Novel inhibitory activity of the Staphylococcus aureus NorA efflux pump by a kaempferol rhamnoside isolated from Persea lingue Nees.
OBJECTIVE
To isolate a plant-derived compound with efflux inhibitory activity towards the NorA transporter of Staphylococcus aureus.


METHODS
Bioassay-guided isolation was used, with inhibition of ethidium bromide efflux via NorA as a guide. Characterization of activity was carried out using MIC determination and potentiation studies of a fluoroquinolone antibiotic in combination with the isolated compound. Everted membrane vesicles of Escherichia coli cells enriched with NorA were prepared to study efflux inhibitory activity in an isolated manner.


RESULTS
The ethanolic extract of Persea lingue was subjected to bioassay-guided fractionation and led to the isolation of the known compound kaempferol-3-O-α-L-(2,4-bis-E-p-coumaroyl)rhamnoside (compound 1). Evaluation of the dose-response relationship of compound 1 showed that ethidium bromide efflux was inhibited, with an IC(50) value of 2 μM. The positive control, reserpine, was found to have an IC(50) value of 9 μM. Compound 1 also inhibited NorA in enriched everted membrane vesicles of E. coli. Potentiation studies revealed that compound 1 at 1.56 mg/L synergistically increased the antimicrobial activity of ciprofloxacin 8-fold against a NorA overexpresser, and the synergistic activity was exerted at a fourth of the concentration necessary for reserpine. Compound 1 was not found to exert a synergistic effect on ciprofloxacin against a norA deletion mutant. The 2,3-coumaroyl isomer of compound 1 has been shown previously not to cause acute toxicity in mice at 20 mg/kg/day.


CONCLUSIONS
Our results show that compound 1 acts through inhibition of the NorA efflux pump. Combination of compound 1 with subinhibitory concentrations of ciprofloxacin renders a wild-type more susceptible and a NorA overexpresser S. aureus susceptible.
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark. jgh@farma.ku.dk
Jes Gitz
JG
Holler
S Brøgger
SB
Christensen
Hans-Christian
HC
Slotved
Hasse B
HB
Rasmussen
Alfonso
A
Gúzman
Carl-Erik
CE
Olsen
Bent
B
Petersen
Per
P
Mølgaard
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D001681:Biological Assay; D002939:Ciprofloxacin / Q000494:pharmacology; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D004996:Ethidium / Q000378:metabolism; D020128:Inhibitory Concentration 50; D044949:Kaempferols / Q000302:isolation & purification / Q000494:pharmacology*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors*; D027421:Persea / Q000737:chemistry*; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology*; D013211:Staphylococcus aureus / Q000187:drug effects*
D001426:Bacterial Proteins; D002939:Ciprofloxacin; D004791:Enzyme Inhibitors; D004996:Ethidium; D044949:Kaempferols; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010936:Plant Extracts; C075040:NorA protein, Staphylococcus; C006552:kaempferol