Device for overall characterization of skin-microbiota complex and cosmetic recommendation method

Abstract

The present invention relates to the field of characterizing microbiota present on human skin. More specifically, the present invention relates to a device for revealing a skin-microbiota complex in an immunochromatographic manner, the device allows for rapid and meaningful biological determination of two members of skin-microbiota symbiosis by means of immunological characterization of biomarkers available on the surface of the microbiota-skin interacting group. Artificial intelligence is then used to recommend optimal care that is suitable for the skin type on the one hand and compatible with its microbiota on the other hand, thereby reducing the risk of adverse effects. Similarly, in the case of skin flaws and / or microbiota imbalances, the present invention will recommend optimal corrective treatments to ensure compatibility with or be able to modulate microbiota.

Description

[0001] The present invention relates to the field of characterizing the microbiota present on human skin (or the human skin "microbiome"). More specifically, the present invention relates to a device for immunochromatographically revealing the skin-microbiota complex, which allows for rapid and meaningful biological determination of both members of the skin-microbiota symbiosis by immunologically characterizing the ostensibly available biomarkers of the microbiota-skin interactome. Artificial intelligence is then used to recommend optimal treatments that are appropriate to the skin type on the one hand and compatible with its microbiota on the other, thereby reducing the risk of adverse effects. Similarly, in the case of skin blemishes and / or microbiota imbalances, the present invention recommends the optimal corrective treatment to ensure compatibility with or ability to modulate the microbiota. Technical Field

[0002] The field of the present invention is that of cosmetic care recommendations. Without extrapolating to the health sector, cosmetic products must fulfill their role as care products to maintain or improve the user's appearance. They must not present any health risks when in contact with the various surface areas of the human body (epidermis, hair and capillary system, nails, lips, and external genitalia). To provide personalized recommendations, the present invention proposes providing information on the skin and the associated microbiome, corresponding to the symbiosis, by taking into account the product's suitability for the skin's needs in terms of hydration, hygiene, protection, and appearance modification (skin tone, color, etc.), as well as the susceptibility of the microbiome. For example, this can be achieved by recommending products containing active ingredients compatible with maintaining or regenerating the microbiome and by avoiding active ingredients that destabilize the microbiome (favoring microbial species that may induce opportunistic proliferation risks). By extension, the field of the present invention can be applied to the medical diagnosis of a patient's skin, enabling the consolidation of clinical diagnoses related to, for example, inflammatory conditions, allergic reactions (results of immune system reactions), and microbial conditions (a balanced microbiome or dysbiosis with a high presence of opportunistic pathogens). The present invention can then be transformed into an in vitro diagnostic medical device. Similarly, by extension, the present invention may be used to monitor the condition of the skin over a given period of time and to monitor the effectiveness of cosmetic routines as well as the effectiveness of preventative and / or curative therapeutic treatments.

[0003] The field of the present invention applies to humans as a "mammal-microbe hybrid," a "superorganism," because our microbiome exceeds the number of cells that make up our bodies by a factor of 10. Therefore, the microbiome is understood to include all microorganisms that come into contact with the cells that make up the human body: viruses, bacteriophages, bacteria, yeasts, fungi, etc., all the way to dust mites. If we narrow it down to the bacterial microbiome, the order of magnitude drops to 1.3 times. This situation of human-microbe symbiosis is common to the entire animal and plant kingdoms: in the course of evolution, complex eukaryotic-prokaryotic relationships had to be established, which were refined, diversified, and became more 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, and also on the skin itself, and makes a major metabolic and nutritional contribution by providing the ability to hydrolyze complex plant sugars and produce nutrients (such as short-chain fatty acids and certain vitamins) that are not present in mammals.

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

[0005] microbiome

[0006] The "skin flora" constitutes the external part of the human microbiome (together with the microbiome of the digestive tract (most important in terms of biomass) and the microbiome of the oral cavity, nasal cavity, ear and female vaginal cavity). This flora acts as a barrier against external physical, chemical and biological attacks and, depending on the site, usually interacts with the host organism via cells of the immune system. In general, bacterial biodiversity limits the risk of pathogenic bacteria colonizing the skin and protects against inflammation, an allergic skin reaction.

[0007] Due to the microscopic size of the organisms that make it up, the human microbial flora is invisible to our eyes. Adult skin contains an average of 1 million bacteria per cm2, belonging to more than 500 different species. The skin naturally supports and maintains its own "micro-ecosystem". This is formed at birth and then evolves until death. The skin microbiome is physically formed on the surface and is a consortium of microorganisms organized in a biofilm. It feeds on both molecules and compounds secreted by the skin itself, and on compounds secreted by more or less symbiotic microbial communities, which are mainly bacteria organized in biofilms. The composition of the skin microbiome varies according to the individual, its age (neonatal, adolescence, adulthood, old age), its gender (developmental stage, female menopause), its activity, its behavior and environment. The skin biofilm constantly renews itself, adapting to the natural peeling and differentiation erosion of the skin, as well as personal hygiene habits (which must not be excessive and fail to protect the "barrier" function of the skin, nor can it reduce the diversity of the microbiome that protects against inflammation). In addition, it evolves with age. In children, bacterial biofilms differ depending on the delivery mode: via the genital route, children are colonized with a community similar to the mother's vaginal microbiome, whereas via cesarean section, they are colonized with a community similar to the skin microbiome. After one month, the skin community profile steadily changes, gradually establishing taxonomic specificity (viruses, bacteria, yeasts, fungi, and even mites) for various differentiated sites on the body: external skin sites (face, scalp, etc.), cavities (oral cavity, nasal cavity, vagina), and internal sites (primarily the digestive tract). Recent research (Huang, 2020) even determined which body region (digestive tract, oral cavity, skin) best predicts age, finding that the skin was the best, providing accurate predictions within a mean error of four years. The importance of the level of symbiosis between the microbiome and the skin according to age is increasingly being demonstrated (Kim, 2019; Trojahn, 2015), confirming the need to ensure that cosmetic products applied to the skin are compatible with the resident microbiome and the potential for monitoring the evolution of human-microbiome symbiosis throughout life.

[0008] The microbiome plays a role in human skin odor, which also evolves throughout life. Odor production may originate from the degradation of sweat compounds such as volatile fatty acids or odorous steroids (James et al., FEMS Microbiology Ecology, 2013), involving certain bacterial genera such as Corynebacterium and strains of Streptococcus. Short-chain fatty acids, such as caprylic acid, capric acid, valeric acid, or propionic acid, are also involved in the formation of body odor. These are products of the degradation of amino acids and long-chain fatty acids derived from sebum by bacteria of the genera Brevibacterium and Propionibacterium; propionic acid gives the skin a hazelnut odor at low concentrations (usually after showering) or a cheesy smell of dirty skin at high concentrations. These body odors vary based on gender, age, or menstrual cycle.

[0009] Skin biofilms play both positive and negative roles. The formation of a commensal microbiome on the skin corresponds to the colonization of a microbiome with protective effects against other pathogenic microorganisms that are associated with infection and can be identified using next-generation sequencing analysis technologies. For example, in the case of wound infections (an extreme case where the skin presents a discontinuous, fissured surface rather than a uniform, continuous surface), a group of bacteria known as the "pathogenic microbiota" (Proteus, Morganella, Anaerobic Cocci, and Peptococcus) has been described, the presence of which is associated with worsening, non-healing wounds (Dunyach-Rémi et al., 2020). On the other hand, also in the context of wound infections, bacteria have been identified that exert antimicrobial 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 microbiota."

[0010] The microbiome that resides on the skin is invisible except when it provokes a skin response from the immune system, such as acne breakouts associated with the proliferation of the bacteria Cutibacterium acnes, the redness associated with the inflammatory state of eczema, or atopic dermatitis exacerbated by colonization with the bacterium Staphylococcus aureus. There is also a correlation between spots on the skin and the presence of certain microorganisms, such as Kocuria and Aerococcus (Zanchetta et al., 2022).

[0011] Microscopy has long been the main method used to observe the skin microbiome. Advances in biomolecular technologies have opened up new areas of research through genomics, molecular genetics, metagenomics, high-throughput sequencing and "culturomics". By directly sequencing the DNA present in a sample, this approach not only provides a genomic description of the sample's contents, but also provides insights into the functional potential of the environment. Biologists use high-throughput sequencing technologies to study metagenomic samples. The DNA sequences obtained are then analyzed using bioinformatics techniques. Metagenomic studies require the logistics of transporting samples taken from a person (usually with a simple cotton swab) to an analytical laboratory, which itself must be equipped with a range of equipment for extracting genomic material (DNA) and sequencing it.

[0012] To reveal the presence of these microorganisms in biofilms as a characteristic of skin conditions, biomarkers can also be used: molecules present on the surface of the membrane, skin cell adhesion molecules, and secreted molecules (exopolysaccharides, DNA, proteins, etc.) that form the matrix of the biofilm. These biomarkers can be obtained from humans and immediately analyzed using lateral flow immunoassay (LFIA) technology.

