1株跨种裂解李氏杆菌噬菌体的分离鉴定

本研究旨在获得天然李氏杆菌噬菌体，提供防治李氏杆菌病新型生物制剂，减少抗微生物药物的使用，抑制病原菌耐药性的产生。利用产单核细胞李氏杆菌作为宿主菌对屠宰场污水进行双层琼脂平板法筛选，获得噬菌体，并对其进行透射电镜观察、生长特性检测(温度、pH、一步生长曲线、最佳感染复数、有机溶剂影响)、基因组酶切鉴定和全基因组测序分析。分离出的产单核细胞李氏杆菌噬菌体中，选取1株裂解性强，遗传稳定的噬菌体进行后续试验，并命名为LP8；经电镜观察为肌尾科噬菌体，可以跨种裂解18株产单核细胞李氏杆菌和5株威尔斯李氏杆菌，确定LP8的最佳感染复数为1、最适生长温度为45 ℃、最适pH为7；在6种有机溶剂中，仅异戊醇可导致LP8活性丧失；对提取的基因组进行酶切鉴定，确定为双链DNA；LP8全基因组测序结果表明，基因组大小为87 038 bp、含有120个编码基因、编码基因的累计长度为76 326 bp、编码基因的平均长度为636 bp、编码区域长度占基因组的比例为87.69%。本试验分离出产单核细胞李氏杆菌噬菌体，并对其噬菌能力和应用价值进行鉴定。分离出的LP8噬菌体相较于李氏杆菌的噬菌体，细菌裂解能力更强，适应环境范围更广。本研究为实验室后续建立产单核细胞李氏杆菌噬菌体库和产单核细胞李氏杆菌噬菌体的其他应用提供了良好的基础。

Isolation and Identification of a Cross-species Phage Against Listeria

The purpose of this study is to obtain natural Listeria monocytogenes phage, provide new biological agents for the prevention and treatment of listeriosis, reduce the use of antimicrobial agents, and inhibit the production of antimicrobial resistance of pathogens. Using L. monocytogenes as the host, the slaughterhouse sewage was screened by double-layer agar plate method, and the phage was obtained. Application value of the phage were evaluated by transmission electron microscope, growth characteristics detection (temperature, pH, one-step growth curve, multiplicity of infection, the effect of organic solvents), genome restriction endonuclease digestion and whole genome sequencing. In this study, several strains of L. monocytogenes phage were isolated, and one of the phages with the stronger lytic ability and genetic stability was selected, and named LP8; as myotail phage under electron microscope, can lyse 18 strains of L. monocytogenes and 5 strains of Listeria welshimeri, it was determined that the multiplicity of infection of LP8 was 1, the optimum growth temperature was 45℃, and the pH was 7. During 6 kinds of organic solvents, only isoamyl alcohol could inactivate LP8 completely. The extracted genome was identified by restriction endonuclease digestion and identified as double-stranded DNA, the genome size was 87 038 bp, contained 120 coding genes, the cumulative length of coding genes was 76 326 bp, the average length of coding genes was 636 bp, and the proportion of coding region was 87.69%. In this study, the phage of L. monocytogenes was isolated, and its phage ability and application value were identified. Compared with other phages, the isolated LP8 stronger ability to adapt environmental changes and lyse bacterial. It provides a basis for the subsequent establishment of L. monocytogenes phage library and other applications of L. monocytogenes phage.