一株新的变形杆菌噬菌体的分离、鉴定与生物学特性分析

摘要/Abstract

摘要： 目的：一株新的变形杆菌噬菌体的分离、鉴定与生物学特性及基因组分析。方法：将实验室从牦牛屠宰环节中分离到的菌株纯培养后，对其DNA提取进行16S rRNA测序，鉴定获得宿主菌，采集成都牦牛屠宰场环境污水，以获得的宿主菌进行噬菌体分离；运用双层琼脂平板法进行噬菌体的分离纯化，测定噬菌体的最佳感染复数，一步生长曲线和噬菌体耐受性等；通过磷酸负染、透射电镜观察噬菌体的形态；采用试剂盒提取噬菌体全基因组DNA，使用Iumina Miseq测序平台进行噬菌体全基因组测序，对噬菌体全基因组序列进行组装、注释和基因组学分析。结果：从67株分离菌株中鉴定出10株变形杆菌，并从污水中分离得到一株可裂解变形杆菌和粪肠球菌并且裂解性较强的噬菌体，命名为Proteus phage PV66；其最佳感染复数为0.1，一步生长曲线结果显示，感染宿主菌的潜伏期约为10 min，裂解期约为80 min，平均裂解量为321 PFU/cell ；耐受pH范围为3-12，耐受温度为65 ℃。电镜观察该噬菌体为C3形态噬菌体，其头部长 (140±1) nm，宽 (54±2) nm，尾部长为（36±1）nm。基因组测序结果表明PV66全序列长度为90 492 bp，系统发育树分析发现该噬菌体与奇异变形杆菌噬菌体和克诺罗杆菌噬菌体同源性较高，与 Non-Kuravirus属处于同一分支。结论：本研究分离到一株新的变形杆菌噬菌体并具有良好的应用潜力，为进一步开发噬菌体类新型抗菌剂提供基础，同时本研究也丰富了变形杆菌噬菌体的数据库。

关键词: 变形杆菌, 噬菌体, 生物学特性, 基因组分析

Abstract: Objective To isolate, identify, characterize and analyze the genome of a new Proteus phage. Methods The strains isolated from yak slaughtering in the laboratory were cultured, their DNA was extracted and sequenced with 16S rRNA, and the host bacteria were identified. The environmental sewage of Chengdu Yak slaughterhouse was collected, and the host bacteria were used for bacteriophage separation; The bacteriophage was separated and purified by double agar plate method, and the optimal multiplicity of infection, one-step growth curve and bacteriophage tolerance were determined; The morphology of phage was observed by negative staining with phosphoric acid and transmission electron microscopy; The whole genome DNA of bacteriophage was extracted with the kit, and the whole genome of bacteriophage was sequenced using the Iumina Miseq sequencing platform. The whole genome sequence of bacteriophage was assembled, annotated, and genomically analyzed. Results 10 Proteus strains were identified from 67 isolates, and a phage with strong lytic activity, named Proteus phase PV66, was isolated from sewage; The optimal multiplicity of infection was 0.1. The results of one-step growth curve showed that the incubation period of the infected host bacteria was about 10 min, the lytic period was about 80 min, and the average lytic amount was 321 PFU/cell; The tolerance pH range is 3-12, and the tolerance temperature is 65 ℃. The bacteriophage was C3 shaped under electron microscope, with a head length of (140 ± 1) nm, a width of (54 ± 2) nm, and a tail length of (36 ± 1) nm. The genome sequencing results showed that the whole sequence length of PV66 was 90492 bp. Phylogenetic tree analysis showed that the phage had high homology with Proteus mirabilis phage and Cronobacter bacteriophage, and was in the same branch as Non-Kuravirus. Conclusion A new Proteus phage was isolated in this study and has good application potential, which provides a basis for further development of new bacteriophages. At the same time, this study also enriches the database of Proteus phages.

Key words: Proteus, Phage, Biological Characteristics, Genome Analysis

中图分类号:

TS252.1

苏雅航侯忠余朱成林陈娟刀筱芳唐俊妮. 一株新的变形杆菌噬菌体的分离、鉴定与生物学特性分析[J]. 食品科学, 0, (): 0-0.

Junni Tang. Isolation, Identification and Biological Characteristics Analysis of a new Proteus Phage[J]. FOOD SCIENCE, 0, (): 0-0.

