从猪肉中分离的两株携带mcr-1肺炎克雷伯菌分子特征研究

Molecular characteristics of two mcr-1-producing Klebsiella pneumoniae strains from pork

目的 对2株食源性mcr-1阳性的肺炎克雷伯菌的耐药分子特征进行研究,为畜禽动物养殖过程中抗生素的合理使用及耐药菌株防控提供依据。方法 采用微量肉汤稀释法对2016年从猪肉样品中分离的2株肺炎克雷伯菌进行药敏试验,并通过聚合酶链式反应(PCR)对2株多黏菌素B耐药的肺炎克雷伯菌(编号为CP143和SJP177)进行mcr耐药基因检测,运用全基因组测序对2株肺炎克雷伯菌进行耐药基因、质粒复制子类型识别。结果 两株菌均为mcr-1阳性肺炎克雷伯菌,CP143对多黏菌素B、复方磺胺、氯霉素、四环素抗生素耐药,SJP177对多黏菌素B、氨苄西林、头孢噻肟、头孢他啶、庆大霉素、氯霉素、阿奇霉素、四环素、环丙沙星和萘啶酸抗生素耐药;脉冲场电泳(Pulse Field Gel Electrophoresis,PFGE)显示菌株之间的亲缘关系较远;全基因组序列分析结果显示CP143菌株含有IncFIB,IncFII,IncHI1B和IncX4 4个不相容群质粒,SJP177菌株含有Col,Col156,Col RNAI,IncFI,IncHI2,IncHI2A,IncR和pENTAS02 8个不相容群质粒。此外,CP143菌株携带mcr-1、sul1、flo R、tet(A)耐药相关基因,SJP177菌株携带mcr-1、bla、bla、armA、floR、mph(A/E)、msr(E)、tet(A)、qnrS1耐药相关基因。结论 2株mcr-1阳性肺炎克雷伯菌都为多重耐药菌株,并携带相关的耐药基因,研究结果提示应加强对食用畜禽养殖过程中抗生素合理使用的指导和监控。

Objective The molecular characteristics of two foodborne mcr-1-producing Klebsiella pneumoniae strains were studied to provide a basis for the rational use of antibiotics and the prevention and control of drug-resistant strains in livestock and poultry breeding.Methods The antimicrobial susceptibility testing of two Klebsiella pneumoniae isolates(CP143 and SJP177) that were collected from pork in 2011 was conducted by antimicrobial susceptibility test and PCR.The molecular typing of mcr-1 positive Klebsiella pneumoniae was carried out by PFGE.Then two strains were sequenced using the illumina HiSeq X ten platform,based on the assembled genome sequences of two strains,antimicrobial resistance genes and plasmid replicon type were performed.Results The two strains were identified to be mcr-1 positive Klebsiella pneumoniae.Drug susceptibility tests showed that CP143 resistance to Polymyxin B,Trimethoprim-sulfamethoxazole,Chloramphenicol,Tetracycline,SJP177 resistance to Polymyxin B,Ampicillin,Cefotaxime,Ceftazidime,Gentamicin,Chloramphenicol,Azithromycin,Tetracycline,Ciprofloxacin,and Nalidixic acid.PFGE clustering map showed that the genetic relationship between two strains was far from each other.The genomic.analysis results showed that the plasmid of CP143 belonged to 4 incompatible groups including IncFIB,IncFII,IncHI1 B and IncX4,and that of SJP177 belonged to 8 incompatible groups including Col,Col156,ColRNAI,IncFIA,IncHI2,IncHI2A,IncR and pENTAS02.Additionally,the multi-antimicrobial resistance genes mcr-1,sul1,floR,and tet( A) were identified on the genome of strain CP143,while mcr-1,bla,bla,armA,floR,mph(A/E),msr(E),tet(A),and qnrS1 were identified on the genome of strain SJP177.Conclusion The mcr-1-producing Klebsiella pneumoniae strains were multi-drug resistant and carried related drug resistance genes,The results suggest that the guidance and monitoring on rational use of antibiotics in food livestock and poultry production should be strengthened.