<p>Streptococcus pneumoniae is a gram-positive bacterium that can be found in both healthy carriers as well as in people that have developed disease. One of the major virulence factors of pneumococci is their polysaccharide capsule. Based on the capsule that surrounds the bacteria, pneumococci are divided into at least 90 different serotypes. Some serotypes seem to be more related to virulence than others.</p><p>I have with comparative genome hybridization microarray technique, studied gene differences between 18 epidemiological well-characterised pneumococcal strains with different potential to cause disease. A microarray chip based on two sequenced pneumococcal genomes, R6 and TIGR4, has already been designed. According to Hierarchical clustering, both the serotype and the genetic type as assessed by MLST (sequence type or ST) seem to have impact on the relationship of clinical isolates. Most clinical isolates of the same serotype are clustered together except for serotype 14 isolates that seem to be more divergent. Further more the number of genes that are divergent between clinical isolates compared to R6 and TIGR4 differ from 65 to 289. Preliminary results indicate that although there is diversity among clinical isolates some are more closely related to each other then others. Absent genes seem to be evenly distributed among all 18 clinical isolates tested but hypothetical genes and genes for cell envelope are two groups of role categories that are absent to the largest extent in all isolates.</p><p>According to results from microarray analysis, a gene region, spr0112-spr1015- is present in all type 9V isolates and absent in many isolates of serotype 14, 19F and 7F. These results have been confirmed by polymerase chain reaction (PCR).</p><p>Conserved genes in a region around the capsule genes have been sequenced to identify marker genes for a capsulular switch between serotype 9V and 14. Preliminary results of the sequencing showed that as much as 750kb might have been transferred in the event of capsular switch.</p>

The biochemical activity of Streptococcus thermophilus and Lactobacillus bulgaricus in a 1:1 ratio (Chr. Hansen's Lab - CH-III) were studied at 43 C in goat and cow milk during the manufacture and storage of yogurt. Determination of pH, titratable acidity, lactose content, proteolytic activity, and enumeration of the starters were performed on samples taken at hourly intervals until the yogurt became set, and after 8, 16, 24 hours, 11 and 22 days of storage at 4 C. In goat milk the number of starter culture organisms showed higher values than those in cow milk. Fermentation was considered to be finished when the pH decreased to 4.7 and 0.7% of lactic acid was produced. Those values were reached after 3 and 4 hours for goat and cow milk respectively. The values for pH in both milks were stable at 4.7 during storage at 4 C. Goat milk and goat milk yogurt had higher levels of free amino acids (12.65 mg/dl and 24.39 mg/dl respectively) than cow milk (10.31 mg/dl) or cow milk yogurt (20.20 mg/dl). During storage at 4 C free amino acids in goat milk decreased by 20%, in cow milk they increased by 70% from values found in fresh yogurt. The proteinase activity in goat milk during elaboration of yogurt was from 2 to 3 hours after incubation, was then stable until 24 hours of storage at 4 C and then decreased. No change in the proteinase activity in cow milk yogurt was seen until 4 hours of incubation for experiments 1 and 2 and 3 hours in experiment 3. During the storage at 4 C, proteinase activity in cow milk yogurt was stable. Polyacrylamide gel electrophoresis of caseins failed to reveal marked changes during the manufacture of yogurt and storage after 22 days at 4 C. However, a scanning gel densitometer showed that the area corresponding to alpha-casein decreased by 22% and for beta-casein 13.32% during the elaboration of goat milk yogurt. After 22 days of storage at 4 C, the alpha-casein fraction decreased an additional 14%, but no change was observed in beta-casein. The area corresponding to alpha- and beta-caseins decreased by 7.16 and 14.48% respectively during yogurt manufacture and an additional 6.74 and 8% during storage of cow milk yogurt. The overall results found in this study suggest that the metabolic activity of S. thermophilus and L. bulgaricus is greater in goat milk than in cow milk during elaboration and storage of yogurt.

