Combining Diclofenac and Cannabidiol to Enhance the Antibacterial Capacity of Nonantibiotics Drugs Through Potentiation

1. Introduction

Rapidly increasing antimicrobial resistance (AMR) is a significant challenge as bacteria evolve mechanisms to evade therapeutic agents, complicating the management of viral, bacterial, parasitic, and fungal diseases, jeopardising public health systems, exacerbating social inequalities, especially in resource-limited areas, impacting the health of animals and plants, and endangering food security [1]. Since AMR-related mortality is projected to rise to over 39 million between 2025 and 2050, the economic implications are enormous, with additional healthcare costs projected at US$1 trillion by 2050 and GDP losses up to US$3.4 trillion annually by 2030 [2,3]. The 2022 Global Antimicrobial Resistance and Use Surveillance System (GLASS) highlighted alarming levels of resistance among bacterial pathogens, including E. coli and Staphylococcus aureus [4]. Leveraging plants that produce secondary metabolites could provide innovative antibiotics to combat this resistance [5]. Thus, exploring natural products [6] and drug repurposing [7] could support the strategies to tackle the pressing threat of multidrug resistance. In this context, some of the FDA-approved nonantibiotic medication classes, including nonsteroidal anti-inflammatory drugs (NSAIDs), antidepressants, antiplatelets, antipsychotics, and statins, have antibacterial effects [8,9] against both Gram-positive and Gram-negative bacteria [10,11,12] and can play essential parts.

Diclofenac sodium (DFNAC), a potent nonsteroidal anti-inflammatory agent, also exhibits antibacterial activity [13] and has various applications [14]. DFNAC alone exhibited activity against drug-sensitive and drug-resistant clinical isolates of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Mycobacterium spp. [15]. Also, combined with streptomycin [15], curcumin [16], gentamicin or ampicillin [17], streptomycin [18] has potential for the management of problematic antibiotic-resistant Gram-positive and Gram-negative bacterial infections. Furthermore, DFNAC-kanamycin and DFNAC-tetracycline demonstrated additive interactions and promising control of the Escherichia coli and Staphylococcus aureus biofilms [19]. Notably, synergistic effects have been observed between DFNAC and echinocandin anidulafungin on the tested biofilms [20], DFNAC and Pelargonium graveolens against C. parapsilosis [21], and DFNAC and Colistin against Multidrug-Resistant Acinetobacter baumannii [22]. DFNAC, in combination with β-lactams, inhibits MRSA-associated biofilm formation on implants [23] and can be used in combination with antibiotics as an anti-virulence agent against MDR-MRSA [24]. However, no potentiation was observed when DFNAC was tested as an adjuvant to antibiotic therapy (cefuroxime, chloramphenicol) against 10 strains of S. aureus [25].

Meantime, the discovery of phyto-cannabinoids and the identification of their chemical structure, characterised by a lipid structure featuring alkyl-resorcinol and monoterpene moieties, led to the exploration of the endocannabinoid system as the main pharmacological target for cannabinoids [26,27]. Despite ongoing debates about the legal status of Cannabis, there is evidence for its useful therapeutic applications in modern medicine, from nausea [28], pain [29,30] Parkinson’s disease and multiple sclerosis-related symptoms [31,32], to sleep disorders [33] and antibacterial and antifilm activities against a broad spectrum of Gram-positive bacteria and some Gram-negative bacteria, including multidrug-resistant (MDR) strains [27,34]. Therefore, distinct Cannabis-derived molecules expand the repertoire of antibiotic-independent treatment options [35]. For instance, CBD-polymyxin B exerted an antimicrobial effect against E. coli [36], ultrapure CBD-polymyxin B showed potentiated antibacterial activity against Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii, including four antibiotic-resistant K. pneumoniae isolates [37], CBD-ampicillin and CBD-polymyxin B showed potential synergism on S. Typhimurium [38] and CBD-colistin on A. baumannii [27,34], and additive and/or synergistic effect against LOS-expressing Gram-negative diplococci; however, no potential synergism between CBD and kanamycin [27,34]. While some studies recognised CBD as a powerful, low-resistance-inducing antibiotic against Gram-positive bacteria, others expressed caution due to inconsistencies in bacterial sensitivity and limitations in treating Gram-negative infections [27,34,39] Since dosage is crucial when therapeutic windows are narrow, potentiation has been considered, and “drug repurposing” [11] leverages synergism with nonantibiotics to extend the spectrum.

