Дисертації з теми "Staphylococcus aureus biofilms"

Orientadores: Karina Cogo Müller, Francisco Carlos Groppo
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: As estatinas são um grupo de fármacos que atuam como inibidores competitivos da enzima 3-Hidroxi-3-MetilGlutaril Coenzima-A Redutase (HMG-CoA redutase). Além de atuarem como importantes agentes hipolipemiantes, também apresentam outros efeitos, chamados de pleiotrópicos. Diversos estudos têm explorado um possível efeito protetor das estatinas atuando na redução na morbidade e mortalidade de várias doenças infecciosas. A atividade antimicrobiana das estatinas tem sido reportada por estudos in vivo e in vitro. O objetivo desse estudo foi avaliar os efeitos das estatinas sobre o crescimento e viabilidade de bactérias aeróbias patogênicas, e o efeito da sinvastatina sobre o biofilme de Staphylococcus aureus. Culturas das espécies de Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli e Enterococcus faecalis foram avaliadas na forma planctônica quanto à sensibilidade à atorvastatina, pravastatina e sinvastatina, através do teste de Concentração Inibitória Mínima (CIM). Além disso, diante da atividade apresentada pela sinvastatina contra S. aureus, foi determinada a ação dessa droga sobre a viabilidade celular através dos testes de Time-kill e Efeito pós-antibiótico (EPA). Também foi verificado um possível efeito sinérgico entre a sinvastatina e vancomicina. Por fim, a ação da sinvastatina foi avaliada contra biofilmes de S. aureus. Os valores de CIM da sinvastatina para o microrganismo S. aureus foram: 15,65 µg/ml (ATCC 29213) e 31,25 µg/ml (ATCC 33591, 43300, 14458 e 6538). A sinvastatina apresentou um perfil bacteriostático, e na concentração de 4xCIM seu EPA foi similar ao da vancomicina. Não foi encontrado nenhum tipo de interação entre a associação de sinvastatina e vancomicina. Entretanto, a sinvastatina foi capaz de reduzir a formação do biofilme nas concentrações entre 1/8CIM à 4xCIM. Além disso, na concentração 4xMIC foi capaz de diminuir a viabilidade, biomassa e a produção de polissacarídeos extracelulares e aumentar a produção de polissacarídeos intracelulares de biofilmes maduros de S. aureus. A produção de proteínas pelo biofilme não foi alterada. Em conclusão, os resultados encontrados mostram que a sinvastatina possui um grande potencial a ser explorado, principalmente em relação ao descobrimento de novos antimicrobianos
Abstract: Statins are drugs that competitively inhibit the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA). Besides their important lipid-lowering action, they also are pleiotropic agents. Several studies have explored a possible protective effect of statins to reduce the morbidity and mortality of various infectious diseases. The antimicrobial activity of statins has been reported by in vivo and in vitro studies. The aim of this study was to evaluate the effects of statins on the growth, viability and biofilm formation of pathogenic aerobic bacteria. The Minimum Inhibitory Concentrations (MIC) of atorvastatin, pravastatin and simvastatin against planktonic cells of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis strains were obtained. Since simvastatin showed activity against S. aureus, its effects on cell viability were evaluated in a time-kill and post-antibiotic effect (PAE) assays. A possible synergistic effect between simvastatin and vancomycin was also assessed. In addition, the effect of simvastatin against biofilms of S. aureus was tested. The MIC values of simvastatin for S. aureus were: 15.65 µg/ml (ATCC 29213) and 31.25 µg/ml (ATCC 33591, 43300, 14458 and 6538). Simvastatin showed a bacteriostatic profile, and in a 4x>MIC concentration the PAE was similar to vancomycin. No synergistic effect was found between simvastatin and vancomycin. Simvastatin was able to reduce the formation of biofilms in concentrations ranging from 1/8MIC to 4xMIC. In addition, the 4xMIC was able to decrease the viability, biomass and production of extracellular polysaccharides and increase the production of intracellular polysaccharides on mature biofilm of S. aureus. The protein production on biofilm was not altered in the presence of simvastatin . In conclusion, our results showed that simvastatin has a great potential to be explored, especially in relation to the development new antimicrobial agents
Mestrado
Farmacologia, Anestesiologia e Terapeutica
Mestra em Odontologia

Les infections ostéo-articulaires requièrent souvent un acte chirurgical couplé à une antibiothérapie prolongée, en lien avec la capacité des bactéries en cause à former des biofilms sur les prothèses. La présence d’antibiotiques à des concentrations sub-inhibitrices (sub-CMI) peut stimuler la capacité des bactéries à former des biofilms, ce qui complexifie la problématique. Notre étude avait pour but de caractériser in vitro le comportement biofilm d’une souche clinique de Staphylococcus aureus issue d’une infection ostéo-articulaire en présence de 12 antibiotiques préconisés dans le traitement des infections à staphylocoques, à différentes concentrations (dont des concentrations sub-CMI).Un dénombrement des bactéries viables a été effectué à partir de biofilms formés en présence de chacun des 12 antibiotiques à différentes concentrations (incluant des concentrations sub-CMI), ainsi que dans les suspensions présentes autour de ces mêmes biofilms. La présence de 7 d'entre eux (ceftaroline, daptomycine, gentamicine, fosfomycine, ofloxacine, rifampicine et vancomycine) entraînait une inhibition de la formation de biofilm à des concentrations inférieures ou égales aux concentrations critiques. L'action de la fosfomycine s'étendait même à des concentrations sub-CMI. Seules la daptomycine et la gentamicine étaient capables d’agir à la fois sur les bactéries sessiles et sur les bactéries non-adhérentes en suspension.Quant aux biofilms mâtures de cette même souche préalablement formés en absence d'antibiotiques, seules des concentrations 800 à 51200 fois supérieures à la CMI -concentrations largement incompatibles avec une utilisation thérapeutique - entraînaient leur éradication.Dans la deuxième partie de ce travail, nous avons concentré nos efforts sur l'action de la fosfomycine, choisie en fonction de son effet sur la formation de biofilm et de son intérêt thérapeutique. Une analyse transcriptomique des cellules présentes dans les biofilms formés en présence de fosfomycine et de cellules issues de biofilms matures traités ultérieurement à la fosfomycine a montré que la présence de cet antibiotique induisait majoritairement une sous expression de gènes codant des protéines impliquées dans le métabolisme et le transport des nucléotides, des acides aminés et des carbohydrates. Des gènes codant des adhésines et des protéines impliquées dans la synthèse de la capsule (ScdA) étaient également sous-exprimés. De manière moins importante, l'expression de gènes codant des molécules impliquées dans la synthèse du peptidoglycane (MGT et MurA) et des autolysines était également diminuée. Le ralentissement métabolique et les modifications induites au niveau des membranes par la présence de l'antibiotique seraient responsables du changement des capacités d'adhésion de la bactérie.L'impact de la fosfomycine à une concentration sub-CMI sur les caractéristiques des bactéries viables isolées dans la suspension autour des biofilms a également été déterminé. De façon surprenante, ces bactéries montrent une capacité accrue à former du biofilm par rapport à celles issues de l'environnement d'un biofilm non soumis à l'action de l'antibiotique, en lien probablement avec un accroissement d'épaisseur de leur couche de peptidoglycane.En conclusion, ces données obtenues in vitro devront être confirmées dans des modèles d'infections expérimentales in vivo. Malgré tout, elles soulignent la pertinence de l’utilisation de la fosfomycine dans la prévention des infections ostéo-articulaires liées à S. aureus, à condition d'éradiquer en parallèle les formes non adhérentes
Osteoarticular infections (OAI) often require a surgical procedure with prosthesis removal followed by long-term complex antibiotherapy. The ability of Staphylococcus aureus to adhere and produce biofilm on the surface of implanted material contributes to treatment failures and microbiological relapses. In addition, biofilm formation can be induced by some antibiotics at sub-minimal inhibitory concentrations (sub-MICs). The present study characterizes in vitro the effects of 12 antibiotics on biofilm formed by a strain of methicillin-susceptible Staphylococcus aureus isolated from an osteo-articular infection.The influence of these antibiotics was assessed on biofilm formation at concentrations including the breakpoints, by numbering viable cells in the biofilm biomass and in the suspensions (unattached cells) surrounding the biofilm. Biofilm formation was prevented in presence of ceftarolin, daptomycin, fosfomycin, gentamicin, ofloxacin, rifampicin and vancomycin at the highest concentrations tested. Only fosfomycin showed inhibition properties also at sub-MICs. Unattached and sessile viable bacteria were undetectable to daptomycin and gentamicin at the highest concentrations tested.Determination of the minimum biofilm eradication concentrations (MBECs) indicated that in vitro eradication of 24h-old biofilms required concentrations at least 800 times higher than the planktonic MIC, concentrations obviously not compatibles with classical therapeutic doses.In the second part of this work, we focused our study on the action of fosfomycin, because of its effect on biofilm formation and its therapeutic interest. A transcriptome analysis was performed with sessile cells from both biofilm formed in the presence of sub-MIC of fosfomycin and cells from pre-formed 24h-old biofilm treated by fosfomycin at sub-MBEC. Fosfomycin induced mostly down regulation of genes assigned to nucleotide, amino acid and carbohydrate transport and metabolism. Adhesins and capsular biosynthesis proteins (ScdA) encoding genes were also down regulated. To a lesser extent, peptidoglycan biosynthesis proteins (MGT and MurA) and autolysins encoding genes were found down regulated. Metabolic slowdown and cell membrane modifications induced by fosfomycin are likely to be responsible for the impairment of bacterial adhesion capacity.The action of fosfomycin at sub-MIC on unattached cells surrounding biofilm was also analyzed. Surprisingly, they displayed higher capacity to form new biofilm than their counterparts obtained without fosfomycin, probably associated with their large peptidoglycan layer.In conclusion, these data underline the relevance of the use of fosfomycine in preventing osteo-articular infections due to S. aureus and should be assessed in in vivo experiments. However, simultaneous eradication of unattached cells should also be considered due to the high capacities of these cells to disseminate and establish new biofilm, a real risk of treatment failure

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A necessidade de superar o desafio criado pelos biofilmes resistentes aos tratamentos antimicrobianos convencionais tem levado à busca por tratamentos alternativos, como terapia fotodinâmica antimicrobiana (aPDT). Este estudo avaliou in vitro a eficácia da aPDT na inativação de biofilmes de Staphylococcus aureus suscetíveis e resistentes à meticilina (MRSA e MSSA), mediado pelos fotossensibilizadores (PSs) Curcumina (Cur) e Photodithazine® (PDZ). Biofilmes foram formados e tratados com diferentes concentrações de Cur (0, 20, 40 e 80 μM) e PDZ (0, 50 e 75 mg/L), e iluminados ou não por fonte de luz LED (Cur 455 ± 3 nm/ 5,28 J/cm2; PDZ 660 ± 3 nm/ 5,28 J/cm2 ou 50 J/cm²). Os grupos Controle Positivo (CP) não receberam nenhum PS e também não foram iluminados. A viabilidade dos micro-organismos após a aPDT foi avaliado pelo número de colônias viáveis, pelo ensaio de XTT e pela utilização do kit LIVE/DEAD® na Microscopia Confocal de Varredura à Laser (MCVL). Os resultados foram avaliados por análises de variância de dois fatores de efeitos fixos (ANOVA) e complementados por comparações múltiplas de médias pelo teste de Tukey. Para ambas as cepas, todas as concentrações de Cur e PDZ testadas reduziram significativamente a atividade metabólica e o UFC/mL para ambos micro-organismos quando comparado com os grupos CN (p0,05). Os resultados foram otimizados para a Cur quando utilizou-se a maior concentração (80 μM), para a PDZ, a maior redução nos micro-organismos foi observada quando associou-se a maior concentração de PDZ (75 mg/L) com a maior dose de luz (50 J/cm²). Os biofilmes submetidos a aPDT demostraram pela MCVL um maior número de células coradas em vermelho, indicando que a aPDT foi eficaz para promover danos ou morte às células bacterianas. Assim, a aPDT pode ser considerada promissora para atuar de forma sinérgica no tratamento de infecções bacterianas
The need to overcome the challenge created by biofilms regarding conventional antimicrobial approaches has lead to search of alternative treatments such as Antimicrobial Photodynamic Therapy (aPDT). This in vitro study evaluated the efficacy of aPDT using the photosensitizer (PS) Curcumin (Cur) and Photodithazine® (PDZ) in the inactivation of biofilms of methicillin susceptible and resistant S. aureus (MSSA and MRSA). Biofilms were treated with different Cur (0, 20, 40 or 80 μM of Cur) and PDZ concentrations (0, 50 or 75 mg/L) and illuminated or not with LED source (Cur 455 ± 3 nm/ 5.28 J/cm2; PDZ 660 ± 3 nm/ 5.28 J/cm2 or 50 J/cm²). Positive control samples were not exposed to PS or light. The microorganisms viability after aPDT were evaluated by counting the number of colonies, the XTT assay and LIVE/DEAD® staining using confocal laser scanning microscopy (CLSM). The results were evaluated by analysis of variance, two-factor fixed effects (ANOVA) and complemented by multiple comparisons by Tukey test. For both strains, all the tested Cur and PDZ concentrations reduced significantly both biofilm metabolic activity and CFU/mL compared to the negative control (p0.05). Moreover, the results were optimized for Cur when the higher concentration was used (80 μM); For PDZ, the best results were obtained when it was associated a higher concentration of PDZ (75 mg/L) with the higher dose of light (50 J/cm²). Biofilms submitted to aPDT showed a large number of red-stained colonies, indicating that this therapy was efficient in disrupting the bacterial membrane. It can be concluded that PS was efficient in reducing viable colonies of both S. aureus strains by damaging cell membrane and causing cell death. Thus, the aPDT is can be considered promising to act synergistically in the treatment of bacterial infections

Orientador: Ana Claúdia Pavarina
Banca: Eunice Teresinha Giampaolo
Banca: Ana Paula Dias Ribeiro
Resumo: A necessidade de superar o desafio criado pelos biofilmes resistentes aos tratamentos antimicrobianos convencionais tem levado à busca por tratamentos alternativos, como terapia fotodinâmica antimicrobiana (aPDT). Este estudo avaliou in vitro a eficácia da aPDT na inativação de biofilmes de Staphylococcus aureus suscetíveis e resistentes à meticilina (MRSA e MSSA), mediado pelos fotossensibilizadores (PSs) Curcumina (Cur) e Photodithazine® (PDZ). Biofilmes foram formados e tratados com diferentes concentrações de Cur (0, 20, 40 e 80 μM) e PDZ (0, 50 e 75 mg/L), e iluminados ou não por fonte de luz LED (Cur 455 ± 3 nm/ 5,28 J/cm2; PDZ 660 ± 3 nm/ 5,28 J/cm2 ou 50 J/cm²). Os grupos Controle Positivo (CP) não receberam nenhum PS e também não foram iluminados. A viabilidade dos micro-organismos após a aPDT foi avaliado pelo número de colônias viáveis, pelo ensaio de XTT e pela utilização do kit LIVE/DEAD® na Microscopia Confocal de Varredura à Laser (MCVL). Os resultados foram avaliados por análises de variância de dois fatores de efeitos fixos (ANOVA) e complementados por comparações múltiplas de médias pelo teste de Tukey. Para ambas as cepas, todas as concentrações de Cur e PDZ testadas reduziram significativamente a atividade metabólica e o UFC/mL para ambos micro-organismos quando comparado com os grupos CN (p0,05). Os resultados foram otimizados para a Cur quando utilizou-se a maior concentração (80 μM), para a PDZ, a maior redução nos micro-organismos foi observada quando associou-se a maior concentração de PDZ (75 mg/L) com a maior dose de luz (50 J/cm²). Os biofilmes submetidos a aPDT demostraram pela MCVL um maior número de células coradas em vermelho, indicando que a aPDT foi eficaz para promover danos ou morte às células bacterianas. Assim, a aPDT pode ser considerada promissora para atuar de forma sinérgica no tratamento de infecções bacterianas
Abstract: The need to overcome the challenge created by biofilms regarding conventional antimicrobial approaches has lead to search of alternative treatments such as Antimicrobial Photodynamic Therapy (aPDT). This in vitro study evaluated the efficacy of aPDT using the photosensitizer (PS) Curcumin (Cur) and Photodithazine® (PDZ) in the inactivation of biofilms of methicillin susceptible and resistant S. aureus (MSSA and MRSA). Biofilms were treated with different Cur (0, 20, 40 or 80 μM of Cur) and PDZ concentrations (0, 50 or 75 mg/L) and illuminated or not with LED source (Cur 455 ± 3 nm/ 5.28 J/cm2; PDZ 660 ± 3 nm/ 5.28 J/cm2 or 50 J/cm²). Positive control samples were not exposed to PS or light. The microorganisms viability after aPDT were evaluated by counting the number of colonies, the XTT assay and LIVE/DEAD® staining using confocal laser scanning microscopy (CLSM). The results were evaluated by analysis of variance, two-factor fixed effects (ANOVA) and complemented by multiple comparisons by Tukey test. For both strains, all the tested Cur and PDZ concentrations reduced significantly both biofilm metabolic activity and CFU/mL compared to the negative control (p0.05). Moreover, the results were optimized for Cur when the higher concentration was used (80 μM); For PDZ, the best results were obtained when it was associated a higher concentration of PDZ (75 mg/L) with the higher dose of light (50 J/cm²). Biofilms submitted to aPDT showed a large number of red-stained colonies, indicating that this therapy was efficient in disrupting the bacterial membrane. It can be concluded that PS was efficient in reducing viable colonies of both S. aureus strains by damaging cell membrane and causing cell death. Thus, the aPDT is can be considered promising to act synergistically in the treatment of bacterial infections
Mestre

The glycopeptide vancomycin is commonly used to treat a variety of bacterial infections, especially multi-drug resistant species of bacteria such as Staphylococcus aureus. While vancomycin remains an effective treatment for Staphylococcal infections, strains of vancomycin-intermediate Staphylococcus aureus (VISA) and vancomycin-resistant Staphylococcus aureus (VRSA) strains have emerged. One mechanism for the increased antibiotic (vancomycin-intermediate) resistance is due to acquisition of various mutations within different genes that alter the cell wall physiology making vancomycin ineffective. Biofilm development is a bacterial mode of growth that can lead to mutations within the bacterial genome and allow for advantageous traits such as increased antibiotic resistance. The biofilm environment can be harsh, having niches that are often nutrient and oxygen deficient, leading to damaged DNA. This DNA damage induces the SOS response to repair double-stranded breaks in DNA, and enables bacterial survival. However, DNA repair via the SOS response is an error-prone system that often results in mutations within the genome. We hypothesize that the acquisition of vancomycin intermediate resistance is an unintended consequence within the S. aureus biofilm environment. To assess the hypothesis, both wildtype and RecA/LexA deficient biofilms were grown in microtiter assays with and without the addition of sub-inhibitory concentrations of vancomycin. Efficiency of plating techniques were used to quantify the subpopulation of biofilm-derived S. aureus cells that developed vancomycin intermediate resistance. Microtiter assays and efficiency of plating techniques were repeated using multiple strains of S. aureus. Experimentation was repeated by comparing the subpopulation of biofilm-derived and planktonic culture cells that grew in intermediate-concentrations of vancomycin with three additional strains of S. aureus. Mutagenesis that occurs within the biofilm environment was further assessed by plating both biofilm-derived and planktonic culture cells on sheep blood agar and tryptic soy agar supplemented with streptomycin, novobiocin, or rifampicin, and quantifying the non-hemolytic variants that grew on blood agar, or the number of colonies that grew in the presence of an antibiotic, respectively. The biofilm results were then compared to the results from wildtype and RecA/LexA deficient planktonic cultures and used to determine the impact of the S. aureus biofilm environment in the acquisition of vancomycin intermediate resistance. The results indicate that a larger subpopulation of cells derived from wildtype biofilms grew in increased concentrations of vancomycin (4 µg/ml) as compared to the planktonic counterpart. The subpopulation of cells derived from wildtype biofilms was also higher than all subpopulations of RecA/LexA deficient biofilm and planktonic cultures. Further experimentation indicates that this phenomenon may not be specific to all strain backgrounds of S. aureus. Additionally, growth with sub-inhibitory concentrations of vancomycin did not exhibit an exaggerated subpopulation of cells in biofilm environments or planktonic cultures that could grow in intermediate-concentrations of vancomycin, however standard antibiotic testing suggests that the mechanism by which point mutations occur in planktonic conditions may be mediated by the RecA and SOS response system. Bacteria that live in a biofilm community are often subjected to harsh environments. In order to survive, the SOS response system will be activated to repair damaged DNA. This error prone process will result in mutational changes and increased genetic diversity. The VISA phenotype may be a result of the diversity that occurs within the biofilm environment. While the VISA phenotype would be an unintended consequence of genetic diversity and gene transfer in the biofilm setting, it demonstrates that mutations that occur within the biofilm environment allow for S. aureus to better adapt to new environments, including the presence of widely used antibiotics such as vancomycin.

