Methods for diagnosing long-term exposure to pollution

Abstract

The present invention relates to a method for diagnosing, in particular in vitro, chronic exposure of a subject, in particular the skin of a subject, to pollution, comprising the step (a) of determining the level of at least one marker in a skin sample of the subject, the at least one marker being selected from the group consisting of the following bacterial species: Corynebacterium lipophiloflavum, Corynebacterium durum, Corynebacterium propinquum, Kytococcus sedentarius, Brevibacterium sp., Haemophilus parainfluenzae, Actinomyces oris, Neisseria sp., Abiotrophia defectiva, Alicycliphilus sp., Micrococcus luteus, Caulobacter sp., Streptococcus mitis / oral / pneumoniae and Staphylococcus epidermidis species.

Description

Technical Field

[0001] The present invention relates to diagnosing chronic exposure of a subject, particularly the subject's skin, to pollution. Background Art

[0002] Pollution peaks are often seen in certain urban areas. In daily life, especially in urban areas, individuals may be exposed to multiple attacks on their keratin materials, especially various types of air pollution on the skin.

[0003] Among the known pollutants we mention first of all exhaust gases, heavy metals, which have become a major problem in large cities, but also particulates containing polycyclic aromatic hydrocarbons such as benzo[a]pyrene and benzanthracene.

[0004] These pollutants will significantly cause, at the skin level, the deposition of particles on the surface of the epidermis and, among other consequences, can induce premature skin aging, marked by the presence of wrinkles and / or fine lines, extended spots, freckles simplex, redness and / or a dull and / or uneven appearance of the skin tone, as well as imperfections of the skin and its skin tone level.

[0005] Therefore, it is important to be able to demonstrate long-term exposure to pollution.

[0006] Therefore, methods for diagnosing aesthetic degenerations of the skin, especially those related to pollution, are highly desirable. Summary of the invention

[0007] The present invention satisfies this need.

[0008] The present invention arises from the unexpected discovery by the inventors that the relative abundance of specific bacterial species in individual skin samples is associated with exposure to specific pollutants, particularly certain PAHs (polycyclic aromatic hydrocarbons), as demonstrated by the detection of these pollutants in hair samples from the study subjects.

[0009] The object of the present invention is therefore a method for diagnosing (in particular in vitro) a subject, in particular a subject's skin, long-term exposure to pollution, comprising the step (a) determining the level of at least one marker in a skin sample of said subject, in particular a sample of the skin surface of said subject, said marker being selected from the group consisting of the following bacterial species: Corynebacterium lipophiloflavum, Corynebacterium durum, Corynebacterium propinquum, Kytococcus sedentarius, Brevibacterium sp., Haemophilus parainfluenzae, Actinomyces oris, Neisseria sp., Abiotrophia defectiva, Alicycliphilus sp., Micrococcus luteus, Caulobacter sp., Streptococcus mitis / oralis / pneumoniae and Staphylococcus epidermidis. DETAILED DESCRIPTION

[0010] Markers

[0011] The markers used in the context of the present invention are selected from the group consisting of the following bacterial species: Corynebacterium lipophiloflavum, Corynebacterium durum, Corynebacterium propinquum, Kytococcus sedentarius, Brevibacterium sp., Haemophilus parainfluenzae, Actinomyces oris, Neisseria sp., Abiotrophia defectiva, Alicycliphilus sp., Micrococcus luteus, Caulobacter sp., Streptococcus mitis / oral / pneumoniae and Staphylococcus epidermidis.

[0012] "Bacterial species Corynebacterium lipophiloflavum" refers herein to a Gram-positive bacterium of the genus Corynebacterium, the type strain of which is strain DSM 44291 described in Funke et al. (1997) FEMS Microbiol. Lett. 150: 219-224.

[0013] "Bacterial species Corynebacterium durum" refers herein to a Gram-positive bacterium of the genus Corynebacterium, the type strain of which is the DSM 45333 strain described in Riegel et al. (1997) Int. J. Syst. Bacteriol. 47: 1107-1111.

[0014] "Bacterial species Corynebacterium propinquum" refers herein to a Gram-positive bacterium of the genus Corynebacterium, the type strain of which is strain DSM 44285 described in Riegel et al. (1993) FEMS Microbiol. Lett. 113: 229-234.

[0015] "Bacterial species Kytococcus sedentarius" refers herein to a Gram-positive bacterium of the genus Kytococcus, formerly known as Micrococcus sedentarius, the type strain of which is strain DSM 20547 described in Stackebrandt et al. (1995) Int. J. Syst. Bacteriol. 45: 682-692.

[0016] "Bacterial species Brevibacterium sp." herein refers to a Gram-positive bacterium of the order Actinomycetales, the type species of which is Brevibacterium linens.

[0017] "The bacterial species Haemophilus parainfluenzae" refers herein to a commensal bacterium of the genus Haemophilus, the type strain of which is strain DSM 8978 described in Rivers (1922) Johns Hopkins Hospital Bulletin 33: 429-431.

