2025年7月24日 周四
首页 > 过刊浏览>2023年第63卷第8期 >3110-3128. DOI:10.13343/j.cnki.wsxb.20220878
PDF HTML阅读 XML下载 导出引用 引用提醒
五株克雷伯氏菌噬菌体的生物学特性及比较基因组学研究
作者:
基金项目:

广东省科学院人才专项(2016GDASRC-0205)；北京动物园圈养野生动物技术北京市重点实验室开放课题(ZDK202105)；河北省重点研发计划(20326603D)；河北省现代农业产业技术体系奶牛产业创新团队奶牛疫病防控岗位(HBCT2018120205)


Biological characteristics and comparative genomics of five Klebsiella phage isolates
Author:
  • 摘要
    • | |
  • 访问统计
    • |
  • 参考文献 [51]
    • |
  • 相似文献
    • | | |
  • 文章评论
    摘要:

    【目的】耐药性克雷伯氏菌属(Klebsiella)的细菌作为人类感染的重要病原，是临床治疗重要的挑战。本研究对多株克雷伯氏菌裂解性噬菌体的生物学特性和基因组特征进行比较分析，为其应用提供更多科学数据。【方法】使用双层平板法从人类和动物新鲜粪便、污水中分离纯化裂解性克雷伯氏菌噬菌体；通过磷钨酸染色和透射电镜观察其形态；采用双层平板噬菌斑法确定其宿主范围，测定温度和pH稳定性、一步生长曲线和体外抑菌效果等生物学特性；基于全基因组测序对分离株进行比较基因组学分析；通过体内抑菌试验评估噬菌体对多重耐药变栖克雷伯氏菌(Klebsiella variicola) BS375-3感染的大蜡螟(Galleria mellonella)幼虫的保护作用。【结果】5株噬菌体分别属于Schitoviridae (pKP-BM327-1.2)、Autographiviridae (pKP-M186-2.1、pKP-M186-2.2和pKV-BS375-3.1)、Drexlerviridae (pKP-BS317-1.1)家族；噬菌体pKV-BS375-3.1可裂解受试菌中的8株，pKP-BM327-1.2可裂解受试菌中的3株，pKP-M186-2.1、pKP-M186-2.2和pKP-BS317-1.1则分别裂解受试菌中的1株；5株噬菌体感染10-20 min后即进入指数增长期，在-20-37 ℃、pH 6-10环境下均能够保持稳定活性；感染变栖克雷伯氏菌BS375-3后经噬菌体pKV-BS375-3.1处理[感染复数(multiplicity of infection, MOI)=100]的大蜡螟幼虫96 h内存活率达到80% (8/10)；5株噬菌体基因组长度在42-77 kb之间，未携带抗生素抗性基因和毒力基因，基于内溶素(endolysin)的溯源分析显示该蛋白在克雷伯氏菌噬菌体中呈现多样性，属内呈保守性。【结论】5株克雷伯氏菌噬菌体均具有较好的体外抑菌活性，生物学特性稳定，endolysin在噬菌体属内呈现保守性。宿主谱宽、潜伏期短的噬菌体pKV-BS375-3.1在治疗Klebsiella pneumoniae和K. variicola临床感染方面具有潜在应用前景。

    Abstract:

