacks sensitivity. Approximately half of patients later confirmed culture positive are not detected by IHA at presentation and a subset of patients persistently continue to be IHA negative. More rapid and reliable serologic testing for melioidosis is essential and will improve diagnosis and patient outcome. We have developed an ELISA and a quantitative immuno-polymerase chain reaction assay capable of detecting melioidosis-specific antibodies and demonstrate their validity with IHA-negative sera from patients with melioidosis. These new sensitive assays are based upon a secreted antigenic fraction from B. pseudomallei and will be ideal for the diagnosis of melioidosis in patients in nonendemic regions returning from endemic tropical areas and for seroepidemiologic surveys. PMID:23177220

Cooper, Alanna; Williams, Natasha L; Morris, Jodie L; Norton, Robert E; Ketheesan, Natkunam; Schaeffer, Patrick M

2012-11-21

272

Melioidosis and the kidney.  

PubMed

Melioidosis, caused by the saprophytic soil and freshwater Gram-negative aerobic bacillus Burkholderia pseudomallei, is classically characterized by pneumonia, sometimes with multiple organ abscesses, usually in patients with defined risk factors and with a mortality rate of up to 40%. It is a major cause of community-acquired sepsis in Southeast Asia and tropical northern Australia with an expanding global geographical distribution. It is increasingly recognized as an opportunistic infectious disease of importance to physicians, who may need to suspect it in at-risk patients that may come from or visit endemic areas, and could be fatal if treated late or inappropriately. Mortality could be prevented by early institution of specific antimicrobial therapy. Epidemiology, clinical features, overall management, and aspects of melioidosis particularly relevant to kidney disease and immunosuppression are discussed in this review. PMID:23279670

Jabbar, Zulfikar; Currie, Bart J

2013-03-01

273

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

PubMed

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

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

2011-11-12

274

Genomic transcriptional profiling identifies a candidate blood biomarker signature for the diagnosis of septicemic melioidosis  

Microsoft Academic Search

BACKGROUND: Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei, a Gram-negative bacillus classified by the National Institute of Allergy and Infectious Diseases (NIAID) as a category B priority agent. Septicemia is the most common presentation of the disease with a 40% mortality rate even with appropriate treatments. Better diagnostic tests are therefore needed to improve therapeutic efficacy and

Rungnapa Pankla; Surachat Buddhisa; Matthew Berry; Derek M Blankenship; Gregory J Bancroft; Jacques Banchereau; Ganjana Lertmemongkolchai; Damien Chaussabel

2009-01-01

275

Nasal Acai polysaccharides potentiate innate immunity to protect against pulmonary Francisella tularensis and Burkholderia pseudomallei Infections.  

PubMed

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

2012-03-15

276

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

PubMed Central

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

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

2009-01-01

277

Expression and Function of Macrophage Migration Inhibitory Factor (MIF) in Melioidosis  

PubMed Central

Background Macrophage migration inhibitory factor (MIF) has emerged as a pivotal mediator of innate immunity and has been shown to be an important effector molecule in severe sepsis. Melioidosis, caused by Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast-Asia. We aimed to characterize the expression and function of MIF in melioidosis. Methodology and Principal Findings MIF expression was determined in leukocytes and plasma from 34 melioidosis patients and 32 controls, and in mice infected with B. pseudomallei. MIF function was investigated in experimental murine melioidosis using anti-MIF antibodies and recombinant MIF. Patients demonstrated markedly increased MIF mRNA leukocyte and MIF plasma concentrations. Elevated MIF concentrations were associated with mortality. Mice inoculated intranasally with B. pseudomallei displayed a robust increase in pulmonary and systemic MIF expression. Anti-MIF treated mice showed lower bacterial loads in their lungs upon infection with a low inoculum. Conversely, mice treated with recombinant MIF displayed a modestly impaired clearance of B. pseudomallei. MIF exerted no direct effects on bacterial outgrowth or phagocytosis of B. pseudomallei. Conclusions MIF concentrations are markedly elevated during clinical melioidosis and correlate with patients' outcomes. In experimental melioidosis MIF impaired antibacterial defense.

Wiersinga, W. Joost; Calandra, Thierry; Kager, Liesbeth M.; van der Windt, Gerritje J. W.; Roger, Thierry; le Roy, Didier; Florquin, Sandrine; Peacock, Sharon J.; Sweep, Fred C. G. J.; van der Poll, Tom

2010-01-01

278

Structural Characterization of the Lipopolysaccharide O Antigens ofBurkholderia pseudomallei  

Microsoft Academic Search

A serologically typical strain of Burkholderia pseudomallei (strain 304b) was found to produce two S-type lipopolysaccharides (LPS) differing in the chemical structures of their O-polysaccharide (O-PS) components. Structural analysis revealed that one O-antigenic polysaccharide (O-PS I) is an unbranched high-molecular- weight polymer of 1,3-linked 2-O-acetyl-6-deoxy-b-D-manno-heptopyranose residues. The other LPS O antigen (O-PS II) is an unbranched polymer of repeating disaccharide

MALCOLM B. PERRY; LEANN L. MACLEAN; TINEKE SCHOLLAARDT

279

A Burkholderia pseudomallei ?purM Mutant Is Avirulent in Immunocompetent and Immunodeficient Animals: Candidate Strain for Exclusion from Select-Agent Lists?  

PubMed Central

Burkholderia pseudomallei causes the disease melioidosis in humans and is classified as a category B select agent. Research utilizing this pathogen is highly regulated in the United States, and even basic studies must be conducted in biosafety level 3 (BSL-3) facilities. There is currently no attenuated B. pseudomallei strain available that is excluded from select-agent regulations and can be safely handled at BSL-2 facilities. To address this need, we created Bp82 and Bp190, which are ?purM derivatives of B. pseudomallei strains 1026b and K96243 that are deficient in adenine and thiamine biosynthesis but replication competent in vitro in rich medium. A series of animal challenge studies was conducted to ensure that these strains were fully attenuated. Whereas the parental strains 1026b and K96243 and the complemented mutants Bp410 and Bp454 were virulent in BALB/c mice following intranasal inoculation, the ?purM mutants Bp82 and Bp190 were avirulent even when they were administered at doses 4 logs higher than the doses used for the parental strains. Animals challenged with high doses of the ?purM mutants rapidly cleared the bacterium from tissues (lung, liver, and spleen) and remained free of culturable bacteria for the duration of the experiments (up to 60 days postinfection). Moreover, highly susceptible 129/SvEv mice and immune incompetent mice (IFN-??/?, SCID) were resistant to challenges with ?purM mutant Bp82. This strain was also avirulent in the Syrian hamster challenge model. We concluded that ?purM mutant Bp82 is fully attenuated and safe for use under BSL-2 laboratory conditions and thus is a candidate for exclusion from the select-agent list.

Propst, Katie L.; Mima, Takehiko; Choi, Kyoung-Hee; Dow, Steven W.; Schweizer, Herbert P.

2010-01-01

280

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

PubMed

The nitrogen-related phosphoenolpyruvate phosphotransferase system (PTS(Ntr) ) is involved in controlling ammonia assimilation and nitrogen fixation. The additional role of PTS(Ntr) as a regulatory link between nitrogen and carbon utilization in Escherichia coli is assumed to be closely related to molecular functions of IIA(Ntr) in potassium homeostasis. We have determined the crystal structure of IIA(Ntr) from Burkholderia pseudomallei (BpIIA(Ntr) ), which is a causative agent of melioidosis. The crystal structure of dimeric BpIIA(Ntr) determined at 3.0 Å revealed that its active sites are mutually blocked. This dimeric state is stabilized by charge and weak hydrophobic interactions. Overall monomeric structure and the active site residues, Arg51 and His67, of BpIIA(Ntr) are well conserved with those of IIA(Ntr) enzymes from E. coli and Neisseria meningitides. Interestingly, His113 of BpIIA(Ntr) , which corresponds to a key residue in another phosphoryl group relay in the mannitol-specific enzyme EIIA family (EIIA(Mtl) ), is located away from the active site due to the loop connecting ?5 and ?3. Combined with other differences in molecular surface properties, these structural signatures distinguish the IIA(Ntr) family from the EIIA(Mtl) family. Since, there is no gene for NPr in the chromosome of B. pseudomallei, modeling and docking studies of the BpIIA(Ntr) -BpHPr complex has been performed to support the proposal on the NPr-like activity of BpHPr. A potential dual role of BpHPr as a nonspecific phosphocarrier protein interacting with both sugar EIIAs and IIA(Ntr) in B. pseudomallei has been discussed. PMID:23483653

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

2013-09-01

281

Expression and Function of Transforming Growth Factor ? in Melioidosis  

PubMed Central

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast Asia and northern Australia. An important controller of the immune system is the pleiotropic cytokine transforming growth factor ? (TGF-?), of which Smad2 and Smad3 are the major signal transducers. In this study, we aimed to characterize TGF-? expression and function in experimental melioidosis. TGF-? expression was determined in 33 patients with culture-proven infection with B. pseudomallei and 30 healthy controls. We found that plasma TGF-? concentrations were strongly elevated during melioidosis. In line with this finding, TGF-? expression in C57BL/6 mice intranasally inoculated with B. pseudomallei was enhanced as well. To assess the role of TGF-?, we inhibited TGF-? using a selective murine TGF-? antibody. Treatment of mice with anti-TGF-? antibody resulted in decreased lung Smad2 phosphorylation. TGF-? blockade appeared to be protective: mice treated with anti-TGF-? antibody and subsequently infected with B. pseudomallei showed diminished bacterial loads. Moreover, less distant organ injury was observed in anti-TGF-? treated mice as shown by reduced blood urea nitrogen (BUN) and aspartate transaminase (AST) values. However, anti-TGF-? treatment did not have an effect on survival. In conclusion, TGF-? is upregulated during B. pseudomallei infection and plays a limited but proinflammatory role during experimental melioidosis.

Wieland, Catharina W.; van der Windt, Gerritje J. W.; Duitman, Jan-Willem; Boon, Louis; Day, Nicholas P. J.; Peacock, Sharon J.; van der Poll, Tom; Wiersinga, W. Joost

2012-01-01

282

Morbidity and Mortality Weekly Report, Vol. 55, No. 32, August 18, 2006. Imported Melioidosis, South Florida, 2005.  

National Technical Information Service (NTIS)

In 2005, two cases of melioidosis (one in August, one in October) were reported to the Florida Department of Health, the first cases since reporting the disease became mandatory in Florida in 2003. In one case, Burkholderia pseudomallei was not recognized...

2006-01-01

283

Burkholderia pseudomallei infection induces the expression of apoptosis-related genes and proteins in mouse macrophages.  

PubMed

BACKGROUND/PURPOSE: In this study, we addressed whether the production of apoptosis-related genes and proteins is induced in mouse macrophages infected with Burkholderia pseudomallei cells. METHODS: Mouse macrophages were infected with B. pseudomallei cells at 0.5 hours, 1 hour, 2 hours, 4 hours, and 6 hours, respectively, followed by real-time polymerase chain reaction (PCR) array analysis. The amount of apoptosis-related proteins (caspase-3, caspase -8, caspase -9, Bax, and Bcl-2) was confirmed by Western blot. RESULTS: After infection, an increase of these proteins was observed. The expression levels of other apoptosis-related genes were also determined by PCR array. Experimental results revealed that the messenger RNA levels of tumor necrosis factor ligand (e.g., tnfsf10 and tnfrs10b) and fas were increased, whereas the expression levels of some antiapoptosis genes such as Birc5, Hells, and Bnip3 were decreased. CONCLUSION: Our study results demonstrate that the apoptosis-related genes and proteins in mouse macrophages were modulated by B. pseudomallei. PMID:23751765

Hseu, You-Cheng; Sung, Jia-Chuen; Shieh, Bao-Sen; Chen, Ssu Ching

2013-06-01

284

Identification of Burkholderia pseudomallei genes required for the intracellular life cycle and in vivo virulence.  

PubMed

The bacterial pathogen Burkholderia pseudomallei invades host cells, escapes from endocytic vesicles, multiplies intracellularly, and induces the formation of actin tails and membrane protrusions, leading to direct cell-to-cell spreading. This study was aimed at the identification of B. pseudomallei genes responsible for the different steps of this intracellular life cycle. B. pseudomallei transposon mutants were screened for a reduced ability to form plaques on PtK2 cell monolayers as a result of reduced intercellular spreading. Nine plaque assay mutants with insertions in different open reading frames were selected for further studies. One mutant defective in a hypothetical protein encoded within the Bsa type III secretion system gene cluster was found to be unable to escape from endocytic vesicles after invasion but still multiplied within the vacuoles. Another mutant with a defect in a putative exported protein reached the cytoplasm but exhibited impaired actin tail formation in addition to a severe intracellular growth defect. In four mutants, the transposon had inserted into genes involved in either purine, histidine, or p-aminobenzoate biosynthesis, suggesting that these pathways are essential for intracellular growth. Three mutants with reduced plaque formation were shown to have gene defects in a putative cytidyltransferase, a putative lipoate-protein ligase B, and a hypothetical protein. All nine mutants proved to be significantly attenuated in a murine model of infection, with some mutants being essentially avirulent. In conclusion, we have identified a number of novel major B. pseudomallei virulence genes which are essential for the intracellular life cycle of this pathogen. PMID:16714590

Pilatz, Sabine; Breitbach, Katrin; Hein, Nadine; Fehlhaber, Beate; Schulze, Jessika; Brenneke, Birgit; Eberl, Leo; Steinmetz, Ivo

2006-06-01

285

Biodefense-Driven Murine Model of Pneumonic Melioidosis  

Microsoft Academic Search

A whole-body mouse model of pneumonic melioidosis was established for future evaluation of biodefense vaccine candidates. The aerosol 50% lethal doses of Burkholderia pseudomallei strain 1026b for BALB\\/c and C57BL\\/6 mice and the times to death, dissemination in organs, and tissue loads after exposure of the mice to low- and high-dose aerosols are reported. In addition, rpsL mutant backgrounds were

J. A. Jeddeloh; D. L. Fritz; D. M. Waag; J. M. Hartings; G. P. Andrews

2003-01-01

286

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

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; Utaisincharoen, Pongsak

2013-07-08

287

Identification of type II toxin-antitoxin modules in Burkholderia pseudomallei.  

