Thesis submitted in fulfilment of the requirements for the degree DOCTOR TECHNOLOGIAE: ENGINEERING: CHEMICAL In the FACULTY OF ENGINEERING At the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY 2009 / The white rot fungus, Phanerochaete chrysosporium strain BKMF-1767 (ATCC 24725), produces the extracellular enzymes, Lignin peroxidase (LiP) and Manganese peroxidase (MnP), that constitute the major route for lignin degradation by this organism. LiP and MnP have also been shown to play a major role in aromatic pollutant degradation. Due to the need for continuous production of LiP and MnP, a fixed-film bioreactor, classified as a membrane gradostat reactor (MGR), was developed. The implementation of batch-reactor operational parameters to the MGR system was found to be ineffective, thus creating the need for further research to improve the operational aspects of the MGR system to optimise its capabilities for continuous and industrial-scale operations. The research undertaken in this study, provides information that can be used to classify the dissolved oxygen (DO) transport kinetics into immobilised fixed-films of P. chrysosporium. Operational limitations of the MGR relating to environmental stresses in the bioreactor during operation and to biofilm deterioration, including limitations of DO mass transport, oxidative stress, trace element accumulation and polysaccharide storage in the fungal biomass, were evaluated in single capillary MGR systems (SCMGRs). These conditions were identified as existing in the continuous MGR systems. From DO profiles, the oxygen consumption and flux into the biofilms, including the distribution of DO, was determined to be dependent on the immobilised biofilm’s age. Younger biofilms showed higher DO distribution than older biofilms even when aeration was directed to the extracapillary space (ECS) of the reactor against the biofilm’s surface. An increase in anaerobic zone thickness was observed to be increasing with an increase in biofilm thickness. Although, DO kinetic parameters were comparable with those obtained in submerged mycelia pellets, higher oxygen consumption values were observed in biofilms grown in the SCMGRs. The limitations of MGR were identified as: 1) poor DO distribution in immobilised biofilms because of b-glucan production and storage in the immobilised biomass, resulting in ethanol production; 2) the peroxidation of lipids of the biofilms, which in turn will affect the long-term performance of the biomass caused by oxygenation and 3) trace element ion accumulation enhanced by b-glucan production. Furthermore, trace element ion accumulation was higher in the MGRs than in batch cultures using the same nutrient medium. The development of a perfluorocarbon (PFC) emulsion for the MGRs to counteract these limitations was investigated. The compatibility of the emulsion with oxygen-carrying capacity was shown with an improvement in biomass generation, LiP/MnP production and overall consumption of primary substrates, mainly glucose and ammonium tartrate, in batch cultures. The emulsions investigated were based on the addition of oxygen carriers: Perfluorooctyl bromide (PFOB), Bis-(Perfluorobutyl) ethene (PFBE) and Perfluoropropylamine (PFPA), using Pluronic F 68 (PF 68) as the surfactant. Concentrations of 10 to 30% (w/v) PFC and 8.5% (w/v) PF 68 were tested successfully in batch cultures. The emulsions containing 10% (w/v) PFCs resulted in improved biomass performance as opposed to emulsions with higher PFC oil concentrations. An emulsion containing 10% (w/v) PFOB was used to evaluate its efficacy in the SCMGRs, as the biomass yield and overall enzyme production were superior to PFPA and PFBE-based emulsions with similar oil concentrations. After successfully applying PFOB and PF 68 to the SCMGRs, the following results were obtained: 1) reduced ethanol production; 2) reduced trace element accumulation; 3) lower b-glucan production and 4) improved DO-penetration ratio in immobilised biofilms.

