Novel and reported compensatory mutations in genes found in drug resistant tuberculosis outbreaks.

Background: Rifampicin (RIF) is a key first-line drug used to treat tuberculosis, a primarily pulmonary disease caused by . RIF resistance is caused by mutations in , at the cost of slower growth and reduced transcription efficiency. Antibiotic resistance to RIF is prevalent despite this fitness cost. Compensatory mutations in genes have been shown to alleviate the fitness cost of :S450L, explaining how RIF resistant strains harbor this mutation can spread so rapidly. Unfortunately, the full set of RIF compensatory mutations is still unknown, particularly those compensating for rarer RIF resistance mutations.

Objectives: We performed an association study on a globally representative set of 4,309 whole genome sequenced clinical isolates to identify novel putative compensatory mutations, determine the prevalence of known and previously reported putative compensatory mutations, and determine which RIF resistance markers associate with these compensatory mutations.

Results And Conclusions: Of the 1,079 RIF resistant isolates, 638 carried previously reported putative and high-probability compensatory mutations. Our strict criteria identified 46 additional mutations in for which no strong prior evidence of their compensatory role exists. Of these, 35 have previously been reported. As such, our independent corroboration adds to the mounting evidence that these 35 also carry a compensatory role. The remaining 11 are novel putative compensatory markers, reported here for the first time. Six of these 11 novel putative compensatory mutations had two or more mutation events. Most compensatory mutations appear to be specifically compensating for the fitness loss due to :S450L. However, an outbreak of 22 closely related isolates each carried three mutations, the rare RIF markers D435G and L452P and the putative compensatory mutation I1106T. This suggests compensation may require specific combinations of mutations. Here, we report only mutations that met our very strict criteria. It is highly likely that many additional mutations compensate for rare resistance-causing mutations and therefore did not carry the statistical power to be reported here. These findings aid in the identification of RIF resistant strains with restored fitness, which pose a greater risk of causing resistant outbreaks.