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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>0.1),作用时间对BF厚度的影响差异有统计学意义(F=985.81,P<0.001)。(3)ISA软件定量化分析显示,银离子含量、作用时间对BF空间结构均有明显影响,不同时间组含有纳米银离子材料上BF的区域孔率(Areal Porosity,AP)、平均扩散距离(Average Diffusion Distance,ADD)及结构熵(Textural Entropy,TE)影响的均显著高于空白对照组(P<0.05),但2种纳米银离子材料上BF的AP、ADD及TE的影响差异无统计学意义(P>0.05)。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。
Objective To investigate the effect of nano silver ions on the spatial structure of biofilm (BF). Methods Shaker method was used to simulate the formation of P. aeruginosa (PA) BF in bacteria with bacterial adhesion carrier of ethylene-vinyl acetate (EVA) with different nano-silver ion content. Microenvironment was established to establish BF model in vitro. The blank specimens cultured for 3 days were observed under scanning electron microscopy (SEM) and FITC-ConA staining to observe the formation of BF in non-nano-silver EVA under fluorescence microscope. The BF models of 0.5, 1, 2, 3 and 5 d were stained with SYTO9 / PI and confocal laser scanning microscopy (CLSM) images of different layers were taken. Then the laser scanning confocal microscope TCS SP2 With the analysis software and ISA analysis software to obtain PAO1 strain BF related spatial structure parameters of quantitative data. Results (1) Using the SEM and fluorescence microscopy methods, flow-like BF formation was observed in the specimens cultured for 3 days on non-nano-silver EVA plastic as bacterial adhesion carrier. (2) Quantitative analysis of the analysis software of TCS SP2 by laser scanning confocal microscope showed that with the extension of time, the average thickness of BF in PAO1 containing nano-silver ion material increased first and then decreased, (F = 2.11, P> 0.1). The effect of action time on BF thickness was statistically significant (F = 985.81, P < 0.001). (3) The quantitative analysis of ISA software showed that the content of silver ions and the action time had a significant effect on the spatial structure of BF. The different time groups contained the regional porosity (BF) of Areal Porosity (AP) and the average diffusion distance Average Diffusion Distance, ADD and Textural Entropy (TE) were significantly higher than those of the blank control group (P <0.05). However, there was no significant difference in the AP, ADD and TE of the two silver nanosized materials Significance (P> 0.05). With the prolongation of culture time, the AP value and ADD value of PAO1 strain BF in each group containing nano-silver ion material had no obvious change trend. The AP value of PAO1 strain BF containing nano-silver ion material group in the same time group was higher than that of blank control Group AP value; the same time group containing nano silver ion material group PAO1 strain BF ADD values were lower than the control group ADD value. With the extension of time, the TE value of PAO1 strain BF in each nano-silver ion-containing material group increased first and then decreased, with the highest values in both groups at the same time; Increase all showed a downward trend. Conclusion The BF model of in vitro PAO1 strain was successfully established by shaker method. The nano silver ion had a significant effect on the spatial structure of PAO1 strain BF.