About this Biochemistry, Genetics and Molecular Biology article

Development of a liquid chromatography/mass spectrometry-based drug accumulation assay in Pseudomonas aeruginosa by Hongliang Cai; Kelly Rose; Lan-Hsin Liang; Steve Dunham; Charles Stover is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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 ciproflo

Author

Hongliang Cai; Kelly Rose; Lan-Hsin Liang; Steve Dunham; Charles Stover

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2009

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)