Enhanced Tolerance of Oral Biofilms against Chlorhexidine

Objective: Virulence of oral microorganisms, such as resistance against antibacterial drugs, seems to be enhanced when they produce biofilm structure. This study aimed to compare ratios of dead/live bacteria taken from dental plaque, with or without biofilm structure, after exposure to chlorhexidine (CHX). Methods: Dental plaque masses (natural biofilms) of more than one-week-old were taken from volunteers and transferred into an anaerobic glove box. Some bacterial masses were dispersed under anaerobic conditions and suspended in phosphate buffer. Artificial biofilms were reconstructed by culturing the dispersed bacteria on filter-membranes for 3 days, using the method we established. Some bacteria were exposed to CHX at the concentrations of 0.1 or 1.0% for 1 minute. Dead microorganisms were identified by propidium-iodide staining, and determined with a fluorospectrophotometer. For statistic analysis, paired t-test was applied. Results: It should be noted that 62% and 80% of plaque bacteria were sensitive against 0.1% (n=3, each) and 1.0% CHX, respectively, when they are dispersed and suspended. On the contrary, dental plaque bacteria of natural and artificial biofilms were significantly (p<0.05) less sensitive (Death rate=33% and 9.5%, respectively) against 0.1% (n=3, each) CHX, suggesting that biofilm structure protected bacteria from CHX. A high proportion of bacteria in dental plaque masses survived even after exposure to 1.0% CHX (death rate=35%, n=3), while bacteria in artificial biofilm were more sensitive to 1.0% CHX (death rate=71%, n=3). Conclusion: Dental plaque structure, referred as biofilm, protected plaque bacteria from CHX. (Grant: Grants in Aid for Scientific Research 17791354, Grant for promotion of Niigata University Research Projects).