Comparative pathogenomic analysis reveals a highly tetanus toxin-producing clade of Clostridium tetani isolates in Japan

Clostridium tetani is the etiological agent of the disease tetanus, resulting in various symptoms, including neck tension, tongue-twisting, a decline in walking ability, and death in severe cases. To date, publicly available genome sequences of C. tetani are limited. C. tetani was isolated from soil in Kumamoto Prefecture, Japan, between 2020 and 2022. Subsequently, bacteriological, biochemical, genetic, and immunological tests were conducted. Among the 46 soil sampling sites, 151 C. tetani strains were isolated from 33 sites, with a 71.7% positivity rate. Comparative genomic analysis subdivided clade 1 into clades 1-1, 1-2, and 1-3. The enzyme-linked immunosorbent assay revealed that partial clade 1-3 strains produced over sevenfold more tetanus neurotoxin (TeNT) than the reference strain (first isolate of C. tetani KHSU-154301-001). RNA-Seq analysis supported this elevated tetanus toxin tetX gene expression. Additionally, tetX-negative strains were isolated from the clade, indicating that TeNT may not be essential for ensuring the survival of their offspring without its representative toxin-causing tetanus. Five strains acquired the tetracycline resistance gene tet(M) in the tetX plasmid, resulting in significantly reduced susceptibility to tetracycline. In conclusion, although we investigated C. tetani isolates from a specific region of the Kumamoto Prefecture, we demonstrated that these isolates exhibit variable genomic features, toxin productivity, and substantial acquired antimicrobial resistance. These findings indicate that multiple C. tetani lineages have successfully adapted to the soil in Japan, as evidenced by the additional genome information and toxin productivity assays in this study.