Metabolomic Insights into the Anti-Infective Potential of Azadirachta indica: An In Vivo and In Vitro Investigation

Background: The escalating threat of multidrug-resistant (MDR) pathogens underscores the urgent need for novel anti-infective therapeutics. Azadirachta indica (Neem), a traditional medicinal plant, is rich in bioactive limonoids, quercetin derivatives, and azadirachtin with demonstrated antimicrobial activity. However, its metabolomic signature and translational anti-infective potential remain underexplored.

Methods: A combined in vitro and in vivo approach was employed. Methanolic Neem leaf extract was fractionated by LC-MS/MS–based untargeted metabolomics, identifying key metabolites linked to antimicrobial bioactivity. In vitro susceptibility was assessed against MDR Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa using CLSI-standard broth microdilution assays. Synergy with ciprofloxacin and meropenem was evaluated through checkerboard assays. For in vivo validation, BALB/c mice (n = 48) were challenged intraperitoneally with MDR E. coli. Neem extract nanoparticles (50 mg/kg, oral gavage) were administered daily for 10 days, with ciprofloxacin (20 mg/kg) as positive control.

Results: Metabolomic profiling revealed abundant nimbolide, azadirachtin, and quercetin glycosides as major antimicrobial metabolites. Neem extract demonstrated potent in vitro activity with MICs of 64 µg/mL (E. coli), 32 µg/mL (S. aureus), and 128 µg/mL (P. aeruginosa). Checkerboard assays revealed synergistic interactions with ciprofloxacin (FICI = 0.28) and meropenem (FICI = 0.32). In vivo, Neem-treated mice exhibited a 65% survival rate compared to 25% in untreated controls (p < 0.01). Bacterial load in spleen and liver decreased by >1.8 log CFU, with concomitant reduction in serum TNF-α (↓42%) and IL-6 (↓37%).

Conclusion: Neem demonstrates potent anti-infective efficacy through synergistic interactions with antibiotics, immune modulation, and direct antimicrobial activity. Metabolomics-guided identification of active compounds positions Azadirachta indica as a promising medicinal lead for combating MDR infections.