少见CTX-M基因型鉴定及其耐药机制研究

Since the application of the extended spectrum β-lactam antibiotics to the treatment of clinical infectious diseases, the resistant strains of ESBLs have been observed immediately. Resistant strains which produce ESBLs have recently become the common resistance in Gram-negative pathogens, of which the CTX-M-type ESBLs are the most widespread enzymes. As is shown previously, more powerful genetic systems that are capable of mobilizing CTX-M-type ESBLs genes have emerged in clinical bacteria as ISEcp and ISCR elements. And these resistance gene movement mechanisms have become common place in Gram-negative pathogens, enabling the collection of genes that together mediate cross-class antibiotic resistance. In this study, a lot of drug-resistant strains have been collected domestically, a rare CTX-M-ESBLs subtype (CTX-M-64) and a multiple resistant strain which is shared by KPC-2 and ESBLs (CTX-M-55) has been obtained by sequencing; and then combined transfer, primer extension, gene locus and removable elements annotation will be carried out to trace the source and transmission mode of the resistance gene; then enzymatic analysis will be done by means of the crystal structure and enzymatic hydrolysis ability assay. Finally, on the basis of the results we will gain a deeper understanding of molecular genetic characteristics and molecular mechanism of acquired drug-resistant genes of clinical strains, lay a foundation for epidemiological studies of clinical resistant strains, provide evidence-based basis for early experiential medication of infectious diseases, and improve the prevention and control consciousness of clinical workers in slowing down bacteria’s drug resistance.

自超广谱β-内酰胺类抗生素应用于临床感染性疾病的治疗，产超广谱β-内酰胺酶（ESBLs）的耐药菌株即迅速出现。目前产ESBLs已成为革兰阴性（G-）杆菌重要的耐药形式，其中CTX-M为ESBLs最主要的耐药基因型，移动元件ISEcp和ISCR介导了CTX-M-ESBLs耐药基因的传播与转移，促进耐药基因的聚集，介导多重耐药菌株的出现。本研究大量收集国内耐药菌株，并且在全序测定基础上发现少见型CTX-M亚型，CTX-M-64及碳青霉烯酶（KPC-2）与ESBLs（CTX-M-55）共存的多重耐药菌；试图通过接合转移实验、引物延伸实验、基因座位以及可移动元件注释追溯耐药基因的来源及传播方式；通过晶体结构和底物水解能力实验进行酶学分析；最终阐明此临床菌株获得性耐药的分子遗传特征及耐药的分子机制，为临床耐药菌株的流行病学研究奠定基础，为临床感染性疾病的早期经验用药提供循证医学依据，延缓细菌耐药。

细菌耐药是全球公共健康领域的重大挑战，已成为全球关注的公共卫生问题。而产超广谱β-内酰胺酶（ESBLs）是革兰阴性（G-）杆菌重要的耐药形式，其中CTX-M为ESBLs最主要的耐药基因型。本研究主要研究CTX-M-ESBLs介导的临床多重耐药菌株的耐药分子机制，通过耐药基因扩增及CTX-M全序测定实验鉴定了耐药基因亚型；利用接合转移实验验证质粒介导的耐药基因转移和传播方式，结果表明CTX-M基因位于耐药性质粒上，可以通过性菌毛以接合转移的方式将耐药质粒和耐药表型转移至受体菌；通过引物延伸实验验证耐药基因转录水平和调控方式，结果表明CTX-M基因的表达受上游转座子ISEcp1的影响，且其转录水平是恒定的，不受抗生素的诱导作用；最后通过耐药质粒全序测定对耐药基因座位以及可移动元件进行了注释。.从表型分析到分子试验对编码CTX-M-ESBLs的多重耐药菌进行了耐药机制研究，阐述了耐药基因传播的分析遗传学特征，验证了耐药基因转录和表达的模式；为临床感染性疾病的早期经验用药提供循证医学依据，为临床耐药菌株的防控提供了理论基础，延缓细菌耐药.