Evaluation of the antimicrobial effect of cannabidiol (CBD) in a multispecies subgingival biofilm model
Department of Periodontology, Dental Research Division, Guarulhos University, Guarulhos, SP, Brazil
Departamento de Periodoncia, Facultad de Odontología, Universidad Antonio Nariño, Bogotá, Colombia
Departamento de Ciencias Básicas y Medicina Oral, Facultad de OdontologiaUniversidad Nacional de Colombia, Bogotá, Colombia
Programa Doctorado en Ingeniería, Facultad de Ingeniería, Pontificia Universidad Javeriana, Bogotá, Colombia
Departamento de Ingeniería Industrial, Facultad de Ingeniería, Pontificia Universidad Javeriana, Bogotá, Colombia
Centro de Investigaciones Odontológicas, Facultad de Odontología, Pontifícia Universidad Javeriana, Bogotá, Colombia.
Division of Periodontics, Faculdade São Leopoldo Mandic, Instituto e Centro de Pesquisas São Leopoldo Mandic, Campinas, Brazil
Departamento de Biociências, Faculdade de Odontologia de Piracicaba, Universidade de Campinas, Piracicaba, Brasil
ABSTRACT
Background
This study evaluated the antimicrobial effect of cannabidiol (CBD) on a multi-species subgingival biofilm model.
Materials and methods
Biofilms were formed using 33 bacterial species on a Calgary device. Two protocols were tested: (A) biofilm in contact with CBD (125, 250 and 500 µg/mL) and chlorhexidine 0.12% (CHX) for the entire period; (B) treatments with CBD (500 and 1000 µg/mL) and CHX started on day 3, twice a day, for 1 minute. The total biofilm counts, the proportion of complexes, and the counts of each species were evaluated by DNA-DNA hybridization (Checkerboard).
Results
In Experiment A, CBD at concentrations of 250 and 500 µg/mL, as well as CHX, significantly reduced the total biofilm count. At 500 µg/mL, CBD also decreased the proportion of the red complex and reduced the counts of 10 bacterial species, whereas CHX affected 20 species. In Protocol B, both CBD at 1000 µg/mL and CHX reduced the total biofilm count and the proportion of the red complex, while increasing the proportion of the green complex. Both protocols led to a reduction in Porphyromonas gingivalis and Tannerella forsythia.
Conclusion
CBD reduced the total bacterial count and the red complex, inhibiting known periodontal pathogens. Within the limitations, the results provide exploratory evidence that CBD may reduce the total bacterial count in the proposed polymicrobial biofilm model, including the red complex bacteria, and may thus be postulated as an inhibitor of known periodontal pathogens. However, future in vivo studies with robust sample sizes and standardized CFU-based quantification are required to confirm these findings.
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KEYWORDS: Multispecies biofilm, periodontal disease, natural agents, cannabidiol, antimicrobial
Article notes
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Received 2025 Sep 6; Accepted 2025 Dec 9; Collection date 2026.
Introduction
Dental biofilm is a complex, structured community of microorganisms embedded in an extracellular matrix that provides enhanced resistance to adverse environmental conditions, which free-floating (planktonic) bacteria typically cannot withstand. These conditions include nutrient scarcity, fluctuations in pH and oxygen levels, and resistance to chemical and mechanical biofilm control methods [1].
Biofilm formation begins with the adhesion of initial colonisers to the acquired pellicle. These early colonisers primarily consist of bacteria from the purple, yellow, and green complexes, as classified by Socransky et al. [2], and are generally associated with periodontal health. Subsequently, secondary colonisers from the orange complex integrate into the developing biofilm, acting as bridge organisms that facilitate the attachment of late colonisers. These late colonisers, comprising the red complex, are strongly associated with the pathogenesis of periodontal disease and are linked to poorer clinical periodontal outcomes [2,3].
