Bacterium of the genus brevundimonas and cosmetic uses thereof

Abstract

The present invention relates to a novel bacterium of the genus Brevundimonas. A method for screening candidate substances for the cosmetic effect of same is also provided.

Description

[0001] The present invention relates to the cosmetic use of a bacterial species of the genus Brevundimonas, a strain of which has recently been isolated.

[0002] State of the art

[0003] The skin is an ecosystem inhabited by a plethora of microorganisms as diverse as bacteria, archaea, fungi, and small arthropods. Together, they constitute the skin microbiota. This microbiota is subject to numerous parameters of variation, both intrinsic and extrinsic. Recent studies have identified correlations between changes in the composition of commensal skin populations and the physiological state of this environment, influenced by factors such as aging.

[0004] Metagenomic studies have revealed a large number of bacteria, notably demonstrating the decline, during aging, of Cutibacterium acnes, one of the most abundant species in the skin microbiota. However, these metagenomic studies are limited to characterizing abundant bacteria, which introduces biases into the analysis of microbiota composition. Used alone, metagenomics is therefore insufficient to reflect the complexity and diversity of the skin microbiota.

[0005] Culturomics can help overcome the limitations of metagenomics. This technique relies on multiplying the culture conditions of microorganisms, thus enabling the growth of a greater diversity of bacteria, yeasts, and fungi, some of which are considered unculturable. These species are not necessarily in the minority in this habitat, and their influence is largely unknown. One of the main difficulties of culturomics, however, is determining the isolation and culture conditions favorable to the growth of certain previously unknown bacterial strains. Indeed, depending on the skin area considered, there are differences in temperature, humidity, and pH that can influence the bacterial composition of the microbiota.

[0006] Summary of the invention

[0007] The inventors succeeded in isolating a strain of a new bacterial species, the presence of which is correlated with the appearance of signs of premature or accelerated skin aging, particularly photo-induced aging. The inventors also observed that the presence of this strain was correlated with improved skin tone uniformity and increased sebum production, contributing to the lipid barrier, resulting in more hydrated skin and better protection against external aggressors.

[0008] This strain was deposited at the National Collection of Microorganism Cultures (CNCM, Institut Pasteur, 25-28 rue du Docteur Roux, 75015 Paris, France) on January 13, 2021 under number 1-5641.

[0009] An object of the invention is a bacterium, said bacterium belonging to the species Brevundimonas massiliensis sp. nov. characterized in that its genome i) exhibits an average nucleotide identity (ANI) of at least 95% with respect to the complete genome of the strain deposited with the CNCM on January 13, 2021 under number 1-5641 and / or ii) exhibits at least 98.65% identity with the nucleotide sequence SEQ ID NO: 1 (gene 16S B. massiliensis sp. nov.).

[0010] Preferably, the bacterium is the strain isolated and deposited at the National Collection of Microorganism Cultures (CNCM, Institut Pasteur, 25-28 rue du Docteur Roux, 75015 Paris, France) on January 13, 2021 under number 1-5641.

[0011] The invention also provides a method for screening substances that may have a cosmetic effect, said method comprising bringing healthy human skin into contact with a candidate substance, and monitoring the presence and / or quantification of a bacterium of the species Brevundimonas massiliensis sp. nov. on the skin surface, whereby a substance that stimulates the proliferation of the bacterium on the skin surface is identified as having a cosmetic effect, the species Brevundimonas massiliensis sp. nov. being characterized in that its genome i) exhibits an average nucleotide identity (ANI) of at least 95% with respect to the complete genome of the strain filed with the CNCM on January 13, 2021 under number 1-5641 and / or ii) exhibits at least 98.65% identity with the nucleotide sequence SEQ ID NO: 1 (gene 16S B. massiliensis sp. nov.); said bacterium being preferably the strain deposited at the CNCM on January 13, 2021 under number 1-5641.

[0012] More specifically, the cosmetic effect is an effect of preventing premature or accelerated skin aging, skin aging induced by the environment, including stress, diet or pollution, photo-induced skin aging, preventing the appearance of pigment spots, improving the homogeneity of the complexion and / or improving sebum production.

