Epidemiology and genetic diversity of Burkholderia pseudomallei from Riau Province, Indonesia.

Melioidosis is a bacterial infection caused by Burkholderia pseudomallei, that is common in tropical and subtropical countries including Southeast Asia and Northern Australia. The magnitude of undiagnosed and untreated melioidosis across the country remains unclear. Given its proximity to regions with high infection rates, Riau Province on Sumatera Island is anticipated to have endemic melioidosis. This study reports retrospectively collected data on 68 culture-confirmed melioidosis cases from two hospitals in Riau Province between January 1, 2009, and December 31, 2021, with full clinical data available on 41 cases. We also describe whole genome sequencing and genotypic analysis of six isolates of B. pseudomallei. The mean age of the melioidosis patients was 49.1 (SD 11.5) years, 85% were male and the most common risk factor was diabetes mellitus (78%). Pulmonary infection was the most common presentation (39%), and overall mortality was 41%. Lung as a focal infection (aOR: 6.43; 95% CI: 1.13-36.59, p = 0.036) and bacteremia (aOR: 15.21; 95% CI: 2.59-89.31, p = 0.003) were significantly associated with death. Multilocus sequence typing analysis conducted on six B.pseudomallei genomes identified three sequence types (STs), namely novel ST1794 (n = 3), ST46 (n = 2), and ST289 (n = 1). A phylogenetic tree of Riau B. pseudomallei whole genome sequences with a global dataset of genomes clearly distinguished the genomes of B. pseudomallei in Indonesia from the ancestral Australian clade and classified them within the Asian clade. This study expands the known presence of B. pseudomallei within Indonesia and confirms that Indonesian B. pseudomallei are genetically linked to those in the rest of Southeast Asia. It is anticipated that melioidosis will be found in other locations across Indonesia as laboratory capacities improve and standardized protocols for detecting and confirming suspected cases of melioidosis are more widely implemented.

Multiple phylogenetically-diverse, differentially-virulent Burkholderia pseudomallei isolated from a single soil sample collected in Thailand.

Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and northern Australia that causes the disease, melioidosis. Although the global genomic diversity of clinical B. pseudomallei isolates has been investigated, there is limited understanding of its genomic diversity across small geographic scales, especially in soil. In this study, we obtained 288 B. pseudomallei isolates from a single soil sample (~100g; intensive site 2, INT2) collected at a depth of 30cm from a site in Ubon Ratchathani Province, Thailand. We sequenced the genomes of 169 of these isolates that represent 7 distinct sequence types (STs), including a new ST (ST1820), based on multi-locus sequence typing (MLST) analysis. A core genome SNP phylogeny demonstrated that all identified STs share a recent common ancestor that diverged an estimated 796-1260 years ago. A pan-genomics analysis demonstrated recombination between clades and intra-MLST phylogenetic and gene differences. To identify potential differential virulence between STs, groups of BALB/c mice (5 mice/isolate) were challenged via subcutaneous injection (500 CFUs) with 30 INT2 isolates representing 5 different STs; over the 21-day experiment, eight isolates killed all mice, 2 isolates killed an intermediate number of mice (1-2), and 20 isolates killed no mice. Although the virulence results were largely stratified by ST, one virulent isolate and six attenuated isolates were from the same ST (ST1005), suggesting that variably conserved genomic regions may contribute to virulence. Genomes from the animal-challenged isolates were subjected to a bacterial genome-wide association study to identify genomic regions associated with differential virulence. One associated region is a unique variant of Hcp1, a component of the type VI secretion system, which may result in attenuation. The results of this study have implications for comprehensive sampling strategies, environmental exposure risk assessment, and understanding recombination and differential virulence in B. pseudomallei.

Genomic Diversity of Burkholderia pseudomallei in Ceara, Brazil.

