Isolation and identification of four strains of Mycobacterium smegmatis bacteriophages

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.

Key words:Mycobacterium;bacteriophage;isolation and identification;bacteriophage cocktail