Enterococcus faecium MBBL3 Exhibits Promising Probiotic Potential and Antimicrobial Efficacy Against Bovine Mastitis-Associated Escherichia coli and Klebsiella pneumoniae

Enterococcus faecium, a promising probiotic, combats pathogens, supports gut health, strengthens immunity, and provides a natural approach to address the escalating global challenge of antimicrobial resistance. This study aimed to investigate the genome of E. faecium MBBL3, isolated from healthy cow milk, to assess its probiotic potential and antimicrobial activity against pathogens causing bovine mastitis. The strain was analyzed through whole genome sequencing, along with in-vitro and in-silico assessments were conducted to determine its antimicrobial efficacy against bovine mastitis pathogens, Klebsiella pneumoniae MBBL2 (Kp MBBL2) and Escherichia coli MBBL4 (Ec MBBL4). The genome assembly and functional annotations uncovered many important probiotic traits in MBBL3, where genome comparison revealed its high genetic similarity with other Enterococcus strains. MBBL3 demonstrated the ability to ferment a wide range of carbohydrates and possessed 76 carbohydrate-active enzyme-related genes, including five key CAZy families namely GH73, GH18, CBM50, CE4, and AA10. It also possessed importance genes for bile salt and acid tolerance, stress resistance, and surface adhesion. Additionally, MBBL3 contained metabolite regions involved in the biosynthesis of antimicrobial compounds such as 2,4-DAPG, aborycin, enterocin NKR-5-3B, and sodorifen, and bacteriocin gene clusters for sactipeptides, Enterolysin_A, and UviB. Safety assessments indicated low pathogenic potential, while in-vitro assays demonstrated antibiotic susceptibility and suppressed the growth of Kp MBBL2 and Ec MBBL4, respectively. Its bacteriocin compound Enterolysin_A exhibited strong molecular interactions with virulence proteins of these mastitis pathogens. Therefore, the promising probiotic potential and antimicrobial efficacy of E. faecium MBBL3, especially against mastitis pathogens combined with its safety, position it as a valuable candidate for therapeutic applications. Key pointsO_LIE. faecium MBBL3 genosme showed high similarity with other species of this genera. C_LIO_LIGenetic makeup of MBBL3 revealed its ability to survival and adaptation in different niches including hosts gut. C_LIO_LIIn-vitro and in-silico study results, along with several genes linked to antimicrobials demonstrated its ability to combat against mastitis pathogens. C_LI