同时产碳青霉烯酶IMP与NDM的肺炎克雷伯菌和霍氏肠杆菌的分离及耐药性研究

Isolation of carbapenemases IMP-and NDM-producing Klebsiella pneumoniae and Enterobacter hormaechei strains and their drug resistance

目的对分别分离自我国两家医院的多药耐药菌株肺炎克雷伯菌10677和霍氏肠杆菌128379进行耐药机制研究。方法通过16SrDNA鉴定菌种,PCR筛查菌株携带的耐药基因,应用改良Carba NP法检测菌株产碳青霉烯酶的类型,接合转移和电转化试验验证携带IMP和NDM的质粒是否具有可转移性,全基因组测序获得细菌所携带的所有质粒序列,应用RAST、BLAST等对序列进行生物信息学分析。结果 10677和128379两株菌均产B类酶,且均携带碳青霉烯酶基因blaIMP和blaNDM。全基因测序结果显示10677和128379各包含两个分别携带blaIMP-4和blaNDM-1的质粒,分别为p10677-IMP、p10677-NDM、p128379-IMP和p128379-NDM。其中p10677-IMP、p10677-NDM和p128379-NDM可通过接合转移得到相应的接合子,p128379-IMP仅可通过电转的方式得到其电转化子。p10677-IMP和p128379-IMP均为IncN1型质粒,有三个共有的外源插入区,分别为包含有携带blaIMP-4的整合子In823b,IS1插入区,携带qnrS1基因的转座子Tn6292,除此外,p10677-IMP还包括携带fosA5基因的转座子Tn6412区以及IS26插入区。p10677-NDM和p128379-NDM为IncX3型质粒,包含两个共有的外源插入区,分别为ISKox3和携带blaNDM-1的耐药区,此外,p10677-NDM还包含一个独有的插入区ISEhe3。结论质粒p10677-IMP、p10677-NDM和p128379-IMP、p128379-NDM分别介导10677和128379多药耐药且均具有转移性,是这两株耐药菌传播的重要载体。

OBJECTIVE To explore the drug resistance mechanisms of multidrug-resistant Klebsiella pneumoniae10 677 and Enterobacter hormaechei 128 379 isolated from two hospitals in China.METHODS The strains were identified by 16 SrDNA,the drug resistance genes were screened by,the types of carbapenemases were detected by using modified Carba NP method.Conjugation and electrotransformation experiments were performed to verify the transferability of plasmids carrying IMP and NDM.All of the plasmid sequences in the bacteria were obtained through whole genome sequencing.The bioinformatics analysis of the plasmid sequences was performed with the use of RAST and BLAST.RESULTS The modified Carba NP test showed that both 10 677 and 128 379 produced type B carbapenemase and harbored the carbapenemases genes blaIMPand blaNDM.The result of the whole genome sequencing showed that each of 10 677 and 128 379 contained two plasmids carrying with blaIMP-4 and blaNDM-1,namely p10677-IMP,p10677-NDM,p128379-IMP and p128379-NDM.p10677-IMP,p10677-NDM and p128379-NDM could be transferred into EC600 through conjugational transfer,but p128379-IMP transferred into TOP10 through eletrotransformation rather than conjugation.Plasmids p10677-IMP and p128379-IMP were IncN1 plasmids,and each of them carried at least three accessory regions including the blaIMP-4-carrying In823 b,the IS1 remnant and the qnrS1-carrying Tn6292 region.Besides,p10677-IMP contained two additional accessory regions：the fosA5-carrying transposon Tn6412 and IS26.p10677-NDM and p128379-NDM were IncX3 plasmids.There were two accessory regions including an ISKox3 region and a blaNDM-1 region in both p10677-NDM and p128379-NDM.Besides,p10677-NDM had an additional ISEhe3.CONCLUSION The coexistence of p10677-IMP and p10677-NDM in 10677 and p128379-IMP and p128379-NDM in 128379 contribute most to their high resistance to many antibiotics.The two plasmids play important roles for transmission of drug-resistant bacteria.