High-Level Primary Pretomanid-Resistant with ddn In-Frame Deletion of Mycobacterium tuberculosis and Its Association with Lineage 4.5 in China

Pretomanid (PMD) is a key antibiotic of the newest multidrug-resistant or rifampicin-resistant tuberculosis treatment regimen "BPaL", but knowledge of its resistance mutations is still limited, especially in China, as it was approved only in late 2024. We analyzed a collection of (MTB) isolates from China including the whole-genome sequencing and drug susceptibility testing data and extended analysis to other 2165 isolates from lineage 4. We found in-frame deletion variants in the ddn genome sequence in the isolates collected in Xinjiang Uyghur Autonomous Region, China, with a high level of PMD resistance without pre-exposure to PMD belonging to sublineage 4.5. The extended analysis found that in-frame deletion variants occurred more frequently in sublineage 4.5. Some isolates contain multiple ancestral components after historical evolution, which may cause in-frame deletion variants to spread in some settings. Furthermore, molecular dynamics simulations and free energy calculations of the key mutants indicated that the impaired mutant structures result in unfavorable domination binding, which poses less probability for PMD activation for targeting other critical MTB targets and also leads to insufficient generation of NO (nitric oxide) to kill MTB. Currently, PMD resistance is mainly due to ddn gene mutations, especially frameshift mutations. However, our findings underscore the importance of surveillance for in-frame deletions, especially in regions with a high prevalence of sublineage 4.5, and the high level of PMD resistance conferred by deletions raises crucial concerns about the future effectiveness of the BPaL regimen.