    [Objective] As a major pathogen of human infection, multidrug-resistant Klebsiella poses a great challenge in clinical treatment. To provide more scientific data for the application of phages, we analyzed and compared the biological and genomic characteristics of several strains of Klebsiella phages. [Methods] Klebsiella phages were isolated from human and animal fresh feces and sewage by the double plate method. The phage morphology was observed by phosphotungstic acid staining and transmission electron microscopy. The host spectrum, thermal stability, and pH stability of the phages were measured, and the one-step growth curve and in vitro bacteriostatic curve were established. The comparative genomics analysis was carried out for the whole genomes of the phage isolates. We further evaluated the protective effect of the phage isolates by the survival rate of Galleria mellonella larvae infected with the multidrug-resistant mutant Klebsiella variicola BS375-3 in vivo. [Results] The five phage isolates belonged to Schitoviridae (pKP-BM327-1.2), Autographiviridae (pKP-M186-2.1, pKP-M186-2.2, and pKV-BS375-3.1), and Drexlerviridae (pKP-BS317-1.1). The phage isolates pKV-BS375-3.1 and pKP-BM327-1.2 could lyse eight and three bacterial hosts, respectively, while pKP-M186-2.1, pKP-M186-2.2, and pKP-BS317-1.1 could only lyse one bacterial host. The five phage isolates entered the exponential growth stage 10-20 min after inoculation and maintained stable activity at -20 ℃-37 ℃ and pH 6-10. The survival rate of G. mellonella larvae infected with the K. variicola BS375-3 treated with pKV-BS375-3.1 (MOI=100) reached 80% (8/10) after 96 h. The genomes of the five phage isolates had the length of 42-77 kb and did not carry resistance genes or virulence genes. The traceability analysis based on endolysin showed that the protein demonstrated diversity in Klebsiella phages and was conserved within the genus. [Conclusion] All the five Klebsiella phage isolates had good antibacterial activity in vitro and stable biological characteristics. The endolysins of the phage isolates were conserved within the genus. The phage pKV-BS375-3.1 with a wide host range and short latency has a potential application prospect in the clinical treatment of Klebsiella pneumoniae and K. variicola infections.

