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.
We identified 10 patients in Thailand with culture-confirmed melioidosis who had Burkholderia pseudomallei isolated from their drinking water. The multilocus sequence type of B. pseudomallei from clinical specimens and water samples were identical for 2 patients. This finding suggests that drinking water is a preventable source of B. pseudomallei infection.
Wongsuvan, Gumphol; Aanensen, David; Ngamwilai, Sujittra; Saiprom, Natnaree; Rongkard, Patpong; Thaipadungpanit, Janjira; Kanoksil, Manas; Chantratita, Narisara; Day, Nicholas P.J.; Peacock, Sharon J.
Burkholderia pseudomallei, the etiologic agent of melioidosis, is a CDC tier 1 select agent that causes severe disease in both humans and animals. Diagnosis and treatment of melioidosis can be challenging, and in the absence of optimal chemotherapeutic intervention, acute disease is frequently fatal. Melioidosis is an emerging infectious disease for which there are currently no licensed vaccines. Due to the potential malicious use of B. pseudomallei as well as its impact on public health in regions where the disease is endemic, there is significant interest in developing vaccines for immunization against this disease. In the present study, type A O-polysaccharide (OPS) and manno-heptose capsular polysaccharide (CPS) antigens were isolated from nonpathogenic, select-agent-excluded strains of B. pseudomallei and covalently linked to carrier proteins. By using these conjugates (OPS2B1 and CPS2B1, respectively), it was shown that although high-titer IgG responses against the OPS or CPS component of the glycoconjugates could be raised in BALB/c mice, only those animals immunized with CPS2B1 were protected against intraperitoneal challenge with B. pseudomallei. Extending upon these studies, it was also demonstrated that when the mice were immunized with a combination of CPS2B1 and recombinant B. pseudomallei LolC, rather than with CPS2B1 or LolC individually, they exhibited higher survival rates when challenged with a lethal dose of B. pseudomallei. Collectively, these results suggest that CPS-based glycoconjugates are promising candidates for the development of subunit vaccines for immunization against melioidosis. PMID:24866807
Scott, Andrew E; Burtnick, Mary N; Stokes, Margaret G M; Whelan, Adam O; Williamson, E Diane; Atkins, Timothy P; Prior, Joann L; Brett, Paul J
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
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
While Southeast Asia and northern Australia are well recognized as the major endemic regions for melioidosis, recent reports have expanded the endemic zone. Severe weather events and environmental disasters such as the 2004 Asian tsunami have unmasked locations of sporadic cases and have reconfirmed endemicity in Indonesia. The endemic region now includes the majority of the Indian subcontinent, southern China, Hong Kong and Taiwan. Sporadic cases have occurred in Brazil and elsewhere in the Americas and in island communities such as New Caledonia, in the Pacific Ocean, and Mauritius in the Indian Ocean. Some of the factors that are critical to further elucidating the global distribution of Burkholderia pseudomallei and melioidosis include improved access to diagnostic laboratory facilities and formal confirmation of the identity of bacterial isolates from suspected cases. PMID:19121666
Currie, Bart J; Dance, David A B; Cheng, Allen C
Major advances have been made in molecular studies of Burkholderia pseudomallei and the immunology of melioidosis. However, there remain large gaps in understanding of the epidemiology of this enigmatic disease. Identified global distribution boundaries of melioidosis continue to expand. Recent data suggest Australian strains of B. pseudomallei may be ancestral to those from Southeast Asia, but the ecology of this environmental bacterium remains elusive. Despite the potential for rapidly progressive septicaemia, the critical virulence factors in B. pseudomallei remain to be clarified. Inhalation following aerosolization of B. pseudomallei may account for the high mortality when melioidosis occurs after severe weather events. PMID:18166205
Currie, Bart J
A cross-sectional serological survey of 2,214 children living in northeast Thailand was conducted to define the antibody response to Burkholderia pseudomallei from birth to 14 years. There was a sharp rise in detectable antibodies from birth to 4 years followed by reactivity in approximately 60-70% of children thereafter. Burkholderia pseudomallei is a soil saprophyte and the cause of melioidosis.1 This
VANAPORN WUTHIEKANUN; SAYAN LANGA; CHANATHIP PANPITPAT; NICHOLAS P. DAY; SHARON J. PEACOCK
A prospective study was performed to determine the rate at which patients with melioidosis are infected with more than one strain of Burkholderia pseudomallei. Genotyping of 2,058 bacterial colonies isolated from 215 samples taken from 133 patients demonstrated that mixed infection is uncommon (2/133 cases [1.5%; 95% confidence interval, 0.2 to 5.3%]).
Limmathurotsakul, Direk; Wuthiekanun, Vanaporn; Chantratita, Narisara; Wongsuvan, Gumphol; Thanwisai, Aunchalee; Biaklang, Mayurachat; Tumapa, Sarinna; Lee, Sue; Day, Nicholas P. J.; Peacock, Sharon J.
Background The soil-dwelling saprophyte bacterium Burkholderia pseudomallei is the cause of melioidosis, a severe disease of humans and animals in southeast Asia and northern Australia. Despite the detection of B. pseudomallei in various soil and water samples from endemic areas, the environmental habitat of B. pseudomallei remains unclear. Methodology/Principal Findings We performed a large survey in the Darwin area in tropical Australia and screened 809 soil samples for the presence of these bacteria. B. pseudomallei were detected by using a recently developed and validated protocol involving soil DNA extraction and real-time PCR targeting the B. pseudomallei–specific Type III Secretion System TTS1 gene cluster. Statistical analyses such as multivariable cluster logistic regression and principal component analysis were performed to assess the association of B. pseudomallei with environmental factors. The combination of factors describing the habitat of B. pseudomallei differed between undisturbed sites and environmentally manipulated areas. At undisturbed sites, the occurrence of B. pseudomallei was found to be significantly associated with areas rich in grasses, whereas at environmentally disturbed sites, B. pseudomallei was associated with the presence of livestock animals, lower soil pH and different combinations of soil texture and colour. Conclusions/Significance This study contributes to the elucidation of environmental factors influencing the occurrence of B. pseudomallei and raises concerns that B. pseudomallei may spread due to changes in land use.
Kaestli, Mirjam; Mayo, Mark; Harrington, Glenda; Ward, Linda; Watt, Felicity; Hill, Jason V.; Cheng, Allen C.; Currie, Bart J.
Background Melioidosis is a severe bacterial infection caused by Burkholderia pseudomallei with a high case-fatality rate. Epidemiological and animal studies show the possibility of inhalation transmission. However, no B. pseudomallei concentrations in ambient air have been researched. Here, we developed a method to quantify ambient B. pseudomallei and then measured concentrations of ambient B. pseudomallei during the typhoon season and the non-typhoon season to determine the factors influencing ambient B. pseudomallei levels. Methods We quantified ambient B. pseudomallei by using a filter/real-time qPCR method in the Zoynan Region in Kaohsiung, southern Taiwan. Twenty-four hour samples were collected at a sampling rate of 20 L/min every day from June 11 to December 21, 2012 including during the typhoon season (June to September) and reference season (October to December). Results We successfully developed a filtration/real-time qPCR method to quantify ambient B. pseudomallei. To our knowledge, this is the first report describing concentrations of ambient B. pseudomallei. Ambient B. pseudomallei were only detected during the typhoon season when compared to the reference season. For the typhoons affecting the Zoynan Region, the positive rates of ambient B. pseudomallei were very high at 80% to 100%. During June to December, rainfall was positively correlated with ambient B. pseudomallei with a statistical significance. Sediment at a nearby pond significantly influenced the concentration of ambient B. pseudomallei. During the typhoon month, the typhoon was positively correlated with ambient B. pseudomallei whereas wind speed was reversely correlated with ambient B. pseudomallei. Conclusions Our data suggest the possibility of transmission of B. pseudomallei via inhalation during the typhoon season.
Yang, Chun-Yuh; Lee, Min Sheng; Ho, Chi-Kung; Mena, Kristina D.; Wang, Peng-Yau; Chen, Pei-Shih
Burkholderia pseudomallei causes a potentially fatal infection called melioidosis. We have developed a nonfluorescent, colorimetric in situ hybridization assay using a specific probe to target 16s rRNA of B. pseudomallei in formalin-fixed, paraffin-embedded infected tissues for diagnostic purposes and to study infectious disease pathology. A 63-base pair DNA probe was synthesized and labeled with digoxigenin by PCR. Probe specificity was confirmed by BLAST analysis and by testing on appropriate microbial controls. The in situ hybridization assay was specifically and consistently positive for B. pseudomallei, showing strongly and crisply stained, single bacillus and bacilli clusters in mainly inflamed tissues in seven human acute melioidosis cases and experimentally infected mouse tissues. Intravascular and extravascular bacilli were detected in both intracellular and extracellular locations in various human organs, including lung, spleen, kidney, liver, bone marrow, and aortic mycotic aneurysm, particularly in the inflamed areas. Intravascular, intracellular bacteria in melioidosis have not been previously reported. Although the identity of infected intravascular leukocytes has to be confirmed, extravascular, intracellular bacilli appear to be found mainly within macrophages and neutrophils. Rarely, large intravascular, extracellular bacillary clusters/emboli could be detected in both human and mouse tissues. B. cepacia and non-Burkholderia pathogens (16 microbial species) all tested negative. Nonpathogenic B. thailandensis showed some cross-hybridization but signals were less intense. This in situ hybridization assay could be usefully adapted for B. pseudomallei identification in other clinical specimens such as pus and sputum. PMID:24186135
Eu, Lin Chuan; Ong, Kien Chai; Hiu, Jessie; Vadivelu, Jamunarani; Nathan, Sheila; Wong, Kum Thong
We analyzed water parameters and the occurrence of the melioidosis agent Burkholderia pseudomallei in 47 water bores in Northern Australia. B. pseudomallei was associated with soft, acidic bore water of low salinity but high iron levels. This finding aids in identifying water supplies at risk of contamination with this pathogenic bacterium. PMID:20543039
Draper, A D K; Mayo, M; Harrington, G; Karp, D; Yinfoo, D; Ward, L; Haslem, A; Currie, B J; Kaestli, M
We analyzed water parameters and the occurrence of the melioidosis agent Burkholderia pseudomallei in 47 water bores in Northern Australia. B. pseudomallei was associated with soft, acidic bore water of low salinity but high iron levels. This finding aids in identifying water supplies at risk of contamination with this pathogenic bacterium.
Draper, A. D. K.; Mayo, M.; Harrington, G.; Karp, D.; Yinfoo, D.; Ward, L.; Haslem, A.; Currie, B. J.; Kaestli, M.
Little is currently known about bacterial pathogen evolution and adaptation within the host during acute infection. Previous studies of Burkholderia pseudomallei, the etiologic agent of melioidosis, have shown that this opportunistic pathogen mutates rapidly both in vitro and in vivo at tandemly repeated loci, making this organism a relevant model for studying short-term evolution. In the current study, B. pseudomallei isolates cultured from multiple body sites from four Thai patients with disseminated melioidosis were subjected to fine-scale genotyping using multilocus variable-number tandem repeat analysis (MLVA). In order to understand and model the in vivo variable-number tandem repeat (VNTR) mutational process, we characterized the patterns and rates of mutations in vitro through parallel serial passage experiments of B. pseudomallei. Despite the short period of infection, substantial divergence from the putative founder genotype was observed in all four melioidosis cases. This study presents a paradigm for examining bacterial evolution over the short timescale of an acute infection. Further studies are required to determine whether the mutational process leads to phenotypic alterations that impact upon bacterial fitness in vivo. Our findings have important implications for future sampling strategies, since colonies in a single clinical sample may be genetically heterogeneous, and organisms in a culture taken late in the infective process may have undergone considerable genetic change compared with the founder inoculum.
Limmathurotsakul, Direk; Max, Tamara L.; Sarovich, Derek S.; Vogler, Amy J.; Dale, Julia L.; Ginther, Jennifer L.; Leadem, Benjamin; Colman, Rebecca E.; Foster, Jeffrey T.; Tuanyok, Apichai; Wagner, David M.; Peacock, Sharon J.; Pearson, Talima; Keim, Paul
Little is currently known about bacterial pathogen evolution and adaptation within the host during acute infection. Previous studies of Burkholderia pseudomallei, the etiologic agent of melioidosis, have shown that this opportunistic pathogen mutates rapidly both in vitro and in vivo at tandemly repeated loci, making this organism a relevant model for studying short-term evolution. In the current study, B. pseudomallei isolates cultured from multiple body sites from four Thai patients with disseminated melioidosis were subjected to fine-scale genotyping using multilocus variable-number tandem repeat analysis (MLVA). In order to understand and model the in vivo variable-number tandem repeat (VNTR) mutational process, we characterized the patterns and rates of mutations in vitro through parallel serial passage experiments of B. pseudomallei. Despite the short period of infection, substantial divergence from the putative founder genotype was observed in all four melioidosis cases. This study presents a paradigm for examining bacterial evolution over the short timescale of an acute infection. Further studies are required to determine whether the mutational process leads to phenotypic alterations that impact upon bacterial fitness in vivo. Our findings have important implications for future sampling strategies, since colonies in a single clinical sample may be genetically heterogeneous, and organisms in a culture taken late in the infective process may have undergone considerable genetic change compared with the founder inoculum. PMID:20090837
Price, Erin P; Hornstra, Heidie M; Limmathurotsakul, Direk; Max, Tamara L; Sarovich, Derek S; Vogler, Amy J; Dale, Julia L; Ginther, Jennifer L; Leadem, Benjamin; Colman, Rebecca E; Foster, Jeffrey T; Tuanyok, Apichai; Wagner, David M; Peacock, Sharon J; Pearson, Talima; Keim, Paul
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.
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.
James, Gemma L.; Delaney, Ben; Ward, Linda; Freeman, Kevin; Mayo, Mark
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
Burkholderia pseudomallei, the cause of the severe disease melioidosis in humans and animals, is a gram-negative saprophyte living in soil and water of areas of endemicity such as tropical northern Australia and Southeast Asia. Infection occurs mainly by contact with wet contaminated soil. The environmental distribution of B. pseudomallei in northern Australia is still unclear. We developed and evaluated a direct soil B. pseudomallei DNA detection method based on the recently published real-time PCR targeting the B. pseudomallei type III secretion system. The method was evaluated by inoculating different soil types with B. pseudomallei dilution series and by comparing B. pseudomallei detection rate with culture-based detection rate for 104 randomly collected soil samples from the Darwin rural area in northern Australia. We found that direct soil B. pseudomallei DNA detection not only was substantially faster than culture but also proved to be more sensitive with no evident false-positive results. This assay provides a new tool to detect B. pseudomallei in soil samples in a fast and highly sensitive and specific manner and is applicable for large-scale B. pseudomallei environmental screening studies or in outbreak situations. Furthermore, analysis of the 104 collected soil samples revealed a significant association between B. pseudomallei-positive sites and the presence of animals at these locations and also with moist, reddish brown-to-reddish gray soils. PMID:17873073
Kaestli, Mirjam; Mayo, Mark; Harrington, Glenda; Watt, Felicity; Hill, Jason; Gal, Daniel; Currie, Bart J
Background The gram-negative organism, Burkholderia pseudomallei, is responsible for the disease melioidosis. Septic arthritis and osteomyelitis due to B. pseudomallei are rare but recognised presentations of the disease. Methods A prospective database of all cases of melioidosis in the Northern Territory of Australia has been kept since October 1989. Entries to April 2009 were reviewed and cases involving bone and/or joint were investigated. We also present in detail the case reports of 3 presentations of bone and joint melioidosis. Results There were 536 presentations of melioidosis during the 20-year study period. Amongst these, there were 13 patients with primary septic arthritis and 7 cases of primary osteomyelitis. Septic arthritis and osteomyelitis were secondary to primary melioidosis elsewhere in 14 and 7 patients respectively. Melioidosis patients with bone/joint involvement were more likely to be Indigenous (p = 0.006) and female (p = 0.023) compared to patients with other presentations of disease. Conclusions Timely microbiological diagnosis and prompt treatment of melioidosis involving bone and/or joint with appropriate intravenous antibiotics is important, as is adequate surgical drainage and debridement where indicated. A subsequent protracted course of antibiotic eradication therapy is important to avoid relapse of disease.
Morse, Levi P.; Smith, Jonathan; Mehta, Janak; Ward, Linda; Cheng, Allen C.; Currie, Bart J.
Melioidosis is a severe suppurative to granulomatous infection caused by Burkholderia pseudomallei. The disease is endemic to South-East Asia and Northern Australasia and is also of interest as a potential biological weapon. Natural infection can occur by percutaneous inoculation, inhalation or ingestion, but the relative importance of each route is unknown. Experimental infection models using mice have shown inhalation to be the most lethal route of exposure, but few studies have examined the pathogenesis of percutaneous infection despite its presumptive importance in natural disease. Caprine models are useful in the study of melioidosis because goats are susceptible to natural infection by B. pseudomallei, display similar epizootiology/epidemiology to that of humans within the endemic range and develop similar pathologic lesions. Percutaneous inoculation with 10(4) CFU of B. pseudomallei produced disease in all experimental animals with rapid dissemination to the lungs, spleen and kidneys. Initial fever was brief, but temperatures did not return to pre-infection levels until day 18, concurrent with a dramatic lymphocytosis and the transition to chronic disease. Distribution and appearance of gross pathologic and radiographic lesions in goats were similar to caprine aerosol infection and to reported human disease. The similarities seen despite different routes of infection suggest that host or bacterial factors may be more important than the route of infection in disease pathogenesis. The nature of melioidosis in goats makes it amenable for modelling additional risk factors to produce acute clinical disease, which is important to the study of human melioidosis. PMID:24571408
Soffler, Carl; Bosco-Lauth, Angela M; Aboellail, Tawfik A; Marolf, Angela J; Bowen, Richard A
ABSTRACT Melioidosis is a potentially fatal disease that is endemic to tropical northern Australia and Southeast Asia, with a mortality rate of 14 to 50%. The bacterium Burkholderia pseudomallei is the causative agent which infects numerous parts of the human body, including the brain, which results in the neurological manifestation of melioidosis. The olfactory nerve constitutes a direct conduit from the nasal cavity into the brain, and we have previously reported that B. pseudomallei can colonize this nerve in mice. We have now investigated in detail the mechanism by which the bacteria penetrate the olfactory and trigeminal nerves within the nasal cavity and infect the brain. We found that the olfactory epithelium responded to intranasal B. pseudomallei infection by widespread crenellation followed by disintegration of the neuronal layer to expose the underlying basal layer, which the bacteria then colonized. With the loss of the neuronal cell bodies, olfactory axons also degenerated, and the bacteria then migrated through the now-open conduit of the olfactory nerves. Using immunohistochemistry, we demonstrated that B. pseudomallei migrated through the cribriform plate via the olfactory nerves to enter the outer layer of the olfactory bulb in the brain within 24 h. We also found that the bacteria colonized the thin respiratory epithelium in the nasal cavity and then rapidly migrated along the underlying trigeminal nerve to penetrate the cranial cavity. These results demonstrate that B. pseudomallei invasion of the nerves of the nasal cavity leads to direct infection of the brain and bypasses the blood-brain barrier.
St. John, James A.; Ekberg, Jenny A. K.; Dando, Samantha J.; Meedeniya, Adrian C. B.; Horton, Rachel E.; Batzloff, Michael; Owen, Suzzanne J.; Holt, Stephanie; Peak, Ian R.; Ulett, Glen C.; Mackay-Sim, Alan; Beacham, Ifor R.
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.
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 m 2 of disused land in northeast Thailand. B. pseudomallei was present on
Narisara Chantratita; Vanaporn Wuthiekanun; Direk Limmathurotsakul; Mongkol Vesaratchavest; Aunchalee Thanwisai; Premjit Amornchai; Sarinna Tumapa; Edward J. Feil; Nicholas P. Day; Sharon J. Peacock
Background At present, very little is known about how Burkholderia pseudomallei (B. pseudomallei) interacts with its host to elicit melioidosis symptoms. We established a murine acute-phase melioidosis model and used DNA microarray technology to investigate the global host/pathogen interaction. We compared the transcriptome of infected liver and spleen with uninfected tissues over an infection period of 42 hr to identify genes whose expression is altered in response to an acute infection. Results Viable B. pseudomallei cells were consistently detected in the blood, liver and spleen during the 42 hr course of infection. Microarray analysis of the liver and spleen over this time course demonstrated that genes involved in immune response, stress response, cell cycle regulation, proteasomal degradation, cellular metabolism and signal transduction pathways were differentially regulated. Up regulation of toll-like receptor 2 (TLR2) gene expression suggested that a TLR2-mediated signalling pathway is responsible for recognition and initiation of an inflammatory response to the acute B. pseudomallei infection. Most of the highly elevated inflammatory genes are a cohort of "core host immune response" genes commonly seen in general inflammation infections. Concomitant to this initial inflammatory response, we observed an increase in transcripts associated with cell-death, caspase activation and peptidoglysis that ultimately promote tissue injury in the host. The complement system responsible for restoring host cellular homeostasis and eliminating intracellular bacteria was activated only after 24 hr post-infection. However, at this time point, diverse host nutrient metabolic and cellular pathways including glycolysis, fatty acid metabolism and tricarboxylic acid (TCA) cycle were repressed. Conclusions This detailed picture of the host transcriptional response during acute melioidosis highlights a broad range of innate immune mechanisms that are activated in the host within 24 hrs, including the core immune response commonly seen in general inflammatory infections. Nevertheless, this activation is suppressed at 42 hr post-infection and in addition, suboptimal activation and function of the downstream complement system promotes uncontrolled spread of the bacteria.
Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif), that is injected into host cells via a Type III secretion system (T3SS) leading to arrest of cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385), which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7%) harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15) of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE) was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro.
Pumirat, Pornpan; Broek, Charles Vander; Juntawieng, Niramol; Muangsombut, Veerachat; Kiratisin, Pattarachai; Pattanapanyasat, Kovit; Stevens, Joanne M.; Stevens, Mark P.; Korbsrisate, Sunee
Burkholderia pseudomallei was isolated from environmental specimens 1 year after an outbreak of acute melioidosis in a remote coastal community in northwestern Australia. B. pseudomallei was isolated from a water storage tank and from spray formed in a pH-raising aerator unit. Pulsed-field gel electrophoresis confirmed the aerator and storage tank isolates were identical to the outbreak strain, WKo97.
Inglis, T. J.; Garrow, S. C.; Henderson, M.; Clair, A.; Sampson, J.; O'Reilly, L.; Cameron, B.
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
BackgroundThe 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 FindingsWe 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
Narisara Chantratita; Vanaporn Wuthiekanun; Direk Limmathurotsakul; Mongkol Vesaratchavest; Aunchalee Thanwisai; Premjit Amornchai; Sarinna Tumapa; Edward J. Feil; Nicholas P. Day; Sharon J. Peacock
Burkholderia pseudomallei is a Gram-negative bacterium which is the causative agent of melioidosis, a disease which carries a high mortality and morbidity rate in endemic areas of South East Asia and Northern Australia. At present there is no available human vaccine that protects against B. pseudomallei, and with the current limitations of antibiotic treatment, the development of new preventative and therapeutic interventions is crucial. This review considers the multiple elements of melioidosis vaccine research including: (i) the immune responses required for protective immunity, (ii) animal models available for preclinical testing of potential candidates, (iii) the different experimental vaccine strategies which are being pursued, and (iv) the obstacles and opportunities for eventual registration of a licensed vaccine in humans.
Patel, Natasha; Conejero, Laura; De Reynal, Melanie; Easton, Anna; Bancroft, Gregory J.; Titball, Richard W.
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
Gernot Schmoock; Ralf Ehricht; Falk Melzer; Astrid Rassbach; Holger C. Scholz; Heinrich Neubauer; Konrad Sachse; Rinaldo Aparecido Mota; Muhammad Saqib; Mandy Elschner
The Gram-negative bacterium Burkholderia pseudomallei is the aetiological agent of melioidosis, which is an endemic disease in tropical areas of Southeast Asia and Northern Australia. Burkholderia pseudomallei has intrinsic resistance to a number of commonly used antibiotics and has also been reported to develop a biofilm. Resistance to killing by antimicrobial agents is one of the hallmarks of bacteria grown
Sakawrat Kanthawong; Jan G. M. Bolscher; Enno C. I. Veerman; Jan van Marle; Hans J. J. de Soet; Kamran Nazmi; Surasakdi Wongratanacheewin; Suwimol Taweechaisupapong
The Gram-negative facultative intracellular rod Burkholderia pseudomallei causes melioidosis, an infectious disease with a wide range of clinical presentations. Among the observed visceral abscesses, the liver is commonly affected. However, neither this organotropism of B. pseudomallei nor local hepatic defense mechanisms have been thoroughly investigated so far. Own previous studies using electron microscopy of the murine liver after systemic infection of mice indicated that hepatocytes might be capable of killing B. pseudomallei. Therefore, the aim of this study was to further elucidate the interaction of B. pseudomallei with these cells and to analyze the role of hepatocytes in anti-B. pseudomallei host defense. In vitro studies using the human hepatocyte cell line HepG2 revealed that B. pseudomallei can invade these cells. Subsequently, B. pseudomallei is able to escape from the vacuole, to replicate within the cytosol of HepG2 cells involving its type 3 and type 6 secretion systems, and to induce actin tail formation. Furthermore, stimulation of HepG2 cells showed that IFN? can restrict growth of B. pseudomallei in the early and late phase of infection whereas the combination of IFN?, IL-1?, and TNF? is required for the maximal antibacterial activity. This anti-B. pseudomallei defense of HepG2 cells did not seem to be mediated by inducible nitric oxide synthase-derived nitric oxide or NADPH oxidase-derived superoxide. In summary, this is the first study describing B. pseudomallei intracellular life cycle characteristics in hepatocytes and showing that IFN?-mediated, but nitric oxide- and reactive oxygen species-independent, effector mechanisms are important in anti-B. pseudomallei host defense of hepatocytes.
Bast, Antje; Schmidt, Imke H. E.; Brauner, Paul; Brix, Bettina; Breitbach, Katrin; Steinmetz, Ivo
Cases of melioidosis and glanders are rare in the United States, but the etiologic agents of each disease (Burkholderia pseudomallei and Burkholderia mallei, respectively) are classified as Tier 1 select agents because of concerns about their potential use as bioterrorism agents. A rapid, highly sensitive, and portable assay for clinical laboratories and field use is required. Our laboratory has further evaluated a latex agglutination assay for its ability to identify B. pseudomallei and B. mallei isolates. This assay uses a monoclonal antibody that specifically recognizes the capsular polysaccharide produced by B. pseudomallei and B. mallei, but is absent in closely related Burkholderia species. A total of 110 B. pseudomallei and B. mallei were tested, and 36 closely related Burkholderia species. The latex agglutination assay was positive for 109 of 110 (99.1% sensitivity) B. pseudomallei and B. mallei isolates tested. PMID:24710616
Duval, Brea D; Elrod, Mindy G; Gee, Jay E; Chantratita, Narisara; Tandhavanant, Sarunporn; Limmathurotsakul, Direk; Hoffmaster, Alex R
Cases of melioidosis and glanders are rare in the United States, but the etiologic agents of each disease (Burkholderia pseudomallei and Burkholderia mallei, respectively) are classified as Tier 1 select agents because of concerns about their potential use as bioterrorism agents. A rapid, highly sensitive, and portable assay for clinical laboratories and field use is required. Our laboratory has further evaluated a latex agglutination assay for its ability to identify B. pseudomallei and B. mallei isolates. This assay uses a monoclonal antibody that specifically recognizes the capsular polysaccharide produced by B. pseudomallei and B. mallei, but is absent in closely related Burkholderia species. A total of 110 B. pseudomallei and B. mallei were tested, and 36 closely related Burkholderia species. The latex agglutination assay was positive for 109 of 110 (99.1% sensitivity) B. pseudomallei and B. mallei isolates tested.
Duval, Brea D.; Elrod, Mindy G.; Gee, Jay E.; Chantratita, Narisara; Tandhavanant, Sarunporn; Limmathurotsakul, Direk; Hoffmaster, Alex R.
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
A previously healthy Chinese male returned from working in the Malaysian jungle with a fever. A blood culture grew Gram-negative bacilli that were initially identified as Burkholderia cepacia by the VITEK 2 system but were subsequently found to be Burkholderia pseudomallei by partial sequencing of the 16S rRNA gene. The identification of B. pseudomallei using commercially available automated systems is problematic and clinicians in non-endemic areas should be aware of the possibility of melioidosis in patients with a relevant travel history and blood cultures growing Burkholderia spp. PMID:22820689
Zong, Zhiyong; Wang, Xiaohui; Deng, Yiyun; Zhou, Taoyou
IgM and IgG based ELISA systems were developed using the culture filtrate antigen (CFA) of Burkholderia pseudomallei. The assays were evaluated using 95 sera from 66 septicemic cases and 47 sera from 20 cases with localized melioidosis. In addition 65 sera from culture negative cases that were also serologically negative for other endemic infections clinically suspected of melioidosis were included.
V. Chenthamarakshan; J. Vadivelu; S. D. Puthucheary
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
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
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.
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
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.
Campos, Cristine G.; Byrd, Matthew S.
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, and the Pasteur Institute (Vietnam). The 207 isolates of the collection were resolved into 80 sequence types (STs); 56 of these were novel. eBURST diagrams predict that the historical-collection STs segregate into three complexes when analyzed separately. When added to the 760 isolates and 365 STs of the B. pseudomallei MLST database, the historical-collection STs cluster significantly within the main complex of the eBURST diagram in an ancestral pattern and alter the B. pseudomallei “population snapshot.” Differences in colony morphology among reference isolates were found not to affect the STs assigned, which were consistent with the original isolates. Australian ST84 is likely characteristic of B. pseudomallei isolates of Southeast Asia rather than Australia, since multiple environmental isolates from Thailand and Malaysia share this ST with the single Australian clinical isolate in the MLST database. Phylogenetic evidence is also provided suggesting that Australian isolates may not be distinct from those of Thailand, since ST60 is common to environmental isolates from both countries. MLST and eBURST are useful tools for the study of population biology and epidemiology, since they provide methods to elucidate new genetic relationships among bacterial isolates.
McCombie, Roberta L.; Finkelstein, Richard A.; Woods, Donald E.
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.
Silva, Ediane B.; Dow, Steven W.
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
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.
Ashdown's medium, Burkholderia pseudomallei selective agar (BPSA), and a commercial Burkholderia cepacia medium were compared for their abilities to grow B. pseudomallei from 155 clinical specimens that proved positive for this organism. The sensitivity of each was equivalent; the selectivity of BPSA was lower than that of Ashdown's or B. cepacia medium. Burkholderia pseudomallei is a gram-negative soil saprophyte and
Sharon J. Peacock; Grace Chieng; Allen C. Cheng; David A. B. Dance; Gumphol Wongsuvan; Nittaya Teerawattanasook; Nicholas P. J. Day
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
Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacillus endemic to areas of southeast Asia and northern Australia. Presently, there is no licensed vaccine for B. pseudomallei and the organism is refractive to antibiotic therapy. The bacterium is known to survive and multiply inside both phagocytic and nonphagocytic host cells and may be able to spread directly from
Gareth D. Healey; Stephen J. Elvin; Margaret Morton; E. Diane Williamson
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
Justin A. Boddey; Cameron P. Flegg; Chris J. Day; Ifor R. Beacham; Ian R. Peak
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
Jaruek Charoensap; Pongsak Utaisincharoen; Anneke Engering; Stitaya Sirisinha
BackgroundThe soil-dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis. Extreme structuring of genotype and genotypic frequency has been demonstrated for B. pseudomallei in uncultivated land, but its distribution and genetic diversity in agricultural land where most human infections are probably acquired is not well defined.MethodsFixed-interval soil sampling was performed in a rice paddy in northeast Thailand in which
Vanaporn Wuthiekanun; Direk Limmathurotsakul; Narisara Chantratita; Edward J. Feil; Nicholas P. J. Day; Sharon J. Peacock
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.
Stone, Joshua K.; Johnson, Shannon L.; Bruce, David C.; Detter, J. Chris; Mayo, Mark; Currie, Bart J.; Gelhaus, H. Carl; Keim, Paul
BACKGROUND: Burkholderia pseudomallei are the causative agent of melioidosis. Increasing resistance of the disease to antibiotics is a severe problem in treatment regime and has led to intensification of the search for new drugs. Antimicrobial peptides are the most ubiquitous in nature as part of the innate immune system and host defense mechanism. METHODS: Here, we investigated a group of
R Perumal Samy; A Pachiappan; P Gopalakrishnakone; Maung M Thwin; Yap E Hian; Vincent TK Chow; Ho Bow; Joseph T Weng
The Gram-negative bacterium Burkholderia pseudomallei is the etiological agent of melioidosis and is remarkably resistant to most classes of antibacterials. Even after months of treatment with antibacterials that are relatively effective in vitro, there is a high rate of treatment failure, indicating that this pathogen alters its patterns of antibacterial susceptibility in response to cues encountered in the host. The pathology of melioidosis indicates that B. pseudomallei encounters host microenvironments that limit aerobic respiration, including the lack of oxygen found in abscesses and in the presence of nitric oxide produced by macrophages. We investigated whether B. pseudomallei could survive in a nonreplicating, oxygen-deprived state and determined if this physiological state was tolerant of conventional antibacterials. B. pseudomallei survived initial anaerobiosis, especially under moderately acidic conditions similar to those found in abscesses. Microarray expression profiling indicated a major shift in the physiological state of hypoxic B. pseudomallei, including induction of a variety of typical anaerobic-environment-responsive genes and genes that appear specific to anaerobic B. pseudomallei. Interestingly, anaerobic B. pseudomallei was unaffected by antibacterials typically used in therapy. However, it was exquisitely sensitive to drugs used against anaerobic pathogens. After several weeks of anaerobic culture, a significant loss of viability was observed. However, a stable subpopulation that maintained complete viability for at least 1 year was established. Thus, during the course of human infection, if a minor subpopulation of bacteria inhabited an oxygen-restricted environment, it might be indifferent to traditional therapy but susceptible to antibiotics frequently used to treat anaerobic infections. PMID:21537012
Hamad, Mohamad A; Austin, Chad R; Stewart, Amanda L; Higgins, Mike; Vázquez-Torres, Andrés; Voskuil, Martin I
The Gram-negative bacterium Burkholderia pseudomallei is the etiological agent of melioidosis and is remarkably resistant to most classes of antibacterials. Even after months of treatment with antibacterials that are relatively effective in vitro, there is a high rate of treatment failure, indicating that this pathogen alters its patterns of antibacterial susceptibility in response to cues encountered in the host. The pathology of melioidosis indicates that B. pseudomallei encounters host microenvironments that limit aerobic respiration, including the lack of oxygen found in abscesses and in the presence of nitric oxide produced by macrophages. We investigated whether B. pseudomallei could survive in a nonreplicating, oxygen-deprived state and determined if this physiological state was tolerant of conventional antibacterials. B. pseudomallei survived initial anaerobiosis, especially under moderately acidic conditions similar to those found in abscesses. Microarray expression profiling indicated a major shift in the physiological state of hypoxic B. pseudomallei, including induction of a variety of typical anaerobic-environment-responsive genes and genes that appear specific to anaerobic B. pseudomallei. Interestingly, anaerobic B. pseudomallei was unaffected by antibacterials typically used in therapy. However, it was exquisitely sensitive to drugs used against anaerobic pathogens. After several weeks of anaerobic culture, a significant loss of viability was observed. However, a stable subpopulation that maintained complete viability for at least 1 year was established. Thus, during the course of human infection, if a minor subpopulation of bacteria inhabited an oxygen-restricted environment, it might be indifferent to traditional therapy but susceptible to antibiotics frequently used to treat anaerobic infections.
Hamad, Mohamad A.; Austin, Chad R.; Stewart, Amanda L.; Higgins, Mike; Vazquez-Torres, Andres; Voskuil, Martin I.
