Haemophilus influenzae tipo b (Hib) é uma patógeno causador de doenças como meningite. O polissacarídeo capsular (PRP) é usado como antígeno vacinal. O alto custo da vacina provém da instabilidade do PRP. Este trabalho tem por finalidade otimizar o processo de produção do PRP. Primeiro, foi escolhido um modelo matemático para a descrição da cinética de Hib. Em seguida, foi realizado um desenho experimental para avaliar os efeitos de pH e temperatura sobre a cinética. Tanto a produtividade de PRP quanto sua massa molecular foram máximas em pH 7,1, porém em temperaturas mais altas a produtividade foi maior e a massa molecular menor. As condições otimizadas foram definidas como pH 7,1 e 30 °C. Nestas condições, a massa molecular foi cerca de 78 % maior que nas condições da literatura de 37°C e pH 7,5, com uma produção de PRP equivalente. Isto resultou em maior recuperação na primeira etapa de purificação, comprovando que a condição de cultivo otimizada pode contribuir em melhores rendimentos durante o processo e assim impactar na redução do custo final da vacina. / Haemophilus influenzae type b (Hib) is a pathogen responsible for deseases like meningitis. The capsular polysaccharide (PRP) is used as antigen in vaccines. The high cost of the vaccine is due to instability of PRP. The goal of this work is to optimize the production of PRP. First, a mathematical model was selected to describe Hib kinetics. Then, an experimental design was carried out to evaluate the effects of pH and temperature. Both PRP productivity and molecular mass were optimal in pH 7.1, but at higher temperature productivity increased and molecular mass decreased. The optimal conditions were set at pH 7.1 and 30 °C. In these conditions, the molecular mass was 78 % higher, with the same amount of PRP produced. This resulted in higher recovery at the first step of purification, ensuring that the improved conditions may contribute with processes efficiency and enable reduction of the final cost of the vaccine.

Haemophilus influenzae b (Hib) é uma bactéria patogênica causadora de pneumonia e meningite. Sua cápsula polissacarídica (PRP) é considerada como principal fator de virulência e utilizada como antígeno vacinal. Hib é fastidioso e requer micronutrientes para seu desenvolvimento. A finalidade deste trabalho é estabelecer o meio quimicamente definido para desenvolvimento de Hib e produção de PRP. Inicialmente, definiu-se um meio a partir de dados da literatura. Este meio foi estudado através do delineamento de Plackett-Burman de 44 ensaios, obtendo-se valores máximos de DO540nm de 5,0 UA, e 227,7 mg/L de PRP. A análise estatística revelou que EDTA, NH4Cl, Cys e PVA podem ser removidos do meio sem impactar os parâmetros estudados e que Glm, Hipoxantina, Inosina, Tiamina, Hemina e Tween 80 apresentam efeito significativo positivo para produção de PRP. Analisando os meios estudados, foi possível verificar que a composição do E44 possibilitou produzir o PRP a US$ 16,50/g, sendo considerado o meio quimicamente definido estabelecido neste trabalho. / Haemophilus influenzae b (Hib) is a pathogenic bacterium that causes pneumonia and meningitis. Its capsular polysaccharide (PRP) is considered as a major virulence factor and used as vaccine antigen. Hib is fastidious and requires micronutrients for its development. The purpose of this study is to establish the chemically defined medium for Hib development and PRP production. Initially, a medium was defined based in the literature. This medium was studied by the Plackett-Burman design of 44 trials, achieving maximum values of DO540nm of 5.0 AU and 227.7 mg / L of PRP. Statistical analysis revealed that EDTA, NH4Cl, Cys and PVA can be removed from the medium without impacting the parameters studied and Glm, Hypoxanthine, Inosine, Thiamine, Tween 80 and Hemin exhibit significant positive effect on the PRP production. Analyzing the studied media, it was possible to verify that the composition of E44 enabled to produce PRP to $ 16.50/g, being considered the chemically defined medium established in this work.

