鲍曼不动杆菌噬菌体SWH-Ab-3的生物学特性及其全基因组初步研究
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摘要
目的从医院污水中分离鲍曼不动杆菌裂解性噬菌体,探讨其生物学特性及基因组基本信息。方法以临床分离的鲍曼不动杆菌为指示菌,采用双层琼脂平板法分离、纯化噬菌体,观察噬菌斑特征;利用透射电子显微镜观察噬菌体的形态特征;采用点板试验测定噬菌体的裂菌谱,并观察其最佳感染复数(MOI)、一步生长曲线以及对热、酸碱等理化因素的耐受性。提取噬菌体全基因组DNA,采用Illumina Hiseq2000高通量测序,并对其基因组序列进行注释、比对分析。结果从污水中成功分离出1株鲍曼不动杆菌的裂解性噬菌体,命名为SWH-Ab-3;噬菌体头部直径约为70 nm,尾长约80 nm,初步判断其属肌尾噬菌体科;该噬菌体裂菌谱较窄(6株/42株),最佳感染复数为0.1,潜伏期为40 min,爆发期为50 min,裂解量为31.4 PFU/cell,对热和酸碱环境的耐受性较高。SWH-Ab-3基因组长度为41 730 bp,G+C%含量为39.38%,共编码53个ORF(33个已知功能基因,20个假想蛋白),其序列与Abp1和vB_AbaP_B3的同源性最高(≥94.0%),但基因组长度、G+C%含量和形态特征差异显著。结论 SWH-Ab-3为1株新分离的鲍曼不动杆菌裂解性噬菌体,属肌尾噬菌体科,对热、酸碱等理化因素较耐受,可能通过穿孔素和裂解酶组成的"二元裂解系统"发挥裂菌作用,裂解能力较强,但裂菌谱较窄。
Objective To investigate the biological characteristics and basic genomic information of Acinetobacter baumannii(A.baumannii) lytic bacteriophage isolated from hospital sewage. Methods Bacteriophage was isolated and purified with indicator of A.baumannii by double-layer agar plate method, and plaque characteristics of the phage were observed. The structure of purified phage was observed with transmission electron microscopy. The host spectrum was determined by point plate test, and optimal multiplicity of infection(MOI), one-step growth curve, temperature and pH stability of the phage were investigated. The whole genome DNA of phage was extracted and sequenced by Illumina Hiseq2000 high-throughput sequencing technology, and then the genome sequence was annotated and analyzed by alignment. Results A strain of lytic bacteriophage against the A.baumannii, named SWH-Ab-3, was isolated from sewage. The phage had a head of about 70 nm in diameter and a tail of about 80 nm in length. SWH-Ab-3 was identified to belong to Myoviridae family. The bacteriophage had a narrower lytic spectrum of A.baumannii(6/42), optimal MOI of 0.1, latent period of 40 min and burst period of 50 min, and the burst size about 31.4 PFU/cell, and could stand the temperature up to 60 ℃ and keep stable titer under acid or alkali environment. Its genome size was 41 730 bp with G+C content of 39.38%. There were 53 ORF encoded, of which 33 were known functional genes and 20 were hypothetical proteins. The BLASTn alignment found that SWH-Ab-3 had the highest homology with Abp1 and vB_AbaP_B3(> 94%), but the length of the genome, the G+C% content and the morphological characteristics of the phage were significant. Conclusion SWH-Ab-3 is a new lytic phage of A.baumannii, belonging to the Myoviridae family. It is tolerant to heat, acid and alkali. It may lyse the host bacteria through "two-element lysis system", composed of holin and lyase. It has stronger lytic ability, but narrower lytic spectrum.
