Isolation, identification, drug resistance and histopathology of Citrobacter freundii from bullfrog (Rana catesbeiana) in Guangxi

This study was conducted to investigate the cause of a disease outbreak of bullfrog (Rana catesbeiana) at a breeding base in Nanning, Guangxi in May 2022. The dominant bacterial colonies were isolated from the liver, spleen, kidney and intestine tissues of diseased R. catesbeiana, and identified using morphological observation, physiological and biochemical experiments, 16S rRNA sequencing and phylogenetic analysis. To determine the pathogenic mechanism of these dominant bacteria, artificial infection experiments, histopathological observations, drug sensitivity experiments, virulence gene and resistance gene testing were conducted. The results showed that a single dominant strain was isolated from the liver, spleen, kidneys, and intestines of the critically ill R. catesbeiana. The dominant strain isolated from the intestine was named NFCF-02 and identified as Citrobacter freundii. This strain carried six virulence genes, including viaB, ompX, ureE, ureD, ureG, ureF, with a half-lethal concentration of 3.12×10⁶ CFU/mL. The R. catesbeiana infected with C. freundii presented the symptoms of liver changing to yellow and necrosis, kidney congestion and redness; blood streaks on the surface of the stomach; and blood pus in intestines. Histopathological observation revealed degeneration and necrosis of liver cells, with disordered arrangement of liver cords. Meanwhile swelling and necrosis of renal tubular epithelial cells, severe tubular collapse and necrosis in severe areas, and splenic central vein dilation and congestion with an increase in hemosiderin and pigment cells was also appeared. Besides, intestinal mucosal detachment, necrosis and detachment of intestinal epithelial cells, and necrosis of goblet cells were also the symptoms in R. catesbeiana infected with C. freundii. C. freundii was sensitive to neomycin, polymyxin B, cefradine, and doxycycline, which has developed resistance to 14 antibiotics, including lincomycin, and carries 7 resistance genes such as gyrA and Sul1. In summary, C. freundii has high pathogenicity to bullfrogs, causing pathological damage to multiple organ tissues even death of the R. catesbeiana. This study is the first to determine the pathogenicity of C. freundii to R. catesbeiana from the perspective of histopathological analysis and virulence factor carrying, which can provide reference for the diagnosis and drug control of C. freundii disease in farmed R. catesbeiana.