黄鳝藤黄微球菌的分离鉴定及药敏试验

Isolation and identification of Micrococcus luteus in rice field eel(Monopterus albus) in Sichuan Province

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。

This experiment was conducted to analyse the species and drug resistance of hemorrhagic pathogens from Monopterus albus.One hemorrhagic disease pathogens（HM1） of M.albus from Mingshan in Sichuan was isolated using bacteria classification and identification technique,and was identified according to the morphological,physiological and biological characteristics and the phylogenetic analysis.On pathogenicity test of animals,the experimental Ictalurus nebulosus demonstrated weak pathogenic symptom,but not any pahthogenic symptom in mice and Misgurnus anguillicaudatus.Morphological and physiological biochemical character homophyly of HM1 strain was in accordance with Micrococcus luteus,which presented gram positive coccus,non-motility,arranged in couples or quadruple or clumps,non-chain-like conformation,non-sporulation;catalase-positive,gelatin liquefaction-positive,lactose-negative,mannitol-negative,glucose-negative,aesculin hydrolysis-negative,nitrate reduction-negative,arginine hydrolytic enzyme-negative and so on.The 16S rDNA gene of HM1 was amplified by PCR using the universal primers.Then,the 16S rDNA gene was cloned and sequenced.A sequence of 1 382 base pair（bp） was obtained from HM1（GenBank accession number： HM044913）.The analysis using online Classifer software form the Ribosomal Database Project（RDP） database showed that the strain HM1 was classified in Micrococcus.The nucleotide sequences homology of 16S rDNA from HM1 strain had 99% identity with 7 strains M.luteus in GenBank.Phylogenetic trees reconstructed based on genes 16S rDNA indicate that the strains of HM1 and other M.luteus in GenBank are clustered in the same clade.The results suggest that the isolated strain（HM1） was classified in M.luteus.Antibiotic susceptibility tests showed that the isolated strain（HM1） was sensitive to 18 kinds of drugs inculding norfloxacin,cefazolin sodium and carbenicillin,etc;and was medium sensitive to ceftazidime;and insusceptible to 5 kind of drugs such as nalidixic acid,gatifloxacin and sulfafurazole,etc.