嗜水气单胞菌对四环素类药物诱导耐药表型及机理研究

Tetracyclines-induced phenotype and resistance mechanism in Aeromonas hydrophila

摘要:

[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tetA和tetE;在诱导前后的2号菌株中检测到tetC;在诱导前后的1、3、4、5、6、7号菌株中均检测到tetE。[结论] 本研究表明tetE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。

Abstract:

[Objective] The aim of this study is to explore the changes of sensitivity to tetracyclines and the resistance mechanism of Aeromonas hydrophila (Ah) after in vitro induction by exposure Ah to the increasing concentration of Doxycycline (DO). [Methods] The sensitive Ah isolates were first screened from clinical isolates and then incubated on TSA solid medium containing DO of 1/4×MIC value. The resistant strains were obtained through continuous subculture by increasing the concentration of DO in the medium for geometric series. The minimal inhibitory concentrations (MICs) of induced strains to DO and 16 other non-selected antimicrobials were determined. Meanwhile, MICs were also determined after adding efflux pump inhibitor 1-methyl-2-pyrrolidinone (NMP) to the medium. The relationship between sensitivity changes and efflux function were analyzed. Then five genes of tet from induced strains were amplified and sequenced. [Results] The MICs of induced strains to DO increased significantly after induction, whereas the MICs of strains to those non-selected tetracyclines also increased. The MICs of induced strains to fluoroquinolone increased much more than that of control. The induced strains exhibited a little higher sensitivity to aminoglycoside and rifampicin. However, the MICs of all induced strains to DO decreased after adding NMP to the medium. The detection of tet genes indicated that tetA and tetE were positive in No. 7 after induction and the tetC gene was positive in No. 2 before and after induction. The tetE gene was detected in strains No. 1, 3, 4, 5, 6 and 7 no matter whether it was induced or not. [Conclusion] This study suggested that tetE gene may be the predominant gene mediating tetracyclines-resistance of Ah, which would provide theoretical basis to clarify the resistance mechanism of Ah to tetracyclines.