Genetic diversity, determinants, and dissemination of Burkholderia pseudomallei lineages implicated in melioidosis in Northeast Thailand.

Melioidosis is an often-fatal neglected tropical disease caused by an environmental bacterium Burkholderia pseudomallei. However, our understanding of the disease-causing bacterial lineages, their dissemination, and adaptive mechanisms remains limited. To address this, we conduct a comprehensive genomic analysis of 1,391 B. pseudomallei isolates collected from nine hospitals in northeast Thailand between 2015 and 2018, and contemporaneous isolates from neighbouring countries, representing the most densely sampled collection to date. Our study identifies three dominant lineages, each with unique gene sets potentially enhancing bacterial fitness in the environment. We find that recombination drives lineage-specific gene flow. Transcriptome analyses of representative clinical isolates from each dominant lineage reveal increased expression of lineage-specific genes under environmental conditions in two out of three lineages. This underscores the potential importance of environmental persistence for these dominant lineages. The study also highlights the influence of environmental factors such as terrain slope, altitude, and river direction on the geographical dispersal of B. pseudomallei. Collectively, our findings suggest that environmental persistence may play a role in facilitating the spread of B. pseudomallei, and as a prerequisite for exposure and infection, thereby providing useful insights for informing melioidosis prevention and control strategies.

Diabetes and Infectious Diseases with a Focus on Melioidosis.

Diabetes mellitus (DM) leads to impaired innate and adaptive immune responses. This renders individuals with DM highly susceptible to microbial infections such as COVID-19, tuberculosis and melioidosis. Melioidosis is a tropical disease caused by the bacterial pathogen Burkholderia pseudomallei, where diabetes is consistently reported as the most significant risk factor associated with the disease. Type-2 diabetes is observed in 39% of melioidosis patients where the risk of infection is 13-fold higher than non-diabetic individuals. B. pseudomallei is found in the environment and is an opportunistic pathogen in humans, often exhibiting severe clinical manifestations in immunocompromised patients. The pathophysiology of diabetes significantly affects the host immune responses that play a critical role in fighting the infection, such as leukocyte and neutrophil impairment, macrophage and monocyte inhibition and natural killer cell dysfunction. These defects result in delayed recruitment as well as activation of immune cells to target the invading B. pseudomallei. This provides an advantage for the pathogen to survive and adapt within the immunocompromised diabetic patients. Nevertheless, knowledge gaps on diabetes-infectious disease comorbidity, in particular, melioidosis-diabetes comorbidity, need to be filled to fully understand the dysfunctional host immune responses and adaptation of the pathogen under diabetic conditions to guide therapeutic options.

Burkholderia pseudomallei BipD modulates host mitophagy to evade killing.

Mitophagy is critical for mitochondrial quality control and function to clear damaged mitochondria. Here, we found that Burkholderia pseudomallei maneuvered host mitophagy for its intracellular survival through the type III secretion system needle tip protein BipD. We identified BipD, interacting with BTB-containing proteins KLHL9 and KLHL13 by binding to the Back and Kelch domains, recruited NEDD8 family RING E3 ligase CUL3 in response to B. pseudomallei infection. Although evidently not involved in regulation of infectious diseases, KLHL9/KLHL13/CUL3 E3 ligase complex was essential for BipD-dependent ubiquitination of mitochondria in mouse macrophages. Mechanistically, we discovered the inner mitochondrial membrane IMMT via host ubiquitome profiling as a substrate of KLHL9/KLHL13/CUL3 complex. Notably, K63-linked ubiquitination of IMMT K211 was required for initiating host mitophagy, thereby reducing mitochondrial ROS production. Here, we show a unique mechanism used by bacterial pathogens that hijacks host mitophagy for their survival.

Tracking melioidosis in Iran: Utilizing abattoir-based surveillance as a One Health approach.

