Antibacterial activity of Ayapana triplinervis against multidrug-resistant Aeromonas in aquaculture

Introduction: Motile Aeromonas septicaemia (MAS), caused by multidrug-resistant (MDR) Aeromonas hydrophila and A. veronii, poses a significant threat to global aquaculture, necessitating sustainable alternatives to antibiotics. Materials and methods: This study characterized MDR Aeromonas isolates from diseased Labeo rohita in West Bengal, India, and evaluated the in vitro antibacterial potential of Ayapana triplinervis ethanol extract. Thirty bacterial isolates were phenotypically characterized, with three MDR isolates selected for molecular identification as A. hydrophila (BAH; 99.86% identity) and A. veronii (RK, RS1; 99.60% and 99.37% identity). Pathogenicity was confirmed through challenge trials, with LD50 values of 1.28 × 106 colony forming unit (CFU)/fish for BAH, 8.9 × 106 CFU/fish for RK, and 1 × 107 CFU/fish for RS1. Gas chromatography-mass spectrometry (GC-MS) analysis of the extract identified 17 phytoconstituents, predominantly β-selinene (17.16%), thymohydroquinone dimethyl ether (12.44%), n-hexadecanoic acid (13.79%), and α-linolenic acid (14.04%). Results: The extract exhibited potent bactericidal activity against all MDR isolates, with minimum inhibitory concentration (MIC) values of 1.0 µl/ml (910 µg/ml) for A. veronii strains and 4.0 µl/ml (3640 µg/ml) for A. hydrophila, as well as minimum bactericidal concentration (MBC) values ranging from 4.0 to 16.0 µl/ml (3640–14,560 µg/ml). The MBC/MIC ratio of three for all strains confirmed bactericidal activity according to established criteria. A comparative analysis with oxytetracycline revealed expected potency differences between the crude extract and purified antibiotic, with species-specific susceptibility patterns observed. Conclusions: This first report on A. triplinervis against piscine MDR pathogens highlights its potential as a source of bioactive compounds for eco-friendly phytotherapeutic development, aligning with sustainable aquaculture and One Health principles. Further bioassay-guided fractionation, mechanistic studies, and in vivo trials are warranted to fully validate its therapeutic application.