Anandamide Alters Glycolytic Activity in Streptococcus mutans: Metabolomics and Stable Isotope Labeling Study

<i>Streptococcus mutans (S. mutans)</i> 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 <i>S. mutans.</i> Here, we aimed to investigate its effects on <i>S. mutans</i> through metabolomics analyses. <i>S. mutans</i> 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 <i>S. mutans</i> 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 <i>S. mutans</i>.