Abstract
Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis. Inhalational exposure leading to pulmonary melioidosis is the most common clinical manifestation with significant mortality. However, the role of B. pseudomallei biofilm phenotype during bacterial-host interaction remains unclear. We hypothesize that biofilm phenotype may play a role in such interactions. In this study, B. pseudomallei H777 (biofilm wild type), B. pseudomallei M10 (biofilm mutant) and B. pseudomallei C17 (biofilm-complemented) strains were used to assess the contribution of biofilm to adhesion to human lung epithelial cells (A549), intracellular interactions, apoptosis/necrosis and impact on proinflammatory responses. Confocal laser scanning microscopy demonstrated that B. pseudomallei H777 and C17 produced biofilm, whereas M10 did not. To determine the role of biofilm in host interaction, we assessed the ability of each of the three strains to interact with the A549 cells at MOI 10. Strain H777 exhibited higher levels of attachment and invasion compared to strain M10 (p < 0.05). In addition, the biofilm-complemented strain, C17 exhibited restored bacterial invasion ability. Flow cytometry combined with a double-staining assay using annexin V and propidium iodide revealed significantly higher numbers of early apoptotic and late apoptotic A549 cells when these were infected with strain H777 (1.52%) and C17 (1.43%) compared to strain M10 (0.85%) (p < 0.05). Strains H777 and C17 were able to stimulate significant secretion of IL-6 and IL-8 compared with the biofilm mutant (p < 0.05). Together, these findings demonstrated the role of biofilm-associated phenotypes of B. pseudomallei in cellular pathogenesis of human lung epithelial cells with respect to initial attachment and invasion, apoptosis and proinflammatory responses.
Highlights
Melioidosis, caused by the bacterium Burkholderia pseudomallei, predominantly affects people in regular contact with contaminated dust/soil or stagnant water in endemic areas e.g. northern Australia and north-east Thailand [1, 2]
Attachment and invasion into the epithelial lining is a prerequisite of bacterial pathogenesis
We demonstrated the biofilm phenotypes of B. pseudomallei strains H777 and C17 grown in LB at 37°C and that the biofilm mutant, strain M10, was deficient in this regard
Summary
Melioidosis, caused by the bacterium Burkholderia pseudomallei, predominantly affects people in regular contact with contaminated dust/soil or stagnant water in endemic areas e.g. northern Australia and north-east Thailand [1, 2]. During heavy monsoonal rain and winds, appears to be the predominant form of transmission of B. pseudomallei and most cases have pulmonary involvement, leading to fulminant pneumonia and septic shock [1, 4, 7,8,9]. Biofilm formation by Burkholderia species, induced by lung environmental conditions, correlated with bacterial penetration and intracellular survival in host cells. This can lead to severe infections and frequent failures of antibiotic treatment in cystic fibrosis patients [15]
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