estimated that almost 25% of the global population is infected with
“latent” tuberculosis disease, which is highly drug-resistant and
could rapidly progress to an active infection, with the bacteria
able to resuscitate from their NRP state [10, 11].
It is therefore becoming a priority to be able to accurately
model NRP conditions in vitro, for a variety of analyses, from
monitoring transcriptomic changes through to drug screening.
Due to the incredibly long-lived nature of NRP, individuals could
be latently infected for their lifetime and at risk of reactivation to
active Tuberculosis. Therefore, any assay used to represent this state
should reflect this longevity and the model of NRP used should
maintain cells as non-growing but viable, with the ability to resuscitate. Traditionally, the model used to study NRP utilizes hypoxic
conditions to induce the associated metabolic and physiological
changes. The Wayne model employs a slow reduction in oxygen
saturation to drive entry into the first stage of NRP, known as NRP
stage I [12]. This stage is defined by a cessation of replication and
DNA synthesis, but with unaltered intracellular ATP levels and
some active DNA repair mechanisms [1]. The oxygen levels at
this stage are microaerophilic (2–10% O 2 ); however, with the slow
reduction of oxygen due to respiration of mycobacterial cells, the
environment soon becomes anaerobic (>0.06% O 2 ) and the bacteria enter NRP stage II [4, 12]. It is necessary to drive this transition
slowly through NRP stage I and into NRP stage II, the phenotype
most commonly investigated in terms of NRP, since Mycobacterium
spp. cannot survive direct entry into NRP stage II [4, 13].
Entry into NRP creates a differing drug susceptibility phenotype than that of aerobic, actively growing mycobacteria. Notably,
the frontline antituberculosis drug isoniazid has no effect on
M. tuberculosis cells that are NRP within the Wayne model, whereas
they gain susceptibility to metronidazole, a classical anaerobic drug
[4, 12]. This is in stark contrast to the drug phenotype of aerobic,
replicating M. tuberculosis, which shows the opposite profile of
isoniazid susceptibility and metronidazole resistance [14]. It is
highly concerning that this NRP-associated loss of susceptibility is
also seen for other frontline antituberculosis drugs [15]. Interestingly, if nutrient deprivation is the NRP-inducing factor, rather
than hypoxia, this drug susceptibility phenotype is different once
again; there is an overarching resistance to all frontline
M. tuberculosis drugs and susceptibility to metronidazole is lost
[5]. Therefore, there is an impetus to discover new drugs that
have conserved activity against these different NRP phenotypes to
enable us to better treat latent tuberculosis infection and eradicate
the vast reservoirs of disease present within the population.
In this chapter, we describe methods for inducing hypoxia and
drug susceptibility testing against Mycobacterium spp. using either
rich media (Wayne model) or minimal cholesterol media (utilizing
both hypoxia and nutrient deprivation). We also describe methods
of preparing NRP cultures for further investigation.
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Savannah E. R. Gibson et al.
“latent” tuberculosis disease, which is highly drug-resistant and
could rapidly progress to an active infection, with the bacteria
able to resuscitate from their NRP state [10, 11].
It is therefore becoming a priority to be able to accurately
model NRP conditions in vitro, for a variety of analyses, from
monitoring transcriptomic changes through to drug screening.
Due to the incredibly long-lived nature of NRP, individuals could
be latently infected for their lifetime and at risk of reactivation to
active Tuberculosis. Therefore, any assay used to represent this state
should reflect this longevity and the model of NRP used should
maintain cells as non-growing but viable, with the ability to resuscitate. Traditionally, the model used to study NRP utilizes hypoxic
conditions to induce the associated metabolic and physiological
changes. The Wayne model employs a slow reduction in oxygen
saturation to drive entry into the first stage of NRP, known as NRP
stage I [12]. This stage is defined by a cessation of replication and
DNA synthesis, but with unaltered intracellular ATP levels and
some active DNA repair mechanisms [1]. The oxygen levels at
this stage are microaerophilic (2–10% O 2 ); however, with the slow
reduction of oxygen due to respiration of mycobacterial cells, the
environment soon becomes anaerobic (>0.06% O 2 ) and the bacteria enter NRP stage II [4, 12]. It is necessary to drive this transition
slowly through NRP stage I and into NRP stage II, the phenotype
most commonly investigated in terms of NRP, since Mycobacterium
spp. cannot survive direct entry into NRP stage II [4, 13].
Entry into NRP creates a differing drug susceptibility phenotype than that of aerobic, actively growing mycobacteria. Notably,
the frontline antituberculosis drug isoniazid has no effect on
M. tuberculosis cells that are NRP within the Wayne model, whereas
they gain susceptibility to metronidazole, a classical anaerobic drug
[4, 12]. This is in stark contrast to the drug phenotype of aerobic,
replicating M. tuberculosis, which shows the opposite profile of
isoniazid susceptibility and metronidazole resistance [14]. It is
highly concerning that this NRP-associated loss of susceptibility is
also seen for other frontline antituberculosis drugs [15]. Interestingly, if nutrient deprivation is the NRP-inducing factor, rather
than hypoxia, this drug susceptibility phenotype is different once
again; there is an overarching resistance to all frontline
M. tuberculosis drugs and susceptibility to metronidazole is lost
[5]. Therefore, there is an impetus to discover new drugs that
have conserved activity against these different NRP phenotypes to
enable us to better treat latent tuberculosis infection and eradicate
the vast reservoirs of disease present within the population.
In this chapter, we describe methods for inducing hypoxia and
drug susceptibility testing against Mycobacterium spp. using either
rich media (Wayne model) or minimal cholesterol media (utilizing
both hypoxia and nutrient deprivation). We also describe methods
of preparing NRP cultures for further investigation.
248
Savannah E. R. Gibson et al.