Molecular Characteristics And Virulence Analysis Of Carbapen Em-resistant Klebsiella Pneumoniae Of Bloodstream Infection

Objective:From June to December 2018,62 strains of Carbapenem-Rsistant Klebsiella pneumoniae(CRKP),were collected from a sterile blood specimen of Zhengzhou University,which provides data support for the futher prevention and control of CRKP strains and also provides theoretical basis for clinical treatment.Methods:(1)The microbial dilution and agar dilution method were used to determine the minimum inhibitory concentration of 16 different antibiotics commonly used in clinic to determine,multi-site sequence typing and serotype detection and analysis of ST and serotype strains.(2)Detection of drug resistance genes including carbapenemase genes based on drug resistance phenotype by PCR,and detection of virulence marker genes rmpA2,rmpA,peg344,iroB,iucA.(3)Screen suspected high-virulence CRKP strains with double positive blaKPC-2 and rmpA2 genes,and determine the size of drug-resistant and virulence plasmids where blaKPC-2 and rmpA2 genes are located by S1-PFGE and southern hybridization techniques.(4)The PCR mapping method was used to analyze the molecular characteristics of the virulence plasmid with pVir-CR-hvKP4 plasmid as the reference.Whole-genome sequencing analysis was performed on the strain KP18-2079,which is suspected to be on the same plasmid as blaKPC-2 and rmpA2.(5)In the string test experiment,the viscous phenotype of the target strain was determined and a mouse model of intraperitoneal infection was established to evaluate the virulence of the strain.Results:(1)62 CRKP clinical isolates with bloodstream infection have multiple drug resistance phenotypes,which 54%were isolated from the ICU,and the age was concentrated between 40-80 years old,mainly males.Among them,ST type and serotype are mainly ST11-K64.(2)62 CRKP strains mainly carry KPC carbapenemase,accounting for 89%(55/62).A total of 35 CRKP strains carry virulence marker genes,accounting for 56%(35/62),of which 24 strains carry KPC plsmid and pVir-CR-HvKP4-like virulence plasmids,the virulence plasmid size is between 190-300 kb.(3)Only one of the 62 strains has a high-viscosity phenotype and five ST 11-K47 CRKP strains carrying the virulence marker genes rmpA2 and iucA have a virulence-enhancing phenotype in the intraperitoneal infection model of mice.(4)The plasmids pKP18-2079-KPC and pKP18-2079-vir carried by Klebsiella pneumoniae KP18-2079 are formed by the exchange and recombination of plasmids p1-L388 and pKPC-L388 respectively,resulting in the integration of blaKPC-2 in the virulence plasmid and the pKP18-2079-vir plasmid carries multiple drug resistance genes simultaneously.Conclusion:The analysis of the drug resistance and virulence characteristics of 62 strains of CRKP infected by bloodstream showed that the clinically isolated CRKP multi-drug resistance is serious,which greatly limits the use of antibiotics.ST11-K64 is the main popular clone of 62 CRKP strains,CRKP carrying the virulence marker gene ST11-K47 has a potential virulence enhancement phenotype.Under the pressure of antibiotic selection,multi-drug resistant strains make the resistance genes and virulence genes on the same plasmid,which leads to the cooperative transmission,so attention is needed.