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Title:
DERMATOLOGICAL PREPARATIONS FOR MAINTAINING AND/OR RESTORING HEALTHY SKIN MICROBIOTA
Document Type and Number:
WIPO Patent Application WO/2017/220525
Kind Code:
A1
Abstract:
The present invention is directed to the direct application of beneficial or probiotic bacteria to the skin for maintenance of a healthy skin microbiota and to help restore an unbalanced skin microbiota. The application is based on the use of selected Lactobacillus strains as anti-5 pathogenic agents, in particular L. plantarum, L. pentosus and/or L. rhamnosus, against common skin pathogens, whereby produced acids such as lactic acid are important antimicrobial factors.

Inventors:
LEBEER, Sarah (Groenenborgerlaan 171, 2020 Antwerpen, 2020, BE)
CLAES, Ingmar (Groenenborgerlaan 171, 2020 Antwerpen, 2020, BE)
OERLEMANS, Eline (Groenenborgerlaan 171, 2020 Antwerpen, 2020, BE)
VAN DEN BROEK, Marianne (Groenenborgerlaan 171, 2020 Antwerpen, 2020, BE)
Application Number:
EP2017/065006
Publication Date:
December 28, 2017
Filing Date:
June 20, 2017
Export Citation:
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Assignee:
YUN NV (Berkenlaan 4, 2630 Aartselaar, 2630, BE)
UNIVERSITEIT ANTWERPEN (Prinsstraat 13, 2000 Antwerpen, 2000, BE)
International Classes:
A61Q19/00; A61K8/99; A61K35/747; A61P17/00; C12R1/225; C12R1/25
Domestic Patent References:
WO2001013956A22001-03-01
WO2013188626A22013-12-19
WO2016023688A12016-02-18
WO2011052996A22011-05-05
Foreign References:
EP2149368A12010-02-03
EP1736537A12006-12-27
EP2332521A12011-06-15
KR20120089530A2012-08-13
KR20150075447A2015-07-06
Other References:
BEYLOT, C. ET AL.: "Propionibacterium acnes: an update on its role in the pathogenesis of acne", JOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY AND VENEREOLOGY: JEADV, vol. 28, 2014, pages 271 - 8
CHAN, R.C. ET AL.: "Competitive exclusion of uropathogens from human uroepithelial cells by Lactobacillus whole cells and cell wall fragments", INFECTION AND IMMUNITY, vol. 47, no. 1, 1985, pages 84 - 9
CHAN, R.C.; BRUCE, A.W.; REID, G: "Adherence of cervical, vaginal and distal urethral normal microbial flora to human uroepithelial cells and the inhibition of adherence of gram-negative uropathogens by competitive exclusion", THE JOURNAL OF UROLOGY, vol. 131, no. 3, 1984, pages 596 - 601
GRICE, E.A.; SEGRE, J.A.: "The skin microbiome", NATURE REVIEWS. MICROBIOLOGY, vol. 9, no. 4, 2011, pages 244 - 53, XP055344752, DOI: doi:10.1038/nrmicro2537
REID, G.: "The Scientific Basis for Probiotic Strains of Lactobacillus", APPL. ENVIR. MICROBIOL., vol. 65, no. 9, 1999, pages 3763 - 3766
REID, G.; BRUCE, A.W.: "Selection of lactobacillus strains for urogenital probiotic applications", THE JOURNAL OF INFECTIOUS DISEASES, vol. 183, 2001, pages S77 - 80
SUGITA, T. ET AL.: "Temporal changes in the skin Malassezia microbiota of members of the Japanese Antarctic Research Expedition (JARE): A case study in Antarctica as a pseudo-space environment.", MEDICAL MYCOLOGY, vol. 53, no. 7, 2015, pages 717 - 24
WANG, L. ET AL.: "Characterization of the major bacterial-fungal populations colonizing dandruff scalps in Shanghai, China, shows microbial disequilibrium", EXPERIMENTAL DERMATOLOGY,, vol. 24, no. 5, 2015, pages 398 - 400
Attorney, Agent or Firm:
LC PATENTS (Kempische Steenweg 542A, 3500 Hasselt, 3500, BE)
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Claims:
CLAIMS

A topical dermatological composition comprising one or more live Lactobacillus species; wherein at least one of said Lactobacillus species is a L. plantarum strain having at least 97% sequence similarity with SEQ ID N° 4 in its 16S rRNA gene.

A live Lactobacillus species for use in restoring and/or maintaining a healthy skin microbiota, by topical route, said Lactobacillus species being a L. plantarum strain having at least 97% sequence similarity with SEQ ID N° 4 in its 16S rRNA gene.

Use of one or more live Lactobacillus species, in the preparation of a topical dermatological composition for restoring and/or maintaining a healthy skin microbiota; wherein at least one of said Lactobacillus species is a L. plantarum strain having at least 97% sequence similarity with SEQ ID N° 4 in its 16S rRNA gene.

Method for restoring and/or maintaining a healthy skin microbiota; comprising at least one step of administering by topical route, to an individual, an effective amount of one or more live Lactobacillus species; wherein at least one of said Lactobacillus species is a L. plantarum strain having at least 97% sequence similarity with SEQ ID N° 4 in its 16S rRNA gene.

