Inhibiting antimicrobial resistance in Staphylococcus aureus using natural products

Since the treatment of infections with antibiotics, Staphylococcus aureus has developed resistance to antimicrobial agents. Methicillin-resistant S aureus (MRSA) contributes significantly to the healthcare burden of both hospital and community infections. Investigation into the resistance mechanism and their inhibition has the potential to increase the effectiveness in treating infection. It is hypothesized that the diversity of compounds and efficacy found in complex natural product extracts would lead to active leads that inhibit bacterial resistance. Using synergistic activity testing as a guide to a fractionation process, 3 flavonoids (8-desmethyl-sideroxylin, sideroxylin, and 6-desmethyl-sideroxlyn) were identified as synergists within a Hydrastis canadensis extract. These compounds were further characterized as efflux pump inhibitors using fluorescence in real-time and under confocal microscopy. In addition to disrupting efflux, the ability of S. aureus to cause disease was investigated. By detecting auto-inducing peptide I (AIP I), a direct product in the activation of this pathway, inhibition via natural products could be determined. Disruption in the intercellular communication via the Agr quorum sensing pathway in MRSA was observed in a Penicillium fungus and in H. canadensis. This quorum sensing inhibition (quorum quenching) limits virulence and toxin production making it easier for the host to manage infection

A Mass Spectrometry-Based Assay for Improved Quantitative Measurements of Efflux Pump Inhibition

<div><p>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 <i>Staphylococcus aureus</i>. 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 <i>Hydrastis Canadensis</i>, 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.</p></div

Efflux pump inhibitory activity and antimicrobial activity of flavonoids.

<p>a: Efflux pump inhibition was measured via LC-MS analysis of ethidium in spent, filtered culture supernatant after a 30 min incubation in triplicate wells.</p><p>b: Growth inhibition was measured by optical density at 600nm (in triplicate) after an 18 hr incubation.</p><p>c: CCCP is an abbreviation for the compound carbonyl cyanide m-chloro-phenylhydrazone</p><p>Efflux pump inhibitory activity and antimicrobial activity of flavonoids.</p

Representative selected chromatograms of filtered, spent broth showing a peak for the ethidium ion (MS-MS transition of <i>m/z</i> 314 to 286).

<p>Sample 1 is the negative control (<i>S</i>. <i>aureus</i> cultured for 30 min in Mueller Hinton broth with 1.25 μg/mL ethidium bromide and 10% DMSO), samples 2 and 3 were cultured under the same conditions as sample 1 with the addition of 75 μg/mL and 300 μg/mL piperine, respectively. All three peaks are normalized to a signal intensity of 3.45 x 10⁶. As piperine is added, efflux pumps are blocked, trapping the ethidium inside the cells and decreasing the quantity of ethidium (as indicated by the area of the ethidium peak) in the spent broth.</p

Efflux pump inhibition in <i>S</i>. <i>aureus</i> by a goldenseal (<i>Hydrastis canadensis</i>) extract.

<p>(A) Data collected using the fluorescence-based ethidium accumulation assay for a range of <i>H</i>. <i>canadensis</i> extract concentrations. (B) Data collected using the mass spectrometry-based ethidium accumulation assay. Incubation time was 30 min for both A and B, data represents mean of 3 samples, error bars represent standard deviation.</p

Change in absolute fluorescent intensity over time for <i>Staphylococcus aureus</i> exposed to ethidium bromide in the efflux pump inhibitor piperine.

<p>Fluorescence increases over time due to intracellular accumulation of ethidium bromide. The increase is more pronounced in the presence of piperine, which enhances intracellular accumulation of ethidium bromide by blocking efflux. Data points represent the mean of 3 samples, error bars represent standard deviation.</p

Efflux pump inhibitory activity of apigenin, piperine, and quercetin as indicated by LC-MS measurement of residual ethidium bromide in spent broth after a 30 min incubation.

<p>Relative peak area (expressed as a percentage) for ethidium is plotted as a function of concentration of the putative inhibitor. Data points represent the mean of 3 measurements (biological replicates), with error bars representing standard deviation.</p