Brenda Govan

The molecular and cellular basis of pathogenesis in melioidosis: how does Burkholderia pseudomallei cause disease?

Melioidosis, a febrile illness with disease states ranging from acute pneumonia or septicaemia to chronic abscesses, was first documented by Whitmore & Krishnaswami (1912). The causative agent, Burkholderia pseudomallei, was subsequently identified as a motile, gram-negative bacillus, which is principally an environmental saprophyte. Melioidosis has become an increasingly important disease in endemic areas such as northern Thailand and Australia (Currie et al., 2000). This health burden, plus the classification of B. pseudomallei as a category B biological agent (Rotz et al., 2002), has resulted in an escalation of research interest. This review focuses on the molecular and cellular basis of pathogenesis in melioidosis, with a comprehensive overview of the current knowledge on how B. pseudomallei can cause disease. The process of B. pseudomallei movement from the environmental reservoir to attachment and invasion of epithelial and macrophage cells and the subsequent intracellular survival and spread is outlined. Furthermore, the diverse assortment of virulence factors that allow B. pseudomallei to become an effective opportunistic pathogen, as well as to avoid or subvert the host immune response, is discussed. With the recent increase in genomic and molecular studies, the current understanding of the infection process of melioidosis has increased substantially, yet, much still remains to be elucidated.

Immunological mechanisms contributing to the double burden of diabetes and intracellular bacterial infections

Diabetes has been recognized as an important risk factor for a variety of intracellular bacterial infections, but research into the dysregulated immune mechanisms contributing to the impaired host–pathogen interactions is in its infancy. Diabetes is characterized by a chronic state of low‐grade inflammation due to activation of pro‐inflammatory mediators and increased formation of advanced glycation end products. Increased oxidative stress also exacerbates the chronic inflammatory processes observed in diabetes. The reduced phagocytic and antibacterial activity of neutrophils and macrophages provides an intracellular niche for the pathogen to replicate. Phagocytic and antibacterial dysfunction may be mediated directly through altered glucose metabolism and oxidative stress. Furthermore, impaired activation of natural killer cells contributes to decreased levels of interferon‐γ, required for promoting macrophage antibacterial mechanisms. Together with impaired dendritic cell function, this impedes timely activation of adaptive immune responses. Increased intracellular oxidation of antigen‐presenting cells in individuals with diabetes alters the cytokine profile generated and the subsequent balance of T‐cell immunity. The establishment of acute intracellular bacterial infections in the diabetic host is associated with impaired T‐cell‐mediated immune responses. Concomitant to the greater intracellular bacterial burden and potential cumulative effect of chronic inflammatory processes, late hyper‐inflammatory cytokine responses are often observed in individuals with diabetes, contributing to systemic pathology. The convergence of intracellular bacterial infections and diabetes poses new challenges for immunologists, providing the impetus for multidisciplinary research.

Altered macrophage function is associated with severe Burkholderia pseudomallei infection in a murine model of type 2 diabetes

This study used a murine model of type 2 diabetes (BKS.Cg-Dock7(m) +/+Lepr(db)/J mice) to investigate the inflammatory and cellular mechanisms predisposing to Burkholderia pseudomallei infection and co-morbid diabetes. Homozygous db/db (diabetic) mice developed extreme obesity, dyslipidaemia and glucose intolerance leading to hyperglycaemia and overt type 2 diabetes. Compared to their heterozygous db/+ (non-diabetic) littermates, diabetic mice rapidly succumbed to subcutaneous B. pseudomallei infection, paralleled by severe hypoglycaemia and increased expression of the proinflammatory cytokines, tumour necrosis factor (TNF)-α and interleukin (IL)-1β, in the spleen, despite comparable bacterial loads in the spleen of non-diabetic mice. Neutrophil oxidative burst and dendritic cell uptake and killing of B. pseudomallei were similar between diabetic and non-diabetic mice. Compared to peritoneal macrophages from non-diabetic mice, macrophages from diabetic mice were unable to contain and kill B. pseudomallei. Functional differences between macrophages of diabetic and non-diabetic mice toward B. pseudomallei may contribute to rapid dissemination and more severe disease progression in hosts with co-morbid type 2 diabetes.

