A luminescent Escherichia coli biosensor for the high throughput detection of beta-lactams.
A group-specific bioluminescent Escherichia coli strain for studying the action of beta-lactam antibiotics is described. The strain contains a plasmid, pBlaLux1, in which the luciferase genes from Photorhabdus luminescens are inserted under the control of the beta-lactam-responsive element ampR/ampC from Citrobacter freundii. In the presence of beta-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 beta-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 beta-lactams were studied. The dose-response curves were bell shaped, indicating toxic effects for the sensor strain at high concentrations of beta-lactams. Various beta-lactams had fairly different assay ranges: ampicillin, 0.05-1.0 microg/ml; piperacillin, 0.0025-25 microg/ml; imipenem, 0.0025-0.25 microg/ml; cephapirin, 0.025-2.5 microg/ml; cefoxitin, 0.0025-1.5 microg/ml; and oxacillin, 25-500 microg/ml. Also, the induction coefficients (signal over background noninduced control) varied considerably from 3 to 158 in a 2-hour assay. Different non-beta-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 beta-lactam action.
Valtonen
Satu J
SJ
Karolinska Institutet, Center for Genomics and Bioinformatics, Stockholm, Sweden.
Kurittu
Jussi S
JS
Karp
Matti T
MT
D000667:Ampicillin / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015374:Biosensing Techniques / Q000379:methods*; D002440:Cefoxitin / Q000494:pharmacology; D002511:Cephalosporins / Q000494:pharmacology; D002513:Cephamycins / Q000494:pharmacology; D002514:Cephapirin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D015378:Imipenem / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008027:Light; D008156:Luciferases / Q000378:metabolism; D010068:Oxacillin / Q000494:pharmacology; D010406:Penicillins / Q000494:pharmacology; D010878:Piperacillin / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D013845:Thienamycins / Q000494:pharmacology; D013997:Time Factors
D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D002513:Cephamycins; D010406:Penicillins; D013845:Thienamycins; D002440:Cefoxitin; D015378:Imipenem; D000667:Ampicillin; D002514:Cephapirin; D008156:Luciferases; D010068:Oxacillin; D010878:Piperacillin