[0013] Another object of the invention is the use of a bacterium of the species Brevundimonas massiliensis sp. nov., as a marker of premature or accelerated skin aging, it being understood that the presence of said bacterium is correlated with skin protected against premature or accelerated skin aging; the species Brevundimonas massiliensis sp. nov. being characterized in that its genome i) exhibits an average nucleotide identity (ANI) of at least 95% with respect to the complete genome of the strain filed with the CNCM on January 13, 2021 under number 1-5641 and / or ii) exhibits at least 98.65% identity with the nucleotide sequence SEQ ID NO: 1 (16S gene B. massiliensis sp. nov.); said bacterium preferably being the strain filed with the CNCM on January 13, 2021 under number 1-5641.

[0014] Detailed description of the invention

[0015] The inventors succeeded in isolating a strain belonging to a new bacterial species. Through a clinical study on young and elderly volunteers, they demonstrated a significant decrease in the presence of this species in cases of premature or accelerated skin aging. Its presence is also significantly associated with an even skin tone and / or sufficient sebum production to combat external aggressors.

[0016] New bacterial species

[0017] This bacterium, of the genus Brevundimonas, is the species Brevundimonas massiliensis sp. nov., a newly isolated strain of which was deposited at the CNCM on January 13, 2021 under number 1-5641.

[0018] The genome of the bacterium isolated and deposited at the CNCM on January 13, 2021 under number I-5641 has been fully sequenced.

[0019] The membership of a bacterium in this species is defined as its genome i) exhibiting an average nucleotide identity (ANI) of at least 95% with respect to the complete genome of the strain deposited with the CNCM on January 13, 2021 under number 1-5641 and / or ii) exhibiting at least 98.65% identity with the nucleotide sequence SEQ ID NO: 1 (gene 16S B. massiliensis sp. nov.).

[0020] The term "sequence identity" or simply "identity" here refers to the percentage of matching nucleotides in the positions of an alignment of two nucleotide sequences. Sequence identity is determined by comparing the sequences when aligned in a way that maximizes overlap and identity while minimizing sequence discrepancies. Specifically, sequence identity can be determined using a number of global or local alignment algorithms, depending on the length of the two sequences.Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, J Mol Biol, 1970;48(3):443-53) which optimally aligns sequences along their entire length, while sequences of significantly different lengths are preferably aligned using a local alignment algorithm (e.g., the Smith and Waterman algorithm (Smith and Waterman, J Mol Biol, 1981;147(1):195-7) or the Altschul algorithm (Altschul et al., Nucleic Acids Res. 1997;25(17):3389-402; Altschul et al., FEBS J, 2005;272(20):5101-9). Alignment is used for determining the percentage of sequence identity. nucleotides can be produced in various ways which are artful, for example by using publicly available software available on websites such as https: / / blast.ncbi.nlm.nih.gov / Blast.cgi or http: / / www.ebi.ac.uk / Tools / emboss / .A person skilled in the art can determine the appropriate parameters for measuring alignment, including the algorithms needed to achieve maximum alignment over the entire length of the compared sequences.

[0021] 16S rRNA gene sequencing is a commonly used method for identifying bacteria, specifically for discriminating between bacterial species (Woo, PCY et al. Clinical Microbiology and Infection, 2008 Volume 14, Issue 10, 908 - 934).

[0022] The 16S rRNA gene sequence can be amplified, notably using universal primers known to those skilled in the art. Preferably, the bacterium contains a 16S rRNA gene exhibiting at least 98.65%, preferably at least 99%, or at least 99.5%, at least 99.7%, or even at least 99.8% or 99.9% identity with the nucleotide sequence SEQ ID NO: 1.

[0023] ANI is a reference method for pairwise comparison of whole-genome bacterial sequences, working on both complete and partially completed genomes, to determine the level of genetic relatedness between bacterial strains and define bacterial species. In particular, the ANI between two bacterial genomes is calculated by pairwise comparison of all similar sequences between the two genomes and can be determined, for example, using any of the publicly available ANI calculation software, including, but not limited to, OrthoANI (Lee et al, Int J Syst Evol Microbiol. 2016, 66(2):1100-1103) and JSpeciesWS (Richter et al, Bioinformatics, 2016, 32(6):929-931). Typically, the genomes of bacterial strains belonging to the same species exhibit an ANI greater than or equal to 95% while the genomes of bacterial strains belonging to the same genus exhibit an ANI greater than or equal to 80% (Jain et al, Nat Commun.2018; 9: 5114).