Burkholderia pseudomallei is a Gram-negative bacterium that causes the sapronotic disease melioidosis. An outbreak in 2003 in the state of Ceara, Brazil, resulted in subsequent surveillance and environmental sampling which led to the recognition of B. pseudomallei as an endemic pathogen in that area. From 2003 to 2015, 24 clinical and 12 environmental isolates were collected across Ceara along with one from the state of Alagoas. Using next-generation sequencing, multilocus sequence typing, and single nucleotide polymorphism analysis, we characterized the genomic diversity of this collection to better understand the population structure of B. pseudomallei associated with Ceara. We found that the isolates in this collection form a distinct subclade compared to other examples from the Western Hemisphere. Substantial genetic diversity among the clinical and environmental isolates was observed, with 14 sequence types (STs) identified among the 37 isolates. Of the 31,594 core single-nucleotide polymorphisms (SNPs) identified, a high proportion (59%) were due to recombination. Because recombination events do not follow a molecular clock, the observation of high occurrence underscores the importance of identifying and removing recombination SNPs prior to evolutionary reconstructions and inferences in public health responses to B. pseudomallei outbreaks. Our results suggest long-term B. pseudomallei prevalence in this recently recognized region of melioidosis endemicity.IMPORTANCEB. pseudomallei causes significant morbidity and mortality, but its geographic prevalence and genetic diversity are not well characterized, especially in the Western Hemisphere. A better understanding of the genetic relationships among clinical and environmental isolates will improve knowledge of the population structure of this bacterium as well as the ability to conduct epidemiological investigations of cases of melioidosis.

Whole-genome comparative analysis of Malaysian Burkholderia pseudomallei clinical isolates.

Burkholderia pseudomallei, a soil-dwelling Gram-negative bacterium, is the causative agent of the endemic tropical disease melioidosis. Clinical manifestations of B. pseudomallei infection range from acute or chronic localized infection in a single organ to fulminant septicaemia in multiple organs. The diverse clinical manifestations are attributed to various factors, including the genome plasticity across B. pseudomallei strains. We previously characterized B. pseudomallei strains isolated in Malaysia and noted different levels of virulence in model hosts. We hypothesized that the difference in virulence might be a result of variance at the genome level. In this study, we sequenced and assembled four Malaysian clinical B. pseudomallei isolates, UKMR15, UKMPMC2000, UKMD286 and UKMH10. Phylogenomic analysis showed that Malaysian subclades emerged from the Asian subclade, suggesting that the Malaysian strains originated from the Asian region. Interestingly, the low-virulence strain, UKMH10, was the most distantly related compared to the other Malaysian isolates. Genomic island (GI) prediction analysis identified a new island of 23 kb, GI9c, which is present in B. pseudomallei and Burkholderia mallei, but not Burkholderia thailandensis. Genes encoding known B. pseudomallei virulence factors were present across all four genomes, but comparative analysis of the total gene content across the Malaysian strains identified 104 genes that are absent in UKMH10. We propose that these genes may encode novel virulence factors, which may explain the reduced virulence of this strain. Further investigation on the identity and role of these 104 proteins may aid in understanding B. pseudomallei pathogenicity to guide the design of new therapeutics for treating melioidosis.

Burkholderia pseudomallei septic arthritis in Type-2 diabetes mellitus patients: Report of two cases.

Two cases of Burkholderia pseudomallei septic arthritis are presented with a brief review of the literature. B. pseudomallei septic arthritis most commonly occurs in diabetics and other immunocompromised patients and may prove fatal despite appropriate therapy. Clinical and microbiological suspicion of B. pseudomallei infection may help in providing appropriate empirical therapy.

Pathogen to commensal? Longitudinal within-host population dynamics, evolution, and adaptation during a chronic >16-year Burkholderia pseudomallei infection.

Although acute melioidosis is the most common outcome of Burkholderia pseudomallei infection, we have documented a case, P314, where disease severity lessened with time, and the pathogen evolved towards a commensal relationship with the host. In the current study, we used whole-genome sequencing to monitor this long-term symbiotic relationship to better understand B. pseudomallei persistence in P314's sputum despite intensive initial therapeutic regimens. We collected and sequenced 118 B. pseudomallei isolates from P314's airways over a >16-year period, and also sampled the patient's home environment, recovering six closely related B. pseudomallei isolates from the household water system. Using comparative genomics, we identified 126 SNPs in the core genome of the 124 isolates or 162 SNPs/indels when the accessory genome was included. The core SNPs were used to construct a phylogenetic tree, which demonstrated a close relationship between environmental and clinical isolates and detailed within-host evolutionary patterns. The phylogeny had little homoplasy, consistent with a strictly clonal mode of genetic inheritance. Repeated sampling revealed evidence of genetic diversification, but frequent extinctions left only one successful lineage through the first four years and two lineages after that. Overall, the evolution of this population is nonadaptive and best explained by genetic drift. However, some genetic and phenotypic changes are consistent with in situ adaptation. Using a mouse model, P314 isolates caused greatly reduced morbidity and mortality compared to the environmental isolates. Additionally, potentially adaptive phenotypes emerged and included differences in the O-antigen, capsular polysaccharide, motility, and colony morphology. The >13-year co-existence of two long-lived lineages presents interesting hypotheses that can be tested in future studies to provide additional insights into selective pressures, niche differentiation, and microbial adaptation. This unusual melioidosis case presents a rare example of the evolutionary progression towards commensalism by a highly virulent pathogen within a single human host.

