Secretome-Mediated Antimicrobial and Immunomodulatory Activity of Lactobacillus johnsonii Against Multidrug-Resistant Enteroaggregative Escherichia coli

Abstract Background: Enteroaggregative Escherichia coli (EAEC) is a leading cause of persistent diarrhea in children in low- and middle-income countries, and the emergence of multidrug-resistant (MDR) strains necessitates non-antibiotic therapeutic strategies. This study evaluated Lactobacillus johnsonii , previously characterized by our group, as a probiotic candidate against a clinically confirmed MDR EAEC isolate resistant to ampicillin, ciprofloxacin, azithromycin, amoxicillin, and gentamicin. Methods: Probiotic properties of L. johnsonii were assessed through gastrointestinal stress tolerance, cell surface hydrophobicity, phenol tolerance, and autoaggregation assays. Antimicrobial and anti-biofilm activity against MDR EAEC were evaluated using agar overlay and crystal violet biofilm assays. Epithelial interaction was assessed through adherence and protection assays on HCT-116 intestinal epithelial cells. Gas production suppression, macrophage nitric oxide modulation in RAW 264.7 cells, and nutrient competition were examined as mechanistic endpoints. The L. johnsonii cell-free supernatant was fractionated by fast protein liquid chromatography (FPLC) and active fractions confirmed by Gram staining. Statistical analyses were performed using unpaired Student's t-test, one-way ANOVA, or two-way ANOVA with Dunnett's multiple comparisons test. Results: L. johnsonii demonstrated robust gastrointestinal resilience, high cell surface hydrophobicity, and rapid autoaggregation reaching 80.4 ± 2.3% by 4 hours, collectively supporting mucosal colonization potential. It produced zones of inhibition against MDR EAEC substantially exceeding those of gentamicin, reduced viable biofilm-associated EAEC by over 80%, and displaced pre-adhered EAEC from intestinal epithelial cells in a time-dependent manner requiring prior epithelial establishment. L. johnsonii attenuated EAEC-induced gas production and reduced macrophage nitric oxide levels by 67.7%. Inhibition was predominantly secretome-dependent, as nutrient competition did not contribute to pathogen suppression. FPLC fractionation identified fractions S5 and S6 as the principal bioactive components with sustained bactericidal activity at 6 hours, with S6 exhibiting greater killing potency than S5. Conclusions: L. johnsonii exerts potent secretome-mediated antimicrobial and immunomodulatory activity against MDR EAEC in vitro through multiple complementary mechanisms, supporting its potential as a non-antibiotic biotherapeutic strategy for antibiotic-resistant enteric infections. In vivo validation and molecular characterization of the active fractions are important next steps.