Anti-Biofilm Effects of Alcohol-Free Mouthwash against <i>Streptococcus mutans</i>

Background: Various commercial mouthwashes are used in Korea.However, comparative data on how alcohol-containing and alcohol-free formulations differ in their ability to inhibit Streptococcus mutans biofilm formation remain limited.Few studies have assessed both planktonic antimicrobial activity and antibiofilm efficacy under conditions that simulate clinical use.This study aimed to evaluate these effects by comparing alcohol-containing and alcohol-free mouthwashes in a single species, S. mutans.Methods: Four commercially available mouthwashes were tested: two cetylpyridinium chloride-based formulations (Garglin Original , Garglin Zero ), two essential oil (EO)-based formulations, one alcohol-containing (Listerine Cool Mint ), and one alcohol-free (Listerine Cool Mint Mild [LM]).Sterile distilled water and 0.1% chlorhexidine (CHX) served as the negative and positive controls, respectively.The planktonic antimicrobial activity was evaluated after a 1-minute exposure.For the biofilm experiment, S. mutans biofilms were formed on hydroxyapatite discs for 16 hours and subsequently treated with each mouthwash three times per day for 1 minute, totaling six treatments during a 64-hour maturation period that simulated clinical rinsing.Biofilm viability (log CFU/ml), thickness, and live/dead cell ratios were assessed using culture-based methods and confocal laser scanning microscopy with COMSTAT software.Results: All commercial mouthwashes demonstrated complete bactericidal activity below the detection limit against planktonic S. mutans.In contrast, only LM and CHX showed significant antibiofilm activity.LM reduced biofilm colony-forming unit (CFU) by approximately 0.7 log and decreased biofilm thickness and live/dead ratios by 39.0% and 30.8%, respectively, compared with the control (p0.01).CHX exhibited the greatest reduction across all parameters.Conclusion: LM exhibited the strongest antibiofilm potential among all tested mouthwashes.These findings indicate that alcohol-free EO rinses may be a clinically useful alternative for patients who cannot tolerate alcohol-containing products or long-term CHX use.Incorporating such formulations into daily oral hygiene routines may help prevent dental caries.