The Arabinose 5-Phosphate Isomerase KdsD Is Required for Virulence in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, which is endemic primarily in Southeast Asia and northern Australia but is increasingly being seen in other tropical and subtropical regions of the world. Melioidosis is associated with high morbidity and mortality rates, which is mediated by the wide range of virulence factors encoded by B. pseudomallei. These virulence determinants include surface polysaccharides such as lipopolysaccharide (LPS) and capsular polysaccharides (CPS). Here, we investigated a predicted arabinose-5-phosphate isomerase (API) similar to KdsD in B. pseudomallei strain K96243. KdsD is required for the production of the highly conserved 3-deoxy-d-manno-octulosonic acid (Kdo), a key sugar in the core region of LPS. Recombinant KdsD was expressed and purified, and API activity was determined. Although a putative API paralogue (KpsF) is also predicted to be encoded, the deletion of kdsD resulted in growth defects, loss of motility, reduced survival in RAW 264.7 murine macrophages, and attenuation in a BALB/c mouse model of melioidosis. Suppressor mutations were observed during a phenotypic screen for motility, revealing single nucleotide polymorphisms or indels located in the poorly understood CPS type IV cluster. Crucially, suppressor mutations did not result in reversion of attenuation in vivo. This study demonstrates the importance of KdsD for B. pseudomallei virulence and highlights further the complex nature of the polysaccharides it produces. IMPORTANCE The intrinsic resistance of B. pseudomallei to many antibiotics complicates treatment. This opportunistic pathogen possesses a wide range of virulence factors, resulting in severe and potentially fatal disease. Virulence factors as targets for drug development offer an alternative approach to combat pathogenic bacteria. Prior to initiating early drug discovery approaches, it is important to demonstrate that disruption of the target gene will prevent the development of disease. This study highlights the fact that KdsD is crucial for virulence of B. pseudomallei in an animal model of infection and provides supportive phenotypic characterization that builds a foundation for future therapeutic development.

Case Report: Vagal Nerve Neuritis Associated with Pulmonary Melioidosis Provides Potential Insights into the Pathophysiology of Neuromelioidosis.

Encephalomyelitis is the most frequent manifestation of neuromelioidosis in Australia. It is hypothesized that Burkholderia pseudomallei causes encephalomyelitis after entering the brain directly, if complicating a scalp infection, or after traveling to the brain within peripheral or cranial nerves. A 76-year-old man presented with fever, dysphonia, and hiccups. Chest imaging demonstrated extensive bilateral pneumonia with mediastinal lymphadenopathy, blood cultures isolated B. pseudomallei, and nasendoscopy confirmed a left vocal cord palsy. Magnetic resonance imaging identified no intracranial abnormality but demonstrated an enlarged, enhancing left vagus nerve, consistent with neuritis. We hypothesize that B. pseudomallei invaded the vagus nerve in the thorax, was traveling proximally-involving the left recurrent laryngeal nerve and causing the left vocal cord palsy, but had not yet reached the brainstem. Given the frequency of pneumonia in cases of melioidosis, the vagus nerve may represent an alternative, and indeed common, route for B. pseudomallei to enter the brainstem in cases of melioidosis-related encephalomyelitis.

Corticospinal Tract Involvement in MRI of Neuromelioidosis: Report of Three Cases with a Review of Clinicoradiological Features.

Melioidosis is gram-negative bacterial infection endemic in parts of Australia and Asia with significant morbidity and mortality. It is acquired in wet rainy seasons through occupational and recreational activities. Although central nervous system (CNS) involvement is seen in less than 10%, it can have severe sequelae. MRI (magnetic resonance imaging) findings in reported cases have shown predominant brain stem and frontoparietal involvement. We present three pediatric cases of neuromelioidosis in which corticospinal tract involvement was a characteristic finding.

Skin models for cutaneous melioidosis reveal Burkholderia infection dynamics at wound's edge with inflammasome activation, keratinocyte extrusion and epidermal detachment.

ABSTRACTMelioidosis is a serious infectious disease endemic in Southeast Asia, Northern Australia and has been increasingly reported in other tropical and subtropical regions in the world. Percutaneous inoculation through cuts and wounds on the skin is one of the major modes of natural transmission. Despite cuts in skin being a major route of entry, very little is known about how the causative bacterium Burkholderia pseudomallei initiates an infection at the skin and the disease manifestation at the skin known as cutaneous melioidosis. One key issue is the lack of suitable and relevant infection models. Employing an in vitro 2D keratinocyte cell culture, a 3D skin equivalent fibroblast-keratinocyte co-culture and ex vivo organ culture from human skin, we developed infection models utilizing surrogate model organism Burkholderia thailandensis to investigate Burkholderia-skin interactions. Collectively, these models show that the bacterial infection was largely limited at the wound's edge. Infection impedes wound closure, triggers inflammasome activation and cellular extrusion in the keratinocytes as a potential way to control bacterial infectious load at the skin. However, extensive infection over time could result in the epidermal layer being sloughed off, potentially contributing to formation of skin lesions.

