Dual-Reporter Mycobacteriophages (Φ ² DRMs) Reveal Preexisting Mycobacterium tuberculosis Persistent Cells in Human Sputum

Abstract

Tuberculosis (TB) is again the leading cause of death from a single infectious disease, having surpassed HIV. The recalcitrance of the TB pandemic is largely due to the ability of the pathogen Mycobacterium tuberculosis to enter a persistent state in which it is less susceptible to antibiotics and immune effectors, necessitating lengthy treatment. It has been difficult to study persister cells, as we have lacked tools to isolate these rare cells. In this article, we describe the development of dual-reporter mycobacteriophages that encode a green fluorescent marker of viability and in which the promoters of genes we have identified as induced in the persister state are fused to a gene encoding a red fluorescent protein. We show that these tools can identify heterogeneity in a cell population that correlates with propensity to survive antibiotic treatment and that the proportions of these subpopulations change in M. tuberculosis cells within human sputum during the course of treatment.