Bioluminescent Mycobacterium aurum expressing firefly luciferase for rapid and high throughput screening of antimycobacterial drugs in vitro and in infected macrophages.
The slow growth and highly infectious nature of Mycobacterium tuberculosis is a limiting factor in its use as test organism in high throughput screening for inhibitory compounds. To overcome these problems, use of surrogate strains and reporter genes have been considered. In this study, we have investigated the application of a fast growing nonpathogenic M. aurum expressing firefly luciferase in rapid screening of antituberculosis compounds in vitro and in infected macrophages using bioluminescence assay. The assay is based on luminescence determination using luciferin as substrate. Inhibition of bioluminescence was obtained with frontline antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, ofloxacin, and sparfloxacin at their reported MICs. Inhibition could be observed as early as 2 h in vitro and within 24 h in infected macrophages. The system can reliably be used in high throughput screening.
Deb
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DK
Division of Microbiology, Central Drug Research Institute, Lucknow, 226001, India.
Srivastava
K K
KK
Srivastava
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R
Srivastava
B S
BS
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000890:Anti-Infective Agents / Q000494:pharmacology; D000995:Antitubercular Agents / Q000494:pharmacology*; D002460:Cell Line; D001517:Coleoptera; D024841:Fluoroquinolones; D017930:Genes, Reporter; D008156:Luciferases / Q000032:analysis* / Q000235:genetics; D008163:Luminescent Measurements; D008264:Macrophages; D051379:Mice; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009161:Mycobacterium / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D015242:Ofloxacin / Q000494:pharmacology; D011994:Recombinant Proteins / Q000032:analysis; D012293:Rifampin / Q000494:pharmacology; D013307:Streptomycin / Q000494:pharmacology; D014162:Transfection
D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; D000995:Antitubercular Agents; D024841:Fluoroquinolones; D011994:Recombinant Proteins; D015242:Ofloxacin; D008156:Luciferases; C061363:sparfloxacin; D012293:Rifampin; D013307:Streptomycin