基于CRISPR-Cas9基因编辑及微流控芯片系统研究噬菌体介导的肺炎克雷伯菌耐药性沉默机制

Drug-resistant Kelebsiella pneumoniae (KP) is one of the major culprit pathogens causing refractory community-acquired and nosocomial infections with the advent of enhancement and extensive spread of its antibiotic resistant capacity. In previous research, we found that the chromosomes and plasmids of pan-drug resistant K. pneumoniae carried different types of antibiotic resistance genes, which mainly caused antibiotic resistance and intractable infections. In addition, our investigation data suggests that antibiotic resistance of Extended-Spectrum β-Lactamases (ESBL) could be silenced by modifying ESBL gene operon element and the antibiotic resistant phenotype of K. pneumoniae could be transformed as antibiotic sensitive status. In the current proposal project, optimizing sequences are applied to effectively silence drug-resistant phenotype with CRIPSR-Cas9 gene editing technology. Then, K. pneumoniae lysogenic phage carrying antibiotic resistance silencing system will be constructed to explore the efficiency and mechanism of silencing the drug-resistant phenotypes of K. pneumoniae. Furthermore, the capacity of lysogenic phage extinguishing and silencing antibiotic resistance and its adjuvant therapeutic traits will be validated by the drug-resistant microfluidic chip detection system in vitro and infected animal models in vivo respectively. This project will provide an innovative management pathway and strategies to deal with refractory pulmonary infections caused by extensive- or pan-drug resistant K. pneumoniae.

肺炎克雷伯菌（简称肺克）是临床上引起院内外感染的重要致病菌之一，其耐药性不断增强和广泛传播导致临床管理难度不断增大。本项目组前期研究发现泛耐药肺克基因组和质粒上同时携带多种类型抗生素抗性基因是其引起对抗菌药物耐药和导致难治性感染的主要原因，并通过改变超广谱β-内酰胺酶（ESBL）耐药基因操纵子元件可以沉默ESBL的耐药性，可消减肺克的耐药表型。本研究拟利用CRISPR-Cas9基因编辑技术，设计能够有效沉默其耐药表型的基因序列，以此构建携带此序列的肺克溶原性噬菌体耐药沉默转导体系，通过感染临床分离的全耐药肺克菌株，探讨其沉默肺克耐药性的效果和机制；其次，通过微流控芯片细菌耐药检测系统的体外实时监测结果及动物感染模型的体内辅助治疗作用，研究溶原性噬菌体耐药沉默转导体系消减和沉默肺克耐药性的能力及其对机体炎症反应的影响，从而为应对广泛耐药或全耐药肺克引起的难治性肺部感染提供新的路径和治疗策略。

肺炎克雷伯菌是临床上引起院内外感染的重要致病菌之一，其耐药性不断增强导致广泛传播和临床管理难度增大。新抗生素发现越来越难，开发新的治疗策略具有广阔的应用前景。.本项目组前期研究发现泛耐药肺克基因组和质粒上同时携带多种类型抗生素抗性基因，是其引起抗菌药物耐药和导致难治性感染的主要原因。我们首先探讨了噬菌体解聚酶对肺炎克雷伯菌的辅助治疗作用。鉴于噬菌体靶向肺炎克雷伯菌具有菌株特异性，我们创新性地应用仿生纳米囊泡递送系统递送靶向耐药基因的CRISPR-Cas9质粒，该系统不仅可以应用于所有耐药性肺炎克雷伯菌，也可以应用于其它革兰氏阴性耐药菌，具有更广阔的应用前景。通过体外和体内实验，证实了该系统可以有效沉默肺炎克雷伯菌耐药基因并且逆转其耐药表型。这些研究为应对广泛耐药或全耐药肺炎克雷伯菌引起的难治性肺部感染提供新的路径和治疗策略。