一株新型杀鲑气单胞菌烈性类T4噬菌体Asfd-1的分离鉴定和生物学特性分析
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摘要
噬菌体是自然界中最普遍的生物之一,其专一性裂解细菌的能力在针对耐药菌感染治疗方面具有很大的应用前景。杀鲑气单胞菌(Aeromonas salmonicide)是水产养殖业中一种主要病原菌,其引发的疖疮病是一种常见的水产养殖动物疾病,具有高死亡率和高发病率,一旦爆发,将给水产养殖业带来重大的经济损失。该研究以杀鲑气单胞菌MF663675.1为宿主菌,从深圳海鲜市场的污水中,采用双层琼脂平板法分离纯化得到单个噬菌体,通过电镜对其表观形态特征进行观察,并运用生物信息的方法进一步确定其基因型特征及遗传分类地位。研究结果表明,分离出的烈性噬菌体命名为Asfd-1,噬菌斑呈圆形,轮廓分明、透明,直径约1mm。电镜观察显示,Asfd-1具有对称的二十面体头部,以及长度为51 nm的收缩尾部。基因组测序和基因对比分析表明,Asfd-1含双链脱氧核糖核酸(dsDNA),全基因组大小为168 962 bp,鸟嘌呤(G)和胞嘧啶(C)所占的比例为41.53%,共编码263个开放阅读框和16个转移核糖核酸(tRNA)序列,Genbank登录号为MK577502。根据国际病毒分类委员会规定的病毒分类方法,与同源的Aeromonas phage 31 GCA_000862645.1和Aeromonas phiSA4GCA_002710125.1全基因组对比结果显示,Asfd-1是新型噬菌体。综合电镜观察结果及系统进化树分析,Asfd-1属于肌尾科下的T4噬菌体属、肌尾噬菌体科、有尾噬菌体目的新成员。该研究可为应用噬菌体治疗细菌感染提供进一步的参考。
Bacteriophages are one of the most common microorganisms on the earth, and are potential biocontrol agents for multidrug-resistant bacteria. Aeromonas salmonicide is a major bacterial pathogen in aquaculture. It can be responsible for furunculosis, which is a ubiquitous disease in aquaculture operation and featured by high mortality and morbidity, causing significant economic loss of aquaculture. In this study,Aeromonas salmonicida MF663675.1 was used as host to isolate bacteriophage from the sewage of a seafood market in Shenzhen, China. A novel lytic phage Asfd-1 was isolated and purified by double-layer agar plate.The morphological characteristics were observed by transmission electron microscope(TEM). The plaque characteristics were observed as well. The whole genome and phylogenetic analysis of phage Asfd-1 were carried out by bioinformatics methods. The results show that plaque of the phage was round and transparent with size of about 1 mm in diameter. TEM shows that Asfd-1 has an isometric, elongated and icosahedral head of 100 nm in diameter by 71 nm in width and a 51 nm contracted tail. Comparative genomics analysis indicates that Asfd-1 has a double-stranded DNA of 168 962 bp in length, and the content ratio of guanine and cytosine is 41.53%. Asfd-1 genome encodes 263 open reading frames and 16 tRNA genes, indicating that Asfd-1 is not totally dependent on host cell transcription machinery. Combined with TEM and phylogenetic analysis, it proposes that Asfd-1 belongs a new member of the T4 virus, Myoviridae family, Caudovirales order. Based on its microbiological traits, phage Asfd-1 is a promising agent that can be used for controlling Aeromonas salmonicida infection.
