version of the assay limits the dilution to fivefold and carries out
both phases in the same plate. In either format, the binary readout
is quantified by optical density after a 7- to 10-day outgrowth,
allowing one to distinguish inactive from bactericidal compounds.
There are two drawbacks of this two-stage assay. First, the
3-day exposure to nonreplicating conditions followed by a 7- to
10-day outgrowth in replicating conditions makes it imperative to
limit evaporation and to adhere to strict sterility techniques. Second, compounds that are only active against replicating
M. tuberculosis but are potent enough to kill M. tuberculosis after
dilution into the outgrowth phase generate false-positive results.
Thus, we typically run two assays in parallel: the two-phase nonreplicating assay and a one-phase assay in the outgrowth conditions
alone (replicating assay). As a first step to distinguish the true dualactives, we compare MIC 90 values and calculate if the compound’s
potency in the replicating assay is high enough to potentially
account for its apparent activity in the nonreplicating assay. However, it is difficult to distinguish the activities of compounds that
potently kill both replicating and nonreplicating M. tuberculosis
using standard broth MIC assays. The charcoal agar resazurin
assay [31, 32] was developed to mitigate compound carryover
effects and provide further evidence of bona fide nonreplicating
activity. Nonreplicating activity is then unambiguously confirmed
in a two-stage assay conducted in concentration-response format,
where the second stage is to conduct serial dilutions in PBS followed by plating on agar for determination of CFU. The extensive
dilution preceding and accompanying the plating markedly
diminishes the carryover effect of a compound that is only active
against replicating M. tuberculosis.
wild-type or
BSL2+ (∆panCD∆lysA)
Mycobacterium tuberculosis
wash replicating
cells 2x in
PBS-tyloxapol
resuspend in NR medium
- pH 5.0
- 0.5 mM NaNO 2
- butyrate C-source
dilute to
OD 580 = 0.1
add compounds
incubate 3-7d at 37 degrees
1% O 2 and 5% CO 2
CFU-based
enumeration
384-well
96-well
dose-response
for MIC 90
multi-stress model
of nonreplication
dose-response for MIC 90
or single point HTS
mix cells:
add 2 x 30 μL R medium
with plate filler
mix cells:
pipette up/down
with multichannel
mix cells:
pipette up/down
with multichannel
spread dilutions
on 7H11-OADC
agar plates
dilute 1:21-fold
in 7H9-ADN
liquid medium
Plate format & type of assay
dilute 1:5-fold
in 7H9-ADN
liquid medium
read OD 580
incubate 37 deg.
7-10 d
incubate 37 deg.
~ 21 d
count colonies
dilute to
OD 580 = 0.01
add compounds
incubate 7d at 37 degrees
20% O 2 and 5% CO 2
replicating model
*
*
high-througput screening, MIC determination, and CFU assays
Fig. 1 Overview of the replicating and nonreplicating assays. The red asterisks indicate the points of the
nonreplicating assays in which diluted test compound is still present during the replicating outgrowth phase
and may result in false-positive hits
Multi-stress Model for Nonreplicating M. tuberculosis
613
both phases in the same plate. In either format, the binary readout
is quantified by optical density after a 7- to 10-day outgrowth,
allowing one to distinguish inactive from bactericidal compounds.
There are two drawbacks of this two-stage assay. First, the
3-day exposure to nonreplicating conditions followed by a 7- to
10-day outgrowth in replicating conditions makes it imperative to
limit evaporation and to adhere to strict sterility techniques. Second, compounds that are only active against replicating
M. tuberculosis but are potent enough to kill M. tuberculosis after
dilution into the outgrowth phase generate false-positive results.
Thus, we typically run two assays in parallel: the two-phase nonreplicating assay and a one-phase assay in the outgrowth conditions
alone (replicating assay). As a first step to distinguish the true dualactives, we compare MIC 90 values and calculate if the compound’s
potency in the replicating assay is high enough to potentially
account for its apparent activity in the nonreplicating assay. However, it is difficult to distinguish the activities of compounds that
potently kill both replicating and nonreplicating M. tuberculosis
using standard broth MIC assays. The charcoal agar resazurin
assay [31, 32] was developed to mitigate compound carryover
effects and provide further evidence of bona fide nonreplicating
activity. Nonreplicating activity is then unambiguously confirmed
in a two-stage assay conducted in concentration-response format,
where the second stage is to conduct serial dilutions in PBS followed by plating on agar for determination of CFU. The extensive
dilution preceding and accompanying the plating markedly
diminishes the carryover effect of a compound that is only active
against replicating M. tuberculosis.
wild-type or
BSL2+ (∆panCD∆lysA)
Mycobacterium tuberculosis
wash replicating
cells 2x in
PBS-tyloxapol
resuspend in NR medium
- pH 5.0
- 0.5 mM NaNO 2
- butyrate C-source
dilute to
OD 580 = 0.1
add compounds
incubate 3-7d at 37 degrees
1% O 2 and 5% CO 2
CFU-based
enumeration
384-well
96-well
dose-response
for MIC 90
multi-stress model
of nonreplication
dose-response for MIC 90
or single point HTS
mix cells:
add 2 x 30 μL R medium
with plate filler
mix cells:
pipette up/down
with multichannel
mix cells:
pipette up/down
with multichannel
spread dilutions
on 7H11-OADC
agar plates
dilute 1:21-fold
in 7H9-ADN
liquid medium
Plate format & type of assay
dilute 1:5-fold
in 7H9-ADN
liquid medium
read OD 580
incubate 37 deg.
7-10 d
incubate 37 deg.
~ 21 d
count colonies
dilute to
OD 580 = 0.01
add compounds
incubate 7d at 37 degrees
20% O 2 and 5% CO 2
replicating model
*
*
high-througput screening, MIC determination, and CFU assays
Fig. 1 Overview of the replicating and nonreplicating assays. The red asterisks indicate the points of the
nonreplicating assays in which diluted test compound is still present during the replicating outgrowth phase
and may result in false-positive hits
Multi-stress Model for Nonreplicating M. tuberculosis
613