OT-CATH, a conserved antimicrobial peptide in Otariidae species, exhibits potent activity against gram-negative bacteria.

The rapid emergence of multidrug-resistant gram-negative bacteria necessitates the development of effective antimicrobial agents to combat antimicrobial resistance. Here, we identified and characterized OT-CATH, a conserved cathelicidin-family antimicrobial peptide derived from Otariidae species. OT-CATH exhibited potent bactericidal activity against gram-negative bacteria, particularly extended-spectrum β-lactamase-producing Escherichia coli, with rapid killing kinetics, potent antibiofilm activity, and no detectable resistance after 40 serial passages. Mechanistically, OT-CATH bound lipopolysaccharide, disrupted both the outer and inner bacterial membranes, and translocated into the cytoplasm to interact with bacterial DNA. Membrane disruption further triggered intracellular reactive oxygen species accumulation, proton motive force dissipation, ATP depletion, and metabolic collapse, collectively leading to bacterial death. OT-CATH also demonstrated favorable stability, low cytotoxicity, and favorable pharmacokinetic properties with measurable pulmonary distribution. In a murine pneumonia model, OT-CATH significantly improved survival, reduced bacterial burdens in the lungs and bronchoalveolar lavage fluid, and alleviated lung injury. These findings identify OT-CATH as a promising membrane-targeting antimicrobial peptide for the treatment of multidrug-resistant gram-negative bacterial infections.