A tetrahydrocannabinol-rich variety of Cannabis sativa L. (Cannabaceae) was kindly donated by the Czech University of Life Sciences in Prague. The cannabigerol- and cannabidiol-rich cultivars of C. sativa were both generously supplied by the Cannilav (Brno, Czech Republic).

The Vibrio harveyi mutant strain MM30 was kindly donated by the Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana [28]. The V. harveyi strain MM30 is deficient in the luxS gene, and therefore cannot synthesize its own AI-2 [11,29]. Although the AI-1 system remains functional and capable of independently generating luminescence [30], the intensity of the light produced is significantly lower than that observed with the addition of exogenous AI-2. This strain is widely used to evaluate AI-2 production in other bacteria [28], and is also useful for determining the activity of test compounds on the LuxPQ receptor, including the inhibition or induction of AI-2 binding to the receptor site [12].

The methicillin-resistant Staphylococcus aureus (MRSA) strain 7112 was purchased form the Czech Collection of Microorganisms, Masaryk University, Brno. Staphylococcus aureus bacteria, in general, are known to produce AI-2 molecules [31].

We used a slightly modified version of the official method recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2024). V. harveyi mutant strain MM30 had been cultured overnight in medium at 30 °C in the Environmental Shaker-Incubator: ES-20 (Biosan, Riga. Latvia) and was diluted with the same medium to a density of 0.1 McFarland units. Subsequently, this diluted suspension was dispensed into a 96-well microtiter plate. The test compounds, dissolved in DMSO to a concentration of 1.28 mg/mL, were introduced to the bacterial culture in the first row of wells, resulting in a final concentration of 128 μg/mL. In each subsequent row, the concentration was halved, continuing this serial dilution process until a concentration of 2 μg/mL was reached. The last row of wells served as a control of growth and contained the bacterial suspension only. The plate was then incubated for 24 h at a temperature of 30 °C. After incubation, the absorbance of the samples was measured (at λ = 600 nm), to assess the effects of the test compounds on bacterial growth and viability.

The MRSA strain 7112 was cultured overnight at 37 °C on a solid medium containing agar and Mueller–Hinton broth. A small portion of bacteria was then scraped from the Petri dish and diluted with liquid Mueller–Hinton (MH) medium to achieve a density of 0.5 McFarland units. Next, pure sterile MH medium was added to a 96-well microtiter plate. The test compounds, dissolved in DMSO, were added to the wells and diluted using the same procedure as described for V. harveyi. The plate was then inoculated with the diluted culture of MRSA 7112 and incubated at 37 °C for 24 h. Following incubation, the absorbance was measured analogously to the procedure used for V. harveyi. To ensure the accuracy and reliability of the results, the experiment was conducted in triplicate, incorporating both a positive control (17) and a negative control (DMSO).

The experimental protocol followed the methodology outlined by Ramić et al. (Ramić, 2022). In this experiment, we initially cultured the MRSA 7112 strain overnight at 37 °C in MH medium. The bacterial culture was then centrifuged, and the supernatant was filtered using a 0.2 μm microfilter syringe to remove all bacterial cells, leaving behind a medium rich in AI-2. The AI-2-rich medium was then added at a ratio of 1 part of AI-2 medium to 8.5 parts of AB medium containing an overnight culture of V. harveyi MM30 (grown at 30 °C, diluted to a density of 0.1 McFarland units). A total of 9.5 millilitres of the mixture was thoroughly shaken and then transferred to a microtiter plate with white walls and a transparent bottom. The test compounds were added to the plate and subsequently diluted, using the same method employed in the MIC experiments. The highest concentration used was half of the MIC. The total volume of the experimental mixture dispensed into each well was standardized to 100 µL. The thermostat of the reader was set to 30 °C, to maintain consistent temperature conditions throughout the experiment. Absorbance (at λ = 600 nm) and luminescence were measured every 30 min for 24 h with spectrophotometric reader FLUOstar Omega (BMG Labtech).

The assay was repeated independently, four times. The results are expressed as the ratio of luminescence to absorbance, each compared to the negative control (DMSO). Chlorogenic acid (19) was used as a positive control. GraphPad Prism 10 was used to calculate standard errors of the mean, while IBM SPSS Statistics 26.0 was employed to evaluate the significance of the results.

The method for this assay was identical to that of the previous experiment, except that the AI-2-rich medium was not included in this experiment and V. harveyi MM30 was incubated in pure AB medium. instead.

