Probiotic potential of lactic acid bacteria isolated from canine and feline microbiota: functional profiling and host-adapted benefits

The global rise in pet ownership has increased demand for health-promoting products, particularly probiotics designed to support gastrointestinal and immune health in companion animals. However, most commercial products rely on non-host-adapted strains, which may limit gastrointestinal colonization and host-specific benefits. To address this gap, 56 bacterial isolates were obtained from the fecal and milk microbiota of clinically healthy dogs and cats. Among these, Limosilactobacillus reuteri DF/KS2, derived from the fecal microbiota of a Kangal Shepherd dog, and Enterococcus faecium CM/BS2 derived from the milk microbiota British Shorthair cat, were selected based on their broad-spectrum antimicrobial activity. Both strains exhibited a safe profile, as evidenced by γ-hemolysis and susceptibility to a panel of clinically relevant antibiotics. Under simulated gastrointestinal conditions, CM/BS2 and DF/KS2 tolerated highly acidic environments and demonstrated resilience against digestive enzymes and bile salts. Furthermore, both isolates displayed strong auto-aggregation and co-aggregation abilities with key pathogens, while adhesion assays using Caco-2 cells confirmed their capacity to inhibit pathogen attachment. Immunomodulatory evaluations further revealed that both strains effectively reduced pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and enhanced anti-inflammatory IL-10 production in canine and feline macrophages. Optimal growth occurred at 37 °C after 24 h in 2% molasses medium, and shelf-life studies demonstrated that freeze-dried cultures retained high viability over six months at - 20 °C. Collectively, these findings highlight the probiotic potential of host-adapted L. reuteri DF/KS2 and E. faecium CM/BS2, emphasizing their suitability for inclusion in species-specific probiotic formulations aimed at supporting gastrointestinal and immune health in dogs and cats.