Bacteria Isolation, Identification And Drug Sensitive Analysis Of Strains From Clinical Sending Brown Frogs

Because of its unique medicinal and nutritional value,brown frogs are now getting more attention.However,the number of wild brown frogs is limited,along with their habitats are destroying and captured by humans without scruple, making the number decrease sharply and wild brown frogs are endangered. The brown frog products are supply falls shout of demand.Therefore, the best way to settle this situation is artificial feeding brown frogs.But there are still many problems reveal from current breeding techniques,such as the large breed density,serious environment pollution,unitary food and so on.Leading to a variety of diseases, for instance red legs disease,meningitis,gastroenteritis,lousy skin disease.For once one of these diseases takes place,large quantities of brown frogs would die,and there is no effective treatment methods and drug standards.These diseases would seriously influence the growth and survival rates of brown frogs,which cause huge economic losses.In order to offer some advises to prevent and treat diseases,make the losses minimum and protect the wild brown frog species. Our research get information about bacterium groups,which brown frog carried and has detected the drug sensitivity of the commonly clinical pathogenic bacterial strains.During three years, we collected live brown frogs and frozen dead brown frogs which came from different areas of the Northeast China.From these two kinds of samples,some different bacterial strains were isolated from the liver or edema fluid by sterile operation.After culturing and identification of these bacteria using morphological preliminary analysis,the genome of the bacteria were extracted as the PCR template.A1500bp genetic fragment was amplified by16S rRNA primers.The PCR products were purified before subcloning into the PMD18-T vector and afterward transforming into DH-5a.The positive clones were selected and sequenced. Sequences were compared with other bacterial strains available sequences which were obtained from GenBank, analyzed through the basic local alignment search tool(BLAST), and phylogenetic tree analysis were performed in order to find out the evolutionary relation of these sequences. Also, biochemistry identification and drug sensitivity analysis of a few isolated strains were performed with VITEK2Compact automatic microbial analysis system to evidence the BLAST results. Totally42strains of bacteria were isolated, of which2strains were isolated from live brown frog. Alignment result of16S rRNA showed that the2strains were Aeromonas hydrophila and Acinetobacter sp, which the similarity is100%and99%, respectively. The results analyzed with VITEK2Compact automatic microbial analysis system amount to the same thing.Except that the Acinetobacter sp strain was finally identified to be Acinetobacter lwoffi. Ultimately, the2strains were named bf003and bfzhl2. The rest strains were isolated from frozen dead brown frogs and identification result was: Acinetobacter11strains,Pseudomonas3strains,Aeromonas.sp.2strains and so on.The study revealed the bacterial population in brown frogs and provided information usefully in disease prevention and diagnosis.