PubMed

Type II toxin-antitoxin (TA) systems are believed to be widely distributed amongst bacteria although their biological functions are not clear. We have identified eight candidate TA systems in the genome of the human pathogen Burkholderia pseudomallei. Five of these were located in genome islands. Of the candidate toxins, BPSL0175 (RelE1) or BPSS1060 (RelE2) caused growth to cease when expressed in Escherichia coli, whereas expression of BPSS0390 (HicA) or BPSS1584 (HipA) (in an E. coli ?hipBA background) caused a reduction in the number of culturable bacteria. The cognate antitoxins could restore growth and culturability of cells. PMID:23082999

Butt, Aaron; Müller, Claudia; Harmer, Nicholas; Titball, Richard W

2012-11-16

288

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.

Burtnick, Mary N.; Brett, Paul J.

2013-01-01

289

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

PubMed

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

2013-10-11

290

Epidemiological Tracking and Population Assignment of the Non-Clonal Bacterium, Burkholderia pseudomallei  

PubMed Central

Rapid assignment of bacterial pathogens into predefined populations is an important first step for epidemiological tracking. For clonal species, a single allele can theoretically define a population. For non-clonal species such as Burkholderia pseudomallei, however, shared allelic states between distantly related isolates make it more difficult to identify population defining characteristics. Two distinct B. pseudomallei populations have been previously identified using multilocus sequence typing (MLST). These populations correlate with the major foci of endemicity (Australia and Southeast Asia). Here, we use multiple Bayesian approaches to evaluate the compositional robustness of these populations, and provide assignment results for MLST sequence types (STs). Our goal was to provide a reference for assigning STs to an established population without the need for further computational analyses. We also provide allele frequency results for each population to enable estimation of population assignment even when novel STs are discovered. The ability for humans and potentially contaminated goods to move rapidly across the globe complicates the task of identifying the source of an infection or outbreak. Population genetic dynamics of B. pseudomallei are particularly complicated relative to other bacterial pathogens, but the work here provides the ability for broad scale population assignment. As there is currently no independent empirical measure of successful population assignment, we provide comprehensive analytical details of our comparisons to enable the reader to evaluate the robustness of population designations and assignments as they pertain to individual research questions. Finer scale subdivision and verification of current population compositions will likely be possible with genotyping data that more comprehensively samples the genome. The approach used here may be valuable for other non-clonal pathogens that lack simple group-defining genetic characteristics and provides a rapid reference for epidemiologists wishing to track the origin of infection without the need to compile population data and learn population assignment algorithms.

Dale, Julia; Price, Erin P.; Hornstra, Heidie; Busch, Joseph D.; Mayo, Mark; Godoy, Daniel; Wuthiekanun, Vanaporn; Baker, Anthony; Foster, Jeffrey T.; Wagner, David M.; Tuanyok, Apichai; Warner, Jeffrey; Spratt, Brian G.; Peacock, Sharon J.; Currie, Bart J.; Keim, Paul; Pearson, Talima

2011-01-01

291

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

PubMed

Burkholderia pseudomallei is the causative agent of the disease melioidosis, which is prevalent in tropical countries and is intractable to a number of antibiotics. In this study, the antibiotic co-trimoxazole (trimethoprim/sulfamethoxazole) was assessed for the post-exposure prophylaxis of experimental infection in mice with B. pseudomallei and its close phylogenetic relative Burkholderia mallei, the causative agent of glanders. Co-trimoxazole was effective against an inhalational infection with B. pseudomallei or B. mallei. However, oral co-trimoxazole delivered twice daily did not eradicate infection when administered from 6h post exposure for 14 days or 21 days, since infected and antibiotic-treated mice succumbed to infection following relapse or immunosuppression. These data highlight the utility of co-trimoxazole for prophylaxis both of B. pseudomallei and B. mallei and the need for new approaches for the treatment of persistent bacterial infection. PMID:23517714

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

2013-03-19

292

CpG oligodeoxyribonucleotides protect mice from Burkholderia pseudomallei but not Francisella tularensis Schu S4 aerosols.  

PubMed

Studies have shown that CpG oligodeoxyribonucleotides (ODN) protect mice from various bacterial pathogens, including Burkholderia pseudomallei and Francisella tularensis live vaccine strain (LVS), when administered before parenteral challenge. Given the potential to develop CpG ODN as a pre-treatment for multiple bacterial biological warfare agents, we examined survival, histopathology, and cytokine data from CpG ODN-treated C57BL/6 mice to determine whether previously-reported protection extended to aerosolized B. pseudomallei 1026b and highly virulent F. tularensis Schu S4 infections. We found that, although CpG ODN protected mice from aerosolized B. pseudomallei challenges, the immunostimulant failed to benefit the animals exposed to F. tularensis Schu S4 aerosols. Our results, which contrast with earlier F. tularensis LVS studies, highlight potential differences in Francisella species pathogenesis and underscore the need to evaluate immunotherapies against human pathogenic species. PMID:20181102

Rozak, David A; Gelhaus, Herbert C; Smith, Mark; Zadeh, Mojgan; Huzella, Louis; Waag, David; Adamovicz, Jeffrey J

2010-02-05

293

Identification of specific antigens of Pseudomonas pseudomallei and evaluation of their efficacies for diagnosis of melioidosis.  

PubMed Central

Current methods for the diagnosis of melioidosis are based on bacteriological culture. A number of serological tests currently available lack specificity and sensitivity. This is largely due to the use of crude antigens which results in a significant cross-reactivity with sera from individuals infected with other bacteria. In this study five different antigens were prepared and evaluated for their potential usefulness in diagnosis of melioidosis. These included a 19.5-kDa antigen which was previously shown to be specific by Western blotting (immunoblotting), a crude cell extract, a veronal extract, a 39.0-kDa antigen, and an immunoaffinity-purified antigen. All antigens were used for detecting antibody in sera from patients with septicemic melioidosis by indirect enzyme-linked immunosorbent assay. The results were compared with those obtained with sera from patients with other bacterial infections and normal sera from areas where the infection is and is not endemic. The 19.5-kDa antigen exhibited the most satisfactory results, with 92% sensitivity, 91% specificity, 81% positive predictive value, and 96% negative predictive value based on a background obtained with normal sera from the area where the infection is nonendemic. These values were 82% sensitivity, 96% specificity, 94% positive predictive value, and 87% negative predictive value based on results with normal sera from the area where the infection is endemic. Results from this study showed that the 19.5-kDa antigen was potentially useful in the diagnosis of melioidosis and deserves further investigation. Images

Anuntagool, N; Rugdech, P; Sirisinha, S

1993-01-01

294

The Burkholderia pseudomallei BpeAB-OprB Efflux Pump: Expression and Impact on Quorum Sensing and Virulence  

Microsoft Academic Search

BpeAB-OprB is a multidrug efflux pump of the bacterial pathogen Burkholderia pseudomallei and is respon- sible for conferring antimicrobial resistance to aminoglycosides and macrolides. Expression of bpeAB-oprB is inducible by its substrate erythromycin and upon entry into stationary phase. BpeR, a member of the TetR family, functions as a repressor of the bpeAB-oprB operon. bpeR expression was similarly induced at

Ying Ying Chan; Kim Lee Chua

2005-01-01

295

A traveller presenting with severe melioidosis complicated by a pericardial effusion: a case report  

PubMed Central

Background Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic to tropic regions, mainly in Southeast Asia and northern Australia. Melioidosis occurs only sporadically in travellers returning from disease-endemic areas. Severe clinical disease is seen mostly in patients with alteration of immune status. In particular, pericardial effusion occurs in 1-3% of patients with melioidosis, confined to endemic regions. To our best knowledge, this is the first reported case of melioidosis in a traveller complicated by a hemodynamically significant pericardial effusion without predisposing disease. Case presentation A 44-year-old Caucasian man developed pneumonia, with bilateral pleural effusions and complicated by a hemodynamically significant pericardial effusion, soon after his return from Thailand to Switzerland. Cultures from different specimens including blood cultures turned out negative. Diagnosis was only accomplished by isolation of Burkholderia pseudomallei from the pericardial aspirate, thus finally enabling the adequate antibiotic treatment. Conclusions Melioidosis is a great mimicker and physicians in non-endemic countries should be aware of its varied manifestations. In particular, melioidosis should be considered in differential diagnosis of pericardial effusion in travellers , even without risk factors predisposing to severe disease.

2012-01-01

296

Impaired TLR5 functionality is associated with survival in melioidosis.  

PubMed

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

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

2013-02-27

297

Impaired TLR5 Functionality Is Associated with Survival in Melioidosis  

PubMed Central

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

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

2013-01-01

298

Novel pan-genomic analysis approach in target selection for multiplex PCR identification and detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia complex species: a proof-of-concept study.  

PubMed

Burkholderia pseudomallei, Burkholderia thailandensis, and the Burkholderia cepacia complex differ greatly in pathogenicity and epidemiology. Yet, they are occasionally misidentified by biochemical profiling, and even 16S rRNA gene sequencing may not offer adequate discrimination between certain species groups. Using the 23 B. pseudomallei, four B. thailandensis, and 16 B. cepacia complex genome sequences available, we identified gene targets specific to each of them (a Tat domain protein, a 70-kDa protein, and a 12-kDa protein for B. pseudomallei, B. thailandensis, and the B. cepacia complex, respectively), with an in-house developed algorithm. Using these targets, we designed a robust multiplex PCR assay useful for their identification and detection from soil and simulated sputum samples. For all 43 B. pseudomallei, seven B. thailandensis, and 20 B. cepacia complex (B. multivorans, n = 6; B. cenocepacia, n = 3; B. cepacia, n = 4; B. arboris, n = 2; B. contaminans, B. anthina, and B. pyrrocinia, n = 1 each; other unnamed members, n = 2) isolates, the assay produced specific products of predicted size without false positives or negatives. Of the 60 soil samples screened, 19 (31.6%) and 29 (48.3%) were positive for B. pseudomallei and the B. cepacia complex, respectively, and in four (6.7%) soil samples, the organisms were codetected. DNA sequencing confirmed that all PCR products originated from their targeted loci. This novel pan-genomic analysis approach in target selection is simple, computationally efficient, and potentially applicable to any species that harbors species-specific genes. A multiplex PCR assay for rapid and accurate identification and detection of B. pseudomallei, B. thailandensis, and the B. cepacia complex was developed and verified. PMID:21177905

Ho, Chi-Chun; Lau, Candy C Y; Martelli, Paolo; Chan, San-Yuen; Tse, Cindy W S; Wu, Alan K L; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y

2010-12-22

299

Melioidosis presenting as pseudomyxoma peritonei: yet another pretense of the great mimicker: an unreported entity.  

PubMed

Abstract Background: Melioidosis, a lethal infectious disease caused by Burkholderia pseudomallei, an important human pathogen in tropical regions, is notorious for its diverse clinical presentations. Methods: We report a case of a 55-year-old woman with a history of total abdominal hysterectomy with bilateral salpingo-oophorectomy for ovarian mucinous cystadenocarcinoma five years back, who presented with complaints of chest pain, abdominal distention, and breathlessness for one week. Ultrasound-guided aspiration of the peritoneal free fluid revealed a thick gelatinous material consistent with pseudomyxoma peritonei. Cytologic analysis of the aspirate was negative for malignant cells, but bacterial culture proved positive for Burkholderia pseudomallei. Results: She was started on ceftazidime, and she improved symptomatically and was discharged on oral doxycycline and chloramphenicol after three weeks of intravenous antibiotic therapy. Conclusion: This case is being reported to emphasize an unusual presentation of melioidosis and the significance of timely appropriate antibiotic therapy. PMID:23965152

Sugi Subramaniam, Raghavan Velayutham; Karthikeyan, Vilvapathy Senguttuvan; Sistla, Sarath Chandra; Ali, S Manwar; Sistla, Sujatha; Ram, Duvuru; Sudhagar, Rengasamy

2013-08-01

300

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

PubMed

The genetic similarity between Burkholderia mallei (glanders) and Burkholderia pseudomallei (melioidosis) had led to the general assumption that pathogenesis of each bacterium would be similar. In 2000, the first human case of glanders in North America since 1945 was reported in a microbiology laboratory worker. Leveraging the availability of pre-exposure sera for this individual and employing the same well-characterized protein array platform that has been previously used to study a large cohort of melioidosis patients in southeast Asia, we describe the antibody response in a human with glanders. Analysis of 156 peptides present on the array revealed antibodies against 17 peptides with a > 2-fold increase in this infection. Unexpectedly, when the glanders data were compared with a previous data set from B. pseudomallei infections, there were only two highly increased antibodies shared between these two infections. These findings have implications in the diagnosis and treatment of B. mallei and B. pseudomallei infections. PMID:23076276

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

2012-10-01

301

Distinct human antibody response to the biological warfare agent Burkholderia mallei  

PubMed Central

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

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

2012-01-01

302

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

Microsoft Academic Search

BACKGROUND: Burkholderia pseudomallei is a saprophyte in tropical environments and an opportunistic human pathogen. This versatility requires a sensing mechanism that allows the bacterium to respond rapidly to altered environmental conditions. We characterized a two-component signal transduction locus from B. pseudomallei 204, mrgR and mrgS, encoding products with extensive homology with response regulators and histidine protein kinases of Escherichia coli,

Magdy E Mahfouz; T Hilton Grayson; David AB Dance; Martyn L Gilpin

2006-01-01

303

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

PubMed Central

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

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

2011-01-01

304

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

PubMed Central

Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species.

