【作者】 林雅茵；

【导师】 余加林；

【作者基本信息】 重庆医科大学 ， 儿科学， 2009， 硕士

【摘要】 第一部分:依地酸与抗菌药联合应用对粘液型铜绿假单胞菌生物被膜的作用目的研究金属离子螯合剂依地酸(ethylenediaminotetraacetic acid,EDTA)联合三种抗菌药对黏液型铜绿假单胞菌(Pseudomonas aeruginosa,PA)生物被膜(biofilm,BF)结构的影响和对其杀菌作用。方法平板法培养铜绿假单胞菌生物被膜,微量肉汤稀释法测量环丙沙星、庆大霉素、氨苄西林单独及与EDTA联合对铜绿假单胞菌的最低抑菌浓度(minimal inhibitory concentration,MIC)。生物被膜中单独或联合加入抗菌药与EDTA作用24小时后,超声清洗,琼脂板计数生物被膜内菌量。荧光探针异硫氰酸荧光素-刀豆凝集素A (Fluorescein isothiocyanate concanavalin A , FITC-ConA)染生物被膜细菌胞外多糖(Extracellular Polymeric Substances,EPS)、荧光显微镜下观察EDTA作用前后生物被膜多糖差别;荧光探针PI/SYTO9标记生物被膜内细菌、激光共聚焦显微镜(Confocal Laser Scanning Microscopy,CLSM)观察药物作用前后生物被膜的结构变化,结合BF图象结构分析软件(Image Structure Analyzer ,ISA)对EDTA作用前后的生物被膜结构参数进行定量分析。结果0.5mg/ml EDTA与抗菌药联合可使环丙沙星和氨苄西林对浮游菌PA的MIC降低约30倍、庆大霉素的MIC降低2倍。当EDTA浓度为5MIC时达到对PA生物被膜的最大杀菌效应,可使菌落数由107 CFU/ml降至104 CFU/ml(P<0.05),0.1MIC的EDTA则对BF菌落数没有影响。然而0.1MIC、5MIC的EDTA均与环丙沙星对生物被膜有协同杀菌效应。5MIC的EDTA与三种抗菌药联合用后于BF后,空白组和联合作用组的菌落数分别为107CFU/ml和103CFU/ml(P<0.05)。荧光显微镜和激光共聚焦显微镜下可见:EDTA作用后生物被膜多糖减少、BF厚度降低、死菌比例增加、菌落变的稀疏,ISA软件分析结果显示:EDTA作用后BF厚度、平均扩散距离(Average Diffusion Distance,ADD)和结构熵(Textual Entropy,TE)均减少(P<0.05);区域孔率(areal porosity,AP)增加(P<0.05)。结论EDTA与三种抗菌药联合对浮游状态下的铜绿假单胞菌均有协同杀菌效应。EDTA可以破坏粘液型铜绿假单胞菌生物被膜结构,与三种抗菌药联合对生物被膜均有显著的清除作用。第二部分:镁离子对粘液型铜绿假单胞菌生物被膜形成过程的影响目的探讨镁离子对黏液型铜绿假单胞菌早期黏附和生物被膜形成生过程的影响。方法荧光多功能酶标仪检测各组不同时间点96孔板底部黏附细菌的荧光强度,荧光探针FITC-ConA染细菌胞外多糖(EPS)、荧光显微镜下观察各组多糖差别;SYTO9/PI染生物被膜内细菌、激光共聚焦显微镜观察结合BF图象结构分析软件(ISA)对各组生物被膜结构参数进行定量分析。结果2天时,空白组和1mM镁组的黏附细菌的荧光强度分别为1845.67±45.39和2254.78±42.45,t=–9.96 , P <0.05; 0.1mM的镁浓度下荧光强度也有增加,其余各时间组趋势与2天组相似;在荧光显微镜下观察可见随着镁浓度增加,EPS增多;激光共聚焦显微镜下可见随着镁浓度增加,生物被膜活菌增加、菌落变密集;ISA软件分析结果示:空白组和1mM镁组的6天生物被膜厚度(d)分别为(25.80±1.16)um和(34.87±1.59)um,t=–13.85, P<0.05;区域孔率(AP)分别为0.96±0.05和0.90±0.04,t=2.48, P<0.05;平均扩散距离(ADD)分别为1.54±0.15和1.92±0.16,t=5.23 ,P<0.05;结构熵(TE)分别为3.64±0.57和4.70±1.09,t=–2.6, P<0.05,3天组BF也有相同的趋势。结论镁离子可以增强黏液型铜绿假单胞菌的早期黏附,影响随后生物被膜的形成及结构。第三部分:依地酸与环丙沙星联合应用对豚鼠铜绿假单胞菌生物被膜感染模型的作用目的探讨EDTA与环丙沙星联合应用对豚鼠肺部黏液型铜绿假单胞菌生物被膜感染的清除作用。方法吸入法建立豚鼠肺部铜绿假单胞菌生物被膜感染模型;EDTA(30mg/kg.d×7天)腹腔注射、环丙沙星(4ug/ml×15分钟×7天)雾化吸入,单独及联合作用于感染后豚鼠;平板计数法对比药物作用前后豚鼠肺部生物被膜菌落数变化,苏木素/伊红(hematoxylin and eosin,HE)染色及扫描电镜观察肺部病理变化。结果空白对照组、EDTA组、环丙沙星组和联合用药组的肺部BF菌落计数分别为5.4±0.43 (lgCFU/g)、4.4±0.57 (lgCFU/g)、4.2±0.47 (lgCFU/g )和1.3±0.19 (lgCFU/g ),联合组与各单独用药组比较差异有统计学意义,P<0.05。且联合用药组肺部组织病理改变明显减轻。结论EDTA与环丙沙星联合应用对豚鼠肺部黏液型铜绿假单胞菌生物被膜感染有显著清除作用。更多还原

