Streptococcus mutans is a cariogenic bacterium in the oral cavity involved in plaque formation and dental caries. The endocannabinoid anandamide (AEA), a naturally occurring bioactive lipid, has been shown to have anti-bacterial and anti-biofilm activities against Staphylococcus aureus. We aimed here to study its effects on S. mutans viability, biofilm formation and extracellular polysaccharide substance (EPS) production. S. mutans were cultivated in the absence or presence of various concentrations of AEA, and the planktonic growth was followed by changes in optical density (OD) and colony-forming units (CFU). The resulting biofilms were examined by MTT metabolic assay, Crystal Violet (CV) staining, spinning disk confocal microscopy (SDCM) and high-resolution scanning electron microscopy (HR-SEM). The EPS production was determined by Congo Red and fluorescent dextran staining. Membrane potential and membrane permeability were determined by diethyloxacarbocyanine iodide (DiOC2(3)) and SYTO 9/propidium iodide (PI) staining, respectively, using flow cytometry. We observed that AEA was bactericidal to S. mutans at 12.5 µg/mL and prevented biofilm formation at the same concentration. AEA reduced the biofilm thickness and biomass with concomitant reduction in total EPS production, although there was a net increase in EPS per bacterium. Preformed biofilms were significantly affected at 50 µg/mL AEA. We further show that AEA increased the membrane permeability and induced membrane hyperpolarization of these bacteria. AEA caused S. mutans to become elongated at the minimum inhibitory concentration (MIC). Gene expression studies showed a significant increase in the cell division gene ftsZ. The concentrations of AEA needed for the anti-bacterial effects were below the cytotoxic concentration for normal Vero epithelial cells. Altogether, our data show that AEA has anti-bacterial and anti-biofilm activities against S. mutans and may have a potential role in preventing biofilms as a therapeutic measure.

Streptococcus mutans (S. mutans) is a cariogenic bacterium in the oral cavity that plays a significant role in plaque formation and dental caries. In previous research by our group, we showed that the endocannabinoid anandamide (AEA) has anti-bacterial and anti-biofilm activities against S. mutans. Here, we aimed to investigate its effects on S. mutans through metabolomics analyses. S. mutans was cultivated in the absence or presence of AEA at a sub-minimum inhibitory concentration (MIC), and changes in metabolites and metabolic pathways were assessed through liquid chromatography–mass spectrometry (LC-MS). Treatment of S. mutans using AEA at 10 µg/mL significantly disturbed the glycolytic flux in the bacteria, which was indicated by a reduced glucose uptake into the cell, suppression of key glycolytic intermediates, reduced acid production into the media, imbalance of NAD⁺/NADH, and decreased adenosine triphosphate (ATP) production. The disruption of carbohydrate metabolism impacts critical cellular processes, including energy production, redox balance, and biosynthetic pathways, leading to metabolic stress and impaired cellular function. These results highlight the mode of action of AEA as an antimicrobial agent. Altogether, these findings suggest that AEA has potential as a novel antimicrobial agent in the development of therapeutics against S. mutans.

Fungal infections remain a major threat to human health, with non-albicans Candida (NAC) species causing more than half of all clinical cases and many strains gaining resistance to current treatments rapidly. Previously, N-arachidonoyl ethanolamine (anandamide, AEA) has been studied and shown to possess antibacterial and antifungal properties against various bacteria and Candida albicans. Given the previous findings, we aim here to expand the current preliminary research on AEA to investigate its antifungal activities against clinically relevant NAC species in vitro: Candida glabrata, Candida parapsilosis, and Candidaozyma auris. The minimum inhibitory concentration (MIC) and growth curve analysis determined planktonic inhibition. MTT metabolic assay and ATP production via BacTiter-Glo luminescence assay evaluated biofilm formation. Membrane fluidity, polarization and efflux pump activity were examined using fluorescence probes Laurdan, DiS-C3(3), and Rhodamine 6G, respectively. Reactive oxygen species (ROS) were assessed using DCFH-DA. Biofilm architecture and cell viability were analyzed by spinning disk confocal microscopy (SDCM). AEA reduced MIC values and slowed planktonic growth, while MTT and ATP assays demonstrated a pronounced dose-dependent reduction in biofilm metabolic activity. Membrane-targeted effects revealed increased fluidity and permeability at 125 µg/mL. Notably, AEA rapidly impaired efflux pump activity and induced intracellular ROS production. This effect was accompanied by reduced cell viability, increased proportions of PI-positive cells, and enhanced intracellular dye retention, as confirmed by SDCM. Together, these findings demonstrate that AEA exerts antifungal activity by disrupting membrane integrity and associated cellular functions and provide the first comparative characterization of species-specific membrane and oxidative stress responses to AEA across three major clinically relevant multidrug-resistant NAC species.