Objective To investigate the biological characteristics and basic genomic information of Acinetobacter baumannii(A.baumannii) lytic bacteriophage isolated from hospital sewage. Methods Bacteriophage was isolated and purified with indicator of A.baumannii by double-layer agar plate method, and plaque characteristics of the phage were observed. The structure of purified phage was observed with transmission electron microscopy. The host spectrum was determined by point plate test, and optimal multiplicity of infection(MOI), one-step growth curve, temperature and pH stability of the phage were investigated. The whole genome DNA of phage was extracted and sequenced by Illumina Hiseq2000 high-throughput sequencing technology, and then the genome sequence was annotated and analyzed by alignment. Results A strain of lytic bacteriophage against the A.baumannii, named SWH-Ab-3, was isolated from sewage. The phage had a head of about 70 nm in diameter and a tail of about 80 nm in length. SWH-Ab-3 was identified to belong to Myoviridae family. The bacteriophage had a narrower lytic spectrum of A.baumannii(6/42), optimal MOI of 0.1, latent period of 40 min and burst period of 50 min, and the burst size about 31.4 PFU/cell, and could stand the temperature up to 60 ℃ and keep stable titer under acid or alkali environment. Its genome size was 41 730 bp with G+C content of 39.38%. There were 53 ORF encoded, of which 33 were known functional genes and 20 were hypothetical proteins. The BLASTn alignment found that SWH-Ab-3 had the highest homology with Abp1 and vB_AbaP_B3(> 94%), but the length of the genome, the G+C% content and the morphological characteristics of the phage were significant. Conclusion SWH-Ab-3 is a new lytic phage of A.baumannii, belonging to the Myoviridae family. It is tolerant to heat, acid and alkali. It may lyse the host bacteria through "two-element lysis system", composed of holin and lyase. It has stronger lytic ability, but narrower lytic spectrum.
引文
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[15] VERBEKEN G,DE VOS D,VANEECHOUTTE M,et al.European regulatory conundrum of phage therapy[J].Future Microbiol,2007,2(5):485-491.DOI:10.2217/17460913.2.5.485.
[16] MATSUZAKI S,RASHEL M,UCHIYAMA J,et al.Bacteriophage therapy:a revitalized therapy against bacterial infectious diseases[J].J Infect Chemother,2005,11(5):211-219.DOI:10.1007/s10156-005-0408-9.
[17] JEON J,D’SOUZA R,PINTO N,et al.Complete genome sequence of the siphoviral bacteriophage Β?-R3177,which lyses an OXA-66-producing carbapenem-resistant Acinetobacter baumannii isolate[J].Arch Virol,2015,160(12):3157-3160.DOI:10.1007/s00705-015-2604-y.
[18] 余丹静,史红艳,王丹,等.鲍曼不动杆菌噬菌体生物学特性的研究[J].微生物学杂志,2013,33(2):17-23.DOI:10.3969 /j.issn.1005-7021.2013.02.004.YU D J,SI H Y,WANG D,et al.Biologic characteristics of Bacteriophages specific to Acinetobacter baumannii[J].J Microbiol,2013,33(2):17-23.DOI:10.3969/j.issn.1005-7021.2013.02.004.
[19] 彭帆,童贻刚,柏长青.多耐药鲍曼不动杆菌噬菌体IME-AB6的分离、生物特性及保存方法研究[J].生物技术通讯,2013,24(5):641-644.DOI:10.3969/j.issn.1009-0002.2013.05.010.PENG F,TONG Y G,BO C Q.Isolation,biological characteristics and preservation methods of multidrug-resistant Acinetobacter baumannii bacteriophage IME-AB6 [J].Lett Biotech,2013,24(5):641-644.DOI:10.3969/j.issn.1009-0002.2013.05.010.
[20] 马翔宇,熊鸿燕,朱才众,等.新型多价大肠埃希菌噬菌体285P生物学特性研究[J].微生物学杂志,2008,28(5):12-15.DOI:10.3969/j.issn.1005-7021.2008.05.003.MA X Y,XIONG H Y,ZHU C Z,et al.Biological characteristics of a neo-polyvalent e.coli phage 285P[J].J Microbiol,2008,28(5):12-15.DOI:10.3969/j.issn.1005-7021.2008.05.003.
