3.2.7. Cannabinol (CBN)

CBN is formed during the degradation of Δ9-THC and has a lower binding affinity to CB1 and CB2 receptors than Δ9-THC [117]. CBN is an agonist of the TRPV1, TRPV2, TRPV3, TRPV4, and TRPA1 cation channels [210]. CBN is a non-psychotropic phytocannabinoid with analgesic and anti-inflammatory properties and acts as an appetite stimulant [117]. CBN has neuroprotective activity that is associated with its anti-oxidative actions, trophic support, and elimination of intraneuronal β-amyloid in neuronal cells [241]. CBN preserves mitochondrial functions, such as redox regulation, calcium uptake, mitochondrial membrane potential, and bioenergetics [242]. CBN promotes endogenous antioxidant defense mechanisms and triggers AMP-activated protein kinase (AMPK) signaling pathways [242].

#### *3.3. Anti-Microbial Effects of Phytocannabinoids*

Several phytocannabinoids have been shown to have anti-bacterial activities, especially on Gram-positive bacteria, including various antibiotic-resistant strains [58,59,62,63,101, 220,243–247] (Table 1). Phytocannabinoids have been shown to exert both bactericidal and bacteriostatic effects [61,62,244,247]. Most of the studies have analyzed the half maximal inhibitory concentration (IC50) or minimum inhibitory concentration (MIC) for each of the compounds against different bacterial species, fungi, and protozoa, while only a few studies have looked at the underlying mechanisms [61,243,244,247–250] (Figure 2).

**Table 1.** Examples of *Cannabis sativa* constituents that have been documented to possess anti-bacterial, anti-fungal, and/or anti-protozoal activities \*.





concentration that causes 50% growth inhibition in comparison to control cells. MBEC = Minimum biofilm eradication concentration is the lowest concentration that completely eradicates preformed biofilm. MBIC = Minimum biofilm inhibitory concentration is the lowest concentration that is required to completely prevent any biofilm formation. MIC = Minimum inhibitory concentration is the lowest concentration that completely inhibits bacterial growth (when no turbidity is observed).

#### 3.3.1. Bacterial Growth Inhibitory Effects of Phytocannabinoids

The minimum inhibitory concentration (MIC) of Δ9-THC and CBD on various *Staphylococcus aureus* strains, including MRSA and *Streptococci* species (e.g., *Streptococcus pyogenes* and *Streptococcus. faecalis*) was found to be in the range of 1–5 μg/mL [58,62,245,246]. There was no significant difference between the anti-bacterial effect of Δ9-THC and CBD [58,245,246]. The anti-microbial effect was attenuated by the presence of either serum or blood, suggesting that serum components can bind the compounds and prevent them from acting on the microorganisms [245]. CBG shows anti-bacterial activity against Gram-positive bacteria, including MSSA, MRSA, and the oral cariogenic *Streptococcus mutans* at low concentrations similar to CBD [58,61,244,247]. CBC and CBDA showed a MIC of 1–2 μg/mL against *Staphylococcus aureus* and *Staphylococcus epidermidis* [62,220]. In these studies, CBDA was less active than CBD [62]. Cannabichromenic acid (CBCA) caused a rapid reduction in the colony-forming units (CFUs) of a clinical MRSA isolate both during the exponential and stationary growth phase, suggesting a bactericidal activity that is independent of the metabolic state of the bacteria [254]. None of the phytocannabinoids had any significant anti-bacterial activity against Gram-negative bacteria, such as *Escherichia coli*, *Salmonella typhi*, *Pseudomonas aeruginosa*, and *Proteus vulgaris* [61,62,220,245,247]. This might be due to the inability of these compounds to penetrate the outer membrane of the Gram-negative

bacteria [61], or the outer membrane protects the bacteria from cell death caused by damage to the inner membrane.

