摘要
目的为了在抗生素使用效果不佳的现状下减少高毒力肺炎克雷伯菌感染而造成的损失,本研究旨在探究使用噬菌体替代抗生素替代疗法。方法使用实验室分离得到的一株高毒力肺炎克雷伯菌为宿主菌经双层平板法分离纯化得到噬菌体并通过透射电镜观察其状态;使用全基因组测序和注释并进行开放阅读框预测和进化树分析;使用不同条件处理噬菌体并铺设双层平板评估其生化特性;最后使用噬菌体鸡尾酒治疗感染肺炎克雷伯菌小鼠,观察小鼠内脏状态和组织切片。结果分离得到的噬菌体13g和15e能够有效地裂解Z18菌株,透射电镜显示二者均于属短尾噬菌体科,基因组分析结果显示二者与已知的噬菌体同源性最高分别为98.89%和99.31%,与多株不同地区分离得到的噬菌体均有亲缘关系;两株噬菌体的最佳感染复数均在0.01~0.001之间;在-20℃~90℃处理1 h以及在1~14的pH值范围内处理1 h,噬菌体的感染活力均有不同程度的下降;两株噬菌体的裂解周期均较长,潜伏期均在10 min左右,在120 min左右达到最大值,爆发量分别为90 CFU·cell^(-1)和59 CFU·cell^(^(-1));噬菌体在体内实验中对比抗生素组和对照组能够显著降低小鼠的死亡率以及大幅度减少器官病变。结论本研究中分离得到的噬菌体在抗极端环境中的能力较好,在裂解细菌方面展示出了优秀的能力。噬菌体鸡尾酒在提高肠道感染肺炎克雷伯菌小鼠模型的生存率、减轻病理损伤等方面显示出较好的体内抗菌效果和应用潜力,为进一步的实验和应用提供了理论基础和实验依据。
Objective In order to reduce the loss caused by hypervirulent Klebsiella pneumoniae(hvKp)infection in the current situation of poor antibiotic use,this study aims to explore the use of phages instead of antibiotic replacement therapy.Methods The phage was isolated and purified by double-layer plate method using a hvKp isolated in the laboratory as the host strain,and its state was observed by transmission electron microscopy.Using whole genome sequencing and annotation and predict their open reading fragment and phylogenetic tree analysis;bacteriophages were treated under different conditions and double-layer plates were laid to evaluate their biochemical characteristics.Finally,phage cocktail was used to treat mice infected with K.pneumoniae,and the visceral state and tissue sections of mice were observed.Results The isolated phages 13 g and 15 e could effectively lyse the Z18 strain.Transmission electron microscopy showed that both of them belonged to the family Podoviridae.The genome analysis showed that the highest homology with known phages was 98.89%and 99.31%,respectively,and they were related to many phages isolated from different regions.The MOI of the two phages was between 0.01 and 0.001.When treated at-20℃~90℃for 1 h or at pH 1~14 for 1 h,the infection activity of phages decreased to varying degrees.The lysis cycle of the two phages was longer,the incubation period was about 10 min,and the maximum value was about 120 min.The bursts were 90 CFU·cell^(-1)and 59 CFU·cell^(-1),respectively.Compared with the antibiotic group and the control group,the phage can significantly reduce the mortality of mice and significantly reduce organ lesions in vivo experiments.Conclusion The phages isolated in this study have a good ability to resist extreme environments,and show excellent ability to lyse bacteria.The phage cocktail showed good in vivo antibacterial effect and application potential in improving the survival rate of the mouse model of intestinal infection with K.pneumoniae and reducing pathological damage,which provided a theoretical basis and experimental basis for further experiments and applications.
作者
叶浏渊
宁思奇
司娟娟
张奇
马赫迪
吴剑桥
薛世隆
胡圣伟
YE Liuyuan;NING Siqi;SI Juanjuan;ZHANG Qi;MA Hedi;WU Jianqiao;XUE Shilong;HU Shengwei(School of Life Science,Shihezi University,Shihezi,Xinjiang 832003,China)
出处
《石河子大学学报(自然科学版)》
北大核心
2025年第6期757-766,共10页
Journal of Shihezi University(Natural Science)
基金
新疆维吾尔自治区天山英才项目(2022TSYCCX0125)。
关键词
噬菌体
肺炎克雷伯菌
噬菌体鸡尾酒
抗菌活性
Bacteriophage
Klebsiella pneumoniae
phage cocktail
antibacterial therapy
