【摘要】 目的:研究大肠杆菌耐药菌株蓄积亲水性氟喹诺酮药物氧氟沙星和疏水性药物托氟沙星的机制有无差异。方法:荧光测定法检测菌体内的药物蓄积量和主动外排;SDS-PAGE法分析细菌外膜蛋白。结果:氧氟沙星在Ecs与JF701的蓄积量基本一致,在JF703中较JF701低1/2,但在4株耐药菌的蓄积量较这些敏感菌低5—7倍;托氟沙星在氟喹诺酮耐药和敏感菌株中的蓄积则未见显著差异。加入质子载体DNP后5,10min,细菌中托氟沙星的蓄积缓慢下降,氧氟沙星的蓄积增加,尤其在耐药菌株明显。氧氟沙星的蓄积增加与其对细菌的MIC呈正相关(r分别为0.9623和0.8006)。此外,Ecs中OmpF和OmpC完整;R2、R256和R5、R6分别发生OmpF和OmpC缺失。结论:OmpF缺失和药物主动外排可导致氧氟沙星,而不是托氟沙星在菌体内的蓄积减少。更多还原

【Abstract】 AIM: To make sure whether there is a difference in mechanism existed in the resistant E coli strains accumulated hydrophilic fluoroquinolone ofloxacin and hydropho-bic fluoroquinoline tosufioxacin. METHODS: Fluoroquinolone accumulation in bacteria and effect of active efflux were measured by fluorescence method. Analysis of outer membrane proteins was made by SDS-PAGE. E coli strains included JF701 and JF703 that are OmpC- or OmpF-deficient mutants of E coli K-12 respectively, and the susceptible strain Ecs and its in-vitro selected resistant strains R2, R256, and clinical resistant isolates R5, R6. RESULTS: Ecs accumulated ofloxacin almost at the same concentration as JF701, but JF703 did about 1/2 of that lower than JF701. However, four resistant strains accumulated ofloxacin about 5 to 7-fold lower than those susceptible strains. On the other hand, there was no significant difference for the accumulation of tosufloxacin between fluoroquinolone-resistant and -susceptible strains. After addition of proton ionophore DNP for 5 min and 10 min, the accumulation of tosufloxacin slowly decreased in E coli strains, whereas the accumulation of ofloxacin was increased, especially in the resistant strains. A good relevance exists between the accumulation increment of ofloxacin and its MIC for each E coli strain after additionof DNP for 5 min and 10 min ( r = 0.9623 and 0.8006 respectively). Furthermore, both OmpF and OmpC in Ecs, OmpF-deficiency in R2, R256 and OmpC-deficien-cy in R5, R6 were observed. CONCLUSION: The accumulation of ofloxacin other than tosufloxacin could be reduced by OmpF-deficiency and active efflux, and the latter may be an important factor in the development of resistance to hydrophilic fluoroquinolone in E coli.更多还原