Burkholderia pseudomallei is a Gram-negative environmental bacterium found in tropical climates that causes melioidosis. Culture remains the diagnostic gold standard, but isolation of B. pseudomallei from heavily contaminated sites, such as fecal specimens, can be difficult. We recently reported that B. pseudomallei is capable of infecting the gastrointestinal tract of mice and suggested that the same may be true in humans. Thus, there is a strong need for new culture techniques to allow for efficient detection of B. pseudomallei in fecal and other specimens. We found that the addition of norfloxacin, ampicillin, and polymyxin B to Ashdown's medium (NAP-A) resulted in increased specificity without affecting the growth of 25 B. pseudomallei strains. Furthermore, recovery of B. pseudomallei from human clinical specimens was not affected by the three additional antibiotics. Therefore, we conclude that NAP-A medium provides a new tool for more sensitive isolation of B. pseudomallei from heavily contaminated sites. PMID:24062483
Goodyear, Andrew; Strange, Linda; Rholl, Drew A; Silisouk, Joy; Dance, David A B; Schweizer, Herbert P; Dow, Steven
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. PMID:22521523
Jones-Carson, Jessica; Laughlin, James R; Stewart, Amanda L; Voskuil, Martin I; Vázquez-Torres, Andrés
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
Burkholderia species, notably Burkholderia cepacia and Burkholderia gladioli, are important pathogens in patients with chronic granulomatous disease (CGD). Burkholderia pseudomallei, the causative agent of melioidosis, is endemic in Southeast Asia and northern Australia but is a rare pathogen in other parts of the world. We describe the occurrence of B. pseudomallei infection in a Puerto Rican patient with CGD. This is one of only a small number of documented cases of melioidosis autochthonous to the Americas and is the first reported case of B. pseudomallei infection in a CGD patient from the Americas. We conclude that B. pseudomallei, like B. cepacia and B. gladioli, should be considered a potential pathogen in patients with CGD and that melioidosis should be considered in the differential diagnosis for ill residents of or travelers to Puerto Rico. PMID:9564470
Dorman, S E; Gill, V J; Gallin, J I; Holland, S M
Melioidosis is a disease in tropical and subtropical regions of the world that is caused by Burkholderia pseudomallei. In endemic regions the disease occurs primarily in humans and goats. In the present study, we used the goat as a model to dissect the polar lipids of B. pseudomallei to identify lipid molecules that could be used for adjuvants/vaccines or as diagnostic tools. We showed that the lipidome of B. pseudomallei and its fractions contain several polar lipids with the capacity to elicit different immune responses in goats, namely rhamnolipids and ornithine lipids which induced IFN-?, whereas phospholipids and an undefined polar lipid induced strong IL-10 secretion in CD4+ T cells. Autologous T cells co-cultured with caprine dendritic cells (cDCs) and polar lipids of B. pseudomallei proliferated and up-regulated the expression of CD25 (IL-2 receptor) molecules. Furthermore, we demonstrated that polar lipids were able to up-regulate CD1w2 antigen expression in cDCs derived from peripheral blood monocytes. Interestingly, the same polar lipids had only little effect on the expression of MHC class II DR antigens in the same caprine dendritic cells. Finally, antibody blocking of the CD1w2 molecules on cDCs resulted in decreased expression for IFN-? by CD4+ T cells. Altogether, these results showed that polar lipids of B. pseudomallei are recognized by the caprine immune system and that their recognition is primarily mediated by the CD1 antigen cluster.
Gonzalez-Juarrero, Mercedes; Mima, Naoko; Trunck, Lily A.; Schweizer, Herbert P.; Bowen, Richard A.; Dascher, Kyle; Mwangi, Waithaka; Eckstein, Torsten M.
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 after 4 h of coculturing using antibiotic protection assay. Adherence was demonstrated by coculturing the cells with fluorescein-labeled bacteria for 1 h and the number of positive cells was analyzed by flow cytometry. The results obtained with this in vitro study demonstrated that compared with its avirulent counterpart, B. pseudomallei is significantly more efficient (P<0.01) in invasion, adherence and inducing cellular damage, as represented by plaque formation. PMID:15043863
Kespichayawattana, Wannapa; Intachote, Pakamas; Utaisincharoen, Pongsak; Sirisinha, Stitaya
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 activity to murine splenocytes than SAE-LPS; moreover, it stimulated even the splenocytes of LPS-resistant C3H/HeJ mice. Unusual chemical structures in the acid-stable inner core region attached to the lipid A moiety of BP-LPS may be responsible for this strong mitogenic activity. PMID:8935661
Matsuura, M; Kawahara, K; Ezaki, T; Nakano, M
Melioidosis is caused by the gram-negative saprophytic bacterium Burkholderia pseudomallei, which is endemic to southeast Asia and northern Australia. We have previously found evidence of geographic localization of strains based on multilocus sequence typing (MLST). In this study, we examined the diversity of 277 isolates from northern Australia, which were resolved into 159 different sequence types. No sequence types were common to both Queensland and the Northern Territory, and there was significant differentiation between the alleles present in the two regions. The considerable diversity in sequence types contrasts with the limited diversity of alleles at MLST loci, supporting previous work suggesting a high rate of recombination relative to mutation in B. pseudomallei, where new sequence types are primarily generated by reassortment of existing alleles.
Cheng, Allen C.; Ward, Linda; Godoy, Daniel; Norton, Robert; Mayo, Mark; Gal, Daniel; Spratt, Brian G.; Currie, Bart J.
Burkholderia pseudomallei is a Gram negative soil saprophyte that causes the disease melioidosis where clinical symptoms can vary from localised infection to pneumonia and septic shock. Ecotin is a potent periplasmic serine protease inhibitor first identified in Escherichia coli. Ecotin, although present in only a small subset of genera, can inhibit a broad range of serine proteases including those typically associated with the innate immune system such as neutrophil elastase and cathepsin G. An Ecotin orthologue identified in B. pseudomallei was recombinantly expressed and found to inhibit elastase. To study the role of Ecotin in B. pseudomallei virulence an in-frame unmarked deletion mutant was created. Infection of a murine macrophages-like cell line revealed Ecotin was necessary for the early stages of colonisation allowing replication following cell entry. Attenuation of the ?eco mutant strain in the murine model of melioidosis further supported Ecotin as a virulence factor of B. pseudomallei. PMID:24462575
Ireland, Philip M; Marshall, Laura; Norville, Isobel; Sarkar-Tyson, Mitali
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.
Asakrah, Saja; Nieves, Wildaliz; Mahdi, Zaid; Agard, Mallory; Zea, Arnold H.; Roy, Chad J.; Morici, Lisa A.
Melioidosis is an infectious disease caused by Burkholderia pseudomallei and endemic in Southeast Asia. One hundred and forty six clinical isolates of B. pseudomallei from different states in Malaysia were obtained and molecular typing was carried out using pulsed-field gel electrophoresis (PFGE). Overall, nine clusters were successfully identified. Burkholderia pseudomallei isolates used in this study were found to be genetically diverse and there were differences in the clusters of isolates from peninsular and east Malaysia. BS9 cluster was the most common cluster and found in all the states while BS2 cluster only existed in a particular state. Based on the PFGE analysis, the distribution of different B. pseudomallei clinical isolates in Malaysia was mapped. PMID:21399594
Chua, K H; See, K H; Thong, K L; Puthucheary, S D
Melioidosis is a potentially fatal disease caused by the saprophytic bacterium Burkholderia pseudomallei. Resistance to gentamicin is generally a hallmark of B. pseudomallei, and gentamicin is a selective agent in media used for diagnosis of melioidosis. In this study, we determined the prevalence and mechanism of gentamicin susceptibility found in B. pseudomallei isolates from Sarawak, Malaysian Borneo. We performed multilocus sequence typing and antibiotic susceptibility testing on 44 B. pseudomallei clinical isolates from melioidosis patients in Sarawak district hospitals. Whole-genome sequencing was used to identify the mechanism of gentamicin susceptibility. A novel allelic-specific PCR was designed to differentiate gentamicin-sensitive isolates from wild-type B. pseudomallei. A reversion assay was performed to confirm the involvement of this mechanism in gentamicin susceptibility. A substantial proportion (86%) of B. pseudomallei clinical isolates in Sarawak, Malaysian Borneo, were found to be susceptible to the aminoglycoside gentamicin, a rare occurrence in other regions where B. pseudomallei is endemic. Gentamicin sensitivity was restricted to genetically related strains belonging to sequence type 881 or its single-locus variant, sequence type 997. Whole-genome sequencing identified a novel nonsynonymous mutation within amrB, encoding an essential component of the AmrAB-OprA multidrug efflux pump. We confirmed the role of this mutation in conferring aminoglycoside and macrolide sensitivity by reversion of this mutation to the wild-type sequence. Our study demonstrates that alternative B. pseudomallei selective media without gentamicin are needed for accurate melioidosis laboratory diagnosis in Sarawak. This finding may also have implications for environmental sampling of other locations to test for B. pseudomallei endemicity.
Podin, Yuwana; Sarovich, Derek S.; Price, Erin P.; Kaestli, Mirjam; Mayo, Mark; Hii, KingChing; Ngian, HieUng; Wong, SeeChang; Wong, IngTien; Wong, JinShyan; Mohan, Anand; Ooi, MongHow; Fam, TemLom; Wong, Jack; Tuanyok, Apichai; Keim, Paul; Giffard, Philip M.
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.
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
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
Burkholderia pseudomallei and B. mallei, the causative agents of melioidosis and glanders, respectively, are designated category B biothreat agents. Current methods for identifying these organisms rely on their phe- notypic characteristics and an extensive set of biochemical reactions. We evaluated the use of 16S rRNA gene sequencing to rapidly identify these two species and differentiate them from each other as
Jay E. Gee; Claudio T. Sacchi; Mindy B. Glass; Barun K. De; Robbin S. Weyant; Paul N. Levett; Anne M. Whitney; Alex R. Hoffmaster; Tanja Popovic
The US Public Health Emergency Medical Countermeasures Enterprise convened subject matter experts at the 2010 HHS Burkholderia Workshop to develop consensus recommendations for postexposure prophylaxis against and treatment for Burkholderia pseudomallei and B. mallei infections, which cause melioidosis and glanders, respectively. Drugs recommended by consensus of the participants are ceftazidime or meropenem for initial intensive therapy, and trimethoprim/sulfamethoxazole or amoxicillin/clavulanic acid for eradication therapy. For postexposure prophylaxis, recommended drugs are trimethoprim/sulfamethoxazole or co-amoxiclav. To improve the timely diagnosis of melioidosis and glanders, further development and wide distribution of rapid diagnostic assays were also recommended. Standardized animal models and B. pseudomallei strains are needed for further development of therapeutic options. Training for laboratory technicians and physicians would facilitate better diagnosis and treatment options.
Garges, Susan; Aurigemma, Rosemarie; Baccam, Prasith; Blaney, David D.; Cheng, Allen C.; Currie, Bart J.; Dance, David; Gee, Jay E.; Larsen, Joseph; Limmathurotsakul, Direk; Morrow, Meredith G.; Norton, Robert; O'Mara, Elizabeth; Peacock, Sharon J.; Pesik, Nicki; Rogers, L. Paige; Schweizer, Herbert P.; Steinmetz, Ivo; Tan, Gladys; Tan, Patrick; Wiersinga, W. Joost; Wuthiekanun, Vanaporn; Smith, Theresa L.
BackgroundBurkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis, was reported to produce biofilm. As the disease causes high relapse rate when compared to other bacterial infections, it therefore might be due to the reactivation of the biofilm forming bacteria which also provided resistance to antimicrobial agents. However, the mechanism on how biofilm can provide tolerance to antimicrobials is still unclear.Methodology\\/Principal
Chakrit Sawasdidoln; Suwimol Taweechaisupapong; Rasana W. Sermswan; Unchalee Tattawasart; Sumalee Tungpradabkul; Surasakdi Wongratanacheewin; Stefan Bereswill
Increasing evidence has shown that small-molecule chemistry in microbes (i.e., secondary metabolism) can modulate the microbe-host response in infection and pathogenicity. The bacterial disease melioidosis is conferred by the highly virulent, antibiotic-resistant pathogen Burkholderia pseudomallei (BP). Whereas some macromolecular structures have been shown to influence BP virulence (e.g., secretion systems, cellular capsule, pili), the role of the large cryptic secondary metabolome encoded within its genome has been largely unexplored for its importance to virulence. Herein we demonstrate that BP-encoded small-molecule biosynthesis is indispensible for in vivo BP pathogenicity. Promoter exchange experiments were used to induce high-level molecule production from two gene clusters (MPN and SYR) found to be essential for in vivo virulence. NMR structural characterization of these metabolites identified a new class of lipopeptide biosurfactants/biofilm modulators (the malleipeptins) and syrbactin-type proteasome inhibitors, both of which represent overlooked small-molecule virulence factors for BP. Disruption of Burkholderia virulence by inhibiting the biosynthesis of these small-molecule biosynthetic pathways may prove to be an effective strategy for developing novel melioidosis-specific therapeutics. PMID:24884988
Biggins, John B; Kang, Hahk-Soo; Ternei, Melinda A; DeShazer, David; Brady, Sean F
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.
Conejero, Laura; Spink, Natasha; Wand, Matthew E.; Bancroft, Gregory J.; Titball, Richard W.
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
Background Burkholderia pseudomallei and Burkholderia mallei cause the diseases melioidosis and glanders, respectively. A well-studied aspect of pathogenesis by these closely-related bacteria is their ability to invade and multiply within eukaryotic cells. In contrast, the means by which B. pseudomallei and B. mallei adhere to cells are poorly defined. The purpose of this study was to identify adherence factors expressed by these organisms. Results Comparative sequence analyses identified a gene product in the published genome of B. mallei strain ATCC23344 (locus # BMAA0649) that resembles the well-characterized Yersinia enterocolitica autotransporter adhesin YadA. The gene encoding this B. mallei protein, designated boaA, was expressed in Escherichia coli and shown to significantly increase adherence to human epithelial cell lines, specifically HEp2 (laryngeal cells) and A549 (type II pneumocytes), as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, disruption of the boaA gene in B. mallei ATCC23344 reduced adherence to all three cell types by ~50%. The genomes of the B. pseudomallei strains K96243 and DD503 were also found to contain boaA and inactivation of the gene in DD503 considerably decreased binding to monolayers of HEp2 and A549 cells and to NHBE cultures. A second YadA-like gene product highly similar to BoaA (65% identity) was identified in the published genomic sequence of B. pseudomallei strain K96243 (locus # BPSL1705). The gene specifying this protein, termed boaB, appears to be B. pseudomallei-specific. Quantitative attachment assays demonstrated that recombinant E. coli expressing BoaB displayed greater binding to A549 pneumocytes, HEp2 cells and NHBE cultures. Moreover, a boaB mutant of B. pseudomallei DD503 showed decreased adherence to these respiratory cells. Additionally, a B. pseudomallei strain lacking expression of both boaA and boaB was impaired in its ability to thrive inside J774A.1 murine macrophages, suggesting a possible role for these proteins in survival within professional phagocytic cells. Conclusions The boaA and boaB genes specify adhesins that mediate adherence to epithelial cells of the human respiratory tract. The boaA gene product is shared by B. pseudomallei and B. mallei whereas BoaB appears to be a B. pseudomallei-specific adherence factor.
Background Burkholderia pseudomallei and B. mallei are closely related Category B Select Agents of bioterrorism and the causative agents of the diseases melioidosis and glanders, respectively. Rapid phage-based diagnostic tools would greatly benefit early recognition and treatment of these diseases. There is extensive strain-to-strain variation in B. pseudomallei genome content due in part to the presence or absence of integrated prophages. Several phages have previously been isolated from B. pseudomallei lysogens, for example ?K96243, ?1026b and ?52237. Results We have isolated a P2-like bacteriophage, ?X216, which infects 78% of all B. pseudomallei strains tested. ?X216 also infects B. mallei, but not other Burkholderia species, including the closely related B. thailandensis and B. oklahomensis. The nature of the ?X216 host receptor remains unclear but evidence indicates that in B. mallei ?X216 uses lipopolysaccharide O-antigen but a different receptor in B. pseudomallei. The 37,637 bp genome of ?X216 encodes 47 predicted open reading frames and shares 99.8% pairwise identity and an identical strain host range with bacteriophage ?52237. Closely related P2-like prophages appear to be widely distributed among B. pseudomallei strains but both ?X216 and ?52237 readily infect prophage carrying strains. Conclusions The broad strain infectivity and high specificity for B. pseudomallei and B. mallei indicate that ?X216 will provide a good platform for the development of phage-based diagnostics for these bacteria.
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.
Burkholderia pseudomallei is the causative agent of melioidosis, a severe disease of humans and animals. At present, no effective vaccine against melioidosis exists. Bacterial type IV pilin proteins have been used successfully as subunit vaccines. In this study, we evaluated a heterologously expressed and purified type IV pilus protein (PilV) of B. pseudomallei strain K96243 as a candidate subunit vaccine. PilV protein was assessed for its ability to protect BALB/c mice against B. pseudomallei strain G207 infection. Mice subcutaneously immunized with purified PilV protein produced high titers of IgG antibodies, which were strongly biased towards IgG1, with lower levels of IgG2a. Even though the PilV protein was highly immunogenic, it could not induce protection against a lethal B. pseudomallei challenge. Possible mechanisms of this non-protection phenomenon are discussed. PMID:22299445
Sangdee, Kusavadee; Waropastrakul, Srivilai; Wongratanachewin, Surasak; Homchampa, Preecha
Melioidosis is a tropical disease caused by ingestion, percutaneous inoculation or inhalation of the Gram-negative soil saprophyte Burkholderia pseudomallei. We developed a reproducible experimental murine model of pneumonic melioidosis induced by inhalation of aerosolized B. pseudomallei 1026b. In a series of experiments performed to bracket the lethal dose, we found that C57BL/6 mice were modestly more resistant than BALB/c mice (median lethal dose 334 CFU/lung vs 204 CFU/lung). We further characterized infection and pulmonary inflammation in C57BL/6 mice infected with a sublethal dose. We observed pulmonary replication and dissemination of bacteria to distant organs in the first days after infection, followed by bacterial containment by day 4 and no evidence of recrudescent infection for up to 2 months. We measured a robust host inflammatory response notable for a neutrophilic bronchoalveolar lavage fluid profile, elevated cytokines and chemokines in the lung and serum and scattered foci of neutrophilic infiltrates in the alveoli and in a perivascular distribution on histological analysis. We previously noted a similar pattern of inflammation in mice infected with aerosolized B. thailandensis. This report builds on the limited literature describing experimental murine pneumonic melioidosis induced by aerosol and characterizes pulmonary infection and resultant inflammation in C57BL/6 mice infected with aerosolized B. pseudomallei. This model has utility for the study of bacterial and host factors that contribute to the virulence of melioidosis. PMID:23136994
West, T Eoin; Myers, Nicolle D; Liggitt, H Denny; Skerrett, Shawn J
The environmental Gram-negative encapsulated bacillus Burkholderia pseudomallei is the causative agent of melioidosis, a disease associated with high morbidity and mortality rates in areas of Southeast Asia and northern Australia in which the disease is endemic. B. pseudomallei is also classified as a tier I select agent due to the high level of lethality of the bacterium and its innate resistance to antibiotics, as well as the lack of an effective vaccine. Gram-negative bacteria, including B. pseudomallei, secrete outer membrane vesicles (OMVs) which are enriched with multiple protein, lipid, and polysaccharide antigens. Previously, we demonstrated that immunization with multivalent B. pseudomallei-derived OMVs protects highly susceptible BALB/c mice against an otherwise lethal aerosol challenge. In this work, we evaluated the protective efficacy of OMV immunization against intraperitoneal challenge with a heterologous strain because systemic infection with phenotypically diverse environmental B. pseudomallei strains poses another hazard and a challenge to vaccine development. We demonstrated that B. pseudomallei OMVs derived from strain 1026b afforded significant protection against septicemic infection with B. pseudomallei strain K96243. OMV immunization induced robust OMV-, lipopolysaccharide-, and capsular polysaccharide-specific serum IgG (IgG1, IgG2a, and IgG3) and IgM antibody responses. OMV-immune serum promoted bacterial killing in vitro, and passive transfer of B. pseudomallei OMV immune sera protected naive mice against a subsequent challenge. These results indicate that OMV immunization provides antibody-mediated protection against acute, rapidly lethal sepsis in mice. B. pseudomallei-derived OMVs may represent an efficacious multivalent vaccine strategy against melioidosis. PMID:24671550
Nieves, Wildaliz; Petersen, Hailey; Judy, Barbara M; Blumentritt, Carla A; Russell-Lodrigue, Kasi; Roy, Chad J; Torres, Alfredo G; Morici, Lisa A
Infections with the Gram-negative bacterium Burkholderia pseudomallei (melioidosis) are associated with high mortality, and there is currently no approved vaccine to prevent the development of melioidosis in humans. Infected patients also do not develop protective immunity to reinfection, and some individuals will develop chronic, subclinical infections with B. pseudomallei. At present, our understanding of what constitutes effective protective immunity against B. pseudomallei infection remains incomplete. Therefore, we conducted a study to elucidate immune correlates of vaccine-induced protective immunity against acute B. pseudomallei infection. BALB/c and C57BL/6 mice were immunized subcutaneously with a highly attenuated, Select Agent-excluded purM deletion mutant of B. pseudomallei (strain Bp82) and then subjected to intranasal challenge with virulent B. pseudomallei strain 1026b. Immunization with Bp82 generated significant protection from challenge with B. pseudomallei, and protection was associated with a significant reduction in bacterial burden in lungs, liver, and spleen of immunized mice. Humoral immunity was critically important for vaccine-induced protection, as mice lacking B cells were not protected by immunization and serum from Bp82-vaccinated mice could transfer partial protection to nonvaccinated animals. In contrast, vaccine-induced protective immunity was found to be independent of both CD4 and CD8 T cells. Tracking studies demonstrated uptake of the Bp82 vaccine strain predominately by neutrophils in vaccine-draining lymph nodes and by smaller numbers of dendritic cells (DC) and monocytes. We concluded that protection following cutaneous immunization with a live attenuated Burkholderia vaccine strain was dependent primarily on generation of effective humoral immune responses.
Silva, Ediane B.; Goodyear, Andrew; Sutherland, Marjorie D.; Podnecky, Nicole L.; Gonzalez-Juarrero, Mercedes; Schweizer, Herbert P.
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.
Background The sRNAs of bacterial pathogens are known to be involved in various cellular roles including environmental adaptation as well as regulation of virulence and pathogenicity. It is expected that sRNAs may also have similar functions for Burkholderia pseudomallei, a soil bacterium that can adapt to diverse environmental conditions, which causes the disease melioidosis and is also able to infect a wide variety of hosts. Results By integrating several proven sRNA prediction programs into a computational pipeline, available Burkholderia spp. genomes were screened to identify sRNA gene candidates. Orthologous sRNA candidates were then identified via comparative analysis. From the total prediction, 21 candidates were found to have Rfam homologs. RT-PCR and sequencing of candidate sRNA genes of unknown functions revealed six putative sRNAs which were highly conserved in Burkholderia spp. and two that were unique to B. pseudomallei present in a normal culture conditions transcriptome. The validated sRNAs include potential cis-acting elements associated with the modulation of methionine metabolism and one B. pseudomallei-specific sRNA that is expected to bind to the Hfq protein. Conclusions The use of the pipeline developed in this study and subsequent comparative analysis have successfully aided in the discovery and shortlisting of sRNA gene candidates for validation. This integrated approach identified 29 B. pseudomallei sRNA genes - of which 21 have Rfam homologs and 8 are novel.
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°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 73.6% (67/91) 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.
Background The soil-dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis. Extreme structuring of genotype and genotypic frequency has been demonstrated for B. pseudomallei in uncultivated land, but its distribution and genetic diversity in agricultural land where most human infections are probably acquired is not well defined. Methods Fixed-interval soil sampling was performed in a rice paddy in northeast Thailand in which 100 grams of soil was sampled at a depth of 30 cm from 10×10 sampling points each measuring 2.5 m by 2.5 m. Soil was cultured for the presence of B. pseudomallei and genotyping of colonies present on primary culture plates was performed using a combination of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Principal Findings B. pseudomallei was cultured from 28/100 samples. Genotyping of 630 primary colonies drawn from 11 sampling points demonstrated 10 PFGE banding pattern types, which on MLST were resolved into 7 sequence types (ST). Overlap of genotypes was observed more often between sampling points that were closely positioned. Two sampling points contained mixed B. pseudomallei genotypes, each with a numerically dominant genotype and one or more additional genotypes present as minority populations. Conclusions Genetic diversity and structuring of B. pseudomallei exists despite the effects of flooding and the physical and chemical processes associated with farming. These findings form an important baseline for future studies of environmental B. pseudomallei.
Wuthiekanun, Vanaporn; Limmathurotsakul, Direk; Chantratita, Narisara; Feil, Edward J.; Day, Nicholas P. J.; Peacock, Sharon J.
Melioidosis is a major cause of morbidity and mortality in Southeast Asia, where the causative organism (Burkholderia pseudomallei) is present in the soil. In the Lao People's Democratic Republic (Laos), B. pseudomallei is a significant cause of sepsis around the capital, Vientiane, and has been isolated in soil near the city, adjacent to the Mekong River. We explored whether B. pseudomallei occurs in Lao soil distant from the Mekong River, drawing three axes across northwest, northeast, and southern Laos to create nine sampling areas in six provinces. Within each sampling area, a random rice field site containing a grid of 100 sampling points each 5 m apart was selected. Soil was obtained from a depth of 30 cm and cultured for B. pseudomallei. Four of nine sites (44%) were positive for B. pseudomallei, including all three sites in Saravane Province, southern Laos. The highest isolation frequency was in east Saravane, where 94% of soil samples were B. pseudomallei positive with a geometric mean concentration of 464 CFU/g soil (95% confidence interval, 372 to 579 CFU/g soil; range, 25 to 10,850 CFU/g soil). At one site in northwest Laos (Luangnamtha), only one sample (1%) was positive for B. pseudomallei, at a concentration of 80 CFU/g soil. Therefore, B. pseudomallei occurs in Lao soils beyond the immediate vicinity of the Mekong River, alerting physicians to the likelihood of melioidosis in these areas. Further studies are needed to investigate potential climatic, soil, and biological determinants of this heterogeneity. PMID:21075883
Rattanavong, Sayaphet; Wuthiekanun, Vanaporn; Langla, Sayan; Amornchai, Premjit; Sirisouk, Joy; Phetsouvanh, Rattanaphone; Moore, Catrin E; Peacock, Sharon J; Buisson, Yves; Newton, Paul N
Burkholderia pseudomallei is a Gram-negative soil bacillus that is the etiological agent of melioidosis and a biothreat agent. Little is known about the biogeography of this bacterium in Australia, despite its hyperendemicity in the northern region of this continent. The population structure of 953 Australian B. pseudomallei strains representing 779 and 174 isolates of clinical and environmental origins, respectively, was analyzed using multilocus sequence typing (MLST). Bayesian population structure and network SplitsTree analyses were performed on concatenated MLST loci, and sequence type (ST) diversity and evenness were examined using Simpson's and Pielou's indices and a multivariate dissimilarity matrix. Bayesian analysis found two B. pseudomallei populations in Australia that were geographically distinct; isolates from the Northern Territory were grouped mainly into the first population, whereas the majority of isolates from Queensland were grouped in a second population. Differences in ST evenness were observed between sampling areas, confirming that B. pseudomallei is widespread and established across northern Australia, with a large number of fragmented habitats. ST analysis showed that B. pseudomallei populations diversified as the sampling area increased. This observation was in contrast to smaller sampling areas where a few STs predominated, suggesting that B. pseudomallei populations are ecologically established and not frequently dispersed. Interestingly, there was no identifiable ST bias between clinical and environmental isolates, suggesting the potential for all culturable B. pseudomallei isolates to cause disease. Our findings have important implications for understanding the ecology of B. pseudomallei in Australia and for potential source attribution of this bacterium in the event of unexpected cases of melioidosis. PMID:24657869
McRobb, Evan; Kaestli, Mirjam; Price, Erin P; Sarovich, Derek S; Mayo, Mark; Warner, Jeffrey; Spratt, Brian G; Currie, Bart J
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.
Background Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. There is no vaccine towards the bacterium available in the market, and the efficacy of many of the bacterium's surface and secreted proteins are currently being evaluated as vaccine candidates. Methodology/Principal Findings With the availability of the B. pseudomallei whole genome sequence, we undertook to identify genes encoding the known immunogenic outer membrane protein A (OmpA). Twelve OmpA domains were identified and ORFs containing these domains were fully annotated. Of the 12 ORFs, two of these OmpAs, Omp3 and Omp7, were successfully cloned, expressed as soluble protein and purified. Both proteins were recognised by antibodies in melioidosis patients' sera by Western blot analysis. Purified soluble fractions of Omp3 and Omp7 were assessed for their ability to protect BALB/c mice against B. pseudomallei infection. Mice were immunised with either Omp3 or Omp7, subsequently challenged with 1×106 colony forming units (cfu) of B. pseudomallei via the intraperitoneal route, and examined daily for 21 days post-challenge. This pilot study has demonstrated that whilst all control unimmunised mice died by day 9 post-challenge, two mice (out of 4) from both immunised groups survived beyond 21 days post-infection. Conclusions/Significance We have demonstrated that B. pseudomallei OmpA proteins are immunogenic in mice as well as melioidosis patients and should be further assessed as potential vaccine candidates against B. pseudomallei infection.
Hara, Yuka; Mohamed, Rahmah; Nathan, Sheila
Burkholderia pseudomallei is the causative agent of melioidosis, a rare disease of biodefense concern with high mortality and extreme difficulty in treatment. No human vaccines are available that protect against B. pseudomallei infection, and with the current limitations of antibiotic treatment, the development of new preventative and therapeutic interventions is crucial. Although clinical trials could be used to test the efficacy of new medical countermeasures (MCMs), the high mortality rates associated with melioidosis raises significant ethical issues concerning treating individuals with new compounds with unknown efficacies. The US Food and Drug Administration (FDA) has formulated a set of guidelines for the licensure of new MCMs to treat diseases in which it would be unethical to test the efficacy of these drugs in humans. The FDA “Animal Rule” 21 CFR 314 calls for consistent, well-characterized B. pseudomallei strains to be used as challenge material in animal models. In order to facilitate the efficacy testing of new MCMs for melioidosis using animal models, we intend to develop a well-characterized panel of strains for use. This panel will comprise of strains that were isolated from human cases, have a low passage history, are virulent in animal models, and are well-characterized phenotypically and genotypically. We have reviewed published and unpublished data on various B. pseudomallei strains to establish an objective method for selecting the strains to be included in the panel of B. pseudomallei strains with attention to five categories: animal infection models, genetic characterization, clinical and passage history, and availability of the strain to the research community. We identified 109 strains with data in at least one of the five categories, scored each strain based on the gathered data and identified six strains as candidate for a B. pseudomallei strain panel.
Van Zandt, Kristopher E.; Tuanyok, Apichai; Keim, Paul S.; Warren, Richard L.; Gelhaus, H. Carl
Background Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. Much remains to be known about the contributions of genotypic variations within the bacteria and the host, and environmental factors that lead to the manifestation of the clinical symptoms of melioidosis. Methodology/Principal Findings In this study, we showed that different isolates of B. pseudomallei have divergent ability to kill the soil nematode Caenorhabditis elegans. The rate of nematode killing was also dependent on growth media: B. pseudomallei grown on peptone-glucose media killed C. elegans more rapidly than bacteria grown on the nematode growth media. Filter and bacteria cell-free culture filtrate assays demonstrated that the extent of killing observed is significantly less than that observed in the direct killing assay. Additionally, we showed that B. pseudomallei does not persistently accumulate within the C. elegans gut as brief exposure to B. pseudomallei is not sufficient for C. elegans infection. Conclusions/Significance A combination of genetic and environmental factors affects virulence. In addition, we have also demonstrated that a Burkholderia-specific mechanism mediating the pathogenic effect in C. elegans requires proliferating B. pseudomallei to continuously produce toxins to mediate complete killing.
Lee, Song-Hua; Ooi, Soon-Keat; Mahadi, Nor Muhammad; Tan, Man-Wah; Nathan, Sheila
The Gram-negative bacterium Burkholderia pseudomallei is the aetiological agent of melioidosis, which is an endemic disease in tropical areas of Southeast Asia and Northern Australia. Burkholderia pseudomallei has intrinsic resistance to a number of commonly used antibiotics and has also been reported to develop a biofilm. Resistance to killing by antimicrobial agents is one of the hallmarks of bacteria grown in biofilm. The aim of this study was to determine the antimicrobial activity and mechanisms of action of LL-37 and its truncated variants against B. pseudomallei both in planktonic and biofilm form, as LL-37 is an antimicrobial peptide that possessed strong killing activity against several pathogens. Antimicrobial assays revealed that LL-31, a truncated variant of LL-37 lacking the six C-terminus residues, exhibited the strongest killing effect. Time-kill experiments showed that 20 ?M LL-31 can reach the bactericidal endpoint within 2h. Freeze-fracture electron microscopy of bacterial cells demonstrated that these peptides disrupt the membrane and cause leakage of intracellular molecules leading to cell death. Moreover, LL-31 also possessed stronger bactericidal activity than ceftazidime against B. pseudomallei grown in biofilm. Thus, LL-31 should be considered as a potent antimicrobial agent against B. pseudomallei both in planktonic and biofilm form. PMID:22005071
Kanthawong, Sakawrat; Bolscher, Jan G M; Veerman, Enno C I; van Marle, Jan; de Soet, Hans J J; Nazmi, Kamran; Wongratanacheewin, Surasakdi; Taweechaisupapong, Suwimol
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
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.
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
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.
Baker, Anthony; Mayo, Mark; Owens, Leigh; Burgess, Graham; Norton, Robert; McBride, William John Hannan; Currie, Bart J.
Background Burkholderia pseudomallei (Bps) is a Gram-negative bacterium that causes frequently lethal melioidosis, with a particularly high prevalence in the north and northeast of Thailand. Bps is highly resistant to many antimicrobial agents and this resistance may result from the low drug permeability of outer membrane proteins, known as porins. Principal Findings Microbiological assays showed that the clinical Bps strain was resistant to most antimicrobial agents and sensitive only to ceftazidime and meropenem. An E. coli strain defective in most porins, but expressing BpsOmp38, exhibited considerably lower antimicrobial susceptibility than the control strain. In addition, mutation of Tyr119, the most prominent pore-lining residue in BpsOmp38, markedly altered membrane permeability, substitution with Ala (mutant BpsOmp38Y119A) enhanced uptake of the antimicrobial agents, while substitution with Phe (mutant BpsOmp38Y119F) inhibited uptake. Channel recordings of BpsOmp38 reconstituted in a planar black lipid membrane (BLM) suggested that the higher permeability of BpsOmp38Y119A was caused by widening of the pore interior through removal of the bulky side chain. In contrast, the lower permeability of BpsOmp38Y119F was caused by introduction of the hydrophobic side chain (Phe), increasing the ‘greasiness’ of the pore lumen. Significantly, liposome swelling assays showed no permeation through the BpsOmp38 channel by antimicrobial agents to which Bps is resistant (cefoxitin, cefepime, and doripenem). In contrast, high permeability to ceftazidime and meropenem was observed, these being agents to which Bps is sensitive. Conclusion/Significance Our results, from both in vivo and in vitro studies, demonstrate that membrane permeability associated with BpsOmp38 expression correlates well with the antimicrobial susceptibility of the virulent bacterium B. pseudomallei, especially to carbapenems and cephalosporins. In addition, substitution of the residue Tyr119 affects the permeability of the BpsOmp38 channel to neutral sugars and antimicrobial agents.
Aunkham, Anuwat; Schulte, Albert; Winterhalter, Mathias; Suginta, Wipa
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
The identification of Burkholderia pseudomallei, the causative agent of melioidosis, is usually not difficult in laboratories in areas where it is endemic. With the increase in international travel and the threat of bioterrorism, it has become more likely that laboratories in areas where it is not endemic could encounter this organism. The increase in the use of and dependence upon automated identification systems makes accurate 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 B. pseudomallei was largely due to differences in the biochemical reactions achieved compared to expected values in the database. It is suggested that this deficiency in the Vitek 2 may be due to the large number of uncertain results reported for these isolates. These results reduce the discriminating ability of the instrument to distinguish between uncommonly encountered isolates such as those of B. pseudomallei.
Lowe, Peter; Engler, Catherine; Norton, Robert
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
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.
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.
Tandhavanant, Sarunporn; Wongsuvan, Gumphol; Wuthiekanun, Vanaporn; Teerawattanasook, Nittaya; Day, Nicholas P. J.; Limmathurotsakul, Direk; Peacock, Sharon J.; Chantratita, Narisara
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
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
Burkholderia pseudomallei and B. mallei, the causative agents of melioidosis and glanders, respectively, are designated category B biothreat agents. Current methods for identifying these organisms rely on their phenotypic characteristics and an extensive set of biochemical reactions. We evaluated the use of 16S rRNA gene sequencing to rapidly identify these two species and differentiate them from each other as well as from closely related species and genera such as Pandoraea spp., Ralstonia spp., Burkholderia gladioli, Burkholderia cepacia, Burkholderia thailandensis, and Pseudomonas aeruginosa. We sequenced the 1.5-kb 16S rRNA gene of 56 B. pseudomallei and 23 B. mallei isolates selected to represent a wide range of temporal, geographic, and origin diversity. Among all 79 isolates, a total of 11 16S types were found based on eight positions of difference. Nine 16S types were identified in B. pseudomallei isolates based on six positions of difference, with differences ranging from 0.5 to 1.5 bp. Twenty-two of 23 B. mallei isolates showed 16S rRNA gene sequence identity and were designated 16S type 10, whereas the remaining isolate was designated type 11. This report provides a basis for rapidly identifying and differentiating B. pseudomallei and B. mallei by molecular methods. PMID:14532197
Gee, Jay E; Sacchi, Claudio T; Glass, Mindy B; De, Barun K; Weyant, Robbin S; Levett, Paul N; Whitney, Anne M; Hoffmaster, Alex R; Popovic, Tanja
Burkholderia pseudomallei, the etiologic agent of melioidosis, is responsible for a broad spectrum of illnesses in humans and animals particularly in Southeast Asia and northern Australia, where it is endemic. Burkholderia thailandensis is a nonpathogenic environmental organism closely related to B. pseudomallei. Subtractive hybridization was carried out between these two species to identify genes encoding virulence determinants in B. pseudomallei. Screening of the subtraction library revealed A-T-rich DNA sequences unique to B. pseudomallei, suggesting they may have been acquired by horizontal transfer. One of the subtraction clones, pDD1015, encoded a protein with homology to a glycosyltransferase from Pseudomonas aeruginosa. This gene was insertionally inactivated in wild-type B. pseudomallei to create SR1015. It was determined by enzyme-linked immunosorbent assay and immunoelectron microscopy that the inactivated gene was involved in the production of a major surface polysaccharide. The 50% lethal dose (LD50) for wild-type B. pseudomallei is <10 CFU; the LD50 for SR1015 was determined to be 3.5 × 105 CFU, similar to that of B. thailandensis (6.8 × 105 CFU). DNA sequencing of the region flanking the glycosyltransferase gene revealed open reading frames similar to capsular polysaccharide genes in Haemophilus influenzae, Escherichia coli, and Neisseria meningitidis. In addition, DNA from Burkholderia mallei and Burkholderia stabilis hybridized to a glycosyltransferase fragment probe, and a capsular structure was identified on the surface of B. stabilis via immunoelectron microscopy. Thus, the combination of PCR-based subtractive hybridization, insertional inactivation, and animal virulence studies has facilitated the identification of an important virulence determinant in B. pseudomallei.
