GAO Mingming,LIU Huiying,LI Puyuan,et al.Isolation and complete genome analysis of Staphylococcus aureus bacteriophage vB_SauH_IME522[J].J Third Mil Med Univ,2020,42(03):229-240.

金黄色葡萄球菌噬菌体vB_SauH_IME522的分离鉴定及全基因组分析(/HTML )




Isolation and complete genome analysis of Staphylococcus aureus bacteriophage vB_SauH_IME522
GAO Mingming LIU Huiying LI Puyuan ZHANG Zhihua QIN Yanhong HUANG Yong CUI Yujun PEI Guangqian TONG Yigang BAI Changqing
Department of Graduate, Hebei North University, Zhangjiakou, Hebei Province, 075000; 2Department of Respiratory and Critical Care Diseases, the Fifth Medical Center,  PLA Chinese General Hospital, Beijing, 100071; 3State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, 100071; 4College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China
Q939.48; R372; R378.11

目的 从医院污水中分离到一株金黄色葡萄球菌噬菌体vB_SauH_IME522,对其进行分离鉴定、测序及进化和基因组学的分析。方法以一株从临床分离到的多重耐药的金黄色葡萄球菌为宿主菌分离噬菌体,应用双层琼脂平板法进行噬菌体最佳感染复数(optimal MOI)、一步生长曲线(one-step growth curve)实验,使用蔗糖密度梯度离心纯化病毒,并通过透射电镜观察噬菌体形态;应用SDS/蛋白酶裂解方案提取噬菌体全基因组,使用Illumina Miseq测序平台进行噬菌体全基因组测序,测序后对噬菌体全基因组序列进行组装、注释、进化和比较基因组学分析。结果分离到一株金黄色葡萄球菌噬菌体,命名为vB_SauH_IME522;其最佳感染复数为0.01,一步生长曲线结果显示,其感染宿主菌的潜伏期约为20 min,裂解期约为200 min,平均裂解量89 PFU/cell;电镜观察该噬菌体,噬菌体vB_SauH_IME522具有典型的二十面体结构和收缩的尾部,其头部直径为(70±1)nm,尾部长度为(135±2)nm。基因组测序结果表明,噬菌体基因组全长为140 246 bp,G+C含量为30.2 %。基因组注释显示噬菌体vB_SauH_IME522含有223个蛋白编码基因、164个启动子、66个终止子、4个tRNAs及7个耐药相关位点。其中52个基因的编码产物有预测的功能,其余基因的编码产物均为功能未知的推定蛋白。系统发生分析表明,该噬菌体属于Twortvirinae亚科的Kayvirus属。结论烈性噬菌体vB_SauH_IME522为Twortvirinae亚科的新成员,具有较强的裂解能力和短的潜伏期。


Objective To isolate, sequence, and identify biological characterization and complete genome sequencing of a virulent Staphylococcus aureus (S. aureus) bacteriophage vB_SauH_IME522. MethodsA phage was isolated from sewage of  PLA Chinese General Hospital using a clinically isolated S. aureus strain as host bacteria. The double-layer agar plate was used to determine the optimal multiplicity of infection (optimal MOI) and one-step growth curve. Phage morphology was observed by transmission electron microscopy after purification using sucrose gradient separation. Phage DNA was sequenced using the Illumina Miseq sequencing platform. The complete genome sequence was annotated, then comparative genomics and evolutionary analyses were performed. ResultsA lytic phage of S. aureus was isolated from hospital sewage and named as vB_SauH_IME522. The optimal MOI of the phage was 0.01. One step growth curve showed that its latent period and burst period were 20 and 200 min, respectively. The burst size was about 89 PFU/cell. Electron microscopy displayed the phage having an icosahedral head (70±1) nm and a long contractile tail (135±2) nm. Genome sequencing results showed that the total length of the phage genome was 140 246 bp and the G+C content accounted for 30.2%. The genomic annotation revealed that the phage contained 223 genes coding proteins, 164 putative promoters, 66 terminators, 4 tRNAs, and 7 resistance-related regions. Phylogenetic analysis suggested that the phage was a member of the genus Kayvirus within the subfamily Twortvirinae. ConclusionA lytic phage of S. aureus vB_SauH_IME522 is identified as a new member of the Twortvirinae subfamily, with strong lytic ability and short latent period.


