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).
Conclusion
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.
