Abstract
BackgroundThe facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing ~18,000 generations.ResultsB. pseudomallei contains a preponderance of tandem repeat loci throughout its genome, many of which are duplicated elsewhere in the genome. The majority of these loci are composed of repeat motif lengths of 6 to 9 bp with 4 to 10 repeat units and are predominately located in intergenic regions of the genome. Across geographically diverse B. pseudomallei and B.mallei isolates, the 32 VNTR loci displayed between 7 and 28 alleles, with Nei's diversity values ranging from 0.47 and 0.94. Mutation rates for these loci are comparable (>10-5 per locus per generation) to that of the most diverse tandemly repeated regions found in other less diverse bacteria.ConclusionThe frequency, location and duplicate nature of tandemly repeated regions within the B. pseudomallei genome indicate that these tandem repeat regions may play a role in generating and maintaining adaptive genomic variation. Multiple-locus VNTR analysis revealed extensive diversity within the global isolate set containing B. pseudomallei and B. mallei, and it detected genotypic differences within clonal lineages of both species that were identical using previous typing methods. Given the health threat to humans and livestock and the potential for B. pseudomallei to be released intentionally, MLVA could prove to be an important tool for fine-scale epidemiological or forensic tracking of this increasingly important environmental pathogen.
Highlights
The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals
The estimation of mutation rates will allow for future epidemiological studies that model the transmission of melioidosis in natural populations, similar to published studies on plague [26]. In this manuscript we describe a multiple-locus variablenumber tandem repeat (VNTR) analysis (MLVA) genotyping system in which 32 independent, tandemly inserted repeated motifs identified in the B. pseudomallei K96243 genome are amplified using fluorescently labeled primers in multiplexed PCRs and separated using capillary electrophoresis
Distribution and location of tandem repeats A χ2 goodness-of-fit test of the "observed" B. pseudomallei tandem repeat distribution to an "expected" Poisson distribution was significant for both the large (p < 0.001) and small chromosomes (p < 0.001) using 10 Kb intervals (Figure 3)
Summary
The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. The environmental saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to tropical regions of Southeast Asia and northern Australia. Due to its highly infectious nature and ability to infect via aerosol, it was used as a biological weapon during World War I and World War II [10,11]. It is listed as a Category B biothreat agent by the CDC [2]
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