Development of a liquid chromatography/mass spectrometry-based drug accumulation assay in Pseudomonas aeruginosa
| Autor: | Steve Dunham, Hongliang Cai, Charles K. Stover, Kelly Rose, Lan-Hsin Liang |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Lysis
Biophysics Pyridinium Compounds medicine.disease_cause Tandem mass spectrometry Biochemistry Fluorescence Ciprofloxacin Tandem Mass Spectrometry Liquid chromatography–mass spectrometry medicine Molecular Biology Chromatography biology Pseudomonas aeruginosa Chemistry Biological Transport Cell Biology biology.organism_classification Pharmaceutical Preparations Efflux Steady state (chemistry) Bacteria Chromatography Liquid medicine.drug |
| Zdroj: | Analytical Biochemistry. 385:321-325 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2008.10.041 |
| Popis: | Bacterial resistance to antibiotic therapy remains a worldwide problem. In Pseudomonasaeruginosa, rates of efflux confer inherent resistance to many antimicrobial agents, including fluoroquinolones, due to a high level of expression and a relatively high turnover number of the efflux pumps in gram-negative bacteria. To understand the roles of efflux pumps in both the influx and efflux of compounds in P. aeruginosa and to aid the chemistry compound design by bridging in vitro enzymatic binding data (IC(50) values) with whole cell results (MIC numbers), a collaborative effort was put forward to validate a series of bacterial penetration/accumulation assays for assessment of intracellular drug concentration. Initially, using 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP) as the tracer, a 96-well fluorescence assay was established to measure the time-dependent accumulation of DMP in wild-type (PAO1), MexABOprM deletion (PAO200), and MexABOprM-MexCDOprJ-MexJKL:FRT deletion mutants (PAO314). At steady state, the order of DMP accumulation was PAO314>PAO200>PAO1. Subsequently, the established assay conditions were applied to a radiolabeled assay format using (3)H-labeled ciprofloxacin. At the concentration tested, the accumulation of [(3)H]ciprofloxacin approached a plateau after 15 min and the amount of accumulation in PAO314 was higher (~2- to 10-fold) than that in PAO1. Finally, with an additional step of cell lysis, a liquid chromatography/mass spectrometry-based assay was established with ciprofloxacin with (i) superior sensitivity (the detection limit can be as low as 0.24 ng/ml for ciprofloxacin) and (ii) the ability to monitor cold or nonfluorescent compounds in a drug discovery setting. |
| Databáze: | OpenAIRE |
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