Philosophiae Doctor - PhD / The aim of the study was to detect the prevalence of AV and its associated bacteria with preterm delivery in the Western Cape, South Africa. Furthermore, it sought particularly to examine and investigate the predictive value of GBS and E. faecalis for preterm delivery (PTD). It also aimed to establish other factors which may predict adverse pregnancy outcomes. Three hundred and one pregnant women were recruited from four different antenatal in the Western Cape, South Africa. The study conformed with the Declaration of Helsinki (2013). Maternal data was collected from a questionnaire and maternal medical records. Vaginal and rectal swabs were collected and microscopically examined for AV, followed by culture characterization of GBS and E. faecalis. Antimicrobial susceptibility testing was also performed. In this study, AV was detected in 79 (26.2%) of the 301 pregnant women, and GBS and E. faecalis isolated from 50 (16.6%) and 21 (7.0%) respectively. GBS serotype V was the predominant serotype, followed by serotype III. Pulse field gel electrophoresis (PFGE) profile analysis for both GBS and E. faecalis yielded a total of 24 restrictions profiles for GBS and 16 for E. faecalis. Multivariable analysis revealed that parity, gravidity, vaginal discharge, urinary tract infection, and smoking were significantly associated with PTD. The results from the study provides improved guidelines maternal screening of pregnant women. The early detection of AV-related bacteria may significantly reduce maternal and neonatal morbidity.

Les bactéries des microbiotes vivent avec l'homme une interaction mutualiste et tout facteur perturbant l'une ou l'autre des parties peut rompre l'équilibre établi engendrant diverses modifications des interactions. Les staphylocoques sont des bactéries opportunistes prévalentes et diverses au sein du microbiote des enfants. Deux modèles complémentaires et d'intérêt clinique majeur ont été utilisés dans le but de 1) décrire la dynamique d'implantation du microbiote digestif chez le très grand prématuré et la transmission de bactéries pathogènes dont les staphylocoques à coagulase négative par voie digestive via le lait maternel, 2) rechercher et caractériser des bactéries opportunistes présentant une résistance aux antibiotiques émergente dans une niche écologique complexe en prenant l'exemple de Staphylococcus aureus de sensibilité diminuée aux glycopeptides (GISA/hGISA) dans les voies respiratoires au cours de la mucoviscidose.Staphylococcus epidermidis est l'espèce prédominante dans les selles des nouveau-nés prématurés. Le lait maternel même pasteurisé est porteur de staphylocoques à coagulase négative. Au niveau des populations, un répertoire d'espèces similaire est retrouvé dans le lait cru, le lait pasteurisé, les selles et le sang. L'étude des cas montre une clonalité entre des souches du lait avant pasteurisation, des selles et du sang. La transmission du streptocoque du groupe B par le biais du lait maternel lors des infections tardives est un exemple de l'hypothèse physiopathologique proposée.Cinq cas de souches S. aureus GISA/hGISA dans le microbiote respiratoire des sujets atteints de mucoviscidose sont décrits. Ces résistances retrouvées chez 4,7% des patients atteints de mucoviscidose et colonisés/infectés par S. aureus ont des caractéristiques atypiques, 3 souches sur 8 étant méticillinosensibles. De plus, les facteurs de risque d'acquisition et de sélection classiquement décrits pour S. aureus GISA/hGISA ne sont pas systématiquement présents, suggérant un rôle particulier de la niche pathologique constituée par les voies respiratoires des patients atteints de mucoviscidose dans l'émergence de ces souches.La dynamique de transmission et d'émergence de pathogènes opportunistes au sein des microbiotes est un élément de connaissance considérable pour améliorer la prévention des infections et anticiper de la prise en charge chez de patients de plus en plus fragiles. / Staphylococci are prevalent opportunistic bacteria in children' microbiota. Two models were used in order to 1) describe the dynamic of colonization of the digestive microbiota in premature neonate and the transmission of pathogenic bacteria as coagulase negative staphylococci from the gastrointestinal tract via breast milk, 2) search and characterize opportunistic bacteria with emerging antibiotic resistance in a complex microbiota with the example of Staphylococcus aureus with reduced susceptibility to glycopeptides in respiratory tract in cystic fibrosis.

