Resveratrol Demonstrates Antimicrobial Effects Against Propionibacterium acnes In Vitro

Resveratrol and benzoyl peroxide were obtained from Sigma-Aldrich (St. Louis, MO, USA). The compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted to 1% DMSO in experiments, to minimize the effect of DMSO.

Antibacterial activity of resveratrol was determined by colony-forming unit (CFU) assays. P. acnes ATCC (American type cell culture) strain 6919 (a ribotype 1 [18] MLST4 1A₁ strain [19]) was grown anaerobically at 37 °C in reinforced clostridial media (Oxoid, Basingstoke, Hampshire, UK) for 3 days and collected in the late exponential phase of growth by centrifugation. Bacteria were washed with pH 7 sodium phosphate buffer supplemented by 0.03% trypticase soy media and quantified by reading with a spectrophotometer at 600 nm and applying a conversion of 1 × 10⁸ bacteria = 1 absorbance unit. Approximately, 1.33 × 10⁶ CFUs of bacteria were then added to 1 mL reinforced clostridial media. Samples were incubated anaerobically at 37 °C, with aliquots periodically withdrawn and plated on brucella agar with 5% sheep blood supplemented with hemin and vitamin K (Remel, Lenexa, KS, USA). Plates were incubated anaerobically at 37 °C for 3 days, and individual P. acnes colonies were counted to determine the concentration.

Propionibacterium acnes at 10⁷ CFU/mL were incubated with either 1 mg/mL of resveratrol or benzoyl peroxide for 24 h. Bacteria were washed three times with phosphate buffered saline (PBS) and resuspended in PBS with 2% glutaraldehyde. Samples were fixed for 5 min with 0.05% OsO₄, dehydrated in graded ethanol, and embedded in Eponate 12 (Ted Pella, Redding, CA, USA). A Reichert-Jung Ultracut E ultramicrotome™ (Leica, Buffalo Grove, IL, USA) was used to cut 60–70 nm slices which were picked up on formvar-coated copper grids. Uranyl acetate and Reynolds lead citrate were used for staining, and stained samples were visualized at 80 kV on a JEOL 100CX electron microscope™ (Peabody, MI, USA).

Blood was drawn from healthy human volunteers recruited by the laboratory with no skin conditions, including acne, and who had not suffered any illness within 2 weeks of the blood draw date. Peripheral blood mononuclear cells were isolated by use of a Ficoll-Paque (Pharmacia, New York, NY, USA) gradient and allowed to adhere for 2 h in RPMI media (Gibco, Grand Island, NY, USA) supplemented with 1% fetal bovine serum (FBS) (Omega Scientific, Tarzana, CA, USA) in 96-well plates (Costar, Tewksbury, MA, USA). Cells were washed 3× with Roswell Park Memorial Park Institute (RPMI) media to obtain adherent monocytes. Monocytes were then incubated at 37 °C in 100 μL RPMI media supplemented with 10% FBS. The HaCaT cell line of human keratinocytes was cultured in 100 μL HaCaT media and incubated at 37 °C in 96-well plates (Costar). To evaluate human monocyte and human keratinocyte viability after incubation with resveratrol or benzoyl peroxide, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cytotoxicity assays were performed. After 16 h of incubation with resveratrol or benzoyl peroxide, 20 μL MTS assay reagent (Promega, Madison, WI, USA) was added to each well, and the monocyte and keratinocyte cells were allowed to incubate for ~4 h at 37 °C. The 490 nm absorbance of each well was then determined using a microtiter plate reader, with absorbance proportional to the number of viable monocyte and keratinocyte cells in each treatment, as previously described [20]. Student’s T test was used for statistical analysis to determine if differences between resveratrol and benzoyl peroxide-treated cells were significant.

Study protocol for withdrawal of blood from healthy volunteers was approved by the Institutional Review Board at the University of California, Los Angles. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki declaration of 1975, as revised in 2000 and 2008. Informed consent was obtained from all patients for being included in the study.

The effect of resveratrol on P. acnes growth was visually demonstrated by incubating P. acnes bacteria with various concentrations of resveratrol for 48 h in reinforced clostridial media before spot plating. At a concentration of at least 50 μg/mL, resveratrol demonstrated significant inhibition of P. acnes growth (Fig. 1). Resveratrol at 25 μg/mL had only a small inhibitory effect.

To determine the antibacterial kinetics of resveratrol, benzoyl peroxide, and a combination of both on P. acnes growth, P. acnes was incubated with these treatments in reinforced clostridial media, and the concentration of bacteria was determined after 1, 2, 3, 7, and 10 days. Resveratrol demonstrated low bactericidal activity, but significant and sustained growth inhibition at concentrations of 100 μg/mL and shorter term growth inhibition at 50 μg/mL (Fig. 2a).

In contrast, benzoyl peroxide demonstrated significant differences in bactericidal kinetics when compared to resveratrol. High bactericidal activity was noted initially, but there was no antibacterial activity observed after the first 24 h (Fig. 2b). P. acnes recovered from benzoyl peroxide treatment and achieved maximum growth rate by the second day, irrespective of benzoyl peroxide concentration. Results from the combination therapy of resveratrol and benzoyl peroxide reflected the antibacterial kinetics of each individual treatment (Fig. 2c). As demonstrated with benzoyl peroxide alone, the combination treatment showed high initial antibacterial activity. Combination treatment also demonstrated the longer term inhibitory effects shown by resveratrol monotherapy. Combination therapy, therefore, resulted in a much lower concentration of P. acnes over the course of the study than with either treatment alone.

A comparison of each treatment group at a concentration of 75 μg/mL highlighted the short-term bactericidal activity of benzoyl peroxide, the sustained inhibitory activity of resveratrol, and the enhanced activity of resveratrol and benzoyl peroxide combination therapy (Fig. 2d).

To further investigate the mechanism by which resveratrol inhibits P. acnes growth, this study examined the bacterium using transmission electron microscopy (Fig. 3). Structural alterations were noted in the bacteria treated with resveratrol, with loss of membrane definition due to intramembranous edema and loss of well-defined extracellular fimbrial structures. Intracellular buildup of a dense substance was also found in some bacteria treated with resveratrol.

This study evaluated the cytotoxic effects of benzoyl peroxide compared to resveratrol via the MTS assay for human monocytes (Fig. 4a) and keratinocytes (Fig. 4b). Benzoyl peroxide was significantly more toxic than resveratrol to monocytes (p < 0.001 for all concentrations tested, Student’s t test), resulting in over 90% cell death at 10 μg/mL, while resveratrol resulted in less than 40% cell death at the same concentration. These effects were less pronounced in keratinocytes (p < 0.01 for all concentrations tested, Student’s t test), where both compounds had lower cytotoxicity. Nonetheless, benzoyl peroxide treatment resulted in 20–30% more cell death at all concentrations tested in keratinocytes.