Synergistic antibacterial activity of shikonin compounds combined with conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA).

Methicillin-resistant Staphylococcus aureus (MRSA) infections pose a major threat to public health due to multi-drug resistance, necessitating the urgent need for novel therapeutic approaches. Synergistic combinations of plant-derived bioactive compounds with conventional antibiotics offer a promising therapeutic alternative. Leveraging the structural diversity and inherent anti-MRSA activity of naphthoquinones from Arnebia euchroma, this study aimed to screen their derivatives for synergistic effects with clinical antibiotics. We investigated the synergy between 5,8-dihydroxy-1,4-naphthoquinone (DHNQ), the most potent derivative among 35 screened shikonin compounds, and eight clinical antibiotics against MRSA. DHNQ exhibited synergistic activity with ursolic acid (UA), Cefazolin (CZ) and gentamicin (GM) against MRSA (FICI≤0.5). Remarkably, the DHNQ-GM combination reduced the MIC of DHNQ by 40-fold (from 8 to 0.2 μg/mL) and that of GM from 64 to 28.8 μg/mL. In vitro, this combination achieved over 80% growth inhibition of MRSA, with negligible cytotoxicity toward HaCaT human keratinocytes and L929 murine fibroblasts, and hemolysis rates below 5%. In a murine model of MRSA-infected wounds, DHNQ-GM combination therapy accelerated wound healing, reduced bacterial loads by 4 log10 CFU, and alleviated systemic pathology. Mechanistically, the combination disrupted MRSA biofilm integrity, as directly visualized by electron microscopy, which revealed characteristic cell shrinkage and membrane fragmentation, accompanied by extensive leakage of intracellular contents. Furthermore, the treatment suppressed arginine and pyrimidine biosynthesis, as indicated by downregulation of key enzymes (ArcC, ArgF, CarB, and PyrF), impaired energy metabolism, evidenced by reduced ATP levels and diminished glucose uptake, and perturbed enzymatic activity, reflected in decreased lactate dehydrogenase (LDH) and increased alkaline phosphatase (AKP) activities. Collectively, these findings identify DHNQ-GM as a promising synergistic regimen against MRSA, combining potent antibacterial efficacy with a favorable safety profile and multi-target mechanisms, thereby offering a potential therapeutic alternative to combat antibiotic resistance.