Orientador: Debora de Barros Barbosa
Coorientadora: Andresa Aparecida Berretta e Silva
Banca: Alberto Carlos Botazzo Delbem
Banca: Denise Pedrini Ostini
Banca: Luiz Fernando Gorup
Banca: Elaine Cristina Guerbach Conti
Resumo: O objetivo deste estudo foi avaliar a capacidade de produção de nanopartículas de prata através do extrato da casca de romã, e produzir formulações contendo estas nanopartículas para uso em feridas. Elas foram testadas quanto à ação antimicrobiana, citotóxica e potencial cicatrizante. Para a produção das nanopartículas de prata propôs-se uma síntese utilizando-se como base duas metodologias já estabelecidas na literatura, utilizando-se para reação carboximetilcelulose, propilenoglicol, nitrato de prata, água e extrato da casca de romã como agente redutor. O extrato da casca de romã foi caracterizado em parâmetros como pH, massa seca e quantidade de taninos bioativos (ácido elágico e totais fenólicos expressos em ácido gálico). Os totais fenólicos do extrato foram também dosados após seu aquecimento nas diferentes condições de tempo e temperatura propostos para as sínteses das nanopartículas de prata (12 minutos, 1 hora e 2 horas, à 50ºC e 100ºC). As nanopartículas de prata produzidas foram, então, adicionadas a uma solução contendo compostos para produção de formulações para serem utilizadas no tratamento de feridas. Elas foram caracterizadas através de espectroscopia UV-Visível, microscopia eletrônica de varredura (MEV), potencial zeta e dosagem de íons remanescentes após as reações. A atividade antimicrobiana tanto das nanopartículas como de suas formulações contra Candida albicans SC 5314 e Staphylococcus aureus ATCC 25923 foi avaliada por meio do método da microdiluição. ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The aim of this study was to investigate the production of silver nanoparticles through peel extract of pomegranate, and produce formulations containing these particles to be used in wound healings. Its antimicrobial action, cytotoxicity and healing potential were tested. The synthesis of silver nanoparticles were based on two methods proposed in the literature with some modifications, which were used carboxymethylcellulose, propylene glycol, silver nitrate, water and peel extract of pomegranate as reducing agent. The peel extract was characterized by pH, dry mass and bioactive tannins (elagic acid and total phenols expressed as galic acid). The total phenols were also quantified after being heated at 50ºC and 100ºC for 12 minutes, 1 hour and 2 hours. Then, silver nanoparticles were added in a solution containing products to develop a formulation to be tested in wound healing. They were characterized by UV-Vis spectroscopy, scanning electron microscopy (SEM), zeta potential and the quantification of remaining silver ions after the synthesis reaction. The antimicrobial activity of the nanoparticles and formulations were tested against Candida albicans (SC 5314) e Staphylococcus aureus (ATCC 25923) by microdilution method. After submitting the peel extract to different conditions of temperature and times (50ºC and 100ºC for 12 minutes, 1 hour and 2 hours), it was noted that the values of the minimum inhibitory concentration was not affected and were 391 μg/ml and 781 μg/ml for S. aureus and C. albicans. The formation of silver nanoparticles was confirmed through the formation of characteristic peaks in the UV-Vis spectroscopy and SEM images, and it was observed that the reaction at 50ºC for 12 min produced silver nanoparticles with regular forms and better dispersed in the formulation. The synthesis proposed promoted...
Doutor

Staphylococcus aureus est l’une des causes majeures des infections communautaires et nosocomiales. Ce germe est responsable des infections aiguës et chroniques dont la plupart sont dues à sa capacité à adhérer sur les implants médicaux et à former un biofilm. D’après le Center for Disease Control and Prevention (CDC), 65% des infections bactériennes sont dues à la présence des biofilms. En outre, les infections associées aux biofilms constituent un problème majeur en clinique et sont la cause de l’augmentation de la mortalité et du coût de traitement.

Chez S. aureus, la formation du biofilm se déroule en deux phases principales: la première phase est l’attachement initial des cellules sur une surface, et la seconde est la multiplication et la formation d’une communauté structurée, mature et multicouche des cellules bactériennes. A l’intérieur du biofilm, les bactéries développent plusieurs types d’interactions et accroissent leur résistance aux agents antimicrobiens et aux défenses immunitaires de l’hôte, ce qui constitue un véritable problème de santé publique.

Les objectifs de ce travail étaient: (1) de caractériser des souches cliniques de S. aureus sensibles et résistantes à la méticilline (SASM et SARM) par une analyse phénotypique et génotypique; (2) d’étudier la répercussion des propriétés de membranes sur l’adhésion et la formation du biofilm; (3) de rechercher un moyen pour la prévention de l’adhésion et de la formation d’un biofilm sur une surface abiotique.

Deux souches de référence et 12 souches cliniques de S. aureus (4 SARM et 8 SASM) collectées à Kinshasa ont été caractérisées par la résistance aux antibiotiques, par le typage d’une région X du gène spa codant pour la protéine A de S. aureus et par la détermination des propriétés de la surface cellulaire. L’adhésion à une surface et la formation du biofilm ont été respectivement étudiées par la méthode de Biofilm Ring Test® (BFRT®) et par celle de coloration au cristal violet. Ces deux méthodes ont été utilisées pour l’évaluation de l’activité de l’acide éthylèneglycol tétraacétique (EGTA) sur l’adhésion et la formation du biofilm.

L’amplification par PCR (Polymerase Chain Reaction) d’un fragment du gène mecA a confirmé l’appartenance des souches étudiées au phénotype SARM ou SASM. L’analyse par PCR des répétitions présentes dans la séquence codante de la protéine A de S. aureus (spa typing) a permis d’identifier 7 types spa pour toutes les souches SARM et SASM2 dont un nouveau type spa t10715.

Les résultats du test MATS (Microbial Adhesion to Solvents) ont montré que les souches de S. aureus sensibles et résistantes à la méticilline possédaient des propriétés membranaires différentes susceptibles de modifier l’adhésion ou la formation d’un biofilm. Les souches sensibles à la méticilline avaient une paroi plus hydrophobe que celle de souches résistantes dont la paroi était acide, acceptrice d’électrons.

Les études sur l’interaction entre des souches cliniques de S. aureus et des surfaces abiotiques ont montré que les souches SARM adhéraient moins vite à une surface et formaient moins de biofilms que les souches SASM.

Les études de l’activité de l’EGTA, un chélateur des cations divalents, ont montré que ce dernier inhibait l’adhésion de souches SARM à une surface abiotique comme un tube de cathéter et empêchait la formation d’un biofilm par toutes les souches sensibles et résistantes à la méticilline. Cette action inhibitrice sur la formation du biofilm était réversible en présence d’un cation divalent (magnésium, calcium ou manganèse).

L’ensemble des données obtenues sur l’adhésion et la formation du biofilm par la méthode de BFRT® et par celle de coloration au cristal violet ont montré que le BFRT® était la méthode de choix dans les études de l’adhésion initiale des souches de S. aureus sur une surface abiotique. Le BFRT® pourrait être utilisée dans le screening rapide de produits contre l’adhésion bactérienne à la surface des implants médicaux à base de polystyrène ou de silicone.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Antibiotic resistance is an ever-growing topic of concern within the medical field causing researchers to examine the mechanisms of resistance to develop new antimicrobials. Bacteria’s ability to form biofilms is one mechanism which aids in antimicrobial resistance. Staphylococcus aureus is of special interest as it is one of the most frequent biofilm-forming bacteria found on medical devices causing infections and posing dangerous threats in a clinical setting. A recently developed antimicrobial gel has been shown to have profound effects on treating bacterial infections and wound healing. This research is centered upon examining the antimicrobial effects of this gel on the three different stages of biofilm formation in clinical and laboratory strains of S. aureus. Through a series of experiments examining the effects this gel has on S. aureus at the stages of biofilm attachment, maturation, and dispersion, the gel has shown significant levels of inhibition. These findings indicate that the novel gel disrupts biofilm forming processes of S. aureus, which provides useful information for fighting infections in the medical field. Further research on the uses and effects of this new gel could lead possibility using the antimicrobial compound for a variety of clinical purposes.

Intoxicações alimentares são uma das causas mais significativas de mortalidade em países desenvolvidos e em desenvolvimento, sendo as contaminações bacterianas as responsáveis pela maioria dos casos. Dentre elas destacam-se as causadas por espécies de Staphylococcus, que são nocivas tanto pela infecção do organismo hospedeiro, quanto pela intoxicação por enterotoxinas termoestáveis presentes em alimentos contaminados e mal acondicionados. Além disso, a capacidade de S. aureus formarem biofilmes confere maior proteção contra agentes de controle. Neste contexto, torna-se importante desenvolver novos métodos visando inibir estes agentes patogênicos. Uma alternativa ao emprego de conservantes sintéticos é a utilização de biossurfatantes, como os ramnolipídeos (RL) que, além de apresentarem alta estabilidade a temperatura, pH e concentração salina, apresentam baixa toxicidade e são biodegradáveis. O objetivo deste trabalho foi avaliar o efeito do pH na atividade antimicrobiana dos RL sobre células planctônicas e sésseis de Staphylococcus aureus (ATCC 8095) e comparar seu efeito ao do dodecil sulfato de sódio (SDS). Os resultados mostraram que o pH exerce grande influência na ação dos surfatantes, sendo mais efetiva em valores de pH menores. Em pH 5, os RL apresentaram ação bactericida (CBM=19,5 mg L-1) superior ao SDS (CBM=39,1 mg L-1), sendo capazes de inibir e remover 80% dos biofilmes em concentrações de 156,2 mg L-1 e reduzir em cerca de 40 % a hidrofobicidade da superfície celular de S. aureus. Em valores de pH maiores a ação dos RL não superou a do SDS, porém estes ainda mostraram resultados promissores, principalmente na remoção de biofilmes evidenciado pela microscopia confocal. A espectroscopia FTIR revelou que quando em pH 6, 7 e 8 S. aureus, possivelmente, induz alterações em sua membrana celular a fim de diminuir a sensibilidade aos surfatantes. As imagens de MEV mostraram que os RL promovem deformações nas células, podendo levá-las a ruptura. O aumento da força iônica, promovido pela adição de NaCl no meio, favoreceu a ação antimicrobiana dos RL, o que torna a aplicação dos RL em alimentos bastante promissora.
Food poisoning can be considered one of the most significant causes of mortality in developed and developing countries with bacterial contamination being responsible for the majority of cases. Among them are those caused by Staphylococcus species that can beharmful, both from the infection of the host organism as the intoxication by thermostable enterotoxins present in contaminated and poorly conditioned food. In addition, the ability to form biofilms gives S. aureus greater protection against control agents. Under this context, it becomes important to develop new methods to inhibit those pathogens. An alternative to the use of synthetic preservatives are biosurfactants such as rhamnolipids (RL), which shows high stability to temperature, pH and salt concentration along with the fact that they have low toxicity and are biodegradable. The aim of this study was to evaluate the effect of pH on the antimicrobial activity of RL on planktonic and sessile Staphylococcus aureus cells (ATCC 8095) and compare its effect to that of the sodium dodecyl sulfate (SDS). The results showed that pH exerts a great influence on the action of surfactants, with greater effectiveness at lower pH. At pH 5, RL presented higher bactericidal action (CBM = 19.5 mg L-1) than SDS (CBM = 39.1 mg L-1), also being able to inhibit and remove 80% of biofilms at concentrations of 156. 2 mg L- 1 and reduce the hydrophobicity of S. aureus cell surface by about 40%. At higher pH values, the action of RL did not exceed that of SDS, neverthelessthey showed promising results, mainly on the removal of biofilms evidenced by confocal microscopy. FTIR spectroscopy revealed that at pH 6, 7 and 8,S. aureus possibly induces changes in its cell membrane in order to decrease the sensitivity to surfactants. The MEV images showed that the RL cause deformations in the cells, which can lead to rupture. The increase in ionic strength, promoted by the addition of NaCl in the medium, favored the antimicrobial action of RL, which makes the application of RL in foods very promising.

Biofilmes de estafilococos têm se tornado uma das grandes preocupações da indústria de lácteos, principalmente em decorrência do ritmo de produção intenso, automação das plantas de processamento e exigência cada vez maior quanto à qualidade microbiológica de leite e derivados. O presente estudo teve por objetivo identificar cepas de S. aureus potencialmente produtoras de biofilmes isoladas de 3 laticínios produtores de queijo Minas frescal, avaliar a influência da temperatura e da superfície de contato (aço inoxidável e polipropileno) no processo de adesão bacteriana, bem como a eficácia de um protocolo simulado de limpeza e sanificação na remoção das células aderidas. Para as análises genotípicas pesquisaram-se os genes icaA e icaD nos isolados, relacionados à produção de polissacarídeos de adesão celular e exopolissacarídeos da matriz de biofilmes. Os ensaios de biofilmes foram realizados em cupons incubados em um reator de biofilmes, comparando-se a adesão frente a duas temperaturas (5°C e 35°C), duas superfícies (aço inoxidável e polipropileno) e quatro tempos de contato (3, 6, 12 horas e após processo de limpeza e sanificação). Para avaliação da eficácia do processo na remoção das células aderidas, foram utilizados detergente neutro (3,5% v/v) e sanificante à base de hipoclorito de sódio (1000 mg.L-1), de modo a simular a situação observada em um dos laticínios estudados. Foi detectada a presença dos genes icaA e icaD em 74% e 77% dos isolados, respectivamente; 70% dos isolados apresentaram ambos os genes, enquanto 19% não apresentaram nenhum. O número de células aderidas em ambas as superfícies foi em torno de 3 e 6 log10 UFC.cm-2 nas temperaturas de 5°C e 35°C, respectivamente, para a maioria das situações avaliadas, com aumento significativo no decorrer dos períodos avaliados. De maneira geral, a temperatura de 35°C favoreceu uma maior adesão de S. aureus. A 5°C, houve considerável número de células aderidas, mas em populações significativamente inferiores às observadas a 35°C. O protocolo de limpeza e sanificação mostrou-se ineficaz na remoção das células aderidas; uma melhor atuação do hipoclorito de sódio foi observada a 5°C, o que deve estar relacionado à menor adesão observada nessa temperatura. De qualquer forma, o processo não foi capaz de reduzir a níveis seguros a quantidade de S. aureus aderida a ambas as superfícies, nas condições avaliadas. O estudo demonstrou a capacidade de adesão de S. aureus isolados de laticínios em superfícies comumente encontradas na produção de queijo Minas frescal, situação que pode favorecer o desenvolvimento de biofilmes em equipamentos e utensílios, conferindo risco à saúde dos consumidores.
Staphylococci biofilms have become a major concern for the dairy industry, mainly due to the intensive production flow, automation of processing plants and increased demand on the microbiological quality of dairy products. This study aims to identify isolated S. aureus strains potentially producers of biofilms from 3 dairies of Minas fresh cheese, evaluate the influence of temperature and the contact surface (stainless steel and polypropylene) in the bacterial adhesion process, as well as the efficacy of an hygiene and sanitation simulated protocol in removing the adhered cells. For genotypic analyzes, the presence of icaA and icaD in strains were sought related to polysaccharide production in cell adhesion and exopolysaccharide of biofilm matrix. Biofilms trials were performed in biofilm coupons incubated in biofilm reactor, comparing the adhesion with two temperatures (5°C and 35°C), two surfaces (stainless steel and polypropylene) and four contact times (3, 6, 12 hours and after cleaning and sanitizing process). To evaluate the process effectiveness in removing the adhered cells, neutral detergent (3.5% v/v) and sanitizing based on sodium hypochlorite (1000 mg.L-1) were used in order to simulate the observed situation in one of the dairy products studied. The presence of icaA and icaD genes was detected in 74% and 77% of strains, respectively; 70% of the isolates showed both genes, whereas 19% of it showed no genes. The number of cells adhered on both surfaces was about 3 and 6 log10 FCU.cm-2 in temperatures of 5 °C and 35 °C, respectively, for most situations evaluated, with a significant increase over the evaluation period. In general, the temperature of 35°C favored a greater adherence of S. aureus. At 5°C, there was a considerable number of adhered cells, but in populations significantly lower than those observed at 35°C. The cleaning and sanitizing protocol was ineffective in removing adhered cells; a better performance of sodium hypochlorite was observed at 5°C, which should be related to lower adherence observed at this temperature. At all, the process was not able to reduce the amount of S. aureus adhered on both surfaces to safe levels under the conditions evaluated. The study demonstrated the ability of adhesion of isolated S. aureus from dairy on surfaces commonly found in Minas fresh cheese production, a situation that may favor the development of biofilms on equipment and utensils, indicating health risk to the consumers.

