Multifunctional antimicrobial effects of Lactobacillus johnsonii against A/E pathogens Enteropathogenic E. coli and Citrobacter rodentium.

INTRODUCTION

Enteropathogenic Escherichia coli (EPEC) remains a leading cause of childhood diarrhea in low-resource settings, and escalating antimicrobial resistance necessitates non-antibiotic therapeutic approaches. This study investigates Lactobacillus johnsonii as a probiotic candidate capable of limiting A/E-pathogen colonization and attenuating infection-associated intestinal inflammation.

METHODS

The probiotic properties of L. johnsonii were evaluated through assays of gastrointestinal tolerance, epithelial adhesion, antimicrobial activity, biofilm inhibition, and pathogen exclusion. Secreted antimicrobial activity was investigated by fractionation and untargeted metabolomic profiling. Therapeutic efficacy was further assessed in an antibiotic-perturbed Citrobacter rodentium mouse infection.

RESULTS

L. johnsonii exhibited robust gastrointestinal resilience (acid pH 1.5-2.5; 0.3% bile) and strong adhesion to human intestinal epithelial cells. In vitro, live L. johnsonii markedly inhibited EPEC growth, disrupted pre-formed biofilms, and displaced adherent pathogens from epithelial surfaces. In an antibiotic-perturbed Citrobacter rodentium infection model, oral L. johnsonii administration reduced pathogen loads in feces and colonic tissues by 3-4 log units, restored colon length, and alleviated epithelial ulceration and inflammatory infiltration. Mechanistic analyses revealed dual antimicrobial actions: nutrient competition and secretion of low-molecular-weight (<75 kDa) bactericidal factors active at ~30 µg mL-1. Untargeted metabolomic profiling of the active fraction generated putative annotations of chemically diverse small molecules, including fatty-acid and hydroxy-acid class compounds, as well as candidate metabolites such as quinine hydrochloride, aloperine, and γ-glutamylglutamine, thereby providing a foundation for future targeted validation of individual antimicrobial components. Notably, probiotic treatment reduced mucosal neutrophil infiltration and preserved epithelial architecture, suggesting attenuation of infection-associated inflammation.

DISCUSSION: Collectively, these findings support L. johnsonii as a multifunctional probiotic that integrates biofilm disruption, metabolic competition, and immune-protective activity. The study highlights its translational potential as a probiotic-based intervention to manage attaching-and-effacing enteric infections and mitigate antibiotic reliance in vulnerable populations.