---
title: "Mycobacterium tuberculosis genomic variants linked to delamanid and pretomanid MICs"
id: "plos-one-18-associating-mycobacterium-tuberculosis-genomic-variants-with-minimum-inhibitory"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-18-associating-mycobacterium-tuberculosis-genomic-variants-with-minimum-inhibitory"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0359717"
published_at: "2026-10-05T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Mycobacterium tuberculosis genomic variants linked to delamanid and pretomanid MICs
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/plos-one-18-associating-mycobacterium-tuberculosis-genomic-variants-with-minimum-inhibitory
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0359717)
- **Published At:** 2026-10-05T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- Emerging resistance to the nitroimidazoles **delamanid** (DLM) and **pretomanid** (Pa) threatens drug-resistant TB treatment; molecular diagnostics are limited by incomplete knowledge of resistance mutations and pDST standardization challenges. - The study examined 17 M. tuberculosis isolates from Georgia (2021–2024) that were phenotypically DLM-resistant by routine MGIT testing and available for REMA MIC testing and whole genome sequencing (WGS). - REMA used DLM concentrations 0.008–2 μg/ml and Pa 0.125–32 μg/ml; WHO-recommended MGIT CC for DLM is 0.06 μg/ml and WHO/EUCAST guidance for Pa varies (WHO two concentrations 0.5/2 μg/ml; EUCAST breakpoint 2 μg/ml). - REMA results: 8/17 (47%) isolates had DLM MICs below the DLM CC (≤0.06 μg/ml), indicating susceptibility; 9 isolates had MICs above CC, with DLM MICs ranging 0.125–2 μg/ml and Pa MICs 2–8 μg/ml. - WGS identified mutations in DLM/Pa-associated genes (ddn, fgd1, fbiC, others): 9/17 (53%) isolates carried catalogue-listed resistance-associated gene variants; ddn mutations predominated, especially the premature stop codon **W88*** found in five isolates and associated with elevated DLM and Pa MICs. - Novel or previously undescribed variants were detected exclusively within known resistance-linked genes: ddn G35fs, fgd1 Q317R and Q279H, and fbiC C565W; allele frequencies varied and some variants are not present in the WHO catalogue. - Some isolates initially classified as DLM-resistant by routine MGIT were reclassified after repeat testing; of 32 initially called resistant, 11 were reclassified as susceptible and 17 underwent REMA and WGS. - Lineage distribution: 14 isolates (82%) lineage 2; 3 isolates (18%) lineage 4; W88* occurred in the W148 outbreak cluster and resistance-associated variants predominated in sub-lineage L2.2.1. - The study supports associations between specific mutations (ddn W88* and fbiC W678G) and elevated DLM/Pa MICs, notes cross-resistance patterns, and reports novel variants that merit further investigation for their role in nitroimidazole resistance. - Data and sequencing reads are deposited in the European Nucleotide Archive under PRJEB39561; study had IRB approval, used biobanked isolates, and was funded in part by a Fogarty/NIH grant.