Staphylococcus aureus est responsable de nombreuses infections nosocomiales etcommunautaires dont la diversité est liée à l’expression de nombreux facteurs de virulence.L’expression de ces facteurs est temporellement coordonnée au cours de l’infection par desfacteurs de transcription, des systèmes à 2 composants et des ARN régulateurs (ARNnc). Ungrand nombre d’ARNnc potentiels a été prédit dans le génome de S. aureus mais leur fonctionreste méconnue.L’objectif premier de ce travail a été de caractériser le rôle dans la régulation de la virulence del’un des ARNnc identifiés par notre équipe, RsaA. Des approches phénotypiques in vitro ontpermis de démontrer que RsaA active la formation du biofilm et réprime la synthèse de lacapsule bactérienne, avec une incidence sur l’opsonophagocytose de la bactérie et sur soninternalisation dans les macrophages. Au niveau moléculaire, RsaA réprime la traduction dufacteur de transcription MgrA, répresseur de la formation de la biofilm et activateur de lacapsule, par un mécanisme de type antisens entre RsaA et l’ARNm mgrA. Parallèlement, RsaAréprimerait l’opéron yabJ-spoVG, activateur de la synthèse de capsule. Ainsi, RsaA active laformation de biofilm et réprime la synthèse de capsule.Le second objectif de ce travail a été de caractériser l’expression in vivo de RsaA, E, G, H ainsique l’ARN III dans des prélèvements d’infections aiguës (abcès cutanés), chroniques (crachatsde patients mucoviscidiques) ou de portages nasales. L’étude de l’expression de ces ARN parRT-PCR, a permis de montrer que tous ces ARN sont exprimés avec une grande variabilité dansles prélèvements infectieux par rapport aux prélèvements de portages
Staphylococcus aureus is responsible for many nosocomial and communityinfections with a diversity linked to the expression of many virulence factors. Their expression istemporally coordinated during infection by transcription factors, two- component systems but alsoregulatory RNAs called non coding RNAs (ncRNAs). A large number of potential ncRNAs waspredicted in the genome of S. aureus but their function remains unknown.The first aim of this thesis was to characterize the role of one ncRNA identified by our team,RsaA, in the regulation of bacterial virulence. Several in vitro approaches allowed todemonstrate that RsaA activates biofilm formation and inhibits the bacterial capsule synthesis,with an impact on the bacterial opsonophagocytosis and internalization into macrophages. RsaArepresses the translation of mgrA mRNA by an antisense mechanism. mgrA codes for atrancriptional regulator known to repress biofilm formation but to activate capsule synthesis. Inparallel, RsaA represses the translation of the yabJ-spoVG operon, an activator of capsuleproduction. In this way, RsaA enhances biofilm formation and represses capsule synthesis.The second objective was to characterize the in vivo expression of RNA III and RsaA, E, G, HsRNAs in samples of acute infections (cutaneous absesses), chronicle infections (cystic fibrosis)and nasal carriage. The expression study (quantitative RT-PCR) allowed to demonstrate that allsRNA are expressed into samples but they have a high expression variability depending ofinfectious samples compared to asymptomatic carriage

Orientadores: José Francisco Höfling, Mary Ann Foglio
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Pesquisas que visam extrair e identificar compostos isolados de plantas tem sido levada a efeito há muitos anos, com o intuito de descobrir compostos com ação antimicrobiana e produzir medicamentos devido ao aumento da resistência apresentada pelos microrganismos aos antimicrobianos disponíveis no mercado. A ação dos compostos presentes na semente, casca, folha e frutos das plantas do gênero Pterodon spp. tem sido estudadas em bactérias, leveduras e protozoários, estando relacionada à ação antimicrobiana, antiinflamatória, antiproliferativa e contra cercárias de Schistossoma mansoni. Leveduras do gênero Candida spp., bactérias do gênero Streptococcus spp. e Staphylococcus spp. estão associadas à formação do biofilme em dispositivos médicos, sendo esta uma estratégia para resistir aos agentes antimicrobianos e às células do sistema imunológico. O composto denominado FrB, identificado através de espectrometria de massas com esqueleto vouacapano, e os compostos geranilgeraniol, éster 6?,7?-diidroxivouacapano-17? oato de metila e os isômeros éster 6?-hidroxi-7?-acetoxi-vouacapano-17?-oato de metila e acetoxi-7?- hidroxi-vouacapano-17?-oato de metila, foram testados através da técnica de concentração inibitória mínima (CIM) em quatro espécies de Candida spp, e nas bactérias Streptococcus mutans e Staphylococcus aureus. Foi observada CIM de 2mg/mL apenas nas quatro cepas de Candida spp., não sendo ativo contra as bactérias. Através da microscopia eletrônica de varredura e de transmissão, observou-se que FrB provocou modificações na parede celular fúngica. O composto diterpeno com esqueleto vouacapano foi testado no biofilme de Candida albicans SC 5314 e verificou-se uma inibição de 70% do biofilme em formação na concentração de 1mg/mL. No entanto, nenhum dos quatro compostos foram ativos contra bactérias. A partir dos resultados obtidos, podemos afirmar que o vouacapano, isolado do óleo da semente de Pterodon pubescens, apresenta atividade antifúngica, sendo uma potencial fonte para produção de medicamentos a base de plantas
Abstract: Research aimed at identifying and extracting compounds isolated from plants have been under a many years ago with the aim of discovering bioactive compounds and produce phyto due to the increased resistance of microorganisms to the antimicrobial commercially used. The action of bioactive compounds present in the seeds, bark, leaves and fruits of plants of the genus Pterodon spp. has been studied in bacteria, yeasts and protozoa, is related to antimicrobial, antiinflammatory, antiproliferative and against infection by Schistosoma mansoni. Yeasts of the genus Candida spp. and bacteria of the genus Streptococcus spp. and Staphylococcus spp. are associated with biofilm formation in medical devices, which is a strategy to resist antimicrobial agents and cells of the immune system. The compound FrB named, isolated from the seed oil Pterodon pubescens and identified by mass spectrometry with skeleton vouacapan, and the compounds geranylgeraniol, éster 6?,7?-dihydroxyvouacapan-17? oato de metila and ester 6?-hidroxi-7?-acetoxi-vouacapan-17?-oate metil e acetoxi-7?- hidroxi-vouacapan-17?-oato metil were tested using the technique of minimal inhibitory concentration (MIC) in four species of Candida spp, and bacteria Streptococcus mutans and Staphylococcus aureus. Was observed MIC of 2 mg/mL to the four strains of Candida spp., although was not active against bacteria. By scanning and transmission electron microscopy, it was observed that FrB caused changes in the fungal cell wall. The diterpen vouacapan was tested on Candida albicans SC 5314 biofilm and there was a 70% inhibition of biofilm formation in a concentration of 1mg/mL. From the results obtained, we can affirm that diterpen skeleton with vouacapan, isolated from the seed oil of Pterodon pubescens, has antifungal activity and is a potential source for the production of plant based medicinal
Mestrado
Microbiologia e Imunologia
Mestra em Biologia Buco-Dental

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Staphylococcus aureus é o microrganismo que mais prevalece em casos de mastite em rebanhos bovinos leiteiros e isto ocorre devido a produção de vários fatores de virulência, inclusive aqueles relacionados a sua capacidade de se instalar no parênquima mamário. Sendo assim, as condições microbiológicas do leite cru que é fornecido às indústrias pode refletir no seu produto final, determinando riscos à saúde do consumidor. Diante do exposto, objetivou-se realizar o isolamento e identificação das estirpes de S. aureus de amostras de leite bubalino da raça Murrah, dos insufladores, água das mangueiras da sala de ordenha e mãos dos ordenhadores em uma propriedade rural em Analândia – SP nos meses de abril, junho, outubro e novembro. Foi avaliada, a capacidade produtora de biofilme in vitro” pelo método de aderência em microplacas e pelo cultivo em Agar Vermelho Congo (CRA), o perfil de sensibilidade dos S. aureus frente a antimicrobianos pelo método da difusão em disco além de identificar os genes que participam na formação de biofilme por Reação em Cadeia da Polimerase (PCR). Das 147 estirpes de Staphylococcus spp isoladas, 32 foram identificadas como S. aureus. Dos isolados, 28,1% foram positivas para o gene icaA, 21,8% para o gene icaD, 28,1% para clfA, 50% clfB, 53,1% sarA e 50% para o gene hla. Todas os isolados demonstraram habilidade de produção de slime em Agar Vermelho Congo (CRA) e formação de biofilme em microplaca. Embora a frequência de isolamento de S. aureus foi baixa, essas estirpes podem ser consideradas potenciais patógenos zoonóticos
Staphylococcus aureus is the most common cause of mastitis in dairy herds due to its production of several virulence factors, including those related to its ability to colonize the teat parenchyma. The microbiological status of raw milk supplied to manufacturers may reflect on the final product and the associated risks to consumer health. Accordingly, the aim of this study was to isolate and characterize S. aureus strains from samples of Murrah buffalo milk, milking machines, water hoses from milking room and from the hands of milkers on a dairy farm located in Analândia – SP during the months of April, July, October, and November. Biofilm-producing capacity was evaluated in vitro” using a microtiter method and cultivation on Congo Red Agar (CRA). Antibiotic sensitivity testing was performed by the disk diffusion method and the identification of the genes involved in biofilm formation by Polymerase Chain Reaction (PCR). One hundred and forty-seven Staphylococcus spp. were isolated and 32 were identified as S. aureus. Of those isolates, 28.1% were positive for the icaA gene, 21.8% for the icaD gene, 28.1% for the clfA, 50.0% for the clfB, 53.1% for sarA and 50.0% for hla. All isolates produced slime on Congo Red Agar (CRA) and formed biofilms in a microtiter assay. Although the frequency of S. aureus was low, the stains isolated can be considered as potential zoonotic agents

Orientador: João Martins Pizauro Junior
Coorientador: Oswaldo Durival Rossi Junior
Banca: Tiago Santana Balbuena
Banca: Poliana de Castro Melo
Resumo: Staphylococcus aureus é o microrganismo que mais prevalece em casos de mastite em rebanhos bovinos leiteiros e isto ocorre devido a produção de vários fatores de virulência, inclusive aqueles relacionados a sua capacidade de se instalar no parênquima mamário. Sendo assim, as condições microbiológicas do leite cru que é fornecido às indústrias pode refletir no seu produto final, determinando riscos à saúde do consumidor. Diante do exposto, objetivou-se realizar o isolamento e identificação das estirpes de S. aureus de amostras de leite bubalino da raça Murrah, dos insufladores, água das mangueiras da sala de ordenha e mãos dos ordenhadores em uma propriedade rural em Analândia - SP nos meses de abril, junho, outubro e novembro. Foi avaliada, a capacidade produtora de biofilme "in vitro" pelo método de aderência em microplacas e pelo cultivo em Agar Vermelho Congo (CRA), o perfil de sensibilidade dos S. aureus frente a antimicrobianos pelo método da difusão em disco além de identificar os genes que participam na formação de biofilme por Reação em Cadeia da Polimerase (PCR). Das 147 estirpes de Staphylococcus spp isoladas, 32 foram identificadas como S. aureus. Dos isolados, 28,1% foram positivas para o gene icaA, 21,8% para o gene icaD, 28,1% para clfA, 50% clfB, 53,1% sarA e 50% para o gene hla. Todas os isolados demonstraram habilidade de produção de slime em Agar Vermelho Congo (CRA) e formação de biofilme em microplaca. Embora a frequência de isolamento de S. aureus foi baixa, essas estirpes podem ser consideradas potenciais patógenos zoonóticos
Abstract: Staphylococcus aureus is the most common cause of mastitis in dairy herds due to its production of several virulence factors, including those related to its ability to colonize the teat parenchyma. The microbiological status of raw milk supplied to manufacturers may reflect on the final product and the associated risks to consumer health. Accordingly, the aim of this study was to isolate and characterize S. aureus strains from samples of Murrah buffalo milk, milking machines, water hoses from milking room and from the hands of milkers on a dairy farm located in Analândia - SP during the months of April, July, October, and November. Biofilm-producing capacity was evaluated "in vitro" using a microtiter method and cultivation on Congo Red Agar (CRA). Antibiotic sensitivity testing was performed by the disk diffusion method and the identification of the genes involved in biofilm formation by Polymerase Chain Reaction (PCR). One hundred and forty-seven Staphylococcus spp. were isolated and 32 were identified as S. aureus. Of those isolates, 28.1% were positive for the icaA gene, 21.8% for the icaD gene, 28.1% for the clfA, 50.0% for the clfB, 53.1% for sarA and 50.0% for hla. All isolates produced slime on Congo Red Agar (CRA) and formed biofilms in a microtiter assay. Although the frequency of S. aureus was low, the stains isolated can be considered as potential zoonotic agents
Mestre

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Estudou-se 94 estirpes de Staphylococcus aureus obtidas do leite de vacas com mastite subclínica em duas propriedades rurais no estado de São Paulo. Essas estirpes foram caracterizadas fenotipicamente quanto a produção de biofilmes pelos testes do agar vermelho congo e pelo teste de aderência em microplacas e também foram genotipicamente identificadas pela presença dos genes icaA e icaD responsáveis pela produção do polissacarídeo de adesão intercelular. Além disso, todas as estirpes foram também submetidas ao teste de sensibilidade aos antimicrobianos. Os resultados obtidos revelaram que 85% das estirpes produziram biofilmes “in vitro” para o teste do agar vermelho congo. No teste de aderência em placas foi verificado que 98,9% das estirpes produziram biofilme, devido a forte aderência nas placas de polietileno. A presença dos genes icaA e icaD foi encontrada em 95,7% das estirpes de S. aureus. No antibiograma foi observado que sete estirpes foram resistentes aos seguintes antimicrobianos: cloranfenicol, clindamicina, eritromicina, gentamicina, oxacilina e penicilinaG, sendo que as maiores resistências ocorreram frente a gentamicina (2,2%) e a penicilina G (6,6%). Na avaliação dos testes fenotípicos com os genotípicos o teste de aderência em microplacas foi o mais sensível (100%), sendo indicado para identificar estirpes produtoras de biofilmes. Todos os testes, com exceção do antibiograma, foram estatisticamente significativos (p<0,05).
94 Staphylococcus aureus strains obtained from milk samples of cows suffering from subclinical mastitis in dairy herd, in two properties, in the state of São Paulo were evaluated. These strains were characterized by the in vitro slime production on the Congo red agar, biofilm formation and by the presence of icaA and icaD genes which are responsible for the intercellular adhesion. All strains were too subjected to in vitro susceptibility to 12 different antibiotics. The results revealed that 85% of isolates tested produced slime on the Congo red agar and 98,9% of the isolates produced biofilm in vitro by the adherence in sterile 96-well “U” bottom polystyrene tissue culture plates. 95,7% of isolates possessed the icaA and icaD genes. The results of in vitro antibiotic susceptibility assay to 12 different antibiotics revealed that seven isolates were resistance to follow antibiotics: clorphenicol, clindamicin, erythromycin, gentamicin, oxacillin and penicillin, the higher resistance occurred to penicillin (6,6%) and gentamicin (2,2%). In the study of phenotypic tests compared with genotypic test, the 96-well polystyrene tissue culture plates assay was the most sensitivity (100%) and is recommended with genotypic test for the investigation biofilm formation in S. aureus. All the tests, exception of antibiotic susceptibility assay were statistically significant (p<005).

Dans le cadre d'infections ostéo-articulaire (IOA), l'utilisation de matériels étrangers peut, en cas de contamination, aboutir à la formation d'un biofilm associé à un risque plus important d'échec du traitement et de récidive. Les bactéries sous forme de biofilm sont en effet protégées de l'action du système immunitaire et ont une tolérance plus importante aux antibiotiques. A l'heure actuelle, l'activité des antibiotiques est déterminée par la CMI (Concentration Minimale Inhibitrice), mais cette valeur ne tient pas compte de la forme sessile des bactéries. C'est pourquoi, la société BioFilm Control a développé un nouveau test, l'Antibiofilmogramme®, permettant de déterminer la CMI biofilm (CMIb) reflétant la capacité préventive des antibiotiques sur l'installation des microorganismes en biofilm. L'objectif de ma thèse a été dans un premier temps de participer à la démonstration de la valeur clinique de ce nouveau test dans le cadre des IOA à Staphylococcus aureus. Nous avons pu mettre en place un recueil prospectif et réaliser les premiers essais in vitro. Nos résultats obtenus pour la cloxacillin ont pu par la suite être confirmés sur un modèle in vivo d'infection sur matériel. Dans un second temps, nous avons pu caractériser la capacité de formation de biofilm des souches cliniques en fonction des profils de résistance obtenus en Antibiofilmogramme®. Nous avons pu mettre en évidence des profils différents liés à la clonalité des souches. Enfin, nous avons pu mettre au point une nouvelle méthode de rinçage et de quantification des biofilms pour les modèles en microplaque via l'utilisation de vapeur. Cette approche simple améliore grandement la reproductibilité des résultats et préserve l'intégrité structurelle des biofilms
In the context of Bone and Joint Infections (BJIs), the orthopedic devices are preferential surface for microorganisms to adhere and form biofilm associated with high rates of failures and relapses. Within biofilm, bacteria are protected from the host immune response and are able to survive in the presence of high concentration of antibiotics. The standard Minimal Inhibitory Concentration (MIC) informs on the antibiotic susceptibility of planktonic bacteria, but is not suited for biofilm. The company BioFilm Control developed a new test named Antibiofilmogram® which measures early-stage biofilm growth in presence of antibiotics, and provides a biofilm Minimal Inhibitory Concentration (bMIC). The aim of my PhD research was first to take part in the demonstration of the clinical value of this new test for Staphylococcus aureus BJIs. We established a prospective collection of data and strains and realized the first in vitro assays. Our results for cloxacillin were confirmed in an in vivo model of catheter-associated infection. Second, we characterized the biofilm formation capacity of various clinical isolates based on the Antibiofilmogram® resistance profile. We showed that the biofilm formation capacity is correlated with clonal lineage. Finally, we were able to develop a new method of washing and quantifying biofilms for microplate system using steam. This simple approach preserves the biofilm integrity and lead to highly reproducible data

L’infection prothétique à Staphylococcus aureus reste un des problèmes majeurs de la chirurgie cardio-vasculaire. Cette pathologie, présente dans 5% des cas, est grevée d’une lourde morbi-mortalité. L’amélioration des connaissances des facteurs favorisant l’adhésion bactérienne, ainsi que les moyens de lutte contre cette infection apparaissent dès lors d’un intérêt majeur. Ce travail a donc porté sur l’influence de l’espèce bactérienne et des paramètres physico-chimiques du support, lors de l’adhésion au carbone pyrolytique et au polyester, éléments constitutifs des prothèses implantées en chirurgie cardiaque. Une deuxième partie est consacrée à l’étude des variations protéomiques et à l’identification des protéines d’intérêt du S. Aureus lors de son adhésion au carbone pyrolytique, afin de découvrir de nouvelles cibles thérapeutiques. Enfin, une dernière partie est consacrée à la lutte contre l’infection prothétique à S. Aureus par l’utilisation d’allogreffes cryopréservées
Prosthetic infection with Staphylococcus aureus remains one of the major problems of the cardio-vascular surgery with a high morbidity and mortality rate. The management and the treatment of these infections remain controversial. Therefore, a better knowledge of the mechanisms involved in biomaterial adhesion is essential. The aim of this study was to analyze the interaction of surface free energy and roughness characteristics of different pyrolytic carbon heart valves with three bacterial species on biofilm formation. A second part is devoted to the study of proteomic changes occurring during adhesion of S. Aureus on pyrolytic carbon and to the identification of proteins of interest. A final section focused on the treatment of S. Aureus prosthetic infection with the used of cryopreserved allografts

