The saliva sample used in this study was collected from healthy volunteers after obtaining written informed consent. The protocol for experimentation and the use of saliva was assessed and approved by the Institutional Ethical Committee, Alagappa University, Karaikudi (IEC Ref No: IEC/AU/2018/5). Methods followed were carried out in accordance with the appropriate guidelines and regulations.

Streptococcus mutans UA159 and Candida albicans (ATCC 90028) were used in this study. Culturing mono species of S. mutans and C. albicans (2 × 10⁶ cfu/ml) was performed using THYES (Todd Hewitt broth supplemented with 1% of yeast extract and sucrose) (HiMedia, India) and YPD (1% yeast extract, 2% peptone, and 2% dextrose) broth (HiMedia, India), respectively. For culturing of dual species (equal volume of each culture), TSBS (soybean casein digest medium supplemented with 1% sucrose) medium (HiMedia, India) was used. Cultures were incubated at 37°C for 24 h.

Thymol was commercially procured from Alfa Aesar, India. Stock solution of thymol was prepared as 50 mg/ml concentration in methanol (Sigma-Aldrich, India) and stored at room temperature. The highest volume of the compound used was chosen as the volume of methanol to be added for vehicle control in each assay.

The MIC of thymol against C. albicans and S. mutans was evaluated through microbroth dilution method according to CLSI guidelines (Wikler, 2006; Barbara et al., 2017). For determination of MMC, single and dual species culture of C. albicans and S. mutans was cultured in the absence and presence of thymol at MIC and sub-MICs. After 24 h of incubation at 37°C, the control and treated groups were subjected to serial dilution followed by spotting and spread-plating on appropriate agar plates (Priya et al., 2021).

Time kill assay was performed to analyze the short-term microbicidal effect of thymol on single and dual species culture of C. albicans and S. mutans as described by Niu et al. (2020) with slight modifications. Briefly, 2 × 10⁶ cells were taken for mono species, and an equal volume of C. albicans and S. mutans was taken for dual species. Various concentrations of thymol (1X, 2X, 5X, and 10X MIC) were added separately. After 2-min exposure, the compound activity was restrained by removal through two rounds of centrifugation. Cells were resuspended in phosphate buffered saline (PBS) and serially diluted, and 5 µl each from all the serial dilutions was also spotted on agar plates.

C. albicans, S. mutans, and dual species cultures in the absence and presence of thymol at MIC and sub-MICs were allowed to form biofilm on 1 cm × 1 cm glass surface for 24 h at 37°C. Post incubation, the glass slides were carefully removed from the medium, dip-washed in sterile PBS to remove loosely bound cells, air-dried, and stained with 0.4% crystal violet. Biofilm cells in the stained glass sections were visualized under a light microscope (Nikon Eclipse 80i, United States) at a magnification of ×400 and documented.

In addition to microscopic observation of the single and dual species C. albicans and S. mutans biofilm under the influence and absence of thymol, cell viability assay was performed with resazurin dye (Alamar blue). Alamar blue is a versatile metabolic dye, which is a redox indicator that is reduced within the cell due to cellular metabolism. Single and dual species cultures of C. albicans and S. mutans were allowed to form biofilm in the presence and absence of thymol at MIC and sub-MICs (32.5, 75, 150, and 300 μg/ml) on polystyrene surface. At the end of 24 h, the planktonic cells were discarded, and loosely bound cells were removed by careful washing with PBS. Surface-attached cells were then resuspended in PBS solution. Stock solution of Alamar blue (Sigma-Aldrich, India) at a concentration of 6.5 mg/ml was prepared in 1× PBS. To 0.9 ml of cell suspension in PBS, 0.1 ml of Alamar blue was added and incubated in the dark for 4 h at 37°C. Sterile PBS added with Alamar blue substrate alone was maintained as blank. Samples were centrifuged at 8,000 rpm for 10 min after incubation. Supernatant was collected, and the fluorescent intensity was measured at 590-nm emission and 560-nm excitation wavelengths (Muthamil et al., 2020).

Unstimulated whole saliva (UWS) was collected from healthy individuals with good oral hygiene. Prior to the collection of saliva, the volunteers were refrained from eating, drinking, and brushing for 2 h. The saliva sample was collected by the method of spitting into a sterile tube, which was immediately clarified by centrifugation at 4,000 × g for 10 min. The cell debris were removed, and the supernatants were pooled and stored at −20°C until use. For biofilm formation, 200 µl of cell suspensions (single and dual species) was added with 20 µl of clarified saliva in the absence and presence of thymol. After 24 h of incubation, the planktonic cells were discarded, and loosely bound cells were washed off with sterile PBS. Surface-bound biofilm cells were stained with 0.4% crystal violet and subsequently destained with 15% glacial acetic acid solution, the absorbance of which was read at 570 nm using a multifunctional spectrophotometer (Spectra Max 3, Molecular Devices, United States) (Ahn et al., 2008).

The impact of thymol on fungal morphogenesis between yeast and hyphal forms was analyzed through the following assays (Priya and Pandian, 2020).

