<i>B</i>. <i>pseudomallei</i> is internalized into amoebae but could not resist digestion.

<p>CLSM micrographs show the internalized <i>B</i>. <i>pseudomallei</i> in <i>P</i>. <i>ustiana</i> (A-C), <i>Acanthamoeba</i> sp. (D-F) and isolate A-ST39-E1 (G-I) at 0, 3 and 6 h after kanamycin treatment. Orange fluorescence represents CellTracker^™ Orange-<i>B</i>. <i>pseudomallei</i> and green fluorescence indicates the amoebae stained with FITC-ConA for visualization.</p

Numbers of <i>Acanthamoeba</i> sp. and isolate A-ST39-E1 over time (A-B and C-D respectively) after feeding with <i>B</i>. <i>pseudomallei</i> (▲) or <i>E</i>. <i>coli</i> (positive control) (■) or deprived of bacteria as a negative control (●).

<p>Graphs and figures show no significant differences between amoebae fed on <i>B</i>. <i>pseudomallei</i> and <i>E</i>. <i>coli</i>. However, numbers of amoebae in the negative control group were significantly lower than in the pother groups (<i>p</i> ≤ 0.0001). Data are mean ± SD from duplicates of the three independent experiments.</p

Intracellular survival through time of <i>B</i>. <i>pseudomallei</i> in <i>P</i>. <i>ustiana</i> (A), <i>Acanthamoeba</i> sp. (B) and isolate A-ST39-E1 (C).

<p>Time zero represents 3 hours after <i>B</i>. <i>pseudomallei</i> feeding. Bars represent the standard errors of the means of duplicate, three times independent experiments, * <i>p</i> < 0. 0001 using ANOVA.</p

Specific primers for the 18s rRNA gene of amoebae.

<p>Specific primers for the 18s rRNA gene of amoebae.</p

<i>Burkholderia pseudomallei</i> Biofilm Promotes Adhesion, Internalization and Stimulates Proinflammatory Cytokines in Human Epithelial A549 Cells - Fig 1

<p><b>(A)</b> Fluorescence microscopic views of the 2-day biofilms of <i>B</i>. <i>pseudomallei</i> 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 <i>B</i>. <i>pseudomallei</i> 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).</p

Intracellular survival and multiplication of <i>B</i>. <i>pseudomallei</i> H777, M10 and C17 strains in human lung epithelial cells at MOI 10.

<p>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.</p

Adhesion of <i>B</i>. <i>pseudomallei</i> H777, M10 and C17 to human lung epithelial cells at MOI 10.

<p>(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 (<i>p</i> < 0.05). (B) Light microscopic images demonstrating <i>B</i>. <i>pseudomallei</i> H777, M10 and C17 adhesion to A549 cells. Bar represents 10 μm. Representative images from Giemsa stained specimens and visualized under 100× magnification.</p

Cytokine production by human lung epithelial cells (A549) in response to infection with <i>B</i>. <i>pseudomallei</i> H777, M10 and C17 strains.

<p>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 (<i>p</i> < 0.05).</p