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. 2026 Jan 22;76(2):109386. doi: 10.1016/j.identj.2025.109386

In Vitro Antimicrobial Effect of Tetrahydrocannabinol on Streptococcus mutans and Its Anticariogenic Potential

Haoyan Zhai a,b, Yixuan Zhang a,b, Minki Kim a, Xintian Zhou c, Joseph Ferracciolo d, Eric Krukonis d, Chunyan Liu b,⁎, Zheng Zhou a,⁎
PMCID: PMC12859778  PMID: 41576726

Abstract

Introduction and aims

With the increasing use of marijuana, it is vital to understand the effect of tetrahydrocannabinol (THC) on oral microbiota, especially the primary carious pathogen Streptococcus mutans.

Methods

The minimum inhibitory concentration (MIC) of THC against S mutans was determined by antimicrobial susceptibility testing. Bacterial acid production was evaluated. The effect of THC on S mutans biofilm formation and preformed biofilms was determined by crystal violet assay. The metabolic activity and viability of the biofilm were assessed using the methylthiazolyldiphenyl tetrazolium bromide assay and live/dead assay, respectively. Extracellular polysaccharide (EPS) was examined by Cascade Blue Dextran staining. S mutans membrane potential was detected by the Baclight Bacterial Membrane Potential Kit.

Results

The MIC of THC against S mutans was 2 µg/mL (P < .0001). A total of ≥2 µg/mL THC reduced bacterial acidogenicity and inhibited over 90% of biofilm formation (P < .0001). Additionally, ≥1 µg/mL THC reduced biofilm viability and EPS production (P < .0001), as assessed by fluorescence measurements and microscopy. While 1 to 64 µg/mL THC did not degrade preformed biofilm, metabolic activity was reduced by 16 to 64 µg/mL THC (P < .01), and 8 to 32 µg/mL THC reduced biofilm viability in a time- and dose-dependent manner (P < .001). Moreover, 2 to 8 µg/mL THC promoted membrane hyperpolarization after a 5-minute treatment (P < .01).

Conclusion

THC inhibits S mutans growth and biofilm formation while also reducing bacterial viability, EPS production, and acid production. Although it does not degrade preformed biofilm biomass, THC diminishes its metabolic activity and viability. These effects may be linked to THC-induced membrane hyperpolarization. This in vitro study suggests that THC may reduce the cariogenic capacity of S mutans.

Clinical Relevance

This study shows that THC inhibits S mutans growth, biofilm formation, properties of preformed biofilms, and acid production. It provides preliminary scientific evidence on the impact of THC on oral health, specifically cannabinoid consumption on cariogenesis, and a potential new avenue for developing a new anticariogenic agent.

Key words: Cannabinoids, Tetrahydrocannabinol (THC), Streptococcus mutans, Antimicrobial effect, Cariogenesis

Introduction

With the global trend of the legalization of the recreational use of cannabis,1 there has been a significant increase in its consumption.2, 3, 4 Cannabis has been historically used for its medicinal properties5 but is also reported to be associated with various potential adverse health effects, including cardiovascular disease,6 mental health issues, and addiction.7,8

Cannabinoids, including tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol, and cannabigerol (CBG), are the major natural components of the Cannabis sativa plant. Among the cannabinoids, THC is the most abundant and exhibits a range of therapeutic effects, including analgesic, antiemetic, anti-inflammatory, anticancer, and antiseizure properties, as well as offering neuroprotective benefits in cases of neurodegeneration.5,9,10 Nevertheless, THC also carries notable adverse effects, such as its psychoactive property and its association with depression and anxiety.11 The usage of oral THC and its derivatives is rapidly increasing due to various THC delivery methods for medical and recreational purposes, including inhalation of combusted plant material, vaping, and THC-containing edibles,12 which might increase the risk of adverse health outcomes to cannabis users,4 but evidence of its effects on oral health is lacking and controversial.13, 14, 15, 16 Therefore, it is crucial to understand its effects on the oral microbiota and potential impact on overall oral health.