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

2011-01-01

305

Multiple-antigen ELISA for melioidosis - a novel approach to the improved serodiagnosis of melioidosis  

PubMed Central

Background Burkholderia pseudomallei, the causative agent of melioidosis, is endemic to Southeast Asia and northern Australia. Clinical manifestations of disease are diverse, ranging from chronic infection to acute septicaemia. The current gold standard of diagnosis involves bacterial culture and identification which is time consuming and often too late for early medical intervention. Hence, rapid diagnosis of melioidosis is crucial for the successful management of melioidosis. Methods The study evaluated 4 purified B. pseudomallei recombinant proteins (TssD-5, Omp3, smBpF4 and Omp85) as potential diagnostic agents for melioidosis. A total of 68 sera samples from Malaysian melioidosis patients were screened for the presence of specific antibodies towards these proteins using enzyme-linked immunosorbent assay (ELISA). Sera from patients with various bacterial and viral infections but negative for B. pseudomallei, as well as sera from healthy individuals, were also included as non-melioidosis controls. The Mann Whitney test was performed to compare the statistical differences between melioidosis patients and the non-melioidosis controls. Results TssD-5 demonstrated the highest sensitivity of 71% followed by Omp3 (59%), smBpF4 (41%) and Omp85 (19%). All 4 antigens showed equally high specificity (89-96%). A cocktail of all 4 antigens resulted in slightly reduced sensitivity of 65% but improved specificity (99%). Multiple-antigen ELISA provided improved sensitivity of 88.2% whilst retaining good specificity (96%). There was minimal reactivity with sera from healthy individuals proposing the utility of these antigens to demarcate diseased from non-symptomatic individuals in an endemic country. Conclusions TssD-5 demonstrated high detection sensitivity and specificity and the results were obtained within a few hours compared to time consuming culture and IFAT methods commonly used in a clinical setting. The use of multiple-antigens resulted in improved sensitivity (88.2%) whilst maintaining superior specificity. These data highlight the use of TssD-5 and other recombinant antigens tested in this study as potential serodiagnostic agents for melioidosis.

2013-01-01

306

Melioidosis in southern India: epidemiological and clinical profile.  

PubMed

Melioidosis, which is mainly prevalent in Thailand and Australia, has shown an increasing trend in India in the last few years. We carried out a retrospective study of 25 culture-proven adult cases of melioidosis who were admitted to a tertiary care hospital in southern India during June 2001 to September 2007. There was a six-fold increase in the number of cases in 2006 and 2007 as compared to 2001. Diabetes mellitus was the predisposing factor in 68% of cases, followed by alcoholism (28%). The clinical presentations were fever (80%), pneumonia and/or pleural effusion (48%), hepatomegaly (56%), joint involvement, and/or osteomyelitis (48%), splenomegaly (40%), splenic abscess (24%) and septicemia (28%). The organism, Burkholderia pseudomallei, was sensitive to co-amoxiclav, cotrimoxazole, ceftazidime, and carbapenem. The study suggests that melioidosis is an emerging infectious disease in the southwestern coastal belt of India, and it is likely to happen at much higher incidence. PMID:20578524

Saravu, K; Mukhopadhyay, C; Vishwanath, S; Valsalan, R; Docherla, M; Vandana, K E; Shastry, B A; Bairy, I; Rao, S P

2010-03-01

307

Burkholderia vaccines: are we moving forward?  

PubMed

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

2013-02-05

308

Preliminary Evaluation of the API 20NE and RapID NF Plus Systems for Rapid Identification of Burkholderia pseudomallei and B. mallei  

PubMed Central

We evaluated the API 20NE and the RapID NF Plus systems with 58 Burkholderia pseudomallei and 23 B. mallei strains for identification of these agents, but neither was reliable for confirmatory identification, with only 0 to 60% strains identified accurately. A greater diversity of strains in the system databases would be beneficial.

Glass, Mindy B.; Popovic, Tanja

2005-01-01

309

Cutaneous Melioidosis in a Man Who Was Taken as a Prisoner of War by the Japanese during World War II  

PubMed Central

Melioidosis, an infection caused by the gram-negative bacillus Burkholderia pseudomallei, is endemic to Southeast Asia and Northern Australia. Human infection is acquired through contact with contaminated water via percutaneous inoculation. Clinical manifestations range from skin and soft tissue infection to pneumonia with sepsis. We report a case of a man who was taken as a prisoner of war by the Japanese during World War II who presented with a nonhealing ulcer on his right hand 62 years after the initial exposure.

Ngauy, Viseth; Lemeshev, Yan; Sadkowski, Lee; Crawford, George

2005-01-01

310

A sensitive & specific multiplex PCR assay for simultaneous detection of Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei & Brucella species  

PubMed Central

Background & objectives: Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei and Brucella species are potential biowarfare agents. Classical bacteriological methods for their identification are cumbersome, time consuming and of potential risk to the handler. Methods: We describe a sensitive and specific multiplex polymerase chain reaction (mPCR) assay involving novel primers sets for the simultaneous detection of B. anthracis, Y. pestis, B. pseudomallei and Brucella species. An additional non-competitive internal amplification control (IAC) was also included. Results: The mPCR was found to be specific when tested against closely related organisms. The sensitivity of the assay in spiked blood samples was 50 colony forming units (cfus)/25 ?l reaction, for the detection of B. anthracis, Y. pestis and Brucella species; and 150 cfus/25 ?l reaction, for B. pseudomallei. The assay proved useful in correctly and promptly identifing the clinical isolates of the targeted agents recovered from patients, compared to the gold standard culture methods. Interpretation & conclusion: The assay described in this study showed promise to be useful in application as a routine detection cum diagnostic method for these pathogens.

Batra, Sai Arun; Krupanidhi, S.; Tuteja, Urmil

2013-01-01

311

A sensitive & specific multiplex PCR assay for simultaneous detection of Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei & Brucella species.  

PubMed

Background & objectives: Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei and Brucella species are potential biowarfare agents. Classical bacteriological methods for their identification are cumbersome, time consuming and of potential risk to the handler. Methods: We describe a sensitive and specific multiplex polymerase chain reaction (mPCR) assay involving novel primers sets for the simultaneous detection of B. anthracis, Y. pestis, B. pseudomallei and Brucella species. An additional non-competitive internal amplification control (IAC) was also included. Results: The mPCR was found to be specific when tested against closely related organisms. The sensitivity of the assay in spiked blood samples was 50 colony forming units (cfus)/25 ?l reaction, for the detection of B. anthracis, Y. pestis and Brucella species; and 150 cfus/25 ?l reaction, for B. pseudomallei. The assay proved useful in correctly and promptly identifing the clinical isolates of the targeted agents recovered from patients, compared to the gold standard culture methods. Interpretation & conclusion: The assay described in this study showed promise to be useful in application as a routine detection cum diagnostic method for these pathogens. PMID:24056564

Batra, Sai Arun; Krupanidhi, S; Tuteja, Urmil

2013-07-01

312

Comparison of Automated and Nonautomated Systems for Identification of Burkholderia pseudomallei  

Microsoft Academic Search

identification of uncommonly encountered organisms such as B. pseudomallei critically important. This study compares the manual API 20NE and 20E identification systems with the automated Vitek 1 and 2 systems. A total of 103 B. pseudomallei isolates were tested and correctly identified in 98%, 99%, 99%, and 19% of cases, respectively. The failure of the Vitek 2 to correctly identify

Peter Lowe; Catherine Engler; Robert Norton

2002-01-01

313

Glyburide Reduces Bacterial Dissemination in a Mouse Model of Melioidosis  

PubMed Central

Background Burkholderia pseudomallei infection (melioidosis) is an important cause of community-acquired Gram-negative sepsis in Northeast Thailand, where it is associated with a ?40% mortality rate despite antimicrobial chemotherapy. We showed in a previous cohort study that patients taking glyburide (?=?glibenclamide) prior to admission have lower mortality and attenuated inflammatory responses compared to patients not taking glyburide. We sought to define the mechanism underlying this observation in a murine model of melioidosis. Methods Mice (C57BL/6) with streptozocin-induced diabetes were inoculated with ?6×102 cfu B. pseudomallei intranasally, then treated with therapeutic ceftazidime (600 mg/kg intraperitoneally twice daily starting 24 h after inoculation) in order to mimic the clinical scenario. Glyburide (50 mg/kg) or vehicle was started 7 d before inoculation and continued until sacrifice. The minimum inhibitory concentration of glyburide for B. pseudomallei was determined by broth microdilution. We also examined the effect of glyburide on interleukin (IL) 1? by bone-marrow-derived macrophages (BMDM). Results Diabetic mice had increased susceptibility to melioidosis, with increased bacterial dissemination but no effect was seen of diabetes on inflammation compared to non-diabetic controls. Glyburide treatment did not affect glucose levels but was associated with reduced pulmonary cellular influx, reduced bacterial dissemination to both liver and spleen and reduced IL1? production when compared to untreated controls. Other cytokines were not different in glyburide-treated animals. There was no direct effect of glyburide on B. pseudomallei growth in vitro or in vivo. Glyburide directly reduced the secretion of IL1? by BMDMs in a dose-dependent fashion. Conclusions Diabetes increases the susceptibility to melioidosis. We further show, for the first time in any model of sepsis, that glyburide acts as an anti-inflammatory agent by reducing IL1? secretion accompanied by diminished cellular influx and reduced bacterial dissemination to distant organs. We found no evidence for a direct effect of glyburide on the bacterium.

Koh, Gavin C. K. W.; Weehuizen, Tassili A.; Breitbach, Katrin; Krause, Kathrin; de Jong, Hanna K.; Kager, Liesbeth M.; Hoogendijk, Arjan J.; Bast, Antje; Peacock, Sharon J.; van der Poll, Tom; Steinmetz, Ivo; Wiersinga, W. Joost

2013-01-01

314

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

PubMed Central

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 drugs therapies against infectious bacterial agents. Here we report the 2.1 Å resolution crystal structure for the 220-residue Adk from Burkholderia pseudomallei (BpAdk), the etiological agent responsible for the infectious disease meliodosis. The general structure of apo BpAdk is similar to other Adk structures, composed of a CORE subdomain with peripheral ATP-binding (ATPbd) and LID subdomains. The two molecules in the asymmetric unit have significantly different conformations, with a backbone RMSD of 1.46 Å. These two BpAdk conformations may represent ‘open’ Adk sub-states along the preferential pathway to the ‘closed’ substrate-bound state.

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

2010-01-01

315

Melioidosis with a Pericardial Effusion, which Relapsed as a Chest Wall Abscess: A Rare Presentation.  

PubMed

Melioidosis, which is caused by a soil saprophyte, Burkholderia pseudomallei, is most prevalent in the south-west coast of India. Although it is frequently seen in immunocompromised patients, melioidosis can occur in apparently normal individuals. Melioidosis can involve almost any organ. A relapse of melioidosis is usually associated with a poor adherence to the eradication therapy, a multifocal involvement and bacteraemia. A relapsing melioidosis is usually known to follow a similar pattern of organ involvement in the first and second episodes of the infection. We are discussing here, a rare case of melioidosis in a 38-year-old construction-worker, with no risk factors, who presented initially with a pericardial effusion. It relapsed 6 months after he completed the prescribed eradication therapy for 3 months, as an anterior chest wall abscess. The author recommends a high index of suspicion for the relapsed melioidosis cases, inspite of the primary episode being non-bacteraemic and compliant with the recommended therapy, in order to avoid further complications. PMID:23730667

Mathai K, Rashmi Teresa; Bhat, K Sundara; Ashraf, Mohammed; Sarawag, Mayank; K P, Kumar

2013-04-01

316

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

317

Development and Characterization of a Caprine Aerosol Infection Model of Melioidosis  

PubMed Central

Infection with Burkholderia pseudomallei causes the disease melioidosis, which often presents as a serious suppurative infection that is typically fatal without intensive treatment and is a significant emerging infectious disease in Southeast Asia. Despite intensive research there is still much that remains unknown about melioidosis pathogenesis. New animal models of melioidosis are needed to examine novel aspects of pathogenesis as well as for the evaluation of novel therapeutics. The objective of the work presented here was to develop a subacute to chronic caprine model of melioidosis and to characterize the progression of disease with respect to clinical presentation, hematology, clinical microbiology, thoracic radiography, and gross and microscopic pathology. Disease was produced in all animals following an intratracheal aerosol of 104 CFU delivered, with variable clinical manifestations indicative of subacute and chronic disease. Bronchointerstitial pneumonia was apparent microscopically by day 2 and radiographically and grossly apparent by day 7 post infection (PI). Early lesions of bronchopneumonia soon progressed to more severe bronchointerstitial pneumonia with pyogranuloma formation. Extrapulmonary dissemination appeared to be a function of pyogranuloma invasion of pulmonary vasculature, which peaked around day 7 PI. Histopathology indicated that leukocytoclastic vasculitis was the central step in dissemination of B. pseudomallei from the lungs as well as in the establishment of new lesions. While higher doses of organism in goats can produce acute fatal disease, the dose investigated and resulting disease had many similarities to human melioidosis and may warrant further development to provide a model for the study of both natural and bioterrorism associated disease.

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

2012-01-01

318

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

PubMed Central

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

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

2011-01-01

319

Survey of Antimicrobial Resistance in Clinical Burkholderia pseudomallei Isolates over Two Decades in Northeast Thailand ? †  

PubMed Central

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

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

2011-01-01

320

In Vitro Activities of Amoxicillin-Clavulanate, Doxycycline, Ceftazidime, Imipenem, and Trimethoprim-Sulfamethoxazole against Biofilm of Brazilian Strains of Burkholderia pseudomallei.  

