Clostridium difficile ribotype 017 - characterization, evolution and epidemiology of the dominant strain in Asia

Abstract

Clostridium difficile ribotype (RT) 017 is an important toxigenic C. difficile RT which, due to a deletion in the repetitive region of the tcdA gene, only produces functional toxin B. Strains belonging to this RT were initially dismissed as nonpathogenic and circulated largely undetected for almost two decades until they rose to prominence following a series of outbreaks in the early 2000s. Despite lacking a functional toxin A, C. difficile RT 017 strains have been shown subsequently to be capable of causing disease as severe as that caused by strains producing both toxins A and B. While C. difficile RT 017 strains can be found in almost every continent today, epidemiological studies suggest that the RT is endemic in Asia and that the global spread of this MLST clade 4 lineage member is a relatively recent event. C. difficile RT 017 transmission appears to be mostly from human to human with only a handful of reports of isolations from animals. An important feature of C. difficile RT 017 strains is their resistance to several antimicrobials and this has been documented as a possible factor driving multiple outbreaks in different parts of the world. This review summarizes what is currently known regarding the emergence and evolution of strains belonging to C. difficile RT 017 as well as features that have allowed it to become an RT of global importance.

Keywords: epidemiology; ribotype 017.

Figure 1.

Comparative analysis of the PaLoc from C. difficile RT 017 and A + B + C. difficile strains. Arrows indicate open reading frames (ORFs) and the direction of transcription. The different enzymatic domain of the tcdB gene is responsible for the different CPE [48]. The nonsense mutation near the 5′ terminal of the tcdA gene is responsible for the loss of function of TcdA [49]. The 1.8 kb deletion near the 3′ terminal of the tcdA gene makes TcdA undetectable by many toxin EIAs [47].

Figure 2.

The cytotoxic effect of TcdB and TcdB-F on VERO cells. VERO cells were treated with the supernatant of 72-hour-old cultures of C. difficile strain 2149 (RT 014/020 which produces TcdB), C. difficile strain 1470 (RT 017 which produce TcdB-F), and C. difficile ATCC 700057 (RT 038 which is non-toxigenic) and incubated at 37°C for 24 hours before inspection under a light microscope. TcdB glycosylates Rho, Rac, and Cdc42 targets resulting in arborization of cells while TcdB-F glycosylates Rac and Ras targets resulting in rounding of cells without arborization.

Figure 3.

Sequence type diversity in evolutionary clade 4. Maximum-likelihood MLST phylogeny. Sequences were aligned using MUSCLE and tree was generated in MEGA7 with evolutionary distances calculated using the Tajima-Nei model. The scale shows the number of nucleotide substitution per site, based on concatenated MLST allele sequences (7 loci, 3501 bp). The tree is mid-point rooted and supported by 500 bootstrap replicates (only values >50 are shown). For global phylogenetic context, well-characterised representatives of MLST clade 1 (ST 54), 2 (ST 1), 3 (ST 22), 5 (ST 11), C1 (ST 181), C2 (ST 200), and C3 (ST 204) are also shown (*). Branches for clade 4 are shown in blue. Known toxin profiles of clade 4 strains are indicated by orange (A-B+CDT-) and green (A-B-CDT-) colour. RT 017 (ST 37) is indicated with a red arrowhead.

Figure 4.

A. Circular representation of the genome of C. difficile strain M68 (RT 017, ST 37, GenBank accession number NC017175.1). From outside to inside, the concentric circles represent (1) and (2) all coding sequences (CDS) transcribed in clockwise and counter-clockwise, (3) all rRNA, (4) all tRNA, (5) transposons (Tn6194 containing ermB gene represented in red and Tn6190 containing tetM gene represented in purple) and prophages (counterclockwise from top; ΦCDHM19 [58,163 bp, GC% = 31.34%], ΦCDHM13 [39,325 bp, GC% = 29.34%], and ΦMMP01 [55,106 bp, GC% = 28.87%]), and (6) GC content. B. Key characteristics of the genome.

Figure 5.

Timeline of C. difficile RT 017 reports around the world. Outbreaks refer to an increase in the regional prevalence of RT 017, which is confirmed either to be clonal or with evidence suggesting that isolates came from the same source. Endemic presence refers to prevalence reports that were not associated with outbreaks.