Xiao Xia, Huan Quanmin, Huang Yanhu, Liu Ziyi, Liu Yuan, Li Ruichao, Wang Mianzhi, Wang Zhiqiang
College of Veterinary Medicine, Yangzhou University, Yangzhou, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, 225009, China.
College of Veterinary Medicine, Yangzhou University, Yangzhou, China.
Phytomedicine. 2024 Apr;126:155421. doi: 10.1016/j.phymed.2024.155421. Epub 2024 Feb 6.
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.
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.
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).
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.
质粒介导的耐药-结节-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耐药性增强有关，从而降低了其有效性。在本研究中，我们探索了天然存在的吲哚生物碱禾本科碱作为一种创新佐剂，以增强对携带tmexCD-toprJ样基因簇的肺炎克雷伯菌引起的感染的治疗效果。
使用棋盘微量肉汤稀释技术和时间杀菌曲线分析评估禾本科碱与抗生素联合对浮游和耐多药肠杆菌科细菌的协同潜力。之后，通过荧光测定和RT-PCR测定细胞膜的质子动力势（PMF）、外排泵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蓄积。研究呼吸速率、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禾本科碱的安全性。使用两种动物感染模型评估体内协同作用。
禾本科碱显著增强了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，在生物膜和持留菌等特殊生理状态的细菌中也观察到了协同活性。机制研究表明，禾本科碱具有通过破坏质子动力势（PMF）和抑制外排泵功能来增加替加环素在细胞内蓄积的能力。此外，在禾本科碱处理下，细菌呼吸速率、细胞内ATP水平和三羧酸循环（TCA）得到促进。值得注意的是，禾本科碱在由tmexCD1-toprJ1阳性肺炎克雷伯菌（RGF105-1）感染的多个动物感染模型中有效恢复了替加环素的活性。
本研究首次提供了证据，证明禾本科碱作为一种新型替加环素佐剂，在治疗携带tmexCD-toprJ样基因簇的肺炎克雷伯菌引起的感染方面具有治疗潜力。