BACKGROUND: Burkholderia pseudomallei, an environmental saprophyte bacterium, causes melioidosis in humans and animals. It was first discovered in Iran between 1967 and 1976 in small ruminants, equines, environments and humans. No subsequent studies have been conducted to determine the existence and prevalence of this pathogen in the country. OBJECTIVES: The present study aims to monitor the presence of B. pseudomallei in the ruminant population of the Golestan province of Iran, which largely depends on pastures. The ruminants can serve as sentinels to indicate the presence of the bacteria in the environment and its potential impact on human health in the One Health triad. METHODS: Liver and lung abscesses from domestic sheep, cattle and goats in three industrial and three conventional slaughterhouses were sampled and analysed using 23S ribosomal DNA polymerase chain reaction (rDNA PCR) with primers CVMP 23-1 and CVP-23-2 for B. pseudomallei, Burkholderia cepacia and Burkholderia vietnamiensis, as well as B. pseudomallei-specific TTS1 real-time PCR, along with microbiological and biochemical assays. RESULTS: Out of the 97 animals sampled, only 14 (15%) tested positive for 23S rDNA PCR. However, the follow-up evaluation using TTS1 real-time PCR and microbiological and biochemical assays did not confirm the presence of B. pseudomallei in the samples. CONCLUSIONS: Although B. pseudomallei was not detected in the current survey, conducting abattoir-based surveillance of ruminants is a cost-effective One Health approach to monitor pathogenic Burkholderia. Developing standards of clinical and laboratory good practices for Burkholderia infections is crucial for One Health surveillance.

Melioidosis and Activation from Latency: The "Time Bomb" Has Not Occurred.

Burkholderia pseudomallei, the causative agent of melioidosis has long been considered able to exist in a latent form. Seropositivity among U.S. soldiers returning from the Vietnam conflict led to melioidosis being dubbed "the Vietnamese time bomb." Cases assigned to "(re)activation from latency" over 30 years of the Darwin Prospective Melioidosis Study (DPMS) were reviewed and reassessed and additional cases from DPMS years 31-34 were added. Historical reports of melioidosis attributed to activation from latency were reviewed. Some earlier DPMS cases and most historical cases described as activation from latency more accurately reflect undiagnosed chronic melioidosis, often with relapsing-remitting courses, rather than truly latent, asymptomatic infection. Such protracted disease should now be diagnosable much earlier, provided melioidosis is considered and laboratory facilities are available. The longest plausible duration of asymptomatic latency remains 29 years. In conclusion, activation from latency is a rare event in melioidosis, accounting in our analysis for under 3% of DPMS cases, consistent with why the Vietnamese time bomb never eventuated.

Exploring the Relationship between Melioidosis Morbidity Rate and Local Environmental Indicators Using Remotely Sensed Data.

Melioidosis is an endemic infectious disease caused by Burkholderia pseudomallei bacteria, which contaminates soil and water. To better understand the environmental changes that have contributed to melioidosis outbreaks, this study used spatiotemporal analyses to clarify the distribution pattern of melioidosis and the relationship between melioidosis morbidity rate and local environmental indicators (land surface temperature, normalised difference vegetation index, normalised difference water index) and rainfall. A retrospective study was conducted from January 2013 to December 2022, covering data from 219 sub-districts in Northeast Thailand, with each exhibiting a varying morbidity rate of melioidosis on a monthly basis. Spatial autocorrelation was determined using local Moran's I, and the relationship between the melioidosis morbidity rate and the environmental indicators was evaluated using a geographically weighted Poisson regression. The results revealed clustered spatiotemporal patterns of melioidosis morbidity rate across sub-districts, with hotspots predominantly observed in the northern region. Furthermore, we observed a range of coefficients for the environmental indicators, varying from negative to positive, which provided insights into their relative contributions to melioidosis in each local area and month. These findings highlight the presence of spatial heterogeneity driven by environmental indicators and underscore the importance of public health offices implementing targeted monitoring and surveillance strategies for melioidosis in different locations.

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.

Electrochemical Immunoassay for Capturing Capsular Polysaccharide of Burkholderia pseudomallei: Early Onsite Detection of Melioidosis.

