Preliminary Study On The Resistant Genotypes Of Carbapenem-resistant Enterobacteriaceae

Objective: To evaluate the drug susceptibility and clinical distribution characteristics of clinically isolated carbapenem-resistant Enterobacteriaceae from the Fourth Hospital of Hebei Medical University,to provide a basis for the study of molecular epidemic characteristics and clinical guidance of drug use.Describe the carbapenemase phenotype and genotype distribution of CRE,and use gene sequencing as the gold standard to compare the differences in the detection rate of carbapenemase genotypes between Immunochromatography assay and Real-time PCR method.Methods:1.Collect 4031 strains of Enterobacteriaceae from our hospital from January 2015 to December 2019 as the research object,use Bio Mérieux VITEK-2 automatic microbial analysis system for strain identification and drug susceptibility test,and CRE strain uses ceftazidime/avibactam and tigecycline for drug sensitivity test.2.Collect 42 CRE strains sterile body fluids from 2015 to 2019 as research objects,use m CIM combined with e CIM test to detect carbapenemase phenotype,use gene sequencing as the gold standard,and use Immunochromatography assay and Real-time PCR to detect the carbapenemase genotypes of 42 strains CRE and 50 strains CSE.3.Using whole genome resequencing,we sequenced the genomes of 1MBL(Metal-β-lactamase)producing E.coli strain to obtain individual or population differences by aligning to known reference genomes.These differences mainly include: Single Nucleotide Polymorphism,Insertion &Deletion,and Structural Variation.Use MLST 2.0,Res Finder 3.2,Virulence Finder 2.0 software for MLST typing,drug resistance gene analysis and virulence factor analysis.Results:1.A total of 235 non repeat carbapenem resistant Enterobacteriaceae were isolated from our hospital during 2015-2019,and the predominant bacteria were K.pneumoniae(48%)and E.coli(24%),mainly from sputum(45%)and urine(13%)specimens,and the most frequently isolated department was the ICU,which accounted for 48%;K.pneumoniae was 80% sensitive to both ceftazidime/avibactam and tigecycline;E.coli was 78% resistant to ceftazidime/avibactam,whereas 100% susceptible to tigecycline.2.The combination of m CIM and e CIM test showed that among 42 CRE strains,except for one strain that didn’t produce carbapenemase,the remaining 41 strains all produced carbapenemase.Among them,66% of CRE bacteria were metal-β-lactamase-producing strains,and 34% of CRE strains produced serine carbapenemase;the sensitivity of Immunochromatography assay was 90%,the specificity was 100%,and the positive predictive value was 100%,the negative predictive value was 93%,and the Kappa value was0.911.The sensitivity of Real-time PCR method was 97%,the specificity was100%,the positive predictive value was 100%,the negative predictive value was 98%,and the Kappa value was 0.978.The gene sequencing method showed that except for one strain that didn’t produce carbapenemase,the other41 strains were all tested for carbapenem resistance genes,with a KPC detection rate of 51% and a NDM detection rate of 49%.3.The GC content of this MBL producing strain was 51.42%,the total number of SNP of this strain was 78722,the total number of Indel was 811,and the total number of SV was 225.The MLST typing was ST167,and it produced β-lactams(such as NDM-5 、 bla CMY-42 and bla CTX-M-14),fluoroquinolones(gyr A)and other drug-resistant genes;the virulence factors were fyu A,irp2,iuc C,iut A,kps E,kps MII-K5,omp T,sit A,tra T,and yfc V.Conclusions:1.The main isolated types of carbapenem resistant Enterobacteriaceae were K.pneumoniae and E.coli,which were distributed in different specimens and departments,and the clinical treatment should be standardized and optimized for healthcare related operations,and timely monitoring to control CRE nosocomial infection and transmission.2.The carbapenem phenotype results showed that 66% of the strains produced metals β-Lactamases and the serine carbapenemase-producing strains accounted for 34%.Using gene sequencing results as the gold standard,Real-time PCR and Immunochromatography assay had different positive rates for carbapenemase genotypes.3.The results of whole genome sequencing showed that this CRE strain was an NDM-5-producing multi drug resistant E.coli,which should be managed with enhanced clinical antimicrobial applications and nosocomial control.