[0018] "Bacterial species Actinomyces oris" herein refers to a Gram-positive bacterium of the genus Actinomyces, the type strain of which is the ATCC27044 strain described in Henssge et al. (2009) Int. J. Syst. Evol. Microbiol. 59: 509-516.

[0019] "Bacterial species Neisseria sp." herein refers to Gram-negative bacteria of the order Neisseriales, the type species of which is Neisseria gonorrhoae.

[0020] "Bacterial species Abiotrophia defectiva" herein refers to Gram-positive bacteria of the genus Abiotrophia, the type strain of which is the DSM9849 strain described in Kawamura et al. (1995) Int. J. Syst. Bacteriol. 45: 798-803.

[0021] "Bacterial species Alicycliphilus sp." herein refers to bacteria of the order Burkholderiales, the type species of which is Alicycliphilus denitrificans.

[0022] "The bacterial species Micrococcus luteus" refers herein to a Gram-positive, spherical, saprophytic bacterium of the family Micrococcaceae, the type strain of which is the DSM 20030 strain described in Wieser et al. (2002) Int. J. Syst. Evol. Microbiol. 52: 629-637.

[0023] "Bacterial species Caulobacter sp." herein refers to bacteria of the Order Caulobacterales, the type species of which is Caulobacter vibrioides.

[0024] "Bacterial species mitis / oral / pneumococcus" herein refers to bacteria of the genus Streptococcus, namely, the species Mitis / oral / pneumococcus (the type strain of which is the NCTC3165 strain described in Andrewes et al. (1906) Lancet 2:708-713), the species Oral Streptococcus (the type strain of which is the DSM20627 strain described in Bridge et al. (1982) Int. J. Syst. Bacteriol. 32:410-415) and the species Pneumococcus (the type strain of which is the DSM20566 strain described in Klein (1884) Practitioner 32:321-352).

[0025] The "bacterial species Staphylococcus epidermidis" herein refers to a Gram-positive, facultative anaerobic bacterium of the genus Staphylococcus, the type strain of which is the DSM 20044 strain described in Evans (1916) J. Infect. Dis. 18: 437-476.

[0026] In a specific embodiment, the at least one marker is selected from the group consisting of the following bacterial species: Corynebacterium lipophiloflavum, Corynebacterium durum, Kytococcus sedentarius, Brevibacterium sp., Haemophilus parainfluenzae, Actinomyces oris, Neisseria sp., Abiotrophiadefectiva, Alicycliphilus sp., Micrococcus luteus, Caulobacter sp. and Streptococcus mitis / oral / pneumoniae.

[0027] In an alternative embodiment, the at least one marker is selected from the group consisting of Corynebacterium propinquum and Staphylococcus epidermidis.

[0028] Diagnostic Methods

[0029] The diagnostic method according to the present invention is a method of diagnosing chronic exposure of a subject, particularly the skin of a subject, to pollution.

[0030] "Pollution" as used herein refers to exposure to particles of matter, particularly polycyclic aromatic hydrocarbons (PAHs).

[0031] Thus, in certain embodiments, the contamination is PAH contamination.

[0032] As is well known to those skilled in the art, PAHs are a subfamily of aromatic hydrocarbons whose structure contains at least two fused aromatic rings. They are produced by the incomplete combustion of organic matter (coal, oil, natural gas, waste and biomass) and are released as environmental pollutants.

[0033] PAHs generally include acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chryzene, benzo-b-fluoranthene, benzo-k-fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, dibenzo-ah-anthracene, and benzo-ghi-perylene.

[0034] In specific embodiments, the PAHs are selected from acenaphthylene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, and benzo[a]pyrene.

[0035] "Long-term exposure" here means regular exposure, in particular daily exposure over a given period of time, preferably a period of more than one year, in particular a period of twelve months (usually corresponding to 12 cm of hair growth), possibly interrupted one or more times for short durations, generally for a duration of less than one month, preferably less than one week.

[0036] The diagnostic method according to the invention comprises the step (a) of determining the level of at least one marker selected from the group consisting of bacteria defined in the "Markers" section above in a skin sample of a subject, in particular a skin surface sample of a subject.

[0037] In specific embodiments, the level of at least one marker in the sample is the relative abundance of the at least one marker in the sample.

[0038] "Relative abundance" herein refers to the relative amount as a percentage of a given taxon relative to the total number of taxa in a sample.

[0039] The level of at least one marker may be determined by any suitable technique.

[0040] In a specific embodiment, the level of at least one marker is determined by sequencing, in particular when the level of at least one marker is usually the relative abundance of at least one marker in the sample, the level of at least one marker is determined by metagenomic sequencing, in particular shotgun sequencing.

[0041] Typically, bacterial genomic DNA present in skin samples is extracted and then sequenced and quantified by shotgun metagenomics.

[0042] In a specific embodiment, the diagnostic method according to the invention further comprises the steps consisting of:

[0043] (b) comparing the level of the at least one marker measured in step (a) with a control, and

[0044] (c) determining, based on the comparison in step (b), whether the subject, in particular the subject's skin, has experienced long-term exposure to pollution.

[0045] In specific embodiments, the control is a reference value.

[0046] In a specific embodiment, the reference value is determined by the mean value of the level of the marker in a defined population, such as a population in a defined age group and / or a population with a specific skin type.

[0047] In a specific embodiment, the reference value is the mean value of the level of said marker in a population of subjects, in particular subjects as defined below, living in a less polluted city, in particular a city where the number of days per year with an air quality index below 100 is less than 100, in particular less than 85.

[0048] In a specific embodiment, the at least one marker is selected from the group consisting of the following bacterial species: Corynebacterium lipophiloflavum, Corynebacterium durum, Kytococcus sedentarius, Brevibacterium sp., Haemophilus parainfluenzae, Actinomyces oris, Neisseria sp., Abiotrophiadefectiva, Alicycliphilus sp., Micrococcus luteus, Caulobacter sp. and Streptococcus mitis / oral / pneumoniae, and if the level of the at least one marker measured in step (a), in particular the relative abundance of the at least one marker, is higher than the control, in particular significantly higher than the control, the subject is diagnosed as having experienced long-term exposure to pollution. The level of the control is typically the level of the marker (in particular the relative abundance of the marker) in a skin sample of a subject living in a less polluted city, in particular a city with an air quality index below 100 days per year of less than 100 (in particular less than 85), and the level of the marker is typically determined by sequencing, in particular by shotgun metagenomic sequencing as generally described above.

[0049] In another specific embodiment, the at least one marker is selected from the group consisting of the following bacterial species: Corynebacterium propinquum and Staphylococcus epidermidis, and if the level of the at least one marker measured in step (a) (particularly the relative abundance of the at least one marker) is lower (particularly significantly lower) than the control, the subject is diagnosed as having experienced long-term exposure to pollution. The control level is typically the marker level (particularly the relative abundance of the marker) in a skin sample of a subject living in a less polluted city, especially a city with less than 100 days (particularly less than 85) of the air quality index below 100 in a year, and the marker level is typically determined by sequencing, especially by shotgun metagenomic sequencing as generally described above.

[0050] According to the present invention, "significantly lower than" means that the level of the marker is statistically significantly reduced compared to the level of the control.

[0051] According to the present invention, "significantly higher than" means that the level of the marker is statistically significantly increased compared to the level of the control.

[0052] In a specific embodiment, the contamination is benzo[a]pyrene contamination and the at least one marker is selected from the group consisting of the following bacterial species: Corynebacterium lipophiloflavum and Kytococcus sedentarius.

[0053] In this specific embodiment, if the level of at least one marker measured in step (a) (in particular the relative abundance of at least one marker) is higher than the control (in particular significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to benzo[a]pyrene pollution, the control level is usually the level of the above marker (in particular the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, in particular a city where the number of days per year with an air quality index below 100 is less than 100 (in particular less than 85), and the level of the marker is usually determined by sequencing, in particular by shotgun metagenomic sequencing as generally described above.

[0054] In another specific embodiment, the contamination is benz[a]anthracene contamination and the at least one marker is the bacterial species Corynebacterium durum.

[0055] In this specific embodiment, if the level of at least one marker measured in step (a) (in particular the relative abundance of at least one marker) is higher than the control (in particular significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to benz[a]anthracene pollution, the control level is usually the level of the above marker (in particular the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, in particular a city where the number of days per year with an air quality index below 100 is less than 100 (in particular less than 85), and the level of the marker is usually determined by sequencing, in particular by shotgun metagenomic sequencing as generally described above.

[0056] In another specific embodiment, the contamination is fluoranthene contamination and said at least one marker is selected from the group consisting of the following bacterial species: Corynebacterium lipophiloflavum and Alicycliphilus sp.

[0057] In this specific embodiment, if the level of at least one marker measured in step (a) (in particular the relative abundance of at least one marker) is higher than the control (in particular significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to fluoranthene pollution, the control level is usually the level of the marker (in particular the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, in particular a city where the number of days per year with an air quality index below 100 is less than 100 (in particular less than 85), and the level of the marker is usually determined by sequencing, in particular by shotgun metagenomic sequencing as generally described above.

[0058] In another specific embodiment, the contamination is fluorene contamination and the at least one marker is selected from the group consisting of the following bacterial species: Corynebacterium lipophiloflavum, Kytococcus sedentarius, Brevibacterium sp., Haemophilus parainfluenzae, Actinomyces oris, Caulobacter sp. and Staphylococcus epidermidis.

[0059] In this specific embodiment, a subject is diagnosed as having experienced chronic exposure to fluorene contamination if:

[0060] - the level (in particular their relative abundance) of the species Corynebacterium lipophiloflavum, Kytococcus sedentarius, Brevibacterium sp., Haemophilus parainfluenzae, Actinomyces oris and / or Caulobacter sp. measured in step (a) is higher than in the control (in particular significantly higher than in the control), and / or

[0061] - the level of Staphylococcus epidermidis measured in step (a) (in particular its relative abundance) is lower than that of the control (in particular significantly lower than that of the control).

