| Objective:1.To investigate the carbapenemase phenotype and carbapenemase genotype characteristics of carbapenem-resistant Klebsiella pneumoniae and their relationship with antimicrobial sensitivity.2.To investigate the characteristics and relationship between the virulence phenotype,high virulence capsule serotype and virulence gene of Klebsiella pneumoniae,and the relationship between hypervirulent Klebsiella Pneumoniae（HvKP）infection and clinical prognosis,and the impact of virulence on drug resistance.3.Analysis of homology and major epidemics of CRKP strains.4.To analyze the prognostic factors of patients with CRKP infection,and provide evidence for clinically effective prevention and control measures.Methods:1.The clinically isolated CRKP strain and carbapenem-sensitive Klebsiella pneumoniae（CSKP）were collected from the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine from January 2016 to September 2018,using Vitek 2 Compact automatic system and supplementary susceptibility card to identificate bacteria and test the antimicrobial susceptibility of strains,and confirmed with disk diffusion method for carbapenem.2.Retrospect the clinical characteristics of the infected patients,incluing gender,age,department,length of hospital stay,separation site,basic diseases,antibiotic treatment,invasive operation and prognosis.3.The modified Hodge test,Carba NP test,Modified Carbapenem Inactivation Method test and EDTA Carbapenem Inactivation Method test were used to detect carbapenemases phenotypes.4.PCR was used to detect the carbapenem resistance gene,including group A(blaKPC,blaGES,blaSME,blaIMI,blaNMC),group B(blaNDM,blaVIM,blaIMP,blaSPM,blaGIM,blaSIM)and group D(blaOXA),PCR amplification positive sequencing results submitted to the GenBank database comparison.5.Detecte the virulence phenotype by mucus test,and using Polymerase chain reaction（PCR）to detect the high virulence capsule serotypes（K1,K2,K3,K5,K20,K54and K57）and virulence genes（magA,rmpA,aerobactin and fim H）.6.Using multilocus sequence typing（MLST）to analyze the genotype and homology of CRKP strains,and combining clinical data of patients to explore the epidemic characteristics of local CRKP molecules.Results:1.Between January 2016 and September 2018,a total of 756 strains of Klebsiella pneumoniae were isolated from the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine,including 124 strains of CRKP and 632 strains of CSKP.Excluding the clinical data unknown and the failed strains,74 strains of CRKP were collected in this experiment,and 71 strains of CSKP were collected as control group（excluding duplicates of the same patient at the same site）.2.CRKP is resistant to most of the antibiotics commonly used in clinical practice,and the resistance rate to cephalosporins,carbapenems andβ-lactamase inhibitors is close to 100%,and the resistance rate to aztreonam and Quinolone antibiotics is≥80%,the resistance rate to aminoglycoside antibiotics is low,but it is increasing year by year,among them,the resistance rate of amikacin in 2016 was only 12.5%,up to 74.1%in2018,with a growth rate of 6 times,the resistance rate of gentamicin in 2016 was only13.0%,and it increased to 59.3%in 2018,an increase 4.5 times.3.Of the 74 strains of CRKP,71 strains were positive for the Hodge test,72 for the carba NP test,73 for the mCIM test,and 8 for the eCIM test.4.73 strains of Carb NP and mCIM positive CRKP strains were positive for PCR amplification.Of the sequence alignment,61 strains carried blaKPC-2PC-2 gene,4 strains carr-ied bla KPC-19PC-19 gene,and 8 strains carried blaNDM-1DM-1 gene.The CRKP strain carrying both drug resistance genes was not detected.Eight strains of eCIM-positive CRKP strains carried blaNDM-1DM-1 gene;71 strains of modified Hodge test positive CRKP detected blaKPCPC or blaNDMDM gene,3 modified Hodge test-negative CRKP,2 strains were blaNDMDM positive,1strain was not detected Out of resistance genes;5.Of the 74 strains of CRKP,4 were positive for mucus silk,but none of the high virulence capsule serotypes and virulence genes were detected;6.Among the 71 strains of CSKP,15 strains were positive for mucus test,and the main capsular serotype was K57（6 strains）,followed by K1 and K2.3 virulence genes were positive for magA,9 were positive for rmpA,13 were positive for aerobactin gene,and no fim-H gene was detected;7.The results of MLST showed that the CRKP strain was mainly ST11 type（43/74）,followed by ST147 type（20/74）,ST307 type was 8 strains,ST20 type and ST1197 type were 1 strain;8.Patients with CRKP have poor clinical prognosis and mortality is significantly higher than those with CSKP infection.The pioneering operation has an impact on prognosis.Conclusion:The CRKP strain isolated in our hospital has high resistance rate to common clinical antibiotics including cephalosporins,quinolones andβ-lactams and their enzyme inhibitors,and the resistance rate to aminoglycosides is relatively low,but it is increasing year by year.The MHT test detects that NDM-type carbapenemase is prone to false-negative,the carba NP test and mCIM test have high sensitivity to detect carbapenemase,which is consistent with PCR detection gene results;mCIM and eCIM combined detection can initially determine production A or Class B enzyme.CRKP strain has multiple drug resistance mechanisms,and carbapenemase producing is one of the most important drug resistance mechanisms.CRKP strains mainly carry blaKPC-2,blaKPC-19and blaNDM-1DM-1 resistance genes in this region.The results of MLST show that the main strains in our hospital are ST11 and ST147,which should be highly valued by the clinical department and the hospital department to avoid large-scale outbreaks. |