This study presented the detection and quantification of capsular polysaccharide (CPS) as a biomarker for the diagnosis of melioidosis. After successfully screening four monoclonal antibodies (mAbs) previously determined to bind CPS molecules, the team developed a portable electrochemical immunosensor based on antibody-antigen interactions. The biosensor was able to detect CPS with a wide detection range from 0.1pg/mL to 1 µg/mL. The developed biosensor achieved high sensitivity for the detection of CPS spiked into both urine and serum. The developed assay platform was successfully programmed into a Windows app, and the sensor performance was evaluated with different spiked concentrations. The rapid electro-analytical device (READ) sensor showed great unprecedented sensitivity for the detection of CPS molecules in both serum and urine, and results were cross-validated with ELISA methods.

Subtractive proteomics-guided vaccine targets identification and designing of multi-epitopes vaccine for immune response instigation against Burkholderia pseudomallei.

In this study, a methodical workflow using subtractive proteomics, vaccine designing, molecular simulation, and agent-based modeling approaches were used to annotate the whole proteome of Burkholderia pseudomallei (strain K96243) for vaccine designing. Among the total 5717 proteins in the whole proteome, 505 were observed to be essential for the pathogen's survival and pathogenesis predicted by the Database of Essential Genes. Among these, 23 vaccine targets were identified, of which fimbrial assembly chaperone (Q63UH5), Outer membrane protein (Q63UH1), and Hemolysin-like protein (Q63UE4) were selected for the subsequent analysis based on the systematic approaches. Using immunoinformatic approaches CTL (cytotoxic T lymphocytes), HTL (helper T lymphocytes), IFN-positive, and B cell epitopes were predicted for these targets. A total of 9 CTL epitopes were added using the GSS linker, 6 HTL epitopes using the GPGPG linker, and 6 B cell epitopes using the KK linker. An adjuvant was added for enhanced antigenicity, an HIV-TAT peptide for improved delivery, and a PADRE sequence was added to form a 466 amino acids long vaccine construct. The construct was classified as non-allergenic, highly antigenic, and experimentally feasible. Molecular docking results validated the robust interaction of MEVC with immune receptors such as TLR2/4. Furthermore, molecular simulation revealed stable dynamics and compact nature of the complexes. The binding free energy results further validated the robust binding. In silico cloning, results revealed GC contents of 50.73 % and a CIA value of 0.978 which shows proper downstream processing. Immune simulation results reported that after the three injections of the vaccine a robust secondary immune response, improved antigen clearance, and effective immune memory generation were observed highlighting its potential for effective and sustained immunity. Future directions should encompass experimental validations, animal model studies, and clinical trials to substantiate the vaccine's efficacy, safety, and immunogenicity.

Understanding The Mimicker: Epidemiological Pattern and Determinant of Melioidosis Mortality in Negeri Sembilan, Malaysia.

BACKGROUND: Melioidosis, a tropical infectious disease caused by Burkholderia pseudomallei, is epidemic in most region in Southeast Asia with high case fatality. However, there is scanty information regarding the disease's epidemiological pattern, demographics, and underlying risk factors. METHOD: This 5-year retrospective study of 185 confirmed cases which were taken from the Negeri Sembilan Melioidosis Registry between 2018 and 2022. We aim to describe the incidence, mortality rate, case fatality, relationship with meteorology, and factors that influence mortality in this central region of Peninsular Malaysia. RESULTS: Incidence rate (IR) of melioidosis in Negeri Sembilan is varied at 1.9 to 5.1 with mean of 3.1 in 100,000 population per year. IR varied between districts in the state from zero to 22.01 in 100,000 population per year. Mortality rate were ranged from 0.17 to 0.74 cases with mean of 0.44 cases in 100,000 population per year. The case fatality rate of this state scattered from 8.70% to 16.67%. There were no significant linear associations between cases and deaths with monthly rainfall and humidity. The mean age of patients was 52.8 years, predominated with age around 41-60 years old. Males (77.8%) predominated, and the majority of cases were Malays (88.9%) and had exposed to soil related activities (74.6%). Mortality from melioidosis was more likely in Bumiputera and non-Malaysians (p<0.05). Patients who had at least one comorbidity were at a higher risk of death from melioidosis (p<0.05). Diabetes mellitus was found in 41.1% of all identified cases, making it a major underlying risk factor for both developing and dying from melioidosis (aOR:19.32, 95%CI:1.91-195.59, p<0.05). Hypertension and mortality status in melioidosis are also significantly correlated (aOR: 7.75, 95% CI: 2.26-26.61, p<0.05). CONCLUSION: The epidemiological patterns of cases reported from Negeri Sembilan are consistent for the most part from previous studies in other states in Malaysia and global with regard to its incidence, case fatality, demographic and predisposing chronic diseases. Diabetes mellitus and hypertension were significantly linked to increased mortality among all determinants.

