可转移遗传元件在NDM-5基因启动子的整合对其表达的影响

The emergence and spread of carbapenem resistance gene blaNDM in the world pose a serious threat to public health. To date, the study about NDM gene was mainly focused on the mechanism of transfer, the mobile genetic elements play an important role in the spread of NDM gene. When some Enterobacteriaceae and their transconjugants acquired the NDM gene, the level of carbapenem resistance was high disparity, whether the distinction was due to the integration of mobile genetic elements in the promoter of NDM gene or not? It is still unclear. To date, NDM-5 is one of the NDM variants that is the most prevalent in carbapenem resistance Enterobacteriaceae of animal origin in China. In our previous study, we have collected some NDM-5 positive Enterobacteriaceae from animals and these isolates showed difference in the level of carbapenem resistance. Thus, the aim of this study was further to screen NDM-5 positive Enterobacteriaceae with mutation in the promoter of NDM-5 gene, to clarify the characterization of the integration of mobile genetic elements in the promoter of NDM-5 gene using high-throughput sequencing technology; to elucidate the expression of NDM-5 gene influenced by the mutation in the promoter using molecular biology methods; to recover the mutated oligonucleotides in the mutation promoter and identify the key mutated position influenced the carbapenem resistance level. This study will provide important scientific evidence for the spread, control and prevention of NDM-5 gene.

碳青霉烯耐药基因blaNDM的出现和传播已对全球公共卫生造成了严重的威胁。目前关于NDM基因的研究主要聚焦在其传播机制上，可转移遗传元件对NDM基因的传播起到了重要的作用。一些肠杆菌科细菌及其接合子获得NDM基因后碳青霉烯耐药水平却表现出较大差异，是否因为可转移遗传元件在NDM基因启动子的整合引起的？无明确结论。目前NDM-5基因是国内动物源碳青霉烯耐药肠杆菌中分离率最高的一个变体。在前期研究中，我们收集了一些碳青霉烯耐药水平差异较大的动物源NDM-5阳性肠杆菌。因此本项目将进一步筛选NDM-5基因启动子突变株，通过高通量测序解析可转移遗传元件在NDM-5基因启动子的整合特征；运用分子生物学技术阐明启动子突变对NDM-5基因表达水平的影响；通过构建启动子突变回复株，寻找NDM-5基因启动子中对菌株耐药水平产生影响的关键突变位点。本研究将为NDM-5基因的转移扩散和防控提供重要的科学依据。

碳青霉烯耐药基因blaNDM的出现和传播已对全球公共卫生造成了严重的威胁。目前关于NDM基因的研究主要聚焦在其传播机制上，可转移遗传元件对NDM基因的转移起到了重要的作用。一些肠杆菌科细菌及其接合子获得NDM基因后碳青霉烯耐药水平却表现出较大差异，是否因为可转移遗传元件在NDM基因启动子的整合引起的？无明确结论。.为了探索该科学问题，本研究不但线下寻找NDM基因启动子突变株，还从线上GenBank数据库中下载了3,354株NDM阳性大肠杆菌，通过生物信息学分析，发现大部分启动子区域上游104 bp覆盖启动子的区域序列相同，仅有2株NDM-1阳性大肠杆菌的blaNDM启动子-10区序列分别为“TATAGT”以及“TACAAT”，与NDM-1上游启动子“-10”区的保守序列“TACAGT”仅有一个碱基的突变，再加上本实验室前期发现一株鸡源大肠杆菌携带的blaNDM-1正常启动子区域上游存在一个多余-35区的，共发现三株blaNDM-1启动子异常的NDM阳性大肠杆菌。把上述异常启动子及其下游的blaNDM-1开放阅读框一起连接到载体pHSG398上的EcoRI以及PstI酶切位点，转移到大肠杆菌工程菌JM109，构建三株大肠杆菌分别命名为JM109+pHSG398+P1+NDM-1，JM109+pHSG398+P2+NDM-1和JM109+pHSG398+P3+NDM-1。再构建正常启动子NDM阳性大肠杆菌作为对照，命名为JM109+pHSG398+P0+NDM-1。对上述构建的NDM阳性启动子突变株（n=3）和正常株（n=1）进行耐药表型检测，这三株启动子有差异的工程菌与对照株对美罗培南的最小抑菌浓度均为256 ug/ml，并无差异。为了进一步分析NDM阳性菌产生不同MIC的原因，本研究挑选4株对美罗培南的MIC为32 ug/ml的临床大肠杆菌以及上述四株构建的NDM-1阳性菌进行NDM质粒拷贝数的绝对定量，分析结果发现美罗培南低耐药菌株（32 ug/ml）所含质粒绝对拷贝数明显低于美罗培南高耐药菌株（256 ug/ml），初步推测NDM质粒拷贝数是导致NDM阳性菌MIC产生差异的原因。