γδ T Cells Mediate Protection Against Neutrophil-associated Lung Inflammation in Pulmonary Melioidosis.

Pulmonary melioidosis is a severe tropical infection caused by Burkholderia pseudomallei and is associated with high mortality despite early antibiotic treatment. γδ T cells have been increasingly implicated as drivers of the host neutrophil response during bacterial pneumonia, but their role in pulmonary melioidosis is unknown. Here, we report that in patients with melioidosis, a lower peripheral blood γδ T cell concentration is associated with higher mortality even when adjusting for severity of illness. γδ T cells were also enriched in the lung and protected against mortality in a mouse model of pulmonary melioidosis. γδ T cell deficiency in infected mice induced an early recruitment of neutrophils to the lung, independent of bacterial burden. Subsequently, γδ T cell deficiency resulted in increased neutrophil-associated inflammation in the lung as well as impaired bacterial clearance. Additionally, γδ T cells influenced neutrophil function and subset diversity in the lung after infection. Our results indicate that γδ T cells serve a novel protective role in the lung during a severe bacterial pneumonia by regulating excessive neutrophil-associated inflammation.

Layering vaccination with antibiotic therapy results in protection and clearance of Burkholderia pseudomallei in Balb/c mice.

Melioidosis is a disease that is difficult to treat due to the causative organism, Burkholderia pseudomallei being inherently antibiotic resistant and it having the ability to invade, survive, and replicate in an intracellular environment. Combination therapy approaches are routinely being evaluated in animal models with the aim of improving the level of protection and clearance of colonizing bacteria detected. In this study, a subunit vaccine layered with the antibiotic finafloxacin was evaluated in vivo against an inhalational infection with B. pseudomallei in Balb/c mice. Groups of mice vaccinated, infected, and euthanized at antibiotic initiation had a reduced bacterial load compared to those that had not been immunized. In addition, the subunit vaccine provided a synergistic effect when it was delivered with a CpG ODN and finafloxacin was initiated at 48 h post-challenge. Vaccination was also shown to improve the outcome, in a composite measure of survival and clearance. In summary, layering a subunit vaccine with the antibiotic finafloxacin is a promising therapeutic alternative for use in the treatment of B. pseudomallei infections.

Decoration of Burkholderia Hcp1 protein to virus-like particles as a vaccine delivery platform.

Virus-like particles (VLPs) are protein-based nanoparticles frequently used as carriers in conjugate vaccine platforms. VLPs have been used to display foreign antigens for vaccination and to deliver immunotherapy against diseases. Hemolysin-coregulated proteins 1 (Hcp1) is a protein component of the Burkholderia type 6 secretion system, which participates in intracellular invasion and dissemination. This protein has been reported as a protective antigen and is used in multiple vaccine candidates with various platforms against melioidosis, a severe infectious disease caused by the intracellular pathogen Burkholderia pseudomallei. In this study, we used P22 VLPs as a surface platform for decoration with Hcp1 using chemical conjugation. C57BL/6 mice were intranasally immunized with three doses of either PBS, VLPs, or conjugated Hcp1-VLPs. Immunization with Hcp1-VLPs formulation induced Hcp1-specific IgG, IgG1, IgG2c, and IgA antibody responses. Furthermore, the serum from Hcp1-VLPs immunized mice enhanced the bacterial uptake and opsonophagocytosis by macrophages in the presence of complement. This study demonstrated an alternative strategy to develop a VLPs-based vaccine platform against Burkholderia species.

Burkholderia pseudomallei Bacteria in Ornamental Fish Tanks, Vientiane, Laos, 2023.

In 2019, a melioidosis case in Maryland, USA, was shown to have been acquired from an ornamental fish tank contaminated with Burkholderia pseudomallei bacteria, likely derived from Southeast Asia. We investigated the presence of B. pseudomallei in ornamental fish tanks in the endemic area of Vientiane, Laos.

Melioidosis in Patients with COVID-19 Exposed to Contaminated Tap Water, Thailand, 2021.

