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

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

Accurate and Rapid Identification of the Burkholderia pseudomallei Near-Neighbour, Burkholderia ubonensis, Using Real-Time PCR

Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc), a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdown’s medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdown’s agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdown’s-positive colonies that are not B. pseudomallei

BurkDiff: A Real-Time PCR Allelic Discrimination Assay for Burkholderia Pseudomallei and B. mallei

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

Within-Host Evolution of Burkholderia pseudomallei in Four Cases of Acute Melioidosis

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

The academy for future science faculty:randomized controlled trial of theory-driven coaching to shape development and diversity of early-career scientists

Background: Approaches to training biomedical scientists have created a talented research community. However, they have failed to create a professional workforce that includes many racial and ethnic minorities and women in proportion to their representation in the population or in PhD training. This is particularly true at the faculty level. Explanations for the absence of diversity in faculty ranks can be found in social science theories that reveal processes by which individuals develop identities, experiences, and skills required to be seen as legitimate within the profession. Methods/Design: Using the social science theories of Communities of Practice, Social Cognitive Career Theory, identity formation, and cultural capital, we have developed and are testing a novel coaching-based model to address some of the limitations of previous diversity approaches. This coaching intervention (The Academy for Future Science Faculty) includes annual in-person meetings of students and trained faculty Career Coaches, along with ongoing virtual coaching, group meetings and communication. The model is being tested as a randomized controlled trial with two cohorts of biomedical PhD students from across the U.S., one recruited at the start of their PhDs and one nearing completion. Stratification into the experimental and control groups, and to coaching groups within the experimental arms, achieved equal numbers of students by race, ethnicity and gender to the extent possible. A fundamental design element of the Academy is to teach and make visible the social science principles which highly influence scientific advancement, as well as acknowledging the extra challenges faced by underrepresented groups working to be seen as legitimate within the scientific communities. Discussion: The strategy being tested is based upon a novel application of the well-established principles of deploying highly skilled coaches, selected and trained for their ability to develop talents of others. This coaching model is intended to be a complement, rather than a substitute, for traditional mentoring in biomedical research training, and is being tested as such

Diversity of 16S-23S rDNA Internal Transcribed Spacer (ITS) Reveals Phylogenetic Relationships in Burkholderia pseudomallei and Its Near-Neighbors

Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species

Colony morphology of MSMB isolates on Ashdown’s Agar (ASA) compared to <i>B</i>. <i>pseudomallei</i> MSHR305.

<p>ASA is considered a selective growth medium for <i>Burkholderia pseudomallei</i>. In this study, at least 20 different species demonstrated the ability to grow on ASA including multiple <i>Burkholderia</i> speices as well as <i>Ralstonia</i>, <i>Cupriavidus</i>, and <i>Panoraea</i>. Many different colony morphologies were observed during this study as evident in the pictures (a) proposed <i>B</i>. <i>humptydooensis</i>MSMB43, (b) unknown <i>Burkholderia spp</i>. MSMB 175, (c) <i>B</i>. <i>multivorans</i> MSMB105, (d) <i>B</i>. <i>thailandensis</i> MSMB60, (e) <i>B</i>. <i>ubonensis</i> MSMB153, (f) <i>B</i>. <i>pseudomallei</i> MSHR 305. Each strain was grown on ASA for 72 hours at 37°C aerobically.</p