569 COSMETIC INGREDIENTS THAT RESPECT SKIN MICROBIOTA
The species proportion (%)in the consortium was calculated by dividing the species
concentration (in CFU/mL) with the concentration of total bacteria (in CFU/mL) and
multiplying by 100.
For each species, the logarithmic reduction (LR) was also calculated from the logarithmic
bacterial concentrations in the exposed consortium versus the non-exposed consortium
(control), according to the following calculation:
LR =log (concentration after contact with the ingredient (log CFU/mL)/concentration of
the control without ingredient
(log CFU/mL)
The LR ​​obtained for the five bacterial species were added, in order to obtain a score (Sc)
per ingredient:
Sc =LR
C.acnes +LR
S.epidermidis +LR
S.mitis +LR
M.luteus +LR
C.xerosis
STATISTICAL ANALYSIS
Each condition (control, ingredients) was analyzed in triplicates. Bacterial concentrations
were expressed as mean values and standard deviation. In Figure 2, percentages were
expressed as mean values and confidence intervals (CI 95%). The coefficient of variation
(CV) was calculated according to the following equation: CV =(σ/μ) × 100, where σ is
the standard deviation and μ the mean value.
RESULTS
VALIDATION OF THE IDENTIFICATION OF WILD TYPE STRAINS
To be as representative of the cheek microbiota as possible, five species were sampled from
healthy volunteers with sterile swabs. After strains isolation, the precise characterization
of each isolate was undertaken, by MALDI-TOF and/or 16S rRNA gene sequencing. The
MALDI-TOF technique provides a mass spectrum fingerprint of the biopolymers from an
isolated microbial colony. Each species has a unique fingerprint that can be compared with
those of the bioMérieux database for precise identification. Similarly, the full sequence of
the 16S rRNA encoding gene of each bacterial isolate was compared to sequences from
the 16S rDNA 500 microbial library to obtain identification at the species level. Results
confirmed the characterization of seven isolates of C acnes, seven isolates of S epidermidis,
seven isolates of S mitis, seven isolates of C xerosis, and seven isolates of M luteus. Isolated
strains were stored either alone or mixed in equal proportion (pools of species). The pools
were used for the development of the coculture model (Figure 1).
COCULTURE OPTIMIZATION
The coculture protocol was optimized before testing ingredients. More specifically, the
nutrient concentration in the growth medium was lowered and the incubation temperature

570 JOURNAL OF COSMETIC SCIENCE
was set at 32°C. The pH (6.5) was the minimal acidity that all bacteria could tolerate
(data not shown). The incubation time was adjusted to obtain the correct proportions of
bacteria. The evolution of the five species in the consortium was monitored for 24 hours,
with sampling at 2, 4, 8, and 24 hours (Figure 2).
After 2 hours of incubation, C xerosis and S epidermidis concentrations were inferior to the
detection limit (102 CFU/mL). However, they rapidly grew (4 × 107 and 2.1 × 108 CFU/
mL respectively after 8 hours) so that their proportions reached 4% for C xerosis and 21%
for S epidermidis. The proportion of S mitis also increased steadily from 2.0% to 30.2%
at 24 hours. The concentration of C acnes increased from 2 to 8 hours (from 4.1 × 107
and 5.2 × 108 CFU/mL respectively), but its proportion decreased during that incubation
time due to the strong increase of the other species. More specifically, after 24 hours, the
proportion of C acnes was only 8.9%.
In general, after an incubation of 8 hours, the proportion of the five species was close to
what is generally observed on the skin of the cheek: C acnes 50%, S epidermidis 20%, S mitis
20%, and M luteus and C xerosis 10% each. Since 8 hours is also the daytime duration,
and thus a relevant contact time with cosmetic products on the skin, it was used to test
cosmetic ingredients.
Next, the optimal atmosphere for the growth of all species was selected by incubating the
consortium in aerobiosis or in anaerobiosis. The proportion of bacteria was calculated based
on their concentrations and results are presented in Figure 3.
With oxygen, the proportion of C acnes was 6% compared to 41% without oxygen. On the
contrary, S epidermidis and S mitis represent 50% and 33% of the population respectively
in the presence of oxygen. Anaerobiosis offers better condition for C acnes while preserving
the growth of the other strains. This condition was chosen to grow the model consortium
in subsequent assays.
Figure 2. Proportions evolution of the five species in the consortium according to time (total bacteria
concentration 1.0 × 109 CFU/mL at 8 h, 2.5 × 109 CFU/mL at 24 h). Species proportion (%)in the consortium
is the ratio between a species concentration (in CFU/mL) and the concentration of total bacteria (in CFU/mL)
multiplied by 100.