The presented master thesis deals with the monitoring of the influence of polysaccharides which are used as an additive in the cosmetic products, on the monitored types of bacteria which are part of the skin microbiome. And it also deals with the study the effect of polysaccharides as prebiotics on selected species of bacteria that are part of the skin microbiome. Two polysaccharides were selected to determine the effects on the skin microbiome: Nanomoist and PoLevan S. The first part of the thesis focuses on the literature search which deals with skin anatomy, skin diseases and skin microbiome and its function. The experimental part is focused on monitoring changes in the quantity of selected microorganisms of the skin microbiome, before and after application of polysaccharides to the skin using qPCR. Staphylococcus epidermidis, Cutibacterium acnes, Escherichia coli and Micrococcus luteus were monitored. The PCR products were detected by agarose gel electrophoresis. The bacterium Staphylococcus epidermidis was detected on the skin to the greatest extent, especially after the application of the polysaccharides Nanomoist and PoLevan S. Thus, a positive effect of both polysaccharides on the growth of this bacterium was found.

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Hudens mikrobiom utgör ett skydd mot patogener och domineras framförallt av grampositiva bakterier som Staphylococcus, där S. epidermidis är den mest förekommande arten. På huden är S. epidermidis en kommensal art och har en aktiv roll i att begränsa tillväxten och koloniseringen av patogener som S. aureus. S. epidermidis är en viktig art i att bibehålla stabiliteten av hudens mikrobiom och en reduktion av S. epidermidis innebär ökade risker för dysbios. S. aureus är en vanlig orsak till hudinfektioner och har upptäckts finnas i en hög utsträckning på patienter med atopisk dermatit samt hos diabetiker med risk för att utveckla fotsår. Syftet med denna studie var att optimera qPCR och undersöka om metoden kunde användas för att uppskatta förhållandet mellan S. aureus och S. epidermidis, vilket skulle kunna identifiera patienter med risk för hudsjukdomar. Tre primerpar undersöktes. Två var specifika mot S. aureus och en var specifik mot S. epidermidis. Flera tester genomfördes för att optimera qPCR och specificiteten av primerparen. Den annealingtemperatur och primerkoncentration som gav mest adekvata resultat användes vid den relativa kvantifieringen. Den utvecklade metoden kunde uppskatta förhållandet mellan S. aureus och S. epidermidis vid de lägre koncentrationerna (80–280 kopior/μl) bättre än vid de högre koncentrationerna (80 000–280 000 kopior/μl), men vidare optimering av metoden behövs.

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O objetivo deste estudo foi avaliar por métodos microbiológicos e microscópia eletrônica de varredura a aderência bacteriana sobre superfície lisa e rugosa (modificada por irradiação a laser) de titânio comercialmente puro (\'Ti\' cp). Os corpos-de-prova eram em forma de discos (12,0 mm x 2,0 mm). Streptococcus mutans ATCC 25175 e Staphylococcus epidermidis ATCC 12228 RP 62A foram às bactérias selecionadas. A topografia das superfícies foram avaliadas por meio de microscópio eletrônico de varredura (MEV). As medidas do ângulo de contato permitiram conhecer a molhabilidade (hidrofobicidade) das superfícies. Os discos de \'Ti\' cp foram incubados em meio de cultura caldo Mueller Hinton inoculado com suspensão bacteriana da ordem de \'10 POT.8\' (UFC)/mL, durante 1, 6, 24, 48 e 72 horas. A cada intervalo de tempo, os discos foram retirados, lavados em solução salina fisiológica e após este procedimento foram colocados em novos tubos contendo 5,0 mL de solução salina fisiológica esterilizada e submetidos ao banho ultrassônico de 40 kHz por oito minutos. A seguir, da suspensão bacteriana resultante foram realizadas diluições seriadas (\'10 POT.-1\'-\'10 POT.