The present work explored the combination of DFNAC and CBD and their potential as an alternative antibiotic-independent treatment and prevention strategy in the combat of bacterial infections caused by Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. With acceptable biocompatibility and potentiation of antibiotic activity through complementary MOAs, the combination of DFNAC and CBD shows potential for further studies and targeted applications.

2. Results

The proposed formulation combining DFNAC and CBD has been evaluated for its antimicrobial effects against Gram-positive and Gram-negative bacteria representative as well as its biocompatibility.

2.1. The Antibacterial Activity of DFNAC and CBD Formulation

2.1.1. The Antimicrobial Effect Against Gram-Positive Bacteria

The antibacterial activity of CBD, DFNAC, and their combination was evaluated against Staphylococcus aureus and Staphylococcus epidermidis using broth microdilution and checkerboard assays (Figure 1).

For S. aureus, CBD alone exhibited antibacterial activity with a MIC of 3.906 µg/mL (Figure 1A), while DFNAC showed inhibitory effects only at high concentrations (MIC = 500 µg/mL; Figure 1B). The combination of CBD and DFNAC (Figure 1C) resulted in a modest improvement in antibacterial activity, reflected by reduced optical density values at lower concentrations. The checkerboard analysis (Figure 1G) confirmed an additive interaction, with a FICI value of approximately 0.5, indicating partial cooperation between the two compounds without true synergism.

In contrast, S. epidermidis displayed a different susceptibility profile. CBD alone showed a higher MIC (15.625 µg/mL; Figure 1D), while DFNAC again exhibited weak antibacterial activity (Figure 1E). However, the combination (Figure 1F) significantly enhanced bacterial inhibition, reducing the effective concentration required to achieve complete growth suppression. The checkerboard heatmap (Figure 1H) clearly demonstrates a shift toward lower OD values across combined concentrations, with the MIC of the combination identified at 3.906 µg/mL for both compounds. The calculated FICI value of 0.258 indicates a strong synergistic interaction, corresponding to a 4-fold reduction in the CBD MIC. These results highlight a strain-dependent response to the DFNAC – CBD combination. While S. aureus showed only additive effects, S. epidermidis exhibited pronounced synergism, suggesting differences in cell envelope structure, membrane permeability, or metabolic sensitivity between the two species. The enhanced efficacy observed in S. epidermidis may result from complementary mechanisms of action, including CBD-induced membrane disruption combined with DFNAC-mediated interference with bacterial metabolic processes.

Overall, the data demonstrate that the DFNAC– CBD combination is particularly effective against S. epidermidis, with synergistic activity, whereas only additive effects are observed against S. aureus. The result highlights the potential of such combinations for selectively targeting Gram-positive pathogens.

2.1.2. The Antimicrobial Effect Against Gram-Negative Bacteria

Both E. coli and P. aeruginosa exhibited resistance to CBD and the DFNAC–CBD combination, with no MIC reached and no evidence of synergistic interaction (Figure 2). Indeed, markedly different behaviour was observed for Pseudomonas aeruginosa and E. coli, in which neither CBD alone nor the DFNAC–CBD combination achieved a minimum inhibitory concentration within the tested range. Consequently, FICI values could not be determined. These results indicate a lack of antibacterial efficacy and absence of synergistic interaction against this strain. The intrinsic resistance of P. aeruginosa is well documented. It can be attributed to its highly impermeable outer membrane, efficient efflux pump systems, and adaptive resistance mechanisms, which collectively limit the penetration and activity of many antimicrobial compounds.

Overall, these findings demonstrate that the effectiveness of the DFNAC–CBD combination is strongly dependent on bacterial species. The observed synergistic effect against S. epidermidis, combined with additive activity against S. aureus and lack of efficacy against P. aeruginosa, underscores the importance of considering bacterial physiology and resistance mechanisms when evaluating combination therapies. This strain-specific response suggests that such combinations may be particularly valuable against susceptible Gram-positive pathogens, while alternative strategies are required to target inherently resistant Gram-negative bacteria.