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / INTRODUCCIÓN: En el biofilm dental se encuentran diversas bacterias, de las cuales Streptococcus mutans (S. mutans) es considerada como una de las de mayor potencial cariogénico. Existen diversos métodos para el control del biofilm, entre los que se encuentran los métodos químicos. Estos métodos se basan en el uso de sustancias antisépticas y/o antibióticas que son utilizados como colutorios orales y que permiten disminuir o retardar la formación de la placa bacteriana. En la actualidad no existe un agente químico antimicrobiano ideal. Es por esto que constantemente se están realizando cambios a los agentes terapéuticos actualmente conocidos, ya sea mejorando sus propiedades antimicrobianas, sus propiedades de adhesión (sustantividad) o disminuyendo sus efectos adversos. Es aquí donde la Nanotecnología y el desarrollo de nanomateriales han adquirido especial importancia debido a la posibilidad de sintetizar materiales con propiedades antimicrobianas. Es sabido que nanopartículas de quitosano (QuitNP) y nanopartículas de cobre (CuNP) presentan propiedades antimicrobianas frente a diversas bacterias, tanto Gram negativas como Gram positivas. Sin embargo no se ha probado la efectividad de las CuNP sobre S. mutans. Por otra parte estudios demuestran que nanopartículas metálicas, como el cobre, soportadas en una matriz de quitosano, presentarían mejoras en sus propiedades de adhesión. El objetivo de este trabajo de tesis es la síntesis de nanopatículas de cobre y quitosano, y evaluar sus propiedades antimicrobianas frente a S. mutans. MATERIAL Y METODOS: Se sintetizaron CuNP c/a utilizando almidón como agente reductor y estabilizante, CuNP s/a sin utilizar almidón, QuitNP y una nanopartícula hibrida (CuNP/QuitNP). Para esto se usaron reactivos biocompatibles, que permitieron la reducción a nanopartículas metálicas, y la gelificación iónica de nanopartículas poliméricas. Las muestras fueron caracterizadas mediante espectrofotometría de absorción molecular, microscopia electrónica de barrido (SEM), difracción de rayos X (DRX), espectroscopia infrarroja de reflectancia total atenuada (FTIR-ART) y análisis termogravimétricos (TGA). Se realizaron ensayos de actividad bactericida, para lo cual se determinó la concentración inhibitoria mínima (CIM) utilizando el método de incubación de medio de cultivo líquido, y la concentración bactericida mínima (CBM) realizando el recuento de unidades formadoras de colonias (UFC) en placas de agar BHI, para las nanopartículas sintetizadas. Además se realizaron pruebas de actividad bactericida sobre un biofilm formado en superficies dentarias. Para esto se utilizaron fragmentos de terceros molares, con un biofilm formado, los que fueron tratados con distintas soluciones antimicrobianas y suspensiones de nanopartículas. RESULTADOS: Utilizando el concepto de “química verde”, se sintetizaron y caracterizaron las partículas de carácter nanométrico; CuNP c/a, QuitNP y CuNP/QuitNP. Por otro lado, las partículas CuNP s/a presentaron dimensiones micrométricas. En los ensayos de actividad bactericida, las CuNP c/a fueron las que presentaron la menor CIM (20 µg/ml) y CBM (30 µg/ml) en comparación a las otras suspensiones. En los ensayos sobre un biofilm formado sobre una superficie dentaria, aquellas nanopartículas que tenían quitosano como parte de su composición fueron las que presentaron mejores resultados, siendo la nanopartícula hibrida (CuNP/QuitNP), que al probarla frente a S. mutans, se obtuvo el menor recuento de UFC. CONCLUSIÓN: El método estudiado en este trabajo, utilizando reactivos biocompatibles permitió la formación de nanopartículas de cobre y quitosano de manera satisfactoria y con características compatibles para futuros estudios en control de biofilm. La incorporación de CuNP c/a durante el proceso de formación de las QuitNP, permitió la síntesis de una nanopartícula hibrida cobre-quitosano (CuNP/QuitNP). Tanto las nanopartículas de cobre, de quitosano y sus combinaciones presentaron actividad antimicrobiana frente al patógeno Streptococcus mutans. Por otra parte, todas las suspensiones de nanopartículas presentaron un efecto antibacteriano al tratar un biofilm de S. mutans previamente formado sobre una superficie dental.

