Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria

Abstract Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determin...

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Autores principales:	David A. Barr, Charles Omollo, Mandy Mason, Anastasia Koch, Robert J. Wilkinson, David G. Lalloo, Graeme Meintjes, Valerie Mizrahi, Digby F. Warner, Gerry Davies
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/74eeee5f5254448cad1a301799e30ef2
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spelling	oai:doaj.org-article:74eeee5f5254448cad1a301799e30ef22021-12-02T18:14:08ZFlow cytometry method for absolute counting and single-cell phenotyping of mycobacteria10.1038/s41598-021-98176-52045-2322https://doaj.org/article/74eeee5f5254448cad1a301799e30ef22021-09-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-98176-5https://doaj.org/toc/2045-2322Abstract Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology—including the tendency to clump, and “differential” culturability—and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA + cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis bacillus Calmette-Guérin (BCG) time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique.David A. BarrCharles OmolloMandy MasonAnastasia KochRobert J. WilkinsonDavid G. LallooGraeme MeintjesValerie MizrahiDigby F. WarnerGerry DaviesNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-17 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q David A. Barr Charles Omollo Mandy Mason Anastasia Koch Robert J. Wilkinson David G. Lalloo Graeme Meintjes Valerie Mizrahi Digby F. Warner Gerry Davies Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria
description	Abstract Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology—including the tendency to clump, and “differential” culturability—and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA + cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis bacillus Calmette-Guérin (BCG) time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique.
format	article
author	David A. Barr Charles Omollo Mandy Mason Anastasia Koch Robert J. Wilkinson David G. Lalloo Graeme Meintjes Valerie Mizrahi Digby F. Warner Gerry Davies
author_facet	David A. Barr Charles Omollo Mandy Mason Anastasia Koch Robert J. Wilkinson David G. Lalloo Graeme Meintjes Valerie Mizrahi Digby F. Warner Gerry Davies
author_sort	David A. Barr
title	Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria
title_short	Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria
title_full	Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria
title_fullStr	Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria
title_full_unstemmed	Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria
title_sort	flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/74eeee5f5254448cad1a301799e30ef2
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Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria

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