一株从草鱼中分离的嗜水气单胞菌的病原学及基因组特征
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摘要
为确定南昌地区某渔场草鱼出血性败血症的病原体及病原特征,从患病草鱼的肝脏病灶中分离出一株致病菌A1310。对分离菌进行了形态特征、理化特性等表型生物学检验、人工感染实验及对抗菌药物的敏感性实验,并对其进行了全基因组测序、基于多序列位点分型(multilocus sequence typing,MLST)的系统进化分析及毒力基因分析。结果显示,生理生化鉴定证明该菌为嗜水气单胞菌,当浓度达1.0×10~6 CFU/mL时,对草鱼有致病性;对供试20种抗菌药物中的青霉素等7种耐药,对卡那霉素等5种敏感;A1310株基因组框架序列共包含96个与毒力和防御相关基因,其中多药耐药性外排泵基因占大多数,约为22%;与美国斑点叉尾鮰分离株(S15-242、S15-458、S15-591、S15-700)聚类为同一进化分支;比较基因组分析发现,A1310具有一个删减版的Ⅵ型分泌系统(typeⅥsecretion system,T6SS),基因数量为标准Ⅵ型分泌系统的80%,缺少vgr G和vca0109基因的部分片段。综上所述,来源于草鱼的嗜水气单胞菌菌株A1310,与美国斑点叉尾鮰分离株具有亲缘关系,含有多个已报道的嗜水气单胞菌的毒力基因。本研究不仅丰富了嗜水气单胞菌的生物学性状内容,也为嗜水气单胞菌的有效检验、防治和深入研究提供一定的参考,对草鱼的疾病防控具有一定意义。
In order to determine the pathogen and pathogenic characteristics of Ctenopharyngodon idella haemorrhagic septicemia in a farm of Nanchang, a strain of pathogenic bacteria A1310 was isolated from the liver lesions of the diseased C. idella. The phenotypic characteristics of the isolated strain A1310 included the morphological characteristics, physiological and biochemical characteristics, pathogenicity, susceptibility test. In addition, the whole genome sequencing, phylogeny-based on multilocus sequence typing(MLST) and virulence gene were examined. According to morphological, biochemical characteristics, the pathogen was identified as Aeromonas hydrophilia. Artificial infection experiments showed that it is pathogenic to C. idella after being infected with the 1.0×106 CFU/mL bacteria strains culture. Antibiotic sensitivity test using 20 antimicrobial agents showed that the isolate was resistant to 7 antibiotics including penicillin, but was sensitive to 5 antibiotics including Kanamycin. The A1310 strain genome sequence contains a total of 96 genes related to toxicity and defense, and most of them are multidrug resistance efflux pump genes, about 22%. MLST typing showed that this isolate was genetically related to the American channel catfish isolates(S15-242, S15-458, S15-591, S15-700).Moreover, the comparative genomic analysis showed that A1310 has at least 80% of the core proteins necessary for a complete T6 SS, lacking a segment of vgr G and a segment of vca0109. In conclusion, A. hydrophila A1310 from C. idella was genetically related to the American channel catfish isolates genetically and contains multiple reported virulence genes of A. hydrophilia. This study broadened our knowledge to understand the biological traits of A. hydrophlia. This can provide some reference for the effective inspection, prevention, and in-depth study of A.hydrophilia, and it has some significance for the prevention and control of C. idella disease and will be a basis for the disease control during C. idella culture.
In order to determine the pathogen and pathogenic characteristics of Ctenopharyngodon idella haemorrhagic septicemia in a farm of Nanchang, a strain of pathogenic bacteria A1310 was isolated from the liver lesions of the diseased C. idella. The phenotypic characteristics of the isolated strain A1310 included the morphological characteristics, physiological and biochemical characteristics, pathogenicity, susceptibility test. In addition, the whole genome sequencing, phylogeny-based on multilocus sequence typing(MLST) and virulence gene were examined. According to morphological, biochemical characteristics, the pathogen was identified as Aeromonas hydrophilia. Artificial infection experiments showed that it is pathogenic to C. idella after being infected with the 1.0×106 CFU/mL bacteria strains culture. Antibiotic sensitivity test using 20 antimicrobial agents showed that the isolate was resistant to 7 antibiotics including penicillin, but was sensitive to 5 antibiotics including Kanamycin. The A1310 strain genome sequence contains a total of 96 genes related to toxicity and defense, and most of them are multidrug resistance efflux pump genes, about 22%. MLST typing showed that this isolate was genetically related to the American channel catfish isolates(S15-242, S15-458, S15-591, S15-700).Moreover, the comparative genomic analysis showed that A1310 has at least 80% of the core proteins necessary for a complete T6 SS, lacking a segment of vgr G and a segment of vca0109. In conclusion, A. hydrophila A1310 from C. idella was genetically related to the American channel catfish isolates genetically and contains multiple reported virulence genes of A. hydrophilia. This study broadened our knowledge to understand the biological traits of A. hydrophlia. This can provide some reference for the effective inspection, prevention, and in-depth study of A.hydrophilia, and it has some significance for the prevention and control of C. idella disease and will be a basis for the disease control during C. idella culture.