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Staphylococcus aureus é comum em infecções intramamárias, que frequentemente se tornam crônicas, entre outros fatores, pela capacidade dessa bactéria em produzir biofilme. Objetivou-se verificar a capacidade produtora de biofilme em estirpes de S. aureus e Staphylococcus coagulase negativa (SCN), oriundas de mastite bovina clínica e subclínica. Um total de 100 cepas de S. aureus e 100 cepas de SCN foram isoladas de casos de mastites subclínicas e clínicas em duas propriedades do Estado de São Paulo. O teste de triagem da mastite subclínica foi o California Mastitis Test (CMT). Foi realizada Contagem de Células Somáticas (CCS) e cultivo microbiológico das amostras de leite. Pesquisou-se a presença de genes icaA, icaD e bap pela Reação em Cadeia pela Polimerase (Polimerase Chain Reaction - PCR) e caracterização fenotípica no ágar Vermelho Congo (VC) e teste de produção de biofilme em placas. Não houve relação entre a intensidade da reação ao CMT e a CCS. A média de CCS foi de 2,88±0,59 para S. aureus e 2,71±0,35 para SCN. Não houve significância estatísticas entre os valores de CCS para estirpes que continham a presença dos genes pesquisados. Porém as médias dos valores de CCS foram maiores nos isolados que continham tais genes. As frequências de icaA, icaD e bap em S. aureus foram de 82%, 83% e 58%, respectivamente. Observou-se que dos 83 isolados de S. aureus que apresentaram icaD, 82 (98,8%) também apresentaram icaA, com significância estatística no Teste Exato de Fischer (p<0,001). Na associação entre os genes icaA e bap, também foi observada associação significante pois das 82 estirpes que apresentaram icaA, foi possível observar a presença de bap em 56 (68,3%). Das 83 linhagens que continham o gene icaD em 56 (67,5%) delas, também foi detectada presença do gene bap. Não houve concordância entre os testes fenotípicos (K<0,2). A positividade no vermelho congo revelou que 25% das...
Staphylococcus aureus is common in intramammary infections, which often become chronic, among other factors, by the ability of this bacteria to produce biofilm. The objective was to verify the production of biofilm capacity in strains of S. aureus and coagulase negative Staphylococcus (CNS), derived from clinical and subclinical bovine mastitis. A total of 100 strains of S. aureus and 100 strains of CNS were isolated from cases of subclinical and clinical mastitis in two properties of São Paulo State. The screening test of subclinical mastitis was the California Mastitis Test (CMT). Somatic Cell Count (SCC) and microbiological culture of milk samples was performed. We researched the presence of icaA, icaD and bap genes by the Polymerase Chain Reaction (PCR) and phenotypic characterization on congo red agar (CRA) and biofilm production on plates. There was no observed relationship between the intensity of the reaction to the CMT and SCC. The mean of SCC was 2.88 ± 0.59 for S. aureus and 2.71 ± 0.35 for CNS. There was no significant statistical between SCC values for strains containing the presence of those researched genes. But the means of the CCS values were higher in isolates that containing such genes. The frequency icaA, icaD and bap in S. aureus were 82%, 83% and 58%, respectively. It was observed that the 83% of S. aureus isolates that had icaD, 82 (98.8%) also had icaA, with statistical significance in the Fischer's exact test (p <0.001). In association between icaA and bap genes, was also observed significant association, for the 82 strains that showed icaA, we observed the presence of bap in 56 (68.3%). Of the 83 strains that containing the icaD gene, 56 (67.5%) of them was also detected the presence of bap gene. There was no agreement between the phenotypic tests (K<0.2). The positivity on congo red agar revealed that 25% of S. aureus strains produced in vitro biofilm. In the adhesion test plates, this number was lower, with ...
FAPESP: 2013/14383-9

Orientador: Hélio Langoni
Coorientador: Virgínia Bodelão Richini-Pereira
Banca: Paulo Francisco Domingues
Banca: Vera Lucia Mores Rall
Resumo: Staphylococcus aureus é comum em infecções intramamárias, que frequentemente se tornam crônicas, entre outros fatores, pela capacidade dessa bactéria em produzir biofilme. Objetivou-se verificar a capacidade produtora de biofilme em estirpes de S. aureus e Staphylococcus coagulase negativa (SCN), oriundas de mastite bovina clínica e subclínica. Um total de 100 cepas de S. aureus e 100 cepas de SCN foram isoladas de casos de mastites subclínicas e clínicas em duas propriedades do Estado de São Paulo. O teste de triagem da mastite subclínica foi o California Mastitis Test (CMT). Foi realizada Contagem de Células Somáticas (CCS) e cultivo microbiológico das amostras de leite. Pesquisou-se a presença de genes icaA, icaD e bap pela Reação em Cadeia pela Polimerase (Polimerase Chain Reaction - PCR) e caracterização fenotípica no ágar Vermelho Congo (VC) e teste de produção de biofilme em placas. Não houve relação entre a intensidade da reação ao CMT e a CCS. A média de CCS foi de 2,88±0,59 para S. aureus e 2,71±0,35 para SCN. Não houve significância estatísticas entre os valores de CCS para estirpes que continham a presença dos genes pesquisados. Porém as médias dos valores de CCS foram maiores nos isolados que continham tais genes. As frequências de icaA, icaD e bap em S. aureus foram de 82%, 83% e 58%, respectivamente. Observou-se que dos 83 isolados de S. aureus que apresentaram icaD, 82 (98,8%) também apresentaram icaA, com significância estatística no Teste Exato de Fischer (p<0,001). Na associação entre os genes icaA e bap, também foi observada associação significante pois das 82 estirpes que apresentaram icaA, foi possível observar a presença de bap em 56 (68,3%). Das 83 linhagens que continham o gene icaD em 56 (67,5%) delas, também foi detectada presença do gene bap. Não houve concordância entre os testes fenotípicos (K<0,2). A positividade no vermelho congo revelou que 25% das...
Abstract: Staphylococcus aureus is common in intramammary infections, which often become chronic, among other factors, by the ability of this bacteria to produce biofilm. The objective was to verify the production of biofilm capacity in strains of S. aureus and coagulase negative Staphylococcus (CNS), derived from clinical and subclinical bovine mastitis. A total of 100 strains of S. aureus and 100 strains of CNS were isolated from cases of subclinical and clinical mastitis in two properties of São Paulo State. The screening test of subclinical mastitis was the California Mastitis Test (CMT). Somatic Cell Count (SCC) and microbiological culture of milk samples was performed. We researched the presence of icaA, icaD and bap genes by the Polymerase Chain Reaction (PCR) and phenotypic characterization on congo red agar (CRA) and biofilm production on plates. There was no observed relationship between the intensity of the reaction to the CMT and SCC. The mean of SCC was 2.88 ± 0.59 for S. aureus and 2.71 ± 0.35 for CNS. There was no significant statistical between SCC values for strains containing the presence of those researched genes. But the means of the CCS values were higher in isolates that containing such genes. The frequency icaA, icaD and bap in S. aureus were 82%, 83% and 58%, respectively. It was observed that the 83% of S. aureus isolates that had icaD, 82 (98.8%) also had icaA, with statistical significance in the Fischer's exact test (p <0.001). In association between icaA and bap genes, was also observed significant association, for the 82 strains that showed icaA, we observed the presence of bap in 56 (68.3%). Of the 83 strains that containing the icaD gene, 56 (67.5%) of them was also detected the presence of bap gene. There was no agreement between the phenotypic tests (K<0.2). The positivity on congo red agar revealed that 25% of S. aureus strains produced in vitro biofilm. In the adhesion test plates, this number was lower, with ...
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O objetivo deste estudo foi avaliar a capacidade de produção de nanopartículas de prata através do extrato da casca de romã, e produzir formulações contendo estas nanopartículas para uso em feridas. Elas foram testadas quanto à ação antimicrobiana, citotóxica e potencial cicatrizante. Para a produção das nanopartículas de prata propôs-se uma síntese utilizando-se como base duas metodologias já estabelecidas na literatura, utilizando-se para reação carboximetilcelulose, propilenoglicol, nitrato de prata, água e extrato da casca de romã como agente redutor. O extrato da casca de romã foi caracterizado em parâmetros como pH, massa seca e quantidade de taninos bioativos (ácido elágico e totais fenólicos expressos em ácido gálico). Os totais fenólicos do extrato foram também dosados após seu aquecimento nas diferentes condições de tempo e temperatura propostos para as sínteses das nanopartículas de prata (12 minutos, 1 hora e 2 horas, à 50ºC e 100ºC). As nanopartículas de prata produzidas foram, então, adicionadas a uma solução contendo compostos para produção de formulações para serem utilizadas no tratamento de feridas. Elas foram caracterizadas através de espectroscopia UV-Visível, microscopia eletrônica de varredura (MEV), potencial zeta e dosagem de íons remanescentes após as reações. A atividade antimicrobiana tanto das nanopartículas como de suas formulações contra Candida albicans SC 5314 e Staphylococcus aureus ATCC 25923 foi avaliada por meio do método da microdiluição. Na avaliação dos extratos, apesar de ocorrer um aumento na concentração de totais fenólicos com o aumento da temperatura, a concentração inibitória mínima (CIM) manteve-se estável com valores de 391 µg/ml e 781 µg/ml para S. aureus e C. albicans. A formação das nanopartículas de prata foi confirmada com a formação de picos característicos na espectroscopia UV-Visível e pelas imagens de MEV, onde verificou-se que a síntese com tempo de reação de 12 minutos e aquecimento a 50ºC gerou nanopartículas mais uniformes e melhor distribuídas na formulação. As sínteses propostas promoveram a redução iônica da prata de aproximadamente 100%, independente do tempo temperatura utilizados na reação. Valores de CIM para as nanopartículas de prata foram de 67,50 µg/ml e 68,75 µg/ml respectivamente para S. aureus e C. albicans independente das variações das condições de síntese. Após a seleção da síntese das nanopartículas de prata por 12 min à 50ºC, estas partículas foram também caracterizadas por difração de raios-X e microscopia eletrônica de transmissão (TEM), e as respectivas formulações por meio de espalhamento de luz dinâmica, dosagem de íons livres, potencial zeta, MEV e TEM. Para essas formulações foi também realizado um teste de estabilidade variando-se umidade e temperatura. Além da atividade antimicrobiana contra Candida albicans SC 5314 e Staphylococcus aureus ATCC 25923, a citotoxicidade em fibroblastos (L929) das nanopartículas de prata e das formulações destas partículas e do extrato da casca de romã foram também avaliadas. Para os extratos foram observados valores de 3,13, 86,39±0,96% m/m, 3,64±0,03 mg/g, 392,0±9 mg/g respectivamente para pH, massa seca, ácido elágico e totais fenólicos. Como controle, foram produzidas nanopartículas de prata sintetizadas convencionalmente (AgNP química), e observou-se potencial redutor de 99,89% e 99,51% para as sínteses utilizando-se extrato de romã (AgNP green) e um agente redutor químico convencional (AgNP química). Verificou-se a formação de partículas com tamanhos médios de 89 e 19 nm para nanopartículas green e química. A formulação contendo as nanopartículas de prata apresentaram um potencial antimicrobiano expressivamente maior do que o princípio ativo isolado, sendo 255 e 4 vezes mais efetiva contra S. aureus e C. albicans, respectivamente. Os valores de citotoxicidade foram consideravelmente menores para as nanopartículas de prata sintetizadas pelo extrato de romã quando comparadas as produzidas convencionalmente. De acordo com os valores de CIM e da citotoxicidade, a concentração das formulações foi ajustada, gerando assim três formulações: i) AgNP green, ii) AgNP química e iii) extrato de romã, nas concentrações de 337,5 µg/ml, 5,55 µg/ml e 94 µg/ml respectivamente. Para o estudo in vivo, utilizou-se como controle um medicamento comercial contendo prata indicado para tratamento de feridas (Sulfadiazina de prata). Foram selecionados noventa ratos Wistar machos com peso médio de 180 gramas. Foi induzida diabetes nos ratos, e, em seguida, realizou-se duas incisões no dorso dos animais e as lesões foram imediatamente infectadas com S. aureus (ATCC 25923) e C. albicans (SC 5314). Após 24 h, os animais foram divididos em grupos de acordo com as formulações propostas em cada tratamento, seguindo-se um protocolo de duas vezes ao dia por 2, 7 e 14 dias. Após o período de tratamento os animais foram eutanasiados e verificado o potencial reparador através do índice de fechamento de ferida, avaliação do infiltrado inflamatório, angiogenese, mieloperoxidase e hidroxiprolina. Ainda foi verificado o potencial antimicrobiano das formulações através da contagem de células viáveis de cada microrganismo infectado nas feridas. De forma geral, as formulações contendo nanopartículas de prata mostraram os melhores resultados para o fechamento das feridas, apresentando ainda uma atividade anti-inflamatória maior que a do extrato de casca de romã, o qual apresentou atividade pró inflamatória. Todos os tratamentos não foram capazes de reduzir de forma significativa o número de células de C. albicans, enquanto para S. aureus todos os tratamentos apresentaram redução significativa após quatorze dias de tratamento. Independente das nanopartículas de prata serem produzidas quimicamente ou por meio do extrato da casca de romã, ambas apresentaram considerável potencial de reparo de feridas infectadas em modelos in vivo com ratos. Os achados das presentes pesquisas reforçam e estimulam o uso potencial das nanopartículas de prata no tratamento de feridas, com destaque para a síntese green por gerar menos danos ao meio ambiente e às pessoas envolvidas tanto em sua produção como aos pacientes, e por apresentar, ainda, custo inferior quando comparada às sínteses utilizando reagentes químicos e processos convencionais.
The aim of this study was to investigate the production of silver nanoparticles through peel extract of pomegranate, and produce formulations containing these particles to be used in wound healings. Its antimicrobial action, cytotoxicity and healing potential were tested. The synthesis of silver nanoparticles were based on two methods proposed in the literature with some modifications, which were used carboxymethylcellulose, propylene glycol, silver nitrate, water and peel extract of pomegranate as reducing agent. The peel extract was characterized by pH, dry mass and bioactive tannins (elagic acid and total phenols expressed as galic acid). The total phenols were also quantified after being heated at 50ºC and 100ºC for 12 minutes, 1 hour and 2 hours. Then, silver nanoparticles were added in a solution containing products to develop a formulation to be tested in wound healing. They were characterized by UV-Vis spectroscopy, scanning electron microscopy (SEM), zeta potential and the quantification of remaining silver ions after the synthesis reaction. The antimicrobial activity of the nanoparticles and formulations were tested against Candida albicans (SC 5314) e Staphylococcus aureus (ATCC 25923) by microdilution method. After submitting the peel extract to different conditions of temperature and times (50ºC and 100ºC for 12 minutes, 1 hour and 2 hours), it was noted that the values of the minimum inhibitory concentration was not affected and were 391 µg/ml and 781 µg/ml for S. aureus and C. albicans. The formation of silver nanoparticles was confirmed through the formation of characteristic peaks in the UV-Vis spectroscopy and SEM images, and it was observed that the reaction at 50ºC for 12 min produced silver nanoparticles with regular forms and better dispersed in the formulation. The synthesis proposed promoted the reduction of silver ions at about 100%, regardless of the time and temperature used in the reaction, which also did not interfere in antimicrobial activity against C. albicans (68,75 µg/ml) and S. aureus (67,50 µg/ml). After selecting the reaction at 50ºC for 12 min, the silver nanoparticles produced ere also characterized by X-ray diffraction and transmission electron microscopy (TEM), and the respective formulations through dynamic light scattering, free ion dosage, zeta potential, SEM and TEM. The stability test varying humidity and temperature was also performed for those formulations. Besides antimicrobial assays, the cytotoxicity (L929 fibroblasts) of the silver nanoparticles and the formulations of these particles and of the pomegranate peel extract were evaluated. It was observed in the peel extract the values of 3,13, 86,39±0,96% m/m, 3,64±0,03 mg/g and 392,0±9 mg/g respectively for pH, dry mass, elagic acid and total phenols concentrations. Silver nanoparticles produced by conventional chemical method was prepared and used as controls, and it was noted the reduction potential of 99,89% and 99,51% for the synthesis using pomegranate peel extract (AgNP green) and chemical reducing agent (AgNP chemical). The averages sizes of green and chemical AgNP were 89 and 19 nm. The formulation containing silver nanoparticles presented an antimicrobial potential expressively higher than the active input, being 254 and 5-fold more effective against S. aureus and C. albicans. Also, the cytotoxicity was notable reduced when silver nanoparticles were produced using pomegranate peel extract. Based on the MIC values and the cytotoxicity findings, the concentration of the formulations were determined: i) AgNP green at 337.5 µg/ml, ii) AgNP chemical at 5.55 µg/ml, and iii) pomegranate peel extract at 94 µg/ml. In the in vivo study, a commercial form of silver (Sulfadizsine) to the wound healing was used as control. Ninety Wistar male rats were selected, and, after inducing diabetes, two incisions on the dorsum of the animals were made and followed infected with S. aureus (ATCC 25923) and C. albicans (SC 5314). After the infection, the animals were treated with the formulations twice a day for 2, 7 and 14 days. Then, the animals were euthanized and the repair potential was verified through wound closure index, inflammatory infiltrate evaluation, angiogenesis, myeloperoxidase and hydroxyproline. It was also determined the antimicrobial potential by counting the viable cells of each microorganism used to infect the wounds. In general, the formulations containing silver nanoparticles promoted a better closure of the wounds, and a higher anti-inflamatory activity than the peel extract formulation which otherwise presented a pro-inflamatory effect. All formulations could not significantly reduce the viable cells of C. albicans, while for S. aureus they reduced significantly the cells after 14 days of treatment. Silver nanoparticles produced by both green and conventional chemical process present notable potential in repairing infected wounds in in vivo rat model. The findings of the present research strengthen and stimulate the potential application of silver nanoparticles in wound healings, highlighting the green production of these particles which apart from being lower costly, it is ecofriendly and less detrimental to people involved in its production and use.
FAPESP: 2016/04230-9

Orientador: Carlos Eduardo Vergani
Resumo: Esta presente tese foi dividida em quatro estudos que tiveram como objetivos. 1. Validar um protocolo e comparar o efeito dos tampões RPMI/MOPS e RPMI/HEPES no desenvolvimento de biofilmes e na viabilidade celular de queratinócitos (NOK-si e HaCat); 2. Comparar o dano celular e a resposta inflamatória induzidos pelos metabólitos de biofilmes simples e misto de Staphylococcus aureus e Candida albicans; 3. Avaliar o tipo de morte celular (apoptose vs. necrose) e a ativação de caspases relacionadas aos metabólitos desses biofilmes e 4. Caracterizar um tecido oral reconstituído e analisar o dano tecidual causado pelo sobrenadante e biofilme propriamente dito desses microrganismos. No estudo 1, a viabilidade celular foi avaliada pelo método colorimétrico do MTT e por imagens da cultura após 12 horas em contato com os meios de cultura. Ambos os tampões permitiram similar crescimento do biofilme. Efeito citotóxico do MOPS foi verificado após 6 horas de crescimento de NOK-si e HaCat. Houve preservação da viabilidade e morfologia quando as células foram expostas a RPMI/HEPES. Conclui-se que RPMI/HEPES pode ser utilizado como um meio tamponamente viável para estudos que avaliam o efeito do biofilme em cultura de queratinócitos ao longo do tempo. No estudo 2, o sobrenadante dos biofilmes de 36 h de C. albicans e S. aureus, isolados ou em associação, foi colocado em contato com NOK-si, HaCat e macrófagos (J774A.1). O dano celular foi avaliado por meio de ensaios de viabilidade celular ... (Resumo completo, clicar acesso eletrônico abaixo)
The present thesis was divided into four studies with the following objectives. 1. Validate a protocol and compare the effect of RPMI/MOPS and RPMI/HEPES buffers on the development of biofilms and keratinocyte cell viability (NOK-si and HaCat); 2. Compare the cellular damage and the inflammatory response induced by the metabolites of simple and mixed biofilms of Staphylococcus aureus and Candida albicans; 3. Evaluate the type of cell death (apoptosis vs. necrosis) and the activation of caspases related to the metabolites of these biofilms and 4. Characterize the reconstituted oral tissue and analyze the tissue damage caused by the supernatant and biofilm of these microorganisms. In study 1, cell viability was evaluated by the MTT colorimetric method and by culture images after 12 hours in contact with the culture media. Both buffers permitted similar biofilm growth. The cytotoxic effect of MOPS was observed after six hours of NOK-si and HaCat growth. There was preservation of viability and morphology when cells were exposed to RPMI/HEPES. It was concluded that RPMI/HEPES can be used as a buffering medium for studies evaluating the effect of biofilm on keratinocyte culture over time. In study 2, the supernatant of the 36-hour biofilms of C. albicans and S. aureus, isolated or in combination, was placed in contact with NOK-si, HaCat and macrophages (J774A.1). Cell damage was assessed by cell viability assays (MTT) and LDH enzyme release. Cytokine production was analyzed by the ELISA method and evaluation of the type of cell death by the staining of the apoptotic cells with annexin V and the necrotic cells with propidium iodide. The mixed biofilm and biofilm of C. albicans were more cytotoxic, and the mixed biofilm caused greater cellular damage through the release of the LDH enzyme. S. aureus biofilm metabolites stimulated greater production of NO, IL-6 and TNF-α... (Complete abstract electronic access below)
Doutor