Despite these health concerns, cannabinoids have also attracted renewed scientific interest for their antibacterial and antibiofilm properties. Early reports from over a century ago suggested that cannabinoids possess antibacterial activity, but this has drawn extensive attention only recently.17, 18 CBD has shown efficacy against both Gram-positive and Gram-negative bacteria and certain fungal pathogens, as well as strong biofilm inhibition activity.19, 20, 21 In dental applications, CBD and CBG-infused mouthwash products have been found to be as effective as chlorhexidine in reducing the bacterial content in dental plaque samples.22 However, little is known about the antibacterial property of THC, the most abundant and frequently used cannabinoid.

Dental caries is a widespread, multifactorial infectious disease that poses significant public health burdens due to its high prevalence, particularly among children and the elderly.23 It has the potential to cause severe oral and systemic health issues.24 Streptococcus mutans is regarded as the primary cariogenic pathogen, a key producer of extracellular polysaccharides (EPSs) that enhance microbial adhesion and cohesion and further impede the diffusion of antimicrobial agents.25 Additionally, S mutans metabolizes fermentable carbohydrates into organic acids such as lactic acid, which lowers the local pH and leads to enamel demineralization.26 Given its central role in caries pathogenesis, there is growing interest in identifying agents capable of modulating S mutans virulence.

Given the significant role of S mutans in caries development and the growing interest in understanding the impact of cannabis on oral microbiota, this study aims to investigate the antibacterial and antibiofilm properties of THC against S mutans. By exploring THC’s effects on this cariogenic pathogen, the research seeks to understand its impact on dental caries and contribute to the broader understanding of how cannabinoids influence oral health.

Materials and methods

Bacterial strains and reagents

The S mutans strain (ATCC 25175) was purchased from the American Type Culture Collection. Both the planktonic S mutans and the biofilms were cultured in Brain Heart Infusion (BHI) broth and grown in an aerobic chamber at 37 °C, 5% CO2.27 EXO-THC was obtained from Cerilliant, Millipore Sigma. It consists of exo-THC in methanol. Working solutions were prepared by diluting the commercial standard in culture medium immediately before experiments without any further modification or synthesis. Ampicillin, methylthiazolyldiphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide were purchased from Sigma-Aldrich. Methanol was purchased from Fisher Chemical. The BacLight Bacterial Membrane Potential Kit was purchased from Invitrogen.

OD–colony-forming unit curve

The OD–colony-forming unit (CFU) standard curve of S mutans was constructed following established methods, with modifications as described.28 Bacteria were grown overnight in BHI medium at 37 °C in 5% CO2 incubator. Cells were collected by centrifugation at 4500 rpm for 10 minutes and washed with phosphate-buffered saline (PBS), and the OD600 value was adjusted to 1.0 using fresh medium. A 1-mL aliquot of the bacterial suspension was transferred to 9 mL fresh BHI medium and incubated at 37 °C with 5% CO2 for 3 to 4 hours to allow the bacteria to reach log phase. After incubation, the bacterial culture was adjusted to an OD600 value of approximately 0.8. Serial 2-fold dilutions were prepared, and the OD600 values of each dilution were measured. The dilutions were subjected to 10-fold serial dilutions (10–1 to 10–7), and 50 μL from each dilution was plated onto BHI agar plates. Plates were incubated upside down at 37 °C in a 5% CO2 incubator until visible colonies formed. Colony counts from plates with 30 to 300 colonies were used for CFU calculations.

Antimicrobial susceptibility testing

The minimum inhibitory concentration (MIC) assay was performed according to the Clinical and Laboratory Standards Institute guidelines,29 as described below. Serial 2-fold dilutions of THC and methanol were added to the wells of a 96-well plate, and then S mutans suspensions with a final concentration of approximately 5 × 105 CFU/mL were added. Methanol at the same concentration as THC was used as the vehicle control. Then, 4 µg/mL ampicillin (AMP) was used as the positive control. BHI was used as a negative control, and BHI without bacteria was used as a blank control. Plates were incubated at 37 °C in a 5% CO2 incubator for 24 hours. Then, the plates were scanned by a TECAN Spark. The MIC was determined as the lowest concentration of THC that inhibited 90% growth of S mutans. Each experiment was performed in triplicate and repeated 3 times.

Biomass of biofilm cells

The biomass of biofilm cells was detected using the crystal violet staining method.27 To examine the effect of THC on S mutans biofilm formation, the overnight bacterial culture was washed with PBS and adjusted to an OD600 = 0.1. Then, 250 µL of the bacterial suspension, supplemented with 10 µL 50% sucrose, was added to 24-well plates. Serial 2-fold dilutions of THC (0.5-16 µg/mL) were prepared in BHI, and 500 µL of each dilution was added to the wells. The final volume in each well was adjusted to 1 mL with BHI medium. Methanol at the same concentration as THC was used as the vehicle control. AMP and BHI were used as a positive control and a negative control, respectively. The plates were incubated at 37 °C in a 5% CO2 incubator for 24 hours. After incubation, the culture medium was removed, and the wells were washed with PBS. The biofilms were then fixed by heating them at 70 °C for 30 minutes. Next, 0.1% crystal violet solution was added to each well and stained for 30 minutes. The wells were then gently rinsed 3 times with tap water. The remaining crystal violet in the biofilm was solubilized with 1 mL of 33.3% acetic acid, and absorbance was measured at 570 nm using the TECAN Spark. To examine the effect of THC on preformed biofilms, the same protocol was followed, but THC, methanol, and AMP were added after biofilm formation for 1, 3, 6, or 24 hours. Each experiment was performed in triplicate and repeated 3 times.

Viability of biofilm cells

The viability of biofilm cells after THC treatment was detected by a live/dead assay.27 Briefly, the biofilms were incubated on 24-well plates and chamber slides, with 1 µL of 1 mM Cascade Blue Dextran (Invitrogen) added simultaneously to stain the EPS.30 Following treatment, the culture medium was discarded and washed with PBS, followed by staining with 2 μM SYTO9 (Invitrogen) and 20 μM propidium iodide (Sigma-Aldrich) for 15 minutes at room temperature. The staining buffer was removed from the chamber slides, and cover slides were added. Images were captured using an Olympus BX63 microscope. The 24-well plates were scanned using a TECAN Spark with green fluorescence (SYTO9) measured at an excitation of 485 nm and emission of 530 nm, and red fluorescence (PI) was measured at an excitation of 485 nm and an emission of 630 nm. Cascade Blue Dextran was measured at an excitation of 400 nm and emission of 420 nm.

Metabolic activity of S mutans biofilm

The metabolic activity of the biofilm was assessed in biological triplicate with the MTT assay.31 MTT can be reduced by the metabolic enzymes located in the bacteria’s cytoplasm and membrane to form formazan, which reflects the metabolic activity of the bacterial biofilm.32 Biofilms were formed in the 96-well plates and treated as described above. After treatment, the culture medium was removed, and the biofilms were washed with PBS and then incubated with 50 µL MTT (0.5 mg/mL) for 1 hour at 37 °C. Supernatants were then removed, and 100 µL dimethyl sulfoxide was added to solubilize the formazan crystals. The absorbance at 570 nm was read using a TECAN Spark. Each experiment was performed in triplicate and repeated 3 times.

pH drop assay

Bacterial suspension of OD600 = 0.5 in BHI medium with 0.5% sucrose was treated with different concentrations of THC (2-8 μg/mL) and incubated at 37 °C, 5% CO2. At 0, 2, 4, 6, and 24 hours, the pH of the samples was measured using pH indicator paper strips33 (Fisher Scientific). Untreated cultures served as controls. Each experiment was repeated 3 times.

Membrane potential assay

The assay for bacterial membrane potential (BacLight Bacterial Membrane Potential Kit; Molecular Probes, Invitrogen) utilizes the carbocyanine dye 3,3′-diethyloxacarbocyanine iodides (DiOC2) to indicate membrane polarization following the manufacturer’s protocol and modified by Horst’s approach.34 When dispersed, DiOC2 fluoresces green. In the presence of a polarized membrane, the lipophilic and cationic DiOC2 molecules are transported into the electronegative cytoplasm due to the transmembrane electrochemical gradient. Once accumulated in the cytoplasm, DiOC2 aggregates, shifting its fluorescence from green to red. Therefore, a decrease in the red/green fluorescence ratio signals membrane depolarization. The proton ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which depolarizes membranes, was used as a positive control. Although bacterial membrane potential assays have typically been described for flow cytometry, Horst validated their use in a 96-well microplate format. In our study, we followed this validation process with some modifications.

