Mechanism of Action of Zinc Oxide Nanoparticles as an Antibacterial Agent Against Streptococcus mutans

Oral microbial biofilms play a critical role in the development of various oral infectious diseases, including periodontitis and tooth caries, with Streptococcus mutans recognized as a key biofilm-forming bacterium due to its strong adhesion and acidogenic capacity. Zinc oxide nanoparticles (ZnO NPs) have demonstrated antibacterial properties against various bacteria. This study investigated the antibacterial and antibiofilm properties of ZnO NPs on S. mutans and elucidated their mode of action. Bacterial cultures were exposed to increasing concentrations of ZnO NPs, and planktonic growth, biofilm biomass and biofilm metabolic activity were measured. Complementary assays assessed bacterial ATP content, pH shift in the media, reactive oxygen species (ROS) production, membrane integrity (SYTO 9/PI live/dead staining) and membrane potential. Morphological changes were examined by high-resolution scanning electron microscopy (HR-SEM), while gene expression was analyzed by real-time qPCR. We observed that ZnO NPs inhibited S. mutans growth and biofilm formation in a dose-dependent manner, with both the minimum inhibitory and biofilm inhibitory concentration of 0.5 mg/mL. ZnO NP treatment disrupted bacterial membranes, caused cytoplasmic leakage, and induced ROS production. EPS production determined by Congo Red staining was significantly reduced. Gene expression analysis revealed significant upregulation of vicR, luxS, wapA, gtpB, nox and ftsZ, and downregulation of spaP, gtpC and atpB. In conclusion, ZnO NPs compromise S. mutans viability and biofilm development through oxidative stress and membrane disruption, highlighting their potential use as bioactive materials in oral healthcare.