Directed evolution of rifampicin and linezolid resistant Mycobacterium tuberculosis mutants

Tuberculosis, caused by Mycobacterium tuberculosis, is the most predominant infectious disease, highlighted by current in a global tuberculosis pandemic and 1.3 million deaths in 2022. The emergence of antibiotic-resistant cases of tuberculosis risks exacerbating the impact of this disease, with 450 thousand antibiotic resistant cases in 2021 and critical priority status on the World Health Organization priority pathogen list. This study aims to identify stepwise genetic changes that drive antibiotic resistance in M. tuberculosis. Here, directed evolution was applied to evolve M. tuberculosis mutants with increasing levels of resistance to either the first-line treatment, rifampicin, or last-line treatment, linezolid. Whole genome sequencing was then applied to identify genomic variants supporting resistance. Identified genes were further investigated using Escherichia coli homologs to phenotypically validate their contribution to antibiotic resistance. Expected clinically observed resistance mutations were identified (rpoB and rplC), in addition to six low-frequency or secondary rifampicin and linezolid resistance mutations. Preliminary phenotypic validation of ppsD and ppsE observed increased antibiotic tolerance to rifampicin. While rpoB and rplC directly prevent antibiotic target binding, this study highlights the utilisation of indirect resistance mutations in M. tuberculosis, that support resistance before and after acquisition of core resistance mutations. These outcomes will improve identification and diagnosis of antibiotic resistance tuberculosis, and optimise management and treatment of tuberculosis infections, ultimately minimising patient deaths.