Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-08-06T15:45:54Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) DISSERTACAO DE MESTRADO TELMA SOUSA.pdf: 2411234 bytes, checksum: 4b7671237d9a10f6795b86433fc36a82 (MD5) / Made available in DSpace on 2014-08-06T15:45:54Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) DISSERTACAO DE MESTRADO TELMA SOUSA.pdf: 2411234 bytes, checksum: 4b7671237d9a10f6795b86433fc36a82 (MD5) Previous issue date: 2009 / To estimate the prevalence, to asses risk factors for Group B Streptococcus (GBS) colonization and to describe phenotypic and genotypic characteristics of isolated strains from pregnant women in Goiânia, Goiás. Methods: A cross sectional study was carried out among 198 pregnant women, at least at the 32o weeks´ gestation, attending a reference health unit, from March to June 2009. Socio, demographic and obstetric profiles were investigated using a standard questionnaire. Samples of vaginal and rectal secretion were collected and placed into selective enrichment broth Todd-Hewitt. Tests for GBS identification (gram, catalase and CAMP) followed by susceptibility test using antibiotic disk diffusion technique were performed. Genetic diversity was assessed by pulsed-field gel electrophoresis (PFGE). Descriptive and analytic statistical tests were applied (Epi Info e SPSS 13.0). Analyses were performed at the Instituto de Patologia Tropical Saúde Pública/UFG. Results: Thirty pregnant women were colonized by GBS yielding a prevalence of 15.2% (IC95% 10.5 ?? 20.9). Pregnant women younger than 20 years and with low income had higher risk of GBS colonization, in univariate analysis (p<0.05). GBS was isolated from 28 vaginal and 14 rectal specimens. Twelve pregnant were vaginal and rectal colonized. All 42 strains were susceptible to penicillin, vancomycin, ceftriaxone and levofloxacin. Three strains (7.1%) were resistant to erythromycin and two (4.7%) to clindamycin. 19 pulsotypes and four clusters were identified. Nine out 12 pars of positive strains (vaginal and rectal) were genetically identical, two were stricted related and one par was colonized by different strains. The same genetic profile was observed in more than one pregnant. Conclusions: Socioeconomic and obstetrics variables had low predictive value for GBE colonization among pregnant women, reinforcing the need for universal microbiology screening strategy in this population, in order to prevent neonatal sepsis. A high genetic diversity of GBS was found among pregnant women in Goiania. / Estimar a prevalência, identificar fatores associados à colonização pelo Streptococcus do grupo B (EGB), descrever o perfil fenotípico e genotípico das cepas isoladas em gestantes, em Goiânia, Goiás. Metodologia: Estudo transversal envolvendo 198 gestantes a partir da 32ª semana, atendidas entre março e junho de 2009, em um serviço de referência materno-infantil, em Goiás. Características sócio-demográficas e obstétricas foram investigadas utilizando um questionário padronizado. Foram coletadas amostras de sítio vaginal e anal e inoculadas no meio seletivo caldo Todd-Hewitt, posteriormente, foram realizados teste de identificação do agente (gram, catalase, CAMP) e de suscetibilidade pela técnica de disco difusão. A diversidade genética foi avaliada por eletroforese em gel em campo pulsado (PFGE). As análises foram realizadas no Instituto de Patologia Tropical Saúde Pública /UFG. Foram utilizados testes de estatística descrita e analítica (Epi Info e SPSS 13.0). Resultados: Trinta gestantes estavam colonizadas pelo EGB resultando a prevalência de 15,2% (IC95% 10,5 ?? 20,9). Em análise univariada, baixa renda e idade ? 19 anos foram associadas à presença de EGB (p<0,05). O EGB foi isolado em 28 amostras de secreção vaginal e em 14 anal. Doze gestantes apresentavam colonização vaginal e anal. Todos os 42 isolados eram sensíveis à penicilina, vancomicina, ceftriaxona e levofloxacina. Três (7,1%) apresentaram resistência à eritromicina e dois (4,7%) à clindamicina. Foram identificados 19 pulsotipos e quatro clusters. Nove entre 12 pares de cepas positivas (anal e vaginal) eram geneticamente idênticas, dois eram estritamente relacionadas e um par apresentou cepas diferentes. O mesmo perfil genético foi observado em pacientes diferentes. Conclusões: Fatores de risco sócio-demográficos e obstétricos apresentaram baixo poder preditivo para colonização, pelo EGB, reforçando a estratégia rastreamento universal por cultura de secreção anorretal das gestantes no terceiro trimestre. Uma grande variabilidade genética entre as cepas de EGB foi evidenciada nas gestantes colonizadas, em Goiânia.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Streptococcus agalactiae, ou Streptococcus do grupo B (GBS), é um importante patógeno oportunista que causa pneumonia, sepse e meningite em recém-nascidos e infecções em adultos imunocomprometidos. O pulmão aparentemente é o portal de entrada para o EGB na corrente sanguínea o que pode evoluir para uma septicemia. Os mecanismos de virulência relevantes envolve a habilidade do EGB em penetrar e sobreviver intracelularmente em células hospedeiras. Neste trabalho, foram analisados os mecanismos moleculares da apoptose epitelial induzida pelo EGB, e a produção de óxido nítrico (NO) e espécies reativas de oxigênio (ROS) em células epiteliais respiratórias A549 durante a infecção por EGB. Todas as amostras de EGB exibiram a capacidade de aderir e invadir células A549. A sobrevivência intracelular do EGB em células A549 ocorreu durante 24 h de incubação sem replicação do patógeno. No entanto, a amsotra 88641-V isolada de vagina não sobreviveu após 0,5 h de interação. O EGB promoveu a perda de viabilidade do epitélio durante a