Limited contribution of compensatory mutations to MDR/RR-TB clustering in Hunan Province, China: a population-based whole-genome sequencing study

Transmission of rifampicin-resistant and multidrug-resistant tuberculosis (MDR/RR-TB) is a major driver of the global drug-resistant TB epidemic, yet the contribution of compensatory mutations to its spread remains uncertain. We analyzed 206 non-duplicate MDR/RR-TB isolates collected from five surveillance sites in Hunan Province, China, between 2013 and 2020 using whole-genome sequencing. Transmission clusters were defined by ≤12 single-nucleotide polymorphism (SNP) differences, and compensatory mutations in rpoA, rpoB, and rpoC were identified through a curated mutation catalog and phylogenetic evidence. Clusters were further classified based on whether compensatory mutations predated transmission (C-type), arose after transmission (M-type), or were absent (N-type), with M-type clusters excluded to reduce confounding. Among the 206 isolates, 63 (30.6%) carried at least one compensatory mutation, predominantly in rpoC, and 27 clusters (32.5%) were identified, ranging from 2 to 6 isolates. No significant difference was observed in clustering frequency between compensated and non-compensated strains (21.7% vs 32.8%, <i>P</i> = 0.113), nor in cluster size between C-type and N-type clusters (<i>P</i> = 0.961). These findings demonstrate that compe nsatory mutations do not significantly affect transmission clustering at the population level; instead, prolonged infectious periods associated with diagnostic delays and treatment challenges are more likely to sustain MDR/RR-TB spread, underscoring the importance of rapid detection, timely effective therapy, and robust patient management to curb transmission.IMPORTANCEUnderstanding the drivers of multidrug-resistant and rifampicin-resistant tuberculosis (MDR/RR-TB) transmission is critical for global TB control. Compensatory mutations in RNA polymerase subunits have been proposed as key contributors to the success of drug-resistant strains, yet population-level evidence has been limited. In this population-based genomic study of 206 MDR/RR-TB isolates collected over 8 years in Hunan Province, China, we systematically examined the association between compensatory mutations and transmission clustering. Our results show that compensatory mutations did not significantly increase clustering frequency or cluster size, indicating a limited impact on the spread of MDR/RR-TB in the community. These findings challenge the prevailing assumption that compensatory evolution is a major determinant of transmission success and provide robust genomic evidence to refine our understanding of drug-resistant TB epidemiology.