The biogeography of melioidosis

Burkholderia pseudomallei causes melioidosis; an often fatal disease prevalent in tropical regions of Southeast Asia and Australasia, and emerging in other regions of the globe. Seemingly random clustering of clinical cases is linked not only to the population distribution, but also to the environmental prevalence of the organism. Previous studies based on the diversity of representative isolates have concluded that an Australian evolutionary origin of the organism is likely, with distinct populations of the organism each side of the Wallace line. However, the means of dissemination of B. pseudomallei between these regions remain uncertain. A focus of B. pseudomallei endemicity has recently been described in the Balimo region of the Western Province, Papua New Guinea; a feature of the isolates from both clinical and environmental sources is a narrow genetic diversity. This has raised questions as to the origins of the organism from this community and their relationship to those from northern Australia. Through a more thorough investigation of the reservoir and phylogeny of B. pseudomallei from northern Queensland and Papua New Guinea, this thesis aims to make a significant contribution to our understanding of the relationship between and origins of isolates in our region. In an attempt to elucidate the origins of B. pseudomallei in the Balimo region of Papua New Guinea, multi-locus sequence typing was employed to reveal three novel sequence types (Chapter 4). Phylogenetic reconstruction using multi-locus sequence typing data and Structure analysis determined that all isolates were genetically closer to those from Australia than those from Southeast Asia. Fimbrial gene cluster analysis however, identified a Yersinialike fimbrial gene cluster among all Balimo isolates that is found predominantly among isolates recovered from Southeast Asia. Higher resolution multi-locus variable number of tandem repeat analysis of the isolates resolved 24 genotypes with high divergence. These findings are consistent with a long term persistence of the organism in the region and a high level of environmental stability. A more thorough comparison of the Balimo isolates was made against Queensland isolates. A field site for the collection of environmental B. pseudomallei in Queensland, Australia was sought to analyse the diversity of isolates from a region closer to Balimo (Chapter 5). Multi-locus sequence typing of 20 isolates collected from environmental sampling revealed for the first time a clinically implicated reservoir of B. pseudomallei in Townsville. Furthermore, it was discovered that naturally occurring groundwater seeps function as a vehicle for the dispersal of viable B. pseudomallei away from a primary environmental reservoir. Analysis of these isolates supported earlier findings that isolates from Queensland are distinct to those from the Northern Territory, yet no associations with B. pseudomallei from New Guinea were identified. In an attempt to analyse B. pseudomallei from a region adjacent to Balimo, this study collected isolates from the Torres Strait region of northern Queensland, Australia (Chapter 6). The Torres Strait is recognised as an important melioidosis endemic region in northern Queensland, yet no reservoir of infection has been identified. The people of the region have a long-term history of trade and travel between the islands, New Guinea and mainland Australia. Typing of 32 clinical B. pseudomallei isolates from the Torres Strait identified statistically significant non-random distribution of sequence types, with localisation to individual islands. In addition, sequence type matches were identified with the Northern Territory, Port Moresby and Thailand, indicating potential long-distance movement of the organism, but less frequent movements between islands. These findings are consistent with the hypothesis that B. pseudomallei movements have been restricted by short stretches of ocean, and indicate a mechanism responsible for the genetic isolation of populations between Australia and Southeast Asia. The highest diversity was identified on Thursday Island, the commercial and administrative hub of the Torres Strait, indicating that human influences may have had an impact on the dispersal of melioidosis around the region. Environmental sampling on Badu Island recovered a single clone belonging to a novel sequence type, yet the isolate was noted to have closest identity to a clinical isolate from the same island. This was the first recovery of an environmental isolate from the Torres Strait and confirms it’s status as a B. pseudomallei endemic region. Balimo B. pseudomallei were unable to be linked to others from the globe using multi-locus sequence typing and so, the analysis of whole genome sequences was used to determine their ancestry (Chapter 7). Phylogenetic reconstruction indicated that all of the Papua New Guinean and Torres Strait isolates were members of the Australian clade. Clinical isolates from Port Moresby comprised an individual branch, whilst isolates from Balimo formed a unique clade along with two isolates from the Torres Strait. These findings verify that Papua New Guinean B. pseudomallei belong to the Australian clade, and provide support for biogeographical boundaries in agreement with observed macro-flora and fauna partitioning each side of the Wallace Line. In addition, these analyses indicate that populations of B. pseudomallei have been stable in Papua New Guinea for an extended period, and are not the result of recent human importation. The results suggest that B. pseudomallei is diverse in the Port Moresby region and may represent a new foci of clinical melioidosis. Finally, a collection of five non-arabinose assimilating B. pseudomallei-like isolates were collected during environmental sampling in the Torres Strait (Chapter 8). Sequencing of the 16S rDNA, recA genes and multi-locus sequence typing loci revealed that the isolates clustered into thee clades within the B. pseudomallei-group, with closest ancestry to B. oklahomensis. Characterisation and genome sequencing of these isolates is ongoing. The recovery of these diverse organisms from a melioidosis endemic region in Australia may have important implications for serology and biochemical based diagnostics in the region. In conclusion, this work contained within thesis has demonstrated that isolates of B. pseudomallei from Balimo are closely related to those from Australia and the Torres Strait, with a narrow genetic diversity resulting from independent evolution following long term isolation of the Balimo population. These findings indicate that the current distribution of B. pseudomallei throughout Australasia may be linked to the geographical history of the region in a pattern congruent with the biogeography of macro flora and fauna.