Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria

Abstract

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out <i>Staphylococcus aureus</i> (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human <i>P</i>-glycoprotein (<i>P</i>-gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of <i>S. aureus</i> in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against <i>S. aureus</i> and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time-kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four-eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited <i>P</i>-gp with half maximal inhibitory concentration (IC₅₀) of 64.85 μM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of <i>S. aureus.</i> Its <i>P</i>-gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer.