Streptococcus pneumoniae is one of the leading causes of morbidity and mortality in children under 5. Low-income countries are disproportionally affected and data in these settings are lacking. Effective strategies to control disease include infant immunisation with pneumococcal protein-polysaccharide conjugate vaccines. However, ongoing surveillance of carriage and disease are important to understand the impact of vaccination within communities. This thesis evaluated nasopharyngeal (NP) carriage in 3 generations, following introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in the UK. NP carriage was also compared between rural and urban Nepalese children and a novel method of delayed culture and transport was assessed. Finally, the immunogenicity of a 10-valent pneumococcal conjugate vaccine administered in a 2-dose priming schedule without a booster was compared to a 3-dose priming schedule with a booster in Nepalese infants. Key findings include carriage rates in UK children being similar to pre-PCV7 (7 valent pneumococcal conjugate vaccine) carriage rates at 47% with low carriage rates in seen in adults. PCV7 serotypes accounted for 1.5% of carriage isolates in children, 0% in parents and 15.4% in older adults. In Nepalese children carriage was higher in a rural (69.2%) compared to an urban setting (40.9%) and delayed culture and transport using silica desiccant packets (SDP) provided a reliable, albeit underestimated, estimate of carriage. Finally this author demonstrated that following primary immunisation and boosting, there was no difference in immune responses to serotypes 1, 5 and 14 with a 2 dose priming schedule compared to a 3-dose schedule. At 2-4 years of age a significantly higher proportion of vaccinees in the 2+1 group had ≥0·2µg/mL IgG for serotypes 1, 5, 6B and 18C compared to vaccinees in the 3+0 group.

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Introducción: Determinar el riesgo cariogénico del paciente es un requisito fundamental para efectuar un adecuado diagnóstico de salud bucal. Establecer el recuento de Streptococcus mutans (S.mutans) sobre piezas dentarias puede permitir identificar el nivel de riesgo microbiológico en desarrollar caries, y en el caso de piezas restauradas, caries secundarias, principal causa de fallas de restauraciones, evitando el futuro recambio de ellas. Conocer la colonización de microbiota cariogénica en restauraciones dentarias y dientes sanos, podría ser un aspecto a considerar en las decisiones de tratamiento, posibilitando la selección de un material de obturación y medidas preventivas, ajustada con el riesgo cariogénico local y propio de cada paciente. Material y Método: Se seleccionaron 69 pacientes de la clínica de Operatoria 4to año de la Facultad de Odontología de la Universidad de Chile durante el período de septiembre a diciembre del año 2011. En cada uno de ellos se tomó una muestra de placa bacteriana dental de una pieza posterior sana y una restaurada por oclusal con resina compuesta utilizando la técnica de cubeta. Este método consiste en una impresión directa sobre las superficies oclusales de restauraciones, mediante una cubetilla de flúor gel modificada cargada con agar TYCSB. Las cubetas se incubaron en estufa a 37°C por 48 horas, para posteriormente proceder al recuento bacteriano. 2 Resultados: Mediante el método de la cubeta se logró aislar Unidades Formadoras de Colonias (UFC) de S. mutans en dientes con resina compuesta oclusal y en piezas sanas en un 95,6% de las muestras. Se observó una diferencia estadísticamente significativa (p <0,05) donde las muestras de biofilm de placa bacteriana depositada sobre las restauraciones de resina presentaban mayor cantidad de UFC/cm2 que las superficie de piezas sanas. Conclusiones: A partir de muestras de placa bacteriana dental obtenidas mediante la técnica de cubeta existen diferencias significativas en el recuento de S. mutans entre dientes con resina compuesta oclusal y dientes permanentes sanos, siendo mayor en las que presentaban resina compuesta.

