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Elucidating population-wide mycobacterial replication dynamics at the single-cell level

Mycobacterium tuberculosis infections result in a spectrum of clinical outcomes, and frequently the infection persists in a latent, clinically asymptomatic state. The within-host bacterial population is likely to be heterogeneous, and it is thought that persistent mycobacteria arise from a small pop...

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Autores principales: Mouton, Jacoba M., Helaine, Sophie, Holden, David W., Sampson, Samantha L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Microbiology Society 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042079/
https://www.ncbi.nlm.nih.gov/pubmed/27027532
http://dx.doi.org/10.1099/mic.0.000288
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author Mouton, Jacoba M.
Helaine, Sophie
Holden, David W.
Sampson, Samantha L.
author_facet Mouton, Jacoba M.
Helaine, Sophie
Holden, David W.
Sampson, Samantha L.
author_sort Mouton, Jacoba M.
collection PubMed
description Mycobacterium tuberculosis infections result in a spectrum of clinical outcomes, and frequently the infection persists in a latent, clinically asymptomatic state. The within-host bacterial population is likely to be heterogeneous, and it is thought that persistent mycobacteria arise from a small population of viable, but non-replicating (VBNR) cells. These are likely to be antibiotic tolerant and necessitate prolonged treatment. Little is known about these persistent mycobacteria, since they are very difficult to isolate. To address this, we have successfully developed a replication reporter system for use in M. tuberculosis. This approach, termed fluorescence dilution, exploits two fluorescent reporters; a constitutive reporter allows the tracking of bacteria, while an inducible reporter enables the measurement of bacterial replication. The application of fluorescence single-cell analysis to characterize intracellular M. tuberculosis identified a distinct subpopulation of non-growing mycobacteria in murine macrophages. The presence of VBNR and actively replicating mycobacteria was observed within the same macrophage after 48 h of infection. Furthermore, our results suggest that macrophage uptake resulted in enrichment of non- or slowly replicating bacteria (as revealed by d-cycloserine treatment); this population is likely to be highly enriched for persisters, based on its drug-tolerant phenotype. These results demonstrate the successful application of the novel dual fluorescence reporter system both in vitro and in macrophage infection models to provide a window into mycobacterial population heterogeneity.
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spelling pubmed-50420792017-03-27 Elucidating population-wide mycobacterial replication dynamics at the single-cell level Mouton, Jacoba M. Helaine, Sophie Holden, David W. Sampson, Samantha L. Microbiology (Reading) Standard Mycobacterium tuberculosis infections result in a spectrum of clinical outcomes, and frequently the infection persists in a latent, clinically asymptomatic state. The within-host bacterial population is likely to be heterogeneous, and it is thought that persistent mycobacteria arise from a small population of viable, but non-replicating (VBNR) cells. These are likely to be antibiotic tolerant and necessitate prolonged treatment. Little is known about these persistent mycobacteria, since they are very difficult to isolate. To address this, we have successfully developed a replication reporter system for use in M. tuberculosis. This approach, termed fluorescence dilution, exploits two fluorescent reporters; a constitutive reporter allows the tracking of bacteria, while an inducible reporter enables the measurement of bacterial replication. The application of fluorescence single-cell analysis to characterize intracellular M. tuberculosis identified a distinct subpopulation of non-growing mycobacteria in murine macrophages. The presence of VBNR and actively replicating mycobacteria was observed within the same macrophage after 48 h of infection. Furthermore, our results suggest that macrophage uptake resulted in enrichment of non- or slowly replicating bacteria (as revealed by d-cycloserine treatment); this population is likely to be highly enriched for persisters, based on its drug-tolerant phenotype. These results demonstrate the successful application of the novel dual fluorescence reporter system both in vitro and in macrophage infection models to provide a window into mycobacterial population heterogeneity. Microbiology Society 2016-06 /pmc/articles/PMC5042079/ /pubmed/27027532 http://dx.doi.org/10.1099/mic.0.000288 Text en © 2016 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
spellingShingle Standard
Mouton, Jacoba M.
Helaine, Sophie
Holden, David W.
Sampson, Samantha L.
Elucidating population-wide mycobacterial replication dynamics at the single-cell level
title Elucidating population-wide mycobacterial replication dynamics at the single-cell level
title_full Elucidating population-wide mycobacterial replication dynamics at the single-cell level
title_fullStr Elucidating population-wide mycobacterial replication dynamics at the single-cell level
title_full_unstemmed Elucidating population-wide mycobacterial replication dynamics at the single-cell level
title_short Elucidating population-wide mycobacterial replication dynamics at the single-cell level
title_sort elucidating population-wide mycobacterial replication dynamics at the single-cell level
topic Standard
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042079/
https://www.ncbi.nlm.nih.gov/pubmed/27027532
http://dx.doi.org/10.1099/mic.0.000288
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