C. albicans and S. mutans cells in single and mixed state were subjected to a brief exposure of thymol (1X, 2X, 5X, and 10X MIC) for 1 h after which the compound was removed by centrifugation. Appropriate positive controls were maintained in parallel. For mono species of C. albicans and S. mutans, amphotericin B (MIC: 2.5 μg/ml) and chlorhexidine (MIC: 16 μg/ml) were used, respectively. For dual species, both amphotericin B and chlorhexidine were used in combination. Post exposure, 1% culture from each group was used as the inoculum and cultured in an appropriate medium. Changes in the cell density were spectrophotometrically observed for a period of 12 h with 1-h time interval (Taweechaisupapong et al., 2012).

Total RNA from C. albicans, S. mutans, and dual species culture was extracted by the Trizol method. Using a high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, United States), the extracted RNA was converted to cDNA. qPCR analysis was performed with the SYBR Green Master Mix (Applied Biosystems, United States) for candidate genes (list of genes, primer details, and function are provided in Table 1) of C. albicans and S. mutans. Changes in the expression were calculated by the ^{Δ Δ}CT method (Livak and Schmittgen, 2001).

The toxic effect of thymol, if any, and the in vivo efficacy to clear the C. albicans and S. mutans infection were analyzed through the invertebrate animal model Galleria mellonella. Larvae weighing around 0.2–0.4 g were taken for experiments. Ten larvae were taken per group. A total of 2 × 10⁶ and 2 × 10⁴ cells of C. albicans and S. mutans, respectively, were taken for infection. Thymol at 300 mg/kg was injected for both toxicity analysis and treatment groups. Injection was performed with a U-100 insulin syringe (Dispovan, HMD, India) in the last proleg. For survival analysis, nine different groups were segregated. Group I received PBS alone and served as the injection control. Group II received PBS along with 2% methanol and served as the vehicle control. Group III larvae were injected with thymol (300 mg/kg) for analysis of the toxicity. Groups IV–VI were designated as the infection control and received the cultures C. albicans, S. mutans, and dual species, respectively. An appropriate volume of culture was taken in the U-100 syringe and injected on the last left proleg. Groups VII–IX were designated as the treatment group where the larvae received thymol in addition to infection. Thymol was injected on the last right proleg. Larva groups were incubated at 37°C for 5 days. Survival was monitored every 12 h. For in vivo efficacy of thymol in controlling the infection, three larvae from infected and treated groups were cut open with a scalpel; the content was suspended in sterile PBS, and the serial dilutions were plated on a selective medium (HiChrome candida differential medium (HiMedia, India) for C. albicans; Mitis salivarius agar (HiMedia, India) for S. mutans; for dual species, both the plates were used) (Selvaraj et al., 2020).

All the experiments were carried out in at least three biological replicates with at least two technical replicates, and values are presented as mean ± standard deviation (SD). To analyze the significant difference between the value of control and treated samples, one-way analysis of variance (ANOVA) and Duncan’s post hoc test were performed with a significant p-value of <0.05 by the SPSS statistical software package version 17.0 (Chicago, IL, United States).

Initially, the MIC of thymol was assessed against single species of C. albicans and S. mutans through microbroth dilution assay. It was found that for monoculture, thymol at 128 and 256 μg/ml inhibited the visible growth of C. albicans and S. mutans, respectively (Figure 1A). Hence, for dual species, 300 μg/ml of thymol was analyzed for growth inhibitory effect, and the same concentration was found to be effective in inhibiting the growth of dual species. Thus, 300 μg/ml of thymol was considered to be the MIC and MMC for dual species. Through cfu analysis, it was evident that thymol exerts the growth inhibitory effect against single and dual species of C. albicans and S. mutans in a concentration-dependent manner (Figure 1B). Spot assay displays that thymol at 300 μg/ml completely inhibited the growth of single and dual species of C. albicans and S. mutans, and a concentration-dependent growth inhibition can also be witnessed (Figure 1C).

As the end application of this study is directly related to the dentifrice formulation, the impact of limited time exposure of bioactives on these pathogens was analyzed through time kill assay where the microbes were exposed to thymol for 2 min. At MIC, S. mutans cells were completely killed by the action of thymol, whereas for C. albicans and dual species, 2X MIC cleared the viable cells (Figure 1D).

The impact of sub-inhibitory concentrations of thymol was microscopically appraised. Dose-dependent diminution in the surface adherence of cells was observed for thymol treatment. Under single and dual species state, the biofilm formation and surface adherence of C. albicans were impaired in a concentration-dependent manner by the influence of thymol. In addition to reduction in surface adherence, the hyphal form was also found to be arrested. At MIC, the viability of S. mutans was completely lost, and thus, no surface adherence of S. mutans was found (Figure 2A).

Similarly, metabolic viability assay was performed for the biofilm of C. albicans and S. mutans single and dual species under the influence of thymol. Results observed are in line with the microscopic observation, as C. albicans biofilm adherence was found to be diminished in a concentration-dependent manner under both single and dual species condition (Figure 2B).

