Effect of flavonoids identified in grape pomace extract on efflux pump in Staphylococcus aureus

Overexpression of efflux pumps, is a primary mechanism of multidrug resistance in Staphylococcus aureus. This study evaluated grape pomace extracts and their constituent flavonoids as potential efflux pump inhibitors (EPIs) to restore ciprofloxacin susceptibility. RP-HPLC analysis of the extracts identified quercetin, (+)-catechin, and (-)-epicatechin. The synergistic interaction between these agents and ciprofloxacin was assessed using the checkerboard method against three S. aureus strains: NCTC 8325-4 (norA wild type), K2378 (overexpressing norA), and K1902 (norA deletion). Additionally, ethidium bromide accumulation assays were conducted to quantify pump inhibition activity. All extracts and identified flavonoids exhibited synergism with ciprofloxacin, yielding Fractional Inhibitory Concentration Indices (FICI) ranging from 0.076 to 0.281. While crude extracts demonstrated nonspecific pump inhibition, the isolated flavonoid (-)-epicatechin showed inhibitory activity against the NorA effux pump. Conversely, (+)-catechin and quercetin displayed lower, concentration-dependent activity. These findings demonstrate that grape pomace flavonoids, particularly (-)-epicatechin, act as effective EPIs capable of efflux pumps mediated resistance, suggesting their potential utility as adjuvants in antimicrobial therapy.