2.2. The Biocompatibility of DFNAC-CBD Formulation

The cytotoxic effects of the DFNAC–CBD combination (3.906 µg/mL) on HDF cells were further evaluated using the LDH assay, which reflects cell membrane integrity. As shown in Figure 3, all tested samples exhibited low LDH release than the positive control, indicating minimal membrane damage. The DFNAC–CBD combination induced only a slight increase in LDH release relative to the negative control, suggesting limited cytotoxicity. Importantly, the LDH levels remained significantly lower than those observed for the positive control, confirming that the treatment does not cause substantial cell lysis or membrane disruption.

The overall trend observed in the LDH assay is consistent with the MTT results, supporting the conclusion that the tested concentration maintains high cell viability while inducing only minor cytotoxic effects. The result indicates that the antibacterial MIC concentration does not compromise cell membrane integrity to a biologically significant extent. Taken together, the low LDH release and preserved metabolic activity demonstrate that the DFNAC–CBD combination exhibits good cytocompatibility with HDF cells, reinforcing its potential for biomedical applications that require both antimicrobial efficacy and host cell safety.

3. Discussion

The rise of antibiotic resistance in bacterial pathogens is reducing the effectiveness of essential antibiotics used in invasive surgery and chemotherapy, complicating treatment for normally manageable diseases. To address this, developing new antibacterials and identifying molecules that enhance the efficacy of existing antibiotics are critical priorities.

This study evaluated the antimicrobial responses of various bacterial strains, particularly focusing on the potential synergistic effects of DFNAC and CBD while considering drug repurposing [40] and previous research [41,42]. Some findings suggest that CBD acts as a “potentiator,” improving standard antibiotics’ effectiveness against resistant strains, while others indicate it may be an effective standalone antibacterial agent for topical use [43]. Other studies highlight DFNAC’s notable antibacterial and anti-inflammatory effects [44]. Our results demonstrated the synergistic interaction against Staphylococcus epidermidis, when combining DFNAC and CBD, significantly enhancing antibacterial efficacy and substantially reducing the minimum inhibitory concentration (MIC). Conversely, when tested against Staphylococcus aureus, the combination exhibited an additive effect, indicating partial cooperation between DFNAC and CBD.

Indeed, research has been directed at identifying antimicrobial candidates to combat the rising antibiotic resistance in Gram-positive bacteria. Given the serious infections caused by Staphylococcus aureus, investigating natural products like CBD [36,45,46] is a logical step, and further evidence of in vivo efficacy is warranted [47,48]. However, pharmacodynamic studies are required to assess drug-drug interactions for a continuous evaluation of CBD’s benefits [42,43].

The question revolves around its MOA – whether it disrupts the bacterial membranes or targets specific metabolic pathways – raising issue of whether it is bactericidal or bacteriostatic. The proposed mechanism for DFNAC-CBD combination, particularly against Gram-positive bacteria, suggests complementary mechanisms leading to enhanced bacterial damage and growth inhibition: CBD primarily disrupts bacterial membrane integrity, while DFNAC interferes with intracellular metabolic processes. Previous studies also indicated different mechanisms when NSAIDs are paired with antibiotics against S. epidermidis and S. aureus. DFNAC c sodium against S. epidermidis, indicated a high affinity for S. epidermidis proteins and TcaR enzymes and suggested molecular docking [44]. DFNAC combined with Gentamicin against S. aureus showed a synergistic effect (FIC index 0.38) [15], suggesting efflux pump inhibition. Conversely, Ketorolac combined with Gentamicin against S. aureus biofilms demonstrated synergistic activity against S. aureus biofilms (FIC index 0.31), likely through modulation of the biofilm matrix [49].

Despite showing weak to moderate direct antibacterial activity (MICs ranging from 32 to 512 µg/mL), based on the Checkerboard assays - the primary method for testing synergistic activity – NSAIDs exhibited synergistic effects with various antibiotics like Gentamicin, Ciprofloxacin, Oxacillin, Chloramphenicol, Cefuroxime, and Tetracycline (FIC indices generally < 0.5 to 0.625). Efflux pump inhibition is frequently proposed as MOA [40]. Moving forward, more molecular studies are essential to validate these findings and explore the potential of NSAIDs as modulators or antibacterial adjuvants. However, challenges remain, such as achieving effective concentrations without toxicity and confirming mechanisms to ensure pharmacokinetic and pharmacodynamic compatibility, specificity and avoid off-target effects. Furthermore, our exploratory evidence indicates that the combination of DFNAC and CBD may become a significant antimicrobial pairing, aligning with the ongoing efforts to refine applications [50,51,52] and establish a regulatory framework [53] for future pharmacological products. It also aims to address methodological inconsistencies in standardised testing methods, culture media, and CBD sources.