Gingivitis and periodontitis are the most common periodontal diseases associated with dental biofilm [4–7]. They typically result from dysbiosis—an imbalance between the microbial communities on the tooth surface and the host immune response [8,9]. Nonsurgical periodontal treatment, which includes mechanical biofilm control, scaling, root planning (both supra- and subgingival), and patient follow-up, remains essential for disease management. These measures aim to reduce chronic inflammation and lower the bacterial load on contaminated surfaces, thereby helping to control disease progression [10]. However, it is noted that not all patients respond adequately to conventional therapies [11]. This observation highlights the need to explore adjunctive protocols that can enhance the effectiveness of standard periodontal treatments.
Chlorhexidine remains the gold standard for chemical control of dental biofilm [12]. However, its chronic use is associated with potential side effects [13–15]. Considering this, natural products have attracted attention for their antimicrobial and anti-inflammatory properties, often accompanied by minimal side effects, reduced bacterial resistance, and improved patient compliance [16,17]. Réndon et al. [18] specifically noted that cannabinoid compounds may serve as effective adjuncts in the treatment of periodontal disease. Additionally, studies suggest that cannabidiol compounds may modulate the immune system by suppressing pro-inflammatory cytokines linked to elevated bone resorption in periodontal disease [19].
Cannabis sativa contains a wide range of pharmacologically active compounds [20,21], among which delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are the most extensively studied for their therapeutic applications [22]. Other constituents, such as cannabigerol (CBG), also exhibit relevant pharmacological properties, including antioxidant, neuroprotective, anti-inflammatory, and antimicrobial activities, particularly against bacteria and fungi [23,24].
Regarding antimicrobial potential, direct evidence on the effect of cannabis derivatives on the oral microbiota is scarce but promising [25]; recent studies highlight the role of cannabinoids not only in changes in the Alpha diversity of the gut microbiome—evidence that underscores the need to evaluate the potential effects of cannabinoids not only on microbiome composition but also on the metabolic activity of bacteria [26]—especially in light of representative findings such as the fact that (intestinal) bacteria can modulate the endocannabinoid system through mechanisms dependent on different bacterial virulence factors like LPS, which highlights the potential to further intervene in host-microbiome relationships [27].
CBD has demonstrated activity against a variety of microorganisms, including bacteria and fungi [28–30]. These findings highlight the promise of CBD and other cannabinoids in targeting dental biofilm and achieving relevant antimicrobial effects. The present study was designed to test the hypothesis that cannabidiol has antimicrobial efficacy against bacteria associated with periodontal disease in a multi-species biofilm model. Specifically, it will investigate the ability of cannabidiol to reduce overall biofilm formation and suppress key pathogenic microorganisms, comparing its effectiveness with 0.12% chlorhexidine (CHX).
Material and methods
In vitro multispecies biofilm formation
In the laboratory, biofilm samples containing 33 bacterial species were prepared according to the technique recommended by Figueiredo et al. [31]. The chosen species are those most related to periodontal diseases, containing strains of Actinomyces gerencseriae (ATCC23860), Actinomyces israelii (ATCC12102), Actinomyces naeslundii (ATCC12104), Actinomyces oris (ATCC43146), Actinomyces odontolyticus (ATCC17929), Veillonella párvula (ATCC10790), Streptococcus gordonii (ATCC10558), Streptococcus intermedius (ATCC273 35), Streptococcus mitis (ATCC49456), Streptococcus oralis (ATCC35037), Streptococcus sanguinis (ATCC10556), Streptococcus anginosus (ATCC33397), Streptococcus mutans (ATCC25175), Aggregatibacter actinomycetemcomitans (ATCC29523), Capnocytophaga gingivalis (ATCC33624), Capnocytophaga ochracea (ATCC33596), Capnocytophaga sputigena (ATCC33612), Eikenella corrodens (ATCC23834), Campylobacter gracilis (ATCC33236), Campylobacter showae (ATCC51146), Eubacterium nodatum (ATCC33099), Eubacterium saburreum (ATCC33271), Fusobacterium nucleatum subsp. polymorphum (ATCC10953), Fusobacterium nucleatum subsp. vincentii (ATCC49256), Fusobacterium periodonticum (ATCC33693), Parvimonas micra (ATCC33270), Prevotella intermedia (ATCC25611), Streptococcus constellatus (ATCC27823), Tannerella forsythia (ATCC43037), Porphyromonas gingivalis (ATCC33277), Gemella morbillorum (ATCC27824), Propionibacterium acnes (ATCC11827), Selenomonas noxia (ATCC43541).