PubMed

This study aimed at investigating the in vitro activities of amoxicillin-clavulanate, doxycycline, ceftazidime, imipenem, and trimethoprim-sulfamethoxazole against Burkholderia pseudomallei in planktonic and biofilm forms, through broth microdilution and resazurin-based viability staining, respectively. In planktonic growth, the strains were susceptible to the drugs, while in biofilm growth, significantly higher antimicrobial concentrations were required, especially for ceftazidime and imipenem, surpassing the resistance breakpoints. These results highlight the importance of the routine evaluation of biofilm antimicrobial susceptibility. PMID:24002089

Bandeira, Tereza de Jesus Pinheiro Gomes; Moreira, Camila Alencar; Brilhante, Raimunda Sâmia Nogueira; Castelo-Branco, Débora de Souza Collares Maia; Neto, Manoel Paiva de Araújo; Cordeiro, Rossana de Aguiar; Rodrigues, Terezinha de Jesus Santos; Rocha, Marcos Fábio Gadelha; Sidrim, José Júlio Costa

2013-09-03

321

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

2013-02-08

322

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.

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

323

Neurologic Melioidosis in an Imported Pigtail Macaque (Macaca nemestrina).  

PubMed

Burkholderia pseudomallei is the cause of melioidosis in humans and other animals. Disease occurs predominately in Asia and Australia. It is rare in North America, and affected people and animals typically have a history of travel to (in human cases) or importation from (in animal cases) endemic areas. We describe the gross and histopathologic features and the microbiologic, molecular, and immunohistochemical diagnoses of a case of acute meningoencephalomyelitis and focal pneumonia caused by B. pseudomallei infection in a pigtail macaque that was imported from Indonesia to the United States for research purposes. This bacterium has been classified as a Tier 1 overlap select agent and toxin; therefore, recognition of pathologic features, along with accurate and timely confirmatory diagnostic testing, in naturally infected research animals is imperative to protect animals and personnel in the laboratory animal setting. PMID:23576240

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

2013-04-10

324

Toll-Like Receptor 4 Region Genetic Variants are Associated with Susceptibility to Melioidosis  

PubMed Central

Melioidosis is a tropical infection caused by the Gram-negative soil saprophyte Burkholderia pseudomallei. Despite broad exposure of northeast Thais, disease develops in only a small proportion of individuals. Although diabetes is a risk factor, the mechanisms of host susceptibility to melioidosis are still poorly understood. We postulated that Toll-like receptors (TLRs) regulate host susceptibility to disease, and that genetic variation in TLRs is associated with melioidosis. We analyzed the frequency of eight previously described TLR pathway polymorphisms in 490 cases compared to 950 non-hospitalized controls or 458 hospitalized controls. Based on these results, we then analyzed the frequency of additional TLR4 or TLR6-1-10 region polymorphisms in cases and controls. We found that the TLR41196C>T variant was associated with protection from melioidosis when compared to non-hospitalized controls. The TLR1742A>G and TLR1?7202A>G variants were associated with melioidosis when compared to hospitalized controls. In further analyses, we found that two additional TLR4 region polymorphisms were associated with disease. In diabetics, three other TLR6-1-10 region polymorphisms were associated with disease when compared to hospitalized controls. We conclude that TLR genetic variants may modulate host susceptibility to melioidosis. Confirmation of these findings and further investigation of the mechanisms is required.

West, T. Eoin; Chierakul, Wirongrong; Chantratita, Narisara; Limmathurotsakul, Direk; Wuthiekanun, Vanaporn; Emond, Mary J.; Hawn, Thomas R.; Peacock, Sharon J.; Skerrett, Shawn J.

2012-01-01

325

Sinonasal melioidosis in a returned traveller presenting with nasal cellulitis and sinusitis.  

PubMed

We illustrate a case involving a 51-year-old man who presented to a tertiary hospital with sepsis secondary to an abscess of the nasal vestibule and pustular eruptions of the nasal mucosa. Associated cellulitis extended across the face to the eye, and mucosal thickening of the sinuses was seen on computed tomography. The patient underwent incision and drainage and endoscopic sinus surgery. Blood cultures and swabs were positive for a gram-negative bacillus, Burkholderia pseudomallei. He had multiple risk factors including travel to an endemic area. The patient received extended antibiotic therapy in keeping with published national guidelines. Melioidosis is caused by Burkholderia pseudomallei, found in the soil in Northern Australia and Asia. It is transmitted via cutaneous or inhaled routes, leading to pneumonia, skin or soft tissue abscesses, and genitourinary infections. Risk factors include diabetes, chronic lung disease, and alcohol abuse. It can exist as a latent, active, or reactivated infection. A high mortality rate has been identified in patients with sepsis. Melioidosis is endemic in tropical Northern Australia and northeastern Thailand where it is the most common cause of severe community-acquired sepsis. There is one other report of melioidosis in the literature involving orbital cellulitis and sinusitis. PMID:23936707

Lim, Rebecca Sin Mei; Flatman, Sam; Dahm, Markus C

2013-07-07

326

Melioidosis and peritoneal dialysis related peritonitis.  

PubMed

We report a case of melioidosis presenting as peritonitis in a patient on continuous ambulatory peritoneal dialysis (CAPD). A 47-year-old man, a lorry driver, with end-stage renal disease due to diabetes mellitus on CAPD presented in PD-related peritonitis. He was started on intraperitoneal cloxacillin and ceftazidime, and changed to intraperitoneal vancomycin and meropenam after day 5 due to nonresponse. Burkholderia pseudomallei was identified from the dialysate culture. He was treated with intraperitoneal meropenam for two weeks, and IV ceftazidime for 4 weeks. He responded, and the Tenckhoff catheter was not removed. He was discharged well and continued on oral sulfamethoxazole/trimethoprim for six months. This patient had done his PD exchanges in a lorry. PMID:23629573

Wong, K W

2013-04-01

327

Acute Systemic Melioidosis.  

National Technical Information Service (NTIS)

Melioidosis is a disease caused by the Gram-negative bacillus Pseudomonas pseudomallei. It is endemic in Southeast Asia, but is being seen with increasing frequency in the United States as more military personnel return from Vietnam. Four patients with re...

K. A. Greenawald G. Nash F. D. Foley

1968-01-01

328

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

PubMed Central

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

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

2012-01-01

329

Present and future therapeutic strategies for melioidosis and glanders.  

PubMed

Burkholderia pseudomallei and Burkholderia mallei are the causative agents of melioidosis and glanders, respectively. Both Gram-negative pathogens are endemic in many parts of the world. Although natural acquisition of these pathogens is rare in the majority of countries, these bacteria have recently gained much interest because of their potential as bioterrorism agents. In modern times, their potential destructive impact on public health has escalated owing to the ability of these pathogens to cause opportunistic infections in diabetic and perhaps otherwise immunocompromised people, two growing populations worldwide. For both pathogens, severe infection in humans carries a high mortality rate, both species are recalcitrant to antibiotic therapy - B. pseudomallei more so than B. mallei - and no licensed vaccine exists for either prophylactic or therapeutic use. The potential malicious use of these organisms has accelerated the investigation of new ways to prevent and to treat the diseases. The availability of several B. pseudomallei and B. mallei genome sequences has greatly facilitated target identification and development of new therapeutics. This review provides a compilation of literature covering studies in antimelioidosis and antiglanders antimicrobial drug discovery, with a particular focus on potential novel therapeutic approaches to combat these diseases. PMID:20192686

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

2010-03-01

330

Endogenous ?2-Antiplasmin Is Protective during Severe Gram-Negative Sepsis (Melioidosis).  

PubMed

Rationale: ?2-Antiplasmin (A2AP) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit plasmin. Although the fibrinolytic system is strongly affected by infection, the functional role of A2AP in the host response to sepsis is unknown. Objectives: To study the role of A2AP in melioidosis, a common form of community-acquired sepsis in Southeast Asia and Northern Australia caused by the gram-negative bacterium Burkholderia pseudomallei. Methods: In a single-center observational study A2AP was measured in patients with culture-proven septic melioidosis. Wild-type and A2AP-deficient (A2AP(-/-)) mice were intranasally infected with B. pseudomallei to induce severe pneumosepsis (melioidosis). Parameters of inflammation and coagulation were measured, and survival studies were performed. Measurements and Main Results: Patients with melioidosis showed elevated A2AP plasma levels. Likewise, A2AP levels in plasma and lung homogenates were elevated in mice infected with B. pseudomallei. A2AP-deficient (A2AP(-/-)) mice had a strongly disturbed host response during experimental melioidosis as reflected by enhanced bacterial growth at the primary site of infection accompanied by increased dissemination to distant organs. In addition, A2AP(-/-) mice showed more severe lung pathology and injury together with an increased accumulation of neutrophils and higher cytokine levels in lung tissue. A2AP deficiency further was associated with exaggerated systemic inflammation and coagulation, increased distant organ injury, and enhanced lethality. Conclusions: This study is the first to identify A2AP as a protective mediator during gram-negative (pneumo)sepsis by limiting bacterial growth, inflammation, tissue injury, and coagulation. PMID:23992406

Kager, Liesbeth M; Weehuizen, Tassili A; Wiersinga, W Joost; Roelofs, Joris J T H; Meijers, Joost C M; Dondorp, Arjen M; van 't Veer, Cornelis; van der Poll, Tom

2013-10-15

331

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

PubMed

Melioidosis is endemic in the South Asian regions, like Thailand, Singapore Malaysia and Australia. The disease is more pronounced in the southern part of the country. It is caused by Burkholderia pseudomallei which causes systemic involvement, morbidity and mortality associated with the disease is high. Due to highly varied clinical presentation, and low general awareness this infection is largely underdiagnosed and under reported in our country. Most laboratories in the country still rely on conventional culturing methods with their low sensitivity, adding to the under reporting. To enhance physician awareness we describe here two cases who presented to our institute after months of misdiagnosis. PMID:23833477

Barman, Purabi; Kaur, Ravneet; Kumar, Kamlesh

2013-01-01

332

Melioidosis: reactivation during radiation therapy  

SciTech Connect

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

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

1980-05-01

333

Immunospecific responses to bacterial elongation factor Tu during Burkholderia infection and immunization.  

PubMed

Burkholderia pseudomallei is the etiological agent of melioidosis, a disease endemic in parts of Southeast Asia and Northern Australia. Currently there is no licensed vaccine against infection with this biological threat agent. In this study, we employed an immunoproteomic approach and identified bacterial Elongation factor-Tu (EF-Tu) as a potential vaccine antigen. EF-Tu is membrane-associated, secreted in outer membrane vesicles (OMVs), and immunogenic during Burkholderia infection in the murine model of melioidosis. Active immunization with EF-Tu induced antigen-specific antibody and cell-mediated immune responses in mice. Mucosal immunization with EF-Tu also reduced lung bacterial loads in mice challenged with aerosolized B. thailandensis. Our data support the utility of EF-Tu as a novel vaccine immunogen against bacterial infection. PMID:21179405

Nieves, Wildaliz; Heang, Julie; Asakrah, Saja; Höner zu Bentrup, Kerstin; Roy, Chad J; Morici, Lisa A

2010-12-17

334

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

PubMed

LC3-associated phagocytosis (LAP) of Burkholderia pseudomallei by murine macrophage (RAW 264.7) cells is an intracellular innate defense mechanism. Beclin 1, a protein with several roles in autophagic processes, is known to be recruited to phagosomal membranes as a very early event in LAP. We sought to determine whether knockdown of Beclin 1 by small interfering RNA (siRNA) would affect recruitment of LC3 and subsequent LAP of infecting B. pseudomallei. Both starvation and rapamycin treatment can induce Beclin 1-dependent autophagy. Therefore, we analyzed the consequences of Beclin 1 knockdown for LAP in infected cells that had been either starved or treated with rapamycin by determining the levels of bacterial colocalization with LC3 and intracellular survival. Concurrently, we confirmed the location of bacteria as either contained in phagosomes or free in the cytoplasm. We found that both rapamycin and starvation treatment enhanced LAP of B. pseudomallei but that the rapamycin response is Beclin 1 independent whereas the starvation response is Beclin 1 dependent. PMID:23115045

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

2012-10-31

335

Beclin 1 Is Required for Starvation-Enhanced, but Not Rapamycin-Enhanced, LC3-Associated Phagocytosis of Burkholderia pseudomallei in RAW 264.7 Cells  

PubMed Central

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

Li, Xuelei; Prescott, Mark; Adler, Ben

2013-01-01

336

Molecular Investigations of a Locally Acquired Case of Melioidosis in Southern AZ, USA  

PubMed Central

Melioidosis is caused by Burkholderia pseudomallei, a Gram-negative bacillus, primarily found in soils in Southeast Asia and northern Australia. A recent case of melioidosis in non-endemic Arizona was determined to be the result of locally acquired infection, as the patient had no travel history to endemic regions and no previous history of disease. Diagnosis of the case was confirmed through multiple microbiologic and molecular techniques. To enhance the epidemiological analysis, we conducted several molecular genotyping procedures, including multi-locus sequence typing, SNP-profiling, and whole genome sequence typing. Each technique has different molecular epidemiologic advantages, all of which provided evidence that the infecting strain was most similar to those found in Southeast Asia, possibly originating in, or around, Malaysia. Advancements in new typing technologies provide genotyping resolution not previously available to public health investigators, allowing for more accurate source identification.

Engelthaler, David M.; Bowers, Jolene; Schupp, James A.; Pearson, Talima; Ginther, Jennifer; Hornstra, Heidie M.; Dale, Julia; Stewart, Tasha; Sunenshine, Rebecca; Waddell, Victor; Levy, Craig; Gillece, John; Price, Lance B.; Contente, Tania; Beckstrom-Sternberg, Stephen M.; Blaney, David D.; Wagner, David M.; Mayo, Mark; Currie, Bart J.; Keim, Paul; Tuanyok, Apichai

2011-01-01

337

A case report of melioidosis in a diabetic patient in a union territory.  