Genome sequence of two novel virulent clinical strains of Burkholderia pseudomallei isolated from acute melioidosis cases imported to Israel from India and Thailand.

OBJECTIVE: Burkholderia pseudomallei, the etiological cause of melioidosis, is a soil saprophyte endemic in South-East Asia, where it constitutes a public health concern of high-priority. Melioidosis cases are sporadically identified in nonendemic areas, usually associated with travelers or import of goods from endemic regions. Due to extensive intercontinental traveling and the anticipated climate change-associated alterations of the soil bacterial flora, there is an increasing concern for inadvertent establishment of novel endemic areas, which may expand the global burden of melioidosis. Rapid diagnosis, isolation and characterization of B. pseudomallei isolates is therefore of utmost importance particularly in non-endemic locations. DATA DESCRIPTION: We report the genome sequences of two novel clinical isolates (MWH2021 and MST2022) of B. pseudomallei identified in distinct acute cases of melioidosis diagnosed in two individuals arriving to Israel from India and Thailand, respectively. The data includes preliminary genetic analysis of the genomes determining their phylogenetic classification in rapport to the genomes of 131 B. pseudomallei strains documented in the NCBI database. Inspection of the genomic data revealed the presence or absence of loci encoding for several documented virulence determinants involved in the molecular pathogenesis of melioidosis. Virulence analysis in murine models of acute or chronic melioidosis established that both strains belong to the highly virulent class of B. pseudomalleii.

Diagnostic accuracy of automation and non-automation techniques for identifying Burkholderia pseudomallei: A systematic review and meta-analysis.

BACKGROUND: Burkholderia pseudomallei, a Gram-negative pathogen, causes melioidosis. Although various clinical laboratory identification methods exist, culture-based techniques lack comprehensive evaluation. Thus, this systematic review and meta-analysis aimed to assess the diagnostic accuracy of culture-based automation and non-automation methods. METHODS: Data were collected via PubMed/MEDLINE, EMBASE, and Scopus using specific search strategies. Selected studies underwent bias assessment using QUADAS-2. Sensitivity and specificity were computed, generating pooled estimates. Heterogeneity was assessed using I2 statistics. RESULTS: The review encompassed 20 studies with 2988 B. pseudomallei samples and 753 non-B. pseudomallei samples. Automation-based methods, particularly with updating databases, exhibited high pooled sensitivity (82.79%; 95% CI 64.44-95.85%) and specificity (99.94%; 95% CI 98.93-100.00%). Subgroup analysis highlighted superior sensitivity for updating-database automation (96.42%, 95% CI 90.01-99.87%) compared to non-updating (3.31%, 95% CI 0.00-10.28%), while specificity remained high at 99.94% (95% CI 98.93-100%). Non-automation methods displayed varying sensitivity and specificity. In-house latex agglutination demonstrated the highest sensitivity (100%; 95% CI 98.49-100%), followed by commercial latex agglutination (99.24%; 95% CI 96.64-100%). However, API 20E had the lowest sensitivity (19.42%; 95% CI 12.94-28.10%). Overall, non-automation tools showed sensitivity of 88.34% (95% CI 77.30-96.25%) and specificity of 90.76% (95% CI 78.45-98.57%). CONCLUSION: The study underscores automation's crucial role in accurately identifying B. pseudomallei, supporting evidence-based melioidosis management decisions. Automation technologies, especially those with updating databases, provide reliable and efficient identification.

