Microevolutionary models and multi-omics analysis uncover the cross-drug resistance characteristics of capreomycin-selected Mycobacterium tuberculosis.

UNLABELLED: The drug-resistant tuberculosis (TB) is a major public health problem worldwide. Capreomycin (Cm) has been used as an effective drug to treat multidrug-resistant tuberculosis (MDR-TB) since 1960s. Although Cm and amikacin (Am) are historically grouped as second-line injectable drugs (SLIDs) with shared resistance mechanisms (e.g., rrs mutations), recent clinical and genetic data suggests divergent evolutionary pathways. It remains unclear how Mtb evolves from sensitive to resistant phenotypes under Cm pressure. In this study, we generate in vitro drug-resistant Mtb models to systematically study the evolutionary mechanisms of Cm and Am cross-resistance. By integrating transcriptome/proteome/metabolome analyses, we identify both genetic mutations and non-genetic factors contributing to Cm resistance. We show that Cm- and Am-selective pressures trigger independent cross-resistance mechanisms revealed by distinct genetic mutations. Gly232AsptlyA and Trp120fstlyA mutations are directly associated with low level resistance to Cm, whereas Ala48ValmmaA2, Gln19ArgrpmA and variant at the promoter of eis (which encodes an N-acetyltransferase; c.-14c>t) mediate cross-resistance pathways specific to Cm selection. In addition, dysregulations of non-genetic factors including metal ions transportations and lipids metabolism also lead to drug-resistance. In summary, our findings systematically characterize the molecular mechanisms of Cm resistance, demonstrating that the "genetic factors" (DNA mutations) are often the drivers that induce the stable "non-genetic factors" (dysregulated pathways, such as eis overexpression, metal ion transport, and metabolic reprogramming), which ultimately mediate the resistance phenotype. These insights uncover the evolutionary trajectory of Cm and Am cross-resistance and provide potential strategies for optimizing Cm use to treat MDR-TB.

IMPORTANCE: Tuberculosis, caused by Mycobacterium tuberculosis, is responsible for the highest mortality rate worldwide among single pathogen infections. The diagnosis and treatment of drug-resistant tuberculosis pose a global challenge. Capreomycin (Cm) is one of the drugs utilized in the management of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). Currently, the clinical detection of mutations in rrs gene is employed to identify Cm resistance. However, the sensitivity and specificity of this method are suboptimal. To further investigate the mechanisms underlying Cm resistance, this study established a microevolution model in vitro, thereby simulating the progression of MTB from susceptible strains to drug-resistant strains. The genomes of each generation were sequenced throughout the evolutionary process. In addition, the transcriptome-proteome-lipid metabolome analysis of representative strains were performed. Our findings reveal that Cm resistance is a combinatory effect of both genetic and non-genetic changes, providing potential optimization strategies to the molecular drug sensitivity testing used in clinical practice.