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Journal of Biomolecular Screening, Vol. 7, No. 2, 127-134 (2002)
DOI: 10.1177/108705710200700205

A Luminescent Escherichia coli Biosensor for the High Throughput Detection of ß-Lactams

Satu J. Valtonen

Karolinska Institutet, Center for Genomics and Bioinformatics, Stockholm, Sweden and Department of Biotechnology, University of Turku, Turku, Finland

Jussi S. Kurittu

Innotrac Diagnostics Oy, Turku, Finland

Matti T. Karp

Department of Biotechnology, University of Turku, Turku, Finland

A group-specific bioluminescent Escherichia coli strain for studying the action of ß-lactam antibiotics is described. The strain contains a plasmid, pBlaLux1, in which the luciferase genes from Photorhabdus luminescent are inserted under the control of the ß-lactam-responsive element ampR/ampC from Citrobacter freundii. In the presence of ß-lactams, the bacterial cells are induced to express the luciferase enzyme and three additional enzymes generating the substrate for the luciferase reaction. This biosensor for ß-lactams does not need any substrate or cofactor additions, and the bioluminescence can be measured very sensitively in real time by using a luminometer. Basic parameters affecting the light production and induction in the gram-negative model organism E. coli SNO301/pBlaLux1 by various ß-lactams were studied. The dose-response curves were bell shaped, indicating toxic effects for the sensor strain at high concentrations of ß-lactams. Various ß-lactams had fairly different assay ranges: ampicillin, 0.05-1.0 µg/ml; piperacillin, 0.0025-25 µg/ml; imipenem, 0.0025-0.25 µg/ml; cephapirin, 0.025-2.5 µg/ml; cefoxitin, 0.0025-1.5 µg/ml; and oxacillin, 25-500 µg/ml. Also, the induction coefficients (signal over background noninduced control) varied considerably from 3 to 158 in a 2-hour assay. Different non-ß-lactam antibiotics did not cause induction. Because the assay can be automated using microplate technologies, the approach may be suitable for higher throughput analysis of ß-lactam action.


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