Isolation And Identification Of Pseudomonas Aeruginosa In Moschus Berezovskii And Study On Its Virulence

Combining traditional bacteriological identification with molecular biology methods, this study isolated and identified three Pseudomonas aeruginosa strains from the lungs and liver tissue of Moschus berezovskii. The proliferation of lymphocyte was observed by crude toxin effecting on the rabbit peripheral blood lymphocyte.PAsp-1, PAsp-2 and PAsp-3 strains were identified by using 16SrDNA sequence analysis method. The conserved general primers of bacteria 16SrDNA were designed according to the literature.The genomic DNA of PAsp-1, PAsp-2 and PAsp-3 were extracted and amplified the target 16SrDNA sequence. Meanwhile, the sequence was compared with the BLAST database of NCBI. Then, it used the biology software DNA star7.1 to construct the phylogentic tree. At last, combined the bacteriological identification; PAsp-1, PAsp-2 and PAsp-3 strains were identified as Pseudomonas aeruginosa.The culture conditions of strains was studied by using orthogonal design text, the text showed that the optimum liquid culture conditions of PAsp-1, PAsp-2 and PAsp-3 were 40℃, pH 6.5.Culturing Pseudomonas aeruginosa under the optimal conditions, proliferation rate of the three strains were fastest in 6-14h, peaked at 14h.Drug sensitivity test showed that, PAsp-1, PAsp-2 and PAsp-3 were sensitive to gentamycin, amikacin, piperacillin, ceftazidime and cefoperazone; Moderately sensitive to ceftriaxone, ciprofloxacin, tetracycline and carbenicillin; Resistance to ampicillin, cotrimoxazole, cefazolin, penicillin, erythromycin and minocycline. Pathogenicity tests were performed by intraperitoneal injection 1 ml/only, the concentration of bacterial cultures were 2.00×107cfu/ml,2.20×107cfu/ml,2.40×107cfu/ml, 2.60×107cuf/ml and 2.80×1007cfu/ml, the LD50 of PAsp-1, PAsp-2 and PAsp-3 were respectively 2.591×107CFU/ml,2.577×107CFU/ml and 2.634×107CFU/ml.Results of lymphocyte proliferation test, measured the protein content of the crude extract toxins, displayed that the time of the best concentrations of toxin of PAsp-1, PAsp-2, PAsp-3 were 48h, the concentrations of toxin were respectively 7.137mg/ml, 9.021mg/ml and 7.876mg/ml. Cell number of lymphocytes isolated from peripheral blood of rabbits was 2.5×106per milliliter, and the percentage of living cells was 96.8%. The IC50 of crude toxins of PAsp-1, PAsp-2, PAsp-3 were respectively 6.745mg/ml, 6.536mg/ml,7.195mg/ml.