[0024] The genomic sequence of the strain Brevundimonas massiliensis sp. nov. deposited at the CNCM on January 13, 2021 under number 1-5641 was obtained by de novo sequencing allowing the generation of 8 scaffolds, presented in the sequence listing as the SEQ ID NO sequences: 2 to 9. The sequence size obtained by alignment of the 8 scaffolds is compatible with the size of the genomes of the genus Brevundimonas and is therefore representative of the entire genome.

[0025] The SEQ ID NO sequences: 2 to 9 thus allow pairwise comparison of bacterial sequences on whole genome, for the calculation of ANI.

[0026] Preferably, the bacterium exhibits an average nucleotide identity (ANI) of at least 95%, preferably at least 95.5%, preferably at least 96%, or at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or even at least 99.8% compared to the complete genome of the strain filed with the CNCM on January 13, 2021 under number 1-5641.

[0027] The membership of a bacterium in this species can also be confirmed by DNA-DNA hybridization (DDH), in particular in silico, of at least 70% between the genomic DNA of the bacterium and the genomic DNA of the strain B. massiliensis sp. nov. deposited at the CNCM under number 1-5641 (Goris et al, Int. J. of Systematic and Evolutionary Microbiology, 2007, 57, 81-91).

[0028] For the purposes of the present invention, "healthy skin" means an area of ​​skin on which the bacteria according to the invention is applied and which is said to be "non-pathological" by a dermatologist, that is to say, not showing any infection, scar, disease or skin condition such as candidiasis, impetigo, psoriasis, eczema, acne or dermatitis, or any wounds or injuries.

[0029] For the purposes of this invention, from a cosmetic perspective, a product is understood to have a preventative effect against premature or accelerated skin aging. This effect is achieved by combating and preventing signs of skin aging induced by environmental factors, such as stress, diet, or pollution, as well as preventing photo-induced skin aging, the appearance of age spots, improving skin tone evenness, and / or improving sebum production. More generally, it refers to preventing the effects of exposure to one or more internal and / or external factors of skin aging. These exposures typically include environmental factors related to lifestyle, such as exposure to sunlight, air pollution, smoking, and diet.We can also talk about the exposome, which encompasses all the exposures to which an individual is subjected from conception to death, that is, all external and internal factors, their interactions, and the response of the human body resulting in biological and clinical signs.

[0030] In a particular embodiment, aging can typically be photo-induced aging, i.e. due to solar radiation including UV radiation, infrared radiation, and visible light.

[0031] Monitoring and screening methods

[0032] The results obtained highlight the potential of the new species of the invention as a marker of premature skin aging. The presence of said bacterium on the surface of healthy skin is correlated with spotless skin and / or a more even complexion and / or a higher sebum level.

[0033] In this respect, one object of the invention relates to a screening method for substances likely to have a cosmetic effect, in particular an effect of preventing premature skin aging, especially photo-induced.

[0034] These can include, for example, small synthetic molecules, alone or in combination, or natural extracts, particularly natural plant extracts.

[0035] This method includes the topical application of candidate substances to an area of ​​skin, and the determination or monitoring of the presence and / or quantity of bacteria of this new species.

[0036] The determination of the presence and / or quantity of bacteria on the skin surface can be advantageously achieved by amplification of bacterial genomic DNA, for example by quantitative PCR (qPCR).

[0037] According to a particular embodiment, the gene for the sulfur transporter protein ThiS is amplified, for example using the PCR primers described in Example 2.

[0038] A candidate substance that stimulates bacterial proliferation on the skin surface has been identified as having potential cosmetic benefits. Advantageously, this cosmetic benefit includes preventing premature or accelerated skin aging, environmentally induced skin aging (including stress, diet, and pollution), photo-induced skin aging, preventing the appearance of pigment spots, improving skin tone evenness, and / or improving sebum production.