A composition comprising one or more live Lactobacillus species for use in restoring and/or maintaining a healthy skin microbiota, by topical route, said Lactobacillus species being selected from the list comprising L. plantarum, L. pentosus and L. rhamnosus; wherein said L. plantarum is a L. plantarum strain having at least 97% sequence similarity with SEQ ID N° 4 in its 16S rRNA gene, said L. pentosus is a L. pentosus strain having at least 97% sequence similarity with SEQ ID N° 1 in its 16S rRNA gene, and said L. rhamnosus is a L. rhamnosus strain having at least 97% sequence similarity with SEQ ID N° 5 in its 16S rRNA gene.

6. A Lactobacillus strain being L. rhamnosus YUN-S1 .0 deposited under accession number LMG P-2961 1 (deposited at BCCM on May, 12 2016.

7. A composition comprising a Lactobacillus strain as defined in claim 6.

8. The composition as defined in claim 7; wherein said composition is a topical skin composition.

9. The use according to claim 3, or the composition according to anyone of claims 1 , 5, 7 or 8 wherein said composition is a topical skin composition in the form of a gel, cream, ovule, suppository forms, foam, lotion, or ointment.

10. A Lactobacillus strain as defined in claim 6 or a composition as defined in any one of claims 7 or 8; for use in restoring and/or maintaining a healthy skin microbiota, by topical route.

11. Topical use of one or more live Lactobacillus species in probiotherapy of the skin; wherein said Lactobacillus species are selected from the list comprising L. plantarum, L. pentosus and L. rhamnosus.

12. Topical use according to claim 1 1 ; wherein said probiotherapy consists of restoring and/or maintaining a healthy skin microbiota in a subject in need thereof.

Topical use as defined in anyone of claims 1 1 or 12, the use as defined in claim 3, the method as defined in claim 4, the live Lactobacillus species as defined in claim 2, or the composition as defined in claim 5; wherein said Lactobacillus species is a Lactobacillus strain selected from the list comprising L. plantarum YUN-V2.0 deposited under accession number LMG P-29456; L. pentosus YUN-V1 .0 deposited under accession number LMG P-29455; and L. rhamnosus YUN-S1 .0 deposited under accession number LMG P-2961 1 .

Description:
DERMATOLOGICAL PREPARATIONS FOR MAINTAINING AND/OR RESTORING

HEALTHY SKIN MICROBIOTA

FIELD OF THE INVENTION

The present invention is directed to the direct topical application of beneficial or probiotic bacteria to the skin for maintenance of a healthy skin microbiota and to help restore an unbalanced skin microbiota. This restoration of a healthy microbiota falls under the term probiotherapy, defined as the use of beneficial micro-organisms or probiotics to restore a healthy microbiota at a site where microbial dysbiosis occurs. The application is based on the use of selected Lactobacillus strains as anti-pathogenic agents, in particular L. plantarum, L. pentosus and/or L. rhamnosus, against common skin pathogens, whereby produced acids such as lactic acid are important antimicrobial factors.

BACKGROUND TO THE INVENTION

Hence, it was an object of the present invention to provide a solution for subjects suffering from skin conditions due to an aberrant microbial balance on the skin. Thereto, it was found that the topical use of L. plantarum, L pentosus and/or L. rhamnosus species on the skin is very effective in restoring and/or maintaining a healthy skin microbiota, and is thus very suitable in relieving skin conditions in subjects in need thereof.

Oral formulations comp sing Lactobacillus strains have been used before in the treatment of skin conditions like atopic dermatitis. However, oral administration versus direct topical administration are different administration routes and each have a completely different underlying mechanism. In oral administration, in particular a beneficial effect on the general health via immuno-stimulation is intended, whereas by direct dermatological (skin) administration, competition with 'unwanted' microorganisms occurs.

Like the gastrointestinal tract, our skin harbours a unique microbial ecosystem. The type of micro-organisms found on the skin depends on a combination of host factors, environmental factors but also topographical location. The role of this microbiota in skin disorders is still not completely unravelled. However, it seems that, at least, some skin disorders are linked to a disturbed microbiota as antimicrobial treatments can improve clinical symptoms (G ee & Segre 201 1 ). For example, in acne vulgaris a correlation has been found with the presence of Propionibacterium acnes (Beylot et al. 2014). Although acne vulgaris is a multifactorial condition and is, among other factors, influenced by hormonal factors, these P. acnes bacteria seem to induce inflammation resulting in inflamed pimples also called papules or pustels. As P. acnes is also found on a healthy skin not causing acne, this suggests that other factors are involved, tipping the balance of the composition of the skin microbiota towards an overgrowth of this bacteria. Another example of a skin disorder where the microbiota seems to be important is dandruff (Wang et al. 2015; Sugita et al. 2015; G ee & Segre 201 1 ). In people with dandruff, the fungus Malassezia is often overrepresented. Indications that it is this fungus that is a possible cause of the condition, come from the fact that antimycotic treatment improves the symptoms. In contrast, antibacterial therapies do not improve dandruff. Again, other factors are expected to be involved in this skin disorder but the correlation with Malassezia is intruiging.

Similarly as for dandruff, fungal skin infections with Candida albicans or dermatophytes, like Trichophyton spp., seem to be skin disorders linked to a dysbiosis in the skin microbiota as these species are also present on healthy subjects. In the case of Tinea pedis or 'athlete's foot' overgrowth of Trichophyton rubrum or T. mentagrophytes is often observed.

The production of lactic acid in combination with possibly other antimicrobial compounds like bacteriocins seems to give protection against aforementioned infections and dysbiotic conditions and lactic acid seems to be active against bacterial, fungal and even viral pathogens. It is for this reason that lactobacilli are considered to be important in the homeostasis of the dynamical dermatological ecosystem. Potential health promoting mechanisms of lactobacilli are i) to preserve a healthy skin pH (+/- 5.5), mainly by production of lactic acid; ii) production of antimicrobial compounds and competitive exclusion of pathogens; iii) modulation of immune response and iv) strengthening of the epithelial barrier.