In September 2021, a total of 25 patients diagnosed with COVID-19 developed acute melioidosis after (median 7 days) admission to a COVID-19 field hospital in Thailand. Eight nonpotable tap water samples and 6 soil samples were culture-positive for Burkholderia pseudomallei. Genomic analysis suggested contaminated tap water as the likely cause of illness.

Synthesis of BLF1-containing trimethyl chitosan nanoparticles and evaluation of its immunogenicity and protection in syrian mice by oral and subcutaneous injections.

The bacterium Burkholderia pseudomallei is the cause of melioidosis infectious disease. In this bacterium, the BLF1 protein wide inhibits the synthesis of proteins in human cells. This disease is reported to cause a death rate of 40% in some parts of the world. Currently, no effective vaccine is available against this bacterial infection. In this study, therefore, a Nano vaccine was synthesized based on the trimethyl chitosan (TMC) polymer containing the BLF1 recombinant protein, and its immunogenicity and protection in Syrian mice were evaluated by oral and subcutaneous injections. The BLF1 recombinant protein expression was induced in Escherichia coli Bl21 (DE3) and purified by the affinity chromatography technique. Recombinant protein-containing nanoparticles (NPs) were then synthesized by the ionotropic gelation method. After oral and subcutaneous injections, antibody titration was assessed by the indirect ELISA assay. Finally, murine groups were challenged using the BLF1 toxin. The results indicated that the immune system showed more antibody titration in subcutaneous injection than in the oral form. However, the results were reversed in the challenge results, and the survival rate was more significant in the oral injection.

A case of pulmonary melioidosis in Germany: a rare differential diagnosis in returning travelers from South-East Asia.

BACKGROUND: Melioidosis is a bacterial infection associated with high mortality. The diagnostic approach to this rare disease in Europe is challenging, especially because pulmonary manifestation of melioidosis can mimic pulmonary tuberculosis (TB). Antibiotic therapy of melioidosis consists of an initial intensive phase of 2-8 weeks followed by an eradication therapy of 3-6 months. CASE PRESENTATION: We present the case of a 46-year-old female patient with pulmonary melioidosis in Germany. The patient showed chronic cough, a pulmonary mass and a cavitary lesion, which led to the initial suspicion of pulmonary TB. Melioidosis was considered due to a long-term stay in Thailand with recurrent exposure to rice fields. RESULTS: Microbiologic results were negative for TB. Histopathology of an endobronchial tumor showed marked chronic granulation tissue and fibrinous inflammation. Melioidosis was diagnosed via polymerase chain reaction by detection of Burkholderia pseudomallei/mallei target from mediastinal lymph-node tissue. CONCLUSION: This case report emphasizes that melioidosis is an important differential diagnosis in patients with suspected pulmonary tuberculosis and recent travel to South-East Asia.

Clinical and microbiological features of positive blood culture episodes caused by non-fermenting gram-negative bacilli other than Pseudomonas and Acinetobacter species (2020-2023).

INTRODUCTION: Non-fermenting Gram-negative bacilli (NFGNB) other than Pseudomonas aeruginosa and Acinetobacter baumannii complex are pathogens of interest due to their ability to cause health-care associated infections and display complex drug resistance phenotypes. However, their clinical and microbiological landscape is still poorly characterized. METHODS: Observational retrospective study including all hospitalized patients presenting with a positive positive blood culture (BC) episode caused by less common NFGNB over a four-year period (January 2020-December 2023). Clinical-microbiological features and factors associated with mortality were investigated. RESULTS: Sixty-six less common NFGNB isolates other than Pseudomonas and Acinetobacter species causing 63 positive BC episodes were recovered from 60 patients. Positive BC episodes were predominantly sustained by Stenotrophomonas maltophilia (49.2%) followed by Achromobacter species (15.9%) that exhibited the most complex resistance phenotype. Positive BC episodes had bloodstream infection criteria in 95.2% of cases (60 out 63), being intravascular device (30.2%) and respiratory tract (19.1%) the main sources of infection. Fourteen-day, 30-day, and in-hospital mortality rates were 6.4%, 9.5%, and 15.9%, respectively. The longer time from admission to the positive BC episode, older age, diabetes, admission due to sepsis, and higher Charlson Comorbidity Index were identified as the main predictors of in-hospital mortality. CONCLUSIONS: Positive BC episodes sustained by NFGNB other than Pseudomonas and Acinetobacter species were predominantly sustained by Stenotrophomonas maltophilia and Achromobacter species, having bloodstream infection criteria in the vast majority of cases. Factors that have emerged to be associated with mortality highlighted how these species may have more room in prolonged hospitalisation and at the end of life for patients with chronic organ diseases.

