Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation.

The biofilm-forming ability of Burkholderia pseudomallei is crucial for its survival in unsuitable environments and is correlated with antibiotic resistance and relapsing cases of melioidosis. Extracellular DNA (eDNA) is an essential component for biofilm development and maturation in many bacteria. The aim of this study was to investigate the eDNA released by B. pseudomallei during biofilm formation using DNase treatment. The extent of biofilm formation and quantity of eDNA were assessed by crystal-violet staining and fluorescent dye-based quantification, respectively, and visualized by confocal laser scanning microscopy (CLSM). Variation in B. pseudomallei biofilm formation and eDNA quantity was demonstrated among isolates. CLSM images of biofilms stained with FITC-ConA (biofilm) and TOTO-3 (eDNA) revealed the localization of eDNA in the biofilm matrix. A positive correlation of biofilm biomass with quantity of eDNA during the 2-day biofilm-formation observation period was found. The increasing eDNA quantity over time, despite constant living/dead ratios of bacterial cells during the experiment suggests that eDNA is delivered from living bacterial cells. CLSM images demonstrated that depletion of eDNA by DNase I significantly lessened bacterial attachment (if DNase added at 0 h) and biofilm developing stages (if added at 24 h) but had no effect on mature biofilm (if added at 45 h). Collectively, our results reveal that eDNA is released from living B. pseudomallei and is correlated with biofilm formation. It was also apparent that eDNA is essential during bacterial cell attachment and biofilm-forming steps. The depletion of eDNA by DNase may provide an option for the prevention or dispersal of B. pseudomallei biofilm

D-LL-31 enhances biofilm-eradicating effect of currently used antibiotics for chronic rhinosinusitis and its immunomodulatory activity on human lung epithelial cells.

Chronic rhinosinusitis (CRS) is a chronic disease that involves long-term inflammation of the nasal cavity and paranasal sinuses. Bacterial biofilms present on the sinus mucosa of certain patients reportedly exhibit resistance against traditional antibiotics, as evidenced by relapse, resulting in severe disease. The aim of this study was to determine the killing activity of human cathelicidin antimicrobial peptides (LL-37, LL-31) and their D-enantiomers (D-LL-37, D-LL-31), alone and in combination with conventional antibiotics (amoxicillin; AMX and tobramycin; TOB), against bacteria grown as biofilm, and to investigate the biological activities of the peptides on human lung epithelial cells. D-LL-31 was the most effective peptide against bacteria under biofilm-stimulating conditions based on IC50 values. The synergistic effect of D-LL-31 with AMX and TOB decreased the IC50 values of antibiotics by 16-fold and could eliminate the biofilm matrix in all tested bacterial strains. D-LL-31 did not cause cytotoxic effects in A549 cells at 25 μM after 24 h of incubation. Moreover, a cytokine array indicated that there was no significant induction of the cytokines involving in immunopathogenesis of CRS in the presence of D-LL-31. However, a tissue-remodeling-associated protein was observed that may prevent the progression of nasal polyposis in CRS patients. Therefore, a combination of D-LL-31 with AMX or TOB may improve the efficacy of currently used antibiotics to kill biofilm-embedded bacteria and eliminate the biofilm matrix. This combination might be clinically applicable for treatment of patients with biofilm-associated CRS

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

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

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

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

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

Ultrastructural effects and antibiofilm activity of LFchimera against Burkholderia pseudomallei

Lactoferrin chimera (LFchimera), a hybrid peptide containing the two antimicrobial stretches of the innate immunity factor bovine lactoferrin, viz. LFampin265-284 and LFcin17-30, has strikingly high antimicrobial activity against the category B pathogen Burkholderia pseudomallei. The action mechanisms of LFchimera against B. pseudomallei is not fully understood. The aim of this study was to further investigate the effect of treated B. pseudomallei with LFchimera using (immune) electron microscopy. The effects of LFchimera on biofilm formation and against preformed biofilm of B. pseudomallei were also determined. After exposure to LFchimera, transmission electron microscopy revealed swelling of the periplasmic space of B. pseudomallei and a highly inhomogeneous electron density in the intracellular DNA region. Localization of LFchimera in B. pseudomallei using immunoelectron microscopy showed gold particles in intracellular structures without accumulation on the membranes. LFchimera also possessed stronger bactericidal activity than ceftazidime against B. pseudomallei grown in biofilm. Moreover, limited exposure of B. pseudomallei to LFchimera at subcidal concentration could reduce biofilm formation. Altogether, the results indicate that LFchimera possesses antibacterial and antibiofilm activities and can modulate B. pseudomallei colonization. Therefore, the efficacy of LFchimera merits further development of this agent for the therapy of melioidosi

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the tested antibiotics against planktonic <i>B</i>. <i>pseudomallei</i> K96243 and <i>B</i>. <i>thailandensis</i> E264.

<p>Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the tested antibiotics against planktonic <i>B</i>. <i>pseudomallei</i> K96243 and <i>B</i>. <i>thailandensis</i> E264.</p