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1 微生物学通报 Microbiology China Sep. 0, 016, 43(9): DOI: /j.microbiol.china 研究报告粪肠球菌噬菌体 vb_e. faecalis_ime196 的生物学特性及其全基因组分析邢少贞 1, 张湘莉兰舒鹏孙强赵宝华 1* 裴广倩童贻刚 * 米志强 ( ) ( ) 安小平王景林摘要 : 目的从医院污水中分离一株能裂解多耐药粪肠球菌的噬菌体, 分析该噬菌体的生物学特性, 并进行全基因组测序和分析, 为治疗和控制多耐药粪肠球菌感染提供基础方法以耐药粪肠球菌为宿主, 从医院污水分离噬菌体, 双层平板法检测噬菌体效价最佳感染复数 (MOI) 和一步生长曲线, 纯化后负染法电镜观察噬菌体形态 ; 蛋白酶 K/SDS 法提取噬菌体全基因组, 酶切处理后琼脂糖凝胶电泳分析, 使用 Ion Torrent 测序平台进行噬菌体全基因组测序, 测序后进行噬菌体全基因组序列组装注释进化分析和比较分析结果分离到一株粪肠球菌噬菌体, 命名为 vb_e. faecalis_ime196 (IME196); 其最佳感染复数为 0.01, 一步生长曲线显示 IME196 的潜伏期为 30 min, 暴发量为 50 PFU, 电镜观察该噬菌体为长尾噬菌体, 结合 BLASTp 分析确定其属于尾病毒目长尾噬菌体科, 基因测序表明, 噬菌体 IME196 核酸类型为 DNA, 基因组全长为 bp,g+c 含量为 33.9% 结论分离鉴定一株粪肠球菌噬菌体, 进行了全基因组测序和分析, 为以后预防和控制粪肠球菌的感染提供了一个新的途径, 为噬菌体治疗多重耐药细菌奠定了基础关键词 : 噬菌体, 粪肠球菌, 生物学特性, 全基因组测序, 基因组分析 Foundation item: China Mega-Project on Infectious Disease Prevention (No. 013ZX , 013ZX , 013ZX , 011ZX ); National High-Tech Research and Development Program of China (863 Program) (No. 014AA0140, 01AA0-003); National Natural Science Foundation of China (No ) *Corresponding authors: ZHAO Bao-Hua: zhaobaohua@hebtu.edu.cn; TONG Yi-Gang: tong.yigang@gmail.com Received: December, 015; Accepted: March 9, 016; Published online ( April 08, 016 基金项目 : (No. 013ZX ZX ZX ZX ) (863 ) (No. 014AA AA0-003) (No ) * 通讯作者 : zhaobaohua@hebtu.edu.cn; tong.yigang@gmail.com 收稿日期 : 接受日期 : 优先数字出版日期 (

2 : vb_e. faecalis_ime Characterization and complete genome sequence analysis of Enterococcus faecalis bacteriophage vb_e. faecalis_ime196 XING Shao-Zhen 1, ZHANG Xiang-Li-Lan SHU Peng SUN Qiang PEI Guang-Qian MI Zhi-Qiang AN Xiao-Ping WANG Jing-Lin ZHAO Bao-Hua 1* TONG Yi-Gang * (1. Hebei Normal University, Shijiazhuang, Hebei 05004, China) (. Beijing Institute of Microbiology and Epidemiology, Beijing , China) Abstract: [Objective] We isolated a novel virulent bacteriophage specifically infecting antibiotics-resistant Enterococcus faecalis from hospital sewage, to study its morphology and to analyze its genome so as to provide basis for treatment and infection control of antibiotics-resistant E. faecalis. [Methods] We used E. faecalis as the host to isolate phage from raw sewage from the sewage treatment center of a hospital. Transmission electromicroscopy, optimal multiplicity of infection (optimal MOI) and one-step growth curve were conducted. The genome DNA was extracted and sequenced by high-throughput sequencing technology. The feature of whole genome, and evolutionary relationship were analyzed. [Results] A lytic bacteriophage designated as vb_e. faecalis_ime196 (IME196) was isolated. The optimal multiplicity of infection (optimal MOI) of IME196 is One-step growth curve shows that IME196 has a burst size of 50 PFU and a latent period of 30 min. The genome of IME196 is a bp in length, linear, terminally non-redundant double-stranded DNA and has a G+C content of 33.9%. [Conclusion] Isolation and characterization of a lytic Enterococcus faecalis phage will help supply a new way to prevent and control the infection of E. faecalis, and laid a foundation to the treatment of multiple drug resistant bacteria in the future. Keywords: Phage, Enterococcus faecalis, Biological characteristics, Whole genomic sequencing, Genomics [1] [] [3] (Enterococcus faecalis) [4] [5] 1986 (VRE) [6] [7] [8] 1 材料与方法 1.1 材料 10 (Enterococcus faecalis) 10

