Decreasing of Multispecies Biofilm Mass by Highly Active Lactams

Objectives: The objective was to evaluate the effects of 3 different lactams on multispecies biofilm formation.
Methods: Lactams 12e, 13e and 13f (γ-alkylidene-γ-lactams) [175µg/mL solubilized in 3.5% dimethyl sulfoxide (DMSO)] were tested. Firstly, a killing assay was performed on planktonic E. faecalis, S. mutans and C. glabrata cells to ensure that the lactams or 3.5% DMSO did not exert any bactericidal activity. Cytotoxicity of the lactams and 3.5% DMSO was tested on fibroblasts from human periodontal ligament (PDL). Multispecies biofilms were produced in an active-attachment biofilm model. Glass coverslips were conditioned with the lactams or 3.5% DMSO (control) for 1h, inoculated with microbial cultures, and incubated for 48h. The effects of conditioning on biofilm characteristics were evaluated by different tests. The overall effect of lactams on biofilm mass was assessed by a crystal violet staining assay. Then, additional experiments were undertaken to evaluate specific biofilm characteristics in detail. An attachment assay was performed to determine the degree of attached viable cells per strain. The amount of protein (biomass) in the biofilm was quantified according to the Bradford protocol. Finally, the amount of extracellular polymeric substance (EPS) was analyzed. Experiments were performed 3 times, mostly in triplicate. All data were analyzed by One-way ANOVA and Tukey tests (P≤0.05).
Results: The lactams and 3.5% DMSO did not exert bactericidal effect on planktonic cells (P>0.05) and were moderately cytotoxic to PDL fibroblasts (P<0.01). The lactams reduced the amount of biofilm mass compared to control (P<0.01), but only lactam 13f reduced the number of attached E. faecalis (P<0.05). The amounts of protein and EPS decreased after conditioning the coverslips with lactams (P≤0.05).
Conclusions: The effects of lactams 12e, 13e and 13f on multispecies biofilm formation were evident by reducing the amount of biomass and EPS, in general without affecting the number of viable microorganisms.