参考文献

[1]ZYCH K, KO?ODZIEJSKA K, DRZEWIECKA D, et al. Serological classification and epitope specificity of Proteus vulgaris TG 251 from Proteus serogroup O65[J]. Arch Immunol Ther Exp (Warsz). 2007;55(3):187-91. doi: 10.1007/s00005-007-0020-z.
[2] DAI H, LU B, LI Z, et al. Multilocus sequence analysis for the taxonomic updating and identification of the genus Proteus and reclassification of Proteus genospecies 5 O'Hara et al. 2000, Proteus cibarius Hyun et al. 2016 as later heterotypic synonyms of Proteus terrae Behrendt et al. 2015[J]. BMC Microbiol. 2020;20(1):152. doi: 10.1186/s12866-020-01844-1.
[3]SUN Y, FENG JQ, TAN YR, et al. Genomic and biological characterization of vB_PvuS_Pm34, a novel lytic bacteriophage that infects Proteus vulgaris[J]. Genomics. 2022;114(1):38-44. doi: 10.1016/j.ygeno.2021.11.033.
[4]冯嘉琪．一株新噬菌体的分离鉴定及基因组学分析[D]．华南农业大学,2017:20-30.
[5]张宏扬. 食源性多重耐药普通变形杆菌P3M环丙沙星和多粘菌素抗性调控机制研究[D].南开大学,2021.DOI:10.27254/d.cnki.gnkau.2021.000028.
[6]毕水莲, 李琳, 唐书泽,等. 变形杆菌属食物中毒的特点与防控措施[J]. 现代食品科技,2009(06):690-695. DOI:10.3969/j.issn.1673-9078.2009.06.032.
[7]马婷婷,韦显凯,闭璟珊,等.猪源奇异变形杆菌的分离鉴定及其毒力的测定[J].中国兽医科学,2017,47（10）:1234-1239. DOI:CNKI:SUN:ZGSY.0.2017-10-005.
[8]俞赵荣,杨侃侃,王元红,等.1株番鸭源奇异变形杆菌的分离鉴定及其毒力基因分析[J].中国动物传染病学报,2020,28(03):14-20. DOI: CNKI:SUN:ZSJB.0.2020-03-003.
[9]宋军,徐若洋,靳育琦,等.犬源奇异变形杆菌噬菌体分离、鉴定及对生物被膜的作用[J].畜牧兽医学报,2020,51(11):2785-2793.DOI:10.11843/j.issn.0366-6964.2020.11.017.
[10]侯晓,徐结,张雪婧,等.甘肃某牦牛场奇异变形杆菌的分离鉴定及小鼠致病性试验[J].动物医学进展,2019,40(10):44-48. DOI:10.16437/j.cnki.1007-5038.2019.10.009.
[11]曾显成,池雪林,修云芳,等.大熊猫源普通变形杆菌分离鉴定及致病性分析[J].中国兽医科学,2020,50(11):1379-1388. DOI:10.16656/j.issn.1673-4696.2020.0182.
[12]PATHAK P, NOVAK J, SHUKLA PK, et al. Design, synthesis, antibacterial evaluation, and computational studies of hybrid oxothiazolidin-1,2,4-triazole scaffolds[J]. Arch Pharm (Weinheim). 2021:e2000473. DOI: 10.1002/ardp.202000473.
[13]VáRADI L, LUO JL, HIBBS DE, et al. Methods for the detection and identification of pathogenic bacteria :past, present, and future［J］. Chem Soc Rev, 2017, 46(16):4818-4832. DOI: 10.1039/c6cs00693k.
[14]CARVALHO C, COSTA AR, SILVA F, et al. Bacteriophages and their derivatives for the treatment and control of food-producing animal infections [J]. Crit Rev Microbiol, 2017, 43(5):583-601. DOI: 10.1080/1040841X.2016.1271309.
[15]WEI S, CHELLIAH R, RUBAB M, et al. Bacteriophages as potential tools for detection and control of Salmonella spp. in food systems[J]. Microorganisms, 2019, 7(11):570. DOI: 10.3390/microorganisms7110570.
[16]OH JH, PARK MK. Recent trends in Salmonella outbreaks and emerging technology for biocontrol of Salmonella using phages in foods :a review[J]. Journal of microbiology and biotechnology, 2017, 27(12): 2075-2088. DOI: 10.4014/jmb.1710.10049.
[17]KAWACKA I, OLEJNIK-SCHMIDT A, SCHMIDT M, et al. Effectiveness of Phage-Based Inhibition of Listeria monocytogenes in Food Products and Food Processing Environments[J]. Microorganisms. 2020;8(11):1764. doi: 10.3390/microorganisms8111764.
[18]REHMAN S, ALI Z, KHAN M, et al. The dawn of phage therapy[J]. Rev Med Virol. 2019;29(4):e2041. doi: 10.1002/rmv.2041.
[19]WIRJON IA, LAU NS, ARIP YM. Complete Genome Sequence of Proteus mirabilis Phage pPM_01 Isolated from Raw Sewage[J]. Intervirology. 2016;59(5-6):243-253. doi: 10.1159/000468987.
[20]周祥莹,刘慧,孙虎芝,等．貉源奇异变形杆菌的分离鉴定及噬菌体的分离[J]．黑龙江畜牧兽医, 2018（20）:199-202. DOI:10.13881/j.cnki.hljxmsy.2018.02.0126.
[21] MILO S , HATHAWAY H , NZAKIZWANAYO J , et al. Prevention of encrustation and blockage of urinary catheters by Proteus mirabilis via pH-triggered release of bacteriophage[J]. J Mater Chem B. 2017;5(27):5403-5411. doi: 10.1039/c7tb01302g.
