Melioidosis is a disease of humans and animals that is caused by the saprophytic bacterium Burkholderia pseudomallei. Once thought to be confined to certain locations, the known presence of B. pseudomallei is expanding as more regions of endemicity are uncovered. There is no vaccine for melioidosis, and even with antibiotic administration, the mortality rate is as high as 40% in some regions that are endemic for the infection. Despite high levels of recombination, phylogenetic reconstruction of B. pseudomallei populations using whole-genome sequencing (WGS) has revealed surprisingly robust biogeographic separation between isolates from Australia and Asia. T he Gram-negative soil-dwelling bacterium Burkholderia pseudomallei is the etiologic agent of melioidosis, an often deadly tropical disease that can be difficult to diagnose, particularly in nonendemic or resource-poor regions where cases are not expected and appropriate microbiological diagnostic tools are not readily available (1). Diabetics are particularly susceptible to melioidosis. B. pseudomallei infection can be acquired from contaminated soil or water by percutaneous inoculation, inhalation, aspiration, or ingestion, and no vaccine targeting this organism is available (2). In 2012, B. pseudomallei was reclassified by U.S. federal agencies as a tier 1 select agent, the highest risk category for a biological entity, due to concerns that this bacterium would pose a severe threat to humans and animals in the event of its deliberate misuse (3).The B. pseudomallei genome exhibits high homologous recombination rates. On a per-allele basis, recombination is estimated to occur between 18 and 30 times more frequently than mutation (4). This extensive lateral gene transfer can confound population analyses, particularly those that are based on studying limited geographic regions (e.g., the Northern Territory, Australia [5]) due to high rates of homoplasy observed among genetic variants. In contrast, genomic analyses of B. pseudomallei populations on a continental scale have revealed a clear separation of B. pseudomallei isolates between Asia and Australia (4, 6, 7). Bayesian analysis of B. pseudomallei genome variation points to an ancient separation, with migration out of Australia into Asia occurring tens of thousands of years ago during the Pleistocene (4). The rarity of pathogen movement is due largely to one factor: new melioidosis cases almost always result from bacterial infection acquired from the local environment, with human-to-human and zoonotic transmission of this pathogen being exceedingly rare (8). In support of the rarity of B. pseudomallei movement across major biogeographic boundaries, the definitive transmission of B. pseudomallei from Asia into Australia has not yet been observed. Nevertheless, melioidosis cases imported into nonendemic locations via travelers are being increasingly reported, as is recognition of locations that are endemic for melioidosis outside the classical regions of Southeast Asia and Australia (9). With modern global tra...

dBurkholderia pseudomallei is a Gram-negative soil bacillus that is the etiological agent of melioidosis and a biothreat agent. Little is known about the biogeography of this bacterium in Australia, despite its hyperendemicity in the northern region of this continent. The population structure of 953 Australian B. pseudomallei strains representing 779 and 174 isolates of clinical and environmental origins, respectively, was analyzed using multilocus sequence typing (MLST). Bayesian population structure and network SplitsTree analyses were performed on concatenated MLST loci, and sequence type (ST) diversity and evenness were examined using Simpson's and Pielou's indices and a multivariate dissimilarity matrix. Bayesian analysis found two B. pseudomallei populations in Australia that were geographically distinct; isolates from the Northern Territory were grouped mainly into the first population, whereas the majority of isolates from Queensland were grouped in a second population. Differences in ST evenness were observed between sampling areas, confirming that B. pseudomallei is widespread and established across northern Australia, with a large number of fragmented habitats. ST analysis showed that B. pseudomallei populations diversified as the sampling area increased. This observation was in contrast to smaller sampling areas where a few STs predominated, suggesting that B. pseudomallei populations are ecologically established and not frequently dispersed. Interestingly, there was no identifiable ST bias between clinical and environmental isolates, suggesting the potential for all culturable B. pseudomallei isolates to cause disease. Our findings have important implications for understanding the ecology of B. pseudomallei in Australia and for potential source attribution of this bacterium in the event of unexpected cases of melioidosis.

