Abstract
Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC50 values ranging from 19 μg/mL to 75 μg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.
Highlights
Bacterial efflux pumps are active transport proteins that function to extrude toxic compounds, including antimicrobial drugs, from the cell
The first goal of our experiments was to determine the applicability of a fluorescence-based accumulation assay to measure the efflux pump inhibitory activity of various flavonoids
As expected, when S. aureus is exposed to ethidium bromide, fluorescence increases over time (Fig 1)
Summary
Bacterial efflux pumps are active transport proteins that function to extrude toxic compounds, including antimicrobial drugs, from the cell. These pumps serve to protect bacteria from damage by toxins, and can play a role in the development of resistance to antimicrobials [1,2,3,4,5]. Measurement of Efflux Pump Inhibition with Mass Spectrometry strains of many bacteria, including methicillin resistant Staphylococcus aureus [6,7,8,9,10]. Compounds that inhibit bacterial efflux pumps are of interest because of their potential to increase antimicrobial effectiveness [11]. Our laboratory has been engaged in experiments to find new efflux pump inhibitors (EPIs) from natural product sources
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