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Introducción: La caries temprana de la infancia (CTI) es una enfermedad dietobacteriana, siendo Streptococcus mutans (S.mutans) el principal patógeno asociado. Afecta a niños menores de 6 años, generando destrucción y dolor, siendo necesario un tratamiento que controle los factores de riesgo relacionados y recupere el tejido dañado. En Chile, no existen estudios que evalúen el impacto de un tratamiento rehabilitador integral en la microbiota oral de niños con CTI. Objetivo: Determinar el efecto del tratamiento rehabilitador integral de CTI en los niveles de Streptococcus mutans en niños tratados en la Clínica de Odontopediatría de la Escuela de Graduados de la Universidad de Chile Materiales y Métodos: Estudio retrospectivo observacional. Se analizaron 89 fichas clínicas de pacientes diagnosticados con CTI rehabilitados integralmente en el posgrado de Odontopediatría de la Universidad de Chile, las cuales registraban el recuento de S.mutans salival antes de iniciar el tratamiento, después del tratamiento preventivo y después del tratamiento rehabilitador. Se analizaron las diferencias entre los niveles de S.mutans de manera cuantitativa y según categorías (riesgo bajo, moderado o alto), durante las distintas etapas del tratamiento. Se analizaron también otras variables como tipo de material restaurador, edad e índice ceod. Resultados: Durante el análisis cuantitativo, se encontraron diferencias estadísticamente significativas al comparar las concentraciones de S.mutans salival inicial con las concentraciones luego del tratamiento preventivo (4,7±1,03 log UFC/ml vs 4,2 ± 0,96 log UFC/ml p<0,05) y luego del tratamiento rehabilitador (4,7±1,03 log UFC/ml vs 3,74 ± 0,88 log UFC/ml p<0,05), así como al comparar las concentraciones después del tratamiento preventivo con las posteriores al tratamiento rehabilitador (4,2 ± 0,96 log UFC/ml vs 3,74 ± 0,88 log UFC/ml p<0,05). Durante el análisis por categorías, sólo se encontraron diferencias significativas entre las concentraciones previas y después de ambos tratamientos, sin encontrar diferencias entre ambos tratamientos. No hubo diferencias al analizar las variaciones según categorías con el tipo de material restaurador, edad ni índice ceod. Conclusión: El tratamiento rehabilitador integral produce una reducción significativa en la concentración de S.mutans salival tanto en la etapa preventiva como en la etapa rehabilitadora.

Streptococcus pneumoniae ist ein Gram-positives Bakterium und ein Kommensale des humanen Nasenrachenraums. Pneumokokken sind andererseits auch die Verursacher schwerer lokaler Infektionen wie der Otitis media, Sinusitis und von lebensbedrohenden invasiven Erkrankungen. So sind Pneumokokken die wichtigsten Erreger einer ambulant erworbenen Pneumonie und sie sind häufige Verursacher von Septikämien und bakteriellen Meningitiden. Die initiale Phase der Pathogenese ist verbunden mit der Besiedelung der mukosalen Epithelzellen des Rachenraumes. Diese Kolonisierung erleichtert die Aufnahme der Bakterien in die Zelle bzw. deren Dissemination in submukosale Bereiche und den Blutstrom. Die Konversion des Kommensalen zu einem invasiven Mikroorganismus ist assoziiert mit der Anpassung des Krankheitserregers an die verschiedenen Wirtsnischen und wird auf der Wirtsseite durch die Zerstörung der transepithelialen Barriere begleitet. Die Anpassung des Erregers ist vermutlich ein in hohem Grade regulierter Prozess. Die Oberfläche von Streptococcus pneumoniae ist mit Proteinen bedeckt, die kovalent oder nicht kovalent mit der Zellwand verknüpft sind. Eine einzigartige Gruppe von Oberflächenproteinen in der Zellwand der Pneumokokken sind die cholinbindenden Proteine (CBPs). Für einige der CBPs konnte bereits die Bedeutung für die Virulenz gezeigt werden. PspC, auch als SpsA oder CbpA bezeichnet, ist ein multifunktionales Oberflächenprotein, das als Adhesin und Faktor H-Bindungsprotein eine wichtige Rolle in der Pathogenese der Pneumokokken hat. PspC vermittelt als Adhesin die Anheftung der Bakterien an die mukosalen Epithelzellen, indem es human-spezifisch an die sekretorische Komponente (SC) des polymeren Immunoglobulinrezeptors (pIgR) bindet. SC ist die Ektodomäne des pIgR und PspC kann ebenso die freie SC binden oder an die SC des sekretorischen IgA Moleküls binden. PspC interagiert auch mit dem löslichen Komplement Faktor H. Die SC und der Faktor erkennen zwei verschiedene Epitope im bakteriellen PspC