Device and cosmetic recommendation method for holistic characterization of the skin microbiota complex

Abstract

The present invention relates to the field of characterizing the microbiota present on human skin. More specifically, the present invention relates to a device for immunochromatographic detection of skin microbiota complexes, which allows for rapid and meaningful biological determination of two members of the skin microbiota symbiosis through immunological characterization of surface-accessible biological markers of microbiota-skin interaction. Then, using artificial intelligence, the device recommends the best care that is appropriate for the skin type and compatible with the microbiota, thereby reducing the risk of side effects. Similarly, in the case of skin defects and / or microbiota imbalances, the device recommends the best corrective treatment that is either guaranteed to be compatible with the microbiota or can regulate the microbiota.

Description

[Technical Field]

[0001] The present invention relates to the field of characterizing the microbiota (or human skin "microflora") present on human skin. More specifically, the present invention relates to a device for immunochromatographic detection of skin microbiota complexes, which allows for a rapid and meaningful biological determination of two members of the skin microbiota symbiosis through immunological characterization of surface-accessible biological markers of microbiota-skin interaction. Artificial intelligence is then used to recommend the best care that is appropriate for the skin type on the one hand and compatible with the microbiota on the other hand, thereby reducing the risk of side effects. Similarly, in the case of skin defects and / or microbiota imbalances, the present invention recommends the best corrective treatment that is either guaranteed to be compatible with the microbiota or can regulate the microbiota.

[0002] The field of the present invention is that of beauty care advice. While not presumed to be in the field of health, cosmetics must be able to serve as a care product for maintaining or improving the user's appearance. When they come into contact with various surfaces of the human body (epidermis, hair and capillary system, nails, lips, and external genitalia), they must not be harmful to health. For the purpose of providing personalized advice, the present invention proposes providing information on the skin and the associated microbiota that corresponds to the symbiosis, taking into account that the product must be adapted to the skin's needs in terms of hydration, hygiene, protection, appearance modification (complexion, color, etc.), and the sensitivity of the microbiota. For example, by recommending products containing active ingredients compatible with the maintenance or regeneration of the microbiota and by avoiding active ingredients that destabilize the microbiota (favoring microbial species that may induce the risk of opportunistic proliferation). By extension, the field of the present invention can be applied to the medical diagnosis of a patient's skin, making it possible to integrate clinical diagnoses regarding, for example, inflammatory conditions, allergic reactions (resulting from immune system reactions), and microbiological conditions (balanced microbiota or dysbiosis with a strong presence of opportunistic pathogens). The present invention can be an in-vitro diagnostic medical device, and by extension, can be used to monitor the condition of the skin over a period of time, to monitor the effectiveness of cosmetic routines, but also to monitor the effectiveness of preventative and / or curative treatments.

[0003] The field of this invention applies to humans as a "mammal-microbe hybrid," a "superorganism," because our microbiome outnumbers the cells that compose our body by 10 times. Therefore, the microbiome is understood to include all microorganisms, viruses, phages, bacteria, yeast, fungi, and even dust mites, that come into contact with the cells that compose the human body. Narrowing the scope to the bacterial microbiome reduces the size order by 1.3 times. This situation of human-microbe symbiosis is common throughout the animal and plant worlds. Evolution had to establish a eukaryote-prokaryote relationship that has become refined, diversified, and increasingly complex due to mutual selective pressures. The microbiome has multiple roles: it acts as a barrier against colonization by pathogens, has a maturation effect on the immune system as well as the skin itself, and makes a major metabolic and nutritional contribution by providing a capacity absent in mammals for the hydrolysis of complex plant sugars and the production of nutrients such as short-chain fatty acids and certain vitamins.

[0004] The field of the present invention considers together two symbiotic partners that have long been considered separately: the "skin" organ and the microflora that has long been called the "skin flora".

[0005] microbiome The "skin flora" constitutes the external microbiota of the human body (together with the microbiota of the gastrointestinal tract, which is the most important in terms of biomass, as well as the microbiota of the oral cavity, nasal cavity, auricular cavity, and in women, vaginal cavity). This flora acts as a barrier against external physical, chemical, and biological attacks and, depending on the site, interacts with the host organism, generally through cells of the immune system. Typically, bacterial biodiversity limits the risk of skin colonization by pathogenic bacteria and protects against inflammatory and allergic skin reactions.

[0006] Due to the microscopic size of the organisms that compose it, the human microbiome is invisible to our eyes. An adult's skin contains over 500 different species of microorganisms per cm of skin. 2The skin naturally supports and maintains its own "microecosystem," which is formed at birth and then evolves until death. The skin microbiota is a community of microorganisms physically formed on the surface and organized into a biofilm. It feeds on both molecules and compounds excreted by the skin itself and, to a greater or lesser extent, compounds secreted by commensal microorganisms, primarily bacterial communities organized into biofilms. The composition of the skin microbiota varies with the individual, age (neonatal, adolescent, adult, elderly), gender (puberty, menopause in women), activity, behavior, and environment. Skin biofilms constantly regenerate themselves, adapting to the skin's natural desquamation and differentiated erosion, as well as personal hygiene practices (which should not be excessive to protect the skin's "barrier" function and should not reduce the diversity of the microbiota that protects against inflammation). Furthermore, they evolve with age. In children, bacterial biofilms differ according to the mode of delivery. By genital route, infants are colonized by a community similar to the mother's vaginal microbiota, whereas by cesarean section, they are colonized by a community similar to the skin microbiota. After one month, the skin colonization profile steadily changes, gradually establishing taxonomic specificities (viruses, bacteria, yeasts, fungi, and even mites) for various differentiated body sites: external skin sites (face, scalp, etc.), cavities (oral cavity, nasal cavity, vagina), and internal sites (mainly the digestive tract). A recent study (Huang, 2020) determined which body region (gastrointestinal tract, oral cavity, skin) of the microbiota could most accurately predict age and found that skin was the best, providing accurate predictions within an average of four years. The importance of the level of symbiosis between the microbiota and the skin according to age is increasingly substantiated ( Kim, 2019 ; Trojahn, 2015 ), confirming, on the one hand, the need to ensure that cosmetics applied to the skin are compatible with the resident microbiota, and, on the other hand, that it may be advisable to monitor the evolution of the symbiosis of the human microbiota throughout life.

[0007] The microbiota plays a role in human skin odor, which also evolves throughout life. Odor production can result from the breakdown of sweat compounds, such as volatile fatty acids or odorous steroids (James et al., FEMS Microbiology Ecology, 2013), involving specific bacterial genera, such as strains of Corynebacterium and Streptococcus. Short-chain fatty acids, such as caprylic acid, capric acid, pentanoic acid, and propionic acid, also contribute to the development of body odor. These are the products of the breakdown of amino acids and long-chain fatty acids derived from sebum by the bacteria Brevibacterium and Propionibacterium. Propionic acid imparts a hazelnut scent to skin at low concentrations (typically after showering) and a cheese scent to soiled skin at high concentrations. These body odors vary based on gender, age, and menstrual cycle.

[0008] Skin biofilms play both positive and negative roles. The formation of a commensal microbiota on the skin corresponds to the colonization of microbial groups that play a defensive role against other pathogenic microorganisms, which corresponds to infection and can be identified by next-generation sequencing analysis techniques. For example, in the case of wound infection (an extreme case in which the skin is not a homogeneous, continuous surface but exhibits discontinuities and fissures), a group of bacteria known as the "pathogenic group" (Proteus, Morganella, Anaerococcus, and Peptoniphilus) has been described, and its presence correlates with worsening, non-healing wounds (Dunyach-Remi et al., 2020). On the other hand, even in the case of wound infection, bacteria have been confirmed to exert antibacterial activity against pathogens (Nakatsuji et al., 2017) or reduce their virulence (Ngba Essebe et al., 2017), thereby protecting the host and giving rise to the concept of a "positive flora."

[0009] The microbiota present on the skin is invisible except when it triggers a skin reaction from the immune system, such as acne associated with the proliferation of the bacterium Cutibacterium acnes, or redness associated with inflammatory conditions such as eczema or atopic dermatitis exacerbated by colonization with the bacterium Staphylococcus aureus. There is also a correlation between blemishes on the skin and the presence of certain microorganisms, such as those of the genera Kocuria and Aerococcus (Zanchetta et al., 2022).

[0010] Microscopy has long been the primary method for observing the skin microbiome. Advances in biomolecular technologies have opened up new fields of research through genomics, molecular genetics, metagenomics, high-throughput sequencing, and culturomics. Through direct sequencing of the DNA present in a sample, this approach not only provides a genomic description of the sample's contents but also insights into the functional potential of the environment. Biologists study metagenomic samples using high-throughput sequencing techniques. The resulting DNA sequences are then analyzed using bioinformatics techniques. Metagenomic studies require the logistics of transporting samples, typically collected from a person with a simple swab, to an analytical laboratory, which must be equipped with a suite of instruments for extracting and sequencing genomic material (DNA).

[0011] To detect the presence of these microorganisms in biofilms, which are characteristic of skin conditions, it is also possible to use biological markers, molecules present on the surface of membranes, skin cell adhesion molecules, and secreted molecules (exopolysaccharides, DNA, proteins, etc.) that form the matrix of the biofilm. These biological markers can be collected from a person and immediately analyzed using lateral flow immunoassay (LFIA) technology.

