Susceptibility of Streptococcus mutans Biofilms to Oral Antiseptics

Abstract

Dental caries is initiated by bacteria growing as biofilms on the surface of teeth. Evaluation of dental antiseptics should therefore be performed on bacterial biofilms, rather than on planktonic cultures. We report the susceptibilities of biofilms of the cariogenic organism Streptococcus mutans to in-use concentrations of two commonly used oral antiseptics, chlorhexidine gluconate and cetylpyridinium chloride. Biofilms were grown anaerobically at 37°C for 17 or 72 h on cellulose nitrate membranes placed on the surface of tryptone soya agar. They were then immersed in 0.2% chlorhexidine gluconate or 0.05% cetylpyridinium chloride solution at 37°C for up to 60 min, followed by vortexing in inactivating broth to detach the cells. The resulting bacterial suspensions were then diluted and enumerated by viable counting. Exposure of biofilms grown for 72 h to chlorhexidine gluconate or cetylpyridinium chloride for 1 or 5 min had little effect on viability. Similarly, a 1-min exposure of 17-h biofilms to either antiseptic, or a 5-min exposure to cetylpyridinium chloride, had little effect on viable count. However, chlorhexidine gluconate achieved a 2.3 log reduction in count of 17-h biofilms following 5 min exposure, and both antiseptics achieved at least a 3.7 log reduction in viable count of 17- and 72-h biofilms after 60 min. Planktonic cells of S. mutans were much more susceptible than biofilms and were killed rapidly, giving > 3.0 log reductions in viable count within 1 min of exposure to either chlorhexidine gluconate or cetylpyridinium chloride. This study demonstrates that biofilms of S. mutans are considerably less susceptible to both chlorhexidine gluconate and cetylpyridinium chloride than planktonic cells and that older biofilms are more resistant to the antiseptics than younger biofilms. Chlorhexidine gluconate (0.2%) was found to be more effective than 0.05% cetylpyridinium chloride.