Sharon J. Peacock

Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei

Burkholderia pseudomallei is a potential bioterror agent and the causative agent of melioidosis, a severe disease that is endemic in areas of Southeast Asia and Northern Australia. Infection is often associated with bacterial dissemination to distant sites, and there are many possible disease manifestations, with melioidosis septic shock being the most severe. Eradication of the organism following infection is difficult, with a slow fever-clearance time, the need for prolonged antibiotic therapy and a high rate of relapse if therapy is not completed. Mortality from melioidosis septic shock remains high despite appropriate antimicrobial therapy. Prevention of disease and a reduction in mortality and the rate of relapse are priority areas for future research efforts. Studying how the disease is acquired and the host–pathogen interactions involved will underpin these efforts; this review presents an overview of current knowledge in these areas, highlighting key topics for evaluation.

Increasing Incidence of Human Melioidosis in Northeast Thailand

Melioidosis is a serious community-acquired infectious disease caused by the Gram-negative environmental bacterium Burkholderia pseudomallei. A prospective cohort study identified 2,243 patients admitted to Sappasithiprasong Hospital in northeast Thailand with culture-confirmed melioidosis between 1997 and 2006. These data were used to calculate an average incidence rate for the province of 12.7 cases of melioidosis per 100,000 people per year. Incidence increased incrementally from 8.0 (95% confidence interval [CI] = 7.2–10.0) in 2000 to 21.3 (95% CI = 19.2–23.6) in 2006 (P < 0.001; χ2 test for trend). Male sex, age ≥ 45 years, and either known or undiagnosed diabetes were independent risk factors for melioidosis. The average mortality rate from melioidosis over the study period was 42.6%. The minimum estimated population mortality rate from melioidosis in 2006 was 8.63 per 100,000 people (95% CI = 7.33–10.11), the third most common cause of death from infectious diseases in northeast Thailand after human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and tuberculosis.

Bacterial fibronectin-binding proteins and endothelial cell surface fibronectin mediate adherence of Staphylococcus aureus to resting human endothelial cells.

Adhesion of Staphylococcus aureus to human endothelial cells is implicated in the pathogenesis of invasive staphylococcal disease. The adhesion to endothelial cells of isogenic mutants defective in defined surface structures was studied. Three strains of S. aureus defective in fibronectin-binding proteins FnBPA and FnBPB showed reduced adhesion. This was fully restored by complementation of a FnBPA- FnBPB- mutant derived from strain 8325-4 with a multicopy plasmid encoding FnBPA or FnBPB. Adhesion of mutants defective in other surface structures was unaffected. Anti-fibronectin antibodies blocked adhesion of 8325-4 to endothelial cells, while adhesion of strains 8325-4, P1 and five clinical isolates was inhibited by the recombinant form of the binding domain of FnBPB (rFNBD) from Streptococcus dysgalactiae. Adherence of bacterial aggregates resulting from the presence of purified fibrinogen was also inhibited by rFNBD protein. Three strains of S. aureus defective in FnBPA and FnBPB were not internalized by endothelial cells. S. aureus FnBPs mediate adhesion to human endothelial cells and are required for subsequent internalization, interactions of potential relevance to pathogenesis and treatment.

Risk Factors For Hematogenous Complications of Intravascular Catheter—Associated Staphylococcus aureus Bacteremia