[0012] Therefore, pathogenic microorganisms can be distinguished from commensal organisms based on pathogen-associated molecular patterns (PAMPs). These so-called "virulence" markers are the most widely documented and described in the literature. These include secreted molecules such as the Panton-Valentine Leukocidin protein of Staphylococcus aureus, but also fluorescent molecules such as porphyrins produced by Cutibacterium acnes, which are visible under ultraviolet light and induce Staphylococcus aureus aggregation and the expression of pro-inflammatory molecules by keratinocytes (IL-6, IL-8, prostaglandin E2, TNF-α, etc.), with a lower degree of toxicity.

[0013] Other markers are characteristic of natural biofilm formation by microorganisms on the skin surface and in the pilosebaceous follicular ducts (see below). Microorganisms generally interact with a family of skin proteins known as extracellular matrix (ECM) components: plasminogen, fibronectin, laminin, or mucins. For Gram-positive bacteria (single membrane), adhesion proteins are used: Bap family adhesins, type 4 fimbriae (important for initial biofilm formation), SAATs (self-associating autotransporters, which promote aggregation between SAAT-carrying bacteria), and intimin / invasin. For Gram-negative bacteria (double membrane LPS), Bap family adhesins displaying the LPXTG C-terminal domain, IMPs (inner membrane proteins), and type 3 and type 4 fimbriae.

[0014] One example is Cutibacterium acnes, a commensal bacterium that can become an opportunistic pathogen and cause acne. This bacterium can form biofilms on either pilosebaceous follicles or keratinocytes, thanks to proteins present on the surface of the bacterial membrane, such as CAMP factor (Christie-Atkins-Munch-Petersen factor), sialidase, dermatan sulfate adhesin, endoglycoceramidase, groEL chaperonin, "SH3 domain-containing lipoprotein," fimbria / pilus-like protein Flp, and, among others, the DsA1 protein, which forms part of the family of MSCRAMMs (molecular surface components recognizing matrix adhesion molecules). This biofilm is highly immunogenic and induces a strong immune response in acne sufferers.

[0015] Staphylococcus species play a major role in the skin microbiome, with Staphylococcus epidermidis being a commensal and rarely pathogenic species, and Staphylococcus aureus, a commensal species with healthy nasal carriage in 30% of the population, which can become pathogenic, especially in cases of atopic dermatitis or eczema. Staphylococcus species also form biofilms on the skin using MSCRAMMs, such as proteins from the Clf-Sdr family, Bbp (bone sialoprotein-binding proteins), FnBPs (fibronectin-binding proteins), and CNA (collagen adhesion). The SesJ protein was recently identified for S. epidermidis (Arora 2020). For S. aureus, aureusimine (phevalin) appears to be a marker of the biofilm phenotype. It should be noted that Staphylococcus also secrete a polysaccharide intercellular adhesin (PIA) extracellular matrix.Other main representatives of the skin microflora are Corynebacterium tuberculostearicum, Malassezia Globosa; characteristic of oily, dry or moist skin: Staphylococcus capitis, Staphylococcus hominis, Streptococcus mitis, Streptococcus oralis, Micrococcus luteus. luteus, Corynebacterium simulans; or microorganisms characteristic of aged skin: Dermococcus, Actinomycetes; Bacteroidetes (e.g., Bacteroides, Alistipes, Prevotella, Porphyromonas, Sphingobacterium), or Firmicutes (e.g., Lactobacillus, Aerococcus, Oscillospira, Ruminococcus) in young skin, common to all skin types (Byrd et al., 2018).

[0016] Organisms often considered non-pathogenic can cause infection in immunocompromised hosts or those recently treated with antimicrobials. When the immune response is impaired, as is often the case in people with diabetes, it is unable to prevent colonization of injured tissue by pathogenic bacteria. In chronically infected wounds, many bacteria form biofilms, where they irreversibly attach to surfaces, grow, produce extracellular polymers that promote matrix formation, and modify their phenotype.

[0017] The most common bacteria found on human skin are gram-positive and belong to five main genera. The following bacteria have been found: Staphylococcus, often opportunistically as Staphylococcus epidermidis, account for over 90% of the aerobic resident flora present on the stratum corneum. Other staphylococci found in skin biofilms are Staphylococcus aureus (commonly found, e.g., in asymptomatic nasal carriage in 30% of cases) and Staphylococcus hominis. Corynebacterium, Propionibacterium (e.g., Cutibacterium acnes, Propionibacterium granulosum, Propionibacterium avidum). Cutibacterium acnes produces fatty acids from the lipolysis of sebum. In doing so, it acidifies the skin environment, increasing the risk of Streptococcus pyogenes, ●Lactobacillus, ●Inhibits the growth of Streptococcus colonies.

[0018] In general, three types of bacteria are encountered in the majority of cases: 1) Staphylococcus epidermidis, a commensal skin bacterium that acts as a barrier against Staphylococcus aureus; 2) Cutibacterium acnes, a commensal skin bacterium that is generally harmless but can cause acne episodes depending on age (puberty), hormonal infiltration, and immunosuppression (transient, iatrogenic, etc.); and 3) Staphylococcus aureus, a commensal bacterium that is asymptomatically carried by 30% of individuals (nasal carriage) and that causes various skin pathologies (impetigo, atopic dermatitis, etc.).

[0019] To give an overview of the field of the invention, it can be said that skin diseases are largely forgotten in public health because they are rarely life-threatening, but they affect the quality of life of 16 million French people, are often accompanied by an underestimated psychological impact, and are associated with skin defects (blemishes, redness, acne, etc.) that patients attenuate or conceal by using cosmetics.

[0020] According to an extensive epidemiological study carried out by the French Society of Dermatology (SFD) (the "Objectifs Pau" study carried out in 2016 on a representative sample of 20,012 people over the age of 15), in France, one in three people suffers from a skin disease. And 80% of the patients involved even have two skin diseases. Women are more susceptible than men, with 33% of women having a dermatological pathology compared to 28% of men.

[0021] These alarming figures are much higher than previous estimates. The increased stress and pollution of modern life certainly play a significant role in the increasing number of skin disorders.

[0022] Among the most common skin conditions, acne tops the list (3.3 million French people suffer from it even after puberty), followed by eczema (2.5 million affected) and psoriasis (2.4 million). Scalp diseases (excluding alopecia), fungal diseases and nail diseases come second, affecting 2.3 million, 2.2 million and 2.1 million French people respectively.

[0023] For all of these conditions, there is a combination of individual factors that influence the skin condition (genetic susceptibility—mutations in genes encoding skin proteins such as filaggrin, a defective immune system, etc.—lifestyle—stress, UV protection, etc.—diet, etc.) and factors that influence the microbiota (cesarean section, hygiene, environment, etc.). Regarding the effects of UV irradiation, a pilot study (Burns, 2019) showed an increase in the phylum Cyanobacteria, a decrease in the families Lactobacillaceae and Pseudomonadaceae, the reactivity of different species to UVA and UVB, and the potential protective and anti-inflammatory role of Lactobacillaceae. Again, studies to evaluate the effects of UV exposure on the dysbiosis of the human microbiota could help improve advice on, on the one hand, products that rehydrate the skin and regenerate / rebalance the microbiota (therapeutic advice), and, on the other hand, protective sun creams (preventive advice).

[0024] This is why, since it has become clear that the claimed effects of anti-wrinkle and anti-aging creams and various beauty skin care products are not similar in their effects and effectiveness for all individuals, or even for the same individual over all periods, cosmetic companies have been interested for several years in understanding the skin microbiome in order to provide customers with appropriate and customized compositions that allow them to prepare active creams that are suitable for the skin's physiology (oily, dry, irritated, with a certain level of inflammation, etc.) and that are also best suited to the state of each customer's skin microbiome at a given period. Different people and different skin types react differently to cosmetics, and therefore there is a need for a device that can determine an individual's effect or responsiveness to a specific type of skin care product. This requires a reliable and simultaneous method for analyzing skin condition and skin microbiome.

[0025] skin The second member of the symbiosis that is relevant to the field of the present invention is the "skin" organ. The exposed part of the skin is called the stratum corneum, and is made up of overlapping anucleate cells and fully keratinized cells, i.e., corneocytes, which form very elongated lamellae (ROBERT et al., Dermopharmacologie, Edisem, 1985). Its thickness is about 10 μm, except on the palms of the hands and the soles of the feet, where it is about 10 times thicker.

[0026] The stratum corneum is the final external product of this organ, the skin, and is 1.5-2 m thick. 2 The skin is structured into three layers of tissue: the subcutaneous tissue (deepest), the dermis (middle) and the epidermis (most superficial).

[0027] The subcutaneous tissue forms the deepest layer of the skin. It is a richly vascularized connective tissue that contains a lot of adipose tissue, itself made up of cells called adipocytes. This layer has high elasticity, is able to absorb shock, and also insulates the body.