[0062] The control level is typically the level of the marker (particularly the relative abundance of the marker) in skin samples from subjects living in less polluted cities, particularly cities where the number of days per year where the air quality index is below 100 is less than 100 (particularly less than 85), and the level of the marker is typically determined by sequencing, particularly by shotgun metagenomic sequencing as generally described above.

[0063] In another specific embodiment, the contamination is anthracene contamination and said at least one marker is selected from the group consisting of the following bacterial species: Neisseria sp. and Abiotrophia defectiva.

[0064] In this specific embodiment, if the level of at least one marker measured in step (a) (in particular the relative abundance of at least one marker) is higher than the control (in particular significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to anthracene pollution, the control level is usually the level of the marker (in particular the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, in particular a city where the number of days per year with an air quality index below 100 is less than 100 (in particular less than 85), and the level of the marker is usually determined by sequencing, in particular by shotgun metagenomic sequencing as generally described above.

[0065] In another specific embodiment, the contamination is pyrene contamination and said at least one marker is selected from the group consisting of the following bacterial species: Kytococcus sedentarius, Brevibacterium sp., Haemophilus parainfluenzae, and Staphylococcus epidermidis.

[0066] In this specific embodiment, a subject is diagnosed as having experienced chronic exposure to pyrene contamination if:

[0067] - the level of species Kytococcus sedentarius, Brevibacterium sp. and / or Haemophilus parainfluenzae measured in step (a) (in particular their relative abundance) is higher than in the control (in particular significantly higher than in the control), and / or

[0068] - the level of Staphylococcus epidermidis measured in step (a) (in particular its relative abundance) is lower than that of the control (in particular significantly lower than that of the control).

[0069] The control level is typically the level of the marker (particularly the relative abundance of the marker) in skin samples from subjects living in less polluted cities, particularly cities where the number of days per year where the air quality index is below 100 is less than 100 (particularly less than 85), while the level of the marker is typically determined by sequencing, particularly by shotgun metagenomic sequencing as generally described above.

[0070] In another specific embodiment, the contamination is acenaphthylene contamination and said at least one marker is selected from the group consisting of the following bacterial species: Alicycliphilus sp, Actinomyces oris and Staphylococcus epidermidis.

[0071] In this specific embodiment, the subject is diagnosed as having experienced chronic exposure to acenaphthylene contamination if:

[0072] - the level of the species Alicycliphilus sp. and / or Actinomyces oris measured in step (a) (in particular their relative abundance) is higher than in a control (in particular significantly higher than in a control), and / or

[0073] - the level of Staphylococcus epidermidis measured in step (a) (in particular its relative abundance) is lower than that of the control (in particular significantly lower than that of the control).

[0074] The control level is typically the level of the marker (particularly the relative abundance of the marker) in skin samples from subjects living in less polluted cities, particularly cities where the number of days per year where the air quality index is below 100 is less than 100 (particularly less than 85), while the level of the marker is typically determined by sequencing, particularly by shotgun metagenomic sequencing as generally described above.

[0075] In another specific embodiment, the contamination is phenanthrene contamination and said at least one marker is selected from the group consisting of the following bacterial species: Corynebacterium durum, Kytococcus sedentarius, Micrococcus luteus, Streptococcus mitis / oral / pneumoniae and Corynebacterium propinquum.

[0076] In this specific embodiment, a subject is diagnosed as having experienced chronic exposure to phenanthrene contamination if:

[0077] - the level of the species Corynebacterium durum, Kytococcus sedentarius, Micrococcus luteus and / or Streptococcus mitis / oral / pneumoniae measured in step (a) (in particular their relative abundance) is higher than in the control (in particular significantly higher than in the control), and / or

[0078] - the level of the species Corynebacterium propinquum measured in step (a) (in particular its relative abundance) is lower than that of the control (in particular significantly lower than that of the control).

[0079] The control level is typically the level of the marker (particularly the relative abundance of the marker) in skin samples from subjects living in less polluted cities, particularly cities where the number of days a year with an air quality index below 100 is less than 100 (particularly less than 85), while the level of the marker is typically determined by sequencing, particularly by shotgun metagenomic sequencing as generally described above.

[0080] In another specific embodiment, said at least one marker is the bacterial species Corynebacterium lipoflavum and the contamination is with benzo[a]pyrene, fluoranthene and / or fluorene.

[0081] In this specific embodiment, if the level (especially its relative abundance) of the bacterial species Corynebacterium lipoflavum measured in step (a) is higher than the control (especially significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to benzo[a]pyrene, fluoranthene and / or fluorene pollution, the control level is usually the level of the marker (especially the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, especially a city with less than 100 days (especially less than 85) in a year with an air quality index below 100, and the level of the marker is usually determined by sequencing, especially by shotgun metagenomic sequencing as generally described above.

