GC‐MS‐based metabolomics analysis reveals L‐aspartate enhances the antibiotic sensitivity of neomycin sulfate‐resistant Aeromonas hydrophila

: Neomycin sulfate, a kind of drug known as aminoglycoside antibiotic, can be used to treat external or internal bacterial infections. However, there are increasing signs that antibiotics use in aquaculture may have a long-term and permanent potential to select for antibiotic-resistant bacteria in the aquatic environment. In the present study, we aimed to identify key metabolic biomarker whose abundance is correlated with neomycin sulfate resistance in A. hydrophila by gas chromatography-mass spectrometry (GC-MS). Through bioinformatics analysis, L-aspartate was identified as the most crucial biomarker in neomycin sulfate-resistant A. hydrophila and a potential modulator of neomycin sulfate resistance. It was validated as a synergist that incubation of neomycin sulfate-susceptible or -resistant A. hydrophila with exogenous L-aspartate sensitized the bacteria to neomycin sulfate in vitro assay. Moreover, L-aspartate also significantly improved the bactericidal efficacy of neomycin sulfate in Carassius auratus, and increased the survival rate of fish after A. hydrophila challenge. This study presents a novel approach in fighting against antibiotic-resistant pathogens through exploration of antibiotic-resistant biomarkers.