Extracellular polysaccharide expression and the propensity to form biofillms in Burkholderia multivorans

Burkholderia multivorans is a gram-negative bacillus that causes opportunistic pulmonary infections in patients with underlying diseases. The purpose of the present study was to better understand the relationship between extracellular polysaccharide (EPS) expression and the propensity of B. multivorans to form in vitro biofilms. Cell envelope lipid composition of the disparate B. multivorans strains examined were typical of those in the phylogenetically-related bacterium Pseudomonas aeruginosa. Mucoid strain CGD2 underwent colonial dissociation concomitantly with serial subculturing, thereby yielding a butyrous, less capsulated derivative strain for comparative experimentation. The outer cell surfaces of all B. multivorans strains were relatively hydrophilic in nature, relatively impermeable to a panel of mechanistically-disparate hydrophobic antimicrobial agents, and equally inaccessible to the fluorescent hydrophobic compound N-phenyl-1-napthylamine, regardless of EPS phenotype. Supplementation of a sugar-deficient basal growth medium with different sugars revealed mannose to support the most EPS production of all sugars tested. Moreover, the mucoid phenotype was found to be as much a function of EPS secretion as it was cellular capsulation. Butyrous strain BAA-247 failed to produce the mucoid phenotype under any of the conditions tested. Microscopic observation indicated that EPS production was not necessary during the initial cellular adhesion stage of biofilm formation as seen in the lack of capsulated cells for the normally highly capsulated strain CGD2. However, the ability to produce EPS played an important role in maintaining mature biofilms as suggested by the fact that only non-EPS producing strain BAA-247 was unable to maintain its biomass as seen in both microscopic observations and in an in vitro biofilm assay. Strain BAA-247 lacked four of the five bce genes necessary for EPS precursor synthesis, secretion, and polymerization. It was therefore unable to express the EPS-producing phenotype required to form stable biofilms as seen with other strains. These data support the conclusion that while EPS expression in B. multivorans is necessary to create a denser, more stable biomass as the biofilm matures, it is not necessarily always associated with cellular capsulation or the ability of the outer cell surface to associate with nonpolar substances.