Hypervirulent clade 2, ribotype 019/sequence type 67 Clostridioides difficile strain from Japan

Abstract

Background: Clostridioides difficile ribotype (RT) 019/sequence type (ST) 67 strains belong to a hypervirulent lineage closely related to RT027/ST1; however, limited data are available for hypervirulent clade 2 lineages in Japan. Herein, we report the draft genome of a C. difficile strain B18-123 belonging to clade 2, RT019/ST67 for the first time in Japan.

Results: The pathogenicity locus carried by B18-123 (19.6 kb) showed higher homology (97.29% nucleotide identity) with strain R20291 (RT027/ST1) than the reference strain 630 (RT012/ST54), and B18-123 harbored 8-nucleotide substitutions in tcdC. However, it did not contain an 18-base pair (bp) deletion or a single-bp deletion at position 117 in tcdC, which was identified in the previous strain R20291. A cytotoxicity assay revealed similar cytotoxicity levels between strains B18-123 and ATCC BAA-1870 (RT027/ST1). The B18-123 strain was found to be susceptible to metronidazole and vancomycin.

Conclusion: Our findings contribute to the further understanding of the characteristics of hypervirulent clade 2 including RT019/ST67 lineages.

Keywords: Binary toxin; Cdt locus; Clostridioides difficile; Hypervirulent clade 2; Pathogenicity locus; Ribotype 019; Sequence type 67; Toxin A; Toxin B.

Fig. 1

Genetic characteristics of PaLoc in Clostridioides difficile strain B18-123. (a) Comparative genomic structure analysis of PaLoc among C. difficile strains used in this study. (b) Phylogenetic tree of PaLoc among 12 strains deposited in the GenBank by maximum likelihood method based on Tamura-Nei model [24] using MEGA X software [25]. Strains surrounded by dotted line belong to ST67. (c) tcdC sequence alignment for C. difficile strains used in this study. Identical nucleotides among their strains are indicated with an asterisk. (d) Phylogenetic tree of tcdC sequences among 12 strains deposited in the GenBank by maximum likelihood method based on Tamura-Nei model [24] using MEGA X software [25]. Strains indicated with an asterisk belong to ST67

Fig. 2

Cytotoxicity of Clostridioides difficile strain B18-123 on Vero cells. (a) Vero cell viability in B18-123 was evaluated together with positive control ATCC BAA-1870 (NAP1/RT027/ST1; TcdA⁺, TcdB⁺, Cdt⁺) and negative control ATCC 700057 (RT038/ST48; TcdA⁻, TcdB⁻, Cdt⁻) strains, as described previously [16]. The results are presented as means ± standard deviations of cell viability percentages. (b) Cytotoxic effect of C. difficile strains studied under a light microscope (original magnification × 200)