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

Citation: Peddayelachagiri BV, Paul S, Nagaraj S, Gogoi M, Sripathy MH, Batra HV (2016) Prevalence and Identification of Burkholderia pseudomallei and Near-Neighbor Species in the Malabar Coastal Region of India. PLoS Negl Trop Dis 10(9): e0004956. doi:10.1371/journal.pntd.0004956
Published: September 15, 2016

Abstract
bps_closeAccurate 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/μl 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.

Author Summary: With the growing list of Burkholderia species, which represents a genus of bacteria with economical, health and biowarfare importance, the need for specific and efficient detection systems is also growing. Burkholderia pseudomallei is reported to be endemic in India. However, limited prevalence studies have been conducted in the country to determine the presence of this pathogen in environment. The high level of phenotypic and genotypic similarities of this bacterium with other species of the genus stands as a difficult challenge to differentiate them. Several molecular detection systems have been reported in this field, with a range of identification potential restricted to Burkholderia cepacia complex. The present study addresses the former mentioned challenges by performing a surveillance study in Malabar coastal region of South India wherein we encountered 22 strains of Burkholderia. Furthermore, we determined the utility of a novel gene for specific detection of the whole Burkholderia genus by developing a novel bdha based PCR assay. The study design includes extensive bioinformatic analyses and comparative genomics for the development of the described compound detection tool. The study also includes the evaluation of the genus-specific gene of its efficiency in specifically identifying the Burkholderia species by nucleotide sequencing based method. The novel genus-specific gene is also evaluated for its role in phylogeny of Burkholderia species in comparison with that of earlier reported recA and 16S rDNA genes.

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