[0082] In another specific embodiment, said at least one marker is the bacterial species Corynebacterium durum and the contamination is with benzo[a]pyrene and / or phenanthrene.

[0083] In this specific embodiment, if the level of the bacterial species Corynebacteriumdurum measured in step (a) (in particular its relative abundance) is higher than the control (in particular significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to benzo[a]pyrene and / or phenanthrene pollution, the control level is usually the level of the marker (in particular the relative abundance of the marker) in a skin sample of a subject living in a less polluted city, in particular a city with less than 100 days (in particular less than 85) per year with an air quality index below 100, and the level of the marker is usually determined by sequencing, in particular by shotgun metagenomic sequencing as generally described above.

[0084] In another specific embodiment, said at least one marker is the bacterial species Kytococcus sedentarius and the contamination is with benzo[a]pyrene, fluorene, pyrene and / or phenanthrene.

[0085] In this specific embodiment, if the level (especially its relative abundance) of the bacterial species Kytococcus sedentarius measured in step (a) is higher than the control (especially significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to benzo[a]pyrene, fluorene, pyrene and / or phenanthrene pollution, the control level is usually the level of the marker (especially the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, especially a city with an air quality index below 100 for less than 100 days per year (especially less than 85), and the level of the marker is usually determined by sequencing, especially by shotgun metagenomic sequencing as generally described above.

[0086] In another specific embodiment, said at least one marker is the bacterial species Brevibacterium sp. and the contamination is with fluorene and / or pyrene.

[0087] In this specific embodiment, if the level of the bacterial species Brevibacterium sp. measured in step (a) (in particular its relative abundance) is higher than the control (in particular significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to fluorene and / or pyrene pollution, the control level is usually the level of the marker (in particular the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, in particular a city with an air quality index below 100 for less than 100 days per year (in particular less than 85), and the level of the marker is usually determined by sequencing, in particular by shotgun metagenomic sequencing as generally described above.

[0088] In another specific embodiment, said at least one marker is the bacterial species Haemophilus parainfluenzae and the contamination is contamination with fluorene and / or pyrene.

[0089] In this specific embodiment, if the level (especially its relative abundance) of the bacterial species Haemophilus parainfluenzae measured in step (a) is higher than the control (especially significantly higher than the control), the subject is diagnosed to have experienced long-term exposure to fluorene and / or pyrene pollution, the control level is usually the level of the marker (especially the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, especially a city with an air quality index below 100 for less than 100 days per year (especially less than 85), and the level of the marker is usually determined by sequencing, especially by shotgun metagenomic sequencing as generally described above.

[0090] In another specific embodiment, said at least one marker is the bacterial species Actinomyces oris and the contamination is with fluorene and / or acenaphthylene.

[0091] In this specific embodiment, if the level of the bacterial species Actinomyces oris measured in step (a) (in particular its relative abundance) is higher than the control (in particular significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to fluorene and / or acenaphthylene pollution, the control level is usually the level of the marker (in particular the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, in particular a city with an air quality index below 100 for less than 100 days per year (in particular less than 85), and the level of the marker is usually determined by sequencing, in particular by shotgun metagenomic sequencing as generally described above.

[0092] In another specific embodiment, said at least one marker is the bacterial species Neisseria sp. and the contamination is contamination with anthracene.

[0093] In this specific embodiment, if the level of the bacterial species Neisseria sp. measured in step (a) (especially its relative abundance) is higher than the control (especially significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to anthracene pollution, the control level is usually the level of the marker (especially the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, especially a city with an air quality index below 100 for less than 100 days per year (especially less than 85), and the level of the marker is usually determined by sequencing, especially by shotgun metagenomic sequencing as generally described above.

[0094] In another specific embodiment, said at least one marker is the bacterial species Abiotrophia defectiva and the contamination is contamination with anthracene.

[0095] In this specific embodiment, if the level of the bacterial species Abiotrophia defectiva measured in step (a) (especially its relative abundance) is higher than the control (especially significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to anthracene pollution, the control level is usually the level of the marker (especially the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, especially a city with an air quality index below 100 for less than 100 days per year (especially less than 85), and the level of the marker is usually determined by sequencing, especially by shotgun metagenomic sequencing as generally described above.

[0096] In another specific embodiment, said at least one marker is the bacterial species Alicycliphilus sp. and the contamination is contamination with fluoranthene and / or acenaphthylene.

[0097] In this specific embodiment, if the level (especially its relative abundance) of the bacterial species Alicycliphilus sp. measured in step (a) is higher than the control (especially significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to fluoranthene and / or acenaphthylene pollution, the control level is usually the level of the marker (especially the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, especially a city with an air quality index below 100 for less than 100 days per year (especially less than 85), and the level of the marker is usually determined by sequencing, especially by shotgun metagenomic sequencing as generally described above.

