Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium

Abstract

Background: The continued rise of Clostridium difficile infections worldwide has been accompanied by the rapid emergence of a highly virulent clone designated PCR-ribotype 027. To understand more about the evolution of this virulent clone, we made a three-way genomic and phenotypic comparison of an 'historic' non-epidemic 027 C. difficile (CD196), a recent epidemic and hypervirulent 027 (R20291) and a previously sequenced PCR-ribotype 012 strain (630).

Results: Although the genomes are highly conserved, the 027 genomes have 234 additional genes compared to 630, which may contribute to the distinct phenotypic differences we observe between these strains relating to motility, antibiotic resistance and toxicity. The epidemic 027 strain has five unique genetic regions, absent from both the non-epidemic 027 and strain 630, which include a novel phage island, a two component regulatory system and transcriptional regulators.

Conclusions: A comparison of a series of 027 isolates showed that some of these genes appeared to have been gained by 027 strains over the past two decades. This study provides genetic markers for the identification of 027 strains and offers a unique opportunity to explain the recent emergence of a hypervirulent bacterium.

Figure 1

Distribution of orthologues CDSs in C. difficile strains 630, CD196 and R20291. The Venn diagram shows the number of genes unique, shared or core between the three strains. The associated pie charts show the breakdown of the functional categories assigned to these CDS.

Figure 2

Circular representations of C. difficile chromosomes. From the outside (scale in bp): circles 1 and 2 show the position of R20291 CDS transcribed in a clockwise and anti-clockwise direction colored according to predicted function; circle 3 shows CDS unique to R20291; circle 4 shows CDS unique to both R20291 and CD196; circle 5 shows GC content; circle 6 shows GC deviation (> 0%, olive; < 0%, purple). Color coding for CDS functions: dark blue, pathogenicity/adaptation; black, energy metabolism; red, information transfer; dark green, surface-associated; cyan, degradation of large molecules; magenta, degradation of small molecules; yellow, central/intermediary metabolism; pale green, unknown; pale blue, regulators; orange, conserved hypothetical; brown, pseudogenes; pink, phage and IS (Insertion Sequence) elements; grey, miscellaneous.

Figure 3

ACT comparison of flagellin and flagellin glycosylation-associated loci. F1 genes are CD0226-240 (630), CDR20291_0227- 241 (R20291), and CD196_0240-254 (CD196). F2 genes are CD0241-244 (630), CDR20291_0242-247 (R20291), and CD196_0255-260 (CD196). F3 genes are CD0245-271 (630), CDR20291_0248-275 (R20291), and CD196_0261-288 (CD196). Red bars indicate > 84% DNA sequence identity.

Figure 4

Comparative motility assays for C. difficile strains. The motility of strain 630 was compared to that of both recent and historic 027 ribotypes, R20291, BI-16 and CD196; M120 was the non-motile control. Strains were inoculated into 0.05% BHI agar and incubated for 24 hours in an anaerobe chamber. The motility is visualized as stalactite projections.

Figure 5

Autoagglutination of C. difficile strains. C. difficile strains were grown on BHI plates for 1 to 2 days, then inoculated into pre-equilibrated phosphate-buffered saline to an OD600 nm of 1.0 (± 0.1). These were incubated for 24 hours in pre-equilibrated glass tubes, then the OD600 nm was measured. The percentage of autoagglutination was normalized to the starting OD ((Starting OD - Final OD)/Final OD × 100). The bars indicate the percentage of cells autoagglutinating. Significant differences in autoagglutination are marked with an asterisk; P < 0.05, Students t-test. M120 is a non-motile strain thar autoagglutinates to a significantly higher level than 630 (P < 0.05).

Figure 6

Comparison of phage island SMPI (Stoke Mandeville phage island) between C. difficile strains 630, CD196 and R20291.