infecção. As alterações morfológicas em células A549 infectadas com o EGB incluem arredondamento celular, condensação nuclear, encolhimento celular e perda de contato célula-célula e célula-substrato. A dupla marcação AV/IP revelou que amostras de EGB sorotipo III induziram apoptose enquanto amostras do sorotipo V induziram morte celular semelhante a necrose em células A549. Caspase-3 foi ativada durante a apoptose induzida por EGB em células epiteliais. No entanto, a ativação de caspases-8 e -9 foi detectada apenas para a amostra 88641-V e as amostras EGB do sorotipo III, respectivamente. Experimentos comparativos de Immunoblotting revelaram que o EGB induziu um aumento da expressão Bim, uma proteína pró-apoptótica e diminuiu a expressão de Bcl-2 e Bcl-xL, proteínas anti-apoptóticas. As células A549 apresentaram perda de potencial de membrana mitocondrial &#916;&#968;m e co-localização com o Bax. Ensaio de espectrometria de massa identificou a proteína PI-2a, uma proteína estrutural de pili, que exibe atividade carboxipepdidase. Descobrimos que os dois sorotipos (III e V) induziram a produção ROS e NO em células A549. Em conclusão, a apoptose induzida pelo EGB em células A549 é um mecanismo importante de virulência, resultando na destruição de tecidos, escape do sistema imune do hospedeiro com espalhamento bacteriano e, em consequência, a doença invasiva ou uma infecção sistémica. / Streptococcus agalactiae, or group B Streptococcus (GBS), is an important opportunistic pathogen that causes pneumonia, sepsis, and meningitis in neonates and severe diseases in immunocompromised adults. The lung is the apparent portal of entry for GBS into the bloodstream, after which septicemia may ensue. A relevant virulence mechanism involves the ability of GBS to penetrate and to survive intracellularly within these host cells. In this work, we analyzed the molecular mechanisms of GBS-induced epithelial apoptosis, and nitric oxide (NO) and reactive oxygen species (ROS) production by lung epithelial cell line A549 cells during infection with GBS. All GBS exhibited the ability to adhere and to invade A549 cells. The survival of GBS within A549 cells without replication was shown during 24 h incubation. However, the 88641-V strain isolated from vagina did not survive after 0.5 h of interaction. GBS promoted the loss of viability of the epithelium during infection. The morphological changes in A549 cells infected with GBS included cell rounding, nuclear condensation, cellular shrinkage and loss of cell-cell contact and cell-substrate. The double staining AV / IP revealed that GBS serotype III induced apoptosis while GBS serotype V induced like necrosis cell death in A549 cells. Caspase-3 was activated during GBS-induced endothelial apoptosis. However, activation of caspases-8 and -9 was detected only by GBS 88641-V and GBS-III, respectively. Comparative immunoblotting experiments revealed that GBS induced an increasing pro-apoptotic Bim expression and decreasing anti-apoptotic Bcl-2 and Bcl-XL expression. A549 cells exhibited loss of mitochondrial membrane potential &#916;&#968;m with Bax colocalization. Mass spectrometry assay identified protein PI-2a, a structural protein pili, which exhibit carboxipepdidase activity. We found that both serotypes (III and V) induced ROS and NO production in A549 cells. In conclusion, apoptosis of A549 cell induced by GBS is an important virulence mechanism resulting in tissue destruction, escape from the host immune system with bacterial spreading and, in consequence, invasive disease or systemic infection.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Estreptococos do grupo B (EGB), principalmente sorotipo III são a principal causa de pneumonia neonatal, sepse e meningite. O potencial de virulência das amostras de EGB pode determinar a colonização ou a infecção do hospedeiro. Como o pulmão constitui uns dos primeiros órgãos durante o processo de invasão sistêmica por EGB, nós decidimos investigar os mecanismos de adesão e invasão de amostras do sorotipo III (90356-líquor e 80340-vagina) com linhagem de células epiteliais do pulmão humano (A549). Desta forma, o principal objetivo deste estudo foi avaliar a capacidade de aderência e invasão de duas amostras de EGB sorotipo III com células de epiteliais pulmonares A549, a persistência bacteriana intracelular, a fusão com compartimentos acídicos, potencial citotóxico e indução de apoptose. As amostras mostraram capacidade de aderir e invadir o epitélio pulmonar A549, onde a amostra 90356-líquor isolada de paciente a que apresentou maior propriedade adesiva e invasiva que a amostra 80340-vagina (p<0,05). Ambas as cepas mostraram persistência intracelular sem replicação no interior do epitélio respiratório até 24h de incubação. Além disso, verificamos que os EGB são capazes de promover vacuolização celular permanecendo viáveis dentro de vacúolos acídicos, sugerindo a ocorrência de fusão lisossomo-fagossomo. A amostra 90356-líquor também mostrou maior citotoxidade quando comparada com a amostra 80340-vagina. A análise por citometria de fluxo demonstrou, pela primeira vez, que o EGB induz apoptose em epitélio respiratório, podendo representar um mecanismo importante para o desenvolvimento da lesão celular aguda e a patogênese bacteriana. / Group B streptococci (GBS), mainly serotype III, is a major cause of neonatal pneumonia, sepsis and meningitis. Virulence potential of GBS strains may determine the outcome of host colonization or infection. Because the lung constitutes a first step in GBS systemic invasion processes, we have investigates the adherence and invasion mechanisms of GBS-III isolates to human lung epithelial cell line (A549). Thus, the main objective of this