Chapter 10
Drug Susceptibility Screening Using In Vitro Models of
Hypoxic Non-Replicating Persistent Mycobacteria
Savannah E. R. Gibson, James Harrison, and Jonathan A. G. Cox
Abstract
Non-replicating persistence (NRP) is a functional adaptation that mycobacteria undergo in response to the
stresses of the granuloma, facilitating antibiotic tolerance and long-term infection. These stresses, or
NRP-inducing factors, include hypoxia, nutrient deprivation, and nitric oxide assault, which mycobacteria
are well evolved to tolerate through a series of metabolic and physiological adaptations producing the NRP
state. Most attempts to replicate these conditions in vitro have focused on only one of these factors at a time
for ease and simplicity, but as a result, do not necessarily produce physiologically relevant phenotypes. Here,
we provide the methods for two different in vitro NRP strategies that are useful for drug susceptibility
testing and high-throughput screening.
Key words Non-replicating persistence, Hypoxia, Nutrient deprivation, Cholesterol minimal media,
High-throughput screening, Latent tuberculosis infection, Drug discovery, Susceptibility testing
1 Introduction
Pathogenic mycobacteria can evade host immune systems and survive within the harsh environment of a granuloma in a metabolically
altered non-replicating persistent (NRP) state [1]. The conditions
within the granuloma that are responsible for this shift in physiology are largely the reduction in available oxygen and nutrients, as
well as an increase in nitric oxide, with each of these conditions
individually able to induce mycobacteria to enter the NRP state
[2, 3]. During NRP, Mycobacterium spp. enter a state of anaerobiosis, wherein they can survive under these hypoxic conditions and
cope with severe nutrient deprivation and nitric oxide assault. This
is made possible through a number of metabolic and physiological
changes including cessation of replication, regulation of their transcriptome to a minimal number of essential proteins, and limitation
of the metabolome to lipids, such as cholesterol scavenged from
macrophage cell membranes [4–9]. This persistent state is a concern in the case of Mycobacterium tuberculosis, in which it is
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
247
Drug Susceptibility Screening Using In Vitro Models of
Hypoxic Non-Replicating Persistent Mycobacteria
Savannah E. R. Gibson, James Harrison, and Jonathan A. G. Cox
Abstract
Non-replicating persistence (NRP) is a functional adaptation that mycobacteria undergo in response to the
stresses of the granuloma, facilitating antibiotic tolerance and long-term infection. These stresses, or
NRP-inducing factors, include hypoxia, nutrient deprivation, and nitric oxide assault, which mycobacteria
are well evolved to tolerate through a series of metabolic and physiological adaptations producing the NRP
state. Most attempts to replicate these conditions in vitro have focused on only one of these factors at a time
for ease and simplicity, but as a result, do not necessarily produce physiologically relevant phenotypes. Here,
we provide the methods for two different in vitro NRP strategies that are useful for drug susceptibility
testing and high-throughput screening.
Key words Non-replicating persistence, Hypoxia, Nutrient deprivation, Cholesterol minimal media,
High-throughput screening, Latent tuberculosis infection, Drug discovery, Susceptibility testing
1 Introduction
Pathogenic mycobacteria can evade host immune systems and survive within the harsh environment of a granuloma in a metabolically
altered non-replicating persistent (NRP) state [1]. The conditions
within the granuloma that are responsible for this shift in physiology are largely the reduction in available oxygen and nutrients, as
well as an increase in nitric oxide, with each of these conditions
individually able to induce mycobacteria to enter the NRP state
[2, 3]. During NRP, Mycobacterium spp. enter a state of anaerobiosis, wherein they can survive under these hypoxic conditions and
cope with severe nutrient deprivation and nitric oxide assault. This
is made possible through a number of metabolic and physiological
changes including cessation of replication, regulation of their transcriptome to a minimal number of essential proteins, and limitation
of the metabolome to lipids, such as cholesterol scavenged from
macrophage cell membranes [4–9]. This persistent state is a concern in the case of Mycobacterium tuberculosis, in which it is
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
247