[22] MASZEWSKA A, ZYGMUNT M, GRZEJDZIAK I, et al. Use of polyvalent bacteriophages to combat biofilm of Proteus mirabilis causing catheter-associated urinary tract infections[J]. J Appl Microbiol. 2018;125(5):1253-1265. doi: 10.1111/jam.14026.
[23] UJMAJURIDZE A, CHANISHVILI N, GODERDZISHVILI M, et al. Adapted Bacteriophages for Treating Urinary Tract Infections[J]. Front Microbiol. 2018;9:1832. doi: 10.3389/fmicb.2018.01832.
[24]GOMAA S, SERRY F, ABDELLATIF H, et al. Elimination of multidrug-resistant Proteus mirabilis biofifilms using bacteriophages[J]. Archives of Virology. 2019; 164(9):2265–2275 . DOI 10.1007/s00705-019-04305-x.
[25]SAYERS EW, CAVANAUGH M, CLARK K,et al. Database resources of the National Center for Biotechnology Information.[J]. Nucleic acids research, 2020, 48(D1).D84–D86. DOI 10.1093/nar/gkz899.
[26]PROZESKY OW, KLERK HCD, COETZEE JN. The morphology of proteus bacteriophages[J]. Journal of General Microbiology, 2015, 41(1):29–36. DOI 10.1099/00221287-41-1-29.
[27]ALVES DR, NZAKIZWANAYO J, DEDI C, et al. Genomic and Ecogenomic Characterization of Proteus mirabilis Bacteriophages.[J].Frontiers in microbiology,2019,10.:1783. DOI 10.3389/fmicb.2019.01783.
[28]唐俊妮,龙飞,史贤明,等.金黄色葡萄球菌基因组DNA提取方法的比较研究[J].中国卫生检验杂志,2008(08):1467-1469. DOI:10. 3969/j.issn.1004-8685.2008.08.001.
[29]TANG J, CHEN J, LI H, et al. Characterization of adhesin genes, staphylococcal nuclease, hemolysis, and biofilm formation among Staphylococcus aureus strains isolated from different sources[J]. Foodborne Pathogens & Disease, 2013, 10(9):757-763. DOI:10.1089/fpd.2012.1474.
[30]汤婷婷. 金黄色葡萄球菌噬菌体的分离鉴定及对治疗乳腺炎的初步应用[D].东北农业大学,2019:10-20
[31]CZAJKOWSKI R, PéROMBELON M C M, VAN VEEN J A, et al. Control of blackleg and tuber soft rot of potato caused by Pectobacterium and Dickeya species: a review [J]. Plant Pathology, 2011, 60(6): 999-1013. DOI:10.1111/j.1365-3059.2011.02470.x.
[32]宋敬东, 屈建国, 鲁茁壮,等.提高负染法透射电镜检测病毒灵敏度的制样方法及应用[J].病毒学报,2010,26(05):410-413. DOI:10.13242/j.cnki.bingduxuebao.002111.
[33]MINH D H, MINH S H, HONJOH K I, et al. Isolation and biocontrol of extended spectrum beta-lactamase (ESBL) producing Escherichia coli contamination in raw chicken meat by using lytic bacteriophages[J]. LWT-Food Science and Technology, 2016, 71: 339-346. DOI:10.1016/j.lwt.2016.04.013
[34]LU Z, BREIDT F, FLEMING H P, et al. Isolation and characterization of a Lactobacillus plantarum bacteriophage, phiJL-1, from a cucumber fermentation[J]. International Journal of Food Microbiology, 2003, 84(2): 225-235. DOI:10.1016/S0168-1605(03)00111-9
[35]侯忠余,李传友,朱成林,等.1株金黄色葡萄球菌烈性噬菌体的生物学特性及其裂解效果[J].食品科学,2022,43(08):113-120. DOI:10.7506/spkx1002-6630-20210510-098.
[36]LEFKOWITZ EJ, DEMPSEY DM, HENDRICKSON RC, et al. Virus taxonomy: the database of the International Committee on Taxonomy of Viruses (ICTV)[J]. Nucleic acids research,2018,46(D1):D708–D717. DOI 10.1093/nar/gkx932
[37]CORBAN JE, RAMSEY J. Characterization and complete genome sequence of Privateer, a highly prolate Proteus mirabilis podophage[J]. Peer J. 2021; 9:e10645. DOI: 10.7717/peerj.10645.
[38]SCHOCH CL, CIUFO S, DOMRACHEV M, et al. NCBI Taxonomy: a comprehensive update on curation, resources and tools[J]. Database (Oxford). 2020; 2020:baaa062. doi: 10.1093/database/baaa062.
[39]HENDRIX RW. Bacteriophages: evolution of the majority[J]. Theor Popul Biol. 2002; 61(4):471-80. doi: 10.1006/tpbi.2002.1590.
[40]POPE WH, ANDERS KR, BAIRD M, et al. Cluster M mycobacteriophages Bongo, PegLeg, and Rey with unusually large repertoires of tRNA isotypes[J]. J Virol. 2014; 88(5):2461-80. doi: 10.1128/JVI.03363-13.