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

摘要/Abstract

摘要： 【目的】探究从污水样品中分离到的1株肺炎克雷伯菌噬菌体的生物学特性及全基因组特征，为临床肺炎克雷伯菌感染的防控和开发噬菌体相关新型替抗产品提供基础。【方法】以肺炎克雷伯菌TJAU-K11为宿主菌，采用双层平板法从污水样品中分离纯化获得肺炎克雷伯菌噬菌体。通过透射电镜观察噬菌体形态，测定噬菌体宿主谱、最佳感染复数(MOI)、一步生长曲线，评估噬菌体稳定性，探究其对肺炎克雷伯菌TJAU-K11的生物被膜形成的影响以及裂解效果，同时进行全基因组测序，了解噬菌体的生物学特性及全基因组特征。【结果】从污水样品中成功分离获得3株肺炎克雷伯菌噬菌体，分别命名为kp4、kp5和kp6，透射电镜观察结果显示，噬菌体kp6是具有正二十面体形态的短尾噬菌体。一步生长曲线结果显示，噬菌体kp6的潜伏期为20 min，爆发期为30 min。该噬菌体在―20～50 ℃、pH 4.0～10.0和紫外照射12 min内噬菌体效价保持稳定。单一噬菌体的体外抑菌试验结果显示，不同MOI噬菌体kp6组的D_{600 nm}值在7 h内始终＜0.4；噬菌体鸡尾酒制剂的体外抑菌试验结果显示，MOI为1、100时，噬菌体kp6在12 h内的D_{600 nm}值＜0.1。不同MOI噬菌体kp6组宿主菌生物被膜形成量均极显著低于阳性对照组(P＜0.01)，单一噬菌体kp6与噬菌体鸡尾酒制剂均能裂解宿主菌生物被膜。全基因组测序表明噬菌体kp6基因组为双链环状DNA，全长40 971 bp，GC含量为53.13%，49个编码序列中有23个编码已知功能的蛋白，其中包括短尾噬菌体吸附和注入作用的尾纤蛋白gp12、裂解功能蛋白内溶酶等。【结论】本研究分离获得的肺炎克雷伯菌裂解性噬菌体kp6，其基因组中存在1个内溶酶，以其制备的噬菌体鸡尾酒制剂在12 h内能完全抑制宿主菌生长，试验结果为后续裂解酶和噬菌体制剂的开发奠定基础。

关键词: 肺炎克雷伯菌; 噬菌体; 生物学特性; 全基因组

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

中图分类号:

S852.61⁺2

荣睿璠, 郭海悦, 胡鹏程, 左君豪, 张东超, 郭志良, 李颖, 张宇欣, 王心如, 赵瑞利, 刘鼎阔. 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[J]. 中国畜牧兽医, 2024, 51(9): 3990-4001.

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.

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