title: A rapid method for screening antimicrobial agents for activities against a strain of Mycobacterium tuberculosis expressing firefly luciferase.
abstract: We developed a rapid method to screen the efficacy of antimicrobial agents against Mycobacterium tuberculosis. A restriction fragment carrying a promoterless firefly luciferase gene was cloned into a 4,488-bp shuttle vector, pMV261, and luciferase was expressed under the control of a mycobacterial heat shock promoter. The resulting plasmid, pLUC10, was introduced by electroporation into the avirulent strain M. tuberculosis H37Ra. Luciferase assays of sonic lysates of Triton X-100-treated cells of M. tuberculosis H37Ra(pLUC10) yielded bioluminescence in excess of 1,000 relative light units/approximately 10(9) tubercle bacilli, compared with 0.0025 for the same number of parental cells. A 48-h microdilution antimicrobial agent-screening assay using this strain was developed.
authors.affiliation: Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333.
authors.forename: R C
authors.initials: RC
authors.lastname: Cooksey
authors.forename: J T
authors.initials: JT
authors.lastname: Crawford
authors.forename: W R
authors.initials: WR
authors.lastname: Jacobs
authors.forename: T M
authors.initials: TM
authors.lastname: Shinnick
mesh_terms: D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology*; D001517:Coleoptera / Q000201:enzymology; D015870:Gene Expression / Q000235:genetics; D008027:Light; D008156:Luciferases / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000502:physiology; D008163:Luminescent Measurements; D008826:Microbial Sensitivity Tests / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D014169:Transformation, Bacterial / Q000235:genetics
chemical_list: D000995:Antitubercular Agents; D008156:Luciferases