## Clinical Analysis & Structured Key Points
Associating Mycobacterium tuberculosis genomic variants with minimum inhibitory concentrations to delamanid and pretomanid | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Reader Comments Figures Figures Abstract 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. Citation: Tsutsunava A, Tukvadze N, Avaliani Z, Goig GA, Jugheli L, Gagneux S, et al. (2026) Associating Mycobacterium tuberculosis genomic variants with minimum inhibitory concentrations to delamanid and pretomanid. PLoS One 21(10): e0359717. https://doi.org/10.1371/journal.pone.0359717 Editor: Divakar Sharma, Lady Hardinge Medical College, INDIA Received: October 7, 2025; Accepted: September 17, 2026; Published: October 5, 2026 Copyright: © 2026 Tsutsunava et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript. Isolate sequencing data are deposited to the European Nucleotide Archive under the study project PRJEB39561. Funding: This study was financially supported by the Fogarty International Center of the National Institutes of Health (NIH) Global Infectious Diseases in the form of a grant awarded to NT (D43TW007124). No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction Surpassing COVID-19, with an estimated 1.23 million deaths in 2024, TB reclaimed to be the world’s leading cause of death from a single infectious agent [ 1 ]. While the introduction of novel medications against drug-resistant TB, such as bedaquiline (BDQ), delamanid (DLM) and pretomanid (Pa) have increased treatment success rates, the global increase in emerging resistance towards these medications is concerning and threatens their long term-efficacy [ 2 – 4 ]. DLM, approved by the European Medicines Agency (EMA) in 2014 is a part of individualized treatment regimens recommended for all persons with rifampicin resistant and multi-drug resistant (RR/MDR) TB for all age groups. Classified as group C medication, it is a major alternative drug in case of resistance or intolerance towards group A and B agents, moreover it is included in a newly recommended regimen for RR/MDR-TB alongside bedaquiline, linezolid, levofloxacin, and clofazimine (6-month BDLLfxC) [ 5 , 6 ]. Pa – the latest addition to the anti-TB drug arsenal, was approved by the United States Food and Drug Administration (FDA) in 2019 for use in the BPaL regimen, which combines Pa with BDQ and linezolid, later recommended by WHO as regimen of first choice with or without moxifloxacin [ 5 – 7 ] . Both compounds – DLM and Pa, are prodrugs and share the same activation pathway involving the mycobacterial F420-dependent nitro-reductase encoded by the ddn ( Rv3547 ) gene [ 8 ]. Co-factor F420 is synthesized and activated by a group of enzymes encoded by the genes fgd1 (Rv0407), fbiA (Rv3261), fbiB (Rv3262), fbiC (Rv1173) and fbiD (Rv2983) [ 9 ]. With a similar mechanism of action, these two drugs inhibit the mycobacterial cell wall synthesis, leading to cell death. Various mutations in these genes have been associated with an increased MICs to DLM and Pa [ 10 ] additionally confirming the presence of cross-resistance between these two medications [ 11 , 12 ]. Although the exact molecular mechanisms of action of DLM and PA are not yet fully understood, recent studies have described the essential enzyme in cell wall biosynthesis -decaprenylphosphoryl-2-keto-β-D-erythro-pentose reductase ( DprE2 ), as a shared target of both drugs implicating possible role in resistance [ 13 ]. To preserve novel drugs and regimens, precise and prompt detection of resistance to these medications is crucial. The World Health Organization (WHO) recommends performing drug susceptibility testing (DST) to detect resistance to DLM and Pa. For DLM, a critical concentration (CC) of 0.06 μg/ml is currently recommended for MGIT-based testing [ 14 ]. However, interpreting Pa susceptibility remains more complex. Early studies suggested a single critical concentration of 1 μg/ml for pretomanid based on available MIC data [ 15 ]. Nevertheless, subsequent studies showed that MIC values vary by Mycobacterium tuberculosis ( Mtb) lineage, with lineage 1 strains generally exhibiting higher MICs [ 15 – 17 ]. As a result, WHO now recommends testing Pa at two concentrations (0.5 and 2 μg/ml) in MGIT. Growth at 2 μg/ml is used to define resistance, while results at