title: Fluorescent Trimethoprim Conjugate Probes To Assess Drug Accumulation in Wild Type and Mutant Escherichia coli.
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.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Wanida
authors.initials: W
authors.lastname: Phetsang
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Ruby
authors.initials: R
authors.lastname: Pelingon
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Butler
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Sanjaya
authors.initials: S
authors.lastname: Kc
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Miranda E
authors.initials: ME
authors.lastname: Pitt
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Geraldine
authors.initials: G
authors.lastname: Kaeslin
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Cooper
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Mark A T
authors.initials: MA
authors.lastname: Blaskovich
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D053719:Tandem Mass Spectrometry; D014295:Trimethoprim / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D014295:Trimethoprim
keywords: antibiotics; cellular accumulation; efflux pump; fluorescent probes; trimethoprim