Drivers of the differentiation of the human pathogen Clostridioides difficile

Clostridioides difficile is a major causative agent of antibiotic-associated diarrhea and pseudomembranous colitis. However, most research is based on incomplete draft genome sequences mainly with clinical background. Recent findings suggest a large, but untapped genomic variability particularly of the mobilome of non-clinical and environmental isolates. However, a resolution beyond draft genomes is required for better understanding into their evolution and functional implications.

To elucidate the genomic diversity and evolutionary dynamics, our large-scale study comprised a representative dataset consisting of 175 C. difficile isolates which was investigated by de novo complete genome sequencing and comparative genomics. This allowed the prediction of lineage-specific genes, mobile genetic elements and a detailed investigation of the pan-genome. Newly sequenced complete genomes were obtained from various geographic locations and epidemiological contexts and tried to specifically cover previously underrepresented clades. This revealed a high conservation of genomic synteny across different continents and clades, despite a high variability of acquired lineage-specific genes and mobile genetic elements. Among the lineage-specific genes, several virulence factors and antiviral systems were identified. Additionally, a diverse set of phages and phage-like plasmids was detected. This is in agreement with the phylogenetic network analyses of the core-genome, which revealed different evolutionary strategies between the five clades. The selective advantage and genetic function of lineage-specific genes were determined and potential evolutionary mechanisms based on genomic evidence identified.

Based on the dataset of C. difficile different genomic evidences about evolving traits, the mobilome and extrachromosomal elements were identified, which one could not conduct without complete genomes. These findings contribute to our understanding of early genome evolution and the role of mobile elements on the separation, diversification and ultimately the speciation of C. difficile.