Hence, it was an object of the present invention to provide a solution for subjects suffering from dermatological conditions due to an aberrant microbial balance of the skin. Thereto, it was found that the topical dermatological use of L. plantarum, L pentosus and/or L. rhamnosus species is very effective in restoring and/or maintaining a healthy microbiota on the skin, and is thus very suitable in relieving dermatological conditions in subjects in need thereof.

Oral formulations comprising Lactobacillus strains have been used before in the treatment of dermatological disorders. However, oral administration versus direct topical administration are different administration routes and each have a completely different underlying mechanism. In oral administration, in particular a beneficial effect on the general health via immuno-stimulation is intended, whereas by direct administration on the skin, competition with 'unwanted' microorganisms occur. SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a topical skin composition comprising one or more live Lactobacillus species; wherein at least one of said Lactobacillus species is L. plantarum; more in particular a L. plantarum strain having at least 97% sequence similarity with SEQ ID N° 4 in its 16S rRNA gene.

In a further aspect, the present invention provides a live Lactobacillus species for use in restoring and/or maintaining a healthy skin microbiota, by topical route, said Lactobacillus species being L. plantarum; more in particular a L. plantarum strain having at least 97% sequence similarity with SEQ ID N° 4 in its 16S rRNA gene.

In yet a further aspect, the present invention provides the use of one or more live Lactobacillus species, in the preparation of a topical skin composition for restoring and/or maintaining a healthy skin microbiota; wherein at least one of said Lactobacillus species is L. plantarum; more in particular a L. plantarum strain having at least 97% sequence similarity with SEQ ID N° 4 in its 16S rRNA gene.

The present invention also provides a method for restoring and/or maintaining a healthy skin microbiota; comprising at least one step of administering by topical route, to an individual, an effective amount of one or more live Lactobacillus species; wherein at least one of said Lactobacillus species is L. plantarum; more in particular a L. plantarum strain having at least 97% sequence similarity with SEQ ID N° 4 in its 16S rRNA gene.

In yet another aspect, the present invention provides a composition comprising one or more live Lactobacillus species for use in restoring and/or maintaining a healthy skin microbiota, by topical route, said Lactobacillus species being selected from the list comprising L. plantarum, L. pentosus and L. rhamnosus; more in particular a L. plantarum strain having at least 97% sequence similarity with SEQ ID N° 4 in its 16S rRNA gene, a L. pentosus strain having at least 97% sequence similarity with SEQ ID N° 1 in its 16S rRNA gene and a L. rhamnosus strain having at least 97% sequence similarity with SEQ ID N° 5 in its 16S rRNA gene.

The present invention further provides a Lactobacillus strain being L. rhamnosus YUN-S1 .0 deposited under accession number LMG P-2961 1 (deposited at BCCM on May, 12 2016). In a particular aspect, the present invention provides a composition comprising one or more Lactobacillus strains as defined herein above.

In a particular embodiment, the composition of the present invention is a topical skin composition, more in particular in the form of a gel, cream, foam, lotion or ointment. In another particular embodiment, the present invention provides the Lactobacillus strain as defined herein above or the compositions as defined herein above; for use in restoring and/or maintaining a healthy skin microbiota, by topical route. In a particular aspect, the present invention provides a topical use of one or more live Lactobacillus species in probiotherapy of the skin; wherein said Lactobacillus species are selected from the list comprising L. plantarum, L. pentosus and L. rhamnosus; more in particular, said probiotherapy consists of restoring and/or maintaining a healthy skin microbiota in a subject in need thereof.

In another particular embodiment, said Lactobacillus species in the topical uses, methods and compositions as disclosed herein, is a Lactobacillus strain selected from the list comprising L. plantarum YUN-V2.0 deposited under accession number LMG P-29456 (deposited at BCCM on Mar, 09 2016), L. pentosus YUN-V1 .0 deposited under accession number LMG P-29455 (deposited at BCCM on Mar, 09 2016); and L. rhamnosus YUN-S1 .0 deposited under accession number LMG P-2961 1 (deposited at BCCM on May, 12 2016).

BRIEF DESCRIPTION OF THE DRAWINGS

With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

Fig. 1 : Characteristics of lactobacilli in reference to growth, production of D- and L-lactic acid (LA) and lowering of the pH of the medium.

Fig. 2: Time course experiment for the analysis of the antipathogenic effect of spent culture supernatant of lactobacilli against Propionibacterium acnes. Growth of the bacteria (optical density at 600nm; Y-axis) is measured in time (X-axis). Each graph shows replicates of growth of P. acnes. It can be clearly noted that without any addition of antibiotic or SCS, P. acnes quickly starts to grow (NC1 ). Similar as when erythromycin at 50pg/ml is added, SCS of all lactobacilli prevents growth of P. acnes while SCS of streptococci or staphylococci does not inhibit growth. Erythromycine (50ug/ml); # Erythromycine (5ug/ml); Minocycline (20 pg/ml) NC1 =medium control; NC2=MRS at pH4.3; Numbers 1 to 22 = lactobacilli strains (for details see table 1 ); St=Streptococcus thermophilus; Ss=Streptococcus salivarius; Se=Staphylococcus epidermidis; TO.5=0.5% Tween 80; T1 =1 % Tween 80. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of specific Lactobacillus strains that can compete with growth of Propionibacterium acnes, Candida albicans, Malassezia spp., Trichophyton spp. and bacteria or fungi that are linked with skin conditions like acne vulgaris, dandruff, tinea pedis or other fungal skin infections. These selected strains are herein generally termed "YUN" strains and are capable of competing with skin pathogens and thereby restore a healthy skin microbiota. This restoration of a healthy microbiota falls under the term probiotherapy, defined as the use of beneficial micro-organisms or probiotics to restore a healthy microbiota at a site where microbial dysbiosis occurs.

Hence, in a first aspect, the present invention provides a topical skin composition comprising one or more live Lactobacillus species; wherein at least one of said Lactobacillus species is L. plantarum; more in particular a L. plantarum strain having at least 97% sequence simila ty with SEQ ID N° 4 in its 16S rRNA gene.

Said composition according to the present invention may comprise further Lactobacillus species such as for example selected from the non-limiting list comprising L. pentosus, L. gasseri, L. crispatus, L. acidophilus, L. jensenii, L. fermentum, L. rhamnosus.

In the context of the present invention, the term "topical" is meant to be the local delivery at a specified location of the body, in particular the application to a particular place on the body. In particular, it includes the application via non-solid formulations such as creams, foams, gels, lotions or ointments. The term "topical" is not meant to include the delivery in the form of solid preparations such as capsules, tablets, ...

Hence, the term "topical skin" is meant to include the local delivery using non-solid formulations directly onto the skin of the body. Preferably, the compositions according to the present invention are applied over a large area of the skin in order to be most effective.

In the context of the present invention the term "live Lactobacillus species" is meant to be viable Lactobacillus species, and is not meant to be fragments, culture supernatants, or killed forms thereof. In a further aspect, the present invention provides a live Lactobacillus species for use in probiotherapy of the skin, by topical route, said Lactobacillus species being L. plantarum; more in particular a L. plantarum strain having at least 97% sequence similarity with SEQ ID N° 4 in its 16S rRNA gene. As already defined herein above, said probiotherapy is meant to be the restoration and/or maintainance of a healthy skin microbiota in a subject in need thereof. Subjects that may benefit from such probiotherapy are for example people/persons with a skin conditions linked to a disturbed skin microbiota possibly due to bacterial or yeast infections and/or any dysbiosis caused by overgrowth of specific pathogenic micro-organisms, like acne vulgaris, tinea pedis, dandruff, rosaceae, impetigo, ...

Hence, in a further aspect, the present invention provides the use of one or more live Lactobacillus species, in the preparation of a topical skin composition for restoring and/or maintaining a healthy skin microbiota; wherein at least one of said Lactobacillus species is L. plantarum; more in particular a L. plantarum strain having at least 97% sequence simila ty with SEQ ID N° 4 in its 16S rRNA gene.

The present invention also provides a method for restoring and/or maintaining a healthy skin microbiota; comprising at least one step of administering by topical route, to an individual, an effective amount of one or more live Lactobacillus species; wherein at least one of said Lactobacillus species is L. plantarum; more in particular a L. plantarum strain having at least 97% sequence simila ty with SEQ ID N° 4 in its 16S rRNA gene.

In yet another aspect, the present invention provides a composition comprising one or more live Lactobacillus species for use in restoring and/or maintaining a healthy skin microbiota, by topical route, said Lactobacillus species being selected from the list comprising L. plantarum, L. pentosus and L. rhamnosus; more in particular a L. plantarum strain having at least 97% sequence similahty with SEQ ID N° 4 in its 16S rRNA gene; a L. pentosus strain having at least 97% sequence similahty with SEQ ID N° 1 in its 16S rRNA gene and a L. rhamnosus strain having at least 97% sequence similarity with SEQ ID N° 5 in its 16S rRNA gene.

The present invention further provides a Lactobacillus strain selected from the list comprising L. pentosus YUN-V1 .0 deposited under accession number LMG P-29455 (deposited at BCCM on Mar, 09 2016); L. plantarum YUN-V2.0 deposited under accession number LMG P-29456 (deposited at BCCM on Mar, 09 2016); and L. rhamnosus YUN-S1 .0 deposited under accession number LMG P-2961 1 (deposited at BCCM on May, 12 2016)

The microbiological deposits mentioned herein, have been made with the BCCM/LMG Bacteria collection ("Belgian co-ordinated collections of micro-organism") with correspondence address: Laboratorium voor Microbiologie, Universiteit Gent, K.L. Ledeganckstraat 35 - 9000 Gent, Belgium

Lactobacillus pentosus YUN-V1 .0 is a single colony isolate obtained in our lab after subculturing of a strain, that was originally a vaginal isolate of healthy woman. The 16S rRNA gene sequence (SEQ ID N° 1 ) for strain L. pentosus YUN-V1 .0 was determined by PCR using primers 8F (5'-AGAGTTTGATCCTGGCTCAG-3' - SEQ ID N° 2) and 1525R (5'- AAGGAGGTGATCCAGCCGCA-3' - SEQ ID N° 3).

YUN-V2.0 and YUN-V3.0 are single colony isolates obtained in our lab after subculturing of Lactobacillus plantarum strains that were originally isolated from human saliva and a maize silage respectively. The 16S rRNA gene sequence (SEQ ID N° 4) for strain L. plantarum YUN- V2.0 was determined by PCR using primers 8F (5'-AGAGTTTGATCCTGGCTCAG-3' - SEQ ID N° 2) and 1525R (5'-AAGGAGGTGATCCAGCCGCA-3' - SEQ ID N° 3).

