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目的探讨体外阿奇霉素诱导肺炎支原体(MP)耐药的发生机制。方法在含亚抑菌质量浓度阿奇霉素的MP培养基中,诱导培养MP国际标准株FH株对大环内酯类抗生素的耐药株;应用体外药物敏感试验,检测诱导株对红霉素的最小抑菌质量浓度(MIC),并筛选出耐药株;测定耐药株对阿奇霉素和交沙霉素的MIC值;通过PCR扩增与大环内酯类抗生素耐药性有关的23SrRNA结构域V区及核糖体蛋白L4、L22基因,并对扩增产物进行全自动DNA测序,测得序列与美国国立生物信息中心已登录的MP标准株M129的相应基因序列进行对比。结果通过体外诱导得到FH体外诱导株8代(YD1-YD8),其中YD1-YD4为敏感株,YD5-YD8为耐药株。耐药株对红霉素、阿奇霉素、交沙霉素的MIC值均显著升高。在与大环内酯类抗生素耐药相关的23S rRNA结构域V区中YD1-YD6诱导株和FH株均未出现点突变,YD7、YD8诱导株均出现了2067位A→G的点突变;在核糖体蛋白L4中,诱导株和FH株均出现了162位C→A和430位A→G的点突变;在核糖体蛋白L22中,诱导株和FH株均出现了279位T→C和508位T→C的点突变。结论 23S rRNA结构域V区中心环的药物作用靶位基因突变是耐药性产生的主要机制。在使用阿奇霉素药物治疗的过程中,有体内诱导出耐药株的可能。
Objective To investigate the mechanism of azithromycin-induced mycoplasma pneumonia (MP) resistance in vitro. Methods In MP medium with sub-bacteriostasis concentration of azithromycin, the strains resistant to macrolide antibiotics were induced by MP international standard strain FH strain. The drug susceptibility test was used to detect the minimum level of erythromycin Antibacterial concentration (MIC), and screened drug-resistant strains; determination of MIC strains azithromycin and josamycin resistance; PCR amplification of macrolide antibiotic resistance 23SrRNA domain V Region and the ribosomal protein L4 and L22 genes were amplified by PCR and sequenced. The sequences were compared with the corresponding sequences of the MP standard strain M129 registered by the National Center for Bioinformatics (NCBI). Results In vitro induction of FH induced 8 generations (YD1-YD8), of which YD1-YD4 sensitive strains, YD5-YD8 resistant strains. The MIC of erythromycin, azithromycin and jasamycin were all significantly increased in resistant strains. No point mutations were observed in the YD1-YD6 and FH strains in the V region of the 23S rRNA domain associated with macrolide resistance, and point mutations of 2067 A → G occurred in both YD7 and YD8-induced strains. In ribosomal protein L4, point mutations of C → A at position 162 and A → G at position 430 appeared in both induced and FH strains. In ribosomal protein L22, both the induced strain and the FH strain showed a 279 → T → C And a point mutation at T → C at position 508. Conclusion Mutation of drug target in the center of 23S rRNA domain is the main mechanism of drug resistance. In the use of azithromycin drugs in the process, there is the possibility of induction of resistant strains in vivo.