Abstract

Background/Objectives: Tetracycline resistance in Gram-positive bacteria has increased globally through horizontal gene transfer. Antimicrobial resistance genes in Clostridium tetani, the causative agent of tetanus, are rarely reported. Previously, we identified tetracycline resistance gene tet(M)-positive C. tetani strains in Japan, but their evolutionary origin and acquisition mechanism remain unclear. This study aimed to elucidate the evolutionary origin of tet(M)-positive C. tetani and clarify the mechanism of horizontal resistance gene acquisition. Methods: Complete genome sequences of six tet(M)-positive C. tetani strains were determined using hybrid assembly of short-read (Illumina) and long-read (Oxford Nanopore) sequencing. Core genome single-nucleotide variant (SNV) analysis was performed to determine phylogenetic relationships. Tn916 element analysis of the tet(M) gene identified the origin of resistance genes and potential donor bacteria. Results: Based on core genome SNV analysis, all six tet(M)-positive strains belonged to Clade 1-2. The Tn916 element was approximately 18 kb and highly conserved; however, insertion sites differed significantly among strains. Phylogenetic analysis of the tet(M) gene revealed at least three distinct variants with different origins. Conclusions: Multiple independent acquisitions of Tn916-mediated tet(M), rather than clonal propagation, drove resistance emergence in geographically confined C. tetani populations. These findings support the notion that tetracycline resistance emerged through multiple independent horizontal transfer events involving Tn916. This pattern suggests that persistent environmental selective pressure, rather than clonal expansion, has shaped resistance dissemination, highlighting the importance of genomic surveillance and systematic monitoring to track the emergence of antimicrobial resistance in environmental and clinical settings.

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