Autoimmune encephalitic (AE) syndromes represent a unique manifestation of autoimmunity: the immune system recognizes the brain as foreign, and interferes with neuronal function. AE syndromes are characterized by hallucinations, paranoia, anxiety, seizures, and autonomic dysregulation, and progress over a matter of weeks as autoantibodies targeting the brain bind more densely to their CNS targets. Development of AE has been linked to peripheral tumors and infection, both of which provide structural mimetics to CNS antigens to incite an immune response in the periphery. How these brain-specific antibodies reach the CNS remains unclear. In rare cases, Group A Streptococcus (GAS, S. pyogenes) infections can cause CNS autoimmunity targeting the basal ganglia, termed post-infectious basal ganglia encephalitis (BGE), manifesting as motor (Sydenham’s chorea) and psychiatric (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus, PANDAS) abnormalities. In a mouse model of post-infectious BGE, we have shown that repeated intranasal GAS infections generate robust T cell infiltration into the CNS, concomitant with blood-brain barrier breakdown, microglial activation, and olfactory synapse degradation. However, the different T cell subtypes that enter the brain, how they enter the CNS, and their relative contributions to neural dysfunction and neuroinflammation remain unclear. Th17 lymphocytes are heavily implicated in many autoimmune diseases, but whether the inflammatory post-infectious BGE reaction induces functional deficits in odor processing and requires Th17 lymphocytes was unknown. Here we demonstrate that mice lacking Th17 lymphocytes display both reduced BBB impairment and antibody infiltration. Furthermore, multiple GAS infections induce deficits in odor processing, which are partially ameliorated in Th17 cell-deficient mice. Notably, neuroinflammation and some excitatory synaptic loss persist, due to the presence of Th1 lymphocytes. Th17 lymphocytes are therefore critical for both selective CNS entry of autoantibodies and neural circuit impairment during post-infectious BGE. By examining the regulation of chemokine ligand expression in GAS-inoculated mice, we determined that CCL20/CCR6 and CCL2/CCR2 chemokine axes are implicated in T cell homing to the brain. In chemokine receptor mutant CCR6+/- CCR2-/- mice, T cell infiltration is reduced by 86.2%, and microglial activation is blunted. These findings suggest that CCL2/CCR2 and CCL20/CCR6 signaling may play a role in T cell homing to the brain and neuroinflammation. Finally, we first assessed behavioral and immune responses in two different GAS exposure models. It was clear that while behavioral abnormalities can be recapitulated in mice given subcutaneous GAS immunizations, this elicited relatively weak cellular and humoral immune responses. By contrast, mice given intranasal GAS inoculations showed minimal behavioral abnormalities, but elicited robust humoral and cellular immune responses. Taken together, these data demonstrate the pivotal role of Th17 lymphocytes in brain pathology and olfactory processing deficits after recurrent GAS infections in our mouse model. Our intranasal inoculation model supports the conclusion that post-infectious BGE is autoimmune in nature, despite the absence of behavioral symptoms in this model. Using multiple mouse models of post-infectious BGE may allow us to study distinct facets of disease pathogenesis. Finally, this work underscores the ability of T cells to incite neuroinflammation, provides a useful clinical diagnostic test in olfactory functional assessments, and lends support to T cell immunotherapy strategies in patients with post-infectious BGEs.

DSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2009.

