Methods: The effect of Chlorhexidine (CHX) on S. mutans viability in biofilm and biofilm formation was assessed by determining the minimal biofilm inhibition concentration (BIC). The results were analyzed using live/dead stain and Confocal Laser Scanning Microscopy (CLSM). Extracellular FTF expression was analyzed using dot blot. Quorum sensing activity was assessed using bioluminescence assay of V. harveyi.
Results: MIC of CHX on planktonic S. mutans was 10 μg/ml. CHX inhibits biofilm formation at the concentration of 10 μg/ml (BIC). In concentrations lower then BIC, CHX increased the relative amount of live bacteria in the biofilm. At the same sub- lethal concentrations, CHX increased the expression of soluble FTF from immobilized bacteria. The suppernatant fluid of S. mutans biofilm, grown in the presence of sub- lethal CHX concentration, caused the inhibition of Quorum sensing in V. harveyi model.
Conclusions: CHX biofilm inhibition activity was at the same magnitude as the MIC of the planktonic bacteria. In sub- lethal concentrations, CHX increased the ratio of live bacteria immobilized in the biofilm, probably due to its high affinity toward the negatively charged bacteria.
CHX may affect biofilm formation by increasing extracellular FTF expression, which will increase extracellular matrix. The quorum sensing inhibition by sub- lethal concentrations of CHX, results in decreased of biofilm total mass, which may compensate for its effect on extracellular FTF expression.