金黄色葡萄球菌生物膜形成能力比较及盐酸氨溴索协同万古霉素对其干预作用

发布时间：2018-05-16 02:34

本文选题：金黄色葡萄球菌 + 生物膜　；参考：《广西医科大学》2010年硕士论文

【摘要】： 金黄色葡萄球菌(Staphylococcus aureus, S. aureus)是常见的革兰染色阳性条件致病菌之一,是医学相关材料、呼吸机相关肺炎、支气管扩张、肺脓肿等疾病的常见病原体。在急性感染过程中,S. aureus对常用抗生素耐药与其自身特征有关；而在慢性感染过程中,其耐药则与细菌生物膜(bacterial biofilm, BF)形成关系密切。参阅近年众多国内外文献资料,目前尚无MRSA与MSSA,以及同源性S. aureus的BF形成能力差异报道；同时,目前尚无能形成BF的S. aureus国际公认菌株,为减少实验偏倚,使研究结果稳定、可靠、可重复性强,有必要筛选出具有代表性可形成BF的S. aureus,作为后继科学研究用菌株。基于此,我课题组已对广西医科大学第一附属医院临床微生物检验中心2007年12月分离出的21株S. aureus进行了耐药谱分型及蛋白A基因(spa)分型。在此基础上,本课题首先建立S. aureus的BF体外静置模型；然后,比较其BF的形成能力；最后,探讨盐酸氨溴索协同万古霉素对S. aureus生物膜的干预作用。第一部分金黄色葡萄球菌生物膜形成能力比较目的建立S. aureus的BF体外静置模型,比较临床分离21株S.aureus的BF形成能力差异。方法(1)以医学材料(聚氯乙烯)为载体,采用胰蛋白胨大豆肉汤(tryptic soy broth, TSB)培养基,建立S. aureus体外静置BF模型,分别于建模第3天和第7天,用银染、扫描电子显微镜·(scanning electron microscope,SEM)观察载体表面形成的BF。(2)探讨TSB中不同浓度葡萄糖(2.5 g／L5.0 g／L、7.5 g／L、10 g／L)对S. aureus粘附及其BF形成能力的影响。(3)采用刚果红平板法定性能形成BF的菌株,对能形成BF的S. aureus采用结晶紫染色BF半定量法,比较MRSA与MSSA、同源性S. aureus的粘附及其BF形成能力。结果(1)培养3天,载体银染后光镜下观察,可见团块状、棉絮样的BF,SEM观察载体表面可见早期BF；培养7天,载体银染后光镜下观察,可见体积更大的团块状、棉絮样的BF,SEM观察载体表面可见具有立体结构的成熟BF。(2)含5.0g／L葡萄糖的TSB,能明显促进S. aureus粘附于载体表面,并促进其BF形成；但是加入更高浓度(7.5g／L、10g／L)的葡萄糖其促进S. aureus粘附和BF形成能力并不呈递增关系。(3)临床分离21株S. aureus株中,有20株为刚果红平板法定性BF形成阳性菌株,阳性率为95.2%(20／21)。(4)MRSA的粘附及早期BF形成能力明显强于MSSA；但随着培养时间的延长,至形成成熟BF结构时,MSSA的BF形成能力明显强于MRSA。(5)同源性S. aureus之间粘附及早期BF形成能力有差异：与其它S. aureus比较,编号为17546的S. aureus粘附及BF形成能力最强,编号为17422的S. aureus粘附及BF形成能力最弱,差异有统计学意义,P0.001；在成熟BF阶段,与其他S. aureus相比,编号为17546、17642的S. aureus生物膜形成能力最强,差异有统计学意义,P0.001；编号为17422的S. aureus生物膜形成能力弱,与编号为17431、18541-2、18558、18565、18719的S. aureus相比,差异无统计学意义,P0.05。结论1.以聚氯乙烯为载体,含5%葡萄糖的TSB为培养基,能成功建立S. aureus的BF体外静置模型；2.我院形成BF的S. aureus检出率高,且MRSA与MSSA之间,同源性S. aureus之间BF的形成能力有差异。第二部分盐酸氨溴索协同万古霉素对金黄色葡萄球菌生物膜破坏及协同杀菌作用目的探讨盐酸氨溴索协同万古霉素对S. aureus的BF破坏作用,以及协同万古霉素对BF内S. aureus的杀菌作用。方法(1)首先检测万古霉素对S. aureus的MIC、MBC。(2)分为空白对照组、盐酸氨溴索组、万古霉素组、盐酸氨溴索+万古霉素组。于建模第3天、第7天分别加入上述药物及其组合,药物作用24小时后,SEM、银染法观察载体表面BF；药物作用24小时后,采用MTT法行载体表面活菌计数。结果(1)万古霉素对S. aureus的MIC、MBC分别是1μg／mL、2μg／mL。(2)无论是培养3天,还是培养7天,药物作用24小时后,SEM观察发现,万古霉素组、盐酸氨溴索+万古霉素组载体表面BF较空白对照组,BF结构明显破坏,且盐酸氨溴索+万古霉素组明显于万古霉素组。(3)无论是培养3天,还是培养7天,MTT活菌计数法结果显示,药物作用24小时后,盐酸氨溴索+万古霉素组、万古霉素组活菌数少于空白对照组、盐酸氨溴索组,且盐酸氨溴索+万古霉素组少于万古霉素组。结论盐酸氨溴索具有协同万古霉素破坏S. aureus的BF作用,并能协同万古霉素减少BF内的S. aureus。
[Abstract]:Staphylococcus aureus (Staphylococcus aureus, S. aureus) is one of the common pathogens of gram-positive positive conditions. It is a common pathogen of medical related materials, ventilator related pneumonia, bronchiectasis, and lung abscess. In the course of acute infection, the resistance of S. aureus to common antibiotics is related to its own characteristics; but in the slow process, it is slow. In the course of sexual infection, the drug resistance is closely related to the bacterial biofilm (BF).
Referring to many domestic and foreign literature in recent years, there is no MRSA and MSSA, and the difference of BF formation ability of homologous S. aureus. At the same time, there is no internationally recognized S. aureus strain to form BF, in order to reduce experimental bias and make the research result stable, reliable and repeatable, and it is necessary to screen out S. a that can represent BF. UREUS, as a successor scientific research strain.
Based on this, we have classified 21 strains of S. aureus isolated from the clinical microbiology test center of the First Affiliated Hospital of Guangxi Medical University in December 2007. On the basis of this, we first set up BF in vitro S. model of S. aureus; then, compare the formation ability of BF; finally, explore the formation ability of BF. Objective to explore the intervention effect of ambroxol hydrochloride combined with vancomycin on S. aureus biofilm.
Part 1 Comparison of biofilm forming ability of Staphylococcus aureus
Objective to establish a BF static model of S. aureus in vitro and compare the difference in BF formation ability between 21 S.aureus isolates.