Introdução: O Haemophilus influenzae type b (Hib) é a primeira maior causa de meningites e pneumonias provocadas por bactérias encapsuladas. Trabalhos revelam que anticorpos maternos, séricos e secretores, podem proteger recém nascidos (RN) destes patógenos encapsulados e contribuem para a maturação do sistema imune do infante. Objetivo: O presente estudo teve como objetivo investigar a transferência materno-infantil de anticorpos anti-Hib em mães vacinadas e que não receberam a vacina anti-Hib. Materiais e Métodos: Nós avaliamos 29 mulheres saudáveis, das quais 13 foram vacinadas e 16 não receberam a vacina ActHib®. Destas mães foram obtidas amostras de sangue periférico e do cordão umbilical, colostro e leite, sendo determinadas as imunoglobulinas totais (lgG e IgA) e suas subclasses (IgG1 e 2) por Imunodifusão Radial Quantitativa (IDR) e nefelometria. A concentração de anticorpos IgG, as subclasses (lgG1 e 2) e IgA anti-Hib foram analisados por ensaio imunoenzimático (ELlSA), também utilizado para determinar a avidez dos anticorpos IgG e IgA anti-Hib. Avaliação qualitativa destes anticorpos foi realizada a partir de ensaios de immunoblotting (IB). Resultados: As amostras maternas de mães vacinadas não apresentaram diferenças quantitativas de imunoglobulinas secretoras (lgA), séricas (lgG) e suas subclasses (lgG1 e 2) totais, comparadas às amostras de mães que não receberam a vacina anti-Hib. O grupo vacinado mostrou maior concentração e avidez de anticorpos específicos para o Hib quando relacionados ao grupo de mães não vacinado. Os soros de cordões umbilicais de mães imunizadas apresentaram menor taxa de passagem transplacentária que os cordões de mães não vacinadas. Em ambos os grupos, as amostras de colostro apresentaram maior concentração de imunoglobulinas totais e específicas para o Hib que as amostras de leite. O 18 revelou o mesmo padrão de reconhecimento antigênico para o Hib entre as amostras maternas, nas duas populações. Conclusão: Os resultados revelaram que o perfil de resposta humoral de mães vacinadas pode proteger mais o infante que as mães não vacinadas, pois o primeiro grupo transferiu maior quantidade de anlicorpos com melhor avidez para a criança, conferindo proteção eficaz com relação às doenças causadas por Hib. / Background: Haemophilus influenzae, type b (Hib) has been one of the major causes of bacterial meningitis and pneumonia. Recent works show that maternal, seric and secretory antibodies, may protect the newborn and contribute the maturation of the infant immune system. Objective: The present study has as aim to investigates the maternal-infantile transfer of anti-Hib antibodies in immunized and not immunized mothers\' with anti-Hib vaccine. Material and Methods: We evaluated 29 healthy women, from whitch 13 mothers were immunized and 16 not immunized mothers with the ActHib® vaccine. From these mothers it were obtained peripheric and cord serum, colostrum and milk samples, the total immunoglobulin (IgG and IgA) and its subclass (lgG1 and IgG2) was determined by Quantitative Radial Immunodiffusion (IDR) and nephelometry. The concentration of anti-Hib IgG, subclass (lgG1 IgG2) and IgA antibodies were analyzed by immunoenzymatic assay (ELlSA), it also were utilized to determine the antibodies avidities\'. Qualitative evaluation these antibodies were determined by Immunoblotting assays (IB). Results: The results didn\'t show difference between maternal samples of the immunized and not immunized mothers in the concentration of the total secretory and seric imunoglobulins as well as its total immunoglobulins subclasses. The immunized set showed higher avidity and anti-Hib antibody levels comparing to the non-immunized mother sets. The umbilical cord serums\' from immunized mothers revealed lower rate of placental transfer than the cord serum of not immunized mothers. In both sets, the colostrum sample showed higher antibody levels comparing to the milk samples. IB revealed the same recognition pattern of Hib antigens between mother and cord serum IgG and colostrum and milk IgA, in both populations. Conclusion: The results shows that the antibodies profile response of the immunized mother can protect more the infant than the not immunized mother, because the first set transported higher quantity of antibodies with better avidity for the children, these antibodies confere an efficient protection to infections provoked by Hib.

Haemophilus influenzae tipo b é uma bactéria Gram-negativa, patogênica causadora de meningites em crianças. A cápsula polissacarídica (PSb) é o principal fator de virulência e é usado como antígeno vacinal. O método clássico de purificação do PSb envolve várias etapas de precipitação com etanol, fenol e detergente catiônico (inflamável, corrosivo e tóxico), e etapas de ultracentrifugação. O objetivo deste estudo foi substituir total ou parcial as precipitações e/ou uso das centrífugas por cromatografia, digestão enzimática, microfiltração e ultrafiltração tangencial. As cromatografias de troca iônica e de filtração em gel não apresentaram boas purificações, entretanto a hidrofóbica pode eliminar as proteínas contaminantes. As precipitações com etanol foram necessárias para obter a pureza requerida. O etanol de alguma forma favoreceu a ação enzimática e facilitou a posterior ultrafiltração. A separação com etanol em fibra-oca de microfiltração tangencial mostrou melhores purificações do que a centrifugação, mas com uso repetido verificou-se redução na eficiência. / Haemophilus influenzae type b is Gram-negative pathogenic bacterium cause meningitis in children. The capsular polysaccharide (PSb) is the main virulence factor and it is used as vaccine antigen. The classical PSb purification process includes ethanol, phenol and cationic detergent precipitations (explosion prone, corrosive, toxic) and ultracentrifugation steps. The aim of this work was to replace total or partial ethanol precipitations steps and/or elimination of centrifugation by chromatography methods, enzymatic digestion and ultrafiltration (UF) or microfiltration. The results have showed that ion exchange chromatography and gel filtration did not result in good purification, however the hydrophobic can be used for proteins elimination. The ethanol precipitation steps are necessary to achieve the required purity of PSb. In some way ethanol contributed for enzymes action and further improvements in the UF. The ethanol separation with hollow fiber microfiltration exhibited better purification than centrifugation, but after some uses the efficiency has reduced.

