title: Dual-Reporter Mycobacteriophages (Φ2DRMs) Reveal Preexisting Mycobacterium tuberculosis Persistent Cells in Human Sputum.
abstract: Persisters are the minor subpopulation of bacterial cells that lack alleles conferring resistance to a specific bactericidal antibiotic but can survive otherwise lethal concentrations of that antibiotic. In infections with Mycobacterium tuberculosis, such persisters underlie the need for long-term antibiotic therapy and contribute to treatment failure in tuberculosis cases. Here, we demonstrate the value of dual-reporter mycobacteriophages (Φ2DRMs) for characterizing M. tuberculosis persisters. The addition of isoniazid (INH) to exponentially growing M. tuberculosis cells consistently resulted in a 2- to 3-log decrease in CFU within 4 days, and the remaining ≤1% of cells, which survived despite being INH sensitive, were INH-tolerant persisters with a distinct transcriptional profile. We fused the promoters of several genes upregulated in persisters to the red fluorescent protein tdTomato gene in Φ2GFP10, a mycobacteriophage constitutively expressing green fluorescent protein (GFP), thus generating Φ2DRMs. A population enriched in INH persisters exhibited strong red fluorescence, by microscopy and flow cytometry, using a Φ2DRM with tdTomato controlled from the dnaK promoter. Interestingly, we demonstrated that, prior to INH exposure, a population primed for persistence existed in M. tuberculosis cells from both cultures and human sputa and that this population was highly enriched following INH exposure. We conclude that Φ2DRMs provide a new tool to identify and quantitate M. tuberculosis persister cells.
  
  
  
  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.
authors: Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York,New York, USA.
authors: Paras
authors: P
authors: Jain
authors: Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York,New York, USA.|Howard Hughes Medical Institute, Albert Einstein College of Medicine, New York, New York, USA.
authors: Brian C
authors: BC
authors: Weinrick
authors: Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York,New York, USA.|Howard Hughes Medical Institute, Albert Einstein College of Medicine, New York, New York, USA.
authors: Eric J
authors: EJ
authors: Kalivoda
authors: Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York,New York, USA.
authors: Hui
authors: H
authors: Yang
authors: KwaZulu-Natal Research Institute for TB/HIV (K-RITH), Durban, South Africa.
authors: Vanisha
authors: V
authors: Munsamy
authors: Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York,New York, USA.|Howard Hughes Medical Institute, Albert Einstein College of Medicine, New York, New York, USA.
authors: Catherine
authors: C
authors: Vilcheze
authors: Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York,New York, USA.|Howard Hughes Medical Institute, Albert Einstein College of Medicine, New York, New York, USA.
authors: Torin R
authors: TR
authors: Weisbrod
authors: Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York,New York, USA.|Department of Medicine, Albert Einstein College of Medicine, New York, New York, USA.
authors: Michelle H
authors: MH
authors: Larsen
authors: Division of Pulmonary, Columbia University Medical Center, New York, New York, USA.|Department of Epidemiology Allergy and Critical Care Medicine, Columbia University Medical Center, New York, New York, USA.|Centre for AIDS Programme of Research in South Africa (CAPRISA), Durban, South Africa.
authors: Max R
authors: MR
authors: O'Donnell
authors: KwaZulu-Natal Research Institute for TB/HIV (K-RITH), Durban, South Africa.|Africa Centre for Health and Population Studies, University of KwaZuluNatal, Mtubatuba, South Africa.
authors: Alexander
authors: A
authors: Pym
authors: Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York,New York, USA jacobsw@hhmi.org.|Howard Hughes Medical Institute, Albert Einstein College of Medicine, New York, New York, USA.|Department of Genetics, Albert Einstein College of Medicine, New York, New York, USA.
authors: William R
authors: WR
authors: Jacobs
mesh_terms: D001431:Bacteriological Techniques; D004361:Drug Tolerance; D005453:Fluorescence; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D009160:Mycobacteriophages / Q000235:genetics / Q000254:growth & development*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification* / Q000821:virology; D013183:Sputum / Q000382:microbiology*; D013194:Staining and Labeling
chemical_list: D008164:Luminescent Proteins