YUN-S1 .0 is a single colony isolate obtained in our lab after subculturing of a Lactobacillus rhamnosus strain that was originally isolated from a healthy person. The 16S rRNA gene sequence (SEQ ID N° 5) for strain L. rhamnosus YUN-S1 .0 was determined by PCR using primers 8F (5'-AGAGTTTGATCCTGGCTCAG-3' - SEQ ID N° 2) and 1525R (5'- AAGGAGGTGATCCAGCCGCA-3' - SEQ ID N° 3). These particular "YUN" strains can either be used as such, or are preferably formulated in a composition comprising such strains. Said compositions are topical skin compositions more in particular in the form of non-solid formulations such as creams, foams, gels, lotions or ointments. In particular, the present invention provides the above defined "YUN" strains for use in probiotherapy of the skin, i.e. for restoring and/or maintaining a healthy skin microbiota.

In yet a further aspect, the present invention provides a topical use of one or more live Lactobacillus species in probiotherapy of the skin; wherein said Lactobacillus species are selected from the list comprising L. plantarum, L. pentosus and L. rhamnosus; more in particular, said probiotherapy consists of restoring and/or maintaining a healthy skin microbiota in a subject in need thereof.

In a specific embodiment, the Lactobacillus species in the topical uses, methods and compositions as disclosed herein, is a Lactobacillus strain selected from the list comprising L. plantarum YUN-V2.0 deposited under accession number LMG P-29456 (deposited at BCCM on Mar, 09 2016); L. pentosus YUN-V1 .0 deposited under accession number LMG P-29455 (deposited at BCCM on Mar, 09 2016); and L. rhamnosus YUN-S1 .0 deposited under accession number LMG P-2961 1 (deposited at BCCM on May, 12 2016). EXAMPLES

MATERIALS AND METHODS

Bacterial strains and growth conditions

Lactobacillus strains (Table 1 ) were grown at 37°C in de Man, Rogosa and Sharpe (MRS) medium (Carl Roth). All bacteria were grown in non-shaking conditions and inoculated from glycerol stocks (-80°C). Solid media contained 1 .5% (w/v) agar.

Table 1 : Bacterial strains used in this research

Species # Strain Relevant genotype or description Reference and/or Source

LACTOBACILLI

Lactobacillus 1 ATCC334 Single colony isolate obtained in our lab ATCC

casei from a stock culture of ATCC334

Lactobacillus 2 DN-1 14001 Single colony isolate obtained in our lab Commercial casei from a commercially available fermented probiotic product drink (Actimel®) containing L. casei DN- 1 14001 , confirmed by sequencing

Lactobacillus 3 Shirota Single colony isolate obtained in our lab Commercial casei from a commercially available fermented probiotic product drink containing L. casei Shirota

(Yakult®), confirmed by sequencing

Lactobacillus 4 YUN-V1 .0 Single colony isolate

pentosus

Lactobacillus 5 LMG1284 Single colony isolate from L. plantarum ATCC

plantarum ATCC8014 or LMG1284

Lactobacillus 6 RC-14 Single colony isolate obtained in our lab Commercial reuteri from a commercially available probiotic probiotic product supplement containing L. reuteri RC-14,

confirmed by sequencing

Lactobacillus 7 YUN-S1 .0 Clinical isolate

rhamnosus

Lactobacillus 12 GR-1 Single colony isolate obtained in our lab (Chan et al. rhamnosus from a commercially available probiotic 1984; 1985;

supplement containing L. rhamnosus GR- Reid 1999; Reid 1 & Bruce 2001 ),

ATCC

Lactobacillus 14 AMB-2 single colony isolate Commercial helveticus probiotic product

Lactobacillus 15 YUN-V2.0 Single colony isolate

plantarum

Lactobacillus 16 5057 Single colony isolate

plantarum

Lactobacillus 17 LMG12586 Single colony isolate obtained in our lab BCCM/LMG paracasei from a stock culture of LMG12586

Lactobacillus 22 / Single colony isolate

plantarum

Lactobacillus 25 LMG8041 Single colony isolate BCCM/LMG pentosus PATHOGENS

Trichophyton 2 1 Clinical isolate BCCM/LMG rubrum

Malassezia 1 Clinical isolate BCCM/LMG furfur

Candida 1 1 Clinical isolate

albicans

Preparation of spent culture supernatant (SCS) of selected strains

To obtain spent culture supernatant (SCS) containing the secreted active antimicrobial products, growth medium specific for each species was inoculated from a preculture and incubated for 24h. SCS was obtained by centrifugation for 30 min. at 6797 g (8000 rpm) at 4°C. Afterwards, the SCS was filter sterilized (0.20 pm cellulose acetate, VWR).

Antimicrobial activity assays for co-cultures of live lactobacilli against Malassezia furfur, Trichophyton rubrum, Propionibacterium acnes and Candida albican.

The antimicrobial activity of the selected bacteria was explored by standard antimicrobial tests with some minor modifications. The antimicrobial activity of the selected bacteria was explored by spot assay (Schillinger and LQcke 1989). B efly, 1 -3 μΙ_ of each culture was spotted on an agar plate. These plates were incubated for 24h up to 72h depending on the strain. Next, an overnight culture of the pathogen was diluted into 7 ml_ of soft agar of the medium of the pathogen and poured over the plates with the spots of the selected strains. The plates were incubated overnight at 30-37°C, after which the inhibition zones were measured. A spot of miconazole (for fungi) and/or 0.1 % hexetidine and/or tetracycline (for Propionibacterium acnes) was added to the spot plate as positive control before the soft agar was poured. Radial diffusion test of SCS of lactobacilli

In addition, the antimicrobial activity of spent culture supernatant (SCS) was investigated with a protocol as previously described for the competition assays between lactobacilli and gastrointestinal pathogens (Coconnier et al. 1997). Miconazol (for fungi) and tetracycline (for Propionibacterium acnes) was used as a positive control. Sterile growth medium was used as a negative control.

Time course analysis of the antimicrobial activity of SCS of the selected strains against Candida, Propionibacterium acnes, Malassezia furfur and Trichophyton spp (further referred to as 'pathogens').

The time course analysis was performed similarly as described previously (De Keersmaecker et al. 2006) with minor modifications. B efly, an overnight culture of the pathogen was added to the wells of a microplate filled with 50-80% the proper medium supplemented with 50-5% SCS of lactobacilli. MRS at pH 4,3 and antibiotics or antimycotics at the proper concentration were used as a negative and a positive control, respectively. Bacteria or fungi were grown and the optical density (OD) was measured at 590 nm each 30 min during 3 days using a Synergy HTX multi-mode reader (Biotek). Each test was measured at least in triplicate and the average OD was calculated. The antimicrobial activity was expressed as the relative optical density reached after 24h (stationary phase) compared to the negative controls. Antibiotic susceptibility

Antibiotic sensitivity was evaluated using the Kirby-Bauer disc diffusion test. In short, antibiotics were spotted on paper discs and the bacterial inhibition zone was measured on agar plates. The antibiotics tested were erythromycin, normocin, tetracyclin, ampicillin and clindamycin at relevant concentrations.

Proof-of-concept human clinical trial in patients with acne vulgaris

A proof-of-concept clinical trial was performed on 20 patients with acne vulgaris. Patients were men between 12-25 years with mild inflammatory acne. The aim of this proof-of-concept trial was to assess the impact of a topical probiotic cream (containing +-10-8 colony forming units (CFU) of L. pentosus YUN-V1 .0, +-10-8 CFU of L. plantarum YUN-V2.0 and +-10-8 CFU L. rhamnosus YUN-S1 .0 per application of 1 g of the topical cream ACN) on the skin microbiota and on the acne severity. Patients were asked to apply the cream twice daily for 56 days (8 weeks). The patients were seen by a dermatologist at start (before the therapy), week 4, week 8 and week 10. A skin swab was taken at each visit. Bacterial DNA was isolated from these samples by the commercial MoBio Powersoil kit (cfr. Human Microbiome Project). Isolated DNA was analysed via 16S rRNA amplicon sequencing with MiSeq lllumina and a bio-informatical analysis was performed. Moreover, a clinical scoring was performed and a photograph taken at each visit.

Proof-of-concept human clinical trial in patients with tinea pedis (Athlete's foot)

A proof-of-concept clinical trial was performed on 20 patients with tinea pedis. Patients were between 18-65 years having tinea pedis. The aim of this proof-of-concept trial was to assess the impact of a topical probiotic cream (containing +-10-8 colony forming units (CFU) of L. pentosus YUN-V1 .0, +-10-8 CFU of L. plantarum YUN-V2.0 and +-10-8 CFU L. rhamnosus YUN-S1 .0 per application of 1 g of of the topical cream FNG) on the skin microbiota and on the Trichophyton infection. Patients were asked to apply the cream twice daily for 56 days (8 weeks). The patients were seen by a dermatologist at start (before the therapy), week 4, week 8 and week 10. A skin swab was taken at each visit. Bacterial DNA was isolated from these samples by the commercial MoBio Powersoil kit (cfr. Human Microbiome Project). Isolated DNA was analysed via 16S rRNA amplicon sequencing with MiSeq lllumina and a bio-informatical analysis was performed. For analysis of the presence of the fungi, swabs were also plated out on Trichophyton specific medium (medium suggested by BCCM). Colony PCR using universal ITS ('internal transcribed region') primers ITS1 (SEQ ID N°6) (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (SEQ ID N° 7) (5'-TCCTCCGCTTATTGATATGC-3') followed by sequencing was performed to identify the fungi. Moreover, a clinical scoring was performed and a photograph taken at each visit. RESULTS

Growth characteristics and lactate production

Possible beneficial or probiotic strains were characterized in terms of growth characteristics, lactate production and ability of lowering of the pH of the medium. These characteristics are expected to be important for the antipathogenic activity. These data show that Lactobacillus pentosus YUN-V1 .0 and L. plantarum YUN-V2.0 and L. rhamnosus YUN-S1 .0 produce the highest amount of lactic acid (Fig 1 ). Antipathogenic activity against Propionibacterium acnes

Time course experiments were performed analyzing the antimicrobial activity of spent culture supernatant (SCS) of the selected strains against Propionibacterium acnes. SCS of all tested strains inhibited the growth of Propionibacterium acnes while SCS of other bacterial species like Streptococcus thermophilus and S. salivarius, both also lactic acid bacteria, and Staphyloccus epidermidis did not inhibit growth of P. acnes. This suggests species and perhaps strain specific properties of the selected lactobacilli to be important for the antipathogenic activity against P. acnes (Fig 2).

Antipathogenic activity against Malassezia, Trichophyton and Candida

In a next phase, the beneficial or probiotic bacteria were screened for their antipathogenic effect against specific skin pathogens. The results of a spot assays against Malassezia furfur, Trichophyton rubrum and Candida albicans are shown in table 1 , 2 and 3 respectively.

Table 1 : Spot assay of selected lactobacilli against Malassezia furfur.