Melioidosis: Laboratory Investigations and Association with Patient Outcomes.

Melioidosis is an infection caused by the bacterium Burkholderia pseudomallei. The most common presentation is bacteremia occurring in 38-73% of all patients, and the mortality rate ranges from 9% to 42%. Although there is abundant data representing risk factors for infection and patient outcomes, there is limited information regarding laboratory investigations associated with bacteremia and mortality. We assessed a range of baseline and diagnostic investigations and their association with patient outcomes in a retrospective cohort study in Townsville, Australia. 124 patients' medical and laboratory records were reviewed between January 1, 1997 and December 31, 2020. Twenty-seven patients died and 87 patients were bacteremic. The presence of lymphopenia (< 1.5 × 109 cells/L) was the highest risk for bacteremia (relative risk [RR] 2.2; 95% CI: 1.3-3.7, P < 0.001). Factors associated with mortality included lymphopenia, (RR: 1.4; 95% CI: 1.2-1.6, P = 0.004); uremia (RR: 1.7; 95% CI: 1.1-2.5, P = 0.03); and an elevated international normalized ratio (RR: 1.5; 95% CI: 1.2-2.0, P = 0.006). Median incubation to positive blood culture result was 28 hours with 15/82 (18%) positive in ≤ 24 hours. For serological testing during admission only 53/121 (44%) were indirect hemagglutination assay positive, 67/120 (56%) enzyme immunoassay IgG positive, and 23/89 (26%) IgM positive. Simple baseline investigations at time of presentation may be used to stratify patients at high risk for both bacteremia and mortality. This information can be used as a decision aid for early intensive management.

Risk Factors for Mortality in Melioidosis: A Single-Centre, 10-Year Retrospective Cohort Study.

Melioidosis is a tropical infectious disease with diverse clinical presentations. We aimed to investigate the characteristics and mortality risk factors of patients diagnosed with melioidosis in the past 10 years. This was a retrospective cohort study conducted at a quaternary care centre in South India. Clinical, demographic, and biochemical data in patients diagnosed with melioidosis with cultures were collected between January 2011 and December 2020 from medical records. Logistic regression analysis was performed to screen mortality risk factors of melioidosis in addition to descriptive statistics and chi-square analysis. Seventy-three melioidosis patients' records were analysed, and the most common comorbidity was type 2 diabetes mellitus (n = 53, 72.6%). The patients showed diverse presentations: pulmonary involvement, 30 (41.1%); splenomegaly, 29 (39.7%); abscesses and cutaneous involvement, 18 (24.7%); lymph node, 10 (13.7%); arthritis and osteomyelitis, 9 (12.3%); and genitourinary infection, 4 (5.4%). The mortality was noted to be 15 (20.5%). Logistic regression analysis indicated that chronic kidney disease (OR = 14.0), CRP >100 IU/L (OR = 6.964), and S. albumin <3 gm/dl (OR = 8.0) were risk factors associated with mortality and can guide in risk stratification. Hypoalbuminemia is a novel mortality risk factor, detected in this study, and requires further investigation to validate its utility as a prognostic marker and reveal possible therapeutic benefits in clinical correction.

Description of two fatal cases of melioidosis in Mexican children with acute pneumonia: case report.

BACKGROUND: Melioidosis is an infectious disease caused by Burkholderia pseudomallei. In Mexico, the disease is rarely diagnosed in humans and there is no evidence of simultaneous environmental isolation of the pathogen. Here, we describe clinical profiles of fatal cases of melioidosis in two children, in a region without history of that disease. CASE PRESENTATION: About 48 h before onset of symptoms, patients swam in a natural body of water, and thereafter they rapidly developed fatal septicemic illness. Upon necropsy, samples from liver, spleen, lung, cerebrospinal fluid, and bronchial aspirate tissues contained Burkholderia pseudomallei. Environmental samples collected from the locations where the children swam also contained B. pseudomallei. All the clinical and environmental strains showed the same BOX-PCR pattern, suggesting that infection originated from the area where the patients were swimming. CONCLUSIONS: The identification of B. pseudomallei confirmed that melioidosis disease exists in Sonora, Mexico. The presence of B. pseudomallei in the environment may suggest endemicity of the pathogen in the region. This study highlights the importance of strengthening laboratory capacity to prevent and control future melioidosis cases.

