亚抑菌浓度β内酰胺类药物对金黄色葡萄球菌α溶血素的表达调控作用

发布时间：2018-11-11 21:26

【摘要】：目的:探讨亚抑菌浓度β内酰胺类药物对金黄色葡萄球菌(Staphylococcus aureus,S.aureus)致病物质α溶血素(Alpha hemolysin,Hla)表达的调控作用及其调控机制。方法:①实验金黄色葡萄球菌菌株分别收集于安徽医科大学第一附属医院检验科培养临床标本,质控菌株选用金黄色葡萄球菌ATCC29213;选择青霉素、头孢西丁、头孢他啶、头孢唑啉作为β内酰胺类代表药物,肉汤稀释法分别测定实验细菌对选用药物的最小抑菌浓度(minimal inhibitory concentration,MIC);②实验金黄色葡萄球菌菌株经过培养8h后,分别收集细菌与培养上清。荧光定量PCR检测细菌α溶血素mRNA的表达变化,免疫印迹法检测细菌培养上清中α溶血素蛋白的表达变化,红细胞溶血实验检测细菌培养上清中溶血活性的水平变化;③取金黄色葡萄球菌溶血活性表达较强的菌株,分别在培养的同时加入1/8MIC、1/4MIC、1/2MIC亚抑菌浓度的β内酰胺类药物,分为青霉素、头孢西丁、头孢他啶和头孢唑啉作用组;平板菌落计数法计数绘制生长曲线,组设立4h、8h、12h和24h四个培养观察时间点,分别检测细菌与培养上清中α溶血素表达变化,以及细菌培养上清中溶血活性的水平变化;以未加入药物培养的细菌作为正常对照。④荧光定量PCR检测金黄色葡萄球菌转录调控因子AgrA在亚抑菌浓度β内酰胺类药物作用下的表达变化。结果:①不同金黄色葡萄球菌菌株之间α溶血素mRNA转录水平、上清中的蛋白翻译水平的表达量以及培养上清的溶血活性存在差异;②1/8MIC、1/4MIC亚抑菌浓度β内酰胺类药物对金黄色葡萄球菌生长无明显抑制作用,可显著上调细菌α溶血素的表达;作用8h后,即可显著上调α溶血素mRNA、上清中蛋白的表达,提高培养上清对红细胞的溶血作用;③亚抑菌浓度β内酰胺类药物对金黄色葡萄球菌作用后,可显著上调细菌转录调控因子AgrA的表达,结果显示可通过AgrA上调α溶血素mRNA的表达。结论:亚抑菌浓度β内酰胺类药物能够显著上调金黄色葡萄球菌致病物质α溶血素的表达,其机制可能通过细菌转录调控因子AgrA的表达调控。
[Abstract]:Aim: to investigate the regulatory effect of 尾 lactams on the expression of 伪 hemolysin (Alpha hemolysin,Hla) in Staphylococcus aureus (Staphylococcus aureus,S.aureus). Methods: 1 the strains of Staphylococcus aureus were collected from the laboratory of the first affiliated Hospital of Anhui Medical University, and Staphylococcus aureus ATCC29213; was used as the quality control strain. Penicillin, cefoxitin, ceftazidime and cefazolin were selected as representative drugs of 尾 -lactam. The minimal inhibitory concentration (minimal inhibitory concentration,MIC) of experimental bacteria against selected drugs was determined by broth dilution method. 2Staphylococcus aureus strains were collected and supernatant cultured for 8 h. The expression of 伪 -hemolysin mRNA was detected by fluorescence quantitative PCR, the expression of 伪 -hemolysin protein in the supernatant of bacterial culture was detected by Western blot, and the hemolytic activity in the supernatant of bacterial culture was detected by erythrocyte hemolysis test. 3 the strains with strong hemolytic activity of Staphylococcus aureus were cultured with 尾 lactams of 1 / 8 MIC1 / 4 MIC1 / 2 MIC and divided into penicillin and cefoxitin. Ceftazidime and cefazolin group; The growth curve was plotted by plate colony counting method. The four culture observation time points of 4 h, 8 h, 12 h and 24 h were set up to detect the changes of 伪 hemolysin expression in bacteria and culture supernatant, and the level of hemolytic activity in bacteria culture supernatant. The expression of staphylococcus aureus transcription regulator AgrA was detected by fluorescence quantitative PCR under the subinhibitory concentration of 尾 lactams. Results: 1 there were differences in the transcription level of 伪 -hemolysin mRNA, the level of protein translation in supernatant and the hemolytic activity of culture supernatant among different Staphylococcus aureus strains. The growth of Staphylococcus aureus was not inhibited by 尾 -lactams, but the expression of 伪 -hemolysin was significantly up-regulated. After 8 h treatment, the expression of protein in the supernatant of 伪 hemolysin mRNA, could be upregulated significantly, and the hemolytic effect of culture supernatant on erythrocyte was increased. (3) 尾 -lactam could significantly up-regulate the expression of AgrA, the expression of 伪 -hemolysin mRNA, after the treatment of staphylococcus aureus with subinhibitory concentration of 尾 -lactam. The results showed that the expression of 伪 -hemolysin mRNA could be upregulated by AgrA. Conclusion: 尾 lactams can significantly up-regulate the expression of 伪 hemolysin, a pathogenic substance of Staphylococcus aureus, and its mechanism may be regulated by the expression of bacterial transcription regulator AgrA.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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