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.

L'adhésion bactérienne est la première étape du processus de formation d'un biofilm et est un enjeu majeur de la recherche depuis plusieurs dizaines d'années. Les biofilms ont des conséquences parfois dramatiques dans des domaines comme la santé, l'agroalimentaire ou la purification des eaux usées. Toutefois, l'adhésion bactérienne reste un phénomène mal compris. Dans cette thèse, l'adhésion bactérienne est étudiée sur des surfaces modèles très bien organisées et structurées, de chimie de surface variable à l'échelle moléculaire. Une méthodologie de caractérisation adaptée aux monocouches déposées sur wafers de silicium est proposée. Des surfaces modèles composées de monocouches mixtes auto-assemblées de densités variables de NH2 dans un continuum de CH, sont développées et optimisées. Ces surfaces contrôlées, de densités de 0% NH2 à 100% NH2 dans CH3, sont utilisées comme outil pour étudier l'adhésion bactérienne en conditions de culture « batch »et « temps réel ». Les résultats montrent un impact significatif sur l'adhésion bactérienne de faibles différences chimiques à l'échelle moléculaire. Les résultats des expériences menées en conditions « batch » permettent de déterminer deux zones « plateau » dans lesquelles l'adhésion bactérienne ne varie pas significativement malgré des variations importantes de la concentration en groupements amine sur la surface. Une zone de transition entre les zones « plateau » est mise en évidence, dans laquelle une faible modification de la concentration en groupement amine mène à l'augmentation / diminution significative du nombre de bactéries adhérées. Cette tendance est montrée pour deux souches différentes de bactérie. / Bacterial adhesion is the first step of biofilm formation and in the focus of research interest since several decades. Biofilms cause many problems, sometimes dramatic, for example in health, food packing or waste water purification. Despite of high interest, bacterial adhesion process is only poorly understood yet. In this work, bacterial adhesion was investigated on well-organized and structured model surfaces with various chemistries at molecular scale. For that purpose a characterization methodology was developed to sufficiently analyze monolayers on silicon wafers, and controlled mixed monolayers surfaces with different densities of NH 2 backfilled with CH3 were developed and optimized. These controlled surfaces with different densities of 0 % NH2 up to 100% NH2 were eventually used as tool to study bacterial adhesion in batch and real time conditions. The results demonstrate a significant impact on bacterial adhesion of weak difference in the surface chemistry at molecular scale. In the batch experiments, two so-called "plateaus" zones were determined, in which bacterial adhesion is not significantly different despite the change of the amine concentration on the surface. On the contrary, one transition zone exists between the "plateaus" in which a slight chunge.in the amine concentration leads to a significant increase / decrease of the bacterial adhesion. The same trend of bacteria behavior was observed for different bacterial strains.

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 phénomènes de bioadhésion de microorganismes sur les aciers inoxydables son fréquents et peuvent entraîner des problèmes de santé publique tels que des infections toxicologiques. En partenariat avec Ugine-Alz, nous nous sommes intéressés, à l'aide d'une chambre à flux radial (Décavé et al. Biophysical Journal, 2002, 82, 2383-95), à l'étude quantitative des cinétiques de détachement de trois microorganismes après adhésion sur des surfaces d'acier inoxydable. Saccharomyces cerevisiae (levure), Escherichia coli (Gram-), et Staphylococcus epidermidis (Gram+). Les aciers inoxydables utilisés sont des échantillons polis miroir et polis attaqués avec des tailles de grain et des profondeurs d'attaque des joints de grain différentes. 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 la taille de grain accélèrent la cinétique de détachement de S. cerevisiae et ralentissent celle d'E. coli. Enfin, une adhésion préférentielle a été mise en évidence, pour les trois microorganismes, sur les grains d'orientation cristallographique <001> (Demilly et al., Colloids and Surfaces B, in press, 2006).

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.

Coagulase negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet concentrates (PCs), and have been implicated in severe and fatal transfusion reactions. Of this group, Staphylococcus epidermidis is most frequently identified. The preliminary objective of this thesis was to confirm that S. epidermidis could form biofilms under platelet storage conditions. This was achieved using a modified crystal violet staining assay to detect plastic-adherent bacterial cells and examination of attachment processes by scanning electron microscopy. A collection of CoNS isolated from PCs obtained from reportedly healthy donors was then assessed for biofilm-forming potential at the genetic and phenotypic level. Despite the presumable commensal origin of these isolates, a high proportion of S. epidermidis strains displayed a biofilm positive phenotype. The threat of S. epidermidis biofilm formation during platelet storage identified herein signifies that any alterations made to platelet storage protocols should be evaluated with consideration of this risk. The advent of platelet additive solutions (PASs) as an alternative to plasma for PC storage provides a relevant example, since little is known about the effect of PAS on contaminant bacteria, and vice versa. Growth and biofilm formation by S. epidermidis and the Gram-negative bacterium Serratia liquefaciens were measured in PAS- or plasma-PCs over 5 days, simulating standard platelet storage conditions, after initial inoculation with low, clinically relevant bacterial concentrations. Assays for platelet quality were performed simultaneously. Only S. liquefaciens exhibited a slower doubling time in plasma-PCs than in PAS-PCs. Biofilm formation by both species was reduced during storage in PAS-PCs, increasing bacteria availability for detection. Although S. liquefaciens adversely affected platelet quality in both media, S. epidermidis contamination did not. Ultimately, culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs. Lastly, since formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, it was postulated that biofilm formation by contaminant bacteria could be abrogated by receptor shielding. Methoxypoly(ethylene glycol) was applied to covalently modify the platelet surface using a process termed ‘PEGylation’. It is herein demonstrated that PEGylation of PCs inoculated with S. epidermidis results in significantly reduced bacterial binding and biofilm formation during platelet storage.

Coagulase negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet concentrates (PCs), and have been implicated in severe and fatal transfusion reactions. Of this group, Staphylococcus epidermidis is most frequently identified. The preliminary objective of this thesis was to confirm that S. epidermidis could form biofilms under platelet storage conditions. This was achieved using a modified crystal violet staining assay to detect plastic-adherent bacterial cells and examination of attachment processes by scanning electron microscopy. A collection of CoNS isolated from PCs obtained from reportedly healthy donors was then assessed for biofilm-forming potential at the genetic and phenotypic level. Despite the presumable commensal origin of these isolates, a high proportion of S. epidermidis strains displayed a biofilm positive phenotype. The threat of S. epidermidis biofilm formation during platelet storage identified herein signifies that any alterations made to platelet storage protocols should be evaluated with consideration of this risk. The advent of platelet additive solutions (PASs) as an alternative to plasma for PC storage provides a relevant example, since little is known about the effect of PAS on contaminant bacteria, and vice versa. Growth and biofilm formation by S. epidermidis and the Gram-negative bacterium Serratia liquefaciens were measured in PAS- or plasma-PCs over 5 days, simulating standard platelet storage conditions, after initial inoculation with low, clinically relevant bacterial concentrations. Assays for platelet quality were performed simultaneously. Only S. liquefaciens exhibited a slower doubling time in plasma-PCs than in PAS-PCs. Biofilm formation by both species was reduced during storage in PAS-PCs, increasing bacteria availability for detection. Although S. liquefaciens adversely affected platelet quality in both media, S. epidermidis contamination did not. Ultimately, culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs. Lastly, since formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, it was postulated that biofilm formation by contaminant bacteria could be abrogated by receptor shielding. Methoxypoly(ethylene glycol) was applied to covalently modify the platelet surface using a process termed ‘PEGylation’. It is herein demonstrated that PEGylation of PCs inoculated with S. epidermidis results in significantly reduced bacterial binding and biofilm formation during platelet storage.