study was to evaluate the ability of adhesion and invasion of GBS strains serotype III (90356-liquor and 80340-vagina) with A549 lung epithelial cells, intracellular bacterial persistence, fusion with acidic compartments, cytotoxic potential, and induction of apoptosis. The strains showed ability to adhere and invade the epithelial A549 cells; GBS 90356-liquor isolated from a patient showed a more efficient adherence and invasion properties than GBS-III 80340 isolated from vagina (P<0,05). Both strains showed persistent intracellular viability without replication into A549 cells up to 24h incubation. In addition, we found that GBS are able to promote cellular vacuolization and persisted viable inside acidic vacuoles, suggesting lysosome-phagosome fusion. The GBS 90356-líquor also showed higher cytotoxicity when compared with 80340-vagina strain. The present work describe for first time by flow cytometry that GBS induces apoptosis in respiratory epithelium and be an important mechanism for the development of acute cellular injury and bacterial pathogenesis.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Streptococcus agalactiae, ou Streptococcus do grupo B (GBS), é um importante patógeno oportunista que causa pneumonia, sepse e meningite em recém-nascidos e infecções em adultos imunocomprometidos. O pulmão aparentemente é o portal de entrada para o EGB na corrente sanguínea o que pode evoluir para uma septicemia. Os mecanismos de virulência relevantes envolve a habilidade do EGB em penetrar e sobreviver intracelularmente em células hospedeiras. Neste trabalho, foram analisados os mecanismos moleculares da apoptose epitelial induzida pelo EGB, e a produção de óxido nítrico (NO) e espécies reativas de oxigênio (ROS) em células epiteliais respiratórias A549 durante a infecção por EGB. Todas as amostras de EGB exibiram a capacidade de aderir e invadir células A549. A sobrevivência intracelular do EGB em células A549 ocorreu durante 24 h de incubação sem replicação do patógeno. No entanto, a amsotra 88641-V isolada de vagina não sobreviveu após 0,5 h de interação. O EGB promoveu a perda de viabilidade do epitélio durante a infecção. As alterações morfológicas em células A549 infectadas com o EGB incluem arredondamento celular, condensação nuclear, encolhimento celular e perda de contato célula-célula e célula-substrato. A dupla marcação AV/IP revelou que amostras de EGB sorotipo III induziram apoptose enquanto amostras do sorotipo V induziram morte celular semelhante a necrose em células A549. Caspase-3 foi ativada durante a apoptose induzida por EGB em células epiteliais. No entanto, a ativação de caspases-8 e -9 foi detectada apenas para a amostra 88641-V e as amostras EGB do sorotipo III, respectivamente. Experimentos comparativos de Immunoblotting revelaram que o EGB induziu um aumento da expressão Bim, uma proteína pró-apoptótica e diminuiu a expressão de Bcl-2 e Bcl-xL, proteínas anti-apoptóticas. As células A549 apresentaram perda de potencial de membrana mitocondrial &#916;&#968;m e co-localização com o Bax. Ensaio de espectrometria de massa identificou a proteína PI-2a, uma proteína estrutural de pili, que exibe atividade carboxipepdidase. Descobrimos que os dois sorotipos (III e V) induziram a produção ROS e NO em células A549. Em conclusão, a apoptose induzida pelo EGB em células A549 é um mecanismo importante de virulência, resultando na destruição de tecidos, escape do sistema imune do hospedeiro com espalhamento bacteriano e, em consequência, a doença invasiva ou uma infecção sistémica. / Streptococcus agalactiae, or group B Streptococcus (GBS), is an important opportunistic pathogen that causes pneumonia, sepsis, and meningitis in neonates and severe diseases in immunocompromised adults. The lung is the apparent portal of entry for GBS into the bloodstream, after which septicemia may ensue. A relevant virulence mechanism involves the ability of GBS to penetrate and to survive intracellularly within these host cells. In this work, we analyzed the molecular mechanisms of GBS-induced epithelial apoptosis, and nitric oxide (NO) and reactive oxygen species (ROS) production by lung epithelial cell line A549 cells during infection with GBS. All GBS exhibited the ability to adhere and to invade A549 cells. The survival of GBS within A549 cells without replication was shown during 24 h incubation. However, the 88641-V strain isolated from vagina did not survive after 0.5 h of interaction. GBS promoted the loss of viability of the epithelium during infection. The morphological changes in A549 cells infected with GBS included cell rounding, nuclear condensation, cellular shrinkage and loss of cell-cell contact and cell-substrate. The double staining AV / IP revealed that GBS serotype III induced apoptosis while GBS serotype V induced like necrosis cell death in A549 cells. Caspase-3 was activated during GBS-induced endothelial apoptosis. However, activation of caspases-8 and -9 was detected only by GBS 88641-V and GBS-III, respectively. Comparative immunoblotting experiments revealed that GBS induced an increasing pro-apoptotic Bim expression and decreasing anti-apoptotic Bcl-2 and Bcl-XL expression. A549 cells exhibited loss of mitochondrial membrane potential &#916;&#968;m with Bax colocalization. Mass spectrometry assay identified protein PI-2a, a structural protein pili, which exhibit carboxipepdidase activity. We found that both serotypes (III and V) induced ROS and NO production in A549 cells. In conclusion, apoptosis of A549 cell induced by GBS is an important virulence mechanism resulting in tissue destruction, escape from the host immune system with bacterial spreading and, in consequence, invasive disease or systemic infection.

