Ability of Ozone to Inactivate Streptococcus mutans in Artificial Biofilms

Objectives: To evaluate the potential of ozone on inactivation of Streptococcus mutans (S. muatns) in biofilms in vitro. Methods: The antibacterial efficacy of ozone was tested against S. muatns (MT8148) biofilms grown on bovine enamel slab surfaces using 1% sucrose. Artificial biofilms were grown inside an artificial mouth culture system on equal-sized and polished bovine enamel slabs using freshly cultured S. muatns suspended in phosphate buffered saline (PBS) at an OD₅₀₀=2 (approximately 2x10⁷CFU/ml), heart infusion (HI) and PBS for 20 hrs at 37°C. Gaseous ozone (HealOzone, Kavo., Germany) was applied for 30, 60, 90, 120 and 240 s, to the S. muatns biofilms on enamel slabs separately. The biofilms were detached and homogenized in PBS and plated by a spiral machine (EDDY JET, IUL, Spain) on Mitis salivarius agar medium (Difco, BD, USA). After anaerobic culture at 37°C for 48 hrs, the number of CFU/ml on individual slabs was counted. In a separate test, biofilms with 0.25% sucrose were also exposed to gaseous ozone. All experiments were repeated three times (n=4 for each group). Results: Application of HealOzone dose dependently reduced the number of CFU/ml from 60 s exposure time both in 1% and in 0.25% sucrose concentrations. There were no numerical difference between "Control (no ozone)" and 30 s exposure. However, there were significant (p < 0.05) reductions of bacteria in the artificial biofilms (3 log₁₀ reductions from Control) following 120 s applications. Conclusion: Ozone had an antibacterial effect on S. muatns cells embedded in water-soluble glucans of the artificial biofilms and these results indicate that HealOzone may effectively inactivate S. muatns present in early plaque on the tooth surfaces or in the shallow caries if applied clinically as a prophylactic treatment. This study was supported by COE program for FRMDRTB at TMDU and Japan Monbukagaku Research Grant (16390544).