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.

Stress is an important contributing factor in the outbreak of infectious fish diseases. To comprehensively understand the impact of catecholamine stress hormone norepinephrine (NE) on the pathogenicity of Aeromonas hydrophila, we assessed variations in bacterial growth, virulence‐related genes expression and virulence factors activity after NE addition in serum‐SAPI medium. Further, we assessed the effects of NE on A. hydrophila virulence in vivo by challenging fish with pathogenic strain AH196 and following with or without NE injection. The NE‐associated stimulation of A. hydrophila strain growth was not linear‐dose‐dependent, and only 100 μM, or higher concentrations, could stimulate growth. Real‐time PCR analyses revealed that NE notably changed 13 out of the 16 virulence‐associated genes (e.g. ompW, ahp, aha, ela, ahyR, ompA, and fur) expression, which were all significantly upregulated in A. hydrophila AH196 (p < 0.01). NE could enhance the protease activity, but not affect the lipase activity, hemolysis, and motility. Further, the mortality of crucian carp challenged with A. hydrophila AH196 was significantly higher in the group treated with NE (p < 0.01). Collectively, our results showed that NE enhanced the growth and virulence of pathogenic bacterium A. hydrophila.