Associating Mycobacterium tuberculosis genomic variants with minimum inhibitory concentrations to delamanid and pretomanid

Emerging resistance to delamanid (DLM) and pretomanid (Pa) threatens drug-resistant tuberculosis (DR-TB) treatment outcomes. The lack of rapid molecular diagnostics, due to limited knowledge of resistance-conferring mutations, along with standardization challenges for critical concentrations (CC) in phenotypic drug susceptibility testing (pDST) hinder resistance detection. Here, we explore the correlation between minimum inhibitory concentrations (MICs) and genomic variants of DLM and Pa resistance in Mycobacterium tuberculosis ( Mtb ) . Seventeen Mtb isolates phenotypically resistant to DLM detected through routine pDST using the Mycobacterial Growth Indicator Tube (MGIT) system from 2021–2024 were selected from the National Reference Laboratory biobank in Tbilisi, Georgia. DLM/Pa MICs were determined using the Resazurin Microtiter Assay (REMA) with a DLM concentration range of 0.008–2 µg/ml, and a broader range of 0.125–32 µg/ml was utilized for Pa. To identify mutations associated with DLM/Pa resistance, isolates were subjected to whole genome sequencing (WGS). Of the seventeen Mtb isolates phenotypically resistant to DLM, eight (47%) manifested a MIC value below the DLM CC (0.06 µg/ml), with only one carrying a mutation in a gene associated with DLM/Pa resistance ( fbiC ). Among the remaining nine isolates with MICs above the CC, the premature stop codon mutation W88* in ddn gene predominated, present in five cases and associated with elevated DLM and Pa MICs (0.125–2 µg/ml and 2–8 µg/ml, respectively). Three isolates (19%) harbored novel, previously undescribed genetic variants, all within genes previously linked to resistance DLM/Pa (G35fs in ddn , Q317R and Q279H in fgd1 , and C565W in fbiC ). Our results support DLM and Pa resistance association with W88* and W678G mutations in ddn and fbiC genes respectively, currently graded as “uncertain significance” in the WHO catalogue of Mtb drug resistance-conferring mutations. Additionally, we observed previously undescribed mutations exclusively within genes linked to DLM/Pa susceptibility, prompting further investigation of their role in resistance.