3 04 微生物学通报 Microbiol. China 016, Vol.43, No.9 (Enterococcus faecium) 16S rrna 4 (Staphylococcus aureus) (Escherichia coli) BHI (Brain heart infusion) BD : : (5:4:1 ) 8000 (PEG8000) AMRESCO K RNase DNase Fermentas 1. 噬菌体的分离 r/min 10 min 0. μm 100 μl 5 ml 37 C 0 r/min r/min 10 min ( 0. μm) 1.3 噬菌体的纯化与浓缩 BHI [4] 5 ml OD C 0 r/min 6 h r/min 1 min 500 ml OD C 0 r/min 1 h PEG [4] 1.4 透射电子显微镜下观察噬菌体形态及大小 50 μl 50 μl 0.5% 4% 30 μl Formvar 30 min % 10 min Philips TECNAI 噬菌体最佳感染复数测定 [4] (Multiplicity of infection MOI) MOI BHI 8 ml 37 C 0 r/min 5 h r/min 1 min 0. μm MOI 1.6 噬菌体一步生长曲线测定 [4] 007 OD BHI BHI 10 8 CFU/mL MOI 0.01 IME C min IME 噬菌体裂解谱测定 ( ) OD μl 5 min 1.5 μl 0 min 37 C 10 h 1.8 提取噬菌体 IME196 基因组 600 μl [4] K/SDS DNaseⅠ RNase A 1 mg/l 37 C 80 C 15 min 0.5 mol/l EDTA ( 0.0 mol/l) 0 g/l K ( 50 mg/l) 10% SDS ( 0.5%) 56 C 1 h

4 : vb_e. faecalis_ime r/min 5 min 1.5 ml - -(5:4:1 ) r/min 10 min 0 C 4 h r/min 15 min 75% DNA r/min 0 min DNA 1.9 噬菌体 IME196 的全基因组测序 90 bp Life Technologies Ion Torrent PGM DNA DNA Emulsion PCR 1.10 噬菌体 IME196 全基因组序列分析 IME196 Newbler.9 RAST ( BASys (Bacterial Annotation System) BLAST ( NCBI Microbial Genome Submission Check NCBI CGView ( Ion Torrent 58 M 90 bp bp 结果与分析.1 噬菌体 IME196 形态及大小 h 1A ( 1B) 58 nm 30 nm vb_e. faecalis_ime196 (Siphoviridae). 噬菌体 IME196 的最佳感染复数 5 h MOI= Antimicrobial drugs Table 1 MIC (mg/l) 表 1 粪肠球菌 007 抗生素耐药性 Enterococcus faecalis 007 antibiotic resistance Sensitivity Antimicrobial drugs MIC (mg/l) Cotrimoxazole 0 S Quinupristin 4 R Levofloxacin 0.5 S Ceftriaxone 1 S Piperacillin 4 S Cefazolin 64 R High streptomycin 4 R Nitrofurantoin 16 S Ceftazidime 1 S Cefepime 1 S Ampicillin 4 S Gentamicin 1 S Meropenem 0.5 S Tetracycline 64 R S I R. Note: S: Sensitivity; I: Betweeness; R: Drug resistance. Sensitivity

044 微生物学通报 Microbiol. China 016, Vol.43, No.9.3 噬菌体 IME196 的一步生长曲线 30 min 60 min / =1.

