Mycobacterium tuberculosis within-host genetic diversity and acquired drug resistance

Drug-resistant tuberculosis (DR-TB) remains a major global health challenge with increasing numbers of cases reported each year. Whole genome sequencing (WGS) is used increasingly in both research and clinical services to rapidly identify drug resistance. Mycobacterium tuberculosis WGS directly from sputum has been achieved using DNA enrichment but with a higher limit of detection than sequencing from culture. I demonstrate methodological adaptations that improve M. tuberculosis genome coverage for direct sequencing. I then show WGS directly from sputum preserves more within-patient M. tuberculosis genetic diversity than sequencing from culture, where bacterial populations better suited to growth in culture may be selected. Infections caused by multiple M. tuberculosis strains lead to worse outcomes. However, whether higher genetic diversity in single strain infections also does and the clinical importance of unfixed (<100% allele frequency) resistance mutations are not known. I established a prospective cohort of patients with DR-TB followed for six months with monthly sputum culture and WGS, and found no association between baseline genetic diversity and clinical outcome. Macroheteroresistance (variants >5% frequency) mostly persisted or became fixed over time. However, new macroheteroresistant or fixed resistance mutations were rare in the cohort (3.1%) and not predicted by the prior presence of microheteroresistance (<5%). Most of the resistance identified was to bedaquiline, a key drug. In one cohort, 5.4% of patients with baseline positive M. tuberculosis culture had mutations in the Rv0678 gene responsible for bedaquiline resistance, and a further 5.7% acquired resistance during treatment due to new Rv0678 mutations. A temporal dating analysis of global phylogenies incorporating the major M. tuberculosis lineages 2 and 4 indicates that variants in Rv0678 arose in the late 19th century and pre-date the antibiotic era, therefore likely conferring evolutionary advantages beyond drug resistance.