Orientador: Angela Cristina Cilense Zuanon / Banca: Cyneu Aguiar Pansani / Banca: Karina Antunes Neves / Resumo: O objetivo deste trabalho foi avaliar a porosidade, rugosidade superficial e atividade antibiofilme de um cimento de ionômero de vidro (CIV) após a incorporação de gluconato ou diacetato de clorexidina (CLX) em diferentes concentrações. Para os testes de porosidade e rugosidade superficial foram confeccionados 10 corpos de prova com o CIV Ketac Molar EasyMix (KM) divididos nos grupos: controle (C), CIV e diacetato de CHX 0,5% (D1), CIV e diacetato de CHX 1,0% (D2) e CIV e diacetato de CHX 2,0% (D3), CIV e gluconato de CHX 0.5% (G1), CIV e gluconato de CHX 1.0% (G2), CIV e gluconato de CHX 2.0% (G3). Para a avaliação da porosidade, os corpos de prova foram fraturados, os fragmentos foram fotografados em microscópio eletrônico de varredura (MEV) e as imagens, analisadas com auxílio do programa Image J. A rugosidade superficial (Ra) foi obtida por meio da média de três leituras realizadas na superfície de cada espécime, passando sempre pelo centro da mesma. Para a análise do teste de atividade antibiofilme foram utilizadas cepas de S. mutans ATCC 25175 e os grupos foram divididos em: C1 - CIV (1 dia); C2 - CIV (7 dias), C3- CIV (14 dias), C4 - CIV (21 dias); grupo D1: CIV+CLX (1 dia), D2 - CIV+CLX (7 dias), D3: CIV+CLX (14 dias), D4: CIV+CLX (21 dias); G1- CIV+ CLX (1 dia), G2- CIV+ CLX (7 dias), G3- CIV+ CLX (14 dias) e G4- CIV+ CLX (21 dias) sendo 10 corpos de prova por grupo. Para crescimento dos biofilmes, os espécimes foram posicionados verticalmente em placas de 24 poços, contendo 1,5 mL de inóculo fresco preparado em caldo BHI diluindo-se 10 vezes uma suspensão incubada overnight e 0,2% de sacarose. O meio de cultura foi renovado a cada 24 h. Após crescimento por 1, 7, 14 e 21 dias, os biofilmes foram re-suspensos em solução salina. A suspensão foi diluída e semeada em ágar BHI, para a quantificação de bactérias presentes. Para avaliação de todos os testes utilizou-se... / Abstract: The aim of this study was to evaluate the porosity, surface roughness and antibiofilm activity of a glass ionomer cement (GIC) after incorporating gluconate or chlorhexidine diacetate (CHX) at different concentrations. For tests of porosity and surface roughness were prepared 10 specimens with GIC Ketac Molar EasyMix (KM) divided into groups: control (C), and GIC CHX gluconate 0.5% (G1), GIC and 1.0% CHX gluconate (G2), and GIC 2.0% CHX gluconate (G3), GIC and 0.5% CHX diacetate (D1), GIC and 1.0% CHX diacetate (D2) and GIC and 2.0% CHX diacetate (D3). For the evaluation of porosity, the samples were fractured, the fragments were photographed under a microscopy scanning electron (SEM) and the images analyzed with the software Image J. The surface roughness (Ra) was measured by the mean of three readings taken at the surface of each specimen, always passing through the center thereof. For the analysis of the test anti-biofilm activity were used strains of S. mutans ATCC 25175 and were divided into groups: C1 - GIC (1 day); C2 - GIC (7 days), C3- GIC (14 days), C4 - GIC (21 days); group D1: GIC +CHX (1 day), D2 - GIC +CHX (7 days), D3: GIC +CHX (14 days), D4: GIC +CHX (21 days); G1- GIC + CHX (1 days), G2- GIC + CHX (7 days), G3- GIC + CHX (14 days) e G4- GIC + CHX (21 dias), with 10 specimens per group. For growth of biofilms, the specimens were positioned vertically in 24-well plates containing 1.5 mL of inoculum in fresh BHI broth prepared by diluting 10-fold a suspension incubated overnight and 0.2% sucrose. The culture medium was renewed every 24 h. After growth for 1, 7, 14 and 21 days, biofilms were re-suspended in saline. The suspension was diluted and plated on BHI agar for quantitation of bacteria. For evaluation of all tests we used analysis of variance of two factors, and if necessary, we applied the Tukey test, with significance level of 5%.As for the porosity of the GIC, analysis of... / Mestre