The serum of an immunized animal will contain antibodies referred to as agglutinins, precipitins, opsonins, bacteriolysins, or complement-fixing antibodies (Zinsser 1952). The presence of such antibodies may be demonstrated in the laboratory, the type of reaction depending on the circumstance and the laboratory manipulation employed. Regardless of the specific serolological method utilized, the manifestation of the antigen-antibody reaction is the visible observation that such a combination has occurred.

Um novo monômero, brometo de metacriloiloxidodecilpiridínio (MDPB), com efeito antibacteriano e capacidade de co-polimerizar com outros monômeros, foi apresentado por Imazato, Torii e Tsuchitani (1993). Este estudo avaliou a adesão bacteriana, em 16, 40 e 64 horas, à resina composta contendo ou não o monômero antibacteriano MDPB. A adesão foi testada para Streptococcus sanguinis e Streptococcus sobrinus. Após as amostras terem sido submetidas à incubação, o biofilme foi coletado e a contagem de UFCs foi realizada. Os dados foram comparados pelo método ANOVA complementado por teste de Tukey. Os resultados demonstraram que, para o S. sanguinis, a adesão sobre o MDPB foi significativamente maior (p<0.05) quando comparado ao controle em 16 horas, mas diminuiu significativamente em 40 horas, não apresentando diferenças quando comparado ao controle neste tempo (p<0.01). Para o S. sobrinus, o controle apresentou aumento significativo da adesão bacteriana em 64 horas quando comparado com 16 horas (p<0.01), sendo significativamente maior que para o MDPB em 64 horas (p<0.05). Assim, o estudo mostrou que o MDPB é capaz de inibir a adesão de S. sobrinus sem interferir na adesão do S. sanguinis. Portanto, nas condições deste estudo os resultados sugerem que a incorporação do MDPB a resinas compostas pode ser de importância na prevenção de cáries secundárias favorecendo a adesão de bactérias comensais em detrimento de bactérias com potencial cariogênico. / A new antibacterial monomer, Methacryloyloxydodecylpyridinium bromide (MDPB), with antibacterial property and ability to co-polymerize with other monomers, was introduced by Imazato, Torii and Tsuchitani (1993). This study aimed to analyze the effect of MDPB on bacterial adherence to resin composites containing or not MDPB. Streptococcus sanguinis and Streptococcus sobrinus were used. The biofilms were collected from the samples and the colony forming units (CFUs) were counted after 16, 40 e 64 h of incubation. The data were compared by ANOVA complemented by the Tukey’s test. The results showed that the adhesion of S. sanguinis to MDPB-containing resin composite was significantly higher (p<0.05) than to control samples at 16 h, but significantly diminished at 40 h, reaching values similar to those of control samples (p<0.01). The adherence of S. sobrinus to control samples significantly increased throughout the experimental time (p<0.01) and was considerably higher than to MDPB at 64 h (p<0.05). Thus, the study showed that MDPB is capable of inhibit adhesion of S. sobrinus with no interference on the adhesion of S. sanguinis. Thus, at the conditions of this study we suggest that MDPB incorporated to resin composites could be of importance to prevent secondary caries favoring adhesion of commensal bacteria and impairing adhesion of cariogenic bacteria.

