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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\n                    <jats:italic>Naja<\/jats:italic>\n                    Cardiotoxin Peptide-3 (NCP-3), derived from cardiotoxin 1 of the Chinese cobra (\n                    <jats:italic>Naja atra<\/jats:italic>\n                    subsp.\n                    <jats:italic>atra<\/jats:italic>\n                    ), 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\n                    <jats:italic>Malassezia pachydermatis<\/jats:italic>\n                    . NCP-3 was effective against all reference tested strains except\n                    <jats:italic>Proteus mirabilis<\/jats:italic>\n                    . Among field strains, the highest bactericidal activity was observed on\n                    <jats:italic>Staphylococcus pseudintermedius<\/jats:italic>\n                    (MBC\u2085\u2080=3.1\u00a0\u00b5g\/mL; MBC\u2089\u2080=6.3\u00a0\u00b5g\/mL), whereas higher concentrations were required to inhibit Pseudomonadaceae (MBC\u2085\u2080=12.5\u00a0\u00b5g\/mL). Neither MBC\u2085\u2080 nor MBC\u2089\u2080 were achieved for Enterobacteriaceae or\n                    <jats:italic>M. pachydermatis<\/jats:italic>\n                    . 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\u00a0min. Overall, these findings indicate that NCP-3 exhibits stronger antimicrobial activity against Gram-positive bacteria, including resistant strains, than on Gram-negative bacteria and\n                    <jats:italic>M. pachydermatis<\/jats:italic>\n                    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.\n                  <\/jats:p>","DOI":"10.1007\/s11259-026-11284-3","type":"journal-article","created":{"date-parts":[[2026,5,23]],"date-time":"2026-05-23T06:52:57Z","timestamp":1779519177000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["In vitro antimicrobial activity of Naja cardiotoxin peptide-3 against clinical isolates from canine otitis"],"prefix":"10.1007","volume":"50","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0211-5293","authenticated-orcid":false,"given":"Costanza","family":"Spadini","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5652-2922","authenticated-orcid":false,"given":"Nicol\u00f2","family":"Mezzasalma","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-4964-1592","authenticated-orcid":false,"given":"Alessandro","family":"Cupola","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8201-8591","authenticated-orcid":false,"given":"Simone","family":"Taddei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1936-9858","authenticated-orcid":false,"given":"Clotilde Silvia","family":"Cabassi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,5,23]]},"reference":[{"key":"11284_CR1","doi-asserted-by":"publisher","DOI":"10.1017\/S0950268818003278","author":"C Bour\u00e9ly","year":"2019","unstructured":"Bour\u00e9ly C, Cazeau G, Jarrige N, Leblond A, Madec JY, Haenni M, Gay E (2019) Antimicrobial resistance patterns of bacteria isolated from dogs with otitis. 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