Bacteriophages are one of the most common microorganisms on the earth, and are potential biocontrol agents for multidrug-resistant bacteria. Aeromonas salmonicide is a major bacterial pathogen in aquaculture. It can be responsible for furunculosis, which is a ubiquitous disease in aquaculture operation and featured by high mortality and morbidity, causing significant economic loss of aquaculture. In this study,Aeromonas salmonicida MF663675.1 was used as host to isolate bacteriophage from the sewage of a seafood market in Shenzhen, China. A novel lytic phage Asfd-1 was isolated and purified by double-layer agar plate.The morphological characteristics were observed by transmission electron microscope(TEM). The plaque characteristics were observed as well. The whole genome and phylogenetic analysis of phage Asfd-1 were carried out by bioinformatics methods. The results show that plaque of the phage was round and transparent with size of about 1 mm in diameter. TEM shows that Asfd-1 has an isometric, elongated and icosahedral head of 100 nm in diameter by 71 nm in width and a 51 nm contracted tail. Comparative genomics analysis indicates that Asfd-1 has a double-stranded DNA of 168 962 bp in length, and the content ratio of guanine and cytosine is 41.53%. Asfd-1 genome encodes 263 open reading frames and 16 tRNA genes, indicating that Asfd-1 is not totally dependent on host cell transcription machinery. Combined with TEM and phylogenetic analysis, it proposes that Asfd-1 belongs a new member of the T4 virus, Myoviridae family, Caudovirales order. Based on its microbiological traits, phage Asfd-1 is a promising agent that can be used for controlling Aeromonas salmonicida infection.
引文
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[14]Yamamoto KR,Alberts BM,Benzinger R,et al Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to largescale virus purification[J].Virology,1970,40(3):734-744.
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[20]Morita M,Tanji Y,MizoguchiK,et al.Characterization of a virulent bacteriophage specific for Escherichia coli O157:H7 and analysis of its cellular receptor and two tail fiber genes[J].FEMSMicrobiology Letters,2002,211(1):77-83.
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[26]Kao SH,Mcclain WH.Baseplate protein of bacteriophage T4 with both structural and lytic functions[J].Journal of Virology,1980,34(1):95-103.
[27]Bo H,William M,Molineux IJ,et al.Structural remodeling of bacteriophage T4 and host membranes during infection initiation[J]Proceedings of the National Academy of Sciences2015,112(35):4919-4928.
[28]Fan JQ,Chen LM,Mai GQ,et al.Dynamics of the gut microbiota in developmental stages of Litopenaeus vannameireveal its association with body weight[J].Scientific Reports,2018,9:734.
[29]Chen L,Yuan SJ,Liu Q,et al.In vitro design and evaluation of phage cocktails against Aeromonas salmonicida[J].Frontiers in Microbiology,2018,9:1476.
[30]傅强,单文雅,王兆飞,等.一株强裂解性大肠杆菌T1样噬菌体新成员的分离与鉴定[J].微生物学通报,2017(12):18-30.
[2]Rachna T,Mridu D,Shyamasree N,et al.Isolation of Aeromonas salmonicida from human blood sample:a case report[J].Journal of Clinical&Diagnostic Research Jcdr,2014,8(2):139-140.
[3]Mccarthy DH.An experimental model for fish furunculosis caused by Aeromonas salmonicida[J]Journal of Fish Diseases,2010,6(3):231-237.
[4]Scott M.The pathogenicity of Aeromon as salmonicida(Griffin)in sea and brackish waters[J]Journal of General Microbiology,1968,50(2):321.
[5]Dallaire-Dufresne S,Tanaka KH,Trudel MV,et al.Virulence,genomic features,and plasticity of Aeromonas salmonicida subsp.salmonicida,the causative agent of fish furunculosis[J].Veterinary Microbiology,2014,169(1-2):1-7.
[6]Stoffrengen DA,Chako AJ,Backman S,et al Successful therapy of furunculosis in Atlantic salmon,Salmo salar L.,using the fluoroquinolone antimicrobial agent enrofloxacin[J].Journal of Fish Diseases,1993,16(3):219-228.
[7]Flakas K.Sulfonamide therapy of furunculosis in brown trout[J].Transactions of the American Fisheries Society,2011,78(1):117-127.
[8]Liu PF,Du YS,Meng LJ,et al.ALDH7A1 is a protein that protects Atlantic salmon against Aeromonas salmonicida at the early stages of infection[J].Fish&Shellfish Immunology,201770:30-39.
