CRISPR/Cas9系统清除细菌多药耐药基因IMP-4和KPC-2的机制  

Clearance mechanisms of bacterial multidrug resistance gene IMP-4 and KPC-2 by CRISPR/Cas9 system

作　　者：黄俊 刘鸿博 丁康慧 邱少富 杜昕颖 包仁龙 宋宏彬 袁正泉 HUANG Jun;LIU Hong-bo;DING Kang-hui;QIU Shao-fu;DU Xin-ying;BAO Ren-long;SONG Hong-bin;YUAN Zheng-quan(School of Public Health,Anhui Medical University,Hefei,Anhui 230000,China;不详)

机构地区：[1]安徽医科大学公共卫生学院职业卫生与环境卫生学系,安徽合肥230000 [2]中国人民解放军疾病预防控制中心传染病防控科,北京100000 [3]安徽医科大学公共卫生学院流行病与卫生统计学系,安徽合肥230000

出　　处：《中华医院感染学杂志》2024年第12期1767-1772,共6页Chinese Journal of Nosocomiology

基　　金：国家自然科学基金资助项目(32141003)。

摘　　要：目的利用CRISPR/Cas9系统对细菌多药耐药基因IMP-4、KPC-2进行清除,恢复抗菌药物的杀菌效力.方法采用分子克隆方法构建CRISPR/Cas9质粒.制备耐药菌感受态细胞,采用质粒转化的方法将CRISPR/Cas9系统递送至耐药菌.利用菌落聚合酶链式反应(PCR)检测耐药基因携带情况.采用实时荧光定量PCR检测各个CRISPR/Cas9靶点针对耐药基因的清除效率.采用Sensititre药敏板法及E-test药敏试纸条法检测细菌药敏表型.结果针对IM4P-4、KPC-2构建携带目的sapcer的CRISPR/Cas9质粒;菌落计数结果表明耐药基因被CRISPR/Cas9系统清除,清除率为100.00%(5/5);对细菌耐药基因进行相对定量分析,结果显示各个靶点的清除效率在24 h即达到99.9%;药敏鉴定实验结果表明实验组细菌恢复对抗菌药物的敏感性,而对照组细菌依旧对抗菌药物耐药.在药敏Etest试纸条中,与对照组相比,实验组细菌哌拉西林的最小抑菌浓度(MIC)值减少约204.8倍(P<0.05),恢复了细菌对抗菌药物的敏感性;CRISPR/Cas9系统能阻断细菌通过转化途径对耐药质粒的获取,且阻断效率高达99.9%(P<0.05).结论利用CRISPR/Cas9系统快速清除细菌耐药基因IMP-4、KPC-2,恢复抗菌药物的杀菌效力,展现了极大的临床应用价值.OBJECTIVE To restore the bactericidal efficacy of antimicrobial drugs by removing the multi-drug resistance genes IMP-4 and KPC-2 using the CRISPR/Cas9 system.METHODS The CRISPR/Cas9 plasmid was constructed using molecular cloning method.The competent cells of drug-resistant bacteria were prepared,and the CRISPR/Cas9 system was delivered to the drug-resistant bacteria by plasmid transformation.Bacterial colony polymerase chain reaction(PCR)was used to detect the presence of drug resistance genes.The clearance efficiency of each CRISPR/Cas9 target for drug resistance genes was determined by real-time fluorescence quantitative PCR(RT-qPCR).Drug sensitive phenotypes of bacteria were determined by Sensititre drug sensitive plate and E-test drug sensitive strip.RESULTS The CRISPR/Cas9 plasmids carrying the target spacer was constructed for IMP-4 and KPC-2.The results of colony counting showed that the drug-resistant genes were eradicated by CRISPR/Cas9 system,and the clearance rate was 100.00%(5/5).The relative quantitative analysis of bacterial drug resistance genes showed that the clearance efficiency of each target site reached 99.9%within 24 hours.Drug sensitivity test results showed that the bacteria in the experimental group restored their sensitivity to antibiotics,while the bacteria in the control group remained resistant to antibiotics.In the E-test strip experiment,the minimum inhibitory concentration(MIC)of piperacillin in the experimental group was reduced by about 204.8 times compared with the control group(P<0.05),which restored the bacterial susceptibility to the antimicrobial drug.The CRISPR/Cas9 system was able to block bacterial acquisition of drug-resistant plasmids through the transformation pathway,and the efficiency of blockage was as high as 99.9%(P<0.05).CONCLUSION The rapid removal of bacterial drug-resistant genes IMP-4 and KPC-2 to restore the bacterial efficacy of antimicrobial drugs using the CRISPR/Cas9 system demonstrated great clinical application value.

关 键 词：CRISPR/Cas9 细菌耐药性 多药耐药基因 耐药性清除 

分 类 号：R378[医药卫生—病原生物学]