Comparative genomics confirms a rare melioidosis human-to-human transmission event and reveals incorrect phylogenomic reconstruction due to polyclonality.

Human-to-human transmission of the melioidosis bacterium, Burkholderia pseudomallei, is exceedingly rare, with only a handful of suspected cases documented to date. Here, we used whole-genome sequencing (WGS) to characterize one such unusual B. pseudomallei transmission event, which occurred between a breastfeeding mother with mastitis and her child. Two strains corresponding to multilocus sequence types (STs)-259 and -261 were identified in the mother's sputum from both the primary culture sweep and in purified colonies, confirming an unusual polyclonal infection in this patient. In contrast, primary culture sweeps of the mother's breast milk and the child's cerebrospinal fluid and blood samples contained only ST-259, indicating monoclonal transmission to the child. Analysis of purified ST-259 isolates showed no genetic variation between mother and baby isolates, providing the strongest possible evidence of B. pseudomallei human-to-human transmission, probably via breastfeeding. Next, phylogenomic analysis of all isolates, including the mother's mixed ST-259/ST-261 sputum sample, was performed to investigate the effects of mixtures on phylogenetic inference. Inclusion of this mixture caused a dramatic reduction in the number of informative SNPs, resulting in branch collapse of ST-259 and ST-261 isolates, and several instances of incorrect topology in a global B. pseudomallei phylogeny, resulting in phylogenetic incongruence. Although phylogenomics can provide clues about the presence of mixtures within WGS datasets, our results demonstrate that this methodology can lead to phylogenetic misinterpretation if mixed genomes are not correctly identified and omitted. Using current bioinformatic tools, we demonstrate a robust method for bacterial mixture identification and strain parsing that avoids these pitfalls.

A comparative genomic analysis of small-colony variant and wild-type Burkholderia pseudomallei in a patient with bacterial liver abscess.

OBJECTIVE: To understand the genotypic variations of Burkholderia pseudomallei (B. pseudomallei) small-colony variant (SCV). METHODS: A pair of isogenic wild-type (WT) and SCV B. pseudomallei strains (CX1-1 and CX2-1, respectively) were isolated from a patient with a bacterial liver abscess. They were further identified by multilocus sequence typing (MLST) analysis. To compare their growth speed, the time to detection for the two strains was assessed by BacT/Alert 3D. Antibiotic susceptibility tests were performed by disc diffusion method and Etest assay according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The whole genomes of the two strains were sequenced. A comparative genome analysis was performed to determine the genotypic variations of the CX2-1 strain. RESULTS: The CX1-1 and CX2-1 strains were both identified as ST70 by MLST. The CX2-1 grew more slowly than the WT strain CX1-1 and was more resistant to imipenem, meropenem, doxycycline, trimethoprim-sulfamethoxazole, and ceftazidime. The comparative genome analysis revealed 38 variations in 30 genes associated with metabolism, drug resistance and virulence. The mutated genes encoded some cell membrane proteins, membrane transporters and synthetases, including: LolB, HisP, PchF, putative polyketide synthetases, probable non-ribosomal peptide synthetases, putative TonB-dependent outer-membrane receptor protein, and putative type III secretion protein. CONCLUSIONS: The reduced growth speed and increased drug resistance of B. pseudomallei SCV strain may be related to those variations in the genome. This provides some clues to their association between the morphotypic and phenotypic characteristics of colony variants, and the potential association of its colony morphotypes with metabolism, antibiotic resistance and virulence.

Genetic variation associated with infection and the environment in the accidental pathogen Burkholderia pseudomallei.