Methods (1) the S. aureus in vitro BF model was established by using tryptic soy broth (TSB) culture medium as the carrier of medical material (polyvinyl chloride) and the S. aureus in vitro BF model was established. The BF. (2) was studied by silver staining, scanning electron microscope (scanning electron microscope, SEM), respectively. The effect of concentration of glucose (2.5 g / L5.0 g / L, 7.5 g / L, 10 g / L) on the adhesion of S. aureus and its BF formation ability. (3) using the legal performance of Congo red plate to form a BF strain.
Results (1) for 3 days, it was observed under the light microscope of the carrier silver dye that the early BF was visible on the surface of the BF, and the surface of the carrier was observed on the surface of the carrier. For 7 days, the bulk of the bulk, the cotton like BF, the SEM observation carrier showed the mature BF. (2) containing 5.0g / L glucose in the surface of the carrier, and the TSB of 5.0g / L was visible on the surface of the carrier. Obviously, S. aureus adhered to the surface of the carrier and promoted the formation of BF, but the glucose added to the higher concentration (7.5G / L, 10g / L) did not increase the S. aureus adhesion and BF formation ability. (3) in 21 S. aureus strains, 20 strains formed a positive strain for Congo red plate, with a positive rate of 95.2% (20 / 2). 1) (4) the adhesion and early BF formation ability of MRSA is stronger than that of MSSA, but with the prolongation of culture time and the formation of mature BF structure, the BF formation ability of MSSA is significantly stronger than that of MRSA. (5) homologous S. aureus and the ability of early BF formation. The strongest, 17422 S. aureus adhesion and BF formation ability was the weakest, the difference was statistically significant, P0.001; in the mature BF stage, the S. aureus biofilm with the number of 1754617642 was the strongest, and the difference was statistically significant, P0.001; the S. aureus biofilm numbered 17422 was weak, and the number was 17. Compared with 43118541-2185581856518719 S. aureus, the difference was not statistically significant. P0.05. conclusion 1. using polyvinyl chloride as the carrier and the TSB containing 5% glucose as the medium, the BF in vitro static model of S. aureus can be successfully established. 2. the S. aureus detection rate of BF in our hospital is high, and between MRSA and MSSA. There is a difference.
The second part is the destruction and synergistic bactericidal effect of ambroxol hydrochloride combined with vancomycin on Staphylococcus aureus biofilm.
Objective to explore the destruction effect of ambroxol hydrochloride combined with vancomycin on BF of S. aureus and the bactericidal effect of vancomycin combined with vancomycin on BF S. aureus.
Method (1) first test vancomycin S. aureus MIC, MBC. (2) divided into blank control group, ambroxol hydrochloride group, vancomycin group, ambroxol HCl + vancomycin group. After modeling for third days, seventh days respectively added to the drugs and their combination, after 24 hours of drug effect, SEM, silver staining method to observe the carrier surface BF; after 24 hours of drug effect, the use of drugs, the use of the drug The MTT method was used to count the living bacteria on the surface of the carrier.
Results (1) vancomycin for S. aureus MIC, MBC was 1 g / mL, 2 mu g / mL. (2) no matter for 3 days, or 7 days of culture, and after 24 hours of drug effect, SEM observation found that vancomycin group, ambroxol + vancomycin group carrier surface BF compared with the blank control group, BF structure obviously destroyed, and ambroxol + vancomycin group was obvious ten thousand The group of palaeomycin. (3) no matter for 3 days or 7 days of culture, the results of MTT living bacteria count showed that after 24 hours of drug action, the number of ambroxol + vancomycin hydrochloride and vancomycin group was less than that of the blank control group, ambroxol hydrochloride group and ambroxol and vancomycin hydrochloride group were less than the vancomycin group.
Conclusion ambroxol hydrochloride has synergistic effect on vancomycin destroying S. aureus, and can synergy with vancomycin to reduce S. aureus. in BF BF.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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