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.

1. Introduction

Dental plaque is a complex multispecies biofilm growing on the tooth surface, in which bacteria are embedded and proliferate, protected from environmental stresses and host defense [1,2]. Even though several microorganisms contribute to dental plaque development and establishment, Streptococcus mutans (S. mutans) is considered to play a pivotal role in creating an optimal microenvironment in terms of pH and extracellular polysaccharidic matrix, thus promoting the growth of other cariogenic bacteria. S. mutans is able to use a large variety of carbohydrates as an energy source, and glycolysis is its exclusive route for energy production [1]. As a facultative anaerobe, S. mutans is specialized in converting carbohydrates into organic acids through anaerobic fermentation and glycolysis. This feature, defined as acidogenicity, leads to a pH reduction below 4.0 in the microenvironment, ultimately causing tooth demineralization [1]. S. mutans is able to use aerobic respiration and bears detoxification mechanisms of reactive oxygen species generated during oxygen exposure [3]. Genome sequencing and annotation of different S. mutans strains highlighted the presence of two main ABC-type transporters for the uptake of multiple sugars and around 280 genes associated with sugar phosphotransferase systems [4,5]. Sucrose (β-1,2-linked disaccharide of glucose and fructose) is one of the main sugars used as an energy source in human nutrition, and it has been recognized that several S. mutans extracellular glycosyl transferases use this molecule to synthesize extracellular polymers rich in dextran, α-1,3 and α-1,6 glucans [6]. These polysaccharides share a central role in mediating the tooth adhesion and virulence of the bacterium, which renders S. mutans one of the principal actors in virulent plaque formation and one of the model organisms to investigate for the study of dental plaque development.

Daily use of mouthwash helps control dental plaque, mainly by limiting further adhesion of oral bacteria and plaque’s uncontrolled development [7]. Chlorhexidine (CHG), a bis-biguanide compound including two chloroguanide chains carrying positive charges at physiological pH, is currently the most used antiseptic agent in mouthwashes [8,9]. Its mechanism of action relies on its charged moieties, which interact with negatively charged membrane phospholipids in bacteria, ultimately leading to membrane damage and cytoplasmic leakage [10]. Generally, Gram-positive bacteria such as S. mutans show high sensitivity to CHG, while in Gram-negative bacteria, the outer membrane layer acts as a protection, binding chlorhexidine functional groups and shielding the plasma inner membrane from chlorhexidine action. A Chlorhexidine digluconate concentration of around 0.2% is currently used in mouthwashes. This concentration was proven to exert an effective bactericidal action against most microorganisms, and CHG has been considered for a long time by dental clinicians as the gold-standard antiseptic for dental plaque and gingivitis control [11]. However, little is still known about the effect of CHG at sub-inhibitory concentrations against complex oral biofilms. In this case, in fact, a plethora of events may occur, including, among others, osmoregulation impairment and alteration in transport and respiratory activity [12]. All these aspects are worth further consideration since during treatments in the oral cavity with CHG-containing products, drug concentration may easily fall in subinhibitory concentration, thus resulting in the onset and dissemination of resistant strains [12].

Acquired resistance mechanisms toward CHG often involve an increase in multidrug efflux pump activity and cell membrane changes [13]. Inside oral biofilms, the situation is even more favorable for the emergence of resistant strains since lowered CHG concentration in deeper biofilm layers creates subinhibitory microenvironments with a strong selective pressure favoring CHG-resistant strains [14]. In addition, different works are currently suggesting that CHG may be ineffective upon long treatment and is not free from the occurrence of resistance mechanisms [14]. For these reasons, in recent years, other antiseptic agents have been considered for the control of oral infections, in particular, quaternary ammonium compounds such as cetylpyridinium chloride (CPC) [14,15]. Experimental data concerning the efficacy of these molecules against dental plaque microorganisms, especially in biofilm matrixes, are still controversial and not fully consistent [7,16,17]. CHG is, thus, still regarded as the reference compound among oral antiseptics, although undesirable side effects are reported to occur with its long-term use, including teeth yellowing staining and burning sensations [18]. Therefore, different studies in recent years investigated the antibacterial and antibiofilm activity of several natural extracts and essential oils alone and in combination with CHG, suggesting a potential beneficial synergistic effect [19,20,21,22]. Although several studies have been undertaken, results still show high variability and lack of consistency [19,20]. In the literature, a limited number of studies are reported that investigate and compare the efficacy of different CHG-containing mouthwash formulations. In these cases, the antimicrobial efficacy seems to vary extensively when comparable CHG concentrations are combined in different pharmaceutical formulations [23,24,25].

