Whole-genome sequencing and virulence characteristics of a community-acquired lower extremity emphysematous gangrene causing Klebsiella pneumoniae belonged to ST660 / 中华检验医学杂志

ABSTRACT

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% (LD 50) 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 5 377 071 bp, belonging to ST660 type, and the capsular type was K16. A IncFIB(K)/HI1B virulence plasmid (207 506 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 lgLD 50 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.