Submitted by Natalie Mendes (nataliermendes@gmail.com) on 2015-07-28T23:01:37Z No. of bitstreams: 1 COMPOSI??O QU?MICA DO ?LEO ESSENCIAL DE Lippia origanoides Kunth E ATIVIDADE ANTIMICROBIANA FRENTE A DIFERENTES SOROTIPOS~1.pdf: 1434340 bytes, checksum: 8072381e4bdaa1aac1583317e834ac0d (MD5) / Made available in DSpace on 2015-07-28T23:01:37Z (GMT). No. of bitstreams: 1 COMPOSI??O QU?MICA DO ?LEO ESSENCIAL DE Lippia origanoides Kunth E ATIVIDADE ANTIMICROBIANA FRENTE A DIFERENTES SOROTIPOS~1.pdf: 1434340 bytes, checksum: 8072381e4bdaa1aac1583317e834ac0d (MD5) Previous issue date: 2014-03-28 / Pig farming has become increasingly important in recent years in Brazil, because of this, studies for the treatment of diseases that cause the loss of mass of meat animals has increased significantly, such as the Glasser's disease caused by Haemophilus parasuis. Some initial studies have shown human resistance to antibiotics due to the consumption of meat produced with high levels of these substances, and alternatively treatments have been developed from natural products. Lippia origanoides Kunth is presented as a natural source of antimicrobial substances due to the composition of the essential oil obtained, mainly, from the leaves of this plant. In this study the antimicrobial activity of the essential oil from Lippia origanoides Kunth, by determining the minimum inhibitory concentration (MIC) and Minimum Bactericidal Concentration (MBC) against Haemophilus parasuis serotypes 1,2,4,5,9,10,12,13,14 and one untypable was studied. Essential oils were obtained by hydrodistillation of the dried leaves and the chemical composition analysis revealed the presence of carvacrol as the predominant component, which characterizes the chemotype B. The results of the antimicrobial activity demonstrated the inhibitory effect of essential oil samples for all tested bacteria. The best result was 0.005% against the sample MV12315 (serotype 10) while the least satisfactory was 0.078% against the sample MV12196 (serotype 12). Results demonstrate the bactericidal action of the oil against the different serotypes of Haemophilus parasuis. / A suinocultura vem se sobressaindo nos ?ltimos anos no Brasil, por isso aumentam os estudos para tratamento das doen?as que causam perdas de carca?a dos animais, como a doen?a de Gl?sser, provocada pelo Haemophilus parasuis. Alguns trabalhos incipientes demonstram a resist?ncia humana a antibi?ticos devido ao consumo de carnes produzidas com altos ?ndices destas subst?ncias, e tratamento alternativos com produtos naturais vem sendo desenvolvidos. Lippia origanoides Kunth se apresenta como uma fonte natural de subst?ncias antimicrobianas devido ? composi??o do seu ?leo essencial obtido principalmente das folhas desta planta. Este trabalho avaliou a atividade antimicrobiana do ?leo essencial de Lippia origanoides atrav?s da determina??o da Concentra??o Inibit?ria M?nima (CIM) e Concentra??o Bactericida M?nima (CBM) frente a amostras de campo do Haemophilus parasuis com sorotipos 1,2,4,5,9,10,12,13,14 e um n?o sorotip?vel. Os ?leos essenciais foram obtidos por hidrodestila??o das folhas secas ap?s tr?s horas, e na an?lise da composi??o qu?mica, o carvacrol foi identificado como componente predominante, caracterizando-o como quimiotipo B. Os resultados de atividade antimicrobiana demonstram o efeito inibit?rio do ?leo essencial para todas as amostras de bact?rias testadas. O melhor resultado encontrado foi de 0,005% frente a amostra MV12315 (sorotipo 10) enquanto o menos satisfat?rio foi de 0,078% contra a amostra MV12196 (sorotipo 12). Os resultados obtidos demonstram a a??o bactericida do ?leo para os diferentes sorotipos do Haemophilus parasuis.

Ma, Hok Lun. / Thesis submitted in: December 2008. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 233-273). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese version) --- p.v / Tables of contents --- p.vi / Acknowledgement --- p.xvi / List of figures --- p.xvii / List of tables --- p.xxi / List of abbreviations and symbols --- p.xxviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Respiratory illnesses in children --- p.1 / Chapter 1.1.1 --- Worldwide burden of childhood pneumonia --- p.1 / Chapter 1.1.2 --- Further mortality related to childhood pneumonia --- p.4 / Chapter 1.2 --- Etiology agent of childhood respiratory illnesses --- p.5 / Chapter 1.2.1 --- Difficulties in determining etiological agent --- p.5 / Chapter 1.2.2 --- Overall situation of etiological agents in childhood pneumonia --- p.6 / Chapter 1.2.3 --- Relationship between age and pathogens --- p.9 / Chapter 1.2.4 --- "Relationship between serotypes, carriage and invasiveness" --- p.11 / Chapter 1.2.4.1 --- Carriage and Invasiveness --- p.12 / Chapter 1.2.4.2.1 --- Carriage of S. pneumoniae and H. influenzae in children in Hong Kong --- p.12 / Chapter 1.2.4.2.2 --- "Serotypes, carriage and invasiveness in S. pneumoniae" --- p.14 / Chapter 1.2.4.2.3 --- "Serotypes, carriage and invasiveness in H. influenzae" --- p.17 / Chapter 1.3 --- Epidemiology of antibiotic-resistant pathogens --- p.18 / Chapter 1.3.1 --- Molecular typing methods --- p.18 / Chapter 1.3.2 --- Spread of antibiotic-resistant pathogens --- p.20 / Chapter 1.3.2.1 --- Spread of antibiotic-resistant S. pneumoniae --- p.26 / Chapter 1.3.2.1.1 --- Spread of penicillin-resistant S. pneumoniae --- p.26 / Chapter 1.3.2.1.1.1 --- Spread of Spanish-23F-1 --- p.27 / Chapter 1.3.2.1.1.2 --- Spread of Spanish-6B-2 --- p.28 / Chapter 1.3.2.1.1.3 --- Spread of antibiotic-resistant S. pneumoniae clones in Hong Kong --- p.28 / Chapter 1.3.2.1.2 --- Spread of cephalosporin-resistant S. pneumoniae --- p.29 / Chapter 1.3.2.1.3 --- Spread of macrolide-resistant S. pneumoniae --- p.30 / Chapter 1.3.2.1.4 --- Spread of fluoroquinolone-resistant S. pneumoniae --- p.31 / Chapter 1.3.2.2 --- Spread of antibiotic-resistant H. influenzae --- p.32 / Chapter 1.3.2.2.1 --- Spread of β-lactam-resistant H. influenzae --- p.32 / Chapter 1.3.2.2.2 --- Spread of macrolide-resistant H. influenzae --- p.33 / Chapter 1.3.2.2.3 --- Spread of fluoroquinolone-resistant H. influenzae --- p.34 / Chapter 1.4 --- Mechanism of antibiotic-resistance in respiratory pathogens --- p.36 / Chapter 1.4.1 --- Mechanism of antibiotic-resistance in S. pneumoniae --- p.37 / Chapter 1.4.1.1 --- Mechanism of penicillin- and cephalosporin-resistance in S. pneumoniae --- p.37 / Chapter 1.4.1.1.1 --- Penicillin-binding protein (PBP)-mediated mechanism --- p.37 / Chapter 1.4.1.1.2 --- PBP-independent mechanisms --- p.49 / Chapter 1.4.1.1.2.1 --- "Murine peptide branching genes, murMN operon" --- p.49 / Chapter 1.4.1.1.2.2 --- "Two-component system, CiaRH" --- p.50 / Chapter 1.4.1.1.2.3 --- "Putative glycosyltransferase, CpoA" --- p.52 / Chapter 1.4.1.1.3 --- RNA and protein expression studies on S. pneumoniae for β-lactam-resistance --- p.52 / Chapter 1.4.1.1.3.1 --- RNA expression in penicillin-sensitive S. pneumoniae --- p.53 / Chapter 1.4.1.1.3.2 --- Protein expression in penicillin-resistant S. pneumoniae --- p.53 / Chapter 1.4.1.2 --- Mechanism of macrolide- and lincosamide- resistance in S. pneumoniae --- p.54 / Chapter 1.4.1.3 --- Mechanism of tetracycline-resistance in S. pneumoniae --- p.55 / Chapter 1.4.1.4 --- Mechanism of fluoroquinolone-resistance in S. pneumoniae --- p.55 / Chapter 1.4.2 --- Mechanism of antibiotic-resistant in H. influenzae --- p.56 / Chapter 1.4.2.1 --- Mechanism of β-lactam-resistance in H. influenzae --- p.56 / Chapter 1.4.2.1.1 --- β-lactamase-producing H. influenzae --- p.56 / Chapter 1.4.2.1.2 --- β-lactamase-negative ampicillin-resistant (BLNAR) H. influenzae --- p.58 / Chapter 1.4.2.1.2.1 --- Relationship between amino acid substitutions in PBP3 and β-lactam- resistance --- p.58 / Chapter 1.4.2.1.2.2 --- Relationship between amino acid substitutions in AcrR and β-lactam-resistance --- p.60 / Chapter 1.4.2.2 --- Mechanism of macrolide-resistance in H. influenzae --- p.61 / Chapter 1.4.2.3 --- Mechanism of fluoroquinolone-resistance in H. influenzae --- p.64 / Chapter 1.5 --- Impact of vaccination --- p.65 / Chapter 1.5.1 --- H. influenzae type b vaccination --- p.65 / Chapter 1.5.1.1 --- Efficacy of Hib conjugate vaccine --- p.66 / Chapter 1.5.1.2 --- Herd immunity related to Hib conjugate vaccine --- p.66 / Chapter 1.5.2 --- Pneumococcal vaccination --- p.66 / Chapter 1.5.2.1 --- Vaccine efficacy and herd immunity of pneumococcal vaccines --- p.67 / Chapter 1.5.2.2 --- Development of conjugate vaccines with higher valency --- p.67 / Chapter 1.5.2.3 --- Serotype replacement --- p.67 / Chapter 1.5.2.4 --- Development of pneumococcal vaccines with new targets --- p.69 / Chapter 1.6 --- Objectives of this study --- p.70 / Chapter Chapter 2 --- Materials and methods --- p.72 / Chapter 2.1 --- Collection and Identification of microorganisms --- p.72 / Chapter 2.1.1 --- Collection of S. pneumoniae and H. influenzae --- p.72 / Chapter 2.1.2 --- Identification of S. pneumoniae and H. influenzae --- p.73 / Chapter 2.2 --- Serotyping of S. pneumoniae and H. influenzae --- p.74 / Chapter 2.2.1 --- Serotyping by polymerase chain reaction (PCR) --- p.74 / Chapter 2.2.1.1 --- Preparation of crude DNA extract --- p.74 / Chapter 2.2.1.2 --- Screening for common serotypes by multiplex PCR --- p.74 / Chapter 2.2.1.3 --- Composition of PCR Mix --- p.77 / Chapter 2.2.1.4 --- Serotyping PCR conditions --- p.81 / Chapter 2.2.1.5 --- Gel Electrophoresis --- p.81 / Chapter 2.2.2 --- Serotyping by serum agglutination --- p.82 / Chapter 2.3 --- Antimicrobial susceptibility testing --- p.83 / Chapter 2.4 --- Clonal analysis of penicillin- and cephalosporin-resistant S. pneumoniae --- p.87 / Chapter 2.4.1 --- Pulsed-field Gel