[0013] Therefore, pathogenic microorganisms can be distinguished from commensals based on pathogen-associated molecular patterns (PAMPs). These so-called "virulence" markers are the most widely documented and described in the literature. These are secreted molecules, such as the Panton-Valentine Leukocidin protein of Staphylococcus aureus and porphyrins produced by fluorescent molecules of Propionibacterium acnes (visible under ultraviolet light (Wood's lamp)) with lower toxicity, which induce the aggregation of Staphylococcus aureus and the expression of pro-inflammatory molecules (IL-6, IL-8, prostaglandin E2, TNF-α, etc.) of keratinocytes.

[0014] Other markers are characteristic of natural biofilm formation of microorganisms on the skin surface and in the sebaceous ducts of the hair follicles (see below). Typically, microorganisms interact with the following families of skin proteins known as components of the extracellular matrix (ECM): plasminogen, fibronectin, laminin or mucin. For Gram-positive bacteria (single membrane), adhesion proteins are used: Bap family adhesins, type 4 pili (important for early biofilm formation), SAAT (self-associating autotransporter, promoting aggregation of bacteria carrying SAAT), compactins / invasins, etc.; and for Gram-negative bacteria (double membrane - LPS): Bap family adhesins displaying the LPXTG C-terminal domain, IMP (intrinsic membrane protein), type 3 and type 4 pili, etc.

[0015] One example is Propionibacterium acnes, a commensal bacterium that can become an opportunistic pathogen and cause acne. The bacterium can form a biofilm on the sebaceous glands of the hair follicles or on keratinocytes with the help of proteins present on the surface of the bacterial membrane, such as CAMP factor (Christie-Atkins-Munch-Petersen factor), sialidase, dermatan sulfate adhesin, glycosidic endoceramidase, groEL chaperone, "SH3 domain-containing lipoprotein", pili / fimbriae-like protein Flp, and especially DsA1 protein, which forms part of the MSCRAMM (molecular surface component of matrix-recognizing adhesion molecules) family, which is highly immunogenic and evokes a strong immune response in acne sufferers.

[0016] Staphylococcus bacteria play a major role in the skin microbiome, with Staphylococcus epidermidis being a commensal and rarely pathogenic species, and Staphylococcus aureus being a commensal species with healthy nasal carriage in 30% of the population that can become pathogenic, particularly in cases of atopic dermatitis or eczema. Staphylococci also use MSCRAMMs, such as proteins from the Clf-Sdr family, Bbp (bone sialoprotein binding protein), FnBP (fibronectin binding protein), and CNA (collagen adhesin) to form biofilms on the skin, and the SesJ protein has recently been identified for Staphylococcus epidermidis (Arora 2020). For Staphylococcus aureus, aureusimine (Phevalin) appears to be a marker of the biofilm phenotype. Note that Staphylococci also secrete the polysaccharide intercellular adhesin (PIA) extracellular matrix. Other major representatives of the skin microbiota are (Byrd et al., 2018) common to all skin types: Corynebacterium tuberculosis, Malassezia globosa; or more characteristic of oily, dry or wet skin: Staphylococcus capitis, Staphylococcus hominis, Streptococcus mitis, Streptococcus oralis, Micrococcus luteus, Corynebacterium simulans); or as microorganisms characteristic of older skin: Dermococcus, Actinomyces; young skin with Bacteroidetes (such as Bacteroides, Alistipes, Prevotella, Porphyromonas, Sphingobacterium) or with Firmicutes (such as Lactobacillus, Aerococcus, Oscillospira, Ruminococcus).

[0017] Organisms that are generally considered non-pathogenic can cause infections in hosts with weakened immunity or those that have recently received antimicrobial therapy. When immune responses are impaired, as is often the case in diabetic patients, they may not be able to prevent the colonization of damaged tissue by pathogenic bacteria. In chronically infected wounds, many bacteria form biofilms, in which they irreversibly attach to and grow on surfaces, producing extracellular polymers that promote matrix formation and alter their phenotype.

[0018] The most common bacteria found on human skin are Gram-positive and belong primarily to five genera. The following bacteria have been found:

[0019] Staphylococci, usually opportunistic, such as Staphylococcus epidermidis, which accounts for more than 90% of the aerobic resident flora present on the stratum corneum. Other Staphylococci found in skin biofilms are Staphylococcus aureus (common, for example, in asymptomatic carriers in the nasal cavity in 30% of cases) and Staphylococcus hominis;

[0020] Corynebacterium

[0021] Propionibacterium (e.g., Propionibacterium acnes, Cutibacterium granulosum, Propionibacterium vulgaris). Propionibacterium acnes produces fatty acids from lipolysis of sebum. In doing so, it acidifies the skin environment, which inhibits the growth of Streptococcus pyogenes colonies;

[0022] Lactobacillus;

[0023] Streptococcus.

[0024] Typically, three types of bacteria are encountered in most cases: 1) Staphylococcus epidermidis, a commensal skin bacterium that has a barrier effect against Staphylococcus aureus; 2) Propionibacterium acnes, a commensal skin bacterium that is usually harmless but can cause acne attacks depending on age (developmental stage), hormonal immersion, immunosuppression (transient, iatrogenic, etc.); 3) Staphylococcus aureus, a commensal bacterium that is carried asymptomatically by 30% of individuals (nasal carriage), causing various skin pathological changes (impetigo, atopic dermatitis, etc.).

[0025] 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. However, they affect the quality of life of 16 million French people and have a psychological impact that is often underestimated, associated with skin imperfections (spots, redness, acne, etc.) that the affected person will try to reduce or cover up by using cosmetic products.

[0026] According to a large epidemiological study conducted by the French Society of Dermatology (SFD) (the "Objectifs Peau" study, conducted in 2016 on a representative sample of 20,012 people aged 15 and over), 1 in 3 people in France suffer from a skin disease. And 80% of these patients have two skin diseases. Women are more affected than men: 33% of women have dermatological changes, compared to 28% of men.

[0027] These alarming figures are significantly higher than previous estimates. The stress of modern life and increasing pollution undoubtedly play a major role in the rising number of skin conditions.

[0028] Among the most common skin conditions, acne tops the list (3.3 million French people suffer from acne even after puberty), followed by eczema (2.5 million sufferers) and psoriasis (2.4 million). Scalp diseases (excluding hair loss), fungal diseases, and nail diseases come in second, with 2.3 million, 2.2 million, and 2.1 million French people suffering from them, respectively.

[0029] For all of these conditions, there are individual criteria for conditions affecting the skin (genetic susceptibility—mutations in genes encoding skin proteins such as filaggrin, immune system deficiencies, etc., lifestyle—stress, UV exposure, diet, etc.) and a combination of criteria affecting the microbiome (C-section, hygiene, environment, etc.). Regarding the effects of UV radiation, preliminary studies (Burns, 2019) show an increase in Cyanobacteria, a decrease in Lactobacillaceae and Pseudomonadaceae, the reactivity of different species to UVA and UVB, and the potential protective and anti-inflammatory effects of Lactobacillaceae. Here, a test evaluating the effects of UV exposure on human-microbiome symbiosis could also help refine recommendations, both for products that rehydrate the skin and regenerate / rebalance the microbiome (curative recommendations) and for protective sunscreens (preventive recommendations).

[0030] This is why cosmetic companies have been interested in understanding the skin microbiome for many years in order to offer their customers suitable and customized compositions that allow them to prepare active creams that are tailored to both the skin's physiology (oily, dry, irritated, with a certain degree of inflammation, etc.) and the state of each customer's skin microbiome at a given time. It's become clear that the claimed effects of anti-wrinkle and anti-aging creams and various cosmetic skin care products are not similar in their effectiveness and efficacy for all individuals, or even for the same individual over time. Different people and different skin types react differently to cosmetic products, so there's a need for devices that can determine an individual's effectiveness or reactivity to a specific type of skin care product. This requires a reliable and simultaneous method for analyzing skin condition and skin microbiome.

[0031] skin

[0032] The second member of the symbiosis to which the present invention relates is the organ "skin": the exposed part of the skin is called the horny layer (stratum corneum), which is a superposition of anucleated and completely cornified cells (i.e. corneocytes) forming a highly elongated thin layer (ROBERT et al. Dermopharmacologie, Edisem, 1985). Its thickness is about 10 μm, except on the palms and soles of the feet, where it is about 10 times thicker.

[0033] The stratum corneum is the final outgrowth of the organ (i.e., the skin) and covers an average of 1.5 m 2 Up to 2m 2 The skin is structured into three layers of tissue: the hypodermis (deepest), the dermis (middle), and the epidermis (superficial).

[0034] The hypodermis forms the deepest layer of the skin. It is a connective tissue rich in blood vessels that contains a large amount of adipose tissue, itself composed of cells called adipocytes. This layer is highly elastic, capable of absorbing shock and also provides thermal insulation.

[0035] The dermis is the middle layer. It is also the connective tissue that supports the epidermis. It is crisscrossed by a rich network of capillaries and is filled with numerous nerve endings. It is divided into two parts: the papillary dermis (superficial) and the reticular dermis (deep and middle layers). Fibroblasts dispersed throughout the dermis synthesize collagen and elastic fibers, which are immersed in a gel called the extracellular matrix, which is composed of water and glycoproteins. This acts as a water reservoir, which can be affected by environmental influences (dry weather in winter) or excessive exposure to ultraviolet radiation (sunbathing, UV rooms, etc.), leading to 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. It enables the body's first line of defense 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 of the microbiome. The epidermis, without capillaries, draws the necessary energy and nutrients from them to ensure its cellular activity. In addition to sweat glands, which play an important role in body temperature regulation, sweat glands and pilosebaceous glands are epidermal appendages implanted in the dermis.