The proficiency of thymol in inhibiting the biofilm formation of C. albicans and S. mutans in the presence of saliva was also analyzed. The efficiency of thymol continued to be the same even in the presence of saliva (Figure 2C). These results suggest that thymol can be effective against the C. albicans and S. mutans biofilm.

Phenotypic switch between yeast and hyphal forms under the influence of thymol was analyzed. In a concentration-dependent manner, thymol could restrain the shift of yeast to hyphal phase (Figure 3A) and can revert the hyphal cells to yeast morphogenesis (Figure 3B). Hyphal morphogenesis of C. albicans during interaction with S. mutans was found to be diminished, and the same has been evidenced in the present study through the filamentation assay, which when compared to mono species, the filamentation of C. albicans under dual state was found to be less. Despite the single or dual state, thymol significantly impeded the development of filamentous morphology (Figure 3C).

Metabolic breakdown of carbohydrate through glycolysis was interfered by the presence of thymol. In single as well as dual state, the pH of the control was dropped to more acidic condition. For thymol treatment, at MIC, a slight variation in the pH change was noted, whereas at higher MICs a significant change was observed (Figure 4A).

Correspondingly, the aciduric ability of C. albicans and S. mutans was found to be significantly diminished under the impact of thymol. Both adapted and unadapted cells were found to be sensitized to the acidic pH condition under the single and dual species state when treated with thymol (Figure 4B). Unadapted cells were found to be more sensitive to thymol. Irrespective of prior adaptation conditions, thymol reduced the survival of cells under low pH condition, which is an added advantage for the treatment of caries.

As oral pathogens are exposed to dentifrices only for a short duration, the antimicrobial effect after the removal of thymol was analyzed. Compared to the positive controls—chlorhexidine and amphotericin b—thymol exhibited proficient post antimicrobial effect against single and dual species of C. albicans and S. mutans even at MIC by arresting the proliferation of cells (Figure 5).

The possibility of resistance development against thymol by single and dual species of C. albicans and S. mutans was investigated. Spontaneous resistance (Figure 6A) to higher concentration of thymol as well as resistance to successive passage (Figure 6B) in the presence of increasing concentrations of thymol were not acquired by the pathogens. When single and dual species of C. albicans and S. mutans was allowed to grow on a medium supplemented with high concentrations of thymol, no colonies were developed, signifying that the pathogens were unable to outgrow in the presence of thymol. Similarly, when the pathogens were exposed to thymol from lower concentration to higher concentration over a period, no resistance development was observed as complete growth inhibition was observed at sub-MIC of thymol at the 12th day of passage (Figure 6B).

Treatment with thymol influenced the transcriptional level modulations in the virulence genes (Figure 7). Except for the transcriptional repressors nrg1 and tup1, the expression of all other genes of both C. albicans and S. mutans was found to be downregulated. Expressions of nrg1 and tup1 were found to be upregulated, which is in line with the antihyphal activity observed through in vitro assays. Genes involved in the development and maintenance of hyphae in C. albicans such as hwp1, ras1, ece1, and cph1, genes responsible for filamentous morphology such as eap 1, efg1, adhesin als 1, and the transcriptional regulator of filamentous growth such as ume6 and hst7, which is required for biofilm formation, are found to be downregulated. Negative transcriptional regulators of filamentation such as nrg1 and tup1 were upregulated upon thymol treatment in both single and dual species. Downregulation of genes associated with the hyphal development and filamentous morphology and upregulation of negative regulators of the same under the influence of sub-inhibitory concentration of thymol imply that the compound can influence crucial virulence aspects of the pathogen. Similarly, vicR and comDE, the two major two-component regulatory systems of S. mutans, were found to be downregulated. Downregulation of vicR has affected the expression of glucosyltransferases gtfBCD, which are responsible for the synthesis of water-soluble and -insoluble glucans that are elemental bridge molecules between bacteria and acquired pellicle, thereby facilitating the colonization of the microbial biofilm. Decreased expression of ComDE, the two-component signal transduction system allied with the quorum sensing, which is known to regulate the competence and biofilm formation of S. mutans, suggests that the communication between the microbial systems resulting in the increased biofilm amalgamation has been impaired by the action of thymol. Similarly, the expression of two other genes gbpB and Smu0630 that are correlated with biofilm formation are declined.

Thymol at 300 mg/kg concentration does not exert any significant toxic effect to the larvae, whereas infection with C. albicans, S. mutans, and dual species of C. albicans and S. mutans impaired the survival (Figures 8A,B). Treatment with thymol rescued the larvae from the infection and increased the survival rate (Figure 8C). About 70% of larvae survived up to 120 h after administration of thymol. Only 35, 50, and 30% of larvae survived following infection with C. albicans, S. mutans, and dual species, respectively. On the other hand, treatment with thymol increased the survival rate to 70, 80, and 60% in larvae infected with C. albicans, S. mutans, and dual species, respectively. In addition to this, the in vivo infection clearance was also promoted by thymol treatment, which was evidenced through the reduced colony count in CFU analysis.