Effects of marine exopolysaccharides on bacterial adhesion to human skin cells and on biofilm production, applications for cosmetics
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Presented by: Pierre Yves Morvan
In natural conditions, some marine bacteria have the ability to produce biofilms composed by exopolysaccharides (EPS). These bacterial structures provide self-protection against other bacteria and changes of environmental conditions. In addition, they ensure constant humidity around the cells but their role is also to attach themselves to natural supports (rock, wood, algae, …) facilitating adhesion and development. These complex structures are very variable. Some EPS contain simple sugars (glucose, galactose, mannose, rhamnose) but they are also composed by acid groups (glucuronic acid, galacturonic acid), sulfate or acetate groups (N-acetylglucosamine, N-acetylglucuronic acid). Presence of amino acids (alanine, serine, threonine) covalently linked to carbohydrates is also described. The particular shared chemical structures explain recognition, bio-affinity, penetration and efficacies of EPS. Literature related already biological effects on epidermal renewal or inflammatory responses and more physical benefits on skin surface such as tensing or mattifying agents. To mimic bacteria and its support crosstalk due to EPS, we focused on microbiome. We wondered if and how marine EPS could influence the skin microbiota comportment in the skin surface.
In a first step, we measured the adhesion capabilities of different common bacteria in presence of EPS. (1) Staphylococcus epidermidis (S.epid., ref ATCC: 12228) is a commensal bacterium, the more common of the skin surface. (2) In another hand Staphylococcus aureus (S.aureus, ref ATCC: 6538) is a pathogenic bacterium responsible for numerous skin affections. (3) Cutibacterium acnes (C.acnes, ref ATCC: 11827), is a well-known anaerobic bacterium, implicated in acne lesions and acidification of skin surface due to its metabolic release of propionic acid. Even if important inter-individual variations exist, the three bacteria and their ratio are considered as major indicators for healthy skin surface. To quantify bacteria adhesion, the strains, previously labelled with carboxyfluorescein diacetate N-succinimidyl ester (CFSE), were applied on corneocytes (collected with adhesive disposal) during two hours, in presence of different concentrations of different EPS (5 EPS provided by CODIF International). After incubation then washes, residual fluorescence (i.e. bacteria adhesion) were counted and calculated versus the control condition (%). For another previous control, the tested EPS presented no effect on bacterial growth inhibition. Profiles showed very different results from one EPS to another. For example, some of them (EPS1 and EPS3) inhibited S.aureus adhesion in the same manner but EPS1 decreased C.acnes adhesion (EPS3 remained without any effect on C.acnes), while EPS3 inhibited S.epid adhesion (EPS1 presented no effect on S.epid). Another one (EPS5) inhibited the adhesion of all the strains in a dose dependant manner.
To better understand potential interactions between bacteria on the skin surface (corneocytes) we realised glycoprofiling. Bacteria are known to interact with their support towards lectins or glycan binding proteins (GBP), as membranes of human cells wear these molecules. Lectins and GBP are glycoproteins enabled to present specific (and releasable) affinity with glycans or oligosides. They are implicated in cell-cell recognition / communication for different biological process. We performed glycoprofiling of each EPS according to an indirect method that demonstrated the inhibition of lectin/glycans interaction, developed by GLYcoDiag. Thus, thanks to these complementary informations about EPS composition and interactions, we imagined competition in lectin/GBP recognition on the skin surface and/or on the bacteria surface. This competition could explain inhibition of bacteria adhesion.
Additional experiments were made on C.acnes biofilm formation. As for marine bacteria and numerous bacteria, bacteria of the skin surface are able to create specific biofilm, that correspond to a normal step of development with the production of an adhesive and protective matrix. We used the BioFilm Ring Test® technology developed by BiofilmControl. It is based on superparamagnetical microbeads mobility measurement under magnetic field. The biofilm influences the mobility and decreases the capacity of beads movements. The presence of EPS modified the signal. Consequently it modified the production of C.acnes biofilm.
Results of our study show that marine EPS are not only physical fil-formers. They also play an important role in glycobiological interaction processes. They are recognised by some specific receptors for carbohydrates. Thus, they can interfere with human corneocytes and lock competitively bacterial survey (adhesion on skin surface and biofilm production). That are really interesting for cosmetics: for skin that presents unbalanced microbiota (dysbiosis), to maintain healthy conditions or to prevent excessive pathogenic strains invasion.
In a first step, we measured the adhesion capabilities of different common bacteria in presence of EPS. (1) Staphylococcus epidermidis (S.epid., ref ATCC: 12228) is a commensal bacterium, the more common of the skin surface. (2) In another hand Staphylococcus aureus (S.aureus, ref ATCC: 6538) is a pathogenic bacterium responsible for numerous skin affections. (3) Cutibacterium acnes (C.acnes, ref ATCC: 11827), is a well-known anaerobic bacterium, implicated in acne lesions and acidification of skin surface due to its metabolic release of propionic acid. Even if important inter-individual variations exist, the three bacteria and their ratio are considered as major indicators for healthy skin surface. To quantify bacteria adhesion, the strains, previously labelled with carboxyfluorescein diacetate N-succinimidyl ester (CFSE), were applied on corneocytes (collected with adhesive disposal) during two hours, in presence of different concentrations of different EPS (5 EPS provided by CODIF International). After incubation then washes, residual fluorescence (i.e. bacteria adhesion) were counted and calculated versus the control condition (%). For another previous control, the tested EPS presented no effect on bacterial growth inhibition. Profiles showed very different results from one EPS to another. For example, some of them (EPS1 and EPS3) inhibited S.aureus adhesion in the same manner but EPS1 decreased C.acnes adhesion (EPS3 remained without any effect on C.acnes), while EPS3 inhibited S.epid adhesion (EPS1 presented no effect on S.epid). Another one (EPS5) inhibited the adhesion of all the strains in a dose dependant manner.
To better understand potential interactions between bacteria on the skin surface (corneocytes) we realised glycoprofiling. Bacteria are known to interact with their support towards lectins or glycan binding proteins (GBP), as membranes of human cells wear these molecules. Lectins and GBP are glycoproteins enabled to present specific (and releasable) affinity with glycans or oligosides. They are implicated in cell-cell recognition / communication for different biological process. We performed glycoprofiling of each EPS according to an indirect method that demonstrated the inhibition of lectin/glycans interaction, developed by GLYcoDiag. Thus, thanks to these complementary informations about EPS composition and interactions, we imagined competition in lectin/GBP recognition on the skin surface and/or on the bacteria surface. This competition could explain inhibition of bacteria adhesion.
Additional experiments were made on C.acnes biofilm formation. As for marine bacteria and numerous bacteria, bacteria of the skin surface are able to create specific biofilm, that correspond to a normal step of development with the production of an adhesive and protective matrix. We used the BioFilm Ring Test® technology developed by BiofilmControl. It is based on superparamagnetical microbeads mobility measurement under magnetic field. The biofilm influences the mobility and decreases the capacity of beads movements. The presence of EPS modified the signal. Consequently it modified the production of C.acnes biofilm.
Results of our study show that marine EPS are not only physical fil-formers. They also play an important role in glycobiological interaction processes. They are recognised by some specific receptors for carbohydrates. Thus, they can interfere with human corneocytes and lock competitively bacterial survey (adhesion on skin surface and biofilm production). That are really interesting for cosmetics: for skin that presents unbalanced microbiota (dysbiosis), to maintain healthy conditions or to prevent excessive pathogenic strains invasion.