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

Abstract:

:We previously reported the 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 PCR (M. C. Mulvey et al., mBio 3: 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 10(4) to <10(2) M. tuberculosis cells, and correctly identified the susceptibility profile of M. 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.

authors

Mulvey MC,Lemmon M,Rotter S,Lees J,Einck L,Nacy CA

doi

10.1128/AAC.03135-14

subject

Has Abstract

pub_date

2015-01-01 00:00:00

pages

407-13

issue

1

eissn

0066-4804

issn

1098-6596

pii

AAC.03135-14

journal_volume

59

pub_type

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