Protein. Der genaue Mechanismus der jeweiligen Interaktionen unter physiologischen- bzw. wirtspezifischen Bedingungen ist noch nicht vollständig verstanden. In dieser Arbeit wurde die Auswirkung der PspC Interaktion mit dem humanen pIgR (hpIgR) bzw. dem Faktor H auf die Virulenz der Pneumokokken und die Wirtszellantwort, d.h. die induzierten Signalkaskaden in den eukaryotischen Zellen untersucht. Die molekulare Analyse und die Verwendung von spezifischen pharmakologischen Inhibitoren der Signalmoleküle zeigten, dass verschiedene Signalmoleküle an der PspC-pIgR vermittelten Internalisierung beteiligt sind. Die Aktivierung, d.h. die Phosphorylierung der Signalmoleküle wurde in Immunblots demonstriert. Die Studien zeigten, dass das Aktinzytoskelett und die Mikrotubuli für die bakterielle Aufnahme essentiell sind. Es konnte auch zum ersten Mal nachgewiesen werden, dass Cdc42 die entscheidende GTPase für die Invasion der Pneumokokken in die Wirtsepithelzellen, vermittelt über den PspC-hpIgR Mechanismus, ist. Der Einsatz von PI3-kinase und Akt Kinase Inhibitoren reduzierte signifikant die hpIgR-vermittelte Aufnahme der Pneumokokken in die Wirtszelle. Zusätzlich durchgeführte Infektionen von hpIgR exprimierenden Zellen zeigten eine zeitabhängige Phosphorylierung von Akt und der p85&#945; Untereinheit der PI3-Kinase. Damit ist neben der GTPase Cdc42 der PI3K und Akt Signalweg entscheidend für die PspC-pIgR vermittelte Invasion der Pneumokokken. Des Weiteren sind an der Infektion mit Pneumokokken auch die Protein Tyrosin Kinasen Src, ERK1/2 und JNK beteiligt. Dabei wird die Src Kinase unabhängig von der PI3K in hpIgR exprimierenden Zellen aktiviert. Inhibitionsexperimente und genetische Knockdown Versuche mit siRNA bewiesen, dass die Endozytose der Pneumokokken über PspC-pIgR ein Clathrin und Dynamin abhängiger Mechanismus ist. Im weiterenn Teil der Arbeit wurde der Einfluss des PspC gebundenen Faktor H auf die Anheftung an und Invasion in die Epithelzellen analysiert. Die Bindung von Faktor H erfolgte unabhängig vom PspC-Subtyp. Die Bindungsversuche bewiesen, dass die Kapselmenge negativ korreliert mit der Bindung des Faktor H. Der Einsatz von Faktor H aus Maus oder Ratte zeigte keine typische Bindung. Daraus kann abgeleitet werden, dass diese Interaktion humanspezifisch ist. Die Infektionsexperimente demonstrierten, dass Faktor H die Adhärenz und die Invasion der Bakterien in die Nasenrachenraumzellen (Detroit562), alveolären Lungenepithelzellen (A549) und humanen Hirnendothelzellen (HBMEC) steigert. Der Faktor H hat Heparin Bindestellen. Diese Bindestellen vermitteln die Adhärenz der Faktor H gebundenen Pneumokokken mit Epithelzellen. Inhibitionsstudien mit spezifischen monoklonalen Antikörpern, die gegen die short consensus repeats (SCRs) von Faktor H gerichtet waren, konnten die essentielle Bedeutung der SCR19-20 für die Anheftung der Pneumokokken über Faktor H an die Wirtszellen nachweisen. Die Faktor H vermittelte Assoziation der Pneumokokken an polymorphonukleäre Leukozyten (PMNs) erfolgt über das Integrin CD11b/CD18. Die weiteren Inhibitionsstudien zeigten dann auch zum ersten Mal den Einfluss des Aktinzytoskeletts der Wirtszelle auf die Faktor H-vermittelten bakterieller Internalisierung und den dabei bedeutsamen Signaltransduktionswegen in der eukaryotischen Zelle. Dabei wurden insbesondere die Proteintyrosinkinasen und die PI3K als wichtige Signalmoleküle für die Faktor H vermittelte Invasion der Pneumokokken identifiziert. Die in dieser Arbeit erhaltenen Resultate belegen, dass die Faktor H vermittelte Infektion der Zellen mit S. pneumoniae ein konzertierter Mechanismus ist, bei dem Oberflächen-Glycosaminoglycane, Integrine und Signaltransduktionswege der Wirtsepithelzellen involviert sind. Des Weiteren wurde aufgezeigt, dass die PspC-pIgR-vermittelte Invasion in mukosale Epithelzellen unterschiedliche Signalwege wie z.B. den PI3K und Akt Weg induziert und abhängig von Cdc42 und einer Clathrin vermittelten Endozytosemechanismus ist. / Streptococcus pneumoniae (pneumococci) are Gram-positive bacteria and commensals of the nasopharyngeal cavity. Besides colonization, pneumococci are responsible for severe local infections such as otitis media, sinusitis and life-threatening invasive diseases, including pneumonia, sepsis and meningitis. The surface of pneumococci is decorated with proteins that are covalently or non-covalently anchored to the cell wall. The most unique group of cell wall associated