title: Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria.
abstract: There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 °C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to ≥99% bacterial kill (MBC≥99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.
authors.affiliation: Departments of Microbiology & Immunology, Weill Cornell Medical College; bsg2001@med.cornell.edu.
authors.forename: Ben
authors.initials: B
authors.lastname: Gold
authors.affiliation: Departments of Microbiology & Immunology, Weill Cornell Medical College.
authors.forename: Julia
authors.initials: J
authors.lastname: Roberts
authors.affiliation: Departments of Microbiology & Immunology, Weill Cornell Medical College.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ling
authors.affiliation: Departments of Microbiology & Immunology, Weill Cornell Medical College.
authors.forename: Landys
authors.initials: L
authors.lastname: Lopez Quezada
authors.affiliation: Departments of Microbiology & Immunology, Weill Cornell Medical College.
authors.forename: Jou
authors.initials: J
authors.lastname: Glasheen
authors.affiliation: Departments of Microbiology & Immunology, Weill Cornell Medical College.
authors.forename: Elaine
authors.initials: E
authors.lastname: Ballinger
authors.affiliation: Medicine, Weill Cornell Medical College.
authors.forename: Selin
authors.initials: S
authors.lastname: Somersan-Karakaya
authors.affiliation: Departments of Microbiology & Immunology, Weill Cornell Medical College.
authors.forename: Thulasi
authors.initials: T
authors.lastname: Warrier
authors.affiliation: Departments of Microbiology & Immunology, Weill Cornell Medical College.
authors.forename: Carl
authors.initials: C
authors.lastname: Nathan
mesh_terms: D000362:Agar; D000995:Antitubercular Agents; D001681:Biological Assay; D002606:Charcoal / Q000737:chemistry*; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D010078:Oxazines; D010084:Oxidation-Reduction; D014966:Xanthenes
chemical_list: D000995:Antitubercular Agents; D010078:Oxazines; D014966:Xanthenes; D002606:Charcoal; C005843:resazurin; D000362:Agar