Toward a Phage Cocktail for Tuberculosis: Susceptibility and Tuberculocidal Action of Mycobacteriophages against Diverse Mycobacterium tuberculosis Strains.

The global health burden of human tuberculosis (TB) and the widespread antibiotic resistance of its causative agent Mycobacterium tuberculosis warrant new strategies for TB control. The successful use of a bacteriophage cocktail to treat a Mycobacterium abscessus infection suggests that phages could play a role in tuberculosis therapy. To assemble a phage cocktail with optimal therapeutic potential for tuberculosis, we have explored mycobacteriophage diversity to identify phages that demonstrate tuberculocidal activity and determined the phage infection profiles for a diverse set of strains spanning the major lineages of human-adapted strains of the Mycobacterium tuberculosis complex. Using a combination of genome engineering and bacteriophage genetics, we have assembled a five-phage cocktail that minimizes the emergence of phage resistance and cross-resistance to multiple phages, and which efficiently kills the M. tuberculosis strains tested. Furthermore, these phages function without antagonizing antibiotic effectiveness, and infect both isoniazid-resistant and -sensitive strains.IMPORTANCE Tuberculosis kills 1.5 million people each year, and resistance to commonly used antibiotics contributes to treatment failures. The therapeutic potential of bacteriophages against Mycobacterium tuberculosis offers prospects for shortening antibiotic regimens, provides new tools for treating multiple drug-resistant (MDR)-TB and extensively drug-resistant (XDR)-TB infections, and protects newly developed antibiotics against rapidly emerging resistance to them. Identifying a suitable suite of phages active against diverse M. tuberculosis isolates circumvents many of the barriers to initiating clinical evaluation of phages as part of the arsenal of antituberculosis therapeutics.

Mycobacteriophages as Potential Therapeutic Agents against Drug-Resistant Tuberculosis.

The current emergence of multi-, extensively-, extremely-, and total-drug resistant strains of Mycobacterium tuberculosis poses a major health, social, and economic threat, and stresses the need to develop new therapeutic strategies. The notion of phage therapy against bacteria has been around for more than a century and, although its implementation was abandoned after the introduction of drugs, it is now making a comeback and gaining renewed interest in Western medicine as an alternative to treat drug-resistant pathogens. Mycobacteriophages are genetically diverse viruses that specifically infect mycobacterial hosts, including members of the M. tuberculosis complex. This review describes general features of mycobacteriophages and their mechanisms of killing M. tuberculosis, as well as their advantages and limitations as therapeutic and prophylactic agents against drug-resistant M. tuberculosis strains. This review also discusses the role of human lung micro-environments in shaping the availability of mycobacteriophage receptors on the M. tuberculosis cell envelope surface, the risk of potential development of bacterial resistance to mycobacteriophages, and the interactions with the mammalian host immune system. Finally, it summarizes the knowledge gaps and defines key questions to be addressed regarding the clinical application of phage therapy for the treatment of drug-resistant tuberculosis.

SIV Evolutionary Dynamics in Cynomolgus Macaques during SIV-Mycobacterium tuberculosis Co-Infection.

Co-infection with Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV) is a worldwide public health concern, leading to worse clinical outcomes caused by both pathogens. We used a non-human primate model of simian immunodeficiency virus (SIV)-Mtb co-infection, in which latent Mtb infection was established prior to SIVmac251 infection. The evolutionary dynamics of SIV env was evaluated from samples in plasma, lymph nodes, and lungs (including granulomas) of SIV-Mtb co-infected and SIV only control animals. While the diversity of the challenge virus was low and overall viral diversity remained relatively low over 6-9 weeks, changes in viral diversity and divergence were observed, including evidence for tissue compartmentalization. Overall, viral diversity was highest in SIV-Mtb animals that did not develop clinical Mtb reactivation compared to animals with Mtb reactivation. Among lung granulomas, viral diversity was positively correlated with the frequency of CD4+ T cells and negatively correlated with the frequency of CD8+ T cells. SIV diversity was highest in the thoracic lymph nodes compared to other sites, suggesting that lymphatic drainage from the lungs in co-infected animals provides an advantageous environment for SIV replication. This is the first assessment of SIV diversity across tissue compartments during SIV-Mtb co-infection after established Mtb latency.

Genomic and proteomic portrait of a novel mycobacteriophage SWU2 isolated from China.

