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This study aimed to isolate and characterize phages with lytic activity against multidrug-resistant A. baumannii strains to enable antibacterial alternatives. Eight phages (AKO8a, PS118, B612, MCR, IDQ7, 89P13, CRL20, and CIM23) were isolated and subjected to genomic, phylogenetic, and functional analyses. Antibacterial activity was assessed in vitro against A. baumannii strain AbAK04 by measuring optical density over 17 h at multiplicities of infection (MOIs) of 0.1, 1, and 10, using a repeated-measures design with time as a crossed factor and MOI as a nested factor. Tukey\u2019s post-hoc test identified significant bacterial growth reductions of 57\u201372% (p &lt; 0.001). Specifically, phages PS118 and 89P13 reduced growth by 71% at MOI 10; CIM23, B612, and CRL20 achieved 68% reduction at MOI 1; and MCR reduced growth by 64% at MOIs 0.1 and 1. Notably, lytic phage MCR encodes a glycosyl hydrolase family 58 (GH58) enzyme, potentially contributing to its antibacterial activity. Genomic analyses confirmed absence of virulence and antibiotic resistance genes, with all phages classified as novel species within the Kagunavirus genus. These findings support the use of these phages as promising candidates for in vivo evaluation.<\/jats:p>","DOI":"10.3390\/ijms26136141","type":"journal-article","created":{"date-parts":[[2025,6,26]],"date-time":"2025-06-26T06:55:13Z","timestamp":1750920913000},"page":"6141","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Genomic and Functional Characterization of Novel Phages Targeting Multidrug-Resistant Acinetobacter baumannii"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2514-0651","authenticated-orcid":false,"given":"Alma Karen","family":"Orozco-Ochoa","sequence":"first","affiliation":[{"name":"Laboratorio Nacional para la Investigaci\u00f3n en Inocuidad Alimentaria (LANIIA), Centro de Investigaci\u00f3n en Alimentaci\u00f3n y Desarrollo, A.C. (CIAD), Carretera a Eldorado Km 5.5, Campo El Diez, Culiac\u00e1n 80110, Sinaloa, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5010-9889","authenticated-orcid":false,"given":"Beatriz","family":"Qui\u00f1ones","sequence":"additional","affiliation":[{"name":"Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, CA 94710, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2994-4358","authenticated-orcid":false,"given":"Jean Pierre","family":"Gonz\u00e1lez-G\u00f3mez","sequence":"additional","affiliation":[{"name":"Laboratorio Nacional para la Investigaci\u00f3n en Inocuidad Alimentaria (LANIIA), Centro de Investigaci\u00f3n en Alimentaci\u00f3n y Desarrollo, A.C. (CIAD), Carretera a Eldorado Km 5.5, Campo El Diez, Culiac\u00e1n 80110, Sinaloa, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0738-492X","authenticated-orcid":false,"given":"Nohelia","family":"Castro-del Campo","sequence":"additional","affiliation":[{"name":"Laboratorio Nacional para la Investigaci\u00f3n en Inocuidad Alimentaria (LANIIA), Centro de Investigaci\u00f3n en Alimentaci\u00f3n y Desarrollo, A.C. (CIAD), Carretera a Eldorado Km 5.5, Campo El Diez, Culiac\u00e1n 80110, Sinaloa, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9155-6981","authenticated-orcid":false,"given":"Jos\u00e9 Benigno","family":"Valdez-Torres","sequence":"additional","affiliation":[{"name":"Laboratorio Nacional para la Investigaci\u00f3n en Inocuidad Alimentaria (LANIIA), Centro de Investigaci\u00f3n en Alimentaci\u00f3n y Desarrollo, A.C. (CIAD), Carretera a Eldorado Km 5.5, Campo El Diez, Culiac\u00e1n 80110, Sinaloa, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5071-8270","authenticated-orcid":false,"given":"Crist\u00f3bal","family":"Chaidez-Quiroz","sequence":"additional","affiliation":[{"name":"Laboratorio Nacional para la Investigaci\u00f3n en Inocuidad Alimentaria (LANIIA), Centro de Investigaci\u00f3n en Alimentaci\u00f3n y Desarrollo, A.C. (CIAD), Carretera a Eldorado Km 5.5, Campo El Diez, Culiac\u00e1n 80110, Sinaloa, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,26]]},"reference":[{"key":"ref_1","first-page":"135","article-title":"Penicillin\u2019s Discovery and Antibiotic Resistance: Lessons for the Future?","volume":"90","author":"Lobanovska","year":"2017","journal-title":"Yale J. Biol. 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