Spontaneous mutations to delamanid and pretomanid resistance in Mycobacterium tuberculosis: SNPs confer high-level resistance.

OBJECTIVES: Multidrug-resistant tuberculosis (MDR-TB) remains a major global health threat. Delamanid (DLM) and pretomanid (PMD) are core drugs in new multidrug-resistant tuberculosis regimens, but the spectrum of their resistance mutations is poorly defined.

METHODS: To investigate these mutations, five parallel in vitro experiments selected for resistance to DLM or PMD. Mycobacterium tuberculosis H37Rv cultures were plated on twice the critical concentrations (CC) of DLM (0.4 µg/mL) or PMD (4 µg/mL) to select resistant clones. Minimum inhibitory concentration (MIC) testing quantified the level of resistance and whole-genome sequencing (WGS) of pooled and individual clones identified mutations associated with drug resistance.

RESULTS: Each parallel experiment yielded about 150 DLM-resistant and 200 PMD-resistant colonies. Pooled-colony WGS detected 102 mutations across six resistance-associated genes (ddn, fgd1, fbiA, fbiB, fbiC, fbiD). MIC assays of 259 individual colonies showed extensive DLM-PMD cross-resistance, with most isolates exhibiting high-level resistance (MIC ≥32 × CC, ≥6.4 µg/mL for DLM and ≥64 µg/mL for PMD) to both drugs. WGS of 100 randomly selected single colonies revealed that non-synonymous single-nucleotide polymorphisms (SNPs), loss-of-function (LoF) mutations, and inframe indels all confer high-level resistance. Of the 111 mutations identified, only six were reported in previous in vitro selection studies. According to the WHO mutation catalogue, 14 loss-of-function mutations were identified as resistant mutations.

CONCLUSION: In conclusion, in vitro selection demonstrated a high diversity of resistance mutations. Most mutations, including single-nucleotide polymorphisms, confer resistance to both DLM and PMD.