Phage therapy, especially combination with antibiotics, was revitalized to control the antibiotics resistance. Mycobacteriophage, the phage of mycobacterium with the most notorious Mycobacterium tuberculosis (M. tuberculosis), was intensively explored. A novel mycobacteriophage SWU2 was isolated from a soil sample collected at Nanchang city, Jiangxi province, China, by using Mycolicibacterium smegmatis (M. smegmatis) mc2 155 as the host. Phage morphology and biology were characterized. Phage structure proteins were analyzed by LC-MS/MS. The putative functions of phage proteins and multi-genome comparison were performed with bioinformatics. The transmission electron microscopy result indicated that this phage belongs to Siphoviridae of Caudovirales. Plaques of SWU2 appeared clear but small. In a one-step growth test, we demonstrated that SWU2 had a latent period of 30 min and a logarithmic phase of 120 min. Among the 76 predicted Open Reading Frames (ORFs), 9 ORFs were identified as phage structure proteins of SWU2. The assembled phage genome size is 50,013 bp, with 62.7% of G + C content. SWU2 genome sequence shares 88% identity with Mycobacterium phages HINdeR and Timshel, differing in substitutions, insertions and deletions in SWU2. Phylogenetic tree revealed that SWU2 is grouped into A7 sub-cluster. There are several substitutions, insertions and deletions in SWU2 genome in comparison with close cousin phages HINdeR and Timshel. The new phage adds another dimension of abundance to the mycobacteriophages.

Nanoluciferase Reporter Mycobacteriophage for Sensitive and Rapid Detection of Mycobacterium tuberculosis Drug Susceptibility.

Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains.IMPORTANCEMycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

Study of characteristics of mycobacteriophage - A novel tool to treat Mycobacterium spp.

Background: Mycobacteriophages are viruses that infect Mycobacterium spp. Till date, 10427 mycobacteriophages have been isolated and 1670 mycobacteriophage genomes have been sequenced https://phagesdb.org/hosts/genera/1/ (cited on 30th December,2018). In the previous study, 10 different mycobacteriophages from 14 soil samples were isolated, by qualitative plaque formation method using Mycobacterium smegmatis as host. Among these, three phages were found to infect four different species of Mycobacterium, i.e., Mycobacterium fortuitum subsp. fortuitum MTCC993, Mycobacterium kansasii MTCC3058, Mycobacterium avium subsp. avium MTCC1723, and Mycobacterium tuberculosis MTCC300, besides the host M. smegmatis. The phage lysates were concentrated by polyethylene glycol (PEG) precipitation. One of the three phages showing host diversity was selected for further study. The various phage growth parameters such as incubation temperature, time of adsorption, host cell density and effect of cations were standardised. Methods: The studies were done by qualitative and quantitative plaque assay method. Results: The phage selected for further study showed an optimum adsorption time of 15 min. The optimum temperature for propagation was found to be 37°C. The phage was found to be stable at 42°C. In the presence of calcium, the phage showed a higher rate of infectivity. Conclusion: Understanding the biology of mycobacteriophages and their host diversity is the key to understanding mycobacterial systems. This could be the first step toward exploiting the potential of phages as therapeutic agents.

The Mycobacterium tuberculosis CRISPR-Associated Cas1 Involves Persistence and Tolerance to Anti-Tubercular Drugs.

Tuberculosis remains one of the leading causes of death worldwide. Even if new antitubercular drugs are currently being developed, the rapid emergence and spread of drug-resistant strain remain a severe challenge. The CRISPR associated proteins 1 (Cas1), a most conserved endonuclease which is responsible for spacer integration into CRISPR arrays, was found deleted in many specific drug-resistant strains. The function of Cas1 is still unknown in Mycobacterium type III-A CRISPR family. In this study, the Cas1 (Rv2817c) defect was found in 57.14% of clinical isolates. To investigate the function of Cas1 in new spacer acquisition, we challenged Bacillus Calmette-Guérin (BCG) with a mycobacteriophage D29. Newly acquired spacer sequence matches D29 genome was not found by spacer deep-sequencing. We further expressed Cas1 in recombinant Mycobacterium smegmatis. We found that Cas1 increased the sensitivity to multiple anti-tuberculosis drugs by reducing the persistence during drug treatment. We also showed that Cas1 impaired the repair of DNA damage and changed the stress response of Mycobacterium smegmatis. This study provides a further understanding of Cas1 in Mycobacterium tuberculosis complex (MTBC) drug-resistance evolution and a new sight for the tuberculosis treatment.

