Evaluation of Lethal Dose, Minimum Inhibitory Concentration and Antibacterial Properties of Herbal Extracts against Aeromonas hydrophila

Aeromonas hydrophila is a major bacterial pathogen that causes significant economic losses in freshwater aquaculture, highlighting the need for alternatives to conventional antibiotics. This study evaluated the antibacterial activity of neem (Azadirachta indica), tulsi (Ocimum sanctum), and their combined formulations against A. hydrophila using agar well diffusion and resazurin-based broth microdilution assays with aqueous and ethanolic extracts, as well as an in vivo assessment of pathogen virulence in common carp (Cyprinus carpio). All herbal preparations exhibited inhibitory activity, with the combined 5% neem + 5% tulsi aqueous extract producing the largest inhibition zone (14.3 ± 0.3 mm), while the corresponding ethanolic formulation showed significantly greater activity (20.6 ± 0.3 mm), although remaining less potent than oxytetracycline. Minimum inhibitory concentration analysis revealed substantially lower MIC values for ethanolic extracts (0.0391% for neem, 0.0195% for tulsi, and 0.0391% for the combined formulation) compared with aqueous extracts (0.625% for individual herbs and 0.0391% for the aqueous combination), indicating enhanced efficacy of ethanol-extracted phytoconstituents and synergistic effects in combined treatments. Pathogenicity trials demonstrated a dose-dependent mortality response in common carp, with mortality reaching ~60% at 1.0 × 10⁸ cfu/ml and declining sharply at lower doses. The LD₅₀ was estimated to be between 1.0 × 10⁷ and 1.0 × 10⁸ cfu/ml, confirming high pathogen virulence. Overall, neem–tulsi formulations, particularly ethanolic extracts, show promise as phytotherapeutic agents for sustainable aquaculture disease management.