proteins in pneumococci are the choline-binding proteins (CBPs). PspC, also known as SpsA or CbpA, is a multifunctional choline-binding protein that plays an essential role in pneumococcal pathogenesis by functioning as an adhesin. PspC promotes adherence of pneumococci to mucosal epithelial cells by interacting in a human specific manner with the free secretory component (SC) or to SC as part of the secretory IgA (SIgA) or polymeric immunoglobulin receptor (pIgR). PspC also interacts specifically with the soluble complement Factor H. Apparently, PspC uses two different epitopes for binding the soluble host protein Factor H and SC of pIgR. However, the mechanism by which these independent interactions facilitate pneumococcal infections under physiological and host specific conditions have not yet been completely elucidated. This study aims to explore the impact of the PspC interaction with human pIgR (hpIgR) or complement regulator Factor H on pneumococcal virulence. Here the cellular and molecular basis of PspC-mediated adherence to and invasion of host epithelial and endothelial cells was demonstrated. The genetic approach, specific pharmacological inhibitors and immunoblot analysis demonstrated the complexity of the induced signal transduction pathways during PspC-hpIgR mediated pneumococcal uptake by host cells. Inhibition studies with specific inhibitors of actin cytoskeleton and microtubules demonstrated that the dynamics of host cell cytoskeleton are essential for pneumococcal uptake by mucosal epithelial cells. Moreover, this study reports for the first time that the small GTPase Cdc42 is essential for pneumococcal internalization into epithelial cells via the PspC-hpIgR mechanism. In addition, in infection experiments performed in presence of specific inhibitors of PI3-kinase/Akt and protein tyrosine kinase (PTKs), hpIgR-mediated pneumococcal uptake by host cells was significantly blocked. Amongst PTKs the Src kinase pathway, ERK1/2 and JNK pathways were implicated during pneumococcal ingestion by hpIgR expressing cells. In addition, inhibition experiments performed in the presence of individual inhibitors or with a combination of inhibitors suggested the independent activation of PI3-kinase/Akt and Src kinase pathways during pneumococcal infections of hpIgR expressing cells. By employing specific inhibitors and siRNA in cell culture infection experiments it was further demonstrated that pneumococcal endocytosis by host epithelial cells via the PspC-hpIgR mechanism depends on clathrin and dynamin. PspC recruits also Factor H to the pneumococcal cell surface. Consequently, the impact of pneumococcal cell surface bound Factor H on adherence to host cells and the molecular mechanism facilitating the uptake of Factor H bound pneumococci by epithelial cells was investigated. Flow cytometry and immunoblots revealed that S. pneumoniae has evolved the ability to recruit both purified Factor H as well as Factor H from human plasma or serum. Moreover, it was demonstrated that the recruitment of Factor H is independent of the PspC-subtypes and that capsular polysaccharide (CPS) interferes with its recruitment. Factor H bound to pneumococci significantly increased bacterial attachment to and invasion of host epithelial cells including nasopharyngeal cells (Detroit562), lung epithelial cells (A549), and human brain-derived endothelial cells (HBMEC). Blocking experiments demonstrated that bacteria bound Factor H interacts via the heparin binding sites on Factor H with eukaryotic cell surface glycosaminoglycans and that this interaction promotes pneumococcal adherence to host cells. In addition, inhibition studies with mAbs recognizing specifically different short consensus repeats (SCR) of Factor H suggested that SCR 19-20 of Factor H are essential for the pneumococcal interaction with host epithelial cells via Factor H. In the presence of Factor H, attachment of pneumococci to human polymorphonuclear leukocytes (PMNs) is enhanced. The integrin CD11b/CD18 was identified as the cellular receptor on PMNs. By using pharmacological inhibitors the impact of host cell cytoskeleton and signalling molecules, such as PTKs and PI3-kinase, for Factor H-mediated pneumococcal internalization into eukaryotic cells was shown. Taken together, the results revealed that Factor-H mediated pneumococcal infection requires a concerted role of host epithelial cell surface glycosaminoglycans, integrins and host cell signalling pathways.