The MRSA 7112 culture in liquid MH medium, initially at a concentration of 1 McFarland unit, was diluted 100× with a pure MH medium. Next, solutions of the test compounds in DMSO were added to achieve a range of subinhibitory concentrations. Following 24 h incubation, the bacterial culture was transferred to 2 mL Eppendorf tubes and centrifuged for 2 min at 10,800 rpm. Then, 10 μL of the resulting supernatant from each tube was combined with 190 μL of V. harveyi MM30 culture (AB medium) in a microtitration plate with white walls. Absorbance and bioluminescence measurements were conducted using the same method as in the previous experiments.

Statistical analyses were performed using IBM SPSS Statistics for Windows, version 26.0 (Armonk, NY, USA). Data are presented as the mean ± standard error of the mean (SEM). Graphs were generated, and SEM was calculated using GraphPad Prism software, version 10. Group comparisons were conducted using the Mann–Whitney U test.

The antibacterial activity of test compounds 1–17 was tested against both V. harveyi MM30 and MRSA 7112. All of the test compounds exhibited much greater activity against Gram-positive MRSA than against Gram-negative V. harveyi. This more pronounced effect on Gram-positive bacteria has been well reported for both cannabinoids [32] and arylbenzofurans (Naik, 2015). The higher activity against various MRSA strains reported by Appendino et al. [33] for neutral (1,2,3) and acidic cannabinoids (4,5,6) may be attributed to the use of different, potentially more susceptible, strains, compared to those used in our study. The obtained values of MIC are summarized in the Table 1.

Compounds 18–20, considered as potential positive controls for the bioluminescence assay, were tested exclusively for their antibacterial activity against V. harveyi MM30. Doxycycline (17) was employed as a positive control, with an MIC of 8 µg/mL. Among the test compounds, compound 20 exhibited potent activity at 16 µg/mL, while 19 showed moderate activity at 128 µg/mL. Even though the highest concentration (128 µg/mL) of 9, 11, and 13 did cause a decrease in absorbance (by 67%, 67%, and 71%, respectively), noticeable turbidity remained in the wells. The remaining test compounds exhibited no antibacterial effect on V. harveyi MM30 (Table 1).

The test compounds were analysed in an assay evaluating the level of bioluminescence in V. harveyi MM30 after MRSA filtrate (AI-2) application [28]. The most pronounced dose-dependent ability to inhibit the luminescence of V. harveyi MM30 was observed for the compounds 1, 2, 10, and 11 (Figure 2). Statistically significant inhibition was also observed for the compounds 5 and 8 at the highest concentration (128 µg/mL).

Of the compounds we tested as possible positive controls for bioluminescence assays (both with and without the addition of AI-2-containing medium) in V. harveyi MM30, 20 was deemed unsuitable for further testing, due to its pronounced antibacterial effect. Compounds 18 and 19 were subsequently evaluated in bioluminescence assays at concentrations ranging from 128 to 2 µg/mL and 64 to 1 µg/mL, respectively. In an assay with AI-2 supplementation, compound 18 significantly decreased luminescence by 54% ± 12% at the highest test concentration (128 µg/mL). Compound 19 exhibited significant activity at concentrations of 64, 32, and 16 µg/mL, reducing bioluminescence by 35% ± 4%, 19% ± 4%, and 16% ± 2%, respectively.

By comparing the molecules efficient in this assay with structurally similar but less-active counterparts, we inferred potential structure–activity relationships. In our study, we observed that neutral cannabinoids exhibited greater activity in reducing AI-2-controlled bioluminescence compared to their acidic forms. The enhanced activity of neutral cannabinoids could be attributed to their greater lipophilicity. Appendino et al. described how structural modifications of cannabinoids that increase the hydrophilicity of the molecule, such as dihydroxylation of the ω-double bond in cannabigerol (CBG), can negatively impact their antibacterial activity [32]. To date, the effect of such structural modifications of cannabinoids on anti-quorum-sensing activity has not been reported.

The importance of maintaining the correct pH level in AI-2 bioluminescence assays is well documented. For example, an acidic environment is known to decrease light production in V. harveyi, potentially leading to false positive results [34]. Also, the cannabinoid acids are only weakly acidic, and in the concentrations used probably would not be able to significantly change the pH level of the mixture. Therefore, this factor likely does not account for the observed lower activity of cannabinoid acids. Another factor that could explain the reduced activity of acidic cannabinoids is their lower stability in aqueous environments and at higher temperatures [35]. Although they commonly convert to their neutral forms under these conditions, the resulting concentration of the active neutral product would be much lower than if the corresponding neutral cannabinoid were initially present.