The environmental bacterium Burkholderia pseudomallei causes melioidosis, an important endemic human disease in tropical and sub-tropical countries. This bacterium occupies broad ecological niches including soil, contaminated water, single-cell microbes, plants and infection in a range of animal species. Here, we performed genome-wide association studies for genetic determinants of environmental and human adaptation using a combined dataset of 1,010 whole genome sequences of B. pseudomallei from Northeast Thailand and Australia, representing two major disease hotspots. With these data, we identified 47 genes from 26 distinct loci associated with clinical or environmental isolates from Thailand and replicated 12 genes in an independent Australian cohort. We next outlined the selective pressures on the genetic loci (dN/dS) and the frequency at which they had been gained or lost throughout their evolutionary history, reflecting the bacterial adaptability to a wide range of ecological niches. Finally, we highlighted loci likely implicated in human disease.

Fatal deep venous thrombosis and pulmonary embolism secondary to melioidosis in China: case report and literature review.

BACKGROUND: Burkholderia pseudomallei is a gram-negative bacterium and the causative pathogen of melioidosis, which manifests a variety ranges of infection symptoms. However, deep venous thrombosis (DVT) and pulmonary embolism (PE) secondary to bacteremic melioidosis are rarely documented in the literature. Herein, we reported a fatal case of melioidosis combined with DVT and PE. CASE PRESENTATION: A 54-year-old male construction worker and farmer with a history of diabetes was febrile, painful in left thigh, swelling in left lower limb, with chest tightness and shortness of breath for 4 days. He was later diagnosed as DVT of left lower extremity and PE. The culture of his blood, sputum and bone marrow samples grew B. pseudomallei. The subject was administrated with antibiotics (levofloxacin, cefoperazone/tazobactam, and imipenem) according to antimicrobial susceptibility testing and low molecular heparin for venous thrombosis. However, even after appropriate treatment, the patient deteriorated rapidly, and died 2 weeks after admission. CONCLUSIONS: This study enhanced awareness of the risk of B. pseudomallei bloodstream infection in those with diabetes. If a patient has predisposing factors of melioidosis, when DVT is suspected, active investigation and multiple therapeutic interventions should be implemented immediately to reduce mortality rate.

Opportunistic pathogens and large microbial diversity detected in source-to-distribution drinking water of three remote communities in Northern Australia.

In the wet-dry tropics of Northern Australia, drinking water in remote communities is mostly sourced from bores accessing groundwater. Many aquifers contain naturally high levels of iron and some are shallow with surface water intrusion in the wet season. Therefore, environmental bacteria such as iron-cycling bacteria promoting biofilm formation in pipes or opportunistic pathogens can occur in these waters. An opportunistic pathogen endemic to northern Australia and Southeast Asia and emerging worldwide is Burkholderia pseudomallei. It causes the frequently fatal disease melioidosis in humans and animals. As we know very little about the microbial composition of drinking water in remote communities, this study aimed to provide a first snapshot of the microbiota and occurrence of opportunistic pathogens in bulk water and biofilms from the source and through the distribution system of three remote water supplies with varying iron levels. Using 16s-rRNA gene sequencing, we found that the geochemistry of the groundwater had a substantial impact on the untreated microbiota. Different iron-cycling bacteria reflected differences in redox status and nutrients. We cultured and sequenced B. pseudomallei from bores with elevated iron and from a multi-species biofilm which also contained iron-oxidizing Gallionella, nitrifying Nitrospira and amoebae. Gallionella are increasingly used in iron-removal filters in water supplies and more research is needed to examine these interactions. Similar to other opportunistic pathogens, B. pseudomallei occurred in water with low organic carbon levels and with low heterotrophic microbial growth. No B. pseudomallei were detected in treated water; however, abundant DNA of another opportunistic pathogen group, non-tuberculous mycobacteria was recovered from treated parts of one supply. Results from this study will inform future studies to ultimately improve management guidelines for water supplies in the wet-dry tropics.

Case Series Study of Melioidosis, Colombia.

We report 7 cases of melioidosis in Colombia and comparision of 4 commercial systems for identifying Burkholderia pseudomallei. Phoenix systems were not a definitive method for identifying B. pseudomallei. For accurate identification, we recommend including this bacterium in the library databases of matrix-assisted laser desorption/ionization mass spectrometry systems in Latin America.