L'implantation de valves mécaniques et les prothèses vaculaires en polyester est devenue quotidien. Malgrè les dispositions prises pour limiter les contaminations, l'infection prothétique survient dans 5% des cas. Ce type d'infection se caractérise par la présence d'un biofilm évolué très resistant au traitement antibiotique, aboutissant à un sepsis difficilement contrôlable. L'identification des facteurs favorisant l'adhésion bactérienne pourrait permettre de mieux prévenir et diagnostiquer ce type d'infection. Les objectifs de cette étude sont donc de déterminer les changements phénotypiques de S. Aureus , pathogène majeur de l'infection prothétique, inhérents à l'adhésion sur les prothèses cardio-vasculaires. Nous avons donc étudié les interactions physico-chimiques entre la bactérie et le support. Puis, nous avons déterminé les modifications du protéome de S. Aureus lors de l'adhésion sur quatre type de prothèses cardio-vasculaires et identifié les protéines d'intérêt
Mechanical heart valves and vascular protheses are routinely implanted. Despite of rigorous aseptie, prosthetic infection arises in 5% of the cases. Afetr the adhesion phase, a mature biofilm appeared leading to a sepsis, highly resistant to antibiotherapy. Better knowledge of factors promoting bacterial adhesion could allow to prevent and diagnose this tpe of infection. The aims of this study are to determine the phenotypical changes of S. Aureus , a major pathogenn, that occure during the adhesion on cardiovascular prostheses. We have studied the phisico-chemical interactions between bacteria and support. Then, we have determined the proteomic changes of S. Aureux during the adhesion on four types of cardiovascular protheses and we have identified proteins of interest

Les bactéries forment des communautés spatiales adhérentes à des surfaces, appelées biofilms. Ces organisations bactériennes sont omniprésentes dans les milieux naturel, industriel et médical et peuvent porter atteinte à notre santé lorsqu'elles hébergent des agents pathogènes, parmi lesquels le médiatique Staphylococcus aureus sur lequel a porté l'ensemble de ce travail de thèse. Cette bactérie est l'une des principales causes d'infections chroniques, mais également d'infections nosocomiales, impliquant le plus souvent des biofilms. Il est aujourd'hui reconnu qu'une telle biostructure est un véritable bouclier à l'action des antimicrobiens et à celle du système immunitaire. Outre les résistances génétiques des bactéries pathogènes aux antibiotiques, l'hétérogénéité chimique et biologique de la structure tridimensionnelle des biofilms pourrait être à l'origine de ces phénomènes de tolérance et de chronicité d'infections. C'est à cette problématique que se rattache ce travail de thèse concernant l'action de la vancomycine sur des biofilms de S. aureus. Alors que les connaissances sur la réactivité de cet antibiotique clef avec S. aureus proviennent essentiellement d'études réalisées sur des cellules planctoniques, l'originalité de notre approche a été d'étudier la diffusion-réaction de la vancomycine in situ dans l'épaisseur des biofilms en utilisant en particulier des outils avancés de microscopie de fluorescence (Time-Lapse, FLIM, FRAP, et FCS). Nous avons ainsi évalué sa biodisponibilité dans la matrice d'exopolymères, ainsi que l'impact de la physiologie spécifique des bactéries incluses en biofilms sur l'activité de cet antibiotique, utilisé seul ou en association avec la rifampicine. Cette approche multidisciplinaire a permis une meilleure compréhension des mécanismes impliqués dans la singulière tolérance de ces biostructures à l'action des antibiotiques, et de souligner l'urgence de développer des approches préventives telles que le diagnostic précoce des infections impliquant des biofilms.

Staphylococcus aureus has developed resistance to several antibiotics including vancomycin, which is often used as a “last resort” treatment. There is an ever-increasing need to develop novel antimicrobial treatments to combat S. aureus and other drug resistant bacteria. Microorganisms are most often found in polymicrobial communities where they either exhibit synergistic or antagonistic relationships. Competition between microorganisms can lead to the discovery of new antimicrobial targets as the specific mechanisms of resistance are elucidated. In addition, synergistic treatments are being evaluated for their combined effect and potential to decrease the concentration of drugs needed, and thus the side effects also. Alcaligenes faecalis is a microorganism that our lab has previously shown to inhibit S. aureus and other various bacterial species. In this study, we found that A. faecalis reduces the planktonic growth of S. aureus by 94.5% and biofilm growth by 76.6%. A. faecalis also has a synergistic effect when paired with bacitracin to reduce the planktonic growth by 99.9% and biofilm growth by 99.7%. Transposon mutagenesis was successfully performed on A. faecalis, and loss of function mutations were attained. Two mutants were no longer able to inhibit the growth of Staphylococcus aureus, Candida albicans, or Bacillus megaterium. Further analysis and genomic sequencing of these mutants is needed to determine the gene(s) that were interrupted and the mechanism of A. faecalis’ antimicrobial activity. The findings of this study may aid in the identification of new therapeutic targets for novel S. aureus treatments.

La formation des biofilms dans le secteur alimentaire et hospitalier constitue une cause principale soit d’intoxications alimentaires, soit d’infections nosocomiales. Pour prévenir ces infections, les travaux de thèse ont concerné, dans un premier temps, l’étude de l’effet de la température de culture sur la prédiction théorique de l’adhésion bactérienne, de Pseudomonas aeruginosa et de Staphylococcus aureus, sur l’acier et le polycarbonate. Les résultats ont montré que l’historique écologique de la bactérie influence significativement les propriétés de la surface bactérienne et par conséquent l’adhésion cellulaires sur les supports abiotiques. Cependant, les modèles mathématiques étudiés ne semblent pas être adéquats pour prédire les données expérimentales. Dans un deuxième temps, un system statique de formation des biofilms a été mis au point. Ce système a permis d’étudier l’effet de la température de culture, le type de support et l’âge physiologique sur la résistance des biofilms aux désinfectants. La structure tridimensionnelle des biofilms, la production qualitative, et quantitative, de la matrice extracellulaire ont été étudiés pour comprendre les mécanismes de résistance des biofilms. De plus, le profil d’acides gras membranaires des bactéries structurées en biofilm a été caractérisé afin d’étudier les mécanismes de résistance des biofilms à l’échelle cellulaire. Les résultats montrent que la résistance des biofilms aux agents biocides dépend des conditions environnementales de la formation des biofilms. Les résultats montrent aussi que la matrice extracellulaire ne permet pas toujours d’expliquer la résistance des biofilms aux traitements biocides et que l’état physiologie des bactéries structurées en biofilm joue un rôle significatif dans cette résistance
The biofilm formation in food and medical sectors represents a significant source of infections worldwide such as the foodborne and nosocomial ones. To prevent infections, the the first part of this PhD thesis has dealt with the effect of growth temperature of Pseudomonas aeruginosa, and Staphylococcus aureus, cells on the theoretical prediction of bacterial adhesion to stainless steel and polycarbonate. The results showed that the bacterial background influenced the surface properties of bacterial cells and therefore the bacterial adhesion to the two selected surfaces. However, the mathematical theories seem to be inadequate to predict the bacterial adhesion to abiotic surfaces. Thereafter, a static biofilm reactor was established. This system has served to study the effect of the growth temperature, surface type and incubation time on the biofilm resistance to disinfectants. In order to understand the mechanisms of biofilm resistance to disinfectants, the investigations were carried out at a microscopic and macroscopic level. In fact, the three-dimensional structure of biofilms was investigated under the conditions selected for this study. Moreover, qualitative and quantitative studies of the biofilm matrix were also realized. In addition, the membrane fluidity of sessile cells was investigated through the study of membrane fatty acid profiles. The results showed that the biofilm resistance is a complex phenomenon and depends on several parameters. The results also showed that the biofilm matrix cannot always explain the biofilm resistance to antimicrobial agents. In fact, other factors related the physiological states of sessile bacteria are involved in this resistance

Orientador: Antonio Nader Filho
Banca: Luiz Augusto do Amaral
Banca: Elisabeth Loshchagin Pizzolitto
Resumo: Estudou-se 94 estirpes de Staphylococcus aureus obtidas do leite de vacas com mastite subclínica em duas propriedades rurais no estado de São Paulo. Essas estirpes foram caracterizadas fenotipicamente quanto a produção de biofilmes pelos testes do agar vermelho congo e pelo teste de aderência em microplacas e também foram genotipicamente identificadas pela presença dos genes icaA e icaD responsáveis pela produção do polissacarídeo de adesão intercelular. Além disso, todas as estirpes foram também submetidas ao teste de sensibilidade aos antimicrobianos. Os resultados obtidos revelaram que 85% das estirpes produziram biofilmes "in vitro" para o teste do agar vermelho congo. No teste de aderência em placas foi verificado que 98,9% das estirpes produziram biofilme, devido a forte aderência nas placas de polietileno. A presença dos genes icaA e icaD foi encontrada em 95,7% das estirpes de S. aureus. No antibiograma foi observado que sete estirpes foram resistentes aos seguintes antimicrobianos: cloranfenicol, clindamicina, eritromicina, gentamicina, oxacilina e penicilinaG, sendo que as maiores resistências ocorreram frente a gentamicina (2,2%) e a penicilina G (6,6%). Na avaliação dos testes fenotípicos com os genotípicos o teste de aderência em microplacas foi o mais sensível (100%), sendo indicado para identificar estirpes produtoras de biofilmes. Todos os testes, com exceção do antibiograma, foram estatisticamente significativos (p<0,05).
Abstract: 94 Staphylococcus aureus strains obtained from milk samples of cows suffering from subclinical mastitis in dairy herd, in two properties, in the state of São Paulo were evaluated. These strains were characterized by the in vitro slime production on the Congo red agar, biofilm formation and by the presence of icaA and icaD genes which are responsible for the intercellular adhesion. All strains were too subjected to in vitro susceptibility to 12 different antibiotics. The results revealed that 85% of isolates tested produced slime on the Congo red agar and 98,9% of the isolates produced biofilm in vitro by the adherence in sterile 96-well "U" bottom polystyrene tissue culture plates. 95,7% of isolates possessed the icaA and icaD genes. The results of in vitro antibiotic susceptibility assay to 12 different antibiotics revealed that seven isolates were resistance to follow antibiotics: clorphenicol, clindamicin, erythromycin, gentamicin, oxacillin and penicillin, the higher resistance occurred to penicillin (6,6%) and gentamicin (2,2%). In the study of phenotypic tests compared with genotypic test, the 96-well polystyrene tissue culture plates assay was the most sensitivity (100%) and is recommended with genotypic test for the investigation biofilm formation in S. aureus. All the tests, exception of antibiotic susceptibility assay were statistically significant (p<005).
Mestre

Health Care Associated Infections (HCAIs) are a concern especially in regards to antibiotic resistance and effective treatments. Staphylococcus aureus is often the main focus for eradication and prevention procedures, however, other bacterial species are also problematic. These include Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus epidermidis amongst others. Chronic infections caused by these bacteria are often biofilm related, and include dental caries, otitis media, osteomyelitis, burns & chronic wounds, and device related & prosthetic joint infections. Prosthetic joints and indwelling devices, such as catheters, are a prime environment on which biofilms can develop. This thesis aims to look at biofilms, investigating how they are established, the development of resistance against individual antibiotics and the antibiotic concentrations required to reduce biofilm load. A novel biofilm system – the alginate bead method will be used for these experiments, The alginate bead method was developed by a previous student in the Gallagher Laboratory, due to a need to have a reliable, robust and inexpensive technique to examine formation of biofilms and antibiotic resistance. There are devices and assays available, such as the Calgary Biofilm Device, which are extensively used for these purposes. However, the cost is prohibitive. This thesis found that the development of biofilms occurs much earlier than expected, with stable, fixed formation after just four hours of growth. Depending upon the antibiotic, resistance can develop within the first two hours of growth and thereafter steadily increases. By 24 hours the biofilms are fully resistant to all the tested antibiotics. In mixed species biofilms, the two species act synergistically protecting each other against the antibiotics, resulting in a much higher antibiotic concentration required. Common antibiotics used to treat staphylococcal infections are often combined to enhance their destructive effect and prevent the development of resistance. The effects of these antibiotics, when combined was explored. Biofilm resistance against gentamicin, one of the most common antibiotics used to treat staphylococcal infections develops quickly. However, when combined with other antibiotics gentamicin resistance is delayed. As antibiotic concentrations have to be extremely high in order to have any effect on established biofilms, alternative methods need to be investigated. Any alternative approaches would be employed in conjunction with conventional therapies preventing stable biofilm formation and disrupting established biofilms. Such methods may include sugar metabolites, enzymatic disruption, D-amino acids and activation of the quorum sensing system. The main conclusion which can be taken from this work are that firstly the alginate bead method of a viable, suitable alternative to the Calgary Biofilm Device and supports biofilm formation and testing. Secondly that biofilms form and are resistant to antibiotics much earlier than expected, and extreme concentrations of antibiotics are required to have an effect. Thus the inclusion of alternative methods which disrupt biofilms would be beneficial to clinical practice. However, the alternative methods investigated within this thesis (D-amino acids and sugar metabolites) failed to show any inhibition of biofilms. There are other possible choices which would need to be investigated.

Orientador: Luciane Dias de Oliveira
Banca: Antonio Olavo Cardoso Jorge
Banca: Priscila Christiane Susy Liporoni
Banca: Luana M. R. de Vasconcellos
Banca: Cristiane Aparecida Pereira
Resumo: Os micro-organismos estão cada vez mais resistentes aos medicamentos disponíveis tanto na medicina quanto na odontologia, e esta resistência é ainda maior quando estão organizados em biofilmes mono ou multiespécies, de modo que o estudo de antimicrobianos alternativos, como fitoterápicos, estão em crescente ascensão. A interação entre leveduras e bactérias está intimamente presente na cavidade bucal, em que nichos como dentes, língua, mucosa e bolsa periodontal, nutrientes e temperatura adequados promovem condições favoráveis para formação do biofilme. Com isso, o objetivo deste trabalho foi avaliar a atividade antimicrobiana dos extratos de Pfaffia paniculata K (pfaffia), Hamamelis virginiana L. (hamamelis), Stryphnodendron barbatiman (barbatimão) e Gymnema sylvestre (gimena) em biofilmes heterotípicos de Candida albicans (ATCC 18804) com Streptococcus mutans (ATCC 35688), Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (ATCC 4083) e Pseudomonas aeruginosa (ATCC 15442). Para isso, suspensões padronizadas em 107 cels/mL dos micro-organismos testes, foram distribuídos em placas de microtitulação de 96 poços, juntamente 100 µL de caldo BHI. As placas foram incubadas em estufa bacteriológica a 37ºC/48h (5% de CO2 para S. mutans) e, após, os biofilmes foram submetidos ao tratamento com os extratos por 5 min e 24 h, nas respectivas concentrações de 100 mg/mL, 50 mg/mL e 25 mg/mL. Foi utilizado solução salina 0,9% (5 min) ou caldo BHI (24 h) nos grupos controles. Após, os ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Microorganisms are increasingly resistant to drugs available in both medicine and dentistry, and this resistance is even greater when they are organized into mono or multispecies biofilms, so that the study of alternative antimicrobials, such as herbal medicines, are on the rise. The interaction between yeasts and bacteria is intimately present in the oral cavity, in which niches such as teeth, tongue, mucosa and periodontal pocket, food and adequate temperature promote adequate conditions for biofilm formation. The aim of this study was to evaluate the antimicrobial activity of extracts of Pfaffia paniculata K., Hamamelis virginiana L., Gymnema sylvestre and Stryphnodendron barbatiman M. in heterotopic biofilms of Candida albicans (ATCC 18804) with Streptococcus mutans (ATCC 35688). Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (ATCC 4083), and Pseudomonas aeruginosa (ATCC 15442). Standardized suspensions at 107 cells/mL of the test microorganisms were distributed in 96 well microtiter plates together with 100 μL of BHI broth, the plates were incubated in a bacteriological oven at 37°C/48h (5% CO2 for S. mutans). After the incubation time, treatments were performed at 5 min and 24 h times, applying the respective extracts at the concentrations of 100 mg, 50 mg and 25 mg/mL and applying 0.9% saline solution or BHI broth in the control groups. After the biofilms were washed and disaggregated from the bottom of the plate, performing serial dilutions for later seeding... (Complete abstract click electronic access below)
Doutor

Bacterial infections are becoming increasingly difficult to treat due to the emergence of antimicrobial resistance, (AMR), which renders current antimicrobial therapies ineffective. Further complicating matters is the ability of bacteria to form communities called biofilms, which are infamous for their tolerance to antimicrobial therapy. Biofilm mediated tolerance and AMR contribute to disease chronicity. Consequently, there is a need for novel antimicrobial interventions. The aim of this thesis was to characterise a novel antimicrobial, HT61, against biofilms of clinically relevant bacterial pathogens, particularly Pseudomonas aeruginosa and Staphylococcus aureus. Phenotypic studies showed that HT61 was effective against biofilms formed by Gram-positive species with limited efficacy towards biofilms of Gram-negative species. Scanning electron microscopy of HT61 treated biofilms of S. aureus and P. aeruginosa suggested a mechanism of action targeting the cell envelope. Quantitative proteomic analysis of HT61 treated S. aureus cultures supported this, identifying upregulation of proteins associated with the cell wall stress stimulon and division cell wall cluster. To investigate the effect of interspecies interactions on bacterial adaptation to HT61, a dual species biofilm model of P. aeruginosa and S. aureus was developed and characterised. Fluctuation analysis suggested that biofilm co-culture increased the mutation rate of S. aureus over 500-fold compared to planktonic culture and almost 100-fold compared to culture as a single species biofilm. Whole genome sequencing of single and dual species biofilm derived P. aeruginosa and S. aureus isolates revealed significant genomic variation in both coding and intergenic sequences, but no change in evolutionary trajectory between isolates derived from mono- or co-culture biofilms. Following HT61 treatment, mutations in S. aureus were identified in graS and fmtC, which encode products that modulate the cell envelope and may suggest routes to HT61 adaptation. In summary, the data presented in this thesis suggests potential mechanisms of action and adaptation to HT61, which could inform future antimicrobial development. This thesis also reinforces the need to understand the impact of interspecies interactions on bacterial evolution and shows that biofilms are important hubs of genomic diversity, which could have dangerous implications for the emergence of AMR.