[0028] The dermis is the middle layer. It also contains connective tissue supporting the epidermis. It is crisscrossed by a rich network of capillaries and swarmed with numerous nerve endings. The dermis is divided into two layers: the papillary (superficial) dermis and the reticular dermis (deep and middle). Fibroblasts scattered throughout the dermis synthesize collagen and elastic fibers immersed in a type of gel called the extracellular matrix, which consists of water and glycoproteins. This acts as a water reservoir that can be affected by environmental factors (dry winter weather) or excessive exposure to ultraviolet light (sunbathing, UV booths, etc.), resulting in increased evaporation. Immune system cells such as macrophages, dermal dendritic cells, mast cells, lymphocytes, and mature cells are also present. The rich vascularization of this layer supports several functions. This allows the body's first line of defense, using the microbiota, to effectively respond to any danger signals, especially in cases of dysbiosis or when a wound creates a breach in the protective upper outer layer. The epidermis, lacking capillaries, derives from them the energy and nutrients necessary to ensure its cellular activity. Along with sweat glands, it plays an important role in thermoregulation. Sweat glands and pilosebaceous follicles are epidermal appendages implanted in the dermis.

[0029] The epidermis is the outermost avascular structure and is divided into five overlapping layers (from inner to outer). - The basal layer (stratum basale) allows the skin to renew itself by cell division (almost all cells are in mitosis), which allows the cells produced to migrate towards the outermost layer. This layer is mainly composed of keratinocytes. - The stratum spinosum or Malpighian layer is the thickest layer of the epidermis. It is composed of keratinocytes. The thinner granular layer (stratum granulosum) is composed of keratinocytes undergoing apoptosis. Their cytoplasm contains fewer cytoplasmic organelles and less nuclear chromatin. The cells are flatter and are characterized by the presence of keratohyalin granules and lamellar granules (or Odland bodies). -The stratum lucidum (stratum lucidum) has anuclear cells (their nuclei are missing). Keratohyalin granules are converted into a protein called filaggrin. - The stratum corneum (stratum corneum) is the most superficial layer and is composed of flattened dead cells known as corneocytes. They are said to be dead, but remain biologically active. They have lost all organelles and are replaced by dense keratin filaments, bound together by an interlipid cement of fatty acids, cholesterol, and ceramides, as well as corneodesmosomes. This cement is formed from Odland's bodies found in the preceding layers. These tightly packed, connected cells form an impermeable coating on the skin and play an important defensive role.

[0030] The keratinization process ensures the continuous renewal of the skin from the stratum basale to the stratum corneum within 3-4 weeks. Various components are found on the surface, and the microbiota forms a biofilm on the surface of the stratum corneum. Biological markers of skin condition are now found to allow assessment of whether the skin is in a healthy state, whether it is subjected to hormonal penetration (puberty, menstrual cycle, etc.), inflammation (endogenous or exogenous), allergic reactions, or aggressions (physical: UV rays, pollution; chemical: personal care products, cosmetics; mechanical: abrasion during scrubbing, exfoliation, peeling, etc.).

[0031] The stratum corneum is the first line of defense between the human body and the outside world, acting as a barrier to penetration by foreign substances and preventing the passage of harmful microorganisms or chemicals into the body. If this first level of defense fails, other defense mechanisms, including cells of the immune system, are activated.

[0032] The stratum corneum is the first level of the interactome, in direct contact, where the microbiota uses adhesion proteins to form biofilms and secretes exopolysaccharides that allow them to persist on the skin.

[0033] When the physical barrier of the stratum corneum no longer functions, for example, superficially after significant exfoliation or peeling, or more deeply in the case of trauma or surgical wounds, due to physical removal by abrasion, the skin's immune system enters action. Its purpose is to protect the host and, if necessary, restore skin integrity. It is divided into two types: innate immunity and adaptive or specific immunity. In both cases, the primary protectors are the most numerous (90% of skin cells) keratinocytes. They act as immune sentinels, recognizing foreign substances through pattern recognition receptors (PRRs) from the Toll-like receptor family, which, upon recognition, synthesize chemical mediators (cytokines and chemokines), initiating the inflammatory cascade. On the other hand, keratinocytes can also produce antimicrobial peptides that inhibit microorganisms such as Staphylococcus aureus and Candida albicans.

[0034] The epidermis contains two types of immune cells. Dendritic cells, or Langerhans cells, have pseudopodia that allow them to bind to and phagocytose pathogens. These cells are known as APCs (antigen-presenting cells). When a pathogen or antigen is phagocytosed, dendritic cells secrete chemical mediators (prostaglandins or chemokines) that cause local vasodilation, thus increasing blood flow and the local recruitment of other immune cells, such as polymorphonuclear neutrophils and macrophages. Skin immune cells (dermal dendritic cells, macrophages, mast cells) are also recruited to the affected area. The epidermis also contains T lymphocytes activated in lymph nodes by APCs. Thus, after APC activation, a cascade of proliferation and differentiation occurs, ultimately leading to the destruction of the pathogen. These biological markers can be used to assess the condition of the skin surface, particularly the level of inflammation, known as "inflammation," which can be chronic and become increasingly critical with age.

[0035] Comprehensively, biological markers of skin inflammation include interleukins IL-1 beta, IL-4, IL-6, IL-8, IL-11, IL-12, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), and transforming growth factor-beta (TGF-beta). Although not directly considered a marker of inflammation, immunoglobulin E (IgE), whose serum levels are elevated in cases of atopic eczema-dermatitis and which is responsible for the majority of so-called IgE-mediated allergic reactions, must also be mentioned.

[0036] Other skin cells are involved in maintaining a healthy state in the face of solar attack or exposure to ultraviolet light (e.g., in a booth using a UV lamp). These include melanocytes present in the skin, which play a protective role against ultraviolet (UV) radiation. When skin is exposed to UV light, keratinocytes secrete the melanotropic hormone or α-MSH (α-melanocyte-stimulating hormone) by maturing the prohormone POMC (proopiomelanocortin). This α-MSH hormone attaches to membrane receptors called MCRs (melanocortin receptors) expressed by melanocytes and induces melanin biosynthesis, which is responsible for skin tanning. This thus protects the genetic material of epidermal cells exposed to UV radiation.

[0037] The description of the "skin" organ is completed with the appendages contained in its different layers: hair, sebaceous glands, and sweat glands.

[0038] Hairs of various sizes cover almost the entire human body. They play a very important role in thermoregulation. They are anchored in so-called hair follicles in the dermis.

[0039] The sebaceous glands attached to the hair are intradermal glands. They are a major component, along with sweat, of the lipid film that protects the skin and prevents it from drying out. These glands interact with the immune system and, as we will see later, have a subsequent effect on the microflora.

[0040] Sweat glands secrete sweat, a process that allows the body to combat heat. Indeed, when body temperature rises, for example during a sports session, or when ill, the sweat released evaporates on the surface of the skin. This mechanism removes heat and, together with vasodilation, contributes to cooling the body.

[0041] Of these appendages, the sebaceous glands play a central role in regulating the pH of the skin, its more or less obstructive nature and therefore its access to oxygen (influencing the aerobic or anaerobic capacity of the microorganisms of the microflora), its antioxidant effect, its promoting and / or antibacterial effect (variable depending on the microbial species), and the transport of hormones / pheromones. Sebum is composed of triglycerides, diglycerides, free fatty acids, wax esters, squalene and cholesterol.

[0042] Symbiosis of the skin microbiota The symbiosis of the human microbiota at the skin level is becoming increasingly complex, as it has recently been revealed that so-called "innate lymphoid" immune cells (ILCs) play a regulatory role on sebaceous glands (Kobayashi et al., 2019). These ILCs limit sebaceous gland growth (by producing "TNF receptor ligands"). When these ILCs are deficient, sebaceous gland hyperplasia is observed, leading to increased production of antimicrobial lipids, particularly palmitoleic acid and oleic acid, which affects the balance of the skin microbiota. Indeed, palmitoleic acid inhibits the growth of Gram-positive aerobic cocci such as Staphylococcus aureus and Staphylococcus xylosus, but not Gram-negative anaerobic bacteria such as Bacteroides species. Combined with the overproduction of oleic acid, this creates an occlusive film, i.e., anaerobic conditions unfavorable for Gram-positive cocci. This may lead to cosmetics containing these two fatty acids not being recommended if biological markers correlating with the "oily skin + high presence of Staphylococcus" profile are characterized with the immunochromatographic detection device that forms the subject of the present patent.

[0043] The human microbiota dysbiosis is also organized at levels other than that of the epidermis-stratum corneum, but more broadly based on various endogenous factors such as body area (T-zone of the face, scalp, axillae, etc.), age, hormonal penetration (puberty, menstrual cycle, etc.), inflammation (endogenous origin - atopic dermatitis, psoriasis, etc. - or exogenous - contact with irritating products), and insults (physical: UV rays, pollution); chemical: personal care products, cosmetics; mechanical: abrasion during exfoliation or peeling, etc.).

[0044] The body region is segmented into three zones characterized by different physicochemical parameters.

[0045] -Wet Zone The so-called "wet" zones are characterized by a high density of sweat glands. Moisture is due to the secretion of sweat by these glands. Sweat is composed mainly of water, inorganic salts, uric acid and urea. The relevant zones of the human body are characterized by a high density of microorganisms (10 5 ~10 8 bacteria / cm 2 The areas with high levels of colonization by B. difficile are the axillae, perineum, interdigital folds, palms, and axillae.

