Aeromonas hydrophila is a ubiquitous Gram-negative opportunistic pathogen in the freshwater environment and the most common cause of bacterial septicemia in aquaculture. In this study, we investigated the impact of carvacrol, a natural monoterpenoid found in herbs, on the virulence of A. hydrophila in vitro and the antibacterial effect in combination with antibiotics. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of carvacrol against A. hydrophila NJ-35 were 125 µg/mL and 250 µg/mL, respectively. Carvacrol could inhibit the virulence factors (biofilm, protease, exopolysaccharide, and hemolysin) of A. hydrophila, and the antibiofilm potential of carvacrol was further verified by microscopic detection. Transcriptional analyses showed that the gene expression of flaB, ompA, aha, ahp, ela, act, aerA, AhyR, and hly were marked as downregulated. The checkerboard assay results showed that carvacrol did not have an antagonistic effect in combination with antibiotics (florfenicol, enrofloxacin, thiamphenicol, or doxycycline hydrochloride) commonly used in aquaculture but possessed an additive-synergistic effect with neomycin sulfate. In vivo studies demonstrated that carvacrol protected grass carp (Ctenopharyngodon idella) from A. hydrophila infection. Our results indicated that carvacrol possessed significant anti-bacterial and anti-virulence effects on A. hydrophila.

Bacterial quorum sensing (QS) plays a crucial role in chemical communication between bacteria involving autoinducers and receptors and controls the production of virulence factors in bacteria. Therefore, reducing the concentration of signaling molecules in QS is an effective strategy for mitigating the virulence of pathogenic bacteria. In this study, we demonstrated that carvacrol at 15.625 μg/mL (1/4 MIC), a natural compound found in plants, exhibits potent inhibitory activity against QS in Chromobacterium violaceum, as evidenced by a significant reduction (62.46%) in violacein production. Based on its impressive performance, carvacrol was employed as a natural QS inhibitor to suppress the pathogenicity of Aeromonas hydrophila NJ-35. This study revealed a significant reduction (36.01%) in the concentration of N-acyl-homoserine lactones (AHLs), a QS signal molecular secreted by A. hydrophila NJ-35, after 1/4 MIC carvacrol treatment. Moreover, carvacrol was found to down-regulate the expression of ahyR/I, two key genes in the QS system, which further inhibited the QS system of A. hydrophila NJ-35. Finally, based on the above results and molecular docking, we proposed that carvacrol alleviate the pathogenicity of A. hydrophila NJ-35 through QS inhibition. These results suggest that carvacrol could serve as a potential strategy for reducing the virulence of pathogenic bacteria and minimizing the reliance on antibiotics in aquaculture.