Orientador: Elisa Maria Aparecida Giro / Banca: Angela Cristina Cilense Zuanon / Banca: Renata Cristiane da Silva Molina / Resumo: Este estudo teve como objetivo avaliar a resistência de união à dentina e a composição microbiana do biofilme formado sobre um CIV de alta viscosidade ao qual foi adicionado diacetato de CLX em diferentes concentrações. A CLX foi misturada ao pó do CIV nas concentrações de 0,5%, 1% e 2%. O CIV sem CLX foi usado como controle. No primeiro estudo, foram utilizados 80 terceiros molares, que tiveram uma superfície de dentina exposta na face oclusal. Metade dos dentes foi mantida hígida e a outra metade foi submetida à indução artificial de cárie. Em cada superfície dentinária, foi confeccionado um espécime cilíndrico com 1mm de diâmetro x 1 mm de altura (n=10 por grupo). Estes foram mantidos a 37ºC com 100% de umidade por 24 horas, e submetidos ao teste de microcisalhamento. Os resultados foram analisados pelos testes de Kruskall-Wallis e Mann Whitney (α=0,05). No segundo estudo, voluntários (n=8) usaram dispositivos palatinos contendo espécimes (4 mm de diâmetro x 1 mm de altura). Os materiais foram testados por todos os voluntários na ordem crescente de concentração de CLX por um período de 7 dias e com intervalo de descanso de 15 dias entre elas. O biofilme formado sobre os espécimes foi coletado e analisado. Os dados referentes às contagens de microrganismos anaeróbios totais, estreptococos totais, estreptococos do grupo mutans e lactobacilos foram submetidos à análise de variância de medidas repetidas complementada pelo teste de Tukey. O nível de significância adotado foi de 5% . Não houve diferença estatística na resistência de união entre a dentina hígida e afetada (p>0,05). Os grupos CIV, CIV+CLX 0,5% e CIV+CLX 1% apresentaram resistência de união estatisticamente semelhante (p>0,05), e superior ao CIV+CLX 2% (p≤0,025). Houve diferença entre as médias de contagem de microrganismos apenas para o lactobacilos (p<0,05), sendo estas significativamente maiores... / Abstract: The aim of this study was to evaluate the bond strength to dentin and the microbial composition of the biofilm formed over a high-viscosity GIC to which chlorhexidine diacetate (CHX) was added at different concentrations. CHX was mixed with GIC powder at 0.5%, 1% and 2% (w/w). GIC without CHX was used as control. In the first study, eighty human third molars were used. They had an area of dentin exposed on the occlusal surface. Half of the specimens were kept sound and the other half was subjected to artificial caries induction. In each dentin surface a cylindrical specimen with 1 mm in diameter and 1 mm in weight (n=10 per group) was made. They were kept at 37°C and 100% humidity for 24 hours and tested for microshear. The results were analyzed using Kruskal-Wallis and Mann Whitney tests (α = 0.05). In the second study, volunteers (n=8) wore palatal appliances containing specimens with 4mm in diameter and 1mm in weight. The material with increasing concentrations of chlorhexidine were tested by all volunteers for a period of 7 days each, with a wasout period of 15 days. After each step, the biofilm formed on the specimens were collected and analyzed. Data from total anaerobic microorganisms, total streptococci, mutans streptococci and lactobacilli were analyzed by repeated measures ANOVA followed by Tukey test. The significance level was set at 5%. There were no statistical significant differences between bond strength of sound and caries-affected dentin (p> 0.05). Groups GIC, GIC+CHX 0.5% and GIC+CHX 1% showed bond strength statistically similar (p> 0.05) and higher than GIC+CHX 2% group (p ≤ 0.025). Microbial counts showed statistical significant differences among groups only for lactobacilli (p<0.05). GIC+CHX 2% presented higher mean counts than GIC and GIC + CHX 0.5% (p<0.05). In conclusion, the addition of CHX diacetate 0.5% and 1% did not change bond strength of a high-viscosity GIC in... / Mestre

Orientador: Marcia Carneiro Varela / Banca: Claudio Antônio Talge Carvalho / Banca: Caio Casar Randi Ferraz / Resumo: O objetivo desse estudo foi avaliar a ação de medicações intracanal sobre biofilme microbiano localizado em áreas de reabsorção externa de dentes humanos extraídos. Para isto, 90 raízes de dentes humanos extraídos apresentando reabsorção apical externa foram submetidas à instrumentação padronizada. Em seguida foram divididas aleatoriamente em 6 grupos (n=15). As raízes foram expostas a cepa de E. faecalis (ATCC 29212) por 10 dias para induzir a formação de biofilme microbiano nas áreas de reabsorção externa apical. Após a formação do biofilme, foram inseridas medicações intracanal de acordo com os grupos. Grupo (HC): hidróxido de cálcio com solução fisiológica; Grupo (HC-CHX): hidróxido de cálcio com Clorexidina gel 2%; Grupo (HC-GEN) hidróxido de cálcio com extrato glicólico de gengibre 20%; Grupo (CHX): clorexidina gel 2%; Grupo (GEN): extrato glicólico de