Tryptone Soy Agar (Scharlau, Barcelona, Spain) supplemented with 5% sheep blood was used to cultivate most species under anaerobic conditions, consisting of 85% nitrogen, 10% carbon dioxide, and 5% hydrogen. Porphyromonas gingivalis was grown on tryptone soy agar containing yeast extract, enriched with 1% hemin, 5% menadione, and 5% sheep blood. Tannerella forsythia was cultured on tryptone soy agar with yeast extract, enriched with 1% hemin, 5% menadione, 5% sheep blood, and 1% N-acetylmuramic acid. All species were incubated on agar plates for 24 hours and then transferred to glass tubes containing Brain Heart Infusion (BHI) medium (Becton Dickinson, Sparks, MD, USA), supplemented with 1% hemin. After 24 hours of growth in conical tubes, the optical density was adjusted so that the inoculum of each species contained approximately 10⁸ cells/mL. The cell suspensions were diluted, and 100 μL aliquots containing 10⁶ cells of each species were added to 11,700 μL of BHI broth supplemented with 1% hemin and 5% sheep blood, resulting in a final inoculum volume of 15 mL [32].
The multispecies biofilm model was developed using a Calgary Biofilm Device in a 96-well plate (Nunc; Thermo Scientific, Roskilde, Denmark). A 150 μL aliquot of each inoculum was added to the wells, corresponding to approximately 1 × 10⁴ cells of each bacterial strain, except for Porphyromonas gingivalis and Prevotella intermedia, where the inoculum was adjusted to 2 × 10⁴ cells. A lid with polystyrene pins was used to seal the 96-well plate (Nunc TSP system; Thermo Scientific, Roskilde, Denmark). The plates with lids that have pins were incubated at 37 °C under anaerobic conditions [33].
Preparation of CBD
CBD (99% purity), characterised by high-performance liquid chromatography, was purchased from Avicanna (Toronto, ON, Canada). Stock solutions were prepared in DMSO at 0.01% (Thermo Scientific™, Waltham, MA, USA), diluted to the desired concentrations, and stored at 4 °C under light-protected conditions.
Sample treatment
The experimental design was divided into two treatment schemes: Protocol A and Protocol B [25]. In protocol A, CBD was administered to the biofilm from the very start of the experiment. In protocol B, bacteria were allowed to form an initial biofilm for 72 hours before treatments were initiated. Treatments were performed twice daily, with each treatment lasting 1 minute. Protocol A used CBD concentrations of 125, 250, and 500 µg/mL, while protocol B used 500 and 1000 µg/mL. These concentrations were chosen based on previous results showing antimicrobial data over the same concentrations tested here [25]. Because protocol B allows initial biofilm formation and treatments were performed for only 1 minute, we considered that a higher concentration should be used due to the greater challenge (a pre-formed biofilm and shorter treatment time compared with protocol A). The positive control was 0.12% chlorhexidine, a well-recognised gold standard, as described in the Introduction, while biofilms treated with culture medium served as the negative control. At the end of the treatment period, the pins containing the biofilms were collected, and the biofilms were analysed by DNA-DNA hybridisation. Three independent experiments were conducted, each in duplicate.