**Figure 2.** The anti-bacterial activities of phytocannabinoids.

3.3.2. Outer Membrane Permeabilization of Gram-Negative Bacteria Sensitizes Them to Phytocannabinoids

Interestingly, CBD and CBG could act on some Gram-negative bacteria (e.g., *Escherichia coli*, *Acinetobacter baumannii*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*) if the outer membrane was permeabilized with the LPS-binding antibiotic polymyxin B [61,247]. It was shown that an *Escherichia coli* Δ*bamB*Δ*tolC* deletion strain that renders the bacteria hyperpermeable to many small molecules was sensitive to CBG with a MIC of 4 μg/mL, which is in contrast to the parental *Escherichia coli* wild-type strain that showed a MIC above 128 μg/mL [61]. Similarly, a lipo-oligosaccharide-deficient *Acinetobacter baumannii* strain became sensitive to CBG with a MIC of 0.5 μg/mL compared to the parental strain showing a MIC of 64 μg/mL [61].

#### 3.3.3. Combined Treatment of Phytocannabinoids with Antibiotics

No synergistic or antagonistic effects of CBD were observed on MRSA strain USA300 when combined with different conventional antibiotics, such as clindamycin, ofloxacin, meropenem, tobramycin, methicillin, teicoplanin, and vancomycin [62]. These authors concluded that the membrane-perturbing effect of CBD was not sufficient to enhance the uptake of conventional antibiotics [62]. However, Wassmann et al. [251] observed that CBD could reduce the MIC value of bacitracin against several Gram-positive bacteria, including *Staphylococcus* species, *Listeria monocytogenes*, and *Enterococcus faecalis*. The simultaneous use of CBD and bacitracin on MRSA USA300 resulted in the formation of multiple septa during cell division, appearance of membrane irregularities, reduced autolysis, and decreased membrane potential [251]. The combined CBD/bacitracin treatment did not affect the growth of the Gram-negative bacteria *Pseudomonas aeruginosa*, *Salmonella typhimurium*, *Klebsiella pneumoniae*, and *Escherichia coli* [251].

#### 3.3.4. Phytocannabinoids Also Act on Persister Cells and Do Not Induce Drug Resistance

CBG was found to be active against MRSA persister cells, which are dormant, nondividing bacteria [61]. This trait is therapeutically important, since many antibiotics require cell division to be effective, and they are frequently unable to eradicate persister cells that usually recover after antibiotic withdrawal [255–257]. Another obstacle of antibiotic therapy is the development of drug resistance, a frequent reason for treatment failure [258]. Farha et al. [61] attempted to develop CBG-resistant bacteria in the hopes of finding the target molecules. Despite rechallenging the MRSA with 2x and 16x MIC concentration of CBG, they were unable to ge<sup>t</sup> any spontaneously CBG-resistant mutants [61]. Similarly, MRSA that had been daily exposed to sub-lethal concentration of CBD for 20 days were still sensitive to CBD [247]. The authors of these two studies [61,247] concluded that CBD and CBG do not induce drug resistance. However, it should be noted that following exposure to CBD or CBG, the surviving growth-arrested bacteria could regain growth after withdrawal of the drug.

#### 3.3.5. Therapeutic Anti-Microbial Potential of Phytocannabinoids

The hemolytic activity of CBD and CBG was found to be 256 μg/mL and 32 μg/mL, respectively, which is far above the MIC of 1–4 μg/mL for MRSA [61,247]. Additionally, the hemolytic activity of CBDA was found to be above 32 μg/mL [62]. This makes phytocannabinoids potential drugs that can act within a reasonable therapeutic window.

Farha et al. [61] observed that treating MRSA-infected mice with a high dose of 100 mg/kg CBG could reduce the bacterial burden in the spleen by a 2.8 log10 of CFU. Blaskovich et al. [247] tried various CBD-containing ointment formulations that could reduce a 2–3 log10 of CFU of MRSA inoculated on porcine skin after 1 h and a reduction of more than 5 log10 of CFU after a 24 h incubation. CBD, however, failed to significantly reduce the bacterial load of MRSA ATCC 43300 in a thigh infection mouse model [247].