BACKGROUNDnThe role of both host and pathogen characteristics in hematogenous seeding following Staphylococcus aureus bacteremia is incompletely understood.nnnMETHODSnConsecutive patients with intravascular catheter-associated Staphylococcus aureus bacteremia were prospectively recruited over a 91-month period. The corresponding bloodstream isolates were examined for the presence of 35 putative virulence determinants. Patient and bacterial characteristics associated with the development of hematogenous complications (HCs) (i.e., septic arthritis, vertebral osteomyelitis, or endocarditis) were defined.nnnRESULTSnHC occurred in 42 (13%) of 324 patients. Patient characteristics at diagnosis that were associated with HC included community onset (relative risk [RR], 2.25; 95% confidence interval [CI], 1.24-4.07; P=.007), increased symptom duration (odds ratio for each day, 1.14; 95% CI, 1.06-1.2; P<.001), presence of a long-term intravascular catheter or noncatheter prosthesis (RR, 4.02; 95% CI, 1.74-9.27; P<.001), hemodialysis dependence (RR, 3.84; 95% CI, 2.08-7.10; P<.001), and higher APACHE II score (P=.02). Bacterial characteristics included sea (RR, 2.03; 95% CI, 1.16-3.55; P=.011) and methicillin-resistant S. aureus (MRSA) (RR, 2.09; 95% CI, 1.19-3.67; P=.015). Subsequent failure to remove a catheter was also associated with HC (RR, 2.28; 95% CI, 1.22-4.27; P=.011). On multivariable analysis, symptom duration, hemodialysis dependence, presence of a long-term intravascular catheter or a noncatheter device, and infection with MRSA remained significantly associated with HC.nnnCONCLUSIONSnThis investigation identifies 4 host- and pathogen-related risk factors for hematogenous bacterial seeding and reaffirms the importance of prompt catheter removal.

Identification of In Vivo–Expressed Antigens of Staphylococcus aureus and Their Use in Vaccinations for Protection against Nasal Carriage

A spectrum of in vivo-expressed Staphylococcus aureus antigens was identified by probing bacteriophage expression libraries of S. aureus with serum samples from infected and uninfected individuals. Eleven recombinant antigenic proteins were produced, and specific antibody titers in a large collection of human serum samples were determined. Significantly increased concentrations of reactive immunoglobulin G (IgG) to 7 antigens were found in serum samples from ill individuals, compared with those in healthy individuals. Significantly higher concentrations of reactive IgG to 4 antigens, including iron-responsive surface determinant (Isd) A and IsdH, were found in serum samples from healthy individuals who were not nasal carriers of S. aureus, compared with those in healthy carriers. Vaccination of cotton rats with IsdA or IsdH protected against nasal carriage. Also, IsdA is involved in adherence of S. aureus to human desquamated nasal epithelial cells and is required for nasal colonization in the cotton rat model. Thus, vaccination with these antigens may prevent S. aureus carriage and reduce the prevalence of human disease.

Melioidosis: a clinical overview

INTRODUCTIONnMelioidosis, an infection caused by the environmental Gram-negative bacillus Burkholderia pseudomallei, has emerged as an important cause of morbidity and mortality in Southeast Asia and northern Australia.nnnSOURCES OF DATAna review of the literature using PubMed.nnnAREAS OF AGREEMENTnApproaches to diagnosis and antimicrobial therapy.nnnAREAS OF CONTROVERSYnWhether seroconversion signals the presence of a quiescent bacterial focus and an increase in long-term risk of melioidosis.nnnAREAS TIMELY FOR DEVELOPING RESEARCHnMelioidosis is potentially preventable, but there is a striking lack of evidence on which to base an effective prevention programme. An accurate map defining the global distribution of B. pseudomallei is needed, together with studies on the relative importance of different routes of infection. There is a marked difference in mortality from melioidosis in high-income versus lower income countries, and affordable strategies that reduce death from severe sepsis (from any cause) in resource-restricted settings are needed.

Toll-Like Receptor 2 Impairs Host Defense in Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis)

Background Toll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis. Methods and Findings Patient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2), TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO) mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS) of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury. Conclusions Patients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment strategy in melioidosis.

IFN-gamma at the site of infection determines rate of clearance of infection in cryptococcal meningitis.

