Fig 1.
(A) Fluorescence microscopic views of the 2-day biofilms of B. pseudomallei H777, M10 and C17 grown statically on glass slides in LB broth at 37°C. The biofilms were stained with FITC-ConA and monitored under a Nikon Eclipse Ni-U fluorescence microscope (20× magnification). Strains H777 and C17 showed aggregation of surface-adherent bacteria whereas the biofilm mutant, M10, was rarely attached on the glass slide. (B) Confocal laser scanning micrographs of the 2-day biofilms of B. pseudomallei H777, M10 and C17 grown statically on glass slides in LB broth at 37°C. The biofilms were stained with FITC-ConA. The crossing lines in each images (x and y axes) indicate the correspondent vertical CLSM section (Z), indicating the thickness of the biofilm. The bars indicate 5 μm. The images were taken using a Zeiss 500 and a Zeiss 800 CLSM microscope (100× magnification).
Fig 2.
Adhesion of B. pseudomallei H777, M10 and C17 to human lung epithelial cells at MOI 10.
(A) Percentages of bacterial adhesion were determined by comparing the number of adherent bacteria to the inoculum. The numbers of CFU of cell-associated bacteria were counted after 1 h p.i. using the drop plate technique. Data represent the mean ± standard deviation of triplicates from at least three independent experiments. Asterisks denote statistical significance relative to H777 (p < 0.05). (B) Light microscopic images demonstrating B. pseudomallei H777, M10 and C17 adhesion to A549 cells. Bar represents 10 μm. Representative images from Giemsa stained specimens and visualized under 100× magnification.
Fig 3.
Internalization of B. pseudomallei H777, M10 and C17 strains into human lung epithelial cells at MOI 10.
The number of bacteria internalized were enumerated at 4 h p.i. using the drop plate technique. Percentages of bacteria that successfully internalized were calculated from the number of intracellular bacteria compared to the inoculum. Data are represented as the means ± standard deviation from at least three independent experiments, each in triplicate wells. Asterisks denote statistical significance relative to H777 (p < 0.05).
Fig 4.
Intracellular survival and multiplication of B. pseudomallei H777, M10 and C17 strains in human lung epithelial cells at MOI 10.
The number of bacteria present were enumerated at 4, 8 and 12 h p.i. using the drop plate technique. Data are represented as means ± standard deviation from at least three independent experiments in triplicate wells.
Fig 5.
Induction of death among infected human lung epithelial cells with B. pseudomallei H777, M10 and C17 at MOI 10.
At 12 h p.i. the apoptotic and necrotic cells were determined by flow cytometry. (A) Illustrates the percentage ± SD of dead A549 cells infected with B. pseudomallei H777, M10 and C17 strains. (B) Graphical representations of annexin-V/PI Plots. The numbers of viable (AV-/PI-), early apoptotic (AV+/PI-), late apoptotic (AV+/PI+) and necrotic (AV-/PI+) cells are shown. Asterisks denote statistical significance relative to H777 (p < 0.05).
Fig 6.
Cytokine production by human lung epithelial cells (A549) in response to infection with B. pseudomallei H777, M10 and C17 strains.
A549 cells were infected at MOI 10 and MOI 100. The culture supernatants were harvested at 8 h p.i. to measure cytokine levels. Data represent the means ± standard errors for triplicate wells of single representative experiments. Each experiment was performed at least three times. Asterisks denote statistical significance (p < 0.05).