[0046] -Lipid Zone The so-called "lipid" zones are associated with a high presence of sebaceous glands. These glands secrete sebum, which flows to the surface of the skin in the form of a lipid film. The head, especially the cheeks, forehead, nose, chin, trunk and upper back, are the areas with the highest levels of sebaceous glands in the 10 6 ~10 7 bacteria / cm 2 The primary lipid zone has a microbial concentration on the order of 0.1 μm.

[0047] -Drying zone The so-called "dry" zones are poorly endowed with sweat and sebaceous glands and therefore contain less sweat and sebum secretions.

[0048] The backs of the hands and the outside of the extremities are the main dry zones. These are the areas with the lowest concentrations (10 3 ~10 4 bacteria / cm2 ) microorganisms.

[0049] The average pH of skin is 5.5. This is due to the lipid film that covers the entire surface of the skin, produced by secretions from the sweat and sebaceous glands. The relative acidity of this film provides protection, especially against pathogens. Skin pH is a very important parameter for maintaining skin homeostasis and the balance of the biofilm microflora. Changes in pH have been reported in the course of many pathologies, such as atopic eczema-dermatitis, psoriasis, and acne, and in the case of atopic dermatitis, which correlates with the presence of Staphylococcus aureus (Rippke et al., 2004). [Background technology]

[0050] Various solutions for analyzing the skin microbiome are known in the state of the art.

[0051] WO 2014 / 184151 discloses a "point-of-care" diagnostic device based on lateral flow analysis technology, allowing the non-invasive analysis of diffusible factors secreted from the skin surface. This document discloses, inter alia, a diagnostic kit for detecting the presence or amount of one or more analytes in a test sample taken from the skin surface of a mammal, the diagnostic kit comprising: a) a separate insert for a lateral flow device comprising a membrane, the membrane having a defined thickness, width and length, optionally attached to a rigid support, the separate insert configured to obtain a test sample (analyte); b) a lateral flow assay device configured to accept a separate insert; and c) an attachment element configured to releasably attach the separate insert to the skin surface of the mammal.

[0052] US Patent Application Publication No. 2022 / 0178943 provides a kit for detecting the presence or amount of one or more test analytes in a test sample obtained from a mammalian skin surface, the kit comprising: a) a lateral flow analysis (LFIA) device comprising a cassette containing one or more porous elements forming an array of porous supports, the cassette being configured to receive and hold a sample collection pad, the sample collection pad being configured to contact the array of porous supports when the sample pad is inserted into the cassette; b) a blister pack, the blister pack containing a buffer solution, and the cassette configured to receive the blister pack; c) a sample collection pad configured to be used to collect a test sample.

[0053] EP 3691788 also relates to a diagnostic kit for detecting the presence or amount of one or more test analytes in a test sample obtained from the skin surface of a mammal, the diagnostic kit comprising: a) a separate pad configured to be used to collect a test sample, the pad including a sample collection pad attached to a support member; b) a lateral flow analysis device including one or more porous elements, the lateral flow analysis device configured to receive and hold individual swabs, and the sample collection pad configured to form part of an array of porous supports when the individual swabs are inserted into the lateral flow analysis device.

[0054] WO 2019 / 025610 discloses diagnostic kits and methods based on lateral flow assay devices for detecting the presence or amount of one or more test analytes in a test sample taken from the skin of a mammal.

[0055] Disadvantages of the prior art There are either analytical solutions based on high-precision molecular biology techniques that are time-consuming, complex and expensive to implement, or lateral flow analytical solutions that only have one or two markers and are not suitable for skin characterization. Binary LFIA solutions only detect the presence or absence of biomarkers.

[0056] Background of the Invention The methods used to recover the amount of analyte sampled in the solution (e.g., cotton swabs or swabs, D-Squame or Sebutape adhesive strips) and the performance of lateral flow immunochromatographic tests require a minimum of 10 per milliliter. 4 Since analytes of the order of magnitude are required, genomic analysis solutions involving a DNA amplification step are used to obtain meaningful results.

[0057] These solutions are not entirely satisfactory because they require complex post-sampling procedures (such as DNA extraction, PCR, Q-PCR or sequencing) in the biological laboratory, involving personnel skilled in the use of genomics equipment.

[0058] To do this, they require extensive logistics, with processing times between sampling and genomic analysis ranging from days to weeks, and are relatively expensive.

[0059] As a result, the number of studies evaluating the effects of cosmetics or personal care products applied to all or part of the face (or the rest of the body) on the skin and its microbiome remains very limited to date (e.g., according to the clinicaltrial.gov website, in 2017-18, the study "The Effect of Skin Cleansers on the Skin Surface Microbiome" (sponsored by Mundipharma Manufacturing Pte Ltd) included only 12 volunteers, analyzed their skin microbiomes by next-generation sequencing, and compared the effects of three products: 1) 7.5% povidone-iodine, 2) 4% chlorhexidine skin cleanser, and 3) plain non-antibacterial soap).

[0060] Ultimately, the proposed metagenomic testing or analysis of the "microbiome" will lead to the generation of a list of identified microorganisms for recording purposes on a related smartphone application. The ultimate goal of this approach consists of recommending cosmetics and personal care products based on the information collected via the smartphone application when the "skin microbiome" test is ordered (a facial photo to assess parameters such as wrinkles, redness, etc., as well as declarative information such as age, weight, height, smoking habits, alcohol consumption, estimated skin condition (inflammation, dryness, oiliness), and pathologies (declaration of acne, eczema, atopic dermatitis, psoriasis, etc.)). Summary of the Invention [Problem to be solved by the invention]

[0061] The present invention improves upon the drawbacks of the background art by providing the following solutions. -fast -Inexpensive -contains sufficient elements to characterize the skin, and - Several levels of biomarker readings allow for sufficient information to make cosmetic recommendations. [Means for solving the problem]

[0062] The present invention therefore provides a device for immunochromatographically detecting a skin microbiota complex, comprising a support in which a transfer well is arranged intended to receive a solution containing a component of the skin microbiota complex, the well leading to a plurality of N detection zones, each of which has (i) an immunological detection reagent and (ii) an immunological capture reagent, the skin microbiota complex consisting of skin cells and microorganisms as well as other components of the skin microbiota, the device comprising: the number N of detection zones, where N is 5 or greater; at least one immunological detection reagent specific for a skin cell biomarker and at least one immunological detection reagent specific for a biomarker of the skin microbiome; at least one foolproof means for directing the reading of the device; Including, the immunological detection reagents each comprise an antibody specific for a biomarker of skin cells or skin microbiota conjugated to a colorimetric identification system, each capture reagent comprises an antibody bound to a support in each of the N detection zones, capable of recognizing a biomarker present in the zone; - at least three zones for detecting three biomarkers of the skin microbiome, wherein the biomarkers are bacteria from the genera Staphylococcus, Cutibacterium and Corynebacterium, respectively; - at least one zone for detecting a skin cell biomarker selected from (i) a structural protein, (ii) an inflammation biomarker, or (iii) an allergy biomarker.

[0063] The invention also relates to a disposable kit for characterizing skin conditions, a system for implementing personalized cosmetic recommendations, and a method for personalized cosmetic recommendation based on characterizing an individual's skin microbiome complex and implementing a device according to the invention.

[0064] Just as the intestinal microbiota interacts with the digestive tract in a "microbiota-host" symbiosis, the skin microbiota also interacts with the skin in a symbiotic relationship. Similarly, there are as many types of skin as there are microbiota, and damage to one member of the symbiosis affects the other members. This is important for maintaining healthy skin or correcting defects with beauty care products. The present invention allows for rapid and meaningful biological determination of the two members of the symbiosis through immunochromatographic characterization of biological markers accessible on the surface of the microbiota-skin interactome. Then, using artificial intelligence, the best care is recommended that is appropriate for the skin type on the one hand and compatible with its microbiota on the other hand, thereby reducing the risk of side effects. Similarly, in the case of skin defects and / or microbiota imbalances, the present invention recommends the best corrective treatment, which is either guaranteed to be compatible with the microbiota or can regulate the microbiota. [Brief explanation of the drawings]