DNA-DNA hybridisation (checkerboard DNA-DNA)
Three 7-day biofilm-coated pins from each group were transferred to Eppendorf tubes with 150 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.6), followed by the addition of 100 μL of 0.5 M NaOH. These tubes, containing both the pins and solution, were then boiled for 10 minutes. The solution was neutralised by adding 0.8 mL of 5 M ammonium. Each sample was individually analysed to determine the presence and quantity of 33 bacterial species through DNA-DNA hybridisation. Briefly, after lysis, the DNA was transferred onto a nylon membrane using a Minislot apparatus (Immunetics, Cambridge, MA). Once the DNA was attached to the membrane, it was placed in a Miniblotter 45 (Immunetics), and digoxigenin-labelled DNA probes for the full genome of each subgingival species were hybridised onto individual lanes. Following hybridisation, the membranes were washed, and DNA probes were identified using a digoxigenin-specific antibody conjugated to alkaline phosphatase. Signals were detected with AttoPhos substrate (Amersham Life Sciences, Arlington Heights, IL) and visualised with the Typhoon Trio Plus (Molecular Dynamics, Sunnyvale, CA). Each run included two lanes with standards containing 105 and 106 cells per species. The signals from the Typhoon Trio were converted to absolute counts by comparing the observed patterns on the same membrane. The absence of a signal was recorded as zero. Counts below the method detection limit (1 × 104) were recorded as zero for calculating the mean counts of individual bacterial species [34]. Based on the semi-quantitative data obtained, three types of analyses were performed: total biofilm counts, proportions of microbial complexes, and counts of individual bacterial species [31].
Total biofilm counts were determined by summing the values of all bacterial species detected in each biofilm formed on a pin (Figures 1 and 4). For the analysis of microbial complexes, the counts of bacterial species assigned to each Socransky complex were combined, and their relative proportions were calculated and presented as pie charts (Figures 2 and 5). Finally, the counts of individual bacterial species were obtained from the checkerboard assay results and used to construct species-specific graphs (Figures 3 and 6).
Statistical analysis
The data were analysed without any transformation. Since the normality test indicated non-parametric data, statistical differences among all groups within each specific analysis, as presented in the figures, were evaluated using the Kruskal–Wallis test followed by Dunn’s post hoc test (p ≤ 0.05). The Jamovi 2.3.28 (Jamovi Research, Vienna, Austria) was used for the analyses.
Results
Figure 1 shows the total biofilm counts after one week of treatment (experimental design A) with cannabidiol (CBD) at concentrations of 125, 250, and 500 µg/mL, as well as with 0.12% chlorhexidine (CHX). None of the CBD concentrations produced a statistically significant reduction compared with the negative control (CONTROL; p > 0.05), although treatments with 250 and 500 µg/mL CBD achieved an approximate 50% numerical reduction. In contrast, CHX induced a statistically significant decrease in total biofilm counts compared to the control (p ≤ 0.05).
Figure 2 illustrates the proportions of microbial complexes within biofilms treated with CBD at concentrations of 125, 250, and 500 µg/mL, as well as with chlorhexidine (CHX) following protocol A. A significant reduction in the red complex was observed after treatment with both 500 µg/mL CBD and CHX (p ≤ 0.05). No statistically significant differences were found for other microbial complexes across any treatments (p > 0.05).
Since CBD 250 and 125 did not show statistical significance to the control in the two previous analyses, both concentrations were removed from the next assay. In this way, Figure 3 presents the counts of individual bacterial species within the multispecies biofilm. Treatment with CBD500 significantly reduced the counts of 10 species, whereas CHX reduced those of 20 species (p ≤ 0.05), compared with the control. Both treatments exhibited inhibitory effects on 23 species, with notable reductions observed for Porphyromonas gingivalis, Tannerella forsythia, Parvimonas micra, Fusobacterium periodonticum, and Eubacterium saburreum.
Figure 4 shows the results of the total reduction of biofilm bacteria after treatment with CBD1000 and CHX (two daily treatments for five days/seven days—experimental design B). For CBD at a concentration of 500 µg/mL, no statistically significant differences were observed (p > 0,05). In relation to CBD 1000, statistically significant differences were observed in the total reduction of bacteria, as well as for the group treated with CHX, which drastically reduced total counts compared to the control (p < 0.05).
Figure 5 illustrates the proportions of microbial complexes in biofilms treated with CBD at 1000 µg/mL and with CHX (two daily treatments). Both CBD1000 and CHX significantly reduced the proportion of red complex bacteria (p ≤ 0.05). In addition, both treatments led to a significant increase in the proportion of green complex bacteria, associated with periodontal health, compared to the control (p ≤ 0.05).
