驯化噬菌体提高噬菌体对碳青霉烯类耐药肺炎克雷伯菌的杀菌能力

Phage training improves the bactericidal ability against carbapenem-resistant Klebsiella pneumoniae

摘要:

【目的】本研究旨在通过驯化提高噬菌体的裂解能力并降低其宿主菌耐受性产生的速度，从而提高对重要病原菌-碳青霉烯类耐药肺炎克雷伯菌(carbapenem-resistant Klebsiella pneumoniae, CRKp)的杀菌效果。【方法】以临床CRKp菌株Kp2092为宿主菌，利用双层琼脂平板法从污水中分离噬菌体并分析其裂解谱；对其中的广谱强裂解性噬菌体通过透射电镜观察其形态特征并进行全基因组测序；通过噬菌体-宿主连续培养进行噬菌体驯化，并比较驯化前后噬菌体生物学特性的差异。【结果】分离得到的9株肺炎克雷伯菌噬菌体中，噬菌体P55anc裂解能力强且裂解谱广，透射电镜观察发现其为短尾噬菌体。P55anc基因组全长40 301 bp，包含51个编码序列，其中27个具有已知功能，主要涉及核酸代谢、噬菌体结构蛋白、DNA包装和细胞裂解等。噬菌体P55anc经9 d的驯化后，得到3株驯化噬菌体。驯化后噬菌体杀菌能力增强，主要表现为细菌生长曲线显著下降、噬菌体暴发量增多、裂解谱扩大，且宿主菌对其产生抗性的概率显著降低。与此同时，驯化后的噬菌体在热处理、紫外暴露以及血清等环境下保持较好的稳定性。【结论】利用噬菌体-宿主连续培养的方法可对噬菌体进行驯化和筛选，驯化后的噬菌体杀菌效果更强，且在不同压力处理下的稳定性良好，而细菌产生噬菌体抗性的概率也降低。

Abstract:

[Objective] This study aims to improve the bactericidal ability of phages against carbapenem-resistant Klebsiella pneumoniae, a major pathogen in clinical practice, and reduce host resistance to phages by developing a novel method of phage training. [Methods] Phages were isolated from municipal wastewater by the double-layer agar plate method with clinical carbapenem-resistant K. pneumoniae strain Kp2092 as the host, and their host ranges were analyzed. The morphological and genetic characteristics of a phage strain with strong lysis ability and a broad host range were analyzed by transmission electron microscopy and whole-genome sequencing. Phage training was performed by phage-host co-culture for generations, and the phages before and after training were compared in terms of biological properties such as bactericidal activity, optimal multiplicity of infection, one-step growth curve, and stability under different stress conditions. [Results] A total of 9 phage strains were isolated, in which P55anc was a short-tailed phage with the strongest lysis ability and the broadest host range. The genome (40 301 bp) of P55anc included 51 coding sequences, of which 27 showed possible functions involving nucleic acid metabolism, virion morphogenesis, DNA packaging, and host lysis. Three evolved phages of P55anc were obtained after 9 days of phage training. These evolved phages showed significantly enhanced bactericidal activities, manifested by the increased burst size, broadened host range, and reduced host resistance. Moreover, the evolved phages maintained stable when being exposed to heat, ultraviolet, and serum treatments. [Conclusion] Phage training by phage-host co-culture can be employed to obtain evolved phages with enhanced bactericidal effects. Furthermore, the evolved phages reduced the host resistance and remained stable under different stress conditions.