Inhibition Mechanism Of Biochanin A On Efflux System Of Methicillin-resistant Staphylococcus Aureus

Efflux system is an important mechanism of multi-drug resistant bacteria by which theantibiotics are pumped out of the intracellular, reducing the effective concentration of thedrug. Efflux inhibitors can block the efflux of antibiotics and they are through thecombination with some antibiotics to achieve therapeutic purposes and difficult to inducebacterial resistance. Biochanin A is a kind of natural chickpea isoflavones which has a widerange of biological activity and pharmacological effects. As an efflux inhibitor, Biochanin Aalso has been reported, but most of the researches are just preliminary study. In thisexperiment, the mechanism of biochanin A against antibiotic efflux is studied systematically.The results are as follows:(1) This study examines drug resistance, drug efflux phenotype and genotype from fiveclinical isolates by cefoxitin disk diffusion method, double broth dilution method and PCRmethod. MRSA41577proves to be multi-drug resistant strain that contains drug efflux systemand is chosen as downstream experimental strain.(2) The MIC value of ciprofloxacin combining with biochanin A is measured by brothdilution method. Bactericidal rate is measured by bactericidal assay in vitro. The results showthat biochanin A itself has no antibacterial activity, but can significantly reduce the MICvalue of ciprofloxacin after combination with ciprofloxacin. We speculates that biochanin Aand ciprofloxacin have a synergistic effect.(3) Biochanin A inhibition zones is measured by two-plate method. We comparebiochanin A inhibition zones between ciprofloxacin and ciprofloxacin-free plate and concludethat biochanin A is an efflux pump inhibitor preliminarily.(reserpine as a positive control)(4) The influence of ciprofloxacin accumulation concentration in the bacteria forbiochanin A is detected by fluorescence spectrophotometry. The results show that biochaninA can significantly enhance the accumulation of ciprofloxacin concentration, and at15thminute,83%increase is in the experimental group than in the control group (p<0.01). Weconclude that biochanin A is an efflux pump inhibitor indeed and has almost the same effectas reserpine.(5) The influence of biochanin A on efflux gene norA expression is detected by RealtimePCR method. The results show that sub-inhibitory concentrations of ciprofloxacin alone cansignificantly increased the norA expression, however, after adding biochanin A the norA gene expression falls to below the normal level. We conclude that biochanin A can inhibittranscription of norA gene, thereby reducing norA efflux protein synthesis.(6) The impact of biochanin A on bacteria protein synthesis is detected by SDS-PAGEassay and HPLC-MS. The results show that27.9kDa ABC transporter ATP-binding proteindecreases obviously, and this protein is the member of the ABC transporters, which suggestsbiochanin A may also inhibit the synthesis of the ABC transporter to suppress the antibioticsefflux.