The Mechanism Of Resistance To Fluquinolone In ESBLs-producing Escherichia Coli

Fluquinolones is the first completely synthesis antimicrobial agents. They are extensively used in various types of infections due to their strongly activity against gram-negative,gram-positive bacteria and anaerobes bacteria. Extensive use of them has introduced the high fluquinolones-resistance. The mechanism of it is quite complex. There are two main mechanisms implied in the resistance: alterations in the targets of the fluquinolones and decrease in the accumulation of the antibiotic in the bacterial cell by making the membrane impermeable or by expression of active expulsion systems. Fluquinolones target is the type 2 topoisomerases( gyrase) and topoisomerase IV. DNA gyrase consists of two GyrA subunits and two GyrB subunits, which catalyses the negative supercoiling of DNA that is essential for maintenance of DNA topology. Topoisomerase IV consists of two ParC subunits and two ParE subunits which is involved in the segregation of replicated daughter chromosomes during DNA replication. gyrA /gyrB /parC /pareE were their encoding genes. Mutations in the target genes increase the resistance of fluquinolones .In 1998, horizontally transferable resistance to fluquinolones was described for the first time. The qnrA gene is genetically responsible for resistance and is found inside a mobile element. Then qnrB and qnrS were found. It is implied that plasmid-mediated fluquinolone-resistance is another mechanisms.The objective of the study is to clarify the fluquinolones-resistance and its mechanism in Escherichia coli (E.coli) clinical isolates from the first people's Hospital of Kunming.Methods: The isolates ESBLs- producing E coli from 2005.1 to 2005.12 in the hospital were collected. The MICs of fluquinolones were detected by argrose dilution method. Conjugation test was used to determine the plasmid-mediated fluquinolone-resistance, and the gyrA/gyrB/parC/parE genes were detected by Polymerase Chain Reaction (PCR). The point mutations were found by sequencing of the PCR products. The plasmid-mediated fluquinolone-resistance genes (qnrA,qnrS,qnrB,aac(6' )-Ib-cr) were detected by PCR.Results:There are 93(89.4%) isolates of 104 E.coli resisted to Ciprofloxacin, 91(87.5%) were resistant to Levofloxacin, and 93(89.4%) were resistant to Moxifloxacin. 78 isolates of 104 E.coli were tested by conjugation. 2 transconjugants were acquired, and the percent of conjugantion was 2.6%.The gyrA/gyrB/parC/parE genes of highly resistant stains were detected by PCR. The point mutations of the gyrA/ parC gene have been found. they are gyrA 83 Ser→Leu, 87Asp→Asn and parC 55Ser→Leu, 62Gln→Glu. The aac(6' )-Ib-cr gene were detected in 7 of 90 isolates ESBLs- producing and fluquinolone-resistant of E.coli. The qnrA,qnrS,qnrB genes were not found.Conclusion: The fluquinolone-resistance among the clinical isolates of E.coli in this hospital was critical. The mechanism of fluquinolone-resistance in the clinical isolates of E.coli is complex. There are not only point mutations in the fluquinolone resistance determining- region of gyrA or parC gene but also the plasmid-mediated fluquinolone-resistance genes in the isolates ESBLs- producing E coli and the horizontally transferable elements of fluquinolones- resistance. It is necessary that avoiding the extensively use this agents in order to control the fluquinolones-resistance.