Discovery of Citrobacter freundii as a disease-causing agent in Singhi Heteropneustes fossilis : A first report

Objective The purpose of this study was to identify and describe Citrobacter freundii, which was associated with a disease outbreak in Singhi (also known as Stinging Catfish) Heteropneustes fossilis that are cultured under intensive freshwater farming conditions in Tripura, India, and to evaluate its pathogenicity and antimicrobial resistance profile. Methods A bacterial isolate (COF_AHE25; GenBank accession no. ON798627) was recovered from diseased fish that were exhibiting white patches, skin ulcers, hemorrhages, fin rot, and mortality. The isolate was identified using phenotypic and biochemical evaluation, followed by molecular confirmation based on 16S ribosomal RNA gene sequencing and phylogenetic analysis. Pathogenicity was assessed through experimental infection trials, ­daily monitoring of cumulative mortality, and estimation of the median lethal dose for the species. Host responses were evaluated through hematological, biochemical, and immunological analyses. Antimicrobial susceptibility was determined using the Kirby–Bauer disk diffusion method in accordance with the Clinical and Laboratory Standards Institute guidelines. Results Experimental infection confirmed the virulence of the isolate, with infected Singhi developing clinical signs that were comparable to those observed during the natural outbreak. Mortality commenced at 4 d postinfection and progressed in a dose-dependent manner. Significant alterations in hematological, biochemical, and immunological parameters were observed, consistent with systemic bacterial septicemia. The median lethal dose of C. freundii strain COF_AHE25 was estimated at 2.5 × 107 CFU/fish. The isolate exhibited multidrug resistance, including resistance to tetracycline and erythromycin as well as to β-lactam antibiotics (penicillin, oxacillin, and cefoxitin) and vancomycin. Conclusions The findings establish C. freundii as a pathogenic bacterium capable of causing significant disease and mortality in intensively farmed Singhi. The detection of resistance to antimicrobials commonly used in freshwater aquaculture highlights the need for strengthened disease surveillance, responsible antimicrobial stewardship, and improved health management practices in intensive fish farming systems.