Streptococcus pneumoniae is a highly diverse and adaptable opportunistic pathogen that can infect and colonise different niches within the human host to cause a wide range of invasive disease (sepsis and meningitis) and noninvasive disease (pneumonia, otitis media and sinusitis). The molecular mechanisms that contribute to the different patterns of pneumococcal infection remain largely unknown. This thesis aims to determine the physiological and proteomic responses that allow the pneumococcus to survive and adapt to invasive and non-invasive sites. The comparative proteomic analyses of clinical S. pneumoniae isolates recovered from blood cultures (classified as invasive site isolates) and mucosal surfaces such as sputum, skin and ear swabs (classified as non-invasive site isolates) was initiated. The pneumococci were grown in vitro under standard conditions and the total cellular bacterial proteins extracted and analysed using both gel based and non-gel based proteomic approaches. Analysis of the pneumococcal isolates by two-dimensional polyacrylamide gel electrophoresis (2DGE) revealed that a high degree of heterogeneity existed between the pneumococcal isolates particularly among isolates in the invasive site isolates. Differential patterns of protein synthesis were observed that discriminated the pneumococcal isolates according to their sites of isolation. These were proposed to be associated with the bacterial adaptation to invasive and non-invasive sites of infection. Mass spectrometry was used to identify selected significant (ANOVA, p < 0.05) protein spots, which were further categorised into functional groups by Gene Ontology analysis. An extension of the 2DGE data using an integrated approach comprising bioinformatics, surfome analysis and a shotgun proteomic workflow provided a comprehensive qualitative and quantitative analyses of the pneumococcal intracellular and cell-surface proteomes. Proteins potentially involved in pneumococcal niche-specific adaptation and surface proteins with potential for further investigation and inclusion in the pipeline of vaccine candidates were identified. Quantitative regulation of proteins involved in energy metabolism, genetic competence, stress response, surface adhesion and virulence were considered important for pneumococcal adaptation to invasive and non-invasive sites. The anatomical sites colonised by the pneumococcus vary in their V availability for iron. The 2DGE method was also used on selected pneumococcal isolates from the two sites of infection to define the proteome variability linked to the effect of iron starvation that may contribute to the different disease outcomes associated with pneumococcal infections. The iron restricted condition was generated by cation depletion of the growth medium using Chelex-100. Quantitative differences in protein abundance were demonstrated that correlated with pneumococcal adaptation to iron restriction. The identification of selected significant spots by liquid chromatography-mass spectrometry and systems biology analysis of the identified proteins contributed to the elucidation of the molecular mechanisms underlying pneumococcal survival under iron limitation. The expression/repression of proteins functionally associated with metal ion binding, oxidative stress response, translation and virulence mainly constituted the pneumococcal adaptive responses to growth under conditions of limited iron availability. The data presented in this thesis extended our understanding of the molecular events underlying pneumococcal physiological adaptation and provide the basis of future work in this area.

