盐酸氨溴索联合环丙沙星抗铜绿假单胞菌成熟生物膜作用研究

Research on effect of ambroxol combined with ciprofloxacin on biofilm of Pseudomonas aeruginosa

目的构建铜绿假单胞菌发光菌株,建立铜绿假单胞菌生物膜(biofilm,BF)模型,研究盐酸氨溴索对铜绿假单胞菌成熟BF的影响及与环丙沙星合用是否具有协同杀菌作用。方法将携带绿色荧光蛋白(green fluorescent protein,GFP)的质粒转染铜绿假单胞菌,平板培养法培养铜绿假单胞菌BF,建立铜绿假单胞菌BF体外模型,经盐酸氨溴索作用后,扫描电镜(scanning elec-tron microscope,SEM)观察盐酸氨溴索对BF形态结构的影响,荧光显微镜定性观察盐酸氨溴索对BF内活菌的作用,通过发光菌株荧光强度定量分析盐酸氨溴索单独作用以及与环丙沙星联用后BF内活菌量。结果盐酸氨溴索作用后电镜观察可见黏液样物变稀疏,不规则。盐酸氨溴索浓度大于0.49mg/mlBF内活菌数减少(F=18,P<0.05);盐酸氨溴索与环丙沙星存在协同作用(F=15.1,P<0.05),且随着盐酸氨溴索浓度升高协同作用增强。结论盐酸氨溴索破坏铜绿假单胞菌成熟BF形态结构,减少其内活菌数;盐酸氨溴索与环丙沙星存在协同作用,增强其杀菌能力。

Objective To establish a biofilm model of Pseudomonas aeruginosa in vitro and observe the effect of ambroxol on biofilm of Pseudomonas aeruginosa, and study the synergistic effect with ciprofloxacin on tested specimens. Method Plasmids carried with green fluorescent protein （GFP） was tansfected to the strain of Pseudomonas aeruginosa. Plate culture method was used to establish biofilm model of Pseudomonas aeruginosa in vitro. After ambroxol was added to the biofilm, the appearance of the biofilm was detected by scanning electron microscope （SEM）, and the viable counts of bacterial in biofilm after treated by ambroxol alone and together with ciprofloxacin were determined by bio-fluorescence method. Result After adding ambroxol, the biofilm was thin and abnormal compared with the control by SEM detection. Further detection with fluorescence microscope showed the viable bacteria in biofilm was reduced significantly （F=18, P〈0.05） when its concentration was higher than 0.49 mg/ml, destroyed furthermore, there was the synergistic effect between ambroxol and ciprofloxacin （F=15.1, P〈0.05）. The effect enhanced with the increasing concentration of ambroxol. Conclusion Ambroxol destroyed furthermore the biofilm shape of Pseudomonas aeruginosa, restrained its growth, and had the synergistic effect on bactericidal activity with ciprofloxacin.