-6\'), semeadas em ágar Mueller Hinton e as placas incubadas em estufa bacteriológica a 37 graus Celsius para aguardar o desenvolvimento bacteriano. As colônias crescidas foram contadas, enumeradas e o valor expresso em UFC/mL para cada diluição. Os discos removidos do banho ultrassônico foram preparados para observação por MEV. A medida do ângulo de contato foi realizada em equipamento denominado goniômetro. O resultado da avaliação da viabilidade das células de biofilme de S. mutans sobre superfície lisa em média foi de 1,66 \'+ OU -\' 1,67 x \'10 POT.6\' e sobre superfície rugosa 1,06 \'+ OU -\' 1,07 x \'10 POT.6\' a nível 0,05 as médias não são significantemente diferentes. Em média as células viáveis do biofilme de S. epidermidis sobre superfície lisa foram de 6,68 \'+ OU -\' ) 5,83 x \'10 POT.6\' a nível 0,05 as médias não são significativamente diferentes. As células viáveis do biofilme de S. epidermidis, recuperadas em vários intervalos de tempo (1, 6, 24, 48 e 72h) da superfície rugosa 7,16 \'+ OU -\' 2,34 x \'10 POT.6\' a nível 0,05 são significantemente diferentes. Em relação à molhabilidade a superfície lisa é hidrofóbica, com um ângulo de 75 graus e a superfície rugosa hidrofílica, com um ângulo < 7 graus. Apesar da superfície lisa ser hidrofóbica e a superfície rugosa hidrofílica ambas as superfícies permitiram a aderência bacteriana e formação de biofilme, fato comprovado por MEV e por cultura. Comparando-se as superfícies lisa e rugosa - modificada por meio físico (aplicação de feixe de laser de alta intensidade \'Nd\':YAG) não foi observada redução significante no número de bactérias aderidas à superfície rugosa, o que permite concluir que a modificação de superfície por laser de alta intensidade cria superfície favorável para aderência de S. mutans e S. epidermidis, sem reduzir a aderência bacteriana, o que pode ser fator de risco para adquirir infecção. / The objective of this study was assessed by microbiological methods and the scanning electron microscope on bacterial adhesion to smooth and rough (modified by laser irradiation) of commercially pure titanium (\'Ti\' cp). The bodies-of-proof was in the form of discs (12,0 mm x 2,0 mm). Streptococcus mutans ATCC 25175 and Staphylococcus epidermidis ATCC 12228 RP62A were selected bacteria. The topography of the areas were evaluated by scanning electron microscope (SEM). Measures the angle of contact allowed to know the wettability (hydrophobicity) of the areas. Of \'Ti\' cp discs were incubated in culture media Mueller Hinton broth inoculated with bacterial suspension of approximately \'10 POT.8\' (CFU)/mL, for 1, 6, 24, 48 and 72 hours. Each time, the discs were removed, washed in saline solution and after this procedure were placed into new tubes containing 5.0 mL of sterile saline solution and submitted to the ultrasonic bath for eight minutes of 40 kHz. Then, the resulting bacterial suspension were serially diluted (\'10 POT.-1\'-\'10 POT.-6\'), grown in Mueller Hinton agar plates and incubated in bacteriological incubator at 37 Celsius degrees to await the development blight. The grown colonies were counted, listed and the value expressed in CFU/mL for each dilution. Discs removed from the ultrasonic bath were prepared for observation by SEM. The measure of the angle of contact was made in equipment called goniometer. The result of evaluating the viability biofilm cells of S. mutans on smooth surface was on average of 1,66 \'+ OU -\' 1,67 x \'10 POT.6\' at the 0,05 and on area rugosa 1,06 \'+ OU -\' 1,07 x \'10 POT.6\' at the 0,05 average is not significantly different. On average the cells of the biofilm of S. epidermidis on smooth 6,68 \'+ OU -\' 5,83 x \'10 POT.6\' at the 0,05 average is not significantly surface were different. The cells of the biofilm of S. epidermidis, recovered at various at intervals of time (1, 6, 24, 48 and 72h) of the area rugosa 7,16 \'+ OU -\' 2,34 x \'10 POT.6\' 0,05 are significantly different. In relation to the smooth surface wettability is hydrophobic, with an angle of 75 degrees and the hydrophilic surface rough, with an angle < 7 degrees. Despite the smooth surface is rough hydrophobic and hydrophilic surface areas have both the adhesion and formation of bacterial biofilm, a fact evidenced by SEM and by culture. Comparing the smooth and rough surfaces - modified by the physical environment (application of the laser beam of high intensity \'Nd\': YAG) was not observed a significant reduction in the number of bacteria adhered to the surface rough, which indicates that the modification of surface by high-intensity laser creates favorable surface for adhesion of S. mutans and S. epidermidis, without reducing the bacterial adhesion, which may be a risk factor for acquiring infection.

O objetivo deste estudo foi avaliar por métodos microbiológicos e microscópia eletrônica de varredura a aderência bacteriana sobre superfície lisa e rugosa (modificada por irradiação a laser) de titânio comercialmente puro (\'Ti\' cp). Os corpos-de-prova eram em forma de discos (12,0 mm x 2,0 mm). Streptococcus mutans ATCC 25175 e Staphylococcus epidermidis ATCC 12228 RP 62A foram às bactérias selecionadas. A topografia das superfícies foram avaliadas por meio de microscópio eletrônico de varredura (MEV). As medidas do ângulo de contato permitiram conhecer a molhabilidade (hidrofobicidade) das superfícies. Os discos de \'Ti\' cp foram incubados em meio de cultura caldo Mueller Hinton inoculado com suspensão bacteriana da ordem de \'10 POT.8\' (UFC)/mL, durante 1, 6, 24, 48 e 72 horas. A cada intervalo de tempo, os discos foram retirados, lavados em solução salina fisiológica e após este procedimento foram colocados em novos tubos contendo 5,0 mL de solução salina fisiológica esterilizada e submetidos ao banho ultrassônico de 40 kHz por oito minutos. A seguir, da suspensão bacteriana resultante foram realizadas diluições seriadas (\'10 POT.-1\'-\'10 POT.-6\'), semeadas em ágar Mueller Hinton e as placas incubadas em estufa bacteriológica a 37 graus Celsius para aguardar o desenvolvimento bacteriano. As colônias crescidas foram contadas, enumeradas e o valor expresso em UFC/mL para cada diluição. Os discos removidos do banho ultrassônico foram preparados para observação por MEV. A medida do ângulo de contato foi realizada em equipamento denominado goniômetro. O resultado da avaliação da viabilidade das células de biofilme de S. mutans sobre superfície lisa em média foi de 1,66 \'+ OU -\' 1,67 x \'10 POT.6\' e sobre superfície rugosa 1,06 \'+ OU -\' 1,07 x \'10 POT.6\' a nível 0,05 as médias não são significantemente diferentes. Em média as células viáveis do biofilme de S. epidermidis sobre superfície lisa foram de 6,68 \'+ OU -\' ) 5,83 x \'10 POT.6\' a nível 0,05 as médias não são significativamente diferentes. As células viáveis do biofilme de S. epidermidis, recuperadas em vários intervalos de tempo (1, 6, 24, 48 e 72h) da superfície rugosa 7,16 \'+ OU -\' 2,34 x \'10 POT.6\' a nível 0,05 são significantemente diferentes. Em relação à molhabilidade a superfície lisa é hidrofóbica, com um ângulo de 75 graus e a superfície rugosa hidrofílica, com um ângulo < 7 graus. Apesar da superfície lisa ser hidrofóbica e a superfície rugosa hidrofílica ambas as superfícies permitiram a aderência bacteriana e formação de biofilme, fato comprovado por MEV e por cultura. Comparando-se as superfícies lisa e rugosa - modificada por meio físico (aplicação de feixe de laser de alta intensidade \'Nd\':YAG) não foi observada redução significante no número de bactérias aderidas à superfície rugosa, o que permite concluir que a modificação de superfície por laser de alta intensidade cria superfície favorável para aderência de S. mutans e S. epidermidis, sem reduzir a aderência bacteriana, o que pode ser fator de risco para adquirir infecção. / The objective of this study was assessed by microbiological methods and the scanning electron microscope on bacterial adhesion to smooth and rough (modified by laser irradiation) of commercially pure titanium (\'Ti\' cp). The bodies-of-proof was in the form of discs (12,0 mm x 2,0 mm). Streptococcus mutans ATCC 25175 and Staphylococcus epidermidis ATCC 12228 RP62A were selected bacteria. The topography of the areas were evaluated by scanning electron microscope (SEM). Measures the angle of contact allowed to know the wettability (hydrophobicity) of the areas. Of \'Ti\' cp discs were incubated in culture media Mueller Hinton broth inoculated with bacterial suspension of approximately \'10 POT.8\' (CFU)/mL, for 1, 6, 24, 48 and 72 hours. Each time, the discs were removed, washed in saline solution and after this procedure were placed into new tubes containing 5.0 mL of sterile saline solution and submitted to the ultrasonic bath for eight minutes of 40 kHz. Then, the resulting bacterial suspension were serially diluted (\'10 POT.-1\'-\'10 POT.-6\'), grown in Mueller Hinton agar plates and incubated in bacteriological incubator at 37 Celsius degrees to await the development blight. The grown colonies were counted, listed and the value expressed in CFU/mL for each dilution. Discs removed from the ultrasonic bath were prepared for observation by SEM. The measure of the angle of contact was made in equipment called goniometer. The result of evaluating the viability biofilm cells of S. mutans on smooth surface was on average of 1,66 \'+ OU -\' 1,67 x \'10 POT.6\' at the 0,05 and on area rugosa 1,06 \'+ OU -\' 1,07 x \'10 POT.6\' at the 0,05 average is not significantly different. On average the cells of the biofilm of S. epidermidis on smooth 6,68 \'+ OU -\' 5,83 x \'10 POT.6\' at the 0,05 average is not significantly surface were different. The cells of the biofilm of S. epidermidis, recovered at various at intervals of time (1, 6, 24, 48 and 72h) of the area rugosa 7,16 \'+ OU -\' 2,34 x \'10 POT.6\' 0,05 are significantly different. In relation to the smooth surface wettability is hydrophobic, with an angle of 75 degrees and the hydrophilic surface rough, with an angle < 7 degrees. Despite the smooth surface is rough hydrophobic and hydrophilic surface areas have both the adhesion and formation of bacterial biofilm, a fact evidenced by SEM and by culture. Comparing the smooth and rough surfaces - modified by the physical environment (application of the laser beam of high intensity \'Nd\': YAG) was not observed a significant reduction in the number of bacteria adhered to the surface rough, which indicates that the modification of surface by high-intensity laser creates favorable surface for adhesion of S. mutans and S. epidermidis, without reducing the bacterial adhesion, which may be a risk factor for acquiring infection.

