Thymoquinone Combined with Conventional Antibiotics Against Pandrug-Resistant Staphylococcus aureus: A Pharmacodynamic and Molecular Simulation Strategy to Overcome Bioavailability Limitations

Adel Attia M. Ahmad¹El-Sayed Y.M. El-Naenaeey¹Gamal Elmowalid¹Tarek Khamis¹Touka A. Kandel¹Dina Khodeer^2,3*Razan Orfali³Marwa Ibrahim Abd El-Hamid^1*

• ¹Zagazig University, Zagazig, Egypt
• ²Faculty of Pharmacy, Suez Canal University, Ismaïlia, Egypt
• ³Imam Muhammad Ibn Saud Islamic University, Riyadh, Saudi Arabia

Background Infections with pandrug-resistant (PDR) Staphylococcus aureus (S. aureus) present no available treatment alternatives, even when employing most antibacterial combination regimen. Restoring the effectiveness of existing antibiotics especially through the integration of safe plant-derived compounds represents a promising therapeutic approach. Despite extensive in vitro evidence of thymoquinone's (TQ, Nigella sativa extract) bactericidal activity against S. aureus, its clinical application remains limited due to extremely low serum maximum concentration (Cmax), which is far below the minimal inhibitory concentrations (MICs) required for most clinical isolates. This study aimed to overcome this pharmacokinetic barrier by identifying synergistic antibacterial combinations that enhance TQ efficacy at clinically achievable concentrations. Methods Pharmacodynamic interactions between TQ or Nigella sativa essential oil and selected antibiotics were evaluated in treated Staphylococcus aureus strains using disc replacement and modified checkerboard assays. To investigate the molecular response, the expression levels of norA, PBP2a and PBP4 genes were quantified through real-time PCR analysis. Furthermore, molecular dynamics simulations were performed, for the first time, to demonstrate TQ's direct inhibitory action on PBP2a and PBP4 proteins Results Both TQ and individual antibacterials initially exhibited MIC values consistent with bacterial resistance. However, when TQ was combined with ciprofloxacin and one of azithromycin, clindamycin, gentamicin or amoxicillin/clavulanic acid, a marked pharmacodynamic enhancement was observed leading to 8 to 10-fold reductions in TQ-MICs. These combinations successfully restored antibacterial activity as evidenced by fractional inhibitory concentration index values ranging from 0.0002 to 0.01. Additionally, TQ contributed to the 3 prevention of antagonistic interactions between certain dual antibacterial pairings. The combination interactions were closely associated with downregulation of norA, PBP2a and PBP4 genes as well as the direct inhibition of PBP2a and PBP4 proteins through thymoquinone ligands. Conclusion Pharmacodynamic interactions in S. aureus treated with certain dual antibacterial combinations alongside TQ led to a significant reduction in MIC values, downregulated norA, PBP2a and PBP4 genes and directly inhibited PBP2a and PBP4 proteins through TQ ligand binding. These findings provide new perspectives on enhancing the clinical applicability of TQ reviving the efficacy of conventional dual antibacterial therapies.