The antibacterial activity of arylbenzofurans from Morus alba has been studied extensively and confirmed [36,37]. In our study, we report for the first time the anti-QS activity of arylbenzofurans. This finding is not entirely surprising, as arylbenzofurans contain the furan moiety, which is also present in several well-known QS inhibitors, such as furanocoumarins from Citrus sp. [15] and halogenated furanones from the marine algae Delisea pulchra [38]. The furan moiety is also present in both AI-2 and AI-1 of V. harveyi. Interestingly, the introduction of prenyl groups, which increases lipophilicity, resulted in a significant decrease in activity. Among the arylbenzofurans, the non-prenylated compound 10 exhibited the highest activity. The second most active was compound 11. Despite being prenylated, it has a significantly lower molecular weight compared to the other test compounds in this structural class. This observation, along with the superior activity of neutral cannabinoids, suggests that the smaller molecular size of those compounds may contribute to their enhanced bioactivity. Among the compounds evaluated as potential positive controls for this assay, compound 19 demonstrated the highest efficiency. This observation aligns with the existing literature on the effects of compound 19 on various bacterial species, such as P. aeruginosa [26]. Due to its relatively high efficacy in reducing AI-2-mediated bioluminescence and the low standard error of the mean observed in the experimental results, this substance serves as an appropriate positive control for the assay.

Similarly, the influence of the test compounds was evaluated, to analyse the potential decrease in the bioluminescence of V. harveyi without the application of MRSA 7112 filtrate (AI-2). In this assay, none of the test compounds significantly decreased the bioluminescence of V. harveyi MM30 compared to the negative control (Figure 3), and compounds 7 and 8 showed an apparent positive effect on bioluminescence. Substance 19 (the positive control in the previous assay) did not significantly reduce light production in the absence of AI-2 enriched medium. Although it caused an average decrease of 44% in bioluminescence, the large standard error of the mean (±14%) indicated considerable variability in the results. The method used to evaluate non-AI-2-controlled bioluminescence had significant limitations. Given that bioluminescence in V. harveyi MM30 is primarily regulated by the AI-2/lux-S system [12], the observed luminescence values in this experiment were lower in magnitude and exhibited greater variability (Figure 4). This led to larger standard errors of the mean compared to experiments conducted in a medium enriched with AI-2. Additionally, the influence of the solvent used (DMSO) was found to be non-negligible for most of the concentrations. These factors could contribute to the apparent induction of luminescence in some of the compounds, although other causes, including the actual ability of our compounds to support bioluminescence (and quorum sensing), cannot be ruled out. Several natural compounds are known to increase quorum sensing in various strains of bacteria. For instance, Ahmad et al. investigated this phenomenon in natural monoterpenoids [39]. Their findings highlighted the significant role of stereochemistry in QS modulation. Specifically, the (+)- enantiomers of carvone, limonene, and borneol were observed to enhance (QS-regulated) violacein production in Chromobacterium violaceum and pyocyanin production in P. aeruginosa. Conversely, their laevorotary analogues exhibited inhibitory effects on its production.

The influence of the compounds 1, 2, 5, 10, and 11 on the production of AI-2 was tested in MRSA 7112. Among the compounds tested, 10 exhibited a significant reduction in bioluminescence across all concentrations tested (23% ± 2%, 37% ± 7%, 61% ± 7% for 16, 32, and 64 µg/mL, respectively), while 5 demonstrated a significant decrease only at the highest concentration used (64 µg/mL), resulting in a mean reduction of bioluminescence by −51% ± 20%. Although compound 11 also significantly decreased bioluminescence at all tested concentrations, this effect was likely attributable to the pronounced antibacterial activity observed in the assay, as MRSA cultures treated with this compound showed no visible turbidity, suggesting substantial inhibition of bacterial growth. The results are shown in Figure 5, Figure 6 and Figure 7.

The main limitation of the method we employed to evaluate AI-2 production in MRSA is its inability to correlate AI-2 production with the count of viable bacterial cells in the MRSA culture. Despite establishing MIC values for 1, 2, and 5 for MRSA in microtitration plate tests and using sub-MIC concentrations (1/4, 1/2, and 1/8 of their MICs, respectively), some cultures did not exhibit noticeable turbidity. However, this variability was not consistent across all repetitions of the experiment; for instance, in the case of 16 µg/mL of compound 1, turbidity ranged from 0 to 0.5 McFarland units. This inconsistency contributed to significant variations and higher standard errors of the mean, especially at the highest concentration (16 µg/mL). Most importantly, due to this limitation, the potential influence of bacteriostatic effects on MRSA cannot be disregarded.