Reckseidler, Shauna L.; DeShazer, David; Sokol, Pamela A.; Woods, Donald E.
The major predisposing factor for melioidosis is diabetes mellitus, but no immunological mechanisms have been investigated to explain this. In this study, polymorphonuclear neutrophil (PMN) responses to Burkhold- eria pseudomallei, the causative agent of melioidosis, in healthy and diabetic Thai subjects were determined by flow cytometry. The results showed that B. pseudomallei displayed reduced uptake by PMNs compared to Salmonella
Sujin Chanchamroen; Chidchamai Kewcharoenwong; Wattanachai Susaengrat; Manabu Ato; Ganjana Lertmemongkolchai
Understanding the way in which the immune system responds to infection is central to the development of vaccines and many diagnostics. To provide insight into this area, we fabricated a protein microarray containing 1,205 Burkholderia pseudomallei proteins, probed it with 88 melioidosis patient sera, and identified 170 reactive antigens. This subset of antigens was printed on a smaller array and probed with a collection of 747 individual sera derived from 10 patient groups including melioidosis patients from Northeast Thailand and Singapore, patients with different infections, healthy individuals from the USA, and from endemic and nonendemic regions of Thailand. We identified 49 antigens that are significantly more reactive in melioidosis patients than healthy people and patients with other types of bacterial infections. We also identified 59 cross-reactive antigens that are equally reactive among all groups, including healthy controls from the USA. Using these results we were able to devise a test that can classify melioidosis positive and negative individuals with sensitivity and specificity of 95% and 83%, respectively, a significant improvement over currently available diagnostic assays. Half of the reactive antigens contained a predicted signal peptide sequence and were classified as outer membrane, surface structures or secreted molecules, and an additional 20% were associated with pathogenicity, adaptation or chaperones. These results show that microarrays allow a more comprehensive analysis of the immune response on an antigen-specific, patient-specific, and population-specific basis, can identify serodiagnostic antigens, and contribute to a more detailed understanding of immunogenicity to this pathogen.
Felgner, Philip L.; Kayala, Matthew A.; Vigil, Adam; Burk, Chad; Nakajima-Sasaki, Rie; Pablo, Jozelyn; Molina, Douglas M.; Hirst, Siddiqua; Chew, Janet S. W.; Wang, Dongling; Tan, Gladys; Duffield, Melanie; Yang, Ron; Neel, Julien; Chantratita, Narisara; Bancroft, Greg; Lertmemongkolchai, Ganjana; Davies, D. Huw; Baldi, Pierre; Peacock, Sharon; Titball, Richard W.
Background Burkholderia pseudomallei are the causative agent of melioidosis. Increasing resistance of the disease to antibiotics is a severe problem in treatment regime and has led to intensification of the search for new drugs. Antimicrobial peptides are the most ubiquitous in nature as part of the innate immune system and host defense mechanism. Methods Here, we investigated a group of venoms (snakes, scorpions and honey bee venoms) for antimicrobial properties against two strains of Gram-negative bacteria Burkholderia pseudomallei by using disc-diffusion assay for in vitro susceptibility testing. The antibacterial activities of the venoms were compared with that of the isolated L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2s) enzymes. MICs were determined using broth dilution method. Bacterial growth was assessed by measurement of optical density at the lowest dilutions (MIC 0.25 mg/ml). The cell viability was measured using tetrazolium salts (XTT) based cytotoxic assay. Results The studied venoms showed high antimicrobial activity. The venoms of C. adamanteus, Daboia russelli russelli, A. halys, P. australis, B. candidus and P. guttata were equally as effective as Chloramphenicol and Ceftazidime (30 ?g/disc). Among those tested, phospholipase A2 enzymes (crotoxin B and daboiatoxin) showed the most potent antibacterial activity against Gram-negative (TES) bacteria. Naturally occurring venom peptides and phospholipase A2 proved to possess highly potent antimicrobial activity against Burkholderia pseudomallei. The XTT-assay results showed that the cell survival decreased with increasing concentrations (0.05–10 mg/mL) of Crotalus adamanteus venom, with no effect on the cell viability evident at 0.5 mg/mL. Conclusion This antibacterial profile of snake venoms reported herein will be useful in the search for potential antibacterial agents against drug resistant microorganisms like B. pseudomallei.
Perumal Samy, R; Pachiappan, A; Gopalakrishnakone, P; Thwin, Maung M; Hian, Yap E; Chow, Vincent TK; Bow, Ho; Weng, Joseph T
Burkholderia pseudomallei and its host-adapted deletion clone Burkholderia mallei, cause the potentially fatal human diseases melioidosis and glanders, respectively. Their antibiotic resistance profile, ability to infect via aerosol, and the absence of pr...
D. DeShazer L. Lipscomb M. A. Schell
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
The autotransporters are a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play a role in numerous environmental and virulence-associated interactions. As part of a larger systematic study on the autotransporters of Burkholderia pseudomallei, the causative agent of the severe tropical disease melioidosis, we have constructed an insertion mutant in the bpss1439 gene encoding an unstudied predicted trimeric autotransporter adhesin. The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain. This phenotype was complemented to wild-type levels by the introduction of a full-length copy of the bpss1439 gene in trans. Examination of the wild-type and bpss1439 mutant strains under biofilm-inducing conditions by microscopy after 48 hours confirmed that the bpss1439 mutant produced less biofilm compared to wild-type. Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation. In a murine melioidosis model, the bpss1439 mutant strain demonstrated a moderate attenuation for virulence compared to the wild-type strain. This attenuation was abrogated by in trans complementation, suggesting that bpss1439 plays a subtle role in the pathogenesis of B. pseudomallei. Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A.
Lazar Adler, Natalie R.; Dean, Rachel E.; Saint, Richard J.; Stevens, Mark P.; Prior, Joann L.; Atkins, Timothy P.; Galyov, Edouard E.
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.
A Burkholderia pseudomallei-like organism has recently been identified among some soil isolates of B. pseudomallei in an area with endemic melioidosis. This organism is almost identical to B. pseudomallei in terms of morphological and biochemical profiles, except that it differs in ability to assimilate l-arabinose. These Ara+ isolates are also less virulent than the Ara? isolates in animal models. In addition, clinical isolates of B. pseudomallei available to date are almost exclusively Ara?. These features suggested that these two organisms may belong to distinctive species. In this study, the 16S rRNA-encoding genes from five clinical (four Ara? and one Ara+) and nine soil isolates (five Ara? and four Ara+) of B. pseudomallei were sequenced. The nucleotide sequences and phylogenetic analysis indicated that the 16S rRNA-encoding gene of the Ara+ biotype was similar to but distinctively different from that of the Ara? soil isolates, which were identical to the classical clinical isolates of B. pseudomallei. The nucleotide sequence differences in the 16S rRNA-encoding gene appeared to be specific for the Ara+ or Ara? biotypes. The differences were, however, not sufficient for classification into a new species within the genus Burkholderia. A simple and rapid multiplex PCR procedure was developed to discriminate between Ara? and Ara+ B. pseudomallei isolates. This new method could also be incorporated into our previously reported nested PCR system for detecting B. pseudomallei in clinical specimens.
Dharakul, Tararaj; Tassaneetrithep, Boonratn; Trakulsomboon, Suwanna; Songsivilai, Sirirurg
Burkholderia pseudomallei, the causative agent of melioidosis, contains a large pathogen genome (7.2 Mb) with ?2,000 genes of putative or unknown function. Interactions with potential hosts and environmental factors may induce rapid adaptations in these B. pseudomallei genes, which can be discerned through evolutionary analysis of multiple B. pseudomallei genomes. Here we show that several previously uncharacterized B. pseudomallei genes bearing genetic signatures of rapid adaptation (positive selection) can induce diverse cellular phenotypes when expressed in mammalian cells. Notably, several of these phenotypes are plausibly related to virulence, including multinuclear giant cell formation, apoptosis, and autophagy induction. Specifically, we show that BPSS0180, a type VI cluster-associated gene, is capable of inducing autophagy in both phagocytic and nonphagocytic mammalian cells. Following infection of macrophages, a B. pseudomallei mutant disrupted in BPSS0180 exhibited significantly decreased colocalization with LC3 and impaired intracellular survival; these phenotypes were rescued by introduction of an intact BPSS0180 gene. The results suggest that BPSS0180 may be a novel inducer of host cell autophagy that contributes to B. pseudomallei intracellular growth. More generally, our study highlights the utility of applying evolutionary principles to microbial genomes to identify novel virulence genes.
Singh, Arvind Pratap; Lai, Shu-chin; Nandi, Tannistha; Chua, Hui Hoon; Ooi, Wen Fong; Ong, Catherine; Boyce, John D.; Adler, Ben
Burkholderia pseudomallei, the causative agent of melioidosis, is an important intracellular pathogen in tropical regions. TANK-binding kinase (TBK1), part of the pathway that induces transcription of Type I interferon genes, has been demonstrated to play an important role in controlling intracellular bacterial infections. To investigate the role of tbk1 in protecting against B. pseudomallei we developed tbk1-deficient cell lines by using shRNA for transient knockdown of the tbk1 gene in HeLa and RAW 264.7 cells. In tbk1-deficient RAW cells, the replication of invasive and non-invasive Escherichia coli was significantly increased at 48 h after infection compared with wild-type cells. The result was confirmed using Brucella melitensis in tbk1-deficient HeLa cells, which demonstrated a >1.5-2.0 log higher bacterial count at 6-48 h after infection compared to wild-type cells. By contrast, the growth of Burkholderia pseudomallei expressing either typical (A2) or atypical (G207) lipopolysaccharide was not significantly different between the tbk1-deficient and control cells. These results suggest that the tbk1 gene and its activation may be able to control invasive E. coli, non-invasive E. coli and B. melitensis growth but may not be able to control B. pseudomallei infection. The role of the tbk1 gene in proinflammatory cytokine induction and bacterial intracellular infection needs further investigation to identify mechanistic differences among the life cycles of various intracellular bacteria. PMID:19121697
Panomket, Pawana; Splitter, Gary; Harms, Jerome; Sermswan, Rasana W; Chedchotisakd, Pleonchan; Wongratanacheewin, Surasakdi
Background Burkholderia pseudomallei is the causative agent of melioidosis where the highest reported incidence world wide is in the Northeast of Thailand, where saline soil and water are prevalent. Moreover, recent reports indicate a potential pathogenic role for B. pseudomallei in cystic fibrosis lung disease, where an increased sodium chloride (NaCl) concentration in airway surface liquid has been proposed. These observations raise the possibility that high salinity may represent a favorable niche for B. pseudomallei. We therefore investigated the global transcriptional response of B. pseudomallei to increased salinity using microarray analysis. Results Transcriptome analysis of B. pseudomallei under salt stress revealed several genes significantly up-regulated in the presence of 320 mM NaCl including genes associated with the bsa-derived Type III secretion system (T3SS). Microarray data were verified by reverse transcriptase-polymerase chain reactions (RT-PCR). Western blot analysis confirmed the increased expression and secretion of the invasion-associated type III secreted proteins BipD and BopE in B. pseudomallei cultures at 170 and 320 mM NaCl relative to salt-free medium. Furthermore, salt-treated B. pseudomallei exhibited greater invasion efficiency into the lung epithelial cell line A549 in a manner partly dependent on a functional Bsa system. Conclusions B. pseudomallei responds to salt stress by modulating the transcription of a relatively small set of genes, among which is the bsa locus associated with invasion and virulence. Expression and secretion of Bsa-secreted proteins was elevated in the presence of exogenous salt and the invasion efficiency was enhanced. Our data indicate that salinity has the potential to influence the virulence of B. pseudomallei.
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
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
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.
Burkholderia pseudomallei is an opportunistic pathogen and the causative agent of melioidosis. It is able to adapt to harsh environments and can live intracellularly in its infected hosts. In this study, identification of transcriptional factors that associate with the ?? subunit (RpoC) of RNA polymerase was performed. The N-terminal region of this subunit is known to trigger promoter melting when associated with a sigma factor. A pull-down assay using histidine-tagged B. pseudomallei RpoC N-terminal region as bait showed that a hypothetical protein BPSS1356 was one of the proteins bound. This hypothetical protein is conserved in all B. pseudomallei strains and present only in the Burkholderia genus. A BPSS1356 deletion mutant was generated to investigate its biological function. The mutant strain exhibited reduced biofilm formation and a lower cell density during the stationary phase of growth in LB medium. Electron microscopic analysis revealed that the ?BPSS1356 mutant cells had a shrunken cytoplasm indicative of cell plasmolysis and a rougher surface when compared to the wild type. An RNA microarray result showed that a total of 63 genes were transcriptionally affected by the BPSS1356 deletion with fold change values of higher than 4. The expression of a group of genes encoding membrane located transporters was concurrently down-regulated in ?BPSS1356 mutant. Amongst the affected genes, the putative ion transportation genes were the most severely suppressed. Deprivation of BPSS1356 also down-regulated the transcriptions of genes for the arginine deiminase system, glycerol metabolism, type III secretion system cluster 2, cytochrome bd oxidase and arsenic resistance. It is therefore obvious that BPSS1356 plays a multiple regulatory roles on many genes.
Yam, Hokchai; Abdul Rahim, Ainihayati; Mohamad, Suriani; Mahadi, Nor Muhammad; Abdul Manaf, Uyub; Shu-Chien, Alexander Chong; Najimudin, Nazalan
Molecular typing methods for Burkholderia pseudomallei have been successful at assigning isolates into epidemiologically related groups, but have not been able to detect differences and define evolutionary patterns within groups. Our variable number tandem repeat (VNTR) analysis of a set of 121 Australian B. pseudomallei isolates, 104 of which were associated with nine epidemiological groups, provides fine scale differentiation even among very closely related isolates. We used a Bayesian model based upon mutation accumulation patterns to define the close phylogenetic relationships within these epidemiological groups. Our results reveal that genetic diversity can exist within a very small geographic area, and that low levels of diversity can exist even within a single infection. These methods provide the ability to generate robust evolutionary hypotheses that enable tracking of B. pseudomallei in forensic and epidemiological outbreaks at fine phylogenetic scales. PMID:17258514
Pearson, Talima; U'Ren, Jana M; Schupp, James M; Allan, Gerard J; Foster, Peter G; Mayo, Mark J; Gal, Daniel; Choy, Jodie Low; Daugherty, Rebecca Leadem; Kachur, Sergey; Friedman, Christine L Clark; Leadem, Benjamin; Georgia, Shalamar; Hornstra, Heidie; Vogler, Amy J; Wagner, David M; Keim, Paul; Currie, Bart J
Two mechanics working at a garage in tropical northern Australia simultaneously developed upper limb melioidosis ulcers. Both patients had Burkholderia pseudomallei of identical pulsed-field gel electrophoresis (PFGE) type (Spe I). Environmental sampling identified B. pseudomallei in a container of commercial hand wash detergent as the likely source of infection, although there were multiple isolates of different PFGE types to the
DANIEL GAL; MARK MAYO; HEIDI SMITH-VAUGHAN; PALLAVE DASARI; MELITA MCKINNON; SUSAN P. JACUPS; ANDREW I. URQUHART; MARILYN HASSELL; BART J. CURRIE
Burkholderia pseudomallei, a Gram-negative saprophytic bacterium, is the causative agent of the potentially fatal melioidosis disease in humans. In this study, environmental parameters including temperature, nutrient content, pH and the presence of glucose were shown to play a role in in vitro biofilm formation by 28 B. pseudomallei clinical isolates, including four isolates with large colony variants (LCVs) and small colony variants (SCVs) morphotypes. Enhanced biofilm formation was observed when the isolates were tested in LB medium, at 30 °C, at pH 7.2, and in the presence of as little as 2 mM glucose respectively. It was also shown that all SVCs displayed significantly greater capacity to form biofilms than the corresponding LCVs when cultured in LB at 37 °C. In addition, octanoyl-homoserine lactone (C(8)-HSL), a quorum sensing molecule, was identified by mass spectrometry analysis in bacterial isolates referred to as LCV CTH, LCV VIT, SCV TOM, SCV CTH, 1 and 3, and the presence of other AHL's with higher masses; decanoyl-homoserine lactone (C(10)-HSL) and dodecanoyl-homoserine lactone (C(12)-HSL) were also found in all tested strain in this study. Last but not least, we had successfully acquired two Bacillus sp. soil isolates, termed KW and SA respectively, which possessed strong AHLs degradation activity. Biofilm formation of B. pseudomallei isolates was significantly decreased after treated with culture supernatants of KW and SA strains, demonstrating that AHLs may play a role in B. pseudomallei biofilm formation. PMID:22970167
Ramli, Nur Siti K; Eng Guan, Chua; Nathan, Sheila; Vadivelu, Jamuna
Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (CT) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×104 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.
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.
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
Natural isolates of pathogenic bacteria can exhibit a broad range of phenotypic traits. To investigate the molecular mechanisms contributing to such phenotypic variability, we compared the genomes, transcriptomes, and proteomes of two natural isolates of the gram-negative bacterium Burkholderia pseudomallei, the causative agent of the human disease melioidosis. Significant intrinsic genomic, transcriptional, and proteomic variations were observed between the two strains involving genes of diverse functions. We identified 16 strain-specific regions in the B. pseudomallei K96243 reference genome, and for eight regions their differential presence could be ascribed to either DNA acquisition or loss. A remarkable 43% of the transcriptional differences between the strains could be attributed to genes that were differentially present between K96243 and Bp15682, demonstrating the importance of lateral gene transfer or gene loss events in contributing to pathogen diversity at the gene expression level. Proteins expressed in a strain-specific manner were similarly correlated at the gene expression level, but up to 38% of the global proteomic variation between strains comprised proteins expressed in both strains but associated with strain-specific protein isoforms. Collectively, >65 hypothetical genes were transcriptionally or proteomically expressed, supporting their bona fide biological presence. Our results provide, for the first time, an integrated framework for classifying the repertoire of natural variations existing at distinct molecular levels for an important human pathogen.
Ou, Keli; Ong, Catherine; Koh, Shze Yung; Rodrigues, Fiona; Sim, Siew Hoon; Wong, Daniel; Ooi, Chia Huey; Ng, Kim Chong; Jikuya, Hiroyuki; Yau, Chin Chin; Soon, Sou Yen; Kesuma, Djohan; Lee, May Ann; Tan, Patrick
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.
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.
Burkholderia pseudomallei is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a lacZ? gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed gusA indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or sacB-based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an araC-PBAD-I-sceI expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved ?asd Escherichia coli mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI- and sacB-based methods afforded by pEXKm5 in conjunction with E. coli RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory B. pseudomallei strains. Finally, sacB-based counterselection was employed to isolate a defined chromosomal fabD(Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme.
Lopez, Carolina M.; Rholl, Drew A.; Trunck, Lily A.; Schweizer, Herbert P.
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.
Phophetleb, Onanong; Nasompag, Sawinee; Thammasirirak, Sompong; Daduang, Sakda; Taweechaisupapong, Suwimol; Lomize, Andrei L.; Patramanon, Rina
Burkholderia pseudomallei is a Gram-negative rod and the causative agent of melioidosis, an emerging infectious disease of tropical and subtropical areas worldwide. B. pseudomallei harbors a remarkable number of virulence factors, including six type VI secretion systems (T6SS). Using our previously described plaque assay screening system, we identified a B. pseudomallei transposon mutant defective in the BPSS1504 gene that showed reduced plaque formation. The BPSS1504 locus is encoded within T6SS cluster 1 (T6SS1), which is known to be involved in the pathogenesis of B. pseudomallei in mammalian hosts. For further analysis, a B. pseudomallei BPSS1504 deletion (Bp?BPSS1504) mutant and complemented mutant strain were constructed. B. pseudomallei lacking the BPSS1504 gene was highly attenuated in BALB/c mice, whereas the in vivo virulence of the complemented mutant strain was fully restored to the wild-type level. The Bp?BPSS1504 mutant showed impaired intracellular replication and formation of multinucleated giant cells in macrophages compared with wild-type bacteria, whereas the induction of actin tail formation within host cells was not affected. These observations resembled the phenotype of a mutant lacking hcp1, which is an integral component of the T6SS1 apparatus and is associated with full functionality of the T6SS1. Transcriptional expression of the T6SS components vgrG, tssA, and hcp1, as well as the T6SS regulators virAG, bprC, and bsaN, was not dependent on BPSS1504 expression. However, secretion of Hcp1 was not detectable in the absence of BPSS1504. Thus, BPSS1504 seems to serve as a T6SS component that affects Hcp1 secretion and is therefore involved in the integrity of the T6SS1 apparatus. PMID:24595140
Hopf, Verena; Göhler, André; Eske-Pogodda, Kristin; Bast, Antje; Steinmetz, Ivo; Breitbach, Katrin
Burkholderia pseudomallei, the etiologic agent of melioidosis, is an opportunistic pathogen that harbors a wide array of secretion systems, including a type II secretion system (T2SS), three type III secretion systems (T3SS), and six type VI secretion systems (T6SS). The proteins exported by these systems provide B. pseudomallei with a growth advantage in vitro and in vivo, but relatively little is known about the full repertoire of exoproducts associated with each system. In this study, we constructed deletion mutations in gspD and gspE, T2SS genes encoding an outer membrane secretin and a cytoplasmic ATPase, respectively. The secretion profiles of B. pseudomallei MSHR668 and its T2SS mutants were noticeably different when analyzed by SDS-PAGE. We utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins present in the supernatants of B. pseudomallei MSHR668 and B. pseudomallei ?gspD grown in rich and minimal media. The MSHR668 supernatants contained 48 proteins that were either absent or substantially reduced in the supernatants of ?gspD strains. Many of these proteins were putative hydrolytic enzymes, including 12 proteases, two phospholipases, and a chitinase. Biochemical assays validated the LC-MS/MS results and demonstrated that the export of protease, phospholipase C, and chitinase activities is T2SS dependent. Previous studies had failed to identify the mechanism of secretion of TssM, a deubiquitinase that plays an integral role in regulating the innate immune response. Here we present evidence that TssM harbors an atypical signal sequence and that its secretion is mediated by the T2SS. This study provides the first in-depth characterization of the B. pseudomallei T2SS secretome. PMID:24866793
Burtnick, Mary N; Brett, Paul J; DeShazer, David
The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1? and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1? secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1?, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of B. pseudomallei infection.
Schmidt, Imke H. E.; Pudla, Matsayapan; Brakopp, Stefanie; Hopf, Verena; Breitbach, Katrin; Steinmetz, Ivo
The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1? and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1? secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1?, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of B. pseudomallei infection. PMID:24626296
Bast, Antje; Krause, Kathrin; Schmidt, Imke H E; Pudla, Matsayapan; Brakopp, Stefanie; Hopf, Verena; Breitbach, Katrin; Steinmetz, Ivo
In the tropical city of Darwin, Northern Territory, Australia, dry season soil sampling cultured Burkholderia pseudomallei from 7 (70%) of 10 sports fields. However, during the 23 years of the Darwin Prospective Melioidosis Study, only 5 (0.6%) of 785 melioidosis cases have been attributed to infection from sports fields. In one soccer player with cutaneous melioidosis, B. pseudomallei cultured from the player was identical by multilocus sequence typing and multilocus variable-number tandem repeat analysis with an isolate recovered from soil at the location on the sports field where he was injured. Melioidosis is uncommon in otherwise healthy sports persons in melioidosis-endemic regions but still needs consideration in persons with abrasion injuries that involve contact with soil. PMID:23732257
Hill, Audrey A; Mayo, Mark; Kaestli, Mirjam; Price, Erin P; Richardson, Leisha J; Godoy, Daniel; Spratt, Brian G; Currie, Bart J
Burkholderia pseudomallei is a bacterium endemic to Southeast Asia and northern Australia, but it has not been found to occur endemically in the United States. We report an ostensibly autochthonous case of melioidosis in the United States. Despite an extensive investigation, the source of exposure was not identified.
Engelthaler, David M.; Blaney, David D.; Tuanyok, Apichai; Wangsness, Eric; Smith, Theresa L.; Pearson, Talima; Komatsu, Kenneth K.; Keim, Paul; Currie, Bart J.; Levy, Craig; Sunenshine, Rebecca
Trimethoprim-sulfamethoxazole (co-trimoxazole) is the primary drug used for oral eradication therapy of Burkholderia pseudomallei infections (melioidosis). Here, we demonstrate that trimethoprim resistance is widespread in clinical and environmental isolates from northeast Thailand and northern Australia. This resistance was shown to be due to BpeEF-OprC efflux pump expression. No dihydrofolate reductase target mutations were involved, although frequent insertion of ISBma2 was noted within the putative folA transcriptional terminator. All isolates tested remained susceptible to trimethoprim-sulfamethoxazole, suggesting that resistance to trimethoprim alone in these strains probably does not affect the efficacy of co-trimoxazole therapy.
Podnecky, Nicole L.; Wuthiekanun, Vanaporn; Peacock, Sharon J.
Melioidosis is endemic in areas of Southeast Asia, however, there are no published reports from the Thai-Myanmar border. We report the first two documented cases of fatal melioidosis in this region. This is of great public health importance and highlights the need to both increase clinical awareness of melioidosis on the Thai-Myanmar border, and to assess the true burden of disease in the area through improved case detection and Burkholderia pseudomallei prevalence studies.
Melioidosis is endemic in areas of Southeast Asia, however, there are no published reports from the Thai-Myanmar border. We report the first two documented cases of fatal melioidosis in this region. This is of great public health importance and highlights the need to both increase clinical awareness of melioidosis on the Thai-Myanmar border, and to assess the true burden of disease in the area through improved case detection and Burkholderia pseudomallei prevalence studies. PMID:24715973
Chu, Cindy S; Winearls, Stuart; Ling, Clare; Torchinsky, Miriam Beer; Phyo, Aung Phae; Haohankunnathum, Warat; Turner, Paul; Wuthiekanun, Vanaporn; Nosten, François
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.
Three kinds of capsular polysaccharide (CP) were found to be produced by Burkholderia pseudomallei. When the bacterium was grown with the medium without glycerol, CP-1a and CP-1b were produced. CP-1a was mainly 1.4-linked glucan and CP-1b was identified as a polymer composed of galactose and 3-deoxy-D-manno-octulosonic acid, whose chemical structure was recently reported by other laboratories. When the bacterium was grown with the medium containing 5" glycerol. CP-2 was synthesized. CP-2 contained galactose, rhamnose, mannose, glucose and a uronic acid in a ratio of approximately 3:1:0.3:1:1. Methylation analysis of the purified polysaccharides demonstrated that the two acidic polysaccharides. CP-1b and CP-2 shared no common structure, indicating that CP-2 was an acidic capsular polysaccharide whose chemical characters were not reported previously. PMID:10744478
Kawahara, K; Dejsirilert, S; Ezaki, T
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
Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind Arp3.
Stevens, Joanne M.; Ulrich, Ricky L.; Taylor, Lowrie A.; Wood, Michael W.; DeShazer, David; Stevens, Mark P.; Galyov, Edouard E.
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
Background Autotransporters form a large family of outer membrane proteins specifying diverse biological traits of Gram-negative bacteria. In this study, we report the identification and characterization of a novel autotransporter gene product of Burkholderia mallei (locus tag BMA1027 in strain ATCC 23344). Results Database searches identified the gene in at least seven B. mallei isolates and the encoded proteins were found to be 84% identical. Inactivation of the gene encoding the autotransporter in the genome of strain ATCC 23344 substantially reduces adherence to monolayers of HEp-2 laryngeal cells and A549 type II pneumocytes, as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, expression of the autotransporter on the surface of recombinant E. coli bacteria increases adherence to these cell types by 5–7 fold. The gene specifying the autotransporter was identified in the genome of 29 B. pseudomallei isolates and disruption of the gene in strain DD503 reduced adherence to NHBE cultures by 61%. Unlike B. mallei, the mutation did not impair binding of B. pseudomallei to A549 or HEp-2 cells. Analysis of sera from mice infected via the aerosol route with B. mallei and B. pseudomallei revealed that animals inoculated with as few as 10 organisms produce antibodies against the autotransporter, therefore indicating expression in vivo. Conclusions Our data demonstrate that we have identified an autotransporter protein common to the pathogenic species B. mallei and B. pseudomallei which mediates adherence to respiratory epithelial cells and is expressed in vivo during the course of aerosol infection.
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
This study aimed to evaluate the in vitro combination of farnesol and ?-lactams against Burkholderia pseudomallei. A total of 12 ?-lactamase-positive strains were tested according to CLSI standards. All strains were inhibited by farnesol, with MICs ranging from 75 to 150 ?M. The combination of this compound with ?-lactams resulted in statistically significant ?-lactam MIC reduction (P ? 0.05). This study provides new perspectives for the use of farnesol combined with ?-lactam antibiotics against strains of B. pseudomallei.
Valente, L. G. A.; Rocha, M. F. G.; Bandeira, T. J. P. G.; Cordeiro, R. A.; Lima, R. A. C.; Leite, J. J. G.; Ribeiro, J. F.; Pereira, J. F.; Castelo-Branco, D. S. C. M.; Monteiro, A. J.; Sidrim, J. J. C.
Evaluation of a series of MetAP inhibitors in an in vitro enzyme activity assay led to the first identification of potent molecules that show significant growth inhibition against Burkholderia pseudomallei. Nitroxoline analogs show excellent inhibition potency in the BpMetAP1 enzyme activity assay with the lowest IC50 of 30 nM, and inhibit the growth of B. pseudomallei and B. thailandensis at concentrations ? 31 ?M.
Wangtrakuldee, Phumvadee; Byrd, Matthew S.; Campos, Cristine G.; Henderson, Michael W.; Zhang, Zheng; Clare, Michael; Masoudi, Ali; Myler, Peter J.; Horn, James R.; Cotter, Peggy A.; Hagen, Timothy J.
B. pseudomallei is a gram-negative bacterium that causes the tropical infection melioidosis. In northeast Thailand, mortality from melioidosis approaches 40%. As exemplified by the lipopolysaccharide-Toll-like receptor 4 interaction, innate immune responses to invading bacteria are precipitated by activation of host pathogen recognition receptors by pathogen associated molecular patterns. Human melioidosis is characterized by up-regulation of pathogen recognition receptors and pro-inflammatory cytokine release. In contrast to many gram-negative pathogens, however, the lipopolysaccharide of B. pseudomallei is considered only weakly inflammatory. We conducted a study in 300 healthy Thai subjects to investigate the ex vivo human blood response to various bacterial pathogen associated molecular patterns, including lipopolysaccharide from several bacteria, and to two heat-killed B. pseudomallei isolates. We measured cytokine levels after stimulation of fresh whole blood with a panel of stimuli. We found that age, sex, and white blood cell count modulate the innate immune response to B. pseudomallei. We further observed that, in comparison to other stimuli, the innate immune response to B. pseudomallei is most highly correlated with the response to lipopolysaccharide. The magnitude of cytokine responses induced by B. pseudomallei lipopolysaccharide was significantly greater than those induced by lipopolysaccharide from Escherichia coli and comparable to many responses induced by lipopolysaccharide from Salmonella minnesota despite lower amounts of lipid A in the B. pseudomallei lipopolysaccharide preparation. In human monocytes stimulated with B. pseudomallei, addition of polymyxin B or a TLR4/MD-2 neutralizing antibody inhibited the majority of TNF-? production. Challenging existing views, our data indicate that the innate immune response to B. pseudomallei in human blood is largely driven by lipopolysaccharide, and that the response to B. pseudomallei lipopolysaccharide in blood is greater than the response to other lipopolysaccharide expressing isolates. Our findings suggest that B. pseudomallei lipopolysaccharide may play a central role in stimulating the host response in melioidosis.
Chantratita, Narisara; Tandhavanant, Sarunporn; Myers, Nicolle D.; Seal, Sudeshna; Arayawichanont, Arkhom; Kliangsa-ad, Aroonsri; Hittle, Lauren E.; Ernst, Robert K.; Emond, Mary J.; Wurfel, Mark M.; Day, Nicholas P. J.; Peacock, Sharon J.; West, T. Eoin
The Gram-negative proteobacterium Burkholderia pseudomallei can survive and multiply within a variety of eukaryotic cells, including macrophages. This property is believed to be important for its ability to cause the disease melioidosis in a wide range of animal species, including humans. To identify determinants that are important for the ability of B. pseudomallei to survive within macrophages, in vivo expression technology (IVET) was employed. Several putative macrophage-inducible genes were identified that are likely to contribute to the virulence of B. pseudomallei, including three genes (tssH-5, tssI-5 and tssM-5) located within the same type VI secretion system cluster (tss-5), mntH, encoding a natural resistance-associated macrophage protein (NRAMP)-like manganese ion transporter, and a haem acquisition gene, bhuT. The macrophage-inducibility of the tss-5 gene cluster was confirmed by reporter gene analysis. Construction of tssH-5 and bhuT null mutants indicated that expression of the tss-5 unit and the bhu operon were not required for intramacrophage survival. A further five tss units were identified within the B. pseudomallei genome that, together with tss-5, account for approximately 2.3 % of the total genome size. The presence of six type VI secretion systems in this organism is likely to be an important factor in making this bacterium such a versatile pathogen. PMID:17660433
Shalom, Gil; Shaw, Jonathan G; Thomas, Mark S
The O-polysaccharide moiety ofBurkholderia pseudomallei319a lipopolysaccharide was covalently linked to flagellin protein isolated from the same strain. A glycoconjugate incorporating adipic acid dihydrazide as a spacer molecule elicited high-titer immunoglobulin G responses to both the protein and carbohydrate com- ponents of the construct. This immunoglobulin G was capable of protecting diabetic rats from challenge with a heterologousB. pseudomalleistrain. Burkholderia pseudomalleiis
PAUL J. BRETT ANDDONALD E. WOODS
Burkholderia pseudomallei and its host-adapted deletion clone Burkholderia mallei cause the potentially fatal human diseases melioidosis and glanders, respectively. The antibiotic resistance profile and ability to infect via aerosol of these organisms and the absence of protective vaccines have led to their classification as major biothreats and select agents. Although documented infections by these bacteria date back over 100 years,
Mark A. Schell; Lyla Lipscomb; David DeShazer
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
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. PMID:23076282
Ooi, Soon-Keat; Lim, Tian-Yeh; Lee, Song-Hua; Nathan, Sheila
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.
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
Melioidosis is a rare potentially fatal infection caused by the bacterium Burkholderia pseudomallei, which is endemic in Southeast Asia and Northern Australia and an emerging pathogen in India. It is a largely under-diagnosed disease and an imitator of tuberculosis, both clinically and radiologically.
Boruah, Deb K; Prakash, Arjun; Bora, Reeta; Buragohain, Lemanstar
The overall goal of this project is to forensically characterize 100 unknown Burkholderia isolates in the US-Australia collaboration. We will identify genome-wide single nucleotide polymorphisms (SNPs) from B. pseudomallei and near neighbor species including B. mallei, B. thailandensis and B. oklahomensis. We will design microarray probes to detect these SNP markers and analyze 100 Burkholderia genomic DNAs extracted from environmental, clinical and near neighbor isolates from Australian collaborators on the Burkholderia SNP microarray. We will analyze the microarray genotyping results to characterize the genetic diversity of these new isolates and triage the samples for whole genome sequencing. In this interim report, we described the SNP analysis and the microarray probe design for the Burkholderia SNP microarray.
Gardner, S; Jaing, C
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
NOD2 is a cytosolic pathogen recognition receptor that regulates susceptibility to a variety of infections and chronic diseases. Burkholderia pseudomallei, a facultative intracellular bacterium, causes the tropical infection melioidosis. We hypothesized that NOD2 may participate in host defense in melioidosis. We performed a series of in vitro assays and in vivo experiments, and analyzed the association of human genetic variation with infection to delineate the contribution of NOD2 to the host response to B. pseudomallei. We found that transfection with NOD2 mediated NF-?B activation induced by B. pseudomallei stimulation of HEK293 cells. After low dose inoculation with aerosolized B. pseudomallei, Nod2-deficient mice showed impaired clinical responses and permitted greater bacterial replication in the lung and dissemination to the spleen compared to wild type mice. IL-6 and KC levels were higher in the lungs of Nod2-deficient mice. In a cohort of 1,562 Thai subjects, a common genetic polymorphism in the NOD2 region, rs7194886, was associated with melioidosis and this effect was most pronounced in women. rs7194886 was not associated with differences in cytokine production induced by whole blood stimulation with the NOD2 ligand, MDP, or B. pseudomallei. These findings are the first to characterize the role of NOD2 in host defense in mammalian melioidosis.
