Antibacterial and Antibiofilm Potential of Thymol–Benzimidazolium–Chalcone Hybrids Against Clinical MRSA Strains: Insights from Gene Expression Profiling and Molecular Docking

Background/Objectives: Four novel thymol–benzimidazolium–chalcone hybrids were designed based on a molecular hybridization strategy that integrates bioactive scaffolds known for their antimicrobial and antioxidant properties. This approach aims to enhance biological activity through synergistic effects and multi-target interactions, as supported by previous studies on phenolic and benzimidazole derivatives. The inclusion of both antioxidant and antibacterial evaluations was motivated by the well-established role of oxidative stress in bacterial pathogenicity and resistance mechanisms. Methods: Their antibacterial potential was initially screened using the disk diffusion method and subsequently evaluated by determining MIC and MBC values against eight clinical Staphylococcus aureus isolates. Results: Among the tested compounds, compound 3a emerged as the most potent derivative, exhibiting MIC values ranging from 0.25 to 1 µg/mL. Morphological analysis confirmed significant disruption of bacterial cell integrity, and further investigation demonstrated strong antibiofilm activity accompanied by downregulation of key biofilm- and resistance-associated genes (icaA, dltB, and mepA). Molecular docking studies were performed against selected target proteins, including 1MWT, 3VSL, 3ZG5 (sortase A), and 2ZCS, which are associated with bacterial cell wall biosynthesis, DNA replication, virulence, and metabolic pathways. Compound 3a exhibited the highest binding affinity, with a docking score of −11.953 kcal/mol against 2ZCS. Conclusions: Overall, these findings highlight the potential of thymol-based benzimidazolium–chalcone hybrids as promising multifunctional agents with combined antibacterial, antibiofilm, and antioxidant properties.