[0065] [Figure 1] 1 is a depiction of an embodiment of a device for immunochromatographically detecting skin microbiota complexes according to the present invention, comprising a support (0) in which a transfer well intended to receive a solution containing components of the skin microbiota complex is arranged, the well leading to a plurality of N detection zones: (1) a sampling device (swab, cotton applicator, etc.), (2) a solution sampled from the skin, (3) a transfer well, (4) an absorption zone for the sampled solution, (5) a zone with a detection conjugate (or a monoclonal detection antibody), (6) a monoclonal detection antibody, (7) a direction of transfer flow, (8) a zone in which a monoclonal capture antibody is deposited, (9) a monoclonal capture antibody (or antibody fragment, or nanobody), (10) a control zone, and (11) an antibody (or antibody fragment, or nanobody) directed against the conjugate. [Figure 2]1A-1C are depictions of three embodiments of detection strips that can be used in a device for immunochromatographic detection of skin microbiota complexes according to the present invention. (A) One biomarker detected at several detection thresholds (e.g., for one microorganism: threshold 1 = 10 CFU, threshold 2 = 10 CFU, threshold 3 = 10 CFU, threshold 4 = 10 CFU; for one molecule: threshold 1 = 1 ng, threshold 2 = 10 ng, threshold 3 = 100 ng, threshold 4 = 1000 ng). The control corresponds to detection with an antibody (or antibody fragment). (B) Three microbiota biomarkers detected across four zones. The control corresponds to detection with an antibody (or antibody fragment). (C) Two skin biomarkers detected across four zones. The control corresponds to biomarker deposition (verification of the migration of detection conjugates specific for each biomarker). The intensity of the band indicates that the control zone must be saturated, while the intensity of the other zones depends on the amount of migrated analyte, thus allowing semi-quantitative analysis of the biomarkers. [Figure 3] 1 is a representation of an embodiment of a support (0) for a device for immunochromatographic detection of skin microbiota complexes according to the present invention, on which is arranged a (central) transfer well (3) intended to receive a solution containing a composition of skin microbiota complexes, the wells leading to a plurality of N detection zones arranged in strips arranged radially around the central transfer well (here, for illustrative purposes, 8 strips, each containing 4 or 5 detection zones). This support comprises at least one foolproof means for orienting the reading of the device (12), embodied for example by a black triangle, which may be a notch or a pattern, allowing the reading direction of the support to be oriented. [Figure 4]Schematic illustration of the process leading to personalized cosmetic product recommendations: (A) Depiction of the semi-quantitative analysis of the microbiota profile; the same type of analysis is performed for one or more skin biomarkers; (B) Presentation of the results of the combinatorial analysis of biomarkers to establish the characterization of the skin microbiota complex; (C) Recommendation of appropriate cosmetic products related to the state of the skin microbiota complex based on the product database. DETAILED DESCRIPTION OF THE INVENTION

[0066] A first object of the present invention is a device for immunochromatographic detection of a skin microbiota complex, comprising a support in which are arranged transfer wells intended to receive a solution containing a component of the skin microbiota complex, the wells leading to a plurality of N detection zones, each of which has (i) an immunological detection reagent and (ii) an immunological capture reagent, the skin microbiota complex consisting of skin cells and microorganisms as well as other components of the skin microbiota, the device comprising: the number N of detection zones, where N is 5 or greater; at least one immunological detection reagent specific for a skin cell biomarker and at least one immunological detection reagent specific for a biomarker of the skin microbiome; at least one foolproof means for directing the reading of the device; Including, the immunological detection reagents each comprise an antibody specific for a biomarker of skin cells or skin microbiota conjugated to a colorimetric identification system, each capture reagent comprises an antibody bound to a support in each of the N detection zones and capable of recognizing a biomarker present in the zone; - three zones for detecting three biomarkers of the skin microbiome, the biomarkers being three bacteria of the genus Staphylococcus, Cutibacterium and Corynebacterium, respectively; - a zone for detecting skin cell biomarkers selected from (i) structural proteins, (ii) inflammatory biomarkers, or (iii) allergy biomarkers; The present invention is characterized by comprising:

[0067] For the purposes of this invention, the "skin microbiota complex" is composed of two cell types: skin cells of human origin and microorganisms, which interact with each other to affect skin conditions. A symbiotic phenomenon is observed. Therefore, by simultaneously sampling human cells and microbiota present on the surface of the skin, the inventors obtain a representative sample of the skin condition. This complex also contains other components of the skin microbiota complex secreted by the two types of cells. On the one hand, it includes molecules secreted by either epidermal or immune skin cells, as well as secretions from the microorganisms that make up the microbiota.

[0068] The objective herein is to establish a comprehensive characterization of skin conditions based on detecting biomarkers that represent the physiological state of the skin and the composition of the microbiota, and to enable their combinatorial analysis. The number of detection zones N corresponds to the number of biomarkers to be tested. N is greater than 5, so that a sufficient number of biomarkers can be tested to obtain value-added results resulting from the combinatorial analysis of different biomarkers. The device also includes at least one control zone that is not included in the "at least five zones."

[0069] The "at least five zones" of the device detect at least one skin cell biomarker selected from (i) structural proteins, (ii) inflammatory biomarkers, or (iii) allergy biomarkers, and at least three biomarkers of the skin microbiome, where these three biomarkers are bacteria belonging to the genera Staphylococcus, Cutibacterium, and Corynebacterium. Preferred embodiments are described below.

[0070] In certain embodiments of the invention, at least 8, 10, 15, 20, 25, 30 different types of biomarkers are tested simultaneously.

[0071] The device not only detects the absence or presence of the biomarkers under consideration, but also quantifies them in absolute or relative terms, based on the method selected.

[0072] microbiome The skin microbiota constitutes the first component of the symbiotic complex detected by the device according to the invention. It comprises a range of microorganisms, including bacteria, yeasts, fungi and mites.

[0073] To provide information about the state of the skin microflora, the device according to the invention is capable of detecting the presence of bacteria of at least the genera Staphylococcus, Cutibacterium and Corynebacterium.

[0074] Preferably, the species of Staphylococcus investigated are selected from Staphylococcus epidermidis and Staphylococcus aureus. These biomarkers can be detected via antibodies capable of recognizing at least these two species of Staphylococcus (genus-specific antibodies) or via two antibodies specific for the species Staphylococcus epidermidis and Staphylococcus aureus, respectively.

[0075] Preferably, the investigated Cutibacterium species are selected from Cutibacterium acnes and Cutibacterium granulosum. These biomarkers can be detected via antibodies capable of recognizing at least these two Cutibacterium species (genus-specific antibodies) or via two antibodies specific for the species Cutibacterium acnes and Cutibacterium granulosum, respectively.

[0076] Preferably, the species of Corynebacterium investigated are selected from Corynebacterium xerosis and Corynebacterium kloppenstätti. These biomarkers can be detected via antibodies capable of recognizing at least these two species of Corynebacterium (genus-specific antibodies) or via two antibodies specific for the species Corynebacterium xerosis and Corynebacterium kloppenstätti, respectively.

[0077] In the context of the present invention, the skin microbiota is analyzed by detecting epitopes exposed on the surface of the microbiota. In a specific embodiment of the present invention, the epitopes exposed on the surface of the microbiota are associated with proteins present on the surface of the microorganisms that constitute the microbiota. In particular, they may be epitopes associated with the biofilm-like behavior of the microbiota.

[0078] The detection zone can be used to detect "additional biomarkers of the skin microbiome", i.e. biomarkers other than those that detect the presence of bacteria belonging to the genera Staphylococcus, Cutibacterium or Corynebacterium, or bacteria belonging to related species of the aforementioned. These other biomarkers can detect the presence of microorganisms selected from bacteria, yeasts, fungi or mites.

[0079] Preferably, the microorganisms constituting the microbiota investigated to establish a diagnosis of a skin condition within the scope of the present invention are selected from the following bacteria (Byrd et al., 2018): Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus lugdunensis, Staphylococcus hominis, Streptococcus mitis, Streptococcus oralis, Streptococcus pseudopneumoniae, Streptococcus sanguinis, Staphylococcus capitis, Cutibacterium acnes, Corynebacterium simulans, Corynebacterium fastidium fastidiosum, Corynebacterium afermentans, Corynebacterium xerosis, Corynebacterium aurimucosum (G), Corynebacterium kroppenstedtii, Corynebacterium amycolatum, Corynebacterium tuberculostearicum, Veillonella parvula (S), Micrococcus luteus (S, H), Enhydrobacter aerosaccs (H), Epidermophyton floccosum, Nannizzia nana, Nephroselmis, Cyanophora paradoxa, Aureoumbra lagunensis, Pycnococcus provasolii Pyramimonas parkeae, Parachlorella kessleri, Aspergillus tubingensis, Zymoseptoria tritici, Tilletia walkeri, Or bacteria of the genus Dermacoccus, Actinomyces, Bacteroides, Alistipes, Prevotella, Porphyromonas, Sphingobacterium, Lactobacillus, Aerococcus, Oscillospira, and Ruminococcus.

[0080] Other microorganisms that make up the skin microbiome that can be investigated to establish a diagnosis according to the invention are: - Yeasts such as Malassezia globosa, Malassezia restricta, Malassezia furfur, Malassezia sympodialis, Candida parapsilosis, etc. -Aspergillus tubingensis, Zymoseptoria tritici, Tilletia walkeri, Epidermophyton floccosum, Nannizzia nana, Nephroselmis olivacea, Cyanophora paradoxa, Aureoumbra lagunensis, Pycnococcus provasolii, etc. Mites, such as Demodex folliculorum and Demodex brevis.

[0081] The device according to the invention therefore makes it possible to analyze the presence of microorganisms, optionally characterizing their organization in the form of a biofilm, but also to analyze the presence within the complex of other elements whose presence provides interesting information about the state of the skin. If these other elements are immunogenic, it is possible to provide the device with antibodies capable of detecting their presence.

[0082] "Epitopes exposed on the surface of the microbiota" refers to epitopes exposed on the surface of the microorganisms that make up the microbiota through molecules that make up the membrane of the microorganisms, such as proteins, complex fatty acids, and polysaccharides, but also epitopes formed by secreted molecules such as exopolysaccharides that make up the biofilm matrix.

