Fluoroquinolone-resistant Plasmid And GyrA Gene Of E.coli With Multiple And High-level Drug Resistance

DNA gyrase of Escherichia co/i consists of subunits A and B,which are the products of the gyrA and gyrB genes,respectively. Mutations in gyrA gene can cause quinolone resistance. DNA sequence analysis previously done has disclosed that these mutations were located within a relatively small region of the A subunit, which is called a quinlolne resistance-determining region (QRDR). and these mutations are basic and important for quinoloneresistance. Over the last decade there have been sporadic reports of plasmidmediated quinolone-resistance, which described when a mutator plasmid was present .the frequency of resistance for selecting antibiotic-resistant strains always went up,but no quinolone-resistance plasmid was found in E.coli. To study on the mechanisms of Fluoroquinolone resistance of E.coli. the drug susceptibility of E.coli clinical isolates from Jilin province during the past 3year period (1996-1999) were tested. 32 strains of pathogenic E. co/i, which were superhigh fluoroquinolone-resistant and multi-drug resistant, were further studied. After the plasmid DNA extraction, purification and transformation. only one multi-drug resistant strain CEO1 whose fluoroquinolone resistance was associated with its plasmid DNA was obtained, and after drug-resistant plasmid elimination, there were only four strains which levels of fluoroquinolone resistance lowered clearly after elimination . Then plasmids of these four strains were extracted and electrophoresized, a plasmid of strain CEO 1, which can be transferred with fluoroquinolone resistance, had been eliminated by BS or EB while there were no changes of plasmids associated with eliminators in the others. Therefore, the fluoroquinolone-resistant plasmid was found in the strain CEO 1. And the quinolone resistance-determining region (QRDR) of the gyrA gene v%as amplified by PCR with the plasmid DNA and chromosomal DNA templates from the strain CEO 1. The expected 668-bp gy抮A fragments were both obtained from them by PCR. The nucleotide sequences of PCR products58from plsmid DNA and chromosomal DNA of strain CEOI showed significant homology (98.17% identity). When compared to the corresponding sequences of gyrA of F. coil from the nucleotide sequence data reported by Swanberg,S.L. and Wang,J.C., 13 mutant sites were found in the nucleotide sequence of PCR product from plasmid DNA, and 3 amino acids changed:while 12 mutant sites were found in that from chromosomal DNA, and 2 amino acids changed. The amplified products from plasmid DNA and chromosomal DNA of the strain CEO1 were labelled with a -32P dCTP by nick translation reaction. and were used as probes to detect plasmid DNA and chromosomal DNA of the strain CEO1 by southern hybridization and dot-blot hybridization, respectively . Plasmid DNA and chromosomal DNA of the strain CEOI all obtained positive dot-blot to plasmid probe and chromosomal probe correspondingly. As a result, fluoroquinolone-resistant rate of E.coli strains isolated from swine was 56%, that from chicken was 20.5%. Strain CEOI has a plasmid associated with fluoroquinolone resistance. and fluoroquinolone resistant mutant gy抮A gene existed surely both in the plasmid and chromosome of strain CEO 1. Both the plasmid and chromosome are associated with fluoroquinolone resistance of strain CEO 1.