诺卡氏菌烈性噬菌体vB_Ncarnea_KYD1的分离纯化与基因组分析

Isolation, purification, and genomic analysis of the virulent phage vB_Ncarnea_KYD1 from Nocardia spp.

摘要:

【背景】诺卡氏菌是一种广泛分布的好氧放线菌，可在人体内引起局部或播散性感染，尤其是在免疫功能低下的个体中。诺卡氏菌感染在临床上较难鉴定，而且不断有新型诺卡氏菌种被发现。不同类型、不同地域的诺卡氏菌具有流行差异和抗生素敏感性差异，阻碍了适当治疗方式的选择。利用病灶处的宿主菌分离得到噬菌体来控制诺卡氏菌感染的这种方法在近年来受到了各界的关注。【目的】尝试从环境中分离出能够用于临床治疗的针对诺卡氏菌的烈性噬菌体，并研究其基因组学特征。【方法】利用双层平板法分离得到目标噬菌体，观察其噬菌斑形态，并对噬菌体进行分离纯化，在透射电镜下鉴定其特征。提取噬菌体DNA进行全基因组测序与注释，并与数据库内已知噬菌体基因组进行比较，同时构建系统进化树以进行遗传进化分析。【结果】本文以肉色诺卡氏菌为宿主，从环境样本中分离出一株烈性噬菌体vB_Ncarnea_KYD1，在双层平板上可形成直径<2 mm的透亮均匀的噬菌斑。基因组分析表明，vB_Ncarnea_KYD1DNA为环状，大小为66 621 bp，共发现102个蛋白质编码区(coding sequence，CDS)及一个tRNA-Ser编码序列。透射电镜观察与系统进化树综合分析可以确定，vB_Ncarnea_KYD1为长尾噬菌体科的一个新属。其在进化过程中经历了复杂的基因重组过程。暂未发现毒力因子相关基因与抗性基因，具备实用价值。【结论】从环境水体中分离出一株烈性肉色诺卡氏菌噬菌体vB_Ncarnea_KYD1，通过电镜观察与基因组分析可知，此株噬菌体为长尾噬菌体，基因组中暂未发现不利于临床应用的相关基因，是一株相对安全的烈性诺卡氏菌噬菌体。研究结果丰富了国内噬菌体资源库，并为后续诺卡氏菌感染疾病的治疗提供支持。

Abstract:

[Background] Nocardia belongs to aerobic actinomycete. Being widely distributed, Nocardia can cause local or disseminated infection in human, especially in those with low immune function. The Nocardia infection is difficult to be clinically identified, and novel Nocardia strains are constantly being discovered. Different types of Nocardia from different regions have different prevalences and antibiotic sensitivities, which hinders the treatment. The treatment of Nocardia infection by the phage isolated from the host bacteria at the lesion has attracted great attention in recent years. [Objective] To isolate the virulent phage against Nocardia from the environment that can be used in clinic and explore the genomic characteristics. [Methods] The target phage was isolated by the double-layer plate method, and the plaque morphology was observed. The phage was purified, and the characteristics were observed through the transmission electron microscope. The DNA of the phage was extracted, and the whole genome was sequenced, annotated, and compared with the known phage genomes in the database. The phylogenetic tree was constructed for genetic evolution analysis. [Results] The virulent phage vB_Ncarnea_KYD1 with Nocardia carnea as the host, isolated from the environmental samples, formed transparent and uniform plaques with a diameter <2 mm on the double-layer plate. Genome analysis showed that the DNA of vB_Ncarnea_KYD1 was circular with a size of 66 621 bp. A total of 102 proteins and 1 tRNA-Ser were found in coding sequences (CDS). According to the transmission electron microscope observation and phylogenetic tree analysis, vB_Ncarnea_KYD1 was a new phage in the Siphophages, which experienced complex gene recombination in the evolution. vB_Ncarnea_KYD1 had practical value, and no virulence factor-related genes or antibiotic-resistance genes were found. [Conclusion] The novel virulent phage vB_Ncarnea_KYD1 of N. carnea was isolated from the environmental water sample. The transmission electron microscope observation and genome analysis showed that vB_Ncarnea_KYD1 belonged to the Siphophages. Since no relevant genes unfavorable to clinical application were found in the genome, vB_Ncarnea_KYD1 was a relatively safe virulent phage of Nocardia The findings of this study provided references for the follow-up treatment of Nocardia infection, and enriched the domestic phage resources.