Comparing in vitro and in vivo virulence phenotypes of Burkholderia pseudomallei type G strains

Abstract

Burkholderia pseudomallei (Bpm) is a saprophytic rod-shaped gram-negative bacterium and the causative agent of melioidosis. This disease has previously been described as endemic in areas such as northern Australia and Southeast Asia, but, more recently, a better understanding of the epidemiology of melioidosis indicated that the disease is distributed worldwide, including regions of the Americas and Africa. A 16S-23S rDNA internal transcribed spacer (ITS) typing system has been developed for Bpm and has revealed that ITS types C, E, and hybrid CE are mainly associated with Australia and Southeast Asia while type G strains are more associated with cases of melioidosis in the Western Hemisphere. The purpose of the current study was to determine the in vitro and in vivo virulence profiles of the understudied Bpm type G strains Ca2009, Ca2013a, Mx2013, and 724644 and compared such phenotypes to the commonly studied Bpm type C strain K96243. We evaluated virulence by measuring invasion/uptake and survival of these Bpm strains in murine respiratory epithelial LA-4 cells and alveolar macrophage MH-S cells using different multiplicity of infections (MOIs of 1 and 10). We also calculated the lethal dose 50 values (LD50) in BALB/c mice that were inoculated intranasally with either Ca2009, Ca2013a, or Mx2013. Overall, the virulence and lethality phenotypes of Bpm type G strains were similar to the Bpm type C strain K96243. Additional comparative analyses between the Bpm ITS types may lead to a better understanding of the contribution of the ITS type to the epidemiology and ecology of Bpm strains.

Fig 1. Burkholderia pseudomallei invasion and intracellular survival in LA-4 murine lung epithelial cells.

Cells were infected with Bpm at a multiplicity of infection (MOI) of 1 (A) or 10 (B). After 1 hpi, cells were lysed and bacterial invasion was determined by dilution plating. Intracellular survival was determined using an MOI of 1 (C) or 10 (D) at 3 hpi. Intracellular survival percentages were calculated by dividing the number of bacteria recovered at 3 hpi after extracellular killing divided by the average number of cells that invaded in (A) or (B). Individual data points are shown as well as mean ± S.E.M. Levels of significance: *, p < 0.05; ***, p < 0.0005.

Fig 2. Burkholderia pseudomallei uptake and intracellular survival in MH-S murine alveolar macrophages.

Cells were infected with Bpm at an MOI of 1 (A) or 10 (B). After 1 hpi, cells were lysed and bacterial uptake was determined by dilution plating. Intracellular survival was carried out using MOIs of 1 (C) or 10 (D). Intracellular survival percentages were calculated by dividing the number of bacteria recovered 3 hpi after extracellular killing divided by the average number of cells that were taken up in (A) or (B). Individual data points are shown as well as mean ± S.E.M. Levels of significance: *, p < 0.05; ***, p < 0.0005.

Fig 3. LD₅₀ determination of B. pseudomallei CA2009 and colonization of lung, liver, and spleen in BALB/c mice.

(A) Percent survival of BALB/c mice (n = 8) infected via the intranasal route with the listed number of CFU of Bpm CA2009. LD₅₀ = 101 CFU. Colonization of the lungs (B), livers (C), and spleens (D) were determined at 48 hpi and at 23 dpi in surviving mice. Values depicted in (B-D) are the mean ± S.E.M.

Fig 4. LD₅₀ determination of B. pseudomallei CA20013a and colonization of lung, liver, and spleen in BALB/c mice.

(A) Percent survival of BALB/c mice (n = 8) infected via the intranasal route with the listed number of CFU of Bpm CA2013a. LD₅₀ = 4,260 CFU. Colonization of the lungs (B), livers (C), and spleens (D) were determined at 48 hpi and at 23 dpi in surviving mice. Values depicted in (B-D) are the mean ± S.E.M.

Fig 5. LD₅₀ determination of B. pseudomallei MX20013 and colonization of lung, liver, and spleen in BALB/c mice.

(A) Percent survival of BALB/c mice (n = 8) infected via the intranasal route with the listed number of CFU of Bpm MX2013. LD₅₀ = 1,330 CFU. Colonization of the lungs (B), livers (C), and spleens (D) were determined at 48 hpi and at 23 dpi in surviving mice. Values depicted in (B-D) are the mean ± S.E.M.