四株耻垢分枝杆菌噬菌体的分离与鉴定

Isolation and identification of four strains of Mycobacterium smegmatis bacteriophages

摘要:

【背景】分枝杆菌属(Mycobacterium)是一类细长略弯曲、可呈分枝状生长的杆菌，部分严重威胁人类和动物健康；噬菌体因其高效特异及易于制备的特性，成为抗生素替代疗法的研究热点。【目的】分离鉴定耻垢分枝杆菌(Mycobacterium smegmatis)噬菌体并对其进行生物学特性分析，评价其在不同环境下的适应能力及体外抑菌能力。【方法】以耻垢分枝杆菌mc²155为宿主菌，从奶牛场污水、粪便、土壤、垫料中分离纯化烈性噬菌体，并对其进行生物学特性研究。【结果】分离纯化鉴定并获得4株耻垢分枝杆菌噬菌体，分别命名为MP2201、MP2202、MP2203、MP2204，其效价分别为1×10⁸、5×10⁸、3×10⁸、1×10⁸ PFU/mL；透射电子显微镜结果显示，4株噬菌体均属于长尾噬菌体；MP2204最佳感染复数为1，其余3种噬菌体最佳感染复数均为0.1；噬菌体MP2201暴发量最高，可达500 PFU/cell；4株噬菌体在40−60 ℃时均可稳定存活，当pH 3.0−11.0时，效价变化不显著；制备噬菌体鸡尾酒能够显著抑制宿主菌生长；噬菌体全基因组测序结果表明：MP2201、MP2202、MP2203和MP2204的基因组全长分别为69 720、68 282、47 622和70 950 bp，G+C含量分别为59.14%、66.58%、63.22%和59.02%，并且不含耐药基因。【结论】分离鉴定4株耻垢分枝杆菌噬菌体，为开发针对分枝杆菌感染的新型生物防治手段提供了生物材料和理论依据。

Abstract:

[Background] Mycobacterium is a genus of slender, slightly curved, and branching bacilli, some of which seriously threaten human and animal health. Bacteriophages have emerged as a substitute of antibiotics because of their high efficiency, high specificity, and easy preparation. [Objective] To isolate and identify bacteriophages of Mycobacterium smegmatis, analyze their biological characteristics, and evaluate their adaptability in different environments and their antibacterial activity in vitro. [Methods] Using M. smegmatis mc²155 as the host, we isolated virulent bacteriophages from dairy farm wastewater, feces, soil, and bedding and studied their biological characteristics. [Results] Four strains of M. smegmatis bacteriophages were isolated, identified, and named MP2201, MP2202, MP2203, and MP2204, with titers of 1×10⁸, 5×10⁸, 3×10⁸, and 1×10⁸ PFU/mL, respectively. Transmission electron microscopy results showed that the four strains were long-tailed bacteriophages. The optimal multiplicity of infection of MP2204 was 1, while that of the other three strains was 0.1. Strain MP2201 had the highest burst size, which reached 500 PFU/cell. All the four strains remained stable at 40−60 ℃, and their titers did not change significantly within the range of pH 3.0–11.0. Preparing a bacteriophage cocktail significantly inhibited the growth of the host strain. Whole genome sequencing of the phages revealed that MP2201, MP2202, MP2203, and MP2204 had the genome lengths of 69 720, 68 282, 47 622, and 70 950 bp, in which the corresponding G+C content was 59.14%, 66.58%, 63.22%, and 59.02%, respectively. Notably, none of these genomes harbored antibiotic resistance genes. [Conclusion] Four strains of M. smegmatis bacteriophages were successfully isolated and identified, which provided biological materials and a theoretical basis for the development of new biocontrol methods for mycobacterial infection.