GMeissner

1: London School of Hygiene and Tropical Medicine

Resistance to BDQ can emerge through loss-of-function (LOF) mutations in mmpR5, a transcriptional repressor of the mmpL5/mmpS5 efflux pump. In clinical samples, multiple LOF mutations are often found at non-fixed allele frequencies, suggesting independent acquisition events within a single host. While LOF mutations are associated with resistance, the role of single nucleotide polymorphisms (SNPs) remains poorly understood. We hypothesise that SNPs of unknown significance, when co-occurring with mmpR5 LOF mutations at subclonal frequencies, may represent alternative resistance mechanisms and contribute to resistance. To investigate this, we analysed mmpR5 mutations in 173,816 M. tuberculosis genomes. Candidate SNPs were identified by selecting samples containing both a frameshift mutation and a missense SNP in mmpR5, each at subclonal frequencies (<90%). This approach identified 141 unique missense SNPs (observed 284 times) co-occurring with 69 frameshift mutations (303 occurrences) across 246 samples. The missense SNPs were detected in 1 to 59 samples and exhibited allele frequencies ranging from 10% to 97%. Of the candidate SNPs, 45 (31.9%) were observed in more than one sublineage, consistent with repeated selection likely driven by drug exposure. Enrichment analysis revealed that these mutations were significantly associated with multi-drug resistant (MDR; 20.3%) and extensively drug-resistant (XDR; 68.7%) isolates. Our findings suggest that a subset of mmpR5 SNPs—currently unclassified—may be under positive selection in BDQ- or clofazimine-exposed strains. These mutations warrant further functional characterisation, and clinical isolates carrying them should be carefully evaluated for BDQ/CFZ susceptibility.