社区获得性致下肢气性坏疽ST660型肺炎克雷伯菌全基因组测序及毒力特征分析

Whole-genome sequencing and virulence characteristics of a community-acquired lower extremity emphysematous gangrene causing Klebsiella pneumoniae belonged to ST660

目的对1株致下肢气性坏疽肺炎克雷伯菌(KPN)进行全基因组测序及毒力特性分析,为认识致社区获得性重症感染KPN分子毒力特征提供参考。方法患者于2018年3月13日进入急诊科治疗,主要临床症状为高热、左下肢气性坏疽及糖尿病酮症酸中毒。取患者脓液标本进行分离培养、菌株鉴定、高黏表型及药敏检测。对菌株进行全基因组测序并分析其多位点序列分型、荚膜分型、质粒分型、毒力及耐药基因。通过接合及消除试验分析质粒特征。血清杀菌试验及大蜡螟感染模型分析菌株毒力表型,双样本t检验比较待测菌株和对照菌株对大蜡螟幼虫半数致死率(LD50)差异。结果检出菌株KPN41053,仅对氨苄西林耐药,高黏表型阴性。KPN41053染色体总长5 377 071 bp,属于ST660型,荚膜型为K16;携带一个IncFIB(K)/HI1B型毒力质粒(207 506 bp),质粒包含多种毒力因子且序列与pLVPK高度相似,但其中rmpA和rmpA2均为假基因。该质粒接合试验阴性。通过质粒消除获得变异株KPN41053PC。毒力表型分析显示,KPN41053为血清抗性(6级),而KPN41053PC为血清中度敏感(3级),KPN41053对大蜡螟幼虫的lgLD50与高毒力对照株(ST23-K1型)差异无统计学意义(t=0.32,P=0.765),低于KPN41053PC(t=5.97,P=0.004)。结论 KPN41053为1株属于ST660型且高黏表型阴性的非典型高毒力KPN,毒力质粒为其关键毒力因子。KPN可导致糖尿病患者发生社区获得性气性坏疽。

Objective The whole-genome sequencing and virulence characteristics analysis of a Klebsiella pneumoniae isolate that caused lower limb gas gangrene were performed to provide a reference for the comprehensive understanding of molecular virulence characteristics of K.pneumoniae causing severe community-acquired infection.Methods The patient was admitted to the emergency department for treatment on March 13,2018.The main clinical symptoms of the patients were high fever,gas gangrene of the left lower limb,and diabetic ketoacidosis.The pus specimen was collected for the bacterial culture,isolates identification and antimicrobial susceptibility testing.Hypermucoviscous phenotype was detected by string test.The whole genome of the isolate was sequenced and the multi-site sequence typing,capsular serotyping,plasmid characteristics,virulence and antimicrobial resistance genes of the isolate were analyzed.Plasmid curing and conjugation experiments were used to analyze plasmid characteristics.The virulence of the strain was assessed by serum killing and Galleria mellonella lethality assays.A two-sample t-test was used to compare the differences in the lethal dose of 50%(LD50)between the tested strains and reference strains against the G.mellonella larvae.Results K.pneumoniae strain KPN41053 was identified,it was only resistant to ampicillin and was negative for hypermucoviscous phenotype.Whole genome sequencing showed that the length of KPN41053 chromosome was 5377071 bp,belonging to ST660 type,and the capsular type was K16.A IncFIB(K)/HI1B virulence plasmid(207506 bp)with a sequence that was highly similar to pLVPK was harbored by KPN41053.The plasmid carried a variety of virulence genes,among which rmpA and rmpA2 were pseudogenes.The plasmid could not be transferred horizontally by conjugation.The variation strain KPN41053_PC was obtained by plasmid curing.Serum killing analysis showed that KPN41053 was serum resistant(Grade 6),and KPN41053_PC was serum intermediately sensitive(Grade 3).The lgLD50 of KPN41053 had no difference with that of the hypervirulent control strain(ST23-K1 type)(t=0.32,P=0.765),and was significantly lower than that of KPN41053_PC(t=5.97,P=0.004).Conclusions KPN41053 was an atypical hypervirulent K.pneumoniae that belonged to ST660 but without a hypermucoviscous phenotype.The virulence plasmid harbored by KPN41053 was its key virulence factor.Hypervirulent K.pneumoniae can lead to community-acquired gas gangrene in diabetic patient,which deserves clinical attention.