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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

黄斯旖 龚雅利 周大鹏 蒋玲丽 向丽娟 陈泽慧 王达利 黄广涛

黄斯旖, 龚雅利, 周大鹏, 等. 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[J]. 中华烧伤与创面修复杂志, 2022, 38(12): 1140-1147. DOI: 10.3760/cma.j.cn501120-20210930-00343.
引用本文: 黄斯旖, 龚雅利, 周大鹏, 等. 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[J]. 中华烧伤与创面修复杂志, 2022, 38(12): 1140-1147. DOI: 10.3760/cma.j.cn501120-20210930-00343.
Huang SY,Gong YL,Zhou DP,et al.Plasmids carried by carbapenems-resistant Klebsiella pneumoniae in burn patients and its correlation with strain transmission[J].Chin J Burns Wounds,2022,38(12):1140-1147.DOI: 10.3760/cma.j.cn501120-20210930-00343.
Citation: Huang SY,Gong YL,Zhou DP,et al.Plasmids carried by carbapenems-resistant Klebsiella pneumoniae in burn patients and its correlation with strain transmission[J].Chin J Burns Wounds,2022,38(12):1140-1147.DOI: 10.3760/cma.j.cn501120-20210930-00343.

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

doi: 10.3760/cma.j.cn501120-20210930-00343
基金项目: 

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 2020-39

遵义市科技局联合基金 HZ-2019-50

详细信息
    通讯作者:

    黄广涛,Email:haitao3140@sina.com

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

Funds: 

Youth Science Fund Project of National Natural Science Foundation of China 81801916

Regional Science Fund Project of National Natural Science Foundation of China 81960353

China Postdoctoral Science Foundation 2020M670112ZX

Collaborative Innovation Center of Chinese Ministry of Education 2020-39

Zunyi City Science and Technology Bureau Joint Fund HZ-2019-50

More Information
  • 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-1bleMBLT4SS、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。

     

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  • 1  26株从烧伤患者临床相关标本分离的CRKP携带质粒的琼脂糖凝胶电泳图谱。1A. 1~9及11~24号CRKP;1B.10、25、26号CRKP

    注:条带上方M为marker,1~26为耐碳青霉烯类抗生素肺炎克雷伯菌(CRKP)菌株编号

    2  转化菌株与TOP10菌株涂布含有100 μg/mL氨苄西林的固体LB培养基过夜后生长情况及转化菌株和3号CRKP携带质粒的琼脂糖凝胶电泳图谱。2A.转化菌株大量菌落生长;2B.TOP10菌株无菌落生长;2C.电泳图谱

    注:TOP10菌株为感受态大肠埃希菌TOP10菌株,转化菌株为从烧伤患者临床相关标本分离的3号耐碳青霉烯类抗生素肺炎克雷伯菌(CRKP)携带质粒与TOP10菌株共培养后所得;条带上方M为marker,3指3号CRKP,3*指转化菌株

    3  转化菌株TOP10-pKP03携带质粒(命名为pKP03-NDM1)的基因图谱

    注:每条线指示1个开放阅读框,不同颜色代表编码蛋白不同功能分类,未标注颜色部分为未知功能片段;GC中G为鸟嘌呤、C为胞嘧啶,GC偏移+表示G的含量大于C,GC偏移-表示G的含量小于C,其他功能包括单链DNA结合蛋白、松弛酶、甘露糖-6-磷酸异构酶等;TOP10-pKP03由从烧伤患者临床相关标本分离的3号耐碳青霉烯类抗生素肺炎克雷伯菌携带质粒与TOP10菌株共培养后所得

    4  PCR法及琼脂糖凝胶电泳检测26株CRKP的blaNDM-1基因(204 bp)携带情况。4A.3号CRKP;4B.1、2、4~11、13~20号CRKP;4C.12、21~26号CRKP

    注:条带上方M为marker,1~26为耐碳青霉烯类抗生素肺炎克雷伯菌(CRKP)菌株编号,blaNDM-1为新德里金属β-内酰胺酶-1

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  • 收稿日期:  2021-09-30

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