Studying the evolution of Mycobacterium tuberculosis across timescales and its consequences for drug resistance development and patient treatment outcomes

Tuberculosis (TB) remains one of the most important global health problems. With the COVID-19 pandemic receding, TB is once again the number one cause of human mortality due to infection. Moreover, multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) are among the most frequent causes of human death due to antimicrobial resistance. TB is caused by members of the Mycobacterium tuberculosis Complex (MTBC), which exhibit a low genetic diversity compared to other bacteria as well as a highly clonal population structure without any ongoing horizontal gene transfer (HGT). How this lack of HGT affects the evolution of the MTBC on the short and long term is poorly understood. Our limited understanding of MTBC evolution also stems from the fact that most of the comparative genomic data to date has been generated using short-read DNA sequencing technologies. Because of the inherent limitations of short-read sequencing, mobile and repetitive genomic regions, including the PE/PPE genes, are usually excluded during analysis, and consequently, little is known on the role of mobile elements, indels, duplications, rearrangements and gene conversion in MTBC evolution. Despite the absence of HGT, many MTBC strains have acquired resistance to first- and second-line antibiotics, fueling outbreaks of MDR/XDR-TB around the world.

Recently, new regimens have been endorsed by the World Health Organization (WHO) for the treatment of MDR/XDR-TB that rely on the new drugs bedaquiline and pretomanid; these new regimens are now being rolled-out globally. While resistance to these new drugs is already emerging, little is known on the bacterial factors underlying the emergence of drug resistance to these new drugs. In particular, the role of drug tolerance and drug resilience in resistance development, as well as the influence of pre-existing MTBC genomic variation, are poorly understood. Here we will analyze an existing collection of MTBC clinical isolates, together with the associated patient data, that was compiled during clinical trials conducted by the TB Alliance. These trials formed the basis for the regulatory approval and the recent WHO endorsement of the new bedaquiline- and pretomanid-containing regimens. These trials also represent real-world experiments, providing an opportunity to study MTBC evolution under in clinico conditions. This TB Alliance Strain Collection comprises representatives of the four globally most prevalent phylogenetic lineages of the MTBC as well as isolates from transmission chains and sequential isolates from individual patients with drug-susceptible and drug-resistant TB, thus reflecting various evolutionary timescales.

We will use this strain collection and apply long-read genome sequencing, comparative proteomics and other phenotypic assays to i) study the genome evolution of the MTBC across different timescales, ii) investigate the evolution of drug tolerance, drug resilience and drug resistance in serial MTBC isolates from patients who received old or new TB drugs, and iii) explore how MTBC strain diversity influences patient treatment outcomes. Because the TB Alliance Strain Collection comes with a wealth of high-quality clinical data, we will be able to control for potential confounding factors during data analysis. In summary, this project will generate new knowledge on MTBC evolution, and identify MTBC variation emerging within and between patients that modulates the bacterial proteome, phenotypic drug tolerance, and the development of resistance to old and new anti-TB drugs. In addition, this project will link genotypic and phenotypic MTBC diversity to patient treatment outcomes. Together, the results of this project will inform the biology, the epidemiology and the control of one of humankind’s most deadly infectious diseases.