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

Abstract

bWe 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. Multidrug-resistant tuberculosis (MDR-TB) is caused by Mycobacterium tuberculosis strains resistant to at least two of the drugs, isoniazid (INH) and rifampin (RIF), in the recommended four-drug TB treatment regimen (1). The World Health Organization (WHO) estimates the prevalence of MDR-TB to be approximately 50 million people worldwide (2), expanding by nearly 500,000 new cases each year (3). A more disquieting development is the increase in and global distribution of extremely drug-resistant TB (XDR-TB) (M. tuberculosis resistant to INH, RIF, and key second-line drugs) (4). Early recognition of M/XDR-TB through the use of an accurate and sensitive antibiotic susceptibility test (AST) promises to improve patient outcomes and assist in TB control efforts.Due to the notoriously slow doubling time of M. tuberculosis, a standard AST using culture takes several weeks to months. Culture-based assays are the gold standard for AST, however, because they measure every type of drug susceptibility and do not require knowledge of the genetics underlying resistance. However, they are slow or too insensitive to interrogate effects of antibiotics on small numbers of M. tuberculosis present in most clinical samples. Rapid molecular diagnostic tests such as Xpert MTB/RIF (Cepheid) and GenoType MTBDR (Hain Lifescience) are the emerging standard for rapid INH-or RIF-resistant M. tuberculosis detection, markers of MDR-TB, because their detection thresholds from patient specimens are comparable to culture (5, 6). These tests identify M. tuberculosis DNA in a patient sample and detect common mutations that confer resistance to INH (GenoType MTBDR) and/or RIF (GenoType MTBDR and Xpert MTB/RIF) in 2 h (Xpert MTB/RIF) or 2 days (GenoType MTBDR) from the initiation of t...

Phage-Mediated Molecular Detection (PMMD): A Novel Rapid Method for Phage-Specific Bacterial Detection

“…Therefore, upon infection, relatively long incubation times are need it. This was cleverly resolved by Mulvey and collaborators by focusing on the detection of RNA molecules synthetized by a highly-transcribed phage-borne artificial cassette instead of detecting phage DNA [10,16]. Due to the fact that the number of phage RNA molecules produced per infected cell generally far exceeds the number of new phage DNA genomes, we explored the possibility of developing phage RNA detection as a reliable and rapid diagnostic tool.…”

“…Moreover, detection methods based on the reverse transcription of highly abundant RNAs show better LOD and are less prone to false positives than DNA detection assays [15]. Interestingly, Mulvey et al [10,16] reported the use of TM4 genetically engineered phages for the detection and antibiotic susceptibility determination of Mycobacterium tuberculosis. In this assay, the phage delivers heterologous cassette(s), "surrogate marker loci" (SML), designed to produce high RNA levels.…”

A Rapid Molecular Test for Determining Yersinia pestis Susceptibility to Ciprofloxacin by the Quantification of Differentially Expressed Marker Genes

“…Various alternative AST methods have been described in recent years, including nucleic acid-based ASTs. These nucleic acid-based ASTs are performed by determining either the bacterial chromosomal DNA copy number using qPCR or phage-specific mRNA transcript copy number using qRT-PCR (Waldeisen et al, 2011 ; Mulvey et al, 2015 ). However, these assays are still growth dependent and time consuming.…”

Application of Bacteriophages for Mycobacterial Infections, from Diagnosis to Treatment

“…Several assays used enzymes to catalyze the generation of their respective biomarkers. Using this method, two endpoints have been explored: the detection of changes in electric current [28,29] and the detection of unique nucleic acid sequences [30,31]. One approach used reporter phages to introduce the enzyme [31], whereas another used an enzyme already present in mycobacteria [30].…”