Study On The Plasmid-mediated QnrA Gene In Escherichia Coli And Klebsiella Pneumoniae Producing Extended-spectrum β-lactamase

Previous studies demonstrated that bacterial resistance to quinolone principally caused by alterations in the chromosomal genes coding for DNA gyrase and /or topoisomerase IV, which were the targets of quinolone action. In 1998, low level quinolone resistance was found to be transfered by a plasmid firstly discovered in a clinical isolate of Klebsiella pnermoniae. The gene responsible, named qnr, proved to be the quinolone resistance gene, which can protect DNA gyrase and /or topoisomerase IV from inhibition by quinolone. Many analysis of the gene structure indicated that qnr was always located in class 1 integrons which included many cassttes, especially ESBLs gene. Nowadays qnr was reported in worldwide including East Asia, American and Europe, et al.263 clinical strains of Escherichia coli and 99 clinical strains of Klebsiella pneumoniae producing ESBLs were collected from six provinces including Zhejiang, Xinjiang, Henan, Jiangsu, Shenyang and Macao, China. The minimal inhibiton concentratin (MICs) of various antimicrobials were determined by agar dilution method. PFGE was performed to analyse the total DNA from isolates with qnr A. Polymerase chainreaction (PCR), conjugation experiment, Southern blot and shot gun sequencing were carried out for analyzing the resistance gene and flanking sequences.qnrA was detected in 5 of 263 (1.9%) Escherichia coli isolates and 8 of 99 (8.1%) Klebsiella pneumonia isolates. The CTX-M gene presented in all 13 qnrA-positive strains (including CTX-M-9 CTX-M-14 and CTX-M-24) .Transfer of the qnrA determinant by conjugation could be deteced from all 13 qnrA-positive strains. The MICs of ciprofloxacin for transconjugants were 0.06-2 μg/ml, representing an increase of 4- to 133- fold compared to the recipient, E.coli J53. Mution of amino acid 83 and/or 87 in GyrA were found in 11 isolates with resistance to ciprofloxacin (MIC≥4mg/L), while mutations of amino acid 80 in ParC were also found in 8 of 11 isolates with high-level resistant to ciprofloxacin (MIC≥16mg/L). There was no mutation in 2 isolates with low-level resistant to ciprofloxacin. qnrA gene and CTX-M-24 gene were proved to locate in a 50kb conjugative plasmid by southern blot and sequence.In brief, the positive rate of qnrA gene in Klebsiella pneumonia is higher than that in Escherichia coli. The co-presence of qnrA gene and mutation of GyrA and (or) ParC results in the higher level resistance to quinolone. qnrA gene and CTX-M gene can be cotransfered simultaneously by a plasmid.