Mutations in () as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis.

The nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior studies suggest a relatively low barrier to nitroimidazole resistance in , but clinical evidence is limited to date. We selected pretomanid-resistant mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, (56%), (15%), (12%), (4%), and (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in (), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although and mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an mutant with wild-type restored susceptibility to pretomanid and delamanid. By quantifying intracellular F and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F biosynthesis. Finally, mutants and other FH-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.