[0036] The epidermis is the outermost, nonvascular structure and is divided into five superimposed layers (from inner to outer):

[0037] The basal layer (Stratum basale) regenerates the skin by means of cell division (almost all cells are in mitosis), which allows the generated cells to migrate towards the outermost layer. This layer is mainly composed of keratinocytes.

[0038] -The spiny layer (stratum spinosum or Malpighian layer) is the thickest layer of the epidermis. It is composed of keratinocytes.

[0039] The thinner granular layer (stratum granulosum) is composed of keratinocytes that have undergone 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 lamellar bodies).

[0040] -The clear layer (stratum lucidum) has anucleated cells (their nuclei have disappeared). The keratohyalin granules are converted to a protein called filaggrin.

[0041] The horny layer (stratum corneum) is the outermost layer, composed of flattened, dead cells called keratinocytes. Despite being dead, they retain biological activity. They have lost all their organelles and have been replaced by dense keratin filaments, held together by a lipid matrix of fatty acids, cholesterol, and ceramides, as well as desmosomes. This matrix is formed by the lamellar bodies found in the preceding layers. These tightly packed, connected cells form an impermeable coating on the skin and play an important defensive role.

[0042] The cornification process ensures continuous renewal of the skin from the basal layer to the stratum corneum over a period of 3 to 4 weeks. Various constituent elements will be present on the surface and will form the surface of the stratum corneum, on which the microbial community forms a biofilm. Here, biomarkers of skin condition will be discovered, making it possible to assess whether the skin is in a healthy state, hormonal influences (developmental period, menstrual cycle, etc.), inflammation (endogenous or exogenous), allergic reactions, and aggressions (physical: UV radiation, pollution; chemical: personal care products, cosmetics; mechanical: wear during scrubbing, exfoliation or peeling, etc.).

[0043] The stratum corneum is the first line of defense between the human body and the outside world. It acts as a barrier against the penetration of external agents, preventing harmful microorganisms or chemical agents from entering our body. If this first level of protection fails, another protective mechanism comes into play, including cells of the immune system.

[0044] The stratum corneum represents the first level of the interactome, in direct contact, where the microbiota forms biofilms using adhesion proteins and secretes exopolysaccharides that enable their persistence on the skin.

[0045] When the physical barrier of the stratum corneum no longer works because it has been physically removed by wear, for example on the surface after obvious peeling or peeling, or deeper in the case of trauma or surgical wounds, the skin's immune system begins to work. Its purpose is to protect the host and, if necessary, restore the integrity of the skin. It is divided into two types: first, innate immunity, and then adaptive or specific immunity. In both cases, the first defenders are keratinocytes because they are the most numerous (90% of skin cells). They act as immune sentinels and recognize foreign agents with the help of pattern recognition receptors (PRRs) from the Toll-like receptor family. Once recognized, these pattern recognition receptors synthesize chemical modulators (cytokines and chemokines). They can trigger an inflammatory cascade. On the other hand, keratinocytes are also able to produce antimicrobial peptides that inhibit microorganisms (such as Staphylococcus aureus and Candida albicans).

[0046] The epidermis contains two types of immune cells. Dendritic cells, or Langerhans cells, possess pseudopodia that enable them to latch onto pathogens and engulf them. These cells are known as APCs: antigen-presenting cells. Once the pathogen or antigen is engulfed, the dendritic cells secrete chemoattractants (prostaglandins or chemokines), which cause local vasodilation and, consequently, increase blood flow and the local recruitment of other immune cells (such as polymorphonuclear neutrophils and macrophages). Dermal immune cells (dermal dendritic cells, macrophages, mast cells) are also mobilized at the site of action. The epidermis also contains T lymphocytes, which are activated in the lymph nodes with the help of APCs. Therefore, following APC activation, a cascade of proliferation and differentiation ensues, ultimately leading to the destruction of the pathogen. These biomarkers can be used to assess the condition of the skin's surface, particularly the level of inflammation, which can be chronic and becomes increasingly severe with age, a condition known as "inflammaging."

[0047] In general, biomarkers of skin inflammation include interleukins IL-1β, IL-4, IL-6, IL-8, IL-11, IL-12, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), granulocyte macrophage colony-stimulating factor (GM-CSF), and transforming growth factor-β (TGF-β). Although not directly considered as a marker of inflammation, immunoglobulin E (IgE) must also be mentioned, as its serum levels are elevated in atopic eczema-dermatitis and are responsible for most so-called IgE-dependent allergic reactions.

[0048] Other skin cells participate in maintaining a healthy state when facing the sun's attack or being exposed to ultraviolet radiation (for example, in a room using a UV lamp). These include melanocytes present in the skin, which play a protective role against ultraviolet (UV) radiation. When the skin is exposed to UV light, keratinocytes secrete melanocyte-stimulating hormone or α-MSH (α-melanocyte stimulating hormone) by maturing the prohormone POMC (proopiomelanocortin). The α-MSH hormone is attached to the membrane receptor called MCR (melanocortin receptor) expressed by melanocytes and triggers melanin biosynthesis, which is the source of skin tanning. Therefore, this protects the genetic material of epidermal cells exposed to UV radiation.

[0049] A complete description of the organ "skin" is one with appendages included in different layers: hair and sebaceous and sweat glands.

[0050] Hair covers almost the entire human body and comes in various sizes. It plays a crucial role in regulating body temperature. It is anchored in the dermis in so-called hair follicles.

[0051] The sebaceous glands attached to the hair are intradermal glands. They synthesize sebum, which, along with sweat, is the main component of the hydrolipidic film that protects the skin and prevents it from drying out. These glands interact with the immune system and subsequently influence the microbiome, as we will see later.

[0052] Sweat glands secrete sweat, and through this process, the body is able to combat the heat. In fact, when the body temperature rises, such as during exercise or when you are sick, the sweat released evaporates at the surface of the skin: this mechanism removes heat and, together with vasodilation, helps cool the body.

[0053] Among these appendages, the sebaceous glands play a central role in regulating the skin's pH, its more or less occlusive character and therefore the acquisition of oxygen (influencing the aerobic or anaerobic capacity of the microbial flora), its antioxidant effects, its pro- and / or antimicrobial effects (which vary 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.

[0054] Skin-microbiota symbiosis

[0055] The human-microbiome symbiosis at the skin level is further complicated, as it has recently been revealed that so-called "innate lymphoid" immune cells (ILCs) play a regulatory role in the sebaceous glands (Kobayashi et al., 2019). These ILCs (by producing "TNF receptor ligands") restrict the growth of the sebaceous glands. When these ILCs are deficient, sebaceous gland hyperplasia is observed, leading to increased production of antimicrobial lipids, particularly palmitoleic acid and oleic acid, which have an impact on the balance of the skin microbiome. In fact, palmitoleic acid inhibits the growth of Gram-positive aerobic cocci (such as Staphylococcus aureus and Staphylococcus xylosus), but does not inhibit the growth of Gram-negative anaerobic bacteria (such as Bacteroides species). Combined with the overproduction of oleic acid, this creates an occlusive membrane, i.e., anaerobic conditions that are unfavorable for Gram-positive cocci. This may lead to the recommendation of cosmetic products containing these two fatty acids when the biomarkers associated with the "oily skin + high presence of Staphylococci" profile have been characterized using the immunochromatographic disclosure device that forms the subject of this patent.

[0056] The human-microbiota symbiosis is also organized at levels beyond the epidermis-stratum corneum, but more broadly, based on various endogenous factors such as body area (facial T-zone, scalp, underarms, etc.), age, hormonal immersion (developmental period, menstrual cycle, etc.), inflammation (endogenous sources - atopic dermatitis, psoriasis, etc., or exogenous - contact with irritating products), aggression (physical: UV radiation, pollution; chemical: personal care products, cosmetics; mechanical: wear during exfoliation or peeling, etc.).

[0057] The body regions are divided into three zones characterized by different physicochemical parameters:

[0058] -Wet areas:

[0059] The so-called "wet" areas are characterized by a high density of sweat glands. Moisture is due to the secretion of sweat by these glands. Sweat is mainly composed of water, inorganic salts, uric acid and urea. The relevant human body areas are: the axilla, perineum, interdigital folds, palms and underarms, where there is a high level of microbial colonization: 10 5 -10 8 bacteria / cm 2 .

[0060] -Lipid region:

[0061] The so-called "lipid" zone is 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, is a major lipid zone, where the concentration of microorganisms is about 10 6 -10 7 bacteria / cm 2 .

[0062] -Drying area:

[0063] So-called "dry" areas lack sweat and sebaceous glands and therefore contain less sweat and sebum secretions.

[0064] The backs of the hands and the outsides of the limbs are the main dry areas. These areas have the lowest concentrations of microorganisms: 10 3 -10 4 bacteria / cm 2 .

