1株肺炎克雷伯菌噬菌体kp6的生物学特性及全基因组分析

doi:10.16431/j.cnki.1671-7236.2024.09.027

Abstract

Abstract: 【Objective】 The purpose of this study was to explore the biological characteristics and whole genome characteristics of Klebsiella pneumoniae phage isolated from sewage samples, and provide a basis for the prevention and control of clinical Klebsiella pneumoniae infection and the development of new phage related alternative antibody products. 【Method】 A Klebsiella pneumoniae TJAU-K11 was used as the host bacteria, the phage of Klebsiella pneumoniae was isolated and purified from sewage samples by double plate method.The morphology of the isolated phage was observed by transmission electron microscopy, and the host spectrum, optimal multiplicity of infection (MOI) and one-step growth curve were determined，and the stability of phage was evaluated.The effect of phage on the biofilm formation and cleavage of Klebsiella pneumoniae TJAU-K11 was investigated, and the whole genome was sequenced to understand the biological characteristics and whole genome characteristics of phage. 【Result】 3 Klebsiella pneumoniae phage were successfully isolated from sewage samples and named kp4, kp5 and kp6, respectively.Transmission electron microscopy showed that phage kp6 was a short-tailed phage with regular icosahedral morphology.The one-step growth curve showed that the incubation period of phage kp6 was 20 min and the outbreak period was 30 min.The phage titer remained stable at -20-50 ℃, pH 4.0-10.0 and UV irradiation for 12 min.The results of single bacteriophage inhibition test in vitro showed that the D_{600 nm} value of phage kp6 groups with different MOI was always less than 0.4 within 7 h.The results of in vitro bacteriostatic test of phage cocktail showed that the D_{600 nm} value of phage kp6 within 12 h was less than 0.1 when MOI was 1 and 100.The amount of host bacteria biofilm formation in phage kp6 groups with different MOI was extremely significantly lower than that in positive control group (P＜0.01), and both single phage kp6 and bacteriophage cocktail could lysate host bacteria biofilm.The whole genome sequencing showed that the phage kp6 genome was double-linked looped DNA, with a total length of 40 971 bp and GC content of 53.13%.Among 49 coding sequences, 23 encoded proteins with known functions, including gp12, a tail fibrillar protein for short tail iophage adsorption and injection, and lytic functional protein endolysin. 【Conclusion】 In this study, Klebsiella pneumoniae lytic phage kp6 was isolated, and there was an endolysin in its genome.The phage cocktail prepared by phage kp6 could completely inhibit the growth of host bacteria within 12 h.The results laid a foundation for the subsequent development of lytic enzymes and phage preparations.

Key words: Klebsiella pneumoniae; phage; biological characteristics; whole genome

CLC Number:

S852.61⁺2

RONG Ruifan, GUO Haiyue, HU Pengcheng, ZUO Junhao, ZHANG Dongchao, GUO Zhiliang, LI Ying, ZHANG Yuxin, WANG Xinru, ZHAO Ruili, LIU Dingkuo. Biological Characteristics and Whole Genome Analysis of Klebsiella pneumoniae Phage kp6[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(9): 3990-4001.