[0098] In another specific embodiment, said at least one marker is the bacterial species Micrococcus luteus and the contamination is contamination with phenanthrene.

[0099] In this specific embodiment, if the level (especially its relative abundance) of the bacterial species Micrococcus luteus measured in step (a) is higher than the control (especially significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to phenanthrene pollution, the control level is usually the level of the marker (especially the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, especially a city with an air quality index below 100 for less than 100 days per year (especially less than 85), and the level of the marker is usually determined by sequencing, especially by shotgun metagenomic sequencing as generally described above.

[0100] In another specific embodiment, said at least one marker is the bacterial species Caulobacter sp. and the contamination is with fluorene.

[0101] In this specific embodiment, if the level of the bacterial species Bacillus sp. measured in step (a) (especially its relative abundance) is higher than the control (especially significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to fluorene pollution, the control level is usually the level of the marker (especially the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, especially a city with an air quality index below 100 for less than 100 days per year (especially less than 85), and the level of the marker is usually determined by sequencing, especially by shotgun metagenomic sequencing as generally described above.

[0102] In another specific embodiment, said at least one marker is the bacterial species S. pneumoniae / oral / pneumococcus and the contamination is contamination with phenanthrene.

[0103] In this specific embodiment, if the level of bacterial species S. pneumoniae / oral / pneumococcus (especially its relative abundance) measured in step (a) is higher than the control (especially significantly higher than the control), the subject is diagnosed as having experienced long-term exposure to phenanthrene pollution, the control level is usually the level of the marker (especially the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, especially a city with less than 100 days (especially less than 85) in a year with an air quality index below 100, and the level of the marker is usually determined by sequencing, especially by shotgun metagenomic sequencing as generally described above.

[0104] In another specific embodiment, said at least one marker is the bacterial species Corynebacterium propinquum and the contamination is contamination with phenanthrene.

[0105] In this specific embodiment, if the level of the bacterial species Corynebacterium propinquum measured in step (a) (in particular its relative abundance) is lower than the control (in particular significantly lower than the control), the subject is diagnosed as having experienced long-term exposure to phenanthrene pollution, the control level is usually the level of the marker (in particular the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, in particular a city with an air quality index below 100 for less than 100 days per year (in particular less than 85), and the level of the marker is usually determined by sequencing, in particular by shotgun metagenomic sequencing as generally described above.

[0106] In another specific embodiment, said at least one marker is the bacterial species Staphylococcus epidermidis and the contamination is contamination with fluorene, pyrene and / or acenaphthylene.

[0107] In this specific embodiment, if the level (especially its relative abundance) of the bacterial species Staphylococcus epidermidis measured in step (a) is lower than the control (especially significantly lower than the control), the subject is diagnosed as having experienced long-term exposure to fluorene, pyrene and / or acenaphthylene pollution, the control level is usually the level of the marker (especially the relative abundance of the marker) in the skin sample of a subject living in a less polluted city, especially a city with an air quality index below 100 for less than 100 days per year (especially less than 85), and the level of the marker is usually determined by sequencing, especially by shotgun metagenomic sequencing as generally described above.

[0108] According to a preferred embodiment, the skin sample of the subject used in the diagnostic method according to the present invention is a sample collected, preferably in a non-invasive manner, from the skin of the subject, preferably from the face and / or head of the subject, in particular from the cheek and / or scalp of the subject. Thus, in one embodiment, the skin sample is a cheek skin or scalp sample. Preferably, the skin sample is from the stratum corneum, in particular from the surface of the stratum corneum.

[0109] The stratum corneum is the layer farthest from the epidermis and comprises the surface of the skin. It is composed mainly of dead cells.

[0110] According to an embodiment, the diagnostic method according to the invention comprises a step of collecting a skin sample from a subject, in particular from the skin surface of the subject. This step is preferably performed in a non-invasive manner, in particular without the need for local anesthesia. According to a preferred embodiment, the step of collecting a sample is performed by rubbing the skin surface, in particular with a wet cotton swab.

[0111] Thus, in specific embodiments, the skin sample is collected by abrading the surface of the skin.

[0112] "Subject" herein refers to a human being, preferably between 25 and 45 years old. Preferably, the subject is female. Preferably, the subject is of Asian type.

[0113] In a specific embodiment, the subject is between 25 and 45 years old.

[0114] Chronic exposure to pollution can often lead to premature aging of the skin, which is marked by the presence of wrinkles and / or fine lines, extended spots, freckles simplex, redness and / or a dull and / or uneven appearance of skin tone, as well as aesthetic deterioration of the skin such as pigmentation disorders.

[0115] Therefore, the present invention relates to a method for preventing premature skin aging and / or aesthetic skin degradation caused by long-term exposure to pollution in a subject, the method comprising the following steps:

[0116] A) diagnosing that a subject has experienced long-term exposure to pollution by implementing a diagnostic method according to the present invention,

[0117] B) treating the subject with a cosmetic composition capable of slowing down premature skin aging and / or aesthetic degradation of the skin, if the subject is diagnosed as having experienced chronic exposure to pollution.

