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

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.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-01-11T10:54:50Z No. of bitstreams: 2 Dissertação - Robmary Matias de Almeida - 2017.pdf: 3556874 bytes, checksum: 27db72af224144cb18266e9f23af2021 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-01-11T10:55:37Z (GMT) No. of bitstreams: 2 Dissertação - Robmary Matias de Almeida - 2017.pdf: 3556874 bytes, checksum: 27db72af224144cb18266e9f23af2021 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-01-11T10:55:38Z (GMT). No. of bitstreams: 2 Dissertação - Robmary Matias de Almeida - 2017.pdf: 3556874 bytes, checksum: 27db72af224144cb18266e9f23af2021 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-06-26 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Haemophilus influenzae (Hi) and Haemophilus haemolyticus (Hhae) are important microorganisms present in human nasopharyngeal colonization, with rates varying according to locality, sampling frequency, individual and social factors. Hi is a pathological agent that causes diseases such as meningitis, pneumonia, sepsis and otitis media, which presents in encapsulated forms with six serotypes a, b, c, d, e, f, and uncapsulated or non-typeable (HiNT). Hhae is a nasopharyngeal comensal and rarely causes invasive diseases. The objective of this study was to estimate the prevalence of Hi and Hhae in children under five years of age attending public day care centers in the city of Goiânia-GO, to determine the circulating serotypes, to analyze the risk factors associated with the nasopharyngeal carrige, as well as to characterize the antimicrobial resistance of Hi. Were analyzed 1.188 nasopharynx swabs from healthy children between 36 and 59 months of age from October to December 2010. The samples were submitted to bacterial culture for the isolation of Haemophilus spp. For the identification of the species, the Real-Time Polymerase Chain Reaction (TR-PCR) was used. Serotyping, as well as detection of the bla TEM-1 and bla ROB-1 resistance genes, was performed through the conventional Polymerase Chain Reaction. Phenotypic detection for β-lactamase production was performed by the chromogenic cephalosporin test. The database was constructed with the statistical software SPSS (Chicago, IL, USA) version 18.0. Risk factors, children aged 3 years, low maternal schooling and three or more children under 10 years of age living in the same household of the child recruited in the study were evaluated by multivariate Poisson regression. The prevalence of Hi carriers was 54.4% (646 / 1.188), 0.9% (n = 11) of the serotype e, 0.9% (n = 11) of serotype f, 0.2% (n = 2) serotype a, 0.08% (n = 1) serotype d, 0.0% (n = 0) serotype b and c and 52.3% (621 / 1.188) of HiNT. The prevalence of Hhae was 1.2% (14 / 1.188). Among the encapsulated Hi, the prevalence of the bla TEM-1 gene was 4.0% (1/25) and the bla ROB-1 gene was 4.0% (1/25). Among the 20% (124/621) of HiNT analysed, the prevalence of the bla TEM-1 gene was 13,7% (17/124) and the prevalence of the bla TEM-1 gene was 1,6% (2/124). Continuous surveillance of Haemophilus spp. as a colonizer, is necessary to evaluate its transmission and dissemination in the population where there is a higher risk of invasive disease, to control Hib re-emergence after the vaccinacion and to continue to monitor antimicrobial resistance. / Haemophilus influenzae (Hi) e Haemophilus haemolyticus (Hhae) são importantes microrganismos presentes na colonização nasofaríngea humana, com taxas que variam de acordo com a localidade, frequência de amostragem, fatores individuais e sociais. O Hi é um agente patológico causador de doenças como meningite, pneumonia, sepse e otite média que se apresenta sob as formas capsuladas com seis sorotipos a, b, c, d, e, f, e não capsuladas ounão tipáveis (HiNT). O Hhae é comensal nasofaríngeo e raramente causa doenças invasivas. O objetivo do estudo foi estimar a prevalência de Hi e Hhae em crianças menores de cinco anos de idade que frequentam creches públicas no município de Goiânia-GO, determinar os sorotipos circulantes, analisar os fatores de risco associados ao portador nasofaríngeo, bem como caracterizar a resistência antimicrobiana dos Hi. Foram analisados 1.188 swabs de nasofaringe de crianças saudáveis entre 36 e 59 meses de idade, no período de outubro a dezembro de 2010. As amostras foram submetidas à cultura bacteriana para o isolamento do Haemophilus spp. Para identificação da espécie foi utilizada a Reação em Cadeia da Polimerase em Tempo Real (PCR-TR). A sorotipagem, assim como a detecção dos genes de resistência bla TEM-1 e bla ROB-1 , foi realizada através da Reação em Cadeia da Polimerase convencional. A detecção fenotípica para produção da β-lactamase foi executada pelo teste da cefalosporina cromogênica. A base de dados foi construída com o programa estatístico SPSS (Chicago, IL, USA) versão 18.0. Os fatores de risco, crianças com idade de 3 anos, baixa escolaridade da mãe e três ou mais crianças menores de 10 anos de idade convivendo no mesmo domicilio da criança recrutada no estudo, foram avaliados por regressão de Poisson multivariada. A prevalência de portador do Hi foi de 54,4% (646/1.188) sendo 0,9% (n=11) do sorotipo e, 0,9% (n=11) do sorotipo f, 0,2% (n=2) do sorotipo a, 0,08%(n=1) do sorotipo d, 0,0% (n=0) dos sorotipos b e c e 52,3% (621/1.188) de HiNT. A prevalência do Hhae foi de 1,2% (14/1.188). Entre os Hi encapsulados a prevalência do gene bla TEM-1 foi de 4,0% (1/25) e do gene bla ROB-1 foi de 4,0% (1/25). Em 20% (124/621) dos HiNT, a prevalência do gene bla TEM-1 foi de 13,7% (17/124) e do gene bla ROB-1 de 1,6% (2/124). A vigilância contínua do Haemophilus spp. como colonizador, se faz necessária para avaliar sua transmissão e disseminação na população onde há maior risco de incidência de doenças invasivas, controlar a re-emergência do Hib após a vacinação e continuar monitorando a resistência antimicrobiana.

