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...

Descripción completa

Guardado en:
Detalles Bibliográficos
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:
R
Q
Acceso en línea:https://doaj.org/article/74eeee5f5254448cad1a301799e30ef2
Etiquetas: Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
id oai:doaj.org-article:74eeee5f5254448cad1a301799e30ef2
record_format dspace
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
work_keys_str_mv AT davidabarr flowcytometrymethodforabsolutecountingandsinglecellphenotypingofmycobacteria
AT charlesomollo flowcytometrymethodforabsolutecountingandsinglecellphenotypingofmycobacteria
AT mandymason flowcytometrymethodforabsolutecountingandsinglecellphenotypingofmycobacteria
AT anastasiakoch flowcytometrymethodforabsolutecountingandsinglecellphenotypingofmycobacteria
AT robertjwilkinson flowcytometrymethodforabsolutecountingandsinglecellphenotypingofmycobacteria
AT davidglalloo flowcytometrymethodforabsolutecountingandsinglecellphenotypingofmycobacteria
AT graememeintjes flowcytometrymethodforabsolutecountingandsinglecellphenotypingofmycobacteria
AT valeriemizrahi flowcytometrymethodforabsolutecountingandsinglecellphenotypingofmycobacteria
AT digbyfwarner flowcytometrymethodforabsolutecountingandsinglecellphenotypingofmycobacteria
AT gerrydavies flowcytometrymethodforabsolutecountingandsinglecellphenotypingofmycobacteria
_version_ 1718378458906624000