论文部分内容阅读
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SYTO9/PI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5 MIC时达到对PA生物膜的最大杀菌效应,可使菌落数由107CFU/ml降至104CFU/ml,0.1 MIC、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102CFU/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作用后生物膜死菌比例增加,菌落变稀疏。ISA软件分析结果显示:5 MIC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;0.1 MIC的EDTA效果没有5 MIC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。
Objective To investigate the bactericidal effect of the metal chelator sodium edetate (EDTA) on the mature biofilm of mucoid Pseudomonas aeruginosa (PA) and its influence on the structure. Methods The plate of Pseudomonas aeruginosa was used to culture the biofilm. The minimal inhibitory concentrations of EDTA and ciprofloxacin were measured by the broth microdilution method. The counts of EDTA, ciprofloxacin alone and in combination with biofilm colonies The fluorescent probe FITC-ConA was used to stain the exopolysaccharides of polysaccharides. The difference of polysaccharides before and after the effect of EDTA was observed under a fluorescence microscope. The bacteria in the biofilm SYTO9 / PI labeled with fluorescent probe were observed by laser scanning confocal microscopy and BF image structure analysis software (ISA) The biofilm structure parameters before and after EDTA were quantitatively analyzed. Results The maximum bactericidal effect on PA biofilm was achieved when the concentration of EDTA was 5 MIC. The number of colonies decreased from 107 CFU / ml to 104 CFU / ml. EDTA at 0.1 MIC and 5 MIC all enhanced the effect of ciprofloxacin on biofilm Bactericidal effect, high concentration group effect is more obvious, so that the number of colonies dropped to 102CFU / ml. Under the fluorescence microscope, polysaccharides were destroyed and significantly decreased after EDTA treatment. Confocal laser scanning microscope can be seen after the role of EDTA biofilm dead bacteria increased, the colony became sparse. The results of ISA software showed that the biofilm thickness (d) decreased from (22.59 ± 4.13) μm to (8.97 ± 2.45) μm, t = 8.515, P < ± 0.07 increased to 0.97 ± 0.02, t = -2.653, P <0.05; ADD decreased from 3.08 ± 0.96 to 1.59 ± 0.24, t = 4.510, P <0.05; TE increased from 6.25 ± 0.79 Down to 3.02 ± 0.67, t = 9.375, P <0.05; the effect of EDTA on 0.1 MIC was not obvious at 5 MIC. Conclusion EDTA can destroy the structure of Pseudomonas aeruginosa biofilm and enhance the bactericidal activity of antibiotics on the biofilm.