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.

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.

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.

Haemophilus influenzae is a host adapted human mucosal pathogen involved in a variety of acute and chronic respiratory tract infections, including chronic obstructive pulmonary disease and asthma, all of which rely on its ability to efficiently establish continuing interactions with the host. Here we report the characterization of a novel molybdenum enzyme, TorZ/MtsZ that supports interactions of H. influenzae with host cells during growth in oxygen-limited environments. Strains lacking TorZ/MtsZ showed a reduced ability to survive in contact with epithelial cells as shown by immunofluorescence microscopy and adherence/invasion assays. This included a reduction in the ability of the strain to invade human epithelial cells, a trait that could be linked to the persistence of H. influenzae. The observation that in a murine model of H. influenzae infection, strains lacking TorZ/MtsZ were almost undetectable after 72 h of infection, while similar to 3.6 x 10(3) CFU/mL of the wild type strain were measured under the same conditions is consistent with this view. To understand how TorZ/MtsZ mediates this effect we purified and characterized the enzyme, and were able to show that it is an S- and N-oxide reductase with a stereospecificity for S-sulfoxides. The enzyme converts two physiologically relevant sulfoxides, biotin sulfoxide and methionine sulfoxide (MetSO), with the kinetic parameters suggesting that MetSO is the natural substrate of this enzyme. TorZ/MtsZ was unable to repair sulfoxides in oxidized Calmodulin, suggesting that a role in cell metabolism/energy generation and not protein repair is the key function of this enzyme. Phylogenetic analyses showed that H. influenzae TorZ/MtsZ is only distantly related to the Escherichia colt TorZ TMAO reductase, but instead is a representative of a new, previously uncharacterized Glade of molybdenum enzyme that is widely distributed within the Pasteurellaceae family of pathogenic bacteria. It is likely that MtsZ/TorZ has a similar role in supporting host/pathogen interactions in other members of the Pasteurellaceae, which includes both human and animal pathogens.

Acute respiratory tract infections are the most common cause o f illness and death in the pediatric population worldwide. It is estimated that 70 - 80% o f severe pneumonias in Africa are caused by S.pnewnoniae (the pneumococcus) followed by H. influenzae type b. Surveillance reveals that drug resistance is increasing worldwide, South Africa not being an exception. This has considerably complicated the management o f infections caused by both the pneumococcus and H. influenzae type b ( H ib ). It is widely accepted that colonization of the nasopharynx even briefly precedes middle ear infection and invasive pneumococcal disease. Early onset of colonization after birth has been associated with early onset o f middle ear infections. Furthermore, colonized children are able to transmit these organisms to other children. Carriage o f pneumococci commonly occurs in young children. The carriage of resistant pneumococci is usually limited to those serotypes carried in children. N ew conjugate vaccines may be able to reduce colonization o f these serotypes. This study was undertaken to determine the serotypes and susceptibility o f pneumococci and H. influenzae type b, and the proportion o f healthy children colonized at Zola Community Health Centre (ZCHC) in Soweto.

