Reliable delineation of Clostridioides difficile and related members of the family Peptostreptococcaceae using phylogenomics and spore coat protein-specific molecular markers.

Traditional bacterial classification relies on phenotypic traits (e.g., morphology and metabolic profiles), but these methods lack resolution for closely related taxa and are biased by culture conditions. While 16S rRNA gene sequencing is a widely used molecular complement, it fails to resolve closely related Peptostreptococcaceae species, including Clostridioides difficile. These limitations have caused family-level taxonomic confusion and ambiguous Clostridioides genus boundaries, hindering clinical identification of pathogenic strains and posing public health risks. To address these limitations, we developed an integrated approach combining multi-scale phylogenomic and protein-based molecular evidence, adopting a hierarchical workflow: first, constructing a 16S rRNA phylogeny of 151 Firmicutes strains to demonstrate traditional marker inadequacies; second, generating a whole-genome protein phylogeny of 51 representative Peptostreptococcaceae genomes and defining taxonomic boundaries via average amino acid identity (AAI); third, analyzing spore-associated protein patterns across C. difficile isolates and related genomes. Results revealed high conservation of C. difficile spore coat/exosporium proteins and clear genus-level phylogenetic distinctiveness of these proteins. Combined with AAI-validated whole-genome data, our findings support key Peptostreptococcaceae taxonomic revisions: redefining polyphyletic Romboutsia, reassigning Eubacterium tenue to Paraclostridium, and elevating Alkalithermobacter to genus status. This study establishes spore coat proteins as core taxonomic markers for spore-forming bacteria, with our integrated strategy overcoming traditional limitations to improve classification accuracy and C. difficile surveillance.IMPORTANCEConventional classification struggles to resolve closely related Peptostreptococcaceae species (e.g., Clostridioides difficile). We developed an integrated framework combining 16S rRNA sequencing, whole-genome protein analysis, and spore trait assessment, with a key innovation: identifying spore coat/exosporium proteins as robust, conserved taxonomic markers. This approach enabled three pivotal Peptostreptococcaceae revisions-redefining Romboutsia, reassigning Eubacterium tenue to Paraclostridium, and elevating Alkalithermobacter to genus rank. The findings resolve a longstanding microbial systematics bottleneck for spore-forming bacteria, provide critical taxonomic context for C. difficile's precise monitoring and prevention, and expand taxonomic markers beyond nucleic acid-based methods. This advances classification precision, critical for microbial ecology, pathogenesis, and industrial microbiology research.