[1]GRAHAM P L 3rd, LIN S X, LARSON E L. A US population-based survey of Staphylococcus aureus colonization[J]. Ann Intern Med, 2006, 144(5): 318-325. DOI:10.7326/0003-4819-144-5-200603070-00006.
[2]GORWITZ R J, KRUSZON-MORAN D, MCALLISTER S K, et al. Changes in the prevalence of nasal colonization with Staphylococcus aureus in the United States, 2001-2004[J]. J Infect Dis, 2008, 197(9): 1226-1234. DOI:10.1086/533494.
[3]KENGNE M, FOTSING O, NDOMGUE T, et al. Antibiotic susceptibility patterns of Staphylococcus aureus strains isolated at the yaounde central hospital, Cameroon: a retro prospective study[J]. Pan Afr Med J, 2019, 32: 103. DOI:10.11604/pamj.2019.32.103.15743.
[4]NNIS SYSTEM. National nosocomial infections surveillance (NNIS) system report, data summary from January 1990-May 1999, issued June 1999. A report from the NNIS system[J]. Am J Infect Control, 1999, 27(6): 520-532. DOI:10.1016/s0196-6553(99)70031-3.
[5]JULANDER I. Unfavourable prognostic factors in Staphylococcus aureus septicemia and endocarditis[J]. Scand J Infect Dis, 1985, 17(2): 179-187. DOI:10.3109/inf.1985.17.issue-2.09.
[6]SONG J H, HSUEH P R, CHUNG D R, et al. Spread of methicillin-resistant Staphylococcus aureus between the community and the hospitals in Asian countries: an ANSORP study[J]. J Antimicrob Chemother, 2011, 66(5): 1061-1069. DOI:10.1093/jac/dkr024.
[7]OTTER J A, FRENCH G L. Community-associated meticillin-resistant Staphylococcus aureus strains as a cause of healthcare-associated infection[J]. J Hosp Infect, 2011, 79(3): 189-193. DOI:10.1016/j.jhin.2011.04.028.
[8]MARRIS E. Bugs gain vital ground in their battle against drugs[J]. Nat Med, 2005, 11(5): 461. DOI:10.1038/nm0505-461b.
[9]MILLER L G, PERDREAU-REMINGTON F, RIEG G, et al. Necrotizing fasciitis caused by community-associated methicillin-resistant Staphylococcus aureus in los angeles[J]. N Engl J Med, 2005, 352(14): 1445-1453. DOI:10.1056/NEJMoa042683.
[10]DANTES R, MU Y, BELFLOWER R, et al. National burden of invasive methicillin-resistant Staphylococcus aureus infections, United States, 2011[J]. JAMA Intern Med, 2013, 173(21): 1970-1978. DOI:10.1001/jamainternmed.2013.10423.
[11]O’FLAHERTY S, ROSS R P, MEANEY W, et al. Potential of the polyvalent anti-Staphylococcus bacteriophage K for control of antibiotic-resistant staphylococci from hospitals[J]. Appl Environ Microbiol, 2005, 71(4): 1836-1842. DOI:10.1128/aem.71.4.1836-1842.2005.
[12]CAPPARELLI R, NOCERINO N, LANZETTA R, et al. Bacteriophage-resistant Staphylococcus aureus mutant confers broad immunity against staphylococcal infection in mice[J]. PLoS ONE, 2010, 5(7): e11720. DOI:10.1371/journal.pone.0011720.
[13]GUPTA R, PRASAD Y. Efficacy of polyvalent bacteriophage P-27/HP to control multidrug resistant Staphylococcus aureus associated with human infections[J]. Curr Microbiol, 2011, 62(1): 255-260. DOI:10.1007/s00284-010-9699-x.
[14]RHOADS D D, WOLCOTT R D, KUSKOWSKI M A, et al. Bacteriophage therapy of venous leg ulcers in humans: results of a phase Ⅰ safety trial[J]. J Wound Care, 2009, 18(6): 237-238, 240-243. DOI:10.12968/jowc.2009.18.6.42801.
[15]GREISEN K, LOEFFELHOLZ M, PUROHIT A, et al. PCR primers and probes for the 16S rRNA gene of most species of pathogenic Bacteria, including Bacteria found in cerebrospinal fluid[J]. J Clin Microbiol, 1994, 32(2): 335-351.
[16]MI L Y, LIU Y N, WANG C, et al. Identification of a lytic Pseudomonas aeruginosa phage depolymerase and its anti-biofilm effect and bactericidal contribution to serum[J]. Virus Genes, 2019, 55(3): 394-405. DOI:10.1007/s11262-019-01660-4.
[17]ANDERSON T F. Bacteriophages[J]. Annu Rev Microbiol,1950, 4(1): 21-34. DOI:10.1146/annurev.mi.04.100150.000321.
[18]ACKERMANN H W. Basic phage electron microscopy[J]. Methods Mol Biol, 2009, 501: 113-126. DOI:10.1007/978-1-60327-164-6_12.
[19]XING S Z, MA T P, ZHANG X, et al. First complete genome sequence of a virulent bacteriophage infecting the opportunistic pathogen Serratia rubidaea[J]. Arch Virol, 2017, 162(7): 2021-2028. DOI:10.1007/s00705-017-3300-x.
[20]DELBRCK M. The growth of bacteriophage and lysis of the host[J]. J Gen Physiol, 1940, 23(5): 643-660. DOI:10.1085/jgp.23.5.643.
[21]ZHAO F Y, SUN H Z, ZHOU X Y, et al. Characterization and genome analysis of a novel bacteriophage vB_SpuP_Spp16 that infects Salmonella enterica serovar pullorum[J]. Virus Genes, 2019, 55(4): 532-540. DOI:10.1007/s11262-019-01664-0.
[22]LU S G, LE S, TAN Y L, et al. Genomic and proteomic analyses of the terminally redundant genome of the Pseudomonas aeruginosa phage PaP1: establishment of genus PaP1-like phages[J]. PLoS ONE, 2013, 8(5): e62933. DOI:10.1371/journal.pone.0062933.
[23]MARGULIES M, EGHOLM M, ALTMAN W E, et al. Genome sequencing in microfabricated high-density picolitre reactors[J]. Nature, 2005, 437(7057): 376-380. DOI:10.1038/nature03959.
