In Vitro Antimicrobial Potential of Portuguese Propolis Extracts from Gerês against Pathogenic Microorganisms

Abstract

Antimicrobial resistance (AMR) is one of humanity's main health problems today. Despite all the breakthroughs and research over the past few years, the number of microbial illnesses that are resistant to the available antibiotics is increasing at an alarming rate. In this article, we estimated the biomedical potential of Portuguese propolis harvested from the Gerês apiary over five years, evaluating the in vitro antimicrobial effect of five hydroalcoholic extracts prepared from five single propolis samples and of a hydroalcoholic extract obtained from the mixture of all samples. The antimicrobial potential was firstly assessed by determining the minimum inhibitory concentration (MIC) of these extracts against a panel of three Gram-positive (Bacillus subtilis, methicillin-sensitive Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus) and one Gram-negative bacteria (Escherichia coli), as well as two yeasts (Candida albicans and Saccharomyces cerevisiae). As MIC values against each bacterium were consistent across all the evaluated propolis extracts, we decided to further conduct a disk diffusion assay, which included three commercial antibiotics-erythromycin, vancomycin, and amoxicillin/clavulanic acid-for comparison purposes. In addition to displaying a concentration-dependent antibacterial effect, the hydroalcoholic extracts prepared with 70% ethanol exhibited stronger antimicrobial capacity than vancomycin against B. subtilis (% of increase ranged between 26 and 59%) and methicillin-sensitive S. aureus (% of increase ranged between 63 and 77%). Moreover, methicillin-resistant S. aureus (MRSA) showed susceptibility to the activity of the same extracts and resistance to all tested antibiotics. These findings support that propolis from Gerês is a promising natural product with promising antimicrobial activity, representing a very stimulating result considering the actual problem with AMR.

Keywords: Portuguese propolis; antibiotic resistance; antimicrobial activity; pathogenic microorganisms.

Figure 1

Antibacterial effect of the Gerês propolis hydroalcoholic extracts against Bacillus subtilis. Diameter of the inhibition zones displayed by (A) G.EE₇₀ samples, erythromycin, and vancomycin and by (B) G.EE₃₅ samples, erythromycin, and vancomycin. Results are expressed as means ± SD (* 0.05 > p ≥ 0.01; ** 0.01 > p ≥ 0.001). No growth inhibition zones were observed for disks containing ethanol 70% or ethanol 35%. Reference values of inhibition zone diameters above which a microorganism is susceptible to ERY, AMC, and VAN [33] are represented with red, green, and blue dashed lines, respectively. (ERY: erythromycin; AMC: amoxicillin/Clavulanic Acid; VAN: vancomycin; G: Gerês, mG: mixture of propolis samples from Gerês).

Figure 2

Antibacterial effect of Gerês propolis hydroalcoholic extracts against methicillin-sensitive Staphylococcus aureus. Diameter of the inhibition zones displayed by (A) G.EE₇₀ samples, Vancomycin, and Amoxicillin/Clavulanic acid and by (B) G.EE₃₅ samples, Vancomycin, and Amoxicillin/Clavulanic acid. Results are expressed as means ± SD (*** p < 0.001). No growth inhibition zones were observed for disks containing ethanol 70% or ethanol 35%. Reference values of inhibition zone diameters above which Staphylococcus spp. is susceptible to ERY, AMC, and VAN [33] are represented with red, green, and blue dashed lines, respectively. (ERY: erythromycin; AMC: amoxicillin/Clavulanic Acid; VAN: vancomycin; G: Gerês, mG: mixture of propolis samples from Gerês).

Figure 3

Antibacterial effect of Gerês propolis hydroalcoholic extracts against methicillin-resistant Staphylococcus aureus. Diameter of the inhibition zones displayed by G.EE₇₀ samples. Results are expressed as means ± SD. No growth inhibition zones were observed for disks containing ethanol 70%. Reference values of inhibition zone diameters above which Staphylococcus spp. is susceptible to ERY, AMC, and VAN [33] are represented with red, green, and blue dashed lines, respectively. (ERY: erythromycin; AMC: amoxicillin/Clavulanic Acid; VAN: vancomycin; G: Gerês, mG: mixture of propolis samples from Gerês).

Figure 4

Antibacterial effect of Gerês propolis hydroalcoholic extracts against Escherichia coli. Diameter of the inhibition zones displayed by G.EE₇₀ samples and AMC. Results are expressed as means ± SD. No growth inhibition zones were observed for disks containing ethanol 70%. Reference values of inhibition zone diameters above which a microorganism is susceptible to ERY and AMC are represented with red and green dashed lines, respectively [33]. Vancomycin has no reference diameter against Gram-negative bacteria because of its inability to breach the outer membrane barrier [40]. (ERY: erythromycin; AMC: amoxicillin/Clavulanic Acid; G: Gerês, mG: mixture of propolis samples from Gerês).