Nanoluciferase reporter mycobacteriophage for sensitive and rapid detection of mycobacterium tuberculosis drug susceptibility

Paras Jain, Spencer Garing, Deepshikha Verma, Rajagopalan Saranathan, Nicholas Clute-Reinig, Jacob Gadwa, Chelsea Peterson, Gleda Hermansky, Anna Astashkina Fernandez, Emmanuel Asare, Torin R. Weisbrod, Ethan Spencer, Claire V. Mulholland, Michael Berney, David Bell, Kevin P. Nichols, Anne Laure M. Le Ny, Diane Ordway, William R. Jacobs, Akos SomoskoviKyle J. Minch

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

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 drugsensitive 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.

Original languageEnglish (US)
Article numbere00411-20
JournalJournal of Bacteriology
Volume202
Issue number22
DOIs
StatePublished - Nov 2020

Keywords

  • Bacteriophages
  • Drug screening
  • Drug susceptibility testing
  • Mycobacterium tuberculosis
  • Nanoluciferase
  • Phage

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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