Isolation and characterization of efflux pump inhibitors of Escherichia coli from plant sources

Abstract

Rapid increase of multidrug resistance in bacteria has made the discovery of new antimicrobials necessary. From the time of finding of bacterial efflux pumps in 1980, many bacteria have been characterized as multi drug resistant. Efflux pumps are involve in expelling out the antibiotics, other resistance modifying compounds and dyes. Antibiotic resistance develops very quickly due to the changes in expression of efflux pumps. Mechanism of efflux has wildly reported as vital constituent of resistance in many classes of antibiotics. In the present study, 15 plant extracts with 3 antibiotics (tetracycline, ciprofloxacin and erythromycin) were evaluated for their synergistic as well as efflux pump inhibitory activity against four strains of E. coli. Methanolic extracts of all the fifteen plants showed synergistic activity but maximum synergistic activity was shown by Garlic, Eucalyptus, Mint and Turmeric irrespective of the antibiotics. Extracts of 8 plants i.e. Garlic, Ginger, Turmeric, Clove, Rosemary, Neem, Mint and Coriandrum showed efflux pump inhibitory activity but garlic extract showed maximum efflux pump inhibitory activity hence garlic extract was selected for further study. The methanolic extract of garlic showed good synergism in combination with all three antibiotics. Phytochemical analysis of garlic extract revealed the presence of alkaloids, flavonoids, phenols and saponnin. Bioactive compound was isolated from ethyl acetate fraction of garlic extract. Five fractions obtained from column chromatography were subjected for their efflux pump inhibitory activity. The fraction G1 showed efflux pump inhibitory activity by Berberine potentiation assay. Bioactive compound (rutin) was isolated from the active fraction G1 and confirmation of bioactive compound was made by LC-MS and NMR studies. Rutin showed potent efflux pump inhibitory activity by berberine potentiation assay and ethidium bromide accumulation assay. Rutin showed significantly very high synergistic activity with all three antibiotics and significantly decreased the MIC of antibiotics against test strains of E.coli. This study suggested that rutin may be used as a potent EPI against Gram negative bacteria and may be tried for clinical use. As the previously discovered EPIs were mostly against Gram positive bacteria, so this new discovery may prove very helpful to demolish the efflux pumps in Gram negative bacteria. newline