抗生素M331的抗铜绿假单胞菌的作用及对其体外形成生物被膜影响的研究

发布时间：2018-11-14 09:41

【摘要】：目的：本课题通过对铜绿假单胞菌临床株和标准株的生物学特性、其生物被膜形态学的研究,探讨了不同浓度的M331以及其联合大环内酯类抗生素对铜绿假单胞菌及其形成生物被膜的影响,以探讨新的治疗铜绿假单胞菌感染及它的生物被膜的方法；并初步探讨了药物M331的药物毒性。方法：应用四区划线法分离纯化了菌株；通过对比其对细胞的粘附能力,来观察其毒力；采用改良的组织培养平板法,建立了临床株PA1和标准株PA27853生物被膜的模型；应用微量稀释法,测定了不同药物及其联合用药对PA1、PA3、PA5、PA2059, PA27853的MIC值,应用微量稀释法,测定了PA1、PA2059的MBEC值；应用MTT法测定了不同剂量M331及联合用药后PA1、PA27853生物被膜中的存活细菌数；应用银染法及扫描电镜鉴定生物被膜,并观察了不同剂量M331及其联合用药后PA1、PA27853的超微结构,对其菌落形态学进行描述。结果：临床株和标准株不同菌株的产绿色素的情况不一；PA1、PA27853对Hela细胞的粘附性实验显示,PA1、PA27853对Hela细胞的粘附类型为弥散性粘附,且临床株PA1比标准株PA27853更易于粘附Hela细胞。银染法快速鉴定结果显示,都可见浓密黑染交织分布的物质,其间隙中可见聚集的短杆状菌体。扫描电镜观察,铜绿假单胞菌呈短杆状,聚集成团,为浓厚的粘液样物质紧密包绕,菌体之间相互以粘稠的纤维状粘液丝相连。MTT法测定不同剂量M331及联合罗红霉素PA1、PA27853生物被膜中存活的细菌数,结果显示,罗红霉素对M331有增效作用。应用银染法、扫描电镜观察不同剂量M331和联合罗红霉素处理的PA1、PA27853的生物被膜,可见随着M331及罗红霉素的剂量增大,PA1、PA27853生物被膜中的细菌逐渐被清除,细菌量逐渐减少。结论：通过前期的实验研究,发现从贵州省耕土中分离的某种放线菌能产生一种抗生素,经前期的实验证实其对铜绿假单胞菌疗效较确定,对其它常见菌种无效,我们将此代谢产物命名为M331。本课题通过研究发现,不同抗菌药物对PA1、PA27853的浮游菌的杀菌活性存在明显差异,M331的疗效比庆大霉素弱,与多粘菌素B相当,强于羧苄青霉素。使用大环内酯类抗生素能显著增强M331的抗菌活性,酶抑制剂也能增强β-内酰胺酶类抗生素的抗菌活性。国内外研究发现,14、15元环的大环内酯类抗生素能抑制细菌藻酸盐的合成,其作用机制可能是抑制藻酸盐合成的关键酶——甘露糖脱氢酶(GMD)的活性,与其它药物联合应用能够抑制细菌生物被膜的形成。应用不同剂量的M331及联合罗红霉素在生物被膜培养过程中分别处理PA1、PA27853,显示了罗红霉素联合M331可有效抑制铜绿假单胞菌生物被膜的形成,其对Vero细胞无细胞毒性作用,是一个较为理想的针对铜绿假单胞菌的抗生素。
[Abstract]:Objective: to study the biological characteristics of Pseudomonas aeruginosa clinical and standard strains and the morphology of its biofilm. The effects of different concentrations of M331 and its combination with macrolide antibiotics on Pseudomonas aeruginosa and its biofilm formation were studied in order to explore a new method for the treatment of Pseudomonas aeruginosa infection and its biofilm. The drug toxicity of M331 was preliminarily discussed. Methods: the strain was isolated and purified by four-region crossing method, the virulence of the strain was observed by comparing its adhesion to cells, and the models of clinical strain PA1 and standard strain PA27853 biofilm were established by modified tissue culture plate method. The MIC values of different drugs and their combinations for PA1,PA3,PA5,PA2059, PA27853 were determined by microdilution method. The MBEC values of PA1,PA2059 were determined by microdilution method. MTT method was used to determine the number of viable bacteria in PA1,PA27853 biofilm after different doses of M331 and combined treatment. The biofilm was identified by silver staining and scanning electron microscope (SEM). The ultrastructure of PA1,PA27853 with different doses of M331 and its combination was observed and its colony morphology was described. Results: the green hormone production of different clinical strains and standard strains was different. The adhesion test of PA1,PA27853 to Hela cells showed that the adhesion type of PA1,PA27853 to Hela cells was diffuse adhesion, and PA1 was easier to adhere to Hela cells than the standard PA27853 strain. The results of rapid identification by silver staining showed that the interlaced substances with dense black dye could be seen, and the short rod-like bacteria could be found in the gap. Scanning electron microscope observation showed that Pseudomonas aeruginosa was a short rod shaped, agglomerated into a dense mucus-like material tightly wrapped around each other by a thick fibrous mucous filament. MTT method was used to determine the different doses of M331 and roxithromycin PA1,. The number of bacteria surviving in the PA27853 biofilm showed that roxithromycin had a synergistic effect on M331. Silver staining was used to observe the biofilm of PA1,PA27853 treated with different doses of M331 and roxithromycin. With the increase of the dose of M331 and roxithromycin, the bacteria in the biofilm of PA1,PA27853 were gradually eliminated and the amount of bacteria decreased. Conclusion: it was found that some actinomycetes isolated from cultivated soil of Guizhou province could produce an antibiotic. The experimental results showed that the effect on Pseudomonas aeruginosa was more definite, but not on other common strains of Pseudomonas aeruginosa. We named this metabolite M331. In this study, it was found that the bactericidal activity of different antimicrobial agents against PA1,PA27853 was significantly different. The efficacy of M331 was weaker than that of gentamicin and was similar to polymyxin B and stronger than that of carbenicillin. The antimicrobial activity of M331 was significantly enhanced by macrolides, and the antibacterial activity of 尾 -lactamases was also enhanced by enzyme inhibitors. Studies at home and abroad have shown that macrolides of 14 and 15 ring can inhibit the synthesis of bacterial alginate, and its mechanism may be the inhibition of the activity of mannose dehydrogenase (GMD), a key enzyme in the synthesis of alginate. Combination with other drugs can inhibit the formation of bacterial biofilm. The treatment of PA1,PA27853, with different doses of M331 and roxithromycin showed that roxithromycin combined with roxithromycin could effectively inhibit the formation of biofilm in Pseudomonas aeruginosa and had no cytotoxic effect on Vero cells. It is an ideal antibiotic against Pseudomonas aeruginosa.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378.991

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