584 JOURNAL OF COSMETIC SCIENCE
MATERIALS AND METHODS
RECRUITMENT OF PARTICIPANTS AND SAMPLING PROTOCOL
The study was conducted in accordance with the Declaration of Helsinki (statement of
ethical principles applicable to medical research involving human beings, including research
on human biological material and identifiable data) and informed consent was obtained
from all subjects involved in the study. Seventy-eight volunteers assessed by dermatologists
were found to have healthy skin (free of eczema, psoriasis, wounds, and inflammatory
scaring) and were enrolled in this study. We recruited males and females, aged 18–77 years
old, and having the skin phototype ranging from 1–5. Among the 73 volunteers on which
the analysis was completed, 30 reported having thin and sensitive skin, and were tested
for their hypersensitivity to heat prior to sampling. More precisely, recruited panelists of
the sensitive skin cohort had an electrodermal response evaluated at 155% after a heating
stimulation on the cheeks versus before (using skin conductance response rate measured
with a Galvanic Skin Response electrode).
The skin site investigated in this study was the face. Participants were asked not to wash
their face 24 for hours prior to sample collection, as well as not to apply any cosmetics
during this time frame. A cotton swab immersed in 1.5 mL of skin sampling buffer (10%
glycerol, 0.1% Tween80 in 1× phosphate buffer saline) was used for sampling. An area of
4 cm2 on the cheek was vigorously rubbed with the cotton swab for 30 seconds. Swabs
were then vortexed for 30 seconds at maximum speed in a 1.5 mL microtube to resuspend
biological matter in solution. They were squeezed to maximize the volume of SSB (Single-
Stranded Binding buffer Thermo Fisher Scientific, Massachusetts, United States) in the
microtube by centrifugation. The samples were concentrated in 400 microliters (µL) of 1×
phosphate buffer saline and stored at −80°C until DNA extraction.
DNA EXTRACTION
DNA was extracted using the kit Zymo BIOMICS™ DNA miniprep (D4300, Zymo
Research, Irvine, CA, USA) following manufacturer recommendations including a
mechanical lysis step with BashingBead™ Lysis tube. Blank extractions without swab
samples were also performed to check for potential contamination. The final DNA was
eluted in 50 µL of nuclease-free water. DNA samples were kept at −80°C until bacterial
quantification.
DNA QUANTIFICATION
These TaqMan™ probe-based quantitative polymerase chain reaction (qPCR) assay
(Thermo Fisher Scientific) was used to measure the bacterial concentration of the samples.
The TaqMan™ probes have a 5′ fluorescent reporter dye and a 3′ quencher dye. These
probes are target-specific, and only bind to the DNA sequence of interest downstream
of one of the primers during the annealing step. A culture of Escherichia coli (E. coli) was
grown overnight at 37°C in brain heart infusion (BHI) medium, which was followed by
a DNA extraction. The eluted DNA was quantified using Qubit™ dsDNA HS assay kit
(Thermo Fisher Scientific) following manufacturer recommendations using 5 µL of E. coli
DNA solution to generate standard curves.

585 Specificities of Microbiota From Sensitive Skin
Twenty microliter reactions were performed in triplicate with the following reactants: 10 µL
PrimeTime™ Gene Expression Master Mix, 0.8 µL of forward primer, 0.8 µL of reverse
primer, 0.4 µL of probe, 6 µL of nuclease-free water, and 2 µL of DNA template. A reaction
was performed with a blank extraction as a DNA template, and another was performed
with nuclease-free water as a DNA template. The reactions were performed in white, low
profile 96-well plates, sealed with thermoresistant optical films. The qPCR was performed
in Agilent Aria Mx machine with the following cycling conditions: initial denaturation
95°C—3 minutes—1 cycle denaturation 95°C—5 seconds annealing/extension 60°C—
40 cycles final extension 60°C for 5 minutes and 1 cycle (Agilent, Les Ulis, France).
PACBIO LIBRARY PREPARATION
The primers used to amplify the full-length 16S ribosomal ribonucleic acid (rRNA) gene
were composed of the specific regions 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and
1492R (5′-CGGTTACCTTGTTACGACTT-3′) combined to asymmetrical barcodes. PCRs
were performed with the following solution mix: 10 µL of Q5 reaction buffer, 1 µL of
10 mM deoxinucleotides triphosphates (dNTPs), 2 µL of forward primer at 10 µM, 2 µL
of reverse primer at 10 µM, 2 µL of DNA template at 10-2 ng/µL, 0.5 µL of Q5 high-
fidelity DNA polymerase, 32.5 µL of nuclease-free water. The cycling conditions were as
follows: initial denaturation 95°C—during 30 seconds for 1 cycle denaturation at 95°C for
5 seconds during 30 cycles annealing at 59°C during 30 cycles extension at 72°C for 45
seconds and final extension at 72°C for 2 minutes during 1 cycle.
Amplifications were checked by electrophoresis on 1% v/v agarose gel for a 30 minute
migration, using 100 mV to assess the success of PCR reactions. Nucleic acid fragments
were separated by their length while moving through the agarose matrix then visualized
under ultraviolet light after adding intercalating agent like ethidium bromide (a band
was expected around 1,500 base pairs). Samples were then purified using AMPure™ PB
beads (PacBio, SanDiego, CA, USA) following manufacturer recommendations. The entire
PCR volume was mixed in equal ratio with AMPure™ XP beads, and two washes were
performed with 80% ethanol. Finally, DNA was eluted in 25 µL of nuclease-free water.
DNA concentration was measured using Qubit™ dsDNA HS assay kit (Thermofisher
Scientific) following manufacturer recommendations. Three µL of eluted DNA was used
for quantification. Samples were pooled in equal amounts to reach a final DNA mass of
1,000 ng. In total, 3 DNA pools were generated to ensure sufficient sample coverage. Pools
were then kept at −20°C until completion of library preparation.
Pools were sent to Maryland Genomics (Baltimore, United States) to perform a PacBio short-
insert library preparation, which included DNA damage repair, end repair/A-tail, ligation,
and AMPure™ PB bead purifications. Each pool was sequenced in a PacBio Sequel II 8M
run, 30-hour movie, to generate circular consensus sequences. Samples were demultiplexed
and exported in FASTQ format. FASTQ format is a text-based format for storing both a
biological sequence (usually nucleotide sequence) and its corresponding quality scores.
MOCK COMMUNITY PREPARATION
To assure the quality of the sequencing and bioinformatic workflows, as a positive control
a mock community composed of 14 wild species previously isolated from the skin was