Phylogeography of clinical Burkholderia pseudomallei isolates in Cambodia demonstrates diversity, broad dispersal, and novel genes.

Burkholderia pseudomallei, the causative agent of melioidosis, is ubiquitous in Southeast Asia but remains understudied in many endemic countries. The species' high genetic diversity necessitates large-scale sampling to resolve fine-scale population structure. In countries bordering the Mekong River, the geographic range of B. pseudomallei strains may be increased by water-mediated dispersal. Between 2021 and 2023, we collected and sequenced 253 clinical B. pseudomallei isolates from patients admitted to six Cambodian hospitals and geotagged isolates by patient village. Isolates were genetically diverse and grouped into 11 clades, including one characterized by a chromosomal genomic island containing unique genes absent from other isolates, consistent with local evolution. Comparison with 3,234 publicly available global genomes revealed intermixing between Cambodian and other Southeast Asian isolates but clear distinction from Australian strains. Phylogeographic analyses further demonstrated spatial localization along major waterways, suggesting that river systems, including the Mekong, may contribute to bacterial dissemination in Cambodia. Taken together, these findings demonstrate the importance of localized data sets for contextualizing the population structure and evolution of this diverse pathogen in Southeast Asia.IMPORTANCEBurkholderia pseudomallei is a highly diverse bacterial species with genomic features that vary based on geographic sampling location. Existing data predominantly from Thailand show that isolates from Southeast Asia may be closely related to each other. Here, we sequenced isolates associated with human disease in Cambodia and compared these against genomes from public sequence repositories. We identified many distinct bacterial strains and found that some of these were localized either to the south or in the north of the country. We identified a unique group of genes in one cohort strain with putative roles in bacterial metabolism and genomic recombination. Finally, in a global phylogeny constructed from publicly available genomes, we found that Cambodian isolates intermixed with other Southeast Asian isolates. Our study is the largest genomic study of B. pseudomallei in Cambodia to date, with findings that contribute to the global understanding of the bacterium's diversity and evolution.