The purpose of this study was to compare and evaluate the antimicrobial and antibiofilm activity of S. mutans of CHG and three different CHG-containing mouthwash commercial formulations. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal concentration (MBC) were used to evaluate the antimicrobial activity of mouthwashes along with biofilm prevention concentration (BPC) and CHG formulation activity towards mature biofilm.

2. Materials and Methods

2.1. General

Streptococcus mutans Clarke ATCC 25,175 was purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Bacterial growth was followed by measuring optical density at 600 nm (OD/mL). The growth curve was constructed by measuring optical density over time using a Beckman Coulter Life Science DU730 UV/vis spectrophotometer (Brea, CA, USA). Chlorhexidine standard solution (10 μg/mL) in methanol was obtained from LGC standards (LGC^® Group, Teddington, UK) and used as a standard for mass spectrometry analyses. Chlorhexidine Digluconate solution in water (200 mg/mL) was purchased from Supelco^® Analytical (Merck Italy, Milan, Italy) and used in all microbiological experiments. From now on, both standard solutions used will be referred to as CHG. The three mouthwash formulations containing 0.2% CHG (2 mg/mL) were commercially purchased. Some of the active ingredients present in the formulations are summarized in

Table 1. Some of the active principles contained in the three commercial CHG mouthwash formulations (according to the manufacturer’s descriptions).

Active Principles	Formulation 1	Formulation 2	Formulation 3
CHG	0.20%	0.20%	0.20%
CPC	−	+	−
Aloe barbadensis (Aloe Vera) Leaf Extract	−	+	+
Salvia officinalis (Sage) Leaf Extract	−	+	+
Ethyl alcohol	+	−	−

. Formulations 2 and 3 contain an equal amount of the same herbal extracts. Brain Heart Infusion broth (BHI) medium (Dehydrated) Oxoid was used, following ATCC specifications (Oxoid Italy, Rodano, Italy) [26]. Ninety-six-well plates (Thermo Fisher Scientific Italy, Monza, Italy) and a Tecan Infinite 200 Pro multiplate reader (Männedorf, Switzerland) were used in antimicrobial assays. Crystal Violet was purchased from Sigma-Aldrich (St Louis, MO, USA) and MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide was obtained from Life Technologies (Thermo Fisher Scientific Italy, Monza, Italy).