PubMed

Melioidosis is an emerging disease in India. Cases have also been reported from South East Asia, Australia and Japan. Major risk factors for melioidosis are diabetes mellitus, preexisting renal disease and thalassemia. Exposure to contaminated soil and water are also significant occupational hazards associated with the disease. A patient with diabetes of six years duration on regular medication presented with fever, generalised myalgia and headache for a week. Blood and bone marrow culture yielded Burkholderia pseudomallei. A Computed tomography (CT) study of the thorax also revealed multiple scattered nodules in both lungs. The patient was treated with imipenem and doxycycline. His condition improved gradually and he was advised oral sulfamethoxazole/trimethoprim and doxycycline for a period of three months and has been followed up regularly. PMID:24039633

Esther, Paul; Sudhagar, M; Anandhalakshmi, S; Mathias, Shanthi

2013-08-31

338

Protection from pneumonic infection with burkholderia species by inhalational immunotherapy.  

PubMed

Burkholderia mallei and B. pseudomallei are important human pathogens and cause the diseases glanders and melioidosis, respectively. Both organisms are highly infectious when inhaled and are inherently resistant to many antimicrobials, thus making it difficult to treat pneumonic Burkholderia infections. We investigated whether it was possible to achieve rapid protection against inhaled Burkholderia infection by using inhaled immunotherapy. For this purpose, cationic liposome DNA complexes (CLDC), which are potent activators of innate immunity, were used to elicit the activation of pulmonary innate immune responses. We found that mucosal CLDC administration before or shortly after bacterial challenge could generate complete or nearly complete protection from inhalational challenge with 100% lethal doses of B. mallei and B. pseudomallei. Protection was found to be dependent on the CLDC-mediated induction of gamma interferon responses in lung tissues and was partially dependent on the activation of NK cells. However, CLDC-mediated protection was not dependent on the induction of inducible nitric oxide synthase, as assessed by depletion studies. We concluded that the potent local activation of innate immune responses in the lung could be used to elicit rapid and nonspecific protection from aerosol exposure to both B. mallei and B. pseudomallei. PMID:19179415

Goodyear, Andrew; Kellihan, Lisa; Bielefeldt-Ohmann, Helle; Troyer, Ryan; Propst, Katie; Dow, Steven

2009-01-29

339

Comparative genomics and an insect model rapidly identify novel virulence genes of Burkholderia mallei.  

PubMed

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, relatively little is known about their virulence and pathogenicity mechanisms. We used in silico genomic subtraction to generate their virulome, a set of 650 putative virulence-related genes shared by B. pseudomallei and B. mallei but not present in five closely related nonpathogenic Burkholderia species. Although most of these genes are clustered in putative operons, the number of targets for mutant construction and verification of reduced virulence in animal models is formidable. Therefore, Galleria mellonella (wax moth) larvae were evaluated as a surrogate host; we found that B. pseudomallei and B. mallei, but not other phylogenetically related bacteria, were highly pathogenic for this insect. More importantly, four previously characterized B. mallei mutants with reduced virulence in hamsters or mice had similarly reduced virulence in G. mellonella larvae. Site-specific inactivation of selected genes in the computationally derived virulome identified three new potential virulence genes, each of which was required for rapid and efficient killing of larvae. Thus, this approach may provide a means to quickly identify high-probability virulence genes in B. pseudomallei, B. mallei, and other pathogens. PMID:18223084

Schell, Mark A; Lipscomb, Lyla; DeShazer, David

2008-01-25

340

Spectroscopic and Kinetic Investigation of the Reactions of Peroxyacetic Acid with Burkholderia pseudomallei Catalase-Peroxidase, KatG.  

PubMed

Catalase-peroxidases or KatGs can utilize organic peroxyacids and peroxides instead of hydrogen peroxide to generate the high-valent ferryl-oxo intermediates involved in the catalase and peroxidase reactions. In the absence of peroxidatic one-electron donors, the ferryl intermediates generated with a low excess (10-fold) of peroxyacetic acid (PAA) slowly decay to the ferric resting state after several minutes, a reaction that is demonstrated in this work by both stopped-flow UV-vis absorption measurements and EPR spectroscopic characterization of Burkholderia pseudomallei KatG (BpKatG). EPR spectroscopy showed that the [Fe(IV)?O Trp330(•+)], [Fe(IV)?O Trp139(•)], and [Fe(IV)?O Trp153(•)] intermediates of the peroxidase-like cycle of BpKatG ( Colin, J. Wiseman, B. Switala, J. Loewen, P. C. Ivancich, A. ( 2009 ) J. Am. Chem. Soc. 131 , 8557 - 8563 ), formed with a low excess of PAA at low temperature, are also generated with a high excess (1000-fold) of PAA at room temperature. However, under high excess conditions, there is a rapid conversion to a persistent [Fe(IV)?O] intermediate. Analysis of tryptic peptides of BpKatG by mass spectrometry before and after treatment with PAA showed that specific tryptophan (including W330, W139, and W153), methionine (including Met264 of the M-Y-W adduct), and cysteine residues are either modified with one, two, or three oxygen atoms or could not be identified in the spectrum because of other undetermined modifications. It was concluded that these oxidized residues were the source of electrons used to reduce the excess of PAA to acetic acid and return the enzyme to the ferric state. Treatment of BpKatG with PAA also caused a loss of catalase activity towards certain substrates, consistent with oxidative disruption of the M-Y-W adduct, and a loss of peroxidase activity, consistent with accumulation of the [Fe(IV)?O] intermediate and the oxidative modification of the W330, W139, and W153. PAA, but not H2O2 or tert-butyl hydroperoxide, also caused subunit cross-linking. PMID:24044787

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

2013-10-02

341

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

PubMed

A real-time PCR assay, BurkDiff, was designed to target a unique conserved region in the B. pseudomallei and B. mallei genomes containing a SNP that differentiates the two species. Sensitivity and specificity were assessed by screening BurkDiff across 469 isolates of B. pseudomallei, 49 isolates of B. mallei, and 390 isolates of clinically relevant non-target species. Concordance of results with traditional speciation methods and no cross-reactivity to non-target species show BurkDiff is a robust, highly validated assay for the detection and differentiation of B. pseudomallei and B. mallei. PMID:21103048

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

2010-11-12

342

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

PubMed Central

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

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

2009-01-01

343

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

Microsoft Academic Search

A real-time PCR assay, BurkDiff, was designed to target a unique conserved region in the B. pseudomallei and B. mallei genomes containing a SNP that differentiates the two species. Sensitivity and specificity were assessed by screening BurkDiff across 469 isolates of B. pseudomallei, 49 isolates of B. mallei, and 390 isolates of clinically relevant non-target species. Concordance of results with

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

2010-01-01

344

The guanine-nucleotide-exchange factor BopE from Burkholderia pseudomallei adopts a compact version of the Salmonella SopE/SopE2 fold and undergoes a closed-to-open conformational change upon interaction with Cdc42.  

PubMed

BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis, a severe emerging infection. BopE is a GEF (guanine-nucleotide-exchange factor) for the Rho GTPases Cdc42 (cell division cycle 42) and Rac1. We have determined the structure of BopE catalytic domain (amino acids 78-261) by NMR spectroscopy and it shows that BopE(78-261) comprises two three-helix bundles (alpha1alpha4alpha5 and alpha2alpha3alpha6). This fold is similar to that adopted by the BopE homologues SopE and SopE2, which are GEFs from Salmonella. Whereas the two three-helix bundles of SopE(78-240) and SopE2(69-240) form the arms of a 'Lambda' shape, BopE(78-261) adopts a more closed conformation with substantial interactions between the two three-helix bundles. We propose that arginine and proline residues are important in the conformational differences between BopE and SopE/E2. Analysis of the molecular interface in the SopE(78-240)-Cdc42 complex crystal structure indicates that, in a BopE-Cdc42 interaction, the closed conformation of BopE(78-261) would engender steric clashes with the Cdc42 switch regions. This implies that BopE(78-261) must undergo a closed-to-open conformational change in order to catalyse guanine nucleotide exchange. In an NMR titration to investigate the BopE(78-261)-Cdc42 interaction, the appearance of additional peaks per NH for residues in hinge regions of BopE(78-261) indicates that BopE(78-261) does undergo a closed-to-open conformational change in the presence of Cdc42. The conformational change hypothesis is further supported by substantial improvement of BopE(78-261) catalytic efficiency through mutations that favour an open conformation. Requirement for closed-to-open conformational change explains the 10-40-fold lower k(cat) of BopE compared with SopE and SopE2. PMID:18052936

Upadhyay, Abhishek; Wu, Huan-Lin; Williams, Christopher; Field, Terry; Galyov, Edouard E; van den Elsen, Jean M H; Bagby, Stefan

2008-05-01

345

Diabetes does not influence activation of coagulation, fibrinolysis or anticoagulant pathways in Gram-negative sepsis (melioidosis).  

PubMed

Diabetes is associated with a disturbance of the haemostatic balance and is an important risk factor for sepsis, but the influence of diabetes on the pathogenesis of sepsis remains unclear. Melioidosis ( Burkholderia pseudomallei infection) is a common cause of community-acquired sepsis in Southeast Asia and northern Australia. We sought to investigate the impact of pre-existing diabetes on the coagulation and fibrinolytic systems during sepsis caused by B.pseudomallei . We recruited a cohort of 44 patients (34 with diabetes and 10 without diabetes) with culture-proven melioidosis. Diabetes was defined as a pre-admission diagnosis of diabetes or an HbA?c>7.8% at enrolment. Thirty healthy blood donors and 52 otherwise healthy diabetes patients served as controls. Citrated plasma was collected from all subjects; additionally in melioidosis patients follow-up specimens were collected seven and ? 28 days after enrolment where possible. Relative to uninfected healthy controls, diabetes per se (i.e. in the absence of infection) was characterised by a procoagulant effect. Melioidosis was associated with activation of coagulation (thrombin-antithrombin complexes (TAT), prothrombin fragment F?+? and fibrinogen concentrations were elevated; PT and PTT prolonged), suppression of anti-coagulation (antithrombin, protein C, total and free protein S levels were depressed) and abnormalities of fibrinolysis (D-dimer and plasmin-antiplasmin complex [PAP] were elevated). Remarkably, none of these haemostatic alterations were influenced by pre-existing diabetes. In conclusion, although diabetes is associated with multiple abnormalities of coagulation, anticoagulation and fibrinolysis, these changes are not detectable when superimposed on the background of larger abnormalities attributable to B. pseudomallei sepsis. PMID:21979378

Koh, Gavin C K W; Meijers, Joost C M; Maude, Rapeephan R; Limmathurotsakul, Direk; Day, Nicholas P J; Peacock, Sharon J; van der Poll, Tom; Wiersinga, Willem J

2011-10-06

346

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

347

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

PubMed

Burkholderia pseudomallei and Burkholderia mallei, the etiologic agents of melioidosis and glanders, respectively, cause severe disease in humans and animals and are considered potential agents of biological warfare and terrorism. Diagnosis and treatment of infections caused by these pathogens can be challenging and, in the absence of chemotherapeutic intervention, acute disease is frequently fatal. At present, there are no human or veterinary vaccines available for immunization against these emerging/re-emerging infectious diseases. One of the long term objectives of our research, therefore, is to identify and characterize protective antigens expressed by B. pseudomallei and B. mallei and use them to develop efficacious vaccine candidates. Previous studies have demonstrated that the 6-deoxy-heptan capsular polysaccharide (CPS) expressed by these bacterial pathogens is both a virulence determinant and a protective antigen. Consequently, this carbohydrate moiety has become an important component of the various subunit vaccines that we are currently developing in our laboratory. In the present study, we describe a reliable method for isolating CPS antigens from O-polysaccharide (OPS) deficient strains of B. pseudomallei; including a derivative of the select agent excluded strain Bp82. Utilizing these purified CPS samples, we also describe a simple procedure for covalently linking these T-cell independent antigens to carrier proteins. In addition, we demonstrate that high titer IgG responses can be raised against the CPS component of such constructs. Collectively, these approaches provide a tangible starting point for the development of novel CPS-based glycoconjugates for immunization against melioidosis and glanders. PMID:22912938

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

2012-08-15

348

Fate of a Burkholderia pseudomallei Lipopolysaccharide Mutant in the Mouse Macrophage Cell Line RAW 264.7: Possible Role for the O-Antigenic Polysaccharide Moiety of Lipopolysaccharide in Internalization and Intracellular Survival?  

PubMed Central

Burkholderia pseudomallei is a facultative intracellular gram-negative bacterium that can survive and multiply inside macrophages. One of the mechanisms by which B. pseudomallei escapes macrophage killing is by interfering with the expression of inducible nitric oxide synthase (iNOS). However, the bacterial components that modulate antimicrobial activity of the macrophage have not been fully elucidated. In the present study, we demonstrated that B. pseudomallei strain SRM117, a lipopolysaccharide (LPS) mutant that lacks the O-antigenic polysaccharide moiety, was more susceptible to macrophage killing during the early phase of infection than the parental wild-type strain (1026b). Unlike the wild type, the LPS mutant could readily stimulate Y701-STAT-1 phosphorylation (pY701-STAT-1) and interferon-regulatory factor 1 (IRF-1) expression, both of which are essential transcription factors of iNOS. Neutralizing antibody against beta interferon was able to inhibit the phosphorylation of Y701-STAT-1 and the expression of IRF-1 and iNOS, all of which resulted in an increased rate of intracellular replication. These data suggest that the O-antigenic polysaccharide moiety of B. pseudomallei modulates the host cell response, which in turn controls the intracellular fate of B. pseudomallei inside macrophages.

Arjcharoen, S.; Wikraiphat, C.; Pudla, M.; Limposuwan, K.; Woods, D. E.; Sirisinha, S.; Utaisincharoen, P.