La combustion en boucle chimique (CLC) est un procédé du type oxy-combustion où des particules sont utilisées pour fournir de l'oxygène à la combustion. Des études sont nécessaires pour l'extrapolation et l'optimisation du procédé CLC, fonction des propriétés des particules du groupe B et de la technologie CFB. Les études hydrodynamiques ont été faites dans un riser de 18 m de hauteur. Des profils axiaus de pression, ainsi que les profils radiaux de flux et de quantité de mouvement ont été obtenus. Trois types de particules ont été utilisées ayant un diamètre de Sauter entre 250 et 300 μm et une densité entre 2600 et 3300 kg/ m3. Un impact de la sphéricité des particules sur la perte de charge a été révélé. Dans des conditions identiques, les billes de verre génèrent des pertes de charge d'environ 50% inférieures à celles du sable. Dans la zone d'écoulement développée, la présence du régime cœur-anneau a été détectée. Un modèle hydrodynamique 1D du riser qui est à la fois fondé sur des données expérimentales et sur les équations gaz-solide Euler-Euler, a été développé. Une nouvelle corrélation pour la force de traînée moyennée sur la section est proposée. Une nouvelle corrélation des conditions limite dans la partie inférieure du riser a aussi été établie. Le modèle 1D final est en mesure de prédire la perte de charge du riser pour différentes conditions opératoires et en tenant compte des propriétés des particules, comme la densité, la taille et la forme. Une étude sur la pertinence de l'utilisation du logiciel Barracuda CPFD® pour simuler des particules du groupe B en régime de transport a été réalisée. Il a été montré que le code sous-estime la perte de charge pour le sable / Chemical Looping Combustion (CLC) is an oxy-combustion like process where particles are used to supply oxygen to combustion. Further work is still needed for extrapolation and optimization of the CLC process, concerning properties of Group B particles and CFB technology. Hydrodynamic tests were made on a 18 m tall riser. Axial pressure profiles as well as radial flux profiles and radial momentum quantity profiles were obtained. Three types of Group B particles were used with Sauter mean diameters between 250 and 300 μm and densities between 2600 and 3300 kg/m²s. An important impact of particle sphericity on riser pressure drop has been revealed. At identical conditions, glass beads present about half the pressure drop generated by sand. In the developed region of the riser, the core-annulus regime has been found. A 1D model of the riser, based on experimental results and on the Euler-Euler gas-solid equations, has been developed. Moreover, a new cross section averaged drag force correlation is presented. A new boundary condition on the bottom of the riser has been investigated. The final 1D model is capable of predicted riser pressure drop from the operating conditions and it takes into account particle properties such as density, size and shape. A study on the adequacy of the use of the commercial CFD code Barracuda to simulate risers with Group B particles was made. It was shown that the code under estimates pressure drop along the riser for sand simulations

Orientador: Renato Passini Junior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-04T01:25:35Z (GMT). No. of bitstreams: 1 Nomura_MarceloLuis_D.pdf: 838174 bytes, checksum: a795b39457b62a18ecb17c4609ece12d (MD5) Previous issue date: 2004 / Resumo: Objetivos: Identificar a taxa de prevalência e fatores de risco de colonização materna por estreptococo do grupo B (EGB) em gestantes com trabalho de parto prematuro (TPP) e/ou ruptura prematura pré-termo de membranas (RPM). Métodos: Foram colhidos dois swabs anais e vaginais de 203 gestantes atendidas no CAISM-UNICAMP. Um swab de cada local foi colocado em meio de transporte e enviados para cultura em placas de ágar-sangue, os outros dois foram incubados por 24 horas em meio de Todd-Hewitt para posterior semeadura em placas de ágar-sangue. Resultados: A prevalência de colonização materna por EGB foi de 27,6% (56 gestantes). As taxas de colonização por diagnóstico foram 34,7% para RPM, 25,2% para TPP e 17,8% para TPP + RPM. As variáveis raça branca, baixo nível de escolaridade e infecção urinária foram associadas a maiores taxas de colonização na análise multivariada. A presença de infecção urinária foi a única variável significativamente associada à colonização materna na análise multivariada. A taxa de detecção do estreptococo do grupo B foi significativamente maior com o uso do meio seletivo e com a associação de coleta de culturas anais e vaginais. A taxa de colonização neonatal foi de 3,1%. Ocorreram dois casos de sepse precoce por EGB nesta amostra, com prevalência estimada de 10,8 casos por mil nascidos vivos e mortalidade de 50%. Conclusão: A amostra avaliada apresenta altas taxas de colonização materna por Streptococcus agalactiae. É necessário o uso de meio de cultura seletivo e a associação de culturas anorretais e vaginais para aumentar a taxa de detecção do EGB. A incidência de sepse neonatal precoce foi elevada nesta população / Abstract: Objective: to study group B streptococcus maternal colonization rates and risk factors in women with preterm labor (PTL) and preterm premature rupture of membranes (PROM). Methods: Vaginal and anal swabs (two of each) were colected from 203 women followed at CAISM-UNICAMP. One of each swab was placed in transport media and then cultured in blood-agar plates, the other swabs were incubated in Todd-Hewitt selective media for 24 hours and then subcultured in blood-agar plates. Results: Maternal colonization rate was 27.6% (56 women). Colonization rates by admission diagnosis were 34.7% in PROM, 25.2% in PTL and 17.8% in PTL and PROM. White race, less than elementary education level and urinary tract infection were associated with maternal colonization in the univariate analysis. Urinary tract infection was the only variable associated with maternal colonization in a multivariate analysis. GBS detection rates were significantly higher with the use of selective culture media and with sampling of both vaginal and anorectal sites. Neonatal colonization rate was 3.1%. There were two cases of early-onset neoanatal sepsis caused by GBS, with an estimated prevalence of 10.8 cases per thousand live borns and a mortality rate of 50%. Conclusions: This sample of women had high GBS colonization rates. The use of selective culture media and collection of both anal and vaginal samples is necessary in order to maximize GBS detection rates. Early-onset neonatal sepsis incidence was high in this population / Doutorado / Tocoginecologia / Doutor em Tocoginecologia