引文
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[28]李俊,俞盈,王豪举.细菌Ⅵ型分泌系统的研究进展[J].微生物学报,2011,51(3):291-296.Li J,Yu Y,Wang H J.Advances in bacterial typeⅥsecretion system-a review[J].Acta Microbiologica Sinica,2011,51(3):291-296(in Chinese).
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[2]Sahoo P K,Mahapatra K D,Saha J N,et al.Family association between immune parameters and resistance to Aeromonas hydrophila infection in the Indian major carp,Labeo rohita[J].Fish&Shellfish Immunology,2008,25(1-2):163-169.
[3]陆承平.致病性嗜水气单胞菌及其所致鱼病综述[J].水产学报,1992,16(3):282-288.Lu C P.Pathogenic Aeromonas hydrophila and the fish diseases caused by it[J].Journal of Fisheries of China,1992,16(3):282-288(in Chinese).
[4]田甜.鱼源嗜水气单胞菌的分离鉴定及耐药性研究[D].武汉:华中农业大学,2010.Tian T.Isolation and identification of Aeromonas hydrophila strains from freshwater fish and their antibacterial resistance[D].Wuhan:Huazhong Agricultural University,2010(in Chinese).
[5]陆英杰,杜玉东,王志彪,等.疑似草鱼嗜水气单胞菌的分离鉴定及药敏试验[J].中国畜牧兽医,2013,40(7):62-65.Lu Y J,Du Y D,Wang Z B,et al.Identification and drug sensitive test of suspected grass carp Aeromonas hydrophila[J].China Animal Husbandry&Veterinary Medicine,2013,40(7):62-65(in Chinese).
[6]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.Cai M Y,Dong X Z.Common bacterial system identification manual[M].Beijing:Science Press,2001(in Chinese).
[7]杨正时,房海.人及动物病原细菌学[M].石家庄:河北科学技术出版社,2003.Yang Z S,Fang H.Human and animal pathogenic bacteriology[M].Shijiazhuang:Hebei Science and Technology Press,2003(in Chinese).
[8]叶应妩,王毓三.全国临床检验操作规程[M].南京:东南大学出版社,1991.Ye Y F,Wang Y S.National clinical inspection procedures[M].Nanjing:Southeast University Press,1991(in Chinese).
[9]Zerbino D R,Birney E.Velvet.Algorithms for de novo short read assembly using de Bruijn graphs[J].Genome Research,2008,18(5):821-829.
[10]Aziz R K,Bartels D,Best A A,et al.The RAST server:rapid annotations using subsystems technology[J].BMCGenomics,2008,9:75.
[11]Holt J G,Krieg N R,Sneath P H A,et al.Bergy’s manual of determinative bacteriology[M].9th ed.Baltimore:A Waverly Company,1994.
[12]Austin B,Austin D A.Bacterial fish pathogens:disease of farmed and wild fish[M]//Bacterial Fish Pathogens:Disease in Farmed and Wild Fish.E.Horwood,Halsted Press,1999.
[13]Rasmussen-Ivey C R,Hossain M J,Odom S E,et al.Classification of a hypervirulent Aeromonas hydrophila pathotype responsible for epidemic outbreaks in warmwater fishes[J].Frontiers in Microbiology,2016,7:1615.
[14]肖丹,曹海鹏,胡鲲,等.淡水养殖动物致病性嗜水气单胞菌ERIC-PCR分型与耐药性[J].中国水产科学,2011,18(5):1092-1099.Xiao D,Cao H P,Hu K,et al.ERIC-PCR genotyping and drug resistant analysis of pathogenic Aeromonas hydrophila from freshwater animals[J].Journal of Fishery Sciences of China,2011,18(5):1092-1099(in Chinese).
[15]李楠,孙祎敏,田莉瑛,等.嗜水气单胞菌的分离培养及鉴定[J].河北师范大学学报(自然科学版),2009,33(2):240-243.Li N,Sun Y M,Tian L Y,et al.The isolation and identification of Aeromonas hydrophila[J].Journal of Hebei Normal University(Natural Science Edition),2009,33(2):240-243(in Chinese).
