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Journal of Clinical Microbiology, February 1999, p. 304-309, Vol. 37, No. 2
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Development of a Firefly Luciferase-Based Assay for Determining Antimicrobial Susceptibility of Mycobacterium avium subsp. paratuberculosis

Stephanie L. Williams,1 N. Beth Harris,1,* and Raúl G. Barletta1,2,*

Department of Veterinary and Biomedical Sciences1 and Center for Biotechnology,2 University of Nebraska, Lincoln, Nebraska 68583-0905

Received 29 June 1998/Returned for modification 21 October 1998/Accepted 21 October 1998

Paratuberculosis (Johne's disease) is a fatal disease of ruminants for which no effective treatment is available. Presently, no drugs against Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the causative agent 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(pYUB180), a clinical isolate carrying a plasmid with the firefly luciferase gene. The MICs determined by the broth macrodilution method were as follows: amikacin, 2 µg/ml; Bay y 3118, 0.015 µg/ml; clarithromycin, 1.25 µg/ml; D-cycloserine, 25 µg/ml; ethambutol, 20 µg/ml; and rifabutin, 0.5 µg/ml. The strain was resistant to isoniazid and kanamycin. The results obtained by the luciferase assay were identical or fell within 1 doubling dilution. These results suggest that a combination of amikacin, clarithromycin, and rifabutin may be the most efficacious therapy for the treatment of M. paratuberculosis infections and that the use of fluoroquinolone class of antibiotics deserves further consideration. We demonstrate that the luciferase drug susceptibility assay is reliable for M. paratuberculosis and gives results within 7 days, whereas the broth macrodilution method requires 14 days.

* Corresponding author. Mailing address: Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583-0905. Phone: (402) 472-8717. Fax: (402) 472-9690. E-mail for N. Beth Harris: bharris1{at}unl.edu. E-mail for Raúl G. Barletta: braul{at}crcvms.unl.edu.

Journal of Clinical Microbiology, February 1999, p. 304-309, Vol. 37, No. 2
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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