    参考文献
    [1] 梅艳芳. K57荚膜血清型肺炎克雷伯菌的微生物及临床感染特征[D]. 南昌: 南昌大学硕士学位论文, 2018. MEI YF. Microbiological characteristics and clinical features of serotype K57 Klebsiella pneumoniae[D]. Nanchang: Master’s Thesis of Nanchang University, 2018 (in Chinese).
    [2] HOLT KE, WERTHEIM H, ZADOKS RN, BAKER S, WHITEHOUSE CA, DANCE D, JENNEY A, CONNOR TR, HSU LY, SEVERIN J, BRISSE S, CAO HW, WILKSCH J, GORRIE C, SCHULTZ MB, EDWARDS DJ, van NGUYEN K, NGUYEN TV, DAO TT, MENSINK M, et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(27): E3574-E3581.
    [3] PACZOSA MK, MECSAS J. Klebsiella pneumoniae: going on the offense with a strong defense[J]. Microbiology and Molecular Biology Reviews: MMBR, 2016, 80(3): 629-661.
    [4] LI L, YU T, MA YN, YANG ZJ, WANG WJ, SONG XB, SHEN Y, GUO TT, KONG J, WANG MY, XU H. The genetic structures of an extensively drug resistant (XDR) Klebsiella pneumoniae and its plasmids[J]. Frontiers in Cellular and Infection Microbiology, 2019, 8: 446.
    [5] RODRÍGUEZ-MEDINA N, BARRIOS-CAMACHO H, DURAN-BEDOLLA J, GARZA-RAMOS U. Klebsiella variicola: an emerging pathogen in humans[J]. Emerging Microbes & Infections, 2019, 8(1): 973-988.
    [6] GARZA-RAMOS U, SILVA-SANCHEZ J, BARRIOS H, RODRIGUEZ-MEDINA N, MARTÍNEZ- BARNETCHE J, ANDRADE V. Draft genome sequence of the first hypermucoviscous Klebsiella variicola clinical isolate[J]. Genome Announcements, 2015, 3(2): e01352-e01314.
    [7] HOPKINS KL, FINDLAY J, DOUMITH M, MATHER B, MEUNIER D, D’ARCY S, PIKE R, MUSTAFA N, HOWE R, WOOTTON M, WOODFORD N. IMI-2 carbapenemase in a clinical Klebsiella variicola isolated in the UK[J]. Journal of Antimicrobial Chemotherapy, 2017, 72(7): 2129-2131.
    [8] WANG BJ, PAN F, HAN DD, ZHAO WT, SHI YY, SUN Y, WANG C, ZHANG TD, ZHANG H. Genetic characteristics and microbiological profile of hypermucoviscous multidrug-resistant Klebsiella variicola coproducing IMP-4 and NDM-1 carbapenemases[J]. Microbiology Spectrum, 2022, 10(1): e0158121.
    [9] MAATALLAH M, VADING M, KABIR MH, BAKHROUF A, KALIN M, NAUCLÉR P, BRISSE S, GISKE CG. Klebsiella variicola is a frequent cause of bloodstream infection in the Stockholm area, and associated with higher mortality compared to K. pneumoniae[J]. PLoS One, 2014, 9(11): e113539.
    [10] BIKARD D, EULER CW, JIANG WY, NUSSENZWEIG PM, GOLDBERG GW, DUPORTET X, FISCHETTI VA, MARRAFFINI LA. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials[J]. Nature Biotechnology, 2014, 32(11): 1146-1150.
    [11] SALMOND GPC, FINERAN PC. A century of the phage: past, present and future[J]. Nature Reviews Microbiology, 2015, 13(12): 777-786.
    [12] FEDERICI S, KREDO-RUSSO S, VALDÉS-MAS R, KVIATCOVSKY D, WEINSTOCK E, MATIUHIN Y, SILBERBERG Y, ATARASHI K, FURUICHI M, OKA A, LIU B, FIBELMAN M, WEINER IN, KHABRA E, CULLIN N, BEN-YISHAI N, INBAR D, BEN-DAVID H, NICENBOIM J, KOWALSMAN N, et al. Targeted suppression of human IBD-associated gut microbiota commensals by phage consortia for treatment of intestinal inflammation[J]. Cell, 2022, 185(16): 2879-2898.e24.
    [13] WU J, ZENG H, QIAN X, LI Y, XUE F, REN J, DAI J, TANG F. Pre-treatment with phages achieved greater protection of mice against infection with Shiga toxin-producing Escherichia coli than post-treatment[J]. Research in Veterinary Science, 2022, 150: 72-78.
