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BackgroundMelioidosis is a tropical infectious disease which is being increasingly recognised throughout the globe. Infection occurs in humans and animals, typically through direct exposure to soil or water containing the environmental bacteriumBurkholderia pseudomallei. Case clusters of melioidosis have been described in humans following severe weather events and in exotic animals imported into melioidosis endemic zones. Direct transmission ofB. pseudomalleibetween animals and/or humans has been documented but is considered extremely rare. Between March 2015 and October 2016 eight fatal cases of melioidosis were reported in slender-tailed meerkats (Suricata suricatta)on display at a Wildlife Park in Northern Australia. To further investigate the melioidosis case cluster we sampled the meerkat enclosure and adjacent park areas and performed whole-genome sequencing (WGS) on all culture-positiveB. pseudomalleienvironmental and clinical isolates.ResultsWGS confirmed that the fatalities were caused by two differentB. pseudomalleisequence types (STs) but that seven of the meerkat isolates were highly similar on the whole-genome level. Used concurrently with detailed pathology data, our results demonstrate that the seven cases originated from a single original source, but routes of infection varied amongst meerkats belonging to the clonal outbreak cluster. Moreover, in some instances direct transmission may have transpired through wounds inflicted while fighting.ConclusionsCollectively, this study supports the use of high-resolution WGS to enhance epidemiological investigations into transmission modalities and pathogenesis of melioidosis, especially in the instance of a possible clonal outbreak scenario in exotic zoological collections. Such findings from an animal outbreak have important One Health implications.

To investigate the current epidemiology of melioidosis in Yangon, Myanmar, between June 2017 and May 2019 we conducted enhanced surveillance for melioidosis in four tertiary hospitals in Yangon, where the disease was first discovered in 1911. Oxidase-positive Gram-negative rods were obtained from the microbiology laboratories and further analysed at the Department of Medical Research. Analysis included culture on Ashdown agar, the three disc sensitivity test (gentamicin, colistin and co-amoxiclav), latex agglutination, API 20 NE, antibiotic susceptibility testing, and a subset underwent molecular confirmation with a Burkholderia pseudomallei specific assay. Twenty one of 364 isolates (5.7%) were confirmed as B. pseudomallei and were mostly susceptible to the antibiotics used in standard therapy for melioidosis. Ten patients were from Yangon Region, nine were from Ayeyarwaddy region, and one each was from Kayin and Rakhine States. A history of soil contact was given by seven patients, five had diabetes mellitus and one had renal insufficiency. The patients presented with septicaemia (12 cases), pneumonia (three cases), urinary tract infection (two cases) and wound infection (four cases). Eighteen patients survived to hospital discharge. This study highlights the likelihood that melioidosis may be far more common, but underdiagnosed, in more rural parts of Myanmar as in other countries in SE Asia.

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 a Gram-negative saprophytic bacillus and the aetiological agent of melioidosis, a disease of public-health importance throughout Southeast Asia and northern Australia. Infection can occur in humans and a wide array of animal species, though zoonotic transmission and case clusters are rare. Despite its highly plastic genome and extensive strain diversity, fine-scale investigations into the population structure of B. pseudomallei indicate there is limited geographical dispersal amongst sequence types (STs). In the ‘Top End’ of northern Australia, five STs comprise 90 % of the overall abundance, the most prevalent and widespread of which is ST-109. In May 2016, ST-109 was implicated in two fatal cases of melioidosis in juvenile saltwater crocodiles at a wildlife park near Darwin, Australia. To determine the probable source of infection, we sampled the crocodile enclosures and analysed the phylogenetic relatedness of crocodile and culture-positive ST-109 environmental park isolates against an additional 135 ST-109 B. pseudomallei isolates from the Top End. Collectively, our whole-genome sequencing (WGS) and pathology findings confirmed B. pseudomallei detected in the hatchling incubator as the likely source of infection, with zero SNPs identified between clinical and environmental isolates. Our results also demonstrate little variation across the ST-109 genome, with SNPs in recombinogenic regions and one suspected case of ST homoplasy accounting for nearly all observed diversity. Collectively, this study supports the use of WGS for outbreak source attribution in highly recombinogenic pathogens, and confirms the epidemiological and phylogenetic insights that can be gained from high-resolution sequencing platforms.

