肺炎支原体外排泵ABC Transporter在大环内酯类耐药中的作用机制研究

The mutations on 23S ribosomal RNA gene were known to be the major mechanism that gives rise to the macrolide resistance in M.pneumoniae, It is important to find new resistant mechanism. In our previous study, more new SNPs were found focus on ABC Transporter genes by using whole genome sequencing method, and efflux pump inhibitor could reduce the MIC values of macrolide resistant MP, it was speculated that SNPs in ABC Transporter genes may play a great role in M. pneumoniae drug resistance. In this study, we will construct the ABC Transporter gene mutation strains and ABC Transporter gene combined with 23S ribosomal RNA gene mutations strains, observe their macrolide resistance and compare with reference strains, to identify the mechanism of the ABC Transporter genes in M. pneumoniae resistance and explore the relationship between ABC Transporter gene mutations and 23S rRNA mutations. Combining with the clinical data, the SNPs of ABC Transporter genes were detected in clinical specimens for the further research of the role of ABC Transporter genes in M. pneumoniae macrolide resistance.

目前Mp耐药机制研究主要局限于23S rRNA基因点突变。项目组前期在Mp全基因组测序分析中发现Mp耐药株与敏感株的差异SNPs富集于外排泵系统ABC Transporter基因区，并实验证实外排泵抑制剂可显著降低耐药株MIC值，推测ABC Transporter可能在Mp大环内酯类耐药中发挥作用。本研究拟采用同源重组方法构建ABC Transporter基因突变株和ABC Transporter基因与23S rRNA基因同时突变菌株，检测两者耐药性差异，并以标准株做对照，了解ABC Transporter基因突变与菌株耐药及原有23SrRNA基因突变相关性；检测临床标本ABC Transporter基因，探讨其在临床耐药性中的作用，为进一步阐明Mp耐药机制，开辟Mp新的治疗靶点和药物提供新思路。

肺炎支原体是儿童呼吸道感染主要病原体，耐药率逐年攀升。本研究主要针对肺炎支原体外排泵系统ABC Transporter 中重要基因Mpn80和Mpn260与耐药相关的功能进行研究。项目执行期间，完成了（1）肺炎支原体外排泵系统ABC Transporter中Mpn80和Mpn260基因突变株的构建，本部分先后采用了两种方法进行，分别为在含有Tn4001的转座子中插入突变部分的基因，采用电转化的方法转入和将突变部分采用脂质体包埋后化学转入。（2）肺炎支原体的ABC Transporter中Mpn80和Mpn260基因突变株耐药性的检测，采用MIC的方法进行了红霉素、阿奇霉素大环内酯类抗生素的检测。与标准株相比，突变株的MIC值均持续几代升高64-128mg/L），证明Mpn080和Mpn260基因中的突变位点影响了菌株耐药，验证了实验假说。（3）获得了含有ABC Transporter 的Mpn80和Mpn260基因SNPs位点肺炎支原体感染临床菌株的临床特征，分析了65例标本的耐药特征，及其在Mp临床耐药中的意义，并发现这些SNPs与Mp的P1基因型存在密切相关性。本研究证实了外排泵系统ABC Transporter 中Mpn80和Mpn260与Mp耐药相关，为开辟Mp新的治疗靶位和药物提供了新的思路，为临床治疗Mp耐药菌株感染指出了新的治疗途径，会产生较好的社会效益和经济效益。