Genetic variation, structural analysis, and virulence implications of BimA and BimC in clinical isolates of Burkholderia pseudomallei in Thailand.

Melioidosis is a life-threatening tropical disease caused by an intracellular gram-negative bacterium Burkholderia pseudomallei. B. pseudomallei polymerizes the host cell actin through autotransporters, BimA, and BimC, to facilitate intracellular motility. Two variations of BimA in B. pseudomallei have been reported previously: BimABp and BimA B. mallei-like (BimABm). However, little is known about genetic sequence variations within BimA and BimC, and their potential effect on the virulence of B. pseudomallei. This study analyzed 1,294 genomes from clinical isolates of patients admitted to nine hospitals in northeast Thailand between 2015 and 2018 and performed 3D structural analysis and plaque-forming efficiency assay. The genomic analysis identified 10 BimABp and 5 major BimC types, in the dominant and non-dominant lineages of the B. pseudomallei population structure. Our protein prediction analysis of all BimABp and major BimC variants revealed that their 3D structures were conserved compared to those of B. pseudomallei K96243. Sixteen representative strains of the most distant BimABp types were tested for plaque formation and the development of polar actin tails in A549 epithelial cells. We found that all isolates retained these functions. These findings enhance our understanding of the prevalence of BimABp and BimC variants and their implications for B. pseudomallei virulence.

Role of the type 6 secretion system on apoptosis and macrophage polarization during Burkholderia pseudomallei infection.

Burkholderia pseudomallei (Bpm) is the causative agent of the disease melioidosis. As a facultative intracellular pathogen, Bpm has a complex lifestyle that culminates in cell-to-cell fusion and multinucleated giant cells (MNGCs) formation. The virulence factor responsible for MNGC formation is the type 6 secretion system (T6SS), a contractile nanomachine. MNGC formation is a cell-to-cell spread strategy that allows the bacteria to avoid the extracellular immune system and our previous data highlighted cell death, apoptosis, and inflammation as pathways significantly impacted by T6SS activity. Thusly, we investigated how the T6SS influences these phenotypes within the macrophage and pulmonary models of infection. Here we report that the T6SS is responsible for exacerbating apoptotic cell death during infection in both macrophages and the lungs of infected mice. We also demonstrate that although the T6SS does not influence differential macrophage polarization, the M2 polarization observed is potentially beneficial for Bpm pathogenesis and replication. Finally, we show that the T6SS contributes to the severity of inflammatory nodule formation in the lungs, which might be potentially connected to the amount of apoptosis that is triggered by the bacteria.

Virulome and phylogenomic profiling of a novel Burkholderia pseudomallei strain from an Indian clinical isolate.

Highly pathogenic Burkholderia pseudomallei is the causative agent of melioidosis, a neglected tropical disease endemic in Southeast Asian tropical region. This bacterium encompasses diverse virulence factors which further undergo dynamic gene-expression flux as it transits through distinct environmental niches within the host which may lead to manifestation of differential clinical symptoms. B. pseudomallei, is classified as a Tier 1 select agent in the United States and regarded as a risk group 3 organism in India with the potential to be used as bioweapon. Considering these facts, it is vital to uncover both physiological and genetic heterogeneity of B. pseudomallei, particularly to identify any novel virulence factors that may contribute to pathogenicity. B. pseudomallei strain CM000113 was isolated from a clinical case in India, characterized it for its physiological, biochemical, and prominently genetic traits through WGS. It has a type 2 morphotype with faster doubling time and high biofilm producing capacity as compared to Pseudomonas aeruginosa. The genome size is 7.3 Mbp and it is phylogenetically close to B. pseudomallei strain Mahidol 1106a and Burkholderia mallei Turkey 2. We observed genetic heterogeneity, as key virulence factors that were identified shows sequence dissimilarity with reference strains. Additionally, presence of genomic islands, harbouring two virulence factors, GmhA and GmhB2, associated with pathogenesis indicates possibility of horizontal gene transfer. These results emphasize the need for an extensive study focusing the genome of B. pseudomallei and its associated heterogeneity, to identify molecular biomarkers aiding to develop point-of-care diagnostic kits for early diagnosis of melioidosis.

