Rapid phenotypic detection of complex and emergent TB drug-resistance using a next-generation nanoluciferase reporter phage

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Project Summary Mycobacterium tuberculosis (M.tuberculosis) is the causative agent of tuberculosis (TB); although a treatable disease TB remains a leading cause of morbidity and mortality globally. This in large is driven by the unparalleled increase in anti- mycobacterial drug resistance. Approximately 10 million people fell ill with TB in 2019, where 500 000 were new rifampicin resistant cases and 78% of rifampicin cases were multi-drug resistant; compromising the most effective first-line drugs. In line with recent WHO guidance, the majority of these patients are treated with all-oral short-course treatment incorporating bedaquiline and other novel agents. Emerging drug resistance to these novel drugs is a critical threat to expansion of shortened, all-oral, DR-TB treatment regimens. Current diagnostic assays do not incorporate resistance detection to these new drugs, largely as a result of our incomplete understanding of the underlying genetic mechanisms mediating resistance. As a result, a combination of genotypic and phenotypic techniques is required to monitor resistance to these new drugs. Reporter mycobacteriophage assays represent a promising approach for deriving phenotypic data in a replication-independent system, maintaining high sensitivity while reducing the time to result attendant with growth-based methods. We created a TM4 phage vector that delivers a gene cassette of the nanoluciferase (Nluc) reporter enzyme. The TM4-nluc phage was tested on a range of auxotrophic and virulent clinical M. tuberculosis strains, assessing cellular limit of detection and compatibility with drug susceptibility testing on a wide range of antimycobacterial drugs. We found that following a preculture period, we could identify drug susceptibility consistent with WHO-endorsed treatment concentrations to a suite of first-line and second-line drugs as well as novel and repurposed drugs (bedaquiline, pretomanid, and linezolid). The overall objective of this research is to validate the nanoluciferase phage for detection of resistance to novel anti-mycobaterial agents, will be accomplished in two Aims. Aim 1 will establish the utility of the assay in paucibacillary conditions and mixed infection. Aim2 will derive correlations between established minimum inhibitory values around critical concentrations and nanoluciferase fluorescence intensity measures, and validate these cut points in a established prospective cohort study of MDR-TB treatment. An exploratory sub Aim will trial minimally processed sputum rather than MTB culture for MTB detection and drug susceptibility testing. The proposed work will provide additional validation on the TM4-nluc phage system and offer a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.