[0065] The average pH of the skin is 5.5. This is due to the hydrolipidic film covering the entire surface of the skin, which is produced by secretions from the sweat glands and sebaceous glands. The relative acidity of this film provides protection against pathogens in particular. Skin pH is a very important parameter for maintaining skin homeostasis and biofilm microbial population balance. In many pathological changes, such as atopic eczema-dermatitis, psoriasis, acne, etc., pH changes, and it has been documented that pH changes in the case of atopic dermatitis associated with the presence of Staphylococcus aureus (Rippke et al., 2004). Background Art

[0066] Various solutions for analyzing the skin microbiota are known in the prior art.

[0067] WO 2014184151 A1 discloses a "point-of-care" diagnostic device based on lateral flow assay technology, capable of non-invasively analyzing factors secreted and diffused from the skin surface. Specifically, the document discloses a diagnostic kit for detecting the presence or amount of one or more analytes in a test sample obtained from the skin surface of a mammal, characterized in that the diagnostic kit comprises:

[0068] a) a separate insert for a lateral flow device comprising a membrane having a defined thickness, width and length, said membrane optionally being attached to a rigid support and configured to obtain said test sample (analyte),

[0069] b) a lateral flow assay device configured to receive the separate insert, and

[0070] c) an attachment element configured to releasably attach the separate insert to the skin surface of a mammal.

[0071] Patent application US2022 / 0178943 provides a 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 kit comprising:

[0072] a) a lateral flow assay (LFIA) device comprising a cartridge comprising one or more porous elements forming a porous support array, wherein the cartridge is configured to receive and hold a sample collection pad, wherein the sample collection pad is configured to contact the porous support array when the sample pad is inserted into the cartridge,

[0073] b) a blister pack, wherein the blister pack contains a buffer solution, wherein the box is configured to receive the blister pack, and

[0074] c) a sample collection pad configured to collect the test sample.

[0075] Application EP3691788A1 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:

[0076] a) a separate pad configured for collecting the test sample, the pad comprising a sample collection pad attached to a support member,

[0077] b) a lateral flow assay device comprising one or more porous elements, wherein the lateral flow assay device is configured to receive and retain the individual swab, wherein the sample collection pad is configured to form a portion of a porous support array when the individual swab is inserted into the lateral flow assay device.

[0078] Application WO2019025610 discloses a diagnostic kit and method based on a lateral flow assay device to detect the presence or amount of one or more test analytes in a test sample taken from the skin of a mammal.

[0079] Disadvantages of existing technology

[0080] There are analytical solutions based on highly accurate molecular biology techniques, which are lengthy, complex, and expensive to implement, or lateral flow assay solutions that have only one or two markers and are not suitable for skin characterization. Binary LFIA solutions only reveal the presence or absence of a biomarker.

[0081] Since the method for recovering the amount of analyte sampled with background art solutions (swabs or swabs, D-Squame or Sebutape adhesive strips, etc.) and the performance of lateral flow immunochromatographic tests require a minimum of about 10 4 analytes, therefore, a genomic analysis solution with a DNA amplification step is used to achieve significant results.

[0082] These solutions are not entirely satisfactory because they require complex post-sampling procedures (DNA extraction, PCR, Q-PCR or sequencing, etc.) in the biology laboratory and require personnel experienced in using genomic equipment.

[0083] To do so, they require extensive logistics, with processing times between sampling and genomic analysis ranging from days to weeks, and are also relatively costly.

[0084] As a result, to date, 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 (for example, according to clinicaltrial.gov, the 2017-18 study, “The Effect of Skin Cleansers on the Skin Surface Microbiome,” sponsored by Mundipharma Manufacturing Pte Ltd, included only 12 volunteers whose skin microbiomes were analyzed by next-generation sequencing to compare the effects of three products: 1) 7.5% povidone-iodine, 2) 4% chlorhexidine skin cleanser, and 3) plain non-antibacterial soap).

[0085] Ultimately, the proposed metagenomic test or "microbiome" analysis results in the generation of a list of identified microorganisms for documentation purposes on an associated smartphone app. The ultimate goal of the method consists of recommending cosmetic and personal care products based on the information collected via the smartphone app when ordering the "skin microbiome" test (a facial photo to assess parameters such as wrinkles, redness, etc., and declarative information such as age, weight, height, smoking habits, alcohol consumption, estimated skin condition (irritation, dryness, oiliness), declaration of pathological changes (acne, eczema, atopic dermatitis, psoriasis, etc.), etc.).

[0086] The solution provided by the present invention

[0087] The present invention remedies the shortcomings of the background art by providing a solution that

[0088] -fast

[0089] - Cheap

[0090] - Includes sufficient elements to characterize the skin, and

[0091] - Allows for several levels of biomarker readings to give sufficient information to make cosmetic recommendations.

[0092] Therefore, the present invention relates to a device for revealing a skin-microbiota complex in an immunochromatographic manner, said device comprising a support on which a transfer well is arranged for receiving a solution containing components of the skin-microbiota complex, said well leading to a plurality N of revealing zones, each revealing zone having (i) an immunodetection reagent and (ii) an immunocapture reagent, and wherein said skin-microbiota complex consists of skin cells and microorganisms as well as other constituent elements of the skin microbiota, said device comprising:

[0093] ○N number of reveal areas, where N is equal to or greater than 5

[0094] o At least one immunodetection reagent specific for a skin cell biomarker and at least one immunodetection reagent specific for a biomarker of the skin microbiome

[0095] o The immunoassay reagents, each comprising an antibody specific for a biomarker of skin cells or a biomarker of the skin microbiome conjugated to a colorimetric identification system

[0096] o the capture reagents, each of which comprises an antibody bound to the support in each of the N revealing regions, capable of recognizing the biomarker present in said region,

[0097] o At least one anti-tampering tool for orienting the reading of the device

[0098] Characterized in that the device comprises:

[0099] - at least three zones for revealing three biomarkers of the skin microbiota, the biomarkers being bacteria from each of the genera Staphylococcus, Propionibacterium and Corynebacterium;

[0100] - for revealing at least one region of a skin cell biomarker selected from (i) a structural protein, (ii) an inflammatory biomarker, or (iii) an allergy biomarker.

[0101] The invention also relates to a disposable kit for characterizing the condition of the skin, a system for implementing personalized cosmetic recommendations, and a method for personalized cosmetic recommendations based on characterizing the skin-microbiota complex of an individual and implementing the device according to the invention.

[0102] Just as the gut microbiome interacts with the digestive tract in a "microbiome-host" symbiotic relationship, the skin microbiome interacts with the skin in a symbiotic relationship. Similarly, there are as many skin types as there are microbiome types, and damage to one member of the symbiosis will have an impact on the other. This is important for maintaining healthy skin or correcting imperfections with cosmetic care products. The present invention enables rapid and significant biological determination of both members of the symbiosis by immunochromatographic characterization of biomarkers available on the surface of the microbiome-skin interactome. Artificial intelligence is then used to recommend the best care that is appropriate for the skin type on the one hand and compatible with its microbiome on the other, thereby reducing the risk of adverse effects. Similarly, in the case of skin blemishes and / or microbiome imbalance, the present invention will recommend the best corrective treatment to ensure compatibility with or ability to modulate the microbiome. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] [ Figure 1 ] Figure 1 : Description of an embodiment of a device for immunochromatographically revealing the skin-microbiota complex according to the invention, comprising a support (0) on which a transfer well is arranged for receiving a solution containing components of the skin-microbiota complex, said well opening into a plurality N of revealing zones. (1) Sampling device (swab, cotton tip, etc.), (2) Sampling solution from the skin, (3) Transfer well, (4) Absorption zone for the sampling solution, (5) Zone with detection conjugate (or monoclonal detection antibody), (6) Monoclonal detection antibody, (7) Direction of migration flow, (8) Zone on which monoclonal capture antibody has been deposited, (9) Monoclonal capture antibody (or antibody fragment or Nanobody), (10) Control zone, (11) Antibody (or antibody fragment or Nanobody) directed against the conjugate.

[0104] [ Figure 2 ] Figure 2 : Depiction of three embodiments of test strips that can be used in a device for immunochromatographically revealing the skin-microbiota complex according to the present invention. (A) One biomarker detected with several detection thresholds (e.g., for one microorganism: threshold 1 = 10 3 CFU; threshold 2 = 10 5 CFU; threshold 3 = 10 7 CFU; threshold 4 = 10 9CFU; for one molecule: threshold 1 = 1 ng; threshold 2 = 10 ng; threshold 3 = 100 ng; threshold 4 = 1000 ng); control corresponds to 3 microbiota biomarkers detected on 4 zones by detection with antibodies (or antibody fragments) (B); control corresponds to 2 skin biomarkers detected on 4 zones by detection with antibodies (or antibody fragments) (C); control corresponds to biomarker deposition (validation of migration of detection conjugates specific for each biomarker). The intensity of the bands shows that the control zone must be saturated, while the intensity of the other zones depends on the amount of analyte that has migrated, allowing semi-quantitative analysis of the biomarkers.