[0039] The state of premature aging of human skin can also be assessed by determining the presence and / or quantity of bacteria of this new species of the invention on an area of ​​healthy skin. The presence of said bacteria is correlated with youthful, spotless skin and / or a more even complexion and / or a higher sebum level. In other words, the quantity of bacteria found on the skin's surface decreases as the skin exhibits an increasingly pronounced state of premature aging.

[0040] The examples and figures illustrate the invention, without limiting the scope of the invention.

[0041] EXAMPLES

[0042] Example 1: Isolation of a new bacterial species (clinical study)

[0043] Materials and methods

[0044] 1- Inclusion of volunteers

[0045] Skin samples were collected from 47 healthy women living around Marseille in two different age groups: 18 to 30 years old (young group) and over 55 years old (older group).

[0046] Furthermore, for this study, additional recruitment criteria were added: participants must not have taken antibiotics or antihistamines for at least one month, must not have a skin condition such as atopic dermatitis or psoriasis (as determined by a dermatological examination), must not have showered or washed their forehead for at least 12 hours prior to the study, and must not have applied any cosmetic products to the sampled area since the last washing. For the younger group, women had to be using contraception, excluding high-dose pills known to affect acne. Participants in the older group had to be postmenopausal, not using hormone replacement therapy, and exhibit signs of photoaging (spots, yellowing of the skin, pronounced wrinkles, etc.).

[0047] The results obtained from 47 volunteers for the analysis of forehead samples are divided into 23 volunteers from the younger group and 24 volunteers from the older group. 2- Collection of skin microbiota samples

[0048] The sampled frontal zones were 10 cm 2 Samples were collected using sterile swabs soaked in Culture Top® Transport Media (C-top Ae-Ana, Eurobio, France) and in a Z-stroked manner. Two swabs were used simultaneously for culturomics. Air-shaken swabs were performed as a negative control. Samples were immediately cultured for culturomics. After collection, these samples were immediately stored at -80 °C prior to DNA extraction.

[0049] 3- Identification of microorganisms by MALDI-TOF mass spectrometry

[0050] After culturing, the isolated microorganisms were identified using the MALDI-TOF technique. The bacteria were directly loaded onto the MSP96 target (Bruker Daltonics, GmbH, Bremen, Germany) with a positive control (Escherichia coli) and a negative control (the matrix); this method is called "direct loading." Data acquisition was performed on the MALDI Biotyper (Microflex LT system; Bruker Daltonics GmbH, Bremen, Germany) using Flex Control™ software and the MALDI BioTyper RTC identification software (Bruker Daltonics GmbH, Bremen, Germany). When identification was not possible using this technique, three successive subcultures of the bacteria were performed to purify them as much as possible. For each new identification test, a formic acid extraction was carried out, and 1 µL of the supernatant was loaded onto the plate.This method makes it possible to generate a protein spectrum without impurities and maximizes the chances of matching the generated spectrum with the spectra contained in the database.

[0051] 4- Identification of microorganisms by genomic sequencing

[0052] In the event of further failures to identify by the MALDI-TOF technique, sequencing via Next Generation Sequencing methods is carried out with IlluminaMiseq (Illumina Inc., San Diego, CA, USA) and the Oxford Nanopore method (Oxford Nanopore Technologies, Inc, Oxford, UK).

[0053] Genome sequencing of Brevundimonas massiliensis sp. nov.

