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Identification and characterization of near-neighbor species are critical to the development of robust molecular diagnostic tools for biothreat agents. One such agent,  Burkholderia pseudomallei , a soil bacterium and the causative agent of melioidosis, is lacking in this area because of its genomic diversity and widespread geographic distribution. The  Burkholderia  genus contains over 60 species and occupies a large range of environments including soil, plants, rhizospheres, water, animals and humans. The identification of novel species in new locations necessitates the need to identify the true global distribution of  Burkholderia  species, especially the members that are closely related to  B .  pseudomallei . In our current study, we used the  Burkholderia -specific  recA  sequencing assay to analyze environmental samples from the Darwin region in the Northern Territory of Australia where melioidosis is endemic.  Burkholderia recA  PCR negative samples were further characterized using 16s  rRNA  sequencing for species identification. Phylogenetic analysis demonstrated that over 70% of the bacterial isolates were identified as  B .  ubonensis  indicating that this species is common in the soil where  B .  pseudomallei  is endemic. Bayesian phylogenetic analysis reveals many novel branches within the  B .  cepacia  complex, one novel  B .  oklahomensis -like species, and one novel branch containing one isolate that is distinct from all other samples on the phylogenetic tree. During the analysis with  recA  sequencing, we discovered 2 single nucleotide polymorphisms in the reverse priming region of  B .  oklahomensis . A degenerate primer was developed and is proposed for future use. We conclude that the  recA  sequencing technique is an effective tool to classify  Burkholderia  and identify soil organisms in a melioidosis endemic area.

Identification of Burkholderia pseudomallei Near-Neighbor Species in the Northern Territory of Australia