lower concentrations should be interpreted with caution [ 18 ]. In contrast, the European Committee on Antimicrobial Susceptibility Testing (EUCAST), which establishes clinical breakpoints on behalf of EMA, defines the breakpoint for Pa at 2 μg/ml using the MGIT system [ 19 ]. The WHO mutation catalogue (2nd edition) [ 20 ], based on highly conservative selection criteria, describes 24 mutations associated with DLM resistance classified as Group 2 - “associated with resistance interim”. In addition, the WHO recommends that any loss-of-function mutation in the six resistance-associated genes ( ddn , fgd1 , fbiA, fbiB , fbiC , and fbiD ) to be interpreted similarly as a Group 2 mutation. Notably, six such loss-of-function mutations are listed in the catalogue annex [ 20 ]. The catalogue further includes resistance-associated mutations for Pa, including shared mutations such as ddn W88*, which are classified as Group 2 for both nitroimidazoles. In addition, uncertainty around the associations of Mtb genetic variants with phenotypic resistance to nitroimidazoles highlights a key gap that needs to be addressed. In this regard, the combination of the comprehensive MIC data, pDST and whole genome sequencing (WGS) can help identify the genetic determinants of resistance, and in developing rapid diagnostics [ 21 ]. We aimed to link DLM phenotypic susceptibility (BACTEC MGIT 960 system) and DLM/Pa MIC results (REMA assay) with WGS data. Materials and methods Ethics This study was conducted with approval from the Institutional Review Board (IRB) of the National Center for Lung Health (NCLH; formerly, National Center for Tuberculosis and Lung Disease) in Tbilisi, Georgia. While designed and executed independently, it utilized biobanked isolates and genomic data generated through a nationwide genomic epidemiology project funded by the European Research Council (ERC), in collaboration with the Swiss Tropical and Public Health Institute (Swiss TPH), for which the necessary ethical approvals had already been obtained in both Georgia and Switzerland (Ethikkommission Nordwest- und Zentralschweiz). Given the population-wide scope of the genomic study, the Ministry of Labor, Health and Social Affairs of Georgia, along with the local IRB, granted a waiver of written informed consent for both prospective and retrospective TB cases. All clinical and sample-related data used in this study were pseudonymised and the authors had no access to any personally identifiable information at any stage during or after data collection. Mtb isolate selection We identified Mtb isolates with phenotypic resistance to DLM through the National TB Program (NTP) laboratory database that encompasses the TB diagnostic test results from across the country. Routine DST for DLM was implemented by the NCLH in February 11, 2021 using the BACTEC MGIT 960 system with a CC of 0.06 µg/ml, as recommended by WHO [ 14 ]. All routine DLM pDST was performed at the NRL, NCLH, ensuring methodological consistency across the study period, with continuously maintained regular external quality assessment using blinded strains, through participation in the WHO Supranational Reference Laboratory Network (SRLN). Baseline Mtb isolates were selected from TB cases diagnosed between February 11, 2021 and July 31, 2024. Routine DLM phenotypic DST was performed in patients with RR-TB as part of national programmatic practice. Clinical and bacteriological metadata, including previous TB history, TB profile, treatment regimen, and treatment outcomes, were obtained from the NTP surveillance electronic database of the NCLH. MIC assay All available baseline DLM resistant Mtb isolates retrieved from the NCLH Biobank, were sub-cultured on Löwenstein-Jensen (LJ) medium, and subsequently subjected to MIC testing against DLM and Pa using REMA [ 22 ]. Stock solutions of drugs prepared in DMSO were frozen at −20°C in aliquots for up to 6 months and discarded after single thawing for the assay preparation. Plates containing serial dilutions of DLM or Pa were prepared in advance. Before inoculation, bacterial suspensions were adjusted in saline–Tween broth to a turbidity equivalent to McFarland 0.5 using a nephelometer, then added to the plates. The concentration ranges were as follows: 2/1/0.5/0.25/0.125/0.06/0.03/0.016/0.008 μg/ml for DLM and 