#### 3.3.6. Anti-Biofilm Activities of Phytocannabinoids

Biofilms are communities of bacteria embedded in an extracellular matrix that have attached to a biotic surface (e.g., lung tissue, gastrointestinal tract, nasal mucosa, inner ear) or an abiotic surface (e.g., medical devices, such as catheters, heart valves, stents, prostheses) [259]. The majority of infectious diseases involve bacterial biofilms that are usually difficult to eradicate due to reduced antibiotic sensitivity [259,260]. Several studies show that CBD and CBG can prevent biofilm formation of various Gram-positive bacteria (e.g., MSSA, MRSA, *Streptococcus mutans*) [61,243,247]. The extent of anti-biofilm activity of CBD and CBG against these bacteria correlated with their anti-bacterial activity [61,243,244,247]. In most cases, a similar concentration of these compounds was required to achieve both effects, suggesting that some of the anti-biofilm effect is caused by the anti-bacterial activity [61,243,244]. Moreover, CBD was found to be able to eradicate preformed MSSA and MRSA biofilms with a minimum biofilm eradication concentration (MBEC) of 1–4 μg/mL,

indicating that CBD can penetrate the biofilms and act on the biofilm-embedded bacteria [247]. Some cannabinoids (e.g., CBD, CBG, CBC, and CBN) were shown to reduce the bacterial content of dental plaques in an in vitro assay where dental plaques were spread on agar plates coated with the cannabinoids [261]. The anti-biofilm activity of the cannabinoids has significant clinical importance, since the bacteria-embedded bacteria frequently show antibiotic resistance, and some antibiotics are unable to penetrate through the extracellular matrix of the biofilms [259,262,263].

#### 3.3.7. Anti-Fungal Biofilm Activities of Phytocannabinoids

CBD barely affects the viability of*Candida albicans* with a MIC above 50–100 μg/mL [247,253], but it reduces biofilm formation with a biofilm inhibitory concentration 50 (BIC50) at 12.5 μg/mL and a MBIC90 of 100 μg/mL [253]. CBD reduced the metabolic activity of preformed *Candida albicans* biofilms by 50–60% at 6.25 μg/mL with no further reduction at higher concentrations, even at 100 μg/mL [253]. The morphology of the *Candida albicans* biofilm becomes altered in the presence of CBD. While the hyphal form was predominant in control biofilms, the CBD (25 μg/mL)-treated biofilms appeared in clusters mostly in yeas<sup>t</sup> and pseudohyphal forms [253]. CBD caused a dose-dependent reduction in the cell wall chitin content and the intracellular ATP level, while increasing the intracellular reactive oxygen species (ROS) levels [253]. Gene expression studies showed that after a 24 h incubation with 25 μg/mL CBD, there is a significant downregulation of: *ADH5* (Alcohol dehydrogenase 5), involved in extracellular matrix production; *BIG1*, required for synthesis of the extracellular matrix component β-1,6-glucan; *ECE1* (extent of cell elongation protein 1), involved in biofilm formation; *EED1*, involved in filamentous growth; *CHT1* and *CHT3* chitinases, involved in the remodeling of chitin in the fungal cell wall; and *TRR1* (thioredoxin reductase) with anti-oxidant properties. On the other hand, a significant upregulation of *YWP1* (yeast-form wall protein 1) which is expressed predominantly in the yeas<sup>t</sup> form, was observed [253]. These changes in gene expression might explain, at least in part, the reduced biofilm mass of *Candida albicans* in the presence of CBD and the increase in oxidative stress [253].

