Characterization of a novel virulent mycobacteriophage Kashi-SSH1 (KSSH1) depicting genus-specific broad-spectrum anti-mycobacterial activity

Abstract

Aim: Tuberculosis (TB) is one of the leading infectious disease causing mortality in the world and the rise of drug resistance; multi-drug resistance (MDR) and extensive-drug resistance (XDR) has added to extra complicacy of the disease. In this scenario, phage therapy has emerged as a potential treatment option against drug-sensitive/-resistant strains. Materials and methods: The mycobacteriophage Kashi-SSH1 (KSSH1) was isolated from soil sample and was genomically, phenotypically, and functionally characterized. It includes genome assembly/annotation, transmission electron microscopy, multiplicity of infection (MOI), one-step growth curve, temperature/pH stability, confocal microscopy, host range determination and host growth reduction assays. Key findings: KSSH1 is a novel polyvalent virulent mycobacteriophage from the Myoviridae family, classified under cluster C1 with a 155,659 bp genome carrying key lysis genes—Holliday junction resolvase, Holin, Lysin A, and Lysin B, has an optimal MOI of 0.01, a 60-min latent period, and a burst size of 200 phages/bacterial cell. It remains stable up to 55 °C and within pH 7–10, exhibiting broad-spectrum activity against Mycobacterium species, like M. fortuitum (opportunistic pathogen), M. tuberculosis H37Ra (attenuated pathogen), and M. smegmatis, but not non-mycobacterial hosts. KSSH1 exhibits comparable growth inhibition of M. smegmatis like the antibiotics isoniazid and rifampicin as compared to the control, in liquid cultures for over 50 h without regrowth. Significance: KSSH1 exhibits strong lytic activity against various Mycobacterium species, lacks lysogeny-associated genes like integrases/transcriptional repressors, antibiotic resistance and virulence genes and remains stable from 4 °C to 37 °C and pH 8–10 ensuring safety/stability making it an ideal candidate for therapeutic use. © 2025