Le biofilm est à l’origine d’infections nosocomiales et d’intoxications alimentaires dans les secteurs alimentaire et hospitalier. Les bactéries structurées en biofilm peuvent se détacher et coloniser de nouvelles surfaces. Le risque microbiologique associé aux bactéries détachées de biofilm est peu étudié. Les travaux de thèse ont concerné, l’étude de l’effet des conditions de croissance sur les propriétés physicochimiques de surface de Staphylococcus aureus et Pseudomonas aeruginosa sous leurs formes détachées de biofilm et planctoniques, ainsi que leurs adhésion sur l'acier inoxydable (SS) et le polycarbonate (PC). Le pouvoir pathogène des deux populations bactériennes a été étudié. Les résultats ont montré que les conditions et le mode de croissance influencent les propriétés de surface et par conséquent l’adhésion de S. aureus et P. aeruginosa sur le SS et le PC. De plus, la température de croissance, le type de surface et l’âge physiologique des bactéries influencent significativement leur production des facteurs de virulence et leur cytotoxicité envers les cellules HeLa. Dans un deuxième temps, l'effet de température de croissance sur la résistance des cellules détachées de biofilm et planctoniques au chlorure de benzalkonium (BAC) a été évalué. Pour comprendre les mécanismes de résistance à l’échelle cellulaire, les lésions des membranes bactériennes associées au BAC ont été suivies par l’efflux des ions K+ intracellulaires. En outre, la fluidité membranaire de deux populations bactériennes a été caractérisée à travers l'étude de profils d'acides gras membranaires. Les résultats ont montré que la résistance au BAC dépend de la température et de l’état physiologique des bactéries
The biofilm formation in food and medical sectors represents a significant source of foodborne and nosocomial diseases. Bacteria structured in biofilm can detach and colonize new surfaces. The microbiological risk associated with biofilm-detached bacteria is poorly studied. On one hand, the thesis concerned the study of growth conditions effect on the bacterial surface physicochemical properties as well as the adhesion of Staphylococcus aureus and Pseudomonas aeruginosa biofilm-detached and planktonic cells to stainless steel (SS) and polycarbonate (PC). The pathogenicity of both bacterial populations has also been studied. The results showed that the conditions and the mode of growth influence the surface properties and consequently the adhesion of S. aureus and P. aeruginosa on the SS and the PC. In addition, growth temperature, surface type and physiological age of bacterial cells significantly influence their production of virulence factors and their cytotoxicity towards HeLa cells. On the other hand, the effect of growth temperature on the resistance of biofilm-detached and planktonic cells to benzalkonium chloride (BAC) was assessed. In order to understand the mechanisms of resistance at the cellular level, bacterial membrane damage associated with BAC was assessed by the efflux of intracellular K+ ions. In addition, the membrane fluidity of bacterial populations was characterized through the study of membrane fatty acid profiles. The results showed that resistance to BAC depends on the temperature and physiological state of the studied bacteria

Staphylococcus aureus a été classé parmi les bactéries les plus pathogènes du genre Staphylococcus. Ce pathogène est responsable d'infections localisées (plaies chroniques, infections sur prothèses) voire de septicémies et d’infections nosocomiales. L’objectif principal de ce projet est d’élaborer des vecteurs colloïdaux biocompatibles à base de polysaccharides, chargés en principe actif antibactérien, et ciblant spécifiquement des biofilms de S. aureus. La méthode de complexation polyélectrolytes entre polysaccharides de charge opposée (chitosane/alginate et chitosane/dextrane sulfate) a été sélectionnée pour élaborer des particules de taille micrométrique. Ces microparticules n’étant pas stables, elles ont été stabilisées par réticulation chimique. Un antibiotique à large spectre d’activité de la famille des fluoroquinolones, la ciprofloxacine, a été séquestrée dans les microparticules. Des essais microbiologiques ont été réalisés en planctonique et sur biofilms, sur une souche de S. aureus et une souche de Pseudomonas aeruginosa. La ciprofloxacine encapsulée présente une activité antibactérienne (CMI, CMB et CMEB) plus importante que la ciprofloxacine libre. Par ailleurs, les MPs à base de chitosane/alginate sont plus actives que celles constituées de chitosane/dextrane sulfate. Enfin, un greffage d’un anticorps anti-protéine A a été réalisé sur les microparticules chitosane/alginate chargées en ciprofloxacine. Ces microparticules présentent une activité antibactérienne sur le biofilm de S. aureus légèrement améliorée par rapport aux microparticules dépourvues d’anticorps
Staphylococcus aureus has been classified as one of the most pathogenic bacteria of the Staphylococcus genus. This bacterium is responsible for localized infections (chronic wounds, infections on artificial joints) or even septicemia and nosocomial infections. The main objective of this project is to develop biocompatible colloidal vectors based on polysaccharides, loaded with antibacterial active compound, and specifically targeting Staphylococcus aureus biofilms. The polyelectrolyte complexation between polysaccharide of opposite charge (chitosan / alginate and chitosan / dextran sulfate) has been selected to produce micrometric particles. By varying the total concentration of polysaccharide and the charge ratio between polyanion and polycation, it is possible to obtain variable sizes. As these microparticles were not stable, they were stabilized by chemical crosslinking. An antibiotic of the fluoroquinolone family, ciprofloxacin, with a large spectrum of activity, was entrapped in the micoparticles. Microbiological tests were carried out in planktonics and biofilms on different strains of Staphylococcus aureus and Pseudomonas aeruginosa. Loaded ciprofloxacin exhibits greater antibacterial activity (MIC, CMB and CMEB). Moreover, the chitosan / alginate-based MPs are more active than those consisting of chitosan / dextran sulfate. Finally, a grafting of an antiprotein A antibody was carried out on chitosan / alginate microparticles loaded with ciprofloxacin. These modified microparticles exhibit a slightly improved antibacterial activity compared to loaded ciprofloxaxin microparticles whitoutantibody

The persistence of biofilms in hospital settings are associated with Healthcare Associated Infections (HCAI), causing increased morbidity, mortality and healthcare costs. The resistance of biofilms against commonly used hospital disinfectants has been well reported. Metal and metal oxide nanoparticles (NP) such as silver (Ag), copper (Cu), zinc oxide (ZnO) and copper oxide (CuO) exhibit antimicrobial properties against various pathogens. Methods: Biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus in a Centre for Disease Control (CDC) biofilm reactor and a 96 well plate was compared. A three stage approach including Minimum Biofilm Reduction Concentration (MBRC), R2 values and log(10) reductions was used to assess the efficacy of Ag and ZnO NPs both alone and in combination against P. aeruginosa and S. aureus biofilms. Atomic Absorption Spectroscopy (AAS), Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) was used to further assess the antimicrobial ability of the metal and metal oxide NPs. The prevention of P. aeruginosa and S. aureus adherence on Ag and ZnO thin film coating on silicon (Si) surfaces was also investigated, as well as icaC, ebpS and fnbB gene expression in S. aureus biofilms. Results: The CDC biofilm reactor demonstrated to be the most effective method for P. aeruginosa and S. aureus biofilm production in comparison to 96 well plates, with lower standard errors of the mean (SE) and higher replicability. Individual MBRC of ZnO and Ag NPs in suspension were 256 and 50 µg/ml for P. aeruginosa and 16 and 50 µg/ml for S. aureus respectively. The concentrations in combination were reduced by at least a half, with concentrations of 32/25 µg/ml of ZnO/Ag NPs in suspension resulting in a significant (p ≤0.05) reduction of 3.77 log(10) against P. aeruginosa biofilms and 8/12 µg/ml of ZnO/Ag NPs in suspension resulted in a 3.91 log(10) (p ≤0.05) against S. aureus biofilms. Both combinations showed an additive effect. Time point analysis confirmed that a 24 hour treatment is vital for any significant (p ≤0.05) antimicrobial activity. AAS data suggested that the Ag+ ions quenched Zn2+ ions, therefore the antimicrobial efficacy of the combination is mainly due to Ag+ ions. Damage of the biofilms from Ag and ZnO NPs was observed in the SEM imaging and energy dispersive X-ray (EDX) analysis confirmed the adherence of Zn and Ag within the biofilms. CLSM imaging showed dead (red) cells of P. aeruginosa and S. aureus biofilms throughout the depth of the biofilm. P. aeruginosa formation was reduced by 1.41 log(10) and 1.43 log(10) on Ag and ZnO thin film coatings respectively. For S. aureus, a reduction of 1.82 log(10) and 1.65 log(10) was obtained for Ag and ZnO coating respectively. Only low levels of ribonucleic acid (RNA) were achieved so no further gene analysis could occur. Conclusion: Reductions of ≥3 log(10) were observed for P. aeruginosa and S. aureus biofilm treatment with ZnO/Ag NP suspensions. It can be concluded that the ZnO/Ag NP suspensions had greater antimicrobial activity than Ag and ZnO coated surfaces owing to large concentrations of Ag+ and Zn2+ ions acting upon the biofilms. The slower release of ions from coated surfaces suggest an inadequate concentration of ions in the media, which are therefore unable to prevent biofilm formation as rapidly as NP suspensions, however provide a sustained release of ions over time. The results from this investigation propose that Ag and ZnO NPs in suspension could be a potential alternative to disinfectants for use in nosocomial environments against P. aeruginosa and S. aureus biofilms.

Staphylococcus Aureus est un pathogène opportuniste produisant un grand nombre de facteurs de virulence. Parmi ceux-ci, la toxine a excrétée, codée par hla, et la protéine A de surface, codée par spa, sont souvent utilisées comme modèle d'étude de la régulation de l'expression des facteurs de virulence. Le système de quorum sensing (QS), codé par le locus agr, intervient dans leur régulation in vitro. La capacité de S. Aureus à former des biofilm est aussi considéré comme facteur de virulence permettant d'aggraver et de rendre chronique les infections. L'un des composants du biofilm (le PIA) est synthétisé par les produits du locus ica. Le rôle du QS dans la régulation de l'expression de hla, de spa et ica a été étudié chez un isolat clinique issu d'une infection sur prothèse orthopédique et chez son mutant isogénique délété dans le gène agrC, au cours de la croissance en conditions planctonique et sessile dans le biofilm. La technique de PCR quantitative en temps réel associée à la méthode de quantification relative des transcrits desgènes d'interêt, en utilisant le transcrit 16S comme standard interne se sont révélées les mieux adaptées à l'étude de la transcription au cours de la croissance. Dans les deux conditions de culture, nous avons montré que le transcrit RNAIII, molécule effectrice du QS, était exprimée à un niveau basal en absence d'agrC, supposé être absolument requis pour son expression. Notre étude a également mis en évidence, pour la première fois une activation d'ica par le QS, alors que la production de biofilm est supérieure chez le mutant. Enfin, la régulation de l'expression d'hla et spa par le QS est très différente selon les conditions de culture
Staphylococcus aureus is an opportunistic pathogen producing a large number of virulence factors. Among these, the secreted alpha-toxin, encoded by hla, and the surface-associated protein A, encoded by spa, are often considered as reporters of virulence factors regulation studies. The quorum sensing (QS) system, encoded by agr, is involved in their regulation in vitro. The biofilm formation ability of S. Aureus is also considered as a virulence factor, as it is the cause of heavier and chronic infections. One of the biofilm components (PIA) is synthesized by the products of the ica locus. QS involvement in the expression regulation of hla, spa and ica is studied in a clinical strain, isolated from an infected prosthesis, and in an isogenic mutant strain deleted in agrC gene, during planktonic and biofilm sessile growth. The real-time quantitative PCR technique associated with relative quantification method of target genes transcripts compared to 16S transcripts appeared to be the best method for transcription study during growth. In both growth conditions, we have demonstrated that the RNAIII transcript, the QS molecular effector, was expressed in a basal level in agrC mutant. In the same time, our study has for the first time outlined an ica activation by the QS, whereas the biofilm production by the mutant was more important. Finally, hla and spa expression regulation by QS is very different depending on growing conditions

Las infecciones de prótesis articulares causadas por S. aureus son difíciles de curar, y con los tratamientos actuales recomendados siguen habiendo fallos tanto microbiológicos como clínicos. Asimismo, no se han estudiado de forma exhaustiva las posibles alternativas terapéuticas en caso de no poder administrar por alergia o intolerancia a alguno de los antibióticos considerados de elección. Actualmente se están investigando nuevas terapias terapéuticas ya sea con antibióticos de reciente aparición, como es daptomicina, o a partir de terapias anti-biofilm. Daptomicina es un lipopéptido cíclico que ha demostrado tener actividad bactericida frente a bacterias en fase planctónica y en fase estacionaria, y se ha visto que tiene una alta eficacia en combinación con otros antimicrobianos frente a la infección de cuerpo extraño por S. aureus resistente a meticilina (SARM). Por otro lado, la escasa información existente sobre la terapia anti-biofilm con macrólidos frente a S. aureus es contradictoria; si bien algunos estudios defienden que posee una actividad contra el biofilm, otros observaron poca o ninguna actividad. Como se ha comentado, las consideradas como alternativas terapéuticas en la infección por S. aureus (ej., cotrimoxazol y ácido fusídico) no se han estudiado en profundidad y la información que se tiene de estos antibióticos es de hace algunas décadas. El objetivo de esta tesis fue por un lado estandarizar dos modelos, uno in vitro en el CDC biofilm reactor y otro in vivo de infección de cuerpo extraño con cepas S. aureus sensibles a meticilina (SASM) y SARM, para poder evaluar (i) la eficacia de las combinaciones de daptomicina frente a SASM; (ii) el potencial efecto anti-biofilm de claritromicina; y (iii) la actividad de cotrimoxazol, ácido fusídico y las combinaciones de rifampicina con cotrimoxazol y linezolid como posibles alternativas terapéuticas al tratamiento estándar para S. aureus. Los principales hallazgos son: - El modelo in vivo estandarizado con una infección de 3 y 7 días, como modelo de infección aguda de cuerpo extraño, se muestra válido para la evaluación de la eficacia antimicrobiana - El modelo in vitro dinámico de CDC biofilm reactor permite la formación de biofilm de S.aureus, su mantenimiento en el tiempo, así como el estudio de la eficacia de las diferentes pautas terapéuticas. - Daptomicina-cloxacilina ha demostrado tener una actividad similar a cloxacilina- rifampicina (tratamiento estándar) y podría utilizarse como alternativa terapéutica en fase precoz del tratamiento de infecciones de prótesis por SASM - Daptomicina-rifampicina fue el tratamiento más eficaz en la infección in vivo de cuerpo extraño por SASM, incluso tuvo más actividad que el tratamiento estándar levofloxacino- rifampicina. Estos resultados sugieren que daptomicina-rifampicina podría utilizarse como alternativa terapéutica en los casos que no pudiera utilizarse las fluoroquinolonas. - Claritromicina no tuvo actividad anti-biofilm relevante frente a las infecciones por SASM y SARM - Cotrimoxazol fue ineficaz frente a la infección por SASM y SARM en la rata. Los resultados observados sugieren que una de las causas de su inactividad podría ser la interacción con la timidina (que se encuentra en altas concentraciones de forma inherente en la rata, y en infecciones purulentas) . - Ácido fusídico presentó resultados microbiológicos prometedores (aumento de actividad de daptomicina, y protección frente la aparición total o parcialmente). Sin embargo no se pudo estudiar en el modelo in vivo debido a la toxicidad que presentó en los animales. - En el modelo in vitro dinámico del reactor para SASM observamos que la combinación de levofloxacino con rifampicina fue el tratamiento más eficaz, mientras que cotrimoxazol- rifampicina y linezolid-rifampicina no mostraron diferencias significativas entre ellos en términos de eficacia. Sin embargo, linezolid fue capaz de proteger frente a la aparición de resistencias a rifampicina, mientras que cotrimoxazol no lo hizo. En el modelo para SARM, linezolid-rifampicina se presentó como la terapia más eficaz y protegió frente a la aparición de cepas resistentes. De nuevo, la combinación de cotrimoxazol-rifampicina no protegió frente a la aparición de resistencias a rifampicina, que aparecieron de forma sistemática a las 8 horas de iniciar el tratamiento.
Despite optimal therapeutic management for acute prosthetic joint infections (PJI) caused by Staphylocococcus aureus, some clinical and microbiological failures occur, especially when implant retention is attempted. Moreover, occasions arise when drugs often cannot be administered owing to intolerance or allergy, and no adequately evaluated alternatives exist. In response to these difficulties, alternative therapies and other therapeutic approaches to bacterial biofilm are been investigated. Daptomycin combinations had a high efficacy against foreign-body infection by methicillin- resistant S. aureus (MRSA). On the other hand, the information about the potential anti-biofilm effect of macrolides against S. aureus is scarce and controversial. Additionally, the alternative therapies for S.aureus infections (ie cotrimoxazole and fusidic acid) have not been accurately evaluated. The aims of this project was standardize an dynamic in vitro model (CDC biofilm reactor) and an in vivo foreign-body infection (FBI) by methicillin-susceptible S. aureus (SASM) and MRSA strains, in order to evaluate (i) The efficacy of daptomycin and its combinations against SASM; (ii) the potential anti-biofilm effect of clarithromycin; and (iii) the activity of cotrimoxazole, fusidic acid and rifampicin combinations with cotrimoxazole and linezolid as possible therapeutic alternatives to the standard treatment for S. aureus. The main results were: - The In vivo model with a period of infection of 3 and 7 days, as an acute FBI model, and the dynamic in vitro model of CDC biofilm reactor were suitable to study the efficacy of different therapies. -Daptomycin-Cloxacillin therapy was as effective as cloxacillin-rifampicin (standard treatment) and can be considered as alternative anti-MSSA therapy for FBI. -Daptomycin-Rifampicin was the most effective combination, providing higher efficacy than levofloxacin-rifampicin. These results suggest that daptomycin-rifampicin may be useful as a first-line therapy against MSSA PJI. -Clarithromycin did not show anti-biofilm activity against MSSA and MRSA infections. -Cotrimoxazole was ineffective against S.aureus in animal model. The results observed suggest that the lack of activity could be due in part to its inactivation by thymidine. -Fusidic acid had promising microbiological results. However, it was not possible to perform the in vivo studies due to the animal intolerance to antibiotic. - In CDC biofilm reactor, levofloxacin-rifampicin was the most effective treatment against MSSA. Rifampicin combinations with cotrimoxazole and linezolid had similar activity. Against MRSA, linezolid-rifampicin combination was the most effective treatment. In both strains, the cotrimoxazole-rifampicin combination did not protect against rifampicin resistant strains.

Um total de 107 S. aureus isolados de casos de mastite, glândulas portadoras, pele do úbere, ordenhadores e insufladores, em rebanhos de São Paulo e Pernambuco, foram tipados por técnicas moleculares PCR-RFLP do gene coa e PCR de spa distinguiram seis perfis. Todas as amostras amplificaram genes coa, spa, icaA e 69% produziram biofilme glicose-induzido in vitro. PFGE identificou 31 perfis e 12 linhagens. Uma linhagem foi predominante (P < 0,0001) e amplamente disseminada em ambas as regiões. Um mesmo perfil foi isolado de mastite clínica, subclínica e portadoras. Houve heterogeneidade genética entre isolados de fazenda. Isolados de origem humana e animal constituíram populações distintas. Poucos isolados de leite, insufladores e pele do úbere tiveram igual perfil. Uma amostra extramamária, 77% dos isolados de leite e. 99% de S. aureus de portadoras produziram biofilme. Não foi detectada correlação entre produção de biofilme e CCS. O isolamento sucessivo do mesmo perfil de PFGE de glândulas assintomáticas por mais de 16 dias caracterizou o estágio de portador.
A total of 107 S. aureus isolated from bovine milk, udder skin, milkers and milking machine, from São Paulo and Pernambuco herds was typed by molecular techniques. PCR-RFLP coa gene and PCR spa gene distinguished six amplicons. All strains amplified coa, spa, icaA genes and, 69% produced in vitro glucose-induced biofilm. PFGE identified 31 pulsotypes, 12 lineages. One of the lineages was predominantly isolated (P<0.0001) and widely disseminated. A same pulsotype was isolated from clinical and, subclinical mastitis as well as from carriers. There was genetic heterogeneity among isolates from the herds. Strains from human and animal origin were genetically different. Few isolates from milk, milking machine and udder skin showed similar pulsotype. An extramammary strain, 77% of the milk isolates and, 99% of the S. aureus isolated from carriers produced biofilm. It was not detected any correlation between SCC and biofilm production. The successive isolation during more than 16 days of a same pulsotype from the asymptomatic glands characterized the carrier status.

