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

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携带的F型质粒转化TOP10菌株大量菌落生长(转化菌株被分别命名为TOP10-pKP03、TOP10-pKP15), 成功转化比例为1/3。TOP10-pKP03携带质粒琼脂糖凝胶电泳图仅表现为1个条带, 大小与3号CRKP携带质粒最大的条带一致。TOP10菌株对所检测的17种临床常用抗生素全敏感;TOP10-pKP03和TOP10-pKP15对青霉素类、头孢菌素类、碳青霉烯类抗生素耐药, 对单环β-内酰胺类、氨基糖苷类、喹诺酮类抗生素以及替加环素敏感。TOP10-pKP03携带质粒全长为41 190 bp, 该质粒携带blaNDM-1、bleMBL、T4SS、博来霉素抗性基因且包含接合转移元件、松弛酶等片段, 与从大肠埃希菌JN24中提取的质粒pJN24NDM1长度相同且核苷酸相似性为99%。从16株(61.5%)CRKP携带质粒中检测到了blaNDM-1基因, 该16株CRKP的ST型别中, ST11型11株, ST215型、ST260型、ST395型、ST2230型、新ST型各1株;不携带blaNDM-1基因的10株CRKP的ST型别中, ST11型8株, ST395型、ST2230型各1株。结论自烧伤患者CRKP中分离测序了一个高携带率的包含blaNDM-1基因的质粒pKP03-NDM1, 该质粒可能通过介导blaNDM-1基因在CRKP间以及CRKP和大肠埃希菌间的水平转移, 导致耐药性传播。

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 genome sequence of the plasmid carried by the smallest successfully transformed strain,the polymerase chain reaction,agarose gel electrophoresis,and gene sequencing were used to detect the New Delhi metallo-beta lactamase-1(blaNDM-1)of plasmids in the remaining 25 strains of CRKP.The ST typing in multilocus sequence typing of 26 strains of CRKP was analyzed based on the literature.Results Plasmids were successfully extracted from 26 CRKP,with mass concentrations ranging from 19.3 to 189.8 ng/μL.Each of the 26 CRKP carrying plasmids showed at least one band longer than 2500 bp in the agarose gel electrophoresis,which were roughly divided into 6 patterns of A,B,C,D,E,and F.After overnight cultivation,no growth of strains was observed in LB agar medium containing ampicillin inoculated with the TOP10 strain or TOP10 strains transformed by the plasmid of CRKP patterning A,B,D,or E.In contrast,TOP10 strains transformed by the pattern C plasmid from NO.3 CRKP and the pattern F plasmid from NO.15 CRKP resulted in numerous colony growths,and those transformed strains were named as TOP10-pKP03 and TOP10-pKP15,respectively.The proportion of successful transformation was 1/3.The plasmid carried by TOP10-pKP03 showed a single band in the agarose gel electrophoresis,which was the same size as the largest band of the plasmid from NO.3 CRKP.The TOP10 strain was sensitive to the 17 antibiotics commonly used in clinic.TOP10-pKP03 and TOP10-pKP15 were resistant to penicillins,cephalosporins,and carbapenems but remained sensitive to monocyclicβ-lactam,aminoglycosides,quinolones and tigecycline.The full length of the plasmid carried by TOP10-pKP03 was 41190 bp.In addition to blaNDM-1,this plasmid carried bleMBL,T4SS,bleomycin resistance gene,conjugation transfer elements,and relaxase,etc.The plasmid showed 99%nucleotide identity similarity and the same length to the plasmid pJN24NDM1 extracted from an E.coli isolate JN24.Totally 16(61.5%)CRKP were confirmed to carrying blaNDM-1 gene,among the ST typing of the 16 strains,11 strains were ST11,while ST215,ST260,ST395,ST2230,and new ST had 1 strain each.Among the ST typing of 10 blaNDM-1-negative CRKP,8 strains were ST11,while ST395 and ST2230 had 1 strain each.Conclusions A blaNDM-1 gene carrying plasmid pKP03-NDM1 was extracted and sequenced from CRKP isolated from burn patients,with a high plasmid carrying rate.Meanwhile,this plasmid may mediate inter-CRKP and CRKP-E.coli horizontal transfer of blaNDM-1,leading to transmission of antimicrobial resistance.