烧伤患者耐碳青霉烯类抗生素肺炎克雷伯菌携带质粒及其与菌株传播的相关性  

Plasmids carried by carbapenems-resistant Klebsiella pneumoniae in burn patients and its correlation with strain transmission

作　　者：黄斯旖 龚雅利 周大鹏 蒋玲丽 向丽娟 陈泽慧 王达利 黄广涛 Huang Siyi;Gong Yali;Zhou Dapeng;Jiang Lingli;Xiang Lijuan;Chen Zehui;Wang Dali;Huang Guangtao(Department of Burns and Plastic Surgery,Affiliated Hospital of Zunyi Medical University,Zunyi 563003,China;State Key Laboratory of Trauma,Burns and Combined Injury,Institute of Burn Research,the First Affiliated Hospital of Army Medical University(the Third Military Medical University),Chongqing 400038,China;Department of Laboratory Medicine,Affiliated Hospital of Zunyi Medical University,Zunyi 563003,China)

机构地区：[1]遵义医科大学附属医院烧伤整形外科,遵义563003 [2]陆军军医大学(第三军医大学)第一附属医院全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆400038 [3]遵义医科大学附属医院检验科,遵义563003

出　　处：《中华烧伤与创面修复杂志》2022年第12期1140-1147,共8页Chinese Journal of Burns And Wounds

基　　金：国家自然科学基金青年科学基金项目(81801916);国家自然科学基金地区科学基金项目(81960353);中国博士后科学基金(2020M670112ZX);省部共建协同创新中心项目(教科技厅函【2020】39号);遵义市科技局联合基金(HZ-2019-50)。

摘　　要：目的探索烧伤患者耐碳青霉烯类抗生素肺炎克雷伯菌(CRKP)的质粒携带情况, 分析这些质粒与CRKP传播的相关性。方法采用回顾性观察性研究方法。取从陆军军医大学(第三军医大学)第一附属医院2017年1—12月收治的22例烧伤患者[男20例、女2例, 年龄(42±16)岁]临床相关标本中分离保存的26株CRKP, 对其进行编号。采用碱裂解法提取菌株质粒, 用核酸浓度检测仪测定浓度后行琼脂糖凝胶电泳观察条带情况, 并对质粒行大致分型。从各个质粒分型中选取最小编号CRKP携带质粒转化感受态大肠埃希菌TOP10菌株(以下简称TOP10菌株), 观察各转化菌株与TOP10菌株涂布含氨苄西林的固体LB培养基并过夜培养后生长情况, 计算成功转化比例。取成功转化TOP10菌株的最小编号CRKP携带质粒的转化菌株(以下简称最小成功转化菌株)与相应编号CRKP, 提取菌株携带质粒, 行琼脂糖凝胶电泳观察条带情况。取前述各成功转化菌株与TOP10菌株, 采用药物敏感试验检测菌株对17种临床常用抗生素的耐药性。取最小成功转化菌株携带质粒, 使用第二代测序技术进行基因测序, 并完成蛋白质编码基因预测、序列比对等生物信息分析, 后续根据耐药基因携带情况命名为pKP03-NDM1。根据最小成功转化菌株携带质粒全基因组序列, 采用PCR法及琼脂糖凝胶电泳与基因测序检测剩余25株CRKP携带质粒的新德里金属β-内酰胺酶-1(blaNDM-1)基因携带情况, 结合文献分析26株CRKP多位点序列分型中的ST分型。结果从26株CRKP中均提取到质粒, 质粒质量浓度为19.3~189.8 ng/μL。26株CRKP携带质粒的琼脂糖凝胶电泳均可见2 500 bp以上条带, 可大致分为A、B、C、D、E、F型6个型别。过夜培养后, 在含氨苄西林的固体LB培养基上, 未见A、B、D、E型CRKP携带质粒转化TOP10菌株或单纯TOP10菌株菌落生长, 可见3号CRKP携带的C型质粒和15号CRKP携带�Objective To explore the carrier status of carbapenems-resistant Klebsiella pneumoniae(CRKP)plasmids in burn patients and analyze the correlation of these plasmids with the transmission of CRKP.Methods A retrospective observational study was conducted.A total of 26 CRKP strains,which were isolated from the clinic-related samples of 22 burn patients(with 20 males and 2 females,aged(42±16)years)admitted to the First Affiliated Hospital of Army Medical University(the Third Military Medical University)from January to December 2017,were collected and individually numbered.The plasmids of the strains were extracted by alkali lysis.After determination of the plasmid concentration by a nucleic acid concentration detector,the agarose gel electrophoresis was used to visualize the bands,and rough plasmids typing was performed.The plasmid of the smallest numbered CRKP in each plasmid type was transformed into competent Escherichia coli(E.coli)strain Top10(hereinafter referred to as TOP10 strain).The growth of each transformed strains and a Top10 strain cultivated in ampicillin containing Luria-Bertani(LB)agar medium overnight was observed,and the proportion of successful transformation was calculated.The plasmids from the smallest numbered plasmid carrying CRKP strain of successfully transformed Top10 strains(hereinafter referred to as the smallest successfully transformed strain)and correspondingly numbered CRKP were extracted,and then,the agarose gel electrophoresis was used to visualize the bands.Aforementioned successfully transformed strains and a TOP10 strain were used for the antimicrobial susceptibility testing with 17 antibiotics commonly used in clinic.The plasmid from the smallest successfully transformed strain was sequenced using the next-generation sequencing technology.Bioinformatics analyses such as protein-coding gene prediction and protein sequence alignment were performed successively.The sequence was subsequently named pKP03-NDM1 according to the carrying of drug resistance gene.According to the whole ge

关 键 词：烧伤 感染 抗药性 多药 质粒 耐碳青霉烯类抗生素肺炎克雷伯菌 

分 类 号：R644[医药卫生—外科学] R446.5[医药卫生—临床医学]