References

[1] 刘家齐,于新,田爽，等.多重耐药肺炎克雷伯菌裂解性噬菌体Kp-8329的分离鉴定和基因组分析[J].中国兽医杂志,2021,57(7):42-46. LIU J Q,YU X,TIAN S,et al.Isolation,identification,and genomic analysis of the lytic phage Kp-8329 of multidrug-resistant Klebsiella pneumoniae[J].Chinese Journal of Veterinary Medicine,2021,57(7):42-46.(in Chinese)
[2] WAGENLEHNER F M E,DITTMAR F.Re:Global burden of bacterial antimicrobial resistance in 2019:A systematic analysis[J].European Urology,2022,82(6):658.
[3] 解天慧.大肠杆菌O157:H7和沙门氏菌噬菌体及其内溶酶和穿孔素的研究[D].重庆：西南大学,2021. XIE T H.A study on Escherichia coli O157∶H7 and Salmonella phages and endolytic enzymes and perforins[D].Chongqing：Southwest University,2021.(in Chinese)
[4] 刘源平,王兆飞,孙爱喜，等.肺炎克雷伯菌噬菌体解聚酶的研究进展[J].微生物学通报,2021,48(9):3281-3292. LIU Y P,WANG Z F,SUN A X,et al.Research progress on phage depolymerases of Klebsiella pneumoniae[J]Microbiology China,2021,48(9):3281-3292.(in Chinese)
[5] 蔡若鹏,顾敬敏,韩文瑜.噬菌体解聚酶的分类与结构特征及其应用研究进展[J].中国兽医学报,2020,40(6):1258-1264. CAI R P,GU J M,HAN W Y.Classification and structural characteristics of bacteriophage depolymerizing enzymes and their application research progress[J].Chinese Journal of Veterinary Science,2020,40(6):1258-1264.(in Chinese)
[6] LI M,WANG H,CHEN L,et al.Identification of a phage-derived depolymerase specific for KL47 capsule of Klebsiella pneumoniae and its therapeutic potential in mice[J]. Virologica Sinica，2022，37(4):538-546.
[7] PENZINER S,SCHOOLEY R, PRIDE D.Animal models of phage therapy[J]. Frontiers in Microbiology,2021,12:631794.
[8] NICOLA P，ETTORE S，SUSANNA E.Advantages and limitations of bacteriophages for the treatment of bacterial infections[J].Frontiers in Pharmacology,2019,10:513.
[9] 罗书泓,汪璐,徐朝霞，等.耐碳青霉烯类肺炎克雷伯菌噬菌体vB_KpnP1的分离鉴定和生物学特性研究[J].陆军军医大学学报,2023,45(10):1030-1039. LUO S H,WANG L,XU Z X,et al.Isolation，identification and biological characteristics of phage vB_KpnP1 for carbapenem resistant Klebsiella pneumoniae[J].Journal of Army Medical University,2023,45(10):1030-1039.(in Chinese)
[10] 曾君.多重耐药铜绿假单胞菌噬菌体生物学特性及全基因组分析[D].天津：天津农学院,2023. ZENG J.Biological characteristics and genome analysis of multidrug resistant Pseudomonas aeruginosa phage[D].Tianjin：Tianjin Agricultural University,2023.(in Chinese)
[11] 黄旗森.全基因组测序揭示高毒力肺炎克雷伯菌毒力及耐药质粒的遗传多样性与进化[D].南昌：南昌大学，2022. HUANG Q S.Whole genome sequencing reveals genetic diversity and evolution of virulence and resistant plasmids of highly virulent Klebsiella pneumoniae[D].Nanchang:Nanchang University,2022.(in Chinese)
[12] MOHAMED S,MOHAMED M,KHALIL M,et al.Combination of essential oil and ciprofloxacin to inhibit/eradicate biofilms in multidrug-resistant Klebsiella pneumoniae[J]. Journal of Applied Microbiology,2018,125(1):84-95.
[13] 李莉莎,李建辉,何斌,等.噬菌体治疗泛耐药肺炎克雷伯菌肺部感染的临床应用及效果初探[J].上海交通大学学报(医学版),2021,41(9):1272-1276. LI L S,LI J H,HE B,et al.Clinical application and efficacy of phage therapy for pan resistant Klebsiella pneumoniae pulmonary infection[J].Journal of Shanghai Jiaotong University (Medical Science),2021,41(9):1272-1276.(in Chinese)
[14] TURNE D,SHKOPOROV A,LOOD C,et al.Abolishment of morphology-based taxa and change to binomial species names:2022 taxonomy update of the ICTV bacterial viruses subcommittee[J].Archives of Virology，2023，168(2):74.
[15] BARYLSKI J,ENAULT F,DUTILH B,et al.Analysis of Spounaviruses as a case study for the overdue reclassification of tailed phages[J].Systematic Biology,2020,69(1):110-123.
[16] WENDLING C,LANGE J,HEIKO L，et al.Higher phage virulence accelerates the evolution of host resistance[J]. Royal Society,2022,289(1984):20221070.
[17] 王博宇,杨孜孜,SUN F Z，等.基于智能计算噬菌体的细菌宿主范围预测[J].微生物学报,2024,64(2):344-363. WANG B Y,YANG Z Z,SUN F Z,et al.Prediction of bacterial host range based on intelligent computing of bacteriophages[J].Acta Microbiologica Sinica,2024,64(2):344-363.(in Chinese)
[18] 孙佳慧,耿佩玲,万小芙,等.噬菌体吸附及受体结合蛋白(RBP)的研究进展与应用[J].生物工程学报,2021,37(8):2614-2622. SUN J H,GENG P L,WAN X F,et al.Research progress and application of bacteriophage adsorption and receptor binding protein (RBP)[J].Chinese Journal of Biotechnology,2021,37(8):2614-2622.(in Chinese)
[19] 赵日虹.K1型肺炎克雷伯菌噬菌体vB_KpnP_ZK1及其解聚酶的生物学特性研究[D].长春：吉林大学,2022. ZHAO R H.The biological characteristics of Klebsiella pneumoniae phage vB_KpnP_ZK1 and its depolymerase[D].Changchun:Jilin University,2022.(in Chinese)

Biological Characteristics and Whole Genome Analysis of Klebsiella pneumoniae Phage kp6

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