Clinical Clostridium difficile: clonality and pathogenicity locus diversity

Abstract

Clostridium difficile infection (CDI) is an important cause of mortality and morbidity in healthcare settings. The major virulence determinants are large clostridial toxins, toxin A (tcdA) and toxin B (tcdB), encoded within the pathogenicity locus (PaLoc). Isolates vary in pathogenicity from hypervirulent PCR-ribotypes 027 and 078 with high mortality, to benign non-toxigenic strains carried asymptomatically. The relative pathogenicity of most toxigenic genotypes is still unclear, but may be influenced by PaLoc genetic variant. This is the largest study of C. difficile molecular epidemiology performed to date, in which a representative collection of recent isolates (n = 1290) from patients with CDI in Oxfordshire, UK, was genotyped by multilocus sequence typing. The population structure was described using NeighborNet and ClonalFrame. Sequence variation within toxin B (tcdB) and its negative regulator (tcdC), was mapped onto the population structure. The 69 Sequence Types (ST) showed evidence for homologous recombination with an effect on genetic diversification four times lower than mutation. Five previously recognised genetic groups or clades persisted, designated 1 to 5, each having a strikingly congruent association with tcdB and tcdC variants. Hypervirulent ST-11 (078) was the only member of clade 5, which was divergent from the other four clades within the MLST loci. However, it was closely related to the other clades within the tcdB and tcdC loci. ST-11 (078) may represent a divergent formerly non-toxigenic strain that acquired the PaLoc (at least) by genetic recombination. This study focused on human clinical isolates collected from a single geographic location, to achieve a uniquely high density of sampling. It sets a baseline of MLST data for future comparative studies investigating genotype virulence potential (using clinical severity data for these isolates), possible reservoirs of human CDI, and the evolutionary origins of hypervirulent strains.

Figure 1. The C. difficile population is comprised of five clades with evidence of recombination within and among groups.

(A) Phylogenetic network to illustrate relationships among 78 STs comprising the five clades. The networks were constructed using concatenated MLST loci and the program Neighbour-Net . The clades containing well characterised isolates or genotypes, their ribotypes and STs are indicated (ST-89 was excluded as it differs from ST-6 by a single nucleotide deletion in the sodA locus only). (B) eBURST diagram to show the relationships among STs based on alleleic profiles . Circle size for each of the 69 STs identified in the clinical isolate collection (total n = 1290) is proportional to the number of isolates. For completeness, one example of eight additional previously described STs (ST-20, ST-27, ST-29, ST-32, ST-38, ST-39, ST-40, ST-69) , and one (ST-30) identified in a separate study of infants were included to demonstrate their relationship to other members of the population. Well characterised representatives of each clade are indicated as in (A). STs identified in both toxigenic and non-toxigenic form are indicated by a star. (C) ClonalFrame analysis . The five clades are indicated and branch lengths are measured in expected number of mutation and recombination events.

Figure 2. Relative positions of the tcdB and tcdC genes within the PaLoc and the sequences determined in this study.

(A) Organisation of the five genes encoded by the 19.6 kb PaLoc of C. difficile, and two flanking genes containing the lok1/3 primer pair . (B) The four functional domains of the 2366 amino acid TcdB protein, . The region of the receptor binding domain (RBD) sequenced is indicated (horizontal black bar). The sequence determined is located within the C-terminal repetitive region which may bind to enteric cells via carbohydrate receptors . (C) The 233 amino acid negative regulator TcdC. The location of the coiled-coil dimerization domain is indicated; deletions found within the repetitive sequences of this domain are indicated by a triangle. The truncations found in PCR-ribotype 027 and 078 strains are indicated by a vertical bar. The sequence determined is indicated by a black horizontal bar.

Figure 3. The association of tcdB-RBD alleles and clade is congruent.

(A) Neighbour joining tree showing the relationships among the 17 tcdB allele sequences (597nt) and C. sordelii constructed using MEGA with bootstraps calculated using 1000 replicates. Coloured shapes indicate the clade (Fig. 1) with which each tcdB allele is associated. The number of variable nucleotide sites (excluding C. sordelii) was 97/597 (16.2%) and amino acids 42/199 (21.1%). * Indicates putative recombinants derived from clade 2 and clade 3 sequences. C. sordelii was used to root the tree as it encodes the closest known relative to C. difficile tcdB. (B) Alignment of the tcdB-RBD allele variable amino acid sites, relative to the sequence of the CD630 genome strain (clade 1, allele tcdB-2). Alleles tcdB-3, tcdB-4, tcdB-6, and tcdB-17 are not shown as they have an amino acid sequence identical to tcdB-2. The bold box indicates the closely related sequences of clade 3 and clade 5; the yellow shaded box indicates putative clade 2/clade 3 tcdB recombinants; * in (A).

Figure 4. The association of tcdC alleles and clade is almost congruent.

(A) Neighbour joining tree showing the relationship among tcdC variants and the clades. The variants were manually aligned in BioEdit and a neighbour joining tree was computed using MEGA with bootstraps calculated using 1000 replicates. Coloured shapes indicate the clade (Fig. 1) with which the tcdC allele was associated. Alleles appear the same if they differ only in terms of their relative deletion lengths which are indicated in (B). Alleles WTtcdC-8 and WTtcdC-14 appear the same on the tree as they differ by a single nucleotide at position 117, which is deleted in some other alleles and therefore contained an N in some variants in the alignment. (B) Predicted translation products from tcdC variants. Three different TcdC variants were found (i) wild type (WT) (ii) truncated by Δ1 at nucleotide 117 creating a frameshift and a stop codon at amino acid 66, (Δ1stop), or by a CAA to TAA substitution creating a stop codon at amino acid 62, (TAAstop) (iii) 18 nucleotides deleted (Δ18) within the coiled-coil dimerization domain. Further deletions within the untranslated sequence of clade 3 and clade 5 were 36 nucleotides long (Δ36) or 54 nucleotides long (Δ54). Translated regions are represented in the figure by the bold black line; untranslated regions are represented by the light black line.