2007-01-01

349

Immunofluorescence microscopy for the rapid diagnosis of melioidosis  

Microsoft Academic Search

A direct immunofluorescent antibody test (DIF) was developed for the rapid diagnosis of melioidosis, a potentially fatal infection caused by Pseudomonas pseudomallei. In a clinical evaluation of 369 sputum, pus, or urine specimens from 272 patients with suspected melioidosis, the DIF had a sensitivity of 73% and a specificity of 99% compared with culture. Using this DIF, a confident diagnosis

A L Walsh; M D Smith; V Wuthiekanun; Y Suputtamongkol; V Desakorn; W Chaowagul; N J White

1994-01-01

350

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

351

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

PubMed

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

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

2009-01-01

352

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.

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

2010-01-01

353

Development and application of a cellular, gain-of-signal, bioluminescent reporter screen for inhibitors of type II secretion in Pseudomonas aeruginosa and Burkholderia pseudomallei.  

PubMed

The type II secretion (T2S) system in gram-negative bacteria comprises the Sec and Tat pathways for translocating proteins into the periplasm and an outer membrane secretin for transporting proteins into the extracellular space. To discover Sec/Tat/T2S pathway inhibitors as potential new therapeutics, the authors used a Pseudomonas aeruginosa bioluminescent reporter strain responsive to SecA depletion and inhibition to screen compound libraries and characterize the hits. The reporter strain placed a luxCDABE operon under regulation of a SecA depletion-responsive upregulated promoter in a secA deletion background complemented with an ectopic lac-regulated secA copy. Bioluminescence was indirectly proportional to the isopropyl-?-D-thiogalactopyranoside concentration and stimulated by azide, a known SecA ATPase inhibitor. A total of 96 compounds (0.1% of 73,000) were detected as primary hits due to stimulation of luminescence with a z score ?5. Direct secretion assays of the nine most potent hits, representing five chemical scaffolds, revealed that they do not inhibit SecA-mediated secretion of ?-lactamase into the periplasm but do inhibit T2S-mediated extracellular secretion of elastase with IC(50) values from 5 to 25 µM. In addition, seven of the nine compounds also inhibited the T2S-mediated extracellular secretion of phospholipase C by P. aeruginosa and protease activity by Burkholderia pseudomallei. PMID:21602485

Moir, Donald T; Di, Ming; Wong, Erica; Moore, Richard A; Schweizer, Herbert P; Woods, Donald E; Bowlin, Terry L

2011-05-20

354

Impaired early cytokine responses at the site of infection in a murine model of type 2 diabetes and melioidosis comorbidity.  

PubMed

Bacterial infections are a common and serious complication of type 2 diabetes (T2D). The prevalence of melioidosis, an emerging tropical infection caused by the Gram-negative bacterium Burkholderia pseudomallei, is increased in people with T2D. This is the first study to compare murine models of T2D and melioidosis. Susceptibility and disease progression following infection with B. pseudomallei were compared in our diet-induced polygenic mouse model and a leptin receptor-deficient monogenic model of T2D. The metabolic profile of mice with diet-induced diabetes, including body weight, blood glucose, cholesterol, triglycerides, insulin resistance, and baseline levels of inflammation, closely resembled that of clinical T2D. Following subcutaneous infection with B. pseudomallei, bacterial loads at 24 and 72 h postinfection in the blood, spleen, liver, lungs, and subcutaneous adipose tissue (SAT) at the site of infection were compared in parallel with the expression of inflammatory cytokines and tissue histology. As early as 24 h postinfection, the expression of inflammatory (interleukin-1? [IL-1?], tumor necrosis factor alpha [TNF-?], and IL-6) and T(H)1 (IL-12 and gamma interferon [IFN-?]) cytokines was impaired in diabetic mice compared to nondiabetic littermates. Early differences in cytokine expression were associated with excessive infiltration of polymorphonuclear neutrophils (PMN) in diabetic mice compared to nondiabetic littermates. This was accompanied by bacteremia, hematogenous dissemination of bacteria to the lungs, and uncontrolled bacterial growth in the spleens of diabetic mice by 72 h postinfection. The findings from our novel model of T2D and melioidosis comorbidity support the role of impaired early immune pathways in the increased susceptibility of individuals with T2D to bacterial infections. PMID:23208607

Hodgson, Kelly A; Govan, Brenda L; Walduck, Anna K; Ketheesan, Natkunam; Morris, Jodie L

2012-12-03

355

Impaired Early Cytokine Responses at the Site of Infection in a Murine Model of Type 2 Diabetes and Melioidosis Comorbidity  

PubMed Central

Bacterial infections are a common and serious complication of type 2 diabetes (T2D). The prevalence of melioidosis, an emerging tropical infection caused by the Gram-negative bacterium Burkholderia pseudomallei, is increased in people with T2D. This is the first study to compare murine models of T2D and melioidosis. Susceptibility and disease progression following infection with B. pseudomallei were compared in our diet-induced polygenic mouse model and a leptin receptor-deficient monogenic model of T2D. The metabolic profile of mice with diet-induced diabetes, including body weight, blood glucose, cholesterol, triglycerides, insulin resistance, and baseline levels of inflammation, closely resembled that of clinical T2D. Following subcutaneous infection with B. pseudomallei, bacterial loads at 24 and 72 h postinfection in the blood, spleen, liver, lungs, and subcutaneous adipose tissue (SAT) at the site of infection were compared in parallel with the expression of inflammatory cytokines and tissue histology. As early as 24 h postinfection, the expression of inflammatory (interleukin-1? [IL-1?], tumor necrosis factor alpha [TNF-?], and IL-6) and TH1 (IL-12 and gamma interferon [IFN-?]) cytokines was impaired in diabetic mice compared to nondiabetic littermates. Early differences in cytokine expression were associated with excessive infiltration of polymorphonuclear neutrophils (PMN) in diabetic mice compared to nondiabetic littermates. This was accompanied by bacteremia, hematogenous dissemination of bacteria to the lungs, and uncontrolled bacterial growth in the spleens of diabetic mice by 72 h postinfection. The findings from our novel model of T2D and melioidosis comorbidity support the role of impaired early immune pathways in the increased susceptibility of individuals with T2D to bacterial infections.

Govan, Brenda L.; Walduck, Anna K.; Ketheesan, Natkunam; Morris, Jodie L.

2013-01-01

356

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.

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

357

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

PubMed

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

2013-09-30

358

Melioidosis and safety in the clinical laboratory.  

PubMed

Human infection with Pseudomonas pseudomallei, the causative agent of melioidosis, typically produces subclinical disease and an asymptomatic carrier state; occasionally clinical illness, frequently with a fatal outcome, may occur. Consequently, to help protect staff from laboratory-acquired melioidosis, microbiological and biomedical laboratories must have adequate facilities for safe work procedures and laboratory staff must engage in safe work practices. Recommendations from a melioidosis-endemic, diagnostic laboratory for the prevention of laboratory-acquired infection with this bacterium are essentially Category 3 (Advisory Committee on Dangerous Pathogens), Risk Group 3 (Australian Standards) or Biosafety Level 2 (National Institutes of Health) precautions. These include safeguards for centrifugation, prohibiting the 'sniff' test and the use of a biological safety cabinet for sputum processing, for subculture of stock strains, for preparation of antigen and for research studies but not for routine diagnostic techniques with P. pseudomallei. PMID:1355785

Ashdown, L R

1992-08-01

359

Pharmacokinetic-pharmacodynamic evaluation of ceftazidime continuous infusion vs intermittent bolus injection in septicaemic melioidosis  

PubMed Central

Aims Experimental studies have suggested that constant intravenous infusion would be preferable to conventional intermittent bolus administration of beta-lactam antibiotics for serious Gram-negative infections. Severe melioidosis (Burkholderia pseudomallei infection) carries a mortality over 40% despite treatment with high dose ceftazidime. The aim of this study was to measure the pharmacokinetic and pharmacodynamic effects of continuous infusion of ceftazidime vs intermittent bolus dosing in septicaemic melioidosis. Methods Patients with suspected septicaemic melioidosis were randomised to receive ceftazidime 40 mg kg?1 8 hourly by bolus injection or 4 mg kg?1 h?1 by constant infusion following a 12 mg kg?1 priming dose and pharmacokinetic and pharmacodynamic parameters were compared. Results Of the 34 patients studied 16 (59%) died. Twenty patients had cultures positive for B. pseudomallei of whom 12 (60%) died. The median MIC90 of B. pseudomallei was 2 mg l?1, giving a minimum target concentration (4*MIC) of 8 mg l?1. The median (range) estimated total apparent volume of distribution, systemic clearance and terminal elimination half-lives of ceftazidime were 0.468 (0.241–0.573) l kg?1, 0.058 (0.005–0.159) l kg?1 h?1 and 7.74 (1.95–44.71) h, respectively. Clearance of ceftazidime and creatinine clearance were correlated closely (r = 0.71; P < 0.001) and there was no evidence of significant nonrenal clearance. Conclusions Simulations based on these data and the ceftazidime sensitivity of the B. pseudomallei isolates indicated that administration by constant infusion would allow significant dose reduction and cost saving. With conventional 8 h intermittent dosing to patients with normal renal function, plasma ceftazidime concentrations could fall below the target concentration but this would be unlikely with a constant infusion. Correction for renal failure, which is common in patients with meliodosis is Clearance = k * creatinine clearance where k = 0.72. Calculation of a loading dose gives median (range) values of loading dose, DL of 18.7 mg kg?1 (9.5–23) and infusion rate I = 3.5 mg kg?1 h?1 (0.4–13) (which equals 84 mg kg?1 day?1). A nomogram for adjustment in renal failure is given.

Angus, B J; Smith, M D; Suputtamongkol, Y; Mattie, H; Walsh, A L; Wuthiekanun, V; Chaowagul, W; White, N J

2000-01-01

360

Activities of Daily Living Associated with Acquisition of Melioidosis in Northeast Thailand: A Matched Case-Control Study  

PubMed Central

Background Melioidosis is a serious infectious disease caused by the Category B select agent and environmental saprophyte, Burkholderia pseudomallei. Most cases of naturally acquired infection are assumed to result from skin inoculation after exposure to soil or water. The aim of this study was to provide evidence for inoculation, inhalation and ingestion as routes of infection, and develop preventive guidelines based on this evidence. Methods/Principal Findings A prospective hospital-based 1?2 matched case-control study was conducted in Northeast Thailand. Cases were patients with culture-confirmed melioidosis, and controls were patients admitted with non-infectious conditions during the same period, matched for gender, age, and diabetes mellitus. Activities of daily living were recorded for the 30-day period before onset of symptoms, and home visits were performed to obtain drinking water and culture this for B. pseudomallei. Multivariable conditional logistic regression analysis based on 286 cases and 512 controls showed that activities associated with a risk of melioidosis included working in a rice field (conditional odds ratio [cOR]?=?2.1; 95% confidence interval [CI] 1.4–3.3), other activities associated with exposure to soil or water (cOR?=?1.4; 95%CI 0.8–2.6), an open wound (cOR?=?2.0; 95%CI 1.2–3.3), eating food contaminated with soil or dust (cOR?=?1.5; 95%CI 1.0–2.2), drinking untreated water (cOR?=?1.7; 95%CI 1.1–2.6), outdoor exposure to rain (cOR?=?2.1; 95%CI 1.4–3.2), water inhalation (cOR?=?2.4; 95%CI 1.5–3.9), current smoking (cOR?=?1.5; 95%CI 1.0–2.3) and steroid intake (cOR?=?3.1; 95%CI 1.4–6.9). B. pseudomallei was detected in water source(s) consumed by 7% of cases and 3% of controls (cOR?=?2.2; 95%CI 0.8–5.8). Conclusions/Significance We used these findings to develop the first evidence-based guidelines for the prevention of melioidosis. These are suitable for people in melioidosis-endemic areas, travelers and military personnel. Public health campaigns based on our recommendations are under development in Thailand.

Limmathurotsakul, Direk; Kanoksil, Manas; Wuthiekanun, Vanaporn; Kitphati, Rungrueng; deStavola, Bianca; Day, Nicholas P. J.; Peacock, Sharon J.

2013-01-01

361

Bacterial Genome Adaptation to Niches: Divergence of the Potential Virulence Genes in Three Burkholderia Species of Different Survival Strategies.  

National Technical Information Service (NTIS)

Two closely related species -- Burkholderia mallei (Bm) and Burkholderia pseudomallei (Bp) -- are serious human health hazards and are potential bio-warfare agents, whereas another closely related species -- Burkholderia thailandensis (Bt) -- is a non-pat...

H. S. Kim M. A. Schell Y. Yu R. L. Ulrich S. H. Sarria

2005-01-01

362

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.

Pilatova, Martina; Dionne, Marc S.

2012-01-01

363

Glyburide Is Anti-inflammatory and Associated with Reduced Mortality in Melioidosis  

PubMed Central

Background. Patients with diabetes mellitus are more prone to bacterial sepsis, but there are conflicting data on whether outcomes are worse in diabetics after presentation with sepsis. Glyburide is an oral hypoglycemic agent used to treat diabetes mellitus. This KATP-channel blocker and broad-spectrum ATP-binding cassette (ABC) transporter inhibitor has broad-ranging effects on the immune system, including inhibition of inflammasome assembly and would be predicted to influence the host response to infection. Methods. We studied a cohort of 1160 patients with gram-negative sepsis caused by a single pathogen (Burkholderia pseudomallei), 410 (35%) of whom were known to have diabetes. We subsequently studied prospectively diabetics with B. pseudomallei infection (n = 20) to compare the gene expression profile of peripheral whole blood leukocytes in patients who were taking glyburide against those not taking any sulfonylurea. Results. Survival was greater in diabetics than in nondiabetics (38% vs 45%, respectively, P = .04), but the survival benefit was confined to the patient group taking glyburide (adjusted odds ratio .47, 95% confidence interval .28–.74, P = .005). We identified differential expression of 63 immune-related genes (P = .001) in patients taking glyburide, the sum effect of which we predict to be antiinflammatory in the glyburide group. Conclusions. We present observational evidence for a glyburide-associated benefit during human melioidosis and correlate this with an anti-inflammatory effect of glyburide on the immune system.