Myers, Nicolle D.; Chantratita, Narisara; Berrington, William R.; Chierakul, Wirongrong; Limmathurotsakul, Direk; Wuthiekanun, Vanaporn; Robertson, Johanna D.; Liggitt, H. Denny; Peacock, Sharon J.; Skerrett, Shawn J.; West, T. Eoin
Burkholderia pseudomallei is a Gram-negative soil bacterium that infects both humans and animals. Although cell culture studies have revealed significant insights into factors contributing to virulence and host defense, the interactions between this pathogen and its intact host remain to be elucidated. To gain insights into the host defense responses to B. pseudomallei infection within an intact host, we analyzed the genome-wide transcriptome of infected Caenorhabditis elegans and identified ?6% of the nematode genes that were significantly altered over a 12-h course of infection. An unexpected feature of the transcriptional response to B. pseudomallei was a progressive increase in the proportion of down-regulated genes, of which ELT-2 transcriptional targets were significantly enriched. ELT-2 is an intestinal GATA transcription factor with a conserved role in immune responses. We demonstrate that B. pseudomallei down-regulation of ELT-2 targets is associated with degradation of ELT-2 protein by the host ubiquitin–proteasome system. Degradation of ELT-2 requires the B. pseudomallei type III secretion system. Together, our studies using an intact host provide evidence for pathogen-mediated host immune suppression through the destruction of a host transcription factor.
Lee, Song-Hua; Wong, Rui-Rui; Chin, Chui-Yoke; Lim, Tian-Yeh; Eng, Su-Anne; Kong, Cin; Ijap, Nur Afifah; Lau, Ming-Seong; Lim, Mei-Perng; Gan, Yunn-Hwen; He, Fang-Lian; Tan, Man-Wah; Nathan, Sheila
Burkholderia pseudomallei is a Gram-negative soil bacterium that infects both humans and animals. Although cell culture studies have revealed significant insights into factors contributing to virulence and host defense, the interactions between this pathogen and its intact host remain to be elucidated. To gain insights into the host defense responses to B. pseudomallei infection within an intact host, we analyzed the genome-wide transcriptome of infected Caenorhabditis elegans and identified ?6% of the nematode genes that were significantly altered over a 12-h course of infection. An unexpected feature of the transcriptional response to B. pseudomallei was a progressive increase in the proportion of down-regulated genes, of which ELT-2 transcriptional targets were significantly enriched. ELT-2 is an intestinal GATA transcription factor with a conserved role in immune responses. We demonstrate that B. pseudomallei down-regulation of ELT-2 targets is associated with degradation of ELT-2 protein by the host ubiquitin-proteasome system. Degradation of ELT-2 requires the B. pseudomallei type III secretion system. Together, our studies using an intact host provide evidence for pathogen-mediated host immune suppression through the destruction of a host transcription factor. PMID:23980181
Lee, Song-Hua; Wong, Rui-Rui; Chin, Chui-Yoke; Lim, Tian-Yeh; Eng, Su-Anne; Kong, Cin; Ijap, Nur Afifah; Lau, Ming-Seong; Lim, Mei-Perng; Gan, Yunn-Hwen; He, Fang-Lian; Tan, Man-Wah; Nathan, Sheila
Melioidosis which is infection with Burkholderia pseudomallei, is an important cause of sepsis in India, southeast Asia and northern Australia. Mortality is high and treatment is problematic. Neurological melioidosis is unusual but meningoencephalitis, encephalomyelitis and brain microabscess can occur Dural sinus thrombosis is not an uncommon cerebrovascular disorder with various etiologies. Hypercoagulable state, pregnancy, dehydration, certain blood dyscrasia and contraceptive pills are common causes however meningitis and local head & neck infections may lead to this condition. Dural sinus thrombosis complicating septicemic melioidosis has never been reported. The authors report a 42-year-old Thai man suffering from septicemic melioidosis with dural sinus thrombosis. He had high fever, headache, left hemiparesis, focal seizure and increased intracranial pressure. Diabetes and mild alcoholic cirrhosis were diagnosed in this admission. CT scan, MRI brain and MRV revealed superior saggital sinus thrombosis with complicating venous infarction over right posterior parietal lobe. Hemoculture demonstrated Burkholderia pseudomallei and CSF was acellular Investigations for causes of dural sinus thrombosis were all negative. This patient gradually improved after treatment with ceftazidime, antiepileptic drug and heparin without clinical recurrence. Neuromelioidosis is a rare syndrome that may present as brain abscess, encephalitis or meningoencephalitis. The authors report dural sinus thrombosis associated with septicemic melioidosis. The authors' hypothesis of venous thrombosis in the presented case is sepsis induced hypercoagulable state. Physicians should be aware of cerebral venous thrombosis in case of suspicious melioidosis with neurological involvement. Prompt treatment with intravenous heparin and antibiotic is potentially effective. PMID:16579013
Niyasom, Suchada; Sithinamsuwan, Pasiri; Udommongkol, Chesda; Suwantamee, Jithanorm
Background Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and Northern Australia. This Gram-negative pathogen possesses numerous virulence factors including three “injection type” type three secretion systems (T3SSs). B. pseudomallei has been shown to activate NF?B in HEK293T cells in a Toll-like receptor and MyD88 independent manner that requires T3SS gene cluster 3 (T3SS3 or T3SSBsa). However, the mechanism of how T3SS3 contributes to NF?B activation is unknown. Results Known T3SS3 effectors are not responsible for NF?B activation. Furthermore, T3SS3-null mutants are able to activate NF?B almost to the same extent as wildtype bacteria at late time points of infection, corresponding to delayed escape into the cytosol. NF?B activation also occurs when bacteria are delivered directly into the cytosol by photothermal nanoblade injection. Conclusions T3SS3 does not directly activate NF?B but facilitates bacterial escape into the cytosol where the host is able to sense the presence of the pathogen through cytosolic sensors leading to NF?B activation.
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.
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.
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.
We have analysed DNA fingerprinting patterns by pulsed-field gel electrophoresis (PFGE) of 52 unrelated Burkholderia pseudomallei strains isolated from septicemic and localized infections from Malaysian subjects. A total of 38 PFGE types were observed among 36 septicemic and 16 localized strains with no predominant pattern. Type 25 was seen in 2 epidemiologically related strains, suggesting human to human transmission. Twelve PFGE types were shared among 26 strains (21 septicemic and 5 localized) showing close genetic relatedness with coefficient of similarity of 0.81 to 1.0. The other 26 strains (15 septicemic and 11 localized) were unrelated as shown by the similarity coefficient of < 0.8. This study showed that our B. pseudomallei strains in Malaysia were mainly heterogenous with no predominant type both in septicemic or localized strains. PMID:21323173
Azura, M N; Norazah, A; Kamel, A G M; Zorin, S Ahmad
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
Melioidosis became a notifiable disease in Western Australia (WA) 2 years after the West Kimberley melioidosis outbreak. Two cases of melioidosis caused by the outbreak genotype of Burkholderia pseudomallei (National Collection of Type Cultures [NCTC] 13177) occurred in 1998 and 1999 in persons who visited the outbreak location at the time. No other infections caused by the outbreak strain have been recorded in WA since that time, despite an average of four culture-positive cases per year. Sporadic cases of melioidosis often follow tropical storms and cyclones during summer, and they have been detected outside the endemic area when cyclones travel far inland. In 2007, environmental isolates resembling NCTC 13177 were found 500 km east of the outbreak location after unusually severe weather. Recent whole-genome analysis places NCTC 13177 genetically close to other Australian isolates. Additional biogeographic and ecological studies are needed to establish the relative importance of environmental cofactors in disease pathogenesis.
Inglis, Timothy J. J.; O'Reilly, Lyn; Merritt, Adam J.; Levy, Avram; Heath, Christopher
Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (?0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation. PMID:24651568
Houghton, Raymond L; Reed, Dana E; Hubbard, Mark A; Dillon, Michael J; Chen, Hongjing; Currie, Bart J; Mayo, Mark; Sarovich, Derek S; Theobald, Vanessa; Limmathurotsakul, Direk; Wongsuvan, Gumphol; Chantratita, Narisara; Peacock, Sharon J; Hoffmaster, Alex R; Duval, Brea; Brett, Paul J; Burtnick, Mary N; Aucoin, David P
The Burkholderia pseudomallei rpoS gene was identified, and an rpoS null mutant was constructed. The mutant was shown to have an increased sensitivity to carbon starvation and oxidative stress. By using rpoS-lacZ fusions, transcription of rpoS was shown to be growth phase regulated, reaching a peak upon entry into stationary phase.
Subsin, Benchamas; Thomas, Mark S.; Katzenmeier, Gerd; Shaw, Jonathan G.; Tungpradabkul, Sumalee; Kunakorn, Mongkol
Phage amplification detected by MALDI-TOF MS was investigated for rapid and simultaneous Burkholderia pseudomallei identification and ceftazidime resistance determination. B. pseudomallei ceftazidime susceptible and resistant ?purM mutant strains Bp82 and Bp82.3 were infected with broadly targeting B. pseudomallei phage ?X216 and production of the m/z 37.6 kDa phage capsid protein observed by MALDI-TOF MS over the course of 3 h infections. This allowed for repoducible phage-based bacterial ID within 2 h of the onset of infection. MALDI-TOF MS-measured time to detection correlated with in silico modeling, which predicted an approximate 2 h detection time. Ceftazidime susceptible strain Bp82, while detectable in the absence of the drug, owing to the reliance of phage amplification on a viable host, was not detectable when 10 ?g/mL ceftazidime was added at the onset of infection. In contrast, resistant strain Bp82.3 was detected in the same 2 h timeframe both with and without the addition of ceftazidime.
Cox, Christopher R; Saichek, Nicholas R; Schweizer, Herbert P; Voorhees, Kent J
In the present paper, we describe cloning and expression of two outer membrane proteins, BpsOmp38 (from Burkholderia pseudomallei) and BthOmp38 (from Burkholderia thailandensis) lacking signal peptide sequences, using the pET23d(+) expression vector and Escherichia coli host strain Origami(DE3). The 38 kDa proteins, expressed as insoluble inclusion bodies, were purified, solubilized in 8 M urea, and then subjected to refolding experiments. As seen on SDS/PAGE, the 38 kDa band completely migrated to ?110 kDa when the purified monomeric proteins were refolded in a buffer system containing 10% (w/v) Zwittergent® 3-14, together with a subsequent heating to 95 °C for 5 min. CD spectroscopy revealed that the 110 kDa proteins contained a predominant ?-sheet structure, which corresponded completely to the structure of the Omp38 proteins isolated from B. pseudomallei and B. thailandensis. Immunoblot analysis using anti-BpsOmp38 polyclonal antibodies and peptide mass analysis by MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS confirmed that the expressed proteins were BpsOmp38 and BthOmp38. The anti-BpsOmp38 antibodies considerably exhibited the inhibitory effects on the permeation of small sugars through the Omp38-reconstituted liposomes. A linear relation between relative permeability rates and Mr of neutral sugars and charged antibiotics suggested strongly that the in vitro re-assembled Omp38 functioned fully as a diffusion porin.
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
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.
Melioidosis (Burkholderia pseudomallei infection) is a common cause of community-acquired sepsis in Northeast Thailand and northern Australia. B. pseudomallei is a soil saprophyte endemic to Southeast Asia and northern Australia. The clinical presentation of melioidosis may mimic tuberculosis (both cause chronic suppurative lesions unresponsive to conventional antibiotics and both commonly affect the lungs). The two diseases have overlapping risk profiles (e.g., diabetes, corticosteroid use), and both B. pseudomallei and Mycobacterium tuberculosis are intracellular pathogens. There are however important differences: the majority of melioidosis cases are acute, not chronic, and present with severe sepsis and a mortality rate that approaches 50% despite appropriate antimicrobial therapy. By contrast, tuberculosis is characteristically a chronic illness with mortality <2% with appropriate antimicrobial chemotherapy. We examined the gene expression profiles of total peripheral leukocytes in two cohorts of patients, one with acute melioidosis (30 patients and 30 controls) and another with tuberculosis (20 patients and 24 controls). Interferon-mediated responses dominate the host response to both infections, and both type 1 and type 2 interferon responses are important. An 86-gene signature previously thought to be specific for tuberculosis is also found in melioidosis. We conclude that the host responses to melioidosis and to tuberculosis are similar: both are dominated by interferon-signalling pathways and this similarity means gene expression signatures from whole blood do not distinguish between these two diseases.
Koh, Gavin C. K. W.; Schreiber, M. Fernanda; Bautista, Ruben; Maude, Rapeephan R.; Dunachie, Susanna; Limmathurotsakul, Direk; Day, Nicholas P. J.; Dougan, Gordon; Peacock, Sharon J.
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.
Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations. PMID:24455204
Jakupciak, John P; Wells, Jeffrey M; Karalus, Richard J; Pawlowski, David R; Lin, Jeffrey S; Feldman, Andrew B
Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations.
Jakupciak, John P.; Wells, Jeffrey M.; Karalus, Richard J.; Pawlowski, David R.; Lin, Jeffrey S.; Feldman, Andrew B.
TA (toxin–antitoxin) systems are widely distributed amongst bacteria and are associated with the formation of antibiotic tolerant (persister) cells that may have involvement in chronic and recurrent disease. We show that overexpression of the Burkholderia pseudomallei HicA toxin causes growth arrest and increases the number of persister cells tolerant to ciprofloxacin or ceftazidime. Furthermore, our data show that persistence towards ciprofloxacin or ceftazidime can be differentially modulated depending on the level of induction of HicA expression. Deleting the hicAB locus from B. pseudomallei K96243 significantly reduced persister cell frequencies following exposure to ciprofloxacin, but not ceftazidime. The structure of HicA(H24A) was solved by NMR and forms a dsRBD-like (dsRNA-binding domain-like) fold, composed of a triple-stranded ?-sheet, with two helices packed against one face. The surface of the protein is highly positively charged indicative of an RNA-binding protein and His24 and Gly22 were functionality important residues. This is the first study demonstrating a role for the HicAB system in bacterial persistence and the first structure of a HicA protein that has been experimentally characterized.
Butt, Aaron; Higman, Victoria A.; Williams, Christopher; Crump, Matthew P.; Hemsley, Claudia M.; Harmer, Nicholas; Titball, Richard W.
The aim of this study was to analyze the clinical characteristics of diabetes mellitus patients with Burkholderia pseudomallei septicemia and evaluate strategies of diagnosis and treatment. The clinical characteristics, diagnosis, treatment, and prognosis of 39 diabetes mellitus patients with B. pseudomallei septicemia were retrospectively analyzed. Farmers, fishermen and workers were found to be high-risk groups. The clinical manifestations of patients were diverse without specific features, but mainly presented manifestations of acute fulminant septicemia, diabetic ketoacidosis, and abscesses in tissues or/and organs. Patients showed high mortality and misdiagnosis rates and were prone to relapses and long treatment duration as there are currently few effective and sensitive antibiotics for the disease. Consequently, the cost of treatment for the disease was high. Early diagnosis, a prolonged course of heavy doses of sensitive intravenous antibiotics, drainage of abscesses, intensive insulin therapy, and supportive treatment are the keys for successful management of the disease. Regular follow-ups combined with long-term blood glucose control can help reduce the disease recurrence. PMID:24782168
Quan, H B; Li, T Y; Gao, Y Y; Chen, D X
Mips (macrophage infectivity potentiators) are a subset of immunophilins associated with virulence in a range of micro-organisms. These proteins possess peptidylprolyl isomerase activity and are inhibited by drugs including rapamycin and tacrolimus. We determined the structure of the Mip homologue [BpML1 (Burkholderia pseudomallei Mip-like protein 1)] from the human pathogen and biowarfare threat B. pseudomallei by NMR and X-ray crystallography. The crystal structure suggests that key catalytic residues in the BpML1 active site have unexpected conformational flexibility consistent with a role in catalysis. The structure further revealed BpML1 binding to a helical peptide, in a manner resembling the physiological interaction of human TGF?RI (transforming growth factor ? receptor I) with the human immunophilin FKBP12 (FK506-binding protein 12). Furthermore, the structure of BpML1 bound to the class inhibitor cycloheximide N-ethylethanoate showed that this inhibitor mimics such a helical peptide, in contrast with the extended prolyl-peptide mimicking shown by inhibitors such as tacrolimus. We suggest that Mips, and potentially other bacterial immunophilins, participate in protein–protein interactions in addition to their peptidylprolyl isomerase activity, and that some roles of Mip proteins in virulence are independent of their peptidylprolyl isomerase activity.
Norville, Isobel H.; O'Shea, Katherine; Sarkar-Tyson, Mitali; Zheng, Suxin; Titball, Richard W.; Varani, Gabriele; Harmer, Nicholas J.
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
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.
A 35 year-old man was admitted to the hospital for fever upon returning from the Caribbean area. He died 48 hours later, after developing pulmonary lesions that were complicated by multi-organ failure, despite rapid diagnosis of melioidosis by mass spectrometry on blood cultures. Melioidosis is a rare bacterial disease in the traveller that is caused by Burkholderia pseudomallei. Although the clinical presentation is variable, pneumonia is the most frequent finding. Diagnosis may be considered in travellers returning from tropical and subtropical regions, especially during rainy seasons. Accordingly, when confronted with a patient who presents with fever after travelling, it is important to carefully specify the regions visited, potential expositions, and rapidly offer adequate laboratory testing. PMID:21692313
Abbas, M; Emonet, S; Schrenzel, J; Merlani, P; Loutan, L; Gétaz, L
Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a dreadful disease common in South-East Asia and Northern Australia and is characterized by chronic suppurative lesions and pneumonia. Melioidosis may evolve into severe sepsis with multi-organ failure with high mortalities, despite proper antibiotic therapy. Besides activation of a strong pro-inflammatory host response, the coagulation system plays an important role during melioidosis, which is thought to be host-protective. In particular, a procoagulant state together with downregulation of anticoagulant pathways and activation of fibrinolysis are present, all closely interrelated with parameters of inflammation. This review presents an overview of recent studies in which the role of coagulation, anti-coagulation and fibrinolysis during melioidosis was investigated both in patients and in experimental settings. PMID:24962103
Kager, Liesbeth Martine; van der Poll, Tom; Wiersinga, Willem Joost
Melioidosis, caused by the gram-negative saprophyte Burkholderia pseudomallei, is a disease of public health importance in southeast Asia and northern Australia that is associated with high case-fatality rates in animals and humans. It has the potential for epidemic spread to areas where it is not endemic, and sporadic case reports elsewhere in the world suggest that as-yet-unrecognized foci of infection may exist. Environmental determinants of this infection, apart from a close association with rainfall, are yet to be elucidated. The sequencing of the genome of a strain of B. pseudomallei has recently been completed and will help in the further identification of virulence factors. The presence of specific risk factors for infection, such as diabetes, suggests that functional neutrophil defects are important in the pathogenesis of melioidosis; other studies have defined virulence factors (including a type III secretion system) that allow evasion of killing mechanisms by phagocytes. There is a possible role for cell-mediated immunity, but repeated environmental exposure does not elicit protective humoral or cellular immunity. A vaccine is under development, but economic constraints may make vaccination an unrealistic option for many regions of endemicity. Disease manifestations are protean, and no inexpensive, practical, and accurate rapid diagnostic tests are commercially available; diagnosis relies on culture of the organism. Despite the introduction of ceftazidime- and carbapenem-based intravenous treatments, melioidosis is still associated with a significant mortality attributable to severe sepsis and its complications. A long course of oral eradication therapy is required to prevent relapse. Studies exploring the role of preventative measures, earlier clinical identification, and better management of severe sepsis are required to reduce the burden of this disease.
Cheng, Allen C.; Currie, Bart J.
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
Background Burkholderia pseudomallei (Bp), a Gram-negative, motile, facultative intracellular bacterium is the causative agent of melioidosis in humans and animals. The Bp genome encodes a repertoire of virulence factors, including the cluster 3 type III secretion system (T3SS-3), the cluster 1 type VI secretion system (T6SS-1), and the intracellular motility protein BimA, that enable the pathogen to invade both phagocytic and non-phagocytic cells. A unique hallmark of Bp infection both in vitro and in vivo is its ability to induce cell-to-cell fusion of macrophages to form multinucleated giant cells (MNGCs), which to date are semi-quantitatively reported following visual inspection. Results In this study we report the development of an automated high-content image acquisition and analysis assay to quantitate the Bp induced MNGC phenotype. Validation of the assay was performed using T6SS-1 (?hcp1) and T3SS-3 (?bsaZ) mutants of Bp that have been previously reported to exhibit defects in their ability to induce MNGCs. Finally, screening of a focused small molecule library identified several Histone Deacetylase (HDAC) inhibitors that inhibited Bp-induced MNGC formation of macrophages. Conclusions We have successfully developed an automated HCI assay to quantitate MNGCs induced by Bp in macrophages. This assay was then used to characterize the phenotype of the Bp mutants for their ability to induce MNGC formation and identify small molecules that interfere with this process. Successful application of chemical genetics and functional reverse genetics siRNA approaches in the MNGC assay will help gain a better understanding of the molecular targets and cellular mechanisms responsible for the MNGC phenotype induced by Bp, by other bacteria such as Mycobacterium tuberculosis, or by exogenously added cytokines.
Source-Identifying Biomarker Ions between Environmental and Clinical Burkholderia pseudomallei Using Whole-Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)
Burkholderia pseudomallei is the causative agent of melioidosis, which is an endemic disease in Northeast Thailand and Northern Australia. Environmental reservoirs, including wet soils and muddy water, serve as the major sources for contributing bacterial infection to both humans and animals. The whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has recently been applied as a rapid, accurate, and high-throughput tool for clinical diagnosis and microbiological research. In this present study, we employed a whole-cell MALDI-TOF MS approach for assessing its potency in clustering a total of 11 different B. pseudomallei isolates (consisting of 5 environmental and 6 clinical isolates) with respect to their origins and to further investigate the source-identifying biomarker ions belonging to each bacterial group. The cluster analysis demonstrated that six out of eleven isolates were grouped correctly to their sources. Our results revealed a total of ten source-identifying biomarker ions, which exhibited statistically significant differences in peak intensity between average environmental and clinical mass spectra using ClinProTools software. Six out of ten mass ions were assigned as environmental-identifying biomarker ions (EIBIs), including, m/z 4,056, 4,214, 5,814, 7,545, 7,895, and 8,112, whereas the remaining four mass ions were defined as clinical-identifying biomarker ions (CIBIs) consisting of m/z 3,658, 6,322, 7,035, and 7,984. Hence, our findings represented, for the first time, the source-specific biomarkers of environmental and clinical B. pseudomallei.
Srisanga, Kitima; Roytrakul, Sittiruk; Tungpradabkul, Sumalee
Burkholderia pseudomallei, the causative agent of melioidosis, is recognized as a serious health threat due to its involvement in septic and pulmonary infections in areas of endemicity and is recognized by the Centers for Disease Control and Prevention as a category B biothreat agent. An animal model is desirable to evaluate the pathogenesis of melioidosis and medical countermeasures. A model system that represents human melioidosis infections is essential in this process. A group of 10 rhesus macaques (RMs) and 10 African green monkeys (AGMs) was exposed to aerosolized B. pseudomallei 1026b. The first clinical signs were fever developing 24 to 40 h postexposure followed by leukocytosis resulting from a high percentage of neutrophils. Dyspnea manifested 2 to 4 days postexposure. In the AGMs, an increase in interleukin 1? (IL-1?), IL-6, IL-8, gamma interferon (IFN-?), and tumor necrosis factor alpha (TNF-?) was observed. In the RMs, IL-1?, IL-6, and TNF-? increased. All the RMs and AGMs had various degrees of bronchopneumonia, with inflammation consisting of numerous neutrophils and a moderate number of macrophages. Both the RMs and the AGMs appear to develop a melioidosis infection that closely resembles that seen in acute human melioidosis. However, for an evaluation of medical countermeasures, AGMs appear to be a more appropriate model.
Facemire, Paul; Dabisch, Paul A.; Robinson, Camenzind G.; Nyakiti, David; Beck, Katie; Baker, Reese; Pitt, M. Louise M.
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.
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
Identification of the select agent Burkholderia pseudomallei in macaques imported into the United States is rare. A purpose-bred, 4.5-y-old pigtail macaque (Macaca nemestrina) imported from Southeast Asia was received from a commercial vendor at our facility in March 2012. After the initial acclimation period of 5 to 7 d, physical examination of the macaque revealed a subcutaneous abscess that surrounded the right stifle joint. The wound was treated and resolved over 3 mo. In August 2012, 2 mo after the stifle joint wound resolved, the macaque exhibited neurologic clinical signs. Postmortem microbiologic analysis revealed that the macaque was infected with B. pseudomallei. This case report describes the clinical evaluation of a B. pseudomallei-infected macaque, management and care of the potentially exposed colony of animals, and protocols established for the animal care staff that worked with the infected macaque and potentially exposed colony. This article also provides relevant information on addressing matters related to regulatory issues and risk management of potentially exposed animals and animal care staff.
Johnson, Crystal H; Skinner, Brianna L; Dietz, Sharon M; Blaney, David; Engel, Robyn M; Lathrop, George W; Hoffmaster, Alex R; Gee, Jay E; Elrod, Mindy G; Powell, Nathaniel; Walke, Henry
Sequencing of the gene rpsU reliably delineates saprophytic Burkholderia (B.) thailandensis from highly pathogenic B. mallei and B. pseudomallei. We analyzed the suitability of this technique for the delineation of the B. pseudomallei complex from other Burkholderia species. Both newly recorded and previously deposited sequences of well-characterized or reference strains (n = 84) of Azoarcus spp., B. ambifaria, B. anthina, B. caledonica, B. caribensis, B. caryophylli, B. cenocepacia, B. cepacia, B. cocovenenans, B. dolosa, B. fungorum, B. gladioli, B. glathei, B. glumae, B. graminis, B. hospita, B. kururensis, B. mallei, B. multivorans, B. phenazinium, B. phenoliruptrix, B. phymatum, B. phytofirmans, B. plantarii, B. pseudomallei, B. pyrrocinia, B. stabilis, B. thailandensis, B. ubonensis, B. vietnamiensis, B. xenovorans, not further defined Burkholderia spp., and the outliers Cupriavidus metallidurans, Laribacter hongkongensis, Pandorea norimbergensis, and Ralstonia pickettii were included in a multiple sequence analysis. Multiple sequence alignments led to the delineation of four major clusters, rpsU-I to rpsU-IV, with a sequence homology >92%. The B. pseudomallei complex formed the complex rpsU-II. Several Burkholderia species showed 100% sequence homology. This procedure is useful for the molecular confirmation or exclusion of glanders or melioidosis from primary patient material. Further discrimination within the Burkholderia genus requires other molecular approaches. PMID:24883196
Frickmann, H; Neubauer, H; Loderstaedt, U; Derschum, H; Hagen, R M
Sequencing of the gene rpsU reliably delineates saprophytic Burkholderia (B.) thailandensis from highly pathogenic B. mallei and B. pseudomallei. We analyzed the suitability of this technique for the delineation of the B. pseudomallei complex from other Burkholderia species. Both newly recorded and previously deposited sequences of well-characterized or reference strains (n = 84) of Azoarcus spp., B. ambifaria, B. anthina, B. caledonica, B. caribensis, B. caryophylli, B. cenocepacia, B. cepacia, B. cocovenenans, B. dolosa, B. fungorum, B. gladioli, B. glathei, B. glumae, B. graminis, B. hospita, B. kururensis, B. mallei, B. multivorans, B. phenazinium, B. phenoliruptrix, B. phymatum, B. phytofirmans, B. plantarii, B. pseudomallei, B. pyrrocinia, B. stabilis, B. thailandensis, B. ubonensis, B. vietnamiensis, B. xenovorans, not further defined Burkholderia spp., and the outliers Cupriavidus metallidurans, Laribacter hongkongensis, Pandorea norimbergensis, and Ralstonia pickettii were included in a multiple sequence analysis. Multiple sequence alignments led to the delineation of four major clusters, rpsU-I to rpsU-IV, with a sequence homology >92%. The B. pseudomallei complex formed the complex rpsU-II. Several Burkholderia species showed 100% sequence homology. This procedure is useful for the molecular confirmation or exclusion of glanders or melioidosis from primary patient material. Further discrimination within the Burkholderia genus requires other molecular approaches.
Neubauer, H.; Loderstaedt, U.; Derschum, H.; Hagen, R. M.
Melioidosis, an infectious disease caused by Burkholderia pseudomallei is an emerging disease with high impact on animals and man. In different animal species, the clinical course varies and delayed diagnosis poses risks for the dissemination of the agent in non-endemic areas. Not only migration and transport of animals around the world but also tourism increases the risk that melioidosis can leave its endemic boundaries and establish itself elsewhere. Detection of the agent is a major challenge, as the agent has to be handled in laboratories of biosafety level 3 and test kits are not yet commercially available. Veterinarians and doctors should be aware of melioidosis not only as an agent of public interest but also in terms of a bioterrorist attack. The aim of this review is to describe the agent, its aetiology, the manifestation in a variety of animal species as well as to describe diagnostic procedures, typing techniques and countermeasures. PMID:15525357
Sprague, L D; Neubauer, H
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
Background Burkholderia pseudomallei is the causative agent of melioidosis, a frequently occurring disease in northeastern Thailand, where soil and water high in salt content are common. Using microarray analysis, we previously showed that B. pseudomallei up-regulated a short-chain dehydrogenase/oxidoreductase (SDO) under salt stress. However, the importance of SDO in B. pseudomallei infection is unknown. This study aimed to explore the function of B. pseudomallei SDO, and to investigate its role in interactions between B. pseudomallei and host cells. Results Bioinformatics analysis of B. pseudomallei SDO structure, based on homology modeling, revealed a NAD+ cofactor domain and a catalytic triad containing Ser149, Tyr162, and Lys166. This is similar to Bacillus megaterium glucose 1-dehydrogenase. To investigate the role of this protein, we constructed a B. pseudomallei SDO defective mutant, measured glucose dehydrogenase (GDH) activity, and tested the interactions with host cells. The B. pseudomallei K96243 wild type exhibited potent GDH activity under condition containing 300 mM NaCl, while the mutant showed activity levels 15 times lower. Both invasion into the A549 cell line and early intracellular survival within the J774A.1 macrophage cell were impaired in the mutant. Complementation of SDO was able to restore the mutant ability to produce GDH activity, invade epithelial cells, and survive in macrophages. Conclusions Our data suggest that induced SDO activity during salt stress may facilitate B. pseudomallei invasion and affect initiation of successful intracellular infection. Identifying the role of B. pseudomallei SDO provides a better understanding of the association between bacterial adaptation and pathogenesis in melioidosis.
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.
Lim, Rebecca Sin Mei; Flatman, Sam; Dahm, Markus C.
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
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 identifcation 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.
Estes, D Mark; Dow, Steven W; Schweizer, Herbert P; Torres, Alfredo G
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.
Bandeira, Tereza de Jesus Pinheiro Gomes; Moreira, Camila Alencar; Castelo-Branco, Debora de Souza Collares Maia; Neto, Manoel Paiva de Araujo; Cordeiro, Rossana de Aguiar; Rodrigues, Terezinha de Jesus Santos; Rocha, Marcos Fabio Gadelha; Sidrim, Jose Julio Costa
Background Melioidosis is an infection caused by the facultative intracellular gram-negative bacterium; Burkholderia pseudomallei. It gives rise to protean clinical manifestations and has a varied prognosis. Although it was rare in Sri Lanka increasing numbers of cases are being reported with high morbidity and mortality. Here we report a case of melioidosis presenting with lymphadenitis which was diagnosed early and treated promptly with a good outcome. Case presentation A 53-year-old Sinhalese woman with diabetes presented with fever and left sided painful inguinal lymphadenitis for one month. She had undergone incision and drainage of a thigh abscess three months previously and had been treated with a short course of antibiotics. There was no record that abscess material was tested microbiologically. She had neutrophil leukocytosis and elevated inflammatory markers. Initial pus culture revealed a scanty growth of “Pseudomonas sp.” and Escherichia coli which were sensitive to ceftazidime and resistant to gentamicin. Due to the history of diabetes, recurrent abscess formation and the suggestive sensitivity pattern of the bacterial isolates, we actively investigated for melioidosis. The bacterial isolate was subsequently identified as B. pseudomallei by polymerase chain reaction and antibodies to melioidin antigen were found to be raised at a titre of 1:160. The patient was treated with high dose intravenous ceftazidime for four weeks followed by eradication therapy with cotrimoxazole and doxycycline. As the patient was intolerant to cotrimoxazole, the antibiotics were changed to a combination of co-amoxyclav and doxycycline and continued for 12 weeks. The patient was well after 6 months without any relapse. Conclusions Melioidosis is an emerging infection in South Asia. It may present with recurrent abscesses. Therefore it is very important to send pus for culture whenever an abscess is drained. However, it should be noted that the reporting laboratory may be unfamiliar with this bacterium and the isolate may be misidentified as Pseudomonas or even E. coli. Melioidosis should be suspected when an isolate with the typical antibiotic sensitivity pattern of ceftazidime sensitivity and gentamicin resistance is cultured, especially in a patient with diabetes. This will expedite diagnosis and prompt treatment leading to an excellent prognosis.
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.
Type IV pili are surface-exposed filaments and bacterial virulence factors, represented by the Tfpa and Tfpb types, which assemble via specific machineries. The Tfpb group is further divided into seven variants, linked to heterogeneity in the assembly machineries. Here we focus on PilO2Bp, a protein component of the Tfpb R64 thin pilus variant assembly machinery from the pathogen Burkholderia pseudomallei. PilO2Bp belongs to the PF06864 Pfam family, for which an improved definition is presented based on newly derived Hidden Markov Model (HMM) profiles. The 3D structure of the N-terminal domain of PilO2Bp (N-PilO2Bp), here reported, is the first structural representative of the PF06864 family. N-PilO2Bp presents an actin-like ATPase fold that is shown to be present in BfpC, a different variant assembly protein; the new HMM profiles classify BfpC as a PF06864 member. Our results provide structural insight into the PF06864 family and on the Type IV pili assembly machinery.
Manjasetty, Babu A.; Yero, Daniel; Perletti, Lucia; Belrhali, Hassan; Daura, Xavier; Gourlay, Louise J.; Bolognesi, Martino
Type IV pili are surface-exposed filaments and bacterial virulence factors, represented by the Tfpa and Tfpb types, which assemble via specific machineries. The Tfpb group is further divided into seven variants, linked to heterogeneity in the assembly machineries. Here we focus on PilO2(Bp), a protein component of the Tfpb R64 thin pilus variant assembly machinery from the pathogen Burkholderia pseudomallei. PilO2(Bp) belongs to the PF06864 Pfam family, for which an improved definition is presented based on newly derived Hidden Markov Model (HMM) profiles. The 3D structure of the N-terminal domain of PilO2(Bp) (N-PilO2(Bp)), here reported, is the first structural representative of the PF06864 family. N-PilO2(Bp) presents an actin-like ATPase fold that is shown to be present in BfpC, a different variant assembly protein; the new HMM profiles classify BfpC as a PF06864 member. Our results provide structural insight into the PF06864 family and on the Type IV pili assembly machinery. PMID:24728008
Lassaux, Patricia; Conchillo-Solé, Oscar; Manjasetty, Babu A; Yero, Daniel; Perletti, Lucia; Belrhali, Hassan; Daura, Xavier; Gourlay, Louise J; Bolognesi, Martino
Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. It is refractory to antibiotic treatment and there is currently no licensed vaccine. In this report we detail the construction and protective efficacy of a polysaccharide-protein conjugate composed of B. pseudomallei lipopolysaccharide and the Hc fragment of tetanus toxin. Immunisation of mice with the lipopolysaccharide-conjugate led to significantly reduced bacterial burdens in the spleen 48 hours after challenge and afforded significant protection against a lethal challenge with B. pseudomallei. The conjugate generated significantly higher levels of antigen-specific IgG1 and IgG2a than in lipopolysaccharide-immunised mice. Immunisation with the conjugate also demonstrated a bias towards Th1 type responses, evidenced by high levels of IgG2a. In contrast, immunisation with unconjugated lipopolysaccharide evoked almost no IgG2a demonstrating a bias towards Th2 type responses. This study demonstrates the effectiveness of this approach in the development of an efficacious and protective vaccine against melioidosis.
Scott, Andrew E.; Ngugi, Sarah A.; Laws, Thomas R.; Corser, David; Lonsdale, Claire L.; D'Elia, Riccardo V.; Titball, Richard W.; Williamson, E. Diane; Atkins, Timothy P.; Prior, Joann L.