2.2. Development and Validation of LC-MS/MS-Based Methodology

CHG standard was initially used to set up and validate a detection method for CHG. EMA guidelines for the validation of bioanalytical methods were followed. The LC-MS/MS analysis system consisted of a UHPLC (Ultrahigh-Performance Liquid Chromatography) UltiMate 3000 Dioneex^® chromatograph from Thermo Fisher Scientific™ (Waltham, MA, USA) equipped with a binary pump, a refrigerated autosampler and a thermostated chromatographic column coupled to the TSQ Endura™ mass spectrometer (MS) from Thermo Fisher Scientific™ (Waltham, MA, USA), equipped with electrospray ionization (ESI) source and a triple quadrupole analyzer (QqQ). Chromatographic separation was performed using a Kinetex C18 column (50 × 2.1 mm—1.73 μm particle size) (Phenomenex, Torrance, CA, USA). Compound elution was obtained using a two-component mobile phase: 0.1% formic acid in water (eluent A) and methanol (eluent B). The following multi-step gradient was optimized: 30% B at time 0, from 0 to 2.5 min 95% B, 2.5 to 3.5 min at 95% B, at 3.5 return to initial conditions and equilibration of the column at 30% B up to 4.5 min. The flow rate was 0.4 mL/min, and the column temperature was maintained at 20 °C. The injection volume was 5 μL, and all samples were analyzed in triplicate. Mass spectrometric analyses were carried out in selected reaction monitoring (SRM) and electrospray positive mode. A CHG mono-charged precursor ion and a double-charged precursor ion were identified with direct infusion of pure standard (m/z 506 and 253, respectively). For each analyte, a precursor and two products (quantifier and qualifier) were selected and used for the detection and quantification: CHG mono-charged, 506 → 336 and 202; CHG double-charged, 253 → 170 and 153. Selectivity, specificity, absence of carryover, the lower limit of detection (LLOD) and quantification (LLOQ) were also determined. Selectivity was assessed by analyzing the diluent solvent (MeOH 20%, TCA 1%). The absence of interfering components was verified. Specificity was evaluated based on the consistency of retention times across runs and the presence of both products (quantifier and qualifier) in a constant ratio. The LLOD was defined as the lowest concentration at which the analytical test can detect the analyte signal (S), discriminating it from the background noise (N) (LLOD: S/N ≥ 3). The LLOQ is the lowest concentration at which precision and trueness were within 20%, with a peak intensity at least 5 times higher than baseline noise (LLOQ: S/N ≥ 5). Linearity was evaluated using a 6-point calibration curve, built by using the CHG standard, in a range of concentrations from 0.25 to 2 μg/mL, including a zero value. Peak areas obtained were plotted against analyte concentrations, and a linear regression analysis was performed. The slope and the correlation coefficient were calculated for each calibration curve. Quality controls (QCs) of quantifications were used at 0.6 and 1.2 μg/mL. Precision and trueness were evaluated by analyzing LLOQ and the two QCs samples in five replicates to assess the intraday accuracy. Precision was expressed as the percentage coefficient of variation (%CV), and the trueness was evaluated by measuring the relative error, expressed as %bias, between experimental measurements and theoretical concentrations. Mean values within ±15% of the nominal concentration for both precision and trueness were considered acceptable. The potential occurrence of carryover was assessed by injecting a blank diluent sample after a CHG-concentrated sample. CHG concentration in mouthwashes was assessed by diluting the 0.2% CHG formulations (2 mg/mL) in the diluent solvent (MeOH 20%, TCA 1%), up to 0.5, 1, 1.5 and 2 μg/mL final nominal concentrations. Samples were centrifuged, transferred into appropriate vials, and injected. Peak areas were collected using Qual Browser software and used for the quantification with the linear regression equation constructed.

2.3. Streptococcus Mutans Growth Conditions

S. mutans strain was transferred from frozen glycerol stock (−80 °C) and plated on sterile Brain Heart Infusion (BHI) agar for 72 h at 37 °C in a Petri plate. Some colonies were inoculated from the agar plate in 10 mL of BHI broth and incubated at 37 °C in constant shaking overnight (O/N). The overnight culture was then used to measure optical density at 600 nm (OD/mL) and determine the number of colony-forming units per mL (CFU/mL). Indeed, the standardization of vital bacteria inoculated in susceptibility testing is of critical importance for obtaining accurate and reproducible results. Therefore, we defined the number of CFU/mL of our S. mutans Clarke ATCC 25,175 after overnight growth. This latter was diluted in nine different ten-fold serial dilutions in sterile BHI broth. One hundred μL of each dilution was plated on BHI agar and incubated at 37 °C for 48 h. Colonies were counted on plates (considering only plates with 50–400 colonies). CFU/mL was calculated from each plate counted using the following formula:

$$N=\frac{\left(\mathrm{C}\ast 10\right)}{10-D}$$

where N = CFU/mL; C = number of colonies per plate; D = number of 10-fold dilution factor [27]. Results obtained from all counted plates and three replicate experiments performed were averaged and correlated with the OD/mL value. An overnight culture of S. mutans Clarke ATCC 25,175 in 10 mL, diluted at 1 OD/mL, resulted in 1.9 (+/−0.9) × 10⁹ CFU/mL. This relationship holds true for subsequent cultures of the same bacterium grown in the same way.

2.4. MIC (Minimal Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) Determination

The minimal inhibitory concentrations (MICs) of CHG and mouthwash formulations were determined by using the microbroth dilution technique [28]. An overnight culture of S. mutans Clarke ATCC 25,175 was diluted up to 2 × 10⁶ CFU/mL in a fresh BHI medium. CHG stock (200 mg/mL) and CHG formulations (2 mg/mL) were diluted up to 20 μg/mL in sterile BHI broth. Then, two-fold dilutions, 100 µL each, were prepared in a 96-well-microtiter plate in duplicate. One hundred µL of sterile BHI broth was used as a growth medium for positive controls. Negative controls were performed using 100 μg/mL ampicillin in the same broth. Thereafter, 100 µL of the bacterial inoculum prepared at 2 × 10⁶ CFU/mL was added to each well. The final CHG concentrations tested were 10, 5, 2.5, 1.25, 0.6, 0.3 and 0.15 μg/mL in the presence of 10⁶ CFU/mL of S. mutans. Plates were incubated at 37 °C for 24 h, and growth was monitored by measuring optical density at 600 nm. The MIC was defined as the lowest concentration showing clear growth inhibition (OD/mL comparable to sterility controls). Experiments were performed in five independent replicates.

