肺炎克雷伯菌中CRISPR调控耐药机制及其分布特征与分离地点的关系

  目的  了解呼吸道标本肺炎克雷伯菌中成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats，CRISPR）对耐药的调控机制，并分析其CRISPR分布特征与分离地点的关系。  方法  收集并提取120株肺炎克雷伯菌的基因组DNA，通过扩增CRISPR/Cas（clustered regularly interspaced short palindromic repeats/CRISPR-associated）系统相关基因CRISPR 1、CRISPR 2来确定CRISPR阳性菌株。CRISPR阳性菌株的耐药表型用BD Phoenix-100细菌鉴定仪进行检测，利用CRISPR Target寻找间隔序列同源噬菌体或质粒，并在Center for Genomic Epidemiology上查找同源质粒或噬菌体的耐药信息并检测间隔序列所在菌株的耐药基因，分析两者携带耐药基因的关系。采用CRISPR Finder分析CRISPR并运用多序列比对分析间隔序列的一致性。  结果  CRISPR1、CRISPR2阳性率分别为12.50%和13.33%；间隔序列同源质粒与其所在菌株均携带共同的耐药基因，且菌株的耐药表型与其携带的耐药基因高度符合；相同地点菌株的CRISPR分布具有极高相似性。  结论  肺炎克雷伯菌通过将外来质粒的耐药基因片段整合到菌株的基因组中实现对菌株耐药性的调控；CRISPR中间隔序列的分布与菌株分离地点密切相关，为临床治疗和感染控制工作提供理论依据。

The mechanism of CRISPR-mediated drug resistance and the relationship between the characteristics of CRISPR and isolation site in Klebsiella pneumoniae

  Objective  To understand the regulatory mechanism of clustered regularly interspaced short palindromic repeats (CRISPR) to drug resistance of Klebsiella pneumoniae in respiratory specimens, and to analyze the relationship between the characteristics of CRISPR and the location of isolation.  Methods  120 strains of Klebsiella pneumoniae were collected and genomic DNA was extracted. CRISPR-positive strains were identified by amplifying CRISPR/CRISPR-associated(Cas) related genes CRISPR 1 and CRISPR 2. The resistance phenotype of CRISPR-positive strains was detected by the BD Phoenix-100 bacterial identification instrument. CRISPR Target was used to look for homologous bacteriophages or plasmids with spacer sequence, find the drug resistance information of homologous bacteriophages or plasmids in Center for Genomic Epidemiology and detect the drug resistance genes of the strain where the spacer sequence is located, and analyze the relationship between the drug resistance genes of the two. CRISPR Finder was used to analyze CRISPR and multi-sequence alignment was used to analyze the consistency of spacer sequences.  Results  The positive rates of CRISPR 1 and CRISPR 2 were 12.50% and 13.33%; The homologous plasmids of the spacer sequence and the strains in which they were both carried common drug resistance genes, and the resistance phenotype of the strain was highly consistent with the drug resistance genes they carry. At the same location, the CRISPR distribution of the strains was extremely similar.  Conclusions  Klebsiella pneumoniae regulates drug resistance by integrating foreign plasmid resistance gene fragments into the strain's genome. The distribution of spacer sequence in CRISPR is closely related to the location of the strain isolation, which provides a theoretical basis for clinical treatment and infection control work.