期刊
ACS INFECTIOUS DISEASES
卷 2, 期 10, 页码 688-701出版社
AMER CHEMICAL SOC
DOI: 10.1021/acsinfecdis.6b00080
关键词
antibiotics; efflux pump; fluorescent probes; cellular accumulation; trimethoprim
资金
- NHMRC [APP1059354]
- UQ International Scholarship (UQI)
- IMB Postgraduate Award (IMBPA)
- Australian Postgraduate Award (APA)
- Wellcome Trust [WT141107]
- ARC [LIEF LE130100078]
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 (Delta tolC). 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.
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