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J. Cosmet. Sci., 75.6, 565–580 (November/December 2024)
*Address all correspondence to Alicia Roso, alicia.roso@airliquide.com
An Innovative Model Based on Wild Type Bacteria
Co-Culture to Identify Cosmetic Ingredients That Respect
the Skin Microbiota
ALICIA ROSO, SOPHIE PÉCASTAINGS, SOPHIE CAMBOS, RICHARD MARTIN,
LAURA BAUCHET, BENOÎT ROUBINET, LUDOVIC LANDEMARRE AND
CHRISTINE GARCIA
Seppic Research and Innovation, La Garenne Colombes, Franc (A.R., S.P., S.C., C.G.)
Mercurialis, Rochecorbon, France (R.M.)
GLYcoDiag, Orléans, France (L.B., B.R., L.L.)
Accepted for publication October 02, 2024.
Synopsis
Human skin is the home of thousands of microorganisms that are well adapted to that hostile environment.
Divesting from the hygienist theory, microorganisms are not considered as potential pathogens anymore, but
as symbionts that must be preserved. With this consideration in mind, cosmetic formulators have sought
ingredients able to preserve and not interfere with cutaneous bacteria. The aim of the present study is to
design a relevant and representative in vitro model of bacteria coculture able to identify ingredients that do not
harm skin microbiota. A protocol was designed to obtain a coculture of the five main bacterial representatives
of the face’s microbiota: Cutibacterium acnes Staphylococcus epidermidis Streptococcus mitis Micrococcus luteus, and
Corynebacterium xerosis, from wild isolates, and in proportions representative of the cheek microbiota. After
8 hours of contact with the test ingredients, bacteria concentrations were measured. A score establishing
the impact of ingredients was implemented, based on the logarithmic reduction of the five species. This
methodology correctly discriminated between known substances with neutral or bactericidal effects. Finally,
four substances used in cosmetic products were evaluated. The proposed model is adapted to screen for
new “microbiota-friendly” ingredients as a first step to design formulations intended to preserve the skin
microbiota.
INTRODUCTION
The human skin represents an interface of 1.5 –2 m2 between the body and its environment.
This complex surface is naturally colonized by a variety of microorganisms that, together,
constitute the skin microbiota. Bacteria are the main representatives (104–106 Colony
Forming Units, or CFU, per cm2), but depending on the area, yeasts and viruses can also
represent a significant proportion of the skin microbiota.1,2 Microorganisms colonizing the
skin, defined as commensals as opposed to transient microorganisms, are very well adapted

566 JOURNAL OF COSMETIC SCIENCE
to their habitat. Indeed, most of them are able to thrive in poorly nutritious conditions,
essentially dry, characterized by an acidic pH (around five) and exposed to varying climatic
conditions (ultraviolet radiations or UV, temperature, etc.).3 Despite a great variability in
the composition of the skin microbiota from one individual to another, common features
are described. On the face, the most abundant genera are Cutibacterium sp. (formerly
known as Propionibacterium sp.), coagulase-negative Staphylococci (CoNS), Corynebacterium sp.,4
Micrococcus sp. and Streptococcus sp.1
Most microorganisms are not harmful and some are known to benefit their host.5 For
instance, they can inhibit pathogens through nutrient competition or the secretion of
harmful molecules like bacteriocins.5 They can also play a role in the immune system
training, or contribute to the maintenance of the barrier function.6 Hence, the microbiota,
in symbiosis with its host, plays an important role for the skin. Nonetheless, the skin
is constantly exposed to harsh environmental conditions or to pollutants or xenobiotic
substances that can alter the homeostasis of this ecosystem.7,8 It has been reported that
the regular use of hygienic products can alter the skin microbiota.9–11 Most cosmetics also
contain preservatives to prevent the proliferation of potential pathogens.12
To ensure that cosmetic products are not harmful to the skin microbiota, researchers have
designed a variety of assays, as described hereafter. Some commercial solutions (“Microbiome
Friendly” certifications by My Microbiome or Byome Labs “Microbiome-Friendly+” seal
from Labskin, etc.)—more or less complex—are already proposed by contractors. The most
common assays consist in putting into contact a cosmetic ingredient or a formulation with
targeted commensal species of the skin (e.g., Staphylococcus epidermidis, Cutibacterium acnes,
Corynebacterium spp., etc.).14 These methods are easy and rapid to implement.13 However,
they are often achieved by working with microorganisms from collections, such as the
American Type Culture Collection (ATCC) or the Biological Resource Center of Institut
Pasteur (CRBIP) and grown in optimal laboratory conditions that are not relevant to the
conditions found on the skin. Moreover, they do not take into consideration the fact that
the skin microbiota is a consortium of several species interacting with each other.
Some models gain in complexity by depositing tested products on 3D (three-dimensional)
skin models colonized by microorganisms.13,15 Here, not only can the microorganisms
be analyzed following the adjunction of products, but also the impact on the skin
tissues. Another advantage is that several species of microorganisms can be inoculated
simultaneously on a reconstructed human epidermis (RHE), hence mimicking the skin
microbiota and giving more relevance to the test. However, mixing more than three
different species in 3D models is a technical challenge.
Finally, the “friendliness” of a product is sometimes evaluated through clinical assays. With
the use of next generation sequencing techniques, such trials offer accurate data about the
formulas’ effect on the microbiome.9,6 Nevertheless, the screening of multiple ingredients
can hardly be achieved with a single clinical trial, and this method seems more appropriate
for finished products.
The present work aims at designing an in vitro model to test the ability of cosmetic ingredients
to preserve the microbiota, and takes into account the complexity and specificity of the skin
ecosystem. Wild isolates from the main species of the human face microbiota (Cutibacterium
acnes, Staphyloccocus epidermidis, Corynebacterium xerosis, Micrococcus luteus, and Streptococcus
mitis) were collected from healthy individuals. Given the environmental conditions found
on the skin, the aim was to develop a coculture protocol (pH near 5.5, temperature near