罗非鱼源无乳链球菌肠道拮抗芽孢杆菌的筛选及其生物学特性
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摘要
【目的】从健康尼罗罗非鱼(Oreochromis niloticus)肠道中筛选一株对罗非鱼源无乳链球菌等病原菌具有拮抗功能的益生菌。【方法】取健康尼罗罗非鱼肠道,匀浆后进行10倍系列梯度稀释,然后涂布BHI平板,培养1–2d,挑取单克隆菌落。采用点种法初步筛选对罗非鱼源无乳链球菌有拮抗作用的菌株,选取其中一株拮抗效果较好的菌株LF01,通过形态学、生理生化特征以及分子生物学分析,对LF01菌株进行鉴定。然后对LF01菌株的生长特性、水解淀粉和酪蛋白能力、药物敏感特性、抗菌谱和生物安全性进行测定和分析。【结果】根据菌落形态和生长时间的差异,从健康尼罗罗非鱼肠道中筛选出64株细菌,通过拮抗试验筛选出6株具有明显拮抗效果的菌株,其中LF01菌株的拮抗效果最好。根据LF01的形态、生理生化特征和gyr A基因的进化分析,确定该菌株为贝莱斯芽孢杆菌(Bacillus velezensis)。LF01菌株的最适生长温度为30°C,最适p H值为7,最适盐度为5‰,而且该菌株具有水解淀粉和酪蛋白的功能。药敏试验结果显示,LF01菌株对多数抗生素敏感,仅对杆菌肽耐药。拮抗试验结果显示LF01株对无乳链球菌、海豚链球菌、迟缓爱德华氏菌、鮰爱德华氏菌、嗜水气单胞菌、舒氏气单胞菌、维氏气单胞菌、简氏气单胞菌、鰤鱼诺卡氏菌等病原菌均具有拮抗作用,其中对鰤鱼诺卡氏菌的拮抗作用最强,平均抑菌圈直径达28.3 mm。生物安全试验表明,LF01菌株对尼罗罗非鱼、斑马鱼(Danio rerio)和乌鳢(Channa argus)等3种鱼均无致病性,具有良好的安全性。【结论】本研究筛选了一株贝莱斯芽孢杆菌LF01株,该菌的生物安全性良好,而且可拮抗常见的水产病原菌,具有防控多种水产经济动物疾病的潜力,应用前景十分广阔。
[Objective] The aim of this study was to screen a probiotic strain that antagonizes Streptococcus agalactiae isolated from the intestine of healthy Nile tilapia(Oreochromis niloticus). [Methods] The gut of healthy tilapia was collected, homogenized, and diluted by a 10-fold series. Then 0.3 mL suspension was streaked on brain heart infusion plates and incubated for 1–2 d at 30 °C, and then single colonies were selected for purification. Strains had antagonistic function against S. agalactiae were screened by the dot-inoculating method, and a strain named LF01 with a better antagonistic effect was selected. In this study, the strain LF01 was identified by physiological, biochemical characteristics and molecular biological analysis. In addition, some characteristics and biosafety of the strain LF01 were determined and analyzed. [Results] A total of 64 strains were isolated from the intestine of healthy tilapia and 6 strains with obvious antagonistic effects were obtained, with the antagonism of LF01 strain considered the best. Strain LF01 was identified as Bacillus velezensis. The best growth temperature, p H, and salinities of strain LF01 were 30 °C, 7, and 5‰, respectively. Furthermore, the strain LF01 exhibited the ability to hydrolyze starch and casein. Strain LF01 was sensitive to most antibiotics and resistant only to bacitracin, and showed antimicrobial activity against a broad range of fish pathogens including Streptococcus agalactiae, Streptococcus iniae, Edwardsiella tarda, Edwardsiella ictaluri, Aeromonas hydrophila, Aeromonas schubertii, Aeromonas veronii, Aeromonas jandaei and Nocardia seriolae. Biosafety tests suggested that the strain LF01 was not pathogenic to Nile tilapia, Danio rerio, and Channa argus. [Conclusion] In this study, it was found that B. velezensis LF01 strain isolated from the intestine of healthy tilapia has good biosafety and could antagonize the common aquatic pathogens. It has the potential to prevent and control a variety of aquatic animal diseases, and has broad application prospects.
