575 COSMETIC INGREDIENTS THAT RESPECT SKIN MICROBIOTA
MODEL DESIGNING, STRAINS SELECTION
Skin microbiota is composed of more or less 1,000 species per person,17 according to data
from 16S rRNA amplicon sequencing, or Shotgun sequencing. These communities do not
consist in a random combination, but rather in the specific association of microorganisms
particularly fit to their environment, as illustrated by the different consortia colonizing the
different zones of the skin (sebaceous, dry, moist). Considering that microorganisms adhere
to the skin surface, some authors describe these communities as biofilms. Indeed, Jahns
et al. (2014) were able to observe biofilms of C acnes in the pilosebaceous units.18 In complex
consortia, microorganisms have complex relationships.19 Today, there is growing evidence
showing that bacteria in microbial communities behave differently than in axenic culture,20
and produce a wider variety of metabolites.21 Recently, Benitez et al.22 also demonstrated
that some bacteria were more resistant to antibiotics when incubated in a coculture with
resistant strains, highlighting the beneficial impact of interactions on the bacterial behavior.
Therefore, a culture model using multiple wild-type species, in proportions relevant to the
skin microbiota, was designed to study the effect of exogenous substances. This model
could be the first step of a more complex design to study multispecies biofilms.
The methodology used here to collect wild type bacteria on the skin involved an enrichment
and a growth step on laboratory media. Consequently, only cultivable bacteria were collected
and integrated into the model. It also means that the isolated species is the one that grew
faster on artificial media, but not necessarily the most representative of a bacterial genus on
the skin (i.e. for Corynebacteria).
While the proportions in the microorganisms vary in each individual’s microbiota, it is
clear that C acnes is the most prevalent representative of the face microbiota. Cutibacterium
acnes is adapted to thrive in low-oxygen environments and able to metabolize lipids.
Consequently, it is mostly found on sebaceous areas (back, shoulder, face), and more
specifically in the pilosebaceous units. While C acnes is notoriously known to be involved in
the acne pathology, it is now admitted that the loss of balance between the different strains
(phylotypes) is partly responsible for this skin condition, rather than the simple presence
of the bacteria.23 Besides, positive effects have been identified (secretion of antioxidant
molecules,24 maintenance of a protective acidic pH in the pilosebaceous unit against
pathogens25), and C acnes is now recognized as an important part of the microbiota, with
proportions reaching more than 90% of the face microbiota.26,27
S epidermidis is a coagulase negative Staphylococcus, and one of the most studied skin
commensal.28 Its beneficial roles for the skin are now better understood. For instance, the
secretion of bacteriocins like phenol soluble modulins and lantibiotics allow S epidermidis to
control the proliferation of potential pathogens. Other competing commensals like C acnes
can also be the target of S epidermidis, through various mechanisms like the secretion of
deleterious fermentation products or type VII secretion systems.29 The positive roles of
S epidermidis for the skin now extend to the participation in the regulation of inflammation30,31
or, as described recently, in the maintenance of the skin barrier, through the secretion of a
sphingomyelinase triggering ceramide synthesis in the skin.32 The literature indicates that
it is present on all body sites, with a predominance on moist areas.33
Corynebacteria are a diverse phylogenetic group, as illustrated by 16S rRNA amplicon
sequencing studies of the skin microbiota.34 They are composed of lipophilic bacteria4 that
lack essential enzyme activities (proteinase, phospholipase, saccharolytic enzymes) and are
sensitive to low pH, which is unexpected for skin residents. As a result, Kwaszewska et al.35
576 JOURNAL OF COSMETIC SCIENCE
suggested that Corynebacteria must have mutualist trophic relationships with the main skin
colonizers, like Staphylococci. The literature mentions that Corynebacteria colonize all body
sites (dry, sebaceous, moist), even if in low proportions on the skin (10%)1, and Dimitri
et al. (2019) also suggested that some species like C kroppenstedtii are associated with skin
aging and wrinkles. Overall, these data suggest that Corynebacteria are ubiquitous members
of the skin microbiota.
Micrococcus sp. and Streptococci are low abundant species (5%),36,9 yet several studies of the
face microbiota report their presence and describe potentially beneficial roles for the skin.
For instance, Streptococci have been associated to the recovery of the barrier function in vitro
through the secretion of spermidine while Micrococci have been positively associated with
protection against signs of skin aging.37,38
Together, these data indicate that not only C acnes, S epidermidis, Micrococci, Streptococci, and
Corynebacteria are representative of the skin microbiota, but they also play key roles in the
skin homeostasis maintenance. One of the challenges of the present study was to obtain
a consortium with these five species. Despite their different nutritional needs and growth
requirements, an adapted protocol was elaborated that allowed the growth of wild isolates
for these five bacteria.
REPRODUCIBILITY OF THE MODEL
Overall, the species proportions (%)in the coculture after 8 hours of incubation are close
to those that are described in the literature (Figure 4).9 Nevertheless, the total bacterial
concentration in the consortium after 8 h of incubation is of more than 109 CFU/mL.
Thus, percentages (per 102) are not relevant indicators to study the model reproducibility.
Consequently, the concentrations’ variability were analyzed rather than the proportions.
Coefficients of variation are inferior to 4%, indicating a very good reproducibility of the
coculture preparation protocol.
IMPACT OF COSMETIC PRODUCTS ON THE SKIN MICROBIOTA
In order to analyze the effects and compare tested products, the LR on the five species
were calculated. The calculation of LR is common to measure the bactericidal efficacy
of disinfectants or preservatives that indicates the quantity of microorganisms
eliminated by a substance in a given contact time.39,40 When studying biocidal
substances, LR at least equal to five log are requested for a treatment to be effective.
In a challenge test for cosmetic formulations, an LR of three is required on bacteria
to validate the preservative efficacy.41 On the contrary, LRs less than or equal to 1
(LR 1) are not considered significant. In the present study, a score (sum of the 5
LR) was calculated, in order to aggregate the data on the five species. Consequently,
scores between 0 and –5 (LR 1) are obtained with neutral ingredients for the
microbiota (microbiota-friendly). Such scores were observed with proline, glycine,
glycerol, glucose, and inulin. All these substances are potential substrates for bacteria
and could have stimulated bacterial growth. However, the contact time of 8 hours
was not long enough to observe a significant increase of bacteria. We concluded that
the model was stable enough to be well adapted to discriminate between neutral and
harmful substances.
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