Streptococcus (S.) agalactiae sind grampositive, in Ketten gelagerte Kokken, die auf Blutagar eine Hämolyse zeigen. Aufgrund ihrer Zugehörigkeit zur Lancefield-Gruppe B werden sie auch als Gruppe B Streptokokken (GBS) bezeichnet (Hof, 2005). GBS sind die Hauptursache von Sepsis, Meningitis und Pneumonie bei Neugeborenen (Schrag, et al., 2000). Die Arbeit beschäftigte sich mit der Genotypisierung von GBS. Darüber hinaus konnten auch Einblicke in die Phylogenese sowie die Populationsstruktur von GBS gewonnen werden. Ziel war es, einen DNA-Microarray zu entwickeln und zur Genotypisierung von GBS einzusetzen. Während der Evaluierung des DNA Microarray konnten stammspezifische Muster beobachtet werden, diese wurden durch bereits etablierte Typisierungmethoden (MLST) bekannten Genotypen zugeordnet. Die Ergebnisse wurden in einer Datenbank zusammengefasst. Mithilfe der Datenbank konnte die Software zur Auswertung entwickelt werden. (siehe http://alere-technologies.com/en/products/lab-solutions.html). Der DNA-Microarray trägt Sonden für GBS-spezifische Virulenzfaktoren und Oberflächenmarker. Für die auf dem Microarray basierende Typisierung wurden 11 über das ganze Genom verteilte Gene bzw. Gencluster (bac, alp, pil1 locus, pepS8, fBsB, capsule locus, hylB, abiG-I/-II plus Q8DZ34, pil2 locus, nss plus srr plus rogB2 und rgfC/A/D/B) ausgewählt. Ubiquitär vorkommende, konservierte Gene (z. B. cylD/cylE) eignen sich nicht als Marker für eine Typisierung, wurden aber als Kontrollen und zur Normierung eingeschlossen. Zur vollständigen Charakterisierung wurden außerdem Sonden für hochmobile plasmidgebundene Resistenzgene wie z. B. erm(A), erm(B), erm(C), tet(M), emrB/qacA aufgetragen (Aracil, et al., 2002; Betriu, et al., 2003; Uh, et al., 2001). Diese Gene sind nicht für GBS spezifisch. Sie eignen sich z. B. für eine Unterscheidung einzelner Isolate, nicht jedoch für die Unterteilung der GBS Population in verschiedene Stämme. Für die vorliegende Arbeit wurden insgesamt 448 klinische Isolate von GBS ausgewählt und untersucht. Darunter waren Isolate, die schwerwiegende Erkrankungen wie Sepsis und Meningitis verursacht haben, Isolate aus lokalen Infektionen sowie Isolate von asymptomatischen/gesunden Trägern. Zu Vergleichszwecken wurden außerdem Isolate aus der Veterinärmedizin (von bovinen Mastitisfällen) und humane Isolate aus einer geographisch weit entfernten Region (Trinidad und Tobago) genotypisiert. Für 36 ausgewählte Isolate mit repräsentativen Hybridisierungsmustern wurde zusätzlich eine Typisierung mittels Multilocus-Sequenztypisierung (MLST) (Jones, et al., 2003) durchgeführt. Die Hybridisierungsmuster vom Microarray wurden mit Daten aus diesem bereits etablierten Typisierungssystem verbunden. Durch die Verknüpfung beider Methoden konnte eine Einteilung der GBS Isolate in verschiedene Stämme vorgenommen werden. Mit Hilfe des eBURST-Algorithmus wurde gezeigt, dass einige Hybridisierungsmuster sich zu Gruppen zusammenfügen. Dieses Verfahren veranschaulicht die Populationsstruktur und beschreibt die genetische Vielfalt. Mit den 11 definierten Markern konnten die untersuchten Isolate 76 verschiedenen Stämmen bzw. „hybridization profiles“ (HP) zugeordnet werden. Die Einteilung beruht auf dem Fehlen bzw. Vorhandensein einzelner Gene/Gencluster bzw. deren allelischen Varianten. Diese Stämme korrelieren mit den durch MLST definierten klonalen Komplexen (CC). Isolate mit identischen oder ähnlichen Hybridisierungsprofilen gehören zum selben CC. Dagegen können Isolate mit einem ähnlichen MLST-Profil verschiedene Hybridisierungsmuster zeigen. Es konnte außerdem häufig beobachtet werden, dass ansonsten ähnliche Stämme sich in einzelnen Merkmalen, z. B. Kapsel-Genen, alp- oder pili-Genen, voneinander unterscheiden, und dass diese Gene unabhängig voneinander variieren. Zusätzlich zeigten einige ubiquitäre Gene/Gencluster, die sich in den publizierten Genomsequenzen immer an derselben Position befinden, zahlreiche verschiedene Allele. Welches Allel in einem gegebenen Stamm gerade vorliegt, scheint dabei eher zufällig zu sein. Eine Erklärung dieses Phänomens könnte in vergangenen Rekombinationsereignissen liegen. Auch eine konvergente Evolution könnte diskutiert werden. Ähnliche Stämme/ „hybridization profiles“ wurden in Analogie zu den MLST-definierten klonalen Komplexen zu Gruppen zusammengefügt. Das bedeutet jedoch nicht notwendigerweise eine direkte Verwandtschaft der Isolate im Sinne des Vorhandenseins eines unmittelbaren gemeinsamen Vorfahren. Die Typisierung sowohl über den DNA-Microarray als auch über die MLST kann nicht die „wahre“ Phylogenese im Rahmen der Evolutionsgeschichte und Herkunft widerspiegeln. Sie stellt lediglich ein zufälliges Modell dar, ein Ordnungssystem im Sinne eines genetischen Fingerabdrucks, das einen Vergleich von Isolaten, aber keine Rückschlüsse über deren Abstammung und Herkunft erlaubt.Die untersuchten GBS Isolate konnten in fünf klonale Komplexe (CC19, CC23, CC26, CC103, CC130) eingeteilt werden, deren Häufigkeit unterschiedlich war. Deutsche humanmedizinische Isolate konnten vorwiegend CC19 zugeordnet werden. Karibische humanmedizinische Isolate sind zumeist CC19 und CC23 zugehörig. Bovine Isolate gehören meist zu CC19 und CC103. Unter humanen Isolaten ist CC103 rar. Vermutlich basierend auf der geografischen und wirtsspezifischen Herkunft der untersuchten GBS Isolate gibt es Unterschiede in der Populationsstruktur. In der vorliegenden Arbeit war CC19 der am häufigsten gefundene und außerdem ein genetisch besonders inhomogener CC. Er besteht aus mehreren unterschiedlichen, bisher als eigenständig angesehenen CCs (darunter CC1, CC17, CC19 und CC22). Diese werden von dem zur MLST-Verwandtschaftsanalysen verwendeten eBURST-Algorithmus zu CC19 zusammengefasst, seit die MLST-Profile von 'missing links' zwischen den CCs identifiziert wurden, da eBURST „gemeinsame Vorfahren“ nicht von durch horizontalem Gentransfer bzw. durch Hybridisierungen