Disseminated melioidosis-challenge to routine antibiotic therapy: a case report.

INTRODUCTION: Melioidosis caused by Burkholderia pseudomallei, often referred to as a great mimicker or escapist, evades not only the immune system, but also all manual identification methods in an under-equipped clinical microbiology laboratory due to its tedious identification process. This is a case report of disseminated melioidosis with septic arthritis, misdiagnosed both clinicoradiologically and microbiologically as disseminated tuberculosis or other bacterial infection. CASE HISTORY: A middle-aged Asian diabetic male presented with high-grade fever and breathlessness for 4 days along with left knee and ankle swelling for 40 days. Previous hospitalization records revealed growth of pan-sensitive Acinetobacter spp. from ankle and a chest X-ray suspecting tuberculosis for which antibiotic and antitubercular regimen were initiated. After admission, repeated blood cultures and pus culture (ankle and knee joint) confirmed Burkholderia pseudomallei with VITEK-II automated identification system. Recommended therapy was initiated according to revised Darwin's guideline, leading to gradual cure of the patient. CONCLUSION: Misidentification leads to inadequate treatment, as melioidosis medication is different from other bacterial infections. Here initiation of meropenem- and cotrimoxazole-intensive therapy for 4 weeks, and 6-month eradication phase with cotrimoxazole, resulted in gradual recovery of the patient. It took around 21 days of intensive antibiotic therapy to get bacteriological clearance from blood, which signifies the tenacious nature of this infection.

High Affinity Inhibitors of the Macrophage Infectivity Potentiator Protein from Trypanosoma cruzi, Burkholderia pseudomallei, and Legionella pneumophila─A Comparison.

Since Chagas disease, melioidosis, and Legionnaires' disease are all potentially life-threatening infections, there is an urgent need for new treatment strategies. All causative agents, Trypanosoma cruzi, Burkholderia pseudomallei, and Legionella pneumophila, express a virulence factor, the macrophage infectivity potentiator (MIP) protein, emerging as a promising new therapeutic target. Inhibition of MIP proteins having a peptidyl-prolyl isomerase activity leads to reduced viability, proliferation, and cell invasion. The affinity of a series of pipecolic acid-type MIP inhibitors was evaluated against all MIPs using a fluorescence polarization assay. The analysis of structure-activity relationships led to highly active inhibitors of MIPs of all pathogens, characterized by a one-digit nanomolar affinity for the MIPs and a very effective inhibition of their peptidyl-prolyl isomerase activity. Docking studies, molecular dynamics simulations, and quantum mechanical calculations suggest an extended σ-hole of the meta-halogenated phenyl sulfonamide to be responsible for the high affinity.

Shared challenges to the control of complex intracellular neglected pathogens.

The complex intracellular pathogens Mycobacterium tuberculosis, Mycobacterium leprae, Leishmania spp., and Burkholderia pseudomallei, which cause tuberculosis, leprosy, leishmaniasis, and melioidosis respectively, represent major health threats with a significant global burden concentrated in low- and middle-income countries. While these diseases vary in their aetiology, pathology and epidemiology, they share key similarities in the biological and sociodemographic factors influencing their incidence and impact worldwide. In particular, their occurrence in resource-limited settings has important implications for research and development, disease prevalence and associated risk factors, as well as access to diagnostics and therapeutics. In accordance with the vision of the VALIDATE (VAccine deveLopment for complex Intracellular neglecteD pAThogeEns) Network, we consider shared challenges to the effective prevention, diagnosis and treatment of these diseases as shaped by both biological and social factors, illustrating the importance of taking an interdisciplinary approach. We further highlight how a cross-pathogen perspective may provide valuable insights for understanding and addressing challenges to the control of all four pathogens.

