Punica granatum rind extract: Antibiotic potentiator and efflux pump inhibitor of multidrug resistant Klebsiella pneumoniae clinical isolates

Abstract

Objective: With a rise in multidrug resistant (MDR) bacterial isolates, search for antibiotics or compounds that could act synergistically with them is a significant area of research. Efflux-mediated resistance, in particular, is a great hurdle that needs to be overcome. In an effort to identify such synergistic compounds and potential efflux pump inhibitors (EPI), we analyzed the rind of Punica granatum (pomegranate) against MDR clinical Klebsiella pneumoniae isolates. Methods: Sequential fractionation of P. granatum rind ethanol (PGR) extract was carried out to obtain hexane, butanol and water fractions. Antibacterial activity of the plant extracts was confirmed, and synergistic interaction with antibiotics was determined by the checkerboard assay. Gas chromatography-mass spectrometry (GC-MS) analysis was performed to identify the phytochemical constituents of the hexane extract. To study EPI activity of the extracts, norfloxacin accumulation assay was carried out. Results: PGR ethanol extract was found to have synergistic activity with ciprofloxacin, levofloxacin, ceftazidime, cefoxitin, meropenem, and gentamicin resulting in fold decrease of minimum inhibitory concentration (MIC) ranging from 2 to 32 fold. The hexane fraction was found to have maximum synergistic activity resulting in a 32-fold reduction of ciprofloxacin MIC followed by butanol and water fractions. The PGR ethanol extract was also found to have efflux inhibition activity by the norfloxacin accumulation assay. Of the sequential fractions, the butanol fraction had maximum efflux inhibition activity. Conclusion: Therefore, our study shows that PGR extract can potentiate the effect of antibiotics on MDR bacteria, and the mode of action is likely to be due to EPI.