[0054] The genomic DNA (gDNA) of Brevundimonas massiliensis sp. nov. (the strain of the invention) was extracted in two steps: first, mechanical processing was performed with acid-washed glass beads (G4649 Sigma, St. Louis, MO, USA) using the FastPrep-24™ 5G Grinder (mpBio, Santa Ana, CA, USA) at maximum rotation speed (6.5 m / s) for 90 seconds. Then, after 30 minutes of lysozyme incubation at 37°C, the DNA was extracted on the EZ1 biorobot (QIAGEN, Valencia, CA, USA) with the EZ1 DNA Tissues kit. The gDNA of Brevundimonas massiliensis sp. nov. was quantified by a high-sensitivity Qubit assay (Life Technologies, Carlsbad, CA, USA) at 0.2 ng / pl. The genomic DNA was then sequenced using MiSeq technology (Illumina Inc, San Diego, CA, USA). To prepare the paired end library, a dilution was performed to have 1 ng of the genome as the input template.The "tagmentation" step fragmented and labeled the DNA by anchoring it to the cohesive end segments of a universal nucleotide sequence. Subsequently, cycle-limited PCR (12 cycles) introduced double-index barcodes. After purification on AM Pure XP beads (Beckman Coulter Inc., Fullerton, CA, USA), the libraries were normalized on specific beads according to the Nextera XT protocol (Illumina Inc., San Diego, CA, USA). The normalized libraries were then combined into a single library for sequencing on the MiSeq instrument. Automated cluster generation and sequencing of the matched end segments with double-index reads were performed in a single 39-hour run at 2x250 bp. Total information of 4.5 Gb was obtained from an aggregate density of 462 K / mm2 with a cluster passing quality control filters of 93.9%.The final 9,045,583 matched reads were filtered based on read quality. To improve the sequence generated by the Illumina technique, the Oxford Nanopore approach was implemented for genomic DNA sequencing using the 1D method with Minlon technology and the SQK-LSK109 kit. The library was constructed from 1 pg of genomic DNA without fragmentation or end repair. Adapters were ligated to both ends of the genomic DNA. After purification on AMPure XP beads (Beckman Coulter Inc., Fullerton, CA, USA), the library was quantified using a high-sensitivity Qubit assay (Life Technologies, Carlsbad, CA, USA). 1,353 active pores were detected for sequencing, and the WIMP workflow was selected for live bioinformatics analysis. 655,000 reads were generated as raw data.

[0055] Genome analysis

[0056] The annotation was obtained using Prokka Galaxy software version 1.14.5+galaxyO with default settings via the Online Galaxy platform. Genome sequence data were uploaded to the Type (Strain) Genome Server (TYGS), a free online bioinformatics platform (https: / / tygs.dsmz.de) for performing comprehensive genome-based taxonomic analysis. The determination of the closest type strain genomes was carried out in two complementary ways. First, the genome of Brevundimonas massiliensis sp. nov. (the invention strain) was compared to all available type strain genomes in the TYGS database using the MASH algorithm, which allows for rapid approximation of intermenomic relatedness and the selection of the ten type strains with the smallest distances.Secondly, an additional set of ten closely related type strains was determined via 16S rDNA genetic sequences. These were extracted from the submitted genome using RNAmmer, and each sequence was then BLASTed against the 16S rDNA gene sequence of each of the 12,983 type strains currently available in the TYGS database. Overlaying the results of these two methods allowed for the ranking of the 50 best matching type strains (based on bit score) and the subsequent calculation of precise distances using the Genome BLAST Distance Phylogeny (GBDP) approach with the "coverage" algorithm and the d5 distance formula. These distances were ultimately used to determine the 10 closest type strain genomes for each of the user genomes.All pairwise comparisons across the genome set were performed using GBDP and precise intergenomic distances derived with the trimming algorithm and the d5 distance formula. The calculations were performed with 100 iterations. DDH values ​​and confidence intervals were calculated using the parameters recommended by GGDC v2.1 software. Additionally, the degree of genomic similarity of the strain of the invention to closely related species was estimated using OrthoANI software with default parameters. Antibiotic resistance genes and the presence of pathogenesis-related proteins were investigated using ABRicate tools integrating the CARD and VFDB databases via the Online Galaxy platform.The analysis of secondary metabolite synthesis was performed using AntiSmash software and then confirmed by the analysis of protein sequences from the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) database using BLASTp with a threshold value of e of 1 E'. 3 .

[0057] RESULTS

[0058] 1- Isolation and identification of a new species of the skin microbiota

[0059] 128 different species were isolated from samples taken from the forehead, distributed across 55 different genera. The major genera identified from the forehead were Staphylococcus (isolated from 100% of volunteers), Cutibacterium (76.60%) and Streptococcus (48.94%), the main representatives of which were Staphylococcus epidermidis, Cutibacterium acnes, and Streptococcus mitis / oralis.

