Evolutionary Epidemiology of the Mycobacterium tuberculosis Complex

Tuberculosis (TB) is an ancient disease, whose control is continuously challenged by emerging threats: the HIV pandemic since the 1980s, the emergence and spread of multidrug-resistant strains since the 1990s, and the SARS-CoV-2 pandemic today. The majority of TB patients are treated by a combination of four different drugs, given over a six months period. However, drug-resistant forms of TB have emerged worldwide, requiring treatments that are not only more toxic, but also longer (up to 30 months). Although TB is a curable disease, it claims the lives of 1.4 million individuals every year. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC), which includes Mycobacterium tuberculosis, the human-adapted ecotype, Mycobacterium bovis, the most important agent of TB in livestock and the most important cause of zoonotic disease in humans, and several other animal-adapted ecotypes. In this thesis we took advantage of the recent technical advances and decreasing costs of whole-genome sequencing (WGS) to examine the macroevolutionary and microevolutionary events that shaped the MTBC populations. We relied on two sources of genomic data: (i) WGS retrieved from public repositories, and (ii) clinical strains of the MTBC, collected from high TB burden countries around the world, and which were then sequenced in Basel. This led to an in-house clinical database of 50,000 MTBC WGS. We developed bioinformatics tools to analyze these genomes, laying the methodological foundation for the rest of the thesis (Chapter 3). In the first part of the thesis, we analysed publicly available WGS and explored the diversity of the MTBC. This led to the discovery of a new MTBC lineage, which shared a most recent common ancestor with the rest of the MTBC. This lineage was restricted to East Africa, providing additional evidence for an East-African origin of the MTBC (Chapter 4). We then compiled a global collection of M. bovis genomes, and showed M. bovis likely originated in East Africa. We found eight previously uncharacterized clades, one of which was of particular clinical interest, as it lacked the mutation responsible for intrinsic pyrazinamide resistance of canonical M. bovis (Chapter 5). We then assessed the performance of a widely used diagnostic assay, and propose new markers to better distinguish MTBC lineages and ecotypes, which will be implemented in the new version of the assay (Chapter 6). In the second part of the thesis, we explored two other important aspects of TB epidemiology: drug-resistance and HIV co-infection. We analyzed a global dataset of rifampicin-resistant M. tuberculosis genomes collected from HIV-negative and HIV-positive patients, and found that low fitness rifampicin resistance-conferring variants can thrive in the context of reduced immunity (Chapter 7). We conducted a nation-wide genomic epidemiological study in Georgia, a country that has one of the highest rates of multidrug- resistant TB (MDR-TB) in the world. We studied the bacterial and environmental determinants that characterize the epidemiological success of MDR-TB (Chapter 8 and 9), and found that 63% of MDR-TB in Georgia was due to recent transmission of highly drug resistant strains, including resistance to newly-developed antituberculous agents. We demonstrate the role of compensatory mutations in mitigating the fitness cost associated with drug resistance, and found that prisons played a central role in fuelling the epidemic of MDR-TB (Chapter 8). Overall, in this doctoral thesis we addressed key aspects of TB epidemiology, and demonstrate how the joint evolutionary and epidemiological investigations of the MTBC can be translated into clinical practices