Isolation and characterization of probiotic candidates with antimicrobial and barrier-protective properties

Antimicrobial resistance (AMR) in pathogens such as multidrug-resistant Salmonella Typhimurium and extended-spectrum β-lactamase (ESBL) Escherichia coli poses a growing threat to human, animal, and environmental health, yet effective non-antibiotic mitigation strategies remain limited. Here, we report the isolation and comprehensive characterization of three lactic acid bacteria from homemade cottage cheese, including Leuconostoc pseudomesenteroides MLS3, Lactococcus lactis MLS12, and Leuconostoc lactis MLS22 as next-generation probiotic candidates targeting these AMR pathogens. Whole-genome sequencing confirmed compact genomes (1.7-2.5 Mb) free of transferable resistance determinants and enriched in secondary metabolite biosynthetic clusters. All three strains survived gastrointestinal challenge conditions (≥ 6 log CFU/mL at pH 1.5, 2% bile salts, and 100 µg/mL lysozyme), exhibited strong mucosal adhesion parameters (auto-aggregation 30-33%, surface hydrophobicity 20-49%), and co-aggregated efficiently with both pathogens (27-64%). Live co-cultures and cell-free supernatants significantly inhibited S. Typhimurium and ESBL E. coli within 24 h, with electron microscopy revealing probiotic-induced structural disruption of pathogen cell integrity. Probiotic pretreatment extended Caenorhabditis elegans lifespan under Salmonella challenge (P < 0.001), and cell-free supernatants attenuated LPS-induced epithelial barrier permeability in chicken enteroids (P < 0.05) without affecting baseline barrier function. Together, these findings demonstrate that MLS3, MLS12, and MLS22 combine genomic safety, gastrointestinal resilience, direct antimicrobial potency, and epithelial barrier protection, supporting their potential as effective probiotics for AMR-associated applications in food safety and gut health.