Genomic Landscape of 340 Virulent Acinetobacter Bacteriophages Reveals Anti-CRISPR–Enriched Candidates for Therapeutic Prioritization

Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a critical global health threat, and the inadequacy of existing antibiotics has accelerated interest in phage-based therapeutic strategies. However, rational phage therapy development demands comprehensive genomic characterization of candidate phages for both efficacy and safety. Here, we performed a large-scale comparative genomic analysis of 340 virulent Acinetobacter bacteriophages integrating phylogenetic reconstruction, pangenome analysis, CRISPR spacer-based host interaction mapping, anti-CRISPR identification, and antimicrobial resistance gene screening. Genome sizes spanned a nearly 20-fold range with a significant negative association between genome size and GC content (R² = 0.139, ρ = −0.630), indicating independent evolutionary pressures governing each property. Phylogenetic analysis revealed extensive divergence across multiple lineages with no dominant clade, while pangenome analysis yielded 20,982 unique protein families, of which 76.2% were cloud genes, confirming a highly open genome architecture. CRISPR spacer matching identified 1,480 high-confidence matches across 100 phage genomes, providing molecular evidence of broad historical infectivity. Anti-CRISPR profiling highlighted phage XC1 as an exceptional candidate harboring 55 predicted anti-CRISPR proteins with canonical regulatory locus architecture. Systematic AMR gene screening identified 23 resistance-associated gene families distributed heterogeneously across the dataset, confirming abundant therapeutically clean candidates while flagging a subset carrying transferable resistance determinants. These findings provide a multi-criteria genomic framework for the rational prioritization of phage candidates against multidrug-resistant Acinetobacter infections.