[0105] [ Figure 3 ] Figure 3 : Representation of an embodiment of a support (0) of a device for immunochromatographically revealing a skin-microbiota complex according to the invention, on which a (central) transfer hole (3) is arranged, intended to receive a solution containing components of the skin-microbiota complex, said hole opening into a plurality N of revealing areas (herein, for illustrative purposes, there are 8 strips, each comprising 4 or 5 revealing areas) arranged in strips arranged radially around said central transfer hole. The support comprises at least one anti-tampering means (12) for orienting the reading of the device, embodied as a black triangle, which may be, for example, a notch or a pattern, and which allows orienting the reading direction of the support.

[0106] [ Figure 4 ] Figure 4 Schematic diagram of the process for achieving personalized cosmetic recommendations. (A) Depicts a semiquantitative analysis of the microbiome profile, the same type of analysis performed on one or more skin biomarkers, (B) presents the results of the combined biomarker analysis to establish a profile of the skin-microbiome complex, and (C) recommends suitable cosmetic products based on a product database and correlated with the status of the skin-microbiome complex. DETAILED DESCRIPTION

[0107] A first object of the present invention relates to a device for revealing a skin-microbiota complex in an immunochromatographic manner, the device comprising a support on which a transfer well is arranged for receiving a solution containing components of the skin-microbiota complex, the well leading to a plurality of N revealing areas, each revealing area having (i) an immunodetection reagent and (ii) an immunocapture reagent, and the skin-microbiota complex is composed of skin cells and microorganisms and other constituent elements of the skin microbiota, the device comprising:

[0108] ○N number of reveal areas, where N is equal to or greater than 5

[0109] o At least one immunodetection reagent specific for a skin cell biomarker and at least one immunodetection reagent specific for a biomarker of the skin microbiome

[0110] o The immunoassay reagents, each comprising an antibody specific for a biomarker of skin cells or a biomarker of the skin microbiome conjugated to a colorimetric identification system

[0111] o the capture reagents, each of which comprises an antibody bound to the support in each of the N revealing regions, capable of recognizing the biomarker present in said region,

[0112] o At least one anti-tampering tool for orienting the reading of the device

[0113] Characterized in that the device comprises:

[0114] - three zones for revealing three biomarkers of the skin microbiome, the biomarkers being three bacteria of the genera Staphylococcus, Propionibacterium and Corynebacterium;

[0115] - a region for revealing a skin cell biomarker selected from (i) a structural protein, (ii) an inflammatory biomarker, or (iii) an allergy biomarker.

[0116] For the purposes of this invention, the "skin-microbiota complex" consists of two cell types: skin cells of human origin and microorganisms; these two cell types participate in the skin's condition through their interaction. A symbiotic phenomenon is observed. Thus, by simultaneously sampling human cells and the microbiome present on the skin's surface, we obtain a representative sample of the skin's condition. This complex also contains other components of the skin-microbiota complex secreted by these two cell types. These include, on the one hand, molecules secreted by skin cells (whether epidermal or immune cells), but also secretions from the microorganisms that make up the microbiota.

[0117] The goal here is to establish a holistic representation of skin condition based on revealing biomarkers representing the physiological state of the skin and the composition of its microbiome, enabling their combined analysis. The number of revealing zones, N, corresponds to the number of biomarkers tested. N is greater than 5, allowing for testing a sufficient number of biomarkers to obtain value-added results from the combined analysis of different biomarkers. The device also includes at least one control zone, which is not included in the "at least 5 zones."

[0118] The "at least five zones" of the device reveal at least one skin cell biomarker selected from (i) structural proteins, (ii) inflammatory biomarkers, or (iii) allergy biomarkers, and at least three biomarkers of skin microbiota, the three biomarkers being bacteria belonging to the genera Staphylococcus, Propionibacterium, and Corynebacterium. Preferred embodiments are described below.

[0119] In specific embodiments of the invention, at least 8, 10, 15, 20, 25, 30 different types of biomarkers are tested simultaneously.

[0120] The device not only reveals the absence or presence of the biomarkers in question, but also quantifies them in absolute or relative terms based on the chosen method.

[0121] microbiome

[0122] The skin microbiome constitutes the first element of the symbiotic complex revealed by the device according to the invention. It comprises a range of microorganisms including bacteria, yeasts, fungi and mites.

[0123] In order to provide information about the status of the skin microbiota, the device according to the invention can reveal at least the presence of bacteria of the genera Staphylococcus, Propionibacterium and Corynebacterium.

[0124] Preferably, the Staphylococcus species studied will be selected from Staphylococcus epidermidis and Staphylococcus aureus. These biomarkers can be revealed by antibodies that can recognize at least these two species of the genus Staphylococcus (genus-specific antibodies), or by two antibodies specific for the species Staphylococcus epidermidis and Staphylococcus aureus, respectively.

[0125] Preferably, the Propionibacterium species studied will be selected from P. acnes and P. granulosum. These biomarkers can be revealed via antibodies that are able to recognize at least these two species of the Propionibacterium genus (genus-specific antibodies), or via two antibodies specific for the species P. acnes and P. granulosum, respectively.

[0126] Preferably, the coryneform bacteria species studied is selected from dry coryneform bacteria (Corynebacterium xerosis) and Corynebacterium kroppenstedtii (Corynebacterium kroppenstedtii).These biomarkers can reveal via the antibody (genus-specific antibody) of at least these two species that can identify Corynebacterium or via respectively species dry coryneform bacteria and Corynebacterium kroppenstedtii have two kinds of specific antibodies (species-specific antibodies).

[0127] In the context of the present invention, the skin microbiome is analyzed by revealing epitopes exposed on the surface of the microbiome. In a specific embodiment of the present invention, the epitope exposed on the surface of the microbiome is associated with a protein present on the surface of the microorganisms that make up the microbiome. In particular, it can be an epitope associated with the biofilm-like behavior of the microbiome.

[0128] The disclosure area can be used to disclose "additional biomarkers of the skin microbiome," i.e., biomarkers other than those that disclose the presence of bacteria belonging to the genera Staphylococcus, Propionibacterium, or Corynebacterium, or to the related species mentioned previously. These other biomarkers may disclose the presence of microorganisms selected from bacteria, yeasts, fungi, or mites.

[0129] Preferably, the microorganisms constituting the microbiota to be studied to establish the diagnosis of skin conditions within the scope of the present invention are chosen from the following bacteria (Byrd et al., 2018):

[0130] Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus lugdunensis, Staphylococcus hominis (H, G), Streptococcus mitis (H, G), Streptococcus oralis (S), Streptococcus pseudopneumoniae (S), Streptococcus sanguinis (S), Staphylococcus capitis, Propionibacterium acnes, Corynebacterium pseudobacterium, Corynebacterium fastidiosums (H), Corynebacterium afermentans (H), Corynebacterium dry bacteria, Corynebacterium aurimucosum (G), Corynebacterium krebs (G), Corynebacterium amycolatum (G), Corynebacterium tuberculostearicum, Veillonella parvula (S), Micrococcus luteus (S, H), Enhydrobacter aerosaccus (H), Epidermophyton floccosum, Nannizzia nana, Nephroselmis olivacae, Cyanophora paradoxa, Aureoumbra lagunensis, Pycnococcus provasolii, Pyramimonas parkeae, Parachlorella kessleri, Aspergillus tubingensis, Zymoseptoria tritici,

[0131] Tilletia walkeri or bacteria of the genera Dermococcus, Actinomyces, Bacteroides, Alternaria, Prevotella, Porphyromonas, Sphingobacillus, Lactobacillus, Aeromonas, Oscillospira, and Ruminococcus.

[0132] Other microorganisms that make up the skin microbiota that may be studied to establish a diagnostic according to the invention are:

[0133] Yeasts such as Malassezia globosa, Malassezia restricta, Malassezia furfur, Malassezia sympodialis, Candida parapsilosis, and the like.

[0134] -Fungi, such as Aspergillus tubingensis, Triticum aestivum, Tilletia perenne, Epidermophyton floccosum, Nepenthes dwarfus, Nephrolepis ovata, Diatomaceous blue, Brown tide algae, Provasoli microcystis, etc.

[0135] - mites, such as Demodex folliculorum and Demodex brevis.

[0136] The device according to the invention thus 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 of other elements within the complex that provide interesting information about the condition of the skin. If these other elements are immunogenic, the device can be supplied with antibodies capable of revealing their presence.

[0137] By “epitopes exposed on the surface of the microbiota”, it is meant epitopes exposed on the surface of the microorganisms constituting the microbiota via molecules constituting the membrane of the microorganisms (such as proteins, complex fatty acids and polysaccharides), as well as epitopes formed by secreted molecules constituting the biofilm matrix (such as exopolysaccharides).

[0138] The skin cells harvested superficially are cells of the stratum corneum or, depending on the intensity of the sampling (abrasion) or the condition of the skin (exposed after scrubbing or intense exfoliation or peeling), cells from the underlying stratum lucidum or even the stratum granulosum, which is normally even more deeply buried.

