In Vitro Evaluation of Antibacterial and Antibiofilm Activity of Different Chlorhexidine-Containing Mouthwash Formulations against Streptococcus mutans

Abstract

Daily use of mouthwash is generally recommended to control dental plaque development and limit further adhesion of oral bacteria. To this purpose, Chlorhexidine di-gluconate (CHG)-containing products are still the most used antiseptic in oral health. The aim of this study was to compare the antimicrobial and antibiofilm activity of three different mouthwash commercial formulations containing CHG at the same concentration (0.2%) on the main etiological agent of dental plaque, Streptococcus mutans (S. mutans). Liquid Chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to confirm the CHG concentration in the commercial formulations. The Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal concentration (MBC) were measured to evaluate the antimicrobial activity of mouthwashes on planktonic cells. The biofilm prevention concentration (BPC) and CHG formulation activity towards mature biofilm were investigated. Both total cell titer and viability in S. mutans pregrown biofilms were evaluated after treatment with formulations at different time points and concentrations, using Crystal Violet (CV) and tetrazolium dye assay (MTT). The Shapiro–Wilk and Levene tests were used to evaluate the normality and overall homogeneity of the datasets, respectively. The chi-square test, Fischer’s exact test and Student’s t-test were used to evaluate the inhibitory capacity of the commercial formulations in CV and MTT assays. Results showed that even though only slight differences in MIC and MBC values were found on planktonic bacteria, the antibiofilm effect of the different formulations exhibited wide variation when mature biofilms were treated. In addition, results were discussed based on the different compositions of the three formulations, suggesting that herbal extracts, when present in mouthwash formulations, may not always have a beneficial effect, especially when mature biofilms are concerned.