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Fluorescent Trimethoprim Conjugate Probes To Assess Drug Accumulation in Wild Type and Mutant Escherichia coli.

Authors :
Phetsang W
Pelingon R
Butler MS
Kc S
Pitt ME
Kaeslin G
Cooper MA
Blaskovich MA
Source :
ACS infectious diseases [ACS Infect Dis] 2016 Oct 14; Vol. 2 (10), pp. 688-701. Date of Electronic Publication: 2016 Aug 16.
Publication Year :
2016

Abstract

Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment.

Details

Language :
English
ISSN :
2373-8227
Volume :
2
Issue :
10
Database :
MEDLINE
Journal :
ACS infectious diseases
Publication Type :
Academic Journal
Accession number :
27737551
Full Text :
https://doi.org/10.1021/acsinfecdis.6b00080