Resistance Mechanism Of Carbapenems In Klebsiella Pneumoniae And Proteus Mirabilis

BACKGROUNDS The discovery and use of antibiotics have improved the therapeutic effect of clinical anti-infections and made important contributions to the human health.The over-range and over-dose antibiotics use has led to the emergence of bacterial resistance and brought great difficulties to the treatment of bacterial infectious diseases.In the antibiotic pressure,Bacteria get the ability against the drug by different methods like gene mutation,gene recombination,and plasmid transformation.Now,carbapenems resistance has attracted much attention.Researchers have found that carbapenems have the following resistance mechanisms such as changes in cell membrane permeability,plasmid transduction,efflux pump system,carbapenems production and integron-mediated changes.Drug-resistant genes on bacterial chromosomes can be integrated into bacterial plasmids by transposon.This biological phenomenon can lead to large-scale spread of drug-resistant genes.Plasmids can transmit gene through contact with the sexual pili of bacteria.So,it is very important to clarify the resistance genes,plasmids,mobile prototypes,insertion sequences,transposons,and the molecular mechanism of drug resistance gene transmission and diffusion in bacterial genome.OBJECTIVE Based on previous studies,this paper studies the resistance mechanism of carbapenems of Klebsiella pneumoniae and Proteus mirabilis in Enterobacteriaceae,and wish to make rational use of antibiotics in clinical practice to avoid Drug resistance.METHODS 1.Collect clinical isolates from different sources,carry out drug sensitivity test and isolate drug-resistant strains.2.Minimum inhibitory concentration(MIC)of drug-resistant strains against commonly of clinical antibiotics was determined by broth dilution method.3.Carba NP method was used to determine whether the strain produced carbapenem enzyme and the type of the enzyme.4.High-throughput genome sequencing and polymerase chain reaction(PCR)were used to study the drug-resistant genes carried by isolated drug-resistant strains,such as extended-spectrum beta-lactamase resistance gene,quinolone resistance gene,tetracycline resistance gene,aminoglycoside resistance gene and so on.5.Horizontal transfer of drug resistance genes was determined by plasmid conjugation and transduction experiments.6.Molecular mechanism of efflux pumps mediating bacterial resistance was also tested.7.The second generation genome sequencing of bacteria was used to confirm the location of drug-resistant genes in the whole genome.RESULTS 1.The MIC values of Klebsiella pneumoniae and Proteus mirabilis against carbapenem antibiotics,imipenem,meropenem and biapenem were all above 10?g/mL.2.Through second-generation genome sequencing combined with PCR amplification,Klebsiella pneumoniae was found containing resistance genes such as IMP-4,DHA-1,qnrs1,TetA.3.The results of plasmid extraction showed that Klebsiella pneumoniae has plasmid,which could mediate carbapenems resistance of host bacteria through the experiments of E.coli conjugation and transduction.4.The results of second-generation plasmid sequencing of Klebsiella pneumoniae showed that the plasmid structure of Klebsiella pneumoniae included the conserved skeleton region of IncN1 type and the repA and tra genes.Many exogenous insertion sequences were integrated in the cytoskeleton of the plasmid,and most of the resistance genes were found in the insertion sequence.CONCLUSIONS 1.Klebsiella pneumoniae is resistant to carbapenems by expressing carbapenemases IMP-4 and DHA-1,which could hydrolyze the beta-lactam ring of carbapenems.2.Plasmid conjugation and transduction experiments demonstrated that carbapenemase resistance genes could spread through plasmids,and alerting us the risk of antibiotic resistance gene diffusion..3.The basic structure of drug-resistant plasmids was constructed by using second-generation genome-wide sequencing and scanning technology.The initial gene of plasmid replication,plasmid skeleton region and plasmid exogenous insertion region were analyzed.The location of resistant genes in plasmid was also analyzed,and the mechanism of plasmid-mediated transmission and diffusion of multi-drug-resistant genes was preliminarily clarified.In conclusion,our work give an insight of the mechanism of drug-resistant gene transmission in drug-resistant bacteria and even pan-drug-resistant bacteria.