Genome-based phylogenomic reassessment of Clostridium with proposals for species synonymization and new subspecies

The genus Clostridium encompasses phylogenetically diverse, obligately anaerobic, Gram-positive, spore-forming bacteria, yet taxonomic resolution for several members remains limited by the conservatism of the 16S rRNA gene. We reassessed four closely related species pairs: Clostridium baratii and Clostridium nitritogenes ; Clostridium thermopalmarium and Clostridium colicanis ; Clostridium botulinum and Clostridium combesii ; and Clostridium estertheticum subsp. estertheticum and Clostridium estertheticum subsp. laramiense . A polyphasic framework was applied, integrating 16S rRNA and core-genome phylogenies, whole-genome relatedness metrics (digital DNA–DNA hybridization, average nucleotide identity and average amino acid identity), phenotypic characterization and carbohydrate-active enzyme profiling. Concordant genomic and phenotypic evidence supported the synonymization of C. nitritogenes (Prévot 1940) Bernard et al . 2018 with C. baratii (Prévot 1938) Holdeman and Moore 1970, C. colicanis Greetham et al . 2003 with C. thermopalmarium Soh et al . 1991 and C. estertheticum subsp. laramiense (Kalchayanand et al . 1993) Spring et al . 2003 with C. estertheticum subsp. estertheticum (Collins et al . 1993) Spring et al . 2003. By contrast, despite high genomic relatedness, C. botulinum (van Ermengem 1896) Bergey et al . 1923 and C. combesii (Prévot and Laplanche 1947) Bernard et al . 2018 displayed reproducible phenotypic and functional distinctions consistent with subspecies status. We, therefore, propose C. botulinum subsp. combesii comb. nov., alongside C. botulinum subsp. botulinum comb. nov., thereby refining genome-based classification within the genus.