The lymphatic system as a potential mechanism of spread of melioidosis following ingestion of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, which is a Gram negative, facultative intracellular bacterium. Disease is prevalent in SE Asia and in northern Australia, as well as in other tropical and subtropical regions. Recently, there is an increasing awareness of the importance of bacterial ingestion as a potential route of infection, particularly in cases of unexplained origin of the disease. The marmoset is a New World Monkey (NWM) species that is being developed as an alternative NHP model to complement the more traditionally used Old World Monkeys (OWM). Models have been developed for the traditional routes of disease acquisition, subcutaneous and inhalational. This manuscript details the development and characterisation of an ingestion model of melioidosis. Dose-ranging study assessed the lethality of B. pseudomallei and disease progression was assessed by euthanizing animals at predetermined time points, 12, 36, 48 and 54 hours post-challenge. Challenge doses of greater than 6.2 x 106 cfu resulted in an acute, lethal, febrile disease. Following challenge the lung was the first organ, outside of the gastrointestinal tract, to become colonised. Enteritis (duodenitis, ileitis and/or jejunitis) was observed in sections of the small intestine from animals that succumbed to disease. However, the most severe pathological features were observed in the mesenteric lymph nodes from these animals. These findings are consistent with lymphatic draining as route of dissemination.

Comparative virulence of three different strains of Burkholderia pseudomallei in an aerosol non-human primate model.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a major cause of sepsis and mortality in endemic regions of Southeast Asia and Northern Australia. B. pseudomallei is a potential bioterrorism agent due to its high infectivity, especially via inhalation, and its inherent resistance to antimicrobials. There is currently no vaccine for melioidosis and antibiotic treatment can fail due to innate drug resistance, delayed diagnosis and treatment, or insufficient duration of treatment. A well-characterized animal model that mimics human melioidosis is needed for the development of new medical countermeasures. This study first characterized the disease progression of melioidosis in the African green monkey (AGM) and rhesus macaque (RM) for non-human primate model down-selection. All AGMs developed acute lethal disease similar to that described in human acute infection following exposure to aerosolized B. pseudomallei strain HBPUB10134a. Only 20% of RMs succumbed to acute disease. Disease progression, immune response and pathology of two other strains of B. pseudomallei, K96243 and MSHR5855, were also compared using AGMs. These three B. pseudomallei strains represent a highly virulent strain from Thailand (HBPUB101034a), a highly virulent strains from Australia (MSHR5855), and a commonly used laboratory strains originating from Thailand (K96243). Animals were observed for clinical signs of infection and blood samples were analyzed for cytokine responses, blood chemistry and leukocyte changes in order to characterize bacterial infection. AGMs experienced fever after exposure to aerosolized B. pseudomallei at the onset of acute disease. Inflammation, abscesses and/or pyogranulomas were observed in lung with all three strains of B. pseudomallei. Inflammation, abscesses and/or pyogranulomas were observed in lymph nodes, spleen, liver and/or kidney with B. pseudomallei, HBPUB10134a and K96243. Additionally, the Australian strain MSHR5855 induced brain lesions in one AGM similar to clinical cases of melioidosis seen in Australia. Elevated serum levels of IL-1ß, IL-1 receptor antagonist, IL-6, MCP-1, G-CSF, HGF, IFNγ, MIG, I-TAC, and MIP-1ß at terminal end points can be significantly correlated with non-survivors with B. pseudomallei infection in AGM. The AGM model represents an acute model of B. pseudomallei infection for all three strains from two geographical locations and will be useful for efficacy testing of vaccines and therapeutics against melioidosis. In summary, a dysregulated immune response leading to excessive persistent inflammation and inflammatory cell death is the key driver of acute melioidosis. Early intervention in these pathways will be necessary to counter B. pseudomallei and mitigate the pathological consequences of melioidosis.

Burkholderia pseudomallei triggers canonical inflammasome activation in a human primary macrophage-based infection model.