Notably, we found no antibacterial activity against the Gram-negative strains Escherichia coli and Pseudomonas aeruginosa, which reflects their intrinsic resistance and highlights the combination’s inability to overcome the permeability barrier of their outer membranes. While it is widely accepted that CBD is highly effective against Gram-positive bacteria, including MRSA, there is a debate about its efficacy against Gram-negative bacteria. Many studies support the view that CBD is inactive against Gram-negative bacteria due to their protective outer membrane. However, a conflicting hypothesis suggests that certain Gram-negative bacteria—specifically Neisseria gonorrhoeae, Neisseria meningitidis, and Legionella pneumophila—are highly sensitive to CBD. This observation raises the question of whether CBD is a niche Gram-positive agent or a broad-spectrum candidate with tailored applications [27,34,54,55]. In the meantime, dose–response studies against E. coli and S. aureus indicated that DFNAC reduced metabolic activity without removing biofilm mass at therapeutic concentrations, resulting in significant reductions in colony-forming units within the biofilm as confirmed by Scanning Electron Microscopy [56]. However, the bactericidal effect against Gram-negative and Gram-positive bacteria – evident through inhibition of biofilm formation or reduction of metabolic viability – is limited at clinically relevant concentrations with standardised MBIC or MBEC values missing. The minimum bactericidal concentrations (MBCs) were consistently above 2000 µg/mL, indicating limited bactericidal potential [40]. Therefore, direct and consistent data on membrane disruption as well as in vivo or toxicity study results are still needed for conclusive mapping. To aid in drug formulation profiling, our study provided information on drug-drug interactions and confirmed the biocompatibility of the proposed combination of DFNAC and CBD. We highlighted the biocompatibility profile of the combination on human dermal fibroblasts (HDF). Both MTT and LDH assays demonstrated that the antibacterial concentration (3.906 µg/mL) preserves cell viability and membrane integrity. Live/Dead staining confirmed that most cells remained viable and exhibited normal morphology. The overall results indicate that the combination operates within a therapeutic window, achieving antimicrobial efficacy without inducing significant cytotoxicity. Moreover, the combination shows high biocompatibility. Investigations reported that CBD decreased the overall acidity levels in rats treated with DFNAC, suggesting that cannabidiol effectively treated the condition [57], or provided hepatoprotection against DFNAC sodium-induced hepatotoxicity [58]. However, the increasing interest in cannabis also requires a deeper understanding of the potential interactions between CBD with NSAIDs based on current knowledge and evidence [59,60].

Therefore, further rigorous quantitative biofilm assays, mechanistic membrane studies, and validation through clinical or animal models are essential before NSAIDs [41] and CBD [61] can be considered viable anti-biofilm adjuvants. Although DFNAC was tested in clinical trials involving patients with uncomplicated urinary tract infections and cellulitis [43], additional studies are necessary to complete its antimicrobial profile. Recent developments in plant-derived and synthetic cannabinoids [51] and NSAIDs [62] support the hypothesis that new therapeutic formulations [63,64] can be designed [65] specifically to address the antimicrobial crisis while meeting clinical applications [66] and regulatory requirements. Moreover, addressing antibiotic resistance through drug reprofiling will enhance formulations for personalised medicine.

4. Materials and Methods

The antimicrobial interaction between CBD and DFNAC was evaluated against ATCC bacterial strains cultured in tryptic soy broth (TSB). Bacterial suspensions were prepared from overnight cultures and adjusted to the required inoculum density. Serial two-fold dilutions of CBD, DFNAC, and CBD–DFNAC combinations were prepared in TSB in sterile 96-well microplates. Wells containing bacteria without treatment served as growth controls, while wells containing TSB and tested compounds without bacteria were used as blanks. Plates were incubated at 37 °C for 24 h, after which bacterial growth was evaluated by measuring optical density at 620 nm. The minimum inhibitory concentration (MIC) was defined as the lowest concentration showing no visible bacterial growth and OD values comparable to the blank control. The interaction between CBD and DFNAC was assessed using the fractional inhibitory concentration index (FICI), calculated as follows:

FICI= (MIC CBD in combination/MIC CBD alone) x (MIC CBD in combination / MIC DFNAC alone)

(1)

The interaction was interpreted as synergistic when FICI ≤ 0.5, additive when 0.5 < FICI ≤ 1, indifferent when 1 < FICI ≤ 4, and antagonistic when FICI > 4.