32/16/8/4/2/1/0.5/0.25/0.125 μg/ml for Pa. A broader concentration range was explored for Pa due to the absence of routine pDST data. Isolates were tested in batches under standardized conditions. Each batch included well characterized laboratory reference susceptible strain H37Rv as a quality control, drug-free wells as positive growth controls, and wells without inoculum as negative controls. H37Rv MICs remained consistently ≤0.008 μg/mL for DLM and ≤0.125 μg/mL for Pa. Because these values corresponded to the lowest concentrations tested, exact MIC endpoints could not be determined. Alamar blue was added to the plates, which were incubated at 37 ºC. After 21 days, the MIC was defined as the lowest drug concentration where the growth was observed in triplicate wells, as determined by visual color change. Growth and contamination were monitored throughout the incubation period, and results were recorded accordingly. Genomic data DNA from study isolates were extracted using phenol-CTAB method, measured on Nanodrop spectrophotometer for assessing quantity and purity and sent for whole genome sequencing at the genomic core facility of the University of Basel and the Department of Biosystems Science and Engineering at ETHZ in Basel, Switzerland. For sequencing the NEBNext Ultra II FS DNA Library Prep Kit with an Illumina NovaSeq 6000 and an Illumina HiSeq 2500 platform were used. We identified nonsynonymous mutations, frameshifts, indels, or nonsense mutations in the DLM/Pa resistance-associated genes ddn, fgd1, fbiA, fbiB, fbiC, and fbiD. In our study we also explored mutations within dprE2 as a shared target for both drugs. Sequencing reads from samples passing quality criteria were taxonomically classified with Kraken [ 23 ] and non-MTBC reads were removed. The resulting reads were aligned to the genome of the inferred MTBC ancestor (10.5281/zenodo.3497110), and variants were called using the microbial mode of GATK Mutect2. Variants in repetitive regions such as PE, PPE, and PGRS genes were removed. Resistance profiles for each isolate were assigned based on the presence of mutations in resistance-associated genes in accordance with the latest WHO catalogue and definitions. Phylogenetic analysis was performed to explore the evolutionary relationships among the Mtb isolates and emergence of the mutations, assess whether specific resistance-associated variants were lineage-specific, providing insights into the potential spread and genetic context of DLM/Pa resistance. To infer a maximum-likelihood phylogeny we built a multiple alignment of all non-redundant single-nucleotide polymorphisms (SNPs). In this alignment, we only considered SNPs with a minimum intra-sample allele frequency of 90%. The phylogeny was inferred using IQ-TREE 2 [ 24 ], specifying the general time-reversible model of sequence evolution, indicating the number of invariant sites of each nucleotide, and rooted using M. canettii as an outgroup. Results Isolate selection During the study period (2021–2024), a total of 469 patients were diagnosed with RR-TB by Xpert MTB/RIF Ultra countrywide. Routine DLM pDST was performed for 425 (90.6%) patients as part of the NTP, whereas DLM pDST was unavailable for 44 (9.4%) patients (overlap with validation of the DLM pDST workflow, lack of a clinician request, or drug unavailability). Of the 425 patients tested, 32 (7.5%) isolates were initially classified as DLM resistant. Although repeat testing is not routinely performed, it may be undertaken based on the overall drug resistance profile and clinical context. All 32 isolates underwent repeat MGIT testing, after which 11 isolates were reclassified as susceptible and excluded from further analysis. This resulted in 21 isolates (4.9%) of the total tested being reported as DLM-resistant. Among these, one isolate failed regrowth and three lacked WGS data, leaving 17 isolates available for further characterization using REMA MIC and WGS ( Fig 1 ). Download: PNG larger image TIFF original image Fig 1. Flow diagram of Mtb isolate selection. Flow diagram of baseline Mycobacterium tuberculosis isolate selection from rifampicin-resistant tuberculosis (RR-TB) patients, including routine delamanid (DLM) phenotypic drug susceptibility testing (pDST), repeat testing of initially resistant isolates, and the final study isolates analyzed