Comparative genomics reveals diversity and taxonomic relationships among Clostridioides difficile phages

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. This is the first comprehensive comparative study regarding C. difficile phage genomes. Our study provides valuable genomic insights that add to the current understanding of the phages. Our taxonomic analysis may improve the classification scheme of C. difficile phages and aid in the future classification of newly isolated C. difficile phage genomes.