Genetic mechanisms of co-emergence of INH-resistant
Mycobacterium tuberculosis
strains during the standard course of antituberculosis therapy
Authors: Tafess Ketema , Ng Timothy Ting-Leung , Tam Kingsley King-Gee , Leung Kenneth Siu-Sing , Leung Jake Siu-Lun , Lee Lam-Kwong , Lao Hiu Yin , Chan Chloe Toi-Mei , Yam Wing-Cheong , Wong Samson Sai Yin , Lau Terrence Chi-Kwong , Siu Gilman Kit-Hang 
Published: March 2024

The incidence of isoniazid (INH) resistant
Mycobacterium tuberculosis
is increasing globally. This study aimed to identify the molecular mechanisms behind the development of INH resistance in
M. tuberculosis
strains collected from the same patients during the standard course of treatment. Three
M. tuberculosis
strains were collected from a patient before and during antituberculosis (anti-TB) therapy. The strains were characterized using phenotypic drug susceptibility tests, Mycobacterial Interspersed Repeated Unit-Variable-Number Tandem Repeats (MIRU-VNTR), and whole-genome sequencing (WGS) to identify mutations associated with INH resistance. To validate the role of the novel mutations in INH resistance, the mutated
katG
genes were electroporated into a KatG-deleted
M. tuberculosis
strain (GA03). Three-dimensional structures of mutated KatG were modeled to predict their impact on INH binding. The pre-treatment strain was susceptible to INH. However, two INH-resistant strains were isolated from the patient after anti-TB therapy. MIRU-VNTR and WGS revealed that the three strains were clonally identical. A missense mutation (P232L) and a nonsense mutation (Q461Stop) were identified in the
katG
of the two post-treatment strains, respectively. Transformation experiments showed that
katG
of the pre-treatment strain restored INH susceptibility in GA03, whereas the mutated
katG
genes from the post-treatment strains rendered negative catalase activity and INH resistance. The protein model indicated that P232L reduced INH-KatG binding affinity while Q461Stop truncated gene transcription. Our results showed that the two
katG
mutations, P232L and Q461Stop, accounted for the co-emergence of INH-resistant clones during anti-TB therapy. The inclusion of these mutations in the design of molecular assays could increase the diagnostic performance.

IMPORTANCE

The evolution of drug-resistant strains of
Mycobacterium tuberculosis
within the lung lesions of a patient has a detrimental impact on treatment outcomes. This is particularly concerning for isoniazid (INH), which is the most potent first-line antimycobacterial drug. However, the precise genetic factors responsible for drug resistance in patients have not been fully elucidated, with approximately 15% of INH-resistant strains harboring unknown genetic factors. This raises concerns about the emergence of drug-resistant clones within patients, further contributing to the global epidemic of resistance. In this study, we revealed the presence of two novel
katG
mutations, which emerged independently due to the stress exerted by antituberculosis (anti-TB) treatment on a parental strain. Importantly, we experimentally demonstrated the functional significance of both mutations in conferring resistance to INH. Overall, this research sheds light on the genetic mechanisms underlying the evolution of INH resistance within patients and provides valuable insights for improving diagnostic performance by targeting specific mutations.