Drug Resistance And Molecular Epidemical Research Of KPC-producing Klebsiella.pneumoniae

Objective:Carbapenem-resistant Klebsiella pneumoniae(CRKP)infection has become an independent risk factor for the death of nosocomial infection patients because of the difficulty in prevention and treatment.In this study,90 CRKP strains were collected in the past two and a half years from the People’s Hospital of Jiangsu University and the phenotypic study and molecular typing methods were applied to investigate the drug resistance mechanism and clinical epidemic status of carbapenemase-producing K.pneumoniae,and to track the source of infection and possible transmission routes,so as to guide the preventions and controls of nosocomial infections.Methods:1.Cultivation and preservation of strains: K.pneumoniae strains resistant to imipenem were collected from July 2015 to January 2018 in the People’s Hospital of Jiangsu University.After isolation and purification,single colony was taken and frozen in magnetic beads Tubes respectively in-80 ° C.2.Antibiotic susceptibility testing: VITEK 2 COMPACT automatic microbial identification system was used to identify strains and determine the minimum inhibitory concentration of common antibiotics.3.Collection and analysis of drug resistance data: Software was used to analyze the detection rate of K.pneumoniae in the same period and the resistance rate of antibiotics to imipenem and other antibiotics.4.modified Carbapenem inactivation method(m CIM): After incubation with isolation samples,meropenem drug resistant papers were applied for drug sensitive test.Carbapenemase phenotype of was identified according to diameter of inhibition zone.5.Multilocus sequence typing(MLST): DNA of carbapenem-producing strains were extracted by boiling method separately,and 7 housekeeping genes wereamplified by PCR.The sequencing result was uploaded to the relevant website to obtain the sequencing type of each strain.6.Pulsed-field gel electrophoresis(PFGE): Single colonies were subjected to pulsed-field gel electrophoresis after gelatinized and enzyme digested.The obtained spectra were analyzed by software,and the homology of the strains were determined according to the similarity of the bands.7.Detection of carbapenemase-producing gene: The DNAs were extracted by boiling method from carbapenem-producing strains as template of PCR.Six common genes encoding carbapenemase were amplified such as kpc,ndm,vim,oxa-48,ges and imp.8.Whole genome sequencing(WGS): According to the results of MLST and PFGE,the tested strains were screened for whole genome sequencing,and the genetic evolution relationship of drug-resistant strains was analyzed according to the single nucleotide polymorphism of the strains.Results:1.Identification by the Vitek 2 compact automatic microbial identification system,90 strains of K.pneumoniae resistant to imipenem were obtained.2.After removing repeated patient strains,the results of Whonet software analysis showed that the detection rate of carbapenem-resistant strains decreased slightly from 2016 to 2018,and resistant rate to carbapenem was obviously lower than the national average.We didn’t find tigecycline-resistant strains.The resistance rate to aminoglycosides is relatively low,ranging from 68% to 85%.3.Results of m CIM showed that all the tested strains were able to synthesize carbapenemase.4.The results of MLST typing showed that the majority of the sample strains were ST11 type K.pneumoniae.No new ST type has appeared.5.PFGE showed that 90 strains of CRKP can be divided into 12 clones,of which 82 have more than 80% homology and 69 belong to the same clone.6.By PCR amplification and sequencing,all carbapenemase-producing strainscarries kpc gene,and no strain carrying gene ndm,vim,oxa-48,ges or imp has been found.7.According to MLST and PFGE results,8 highly homologous resistant strains were screened for whole genome sequencing.The results showed that the number of homozygous SNPs of 8 resistant strains ranged from 924 to 2002,which can be divided into 3 clusters,and the INDELs mainly occurred in regions of exonic.These strains show far-reaching genetic evolution between different clusters.Conclusions:1.The resistance rate of K.pneumoniae to imipenem in the hospital is far lower than the national average,it shows a downward trend from 2016 to 2018,and the drug resistance control is well-conducted.However,resistance to common antibiotics such as cephalosporins is still on the rise.The resistance to carbapenems is mainly due to the synthesis of KPC enzyme,which is 100% sensitive to tigecycline.2.The drug-resistant strains in the hospital mainly existed in the ICU department.According to results of MLST,PFGE and WGS,most CRKP strains are belong to ST11 type.We Considered that in addition to the in-hospital epidemic caused by a single cloned strain,there may be long-term extensive colonization of monoclonal drug-resistant strains,and different strains formed by evolutionary mutations in different directions may occur in a long time and cause in-hospital transmission.It is necessary to further strengthen the monitoring of the epidemic of drug-resistant strains and the prevention and control of in-hospital infection.