The Antimicrobial Peptide CRAMP-34 Eradicates Escherichia coli Biofilms by Interfering with the kduD-Dependent Network

<b>Background/Objectives</b>: Bacterial biofilms formed by <i>Escherichia coli</i> pose 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. <b>Methods</b>: 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 a <i>kduD</i> mutant. 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. <b>Results:</b> CRAMP-34 effectively eradicated pre-formed biofilms of a clinically relevant, porcine-origin <i>E. coli</i> strain and promoted wound healing in the murine infection model. We conducted a genome-wide transposon mutagenesis screen, which identified <i>kduD</i> as a critical gene for robust biofilm formation. Functional characterization revealed that <i>kduD</i> deletion 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 the <i>kduD</i> deletion, including motility inhibition and transcriptional repression of a common set of biofilm-related genes. <b>Conclusions:</b> This research highlights CRAMP-34 as a potent anti-biofilm agent and unveils <i>kduD</i> as a previously unrecognized regulator of <i>E. coli</i> biofilm development, which is also targeted by CRAMP-34.