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

Abstract

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 {micro}g 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. Author SummaryDiarrheal disease still harms millions of children, and growing antibiotic resistance makes treatment harder. We asked whether a friendly gut bacterium, Lactobacillus johnsonii, could protect against harmful microbes without relying on antibiotics. First, we tested simple but key questions: can this probiotic survive the harsh journey through the stomach and small intestine, can it attach to the gut lining, and can it push back against disease-causing bacteria? We found that Lactobacillus johnsonii survives strong acid and bile and sticks well to human intestinal cells. In lab dishes, live cells slowed pathogen growth, broke up their protective biofilms (the sticky layers that help microbes persist), and even dislodged bacteria that had already attached to cells. We then moved to a mouse model of diarrheal infection using Citrobacter rodentium, which mimics important features of human disease. Giving Lactobacillus johnsonii by mouth lowered bacterial counts in the gut and tissues, reduced tissue damage, and improved overall colon health. Finally, we explored how it works. We found two main actions: the probiotic competes with pathogens for nutrients, and it releases small natural compounds that can directly kill them. Together, these results support Lactobacillus johnsonii as a practical, affordable, non-antibiotic option that merits careful testing in people, especially in communities with the greatest burden of diarrheal disease. Graphical AbstractSchematic overview of the study design and key findings. A high-resolution image and legend are provided. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/682759v1_figa1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@21f059org.highwire.dtl.DTLVardef@2a7bbforg.highwire.dtl.DTLVardef@1223abcorg.highwire.dtl.DTLVardef@3e0d38_HPS_FORMAT_FIGEXP M_FIG Mechanism of Lactobacillus johnsonii-mediated protection against EPEC and Citrobacter rodentium in the gut Left (Pathogenic Effects): Colonization by EPEC or C. rodentium disrupts the intestinal mucus barrier, induces attaching and effacing (A/E) lesions on epithelial cells, and triggers pro-inflammatory cytokine release. These events promote biofilm formation, immune cell infiltration (including dendritic cells, macrophages, neutrophils, and T cells), and epithelial ulceration, ultimately exacerbating intestinal inflammation. Right (Protective Effects): L. johnsonii counteracts these pathogenic effects through secretion of antimicrobial metabolites, competition for nutrients, and enhanced mucosal adherence. Collectively, these mechanisms inhibit biofilm formation, reduce pathogen burden, and maintain epithelial barrier integrity. C_FIG