Between 1996 and 2002, 162 cases of pulmonary melioidosis were reported from Srinagarind Hospital, Khon Kaen, northeast Thailand, 90 acute vs 72 subacute/chronic. Patients averaged 50 years of age and half worked as farmers. The male to female ratio was between 2 and 3 to 1 depending on the subgrouping. Burkholderia pseudomallei was confirmed by a culture or a four-fold rise in titer in the majority of cases, while the others were presumptive diagnoses based on response to treatment. Pulmonary melioidosis presented as either acute fulminant pneumonia or as an indolent disease. The common concurrent medical illness was diabetes mellitus. Mean incubation of the acute vs the sub-acute/chronic form was 8.7 vs 54.4 days, respectively. Leukocytosis was detected in 70% of cases. Sputum Gram's stain was not sensitive for diagnosis. Sputum culture and blood culture were diagnostic for 31.1 vs 22.2 and 40 vs 37.5% of the acute vs subacute/chronic forms, respectively. The common radiographic patterns for acute pneumonia were localized patchy alveolar infiltrate or hematogenous pattern. A bilateral diffuse patchy alveolar infiltration or multiple nodular lesions characterized the latter. Upper-lobe involvement with early cavitation and rapid progression were common. In the subacute/chronic forms, the radiographic pattern sometimes mimicked tuberculosis, with upper lobe involvement, patchy alveolar infiltrate with cavities or fibroreticular lesions. In approximately 30% of cases, liver and/or splenic abscess were common sites of extrapulmonary infection. Respiratory failure and septic shock from acute pulmonary melioidosis was 20% fatal. Early empirical antibiotic therapy should be given for severe pneumonia. PMID:15689084
The human pathogen, Burkholderia pseudomalle, is able to survive and multiply in hostile environments such as within macrophages. In an attempt to understand its strategy to cope with oxidative stress, the physiological role and gene regulation of a nonspecific DNA-binding protein (DpsA) was investigated. Expression of dpsA increases in response to oxidative stress through increased transcription from the upstream katG
Suvit Loprasert; Wirongrong Whangsuk; Ratiboot Sallabhan; Skorn Mongkolsuk
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
Background Burkholderia pseudomallei is a Category B select agent and the cause of melioidosis. Research funding for vaccine development has largely considered protection within the biothreat context, but the resulting vaccines could be applicable to populations who are at risk of naturally acquired melioidosis. Here, we discuss target populations for vaccination, consider the cost-benefit of different vaccination strategies and review potential vaccine candidates. Methods and Findings Melioidosis is highly endemic in Thailand and northern Australia, where a biodefense vaccine might be adopted for public health purposes. A cost-effectiveness analysis model was developed, which showed that a vaccine could be a cost-effective intervention in Thailand, particularly if used in high-risk populations such as diabetics. Cost-effectiveness was observed in a model in which only partial immunity was assumed. The review systematically summarized all melioidosis vaccine candidates and studies in animal models that had evaluated their protectiveness. Possible candidates included live attenuated, whole cell killed, sub-unit, plasmid DNA and dendritic cell vaccines. Live attenuated vaccines were not considered favorably because of possible reversion to virulence and hypothetical risk of latent infection, while the other candidates need further development and evaluation. Melioidosis is acquired by skin inoculation, inhalation and ingestion, but routes of animal inoculation in most published studies to date do not reflect all of this. We found a lack of studies using diabetic models, which will be central to any evaluation of a melioidosis vaccine for natural infection since diabetes is the most important risk factor. Conclusion Vaccines could represent one strand of a public health initiative to reduce the global incidence of melioidosis.
Lubell, Yoel; Koh, Gavin C. K. W.; White, Lisa J.; Day, Nicholas P. J.; Titball, Richard W.
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.
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.
Background Infections due to Mycobacterium tuberculosis, Burkholderia pseudomallei and non-typhoidal Salmonella cause significant morbidity and mortality throughout the world. These intracellular pathogens share some common predisposing factors and clinical features. Co-infection with two of these organisms has been reported previously but, to our knowledge, this is the first time that infection with all three has been reported in one person. Case presentation In September 2010, a 58-year-old diabetic Malaysian male presented with fever and a fluctuant mass on the right side of his neck. B. pseudomallei was isolated from an aspirate of this lesion and there was radiological evidence of disseminated infection in the liver and spleen. The recurrence of clinical symptoms over ensuing months prompted further aspiration and biopsy of a cervical abscess and underlying lymph nodes. Salmonella enterica serovar Stanley and then M. tuberculosis were identified from these specimens by culture and molecular methods. The patient responded to targeted medical management of each of these infections. Conclusion In endemic settings, a high index of suspicion and adequate tissue sampling are imperative in identifying these pathogenic organisms. Diabetes was identified as a predisposing factor in this case while our understanding of other potential risk factors is evolving.
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
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.
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 survival rates. Results We have used microarray technology to generate genome-wide transcriptional profiles (>48,000 transcripts) from the whole blood of patients with septicemic melioidosis (n = 32), patients with sepsis caused by other pathogens (n = 31), and uninfected controls (n = 29). Unsupervised analyses demonstrated the existence of a whole blood transcriptional signature distinguishing patients with sepsis from control subjects. The majority of changes observed were common to both septicemic melioidosis and sepsis caused by other infections, including genes related to inflammation, interferon-related genes, neutrophils, cytotoxic cells, and T-cells. Finally, class prediction analysis identified a 37 transcript candidate diagnostic signature that distinguished melioidosis from sepsis caused by other organisms with 100% accuracy in a training set. This finding was confirmed in 2 independent validation sets, which gave high prediction accuracies of 78% and 80%, respectively. This signature was significantly enriched in genes coding for products involved in the MHC class II antigen processing and presentation pathway. Conclusions Blood transcriptional patterns distinguish patients with septicemic melioidosis from patients with sepsis caused by other pathogens. Once confirmed in a large scale trial this diagnostic signature might constitute the basis of a differential diagnostic assay.
Background Melioidosis is a problem in the developing tropical regions of Southeast Asia and Northern Australia where the the Gram negative saprophytic bacillus Burkholderia pseudomallei is endemic with the risk of fulminant septicaemia. While diabetes mellitus is a well-established risk factor for melioidiosis, little is known if specific hypoglycemic agents may differentially influence the susceptibility and clinical course of infection with B. pseudomallei (Bp). Methodology/Principal Findings In this cohort study, patients with pre-existing diabetes and melioidosis were retrospectively studied. Outcome measures: mortality, length of stay and development of complications (namely hypotension, intubation, renal failure and septicaemia) were studied in relation to prior diabetic treatment regimen. Peripheral blood mononuclear cells (PBMC) from diabetic patients and healthy PBMC primed with metformin, glyburide and insulin were stimulated with purified Bp antigens in vitro. Immune response and specific immune pathway mediators were studied to relate to the clinical findings mechanistically. Of 74 subjects, 44 (57.9%) had sulphonylurea-containing diabetic regimens. Patient receiving sulphonylureas had more severe septic complications (47.7% versus 16.7% p?=?0.006), in particular, hypotension requiring intropes (p?=?0.005). There was also a trend towards increased mortality in sulphonylurea-users (15.9% versus 3.3% p?=?0.08). In-vitro, glyburide suppressed inflammatory cytokine production in a dose-dependent manner. An effect of the drug was the induction of IL-1R-associated kinase-M at the level of mRNA transcription. Conclusion/Significance Sulphonylurea treatment results in suppression of host inflammatory response and may put patients at higher risk for adverse outcomes in melioidosis.
Liu, Xiang; Foo, Geraldine; Lim, Wan Peng; Ravikumar, Sharada; Sim, Siew Hoon; Win, Mar Soe; Goh, Jessamine Geraldine; Lim, Joan Hui Juan; Ng, Ying Hui; Fisher, Dale; Khoo, Chin Meng
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.
Background Melioidosis is an important cause of morbidity and mortality in East Asia. Recurrent melioidosis occurs in around 10% of patients following treatment either because of relapse with the same strain or re-infection with a new strain of Burkholderia pseudomallei. Distinguishing between the two is important but requires bacterial genotyping. The aim of this study was to develop a simple scoring system to distinguish re-infection from relapse. Methods In a prospective study of 2,804 consecutive adult patients with melioidosis presenting to Sappasithiprasong Hospital, NE Thailand, between1986 and 2005, there were 141 patients with recurrent melioidosis with paired strains available for genotyping. Of these, 92 patients had relapse and 49 patients had re-infection. Variables associated with relapse or re-infection were identified by multivariable logistic regression and used to develop a predictive model. Performance of the scoring system was quantified with respect to discrimination (area under receiver operating characteristic curves, AUC) and categorization (graphically). Bootstrap resampling was used to internally validate the predictors and adjust for over-optimism. Findings Duration of oral antimicrobial treatment, interval between the primary episode and recurrence, season, and renal function at recurrence were independent predictors of relapse or re-infection. A score of <5 correctly identified relapse in 76 of 89 patients (85%), whereas a score ?5 correctly identified re-infection in 36 of 52 patients (69%). The scoring index had good discriminative power, with a bootstrap bias-corrected AUC of 0.80 (95%CI: 0.73–0.87). Conclusions A simple scoring index to predict the cause of recurrent melioidosis has been developed to provide important bedside information where rapid bacterial genotyping is unavailable.
Limmathurotsakul, Direk; Chaowagul, Wipada; Chantratita, Narisara; Wuthiekanun, Vanaporn; Biaklang, Mayurachat; Tumapa, Sarinna; White, Nicholas J.; Day, Nicholas P. J.; Peacock, Sharon J.
Background During severe (pneumo)sepsis inflammatory and coagulation pathways become activated as part of the host immune response. Thrombomodulin (TM) is involved in a range of host defense mechanisms during infection and plays a pivotal role in activation of protein C (PC) into active protein C (APC). APC has both anticoagulant and anti-inflammatory properties. In this study we investigated the effects of impaired TM-mediated APC generation during melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia caused by Burkholderia (B.) pseudomallei. Methodology/Principal Findings (WT) mice and mice with an impaired capacity to activate protein C due to a point mutation in their Thbd gene (TMpro/pro mice) were intranasally infected with B. pseudomallei and sacrificed after 24, 48 or 72 hours for analyses. Additionally, survival studies were performed. When compared to WT mice, TMpro/pro mice displayed a worse survival upon infection with B. pseudomallei, accompanied by increased coagulation activation, enhanced lung neutrophil influx and bronchoalveolar inflammation at late time points, together with increased hepatocellular injury. The TMpro/pro mutation had limited if any impact on bacterial growth and dissemination. Conclusion/Significance TM-mediated protein C activation contributes to protective immunity after infection with B. pseudomallei. These results add to a better understanding of the regulation of the inflammatory and procoagulant response during severe Gram-negative (pneumo)sepsis.
Kager, Liesbeth M.; Wiersinga, W. Joost; Roelofs, Joris J. T. H.; de Boer, Onno J.; Weiler, Hartmut; van 't Veer, Cornelis; van der Poll, Tom
The type II secretion (T2S) system in Gram-negative bacteria is comprised of 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, we 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 up-regulated promoter in a secA deletion background complemented with an ectopic lac-regulated secA copy. Bioluminescence was indirectly proportional to the IPTG 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 9 most potent hits, representing 5 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 IC50 values from 5 – 25 ?M. In addition, 7 of the 9 compounds also inhibited the T2S-mediated extracellular secretion of phospholipases C by P. aeruginosa and of protease activity by Burkholderia pseudomallei.
Moir, Donald T.; Di, Ming; Wong, Erica; Moore, Richard A.; Schweizer, Herbert P.; Woods, Donald E.; Bowlin, Terry L.
4-Hydroxy-2-alkylquinolines (HAQs), especially 3,4-dihydroxy-2-heptylquinoline (Pseudomonas quinolone signal) and its precursor, 4-hydroxy-2-heptylquinoline, are attracting much attention, mainly because of their role as signaling molecules in Pseudomonas aeruginosa. The pqsABCDE operon is centrally involved in their biosynthesis. The presence of a homologous operon in Burkholderia pseudomallei and B. thailandensis was recently reported. Thus, we have investigated the abilities of 11 Burkholderia species to produce HAQ-like molecules by liquid chromatography/mass spectrometry. We have identified 29 different HAQ derivatives produced by the only three Burkholderia species where a pqsABCDE homologue was found among available sequenced Burkholderia species genomes, including B. ambifaria, a member of the Burkholderia cepacia complex. In contrast with those of P. aeruginosa, Burkholderia HAQs typically bear a methyl group, hence their designation as 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs). We identified three families of HMAQs with a saturated or unsaturated alkyl chain at the 2? position, in contrast with the 1? position of P. aeruginosa, including one with an N-oxide group. Furthermore, the operon in these species contains two more genes downstream of the pqsE homologue, resulting in the hmqABCDEFG operon. While the inactivation of hmqA inhibits the production of HMAQs, the methylation of the quinoline ring requires a putative methyltransferase encoded by hmqG. Interestingly, hmqA or hmqG mutations increase the production of acyl homoserine lactones and, consequently, phenotypes under the control of quorum sensing in B. ambifaria: antifungal activity, siderophore production, and proteolytic activity. These results indicate that only HAQs bearing a methyl group (HMAQs) are involved in quorum-sensing regulation.
Vial, Ludovic; Lepine, Francois; Milot, Sylvain; Groleau, Marie-Christine; Dekimpe, Valerie; Woods, Donald E.; Deziel, Eric
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
For nearly 80 years clinical melioidosis has been considered a rare disease. This bacterial infection is caused by Pseudomonas pseudomallei, a saprophyte found in soil and surface water of endemic areas. Consequently, those who have most contact with soil, the rural poor, are likely to be at greatest risk of infection. Since the diversity of clinical manifestations necessitates the isolation and identification of the causative organism for a definitive diagnosis of melioidosis and the population at greatest risk within endemic areas rarely have access to an appropriate level of health care, the disease has probably been underrecognized. Melioidosis is now known to be an important cause of human morbidity and mortality in Thailand, and this may be true throughout Southeast Asia, which is usually regarded as the main endemic area for the disease. In Australia, melioidosis causes a smaller number of human infections, while disease among livestock has important economic and possible public health implications. Sporadic reports of the infection indicate its presence in several other tropical regions: in the Indian subcontinent, Africa, and Central and South America. Clinical melioidosis may be highly prevalent in these areas, but underdiagnosed as a result of a lack of awareness of the clinical and microbiological features of the disease, or simply because of a lack of health care facilities. Furthermore, during the last two decades the importation and transmission of melioidosis within nontropical zones have been documented. The causative organism is not difficult to grow, and modern antibiotics have improved disease prognosis. Further studies are needed to determine the true worldwide distribution and prevalence of melioidosis so that improved therapeutic and preventive measures can be developed and applied.
Dance, D A
Background Burkholderia pseudomallei is the causative agent of melioidosis, a tropical disease of humans with a variable and often fatal outcome. In murine models of infection, different strains exhibit varying degrees of virulence. In contrast, two related species, B. thailandensis and B. oklahomensis, are highly attenuated in mice. Our aim was to determine whether virulence in mice is reflected in macrophage or wax moth larvae (Galleria mellonella) infection models. Results B. pseudomallei strains 576 and K96243, which have low median lethal dose (MLD) values in mice, were able to replicate and induce cellular damage in macrophages and caused rapid death of G. mellonella. In contrast, B. pseudomallei strain 708a, which is attenuated in mice, showed reduced replication in macrophages, negligible cellular damage and was avirulent in G. mellonella larvae. B. thailandensis isolates were less virulent than B. pseudomallei in all of the models tested. However, we did record strain dependent differences. B. oklahomensis isolates were the least virulent isolates. They showed minimal ability to replicate in macrophages, were unable to evoke actin-based motility or to form multinucleated giant cells and were markedly attenuated in G. mellonella compared to B. thailandensis. Conclusions We have shown that the alternative infection models tested here, namely macrophages and Galleria mellonella, are able to distinguish between strains of B. pseudomallei, B. thailandensis and B. oklahomensis and that these differences reflect the observed virulence in murine infection models. Our results indicate that B. oklahomensis is the least pathogenic of the species investigated. They also show a correlation between isolates of B. thailandensis associated with human infection and virulence in macrophage and Galleria infection models.
Background The genus Burkholderia includes pathogenic gram-negative bacteria that cause melioidosis, glanders, and pulmonary infections of patients with cancer and cystic fibrosis. Drug resistance has made development of new antimicrobials critical. Many approaches to discovering new antimicrobials, such as structure-based drug design and whole cell phenotypic screens followed by lead refinement, require high-resolution structures of proteins essential to the parasite. Methodology/Principal Findings We experimentally identified 406 putative essential genes in B. thailandensis, a low-virulence species phylogenetically similar to B. pseudomallei, the causative agent of melioidosis, using saturation-level transposon mutagenesis and next-generation sequencing (Tn-seq). We selected 315 protein products of these genes based on structure-determination criteria, such as excluding very large and/or integral membrane proteins, and entered them into the Seattle Structural Genomics Center for Infection Disease (SSGCID) structure determination pipeline. To maximize structural coverage of these targets, we applied an “ortholog rescue” strategy for those producing insoluble or difficult to crystallize proteins, resulting in the addition of 387 orthologs (or paralogs) from seven other Burkholderia species into the SSGCID pipeline. This structural genomics approach yielded structures from 31 putative essential targets from B. thailandensis, and 25 orthologs from other Burkholderia species, yielding an overall structural coverage for 49 of the 406 essential gene families, with a total of 88 depositions into the Protein Data Bank. Of these, 25 proteins have properties of a potential antimicrobial drug target i.e., no close human homolog, part of an essential metabolic pathway, and a deep binding pocket. We describe the structures of several potential drug targets in detail. Conclusions/Significance This collection of structures, solubility and experimental essentiality data provides a resource for development of drugs against infections and diseases caused by Burkholderia. All expression clones and proteins created in this study are freely available by request.
Baugh, Loren; Gallagher, Larry A.; Patrapuvich, Rapatbhorn; Clifton, Matthew C.; Gardberg, Anna S.; Edwards, Thomas E.; Armour, Brianna; Begley, Darren W.; Dieterich, Shellie H.; Dranow, David M.; Abendroth, Jan; Fairman, James W.; Fox, David; Staker, Bart L.; Phan, Isabelle; Gillespie, Angela; Choi, Ryan; Nakazawa-Hewitt, Steve; Nguyen, Mary Trang; Napuli, Alberto; Barrett, Lynn; Buchko, Garry W.; Stacy, Robin; Myler, Peter J.; Stewart, Lance J.; Manoil, Colin; Van Voorhis, Wesley C.
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
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 bacterio- phage, 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
Donald E. Woods; Jeffrey A. Jeddeloh; David L. Fritz; David DeShazer
The genus Burkholderia contains over 30 species, many of which are important human pathogens. In addition to the primary pathogens Burkholderia pseudomallei and Burkholderia mallei, several species have emerged as opportunistic pathogens in persons suffering from cystic fibrosis (CF) and immunocompromised individuals. All Burkholderia species investigated so far employ quorum-sensing (QS) systems that rely on N-acyl-homoserine lactone (AHL) signal molecules to express certain phenotypic traits in a population density-dependent manner. Whilst many Burkholderia strains only contain the CepI/CepR QS system, which relies on C8-HSL, some strains, in particular isolates of B. pseudomallei and B. mallei, harbour multiple LuxI/LuxR homologues and produce numerous AHL signal molecules. Evidence has accumulated over the past few years that the QS systems operating in Burkholderia are crucial for full virulence in various animal models. However, only few QS-regulated functions required for virulence in the different infection models have so far been identified. Given the essential role of QS in the expression of pathogenic traits in Burkholderia these regulatory systems represent attractive targets for the development of novel therapeutics. PMID:16490397
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
Melioidosis is a complex bacterial infection, treatment of which combines the urgency of treating rapidly fatal Gram negative septicaemia with the need for eradication of long-term persistent disease in pulmonary, soft tissue, skeletal and other organ systems. Incremental improvements in treatment have been made as a result of multicentre collaboration across the main endemic region of Southeast Asia and northern Australia. There is an emerging consensus on the three main patterns of antimicrobial chemotherapy; initial (Phase 1) treatment, subsequent eradication (Phase 2) therapy and most recently post-exposure (Phase 0) prophylaxis. The combination of agents used, duration of therapy and need for adjunct modalities depends on the type, severity and antimicrobial susceptibility of infection. New antibiotic and adjunct therapies are at an investigational stage but on currently available data are unlikely to make a significant impact on this potentially fatal infection.
Inglis, Timothy J.J.
Melioidosis is an infective condition which is common in South East Asia. It can present in various forms like cutaneous abscess, pneumonia and severe septicaemia. However, melioidosis causing abdominal aortic pseudoaneurysms is extremely rare and a difficult condition to diagnose and treat. We present our management of two cases of abdominal aortic pseudoaneurysms secondary to melioidosis and their subsequent outcomes. PMID:19321406
Rao, Jaideepraj; Kaushal, A S; Hoong, Chia Kok
A procedure for molecular identification of Burkholderia gladioli is described. Specific 16S and 23S rRNA gene signature sequences were defined as primers for PCR. The method allows rapid and specific discrimination of B. gladioli from related species (B. cepacia, B. multivorans, B. vietnamiensis, B. mallei, B. pseudomallei, Ralstonia pickettii, and R. eutropha) and should contribute to the clarification of its role as a human pathogen, e.g., in cystic fibrosis. PMID:9705429
Bauernfeind, A; Schneider, I; Jungwirth, R; Roller, C
AIMS: To determine whether quantitative blood culture methods could improve the diagnosis of septicaemic melioidosis. METHODS: A comparison of conventional broth based blood cultures, a pour plate method, and a commercial lysis centrifugation (Isolator 10) blood culture system was conducted in 71 Thai patients with severe melioidosis. The time to identification of B pseudomallei was recorded for each method. RESULTS: 42 patients (59%) were septicaemic. Compared with conventional blood culture, the Isolator and pour plate methods had sensitivities of 81% and 61%, respectively. The median times to a positive culture were: Isolator 39.3 hours, pour plates 45.5 hours, broth culture 61.8 hours (p < 0.001 Isolator v broth). There was a significant inverse correlation between Isolator tube or pour plate quantitative counts and time to detection (r = -0.44 and -0.57, respectively). Mortality was higher in patients who were septicaemic. CONCLUSIONS: Routine use of one of these quantitative methods, in addition to conventional broth culture, may lead to earlier diagnosis of septicaemic melioidosis.
Simpson, A J; Howe, P A; Wuthiekanun, V; White, N J
Melioidosis was first described in Australia in an outbreak in sheep in 1949 in north Queensland (22°S). Human melioidosis was first described from Townsville (19°S) in 1950. Melioidosis is hyperendemic in the Top End of the Northern Territory (NT) and as in parts of northeastern Thailand it is the commonest cause of fatal community-acquired septicemic pneumonia. In the 9 years
Bart J Currie; Dale A Fisher; Diane M Howard; James N. C Burrow; Sudarshan Selvanayagam; Paul L Snelling; Nicholas M Anstey; Mark J Mayo
Burkholderia species are bacterial soil inhabitants that are capable of interacting with a variety of eukaryotes, in some cases occupying intracellular habitats. Pathogenic and nonpathogenic Burkholderia spp., including B. vietnamiensis, B. cepacia, and B. pseudomallei, were grown on germinating spores of the arbuscular mycorrhizal fungus Gigaspora decipiens. Spore lysis assays revealed that all Burkholderia spp. tested were able to colonize the interior of G. decipiens spores. Amplification of specific DNA sequences and transmission electron microscopy confirmed the intracellular presence of B. vietnamiensis. Twelve percent of all spores were invaded by B. vietnamiensis, with an average of 1.5 × 106 CFU recovered from individual infected spores. Of those spores inoculated with B. pseudomallei, 7% were invaded, with an average of 5.5 × 105 CFU recovered from individual infected spores. Scanning electron and fluorescence microscopy provided insights into the morphology of surfaces of spores and hyphae of G. decipiens and the attachment of bacteria. Burkholderia spp. colonized both hyphae and spores, attaching to surfaces in either an end-on or side-on fashion. Adherence of Burkholderia spp. to eukaryotic surfaces also involved the formation of numerous fibrillar structures.
Levy, Avram; Chang, Barbara J.; Abbott, Lynette K.; Kuo, John; Harnett, Gerry; Inglis, Timothy J. J.
The aim of this study was to develop a typing scheme for Pseudomonas pseudomallei by comparison of patterns of restriction fragment length polymorphisms in rRNA genes (ribotyping). BamHI restriction digests of 100 isolates from various animal (34), human (58), and environmental (6) sources, including six reference strains, were hybridized to Escherichia coli 16S and 23S rRNAs. A chemiluminescent labelling and detection system was used to visualize bands. On the basis of patterns, the strains were classified into 22 different groups, with the largest containing 29 isolates. While most of the ribotypes were not exclusive to a particular source, some ribotypes were restricted to a particular geographic area or to either a human or a particular animal species. Application of the typing scheme to isolates of four independent outbreaks among animals showed that certain ribotypes predominated. The study demonstrated ribotyping to be a useful tool in epidemiological investigations of melioidosis. Images
Lew, A E; Desmarchelier, P M
Monoclonal antibodies (MAbs) to Burkholderia cepacia were produced from mice immunized with inactivated whole-cell antigen. For screening of resulting MAbs an enzyme-linked immunosorbent assay (ELISA) was used. A stable hybridoma cell line (BC-2) producing specific antibodies to a 64 kDa somatic antigen from B. cepacia was established. In ELISA and immunoblotting analysis the MAb BC-2 recognized all tested strains of B. cepacia whereas no cross-reaction with 32 Pseudomonas aeruginosa strains was found. From a wide range of other bacteria only strains of the species Burkholderia mallei, Burkholderia pseudomallei, and Burkholderia gladioli showed cross-reactions. The MAb BC-2 will be used to develop a diagnostic assay for the identification of B. cepacia and B. gladioli, important agents of nosocomial infections in immunocompromised patients suffering especially from cystic fibrosis (CF). PMID:9627054
Otterbein, C K; Splettstoesser, W D; Linde, H J; Grunow, R; Wolf, H; Finke, E J; Neubauer, H
Bacteria belonging to the genus Burkholderia are highly versatile with respect to their ecological niches and lifestyles, ranging from nodulating tropical plants to causing melioidosis and fatal infections in cystic fibrosis patients. Despite the clinical importance and agronomical relevance of Burkholderia species, information about the factors influencing their occurrence, abundance and diversity in the environment is scarce. Recent findings have demonstrated that pH is the main predictor of soil bacterial diversity and community structure, with the highest diversity observed in neutral pH soils. As many Burkholderia species have been isolated from low pH environments, we hypothesized that acid tolerance may be a general feature of this genus, and pH a good predictor of their occurrence in soils. Using a combination of environmental surveys at trans-continental and local scales, as well as in vitro assays, we show that, unlike most bacteria, Burkholderia species have a competitive advantage in acidic soils, but are outcompeted in alkaline soils. Physiological assays and diversity analysis based on 16S rRNA clone libraries demonstrate that pH tolerance is a general phenotypic trait of the genus Burkholderia. Our results provide a basis for building a predictive understanding of the biogeographical patterns exhibited by Burkholderia sp. PMID:23945027
Stopnisek, Nejc; Bodenhausen, Natacha; Frey, Beat; Fierer, Noah; Eberl, Leo; Weisskopf, Laure
Neurological melioidosis is a very rare and very few cases have been reported from India. Presentation is an extremely varied and as this disease is associated with high mortality, high index of suspicion is needed to diagnose and treat. In this context, we report a patient presenting as Guillain Barre syndrome evaluated as melioidosis.
Krovvidi, Rajesh; Mridula, Rukmini K.; Jabeen, S. A.; Meena, A. K.
Melioidosis is a zoonosis caused by the accidental pathogen Burkholderia pseudomallei, which is endemic in Southeast Asia and northern Australia. The mortality of melioidosis is 20-50% even with treatment. Suppurative lymphadenitis caused by melioidosis has been rarely encountered by clinicians practicing in endemic areas. In the majority of previously described patients, the infected lymph nodes were in the head and neck region, except for four patients who presented with unilateral, inguinal lymphadenitis. Hence, we report a case of unilateral suppurative inguinal lymphadenitis caused by B. pseudomallei in a 48-year-old lady who presented with groin swelling of 2 months duration.
Jakribettu, RP; Boloor, R; D'Souza, R; Aithala, S
Bacterial response regulators (RR) that function as transcription factors in two component signaling pathways are crucial for ensuring tight regulation and coordinated expression of the genome. Currently, consensus DNA binding sites in the promoter for very few bacterial RRs have been identified. A systematic method to characterize these DNA binding sites for RRs would enable prediction of specific gene expression patterns in response to extracellular stimuli. To identify RR DNA binding sites, we functionally activated RRs using beryllofluoride and applied them to a protein-binding microarray (PBM) to discover DNA binding motifs for RRs expressed in Burkholderia, a Gram-negative bacterial genus. We identified DNA binding motifs for conserved RRs in Burkholderia thailandensis, including KdpE, RisA, and NarL, as well as for a previously uncharacterized RR at locus BTH_II2335 and its ortholog in the human pathogen Burkholderia pseudomallei at locus BPSS2315. We further demonstrate RR binding of predicted genomic targets for the two orthologs using gel shift assays and reveal a pattern of RR regulation of expression of self and other two component systems. Our studies illustrate the use of PBMs to identify DNA binding specificities for bacterial RRs and enable prediction of gene regulatory networks in response to two component signaling. PMID:22521922
Nowak-Lovato, Kristy L; Hickmott, Alexana J; Maity, Tuhin S; Bulyk, Martha L; Dunbar, John; Hong-Geller, Elizabeth
Burkholderia is a bacterial genus comprising several pathogenic species, including two species highly pathogenic for humans, B. pseudomallei and B. mallei. B. thailandensis is a weakly pathogenic species closely related to both B. pseudomallei and B. mallei. It is used as a study model. These bacteria are able to exhibit multiple resistance mechanisms towards various families of antibiotics. By sequentially plating B. thailandensis wild type strains on chloramphenicol we obtained several resistant variants. This chloramphenicol-induced resistance was associated with resistance against structurally unrelated antibiotics including quinolones and tetracyclines. We functionally and proteomically demonstrate that this multidrug resistance phenotype, identified in chloramphenicol-resistant variants, is associated with the overexpression of two different efflux pumps. These efflux pumps are able to expel antibiotics from several families, including chloramphenicol, quinolones, tetracyclines, trimethoprim and some ?-lactams, and present a partial susceptibility to efflux pump inhibitors. It is thus possible that Burkholderia species can develop such adaptive resistance mechanisms in response to antibiotic pressure resulting in emergence of multidrug resistant strains. Antibiotics known to easily induce overexpression of these efflux pumps should be used with discernment in the treatment of Burkholderia infections.
Biot, Fabrice V.; Valade, Eric; Garnotel, Eric; Chevalier, Jacqueline; Villard, Claude; Thibault, Francois M.; Vidal, Dominique R.; Pages, Jean-Marie
The development and regulatory approval of medical countermeasures (MCMs) for the treatment and prevention of bacterial threat agent infections will require the evaluation of products in animal models. To obtain regulatory approval, these models must accurately recapitulate aspects of human disease, including, but not necessarily limited to, route of exposure, time to disease onset, pathology, immune response, and mortality. This article focuses on the state of animal model development for 3 agents for which models are largely immature: Francisella tularensis, Burkholderia mallei, and Burkholderia pseudomallei. An overview of available models and a description of scientific and regulatory gaps are provided. PMID:23628693
Stundick, M V; Albrecht, M T; Houchens, C R; Smith, A Pierce; Dreier, T M; Larsen, J C
The aim of this work was to catalog the clinical and ecoepidemiological characteristics of melioidosis in Brazil. The clinical-epidemiological features of melioidosis in Ceará are similar to those in other regions where the disease is endemic. These findings support the inclusion of this Brazilian state as part of the zone of endemicity for melioidosis.
Bandeira, Tereza J. P. G.; Cordeiro, Rossana A.; Grangeiro, Thalles B.; Lima, Rita A. C.; Ribeiro, Joyce F.; Castelo-Branco, Debora S. C. M.; Rodrigues, Jorge L. N.; Coelho, Ivo C. B.; Magalhaes, Francisco G.; Rocha, Marcos F. G.; Sidrim, Jose J. C.
Burkholderia thailandensis is used in the laboratory as a surrogate of the more virulent B. pseudomallei. Since inhalation is believed to be a natural route of infection for B. pseudomallei, many animal studies with B. pseudomallei and B. thailandensis utilize the inhalation route of exposure. The aim of the present study was to quantify the recovery efficiency of culturable B. thailandensis from several common aerosol sampling devices to ensure that collected microorganisms could be reliably recovered post-collection. The sampling devices tested included 25 mm gelatin filters, 22 mm stainless steel disks used in Mercer cascade impactors, and two types of glass impingers. The results demonstrate that while several processing methods tested resulted in significantly lower physical recovery efficiencies than other methods, it was possible to obtain culturable recovery efficiencies for B. thailandensis and physical recovery efficiencies for 1 ?m fluorescent spheres of at least 0.95 from all of the sampling media tested given an appropriate sample processing procedure. The results of the present study also demonstrated that the bubbling action of liquid media in all-glass impingers (AGIs) can result in physical loss of material from the collection medium, although additional studies are needed to verify the exact mechanisms involved. Overall, the results of this study demonstrate that the collection mechanism as well as the post-collection processing method can significantly affect the recovery from and retention of culturable microorganisms in sampling media, potentially affecting the calculated airborne concentration and any subsequent estimations of risk or dose derived from such data. PMID:22919669
Dabisch, Paul; Bower, Kristin; Dorsey, Brandi; Wronka, Loni
There are few appropriate single-copy genetic tools for most Burkholderia species, and the high level of antibiotic resistance in this genus further complicates the development of genetic tools. In addition, the utilization of resistance genes for clinically important antibiotics is prohibited for the bioterrorism agents Burkholderia pseudomallei and Burkholderia mallei, necessitating the development of additional nonantibiotic-based genetic tools. Three single-copy systems devoid of antibiotic selection based on two nonantibiotic selectable markers, tellurite resistance (Telr) and Escherichia coli aspartate-semialdehyde dehydrogenase (asdEc), were developed to facilitate genetic manipulation in Burkholderia species. These systems include one mariner transposon, a mini-Tn7-derived site-specific transposon, and six FRT reporter fusion vectors based on the lacZ, gfp, and luxCDABE reporter genes. Initially, we showed that the random mariner transposon pBT20-?bla-Telr-FRT efficiently transposed within Burkholderia cenocepacia, Burkholderia thailandensis, B. pseudomallei, and B. mallei. We then utilized the mini-Tn7-Telr-based transposon vector (mini-Tn7-Telr-betBA) and a transposase-containing helper plasmid (pTNS3-asdEc) to complement the B. thailandensis ?betBA mutation. Next, one of the FRT-lacZ fusion vectors (pFRT1-lacZ-Telr) was integrated by Flp (encoded on a helper plasmid, pCD13SK-Flp-oriT-asdEc) to construct the B. thailandensis ?betBA::FRT-lacZ-Telr reporter fusion strain. The betBA operon was shown to be induced in the presence of choline and under osmotic stress conditions by performing ?-galactosidase assays on the B. thailandensis ?betBA::FRT-lacZ-Telr fusion strain. Finally, we engineered B. thailandensis ?betBA::FRT-gfp-Telr and ?betBA::FRT-lux-Telr fusion strains by utilizing fusion vectors pFRT1-gfp-Telr and pFRT1-lux-Telr, respectively. The induction of the betBA operon by choline and osmotic stress was confirmed by performing fluorescent microscopy and bioluminescent imaging analyses.
Kang, Yun; Norris, Michael H.; Barrett, Ashley R.; Wilcox, Bruce A.; Hoang, Tung T.
Acyl-homoserine lactone (acyl-HSL) quorum-sensing signaling is common to many Proteobacteria. Acyl-HSLs are synthesized by the LuxI family of synthases, and the signal response is mediated by members of the LuxR family of transcriptional regulators. Burkholderia thailandensis is a member of a closely related cluster of three species, including the animal pathogens Burkholderia mallei and Burkholderia pseudomallei. Members of this group have similar luxI and luxR homologs, and these genes contribute to B. pseudomallei and B. mallei virulence. B. thailandensis possesses three pairs of luxI-luxR homologs. One of these pairs, BtaI2-BtaR2, has been shown to produce and respond to 3OHC10-HSL and to control the synthesis of an antibiotic. By using a markerless-exhange method, we constructed an assortment of B. thailandensis quorum-sensing mutants, and we used these mutants to show that BtaI1 is responsible for C8-HSL production and BtaI3 is responsible for 3OHC8-HSL production. We also show that a strain incapable of acyl-HSL production is capable of growth on the same assortment of carbon and nitrogen sources as the wild type. Furthermore, this mutant shows no loss of virulence compared to the wild type in mice. However, the wild type self-aggregates in minimal medium, whereas the quorum-sensing mutant does not. The wild-type aggregation phenotype is recovered by addition of the BtaI1-R1 HSL signal C8-HSL. We propose that the key function of the BtaR1-BtaI1 quorum-sensing system is to cause cells to gather into aggregates once a sufficient population has been established.
Chandler, Josephine R.; Duerkop, Breck A.; Hinz, Aaron; West, T. Eoin; Herman, Jake P.; Churchill, Mair E. A.; Skerrett, Shawn J.; Greenberg, E. Peter
Burkholderia cepacia complex and Burkholderia pseudomallei are opportunistic human pathogens. Resistance to ?-lactams among Burkholderia spp. is attributable to expression of ?-lactamases (e.g. PenA in B. cepacia complex and PenI in B. pseudomallei). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor-resistant carbapenemase, most similar to KPC-2 (the most clinically significant serine carbapenemase), whereas PenI is an extended spectrum ?-lactamase. PenA hydrolyzes ?-lactams with kcat values ranging from 0.38 ± 0.04 to 460 ± 46 s?1 and possesses high kcat/kinact values of 2000, 1500, and 75 for ?-lactamase inhibitors. PenI demonstrates the highest kcat value for cefotaxime of 9.0 ± 0.9 s?1. Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explain the lower kcat values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. Conversely, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of ?-lactamases, which can lead to the rational design of novel agents against these pathogens.
