Long-read sequencing of clinical multidrug-resistant Mycobacterium tuberculosis reveals a novel ethA structural variant conferring ethionamide resistance

Multidrug-resistant Mycobacterium tuberculosis (MDR-MTB) poses a major obstacle to global tuberculosis control. Although whole-genome sequencing (WGS) has advanced the detection of resistance-associated mutations, the contribution of structural variants (SVs) remains inadequately characterized due to the inherent limitations of short-read sequencing technologies in resolving repetitive genomic regions and large insertions or deletions. We applied Oxford Nanopore long-read sequencing to clinical MDR-MTB isolates and susceptible controls, complemented by Illumina short-read sequencing. Variants were identified using BCFtools and independently validated with SVIM-asm. Candidate resistance mutations were mapped against the 2023 World Health Organization (WHO) mutation catalogue. Functional validation of identified SVs was performed through heterologous expression in Mycobacterium smegmatis , minimum inhibitory concentration determination, Western blotting, and AlphaFold 3-based structural modeling. We identified two unclassified SVs within WHO-catalogued resistance genes: an IS6110 insertion in Rv0678 (c.59_60ins1358bp, p.Gln22fs) and a 131-bp deletion in ethA (c.135_265del, p.Trp45fs). While IS6110 -mediated disruption of Rv0678 has been previously associated with bedaquiline resistance, the ethA deletion represents a previously undescribed SV. Heterologous expression of the ethA deletion in Mycobacterium smegmatis conferred a 16-fold increase in the minimum inhibitory concentration of ethionamide (256 μg/mL vs. 16 μg/mL for wild-type), and AlphaFold 3 structural prediction provided a structural rationale for the loss of the C-terminal domain essential for monooxygenase activity. This study identifies a novel ethA SV conferring ethionamide resistance and provides functional validation of a specific Rv0678 IS6110 insertion allele, demonstrating the power of long-read sequencing to uncover both known and novel mechanisms of drug resistance in MDR-MTB.