Paediatric melioidosis.

Melioidosis is a tropical infectious disease caused by the saprophytic gram-negative bacterium Burkholderia pseudomallei. Despite the infection being endemic in southeast Asia and northern Australia, the broad clinical presentations and diagnostic difficulties limit its early detection, particularly in children. Melioidosis more commonly affects the immunocompromised and adults. Melioidosis is increasingly being diagnosed around the world and whole-genome sequencing indicates that these cases are not linked with travel to endemic areas. Research has concentrated on the adult population with limited experience reported in the care of this uncommon, but potentially fatal condition in children presenting with bacteraemia and pneumonia.

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.

Hemophagocytic lymphohistiocytosis complicating septicemic melioidosis: A case report.

Melioidosis is an infectious disease caused by Burkholderia pseudomallei. People infected with B. pseudomallei experience fever and skin changes, pneumonia, abscesses, and septic shock that could cause death. Hemophagocytic lymphohistiocytosis is a severe inflammatory syndrome due to the excess activation of macrophages and T cells. We report a 50-year-old hypertensive and diabetic male patient presented with high-grade intermittent fever with loss of appetite and weight loss for two months and a history of jaundice, backache and swelling of both feet for 15 days. Blood and bone marrow culture grew Burkholderia pseudomallei. A liver biopsy revealed Kupffer cell hyperplasia and hemophagocytosis. The patient was treated with an injection of dexamethasone 4mg intravenous three times a day for five days and tapered over 15 days with ceftazidime 2 gm intravenous three times a day for six weeks. Early suspicion in the diagnosis of hemophagocytic lymphohistiocytosis in septicemia can prevent severe complications, even death.

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.

Burkholderia pseudomallei Laboratory Exposure, Arizona, USA.

We describe an incidental Burkholderia pseudomallei laboratory exposure in Arizona, USA. Because melioidosis cases are increasing in the United States and B. pseudomallei reservoirs have been discovered in the Gulf Coast Region, US laboratory staff could be at increased risk for B. pseudomallei exposure.

Presence of Burkholderia pseudomallei in Soil, Nigeria, 2019.

Melioidosis, caused by the soil-dwelling bacterium Burkholderia pseudomallei, is predicted to be endemic in Nigeria but is only occasionally reported. This report documents the systematic identification of the presence of B. pseudomallei and B. thailandensis in the soil across multiple states in Nigeria.

Clinical Manifestations and Genomic Evaluation of Melioidosis Outbreak among Children after Sporting Event, Australia.

Melioidosis, caused by the environmental gram-negative bacterium Burkholderia pseudomallei, usually develops in adults with predisposing conditions and in Australia more commonly occurs during the monsoonal wet season. We report an outbreak of 7 cases of melioidosis in immunocompetent children in Australia. All the children had participated in a single-day sporting event during the dry season in a tropical region of Australia, and all had limited cutaneous disease. All case-patients had an adverse reaction to oral trimethoprim/sulfamethoxazole treatment, necessitating its discontinuation. We describe the clinical features, environmental sampling, genomic epidemiologic investigation, and public health response to the outbreak. Management of this outbreak shows the potential benefits of making melioidosis a notifiable disease. The approach used could also be used as a framework for similar outbreaks in the future.