Papp-Wallace, Krisztina M.; Taracila, Magdalena A.; Gatta, Julian A.; Ohuchi, Nozomi; Bonomo, Robert A.; Nukaga, Michiyoshi
Burkholderia pseudomallei and Burkholderia thailandensis are related pathogens that invade a variety of cell types, replicate in the cytoplasm, and spread to nearby cells. We have investigated temporal and spatial requirements for virulence determinants in the intracellular life cycle, using genetic dissection and photothermal nanoblade delivery, which allows efficient placement of bacterium-sized cargo into the cytoplasm of mammalian cells. The conserved Bsa type III secretion system (T3SSBsa) is dispensable for invasion, but is essential for escape from primary endosomes. By nanoblade delivery of B. thailandensis we demonstrate that all subsequent events in intercellular spread occur independently of T3SSBsa activity. Although intracellular movement was essential for cell–cell spread by B. pseudomallei and B. thailandensis, neither BimA-mediated actin polymerization nor the formation of membrane protrusions containing bacteria was required for B. thailandensis. Surprisingly, the cryptic (fla2) flagellar system encoded on chromosome 2 of B. thailandensis supported rapid intracellular motility and efficient cell–cell spread. Plaque formation by both pathogens was dependent on the activity of a type VI secretion system (T6SS-1) that functions downstream from T3SSBsa-mediated endosome escape. A remarkable feature of Burkholderia is their ability to induce the formation of multinucleate giant cells (MNGCs) in multiple cell types. By infection and nanoblade delivery, we observed complete correspondence between mutant phenotypes in assays for cell fusion and plaque formation, and time-course studies showed that plaque formation represents MNGC death. Our data suggest that the primary means for intercellular spread involves cell fusion, as opposed to pseudopod engulfment and bacterial escape from double-membrane vacuoles.
French, Christopher T.; Toesca, Isabelle J.; Wu, Ting-Hsiang; Teslaa, Tara; Beaty, Shannon M.; Wong, Wayne; Liu, Minghsun; Schroder, Imke; Chiou, Pei-Yu; Teitell, Michael A.; Miller, Jeff F.
Many Proteobacteria use acyl-homoserine lactone-mediated quorum-sensing (QS) to activate specific sets of genes as a function of cell density. QS often controls the virulence of pathogenic species, and in fact a previous study indicated that QS was important for Burkholderia mallei mouse lung infections. To gain in-depth information on the role of QS in B. mallei virulence, we constructed and characterized a mutant of B. mallei strain GB8 that was unable to make acyl-homoserine lactones. The QS mutant showed virulence equal to that of its wild-type parent in an aerosol mouse infection model, and growth in macrophages was indistinguishable from that of the parent strain. Furthermore, we assessed the role of QS in B. mallei ATCC 23344 by constructing and characterizing a mutant strain producing AiiA, a lactonase enzyme that degrades acyl-homoserine lactones. Although acyl-homoserine lactone levels in cultures of this strain are very low, it showed full virulence. Contrary to the previous report, we conclude that QS is not required for acute B. mallei infections of mice. QS may be involved in some stage of chronic infections in the natural host of horses, or the QS genes may be remnants of the QS network in B. pseudomallei from which this host-adapted pathogen evolved.
Majerczyk, Charlotte; Kinman, Loren; Han, Tony; Bunt, Richard
BackgroundOver 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 FindingsThe 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
Bart J. Currie; Linda Ward; Allen C. Cheng
The genus Burkholderia includes over 60 species isolated from a wide range of environmental niches and can be tentatively divided into two major species clusters. The first cluster includes pathogens such as Burkholderia glumae, B. pseudomallei, and B. mallei and 17 well-studied species of the Burkholderia cepacia complex. The other recently established cluster comprises at least 29 nonpathogenic species, which in most cases have been found to be associated with plants. It was previously established that Burkholderia kururiensis, a member of the latter cluster, possesses an N-acyl homoserine lactone (AHL) quorum-sensing (QS) system designated “BraI/R,” which is found in all species of the plant-associated cluster. In the present study, two other BraI/R-like systems were characterized in B. xenovorans and B. unamae and were designated the BraI/RXEN and BraI/RUNA systems, respectively. Several phenotypes were analyzed, and it was determined that exopolysaccharide was positively regulated by the BraIR-like system in the species B. kururiensis, B. unamae, and B. xenovorans, highlighting commonality in targets. However, the three BraIR-like systems also revealed differences in targets since biofilm formation and plant colonization were differentially regulated. In addition, a second AHL QS system designated XenI2/R2 and an unpaired LuxR solo protein designated BxeR solo were also identified and characterized in B. xenovorans LB400T. The two AHL QS systems of B. xenovorans are not transcriptionally regulating each other, whereas BxeR solo negatively regulated xenI2. The XenI2/R2 and BxeR solo proteins are not widespread in the Burkholderia species cluster. In conclusion, the present study represents an extensive analysis of AHL QS in the Burkholderia plant-associated cluster demonstrating both commonalities and differences, probably reflecting environmental adaptations of the various species.
Suarez-Moreno, Zulma Rocio; Devescovi, Giulia; Myers, Mike; Hallack, Leticia; Mendonca-Previato, Lucia; Caballero-Mellado, Jesus; Venturi, Vittorio
Melioidosis is an infectious disease with a propensity for relapse, despite prolonged antibiotic eradication therapy for 12 to 20 weeks. A pharmacokinetic (PK) simulation study was performed to determine the optimal dosing of cotrimoxazole (trimethoprim-sulfamethoxazole [TMP-SMX]) used in current eradication regimens in Thailand and Australia. Data for bioavailability, protein binding, and coefficients of absorption and elimination were taken from published literature. Apparent volumes of distribution were correlated with body mass and were estimated separately for Thai and Australian populations. In vitro experiments demonstrated concentration-dependent killing. In Australia, the currently used eradication regimen (320 [TMP]/1,600 [SMX] mg every 12 h [q12h]) was predicted to achieve the PK-pharmacodynamic (PD) target (an area under the concentration-time curve from 0 to 24 h/MIC ratio of >25 for both TMP and SMX) for strains with the MIC90 of Australian strains (< or = 1/19 mg/liter). In Thailand, the former regimen of 160/800 mg q12h would not be expected to attain the target for strains with an MIC of > or = 1/19 mg/liter, but the recently implemented weight-based regimen (<40 kg [body weight], 160/800 mg q12h; 40 to 60 kg, 240/1,200 mg q12h; >60 kg, 320/1,600 mg q12h) would be expected to achieve adequate concentrations for strains with an MIC of < or = 1/19 mg/liter. The results were sensitive to the variance of the PK parameters. Prospective PK-PD studies of Asian populations are needed to optimize TMP-SMX dosing in melioidosis. PMID:19620336
Cheng, Allen C; McBryde, Emma S; Wuthiekanun, Vanaporn; Chierakul, Wirongrong; Amornchai, Premjit; Day, Nicholas P J; White, Nicholas J; Peacock, Sharon J
We have used CHEF gel electrophoresis to screen preparations of large DNA from different Burkholderia cepacia isolates for the presence of DNA species corresponding to the linearized forms of the three chromosomes of 3.4,2.5, and 0.9 Mb identified in B. cepacia strain 17616. DNA ...
Background Rhamnolipids are surface active molecules composed of rhamnose and ?-hydroxydecanoic acid. These biosurfactants are produced mainly by Pseudomonas aeruginosa and have been thoroughly investigated since their early discovery. Recently, they have attracted renewed attention because of their involvement in various multicellular behaviors. Despite this high interest, only very few studies have focused on the production of rhamnolipids by Burkholderia species. Results Orthologs of rhlA, rhlB and rhlC, which are responsible for the biosynthesis of rhamnolipids in P. aeruginosa, have been found in the non-infectious Burkholderia thailandensis, as well as in the genetically similar important pathogen B. pseudomallei. In contrast to P. aeruginosa, both Burkholderia species contain these three genes necessary for rhamnolipid production within a single gene cluster. Furthermore, two identical, paralogous copies of this gene cluster are found on the second chromosome of these bacteria. Both Burkholderia spp. produce rhamnolipids containing 3-hydroxy fatty acid moieties with longer side chains than those described for P. aeruginosa. Additionally, the rhamnolipids produced by B. thailandensis contain a much larger proportion of dirhamnolipids versus monorhamnolipids when compared to P. aeruginosa. The rhamnolipids produced by B. thailandensis reduce the surface tension of water to 42 mN/m while displaying a critical micelle concentration value of 225 mg/L. Separate mutations in both rhlA alleles, which are responsible for the synthesis of the rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid, prove that both copies of the rhl gene cluster are functional, but one contributes more to the total production than the other. Finally, a double ?rhlA mutant that is completely devoid of rhamnolipid production is incapable of swarming motility, showing that both gene clusters contribute to this phenotype. Conclusions Collectively, these results add another Burkholderia species to the list of bacteria able to produce rhamnolipids and this, by the means of two identical functional gene clusters. Our results also demonstrate the very impressive tensio-active properties these long-chain rhamnolipids possess in comparison to the well-studied short-chain ones from P. aeruginosa.
We report a case of continuous ambulatory peritoneal dialysis (CAPD)-related peritonitis. Burkholderia gladioli, as a plant pathogen, were cultured from peritoneal effluent. Peritonitis healed after intraperitoneal cefazolin and gentamicin. This case indicated that CAPD effluent may be contaminated by B. gladioli, causing CAPD-related peritonitis. On the other hand, it can be successfully treated with intraperitoneal antimicrobials without removal of the Tenckhoff catheter. PMID:23886791
Tong, YanQing; Dou, LiLi; Wang, ChuFeng
Burkholderia humi sp. nov., Burkholderia choica sp. nov., Burkholderia telluris sp. nov., Burkholderia terrestris sp. nov. and Burkholderia udeis sp. nov.: Burkholderia glathei-like bacteria from soil and rhizosphere soil.
Analysis of partial gyrB gene sequences revealed six taxa in a group of 17 Burkholderia glathei-like isolates which were further examined by (GTG)5-PCR fingerprinting, 16S rRNA gene sequence analysis, DNA-DNA hybridizations, determination of the DNA G+C content, whole-cell fatty acid analysis and an analysis of cell and colony morphology and more than 180 biochemical characteristics. The results demonstrated that one taxon consisting of three human clinical isolates represented Burkholderia zhejiangensis, a recently described methyl-parathion-degrading bacterium isolated from a wastewater-treatment system in China. The remaining taxa represented five novel species isolated from soil or rhizosphere soil samples, and could be distinguished by both genotypic and phenotypic characteristics. We therefore propose to formally classify these bacteria as Burkholderia humi sp. nov. (type strain, LMG 22934(T)?=?CCUG 63059(T)), Burkholderia choica sp. nov. (type strain, LMG 22940(T)?=?CCUG 63063(T)), Burkholderia telluris sp. nov. (type strain, LMG 22936(T)?=?CCUG 63060(T)), Burkholderia udeis sp. nov. (type strain, LMG 27134(T)?=?CCUG 63061(T)) and Burkholderia terrestris sp. nov. (type strain, LMG 22937(T)?=?CCUG 63062(T)). PMID:23959831
Vandamme, Peter; De Brandt, Evie; Houf, Kurt; Salles, Joana Falcão; Dirk van Elsas, Jan; Spilker, Theodore; Lipuma, John J
Glanders is a contagious disease caused by the Gram-negative bacillus Burkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e., crude preparations of whole cells. False-positive results obtained from other diagnostic tests utilizing crude antigens lead to financial losses to animal owners, and false-negative results can turn a risk into a possible threat. In this study, we report on the identification of diagnostic targets using bioinformatics tools for serodiagnosis of glanders. The identified gene sequences were cloned and expressed as recombinant proteins. The purified recombinant proteins of B. mallei were used in an indirect ELISA format for serodiagnosis of glanders. Two recombinant proteins, 0375H and 0375TH, exhibited 100% sensitivity and specificity for glanders diagnosis. The proteins also did not cross-react with sera from patients with the closely related disease melioidosis. The results of this investigation highlight the potential of recombinant 0375H and 0375TH proteins in specific and sensitive diagnosis of glanders.
Kumar, Subodh; Malik, Praveen
The twin arginine translocation (Tat) system in bacteria is responsible for transporting folded proteins across the cytoplasmic membrane, and in some bacteria, Tat-exported substrates have been linked to virulence. We report here that the Tat machinery is present in Burkholderia pseudomallei, B. mallei, and B. thailandensis, and we show that the system is essential for aerobic but not anaerobic growth. Switching off of the Tat system in B. thailandensis grown anaerobically resulted in filamentous bacteria, and bacteria showed increased sensitivity to some ?-lactam antibiotics. In Galleria mellonella and zebrafish infection models, the Tat conditional mutant was attenuated. The aerobic growth-restricted phenotype indicates that Tat substrates may play a functional role in oxygen-dependent energy conservation. In other bacteria, aerobic growth restriction in Tat mutants has been attributed to the inability to translocate PetA, the Rieske iron-sulfur protein which forms part of the quinol-cytochrome c oxidoreductase complex. Here, we show that PetA is not responsible for aerobic growth restriction in B. thailandensis. However, we have identified an operon encoding 2 proteins of unknown function (BTH_I2176 and BTH_I2175) that play a role in aerobic growth restriction, and we present evidence that BTH_I2176 is Tat translocated.
Wagley, Sariqa; Hemsley, Claudia; Thomas, Rachael; Moule, Madeleine G.; Vanaporn, Muthita; Andreae, Clio; Robinson, Matthew; Goldman, Stan; Wren, Brendan W.; Butler, Clive S.
Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors.
Peano, Clelia; Chiaramonte, Fabrizio; Motta, Sara; Pietrelli, Alessandro; Jaillon, Sebastien; Rossi, Elio; Consolandi, Clarissa; Champion, Olivia L.; Michell, Stephen L.; Freddi, Luca; Falciola, Luigi; Basilico, Fabrizio; Garlanda, Cecilia; Mauri, Pierluigi; De Bellis, Gianluca; Landini, Paolo
Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors. PMID:24671187
Peano, Clelia; Chiaramonte, Fabrizio; Motta, Sara; Pietrelli, Alessandro; Jaillon, Sebastien; Rossi, Elio; Consolandi, Clarissa; Champion, Olivia L; Michell, Stephen L; Freddi, Luca; Falciola, Luigi; Basilico, Fabrizio; Garlanda, Cecilia; Mauri, Pierluigi; De Bellis, Gianluca; Landini, Paolo
Annealing experiments on membrane filters were carried out with deoxyribonucleic acids (DNA) from selected strains of the nomen-species of Pseudomonas, Actinobacillus, Chromobacterium, and Micrococcus, with the use of DNA of Pseudomonas pseudomallei and A...
M. Rogul J. J. Brendle D. K. Haapala A. D. Alexander
We performed an integrated genotypic and phenotypic analysis of 128 strains of the genera Burkholderia, Rabtonia, and Pseudomonas in order to study the taxonomic structure of Burkholderia cepacia and its relation- ships with other Burkholderia species. Our data show that presumed B. cepacia strains isolated from cystic fibrosis patients belong to at least five distinct genomic species, one of which
P. VANDAMME; B. HOLMES; M. VANCANNEYT; T. COENYE; B. HOSTE; R. COOPMAN; H. REVETS; S. LAUWERS; M. GILLIS; K. KERSTERS; J. R. W. GOVAN
Infection with Burkholderia species is typically considered a contraindication leading to transplantation in cystic fibrosis (CF). However, the risks posed by different Burkholderia species on transplantation outcomes are poorly defined. We present the case of a patient with CF who underwent lung transplantation due to a severe respiratory failure from chronic airways infection with Burkholderia pyrrocinia (B. cepacia genomovar IX) and pan-resistant Pseudomonas aeruginosa. The postoperative course was complicated by recurrent B. pyrrocinia infections, ultimately lea ding to uncontrollable sepsis and death. This is the first case report in CF of Burkholderia pyrrocinia infection and lung transplantation, providing further evidence of the high risk nature of the Burkholderia species. PMID:24507071
Savi, D; De Biase, R Valerio; Amaddeo, A; Anile, M; Venuta, F; Ruberto, F; Simmonds, N; Cimino, G; Quattrucci, S
Further characterization of 32 clinical isolates originally identified as Burkholderia cepacia by biochemical and fatty acid profiling revealed the presence of 12 strains bearing partial resemblance to the closely related species Burkholderia gladioli. These strains were highly resistant to a wide range of antibiotics including ticarcillin (with or without clavulanic acid), cefsulodin, imipenem, the aminoglycosides, colistin and fosfomycin. They typically produced a red-brown pigment and possessed distinct, although quite diverse biochemical profiles. This reinforces the previous opinion that hybrids between B. cepacia and B. gladioli exist and may possess a significant pathogenic role. It also suggests that further taxonomic clarification is required in the genus Burkholderia. PMID:9069537
Baxter, I A; Lambert, P A; Simpson, I N
Phenotypic and genotypic studies revealed new tools for differentiating Burkholderia cepacia genomovar VI from Burkholderia multivorans and other B. cepacia-complex species. Hence, the name Burkholderia dolosa sp. nov. is proposed, with LMG 18943(T) (=CCUG 47727(T)) as the type strain. B. dolosa can be differentiated from other B. cepacia-complex bacteria by its inability to assimilate tryptamine, azelaic acid and salicin and by its failure to grow on the B. cepacia-selective medium PCAT. Both 16S rDNA and recA RFLP analysis revealed unique B. dolosa restriction patterns. In addition, new 16S rDNA- and recA-based PCR assays allowed its specific identification. PMID:15143009
Vermis, Karen; Coenye, Tom; LiPuma, John J; Mahenthiralingam, Eshwar; Nelis, Hans J; Vandamme, Peter
Burkholderia cenocepacia and Burkholderia multivorans are opportunistic drug-resistant pathogens that account for the majority of Burkholderia cepacia complex infections in cystic fibrosis patients and also infect other immunocompromised individuals. While they share similar genetic compositions, B. cenocepacia and B. multivorans exhibit important differences in pathogenesis. We have developed reconciled genome-scale metabolic network reconstructions of B. cenocepacia J2315 and B. multivorans ATCC 17616 in parallel (designated iPY1537 and iJB1411, respectively) to compare metabolic abilities and contextualize genetic differences between species. The reconstructions capture the metabolic functions of the two species and give insight into similarities and differences in their virulence and growth capabilities. The two reconstructions have 1,437 reactions in common, and iPY1537 and iJB1411 have 67 and 36 metabolic reactions unique to each, respectively. After curating the extensive reservoir of metabolic genes in Burkholderia, we identified 6 genes essential to growth that are unique to iPY1513 and 13 genes uniquely essential to iJB1411. The reconstructions were refined and validated by comparing in silico growth predictions to in vitro growth capabilities of B. cenocepacia J2315, B. cenocepacia K56-2, and B. multivorans ATCC 17616 on 104 carbon sources. Overall, we identified functional pathways that indicate B. cenocepacia can produce a wider array of virulence factors compared to B. multivorans, which supports the clinical observation that B. cenocepacia is more virulent than B. multivorans. The reconciled reconstructions provide a framework for generating and testing hypotheses on the metabolic and virulence capabilities of these two related emerging pathogens.
Bartell, Jennifer A.; Yen, Phillip; Varga, John J.; Goldberg, Joanna B.
Nitroaromatic compounds are toxic and potential carcinogens. In this study, a drop assay was used to detect chemotaxis toward nitroaromatic compounds for wild-type Burkholderia cepacia R34, wild-type Burkholderia sp. strain DNT, and a 2,4-dinitrotoluene (2,4-DNT) dioxygenase mutant strain (S5). The three strains are chemotactic toward 2,4,6-trinitrotoluene (TNT), 2,3-DNT, 2,4-DNT, 2,5-DNT, 2-nitrotoluene (NT), 4NT, and 4-methyl-5-nitrocatechol (4M5NC), but not toward 2,6-DNT.
Thammajun Leungsakul; Brendan G. Keenan; Barth F. Smets; Thomas K. Wood
The genomic libraries of P. mallei and P. pseudomallei species were constructed in Escherichia coli. The chromosomal DNA of P. pseudomallei C-141 strain has been cloned into the cosmid vector pHC79 and the broad host range plasmid vector pES154. The chromosomal DNA of P. mallei [symbol: see text]-5 strain has been cloned into the plasmid vector pSUP202. The recombinant clones of the genomic libraries were screened by the enzyme-linked immunoadsorbent assay (ELISA) to detect the production of Pseudomonas antigens: 28 clones were positive. Twelve recombinant strains demonstrated specific antigenic determinants of P. mallei and P. pseudomallei by immunoblotting. Cloned proteins of P. mallei and P. pseudomallei have molecular weights from 30 to 70 kD. A new method for introducing foreign genes into Pseudomonas genomes is offered. P. mallei strains with the chromosomally integrated plasmids pSM are universal recipients for ColEI-based cloning vectors. PMID:9082182
Abaev, I V; Pomerantseva, O M; Astashkin, E I; Pachkunov, D M; Rudnitski?, V Iu; Stagis, I I; Negri?, N V; Bannov, V A; Svetoch, E A
Burkholderia gladioli, a bacterial pathogen, causes a variety of infectious ailments in immunocompromised individuals. We present a case report of foreign body associated Burkholderia gladioli related abscess in an immunocompetent type 1 diabetic patient. Relevant medical literature is reviewed to understand this unusual mode of presentation of this bacterium in our patient. PMID:18626540
Waseem, Mohiuddin; Al-Sherbeeni, Suphia; Al-Malki, Mussa H; Al-Ghamdi, Mubarak S
Burkholderia gladioli is an unusual organism that has become increasingly responsible for infections in patients who are immunosuppressed, including patients who have undergone solid organ transplantation. This article presents a patient in whom a mediastinal mass due to Burkholderia gladioli developed after lung transplantation. A review of the literature is also presented. PMID:19416783
Church, Alistair C; Sivasothy, Pasupathy; Parmer, Jasvir; Foweraker, Juliet
Objectives The Burkholderia cepacia complex (Bcc) species are important opportunistic pathogens with intrinsic antibiotic resistance. They are also well known as contaminants of disinfectants, yet their biocide susceptibility has not been studied in detail. We investigated Bcc biocide susceptibility and correlated it to their taxonomy, antibiotic susceptibility and ability to form biofilms. Methods Genetically distinct Bcc strains belonging to 12 of the defined species were examined. Biocide susceptibility was assessed by (i) broth dilution MIC assays, (ii) agar growth-based MBC screens and (iii) suspension tests. Antibiotic MIC was determined by Etest® strips, and the ability to form biofilms was examined in a 96-well plate assay. Results Biocide susceptibility varied across the Bcc complex with high MIC recorded for chlorhexidine (>100 mg/L), cetylpyridinium chloride (>200 mg/L), triclosan (>500 mg/L), benzalkonium chloride (>400 mg/L) and povidone (>50?000 mg/L). Species-dependent differences were apparent only for cetylpyridinium chloride. There was no correlation between biocide susceptibility and (i) antibiotic susceptibility or (ii) the ability to form biofilms. Biocide MBC was considerably higher than the MIC (chlorhexidine, 6-fold greater; cetylpyridinium chloride, 20-fold greater). Cystic fibrosis outbreak strains (Burkholderia multivorans Glasgow strain and Burkholderia cenocepacia ET12) possessed elevated chlorhexidine resistance, and Bcc bacteria were also shown to remain viable in current commercial biocide formulations. Conclusions Bcc bacteria are resistant to a wide range of biocides and further representatives of this group should be included as reference strains in the development of new anti-infectives and commercial formulations.
Rose, Helen; Baldwin, Adam; Dowson, Christopher G.; Mahenthiralingam, Eshwar
...Clostridium, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, Ebola viruses, Marburg virus, Variola major virus (Smallpox virus), Variola minor (Alastrim), or Yersinia pestis. The final disposition...
Pseudomonas pseudomallei EBN-10 strain, previously isolated from a local pharmaceutical industry's wastewater, was spontaneously adapted to higher p-nitrophenol (PNP) levels, which then was subjected to gamma ray-induced mutagenesis; the efficient isolates hence obtained were designated as EBN-11 and EBN-12, respectively. EBN-12 mutant strain could completely mineralize PNP (100 mg\\/L) on the minimal media in 24 h while, the parent strain
Asma Rehman; Zulfiqar A. Raza; Muhammad Afzal; Zafar M. Khalid
...ACT REPORTING REQUIREMENTS AND REVIEW PROCESSES FOR MICROORGANISMS Significant New Uses for Specific Microorganisms Â§ 725.1075 Burkholderia cepacia complex. (a) Microorganism and significant new uses subject to reporting....
Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs) of Burkholderia thailandensis (B. thai) in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ?T6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans—leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.
Schwarz, Sandra; West, T. Eoin; Boyer, Frederic; Chiang, Wen-Chi; Carl, Mike A.; Hood, Rachel D.; Rohmer, Laurence; Tolker-Nielsen, Tim; Skerrett, Shawn J.; Mougous, Joseph D.
Burkholderia cepacia: current clinical issues, environmental controversies and ethical dilemmas. A.M. Jones, M.E. Dodd, A.K. Webb. #ERS Journals Ltd 2001. ABSTRACT: Burkholderia cepacia is a plant phytogen and is known as a hardy and versatile organism. Over the past two decades it has emerged as a pathogen in the cystic fibrosis (CF) community, with devastating effects. Pulmonary colonisation can lead
A. M. Jones; M. E. Dodd; A. K. Webb
...Teerawattanasook, et al, ``Increasing Incidence of Human Melioidosis in Northeast Thailand,'' American Journal of Tropical Medicine and Hygiene, vol. 82(6), pp. 1113-1117, 2010. 3. Wiersinga W.J., B.J. Currie, and S.J....
A polyphasic taxonomic study was performed on 23 strains isolated from cystic fibrosis (CF) patients in the USA. These strains were tentatively identified as Burkholderia cepacia, Burkholderia vietnamiensis and Burkholderia or Ralstonia sp. using biochemical tests and 16S rDNA-based PCR assays. Visual comparison of protein profiles indicated that they belonged to a single new group ('group 13'). The polyphasic taxonomic data showed that 18 of these strains represent a new member of the B. cepacia complex, referred to in this report as B. cepacia genomovar VI, whereas the other five strains belonged to Burkholderia multivorans. By means of biochemical tests, B. cepacia genomovar VI strains can be separated from B. cepacia genomovars I and III, Burkholderia stabilis, B. vietnamiensis and Burkholderia gladioli, but not from B. multivorans. Separation of B. cepacia genomovar VI and B. multivorans is possible using AFLP (amplified fragment length polymorphism) fingerprinting and DNA-DNA hybridizations. Retrospective analysis of epidemiological and genotypic data suggests that strains of B. cepacia genomovar VI have been involved in chronic colonization of CF patients and have been spread from person to person. PMID:11321071
Coenye, T; LiPuma, J J; Henry, D; Hoste, B; Vandemeulebroecke, K; Gillis, M; Speert, D P; Vandamme, P
Gold blot tests for rapid serodiagnosis of melioidosis were developed and evaluated with sera from 40 melioidosis patients and 159 normal controls. The sensitivity and specificity were 87.5 and 88%, respectively, for the immunoglobulin M (IgM) test and 100 and 91%, respectively, for the protein A test for IgG. Combination of the IgM gold blot and protein A gold blot yielded 97.5% sensitivity and 94.3% specificity. The tests were rapid and simple.
Kunakorn, M; Petchclai, B; Khupulsup, K; Naigowit, P
The Burkholderia cepacia complex currently comprises five genomic species, i.e., B. cepacia genomovar I, B. multivorans (formerly known as B. cepacia genomovar II), B. cepacia genomovar III, B. cepacia genomovar IV, and B. vietnamiensis (also known as B. cepacia genomovar V). In the absence of straightforward diagnostic tests for the identification of B. cepacia genomovars I, III, and IV, the last two genomic species were not formally classified as novel Burkholderia species (genomovar I contains the type strain and therefore retains the name B. cepacia). In the present study, we describe differential biochemical tests and a recA gene-based PCR assay for the routine identification of strains currently known as B. cepacia genomovar IV and propose formal classification of this organism as Burkholderia stabilis sp. nov. B. stabilis can indeed be differentiated from all other B. cepacia complex strains by the absence of beta-galactosidase activity, from strains of B. cepacia genomovars I and III and B. vietnamiensis by the inability to oxidize sucrose, and from B. multivorans by the lack of growth at 42°C. In addition, analysis with the recA gene-derived primers BCRG41 (5?-ACCGGCGAGCAGGCGCTT-3?) and BCRG42 (5?-ACGCCATCGGGCATGGCA-3?) specifically allows the detection of B. stabilis strains in a conventional PCR assay. Examination of a set of 21 B. stabilis strains by means of random amplified polymorphic DNA analysis and pulsed-field gel electrophoresis typing suggested that the genome of this organism is highly conserved, which is in sharp contrast to the generally accepted genomic diversity, variability, and plasticity among B. cepacia strains.
Vandamme, P.; Mahenthiralingam, E.; Holmes, B.; Coenye, T.; Hoste, B.; De Vos, P.; Henry, D.; Speert, D. P.
Several species belonging to the genus Burkholderia are clinically relevant, opportunistic pathogens that inhabit major environmental reservoirs. Consequently, the availability of means for adequate identification and epidemiological characterization of individual environmental or clinical isolates is mandatory. In the present communication we describe the use of the Riboprinter microbial characterization system (Qualicon, Warwick, United Kingdom) for automated ribotyping of 104 strains of Burkholderia species from diverse sources, including several publicly accessible collections. The main outcome of this analysis was that all strains were typeable and that strains of Burkholderia gladioli and of each species of the B. cepacia complex, including B. multivorans, B. stabilis, and B. vietnamiensis, were effectively discriminated. Furthermore, different ribotypes were discerned within each species. Ribotyping results were in general agreement with strain classification based on restriction fragment analysis of 16S ribosomal amplicons, but the resolution of ribotyping was much higher. This enabled automated molecular typing below the species level. Cluster analysis of the patterns obtained by ribotyping (riboprints) showed that within B. gladioli, B. multivorans, and B. cepacia genomovar VI, the different riboprints identified always clustered together. Riboprints of B. cepacia genomovars I and III, B. stabilis, and B. vietnamiensis did not show distinct clustering but rather exhibited the formation of loose assemblages within which several smaller, genomovar-specific clusters were delineated. Therefore, ribotyping proved useful for genomovar identification. Analysis of serial isolates from individual patients demonstrated that infection with a single ribotype had occurred, despite minor genetic differences that were detected by pulsed-field gel electrophoresis of DNA macrorestriction fragments. The automated approach allows very rapid and reliable identification and epidemiological characterization of strains and generates an easily manageable database suited for expansion with information on additional bacterial isolates. PMID:10790116
Brisse, S; Verduin, C M; Milatovic, D; Fluit, A; Verhoef, J; Laevens, S; Vandamme, P; Tümmler, B; Verbrugh, H A; van Belkum, A
Three strains of Gram-stain-negative, rod-shaped bacteria were isolated from Lebeckia ambigua root nodules and authenticated on this host. Based on the 16S rRNA gene sequence phylogeny, they were shown to belong to the genus Burkholderia, with the representative strain WSM3556(T) being most closely related to Burkholderia caledonica LMG 23644(T) (98.70?% 16S rRNA gene sequence similarity) and Burkholderia rhynchosiae WSM3937(T) (98.50?%). Additionally, these strains formed a distinct group in phylogenetic trees of the housekeeping genes gyrB and recA. Chemotaxonomic data, including fatty acid profiles and analysis of respiratory quinones, supported the assignment of our strains to the genus Burkholderia. Results of DNA-DNA hybridizations, MALDI-TOF MS analysis and physiological and biochemical tests allowed genotypic and phenotypic differentiation of our strains from their nearest neighbour species. Therefore, these strains represent a novel species, for which the name Burkholderia dilworthii sp. nov. is proposed, with the type strain WSM3556(T) (?=?LMG 27173(T)?=?HAMBI 3353(T)). PMID:24368690
De Meyer, Sofie E; Cnockaert, Margo; Ardley, Julie K; Van Wyk, Ben-Erik; Vandamme, Peter A; Howieson, John G
Pseudomonas aeruginosa and members of the Burkholderia cepacia complex often coexist in both the soil and the lungs of cystic fibrosis patients. To gain an understanding of how these different species affect each other's physiology when coexisting, we performed a screen to identify P. aeruginosa genes that are induced in the presence of Burkholderia: A random gene fusion library was constructed in P. aeruginosa PA14 by using a transposon containing a promoterless lacZ gene. Fusion strains were screened for their ability to be induced in the presence of Burkholderia strains in a cross-streak assay. Three fusion strains were induced specifically by Burkholderia species; all three had transposon insertions in genes known to be iron regulated. One of these fusion strains, containing a transposon insertion in gene PA4467, was used to characterize the inducing activity from Burkholderia: Biochemical and genetic evidence demonstrate that ornibactin, a siderophore produced by nearly all B. cepacia strains, can induce P. aeruginosa PA4467. Significantly, PA4467 is induced early in coculture with an ornibactin-producing but not an ornibactin-deficient B. cepacia strain, indicating that ornibactin can be produced by B. cepacia and detected by P. aeruginosa when the two species coexist. PMID:15060040
Weaver, Valerie B; Kolter, Roberto
In order to develop a detection method for the rice pathogens Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli, the phylogeny of six plant-pathogenic Burkholderia species was analysed using the combined nucleotide sequences of gyrB and rpoD. B. plantarii, B. glumae and B. gladioli formed tight monophyletic branches supported by high bootstrap probabilities. The high sequence similarity revealed a close phylogenetic relationship between B. glumae and B. plantarii. B. plantarii strains were divided into three subclusters comprising rice strains, whereas the single Vanda strain occupied a unique position in the phylogenetic tree. The gyrB and rpoD sequences of all B. glumae strains examined were highly conserved. In contrast, B. gladioli strains demonstrated a far greater sequence diversity, but this diversity did not correlate with pathovar, host plant or geographical origin of the strains. A multiplex-PCR protocol using specific primers from the gyrB sequences was designed that allowed the specific detection and identification of B. plantarii, B. glumae and B. gladioli in rice seeds infected with these pathogenic species. PMID:16627650
Maeda, Yukiko; Shinohara, Hirosuke; Kiba, Akinori; Ohnishi, Kouhei; Furuya, Naruto; Kawamura, Yoshiaki; Ezaki, Takayuki; Vandamme, Peter; Tsushima, Seiya; Hikichi, Yasufumi
Comparison of the 16S rDNA sequence of Pseudomonas antimicrobica LMG 18920T with published 16S rDNA sequences from other pseudomonads indicated that Pseudomonas antimicrobica belongs to the genus Burkholderia, with Burkholderia gladioli, Burkholderia glumae and Burkholderia plantarii as its closest neighbours. DNA-DNA hybridizations confirmed that Pseudomonas antimicrobica and Burkholderia gladioli represent the same species. Strain LMG 18920T and other Burkholderia gladioli strains were also indistinguishable by SDS-PAGE of whole-cell proteins and had similar biochemical characteristics. The whole-cell fatty acid composition, however, was different from that of other Burkholderia gladioli strains. It is concluded that Pseudomonas antimicrobica is a later synonym of Burkholderia gladioli. As Burkholderia gladioli is known to cause infections in patients with cystic fibrosis and chronic granulomatous disease, the eventual use of strain LMG 18920T as a biological control agent should be approached with caution. PMID:11155989
Coenye, T; Gillis, M; Vandamme, P
Comparative analysis of recipient activity of Pseudomonas mallei, Pseudomonas pseudomallei, and Pseudomonas cepacia strains towards naturally occurring and recombinant plasmid replicons was carried out. Autonomic broad host range vector plasmids based on RSF1010(IncQ) and pSa(IncW) replicons as well as integrative vectors based on pSUP202(Co1E1) replicon have been constructed. The study has shown that naturally occurring plasmids RSF1010(IncQ), pSa(IncW), R15(IncN), and RP4(IncP) are being efficiently transferred and stably maintained in investigated Pseudomonas strains. However, recombinant plasmids with the mini-replicon pSa which are stable in Escherichia coli have shown segregative instability in Pseudomonas strains, whereas derivatives of plasmid RSF1010 demonstrated different stability depending on the type of insertion. Plasmid pSUP202 derivative integrative vector pSM525 is efficiently introduced and stably maintained in P. mallei C-5 strain. Two vector systems for genetic manipulations in P.mallei and P.pseudomallei cells have been developed. PMID:7541512
Abaev, I V; Astashkin, E I; Pachkunov, D M; Stagis, N I; Shitov, V T; Svetoch, E A
Organisms from the Burkholderia cepacia complex are important pathogens in cystic fibrosis and are associated with increased rates of sepsis and death. These organisms comprise nine closely related species known as genomovars. B. cenocepacia (genomovar III) is the most prevalent and appears the most virulent. We investigated the biological activity of a reference panel of strains using whole-cell lysates to
Anthony De Soyza; Charles D. Ellis; C. M. Anjam Khan; Paul A. Corris; Raquel Demarco de Hormaeche
Members of the Burkholderia cepacia complex (Bcc), found in many environments, are associated with clinical infections. Examining diverse species and strains from dif- ferent environments with multilocus sequence typing, we identified >20% of 381 clinical isolates as indistinguishable from those in the environment. This finding links the natural environment with the emergence of many Bcc infections.
