Isolation, identification and the pathogenicity characterization of Pseudomonas putida 1C3 and its activation on immune responses in Japanese flounder (Paralichthys olivaceus).

The outbreak of mass mortality of Japanese flounder occurred in an aquaculture farm in Hebei province of China. This study isolated and identified Pseudomonas putida as the dominant bacterium from diseased Japanese flounder (Paralichthys olivaceus) based on morphological, physiological, biochemical characteristics, 16S rRNA gene sequencing, and whole-genome sequencing. Pathogenicity assessment, histopathological analysis, and host immune response were investigated. Results demonstrated that P. putida was pathogenic, causing acute enteritis and multiple organ damage in infected fish. The median lethal dose (LD50) was determined as 2.66 × 106 CFU/g. Transcriptome analysis of the spleen at three post-infection timepoints revealed a robust immune response, with significantly upregulation of immune pathways and downregulation of metabolic functions. Key cytokines (il-1β, il-6, tnf, il-8, il-12, cxcl10, ccl2) were significantly upregulated, indicating intense immune activation. Notably, the P. putida strain exhibited a multidrug-resistant phenotype and harbored multiple drug resistance genes and virulence factors. This is the first report linking P. putida to disease in P. olivaceus, comprehensively elucidating its causative role and the host immune response in Japanese flounder culture.