The C-terminal head domain of Burkholderia pseudomallei BpaC has a striking hydrophilic core with an extensive solvent network.

Gram-negative pathogens like Burkholderia pseudomallei use trimeric autotransporter adhesins such as BpaC as key molecules in their pathogenicity. Our 1.4 Å crystal structure of the membrane-proximal part of the BpaC head domain shows that the domain is exclusively made of left-handed parallel ß-roll repeats. This, the largest such structure solved, has two unique features. First, the core, rather than being composed of the canonical hydrophobic Ile and Val, is made up primarily of the hydrophilic Thr and Asn, with two different solvent channels. Second, comparing BpaC to all other left-handed parallel ß-roll structures showed that the position of the head domain in the protein correlates with the number and type of charged residues. In BpaC, only negatively charged residues face the solvent-in stark contrast to the primarily positive surface charge of the left-handed parallel ß-roll "type" protein, YadA. We propose extending the definitions of these head domains to include the BpaC-like head domain as a separate subtype, based on its unusual sequence, position, and charge. We speculate that the function of left-handed parallel ß-roll structures may differ depending on their position in the structure.

Role of type VI secretion system protein TssJ-3 in virulence and intracellular survival of Burkholderia pseudomallei.

TssJ-3 is an outer-membrane lipoprotein and is one of the key components of the type VI secretion system in Burkholderia pseudomallei. TssJ translocates effector proteins to target cells to induce innate immune response in the host. However, the tssJ gene has not been identified in B. pseudomallei and its function in this bacterium has not yet been characterized. tssJ-3 knockout and tssJ-3-complemented B. pseudomallei strains were constructed to determine the effects of tssJ-3 on bacterial growth, biofilm formation, flagellum synthesis, motility, host cell infection, and gene expression in B. pseudomallei. We found that the ΔtssJ-3 mutant strain of B. pseudomallei showed significantly suppressed biofilm formation, flagellum synthesis, bacterial growth, motility, and bacterial invasion into host cells (A549 cells). Furthermore, the ΔtssJ-3 mutation downregulated multiple key genes, including biofilm and flagellum-related genes in B. pseudomallei and induced interleukin-8 gene expression in host cells. These results suggest that tssJ-3, an important gene controlling TssJ-3 protein expression, has regulatory effects on biofilm formation and flagellum synthesis in B. pseudomallei. In addition, B. pseudomallei-derived tssJ-3 contributes to cell infiltration and intracellular replication. This study provides a molecular basis of tssJ-3 for developing therapeutic strategies against B. pseudomallei infections.

Prospective Analysis of Antibody Diagnostic Tests and TTS1 Real-Time PCR for Diagnosis of Melioidosis in Areas Where It Is Endemic.

Melioidosis is a tropical infectious disease caused by Burkholderia pseudomallei. Melioidosis is associated with diverse clinical manifestations and high mortality. Early diagnosis is needed for appropriate treatment, but it takes several days to obtain bacterial culture results. We previously developed a rapid immunochromatography test (ICT) based on hemolysin coregulated protein 1 (Hcp1) and two enzyme-linked immunosorbent assays (ELISAs) based on Hcp1 (Hcp1-ELISA) and O-polysaccharide (OPS-ELISA) for serodiagnosis of melioidosis. This study prospectively validated the diagnostic accuracy of the Hcp1-ICT in suspected melioidosis cases and determined its potential use for identifying occult melioidosis cases. Patients were enrolled and grouped by culture results, including 55 melioidosis cases, 49 other infection patients, and 69 patients with no pathogen detected. The results of the Hcp1-ICT were compared with culture, a real-time PCR test based on type 3 secretion system 1 genes (TTS1-PCR), and ELISAs. Patients in the no-pathogen-detected group were followed for subsequent culture results. Using bacterial culture as a gold standard, the sensitivity and specificity of Hcp1-ICT were 74.5% and 89.8%, respectively. The sensitivity and specificity of TTS1-PCR were 78.2% and 100%, respectively. The diagnostic accuracy was markedly improved if the Hcp1-ICT results were combined with TTS1-PCR results (sensitivity and specificity were 98.2% and 89.8%, respectively). Among patients with initially negative cultures, Hcp1-ICT was positive in 16/73 (21.9%). Five of the 16 patients (31.3%) were subsequently confirmed to have melioidosis by repeat culture. The combined Hcp1-ICT and TTS1-PCR test results are useful for diagnosis, and Hcp1-ICT may help identify occult cases of melioidosis.