Coagulase negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet concentrates (PCs), and have been implicated in severe and fatal transfusion reactions. Of this group, Staphylococcus epidermidis is most frequently identified. The preliminary objective of this thesis was to confirm that S. epidermidis could form biofilms under platelet storage conditions. This was achieved using a modified crystal violet staining assay to detect plastic-adherent bacterial cells and examination of attachment processes by scanning electron microscopy. A collection of CoNS isolated from PCs obtained from reportedly healthy donors was then assessed for biofilm-forming potential at the genetic and phenotypic level. Despite the presumable commensal origin of these isolates, a high proportion of S. epidermidis strains displayed a biofilm positive phenotype. The threat of S. epidermidis biofilm formation during platelet storage identified herein signifies that any alterations made to platelet storage protocols should be evaluated with consideration of this risk. The advent of platelet additive solutions (PASs) as an alternative to plasma for PC storage provides a relevant example, since little is known about the effect of PAS on contaminant bacteria, and vice versa. Growth and biofilm formation by S. epidermidis and the Gram-negative bacterium Serratia liquefaciens were measured in PAS- or plasma-PCs over 5 days, simulating standard platelet storage conditions, after initial inoculation with low, clinically relevant bacterial concentrations. Assays for platelet quality were performed simultaneously. Only S. liquefaciens exhibited a slower doubling time in plasma-PCs than in PAS-PCs. Biofilm formation by both species was reduced during storage in PAS-PCs, increasing bacteria availability for detection. Although S. liquefaciens adversely affected platelet quality in both media, S. epidermidis contamination did not. Ultimately, culture-based detection remains the earliest indicator of bacterial presence in PAS-PCs. Lastly, since formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, it was postulated that biofilm formation by contaminant bacteria could be abrogated by receptor shielding. Methoxypoly(ethylene glycol) was applied to covalently modify the platelet surface using a process termed ‘PEGylation’. It is herein demonstrated that PEGylation of PCs inoculated with S. epidermidis results in significantly reduced bacterial binding and biofilm formation during platelet storage.