Staphylococcus aureus é uma bactéria que pode ser encontrada colonizando diversas partes do corpo humano, entretanto os diversos fatores de virulência que a bactéria possui, ancorados a sua superfície ou excretados para o meio extracelular, tornam essa bactéria um potencial patógeno, causando infecções de pele e tecidos moles, osteomielite, infecções respiratórias, infecções relacionadas a cateteres e outros dispositivos e bacteremia. Um dos fatores de virulência da bactéria, é a habilidade em formar biofilmes. Biofilmes são comunidades bacterianas tridimensionais complexas, que vivem organizadas e aderidas a uma superfície biótica ou abiótica, embebidas em uma matriz exopolimérica. Cerca de 80% das bactérias vivem organizadas na forma de biofilme, pois nestas estruturas são menos sensíveis aos antibióticos e à resposta imune do hospedeiro. A habilidade de S. aureus em formar biofilme é importante pois o torna uma das principais bactérias que infecta dispositivos médicos e implantes, aumentando a morbidade e mortalidade dos pacientes que apresentam esse tipo de infecção. Os medicamentos da classe dos β-lactâmicos eram a principal escolha para o tratamento de S. aureus, entretanto nos últimos anos essa bactéria adquiriu resistência a esses antimicrobianos, através da aquisição do gene mecA, tornando escassa as opções terapêuticas. Como se não bastasse, os biofilmes bacterianos são particularmente mais resistentes a tratamentos com antibióticos, não só devido ao aumento da transmissão de mecanismos de resistência dentro da comunidade, mas também por causa das limitações de difusão da droga colocados pela matriz extracelular, inativação de antibióticos pela alta concentração de íons de metal e baixo pH, entre outros fatores. Combinados, esses atributos tornam o biofilme bacteriano em torno de 1000 vezes mais tolerante e/ou resistente aos antimicrobianos comparado às células planctônicas. A investigação de estudos epidemiológicos para prevenção dessas infecções, bom como de novas estratégias para prevenção e tratamento de infecções por biofilmes, especialmente em isolados clínicos sabidamente multirresistentes, é urgentemente necessária. Dentre estas estratégias estão a pesquisa de diferentes mecanismos ou substâncias capazes de provocar a inibição da formação ou a erradicação do biofilme formado. Neste contexto, 8 os sistemas de bombas de efluxo e inibidores de bombas de efluxo representam uma fonte promissora de erradicação do biofilme formado. O principal objetivo deste estudo é investigar características clínico-epidemiológicas em isolados clínicos que estejam associadas a formação de biofilme, bem como investigar o papel de bombas de efluxo, inibidores dessas bombas e novos genes envolvidos na habilidade de isolados clínicos de S. aureus em formar biofilme. O capítulo 1 associa características clínicas e epidemiológicas com a habilidade de formação de biofilme. O capítulo 2 mostra o papel da adição de antimicrobianos na inibição e erradicação de biofilmes, a associação com inibidores de bomba de efluxo para melhor entender os sistemas de bomba de efluxo na capacidade desses isolados em formar biofilme e por último, novos genes que participam desse processo, em isolados clínicos de MRSA. Este estudo permite planejar ações preventivas para essas infecções relacionadas a biofilmes. Além disso, demonstra que os sistemas de bombas de efluxo parecem ser alvos promissores para erradicar infecções associadas a biofilmes bacterianos.
Staphylococcus aureus can be found colonizing the human body, however its virulence factors anchored to its surface or secreted into the extracellular medium, makes this bacteria as a potential pathogenic, causing skin and soft tissue infections, osteomyelitis, respiratory infections, catheter-related and other devices infections and bacteremia. One of the virulence factors that bacteria produce is the ability to form biofilms. Biofilms are complex three-dimensional bacterial communities, living organized and attached on a biotic or abiotic surface, embedded in a matrix exopolimérica. About 80% of live bacteria are organized in the form of biofilms because in these structures are less sensitive to antibiotic and the host immune response. The ability of S. aureus to form biofilms is important because it makes it one of the main bacteria that infects medical devices and implants, increasing patient morbidity and mortality. The class of β-lactam drugs used to be main choice for the treatment of S. aureus infections, however in recent years the bacteria acquired resistance to these antibiotics by acquiring mecA gene, so therapeutic options becoming scarce. Besides that, bacterial biofilms are particularly resistant to antibiotic treatments, not only due to increased transmission resistance mechanisms within the community, but also because limitations in drug diffusion by extracellular matrix, inactivation of antibiotics due to high concentration of metal ions and low pH, and other factors. Combined, these attributes make the bacterial biofilm around 1000 times more tolerant and / or resistant to antimicrobial compared to planktonic cells. Investigation of epidemiological studies to prevent such infections, as well as new strategies for prevention and treatment of biofilm infections, especially in known multidrug-resistant clinical isolates, is urgently needed. Among these strategies we could list the different search engines or substances capable of causing or inhibiting the formation of biofilm eradication. In this context, system efflux pumps and efflux pump inhibitors represent a promising source of biofilm eradication. The aim of this study is to investigate the clinical and epidemiological characteristics in clinical isolates that are associated with biofilm formation and investigate the role of efflux pumps and inhibitors of these pumps in the ability of S. 10 aureus clinical isoltes to form biofilms. The chapter 1 associates clinical and epidemiological characteristics with biofilm formation ability. Chapter 2 shows the role of the addition of antimicrobials in inhibition and eradication of biofilms, the association with efflux pump inhibitors to better understand the efflux pump systems in the ability of these isolates to form biofilm and, finally, new genes important in MRSA clincal isolates biofilm formation. This study allows planning preventive actions for these biofilm-related infections. In addition, it demonstrates that efflux pump systems appear to be promising targets for eradicating infections associated with bacterial biofilms.

@On appelle infections nosocomiales, les maladies infectieuses contractées au cours d'une hospitalisation qui n'étaient ni en incubation ni présentes à l'admission du patient. Elles peuvent être d'origine endogène ou exogène : le patients lui-même, le personnel, le matériel, les surfaces et l'environnement. Cette contamination peut résulter d'un manque d'efficacité des procédures de nettoyage-désinfection, déficience souvent attribuée à l'état dans lequel se trouve ces germes indésirables (biofilms). Pour réduire ces risques de contamination deux axes ont été abordés : d'une part les prédictions d'adhésion entre le support en acier inoxydable AISI 304 (constituant majeur du matériel en bloc opératoire) et quatre souches responsables d'infections nosocomiales (E. Coli, S. Aureus, P. Aeruginosa et E. Hirae) et d'autres parts, l'évaluation de l'efficacité ainsi que les conditions d'utilisation d'un nouveau désinfectant OXSILÒ 320N sur des cellules planctoniques et des biofilms. L'étude physicochimique a montré toute la complexité des interactions impliquées dans la phase initiale d'adhésion. Si en théorie, il est possible d'enrayer l'adhésion bactérienne à un support en modifiant ses caractéristiques de surface, notre étude montre qu'il est impossible de limiter l'adhésion simultanée des 4 souches bactériennes étudiées au support AISI 304. Face à une telle situation, il est donc impératif d'optimiser les procédures de nettoyage-désinfection. OXSILÒ320N possède une activité de spectre 4 selon la norme AFNOR NF T 72-150 sur des cellules planctoniques à la concentration de 3,13%. Par contre les biofilms sont plus résistants que leurs homologues planctoniques. Ceci serait dû à la présence de mécanismes de défense connus. Selon les recommandations d'utilisation de SODIFRA les conditions optimales pour obtenir un niveau "zéro" de contamination sont : 12,52% d'OXSILÒ320N et un temps de contact de 10 min évitant tout risque infectieux entre deux opérations de courtes ou de longues durées en blocs opératoires. Ces résultats ont mis en évidence la nécessité d'utiliser ce désinfectant à une concentration appropriée et de ne pas négliger les conséquences d'une utilisation en infradose même si celle-ci autorise selon les normes AFNOR une activité bactéricide
@Nosocomial infections are hospital acquired infections. They are not present or incubating when the patient is admitted to the hospital, and are either endogenous or exogenous. Endogenous ones are caused by organisms present in the patient own flora and exogenous infections are caused by organisms originating from medical devices, hospital staff, or environment. This contamination might be the result of a lack of efficiency in cleaning and disinfection procedures, those deficiencies being often attributed to the state in which these harmful microorganisms are found (biofilms). To reduce the risks of contamination, two parts have been developed in this thesis: at first, predictions of adhesion between stainless steel AISI 304 (major component of equipment in operating room) and four nosocomial strains (E. Coli, S. Aureus, P. Aeruginosa et E. Hirae), and in the second part, we determine, the efficient concentration of a new disinfectant OXSILÒ 320N in order to eliminate biofilms. Physico-schemical studies demonstrated the complexity of interactions involved in the initial phase of adhesion. We suggest that a change of the stainless steel surface properties could theoretically limit bacterial adhesion. However it was nearly impossible to limit adhesion between the four studied bacterial strains and the support AISI 304. It is then necessary to optimise preventive hygienic conditions. The bactericidal concentration of OXSILÒ 320N against planktonic cells, according to AFNOR Norm NF T 72-150, was 3,13%. Furthermore, biofilms were commonly more resistant than their planktonic counterparts due to the presence of known resistance mechanisms. According to the SODIFRA recommendations, the optimal conditions, required to avoid any contamination, were a concentration 12,52% and 10 min contact. These conditions can eliminate infection risks during short or long laps between two interventions in an operating room. These results revealed the necessity to use this disinfectant at appropriate concentration and but also the consequences of using it in infradose, although this concentration is considered as an efficient concentration by AFNOR Norm

O objetivo do presente estudo foi avaliar a ocorrência de cepas de Staphylococcus aureus no ambiente de ordenha, analisar seu perfil genético e a produção de biofilme, provenientes de 10 propriedades localizadas nas regiões de Franca e Ribeirão Preto, estado de São Paulo, Brasil. Foram analisadas 220 amostras de leite individual de vacas positivas no teste CMT (California Mastitis Test), 120 amostras de leite de tanque de expansão, 389 swabs de utensílios e equipamento relacionados com ordenha e 120 swabs de mãos de manipuladores. As coletas de amostras foram realizadas mensalmente durante o período de agosto/2010 a janeiro/2011. Das 849 amostras analisadas, 56 cepas de S. aureus (6,6%) foram isoladas, sendo 12 (5,4%) de leite individual de vacas, 26 (21,6%) de leite de tanque de expansão, 14 (3,6%) de utensílios e equipamentos e 4 (6,9%) de mãos de manipuladores. Os resultados indicam uma baixa prevalência de S. aureus nas propriedades analisadas, não havendo diferença significativa entre as frequências encontradas nas duas regiões analisadas. A técnica de PFGE (pulsed-field gel electrophoresis) permitiu identificar 31 perfis genéticos (pulsotipos), utilizando-se a enzima de restrição SmaI. Nos ensaios de produção de biofilmes em microplaca, 19 (63,3%) de 30 pulsotipos avaliados foram considerados produtores de biofilme. Nos ensaios conduzidos em aço inox, 13 (43,3%, N=30) foram positivos e, para o silicone, não houve produção de biofilme. O elevado percentual de isolados no leite de tanque de expansão evidencia um problema de saúde pública, considerando que no Brasil muitas vezes o leite é consumido sem pasteurização. A ocorrência de pulsitipos de S. aureus formadores de biofilmes indica a persistência do patógeno em diversos pontos no ambiente de ordenha, bem como provável envolvimento dos mesmos com casos de mastite e sua eliminação no leite, ressaltando a necessidade de práticas higiênicas para prevenir a formação de biofilmes nas propriedades estudadas.
The objective of the present study was to evaluate the occurrence of biofilm-producing strains of Staphylococcus aureus in the milking environment from 10 farms located in the regions of Franca and Ribeirão Preto, state of São Paulo, Brazil. Twohundred twenty samples of milk from individual cows previously tested for CMT (California Mastitis Test), 120 samples of bulk tank milk, 389 swabs of equipments and utensils related to milking and 120 swabs of milk\'s handlers hands were analyzed. A total of 56 (6.6%) S. aureus strains were isolated out of 849 samples analyzed, being 12 (5.4%) from milk samples from individual cows, 26 (21.6%) from bulk tank milk, 14 (3.6%) from swabs of equipments and utensils, and 4 (3.3%) from swabs hands of milk\'s handlers. Results indicated a low prevalence of S. aureus in the dairy farms analyzed, and there was no significant difference between the percentages found in the two regions evaluated. On the basis of PFGE results (using SmaI enzyme), 31 profiles (pulsotypes) were found. In the biofilm-production assay using microplates, 19 (63.3%) of 30 pulsotypes tested were considered positive (biofilm producers). For assays conducted in stainless steel, 13 (43.3%) of pulsotypes were biofilm producers, although no pulsotype was able to produce biofilms on the surface of silicon. Results of this trial showed a high percentage of bulk tank milk samples contaminated with S. aureus, hence indicating a public health problem especially in Brazil were milk is often consumed without pasteurization. The occurrence of S. aureus pulsotypes which were also biofilm-producers suggests the persistence of the pathogen in several sites at the milking environment, as well the probable involvement of S. aureus biofilms with mastitis and its excretion in the milk. The need for adoption of hygienic practices to prevent the formation of biofilms of S. aureus in the dairy farms evaluated is stressed.

Nous avons effectué une étude bibliographique intitulée “The diabetic foot microbiota: A review”. Cette revue a permis une réactualisation des données connues sur le microbiote du pied diabétique. Nous avons discuté le rôle des bactéries dans la pathogénèse de l’ulcère du pied diabétique et la différenciation entre l’infection et la colonisation bactérienne au niveau de la plaie ainsi que la superposition de l’approche moléculaire et la culture. Nous avons étudié le microbiote du pied diabétique infecté par culturomic. Il s’agit de varier les conditions de culture, puis identifier les colonies par MALDI-TOF ou par amplification et séquençage ARNr 16S. Cette technique s’est montrée efficace dans l’élargissement de l’éventail des bactéries identifiées. 53 espèces bactériennes différentes ont été identifiées à partir des échantillons de 43 patients. L’hétérogénéité et la richesse des plaies se sont montrées importantes. Staphylococcus aureus était l’espèce dominante. Nous nous sommes servis de tests statistiques pour évaluer l’influence que les facteurs cliniques chez les patients pourraient avoir sur la composition de cette flore et l’évolution de la plaie. On s’est orienté vers des souches spéciales « ExPEC » isolées des pieds diabétiques reconnues par leur virulence et impliquées dans des infections extra intestinales sévères. Nous avons disposé d’une collection de souches d’E Coli isolées des pieds diabétiques afin de mener une étude génétique. Nous sommes parvenus à amplifier les gènes de virulence, classer les souches par phylogroupes et les assembler dans des clones. En plus, nous avons réalisé un typage des souches ainsi qu’une exploitation des gènes de résistance
We conducted a bibliographic study entitled "The Diabetic Foot Microbiota: a review". This review has offered an update of the data concerning the diabetic foot microbiota. We discussed the role of bacteria in the pathogenesis of diabetic foot ulcer, the differentiation between infection and bacterial colonization of the wound and the importance of superposing culture and molecular Tools.We studied the diabetic foot microbiota using the culturomics. It consists in varying the culture conditions and then to identify the colonies by MALDI-TOF or 16s rRNA amplification and sequencing. We decided to practice this technique on the diabetic foot microbiota because it has proven to be effective in broadening the range of identified bacteria. 53 different bacterial species were identified from the samples of 43 patients. The heterogeneity and richness of the wounds were elevated. Staphylococcus aureus was the dominant species. We used statistical tests to evaluate the influence that clinical factors in patients might have on the composition of this flora and the evolution of the wound. We targeted special strains « ExPEC »isolated from diabetic feet known to their virulance and involved in severe extra intestinal infections. We disposed of a collection of E Coli strains isolated from diabetic wounds to conduct a genetic study. We have managed to amplify the virulence genes, classify the strains by phylogroupes and assemble them in clones. In addition, we performed a strain typing as well as an exploitation of the resistance genes

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
This study aimed to evaluate the ability of adhesion, the kinetics of separation, the pattern of biofilm formation of strains of S. aureus isolated from different Food and Nutrition Services from João Pessoa - PB, when cultured in meat-based broth and vegetable-based broth, and incubated at temperatures of 28ºC and 7°C for an extended time (24 72 h). Still, we evaluated the effect of applying sanitizers sodium hypochlorite (250 mg/L) and peracetic acid (30 mg/L) at inactivating bacterial cells in the biofilm matrix formed previously. These experiments were used as coupons (2 x 2 cm) of polypropylene and stainless steel AISI 304, whereas these materials are widely used in the composition of surfaces, equipment and / or utensils used in various types of food services. The results showed a high adhesion capacity of the strains tested in meat-based broth and vegetable-based broth with counts above 5 log CFU/cm2, regardless of surface type and incubation temperature. The detachment of cells on the surface was at least 103 during the first 6 CFU/cm2 contact with agar for both types of substrate used, featuring a high persistence over a prolonged incubation time (24 to 72h). There was not a clear influence from the surface and the temperature used to evaluate the adhesion. In general, for both types of substrate was shown a similar pattern of biofilm formation when strains were subjected to different combinations of surface types and growth temperatures. The number of cells (105-107 CFU/cm2) required for biofilm formation was observed in all experimental systems already after 3 days of incubation followed by a linear decrease after the 6th day, with the exception of strain S28 grown in vegetablebased broth, which showed values around 104 CFU/cm2 the first 24 hours of incubation. A range from 2.6 to 3.7 log CFU/cm2 for strains incubated meat-based broth and 2.0 to 3.3 log CFU/cm2 for strains incubated in vegetable-based broth was observed in reducing cells in the matrix of the biofilm caused by peracetic acid while the sodium hypochlorite reduction was about 2.1 to 2.7 log CFU/cm2 and 1.5 to 2.1 CFU/cm2 for strains grown in meat-based broth and vegetables, respectively. From the results obtained, it can be concluded that the strains tested showed a high capacity for adhesion and biofilm formation on food contact surfaces when exposed to different culture media and environmental characteristics. Furthermore, the sanitizers used, although reducing the number of adhered cells, demonstrate a certain ineffectiveness in removing cells from the biofilm matrix considering the number of remaining cells found after the process of applying sanitizers.
O presente estudo teve como objetivo avaliar a capacidade de adesão, a cinética de separação, a formação de biofilmes de cepas de S. aureus isoladas de diferentes Serviços de Alimentação e Nutrição da cidade de João Pessoa PB, quando cultivadas em caldo substrato base carne e caldo substrato base vegetais, e incubadas em temperaturas de 28ºC e 7ºC por um tempo prolongado (24 72 h) além de avaliar o efeito da aplicação dos sanitizantes hipoclorito de sódio (250 mg/L) e ácido peracético (30 mg/L) em inativar células bacterianas na matriz do biofilme previamente formado. Nestes experimentos foram utilizadas como superfícies-testes minisuperfícies (3 x 3 cm) de polipropileno e de aço inoxidável AISI 304, visto que tais materiais são amplamente utilizados na composição de superfícies, equipamentos e/ou utensílios utilizados nos variados tipos de serviços de alimentação. Os resultados mostraram uma alta capacidade de adesão das cepas avaliadas em caldo substrato base carne e em caldo substrato base vegetal com contagens superiores a 5 log UFC/cm2, independente do tipo de superfície e a temperatura de incubação. O descolamento das células nas superfícies foi de pelo menos 103 UFC/cm2 durante os 6 primeiros contatos com o ágar para os dois tipos de substrato utilizados, caracterizando uma alta persistência durante um tempo prolongado de incubação (24 a 72h). Não foi encontrada uma clara influência em relação à superfície e a temperatura utilizada na avaliação da capacidade de adesão. Em geral, para os dois tipos de substratos utilizados foi demonstrado um padrão de formação de biofilme semelhante, ao final de 15 dias de incubação, quando as cepas foram submetidas às diferentes combinações de tipos de superfícies e temperaturas de crescimento. O número de células (105 107 UFC/cm2) necessários para a formação do biofilme foi observado em todos os sistemas experimentais já após 3 dias de incubação seguidos de uma redução linear após o 6º dia, com exceção da cepa S28 cultivada em caldo substrato base vegetais, que apresentou valores em torno de 104 UFC/cm2 nas primeiras 24h de incubação. Um intervalo de 2,6 3,7 log UFC/cm2 para as cepas incubadas em caldo substrato base carne e de 2,0 3,3 log UFC/cm2 para as cepas incubadas em caldo substrato base vegetal foi observado na redução das células na matriz do biofilme causado pelo ácido peracético enquanto que pelo hipoclorito de sódio a redução foi da ordem de 2,1 2,7 log UFC/cm2 e de 1,5 2,1 UFC/cm2 para cepas cultivadas em caldos substrato base carne e vegetais, respectivamente. A partir dos resultados obtidos, pode-se concluir que as cepas testadas evidenciam uma elevada capacidade de adesão e formação de biofilme em superfícies de contato de alimentos quando expostas a diferentes meios de cultura e características ambientais. Alem disso, os sanitizantes utilizados, apesar de reduzirem o número de células aderidas às superfícies, demonstraram ineficácia na remoção de células da matriz do biofilme tendo em vista o número de células viáveis encontradas após o processo de aplicação dos sanitizantes.

