Evolutionary dynamics of Clostridium difficile over short and long time scales

Abstract

Clostridium difficile has rapidly emerged as the leading cause of antibiotic-associated diarrheal disease, with the transcontinental spread of various PCR ribotypes, including 001, 017, 027 and 078. However, the genetic basis for the emergence of C. difficile as a human pathogen is unclear. Whole genome sequencing was used to analyze genetic variation and virulence of a diverse collection of thirty C. difficile isolates, to determine both macro and microevolution of the species. Horizontal gene transfer and large-scale recombination of core genes has shaped the C. difficile genome over both short and long time scales. Phylogenetic analysis demonstrates C. difficile is a genetically diverse species, which has evolved within the last 1.1-85 million years. By contrast, the disease-causing isolates have arisen from multiple lineages, suggesting that virulence evolved independently in the highly epidemic lineages.

Fig. 1.

Phylogenetic trees of C. difficile based on whole-genome sequences. Arrows and unfilled circles denote insertion and deletion events, respectively. Genomic islands carrying drug resistance genes are shown with asterisks. (A) Deep-branching phylogeny that illustrates the relationships between different lineages/ribotypes are shown by different colors. The four 027 ribotype isolates are collectively represented as node “027s”. Scale bar indicates number of substitutions per site. The root connects to C. bartlettii and C. hiranonis. (B) Split decomposition network indicating microevolution within the hypervirulent lineage. Strain names are colored according to countries of isolation (blue, United States; red, United Kingdom; green, France). Bootstrap values are labeled along branches. The root connects to strain 630.

Fig. 2.

SNPs between CD196 and 24 other hypervirulent C. difficile isolates. Outer circle: CDSs of C. difficile CD196 genome, shown on a pair of concentric rings representing both coding strands; two inner circles: G+C% content plot and GC deviation plot (>0% olive, <0% purple); in between: SNPs (blue and red) between CD196 and other isolates, from outer to inner: 2004013, 2004102, 2004118, 2004163, 2006439, 2007140, 2007218, 2007825, 2007833, 2007837, 2007855, BI-1, BI-2, BI-3, BI-4, BI-5, BI-6, BI-6p, BI-7, BI-10, BI-11, BI-13, BI-15, and R20291. The rings representing isolates with large homologous recombination blocks (BI–4 and BI–11) are shown in red.

Fig. 3.

Signatures of recombination in the deep-branching phylogeny. The genome-wide distribution of SNPs is shown for each strain against the core genome (excluding repetitive sequences) of strain CF5, which is indicated along the x axis. The y axis gives the number of SNPs in each 500-bp window.

Fig. 4.

Trajectory of 1/(dN/dS) within the C. difficile phylogeny over time. The number of intergenic SNPs (A) and synonymous changes (B) serve as measures of time since divergence.