The Antimicrobial Peptide CRAMP-34 EradicatesBiofilms by Interfering with the-Dependent Network.

: Bacterial biofilms formed bypose a significant challenge in veterinary medicine due to their intrinsic resistance to antibiotics. Antimicrobial peptides (AMPs) represent a promising alternative. AMPs exert their bactericidal activity by binding to negatively charged phospholipids in bacterial membranes via electrostatic interactions, leading to membrane disruption and rapid cell lysis.: In vitro assays including MIC determination, biofilm eradication testing (crystal violet, colony counts, and CLSM), swimming motility, and EPS quantification were performed. CRISPR/Cas9 was used to construct and complement amutant. A transposon mutagenesis library was screened for biofilm-defective mutants. In an in vivo murine excisional wound infection model treated with the mouse cathelicidin-related antimicrobial peptide (CRAMP-34), wound closure and bacterial burden were monitored. Gene expression changes were analyzed via RT-qPCR.CRAMP-34 effectively eradicated pre-formed biofilms of a clinically relevant, porcine-originstrain and promoted wound healing in the murine infection model. We conducted a genome-wide transposon mutagenesis screen, which identifiedas a critical gene for robust biofilm formation. Functional characterization revealed thatdeletion drastically impairs flagellar motility and alters exopolysaccharide production, leading to defective biofilm architecture without affecting growth. Notably, the anti-biofilm activity of CRAMP-34 phenocopied aspects of thedeletion, including motility inhibition and transcriptional repression of a common set of biofilm-related genes.This research highlights CRAMP-34 as a potent anti-biofilm agent and unveilsas a previously unrecognized regulator ofbiofilm development, which is also targeted by CRAMP-34.