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.

Fig 1. B. pseudomallei LPS profiles.

A. Purified LPS samples from 11 B. pseudomallei strains were separated by SDS-PAGE. The gel was electroblotted, and the western blot was developed using a monoclonal antibody directed against B. pseudomallei LPS (11G3-1). B. Results were confirmed by silver staining. B. pseudomallei 576 displaying the LPS type B banding pattern is shown for comparison. M = molecular weight marker.

Fig 2. Statistical analyses of the LD₅₀ determinations.

Kernel density plot of the posterior distributions of the LD₅₀ values of each B. pseudomallei isolate in BALB/c mice through A. 21 day survival challenge and B. 60 day survival challenge.

Fig 3. Representative histological lesions.

A. Multifocal random areas of necrosis in the liver admixed with pyogranulomatous inflammation (BALB/c mouse challenged with 1.9 x 10³ CFU K96243, H&E 200X). The * identifies a necrotic area. B. Pyogranulomas and abscesses closely associated with lymphatic vessels within the haired skin which were markedly distended with and occasionally obliterated by large numbers of neutrophils (BALB/c mouse challenged with 1.9 x 10³ CFU K96243, H&E 40X). The arrow identifies the lumen of the lymphatic vessel containing inflammation. C. Pyogranulomatous inflammation (which originated from the haired skin and nasal sinuses) extending through the skull (S) into the meninges and cerebrum (C) (BALB/c mouse challenged with 1.4 x 10⁴ CFU K96243, H&E 40X). The * identifies an abscess. D. Significant area of the lung parenchyma obliterated by pyogranulomatous inflammation (BALB/c mouse challenged with 1.1 x 10² CFU HBPUB10303a, H&E 20X). The * identifies an abscess.

Fig 4. Several noteworthy clinical/gross observations and their histopathological correlates.

A. Swelling of or around the eye (white arrow). Histologic evaluation revealed abscesses and pyogranulomatous inflammation filling the retro-orbital space causing the globe of the eye (E) to protrude from the socket (BALB/c mouse challenged with 4.2 x 10⁴ CFU 1106a, H&E 2X). The nasal cavity (N) is also marked for orientation. B. Large swellings on the tail (often seen in conjunction with hind limb swelling and/or paralysis). Histologic evaluation revealed pyogranulomatous inflammation from the haired skin of the tail extending into adjacent bone, skeletal muscle, and adipose tissue (BALB/c mouse challenged with 1.4 x 10⁴ CFU K96243, H&E 20X). The tail vertebra (V) is denoted for orientation. C. Firm white nodules in the spleen. Histologic evaluation revealed marked pyogranulomatous inflammation and necrosis (identified with an *) in the spleen which effaces normal white and red pulp (BALB/c mouse challenged with 9.0 x 10² MSHR668, H&E 20X).

Fig 5. Comparisons of viable counts recovered from J774.A1 macrophages infected with different strains of B. pseudomallei.

A. The MOIs for strains 1106a and HBPUB10134a were 17.2 and 13.7, respectively. Strain 1106a was phagocytosed to a greater extent than HBPUB10134a, as shown by the 1 h and 3 h viable counts and multiplied to a greater extent during the 5 h period between phagocytosis and the 8 h collections. The viable counts recovered from the macrophages of 1106a for all three time points were greater than those of HBPUB10134a: P = 0.0214 (1 h), P < 0.0001 (3 h), and P = 0.0018 (8 h). B. The MOIs for strains K96243 and HBPUB10134a were 27.5 and 26.5, respectively. The viable counts recovered from the macrophages of K96243 for all three time points were greater than those of HBPUB10134a: P < 0.0001 (1 h), 0.0006 (3 h), and 0.0052 (8 h). C. The MOIs for strains MSHR668 and 1106a were 8.9 and 5.8, respectively. The viable counts recovered from the macrophages of 1106a for all three time points were nearly or significantly greater than those of MSHR668: P < 0.0001 (1 h), P = 0.070 (3 h), and P = 0.0003 (8 h). D. The MOIs for strains 1106a and MSHR5855 were 12.4 and 7.7, respectively. The viable counts recovered from the macrophages of 1106a for all three time points were greater than those of MSHR5855: P = 0.0002 (1 h), 0.0019 (3 h), and < 0.0001 (8 h) E. The MOIs for strains 1026b and MSHR305 were 16.2 and 16.9, respectively. The viable counts recovered from the macrophages of 1026b were greater than those of MSHR5855 at the 3 h (P = 0.0015) and 8 h time points (P = 0.0097); the 1 h viable counts were not significantly different (P = 0.077). F. The MOI for both the 1026b and 1106a strains was 16.2. The viable counts recovered from the macrophages of 1106a for all three time points were greater than those of 1026b: P = 0.0001 for 1 h and 3 h time points, and P = 0.0029 for the 8 h time point.

Fig 6. Comparative phenotypes of macrophages infected with strain 1106a or HBPUB10134a.

Morphological changes in cells and in the extent of cell death/detachment as observed in Diff-Quik stains of cultures at the 8 h time point. The typical normal appearance of uninfected semi-confluent J774.A1 cells at low power, 100x (A) and high power, 600x (D). B, E: Cells infected with 1106a, showing loss of monolayer and presence of numerous MNGCs and clusters of fused necrotic cells at 100x (B) and 600x (E). C, F: Cells infected with HBPUB10134a and viewed at 100x (C) and 600x (F) demonstrated cell loss to a lesser extent than those with 1106a and promoted MNGC formation, albeit fewer in number and smaller in size. The arrows indicate examples of MNGCs.