Honokiol enhances the antibacterial efficacy of doxycycline against Vibrio parahaemolyticus by membrane disruption and confers protective efficacy in Pacific white shrimp (Penaeus vannamei)

Honokiol (HKL) sensitizes Vibrio parahaemolyticus to doxycycline. • HKL–DOX shows additive interaction and reduces culturable bacteria. • HKL–DOX is associated with membrane perturbation and ATP depletion. • Dietary HKL–DOX improves shrimp survival and lowers bacterial burden. • Host and microbiota changes accompany HKL–DOX-mediated protection. Vibrio parahaemolyticus is a major bacterial pathogen in shrimp aquaculture, and the development of new anti-infective strategies is urgently needed due to limited protective options. In this study, we report that the plant-derived compound honokiol (HKL) acts as an effective adjuvant to doxycycline (DOX) against V. parahaemolyticus . Checkerboard assays revealed an additive interaction between HKL and DOX (FICI = 0.5625), accompanied by a 16-fold reduction in the minimum inhibitory concentration (MIC) of DOX from 64 μg/mL to 4 μg/mL. Plate-count-based time-kill analysis showed that the HKL–DOX combination reduced recoverable culturable bacteria to below the detection limit under the tested plating conditions. Mechanistic investigations indicated that HKL acted as an important membrane-active component of the combination, increasing bacterial membrane permeability and perturbing membrane integrity. Under combination conditions, HKL and DOX induced membrane-associated damage and intracellular ATP depletion. In a shrimp infection model, the HKL-DOX combination therapy significantly improved shrimp survival, reduced bacterial load in the hepatopancreas, partially preserved intestinal and hepatopancreatic histoarchitecture, was accompanied by changes in immune- and mucosal barrier-related gene expression and intestinal microbiota diversity and composition. Overall, HKL acts as an effective antibiotic adjuvant, highlighting a promising combination strategy for controlling V. parahaemolyticus infections in aquaculture.