Study on the effects and mechanisms of the antagonistic bacterium Bacillus subtilis JSHY-K3 against Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease in shrimp Penaeus vannamei

Acute hepatopancreatic necrosis disease (AHPND), caused by certain strains of Vibrio, has resulted in substantial economic losses in global shrimp industries. Hence, developing effective and sustainable alternatives to antibiotics to control this disease is of profound importance. In this study, we isolated a strain of antagonistic bacterium, JSHY-K3, from shrimp pond sediment, evaluated its efficacy in preventing AHPND in shrimp and tentatively investigated its mechanism of defence and control against AHPND. The isolated strain exhibited strong antagonistic activity against Vibrio parahaemolyticus, the etiological agent of AHPND (VpAHPND) harboring toxin genes pirA and pirB. JSHY-K3 was identified as B. subtilis based on its morphological, physiological, biochemical characteristics and 16 S rDNA sequence analysis. In vivo challenge assays demonstrated that JSHY-K3 could significantly mitigate cumulative mortality of shrimps infected with VpAHPND, suppress the expression of VpAHPND virulence genes and modulate shrimp immune responses. To elucidate the mechanistic basis of JSHY-K3 in preventing and controlling AHPND, whole-genome sequencing and untargeted metabolomics-LCMS-EMDB analyses were conducted. Whole-genome analysis revealed 12 gene clusters involved in synthesizing of secondary metabolites including the bacteriostatic agents surfactin, aurantinin B/C/D, fengycin, sublancin 168, bacillibactin, subtilosin A and bacilysin. Untargeted metabolomics uncovered 3691 known metabolites, with caffeic acid, fosfomycin, minocycline and surfactin A documented as V. parahaemolyticus growth inhibitors. We hypothesize that the production of these bacteriostatic metabolites by JSHY-K3 underlies its antagonistic effects against VpAHPND. Collectively, these findings suggest B. subtilis JSHY-K3 could be applied as a probiotic to prevent AHPND in shrimp aquaculture.