Genome-Guided Analysis of Functional Potential and Safety Signals in Three Snake-Gut Bacterial Isolates

Snake-gut bacterial isolates may have distinctive functional potential, but their application requires consideration of both strain-level functional evaluation and safety risk screening. This study combined in vitro phenotypic data, long-read whole-genome sequencing, species-matched comparative genomics, and multi-database screening to comprehensively characterize the previously high-performing Enterococcus hirae SA0215 and Lactococcus lactis SA0220 from Trimerodytes annularis and Enterococcus faecalis TP0208 from Trimerodytes percarinatus. All three strains yielded negative results in the gelatinase and catalase tests, and none could effectively use mucin as a carbon source to support vigorous growth; E. hirae SA0215 and L. lactis SA0220 tested qualitatively positive for bile salt hydrolase, and L. lactis SA0220 also exhibited significant auto-aggregation and co-aggregation capacities. Genomic analysis suggested that E. hirae SA0215 possessed features related to nutrient utilization and cell-surface homeostasis, together with features potentially related to antibacterial activity, whereas L. lactis SA0220 showed relatively prominent acid-stress-related modules and carbon-source utilization strategies under nutrient fluctuations. Although E. faecalis TP0208 possessed diverse metabolism-related modules, it also carried risk signals related to adhesion, biofilm formation, extracellular proteolysis, and antimicrobial resistance. Based on the combined phenotypic and genomic evidence, E. hirae SA0215 and L. lactis SA0220 may be further evaluated for in vivo safety and efficacy, whereas E. faecalis TP0208, as a subject for safety risk characterization, requires further targeted safety assessment. This study provides a strain-level framework for functional evaluation and risk stratification of isolates from nontraditional hosts.