抗菌肽17BIPHE2抑制金黄色葡萄球菌及铜绿假单胞菌生物被膜作用的研究

发布时间：2018-04-17 02:01

本文选题：抗菌肽 + 17BIPHE2　；参考：《宁夏医科大学》2017年硕士论文

【摘要】：目的研究抗菌肽17BIPHE2单独使用及联合抗生素对金黄色葡萄球菌(Staphylococcus aureus,SA)和铜绿假单胞菌(Pseudomonas aeruginosa,PA)生物被膜的抑制作用,并初步探讨抗菌肽17BIPHE2作用细菌后对生物被膜相关基因转录水平的影响,为治疗金黄色葡萄球菌、铜绿假单胞菌生物被膜相关感染及药物研发提供了一个新思路。方法1.采用刚果红平板测试法(金黄色葡萄球菌)和结晶紫染色法评估受试菌形成生物被膜的能力;2.微量肉汤稀释法和琼脂平板测试法测定金黄色葡萄球菌、铜绿假单胞菌最小抑菌浓度(MIC)、最小杀菌浓度(MBC);3.利用生物被膜早期黏附实验观察17BIPHE2单独使用及联合抗生素对生物被膜黏附阶段的影响;4.利用生物被膜形成抑制实验观察17BIPHE2单独使用及联合抗生素对生物被膜形成阶段的影响;5.通过扫描电子显微镜(SEM)观察17BIPHE2单独使用及联合抗生素是否具有清除成熟阶段生物被膜的作用;6.采用激光共聚焦激光扫描显微镜(LSCM)观察17BIPHE2是否具有杀伤成熟铜绿假单胞菌生物被膜内细菌的作用;7.利用Real-Time PCR分析17BIPHE2作用细菌后对生物被膜相关基因转录水平的影响。结果1.经刚果红平板测试法和结晶紫染色法评估临床分离株SA 05具有较强形成生物被膜能力,临床分离株SA 07不具有形成生物被膜的能力;经结晶紫染色法评估临床分离株PA 03具有较强的形成生物被膜能力,临床分离株PA 07不具有形成生物被膜的能力;2.17BIPHE2对金黄色葡萄球菌MIC值为8μmol/L,1/2×MIC就可以有效抑制浮游菌的生长。17BIPHE2对铜绿假单胞菌MIC值为48μmol/L,亚抑菌浓度也可以有效抑制浮游菌的生长。17BIPHE2联合抗生素使用较单一使用MIC值均有所下降;3.单独使用17BIPHE2在金黄色葡萄球菌黏附阶段抑制率为40%,17BIPHE2联合抗生素使用较单独使用抗生素其抑制效果有所提升。单独使用17BIPHE2在铜绿假单胞菌黏附阶段抑制率为60%,17BIPHE2联合抗生素使用与上述结果相似,较单独使用抗生素抑制率有所提高;4.在金黄色葡萄球菌生物被膜形成阶段17BIPHE2单独使用抑制率达到35%,而在铜绿假单胞菌生物被膜形成阶段17BIPHE2单独使用抑制率达到80%。值得注意的是,17BIPHE2在1/4×MIC也可以显著抑制两种细菌产生生物被膜。17BIPHE2联合抗生素结果显示较单独使用抗生素其抑制生物被膜产生能力均有所提高;5.经扫描电子显微镜观察,17BIPHE2在1/4×MIC浓度即可促进金黄色葡萄球菌成熟生物被膜消散,1×MIC生物被膜消散同时细菌黏附量有所下降,联合万古霉素促进生物被膜消散同时细菌胞质大量外泄。17BIPHE2促进铜绿假单胞菌成熟生物被膜消散效果与上述结果相似,亚抑菌浓度即可促进铜绿假单胞菌生物被膜消散;随着药物浓度提高细菌黏附量急剧下降,并且呈现剂量依赖关系。当17BIPHE2浓度达到1×MIC时细菌完整形态几乎消失,生物被膜消散同时细胞质外泄;抗菌肽联合环丙沙星清除生物被膜作用尤为显著;6.采用激光共聚焦扫描显微镜观察单独17BIPHE2处理铜绿假单胞菌生物被膜后发现,随着药物浓度的提高,细菌黏附量逐渐下降,死菌数逐渐增多,并且呈现明显的剂量依赖关系。当抗菌肽浓度提升至1×MIC,活菌数和死菌数均明显下降。17BIPHE2联合抗生素处理组较单独使用抗生素处理组细菌黏附量显著下降,杀菌作用明显提高;7.Real-Time PCR结果表明,随着17BIPHE2浓度提高,金黄色葡萄球菌和铜绿假单胞菌生物被膜相关基因在转录水平表达量均出现不同程度的抑制,且高浓度处理组抑制作用更为明显。结论抗菌肽17BIPHE2具有良好的抑制金黄色葡萄球菌及铜绿假单胞菌生物被膜黏附、聚集作用,并可促进成熟生物被膜消散。17BIPHE2辅助抗生素抗生物被膜作用进一步提高。17BIPHE2可以有效抑制生物被膜相关基因的转录,这将为治疗由金黄色葡萄球菌及铜绿假单胞菌生物被膜引起的相关感染提供了一个新思路。
[Abstract]:Objective to study the antibacterial peptide 17BIPHE2 used alone and combined with antibiotics against Staphylococcus aureus (Staphylococcus, aureus, SA) and Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA) by inhibition of biological membranes, and to investigate the effect of antibacterial peptide 17BIPHE2 bacteria effect on biofilm associated gene transcription, for the treatment of Staphylococcus aureus, Pseudomonas Areuginosa provides a new idea of membrane associated infection and drug development. Methods 1. Congo red plate test method (Staphylococcus aureus) and crystal violet staining method to assess the bacteria the ability to form biofilm; Determination of 2. Staphylococcus aureus broth dilution method and agar the test method, the minimum inhibitory concentration of Pseudomonas aeruginosa (MIC), minimum bactericidal concentration (MBC); 3. with biofilm adhesion experimental observation of 17BIPHE2 used alone and combined with antibiotics on Students Is the 4. stage of membrane adhesion; the biofilm formation inhibition of experimental observation of 17BIPHE2 used alone and combined with antibiotics on biofilm formation stage; 5. by scanning electron microscopy (SEM) observation of 17BIPHE2 used alone and combined with antibiotics is clear the mature stage of biofilm; 6. by laser confocal laser scanning electron microscopy (LSCM) observed whether 17BIPHE2 could kill the mature biofilm of Pseudomonas aeruginosa in bacteria; 7. using Real-Time PCR 17BIPHE2 analysis of bacteria on biofilm related genes affecting transcriptional level. Results 1. by Congo red plate test method and crystal violet staining method to evaluate clinical isolates with SA 05 strong ability of biofilm formation, clinical isolates of SA 07 does not have the ability to form biofilm by crystal violet staining; evaluation of clinical isolates of PA 03 has Strong ability of biofilm formation, clinical isolates of PA 07 does not have the ability to form biofilm; 2.17BIPHE2 of Staphylococcus aureus MIC value is 8 mol/L, the growth of.17BIPHE2 1/2 * MIC can effectively inhibit bacteria of Pseudomonas aeruginosa MIC was 48 mol/L, compared with single use MIC value decreased use of subinhibitory concentration can effectively inhibit the growth of planktonic bacteria.17BIPHE2 combined with antibiotics; 3. 