For the determination of the minimum bactericidal concentration (MBC), the culture broths of the wells showing no visible growth were plated on BHI agar plates and incubated for 48 h at 37 °C. The concentration at which 99.9% of bacteria were killed was identified as the MBC.

2.5. Biofilm Prevention Concentration (BPC) Assay

To analyze the CHG effect in preventing S. mutans biofilm formation, an overnight culture of S. mutans Clarke ATCC 25,175 was diluted up to 2 × 10⁸ CFU/mL in a fresh BHI medium containing 2% glucose to induce biofilm formation. CHG stock (200 mg/mL) and CHG formulations (2 mg/mL) were diluted up to 20 μg/mL in sterile 2% glucose BHI broth. Then, two-fold dilutions, 100 µL each, were prepared in a 96-well-microtiter plate in duplicate. One-hundred microliters of sterile 2% glucose BHI broth was used as a growth medium for positive controls. Negative controls were performed using 100 μg/mL ampicillin in the same broth. Thereafter, 100 µL of the bacterial inoculum prepared at 2 × 10⁸ CFU/mL was added to each well. The final CHG concentrations tested were 10, 5, 2.5, 1.25, 0.6, 0.3 and 0.15 μg/mL in the presence of 10⁶ CFU/mL of S. mutans. Plates were incubated at 37 °C for 48 h, and growth was monitored by measuring optical density at 600 nm.

The plate was incubated at 37 °C overnight. The day after, supernatants were removed, and each well was washed 3 times with 150 μL of (phosphate buffered saline) PBS 1X to remove nonadherent cells. An aliquot of 100 μL of 0.1% CV was added to each well (CV was diluted in MilliQ water), and the plate was incubated at RT for 40 min in the dark. The CV was then removed, and 100 μL of 95% ethanol was added to each well and mixed by pipetting. The absorbance of the samples was read at λ = 540 nm.

2.6. Streptococcus Mutans Biofilm Growth Conditions

All other experiments were carried out on pre-established biofilms grown in a 96-well-microtiter plate as follows: An overnight culture of S. mutans Clarke ATCC 25,175 was diluted up to 10⁸ CFU/mL in fresh BHI medium containing 2% glucose to induce biofilm formation. An aliquot of 200 µL of bacterial inoculum was incubated in each well. Negative controls were performed using 100 μg/mL ampicillin in the same broth. Plates were incubated at 37 °C for 48 h to allow biofilm growth.

2.7. CV Assay

S. mutans biofilms were grown as described above. After 48 h, plates were washed 3 times with 150 µL of sterile PBS 1X to remove nonadherent cells. CHG stock (200 mg/mL) and CHG formulations (2 mg/mL) were diluted up to 480 μg/mL in sterile 2% glucose BHI broth. Then, two-fold dilutions, 200 µL each, were prepared in the same broth in a sterile 96-well-microtiter plate and then added to the wells containing S. mutans biofilm. The final CHG concentrations tested on mature biofilms were 480, 240, 120, 60, 30 and 15 μg/mL in duplicate. Two hundred µL of sterile 2% glucose BHI broth was added to mature biofilms for positive controls. Plates were incubated at 37 °C for 24 h. Then, CHG and mouthwash-containing media were removed, and biofilm was washed 3 times with 150 µL of sterile PBS 1X. One hundred µL of 0.1% CV in deionized water was added to each well, and plates were incubated at RT for 40 min in the dark. CV was then removed, and biofilms were washed 3 times with 150 µL of sterile PBS 1X to remove the excess staining. Finally, 100 µL of 95% ethanol was added to each well and repeatedly pipetted to solubilize CV staining. Absorbance was registered at λ = 540 nm. The same protocol was also used to evaluate the effect of undiluted mouthwash formulations on the preformed biofilm after 48 h incubations (Results in paragraph 3.6). CHG stock (2 mg/mL in PBS) and CHG formulations (2 mg/mL), 100 µL each, were added to the wells containing S. mutans biofilm. Plates were incubated at 37 °C for 5 min. Then, the CHG and mouthwashes were removed, and the biofilm was washed 3 times with 150 µL of sterile PBS 1X. From this point on, the experimental procedures were repeated as described above.