[0060] Among them, a new species was isolated from the skin of a 60-year-old volunteer. Sequencing its genome revealed that it was a new species of the genus Brevundimonas, never before described, and named Brevundimonas massiliensis sp. nov.

[0061] The cultivable strain of Brevundimonas massiliensis sp. nov. was deposited at the CNCM (national collection of microorganism culture of the Pasteur Institute) under the number CNCM-l-5641. Example 2: Study of correlations between the presence of Brevundimonas massiliensis sp. Nov. and clinical parameters

[0062] Objective

[0063] The primary objective of the study was to analyze the presence of Brevundimonas massiliensis sp. nov. bacteria on the facial skin of adult women. The secondary objective was to study the variations of this bacterial species according to age and clinical skin characteristics.

[0064] Materials and methods

[0065] Clinical study

[0066] Skin microbiota samples were collected from the foreheads of 108 volunteers, divided into 52 from the younger group (18-30 years) and 56 from the older group (55-70 years). These samples were then analyzed using quantitative PCR for the bacteria species Brevundimonas massiliensis sp. nov.

[0067] Clinical assessments of skin condition and measurements of clinical parameters such as sebum level, hydration level, transepidermal water loss and measurements of skin biomechanical properties were carried out on the face of all volunteer subjects.

[0068] Correlation analyses were performed between clinical data, the age of volunteer subjects and the quantity of bacteria of the species Brevundimonas massiliensis sp. nov. found on the surface of facial skin.

[0069] Evaluation parameters

[0070] Skin microbiota sampling

[0071] The skin microbiota was collected from a 25 cm² area 2 at the level of the forehead. Samples were taken using a swab (Transwab®) for each area, by gently rubbing the swab on the skin of the forehead.

[0072] Taking photos

[0073] High-resolution digital facial photographs were acquired using a HeadScan repositioning table and CameraScan software (Orion Concept) at the COSNAT Provence laboratory. Standardized acquisitions of volunteer subjects were performed by a clinical technician, in frontal and profile views (45° left and 45° right), and with cross-polarized and parallel-polarized filters, during the evaluation visit. These acquisitions then served as visual support for clinical scoring performed at the end of the study. Clinical measurements

[0074] Some clinical measurements are non-invasive instrumental measurements requiring skin contact. They were performed on a separate area of ​​the forehead from the area sampled for the microbiota, or on the temple for mechanical properties:

[0075] Hydration by Corneometry (Corneometer® CM825 - COURAGE & KHAZAKA) Sebum level by Sebumetry (Sebumeter® SM 815 - COURAGE & KHAZAKA) Insensible water loss by Tewametry (Tewametre® TM300)

[0076] Biomechanical properties of the skin by Cutometry (Cutometer® MPA 580 - COURAGE & KHAZAKA)

[0077] Clinical scoring

[0078] Clinical scoring was performed by dermatologist investigators based on standardized photographs taken during the baseline visit. The scoring scales used were as follows:

[0079] Crow's feet wrinkles according to the BAZIN Atlas, Atlas of Skin Aging, Volume 1 p. 40, MED'COM editions, 2007,

[0080] Fine lines under the eyes according to the BAZIN Atlas, Atlas of Skin Aging, Volume 1 p. 40, MED'COM editions, 2007,

[0081] Pigmentation spots according to the BAZIN Atlas, Skin Aging Atlas, Volume 2 p.93, MED'COM publishing 2010 for the face,

[0082] Evenness of skin tone (internal scale) according to the following 5-point scale: 1- very uneven skin tone, 2- slightly uneven skin tone, 3- moderately even skin tone, 4- even skin tone, 5- very even skin tone

[0083] Complexion radiance (internal scale) according to the following 5-point scale: 1- dull, lifeless complexion, 2- slightly radiant and luminous complexion, 3- moderately radiant and luminous complexion, 4- radiant and luminous complexion, 5- very radiant and luminous complexion

[0084] Analysis of Brevundimonas massiliensis sp. nov. by quantitative PCR

[0085] All skin microbiota samples were frozen and stored at -80 °C immediately after collection. After thawing, 20 µl of Proteinase K at 20 mg / ml were added to each sample and heated for 1 h at 50 °C in a water bath. The solution was then concentrated using a SpeedVac before DNA extraction with the QIAamp® PowerFecal® Pro DNA kit (Qiagen). Quantitative PCR was then performed using the Brevundimonas massiliensis sp. nov. genome-specific PCR systems (Table 1) and the TaqMan® Fast Advanced Master Mix (Applied Biosystems). The PCR reaction was performed using the QuantStudio™ 7 Pro RT-PCR instrument (Applied Biosystems) with 50 amplification cycles.

