Measurement of effects of antibiotics in bioluminescent Staphylococcus aureus RN4220

The spread of antibiotic resistance among pathogenic bacteria is a serious threat to humans and animals. Therefore, unnecessary use should be minimize d, and new antimicrobial agents with novel mechanisms of action are needed. We have developed an efficient method for measuring the action of antibiot ics which is applied to a gram-positive strain, Staphylococcus aureus RN422 0. The method utilizes the firefly luciferase reporter gene coupled to the metal-inducible cadA promoter in a plasmid, pTOO24. Correctly timed inducti on by micromolar concentrations of antimonite rapidly triggers the lucifera se gene transcription and translation. This sensitizes the detection system to the action of antibiotics, and especially for transcriptional and trans lational inhibitors. We show the results for 11 model antibiotics with the present approach and compare them to an analytical setup with a strain wher e luciferase expression is under the regulation of a constitutive promoter giving only a report of metabolic inhibition. The measurement of light emis sion from intact living cells is shown to correlate extremely well (r = 0.9 9) with the conventional overnight growth inhibition measurement. Four of t he antibiotics were within a 20% concentration range and four were within a 60% concentration range of the drugs tested. This approach shortens the as say time needed, and it can be performed in 1 to 4 h, depending on the sens itivity needed. Furthermore, the assay can be automatized for high-throughp ut screening by the pharmaceutical industry.