基于全基因组测序的院内耐碳青霉烯肺炎克雷伯菌耐药性及同源性分析  

Analysis of carbapenem⁃resistant and homology of hospital⁃acquired Klebsiella pneumoniae based on whole⁃genome sequencing

作　　者：余清[1] 徐敏 史桂兰[1] 曹慧玲[1] 李岷[1] 吴倩[2] 赵苏瑛[1] YU Qing;XU Min;SHI Guilan;CAO Huiling;LI Min;WU Qian;ZHAO Suying(Department of Laboratory Medicine,Jiangsu Province Hospital of Chinese Medicine,Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing,210029;School of Public Health,Nanjing Medical University,Nanjing,211166,China)

机构地区：[1]南京中医药大学附属医院检验科,江苏南京210029 [2]南京医科大学公共卫生学院,江苏南京211166

出　　处：《南京医科大学学报（自然科学版）》2023年第12期1729-1736,1763,共9页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(82073630);江苏省中医院“益中”项目(2019Y48)。

摘　　要：目的:基于基因组学研究医院内碳青霉烯耐药肺炎克雷伯菌的耐药基因分布,毒力情况及同源性分析,深入阐明院内感染的分子机制,为多重耐药菌株临床治疗和预防提供实验室依据。方法:收集1株临床分离的碳青霉烯耐药的泛耐药肺炎克雷伯菌,经培养鉴定,测定对常用抗生素的最低抑菌浓度(minimum inhibitory concentration,MIC),多位点序列分型(multi⁃locus sequence typing,MLST)进行基因分型并行全基因组测序。根据基因测序结果,选择相关耐药基因在临床收集的另外50株碳青霉烯耐药的肺炎克雷伯菌中进行扩增检测,验证其携带情况,对测序结果进行进一步的阐述。结果:药敏结果显示该菌对除粘菌素和替加环素外的抗生素全部耐药,测序结果显示该菌染色体基因序列全长5468925 bp,含有4个质粒(179972 bp、141377 bp、85181 bp和20247 bp),共有5984个蛋白质编码基因,85个tRNA基因和25个rRNA操纵子。此外,该菌携带有大量参与编码多种抗生素的耐药基因。MLST结果显示,该菌基因型为ST11型,该菌大部分序列与之前在四川和杭州报道的菌株类似,最接近的是一株编号SCKP020029(NCBI Accession number:CP029384)的肺炎克雷伯菌。收集的另外50株碳青霉烯耐药的肺炎克雷伯菌确认均为产超广谱β内酰胺酶(extented⁃spectrum beta⁃bactamases,ESBLs),其中97%为ST11型。脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)结果显示分属3个克隆。在这些菌株中,耐药基因检出率分别为KPC⁃2(98%),SHV⁃11(98%),TEM⁃1(76%),CTX⁃M(76%),Oqxb1(66%),qnrS(70%),Int1(42%),sul1(82%),sul2(96%),iutA(88%),iucABCD(10%)和rmpA2(100%)。结论:实验结果揭示了院内碳青霉烯类耐药肺炎克雷伯菌的基因组学具有高度相似性,流行病学具有聚集性和散发性交叉的特点。抗生素耐药结果提示了细菌耐药中选择性抗生素耐药压力的作用效应,同时质粒耐药基因在细菌中的传播提示这�Objective:Based on genomics,this study aims to investigate the distribution of drug⁃resistant genes,virulence factors,and homology analysis of the hospital isolated carbapenem resistant Klebislla.pneumoniae strains.The molecular mechanisms of nosocomial infections will be elucidated to provide laboratory evidence for the clinical treatment and prevention of multidrug⁃resistant strains.Methods:A clinical isolate of carbapenem⁃resistant pan⁃drug⁃resistant Klebislla.pneumoniae(KPX)was collected,cultured,and identified.The minimum inhibitory concentrations(MICs)of commonly used antibiotics were determined,and multilocus sequence typing(MLST)was performed for genetic typing,followed by whole⁃genome sequencing.Based on the results of the genetic sequencing,selected relevant resistance genes for amplification detection in an additional 50 clinical isolates of carbapenem⁃resistant KPX,verified their carriage status,and further elaborate on the sequencing results.Results:The drug sensitivity test showed that the KPX strain was resistant to all antibiotics except colistin and tigecycline.The sequencing results relevant that the chromosomal gene sequence of this bacterium is 5468925 bp in length and contains four plasmids(179972 bp,141377 bp,85181 bp,and 20247 bp).It had a total of 5984 protein⁃coding genes,85 tRNA genes,and 25 rRNA operons.Additionally,this bacterium carried a large number of resistant genes involved in encoding multiple antibiotics.MLST results indicated that the genetic type of this bacterium is ST11,and most of its sequences are similar to previously reported strains in Sichuan and Hangzhou.The closest match was a strain with the code SCKP020029(NCBI accession number:CP029384)of Klebislla.pneumoniae.The additional 50 carbapenem⁃resistant Klebislla.pneumoniae collected were confirmed to produce extended⁃spectrum beta⁃lactamases(ESBLs),with 97%being of ST11.Pulsed⁃field gel electrophoresis(PFGE)results showed that they belonged to three different clones.Among these strains,the dete

关 键 词：碳青霉烯耐药 肺炎克雷伯菌 基因组 测序 

分 类 号：R446[医药卫生—诊断学]