Electrophoresis (PFGE) --- p.87 / Chapter 2.4.1.1 --- Preparation of agarose plugs for PFGE --- p.87 / Chapter 2.4.1.2 --- Lysis of bacteria in agarose plugs --- p.89 / Chapter 2.4.1.3 --- Digestion of chromosomal DNA by restriction enzyme --- p.89 / Chapter 2.4.2 --- Multi-locus sequence typing (MLST) --- p.90 / Chapter 2.4.2.1 --- PCR amplification of house-keeping genes in MLST --- p.90 / Chapter 2.4.2.1.1 --- Preparation of DNA from agarose plugs --- p.92 / Chapter 2.4.2.1.2 --- Composition of PCR Mix --- p.92 / Chapter 2.4.2.1.3 --- MLST PCR conditions --- p.92 / Chapter 2.4.2.1.4 --- Gel Electrophoresis of MLST PCR products --- p.92 / Chapter 2.4.2.1.5 --- MLST PCR products purification --- p.93 / Chapter 2.4.2.2 --- Sequencing of housekeeping genes in MLST --- p.93 / Chapter 2.4.2.3 --- Sequencing analysis and sequence type (ST) determination in MLST --- p.94 / Chapter 2.4.3 --- Extended panel of antibiotic susceptibility testing on S. pneumoniae with known STs --- p.94 / Chapter 2.5 --- Analysis on potential penicillin- and cephalosporin-resistance mechanisms in S. pneumoniae --- p.96 / Chapter 2.5.1 --- Sequencing of potnetial penicillin- and cephalosporin- resistance determinants in S. pneumoniae --- p.96 / Chapter 2.5.1.1 --- Primer design of penicillin-binding protein (PBP) genes --- p.96 / Chapter 2.5.1.2 --- Primer design of non-PBP resistance determinants --- p.100 / Chapter 2.5.1.3 --- PCR amplification and sequencing of resistant determinants --- p.100 / Chapter 2.5.1.4 --- Sequence analysis --- p.100 / Chapter 2.5.2 --- Study on efflux mechanism of S. pneumoniae --- p.103 / Chapter 2.5.2.1 --- Modification of macrodilution for efflux assay --- p.103 / Chapter 2.5.2.2 --- Cefotaxime MIC determination with efflux inhibitors --- p.104 / Chapter 2.5.2.3 --- Determination of appropriate CCCP concentration --- p.105 / Chapter 2.5.2.4 --- Growth curve with efflux inhibitor --- p.105 / Chapter 2.5.3 --- Heteroresistance assay of S. pneumoniae --- p.106 / Chapter 2.5.4 --- "RNA expression study on penicillin- and cefotaxime-resistance determinants (pbp2x, pbpla and pbp2a) of S. pneumoniae" --- p.107 / Chapter 2.5.4.1 --- Growth of S. pneumoniae for RNA extraction --- p.107 / Chapter 2.5.4.2 --- RNA extraction and DNase digestion --- p.107 / Chapter 2.5.4.3 --- cDNA synthesis and real-time PCR --- p.108 / Chapter 2.6 --- Analysis on cephalosporin- and macrolide-resistance mechanisms in H. influenzae --- p.111 / Chapter 2.6.1 --- β-lactamase production of H. influenzae --- p.111 / Chapter 2.6.1.1 --- Nitrocefin Hydrolysis --- p.111 / Chapter 2.6.1.2 --- Screening for the presence of p-lactamase gene (blaTEM-1 and blaROB-1) by multiplex PCR --- p.111 / Chapter 2.6.2 --- PCR detection and sequencing of β-lactam- and macrolide- resistance determinants in H. influenzae --- p.113 / Chapter Chapter 3 --- Results of S. pneumoniae and H. influenzae children study --- p.116 / Chapter 3.1 --- Patient demographics of children study --- p.116 / Chapter 3.2 --- Serotype distributions --- p.117 / Chapter 3.2.1 --- Serotypes / serogroup distribution in S. pneumoniae --- p.117 / Chapter 3.2.2 --- Serotype distribution in H. influenzae children study --- p.120 / Chapter 3.3 --- Antibiotic susceptibilities and resistance antibiograms --- p.122 / Chapter 3.3.1 --- Antibiotic susceptibilities of S. pneumoniae --- p.122 / Chapter 3.3.2 --- Relationship between antibiotic resistance profiles and serotypes in S.pneumoniae --- p.126 / Chapter 3.3.3 --- Antibiotic susceptibilities of H. influenzae --- p.135 / Chapter 3.3.4 --- Antibiotic resistance profiles of H. influenzae --- p.138 / Chapter 3.4 --- Clonal analysis of penicillin- and cephalosporin-resistant S.pneumoniae --- p.139 / Chapter 3.4.1 --- Pulsed-field gel electrophoresis (PFGE) of S. pneumoniae --- p.139 / Chapter 3.4.2 --- Multi-locus sequence typing of S. pneumoniae --- p.141 / Chapter 3.5 --- Analysis of the penicillin- and cephalosporin-resistance determinants in S. pneumoniae --- p.143 / Chapter 3.5.1 --- "Sequence analysis of major pbp genes (pbp2x, pbpla and pbp2a)" --- p.143 / Chapter 3.5.2 --- "Sequence analysis of other potential penicillin- and cephalosporin- resistance determinants (pbp 1 b, pbp2b, pbp3, cpoA, ciaRH and murMN)" --- p.152 / Chapter 3.5.3 --- Sequence analysis of putative promoter sequences of pbp genes --- p.167 / Chapter 3.5.4 --- Efflux Inhibition Assay --- p.171 / Chapter 3.5.5 --- Heteroresistance Assay --- p.177 / Chapter 3.5.6 --- "RNA expression study on penicillin- and cephalosporin resistance determinants (pbp2x, pbpla and pbp2a)" --- p.179 / Chapter 3.6 --- Analysis of β-lactam-resistance determinants in H. influenzae --- p.185 / Chapter 3.6.1 --- β-lactamase production and blaTEM-1 promoter study --- p.185 / Chapter 3.6.2 --- "Sequence analysis of β-lactam-resistance determinants (ftsl, acrR genes, AcrAB-TolC