In the initial optimization experiments (without THC), S mutans and BHI medium were combined to make 200-μL suspensions with final S mutans cell densities of OD600 = 0.003, 0.03, or 0.1. For the negative control, DiOC2(3) was added at final concentrations of 5 μM, 10 μM, and 30 μM per well. For the positive control, a final concentration of 5 μM CCCP was used. Fluorescence was measured hourly over an 8-hour period with green fluorescence (excitation λ = 485 nm; emission λ = 530 nm) and red fluorescence (excitation λ = 485 nm; emission λ = 630 nm).

To test the effects of methanol and THC on membrane potential, the protocol was similar to drug-free optimization experiments, except 100 μL of methanol or THC suspensions was added to the S mutans suspension instead of BHI, resulting in final methanol or THC concentrations of 1 to 8 µg/mL with S mutans at OD600 = 0.03. Fluorescence was measured after 5 minutes to assess indirect interference. Each experiment was performed in triplicate and repeated 3 times.

Statistical analysis

All the experimental results were normalized according to controls and analyzed by analysis of variance and presented as mean ± SEM. P < 0.05 was considered statistically significant.

Results

THC suppressed planktonic S mutans growth and function in vitro

To assess whether THC has any effects on S mutans, an antimicrobial susceptibility test was performed using a plated microdilution assay. THC concentrations over 2 µg/mL were able to inhibit >90% S mutans growth, while 1.5 µg/mL and 1.25 µg/mL THC inhibited 70.53% and 28.65% bacteria growth, respectively (Figure 1A). THC at a concentration lower than 1 µg/mL did not exert a significant effect on S mutans. These results showed that the MIC90 (minimum inhibitory concentration for 90% of organisms tested) of THC was 2 µg/mL.

Figure 1.

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The effect of tetrahydrocannabinol (THC) on planktonic S mutans. (A) Growth inhibition rate (%) of S mutans cells after THC treatment. THC concentrations of 2 μg/mL and above inhibited 90% of S mutans cell growth. The experiment was performed in triplicate and repeated 3 times. AMP, ampicillin. (B) The pH of the culture medium of planktonic growing S mutans in Brain Heart Infusion with increasing concentrations of THC. The experiment was repeated 3 times but no triplicates. **P < .01, ****P < .0001.

To investigate whether THC can inhibit the acidogenic ability of S mutans, we measured the dynamic pH levels in the S mutans culture medium hourly. The results showed that in the control group, the pH value reduced to 4.5 within the first 2 hours, whereas 2 µg/mL THC delayed this effect to 3 hours. As THC concentration increased, the pH decreased at a progressively slower rate. After treatment with 8 µg/mL THC, it took 5 hours for the pH value to drop to 5.5, the critical threshold for enamel demineralization (Figure 1B).

THC inhibited the formation of S mutans biofilm

As S mutans exists as a biofilm intraorally, we further investigated the effect of THC on S mutans biofilm formation. First, the biofilm biomass was quantified using a crystal violet assay. It was found that 2 µg/mL THC was able to inhibit 90% biofilm formation, and 1 µg/mL THC inhibited 87.89% biofilm formation (P < .0001) (Figure 2A, B). Second, we explored if the inhibition of biofilm formation was caused by reducing the viability of S mutans. A live/dead assay was performed to detect the viability of biofilm following a 1- to 8-µg/mL THC treatment. Indeed, THC concentrations over 1 µg/mL can significantly reduce the number of live and dead cells in the biofilm, as well as the EPS. The number of viable cells and the amount of EPS decreased as THC concentration increased (Figure 2C). However, quantitative analysis of the immunofluorescence images showed that the proportion of EPS relative to the ratio of live and dead cells was not correlated with the increase in drug concentration (Figure 3). The fluorescence microscopic observations (Figure 3) were consistent with the TECAN results, showing the S mutans biofilm became sparser with elevated THC concentration and more red (merged cells).