Leptospirosis and melioidosis coinfection presenting as acute respiratory distress syndrome and osteomyelitis: Case report and systematic review.

INTRODUCTION: Leptospirosis and melioidosis are common in tropical and temperate climates and can be acquired by exposure to contaminated water and soil. However, concomitant leptospirosis and melioidosis infection is rarely described in the literature. We report a case of leptospirosis-melioidosis coinfection and systematically review the literature. CASE PRESENTATION: A 42-year-old male presented with fever associated with chills and rigor, dull aching pain in the right thigh, myalgia, progressive breathlessness, and dry cough for 10 days. At presentation, he was tachypneic and had tachycardia, and oxygen saturation was 46% in room air. Chest radiography and computed tomography scan showed interstitial involvement. Magnetic resonance imaging for thigh pain revealed right femur osteomyelitis. Leptospira serology was positive, and blood culture grew Burkholderia pseudomallei, confirming the diagnosis of melioidosis. Thus, a diagnosis of presumptive leptospirosis based on modified Faine's criteria and systemic melioidosis was made. He received doxycycline and intravenous meropenem and improved. RESULTS: We performed a systematic review to understand the spectrum of leptospirosis-melioidosis coinfection. We identified only nine cases of coinfection described in literature. Only one patient had septic arthritis, and our case is the only one presenting with osteomyelitis. Serology diagnosed leptospirosis, whereas melioidosis was confirmed by blood culture in most patients. The majority of coinfected patients developed some complications, and six died. CONCLUSIONS: Leptospirosis-melioidosis coinfection is rarely reported in the literature. Physicians should maintain a high index suspicion of leptospirosis-melioidosis coinfection in patients presenting with acute febrile illness following exposure to soil or freshwater, particularly in tropical and endemic regions.

Virulence of Burkholderia pseudomallei ATS2021 Unintentionally Imported to United States in Aromatherapy Spray.

In the United States in 2021, an outbreak of 4 cases of Burkholderia pseudomallei, the etiologic agent of melioidosis and a Tier One Select Agent (potential for deliberate misuse and subsequent harm), resulted in 2 deaths. The causative strain, B. pseudomallei ATS2021, was unintentionally imported into the United States in an aromatherapy spray manufactured in India. We established that ATS2021 represents a virulent strain of B. pseudomallei capable of robust formation of biofilm at physiologic temperatures that may contribute to virulence. By using mouse melioidosis models, we determined median lethal dose estimates and analyzed the bacteriologic and histopathologic characteristics of the organism, particularly the potential neurologic pathogenesis that is probably associated with the bimABm allele identified in B. pseudomallei strain ATS2021. Our data, combined with previous case reports and the identification of endemic B. pseudomallei strains in Mississippi, support the concept that melioidosis is emerging in the United States.

Protein expression, purification, crystallization and crystallographic studies of BPSL0741 from Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of the lethal disease melioidosis. This bacterium infects animals and humans and is increasingly resistant to multiple antibiotics. Recently, genes associated with survival of the bacterium in the infected host have been identified. One of these genes, bpsl0741, is annotated as a hypothetical protein of 185 amino acids. Here, recombinant BPSL0741 (rBPSL0741) protein was expressed, purified, verified by mass spectrometry, crystallized and analyzed by X-ray diffraction. rBPSL0741 was crystallized by vapor diffusion using a reservoir solution consisting of 0.2 M ammonium acetate, 0.1 M sodium acetate trihydrate pH 4.6, 30% PEG 4000. The crystals diffracted to 2.1 Šresolution using an in-house X-ray diffractometer and belonged to an orthorhombic space group, with unit-cell parameters a = 62.92, b = 64.57, c = 89.16 Å. The Matthews coefficient (VM) was calculated to be 2.18 Å3 Da-1, suggesting the presence of two molecules per asymmetric unit and an estimated solvent content of 43.5%. The crystal was deemed to be suitable for further structural studies, which are currently ongoing.