Development of a novel sequence based real-time PCR assay for specific and sensitive detection of Burkholderia pseudomallei in clinical and environmental matrices.

BACKGROUND: Melioidosis, caused by the category B biothreat agent Burkholderia pseudomallei, is a disease with a high mortality rate and requires an immediate culture-independent diagnosis for effective disease management. In this study, we developed a highly sensitive qPCR assay for specific detection of Burkholderia pseudomallei and melioidosis disease diagnosis based on a novel target sequence. METHODS: An extensive in-silico analysis was done to identify a novel and highly conserved sequence for developing a qPCR assay. The specificity of the developed assay was analyzed with 65 different bacterial cultures, and the analytical sensitivity of the assay was determined with the purified genomic DNA of B. pseudomallei. The applicability of the assay for B. pseudomallei detection in clinical and environmental matrices was evaluated by spiking B. pseudomallei cells in the blood, urine, soil, and water along with suitable internal controls. RESULTS: A novel 85-nucleotide-long sequence was identified using in-silico tools and employed for the development of the highly sensitive and specific quantitative real-time PCR assay S664. The assay S664 was found to be highly specific when evaluated with 65 different bacterial cultures related and non-related to B. pseudomallei. The assay was found to be highly sensitive, with a detection limit of 3 B. pseudomallei genome equivalent copies per qPCR reaction. The detection limit in clinical matrices was found to be 5 × 102 CFU/mL for both human blood and urine. In environmental matrices, the detection limit was found to be 5 × 101 CFU/mL of river water and 2 × 103 CFU/gm of paddy field soil. CONCLUSIONS: The findings of the present study suggest that the developed assay S664 along with suitable internal controls has a huge diagnostic potential and can be successfully employed for specific, sensitive, and rapid molecular detection of B. pseudomallei in various clinical and environmental matrices.

Outcomes of Adjunctive Therapy with Open Arthrotomy for Patients with Septic Arthritis Due to Melioidosis: A Retrospective Study.

Melioidosis is a potentially fatal infection caused by the bacterium Burkholderia pseudomallei. Septic arthritis caused by this infection is uncommon and difficult to treat. The role of adjunctive open arthrotomy in this type of infection has not yet been elucidated. We conducted a retrospective study of patients with microbiologically confirmed melioidosis between January 2002 and December 2022. Patients with a clinical condition of septic arthritis and positive cultures for B. pseudomallei were included. Comparisons were made between patients who received adjunctive therapy with open arthrotomy with conventional standard treatment and those who did not in terms of clinical outcomes and hospital expenditures. Of the 478 patients diagnosed with melioidosis microbiological confirmation, 81 patients had septic arthritis, accounting for 17% of cases. Among these patients, only 36 (44%) underwent adjunctive therapy with open arthrotomy. The 14-day and 30-day in-hospital mortality and length of hospital stays of patients who underwent adjunctive therapy with open arthrotomy were more favorable than those of patients who did not receive adjunctive therapy with open arthrotomy; however, the difference was not statistically significant. Patients who underwent adjunctive therapy with open arthrotomy had lower hospital expenditures (antimicrobial and non-antimicrobial costs) than those who did not undergo open arthrotomy. Adjunctive therapy with open arthrotomy for patients with septic arthritis due to melioidosis was associated with favorable clinical outcomes and significantly lower hospital expenditures.

Magnetically modified bacteriophage-triggered ATP release activated EXPAR-CRISPR/Cas14a system for visual detection of Burkholderia pseudomallei.