    [14] NIR-PAZ R, GELMAN D, KHOURI A, SISSON BM, FACKLER J, ALKALAY-OREN S, KHALIFA L, RIMON A, YERUSHALMY O, BADER R, AMIT S, COPPENHAGEN-GLAZER S, HENRY M, QUINONES J, MALAGON F, BISWAS B, MOSES AE, MERRIL G, SCHOOLEY RT, BROWNSTEIN MJ, et al. Successful treatment of antibiotic-resistant, poly-microbial bone infection with bacteriophages and antibiotics combination[J]. Clinical Infectious Diseases, 2019, 69(11): 2015-2018.
    [15] YEHL K, LEMIRE S, YANG AC, ANDO H, MIMEE M, TORRES MT, deLa FUENTE-NUNEZ C, LU TK. Engineering phage host-range and suppressing bacterial resistance through phage tail fiber mutagenesis[J]. Cell, 2019, 179(2): 459-469.e9.
    [16] WANG ZF, KONG LC, LIU Y, FU Q, CUI ZL, WANG J, MA JJ, WANG HG, YAN YX, SUN JH. A phage lysin fused to a cell-penetrating peptide kills intracellular methicillin-resistant Staphylococcus aureus in keratinocytes and has potential as a treatment for skin infections in mice[J]. Applied and Environmental Microbiology, 2018, 84(12): e00380-18.
    [17] 巩倩雯, 李一昊, 曾颃, 于沛欣, 钱新杰, 王瑜欣, 戴建君, 汤芳. 特异性识别K1荚膜大肠杆菌的噬菌体PNJ1809-36生物学特性及全基因组分析[J]. 畜牧兽医学报, 2021, 52(6): 1677-1688. GONG QW, LI YH, ZENG H, YU PX, QIAN XJ, WANG YX, DAI JJ, TANG F. Biological characteristics and whole genome analysis of phage PNJ1809-36 target Escherichia coli K1[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(6): 1677-1688 (in Chinese).
    [18] LI M, LI P, CHEN L, GUO GL, XIAO YY, CHEN L, DU H, ZHANG W. Identification of a phage-derived depolymerase specific for KL64 capsule of Klebsiella pneumoniae and its anti-biofilm effect[J]. Virus Genes, 2021, 57(5): 434-442.
    [19] SOFY AR, EL-DOUGDOUG NK, REFAEY EE, DAWOUD RA, HMED AA. Characterization and full genome sequence of novel KPP-5 lytic phage against Klebsiella pneumoniae responsible for recalcitrant infection[J]. Biomedicines, 2021, 9(4): 342.
    [20] BERTELS F, SILANDER OK, PACHKOV M, RAINEY PB, van NIMWEGEN E. Automated reconstruction of whole-genome phylogenies from short-sequence reads[J]. Molecular Biology and Evolution, 2014, 31(5): 1077-1088.
    [21] ESTRADA BONILLA B, COSTA AR, van den BERG DF, van ROSSUM T, HAGEDOORN S, WALINGA H, XIAO MF, SONG WC, HAAS PJ, NOBREGA FL, BROUNS SJJ. Genomic characterization of four novel bacteriophages infecting the clinical pathogen Klebsiella pneumoniae[J]. DNA Research, 2021, 28(4): dsab013.
    [22] PU MF, HAN PJ, ZHANG GY, LIU YC, LI YH, LI F, LI MZ, AN XP, SONG LH, CHEN YM, FAN HH, TONG YG. Characterization and comparative genomics analysis of a new bacteriophage BUCT610 against Klebsiella pneumoniae and efficacy assessment in Galleria mellonella larvae[J]. International Journal of Molecular Sciences, 2022, 23(14): 8040.
    [23] ADRIAENSSENS E, BRISTER JR. How to name and classify your phage: an informal guide[J]. Viruses, 2017, 9(4): 70.
    [24] SUN QG, KUTY GF, AROCKIASAMY A, XU M, YOUNG R, SACCHETTINI JC. Regulation of a muralytic enzyme by dynamic membrane topology[J]. Nature Structural & Molecular Biology, 2009, 16(11): 1192-1194.
    [25] LU B, YAO XP, HAN GL, LUO ZD, ZHANG JR, YONG K, WANG Y, LUO Y, YANG ZX, REN MS, CAO SZ. Isolation of Klebsiella pneumoniae phage vB_KpnS_MK54 and pathological assessment of endolysin in the treatment of pneumonia mice model[J]. Frontiers in Microbiology, 2022, 13: 854908.
