氟喹诺酮类耐药表皮葡萄球菌的筛选及其特点

Screening of Fluoroquinolones-Resistant Staphylococcus epidermidis and Its Characteristics

目的 建立应用四环素筛选氟喹诺酮类 (FQNs)非特异性耐药表皮葡萄球菌的方法 ,并探讨耐药性经质粒介导传播的可能性。方法 选择培养对四环素高度耐药的突变菌株 ,并从中选择环丙沙星 (CPL X)和司帕沙星(SPL X)耐药菌株 ,用 K- B纸片法评价耐药性的遗传稳定性 ;提取细菌质粒 DNA进行琼脂糖凝胶电泳 ,结合细菌对 FQNs的敏感性分析质粒与 FQNs耐药的联系。结果 经过 4次筛选后 ,所获得的突变菌株全部对两种 FQNs耐药 (MIC≥ 1μg/ m l) ,其中有 1/ 3菌株达到中等程度耐药水平 (MIC≥ 4μg/ ml) ,在空白 MH琼脂平板上连续培养 10代后 ,细菌对两种 FQNs的耐药性虽有下降但仍维持在中度耐药水平 ;耐药菌中含有 17kbp的可自主复制质粒 ,敏感菌和质粒消除菌则未检测到质粒存在。结论 敏感的表皮葡萄球菌经四环素选择培养后可获得对FQNs非特异性耐药的菌株 ,与耐药性相关的基因定位于细菌质粒 。

OBJECTIVE To build an in vitro method which could isolate FQNs resistante mutant from clinical wild type isolated after exposing to high concentration tetracycline. The possibility that bacteria′s fluoroquinolones resistance transfered via plasmids was also involved in this study. METHODS FQNs sensitive isolates were inoculated stepwise in Mueller Hinton agar culture medium containing increasing concentration of tetracycline. Those mutants which were resistant to ciprofloxacin (CPLX) and sparfloxocin (SPLX) were collectcd for the following experiments. All the mutants were incubated in MH agar culture medium without antibiotics for ten generations to see whether they could preserve FQNs resistance. Bacteria′s plasmid DNA was extrated and analyzed by agarose gel electrophoresis method. RESULTS Selected by tetracyoline, all the mutants were resistant to CPLX and SPLX (MID≥1 μg/ml), of which about 1/3 strains showed moderate resistance (MIN≥4 μg/ml). After incubated in bland MH agar culture medium, these strains could preserve their resistance to FQNs for a long time. Agarose gel electrophoresis assay of bacteria plasmid DNA suggested that FQNs resistance mutants contain a 17 kbp plasmid which could replicate independently. There was no plasmid detected in FQNs susceptible isolates and plasmid elimination mutants. CONCLUSIONS After exposing to high concentration of tetracycline FQNs resistant Staphylococcus epidermidis mutants could be isolated, which showed medium level no structure related resistance to CPLX and SPLX. Persistance genes were encoded in an independen replication plasmid through which sensitive bacteria might acquire tolerance to FQNs.