纳米银离子对铜绿假单胞菌生物被膜结构的影响的分析

目的探讨纳米银离子对细菌生物被膜（biofilm,BF）的空间结构的影响。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.aeruginosa,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（scanning electron microscopy,SEM）下及用FITC-ConA染色后荧光显微镜下观察不含纳米银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（confocal laser scanning microscopy,CLSM）下摄取不同层面的图像,然后应用激光共聚焦显微镜TCS SP2自身具有的分析软件及ISA分析软件获得PAO1菌株BF的相关空间结构参数定量化数据。结果 （1）运用SEM及荧光显微镜的方法,在以不含纳米银EVA塑料为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。（2）激光共聚焦显微镜TCS SP2自身具有的分析软件定量化分析显示,随着时间的延长,各含纳米银离子材料组PAO1菌株BF的平均厚度都呈先升高后降低的趋势,3天组都达最高值;纳米银离子的含量对BF厚度的影响差异无统计学意义（F=2.11,P＆gt;0.1）,作用时间对BF厚度的影响差异有统计学意义（F=985.81,P0.05）。随着培养时间的延长,各含纳米银离子材料组PAO1菌株BF的AP值、ADD值无明显的变化趋势,同一时间组的含有纳米银离子材料组PAO1菌株BF的AP值都高于空白对照组的AP值;同一时间组的含有纳米银离子材料组PAO1菌株BF的ADD值都低于空白对照组的ADD值。各含纳米银离子材料组PAO1菌株BF的TE值随着时间的延长,都呈先升高后降低的趋势,2天组都为最高值;同一时间组的TE值随着含纳米银离子的增加都呈降低趋势。结论运用摇床法成功建立了体外PAO1菌株BF模型;纳米银离子对PAO1菌株BF空间结构有显著的影响。

Effect of silver nanoparticle on the spatial structure of Pseudomonas aeruginosa biofilms

Objective To analyze the effect of silver nanoparticle on the spatial structure of Pseudomonas aeruginosa （P. aeruginosa） biofilms. Methods Ethylene-vinyl acetate （EVA） plastics with different concentration of silver nanoparticle were used as the carriers for the bacteria adhesion. The biofilm （BF） models in vitro were built by shanking method, simulating the micro-environment of P. aeruginosa in vivo. The bacteria in control group were cultured for 3 days and then respectively examined by SEM and by fluorescence microscope after being stained with FITC-ConA. The specimens cultured for 0.5 day, 1 day, 2 days, 3 days and 5 days were respectively stained with SYTOg/PI and then examined with CLSM to get the images of different levels. These images were analyzed by using the software from CLSM to compare the thickness of P. aeruginosa biofilms under different intervention conditions, and the software named Image Structure Analyer （ISA） were used to get quantitative data on spatial structure of P. aeruginosa biofilms. Results （ 1 ） The treamline-like BF were detected in the 3-day-cuhured controls without intervention by EVA. （2） The mean thickness of P. aeruginosa BF in all intervention groups reached to peak value at Day 3. The action time showed an effect on BF thickness （F = 985.81, P 〈 0.001 ） while the concentration of silver nanoparticle did not （ F = 2.11, P 〉 0.1 ）. （ 3 ） The spatial structure of the biofilm was significantly affected by the concentration of silver nanoparticle and action time. The Areal Porosity （AP） , Average Diffusion Distance （ADD） and Textural Entropy （TE） in silver nanopartiele intervention group were significantly higher than those in control group at different time points （ P 〈 0.05 ）. However the intervention group with different concentration of silver nanoparticle showed no significant differences in regard with the above indice（ P 〉 0.05 ）. And these indice showed no changes along with the culturing time. At the same t