Integration of transcriptomics and metabolomics reveals the mechanism of enrofloxacin resistance in Aeromonas schubertii.

Aeromonas schubertii infections has caused severe economic losses in aquaculture in China. In this study, we first induced enrofloxacin (ENR) resistance in A. schubertii strains and then analyzed the mechanisms of drug resistance using transcriptomics and metabolomics. We found that the minimal inhibitory concentration (MIC) was 0.03125 μg/mL for the sensitive strain (WL23S) and 32 μg/mL for the resistant strain (WL23R), which is a 1024-fold increase. After 40 serial passages, the WL23R strain maintained a MIC of 32 μg/mL, even in the absence of ENR-induced stress. Notably, it had also developed resistance to several other antibiotics, such as neomycin sulfate and flumequine. There was no significant difference in the growth rates of the two strains, highlighting the strong adaptability and growth characteristics of the WL23R strain. Comparison of the transcriptome data between the WL23R and WL23S strains identified 579 differentially expressed genes. Expression of the efflux pump-related genes (e.g., acrA, acrB, pstB, pstC, pstS) was significantly upregulated in the WL23R strain (P < 0.05). The highest enrichment of differential genes in the Gene Ontology analysis was in the catabolism of various amino acids, and that in the Kyoto Encyclopedia of Genes and Genomes pathway was in ATP-binding cassette (ABC) transport. Comparison of the metabolomics data between the WL23R and WL23S strains revealed 1, 059 differentially expressed metabolites. Metabolomics analysis revealed the impact of drug resistance on the levels of amino acids, the activity of amino acid biosynthesis/metabolism pathways, and the ABC transport protein pathway, which confirmed the transcriptomics results. The joint analysis results showed that ABC transporters were most prominent in the shared pathways between enriched differentially expressed genes and metabolites. To further validate the resistance mechanism of A. schubertii, we exposed the WL23R strain to the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone. The minimal inhibitory concentration of the induced resistant strain decreased by 4-fold after the addition of the inhibitor, indicating the overexpression of active efflux pumps in WL23R. Our results indicate that the efflux system and ABC transporters play crucial roles during the development of multidrug resistance in A. schubertii. This study will serve as an important reference for understanding bacterial resistance to quinolones and multidrug resistance in aquatic environments.