Peng-cheng Li Yin-Chao Tong Xing-Lan Xiao Shao-Qiang Sun ^* Yun-Peng Fan Wu-Ren Ma Ying-Qiu Liu ^* Shen Zhuang ^* Su-Zhu Qing ^* Wei-Min Zhang ^*

• College of Veterinary Medicine, Northwest A&F University, Xianyang, Shaanxi, China

The development of extended-spectrum-beta-lactamase (ESBLs)Escherichia coli (E. coli) has become a global threat to public health. An alternative strategy to alleviate this is identifying potential natural compounds to restore antibiotic activity against ESBLs E. coli. This study aimed to find a possible compound to restore ESBLs E. coli sensitivity to ceftiofur. The synergistic effect of kaempferol and ceftiofur against ESBLs E. coli was investigated by checkerboard assays, time-kill, growth curves, and scanning electronic microscope. And unexpectedly, we found that the combination of kaempferol and ceftiofur showed inhibitory effects on the biofilm of ESBLs E. coli through crystal violet staining and laser scanning confocal microscope. We also evaluated the impact of kaempferol on the initial adhesion and aggregation of E. coli (SY20) by examining motility, adhesion, and surface characteristics, and our findings revealed that the combination of kaempferol and ceftiofur demonstrated inhibitory effects in this regard. In addition, with the treatment of kaempferol and ceftiofur, the relative expression of LuxS /Autoinducer-2 (AI-2) system genes declined, the molecular weight of the AI-2 signal was reduced, the Molecular docking showed that kaempferol could interact with the active center of LuxS protein, which indicated that kaempferol may affect the LuxS /AI-2 system and biofilm formation to restore the susceptibility of ESBLs E. coli to ceftiofur. Furthermore, we found that kaempferol can be stably bound to the active center of the blaCTX-M-27 protein, reduce the relation expression of the blaCTX-M-27 gene, and inhibit the hydrolytic activity of the blaCTX-M-27 protein by RT-qPCR, Molecular docking, and the nitrocefin test. The results of transcriptome analysis further suggest our hypothesis. Last, in vivo, kaempferol protected the small intestinal villi from the damage of ESBLs E. coli. Furthermore, kaempferol fully restores the activity of ceftiofur in animal infection models by relieving the TLR4/NF-.κb pathway. In conclusion, the sensitivity of ESBLs E. coli to ceftiofur in vitro and in vivo could be enhanced by kaempferol, which showed that kaempferol may be a kind of antibiotic adjuvant.