Figure 6 presents the counts of individual bacterial species within the multispecies biofilm. Treatment with CBD1000 significantly reduced the counts of nine species, whereas CHX reduced those of 17 species (p ≤ 0.05) compared to the control. Both treatments exhibited inhibitory effects on 19 species, notably Porphyromonas gingivalis, Tannerella forsythia, Eubacterium notatum, Eubacterium saburreum, and Selenomonas noxia.
Discussion
Periodontal diseases are multifactorial, primarily resulting from an imbalance (dysbiosis) between pathogenic bacteria within dental biofilm and the patient’s immune system [35]. Initially, "beneficial" bacteria, predominantly Gram-positive aerobic species, dominate this biofilm. However, as periodontal disease progresses, there is a shift towards Gram-negative anaerobic species, such as P. gingivalis and T. forsythia, both recognised periodontopathogens [36]. This shift in microbial composition underlies disease pathogenesis and results in clinical manifestations that, if left untreated, can progress to irreversible outcomes, including alveolar bone loss, clinical attachment loss, and, in advanced stages, tooth loss [37].
This understanding highlights the role of specific microbial groups in the health-disease process, emphasising the importance of interventions that target pathogenic bacteria within periodontal treatment protocols. Given the high invasive potential of these pathogens in periodontal tissues, traditional therapeutic protocols may sometimes prove insufficient, particularly over the medium and long term. Consequently, there is a growing need to explore adjunctive therapies that can help stabilise periodontal disease [36,38].
In this context, compounds from Cannabis sativa have demonstrated promising anti-inflammatory and antimicrobial properties [18,39,40]. CBD modulates inflammation by reducing the production of cytokines and other inflammatory mediators, primarily through CB2 receptor activation. Regarding its antimicrobial activity, CBD has shown efficacy against highly resistant bacteria, such as Staphylococcus aureus [41], as well as other pathogens, including Candida albicans [42]. Moreover, recent studies have reported that both CBD and cannabigerol (CBG) are effective when formulated in a mouthwash [43].
In this context, several studies have investigated the antimicrobial potential of natural products, including propolis [44,45], Aloe vera [46–48], green tea [49–51], cranberry [52–55], and calendula [56,57]. These compounds have been shown to reduce the abundance of species associated with periodontal diseases, such as Tannerella forsythia, in multispecies biofilm models similar to the one used in this study [17]. Additionally, a commercial formulation containing Aloe vera, propolis extract, green tea, cranberry, and calendula has demonstrated comparable antimicrobial effects.
This study assessed two chemical therapy protocols for their effectiveness in reducing total bacterial counts, microbial complexes, and specific bacterial species. In experiment A, the antimicrobial agents CBD at 500 µg/mL and CHX (positive control) were applied at the onset of biofilm formation, simulating potential clinical use immediately following scaling and root planing. Both treatments reduced total bacterial counts to similar levels and demonstrated activity against key periodontopathogens, such as P. gingivalis and T. forsythia, although CHX affected a greater number of bacterial species than CBD.
In experiment B, treatment began on day three, after biofilm maturation, and was applied twice daily for 1 minute over 7 days. A higher concentration of CBD (1000 µg/mL) was tested alongside CHX. Both treatments targeted P. gingivalis and T. forsythia, with CHX showing broader efficacy against periodontal pathogens. CBD1000 demonstrated a tendency to preserve bacterial species commonly associated with periodontal health within the multispecies biofilm model. This pattern suggests that the compound may promote a selective reduction of pathogenic microorganisms while maintaining those beneficial to the oral microbiota. Consequently, these preliminary findings point to the possible development of therapeutic strategies focused on restoring microbial balance (rebiosis) in dysbiotic communities related to oral infectious and inflammatory diseases. However, given the small sample size, limited number of replicates, and the in vitro setting, these shifts must be interpreted cautiously and cannot be overgeneralized to the clinical microbiome.
