依地酸钠对黏液型铜绿假单胞菌生物膜的作用

目的探讨金属螯合剂依地酸钠（EDTA）对黏液型铜绿假单胞菌（PA）成熟生物膜的杀菌作用和对其结构的影响。方法平板法培养成熟铜绿假单胞菌生物膜，微量肉汤稀释法测量EDTA、环丙沙星的最低抑菌浓度，平板计数法计算EDTA、环丙沙星单独及联合对生物膜菌落数的影响，荧光探针FITC-ConA染细菌胞外多糖、荧光显微镜下观察EDTA作用前后多糖差别，荧光探针SYT09／H标记生物膜内细菌、激光共聚焦显微镜观察结合BF图像结构分析软件（ISA）对EDTA作用前后的生物膜结构参数进行定量分析。结果当EDTA浓度为5MIC时达到对PA生物膜的最大杀菌效应，可使菌落数由10^7CFU／ml降至10^4CFU／ml，0．1MIC、5 MIC的EDTA均可增强环丙沙星对生物膜的杀菌作用，高浓度组效果更明显、使菌落数降至10^2CFU／ml。EDTA作用后荧光显微镜下可见多糖被破坏，明显减少。激光共聚焦显微镜下可见EDTA作用后生物膜死茵比例增加，菌落变稀疏。ISA软件分析结果显示：5MIC的EDTA作用后生物膜厚度（d）由（22．59±4．13）μm降至（8．97±2．45）μm，t=8．515，P〈0．05；AP（区域孔率）由0．89±0．07增加至0．97±0．02，t=-2．653，P〈0．05；ADD（平均扩散距离）由3．08±0．96降至1．59±0．24，t=4．510，P〈0．05；TE（结构熵）由6．25±0．79降至3．02±0．67，t=9．375，P〈0．05；0．1MIC的EDTA效果没有5MIC明显。结论EDTA可以破坏铜绿假单胞菌生物膜的结构，增强抗生素对生物膜杀菌活性。

Effects of EDTA on the mucoid Pseudomonas aeruginosa biofilm

Objective To investigate the effects of EDTA on the structure of mucoid Pseudomonas aeruginosa （PA） biofilm. Method Plate culture method was used to establish muture bioflim, tube dilusion method to measure the MICs of EDTA and ciprofloxacin. The biofilm was treated by EDTA, ciprufloxacin alone or combined, and the number of the bacteria in biofilm was measured by agar plate. The biofilm was stained by FITC-ConA, observed by fluorescence microscope. The treated biofilm was stained by SYT09/PI,monitored by Confocal Laser Scanning Microscope （CLSM） ,quantification of the effect was calculated by Image Structure Analyzer （ISA） sofeware. Result 5 MIC EDTA could exert the best effect upon PA biofilm and reduce the number of colonies from 107 CFU/ml to 104 CFU/ml. 0. 1 MIC and 5 MIC EDTA could enhance the bactericidal ability of ciprofloxacin. EDTA could break the ESP of biofilm,change the structure of biofilm and raise the ratio of dead bacteria. It decreased the depth of biofilm from （22.59 ±4.13）μm to （8.97 ±2.45 ） μm （ t = 8.515, P 〈0. 05 ）, Average Diffusion Distance （ADD） from 3.08 ± 0.96 to 1.59 ± 0.24 （ t = 4.510, P 〈 0.05 ）, Textual Entropy （TE） from 6.25 ± 0.79 to 3.02 ± 0.67 （ t = 9.375 ,P 〈 0.05）, and increased areal porosity （AP） from 0.89 ± 0. 07 to 0.97 ± 0.02 （t = -2. 653 ,P 〈 0.05 ）. Conclusion EDTA can destroy the structure of BF and enhance the bactericidal ability of antibiotics.