Figure 2.

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The effect of tetrahydrocannabinol on S mutans biofilm formation. (A, B) The crystal violet staining was used to detect the biomass of the S mutans biofilm. (C) The live/dead assay was used to determine the viability of the S mutans biofilm. Relative fluorescent units indicate the value of each treatment group normalized to the solvent control. Those results were determined by the TECAN Spark. Each experiment was performed in triplicate and repeated 3 times. ****P < .0001.

Figure 3.

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The effect of tetrahydrocannabinol (THC) inhibited S mutans biofilm formation, and viability was examined by immunofluorescence. Each experiment was performed twice, and representative images were used. Pie charts show the proportions of integrated fluorescence intensity (IntDen, analyzed with ImageJ) corresponding to PI (red), SYTO 9 (green), and Cascade Blue Dextran (blue) signals. Scale bars represent 20 µm.

THC reduced the viability and metabolic activity of preformed biofilm

To investigate whether cannabinoids can affect the amount of preformed biofilm biomass, we treated the preformed biofilm with 1 to 64 µg/mL THC and detected it by using the crystal violet assay. It was found that 4 to 64 µg/mL THC can reduce the biomass of the biofilm up to 10.43% but not significantly (Figure 4A, B). To verify these data, we further tested the biofilm viability by the live/dead assay. The results showed that THC concentrations of 8 µg/mL, 16 µg/mL, and 32 µg/mL significantly reduced the number of live cells compared to the control group (Figure 4C-F) but did not affect the dead cell number in the S mutans biofilm, demonstrating no significant changes after a 6-hour treatment.

Figure 4.

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The effect of tetrahydrocannabinol (THC) on the preformed S mutans biofilm. (A, B) The crystal violet staining was used to detect the biomass of the S mutans biofilm. (C-F) The live/dead assay was used to determine the viability of the S mutans biofilm after 1, 3, 6, and 24 hours of treatment. (G) The methylthiazolyldiphenyl tetrazolium bromide (MTT) assay was used to detect the metabolic activity of the S mutans biofilm. (H, I) The timeline of the live cells and dead cells after THC treatment for 1, 3, 6, and 24 hours. Those results were determined by the TECAN Spark. Each experiment was performed 3 times in triplicate. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Next, we asked whether THC impacted the metabolic activity of the S mutans biofilm after 6 hours of treatment. The MTT results showed that 16 to 64 µg/mL THC can significantly reduce the metabolic activity of preformed biofilm (Figure 4G). Subsequently, we reanalyzed the data of live/dead staining results using time as the x-axis (Figure 4H, I). The results showed that although the effect of THC can last for 24 hours, the ratio of live cells at 24 hours was higher than at 6 hours, indicating that the maximum impact of THC on S mutans biofilm viability lasts for 6 hours when compared to the control group (Figure 4H). However, the number of dead cells did not show a significant change, except for the 32-µg/mL group after a 24-hour treatment, suggesting that THC may have inhibited cell growth or metabolic activity without directly causing cell death. The images of the live/dead assay were consistent with the results scanned using the TECAN Spark (Figure 5).

Figure 5.

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The effect of tetrahydrocannabinol (THC) on the preformed S mutans biofilm viability. Biofilms were photographed under a fluorescence microscope after live/dead staining. The live cells are shown in green, and the dead cells in red. The figure shows a merged color of green and red, evaluating the viability of S mutans biofilm. At the 1-hour, 3-hour, and 6-hour time points, compared with the methanol group, the biofilm was more yellow and even red in the THC groups, and as the THC concentration increased, the color of the biofilm became redder, indicating a dose-dependent change. In the different concentration groups, as time increased, the color of the biofilm became increasingly red from 1 to 6 hours, while there was no obvious change in the methanol group, illustrating a time-dependent change. In the 24-hour group, the color of the biofilms in the THC groups was not as red as at 6 hours, consistent with the TECAN results. As shown in Figure 4H-I, the live cells increased, and the dead cells did not change much, suggesting that the effect of THC gradually diminished over 6 to 24 hours. Each experiment was performed twice, and representative images were used. Pie charts show the proportions of integrated fluorescence intensity (IntDen, analyzed with ImageJ) corresponding to PI (red) and SYTO 9 (green) signals. Scale bars represent 20 µm.

