Comparative phylogenomics of Clostridium difficile reveals clade specificity and microevolution of hypervirulent strains

Abstract

Clostridium difficile is the most frequent cause of nosocomial diarrhea worldwide, and recent reports suggested the emergence of a hypervirulent strain in North America and Europe. In this study, we applied comparative phylogenomics (whole-genome comparisons using DNA microarrays combined with Bayesian phylogenies) to model the phylogeny of C. difficile, including 75 diverse isolates comprising hypervirulent, toxin-variable, and animal strains. The analysis identified four distinct statistically supported clusters comprising a hypervirulent clade, a toxin A(-) B(+) clade, and two clades with human and animal isolates. Genetic differences among clades revealed several genetic islands relating to virulence and niche adaptation, including antibiotic resistance, motility, adhesion, and enteric metabolism. Only 19.7% of genes were shared by all strains, confirming that this enteric species readily undergoes genetic exchange. This study has provided insight into the possible origins of C. difficile and its evolution that may have implications in disease control strategies.

FIG. 1.

Phylogenetic relationship of strains associated with different clinical outcomes and animal sources represented as four major clades (HY, A⁻ B⁺, HA1, and HA2). Strains are designated at the end of the branches and are colored according to the animal source from which the C. difficile strain was isolated. Black, human; blue, mouse; green, bovine; red, swine; light blue, equine. Branches with ** have a P value of 1.0 and represent 100% of all phylogenies showing a given topology. * indicates a P value of ≥0.98.

FIG. 2.

Selected gene map on toxin PaLoc (tcdD, tcdB, tcdE, tcdA, and tcdC). A horizontal bar indicates array competitive genomic hybridization of a single strain, and a vertical color bar represents the presence (yellow lines) or absence/high divergence (blue lines) of each gene from CD0659 (tcdD), on the left, to CD0664 (tcdC), on the right. In the clade blocks, dark blue represents strains in the A⁻ B⁺ clade, light blue represents strains in the HY clade, yellow represents strains in the HA1 clade, and red represents strains in the HA2 clade.

FIG. 3.

Whole-genome analysis of all 75 strains. A vertical color bar indicates array competitive genomic hybridization of a single strain, and a horizontal line represents the presence (yellow lines) or absence/high divergence (blue lines) of each gene from CD0001 (top) to CD3679 (bottom). Selected genomic islands of interest are labeled at the sides. In the clade blocks, dark blue represents strains in the A⁻ B⁺ clade, light blue represents strains in the HY clade, yellow represents strains in the HA1 clade, and red represents strains in the HA2 clade.

FIG. 4.

Selected gene map on flagellin-associated genes. A vertical color bar indicates array competitive genomic hybridization of a single strain, and a horizontal line represents the presence (yellow lines) or absence/high divergence (blue lines) of each gene from CD0226 (top) to CD0271 (bottom). F1 indicates flagellar loci CD0226-CD0240, F2 indicates interflagellar loci CD0241-CD0244, and F3 indicates flagellar loci CD0245-CD0271. In the clade blocks, dark blue represents strains in the A⁻ B⁺ clade, light blue represents strains in the HY clade, yellow represents strains in the HA1 clade, and red represents strains in the HA2 clade.