碳青霉烯酶新基因IMP-38生物学特性、水平传播及耐药机制研究

Carbapenem-resistant Enterobacteriaceae(CRE) are being increasingly reported worldwide, representing a significant threat to public health; great efforts toward molecular mechanism study of horizontal gene transfer and antimicrobial resistance are required. IMP-38 is a novel carbapenemase gene, discovered from Klebsiella pneumoniae isolates, isolated from sputum samples from the trachea cannula of 9 newborn patients who were suffering from respiratory distress syndrome in the neonatal ward of our hospital. Dissemination of carbapenemase-producing Klebsiella pneumoniae has caused a public health crisis because carbapenemase-producing Klebsiella pneumoniae isolates can hydrolyze most of β-lactam antibiotics, including the carbapenems. What is worse, the blaIMP can be transferred to other bacterial species leading to their horizontal transmission and worldwide distribution through the horizontal gene transfer. Our previous study has verified the IMP-38 gene sequence by subcloning technique .The main contents of our study are summarized as follows:IMP-38 is expressed and purified by the construction of recombinant plasmid PET-28a(+)/IMP-38. Biological characteristics of IMP-38 is investigated using Enzyme Kinetics experiment and isoelectric focusing. The surrounding structure of IMP-38 is studied with sequencing.In order to elucidate the molecular mechnisms of carbapenem resistance in KP and provide the right information for the clinics and slow down the emergence of resistant strains, molecular biology techniques including FQ-PCR and Western blot are adpoted to analyze not only the epidemiological characteristics for carbapenem resistance, but also the differential expression of the resistance genes and the regulation sequences..The study is expected to provide scientific basis and novel perspective for clinical treatment and nosocomial infection control of multidrug-resistant and carbapenemase -producing bacterial infections, as well as the new antibiotic development.

碳青霉烯类耐药肠杆菌科细菌是全球抗感染界关注的焦点问题之一，给临床抗感染治疗带来极大挑战，抗生素长期应用难以避免产生抗药性问题，因此深入研究细菌耐药机制是当务之急。IMP-38是本课题组从肺炎克雷伯菌（KPN）中发现的新型碳青霉烯酶基因，产碳青霉烯酶可使细菌对大多数临床常用抗菌药物耐药，并可通过基因水平转移进行耐药性传播。IMP-38与其他亚型的IMP氨基酸结构不同，我们推测：该亚型的等电点及对抗菌药物的水解活性等生物学特性也不同。本项目拟采用酶动力学实验及等电点技术进行新型酶生物学特性研究；接合试验、全质粒测序、southern blot等方法分析IMP-38基因环境和水平传播机制；采用FQ-PCR和Western blot 等方法，探讨产碳青霉烯酶KPN耐药基因及调控序列等分子机制，为阐明细菌耐药过程及机制、开发治疗产碳青霉烯酶细菌感染的新型药物奠定理论基础。

碳青霉烯类耐药肠杆菌科细菌给临床抗感染治疗带来极大挑战，IMP-38是本课题组从肺炎克雷伯菌中发现的新型碳青霉烯酶基因。本课题构建原核表达质粒pET-28a(+)-IMP-38和pCold TF-IMP-38转化大肠埃希菌感受态BL21（DE3），优化可溶性重组蛋白表达条件，并通过SDS-PAGE和Western Blotting方法检测蛋白表达情况，蛋白水解实验检测IMP-38重组蛋白对碳青霉烯类抗菌药物美罗培南、亚胺培南和厄他培南的水解活性以及EDTA对这些药物的相对抑酶保护率。对携带IMP-38基因的肺炎克雷伯菌进行全基因组测序，以研究IMP-38基因遗传环境，耐药机制及基因组特征。构建BL21（DE3）-pCold TF /IMP-38-IS6100转化子,比较上述转化菌与课题组前期构建的BL21（DE3）-pCold TF/IMP-38转化子及临床分离的blaIMP-38阳性肺炎克雷伯菌其耐药基因DNA拷贝数、mRNA拷贝数、蛋白表达量及产碳青霉烯酶表型和MIC值的差异。收集本院近年分离自临床标本的对碳青霉烯类抗菌药物耐药或中介肺炎克雷伯菌，PCR和测序筛选出blaIMP-38阳性的菌株，通过多位点序列分型技术（MLST）进行分子分型。最终成功构建原核表达质粒pET-28a(+)-IMP-38和pCold TF-IMP-38，且pCold TF-IMP-38在诱导剂IPTG浓度为1mM时，15℃条件下诱导24h蛋白表达量最高； IMP-38重组蛋白对美罗培南、亚胺培南和厄他培南的水解活性分别为73.98%、65.04%和82.13%。EDTA对这三种药物的相对抑酶保护率分别为87.41%、85.17%和87.24%。BL21（DE3）-pCold TF /IMP-38-IS6100与BL21（DE3）-pCold TF/IMP-38在DNA拷贝数、mRNA拷贝数、蛋白表达量、产碳青霉烯酶表型及MIC值上无显著差异，两者的mRNA转录水平明显高于临床分离的产IMP-38菌株。515株碳青霉烯类耐药或中介的肺炎克雷伯菌中有14株携带新基因IMP-38，总检出率为2.72%，MLST分型结果显示全为ST307型。说明新型碳青霉烯酶IMP-38对碳青霉烯类抗菌药物均有较强的水解活性。本课题为阐明细菌耐药过程及机制、开发治疗产碳青霉烯酶细菌感染的新型药物奠定基础。