β-桧木醇联合替加环素对tet(X4)阳性大肠杆菌的体外协同抗菌作用

Synergistic effect of β-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli in vitro

摘要:

质粒介导tet(X4)基因的出现和流行，严重影响替加环素(tigecycline)对临床多重耐药细菌感染的治疗效果，急需寻找有效的佐剂遏制替加环素耐药性。本研究采用微量棋盘稀释法、时间-杀菌曲线测定β-桧木醇(β-thujaplicin)和替加环素的体外联合抗菌表征，通过测定细菌细胞膜通透性、细菌胞内活性氧(reactive oxygen species, ROS)含量、铁含量以及替加环素含量等指标，来探究β-桧木醇和替加环素联合使用对tet(X4)基因阳性大肠杆菌(Escherichia coli)的抗菌作用机制。结果表明，β-桧木醇联合替加环素对tet(X4)基因阳性大肠杆菌具有体外协同抗菌效果，且β-桧木醇在抗菌作用浓度范围内无显著溶血性和细胞毒性。机制研究发现，β-桧木醇能显著增大细菌细胞膜的通透性，降低细菌胞内铁含量，干扰铁稳态，诱导细胞内ROS显著增加，从而发挥抗菌效果。进一步研究发现，β-桧木醇可通过干扰细菌铁代谢和增大细菌细胞膜通透性，协同增强替加环素的抗菌效果。本研究结果为β-桧木醇联合替加环素治疗tet(X4)基因阳性大肠杆菌感染提供了理论和实践基础。

关键词:β-桧木醇;替加环素;tet(X4);协同抗菌;铁稳态

Abstract:

The widespread of tigecycline resistance gene tet(X4) has a serious impact on the clinical efficacy of tigecycline. The development of effective antibiotic adjuvants to combat the looming tigecycline resistance is needed. The synergistic activity between the natural compound β-thujaplicin and tigecycline in vitro was determined by the checkerboard broth microdilution assay and time-dependent killing curve. The mechanism underlining the synergistic effect between β-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli was investigated by determining cell membrane permeability, bacterial intracellular reactive oxygen species (ROS) content, iron content, and tigecycline content. β-thujaplicin exhibited potentiation effect on tigecycline against tet(X4)-positive E. coli in vitro, and presented no significant hemolysis and cytotoxicity within the range of antibacterial concentrations. Mechanistic studies demonstrated that β-thujaplicin significantly increased the permeability of bacterial cell membranes, chelated bacterial intracellular iron, disrupted the iron homeostasis and significantly increased intracellular ROS level. The synergistic effect of β-thujaplicin and tigecycline was identified to be related to interfere with bacterial iron metabolism and facilitate bacterial cell membrane permeability. Our studies provided theoretical and practical data for the application of combined β-thujaplicin with tigecycline in the treatment of tet(X4)-positive E. coli infection.

Key words:β-thujaplicin;tigecycline;tet(X4);synergistic antibacterial effect;iron homeostasis