Background: Multidrug-resistant (MDR) pathogens pose a major global threat, underscoring the need for plant-derived alternatives. Azadirachta indica (Neem), rich in azadirachtin and nimbolide, offers untapped antibacterial potential.
Methods: Shade-dried Neem leaves were extracted with 80% ethanol (1:10 w/v, Soxhlet, 60 °C, 6 h; yield 14.7%). HPLC (acetonitrile–water 70:30, 1.0 mL/min) identified azadirachtin (1.82 mg/g, Rt 8.3 min) and nimbolide (2.36 mg/g, Rt 11.7 min). Antibacterial activity was assessed against Staphylococcus aureus ATCC 43300, Escherichia coli ATCC 35218, and Pseudomonas aeruginosa ATCC 27853 using broth microdilution (16–1024 µg/mL). Biofilm inhibition was quantified by crystal violet assay, and synergy with ciprofloxacin by checkerboard/FICI. In vivo efficacy was tested in BALB/c mice (n = 48) with MRSA-infected excisional wounds (8 mm, 1×10⁷ CFU), treated with ciprofloxacin (20 mg/kg), Neem extract (100 mg/kg), or both. Environmental assays examined water decontamination and biofilm removal from stainless steel.
Results: MICs were 64 µg/mL (S. aureus), 128 µg/mL (E. coli), and 128 µg/mL (P. aeruginosa), with MBCs 2-fold higher. Neem inhibited biofilm formation by 78%, 62%, and 59% in the respective strains, and eradicated up to 66% of mature biofilms at 2×MIC. Synergy with ciprofloxacin yielded FICI values of 0.38–0.44. In vivo, Neem accelerated wound closure (76% by day 10, complete by day 14) with a 3.1 log₁₀ reduction in MRSA load, while the combination group achieved 90% closure by day 10 and 4.2 log₁₀ bacterial reduction (p < 0.05 vs ciprofloxacin alone). Environmental assays showed >99% bacterial reduction in contaminated water within 2 h (500 µg/mL) and 72% biofilm removal from stainless steel.
Conclusion: A. indica ethanol extract exhibits strong antibacterial, antibiofilm, and synergistic effects against MDR pathogens, confirmed by in vivo wound healing and environmental assays. Neem represents a promising scaffold for plant-derived antibacterial therapeutics and infection-control strategies.
