Lactobacillus johnsonii Limits Enteropathogenic Eschericia coli and Citrobacter rodentium Through Biofilm Disruption, Nutrient Competition, and Antimicrobial Metabolites

Enteropathogenic Escherichia coli is a major cause of childhood diarrhea in underprivileged regions, and its increasing antibiotic resistance underscores the need for non-antibiotic interventions. This study evaluates Lactobacillus johnsonii as a probiotic candidate to counter enteropathogenic Escherichia coli and its murine surrogate, Citrobacter rodentium. Lactobacillus johnsonii exhibited robust gastrointestinal resilience, tolerating strong acidity and bile salts (0.3%), and showed enhanced adhesion to human intestinal epithelial cells. Across in vitro assays, live Lactobacillus johnsonii inhibited pathogen growth in agar overlay assays more strongly than gentamicin, disrupted biofilms, and displaced adherent enteropathogenic Escherichia coli from epithelial surfaces. In antibiotic-treated mice, oral Lactobacillus johnsonii reduced Citrobacter rodentium burdens in feces, colon, cecum, and spleen by approximately three to four log10 units, mitigated colon shortening, and alleviated histopathological damage, including edema, lymphocyte infiltration, and ulceration. Mechanistic studies revealed complementary modes of action: nutrient competition that reduced pathogen growth by more than fifty percent, and contact-independent killing mediated by secreted, low-molecular-weight factors. Fractionation of cell-free supernatant by fast protein liquid chromatography yielded fractions smaller than seventy-five kilodaltons with potent bactericidal activity; one fraction retained activity for six hours and inhibited enteropathogenic Escherichia coli at 30?µg per mL. Untargeted metabolomic profiling of active fractions identified distinct antimicrobial metabolites, including quinine hydrochloride, aloperine, and gamma-glutamylglutamine, alongside chemical classes such as fatty acyls, hydroxy acid derivatives, and carboxylic acids consistent with membrane-disruptive activity. By integrating biofilm disruption, competitive exclusion, and metabolite-mediated killing with demonstrated efficacy in vivo, Lactobacillus johnsonii emerges as a promising biotherapeutic for managing diarrheal diseases caused by attaching-and-effacing pathogens and merits further characterization of its active small molecules and translational evaluation.