Bacteriophage gene products as potential antimicrobials against tuberculosis.

Tuberculosis (TB) is recognised as one of the most pressing global health threats among infectious diseases. Bacteriophages are adapted for killing of their host, and they were exploited in antibacterial therapy already before the discovery of antibiotics. Antibiotics as broadly active drugs overshadowed phage therapy for a long time. However, owing to the rapid spread of antibiotic resistance and the increasing complexity of treatment of drug-resistant TB, mycobacteriophages are being studied for their antimicrobial potential. Besides phage therapy, which is the administration of live phages to infected patients, the development of drugs of phage origin is gaining interest. This path of medical research might provide us with a new pool of previously undiscovered inhibition mechanisms and molecular interactions which are also of interest in basic research of cellular processes, such as transcription. The current state of research on mycobacteriophage-derived anti-TB treatment is reviewed in comparison with inhibitors from other phages, and with focus on transcription as the host target process.

A small mycobacteriophage-derived peptide and its improved isomer restrict mycobacterial infection via dual mycobactericidal-immunoregulatory activities.

Mycobacteriophages express various peptides/proteins to infect Mycobacterium tuberculosis (M. tb). Particular attention has been paid to mycobacteriophage-derived endolysin proteins. We herein characterized a small mycobacteriophage-derived peptide designated AK15 with potent anti-M. tb activity. AK15 adopted cationic amphiphilic α-helical structure, and on the basis of this structure, we designed six isomers with increased hydrophobic moment by rearranging amino acid residues of the helix. We found that one of these isomers, AK15-6, exhibits enhanced anti-mycobacterial efficiency. Both AK15 and AK15-6 directly inhibited M. tb by trehalose 6,6'-dimycolate (TDM) binding and membrane disruption. They both exhibited bactericidal activity, cell selectivity, and synergistic effects with rifampicin, and neither induced drug resistance to M. tb They efficiently attenuated mycobacterial load in the lungs of M. tb-infected mice. We observed that lysine, arginine, tryptophan, and an α-helix are key structural requirements for their direct anti-mycobacterial action. Of note, they also exhibited immunomodulatory effects, including inhibition of proinflammatory response in TDM-stimulated or M. tb-infected murine bone marrow-derived macrophages (BMDMs) and M.tb-infected mice and induction of only a modest level of cytokine (tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6)) production in murine BMDMs and a T-cell cytokine (interferin-γ (IFN-γ) and TNF-α) response in murine lung and spleen. In summary, characterization of a small mycobacteriophage-derived peptide and its improved isomer revealed that both efficiently restrain M. tb infection via dual mycobactericidal-immunoregulatory activities. Our work provides clues for identifying small mycobacteriophage-derived anti-mycobacterial peptides and improving those that have cationic amphiphilic α-helices.

Mycobacteriophage CRB2 defines a new subcluster in mycobacteriophage classification.

Mycobacteriophages are viruses -mostly temperates- that infect Mycobacterium smegmatis and sometimes Mycobacterium tuberculosis. Mycobacteriophages are grouped in clusters on the basis of the overall nucleotide sequence homology, being further divided in subclusters as more mycobacteriophage genomes are sequenced and annotated. As part of our on-going screening for novel isolates, we herein report the bioinformatics analysis of CRB2, a mycobacteriophage belonging into the Siphoviridae family that propagates at 30°C. CRB2 has a 72,217 bp genome with a 69.78% GC content that belongs to Cluster B; nucleotide comparison with other B cluster members positions CRB2 as the sole member of a new subcluster, B9, being mycobacteriophage Saguaro (belonging into subcluster B7) its closest relative. Sequencing and annotation of 14 mycobacteriophages isolated by our group has yielded six cluster A members, a singleton, four of the five members of subcluster B6, one of the three reported members of subcluster G4, and CRB2 which defines subcluster B9. Considering the massive mycobacteriophage search performed in USA and the relatively rarity of our phages, we propose that factors other than size of the sampling determine the variability of mycobacteriophage distribution, and thus a world-wide concerted mining would most likely bring extremely rare and yet undiscovered mycobacteriophages.

Fluoromycobacteriophages for Drug Susceptibility Testing (DST) of Mycobacteria.

