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Certain strains of K. pneumoniae are highly resistant to most antibiotics available in clinical practice, exacerbating the challenge of bacterial infections. Methods: Phage vB_KpnP_PW7 (vKPPW7) was isolated and characterized. Its morphology, stability, adsorption rate, one-step growth curve, lytic activity, whole-genome sequence analysis, and antibacterial and antibiofilm activities were evaluated. Results: The virulent phage has a 73,658 bp linear dsDNA genome and was classified as a new species of the genus Kaypoctavirus, subfamily Enquatrovirinae, and family Schitoviridae. Phage vKPPW7 has a high adsorption rate, a short latent period, and a large burst size. The phage showed activity against 18 K. pneumoniae isolates with the K24 capsular type but was unable to lyse K. pneumoniae isolates whose capsular type was not classified as K24. Additionally, phage vKPPW7 demonstrated strong stability across various temperatures and pH values. The phage exhibited antibacterial activity, and scanning electron microscopy (SEM) confirmed its ability to lyse MDR K. pneumoniae with the K24 capsular type. Furthermore, phage vKPPW7 effectively removed preformed biofilm and prevented biofilm formation, resulting in reduced biofilm biomass and biofilm viability compared to controls. The architecture of phage-treated biofilms was confirmed under SEM. Conclusions: These findings suggest that phage vKPPW7 holds promise for development as a therapeutic or biocontrol agent.<\/jats:p>","DOI":"10.3390\/antibiotics14020157","type":"journal-article","created":{"date-parts":[[2025,2,5]],"date-time":"2025-02-05T07:44:59Z","timestamp":1738741499000},"page":"157","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Isolation, Characterization, and Anti-Biofilm Activity of a Novel Kaypoctavirus Against K24 Capsular Type, Multidrug-Resistant Klebsiella pneumoniae Clinical Isolates"],"prefix":"10.3390","volume":"14","author":[{"given":"Phitchayapak","family":"Wintachai","sequence":"first","affiliation":[{"name":"Bacteriophage Laboratory, Walailak University, Thasala, Nakhon Si Thammarat 80161, Thailand"},{"name":"School of Science, Walailak University, Thasala, Nakhon Si Thammarat 80161, Thailand"},{"name":"Functional Materials and Nanotechnology Center of Excellence, Walailak University, Thasala, Nakhon Si Thammarat 80161, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1808-898X","authenticated-orcid":false,"given":"Joanne","family":"Santini","sequence":"additional","affiliation":[{"name":"Department of Structural and Molecular Biology, Division of Biosciences, University College London, London WC1E 6BT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Renuka","family":"Thonguppatham","sequence":"additional","affiliation":[{"name":"Bacteriophage Laboratory, Walailak University, Thasala, Nakhon Si Thammarat 80161, Thailand"},{"name":"School of Science, Walailak University, Thasala, Nakhon Si Thammarat 80161, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-1775-9873","authenticated-orcid":false,"given":"Maria","family":"Stroyakovski","sequence":"additional","affiliation":[{"name":"Department of Structural and Molecular Biology, Division of Biosciences, University College London, London WC1E 6BT, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7793-7561","authenticated-orcid":false,"given":"Komwit","family":"Surachat","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences and Biomedical Engineering, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand"},{"name":"Translational Medicine Research Center, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90110, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2280-6649","authenticated-orcid":false,"given":"Apichart","family":"Atipairin","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Walailak University, Thasala, Nakhon Si Thammarat 80161, Thailand"},{"name":"Drug and Cosmetics Excellence Center, Walailak University, Thasala, Nakhon Si Thammarat 80161, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1283","DOI":"10.1016\/j.micinf.2008.07.040","article-title":"Host and microbiota factors that control Klebsiella pneumoniae mucosal colonization in mice","volume":"10","author":"Lau","year":"2008","journal-title":"Microbes Infect."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1017\/S0195941700062639","article-title":"The epidemiology of nosocomial infections caused by Klebsiella pneumoniae","volume":"6","author":"Jarvis","year":"1985","journal-title":"Infect. 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