Table Determination of optimal multiplicity of infection (MOI) 6 h Bacteria Phage 6 h Titer Number MOI (CFU/mL) (PFU/mL) (PFU/mL) 1 10 8 10 9 0.01 4.8 10 9 10 8 10 8 0.1 1.0 10 9 3 10 8 10 7 1.

5 044 微生物学通报 Microbiol. China 016, Vol.43, No.9.3 噬菌体 IME196 的一步生长曲线 30 min 60 min / = / =50 PFU 图 1 噬菌体 IME196 形成的噬菌斑 (A) 及透射电镜下噬菌体 IME196 的形态 (B) ( ) Figure 1 The plaque formed by IME196 (A) and the morphology of phage IME196 (B) (TEM ) 表噬菌体最佳感染复数 (MOI) 的测定 Table Determination of optimal multiplicity of infection (MOI) 6 h Bacteria Phage 6 h Titer Number MOI (CFU/mL) (PFU/mL) (PFU/mL) 图噬菌体 IME196 的一步生长曲线 Figure One-step growth curve of phage IME196.4 噬菌体裂解谱的测定 IME196 3 IME196.5 噬菌体 IME196 全基因组高通量测序和组装及高频序列分析 100 μg DNA bp (Reads) bp Reads ( ) IME kb (Contig) bp [9] Reads (Mapping) Reads Reads 00 Reads 3 1 (5 -ATTGTGTTGAGTAAG AATTGGAGGGGGGGCAAAAATTTTT-3 ) (5 -CTTTCGCTTAAACGAATTTCTAAAAAC GAAAGTTGAAAAC-3 ) IME196 ( 4).6 噬菌体 IME196 的全基因组概述 IME bp DNA G+C 33.9% vb_e. faecalis_ime196 GenBank KT93701 BLASTn IME196 EfaCPT1 (40 kb)

: vb_e. faecalis_ime196 045 图 3 噬菌体 IME196 末端的 Reads 发生频率 Figure 3 Frequencies of sequence reads representing the genomic termini of IME196 X Reads Y Reads.

6 : vb_e. faecalis_ime 图 3 噬菌体 IME196 末端的 Reads 发生频率 Figure 3 Frequencies of sequence reads representing the genomic termini of IME196 X Reads Y Reads. Note: X axis is the span of Reads frequency; Y axis is the statistic of the number of Reads. IME-EF4 (40 kb) 9% GenBank JX KF RAST NCBI BLASTp 57 (ORFs) 11 ORFs (Hypothetical protein)46 ORFs 3 ORF1 () ORF () ORF0 () 3 ORF 图 4 噬菌体 IME196 的末端分析 Figure 4 The genomic termini of phage IME196 ORF6 () ORF7 ORF16 DNA ORF4 (DNA ) ORF45 (DNA ) ORF187 (DNA ) ORF51 ( DNA ) ORF38 (DNA ) ORF184 ( ) ORF1 () ORF0 ( ) ORF0 CGview 5 4 IME196 CDS CDS G+C G+C G+C G+C G C/G+C 讨论 [10] 0 90 [9]

