Abstract
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.
Highlights
Biofilm provides shelter for various pathogens and its formation is clearly essential for microbial survival in diverse environments, potentially leading to increased virulence [1, 2]
The static 2-day biofilm and Extracellular DNA (eDNA) associated with that biofilm of 10 different B. pseudomallei isolates (Table 1), stained with crystal violet and QuantiFluor dsDNA reagent, demonstrated the variation of biofilm formation (Fig 1A) and eDNA quantity (Fig 1B)
confocal laser scanning microscopy (CLSM) images clearly demonstrated the localization of eDNA within the biofilm matrix of B. pseudomallei
Summary
Biofilm provides shelter for various pathogens and its formation is clearly essential for microbial survival in diverse environments, potentially leading to increased virulence [1, 2]. Microorganisms in biofilms are encased in a hydrated extracellular matrix of biopolymers, mainly polysaccharides, proteins, lipids and extracellular DNA (eDNA) [3] These various components provide biofilm stabilization, ability to adhere to surfaces, and resistance to harmful effects of antimicrobial agents and host immune responses [3, 4]. In the case of a food-borne pathogen, Listeria monocytogenes, eDNA is a key component of the biofilm matrix during both initial attachment and early biofilm formation. The aim of our study was to demonstrate the role of eDNA during three stages of B. pseudomallei biofilm development: initial attachment of cells to the surface, early development of biofilm architecture and maturation of biofilm architecture [30] using DNase I treatment. Biofilm dispersal using DNase enzymes may be appropriate for B. pseudomallei biofilm control
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