Maude, Rapeephan R.; Schreiber, M. Fernanda; Limmathurotsakul, Direk; Wiersinga, W. Joost; Wuthiekanun, Vanaporn; Lee, Sue J.; Mahavanakul, Weera; Chaowagul, Wipada; Chierakul, Wirongrong; White, Nicholas J.; van der Poll, Tom; Day, Nicholas P. J.; Dougan, Gordon; Peacock, Sharon J.

2011-01-01

364

Improved diagnosis of melioidosis using a 2-dimensional immunoarray.  

PubMed

Melioidosis is caused by the Gram negative bacterium Burkholderia pseudomallei. The gold standard for diagnosis is culture, which requires at least 3-4 days obtaining a result, hindering successful treatment of acute disease. The existing indirect haemagglutination assay (IHA) has several disadvantages, in that approximately half of patients later confirmed culture positive are not diagnosed at presentation and a subset of patients are persistently seronegative. We have developed 2 serological assays, an enzyme-linked immunosorbent assay (ELISA), and a 2-dimensional immunoarray (2DIA), capable of detecting antibodies in patient sera from a greater proportion of IHA-negative patient subsets. The 2DIA format can distinguish between different LPS serotypes. Currently, the 2DIA has a sensitivity and specificity of 100% and 87.1%, respectively, with 100% of culture-positive, IHA-negative samples detected. The ELISA has a sensitivity and specificity of 86.2% and 93.5%, respectively, detecting 67% of culture-positive, IHA-negative samples. The ELISA and 2DIA tests described here are more rapid and reliable for serological testing compared to the existing IHA. PMID:24041552

Sorenson, Alanna E; Williams, Natasha L; Morris, Jodie L; Ketheesan, Natkunam; Norton, Robert E; Schaeffer, Patrick M

2013-09-14

365

Development of Real-Time PCR Assays and Evaluation of Their Potential Use for Rapid Detection of Burkholderia pseudomallei in Clinical Blood Specimens  

Microsoft Academic Search

The early initiation of appropriate antimicrobial therapy is critical for improving the prognosis of patients with septicemic melioidosis. Thus, the use of a rapid molecular diagnosis may affect the outcome of this disease, which has a high mortality rate. We report the development of two TaqMan real-time PCR assays (designated 8653 and 9438) that detect the presence of two novel

Chonthida Supaprom; Dongling Wang; Chanvit Leelayuwat; Wisansanee Thaewpia; Wattanachai Susaengrat; Victor Koh; Eng Eong Ooi; Ganjana Lertmemongkolchai; Yichun Liu

366

Complement fixation test in experimental clinical and subclinical melioidosis.  

PubMed

Nigg, Clara (University of California, Berkeley), and Margaret M. Johnston. Complement fixation test in experimental clinical and subclinical melioidosis. J. Bacteriol. 82:159-168. 1961.-Soluble stable antigens prepared from Pseudomonas pseudomallei gave 4+ complement fixation reactions in a dilution of 1 to 8,000 when tested with specific rabbit antiserum diluted 1 to 10,000. The complement fixation reaction was positive in 100% of experimentally infected rabbits 9 to 11 days postinfection. Infected guinea pigs and monkeys showed similar results. Monkeys inoculated with very small infecting doses of P. pseudomallei developed positive complement fixation reactions in the absence of clinical manifestation of infection. An anamnestic complement-fixing antibody response could be induced in such monkeys, after the titer had dropped to approximately the preinfection level, by inoculating very small doses of viable P. pseudomallei or larger doses of killed melioidosis vaccine. The complement fixation test described appeared to be both sensitive and specific, and should be of value in human melioidosis which cannot be diagnosed on the basis of clinical manifestations alone. It is suggested that subclinical infections may play a role in the epidemiology of human meliodosis. The potential application of the complement fixation test to serological surveys in areas where melioidosis occurs endemically is discussed. PMID:13729013

NIGG, C; JOHNSTON, M M

1961-08-01

367

COMPLEMENT FIXATION TEST IN EXPERIMENTAL CLINICAL AND SUBCLINICAL MELIOIDOSIS  

PubMed Central

Nigg, Clara (University of California, Berkeley), and Margaret M. Johnston. Complement fixation test in experimental clinical and subclinical melioidosis. J. Bacteriol. 82:159–168. 1961.—Soluble stable antigens prepared from Pseudomonas pseudomallei gave 4+ complement fixation reactions in a dilution of 1 to 8,000 when tested with specific rabbit antiserum diluted 1 to 10,000. The complement fixation reaction was positive in 100% of experimentally infected rabbits 9 to 11 days postinfection. Infected guinea pigs and monkeys showed similar results. Monkeys inoculated with very small infecting doses of P. pseudomallei developed positive complement fixation reactions in the absence of clinical manifestation of infection. An anamnestic complement-fixing antibody response could be induced in such monkeys, after the titer had dropped to approximately the preinfection level, by inoculating very small doses of viable P. pseudomallei or larger doses of killed melioidosis vaccine. The complement fixation test described appeared to be both sensitive and specific, and should be of value in human melioidosis which cannot be diagnosed on the basis of clinical manifestations alone. It is suggested that subclinical infections may play a role in the epidemiology of human meliodosis. The potential application of the complement fixation test to serological surveys in areas where melioidosis occurs endemically is discussed.

Nigg, Clara; Johnston, Margaret M.

1961-01-01

368

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.

2010-01-01

369

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

National Technical Information Service (NTIS)

Studies have shown that CpG oligodeoxyribonucleotides (ODN) protect mice from various bacterial pathogens, including Burkholderia pseudomallei and Francisella tularensis live vaccine strain (LVS), when administered before parenteral challenge. Given the p...

D. A. Rozak H. C. Gelhaus L. Huzella M. Smith M. Zadeh

2010-01-01

370

Development and clinical evaluation of a PCR assay targeting the metalloprotease gene (mprA) of B. pseudomallei.  

PubMed

A PCR assay targeting the metalloprotease gene (mprA) of Burkholderia pseudomallei was developed for the specific detection of this organism in pure cultures and clinical samples. All other closely related organisms including B. mallei the causative agent of glanders, and B. thailandensis tested negative. Burkholderia pseudomallei DNA was successfully amplified from paraffin-embedded lung tissue of a camel with a generalized B. pseudomallei infection. The developed PCR assay can be used as a simple tool for the specific and sensitive detection of B. pseudomallei. PMID:17359446

Neubauer, H; Sprague, L D; Joseph, M; Tomaso, H; Al Dahouk, S; Witte, A; Kinne, J; Hensel, A; Wernery, R; Wernery, U; Scholz, H C

2007-01-01

371

Continuing evolution of Burkholderia mallei through genome reduction and large-scale rearrangements.  

PubMed

Burkholderia mallei (Bm), the causative agent of the predominately equine disease glanders, is a genetically uniform species that is very closely related to the much more diverse species Burkholderia pseudomallei (Bp), an opportunistic human pathogen and the primary cause of melioidosis. To gain insight into the relative lack of genetic diversity within Bm, we performed whole-genome comparative analysis of seven Bm strains and contrasted these with eight Bp strains. The Bm core genome (shared by all seven strains) is smaller in size than that of Bp, but the inverse is true for the variable gene sets that are distributed across strains. Interestingly, the biological roles of the Bm variable gene sets are much more homogeneous than those of Bp. The Bm variable genes are found mostly in contiguous regions flanked by insertion sequence (IS) elements, which appear to mediate excision and subsequent elimination of groups of genes that are under reduced selection in the mammalian host. The analysis suggests that the Bm genome continues to evolve through random IS-mediated recombination events, and differences in gene content may contribute to differences in virulence observed among Bm strains. The results are consistent with the view that Bm recently evolved from a single strain of Bp upon introduction into an animal host followed by expansion of IS elements, prophage elimination, and genome rearrangements and reduction mediated by homologous recombination across IS elements. PMID:20333227

Losada, Liliana; Ronning, Catherine M; DeShazer, David; Woods, Donald; Fedorova, Natalie; Kim, H Stanley; Shabalina, Svetlana A; Pearson, Talima R; Brinkac, Lauren; Tan, Patrick; Nandi, Tannistha; Crabtree, Jonathan; Badger, Jonathan; Beckstrom-Sternberg, Steve; Saqib, Muhammad; Schutzer, Steven E; Keim, Paul; Nierman, William C

2010-01-22

372

Pseudomonas pseudomallei infection from drowning: the first reported case in Taiwan.  

PubMed Central

We report a case of Pseudomonas pseudomallei infection, in which the patient acquired the bacteria by aspiration of river water after a drowning incident near Manila, the Philippines. The pulmonary form of melioidosis was noted at the onset, but septicemia developed at a later stage. Positive blood cultures were obtained 17 days after the accident. The patient was treated successfully with a combination of amikacin and cephalothin. This is the first report of P. pseudomallei infection documented in Taiwan. Images

Lee, N; Wu, J L; Lee, C H; Tsai, W C

1985-01-01

373

Melioidosis in a returning traveller.  

PubMed

A 66-year-old man returned to the UK from Thailand with a 2-week history of new confusion, hallucinations, fever with rigours and productive cough. He had not responded to (unspecified) antibiotic treatment in Thailand. On examination he was afebrile, with an abbreviated mental test score of 8/10 and no other findings on systemic examination. He was treated with ceftriaxone in response to discovery of a Gram-negative organism in blood. This was converted to meropenem on the clinical suspicion of our microbiologist, on the basis of a history of contact with surface water in the Far East. A blood culture subsequently confirmed Burkholderia pseudomallei. His condition remained stable for approximately 4 days, but then deteriorated over the course of the next 2 weeks with pneumonia and subsequent formation of disseminated abscesses. Treatment was withdrawn as his condition deteriorated to the point at which survival was deemed impossible and he subsequently died. PMID:23605844

Ismail, Alaa; Buckley, Adam; Dubrey, Simon William

2013-04-18

374

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

PubMed

Glanders and melioidosis are severe infectious diseases of people and animals. The causative agents of these infections refer to the potential agents of bioterrorism of group B. In this work the possibility of use of flagellin-based primers for the identification of B. mallei and B. pseudomallei and for diagnosis of experimental glanders and melioidosis was studied. The obtained results permit to make a conclusion that PCR using the developed primers may be recommended for the incorporation in the scheme of laboratory diagnosis of glanders and melioidosis both for the identification of clean cultures and in experimental clinical material. PMID:17891849

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

2007-01-01

375

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

376

[Molecular methods of detection and identification of pathogenic Burkholderia].  

PubMed

Molecular diagnostic kits for detection and identification of agents of melioidosis and glanders on environmental objects and in clinical material are described. It was demonstrated that PCR with use of specific primers on the basis of different genetic targets could be useful for determination of generic, inter- and intraspecies belonging of pathogenic Burkholderia as well as for epidemiologic inspection of territories where melioidosis is enzootic. PMID:21064227

Grishkina, T A; Samygin, V M

377

Human Melioidosis. A Histopathologic Study of Acute and Chronic Melioidosis.  

National Technical Information Service (NTIS)

Sixteen bacteriologically confirmed cases of melioidosis are reviewed, with particular emphasis on the variety of histopathologic lesions that may be encountered. Ten patients with acute melioidosis, all of whom died of their disease and were autopsied, h...

J. A. Piggott L. Hochholzer

1970-01-01

378

Melioidosis: A Case Report  

PubMed Central

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

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

2011-01-01

379

Isolation of Pseudomonas pseudomallei from soil in north-eastern Thailand  

Microsoft Academic Search

In order to optimize the recovery from soil of Pseudomonas pseudomallei, the cause of melioidosis, 3 selective broths were compared. A basal salt solution containing l-threonine (TBSS) performed significantly better than trypticase soy broth containing crystal violet and colistin 50 mg\\/L (CVC50), both in isolation rate and suppression of overgrowth of other organisms, but the addition of colistin to TBSS

Vanaporn Wuthiekanun; Michael D. Smith; David A. B. Dance; Nicholas J. White

1995-01-01

380

The use of selective media for the isolation of Pseudomonas pseudomallei in clinical practice.  

PubMed

Ashdown's selective-differential agar medium, with or without preenrichment in selective broth, was evaluated for the isolation of Pseudomonas pseudomallei from 1972 clinical specimens obtained from 643 subjects in Northeast Thailand; 226 patients proved to have meliodosis. The use of Ashdown's medium significantly increased the frequency of recovery of P. pseudomallei from sites or specimens with an extensive normal flora (throat, rectum, wounds and sputum) as compared to the recovery on blood and MacConkey agars (p less than 0.01). The isolation frequency from throat, rectal and wound swabs was further increased by the use of the broth pre-enrichment. The colonial morphology of P. pseudomallei on Ashdown's medium was sufficiently characteristic to allow presumptive identification. With the use of these selective media it was possible to culture P. pseudomallei from throat swabs taken from 87% of the patients from whom the organism could also be isolated from corresponding tracheal aspirates or sputum specimens. P. pseudomallei was isolated from rectal swabs taken from 51 patients, the first time that faecal excretion of the organism has been demonstrated in man. The diagnosis of melioidosis would not have been confirmed bacteriologically in eight patients (3.5%) without the use of the selective media. It is suggested that, in areas endemic for melioidosis, all sputum specimens should be cultured on selective media, such as Ashdown's. For the investigation of clinically suspected cases of melioidosis, and for follow-up during treatment of the disease, the use of broth pre-enrichment is recommended for specimens obtained from sites with an extensive normal flora. PMID:2231678

Wuthiekanun, V; Dance, D A; Wattanagoon, Y; Supputtamongkol, Y; Chaowagul, W; White, N J

1990-10-01

381

Pseudomonas pseudomallei isolates collected over 25 years from a non-tropical endemic focus show clonality on the basis of ribotyping.  

