most probable number calculation. The nonreplicating assay
system we describe does not detect DD M. tuberculosis.
Instead, M. tuberculosis in the four-stress nonreplicating assay
yields nearly the same CFU at the beginning of the assay as at
the end, except in the presence of nonreplicating active drugs
and compounds.
28. To determine if a compound synergizes with nitric oxide and
reactive oxygen species (formed by mixing NaNO 2 in acid),
instead of using nitrite as a terminal electron acceptor, the
activity of a compound should be tested against
M. tuberculosis in nonreplicating medium ÆNaNO 2 (nitrite)
and Æ NaNO 3 (nitrate).
29. Due to the slightly more acidic pH, the half-life of DETA-NO
at pH 6.6 will be shorter than at pH 7.4 (~22 h). To maintain a
flux of nitric oxide, DETA-NO should be replenished daily.
30. Alternatively, one can use DETA-NO that was decomposed for
7 days at RT.
31. We favor using filter tips and adding one mix step (~7 μL up/
down once) to ensure the compound has been completely
dispensed from the tip. Alternatively, one may use microplates
that already contain the acoustically dispensed compound on
the well bottoms.
32. Alternative readouts may be used as a surrogate for viability,
including Alamar blue, strains encoding reports for GFP, luciferase, etc. However, to make an accurate comparison of the
activity of a compound against M. tuberculosis in replicating
and nonreplicating conditions, we favor using OD 580 for both
assays.
33. It is critical to ensure the M. tuberculosis strain displays susceptibility to TB antibiotics targeting a variety of cell processes
such as the biosynthesis of RNA, DNA, lipids, folate, etc. This
can be more extensively characterized by comparing the MIC 90
values of these antibiotics with literature values [30, 56, 57].
34. Since a compound is diluted fivefold in the outgrowth phase, a
potent replicating active molecule will appear to kill nonreplicating cells during the replicating outgrowth phase (steps highlighted by red asterisks in Fig. 1) [31]. We have observed that
numerous compounds are modified as a result of incubation in
nonreplicating medium containing NaNO 2 . For example, oxyphenbutazone is hydroxylated on the pyrazolidinedione ring at
the carbon atom that also serves to attach the butyl chain. The
resulting 4-OH-oxyphenbutazone (4-OH-OPB) is an intermediate to forming an electrophilic, reactive species,
4-OH-OPB quinone imine, with a propensity to readily react
with an available nucleophile such as N-acetyl-cysteine or
mycothiol [49]. A medicinal chemist can determine if there
Multi-stress Model for Nonreplicating M. tuberculosis
631
system we describe does not detect DD M. tuberculosis.
Instead, M. tuberculosis in the four-stress nonreplicating assay
yields nearly the same CFU at the beginning of the assay as at
the end, except in the presence of nonreplicating active drugs
and compounds.
28. To determine if a compound synergizes with nitric oxide and
reactive oxygen species (formed by mixing NaNO 2 in acid),
instead of using nitrite as a terminal electron acceptor, the
activity of a compound should be tested against
M. tuberculosis in nonreplicating medium ÆNaNO 2 (nitrite)
and Æ NaNO 3 (nitrate).
29. Due to the slightly more acidic pH, the half-life of DETA-NO
at pH 6.6 will be shorter than at pH 7.4 (~22 h). To maintain a
flux of nitric oxide, DETA-NO should be replenished daily.
30. Alternatively, one can use DETA-NO that was decomposed for
7 days at RT.
31. We favor using filter tips and adding one mix step (~7 μL up/
down once) to ensure the compound has been completely
dispensed from the tip. Alternatively, one may use microplates
that already contain the acoustically dispensed compound on
the well bottoms.
32. Alternative readouts may be used as a surrogate for viability,
including Alamar blue, strains encoding reports for GFP, luciferase, etc. However, to make an accurate comparison of the
activity of a compound against M. tuberculosis in replicating
and nonreplicating conditions, we favor using OD 580 for both
assays.
33. It is critical to ensure the M. tuberculosis strain displays susceptibility to TB antibiotics targeting a variety of cell processes
such as the biosynthesis of RNA, DNA, lipids, folate, etc. This
can be more extensively characterized by comparing the MIC 90
values of these antibiotics with literature values [30, 56, 57].
34. Since a compound is diluted fivefold in the outgrowth phase, a
potent replicating active molecule will appear to kill nonreplicating cells during the replicating outgrowth phase (steps highlighted by red asterisks in Fig. 1) [31]. We have observed that
numerous compounds are modified as a result of incubation in
nonreplicating medium containing NaNO 2 . For example, oxyphenbutazone is hydroxylated on the pyrazolidinedione ring at
the carbon atom that also serves to attach the butyl chain. The
resulting 4-OH-oxyphenbutazone (4-OH-OPB) is an intermediate to forming an electrophilic, reactive species,
4-OH-OPB quinone imine, with a propensity to readily react
with an available nucleophile such as N-acetyl-cysteine or
mycothiol [49]. A medicinal chemist can determine if there
Multi-stress Model for Nonreplicating M. tuberculosis
631