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

Barr, David, Omollo, Charles, Mason, Mandy, Koch, Anastasia, Wilkinson, Robert J, Lalloo, David ORCID:, Meintjes, Graeme, Mizrhai, Valerie, Warner, Digby F and Davies, Gerry (2021) 'Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria'. Scientific Reports, Vol 11, p. 18661.

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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.

Item Type: Article
Subjects: QW Microbiology and Immunology > Bacteria > QW 125 Actinibacteria, Actinomycetales.
WF Respiratory System > Tuberculosis > WF 200 Tuberculosis (General)
Faculty: Department: Clinical Sciences & International Health > Clinical Sciences Department
Digital Object Identifer (DOI):
Depositing User: Debbie Jenkins
Date Deposited: 15 Oct 2021 09:02
Last Modified: 15 Oct 2021 09:02


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