In genome evolution, genetic variants are the source of diversity, which natural selection acts upon. Treatment of human tuberculosis (TB) induces a strong selection pressure for the emergence of antibiotic resistance in the infecting Mycobacterium tuberculosis (MTB) strains. MTB evolution in response to treatment has been intensively studied and mainly attributed to point substitutions. However, the contribution of insertions and deletions (indels) to MTB genome evolution remains poorly understood. Here, we analyzed a multi-drug resistant MTB outbreak for the presence of high-quality indels and substitutions. We find that indels are significantly enriched in genes conferring antibiotic resistance. Furthermore, we show that indels are inherited during the outbreak and follow a molecular clock with an evolutionary rate of 5.37e-9 indels/site/year, which is 23x lower compared to the substitution rate. Inherited indels may co-occur with substitutions in genes along related biological pathways; examples are iron storage and resistance to second-line antibiotics. This suggests that epistatic interactions between indels and substitutions affect antibiotic resistance and compensatory evolution in MTB.Author summary Mycobacterium tuberculosis (MTB) is a human pathogen causing millions of deaths every year. Its genome evolution has been intensively characterized through point substitutions, i.e., nucleotide exchanges that are inherited. Additional mutations are short or long insertions and deletions of nucleotides, termed indels. Short indels in genes might change the reading frame and disrupt the gene product. Here we show that antibiotic treatment has a strong impact on indel evolution in an MTB outbreak. Namely, indels occur frequently in genes causing antibiotic resistance upon disruption. Furthermore, we show that the molecular clock, i.e., the temporal emergence of variants over time, holds for short indels in MTB genomes. Finally, we observe that indels may co-occur with substitutions in genes along related biological pathways. These results support the notion that indels are important contributors to MTB evolution. We anticipate that including indels in the analyses of MTB outbreaks will improve our understanding of antibiotic resistance evolution.

Insertion and deletion evolution reflects antibiotics selection pressure in a Mycobacterium tuberculosis outbreak