gengibre; Grupo controle (CC): solução salina fisiológica. Cada grupo permaneceu com medicação no interior dos canais radiculares por um período de 15 (quinze) dias. Ao final desse período as raízes foram avaliadas através do cálculo de unidades formadoras de colônias por superfície do espécime (UFC/ml) e através de análise em Microscópio Eletrônico de Varredura (MEV). Os resultados foram analisados pelos testes estatísticos de ANOVA e Tukey. Verificouse que nenhuma medicação intracanal eliminou completamente o biofilme, mas houve redução dos microrganismos pela contagem de UFC/mL no grupo CHX, seguido pelo grupo HC. Os demais grupos apresentaram resultados semelhantes ao grupo controle. As imagens por MEV confirmaram a permanência do biofilme nas áreas de reabsorção apical. Conclui-se que as medicações intracanal não foram capazes de eliminar completamente o biofilme apical de E. faecalis in vitro(AU) / Abstract: The aim of this study is to evaluate the effects of intracanal medication on microbial biofilm located in apical external root resorptions of extracted human teeth. 90 roots of extracted human teeth with apical external root surface were submitted to standardized instrumentation. The roots will be randomly divided into 6 groups of 15 roots each (n=15).The roots were exposed to E.faecalis strains (ATCC 29212) for 10 days to induce microbial biofilm formation in resorptions of apical external root surface. After biofilm formation, intracanal medications were inserted in each group. Group (HC): calcium hydroxide with sterile saline solution; Group (HC-CHX): calcium hydroxide with Chlorhexidine gel 2%; Group (HCGEN): calcium hydroxide with glycolic extract of ginger 20%; Group (CHX): chlorhexidine gel 2%; Group (GEN): extract of ginger 20%; Control group (CC): sterile saline solution. Each group remained with medication in the root canal for fifteen (15) days. After this period the roots were evaluated by calculating the colony-forming units per surface of the specimen (CFU / ml) and by analysis of Scanning Electron Microscope. The results will be analyzed using statistical tests of ANOVA and Tukey. It was found that no dressing completely eliminated biofilm, but it was a decrease in microorganisms number according to CFU / mL in the CHX group, followed by HC group. The other groups showed similar results to the control group. The SEM images confirmed the permanence of biofilm in areas of apical resorption. It is concluded that the dressing is not able to completely eliminate the biofilm apical E. faecalis in vitro(AU) / Mestre

Orientador: Maria de Lourdes Ribeiro de Souza da Cunha / Banca: Elizabeth Pizzollito / Banca: Carlos Magno Castelo Branco Fortaleza / Resumo: Staphylococcus epidermidis e Staphylococcus haemolyticus são considerados patógenos oportunistas, cujas infecções estão principalmente associadas à produção de biofilme. As enterotoxinas, superantígenos relacionados a intoxicações alimentares, e as hemolisinas, moléculas capazes de causar lise de membranas celulares, parecem estar associadas à patogênese das infecções estafilocócicas, apesar de ainda ser questionado se elas constituem fatores de virulência importantes nesses organismos. O uso indiscriminado de antimicrobianos vem selecionando cepas resistentes à oxacilina com menor suscetibilidade à vancomicina. A resistência à oxacilina é codificada pelo gene mecA, contido em um elemento genético móvel, denominado SCCmec. Novos antimicrobianos, como a linezolida, tigeciclina, daptomicina e quinupristina/dalfopristina vêm sendo empregados no tratamento de infecções por estafilococos multirresistentes. Este estudo objetivou caracterizar S. epidermidis e S. haemolyticus isolados de hemoculturas quanto à presença de genes de hemolisinas, enterotoxinas, suscetibilidade aos antimicrobianos e perfil clonal. Cento e sessenta e nove isolados foram pesquisados quanto à presença do gene mecA por PCR em tempo real, estando presente em 100% dos S. haemolyticus e 92,9% dos S. epidermidis, cujos tipos de SCCmec mais frequentes, determinados por PCR Multiplex, foram, respectivamente, I e III. Os MICs (Concentração Inibitória Mínima) de linezolida, tigeciclina, daptomicina, quinupristina/dalfopristina e vancomicina foram obtidos por Etest® e revelaram 4,7% de isolados resistentes à tigeciclina, 1,2% resistentes ou com resistência intermediária à quinupristina/dalfopristina, sendo o MIC máximo de vancomicina igual a 3 μg/ml. A microdiluição