Nontypeable Haemophilus influenzae (NTHi), Streptococcus pneumoniae and Moraxella catarrhalis are common bacterial agents of otitis media which is a major cause of morbidity in young children. Mucosal immune responses are an integral part of the immune defense against middle ear infection and it is known that certain populations, including Australian Aboriginal children, are highly susceptible to disease. The current study focussed on the development of the mucosal immunity to the three bacterial pathogens in Aboriginal and non-Aboriginal children from birth to two years of age, living in the Kalgoorlie-Boulder region of Western Australia. Salivary and breast milk IgA levels were measured by the enzyme Linked immunosorbent assay. The measured IgA levels, combined with socio-economic, demographic and bacteriological data were analyzed statistically to determine the influential factors on the mucosal IgA response in these children over time. This study found that each antigen-specific IgA examined followed a distinct ontogeny pattern and IgA responses differed significantly according to age, indigenous status and feeding type. Indoors smoke exposure, maternal smoking, and sibling day care attendance had some impact on salivary IgA levels in the children. However, household crowding and the presence of older siblings had the most significant impact on salivary IgA levels for children of different age groups. These two factors were correlated to increased nasophayrngeal colonization by H. influenzae, S. pneumoniae and M. catarrhalis and colonization status was also found to influence salivary IgA levels in the children. No correlation between maternal breast milk IgA levels and child salivary IgA levels was observed. The results suggest that the degree of exposure to environmental factors rather than immunological deficit is responsible for the observed differences in salivary IgA responses between Aboriginal and non-Aboriginal children and modifying these factors could lead to a reduction in the burden of otitis media experienced by the children. Further studies correlating specific salivary IgA levels to diseases such as otitis media will reveal the role of specific salivary IgA responses in the prevention of infection by respiratory pathogens.

Nontypeable Haemophilus influenzas (NTHi) is recognised as a significant human pathogen causing mild to severe respiratory tract infections. At present, no vaccine is available for prevention of infection caused by this pathogen. Several outer membrane proteins (OMPs) of NTHi and its lipooligosaccharide have been investigated as possible vaccine antigens against NTHi infections. Previous investigations in our laboratory have shown that OMP26 from an NTHi 289 strain was able to significantly enhance pulmonary clearance of NTHi in a rat model in which animals were immunised via intestinal Peyer's patches and then boosted intratracheally (Kyd and Cripps, 1998; El- Adhami et al., 1999). In recent studies, the OMP26, when used as a parenteral immunogen, was also highly effective at inducing immune responses that led to significantly enhanced clearance of the chinchilla nasopharynx (Kyd et al., 2003). These studies indicate significant potential of the OMP26 as a candidate vaccine antigen and warrant further investigations for development of a vaccine against NTHi. This thesis focussed on the immunological and structural characterisation of the NTHi vaccine candidate, OMP26. Peptides of OMP26 were used as tools to localise the immunologically important regions of the OMP26. Two different E. coli expression systems, the GST gene fusion and the 6xHis tagged systems, were employed to construct the OMP26 peptides. It was found in this study that, despite efforts to optimise the system, the GST-fusion protein system failed to produce consistent results for the purification and storage of the OMP26 peptides. In contrast, the 6xHis tagged system exhibited more reliable outcomes in the production of the recombinant OMP26 peptides and the stability of the stored purified peptides. As such, the purified OMP26 peptides from the 6xHis tagged system were chosen to map major regions of immunological significance for the OMP26 protein. The regions of the OMP26 which are involved in the induction of the acquired immune responses have been identified in the present study. Based on the antigen specific lymphocyte proliferation assay, the dominant T cell epitopes for OMP26 were located between amino acid residues 95 and 197 (T3+T4 region). These identified T cell epitopes exhibited the capability of efficient T cell activation, suggesting that the epitopes within the T3+T4 region potentially had the highest affinity for binding to the MHC molecules than did any other OMP26 region. Using two different assay systems, ELISA and BIA, the predominant B cell epitopes of OMP26 were located between amino acid residues 45 and 145 (T2+T3 region). This region was also found to be immunodominant across all animal species tested, and with all immunisation regimens used. Flow cytometry analysis also revealed that these particular epitopes were expressed on the surface of NTHi cells. By integration of the data obtained from these current experimental studies and the computational analysis of the OMP26 sequence, two hypothetical models of the OMP26 were also proposed in this study. The significant outcomes obtained in this thesis provide a better understanding of the specificity of the host immune responses to the OMP26 protein These findings provide great benefit not only for the development of a future NTHi vaccine but for the development of the peptide-based immunodiagnostic reagents as well. These diagnostic reagents will be valuable, in particular, for the evaluation of efficacy of an NTHi vaccine in humans that may include OMP26 or specific conformational structures. Future studies are still required to further define the minimum epitope length required for the B and T cell responses identified in this study. The significance of these responses in immune protection against NTHi infection also requires further investigations. Human immune responses also need to be determined, but this can only be achieved following clinical trial studies.