c Melioidosis, a disease of public health importance in Southeast Asia and northern Australia, is caused by the Gram-negative soil bacillus Burkholderia pseudomallei. Melioidosis is typically acquired through environmental exposure, and case clusters are rare, even in regions where the disease is endemic. B. pseudomallei is classed as a tier 1 select agent by the Centers for Disease Control and Prevention; from a biodefense perspective, source attribution is vital in an outbreak scenario to rule out a deliberate release. Two cases of melioidosis within a 3-month period at a residence in rural northern Australia prompted an investigation to determine the source of exposure. B. pseudomallei isolates from the property's groundwater supply matched the multilocus sequence type of the clinical isolates. Whole-genome sequencing confirmed the water supply as the probable source of infection in both cases, with the clinical isolates differing from the likely infecting environmental strain by just one single nucleotide polymorphism (SNP) each. For the first time, we report a phylogenetic analysis of genomewide insertion/deletion (indel) data, an approach conventionally viewed as problematic due to high mutation rates and homoplasy. Our whole-genome indel analysis was concordant with the SNP phylogeny, and these two combined data sets provided greater resolution and a better fit with our epidemiological chronology of events. Collectively, this investigation represents a highly accurate account of source attribution in a melioidosis outbreak and gives further insight into a frequently overlooked reservoir of B. pseudomallei. Our methods and findings have important implications for outbreak source tracing of this bacterium and other highly recombinogenic pathogens. Melioidosis is an underrecognized disease of significant public health burden in many tropical regions across the globe, especially northern Australia and Southeast Asia, where the greatest number of cases are reported annually (1). Melioidosis is caused by the environmental dwelling Gram-negative bacterium Burkholderia pseudomallei, an opportunistic pathogen that most commonly affects people with underlying disease or risk factors, particularly diabetes and hazardous alcohol use (2). Disease severity varies widely and depends on the strain, host immunity, and inoculum size. The highest case fatality rates exceed 90% in septic shock or untreated septic cases (3). Even when appropriate therapy is administered, mortality ranges from 13% in northern Australia (4, 5) to 50% in Southeast Asia (2). In October 2012, B. pseudomallei was upgraded to a tier 1 select agent by the Centers for Disease Control and Prevention (www.selectagents.gov) owing to fears of a deliberate release coupled with the high mortality rate, lack of a vaccine, intrinsic resistance to standard antimicrobial agents, and protean disease presentations that confound diagnosis, particularly in regions where the disease is not endemic.B. pseudomallei infection primarily occurs via percutaneous inoculati...

Burkholderia pseudomallei is a Gram-negative environmental bacterium that causes melioidosis, a disease of high mortality in humans and animals. Multilocus sequence typing (MLST) is a popular and portable genotyping method that has been used extensively to characterise the genetic diversity of B. pseudomallei populations. MLST has been central to our understanding of the underlying phylogeographical signal present in the B. pseudomallei genome, revealing distinct populations on both the intra- and the inter-continental level. However, due to its high recombination rate, it is possible for B. pseudomallei isolates to share the same multilocus sequence type (ST) despite being genetically and geographically distinct, with two cases of ‘ST homoplasy’ recently reported between Cambodian and Australian B. pseudomallei isolates. This phenomenon can dramatically confound conclusions about melioidosis transmission patterns and source attribution, a critical issue for bacteria such as B. pseudomallei that are of concern due to their potential for use as bioweapons. In this study, we used whole-genome sequencing to identify the first reported instances of intracontinental ST homoplasy, which involved ST-722 and ST-804 B. pseudomallei isolates separated by large geographical distances. In contrast, a third suspected homoplasy case was shown to be a true long-range (460 km) dispersal event between a remote Australian island and the Australian mainland. Our results show that, whilst a highly useful and portable method, MLST can occasionally lead to erroneous conclusions about isolate origin and disease attribution. In cases where a shared ST is identified between geographically distant locales, whole-genome sequencing should be used to resolve strain origin.