Molecular Investigation Of Quinolone Resistance In QnrS-Positive Escherichia Coli Strains

Objectives The aim of the study was to investigate the molecular characterization of quinolone resistance in qnr S-positive Escherichia coli strains. Furthermore, in order to have better understand the qnr S mediated quinolone resistance.Methods 1. QRDRs mutation mediated quinolone resistance in qnr S-positive Escherichia coli. The QRDR genes and their genetic environment were characterized using polymerase chain reaction(PCR) and sequencing. The results were analysis by BLAST. 2. Study on Molecular mechanisms of qnr S-positive Escherichia coli conjugants.The Minimum Inhibitory Concentration(MIC) of the transconjugant strains was detected by agar dilution method, and the results were compared with the donor strains. The plasmids of qnr S-positive Escherichia coli conjugants were obtained by alkaline lysis method. The transconjugant plasmids were assigned to incompatibility groups by PCR-base replicon typing. 3. The qnr S-positive plasmid Sequencing. One selected plasmid was obtained by Giagen plasmid plus maxi kit, and the product was sequenced completely.The results were analyzed for its genetic environment and distribution of resistance genes.Results 1. 56 /57 qnr S-positive Escherichia coli strains contained mutations in the Gyr A,4/57 carried mutations in the Gyr B, 33/57 carried mutations in Par C, 27/57 had Par E mutations.The mutation rate of the Gyr A was 98.24%,and all the strains contained Par C mutation also had Gyr A mutation.One strain with Gyr A mutation would show resistance to CIP by mutating in Par C Ser80-Ile point.All the strains contained Ser458-Ala mutation in Par E also had double mutation of Gyr A and Par C,and one strain contained Ser458-Ala mutation in Par E had higher MIC value of CIP than which did not. 2. Antibiotic susceptibility test of 13 qnr S-positive Escherichia coli donor stains was performed using the CLSI(Clinical and Laboratory Standards Institute) criteria, and 13 strains showed resistance to 5 antibiotics,with Nalidixie acid(100%), Ciprofloxacin and levofloxacin(69.2%), Cefotaxime and Gentamycin(53.8%). And all strains were sensitive to in Amikacin and Imipenem agents. All of the 13 qnr S-positive Escherichia coli conjugons were sensitive to 8 antibiotics; Comparison with E.coli J53,the quinolone MICs value of qnr S-positive Escherichia coli conjugons had slightly increased.And the MICs value of qnr S-positive Escherichia coli conjugons had significantly decreased,by comparison with donor stains;8 of 10 Escherichia coli conjugons have been typed by PCR-base replicon typing,and divide to Inc I1,Inc N,Inc FIB,Inc Frep,Inc Col E groups. 3. p FJUM was a multidrug plasmid. It consisted of the replication regions, the conjugal transfer regions, the transposons and the resistance genes. The plasmid contained several genes that mediated resistance to quinolone, cephalosporins, tetracyclines, penicillins, sulfonamides, aminoglycosides, and also encode efflux pump protein. No integron structure was found in upstream, or downstream of qnr S gene.Conclusion 1. Mutation of the Gyr A subunit is the prior condition leads to quinolone resistance and the Par C subunit mutation. Ser80-Ile of the Par C subunit is a crucial mutation point that induces resistance to CIP. Ser458-Ala mutation of the Par E subunit might mediates high resistance to quinolone antibiotic. 2. qnr S gene only induced slightly increase of the MICs value of quinolone,and chromosome mutation in QRDRs played an important role in mediating high-level quinolones resistance. 3. qnr S-positive plasmids replicon types were various, and most of the plasmids were more than 40-kb. 4. The p FJUM was a classical multi-drug resistant plasmid, and mediated to several antibiotics.The p FJUM plasmid could transfer via conjugation. The p FJUM was formed of an original plasmid that captured resistance gene in the progress of evolving.