Mutations infbiD (Rv2983)as a novel determinant of resistance to pretomanid and delamanid inMycobacterium tuberculosis

Abstract

The nitroimidazole pro-drugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 91% of which occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance: fbiC (56%), fbiA (15%), ddn (12%), fgd (4%) and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983, a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance, but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-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.

Comparative efficacy of the novel diarylquinoline TBAJ-587 and bedaquiline against a resistantRv0678mutant in a mouse model of tuberculosis

“…In stark contrast, no PMD-resistant mutants were isolated from wild-type-infected mice treated with BPaL. These results raise concerns that inadvertent treatment of patients infected with an Rv0678 mutant with BPaL could lead to dangerous new form of multidrug resistance defined by resistance to BDQ and PMD, which would likely extend to delamanid (27, 28).…”

Comparative Efficacy of the Novel Diarylquinoline TBAJ-587 and Bedaquiline against a Resistant Rv0678 Mutant in a Mouse Model of Tuberculosis

“…In stark contrast, no PMD-resistant mutants were isolated from wild-type-infected mice treated with BPaL. These results raise concerns that inadvertent treatment of patients infected with an Rv0678 mutant with BPaL could lead to dangerous new form of multidrug resistance defined by resistance to BDQ and PMD, which would likely extend to delamanid (28,29). Surprisingly, the BPaMZ regimen, like the PaMZ regimen, had similar bactericidal effects against both the wild-type and mutant infections, indicating that the contribution of BDQ to the efficacy of BPaMZ was not affected by the Rv0678 mutation.…”

Targeted next-generation sequencing: a Swiss army knife for mycobacterial diagnostics?

“…line probe assays and real-time PCR) typically analyse fewer than 200 base pairs (bp), the number of diagnostic questions that can be addressed with a single assay is limited. In fact, these assays are not suitable for genotypic AST for delamanid and pretomanid as resistance to these nitroimidazoles can arise by mutations in six genes that span approximately 7500 bp, including promoters [11,12]. These constraints notwithstanding, over 45 first-generation NAATs are currently approved for clinical use to simultaneously identify MTBC and perform genotypic AST directly from the clinical sample (S. Mohamed and co-workers, poster presented at the 50th Union World Conference on Lung Health; available on request).…”