The Studies Of Plasmid-mediated Quinolone Resistance Mechanism In Enterobacteriaceae Producing ESBLs Of Kunming

Quinolones are a kind of synthetic broad-spectrum and powerful antibiotics,which are the most widely used antibacterial drugs.Enterobacteriaceae are the most common clinical isolates,mainly including E.coli and K.pneumoniae,in recent years,the resistance of Enterobacteriaceae to quinolones has become more and more serious,which often leads to delay or failure in clinical treatment.The previous view suggests that quinolone resistance in Enterobacteriaceae is mediated by chromosomes,including changes in cell membrane permeability,active efflux systems and target genes mutation.Subsequent studies have found that plasmid-mediated quinolone resistance pathways,which explains the horizontal transfer of drug-resistant gene.The plasmid-mediated quinolone-resistant genes,including the qnr gene family and the aac(6')-Ib-cr gene,are often propagated with extended-spectrum ?-lactamases(ESBLs),and the distribution of these genes is locally different,Study on the mechanism of plasmid-mediated quinolone resistance in the region is of great importance to the study of clinical experience and the development of appropriate control measures.The main research work of this paper is the mechanism of plasmid-mediated quinolone resistance in E.coli and K.pneumoniae producing ESBLs in Kunming,including the following three aspects(1)Detect the epidemic of plasmid-mediated quinolone resistance geneFrom May 2015 to July 2016,50 strains of Enterobacteriaceae bacteria producing ESBLs were isolated from different departments,different age patients and different infected parts of Yunnan First People's Hospital,including 26 strains of E.coli and 24 strains of K.pneumoniae.Detection of plasmid resistance genes in different strains by PCR,We found that the epidemic resistance genes in E.coli and K.pneumoniae were different.In E.coli the most popular PMQR gene was qnrB,the detection rate was 88.4%.followed by aac(6')-Ib-cr gene,the detection rate was 61.5%,both the qnrD and qnrS genes detected only one case.The qnrB,qnrS and aac(6')-Ib-cr genes were found in all K.pneumoniae,the detection rate was 100%.We also found that the same strain may carry several different plasmid-resistance genes.42.3%of E.coli strains carry two resistance genes,7.6%of the strains carrying three drug resistance genes,all the strains of K.pneumoniae carried three same PMQR genes.(2)Conjugation experiment and the study of multi-drug resistancePlasmid conjugation experiments were performed to Enterobacteriaceae which carried the PMQR gene,all the E.coli was successes to conjugation,two strains of K.pneumoniae failed to conjugate.This shows that the plasmid carrying of quinolone resistance gene has a high level of ability to transfer,the plasmid no joint success probably because lack of conjugative transfer operon.The MIC values of quinolones,?-lactams and aminoglycosides were measured by E-test method for donor,recipient and transconjugants.We found that donor and transconjugants are resistant to quinolones,which indirectly indicate that the resistance gene is located on the plasmid.Most of the donor and transconjugants have a resistance to aminoglycosides,there is one E.coli donor has a drug resistance,but its promoter is sensitive to the drug,indicating that the drug resistance gene is located on the genome.The donor and transconjugants are resistant to beta-lactam drugs except carbapenems,this indicating that the plasmids also carry the resistance genes for these drugs.Based on these data,we can conclude that the plasmid carrying the quinolone resistance gene,also carrying aminoglycosides and P-lactam drug resistance gene.resulting in Enterobacteriaceae bacteria multi-drug resistance.(3)Enterobacteriaceae bacterial homology analysisThe homology of Enterobacteriaceae carrying PMQR gene was analyzed by pulsed field electrophoresis,there are 14 strains of E.coli homology of more than 85%,Among them,the homology of two strains was 100%.There were 16 strains of K.pneumoniae with homology of more than 85%,accounting for a total of 66.6%(16/24),Among them,5 groups of 10 genomic bands were identical for the same strains that were recently transmitted,accounting for 41.6%(10/24),Four of the five strains were from the intensive care unit,indicating that the drug-resistant bacteria spread more rapidly in immunocompromised patients.Combined of these data we can find that the outbreak of plasmid-mediated quinolone resistance in K.pneumoniae more concentrated than E.coli,drug-resistant bacteria in immunocompromised patients spread rapidly.In summary,this study found that the most popular PMQR genes in the clinical isolates of Enterobacteriaceae in Kunming were qnrB,qnrS and aac(6')-Ib-cr,and the plasmid carrying of quinolone resistance gene has a high level of ability to transfer,the plasmid Carrying the PMQR gene also carries a variety of other drug resistance genes.Homology analysis found that the outbreak of K.pneumoniae was more concentrated than E.coli,in the low immunity of patients spread more quickly..These results provide experimental basis for the establishment of drug-resistant gene pool in the region and the control of clinical drug-resistant bacteria.