Infektionen mit Streptokokken der Gruppe B (GBS) sind immer noch die häufigste Ursache für early-onset Erkrankungen des Neugeborenen in den Industrieländern, die zu erhöhter neonataler Mortalität und Morbidität führen. Bereits bekannt ist, dass neben verschiedenen anderen Bakterien GBS durch die direkte Interaktion mit Thrombozyten eine Aggregation verursachen können. In dieser Arbeit wurde das Verhalten von septischen und kolonisierenden GBS-Stämmen verglichen. Es konnte gezeigt werden, dass alle GBS-Stämme eine thrombozytäre Formänderung veranlassen; jedoch führen nur von septischen Patienten isolierte Stämme zur Thrombozytenaggregation sowie zur P-Selektinexpression. Septische GBS-Stämme binden Fibrinogen auf ihrer Oberfläche und induzieren die thrombozytäre Thromboxansynthese und Granulasekretion, während kolonisierende GBS-Stämme dies nicht können. p38-mitogen-aktivierte Proteinkinase (p38-MAPK) wurde bevorzugt durch aus septischen Patienten isolierte GBS-Stämme aktiviert, ebenfalls scheint Proteinkinase C (PKC) durch diese aktiviert zu werden. Alle GBS-Stämme aktivierten Phospholipase CgammaII (PLCgammaII), Calzium-Calmodulin-abhängige Kinase II (CaMKII) und bewirken Myosinleichtketten (MLC)-Phosphorylierung über Fcgamma-Rezeptor IIA abhängige Signalwege. Es gibt weder einen Unterschied noch einen additiven Effekt zwischen der Aggregation induziert durch GBS und der Aggregation durch GBS und einer geringen Menge Adenosindiphosphat (ADP). Diese Kenntnisse der molekularen Mechanismen von GBS-induzierten Signalwegen in menschlichen Thrombozyten werden zu einem besseren Verständnis von Bakterieneffekten auf die Thrombozytenaktivierung beitragen und können deshalb neue molekulare Ziele für die pharmakologische Behandlung von GBS sein. / Infections with group B streptococci (GBS) are the most common cause of early-onset sepsis of the newborn, leading to increased neonatal mortality and morbidity. It is well known that, among several other bacteria, GBS can cause an aggregation by direct interaction with platelets. The present dissertation compares the behaviour of septic and colonizing GBS strains. It is shown that, all GBS strains induce a platelet shape change; but only septic GBS strains cause platelet aggregation and P-selectin expression. Septic strains bind fibrinogen on their surface and induce synthesis of thromboxan and granula secretion, while colonizing strains cannot do this. p38 mitogen activated protein kinase (p38-MAPK) is preferentially activated by septic GBS strains, the same applies to protein kinase C (PKC). All GBS strains activate phospholipase CgammaII (PLCgammaII), calcium/calmodulin-dependent myosin kinase II (CaMKII) and cause phosphorylation of myosin light chain (MLC) by FcgammaRIIA receptor dependent signalling pathways. There is no difference between platelet aggregation of GBS and platelet aggregation of GBS and small quantities of adenosine diphosphate (ADP). This knowledge of the molecular mechanism of GBS induced signalling pathways in human platelets contributes to our understanding of the bacterial induced platelet activation and possibly offers new molecular targets of the pharmalogical treatment of GBS infections.

