Abstract: The rising prevalence of drug-resistant Gram-positive pathogens, particularly methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), poses a substantial clinical challenge. Biofilm-associated infections exacerbate this problem due to their inherent antibiotic resistance and complex structure. Current antibiotic treatments struggle to penetrate biofilms and eradicate persister cells, leading to prolonged antibiotic use and increased resistance. Host defense peptides (HDPs) have shown promise, but their clinical application is limited by factors such as enzymatic degradation and difficulty in large-scale preparation. Synthetic HDP mimics, such as poly(2-oxazoline), have emerged as effective alternatives. Herein, we found that the poly(2-oxazoline), Gly-POX₂₀, demonstrated rapid and potent activity against clinically isolated multidrug-resistant Gram-positive strains. Gly-POX₂₀ showed greater stability under physiological conditions compared to natural peptides, including resistance to protease degradation. Importantly, Gly-POX₂₀ inhibited biofilm formation and eradicated mature biofilm and demonstrated superior in vivo therapeutic efficacy to vancomycin in a MRSA biofilm-associated mouse keratitis model, suggesting its potential as a novel antimicrobial agent against drug-resistant Gram-positive bacteria, especially biofilm-associated infections.

Key words: Poly(2-oxazoline)s, Host defense peptide, Antibiofilm, MRSA, Drug-resistance