17BIPHE2 alone in the adhesion stage of Staphylococcus aureus inhibitory rate was 40%, the use of 17BIPHE2 combined with antibiotics than single use of antibiotics which inhibit effect is improved. The single use of 17BIPHE2 in Pseudomonas aeruginosa adhesion stage inhibition rate was 60% 17BIPHE2, combined with the use of antibiotics and the results were similar, compared with antibiotic inhibition rate increased; 4. in Staphylococcus aureus biofilm formation stage using 17BIPHE2 alone The inhibition rate reached 35%, while in the Pseudomonas Areuginosa stage 17BIPHE2 membrane formation alone inhibition rate was 80%. it is worth noting that 17BIPHE2 can also inhibit two kinds of bacteria biofilm.17BIPHE2 combined with antibiotics showed than single antibiotics which inhibit biofilm production capacity were improved in 1/4 * MIC; 5. by scanning electron microscopy, 17BIPHE2 in 1/4 * MIC concentration can promote Staphylococcus aureus biofilms dissipated 1 * MIC biofilm and dissipation of bacterial adhesion decreased, combined with vancomycin promote biofilm and bacterial cytoplasm dissipate a lot of leakage of.17BIPHE2 promoting Pseudomonas aeruginosa biofilm maturation effect the film dissipation and similar results, subinhibitory concentration could promote Pseudomonas aeruginosa biofilm dissipation; with the increasing of drug concentration of bacterial adhesion sharply under Fall, and in a dose-dependent manner. When the 17BIPHE2 concentration reached 1 * MIC intact bacterial biofilm morphology almost disappeared, while the cytoplasm leakage dissipation of antibacterial peptide; ciprofloxacin combined biofilm removal effect is particularly significant; 6. using confocal laser scanning microscopy 17BIPHE2 alone treatment of Pseudomonas aeruginosa biofilm found with the increase of drug concentration, bacterial adhesion decreased, the number of dead bacteria increased gradually, and showed a dose-dependent manner. When the concentration of antibiotic peptide increased to 1 x MIC, down.17BIPHE2 combined with antibiotic group compared with antibiotics alone treatment group of bacterial adhesion was significantly decreased the number of viable cells and dead cells were significantly lower, sterilization the role of 7.Real-Time increased significantly; PCR results showed that with the increase of 17BIPHE2 concentration, Staphylococcus aureus and Pseudomonas aeruginosa biofilm related genes at the transcriptional level The expression was inhibited, and the higher concentration was inhibited obviously. Conclusion the antibacterial peptide 17BIPHE2 has good inhibition of Staphylococcus aureus and Pseudomonas aeruginosa biofilm adhesion, aggregation, and can promote the mature biofilm dissipation.17BIPHE2 assisted.17BIPHE2 was improved further biological antimicrobial film can effectively inhibit the biological membrane by transcription related genes, which will provide a new way for treatment of infection by Staphylococcus aureus and Pseudomonas aeruginosa biofilm caused.

【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

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