The influence of the lower airway microbiome on the dynamic Mycobacterium tuberculosis populations in active pulmonary Tuberculosis

Tuberculosis (TB) is a leading cause of death worldwide, despite being considered a curable and preventable disease. Pulmonary TB is a heterogeneous disease both in terms of clinical expression, pathology and treatment response. Many factors related to patient genetics, immunology and lifestyle have been studied and shown to contribute to TB progression and prognosis. However, these factors cannot fully explain the variability in disease outcomes. Mycobacterium tuberculosis (Mtb) is a highly successful pathogen that can adopt various physiological states. These include a distinct subpopulation of persister-like bacteria known as “Differentially Culturable Mtb” (DCMtb). They are more resistant to chemotherapy and cannot be cultured in standard media. Importantly, DCMtb have been implicated in unfavourable treatment outcomes and disease relapse in patients after completing treatment. Nitric oxide and reactive oxygen species have been proposed as triggers for DCMtb during infection. Intriguingly, DCMtb are also formed in macrophages activated with lipopolysaccharide, a component of the bacterial cell wall missing in Mtb, suggesting a previously unanticipated role of the wider microbiome in Mtb physiology, disease progression and treatment outcomes. This suggestion is further supported by recently published data indicating that complex and diverse communities in the lung of TB patients may influence treatment success. As yet no studies have explored the respiratory microbiome in relation to the phenotypic characteristics of Mtb strains, in particular their ability to form DCMtb. Therefore, in this study we aim to explore the relationship between the respiratory microbiome and the formation of DCMtb in patients with pulmonary TB (PTB) and their relationship to treatment outcomes. Objectives We hypothesise that bacterial communities within the lung play a key role in the M. tuberculosis phenotype during PTB infection. To explore this hypothesis, we propose to undertake a prospective cohort study in a well characterised cohort of microbiologically confirmed, drug sensitive PTB patients. Patients will undergo CT directed bronchoscopy and sampling (bronchoalveolar lavage (BAL), bronchial brushings) pre-treatment and after completing 6 months of anti-TB treatment. This will provide a unique opportunity to investigate our hypotheses in 3 work packages. WP1. In-depth characterisation of DCMtb subpopulations from representative cohort of treatment naïve pulmonary TB patients from Leicester, UK WP2. Characterisation of the microbial community and biogeography of the lower respiratory tract in PTB pre- and post-treatment WP 3. Investigation of mechanisms underpinning the role of the respiratory microbiome in driving the formation of DCMtb. Direct outputs/impacts Despite increasing evidence of the role of the respiratory microbiome for mediating disease outcomes, exploration of the respiratory microbiome is TB is in its infancy. In this project, we will establish protocols for describing and characterising microbial communities, including DCMtb from patients with PTB. Using these methodologies, we aim to describe the biogeography of the lung during PTB infection to understand how the microbial communities within the lung impact disease progression both directly and indirectly. This work will allow us to further address how microenvironments within the lung drives DCMtb differentially and assess how this impacts treatment response. Through the in-depth characterisation of the DCMtb population in a UK TB population, this study will improve our understanding of Mtb phenotypes and postulate clinically relevant biomarkers of treatment response.