Genomic and In Vitro Analysis of Pediococcus pentosaceus MBBL4 Implicated Its Therapeutic Use Against Mastitis Pathogens and as a Potential Probiotic.

Pediococcus pentosaceus has the potential to be used as probiotics and biologics amid rising trends of global antimicrobial resistance (AMR) and non-communicable diseases. This study analyzed the genome of P. pentosaceus MBBL4, isolated from healthy cow milk, to assess its probiotic properties and antimicrobial efficacy. The strain was subjected to whole genome sequencing (WGS), assembly, and annotations, alongside phylogenetic and comparative genomic analyses. Additionally, carbohydrate utilization, metabolic pathways, genomic safety, and probiotic potential of MBBL4 were assessed. Its in vitro antimicrobial efficacy against mastitis pathogens was also evaluated. The WGS analysis uncovered many important probiotic traits in MBBL4. Phylogenetic analysis demonstrated a close genetic link with other 15 P. pentosaceus strains, sharing more than 99% of core genes within the pan-genome matrix. MBBL4 demonstrated extensive range of carbohydrate metabolism activity, supported by the presence of several genes encoded enzymes, including a complete elucidated lactose metabolism pathway along with 28 additional metabolic pathway modules. Notably, its genome contains regions associated with gallic acid metabolism and related genes. MBBL4 also harbored genes encoding immunity proteins like enterocin A and lactococcin, and antimicrobial compounds including penocin A, lysozymes, laccase, colicin V, and viguiepinol. Comparative analysis with other probiotic strains revealed seven novel exopolysaccharide biosynthesis proteins and one biofilm-related protein. Moreover, MBBL4 remained sensitive to 90% of the tested antibiotics and carried only a single lincosamide resistance gene (lnuA). It effectively inhibited the growth of two important bovine mastitis pathogens, Staphylococcus aureus D4C4 and Escherichia coli G1C5. These results, along with its low pathogenicity score, support the safety profile of MBBL4 and highlight its potential as bioactive natural therapeutic for mastitis and a promising probiotic candidate.