Fluoromycobacteriophages are a new class of reporter phages that contain Laboratorio fluorescent reporter genes (gfp, ZsYellow, and mCherry) and provide a simple means of revealing the metabolic state of mycobacterial cells and therefore their response to antibiotics. Here we described a simple and rapid method for drug susceptibility testing (DST) of Mycobacterium spp using a fluorescence microscope, a flow cytometer, or a fluorimeter in a convenient multiwell format.

Mycobacteriophage Lysis Enzymes: Targeting the Mycobacterial Cell Envelope.

Mycobacteriophages are viruses that specifically infect mycobacteria, which ultimately culminate in host cell death. Dedicated enzymes targeting the complex mycobacterial cell envelope arrangement have been identified in mycobacteriophage genomes, thus being potential candidates as antibacterial agents. These comprise lipolytic enzymes that target the mycolic acid-containing outer membrane and peptidoglycan hydrolases responsive to the atypical mycobacterial peptidoglycan layer. In the recent years, a remarkable progress has been made, particularly on the comprehension of the mechanisms of bacteriophage lysis proteins activity and regulation. Notwithstanding, information about mycobacteriophages lysis strategies is limited and is mainly represented by the studies performed with mycobacteriophage Ms6. Since mycobacteriophages target a specific group of bacteria, which include Mycobacterium tuberculosis responsible for one of the leading causes of death worldwide, exploitation of the use of these lytic enzymes demands a special attention, as they may be an alternative to tackle multidrug resistant tuberculosis. This review focuses on the current knowledge of the function of lysis proteins encoded by mycobacteriophages and their potential applications, which may contribute to increasing the effectiveness of antimycobacterial therapy.

A transcriptomic analysis of the mycobacteriophage D29 genome reveals the presence of novel stoperator-associated promoters in its right arm.

Mycobacteriophage D29 is a lytic phage that infects various species of Mycobacterium including M. tuberculosis. Its genome has 77 genes distributed almost evenly between two converging operons designated as left and right. Transcription of the phage genome is negatively regulated by multiple copies of an operator-like element known as stoperator that acts by binding the phage repressor Gp71. The function of the D29 genes and their expression status are poorly understood and therefore we undertook a transcriptome analysis approach to address these issues. The results indicate that the average transcript intensity of the right arm genes was higher than of those on the left, at the early stage of infection. Moreover, the fold increase from early to the late stage was found to be less for the right arm genes than for the left. Both observations support the prediction that the right arm genes are expressed early whereas the left arm ones are expressed late. The analysis further revealed a break in the continuity of the right arm operon between 89, the first gene in it, and 88, the next. Gene 88 was found to be expressed from a newly identified promoter located between 88 and 89. Another new promoter was found upstream of 89. Thus, the promoter Pleft, identified earlier, is not the only one that drives expression of the right arm genes. All these promoters overlap with stoperators, with which they share a conserved sequence motif, TTGACA, commonly known as the -35 promoter element. We demonstrate mutually exclusive binding of RNA polymerase and Gp71 to the stoperator-promoters and conclude that stoperators can function as -35 promoter elements and that they can control gene expression not only negatively as was believed earlier but in many cases positively as well.

Isolation and characterization of bacteriophages from India, with lytic activity against Mycobacterium tuberculosis.

Bacteriophages are being considered as a promising natural resource for the development of alternative strategies against mycobacterial diseases, especially in the context of the wide-spread occurrence of drug resistance among the clinical isolates of Mycobacterium tuberculosis. However, there is not much information documented on mycobacteriophages from India. Here, we report the isolation of 17 mycobacteriophages using Mycobacterium smegmatis as the bacterial host, where 9 phages also lyse M. tuberculosis H37Rv. We present detailed analysis of one of these mycobacteriophages - PDRPv. Transmission electron microscopy and polymerase chain reaction analysis (of a conserved region within the TMP gene) show PDRPv to belong to the Siphoviridae family and B1 subcluster, respectively. The genome (69 110 bp) of PDRPv is circularly permuted double-stranded DNA with ∼66% GC content and has 106 open reading frames (ORFs). On the basis of sequence similarity and conserved domains, we have assigned function to 28 ORFs and have broadly categorized them into 6 groups that are related to replication and genome maintenance, DNA packaging, virion release, structural proteins, lysogeny-related genes and endolysins. The present study reports the occurrence of novel antimycobacterial phages in India and highlights their potential to contribute to our understanding of these phages and their gene products as potential antimicrobial agents.

Evaluation of the Efficiency of Lytic Mycobacteriophage D29 on the Model of M. tuberculosis-Infected Macrophage RAW 264 Cell Line.

