Secretome-dependent antimicrobial and immunomodulatory activity of Lactobacillus johnsonii against multidrug-resistant Enteroaggregative Escherichia coli

Abstract

Abstract 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. 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. L. johnsonii demonstrated robust gastrointestinal resilience, high cell surface hydrophobicity, and rapid autoaggregation reaching 80.4 ± 2.3% by 4 h, 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 h, with S6 exhibiting greater killing potency than S5. L. johnsonii exhibited potent secretome-dependent antimicrobial activity against MDR EAEC in vitro and significantly suppressed macrophage nitric oxide production, indicating its potential to modulate inflammatory responses. These findings support its potential as a non-antibiotic biotherapeutic strategy for antibiotic-resistant enteric infections. However, further in vivo validation and molecular characterization of the active antimicrobial components are required before clinical translation.