于士翔^1,2, 纪元³, 李新^1,3, 刘峰³, 刘芳华¹
1.中国科学院烟台海岸带研究所 海岸带生物学与生物资源利用所重点实验室, 山东 烟台 264003;2.中国科学院大学, 北京 100049;3.中国农业大学烟台研究院, 山东 烟台 264670

摘要:
对患病的单环刺螠幼体3 000~4 000只/kg肠道内病原菌进行分离鉴定、生长特性研究及药敏分析，为人工养殖单环刺螠幼体过程中出现的疾病提供治疗依据。对患病幼体肠道内的病原菌进行分离纯化、生理生化鉴定、16S rRNA基因序列分析、人工回接感染试验、生长曲线测定、最适生长温度、pH、盐度的探究；采用K-B纸片扩散法对米诺环素、庆大霉素、卡那霉素、头孢唑林、四环素中度敏感，对阿奇霉素、新霉素、万古霉素、利福平、红霉素、克林霉素、阿莫西林进行药敏试验；并对抑菌效果最好的药品进行安全性检测。分离纯化后经生理生化鉴定获得一种弧菌命名为菌株SX-1，16S rRNA基因序列分析极有可能为新喀里多尼亚弧菌（Vibrio neocaledonicus），人工回接感染试验表明SX-1是单环刺螠幼体从沙底钻出，活动能力减弱，体表变红症状的致病菌，生长特性研究表明其最适生长温度、pH、盐度分别为30℃，6.0，35；药敏试验结果表明，SX-1对氯霉素、羧苄西林、氧氟沙星、头孢曲松高度敏感，安全性检测实验表明，氧氟沙星抑菌治疗效果明显，并对单环刺螠无伤害。从单环刺螠肠道内分离获得了一种极有可能为新喀里多尼亚弧菌（Vibrio neocaledonicus）的致病菌SX-1。对于感染该种弧菌的单环刺螠，氧氟沙星抑菌治疗效果明显，并对单环刺螠无伤害。我们的实验结果对于预防并治疗人工养殖过程中单环刺螠患病提供理论依据和治疗模式，因而具有实际指导意义。
关键词:单环刺螠弧菌分离鉴定生长特性药敏试验
DOI：10.11759/hykx20190123001
分类号:
基金项目:国家自然科学基金（91751112，41573071）；“科学”号高端用户项目（KEXUE2018G01）；山东省自然科学****基金（JQ201608）；“泰山****”青年专家计划（tsqn20161054）；山东省重点研发计划（公益类专项，2018GHY115028）

Isolation, identification, characterization, and sensitivity analysis of gut pathogenic Vibrio of Urechis unicinctus
YU Shi-xiang^1,2, JI Yuan³, LI Xin^1,3, LIU Feng³, LIU Fang-hua¹
1.Key Laboratory of Coastal Biology and Biological Resources Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China;2.University of Chinese Academy of Sciences, Beijing 100049, China;3.Yantai Research Institute, China Agricultural University, Yantai 264670, China
Abstract:
The larvae of the diseased intestinal pathogenic bacterium Urechis unicinctus (3000to 4000 kg) were isolated and identified, and their growth characteristics and drug sensitivities were evaluated to provide a treatment basis for diseases that occur in an artificial culture of the larva. Isolation and purification, biochemical identification, 16S rRNA gene sequence analysis, artificial resumption infection, and growth curve assay as well as studies on optimum growth temperature, pH, and salt content were conducted on the pathogenic bacteria isolated from human intestines. The K-B disk diffusion method was applied for drug sensitivity testing, whereas ofloxacin was used for security detection testing. For pathogenic Vibrio, the optimum growth temperature, pH, and salt content were 30℃, 6.0, and 35, respectively. Drug sensitivity tests demonstrated that Vibrio is highly sensitive to chloramphenicol, carbenicillin, ofloxacin, and ceftriaxone; moderately sensitive to minocycline, gentamicin, kanamycin, cefazolin, and tetracycline; but resistant to azithromycin, neomycin, vancomycin, rifampicin, erythromycin, clindamycin, and amoxicillin. Security detection tests revealed that although ofloxacin exerted a clear antibacterial effect, it did not harm U. unicinctus. Based on the results of the drug sensitivity test, Vibrio-infected U. unicinctus can be effectively treated using targeted antibiotics. These experimental results could provide a theoretical basis and treatment pattern for the prevention and treatment of U. unicinctus in artificial cultures.
Key words:Urechis unicinctusVibrioisolation and identificationgrowth characteristicsdrug sensitivity test





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