Cheuk Shing Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 144-161). / Abstracts in English and Chinese. / ACKNOWLEDGMENTS --- p.I / 內容摘要 --- p.II / ABSTRACT --- p.IV / CONTENTS --- p.XI / LIST OF TABLES --- p.XI / LIST OF FIGURES --- p.XI / ABBREVIATIONS --- p.XII / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Taxonomy of Streptococcus agalactiae --- p.1 / Chapter 1.2 --- Characteristics of Streptococcus agalactiae --- p.1 / Chapter 1.3 --- Epidemiology of GBS --- p.3 / Chapter 1.3.1 --- Risk groups --- p.3 / Chapter 1.3.1.1 --- Neonates --- p.3 / Chapter 1.3.1.2 --- Pregnant women --- p.5 / Chapter 1.3.1.3 --- Non-pregnant adult --- p.6 / Chapter 1.3.2 --- World wide distribution --- p.7 / Chapter 1.3.2.1 --- Serotypes --- p.7 / Chapter 1.3.2.2 --- Antibiotic susceptibility --- p.8 / Chapter 1.3.3 --- GBS diseases in Hong Kong --- p.10 / Chapter 1.4 --- Putative virulence factors and pathogenesis --- p.10 / Chapter 1.4.1 --- Capsular polysaccharide --- p.10 / Chapter 1.4.2 --- C5a peptidase --- p.11 / Chapter 1.4.3 --- β-haemolysin/cytolysin --- p.12 / Chapter 1.4.4 --- C protein and C a-like protein --- p.12 / Chapter 1.4.4.1 --- C protein --- p.12 / Chapter 1.4.4.1.1 --- C α protein --- p.13 / Chapter 1.4.4.1.2 --- Cβ protein --- p.14 / Chapter 1.4.4.2 --- C α-like protein --- p.15 / Chapter 1.4.5 --- Hyaluronate lyase --- p.16 / Chapter 1.4.6 --- CAMP factor --- p.17 / Chapter 1.4.7 --- Others --- p.17 / Chapter 1.5 --- Antibiotic resistance and resistance genes --- p.18 / Chapter 1.5.1 --- Macrolides --- p.18 / Chapter 1.5.2 --- Tetracyclines --- p.18 / Chapter 1.5.3 --- Aminoglycosides --- p.19 / Chapter 1.5.4 --- Fluoroquniolones --- p.20 / Chapter 1.5.5 --- Others --- p.20 / Chapter 1.6 --- Mobile genetic elements --- p.21 / Chapter 1.7 --- Typing methods --- p.22 / Chapter 1.7.1 --- Phenotypic methods --- p.23 / Chapter 1.7.1.1 --- Serotyping --- p.23 / Chapter 1.7.1.2 --- Multilocus enzyme electrophoresis (MLEE) --- p.23 / Chapter 1.7.2 --- Genotypic methods --- p.24 / Chapter 1.7.2.1 --- Restriction endonuclease analysis (REA) / restriction fragment-length polymorphism (RFLP) --- p.24 / Chapter 1.7.2.2 --- Pulsed-field gel electrophoresis (PFGE) --- p.25 / Chapter 1.7.2.3 --- Random amplified polymorphic DNA (RAPD) --- p.26 / Chapter 1.7.2.4 --- Sequencing --- p.26 / Chapter 1.8 --- Prevention --- p.29 / Chapter 1.8.1 --- Intrapartum antibiotic prophylaxis (IAP) --- p.29 / Chapter 1.8.2 --- GBS Vaccine --- p.33 / Chapter 1.9 --- Objectives --- p.34 / Chapter CHAPTER 2 --- METHODS AND MATERIALS --- p.35 / Chapter 2.1 --- Bacterial isolates --- p.35 / Chapter 2.2 --- Antibiotic susceptibility test --- p.37 / Chapter 2.2.1 --- Antibiotic preparation --- p.37 / Chapter 2.2.2 --- Microbroth dilution method --- p.39 / Chapter 2.2.2.1 --- Microtitre plate preparation --- p.39 / Chapter 2.2.2.2 --- Suspension preparation and inoculation --- p.39 / Chapter 2.2.2.3 --- End points determination --- p.40 / Chapter 2.2.3 --- Inducible lincomycin resistance determination --- p.40 / Chapter 2.3 --- Serotyping --- p.41 / Chapter 2.3.1 --- Preparation of antigens --- p.41 / Chapter 2.3.2 --- Typing of isolates --- p.42 / Chapter 2.4 --- Pulsed-field Gel Electrophoresis (PFGE) --- p.42 / Chapter 2.4.1 --- Preparation of DNA plug for PFGE --- p.43 / Chapter 2.4.2 --- Restriction enzyme digestion of GBS DNA --- p.43 / Chapter 2.4.3 --- Running of PFGE gel --- p.44 / Chapter 2.5 --- Molecular characterization --- p.44 / Chapter 2.5.1 --- Target genes --- p.44 / Chapter 2.5.2 --- DNA preparation --- p.51 / Chapter 2.5.3 --- Master mix preparation --- p.51 / Chapter 2.5.4 --- Polymerase chain reaction --- p.51 / Chapter 2.5.5 --- PCR product analysis by agarose gel electrophoresis --- p.52 / Chapter 2.5.6 --- DNA sequencing --- p.52 / Chapter 2.6 --- Data analysis --- p.53 / Chapter 2.6.1 --- PFGE and molecular characters analysis --- p.53 / Chapter 2.6.2 --- Sequences analysis --- p.53 / Chapter 2.7 --- Molecular identification by real-time PCR --- p.54 / Chapter 2.7.1 --- Bacterial strains --- p.54 / Chapter 2.7.2 --- DNA isolation for specimens --- p.56 / Chapter 2.7.3 --- Design of TaqMan primers and probes --- p.56 / Chapter 2.7.4 --- Cloning of target sequences --- p.59 / Chapter 2.7.5 --- Master mix of real-time PCR --- p.59 / Chapter 2.7.6 --- Specificity and detection limit --- p.60 / Chapter CHAPTER 3 --- RESULTS --- p.62 / Chapter 3.1 --- Serotype distribution of GBS --- p.62 / Chapter 3.1.1 --- Serotyping using antisera --- p.62 / Chapter 3.1.2 --- Serotyping by molecular method --- p.64 / Chapter 3.1.3 --- Molecular subtype of GBS serotype III --- p.66 / Chapter 3.1.4 --- Correlation of serotypes with diseases --- p.69 / Chapter 3.2 --- Antimicrobial susceptibility --- p.71 / Chapter 3.2.1 --- Phenotypic method --- p.71 / Chapter 3.2.2 --- Detection and distribution of resistance genes --- p.76 / Chapter 3.2.2.1 --- Tetracycline resistance --- p.76 / Chapter 3.2.2.2 --- Macrolide and lincosamide resistance --- p.77 / Chapter 3.2.2.3 --- Aminoglycoside resistance --- p.78 / Chapter 3.3 --- Molecular typing --- p.83 / Chapter 3.3.1 --- Pulsed-field gel electrophoresis (PFGE) --- p.83 / Chapter 3.3.2 --- Distribution of GBS surface protein genes profiles --- p.89 / Chapter 3.3.3 --- Distribution of mobile genetic elements --- p.92 / Chapter 3.4 --- "Analysis based on PFGE, surface protein genes, mobile genetic elements and antibiotic resistance genes" --- p.95 / Chapter 3.4.1 --- Intra-molecular serotype --- p.95 / Chapter 3.4.1.1 --- Molecular serotype Ia --- p.95 / Chapter 3.4.1.2 --- Molecular serotype Ib --- p.99 / Chapter 3.4.1.3 --- Molecular serotype II --- p.101 / Chapter 3.4.1.4 --- Molecular serotype III --- p.103 / Chapter 3.4.1.5 --- Molecular serotype V --- p.107 / Chapter 3.4.1.6 --- Molecular serotype VI --- p.110 / Chapter 3.4.1.7 --- "Molecular serotype IV, VII and VIII" --- p.110 / Chapter 3.4.1.8 --- Non-typeable isolate (NT) --- p.111 / Chapter 3.4.2 --- Analysis of Maternal and neonatal strains --- p.115 / Chapter 3.4.3 --- Comparison of GBS strains from Hong Kong to Australia and Korea --- p.118 / Chapter 3.5 --- Molecular identification of GBS by real-time PCR --- p.120 / Chapter 3.5.1 --- Specificity --- p.120 / Chapter 3.5.2 --- Detection limits --- p.122 / Chapter CHAPTER 4 --- DISCUSSION --- p.125 / Chapter 4.1 --- Laboratory methods for typing and characterization of GBS --- p.125 / Chapter 4.1.1 --- Serotyping by agglutination and molecular method --- p.125 / Chapter 4.1.2 --- Antibiotic susceptibility testing and resistance genes --- p.129 / Chapter 4.1.3 --- PFGE --- p.130 / Chapter 4.1.4 --- Surface protein genes --- p.131 / Chapter 4.1.5 --- Mobile genetic elements --- p.132 / Chapter 4.1.6 --- Real-time PCR --- p.133 / Chapter 4.2 --- Characterization of GBS in Hong Kong --- p.135 / Chapter 4.2.1 --- GBS in Hong Kong --- p.135 / Chapter 4.2.2 --- GBS from Australia and Korea --- p.141 / Chapter 4.3 --- Future research --- p.142 / Chapter 4.4 --- Conclusions --- p.143 / REFERENCES --- p.144 / APPENDIX I: MATERIALS AND REAGENTS --- p.162 / APPENDIX II: DENDROGRAMS --- p.168

