Rapid, Semiquantitative Assay To Discriminate among Compounds with Activity against Replicating or Nonreplicating Mycobacterium tuberculosis

Abstract

The search for drugs that can kill replicating and nonreplicating Mycobacterium tuberculosis faces practical bottlenecks. Measurement of CFU and discrimination of bacteriostatic from bactericidal activity are costly in compounds, supplies, labor, and time. Testing compounds against M. tuberculosis under conditions that prevent the replication of M. tuberculosis often involves a second phase of the test in which conditions are altered to permit the replication of bacteria that survived the first phase. False-positive determinations of activity against nonreplicating M. tuberculosis may arise from carryover of compounds from the nonreplicating stage of the assay that act in the replicating stage. We mitigate these problems by carrying out a 96-well microplate liquid MIC assay and then transferring an aliquot of each well to a second set of plates in which each well contains agar supplemented with activated charcoal. After 7 to 10 days—about 2 weeks sooner than required to count CFU—fluorometry reveals whether M. tuberculosis bacilli in each well have replicated extensively enough to reduce a resazurin dye added for the final hour. This charcoal agar resazurin assay (CARA) distinguishes between bacterial biomasses in any two wells that differ by 2 to 3 log10 CFU. The CARA thus serves as a pretest and semiquantitative surrogate for longer, more laborious, and expensive CFU-based assays, helps distinguish bactericidal from bacteriostatic activity, and identifies compounds that are active under replicating conditions, nonreplicating conditions, or both. Results for 14 antimycobacterial compounds, including tuberculosis (TB) drugs, revealed that PA-824 (pretomanid) and TMC207 (bedaquiline) are largely bacteriostatic.

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations

“…We cannot exclude the possibility that starvation enables Mtb to survive RIF treatment while in liquid media but then RIF proceeds to kill the cells once they have been washed and plated on solid agar with media of similar richness. Arguing against the latter interpretation, results were unaffected by including charcoal in the agar under conditions in which we have documented charcoal's ability to neutralize the effect of antibiotics that are carried into cfu plates in association with washed bacteria (23). Furthermore, the relative specificity to rifamycins as a class and the fact that the phenomenon does not occur with log-phase bacteria exposed to RIF both suggest that a more complex explanation is required than a delayed drug effect on cfu.…”

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

“…By convention, the threshold that distinguishes bactericidal from bacteriostatic activity for slow-growing mycobacteria is approximately 2-3 log 10 kill over 7 days 7,26 . Since the dynamic range of the CARA is also 2-3 log 10 kill, the CARA can provide an estimate of bactericidal or bacteriostatic activity.…”

“…In buffer solutions in vitro, activated charcoal rapidly sequesters most of the standard drugs used to treat tuberculosis 7 . Activated charcoal in CARA microplates binds compounds that may carry over from an assay microplate, and this feature of the CARA eliminates the requirement to serially dilute assay well contents prior to enumeration 7,21,22 . The number of mycobacterial microcolonies on the agar surface of CARA microplates is estimated by the addition of resazurin, a blue dye, whose reduced form, resorufin, is a pink molecule whose fluorescence is measured by a spectrophotometry 23 .…”

“…We recently developed an in vitro model of Class II non-replicating persistence to mimic conditions confronted by M. tuberculosis in activated macrophages and granulomas. The multi-stress model of Class II persistence includes conditions that slow growth, such as a fatty acid carbon source, and completely halt growth, such as mild acidity (pH 5.0), hypoxia (1% O 2 ), and nitric oxide and other reactive nitrogen intermediates [5][6][7][8][9] . Large-scale screening employing this multi-stress model of non-replication, and other Class II non-replicating models, has yielded diverse molecules whose activity is directed against the non-replicating state [5][6][7][9][10][11][12][13][14][15][16][17][18] .…”

“…To address the issues described above, we developed the charcoal agar resazurin assay (CARA) for rapid, semi-quantitative enumeration of mycobacterial species such as M. tuberculosis, M. bovis BCG, and M. smegmatis 7 (Figures 1 and 2). In buffer solutions in vitro, activated charcoal rapidly sequesters most of the standard drugs used to treat tuberculosis 7 .…”