The intense use of antibiotics in human and veterinary medicine, and food production has resulted in significant public health problems including the emergence and spread of antibiotic-resistant pathogens, the presence of drug residues in livestock products (e.g. eggs, meat, and milk), dysbiosis of the gut microbiome, and allergies. Furthermore, with the rising demand for organic and antibiotic-free livestock products, identifying natural, safe, and economical alternatives such as probiotics (live microorganisms that confer health benefits on their host) or their metabolites (e.g. bacteriocins) for sustainable food production and therapeutics is imperative. This project seeks to develop and evaluate novel probiotics and bacteriocins as sustainable alternatives for the biocontrol of pathogens in human and veterinary medicine, and for food/livestock production. Strains of Enterococcus and Staphylococcus spp. from different sources (animals, humans, food, and environment) will be evaluated for inhibitory activities against 14 indicator pathogens (Methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, Methicillin-susceptible S. aureus, Methicillin-resistant S. pseudintermedius, Methicillin-susceptible S. pseudintermedius, Enterococcus faecium, Micrococcus luteus, S. sciuri, S. delphini, S. lugdunensis, Streptococcus suis, Escherichia coli, and Listeria monocytogenes). The cell-free supernatant (CFS) enterococcal and staphylococcal strains showing antimicrobial activity against the indicator pathogens would be evaluated for the production of antimicrobial substances including bacteriocin, organic acids, and hydrogen peroxide. Bacteriocin-producing strains would be further characterized for their carriage of known bacteriocin genes using PCR as well as whole genome sequencing (WGS) to determine their bacteriocin-encoding genes. Crude bacteriocins will be extracted from the producing strains purified, characterized, and identified using high throughput membrane dialysis tubing and lyophilization, solid phase extraction (SPE) on reverse-phase cartridges, MALDI-TOF mass spectrometry, and ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS). Furthermore, bacteriocin-producing strains will be evaluated for probiotic/ protective cultures properties including survivability in simulated gastric juice, adhesion to ileum cells, cell surface properties (auto and co-aggregation, hydrophobicity), tolerance to bile salts and phenol. The safety of both the bacteriocins and producing strains, especially haemolytic activity, cytotoxicity, and virulence will be evaluated. Finally, the identified bacteriocins and probiotic/protective culture potential in the downregulation of the expressions of antibiotic-resistant, biofilms forming and virulence genes would be carried out using qPCR and shotgun metagenomics. This project will lead to the discovery and identification of novel bacteriocins and probiotics/protective cultures for application as suitable alternatives to antibiotics in the control of pathogens in both humans and livestock and for safe and sustainable food production. The findings of the research will be highly significant in the design and planning of future research involving in vivo and lab models of infections as well as large-scale field and clinical trials