The overuse or abuse of antimicrobial drugs in aquaculture, aggravates the generation of drug-resistant bacteria, which has caused potential risks to human health and the aquaculture industry. Flavonoid–antibiotic combinations have been shown to suppress the emergence of resistance in bacteria, and sometimes even reverse it. Here, the antibacterial activity of florfenicol in combination with quercetin, a potential drug to reverse multidrug resistance, was tested against Aeromonas hydrophila (A. hydrophila). Of eleven selected antimicrobial agents, quercetin and florfenicol showed the strongest bactericidal effect, and fractional inhibitory concentration (FIC) indices were 0.28, showing a highly synergistic effect. Then, the antibacterial activities of quercetin and florfenicol against A. hydrophila were further tested in vitro and in vivo. Bacterial viability of A. hydrophila decreased in a florfenicol dose-dependent manner, about 16.3–191.4-fold lower in the presence of 15 μg/mL quercetin and 0.156 to 1.25 μg/mL florfenicol than in the absence of quercetin, respectively. The cell killing was maximum at 45 μg/mL quercetin in the dose range tested plus 0.156 μg/mL florfenicol. The viability decreased over time during the combined treatment with quercetin and florfenicol by 60.5- and 115-fold in 0.156 μg/mL florfenicol and 0.625 μg/mL florfenicol, respectively. Additionally, the synergistic effect was confirmed by the bacterial growth curve. Furthermore, quercetin and florfenicol had an obvious synergistic activity in vivo, reducing the bacterial load in the liver, spleen and kidney tissues of Cyprinus carpio up to 610.6-fold compared with the florfenicol group, and improving the survival rate of infected fish from 10% in the control group to 90% in drug combinations group. These findings indicated that quercetin could potentiate the antibacterial activity of florfenicol against A. hydrophila infection and may reduce the use of antimicrobial drugs and improve the prevention and control capability of bacterial resistance.

Host innate immunity modulated by metabolites is a powerful strategy that can be used to defend against microbial infections. However, the mechanisms underlying the metabolic processes in controlling bacterial infections remain unexplored. Here, a GC-MS based metabolomics approach was adopted to investigate the metabolic characteristics between the surviving and dying Cyprinus carpio upon Aeromonas hydrophila infection.Aspartate was identified as an essential biomarker whose abundance significantly decreased in the dying group while increasing in the surviving group when compared to the control. Exogenous aspartate increased fish survival rate in a dose-dependent manner. Concurrently, we also found that aspartate regulated the expressions of inflammatory and anti-inflammatory factors, and alleviated oxidative damage based on the evaluations of the activities of antioxidant enzymes and histopathology. Interestingly, the results revealed that exogenous aspartate promoted arginine and proline metabolism and arginine biosynthesis, but attenuated the TCA cycle, thereby enhancing the disease resistance against A. hydrophila. We further demonstrated that aspartate induced iNOS expression and NO production, and L-NAME treatment inhibited iNOS expression and blocked NO production, which significantly increased the cumulative mortality of infecting A. hydrophila. These findings indicated that exogenous aspartate could reprogram C. carpio metabolome and improve the host's ability to fight against A. hydrophila infection by inducing of NO release, and demonstrated the metabolitemediated immunomodulatory as a therapeutic intervention for A. hydrophila infection.