entstandenen „Chimären“ unterscheiden kann. Da diese Komplexe klar unterscheidbare Hybridisierungsmuster aufweisen, wurden sie hier als CC19/01, CC19/17, CC19/19 und CC19/22 bezeichnet. Einzelne Gene traten in Gruppen von Isolaten aus verschiedener Herkunft unterschiedlich häufig auf. So fand sich der Virulenzfaktor scpB in 412 von 418 humanen Isolaten (98,6 %), aber nur in 10 von 21 Rinderisolaten (48 %). Ferner ließ sich beobachten, dass invasive Isolate weniger wahrscheinlich abiGI-/II und Q8DZ34 tragen, jedoch häufiger pil1 locus, fbsB (515) und Kapseltyp III sowie pil2b, nss/srr und rgf (COH1 like) aufweisen. Einige dieser Marker erscheinen zusammen in CC19/17-Stämmen, welche häufig bei invasiven Krankheitsverläufen beobachtet werden. CC19 (incl. ST01, ST17, ST19) konnte bei neonatalen Sepsis-Fällen in verschiedenen geografischen Regionen isoliert werden (Brzychczy-Wloch, et al., 2012; Ryu, et al., 2014; Sorensen, et al., 2010; Strakova, et al., 2010; Tien, et al., 2011). Zusätzlich wurden andere Virulenzfaktoren wie speM (Exotoxin M) und das cyl-Operon (beta-Hämolysin) untersucht. In lediglich sieben Isolaten wurde speM nachgewiesen. Das cyl-Operon konnte in allen Isolaten gefunden werden, sein Nachweis ist daher für eine Vorhersage der Virulenz eines gegebenen Isolates nicht hilfreich. Es konnte kein einzelner Faktor zur definitiven Unterscheidung zwischen invasiven Isolaten und Trägerisolaten bestimmt werden. Für die Virulenz eines Isolates ist wahrscheinlich nicht das bloße Vorhandensein oder Fehlen eines bestimmten Genes ausschlaggebend, sondern dessen Expression in vivo. Wichtig wäre in diesem Zusammenhang auch die genaue Betrachtung der Sequenz eines als Virulenzfaktor angesehenen Genes sowie die Untersuchung der zugehörigen regulatorischen Gene. Über den Nachweis der Gene erm(A), erm(B) und erm(C) konnte eine Aussage über die Macrolid-/Clindamycinresistenz eines GBS Isolates getroffen werden. Bei keinem der karibischen Isolate wurden erm Gene nachgewiesen. Innerhalb der deutschen GBS Population wurde erm(B) am häufigsten beobachtet. Die Gene erm(A) und erm(C) waren in humanen Isolaten selten und wurden in bovinen Isolaten überhaupt nicht gefunden. Das Tetracyclinresistenzgen tet(M) wurde häufig in humanen Isolaten und sehr selten in veterinärmedizinischen Isolaten gefunden. Für weiterführende Untersuchungen könnte die beschriebene Typisierungsmethode verfeinert werden. So lassen sich z. B. die oben beschriebenen 11 ausgewählten Typisierungsmarker des Microarrays mit denen der MLST zu einem 18 Marker-System verknüpfen. Daneben können auch erm-, cad-, mer- oder tet-Gene zur Feststellung oder zum Ausschluss der Identität verwandter Isolate in vitro oder in silico verwendet werden. Mit der nun einsatzbereiten Genotypisierungsmethode können in Zukunft weitere Studien zur Untersuchung regionaler und wirtsspezifischer Unterschiede der GBS Population durchgeführt werden. In dieser Arbeit konnte gezeigt werden, dass der DNA-Microarray stabile und reproduzierbare Resultate erbringt. Es kann ein detaillierter Befund erstellt werden, die Ergebnisse sind mit denen anderer Typisierungsmethoden und der Genomsequenzierung vergleichbar. Jedoch steht mit dem DNA-Microarray ein wesentlich unkomplizierteres und schnelleres Procedere zur Verfügung, welches zudem geringere Kosten verursacht.:1. Einleitung 5 1.1 Gegenstand der Untersuchung 5 1.2 Entdeckungsgeschichte 6 1.3 Klinische Bedeutung 7 1.4 Veterinärmedizinische Bedeutung 9 1.5 Stand der Forschung mit Hinblick auf Typisierung von GBS 9 1.6 Ziele der vorliegenden Untersuchung 10 1.7 Vorgehensweise 11 1.7.1 Untersuchungsmaterial 11 1.7.2 Untersuchungsmethode 11 1.8 Zur Typisierung ausgewählte Marker 12 2. DNA Microarray-Based Typing of Streptococcus agalactiae Isolates 15 2.1 Abstract 16 2.2 Introduction 17 2.3 Materials and methods 18 2.3.1 Bacterial isolates 18 2.3.2 Ethics statement 18 2.3.3 Preparation of genomic DNA 19 2.3.4 MLST 19 2.3.5 Microarray design and protocol optimization 19 2.3.6 Microarray procedures 20 2.3.7 eBURST 21 2.4 Results 22 2.4.1 Typing GBS by MLST 22 2.4.2 Genotyping GBS by microarray hybridization 22 2.4.3 Population structure 25 2.4.4 Detection of antibiotic resistance markers 25 2.4.5 Detection of heavy metal resistance markers 26 2.5 Discussion 27 2.6 Acknowledgments 30 2.7 References 31 2.8 Tables and figures 33 3. Zusammenfassung 45 3.1 Zusammenfassung 45 3.2 Summary 49 4. Korrespondenz mit dem Editor 53 4.1 Hinweise des Editors und der Gutachter 53 4.2 Antworten an den Editor 56 4.3 Endgültige Annahme 58 Anhang 61 / Streptococcus (S.) agalactiae are Gram-positive, chain-forming cocci, which show hemolysis on blood agar. They are also referred to group B streptococci (GBS) because of their affiliation to Lancefield-group B (Hof, 2005). GBS are the main cause of sepsis, meningitis and pneumonia in newborns (Schrag, et al., 2000). The work focused on genotyping of GBS. The aim was to develop a DNA microarray and to use it for epidemiological typing of GBS. During the evaluation of the microarray, strain-specific patterns could be observed and these patterns assigned to genotypes as defined by other typing methods (MLST). The results were summarized in a database that subsequently was developed into software for automated analysis of experiments (http://alere-technologies.com/en/products/lab-solutions.html). The DNA microarray carries probes for GBS specific virulence factors and surface markers. For the microarray-based typing, 11 genes or gene clusters were selected that are distributed across the entire genome (bac, alp, pil1 locus, pepS8, fBsB, capsule locus, hylB, abiG-I/-II plus Q8DZ34, pil2 locus, nss plus srr plus rogB2 and rgfC/A/D/B). Ubiquitous, conserved genes (e.g. cylD/cylE) were included to be used as species markers and controls. Furthermore, probes for highly mobile plasmid-borne resistance genes such as erm(A), erm(B), erm(C), tet(M), emrB/qacA were also included (Aracil, et al., 2002; Betriu, et al., 2003; Uh, et al., 2001). These genes are not specific to GBS, but they are found in some isolates. They can be used to distinguish individual, related isolates, rather than for a definition of distinct strains. A total of 448 isolates of GBS was selected and examined for the present work. Among them were isolates from severe