Long-read sequencing of clinical multidrug-resistant Mycobacterium tuberculosis reveals a novel ethA structural variant conferring ethionamide resistance
5 affiliations
- 1Department of Laboratory Medicine, The First Affiliated Hospital of Wenzhou Medical University
- 2School of Laboratory Medicine and Life Sciences, Wenzhou Medical University
- 3Department of Clinical Laboratory, Wenzhou Central Hospital
- 4Department of Infectious Diseases, Wenzhou Central Hospital
- 5Key Laboratory of Laboratory Medicine, Ministry of Education
Abstract
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.
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BibTeX
@article{wang2026longread,
title = {{Long-read sequencing of clinical multidrug-resistant Mycobacterium tuberculosis reveals a novel ethA structural variant conferring ethionamide resistance}},
author = {Qiongdan Wang and Yaya Geng and Lianpeng Wu and Dandan Xia and Ruini Li and Baozhu Hu and Shuai Gao and Jinfang Lu and Jianyi Dai and Feng Xu and Xiaoqun Zheng and Guangxin Xiang},
journal = {Frontiers in Microbiology},
year = {2026},
month = oct,
doi = {10.3389/fmicb.2026.1929307},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2026.1929307/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Long-read sequencing of clinical multidrug-resistant Mycobacterium tuberculosis reveals a novel ethA structural variant conferring ethionamide resistance
AU - Qiongdan Wang
AU - Yaya Geng
AU - Lianpeng Wu
AU - Dandan Xia
AU - Ruini Li
AU - Baozhu Hu
AU - Shuai Gao
AU - Jinfang Lu
AU - Jianyi Dai
AU - Feng Xu
AU - Xiaoqun Zheng
AU - Guangxin Xiang
PY - 2026
DA - 2026/10/27
JO - Frontiers in Microbiology
DO - 10.3389/fmicb.2026.1929307
UR - https://www.frontiersin.org/articles/10.3389/fmicb.2026.1929307/full
AB - 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.
SN - 1664-302X
ER -