#### 3.3.8. Anti-Viral Activities of Phytocannabinoids

There are some lines of evidence for an anti-viral activity of phytocannabinoids [60,264]. Some phytocannabinoids, especially Δ9-THC and CBD, bind to the Mpro protease of SARS-CoV-2, which plays a role in viral replication [60,264]. CBGA and CBDA were found to be allosteric and orthosteric ligands for the spike protein of SARS-CoV-2 and prevented infection of human epithelial cells by a pseudovirus expressing the SARS-CoV-2 spike protein [265]. Phytocannabinoids might indirectly relieve the disease progress of COVID-19 patients through their anti-inflammatory properties [266]. However, CBD failed to alter the clinical disease development of COVID-19 when given at a daily dose of 300 mg for 14 days [267]. Additionally, caution should be taken into account due to the immunosuppressive activities of phytocannabinoids that can prevent proper anti-viral immune responses [268]. Notably, the use of *Cannabis* was increased in U.S. and Canada by 6–8% during the COVID-19 pandemic in comparison to the pre-pandemic period [269], with a special increase among people with mental health [270]. Vulnerability to COVID-19 was correlated with genetic liability to *Cannabis* use disorder (CUD) [271].

#### *3.4. Some Mechanistic Insight into the Anti-Bacterial Activity of Phytocannabinoids*

The ability of phytocannabinoids such as CBD and CBG, to kill MRSA, NorA-overexp ressing *Staphylococcus aureus*, vancomycin-resistant *Staphylococcus aureus* (VRSA), vancomycinresistant*enterococci*(VRE) to a similar extent as the respective antibiotic-sensitive strains [58,245,247], suggests that its action mechanism is not hindered by the common antibiotic-resistance mechanisms. Thus, phytocannabinoids can be used as an alternative drug or an antibiotic adjuvant for infectious diseases caused by drug-resistant Gram-positive bacteria.

3.4.1. CBD and CBG Target the Cytoplasmic Membrane, Increase Membrane Permeability, and Reduce Metabolic Activity

There is evidence that CBD and CBG act by targeting the cytoplasmic membrane of the Gram-positive bacteria [61,247]. Exposure of MSSA and MRSA to CBD or CBG caused a dose-dependent increase in the fluorescence of the potentiometric probe 3,3- dipropylthiadicarbocyanine iodide [DiSC3(5)], suggesting a CBG-induced membrane depolarization [61,247]. CBD inhibited protein, DNA, RNA, and peptidoglycan synthesis in a *Staphylococcus aureus strain* when using concentrations close to the MIC [247]. At sub-MIC levels, CBD inhibited lipid synthesis [247]. CBG was found to inhibit the enzyme enoyl acyl carrier protein reductase (InhA) [272], which is involved in type II fatty acid biosynthesis in *Mycobacterium tuberculosis*. The rapid uptake of the SYTOX green dye into *Staphylococcus aureus* and *Bacillus subtilis* by CBD at MIC, suggests that CBD causes an increase in membrane permeability [247].

CBG prevents the growth of oral cariogenic *Streptococcus mutans* in a concentration and bacterial cell density manner [243]. At a MIC of 2.5 μg/mL, CBG exhibited a bacteriostatic effect on *Streptococcus mutans*, while at 2x MIC and 4x MIC, a bactericidal activity was observed [243]. CBG treatment was found to alter the morphology of *Streptococcus mutans* and cause intracellular accumulation of membrane-like structures [243]. CBG induced an immediate membrane hyperpolarization, followed by increased uptake of propidium iodide, suggesting increased membrane permeabilization [243]. At the same time, Laurdan incorporation into the membranes was reduced in a dose-dependent manner [243], indicative of a more rigid membrane structure. The metabolic activity was decreased in a dose-dependent manner, which might contribute to the growth inhibitory effect [243].

#### 3.4.2. CBD Inhibits the Release of Membrane Vesicles from *Escherichia coli*

Kosgodage et al. [250] observed that CBD inhibits the release of membrane vesicles from the Gram-negative *Escherichia coli* VCS257, while having negligible effect on the membrane vesicle release from the Gram-positive *Staphylococcus aureus* subsp. *aureus* Rosenbach. Membrane vesicles participate in inter-bacterial communication by the transfer of cargo molecules and virulence factors [273]. CBD was found to enhance the anti-bacterial effect of erythromycin, rifampicin, and vancomycin against the tested *Escherichia coli* strain [250].

