Vibrio alginolyticus is a waterborne pathogen that infects a wide variety of hosts including fish and human, and the outbreak of this pathogen can cause a huge economic loss in aquaculture. Thus, enhancing host's capability to survive from V. alginolyticus infection is key to fighting infection and this remains still unexplored. In the present study, we established a V. alginolyticus-zebrafish interaction model by which we explored how zebrafish survived from V. alginolyticus infection. We used GC-MS based metabolomic approaches to characterize differential metabolomes between survival and dying zebrafish upon infection. Pattern recognition analysis identified the TCA cycle as the most impacted pathway. The metabolites in the TCA cycle were decreased in the dying host, whereas the metabolites were increased in the survival host. Furthermore, the enzymatic activities of the TCA cycle including pyruvate dehydrogenase (PDH), α-ketoglutaric dehydrogenase (KGDH) and succinate dehydrogenase (SDH) also supported this conclusion. Among the increased metabolites in the TCA cycle, malic acid was the most crucial biomarker for fish survival. Indeed, exogenous malate promoted zebrafish survival in a dose-dependent manner. The corresponding activities of KGDH and SDH were also increased. These results indicate that the TCA cycle is a key pathway responsible for the survival or death in response to infection caused by V. alginolyticus, and highlight the way on development of metabolic modulation to control the infection.

Summary The development of antibiotic resistance in Vibrio alginolyticus represents a threat to human health and fish farming. Environmental NaCl regulation of bacterial physiology is well documented, but whether the regulation contributes to antibiotic resistance remains unknown. To explore this, we compared minimum inhibitory concentration (MIC) of V. alginolyticus cultured in different media with 0.5%–10% NaCl, and found that the MIC increased as the NaCl concentration increased, especially for aminoglycoside antibiotics. Consistent with this finding, internal NaCl also increased, while intracellular gentamicin level decreased. GC–MS‐based metabolomics showed different distributions of pyruvate cycle intermediates among 0.5%, 4% and 10% NaCl. Differential activity of enzymes in the pyruvate cycle and altered expression of Na(+)‐NQR led to a reducing redox state, characterized by decreased levels of NADH, proton motive force (PMF) and ATP. Meanwhile, NaCl negatively regulated PMF as a consequence of the reducing redox state. These together are responsible for the decreased intracellular gentamicin level with the increased external level of NaCl. Our study reveals a previously unknown redox state‐dependent mechanism regulated by NaCl in V. alginolyticus that impacts antibiotic resistance.

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