efflux pump)" --- p.188 / Chapter 3.6.2.1 --- Sequence analysis offtsl --- p.188 / Chapter 3.6.2.2 --- Analysis of acrR and AcrAB-TolC efflux pump --- p.189 / Chapter 3.7 --- "Analysis of macrolide-resistance determinants in H, influenzae (AcrAB-TolC efflux pump, 23SrRNA, Ribosomal proteins L4 and L22)" --- p.199 / Chapter Chapter 4 --- Discussion on S. pneumoniae and H. influenzae children study --- p.204 / Chapter 4.1 --- Carriage rate of S. pneumoniae children collection --- p.204 / Chapter 4.2 --- Serotype distribution --- p.205 / Chapter 4.2.1 --- Serotype distribution and potential vaccine coverage in S. pneumoniae --- p.205 / Chapter 4.2.2 --- Serotype distribution in H. influenzae --- p.209 / Chapter 4.3 --- Antimicrobial resistance --- p.210 / Chapter 4.3.1 --- Antimicrobial resistance in S. pneumoniae --- p.210 / Chapter 4.3.2 --- Antimicrobial resistance in H. influenzae --- p.214 / Chapter 4.4 --- "Clonal analysis of high-level β-lactam-resistant S, pneumoniae" --- p.217 / Chapter 4.5 --- "β-lactam-resistance mechanisms in S, pneunomiae" --- p.220 / Chapter 4.6 --- Antimicrobial resistance mechanisms in H. influenzae --- p.224 / Chapter 4.6.1 --- β-lactam-resistance mechanism in β-lactamase-producing H. influenzae --- p.224 / Chapter 4.6.1.1 --- Variations in blaTEM-1 promoters in β-lactamase-producing H.influenzae --- p.224 / Chapter 4.6.1.2 --- β-lactam-resistance in β-lactamase-nonproducing H. influenzae --- p.225 / Chapter 4.6.2 --- Macrolide-resistance mechanisms in H. influenzae --- p.228 / Chapter Chapter 5 --- Conclusion and future studies --- p.230 / Chapter 5.1 --- "S, pneumoniae children study" --- p.230 / Chapter 5.2 --- H. influenzae children study --- p.231 / Chapter 5.3 --- Future studies --- p.232 / Bibliography --- p.233 / Appendix I 一 Sequence alignments and Tables --- p.274 / Appendix II 一 Materials and Methods --- p.313

Haemophilus parasuis est un pathogène porcin causant la maladie de Glässer caractérisée par de la polysérosite fibrineuse, polyarthrite, méningite et septicémie. La pathogenèse de l’infection et les facteurs de virulence sont encore mal connus. Le site de colonisation de Haemophilus parasuis dans le tractus respiratoire supérieur est controversé. Pour accéder à la circulation sanguine, H. parasuis doit envahir la muqueuse. H. parasuis adhère à des cellules épithéliales porcines de trachée (NPTr). Pour accéder au système nerveux central et causer la méningite, H. parasuis doit traverser la barrière hémato-méningée. H. parasuis adhère à et envahit des cellules endothéliales porcines de microvaisseaux cérébraux (PBMEC) provenant de la BBB. Le but de cette étude était d’étudier certaines interactions entre H. parasuis et son lipooligosccharide (LOS), et des cellules endothéliales et épithéliales porcines. Les résultats démontrent que l’adhésion de H. parasuis Nagasaki aux NPTr et aux PBMEC est en partie médiée par son LOS. H. parasuis induit l’apoptose des NPTr et des PBMEC, mais le LOS ne semble pas impliqué. H. parasuis, et à un niveau moindre son LOS, stimulent la sécrétion d’interleukine- (IL) 6 et d’IL-8. Différentes souches de H. parasuis sérotypes 4 et 5 (sérotypes les plus prévalents en Amérique du Nord) stimulent également les NPTr et PBMEC à produire IL-6 et IL-8. Les résultats suggèrent que le LOS de H. parasuis joue un certain rôle dans la pathogenèse de l’infection, mais d’autres composantes bactériennes sont également impliquées. / Haemophilus parasuis is a swine pathogen that causes Glässer’s disease characterized by fibrinous polyserositis, polyarthritis, meningitis and septicemia. The pathogenesis of the infection and virulence factors are not well known. Whether the upper respiratory tract is the site of colonization of H. parasuis is still a controversial issue. H. parasuis must invade the mucosa to gain access to the bloodstream. H. parasuis is able to adhere to newborn pig trachea cells (NPTr). H. parasuis must then cross the blood-brain barrier to gain access to the central nervous system in cases of meningitis. H. parasuis is able to adhere to and invade porcine brain microvascular endothelial cells (PBMEC). The aim of this work was to study the interactions between H. parasuis, its lipooligosccharide (LOS), and porcine endothelial and epithelial cells. Results showed that adhesion of H. parasuis Nagasaki to NPTr and PBMEC was partially mediated by its LOS. H. parasuis induced NPTr and PBMEC apoptosis, although purified LOS does not seem to be involved. H. parasuis, and to a lesser extent its LOS, stimulated the release of interleukin- (IL) 6 and IL-8. Field strains of H. parasuis serotypes 4 and 5 (the most prevalent serotypes in North America) also induced the production of IL-6 and IL-8. Results suggest that H. parasuis LOS plays a role in the pathogenesis of the infection, but other bacterial components are also involved.

