Dual Mechanisms of Action of a Halogenated Allyl Fatty Acid against Methicillin-Resistant <i>Staphylococcus aureus</i>

Antimicrobial resistance in MRSA demands agents with new mechanisms. We evaluated two synthetic unsaturated fatty acids, 2-hexadecynoic acid (2-HDA) and (<i>Z</i>)-2-allyl-3-bromo-2-hexadecenoic acid (DAT-51), across clinical and reference MRSA strains. Both compounds reduced the viability in a dose-dependent manner. Fluorescence microscopy and nucleic acid leakage demonstrated rapid membrane permeabilization; flow cytometry revealed strong depolarization, and SEM showed surface lesions consistent with pore formation. DAT-51 additionally induced Lipid II accumulation and inhibited MurA, whereas lipidomics indicated the selective incorporation of 2-HDA into bacterial phospholipids. Vero-cell assays showed a low cytotoxicity at concentrations exceeding MICs. Together, the data support a dual-action paradigm: 2-HDA primarily disrupts membranes via insertion and pore formation, while DAT-51 combines moderate membrane perturbation with the inhibition of peptidoglycan biosynthesis. These findings define complementary mechanisms for uFA-based agents and identify 2-HDA and DAT-51 as promising scaffolds against drug-resistant Gram-positive pathogens.