PubMed Central

Between 1966 and 1991, melioidosis, a disease caused by Pseudomonas pseudomallei that is mostly confined to tropical regions, occurred in farm animals and a farmer in temperate south-west Western Australia. Using an Escherichia coli probe containing a ribosomal RNA operon, P. pseudomallei DNA from isolates from 8 animals, a soil sample and the human case showed an identical ribotype on Southern blotting. The ribotype was different from the 3 commonest ribotypes seen in tropical Australia. This molecular typing supports the theory of clonal introduction of P. pseudomallei into a non-endemic region, with environmental contamination, local dissemination and persistence over 25 years. As melioidosis is often fatal in humans, such persistence in a temperate region is cause for concern. Images Fig. 2

Currie, B.; Smith-Vaughan, H.; Golledge, C.; Buller, N.; Sriprakash, K. S.; Kemp, D. J.

1994-01-01

382

Development of reagents and assays for the detection of pathogenic Burkholderia species.  

PubMed

Rapid detection of the category B biothreat agents Burkholderia pseudomallei and Burkholderia mallei in acute infections is critical to ensure that appropriate treatment is administered quickly to reduce an otherwise high probability of mortality (ca. 40% for B. pseudomallei). We are developing assays that can be used in clinical laboratories or security applications for the direct detection of surface-localized and secreted macromolecules produced by these organisms. We present our current medium-throughout approach for target selection and production of Burkholderia macromolecules and describe the generation of a Fab molecule targeted to the B. mallei BimA protein. We also present development of prototype assays for detecting Burkholderia species using anti-lipopolysaccharide antibodies. PMID:21413172

Qazi, Omar; Rani, Mridula; Gnanam, Annie J; Cullen, Thomas W; Stead, Christopher M; Kensing, Haley; McCaul, Kate; Ngugi, Sarah; Prior, Joann L; Lipka, Alexandria; Nagy, Judit M; Gregory, C Whitlock; Judy, Barbara M; Harding, Sarah V; Titball, Richard W; Sidhu, Sachdev S; Trent, M Stephen; Kitto, G Barrie; Torres, Alfredo; Estes, D Mark; Iverson, Brent; Georgiou, George; Brown, Katherine A

2011-01-01

383

Development of reagents and assays for the detection of pathogenic Burkholderia species  

PubMed Central

Rapid detection of the category B biothreat agents Burkholderia pseudomallei and Burkholderia mallei in acute infections is critical to ensure that appropriate treatment is administered quickly to reduce an otherwise high probability of mortality (ca. 40% for B. pseudomallei). We are developing assays that can be used in clinical laboratories or security applications for the direct detection of surface-localized and secreted macromolecules produced by these organisms. We present our current medium-throughout approach for target selection and production of Burkholderia macromolecules and describe the generation of a Fab molecule targeted to the B. mallei BimA protein. We also present development of prototype assays for detecting Burkholderia species using anti-lipopolysaccharide antibodies.

Qazi, Omar; Rani, Mridula; Gnanam, Annie J.; Cullen, Thomas W.; Stead, Christopher M.; Kensing, Haley; McCaul, Kate; Ngugi, Sarah; Prior, Joann L; Lipka, Alexandria; Whitlock, Gregory C.; Judy, Barbara M.; Harding, Sarah V.; Titball, Richard W.; Sidhu, Sachdev S.; Trent, M. Stephen; Kitto, G Barrie; Torres, Alfredo; Estes, D. Mark; Iverson, Brent; Georgiou, George; Brown, Katherine A.

2013-01-01

384

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

2013-06-05

385

A Zoonosis Gaining Ground: Melioidosis.  

National Technical Information Service (NTIS)

Melioidosis is a nosological entity which was revealed less than half a century ago. Considered at first as a variety restricted to the human species, it was soon found to be more widespread than it had first been supposed. At the same time, it has become...

J. Fournier

1968-01-01

386

Case report of orbital cellulitis and necrotizing fasciitis from melioidosis.  

PubMed

A 48-year-old Thai male farmer presented with progressive swelling of the right eyelid and high-grade fever. His visual acuity declined and the right side of his forehead developed a necrotic skin lesion with a purulent discharge. CT of the orbits suggested orbital cellulitis with subperiosteal abscess. Intravenous ceftriaxone and clindamycin were given empirically but then switched to vancomycin and meropenem because of rapid deterioration together with clinical sepsis. Burkholderia pseudomallei was isolated from the blood 3 days after the treatment, and the antibiotics were then switched to intravenous ceftazidime. The liver enzymes were elevated, and imaging of the abdomen revealed liver and splenic abscesses. After 14 days, the patient showed marked clinical improvement, became afebrile, and regained his OD visual acuity. A repeat CT of the orbit showed improvement with no subperiosteal abscess. The antibiotic was then switched to oral trimethoprim/sulfamethoxazole in combination with doxycycline for 6 months. PMID:23303132

Saonanon, Preamjit; Tirakunwichcha, Suppapong; Chierakul, Wirongrong

387

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.

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

2008-01-01

388

40 CFR 725.1075 - Burkholderia cepacia complex.  

Code of Federal Regulations, 2010 CFR

...Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia pyrrocinia, Burkholderia cepacia genomovar VIII (Burkholderia anthina ), and Burkholderia cepacia genomovars III and VI are subject to reporting under this section...

2010-07-01

389

40 CFR 725.1075 - Burkholderia cepacia complex.  

Code of Federal Regulations, 2010 CFR

...Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia pyrrocinia, Burkholderia cepacia genomovar VIII (Burkholderia anthina ), and Burkholderia cepacia genomovars III and VI are subject to reporting under this section...

2009-07-01

390

Melioidosis Imported from West Africa to Europe  

PubMed Central

We report the first case of imported melioidosis in Spain from a diabetic immigrant who visited West Africa during the rainy season. Because of the unusual presentation of this disease in Africa, clinical and microbiological diagnosis of imported melioidosis from this continent can be very elusive.

Cuadros, Juan; Gil, Horacio; Miguel, Julio De; Marabe, Graciela; Gomez-Herruz, Teresa Arroyo Pena; Lobo, Bruno; Marcos, Ruth; Anda, Pedro

2011-01-01

391

Melioidosis as an emerging global problem  

Microsoft Academic Search

There is remarkably little known about the incidence of melioidosis outside a few countries (Thailand, Australia, Singapore and Malaysia). Presumably it is widespread in tropical south east Asia. Elsewhere there are tantalising glimpses of the tip of what may be a large iceberg. Since a specific diagnosis of melioidosis requires awareness on the part of clinicians, and the existence of

David A. B Dance

2000-01-01

392

Cranial melioidosis with extradural extension after a fall in the bathroom  

PubMed Central

A 32-year-old diabetic male, with a past history of head injury and seizures, presented with a painful swelling over his forehead present for the past three months. Cranial MRI demonstrated the presence of a scalp collection with extradural extension through a bony defect. Biopsy from the area showed caseating necrosis suggestive of tuberculosis. Although the patient failed to return for initiation of anti-tubercular therapy for the next 11 months, the swelling did not progress, and there were no constitutional symptoms. The indolent nature of the swelling prompted re-evaluation and delayed cultures of pus from the collection grew Burkholderia pseudomallei.

Naha, Kushal; Dasari, Sowjanya; Kusugodlu, Ramamoorthi; Prabhu, Mukhyaprana

2012-01-01

393

Cranial melioidosis with extradural extension after a fall in the bathroom.  

PubMed

A 32-year-old diabetic male, with a past history of head injury and seizures, presented with a painful swelling over his forehead present for the past three months. Cranial MRI demonstrated the presence of a scalp collection with extradural extension through a bony defect. Biopsy from the area showed caseating necrosis suggestive of tuberculosis. Although the patient failed to return for initiation of anti-tubercular therapy for the next 11 months, the swelling did not progress, and there were no constitutional symptoms. The indolent nature of the swelling prompted re-evaluation and delayed cultures of pus from the collection grew Burkholderia pseudomallei. PMID:23024720

Naha, Kushal; Dasari, Sowjanya; Kusugodlu, Ramamoorthi; Prabhu, Mukhyaprana

2012-09-09

394

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

PubMed

ADP-L-glycero-D-manno-heptose 6-epimerase (AGME), the product of the rfaD gene, is the last enzyme in the heptose-biosynthesis pathway; it converts ADP-D-glycero-D-manno-heptose (ADP-D,D-Hep) to ADP-L-glycero-D-manno-heptose (ADP-L,D-Hep). AGME contains a catalytic triad involved in catalyzing hydride transfer with the aid of NADP(+). Defective lipopolysaccharide is found in bacterial mutants lacking this gene. Therefore, it is an interesting target enzyme for a novel epimerase inhibitor for use as a co-therapy with antibiotics. The crystal structure of AGME from Burkholderia thailandensis (BtAGME), a surrogate organism for studying the pathogenicity of melioidosis caused by B. pseudomallei, has been determined. The crystal structure determined with co-purified NADP(+) revealed common as well as unique structural properties of the AGME family when compared with UDP-galactose 4-epimerase homologues. They form a similar architecture with conserved catalytic residues. Nevertheless, there are differences in the substrate- and cofactor-binding cavities and the oligomerization domains. Structural comparison of BtAGME with AGME from Escherichia coli indicates that they may recognize their substrate in a `lock-and-key' fashion. Unique structural features of BtAGME are found in two regions. The first region is the loop between ?8 and ?9, affecting the binding affinity of BtAGME for the ADP moiety of ADP-D,D-Hep. The second region is helix ?8, which induces decamerization at low pH that is not found in other AGMEs. With the E210G mutant, it was observed that the resistance of the wild type to acid-induced denaturation is related to the decameric state. An in silico study was performed using the Surflex-Dock GeomX module of the SYBYL-X 1.3 software to predict the catalytic mechanism of BtAGME with its substrate, ADP-D,D-Hep. In the in silico study, the C7'' hydroxymethyl group of ADP-D,D-Hep is predicted to form hydrogen bonds to Ser116 and Gln293. With the aid of these interactions, the hydroxyl of Tyr139 forms a hydrogen bond to O6? of ADP-D,D-Hep and the proton at C6? orients closely to C4 of NADP(+). Therefore, the in silico study supports a one-base mechanism as a major catalytic pathway, in which Tyr139 solely functions as a catalytic acid/base residue. These results provide a new insight into the development of an epimerase inhibitor as an antibiotic adjuvant against melioidosis. PMID:23519675

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

2013-03-14

395

A case of prostatitis due to Burkholderia pseudomallei  

Microsoft Academic Search

Background A 67-year-old male, with a history of stable lower urinary tract symptoms, diabetes mellitus, benign prostatic hyperplasia, gonococcal urethritis, and excessive alcohol consumption, presented to the emergency room with sepsis and acute bacterial prostatitis. He had recently returned from a visit to Indonesia, where he had been a first-hand witness to the 2004 tsunami.Investigations Complete blood cell count, urine

Joshua S Hawley; Crystal Oakman; Rafael V Mora; Jorge M Arzola

2007-01-01

396

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

PubMed

Pathogenic Burkholderia are considered as a cause of dangerous infections and potential agents of bioterrorism. Comparative assessment of different methods of extraction and purification of DNA for PCR analysis of pure cultures and samples contaminated by etiological agents of glanders and melioidosis was performed. Samples of soil and food artificially contaminated by pathogenic Burkholderia as well as organs of infected animals were tested. DNA was extracted by methods of boiling, nucleosorption with presence of guanidine thiocyanate, guanidine thiocyanatephenol extraction, guanidine thiocyanate-phenol extraction with additional purification of DNA by nucleosorption. Amplification was performed by "Flash" technique and detector of fluorescence was used for analysis of PCR products. Utilization of the recommended methods of preparation depending on the nature of sample let to detect by the "Flash" technique the etiological agents of glanders and melioidosis in concentration =10(3) microbial cells per ml. Choice of DNA extraction and purification methods is determined by type of a sample and presence in it of admixtures inhibiting PCR. PMID:18368754

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

397

Burkholderia thailandensis E125 Harbors a Temperate Bacteriophage Specific for Burkholderia mallei  

PubMed Central

Burkholderia thailandensis is a nonpathogenic gram-negative bacillus that is closely related to Burkholderia mallei and Burkholderia pseudomallei. We found that B. thailandensis E125 spontaneously produced a bacteriophage, termed ?E125, which formed turbid plaques in top agar containing B. mallei ATCC 23344. We examined the host range of ?E125 and found that it formed plaques on B. mallei but not on any other bacterial species tested, including B. thailandensis and B. pseudomallei. Examination of the bacteriophage by transmission electron microscopy revealed an isometric head and a long noncontractile tail. B. mallei NCTC 120 and B. mallei DB110795 were resistant to infection with ?E125 and did not produce lipopolysaccharide (LPS) O antigen due to IS407A insertions in wbiE and wbiG, respectively. wbiE was provided in trans on a broad-host-range plasmid to B. mallei NCTC 120, and it restored LPS O-antigen production and susceptibility to ?E125. The 53,373-bp ?E125 genome contained 70 genes, an IS3 family insertion sequence (ISBt3), and an attachment site (attP) encompassing the 3? end of a proline tRNA (UGG) gene. While the overall genetic organization of the ?E125 genome was similar to ?-like bacteriophages and prophages, it also possessed a novel cluster of putative replication and lysogeny genes. The ?E125 genome encoded an adenine and a cytosine methyltransferase, and purified bacteriophage DNA contained both N6-methyladenine and N4-methylcytosine. The results presented here demonstrate that ?E125 is a new member of the ? supergroup of Siphoviridae that may be useful as a diagnostic tool for B. mallei.

Woods, Donald E.; Jeddeloh, Jeffrey A.; Fritz, David L.; DeShazer, David

2002-01-01