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.