泛耐药肺炎克雷伯菌毒力和耐药性分析

Analysis of virulence and drug resistance of pan drug resistant Klebsiella pneumoniae

目的明确2株不同来源泛耐药肺炎克雷伯菌耐药基因、毒力因子及同源性。方法2株泛耐药菌均于2019年1月从2位绍兴市人民医院住院患者的尿液中分离得到,采用微量肉汤稀释法对菌株进行最低抑菌浓度(MIC)检测,EDTA和PBA协同试验筛查碳青霉烯酶表型。通过三代测序平台进行基因组测序并对耐药基因和毒力因子筛选,对测序结果进行拼接、组装以及注释等。多位点序列分型(MLST)技术对菌株进行基因分型。结果除了替加环素和多黏菌素,2株菌对包括碳青霉烯类在内的大部分抗菌药物均耐药,EDTA和PBA协同试验提示可能携带碳青霉烯酶。2株菌均携带有1个染色体及2个质粒,染色体基因相差1192 bp,质粒基因数量完全相同。检测到SHV-28、DHA-18、NMC、SPM、AIM等β内酰胺类耐药基因,AcrA-AcrB-TolC等外排系统及OmpK35和OmpK36外膜蛋白缺失,毒力因子包括铁摄取系统、菌毛、荚膜、内毒素、二元调控系统等。MLST分型均为ST15。结论β内酰胺类耐药基因合并外膜蛋白丢失是细菌对碳青霉烯类耐药的主要原因,首次在本地区肺炎克雷伯菌中检测到NMC、SPM、AIMβ内酰胺类耐药基因。院内可能存在泛耐药肺炎克雷伯菌克隆传播。

Objective To identify resistance genes,virulence factors and homology of two strains of pan drug resistant Klebsiella pneumoniae with different origins.Methods Two strains of pan drug resistant bacteria were isolated from the urine of two inpatients in early 2019.The minimal inhibitory concentration of the strain was determined by broth dilution,and the carbapenemase phenotype was screened by EDTA and PBA.Using the third-generation sequencing platform for genome sequencing and screening of drug-resistant genes and virulence factors,the sequencing results were spliced,assembled and annotated.Multilocus sequence typing(MLST)was conducted for genotyping of strains.Results The two strains were resistant to most antibiotics including carbapenems except tigecycline and polymyxin.The synergistic test of EDTA and PBA suggested that carbapenemase might be carried.The two strains all carried one chromosome and two plasmids.The difference of chromosome genes was 1192 bp,and the number of plasmids was the same.β-lactam resistance genes such as SHV-28,DHA-18,NMC,SPM and AIM,efflux system such as AcrA-AcrB-TolC,and outer membrane protein deletions of OmpK35 and OmpK36 were detected.The virulence factors included iron uptake system,pili,capsule,endotoxin and binary regulatory system.All MLST types were ST15.Conclusionβ-lactam resistance genes combined with the loss of outer membrane proteins are the main reasons for the resistance of bacteria to carbapenems.NMC,SPM and AIMβ-lactam resistance genes are detected in Klebsiella pneumoniae for the first time.There may be clonal transmission of pan drug resistant Klebsiella pneumoniae.