Synergistic effects of DHA27 combined with gentamicin against methicillin-resistant Staphylococcus aureus in vitro and in vivo.

Objectives: To explore the antibacterial sensitizing effect of DHA27, a derivative of artesunate, on the effect of gentamicin (GEN) against methicillin-resistant Staphylococcus aureus (MRSA) and its molecular mechanism.

Materials And Methods: MICs of drugs against 33 MRSA strains were determined by serial 2-fold dilutions. Fractional inhibitory concentration index (FICI) was determined by chessboard method. The changes of bacterial morphology and internal structures were observed under scanning electron microscopy and transmission electron microscopy. The drug concentrations within cells were tested using daunorubicin accumulation test and LC-MS analysis. The mRNA expressions such as β-lactamase, mecA (encoding PBP2a) and efflux pumps were tested using real-time quantitative reverse transcription PCR. The protective effect of DHA27 combined with GEN was investigated in a MRSA challenged mouse sepsis model.

Results: The 33 MRSA strains were resistant to seven antibiotics including OXA and GEN. DHA27 alone had no antibacterial activity, but DHA27 combined with GEN exhibited antibacterial synergy against eight MRSA strains in vitro. The electron microscope observation revealed that the combination disrupts MRSA's cell wall. DHA27 increased the intracellular accumulation of daunorubicin and GEN in MRSA. DHA27 only inhibited norA and norC mRNA expressions of efflux pumps. DHA27 combined with GEN significantly reduced bacterial load in blood and lungs, decreased serum levels of TNF-α and IL-6, and mitigated the damage of lung tissues.

Conclusions: DHA27 enhances the antibacterial effect of GEN against MRSA in vitro and in vivo likely by inhibiting the expression of efflux pump genes, and then increasing GEN accumulation within MRSA and restoring its antibacterial activity.