肺炎克雷伯菌K7R噬菌体生物学特性及其抗性菌株的抗性机制  

Biological characteristics of Klebsiella pneumoniae K7R phage and its resistance mechanism of resistant strains

作　　者：付瑶 张昊 袭恒豫 冀亚路 徐莉 顾敬敏 韩文瑜 FU Yao;ZHANG Hao;XI Hengyu;JI Yalu;XU Li;GU Jingmin;HAN Wenyu(College of Veterinary Medicine,Jilin University,Changchun 130062,China;Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses,Yangzhou,Jiangsu 225000,China;Changchun Animal Disease Prevention and Control Center,Changchun 130061,China)

机构地区：[1]吉林大学动物医学学院,吉林长春130062 [2]江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225000 [3]长春市动物疫病预防控制中心,吉林长春130061

出　　处：《中国兽医学报》2024年第3期504-513,共10页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(U19A2038)。

摘　　要：以肺炎克雷伯菌K7R为宿主菌分离得到1株长尾噬菌体vB_KpnS_ZH01(简称P-K7R),其最佳感染复数为0.01,潜伏期约为10 min,暴发量为169 PFU/cell,且具有稳定、良好的杀菌活性。全基因组测序结果显示,P-K7R全长为52111 bp,共编码86个开放阅读框。经过诱导,获得P-K7R抗性菌K7R-5P,尽管K7R-5P和K7R在显微镜下菌株、菌落形态以及生长曲线无明显差异,但在两者转录组学水平上检测到ompC、ompF、N5、N102及N103五个差异基因。此外,K7R-5P与K7R对噬菌体在吸附率上有明显差异,K7R的吸附效率达到80%以上,而K7R-5P的吸附效率不足20%。最后,利用qRT-PCR对转录组学结果进行验证,结果显示K7R-5P的ompC基因表达量恢复之后,菌株对噬菌体P-K7R的抗性消失,表明ompC基因在噬菌体P-K7R吸附肺炎克雷伯菌过程中起着关键作用。结果表明,成功获得1株裂解性肺炎克雷伯菌噬菌体P-K7R并在体外探究了细菌对噬菌体K7R-5P产生抗性的机制,为噬菌体治疗的进一步高效应用奠定了基础。The aim of this study is to isolate bacteriophages with good bactericidal activity against Klebsiella pneumoniae and explore the resistance mechanism of their resistant strains.In this study,the Siphoviridae phage vB_KpnS_ZH01(P-K7R)was isolated using Klebsiella pneumoniae K7R as the host bacteria,with a multiplicity of infection of 0.01,a latent period of about 10min,a burst rate of 169 PFU/cell,and stable and good bactericidal activity.Whole genome sequencing showed that the full length of P-K7R was 52111 bp and encodes 86 open reading frames.After induction,P-K7R resistant strain K7R-5P was obtained.Although there were no significant differences in morphology,colony morphology and growth curve between K7R-5P and K7R,five different genes ompC,ompF,N5,N102 and N103 were detected at the transcriptome level.There was a significant difference in the adsorption rate of K7R-5P and K7R to phage,with an adsorption efficiency of over 80%for K7R and less than 20%for K7R-5P.Finally,RT-qPCR was utilized to verify the transcriptome results,which showed that the phage resistance of stain P-K7R disappeared after the restoration of gene expression of ompC in K7R-5P,indicating that the ompC gene plays a crucial role in the process of phage P-K7R adsorption of Klebsiella pneumoniae.In this study,a strain of Klebsiella pneumoniae phage P-K7R was obtained,and the mechanism of phage K7R-5P resistance generation in vitro was explored,which lays the foundation for further efficient application of phage therapy.

关 键 词：肺炎克雷伯菌 噬菌体 噬菌体抗性 转录组学 吸附受体 

分 类 号：S852[农业科学—基础兽医学]