This study assessed the therapeutic potential of four rumen microbiome-derived antimicrobial peptides (AMPs) against *Pseudomonas aeruginosa*. All AMPs exhibited antimicrobial activity, with MICs ranging from 4–512 µg/mL. Time-kill kinetics showed complete kill within 10 min to 4 h. Biofilm formation was significantly inhibited, and no resistance development was observed. AMP cytotoxicity was minimal. The AMPs permeabilized the *P. aeruginosa* membrane, and transcriptome/metabolome analysis revealed increased catalytic activity at the cell membrane and β-oxidation of fatty acids. Lynronne 1 and 2 were efficacious in vivo, showing 100% survival in a *Galleria mellonella* infection model.

Adam J. Mulkern, Linda B. Oyama, Alan R. Cookson, Christopher J. Creevey, Toby J. Wilkinson, Hamza Olleik, Marc Maresca, Giarla C. da Silva, Patricia P. Fontes, Denise M. S. Bazzolli, Hilario C. Mantovani, Bamu F. Damaris, Luis A. J. Mur, Sharon A. Huws