鹰嘴豆芽素A对耐甲氧西林金黄色葡萄球菌外排系统的抑制作用

Inhibitory effects of biochanin A on the efflux pump of methicillin-resistant Staphylococcus aureus( MRSA)

【目的】MRSA外排系统是其对多种药物耐药性的主要原因,为此研究鹰嘴豆芽素A对MRSA外排系统的抑制作用。【方法】以MRSA41577为供试菌株,通过二倍稀释法、双平板法和荧光分光光度法测定鹰嘴豆芽素A对MRSA41577的抑制作用及其对外排系统的影响;通过RT-PCR检测加药前后MRSA41577外排蛋白norA的表达量;SDS-PAGE检测加药前后与MRSA41577外排相关蛋白的变化,并用液相色谱质谱仪进行鉴定确认。【结果】鹰嘴豆芽素A本身无抑菌活性,但鹰嘴豆芽素A和环丙沙星联合作用后,MRSA41577对环丙沙星的敏感性显著提高,其中40μg/mL的鹰嘴豆芽素A能使环丙沙星对MRSA41577的MIC从64μg/mL降低到8μg/mL,且作用强度与药物浓度呈正相关。经鹰嘴豆芽素A处理MRSA41577后,菌体内环丙沙星的蓄积量随着药物处理时间的增加而增加,在处理15min时与对照组相比,菌体内环丙沙星蓄积量增加了83%(P﹤0.01),与阳性对照利血平的作用效果相当。鹰嘴豆芽素A能降低MRSA41577的norA表达量,与对照组相比,鹰嘴豆芽素A和环丙沙星作用MRSA41577 16h后,norA的相对表达量降低了65%,其抑制效果优于阳性对照利血平的48%。SDS-PAGE电泳结果显示,鹰嘴豆芽素A作用MRSA4157716h后,与对照组相比菌体的蛋白谱带发生了明显的变化,其中与外排系统相关的蛋白除norA蛋白明显减少外,ABC转运体ATP结合蛋白也明显减少。【结论】鹰嘴豆芽素A是MRSA41577的外排泵抑制剂,其作用机制是通过降低MRSA41577菌体内norA基因的表达量,进而减少norA蛋白的表达量,来抑制MRSA41577对环丙沙星等药物的排出而恢复其对药物的敏感性。

[Objective] To study the inhibitory effect of biochanin A on efflux system of Methicillin-resistant Staphylococcus aureus （MRSA）. [ Methods] Inhibitory effects of biochanin A on efflux system of Strain MRSA41577 were evaluated using double dilution method, two plate method and fluorescence spectrophotometry. Real time PCR and SDS-PAGE were applied to detect the expression of MRSA41577 norA and to analyze the changes of MRSA41577 efflux protein before and after dosing biochanin A in association with liquid chromatography mass spectrometry to determinate protein variation. [ Results] Biochanin A alone had no inhibitory effect on MRSA41577, but it showed synergy effect with ciprofloxacin in inhibition MRSA41577 in which 40p^g/mL biochanin A decreased the minimum inhibitory concentration （MIC） value of ciprofloxacin from 64 Ixg/mL to 8 Ixg/mL. Biochanin A significantly increased the accumulation of ciprofloxacin in MRSA41577 in a time-dependent manner. At 15 min, biochanin A increased ciprofloxacin in MRSA41577 by 83% , which is similar to that of reserpine （positive control）. Further mechanism studies indicated that biochanin A could reduce the expression of norA in ciprofloxacin-treated MRSA41577. After incubated with biochanin A and ciprofloxacin for 16 h, the relative expression of nora of MRSA41577 was reduced by 65%. SDS-PAGE analysis showed that the total protein profiles of MRSA41577 were significantly changed after treatment with biochanin A for 16h, in which both norA protein and efflux system ABC transporter ATP-binding protein were significantly decreased. [ Conclusion] Biochanin A could inhibit Methicillin- resistant Staphylococcus aureus efflux system through reducing pathogen＇ s expression of norA and norA protein.