Introduction
Biofilms are a self-secreted extracellular matrix produced by microbes to mediate adhesion and survival of microbial cells, especially in hostile environments. Biofilms can either be formed on an abiotic surface (surface adherent biofilm) or at the air-liquid interface (pellicle) [1]. Some functions of a biofilm include cell to cell communication, storage and recycle of nutrients, horizontal gene transfer, protection from environmental stressors and, protection of bacterial cells from phagocytic engulfment by host mammalian cells [2]. To form a biofilm, single cells adhere to a surface, aggregate into colonies, and export components of the biofilm. Single cells are dispersed from a mature biofilm to aggregate and form new biofilms [2]. Biofilms can colonize or thrive on most surfaces and is harmful to the host organism accounting for about 80% of microbial infection [3]. Biofilms are extremely difficult to eradicate due to their increased tolerance for antimicrobials and antibiotics [4, 5].
Burkholderia pseudomallei (B. pseudomallei ) is a Gram-negative bacteria known to form both surface adherent biofilms and pellicles [6-8]. It is the causative agent of melioidosis, a disease endemic throughout Southeast Asia and northern Australia with an overall death rate of 16%-18.4% [9]. B. pseudomallei is found in a wide range of ecological niches such as soil, surface water and roots of legumes and can survive under a variety of environmental conditions indicating the ability to quickly sense and respond to environmental changes such as temperature, through specific survival mechanisms. [10, 11]. Human inoculation is typically through inhalation or inoculation through skin abrasions but a handful of human-human transmission cases have been reported [12]. There is no approved melioidosis vaccine and treatments rely on high doses of antibiotics which may be ineffective [13-15]. About 10% of melioidosis patients relapse [16]. A typical relapse in bacteremia is linked to the ability of the bacteria to persist in a colonized niche like a biofilm within the host. Higher mortality rate and chronic inflammation infections in host cells are also linked to biofilm-forming strains [17, 18]. To survive under stressed environments like the human host, B. pseudomallei modifies the expression of virulence genes like the Bsa type III secretion system [19], metabolic and motility genes [20], and biofilm genes [21]. A variety of genes have been implicated in B. pseudomallei biofilm processes like adhesion [22] and eDNA secretion which facilitate biofilm formation [23, 24]. However, the complete pathways for genes involved in the formation of biofilms and/or the secretion of molecules are not clearly understood. Numerous studies have shown that eDNA is released from viable cells and required for biofilm formation and structure inBurkholderia . [23, 25, 26]. Burkholderia species also produce and export numerous outer membrane polysaccharides and proteins [27-29]. Recent studies have suggested that biofilms can be dispersed by enzymes that target biofilm constituents [30]. Biofilm dispersal using enzymes is also suggested as the mechanism by which planktonic cells are liberated from a mature biofilm to colonize a new site/surface [31]. Supernatants of non-dispersed biofilms lack degradative enzymes and endonucleases were expressed in dispersedPseudomonas aeruginosa cells [31, 32]. These enzymes target polysaccharides, proteins, lipids, and eDNA, the core architectural composition of biofilms [2, 30]. Thus, degradative enzymes have been employed to degrade biofilm components in bacteria to initiate dispersal and liberate planktonic cells which are susceptible antibiotics. Dispersin B, an enzyme that hydrolyzes poly-N -acetyl glucosamine (PNAG) polysaccharide, inhibited Acinetobacter baumannii pellicle formation, suggesting that polysaccharides contribute to pellicle formation [33]. DNase and proteinase K are known to decrease the amount of biofilms formed by B. pseudomallei and P. aeruginosa, respectively [25, 34].
In this study, we investigated the biofilms produced by virulent and avirulent Burkholderia strains, including B. thailandensisE264 [35], B. pseudomallei 1026b [36] and four mutants derived from 1026b (1026b ∆asd [37], Bp82 [38], DD503 [39] and JW270 [40]). B. thailandensis E264 is a nonpathogenic soil saprophyte that can be grown and manipulated at biosafety level 2 (BSL-2), but B. pseudomallei 1026b is a virulent human isolate that must be handled in a high containment BSL-3 laboratory. B. pseudomallei 1026b ∆asd harbors a deletion mutation in the gene encoding aspartate-semi aldehyde dehydrogenase and is unable to grow on media without diaminopimelate (DAP) supplementation [37]. B. pseudomallei Bp82 is auxotrophic for adenine and thiamine due to a deletion mutation in the purM gene [38]. 1026b ∆asd and Bp82 are both avirulent in mice and have been removed from the CDC select agent list. DD503 is a 1026b derivative that harbors a deletion of the genes encoding the AmrAB-oprAantibiotic efflux pump and is virulent in animal models of infection and must be worked with at BSL-3 [39]. B. pseudomallei JW270, a DD503 derivative, contains a deletion of the wcb gene cluster encoding capsular polysaccharide CPS I, a homopolymeric polysaccharide required for virulence [40].
We employed glucosidase, proteinase K and DNase to disperse/eradicate the biofilms produced by these Burkholderia strains and examined the molecular composition of both surface-adherent and pellicle biofilms. While we found no difference in biofilm formation betweenB. pseudomallei strains and B. thailandensis , there were striking differences in biofilm polysaccharide, protein and eDNA content. We propose that the different biofilm composition observed between the B. pseudomallei derivatives and B. thailandensis may allow a better understanding of Burkholderiabiofilm biogenesis and regulation. We propose that the genetic manipulations between the 1026b derivatives is responsible for the difference in biofilm composition.