Orientador: Osmir Batista de Oliveira Junior / Banca:Rosane de Fatima Zanirato Lizarelli / Banca: Sergio Luiz de Souza Salvador / Banca: Rita de Cassia Loiola Cordeiro / Banca: Marcelo Ferrarezi de Andrade / Resumo: O uso indiscriminado dos antibióticos e seu mecanismo de ação levaram ao desenvolvimento de cepas bacterianas altamente resistentes e de maior virulência. Estas cepas causam doenças muito mais agressivas e de difícil tratamento, constituindo-se um dos principais desafios enfrentados por profissionais da área da saúde. Como a terapia fotodinâmica antimicrobiana (TFDA) baseia seu mecanismo de ação em reações oxidativas não especificas, esta passou a ser uma alternativa interessante para o tratamento de todas as doenças de origem microbiana, uma vez que, nem bactérias, nem vírus ou fungos tem capacidade de desenvolver resistência a TFDA. Considerando que a carie dental é uma doença de origem bacteriana especifica e que a eficácia da TFDA depende do tipo e dose de energia luminosa utilizada, do fotossensibilizador e da taxa de oxigênio nos tecidos a serem tratados, decidimos investigar a viabilidade da TFDA para prevenção e tratamento da cárie dental. Para tal, foram realizados 3 estudos: 1) Revisão critica da literatura sobre fotossensibilizadores utilizados para controle antimicrobianos de Streptococcus mutans. 2) Avaliação do efeito antibacteriano de três fotossensibilizadores (curcumina, hipericina e hematoporfirina) sobre Streptococcus mutans em suspensões planctônicas e 3) Avaliação do efeito antibacteriano de três fotossensibilizadores (curcumina, hipericina e photogem®) Esteban Florez FL. Terapia Fotodinâmica em microrganismos cariogênicos - Estudo in vitro. [Tese de doutorado]. Araraquara: sobre biofilme maduro formado a partir de cepa de Streptococcus mutans. Dos fotossensibilizadores utilizados a hipericina foi a que apresentou maior efeito antimicrobiano sobre suspensão planctônica. Foi comprovado que a eficácia da TFDA esta diretamente relacionada com o tipo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The indiscriminate use of antibiotics and its mechanisms of action led to the development of highly resistant bacterial strains and more virulent ones. This strains can cause diseases much more aggressive and difficult to treat, and in that way they consist in one of the major challenges to the health care professionals. As the antimicrobial photodynamic therapy (APDT) it is based on non-specific oxidative reaction, this is now considered as an interesting alternative to treat all diseases from bacterial origins, once that, neither bacterias, neither viruses nor fungi can develop acquired resistance from the therapy. Taking into consideration that dental caries is a specific bacterial disease and that the APDT's efficiency is directed related to factors as, wavelength, energy dose, photosensitizer used, and with the oxygen present in the target tissue, we have decided to investigate the APDT to prevent and treat dental decay. To accomplish that, it was realized three studies: 1) Critical Literature revision about photosensitizers used to control S.mutans. 2) Evaluation of the antimicrobial effect of three different photosensitizers (Curcumin, Hypericin and Hematoporfirin) over Streptococcus mutans in planktonic cultures and 3) Evaluation of the antimicrobial effect of three photosensitizers (Curcumin, Hypericin and Hematoporfirin) over mature biofilms obtained from Streptococcus mutans strains. From the photosensitizers used Hypericin was the one that showed the most antibacterial observed effect on the planktonic cultures. It was demonstrated by our data that the APDT efficacy is directly related to the time of irradiation of the samples, with the kind of photosensitizer used, its concentration and with the energy dose delivered. None of the proposed protocols were able to show any significant effect over the microorganisms when in biofilm... (Complete abstract click electronic access below) / Doutor