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detection extremely challenging for clinical studies, and explains
the paucity of effective therapeutic agents. These “invisible” persis-
tent bacteria, therefore, represent an underexplored therapeutic
target, which if successfully detected and eliminated, would not
only shorten anti-TB chemotherapy duration but also reduce dis-
ease relapse.
Essentially, to eliminate these persisters, one must first detect
them. One of the most intuitive and promising methods is to
“wake up” the persistent bacteria from their dormant state, and
induce them to recommence multiplication. Previous studies have
showed that culture supernatant from M. tuberculosis young cul-
ture contains resuscitation promoting factors (RPF), 5 secreted
proteins RPF-A, RPF-B, RPF-C, RPF-D, and RPF-E. These RPF
proteins are able to stimulate persistent bacteria to initiate multipli-
cation [ 5 , 6 ]. This generates bacterial counts from conventionally
culture-negative samples, which enables quantitative persister
detection [ 5 , 7 ]. In our recent study [ 7 ], we demonstrated that
persistent M. tuberculosis which depended on culture supernatant
to replicate were present in stationary phase cultures in vitro and in
M. tuberculosis infected mouse organs. The long duration of tuber-
culosis treatment is due to the presence of RPF-dependent persist-
ers [ 8 ]. We also demonstrated for the first time that high-dose
rifampicin drug regimen was able to kill RPF-dependent persistent
bacteria, enabling a shortened treatment duration in mice without
subsequent disease relapse [ 7 ].
M. tuberculosis rpf-like genes are expressed in exponential
growth phase in vitro [ 9 , 10 ], in infected animals [ 10 ] and in
humans [ 11 ]. Using self-generated M. tuberculosis RPF to stimu-
late persistent bacteria is accurate and efficient with reproducible
experimental results [ 7 , 8 ]. Culture supernatant containing RPF
are essentially collected from exponential growth phase cultures
which can be achieved under aerobic and microaerophilic condi-
tions [ 5 , 7 ]. The mechanisms by which RPF proteins resuscitate
dormant cells remain unknown although peptidoglycan hydrolysis
by RPF has been proposed [ 12 ].
In this chapter, we describe methods for the production of M.
tuberculosis culture filtrates containing RPF and resuscitation of
RPF-dependent persisters in an in vitro hypoxic model and in the
Cornell model of tuberculosis.2 Materials
- Middlebrook 7H9 Broth: add 4.7 g of powder 7H9 medium
(Becton Dickinson, UK), 2 mL of glycerol and Tween 80 to
the final concentration of 0.05% (v/v) to 900 mL of distilled
water (see Note 1) followed by autoclaving at 121 °C for
15 min. After sterilization, cool the medium to room
2.1 Growth
and Preparation
of Mycobacteria
Yanmin Hu and Anthony Coates