红霉素体外诱导肺炎链球菌耐药的研究

Study on the Streptococcus pneumoniae resistance induced by erythromycin in vitro

目的通过体外诱导获得肺炎链球菌(Streptococcus pneumoniae,S.pn)耐药菌株,对比分析S.pn诱导耐药前后红霉素结合域的变异。方法采用最低抑菌浓度(MIC)递增方法对S.pn标准菌株tigr4、tigr2临床分离敏感株进行红霉素体外诱导耐药,PCR与RT-PCR对诱导前后rplD、rplV基因以及23SrRNA扩增并测序,CPHmodels-2.0蛋白质空间构象预测系统对诱导前后rplD、rplV基因编码的核糖体蛋白L4和L22进行空间构象预测分析。结果诱导后tigr4MIC由0.0312mg/L增加到256mg/L,而临床分离敏感株MIC均由0.0312mg/L增加到32mg/L。诱导前后测序结果对比显示标准菌株tigr4核糖体蛋白L4发生V32A变异,L22发生D35G变异,而临床分离敏感株核糖体蛋白L4发生Q67R、K68E变异。空间构象分析L22的D35G及L4的Q67R、K68E变异导致其相应的表面空间构象发生明显改变。结论S.pn可通过红霉素体外诱导而导致耐药,其耐药机制与红霉素结合域发生新的变异有关。

Objective To establish erythromycin resistance models of Streptococcus pneumoniae for analysis of the mutations in the binding domain of erythromycin. Methods Streptococcus pneumonia tigr4 and two clinical sensitive isolates were induced with erythromycin by minimum inhibitory concentration （MIC） method for antimicrobial susceptibility, and then the rplD, rplV genes, and 23S rRNA in sensitive and resistant strains were amplified by PCR and RT-PCR for sequencing. CPHmodels-2.0 was used to predict the spatial structures of ribosomal protein L4 and L22 coded by rplD and rplV genes. Results MIC of Streptococcus pneumonia tigr4 increased from 0. 0312 mg/L to 256mg/L while MICs of the two clinical isolates increased from 0. 0312 mg/L to 32 mg/L. Comparison of the pre- and post-induction results showed V32A mutation in ribosomal protein L4, D35G mutation in ribosomal protein L22, and 67 QK68 to 67 RE6s mutations in ribosomal protein IA. Changes in spatial structure was found in SiThe ribosomal protein L22 and ribosomal protein L4 due to D35G, Q67R, and K68E mutations respectively. Conclusion Streptococcus pneumonia can be induced to resistant to erythromycin in vitro. The mechanisms of resistance may be relate to the new mutations in the binding domain of erythromycin.