Burkholderia pseudomallei, widely distributed in tropical and subtropical ecosystems, is capable of causing the fatal zoonotic disease melioidosis and exhibiting a global trend of dissemination. Rapid and sensitive detection of B. pseudomallei is essential for environmental monitoring as well as infection control. Here, we developed an innovative biosensor for quantitatively detecting B. pseudomallei relies on ATP released triggered by bacteriophage-induced bacteria lysis. The lytic bacteriophage vB_BpP_HN01, with high specificity, is employed alongside magnetic nanoparticles assembly to create a biological receptor, facilitating the capture and enrichment of viable target bacteria. Following a brief extraction and incubation process, the captured target undergoes rapid lysis to release contents including ATP. The EXPAR-CRISPR cascade reaction provides an efficient signal transduction and dual amplification module that allowing the generated ATP to guide the signal output as an activator, ultimately converting the target bacterial amount into a detectable fluorescence signal. The proposed bacteriophage affinity strategy exhibited superior performance for B. pseudomallei detection with a dynamic range from 10^2 to 10^7 CFU mL-1, and a LOD of 45 CFU mL-1 within 80 min. Moreover, with the output signal compatible across various monitoring methods, this work offers a robust assurance for rapid diagnosis and on-site environmental monitoring of B. pseudomallei.

The Predictive Factors Associated with In-Hospital Mortality of Melioidosis: A Cohort Study.

Background and Objectives: Melioidosis is an infectious disease caused by Burkholderia pseudomallei, and it has a wide range of clinical symptoms. It is endemic in tropical areas, including Southeast Asia. Despite the availability of effective treatment, the mortality rate is still high, especially in patients presenting with septic shock. The aim of this study was to determine and explore clinical characteristics, microbiology, treatment outcomes, and factors associated with in-hospital mortality which could predict prognosis and provide a guide for future treatment. Materials and Methods: The population in this retrospective cohort study included all 262 patients with a diagnosis of melioidosis who were hospitalized at Surin Hospital, Surin, Thailand, from April 2014 to March 2017. We included patients older than 15 years with a positive culture for B. pseudomallei. Data regarding the clinical characteristics, microbiology, and treatment outcomes of the patients were collected and analyzed. The patients were divided into two groups dependent on outcome, specifically non-survival and survival. Logistic regression was performed to determine the risk factors associated with in-hospital mortality. Results: Out of the 262 patients with melioidosis during the study period, 117 (44.7%) patients died. The mean age was 57.2 ± 14.4 years, and 193 (73.7%) patients were male. The most common comorbidity was diabetes (123, 46.9%), followed by chronic kidney disease (35, 13.4%) and chronic liver disease (31, 11.8%). Four risk factors were found to be associated with in-hospital mortality, including age (adjusted odds ratio (aOR) 1.04, 95%CI: 1.01-1.07), respiration rate (aOR 1.18, 95%CI: 1.06-1.32), abnormal chest X-ray finding (aOR 4.79, 95%CI: 1.98-11.59), and bicarbonate levels (CO2) (aOR 0.92, 95%CI: 0.85-0.99). Conclusions: Our study identified age, respiration rate, abnormal chest X-ray finding, and CO2 levels are predictive factors associated with in-hospital mortality in melioidosis patients. Physicians should be aware of these factors, have access to aggressive treatment options, and closely monitor patients with these risk factors.

Melioidosis masquerading as malignancy in tropical Australia; lessons for clinicians and implications for clinical management.

Melioidosis is a life-threatening, emerging infectious disease caused by the environmental bacterium Burkholderia pseudomallei. Melioidosis is hyperendemic in tropical Australia and southeast Asia, however the disease is increasingly encountered beyond these regions. Early diagnosis is essential as the infection has a case-fatality rate of up to 50 %. Melioidosis most commonly involves the lungs, although almost any organ can be affected. Most patients present acutely but an insidious presentation over weeks to months is also well described. We present a case series of 7 patients from tropical Australia whom local clinicians initially believed to have cancer ‒ most commonly lung cancer ‒ only for further investigation to establish a diagnosis of melioidosis. All 7 patients had comorbidities that predisposed them to developing melioidosis and all survived, but their delayed diagnosis resulted in 3 receiving anti-cancer therapies that resulted in significant morbidity. The study emphasises the importance of thorough diagnostic evaluation and repeated collection of microbiological samples. It is hoped that our experience will encourage other clinicians ‒ in the appropriate clinical context ‒ to consider melioidosis as a potential explanation for a patient's presentation, expediting its diagnosis and the initiation of potentially life-saving therapy.

