title: A high-throughput cidality screen for Mycobacterium tuberculosis.
abstract: Exposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well 'spot-assay' for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm) and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets). The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808). An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA) or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process.
authors.affiliation: AstraZeneca India Private Limited, Bangalore, Karnataka, India.
authors.forename: Parvinder
authors.initials: P
authors.lastname: Kaur
authors.affiliation: Department of Microbial and Molecular Systems, KU Leuven, Kasteelpark, Arenberg 20, 3001 Leuven, Belgium.
authors.forename: Anirban
authors.initials: A
authors.lastname: Ghosh
authors.affiliation: Gangagen Biotech Private Limited, Bangalore, Karnataka, India.
authors.forename: Ramya Vadageri
authors.initials: RV
authors.lastname: Krishnamurthy
authors.affiliation: AstraZeneca India Private Limited, Bangalore, Karnataka, India.
authors.forename: Deepa Gagwani
authors.initials: DG
authors.lastname: Bhattacharjee
authors.affiliation: AstraZeneca India Private Limited, Bangalore, Karnataka, India.
authors.forename: Vijayashree
authors.initials: V
authors.lastname: Achar
authors.affiliation: Cellworks India, Bangalore, Karnataka, India.
authors.forename: Santanu
authors.initials: S
authors.lastname: Datta
authors.affiliation: AstraZeneca India Private Limited, Bangalore, Karnataka, India.
authors.forename: Shridhar
authors.initials: S
authors.lastname: Narayanan
authors.affiliation: Medical & Biological Computing Laboratory, School of Biosciences & Technology, VIT University, Vellore, Tamil Nadu, India.
authors.forename: Anand
authors.initials: A
authors.lastname: Anbarasu
authors.affiliation: Medical & Biological Computing Laboratory, School of Biosciences & Technology, VIT University, Vellore, Tamil Nadu, India.
authors.forename: Sudha
authors.initials: S
authors.lastname: Ramaiah
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods* / Q000592:standards; D057166:High-Throughput Screening Assays / Q000379:methods* / Q000592:standards; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D012015:Reference Standards; D012449:Safety
chemical_list: D000995:Antitubercular Agents