Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in Mycobacterium tuberculosis

We designed, constructed, and evaluated a prototype novel reporter system comprised of two functional cassettes: (i) the SP6 RNA polymerase gene under transcriptional control of a promoter active in mycobacteria and (ii) the consensus SP6 polymerase promoter that directs expression of an otherwise unexpressed sequence. We incorporated the reporter system into a mycobacteriophage for delivery into viable Mycobacterium tuberculosis, and introduction led to synthesis of an SP6 polymerase-dependent surrogate marker RNA that we detected by reverse transcriptase PCR (RT-PCR). The reporter confirmed the susceptibility profile of both drug-susceptible and drug-resistant M. tuberculosis strains exposed to first-line antitubercular drugs and required as little as 16 h of exposure to antibacterial agents targeting bacterial metabolic processes to accurately read the reaction. The reporter system translated the bacterial phenotype into a language interpretable by rapid and sensitive nucleic acid detection. As a phenotypic assay that works only on viable M. tuberculosis, it could be used to rapidly assess resistance to any drug, including drugs for which the mechanism of resistance is unknown or which result from many potential known (and unknown) genetic alterations