## 4 Discussion  
The three mouthwashes analyzed in this study were characterized by three different formulations that shared the same CHG concentration (0.2%--2 mg/mL). The aim of this work was to investigate how the chlorhexidine antimicrobial and antibiofilm effect on _S. mutans_ was affected by the three different formulations. The data reported here showed only minor differences between the tested formulations in terms of the MIC and MBC values for planktonic cells. Values retrieved were highly similar to others reported for CHG in the literature for the same ATCC strain [32; 33; 34]. However, the reference compound (CHG) and formulation 1 were the more efficient ones, showing lower MIC and MBC compared to the other tested solutions. Other studies described different MIC and MBC values found for _S. mutans_ in different CHG-based mouthwashes. In some cases, differences in MIC higher than 10-fold in concentration were found [35; 36]. Noteworthy, in these cases, was that even though the same mouthwashes were tested in different studies, discordant  
Figure 5: Efflux pump ethidium bromide assay. (**A**) Ethidium bromide fold increase in fluorescence intensity was measured over 30 min. Mouthwash formulations were incubated at half MIC values, and values of four independent experiments were averaged. (**B**) Linear regression equations of EtBr accumulation curves. The ratio of the slope values over control are also reported. results were found in defining the best formulation, possibly depending on the different testing procedures used. Therefore, great variability of the results is observed for these assays, a procedure harmonization is far from being met, and a results comparison is even more difficult when different formulations are analyzed. In this work, more evident differences were obtained in the antibiofilm activity when treatments were performed against the same bacteria in mature biofilms. The latter are complex structures produced by some bacteria in order to provide protection from hostile agents and create an environment suitable for their proliferation. Indeed, concentrations of antibacterial agents more than 200 times higher than the MIC may be required to achieve complete eradication of viable cells in mature biofilms [32]. Therefore, in the case of oral strains, it is of particular importance to test mouthwash efficacy against bacteria embedded in biofilm. In this work, CV and MTT assays were used to assess total biofilm biomass and cell viability in _S. mutans_ mature biofilms. Both these assays are currently used to assess biofilm growth; however, they give slightly different information, thus complementing each other's data. CV provides information concerning all cells present in a biofilm, thus giving insights into biofilm integrity, while MTT allows identification of the rate of living cells present in the matrix [29; 30]. The two assays highlighted a significant difference in the antibiofilm effect of the different CHG-based formulations: reference compound (CHG) and formulation 1 showed comparable and higher antibiofilm efficacy, considering both cell viability (MTT assay) and biofilm matrix integrity (CV assay). Formulations 2 and 3 needed around four times higher concentrations to reach the same decrease in terms of viable cells and did not have any significant effect on the mature biofilm matrix. Even though no CHG concentration used was ever sufficient to completely eradicate the biofilm matrix, the MTT assay allowed evaluation of the concentration of each formulation at which the number of living cells was negligible. This evidence was not surprising and was in line with what was already known about mouthwash activity. In fact, mouthwash is typically used to prevent biofilm formation and does not allow the removal of the dental plaque already established. For this reason, BPC, representing the minimal concentration that prevents biofilm formation, was also evaluated in this work.