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We previously reported the development of a prototype antibiotic sensitivity assay to detect drug-resistantMycobacterium tuberculosis using infection by mycobacteriophage to create a novel nucleic acid transcript, a surrogate marker of mycobacterial viability, detected by reverse transcriptase PCR (M. C. Mulvey et al., mBio3: e00312-11, 2012). This assay detects antibiotic resistance to all drugs, even drugs for which the resistance mechanism is unknown or complex: it is a phenotypic readout using nucleic acid detection. In this report, we describe development and characteristics of an optimized reporter system that directed expression of the RNA cyclase ribozyme, which generated circular RNA through an intramolecular splicing reaction and led to accumulation of a new nucleic acid sequence in phage-infected bacteria. These modifications simplified the assay, increased the limit of detection from 104 to &lt;102 M. tuberculosis cells, and correctly identified the susceptibility profile ofM. tuberculosis strains exposed for 16 h to either first-line or second-line antitubercular drugs. In addition to phenotypic drug resistance or susceptibility, the assay reported streptomycin MICs and clearly detected 10% drug-resistant cells in an otherwise drug-susceptible population.

Optimization of a Nucleic Acid-Based Reporter System To Detect Mycobacterium tuberculosis Antibiotic Sensitivity