In vitro antimicrobial activity of Naja cardiotoxin peptide-3 against clinical isolates from canine otitis

Canine otitis externa is a common multifactorial condition frequently complicated by bacterial and fungal infections. The increasing prevalence of antimicrobial resistance has reduced the effectiveness of conventional therapies, underscoring the need for alternative treatment strategies. Antimicrobial peptides represent a promising option due to their broad-spectrum activity and low propensity to induce resistance. Among these, the Naja Cardiotoxin Peptide-3 (NCP-3), derived from cardiotoxin 1 of the Chinese cobra (Naja atra subsp. atra), has demonstrated potent bactericidal and fungicidal activity with minimal cytotoxicity. In this study, the in vitro antimicrobial activity of NCP-3 was evaluated against clinical isolates obtained from dogs with otitis externa, including multidrug-resistant bacteria and Malassezia pachydermatis. NCP-3 was effective against all reference tested strains except Proteus mirabilis. Among field strains, the highest bactericidal activity was observed on Staphylococcus pseudintermedius (MBC₅₀=3.1 µg/mL; MBC₉₀=6.3 µg/mL), whereas higher concentrations were required to inhibit Pseudomonadaceae (MBC₅₀=12.5 µg/mL). Neither MBC₅₀ nor MBC₉₀ were achieved for Enterobacteriaceae or M. pachydermatis. Time-kill assays demonstrated rapid bactericidal activity of NCP-3 against both reference and clinical Gram-positive strains, with complete inhibition achieved within five minutes. In contrast, Gram-negative reference strains required longer exposure times, with a partial inhibition only at 120 min. Overall, these findings indicate that NCP-3 exhibits stronger antimicrobial activity against Gram-positive bacteria, including resistant strains, than on Gram-negative bacteria and M. pachydermatis field strains. NCP-3 may therefore represent a promising candidate as therapeutic agent for canine otitis externa caused by Gram-positive pathogens, supporting further investigation into its potential applications in veterinary medicine.