A novel contact-independent T6SS that maintains redox homeostasis via Zn2+ and Mn2+ acquisition is conserved in the Burkholderia pseudomallei complex.

The Burkholderia pseudomallei complex consists of six phylogenetically related Gram-negative bacterial species that include environmental saprophytes and mammalian pathogens. These microbes possess multiple type VI secretion systems (T6SS) that provide a fitness advantage in diverse niches by translocating effector molecules into prokaryotic and eukaryotic cells in a contact-dependent manner. Several recent studies have elucidated the regulation and function of T6SS-2, a novel contact-independent member of the T6SS family. Expression of the T6SS-2 gene cluster is repressed by OxyR, Zur and TctR and is activated by GvmR and reactive oxygen species (ROS). The last two genes of the T6SS-2 gene cluster encode a zincophore (TseZ) and a manganeseophore (TseM) that are exported into the extracellular milieu in a contact-independent fashion when microbes encounter oxidative stress. TseZ and TseM bind Zn2+ and Mn2+, respectively, and deliver them to bacteria where they provide protection against the lethal effects of ROS. The TonB-dependent transporters that interact with TseZ and TseM, and actively transport Zn2+ and Mn2+ across the outer membrane, have also been identified. Finally, T6SS-2 provides a contact-independent growth advantage in nutrient limited environments and is critical for virulence in Galleria mellonella larvae, but is dispensable for virulence in rodent models of infection.

Tracing the environmental footprint of the Burkholderia pseudomallei lipopolysaccharide genotypes in the tropical "Top End" of the Northern Territory, Australia.

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 bimABm 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.

Implications of environmental and pathogen-specific determinants on clinical presentations and disease outcome in melioidosis patients.

BACKGROUND: Melioidosis is gaining recognition as an emerging infectious disease with diverse clinical manifestations and high-case fatality rates worldwide. However, the molecular epidemiology of the disease outside the endemic regions such as northeast part of Thailand and northern Australia remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Clinical data and B. pseudomallei isolates obtained from 199 culture-confirmed cases of melioidosis diagnosed during 2006-2016 in South India were used to elucidate the host and pathogen specific variable virulence determinants associated with clinical presentations and disease outcome. Further, we determined the temporal variations and the influence of ecological factors on B.pseudomallei Lipopolysaccharide (LPS) genotypes causing infections. Severe forms of the disease were observed amongst 169 (85%) patients. Renal dysfunction and infection due to B.pseudomallei harboring BimABm variant had significant associations with severe forms of the disease. Diabetes mellitus, septicemic melioidosis and infection due to LPSB genotype were independent risk factors for mortality. LPSB (74%) and LPSA (20.6%) were the prevalent genotypes causing infections. Both genotypes demonstrated temporal variations and had significant correlations with rainfall and humidity. CONCLUSION/SIGNIFICANCE: Our study findings suggest that the pathogen specific virulence traits under the influence of ecological factors are the key drivers for geographical variations in the molecular epidemiology of melioidosis.

Identification of Burkholderia pseudomallei by Use of the Vitek Mass Spectrometer.

Burkholderia pseudomallei is the causative agent of melioidosis. This condition most often presents as pneumonia and bacteremia, with mortality rates of 9% to 70%. Therefore, early identification of this organism may aid in directing appropriate management. This study aimed to use the Vitek matrix-assisted laser desorption ionization-time of flight mass spectrometer to create a spectrum for the rapid identification of B. pseudomallei Spectra from 85 isolate cultures were acquired using the Vitek mass spectrometer research mode. A SuperSpectrum was created using peak matching and subsequently activated for analysis of organism identification. All 85 isolates were correctly identified as B. pseudomallei A total of 899 spectra were analyzed and demonstrated a specificity of 99.8%. Eighty-one clinical isolates were used, of which 10 were neuromelioidosis, and no discernible spectrum difference was appreciated. Spectrum acquisition from a single spot was only successful in 374/899 (41%) of isolates. This increased to 100% when 3 spots of the same extract were analyzed. The Vitek mass spectrometer can be used for the rapid identification of B. pseudomallei with a high level of specificity.

Burkholderia pseudomallei ΔtonB Δhcp1 Live Attenuated Vaccine Strain Elicits Full Protective Immunity against Aerosolized Melioidosis Infection.