*three independent repeats are shown Table 2: Spot assay of selected lactobacilli against Trichophyton rubrum.

* three independent repeats are shown Table 3: Radial diffusion assay of selected lactobacilli against Candida albicans.

* three independent repeats are shown

Spent culture supernatant from L. pentosus YUN-V1 .0 and L. plantarum YUN-V2.0 was also tested in radial diffusion assays and demonstrated to be efficient in inhibiting Malassezia, Trichophyton and Candida growth. L. rhamnosus YUN-S1 .0 was not as efficient in inhibiting growth of Malassizia but was able to inhibit growth of Trichophyton and Candida. Antibiotic susceptibility

The selected bacteria were also tested for their antibiotic susceptibility as to prevent spreading of antibiotic resistance genes. All lactobacilli were susceptible to erythromycin, normocin, tetracyclin, ampicillin and clindamycin, except for L. plantarum 5057, which was susceptible to tetracyclin. For this reason, strain L. plantarum 5057 was found not to be suitable to use as a strain for probiotherapy.

REFERENCES

Beylot, C. et al., 2014. Propionibacterium acnes: an update on its role in the pathogenesis of acne. Journal of the European Academy of Dermatology and Venereology : JEADV, 28(3), pp.271-8.

Chan, R.C. et al., 1985. Competitive exclusion of uropathogens from human uroepithelial cells by Lactobacillus whole cells and cell wall fragments. Infection and immunity, 47(1 ), pp.84- 9.

Chan, R.C, Bruce, A.W. & Reid, G., 1984. Adherence of cervical, vaginal and distal urethral normal microbial flora to human uroepithelial cells and the inhibition of adherence of gram- negative uropathogens by competitive exclusion. The Journal of urology, 131 (3), pp.596- 601 .

G ee, E.A. & Segre, J.A., 201 1 . The skin microbiome. Nature reviews. Microbiology, 9(4), pp.244-53.

Reid, G., 1999. The Scientific Basis for Probiotic Strains of Lactobacillus. Appl. Envir. Microbiol., 65(9), pp.3763-3766.

Reid, G. & Bruce, A.W., 2001 . Selection of lactobacillus strains for urogenital probiotic applications. The Journal of infectious diseases, 183 Suppl , pp.S77-80.

Sugita, T. et al., 2015. Temporal changes in the skin Malassezia microbiota of members of the Japanese Antarctic Research Expedition (JARE): A case study in Antarctica as a pseudo- space environment. Medical mycology, 53(7), pp.717-24.

Wang, L. et al., 2015. Characterization of the major bacterial-fungal populations colonizing dandruff scalps in Shanghai, China, shows microbial disequilibrium. Experimental dermatology, 24(5), pp.398^00.

SEQUENCE LISTING

<110> YUN NV

<120> DERMATOLOGICAL PREPARATIONS FOR MAINTAINING AND/OR RESTORING HEALTHY SKIN MICROBIOTA

<130> YUN-002

<150> BE2016/5454

<151> 2016-06-21

<170> BiSSAP 1.3.6

<210> 1

<211> 1406

<212> RNA

<213> Lactobacillus pentosus

<223> 16S rRNA sequence

<400> 1

cttaggcggc tggttcctaa aaggttaccc caccgacttt gggtgttaca aactctcatg 60 gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg ctgatccgcg 120 attactagcg attccgactt catgtaggcg agttgcagcc tacaatccga actgagaatg 180 gctttaagag attagcttac tctcgcgagt tcgcaactcg ttgtaccatc cattgtagca 240 cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac cttcctccgg 300 tttgtcaccg gcagtctcac cagagtgccc aacttaatgc tggcaactga taataagggt 360 tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac aaccatgcac 420 cacctgtatc catgtccccg aagggaacgt ctaatctctt agatttgcat agtatgtcaa 480 gacctggtaa ggttcttcgc gtagcttcga atgctccacc gcttgtgcgg 540 gcccccgtca attcctttga gtttcagcct tgcggccgta ctccccaggc ggaatgctta 600 atgcgttagc tgcagcactg aagggcggaa accctccaac acttagcatt catcgtttac 660 ggtatggact accagggtat ctaatcctgt ttgctaccca tactttcgag cctcagcgtc 720 agttacagac cagacagccg ccttcgccac tggtgttctt ccatatatct acgcatttca 780 ccgctacaca tggagttcca ctgtcctctt ctgcactcaa gtttcccagt ttccgatgca 840 cttcttcggt tgagccgaag gctttcacat cagacttaaa aaaccgcctg cgctcgcttt 900 acgcccaata aatccggaca acgcttgcca cctacgtatt accgcggctg ctggcacgta 960 gttagccgtg gctttctggt taaataccgt caatacctga acagttactc tcagatatgt 1020 tcttctttaa caacagagtt ttacgagccg aaacccttct tcactcacgc ggcgttgctc 1080 catcagactt tcgtccattg tggaagattc cctactgctg cctcccgtag gagtttgggc 1140 cgtgtctcag tcccaatgtg gccgattacc ctctcaggtc ggctacgtat cattgccatg 1200 gtgagccgtt accccaccat ctagctaata cgccgcggga ccatccagaa gtgatagccg 1260 aagccatctt tcaaactcgg accatgcggt ccaagttgtt atgcggtatt agcatctgtt 1320 tccaggtgtt atcccccgct tctgggcagg tttcccacgt gttactcacc agttcgccac 1380 tcactcaaat gtaaatcatg atgcaa 1406 <210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<223> Primer 8F

