Experimental trial
The present study aimed to evaluate the effects of scFOS (FOSbeauty®Beghin-Meiji) on the in vitro growth and competitive activity of bacterial strains representative of the human skin microbiota (Staphylococcus epidermidis, Cutibacterium acnes and Staphylococcus aureus).
Bacterial strains
Staphylococcus epidermidis (ATCC® 12228) and S. aureus (ATCC® 6538) were acquired from the American Type Culture Collection (ATCC), while C. acnes (CCUG 1794T) was acquired from the Cultural Collection of the University of Gothenburg (CCGU). The bacterial strains, supplied in freeze-dried form, were resuspended and inoculated into sterile tubes containing their respective selective medium [tryptic soy broth (TSB) for Staphylococcus strains, and TSB + 5% defibrinated sheep blood for C. acnes (BTSB)]and allowed to grow under optimal conditions for each bacterium (i.e. 24 h at 37°C under aerobic conditions for Staphylococcus strains, and 48 h at 37°C in anaerobiosis for C. acnes). The resulting bacterial suspension was stored at -80°C, following the freezing procedure indicated by cell banks. At the beginning of the experimental phase, a vial of each bacterium was taken at -80°C, partially thawed and a small aliquot taken with a sterile loop and streaked on agar plates based on selective medium. The streaked bacteria were then allowed to grow under optimal conditions for each strain and, once visible colonies had formed, a single isolated colony was collected and inoculated into selective media. To ensure that all experiments were performed on bacteria from the same selected colony after streaking, fresh inocula for each bacterial strain were generated from previous inocula. The bacterial concentration (CFU/mL) of each inoculum was determined in the mid-logarithmic growth phase by densitometry and CFU counting. The consistency between the different experiments was to prepare fresh inoculums before each experiment.
It should be noted that, although primarily known as an anaerobiotic bacterium, C. acnes is an aerotolerant anaerobe because it has enzymatic systems capable of detoxifying oxygen, allowing it to remain on the surface of the skin. As such, C. acnes can develop under conditions compatible with those found in the skin.
Preparation of scFOS
The solution composed of scFOS was prepared according to Rossi et al..200516. Briefly, depending on the experiment, the scFOS were weighed and dissolved at a concentration of 20% (w/v) either in TSB or in minimal medium. [0.9% NaCl in sterile water + 0.003% of tryptic phosphate broth (TPB)1]. After dissolution, the sterilization of the media was carried out by autoclave.
Bacteriostatic and bactericidal activity of scFOS
The ability of scFOS to inhibit the growth of bacterial strains (i.e., bacteriostatic activity) and/or exert bactericidal activity was assessed by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration ( MBC). MIC was defined as the lowest concentration of an agent preventing bacterial growth, while MBC was the lowest concentration of an antibacterial agent needed to kill them. 10×106 CFU/mL of each strain were exposed to increasing concentrations of scFOS [from 0 to 15% (w/v)] under aerobic conditions, experienced by skin bacteria in vivo. The impact of scFOS concentrations on bacterial growth was assessed at 0, 8 and 24 h and determined by colony count. When corrected for the dilution factor, bacterial growth was calculated as a fold change from the control (t = 0 h). Values greater than 1 indicate bacterial growth, equal to 1 bacteriostatic activity (no bacterial growth), less than 1 bactericidal activity.
Impact of scFOS on bacterial growth kinetics
The ability of the tested strains to metabolize scFOS to support their growth was explored. After centrifugation and washing to remove growth medium, 10 × 106 CFU/mL were exposed to increasing concentrations of scFOS (0-15%) in minimal medium at 37°C for 24 h under shaking and aerobic conditions. After 8 h and 24 h of exposure, aliquots were taken and spread on selective agar plates. Colonies were then counted and corrected for the appropriate dilution factor. Bacterial growth was expressed as a fold-change relative to the bacterial load of the initial inoculum (t=0 h).
Competition between bacterial strains for scFOS
The bacterial ability to compete for scFOS as an energy source was assessed by comparing S. epidermidis versus C. acnes and S. epidermidis versus S. aureus, at increasing concentrations of scFOS (from 0 to 5%). After removal of residual growth medium, 10 × 106 CFU/mL were exposed to increasing concentrations of scFOS in minimal medium at 37°C for 48 h under shaking and aerobic conditions. At 0 h (initial bacterial load), 4, 8, 24 and 48 h, aliquots were taken and inoculated onto selective agar plates. For S. epidermidis versus S. aureus competition, inoculation was carried out on mannitol phenol red salt agar (MSARP), a differential medium capable of discriminating between the fermentation of mannitol (S. aureus) and fermenting without mannitol (S. epidermidis) strains.
For S. epidermidis versus C. acnes competition, a differential colony counting strategy was applied. Collected aliquots were streaked onto MSARP and blood agar plates, and C. acnes calculates as the difference between colonies grown on blood and MSARP agar.
When corrected for the appropriate dilution factor, bacterial growth was expressed as a fold change. The growth rate was expressed as the ratio between the factor of variation of S. epidermidis vs the fold change of C. acnes and between the change of fold of S. epidermidis vs the fold change of S. aureus.
Competition of bacterial strains for scFOS in an in vitro model of reconstructed human skin (RHE)
The impact of scFOS on bacterial growth was also evaluated on an in vitro model of reconstructed human epidermis (RHE) (Epiderm™, MatTek), characterized by a stratified epithelium and endowed with a morpho-functional characteristic similar to the human epidermis. As such, this model can be effectively applied in bacterial strain adhesion, colonization, and competition experiments. Briefly, the same number of bacteria (10 × 106 CFU) for each strain was added to the RHE surface. For competition experiences, S. epidermidis versus C. acnes and S. epidermidis versus S. aureus comparisons were assessed. After adhesion for 3 h at 37°C in a controlled atmosphere incubator (5% CO2 and 85% relative humidity), RHEs were thoroughly rinsed with phosphate-buffered saline (PBS) to remove non-adherent bacteria. Adherent bacteria were then allowed to colonize the RHE for 18 h at 37°C, and colonization was calculated as follows: Colonization (factor change) = tX (CFU/mL)/t0 (CFU/mL).
whereX was the number of CFU/mL following the colonization process and t0 was the bacterial load (CFU/ml).
RHEs colonized with bacteria were exposed either to minimal medium only (negative control) or to increasing concentrations of scFOS. At 0, 8, and 24 h, scFOS-treated and non-scFOS-treated RHEs were rinsed with Hank’s balanced salt solution (HBSS) and adhered bacteria were detached with a modified wash approach. The bacterial suspensions obtained were serially diluted and plated according to the specifications mentioned above. Bacterial growth was expressed as fold relative to the initial bacterial load. The growth rate was expressed as the ratio between the factor of variation of S. epidermidis against the change of fold C. acnes and between the change of fold of S. epidermidis against the change of fold S. aureus.
statistical analyzes
All statistical analyzes were performed with OriginLab software. To determine if statistically significant differences between treatments were present, a t-test analysis was performed. All data were presented as the mean ± standard deviation (SD) of three independent experiments. Differences between groups were considered significant at P