    [26] ESKENAZI A, LOOD C, WUBBOLTS J, HITES M, BALARJISHVILI N, LESHKASHELI L, ASKILASHVILI L, KVACHADZE L, van NOORT V, WAGEMANS J, JAYANKURA M, CHANISHVILI N, de BOER M, NIBBERING P, KUTATELADZE M, LAVIGNE R, MERABISHVILI M, PIRNAY JP. Combination of pre-adapted bacteriophage therapy and antibiotics for treatment of fracture-related infection due to pandrug-resistant Klebsiella pneumoniae[J]. Nature Communications, 2022, 13: 302.
    [27] LUONG T, SALABARRIA AC, ROACH DR. Phage therapy in the resistance era: where do we stand and where are we going?[J]. Clinical Therapeutics, 2020, 42(9): 1659-1680.
    [28] 李金花, 白雨凡, 马春兰, 季秀玲, 魏云林. 噬菌体温度适应性的研究进展[J]. 中国生物工程杂志, 2022, 42(S1): 139-145. LI JH, BAI YF, MA CL, JI XL, WEI YL. Research progress on temperature adaptation of bacteriophage[J]. China Biotechnology, 2022, 42(S1): 139-145 (in Chinese).
    [29] COX J, SCHUBERT AM, TRAVISANO M, PUTONTI C. Adaptive evolution and inherent tolerance to extreme thermal environments[J]. BMC Evolutionary Biology, 2010, 10(1): 75.
    [30] KERING KK, ZHANG XX, NYARUABA R, YU JP, WEI HP. Application of adaptive evolution to improve the stability of bacteriophages during storage[J]. Viruses, 2020, 12(4): 423.
    [31] WHITTINGTON AC, ROKYTA DR. Biophysical spandrels form a hot-spot for kosmotropic mutations in bacteriophage thermal adaptation[J]. Journal of Molecular Evolution, 2019, 87(1): 27-36.
    [32] CATALÃO MJ, GIL F, MONIZ-PEREIRA J, PIMENTEL M. Functional analysis of the holin-like proteins of mycobacteriophage Ms6[J]. Journal of Bacteriology, 2011, 193(11): 2793-2803.
    [33] REDDY BL, SAIER MH Jr. Topological and phylogenetic analyses of bacterial holin families and superfamilies[J]. Biochimica et Biophysica Acta, 2013, 1828(11): 2654-2671.
    [34] 李艳. 猪链球菌噬菌体穿孔素(holin)的亚细胞定位及拓扑结构[D]. 南京: 南京农业大学硕士学位论文, 2013. LI Y. Subcellular localization and topology of holin encoded by Streptococcus suis bacteriophage[D]. Nanjing: Master’s Thesis of Nanjing Agricultural University, 2013 (in Chinese).
    [35] DOWAH ASA, CLOKIE MRJ. Review of the nature, diversity and structure of bacteriophage receptor binding proteins that target Gram-positive bacteria[J]. Biophysical Reviews, 2018, 10(2): 535-542.
    [36] BERTOZZI SILVA J, STORMS Z, SAUVAGEAU D. Host receptors for bacteriophage adsorption[J]. FEMS Microbiology Letters, 2016, 363(4): fnw002.
    [37] DUNSTAN RA, BAMERT RS, BELOUSOFF MJ, SHORT FL, BARLOW CK, PICKARD DJ, WILKSCH JJ, SCHITTENHELM RB, STRUGNELL RA, DOUGAN G, LITHGOW T. Mechanistic insights into the capsule-targeting depolymerase from a Klebsiella pneumoniae bacteriophage[J]. Microbiology Spectrum, 2021, 9(1): e0102321.
    [38] SQUEGLIA F, MACIEJEWSKA B, ŁĄTKA A, RUGGIERO A, BRIERS Y, DRULIS-KAWA Z, BERISIO R. Structural and functional studies of a Klebsiella phage capsule depolymerase tailspike: mechanistic insights into capsular degradation[J]. Structure (London, England: 1993), 2020, 28(6): 613-624.e4.
    [39] LATKA A, LEMIRE S, GRIMON D, DAMS D, MACIEJEWSKA B, LU T, DRULIS-KAWA Z, BRIERS Y. Engineering the modular receptor-binding proteins of Klebsiella phages switches their capsule serotype specificity[J]. mBio, 2021, 12(3): e00455-e00421.
