Comparative genomics reveals diversity and taxonomic relationships among Clostridioides difficile phages

ABSTRACT Clostridioides difficile is associated with life-threatening antibiotic-associated diarrhea, colitis, and toxin-mediated infections. While antibiotics are the primary treatment against C. difficile infections, increasing resistance necessitates alternatives. Bacteriophages and bacteriophage-derived proteins, such as endolysins, hold promise as potential solutions. Understanding phage biology at the genomic level is crucial for their therapeutic use. We conducted a comparative genomic analysis of 44 C . difficile phage genomes from public databases, examining both whole-genome and proteome levels and grouping them by shared protein content. Relationships within each group were observed, and core and highly conserved genes were identified. Using genome and proteome phylogeny, average nucleotide identity, and core gene identification, we proposed an updated taxonomic classification. Nine distinct clusters were identified, without any singleton. Cluster members exhibited similar genome architecture, genome sizes, GC content, number of coding sequences, presence of core genes, and high nucleotide identity. Additionally, we propose 23 new genera, three families, and the elevation of currently assigned genera to subfamilies. The lytic module proteins, endolysins, and holins were also characterized, revealing four distinct endolysin organizations with diverse domain architectures. Notably, the amidase_3 and LysM domains were highly conserved and subjected to purifying selection within the C. difficile phage genomes. Our extensive comparative analyses of C. difficile phage genomes provide valuable genomic insights into the current understanding of the phages. The taxonomic analysis could refine the classification scheme for these phages and facilitate the future categorization of newly isolated C. difficile phage genomes. IMPORTANCE Clostridioides difficile is a significant healthcare concern due to its role in causing severe infections and its increasing resistance to antibiotics. Phages present a promising alternative to antibiotics for combating bacterial pathogens, including C. difficile . Our study is the first comprehensive comparative genomic analysis of C. difficile phage genomes. We analyzed all available C. difficile phage genomes and clustered them into genetically distinct groups based on protein sharing. We also propose major taxonomic revisions to facilitate the accurate classification of C. difficile phages. Additionally, we investigated the lytic module of these phages, revealing diverse endolysin organizations and strong purifying selection acting on the catalytic and cell wall binding domains. Our findings contribute valuable insights into the biology of C. difficile phages and provide a framework that can aid in the classification and genomic analysis of newly isolated C. difficile phages in the future.