Isolation, identification and drug resistance analysis of Citrobacter freundii from Micropterus salmoides

【Objective】The California perch(Micropterus salmonoides) broke out with surface hemorrhage and ulceration at the breeding base of an aquatic seedling company in Sanshui, Foshan.The pathogenic bacteria and drug sensitivity were clarified, which provided scientific evidence for the clinical diagnosis and scientific prevention and control of the disease.【Method】The pathogenic bacteria were isolated from the tissues of diseased M.salmonoides, such as the liver, spleen, kidney, by using conventional bacterial isolation methods.After Gram staining, phosphotungstic acid staining, observation of morphology and staining characteristics of bacteria using optical microscope and transmission electron microscope was conducted.ATB Expression biochemical identification instrument and Biolog Microbes Automatic Identification System were used to identify the physiological and biochemical characteristics, and molecular biology identification was conducted based on 16S rRNA sequence and gyrB gene sequence.The artificial regression test and histopathology analysis were applied to further determine the pathogenicity of the bacteria and tissue damage, and its antibiotic susceptibility was tested by Kirby-Bauer disc diffusion.【Result】A dominant strain 180803 bj_jzl was isolated from the liver of diseased M.salmoides.It was gram-negative rod-shaped bacteria with periflagellum.The biochemical characteristics were identified as the same as Citrobacter freundii up to 99.4% by the Biolog Microbes Automatic Identification System.Phylogenetic tree based on homology of 16S rRNA and gyrB gene sequences revealed that isolated strain 180803bj_jzl clustered with the same branch of C.freundii.The 16S rRNA sequence similarity with the reference strains of C.freundii published by GenBank was more than 99.00%, and the similarity of the gyrB gene sequence was more than 91.00%.The cumulative mortality rate within 7 d of artificial recurrent infection was 60%, and its symptoms were basically the same as those of naturally-occurring M.salmoides.There were the erythrocytes congestion in the central vein and hepatic sinusoids of liver tissue, and a large number of vacuoles in the hepatocyte cytoplasm.The degeneration and necrosis were scattered in liver tissue.The white pulp and red pulp had unclear boundaries.And there were ferritinophilic hemoglobin centers in the splenic sinusoids and splenic small vessels.The epithelial cells of the gill lamellae were separated from the capillaries in a racket-shaped manner, and some epithelial cells of the gill lamellae were necrotic and detached, and only a small amount of gill lamellae skeleton remained.The drug sensitivity tests showed that 180803bj_jzl isolate was sensitive to 7 antimicrobial drugs such as fleroxacin, ofloxacin, macromycin, gentamicin, enrofloxacin and norfloxacin, and resistant to 15 antimicrobial drugs such as doxycycline, tetracycline, acetylcholine, roxithromycin, cefalexin and amoxicillin.【Conclusion】C.freundii can cause the body surface ulceration of M.salmoides and the histopathological damage to the liver, spleen and gills, and it has strong toxicity.The fishery antibiotics, such as neomycin and enrofloxacin, can be used to prevent and treat the disease in actual production,.