| Background:Quinolone is a kind of synthetic antibacterial agents . Rapid development has been made since the first quinolone-nalidixic acid was synthesized successfully in 1962 . As a new class of broad-spectrum antibacterial agents , now fluoroquinolones has become the main stream of quinolones. Fluoroquinolones has many advantages such as broad antibacterial spectrum, high antibiosis activity, slight adverse reaction, extensive tissue distribution, convenient administration and so on, so they are commonly used in clinical medical science and hippiatrics . Esherichia coli (E. coli) is an important pathogen of nosocomial infection and has many complicated mechanisms of drug resistance. With the fluoroquinolones was widely used, especially was abused in clinical, which lead to an escalating rate and increasing level of resistance to quinolones in E. coli. Fluoroquinolone resistance has become a serious problem in clinical treat.The mechanism of resistance to fluoroquinolones in E. coli has become a extensive concerned problem. Three mechanisms of resistance to quinolones in E.coli are currently recognized: mutations that alter the drug targets-DNA gyrase and topoisomerase IV, mutations that reduce drug accumulation, plasmid-mediated resistance. The target-enzyme alterations is main mechanism of resistance to quinolones in E. coli. Investigating the situation of resistance to fluoroquinolones in E. coli in local current, intensive study on the emergence and spread of E. coli resistance, accurate identification of phenotype and genotype of resistance are of importance for restricting the emergence and development of resistance and nosocomial infection, administrating antibacterial agents reasonably. For this purpose, the resistance monitoring data of E. coli isolated in clinical were analyzed, E. coli strains were induced to be highly resistant strains to fluoroquinolones by induce resistance experiment in vitro, gene mutations of the quinolones resistance-determining region(QRDR) of gyrA and parC of the induced resistant strains were detected, in order to deep research the resistance of E. coli to fluoroquinolones.Objective:To investigate the characteristics of resistance to fluoroquinolones in E. coli by analysis of the resistance monitoring data of E. coli in clinical. To measure the susceptibility and the cross-resistance of E. coli to fluoroquinolones ,measure the ESBLs of E. coli and the associated resistance of ESBL-producing E. coli to fluoroquinolones by induce resistance experiment of multiple-step method , in order to provide guidance to clinical reasonable administrate antibacterial agents. To research mechanisms of fluoroquinolones resistance in E. coli by research on the relationships between gene mutation of topoismerase II and resistance to fluoroquinolones.Methods:The resistance monitoring data of E. coli isolated in the First Affiliated Hospital of Henan College of TCM during 2008 were analysed by whonet 5.4. Some clinical isolates of E. coli were primarily selected and identified to species level correctly according to the morghological and biochemical characteritics determined by Phoenix-100. The minimal inhibitory concentration (MIC) of ciprofloxacin, levofloxacin, gatifloxacin was measured by using agar dilution method. Then 37 of clinical isolated E. coli were choosed as the test strains according to the result of strain identification and MIC determination. These test srains were divided into three groups for inducing the resistance to ciprofloxacin (CE1-CE10), levofloxacin (LE1-LE11) and gatifloxacin (GE1-GE12) respectively. E. coli ATCC25922 was used as control strains.The test strains (CE1-CE10 for ciprofloxacin, LE1-LE11 for levofloxacin, GE1-GE12 for gatifloxacin) were performed induce resistance experiment of multiple-step method respectively. The procedure was as follows : The E. colisuspension with 1×10~8 CFU/ml was prepared , 10μl of the prepared suspension was added into 2ml of the MH broth containing 1/4MIC antibacterial agents , and incubated for 24 hours at 35℃.10μl of the suspension with 1×10~8CFU/ml prepared with the culture of 24 hours was added again into 2ml of MH broth containing 1/2MIC antibacterial agents,incubated for 24 hours at 35℃. As the above procedure, the test strains were cultured in MH broth cotaining stepwise graded concentration of antibacterial agents up to containing 128-fold MIC antibacterial agents. The induced resistance strains were incubated drug-free MH broth per day, for five continuous days at 35℃.The ESBLs of clinical isolated strains and test strains were measured with confirmatory test. The MIC of test strains by agar dilution method, gyrA and parc were amplified by PCR, then the nucleotide sequence of these genes was analysed.Results:223 strains E. coli were isolated in 2008, including 94 ESBLs-producing E. coli (42.2%).The resistance rate of ESBLs-producing E. coli to ciprofloxacin, levofloxacin, gatifloxacin is 87.8%, 84.9%, 84% respectively. The resistance rate of ESBLs-nonproducing E. coli to ciprofloxacin, levofloxacin, gatifloxacin is 71.2% , 73%, 72.3% respectively. 12 strains of 14 test strains (CE1-CE6, CE8-CE12, CE14)were obtained highly resistant to ciprofloxacin by continuously culturing in an increasing concentration of ciprofloxacin. The MIC of these induced strains resistant to ciprofloxacin varied from 128 to 512μg/ml, with 128-32000-fold increase when compared with pre-induce strains. 7 strains of 11 test strains (LE1, LE2, LE4, LE6, LE8-LE10) were obtained highly resistant to levofloxacin by continuously culturing in an increasing concentration of levofloxacin. The MIC of these induced strains resistant to levofloxacin varied from 128 to 256μg/ml, with 32-64-fold increase when compared with pre-induce strains. 5 strains of 12 test strains (GE3, GE5, GE8, GE9, GEll)were induced highly resistant to gatifloxacin by continuously culturing in an increasing concentration of gatifloxacin. The MIC of these induced strains resistant to gatifloxacin varied from 128 to 512μg/ml, with 32-128-fold increase when compared with pre-induce strains. The cross-resistance of induced high resistant strains to fluoroquinolones exists. The ESBLs of all strains aren't altered during induction. The associated resistance of ESBLs-producing E. coli to fluoroquinolones exists.E. coli ATCC25922 and the strain of CE1 and partial induced resistant strains including CE1I, CE6I, LEU, LE2I, LE8I, GE3I, GE5I, GE8I were amplified by PCR in gyrA and parC.648 base pairs and 417 base pairs emerge from the Gel electrophoresis map of PCR products of all strains . Neucleotide sequence analysis and comparison of the derived amino acid sequence revealed that QRDR of gyrA had substitution of Ser residue to Leu residue at position 83 and Asp residue to Asn residue at position 87 in 8 resistant strains. The QRDR of parC alterations of Ser to Ile at position 80 were observed in 8 resistant strains. E. coli ATCC25922 and the strain of CE1 aren't found alteration of amino acids in QRDR of gyrA and parC.Conclusion:1. In the long-term selective pressure of antibacterials, E. coli can be acquired resistance to fluoroquinolones.2. The cross-resistance of E. coli to fluoroquinolones exists and the associated resistance of ESBLs-producing E. coli to fluoroquinolones exists.3. The alteration of DNA gyrase and DNA topoisomerase IV is the main mechanism of resistance to fluoroquinolones in E. coli. |
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