取代苯胍衍生物逆转多黏菌素耐药性的作用机制研究

In this century, bacterial resistance to antibiotics has become a global medical problem, especially multidrug-resistant Gram-negative bacteria. Polymyxin is the last treatment option for severe infections caused by multidrug resistant Gram-negative bacteria. However, in recent years, polymyxin resistance has become increasingly serious, especially the emergence of the first plasmid-mediated transferable polymyxin resistance gene mcr-1, which was identified in China last year and soon detected among many other countries, has resulted in almost no treatment options available in clinic. Our previous study showed that substituted phenyl guanidine derivatives could not only reverse the drug resistance of polymyxin-resistant Escherichia coli, but also showed a synergistic effect on polymyxin-susceptible bacteria. In this study, substituted phenyl guanidine derivatives are firstly investigated as polymyxin resistance reversal agents, aimed at screening best compound with excellent activity to reverse polymyxin resistance in vitro, great activity and safety in vivo as well. Then the possible mechanism of this compound to reverse polymyxin resistance will be investigated through plasmid elimination, efflux pump expression, regulation of two-component system, modification of lipid A, ion and pH changes, in order to better understand polymyxin resistance mechanisms, and provide experimental and theoretical basis for the development of polymyxin reversal agent and control of polymyxin resistance.

细菌耐药性已经成为全球性的医学难题，尤其是革兰氏阴性菌的多重耐药性问题最为突出。多黏菌素作为治疗多重耐药革兰氏阴性菌引起严重感染的最后一道防线，近年来耐药现象非常严重，尤其是去年发现mcr-1多黏菌素耐药基因可介导耐药性的水平传播，导致临床几乎无药可用。我们的初步研究发现取代苯胍衍生物不仅可以逆转多黏菌素耐药大肠杆菌的耐药性，还对多粘菌素敏感菌具有协同增效作用。本申请课题是国内外首次将取代苯胍衍生物作为多黏菌素耐药性逆转剂，旨在从中筛选出对质粒和染色体介导多黏菌素耐药大肠杆菌具有最佳逆转效果的化合物，且证实其在体内对多黏菌素耐药有良好的逆转活性和安全性，然后从耐药质粒消除、外排泵的表达、二元调控系统的调节和类脂A修饰等方面研究其对多黏菌素耐药性逆转的可能作用机制，从而更好地了解多黏菌素耐药机制的产生，为多黏菌素耐药逆转剂的研发和减缓多黏菌素耐药性的产生提供实验和理论依据。

细菌耐药性已经成为全球性的医学难题，尤其是质粒介导的黏菌素耐药基因mcr-1的出现，导致临床对该耐药革兰氏阴性菌陷入几乎无药可用的困境。近年来，联合用药已成为药物研发和减缓细菌耐药性的重要途径。本项目发现取代苯胍衍生物与黏菌素联合应用能够逆转多黏菌素耐药菌的耐药水平及降低敏感株的最小抑菌浓度。通过活性筛选和稳定性实验等选出最优化合物异丙氧苯胍。通过棋盘法、体外联合杀菌曲线评估异丙氧苯胍与黏菌素联合应用对大肠杆菌和鲍曼不动杆菌的作用，结果表明异丙氧苯胍与黏菌素联合应用对黏菌素耐药大肠杆菌和多重耐药鲍曼不动杆菌均具有协同作用。通过建立黏菌素耐药大肠杆菌定植小鼠肠道模型来评估联合用药的体内抗菌活性，结果显示联合用药组中黏菌素耐药大肠杆菌在小鼠肠道的定植菌量显著降低。通过实时荧光定量PCR、透射电镜实验发现，联合用药组能够破坏细菌细胞膜完整性，且显著降低二元调控系统中pmrA基因的表达。透射电镜、整合转录组学和代谢组学结果表明其协同作用机制是，异丙氧苯胍与黏菌素共同增加大肠杆菌细胞膜的膜通透性，从而使异丙氧苯胍进入细胞内干扰细菌氨基酸、能量和脂肪酸代谢从而发挥协同抗菌作用。