em caldo para vancomicina mostrou MICs de 0,25 a 2 μg/ml, concordando em 88,2% com os resultados obtidos com Etest®. A tipagem por PFGE... / Abstract: Staphylococcus epidermidis and Staphylococcus haemolyticus are opportunistic pathogens that cause infections which are mainly related to the formation of a biofilm. Enterotoxins, superantigens related to food poisoning and hemolysins, molecules able to lyse mammalian cells, seem to be involved in the pathogenesis of staphylococcal infections, although the question remains whether they represent important virulence factors in these organisms. The indiscriminate use of antimicrobial drugs has selected strains that are resistant to oxacillin, in addition to isolates with reduced vancomycin susceptibility. Oxacillin resistance is encoded by the mecA gene which is carried on a mobile genetic element, SCCmec. New antimicrobial drugs such as linezolid, tigecycline, daptomycin and quinupristin/dalfopristin are being used for the treatment of infections caused by multidrug-resistant staphylococci. The objective of this study was to characterize S. epidermidis and S. haemolyticus strains isolated from blood cultures regarding the presence of hemolysin and enterotoxin genes, antimicrobial susceptibility, and clonal profile. Investigation of the mecA gene by real-time PCR in 169 isolates revealed the presence of the gene in 100% of S. haemolyticus isolates and 92.9% of S. epidermidis. The most frequent SCCmec types determined by multiplex PCR were types I and III, respectively. The minimum inhibitory concentrations (MICs) of linezolid, tigecycline, daptomycin, quinupristin/dalfopristin and vancomycin determined by the Etest® showed that 4.7% of the isolates were resistant to tigecycline and 1.2% were resistant or intermediate resistant to quinupristin/dalfopristin. The maximum MIC for vancomycin was 3 μg/ml. Broth microdilution revealed vancomycin MICs of 0.25 to 2 μg/ml, showing 88.2% agreement with the Etest results. Pulsed-field gel electrophoresis (PFGE) typing revealed the presence of S. epidermidis ... / Mestre

Orientador: Theo Guenter Kieckbusch / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-11T12:31:13Z (GMT). No. of bitstreams: 1 Turbiani_FrancieleRezendeBarbosa_M.pdf: 2355251 bytes, checksum: 54057feae41bd47bee56d5615dbbfd3d (MD5) Previous issue date: 2007 / Resumo: Filmes biodegradáveis são produzidos a partir de polímeros naturais, principalmente polissacarídeos e proteínas, e tem potencial aplicação na área médica, farmacêutica e alimentícia. A incorporação de agentes ativos pode ampliar suas funções como embalagens antimicrobianas, por exemplo. Filmes foram confeccionados à base de alginato de sódio usando cloreto de cálcio como agente reticulante e glicerol como plastificante. O uso de benzoato de cálcio foi investigado como agente ativo (íons benzoato) e como auxiliar na reticulação (íons cálcio). Devido ao alto poder gelificante do Ca++, confeccionou-se, inicialmente, um filme de baixa reticulação a partir de soluções filmogênicas contendo até 0,54% de Ca++ (1º estágio). Esse filme sofreu uma reticulação complementar com excesso de Ca++ (2º estágio). Dentre os vários procedimentos avaliados (contato do filme com tecido e/ou esponja umidecidas, pincel ou rolo de pintura e imersão do filme em solução reticuladora), a simples imersão em solução contendo de 3 a 7% de CaCl2 no 2º estágio produziu filmes com alto grau de reticulação. O aumento da concentração de glicerol nessa solução melhora a manuseabilidade e plasticidade dos filmes, porém aumenta a solubilidade em água e o conteúdo de umidade dos mesmos e um adequado compromisso foi obtido usando 5% desse plastificante. Ensaios nos quais o CaCl2 foi substituído, total ou parcialmente, por benzoato de cálcio indicou que o mesmo não pode ser usado na solução do 2º estágio por favorecer a precipitação de cristais sobre o filme. Filmes ativos de 0,06 mm de espessura, pré-reticulados apenas com benzoato de cálcio e 0,7% de glicerol na solução do 1º estágio e imersos por 30 minutos em banho contendo 3% de CaCl2 e 5% de glicerol no 2º estágio, apresentaram baixa solubilidade em água (até 20% da matéria seca). Estes filmes têm baixo grau de intumescimento (< 50% da massa inicial), boa resistência mecânica à tração, mas baixa elasticidade. A permeabilidade ao vapor de água é moderada e os valores encontrados são típicos de biofilmes hidrofílicos. Ensaios de liberação de benzoato