低浓度中药促进耐药质粒在异种菌株间接合转移的分子机制研究

We found in previous research that, the herb-resistance plasmid, R45, as an independent conjugative plasmid, can transfer between heterologous strains efficiently. The conjugative efficiency showed extreme improvement in small molecular Chinese herbal substances of low concentration (1/2MIC), between E. coli CP9 with R45 and S. aureus RN450RF. Therefore, we speculated that, Chinese herbal decoction of low concentration may have an effect on R45 encoding gene replication, conjugative transfer and its control system. In this project, E. coli CP9 with R45 was assigned as the donor with S. aureus RN450RF as the receptor. The research was carried out in the light of promoter trfAp and trbBp with decisive role on plasmid replication, conjugation and its regulation, as well as the overall regulatory factors. The influence pattern of Chinese herbal decoction of low concentration on conjugative transfer of R45 in liquid phase and its environmental impact factors were analyzed. The influence mechanisms of Chinese herbal decoction of low concentration on conjugative transfer of R45 were investigated by means of morphology, biochemistry, molecular biology and proteomic approaches. Then, our hypothesis was proved and experimental basis for reducing the spread of resistance genes was provided.

我们的研究发现，作为自主接合质粒的中药耐药质粒R45可在不同种间高效传递，而含有R45质粒的E. coli CP9与S. aureus RN450RF的接合效率在低浓度（1/2MIC）中药小分子物质存在下有极大的提高。因此我们推测，低浓度中药干预可能对R45质粒编码基因的复制、接合转移以及调控系统产生影响。本项目以携带R45的E. coli CP9为供体菌，S. aureus RN450RF为受体菌，针对在质粒复制、接合转移及其调控中分别起决定作用的启动子trfAp和trbBp以及整体调节因子展开研究，分析低浓度中药对液相接合条件下R45接合转移的影响规律及其环境影响因素，采用形态学、生物化学、分子生物学以及蛋白质组学的方法和手段，探讨低浓度中药对R45接合转移的影响机制，从而证明我们的假说，为减少耐药基因传播提供实验依据。

我们之前的研究发现，作为中药耐药质粒R45可在异种菌株间高效传递，而含有R45质粒的E. coli CP9与S. aureus RN450RF的接合效率在低浓度（1/2MIC）中药存在下有极大的提高。因此我们推测，低浓度中药可能对R45质粒编码基因的复制、接合转移以及调控系统产生影响。本项目针对在质粒复制、接合转移及其调控中分别起决定作用的启动子trfAp和trbBp以及mRNA展开研究，结果发现：（1）药物最低抑菌浓度实验显示：大肠杆菌的MIC为400g/L，金葡临床菌的MIC为200g/L。（2）当培养液中药物浓度分别为200g/L、100g/L时，接合子数分别为（3.47±0.20）×107、（2.77±0.58）×107个。（3）接合8h、32h、90h后，接合子数分别为（3.47±0.20）×107、（5.97±0.21）×107、（1.15±0.06）×108个。（4）初始菌液浓度为104~108cfu/ml，得到的接合子数目分别为（2.67±0.33）×102、（3.67±0.33）×103、（5.67±0.88）×104、（6.00±0.06）×105、（3.47±0.20）×107。（5）当药物浓度分别为0g/L、100g/L、200g/L时，trfAp的半乳糖苷酶活力分别为100.29±3.70、160.48±8.10、225.11±6.40，trbBp的半乳糖苷酶活力分别为86.69±3.15、117.50±3.85、151.93±4.03。（6）当药物浓度分别为0 g/L、100 g/L、200 g/L时，trbA的荧光定量值分别为1、0.88±0.01、0.39±0.02，korA的荧光定量值分别为1、0.60±0.01、0.32±0.01，korB的荧光定量值分别为1、0.67±0.01、0.22±0.01。以上研究结果提示：1、长期低浓度应用中药可诱导大肠杆菌产生耐药性。2、低浓度中药的存在对耐药质粒 R45 在异种菌株间接合转移具有促进作用。3、在1/2MIC药物浓度作用下，得到的接合子数较多；耐药质粒接合转移并非随作用时间的延长呈现持续增加趋势，而是先增加后减少；初始菌液浓度越高，耐药质粒接合子的数量越多。4、低浓度中药的存在对标本中大肠杆菌阳性克隆菌数量的增长具有促进作用。5、低浓度中药的存在对整体调节因子mRNA表达有负向作用。