Honokiol potentiates low-dose sulfamonomethoxine sodium against antimicrobial-resistant AHPND-causing Vibrio parahaemolyticus through bacterial membrane disruption and host recovery.

Acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio parahaemolyticus, poses a severe threat to global shrimp aquaculture. Reduced susceptibility of aquaculture-associated bacterial pathogens to conventional antibiotics, together with concerns regarding antimicrobial input and environmental exposure, highlights the need for dose-sparing therapeutic strategies. This study investigated the antibiotic-potentiating activity of honokiol (HKL), a plant-derived lignan, in combination with sulfamonomethoxine sodium (SMM-Na) against AHPND. Checkerboard and time-kill assays demonstrated that sub-inhibitory HKL concentrations (32-64 μg/mL) markedly enhanced SMM-Na activity, reducing its minimum inhibitory concentration by 8-32-fold and achieving bactericidal effects in combination. The SMM-Na + HKL combination also significantly inhibited biofilm formation and compromised bacterial membrane integrity, as evidenced by enhanced propidium iodide uptake and scanning electron microscopy, while inducing severe ATP depletion. In Penaeus vannamei challenged with V. parahaemolyticus, dietary administration of low-dose SMM-Na (16 mg/kg) and HKL (32 mg/kg) yielded the highest survival rate (86.6%), substantially reduced hepatopancreatic bacterial load, attenuated histopathological damage, and restored total hemocyte counts. The treatment also upregulated a broad spectrum of immune-related genes (e.g., Alf, Tlr, Lec, Crustin, Lzm, CatB) in the hepatopancreas and mucosal barrier genes (e.g., Muc-1, Muc-4, Muc-5AC, Muc-19) in the intestine. Furthermore, 16S rRNA sequencing indicated that the combination therapy was associated with treatment-related shifts in the intestine microbiota, including increased alpha diversity indices, reduced Vibrio abundance, and altered predicted KEGG functional profiles. Collectively, our results identify HKL as a natural-origin antibiotic potentiator that enhances the efficacy of low-dose SMM-Na against AHPND-causing V. parahaemolyticus. These findings provide proof-of-concept evidence for a dose-sparing combination strategy that may reduce therapeutic SMM-Na input while supporting pathogen control and host recovery in shrimp aquaculture.