## 4 Discussion
The values retrieved were between 2.5 and 10 mg/mL, much closer to MIC concentration but still higher by one or two dilution factors. Once again, even though MIC values were highly similar, a more pronounced difference emerged in terms of BPC, highlighting, also, in this case, the higher efficacy of formulation 1. The antibiofilm activity of the different formulations was also evaluated after 5 min of incubation in the presence of mature biofilms. This approach does not give information about the formulations' dose-dependent efficacy but allows evaluation of mouthwashes' antibiofilm effect after a short time exposure, similar to a real application. In this case, also, the effect on the overall biofilm biomass, assessed through CV assay, was negligible. On the other hand, a pronounced drop in cell viability was observed for CHG and formulation 1, in line with what was previously observed with the 24 h exposure. Results in terms of survival rates are in line with other works carried out for CHG and mouthwashes incubated on _S. mutans_ biofilm [34]. Conversely, almost no effect was found for formulations 2 and 3, further highlighting that the antibiofilm efficacy of these could be even lower than what was observed with prolonged exposure. Statistical data analysis confirmed that only formulation 1 showed significant bacterial growth inhibition for the MTT test, while no statistically significant differences were observed between the four formulations in the CV tests. Finally, we investigated the effect of mouthwash formulations on the bacterial efflux pump system through the indirect EtBr assay. Multidrug efflux pumps are key tools involved in bacterial self-defense and resistance mechanisms. The intrinsic activity of these transporters allows the rapid export of drugs and xenobiotics outside the cell and contributes to antimicrobial resistance [37]. An increase in efflux activity was observed in the presence of all mouthwashes, and a more pronounced drop in EtBr intracellular levels compared to CHG alone was obtained. This evidence was unexpected, considering that CHG always exerted a higher antimicrobial efficacy compared to mouthwashes in all testsperformed. However, there is a paucity of information in the literature about efflux pump perturbations in the presence of mouthwashes for oral bacteria. Possibly, the boost observed in EtBr efflux may be related to additional membrane-perturbing events taking place in the presence of mouthwash formulations, and a straightforward relationship between the increase in the efflux pump system and the antimicrobial efficacy of a biologically active oral formulation has not been described. To the best of our knowledge, this is the first time that a bacterial efflux pump assay was performed comparing different mouthwash formulations. More investigations are undoubtedly needed to shed light on the reasons underlying the efflux boost effect observed for the different compounds present in the pharmaceutical formulations. In conclusion, the three mouthwash formulations with the same CHG concentration showed different efficacy in terms of antimicrobial and antibiofilm effects, which became more significant when evaluated on mature biofilms. The different effect was probably due to the different formulations of the three mouthwashes and, potentially, their different penetration potential in biofilm. Noteworthy, the same herbal extracts (_Aloe barbadensis_ (Aloe vera) Leaf Extract and _Salvia officinalis_ (Sage) Leaf Extract) are present in mouthwash formulations 2 and 3 and are absent in formulation 1. Overall, these extracts do not seem to give any effective improvement in the antibiofilm activity against _S. mutans_. It is also possible that the presence of anionic agents as extract components in the formulations alters the CHX charge state and, therefore, its efficacy. Even though there is some evidence in the literature showing the antibiofilm effect of these herbal extracts, their effective concentrations always range between hundreds and thousands of micrograms per mL [38; 39].