[0118] In a specific embodiment, the cosmetic composition used in the treatment method of the present invention comprises probiotics and / or prebiotics, in particular those which promote the presence of commensal flora.

[0119] The present invention is described in more detail by the following examples.

[0120] Example

[0121] The following example shows the characteristics of 14 bacterial species that were significantly regulated in skin samples from individuals exposed to chronic pollution (based on high levels of pollutants detected in their hair samples).

[0122] Materials and methods

[0123] Main steps:

[0124] - Skin sampling

[0125] - Comprehensive metagenomics known as "shotgun"

[0126] -Correlation analysis between PAH and bacterial species

[0127] Description of the subjects

[0128] All subjects from each city visited facilities in Baoding and Dalian (China). Cheek skin samples were collected from 204 healthy Chinese women aged 25 to 45 years, 102 of whom lived in Baoding, a relatively rural and industrialized city in Hebei Province in northern China with high levels of air pollution (PM 2.5 / m 3 About 90 μg), 102 of whom lived in Dalian, an urbanized and modern city in Liaoning Province in northern China with low air pollution (PM 2.5 / m 3 The two cities are located at the same latitude and have had similar climates and comparable UV exposure (UV index) over the past 15 years.

[0129] Participants living in the two cities were assessed for PAHs (polycyclic aromatic hydrocarbons) exposure in 12 cm hair samples (reflecting exposure over a one-year period) (Palazzi et al. (2018) Env. Iht. 121: 1341-1354). Specifically, the median concentrations of parent PAHs in Baoding were 1.5-2.8 times that of Dalian, and the median concentrations of PAH metabolites were 1.1-2.3 times that of Dalian. Among the quantified parent PAHs, levels of phenanthrene, fluoranthene, pyrene, fluorene, acenaphthylene, and anthracene were high, while levels of PAH metabolites, 9-OH-fluorene, 2-OH-naphthalene, and 1-OH-anthracene were high (Palazzi et al. (2018) Env. Iht. 121: 1341-1354).

[0130] Subjects and sample collection

[0131] Participants had not received any systemic antibiotics or antifungals in the month before sampling, had no serious skin diseases, had not used skin or systemic depigmentation / bleaching treatments in the 3 months before sampling, or had not used exfoliating products in the month before sampling. They were asked to wash their faces with the provided neutral soap without antimicrobial compounds for 3 days (once a day) before sampling. The last application of shampoo and soap was 48 hours and 24 hours before sampling, respectively. No other products were allowed on the scalp, hair, and face until sampling was completed.

[0132] Microbiome sampling was performed in a controlled environment at 22°C and 60% humidity. Samples for microbiome analysis were collected using sterile dry cotton swabs heated to 150°C and pre-moistened with ST solution (0.15 M NaCl with 0.1% Tween 20). For cheek samples, the cotton swab was soaked in collection buffer and rubbed vigorously on the cheek for 60 seconds to cover an area of ​​1 cm × 2 cm. For scalp samples, the cotton swab was rubbed firmly on the scalp surface along one line 4 times, and then the cotton swab was moved to another line using a comb. These steps were performed 6 times to cover 4 cm 2 After sampling, each swab was placed in a microtube and immediately frozen in liquid nitrogen and stored at -80°C before genomic DNA (gDNA) extraction.

[0133] Preparation and analysis of metagenomic sequencing libraries

[0134] Sixteen samples (9 from cheeks and 7 from scalp) were selected for analysis by metagenomic sequencing. The samples were selected based on the extreme levels of individual PAH exposure (low vs. high) in the two cities.

[0135] A sample of these is shown in Table 1 below.

[0136]

[0137]

[0138] PowerSoil was used according to the manufacturer's instructions and with modifications as described in Leung et al. (2014) Appl. About. Microbiol. 80: 6760-6770. gDNA was extracted using the ELISA kit (MO BIO Laboratories, Carlsbad, CA, USA). In addition, after C6 elution, the eluate was passed through the same filter column again to increase the yield. A negative DNA-free water control was extracted in parallel.

[0139] Before library preparation, gDNA of each sample was normalized to 10 ng / μl. Library preparation was performed using standard Illumina protocols, and the final library was prepared using TapeStation Quantification was performed by Agilent (Santa Clara, CA, USA) and by SeqMatic LLC (Fremont, CA, USA) using Illumina The platform performs sequencing to generate 150 bp paired-end reads.

