泥鳅(Misgurnus anguillicaudatus)病原温和气单胞菌(Aeromonas sobria)分子鉴定及耐药性研究

采用常规生理生化特性测定及16SrRNA、gyrB及rpoD基因同源性检索与系统发育学分析等分子生物学方法,对引起养殖泥鳅大量死亡的病原细菌进行了综合鉴定。生理生化特性结果表明,分离菌株NQ090701与气单胞菌属的温和气单胞菌相近;16SrRNA、gyrB及rpoD基因Blast序列同源性检索与系统发育学分析均与温和气单胞菌相似性最高;综合形态与生理生化特征及16SrRNA、gyrB与rpoD基因的分子特征,确认分离菌株NQ090701为温和气单胞菌(Aeromonas sobria)。同时,采用试管稀释法测定了养殖生产中常用的10种抗菌药物对病原温和气单胞菌的体外最小杀菌浓度(Minimal Bactericidal Concentration,MBC),结果表明氟哌酸杀菌作用最好,其次为盐酸沙拉沙星;甲砜霉素和复方磺胺甲恶唑在所试验的药物浓度范围对温和气单胞菌无杀菌效果。

MOLECULAR IDENTIFICATION AND DRUG RESISTANCE OF PATHOGENIC AEROMONAS SOBRIA ISOLATED FROM MISGURNUS ANGUILLICAUDATUS

Outbreaks of mass mortality among cultured loach Misgurnus anguillicaudatus occurred on some farms of Dunshang county, Jiangsu province. A predominant strain (designated as NQ090701) was highly virulent to loach; all fish injected with strain NQ090701 died from the 2th to 3th day post-injection, and all the moribund/dead loach exhibited the same gross symptoms as that observed in natural outbreaks. Morphological, physiological and biochemical characteristics of strain NQ090701 were very similar to Aeromonas sobria. Sequencing analyses of 16S rRNA, gyrB and rpoD also revealed strain NQ090701 to be highly homologous to A. sobria; in the phylogenetic trees constructed using the neighbor-joining method based on 16S rRNA, gyrB and rpoD gene, strain NQ090701 all clustered with the A. sobria. Therefore, strain NQ090701 was identified as A. sobria based on the phenotypes and molecular characteristics. In addition, ten antimicrobial drugs were examined for their minimum bactericidal concentrations to strain NQ090701 by the tube dilution. Norfloxacin had the preferable bactericidal effects, because all the tested drug concentrations can kill the strain NQ090701; and sarafloxacin hydrochloride is the second preferable drug, of which the minimal bactericidal concentration was 4mg/L. Strain NQ090701 was resistant to thiamphnicol and sulfamethoxazole in the range of 1mg/ml to 30mg/ml. Results in this study reveal that norfloxacin and sarafloxacin hydrochloride can effectively control the disease of loach caused by pathogenic Aeromonas sobria in aquaculture.