| [OBJECTIVE] (1)The paper was designed to establish a method to determine mutant prevention concentration(MPC) in vitro. The potential for restricting the selection of resistant mutants of five fluoroquinolones (ciprofloxacin, pazaufloxacin, levofloxacin, gatifloxacin, moxifloxacin) with different structures against Escherichia coli were compared. (2) The effect of drug concentration, drug structure on the resistant gene was discussed and the process of Escherichia coli from sensitivity to resistance was studied. (3) The rationality of clinical use of fluoroquinolones was dicussed according to the MSW theory.[METHOD] (l)The agar dilution method was carried out on Mueller-Hinton agar containing fluoroquinolones according to NCCLS guidelines to determine minimal inhibition concentration(MIC) of ciprofloxacin, pazaufloxacin, levofloxacin, gatifloxacin and moxifloxacin (CPLX, PZLX, LVLX, GTLX, MXLX)against ATCC25922. Mutant prevention concentration was determined by the agar dilution according to MIC determining except that ATCC25922 were enriched in broth, and the bacterial concentrations were adjusted to 1010 colony form units per milliliter. The fraction of bacteria recovered curve was traced by colony counting method. (2)The two quinolone resistance-determining regions of target genes, gyrA and parC of mutants selected in the MSW were obtained by PCR method and sequenced by DNA sequencing. (3) Pharmacokinetic study of single-dose and multi-dose pazufloxacin mesilate were made in Chinese healthy volunteers and pharmacokinetic parameters were determined by 3p97. MPC of Escherichia coli clinical isolates were determined by agar dilution method.[RESULTS] (1) MPC of CPLX,PZLX,LVLX,GTLX and MXLX against ATCC25922 was 0.06, 0.09, 0.09, 0.048, 0.072ug/ml. The MPI of five fluoroquinolones against ATCC25922 were CPLX>PZLX>LVLX-GTLX>MXLX; the fraction of bacteria recovered curve showed that the MSW of MXLX and GTLX was narrower than that of CPLX, LVLX and PZLX; for the mutant frequency of ATCC25922, MXLX was the smallest, CPLX and GTLX was the smaller and LVLX and PZLX was the largest. The third mutant selected by CPLX> LVLX and PZLX was middle-resistant, while the third mutants selected by MXLX and GTLX were still sensitive. MPI of mutants selected by LVLX and PZLX was 2-4 fold greater than that of ATCC25922. (2)Among 53 first-step mutants, 79% was a mutation from Ser to a Leu residue at position 83( a Ser-83—Leu mutation) detected in the quinolone resistant-determining region of the gyrA gene, 19% was a mutation from Asp to a Asn residue at position 87( a Asp—87Asn mutation), 2% was a mutation from Gly to a Asp residue at position 81( a Gly81—Asp mutation), and no parC mutation was detectable. MIC of mutation at position 83 was 2~8 fold larger than that of mutation at position 81 and 1-2 fold larger than that of mutation at position 87. Mutation at position 83 was the most important factor to influence the sensitivity of Escherichia coli. DNA gyrase is the primary target, mutation at position 83 and 87 was the most frequent and no-target mutation was also involved in the resistance .(3) Pharmacokinetic parameters of 25Omg, 500mg> 750mg pazufloxacin mesilate infusion were: Cmax: 4.89 ± l.llmg.L"1^ 9.94 ±2.62mg.L"', 17.43±3.22mg.L"'; ti/2e: 1.50 ± 0.19h? 1.38 + 0.27h> 1.53±0.26h; AUC0.t: 9.53 ± 2.01mg-h-L"1, 21.47 ± e^mg-h-L"1? 36.12 + 6.40mg-h-L"'. MPC90 of CPLX ^ GTLX> PZLX, MXLX against 25 clinical isolates of Escherichia coli were 2> 1, 2, lug/ml; MPCpr9o/MIC9o of four fluoroquinolones were 16, 4, 16, 2 respectively. PK/PD parameters Cmax/MIC90 of 250mg, 500mg, 750mg PZLX against E.coli clinical isolates were 39.12, 79.52, 139.44; AUC0-i2/ MIC90 of 250mg, 500mg, 750mg PZLX against E.coli clinical isolates were 76.24, .171.76, 289; TMswof 250mg, 500mg, 750mg PZLX against E.coli clinical isolates were 50%, 45.6%, 51%. The...
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