TY - JOUR
T1 - Nanoluciferase reporter mycobacteriophage for sensitive and rapid detection of mycobacterium tuberculosis drug susceptibility
AU - Jain, Paras
AU - Garing, Spencer
AU - Verma, Deepshikha
AU - Saranathan, Rajagopalan
AU - Clute-Reinig, Nicholas
AU - Gadwa, Jacob
AU - Peterson, Chelsea
AU - Hermansky, Gleda
AU - Fernandez, Anna Astashkina
AU - Asare, Emmanuel
AU - Weisbrod, Torin R.
AU - Spencer, Ethan
AU - Mulholland, Claire V.
AU - Berney, Michael
AU - Bell, David
AU - Nichols, Kevin P.
AU - Le Ny, Anne Laure M.
AU - Ordway, Diane
AU - Jacobs, William R.
AU - Somoskovi, Akos
AU - Minch, Kyle J.
N1 - Publisher Copyright:
© 2020 Jain et al.
PY - 2020/11
Y1 - 2020/11
N2 - 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.
AB - 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.
KW - Bacteriophages
KW - Drug screening
KW - Drug susceptibility testing
KW - Mycobacterium tuberculosis
KW - Nanoluciferase
KW - Phage
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U2 - 10.1128/JB.00411-20
DO - 10.1128/JB.00411-20
M3 - Article
C2 - 32900827
AN - SCOPUS:85094219463
SN - 0021-9193
VL - 202
JO - Journal of Bacteriology
JF - Journal of Bacteriology
IS - 22
M1 - e00411-20
ER -