Imperial College London
Professor Robert Wilkinson

ProfessorRobertWilkinson

Faculty of Medicine, Department of Infectious Disease

Professor in Infectious Diseases
 
 
 
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Contact

 

r.j.wilkinson Website

 
 
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Location

 

Commonwealth BuildingHammersmith Campus

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Summary

 

Publications

Citation

BibTex format

@unpublished{Barr:2021:10.1101/2021.05.01.442251,
author = {Barr, DA and Omollo, CO and Mason, M and Koch, A and Wilkinson, R and Lalloo, D and Meintjes, G and Mizrahi, V and Warner, DF and Davies, G},
doi = {10.1101/2021.05.01.442251},
publisher = {Cold Spring Harbor Laboratory},
title = {Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria},
url = {http://dx.doi.org/10.1101/2021.05.01.442251},
year = {2021}
}
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RIS format (EndNote, RefMan)

TY  - UNPB
AB  - 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.
AU  - Barr,DA
AU  - Omollo,CO
AU  - Mason,M
AU  - Koch,A
AU  - Wilkinson,R
AU  - Lalloo,D
AU  - Meintjes,G
AU  - Mizrahi,V
AU  - Warner,DF
AU  - Davies,G
DO  - 10.1101/2021.05.01.442251
PB  - Cold Spring Harbor Laboratory
PY  - 2021///
TI  - Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria
UR  - http://dx.doi.org/10.1101/2021.05.01.442251
UR  - https://www.biorxiv.org/content/10.1101/2021.05.01.442251v2
UR  - http://hdl.handle.net/10044/1/91366
ER  -
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