The Tier 1 select agentBurkholderia pseudomalleiis an environmental bacterium that causes melioidosis, a high mortality disease. Variably present genetic markers used to elucidate strain origin, relatedness and virulence inB. pseudomalleiinclude theBurkholderiaintracellular motility factor A (bimA) and filamentous hemagglutinin 3 (fhaB3) gene variants. Three lipopolysaccharide (LPS) O-antigen types inB. pseudomalleihave 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 clinicalB. pseudomalleiisolates 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;pbimAgenotypebimABmhas 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-basedB. pseudomalleiLPS types, and their relationships to regional and global dispersal of melioidosis, require global collaborations with further analysis of larger clinically and geospatially-linked datasets.Author SummaryBurkholderia pseudomalleiis a pathogenic soil bacterium that causes the disease melioidosis, which occurs in many tropical regions globally and in recent years has emerged in non-tropical regions. Melioidosis has been predicted to affect 165,000 people every year resulting in an estimated 89,000 deaths. Person to person transmission is rare with most cases linked to exposure to the bacterium from the environment. The genetic background ofB. pseudomalleihas been well studied and variably present genes have been linked to distinct melioidosis disease states and geographic regions, however we still need a stronger understanding of the association of genes with geography. Three lipopolysaccharide types exist inB. pseudomalleiand the prevalence of the lipopolysaccharide genes vary between melioidosis endemic regions, but it is unknown if the lipopolysaccharide genes can be used as geographical markers in a single melioidosis-endemic region. In this study, we used a combination of whole-genome sequencing, statistics and geographical mapping to elucidate if the three lipopolysaccharide genes can be used as geographical markers within the Northern Territory, Australia. We show that the three LPS types have distinct but overlapping geographical footprints within a single melioidosis region and can be used as geographic markers alongside a number of other important variably presentB. pseudomalleigenes.

BackgroundInfection with the gram-negative bacterium Burkholderia pseudomallei can result in melioidosis, a life-threatening disease that can be difficult to diagnose. Culture remains the gold standard for diagnosis but requires laboratory resources not available in many endemic regions. A lateral flow immunoassay has shown promise for POC diagnostics but suffers from low sensitivity when used on blood samples. PCR also has low sensitivity on blood, attributed to the low bacterial numbers in blood observed in melioidosis patients, even when bacteraemic.MethodsA prototype i-STAT cartridge was developed to utilize the monoclonal antibody specific for the capsule of pathogenic Burkholderia species employed on the LFI. The resulting POC assay was evaluated on 414 clinical specimens from Darwin, Australia and Cambodia.ResultsThe i-STAT assay accurately distinguished Australian blood culture positive melioidosis patients from Australian patients hospitalized with other infections (AUC = 0.91, 95% CI 0.817 - 1.0). We derived an assay cutoff with 76% sensitivity and 94% specificity that correctly classified 88% (n = 74) of the Australian patients. Interestingly, only 46% (6/13) of the culture-positive melioidosis patients in Cambodia were classified correctly. Of great importance however, the assay detected capsule from blood samples for 32% of blood culture negative melioidosis patients in both cohorts and previously undiagnosed melioidosis patients in Cambodia. In addition the assay showed high sensitivity and specificity for urine, pus and sputum.ConclusionsDiagnostic tools that are not dependent upon the growth kinetics or the levels of bacteremia of B. pseudomallei represent the next-generation of diagnostics and must be pursued further.

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, Author summary Burkholderia pseudomallei is a pathogenic soil bacterium that causes the disease melioidosis, which occurs in many tropical regions globally and in recent years has emerged in non-tropical regions. Melioidosis has been predicted to affect 165,000 people every year resulting in an estimated 89,000 deaths. Person to person transmission is rare with most cases linked to exposure to the bacterium from the environment. The genetic background of B. pseudomallei has been well studied and variably present genes have been linked to distinct melioidosis disease states and geographic regions, however we still need a stronger understanding of the association of genes with geography. Three lipopolysaccharide types exist in B. pseudomallei and the prevalence of the lipopolysaccharide genes vary between melioidosis endemic regions, but it is unknown if the lipopolysaccharide genes can be used as geographical markers in a single melioidosis-endemic region. In this study, we used a combination of whole-genome sequencing, statistics and geographical mapping to elucidate if the three lipopolysaccharide genes can be used as geographical markers within the Northern Territory, Australia. We show that the three LPS types have distinct but overlapping geographical footprints within a single melioidosis region and can be used as geographic markers alongside a number of other important variably present B. pseudomallei genes.

Background Melioidosis 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. Methods A part prospective, part retrospective study of children aged, Author summary Melioidosis is a serious, and often fatal community-acquired infection endemic to Southeast Asia and northern Australia. It is caused by the environmental saprophyte Burkholderia pseudomallei, a bacterium that is intrinsically resistant to many commonly used antibiotics. Its presence in Sarawak, Malaysian Borneo, has been documented, and recently, a novel gentamicin-susceptible strain discovered. However, there are limited data on the burden and clinical characteristics of melioidosis in Sarawak, both in adults and in children. In this study, we comprehensively investigated all pediatric melioidosis cases in Central Sarawak over a 6-year period. We found that this region has a very high incidence of pediatric melioidosis, and that children frequently presented with disseminated disease and had high fatality rates, despite the absence of any predisposing risk factor. We confirmed that these infections were caused predominantly by gentamicin-susceptible B. pseudomallei strains. We also highlighted other undescribed epidemiological, clinical and microbiological features, which may help in the overall understanding of B. pseudomallei infections. We emphasized the importance of improving the awareness and recognition of melioidosis in children, both in Sarawak and in other endemic regions.