鲍曼不动杆菌突变株中耐多粘菌素相关基因的定位与验证

发布时间：2018-12-13 19:09

【摘要】：鲍曼不动杆菌(Acinetobacter baumannii)是今年WHO刚刚公布的“耐药性最强的TOP12”中的第一位,其对几乎所有结构类别的抗生素产生耐药。多粘菌素被认为是治疗鲍曼不动杆菌的“最后一道防线”,但多粘菌素治疗鲍曼不动杆菌感染亦存在诸多问题。尽管临床分离到的鲍曼不动杆菌均表现为多粘菌素敏感型,但采用多粘菌素治疗鲍曼不动杆菌感染的失败率高达35%,表明医院内的治疗失败与传统的耐药菌无关,可能与多粘菌素剂量不足引起细菌产生适应性耐药相关。因此,我们猜测鲍曼不动杆菌基因组可能存在“耐药抑制基因”,在改变外在环境的情况下,“耐药抑制基因”被“沉默”了,所以细菌变得耐药。为了验证“耐药抑制基因”这个想法,我们联合mariner转座和多粘菌素筛选到一些耐药突变株,定位并证明“耐药抑制基因”的存在,为后续耐药机制的研究打下基础。本研究以利用mariner转座系统联合多粘菌素构建的耐多粘菌素的突变株为研究样本,首先采用反向PCR技术定位出部分耐多粘菌素突变株的mariner转座子插入位点,将被中断的耐药相关基因克隆入回补载体(pSUTetAB)中,然后将各自的回补质粒转入相应的突变株中,检测突变株和回补突变株对多粘菌素的最低抑菌浓度(MIC)的变化,找出耐药相关性较好的基因,采用RecETab重组系统定点敲除该基因,进一步通过表型验证该基因位点与耐药相关性。最后采用细菌双杂交技术对该位点进行耐药机理的初步探索。本研究成功采用反向PCR技术定位出耐多粘菌素突变株的6个转座插入位点,通过构建相应的回补质粒,比较突变株和回补突变株的多粘菌素MIC变化,找出与耐药相关性较好的DJ41_1093(anti-anti-σ)位点,并进一步利用RecETab重组系统定点敲除该位点基因验证其耐药相关性。此外,通过基因组标注信息,找出与DJ41_1093可能互作的位点DJ41_848(anti-σ),利用细菌双杂交实验验证密码子优化前后两个基因之间的互作关系。初步实验结果显示,两个基因之间未产生关联性。
[Abstract]:Acinetobacter baumannii (Acinetobacter baumannii) was the first of the "most resistant TOP12" recently announced by WHO this year, producing resistance to almost all structural classes of antibiotics. Polymyxin is considered to be the "last line of defense" in the treatment of Acinetobacter baumannii, but there are many problems in the treatment of Acinetobacter baumannii infection by polymyxin. Although the clinical isolates of Acinetobacter baumannii showed polymyxin sensitive type, the failure rate of treatment of Acinetobacter baumannii infection with polymyxin was as high as 35%, indicating that the failure of hospital treatment was not related to the traditional drug-resistant bacteria. It may be related to the adaptive resistance of bacteria caused by insufficient dose of polymyxin. Therefore, we speculate that Acinetobacter baumannii genome may have a "drug resistance suppressor gene," in changing the external environment, the "drug resistance suppressor gene" is "silenced", so the bacteria become resistant. In order to verify the idea of "drug resistance suppressor gene", we combined mariner transposition and polymyxin to screen some drug resistant mutants, localize and prove the existence of "drug resistance suppressor gene", and lay a foundation for the further study of drug resistance mechanism. In this study, polymyxin resistant mutants constructed by mariner transposition system combined with polymyxin were used as the study samples. The mariner transposon insertion sites of some polymyxin resistant mutants were located by reverse PCR technique. The interrupted drug-resistance-related genes were cloned into the complementary vector (pSUTetAB), and then the respective complementary plasmids were transferred into the corresponding mutants to detect the changes of the minimum inhibitory concentration (MIC) of polymyxin in mutants and mutants. The gene with good correlation with drug resistance was identified, and the gene was knockout by RecETab recombination system, and the relationship between the gene locus and drug resistance was further verified by phenotypic analysis. Finally, bacterial two-hybrid technique was used to explore the drug resistance mechanism of this site. In this study, the reverse PCR technique was used to locate the six transposin insertion sites of polymyxin resistant mutants, and to compare the polymyxin MIC changes between mutants and mutants by constructing corresponding repair plasmids. The DJ41_1093 (anti- 蟽) locus, which has good correlation with drug resistance, was identified, and the drug resistance correlation was verified by knockout of the locus gene by RecETab recombination system. In addition, the possible interaction site DJ41_848 (anti- 蟽) with DJ41_1093 was found by genome tagging information, and the interaction between the two genes before and after codon optimization was verified by bacterial two-hybrid experiment. Preliminary results showed that there was no correlation between the two genes.
【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378

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