7 046 微生物学通报 Microbiol. China 016, Vol.43, No.9 表 3 噬菌体 IME196 的 ORF 功能预测 Table 3 ORF analysis of the IME196 genome ORF Top BLAST hit a IDENTITY b (%) (aa) E value Accession No. 1 Terminase small subunit [Enterococcus phage EFAP-1] 98 (154/157).00E 108 YP_ Terminase large subunit [Enterococcus phage IME-EF4] 98 (56/57) 0 YP_ Hypothetical protein [Enterococcus phage IME_EF3] 93 (50/54).00E 3 YP_ Putative portal protein [Enterococcus phage EfaCPT1] 98 (377/383) 0 YP_ Head maturation protease [Enterococcus phage IME_EF3] 99 (186/187) 3.00E 131 YP_ HK97 family major capsid protein [Enterococcus phage AUEF3] 89 (370/418) 0 AHN Major tail protein [Enterococcus phage IME-EF4] 100 (66/66).00E 37 YP_ Head-tail joining protein [Enterococcus phage EFRM31] 99 (97/98).00E 63 YP_ Head-tail adaptor protein [Enterococcus phage EFRM31] 48 (8/585) 1.00E 73 YP_ Head-tail joining protein [Enterococcus phage EFRM31] 98 (13/135) 4.00E 93 YP_ Head-tail joining protein [Enterococcus phage EFRM31] 98 (118/11) 9.00E 83 YP_ Major tail protein [Enterococcus phage EFRM31]] 99 (187/188).00E 19 YP_ Hypothetical protein EFAP1_gp09 [Enterococcus phage EFAP-1] 99 (10/103) 3.00E 66 YP_ Putative tail tape measure chaperone frameshift protein 90 (35/39) 4.00E 16 YP_ [Enterococcus phage efacpt1] 15 Putative tail tape measure protein [Enterococcus phage efacpt1] 86 (1 53/1 456) 0 YP_ Tail family protein [Enterococcus phage AUEF3] 94 (653/693) 0 AHN Minor structural N-terminal region domain protein [Enterococcus 9 (619/676) 0 AHN phage AUEF3] 18 Hypothetical protein efacpt1_gp17 [Enterococcus phage efacpt1] 93 (88/95) 6.00E 59 YP_ Hypothetical protein IME-EF4_44 [Enterococcus phage IME-EF4] 94 (76/81) 5.00E 37 YP_ Holin class II [Enterococcus phage IME_EF3] 99 (77/78) 9.00E 47 YP_ Lysin, N-acetylmuramoyl-L-alanine amidase [Enterococcus phage 98 (30/38) 0 YP_ IME_EF3] Putative glutaredoxin [Enterococcus phage efacpt1] 87 (65/75) 4.00E 39 YP_ HNH endonuclease family protein [Enterococcus phage EFRM31] 50 (75/150) 1.00E 31 YP_ DNA polymerase [Enterococcus phage IME-EF4] 96 (735/763) 0 YP_ Hypothetical protein CO47_0108 [Enterococcus phage AUEF3] 99 (19/0) 9.00E 156 AHN Hypothetical protein IME-EF4_36 [Enterococcus phage IME-EF4] 54 (45/83).00E 0 YP_ Hypothetical protein [Enterococcus phage IME_EF3] 83 (38/46).00E 17 YP_ Hypothetical protein CO47_0111 [Enterococcus phage AUEF3] 98 (59/65) 0 AHN Hypothetical protein IME-EF4_34 [Enterococcus phage IME-EF4] 100 (6/6) 9.00E 36 YP_ Beta-lactamase superfamily domain protein [Enterococcus phage 97 (50/58) 0 AHN AUEF3] 31 Putative HNH homing endonuclease-like protein [Enterococcus 98 (156/159) 1.00E 110 YP_ phage IME_EF3] 3 Hypothetical protein [Enterococcus phage IME_EF3] 99 (71/7) 6.00E 180 YP_ Hypothetical protein IME-EF4_30 [Enterococcus phage IME-EF4] 98 (165/168) 3.00E 118 YP_ Hypothetical protein IME-EF4_9 [Enterococcus phage IME-EF4] 94 (64/68) 1.00E 140 YP_ Putative prim-pol domain protein [Enterococcus phage efacpt1] 81 (35/90) 9.00E 164 YP_ Hypothetical protein IME-EF4_6 [Enterococcus phage IME-EF4] 98 (61/6) 8.00E 35 YP_ Hypothetical protein IME-EF4_5 [Enterococcus phage IME-EF4] 100 (65/65) 4.00E 39 YP_ DNA helicase [Enterococcus phage IME_EF3] 88 (383/434) 0 YP_ ( )