[0139] By "proteins present on the surface of microorganisms", it is meant proteins exposed on the membrane: secretory proteins and structural proteins synthesized by the cell, which pass through or are attached to the cell membrane, as in the case of secretory proteins. As an illustration, Staphylococcus aureus produces the Panton-Valentin leukocidin protein and the extracellular matrix "polysaccharide intercellular adhesin" (PIA), Propionibacterium acnes produces porphyrins, Pseudomonas produces a matrix based on alginate, Escherichia coli produces a matrix based on colanic acid, and Propionibacterium acnes produces a matrix based on β-1,6-linked N-acetylglucosamine (PNAG); for structural or adhesive proteins on the surface of the skin: for Gram-positive bacteria (monomembranous): Bap family adhesins, type 4 pili (important for early biofilm formation), SAAT (self-associating autotransporter, promoting the aggregation of bacteria carrying SAAT), compactins / invasins, etc.; more specifically, for Propionibacterium acnes, proteins present on the surface of the bacterial membrane, such as CAMP factor (Christie-Atki ns-Munch-Petersen factor), sialidases, dermatan sulfate adhesins, glycosidic endoceramidases, GroEL chaperones, SH3 domain-containing lipoproteins, Flp fimbriae / fimbriae-type proteins, and especially DsA1 proteins; more specifically, for Staphylococcus bacteria, MSCRAMMs (molecular surface components of adhesion molecules that recognize matrix), such as proteins of the Clf-Sdr family, as well as Bbp (bone sialoprotein binding protein), FnBP (fibronectin binding protein) and CNA (collagen adhesin), SesJ protein has recently been identified for Staphylococcus epidermidis (Arora et al., 2020), while for Staphylococcus aureus, chlortetracycline (Phevalin) appears to be a biofilm phenotypic marker; and for Gram-negative bacteria (double membrane-LPS): adhesins of the Bap family exhibiting the LPXTG C-terminal domain, IMP (intrinsic membrane protein), type 3 and type 4 fimbriae, etc.

[0140] Thus, the proteins exposed on the membranes of the microorganisms constituting the microbiota are selected from the group consisting of Panton-Valentin leukocidin, porphyrins, alginates, β-1,6-linked N-acetylglucosamine (PNAG), Bap family adhesins, type 4 fimbriae, self-associating autotransporters, compactins / invasins, CAMP factors, sialidases, dermatan sulfate adhesins, glycosidic endoceramidase, GroEL chaperones, SH3 domain-containing lipoproteins, Flp fimbriae / fimbriae proteins, DsA1 proteins; Clf-Sdr family proteins such as Bbp, FnBP and CNA, SesJ proteins, chlortetracycline, Bap family adhesins displaying the LPXTG C-terminal domain, IMPs, type 3 and type 4 fimbriae.

[0141] In the case of Gram-positive bacteria, the complex fatty acids exposed on the membranes of the microorganisms that constitute the microbiota are chosen from teichoic acids or even lipoteichoic acids, which may contain long chains of ribitol phosphate or glycerol-3-phosphate; whereas in the case of Gram-negative bacteria, they are chosen from lipopolysaccharides, which are glycolipids containing a lipid region called lipid A, which is most often made of a disaccharide of phosphorylated glucosamine and contains fatty acids with ester or amide bonds.

[0142] The sugars exposed on the membranes of the microorganisms constituting the microbiota are selected from the group consisting of sugar polymers (e.g. based on rhamnose) and peptidoglycans.

[0143] In a preferred embodiment of the present invention, at least one of the skin microbiota-specific biomarkers corresponds to a biomarker associated with biofilm-like behavior of the microbiota. Bacteria and other microorganisms in biofilm form are defined as opposed to mobile, non-adherent bacteria and other planktonic microorganisms. Biofilm-specific biomarkers may correspond to proteins present on the surface of the microbial membrane or to another element of the microbiota.

[0144] When we want to study the presence of bacteria or other microorganisms in the form of biofilms, the biomarkers are chosen from adhesion proteins such as fimbriae, curli pili, fimbriae, etc., which are characteristic of the presence of biofilms of microflora that form naturally on the skin.

[0145] skin

[0146] The condition of the skin constitutes the second element of the symbiotic complex revealed by the device according to the invention. The skin condition is analyzed with the aid of at least one skin cell biomarker selected from (i) structural proteins, (ii) inflammatory biomarkers or (iii) allergy biomarkers.

[0147] Structural proteins are targeted when we want to reveal the presence of skin cells and / or assess skin quality. The use of structural protein-like biomarkers can also be used to quantify the number of skin cells present in a sample or to normalize biomarker levels. Structural proteins of interest include keratin, filaggrin, and loricrin.

[0148] Inflammatory biomarkers are selected from C-reactive protein (CRP), interleukins IL-1β, IL-4, IL-6, IL-8, IL-11, IL-12, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), granulocyte macrophage colony-stimulating factor (GM-CSF), and transforming growth factor-β (TGF-β).

[0149] The allergy biomarker is selected from the group consisting of immunoglobulins IgE, IgA and IgG, preferably IgE. In fact, in cases of atopic eczema or dermatitis, serum IgE levels are elevated.

[0150] In a preferred embodiment of the present invention, the "at least one" skin cell biomarker studied is IL-1β. As for the other two types of skin cell biomarkers, the structural protein is preferably keratin, and the allergy biomarker is preferably IgE.

[0151] In a preferred embodiment of the invention, the device comprises a zone for specifically revealing the presence of bacteria of the genera Staphylococcus, Propionibacterium and Corynebacterium, and IL-1β. In a more specific embodiment, the device further comprises a zone for revealing the presence of IgE. In an even more specific embodiment, it further comprises a zone for revealing the presence of keratin.

[0152] Devices as biomarker supports

[0153] The disposable immunochromatographic revealing device is sterilely packaged and dehydrated. The module adopts the form of a support body usually made of plastic, which has a hole for a solubilizing solution containing the biomaterial of the harvest, thereby producing a series of channels (such as nitrocellulose strips or lateral flow strips), which contain multiple recognition zones and revealing zones specific for the biomarker of interest. These channels can, for example, receive a nitrocellulose strip carrying an antibody, wherein the solubilizing solution containing the biomaterial of the harvest migrates on the strip by capillary action.

[0154] In a preferred embodiment, the device according to the present invention is a lateral flow immunochromatographic device.

[0155] The detection antibodies can be conjugated to nanoscale gold particles (allowing only one red label color per strip, but different capture lines) or to colored particles (multiplying the number of detections per strip) or e.g. latex (blue).

[0156] Depending on the biomarker disclosure mode, several different configurations of the disclosure zone are possible.

[0157] A revealing zone can be provided on a nitrocellulose strip with a single uniform concentration of capture antibody across the width of the strip (homogeneous zone) to form a line that is revealed by interaction with the analyte (becomes visible by the detection antibody-conjugate). Several detection zones for different analytes can be combined on one strip, each analyte being detected with its own capture antibody threshold concentration ( Figure 2 ). In another arrangement, the revealing zone may also be provided with at least two capture antibodies of varying concentrations. In alternative embodiments, the zone comprises 2, 3 or more capture antibodies of varying concentrations.

[0158] In addition, the detection thresholds of the various biomarkers can be normalized so that relative quantification can be established between the different biomarkers. This normalization can be performed relative to a given common marker (e.g., keratin) used as a reference, or by establishing relative quantification of all markers relative to each other (e.g., bringing them to a cumulative total of 100, where each marker obtains its own proportional value).

[0159] The device must include a mishandling prevention tool, which is a marking necessary to correctly orient the device when observing the signal revealing the biomarker (the visible line on the strip). The mishandling prevention tool is located directly on the device, on the reading surface. It can take a graphical form, such as a picture, or a physical form, such as a notch, a hole, or any other marking system. It can be composed of one, two, or more marking elements.

[0160] The device generally consists of a disc (a support for the revealing area) enclosed in a case.

[0161] Figures 1 to 3 Different embodiments of the device according to the invention are shown.

[0162] A second object of the present invention relates to a disposable kit for characterizing skin conditions, comprising:

[0163] - a device for sampling the skin-microbiota complex, comprising means for sampling said complex and a receptacle containing a solubilizing solution;

[0164] - A device as previously described for immunochromatographically revealing the skin-microbiota complex.

[0165] The sampling tool may, for example, consist of a patch, a spatula, a swab soaked in a buffer solution or a sponge soaked in a buffer solution, a silk mesh or gauze soaked in a buffer solution, or any other suitable tool capable of physical exfoliation, soaking or adsorption behavior.

[0166] The number of microorganisms (especially bacteria) per square centimeter of skin varies greatly depending on the skin area, especially the sebum content. Therefore, in order to ensure representative sampling, it is necessary to have a sampling device that is suitable for the skin type and the sampling area so that there is enough biological material to analyze the biomarkers of the skin cells and the microbiome. The sampled biological material includes the constituent elements of the exposed skin area (cells of the stratum corneum and / or the stratum lucidum and / or the stratum granulosum) and the constituent elements of the microbiome (bacteria, yeasts, fungi, and even mites such as Demodex).

[0167] Typically, sampling should be performed to yield a sufficient number of bacteria to obtain at least 10 3 The concentration of CFU / mL bacteria. Preferably, for optimal detection, the concentration of the solution containing the harvested microbial population should be 10 4 CFU / mL bacteria. However, these concentrations are given for guidance purposes only and depend on the detection sensitivity of the disclosed method.