THC promoted the membrane hyperpolarization of S mutans

To investigate the mechanism of THC’s impact on S mutans, we tested whether THC could alter the membrane potential of S mutans. We tested various S mutans concentrations of OD600 = 0.003, OD600 = 0.03, and OD600 = 0.1 based on prior studies. The fluorescence readings for all 3 concentrations were comparable, and we selected OD600 = 0.03, the manufacturer’s recommended concentration, for subsequent experiments. We found that the DiOC2(3) concentrations of 5 µM, 10 µM, and 30 µM can produce interpretable data. Therefore, we selected 5 µM DiOC2(3) for the following experiments (Figure 6A). The results showed that 2 to 8 µg/mL THC induced membrane hyperpolarization (Figure 6B).

Figure 6.

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The effect of tetrahydrocannabinol (THC) on S mutans membrane potential. (A) Performance and validation of negative (black bars) and positive (gray bars) controls for the membrane potential assays performed simultaneously in a 96-well microplate under harmonized conditions (Brain Heart Infusion medium, cellular density OD600 = 0.03) using S mutans in the absence of cannabinoids. (B) The effect of different concentrations of THC on S mutans after a 5-minute treatment. **P < .01, ****P < .0001.

Discussion

The cannabinoids derived from cannabis have a long history of use as a medicinal agent, particularly for alleviating pain and seizures.10 Studies have shown that cannabinoids such as CBD, cannabinol, CBG, and Cannabichromene exhibit antibacterial properties.17,19,20,22,35 They have demonstrated antibacterial activity against Gram-positive bacteria, including Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and Streptococcus mutans.19,20 However, there is limited work done regarding THC’s antibacterial effects. Van Klingeren et al found that THC inhibits S aureus, S pyogenes, Streptococcus milleri, and Enterococcus faecalis in broth at an MIC ranging from 2 to 5 µg/mL.35

We first reported that THC possesses antibacterial activity against S mutans. Our results showed that ≥2 µg/mL THC was able to inhibit 90% of S mutans growth (Figure 1A). This is consistent with previous reports of other cannabinoids, where 2.5 µg/mL CBG and 5 µg/mL CBD reduced the growth of the cariogenic bacteria S mutans.33,36

Second, THC demonstrated the ability to inhibit S mutans biofilm formation (Figure 2), although the biofilm structure may hinder antimicrobial penetration.37 S mutans biofilm formation was significantly inhibited by over 1 µg/mL THC, and we observed that the biofilm became increasingly sparse as the THC concentration increased. Bacterial EPS is vital to microbial biofilms, aiding bacterial attachment to surfaces and supporting the formation and stability of microcolonies. EPS enhances biofilm resistance to environmental stress and antimicrobial agents, as well as provides nutrients.38 In S mutans, EPS produced by exoenzymes promotes the accumulation of microbes on tooth surfaces, forming a heterogeneous, diffusion-limiting matrix that shields the embedded bacteria.25 In our study, we found that ≥2 µg/mL THC reduced EPS production by over 90%, while 16.7% to 27% of the cells within the biofilm remained viable. Aqawi et al31 also found that CBG decreased EPS production by S mutans.39 However, a sub-MIC concentration of CBD might increase S mutans’ EPS production.40 The effects of these cannabinoid compounds on bacterial EPS production and their potential mechanisms require further investigation. However, in our study, the quantitative analysis of the immunofluorescence images showed that the proportion of EPS relative to the ratio of live and dead cells was not correlated with the increase in drug concentration (Figure 3). This suggests that the reduction in EPS may be due to the decrease in the number of bacteria, rather than a direct inhibitory effect of THC on EPS production.

