Detection of inhibitors of phenotypically drug-tolerant Mycobacterium tuberculosis using an in vitro bactericidal screen

Abstract

Many whole cell screens of chemical libraries currently in use are based on inhibition of bacterial growth. The goal of this study was to develop a chemical library screening model that enabled detection of compounds that are active against drug-tolerant non-growing cultures of Mycobacterium tuberculosis. An in vitro model of low metabolically active mycobacteria was established with 8 and 30 day old cultures of M. smegmatis and M. tuberculosis, respectively. Reduction of resazurin was used as a measure of viability and the assay was applied in screens of chemical libraries for bactericidal compounds. The model provided cells that were phenotypically-resilient to killing by first and second-line clinical drugs including rifampicin. Screening against chemical libraries identified proteasome inhibitors, NSC310551 and NSC321206, and a structurally-related series of thiosemicarbazones, as having potent killing activity towards aged cultures. The inhibitors were confirmed as active against virulent M. tuberculosis strains including multi- and extensively-drug resistant clinical isolates. Our library screen enabled detection of compounds with a potent level of bactericidal activity towards phenotypically drug-tolerant cultures of M. tuberculosis.

The use of resazurin as a novel antimicrobial agent against Francisella tularensis

“…Resazurin has been used previously as an indicator of cell growth for various bacterial species (Mendoza-Aguilar et al, 2012; Bassett et al, 2013; Bauer et al, 2013; Lall et al, 2013). We were interested in using this compound to monitor viability of F. tularensis in broth culture over time.…”

Development of a Mycobacterium smegmatis transposon mutant array for characterising the mechanism of action of tuberculosis drugs: Findings with isoniazid and its structural analogues

“…For logistical reasons, the transposon mutant collection was screened as two distinct 16 plate libraries (library A and library B). Isoniazid was prepared as a 10 mM stock in sterile distilled water and stored at À20 C. The mutant library plates were grown either with isoniazid or in media alone for 48 h, at which point mutant cell number was quantified using a resazurin reduction assay as previously described [28]. Half of the on-plate MRC10 control wells received no treatment (untreated-MRC10 control) while the remaining half received the same treatment as the mutants (treated-MRC10 control).…”

Targeting Phenotypically TolerantMycobacterium tuberculosis

“…There have been numerous whole cell screens to identify small molecules in academic and industrial collections that kill nonreplicating mycobacteria (29, 53, 54, 94, 96, 101–105). Compounds arising from whole cell screening are presumably taken up into the cell to exert bactericidal activity, without any preconceptions about suitable targets.…”

“…The most commonly used models for nonreplicating mycobacteria are: hypoxia (the “Wayne model”) and the “low oxygen recovery assay” (“LORA”) (29, 95, 96); carbon starvation (54, 122); nutrient starvation (12, 52); stationary phase (105); maintenance of intrabacterial pH under acidic culture conditions (120, 123–127); biofilms (102, 128–131); depleting strain SS18b of streptomycin (103, 104, 132, 133); and a multi-stress model that combines acidic pH (pH 5.0), mild hypoxia (1% O 2 ), nitric oxide and other reactive nitrogen intermediates (0.5 mM NaNO 2 ), and a fatty acid carbon source (0.05% butyrate) (53, 94, 100, 134, 135). There are variations of these models, including an “acidic Wayne model” that combines hypoxia with mild acidity (131), and a nutrient-poor, multi-stress model in which cells are cultured at low pH (pH 5.0) under mild hypoxia or tissue-level normoxia (5% O 2 ) and supra-physiologic levels of CO 2 (10% CO 2 ) (136).…”