Evaluation of Limosilactobacillus reuteri ATCC PTA 6127 Reveals Multilayered Antimicrobial and Epithelial Barrier-Supportive Effects in a Canine Epithelial Model

Good canine gastrointestinal health depends on the suppression of enteric pathogens and maintenance of epithelial barrier integrity. Limosilactobacillus reuteri ATCC PTA 6127 (Lr6127) is a dog-derived probiotic, but evidence supporting its functional properties remains limited. Here, we evaluated the antimicrobial and epithelial-supportive effects of Lr6127 using a canine epithelial cell model. Cell-free supernatant (CFS) from Lr6127 significantly inhibited the growth of canine-relevant pathogens, including Enterotoxigenic Escherichia coli (52.0 ± 1.3%), Clostridium perfringens (54.0 ± 2.7%), and Salmonella enterica subsp. enterica serovar Typhimurium (48.6 ± 1.2%), compared with the medium control (p < 0.0001). Pathogen inhibition increased in a dose-dependent manner with increasing CFS concentration. Untargeted metabolomic analysis revealed enrichment of multiple antimicrobial-associated metabolites, indicating a multi-component profile consistent with pathogen suppression, with genomic analysis supporting the aromatic amino acid-derived metabolite findings. In addition, viable Lr6127 significantly reduced the epithelial adhesion of all the tested pathogens (p < 0.01). Beyond direct antimicrobial effects, Lr6127 CFS promoted epithelial wound healing at later time points, accompanied by the coordinated modulation of proteins associated with cytoskeletal remodeling and barrier repair. Collectively, these findings support the idea that Lr6127 is associated with antimicrobial and epithelial-related effects, highlighting its potential to contribute to epithelial function under controlled in vitro conditions.