In the past 15 years, many efforts were made to create functionalized artificial surfaces showing special anti-bacterial and anti-biofouling properties. Thereby, the topography of medical relevant materials plays an important role. However, the targeted fabrication of promising surface structures like hole-, lamella- and pyramid-like patterns with feature sizes in the sub-micrometer range in a one-step process is still a challenge. Optical and e-beam lithography, molding and selfassembly layers show a great potential to design topographies for this purpose. At the same time, most of these techniques are based on sequential processes, require masks or molds and thus are very device relevant and time consuming. In this work, we present the Direct Laser Interference Patterning (DLIP) technology as a capable method for the fast, flexible and direct fabrication of periodic micrometer- and submicrometer structures. This method offers the possibility to equip large plain areas and curved devices with 1D, 2D and 3D patterns. Simple 1D (e.g. lines) and complex 3D (e.g. lamella, pillars) patterns with periodic distances from 0.5 μm to 5 μm were fabricated on polymeric materials (polyimide, polystyrene). Subsequently, we characterized the adhesion behavior of Staphylococcus epidermidis and S. aureus bacteria under in vitro and in vivo conditions. The results revealed that the topographies have a significant impact on bacteria adhesion. On the one side, one-dimensional line-like structures especially with dimensions of the bacteria enhanced microbe attachment. While on the other hand, complex three-dimensional patterns prevented biofilm formation even after implantation and contamination in living organisms.

Les aciers inoxydables sont des matériaux couramment utilisés dans les industries agroalimentaires, la restauration collective mais également dans le milieu médical et hospitalier. Dans ces domaines, les phénomènes de bioadhésion de microorganismes sur ces substrats sont fréquents et peuvent entraîner des problèmes de santé publique tels que des infections toxicologiques ou des contaminations chez des sujets porteurs de matériau étranger (cathéter, prothèse).<br />Dans ce travail, effectué en partenariat avec le groupe sidérugique Ugine-Alz, nous nous sommes intéressés, à l'aide d'une chambre à flux radial, à l'étude quantitative des cinétiques de détachement de trois microorganismes après adhésion sur des surfaces d'acier inoxydable. Saccharomyces cerevisiae, une levure, est représentative des eucaryotes, Escherichia coli est une bactérie Gram- et Staphylococcus epidermidis, une bactérie Gram+. Les états de surface des aciers inoxydables utilisés sont des échantillons polis miroir (PM) et polis attaqués (PA) avec des tailles de grain différentes (20, 40 et 100 µm) et différentes profondeurs d'attaque des joints de grain (100 ± 50 nm, 150 ± 50 nm, 650 ± 150 nm). L'utilisation de ces différents états de surface ne modifie pas la valeur de la contrainte nécessaire pour détacher 50 % des microorganismes (seuil de détachement). Par contre, la profondeur d'attaque des joints de grain et, dans une moindre mesure, la taille de grain (20, 40 et 100 µm) accélèrent la cinétique de détachement de S. cerevisiae, grosse cellule non déformable et ralentissent celle d'E. coli, petite cellule déformable. Enfin, une adhésion préférentielle a été mise en évidence, pour les trois types de cellules, sur les grains d'orientation cristallographiques <001>.<br />Les observtions expérimentales ont par ailleurs été rationalisées à l'aide du modèle de B. Fourcade, D. Garrivier et E. Décavé (Décavé et al., 2002) et par le biais d'une analyse qualitative des interactions cellule-substrat en fonction de la taille et de la déformabilité des cellules.