K2血清型肺炎克雷伯菌毒力及分子流行病学特征

Virulence and molecular epidemiology of K2 Klebsiella pneumoniae

目的 探讨K2血清型肺炎克雷伯菌株(K2-Kpn)的多位点序列分型(MLST)、临床特点、毒力和分子流行病学特征。方法 收集南昌大学第一附属医院2014-2020年临床标本分离的非重复肺炎克雷伯菌1 603株,使用Vitek-2 Compact全自动微生物系统进行菌株鉴定及药敏试验,“拉丝”试验检测菌株高黏液表型(HM);聚合酶链式反应(PCR)检测6种荚膜血清型、9种毒力基因和13种常见耐药基因;脉冲场凝胶电泳(PFGE)和MLST分析同源性;血清抗性、生物膜和大蜡螟试验分析毒力表型。结果 检出18株K2-Kpn, HM试验阳性14株,均不携带magA基因,其他8种毒力基因的检出率均>70.00%;K2-Kpn检出耐碳青霉烯类和产超广谱β-内酰胺酶(ESBLs)的耐药株数均为4株,PCR检出产KPC型碳青霉烯酶1株、NDM型3株,产bla型β内酰胺酶5株、bla型7株和bla型1株,另有16株携带PMQR基因;在18株K2-Kpn中发现7种MLST型别(ST14、25、65、86、374、375和4025),类ST65是最主要的MLST组别,PFGE结果显示K2-Kpn具有多克隆性;K2-Kpn对血清杀伤均有抗性(除ST374和375外),表现不同的生物膜形成能力和大蜡螟毒力。结论 K2-Kpn在医院具有多克隆流行性,类ST65为其主要的临床流行株;血清抗性,生物膜形成和毒力基因携带是预测K2-Kpn高毒力菌株的主要参数;对老年且患有严重基础疾病患者,应积极地防控K2-Kpn感染和传播。

OBJECTIVE To explore the multilocus sequencing type(MLST), clinical characteristics, virulence and molecular epidemiological characteristics of K2 serotype Klebsiella pneumoniae(K2-Kpn). METHODS Totally 1 603 strains of non-repetitive K.pneumoniae were isolated from clinical specimens of the First Affiliated Hospital of Nanchang University between 2014 and 2020, the strains were identified by Vitek-2 Compact automatic microorganism systems, the drug susceptibility testing was performed, the hypermucinous(HM) phenotypes of the strains were detected by string test, 6 capsular serotypes, 9 types of virulence genes and 13 types of common drug resistance genes were detected by polymerase chain reaction(PCR). The homology of the strains was analyzed by pulse field gel electrophoresis(PFGE) and MLST;the virulence phenotypes were analyzed by human serum bactericidal assay, biofilm formation test and G. mellonella test. RESULTS Totally 18 strains of K2-Kpn were isolated, 14 of which were positive for HM test and did not carry with magA gene, the detection rates of other 8 virulence genes were more than 70.00%. There were 4 strains of K2-Kpn that were resistant to carbapenems and 4 strains that were resistant to extended-spectrum β-lactamases(ESBLs). There were 1 strain of KPC type carbapenemase-producing K.pneumoniae, 3 strains of NDM type, 5 strains of blatype β-lactamase-producing K.pneumoniae, 7 strains of blatype and 1 strain of blatype as well as 16 strains of K.pneumoiae carrying with PMQR gene that were detected by PCR. Totally 7 MLST types( ST14, 25, 65, 86, 374, 375 and 4025) were found among the 18 strains of K2-Kpn, ST65-like type was dominant among the MLST types. The result of PFGE showed that the K2-Kpn has been polyclonal;the K2-Kpn showed resistance to serum killing(except for ST374 and 375) and varied in the biofilm formation ability and virulence of galleria mellonella. CONCLUSION The K2-Kpn has been polyclonal in the hospital, the ST65-like type is the major MLST type. The resistance to serum killing, biofilm formation, and virulence genes are the major parameters for prediction of hypervirulent K2-Kpn strains. It is necessary to actively prevent the K2-Kpn infection and spread among the elderly patients with severe underlying diseases.