Indolenine-substituted pyrazole derivative 4e inhibits planktonic growth and biofilm formation by disrupting purine biosynthesis and compromising cell wall and membrane integrity.

is an emerging nosocomial pathogen responsible for biofilm-related infections. Here, we explored the antibacterial and antibiofilm properties of the novel indolenine derivative 4e against and investigated its mechanisms of action. Its antibacterial and antibiofilm activities were assessed against oxacillin-resistant CGMH-SL131 using and models, including human cell lines, larvae, and mice. Mechanistic insights were explored via untargeted metabolomics. 4e exhibited bacteriostatic activity against a panel of gram-positive bacteria, with a 1× minimum inhibitory concentration (MIC) of 62.5 µg/mL. Scanning electron microscope observations of cells treated with 0.5% SDS and 1× MIC 4e displayed signs of cell shape distortion, including complete shrinkage and bursting. 4e effectively inhibited biofilm formation by 54.3% at 1.56 µg/mL, and the minimum biofilm inhibition concentration 80% (MBIC) was 3.125 µg/mL. In addition, 70.3% of 1-day preformed biofilms were dispersed at 1× MBIC. 4e exhibited low cytotoxicity (>85% survival) in HaCaT, H10975, and Caco-2 cells at 1× MIC. When administered 1 hour post-infection, 4e (3.125 mg/kg) improved larval survival to 90%, matching tigecycline (2 mg/kg), whereas untreated larvae had only 20% survival after 7 days. In C57BL/6 mice, 4e (2.5 mg/kg) reduced kidney bacterial loads from 10⁷ to 5.3 × 10⁴ CFU. Untargeted metabolomics suggests that 4e's antibacterial and antibiofilm effects result from disrupting purine biosynthesis and compromising cell wall and membrane integrity. These findings highlight 4e as a promising new antibiofilm agent and potential alternative treatment for biofilm-related infections caused by and multidrug-resistant species.