In silico prediction of host-pathogen protein interactions in melioidosis pathogen Burkholderia pseudomallei and human reveals novel virulence factors and their targets.

The aerobic, Gram-negative motile bacillus, Burkholderia pseudomallei is a facultative intracellular bacterium causing melioidosis, a critical disease of public health importance, which is widely endemic in the tropics and subtropical regions of the world. Melioidosis is associated with high case fatality rates in animals and humans; even with treatment, its mortality is 20-50%. It also infects plants and is designated as a biothreat agent. B. pseudomallei is pathogenic due to its ability to invade, resist factors in serum and survive intracellularly. Despite its importance, to date only a few effector proteins have been functionally characterized, and there is not much information regarding the host-pathogen protein-protein interactions (PPI) of this system, which are important to studying infection mechanisms and thereby develop prevention measures. We explored two computational approaches, the homology-based interolog and the domain-based method, to predict genome-scale host-pathogen interactions (HPIs) between two different strains of B. pseudomallei (prototypical, and highly virulent) and human. In total, 76 335 common HPIs (between the two strains) were predicted involving 8264 human and 1753 B. pseudomallei proteins. Among the unique PPIs, 14 131 non-redundant HPIs were found to be unique between the prototypical strain and human, compared to 3043 non-redundant HPIs between the highly virulent strain and human. The protein hubs analysis showed that most B. pseudomallei proteins formed a hub with human dnaK complex proteins associated with tuberculosis, a disease similar in symptoms to melioidosis. In addition, drug-binding and carbohydrate-binding mechanisms were found overrepresented within the host-pathogen network, and metabolic pathways were frequently activated according to the pathway enrichment. Subcellular localization analysis showed that most of the pathogen proteins are targeting human proteins inside cytoplasm and nucleus. We also discovered the host targets of the drug-related pathogen proteins and proteins that form T3SS and T6SS in B. pseudomallei. Additionally, a comparison between the unique PPI patterns present in the prototypical and highly virulent strains was performed. The current study is the first report on developing a genome-scale host-pathogen protein interaction networks between the human and B. pseudomallei, a critical biothreat agent. We have identified novel virulence factors and their interacting partners in the human proteome. These PPIs can be further validated by high-throughput experiments and may give new insights on how B. pseudomallei interacts with its host, which will help medical researchers in developing better prevention measures.

Cutaneous melioidosis: An updated review and primer for the dermatologist.

Melioidosis is an emerging infection with increasing endemic foci and global distribution. It is underrecognized and underdiagnosed because of factors including limited awareness of the disease, nonspecific clinical presentation, lack of diagnostic facilities in some locations, misidentification in laboratories inexperienced with culture, and identification of Burkholderia pseudomallei. Cutaneous findings are reported in approximately 10% to 20% of melioidosis cases and dermatologists may play a significant role in its recognition and management. The most dynamic situation of melioidosis recognition and/or expansion currently is in the United States. Global modeling had predicted that B. pseudomallei were potentially endemic in the southern United States and endemicity with local cases of melioidosis was confirmed in 2022. With the distribution and prevalence of melioidosis increasing globally and with this recent recognition that melioidosis is now endemic in the southern United States, it is important for dermatologists to maintain high clinical suspicion in appropriate patients and be familiar with its diagnosis and treatment. Here we review the available literature on cutaneous melioidosis to evaluate its epidemiology, etiology, pathophysiology and clinical presentation and provide guidance for diagnosis and management in dermatology practice.