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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2012-05-01
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| Series: | mBio |
| Online Access: | https://journals.asm.org/doi/10.1128/mBio.00312-11 |
| _version_ | 1818936004962156544 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-20T05:29:11Z |
| format | Article |
| id | doaj.art-dfb864677f93400f8522ffe52cbab75b |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-20T05:29:11Z |
| publishDate | 2012-05-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-dfb864677f93400f8522ffe52cbab75b2022-12-21T19:51:48ZengAmerican Society for MicrobiologymBio2150-75112012-05-013210.1128/mBio.00312-11Generation 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>Matthew C. Mulvey0Katherine A. Sacksteder1Leo Einck2Carol A. Nacy3Sequella, Inc., Rockville, Maryland, USASequella, Inc., Rockville, Maryland, USASequella, Inc., Rockville, Maryland, USASequella, Inc., Rockville, Maryland, USAABSTRACT 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.https://journals.asm.org/doi/10.1128/mBio.00312-11 |
| spellingShingle | 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> mBio |
| 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_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_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_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 |
| url | https://journals.asm.org/doi/10.1128/mBio.00312-11 |
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