Medicinal plant extracts with efflux inhibitory activity against Gram-negative bacteria

It was hypothesised that extracts from plants that are used as herbal medicinal products contain inhibitors of efflux in Gram-negative bacteria. Extracts from 21 plants were screened by bioassay for synergy with ciprofloxacin against Salmonella enterica serotype Typhimurium, including mutants in which acrB and tolC had been inactivated. The most active extracts, fractions and purified compounds were further examined by minimum inhibitory concentration testing with five antibiotics for activity against Enterobacteriaceae and Pseudomonas aeruginosa. Efflux activity was determined using the fluorescent dye Hoechst 33342. Eighty-four extracts from 21 plants, 12 fractions thereof and 2 purified molecules were analysed. Of these, 12 plant extracts showed synergy with ciprofloxacin, 2 of which had activity suggesting efflux inhibition. The most active extract, from Levisticum officinale, was fractionated and the two fractions displaying the greatest synergy with the five antibiotics were further analysed. From these two fractions, falcarindiol and the fatty acids oleic acid and linoleic acid were isolated. The fractions and compounds possessed antibacterial activity especially for mutants lacking a component of AcrAB-TolC. However, no synergism was seen with the fractions or purified molecules, suggesting that a combination of compounds is required for efflux inhibition. These data indicate that medicinal plant extracts may provide suitable lead compounds for future development and possible clinical utility as inhibitors of efflux for various Gram-negative bacteria.