The role of exopolysaccharides in dual species biofilm development

A plasmid encoding the green fluorescent protein (GFP) of Aequorea victoria was transformed into a biofilm-forming strain of Enterobacter agglomerans originally isolated from an industrial environment. The transformed strain, EntGFP, could then be identified in dual species biofilms by direct visual ization, plate counts and quantitiative fluorescence measurements. A variet y of cell constituents and products mal be involved in the adhesion and acc umulation process and exopolysaccharides (EPS) represent one of these facto rs. The involvement of EPS in the initial adhesion events and the role in d ual species biofilm development was investigated. Cells of EntGFP and Klebs iella pneumoniae G1 interact forming biofilms more successfully in a mixtur e than in isolation. The co-resistance results in enhanced biofilm formatio n and increased resistance to disinfection. Microscopic examination showed that the two species were often closely juxtaposed in microcolonies, sugges ting the interactions involve surface-associated macromolecules. Fluorescen ce was used to measure the adhesion of EntGFP cells to Kleb. pneumoniae G1 (G1) EPS. The results showed EntGFP adhered better to GI EPS that Ent EPS. Polysaccharde depolymerases isolated from a bacteriophage for Ent. agglomer ans were used to degrade Ent EPS specifically. Following polysaccharase tre atment, the adhaesion of EntGFP to G1 cells was reduced. This suggests both types of EPS mediate adhesion. The two types of EPS were dissolved in dime thylsulphoxide and when mixed, their viscosity increased, reaching a maximu m after approximate to 40 min. This may partially explain the increased pro tection of dual species biofilms from disinfectants. The depolymerases were used to treat dual species biofilms and this resulted in the effective rem oval of both species from the surface. This mar suggest Ent contributes mor e EPS to the biofilm matrix. The EPS play an important role in EntGFP and G 1 dual species biofilm formation both as adhesins and as the EPS interact, changing their physical properties.