Characterization of Burkholderia pseudomallei Strains Using a Murine Intraperitoneal Infection Model and In Vitro Macrophage Assays

Abstract

Burkholderia pseudomallei, the etiologic agent of melioidosis, is a gram-negative facultative intracellular bacterium. This bacterium is endemic in Southeast Asia and Northern Australia and can infect humans and animals by several routes. It has also been estimated to present a considerable risk as a potential biothreat agent. There are currently no effective vaccines for B. pseudomallei, and antibiotic treatment can be hampered by nonspecific symptomology, the high incidence of naturally occurring antibiotic resistant strains, and disease chronicity. Accordingly, there is a concerted effort to better characterize B. pseudomallei and its associated disease. Before novel vaccines and therapeutics can be tested in vivo, a well characterized animal model is essential. Previous work has indicated that mice may be a useful animal model. In order to develop standardized animal models of melioidosis, different strains of bacteria must be isolated, propagated, and characterized. Using a murine intraperitoneal (IP) infection model, we tested the virulence of 11 B. pseudomallei strains. The IP route offers a reproducible way to rank virulence that can be readily reproduced by other laboratories. This infection route is also useful in distinguishing significant differences in strain virulence that may be masked by the exquisite susceptibility associated with other routes of infection (e.g., inhalational). Additionally, there were several pathologic lesions observed in mice following IP infection. These included varisized abscesses in the spleen, liver, and haired skin. This model indicated that commonly used laboratory strains of B. pseudomallei (i.e., K96243 and 1026b) were significantly less virulent as compared to more recently acquired clinical isolates. Additionally, we characterized in vitro strain-associated differences in virulence for macrophages and described a potential inverse relationship between virulence in the IP mouse model of some strains and in the macrophage phagocytosis assay. Strains which were more virulent for mice (e.g., HBPU10304a) were often less virulent in the macrophage assays, as determined by several parameters such as intracellular bacterial replication and host cell cytotoxicity.

Comparison of the early host immune response to two widely diverse virulent strains of Burkholderia pseudomallei that cause acute or chronic infections in BALB/c mice

“…In the initial survival study, there were 10 mice per group with 5 groups challenged IP with different amounts of each strain that were quantified via OD 620 estimations. The IP 21 day LD 50 for Bp 1106a and Bp HBPUB10134a were approximately 42,000 colony forming units (CFU) and 10 CFU, respectively, and had been reported previously [22]. In the early host innate response study approximately 6 LD 50 equivalents of each strain was delivered IP to each mouse in 200 ml of GTB.…”

“…It is unclear if this was a true relapse or possible reinfection [24,25]. Bp HBPUB10134a was isolated from a tracheal suction specimen in 2010 [22]; April Shea, USAMRIID, personnel communication. In addition to the significantly different clinical origins of these two strains, Bp HBPUB10134a and Bp 1106a exhibited differences in their virulence/pathogenicity in BALB/c mice, as shown in Fig.…”

“…Because of the significant differences in the LD 50 values between the more virulent Bp HBPUB10134a strain (1LD 50 is~10 CFU) and less virulent Bp 1106a strain (1LD 50 is~42,000 CFU) as determined by the IP route (22), we wanted to examine early cell-mediated immune events in BALB/c mice to see if we could detect other differences between the two Bp strains. Two groups of twenty-five mice were each infected with approximately 6 LD 50 equivalents of either Bp HBPUB10134a (57 CFU) or Bp 1106a (2.7 Â 10 5 CFU) via IP injection.…”

“…It is for these reasons that effective therapeutics and vaccines are needed. We have previously reported LD 50 values for 11 strains of B. pseudomallei which comprised a strain panel agreed to by the Biomedical Advanced Research and Development Authority (BARDA) and the Defense Threat Reduction Agency (DTRA) [21,22]. In this current study, we further explored the dissemination kinetics and resulting cellular immune response to two diverse strains in BALB/c mice.…”

Burkholderia pseudomallei: Challenges for the Clinical Microbiology Laboratory

“…Clinical manifestations of the disease vary from acute septicemia to chronic infection (88% and 12% of cases in Australia, respectively) (24). Intracellular survival and cell-to-cell spread may have contributed to the organism's ability to evade the immune response, causing a persistent infection (26). The Darwin prospective melioidosis study examined 540 cases of culture-confirmed melioidosis from the Top End of Australia between 1989 and 2009 and identified pneumonia as the most common clinical manifestation (51%, 278 cases), followed by genitourinary infection (14%), skin infection (13%), bacteremia without evident focus (11%), septic arthritis or osteomyelitis (4%), and neurological melioidosis (meningoencephalitis, myelitis, and cerebral abscesses, 3%) (27).…”