An avirulent Burkholderia pseudomallei ∆purM strain with atypical type B LPS: expansion of the toolkit for biosafe studies of melioidosis.

BACKGROUND: The work was undertaken to expand the tools available for researching Burkholderia pseudomallei (Bp), the etiological agent of the tropical disease melioidosis. Melioidosis has the potential to pose a severe threat to public health and safety. In the United States, Bp is listed as a Tier-1 select agent by the Centers for Disease Control and Prevention (CDC), thus requiring high levels of regulation and biosafety level 3 (BSL3) facilities for experimental manipulation of live organisms. An avirulent ∆purM derivative of strain 1026b (Bp82) has proven to be a valuable tool for biosafe research as a select-agent excluded strain, but the high level of genetic diversity between Bp strains necessitates an expansion of the biosafe toolset. RESULTS: The ∆purM mutation was recapitulated in the Bp 576a strain, a serotype B background. An important difference between strains 1026b and 576a is the lipopolysaccharide (LPS), a major virulence factor and protective antigen. Polyclonal sera from 1026b-challenged non-human primates showed no cross reactivity with strain 576a LPS and low reactivity with whole cell lysate. Strain 576a replicates to higher levels in mouse organs and induces more TNF-α in the lungs of BALB/c mice compared to 1026b. The newly created Bp 576a ∆purM strain, designated 576mn, was auxotrophic for adenine in minimal media, capable of wild-type growth in rich media with addition of adenine, and auxotrophy was abrogated with single-copy complementation. Bp 576mn was unable to replicate in human cells and was avirulent in BALB/c mice following high-dose intranasal inoculation, similar to Bp82. Organ loads indicated a significant reduction in bacterial replication. CONCLUSIONS: In this work, the new biosafe strain 576mn with atypical type B LPS was generated. This strain should prove a valuable addition to the toolkit for biosafe studies of Bp and development of therapeutic and preventative strategies aimed at combatting melioidosis. Strain 576mn is an ideal candidate for select-agent exclusion.

Cross-reactivity of latex agglutination assay complicates the identification of Burkholderia pseudomallei from soil.

The monoclonal antibody-based latex agglutination tests targeting a high molecular weight exopolysaccharide antigen of Burkholderia pseudomallei are commercially available. The tests are primarily used in routine diagnosis of melioidosis in major hospitals in Thailand and some endemic countries. Being a rapid test, this technique was employed as a presumptive test to identify colonies of B. pseudomallei among many others grown from soil specimens collected from southern Thailand. Cross-reactivity of these tests with other soil bacteria was a concern since it complicated the identification of B. pseudomallei. Here, we describe the cross-reactivity of two commercial latex agglutination tests for melioidosis with B. territorii, B. pseudomultivorans, B. multivorans and B. cenocepacia isolates from soil.

Seroepidemiology of Burkholderia pseudomallei, Etiologic Agent of Melioidosis, in the Ouest and Sud-Est Departments of Haiti.

Burkholderia pseudomallei, the etiological agent of melioidosis, has been hypothesized to be endemic throughout the Caribbean, including the impoverished nation of Haiti. However, because of the protean clinical manifestations, presence of asymptomatic infections, and limited medical diagnostic capacity, the identification of active melioidosis cases remains challenging. A seroepidemiological study was conducted using a novel enzyme-linked immunosorbent assay (ELISA) to detect antibodies toward B. pseudomallei in the native population. The performance of an indirect ELISA with purified lipopolysaccharide (LPS) from B. pseudomallei was evaluated using serum collected from rhesus macaques exposed to aerosolized B. pseudomallei. After optimization, serum collected from asymptomatic population members (n = 756) was screened for polyvalent (immunoglobulin M [IgM]/ immunoglobulin G [IgG]/ immunoglobulin A) and monoclonal (IgG or IgM) immunoglobulins against B. pseudomallei LPS. The population seroprevalence was 11.5% (95% confidence interval [CI]: 9.2, 13.8) for polyvalent immunoglobulins, 9.8% (95% CI: 7.7, 11.9) for IgG, and 1.7% (95% CI: 0.8, 2.6%) for IgM. The seroprevalence was not significantly different by gender (P = 0.16), but increased significantly (P < 0.001) with age, yielding an estimated annual seroconversion rate of 1.05% (95% CI: 0.81, 1.3). The detection of both recent (IgM+) and previous (IgG+) exposure to B. pseudomallei provides serological evidence that melioidosis is endemic in Haiti.