[0083] The skin cells collected at the surface are either cells of the stratum corneum or, depending on the intensity of the sampling (abrasion) or the condition of the skin (exposed after scrubbing or vigorous exfoliation or peeling), cells from the underlying stratum lucidum or even the stratum granulosum, which are buried deeper under normal circumstances.

[0084] By "proteins present on the surface of a microorganism" is meant proteins that are exposed on the membrane, i.e., secreted proteins, and structural proteins synthesized by the cell that, as in the case of secreted proteins, pass through or are attached to the cell membrane. For example, Staphylococcus aureus produces Panton-Valentine Leukocidin proteins, extracellular matrix "polysaccharide intercellular adhesins" (PIAs), Cutibacterium acnes produces porphyrins, Pseudomonas alginate-based matrices, Escherichia coli colanic acid-based matrices, Cutibacterium β-1,6-linked N-acetylglucosamine (PNAG)-based matrices, and for structural or adhesive proteins on the skin surface: for Gram-positive bacteria (single membrane): Bap family adhesins, type 4 fimbriae (important for initial biofilm formation), SAATs (self-associating autotransporters, promoting aggregation between SAAT-carrying bacteria), intimin / invasin, etc.; more specifically, for Cutibacterium acnes For P. acnes, proteins present on the surface of the bacterial membrane, such as CAMP factors (Christie-Atkins-Munch-Petersen factors), sialidases, dermatan sulfate adhesins, endoglycoceramidases, GroEL chaperonins, SH3 domain-containing lipoproteins, Flp fimbriae / fimbrial proteins, and DsA1 proteins among others; more specifically, for Staphylococcus, they produce MSCRAMMs (molecular surface components recognizing matrix adhesion molecules), such as Bbp (bone sialoprotein-binding proteins), FnBP (fibronectin-binding proteins), and proteins of the Clf-Sdr family with CNA (collagen adhesion). SesJ proteins were recently identified for S. epidermidis (Arora et al., 2020), while for S. aureus, aureusmin (fevalin) appears to be a biofilm phenotype marker.On the other hand, for Gram-negative bacteria (double-membrane LPS), these include the Bap family of adhesins, which exhibit the LPXTG C-terminal domain, IMPs (inner membrane proteins), and type 3 and type 4 fimbriae.

[0085] Therefore, the membrane-exposed proteins of the microorganisms that make up the microbiota are selected from Panton-Valentine leukocidin, porphyrin, alginate, β-1,6-linked N-acetylglucosamine (PNAG), Bap family adhesins, type 4 fimbriae, self-associating autotransporters, intimin / invasin, CAMP factors, sialidases, dermatan sulfate adhesins, endoglycoceramidase, GroEL chaperonin, SH3 domain-containing lipoproteins, Flp fimbriae / fimbriae proteins, DsA1 proteins; Clf-Sdr family proteins, such as Bbp, FnBPs and CNAs, SesJ proteins, aureusimine, Bap family adhesins exhibiting the LPXTG C-terminal domain, IMPs, type 3 and type 4 fimbriae.

[0086] In the case of Gram-positive bacteria, the complex fatty acids exposed on the membranes of the microorganisms that make up the microbiota are selected from teichoic or lipoteichoic acids, which may contain long chains of ribitol phosphate or glycerol-3-phosphate, while in the case of Gram-negative bacteria, they are selected from lipopolysaccharides, glycolipids containing a lipid region called lipid A, which is most commonly made from a disaccharide of phosphorylated glucosamine and contains fatty acids with ester or amide bonds.

[0087] The sugars exposed on the membranes of the microorganisms that make up the microbiota are selected from glycopolymers (eg, based on rhamnans) and peptidoglycans.

[0088] In a preferred embodiment of the present invention, at least one of the biomarkers specific to skin microbiota corresponds to a biomarker related to the biofilm-like behavior of the microbiota.Biofilm-like bacteria and other microorganisms are defined as opposed to mobile, non-adherent bacteria and other planktonic microorganisms.Biofilm-specific biomarkers can correspond to proteins present on the surface of the membrane of microorganisms or other components of the microbiota.

[0089] If it is desired to investigate the presence of bacteria or other microorganisms in biofilm form, the biomarkers are selected from adhesive proteins such as pili, curli, and fimbriae, which are characteristic of the presence of naturally formed microbiota biofilms on the skin.

[0090] skin The skin condition constitutes the second component of the symbiotic complex detected by the device according to the invention, which is analyzed by at least one skin cell biomarker selected from (i) structural proteins, (ii) inflammatory biomarkers, or (iii) allergy biomarkers.

[0091] When detecting the presence of skin cells and / or assessing skin quality, structural proteins are targeted.The use of structural protein-like biomarkers can also be used to quantify the number of skin cells present in collected samples or to normalize the level of biomarkers.Targeted structural proteins include keratin, filaggrin, loricrin, etc.

[0092] The inflammatory biomarker is selected from C-reactive protein (CRP), interleukin IL-1 beta, IL-4, IL-6, IL-8, IL-11, IL-12, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), and transforming growth factor beta (TGF-beta).

[0093] The allergy biomarker is selected from immunoglobulins, IgE, IgA and IgG, preferably IgE. Indeed, serum IgE levels are elevated in cases of atopic eczema or dermatitis.

[0094] In a preferred embodiment of the present invention, the "at least one" skin cell biomarker investigated is IL-1 beta. Two other types of skin cell biomarkers are preferably structural proteins, keratins, and allergy biomarkers, IgE.

[0095] In a preferred embodiment of the invention, the device comprises zones for specifically detecting the presence of bacteria of the genera Staphylococcus, Cutibacterium and Corynebacterium, and IL-1 beta. In a more particular embodiment, the device further comprises a zone for detecting the presence of IgE. In an even more particular embodiment, it further comprises a zone for detecting the presence of keratin.

[0096] Devices as biomarker supports Disposable immunochromatographic detection devices are sterile-packaged and dehydrated. This module typically takes the form of a plastic support with wells for the solubilized solution containing the collected biological material, leading to a series of channels (e.g., nitrocellulose strips or lateral flow strips) containing multiple recognition and detection zones specific to the biomarkers of interest. These channels can, for example, accept antibody-loaded nitrocellulose strips, where the solubilized solution containing the collected biological material migrates by capillary action over the strip.

[0097] In a preferred embodiment, the device according to the present invention is a lateral flow immunochromatography device.

[0098] The detection antibodies can be conjugated to either nano-sized gold particles (allowing only one red marking color per strip, but allowing for different capture lines), or colored particles (increasing the number of detections per strip), or e.g., latex (blue).

[0099] Depending on the biomarker detection mode, several different configurations of the detection zone are possible.

[0100] A detection zone can be established on a nitrocellulose strip with a single, uniform concentration of capture antibody across the entire width of the strip (uniform zone), forming a line that is detected by interaction with the analyte (visualized by a detection antibody conjugate). Several detection zones for different analytes can be combined on one strip, with each analyte being detected with its own threshold concentration of capture antibody (Figure 2). In another arrangement, the detection zone can be established with at least two different concentrations of capture antibody. In alternative embodiments, the zone contains two, three, or more different concentrations of capture antibody.

[0101] Furthermore, the detection thresholds of the various biomarkers can be normalized, thereby establishing relative quantification between different biomarkers. This normalization can be performed with respect to a given common marker (e.g., keratin) that serves as a reference, or by establishing the relative quantification of all markers relative to each other (e.g., each marker gets its own proportional value, which adds up to a cumulative total of 100).

[0102] The device must be equipped with a foolproof means, which is a marking necessary to correctly orient the device when viewing the signal (the line visible on the strip) that detects the biomarker. This foolproof means is placed directly on the reading surface of the device. It can take the form of a graphic, such as a drawing, or a physical form, such as a notch, hole, or any other marking system. It can consist of one, two, or more marking elements.

[0103] The device generally consists of a disk, ie, a support for the detection zone, enclosed in a cassette.

[0104] 1 to 3 show different embodiments of the device according to the invention.

[0105] A second object of the present invention is a disposable kit for characterizing a skin condition, comprising: a sampling device for a skin microbiota complex, the sampling device comprising a means for sampling the complex and a container containing a solubilization solution; - a device for immunochromatographic detection of skin microbiota complexes as described above.

[0106] The sampling means may consist, for example, of a patch, a scraper, a swab soaked in buffer, or a sponge soaked in buffer, a tulle or gauze cloth soaked in buffer, or any other suitable means that allows for physical peeling, soaking, or adsorption.

[0107] The number of microorganisms, especially bacteria, per square centimeter of skin varies greatly depending on the skin zone, especially depending on the sebum content. Therefore, to ensure a representative sample, it is necessary to have a sampling device adapted to the type of skin and the area to be sampled, so as to have enough biological material to carry out the analysis of biomarkers of skin cells and microbiota. The sampled biological material includes components of the exposed parts of the skin (cells of the stratum corneum and / or stratum lucidum and / or stratum granulosum) and components of the microbiota (bacteria, yeast, fungi, and even mites such as Demodex).

[0108] Typically, sampling is performed by contacting the collected biological material with a solubilizing solution intended to receive the collected biological material for at least 10 minutes. 3It should be possible to collect a sufficient number of bacteria to obtain a concentration of bacteria in CFU / mL. Preferably, for optimal detection, the concentration of the solution containing the collected microbiota should be less than 10 bacteria. 4 CFU / mL. However, these concentrations are given for guidance only and depend on the sensitivity of the detection method.