Performance of MALDI-TOF MS, real-time PCR, antigen detection, and automated biochemical testing for the identification of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease highly endemic to Southeast Asia and northern Australia, though the area of endemicity is expanding. Cases may occur in returning travelers or, rarely, from imported contaminated products. Identification of B. pseudomallei is challenging for laboratories that do not see this organism frequently, and misidentifications by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and automated biochemical testing have been reported. The in vitro diagnostic database for use with the Vitek MS has recently been updated to include B. pseudomallei and we aimed to validate the performance for identification in comparison to automated biochemical testing with the Vitek 2 GN card, quantitative real-time polymerase chain reaction (qPCR) targeting the type III secretion system, and capsular polysaccharide antigen detection using a lateral flow immunoassay (LFA). We tested a "derivation" cohort including geographically diverse B. pseudomallei and a range of closely related Burkholderia species, and a prospective "validation" cohort of B. pseudomallei and B. cepacia complex clinical isolates. MALDI-TOF MS had a sensitivity of 1.0 and specificity of 1.0 for the identification and differentiation of B. pseudomallei from related Burkholderia species when a certainty cutoff of 99.9% was used. In contrast, automated biochemical testing for B. pseudomallei identification had a sensitivity of 0.83 and specificity of 0.88. Both qPCR and LFA correctly identified all B. pseudomallei isolates with no false positives. Due to the high level of accuracy, we have now incorporated MALDI-TOF MS into our laboratory's B. pseudomallei identification workflow.IMPORTANCEBurkholderia pseudomallei causes melioidosis, a disease associated with high morbidity and mortality that disproportionately affects rural areas in Southeast Asia and northern Australia. The known area of endemicity is expanding and now includes the continental United States. Laboratory identification can be challenging which may result in missed or delayed diagnoses and poor patient outcomes. In this study, we compared mass spectrometry using an updated spectral database with multiple other methods for B. pseudomallei identification and found mass spectrometry highly accurate. We have therefore incorporated this fast and cost-effective method into our laboratory's workflow for B. pseudomallei identification.

Isolation and characterization of ssDNA aptamers against BipD antigen of Burkholderia pseudomallei.

BACKGROUND: Melioidosis is difficult to diagnose due to its wide range of clinical symptoms. The culture method is time-consuming and less sensitive, emphasizing the importance of rapid and accurate diagnostic tests for melioidosis. Burkholderia invasion protein D (BipD) of Burkholderia pseudomallei is a potential diagnostic biomarker. This study aimed to isolate and characterize single-stranded DNA aptamers that specifically target BipD. METHODS: The recombinant BipD protein was produced, followed by isolation of BipD-specific aptamers using Systematic Evolution of Ligands by EXponential enrichment. The binding affinity and specificity of the selected aptamers were evaluated using Enzyme-Linked Oligonucleotide Assay. RESULTS: The fifth SELEX cycle showed a notable enrichment of recombinant BipD protein-specific aptamers. Sequencing analysis identified two clusters with a total of seventeen distinct aptamers. AptBipD1, AptBipD13, and AptBipD50 were chosen based on their frequency. Among them, AptBipD1 exhibited the highest binding affinity with a Kd value of 1.0 µM for the recombinant BipD protein. Furthermore, AptBipD1 showed significant specificity for B. pseudomallei compared to other tested bacteria. CONCLUSION: AptBipD1 is a promising candidate for further development of reliable, affordable, and efficient point-of-care diagnostic tests for melioidosis.

Dysregulated immunologic landscape of the early host response in melioidosis.