Orientador: Maria Luiza Moretti / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-05T06:00:21Z (GMT). No. of bitstreams: 1 Menezes_DulcineaBlum_D.pdf: 1620753 bytes, checksum: 0d62ea2ae59bfb2f69d0243a4b9e4be5 (MD5) Previous issue date: 2005 / Resumo: A inserção de cateter venoso central (CVC) representa um importante risco para as infecções sistêmicas nosocomiais, e para estas infecções, Staphylococcus epidermidis é o patógeno mais importante. Com o objetivo de analisar os perfis de DNA genômico, detectar a presença e expressão de gene responsável pela produção de biofilme e estudar a dinâmica da colonização, cepas de S. epidermidis obtidas de episódios de isolamento deste microrganismo em culturas microbiológicas de ponta de CVC e/ou hemoculturas foram comparadas com cepas coletadas da microbiota do paciente hospitalizado no Hospital das Clínicas da UNICAMP. Este estudo também objetivou analisar os procedimentos médico-hospitalares intervencionais destes pacientes. Pacientes com culturas microbiológicas de ponta de CVC (>15 UFC) e/ou hemoculturas positivas para S. epidermidis foram selecionados para a coleta de microbiota presente na pele e mucosa nasal, através de coleta local com zaragatoas umedecidas. As cepas de S. epidermidis foram analisadas através do método de PFGE; teste de sensibilidade a antimicrobianos; detecção da presença do gene ica, através da técnica de PCR e detecção de biofilme, através do método CRA. Fizeram parte deste estudo 247 cepas obtidas de 12 pacientes selecionados em 18 episódios estudados. Foram encontrados 26 distintos perfis genotípicos e 4 perfis fortemente relacionados. Em 10 episódios o mesmo perfil genotípico de DNA foi detectado simultaneamente em cepas clínicas e de microbiota, onde 6 destes episódios ocorreram quando o período de implantação da CVC foi superior a 15 dias. Nos 7 episódios em que não houve concordância entre os perfis genotípico de DNA em cepas clínicas e de microbiota, 5 destes episódios ocorreram igualmente em período inferior a 15 dias, não havendo diferença estatística entre os grupos. Por PFGE foram identificados 6 perfis genotípicos predominantes nas cepas de microbiota. Estes perfis representaram 68% (132/193) das cepas de microbiota, e um destes perfis se mostrou prevalente (77/193) nas cepas de microbiota. Em 10 episódios (8 pacientes), o perfil genotípico prevalente foi identificado compondo a microbiota. Foi comprovado, por comparação da diversidade dos perfis genotípicos, que durante o período de hospitalização o perfil geral da microbiota sofre mudanças de um perfil de diversidade genotípica policlonal para um perfil de diversidade oligoclonal, com predominância de um perfil genotípico. A mudança de diversidade genotípica foi relacionando a administração prévia de ciprofloxacina. As cepas com perfis genotípicos predominantes não apresentaram maior prevalência da presença do gene ica, em relação às cepas não predominantes, o que não foi justificado que cepas potencialmente produtoras de biofilme se sobrepusessem em relação às cepas desprovidas deste gene. Oito dos 12 pacientes apresentaram concomitante ou posterior infecção por bacilos Gram negativos, destes 2 foram a óbito por septicemia. De acordo com os resultados, nós concluímos que pacientes submetidos a longos períodos de hospitalização são colonizados por microbiota de diversidade oligoclonal de S. epidermidis e a colonização ou infecção de CVC por destas cepas, potencialmente produtoras de biofilme em contato com a corrente sanguínea, pode ser uma oportunidade para infecções posteriores por outros microrganismos devido a potencial produção de biofilme inerente a S. epidermidis / Abstract: Central vascular catheters (CVC) represent an important risk for nosocomial bloodstream infections and Staphylococcus epidermidis is the most important pathogen of these systemic infections. To analyze the genomic DNA profiles, to detect the presence and expression of the responsible gene for biofilm production and to study the colonization dynamic, S. epidermidis strains isolated from tip CVC and blood positive cultures were compared with the strains isolated from skin and nasal swab in patients hospitalized in a tertiary care university hospital, the Hospital das Clínicas of UNICAMP. It was analyzed the previous medical care proceedings that the same patients underwent. Patients with microbiologic cultures for S. epidermidis from blood and/or catheter tip (>15 CFU) were selected to have swabs from skin and nasal. S. epidermidis were typed using PFGE, antibiotic susceptibility testing, presence of ica gene detection, by PCR, and biofilm detection, by Congo red method, were performed. Twelve patients with 18 episodes of colonization or catheter-related infection were included in this study and 247 strains were analyzed. It was found 26 distinct genotypic profiles and 4 strongly related genotypic profiles. In 10 episodes, the same DNA profile was detected in clinical and in microbiota strains, 6 of them occurred when the period of catheter implantation were higher than 15 days. In 7 episodes, there was not concordance among genotypic profiles from clinical and microbiota strain, and 5 of them occurred when the period of catheter implantation were lower than 15 days, too. It was not found statistic difference between the groups. PFGE identified six predominant genotypic profiles that were present in 68 % (132/193) of microbiota strain, and one of them was prevalently present (77/193). The prevalent genotypic profile was found compounding the microbiota in 10 episodes (8 patients). It was proofed, by comparison of the diversity of genotypic profiles, that during the hospitalization period the microbiota general profile changes from the diversified genotypic profile (polyclonal) to a poorly diversified genotypic profile (oligoclonal), with a predominant genotypic profile. It found was related with the previous ciprofloxacin administration. The predominant DNA profiles strains did not presented higher prevalence according to the presence of ica gene when comparing to non predominant strains, what it was not justified that potentially biofilm producers can superpose over non ica strains. Eight of 12 patients presented concomitant or posterior infection by negative Gram rots, whose 2 were to obit by sepsis. According to the results, we concluded that patients with long-term hospitalization were previously colonized by oligoclonal-diversified microbiota S. epidermidis and CVC colonization or infection by this agent, potentially biofilm producer present at bloodstream can be an opportunity to other microorganism posterior infections, due to potential biofilm production inherent to S. epidermidis / Doutorado / Ciencias Medicas / Doutor em Clínica Médica