Molecular Epidemiology And Antibiotics Resistance Mechanism Of Hypervilulence-Carbapenem Resistant Klebsiella Pneumoniae In Chongqing

Objective:At present,reports on the molecular epidemiology and antibiotic resistance mechanism of hypervirulent-carbapenem resistant Klebsiella pneumoniae?hv-CRKP?are rare in China.According to the previous research of our group,we made a preliminary exploration on the molecular epidemiology and the main mechanism of drug resistance of hv-CRKP in Chongqing,in order to provide theoretical basis for the treatment of hv-CRKP infection in this area.At the same time,the screening ability of modified carbapenem inactivation method?mCIM?combined with EDTA carbapenem inactivation method?eCIM?to carbapenemase was evaluated to provide basis for its clinical application.Method:1.Two hundred and eighteen strains of carbapenem-resistant Klebsiella pneumoniae?CR-KPN?isolated from 20 county hospitals in Chongqing from September 2015 to September 2017 were collected.The resistance to carbapenem antibiotics was checked by microbroth dilution method,the strains with high virulence were screened by string test?ST?,and the homology of hv-CRKP was analyzed by pulsed field gel electrophoresis?PFGE?and multiple locus sequence typing?MLST?,PCR technique and microbroth dilution method were used to detect the mechanisms of antibiotic resistance,such as enzyme production gene,membrane porin deletion and efflux pump mechanism.At the same time,capsule typing,virulence gene detection and serum killing test were carried out to explore its virulence.2.85 strains of carbapenem-resistant Enterobacteriaceae?CRE?and 51strains of carbapenem-susceptible Enterobacteriaceae?CSE?bacteria isolated from 16 county hospitals in Chongqing from January 2015 to December 2016 were collected.85 strains of CRE and 51 strains of CSE carrying carbapenem gene were obtained by PCR technique.The phenotype and classification of carbapenemase were tested by mCIM and eCIM.Taking the results of PCR as the gold standard,the performance of mCIM combined with eCIM in detecting the phenotype and classification of carbapenemase was evaluated.Result:1.218 strains of CR-KPN showed a high level resistance to carbapenem.The resistance rates of ertapenem,imipenem and meropenem were 100%,96.8%and 95.9%,respectively.MIC₅₀0 was 256mg/mL for ertapenem,128mg/mL for imipenem and 64mg/mL for meropenem.2.6 strains of hv-CRKP were screened by ST,accounting for 2.8%?6/218?.The main types were ST11?3/6?and ST592?2/6?.PFGE typing showed that 2 strains of hv-CRKP were homologous.The main mechanism of carbapenem resistance was carbapenemase production and extended spectrum beta-lactamases?ESBLs??6/6?.No ompk35/ompk36 membrane porin deletion and efflux pump overexpression were found.2 strains of serotype K57 were found by capsular typing of 6 strains of hv-CRKP,but no serotypes K1,K2,K5,K20 and K54 were found.The distribution of virulence genes was as follows:iron carriers?enterobacterin entB,yersenin Irp-1/Irp-2?and adhesion factors?type?fimbrial adhesion gene fimH-1,type?fimbrial adhesion gene mrkD,kpn,encoding outer membrane protein lipopolysaccharide ycfM?were all positive?6/6?,and the genes related to high viscosity?magA,rmpA?and Acrobactin were carried in different proportions.The results of serum killing test showed that 4 strains of hv-CRKP had serum resistance.3.79 of 85 carbapenem-resistant Enterobacteriaceae strains?CRE?were positive for carbapenemase gene by PCR,and 8 strains expressed class A and B carbapenemase genes,accounting for 10.13%?8/79?.78 of 85CRE strains were detected to be positive by mCIM,44 strains were positive by eCIM among them.The carbapenem-resistant genes were not detected and mCIM results were negative in the 51 carbapenem-sensitive strains.Screening the carbapenemase by mCIM,the sensitivity was 98.7%?95%confidence interval[CI],92.2 to 99.9?,the specificity was 100%?95%CI,92.1 to 100?,and the Kappa value was 0.985.Screening metallo-b-lactamase,the sensitivity of eCIM was 85.7%?95%CI,72.1 to93.6?,the specificity was 94.4%?95%CI,80.0 to 99.0?,and the Kappa value was 0.787.Screening serine carbapenemase,the sensitivity of eCIM was 89.5%?95%CI,74.3 to 96.6?,the specificity was 100%?95%CI,90.6 to100?,and the Kappa value was 0.904.Conclusion:1.CR-KPN shows a high level resistance to carbapenem antibiotics,and the carbapenem antibiotic MIC₅₀0 is higher than other areas.2.There is hv-CRKP epidemic in CR-KPN in Chongqing.The main sequence type is ST11,no gathering or outbreak strains across counties have been found,no other antibiotic resistance mechanism except enzyme production has been found,virulence genes are mainly carrying iron carrier related genes and adhesion factor related genes,and most strains have serum resistance.3.The combination testing of mCIM and eCIM could screen carbapenemase-producing Enterobacteriaceae effectively.However,The sensitivity of mCIM combined with eCIM detection to screen metallo-b-lactamase will be reduced with the emergence of a large number of co-expressed class A and B carbapenemase-producing Enterobacteriaceae.