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Methods: Here, we identified clinical MDR-UPEC strains AT82 and AT84 collected from hospitalized patients that display extensive antimicrobial resistance at both genetic and phenotypic levels. Due to their high resistance profile, we systematically customized a phage cocktail from our coliphage library using hierarchical clustering based on host specificity and candidate selection through bacterial suppression profiles. Results: This pipeline yielded four lytic coliphages, designated Phi25-4, Phi25-6, Phi50-4, and Killian. Their genomes are relatively large ranging from 112\u2013169 kbp and cluster into two distinct lineages comprising two closely related groups: Phi25-4\/Phi50-4 and Phi25-6\/Killian. Although each phage exhibited potent antibacterial activity, none alone sustained bacterial suppression during prolonged treatment. To overcome this limitation, we systematically compared the antibacterial activity of all possible phage combinations. Conclusions: The four-phage cocktail outperformed all two- or three-phage formulations, sustaining significant growth inhibition of AT82 and AT84 for up to 16 h and reducing area under the curve by more than 80% relative to controls. Cocktail potency was dose-dependent, with lower phage doses yielding the least viable cells at 48 h. Additionally, this cocktail exerted prophylactic action, significantly reducing UPEC invasion by several orders of magnitude, while the phage cocktail alone induced minimal proinflammatory cytokine responses in human bladder epithelium. Together, these findings provide an effective phage cocktail and a complementary framework for cocktail design against urinary tract infections caused by MDR bacteria.<\/jats:p>","DOI":"10.3390\/antibiotics15090870","type":"journal-article","created":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T13:53:28Z","timestamp":1788789208000},"page":"870","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rational Design of a Phage Cocktail for Effective Control of Multidrug-Resistant Uropathogenic Escherichia coli from Hospitalized Patients"],"prefix":"10.3390","volume":"15","author":[{"given":"Patiphan","family":"Khunti","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, 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