# Thymoquinone combined with conventional antibiotics against pandrug-resistant Staphylococcus aureus: a pharmacodynamic and molecular simulation strategy to overcome bioavailability limitations

## TL;DR

This study explores combining thymoquinone with antibiotics to treat drug-resistant Staphylococcus aureus by improving effectiveness and reducing resistance.

## Contribution

The study introduces a novel pharmacodynamic and molecular strategy to enhance thymoquinone's efficacy against pandrug-resistant S. aureus.

## Key findings

- Combining thymoquinone with antibiotics reduced MIC values by 8 to 10-fold.
- Thymoquinone downregulated norA, PBP2a, and PBP4 genes and inhibited their proteins.
- Molecular simulations confirmed direct inhibition of PBP2a and PBP4 by thymoquinone.

## Abstract

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.

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.

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 pharma-codynamic 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 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.

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.