Undetected Drug resistance and Tolerance in lesions of recurrent TB

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Tuberculosis (TB) kills nearly 1.5 million people in the world. Despite powerful chemotherapeutics, surviving bacteria often go undetected by standard diagnostics, persist in lung lesions, and continue to make patients ill, even after declaration of microbiological “cure” as specified by criteria from the World Health Organization. This project’s objective is to characterize the “(epi)genomic” (genomic and epigenomic) composition of Mycobacterium tuberculosis (Mtb) in patients who require surgical intervention for treatment of tuberculosis (TB), directly at the site of infection, and to create a knowledgebase that helps explain failure of chemotherapy and standard diagnostics. Are these failures due to undetected resistant strains absent in the sputum? Were there persister cells in pulmonary lesions that tolerated the drug pressure? Did drugs not reach bacteria within the lesion in sufficient concentrations? Or were persistent or resistant bacilli in the sputum that went undetected by standard diagnostics? Answering these questions will enable the development of new and more sensitive diagnostics, inform development of novel therapeutics that to more effectively penetrate lesions and target the surviving bacteria, or target all bacterial subpopulations, and enable rational design and development of a vaccine with a more comprehensive understanding of genomic variability of this pathogen in the context of human infection. Mtb is an obligate human pathogen, yet treatment decisions hinge on phenotypic testing and genomic characterization of bacteria isolated from sputum and grown in vitro as a proxy for the Mtb communities driving infection in the lungs. Inconsistencies between the sputum and lung bacterial communities have been hypothesized as reasons behind the mischaracterization of the disease. In this project, we investigate this hypothesis and, additionally, aim to determine whether there are additional bacteria in the sputum that can inform development of novel methods to provide more comprehensive diagnostics. In this process, we will also determine whether persistence or superinfection played a role in treatment failure, and how frequently. To reach these goals, this project will study bacteria residing within pulmonary lesions excised from 200 patients who failed traditional treatment. For the first time, we will directly sequence, and de novo assemble genomes and methylomes of Mtb in pulmonary lesions and sputum, and determine the distinguishing genomic and epigenetic characteristics of each subpopulation in these flora. We will detect hidden resistance or persistence, and, through differential culturing, identify whether bacteria have been damaged by antibiotic treatment and whether they are all detected by standard diagnostics. Our existing project has identified novel rapid diversifying mechanisms that allows the bacterial community to withstand immune and drug pressures. As part of this project, we will investigate the prevalence and nature of these mechanisms in the lung. This will inform new paths to development of more effective and novel therapeutics and vaccines.