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.