[16]王春燕.异育银鲫败血病病原体的诊断及毒力和药敏实验[J].生物学杂志,2002,19(6):30-31.Wang C Y.The pathogenicity and drug sensitivity tests of bacterial septicemia from Carassius auratus gibelio[J].Journal of Biology,2002,19(6):30-31(in Chinese).
[17]陈翠珍,张晓君,房海,等.鱼气单胞菌感染症及其病原的检验与分析[J].华北农学报,2000,15(S1):178-184.Chen C Z,Zhang X J,Fang H,et al.Examination and analysis on the infection by Aeromona of fish and pathogeny[J].Acta Agriculturae Boreali-Sinica,2000,15(S1):178-184(in Chinese).
[18]周剑光,杨先乐,艾晓辉.中华鳖疖疮、红脖子、赤斑病并发症的病原研究[J].水产学报,1999,23(3):270-277.Zhou J G,Yang X L,Ai X H.Pathogen of the furunculosis,red neck and red spot complicating diseases of Trionyx sinensis[J].Journal of Fisheries of China,1999,23(3):270-277(in Chinese).
[19]李雪,金莉莉,王秋雨.病原微生物分子分型技术研究进展[J].中国公共卫生,2007,23(3):373-374.Li X,Jin L L,Wang Q Y.Advances in molecular typing techniques of pathogenic microorganisms[J].Chinese Public Health,2007,23(3):373-374(in Chinese).
[20]毕春霞,闫志勇,王斌.16S rRNA基因及16S~23SrRNA基因区间在临床细菌学检验中的应用[J].青岛大学医学院学报,2003,39(4):493-495,498.Bi C X,Yan Z Y,Wang B.Application of 16S rRNA gene and 16S~23S rRNA gene interval in clinical bacteriology[J].Acta Academiae Medicinae Qingdao Universitatis,2003,39(4):493-495,498(in Chinese).
[21]Gonzalez-Escalona N,Martinez-Urtaza J,Romero J,et al.Determination of molecular phylogenetics of Vibrio parahaemolyticus strains by multilocus sequence typing[J].Journal of Bacteriology,2008,190(8):2831-2840.
[22]王晓伟.不同地区自然发酵乳制品中瑞士乳杆菌的多位点序列分型研究[D].呼和浩特:内蒙古农业大学,2015.Wang X W.Multilocus sequence typing of lactobacillus helveticus from naturally fermented dairy products in different regions[D].Hohhot:Inner Mongolia Agricultural University,2015(in Chinese).
[23]吴亚锋.南京地区气单胞菌的分离鉴定及嗜水气单胞菌菌株分型研究[D].南京:南京农业大学,2015.Wu Y F.Isolation and identification of Aeromonas from Nanjing and typing of A.hydrophila strains[D].Nanjing:Nanjing Agricultural University,2015(in Chinese).
[24]王娜,张小军,刘永杰,等.嗜水气单胞菌Ⅲ型分泌系统研究进展[J].河北科技师范学院学报,2011,25(1):21-24.Wang N,Zhang X J,Liu Y J,et al.Research progress of typeⅢsecretion system in Aeromonas hydrophila[J].Journal of Hebei Normal University of Science&Technology,2011,25(1):21-24(in Chinese).
[25]郝彬.鳗弧菌Ⅵ型分泌系统(T6SS)的功能及调控研究[D].北京:中国科学院研究生院(海洋研究所),2012.Hao B.Research on the function and regulation of type VI secretion system(T6SS)in Vibrio anguillarum[D].Beijing:University of Chinese Academy of Sciences,2012(in Chinese).
[26]Young G M,Schmiel D H,Miller V L.A new pathway for the secretion of virulence factors by bacteria:the flagellar export apparatus functions as a protein-secretion system[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(11):6456-6461.
[27]徐晓蒙.基于Ion Torrent平台测序数据的微生物全基因组序列组装及分析方法[D].合肥:安徽医科大学,2015.Xu X M.Whole microbial genome assembly and analysis based on Ion Torrent sequencing data[D].Hefei:Anhui Medical University,2015(in Chinese).
[28]李俊,俞盈,王豪举.细菌Ⅵ型分泌系统的研究进展[J].微生物学报,2011,51(3):291-296.Li J,Yu Y,Wang H J.Advances in bacterial typeⅥsecretion system-a review[J].Acta Microbiologica Sinica,2011,51(3):291-296(in Chinese).
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