Submitted in fulfilment for the degree of Doctor of Philosophy Department of Clinical Microbiology and Infectious Diseases School of Pathology Faculty of Health Sciences University of Witwatersrand 2017. / Background Vaccination of pregnant women with conserved group B Streptococcus (GBS) surface proteins has the potential to confer serotype independent protection against invasive GBS disease. Susceptibility to early onset (<7 days of age) GBS disease in infants is associated with maternal recto-vaginal colonisation, low circulating maternal antibodies and reduced trans-placental transfer of antibodies specific to GBS capsular polysaccharides (CPS). Additionally, invasive GBS disease beyond infancy has been reported in individuals with underlying conditions associated with immunosuppression including HIV infection. In this study we undertook retrospective analysis of serum IgG titres against select GBS surface proteins in relation to invasive GBS disease risk factors. Methods Multiplex Luminex immunoassay was used to measure serum IgG titres against GBS capsular polysaccharides of serotype Ia, Ib, III and V and surface proteins Fibrinogen binding surface Antigen (FbsA), GBS Immunogenic Bacterial Adhesin (BibA), Surface immunogenic protein (Sip), gbs0393, gbs1356, gbs1539, gbs0392; and lipoproteins gbs0233, gbs2106 and Foldase PsrA. Furthermore, in vitro expression of Sip, gbs2106, gbs0393 and gbs1356 proteins on the surface of clinical GBS isolates was assessed by flow cytometry using protein specific polyclonal antibodies generated in rabbits. Results Retrospective analyses of serum antibody titres against GBS surface proteins and CPS were measured in children between 4-7 years of age who were either HIVinfected (n=68) or HIV-uninfected (n=77). Lower geometric meant titres (GMT, U/mL) against Sip and gbs2106 were detected in HIV-infected children (77.03 and 53.10) compared to HIV-uninfected children (196.41 and 139.11, p<0.001) respectively. Similar results were observed for antibodies against CPS, with HIV-infected children having lower IgG GMC for serotype Ib (p=0.012) and V (p=0.005). Protein specific antibody titres were measured in pregnant women at 20-25 and ≥37 weeks of gestation age who were either non-colonised or colonised with GBS in the rectal and/or vaginal tract. Acquisition of GBS colonisation in the vagina was associated with higher antibody titres compared to colonisation in the rectum for proteins Sip (p=0.049), Foldase (p=0.0094), gbs0233 (p=0.0039), gbs0393 (p=0.027), gbs1539 (p=0.0004) and gbs1356 (p=0.039). The likelihood of acquiring GBS colonisation during pregnancy was lower in women having median IgG titres against gbs0233 ≥200 U/mL (adjusted OR=0.47 [95% CI: 0.25-0.89], p=0.021) and gbs1539 ≥85 U/mL (adjusted OR=0.44 [95% CI: 0.24-0.82], p=0.01). The influence of maternal HIV infection on trans-placental transfer of antibodies against GBS surface proteins was evaluated by comparing IgG titres between 83 HIV-infected and 81 HIV-uninfected mother-newborn dyads. Maternal HIV infection was associated with reduced trans-placental transfer of antibodies, demonstrated by the difference in cord-maternal antibody ratios between HIV-infected and HIV-uninfected mothernewborn pairs for Sip (25.8%, p<0.001), Foldase (30.4%, p<0.001), gba0392 (36.5%, p=0.006), gbs0393 (32.9%, p<0.001), gbs1539 (39.2%, p<0.008), gbs2106 (35.7%, p<0.001) and BibA (19.4%, p=0.004). A case control study was used to evaluate the association of protein specific IgG titres and invasive disease in neonates and young infants born to GBS colonised mothers, including 116 with healthy infants and 66 with invasive GBS disease at <90 days of age. Lower IgG GMT were detected in neonates who developed early-onset disease compared to control infants for Sip (65.48 vs 145.43, p<0.001), Foldase (50.45 vs 109.87, p=0.005), gbs0393 (106.42 vs 221.81, p=0.006) and gbs1356 (45.34 vs 94.52, p=0.01). Similarly, young infants with late-onset disease had significantly lower IgG GMT (U/mL) compared to healthy controls for Sip (43.72 vs 79.69, p=0.04) and gbs2106 (30.46 vs 65.35, p=0.003). Infants born to women with Sip specific antibody titres ≥150 U/mL antibodies titres against Sip were 66% less likely to develop invasive disease. Of the 82 GBS isolates collected from mothers who deliverd term babies (39 with invasive GBS disease and 43 healthy