B- and T-Cell Responses in Group A Streptococcus M-Protein- or Peptide-Induced Experimental Carditis

ABSTRACT The etiology of rheumatic fever and rheumatic heart disease (RF/RHD) is believed to be autoimmune, involving immune responses initiated between streptococcal and host tissue proteins through a molecular mimicry mechanism(s). We sought to investigate the humoral and cellular responses elicited in a Lewis rat model of group A streptococcus M-protein- or peptide-induced experimental valvulitis/carditis, a recently developed animal model which may, in part, represent human rheumatic carditis. Recombinant streptococcal M5 protein elicited opsonic antibodies in Lewis rats, and anti-M5 antisera recognized epitopes within the B- and C-repeat regions of M5. One peptide from the streptococcal M5 protein B-repeat region (M5-B.6, amino acids 161 to 180) induced lymphocytes that responded to both recombinant M5 and cardiac myosin. Rats immunized with streptococcal M5 protein developed valvular lesions, distinguished by infiltration of CD3+, CD4+, and CD68+ cells into valve tissue, consistent with human studies that suggest that RF/RHD are mediated by inflammatory CD4+ T cells and CD68+ macrophages. The current study provides additional information that supports the use of the rat autoimmune valvulitis model for investigating RF/RHD.

The epidemiology of melioidosis in the Balimo region of Papua New Guinea

The distribution of Burkholderia pseudomallei was determined in soil collected from a rural district in Papua New Guinea (PNG) where melioidosis had recently been described, predominately affecting children. In 274 samples, 2.6% tested culture-positive for B. pseudomallei. Pulsed-field gel electrophoresis using SpeI digests and rapid polymorphic DNA PCR with five primers demonstrated a single clone amongst clinical isolates and isolates cultured from the environment that was commonly used by children from whom the clinical isolates were derived. We concluded that individuals in this region most probably acquired the organism through close contact with the environment at these sites. Burkholderia thailandensis, a closely related Burkholderia sp. was isolated from 5.5% of samples tested, an observation similar to that of melioidosis-endemic areas in Thailand. This is the first report of an environmental reservoir for melioidosis in PNG and confirms the Balimo district in PNG as melioidosis endemic.

Serological evidence of Coxiella burnetii infection in dogs in a regional centre

OBJECTIVE Investigate the seroprevalence of the causative agent of Q fever, Coxiella burnetii in domestic dogs in the Townsville region, North Queensland, Australia. METHOD Blood samples were collected from dogs attending veterinary clinics for routine procedures. RESULTS An overall seropositivity of 21.8% (95% confidence interval (CI) 21.6-22.1%) was observed. A retrospective study of samples collected in the same region during 1984-85 was also performed, with an overall seropositivity of 16.0% (95% CI 15.9-16.2). CONCLUSION Evidence of C. burnetii infection in domestic dogs may have public health implications for dog owners, as well as veterinarians because of occupational exposure. This study is the first known investigation of C. burnetii seroprevalence in dogs in Queensland.

Detection of Coxiella burnetii DNA in Wildlife and Ticks in Northern Queensland, Australia

Wild animals and the tick species that feed on them form the natural transmission cycle and reservoir of Coxiella burnetii. The objective of this study was to determine whether C. burnetii was present in the blood of host animals and their ticks in northern Queensland, Australia. Three genomic targets were detected using real-time PCR assays-the Coxiella-specific outer membrane protein coding gene (Com1), the multicopy insertion element (IS1111), and the isocitrate dehydrogenase gene (Icd). Quantification of the single-copy targets identified a range of 1.48×10(1) to 4.10×10(3) C. burnetii genome equivalents per microliter in the ticks tested. The detection of Coxiella based on the presence of the genomic targets indicated the occurrence of C. burnetii in both the ticks and whole blood of a variety of native Australian marsupials and confirms these animals are capable of acting as reservoirs of Q fever in northern Queensland.

