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

<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

Specific primers for the 18s rRNA gene of amoebae.

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

Dead and live cells in 2-day old biofilm of <i>B</i>. <i>pseudomallei</i> K96243 and <i>B</i>. <i>thailandensis</i> E264 after treated with 16×MIC of each antibiotic in MVBM and 0.1×MVBM for 16 h.

<p>(A) Live/Dead ratios of <i>B</i>. <i>pseudomallei</i> K96243 and <i>B</i>. <i>thailandensis</i> E264. Data are the mean value of live/dead ratios from 6 random areas. *<i>p</i> < 0.01 compared to control in the same medium, ^#<i>p</i> < 0.01 compared to the same antibiotic in MVBM. The 3D reconstruction of <i>B</i>. <i>pseudomallei</i> K96243 (B) and <i>B</i>. <i>thailandensis</i> E264 (C) biofilm stained with LIVE/DEAD BacLight Bacterial Viability kit; SYTO 9 showing live cells in green and propidium iodide showing dead cells in red (10× objective).</p

Susceptibility of <i>B</i>. <i>pseudomallei</i> K96243 and <i>B</i>. <i>thailandensis</i> E264 in planktonic and biofilm forms to the tested antibiotics determined by Calgary biofilm device.

<p>Susceptibility of <i>B</i>. <i>pseudomallei</i> K96243 and <i>B</i>. <i>thailandensis</i> E264 in planktonic and biofilm forms to the tested antibiotics determined by Calgary biofilm device.</p

Time-lapse of biofilms under flow conditions.

<p><i>B</i>. <i>thailandensis</i> E264 biofilm after treated with 16×MIC of CAZ in MVBM and 0.1×MVBM under flow condition using Bioflux microfluidics platform with flow rate 0.5 dyn/cm² at 37 °C for 16 h. Black arrows indicated the starting time when changing of bacterial morphology was observed after treated with CAZ. The scale bar indicates 50 μm. Experiments were performed three times and representative examples are shown. The movies were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194946#pone.0194946.s001" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194946#pone.0194946.s004" target="_blank">S4</a> Figs.</p

Biofilm-forming capacity of <i>B</i>. <i>pseudomallei</i> K96243 and <i>B</i>. <i>thailandensis</i> E264 after exposed to 16×MIC of each antibiotic in MVBM and 0.1×MVBM for 16 h.

<p>OD₆₂₀ of the biofilm after staining with crystal violet. Data are the mean value of two independent experiments carried out in quadruplicate. *<i>p</i> < 0.01 compared to control in the same medium. ^#<i>p</i> < 0.01 compared to the same antibiotic in MVBM.</p

Planktonic bacterial growth curve.

<p>Growth curve of <i>B</i>. <i>pseudomallei</i> K96243 (A) and <i>B</i>. <i>thailandensis</i> E264 (B) cultured in MVBM, 0.1×MVBM and PBS at 37°C for 72 h. Data are the mean value of two independent experiments carried out in sextuplicate.</p

Impact of nutritional stress on drug susceptibility and biofilm structures of <i>Burkholderia pseudomallei</i> and <i>Burkholderia thailandensis</i> grown in static and microfluidic systems

<div><p><i>Burkholderia pseudomallei</i> is the causative agent of melioidosis and regarded as a bioterrorism threat. It can adapt to the nutrient-limited environment as the bacteria can survive in triple distilled water for 16 years. Moreover, <i>B</i>. <i>pseudomallei</i> exhibits intrinsic resistance to diverse groups of antibiotics in particular while growing in biofilms. Recently, nutrient-limited condition influenced both biofilm formation and ceftazidime (CAZ) tolerance of <i>B</i>. <i>pseudomallei</i> were found. However, there is no information about how nutrient-limitation together with antibiotics used in melioidosis treatment affects the structure of the biofilm produced by <i>B</i>. <i>pseudomallei</i>. Moreover, no comparative study to investigate the biofilm architectures of <i>B</i>. <i>pseudomallei</i> and the related <i>B</i>. <i>thailandensis</i> under different nutrient concentrations has been reported. Therefore, this study aims to provide new information on the effects of four antibiotics used in melioidosis treatment, <i>viz</i>. ceftazidime (CAZ), imipenem (IMI), meropenem (MEM) and doxycycline (DOX) on biofilm architecture of <i>B</i>. <i>pseudomallei</i> and <i>B</i>. <i>thailandensis</i> with different nutrient concentrations under static and flow conditions using confocal laser scanning microscopy. Impact of nutritional stress on drug susceptibility of <i>B</i>. <i>pseudomallei</i> and <i>B</i>. <i>thailandensis</i> grown planktonically or as biofilm was also evaluated. The findings of this study indicate that nutrient-limited environment enhanced survival of <i>B</i>. <i>pseudomallei</i> in biofilm after exposure to the tested antibiotics. The shedding planktonic <i>B</i>. <i>pseudomallei</i> and <i>B</i>. <i>thailandensis</i> were also found to have increased CAZ tolerance in nutrient-limited environment. However, killing activities of MEM and IMI were stronger than CAZ and DOX on <i>B</i>. <i>pseudomallei</i> and <i>B</i>. <i>thailandensis</i> both in planktonic cells and in 2-day old biofilm. In addition, MEM and IMI were able to inhibit <i>B</i>. <i>pseudomallei</i> and <i>B</i>. <i>thailandensis</i> biofilm formation to a larger extend compared to CAZ and DOX. Differences in biofilm architecture were observed for biofilms grown under static and flow conditions. Under static conditions, biofilms grown in full strength modified Vogel and Bonner’s medium (MVBM) showed honeycomb-like architecture while a knitted-like structure was observed under limited nutrient condition (0.1×MVBM). Under flow conditions, biofilms grown in MVBM showed a multilayer structure while merely dispersed bacteria were found when grown in 0.1×MVBM. Altogether, this study provides more insight on the effect of four antibiotics against <i>B</i>. <i>pseudomallei</i> and <i>B</i>. <i>thailandensis</i> in biofilm under different nutrient and flow conditions. Since biofilm formation is believed to be involved in disease relapse, MEM and IMI may be better therapeutic options than CAZ for melioidosis treatment.</p></div