Third, we found that THC was able to reduce the metabolic activity and viability of the S mutans biofilm, although it could not significantly disrupt preformed biofilms (Figure 4). We have not found any studies that have reported on the effects of cannabinoids on the activities of mature biofilms. After performing live/dead staining on the biofilms, we observed a significant reduction in the number of viable bacteria in the biofilms compared to the growth control group, while the number of dead cells remained unchanged. This suggests that THC at concentrations ≥8 µg/mL may exhibit bacteriostatic effects rather than bactericidal effects. These results indicate that cannabinoids can inhibit the continued growth of preformed biofilms and accelerate the decline in their viability.

It is well known that dental caries is the local degradation of susceptible dental hard tissues caused by acidic by-products from bacterial fermentation of dietary sugars.26 S mutans can reduce the pH below the “critical pH” (pH 5.5) through metabolizing dietary carbohydrates, leading to enamel demineralization and caries formation.41 We found that THC can effectively reduce the rate of pH decline in the S mutans culture medium in a dose-dependent manner (Figure 1B), which delayed attainment of the critical demineralization pH threshold,42 suggesting that THC may reduce the cariogenic potential of S mutans by modulating the acidification process via metabolizing sugars. This is in line with the findings by Barak et al33 with CBD.43

It may be explained by the mechanism of cannabinoids promoting the hyperpolarization of the membrane of S mutans and affecting bacterial viability, ATP synthesis, pH homeostasis, and cell division.44,45 Bacterial membrane potential represents the electrical component of the ion motive force and is essential for multiple cellular functions. Recent studies have demonstrated that membrane potential is highly dynamic and responds rapidly to environmental and physiological stimuli. Fluctuations in membrane potential regulate a broad range of bacterial behaviors, including pH homeostasis, nutrient uptake, motility, antibiotic resistance, cell division, electrical communication, and environmental sensing. Because membrane potential integrates metabolic activity, stress responses, and cell viability, monitoring changes in membrane potential provides an informative readout of how bacteria adapt to antimicrobial compounds.44 Therefore, assessing whether THC can alter the membrane potential of S mutans is critical for understanding its potential antimicrobial mechanisms and determining whether membrane depolarization contributes to its inhibitory effects. Lee et al46 revealed a negative correlation between membrane potential hyperpolarization and bacterial growth rate, as well as cell death. Farha et al47 provided evidence that CBG may exert its bacteriostatic effects by targeting cytoplasmic membrane functions, particularly proteins involved in cellular respiration and the electron transport chain. Some other studies found that CBD and CBG exerted similar effects on S mutans.33,36,40

Taken together, we herein provide evidence of the efficacy of THC in antibacterial and antibiofilm activity against S mutans by reducing planktonic growth of S mutans, inhibiting biofilm formation, and interfering with preexisting biofilm activity and function. These in vitro results may provide some preliminary scientific basis to address the impact of THC on oral health, specifically cannabinoid consumption on cariogenesis. Further studies are needed to explore the role of THC in caries development, especially in marijuana smokers. In addition, it may be a potential new avenue for developing new anticariogenic agents by suppressing the growth of S mutans and decelerating the acidification process that leads to enamel demineralization. However, these potential antimicrobial effects must be interpreted within the broader context of THC’s known adverse health effects, such as its psychoactive property and its association with depression and anxiety.11 These systemic and oral risks limit the clinical applicability of THC itself as an antimicrobial agent. Nevertheless, understanding the mechanistic interactions between THC and oral bacteria remains valuable, as these insights may inform the development of safer, nonpsychoactive cannabinoid derivatives with potential therapeutic utilization.

Author contributions

Haoyan Zhai: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft. Yixuan Zhang: Validation. Minki Kim: Validation. Xintian Zhou: Validation. Joseph Ferracciolo: Methodology. Eric Krukonis: Methodology, Resources, Writing – review and editing. Chunyan Liu: Conceptualization, Funding acquisition, Supervision. Zheng Zhou: Conceptualization, Funding acquisition, Resources, Writing – Reviewing and Editing, Supervision.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This work was funded by the University of Detroit Mercy School of Dentistry Faculty Research Grant #FRG-2019-6 and Natural Science Foundation of Hebei Province #2025206603.

Contributor Information

Chunyan Liu, Email: chunyanliu@hebmu.edu.cn.

Zheng Zhou, Email: zhouzh1@udmercy.edu.

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