[24]AZIZ R K, BARTELS D, BEST A A, et al. The RAST Server: rapid annotations using subsystems technology[J]. BMC Genomics, 2008, 9: 75. DOI:10.1186/1471-2164-9-75.
[25]LOWE T M, EDDY S R. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence[J]. Nucleic Acids Res, 1997, 25(5): 955-964. DOI:10.1093/nar/25.5.955.
[26]TAMURA K, STECHER G, PETERSON D, et al. MEGA6: molecular evolutionary genetics analysis version 6.0[J]. Mol Biol Evol, 2013, 30(12): 2725-2729. DOI:10.1093/molbev/mst197.
[27]GUILLIAM T A, KEEN B A, BRISSETT N C, et al. Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes[J]. Nucleic Acids Res, 2015, 43(14): 6651-6664. DOI:10.1093/nar/gkv625.
[28]CRAIG N L, NASH H A. E. coli integration host factor binds to specific sites in DNA[J]. Cell, 1984, 39(3 Pt 2): 707-716. DOI:10.1016/0092-8674(84)90478-1.
[29]GOOSEN N, VAN DE PUTTE P. The regulation oftranscription initiation by integration host factor[J]. Mol Microbiol, 1995, 16(1): 1-7. DOI:10.1111/j.1365-2958.1995.tb02386.x.
[30]KRAWCZYK B, KUR J. Integration host factor, a histone-like Escherichia coli protein, binds to at least four sites of the DNA fragment containing the recA gene[J]. Acta Microbiol Pol, 1994, 43(2): 145-153.
[31]KROPINSKI A M, WADDELL T, MENG J C, et al. The host-range, genomics and proteomics of Escherichia coli O157: H7 bacteriophage rV5[J]. Virol J, 2013, 10: 76. DOI:10.1186/1743-422X-10-76.
[32]LOESSNER M J. Bacteriophage endolysins: current state of research and applications[J]. Curr Opin Microbiol, 2005, 8(4): 480-487. DOI:10.1016/j.mib.2005.06.002.
[33]ISIDRO A, HENRIQUES A O, TAVARES P. The portal protein plays essential roles at different steps of the SPP1 DNA packaging process[J]. Virology, 2004, 322(2): 253-263. DOI:10.1016/j.virol.2004.02.012.
[34]WANG I N, SMITH D L, YOUNG R. Holins: the protein clocks of bacteriophage infections[J]. Annu Rev Microbiol, 2000, 54: 799-825. DOI:10.1146/annurev.micro.54.1.799.
[35]HERON M. Deaths: leading causes for 2007[J]. Natl Vital Stat Rep, 2011, 59(8): 1-95.
[36]LAXMINARAYAN R, DUSE A, WATTAL C, et al. Antibiotic resistance-the need for global solutions[J]. Lancet Infect Dis, 2013, 13(12): 1057-1098. DOI:10.1016/S1473-3099(13)70318-9.
[37]MANGRAM A J, HORAN T C, PEARSON M L, et al. Guideline for prevention of surgical site infection, 1999. centers for disease control and prevention (CDC) hospital infection control practices advisory committee[J]. Am J Infect Control, 1999, 27(2): 97-132; quiz 133-134; discussion 96.
[38]HAGENS S, LOESSNER M J. Bacteriophage for biocontrol of foodborne pathogens: calculations and considerations[J]. Curr Pharm Biotechnol, 2010, 11(1): 58-68. DOI:10.2174/138920110790725429.
[39]DOMINGO-CALAP P, GEORGEL P, BAHRAM S. Back to the future: bacteriophages as promising therapeutic tools[J]. HLA, 2016, 87(3): 133-140. DOI:10.1111/tan.12742.
[40]GRSKI A, MIEDZYBRODZKI R, WEBER-DABROWSKA B, et al. Phage therapy: combating infections with potential for evolving from merely a treatment for complications to targeting diseases[J]. Front Microbiol, 2016, 7: 1515. DOI:10.3389/fmicb.2016.01515.
[41]NIU H X, YEE R, CUI P, et al. Identification of agents active against methicillin-resistant Staphylococcus aureus USA300 from a clinical compound library[J]. Pathogens, 2017, 6(3): E44. DOI:10.3390/pathogens6030044.
[42]MODI S R, LEE H H, SPINA C S, et al. Antibiotic treatment expands the resistance reservoir and ecological network of the phage metagenome[J]. Nature, 2013, 499(7457): 219-222. DOI:10.1038/nature12212.
[43]CHEN J, NOVICK R P. Phage-mediated intergeneric transfer of toxin genes[J]. Science, 2009, 323(5910): 139-141. DOI:10.1126/science.1164783.
[44]OLIVER K M, DEGNAN P H, HUNTER M S, et al. Bacteriophages encode factors required for protection in a symbiotic mutualism[J]. Science, 2009, 325(5943): 992-994. DOI:10.1126/science.1174463.
[45]LINDELL D, JAFFE J D, JOHNSON Z I, et al. Photosynthesis genes in marine viruses yield proteins during host infection[J]. Nature, 2005, 438(7064): 86-89. DOI:10.1038/nature04111.
[46]BEUMER A, ROBINSON J B. A broad-host-range,generalized transducing phage (SN-T) acquires 16S rRNA genes from different genera of Bacteria[J]. Appl Environ Microbiol, 2005, 71(12): 8301-8304. DOI:10.1128/AEM.71.12.8301-8304.2005.
[47]BALCAZAR J L. Bacteriophages as vehicles for antibiotic resistance genes in the environment[J]. PLoS Pathog, 2014, 10(7): e1004219. DOI:10.1371/journal.ppat.1004219.
[48]ENAULT F, BRIET A, BOUTEILLE L, et al. Phages rarely encode antibiotic resistance genes: a cautionary tale for virome analyses[J]. ISME J, 2017, 11(1): 237-247. DOI:10.1038/ismej.2016.90.
[49]MAGANA M, IOANNIDIS A, MAGIORKINIS E, et al. Therapeutic options and emerging alternatives for multidrug resistant staphylococcal infections[J]. Curr Pharm Des, 2015, 21(16): 2058-2072. DOI:10.2174/138161282 1666150310101851.