[0109] If sampling is performed via a patch, the patch should be at least 5 cm long, depending on the abundance of the skin microbiota and the sampling efficiency. 2 , or even 10 cm 2 , 12cm 2 The sampling (or collection) means has a skin interaction surface (collection surface) of at least 1 mL. The sampling (or collection) means is supplemented by a container (e.g., an extraction tube or equivalent) pre-filled and packaged in a sterile package, which contains a solubilization buffer (typically between 1 mL and 5 mL), e.g., a dissolution / transfer buffer for buffering the pH of the sample, minimizing non-specific binding, neutralizing interferences, and controlling the flow rate by using various salts, surfactants, detergents, stabilizers, or blocking reagents (example composition: PBS 1X with 1% Tween R20). The container has a volume adapted to directly receive the collection surface of the sampling device to dissolve the sample from the surface of the skin.

[0110] The disposable kit requires only simple handling that can be carried out immediately after sampling without biological knowledge and allows samples to be characterized with respect to a large number of reference biomarkers to determine combinations that represent a wide variety of skin / microbiota complexes.

[0111] A third object of the present invention is a system for implementing personalized cosmetic recommendations based on a characterization of the skin microbiota complex, comprising: a sampling device for a skin microbiota complex, the sampling device comprising a means for sampling the complex and an extraction tube containing a solubilization solution; - a device for immunochromatographic detection of the aforementioned skin microbiota complex, wherein the N detection zones constitute zones capable of providing a signal when a biomarker is detected; and a computer that performs image analysis processing on an image consisting of all signals from the N zones detected by the immunochromatographic detection device to determine the nature of the biomarkers recognized in the N detection zones and classify the skin condition based on the combination of identified biomarkers.

[0112] The computer acquires images of the various detection zones after a predetermined reaction time and transmits the images in digital form to a processing center for automated analysis to characterize the type of biomarker that reacted with the antibody present in the detection zone of the detection device.

[0113] The image can be acquired in a single shot for a one-off analysis. This analysis mode is particularly suitable for use with devices where the antibody detection threshold is standardized or the detection zone contains at least two different antibody concentrations.

[0114] Images can also be acquired in several successive shots to perform dynamic analysis. This method allows for monitoring the emergence of signals and for relative quantification of biomarkers. It can be implemented using devices with or without standardized antibody detection thresholds. One configuration, for example, provides real-time monitoring of changes in signal intensity (the intensity of the lines in each zone on the strip). The first shot can be triggered when the reference biomarker reaches an intensity corresponding to a saturation level (e.g., after 5 minutes of migration), and the second shot can be triggered when the biomarker with the weakest signal reaches an intensity corresponding to the minimum detection threshold (e.g., after 10 minutes) or when the migration process is considered to be over (e.g., after 15 minutes).

[0115] The shot may be taken using a smartphone or equivalent combined with image analysis software to process it by leveraging the accumulated data (this analysis may involve software trained on the basis of an AI-type learning process that includes both the user's age, weight, height, tobacco and alcohol consumption, sugar sensitivity (diabetes), data provided by UV booth sessions, etc.) It is then possible to scan the barcodes of products used in the beauty routine and link them to information about skin biomarkers and microbiome biomarkers in order to establish recommendations for products (nutraceuticals, cosmetics, etc.) best suited to the condition of the skin and microbiome.

[0116] These three means form an inseparable whole: the sampling device allows to collect a sufficient amount of biological material (skin cells and microbiota) so that biomarker analysis via immunochromatographic detection devices is only possible.

[0117] Due to the large number of biomarkers analyzed in both skin cells and skin microbiome, and the combinatorial approach corresponding to the large number of configurations necessary to classify biological material, the use of this kit for complex analyses that prevent conclusions from being drawn simply by reading the results, as is the case with Covid or pregnancy tests, is only possible due to the simplicity of acquiring images and transmitting them in digital format for processing on a computer that pools the processing of the kit for multiple users.

[0118] The channel providing the detection zone defines a plane closed by a transparent window through which the user takes a photograph after the reaction time has elapsed, for example using a smartphone running a dedicated application that instructs, among other things: a) image acquisition, b) local verification on the smartphone processor of the conformity of the acquired image with respect to a test pattern provided on the imaging device, for example; c) Enter additional information; d) time stamping and optional geolocation by smartphone; e) Transmission of images and related information to a remote server.

[0119] Image use The images received on the server are then automatically processed to recognize detection zones that have reacted to codify the combination of biomarkers present in the analyzed skin microbiome complex, and by processing this combination, to classify the skin, in particular by supervised learning from a reference base of data collected from a panel of people characterized by experts.

[0120] A fourth object of the present invention relates to a method for recommending cosmetics to a person, comprising the steps of: - taking a sample of biological material from the surface of the skin using a sampling device; - suspending the skin cells and microorganisms that constitute the biological material incorporated in a solubilizing solution; - pouring the solubilization solution into a transfer well of the immunological detection device defined above; - incubating the cells and microorganisms on each of the N zones of the immunological detection device for 1 to 10 minutes to allow them to react with the reagent; - processing the image consisting of the set of signals detected in each of the N zones through a computer that makes it possible to classify the skin by processing this combination of signals, in particular by supervised learning from a reference base; - recommending one or more cosmetic products suitable for the individual's skin based on the combination of biomarkers detected.

[0121] The recommendations are established based on a decision tree that takes into account information about the inflamed and / or infected skin condition, which is translated into, for example, the following criteria: 1- Information on the level of skin irritation or inflammation, such as the presence of atopic dermatitis, eczema, rosacea, etc., according to the following "skin" biomarkers: -Biological markers: keratin (sample quality control), filaggrin, etc., IgE, CRP, IL-6, etc. -CRP: can vary based on menstrual cycle, infection markers, -Low keratin marker = recent overly aggressive scrubbing or exfoliation / peeling. 2- Information on the composition of the skin microbiota (level of skin colonization / contamination).

[0122] A microbiota is considered "normal" when colonization is "commensal" and the microbiota is composed of expected microorganisms.

[0123] If the biological marker of the target microorganism is elevated, contamination or infection is detected, e.g. -Presence of acne if Cutibacterium acnes is elevated -Presence of infected atopic dermatitis (eczema) if Staphylococcus aureus (S. aureus) markers are elevated, - presence of rosacea if S. epidermidis or Demodex markers are elevated, - Elevated Malassezia markers indicate a risk of dandruff on the scalp.

[0124] As far as the skin microbiome is concerned, the analysis can provide information about the balance between different populations of microorganisms and detect the presence of dysbiosis.

[0125] Figure 4 shows the steps involved in this recommended method.

[0126] Non-limiting examples of embodiments of the present invention The invention will be better understood on reading the following description of non-limiting exemplary embodiments.

[0127] You notice acne appearing on your face, consider your skin to be quite oily, have a skin care routine that works for you (advice from your mother, friends, hairdresser, etc.), you've heard about the gut microbiome and its effects on your health (stimulating the immune system, protecting against pathogens that can irritate or even attack the digestive tract, etc.), and although tests exist, they are impractical because you have to order a sampling kit from a website that is quite expensive (250-500 euros), take a sample at home, then send it to a laboratory for "metagenomic" analysis, and then wait weeks for the results and advice. You need to be highly motivated and patient.

[0128] With the device according to the present invention, all you have to do is visit a cosmetics store or pharmacy (OTC cosmetics and personal care) or order it via a smartphone application. It is recommended to perform the test in the morning after removing makeup and rinsing the facial skin the day before, but the test can also be performed immediately (at the risk of identifying only three or four major microorganisms in the microbiome, which may be sufficient to confirm, for example, suspected acne). The device is provided in the form of a kit with a simple skin sampling device: a small, spongy, absorbent, slightly abrasive device is applied, immersed in a solution to optimize sampling, and then inserted into a tube containing a so-called "resuspension" (or "solubilization") solution.

[0129] The solution flows into an ICFL device consisting of a sheet of absorbent nitrocellulose (NC) to generate a moving stream, and an inlet zone above which is another sheet of NC on which a "detection conjugate" is deposited. This detection conjugate is generally directed against a target antibody, a monoclonal antibody directed against the analyte being investigated, i.e., a protein characteristic of the skin condition (structural proteins - keratin, filaggrin, etc. -, inflammatory - CRP, etc. -, immune system - IL6, etc. -, etc.) or a protein characteristic of the state of the microbiota (adhesion proteins, pili, curli, fimbriae, etc. - characteristic of the presence of the microbiota as a biofilm that naturally forms on the skin - expressed by the three main bacteria Staphylococcus epidermidi, Cutibacterium acnes, Staphylococcus aureus, and other, less common bacteria - those common to all skin types, and those specific for dry, oily, or moist skin. The detection conjugate is so named because it has attached to it compounds (gold particles, latex microbeads, etc.) that make it possible to visualize the antigen-antibody interaction.

[0130] After this initial interaction at the entrance to the deposition well, the flow proceeds towards the detection zone where monoclonal capture antibodies directed against antigens characteristic of skin and microflora conditions are deposited. Each capture antibody is deposited on a test line, the result of which indicates the presence or absence of the analyte.

