<![CDATA[Abstract. Narulita E, Utami DAW, Izzaturrohmah L, Arrachmi HI, Febrianti RA, Ludfillah RZ, Widianto AH, Addy HS, Utami BR, Kuswati, Fikri K. 2025. Genomic and evolutionary perspectives on bacteriophage ?Afa-NA1 targeting MDR Gram-negative bacteria in diabetic ulcer. Biodiversitas 26: 6014-6024. The rise of Multidrug-Resistant (MDR) bacterial infections poses a significant challenge in clinical settings, particularly for diabetic ulcer patients who are highly susceptible to persistent infections. This study reports the isolation, characterization, and genomic analysis of a novel lytic bacteriophage, ?Afa-NA1, specifically targeting MDR Gram-negative bacteria associated with diabetic ulcers in Indonesia. The bacteriophage was isolated from wound dressings and tested against four clinical isolates: Alcaligenes faecalis T17, Pseudomonas sp. FP1911, and the Gram-positive control Streptomyces violaceoruber S21. ?Afa-NA1 exhibited strong lytic activity against three Gram-negative isolates but none against the Gram-positive control, confirming host specificity. ?Afa-NA1 produced clear plaques with an average diameter of 3.0±0.5 mm on Alcaligenes faecalis T17, indicative of its potent lytic activity. Stability tests showed the phage retained >90% infective titer after 24 hours at -20°C and 4°C, and exhibited robust stability across a wide pH range (pH 6 to 11). One-step growth analysis revealed a short latent period of 21.7±8.1 minutes and a burst size of 27.7±1.5 PFU per infected cell. Genomic analysis showed ?Afa-NA1 possesses a 78,105 bp double-stranded DNA genome with 45.8% G+C content encoding 83 predicted open reading frames. Comparative genomics and phylogenetic reconstruction confirmed its placement among T7-like Podoviruses, consistent with its obligate lytic lifecycle and genomic architecture. However, the potential for therapeutic application remains preliminary, as host range testing was conducted on a limited panel of single clinical isolates, and all assays were performed in vitro.]]>
