| Summary: | (1) Background: The effectiveness of chitosan to improve the action of antimicrobial compounds against planktonic bacteria and young biofilms has been widely investigated in Dentistry, where the biofilm lifecycle is a determining factor for the success of antibacterial treatment. In the present study, mature <i>Streptococcus mutans</i> biofilms were treated with chitosan dispersion (CD) or chitosan microparticles (CM). (2) Methods: CD at 0.25% and 1% were characterized by texture analysis, while CD at 2% was spray-dried to form CM, which were characterized with respect to particle size distribution, zeta potential, and morphology. After determining the minimum inhibitory and bactericidal concentrations, <i>S. mutans</i> biofilms were grown on glass slides exposed 8×/day to 10% sucrose and 2×/day to CD or CM at 0.25% and 1%. Biofilm viability and acidogenicity were determined, using appropriate control groups for each experiment. (3) Results: CD had high viscosity and CM were spherical, with narrow size distribution and positive zeta potential. CM affected bacterial viability and acidogenicity in mature <i>S. mutans</i> biofilms more strongly than CD, especially at 1%. (4) Conclusions: Both chitosan forms exerted antimicrobial effect against mature <i>S. mutans</i> biofilms. CM at 1% can reduce bacterial viability and acidogenicity more effectively than CD at 1%, and thereby be more effective to control the growth of mature biofilms in vitro.
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