Isolation, Identification And Drug Resistance Analysis Of Citrobacter Freundum From

The disease on Andrias davidianus happened frequently recent years. To isolate the pathogen of diseased giant salamander (Andrias davidianus), identify the bacteria species, and determined its several extracellular enzymatic activities, a bacteria stain (TNL12) was separated from the liver of diseased individuals. The morphological, physiological and biochemical characteristics, and 16S rDNA sequence were analyzed for species identification. Its pathogenicity was tested by artificial infection. The extracellular enzymatic activities were detected by plate method. The results showed that the bacteria strain was Gram-negative short bacillus without spores, and the physiological and biochemical characteristics were consistent with Citrobacter freundii. Its 16S rDNA sequence shared high homology of 99% with Citrobacter freundii, and was in the same cluster as Citrobacter freundii on the phylogenetic tree. Artificial infection with the bacteria to healthy frog and Andrias davidianus induced the same diseased symptoms as occurred naturally. The pathogen produced lecithinase and amylase, but no activity of lipases, proteases, gelatinase, and urease were detected. In conclusion, the bacteria stain isolated from diseased Andrias davidianus was Citrobacter freundii, which showed strong pathogenicity to giant salamander.The disc diffusion method was used to study the pathogenic bacteria resistance to 20 antibiotics. The experimental results showed that the pathogen had resistance to macrolide antibiotics, β-lactam antibiotics and quinolone antibiotics.Antibacterial effect in vitro on 45 kinds of Chinese herbs was detected by using the agar diffusion method (punch method of Schisandra Chinensis). The minimal inhibitory concentration and minimal bactericidal concentration were detected by double dilution method on good antibacterial effect of 25 kinds of Chinese herbal medicine. The results showed that the Citrobacter freundii from sick Andrias davidianus is highly sensitive to Schisandra Chinensis and Fructus Mume. And it was moderately sensitive to 7 kinds of Chinese medicinal herbs including Honeysuckle, Rhizoma Coptidis, Clove and so on. To Tree peony bark, Eucommia and other 16 Chinese herbal medicines, the sensitivity was weak. There was no sensitivity to Herba lycopi and other 19 kinds Chinese herbal. Detecting the MIC and MBC of the good inhibitory effect of Chinese herbal medicine, the range was between 7.81 mg/mL～250 mg/mL.In summary, the Citrobacter freundii from Andrias davidianus have developed resistance to many antibiotics. Studying the drug sensitivity and resistance characteristics of the pathogen can provide the theoretical basis for the prevention of disease research on Andrias davidianus.