[21] WON G,HAJAM I A,LEE J H.Improved lysis efficiency and immunogenicity of Salmonella ghosts mediated by co-expression of λ phage holin-endolysin and ?X174 gene E[J].Sci Rep,2017,7:45139.DOI:10.1038/srep45139.
[22] CUI Z L,SHEN W B,WANG Z,et al.Complete genome sequence of Klebsiella pneumoniae phage JD001[J].J Virol,2012,86(24):13843.DOI:10.1128/JVI.02435-12.
[2] KARAGEORGOPOULOS D E,FALAGAS M E.Current control and treatment of multidrug-resistant Acinetobacter baumannii infections[J].Lancet Infect Dis,2008,8(12):751-762.DOI:10.1016/S1473-3099(08)70279-2.
[3] 朱育玮,李玉保,王守荣,等.噬菌体及其治疗细菌感染的研究进展[J].中国畜牧兽医,2015,42(3):769-773.DOI:10.11843/j.issn.1671-7236.2015.03.041.ZHU Y W,LI Y B,WANG S R,et al.Research progress on bacteriophage and the therapy in bacterial infection[J].China Anim Husband Vet Med,2015,42(3):769-773.DOI:10.11843/j.issn.1671-7236.2015.03.041.
[4] 王盛,童贻刚.噬菌体治疗研究进展[J].微生物学通报,2009,36(7):1019-1024.DOI:10.3969/j.issn.1671-6027.2002.04.004.WANG S,TONG Y G.Recent advance in bacteriophage therapy[J].Microbiol China,2009,36(7):1019-1024.DOI:10.3969/j.issn.1671-6027.2002.04.004.
[5] MATSUZAKI S,RASHEL M,UCHIYAMA J,et al.Bacteriophage therapy:A revitalized therapy against bacterial infectious diseases[J].J Infect Chemother,2005,11(5):211-219.DOI:10.1007/s10156-005-0408-9.
[6] VIERTEL T M,RITTER K,HORZ H P.Viruses versus bacteria-novel approaches to phage therapy as a tool against multidrug-resistant pathogens[J].J Antimicrob Chemother,2014,69(9):2326-2336.DOI:10.1093/jac/dku173.
[7] KUTTER E,SULAKVELIDZE A.Bacteriophages:biology and applications[M].Boca Raton:CRC Press,2004.
[8] MERABISHVILI M,VANDENHEUVEL D,KROPINSKI A M,et al.Characterization of newly isolated lytic bacteriophages active against Acinetobacter baumannii[J].PLoS ONE,2014,9(8):e104853.DOI:10.1371/journal.pone.0104853.eCollection 2014.
[9] 罗娟,张然,吴柳,等.鲍曼不动杆菌噬菌体SWH-Ab-1分离鉴定及其重要功能基因的生物信息学分析[J].第三军医大学学报,2018,40(1):23-30.DOI:10.16016/j.1000-5404.201708010.LUO J,ZHANG R,WU L,et al.Isolation and identification of phage SWH-Ab-1 against Acinetobacter baumannii and bioinformatic analysis of its major functional genes[J].J Third Mil Med Univ,2018,40(1):23-30.DOI:10.16016/j.1000-5404.201708010.
[10] 邢少贞,张湘莉兰,舒鹏,等.粪肠球菌噬菌体vB_E.faecalis_IME196的生物学特性及其全基因组分析[J].微生物学通报,2016,43(9):2040-2048.DOI:10.13344/j.microbiol.china.151053.XING S Z,ZHANG X L L,SHU P,et al.Characterization and complete genome sequence analysis of Enterococcus faecalis bacteriophage VBE.faecalisIME196[J].Microbiol China,2016,43(9):2040-2048.DOI:10.13344/j.microbiol.china.151053.
