Isolation, Identification, Physiological Characterization, and Complete Genome Analysis of a Sheep-Derived Enterococcus faecium Strain

Abstract: [Objective] This study aimed to investigate the physiological properties and whole-genome characteristics of a strain of Enterococcus faecalis isolated from the gastrointestinal tract of Large-tailed Han sheep. [Methods] We employed modified GAM anaerobic culture and 16S rRNA technology for the isolation, screening, and identification of the bacterial strain. Growth and acid production curves were plotted to analyze its physiological characteristics. Subsequently, whole-genome sequences were obtained using Illumina and PacBio high-throughput sequencing, and bioinformatics and online resources were utilized to assemble and analyze the sequencing data. Additionally, the antibiotic resistance of the strain was experimentally verified. [Results] Through anaerobic culture, we successfully isolated a non-spore-forming, non-flagellated Gram-positive coccus, identified as Enterococcus faecalis via 16S rRNA gene sequencing. Growth characteristic analysis revealed that the strain entered the logarithmic phase within 2 hours and reached a growth peak at 6 hours, exhibiting acid production activity from the early stages of the experiment. Whole-genome analysis indicated that the strain consists of a circular chromosome and two circular plasmids, with a total length of 2,992,873 bp and a GC content of 37.26%, encoding 2,810 genes. Functional analysis revealed that the strain is primarily involved in key biological processes such as carbohydrate transport and metabolism, translation, ribosomal structure and biogenesis, and transcription. The genome of strain S1 also contains five genomic islands, three prophages, and six CRISPR-Cas systems, which play significant roles in the strain's adaptive evolution. Furthermore, we identified gene clusters related to secondary metabolite synthesis that are closely associated with the strain's production of antimicrobial substances. The strain carries 413 genes that can reduce its virulence by interfering with the expression of antibiotic resistance and pathogenicity genes, thereby inhibiting the strain's pathogenicity. Antibiotic susceptibility testing showed that strain S1 is highly sensitive to multiple antibiotics, including ampicillin, piperacillin, and penicillin, exhibits intermediate sensitivity to streptomycin and erythromycin, and demonstrates tolerance to cephalexin and cefuroxime sodium. [Conclusion] This study successfully isolated and named a new strain of Enterococcus faecalis-LTHS1. The strain's genome is rich in metabolic genes that facilitate the absorption and digestion of nutrients in the gut and achieve antimicrobial effects via secondary metabolites. The results provide a theoretical foundation for the research and application of this strain in the fields of microbiology, medicine, and animal husbandry.