Clostridioides difficile causes antibiotic-associated diarrhea which can progress to pseudomembranous colitis. Most genomic information on this pathogen is based on incomplete draft genome sequences from mainly clinical strains. However, recent findings indicate that the untapped mobilome of non-clinical and environmental isolates is large and highly variable.

To further elucidate the genomic diversity and evolutionary dynamics of C. difficile, we sequenced 163 isolates de novo and compared their closed, high-quality genomes to delimitate their pangenome, lineage-specific genes and mobile genetic elements (MGE). Clade-specific differences in the mobilome and the arsenal of defense mechanisms were observed, suggesting that the five existing clades exploit different evolutionary strategies. While Clades 1 and 4 contain diverse genomes and MGE, Clade 5 remains highly clonal and seems to be undergoing accelerated speciation. Numerous small chromosomal MGE, such as insertion sequence or transposons co-located with lineage-specific resistance genes, defense systems or virulence factors, indicating different mechanisms for gene acquisition. Several prophages and putative phage-plasmids included defense systems and virulence factors, but not resistance genes. Anti-defense systems to bypass the restriction of horizontal gene transfer (HGT) were often observed within the genomes. Remarkably, 94.9% of the CRISPR arrays observed matched C. difficile phages, suggesting they are functional and advantageous. Nearly all detected phages were Caudoviricetes and were consistently targeted by CRISPR-Cas spacer sequences. Genomes from Clade 5 showed an increased number of spacer regions and spacer-phage matches per genome despite having the least number of prophages. Furthermore, type I and II restriction-modification systems have been detected frequently among the genomes, where at least five methylation motifs were successfully matched to their putative methyltransferases.

These findings contribute to our understanding of C. difficile genome evolution and the role of MGE and defense systems on the diversification and, ultimately, speciation of C. difficile.