Genomic profile and infection dynamics of Kashi_RDG1 (KRDG1), a novel Cluster K1 mycobacteriophage infecting mycobacterial hosts

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains one of the leading causes of infectious disease mortality worldwide, with rising incidences of multidrug-resistant (MDR) strains posing major public health challenges. In 2023, TB affected more than 10.8 million individuals and caused approximately 1.25 million deaths globally, highlighting the urgency for effective interventions. Traditional antibiotics often prove inadequate in such cases, prompting the need for novel antimicrobial strategies. Bacteriophage therapy has emerged as a promising alternative, offering species-specific activity and the ability to self-amplify selectively within bacterial hosts. This study reports the isolation and detailed characterization of mycobacteriophage Kashi_RDG1 (KRDG1), isolated using Mycobacterium smegmatis mc²155. Genomic analysis identified KRDG1 as a member of Cluster K1, a group with proven efficacy to target pathogenic Mycobacterium species. The KRDG1 genome is of 58,681 bp with 95 predicted open reading frames (ORFs), of which 39 are functionally annotated. Transmission electron microscopy analysis confirmed its Siphoviridae morphology while host range analysis depicted its polyvalent activity against Mycobacterium fortuitum (opportunistic pathogen) and M. tuberculosis H37Ra (an attenuated Mtb strain) in addition to M. smegmatis. One-step growth curve revealed latent period of 80 minutes and burst size of 100 phage/bacterial cell supporting its efficient infection dynamics. Notably, growth kinetics demonstrated strong host bacterial killing during the logarithmic phase. While KRDG1 exhibits temperate characteristics, its close genomic similarity to previously engineered therapeutic phage (ZoeJ) highlights its potential for possible genetic modifications. Thus, KRDG1 can be a potential candidate for future development of phage therapy targeting pathogenic mycobacterial and non-mycobacterial infections.