[9]Liao WC,Ng WV,Lin IH,et al.T4-like genome organization of the Escherichia coli O157:H7 lytic phage AR1[J].Journal of Virology,2011,85(13):6567-6578.
[10]Sulakvelidze A.Bacteriophage:a new journal for the most ubiquitous organisms on Earth[J].Bacteriophage,2011,1(1):1.
[11]Carlton RM.Phage therapy:past history and future prospects[J].Archivum Immunologiae et Therapiae Experimentalis,1999,47(5):267-274.
[12]Ernst B,Kolb H,Cutting B,et al.Drug discovery today[J].Frontiers in Medicinal Chemistry-Online2005,2(1):533-543.
[13]Yuan SJ,Chen L,Liu Q,et al.Characterization and genomic analyses of Aeromonas hydrophila phages AhSzq-1 and AhSzw-1,isolates representing new species within the T5virus genus[J].Archives of Virology,2018,163(7):1985-1988.
[14]Yamamoto KR,Alberts BM,Benzinger R,et al Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to largescale virus purification[J].Virology,1970,40(3):734-744.
[15]Williams DB,Carter CB.Transmission electron microscopy[J].Springer Science+Business Media1996,4:729.
[16]Luo R,Liu B,Xie Y,et al.SOAPdenovo2:an empirically improved memory-efficient short-read de novo assembler[J].Gigascience,2012,1(1):18.
[17]John B,Mark B.GeneMark:web software for gene finding in prokaryotes,eukaryotes and viruses[J]Nucleic Acids Research,2005,33(33):451-454.
[18]Lavigne R,Seto D,Mahadevan P,et al.Unifying classical and molecular taxonomic classification:analysis of the Podoviridae using BLASTP-based tools[J].Research in Microbiology,2008,159(5):406-414.
[19]Sullivan MJ,Petty NK,Beatson SA.Easyfig:a genome comparison visualizer[J].Bioinformatics2011,27(7):1009-1010.
[20]Morita M,Tanji Y,MizoguchiK,et al.Characterization of a virulent bacteriophage specific for Escherichia coli O157:H7 and analysis of its cellular receptor and two tail fiber genes[J].FEMSMicrobiology Letters,2002,211(1):77-83.
[21]Maniloff J,Ackermann HW.Taxonomy of bacterial viruses:establishment of tailed virus genera and the other Caudovirales[J].Archives of Virology,1998143(10):2051-2063.
[22]Adriaenssens E,Brister JR.How to name and classify your phage:an informal guide[J].Viruses2017,9(4):70.
[23]Rackwitz HR,Zehetner G,Murialdo H,et al Analysis of cosmids using linearization by phage lambda terminase[J].Gene,1985,40(2):259-266.
[24]Martín AC,López R,García P.Functional analysis of the two-gene lysis system of the pneumococcal phage Cp-1 in homologous and heterologous host cells[J].Journal of Bacteriology,1998,180(2):210.
[25]Fouts DE,Rasko DA,Cer RZ,et al.Sequencing bacillus anthracis typing phages gamma and cherry reveals a common ancestry[J].Journal of Bacteriology,2006,188(9):3402-3408.
[26]Kao SH,Mcclain WH.Baseplate protein of bacteriophage T4 with both structural and lytic functions[J].Journal of Virology,1980,34(1):95-103.
[27]Bo H,William M,Molineux IJ,et al.Structural remodeling of bacteriophage T4 and host membranes during infection initiation[J]Proceedings of the National Academy of Sciences2015,112(35):4919-4928.
[28]Fan JQ,Chen LM,Mai GQ,et al.Dynamics of the gut microbiota in developmental stages of Litopenaeus vannameireveal its association with body weight[J].Scientific Reports,2018,9:734.
[29]Chen L,Yuan SJ,Liu Q,et al.In vitro design and evaluation of phage cocktails against Aeromonas salmonicida[J].Frontiers in Microbiology,2018,9:1476.
[30]傅强,单文雅,王兆飞,等.一株强裂解性大肠杆菌T1样噬菌体新成员的分离与鉴定[J].微生物学通报,2017(12):18-30.