Biofilm-associated antibiotic resistance in Pseudomonas aeruginosa (P. aeruginosa) poses a critical global health burden. A promising strategy to deal with resistant P. aeruginosa infections is to interfere with biofilm formation and the production of virulence. In this study, we designed and synthesized a series of novel coumarin derivatives by incorporating long alkyl chains from native QS signal molecules into the coumarin scaffold. Compound XDS-23 emerged as the hit compound with an IC50 of 1.26 ± 0.16 μM to inhibit biofilm in P. aeruginosa PAO1. Furthermore, XDS-23 still exhibited significant efficacy in reducing biofilm and virulence in clinically isolated resistant P. aeruginosa. Mechanistic studies revealed that XDS-23 mainly inhibited the las and pqs systems, thereby suppressing biofilm and multiple virulence factors. Notably, XDS-23 demonstrated synergistic activity with polymyxin B, ciprofloxacin, ceftazidime, and tobramycin against P. aeruginosa both in vitro and in vivo, significantly increasing the survival rate of Galleria mellonella when combined with these antibiotics. Collectively, these findings highlight biofilm inhibitor XDS-23 as a promising biofilm inhibitor to combat resistant P. aeruginosa infections.