[Objective] The aim of this study was to screen a probiotic strain that antagonizes Streptococcus agalactiae isolated from the intestine of healthy Nile tilapia(Oreochromis niloticus). [Methods] The gut of healthy tilapia was collected, homogenized, and diluted by a 10-fold series. Then 0.3 mL suspension was streaked on brain heart infusion plates and incubated for 1–2 d at 30 °C, and then single colonies were selected for purification. Strains had antagonistic function against S. agalactiae were screened by the dot-inoculating method, and a strain named LF01 with a better antagonistic effect was selected. In this study, the strain LF01 was identified by physiological, biochemical characteristics and molecular biological analysis. In addition, some characteristics and biosafety of the strain LF01 were determined and analyzed. [Results] A total of 64 strains were isolated from the intestine of healthy tilapia and 6 strains with obvious antagonistic effects were obtained, with the antagonism of LF01 strain considered the best. Strain LF01 was identified as Bacillus velezensis. The best growth temperature, p H, and salinities of strain LF01 were 30 °C, 7, and 5‰, respectively. Furthermore, the strain LF01 exhibited the ability to hydrolyze starch and casein. Strain LF01 was sensitive to most antibiotics and resistant only to bacitracin, and showed antimicrobial activity against a broad range of fish pathogens including Streptococcus agalactiae, Streptococcus iniae, Edwardsiella tarda, Edwardsiella ictaluri, Aeromonas hydrophila, Aeromonas schubertii, Aeromonas veronii, Aeromonas jandaei and Nocardia seriolae. Biosafety tests suggested that the strain LF01 was not pathogenic to Nile tilapia, Danio rerio, and Channa argus. [Conclusion] In this study, it was found that B. velezensis LF01 strain isolated from the intestine of healthy tilapia has good biosafety and could antagonize the common aquatic pathogens. It has the potential to prevent and control a variety of aquatic animal diseases, and has broad application prospects.
引文
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[2]Geraylou Z,Souffreau C,Rurangwa E,de Meester L,Courtin CM,Delcour JA,Buyse J,Ollevier F.Effects of dietary arabinoxylan-oligosaccharides(AXOS)and endogenous probiotics on the growth performance,non-specific immunity and gut microbiota of juvenile Siberian sturgeon(Acipenser baerii).Fish&Shellfish Immunology,2013,35(3):766-775.
[3]Zhou JM,Wu ZX,Zeng LB,Chen XX,Yang L,Zhao Y.Selection and characterization of pathogen antagonised probiotics from intestinal tract of Yellow catfish(Pelteobagrus fulvidraco).Acta Hydrobiologica Sinica,2012,36(1):78-84.(in Chinese)周金敏,吴志新,曾令兵,陈孝煊,杨丽,赵杨.黄颡鱼肠道病原拮抗性芽孢杆菌的筛选与特性研究.水生生物学报,2012,36(1):78-84.
[4]Lee BY,Coutanceau JP,Ozouf-Costaz C,D’Cotta,H,Baroiller,JF,Kocher,T.Genetic and physical mapping of sex-linked AFLP markers in Nile tilapia(Oreochromis niloticus).Marine Biotechnology,2011,13(3):557-562.
[5]张宗锋.以湛江、茂名海南为例探究我国凡纳滨对虾、罗非鱼养殖现状及面临的问题.上海海洋大学硕士学位论文,2012.
[6]彭民毅.广西罗非鱼无乳链球菌病病原分离、鉴定及毒力基因检测分析.广西大学硕士学位论文,2014.
[7]Lu MX,Li J,Ye X,Deng GC,Jiang XY,Tian YY,Lai CL.Identification and characterizations of Streptococcus agalactiae isolated from tilapia cultured in Guangdong and Hainan provinces.Microbiology China,2010,37(5):766-774.(in Chinese)卢迈新,黎炯,叶星,邓国成,江小燕,田园园,赖翠玲.广东与海南养殖罗非鱼无乳链球菌的分离、鉴定与特性分析.微生物学通报,2010,37(5):766-774.