Burkholderia pseudomallei is a Gram-negative facultative intracellular bacterium and the causative agent of melioidosis, a severe infectious disease found throughout the tropics. This organism is closely related to Burkholderia mallei, the etiological agent of glanders disease which primarily affects equines. These two pathogenic bacteria are classified as Tier 1 select agents due to their amenability to aerosolization, limited treatment options, and lack of an effective vaccine. We have previously successfully demonstrated the immunogenicity and protective efficacy of a live attenuated vaccine strain, B. malleiΔtonB Δhcp1 (CLH001). Thus, we applied this successful approach to the development of a similar vaccine against melioidosis by constructing the B. pseudomalleiΔtonB Δhcp1 (PBK001) strain. C57BL/6 mice were vaccinated intranasally with the live attenuated PBK001 strain and then challenged with wild-type B. pseudomallei K96243 by the aerosol route. Immunization with strain PBK001 resulted in full protection (100% survival) against acute aerosolized melioidosis with very low bacterial burden as observed in the lungs, livers, and spleens of immunized mice. PBK001 vaccination induced strong production of B. pseudomallei-specific serum IgG antibodies and both Th1 and Th17 CD4+ T cell responses. Further, humoral immunity appeared to be essential for vaccine-induced protection, whereas CD4+ and CD8+ T cells played a less direct immune role. Overall, PBK001 was shown to be an effective attenuated vaccine strain that activates a robust immune response and offers full protection against aerosol infection with B. pseudomalleiIMPORTANCE In recent years, an increasing number of melioidosis cases have been reported in several regions where melioidosis is endemic and in areas where melioidosis had not commonly been diagnosed. Currently, the estimated burden of disease is around 165,000 new cases annually, including 89,000 cases that have fatal outcomes. This life-threatening infectious disease is caused by B. pseudomallei, which is classified as a Tier 1 select agent. Due to the high case fatality rate, intrinsic resistance to multiple antibiotic treatments, susceptibility to infection via the aerosol route, and potential use as a bioweapon, we have developed an effective live attenuated PBK001 vaccine capable of protecting against aerosolized melioidosis.

Bacteriophage-associated genes responsible for the widely divergent phenotypes of variants of Burkholderia pseudomallei strain MSHR5848.

PURPOSE: Burkholderia pseudomallei, the tier 1 agent of melioidosis, is a saprophytic microbe that causes endemic infections in tropical regions such as South-East Asia and Northern Australia. It is globally distributed, challenging to diagnose and treat, infectious by several routes including inhalation, and has potential for adversarial use. B. pseudomallei strain MSHR5848 produces two colony variants, smooth (S) and rough (R), which exhibit a divergent range of morphological, biochemical and metabolic phenotypes, and differ in macrophage and animal infectivity. We aimed to characterize two major phenotypic differences, analyse gene expression and study the regulatory basis of the variation. METHODOLOGY: Phenotypic expression was characterized by DNA and RNA sequencing, microscopy, and differential bacteriology. Regulatory genes were identified by cloning and bioinformatics.Results/Key findings. Whereas S produced larger quantities of extracellular DNA, R was upregulated in the production of a unique chromosome 1-encoded Siphoviridae-like bacteriophage, φMSHR5848. Exploratory transcriptional analyses revealed significant differences in variant expression of genes encoding siderophores, pili assembly, type VI secretion system cluster 4 (T6SS-4) proteins, several exopolysaccharides and secondary metabolites. A single 3 base duplication in S was the only difference that separated the variants genetically. It occurred upstream of a cluster of bacteriophage-associated genes on chromosome 2 that were upregulated in S. The first two genes were involved in regulating expression of the multiple phenotypes distinguishing S and R. CONCLUSION: Bacteriophage-associated proteins have a major role in the phenotypic expression of MSHR5848. The goals are to determine the regulatory basis of this phenotypic variation and its role in pathogenesis and environmental persistence of B. pseudomallei.

Isolation of Burkholderia pseudomallei from a Pet Green Iguana, Belgium.

We isolated Burkholderia pseudomallei, the causative agent of melioidosis, from liver granulomas of a pet green iguana (Iguana iguana) in Belgium. This case highlights a risk for imported green iguanas acting as a reservoir for introduction of this high-threat, zoonotic pathogen into nonendemic regions.