Project description:Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei (the Bptm group) are close relatives with very different lifestyles: B. pseudomallei is an opportunistic pathogen, B. thailandensis is a nonpathogenic saprophyte, and B. mallei is a host-restricted pathogen. The acyl-homoserine lactone quorum-sensing (QS) systems of these three species show a high level of conservation. We used transcriptome sequencing (RNA-seq) to define the quorum-sensing regulon in each species, and we performed a cross-species analysis of the QS-controlled orthologs. Our analysis revealed a core set of QS-regulated genes in all three species, as well as QS-controlled factors shared by only two species or unique to a given species. This global survey of the QS regulons of B. pseudomallei, B. thailandensis, and B. mallei serves as a platform for predicting which QS-controlled processes might be important in different bacterial niches and contribute to the pathogenesis of B. pseudomallei and B. mallei.
Project description:We used whole-genome sequencing to evaluate 69 independent colonies of Burkholderia pseudomallei isolated from seven body sites of a patient with acute disseminated melioidosis. Fourteen closely related genotypes were found, providing evidence for the rapid in vivo diversification of B. pseudomallei after inoculation and systemic spread.
Project description:We report a case of melioidosis in China and offer a comparison of 5 commercial detection systems for Burkholderia pseudomallei. The organism was misidentified by the VITEK 2 Compact, Phoenix, VITEK mass spectrometry, and API 20NE systems but was eventually identified by the Bruker Biotyper system and 16S rRNA sequencing.
Project description:Accurate identification of pathogens with biowarfare importance requires detection tools that specifically differentiate them from near-neighbor species. Burkholderia pseudomallei, the causative agent of a fatal disease melioidosis, is one such biothreat agent whose differentiation from its near-neighbor species is always a challenge. This is because of its phenotypic similarity with other Burkholderia species which have a wide spread geographical distribution with shared environmental niches. Melioidosis is a major public health concern in endemic regions including Southeast Asia and northern Australia. In India, the disease is still considered to be emerging. Prevalence surveys of this saprophytic bacterium in environment are under-reported in the country. A major challenge in this case is the specific identification and differentiation of B. pseudomallei from the growing list of species of Burkholderia genus. The objectives of this study included examining the prevalence of B. pseudomallei and near-neighbor species in coastal region of South India and development of a novel detection tool for specific identification and differentiation of Burkholderia species. Briefly, we analyzed soil and water samples collected from Malabar coastal region of Kerala, South India for prevalence of B. pseudomallei. The presumptive Burkholderia isolates were identified using recA PCR assay. The recA PCR assay identified 22 of the total 40 presumptive isolates as Burkholderia strains (22.72% and 77.27% B. pseudomallei and non-pseudomallei Burkholderia respectively). In order to identify each isolate screened, we performed recA and 16S rDNA sequencing. This two genes sequencing revealed that the presumptive isolates included B. pseudomallei, non-pseudomallei Burkholderia as well as non-Burkholderia strains. Furthermore, a gene termed D-beta hydroxybutyrate dehydrogenase (bdha) was studied both in silico and in vitro for accurate detection of Burkholderia genus. The optimized bdha based PCR assay when evaluated on the Burkholderia isolates of this study, it was found to be highly specific (100%) in its detection feature and a clear detection sensitivity of 10 pg/?l of purified gDNA was recorded. Nucleotide sequence variations of bdha among interspecies, as per in silico analysis, ranged from 8 to 29% within the target stretch of 730 bp highlighting the potential utility of bdha sequencing method in specific detection of Burkholderia species. Further, sequencing of the 730 bp bdha PCR amplicon of each Burkholderia strain isolated could differentiate the species and the data was comparable with recA sequence data of the strains. All sequencing results obtained were submitted to NCBI database. Bayesian phylogenetic analysis of bdha in comparison with recA and 16S rDNA showed that the bdha gene provided comparable identification of Burkholderia species.
Project description:The sequences for Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis have been deposited in the DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases under the accession numbers AY312416 and AY312417 respectively. The intracellular pathogen Burkholderia pseudomallei is the causative agent of tropical melioidosis, and Burkholderia thailandensis is a closely-related Gram-negative bacterium that does not cause serious disease. Like other bacteria, the major outer membrane (OM) porins of Burkholderia strains, Bps Omp38 and Bth Omp38 may have roles in antibiotic resistance and immunity. We purified both proteins and found them to be immunologically related, SDS-resistant, heat-sensitive trimers with M (r) of approx. 110000. In functional liposome-swelling assays, both proteins showed similar permeabilities for small sugar molecules, compatible with a pore diameter of between 1.2 and 1.6 nm. Secondary structure analysis by FTIR (Fourier-transform infrared) spectroscopy revealed almost identical spectra with predominantly beta-sheet structures, typical of bacterial porins. MALDI-TOF (matrix-assisted laser-desorption ionization-time of flight) MS and ESI/MS (electrospray ionization MS) analysis of each protein showed extensive sequence similarities to the OpcP1 porin from Burkholderia cepacia (later found to be 76.5% identical). Based on information from the incomplete B. pseudomallei genome-sequencing project, the genes encoding Omp38 were identified and amplified by PCR from B. pseudomallei and B. thailandensis genomic DNA. The nucleotide sequences are 99.7% identical, and the predicted processed proteins are 100% identical. Topology prediction and molecular modelling suggest that this newly-isolated and cloned porin is a 16-stranded beta-barrel and the external loops of the protein could be important determinants of the immune response to infection.
Project description:A PCR procedure for the discrimination of Burkholderia mallei and Burkholderia pseudomallei was developed. It is based on the nucleotide difference T 2143 C (T versus C at position 2143) between B. mallei and B. pseudomallei detected in the 23S rDNA sequences. In comparison with conventional methods the procedure allows more rapid identification at reduced risk for infection of laboratory personnel.
Project description:<h4>Background</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.