Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>

ABSTRACT 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 ot...

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Autores principales:	Matthew C. Mulvey, Katherine A. Sacksteder, Leo Einck, Carol A. Nacy
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2012
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/dfb864677f93400f8522ffe52cbab75b
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spelling	oai:doaj.org-article:dfb864677f93400f8522ffe52cbab75b2021-11-15T15:39:08ZGeneration of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>10.1128/mBio.00312-112150-7511https://doaj.org/article/dfb864677f93400f8522ffe52cbab75b2012-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00312-11https://doaj.org/toc/2150-7511ABSTRACT 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. IMPORTANCE The ability to detect antibiotic resistance of slow-growing bacteria (i.e., Mycobacterium tuberculosis) is hampered by two factors, the time to detection (weeks to months) and the resistance mechanism (unknown for many drugs), delaying the appropriate treatment of patients with drug-resistant or multidrug-resistant tuberculosis (TB). The novel technique described in this article uses a unique surrogate nucleic acid marker produced by phage that infects M. tuberculosis to record phenotypic antibiotic susceptibility in less than a day.Matthew C. MulveyKatherine A. SackstederLeo EinckCarol A. NacyAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 3, Iss 2 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Matthew C. Mulvey Katherine A. Sacksteder Leo Einck Carol A. Nacy Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
description	ABSTRACT 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. IMPORTANCE The ability to detect antibiotic resistance of slow-growing bacteria (i.e., Mycobacterium tuberculosis) is hampered by two factors, the time to detection (weeks to months) and the resistance mechanism (unknown for many drugs), delaying the appropriate treatment of patients with drug-resistant or multidrug-resistant tuberculosis (TB). The novel technique described in this article uses a unique surrogate nucleic acid marker produced by phage that infects M. tuberculosis to record phenotypic antibiotic susceptibility in less than a day.
format	article
author	Matthew C. Mulvey Katherine A. Sacksteder Leo Einck Carol A. Nacy
author_facet	Matthew C. Mulvey Katherine A. Sacksteder Leo Einck Carol A. Nacy
author_sort	Matthew C. Mulvey
title	Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_short	Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_full	Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_fullStr	Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_full_unstemmed	Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_sort	generation of a novel nucleic acid-based reporter system to detect phenotypic susceptibility to antibiotics in <named-content content-type="genus-species">mycobacterium tuberculosis</named-content>
publisher	American Society for Microbiology
publishDate	2012
url	https://doaj.org/article/dfb864677f93400f8522ffe52cbab75b
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Generation of a Novel Nucleic Acid-Based Reporter System To Detect Phenotypic Susceptibility to Antibiotics in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>

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