In animal models, immunity to cryptococcal infection, as in many chronic fungal and bacterial infections, is associated with a granulomatous inflammatory response, intact cell-mediated immunity, and a Th1 pattern of cytokine release. To examine the correlates of human immunity to cryptococcal infection in vivo, we analyzed immune parameters at the site of infection over time and assessed the rate of clearance of infection by serial quantitative cerebrospinal fluid (CSF) fungal cultures in 62 patients in a trial of antifungal therapy for HIV-associated cryptococcal meningitis. CSF IL-6, IFN-γ, TNF-α, and IL-8 were significantly higher in survivors compared with nonsurvivors. There were negative correlations between log TNF-α, IFN-γ, and IL-6 levels and baseline cryptococcal CFU. Log IFN-γ, G-CSF, TNF-α, and IL-6 were correlated positively with the rate of fall in log CFU/ml CSF/day. In a linear regression model including antifungal treatment group, baseline CFU, and these cytokines, only treatment group and log IFN-γ remained independently associated with rate of clearance of infection. The results provide direct in vivo evidence for the importance of quantitative differences in IFN-γ secretion in human immune control of granulomatous infections, and increase the rationale for adjunctive IFN-γ in the treatment of refractory HIV-associated cryptococcosis.

Management of melioidosis

Melioidosis is a serious human infection caused by the environmental Gram-negative bacterium Burkholderia pseudomallei. Outcome following melioidosis remains poor despite 20 years of clinical research. Overall mortality is 50% in north-east Thailand (35% in children) and 19% in Australia. Relapse is common (13% over 10 years), and results from failure to eradicate the organism. Treatment is required to complete 12–20 weeks, or longer if clinically indicated. This is divided into intravenous and oral phases. Clinical trial evidence supports the use of ceftazidime or a carbapenem antibiotic for initial parenteral therapy, which should be administered for at least 10–14 days. This is followed by a prolonged course of oral antimicrobial therapy with trimethoprim–sulfamethoxazole (TMP–SMX) with or without doxycycline. Amoxicillin–clavulanate is an alternative for children, pregnant women and for patients with intolerance to first-line therapy. Resistance of B. pseudomallei to these drugs is rare, with the exception of TMP–SMX; resistance rates are approximately 2.5% in Australia and 13–16% in Thailand. There is a lack of evidence for the value of adjunctive therapies in the treatment of melioidosis. Future studies aim to address whether meropenem is superior to ceftazidime during parenteral therapy, and whether doxycycline is a necessary component of oral treatment.

Melioidosis in 6 Tsunami Survivors in Southern Thailand

BACKGROUNDnSix cases of melioidosis were identified in survivors of the 26 December 2004 tsunami who were admitted to Takuapa General Hospital in Phangnga, a region in southern Thailand where melioidosis is not endemic. All 6 cases were associated with aspiration, and 4 were also associated with laceration.nnnMETHODSnWe compared the clinical, laboratory, and radiographic findings and the outcomes for these 6 patients with those for 22 patients with aspiration-related melioidosis acquired during 1987-2003 in a melioidosis-endemic region in northeast Thailand. Results of tests for detection of Burkholderia pseudomallei in soil specimens from Phangnga and from northeast Thailand were compared.nnnRESULTSnThe 6 patients (age range, 25-65 years) presented with signs and symptoms of pneumonia 3-38 days (median duration, 6.5 days) after the tsunami. Chest radiograph findings at the onset of pneumonia were abnormal in all cases; 1 patient developed a lung abscess. B. pseudomallei was grown in blood cultures in 3 cases and in cultures of respiratory secretions in 4 cases. Two patients required ventilation and inotropes; 1 patient died. Compared with tsunami survivors, patients with aspiration-related melioidosis in northeast Thailand had a shorter interval (median duration, 1 day) between aspiration and onset of pneumonia; were more likely to exhibit shock, respiratory failure, renal failure, and/or altered consciousness (P=.03); and had a higher in-hospital mortality (64% [14 of 22 patients]; P=.07). These differences may be related to the severity of the near-drowning episode, the inhalation of sea water versus fresh water, the size of bacterial inoculum, and the possible acquisition (among tsunami survivors) of B. pseudomallei via laceration. Only 3 (0.8%) of 360 soil samples from Phangnga were positive for B. pseudomallei, compared with 26 (20%) of 133 samples from northeast Thailand (P<.0001).nnnCONCLUSIONSnTsunami survivors are at increased risk of melioidosis if they are injured in an environment containing B. pseudomallei.