[11] HUANG H,DONG Y,YANG Z L,et al.Complete sequence of pABTJ2,a plasmid from Acinetobacter baumannii MDR-TJ,carrying many phage-like elements[J].Genom Proteom Bioinf,2014,12(4):172-177.DOI:10.1016/j.gpb.2014.05.001.
[12] MENDES J J,LEANDRO C,MOTTOLA C,et al.In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infections[J].J Med Microbiol,2014,63(Pt_8):1055-1065.DOI:10.1099/jmm.0.071753-0.
[13] JIN J,LI Z J,WANG S W,et al.Isolation and characterization of ZZ1,a novel lytic phage that infects Acinetobacter baumannii clinical isolates[J].BMC Microbiol,2012,12:156.DOI:10.1186/1471-2180-12-156.
[14] LU T K,KOERIS M S.The next generation of bacteriophage therapy[J].Curr Opin Microbiol,2011,14(5):524-531.DOI:10.1016/j.mib.2011.07.028.
[15] VERBEKEN G,DE VOS D,VANEECHOUTTE M,et al.European regulatory conundrum of phage therapy[J].Future Microbiol,2007,2(5):485-491.DOI:10.2217/17460913.2.5.485.
[16] MATSUZAKI S,RASHEL M,UCHIYAMA J,et al.Bacteriophage therapy:a revitalized therapy against bacterial infectious diseases[J].J Infect Chemother,2005,11(5):211-219.DOI:10.1007/s10156-005-0408-9.
[17] JEON J,D’SOUZA R,PINTO N,et al.Complete genome sequence of the siphoviral bacteriophage Β?-R3177,which lyses an OXA-66-producing carbapenem-resistant Acinetobacter baumannii isolate[J].Arch Virol,2015,160(12):3157-3160.DOI:10.1007/s00705-015-2604-y.
[18] 余丹静,史红艳,王丹,等.鲍曼不动杆菌噬菌体生物学特性的研究[J].微生物学杂志,2013,33(2):17-23.DOI:10.3969 /j.issn.1005-7021.2013.02.004.YU D J,SI H Y,WANG D,et al.Biologic characteristics of Bacteriophages specific to Acinetobacter baumannii[J].J Microbiol,2013,33(2):17-23.DOI:10.3969/j.issn.1005-7021.2013.02.004.
[19] 彭帆,童贻刚,柏长青.多耐药鲍曼不动杆菌噬菌体IME-AB6的分离、生物特性及保存方法研究[J].生物技术通讯,2013,24(5):641-644.DOI:10.3969/j.issn.1009-0002.2013.05.010.PENG F,TONG Y G,BO C Q.Isolation,biological characteristics and preservation methods of multidrug-resistant Acinetobacter baumannii bacteriophage IME-AB6 [J].Lett Biotech,2013,24(5):641-644.DOI:10.3969/j.issn.1009-0002.2013.05.010.
[20] 马翔宇,熊鸿燕,朱才众,等.新型多价大肠埃希菌噬菌体285P生物学特性研究[J].微生物学杂志,2008,28(5):12-15.DOI:10.3969/j.issn.1005-7021.2008.05.003.MA X Y,XIONG H Y,ZHU C Z,et al.Biological characteristics of a neo-polyvalent e.coli phage 285P[J].J Microbiol,2008,28(5):12-15.DOI:10.3969/j.issn.1005-7021.2008.05.003.
[21] WON G,HAJAM I A,LEE J H.Improved lysis efficiency and immunogenicity of Salmonella ghosts mediated by co-expression of λ phage holin-endolysin and ?X174 gene E[J].Sci Rep,2017,7:45139.DOI:10.1038/srep45139.
[22] CUI Z L,SHEN W B,WANG Z,et al.Complete genome sequence of Klebsiella pneumoniae phage JD001[J].J Virol,2012,86(24):13843.DOI:10.1128/JVI.02435-12.