Document Detail


Development of a liquid chromatography/mass spectrometry-based drug accumulation assay in Pseudomonas aeruginosa.
MedLine Citation:
PMID:  19032927     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Hongliang Cai; Kelly Rose; Lan-Hsin Liang; Steve Dunham; Charles Stover
Related Documents :
10321577 - A high throughput colorimetric cell proliferation assay for the identification of human...
23605107 - Effect of tortilla-preparation process on aflatoxins b1 and b2 in corn.
22420557 - Comparison of deproteinization methods used before tlc-db and hplc analysis of flumequi...
23174747 - Combination of flotation and flame atomic absorption spectrometry for determination, pr...
23158317 - Sequential injection spectrophotometric system for evaluation of mushroom tyrosinase-in...
1356447 - Dialysis as an on-line sample-pretreatment technique for column liquid chromatography: ...
Publication Detail:
Type:  Evaluation Studies; Journal Article     Date:  2008-11-05
Journal Detail:
Title:  Analytical biochemistry     Volume:  385     ISSN:  1096-0309     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2009 Feb 
Date Detail:
Created Date:  2009-01-19     Completed Date:  2009-03-10     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  321-5     Citation Subset:  IM    
Affiliation:
Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Michigan Laboratories, Ann Arbor, MI 48105, USA. hongliang.cai@pfizer.com
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Biological Transport
Chromatography, Liquid
Ciprofloxacin / analysis,  metabolism*
Fluorescence
Pharmaceutical Preparations / analysis,  metabolism
Pseudomonas aeruginosa / metabolism*
Pyridinium Compounds
Tandem Mass Spectrometry / methods*
Chemical
Reg. No./Substance:
0/Pharmaceutical Preparations; 0/Pyridinium Compounds; 42457-53-4/2-(dimethylaminostyryl)-1-ethylpyridinium; 85721-33-1/Ciprofloxacin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


Previous Document:  Epidemiology of fracture risk in the Women's Health Initiative.
Next Document:  Peroxisome proliferator-activated receptor gamma is constitutively activated in yeast.