随着抗生素的不断发展与广泛应用，细菌耐药尤其是多重耐药问题日趋严重，耐药菌和超级细菌不断出现，给临床治疗提出很大挑战。中国CHINET 2006-2011年间对临床细菌耐药性监测数据显示，大肠杆菌居于首位，其耐药性的变迁和现状倍受关注。细菌耐药机制的研究是指导药物作用靶点设计、新药研发及正确、合理使用抗生素的重要依据。国内外研究证实，大肠杆菌耐药机制复杂，其多重耐药表型的产生是内源性全局调控网络变化的结果，与自身的耐药调控因子密不可分。因此，深入研究耐药调控基因及其分子调控机制是控制耐药性产生的关键。本研究拟以构建的高容量多重耐药大肠杆菌突变株文库为基础，从中筛选耐药谱发生改变的突变株，进而从全基因组水平上鉴定大肠杆菌多重耐药调控基因，并对其功能及可能参与调节的耐药机制进行探讨，为进一步研究多重耐药调控基因的耐药机制和分子调控机理奠定基础，对药物作用靶点设计及药效增强剂的开发具有重要的意义

Along with the continuous development and extensive use of antibiotics, bacterial resistance especially multi-resistance problem has become increasingly serious, resistant bacteria and super bacteria appear constantly, which put forward a big challenge to the clinical treatment. The monitoring results of bacteria resistance from 2006 to 2011 by China CHINET showed that Escherichia coli was in the first place, and the changes of its resistance status have attracted much attention. At present, the research of bacterial resistance mechanisms has become an important field of bacteriology, and it is also the guidance for the research and development of new antibacterial drugs and the use of antibiotics correctly and rationally. Many studies have confirmed that the resistance mechanism of E. coli is very complex. The appearance of resistant bacteria phenotype is the result of the changed endogenic regulatory network of themselves that closely related to their own resistance genes or regulatory factors. So, further study of the resistance genes and their molecular regulatory mechanisms is the key to control bacteria resistance. In the present study, we take the high capacity transposon mutagenesis library of multi-drug resistant E. coli as the research foundation, then to identify the multiple resistance regulatory genes at whole genome level and to study the gene function and the resistance regulatory mechanism. The study results will lay the foundation for the resistant molecular regulatory mechanism research, and will be of great significance for the design of drug targets and the development of efficacy enhancer for antibacterial agents.

随着抗生素的不断发展与广泛应用，细菌耐药尤其是多重耐药问题日趋严重，耐药菌和超级细菌不断出现，给临床治疗提出很大挑战。国内外研究表明，大肠杆菌耐药机制复杂，其多重耐药表型的产生是内源性全局调控网络变化的结果，与自身的耐药调控因子密不可分。因此，深入研究耐药调控基因及其分子调控机制是控制耐药性产生的关键。为此，本研究以多重耐药大肠杆菌为基础，通过转座子突变技术构建了含4万株突变株的文库，利用抗生素培养板对5567株突变株进行了筛选，获得与亲本菌株相比耐药性发生变化菌株80株，经套式PCR、核苷酸测序及序列比对确定敏感突变株中转座子的插入位点即被破坏的基因共计15个，包括4个转录调控因子，其中DksA和DeoT未见报道。通过Red同源重组系统构建了dksA和deoT基因缺失菌株。MIC测定结果显示，与亲本多重耐药大肠杆菌E8相比，基因缺失株E8ΔdksA和E8ΔdeoT MIC出现不同程度的降低；进而提取了基因缺失株RNA，测定了转录组水平。结果显示，与亲本菌株E8相比，E8ΔdksA株出现168个差异表达基因，其中81个上调基因，87个下调基因；E8ΔdeoT株出现118个差异表达基因，其中76个上调基因，42个下调基因；重点对E8ΔdksA差异表达基因进行功能注释和聚类分析发现，多数基因编码产物为代谢、转录调控因子、二元调控系统、跨膜及转运等相关蛋白，其中包括已经证实的关键多重耐药因子MarR、MdfA、OppA 和OppD 等。因此，本研究从全基因组水平上筛选和鉴定了肠杆菌多重耐药调控基因，并对其功能及可能参与调节的耐药机制进行初步探讨。本研究共发表相关论文3篇，对照项目约定的研究任务书，完成了任务书规定的研究内容、目标和预期指标。