Adam Baldwin; Eshwar Mahenthiralingam; Pavel Drevinek; Peter Vandamme; John R. Govan; David J. Waine; John J. LiPuma; Luigi Chiarini; Claudia Dalmastri; Deborah A. Henry; David P. Speert; David Honeybourne; Martin C. J. Maiden; Chris G. Dowson
Symbiotic ß-proteobacteria not only occur in root nodules of legumes but are also found in leaves of certain Rubiaceae. The discovery of bacteria in plants formerly not implicated in endosymbiosis suggests a wider occurrence of plant-microbe interactions. Several ß-proteobacteria of the genus Burkholderia are detected in close association with tropical plants. This interaction has occurred three times independently, which suggest a recent and open plant-bacteria association. The presence or absence of Burkholderia endophytes is consistent on genus level and therefore implies a predictive value for the discovery of bacteria. Only a single Burkholderia species is found in association with a given plant species. However, the endophyte species are promiscuous and can be found in association with several plant species. Most of the endophytes are part of the plant-associated beneficial and environmental group, but others are closely related to B. glathei. This soil bacteria, together with related nodulating and non-nodulating endophytes, is therefore transferred to a newly defined and larger PBE group within the genus Burkholderia.
Verstraete, Brecht; Janssens, Steven; Smets, Erik; Dessein, Steven
The genetic structure of a population of Burkholderia (Pseudomonas) cepacia isolated from a southeastern blackwater stream was investigated by using multilocus enzyme electrophoresis to examine the allelic variation in eight structural gene loci. Overall, 213 isolates were collected at transect points along the stream continuum, from both the sediments along the bank and the water column. Multilocus enzyme electrophoresis analysis
M. G. Wise; L. J. Shimkets; J. V. McArthur
We describe a case of an adult patient with cystic fibrosis who developed chronic pulmonary infection and multiple episodes of soft tissue abscesses with Burkholderia gladioli; this organism should be added to the list of potential pathogens for individuals with cystic fibrosis. PMID:11243758
Jones, A M; Stanbridge, T N; Isalska, B J; Dodd, M E; Webb, A K
A new strain of bacterium degrading polyaromatic hydrocarbons (PAHs), Burkholderia cepacia 2A-12, was isolated from oil-contaminated soil. Of three PAHs, the isolated strain could utilize naphthalene (Nap) and phenanthrene (Phe) as a sole carbon source but not pyrene (Pyr). However, the strain could degrade Pyr when a cosubstrate such as yeast extract (YE) was supplemented. The PAH degradation rate of
Tae Jung Kim; Eun Young Lee; Youn Jung Kim; Kyung-Suk Cho; Hee Wook Ryu
Strains of the Burkholderia cepacia complex can survive within macrophages by arresting the maturation of phagocytic vacuoles. The bacteria preclude fusion of the phagosome with lysosomes by a process that is poorly understood. Using murine macrophages, we investigated the stage at which maturation is arrested and analyzed the underlying mechanism. Vacuoles containing B. cenocepacia strain J2315, an isolate of the
Kassidy K. Huynh; Jonathan D. Plumb; Gregory P. Downey; Miguel A. Valvano; Sergio Grinstein
Bacterial cell surface hydrophobicity (CSH) is an important factor governing the growth and adhesion behavior of microorganisms on non-aqueous phase liquids (NAPLs). In this work CSH and surface charge was quantified for three oil degrading Burkholderia cultures: aliphatic degrader Burkholderia cepacia (ES1) and two strains of aromatic degrading Burkholderia multivorans (NG1 and HN1) based on contact angle and zeta potential
Sinjini Chakraborty; Soumyo Mukherji; Suparna Mukherji
The main objective of this study was to determine the Burkholderia community structure associated with areas under different agricultural management and to evaluate to which extent this community structure is affected by changes in agricultural management. Two fields with distinct soil history (arable land and permanent grassland) were exposed to three agricultural management regimes (crop rotation, maize monoculture, and grassland). By using a culture-independent approach, based on a Burkholderia-specific polymerase chain reaction-denaturing gradient gel electrophoresis system, it was possible to observe the conversion of Burkholderia communities typical for permanent grassland to those of arable land after four consecutive years. However, the time needed to achieve the reverse transition, i.e., converting the Burkholderia community associated with arable land to that of grassland, was beyond the duration of the field experiment. In addition, by applying principal response curves, the direction and extent of the conversion from grassland to arable land (maize monoculture and to crop rotation) were determined. Hence, the results suggested that agricultural practices, such as fertilization and tillage, were more effective in changing the Burkholderia community structure than agricultural management regime. To determine the effect of agricultural management on the Burkholderia population with biocontrol abilities, the culturable fraction of the Burkholderia community was assessed. The areas under permanent grassland and grassland converted to maize monoculture had the highest percentages of Burkholderia strains with antagonistic activity against Rhizoctonia solani AG-3, mainly Burkholderia pyrrocinia and Burkholderia sp. LMG 22929. The isolation frequency of antagonistic isolates from arable land was extremely low. Our results indicate that (changes in) agricultural management, mainly crop rotation, affect the frequency of isolation of antagonistic Burkholderia strains and that grassland represents a reservoir of Burkholderia species with great potential for agricultural applications. PMID:16897309
Salles, J F; van Elsas, J D; van Veen, J A
We describe a case of insidious small bone osteomyelitis and soft tissue abscess with Burkholderia gladioli in a 6-year-old Caucasian boy with chronic granulomatous disease. DNA sequencing of the 16S ribosomal RNA gene confirmed the bacterial identification. Clinical cure was achieved with a combination of antimicrobial therapy and surgical debridement. A review of infections caused by Burkholderia spp., other than Burkholderia cepacia complex, in pediatric patients with chronic granulomatous disease is provided. PMID:16148855
Boyanton, Bobby L; Noroski, Lenora M; Reddy, Hari; Dishop, Megan K; Hicks, M John; Versalovic, James; Moylett, Edina H
The betaproteobacterial genus Burkholderia is known for its versatile interactions with its hosts that can range from beneficial to pathogenic. A plant-beneficial-environmental (PBE) Burkholderia cluster was recently separated from the pathogen cluster, yet still little is known about burkholderial diversity, distribution, colonization, and transmission patterns on plants. In our study, we applied a combination of high-throughput molecular and microscopic methods to examine the aforementioned factors for Burkholderia communities associated with Sphagnum mosses - model plants for long-term associations - in Austrian and Russian bogs. Analysis of 16S rRNA gene amplicons libraries revealed that most of the Burkholderia are part of the PBE group, but a minor fraction was closely related to B. glathei and B. andropogonis from the pathogen cluster. Notably, Burkholderia showed highly similar composition patterns for each moss species independent of the geographic region, and Burkholderia-specific fluorescent in situ hybridization of Sphagnum gametophytes exhibited similar colonization patterns in different Sphagnum species at multi-geographic scales. To explain these patterns, we compared the compositions of the surrounding water, gametophyte-, and sporophyte-associated microbiome at genus level and discovered that Burkholderia were present in the Sphagnum sporophyte and gametophyte, but were absent in the flark water. Therefore, Burkholderia is a part of the core microbiome transmitted from the moss sporophyte to the gametophyte. This suggests a vertical transmission of Burkholderia strains, and thus underlines their importance for the plants themselves. PMID:24391630
Bragina, Anastasia; Cardinale, Massimiliano; Berg, Christian; Berg, Gabriele
Pseudomonas pseudomallei EBN-10 strain, previously isolated from a local pharmaceutical industry's wastewater, was spontaneously adapted to higher p-nitrophenol (PNP) levels, which then was subjected to gamma ray-induced mutagenesis; the efficient isolates hence obtained were designated as EBN-11 and EBN-12, respectively. EBN-12 mutant strain could completely mineralize PNP (100 mg/L) on the minimal media in 24 h while, the parent strain utilized only 6% of it. Addition of glucose as co-substrate further increased the PNP degradation rate; however, phenol inclusion inhibited the degradation process. Ammonium sulphate was experienced as the best of the nitrogen sources used by EBN-12 mutant strain, while degrading PNP. PMID:17616887
Rehman, Asma; Raza, Zulfiqar A; Afzal, Muhammad; Khalid, Zafar M
Burkholderia species are common soil Betaproteobacteria capable of degrading recalcitrant aromatic compounds and xenobiotics. Burkholderia sp. strain LIG30 was isolated from wet tropical forest soil and is capable of utilizing lignin as a sole carbon source. Here we report the draft genome sequence of Burkholderia sp. strain LIG30.
Woo, Hannah L.; Utturkar, Sagar; Klingeman, Dawn; Simmons, Blake A.; DeAngelis, Kristen M.; Brown, Steven D.
Burkholderia species are common soil Betaproteobacteria capable of degrading recalcitrant aromatic compounds and xenobiotics. Burkholderia sp. strain LIG30 was isolated from wet tropical forest soil and is capable of utilizing lignin as a sole carbon source. Here we report the draft genome sequence of Burkholderia sp. strain LIG30. PMID:24948777
Woo, Hannah L; Utturkar, Sagar; Klingeman, Dawn; Simmons, Blake A; DeAngelis, Kristen M; Brown, Steven D; Hazen, Terry C
A quinoline-biodegrading microorganism was isolated from activated sludge of coke-oven wastewater treatment plant using quinoline as sole carbon and nitrogen source. It is a gram negative, rod-shaped and aerobic strain, which was identified as Burkholderia pickettii. The biodegradation of quinoline was carried out with this isolated strain. Analysis by high performance liquid chromatography and gas chromatography\\/mass spectrum (GC\\/MS) revealed that
Wang Jianlong; Quan Xiangchun; Han Liping; Qian Yi; Werner Hegemann
Biofilm cultures of Burkholderia cepacia complex (BCC) infection have been found to generate the nonvolatile cyanide ion. We investigated if gaseous hydrogen cyanide (HCN) was a marker of BCC infection. Selected ion flow tube mass spectrometry analysis showed HCN was not elevated in the headspace of planktonic or biofilm cultures or in the exhaled breath of adult cystic fibrosis patients with chronic BCC infection. HCN is therefore not an in vitro or in vivo marker of BCC.
Sims, Hayley; Alcock, Alice; Jones, Andrew M.; Bright-Thomas, Rowland J.; Smith, David; Spanel, Patrik; Webb, A. Kevin; Lenney, Warren
The Burkholderia cepacia complex (Bcc) is a group of genetically related environmental bacteria that can cause chronic opportunistic infections in patients with cystic fibrosis (CF) and other underlying diseases. These infections are difficult to treat due to the inherent resistance of the bacteria to antibiotics. Bacteria can spread between CF patients through social contact and sometimes cause cepacia syndrome, a fatal pneumonia accompanied by septicemia. Burkholderia cenocepacia has been the focus of attention because initially it was the most common Bcc species isolated from patients with CF in North America and Europe. Today, B. cenocepacia, along with Burkholderia multivorans, is the most prevalent Bcc species in patients with CF. Given the progress that has been made in our understanding of B. cenocepacia over the past decade, we thought that it was an appropriate time to review our knowledge of the pathogenesis of B. cenocepacia, paying particular attention to the characterization of virulence determinants and the new tools that have been developed to study them. A common theme emerging from these studies is that B. cenocepacia establishes chronic infections in immunocompromised patients, which depend more on determinants mediating host niche adaptation than those involved directly in host cells and tissue damage.
Loutet, Slade A.; Valvano, Miguel A.
A novel aerobic bacterium, designated strain RR11(T), was isolated from peat soil and was characterized by using a polyphasic taxonomic approach and identified in order to determine its taxonomic position. Strain RR11(T) is a Gram-negative, non-sporulating, motile, short-rod-shaped bacterium. 16S rRNA gene sequence analysis identified this strain as a member of the genus Burkholderia of the class Betaproteobacteria. The highest degrees of gene sequence similarity were found with Burkholderia tropica Ppe8(T) (98.0?%), B. bannensis E25(T) (97.3?%), B. ferrariae FeGI01(T) (97.1?%), B. unamae MTI-641(T) (97.1?%) and B. heleia SA41(T) (97.1?%). Strain RR11(T) had the following chemotaxonomic characteristics: the major ubiquinone was Q-8, the DNA G+C content was 60.8 mol%, the major fatty acids were C16?:?0, C19?:?0 cyclo ?8c and C17?:?0 cyclo and the polar lipid profile contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and an unknown aminophospholipid. Based on its morphological, physiological and chemotaxonomic characteristics, together with 16S rRNA gene sequence comparison results, strain RR11(T) represents a novel species, for which the name Burkholderia eburnea sp. nov. is proposed. The type strain is strain RR11(T) (?=?KEMC 7302-065(T)?=?JCM 18070(T)). PMID:24363296
Kang, Sang Rim; Srinivasan, Sathiyaraj; Lee, Sang Seob
Eleven Burkholderia cepacia-like isolates of human clinical and environmental origin were examined by a polyphasic approach including recA and 16S rRNA sequence analysis, multilocus sequence analysis (MLSA), DNA base content determination, fatty acid methyl ester analysis, and biochemical characterization. The results of this study demonstrate that these isolates represent a novel species within the B. cepacia complex (Bcc) for which we propose the name Burkholderia pseudomultivorans. The type strain is strain LMG 26883(T) (=CCUG 62895(T)). B. pseudomultivorans can be differentiated from other Bcc species by recA gene sequence analysis, MLSA, and several biochemical tests including growth at 42°C, acidification of sucrose and adonitol, lysine decarboxylase and ?-galactosidase activity, and esculin hydrolysis. PMID:23867250
Peeters, Charlotte; Zlosnik, James E A; Spilker, Theodore; Hird, Trevor J; LiPuma, John J; Vandamme, Peter
The assessment of Burkholderia diversity in agricultural areas is important considering the potential use of this genus for agronomic and environmental applications. Therefore, the aim of this work was to ascertain how plant species and land use management drive the diversity of the genus Burkholderia. In a greenhouse experiment, different crops, i.e., maize, oat, barley, and grass, were planted in
Joana Falcao Salles; Johannes Antonius van Veen; Elsas Van J. D
The Burkholderia cepacia complex (BCC) is a group of related bacterial species that are commonly isolated from environmental samples. Members of the BCC can cause respiratory infections in cystic fibrosis patients and immunocompromised individuals. We report here the genome sequence of Burkholderia cenocepacia H111, a well-studied model strain of the BCC.
Agnoli, Kirsty; Pessi, Gabriella; Suppiger, Angela; Jenul, Christian; Schmid, Nadine; Tummler, Burkhard; Pinto-Carbo, Marta
We characterized the genome of the antibiotic resistant, caseinolytic and non-hemolytic Burkholderia sp. strain TJI49, isolated from mango trees (Mangifera indica L.) with dieback disease. This isolate produced severe disease symptoms on the indicator plants. Next generation DNA sequencing and short-read assembly generated the 60X deep 7,631,934 nucleotide draft genome of Burkholderia sp. TJI49 which comprised three chromosomes and at least one mega plasmid. Genome annotation studies revealed a total 8,992 genes, out of which 8,940 were protein coding genes. Comparative genomics and phylogenetics identified Burkholderia sp. TJI49 as a distinct species of Burkholderia cepacia complex (BCC), closely related to B. multivorans ATCC17616. Genome-wide sequence alignment of this isolate with replicons of BCC members showed conservation of core function genes but considerable variations in accessory genes. Subsystem-based gene annotation identified the active presence of wide spread colonization island and type VI secretion system in Burkholderia sp. TJI49. Sequence comparisons revealed (a) 28 novel ORFs that have no database matches and (b) 23 ORFs with orthologues in species other than Burkholderia, indicating horizontal gene transfer events. Fold recognition of novel ORFs identified genes encoding pertactin autotransporter-like proteins (a constituent of type V secretion system) and Hap adhesion-like proteins (involved in cell-cell adhesion) in the genome of Burkholderia sp. TJI49. The genomic characterization of this isolate provided additional information related to the 'pan-genome' of Burkholderia species. PMID:23653265
Khan, Asifullah; Asif, Huma; Studholme, David J; Khan, Ishtiaq A; Azim, M Kamran
Expression of heterologous protein in Escherichia coli usually based on the IPTG-inducible expression systems. The use of these systems for membrane protein production, however, usually caused cytotoxic problem that affected the yield and functional characterization of the protein. Optimization of these systems for transporter protein production is time-consuming and is usually ineffective. Here, we described the use of the ribosomal promoters P(s12) from Burkholderia cenocepacia LMG16656 and from Burkholderia cepacia MBA4 for efficient expression of functional transporter protein in E. coli. These promoters were used to drive the expression of a transmembrane protein, Deh4p, which help transport monohaloacetates into B. cepacia MBA4 for metabolism. Production of Deh4p in E. coli using an IPTG-inducible promoter resulted in no expression in uninduced condition and cell lysis in the presence of IPTG. Moreover, it has been reported that IPTG increased the endogenous production of other permeases such as LacZ and MelB. Cells expressing Deh4p from a P(s12) promoter grew normally in rich medium and which did not increase the expression of other permease. Uptake of (14)C-monochloroacetic acid has confirmed the production of the transporter protein in these cells. The results showed that the constitutive ribosomal protein promoters from the Burkholderia sp. could be used for effective expression of transporter protein in E. coli without causing any detrimental and unnecessary effect. PMID:16737826
Yu, Manda; Tsang, Jimmy S H
Burkholderia cepacia complex (Bcc) is an opportunistic bacterial pathogen that causes chronic infections in people with cystic fibrosis (CF). It is a highly antibiotic resistant organism and Bcc infections are rarely cleared from patients, once they are colonized. The two most clinically relevant species within Bcc are Burkholderia cenocepacia and Burkholderia multivorans. The virulence of these pathogens has not been fully elucidated and the virulence proteins expressed during human infection have not been identified to date. Furthermore, given its antibiotic resistance, prevention of infection with a prophylactic vaccine may represent a better alternative than eradication of an existing infection. We have compared the immunoproteome of two strains each from these two species of Bcc, with the aim of identifying immunogenic proteins which are common to both species. Fourteen immunoreactive proteins were exclusive to both B. cenocepacia strains, while 15 were exclusive to B. multivorans. A total of 15 proteins were immunogenic across both species. DNA-directed RNA polymerase, GroEL, 38kDa porin and elongation factor-Tu were immunoreactive proteins expressed by all four strains examined. Many proteins which were immunoreactive in both species, warrant further investigations in order to aid in the elucidation of the mechanisms of pathogenesis of this difficult organism. In addition, identification of some of these could also allow the development of protective vaccines which may prevent colonisation.
Shinoy, Minu; Dennehy, Ruth; Coleman, Lorraine; Carberry, Stephen; Schaffer, Kirsten; Callaghan, Maire; Doyle, Sean; McClean, Siobhan
Burkholderia cocovenenans (van Damme et al. 1960) Gillis et al. 1995 and Burkholderia vandii Urakami et al. 1994 are junior synonyms of Burkholderia gladioli (Severini 1913) Yabuuchi et al. 1993 and Burkholderia plantarii (Azegami et al. 1987) Urakami et al. 1994, respectively.
Reference strains of Burkholderia cocovenenans and Burkholderia vandii were compared with strains of other Burkholderia species using SDS-PAGE of whole-cell proteins, DNA-DNA hybridization and extensive biochemical characterization. Burkholderia gladioli and B. cocovenenans were indistinguishable in the chemotaxonomic and biochemical analyses. Burkholderia plantarii and B. vandii had indistinguishable whole-cell protein patterns but the B. vandii type strain differed from B. plantarii strains in several biochemical tests. The DNA-DNA binding levels (higher than 70%) indicated that (i) B. gladioli and B. cocovenenans, and (ii) B. plantarii and B. vandii each represent a single species. It is concluded that B. cocovenenans and B. vandii are junior synonyms of B. gladioli and B. plantarii, respectively. PMID:10028245
Coenye, T; Holmes, B; Kersters, K; Govan, J R; Vandamme, P
Burkholderia cepacia is an environmental bacterium, capable of colonising vegetal and animal tissues, involved in human opportunist nosocomial infections, and above all, in pulmonary colonisations in patients with cystic fibrosis. In these patients, infection may be followed by a severe deterioration with bacteraemia, leading to death. Moreover, owing to the epidemic spread of some clones within cystic fibrosis communities, strict preventive guidelines have to be instituted. Early detection of Burkholderia cepacia colonisation is therefore essential, and requires the use of selective media. Identification by means of conventional procedures may be problematic, all the more as the previously named Burkholderia cepacia strains have been recently shown to constitute five genomovars (I to V), collectively designated the "cepacia complex", of which only three are classified as new species (II = Burkholderia multivorans; IV = Burkholderia stabilis; V = Burkholderia vietnamiensis). Moreover, closely related species, particularly Burkholderia gladioli, are also involved in cystic fibrosis. Many questions still need clarifications, regarding pathogenic mechanisms and propensity for the cystic fibrosis lung of these organisms. Antimicrobial therapeutic options for B. cepacia complex infections are limited by their innate and acquired antibiotic multiresistance. PMID:11397673
Segonds, C; Chabanon, G
Background The genus Burkholderia includes a variety of species with opportunistic human pathogenic strains, whose increasing global resistance to antibiotics has become a public health problem. In this context a major role could be played by multidrug efflux pumps belonging to Resistance Nodulation Cell-Division (RND) family, which allow bacterial cells to extrude a wide range of different substrates, including antibiotics. This study aims to i) identify rnd genes in the 21 available completely sequenced Burkholderia genomes, ii) analyze their phylogenetic distribution, iii) define the putative function(s) that RND proteins perform within the Burkholderia genus and iv) try tracing the evolutionary history of some of these genes in Burkholderia. Results BLAST analysis of the 21 Burkholderia sequenced genomes, using experimentally characterized ceoB sequence (one of the RND family counterpart in the genus Burkholderia) as probe, allowed the assembly of a dataset comprising 254 putative RND proteins. An extensive phylogenetic analysis revealed the occurrence of several independent events of gene loss and duplication across the different lineages of the genus Burkholderia, leading to notable differences in the number of paralogs between different genomes. A putative substrate [antibiotics (HAE1 proteins)/heavy-metal (HME proteins)] was also assigned to the majority of these proteins. No correlation was found between the ecological niche and the lifestyle of Burkholderia strains and the number/type of efflux pumps they possessed, while a relation can be found with genome size and taxonomy. Remarkably, we observed that only HAE1 proteins are mainly responsible for the different number of proteins observed in strains of the same species. Data concerning both the distribution and the phylogenetic analysis of the HAE1 and HME in the Burkholderia genus allowed depicting a likely evolutionary model accounting for the evolution and spreading of HME and HAE1 systems in the Burkholderia genus. Conclusion A complete knowledge of the presence and distribution of RND proteins in Burkholderia species was obtained and an evolutionary model was depicted. Data presented in this work may serve as a basis for future experimental tests, focused especially on HAE1 proteins, aimed at the identification of novel targets in antimicrobial therapy against Burkholderia species.
We developed an assay to identify Burkholderia cepacia complex genomovars by Pyrosequencing of a variable recA gene segment. Fifteen reference strains and 30 clinical isolates of B. cepacia complex were sequenced. Full 77-base pair target sequences were obtained from 44 of the 45 isolates, and BLAST queries of the sequences correctly identified the genomovar of these 44 isolates. Three Burkholderia multivorans isolates were identified as B. multivorans/Burkholderia ambifaria, indicating that additional identification methods may be needed for some B. multivorans strains. PMID:17509790
Slinger, Robert; Yan, Liying; Myers, Rene; Ramotar, Karam; St Denis, Melissa; Aaron, Shawn D
Macrophage infectivity potentiators (Mips) are immunophilin proteins and essential virulence factors for a range of pathogenic organisms. We applied a structural biology approach to characterize a Mip from Burkholderia pseudomallei (BpML1), the causative agent of melioidosis. Crystal structure and nuclear magnetic resonance analyses of BpML1 in complex with known macrocyclics and other derivatives led to the identification of a key chemical scaffold. This scaffold possesses inhibitory potency for BpML1 without the immunosuppressive components of related macrocyclic agents. Biophysical characterization of a compound series with this scaffold allowed binding site specificity in solution and potency determinations for rank ordering the set. The best compounds in this series possessed a low-micromolar affinity for BpML1, bound at the site of enzymatic activity, and inhibited a panel of homologous Mip proteins from other pathogenic bacteria, without demonstrating toxicity in human macrophages. Importantly, the in vitro activity of BpML1 was reduced by these compounds, leading to decreased macrophage infectivity and intracellular growth of Burkholderia pseudomallei. These compounds offer the potential for activity against a new class of antimicrobial targets and present the utility of a structure-based approach for novel antimicrobial drug discovery. PMID:24366729
Begley, Darren W; Fox, David; Jenner, Dominic; Juli, Christina; Pierce, Phillip G; Abendroth, Jan; Muruthi, Muigai; Safford, Kris; Anderson, Vanessa; Atkins, Kateri; Barnes, Steve R; Moen, Spencer O; Raymond, Amy C; Stacy, Robin; Myler, Peter J; Staker, Bart L; Harmer, Nicholas J; Norville, Isobel H; Holzgrabe, Ulrike; Sarkar-Tyson, Mitali; Edwards, Thomas E; Lorimer, Donald D
A quinoline-biodegrading microorganism was isolated from activated sludge of coke-oven wastewater treatment plant using quinoline as sole carbon and nitrogen source. It is a gram negative, rod-shaped and aerobic strain, which was identified as Burkholderia pickettii. The biodegradation of quinoline was carried out with this isolated strain. Analysis by high performance liquid chromatography and gas chromatography/mass spectrum (GC/MS) revealed that 2-hydroxyquinoline (2-OH-Q) was the first intermediate in the course of quinoline biodegradation. A novel immobilization carrier, that is, polyvinyl alcohol (PVA)-gauze hybrid carrier, was developed. The isolated strain was immobilized by two different immobilizing techniques and used for the quinolinerdegradation. It was found that biodegradation rate of quinoline by the microorganisms immobilized on PVA-gauze hybrid carrier was faster than that by the microorganisms immobilized in PVA gel beads. Kinetics of quinoline biodegradation by cells of Burkholderia pickettii immobilized on PVA-gauze hybrid carrier was investigated. The results demonstrate that quinoline degradation could be described by zero-order reaction rate equation when the initial quinoline concentration was in the range of 50-500 mg l(-1). PMID:12108721
Jianlong, Wang; Xiangchun, Quan; Liping, Han; Yi, Qian; Hegemann, Werner
Prevention and control of infectious diseases remains a major public health challenge and a number of highly virulent pathogens are emerging both in and beyond the hospital setting. Despite beneficial aspects such as use in biocontrol and bioremediation exhibited by members of the Burkholderia cepacia complex (Bcc) some members of this group have recently gained attention as significant bacterial pathogens due to their high levels of intrinsic antibiotic resistance, transmissibility in nosocomial settings, persistence in the presence of antimicrobials and intracellular survival capabilities. The Bcc are opportunistic pathogens and their arsenal of virulence factors includes proteases, lipases and other secreted exoproducts, including secretion system-associated effectors. Deciphering the function of virulence factors and assessment of novel therapeutic strategies has been facilitated by use of diverse non-vertebrate hosts (the fly Drosophila melanogaster, the microscopic nematode Caenorhabditis elegans, the zebrafish and the greater Galleria mellonella wax moth caterpillar larvae). Researchers are now employing sophisticated approaches to dissect the virulence determinants of Bcc with the ultimate goal being the development of novel anti-infective countermeasures. This editorial will highlight selected recent research endeavors aimed at dissecting adaptive responses and the virulence factor portfolio of Burkholderia species. PMID:22546904
Tegos, George P; Haynes, Mark K; Schweizer, Herbert P
The Burkholderia cepacia complex comprises a group of nine closely related species that have emerged as life-threatening pulmonary pathogens in immunocompromised patients, particularly individuals with cystic fibrosis or chronic granulomatous disease. Attempts to explain the genomic plasticity, adaptability and virulence of the complex have paid little attention to bacteriophages, particularly the potential contribution of lysogenic conversion and transduction. In this study, lysogeny was observed in 10 of 20 representative strains of the B. cepacia complex. Three temperate phages and five lytic phages isolated from soils, river sediments or the plant rhizosphere were chosen for further study. Six phages exhibited T-even morphology and two were lambda-like. The host range of individual phages, when tested against 66 strains of the B. cepacia complex and a representative panel of other pseudomonads, was not species-specific within the B. cepacia complex and, in some phages, included Burkholderia gladioli and Pseudomonas aeruginosa. These new data indicate a potential role for phages of the B. cepacia complex in the evolution of these soil bacteria as pathogens of plants, humans and animals, and as novel therapeutic agents. PMID:12748267
Langley, Ross; Kenna, Dervla T; Vandamme, Peter; Ure, Rebecca; Govan, John R W
Burkholderia mallei is a Gram-negative zoonotic pathogen of solipeds that can also secondarily infect human hosts causing glanders. The bacterium is highly infectious and has been considered in the past as a potential biological weapon. In Western Europe ...
D. Carmany M. A. Guelta R. Dorsey W. Swietnicki
Persons with cystic fibrosis (CF) are susceptible to chronic pulmonary infection due to certain Burkholderia species, but it is not clear whether this typically involves persistent infection with the same strain or sequential infection with distinct strains. We analyzed 1095 Burkholderia isolates recovered from serial sputum cultures from 379 patients with CF receiving care in 112 CF treatment centers in the United States. Genotyping was performed by random amplified polymorphic DNA typing or pulsed-field gel electrophoresis. Overall, a change in infecting strain was found in 24 (6.9%) of 347 patients infected with Burkholderia cepacia complex and in 3 (9%) of 32 patients infected with Burkholderia gladioli. Several patients were likely coinfected, at least transiently, with >1 B. cepacia complex strain. The potential for strain replacement during chronic infection may confound studies of the relationship between strain and clinical outcome and must be considered in designing effective infection-control practices. PMID:12955638
Bernhardt, Scott A; Spilker, Theodore; Coffey, Todd; LiPuma, John J
The introduction of bacteria into the environment for bioremediation purposes (bioaugmentation) requires analysis and monitoring of microbial population dynamics to define persistence and activity from both efficacy and risk assessment perspectives, Burkholderia cepacia G4 5223-P...
The introduction of bacteria into the environment for bioremediation purposes (bioaugmentation) requires analysis and monitoring of the persistence and activity of microbial population for efficacy and risk assessment purposes. Burkholderia cepacia G4 PR123 and PR131 constitutive...
Burkholderia cenocepacia is a gram-negative opportunistic pathogen that belongs to the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly within phagocytic cells, and some epidemic strains produce a brown melanin-like pigment that can scavenge free radicals, resulting in the attenuation of the host cell oxidative burst. In this work, we demonstrate that the brown pigment produced by B. cenocepacia C5424
Karen E. Keith; Lauren Killip; Panqing He; Graham R. Moran; Miguel A. Valvano
Burkholderia legume symbionts (also called ?-rhizobia) are ancient in origin and are the main nitrogen-fixing symbionts of species belonging to the large genus Mimosa in Brazil. We investigated the extent of the affinity between Burkholderia and species in the tribe Mimoseae by studying symbionts of the genera Piptadenia (P.), Parapiptadenia (Pp.), Pseudopiptadenia (Ps.), Pityrocarpa (Py.), Anadenanthera (A.) and Microlobius (Mi.), all of which are native to Brazil and are phylogenetically close to Mimosa, and which together with Mimosa comprise the “Piptadenia group”. We characterized 196 strains sampled from 18 species from 17 locations in Brazil using two neutral markers and two symbiotic genes in order to assess their species affiliations and the evolution of their symbiosis genes. We found that Burkholderia are common and highly diversified symbionts of species in the Piptadenia group, comprising nine Burkholderia species, of which three are new ones and one was never reported as symbiotic (B. phenoliruptrix). However, ?-rhizobia were also detected and were occasionally dominant on a few species. A strong sampling site effect on the rhizobial nature of symbionts was detected, with the symbiont pattern of the same legume species changing drastically from location to location, even switching from ? to ?-rhizobia. Coinoculation assays showed a strong affinity of all the Piptadenia group species towards Burkholderia genotypes, with the exception of Mi. foetidus. Phylogenetic analyses of neutral and symbiotic markers showed that symbiosis genes in Burkholderia from the Piptadenia group have evolved mainly through vertical transfer, but also by horizontal transfer in two species.
Bournaud, Caroline; de Faria, Sergio Miana; dos Santos, Jose Miguel Ferreira; Tisseyre, Pierre; Silva, Michele; Chaintreuil, Clemence; Gross, Eduardo; James, Euan K.; Prin, Yves; Moulin, Lionel
The species composition of a Burkholderia cepacia complex population naturally occurring in the maize rhizosphere was investigated by using both culture-dependent and culture-independent methods. B. cepacia complex isolates were recovered from maize root slurry on the two selective media Pseudomonas cepacia azelaic acid tryptamine (PCAT) and trypan blue tetracycline (TB-T) and subjected to identification by a combination of restriction fragment length polymorphism (RFLP) analysis and species-specific polymerase chain reaction (PCR) tests of the recA gene. DNA extracted directly from root slurry was examined by means of nested PCR to amplify recA gene with species-specific B. cepacia complex primers and to obtain a library of PCR amplified recA genes. Using the culture-dependent method the species Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia ambifaria and Burkholderia pyrrocinia were identified, whereas using the culture-independent method also the species Burkholderia vietnamiensis was detected. The latter method also allowed us to highlight a higher diversity within the B. cenocepacia species. In fact, by using the culture-independent method the species B. cenocepacia recA lineages IIIA and IIID besides B. cenocepacia recA lineage IIIB were detected. Moreover, higher heterogeneity of recA RFLP patterns was observed among clones assigned to the species B. cenocepacia than among B. cenocepacia isolates from selective media. PMID:16232288
Pirone, Luisa; Chiarini, Luigi; Dalmastri, Claudia; Bevivino, Annamaria; Tabacchioni, Silvia
This poster presents information on the inactivation of select bioterrorist agents. Information will be presented on chlorine disinfection of vegetative cells of Brucella suis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis and endos...
The Burkholderia cepacia complex (Bcc) is comprised of at least 10 closely related species of Gram-negative proteobacteria that are associated with infections in certain groups of immunocompromised individuals, particularly those with cystic fibrosis. Infections in humans tend to occur in the lungs, which present an iron-restricted environment to a prospective pathogen, and accordingly members of the Bcc appear to possess efficient mechanisms for iron capture. These bacteria specify up to four different types of siderophore (ornibactin, pyochelin, cepabactin and cepaciachelin) that employ the full repertoire of iron-binding groups present in most naturally occurring siderophores. Members of the Bcc are also capable of utilising some exogenous siderophores that they are not able to synthesise. In addition to siderophore-mediated mechanisms of iron uptake, the Bcc possess mechanisms for acquiring iron from haem and from ferritin. The Bcc therefore appear to be well-equipped for life in an iron-poor environment. PMID:17295049
Thomas, Mark S
Burkholderia gladioli has been reported as colonizing the airways of patients with cystic fibrosis (CF) but has not previously been associated with adverse outcome. We describe six patients with CF in whom the same strain of B. gladioli, on the basis of ribotyping and biochemical characteristics, was grown in their sputum. Acquisition of this organism was followed by a fatal outcome in all six patients; one had a rapid decline in respiratory status and another developed fulminant B. gladioli bacteremia. Evidence suggests that patient-to-patient transmission of the organism occurred, and supports nosocomial infection in the ward and/or outpatient clinic despite general and stringent infection-control measures. This is the first report of adverse clinical outcome following sputum colonization with B. gladioli, and the first to demonstrate person-to-person transmission. PMID:9105090
Wilsher, M L; Kolbe, J; Morris, A J; Welch, D F
Mixotrophic metabolism in Burkholderia kururiensis subsp. thiooxydans subsp. nov., a facultative chemolithoautotrophic thiosulfate oxidizing bacterium isolated from rhizosphere soil and proposal for classification of the type strain of Burkholderia kururiensis as Burkholderia kururiensis subsp. kururiensis subsp. nov.
