BackgroundMelioidosis is a serious, and potentially fatal community-acquired infection endemic to northern Australia and Southeast Asia, including Sarawak, Malaysia. The disease, caused by the usually intrinsically aminoglycoside-resistant Burkholderia pseudomallei, most commonly affects adults with predisposing risk factors. There are limited data on pediatric melioidosis in Sarawak.MethodsA part prospective, part retrospective study of children aged <15 years with culture-confirmed melioidosis was conducted in the 3 major public hospitals in Central Sarawak between 2009 and 2014. We examined epidemiological, clinical and microbiological characteristics.FindingsForty-two patients were recruited during the 6-year study period. The overall annual incidence was estimated to be 4.1 per 100,000 children <15 years, with marked variation between districts. No children had pre-existing medical conditions. Twenty-three (55%) had disseminated disease, 10 (43%) of whom died. The commonest site of infection was the lungs, which occurred in 21 (50%) children. Other important sites of infection included lymph nodes, spleen, joints and lacrimal glands. Seven (17%) children had bacteremia with no overt focus of infection. Delays in diagnosis and in melioidosis-appropriate antibiotic treatment were observed in nearly 90% of children. Of the clinical isolates tested, 35/36 (97%) were susceptible to gentamicin. Of these, all 11 isolates that were genotyped were of a single multi-locus sequence type, ST881, and possessed the putative B. pseudomallei virulence determinants bimABp, fhaB3, and the YLF gene cluster.ConclusionsCentral Sarawak has a very high incidence of pediatric melioidosis, caused predominantly by gentamicin-susceptible B. pseudomallei strains. Children frequently presented with disseminated disease and had an alarmingly high death rate, despite the absence of any apparent predisposing risk factor.

Melioidosis is a severe disease caused by the environmental bacterium Burkholderia pseudomallei that affects both humans and animals throughout northern Australia, Southeast Asia and increasingly globally. While there is a considerable degree of genetic diversity amongst isolates, B. pseudomallei has a robust global biogeographic structure and genetic populations are spatially clustered in the environment. We examined the distribution and local spread of B. pseudomallei in Darwin, northern territory, Australia, which has the highest recorded urban incidence of melioidosis globally. We sampled soil and land runoff throughout the city centre and performed whole-genome sequencing (WGS) on B. pseudomallei isolates. By combining phylogenetic analyses, Bayesian clustering and spatial hot spot analysis our results demonstrate that some sequence types (STs) are widespread in the urban Darwin environment, while others are highly spatially clustered over a small geographic scale. this clustering matches the spatial distribution of clinical cases for one St. Results also demonstrate a greater overall isolate diversity recovered from drains compared to park soils, further supporting the role drains may play in dispersal of B. pseudomallei Sts in the environment. collectively, knowledge gained from this study will allow for better understanding of B. pseudomallei phylogeography and melioidosis source attribution, particularly on a local level. Burkholderia pseudomallei is an environmental Gram-negative bacillus and the causative agent of melioidosis, a potentially fatal infection of humans and animals 1. Regions of high B. pseudomallei endemicity predominantly include Southeast Asia and northern Australia, though the bacterium is also increasingly found in other tropical regions including the Indian subcontinent, Africa and the Americas 2. Melioidosis is typically considered noncommunicable with direct person-to-person transmission and zoonotic disease being remarkably rare 3,4. Nearly all B. pseudomallei infections are caused by a single direct exposure event to contaminated soil or surface water in the environment and individual cases of melioidosis are typically the result of infection by different strains of the bacterium 5. A limited geographic dispersal of B. pseudomallei strains has also been identified and the bacterium is now recognised as being ecologically established and spatially clustered in the environment 6-8. This is in spite of the frequent opportunities B. pseudomallei has to spread within the water table, via agricultural and migratory animals, or in transported soil 6,9-11. This restricted geographical distribution has resulted in distinct genetic populations of the bacterium, which are evident despite high levels of gene recombination and sequence type (ST)

The Tier 1 select agent Burkholderia pseudomallei is an environmental bacterium that causes melioidosis, a high mortality disease. Variably present genetic markers used to elucidate strain origin, relatedness and virulence in B . pseudomallei include the Burkholderia intracellular motility factor A ( bimA ) and filamentous hemagglutinin 3 ( fhaB3 ) gene variants. Three lipopolysaccharide (LPS) O-antigen types in B . pseudomallei have been described, which vary in proportion between Australian and Asian isolates. However, it remains unknown if these LPS types can be used as genetic markers for geospatial analysis within a contiguous melioidosis-endemic region. Using a combination of whole-genome sequencing (WGS), statistical analysis and geographical mapping, we examined if the LPS types can be used as geographical markers in the Northern Territory, Australia. The clinical isolates revealed that LPS A prevalence was highest in the Darwin and surrounds (n = 660; 96% being LPS A and 4% LPS B) and LPS B in the Katherine and Katherine remote and East Arnhem regions (n = 79; 60% being LPS A and 40% LPS B). Bivariate logistics regression of 999 clinical B . pseudomallei isolates revealed that the odds of getting a clinical isolate with LPS B was highest in East Arnhem in comparison to Darwin and surrounds (OR 19.5, 95% CI 9.1–42.0; p <0.001). This geospatial correlation was subsequently confirmed by geographically mapping the LPS type from 340 environmental Top End strains. We also found that in the Top End, the minority bimA genotype bimA Bm has a similar remote region geographical footprint to that of LPS B. In addition, correlation of LPS type with multi-locus sequence typing (MLST) was strong, and where multiple LPS types were identified within a single sequence type, WGS confirmed homoplasy of the MLST loci. The clinical, sero-diagnostic and vaccine implications of geographically-based B . pseudomallei LPS types, and their relationships to regional and global dispersal of melioidosis, require global collaborations with further analysis of larger clinically and geospatially-linked datasets.