Browsing by Author "Anis Fadhlina Izyani Awang, Ph.D"

Streptococcus mutans is a key etiological agent of dental caries due to its acid tolerance, biofilm formation, and quorum sensing. Natural-derived bioactive compounds, such as theaflavin-3,3′-digallate (TF3) and farnesol (F), were investigated as safer and promising alternatives to conventional anti-cariogenic agents. This study evaluated the antibacterial activities of TF3 and F, individually and in combination (1:1), focusing on their effects on bacterial growth, biofilm formation and membrane integrity of S. mutans, together with an in silico analysis of their interactions with quorum sensing associated proteins. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and fractional inhibitory concentration (FIC) assays were performed for F, TF3, and their combination (TF3-F). Time-kill assays were conducted over six hours at 1×MIC and 2×MIC of F and TF3-F. Membrane integrity was evaluated using nucleic acid and protein leakage assays, along with crystal violet uptake analysis, followed by assessment of biofilm inhibition using a crystal violet (CV) assay at ½×MIC and ¼×MIC of F and TF3-F. Field Emission Scanning Electron Microscopy (FESEM) analysis was performed to examine the morphological changes of S. mutans at ½×MIC and ¼×MIC of F and TF3-F. Molecular docking was performed using PyRx to evaluate F and TF3 interaction with quorum sensing-associated proteins, with binding energy and hydrogen bond interactions analysed using BIOVIA Discovery Studio. F exhibited MIC and MBC values of 31.25 μg/mL, whereas TF3 showed no inhibitory activity up to 1000 μg/mL. The MIC and MBC for the TF3-F were 62.5 μg/mL and 125 μg/mL, respectively. However, the classification of TF3-F interaction was not determined, as TF3 alone did not exhibit a measurable MIC. In the time-kill assay, all treatments showed growth reduction up to four hours. However, regrowth was observed at six hours for ½×MIC and ¼×MIC of TF3-F, indicating bacteriostatic activities. Biofilm inhibition was highest for ½×MIC TF3-F, followed by ¼×MIC TF3-F, ½×MIC F, and ¼×MIC F. The TF3-F combination significantly disrupted membrane integrity, indicated by an increase in OD260/280 readings and crystal violet uptake. FESEM results provided visual confirmation that the ½×MIC TF3-F, and ¼×MIC TF3-F severely disrupted S. mutans morphology and membrane integrity, exceeding the effects of F alone. Molecular docking revealed that TF3 exhibited the lowest free binding energies and highest number of hydrogen bond interactions with targeted active residues, suggesting that TF3 potentially inhibit the quorum sensing of S. mutans. Overall, the TF3-F combination demonstrated multi-targeted antibacterial activities against S. mutans, encompassing biofilm suppression, membrane disruption, and potential interference with quorum sensing pathways.