Looking to nature for a new concept in antimicrobial treatments: isoflavonoids from Cytisus striatus as antibiotic adjuvants against MRSA

Abstract

The spread of multidrug-resistant Staphylococcus aureus strains, including methicillin-resistant S. aureus (MRSA), has shortened the useful life of anti-staphylococcal drugs enormously. Two approaches can be followed to address this problem: screening various sources for new leads for antibiotics or finding ways to disable the resistance mechanisms to existing antibiotics. Plants are resistant to most microorganisms, but despite extensive efforts to identify metabolites that are responsible for this resistance, no substantial progress has been made. Plants possibly use multiple strategies to deal with microorganisms that evolved over time. For this reason, we searched for plants that could potentiate the effects of known antibiotics. From 29 plant species tested, Cytisus striatus clearly showed such an activity and an NMR-based metabolomics study allowed the identification of compounds from the plant extracts that could act as antibiotic adjuvants. Isoflavonoids were found to potentiate the effect of ciprofloxacin and erythromycin against MRSA strains. For the structure-activity relationship (SAR), 22 isoflavonoids were assessed as antibiotic adjuvants. This study reveals a clear synergy between isoflavonoids and the tested antibiotics, showing their great potential for applications in the clinical therapy of infections with antibiotic-resistant microorganisms such as MRSA.

Figure 1

Potentiating effects of ciprofloxacin and erythromycin obtained when combined with the different extracts of Cytisus striatus against Staphylococcus aureus CECT 976. The activity is expressed as the increase in the inhibition zone diameter (IZD, mm) caused by ciprofloxacin (a) and erythromycin (b) in the presence of the extracts of C. striatus dissolved in Mueller-Hinton agar medium. Bars represent means and SD from at least three independent experiments. The sample parameters (x,y) are shown in Supplementary Table 2.

Figure 2

Orthogonal partial least squares modeling applied to ¹H-NMR data and potentiating activity of Cytisus striatus samples (Fig. 2). OPLS score- and S-plots obtained from the potentiating activity (1: indifferent , 2: additive and 3: potentiation effect ) and ¹H-NMR data in the range of the region between δ 6.00 and 8.60 of the different classes of potentiating activity of extracts of C. striatus on ciprofloxacin (a) and erythromycin (b). The sample preparation and extraction conditions were performed as shown in Supplementary Table 2.

Figure 3

Effect of isoflavonoids on EtBr accumulation in Staphylococcus aureus SA1199B. In (a), fluorescence as a measure of EtBr accumulation is shown for all Staphylococcus aureus strains for 60 min at 37 °C; EtBr was applied at ½ MIC and fluorescence measurement obtained by fluorometric method; in (b), only the isoflavonoids (60 µg/mL) increasing the accumulation of EtBr in SA1199B cultures over control over time (P < 0.05) are represented; the changes in the accumulation of EtBr in SA1199B are also shown for genistein, tectorigenin (both at 60 µg/mL) and orobol (30 µg/mL) (c) and for the flavonoids apigenin and chrysin (d). Reserpine at 20 µg/mL was used as a positive control as efflux pump inhibitor (EPI). Mean values of least three independent experiments are shown.