Most of the current knowledge on Burkholderia pseudomallei-induced inflammasome activation and cell death in macrophages is derived from murine systems. Little is known about the involved bacterial structures and mechanisms in primary human macrophages. This is of particular relevance since murine and human macrophages as well as primary cells and cell lines differ in many aspects of inflammasome activation, including the proteins involved in the recognition of bacterial patterns. In this study, we therefore aimed (i) to establish an in vitro B. pseudomallei infection model with human monocyte-derived primary macrophages from single donors as these cells more closely resemble macrophages in the human host and (ii) to analyze B. pseudomallei-triggered cell death and bacterial elimination in those cells. Our results show that B. pseudomallei-infected primary human macrophages not only release the inflammasome-independent pro-inflammatory cytokines IL-8 and TNF-α, but are also engaged in canonical inflammasome activation as evidenced by caspase-1 and gasdermin D processing. Absence of the B. pseudomallei T3SS-3 needle protein BsaL, a potent activator of the canonical inflammasome, abolished lytic cell death, reduced IL-1ß release, and caspase-1 and gasdermin D processing. IFN-γ, known to promote non-canonical inflammasome activation, did not influence pyroptosis induction or IL-1ß release from infected primary human macrophages. Nevertheless, it reduced intracellular B. pseudomallei loads, an effect which was partially antagonist by the inhibition of NADPH oxidase. Overall, our data implicate T3SS-3 dependent inflammasome activation and IFN-γ induced immune mechanisms as critical defense mechanisms of human macrophages against B. pseudomallei. In addition, our infection model provides a versatile tool to study human host-pathogen interactions and has the potential to elucidate the role of human individual genetic variations in B. pseudomallei infections.

Genomic loss in environmental and isogenic morphotype isolates of Burkholderia pseudomallei is associated with intracellular survival and plaque-forming efficiency.

BACKGROUND: Burkholderia pseudomallei is an environmental bacterium that causes melioidosis. A facultative intracellular pathogen, B. pseudomallei can induce multinucleated giant cells (MNGCs) leading to plaque formation in vitro. B. pseudomallei can switch colony morphotypes under stress conditions. In addition, different isolates have been reported to have varying virulence in vivo, but genomic evolution and the relationship with plaque formation is poorly understood. METHODOLOGY/PRINCIPLE FINDINGS: To gain insights into genetic underpinnings of virulence of B. pseudomallei, we screened plaque formation of 52 clinical isolates and 11 environmental isolates as well as 4 isogenic morphotype isolates of B. pseudomallei strains K96243 (types II and III) and 153 (types II and III) from Thailand in A549 and HeLa cells. All isolates except one environmental strain (A4) and K96243 morphotype II were able to induce plaque formation in both cell lines. Intracellular growth assay and confocal microscopy analyses demonstrated that the two plaque-forming-defective isolates were also impaired in intracellular replication, actin polymerization and MNGC formation in infected cells. Whole genome sequencing analysis and PCR revealed that both isolates had a large genomic loss on the same region in chromosome 2, which included Bim cluster, T3SS-3 and T6SS-5 genes. CONCLUSIONS/SIGNIFICANCE: Our plaque screening and genomic studies revealed evidence of impairment in plaque formation in environmental isolates of B. pseudomallei that is associated with large genomic loss of genes important for intracellular multiplication and MNGC formation. These findings suggest that the genomic and phenotypic differences of environmental isolates may be associated with clinical infection.

Utility of endobronchial ultrasound guided trans-bronchial needle aspiration in diagnosis of melioidosis - case series and review of literature.

Endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA) is routinely performed for diagnostic evaluation of mediastinal lymphadenopathy due to various etiologies with excellent sensitivity and specificity. Melioidosis can have atypical features like isolated mediastinal lymphadenopathy mimicking as tuberculosis or lymphoma. Differentiation of such atypical melioidosis presentation become difficult due to similar clinical, radiological and even similar EBUS lymph node characteristics. Role of EBUS TBNA in diagnosing melioidosis is under investigated and sparsely reported. We describe two cases of melioidosis diagnosed by point of care rapid lateral flow immunoassay antigen testing and culture of EBUS-TBNA samples from necrotic mediastinal lymph nodes.

Melioidosis in Gujarat: a neglected tropical disease.

We report what we believe is the first case in Gujarat of melioidosis in a 67-year-old man. Awareness of this disease is limited, especially in areas supposed not to be endemic.

Burkholderia pseudomallei invades the olfactory nerve and bulb after epithelial injury in mice and causes the formation of multinucleated giant glial cells in vitro.