In contrast, when CBD500 was used in experiment B protocol, no statistically significant differences in total bacterial counts were observed compared to the control group. This outcome may be attributed to the delayed initiation of treatment, as CBD500 was applied after biofilm maturation. The difference observed in experiment A, where CBD500 was applied at the onset of biofilm formation, may be attributed to the increased resistance of mature biofilms to antimicrobial agents. Mature biofilms are well-known for their reduced susceptibility and penetration, which likely limited the efficacy of CBD500 when applied later in the biofilm development process.
Across both experimental protocols, CBD displayed predominantly bacteriostatic activity rather than a bactericidal effect, as complete eradication of bacterial species counts was not achieved for most species, except for P. intermedia in Protocol B. This outcome implies that the minimum inhibitory concentration (MIC) of CBD for P. intermedia is probably below 500 µg/mL (concentration applied in Protocol B) while its minimum bactericidal concentration (MBC) may range between 500 and 1000 µg/mL.
A recent in vitro study also investigated the anti-inflammatory properties of CBD and other cannabis-derived compounds. Furthermore, an increase in gamma interferon (IFN-γ) levels was noted, a cytokine that plays a critical role in the host's immune response to infectious processes [32].
Within the limitations of this study, one key factor is the number of species evaluated (33 species). While these species are representative of periodontal diseases, many others that may play an important role were not included in the experimental model, for example, Treponema denticola, a microorganism belonging to the red complex described by Socransky et al. [2]. T. denticola produces proteolytic enzymes, including collagenase, which can degrade connective tissue and impair the host's immune response, making the disease more complex from a management perspective. Since the Socransky checkerboard method was first introduced approximately three decades ago [34], newer approaches such as next-generation sequencing have provided a far more comprehensive and precise characterisation of the subgingival microbiome. Although the checkerboard technique allows the simultaneous assessment of multiple microbial species across numerous samples and remains suitable for in vitro studies in which the bacterial species are predetermined, its scope is inherently limited. In contrast, sequencing platforms capture the full breadth of microbial diversity, including low-abundance taxa, and therefore should be considered the preferred methodology for clinical trials and ecological investigations [58]. Moreover, the lack of colony-forming unit (CFU) quantification represents a potential limitation of this study. Nevertheless, obtaining reliable CFU counts proved difficult because our experimental model included multiple bacterial species. When these organisms are cultured together on agar plates, their colonies, each with distinct shapes and growth patterns, often merge, making accurate enumeration unreliable.
Another limitation pertains to future clinical applications, particularly in experiment A, where CBD500 was in contact with the biofilm from the moment it was formed. For such a treatment to be feasible in an oral environment, the product could be incorporated into gels and used continuously by the patient through devices like custom trays. Although these in vitro results are promising, translation from the bench to the clinic requires prior animal and human studies to assess potential toxicity or other adverse effects before clinical implementation, along with determining the dose related to the rebiosis process, timing, and action in the presence of inflammation. Nevertheless, the results support the apparently selective antimicrobial effect of CBD.
The primary contribution of this study is that, to our knowledge, it is the first to demonstrate a comparable antimicrobial effect of CBD and CHX in a complex multispecies experimental model comprising 33 species associated with periodontal disease. Notwithstanding the preliminary nature and in vitro origin of the study, the results are particularly encouraging regarding CBD's therapeutic potential. Although the anti-inflammatory potential of CBD has been proven in other models, the preliminary findings of its antimicrobial effects against oral bacteria, coupled with subsequent studies on its anti-inflammatory effects in the context of oral inflammation, could ultimately lead to future applications in the clinic. It is necessary to test the potential clinical benefits that can be obtained with CBD through its impact via CB1 and CB2 receptor modulation.
Conclusion
These exploratory observations demonstrated a notable antimicrobial activity of CBD by reducing red complex bacteria and key periodontopathogens, including Porphyromonas gingivalis and Tannerella forsythia, in a multispecies subgingival biofilm model, comparable to CHX. However, the therapeutic application protocol may influence its efficacy. Drawing from the preceding results, several hypotheses arise concerning the antimicrobial potential of CBD across various oral cavity micronishes, and the extent to which their conditions may influence its therapeutic efficacy.
Funding Statement
Funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.
Disclosure statement
None of the authors has any potential conflict of interest.
References
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