1株牛源耐碳青霉烯K57型肺炎克雷伯菌噬菌体的分离鉴定及其全基因组分析

doi:10.16431/j.cnki.1671-7236.2025.11.042

摘要/Abstract

摘要： 【目的】以牛源K57型耐碳青霉烯肺炎克雷伯菌临床分离株KLB-04为宿主菌，分离该菌的强裂解性噬菌体，并对该噬菌体的生化特性及全基因组进行分析，为后续肺炎克雷伯菌噬菌体临床应用、噬菌体衍生蛋白的开发与使用提供一定的科学依据。【方法】采用双层琼脂平板法从污水中分离肺炎克雷伯菌裂解性噬菌体；通过透射电镜观察、宿主谱测定、pH耐受和温度稳定性评估、最佳感染复数测定、一步生长曲线测定、全基因组测序等方法分析噬菌体的形态学、生物学特性和全基因组特性。【结果】分离到1株裂解性噬菌体并命名为GXKP01，该噬菌体的噬菌斑直径约3 mm，带有约2 mm外圈晕环；透射电镜观察显示，该噬菌体由头部和较短的收缩性尾部组成，头部为正二十面体，直径约60 nm，尾部长15～20 nm，属于Studiervirinae亚科、Przondovirus属。噬菌体GXKP01感染肺炎克雷菌KLB-04的潜伏期约10 min,一个感染周期约持续70 min，每感染一个细菌约可产生136个子代噬菌体病毒颗粒。噬菌体GXKP01仅可裂解宿主菌KLB-04，特异性较强；在4～60 ℃，pH 3.0～12.0条件下稳定性良好。噬菌体GXKP01基因组为双链线性DNA，全长39 795 bp，GC含量为53.13%，共有44个编码基因；不携带毒力因子和耐药基因，显示出遗传的安全性。【结论】本研究分离出1株可裂解K57血清型耐碳青霉烯肺炎克雷伯菌的强裂解性噬菌体，为利用噬菌体预防和治疗高毒力耐药肺炎克雷伯菌感染提供一定的研究基础。

关键词: 噬菌体; 分离鉴定; 耐碳青霉烯肺炎克雷伯菌; 全基因组测序

Abstract: 【Objective】 Using the bovine-derived carbapenem-resistant Klebsiella pneumoniae clinical isolate KLB-04 (K57 type) as the host strain,this study aimed to isolate a highly lytic phage targeting this bacterium and analyze its biochemical characteristics and whole genome,so as to provide a scientific basis for the clinical application of Klebsiella pneumoniae phages and the development of phage-derived proteins. 【Method】 The lytic phage was isolated from sewage using the double-layer agar method.Transmission electron microscopy,host range analysis,pH tolerance and temperature stability tests,determination of the optimal multiplicity of infection,one-step growth curve assay,and whole-genome sequencing were employed to investigate the morphological,biological,and genomic characteristics of the isolated phage. 【Result】 A lytic bacteriophage strain was isolated and designated as GXKP01.GXKP01 formed plaques with an average diameter of approximately 3 mm,surrounded by a halo measuring about 2 mm in width.Transmission electron microscopy revealed that GXKP01 possessed a head-tail structure,with the head being a regular icosahedron approximately 60 nm in diameter and the tail measuring between 15-20 nm in length,which belonged to the subfamily Studiervirinae and the genus Przondovirus.The latent period of GXKP01 infection in KLB-04 was approximately 10 min,with a complete infection cycle lasting around 70 min.Each infected bacterium produced approximately 136 progeny phage particles.Phage GXKP01 could only cleave the host bacterium KLB-04,with strong specificity.Additionally,GXKP01 demonstrated remarkable stability across a wide range of temperatures (4-60 ℃) and pH levels (pH 3.0-12.0).Genomic analysis revealed that GXKP01’s genome consisted of double-stranded linear DNA with a length of 39 795 bp,a GC content of 53.13%,and contained 44 coding genes.Notably,GXKP01 did not carry any virulence factors and antibiotic-resistance genes,confirming its genetic safety. 【Conclusion】 This study isolated a lytic phage capable of targeting K57-type carbapenem-resistant Klebsiella pneumoniae,laying a foundation for future phage-based prevention and treatment of infections caused by hypervirulent,drug-resistant Klebsiella pneumoniae.

Key words: phage; isolation and identification; carbapenem-resistant Klebsiella pneumoniae; whole genome sequencing

中图分类号:

庞舒琪, 韩正慧, 汤昆平, 王乐平, 黎春玲, 白慧丽, 李常挺, 李显, 尹杨燕, 马春霞, 滕翎, 李军, 陈忠伟, 周以林, 彭昊. 1株牛源耐碳青霉烯K57型肺炎克雷伯菌噬菌体的分离鉴定及其全基因组分析[J]. 中国畜牧兽医, 2025, 52(11): 5476-5487.

PANG Shuqi, HAN Zhenghui, TANG Kunping, WANG Leping, LI Chunling, BAI Huili, LI Changting, LI Xian, YIN Yangyan, MA Chunxia, TENG Ling, LI Jun, CHEN Zhongwei, ZHOU Yilin, PENG Hao. Isolation,Identification and Whole Genome Analysis of a Bovine Carbapenem-resistant Klebsiella pneumoniae Phage[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(11): 5476-5487.

