The Mechanism Study Of Heteroresistance To Carbapenem In OXA-48-producing Klebsiella Peumoniae

Objective Bacterial resistance has been a thorny problem in the field of anti infection,and the emergence and prevalence of carbapenem resistant Klebsiella pneumoniae(CRKP)has posed a serious threat to the global public health field.In clinical work,we found that OXA-48 producing K.pneumoniae,usually mediates a low level of drug resistance,but the treatment with carbapenem antibiotics alone can not achieve therapeutic effect,which may be related to heteroresistance.The purpose of this study was to explore the mechanism of heteroresistance to carbapenem of K.pneumoniae producing OXA-48,and to provide a theoretical basis for clinical drug combination.Methods The blaOX-48-like gene was screened from 273 clinical isolates of K.pneumoniae by PCR,and the whole genome of blaOXA-48-like positive strains was analyzed to identify their ST type,drug resistance gene and plasmid gene structure.Through population analysis profile(PAP),the heteroresistance phenotype of carbapenem was clearly studied.Select drug resistant subpopulations,and identify the heterogeneity and stability of drug resistant subpopulations through passage experiments.The relative expression of blaOXA-48-like mRNA of the original clinical strain and its drug resistant subgroup was determined by qRT-PCR,to determine the relationship between heteroresistance of K.pneumoniae and OXA-48.The potential mechanism of heteroresistance to carbapenem of K.pneumoniae producing OXA-48 was discussed by whole genome sequencing and comparative genomic analysis.Results Four strains of K.pneumoniae carrying blaOXA-48-like gene were screened from 273 clinical K.pneumoniae strains.Genomics showed that they were all ST15 type,and the blaOXA-48-ike genotype carried by them was blaOXA-232.The PAP test confirmed that the 4 strains were heteroresistance to imipenem and meropenem.Most strains of drug resistant subgroups are homogeneous drug resistant,a few still have heteroresistance phenotypes,and the drug sensitive phenotypes of drug resistant subgroups are stable.By comparing the relative mRNA expression of the original clinical strain and its drug resistant subgroup blaOXA-48-like,it was found that the relative mRNA expression of the drug resistant subgroup blaOXA-48-like was higher than that of the original clinical strain.Comparative genomic analysis showed that mutations in ompC and metl of D-methionine transport system were found in several drug resistant strains.Conclusion Four strains of K.pneumoniae carrying blaOXA-48-like gene were heteroresistance to imipenem and meropenem.The majority of drug resistant subgroups are homogeneous drug resistant,and their drug sensitive phenotypes and homogeneous phenotypes were stable.Heteroresistance of K.pneumoniae to carbapenem is associated with increased expression of blaOXA-48-like,Mutations of outer membrane porin ompC and D-methionine transport system meta may be another potential mechanism of heteroresistance of K.pneumoniae producing OXA-48 to carbapenem.