纳米银离子对铜绿假单胞菌生物被膜的细菌死亡率的影响

Silver nanoparticles bactericide P.aeruginosa in in vitro biofilm

目的探讨纳米银离子对细菌生物被膜(biofilm,BF)的细菌死亡率的影响。方法采用摇床法,以纳米银离子含量不同的乙烯-醋酸乙烯酯(Ethylene-Vinyl acetate,EVA)塑料为细菌粘附载体,模拟体内铜绿假单胞菌(P.aerugi-nosa,PA)BF形成的微环境,建立体外BF模型;将培养3 d的空白标本分别在扫描电子显微镜(SEM)下及用FITC-ConA染色后荧光显微镜下观察不含纳米银离子EVA中BF的形成情况;将生长0.5、1、2、3、5 d的BF模型行SYTO9/PI染色,激光共聚焦扫描电镜(CLSM)下摄取不同层面的图像,然后采用Image Pro Plus 6.0分析软件分析不同干预条件下BF的细菌的死亡率。结果运用SEM及荧光显微镜的方法,在以不含纳米银离子EVA塑料的细菌粘附载体上培养3 d的标本中均观察到流线状的BF形成;纳米银离子含量、作用时间对BF的细菌死亡率均有明显影响(F值分别为84.62,85.67,P<0.01);不同时间点含有纳米银离子材料的BF细菌死亡率均显著高于空白对照组(P<0.05),但纳米银离子含量不同的2种材料上BF的细菌死亡率无明显影响(P>0.05);纳米银离子含量不同的(0、0.05%、0.1%)材料上的BF,其第1、2、3天细菌死亡率均分别显著高于第0.5、5天的细菌死亡率(P<0.05);含纳米银离子0.1%的材料上BF在作用的第2天其细菌死亡率最高[(88.53±1.88)%]。结论运用摇床法成功建立了体外BF模型,纳米银离子对BF内的细菌有明显杀灭作用。

Objective To explore the bactericidal effect of silver nanoparticle on P. aeruginosa in an in vitro model of biofilms. Methods The ethylene-vinyl acetate （EVA） plastics with 0.01% or 0.05% silver nanoparticle in it were used as carriers for the bacteria adhesion, on which bacterial biofilm models in vitro were built by using shaking method to mimic the micro-environment in vivo. The samples not containing silver nanoparticle which had been cultured for 3 d were scanned by scanning electron microscopy （SEM） and were observed by fluorescence microscope after stained with FITC-ConA to observe biofilm formation. The model specimens which had been cultured for 0.5, 1, 2, 3 or 5 d with shaking method were stained by the fluorescent antibody SYTO9/PI, and then detected under confocal laser scanning microscopy （CLSM） to take the images at different levels. The image were were then analyzed by the software of Image-Pro Plus 6.0. The bacterial mortalities under the different conditions （ concentration of silver nanoparticles and culture times） were calculated. Results SEM and fluorescence microscope displayed the linear flow of bacterial biofilm formation in the 3-daycultured EVA plastic carriers, indicating the establishment of in vitro model of biofilm. Mortality of bacteria was obviously affected by the different concentrations of silver ion, duration of action （F = 84. 62, 85. 67,P 〈 0. 0001 ）. The mortality of bacterial of silver nanoparticle was significantly higher than control group at different time points （P 〈 0.05）. However, there was no significant difference in the mortality between the concentration of 0.05% and 0.1% silver nanoparticle. The mortality of bacteria in the specimens cultured for 1,2 and 3 d was significantly higher than those cultured for 0.5 and 5 d. The mortality rate of bacteria on 0.1% silver nanoparticle EVA plastic cultured for 2 d was the highest among all groups [ （88.53 ± 1.88 ） % ]. Conclusion In vitro model of bacterial biofilm is successfully established by using the shaking method. The bacteria in biofilm can be killed by silver nanoparticle.