背景:质粒介导的抗性结瘤分裂(RND)外排泵基因簇tmexCD1-toprJ1及其相关变体的存在与对替加环素的抗性增强有关,从而降低其效力。在这项研究中,我们探索了gramine的潜力,一种天然存在的吲哚生物碱,作为增强由携带tmexCD-toprJ样基因簇的肺炎克雷伯菌引起的感染的治疗的创新佐剂。
方法:使用棋盘微肉汤稀释技术和时间杀伤曲线分析,评估了与抗生素联合使用的对浮游和耐药性多重耐药肠杆菌的协同潜力。之后,细胞膜的质子动力(PMF),通过荧光分析和RT-PCR测定外排泵的功能和抗氧化系统的活性。通过HPLC-MS/MS评估替加环素的细胞内积累。呼吸率,研究了细菌ATP水平和NAD/NADH比率以揭示代谢状态。最后,通过溶血活性和细胞毒性试验评估了gramine的安全性.使用两种动物感染模型来评估体内协同作用。
结果:草胺可显著增强替加环素和环丙沙星对tmexCD1-toprJ1及其变体阳性病原体的活性。重要的是,在特殊的生理状态,如生物膜和持久细胞中,还观察到了对细菌的协同活性。机制研究表明,gramine具有通过破坏质子动力(PMF)和抑制外排泵功能来增加细胞内替加环素积累的能力。此外,细菌呼吸速率,在gramine处理下促进了细胞内ATP水平和三羧酸循环(TCA)。值得注意的是,在由tmexCD1-toprJ1阳性肺炎克雷伯菌(RGF105-1)感染的多种动物感染模型中,gramine有效恢复了替加环素活性。
结论:这项研究提供了第一个证据,证明了gramine作为新型替加环素佐剂治疗由携带tmexCD-toprJ样基因簇的肺炎克雷伯菌引起的感染的治疗潜力。
BACKGROUND: The presence of plasmid-mediated resistance-nodulation-division (RND) efflux pump gene cluster tmexCD1-toprJ1 and its related variants has been associated with heightened resistance to tigecycline, thus diminishing its effectiveness. In this study, we explored the potential of gramine, a naturally occurring indole alkaloid, as an innovative adjuvant to enhance the treatment of infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters.
METHODS: The synergistic potential of gramine in combination with antibiotics against both planktonic and drug-tolerant multidrug-resistant Enterobacterales was evaluated using the checkerboard microbroth dilution technique and time-killing curve analyses. Afterwards, the proton motive force (PMF) of cell membrane, the function of efflux pump and the activity of antioxidant system were determined by fluorescence assay and RT-PCR. The intracellular accumulation of tigecycline was evaluated by HPLC-MS/MS. The respiration rate, bacterial ATP level and the NAD+/NADH ratio were investigated to reveal the metabolism state. Finally, the safety of gramine was assessed through hemolytic activity and cytotoxicity assays. Two animal infection models were used to evaluate the in vivo synergistic effect.
RESULTS: Gramine significantly potentiated tigecycline and ciprofloxacin activity against tmexCD1-toprJ1 and its variants-positive pathogens. Importantly, the synergistic activity was also observed against bacteria in special physiological states such as biofilms and persister cells. The mechanism study showed that gramine possesses the capability to augment tigecycline accumulation within cells by disrupting the proton motive force (PMF) and inhibiting the efflux pump functionality. In addition, the bacterial respiration rate, intracellular ATP level and tricarboxylic acid cycle (TCA) were promoted under the treatment of gramine. Notably, gramine effectively restored tigecycline activity in multiple animal infection models infected by tmexCD1-toprJ1 positive K. pneumoniae (RGF105-1).
CONCLUSIONS: This study provides the first evidence of gramine\'s therapeutic potential as a novel tigecycline adjuvant for treating infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters.