Case Reports of Citrobacter freundii Infections in Captive Freshwater Species in South Korea

Citrobacter freundii, an opportunistic Gram-negative bacterium belonging to the Enterobacteriaceae family, is emerging as a significant pathogen in diverse aquatic species, often associated with environmental stress and poor husbandry conditions. This study describes four distinct cases of fatal C. freundii-associated lesions in captive freshwater species in South Korea, including an Ocellate river stingray (Potamotrygon motoro), a Royal peacock bass (Cichla intermedia), a Japanese eel (Anguilla japonica), and a Poison dart frog (Dendrobates leucomelas). Bacterial DNA was analyzed using 16S rRNA PCR and sequence analysis, and FFPE tissues were additionally examined by PCR for molecular identification. Tissues were examined using routine hematoxylin and eosin (H&E) staining for detailed histopathology. Results: Molecular analysis detected C. freundii-associated DNA in the liver, kidney, and intestine of all samples. Histopathology consistently revealed severe bacterial enteritis with epithelial necrosis and abundant intraluminal bacterial aggregates (stingray, frog). Systemic manifestations included severe ulcerative skin lesions (peacock bass), hepatic fatty degeneration (peacock bass, eel), renal tubular degeneration (peacock bass), and prominent hepatic melanomacrophage center activation (frog); however, these findings should be interpreted as lesions observed in association with C. freundii detection, as several of these changes are not specific to bacterial infection. To our knowledge, this study provides one of the first comparative histopathological descriptions of naturally occurring C. freundii-associated cases across multiple captive freshwater vertebrate species in South Korea. Although bacterial culture, biochemical identification, and experimental infection were not performed, this retrospective investigation identified an association between molecular detection of C. freundii and histopathological lesions. Therefore, these findings should be interpreted as supportive diagnostic evidence rather than proof of a definitive causal relationship. These observations highlight the complementary value of histopathological examination alongside molecular detection in the diagnostic evaluation of aquatic animal diseases.