Integrated In Vitro and In Silico Evaluation of Benzimidazolium Salts: Antioxidant Activity, Anti-MRSA/MSSA Effects,...

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created an urgent need for the development of novel antimicrobial agents. This study aimed to synthesize and characterize a series of benzimidazolium salts and evaluate their antibacterial, antibiofilm, and molecular interaction properties against MRSA and methicillin-susceptible Staphylococcus aureus (MSSA).

Five benzimidazolium salts bearing different substituents were synthesized and characterized by NMR and FTIR analyses. Their antibacterial activities against MRSA and MSSA were investigated using the resazurin-based minimum inhibitory concentration (MIC) assay and agar diffusion method. Antibiofilm activity was evaluated using crystal violet staining, while scanning electron microscopy (SEM) was employed to examine bacterial morphological changes. Gene expression analysis was performed to assess the effects of the most active compound on virulence- and resistance-related genes. In addition, molecular docking studies were conducted against four target proteins (1MWT, 3ZG5, 1JIJ, and 2Y2M).

Among the synthesized compounds, compound 1c exhibited the strongest antibacterial activity, with MIC values of 0.25 µg/mL against MRSA and 0.5 µg/mL against MSSA. It also produced the largest inhibition zone diameter (31.0 ± 0.5 mm). SEM analysis revealed significant morphological alterations in treated bacterial cells, indicating disruption of cellular integrity. Gene expression studies demonstrated that compound 1c downregulated several virulence- and resistance-associated genes, including icaA, dltA, dltB, sarA, norA, and norB. All compounds displayed antibiofilm activity, with compound 1c showing the highest inhibition rate (68.6 ± 0.6%). Molecular docking analysis revealed that compound 1c exhibited the strongest binding affinity toward the 2Y2M target protein, with a docking score of -5.322 kcal/mol.

The findings demonstrate that benzimidazolium derivatives, particularly compound 1c, possess potent antibacterial and antibiofilm activities against S. aureus strains and effectively modulate virulence- and resistance-related gene expression. Combined with favorable molecular docking results, these compounds represent promising candidates for the development of new therapeutic agents against resistant S. aureus infections.