| Objective: To understand the clinical and molecular characteristics of Carbapenemresistant Klebsiella pneumoniae(CRKP)in a tertiary hospital in Beijing,focusing on the molecular typing,serotypic variation and genomic evolution of CRKP in the blood stream,and to provide scientific prevention and control of CRKP infection in hospitals.Methods: There were 146 CRKP strains and 39 carbapenem-susceptible K.pneumoniae(CSKP)strains isolated from blood samples in a Beijing hospital from 2017 to 2020collected for whole-genome sequencing.Kleborate,prokka and other bioinformatics methods were used to analyze the genomic features of 185 strains.Resistance and virulence phenotypes of the strains were obtained by antibiotic susceptibility test,string test,biofilm formation capacity experiment,serum killing experiment,and Galleria mellonella virulence experiment.Whole genome sequencing data of CRKP from 2011-2020 from different sample sources were downloaded from Pathosystems Resource Integration Center(PATRIC)public database and analyzed using bioinformatics analysis software such as ABRicate.Statistical comparison analysis was conducted using R v4.2.0and IBM SPSS 23.0 software.Chi-square test and Mann-Whitney U test were used to analyze the difference of clinical information between ST11-KL47 and ST11-KL64 serotype strains.Single and multivariate logistic regression were used to analyze the risk factors associated with ST11-KL64 CRKP death outcome.Sequence typing(ST)11CRKP with complete basic information such as sample source was screened out.Chisquare test and Mann-Whitney U test were used to compare the inter-group difference between the two serotypes,and Gubbins and Phandango software were used to explore the evolutionary relationship of the serotypes.Results: A total of 185 K.pneumoniae isolates were included in this study,among which29 ST types were identified.ST11-KL47(n = 81)and ST11-KL64(n = 42)were the predominant serotype types among the CRKP isolates.All of CRKP isolates showed carbapenem-resistance mechanisms,mainly due to the acquisition of carbapenemase genes,mainly harboring KPC-2(n = 127).Patients with ST11-KL47 isolates had a broader age range [79(46,85)],and 56.3% of the ST11-KL64 K.pneumoniae infected patients died during hospitalization,which was significantly higher than the hospitalization mortality rate of ST11-KL47 infected patients(7.0%,P<0.001).Moreover,34.4% of the ST11-KL64 K.pneumoniae isolates showed a hypermucus phenotype,while none of the 71 ST11-KL47 K.pneumoniae isolates presented this phenotype(P<0.001).There were statistically significant differences between ST11-KL47 and ST11-KL64 isolates in terms of age,hospital mortality,use of catheters,tracheotomy,transfusion,and hypermucus phenotype(P<0.05).Some virulence genes,including rmp A2,iuc A,iuc B,iuc C,iuc D,and iut A,as well as the rep B gene potentially involved in DNA replication were only present in ST11-KL64 isolates.Phylogenetic analysis suggested that ST11 CRKP isolates may have evolved from KL47 to KL64.By predicting the transmission and evolution of the isolates,multiple transmission events were identified,especially in the Intensive Care Unit(ICU),where clonal transmission of the isolates occurred more frequently,indicating a high risk of bloodstream infection with CRKP in ICU patients.From the PATRIC database,ST11 K.pneumoniae isolates were mainly ST11-KL47 from 2011 to 2015 and ST11-KL64 from 2016 to 2020.There were 35 virulence genes with statistically significant quantity differences between ST11-KL47 and ST11-KL64.Comparative analysis of the serotype-determining region revealed that ST11-KL64 isolates had more bases than ST11-KL47 isolates in the CD1-VR2-CD2 region,adding about 303 bp to the base sequence and having a higher GC content(58.14%),which may be associated with the evolution of the serotype to KL64.Conclusion: The CRKP strains isolated from blood samples in this hospital carried more virulence genes and had a high mortality rate of infection in hospitalized patients,and ICU patients had a high risk of bloodstream infection.The results of genomic analysis suggest that ST11-KL47 and ST11-KL64 are the predominant serotypes in CRKP from blood samples,and ST11-KL47 may be the evolutionary ancestor of ST11-KL64 type CRKP.This evolution may be associated with genomic recombination occurring in the region near the capsule. |
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