Integrated Phenotypic and Whole-Genome Analysis of Enterococcus hirae HI3 Isolated from Hu Sheep Jejunum Supports Its Potential as a Ruminant Probiotic Candidate

The ban on antibiotic growth promoters in livestock production has created an urgent demand for safe and effective probiotic alternatives derived from indigenous animal microbiota. Here, we isolated Enterococcus hirae HI3 from Hu sheep jejunum and confirmed its species assignment via whole-genome sequencing, with ANI analysis showing 98.92% identity to the E. hirae reference genome. HI3 showed moderate acid tolerance, with survival rates of 19.31% (95% CI: 15.83–22.78%) at pH 2 and 18.40% (95% CI: 17.87–18.94%) at pH 3 after 4 h, and maintained > 50% survival in 2% bile salts. It was susceptible to penicillin, ampicillin, erythromycin, and chloramphenicol. No hemolytic activity was observed, and major enterococcal virulence genes (gelE, cyl, esp, hyl) were absent from the HI3 genome. The HI3 genome (3.06 Mb) consists of one circular chromosome and three circular plasmids. Functional annotation identified 229 carbohydrate metabolism genes (including 65 glycoside hydrolases) and 43 probiotic-related genes involved in acid tolerance (atpA–G, nhaC), bile salt tolerance (cbh), stress responses (clp family, groEL, dnaK, cspA, sod2), and adhesion (ltaS, srtA, eno, epsA). Multiple bacteriocin biosynthetic gene clusters (enterolysin A, class II lanthipeptide, etc.) were identified. Plasmid-borne tetracycline resistance genes tet(M) and tet(L) were detected; however, their functional transferability requires experimental validation. No other known transferable resistance determinants were identified in the genomic analysis. These in vitro and genomic findings represent an initial characterization of E. hirae HI3 and support its potential as a ruminant probiotic candidate. However, in vivo studies are required to validate its colonization capacity, safety, and efficacy in the target ruminant species.