Melioidosis, a neglected tropical infection caused by Burkholderia pseudomallei, commonly presents as pneumonia or sepsis with mortality rates up to 50% despite appropriate treatment. A better understanding of the early host immune response to melioidosis may lead to new therapeutic interventions and prognostication strategies to reduce disease burden. Whole blood transcriptomic signatures in 164 patients with melioidosis and in 70 patients with other infections hospitalized in northeastern Thailand enrolled within 24 hours following hospital admission were studied. Key findings were validated in an independent melioidosis cohort. Melioidosis was characterized by upregulation of interferon (IFN) signaling responses compared with other infections. Mortality in melioidosis was associated with excessive inflammation, enrichment of type 2 immune responses, and a dramatic decrease in T cell-mediated immunity compared with survivors. We identified and independently confirmed a 5-gene predictive set classifying fatal melioidosis (validation cohort area under the receiver operating characteristic curve 0.83; 95% CI, 0.67-0.99). This study highlights the intricate balance between innate and adaptive immunity during fatal melioidosis and can inform future precision medicine strategies for targeted therapies and prognostication in this severe infection.

The genetic diversity and evolution analysis of the Hainan melioidosis outbreak strains.

Melioidosis is a zoonotic disease, with its outbreaks being rare and indicative of an unusual concurrence of extreme climate and natural environmental factors. An outbreak of melioidosis cases emerged in Hainan following Typhoon "Dianmu" from October to December 2021, presenting an opportunity to identify the environmental sources of infection for these cases due to its nature as a well-defined point-source cluster. To investigate the relationship between the occurrence of these melioidosis cases and the environment, we extracted the entire genome of 25 clinical strains and conducted MLST typing, followed by whole genome sequencing and analysis of molecular genetic information for four ST46 genotypes from these strains. Phylogenetic and evolutionary relationships between Hainan sequence types (STs) and those found in other endemic regions were analyzed using IslandPath-DIMO, PHASTER, e-BURST, PHYLOViZ, and the maximum likelihood method. Notably, a total of 25 clinical strains were identified, encompassing 12 STs (ST46, ST1105, ST1991, ST30, ST1992, ST50, ST164, ST55, ST70, ST1993, ST1545, and ST58), with ST1991, ST1992, and ST1993 being newly discovered subtypes. PHYLOViZ clustering analysis divided the strains into two groups (A and B), both closely related to the Asian region. Phylogenetic tree analysis further revealed that most of the strains in this study were closely related to those found in Australia and Thailand. Analysis of patient information and visits to their residences suggested that contaminated water sources might be the primary source of infection during this outbreak. Our findings underscore that extreme weather events, such as typhoons, significantly increase the infection rate of B. pseudomallei, along with its genetic diversity, necessitating additional prevention strategies to control these B. pseudomallei infections.

Predictors of mortality in culture-proven Burkholderia pseudomallei infections in a Singapore Tertiary Hospital.

Burkholderia pseudomallei is a pathogen expanding in geographic range. We performed a retrospective study analyzing the clinical, microbiologic features of culture-proven melioidosis, and predictors of mortality based on data from a Singapore tertiary hospital between 2006- 2016. We found ICU admission, bacteremia, and mechanical ventilation to be associated with mortality.

Analysis of fine-scale phylogeny of Burkholderia pseudomallei in relation to regional geography and drug susceptibility in Thailand.

Melioidosis caused by Burkholderia pseudomallei (Bp) is a public health threat. Genomic-epidemiology research on this deadly disease is scarce. We investigated whole-genome sequences of Bp isolates in relation to environmental source and drug susceptibility. In total, 563 Bp isolates were collected from 11 Northeast Thai provinces during the period 2004-2021. Patients (n = 530 isolates), infected animals (n = 8), and environmental sources (n = 25) provided samples. Phylogenetic analysis revealed genetic diversity among the Bp isolates, including numerous well-supported clusters of varying sizes. Through in-depth analysis of 38 monophyletic clades (MCs), we found eleven associated with province of origin (p-value < 0.001). Closely related clusters (CRCs) within MCs resembled MLST-identified "sequence types" (STs). We found 102 known and 52 novel STs. ST-70 was the most prevalent in this area (n = 78; 13.85%). Sample type (human/environmental) and sampling time intervals were not correlated with genetic distance among clonal Bp isolates. Some members of 12 CRCs had acquired resistance to co-trimoxazole and one against amoxicillin-clavulanic acid. Within Northeast Thailand, there is an association between Bp genotype and geographical origin.