diseases, such as sepsis and meningitis, isolates from local infections as well as isolates from asymptomatic/healthy carriers. For comparison, isolates from veterinary medicine (from cases of bovine mastitis) and human isolates from a geographically distant region (Trinidad and Tobago) were genotyped. For 36 selected isolates with representative hybridization patterns, parallel typing was performed using a second method, multilocus sequence typing (MLST) (Jones, et al., 2003). Hybridization patterns on the Microarray could thus be linked to this already established typing system. With the array based GBS typing isolates could be divided into 76 different strains or 'hybridization profiles', HP. The classification with both methods is based on the absence or presence of individual genes or gene clusters or their allelic variants. Similar isolates were lumped together. The eBURST algorithm was used to group strain-specific patterns into groups of related strains illustrating the population structure and describing the genetic diversity. Groups of similar hybridization patterns largely correlate with the clonal complexes (CC) defined by MLST. While isolates with identical or similar hybridization profiles belong to the same CC, isolates with a similar MLST profile can show different hybridization patterns. It has also often been observed that otherwise similar strains differ from each other in individual traits, e.g. capsule genes, alp or pili genes, and that these genes vary independently of one another. In addition, some ubiquitous genes/gene clusters, which are always localized at the same position in the published genomic sequences, show numerous different alleles and related strains (that belong to one clonal complex) might differ in the presence of one allele. Alleles are thus not linked to clonal complexes, but rather randomly distributed. An explanation of this phenomenon could be a frequent occurrence of recombination events or horizontal gene transfers. A convergent evolution could also be discussed as an alternative explanation. A similarity of hybridization profiles does not necessarily mean a direct phylogenetic relationship between the isolates in the sense of being derived from a direct common ancestor. Typing both the DNA microarray and the MLST cannot reflect the 'true' phylogenesis, evolutionary history and origin. Assuming frequent recombination i.e., random events, the MLST profiles as well as the hybridization patterns can be used as genetic fingerprints, allowing a comparison of isolates, but no conclusions about their phylogeny and origin. The investigated GBS isolates were classified into five clonal complexes (CC19, CC23, CC26, CC103, CC130) with very different relative abundances indicating differences in the population structure with regard to geographic origin and host organisms. German medical isolates were mainly assigned CC19. Caribbean medical isolates mostly were assigned to CC19 and CC23. Bovine isolates usually belonged to CC19 and CC103. Among human isolates, CC103 was rare. In the present work, CC19 was the most abundant and the genetically most inhomogeneous CC. Several different clusters that previously been regarded as CCs (CC1, CC17, CC19 and CC22) have recently been merged to CC19 by the eBURST algorithm since MLST profiles of missing links between the CCs have been identified. Unfortunately, eBURST cannot distinguish whether two MLST types are linked by true common ancestors or by hybrid or chimera strains originating from horizontal gene transfers or hybridization events. Since these complexes within CC19 have clearly distinguishable hybridization patterns, they have been referred to herein as CC19/01, CC19/17, CC19/19 and CC19/22, and we assume that they are linked by hybridizations or gene transfers rather than by shared ancestry. Few differences were found between isolates from different origins. The virulence factor scpB was found in 412 of 418 human isolates (98.6%), but only in 10 of 21 bovine isolates (48%). Furthermore, it was observed that invasive isolates are less likely to carry abiGI-/II and Q8DZ34, but are more likely to have pil1 locus, fbsB (515) and capsule type III as well as pil2b, nss/srr and rgf (COH1 like). Some of these markers appear together in CC19/17 strains, which are often observed in invasive disease. speM (exotoxin M) was also investigated. It was detected only in seven isolates. Contrarily, the cyl (beta-hemolysin) operon was found in all isolates. Thus, it detection is not helpful for a prediction of the virulence of a given isolate. No single factor could be identified that allowed a definitive distinction between invasive isolates and carrier isolates. Probably, the virulence of an isolate does not depend on the presence or absence of one particular gene. In this context, it would be important to investigate the expression in vivo of the various putative virulence factors as well as the allelic variants of the factors and of their associated regulatory genes. Macrolide-/clindamycin resistance genes erm(A), erm(B) and erm(C) can also be detected by the microarray. None of these genes was identified in any of the Caribbean isolates. Within the German GBS population, erm(B) was most frequently observed. The genes erm(A) and erm(C) were rare in human isolates, and they were not found in bovine isolates. The tetracycline resistance gene tet(M) was observed frequently in human isolates but only very rarely in veterinary isolates. With the genotyping method that was developed during the present work, further studies can be carried out to study regional and host-specific differences in the GBS population. For future investigations, the described typing method could further be refined. For example, the 11 selected typing markers on the microarray can be combined with those from MLST to one comprehensive marker system. In addition, it is also possible to use genes on mobile genetic elements such as resistance genes (erm, cad, mer or tet) to prove or to rule out the identity of related isolates in vitro or in silico. In our study, it was shown that the DNA microarray provides stable and reproducible results that are comparable to those of other typing methods and genome sequencing. However, since the DNA microarray offers a much more uncomplicated