Heterotrimeric Gi proteins play a role in signalling activated by lipopolysaccharide (LPS), Staphylococcus aureus (SA) and group B streptococci (GBS), leading to production of inflammatory mediators. We hypothesized that genetic deletion of Gi proteins would alter cytokine and chemokine production induced by LPS, SA and GBS stimulation. LPS-induced, heat-killed SA-induced and heat-killed GBS-induced cytokine and chemokine production in peritoneal macrophages from wild-type (WT), Gαi2-/- or Gαi1/3-/- mice were investigated. LPS induced production of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10 and interferon-γ-inducible protein-10 (IP-10); SA induced TNF-α, and IL-1β production; and GBS induced TNF-α, IL-6, IL-1β, macrophage inflammatory protein-1α (MIP-1α) and keratinocyte chemoattract (KC) production were all decreased (P < 0.05) in Gαi2-/- or Gαi1/3-/- mice compared with WT mice. In contrast to the role of Gi proteins as a positive regulator of mediators, LPS-induced production of MIP-1α and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in macrophages from Gαi1/3-/- mice, and SA-induced MIP-1α production was increased in both groups of Gαi protein-depleted mice. LPS-induced production of KC and IL-1β, SA-induced production of GM-CSF, KC and IP-10, and GBS-induced production of IL-10, GM-CSF and IP-10 were unchanged in macrophages from Gαi2-/- or Gαi1/3-/- mice compared with WT mice. These data suggest that Gi2 and Gi1/3 proteins are both involved and differentially regulate murine inflammatory cytokine and chemokine production in response to both LPS and Gram-positive microbial stimuli.

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 / 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.