The infectious disease melioidosis is caused by the bacterium Burkholderia pseudomallei. Melioidosis is characterised by high mortality and morbidity and can involve the central nervous system (CNS). We have previously discovered that B. pseudomallei can infect the CNS via the olfactory and trigeminal nerves in mice. We have shown that the nerve path is dependent on mouse strain, with outbred mice showing resistance to olfactory nerve infection. Damage to the nasal epithelium by environmental factors is common, and we hypothesised that injury to the olfactory epithelium may increase the vulnerability of the olfactory nerve to microbial insult. We therefore investigated this, using outbred mice that were intranasally inoculated with B. pseudomallei, with or without methimazole-induced injury to the olfactory neuroepithelium. Methimazole-mediated injury resulted in increased B. pseudomallei invasion of the olfactory epithelium, and only in pre-injured animals were bacteria found in the olfactory nerve and bulb. In vitro assays demonstrated that B. pseudomallei readily infected glial cells isolated from the olfactory and trigeminal nerves (olfactory ensheathing cells and trigeminal Schwann cells, respectively). Bacteria were degraded by some cells but persisted in other cells, which led to the formation of multinucleated giant cells (MNGCs), with olfactory ensheathing cells less likely to form MNGCs than Schwann cells. Double Cap mutant bacteria, lacking the protein BimA, did not form MNGCs. These data suggest that injuries to the olfactory epithelium expose the primary olfactory nervous system to bacterial invasion, which can then result in CNS infection with potential pathogenic consequences for the glial cells.

Cranial Melioidosis Presenting as Osteomyelitis and/or Extra-Axial Abscess: Literature Review.

BACKGROUND: Central nervous system (CNS) melioidosis is rare. Clinical presentations depend on the region of endemicity. Despite treatment, neurologic disease has relatively high mortality rates. Less than 80 cases of CNS involvement have been reported. METHODS: A literature review was performed by searching online databases for melioidosis presenting as osteomyelitis or scalp/extra-axial abscess (OSEAA). In addition, 3 similar cases managed at my institute have been presented. RESULTS: Including this report of 3 cases, 20 additional cases have been reported. Of these, 12 cases (60%) were from India. The mean age of patients was 45.5 years (range, 29-74 years), and none were in the pediatric age group. Patients in the fifth to sixth decades were most frequently affected. The male to female ratio was 5.3:1. Eleven patients had predisposing factors. Fever, headache, and scalp swelling were the most common features. Five cases had history of previous melioid infection. Seven cases had systemic disease. Debridement was performed in 11 cases. The average intensive phase treatment duration was 4.6 weeks (range, 2-8 weeks) and 5.5 months (range, 3-12 months) for the maintenance phase. Mean follow-up duration was 13.5 months (range, 2 weeks-40 months). Two deaths (10%) were reported, and 1 case of residual frontal abscess had relapse. CONCLUSIONS: Cranial melioidosis presenting as OSEAA is associated with good outcome, in contrast with other neurologic presentations. Intensive phase for at least 2-3 weeks followed by maintenance phase for 3-6 months is the standard treatment, similar to other melioid presentations. A high degree of suspicion and accurate identification of the organism is crucial. Patients need to be monitored for recurrences, both clinically and radiologically.

Melioidosis fatalities in captive slender-tailed meerkats (Suricata suricatta): combining epidemiology, pathology and whole-genome sequencing supports variable mechanisms of transmission with one health implications.

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

Distinct Pathogenic Patterns of Burkholderia pseudomallei Isolates Selected from Caenorhabditis elegans and Dictyostelium discoideum Models.

Burkholderia pseudomallei is a selective agent that causes septic melioidosis and exhibits a broad range of lethal doses in animals. Host cellular virulence and phagocytic resistance are pathologic keys of B. pseudomallei. We first proposed Caenorhabditis elegans as the host cellular virulence model to mimic bacterial virulence against mammals and second established the resistance of B. pseudomallei to predation by Dictyostelium discoideum as the phagocytosis model. The saprophytic sepsis-causing Burkholderia sp. (B. pseudomallei, Burkholderia thailandensis, Burkholderia cenocepacia, and Burkholderia multivorans) exhibited different virulence patterns in both simple models, but B. pseudomallei was the most toxic. Using both models, attenuated isolates of B. pseudomallei were selected from a transposon-mutant library and a panel of environmental isolates and reconfirmed by in vitro mouse peritoneal exudate cell association and invasion assays. The distinct pathological patterns of melioidosis were inducted by different selected B. pseudomallei isolates. Fatal melioidosis was induced by the isolates with high virulence in both simple models within 4-5 day, whereas the low-virulence isolates resulted in prolonged survival greater than 30 day. Infection with the isolates having high resistance to D. discoideum predation but a low C. elegans killing effect led to 83% of mice with neurologic melioidosis. By contrast, infection with the isolates having low resistance to D. discoideum predation but high C. elegans killing effect led to 20% cases with inflammation in the salivary glands. Our results indicated that individual B. pseudomallei isolates selected from simple biological models contribute differently to disease progression and/or tissue tropism.