一株鹅-鱼养殖环境下罗非鱼无乳链球菌的分离鉴定及药物敏感性分析

    Isolation, Identification and Drug Sensitivity Analysis of a Strain of Streptococcus agalactiae in Tilapia (Oreochromis niloticus) from Goose-fish Polyculture Environment

    • 摘要:
      目的 为确定鹅-鱼立体养殖环境下引发罗非鱼疾病的病原菌,通过分析病原菌的生长特性、致病性与药物敏感性,为罗非鱼病的深入研究与精准防控提供科学依据。
      方法 从患病罗非鱼组织中分离病原菌,采用形态观察、生理生化鉴定、16S rRNA序列分析等方法鉴定菌株;绘制菌株生长曲线,分析温度、盐度及pH值对病原菌生长的影响;通过腹腔注射和拌料口服投喂方式人工回归感染健康罗非鱼,分析病原菌的致病性;采用药敏纸片法测定病原菌对11类共26种抗菌药物的敏感性。
      结果 从患病罗非鱼的脑组织中分离纯化得到一株优势菌株,编号为SAYJN,经形态学观察、生理生化鉴定、16S rRNA序列分析及回归感染试验,鉴定此菌株为无乳链球菌(Streptococcus agalactiae);SAYJN菌株的生长迟缓期为0~5 h,对数期为5~12 h;最适生长温度37 ℃、最适pH值7.0、最适盐度为5‰。腹腔注射和拌料口服投喂SAYJN菌株均可感染健康罗非鱼;水温为29(±1)℃时,腹腔注射SAYJN菌液对罗非鱼有较强的致病性,半致死浓度(LC50)为2.4×108 CFU/mL;SAYJN菌株致病力与其浓度和水温密切相关。SAYJN菌株对甲氧苄啶、庆大霉素、新霉素等6种药物产生耐药性,对恩诺沙星、环丙沙星、阿米卡星等4种药物中度敏感,对多西环素、氟苯尼考、利福平等16种药物敏感。
      结论 无乳链球菌SAYJN菌株是鹅-鱼立体养殖环境下罗非鱼疾病爆发的主要病原菌,该菌的致病力强弱与其浓度和水温密切相关。

       

      Abstract:
      Objective In order to identify the pathogenic bacteria causing the tilapia disease outbreak in the geese-fish polyculture system, the growth characteristics, pathogenicity and drug sensitivity of the pathogenic bacteria were analyzed, with an aim to provide scientific basis for further research and effective prevention and control of tilapia disease.
      Method The pathogenic bacteria were isolated from the organs of diseased tilapia and identified by various methods such as morphological observation, physiological and biochemical identification, and 16S rRNA gene sequence analysis. The impacts of temperature, salinity and pH on the growth of pathogenic bacteria were analyzed, and a growth curve for the strain was plotted. The pathogenicity of the bacterial strain was assessed by intraperitoneal injection and oral feeding of mixed materials. Additionally, the susceptibility of the strain to 26 different antimicrobial agents belonging to 11 classes was determined by the drug sensitive slips method.
      Result A dominant strain SAYJN was isolated and purified from the brain tissue of the diseased tilapia. Through morphological observation, physiological and biochemical identification, 16S rRNA gene sequence analysis and artificial regression infection test, the strain was identified as Streptococcus agalactiae. The growth retardation stage of SAYJN was 0-5 h, and its logarithmic stage was 5-12 h. The optimal conditions for growth were found at a temperature of 37 ℃, pH value of 7.0 and salinity of 5‰. The strain SAYJN with intraperitoneal injection and oral feeding of mixed materials could infect the healthy tilapia. In an artificial infection test by intrabitoneal injection, the strain SAYJN showed high pathogenicity to tilapia at a temperature of 29 (±1) ℃, with a lethal concentration of 2.4×108 CFU/mL. The pathogenicity of the strain SAYJN was found to be closely related to bacterial concentration and water temperature. Further, the strain SAYJN displayed resistance to six drugs such as trimethopridine, gentamicin, neomycin and etc. It showed moderate sensitivity to four drugs such as enrofloxacin, ciprofloxacin and amikacin. Additionally, it was found to be sensitive to 16 drugs such as doxycycline, flufenicol, rifampicin and etc.
      Conclusion The strain SAYJN of S. agalactiae is the primary pathogen responsible for the outbreak of tilapia disease in the goose-fish polyculture environment, and the pathogenicity of this pathogen is closely related to the bacterial concentration and water temperature.

       

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