 SUN Wei-zhong,HU Xiao-mei,RAO Xian-cai,et al.Bioinformatic prediction of endolysin gene’s function in P. aeruginosa bacteriophages[J].J Third Mil Med Univ,2008,30(03):787.
 SUN Wei-zhong,HU Xiao-mei,RAO Xian-cai,et al.Cloning, expression and enzymatic assay of bacteriophage PaP1 endolysin against Pseudomonas aeruginosa[J].J Third Mil Med Univ,2008,30(03):1268.
 Zhu Xiaoyan,Hu Fuquan,Tan Yingling.Identification of endolysin encoded by bacteriophage MmP1 against Morganella morganii[J].J Third Mil Med Univ,2011,33(03):2463.
 Zhang Jie,Liu Xi,Gan Dan.Sequencing and bioinformatic analysis of genome of Acinetobacter baumannii bacteriophage AB3[J].J Third Mil Med Univ,2013,35(03):1562.
 Zhang Jie,Zhou Lirong,Luo Yongai.Expression and antibacterial activity of bacteriophage AB3 endolysin against Acinetobacter baumannii[J].J Third Mil Med Univ,2013,35(03):1823.
[8]张利军.噬菌体宿主特异性变化的分子机制研究[J].第三军医大学学报,2004,26(04):0.[doi:10.16016/j.1000-5404.2004.04.031 ]
[9]张克斌,金晓琳,朱军民,等.铜绿假单胞菌噬菌体PaP3基因组测序[J].第三军医大学学报,2002,24(04):0.[doi:10.16016/j.1000-5404.2002.04.002 ]
 ZHANG Ke bin,JIN Xiao lin,ZHU Jun min,et al.[J].J Third Mil Med Univ,2002,24(03):0.[doi:10.16016/j.1000-5404.2002.04.002 ]
[10]张克斌,金晓琳,朱军民,等.铜绿假单胞菌噬菌体PaP3基因组的初步注释[J].第三军医大学学报,2002,24(04):0.[doi:10.16016/j.1000-5404.2002.04.003 ]
 ZHANG Ke bin,JIN Xiao lin,ZHU Jun min,et al.[J].J Third Mil Med Univ,2002,24(03):0.[doi:10.16016/j.1000-5404.2002.04.003 ]

更新日期/Last Update: 2020-02-06