目的 探索鱼腥草素钠(SH)联合乙二胺四乙酸二钠(EDTA-Na₂)对生物被膜菌的影响,寻找潜在的抗生素替代品.方法 通过微量稀释法以及棋盘法,测定SH和EDTA-Na₂对铜绿假单胞菌(Pseudomonas aeruginosa,PA)、金黄色葡萄球菌(Staphylococcus aureus,SA)、白色念珠菌(Candida albicans,CA)的最小抑菌浓度(MIC)和最小膜清除浓度(MBEC)以及两药协同点;通过平板稀释涂布法测定3种菌在协同点的生存曲线;扫描电子显微镜法(SEM)观察PA、SA和CA生物被膜的形态.结果 SH对PA、SA和CA的MIC分别为2.048、0.064、0.064 mg/mL,MBEC分别为2.048、0.256、0.512 mg/mL;EDTA-Na₂对PA、SA和CA的MIC分别为3.75、0.938、0.117 mg/mL,MBEC分别为15、3.75、30 mg/mL;SH联合EDTA-Na₂(SH/EDTA-Na₂)对PA、SA和CA的MIC(mg/mL)分别为0.256/0.938、0.008/0.233、0.008/0.029,MBEC(mg/mL)分别为0.512/3.722、0.032/0.469、0.064/0.938;SEM观察到两药联合对细菌的生长抑制和生物被膜的清除有明显作用,载体上的细菌显著减少,未见或仅有少量的生物被膜结构,与空白对照组比较差异明显.结论 SH与EDTA-Na₂具有协同作用,二者联合能够显著增强SH对PA、SA和CA的生长抑制和生物被膜的清除作用.

Objective To investigate the effect of sodium houttuyfonate (SH) combined with EDTA-Na₂ (SH/EDTA-Na₂) against biofilm bacteria, and to search for an alternative antibiotics. Methods The microdilution method and checkboard test were adopted to determine the minimal inhibitory concentration (MIC), minimal biofilm eradication concentration (MBEC), and synergistic points of SH and EDTA-Na₂ for Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), and Candida albicans (CA); Time-kill (T-K) curves of three kinds of bacteria in synergistic points were determined by dilution coating method; The morphology of PA, SA, and CA was observed by scanning electronic microscope (SEM). Results The MIC of PA, SA, and CA for SH was 2.048, 0.064, and 0.064 mg/mL, and MBEC was 2.048, 0.256, and 0.512 mg/mL, respectively; The MIC of PA, SA, and CA for EDTA-Na₂ was 3.75, 0.938, and 0.117 mg/mL, and MBEC was 15, 3.75, and 30 mg/mL, respectively; The MIC of PA, SA, and CA for SH/EDTA-Na₂ was 0.256/0.938, 0.008/0.233, and 0.008/0.029 mg/mL, and MBEC was 0.512/3.722, 0.032/0.469, and 0.064/0.938 mg/mL, respectively. Combination of the two drugs could significantly inhibit the growth and eradicate the biofilm of bacteria. The number of bacteria on carriers reduced notably under SEM, with no or little biofilm on carriers, compared with the blank control group. Conclusion EDTA-Na₂ could play a synergistic role in combination with SH against pathogens, and SH could inhibit the growth and eradicate biofilm of PA, SA, and CA more powerfully in combination with EDTA-Na₂.

国家自然科学基金面上项目(81173629);安徽中医药大学人才引进基金(2013RC003);安徽中医药大学青年科学研究基金(2014qn007)