参考文献

[1] DANNI P,JIANKANG Z,KANG C,et al."Superbugs" with hypervirulence and carbapenem resistance in Klebsiella pneumoniae:The rise of such emerging nosocomial pathogens in China[J].Science Bulletin,2023,68(21):2658-2670.
[2] CHENG J,ZHOU M,NOBREGA D B,et al.Genetic diversity and molecular epidemiology of outbreaks of Klebsiella pneumoniae mastitis on two large Chinese dairy farms[J].Journal of Dairy Science,2020,104(1):762-775.
[3] YANG J,XIONG Y,BARKEMA H W,et al.Comparative genomic analyses of Klebsiella pneumoniae K57 capsule serotypes isolated from bovine mastitis in China[J].Journal of Dairy Science,2023,107(5):3114-3126.
[4] WYRES K L,LAM M M C,HOLT K E.Population genomics of Klebsiella pneumoniae[J].Nature Reviews, Microbiology,2020,18(6):344-359.
[5] WONG M H Y,SHUN H P,CHEN J H K,et al.Emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae[J].The Lancet Infectious Diseases,2017,18(1):25.
[6] DIAZ-MUNOZ S L,BRITT K.Bacteria-phage interactions in natural environments[J].Advances in Applied Microbiology,2014,89:135-183.
[7] DION M B,FRANK O,SYLVAIN M.Phage diversity,genomics and phylogeny[J].Nature Reviews.Microbiology,2020,18(3):125-138.
[8] STRATHDEE S A,HATFULL G F,MUTALIK V K,et al.Phage therapy:From biological mechanisms to future directions[J].Cell,2023,186(1):17-31.
[9] GORDILLO ALTAMIRANO F L,BARR J J.Phage therapy in the postantibiotic era[J].Clinical Microbiology Reviews,2019,32(2):e00066-18.
[10] LUONG T,SALABARRIA A,ROACH R D.Phage therapy in the resistance era:Where do we stand and where are we going?[J].Clinical Therapeutics,2020,42(9):1659-1680.
[11] PAL N,SHARMA P,KUMAWAT M,et al.Phage therapy:An alternative treatment modality for MDR bacterial infections[J].Infectious Diseases,2024,56(10):31-33.
[12] 和晋渝.肺炎克雷伯菌的血清分型及毒力基因分布的研究[D].重庆：重庆医科大学,2012.HE J Y.Study on serotyping and virulence gene distribution of Klebsiella pneumoniae[D].Chongqing:Chongqing Medical University,2012.(in Chinese)
[13] CLINICAL AND LABORATORY STANDARDS INSTITUTE. Performance standards for antimicrobial susceptibility testing. 34th ed. CLSI supplment M100[S]. Clinical and Laboratory Standards Institute, 2024.
[14] 王乐平,潘艳,谭奕舟,等.1株大肠杆菌噬菌体vB_EcoP_GN07的分离鉴定及全基因组分析[J].中国畜牧兽医,2023,50(4):1522-1531.WANG L P,PAN Y,TAN Y Z,et al.Isolation,identification and genome analysis of Escherichia coli phage vB_EcoP_GN07[J].China Animal Husbandry & Veterinary Medicine,2023,50(4):1522-1531.(in Chinese)
[15] LIU S,XU H,CAO R,et al.Isolation,identification,and biological characterization of phage vB_KpnM_KpVB3 targeting carbapenem-resistant Klebsiella pneumoniae ST11[J].Journal of Global Antimicrobial Resistance,2024,37:179-184.
[16] PENZINER S,SCHOOLEY R T,PRIDE D T.Animal models of phage therapy[J]. Frontiers in Microbiology,2021,12:631794.
[17] SADDAN M,KHAN M,JAMAL M,et al.Correction:Nutritional analysis and characterization of carbapenemase producing-Klebsiella pneumoniae resistant genes associated with bovine mastitis infected cow’s milk[J].PLoS One,2024,19(1):e0297123.
[18] MUNITA J M,ARIAS C A.Mechanisms of antibiotic resistance[J].Microbiology Spectrum,2016,4(2):10.
[19] 罗书泓.碳青霉烯类耐药肺炎克雷伯菌噬菌体vB＿KonP1的分离鉴定和应用研究[D].泸州：西南医科大学,2023.LUO S H.Isolation,identification and application of carbapenem-resistant Klebsiella pneumoniae bacteriophage vB_KonP1[D].Luzhou:Southwest Medical University,2023.(in Chinese)
[20] ZHU D,KEBEDE B,CHEN G,et al.Effects of the vat pasteurization process and refrigerated storage on the bovine milk metabolome[J].Journal of Dairy Science,2020,103(3):2077-2088.
[21] 王乐平.噬菌体SP02的分离鉴定及其在种鸡蛋消毒的应用研究[D].南宁：广西大学,2023.WANG L P.Isolation and identification of phage SP02 and its application in the sterilization of breeding eggs[D].Nanning:Guangxi University,2023.(in Chinese)
[22] YOSHIKAWA G,ASKORA A,BLANC-MATHIEU R,et al. Xanthomonas citri jumbo phage XacN1 exhibits a wide host range and high complement of tRNA genes[J].Scientific Reports,2018,8(1):4486.
[23] ENAV H,BEJA O,YAEL M.Cyanophage tRNAs may have a role in cross-infectivity of oceanic Prochlorococcus and Synechococcus hosts[J].The ISME Journal,2012,6(3):619-628.
[24] MA R,SHAO S,WEI S,et al.A novel phage infecting the marine photoheterotrophic bacterium Citromicrobium bathyomarinum[J].Viruses,2022,14(3):512.
[25] WENDLIN C C,LANGE J,LIESEGANG H,et al.Higher phage virulence accelerates the evolution of host resistance[J].Proceedings of the Royal Society B,2022,289(1984):20221070.
[26] MONTEIRO R,PIRES D P,COSTA A R,et al.Phage therapy:Going temperate?[J].Trends in Microbiology,2019,27(4):368-378.