Optimization of a nucleic acid-based reporter system to detect Mycobacterium tuberculosis antibiotic sensitivity.

We previously reported development of a prototype antibiotic sensitivity assay to detect drug-resistant Mycobacterium tuberculosis using infection by mycobacteriophage to create a novel nucleic acid transcript, a surrogate marker of mycobacterial viability, detected by reverse transcriptase-polymerase chain reaction (RT-PCR) (1). 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 intra-molecular 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 <102 Mycobacterium tuberculosis cells, and correctly identified the susceptibility profile of Mycobacterium tuberculosis strains exposed for 16 hr to either first-line or second-line anti-tubercular drugs. In addition to phenotypic drug resistance or susceptibility, the assay reported streptomycin minimal inhibitory concentrations and clearly detected 10% drug-resistant cells in an otherwise drug-susceptible population.