A molecular epidemiological analysis of Burkholderia pseudomallei in southern Thailand.

Melioidosis, a severe bacterial illness caused by Burkholderia pseudomallei, is prevalent in most parts of Thailand, including its southern region situated within the Malay Peninsula. Despite a lower reported incidence rate of melioidosis in the South compared to the Northeast, the mortality rate remains persistently high. This study aimed to better understand the epidemiology and investigate the presence of B. pseudomallei in the natural environment of southern Thailand. Using multi-locus sequence typing (MLST), we characterized B. pseudomallei isolates derived from human cases and compared them with previously reported sequence types (STs) from the same region. A total of 263 clinical isolates retrieved from 156 melioidosis patients between 2014 and 2020 were analyzed, revealing 72 distinct STs, with 25 (35%) matching STs from Finkelstein's environmental isolates collected in southern Thailand during 1964-1967. Notably, strains bearing STs 288, 84, 54, 289, and 46 were frequently found among patients. Additionally, we observed strain diversity with multiple STs in 13 of 59 patients, indicating exposure to various B. pseudomallei genotypes in the environmental sources of the infection. Environmental surveys were conducted in Songkhla Province to detect B. pseudomallei in soil and water samples where local patients lived. Of the 2737 soil samples from 208 locations and 244 water samples from diverse sources, 52 (25%) soil sampling locations and 63 (26%) water sources were cultured positive for B. pseudomallei. Positive soil samples were predominantly found in animal farming area and non-agricultural zones like mountains and grasslands, while water samples were frequently positive in waterfalls, streams, and surface runoffs, with only 9% of rice paddies testing positive. Collectively, a significant proportion of recent melioidosis cases in Songkhla Province can be attributed to known B. pseudomallei STs persisting in the environment for at least the past six decades. Further characterization of B. pseudomallei isolates from recent environment surveys is warranted. These findings illuminate the contemporary landscape of B. pseudomallei infections and their environmental prevalence in southern Thailand, contributing to the regional threat assessment in Thailand and Southeast Asia.

Point mutation P174L of the penA gene endowing ceftazidime resistance to Burkholderia pseudomallei in China.

In a clinical isolate of Burkholderia pseudomallei from Hainan, the association between the emergence of ceftazidime resistance and a novel PenA P174L allele was identified for the first time, providing an understanding of one mechanism by which ceftazidime resistance arises in B. pseudomallei.

Medical imaging in melioidosis - 20-year experience in a non-endemic Australian city.

INTRODUCTION: Melioidosis may occasionally be encountered in non-endemic areas and medical imaging is frequently used to identify and characterise sites of disease. The purpose of this study is to describe the spectrum of imaging findings encountered in melioidosis patients treated in the tertiary public hospitals of Perth, Western Australia, between 2002 and 2022. METHODS: A database search and electronic medical record review was used to identify cases. Cases were included if they had Burkholderia pseudomallei isolated on culture and if they had at least one diagnostic imaging study performed at a Perth public tertiary hospital. The relevant imaging studies were reviewed, and imaging findings were recorded. RESULTS: Thirty-six cases were identified. The most common disease manifestation was bacteraemia (72%, 26 cases), followed by pulmonary infection (58%, 21 cases), skin and soft tissue infection (22%, eight cases), prostate abscess (14%, five cases) and septic arthritis (6%, two cases). A previously unreported case of isolated melioid pleural effusion was identified, as was a case of reactivated chronic latent pulmonary melioidosis with an apparent delay of over 20 years between the onset of symptoms and the time of infection. In cases with pulmonary melioidosis, the major lung abnormalities on CT chest could be categorised into one of two distinct patterns: nodular-predominant (78%) or consolidation-predominant (22%). CONCLUSION: Further research is required to assess the utility of the pattern-based categorisation of lung abnormalities on CT chest seen in the pulmonary melioidosis cases of this series.

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.