Study On The Homology And Drug Resistance Mechanism Of Klebsiella Pneumoniae Resistant To Carbapenems And Polymyxin B

Purpose:To study the virulence and drug resistance gene characteristics of clinically isolated carbapenem and polymyxin B-resistant Klebsiella pneumoniae(KP)strains,and analyze the molecular homology characteristics of drug-resistant strains;The mechanism of resistance of polymyxin B and the mechanism of transfer and transmission of mcr-1 gene;explore whether polymyxin B combined with resveratrol has a synergistic effect on the antibacterial activity of drug-resistant strains in vitro.Method:1.Collect 107 non-repetitive carbapenem-resistant Klebsiella pneumoniae(CRKP)clinical isolates from the Second Affiliated Hospital of Nanchang University from January 2018 to June 2019.The Vitek2-compact automatic microbial identification instrument and drug sensitivity analysis system were used to identify bacteria and detect the sensitivity of common antibacterial drugs.Determination of the minimum inhibitory concentration(MIC)of polymyxin B on the strain by micro broth dilution method.Carbapenemase was preliminarily screened by the modified carbapenem inactivation test(m CIM)and the metalloenzyme preliminary screening test.Polymerase chain reaction(PCR)and sequencing to detect carbapenem resistance genes(bla KPC,bla NDM,bla IMP,bla VIM,bla SME,bla GES,bla SPM-1,bla SIM-1,bla GIM-1,bla OXA-48,bla OXA-23);ESBLs genes(bla TEM,bla CTX-M,bla SHV);Amp C enzyme genes(bla AAC,bla CIT,bla DHA,bla EBC,bla FOX,bla MOX);plasmid-mediated quinolone drug resistance genes(acc(6 ’)-Ib-cr)and polymyxin B resistance gene(mcr-1-8).Detection of highly virulent capsular serotype genes(k1,k2,k5,k54,k57)and common virulence genes(rmp A,mag A,alls,wcag,aerobactin,iro N,iut A,fim H-1)of drug-resistant strains.Multi-site sequence typing(MLST)and pulsed field gel electrophoresis(PFGE)were used to analyze the molecular typing and homology of the strains.2.PCR and sequencing were used to detect the mutation of the polymyxin B resistance gene mediated by the PmrA / Pmr B regulatory system,and real-time fluorescent quantitative PCR was used to detect the difference in the expression of the acr A gene in the efflux pump of polymyxin B resistance and sensitive strains.A clinically isolated strain of Klebsiella pneumoniae carrying the mcr-1 gene was taken as the research object,and the mechanism of drug resistance gene transfer and dissemination was elucidated through conjugation and transformation experiments.Plasmid extraction,high-throughput sequencing technology for plasmid sequencing,genetic environment and comparative analysis.3.Taking the colistin-resistant CRKP as the research object,the MIC value of resveratrol on the strain was determined by the broth dilution method,and the MIC value of the polymyxin B combined with resveratrol on the strain was determined by the checkerboard dilution method.,To explore the antibacterial effect of polymyxin B combined with resveratrol.Result:1.Klebsiella pneumoniae,which is simultaneously resistant to polymyxin B and carbapenems,has severe drug resistance and multi-drug resistance;carrying KPC-2 is the main reason for carbapenem resistance,while carrying The SHV and CTX-M-65 genes are responsible for the multi-drug resistance of the strain;the experimental strain carries fewer virulence genes,indicating that its pathogenicity is weak;MLST typing is mainly ST11 type,and an unreported ST Type;PFGE results show that the genetic diversity of the strain.2.The mutation of pmrB gene is involved in the regulation of the resistance of the strain to polymyxin B;the high expression of the efflux pump acr A gene is related to the resistance of the strain to polymyxin B;the results show that the mcr-1 gene is plasmid-mediated It is the first report that this strain carries both mcr-1 and NDM-5genes.The comparison of plasmid sequencing shows that it is consistent with the plasmids MH715959.1 and MK308632.1.3.Polymyxin B combined with resveratrol has a synergistic effect on the in vitro antibacterial activity of Klebsiella pneumoniae.When the two drugs are used in combination,the MIC value is lower than when used alone.Conclusion:1.15 strains of CRKP showed multiple drug resistance,but were more sensitive to drugs such as amikacin,gentamicin,tobramycin,and tigecycline.The carbapenemase-resistant genes carried by KCR-2 Mainly,and also carry SHV gene,8strains also carry CTX-M-65 gene;virulence gene test shows that the common virulence factor is low,no common capsular serum gene is detected;strain MLST ST11 was the main type,and an ST type that had not been reported was found.PFGE results showed that2.Real-time quantitative PCR results showed that resistance to polymyxin B was related to the expression of efflux pump acr A gene;resistance to polymyxin B was related to the plasmid-mediated mcr-1 gene;The mcr-1 and NDM-5 genes carried by this strain are reported for the first time in China,and the plasmid sequencing comparison shows consistency.3.The combination of resveratrol and polymyxin B has a synergistic effect on the antibacterial activity of the strain in vitro.