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



Barr, David ORCID: 0000-0002-2922-9381, Omollo, Charles, Mason, Mandy, Koch, Anastasia, Wilkinson, Robert ORCID: 0000-0002-2753-1800, Lalloo, David ORCID: 0000-0001-7680-2200, Meintjes, Graeme, Mizrahi, Valerie ORCID: 0000-0003-4824-9115, Warner, Digby ORCID: 0000-0002-4146-0930 and Davies, Gerry ORCID: 0000-0002-3819-490X
(2021) Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria. Unknown. 2021.05.01.442251-.

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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 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
Uncontrolled Keywords: Rare Diseases, HIV/AIDS, Vaccine Related, Biotechnology, Tuberculosis, Infectious Diseases, Infection, 3 Good Health and Well Being
Divisions: Faculty of Health and Life Sciences
Faculty of Health and Life Sciences > Institute of Infection, Veterinary and Ecological Sciences
Depositing User: Symplectic Admin
Date Deposited: 13 May 2021 10:52
Last Modified: 15 Mar 2024 00:26
DOI: 10.1101/2021.05.01.442251
Open Access URL: https://www.biorxiv.org/content/10.1101/2021.05.01...
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URI: https://livrepository.liverpool.ac.uk/id/eprint/3122564