Citrobacter freundii induced pathogenicity towards fish: an insight investigation through serum biochemistry and gene expression analysis in Labeo rohita

Citrobacter freundii, a frequent freshwater fish pathogen leads to considerable economic losses in the global aquaculture industry. In this study, the virulence property of C. freundii (NCBI accession number: PP112336) was done through in vitro and in vivo approaches. Scanning electron microscopy revealed that it is an elongated, rod-shaped bacterium having high proficiency in biofilm production. The results of biochemical characterization exhibited high similarities with previously reported biochemical characteristics of C. freundii. Antibiotic susceptibility test highlighted that the isolate has a multiple antibiotic resistance capability (MAR index: 0.25). Haemolytic activity, biofilm assay, swimming motility, and cytotoxicity study indicated its highly pathogenic capability. Next, in vivo virulence screening was done by injecting pure culture of C. freundii in healthy fingerlings of L. rohita. The 96 h LD₅₀ value of this isolate was found to be approximately 1.2 × 10⁴ CFU/fish. Further, 100 times lower sublethal dose to LD₅₀ (1.2 × 10² CFU/fish) of the isolate was administrated in fingerlings to evaluate immunomodulatory effects on the host. Immune and stress-response biomarkers, such as SOD, IgM, and catalase levels were significantly decreased, while cortisol, HSP70, and HSP90 levels were significantly elevated with post injection duration in infected fish. The analysis of mRNA expression of immune-related genes, including TLR4, TNF-α, HSP70, C3, NOD1, and IL-1β showed significant alternations in the liver, kidney, and skin tissues of injected fingerlings over the infection duration. These changes indicated the response of fish to counteract the immunological damage caused by bacterial invasion. This study offers valuable insights to understand the infection process as well as in the development of therapies to control the infection.

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