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

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 evaluates 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. L. johnsonii demonstrated robust gastrointestinal resilience, high cell surface hydrophobicity, phenol tolerance, and rapid autoaggregation reaching 80.4 ± 2.3% by 4 hours, collectively supporting mucosal colonization potential. In antimicrobial assays, L. johnsonii 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 HCT-116 intestinal epithelial cells in a time-dependent manner. L. johnsonii also attenuated MDR EAEC-induced gas production and reduced nitric oxide levels by 67.7% in infected RAW 264.7 macrophages, suggesting immunomodulatory activity. Nutrient competition did not appear to contribute to EAEC suppression under tested conditions, indicating inhibition is predominantly secretome-dependent. Fractionation of the L. johnsonii cell-free supernatant by fast protein liquid chromatography yielded five fractions below 75 kDa; fractions S5 and S6 exhibited sustained bactericidal activity at 6 hours. Gram staining confirmed that both fractions reduced viable EAEC cell numbers, with S6 producing a greater reduction than S5, indicating quantitatively distinct bactericidal potencies. These in vitro findings support the potential of L. johnsonii as a biotherapeutic candidate for antibiotic-resistant enteric infections. In vivo validation and chemical characterization of active fractions remain important next steps.