3.4.3. CBG Reduces the Expression of Biofilm and Quorum Sensing-Related Genes in *Streptococcus mutans*

CBG inhibited sucrose-induced biofilm formation by *Streptococcus mutans* with a minimum biofilm inhibitory concentration (MBIC) of 2.5 μg/mL [243]. Higher concentrations (10 μg/mL) of CBG were required to reduce the metabolic activity of preformed *Streptococcus mutans* biofilms [243]. CBG reduced the expression of various biofilm-related genes (e.g., *gtfB*, *gtfC*, *gtfD*, *ftf*, *gbpA*, *gbpA*, *brpA*, *wapA*) with concomitant reduction in the production of extracellular polymeric substances (EPS) [243]. The quorum sensing-related genes *comE*, *comD*, and *luxS* were downregulated by CBG, while no effect was observed on the gene expression of the stress-associated chaperones *groEL* and *dnaK* [243]. Moreover, CBG induced reactive oxygen species (ROS) production in *Streptococcus mutans*, which might be related to the reduced expression of the oxidative stress defense genes, *sod* and *nox* [243]. Thus, CBG has specific anti-biofilm activity unrelated to its membrane-acting effect. This conclusion is further supported by the study of Aqawi et al. [248] showing that CBG inhibited quorum sensing, bacterial motility, and biofilm formation of the marine Gram-negative *Vibrio harveyi* without affecting the planktonic growth.

#### 3.4.4. CBG and HU-210 Inhibit Quorum Sensing in *Vibrio harveyi*

Quorum sensing is an inter-bacterial communication system mediated by secreted autoinducers that interact with their respective receptors, resulting in the activation of a signal transduction cascade that alters the gene expression repertoire in a cell-density-

dependent manner [274]. CBG prevented the bioluminescence induced by the master quorum sensing regulator LuxR of *Vibrio harveyi* at a concentration of 1 μg/mL [248]. Using a Δ*luxM*, Δ*lusS Vibrio harveyi* mutant that does not produce autoinducers AI-1 and AI-2, CBG was found to prevent the signals delivered by exogenously added autoinducers, with a more profound inhibitory effect on the AI-2-induced than on the AI-1-induced bioluminescence [248]. Further studies show that CBG prevented the expression of several quorum sensing genes in *Vibrio harveyi*, including *luxU*, *luxO*, *qrr1–5*, and *luxR*, which can explain the inhibitory effect of CBG on LuxR-mediated bioluminescence [248]. Altogether, these data demonstrate that CBG can interfere with bacterial quorum sensing.

The synthetic cannabinoid HU-210, which is a dimethylheptyl analog of Δ8-THC (Figure 1) and acts as a high-affinity CB1 and CB2 agonist [275,276], has been shown to inhibit quorum sensing in the *Vibrio harveyi* AI-1<sup>−</sup>, AI-2+ BB152 mutant, but it had barely any effect on the wild-type bacteria or the AI-1+, AI-2− MM30 mutant [249]. This suggests that HU-210 specifically antagonizes the AI-2 pathway [249]. The concentration of HU-210 required to achieve the anti-quorum sensing activity was relatively high (20–200 μg/mL) [249], which is 2–3 magnitudes higher than that of CBG [248]. HU-210 prevented biofilm formation of the AI-1<sup>−</sup>, AI-2+ BB152 mutant with a BIC50 of 2 μg/mL and MBIC90 of 200 μg/mL, while no significant effect was seen on biofilm formation by the wild-type bacteria or the AI-1+, AI-2− MM30 mutant [249]. However, the motility of *Vibrio harveyi* was reduced in all three strains at both 20 and 200 μg/mL HU-210 [249]. Gene expression studies showed that HU-210 at a concentration of 2 μg/mL reduced the expression of the master regulator *luxR* in both wild-type and AI-1<sup>−</sup>, AI-2+ BB152 strain, while it had no effect on the AI-1+, AI-2− MM30 *Vibrio harveyi* mutant strain [249]. The *luxM* gene that encodes for AI-1 was upregulated by HU-210 [249].