<400> 2

agagtttgat cctggctcag

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence

<223> Primer 1525R

<400> 3

aaggaggtga tccagccgca 20

<210> 4

<211> 1425

<212> RNA

<213> Lactobacillus plantarum

<223> 16S rRNA

<400> 4

ggttcctaaa aggttacccc accgactttg ggtgttacaa actctcatgg tgtgacgggc 60 ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga ttactagcga 120 ttccgacttc atgtaggcga gttgcagcct acaatccgaa ctgagaatgg ctttaagaga 180 ttagcttact ctcgcgagtt cgcaactcgt tgtaccatcc attgtagcac gtgtgtagcc 240 caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt ttgtcaccgg 300 cagtctcacc agagtgccca acttaatgct ggcaactgat aataagggtt gcgctcgttg 360 cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc acctgtatcc 420 atgtccccga agggaacgtc taatctctta gatttgcata gtatgtcaag acctggtaag 480 gttcttcgcg tagcttcgaa tt3-3-3.CC3.C3- tgctccaccg cttgtgcggg cccccgtcaa 540 ttcctttgag tttcagcctt gcggccgtac tccccaggcg gaatgcttaa tgcgttagct 600 gcagcactga agggcggaaa ccctccaaca cttagcattc atcgtttacg gtatggacta 660 ccagggtatc taatcctgtt tgctacccat actttcgagc ctcagcgtca gttacagacc 720 agacagccgc cttcgccact ggtgttcttc catatatcta cgcatttcac cgctacacat 780 ggagttccac tgtcctcttc tgcactcaag tttcccagtt tccgatgcac ttcttcggtt 840 gagccgaagg ctttcacatc agacttaaaa aaccgcctgc gctcgcttta cgcccaataa 900 atccggacaa cgcttgccac ctacgtatta ccgcggctgc tggcacgtag ttagccgtgg 960 ctttctggtt aaataccgtc aatacctgaa cagttactct cagatatgtt cttctttaac 1020 aacagagttt tacgagccga aacccttctt cactcacgcg gcgttgctcc atcagacttt 1080 cgtccattgt ggaagattcc ctactgctgc ctcccgtagg agtttgggcc gtgtctcagt 1140 cccaatgtgg ccgattaccc tctcaggtcg getaegtate attgecatgg tgagccgtta 1200 ccccaccatc tagctaatac gccgcgggac catccaaaag tgatagcega agecatcttt 1260 caagctcgga ccatgcggtc caagttgtta tgeggtatta gcatctgttt ccaggtgtta 1320 tcccccgctt ctgggcaggt ttcccacgtg ttactcacca gttcgccact cactcaaatg 1380 taaatcatga tgcaagcacc 3-3-tC3.3-t3.ee agagttcgtt cgact 1425

<210> 5

<211> 1403

<212> RNA

<213> Lactobacillus r amnosus

<223> 16S rRNA <400> 5

gtcgacgagt tctgattatt gaaaggtgct tgcatcttga tttaattttg aacgagtggc 60 ggacgggtga gtaacacgtg ggtaacctgc ccttaagtgg gggataacat ttggaaacag 120 atgetaatae egcataaate caagaaccgc atggttcttg gctgaaagat ggegtaaget 180 atcgcttttg gatggacccg cggcgtatta gctagttggt gaggtaacgg ctcaccaagg 240 caatgatacg tagecgaact gagaggttga tcggccacat tgggactgag acacggccca 300 aactcctacg ggaggcagca gtagggaatc ttccacaatg gaegcaagtc tgatggagca 360 acgccgcgtg agtgaagaag getttegggt egtaaaaetc tgttgttgga gaagaatggt 420 eggcagagta actgttgtcg gcgtgacggt atccaaccag aaagecaegg etaactaegt 480 gccagcagcc gcggtaatac gtaggtggca agcgttatcc ggatttattg ggcgtaaagc 540 gagcgcaggc ggttttttaa gtctgatgtg aaagccctcg gcttaaccga ggaagtgcat 600 eggaaactgg gaaacttgag tgcagaagag gacagtggaa ctccatgtgt agcggtgaaa 660 tgegtagata tatggaagaa caccagtggc gaaggegget gtctggtctg taactgaege 720 tgaggctcga aagcatgggt agegaacagg attagatacc ctggtagtcc atgccgtaaa 780 egatgaatge taggtgttgg agggtttccg cccttcagtg ccgcagctaa egcattaage 840 attccgcctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca 900 caagcggtgg agcatgtggt ttaattcgaa geaacgegaa gaaccttacc aggtcttgac 960 atcttttgat cacctgagag atcaggtttc cccttcgggg gcaaaatgac aggtggtgca 1020 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080 tatgactagt tgecagcatt tagttgggca ctctagtaag actgeeggtg acaaacegga 1140 ggaaggtggg gatgaegtea aatcatcatg ccccttatga cctgggctac acaegtgeta 1200 caatggatgg tacaacgagt tgcgagaccg cgaggtcaag ctaatctctt aaagecatte 1260 tcagttcgga ctgtaggctg caactcgcct acacgaagtc ggaategcta gtaatcgegg 1320 atcagcacgc cgcggtgaat acgttcccgg gecttgtaca caccgcccgt cacaccatga 1380 gagtttgtaa cacccgaagc egg 1403 <210> 6

<211> 19

<212> DNA

<213> Artificial Sequence <223> Primer Sequence

<400> 6

tccgtaggtg aacctgcgg

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence <223> Primer Sequence

<400> 7

tcctccgctt attgatatgc

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