D-enantiomeric β-casein 197 disrupts biofilm-associated infection of MDR Pseudomonas aeruginosa and Acinetobacter baumannii through superior LPS binding and host cell protection

Biofilms formed by multidrug-resistant (MDR) Pseudomonas aeruginosa and Acinetobacter baumannii are a major cause of chronic nosocomial infections and therapeutic failure. L-form antimicrobial peptides (AMPs) are rapidly degraded by proteases, whereas D-enantiomeric peptides offer enhanced resistance to proteases. Here, we synthesized L- and D-β-casein 197, a milk-derived AMP, and systematically compared their antibiofilm activities against eight clinical isolates of P. aeruginosa and A. baumannii. The D-form reduced biofilm biomass by up to 83% during formation and by up to 88% in preformed biofilms. Mechanistic analyses revealed that the D-form caused more extensive membrane disruption, as demonstrated by flow cytometry and transmission electron microscopy. Isothermal titration calorimetry showed that the D-form bound LPS with higher affinity (KD 0.8 μM vs 3.1 μM), and molecular docking identified additional hydrogen bonds with lipid A. The D-form also more effectively inhibited bacterial motility. Both peptides exhibited low cytotoxicity toward cells (>90% viability at 100 μM), and the D-form reduced bacteria-induced cytotoxicity and bacterial internalization in infected macrophages more effectively than the L-form. In a Galleria mellonella infection model, D-β-casein 197 significantly improved survival against P. aeruginosa no. R1 and A. baumannii no. 13 challenge over 96 h compared to the L-form and untreated infected controls, while larval survival remained comparable to PBS controls, indicating negligible host toxicity. These findings identify D-β-casein 197 as a potent antibiofilm peptide against MDR Gram-negative biofilms, with enhanced activity, and LPS-neutralization assays confirmed that its antimicrobial potency is, at least in part, attributable to its stronger LPS binding affinity.