Characterization of Bifidobacterium hominis and proposal of Bifidobacterium catenulatum subsp. puerorum subsp. nov. isolated from human faeces

Bifidobacteria are among the most dominant members of the human gut microbiota throughout life and are associated with host health. Multiple <i>Bifidobacterium</i> species have been isolated from human faeces, with species composition differing according to host age and between individuals. This species- and subspecies-level diversity reflects adaptation to host niches. <i>Bifidobacterium catenulatum</i> currently comprises two subspecies <i>catenulatum</i> and <i>kashiwanohense</i>, which appear to exhibit different ecological adaptations. Our previous study suggested undefined lineages closely related to <i>B. catenulatum</i>. In this study, we investigated these lineages through comparative genomic and phylogenetic analyses of 43 strains, including new isolates from human faeces.Phylogenetic reconstructions based on 16S rRNA gene sequences, multilocus sequence analysis, average nucleotide identity (ANI) and core-gene alignments consistently supported the delineation of two distinct taxa. The first group, consisting of 15 strains, exhibited ANI values of ≥96.41% among themselves but ≤94.79% compared with the known subspecies of <i>B. catenulatum</i>, confirming its classification as a novel species, which was recently validated as <i>Bifidobacterium hominis</i>. The second group, comprising four strains, showed ANI values ranging from 94.38 to 95.65% compared to the existing subspecies of <i>B. catenulatum</i>, indicating that they belong to the same species. However, phylogenetic analyses and distinct substrate utilization profiles supported their designation as a novel subspecies, <i>B. catenulatum</i> subsp. <i>puerorum</i> subsp. nov.Comparative genomic analyses revealed key differences in genes associated with gut adaptation. <i>B. catenulatum</i> subsp. <i>puerorum</i> harboured genes involved in human milk oligosaccharide (HMO) and urea metabolism, consistent with its isolation from infant faeces. In contrast, <i>B. hominis</i> exhibited strain-dependent variation in genes for HMO and xylooligosaccharide utilization. Phenotypic analyses supported these distinctions, including unique inulin utilization by <i>B. catenulatum</i> subsp. <i>puerorum</i>. Based on these findings, we characterized the two clades and propose the novel subspecies <i>B. catenulatum</i> subspecies <i>puerorum,</i> with type strain YIT 11099^T (=JCM 37523^T=DSM 118686^T).