Cell viability and metabolic activity were assessed using the MTT Cell Proliferation Assay Kit (Roche Diagnostics) according to the manufacturer’s protocol. Human dermal fibroblasts (HDFs) were seeded in 24-well plates at a density of 1 × 10⁴ cells/well and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) under standard conditions (37 °C, 5% CO₂, humidified atmosphere). After 24 h to allow cell attachment, the culture medium was replaced and the cells were exposed to the tested CBD–diclofenac mixtures at the selected concentrations.

Following 24 h incubation, the culture medium was removed and the cells were gently washed with phosphate-buffered saline (PBS). MTT reagent was added to each well at a final concentration of 0.5 mg/mL and incubated for 4 h at 37 °C to permit the formation of formazan crystals by metabolically active cells. Subsequently, the crystals were dissolved using the supplied solubilization solution, and absorbance was measured at 550 nm using a Multiskan FC microplate reader (Thermo Scientific). The obtained absorbance values were considered directly proportional to the number of viable cells and overall metabolic activity.

Cytotoxicity and membrane integrity were further evaluated using the Lactate Dehydrogenase (LDH) Cytotoxicity Detection Kit (Roche Diagnostics), following the manufacturer’s instructions. After 24 h exposure to the CBD–diclofenac formulations, 50 µL of culture supernatant from each well was collected and transferred to a 96-well plate. Subsequently, 100 µL of freshly prepared reaction mixture was added to each sample and incubated for 15–20 min at room temperature in the dark. The assay is based on the quantification of LDH released from damaged cells into the extracellular medium, generating a colored product proportional to membrane disruption and cytotoxicity. Absorbance was measured at 490 nm with a reference wavelength of 600 nm using a Tecan Infinite 200 Pro microplate reader. Results were expressed as optical density (OD) values.

Cell viability and morphology were additionally analyzed using the Live/Dead Viability/Cytotoxicity Kit (Invitrogen, Thermo Fisher Scientific). HDF cells were seeded at a density of 1 × 10⁴ cells/well and incubated with the CBD–diclofenac mixtures for 24–72 h under standard culture conditions (37 °C, 5% CO₂). After treatment, the culture medium was removed and the cells were carefully washed with PBS. A staining solution containing calcein-AM, which labels viable cells with green fluorescence, and ethidium homodimer-1, which stains dead cells red, was prepared according to the manufacturer’s recommendations and added to each well. Samples were incubated for 30–60 min in the dark at room temperature prior to imaging. Fluorescence images were acquired using a Zeiss Axioscope fluorescence microscope equipped with an AxioCam imaging system in order to evaluate cell viability, morphology, and cellular attachment following exposure to the tested formulation.

5. Conclusions

Combining DFNAC, a common potent non-steroidal anti-inflammatory drug, with CBD offers a promising approach to enhance antimicrobial capacity against various bacterial strains. This combination targets both the bacterial membrane and cellular mechanisms, thereby reducing antibiotics resistance. Combining complementary MOAs facilitated a synergistic effect against S. epidermidis and an additive effect against S. aureus. The lack of antibacterial activity detected against the Gram-negative strains Escherichia coli and Pseudomonas aeruginosa reflects the need for new complementary MOAs that can simultaneously disrupt bacterial defence mechanisms and prevent the development of resistance. Using a combination of DFNAC and CBD allows for lower, less toxic doses of both substances and co-treatment reduces the development of resistance compared to single-agent treatment. Moreover, the proposed combination provides both direct bacteria killing and anti-inflammatory properties, helping reduce tissue damages during infection and personalised, targeted therapeutic approaches. DFNAC and CBD are excellent candidates for repurposing as adjuncts to enhance the efficacy of conventional antibiotics.