59Stephen H. Gillespie (ed.), Antibiotic Resistance Protocols, Methods in Molecular Biology, vol. 1736,
https://doi.org/10.1007/978-1-4939-7638-6_6, © Springer Science+Business Media, LLC 2018
Chapter 6
Application of Continuous Culture for Assessing Antibiotic
Activity Against Mycobacterium tuberculosis
Charlotte L. Hendon-Dunn, Saba Anwar, Christopher Burton,
and Joanna Bacon
Abstract
There is a proportion of the M. tuberculosis population that is refractory to the bactericidal action of anti-
tuberculosis antibiotics due to phenotypic tolerance. This tolerance can be impacted by environmental
stimuli and the subsequent physiological state of the organism. It may be the result of preexisting popula-
tions of slow growing/non replicating bacteria that are protected from antibiotic action. It still remains
unclear how the slow growth of M. tuberculosis contributes to antibiotic resistance and antibiotic tolerance.
Here, we present a method for assessing the activity of antibiotics against M. tuberculosis using continuous
culture, which is the only system that can be used to control bacterial growth rate and study the impact of
slow or fast growth on the organism’s response to antibiotic exposure.
Key words Mycobacteria, Continuous culture, Chemostat, Antibiotic resistance1 Introduction
An important aim for improving TB treatment is to shorten the
period of antibiotic therapy without increasing relapse rates or
encouraging the development of antibiotic-resistant strains. In any
M. tuberculosis population there is a proportion of bacteria that are
antibiotic-tolerant; this might be because of preexisting popula-
tions of slow growing/nonreplicating bacteria that are protected
from antibiotic action due to the expression of a phenotype that
limits antibiotic activity [ 1 ]. The methods presented here describe
the use of continuous culture for assessing the activity of antibiot-
ics against Mycobacterium tuberculosis, with a particular focus on
the effect of either slow growth rates [69.3 h mean generation time
(MGT)] or fast growth rates (23.1 h MGT) on the response of the
organism to antibiotic exposure. Continuous culture is an ideal
system for growing mycobacteria under defined and controlled
conditions [ 2 – 5 ] and is the only growth system in which bacterial
growth rate can be controlled. During steady-state growth in