A thiosulfate-oxidizing facultative chemolithoautotrophic Burkholderia sp. strain ATSB13(T) was previously isolated from rhizosphere soil of tobacco plant. Strain ATSB13(T) was aerobic, Gram-staining-negative, rod shaped and motile by means of sub-terminal flagellum. Strain ATSB13(T) exhibited mixotrophic growth in a medium containing thiosulfate plus acetate. A phylogenetic study based on 16S rRNA gene sequence analysis indicated that strain ATSB13(T) was most closely related to Burkholderia kururiensis KP23(T) (98.7%), Burkholderia tuberum STM678(T) (96.5%) and Burkholderia phymatum STM815(T) (96.4%). Chemotaxonomic data [G+C 64.0 mol%, major fatty acids, C(18:1) omega7c (28.22%), C(16:1) omega7c/15 iso 2OH (15.15%), and C(16:0) (14.91%) and Q-8 as predominant respiratory ubiquinone] supported the affiliation of the strain ATSB13(T) within the genus Burkholderia. Though the strain ATSB13(T) shared high 16S rRNA gene sequence similarity with the type strain of B. kururiensis but considerably distant from the latter in terms of several phenotypic and chemotaxonomic characteristics. DNA-DNA hybridization between strain ATSB13(T) and B. kururiensis KP23(T) was 100%, and hence, it is inferred that strain ATSB13(T) is a member of B. kururiensis. On the basis of data obtained from this study, we propose that B. kururiensis be subdivided into B. kururiensis subsp. kururiensis subsp. nov. (type strain KP23(T) = JCM 10599(T) = DSM 13646(T)) and B. kururiensis subsp. thiooxydans subsp. nov. (type strain ATSB13(T) = KACC 12758(T)). PMID:19841903
Anandham, Rangasamy; Indira Gandhi, Pandiyan; Kwon, Soon Wo; Sa, Tong Min; Kim, Yong Ki; Jee, Hyeong Jin
Plant roots and shoots harbor complex bacterial communities. Early seed and plantlet colonization plays a key role in determining which bacterial populations will successfully invade plant tissues, yet the mechanisms enabling plants to select for beneficial rather than harmful populations are largely unknown. In this study, we demonstrate a role of oxalate as a determinant in this selection process, using members of the genus Burkholderia as model organisms. Oxalotrophy, i.e., the ability to use oxalate as a carbon source, was found to be a property strictly associated with plant-beneficial species of the Burkholderia genus, while plant pathogenic (B. glumae, B. plantarii) or human opportunistic pathogens (Burkholderia cepacia complex strains) were unable to degrade oxalate. We further show that oxalotrophy is required for successful plant colonization by the broad host endophyte Burkholderia phytofirmans PsJN: an engineered ?oxc mutant, which lost the ability to grow on oxalate, was significantly impaired in early colonization of both lupin and maize compared with the wild-type. This work suggests that in addition to the role of oxalate in heavy metal tolerance of plants and in virulence of phytopathogenic fungi, it is also involved in specifically recruiting plant-beneficial members from complex bacterial communities.
Kost, Thomas; Stopnisek, Nejc; Agnoli, Kirsty; Eberl, Leo
Burkholderia sp. strain RPE67 is a bacterial symbiont isolated from a field-collected bean bug, Riptortus pedestris. To understand the genetic basis of the insect-microbe symbiosis, we performed whole-genome sequencing of the Burkholderia strain, revealing an 8.69-Mb genome consisting of three chromosomes and three plasmids. PMID:24948758
Takeshita, Kazutaka; Shibata, Tomoko F; Nikoh, Naruo; Nishiyama, Tomoaki; Hasebe, Mitsuyasu; Fukatsu, Takema; Shigenobu, Shuji; Kikuchi, Yoshitomo
Burkholderia sp. strain RPE67 is a bacterial symbiont isolated from a field-collected bean bug, Riptortus pedestris. To understand the genetic basis of the insect-microbe symbiosis, we performed whole-genome sequencing of the Burkholderia strain, revealing an 8.69-Mb genome consisting of three chromosomes and three plasmids.
Takeshita, Kazutaka; Shibata, Tomoko F.; Nikoh, Naruo; Nishiyama, Tomoaki; Hasebe, Mitsuyasu; Fukatsu, Takema; Shigenobu, Shuji
The Burkholderia cepacia complex (BCC) is a group of closely related bacteria that are responsible for respiratory infections in immunocompromised humans, most notably those with cystic fibrosis (CF). We report the genome sequences for Burkholderia cenocepacia ET12 lineage CF isolates K56-2 and BC7.
Varga, John J.; Losada, Liliana; Zelazny, Adrian M.; Kim, Maria; McCorrison, Jamison; Brinkac, Lauren; Sampaio, Elizabeth P.; Greenberg, David E.; Singh, Indresh; Heiner, Cheryl; Ashby, Meredith; Nierman, William C.; Holland, Steven M.
Burkholderia sp. strain SJ98 has the chemotactic activity towards nitroaromatic and chloronitroaromatic compounds. Recently our group published draft genome of strain SJ98. In this study, we further sequence and annotate the genome of stain SJ98 to exploit the potential of this bacterium. We specifically annotate its chemotaxis genes and methyl accepting chemotaxis proteins. Genome of Burkholderia sp. SJ98 was annotated using PGAAP pipeline that predicts 7,268 CDSs, 52 tRNAs and 3 rRNAs. Our analysis based on phylogenetic and comparative genomics suggest that Burkholderia sp. YI23 is closest neighbor of the strain SJ98. The genes involved in the chemotaxis of strain SJ98 were compared with genes of closely related Burkholderia strains (i.e. YI23, CCGE 1001, CCGE 1002, CCGE 1003) and with well characterized bacterium E. coli K12. It was found that strain SJ98 has 37 che genes including 19 methyl accepting chemotaxis proteins that involved in sensing of different attractants. Chemotaxis genes have been found in a cluster along with the flagellar motor proteins. We also developed a web resource that provides comprehensive information on strain SJ98 that includes all analysis data (http://crdd.osdd.net/raghava/genomesrs/burkholderia/).
Kumar, Shailesh; Vikram, Surendra; Raghava, Gajendra Pal Singh
We isolated and identified a gibberellin-producing Burkholderia sp. KCTC 11096 from agricultural field soils. The culture filtrate of plant growth promoting rhizobacteria (PGPR) significantly increased the germination and growth of lettuce and Chinese cabbage seeds. The ethyl acetate extract of the PGPR culture showed significantly higher rate of lettuce seed germination and growth as compared to the distilled water treated control. The ethyl acetate fraction of the Burkholderia sp. was subjected to bioassay-guided isolation and we obtained for the first time from a Burkholderia sp. the plant growth promoting compound rhizonin A (1), which was characterized through NMR and MS techniques. Application of various concentrations of 1 significantly promoted the lettuce seed germination as compared to control. PMID:22759911
Kang, Sang-Mo; Khan, Abdul Latif; Hussain, Javid; Ali, Liaqat; Kamran, Muhammad; Waqas, Muhammad; Lee, In-Jung
The genus Burkholderia comprises 19 species, including Burkholderia vietnamiensis which is the only known N2-fixing species of this bacterial genus. The first isolates of B. vietnamiensis were recovered from the rhizosphere of rice plants grown in a phytotron, but its existence in natural environments and its geographic distribution were not reported. In the present study, most N2-fixing isolates recovered from the environment of field-grown maize and coffee plants cultivated in widely separated regions of Mexico were phenotypically identified as B. cepacia using the API 20NE system. Nevertheless, a number of these isolates recovered from inside of maize roots, as well as from the rhizosphere and rhizoplane of maize and coffee plants, showed similar or identical features to those of B. vietnamiensis TVV75T. These features include nitrogenase activity with 10 different carbon sources, identical or very similar nifHDK hybridization patterns, very similar protein electrophoregrams, identical amplified 16S rDNA restriction (ARDRA) profiles, and levels of DNA-DNA reassociation higher than 70% with total DNA from strain TVV75T. Although the ability to fix N2 is not reported to be a common feature among the known species of the genus Burkholderia, the results obtained show that many diazotrophic Burkholderia isolates analyzed showed phenotypic and genotypic features different from those of the known N2-fixing species B. vietnamiensis as well as from those of B. kururiensis, a bacterium identified in the present study as a diazotrophic species. DNA-DNA reassociation assays confirmed the existence of N2-fixing Burkholderia species different from B. vietnamiensis. In addition, this study shows the wide geographic distribution and substantial capability of N2-fixing Burkholderia spp. for colonizing diverse host plants in distantly separated environments.
Estrada-De Los Santos, Paulina; Bustillos-Cristales, Rocio; Caballero-Mellado, Jesus
Performances of several commercial test systems were reviewed to determine their relative levels of accuracy in identifying Burkholderia cepacia complex isolates recovered from cystic fibrosis sputum culture. Positive predictive values ranged from 71 to 98%; negative predictive values ranged from 50 to 82%. All systems misidentified B. cepacia complex. The species most frequently misidentified as B. cepacia was Burkholderia gladioli. These data support the results of previous studies that recommend confirmatory testing, including the use of DNA-based methods, for sputum culture isolates presumptively identified as B. cepacia. PMID:10921992
Shelly, D B; Spilker, T; Gracely, E J; Coenye, T; Vandamme, P; LiPuma, J J
In the present review, we describe and compare the molecular mechanisms that are involved in the regulation of biofilm formation by Pseudomonas putida,?Pseudomonas fluorescens,?Pseudomonas aeruginosa and Burkholderia cenocepacia. Our current knowledge suggests that biofilm formation is regulated by cyclic diguanosine-5'-monophosphate (c-di-GMP), small RNAs (sRNA) and quorum sensing (QS) in all these bacterial species. The systems that employ c-di-GMP as a second messenger regulate the production of exopolysaccharides and surface proteins which function as extracellular matrix components in the biofilms formed by the bacteria. The systems that make use of sRNAs appear to regulate the production of exopolysaccharide biofilm matrix material in all these species. In the pseudomonads, QS regulates the production of extracellular DNA, lectins and biosurfactants which all play a role in biofilm formation. In B.?cenocepacia?QS regulates the expression of a large surface protein, lectins and extracellular DNA that all function as biofilm matrix components. Although the three regulatory systems all regulate the production of factors used for biofilm formation, the molecular mechanisms involved in transducing the signals into expression of the biofilm matrix components differ between the species. Under the conditions tested, exopolysaccharides appears to be the most important biofilm matrix components for P.?aeruginosa, whereas large surface proteins appear to be the most important biofilm matrix components for P.?putida,?P.?fluorescens, and B.?cenocepacia. PMID:24592823
Fazli, Mustafa; Almblad, Henrik; Rybtke, Morten Levin; Givskov, Michael; Eberl, Leo; Tolker-Nielsen, Tim
Burkholderia mallei, the etiologic agent of glanders, are Category B select agents with biothreat potential, and yet effective therapeutic treatments are lacking. In this study, we showed that CpG administration increased survival, demonstrating protection in the murine glanders model. Bacterial recovery from infected lungs, liver and spleen was significantly reduced in CpG-treated animals as compared with non-treated mice. Reciprocally, lungs of CpG-treated infected animals were infiltrated with higher levels of neutrophils and inflammatory monocytes, as compared to control animals. Employing the B. mallei bioluminescent strain CSM001 and the Neutrophil-Specific Fluorescent Imaging Agent, bacterial dissemination and neutrophil trafficking were monitored in real-time using multimodal in vivo whole body imaging techniques. CpG-treatment increased recruitment of neutrophils to the lungs and reduced bioluminescent bacteria, correlating with decreased bacterial burden and increased protection against acute murine glanders. Our results indicate that protection of CpG-treated animals was associated with recruitment of neutrophils prior to infection and demonstrated, for the first time, simultaneous real time in vivo imaging of neutrophils and bacteria. This study provides experimental evidence supporting the importance of incorporating optimized in vivo imaging methods to monitor disease progression and to evaluate the efficacy of therapeutic treatment during bacterial infections.
Mott, Tiffany M.; Johnston, R. Katie; Vijayakumar, Sudhamathi; Estes, D. Mark; Motamedi, Massoud; Sbrana, Elena; Endsley, Janice J.; Torres, Alfredo G.
The Burkholderia cepacia complex (Bcc) comprises strains with a virulence potential toward immunocompromised patients as well as plant growth–promoting rhizobacteria (PGPR). Owing to the link between quorum sensing (QS) and virulence, most studies among Bcc species have been directed toward QS of pathogenic bacteria. We have investigated the QS of B. ambifaria, a PGPR only infrequently recovered from patients. The cepI gene, responsible for the synthesis of the main signaling molecule N-octanoylhomoserine lactone (C8-HSL), was inactivated. Phenotypes of the B. ambifaria cepI mutant we observed, such as increased production of siderophores and decreased proteolytic and antifungal activities, are in agreement with those of other Bcc cepI mutants. The cepI mutant was then used as background strain for a whole-genome transposon-insertion mutagenesis strategy, allowing the identification of 20 QS-controlled genes, corresponding to 17 loci. The main functions identified are linked to antifungal and antimicrobial properties, as we have identified QS-controlled genes implicated in the production of pyrrolnitrin, burkholdines (occidiofungin-like molecules), and enacyloxins. This study provides insights in the QS-regulated functions of a PGPR, which could lead to beneficial potential biotechnological applications.
Chapalain, Annelise; Vial, Ludovic; Laprade, Natacha; Dekimpe, Valerie; Perreault, Jonathan; Deziel, Eric
The genetic structure of a population of Burkholderia (Pseudomonas) cepacia isolated from a southeastern blackwater stream was investigated by using multilocus enzyme electrophoresis to examine the allelic variation in eight structural gene loci. Overall, 213 isolates were collected at transect points along the stream continuum, from both the sediments along the bank and the water column. Multilocus enzyme electrophoresis analysis revealed 164 distinct electrophoretic types, and the mean genetic diversity of the entire population was 0.574. Genetic diversity values did not vary spatially along the stream continuum. From a canonical discriminant analysis, Mahalonobis distances (measurements of genetic similarity between populations) revealed significant differences among the subpopulations at the sediment sampling points, suggesting bacterial adaptation to a heterogeneous (or patchy) microgeographical environment. Multilocus linkage disequilibrium analysis of the isolates revealed only limited association between alleles, suggesting frequent recombination, relative to binary fission, in this population. Furthermore, the dendrogram created from the data of this study and the allele mismatch distribution are typical of a population characterized by extensive genetic mixing. We suggest that B. cepacia be added to the growing list of bacteria that are not obligatorily clonal. 41 refs., 5 figs., 3 tabs.
Wise, M.G.; Shimkets, L.J. [Univ. of Georgia, Athens, GA (United States); McArthur, J.V. [Savannah River Ecology Lab., Aiken, SC (United States)
Background Burkholderia gladioli pathovar cocovenenans (BGC) is responsible for sporadic food-poisoning outbreaks with high morbidity and mortality in Asian countries. Little is known about the regulation of virulence factor and toxin production in BGC, and studies in this bacterium have been hampered by lack of genetic tools. Findings Establishment of a comprehensive antibiotic susceptibility profile showed that BGC strain ATCC33664 is susceptible to a number of antibiotics including aminoglycosides, carbapenems, fluoroquinolones, tetracyclines and trimethoprim. In this study, we established that gentamicin, kanamycin and trimethoprim are good selection markers for use in BGC. Using a 10 min method for preparation of electrocompetent cells, the bacterium could be transformed by electroporation at high frequencies with replicative plasmids containing the pRO1600-derived origin of replication. These plasmids exhibited a copy number of > 100 in BGC. When co-conjugated with a transposase expressing helper plasmid, mini-Tn7 vectors inserted site- and orientation-specifically at a single glmS-associated insertion site in the BGC genome. Lastly, a Himar1 transposon was used for random transposon mutagenesis of BGC. Conclusions A series of genetic tools previously developed for other Gram-negative bacteria was adapted for use in BGC. These tools now facilitate genetic studies of this pathogen and allow establishment of toxin biosynthetic pathways and their genetic regulation.
The genus Burkholderia is a heterogeneous group with extraordinary nutritional versatility and which occupies a diversity of niches. In recent decades, members of Burkholderia have been shown to be active participants in plant-microbe interactions, imparting beneficial effects as plant-growth-promoting bacteria (PGPB) or as pathogens. The study of quorum sensing and cell-density-dependent gene regulation, which play an important role in host colonization and pathogenesis, is extremely important in such a versatile organism. We report the identification and characterization by thin-layer chromatography (TLC) of N-acyl homoserine lactone (HSL) quorum sensing signal molecules by PGP Burkholderia. The Burkholderia spp. strains CBMB40, CBPB-HOD and CBPB-HIM investigated in this study were isolated from rice and possessed one or more PGP characteristics. Culture extracts of these strains contained detectable levels of hexanoyl (C(6)-), heptanoyl (C(7)-) and octanoyl (C(8)-) HSLs. Burkholderia sp. strain CBMB40 produced an additional molecule that migrated along decanoyl (C(10)-) HSL. Inoculation of HSL-producing Burkholderia strains through seed bacterization to canola stimulated root elongation. Signal molecules produced by Burkholderia strains could also be detected in planta, as determined by plate assays and TLC analysis of plant extracts. This study advances the hypothesis that signaling molecules by PGPB in planta might play a substantial role in increasing the pathogen resistance of plants. PMID:17350232
Poonguzhali, Selvaraj; Madhaiyan, Munusamy; Sa, Tongmin
Exposure to microorganisms is considered an environmental factor that can contribute to Type 1 diabetes. Insulin-binding proteins (IBPs) on microorganisms may induce production of antibodies that can react with the human insulin receptor (HIR) with possible consequences in developing a diabetic autoimmune response against HIR and insulin. The interaction of insulin with microorganisms was studied by screening 45 microbial species for their ability to bind insulin. Binding assays were performed using labelled insulin to identify insulin-binding components on the microorganisms. Burkholderia multivorans and Burkholderia cenocepacia isolated from patients with cystic fibrosis (CF) and the fish pathogen Aeromonas salmonicida were the only strains of those tested, which showed insulin-binding components on their cell surfaces. Further work with A. salmonicida suggested that the insulin-binding activity of A. salmonicida is due to the A-layer. A mutant of A. salmonicida lacking the A-layer showed binding, but at a much reduced rate suggesting another insulin-binding component in addition to the high affinity of the A-protein. Soluble protein lysates were subjected to Western ligand blotting using peroxidase-labelled insulin to detect IBPs. Two positive IBPs were apparent at approximately 30 and 20 kDa in lysates from Burkholderia strains, but no IBP was detected in A. salmonicida lysates. PMID:22171975
Nisr, Raid B; Moody, A John; Gilpin, Martyn L
A novel genotype for the initial steps of the oxidative degradation of dibenzothiophene(DBT) is described in a Burkholderia sp. strain isolated from a drain receiving oil refinery wastewater. The strain is capable of transforming DBT with significant efficiency when compared to other microorganisms. Its genotype was discovered by investigating insertional mutants of genes involved in DBT degradation by the Kodama
Simona Di Gregorio; Chiara Zocca; Stephan Sidler; Annita Toffanin; Daniela Lizzari; Giovanni Vallini
The main objective of this study was to determine the Burkholderia community structure associated with areas under different agricultural management and to evaluate to which extent this community structure is affected by changes in agricultural management. Two fields with distinct soil history (arable land and permanent grassland) were exposed to three agricultural management regimes (crop rotation, maize monoculture, and grassland).
J. F. Salles; J. D. van Elsas; J. A. van Veen
The nematode Caenorhabditis elegans may be killed by certain pathogenic bacteria and thus is a model organism for studying interactions between bacteria and animal hosts. However, growing nematodes on prey bacteria may influence their susceptibility to potential pathogens. A method of axenic nematode culture was developed to isolate and quantify interactions between C. elegans and potentially pathogenic strains of the Burkholderia cepacia complex. Studying these dynamics in liquid solution rather than on agar surfaces minimized nematode avoidance behavior and resolved more differences among isolates. Most isolates of B. cenocepacia, B. ambifaria and B. cepacia caused 60–80% mortality of nematodes after 7 days, whereas isolates of B. multivorans caused less mortality (<25%) and supported nematode reproduction. However, some B. cenocepacia isolates recovered from chronic infections were much less virulent (5–28% mortality). As predicted, prior diet altered the outcome of interactions between nematodes and bacteria. When given the choice between Burkholderia and E. coli as prey on agar, axenically raised nematodes initially preferred most lethal Burkholderia isolates to E. coli as a food source, but this was not the case for nematodes fed E. coli, which avoided toxic Burkholderia. This food preference was associated with the cell-free supernatant and thus secreted compounds likely mediated bacterial-nematode interactions. This model, which isolates interactions between bacteria and nematodes from the effects of prior feeding, demonstrates that bacteria can influence nematode behavior and their susceptibility to pathogens.
Cooper, Vaughn S.; Carlson, Wendy A.; LiPuma, John J.
We investigated a cluster of Burkholderia cepacia complex colonization in ventilated pediatric patients. Isolates from 15 patients, 2 sink drains, and several ventilator components were found to belong to a single B cenocepacia clone. Hospital tap water used during oral and tracheostomy care was identified as the most likely mechanism for transmission. PMID:21664002
Lucero, Cynthia A; Cohen, Adam L; Trevino, Ingrid; Rupp, Angela Hammer; Harris, Michelle; Forkan-Kelly, Sinead; Noble-Wang, Judith; Jensen, Bette; Shams, Alicia; Arduino, Matthew J; LiPuma, John J; Gerber, Susan I; Srinivasan, Arjun
The biodegradation rate of hydrocarbons is an important consideration determining the time scale for bioremediation in oil-contaminated environments. Two naturally occurring bacterial cultures, Exiguobacterium aurantiacum and Burkholderia cepacia, were capable of utilizing diesel oil as the sole source of carbon and energy by induction of hydrophobic cell surfaces with water contact angle greater than 70°. The cultures demonstrated good degradation
Gita Mohanty; Suparna Mukherji
Adherence of Burkholderia cepacia to cells of the respiratory tract of patients with cystic fibrosis (CF) appears to be a necessary precondition for colonization and infection. To date, no effective anti-adhesive strategy has been devised for preventing B. cepacia infection in CF patients. It was found in this study that B. cepacia adhered to respiratory epithelial cells both in vitro
Cheng-Hsun Chiu; Simon Wong; Robert E. W. Hancock; David P. Speert; Edward Jenner
Plant growth promoting rhizobacteria (PGPR) has been a focus of research for its potential as an eco-friendly alternative to chemical fertilizers in the agriculture industry. In current study, the effect of culture suspension (CS) of a novel gibberellins (GAs) producing bacterial strain Burkholderia sp. KCTC 11096BP, was observed on shoot length, shoot fresh and dry biomass, root fresh and dry
Sang-Mo Kang; Muhammad Hamayun; Gil-Jae Joo; Abdul Latif Khan; Yoon-Ha Kim; Sang-Kuk Kim; Hyung-Jin Jeong; In-Jung Lee
BACKGROUND: Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed
Jason NR Hamlin; Ruhi AM Bloodworth; Silvia T Cardona
The bean bug Riptortus pedestris is specifically associated with the Burkholderia gut symbiont and acquires the symbiont from the environment every generation. Here, we investigated the infective dose of the symbiont by experimental administration. The 50% infective dose was remarkably low, only 80 cells, indicating efficient colonization of the symbiont. PMID:23291548
Kikuchi, Yoshitomo; Yumoto, Isao
BACKGROUND: Rhamnolipids are surface active molecules composed of rhamnose and ?-hydroxydecanoic acid. These biosurfactants are produced mainly by Pseudomonas aeruginosa and have been thoroughly investigated since their early discovery. Recently, they have attracted renewed attention because of their involvement in various multicellular behaviors. Despite this high interest, only very few studies have focused on the production of rhamnolipids by Burkholderia
Danielle Dubeau; Eric Déziel; Donald E Woods; François Lépine
A 2-Chloro-4-nitrophenol (2C4NP) degrading bacterial strain designated as RKJ 800 was isolated from a pesticide contaminated site of India by enrichment method and utilized 2C4NP as sole source of carbon and energy. The stoichiometric amounts of nitrite and chloride ions were detected during the degradation of 2C4NP. On the basis of thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry, chlorohydroquinone (CHQ) and hydroquinone (HQ) were identified as major metabolites of the degradation pathway of 2C4NP. Manganese dependent HQ dioxygenase activity was observed in the crude extract of 2C4NP induced cells of the strain RKJ 800 that suggested the cleavage of the HQ to ?-hydroxymuconic semialdehyde. On the basis of the 16S rRNA gene sequencing, strain RKJ 800 was identified as a member of genus Burkholderia. Our studies clearly showed that Burkholderia sp. RKJ 800 degraded 2-chloro-4-nitrophenol via hydroquinone pathway. The pathway identified in a gram negative bacterium, Burkholderia sp. strain RKJ 800 was differed from previously reported 2C4NP degradation pathway in another gram-negative Burkholderia sp. SJ98. This is the first report of the formation of CHQ and HQ in the degradation of 2C4NP by any gram-negative bacteria. Laboratory-scale soil microcosm studies showed that strain RKJ 800 is a suitable candidate for bioremediation of 2C4NP contaminated sites.
Arora, Pankaj Kumar
The bean bug Riptortus pedestris is specifically associated with the Burkholderia gut symbiont and acquires the symbiont from the environment every generation. Here, we investigated the infective dose of the symbiont by experimental administration. The 50% infective dose was remarkably low, only 80 cells, indicating efficient colonization of the symbiont.
Burkholderia cepacia DBO1 is able to utilize phthalate as the sole source of carbon and energy for growth. Two overlapping cosmid clones containing the genes for phthalate degradation were isolated from this strain. Subcloning and activity analysis localized the genes for phthala...
Burkholderia pyrrocinia CH-67 was isolated from forest soil as a biocontrol agent to be utilized in agriculture. Here, we report the 8.05-Mb draft genome sequence of this bacterium. Its genome contains genes involved in biosynthesis of secondary metabolites and plant growth promotion, which may contribute to probiotic effects on plants.
Song, Ju Yeon; Kwak, Min-Jung; Lee, Kwang Youll; Kong, Hyun Gi; Kim, Byung Kwon; Kwon, Soon-Kyeong
BACKGROUND: The genus Burkholderia includes a variety of species with opportunistic human pathogenic strains, whose increasing global resistance to antibiotics has become a public health problem. In this context a major role could be played by multidrug efflux pumps belonging to Resistance Nodulation Cell-Division (RND) family, which allow bacterial cells to extrude a wide range of different substrates, including antibiotics.
Elena Perrin; Marco Fondi; Maria Cristiana Papaleo; Isabel Maida; Silvia Buroni; Maria Rosalia Pasca; Giovanna Riccardi; Renato Fani
Burkholderia kururiensis M130 is one of the few characterized rice endophytes and was isolated from surface-sterilized rice roots. This bacterium shows strong growth-promoting effects, being able to increase rice yields. Here we present its draft genome sequence, which contains important traits for endophytic life and plant growth promotion.
Coutinho, Bruna Goncalves; Passos da Silva, Daniel; Previato, Jose Osvaldo
Biosynthetic studies with (13)C-labelled acetates and methionine revealed that the infamous, food-related toxin bongkrekic acid from Burkholderia gladioli is a polyketide with acetate-derived beta-branches and a carboxylate terminus derived from the methyl group of an acetate. PMID:20237660
Rohm, Barbara; Scherlach, Kirstin; Hertweck, Christian
We report two cases of protracted keratitis complicated by corneal ulcer. Burkholderia gladioli, primarily known as a plant pathogen, was cultured from corneal swabs. The epithelial defects healed after extended adequate antibiotic therapy. Despite identical patterns of both strains in restriction fragment length polymorphism analysis, a common infection source was not detectable. PMID:18434558
Lestin, Franka; Kraak, Robert; Podbielski, Andreas
The risk of infection for cystic fibrosis patients from Burkholderia cepacia complex pathogens is of increasing concern to doctors and scientists. This paper reports on how these patients perceive and manage the risk of cepacia infection using Douglas and Calvez's (1990) typology of four cultures of the community (the central community, dissenting enclaves, isolates, and individualists) and Douglas' works on
Karen Lowton; Jonathan Gabe
Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF) pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division) efflux pumps are known to be among the mediators of multidrug resistance in Gram-negative bacteria. Since the significance of the 16 RND efflux systems present in B. cenocepacia (named RND-1 to -16) has been only partially determined, the aim of this work was to analyze mutants of B. cenocepacia strain J2315 impaired in RND-4 and RND-9 efflux systems, and assess their role in the efflux of toxic compounds. The transcriptomes of mutants deleted individually in RND-4 and RND-9 (named D4 and D9), and a double-mutant in both efflux pumps (named D4-D9), were compared to that of the wild-type B. cenocepacia using microarray analysis. Microarray data were confirmed by qRT-PCR, phenotypic experiments, and by Phenotype MicroArray analysis. The data revealed that RND-4 made a significant contribution to the antibiotic resistance of B. cenocepacia, whereas RND-9 was only marginally involved in this process. Moreover, the double mutant D4-D9 showed a phenotype and an expression profile similar to D4. The microarray data showed that motility and chemotaxis-related genes appeared to be up-regulated in both D4 and D4–D9 strains. In contrast, these gene sets were down-regulated or expressed at levels similar to J2315 in the D9 mutant. Biofilm production was enhanced in all mutants. Overall, these results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing additional phenotypic traits important for pathogenesis.
Pasca, Maria Rosalia; Longo, Francesca; Emiliani, Giovanni; Fondi, Marco; Perrin, Elena; Decorosi, Francesca; Viti, Carlo; Giovannetti, Luciana; Leoni, Livia; Fani, Renato; Riccardi, Giovanna; Mahenthiralingam, Eshwar; Buroni, Silvia
The Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacteria that are ubiquitous in the environment and have emerged as opportunistic pathogens in immunocompromised patients. The primary patient populations infected with Bcc include individuals with cystic fibrosis (CF), as well as those with chronic granulomatous disease (CGD). While Bcc infection in CF is better characterized than in CGD, these two genetic diseases are not obviously similar and it is currently unknown if there is any commonality in host immune defects that is responsible for the susceptibility to Bcc. CF is caused by mutations in the CF transmembrane conductance regulator, resulting in manifestations in various organ systems, however the major cause of morbidity and mortality is currently due to bacterial respiratory infections. CGD, on the other hand, is a genetic disorder that is caused by defects in phagocyte NADPH oxidase. Because of the defect in CGD, phagocytes in these patients are unable to produce reactive oxygen species, which results in increased susceptibility to bacterial and fungal infections. Despite this significant defect in microbial clearance, the spectrum of pathogens frequently implicated in infections in CGD is relatively narrow and includes some bacterial species that are considered almost pathognomonic for this disorder. Very little is known about the cause of the specific susceptibility to Bcc over other potential pathogens more prevalent in the environment, and a better understanding of specific mechanisms required for bacterial virulence has become a high priority. This review will summarize both the current knowledge and future directions related to Bcc virulence in immunocompromised individuals with a focus on the roles of bacterial factors and neutrophil defects in pathogenesis.
Porter, Laura A.; Goldberg, Joanna B.
Burkholderia glumae is a motile plant pathogenic bacterium that has multiple polar flagella and one LuxR/LuxI-type quorum sensing (QS) system, TofR/TofI. A QS-dependent transcriptional regulator, QsmR, activates flagellar master regulator flhDC genes. FlhDC subsequently activates flagellar gene expression in B. glumae at 37°C. Here, we confirm that the interplay between QS and temperature is critical for normal polar flagellar morphogenesis in B. glumae. In the wild-type bacterium, flagellar gene expression and flagellar number were greater at 28°C compared to 37°C. The QS-dependent flhC gene was significantly expressed at 28°C in two QS-defective (tofI::? and qsmR::?) mutants. Thus, flagella were present in both tofI::? and qsmR::? mutants at 28°C, but were absent at 37°C. Most tofI::? and qsmR::? mutant cells possessed polar or nonpolar flagella at 28°C. Nonpolarly flagellated cells processing flagella around cell surface of both tofI::? and qsmR::? mutants exhibited tumbling and spinning movements. The flhF gene encoding GTPase involved in regulating the correct placement of flagella in other bacteria was expressed in QS mutants in a FlhDC-dependent manner at 28°C. However, FlhF was mislocalized in QS mutants, and was associated with nonpolar flagellar formation in QS mutants at 28°C. These results indicate that QS-independent expression of flagellar genes at 28°C allows flagellar biogenesis, but is not sufficient for normal polar flagellar morphogenesis in B. glumae. Our findings demonstrate that QS functions together with temperature to control flagellar morphogenesis in B. glumae.
Jang, Moon Sun; Goo, Eunhye; An, Jae Hyung; Kim, Jinwoo; Hwang, Ingyu
Isolation of multiple carbon monoxide (CO)-oxidizing Burkholderia strains and detection by culture-independent approaches suggest that Burkholderia may be an important component of CO-oxidizing communities in Hawaiian volcanic deposits. The absolute and relative abundance of the bacteria in these communities remains unknown, however. In this study, a quantitative PCR (Q-PCR) approach has been developed to enumerate Burkholderia coxL genes (large subunit of carbon monoxide dehydrogenase). This represents the first attempt to enumerate coxL genes from CO oxidizers in environmental samples. coxL copy numbers have been determined for samples from three sites representing a vegetation gradient on a 1959 volcanic deposit that included unvegetated cinders (bare), edges of vegetated sites (edge), and sites within tree stands (canopy). Q-PCR has also been used to estimate copy numbers of Betaproteobacteria 16S rRNA gene copy numbers and total Bacteria 16S rRNA. coxL genes could not be detected in the bare site (detection limit, ?4.7 × 103 copies per reaction) but average 1.0 × 108 ± 2.4 × 107 and 8.6 × 108 ± 7.6 ×107 copies g?1 (dry weight) in edge and canopy sites, respectively, which differ statistically (P = 0.0007). Average Burkholderia coxL gene copy numbers, expressed as a percentage of total Bacteria 16S rRNA gene copy numbers, are 6.2 and 0.7% for the edge and canopy sites, respectively. Although the percentage of Burkholderia coxL is lower in the canopy site, significantly greater gene copy numbers demonstrate that absolute abundance of coxL increases in vegetated sites and contributes to the expansion of CO oxidizer communities during biological succession on volcanic deposits.
Weber, C. F.; King, G. M.
Burkholderia mallei, a category B biothreat agent, is a facultative intracellular pathogen that causes the zoonotic disease glanders. The B. mallei VirAG two-component regulatory system activates the transcription of approximately 60 genes, including a la...
D. M. Waag J. Shanks K. B. Spurgers M. N. Burtnick P. Brett
Background: Burkholderia mallei is an understudied biothreat agent responsible for glanders which can be lethal in humans and animals. Research with this pathogen has been hampered in part by constraints of Select Agent regulations for safety reasons. Who...
B. J. Luft C. M. Ronning D. DeShazer L. R. Schiater S. E. Schutzer
Recent research has provided evidence that interference with bacterial cell-to-cell signaling is a promising strategy for the development of novel antimicrobial agents. Here we report on the computer-aided design of novel compounds that specifically inhibit an N-acyl-homoserine lactone-dependent communication system that is widespread among members of the genus Burkholderia. This genus comprises more than 30 species, many of which are important pathogens of animals and humans. Over the past few years, several Burkholderia species, most notably Burkholderia cenocepacia, have emerged as important opportunistic pathogens causing severe pulmonary deterioration in persons with cystic fibrosis. As efficient treatment of Burkholderia infections is hampered by the inherent resistance of the organisms to a large range of antibiotics, novel strategies for battling these pathogens need to be developed. Here we show that compounds targeting the B. cenocepacia signaling system efficiently inhibit the expression of virulence factors and attenuate the pathogenicity of the organism.
Riedel, Kathrin; Kothe, Manuela; Kramer, Bernd; Saeb, Wael; Gotschlich, Astrid; Ammendola, Aldo; Eberl, Leo
The nutrition-versatility of Burkholderia sp. strain USM (JCM 15050) has initiated the studies on the use of this bacterium for polyhydroxyalkanoate (PHA) production. To date, the Burkholderia sp. has been reported to synthesize 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxy-4-methylvalerate monomers. In this study, the PHA biosynthetic genes of this strain were successfully cloned and characterized. The PHA biosynthetic cluster of this strain consisted of a PHA synthase (phaC), ?-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthesis regulator (phaR). The translated products of these genes revealed identities to corresponding proteins of Burkholderia vietnamiensis (99–100?%) and Cupriavidus necator H16 (63–89%). Heterologous expression of phaCBs conferred PHA synthesis to the PHA-negative Cupriavidus necator PHB¯4, confirming that phaCBs encoded functionally active protein. PHA synthase activity measurements revealed that the crude extracts of C. necator PHB¯4 transformant showed higher synthase activity (243 U/g) compared to that of wild-types Burkholderia sp. (151 U/g) and C. necator H16 (180 U/g). Interestingly, the transformant C. necator PHB¯4 harbouring Burkholderia sp. PHA synthase gene accumulated poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with 4-hydroxybutyrate monomer as high as up to 87?mol% from sodium 4-hydroxybutyrate. The wild type Burkholderia sp. did not have the ability to produce this copolymer.
The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining. PMID:24192802
Velapatiño, Billie; Zlosnik, James E A; Hird, Trevor J; Speert, David P
Using a computational pipeline based on similarity networks reconstruction we analysed the 1133 genes of the Burkholderia vietnamiensis (Bv) G4 five plasmids, showing that gene and operon duplication played an important role in shaping the plasmid architecture. Several single/multiple duplications occurring at intra- and/or interplasmids level involving 253 paralogous genes (stand-alone, clustered or operons) were detected. An extensive gene/operon exchange between plasmids and chromosomes was also disclosed. The larger the plasmid, the higher the number and size of paralogous fragments. Many paralogs encoded mobile genetic elements and duplicated very recently, suggesting that the rearrangement of the Bv plastic genome is ongoing. Concerning the "molecular habitat" and the "taxonomical status" (the Preferential Organismal Sharing) of Bv plasmid genes, most of them have been exchanged with other plasmids of bacteria belonging (or phylogenetically very close) to Burkholderia, suggesting that taxonomical proximity of bacterial strains is a crucial issue in plasmid-mediated gene exchange. PMID:24576463
Maida, Isabel; Fondi, Marco; Orlandini, Valerio; Emiliani, Giovanni; Papaleo, Maria Cristiana; Perrin, Elena; Fani, Renato