体外诱导嗜水气单胞菌对喹诺酮类耐药及其耐药机制研究

Characterization of quinolone resistance mechanism of Aeromonas hydrophila selected in vitro

【目的】探讨在亚抑菌浓度喹诺酮类药物培养后,嗜水气单胞菌Aeromonas hydrophila对喹诺酮类的药物敏感性变化及其耐药机制.【方法】以对喹诺酮类敏感的临床分离嗜水气单胞菌菌株和标准菌ATCC7966为研究对象,分别在含亚抑菌浓度萘啶酸(NAL)和环丙沙星(CIP)的培养基上逐步诱导培养.提取诱导菌的DNA,PCR扩增其gryA和parC基因,测序分析其喹诺酮类耐药决定区(QRDR)突变情况;测定诱导菌对诱导药物和11种非诱导药物的最小抑菌浓度(MIC)及添加外排泵抑制剂羰基氰化氯苯腙(CCCP)后的MIC,分析其敏感性变化与基因突变、外排作用的关系.【结果和结论】诱导后菌株对萘啶酸和环丙沙星的MIC分别提高了1 024和64 000倍,对非诱导药物也有不同程度提高;当萘啶酸和环丙沙星诱导浓度分别达到16和32μg/mL或以上后,诱导菌株gyrA基因编码的氨基酸分别发生Asp87→Tyr和Ser83→Arg的变化,但两者parC基因编码的氨基酸均没有发生突变;添加CCCP后,只有氟喹诺酮类药物的MIC值略有下降,提示嗜水气单胞菌对喹诺酮类耐药存在靶基因突变及主动外排作用等多种耐药机制.

[ Objective ] To investigate the effect of the sub-inhibitory concentration of quinolones on resist- ance to quinolones in Aeromonas hydrophila and its mechanisms. [ Method ] The quinolone sensitive strains of A. hydrophila from clinical and ATCC7966 were selected in vitro stepwise exposure to increasing concentration of nalidixic acid （NAL） and ciprofloxacin （CIP） on solid medium. The quinolone resist- ance-determining region （QRDR） was sequenced to determine the mutation for quinolone resistance. The minimal inhibitory concentrations （MICs） of selected and non-selected antimicrobials as well as MIC by efflux pump inhibition cyanide chlorophenyl hydrazone （CCCP） of each strain were determined by broth microdilution method. The relationship between sensitivity varieties, mutations and efflux pump activities were analyzed. [ Result and conclusion ] The MICs of NAL and CIP increased 1 024-and 64 000-fold af- ter selection, respectively, while the MICs of other antimicrobials increased diversely. When MIC of NAL or CIP in A. hydrophila reached 16 p,g/mL or 32 p,g/mL, Asp87→Tyr or Ser83→Arg, mutations of Gy- rA were identified, but no mutations of ParC were found. After CCCP was added, only MICs of fluoro- quinolones caused a slight decrease, implying that it might include the quinolone mechanisms of target gene mutations and an active efflux system in A. hydrophila.