51Stephen H. Gillespie (ed.), Antibiotic Resistance Protocols, Methods in Molecular Biology, vol. 1736,
https://doi.org/10.1007/978-1-4939-7638-6_5, © Springer Science+Business Media, LLC 2018
Chapter 5
A Flow Cytometry Method for Assessing M. tuberculosis
Responses to Antibiotics
Charlotte L. Hendon-Dunn, Stephen R. Thomas, Stephen C. Taylor,
and Joanna Bacon
Abstract
Traditional drug susceptibility methods can take several days or weeks of incubation between drug exposure
and confirmation of sensitivity or resistance. In addition, these methods do not capture information about
viable organisms that are not immediately culturable after drug exposure. Here, we present a rapid fluores-
cence detection method for assessing the susceptibility of M. tuberculosis to antibiotics. Fluorescent markers
Calcein violet-AM and SYTOX-green are used for measuring cell viability or cell death and to capture infor-
mation about the susceptibility of the whole population and not just those bacteria that can grow in media
postexposure. Postexposure to the antibiotic, the method gives a rapid readout of drug susceptibility, as well
as insights into the concentration and time-dependent drug activity following antibiotic exposure.
Key words Mycobacterium tuberculosis, Antibiotic susceptibility, Calcein violet-AM, SYTOX-green,
Flow cytometry1 Introduction
Current methods for assessing the antibiotic susceptibility of
Mycobacterium tuberculosis are lengthy and do not capture informa-
tion about viable organisms that are not immediately culturable
under standard in vitro conditions; as a result of antibiotic exposure
[ 1 ]. We have developed a rapid dual- fluorescence flow cytometry
method using markers for cell viability and death. The fluorescent
markers we use are Calcein violet-AM (CV-AM; ex/em
400/452 nm) and SYTOX-green (SG; (ex/em 488 nm/523 nm).
CV-AM is a dye used for distinguishing live cells through the action
of intracellular esterase activity, which converts the virtually non-
fluorescent cell-permeant CV-AM to the intensely fluorescent
membrane impermeable Calcein violet (CV), which can be easily
excited with the violet laser (and detected in channel FL6), allowing
other laser lines to be used for conventional fluorochromes. SG,
used for measuring cell death, permeates through damaged bacteria