Research Projects

Rationale: Persistent diarrhea caused by Enteroaggregative Escherichia coli (EAEC) remains a significant public health concern in developing countries, including India, where it predominantly affects children and immunocompromised individuals. The emergence of multidrug-resistant (MDR) EAEC strains resistant to ciprofloxacin, ampicillin, and cotrimoxazole has narrowed treatment options. EAEC’s ability to form drug-tolerant biofilms, resist host clearance, and persist in the gut calls for non-antibiotic, microbiota-safe alternatives. Probiotic-based strategies are promising but understudied against MDR EAEC, especially in India. Preliminary data from our lab highlight Lactobacillus johnsonii (L. johnsonii) as a strong probiotic candidate with high acid/bile resistance, aggregation ability, and epithelial adhesion. It significantly inhibits MDR EAEC growth, disrupts biofilms, and competitively excludes the pathogen in vitro. Its cell-free supernatant (CFS) and <75 kDa FPLC-purified fractions show potent bactericidal effects, suggesting the presence of novel antimicrobial molecules. Scientific Objectives: 1. To evaluate the in vitro antimicrobial, anti-biofilm, and competitive exclusion activities of L. johnsonii against MDR EAEC. 2. To identify and characterize active antimicrobial fractions from L. johnsonii supernatant using FPLC. 3. To validate the therapeutic efficacy of Lactobacillus johnsonii in a preclinical murine model of EAEC-induced diarrhea. 4. To assess microbiome shifts, metabolite profiles, and mucus barrier integrity following L. johnsonii treatment in EAEC-infected mice. Hypothesis: We propose that L. johnsonii functions through antimicrobial secretion, biofilm interference, and gut barrier restoration to combat MDR EAEC Experimental Approach: For Objective 1: Perform co-culture, overlay inhibition, and adhesion assays to assess in vitro antimicrobial and anti-biofilm effects. For Objective 2: Fractionate CFS using FPLC, identify bioactive fractions via SDS-PAGE and LC-MS/MS. For Objective 3: Establish murine MDR EAEC infection model; assess treatment effects on pathogen load, FITC-dextran leakage, tight junction proteins, cytokines (IL-6, IL-8, TNF-α), and histopathology. For Objective 4: Analyze microbiota (16S rRNA sequencing), metabolites (SCFA/TMAO) and Mucus secreting factor (Muc2) to assess gut restoration. Expected Significance: This will be the first detailed preclinical evaluation of L. johnsonii against MDR EAEC. Key outcomes include: • Insight into probiotic-pathogen interactions • Identification of novel bioactives • A murine model for probiotic efficacy testing The project aligns with India’s AMR goals and supports scalable, microbiota-safe therapies for MDR diarrhea.