8 : vb_e. faecalis_ime HNH homing endonuclease [Enterococcus phage IME_EF3] 67 (108/16) 5.00E 67 YP_ Hypothetical protein [Enterococcus phage IME_EF3] 91 (11/13).00E 60 YP_ Hypothetical protein [Enterococcus phage IME_EF3] 83 (54/65) 1.00E 8 YP_ Hypothetical protein IME-EF4_1 [Enterococcus phage IME-EF4] 98 (50/51) 3.00E 4 YP_ Hypothetical protein CO47_018 [Enterococcus phage AUEF3] 88 (79/90) 1.00E 47 AHN Hypothetical protein [Enterococcus phage IME_EF3] 90 (56/6) 6.00E 31 YP_ Putative primase [Enterococcus phage efacpt1] 99 (5/56) 0 YP_ Hypothetical protein [Enterococcus phage IME_EF3] 85 (55/65).00E 30 YP_ Hypothetical protein [Enterococcus phage IME_EF3] 9 (68/74) 6.00E 40 YP_ Hypothetical protein EFLK1_ORF093 [Enterococcus phage EFLK1] 54 (5/46).00E 10 AKC No hits 50 No hits 51 No hits 5 Hypothetical protein efacpt1_gp55 [Enterococcus phage efacpt1] 56 (77/131) 3.00E 33 YP_ APC amino acid-polyamine-organocation transporter [Enterococcus phage EFRM31] 54 No hits 55 Daunorubicin resistance transmembrane protein [Enterococcus phage EFRM31] 56 No hits 57 No hits ( ) 96 (55/57) 3.00E 31 YP_ (101/108).00E 68 YP_ a. No hits. b BLASTp. Note: a : The most closely related genes and names of the organisms. No hits: Indicates no significant homology detected for a particular amino acid sequence. b : Percent identity for top hits in BLASTp searches. 图 5 IME196 全基因组图谱 Figure 5 Genome map of IME vb_e. faecalis_ime196 (Siphoviridae) 0.01 IME min 60 min 50 PFU IME196 Ion Torrent PGM ORF

9 048 微生物学通报 Microbiol. China 016, Vol.43, No.9 IME196 参考文献 [1] Bonilla N, Santiago T, Marcos P, et al. Enterophages, a group of phages infecting Enterococcus faecalis, and their potential as alternate indicators of human faecal contamination[j]. Water Science & Technology, 010, 61(): [] Dejea C, Wick E, Sears CL. Bacterial oncogenesis in the colon[j]. Future Microbiology, 013, 8(4): [3] Lavilla-Lerma L, Pérez-Pulido R, Martínez-Bueno M, et al. Characterization of functional, safety, and gut survival related characteristics of Lactobacillus strains isolated from farmhouse goat's milk cheeses[j]. International Journal of Food Microbiology, 013, 163(/3): [4] Zhang WH, An XP, Fan H, et al. Isolation and characterization of an Enterococcus faecalis phage[j]. Letters in Biotechnology, 013, 4(4): (in Chinese),,,. [J]., 013, 4(4): [5] Takakusagi Y, Kobayashi S, Sugawara F, et al. Camptothecin binds to a synthetic peptide identified by a T7 phage display screen[j]. Bioorganic & Medicinal Chemistry Letters, 005, 15(1): [6] Funatsu T, Taniyama T, Tajima T, et al. Rapid and sensitive detection method of a bacterium by using a GFP reporter phage[j]. Microbiology and Immunology, 00, 46(6): [7] Wang S, Tong YG. Recent advance in bacteriophage therapy[j]. Microbiology China, 009, 36(7): (in Chinese),. [J]., 009, 36(7): [8] Li SS, Fan H, An XP, et al. Utility of high throughput sequencing technology in analyzing the terminal sequence of caudovirales bacteriophage genome[j]. Chinese Journal of Virology, 013, 9(1): 39-4 (in Chinese),,,. [J]., 013, 9(1): 39-4 [9] Abedon ST, Kuhl SJ, Blasdel BG, et al. Phage treatment of human infections[j]. Bacteriophage, 011, 1(): [10] Li XY, Ding P, Han CY. Isolation of a novel Enterococcus faecalis phage IME-EF3 and its potential therapeutic advantages[j]. Biomedical Engineering and Clinical Medicine, 014, 18(): (in Chinese),,,. IME-EF3 [J]., 014, 18():

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