Inhibition Mechanism Of Genistein On Efflux Pump Of Methicillin-resistant Staphylococcus Aureus

Methicillin-resistant Staphylococcus aureus（MRSA）,which has multiple antibiotic resistance to many kinds of antimicrobial agents,has seriously affected the life and health of human beings.Previous studies have shown that the active efflux system of MRSA is one of the main causes of multiple drug resistance.At present,it is an effective way to reverse the drug resistance of bacteria by screening the efflux pump inhibitor from natural products.In this paper,the efflux pump inhibitor of MRSA41577 active efflux system were screened out from 8 kinds of plant compounds,and the mechanism of inhibition of MRSA41577 efflux system was studied.The results are follows:（1）Through the combined chemosensitivity test find no antibacterial activity of genistein,Which can enhance the efficacy of ciprofloxacin.Further determination of genistein on MRSA41577 efflux system inhibition by double-plate method and accumulation dynamics experiment,Results show that genistein can inhibit efflux system of MRSA41577 and restore the sensitivity of ciprofloxacin on MRSA41577.Genistein is the efflux pump inhibitor of MRSA41577;（2）Through the hemolysis experiment testing the safety of genistein.Results show that genistein did not cause red blood cell hemolysis;Genistein is a safe,non-toxic drug;（3）The influence of³¹P NMR experimental determination of genistein on MRSA41577efflux pump energy.The results show that genistein had no effect on the proton motive force of MRSA41577 efflux system,therefore has the potential application in clinical research;（4）Through the Nile Red efflux assay determination of genistein and MRSA41577 efflux substrate competition.The results show that there have a competitive relationship between genistein and substrate.Genistein is the competitive inhibitors of MRSA41577;（5）To detect the expression of norA and agr A/B/C gene by real-time fluorescent quantitative PCR method.The results show that genistein can reduce the expression of norA gene and the quorum sensing system gene agrA/B/C,and the inhibition rates are 40%,44%,56%and 36%,respectively.It can be concluded that genistein not only reduce the expression of MRSA41577 encoded efflux protein by inhibiting the expression of the efflux gene,but also can inhibit the expression of quorum sensing system to reverse the resistance of MRSA41577;（6）Effect of genistein on the expression of MRSA41577 protein by iTRAQ.The results showed that 1312 proteins were detected in the MRSA41577,and the expression of 129proteins were significantly different,the up-regulated and down regulated proteins were 60and 69.Based on the results of comprehensive bioinformatics analysis,14 differentially expressed proteins were identified,which were related to substrate specific transmembrane transport,the up-regulated and down regulated proteins were 6 and 8.It is suggested that genistein can inhibit the active efflux system of MRSA41577 by affecting the protein related to substrate specific transmembrane transport.