Date Published: June 29, 2015
Publisher: Public Library of Science
Author(s): Jennifer L. Ginther, Mark Mayo, Stephanie D. Warrington, Mirjam Kaestli, Travis Mullins, David M. Wagner, Bart J. Currie, Apichai Tuanyok, Paul Keim, Ruifu Yang. http://doi.org/10.1371/journal.pntd.0003892
Abstract: 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.
Partial Text: The Burkholderia genus contains a large number of species, with currently over 60 species identified . Recently, the division of the genus Burkholderia has been proposed that breaks the genus into two genera. Burkholderia includes the clinical and phytopathogenic species whereas Paraburkholderia includes environmental species . This Burkholderia genus occupies a large range of environments including soil, plants, rhizospheres, mammalian hosts, and water . The ability of Burkholderia to occupy various ecological niches is undoubtedly due to the large genome size with up to three chromosomes documented in some B. cepacia complex organisms , and two chromosomes in five members of the Pseudomallei group including B. pseudomallei, B. mallei, B. thailandensis, proposed B. humptydooensis, and B. oklahomensis. Soil is a common habitat for Burkholderia bacteria and microbiologists are only beginning to uncover the complex nature of microbial communities in soil . Continued sampling of the soil has uncovered many novel Burkholderia species in recent years [6–9]. This discovery of novel species across a wide geographic range has caused scientists to begin studying the role of Burkholderia in the soil community, and interactions with co-inhabitants .
In this study, we used a Burkholderia specific recA genotyping scheme to identify unknown environmental isolates from Northern Australia that resemble B. pseudomallei morphology on ASA. Many Burkholderia species throughout the B. cepacia complex and Pseudomallei group were identified during this study. A majority of species identified belong to the B. ubonensis species based upon recA phylogenetics. B. ubonensis was only recently discovered in Australian soil  and our study further supports that this species is found in great abundance in both soil and water throughout the Darwin region of Australia. B. ubonensis was recently found to have antagonistic activity against B.pseudomallei by an unspecified bacteriocin or bacteriocin-like inhibitory substance in Papua New Guinea . An investigation into the B. ubonensis isolates from this study would determine if these isolates contain the same ability to inhibit B. pseudomallei growth as found in Papua New Guinea and further develop our understanding the role of B. ubonensis in the environment. Information from this study and the prevalence of Burkholderia species within endemic regions will continue to advance our knowledge of the bacterial composition of the natural environment. This will allow scientists to develop more specific molecular assays for targeting such agents as B. pseudomallei by understanding the background and potential for false positive results. Continued sampling efforts should be conducted to obtain an even deeper understanding of the geographic distribution of Burkholderia in this region. It is important to note that the methods in this study included using selective media, ASA, to isolate the B. pseudomallei near neighbors. There is a potential some novel near neighbors were not discovered due to the use of this media.