Comparative genomic assessment of probiotic potential across the Anoxybacillaceae family

Thermotolerant and thermophilic bacteria have recently attracted interest as potential next-generation probiotics due to their intrinsic resistance to environmental and technological stresses. Within this context, the family Anoxybacillaceae has emerged as a promising reservoir of candidates. The aim of this study was to assess this potential through a family-level comparative genomics approach. A comparative genomic analysis was conducted on 68 bacterial genomes, including 30 clinically validated probiotic reference strains and 38 Anoxybacillaceae genomes. A curated probiotic functional marker set was assembled and calibrated. From 204 candidate markers, 101 conserved markers were retained following genome-wide screening and lineage-based filtering. Marker detection relied on a consensus-based homology framework integrating DIAMOND and HMMER, generating weighted confidence scores. Complementary analyses of biosynthetic gene clusters, carbohydrate-active enzyme-associated functions (CAZymes), genomic safety features, and mobilome components were performed.Weighted functional marker profiles significantly discriminated probiotics from Anoxybacillaceae (PERMANOVA, Bray-Curtis R² ≈ 0.55, p = 0.001). Probiotic genomes were enriched in markers related to host interaction, adhesion, antimicrobial activity, and transport, whereas Anoxybacillaceae genomes showed higher representation of stress response, energy metabolism, and environmental adaptation markers. Both groups shared a large conserved bacterial functional backbone. Differences in biosynthetic potential, CAZyme-associated profiles, and mobilome composition were consistent with ecological specialisation and were not associated with virulence or antimicrobial resistance.Although predictive, these results demonstrate that probiotic-associated functional signatures are present in the entire Anoxybacillaceae family. By applying complementary homology-based detection approaches, this study provides a rigorous comparative framework for prioritising thermotolerant candidates for targeted experimental validation.