2.8. MTT Assay: Bacterial Viability Test

S. mutans biofilms were grown as previously detailed. After 48 h, plates were washed three times with 150 µL of sterile PBS 1X to remove nonadherent cells. CHG stock (200 mg/mL) and CHG formulations (2 mg/mL) were diluted up to 480 μg/mL in sterile 2% glucose BHI broth. Then, two-fold dilutions, 200 µL each, were prepared in the same broth in a sterile 96-well-microtiter plate and added to the wells containing S. mutans biofilm. The final CHG concentrations tested on mature biofilms were 240, 120, 60, 30 and 15 μg/mL. Experiments were performed in duplicate. Two hundred µL of sterile 2% glucose BHI broth was added to mature biofilms for positive controls. Plates were incubated at 37 °C for 24 h. Afterward, an MTT assay was performed [29,30]. CHG and mouthwash-containing media were removed, and biofilm samples were gently washed 3 times with 150 µL of sterile PBS 1X. One hundred µL of a 0.5 mg/mL MTT solution in PBS 1X was added to each well, and plates were incubated for 3 h at 37 °C in the dark. MTT was then removed, and 100 µL of DMSO was added to each well and pipetted to dissolve the tetrazolium salts. Plates were further incubated at RT for 40 min in the dark. Finally, absorbance was measured at λ = 570 nm. The same experimental procedure was repeated on 48 h preformed biofilm with undiluted formulations (Results in paragraph 3.6). CHG stock (2 mg/mL in PBS) and CHG formulations (2 mg/mL), 100 µL each, were added to the wells containing S. mutans biofilm. Plates were incubated at 37 °C for 5 min. Afterward, CHG and mouthwash-containing media were removed, and the experimental procedure was carried out as described above.

2.9. Efflux Pumps Ethidium Bromide Accumulation Assay

An ethidium bromide (EtBr) accumulation assay for S. mutans was adapted following the procedure of Rodrigues et al. [31]. Several detection methods for evaluating efflux activity use dyes (such as EtBr) that are endowed with fluorescence properties only when present either in the intra- or extracellular environments [32]. One colony of S. mutans was inoculated in 10 mL of BHI medium and grown overnight in constant shaking at 37 °C. Cells were washed twice in PBS buffer to remove the rich BHI medium and resuspended in PBS buffer at 0.6 OD₆₀₀/mL. Cells were incubated in PBS supplemented with glucose at a final concentration of 0.4%. CHG and mouthwash formulations were added at half-MIC concentrations in the presence of EtBr at a final concentration of 2 μg/mL. Negative controls were performed using EtBr in 0.4% glucose in PBS. Samples were prepared into separate wells of a 96-well plate. Ethidium bromide accumulation in cells was followed over time, and fluorescence data were recorded every 60 s for 30 min using an excitation wavelength of 525 nm and an emission wavelength of 605 nm. Fluorescence intensity (FI) was monitored over time.

2.10. Statistical Analysis

S. mutans growth controls were used as a primary outcome variable. For each formulation, means and standard deviations were calculated for the quantitative experiments (MIC, BPC, MTT, CV). For each test, six different concentrations were used and compared to the absence of inhibitor; for each concentration, 6 replicates (n = 6) were analyzed. Statistical analysis was performed using R software for Windows (R version 4.2.1 64 bit, the R foundation for statistical computing). The Shapiro–Wilk and Levene tests were used to evaluate the normality and overall homogeneity of the datasets, respectively. Then, to evaluate the significance of results found in the differential inhibitory effect on mature biofilms, assessed through MTT and CV assays, the Student t-test was used. Eight replicates of each CHG concentration were considered for each formulation and tested to verify that the differences found were statistically representative. A value of p < 0.05 was evaluated as statistically significant. Finally, the chi-square and Fischer exact tests were used to assess if results obtained with undiluted formulations were statistically similar to those obtained with CHG reference compound (2 mg/mL). A value of p < 0.05 was evaluated as statistically significant.