【Abstract】 PartⅠ: Effects of ethylenediaminotetraacetic acid combining with three kinds of antibacterial agents on mucoid Pseudomonas aeruginosa biofilm in vitroObjective To investigate the effects of metal-chelator ethylenediamine tetraacetic acid (EDTA) combination with three kinds of antibacterial agents on mucoid Pseudomonas aeruginosa (PA) biofilm(BF).Methods Plate culture method was used to establish mature bioflim. Mini broth dilution method to measure the minimal inhibitory concentration (MIC) of antibacterial agents and EDTA. The biofilms were treated by EDTA, ciprofloxacin, gentamicin, ampicillin alone or symphysisly for 24 hours, the number of the bacteria colony in biofilm was measured by agar plate method. The EPS in biofilm was stained with FITC-ConA, observed by fluorescence microscope;stained with SYTO9/PI, monitored by Confocal Laser Scanning Microscope (CLSM), quantification of the effect was calculated by Image Structor Analyzer(ISA) software . Results 0.5mg/ml EDTA can reduce the MIC of antibacterial agents against planktonic PA, the MIC of ciprofloxacin and ampicillin were reduced by 30 times. 5MIC EDTA can exerst the best effect against PA biofilm , the number of colonies in biofilm was reduced from 107CFU/ml to 104CFU/ml (p<0.05). 0.1MIC and 5MIC EDTA can enhance the bactericidal activity of ciprofloxacin. EDTA of 5MIC combined with three antibacterial agents make the number of colonies in biofilm reduce from 107 CFU/ml to 103 CFU/ml (P<0.05). Fluorescence microscope and CLSM show: EDTA can reduce the content of extracellular polymeric substances (EPS) and the thickness of the biofilm, raise the ratio of dead bacteria. The result of ISA show that after the administration of EDTA, BF thickness, Average Diffusion Distance (ADD), and Textual Entropy (TE) decreased (P<0.05), while areal porosity (AP) increased(P<0.05).Conclusions EDTA could enhance the activity of antibacterial agents against planktonic PA, and destroy the structure of BF. The combination of EDTA with antibacterial agents has significant activity to remove mucoid PA biofilm.PartⅡ: Effects of Magnesium ions on the mucoid pseudomonas aeruginosa biofilmObjective To investigate the influence of different Mg2+ concentration on biofilm formation by mucoid Pseudomonas aeruginosa.Methods Multifunction fluorometer was used to measure fluorescence of PA at the bottom of 96-well plate. The EPS of PA was stained with FITC-ConA and detected by fluorescence microscope. The biofilm was stained with SYTO9/PI, monitored by Confocal Laser Scanning Microscope(CLSM),quantification of the effect was calculated by Image Structor Analyzer(ISA) sofeware.Results Two days group: after intervention with 1mM mg2+, the fluorescence intensity was increased from 1845.67±45.39 to 2254.78±42.45 ,t=?9.96 ,P<0.05 ; 0.1mM mg2+ could also increase the fluorescence intensity; Other time groups have the approximate tendency as two days group; the EPS in biofilm was significantly increased after intervention with mg2+, which was visualized by fluorescence microscope, based on the FITC-ConA stained . The live bacteria in the biofilm increase, the colony close-up after administration of Mg2+, when observed by CLSM, based on the SYTO9/PI stained. The quantitative data from ISA software showed: after administration of 1 mM Mg2+, for 6 day biofilm ,the depth increase from (25.80±1.16)um to (34.87±1.59)um , t=?13.85 ,P<0.05 , Areal porosity (AP) decrease from 0.96±0.05 to 0.90±0.04,t=2.48, P<0.05 , Average Diffusion Distance (ADD) increase from 1.54±0.15 to 1.92±0.16,t=5.23 ,P<0.05 , Textual Entropy (TE) increase from 3.64±0.57 to 4.70±1.09,t=?2.6 ,P<0.05. The 3 day biofilm have the same tendency. Conclusions Magnesium can increase the initial attachment of PA and alter the subsequent biofilm formation and structure.PartⅢ: Effects of ethylenediaminotetraacetic acid combining with ciprofloxacin on Pseudomonas aeruginosa biofilm in vivoObjective to investigate the effect of EDTA combined with ciprofloxacin on Pseudomonas aeruginose biofilm in vivo.Methods Pseudomonas aeruginosa was inhaled into the lung of guinea pigs and colonized , formed biofilm. After 7 days, the model was treated with ciprofloxacin, EDTA alone , or a combination of them for 7 days. The number of colony in the lungs is measured by agar plate. The pathological change of the lung is observed by hematoxylin and eosin(HE) staining and scanning electron microscope.Results EDTA combined with ciprofloxacin make the number of bacteria colony in the lung reduced from 105CFU/g to 10CFU/g, (t=24.67, P<0.05) , the lung lesion was less-sever histophathologically.Conclusions The combination of EDTA with ciprofloxacin has significant activity to remove mucoid PA biofilm in vivo.更多还原