Abstract
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 SN0301/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 mug/ml; piperacillin, 0.0025-25 mug/ml; imipenem, 0.0025-0.25 mug/ml; cephapirin, 0.025-2.5 mug/ml; cefoxitin, 0.0025-1.5 mug/ml; and oxacillin, 25-500 mug/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.
| Original language | English |
|---|---|
| Pages (from-to) | 127-134 |
| Number of pages | 8 |
| Journal | Journal of Biomolecular Screening |
| Volume | 7 |
| Issue number | 2 |
| Publication status | Published - Apr 2002 |
| Externally published | Yes |
| Publication type | A1 Journal article-refereed |
Keywords
- MICROBIAL BIOSENSORS
- GENE-EXPRESSION
- INDUCTION
- BACTERIA
- BIOLUMINESCENCE
- ARSENITE
- SENSOR
- ASSAY
- MILK
- ANTIBIOTICS