Sub-MIC levels of bedaquiline and clofazimine can select Mycobacterium tuberculosis mutants with increased MIC.
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ABSTRACT:
SUBMITTER: Villellas C
PROVIDER: S-EPMC10989023 | biostudies-literature | 2024 Apr
REPOSITORIES: biostudies-literature
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Sub-MIC levels of bedaquiline and clofazimine can select <i>Mycobacterium tuberculosis</i> mutants with increased MIC.
Villellas Cristina C Stevenaert Frederik F Remmerie Bart B Andries Koen K
Antimicrobial agents and chemotherapy 20240312 4
Multidrug-resistant tuberculosis (MDR-TB) patients not cured at the time of stopping treatment are exposed to Minimum Inhibitory Concentration (MIC) and sub-MIC levels for many months after discontinuing bedaquiline (BDQ) or clofazimine (CFZ) treatment. <i>In vitro</i> cultures treated with BDQ and CFZ sub-MIC concentrations clearly showed enrichment in the <i>Rv0678</i> mutant population, demonstrating that pre-existing <i>Rv0678</i> mutants can be selected by sub-MIC concentrations of BDQ and ...[more]
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Project description:Drug-resistant tuberculosis is a serious global health threat. Bedaquiline (BDQ) is a relatively new core drug, targeting the respiratory chain in Mycobacterium tuberculosis (Mtb). While mutations in the BDQ target gene, atpE, are rare in clinical isolates, mutations in the Rv0678 gene, a transcriptional repressor regulating the efflux pump MmpS5-MmpL5, are increasingly observed, and have been linked to worse treatment outcomes. Nevertheless, underlying mechanisms of (cross)-resistance remain incompletely resolved. Our study aims to distinguish resistance associated variants from other polymorphisms, by assessing the in vitro onset of mutations under drug pressure, combined with their impact on minimum inhibitory concentrations (MICs) and on protein stability. For this purpose, isolates were exposed in vitro to sub-lethal concentrations of BDQ or clofazimine (CFZ). Selected colonies had BDQ- and CFZ-MICs determined on 7H10 and 7H11 agar. Sanger sequencing and additional Deeplex Myc-TB and whole genome sequencing (WGS) for a subset of isolates were used to search for mutations in Rv0678, atpE and pepQ. In silico characterization of relevant mutations was performed using computational tools. We found that colonies that grew on BDQ medium had mutations in Rv0678, atpE or pepQ, while CFZ-exposed isolates presented mutations in Rv0678 and pepQ, but none in atpE. Twenty-eight Rv0678 mutations had previously been described among in vitro selected mutants or in patients' isolates, while 85 were new. Mutations were scattered across the Rv0678 gene without apparent hotspot. While most Rv0678 mutations led to an increased BDQ- and/or CFZ-MIC, only a part of them surpassed the critical concentration (69.1% for BDQ and 87.9% for CFZ). Among the mutations leading to elevated MICs for BDQ and CFZ, we report a synonymous Val1Val mutation in the Rv0678 start codon. Finally, in silico characterization of Rv0678 mutations suggests that especially the C46R mutant may render Rv0678 less stable.
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Project description:BackgroundDrug resistance in tuberculosis (TB) poses a major ongoing challenge to public health. The recent inclusion of bedaquiline into TB drug regimens has improved treatment outcomes, but this advance is threatened by the emergence of strains of Mycobacterium tuberculosis (Mtb) resistant to bedaquiline. Clinical bedaquiline resistance is most frequently conferred by off-target resistance-associated variants (RAVs) in the mmpR5 gene (Rv0678), the regulator of an efflux pump, which can also confer cross-resistance to clofazimine, another TB drug.MethodsWe compiled a dataset of 3682 Mtb genomes, including 180 carrying variants in mmpR5, and its immediate background (i.e. mmpR5 promoter and adjacent mmpL5 gene), that have been associated to borderline (henceforth intermediate) or confirmed resistance to bedaquiline. We characterised the occurrence of all nonsynonymous mutations in mmpR5 in this dataset and estimated, using time-resolved phylogenetic methods, the age of their emergence.ResultsWe identified eight cases where RAVs were present in the genomes of strains collected prior to the use of bedaquiline in TB treatment regimes. Phylogenetic reconstruction points to multiple emergence events and circulation of RAVs in mmpR5, some estimated to predate the introduction of bedaquiline. However, epistatic interactions can complicate bedaquiline drug-susceptibility prediction from genetic sequence data. Indeed, in one clade, Ile67fs (a RAV when considered in isolation) was estimated to have emerged prior to the antibiotic era, together with a resistance reverting mmpL5 mutation.ConclusionsThe presence of a pre-existing reservoir of Mtb strains carrying bedaquiline RAVs prior to its clinical use augments the need for rapid drug susceptibility testing and individualised regimen selection to safeguard the use of bedaquiline in TB care and control.
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Project description:BackgroundBedaquiline is a core drug for the treatment of multidrug-resistant tuberculosis; however, the understanding of resistance mechanisms is poor, which is hampering rapid molecular diagnostics. Some bedaquiline-resistant mutants are also cross-resistant to clofazimine. To decipher bedaquiline and clofazimine resistance determinants, we combined experimental evolution, protein modelling, genome sequencing, and phenotypic data.MethodsFor this in-vitro and in-silico data analysis, we used a novel in-vitro evolutionary model using subinhibitory drug concentrations to select bedaquiline-resistant and clofazimine-resistant mutants. We determined bedaquiline and clofazimine minimum inhibitory concentrations and did Illumina and PacBio sequencing to characterise selected mutants and establish a mutation catalogue. This catalogue also includes phenotypic and genotypic data of a global collection of more than 14 000 clinical Mycobacterium tuberculosis complex isolates, and publicly available data. We investigated variants implicated in bedaquiline resistance by protein modelling and dynamic simulations.FindingsWe discerned 265 genomic variants implicated in bedaquiline resistance, with 250 (94%) variants affecting the transcriptional repressor (Rv0678) of the MmpS5-MmpL5 efflux system. We identified 40 new variants in vitro, and a new bedaquiline resistance mechanism caused by a large-scale genomic rearrangement. Additionally, we identified in vitro 15 (7%) of 208 mutations found in clinical bedaquiline-resistant isolates. From our in-vitro work, we detected 14 (16%) of 88 mutations so far identified as being associated with clofazimine resistance and also seen in clinically resistant strains, and catalogued 35 new mutations. Structural modelling of Rv0678 showed four major mechanisms of bedaquiline resistance: impaired DNA binding, reduction in protein stability, disruption of protein dimerisation, and alteration in affinity for its fatty acid ligand.InterpretationOur findings advance the understanding of drug resistance mechanisms in M tuberculosis complex strains. We have established an extended mutation catalogue, comprising variants implicated in resistance and susceptibility to bedaquiline and clofazimine. Our data emphasise that genotypic testing can delineate clinical isolates with borderline phenotypes, which is essential for the design of effective treatments.FundingLeibniz ScienceCampus Evolutionary Medicine of the Lung, Deutsche Forschungsgemeinschaft, Research Training Group 2501 TransEvo, Rhodes Trust, Stanford University Medical Scientist Training Program, National Institute for Health and Care Research Oxford Biomedical Research Centre, Oxford University Hospitals NHS Foundation Trust, Bill & Melinda Gates Foundation, Wellcome Trust, and Marie Skłodowska-Curie Actions.
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2021-12-15 | GSE166546 | GEO
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