Streptococcus Phage Genomes Reveal Extensive Diversity, New Taxonomic Insights, and Novel Endolysin-Derived Antimicrobial Peptides

The global rise of antibiotic-resistant bacteria, particularly among Streptococcus species, poses an escalating public health threat. Traditional antibiotic development has proven inadequate, making innovative approaches such as bacteriophage-based therapies promising alternatives. A deep understanding of phage biology at the genomic level is essential for advancing therapeutic applications. Here, we analyzed 709 Streptococcus phage genomes to bridge gaps in genomic diversity and propose revisions to Streptococcus phage taxonomy. The phage genomes were clustered based on shared proteins, resulting in 66 clusters and 35 singletons with significant variation in genome characteristics. Through proteome phylogeny, average nucleotide identity, and inter-cluster core genes, we propose 21 new family-level classifications and 296 genus-level subclusters, providing an updated framework for Streptococcus phage taxonomy. Further analysis revealed diverse domain architectures in Streptococcus phage endolysins, including previously unreported structures. Specific domains were associated with distinct streptococcal hosts, suggesting adaptive evolution. We also observed variation in endolysin gene organization, with purifying selection acting on most sites, though some were subject to diversifying selection. Additionally, 182 novel endolysin-derived antimicrobial peptides (AMPs) were identified, some exhibiting antifungal, antiviral, cell-penetrating and non-toxic properties. Molecular dynamics and docking simulations demonstrated high stability and strong binding affinity of peptides EP-39 and EP-121 to the Streptococcus pneumoniae virulence factor autolysin. This is the first comprehensive comparative study of Streptococcus phage genomes, providing critical insights into phage diversity and taxonomy. It also highlights the therapeutic potential of endolysin-derived AMPs against multidrug-resistant Streptococcus strains. Further experimental validation is required to assess their clinical potential.