by REMA minimum inhibitory concentration (MIC) testing and whole genome sequencing (WGS). https://doi.org/10.1371/journal.pone.0359717.g001 MIC distribution REMA testing revealed that 8 (47%) out of the 17 isolates had a DLM MIC value below the CC (0.06 μg/ml), indicating susceptibility. Seven of these isolates exhibited growth only in drug-free wells, with MICs interpreted as ≤0.008 μg/ml for DLM and ≤0.125 μg/ml for Pa, representing the lowest concentrations tested in the REMA assay. All isolates phenotypically susceptible to DLM were also susceptible to Pa, except for one isolate consistently showing a DLM MIC of 0.016 μg/ml, confirmed through repeated testing, that corresponds to the next lowest concentration after 0.008 μg/ml for DLM, with a Pa MIC of 2 μg/ml. The MIC distribution for the remaining 9 isolates was as follows: 6 isolates (67%) exhibited a MIC of 2 μg /ml for DLM, 2 isolates (22%) had a MIC of 0.125 μg /ml and 1 isolate (11%) had MIC of 0.25 μg /ml. All 9 phenotypically resistant DLM strains (by REMA) demonstrated higher Pa MIC values, ranging from 2 to 8 μg /ml. Genomic data Among the 17 isolates analyzed, 9 (53%) harbored mutations in genes associated with DLM/Pa resistance according to the WHO catalogue [ 20 ], specifically ddn , fgd1 , fbiA , fbiB , fbiC , and fbiD . Of these, only one isolate was not confirmed as phenotypically resistant by REMA ( Table 1 ). Within this group, ddn mutations predominated (67%), especially the formerly described [ 9 , 17 , 20 , 25 ] premature stop codon mutation W88* (56%) and occurred in isolates with high MICs for DLM (0.125–2 μg/ml) and Pa (4–8 μg/ml). While fbiA, fbiB , fbiD and dprE2 mutants were not present in our dataset, 3 isolates (33%) carried mutations in the fbiC gene, specifically FbiC C565W and FbiC W678G. Regarding the fgd1 gene, we detected mutations (Q317R, Q279H) in 2 isolates (22%). Out of the detected 6 genomic variants with allele frequency >10%, 4 variants G35fs (33.3%) in ddn , Q317R (22.1%) and Q279H (15.5%) in fgd1 , and C565W detected in two isolates with allele frequencies of 42.7% and 97% in fbiC were exclusively in our sample set and not described elsewhere including WHO mutation catalogue ( Table 1 ). Conversely, among the remaining 8 isolates with DLM MIC values below CC, all showed wild-type (WT) alleles in DLM/Pa resistance-conferring genes, apart from one isolate with fbiC W678G (96.7%) mutation. Two isolates carrying the atpE Ile66Met mutation differed by ≤5 SNPs and belonged to the same sublineage, a genomic relationship compatible with a close epidemiological link. Download: PNG larger image TIFF original image Table 1. Delamanid (DLM) and pretomanid (Pa) minimum inhibitory concentrations (MICs), genomic variants, lineage, and clinical characteristics of 17 Mycobacterium tuberculosis isolates included in the study. https://doi.org/10.1371/journal.pone.0359717.t001 Table 1 represents the data of 17 phenotypically resistant isolates to DLM (BACTEC MGIT 960system) with corresponding MIC results for DLM/Pa via REMA assay, and mutations in resistance conferring genes: ddn (Rv3547), fgd1 (Rv0407), fbiA (Rv3261), fbiB (Rv3262), fbiC (Rv1173), fbiD (Rv2983) in addition with dprE2 (Rv3791); W88*- premature stop codon; ** Variant allele frequency; Mutations in bold are defined as uncertain significance to resistance in WHO mutation catalogue (2nd edition). ***Treatment outcomes are defined according to World Health Organization (WHO) guidelines. In our dataset, 14 isolates (82%) belonged to lineage 2 and three isolates (18%) to lineage 4. Among the eight isolates classified as resistant by MGIT but with MIC values below the critical concentration by REMA, seven belonged to lineage 2 and one to lineage 4, reflecting the overall lineage distribution of the dataset and indicating no lineage-specific clustering of discordant results. Phylogenetic analysis showed the distribution of resistance-associated mutations across Mtb lineages, highlighting their predominance in the L2.2.1 sub-lineage, whereas premature W88 stop codon was detected exclusively in the W148 outbreak cluster ( Fig 2 ). Isolate sequencing data have been deposited in the European Nucleotide Archive under the study project PRJEB39561 and accession numbers are provided in s
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