Membrane Disruption and Charge Neutralization Drive Synergy Between LL37-Derived Peptides and Polymyxins Against E. coli

Background/Objectives: Antimicrobial peptides (AMPs) offer promising strategies for combating drug-resistant bacteria such as Escherichia coli (E. coli). This study evaluated the synergistic effects of the human-derived AMP LL37 and its fragments FK16 and FK13, in combination with polymyxin B, colistin, and vancomycin, against three E. coli strains in vitro. Methods: Because the outer membrane of E. coli acts as a barrier to antimicrobial agents, we assessed the membrane-permeabilizing activity of these AMPs. We further examined electrostatic interactions between the cationic AMPs and bacterial surfaces via zeta potential measurements. To simulate physiological conditions, we investigated how Mg2+ and Ca2+ ions, common in blood, stabilize the bacterial outer membrane and influence AMP activity. Additionally, we assessed cytoplasmic membrane permeabilization as a key mechanism of antibacterial action. Finally, to support clinical translation, we evaluated the cytotoxicity of the AMPs on human dermal fibroblasts. Results: Checkerboard assays revealed that the peptide–antibiotic combinations exerted synergistic effects against the tested E. coli strains in both MHB medium and cation-adjusted MHB medium, with FICI values ranging from 0.3125 to 0.5 and <0.375 to 0.5, respectively. All three AMPs (LL37, FK16, and FK13) exhibited strong outer membrane-permeabilizing activity. Zeta potential measurements revealed a correlation between membrane disruption and surface charge neutralization. The presence of Mg2+ and Ca2+ ions was found to stabilize the bacterial outer membrane. Cytoplasmic membrane permeabilization was confirmed as a key antibacterial mechanism. Cytotoxicity assays confirmed the safety profile of these AMPs on human cells. Conclusions: The study demonstrates that LL37 and its fragments enhance the activity of conventional antibiotics against E. coli through membrane disruption and charge neutralization, as evidenced by the FICI values (0.3125–0.5), membrane disruption and charge neutralization. While divalent cations in physiological conditions influence AMP efficacy, these peptides show potential for clinical application against E.coli.