Synergistic Effects of Thymoquinone, 3-Hydrazinoquinoxaline-2-Thiol, and Amphotericin B Against Methicillin-Resistant Staphylococcus aureus: In vitro and in silico Evaluation

Introduction: Bacterial infections continue to pose a major global health challenge, intensified by the increasing prevalence of antimicrobial-resistant pathogens such as Methicillin-resistant Staphylococcus aureus (MRSA). The emergence of resistance mechanisms, particularly the mecA gene encoding PBP2a, significantly reduces the effectiveness of existing antibiotics, including advanced agents like linezolid and daptomycin. Although vancomycin remains a cornerstone therapy, its variable tissue penetration and growing resistance emphasize the urgent need for alternative treatments. This study explores the potential synergistic effects of 3-hydrazinoquinoxaline-2-thiol (3HTQ), thymoquinone (THQ), and amphotericin B against clinical MRSA isolates. Methods: Antibacterial activity was evaluated using broth microdilution and checkerboard assays. Molecular docking and molecular dynamics (MD) simulations were performed to assess binding interactions and complex stability. Minimum inhibitory concentrations (MICs) were determined for each compound and their combinations. Results: Amphotericin B alone exhibited limited antibacterial activity with baseline MIC values ranging from 16– 64 μg/mL; however, when combined with THQ and 3HTQ, the MIC values were markedly reduced—up to 32-fold in some MRSA isolates. Checkerboard analysis confirmed synergism, with fractional inhibitory concentration index (FICI) values ≤ 0.5. Molecular docking suggested potential interactions with key MRSA proteins, particularly PBP2a, while MD simulations indicated relative stability of these complexes. Conclusion: The proposed mechanisms are based solely on in silico analyses and computational modeling, rather than direct biological or molecular experiments. These findings, supported by in vitro data and computational analyses, suggest that combining 3HTQ, THQ, and amphotericin B could be explored as a potential approach for managing MRSA infections and overcoming emerging antimicrobial resistance.