[0086] The qPCR system was designed using PrimerBlast+ software and verified using the BlastN tool against the nr database. Table 1 below describes the PCR system, comprising a pair of specific primers and a probe, designed to specifically amplify the genomic DNA of Brevundimonas massiliensis sp. nov. by targeting the gene for the sulfur transporter protein ThiS involved in thiamine synthesis.

[0087] Table 1: qPCR system designed to specifically amplify the DNA of Brevundimonas massiliensis sp. nov.

[0088] Target bacterium Target gene Sense primer Antisense primer Probe

[0089] Brevundimonas ThiS (sulfur carrier GCATTCAAGTC CGAACTGAACCAGC TGAAGGCGCTG massiliensis sp. protein ThiS) AACGGCGAT TCGATG TCGCTCGAC nov. strain of (SEQ ID NO: 10) (SEQ ID NO: 11) (SEQ ID NO: 12) the invention

[0090] Statistical analysis

[0091] Clinical data were formatted and error-free using R software version 4.2.1. All statistical analyses were performed in R. Analysis of clinical variation between the younger and older subject groups was conducted for all continuous variables using the Wilcoxon signed-rank test, and p-values ​​were adjusted using the Benjamini-Hochberg method. Correlations between continuous variables were also investigated using Pearson's correlation coefficient, and the results were adjusted using the same method. Potential outliers were identified using the Z-score test.

[0092] A multilinear regression model was then constructed, based on continuous variables (clinical data and quantitative PCR values) using the 'lm' function of the R software and the parameters 'age ~ all continuous variables' in order to observe which variables most explain the differences observed between the different age groups and to take into account any confounding element.

[0093] We then compared the qPCR results with clinical variables (paired test) using the Wilcoxon signed-rank test, adjusting the p-values ​​as before. For continuous variables, we investigated correlations between qPCR and the variable using Spearman's rank correlation coefficient on the overall population.

[0094] Results

[0095] Characterization of the studied population

[0096] Analysis of the clinical characteristics of the study population reveals that the two groups of subjects (young and old) are significantly different for all variables studied, with the exception of tewametry and corneometry values ​​(Table 2). Thus, subjects in the two groups do not show any differences in terms of skin barrier function (tewametry) or skin hydration (corneometry), two clinical characteristics indicative of healthy skin. In contrast, the older subjects exhibit significantly lower sebum production, more pigment spots, a duller and more uneven complexion, and more subocular and crow's feet wrinkles (Table 2).Cutometer measurements also reveal that the skin of subjects in the older group is on average significantly less firm (R6 values ​​on the rise, indicating greater skin viscosity, associated with collagen loss) and less elastic (R1 value on the rise (less good elastic return) and R2, R5 and R7 values ​​(elasticity parameters) on the decline) (Table 2).

[0097] This data is evidence of skin aging.

[0098] Table 2: Statistical analysis of clinical variations between the young and older subject groups. Variations are expressed as a percentage for instrumental measures or as a point for clinical scores (scores ranging from 1 to 5) (Wilcoxon signed-rank test with adjustment of p-values ​​using the Benjamini-Hochberg method). The significance level is set at 0.05 of the p-value. ****p<0.0001.

[0099] Presence, evolution and statistical correlations between the quantity of Brevundimonas massiliensis sp. nov. and clinical parameters

[0100] Samples not reaching the detection threshold (Ct) before 50 qPCR cycles were considered negative (undetectable bacteria).

[0101] The bacterium is considered commensal since it was detected on the skin of 96% of the volunteers in the young group (53 out of 55) and 89% of the volunteers in the older group (49 out of 55).

