Cannabidiol (CBD) Acts as an Antioxidant on Gardnerella vaginalis, Resulting in Reduced Metabolic Activity, Loss of Survivability, and Elimination of Biofilms

<b>Background</b>: <i>Gardnerella vaginalis</i> is a natural inhabitant of the vagina, but when an imbalance occurs in the vaginal microbiota, this bacterium can cause vaginosis, a condition that must be treated when symptomatic and prior to a gynecological intervention. Cannabidiol (CBD) is an anti-inflammatory compound that also has antibacterial activities against several Gram-positive and certain Gram-negative bacteria. <b>Objectives</b>: Since <i>G. vaginalis</i> is an opportunistic pathogenic Gram-variable bacterium, we investigated its response to CBD. <b>Methods</b>: The antibacterial activity of CBD was studied by broth dilution assay, changes in intracellular ATP levels, and the ability of bacteria to recover on chocolate agar plates. The antibiofilm activity was investigated by MTT metabolic assay, crystal violet staining, and HR-SEM. Flow cytometric analyses were performed to measure changes in membrane potential, membrane perforation, and metabolic activity. Reactive oxygen species (ROS) production was analyzed using the nitro blue tetrazolium (NBT) reagent. Gene expression was determined by semi-quantitative real-time PCR, while protein composition was determined by LC-MS/MS analysis. <b>Results</b>: We observed that <i>G. vaginalis</i> clinical isolates exhibited high susceptibility to CBD with a minimum inhibitory concentration (MIC) of 2.5 µg/mL CBD. CBD induced rapid membrane hyperpolarization and caused cytoplasmic leakage of ATP without increasing propidium iodide uptake. This was accompanied by reduced metabolic activity and loss of survivability. Proteomic analysis revealed decreased expression of some ribosomal-associated proteins. CBD exhibited antioxidant activity by reducing intracellular ROS levels in a dose-dependent manner. The antibacterial effect was neutralized by the free radical scavenger α-tocopherol, suggesting the involvement of radicals in executing the antibacterial effect. Importantly, CBD not only prevented the biofilm formation of <i>G. vaginalis</i> but also reduced the metabolic activity and biofilm biomass of preformed, mature biofilms. Real-time PCR analysis of <i>G. vaginalis</i> treated with CBD for 6 h showed an increase in the expression of biofilm-associated genes, suggesting that the antibiofilm activity of CBD is mainly due to its antibacterial effect. CBD did not alter the ability of <i>G. vaginalis</i> to adhere to HeLa cervical carcinoma cells and CBD-treated bacteria were still phagocytosed by RAW264.7 macrophages. <b>Conclusions</b>: Our study shows that CBD exhibits antibacterial and antibiofilm activities against <i>G. vaginalis</i> clinical isolates and is thus a potential drug for the treatment of vaginosis caused by this bacterium.