Introdução: O Haemophilus influenzae type b (Hib) é a primeira maior causa de meningites e pneumonias provocadas por bactérias encapsuladas. Trabalhos revelam que anticorpos maternos, séricos e secretores, podem proteger recém nascidos (RN) destes patógenos encapsulados e contribuem para a maturação do sistema imune do infante. Objetivo: O presente estudo teve como objetivo investigar a transferência materno-infantil de anticorpos anti-Hib em mães vacinadas e que não receberam a vacina anti-Hib. Materiais e Métodos: Nós avaliamos 29 mulheres saudáveis, das quais 13 foram vacinadas e 16 não receberam a vacina ActHib®. Destas mães foram obtidas amostras de sangue periférico e do cordão umbilical, colostro e leite, sendo determinadas as imunoglobulinas totais (lgG e IgA) e suas subclasses (IgG1 e 2) por Imunodifusão Radial Quantitativa (IDR) e nefelometria. A concentração de anticorpos IgG, as subclasses (lgG1 e 2) e IgA anti-Hib foram analisados por ensaio imunoenzimático (ELlSA), também utilizado para determinar a avidez dos anticorpos IgG e IgA anti-Hib. Avaliação qualitativa destes anticorpos foi realizada a partir de ensaios de immunoblotting (IB). Resultados: As amostras maternas de mães vacinadas não apresentaram diferenças quantitativas de imunoglobulinas secretoras (lgA), séricas (lgG) e suas subclasses (lgG1 e 2) totais, comparadas às amostras de mães que não receberam a vacina anti-Hib. O grupo vacinado mostrou maior concentração e avidez de anticorpos específicos para o Hib quando relacionados ao grupo de mães não vacinado. Os soros de cordões umbilicais de mães imunizadas apresentaram menor taxa de passagem transplacentária que os cordões de mães não vacinadas. Em ambos os grupos, as amostras de colostro apresentaram maior concentração de imunoglobulinas totais e específicas para o Hib que as amostras de leite. O 18 revelou o mesmo padrão de reconhecimento antigênico para o Hib entre as amostras maternas, nas duas populações. Conclusão: Os resultados revelaram que o perfil de resposta humoral de mães vacinadas pode proteger mais o infante que as mães não vacinadas, pois o primeiro grupo transferiu maior quantidade de anlicorpos com melhor avidez para a criança, conferindo proteção eficaz com relação às doenças causadas por Hib. / Background: Haemophilus influenzae, type b (Hib) has been one of the major causes of bacterial meningitis and pneumonia. Recent works show that maternal, seric and secretory antibodies, may protect the newborn and contribute the maturation of the infant immune system. Objective: The present study has as aim to investigates the maternal-infantile transfer of anti-Hib antibodies in immunized and not immunized mothers\' with anti-Hib vaccine. Material and Methods: We evaluated 29 healthy women, from whitch 13 mothers were immunized and 16 not immunized mothers with the ActHib® vaccine. From these mothers it were obtained peripheric and cord serum, colostrum and milk samples, the total immunoglobulin (IgG and IgA) and its subclass (lgG1 and IgG2) was determined by Quantitative Radial Immunodiffusion (IDR) and nephelometry. The concentration of anti-Hib IgG, subclass (lgG1 IgG2) and IgA antibodies were analyzed by immunoenzymatic assay (ELlSA), it also were utilized to determine the antibodies avidities\'. Qualitative evaluation these antibodies were determined by Immunoblotting assays (IB). Results: The results didn\'t show difference between maternal samples of the immunized and not immunized mothers in the concentration of the total secretory and seric imunoglobulins as well as its total immunoglobulins subclasses. The immunized set showed higher avidity and anti-Hib antibody levels comparing to the non-immunized mother sets. The umbilical cord serums\' from immunized mothers revealed lower rate of placental transfer than the cord serum of not immunized mothers. In both sets, the colostrum sample showed higher antibody levels comparing to the milk samples. IB revealed the same recognition pattern of Hib antigens between mother and cord serum IgG and colostrum and milk IgA, in both populations. Conclusion: The results shows that the antibodies profile response of the immunized mother can protect more the infant than the not immunized mother, because the first set transported higher quantity of antibodies with better avidity for the children, these antibodies confere an efficient protection to infections provoked by Hib.

