title: A simple whole cell based high throughput screening protocol using Mycobacterium bovis BCG for inhibitors against dormant and active tubercle bacilli.
abstract: This study aimed at developing a whole cell based high throughput screening protocol to identify inhibitors against both active and dormant tubercle bacilli. A respiratory type of nitrate reductase (NarGHJI), which was induced during dormancy, could reflect the viability of dormant bacilli of Mycobacterium bovis BCG in microplate adopted model of in vitro dormancy. Correlation between reduction in viability and nitrate reductase activity was seen clearly when dormant stage inhibitor metronidazole and itaconic anhydride were applied in this in vitro microplate model. Active replicating stage could also be monitored in the same assay by measuring the A(620) of the culture. MIC values of 0.08, 0.075, 0.3 and 3.0 microg/ml, determined through monitoring A(620) in this assay for rifampin, isoniazid, streptomycin and ethambutol respectively, were well in agreement with previously reported by BACTEC and Bio-Siv assays. S/N ratio and Z' factor for the assay were 8.5 and 0.81 respectively which indicated the robustness of the protocol. Altogether the assay provides an easy, inexpensive, rapid, robust and high content screening tool to search novel antitubercular molecules against both active and dormant bacilli.
authors.affiliation: CombiChem Bio Resource Center, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune-411008, India.
authors.forename: Arshad
authors.initials: A
authors.lastname: Khan
authors.forename: Dhiman
authors.initials: D
authors.lastname: Sarkar
mesh_terms: D000818:Animals; D000995:Antitubercular Agents / Q000302:isolation & purification* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002417:Cattle; D003470:Culture Media / Q000737:chemistry; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000191:economics / Q000379:methods; D009163:Mycobacterium bovis / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000254:growth & development; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D003470:Culture Media; D004791:Enzyme Inhibitors; D009565:Nitrate Reductases; D004121:Dimethyl Sulfoxide