D-酪氨酸联合万古霉素对MRSA及其生物膜的消除作用

发布时间：2019-01-04 10:44

【摘要】：研究背景:目前人工关节置换术已成为治疗终末期关节疾患、改善恢复关节功能的最有效方法之一。随着人工关节置换手术总数的不断增加,术后假体周围感染(prosthetic joint infection,PJI)病人的总量也在逐渐增加,而耐甲氧西林金黄色葡萄球菌(methicillin-resistant staphylococcus aureus,MRSA)感染是PJI的治疗难题。目前二期翻修手术联合万古霉素被认为是假体周围MRSA感染治疗的金标准,即便如此,术后仍然有感染复发的可能,而导致感染复发的罪魁祸首就是细菌生物膜(biofilm,BF)。当细菌粘附于物体表面时,细菌能通过分泌粘多糖等物质使彼此聚集在一起,并进一步增殖扩散,最终形成具有屏障作用的生物膜。生物膜的屏障作用有效的阻碍了抗菌药物对膜深层细菌的杀灭作用,在临床中,由于生物膜对细菌的保护作用,导致细菌对抗生素产生耐药,假体周围一旦发生生物膜的感染,抗菌药物很难杀灭致病菌。如果不能彻底消除生物膜,感染的复发将不可避免,因此,生物膜消除的研究越来越受到人们的重视。近年来有研究发现,微生物能够产生D-型氨基酸,这类氨基酸对枯草芽孢杆菌、铜绿假单胞菌、金黄色葡萄球菌生物膜具有分散作用,其中又以D-酪氨酸对细菌生物膜分散作用最强,因此研究对MRSA及其生物膜有消除的方法有重要的临床指导意义。目的:观察D-酪氨酸(D-tyrosine)联合万古霉素对体外培养MRSA及其生物膜的消除作用,旨在为PJI的临床治疗提供可行性依据。方法:1.收集广州市第一人民医院的临床MRSA菌株,微孔板法建立体外生物膜模型,通过结晶紫染色半定量法筛选出产膜能力最强的菌株,该菌株即为实验菌株。2.通过导片法建立体外MRSA生物膜模型,将实验分为四组,分别为空白组,D-酪氨酸组,万古霉素组,联合组(D-酪氨酸+万古霉素),每组干预1小时、6小时、12小时、24小时后分别用结晶紫及荧光染色,分别在高倍镜及激光共聚焦显微镜(CLSM)下观察不同组MRSA及其生物膜的变化情况。结果:1.通过微孔板及结晶紫染色法筛选出编号为2913的MRSA菌株产膜能力最强。2.万古霉素组、D-酪氨酸组、联合组中的细菌密度会随着时间的增加而逐渐减小,其中以D-酪氨酸组及联合组减少明显,且每组间具有统计学差异(P0.05)。3.激光共聚焦显微镜(CLSM)下显示,12小时内万古霉素组及联合组中死菌比例会随着时间增加而增加,其中以联合组增加更明显,且联合组与万古霉素组比较有统计学意义(P0.05),12小时后万古霉素组死菌比例无明显变化,联合组死菌比例进一步增加。结论:1.D-酪氨酸对体外培养MRSA生物膜具有分散作用;2.D-酪氨酸联合万古霉素对生物膜中的MRSA具有更好的消除作用。
[Abstract]:Background: at present, artificial arthroplasty has become one of the most effective methods for the treatment of end-stage joint diseases and the improvement of joint function. With the increase of the total number of artificial joint replacement operations, the total number of patients with periprosthetic infection (prosthetic joint infection,PJI) is also increasing gradually, and methicillin-resistant Staphylococcus aureus (methicillin-resistant staphylococcus aureus,MRSA) infection is a difficult problem in the treatment of PJI. At present, the secondary revision surgery combined with vancomycin is considered to be the gold standard for the treatment of MRSA infection around the prosthesis. Even so, there is still the possibility of recurrence of infection after operation, and the main culprit causing the recurrence of infection is bacterial biofilm (biofilm,BF). When the bacteria adhere to the surface of the object, the bacteria can aggregate each other by secreting mucopolysaccharide, and further proliferate and diffuse, and finally form a barrier biofilm. The barrier effect of biofilm effectively hinders the bactericidal effect of antimicrobial agents on bacteria in deep layer of membrane. In clinic, because of the protective effect of biofilm on bacteria, bacteria become resistant to antibiotics, and once biofilm infection occurs around the prosthesis, Antimicrobial agents are difficult to kill pathogenic bacteria. If biofilm can not be eliminated completely, the recurrence of infection will be inevitable. Therefore, the study of biofilm elimination has been paid more and more attention. In recent years, it has been found that microbes can produce D- amino acids, which can disperse the biofilm of Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus. Among them, Dtyrosine has the strongest effect on bacterial biofilm dispersion, so the study has important clinical significance for the elimination of MRSA and its biofilm. Aim: to observe the effect of D tyrosine (D-tyrosine) combined with vancomycin on the elimination of MRSA and its biofilm in vitro. Methods: 1. Clinical MRSA strains from the first people's Hospital of Guangzhou were collected. The biofilm model in vitro was established by microporous plate method. The strain with the strongest membrane capacity was screened by semi-quantitative method of crystal violet staining. The model of MRSA biofilm in vitro was established by the method of guide tablets. The experiment was divided into four groups: blank group, D-tyrosine group, vancomycin group and combined group (Dtyrosine vancomycin). After 24 hours, the changes of MRSA and biofilm of different groups were observed under high-power microscope and laser confocal microscope (CLSM) with crystal violet and fluorescence staining respectively. Results: 1. Through micropore plate and crystal violet staining method, the MRSA strain numbered 2913 had the strongest membrane production ability. 2. 2. The bacterial density in vancomycin group, D-tyrosine group and combination group decreased gradually with the increase of time, especially in Dtyrosine group and combined group, and there was statistical difference between each group (P0.05). Laser confocal microscope (CLSM) showed that the proportion of dead bacteria in vancomycin group and combined group increased with time within 12 hours, especially in combination group. And the combined group and vancomycin group compared with the statistical significance (P0.05), 12 hours after vancomycin group no significant change in the proportion of dead bacteria, the combined group further increased the proportion of dead bacteria. Conclusion: 1. D- tyrosine can disperse MRSA biofilm in vitro, 2. D- tyrosine combined with vancomycin can eliminate MRSA in biofilm.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R687.4

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