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

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Estreptococos do grupo B (EGB) é a principal causa de sepse e meningite neonatal e tem sido recentemente reconhecido como patógeno responsável por infecções invasivas em adultos imunocomprometidos (idosos ou portadores de doenças crônicas). Os EGB produzem inúmeras enzimas extracelulares, várias das quais interagem com o sistema imune do hospedeiro e são importantes durante a interação EGB-hospedeiro, bem como para o desenvolvimento da doença. Estudos anteriores mostraram que metaloproteases estão envolvidas em várias vias metabólicas em diferentes tipos celulares. Por esta razão, nós decidimos investigar o possível envolvimento de metaloproteases de EGB durante a interação celular e apoptose/necrose induzida pelo micro-organismo em células endoteliais da veia umbilical humana (HUVEC) e da linhagem de epitélio respiratório (A549). Tratamento de EGB com inibidores de metaloproteases (EDTA, EGTA e FEN) não induziu alterações no crescimento bacteriano, mas promoveu alterações na expressão de proteínas de superfície, capacidade adesiva e perfil de sobrevivência intracelular do patógeno. O EGB e o sobrenadante do crescimento bacteriano (meio condicionado; MC) promoveram a morte das células HUVEC e A549. Contudo, o tratamento com inibidores de metaloproteases restauraram a viabilidade celular induzida pelos EGB e o MC, sugerindo que metaloproteases bacteriana estão envolvidas no rompimento da barreira celular, promovendo a disseminação bacteriana. Este trabalho descreve pela primeira vez apoptose e necrose induzidas pelo EGB e MC em HUVEC e células A549 após 24h de incubação, respectivamente. Nós também observamos redução da pró-caspase-3 após infecção das HUVEC com EGB e MC, sugerindo ativação da caspase-3. Além disso, o aumento da expressão da proteína pró-apoptótica Bax e diminuição dos níveis da proteína anti-apoptótica Bcl-2 em HUVEC, demonstram o envolvimento do mecanismo apoptótico mitocondrial (via intrínseca). A melhor compreensão das bases moleculares da patogênese do EGB contribui para identificar novas moléculas bacterianas e hospedeiras que podem representar novos alvos terapêuticos ou imunoprofiláticos contra a doença causada por esse patógeno neonatal. / Group B streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis and has recently been recognized as an increasingly common cause of invasive disease in immunocompromised adults (elderly or chronic diseases). GBS produces a number of extracellular enzymes, several of which interact with the host immune system and are important for the GBS- host interaction and for the development of disease. Previous studies showed that metalloproteases are involved in several metabolic pathways in different cellular types. For this reason, we decided to investigate the possible involvement of GBS metalloproteases during cell interaction and apoptosis/necrosis induced by microorganism in human umbilical vein endothelial cells (HUVEC) and epithelial respiratory cells line (A549). Treatment of GBS with metalloproteases inhibitors (EDTA, EGTA and PHEN) did not induce alteration on bacterial growth, but promoted changes in the expression of surface proteins, adhesive capacity and profile of intracellular survival of the pathogen. The GBS and supernatant of bacterial growth medium (conditioned medium; MC) promoted the death of HUVEC and A549 cells. However, the metalloproteases inhibitors treatment restored the cellular viability induced by GBS and MC, suggesting that GBS metalloproteases are involved in the disruption of cell barrier, promoting bacterial dissemination. This study describes for the first time apoptosis and necrosis induced by GBS and MC in HUVEC and A549 cells after 24h incubation, respectively. We also observe reduction of pro-caspase-3 after infection of HUVEC with GBS and MC, suggesting activation of caspase-3. Moreover, the over-expression of pro -apoptotic protein Bax and decrease of anti-apoptotic protein Bcl-2 levels in HUVEC show the involvement of mitochondrial apoptotic mechanism (intrinsic via). Enhanced understanding of the molecular basis of GBS pathogenesis may pinpoint novel bacterial and host molecules that can represent novel therapeutic or immunoprophylactic targets against disease caused by this foremost of neonatal pathogens.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Estreptococos do grupo B (EGB) é a principal causa de sepse e meningite neonatal e tem sido recentemente reconhecido como patógeno responsável por infecções invasivas em adultos imunocomprometidos (idosos ou portadores de doenças crônicas). Os EGB produzem inúmeras enzimas extracelulares, várias das quais interagem com o sistema imune do hospedeiro e são importantes durante a interação EGB-hospedeiro, bem como para o desenvolvimento da doença. Estudos anteriores mostraram que metaloproteases estão envolvidas em várias vias metabólicas em diferentes tipos celulares. Por esta razão, nós decidimos investigar o possível envolvimento de metaloproteases de EGB durante a interação celular e apoptose/necrose induzida pelo micro-organismo em células endoteliais da veia umbilical humana (HUVEC) e da linhagem de epitélio respiratório (A549). Tratamento de EGB com inibidores de metaloproteases (EDTA, EGTA e FEN) não induziu alterações no crescimento bacteriano, mas promoveu alterações na expressão de proteínas de superfície, capacidade adesiva e perfil de sobrevivência intracelular do patógeno. O EGB e o sobrenadante do crescimento bacteriano (meio condicionado; MC) promoveram a morte das células HUVEC e A549. Contudo, o tratamento com inibidores de metaloproteases restauraram a viabilidade celular induzida pelos EGB e o MC, sugerindo que metaloproteases bacteriana estão envolvidas no rompimento da barreira celular, promovendo a disseminação bacteriana. Este trabalho descreve pela primeira vez apoptose e necrose induzidas pelo EGB e MC em HUVEC e células A549 após 24h de incubação, respectivamente. Nós também observamos redução da pró-caspase-3 após infecção das HUVEC com EGB e MC, sugerindo ativação da caspase-3. Além disso, o aumento da expressão da proteína pró-apoptótica Bax e diminuição dos níveis da proteína anti-apoptótica Bcl-2 em HUVEC, demonstram o envolvimento do mecanismo apoptótico mitocondrial (via intrínseca). A melhor compreensão das bases moleculares da patogênese do EGB contribui para identificar novas moléculas bacterianas e hospedeiras que podem representar novos alvos terapêuticos ou imunoprofiláticos contra a doença causada por esse patógeno neonatal. / Group B streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis and has recently been recognized as an increasingly common cause of invasive disease in immunocompromised adults (elderly or chronic diseases). GBS produces a number of extracellular enzymes, several of which interact with the host immune system and are important for the GBS- host interaction and for the development of disease. Previous studies showed that metalloproteases are involved in several metabolic pathways in different cellular types. For this reason, we decided to investigate the possible involvement of GBS metalloproteases during cell interaction and apoptosis/necrosis induced by microorganism in human umbilical vein endothelial cells (HUVEC) and epithelial respiratory cells line (A549). Treatment of GBS with metalloproteases inhibitors (EDTA, EGTA and PHEN) did not induce alteration on bacterial growth, but promoted changes in the expression of surface proteins, adhesive capacity and profile of intracellular survival of the pathogen. The GBS and supernatant of bacterial growth medium (conditioned medium; MC) promoted the death of HUVEC and A549 cells. However, the metalloproteases inhibitors treatment restored the cellular viability induced by GBS and MC, suggesting that GBS metalloproteases are involved in the disruption of cell barrier, promoting bacterial dissemination. This study describes for the first time apoptosis and necrosis induced by GBS and MC in HUVEC and A549 cells after 24h incubation, respectively. We also observe reduction of pro-caspase-3 after infection of HUVEC with GBS and MC, suggesting activation of caspase-3. Moreover, the over-expression of pro -apoptotic protein Bax and decrease of anti-apoptotic protein Bcl-2 levels in HUVEC show the involvement of mitochondrial apoptotic mechanism (intrinsic via). Enhanced understanding of the molecular basis of GBS pathogenesis may pinpoint novel bacterial and host molecules that can represent novel therapeutic or immunoprophylactic targets against disease caused by this foremost of neonatal pathogens.