Nanoluciferase Reporter Mycobacteriophage for Sensitive and Rapid Detection of Mycobacterium tuberculosis Drug Susceptibility
| Autor: | Paras Jain, Deepshikha Verma, Ethan Spencer, Emmanuel Asare, Chelsea Peterson, Diane J. Ordway, Anne-Laure M. Le Ny, Gleda Hermansky, Spencer Garing, Michael Berney, Kyle J. Minch, Nicholas Clute-Reinig, Claire V. Mulholland, William R. Jacobs, Jacob Gadwa, Torin R. Weisbrod, Rajagopalan Saranathan, Kevin Paul Flood Nichols, Anna Astashkina Fernandez, Akos Somoskovi, Derek Bell |
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| Rok vydání: | 2020 |
| Předmět: |
bacteriophages
Tuberculosis drug susceptibility testing Mycobacteriophage medicine.medical_treatment Antitubercular Agents SQ109 Microbial Sensitivity Tests Drug resistance Biology Microbiology Mycobacterium tuberculosis 03 medical and health sciences chemistry.chemical_compound Drug Resistance Bacterial Tuberculosis Multidrug-Resistant phage medicine Humans drug screening Luciferases Tuberculosis Pulmonary Molecular Biology 030304 developmental biology 0303 health sciences 030306 microbiology Extensively drug-resistant tuberculosis Mycobacteriophages medicine.disease biology.organism_classification Virology chemistry Luminescent Measurements nanoluciferase Tuberculosis management Rifampin Bedaquiline Research Article |
| Zdroj: | Journal of Bacteriology |
| ISSN: | 1098-5530 0021-9193 |
| DOI: | 10.1128/jb.00411-20 |
| Popis: | Mycobacterium 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. 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. IMPORTANCE Mycobacterium 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. |
| Databáze: | OpenAIRE |
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