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Antimicrobial Agents and Chemotherapy, December 2001, p. 3456-3461, Vol. 45, No. 12
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.12.3456-3461.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Measurement of Effects of Antibiotics in Bioluminescent Staphylococcus aureus RN4220

Mervi Tenhami, Kaisa Hakkila, and Matti Karp*

University of Turku, Department of Biotechnology, Turku, Finland

Received 20 October 2000/Returned for modification 30 May 2001/Accepted 17 September 2001

The spread of antibiotic resistance among pathogenic bacteria is a serious threat to humans and animals. Therefore, unnecessary use should be minimized, and new antimicrobial agents with novel mechanisms of action are needed. We have developed an efficient method for measuring the action of antibiotics which is applied to a gram-positive strain, Staphylococcus aureus RN4220. The method utilizes the firefly luciferase reporter gene coupled to the metal-inducible cadA promoter in a plasmid, pTOO24. Correctly timed induction by micromolar concentrations of antimonite rapidly triggers the luciferase gene transcription and translation. This sensitizes the detection system to the action of antibiotics, and especially for transcriptional and translational inhibitors. We show the results for 11 model antibiotics with the present approach and compare them to an analytical setup with a strain where luciferase expression is under the regulation of a constitutive promoter giving only a report of metabolic inhibition. The measurement of light emission from intact living cells is shown to correlate extremely well (r = 0.99) with the conventional overnight growth inhibition measurement. Four of the antibiotics were within a 20% concentration range and four were within a 60% concentration range of the drugs tested. This approach shortens the assay time needed, and it can be performed in 1 to 4 h, depending on the sensitivity needed. Furthermore, the assay can be automatized for high-throughput screening by the pharmaceutical industry.

* Corresponding author. Mailing address: University of Turku, Department of Biotechnology, Tykistökatu 6, 6th floor, FIN-20520 Turku, Finland. Phone: 358-2-3338085. Fax: 358-2-3338050. E-mail: matti.karp{at}utu.fi.

Antimicrobial Agents and Chemotherapy, December 2001, p. 3456-3461, Vol. 45, No. 12
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.12.3456-3461.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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