Wulandari D.Wisastra S.R.Budiharjo A.Setiarto R.H.B.Soowannayan C.Haditomo A.H.C.Ni'matullah Al-Baarri A.Mahidol University2026-06-062026-06-062026-09-15Aquaculture Vol.624 (2026)00448486https://repository.li.mahidol.ac.th/handle/123456789/117115Acute Hepatopancreatic Necrosis Disease is a shrimp bacterial infection caused by particular isolates of Vibrio spp., especially Vibrio parahaemolyticus and Vibrio harveyi, which carry the pVA1 plasmid responsible for expressing the pirAB toxins. These pathogens initially establish biofilms in the shrimp forestomach before releasing toxins that destroy hepatopancreatic tissue. Therefore, biofilm inhibitors are a promising prevention approach to antibiotics. This study evaluated the biofilm-inhibitory potential of cell-free supernatants (CFS) from four Bacillus strains, Bacillus sp., B. amyloliquefaciens, B. licheniformis, and B. safensis isolated from sea cucumbers (Stichopus sp). Using microtiter-plate assays, each CFS was analyzed for its ability to suppress the growth and biofilm development of AHPND-causing V. parahaemolyticus and V. harveyi, employing in vitro assays on uncoated (growth) and 0.4% chitosan-coated (biofilm) 96-well plates. All four Bacillus CFS can significantly reduce biofilm formation up to 90.4% and 74.9% inhibition for V. parahaemolyticus and V. harveyi, respectively. Results also indicate that the probiotic supernatants may exert quorum-quenching effects by reducing AI-2 synthesis and activity. Two out of four bacteria isolates were used for in vivo study by mixing it with feed by 1:1 ratio. Shrimp were given either CFS supplemented test feeds or buffer-supplemented control feed for seven days prior to the immersion challenge with the respective pathogens at 10⁶ CFU/mL. The result showed that the addition of Bacillus CFS can reduce shrimp mortality when infected with V. parahaemolyticus and V. harveyi. Confirmatory histology also showed that it reduces epithelial cells sloughing into the shrimp's hepatopancreas lumen.Agricultural and Biological SciencesArticleSCOPUS10.1016/j.aquaculture.2026.7442482-s2.0-105040197106