The formation of a biofilm is an essential facet of the bacterial life cycle, an adaptive response which provides them protection from various environmental stresses or from combative measures released by the other organisms with which they are cohabitating. From a clinical perspective, biofilms are a well-documented driver of the spread of antimicrobial resistance by providing a protective environment that both limits the effectiveness of antimicrobial therapies and provides a natural reservoir for regeneration of bacterial populations. Owing to their importance, therapies targeting biofilm formation or degradation of biofilm components are of growing interest for their capacity to increase the efficacy of existing treatments in combatting bacterial infections, or as novel antimicrobial therapies.
Biofilms are commonly composed of a mixture of proteins, DNA and polysaccharides. Polysaccharides form the primary macromolecular constituent by mass and consist of either polysaccharides synthesised explicitly for biofilms (e.g. alginate) or of shed components of the cell surface (i.e. O- and/or capsular antigens). There is incredible structural diversity amongst the types of polysaccharides produced, with variability even between related strains. There is a desperate need for new tools or approaches to better study these glycans. Polysaccharide zymograms are a technique that enables visualisation of targeted glycosidase activity. While they have previously been used in the characterisation of enzymes hydrolysing many industrially important polysaccharides, such as hyaluronic acid, cellulose and starch, their routine application to other polysaccharides is largely hampered by the relatively poor sensitivity and specificity of existing stains such as Alcian blue. Stains-all, a carbocyanine dye with the ability to not only broadly stain many acidic polysaccharides, but also to differentiate between them, has the potential to overcome some of the roadblocks to discovering new glycosidases that can hydrolyse polysaccharides in biofilms.
Here we sought to characterise the polysaccharide component of biofilms derived from clinical isolates of two ESKAPE pathogens, Klebsiella pneumoniae and Acinetobacter baumanii. Polysaccharide composition was determined using GC-MS following acid hydrolysis and derivatisation following a methoximation/sialylation strategy. Polysaccharide zymogens using Stains-all based detection was then applied to detect putative hydrolases sourced either endogenously or exogenously from the bacterial proteomes, which were then identified via LC-MSMS. The identification of these novel enzymes capable of degrading biofilms into their oligosaccharide structural units would enable more facile biofilm analysis and has potential use in removal of biofilms.