Host-adapted probiotic potential of Ligilactobacillus agilis 2-2 revealed by comparative genomic and phenotypic analyses

Host-specific adaptation shapes the evolution of safe and effective probiotics. In this study, we performed the first pan-genome analysis of Ligilactobacillus agilis using 40 genomes from poultry and mammalian sources. The species exhibits an open, highly plastic genome with host-driven divergence in carbohydrate metabolism. Glycosyltransferase GT2, bglF_2 and tcyB/C were enriched in mammalian strains, whereas capsule biosynthesis gene epsH and asp2 were predominant in poultry strains. The poultry-derived strain L. agilis 2-2 harbors gene clusters associated with acid and bile tolerance (atpA-atpH), adhesion (mapA), short-chain fatty acid biosynthesis (ldhA, ackA-pta), and antioxidant defense (trxA/B, msrA/B), collectively supporting its intestinal adaptation and probiotic fitness. Phenotypically, L. agilis 2-2 exhibited strong acid (86.9 %) and bile (84.1 %) tolerance, high aggregation (75 %) and hydrophobicity (55.3 %), and potent antimicrobial activity, facilitating gut colonization. Its cell-free supernatant displayed strong antioxidant capacity, scavenging DPPH (79.5 %), hydroxyl (66.2 %), and ABTS⁺ (83.2 %) radicals, and produced abundant lactic acid (7.7 mg/mL), butyrate (1767.2 μg/mL), and propionate (1097.0 μg/mL). Collectively, these findings establish L. agilis 2-2 as a metabolically versatile, host-adapted, and genomically safe probiotic, highlighting its potential for targeted poultry applications and providing mechanistic insights into host-specific adaptation in Ligilactobacillus.