Development of a firefly luciferase-based assay for determining antimicrobial susceptibility of Mycobacterium avium subsp. paratuberculosis

Paratuberculosis (Johne's disease) is a fatal disease of ruminants for whic h no effective treatment is available. Presently, no drugs against Mycobact erium avium subsp. paratuberculosis (M; paratuberculosis), the causative ag ent of Johne's disease, are approved for use in livestock Additionally, M. paratuberculosis has been linked to a human chronic granulomatous ileitis ( Crohn's disease). To assist in the evaluation of antimicrobial agents with potential activity against M. paratuberculosis, we have developed a firefly luciferase-based assay for the determination of drug susceptibilities. The microorganism used was M. paratuberculosis K-10(pYTUB180), a clinical isol ate carrying a plasmid with the firefly luciferase gene. The MICs determine d by the broth macrodilution method were as follows: amikacin, 2 mu g/ml; B ay y 3118, 0.015 mu g/ml; clarithromycin, 1.25 mu g/ml; D-cycloserine, 25 m u g/ml; ethambutol, 20 mu g/ml; and rifabutin, 0,5 mu g/ml. The strain was resistant to isoniazid and kanamycin. The results obtained by the luciferas e assay were identical or fell within 1 doubling dilution. These results su ggest that a combination of amikacin, clarithromycin, and rifabutin may be the most efficacious therapy for the treatment of M. paratuberculosis infec tions and that the use of fluoroquinolone class of antibiotics deserves fur ther consideration. We demonstrate that the luciferase drug susceptibility assay is reliable fur M. paratuberculosis and gives results within 7 days, whereas the broth macrodilution method requires 14 days.