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. 2025 Apr 15;231(4):859-870.
doi: 10.1093/infdis/jiae438.

Engineered Mycobacteriophage TM4::GeNL Rapidly Determines Bedaquiline, Pretomanid, Linezolid, Rifampicin, and Clofazimine Sensitivity in Mycobacterium tuberculosis Clinical Isolates

Saranathan Rajagopalan  1 , Amy K Rourke  2 , Emmanuel Asare  1 , Donna J Kohlerschmidt  2 , Lahari Das  1 , Senamile L Ngema  3 , Claire V Mulholland  1 , Catherine Vilchèze  1 , Vaishnavi Mahalingam  1 , Sashen Moodley  4 , Barry Truebody  4 , Jared Mackenzie  4 , Adrie J C Steyn  4   5 , Rubeshan Perumal  3   6 , Michael Berney  1 , Michelle H Larsen  1 , Max R O'Donnell  7   8 , Vincent E Escuyer  2 , William R Jacobs Jr  1
Affiliations

Engineered Mycobacteriophage TM4::GeNL Rapidly Determines Bedaquiline, Pretomanid, Linezolid, Rifampicin, and Clofazimine Sensitivity in Mycobacterium tuberculosis Clinical Isolates

Saranathan Rajagopalan et al. J Infect Dis. .

Abstract

Background: Drug-resistant tuberculosis is a growing public health threat, and early characterization of the resistance phenotype is essential for guiding treatment and mitigating the high mortality associated with the disease. However, the slow growth rate of Mycobacterium tuberculosis, the causative agent of tuberculosis, necessitates several weeks for conventional culture-dependent drug susceptibility testing (DST). In addition, there are no widely available molecular diagnostic assays for evaluating resistance to newer tuberculosis drugs or drugs with complex resistance mechanisms.

Methods: We have developed a luciferase-based reporter mycobacteriophage assay that can determine drug resistance within 48 hours. We engineered the TM4 mycobacteriophage to express green enhanced nanoluciferase (GeNL) cassette and optimized DST for bedaquiline, pretomanid, linezolid, clofazimine, and rifampicin using clinical M. tuberculosis isolates.

Results: To assess the feasibility of this assay, we conducted a proof-of-principle study using 53 clinical M. tuberculosis isolates. TM4::GeNL phage DST effectively distinguished between sensitive and resistant isolates for bedaquiline and rifampicin at a concentration of 0.125 μg/mL. Optimal differentiation between sensitive and resistant isolates for pretomanid, clofazimine, and linezolid was achieved at concentrations of 0.5 μg/mL, 0.25 μg/mL, and 1 μg/mL, respectively. Additionally, TM4::GeNL DST identified low-level rifampicin resistance in clinical isolates even though they were classified as sensitive by Mycobacteria Growth Indicator Tube DST.

Conclusions: TM4::GeNL reporter phage DST offers a rapid method to identify M. tuberculosis drug resistance, including resistance to newer tuberculosis drugs.

Keywords: Mycobacterium tuberculosis; bedaquiline; drug susceptibility testing; mycobacteriophage; rifampicin.

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Conflict of interest statement

Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

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