Les infeccions bacterianes han esdevingut un greu problema a escala mundial per culpa del mal ús que hom ha fet dels antibiòtics. L’adquisició de resistències als antimicrobians s’ha accelerat i cada cop hi ha més bacteris resistents a tots els antibiòtics coneguts. Aquest problema es veu agreujat quan els bacteris formen un biofilm i les infeccions es tornen cròniques. Els bacteris creixents en forma de biofilm produeixen una matriu extracel·lular protectora i adquireixen així un mecanisme extra de protecció vers els antibiòtics. A més, en l’actualitat no hi ha cap antibiòtic desenvolupat que sigui capaç d’actuar específicament contra els biofilms bacterians madurs i, per tant, es necessiten nous procediments i fàrmacs per a poder tractar-los. En aquest treball s’estudien noves metodologies per al tractament de bacteris creixent en forma de biofilm des de tres punts de vista diferents. En primer lloc, s’intenta buscar noves teràpies i, per això, s’analitza l’acció antimicrobiana i antibiofilm de noves molècules, que han sigut modificades químicament per a millorar el seu potencial contra els biofilms de Staphylococcus aureus. Una part d’aquestes molècules són derivades del triclocarban i unes altres són derivades de l’àcid oleanòlic i l’àcid maslínic. En ambdós casos s’han aconseguit molècules menys tòxiques i més actives que els compostos dels quals deriven, fins i tot contra la soca de S. aureus resistent a meticil·lina (MRSA). En segon lloc, es pretén desenvolupar una tecnologia que permeti determinar, de mantera senzilla i amb precisió, la sensibilitat antibiòtica dels biofilms, ja que els mètodes que es fan servir actualment es basen en el creixement microbià en forma planctònica i no són fiables en bacteris creixent en forma de biofilm. Seguint aquest fil, s’ha desenvolupat el dispositiu “BiofilmChip”, una estructura microfluídica que permet el creixement de biofilms de manera senzilla i l’anàlisi de la seva sensibilitat sense la necessitat d’usar un microscopi confocal. En tercer i últim lloc, es volen desenvolupar noves tecnologies per al tractament de biofilms utilitzant enzims disgregadors del biofilm i fent servir mètodes alternatius d’alliberament de fàrmacs (drug delivery), com són les nanopartícules. Així, s’ha treballat amb els enzims alginat liasa i desoxiribonucleasa I (DNasa I), que degraden components de la matriu extracel·lular dels biofilms i en trenquen l’estructura, fent possible que els antibiòtics travessin aquesta barrera i puguin actuar. Per una banda, s’ha analitzat l’especificitat, l’activitat disgregadora i la sinergia amb antibiòtics de cinc alginat liases contra els biofilms de Pseudomonas aeruginosa. Per altra banda, s’ha treballat amb unes nanopartícules que milloren l’activitat de l’antibiòtic tobramicina, ja que contenen l’enzim DNasa I que permet la penetració d’aquest antibiòtic en els biofilms de S. aureus i P. aeruginosa. Aquestes nanopartícules, a més, estan marcades amb un fluoròfor, la qual cosa ha permès visualitzar la seva interacció amb la matriu extracel·lular del biofilm.

O aparecimento de uma grande variedade de microrganismos patogênicos resistentes aos antimicrobianos tem resultado no aumento do índice de doenças e mortalidade provocadas por infecções que eram facilmente tratadas no passado. Muitas vezes essa resistência está relacionada à formação de biofilme pelos microrganismos, que produzem substâncias poliméricas extracelulares (EPS) dificultando a penetração de agentes antimicrobianos. A Terapia Fotodinâmica antimicrobiana (aPDT, do inglês antimicrobial photodynamic therapy) é uma alternativa promissora para combater infecções microbianas, principalmente aquelas em que apresentam biofilmes. Basicamente esse mecanismo envolve a combinação sinérgica de um fotossensibilizador (FS), oxigênio molecular e luz visível de comprimento de onda adequado para produzir espécies reativas de oxigênio (EROs) que causam oxidação dos componentes da célula levando-a à morte. Devido à natureza multifacetada e não-específica das EROs produzidos na aPDT, os microrganismos têm menos chance de desenvolver mecanismos de resistência. Apesar destas vantagens, a aPDT tem enfrentando o problema da hidrofobicidade que FSs como hipericina (Hy) e ftalocianina de zinco (FcZn) apresentam. Esta hidrofobicidade promove a agregação do FS em meio biológico, reduzindo a sua atividade fotodinâmica. Diante disso, este estudo teve o objetivo avaliar a ação fotodinâmica da Hy, FcZn e seus derivados hidrossolúveis (hipericina-glucamina - HyG, ftalocianina de zinco tetracarboxilada - FcZnTc e ftalocianina de zinco tetracarboxi-N-metilglucamina - FcZnTcG), para inativar as bactérias Staphylococcus aureus e Escherichia coli, tanto em cultura planctônica como em biofilme. Como a aPDT apresenta também a vantagem de seletividade, foi proposto que as condições para fotoinativação destas bactérias provocassem o mínimo de dano às células hospedeiras. Estudos físico-químicos dos novos FSs mostraram menor agregação dos FSs derivados em meio aquoso que seus compostos de origem, bem como um ligeiro aumento no coeficiente de atividade fotodinâmica. Além disso, a hidrofilicidade dos FSs aumentou a acumulação intracelular dos mesmos nas bactérias de estudo S. aureus e E. coli, tanto na forma de células planctônicas quanto em biofilme. Os ensaios de acumulação intracelular dos FSs determinaram os parâmetros de fotoinativação seletiva dos microrganismos, tanto em células planctônicas como em biofilme. Todos os FSs, com exceção de FcZn, foram capazes de promover a seletividade de S. aureus e E. coli na forma planctônica. Entretanto, devido a maior complexidade morfológica de E. coli, os parâmetros de fotoinativação utilizados para inibir esta bactéria foram cerca de duas vezes maiores que para inativar a mesma concentração celular de S. aureus. Dentre todos os FSs, a FcZnTcG apresentou as melhores condições de seletividade tanto para E. coli quanto para S. aureus, uma vez que inibiu aproximadamente 100% destes microrganismos e no máximo 15% de células epiteliais (Vero). A obtenção das condições de seletividade para os biofilmes bacterianos foi mais difícil, pois a acumulação dos FSs por S. aureus e E. coli foi menor, tornando-se assim necessário aumentar os parâmetros de fotoinativação, ou seja, concentração do FS, tempo de incubação e dose de luz, que consequentemente inibiram mais as células epiteliais. Apesar disso, HyG e FcZnTcG foram capazes de promover a seletividade do biofilme de S. aureus em todas as etapas de formação. Entretanto, a seletividade do biofilme de E. coli foi alcançada apenas nas etapas de adesão reversível e irreversível e somente por FcZnTcG. Isso pode ser justificado pela maior concentração de EPS sintetizado por E. coli que por S. aureus, dificultando a acumulação dos FSs nas últimas etapas do biofilme de E. coli (biofilme maduro e dispersão). Portanto, os resultados desse estudo permitem sugerir que a hidrofilicidade é uma característica importante para os FSs fotoinativarem seletivamente os microrganismos S. aureus e E. coli, mesmo na forma de biofilme. Além disso, foi observado que a ação de aPDT no EPS do biofilme bacteriano desempenha um papel fundamental para inibição tanto de S. aureus quanto de E. coli.
The appearance of a large variety of antimicrobial-resistant pathogenic microorganisms has led to increased rates of disease and mortality caused by infections that were easily treated in the past. It has become clear that the biofilm-grown cells increase the bacteria resistance to antibiotics. Antimicrobial photodynamic therapy (aPDT) is a promising alternative way to fight microbial infections, especially the biofilm ones. This technique, basically, involves the synergistic combination of a photosensitizer, molecular oxygen and visible light of an appropriate wavelength to produce highly reactive oxygen species that lead to the oxidation of several cell components and to cell inactivation. The main advantage of the technique is that, given the existence of multiple targets, there is no development of resistance. The hidrophobicity of photosensitizers (PSs) like hypericin (Hy) and zinc phthalocyanine (ZnPc), reduces their photodynamic activity once they form aggregates in biological media. For this reason, was evaluated the effectivenes of Hy, ZnPc and its water-soluble derivatives (glucamine-hypericin-HyG, zinc tetracarboxylated phthalocyanine-ZnTcPc and zinc tetracarboxy-N-metylglucamine phthalocyanine-ZnTcGPc) to photoinactivate S. aureus and E. coli with minimal damage to a model of host cells. Physicochemical studies showed that hidrophilic PSs suffer less aggregation in aqueous media, as well as present a slight increase in photodynamic activity compared to Hy and ZnPC. Furthermore, the hydrophilicity of PSs increased the PS intracellular accumulation in bacteria, either in planktonic culture or biofilms. The accumulation study allowed to determine the parameters of selective photoinactivation of microorganisms. Due the morphologic complexity of E. coli the photodynamic parameters (incubation time, PS concentration and light dose) were twice that used against S. aureus. As a consequence, some more epithelial cells were affected by the process. Despite that, only the ZnPc could not promote the selective inactivation for planktonic cells. By the other hand, HyG and FcZnTcG were able to seletively photoinactivate the biofilm of S. aureus in all its formation stages. However, the selective inactivation of E. coli biofilm was achieved only in the reversible and irreversible adhesion and just for FcZnTcG. This fact can be explained by the higher concentration of exopolysaccharide (1,9 \'mü\'g/mL) synthesized by E. coli compared with S. aureus, making difficult the accumulation of the PSs in the last stages of the E. coli biofilm formation (mature biofilm and dispersion). So, we can suggest that hidrophilicity is an important characteristic for the PSs, improving the selective photoinactivation of S. aureus and E. coli, even as biofilm. Moreover, the effectiveness of aPDT agains EPS plays a key role for inhibition of bacterial biofilms.

Staphylococcus aureus destaca-se como principal micro-organismo associado à mastite bovina contagiosa, sendo que as infecções crônicas podem ser causadas pelo crescimento bacteriano na forma de biofilmes, o que pode estar associado à persistência desta bactéria na glândula mamária e à resistência a diversos antimicrobianos. Estudos epidemiológicos empregando técnicas como a macrorestrição seguida de eletroforese em campo pulsado (PFGE) têm sido realizados, com a finalidade de identificar clones e caracterizar as infecções por S. aureus. Os objetivos deste estudo foram: avaliar a frequência de isolamento de S. aureus em amostras de leite colhidas periodicamente em um grupo de propriedades leiteiras do Vale do Taquari, RS; avaliar o perfil de suscetibilidade aos antimicrobianos dos isolados de S. aureus identificados; classificar esses isolados em grupos clonais; avaliar a distribuição e a permanência dos grupos clonais nas propriedades leiteiras ao longo do tempo, além de verificar a presença de genes relacionados à formação de biofilmes (icaA e icaD). Foram colhidas amostras de leite de todas as vacas em lactação de 21 propriedades, amostradas semestralmente durante dois anos, totalizando 1060 amostras. A presença de S. aureus nas amostras foi detectada por isolamento e a identificação foi realizada de acordo com o National Mastitis Council. Isolados confirmados foram testados, pela técnica de disco difusão em ágar, quanto à suscetibilidade frente a treze antimicrobianos. Os isolados também foram submetidos à macrorrestrição do DNA total – PFGE e posteriormente testados pela PCR para detecção dos genes icaA e icaD. Das 1060 amostras avaliadas, 395 não apresentaram crescimento bacteriano. Staphylococcus sp. coagulase negativa foram identificados em 262 amostras, seguido de 136 amostras em que identificou-se S. aureus. A frequência de isolamento de S. aureus variou de 3,45% a 70,59% nas 17 propriedades em que este agente estava presente. No teste de suscetibilidade aos antimicrobianos, a maioria (75,7%) dos 132 isolados testados apresentaram perfil de sensibilidade, sendo a resistência mais frequente à penicilina (18,2%) e ampicilina (14,4%). Em apenas 27,3% dos isolados detectou-se os genes associados à formação de biofilmes pesquisados, sendo o gene icaD o mais prevalente, seguido da presença de ambos os genes. Os 122 isolados clivados pela enzima SmaI e submetidos à PFGE foram classificados em 38 grupos clonais. Observaram-se poucos grupos clonais persistentes, pois somente seis foram descritos consecutivamente em pelo menos duas coletas. O grupo clonal 16 foi o mais prevantente, apresentando isolados em uma mesma propriedade ao longo de dois anos. Conclui-se que Staphylococcus aureus está presente na glândula mamária de bovinos em lactação em pequenas propriedades da região. Esses isolados apresentam baixa frequência de resistência aos antimicrobianos. Há uma grande variabilidade de pulsotipos entre os isolados presentes nessas propriedades, porém poucos grupos clonais persistem nas propriedades amostradas. Não foi possível associar a permanência dos grupos clonais nos rebanhos à presença dos genes icaA e icaD ou ao perfil de resistência a antimicrobianos.
Staphylococcus aureus stands out as the main microorganism associated with bovine contagious mastitis, whereas chronic infections can be caused by bacterial growth in the form of biofilms, which can be associated with the persistence of the bacteria in the mammary gland and resistance to various antibiotics. Epidemiological studies employing techniques such as macrorestriction followed by pulsed field gel electrophoresis (PFGE) have been carried out, aiming to identify clones and characterize S. aureus infections. The objectives of this study were: to assess the frequency of S. aureus isolation in milk samples collected periodically in a group of dairy farms from Taquari Valley, RS; evaluate the profile of antimicrobial susceptibility of S. aureus identified isolates; classify these isolates in clonal groups; assess the distribution and retention of clonal groups in dairy herds over time and to verify the presence of genes related to biofilm formation (icaA and icaD). Milk samples were collected from all lactating cows from 21 properties that were sampled every six months for two years, totaling 1060 samples. The presence of S. aureus in the samples was detected by isolation and the identification was performed according to National Mastitis Council. Confirmed isolates were tested for susceptibility to thirteen antimicrobial by disk diffusion technique in agar. The isolates also underwent macro-restriction of total DNA - PFGE and were subsequently tested by PCR for detection of genes icaA and icaD. Of the 1060 samples tested, 395 showed no bacterial growth. Staphylococcus sp. coagulase-negative samples were identified at 262, followed by 136 samples in which S. aureus was identified. The frequency of isolation of S. aureus ranged from 3.45% to 70.59% in 17 properties wherein this agent was present. In antimicrobial susceptibility testing, the majority (75.7%) of the 132 isolates tested showed sensitivity profile, being most frequent resistance to penicillin (18.2%) and ampicillin (14.4%). In only 27.3% of the isolates were detected genes associated with biofilm formation surveyed and icaD was the most prevalent, followed by the presence of both genes. The 122 isolates cleaved by SmaI and submitted for PFGE were classified into 38 clonal groups. There were few persistent clonal groups, because only six groups were described consecutively in at least two collections. The clonal group 16 was the most prevalent, presenting isolates at the same property over two years. Is conclusive that Staphylococcus aureus is present in the mammary gland of lactating cattle on small farms in the region. These isolates have low frequency of antimicrobial resistance. There is great variability of pulsotypes among isolates in those properties, but few clonal groups persist in the sampled properties. It was not possible to associate the permanence of clonal groups in herds to the presence of icaA and icaD or to the profile of antimicrobial resistance.

O objetivo foi avaliar a ação do ácido indol-3-acético (AIA) na ausência e presença da peroxidase de raiz forte (HRP) sobre a viabilidade de cepas certificadas de Staphylococcus aureus ATCC 6538 (produtora de biofilme), ATCC 14458 (portadora do gene seb), ATCC 43300 (portadora do gene mecA) e ATCC 29213 (controle). Foi determinada a concentração inibitória mínima (CIM) e, após, escolheu-se uma concentração subinitória para avaliar a capacidade de formação de biofilme, atividade de enzimas antioxidantes, integridade de DNA, integridade de membrana para todas as cepas e avaliação da expressão do gene seb da cepa ATCC 14458. A CIM observada foi de 40 mM de AIA para cepa ATCC 6538, 60 mM para ATCC 43300,50 mM para ATCC 14458 e ATCC 29213. No entanto, houve diferença significativa (P<0,05), na presença de HRP, somente para as cepas ATCC 14458 e 43300. A concentração subinibitória de AIA utilizada foi de 30 mM para ATCC 6538 e ATCC 14458, e de 40 mM para ATCC 43300 e ATCC 29213.mM. A capacidade de formação de biofilme pelo S. aureus(biofilme positivo) não foi alterada pela presença de AIA ou AIA/HRP. A atividade específica de catalase e superóxido dismutase foi aumentada pela ação de AIA/HRP (P<0,05) para todas as cepas, porém, não houve diferença (P>0,05) entre AIA e AIA/HRP. O DNA manteve-se íntegro, porém, houve diminuição na integridade de membrana (P<0,05) para todas as cepas cultivadas na concentração subinibitória de AIA, sendo este efeito potencializado na presença de HRP somente para ATCC 43300. Houve redução na expressão do gene seb da cepa ATCC 14458 (P<0,05). Conclui-se que o AIA, associado à HRP apresenta efeito bacteriostático em cepas de Staphylococcus aureus portadoras de diferentes fatores de virulência.
The objective was to evaluate the action of indole-3-acetic acid (IAA) in the absence and presence of horseradish peroxidase (HRP) on the viability of certified strains of Staphylococcus aureus ATCC 6538 (producer of biofilm), ATCC 14458 (carrying the seb gene), ATCC 43300 (carrying the mecA gene) and ATCC29213 (control). Concentration was determined minimum inhibitory (CIM) and, after, we chose a concentration subinitória to assess the ability of biofilm formation, activity of antioxidant enzymes, integrity DNA, membrane integrity for all strains and evaluation of gene expression seb of strain ATCC 14458. The MIC for the strains ATCC 6538 was 40 mM IAA , for the strains ATCC 14458 and 29213 was 50 mM and for the strain ATCC 43300 was 60 mM IAA, however, significant differences (P<0.05) compared to the addition of HRP to the culture medium, was found only for the doses of MIC of the strains ATCC14458 and 43300. The concentration subinibitória IAA used was 30 mM for ATCC 6538 and ATCC 14458, and 40 mM to ATCC 43300 and ATCC 29213.mM for ATCC 6538 and ATCC 14458, and 50 mM to ATCC 43300 and ATCC 29213.The ability of biofilm formation by S. aureus(biofilm-positive) was not altered by the presence of IAA or IAA/HRP in the culture medium. The activity of catalase and superoxide dismutase was influenced by the action of IAA and IAA/HRP (P<0.05), but there was no difference (P≥0.05) between IAA and IAA/HRP. The DNA remained intact, however, there were changes (P<0.05) in the membrane integrity of microorganisms to sub-inhibitory concentrations of IAA and IAA/HRP, as well as the expression of the sebgene was lower (P<0.05) in microorganisms treated with IAA and IAA/HRP. It is concluded that the IAA has bacteriostatic effect on S. aureus carrying different virulence factors.