    [40] CAHILL J, YOUNG R. Phage lysis: multiple genes for multiple barriers[J]. Advances in Virus Research, 2019, 103: 33-70.
    [41] MARQUES AT, TANOEIRO L, DUARTE A, GONÇALVES L, VÍTOR JMB, VALE FF. Genomic analysis of prophages from Klebsiella pneumoniae clinical isolates[J]. Microorganisms, 2021, 9(11): 2252.
    [42] PAYNE KM, HATFULL GF. Mycobacteriophage endolysins: diverse and modular enzymes with multiple catalytic activities[J]. PLoS One, 2012, 7(3): e34052.
    [43] FANG QQ, FENG Y, McNALLY A, ZONG ZY. Characterization of phage resistance and phages capable of intestinal decolonization of carbapenem-resistant Klebsiella pneumoniae in mice[J]. Communications Biology, 2022, 5: 48.
    [44] MAJKOWSKA-SKROBEK G, MARKWITZ P, SOSNOWSKA E, LOOD C, LAVIGNE R, DRULIS-KAWA Z. The evolutionary trade-offs in phage-resistant Klebsiella pneumoniae entail cross-phage sensitization and loss of multidrug resistance[J]. Environmental Microbiology, 2021, 23(12): 7723-7740.
    [45] CHATTERJEE A, JOHNSON CN, LUONG P, HULLAHALLI K, McBRIDE SW, SCHUBERT AM, PALMER KL, CARLSON PE Jr, DUERKOP BA. Bacteriophage resistance alters antibiotic-mediated intestinal expansion of enterococci[J]. Infection and Immunity, 2019, 87(6): e00085-e00019.
    [46] HO K, HUO WW, PAS S, DAO R, PALMER KL. Loss-of-function mutations in epaR confer resistance to ϕNPV1 infection in Enterococcus faecalis OG1RF[J]. Antimicrobial Agents and Chemotherapy, 2018, 62(10): e00758-18.
    [47] SUMRALL ET, SHEN Y, KELLER AP, RISMONDO J, PAVLOU M, EUGSTER MR, BOULOS S, DISSON O, THOUVENOT P, KILCHER S, WOLLSCHEID B, CABANES D, LECUIT M, GRÜNDLING A, LOESSNER MJ. Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion[J]. PLoS Pathogens, 2019, 15(10): e1008032.
    [48] JEON J, YONG D. Two novel bacteriophages improve survival in Galleria mellonella infection and mouse acute pneumonia models infected with extensively drug-resistant Pseudomonas aeruginosa[J]. Applied and Environmental Microbiology, 2019, 85(9): e02900-e02918.
    [49] OLSZAK T, ZARNOWIEC P, KACA W, DANIS-WLODARCZYK K, AUGUSTYNIAK D, DREVINEK P, de SOYZA A, McCLEAN S, DRULIS-KAWA Z. In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas aeruginosa strains from cystic fibrosis patients[J]. Applied Microbiology and Biotechnology, 2015, 99(14): 6021-6033.
    [50] GHANAIM AM, FOAAD MA, GOMAA EZ, ABDELFATAH EL DOUGDOUG K, MOHAMED GE, ARISHA AH, KHAMIS T. Bacteriophage therapy as an alternative technique for treatment of multidrug-resistant bacteria causing diabetic foot infection[J]. International Microbiology, 2023. 26: 343–359.
    [51] SENEVIRATHNE A, LEE J, de ZOYSA M, NIKAPITIYA C. Genome characterization of bacteriophage KPP-1, a novel member in the subfamily Vequintavirinae, and use of its endolysin for the lysis of multidrug-resistant Klebsiella variicola in vitro[J]. Microorganisms, 2023, 11(1): 207.
    相似文献
    引证文献
引用本文

王猛,王雪,赵佳男,曾君,左君豪,郭志良,季芳,邵建立,张立敏,王承民,秦建华. 五株克雷伯氏菌噬菌体的生物学特性及比较基因组学研究[J]. 微生物学报, 2023, 63(8): 3110-3128

复制
分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2022-11-26
  • 最后修改日期:2023-03-10
  • 在线发布日期: 2023-08-03
  • 出版日期: 2023-08-04
文章二维码

科微学术

微生物学报

您是本站第 访问者

通信地址：北京市朝阳区北辰西路1号院3号 中国科学院微生物研究所   邮编：100101

电话：010-64807516    E-mail：actamicro@im.ac.cn

版权所有：微生物学报 ® 2025 版权所有