铜绿假单胞菌菌毛及羧苄青霉素抗性关系初探

发布时间：2018-03-10 19:53

  本文选题：铜绿假单胞菌　切入点：临床菌株　出处：《西北大学》2017年硕士论文　论文类型：学位论文

【摘要】：近年来,随着多重耐药菌株(Multiple Drug-resistant Strains,MDR)的频频出现,越来越多的现有抗生素逐渐失去效用,细菌性疾病再次严重威胁人类健康。铜绿假单胞菌(Pseudomonas aeruginosa,PA)是人类机会病原菌,可以引起各种严重感染且常常对病人的生命造成威胁。MDR铜绿假单胞菌引起的感染在临床上非常难以治疗,这使其成为对人类健康威胁最大的病原菌之一。研究PA抗药性机理不仅具有重要的理论价值也具有紧迫的临床意义。本实验室前期研究发现,PA四型菌毛(Type Ⅳ Pili,T4P)基因pilY1的转座突变体(PA4554M)对羧苄青霉素(Carbenicillin,Car)抗性比野生型增加了 128倍;而另一个T4P基因pilE的转座突变体对羧苄青霉素的抗性也提高了16倍。这暗示铜绿假单胞菌T4P与羧苄青霉素抗性有着某种联系。本论文在此基础上对T4P与羧苄青霉素抗性的关系进行了初步研究。首先,对实验室已有的PA突变体菌株进行筛选,得到蹭行(Twitching)降低的敲除突变体菌株5株,转座突变体7株,对这12株突变体的羧苄青霉素抗性检测发现,其中5株突变体的羧苄青霉素抗性比野生型有增加,7株突变体的羧苄青霉素抗性比野生型降低。之后,我们挑选了 16种不同类抗生素对这些突变体的抗生素抗性做了滤纸片实验,发现只有羧苄青霉素抗性在12株菌中改变一致,这说明在PA中,T4P特异性影响Car的抗生素抗性,与其它抗生素的抗性没有相关性联系。其次,本研究利用临床菌株,对PA运动性与抗生素抗性的关系进行了分析。我们从西安地区的医院收集了 89株PA临床菌株。对它们的蹭行运动和抗生素抗性表型进行了调查。根据统计有70.78%临床菌株twitching相对于实验室菌株PAO1有所降低;29.22%临床菌株twitching不变;测定Car抗性发现,与PAO1相比,敏感菌株2株,占2.24%,抗性不变的菌株3株,占3.37%,其余84株均表现为抗性,占93.38%,个别临床菌株Car的MIC甚至达到了 8 mg/ml。这些结果再次表明,在临床菌株中,铜绿假单胞菌T4P与羧苄青霉素抗性似乎存在着某种联系,但这种联系的机理需要进一步的研究。最后,为了 了解这种联系的机理,我们对T4P重要的功能、结构基因进行缺失敲除,得到突变体:△pilZ、△pilB、△pilJ、△pilS、△pilE、△fimX、△pilD和△pilY1,所有突变体的twitching运动表型明显丧失,但是,羧苄青霉素抗性却没有变化,我们推测,也许只有特定的pilY1突变体才导致对羧苄青霉素抗性。pilYp的转座突变体对羧苄青霉素的抗性可能取决于转座子插入的特殊位点,造成PilY1特殊的变化。因此T4P与羧苄青霉素抗性之间潜在联系的具体机理还需要后期进一步的研究。本论文的这些结果为进一步研究PA羧苄青霉素抗性的产生机理奠定了初步基础。
[Abstract]:In recent years, with the frequent emergence of multiple Drug-resistant strains, more and more existing antibiotics have gradually lost their efficacy, and bacterial diseases are once again a serious threat to human health. Pseudomonas aeruginosa PAA is a human opportunistic pathogen. Can cause a variety of serious infections and often threaten the lives of patients. Infection caused by Pseudomonas aeruginosa is very difficult to treat clinically. This makes it one of the most dangerous pathogens to human health. Studying the mechanism of PA resistance is not only of important theoretical value but also of urgent clinical significance. The resistance to Carbenicillinus carbenicillinus (Carbenicillin), a transgenic mutant of pilY1, was 128-fold higher than that of wild type. The resistance of another transposed mutant of T4P gene pilE to carbenicillin was also increased by 16 times. This suggests that Pseudomonas aeruginosa T4P has some relationship with resistance to carbenicillin. The relationship of element resistance was studied. First of all, Five knockout mutants and 7 transposed mutants were obtained by screening the PA mutants in the laboratory, and the resistance to carboxylpenicillin of the 12 mutants was detected. The resistance to Carbenicillin of 5 mutants was higher than that of wild type, and the resistance of 7 mutants was lower than that of wild type. We selected 16 different kinds of antibiotics for the antibiotic resistance of these mutants. We found that only the resistance to carbenicillin changed consistently in 12 strains, which indicated that T4P had specific effect on the antibiotic resistance of Car in PA. There is no correlation between the resistance of other antibiotics and the resistance of other antibiotics. Secondly, the clinical strains were used in this study. The relationship between exercise resistance and antibiotic resistance of PA was analyzed. 89 clinical strains of PA were collected from hospitals in Xi'an area. Compared with the laboratory strain PAO1, the clinical strain twitching decreased by 29.22% and the clinical strain twitching did not change. Compared with PAO1, 2 strains (2.24%) were sensitive to Car resistance, 3 strains (3.37%) were resistant, the other 84 strains showed resistance, accounting for 93.38%. The MIC of individual clinical strains Car even reached 8 mg / ml. In clinical strains, Pseudomonas aeruginosa T4P and Carbenicillin resistance seem to have some relationship, but the mechanism of this connection needs further study. Finally, in order to understand the mechanism of this connection, we are important to the function of T4P. The mutant: pilZ, pilB, pilJ, pilS, pilE, FimX, pilD and pilY1, all the mutants have obviously lost their twitching motional surface, but the resistance to Carbenicillin has not changed, we speculate. It may be that only a specific pilY1 mutant causes resistance to Carbenicillin. PilYp's transposable mutants may depend on the particular site of transposon insertion. Therefore, the specific mechanism of the potential relationship between T4P and resistance to carbenicillin needs to be further studied. These results in this paper are useful for further study of the mechanism of resistance to PA Carbenicillin. A preliminary foundation was laid.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378
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本文编号：1594831