Probiotic potential of Enterococcus faecium SWUN5732 isolated from yak yogurt: integrated in vitro and in vivo evaluation for yak health

Background and Aim: The increasing emergence of antimicrobial resistance has intensified the search for safe and effective alternatives to antibiotics in livestock production. Probiotics derived from traditional fermented foods represent a promising strategy due to their adaptability and functional diversity. This study aimed to isolate and characterize a plateau-adapted strain of Enterococcus faecium from traditional yak yogurt and to comprehensively evaluate its probiotic potential through in vitro assays and in vivo validation, with a focus on its applicability in yak health management. Materials and Methods: The strain E. faecium SWUN5732 was isolated and identified using morphological characterization and 16S rRNA gene sequencing. Probiotic properties were assessed through acid (pH 2.0 and 3.0) and bile salt (0.3%) tolerance, hemolytic activity, antibiotic susceptibility, antimicrobial activity against common yak pathogens, and adhesion to Caco-2 cells. Safety evaluation included virulence gene screening targeting cylA, esp, asa1, gelE, agg, and efaAfm, as well as acute oral toxicity testing in mice. Functional efficacy was further examined through a 21-day oral administration study in mice to evaluate body weight gain and intestinal secretory immunoglobulin A levels. Results: E. faecium SWUN5732 demonstrated strong gastrointestinal tolerance, with survival rates of 34.43% at pH 2.0, 60.17% at pH 3.0, and 77.07% in 0.3% bile salts. The strain exhibited no hemolytic activity and was susceptible to clinically relevant antibiotics. It showed broad-spectrum antimicrobial activity against Escherichia coli, Salmonella spp., and Staphylococcus aureus, with inhibition zones ranging from 18 to 37 mm. The adhesion rate to Caco-2 cells was approximately 29%, indicating effective colonization potential. Among the tested virulence determinants, only efaAfm was detected, while other genes (cylA, esp, asa1, gelE, agg) were absent. No acute toxicity or pathological alterations were observed in mice. Notably, oral administration significantly enhanced body weight gain and increased intestinal secretory immunoglobulin A levels (p < 0.01), demonstrating immunomodulatory and growth-promoting effects. Conclusion: The findings indicate that E. faecium SWUN5732 possesses key probiotic attributes, including gastrointestinal resilience, antimicrobial activity, safety, and immunomodulatory capacity. Its origin from high-altitude yak yogurt suggests ecological compatibility with yak production systems. This strain represents a promising candidate as an antibiotic alternative in yak farming; however, further in vivo validation in yaks is required to confirm its practical applicability under field conditions. Keywords: acid tolerance, antibiotic alternative, Enterococcus faecium, high-altitude adaptation, intestinal adhesion, mucosal immunity, probiotic potential, yak yogurt.