controls) that were assessed for in vitro expression of specific proteins, Sip, gbs2106 and gbs0393 were expressed in 70.7% 93.9% and 87.8% GBS isolates respectively. The expression of gbs2106 on the surface of GBS was more frequent in strains of women with infants who developed invasive disease compared to those with healthy infants (100% vs 88.4%, p=0.028). The distribution of Sip and gbs0393 expression between GBS isolates from women of infants with invasive GBS disease and healthy controls was similar, 71.8% vs 69.8% (p=0.84) and 89.7% vs 86.0% (p=0.61) respectively. Among women carrying GBS strains expressing gbs2106, having antibody titres ≥100 U/mL was associated with 90% lower likelihood for delivering infants that develop invasive GBS disease (OR=0.11 [95% CI: 0.02-0.54], p=0.002). Conclusion Vulnerability to invasive GBS disease in HIV-infected immunocompromised individuals is possibly due to reduced antibody levels against CPS, Sip and gbs2106. Also, susceptibility to invasive GBS disease in infants may be exacerbated by maternal HIV-infection, which was associated with reduced transplacental transfer of antibody against GBS surface proteins. Higher maternal antibodies titres against Sip and gbs2106 proteins were associated with reduced odds of their infants developing invasive GBS disease. These data support consideration of Sip and gbs2106 proteins as possible vaccine candidates in pregnant women to protect their infants against invasive GBS disease. Furthermore, reduced GBS colonisation during pregnancy can be achieved by maternal immunisation of gbs0233 and gbs1539, which may subsequently result in lower rates of GBS transmission to newborns and thereby decreasing their risk for invasive GBS disease. / MT2017

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirement for the degree of Master of science Johannesburg. 1996 / An attempt has been made to purify and isolate a starch endo-hydrolase enzyme produced by Streptococcus sanguis, an organism that may be implicated in dental caries. In order to isolate the enzyme by affinity binding,& chemically modified amylopectin Was prepared, similar to the preparation of chromogenic substrate for the assay of a-arnylase, but without dye. The amylopectin was treated with 10 percent ammonium sulphate at 55°C for 45 minutes. A crude enzyme extract was prepared from concentrated culture medium and by precipitation with 60 percent ammonium sulphate. The concentrated culture medium was added to the modified amylopectin substrate and the mixture was incubate for 30 minutes at 37'C and centrifuged. The precipitate was resuspended in 20 mM bepes buffer pH 6.5 which contained 0.02 M KCI to release the enzyme from the enzyme-substrate complex. The suspension was tested for enzyme activity and the presence of proteins, More than 50 percent of the yield of the enzyme was achieved by this process, after three assays, from both crude enzyme extracts and enzyme serum samples. A 5.2 fold purification was obtained from the extraction process of the crude enzyme extract and a protective enzyme activity effect was noticed in the presence of ammonium sulphate. The analytical methods selected for the activity assay were mainly used for the activity evaluation of a-amylases and carbohydrate hydrolysing enzymes. The result showed carbohydrate interference. The isolation method proved sensitive and highly specific for the isolation of a starch metabolizing enzyme produced from Streptococcus sanguis 1MC 204. The purification of the enzyme by gel filtration and its characterization by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that this enzyme was a glycoprotein . SDS-PAGE was performed with mucin and heparin in the. presence of other. proteins as markers, and stained with the periodic acid- aldan prestained silver method. This gave one transparent-band around the phosphorylase b marker and four other more slowly running clear bands. Further, the comparison of scans of several proteins and glycoproteins with the scan of the eluted sample of the amylopectin extracted enzyme showed a similarity with the UV scan of mucin and confirmed that the enzyme was a glycoprotein. It may be further characterized by selecting methods that take into account the ambohydrate content of the protein and by eliminating the carbohydrate interference in the assays. 3

No description available.