[0102] The amount of Brevundimonas massiliensis sp. nov. detected by qPCR was correlated with clinical parameters. These results are summarized in Table 3. They demonstrate that Brevundimonas massiliensis sp. nov. decreases significantly with increasing pigment spot score. A higher amount of Brevundimonas massiliensis sp. nov. is associated with greater skin tone homogeneity and higher sebum production. The amount of Brevundimonas massiliensis sp. nov. is not significantly correlated with variations in hydration parameters (corneometry), skin barrier function (tewametry), or skin biomechanical properties.

[0103] Although Brevundimonas massiliensis sp. nov. shows a decreasing trend with age in volunteers, this correlation is not significant (p=0.061). This observation could be explained by heterogeneity within the older group, with some volunteers exhibiting fewer pigment spots than others, a parameter correlated with the quantity of the bacterium.

[0104] Interestingly, when the statistical analysis is performed within the older group comprising 56 volunteers between 55 and 70 years old and presenting a greater or lesser number of pigment spots, the association between the quantity of the bacterium Brevundimonas massiliensis sp.nov. and a lower pigment spot score is strengthened with a correlation coefficient of -0.41 and a p-value of 0.0018. This result indicates that the presence of pigment spots is the parameter that is most strongly associated with the decrease of the bacterium on the skin surface, regardless of the age of the individuals.

[0105] These results confirm that Brevundimonas massiliensis sp. nov. is associated with skin exhibiting higher sebum production, a more even complexion, and fewer pigment spots. Table 3: Evolution of the quantity of Brevundimonas massiliensis sp. nov. according to clinical parameters. Spearman correlation coefficients are indicated along with their significance for each parameter. ns=not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Claims

Demands 1. Bacteria belonging to the species Brevundimonas massiliensis sp. nov. characterized in that its genome i) exhibits an average nucleotide identity (ANI) of at least 95% with respect to the complete genome of the strain deposited with the CNCM on January 13, 2021 under number 1-5641 and ii) exhibits at least 98.65% identity with the nucleotide sequence SEQ ID NO: 1 (gene 16S B. massiliensis sp. nov.).

2. Bacterium according to claim 1, characterized in that it is the strain deposited with the CNCM on January 13, 2021 under number 1-5641.

3. Method for screening substances likely to have a cosmetic effect, said method comprising bringing healthy human skin into contact with a candidate substance, and monitoring the presence and / or quantification of a bacterium of the species Brevundimonas massiliensis sp. nov. on the skin surface, by which a substance which stimulates the proliferation of the bacterium on the skin surface is identified as likely to have a cosmetic effect, the species Brevundimonas massiliensis sp. nov. being characterized in that its genome i) exhibits an average nucleotide identity (ANI) of at least 95% with respect to the complete genome of the strain deposited with the CNCM on January 13, 2021 under number 1-5641 and ii) exhibits at least 98.65% identity with the nucleotide sequence SEQ ID NO: 1 (gene 16S B. massiliensis sp. nov.); said bacterium being preferably the strain deposited at the CNCM on January 13, 2021 under number I-5641.

4. Screening method according to claim 3, wherein the cosmetic effect is an effect of preventing premature or accelerated skin aging, skin aging induced by the environment, in particular stress, diet or pollution, photo-induced skin aging, preventing the appearance of pigment spots, improving the homogeneity of the complexion and / or improving sebum production.

5. Method according to any one of claims 3 or 4, comprising quantification of said bacterium by quantitative PCR.

6. Use of a bacterium of the species Brevundimonas massiliensis sp. nov., as a marker of premature or accelerated skin aging, it being understood that the presence of said bacterium is correlated with skin protected against premature or accelerated skin aging; the species Brevundimonas massiliensis sp. nov. being characterized in that its genome i) exhibits a mean nucleotide identity (ANI) of at least 95% compared to the complete genome of the strain deposited with the CNCM on January 13, 2021 under number 1-5641 and ii) exhibits at least 98.65% identity with the nucleotide sequence SEQ ID NO: 1 (gene 16S B. massiliensis sp. nov. said bacterium being preferably the strain deposited with the CNCM on January 13, 2021 under number 1-5641.

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