论文部分内容阅读
为探讨黄鳝(Monopterus albus)出血病病原菌的种类及耐药情况,采用动物回归和细菌常规分类鉴定方法及16S rDNA基因系统发育学分析等手段,从四川名山地区黄鳝出血病病料中分离到具有致病性的菌株(HM1),经人工感染实验证实该菌为黄鳝出血病的病原菌。动物致病性试验显示,该分离株对泥鳅和小白鼠不致病,对云斑有较弱的致病力。对该分离株的形态和生理生化指标进行分析,试验结果与报道的藤黄微球菌一致,为革兰氏阳性、球菌,无运动力,多成双、四联或簇状排列,不形成链状排列,不形成芽孢;接触酶和明胶液化阳性;乳糖、甘露醇、葡萄糖、七叶苷、硝酸盐还原、精氨酸双水解阴性等特征。采用细菌16S rDNA基因通用引物对该菌的16S rDNA基因进行PCR扩增、克隆测序,得到1条长度为1 382 bp的核苷酸序列(GenBank登录号为:HM044913)。该序列经RDP(Ribosomal Database Project)数据库在线Classifer分析,结果表明该菌株属于微球菌属细菌。以该菌16SrDNA序列和GenBank数据库内同源性较高的细菌16S rDNA序列进行同源性比对分析和构建系统发育树,结果显示分离菌(HM1)与微球菌属的藤黄微球菌菌株的同源性高达99%,且在系统发育树上与其他不同来源的藤黄微球菌聚为一支。结合形态及生理生化特点将其确定为藤黄微球菌(Micrococcus luteus)。24种药物的药敏试验结果显示,该分离株对诺氟沙星、头孢唑啉、羧苄西林等18种药物敏感;对复达欣中度敏感;但对萘啶酸、加替沙星、磺胺异恶唑等5种药物耐药。研究亮点:采用常规的细菌分离鉴定方法并结合16S rDNA基因系统发育学分析,首次从患出血病的黄鳝体内分离得到了藤黄微球菌(Micrococcusluteus)致病菌株,也是在水生动物体内首次分离获得了藤黄微球菌致病菌株,并完成了其对24种药物的敏感性试验,结果显示该致病菌株对诺氟沙星、头孢唑啉、羧苄西林等18种药物敏感,对萘啶酸、加替沙星、磺胺异恶唑等5种药物耐药,研究结果为黄鳝出血病防治提供了重要参考资料。
In order to explore the species and drug resistance of pathogenic bacteria in monopterus albus, the methods of animal regression and bacterial routine classification and phylogenetic analysis of 16S rDNA gene were used to isolate Pathogenic strains (HM1), the artificial infection experiments confirmed that the bacteria eel pathogenic bacteria. Animal pathogenicity test showed that the isolate did not cause disease to loach and mouse, but had weaker virulence to maculata. The morphological and physiological and biochemical indexes of this isolate were analyzed and the results were consistent with the reported Micrococcus luteus, being Gram-positive, cocci, motile-free, multi-double, quadruple or cluster-like arrangement without chain formation Shaped arrangement, no formation of spores; contact enzymes and positive liquefaction of gelatin; lactose, mannitol, glucose, esculin, nitrate reduction, arginine double hydrolysis negative and so on. The bacterial 16S rDNA gene universal primers were used to amplify the 16S rDNA gene of this bacterium and cloned and sequenced to obtain a 1 382 bp nucleotide sequence (GenBank accession number: HM044913). The sequence was analyzed online by Classifer in RDP (Ribosomal Database Project) database. The results showed that the strain belongs to the genus Micrococcus. The 16S rDNA sequence of this strain was compared with the 16S rDNA sequence of bacteria with high homology in GenBank to construct the phylogenetic tree. The results showed that the isolates of Micrococcus luteus The homology is as high as 99% and is clustered together with other Micrococcus luteus in the phylogenetic tree. Combined with its morphological and physiological and biochemical characteristics identified as Micrococcus luteus (Micrococcus luteus). The drug susceptibility test results of 24 drugs showed that the isolate was sensitive to 18 drugs such as norfloxacin, cefazolin and carbenicillin, moderately sensitive to , Sulfa isoxazole and other five kinds of drug resistance. Research highlights: Using conventional bacterial isolation and identification methods combined with phylogenetic analysis of 16S rDNA gene, Micrococcus luteus pathogenic strains were isolated from eel-infected eel for the first time and were also isolated for the first time in aquatic animals Micrococcus luteus pathogenic strains, and completed its sensitivity test of 24 drugs, the results showed that the pathogenic strains of norfloxacin, cefazolin, carbenicillin and other 18 kinds of drug-sensitive, naphthyridine Acid, gatifloxacin, sulfamethoxazole and other five kinds of drug resistance, the results provide an important reference for prevention and treatment of eel disease.