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Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence

OBJECTIVES: Tuberculosis drug development is hampered by the slow growth of Mycobacterium tuberculosis. Bioluminescence, light produced by an enzymatic reaction, constitutes a rapid and highly sensitive measurement of cell metabolic function that can be used as an indirect marker of cell viability i...

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Autores principales: Andreu, Nuria, Fletcher, Taryn, Krishnan, Nitya, Wiles, Siouxsie, Robertson, Brian D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3254196/
https://www.ncbi.nlm.nih.gov/pubmed/22101217
http://dx.doi.org/10.1093/jac/dkr472
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author Andreu, Nuria
Fletcher, Taryn
Krishnan, Nitya
Wiles, Siouxsie
Robertson, Brian D.
author_facet Andreu, Nuria
Fletcher, Taryn
Krishnan, Nitya
Wiles, Siouxsie
Robertson, Brian D.
author_sort Andreu, Nuria
collection PubMed
description OBJECTIVES: Tuberculosis drug development is hampered by the slow growth of Mycobacterium tuberculosis. Bioluminescence, light produced by an enzymatic reaction, constitutes a rapid and highly sensitive measurement of cell metabolic function that can be used as an indirect marker of cell viability in drug screening assays. The aim of this work was to validate and standardize the use of luminescent M. tuberculosis strains to test the activity of antibacterial drugs in vitro and inside macrophages in a 96-well format. METHODS: We have used strains that express the bacterial lux operon and therefore do not require exogenous substrate to produce light, as well as strains expressing the firefly luciferase that need luciferin substrate. Results were compared with those obtained using the resazurin reduction assay and cfu plating. RESULTS: Using bioluminescence we were able to reduce the time required to measure the MIC and bactericidal concentrations of antimicrobials to just 3 and 6 days, respectively. Furthermore, antibacterial activity against intracellular mycobacteria was detected within 2 days post-infection. Results were comparable to those obtained by conventional methods. CONCLUSIONS: We have developed a simple and rapid method for screening antimycobacterial drugs in culture and in macrophages. The use of autoluminescent bacteria also facilitates the determination of growth and inhibition kinetics. The method is cost-effective, can easily be adapted to a larger scale and is amenable to automation. Current efforts are directed towards applying this technology to drug screening in vivo.
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spelling pubmed-32541962012-01-10 Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence Andreu, Nuria Fletcher, Taryn Krishnan, Nitya Wiles, Siouxsie Robertson, Brian D. J Antimicrob Chemother Original Research OBJECTIVES: Tuberculosis drug development is hampered by the slow growth of Mycobacterium tuberculosis. Bioluminescence, light produced by an enzymatic reaction, constitutes a rapid and highly sensitive measurement of cell metabolic function that can be used as an indirect marker of cell viability in drug screening assays. The aim of this work was to validate and standardize the use of luminescent M. tuberculosis strains to test the activity of antibacterial drugs in vitro and inside macrophages in a 96-well format. METHODS: We have used strains that express the bacterial lux operon and therefore do not require exogenous substrate to produce light, as well as strains expressing the firefly luciferase that need luciferin substrate. Results were compared with those obtained using the resazurin reduction assay and cfu plating. RESULTS: Using bioluminescence we were able to reduce the time required to measure the MIC and bactericidal concentrations of antimicrobials to just 3 and 6 days, respectively. Furthermore, antibacterial activity against intracellular mycobacteria was detected within 2 days post-infection. Results were comparable to those obtained by conventional methods. CONCLUSIONS: We have developed a simple and rapid method for screening antimycobacterial drugs in culture and in macrophages. The use of autoluminescent bacteria also facilitates the determination of growth and inhibition kinetics. The method is cost-effective, can easily be adapted to a larger scale and is amenable to automation. Current efforts are directed towards applying this technology to drug screening in vivo. Oxford University Press 2012-02 2011-11-17 /pmc/articles/PMC3254196/ /pubmed/22101217 http://dx.doi.org/10.1093/jac/dkr472 Text en © The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. http://creativecommons.org/licenses/by-nc/2.5/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons. org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited
spellingShingle Original Research
Andreu, Nuria
Fletcher, Taryn
Krishnan, Nitya
Wiles, Siouxsie
Robertson, Brian D.
Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence
title Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence
title_full Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence
title_fullStr Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence
title_full_unstemmed Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence
title_short Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence
title_sort rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence
topic Original Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3254196/
https://www.ncbi.nlm.nih.gov/pubmed/22101217
http://dx.doi.org/10.1093/jac/dkr472
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