Investigating the Effect of Enzymatically-Derived Blackcurrant Extract on Skin Staphylococci Using an In Vitro Human Stratum Corneum Model

Abstract

Background/Objectives: Numerous intrinsic and extrinsic stressors can disrupt the balance of the skin microbiome, leading to the development of various skin diseases. It has been proven that coagulase-negative staphylococci (CoNS) are important commensals for maintaining skin microbiome homeostasis and fighting cutaneous pathogens such as Staphylococcus aureus (S. aureus). Here, we examined the influence of polyphenol-rich enzymatic blackcurrant extract (EBCE) on pathogenic coagulase-positive S. aureus strains and beneficial CoNS, like Staphylococcus epidermidis (S. epidermidis), to explore its potential for rebalancing the skin microbiota. Methods: The polyphenol profile of EBCE was determined by ultra-high-pressure liquid chromatography-tandem mass spectrometry. Microwell plate assays were employed to study the effect of EBCE on five S. aureus strains isolated from the skin of atopic dermatitis patients. An in vitro human stratum corneum model was used to test its effect on mixed bacterial cultures. Results: EBCE inhibited the growth of all tested S. aureus strains by 80-100% at the highest tested concentration after 7 h. No microbial growth was observed at the highest tested EBCE concentration using the stratum corneum model inoculated with one selected pathogen (S. aureus SA-DUS-017) and one commensal laboratory strain (S. epidermidis DSM 20044). The lowest tested concentration did not interfere with S. aureus growth but strongly stimulated the growth of S. epidermidis (~300-fold colony forming unit increase). In addition, low EBCE concentrations strongly stimulated CoNS growth in microbiome samples taken from the armpits of healthy volunteers that were spiked with S. aureus SA-DUS-017. Conclusions: These preclinical data support further testing of EBCE-enriched topical preparations as potential cutaneous prebiotics in human studies.

Keywords: Staphylococcus aureus; blackcurrant extract; coagulase-negative staphylococci; skin prebiotic; stratum corneum model.

Figure 1

Effect of different EBCE concentrations on the growth of clinical S. aureus strains. Input was ~10⁴ CFU for each S. aureus SA-DUS strain, and bacterial growth was calculated from OD600 values compared to the control sample of each strain without EBCE supplementation (0 µg GAE/mL). A strong reduction growth is seen at the highest EBCE concentration (100 µg GAE/mL). No S. aureus growth-promoting effect was observed for any of the strains. Figures show mean values of two independent experiments with error bars representing standard deviations. p-values are shown with an asterisk (* p < 0.05, ** p < 0.01, or *** p < 0.001) for comparing different dosages of EBCE with those of its own control.

Figure 2

Representative photographs of CoNS (pink) and CoPS (yellow) colonies in a serially diluted (a) control sample without EBCE, (b) a sample supplemented with 3.8 µg GAE/cm² of EBCE, and (c) a sample supplemented with 76.0 µg GAE/cm² of EBCE. These pictures illustrate respectively how S. aureus AD-DUS-017 is outgrowing S. epidermidis DSM 20044 in the control sample, how S. epidermidis DSM 20044 is outgrowing S. aureus SA-DUS-017 in a sample with the low EBCE concentration, and how both strains are completely inhibited in the sample with the high EBCE concentration. Samples represent microbial growth of viable colonies extracted from the stratum corneum model that was inoculated with ~10⁴ CFU of both strains.

Figure 3

Effect of different EBCE concentrations on the growth of S. aureus SA-DUS-017 and S. epidermidis DSM 20044 grown together on the in vitro stratum corneum model. Results represent microbial growth of viable colonies extracted from the stratum corneum model inoculated with ~10⁴ CFU of both strains. (a) the CFU values of both individual strains, and (b) the S. epidermidis/S. aureus ratio calculated from the mean CFU values of the individual strains (log-scale). Figures show the mean values of two independent experiments with error bars representing standard deviations. p-values are shown with an asterisk: * p < 0.05, ** p < 0.01, or *** p < 0.001.

Figure 4

Representative photographs of CoNS (pink) and CoPS (yellow) colonies in (a) a serially diluted control sample without EBCE and (b) a sample supplemented with 3.8 µg GAE/cm² of EBCE. These pictures illustrate how S. aureus SA-DUS-017 is outgrowing CoNS in the control sample, and CoNS are outgrowing S. aureus SA-DUS-017 in the sample with EBCE. Samples represent the growth of viable colonies extracted from the stratum corneum model inoculated with ~10³ CFU of S. aureus SA-DUS-017 and ~10⁴ CFU of aerobic bacteria from the armpits of seven healthy volunteers.

Figure 5

The effect of two different EBCE concentrations on the growth of S. aureus SA-DUS-017 and CoNS bacteria from the skin of healthy volunteers grown together for 24 h on the in vitro stratum corneum model. Results are representing microbial growth of viable colonies extracted from the stratum corneum model inoculated with ~10³ CFU of S. aureus SA-DUS-017 and ~10⁴ CFU of aerobic bacteria from the armpits of seven healthy volunteers. (a) CFU of CoNS and S. aureus SA-DUS-017 and (b) CoNS/S. aureus SA-DUS-017 ratio calculated from mean values of CFU number. Figures are showing mean values of two independent experiments with error bars, which represent standard deviations. p-values are shown with an asterisk: * p < 0.05 or *** p < 0.001.