This thesis focuses on the use of a genomic approach to identify novel molecular markers to differentiate Streptococcus suis (S. suis) isolates into two populations, i) disease-associated and ii) non-disease associated. S. suis is a Gram-positive coccus that is considered one of the most important zoonotic bacterial pathogens of swine responsible for significant economic losses to swine production worldwide. Importantly, S. suis is not only an invasive pathogen but also a very successful coloniser of mucosal surfaces; often endemic in swine populations sampled. The widescale use of antibiotics to control and prevent the various clinical manifestations caused by S.suis has become unsustainable, due to increases in antibiotic resistance and government pressures. Other popular control strategies, such as the development of efficacious vaccines, are hindered by differences in virulence not only between but also within S. suis serotypes, as well as, the lack of a detailed understanding of the role in pathogenesis of many proposed virulence-factors. As a result, the detection of S. suis in asymptomatic swine herds is of little practical value in predicting the likelihood of future clinical relevance. This thesis aims to further understanding of the role the S. suis genome has in pathogenesis. The value of future surveillance and preventative health management lies in the detection of strains that genetically have increased potential to cause disease in presently healthy animals. The first results chapter of this thesis (chapter 3) describes the use of genome-wide associations studies, a so-far unexploited method for S. suis, to identify genetic markers associated with the observed clinical phenotype i) invasive disease or ii) asymptomatic carriage on the palatine tonsils of swine. Chapter 4 then describes the analyses used to select three genetic markers to pathotype S. suis - differentiate isolates of the same species based on their ability to cause disease; going on to describe the design and evaluation of a multiplex-PCR tool targeting the three newly defined "pathotyping markers" in comparison to existing methods used to characterise S. suis. These findings were taken further by using the pathotyping markers to screen material scrapped from the palatine tonsils of swine with no obvious signs of streptococcal disease. This produced an interesting result - the production of both invasive disease-associated and non- disease associated multiplex-PCR amplicons from the same experimental sample. Unsurprising in itself, what was surprising is the frequency with which this observation was found. Picking single colonies from solid agar plates is a crippling bottleneck of existing S. suis diagnostics, and its removal has the potential to improve the sensitivity of surveillance and preventive health management programs. Chapter 5 describes investigation of this surprising observation and indicates that classic culture-based methods of detection are not sensitive enough to confidently report the presence (or absence) of invasive disease-associated S. suis strains. This thesis concludes with the description of efforts to address the lack of a comprehensive understanding of S. suis virulence/'virulence-associated' factors. Chapter 6 describes the design of an isogenic mutant knocking out the invasive disease-associated pathotyping marker, SSU1589 (also known as virA). That is then evaluated in simple in vitro and in vivo experimental models in order to understand the role Type I restriction modification proteins have in S. suis pathogenesis. In conclusion, this thesis furthers our understanding that differences in the S. suis genome are an important factor in S. suis pathogenesis, and describes the identification and evaluation of novel genetic markers for the detection and control of invasive disease-associated S. suis strains in intensive pig production systems.

Bovine mastitis caused by Streptococcus uberis is a common problem in pasture-based dairying systems. This study examines both the ecology of S. uberis and infection of the bovine mammary gland on a New Zealand dairy farm. Initially, the REP-PCR strain typing method was developed and the potential of MALDI-TOF mass spectrometry evaluated as a strain typing method. While strain-specific mass spectra were obtained with MALDI-TOF mass spectrometry, the irreproducibility of spectra was its major downfall. With further work, this rapid method could be very useful for strain typing S. uberis on a large scale. Using optimised REP-PCR and anchored typing methods, multiple S. uberis strains were isolated and strain typed from the dairy environment, including farm races and paddocks, faeces, teat skin, the cow body and from intramammary infections. High strain diversity was observed in all sampled locations; however, some strains were found at more than one site, suggesting transmission may occur between the environment and cows. The most likely means of S. uberis distribution throughout the dairy farm was via excretion with faeces and, although not all cow faeces contained this pathogen, the gastrointestinal tract of some cows appeared to be colonised by specific strains, resulting in persistent shedding of this bacteria in the faeces. Infection of the mammary gland is likely to occur through contamination of the teat skin with highly diverse environmental strains of S. uberis. However, only one or two strains are generally found in milk from mastitis cases, suggesting that, although infection may arise from a random or opportunistic event, a strain selection process may take place. Intramammary challenge with multiple strains of S. uberis revealed that selection of a single infective strain can occur within the mammary gland. The predominance of one strain over others may be related to production of virulence factors allowing enhanced ability to establish in the gland and evade the immune response, or due to direct competition between strains through the production of antimicrobial factors such as bacteriocins. In addition to strain-specific factors, the individual cow and quarter response may play an important role in the development of infection and selection of the infective strain. Using results from this study, a model of S. uberis strain transmission has been proposed, which includes potential mechanisms of infection and persistence of S. uberis within the mammary gland.