and faster procedure, which also results in lower costs, it is more suitable to a routine setting.:1. Einleitung 5 1.1 Gegenstand der Untersuchung 5 1.2 Entdeckungsgeschichte 6 1.3 Klinische Bedeutung 7 1.4 Veterinärmedizinische Bedeutung 9 1.5 Stand der Forschung mit Hinblick auf Typisierung von GBS 9 1.6 Ziele der vorliegenden Untersuchung 10 1.7 Vorgehensweise 11 1.7.1 Untersuchungsmaterial 11 1.7.2 Untersuchungsmethode 11 1.8 Zur Typisierung ausgewählte Marker 12 2. DNA Microarray-Based Typing of Streptococcus agalactiae Isolates 15 2.1 Abstract 16 2.2 Introduction 17 2.3 Materials and methods 18 2.3.1 Bacterial isolates 18 2.3.2 Ethics statement 18 2.3.3 Preparation of genomic DNA 19 2.3.4 MLST 19 2.3.5 Microarray design and protocol optimization 19 2.3.6 Microarray procedures 20 2.3.7 eBURST 21 2.4 Results 22 2.4.1 Typing GBS by MLST 22 2.4.2 Genotyping GBS by microarray hybridization 22 2.4.3 Population structure 25 2.4.4 Detection of antibiotic resistance markers 25 2.4.5 Detection of heavy metal resistance markers 26 2.5 Discussion 27 2.6 Acknowledgments 30 2.7 References 31 2.8 Tables and figures 33 3. Zusammenfassung 45 3.1 Zusammenfassung 45 3.2 Summary 49 4. Korrespondenz mit dem Editor 53 4.1 Hinweise des Editors und der Gutachter 53 4.2 Antworten an den Editor 56 4.3 Endgültige Annahme 58 Anhang 61

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched. / Health Studies / M.Sc. (Life Sciences (Microbiology))

Le Streptocoque de groupe B (GBS) est un important agent d’infection invasive pouvant mener à la mort et demeure la cause principale de septicémie néonatale à ce jour. Neuf sérotypes ont été officiellement décrits basés sur la composition de la capsule polysaccharidique (CPS). Parmi ces sérotypes, le type III est considéré le plus virulent et fréquemment associé aux maladies invasives graves, telle que la méningite. Malgré que plusieurs recherches aient été effectuées au niveau des interactions entre GBS type III et les cellules du système immunitaire innées, aucune information n’est disponible sur la régulation de la réponse immunitaire adaptative dirigée contre ce dernier. Notamment, le rôle de cellules T CD4+ dans l’immuno-pathogenèse de l’infection causée par GBS n’a jamais été étudié. Dans cet étude, trois différents modèles murins d’infection ont été développé pour évaluer l’activation et la modulation des cellules T CD4+ répondantes au GBS de type III : ex vivo, in vivo, et in vitro. Les résultats d’infections ex vivo démontrent que les splénocytes totaux répondent à l’infection en produisant des cytokines de type-1 pro-inflammatoires. Une forte production d’IL-10 accompagne cette cascade inflammatoire, probablement dans l’effort de l’hôte de maintenir l’homéostasie. Les résultats démontrent aussi que les cellules T sont activement recrutées par les cellules répondantes du système inné en produisant des facteurs chimiotactiques, tels que CXCL9, CXCL10, et CCL3. Plus spécifiquement, les résultats obtenus à partir des cellules isolées T CD4+ provenant des infections ex vivo ou in vivo démontrent que ces cellules participent à la production d’IFN-γ et de TNF-α ainsi que d’IL-2, suggérant un profil d’activation Th1. Les cellules isolées T CD4+ n’étaient pas des contributeurs majeurs d’IL-10. Ceci indique que cette cytokine immuno-régulatrice est principalement produite par les cellules de l’immunité innée de la rate de souris infectées. Le profil Th1 des cellules T CD4+ a été confirmé en utilisant un modèle in vitro. Nos résultats démontrent aussi que la CPS de GBS a une role immuno-modulateur dans le développement de la réponse Th1. En résumé, cette étude adresse pour la première fois, la contribution des cellules T CD4+ dans la production d’IFN-γ lors d’une infection à GBS et donc, dans le développement d’une réponse de type Th1. Ces résultats renforcent d’avantage le rôle central de cette cytokine pour un control efficace des infections causées par ce pathogène. / Group B Streptococcus (GBS) is an important agent of life-threatening invasive infections and remains the leading cause of neonatal sepsis to this day. Nine serotypes have been officially described based on capsular polysaccharide (CPS) composition. Among them, capsular type III is considered one of the most virulent and frequently associated with severe invasive diseases, such as meningitis. Although extensive research has been done on the interactions between GBS type III and various cells of the innate immune system, no information is available on the regulation of the adaptive immune response against this pathogen. In particular, the role of CD4+ T cells in the immuno-pathogenesis of the infection caused by GBS has never been assessed. In this study, three different models of murine infection were developed to evaluate activation and modulation of responding CD4+ T cells against GBS type III: ex vivo, in vivo, and in vitro. Ex vivo analysis of total splenocytes showed that GBS induces the release of type-1 pro-inflammatory cytokines. A strong IL-10 production follows this inflammatory cascade, indicating the host effort to maintain homeostasis. Results also indicate that T cells were actively recruited by responding innate immune cells via the release of chemotactic factors such as CXCL9, CXCL10, and CCL3. More specifically, results obtained from isolated CD4+ T cells from ex vivo or in vivo infections showed that they actively participate in the production of IFN-γ and TNF-α, as well as IL-2, suggesting a Th1 profile of activation. On the other hand, isolated CD4+ T cells were not main sources of IL-10. This observation suggests that this immuno-regulatory cytokine is produced mainly by cells of the spleen innate immune system of infected animals. The CD4+ Th1 cell profile was confirmed using an in vitro model of infection. Our results also suggest that the GBS CPS plays an immuno-modulatory role in the development of a Th1 response. In summary, this study addresses for this first time the contribution of CD4+ T cells in IFN-γ production during GBS infection, and thus, in the development of a Th1 response. Our data further highlight the central role of this cytokine for effective control of GBS infections.

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched. / Health Studies / M. Sc. (Life Sciences (Microbiology))