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【目的】初步探讨利用四环素类药物体外诱导嗜水气单胞菌耐药后,嗜水气单胞菌对四环素类药物敏感性的变化及其耐药机制。【方法】筛选临床分离嗜水气单胞菌的四环素类敏感株,从含有1/4×MIC的强力霉素的TSA固体培养基开始,等比2倍提高诱导药物质量浓度对受试菌进行连续传代培养,以获得高耐诱导株;测定诱导菌对强力霉素和16种非诱导药物的最小抑菌浓度(MIC)及添加外排泵抑制剂N-甲基吡咯烷酮(NMP)后的MIC,分析其敏感性变化与外排作用的关系;提取诱导菌的DNA,PCR扩增其5个tet基因并测序。【结果】诱导后菌株对强力霉素的MIC显著升高,对非诱导四环素类药物也有不同程度提高,对氟喹诺酮类药物的MIC比诱导前增加几十至上千倍;对氨基糖苷类药物和利福平的MIC则有不同程度的降低;添加NMP后,所有诱导菌株对强力霉素的MIC值均有不同程度的下降;四环素类耐药基因的检测结果表明,在诱导后7号菌株中同时检测到tet A和tet E;在诱导前后的2号菌株中检测到tet C;在诱导前后的1、3、4、5、6、7号菌株中均检测到tet E。【结论】本研究表明tet E基因可能是介导气单胞菌分离株对四环素类药物耐药的优势基因,为阐明嗜水气单胞菌对四环素类药物耐药机制及耐药性与耐药基因之间的关系提供理论依据。
【Objective】 The purpose of this study was to investigate the changes of the susceptibility of tetracyclines to Aeromonas hydrophila induced by tetracycline-induced drug-resistant Aeromonas hydrophila in vitro and its mechanism of drug resistance. 【Method】 Tetracycline-sensitive strains of Aeromonas hydrophila isolated from clinical isolates were screened from TSA solid medium containing 1/4 × MIC of doxycycline to increase the concentration of induced drug to 2 times Continuous subculturing to obtain highly resistant strains; determining the minimum inhibitory concentration (MIC) of the induced bacteria against doxycycline and 16 non-inducing drugs and the MIC after adding the efflux pump inhibitor N-methyl pyrrolidone (NMP) The relationship between the sensitivity and efflux was analyzed. DNA of the induced bacteria was extracted, and five tet genes were amplified by PCR and sequenced. 【Result】 The results showed that the MICs of the isolates were significantly higher than that of doxycycline and the non-induced tetracyclines were increased to some extent. The MICs of fluoroquinolones were increased by tens to thousands of folds compared with that before induction. For aminoglycosides and MICs of rifampicin decreased to some extent. After adding NMP, the MICs of all the induced strains decreased to different extents. The results of tetracycline resistance genes showed that in the strain 7 after induction Tet A and tet E were detected simultaneously; tet C was detected in strain 2 before and after induction; tet E was detected in strains 1, 3, 4, 5, 6 and 7 before and after induction. 【Conclusion】 This study shows that tet E gene may be the dominant gene that mediates the resistance of Aeromonas isolates to tetracyclines. To elucidate the mechanism of resistance to tetracyclines in Aeromonas hydrophila and its resistance and resistance The relationship between drug genes provide a theoretical basis.