Integrated Phenotypic and Genomic Characterization Reveals Functional Heterogeneity among Four Bifidobacterium...

Members of the genus Bifidobacterium exhibit substantial inter- and intraspecific variation in probiotic-associated functions, emphasizing the need for integrated phenotypic and genomic evaluation. This study comparatively characterized four Bifidobacterium strains from the Thailand Institute of Scientific and Technological Research (TISTR) Culture Collection using phenotypic and genomic approaches. Hybrid genome assembly generated complete circular chromosomes, and genome-based safety assessment detected no recognized virulence-associated genes. All strains were predicted to be non-human pathogens and were phenotypically susceptible to the eight antimicrobials tested. Although tet(W)-associated sequences were identified in TISTR 2591 and TISTR 2893, neither strain exhibited phenotypic tetracycline resistance. Comparative CAZyme analysis identified 71-82 high-confidence CAZyme-encoding genes, with TISTR 2893 possessing the largest and most diverse glycoside hydrolase repertoire. Distinct functional profiles were observed among the four strains. TISTR 2591 exhibited the strongest epithelial adhesion, reaching 19.05% and 10.47% to Caco-2 and HT-29 cells, respectively, together with broad carbohydrate utilization and predominantly L-lactate production. TISTR 2737 showed the greatest gastric-acid tolerance among the TISTR strains, with a 0.34-log₁₀ CFU/mL reduction after 180 min at pH 2.0, whereas TISTR 2986 showed the greatest bile-salt tolerance, with a 0.50-log₁₀ CFU/mL reduction. TISTR 2893 exhibited the broadest antagonistic spectrum, inhibiting all six indicator bacteria. Neutralization of the cell-free supernatants abolished detectable inhibition, while genome mining revealed no convincing complete bacteriocin biosynthetic cluster. Under the trait-weighted functional screening framework, TISTR 2893 obtained the highest numerical score (15/28; 53.57%), whereas the equal-domain sensitivity analysis produced a different numerical ordering, demonstrating the influence of weighting structure on composite evaluation. Collectively, the four strains exhibited distinct functional profiles rather than a uniform hierarchy of probiotic-associated performance, supporting function-specific strain selection based on complementary phenotypic and genomic evidence.