[0168] When sampling is performed via a patch, the patch will have an interaction surface with the skin (collection surface) that is at least 5 cm 2 , or even 10cm 2 , 12cm 2 Or greater, depending on the abundance of the skin microbiota and the sampling efficiency. The sampling (or harvesting) tool is supplemented by a receptacle (such as an extraction tube or equivalent) pre-filled and packaged in sterile packaging, containing a solubilization buffer (typically between 1 mL and 5 mL), such as a lysis / migration buffer, to buffer the pH of the sample, minimize nonspecific binding, neutralize interferences, and control flow rate by using various salts, surfactants, detergents, stabilizers, or blocking agents (exemplary composition: PBS 1X with 1% Tween R20). The receptacle has a volume suitable for directly receiving the collection surface of the sampling device in order to dissolve the material sampled from the surface of the skin.

[0169] The disposable kit requires only simple handling, can be performed immediately after sampling without any biological knowledge, and enables characterization of samples relative to a large number of reference biomarkers to identify combinations representative of various skin / microbiota complexes.

[0170] A third object of the present invention relates to a system for implementing personalized cosmetic recommendations based on the characterization of the skin-microbiota complex, the system comprising:

[0171] - a device for sampling the skin-microbiota complex, comprising a tool for sampling said complex and an extraction tube containing a solubilizing solution;

[0172] - a device for immunochromatographically revealing the skin-microbiota complex as previously described, wherein said N revealing zones constitute zones capable of providing a signal upon detection of a biomarker;

[0173] - A computer that performs analysis processing of an image consisting of all signals from said N zones revealed by means of said immunochromatographic revealing device, in order to determine the properties of the biomarkers that have been identified in said N revealed zones, and to classify the skin condition based on the combination of the identified biomarkers.

[0174] The computer acquires an image of each revealing area after a given reaction time and transmits the image in digital form to a processing center for automatic analysis in order to characterize the type of biomarker that has reacted with the antibody present in the revealing area of the display device.

[0175] The image can be acquired in a single shot for a one-time analysis. This analysis mode is particularly suitable for use with devices where the antibody detection threshold is standardized, or where the revealing zone includes at least two different antibody concentrations.

[0176] It is also possible to obtain images in several continuous shots to perform dynamic analysis. The method makes it possible to monitor the occurrence of signals and to perform relative quantification of biomarkers. It can be achieved using a device, for which the antibody detection threshold may be standardized or not standardized. For example, an arrangement may be provided for real-time monitoring of signal intensity (the intensity of the line in each zone on the bar) changes, with the first shot being triggered when the reference biomarker reaches the intensity corresponding to a saturation level (for example, after migrating for 5 minutes), and the second shot being triggered when the biomarker with the weakest signal reaches the intensity corresponding to a minimum detection threshold (for example, 10 minutes) or when the migration process is considered to have ended (for example, 15 minutes).

[0177] The pictures can be taken with a smartphone or equivalent and processed using the accumulated data in combination with image analysis software (the analysis can involve trained software based on an AI-type learning process, including data provided by the user - age, weight, height, tobacco and alcohol consumption, susceptibility to sugar (diabetes), UV chamber duration, etc.). The barcodes of the products used in the makeup routine can then be scanned and correlated with information related to skin biomarkers and microbiome biomarkers in order to create recommendations for products (nutraceuticals, cosmetics, etc.) that are most suitable for the conditions of the skin and microbiome.

[0178] These three tools form an inseparable whole: biomarker analysis of the device via immunochromatographic revelation is only possible because the sampling device allows the collection of a sufficient volume of biological material (skin cells and microbiota).

[0179] The use of this kit to perform analyses due to the large number of biomarkers analyzed both on skin cells and on the skin microbiome, and due to the complexity of the combinatorial approach corresponding to the large number of arrangements required to classify biological material, prevents drawing conclusions simply by reading the results (as is the case with Covid tests or pregnancy tests), only due to the simplicity of acquiring images and transmitting them in digital form for processing on a computer that aggregates the processing of the kit for a large number of users.

[0180] The passage presenting the detection zone defines a plane enclosed by a transparent window. The user uses this window to take a picture after the reaction time has elapsed, for example using a smartphone running a dedicated application that specifically commands:

[0181] a) Image acquisition,

[0182] b) Local verification on the smartphone's processor of the acquired image's consistency with respect to, for example, a test pattern provided on the imaging device

[0183] c) Input of additional information

[0184] d) Timestamping and optional geolocation by smartphone

[0185] e) Transmission of images and associated information to a remote server

[0186] Image Development

[0187] The images received on the server are then automatically processed to identify the detection zones that have reacted to encode the combination of biomarkers present in the skin-microbiota complex analyzed, and the skin is classified by processing this combination, in particular by supervised learning from a reference library of data collected from a group of people characterized by experts.

[0188] A fourth object of the present invention relates to a personalized cosmetics recommendation method, comprising the following steps:

[0189] - Use a sampling device to take a sample of biological material from the surface of the skin

[0190] -Suspending the skin cells and microorganisms that constitute the extracted biological material in a solubilizing solution

[0191] - pouring the solubilization solution into the transfer well of the immunorevelation device as defined previously

[0192] - Incubate for 1 minute to 10 minutes to allow the cells and microorganisms to react with the reagents on each of the N regions of the immunorevelation device

[0193] - computer processing of an image consisting of a set of signals revealed on each of the N zones, making it possible to classify the skin by processing this combination of signals, in particular by supervised learning from a reference library - recommend one or more cosmetic products suitable for the skin of said individual based on the combination of biomarkers revealed.

[0194] The recommendations are based on a decision tree that takes into account information related to irritated and / or infected skin conditions, which is converted into the following criteria, for example:

[0195] 1- With the help of the following "skin" biomarkers, about the level of skin irritation or inflammation (such as atopic dermatitis, eczema, Information on the presence of rash, rosacea, etc. :

[0196] - Biomarkers: keratin (sample quality control), filaggrin, IgE, CRP, IL-6, etc.;

[0197] -CRP: can vary based on the menstrual cycle and markers of infection;

[0198] - If keratin markers are low = recent and overly aggressive scrubbing or flaking, peeling;

[0199] 2- Information on the composition of the skin microbiota (level of skin colonization / contamination) .

[0200] If colonization is of the “commensal” type, the microbiota is considered “normal” and consists of the expected microorganisms.

[0201] Contamination or infection will be detected if biomarkers for the target microorganism are elevated, for example:

[0202] - If P. acnes is elevated, acne is present

[0203] - If Staphylococcus aureus markers are elevated, infected atopic dermatitis (eczema) is present

[0204] - If Staphylococcus epidermidis or Demodex markers are elevated, rosacea is present

[0205] - Risk of dandruff on the scalp if Malassezia markers are elevated.

[0206] In the case of the skin microbiome, this analysis provides information on the balance between different microbial populations and can detect the presence of dysbiosis.

[0207] Figure 4 The steps involved in the proposed method are shown.

[0208] Non-limiting examples of embodiments of the present invention

[0209] The present invention will be better understood upon reading the following description, which relates to non-limiting exemplary embodiments, in which:

[0210] A person notices breakouts on her face, she considers her skin rather oily, she follows a skincare routine that works for her (advice from her mother, friends, beautician, etc.), she has heard about the gut microbiome and its impact on health (stimulation of the immune system, protection against pathogenic germs that might irritate or even attack the digestive tract, etc.), and while testing exists, it’s not practical because you have to order a rather expensive (€250-€500) sampling kit from a website, take a sample at home, send it to a lab for “metagenomic” analysis, and then wait weeks for the results and recommendations. You need a high degree of motivation and patience.

[0211] To use the device according to the invention, all you need to do is go to a cosmetics store or pharmacy (OTC cosmetics and personal care) or order it via a smartphone app. While it's recommended to test in the morning, after removing makeup and rinsing your facial skin the previous day, the test can also be performed on the spot (with the risk of identifying only three or four key microorganisms in the microbiome, which might be sufficient to confirm a suspicion of acne, for example). The device is presented as a kit with a simple skin sampling device: a small, sponge-like, absorbent, slightly abrasive device is applied, soaked in a solution to optimize sampling, and then inserted into a tube containing a so-called "resuspension" (or "solubilization") solution.

[0212] The solution flows into the ICFL device, which consists of a sheet of absorbent nitrocellulose (NC) to create a migration flow, with an inlet zone above which is another sheet of NC in which a "detection conjugate" has been deposited. This detection conjugate is usually a monoclonal antibody directed against the target antigen, i.e. the analyte of interest, i.e. a protein characteristic of the skin condition (structural proteins - keratin, filaggrin, etc., inflammation - CRP, etc., immune system - IL6, etc., etc.) or a protein characteristic of the microbiome (adhesion proteins fimbriae, curli pili, fimbriae, etc., characterized by the presence of microbial communities as biofilms naturally formed on the skin, expressed by 3 main bacteria Staphylococcus epidermidis, Propionibacterium acnes, Staphylococcus aureus and other smaller bacteria, some bacteria are common to all skin types, while others are specific to dry or oily or wet skin). The detection conjugate is so called because a compound (gold microparticles, latex microbeads, etc.) is bound to it, which allows the antigen-antibody interaction to be visualized.