3. Results

3.1. Determination of CHG Concentrations in Mouthwashes with LC-MS/MS

CHG concentration in mouthwash formulations was measured through a fully validated LC-MS/MS analytical method, as detailed in the Section 2. The results obtained (

Table 2. CHG concentrations in each formulation tested with relative standard errors.

Formulation	Measured Concentration (μg/mL) 1
1	2.24 ± 0.27
2	2.20 ± 0.22
3	2.15 ± 0.17

¹ Concentrations are reported as the average of at least three measurements ± the relative accuracy bias.

) confirmed that the drug concentration in all tested samples was consistent with the nominal ones.

3.2. MIC and MBC Determination

MIC and MBC values of CHG and mouthwash formulations for S. mutans were determined using the microbroth dilution method described in the Section 2. The Shapiro–Wilk and Levine tests confirmed that data obtained at different concentrations did not display a normal distribution and growth inhibition obtained at the same concentration in all cases. Results are reported in

Table 3. MIC and MBC values obtained for S. mutans with CHG and mouthwash formulations.

Formulation	MIC (CHG μg/mL)	MBC (CHG μg/mL)
CHG	1.25	15.00
1	1.25	15.00
2	1.25	25.00
3	2.50	>25.00

and are in line with those previously described for the same ATCC strain [32,33]. However, higher values were measured for formulation 3, suggesting a lower efficacy of this mouthwash in affecting S. mutans proliferation and viability.

3.3. BPC Determination

The Biofilm Prevention Concentration (BPC) is defined as the lowest concentration of active principle hampering biofilm formation. CHG and the three mouthwash formulations were tested to evaluate their inhibitory effect on biofilm formation.

Results reported in Figure 1 show the percentage of biofilm matrix retrieved at different concentrations of active principles tested. A 2.5 μg/mL BPC value was measured for both formulation 1 and CHG, while the BPC of formulation 2 was equal to 5 μg/mL. In the case of formulation 3, BPC was not reached up to 10 μg/mL, although a significant decrease in biofilm amount was observed starting from 5 μg/mL of this preparation.

3.4. Inhibitory Effect on Mature Biofilms

After assessing the effect on preventing biofilm formation, the CHG antibiofilm activity, combined in different formulations, on preformed mature biofilm, was evaluated. In this case, the tested concentrations were higher than those used in MIC and BPC assays (15–480 μg/mL). In fact, established procedures to test the antibiofilm activity on mature biofilms highlighted that MIC concentrations used for planktonic cells are often quite ineffective against bacteria embedded in biofilms. This is probably due to the fact that a higher antimicrobial concentration is needed to penetrate the biofilm matrix [32].

Results shown in Figure 2 highlighted that CHG and formulation 1 mouthwash have a significant effect in destructuring the preformed biofilm, even at low concentrations of active principles (60 μg/mL). However, these preparations failed to fully eliminate the preformed biofilm also when used at higher concentrations, and an average reduction of 50% in biofilm amount was reached. Conversely, no significant differences in the percentage of residual biofilm were observed for the other two mouthwashes, regardless of the concentrations tested. The Student t-test was carried out on data replicates at each concentration by comparing them with the CHG reference compound. Formulation 1 was the only one exhibiting a result statistically comparable to the CHG control.

3.5. Bacterial Viability Evaluation of Mature Biofilm

To evaluate the efficacy of the tested solutions as antibiofilm agents, their influence on the viability of S. mutans cells in the mature biofilms was also measured by MTT assay (Figure 3). The preformed biofilm treated for 24 h with CHG and mouthwash formulation 1 showed a drastic reduction in cell viability, even when using a 30 μg/mL concentration, and an almost complete killing effect was observed when a concentration of 120 μg/mL was reached. On the other hand, a concentration between 240 and 480 μg/mL of formulation 2 was needed to obtain the same bactericidal effect. Again, formulation 3 seemed to be less effective in affecting cell viability in a mature biofilm since 6.3% of metabolically active cells was still retained at a final added concentration of 480 μg/mL. However, the Student t-test revealed that no significant difference was observed between formulations 2 and 3. Conversely, they were significantly different from CHG and formulation 1, which was the only one showing results statistically comparable to the CHG control.

3.6. Inhibitory Effect and Cell Viability Evaluation of Mature Biofilms after 5 min Treatment

To obtain information on the efficacy of the mouthwashes in conditions similar to those of real use [33], the previously described antibiofilm assays were performed using a short incubation time of 5 min on mature biofilms. Then, CV and MTT assays were carried out using undiluted mouthwashes (2 mg/mL CHG). Although no significant decrease in total biofilm biomass was observed and confirmed by Chi-square and Fisher exact tests (Figure 4A), a sharp and significant reduction in viable cells (around 40–50%) was observed when biofilms were incubated in the presence of CHG and formulation 1 (Figure 4B). Conversely, formulations 2 and 3 did not have any relevant effect in reducing cell viability after 5 min exposure (Figure 4B). Again, the Chi-square and Fisher exact tests confirmed that formulation 1 was the only one showing an effect comparable to the CHG reference compound. These results are in line with other antibiofilm assays performed on S. mutans in the literature and highlight how the different experimental conditions tested on mature biofilms do impact the overall antibiofilm effect of a specific formulation, even when the latter is tested in vitro on a single strain [33].

3.7. Efflux Pump Activity Assay

The addition of an antimicrobial or antiseptic agent often determines a boost of intrinsic efflux activity in bacterial cells, which may be monitored through different assays. Here, we used the EtBr assay, following EtBr accumulation in the intracellular environment over time. As shown in Figure 5A, when 0.6 μg/mL CHG was added to the cells, a significant decrease in the EtBr accumulation was observed compared to the control, thus suggesting a boost in the cellular efflux activity. When mouthwashes were tested at half the MIC concentration, a slight additional increase in the efflux activity was observed, and EtBr intracellular concentration dropped. In Figure 5B, the linear regression equations for the different curves in EtBr accumulation assay are reported. The slope ratio of the different curves compared to the control slope highlighted a 1.7-fold decrease in EtBr accumulation when CHG is added and a 2.4-fold decrease when formulation 1 is added. An average 3.3-fold decrease was found when formulations 2 and 3 were added (Figure 5B).

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 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. The values retrieved were between 2.5 and 10 μg/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 tests performed. 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]. In addition, there is a lack of studies focused on the synergistic effect of these extracts with CHG. In formulation 2, along with herbal extracts and CHG, CPC is also present. This latter compound may be responsible for the improvement in formulation 2 antibiofilm activity compared to formulation 3. On the other hand, mouthwash 1, lacking herbal extracts and CPC but characterized by a specific percentage of ethyl alcohol in its composition, was the most active against S. mutans. In this case, the presence of ethanol may be the reason for the higher antimicrobial and antibiofilm activity observed, and the differential effect could be due to the improved penetration potential in the biofilm structure of this mouthwash formulation. Some authors reported the higher effectiveness of nonherbal mouthwashes over herbal ones [40,41]. However, recent works highlighted that the comparison between herbal and nonherbal mouthwashes is inconclusive, displaying discordant results in different studies [42]. The discrepancy observed in the antibiofilm activity of the different formulations could have different causes, which could be hard to reconduct to a single aspect. Other authors investigating the antibiofilm effect of oral formulations, such as toothpastes, point out the difficulty of unraveling the different contributions of the different active principles in complex formulations, even when S. mutans monospecific biofilms are analyzed [43]. Further studies will be needed to better understand the reasons for this variability in terms of antibacterial effect and to better correlate the effect of each of the different compounds in the formulation. Moreover, the results observed in the in vitro study presented here need to be confirmed and evaluated on more complex systems, such as biofilm grown on dentine surface or multispecies biofilms of both commercial strains and clinical isolates. In conclusion, it becomes clear that the lack of studies’ standardization and CLSI official guidelines to assess the antibiofilm activity gave rise to a multitude of different protocols used over the years for this kind of application and, therefore, to discordant and incomparable results. In this work, we compared different methodologies and attempted to give some insights into the importance of considering different approaches in studying mature biofilms.

5. Conclusions

The results obtained in this work highlighted an overall higher efficacy of a mouthwash formulation without herbal extracts compared to two herbal-containing formulations, both in preventing biofilm formation and exerting a bactericidal effect on mature biofilms in vitro. Notably, mouthwashes containing the same CHG concentration displayed a significant difference in their antibiofilm effect against S. mutans, thus pointing out the importance of the pharmaceutical formulation for these applications. In conclusion, the data presented here highlighted the importance of better detail of the antibiofilm effect of antiseptic agents by studying it in different experimental conditions and with different methodological approaches, even in conditions where a single microbial strain is tested.