Impaired Early Cytokine Responses at the Site of Infection in a Murine Model of Type 2 Diabetes and Melioidosis Comorbidity

ABSTRACT Bacterial infections are a common and serious complication of type 2 diabetes (T2D). The prevalence of melioidosis, an emerging tropical infection caused by the Gram-negative bacterium Burkholderia pseudomallei, is increased in people with T2D. This is the first study to compare murine models of T2D and melioidosis. Susceptibility and disease progression following infection with B. pseudomallei were compared in our diet-induced polygenic mouse model and a leptin receptor-deficient monogenic model of T2D. The metabolic profile of mice with diet-induced diabetes, including body weight, blood glucose, cholesterol, triglycerides, insulin resistance, and baseline levels of inflammation, closely resembled that of clinical T2D. Following subcutaneous infection with B. pseudomallei, bacterial loads at 24 and 72 h postinfection in the blood, spleen, liver, lungs, and subcutaneous adipose tissue (SAT) at the site of infection were compared in parallel with the expression of inflammatory cytokines and tissue histology. As early as 24 h postinfection, the expression of inflammatory (interleukin-1β [IL-1β], tumor necrosis factor alpha [TNF-α], and IL-6) and TH1 (IL-12 and gamma interferon [IFN-γ]) cytokines was impaired in diabetic mice compared to nondiabetic littermates. Early differences in cytokine expression were associated with excessive infiltration of polymorphonuclear neutrophils (PMN) in diabetic mice compared to nondiabetic littermates. This was accompanied by bacteremia, hematogenous dissemination of bacteria to the lungs, and uncontrolled bacterial growth in the spleens of diabetic mice by 72 h postinfection. The findings from our novel model of T2D and melioidosis comorbidity support the role of impaired early immune pathways in the increased susceptibility of individuals with T2D to bacterial infections.

Impact of streptozotocin-induced diabetes on functional responses of dendritic cells and macrophages towards Burkholderia pseudomallei

Diabetes mellitus is a documented risk factor for melioidosis, a tropical infection caused by Burkholderia pseudomallei. The increased susceptibility of diabetic individuals to infections with other pathogens has been associated with immune dysregulation. However, the impact of diabetes on the functional responses of dendritic cells (DC) and macrophages during B. pseudomallei infection has not been investigated. This study compared the responses of macrophages and DC towards B. pseudomallei using bone marrow-derived DC (BMDC) and peritoneal elicited macrophages (PEM) isolated from streptozotocin-induced diabetic C57BL/6 mice exhibiting hyperglycaemia for 9 days (acute) or 70 days (chronic) and age-matched nondiabetic C57BL/6 mice. Following coincubation of BMDC and PEM with a highly virulent B. pseudomallei isolate, maturation, bacterial internalization plus intracellular survival and cytokine gene expression profiles were assessed. No significant differences in functional responses of BMDC or PEM isolated from acute diabetic and nondiabetic mice were observed. However, significant differences in BMDC and PEM function were observed when chronic diabetic and nondiabetic mice were compared. This study demonstrates that diabetic mice with extended periods of uncontrolled hyperglycaemia have impaired DC and macrophage function towards B. pseudomallei, which may contribute to the high susceptibility observed in clinical practice.

Comparison of routine bench and molecular diagnostic methods in identification of Burkholderia pseudomallei.

ABSTRACT This study compared the identification of Burkholderia pseudomallei with that of related organisms. Bench tests and latex agglutination were compared with molecular identification. Using bench tests and latex agglutination alone, 100% (30/30) of B. pseudomallei isolates were correctly identified. Amoxicillin-clavulanate susceptibility testing was also a good and simple discriminatory test.