[8]Fang W,Ke BX,Liang HY,He DM,Tan HL,Ke CW,Deng XL,Peng HL,Sun JF,Wang YX.Isolation,identification and characterization of Streptococcus agalactiae isolated from tilapia fish farms in five districts of Guangdong.Journal of Tropical Medicine,2016,16(2):175-178,185,274.(in Chinese)方伟,柯碧霞,梁恒宇,何冬梅,谭海玲,柯昌文,邓小玲,彭华林,孙九峰,王云新.广东五地区养殖罗非鱼无乳链球菌的分离、鉴定与特性分析.热带医学杂志,2016,16(2):175-178,185,274.
[9]Younes AM,Gaafar AY,Awad ES.Virulence determinants and plasmid profile of Aeromonas hydrophila strains isolated from Oreochromis niloticus.Global Veterinaria,2015,15(6):613-617.
[10]Liu C,Chang QQ,Zhang DF,Li KB,Wang F,Lin MH,Shi CB,Jiang L,Wang Q,Bergmann SM.Aeromonas shuberti as a cause of multi-organ necrosis in internal organs of Nile tilapia,Oreochromis niloticus.Journal of Fish Diseases,2018,41(10):1529-1538.
[11]Nagy E,Fadel A,Al-Moghny FA,Ibrahim MS.Isolation,Identification and pathogenicity characterization of Edwardsiella tarda isolated from Oreochromis niloticus fish farms in Kafr-Elshiekh,Egypt.Alexandria Journal of Veterinary Sciences,2018,57(1):171-179.
[12]Currie J,Lin WC,Meng JJ.Addressing antibiotic abuse in China:an experimental audit study.Journal of Development Economics,2014,110:39-51.
[13]Dunstan RA,Heinz E,Wijeyewickrema LC,Pike RN,Purcell AW,Evans TJ,Praszkier J,Robins-Browne RM,Strugnell RA,Korotkov KV,Lithgow T.Assembly of the type IIsecretion system such as found in Vibrio cholerae depends on the novel Pilotin Asp S.PLo S Pathogens,2013,9(1):e1003117.
[14]Li HB,Song XL,Li Y,Wei S.Progress on probiotic bacteria in aquiculture.Progress in Veterinary Medicine,2008,29(5):94-99.(in Chinese)李海兵,宋晓玲,李赟,韦嵩.水产动物益生菌研究进展.动物医学进展,2008,29(5):94-99.
[15]Olsson JC,Westerdahl A,Conway PL,Kjelleberg S.Intestinal colonization potential of turbot(Scophthalmus maximus)-and dab(Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum.Applied and Environmental Microbiology,1992,58(2):551-556.
[16]Zhang XM,Li J,Liu Q.Selection and effect of vibrios-antagnism bacteria.Journal of Fishery Sciences of China,2004,11(4):325-332.(in Chinese)张新明,李健,刘淇.弧菌拮抗菌的筛选及其效果.中国水产科学,2004,11(4):325-332.
[17]布坎南RE,吉本斯NE.伯杰细菌鉴定手册.中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组,译.北京:科学出版社,1984.
[18]东秀珠,蔡妙英.常见细菌系统鉴定手册.北京:科学出版社,2001.
[19]侯宝宏.解淀粉芽孢杆菌TS-1203抑菌活性物质分析及制剂研制.甘肃农业大学硕士学位论文,2016.
[20]Liu ZG,Ke XL,Lu MX,Zhu HP,Gao FY.Effect of temperature on the virulence of Streptococcus agalactiae from Nile tilapia(Oreochromis niloticus).Journal of Fisheries of China,2013,37(11):1733-1741.(in Chinese)刘志刚,可小丽,卢迈新,朱华平,高风英.温度对尼罗罗非鱼无乳链球菌毒力的影响.水产学报,2013,37(11):1733-1741.
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