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

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.