[0213] After this initial interaction at the entrance of the deposition well, flow proceeds toward the detection zone, onto which monoclonal capture antibodies have been deposited, also targeting antigens characteristic of skin conditions and microbiota. Each capture antibody is deposited on the test line, and the result indicates the presence or absence of the analyte.

[0214] In addition, there is always one control line, the results of which serve to verify the correct migration and therefore the test. In order for the reaction to take place in this zone, the antibodies bound to the membrane on the control line are directed against the conjugate.

[0215] Each ICFL device is designed for multiple analyte detection (requiring multiple "detection conjugate and capture antibody" pairs). This allows for the determination of skin condition profiles (healthy, normal, dry, oily, etc.) that correlate with microbiome profiles (healthy, dry, oily skin characteristics, acne prone, dermatitis, etc.).

[0216] The reading and analysis is performed by smartphone image capture and interpreted by AI. The information provided by the biomarkers can also be combined with information provided by the individual.

[0217] Examples of specific implementations of a device for cosmetic recommendations

[0218] Thus, the present invention enables different types of testing:

[0219] i. a one-time test of 15 to 30 minutes (can be done in a store or at home in an "at-home test"), either as a spontaneous process or in response to the use of irritating cosmetic or personal care products, the development of solar erythema after prolonged exposure to sunlight or time in an artificial UV chamber, or after excessive scrubbing or peeling or flaking, or a dermatopathological diagnosis (the most common skin conditions are acne, eczema, psoriasis, scalp disorders (excluding alopecia), fungal diseases, and nail disorders);

[0220] ii. Repeated, serial testing allows us to monitor the overall condition of the skin over time by correlating it with the resident microbiota (skin-microbiota complex), which may allow for better management of exposure to the sun or artificial UV rays, or to space out or attenuate peeling or flaking behaviors;

[0221] iii. Based on the profile obtained, suitable products with associated “microbiota” information are proposed that link the skin condition to a specific treatment (dry skin = moisturizing routine, oily skin = cleansing and exfoliating routine, thin and wrinkled skin = protective and nourishing cream routine, etc.): “probiotic” supplements that stimulate the skin and / or microbiota, or “compatible” products that avoid damaging the microbiota by treating the skin alone (the test activity of said products with the microbiota microorganisms allows the calculation of a compatibility score), or skin care products that can improve unpleasant syndromes (sensation of dryness, oiliness, irritation of the skin, redness, etc.);

[0222] iv. Check compatibility of products used routinely. Modify routine based on the evolution of the skin-microbiota complex with age (as skin thins), climate (hot, humid summers vs. cold, dry winters), pollution, etc.

Claims

1. A device for revealing a skin-microbiota complex by immunochromatography, the device comprising a support on which a transfer well is arranged for receiving a solution containing components of the skin-microbiota complex, the well leading to a plurality of N revealing zones, each revealing zone having (i) an immunodetection reagent and (ii) an immunocapture reagent, and the skin-microbiota complex is composed of skin cells and microorganisms and other constituent elements of the skin microbiota, the device comprising: ○N number of reveal areas, where N is equal to or greater than 5 o At least one immunodetection reagent specific for a skin cell biomarker and at least one immunodetection reagent specific for a biomarker of the skin microbiome o The immunoassay reagents, each comprising an antibody specific for a biomarker of skin cells or a biomarker of the skin microbiome conjugated to a colorimetric identification system o the immunocapture reagents, each comprising an antibody bound to the support in each of the N revealing regions, capable of recognizing the biomarker present in said region, o At least one anti-tampering tool for orienting the reading of the device Characterized in that the device comprises: o Three zones for revealing three biomarkers of the skin microbiome, the biomarkers being bacteria of the genera Staphylococcus, Propionibacterium and Corynebacterium, o A region for revealing a skin cell biomarker selected from (i) a structural protein, (ii) an inflammatory biomarker, or (iii) an allergy biomarker.

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 Propionibacterium are selected from Propionibacterium acnes and Propionibacterium granulosum, and the bacteria of the genus Corynebacterium are selected from Corynebacterium xeroides and Corynebacterium kluyveri. 3 . The device according to claim 1 , wherein at least one of the skin microbiota-specific biomarkers corresponds to an epitope associated with a biofilm-like behavior of the microbiota.

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

5. The device of claim 4, wherein the additional biomarkers of the skin microbiome are selected from the following species: (i) bacteria selected from the group consisting of Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus capitis, Propionibacterium acnes, Propionibacterium granulosum, Propionibacterium avariegatum, Corynebacterium tuberculosis stearic acid, Corynebacterium pseudobacterium, Corynebacterium fastidious, Corynebacterium nonfermentative, Corynebacterium xericidal, Corynebacterium aureum), Corynebacterium kluyveri, Corynebacterium amycolyticum, Streptococcus mitis, Streptococcus oralis), Streptococcus pseudopneumoniae, Streptococcus sanguinis, Veillonella parvula, Micrococcus luteus, Aerocystis spp., Epidermophyton floccosum, Nepenthes dwarfus, Ovum ovatum, Blue miraculous diatom, Brown tide algae, Provasolidella, Parker's pyramidal algae, Chlorella keslerae, Aspergillus tubingensis, Triticum graminis, Tilletia spp., Dermatophytes, Actinomycetes (ii) a yeast selected from Malassezia globosa, Malassezia restricta or Candida parapsilosis (iii) a fungus selected from the group consisting of Aspergillus tubingensis, Aspergillus niger, Tilletia perenne, Epidermophyton floccosum, Nenitziiella dwarfis, Nephrolepis ovata, Diatomaceous blue, Brown tide algae, and Coccidioides provasoli (iv) mites selected from Demodex folliculorum and Demodex lipoderm.

6. The device of claim 4, wherein the biomarker of the microbial population is a protein exposed on the surface of the microorganism, the protein selected from the group consisting of Panton-Valentin leukocidin, porphyrin, alginate, β-1,6-linked N-acetylglucosamine (PNAG), Bap family adhesins, type 4 fimbriae, self-associating autotransporters, compactins / invasins, CAMP factor, sialidase, dermatan sulfate adhesin, glycosidic endoceramidase, GroEL chaperone, SH3 domain-containing lipoprotein, Flp fimbriae / fimbriae protein, DsA1 protein; Clf-Sdr family proteins such as Bbp, FnBP and CNA, SesJ protein, chlortetracycline, Bap family adhesins displaying the LPXTG C-terminal domain, IMP, type 3 and type 4 fimbriae.

7. The device according to one of claims 1 to 6, wherein the biomarkers of skin cells are selected from: (i) structural proteins selected from keratin, filaggrin, loricrin; (ii) inflammatory biomarkers selected from C-reactive protein, interleukin IL-1β, IL-4, IL-6, IL-8, IL-11, IL-12, tumor necrosis factor, interferon-γ, granulocyte-macrophage colony stimulating factor, transforming growth factor-β; 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. The device according to one of claims 1 to 8, characterized in that The device includes a zone for specifically revealing the presence of bacteria of the genera Staphylococcus, Propionibacterium, and Corynebacterium, and IL-1β.

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

11. A disposable kit for characterizing a condition of skin, the disposable kit comprising: A device for sampling the skin-microbiota complex, comprising a tool for sampling said complex and an extraction tube containing a solubilizing solution - Device as defined in one of claims 1 to 10 for immunochromatographically revealing the skin-microbiota complex.

12. The kit according to claim 11, wherein the sampling tool consists of a patch, a spatula, a swab soaked in a buffer solution, a sponge soaked in a buffer solution, a silk mesh soaked in a buffer solution, or gauze.

13. A system for implementing personalized cosmetic recommendations based on characterization of the skin-microbiome complex, the system comprising: A device for sampling the skin-microbiota complex, comprising a tool for sampling said complex and an extraction tube containing a solubilizing solution Device for immunochromatographically revealing the skin-microbiota complex as defined in one of claims 1 to 10, wherein the N revealing zones constitute zones capable of providing a signal upon detection of a biomarker a computer that performs analysis processing of an image consisting of all signals from said N zones revealed by means of said immunorevelation device, in order to determine the properties of said biomarkers that have been identified in said N zones, and to classify the skin condition based on the combination of the identified biomarkers.

14. A method for personalized cosmetic recommendations based on characterization of an individual's skin-microbiome complex, the method comprising the steps of: Taking samples of biological material from the surface of the skin Suspend the skin cells, microorganisms and other components of the biological material in a solubilizing solution Pour the solubilization solution into the transfer well of the immunorevelation device as defined in one of claims 1 to 10 Incubate for 1 to 10 minutes to allow the cells and microorganisms to react with the reagents on each of the N regions of the immunorevelation device Processing by computer an image consisting of a set of signals revealed on each of the N revealing areas, making it possible to classify the skin by processing this combination of signals, in particular by supervised learning from a reference library • Recommending one or more cosmetic products suitable for the individual's skin based on the combination of disclosed biomarkers.

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