[0131] Furthermore, there is always a control line, the result of which is used to confirm correct migration and therefore the test: for the reaction to occur in this zone, the membrane-bound antibody on the control line is directed against the conjugate.

[0132] Each ICFL device is intended for multiple analyte detection (requiring multiple "detection conjugate and capture antibody" pairs), which allows for the determination of skin condition profiles (healthy, normal, dry, oily, acne prone, dermatitis, etc.) associated with microbiota profiles (healthy, dry skin characteristics, oily, acne prone, dermatitis, etc.).

[0133] Reading and analysis is performed by smartphone image capture and interpreted by AI. Information provided by biomarkers can also be combined with information provided by the individual.

[0134] Example of implementation of a device for recommending cosmetics The present invention thus allows for different types of testing. i. A one-time test of 15-30 minutes (possible in-store or at-home "home testing") either as a spontaneous process or event: the appearance of solar erythema after the use of irritating cosmetics or personal care products, after prolonged sun exposure, or after a session in an artificial UV booth, or after scrubbing or peeling or too aggressive peeling, or in response to a diagnosis of a skin lesion (the most common skin conditions are acne, eczema, psoriasis, scalp disorders (excluding alopecia), fungal diseases and nail disorders). ii. Repeated serial testing allows the general condition of the skin to be monitored over time by relating it to the resident microflora (skin microbiota complex), which may lead to better management of exposure to the sun or artificial UV rays, or to spacing out or weakening the practice of exfoliation or peeling. iii. Depending on the profile obtained, appropriate product suggestions relating the skin condition to ad hoc treatments with relevant "microbiome" information (dry skin = moisturizing routine, oily skin = cleansing exfoliation routine, thin and wrinkled skin = protective and nourishing cream routine, etc.): either "probiotic" supplements to stimulate the skin and / or microbiome, or "compatible" products to avoid treating only the skin in a way that harms the microbiome (a testing campaign of products against microbiome microorganisms can make it possible to calculate a compatibility score), or skin care products able to improve unpleasant symptoms (dryness, oiliness, irritated skin, redness, etc.). iv. Check the compatibility of products used on a daily basis. Modify your routine based on the evolution of the skin microbiota complex with age (as the skin thins), climate (hot, humid summers vs. cold, dry winters), pollution, etc.

Claims

1. 1. A device for immunochromatographically detecting a skin microbiota complex, comprising a support in which a transfer well is arranged intended to receive a solution containing a component of said skin microbiota complex, said well providing a plurality of N detection zones each having (i) an immunological detection reagent and (ii) an immunological capture reagent, said skin microbiota complex consisting of skin cells and microorganisms as well as other components of said skin microbiota, said device comprising: the number N of detection zones, where N is 5 or greater; at least one immunological detection reagent specific for a skin cell biomarker and at least one immunological detection reagent specific for a biomarker of the skin microbiome, at least one foolproof means for directing the reading of the device; Including, the immunological detection reagents each comprise an antibody specific for a biomarker of skin cells or skin microbiota conjugated to a colorimetric identification system, each of said immunological capture reagents comprises an antibody bound to said support in each of said N detection zones, said antibody being capable of recognizing said biomarker present in said zone; three zones for detecting three biomarkers of the skin microbiome, wherein the biomarkers are bacteria of the genera Staphylococcus, Cutibacterium and Corynebacterium; a zone for detecting skin cell biomarkers selected from (i) structural proteins, (ii) inflammatory biomarkers, or (iii) allergy biomarkers; An apparatus comprising:

2. 2. The device according to claim 1, wherein the bacteria of the genus Staphylococcus are selected from Staphylococcus epidermidis and Staphylococcus aureus, the bacteria of the genus Cutibacterium are selected from Cutibacterium acnes and Cutibacterium granulosum, and the bacteria of the genus Corynebacterium are selected from Corynebacterium xerosis and Corynebacterium kroppenstedtii.

3. The device according to any one of claims 1 to 2, wherein at least one of the skin microbiota-specific biomarkers corresponds to an epitope associated with biofilm-like behavior of the microbiota.

4. 4. The device according to claim 1, wherein the at least one specific detection zone of the additional biomarker of the skin microbiome is a microorganism selected from bacteria, yeasts, fungi and mites.

5. The additional biomarkers of the skin microbiome include the following species: (i) Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus capitis, Cutibacterium acnes, Propionibacterium, Propionibacterium avidum, Corynebacterium tuberculostearicum tuberculostearicum), Corynebacterium simulans, Corynebacterium fastidium, Corynebacterium afermentans, Corynebacterium xerosis, Corynebacterium auricum, Corynebacterium kloppenstätti, Corynebacterium amycolatum, Streptococcus mitis, Streptococcus oralis, Streptococcus pseudopneumoniae, Streptococcus sanguinis, Veillonella parvula parvula), Micrococcus luteus, Enhydrobacteraerosaccus, Epidermophyton floccosum, Nannizzia nana, Nephroselmis olivacea, Cyanophora paradoxa, Aureumbra lagunensis, Pycnococcus provasolii, Pyramimonas parkeae, Parachlorellaa bacterium selected from the group consisting of S. kessleri, Aspergillus tubingensis, Zymoseptoria tritici, Tilletia Dermacoccus, and Actinomyces; (ii) a yeast selected from Malassezia Globosa, Malassezia restricta, or Candida parapsilosis; (iii) Aspergillus tubingensis, Zymoseptoria tritici, Tilletia walkeri, Epidermophyton floccosum, Nannizzia a fungus selected from Nana, Nephroselmis olivacea, Cyanophora paradoxa, Aureumbra lagunensis, Pycnococcus provasolii; (iv) mites selected from Demodex folliculorum, Demodex brevis.

6. 5. The device of claim 4, wherein the biomarkers of the microbiota are proteins exposed on the surface of the microorganisms selected from Panton-Valentine leukocidin, porphyrin, alginate, β-1,6-linked N-acetylglucosamine (PNAG), Bap family adhesins, type 4 fimbriae, self-associating autotransporters, intimin / invasin, CAMP factors, sialidase, dermatan sulfate adhesins, endoglycoceramidase, GroEL chaperonin, SH3 domain-containing lipoproteins, Flp fimbriae / fimbriae proteins, DsA1 proteins, Clf-Sdr family proteins such as Bbp, FnBPs and CNAs, SesJ proteins, aureusimine, Bap family adhesins exhibiting an LPXTG C-terminal domain, IMPs, type 3 and type 4 fimbriae.

7. 7. The device of claim 1, wherein the biomarkers of skin cells are selected from (i) structural proteins selected from keratin, filaggrin, and loricrin; (ii) inflammatory biomarkers selected from C-reactive protein, interleukin IL-1 beta, IL-4, IL-6, IL-8, IL-11, IL-12, tumor necrosis factor, interferon-gamma, granulocyte-macrophage colony-stimulating factor, and transforming growth factor beta; and (iii) allergy biomarkers selected from immunoglobulins IgE, IgA, and IgG.

8. The device of claim 7, wherein the skin cell biomarker is interleukin 1-beta.

9. 9. A device according to any one of claims 1 to 8, characterized in that it comprises zones for the specific detection of the presence of bacteria of the genera Staphylococcus, Cutibacterium and Corynebacterium, as well as IL-1 beta.

10. 10. The device according to any one of claims 1 to 9, characterized in that it is a lateral flow immunochromatography device.

11. 1. A disposable kit for characterizing a skin condition, comprising: a sampling device for the skin microbiota complex, the sampling device comprising a means for sampling the complex and an extraction tube containing a solubilization solution; a device for detecting a skin microbiota complex by immunochromatography according to any one of claims 1 to 10; A disposable kit comprising:

12. 12. The kit of claim 11, wherein the sampling means comprises a patch, a scraper, a buffer-soaked swab, a buffer-soaked sponge, a buffer-soaked tulle, or a gauze cloth.

13. A system for implementing personalized cosmetic recommendations based on the characterization of the skin microbiota complex, comprising: a sampling device for the skin microbiota complex, the sampling device comprising a means for sampling the complex and an extraction tube containing a solubilization solution; a device for immunochromatographic detection of a skin microbiota complex as defined in any one of claims 1 to 10, wherein the N detection zones constitute zones capable of providing a signal if a biomarker is detected; a computer that performs image analysis processing on an image consisting of all signals from the N zones detected by the immunological detection device to determine the nature of biomarkers recognized in the zones and classify skin conditions based on the combination of identified biomarkers; A system comprising:

14. 1. A method for personalized cosmetic recommendation based on characterization of said skin microbiota complex of an individual, comprising: - taking a sample of biological material from the surface of the skin; - suspending the entrapped skin cells and microorganisms and other components of said biological material in a solubilization solution; - injecting said solubilization solution into a transfer well of an immunological detection device according to any one of claims 1 to 10; Incubating the cells and microorganisms on each of the N zones of the immunological detection device for 1 to 10 minutes to allow them to react with the reagent; processing the image consisting of the set of signals detected in each of the N detection zones through a computer that makes it possible to classify the skin by processing this combination of signals, in particular by supervised learning from a reference base; - recommending one or more cosmetic products suitable for the individual's skin based on the combination of biomarkers detected; A method comprising:

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