utilizando água como sorvedouro, apresentaram bom ajuste as soluções da 2ª Lei de Fick, com valores de difusividade efetiva do benzoato variando de 3 a 5.10-7 cm2/s. Os valores de difusividade diminuiram com o aumento da reticulação e aumentaram com o aumento da concentração de benzoato no filme / Abstract: Biodegradable films are produced from natural polymers, structurized mainly by polysaccharides or proteins, and have potential applications in the medical, pharmaceutical or food area. The incorporation of active agents can extend their application as antimicotic packaging, for instance. Films were manufactured with sodium alginate, using calcium chloride as cross-linking agent and glycerol as plasticizer. The use of calcium benzoate as active agent (benzoate ions) and as crosslinking agent (calcium ions) was investigated. Due to the strong gelling power of Ca++ ions, impeding smooth casting procedures, films with low reticulation are initially manufactured, using less than 0.54% Ca++ in the filmogenic solutions (1st stage). These films are further crosslinked with an excess of Ca++ by immersion in a solution of 3% to 7% of CaCl2 (2nd stage). Increasing the glycerol concentration in this solution improves the handling and plasticity of the films but increase water solubility and moisture content and an adequate compromise was obtained using 5% plasticizer. Tests conducted with partial or total substitution of CaCl2 by calcium benzoate indicated that the later could not be used in the 2nd stage solution since it promoted crystals precipitation on film surface. Active films, 0.06mm thick, pre-reticulated with calcium benzoate only and with 0.7% glycerol in the solution of the 1st stage, immersed for 30 minutes in a 3% CaCl2 and 5% glycerol solution (2nd stage) had around 17% moisture content and low water solubility (up to 20% of total dry mass). These films show low swelling degree (<50% of initial mass), good tension strength but low elongation ability. The water vapor permeability is moderate, typical of highly hydrophilic films. Benzoate liberation tests, using pure water as sink, presented good fit to solutions of Fick¿s 2nd law and effective diffusivities found varied from 4.2 to 6.3 × 10-7 cm2/s. The diffusivity values decreased with the degree of reticulation and increase with benzoate concentration in the film / Mestrado / Engenharia de Processos / Mestre em Engenharia Química

Orientadores: Karina Cogo Müller, Francisco Carlos Groppo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-25T11:18:29Z (GMT). No. of bitstreams: 1 Graziano_TalitaSignoreti_M.pdf: 1603562 bytes, checksum: 9522f75e1f8c1147c84c0fde100549ed (MD5) Previous issue date: 2014 / 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

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Empirical observation of filter beds at South African water treatment plants showed that the filters were insufficiently cleaned by the backwash system and that media losses were unexpectedly high. Specific deposit tests developed by the RAU Water Research Group indicated that the dirtiness correlated with the organic content of the water being treated. This led to the hypothesis that biofilm is present on the media, somehow causing both the media loss and the difficulty to attain efficient backwashing. Biofilm consists of organisms surrounded by a sticky, gelatinous polysaccharide matrix. This matrix, also known as extra-cellular polymeric substances (EPS), is the bulk (50-90%) of the biofilm. Biofilm plays an important role in the establishment and maintenance of organisms in a hostile environment. From the above it doesn¡¦t make sense trying to measure biofilm from the numeration of the organisms. A more reliable direct but tedious measure is quantifying the EPS. A new alternative method developed by the RAU Water Research Group is to mechanically strip the specific deposit off the filter media and then determine the organic fraction by combusting the sample at 500¢XC. Two aspects of mechanical behaviour are deemed important in this study. First, headloss, because an under prediction in headloss will result in a higher than expected backwash frequency. Second, bed expansion, because an under prediction in bed expansion will lead to media washout. Literature indicates that both headloss and bed expansion increase with increasing biofilm growth. However, all those studies were conducted at waste water treatment plants with high