Orientador: Marcelo Lancellotti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T23:59:18Z (GMT). No. of bitstreams: 1 Andrade_AliceTavaresReis_M.pdf: 3292132 bytes, checksum: ee5318f5b87992964c9d97bedc343f00 (MD5) Previous issue date: 2013 / Resumo: Em 1998, foi relatada a transferência lateral do gene sodC do gênero Haemophilus para a espécie Neisseria meningitidis. Sabe-se que, nestes dois grupos a dinâmica deste gene é bastante distinta. Este trabalho tem por objetivo estimar árvores filogenéticas que possam apontar qual a espécie do gênero Haemophilus compartilhou o gene sodC com a espécie N. meningitidis. Testes de seleção positiva foram empregados no intuito de avaliar quais forças evolutivas estão subjacentes ao processo de diversificação molecular do gene nestas espécies ao longo do tempo. Além disso, foi realizada uma modelagem protéica computacional por homogia para avaliar quais substituições de aminoácidos tinham impacto no processo adaptativo da enzima nas espécies consideradas. Ao se reconstruir uma filogenia para o gene sodC, foi constatado que a origem deste gene na espécie H. influenzae é distinta. Um grupo de linhagens recebeu o gene, provavelmente por transferência lateral, da espécie H. haemolyticus, enquanto o outro grupo recebeu o gene da espécie H. parainfluenzae. Neste grupo, o gene sofreu pseudogeneização. Foi observado também que as sequências de N. meningitidis agrupam com as sequências que compartilham um ancestral comum com a espécie H. haemolyticus, porém as sequências do meningococo formam um ramo distinto dentro deste clado. Dada à alta clonalidade das sequências de N. meningitidis, foi constatado que o evento de transferência lateral de genes foi muito recente na escala do tempo. O teste de seleção positiva demonstrou que seleção positiva está atuando especificamente no ramo da árvore que compartilha um ancestral comum com a espécie H. haemolyticus, através da modificação de uma alanina por uma serina na posição 72, embora a nota geral da árvore tenha sido menor que 1. Sabe-se que pseudogenes, por não codificarem uma proteína ativa e, portanto, por não estarem sob nenhum tipo de restrição funcional, estão sob uma ação maior da deriva genética. Portanto, diferentes forças evolutivas estão governando a evolução deste gene nas espécies consideradas. A modelagem protéica concluiu que tal modificação contribuiu para o aumento do potencial redox do sítio ativo. Desta forma, a ação da seleção positiva sob um único resíduo de aminoácido foi benéfica para a função da enzima como um todo / Abstract: In 1998, it was reported the lateral transfer of the sodC gene from the genus Haemophilus to Neisseria meningitidis. It is known that this two groups show a quite distinct dynamics of this gene. This study aims to estimate phylogenetic trees that might point to which species of the genus Haemophilus shared the sodC gene with N. meningitidis. In addition, tests of positive selection were employed in order to assess which evolutionary forces are governing the process of molecular diversification of the gene in these species through time. Moreover, we performed a computational protein modeling by homology to asses which amino acids substitutions had an impact on the adaptative process of the enzyme in the species considered. A phylogeny of the sodC gene was reconstructed and it was found that this gene in H. influenzae has two different origins. A group of lineages has received the gene, probably by lateral transfer, from H. haemolyticus, whereas the other group has received the gene from H. parainfluenzae. In the latter, the gene has become a pseudogene. It was also observed that the sequences from N. meningitides group together with those sequences that share a common ancestor with H. haemolyticus, but they form a distinct branch within this clade. Given the high clonality of the sequences from N. meningitidis, it was found that the lateral gene transfer event is very recent in the time scale. A test of positive selection showed that positive selection is acting specifically in the branch that shares a common ancestor with H. haemolyticus through the substitution of an alanine to a serine at position 72, though the overall score of the tree is less than one. It is known that pseudogenes do not encode active proteins and therefore they are not under any kind of functional constraints, so they are under greater influence of genetic drift. Thus, it was concluded that different forces are driving the evolution of this gene in the species considered here. Protein modeling concluded that this modification contributed to the increase in the redox potencial of the active site. Thus the action of positive selection under a single amino acid residue was beneficial to the function of the enzyme as whole / Mestrado / Bioquimica / Mestra em Biologia Funcional e Molecular

Bakterier har alltid haft en stor inverkan på mänskligheten. För att diagnostisera bakteriella sjukdomar och behandla dem krävs identifiering av bakterien eller bakteriens relevanta egenskaper. Transportmedium har utvecklats för att hålla bakterierna vid liv från provtagning till analys. Syftet med studien var att utvärdera bakteriers viabilitet i det vätskebaserade mediet Copan Eswab jämfört med kolmedium (Copan swab). Bakterierna som ingick i studien var Campylobacter jejuni, Streptococcus pneumoniae, Haemophilus influenzae, Niesseria gonorrhoeae och Fusobacterium nucleatum. Förutom jämförande mellan medierna genomfördes en jämförelse mellan Eswab i kyl och i rumstemperatur. Resultaten för H. influenzae (n=9) och N. gonorrhoeae (n=9) visade att Eswab gav lika många eller fler överlevande bakterier. Gällande F. nucleatum (n=9) visade resultaten att fler överlevde i Copan swab (Copanpinnar) de första 28 timmarna, men även att bakterien inte klarar mer än 28 timmar i rumstemperatur. Gällande S. pneumoniae (n=9) och C. jejuni (n=9) gav båda opålitliga svar. Ytterligare mätpunkter och studier krävs för att erhålla mer pålitliga resultat gällande hur länge bakterierna överlever i Eswab. / Bacteria have always had a great influence on mankind. To diagnose any bacterial disease and treat it it’s necessary to identify the bacteria or any relevant attributes. Different types of specimen transport have been developed to keep the bacteria alive from sampling until the analysis is performed. The purpose of the study was to evaluate the viability of bacteria in the fluid-based media Copan EswabTM compared with charcoal medium (Copan swab). Bacteria included in the study were: Campylobacter jejuni, Streptococcus pneumoniae, Haemophilus influenzae, Niesseria gonorrhoeae and Fusobacterium nucleatum. The study also tried to compare how bacteria survived in Eswab which was refrigerated and in Eswab room temperature. Results for H. influenzae (n=9) and N. gonorrhoeae (n=9) showed that an equal amount or more of the bacteria survived in Eswab. More of F. nucleatum (n=9) survived in Copan swab (Copan swab sticks) for the first 28 hours, additionally they showed that the bacteria won’t survive more than 28 hours in room temperature. Regarding S. pneumoniae (n=9) and C. jejuni (n=9) both displayed unreliable results. Overall more measurements and additional studies are needed for more reliable results.