Culture of mouse macrophages (RAW 264.7 ATCC strain) in wells of a 6-well plate was infected with M. tuberculosis in proportion of 15 mycobacteria per one macrophage and then treated with a lytic strain of mycobacteriophage D29. Antibacterial efficacy of mycobacteriophages was studied using D29 phage (activity 108 plaque-forming units/ml) previously purified by ion exchange chromatography. After single and double 24-h treatment, the lysed cultures of macrophages were inoculated onto Middlebrook 7H10 agar medium. The number of mycobacterial colonies in control and test wells (at least 3 wells in each group) was 300.178±12.500 and 36.0±5.4, respectively (p<0.01).

Dissecting the structure-function relationship in lysozyme domain of mycobacteriophage D29-encoded peptidoglycan hydrolase.

Most bacteriophages rapidly infect and kill bacteria and, therefore, qualify as the next generation therapeutics for rapidly emerging drug-resistant bacteria such as Mycobacterium tuberculosis. We have previously characterized the mycobacteriophage D29-generated endolysin, Lysin A, for its activity against mycobacteria. Here, we present a detailed characterization of the lysozyme domain (LD) of D29 Lysin A that hydrolyzes peptidoglycan of both gram-positive and gram-negative bacteria with high potency. By characterizing an exhaustive LD protein variant library, we have identified critical residues important for LD activity and stability. We further complement our in vitro experiments with detailed in silico investigations. We present LD as a potent candidate for developing phage-based broad-spectrum therapeutics.

Identification of unique essential proteins from a Mycobacterium tuberculosis F15/LAM4/KZN phage secretome library.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis disease (TB), the leading cause of death from bacterial infection worldwide. Although treatable, the resurgence of multidrug-resistant and extensively drug-resistant TB is a major setback for the fight against TB globally. Consequently, there is an urgent need for new Mtb-derived biomarkers for use in the design of new drugs and rapid point-of-care diagnostic or prognostic tools for the management of TB transmission. Therefore, the present study aimed to identify unique Mtb-secreted proteins from the extensively drug-resistant Mtb F15/LAM4/KZN phage secretome library. A whole genome library was constructed using genomic DNA fragments of the Mtb F15/LAM4/KZN strain. A phage secretome sub-library of 8 × 103 clones was prepared and phage DNA was sequenced from 120 randomly selected clones. DNA sequence BLAST analysis identified 86 open reading frames. Using bioinformatics tools and databases, 10 proteins essential for in vivo growth and survival of Mtb (Nrp, PssA, MmpL5, SirA, GatB, EspA, TopA, EccCa1, Rv1634 and Rv3103c) were identified. Proteins essential for the growth and survival of Mtb during infection have potential application in the development of diagnostic tools, new drugs and vaccines. Further studies will be conducted to evaluate their potential application in the fight against TB.

Prophage-mediated defence against viral attack and viral counter-defence.

Temperate phages are common, and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses that infect mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages revealed at least five distinct prophage-expressed viral defence systems that interfere with the infection of lytic and temperate phages that are either closely related (homotypic defence) or unrelated (heterotypic defence) to the prophage. Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defence systems include a single-subunit restriction system, a heterotypic exclusion system and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, which acts as a highly effective counter-defence system. Prophage-mediated viral defence offers an efficient mechanism for bacterial success in host-virus dynamics, and counter-defence promotes phage co-evolution.

Protecting from Envelope Stress: Variations on the Phage-Shock-Protein Theme.

During envelope stress, critical inner-membrane functions are preserved by the phage-shock-protein (Psp) system, a stress response that emerged from work with Escherichia coli and other Gram-negative bacteria. Reciprocal regulatory interactions and multiple effector functions are well documented in these organisms. Searches for the Psp system across phyla reveal conservation of only one protein, PspA. However, examination of Firmicutes and Actinobacteria reveals that PspA orthologs associate with non-orthologous regulatory and effector proteins retaining functions similar to those in Gram-negative counterparts. Conservation across phyla emphasizes the long-standing importance of the Psp system in prokaryotes, while inter- and intra-phyla variations within the system indicate adaptation to different cell envelope structures, bacterial lifestyles, and/or bacterial morphogenetic strategies.

Dual-Reporter Mycobacteriophages (Φ2DRMs) Reveal Preexisting Mycobacterium tuberculosis Persistent Cells in Human Sputum.

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. IMPORTANCE: 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.