Flagellin-modulated inflammasome pathways characterize the human alveolar macrophage response to Burkholderia pseudomallei, a lung-tropic pathogen.

Melioidosis is an emerging tropical infection caused by inhalation, inoculation, or ingestion of the flagellated, facultatively intracellular pathogen Burkholderia pseudomallei. The melioidosis case fatality rate is often high, and pneumonia, the most common presentation, doubles the risk of death. The alveolar macrophage is a sentinel pulmonary host defense cell, but the human alveolar macrophage in B. pseudomallei infection has never been studied. The objective of this study was to investigate the host-pathogen interaction of B. pseudomallei infection with the human alveolar macrophage and to determine the role of flagellin in modulating inflammasome-mediated pathways. We found that B. pseudomallei infects primary human alveolar macrophages but is gradually restricted in the setting of concurrent cell death. Electron microscopy revealed cytosolic bacteria undergoing division, indicating that B. pseudomallei likely escapes the alveolar macrophage phagosome and may replicate in the cytosol, where it triggers immune responses. In paired human blood monocytes, uptake and intracellular restriction of B. pseudomallei are similar to those observed in alveolar macrophages, but cell death is reduced. The alveolar macrophage cytokine response to B. pseudomallei is characterized by marked interleukin (IL)-18 secretion compared to monocytes. Both cytotoxicity and IL-18 secretion in alveolar macrophages are partially flagellin dependent. However, the proportion of IL-18 release that is driven by flagellin is greater in alveolar macrophages than in monocytes. These findings suggest differential flagellin-mediated inflammasome pathway activation in the human alveolar macrophage response to B. pseudomallei infection and expand our understanding of intracellular pathogen recognition by this unique innate immune lung cell.

Inhibition of macrophage infectivity potentiator in Burkholderia pseudomallei suppresses pro-inflammatory responses in murine macrophages.

Introduction: Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a disease endemic in many tropical countries globally. Clinical presentation is highly variable, ranging from asymptomatic to fatal septicemia, and thus the outcome of infection can depend on the host immune responses. The aims of this study were to firstly, characterize the macrophage immune response to B. pseudomallei and secondly, to determine whether the immune response was modified in the presence of novel inhibitors targeting the virulence factor, the macrophage infectivity potentiator (Mip) protein. We hypothesized that inhibition of Mip in B. pseudomallei would disarm the bacteria and result in a host beneficial immune response. Methods: Murine macrophage J774A.1 cells were infected with B. pseudomallei K96243 in the presence of small-molecule inhibitors targeting the Mip protein. RNA-sequencing was performed on infected cells four hours post-infection. Secreted cytokines and lactose dehydrogenase were measured in cell culture supernatants 24 hours post-infection. Viable, intracellular B. pseudomallei in macrophages were also enumerated 24 hours post-infection. Results: Global transcriptional profiling of macrophages infected with B. pseudomallei by RNA-seq demonstrated upregulation of immune-associated genes, in particular a significant enrichment of genes in the TNF signaling pathway. Treatment of B. pseudomallei-infected macrophages with the Mip inhibitor, AN_CH_37 resulted in a 5.3-fold reduction of il1b when compared to cells treated with DMSO, which the inhibitors were solubilized in. A statistically significant reduction in IL-1ß levels in culture supernatants was seen 24 hours post-infection with AN_CH_37, as well as other pro-inflammatory cytokines, namely IL-6 and TNF-α. Treatment with AN_CH_37 also reduced the survival of B. pseudomallei in macrophages after 24 hours which was accompanied by a significant reduction in B. pseudomallei-induced cytotoxicity as determined by lactate dehydrogenase release. Discussion: These data highlight the potential to utilize Mip inhibitors in reducing potentially harmful pro-inflammatory responses resulting from B. pseudomallei infection in macrophages. This could be of significance since overstimulation of pro-inflammatory responses can result in immunopathology, tissue damage and septic shock.