[0140] use v0.6.1 (http: / / huttenhower.sph.harvard.edu / kneaddata) was used to perform quality control on raw reads and process human DNA reads, eliminating reads shorter than 50 bp and reads corresponding to the human reference genome hg38. The filtered reads were subjected to taxonomic alignment using v2.6.0 (Truong et al. (2015) Nat. Methods 12:902-903). Functional assignment was performed using v0.11.1, and the output was expressed as reads per kilobase per million reads (RPKM). HUMAnN2 results were converted to the "KEGGOrthology" (KO) gene family for "heat map" representation and multivariate analysis of functional genes (Abubucker et al. (2012) PLoS Comput. Biol. 8: e1002358). MaAsLin was performed to correlate PAH exposure with MetaPh1An2 and HUMAnN2 data, as shown in Lim et al. (2016) Sci. Rep. 6: 23745 or Knights et al. (2014) Genome Med. 6: 107, and FDR-corrected p-values ​​(q-values) less than or equal to 0.25 were considered significant.

[0141] Winding Analysis

[0142] The nonparametric Mann-Whitney test was performed to test the significance between 2 groups, and the Kruskal-Wallis test was performed to test the significance between 3 or more groups. FDR correction was performed using the "p.adjust" command in R.

[0143] result

[0144] Table 2 below lists the main correlations identified between a -40 unit change in PAH and the percent change in relative abundance (RA) of important species (q-value ≤ 0.25).

[0145] Table 2: The main correlation identified was between a change of -40 units in PAH and changes in the percentage relative abundance (RA) of important species.

[0146]

[0147]

[0148] Correlations for most PAH-species pairs were inferred based on PAH changes of smaller magnitude (i.e., 1.2 units).

[0149] In summary, positive correlations between the percentage of RA and increased PAH were observed for the following species:

[0150] -Abiotrophia defectiva,

[0151] -Actinomyces oris,

[0152] -Alicycliphilus sp.,

[0153] - Brevibacterium sp.,

[0154] - Caulobacter sp.,

[0155] -Corynebacterium durum,

[0156] -Corynebacterium lipophiloflavum,

[0157] -Haemophilus influenzae,

[0158] - Kytococcus sedentarius,

[0159] - Micrococcus luteus,

[0160] -Neisseria sp. and

[0161] - Mild / Oral / Pneumococcal,

[0162] And a negative correlation between the percentage of RA and the increase in PAH was observed for the following species:

[0163] - Staphylococcus epidermidis and

[0164] -Corynebacterium propinquum.

Claims

1. A method for determining long-term exposure of a subject's skin to polycyclic aromatic hydrocarbons (PAHs) contamination, wherein the method is used for purposes other than disease diagnosis, comprising the step of (a) determining the level of at least one marker in a skin sample of the subject, wherein the at least one marker is selected from the group consisting of the following bacterial species: Corynebacterium lipophiloflavum, Corynebacterium durum, Corynebacterium propinquum, Kytococcus sedentarius, Brevibacterium sp., Haemophilus parainfluenzae, Actinomyces oris, Neisseria sp., Abiotrophia defectiva, Alicycliphilus. sp., Streptococcus mitis, Streptococcus oralis, Streptococcus pneumoniae and Staphylococcus epidermidis.

2. The method according to claim 1, wherein: The at least one marker is selected from the group consisting of the following bacterial species: Corynebacterium lipophiloflavum, Corynebacterium durum, Kytococcussedentarius, Brevibacterium sp., Haemophilus parainfluenzae, Actinomyces oris, Neisseria sp., Abiotrophia defectiva, Alicycliphilus sp., Micrococcus luteus, Caulobacter sp., Streptococcus mitis, Streptococcus oralis and Streptococcus pneumoniae.

3. The method according to claim 1, wherein: The at least one marker is selected from the group consisting of the following bacterial species: Corynebacterium propinquum and Staphylococcus epidermidis.

4. The method according to any one of claims 1 to 3, further comprising the steps consisting of: (b) comparing the level of the at least one marker measured in step (a) with a control, and (c) determining whether the subject has experienced long-term exposure to pollution based on the comparison in step (b).

5. The method according to claim 4, wherein: The step (c) is: (c) determining, based on the comparison in step (b), whether the subject's skin has experienced long-term exposure to pollution.

6. The method according to claim 4, wherein: The at least one marker is selected from the group consisting of the following bacterial species: Corynebacterium lipophiloflavum, Corynebacterium durum, Kytococcussedentarius, Brevibacterium sp., Haemophilus parainfluenzae, Actinomyces oris, Neisseria sp., Abiotrophia defectiva, Alicycliphilus sp., Micrococcus luteus, Caulobacter sp., Streptococcus mitis, Streptococcus oralis and Streptococcus pneumoniae, and if the level of the at least one marker measured in step (a) is higher than the control, the subject is diagnosed as having experienced long-term exposure to pollution.

7. The method according to claim 4, wherein: The at least one marker is selected from the group consisting of the following bacterial species: Corynebacterium propinquum and Staphylococcus epidermidis, and if the level of the at least one marker measured in step (a) is lower than the control, the subject is diagnosed as having experienced chronic exposure to pollution.

8. The method according to claim 1, wherein: The level of the at least one marker is determined by metagenomic sequencing.

9. The method according to claim 1, wherein: The skin sample is a cheek sample or a scalp sample.

10. The method according to claim 1, wherein: The subjects were between 25 and 45 years old.

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