organic and solids loading. With the exception of one reference which only discusses headloss, nothing on this topic is available in the literature for potable water treatment. Mathematical models were used to reduce the data from multiple headloss and bed expansion experiments. For the headloss data the Ergun equation was used and the sphericity (ƒÚ) was retained as the only unmeasured calibration constant. For the bed expansion data the Dharmarajah equation was used and the sphericity was retained as the only unmeasured calibration constant. Calibration of the mathematical models was done with least square fitting. The two values of sphericity as determined by Ergun and Dharmarajah are not necessarily the same for the same media sample. The sphericity was used as a calibration constant without any physical meaning, which accounts for different sets of complex unknowns. Samples for experimental work were drawn from full scale operating water treatment plants. The treatment plants were spread over four provincesof South Africa with different raw water sources, but using approximately the same media. The sampling was done on three occasions, Winter 2003, Summer 2003 and Winter 2004, to cover the extreme temperatures experienced in South Africa. Samples collected at the plants were tested for headloss and bed expansion, then transported back to the laboratory and placed in the oven for 24 hours at 110¢XC. The sample was then sieved and the density determined. The headloss and bed expansion tests were then repeated in the laboratory. Parallel to these tests, EPS and volatile fraction quantification tests were done. Direct methods of measuring biofilm, namely EPS and volatile fraction, yielded measurable results, thereby confirming the presence of biofilm. Plants that had large quantities of EPS also had a high volatile fraction, thereby confirming the expectation that the volatile fraction is an excellent method to rapidly quantify biofilm presence. EPS made up 41% of the volatile fraction, which is roughly comparable with the 50-90% quoted in literature. Where large quantities of EPS were found at a plant, a high TOC reduction also occurred through the filters. The indirect methods of measuring biofilm, namely headloss and bed expansion, also yielded measurable results. The filter media with biofilm as sampled from the treatment plants had a higher headloss and bed expansion than the same sample after drying and sieving, which resembles virgin filter media. The sphericity values for headloss decrease by as much as 26% which translates to a headloss gradient increase of 150mm/m at typical filtration rates. The sphericity values for bed expansion decrease by as much as 18% which translates to a bed expansion increase of 17% at normal backwash rates. The conditions at the treatment plants sampled suggest that biofilm growth is stimulated by eutrophic raw water and the presence of pre-ozonation and inhibited when the high pH lime process is used. The mechanism which causes the increased headloss and bed expansion with increased biofilm is hypothesised to be media grains sticking together causing clumping, and not grains which are individually and uniformly covered with a smooth, uniform layer of biofilm. Designers can compensate for this increase in headloss and bed expansion in two ways. They could either apply a correction factor after application of the models to allow for more headloss or bed expansion during eventual plant operation, or they could adjust parameters within the models to account for the larger headloss or bed expansion. As the surface area sphericity was used as a calibration factor in this study and could account for different sets of complex unknowns, it is suggested that this factor is used for adjustment of the model. Operational practice in South Africa often includes in-situ chlorine or acid treatment to alleviate the problem of dirty filter beds. In this study, however, where high and efficient backwash rates were used during tests, no significant improvements in media cleanliness could be attributed to the use of either chlorine or acid. It seems that a good backwash system doesn¡¦t need such remediation, but plants with a backwash system which underperforms might find such remediation useful. / Prof. J. Haarhoff

Please read the abstract in the section 00front of this document / Dissertation (MSc (Microbiology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted