Abstract The purpose was to evaluate the microbiological mechanism of action of xylitol and to assess its use in clinical practice for preventing acute otitis media (AOM). To test whether the effect of xylitol on S. pneumoniae is inhibited by fructose, a total of 20 strains of S. pneumoniae were exposed to xylitol in the presence of fructose and other carbon sources. Addition of 5% xylitol to the media resulted in marked growth inhibition, an effect which was totally eliminated in the presence of 1%, 2.5% or 5% fructose but not in the presence of 1% or 5% glucose, 1% galactose or 1% sucrose. The inhibition of pneumococcal growth is probably mediated via a fructose phosphotransferase system in a similar manner to that seen in mutans streptococci. Sorbitol alone did not affect the growth of pneumococci, and thus sorbitol is unlikely to provide any clinical benefit in the prevention of AOM. To evaluate the effect of xylitol on the ultrastructure of S. pneumoniae and Haemophilus influenzae (H. influenzae) and on the pneumococcal phenotype, five strains of S. pneumoniae and one strain of H. influenzae were examined by electron microscopy after xylitol exposure. Xylitol damaged the ultrastructure of the pneumococci. Some of the bacteria were lysed and the cell wall of the remaining ones became more diffuse and the polysaccharide capsule was ragged. The resulting morphology was identical to that of the transparent pneumococcal phenotypic variant. The properties of the transparent variants of pneumococci could explain the clinical efficacy of xylitol in preventing AOM despite the lack of effect on the nasopharyngeal carriage of pneumococci. The cell wall of H. influenzae became slightly thicker, but the morphology remained otherwise unchanged. To evaluate the pharmacokinetics of xylitol locally in the nasopharynx, xylitol concentrations were measured in the saliva of 65 children by enzymatic assay after giving them xylitol chewing gum or syrup at doses equal to those used in clinical trials. Concentrations high enough to have an antimicrobial effect were attained, but the xylitol disappeared from the saliva within 15 minutes, which indicates that high peak concentrations may be more important for efficacy than the time for which the concentration exceeds the level needed for an antimicrobial effect. To find a more convenient dosing regime for xylitol prophylaxis, xylitol was administered to 1277 children only during an acute respiratory infection (ARI) in a randomised placebo-controlled trial. The occurrence of AOM during ARI was 34/166 (20.5%) in the xylitol mixture group as compared with 32/157 (20.4%) among the children receiving the control mixture. Among older children receiving control chewing gum, xylitol chewing gum or xylitol lozenges, AOM was experienced by 24/218 (11.0%), 31/220 (14.1%) and 34/219 (15.5%) respectively. None of the differences between the groups was statistically significant. Xylitol should be used continuously in AOM prophylaxis, as it proved ineffective when used only during URI.

The role of Spr1851 protein Streptococcus pneumoniae in cell division Human extracellular pathogen S. pneumoniae encodes unique serin/threonine protein kinase of Eucaryotic type StkP and its cognate phosphatase PhpP. This kinase affects number of cellular processes including virulence, competence, cell division and cell wall synthesis by phosphorylating its substrates. Hypothetical protein Spr1851 named Jag was identified as a new StkP substrate in the membrane fraction by comparing the wild-type phosphoproteomes with StkP deleted strain. This protein consists of three domains and an interdomain region that contains T89 phosphorylation site. There is a Jag_N domain with an unknown function at the N-terminus. The C-terminus contains two domains - KH and R3H, which are highly conserved and their expected function is binding of nucleic acid. The main aim of this work is to explain the function of Jag protein, to determine the effect of individual domains on the phenotype and the localization of the protein and to determine the role of phosphorylation on T89. The results confirm that Jag protein could play a role in cell division or cell wall synthesis. Furthermore, the results indicate that the Jag_N domain is essential for the localization of the protein into the membrane, whereas the KH and R3H domains are...

Publicación a texto completo no autorizada por el autor / Determina la presencia de Streptococcus iniae en Tilapia nilótica (Oreochromis niloticus) mediante el aislamiento y análisis bioquímico, caracteriza las lesiones histopatológicas de las tilapias presuntas positivas a S. iniae por bioquímica y confirmar su presencia o no mediante la PCR a tiempo real, en un centro de cultivo intensivo de la zona de Sullana-Piura. Se determina el tamaño de muestra con una prevalencia límite de 2.7%; empleándose en total 150 tilapias de las fases de engorde y pre engorde con signos de enfermedad. La necropsia evidencia lesiones compatibles con Streptococcus spp, como exoftalmia, hifema, congestión y/o hemorragia de meninges, ascitis, esplenomegalia, hepatomegalia y zonas hemorrágicas difusas en todo el cuerpo. La prevalencia media para tilapias positivas al género Streptococcus spp., por microbiología es 26% (21% - 33%) y la prevalencia media de tilapias presuntas positivas a S. iniae por perfil bioquímico es 10.12% (6% – 15.10%). Los hallazgos histopatológicos son: epicarditis, periesplenitis y perihepatitis fibrino supurativa aguda o crónica, meningitis, panoftalmitis, necrosis coagulativa del músculo esquelético y formación de granulomas. Sin embargo, en la prueba confirmatoria de PCR a tiempo real, sondas Taqman, no se obtiene ninguna tilapia positiva a S.iniae. Los resultados son analizados a través de una simulación estocástica de la distribución beta, empleando el programa de incertidumbre @Risk, reportándo una prevalencia media de 0.66 % (0.02-2.41%) en tilapias enfermas. / Tesis

Streptococcus pneumoniae is a common human pathogen, causing severe and often life-threatening respiratory tract infections. Even though most patients receive appropriate antimicrobial chemotherapy, a significant percentage still die. Cigarette smoking is a well-documented risk factor for severe pneumococcal pneumonia; however, the microbiological/ immunological mechanisms which predispose smokers to infection are not yet completely understood. The pneumococcal toxin, pneumolysin, is a major virulence factor of S. pneumoniae; it forms pores in eukaryotic cell membranes, resulting in the influx of extracellular Ca2+. Biofilm is a self-generated polymer matrix, used by microbial pathogens to isolate themselves from both host defences, as well as antibiotics. In this dissertation, the effects of cigarette smoke condensate (CSC) on the bioactivity of pneumolysin, as well as on the growth of, and production of biofilm by Streptococcus pneumoniae are described. A clinical isolate of S. pneumoniae, strain 172, was used for both growth and biofilm determinations in the presence or absence of concentrations of CSC (20-160 μg/ml), representative of the smoking habit. Growth and biofilm determinations were performed using spectrophotometric procedures, viability by standard colony forming unit procedures, and the effect of the condensate on pneumolysin bioactivity using a fura-2/AM-based spectrofluorometric procedure. Exposure of the pneumococcus to CSC resulted in a dose-dependent increase in biofilm formation which achieved statistical significance (P≤0.05) at concentrations of 80 and 160 μg/ml, in the setting of modest effects on bacterial growth. These findings were not unique to S. pneumoniae since exposure of Staphylococcus aureus, a known biofilm former, to CSC showed similar results. Exposure of pneumolysin to CSC (20 and 40 μg/ml) was accompanied by attenuation of the biological activity of the toxin, resulting in impaired pore-forming ability manifest as a considerable reduction in influx of extracellular Ca2+ following exposure of isolated neutrophils to the toxin. It is possible that CSC acts as a stressor to the bacteria, thereby enhancing biofilm formation and consequently persistence in the respiratory tract. These effects of the toxin may be complemented by inactivation of pneumolysin, presumably by pro-oxidative mechanisms, affecting innate cellular host defences. These mechanisms may underpin the predisposition of smokers to develop severe pneumococcal infections. / AFRIKAANS : Streptococcus pneumoniae is ‘n algemene menslike patogeen, wat ernstige en soms lewensbedreigende lugweg infeksies veroorsaak. Alhoewel die meeste pasiënte geskikte antimikrobiese chemoterapie ontvang, sterf ‘n betekenisvolle hoeveelheid steeds. Sigaretrook is ‘n welbekende risikofaktor vir ernstige pneumokokkale pneumonie; die mikrobiologiese/ immunologiese meganisme wat rokers vatbaar maak vir infeksie word egter nog nie heeltemal verstaan nie. Die pneumokokkale toksien, pneumolisien, is ‘n belangrike virulensie faktor van S. pneumoniae; dit vorm porieë in die eukariotiese selmembrane wat ‘n invloei van ekstrasellulêre Ca2+ tot gevolg het. Biofilm is ‘n self-genererende polimeermatriks, wat deur mikrobiese patogene gebruik word om hulself teen gasheerverdedigings meganismes en antibiotika te isoleer. In hierdie verhandeling word die uitwerking van sigaretrook kondensaat (SRK) op die bioaktiwiteit van pneumolisien, asook die effekte op groei en die produksie van biofilm deur S. pneumoniae, beskryf. ‘n Kliniese isolaat van S. pneumoniae, stam 172, is gebruik vir beide groei en biofilm bepalings, in die teenwoordigheid en afwesigheid van SRK (20-160 μg/ml), wat verteenwoordigend van die rook gewoonte is. Groei en biofilm bepalings is uitgevoer deur gebruik te maak van spektrofotometriese prosedures, lewensvatbaarheid, deur standard kolonievormende eenheid prosedures, en die effek van die kondensaat op die pneumolisien bioaktiwiteit, deur fura-2/AM-gebaseerde spektrofluorometriese prosedures. Blootstelling van die pneumokokkus aan SRK het in ‘n dosis-verwante verhoging in biofilm-formasie tot gevolg gehad, wat statistiese betekenisvolheid (P≤0.05) bereik het teen konsentrasies van 80 en 160 μg/ml, teenoor die agtergrond van beskeie effekte op bakteriële groei. Hierdie bevindinge is nie uniek aan S. pneumoniae nie, aangesien blootstelling van Staphylococcus aureus, ‘n bekende biofilm-vormer, aan SRK soortgelyke resultate gelewer het. Blootstelling van pneumolisien aan SRK (20-160 μg/ml) was geassosieer met die verswakking van die biologiese aktiwiteit van die toksien, met verminderde porie-vormende vermoë, wat gemanifesteer het met ‘n ongelooflike verlaging in die invloei van ekstrasellulêre Ca2+ na die blootstelling van geïsoleerde neutrofiele aan die toksien. Dit is moontlik dat die SRK as ‘n stressor vir die bakterieë optree, en daardeur biofilm formasie en gevolglik hardnekkige infeksies in die lugweg tot sellulêre gasheervededing beïnvloed. Hierdie effekte op die toksien mag gekomplimenteer word deur die inaktivering van pneumolisien, heelwaarskynlik deur pro-oksidatiewe meganismes, wat die vatbaarheid van rokers om ernstige pneumokokkale infeksies te ontwikkel, beklemtoon. / Dissertation (MSc)--University of Pretoria, 2011. / Immunology / Unrestricted

MSc., Faculty of Science, University of the Witwatersrand, 2011 / Background: Due to the high cost and limited serotype coverage of pneumococcal conjugate vaccines (PCV), surface proteins of Streptococcus pneumoniae are being investigated for their role as potential vaccine candidates. There are limited data on natural antibody kinetics against pneumococcal surface proteins arising through exposure to pneumococcal nasopharyngeal (NP) colonization in African populations. Objectives: To characterize the natural antibody kinetics and sero-prevalence to 15 pneumococcal proteins with respect to age, PCV vaccination and HIV status as well as to explore the association between antibody titers and pneumococcal nasopharyngeal colonization in infants, older children and adults. Methods: We established a 15-plex Luminex assay for the following proteins: PspA, PspC, LytB, IgA1-proteinase, SP 0082, PdB, PcsB, PsaA, SP 0609, SP 0749, PpmA, SlrA, StkP, SP 2027 and SP 2194, and also validated the Luminex assay comparing it to a standard ELISA method for PspA, PspC, PsaA and PdB. We used the Luminex method to characterize the prevalence and dynamics of serum IgG antibodies against the pneumococcal proteins. The study involved 2 166 human subjects which included: i. A longitudinal cohort of children less than 2 years of age, who were vaccinated with the seven-valent pneumococcal conjugate vaccine (PCV-7) and were either a) HIV-exposed infected, b) HIV-exposed uninfected or c) HIV-unexposed uninfected. ii. A longitudinal cohort of PCV-7 unvaccinated children less than 2 years of age who were either: a) HIV-unexposed uninfected or b) HIV-exposed uninfected. The PCV-7 vaccinated and unvaccinated children were followed up from approximately 4 to 24 months of age. In addition, samples were also analyzed from HIV-uninfected and HIV-infected children Project ID: Pneumococcal protein antigens Student: Zanele Ditse Date: 04 October 2011 - 5 - aged between 4 to 7 years who received either a primary series of PCV-9 or placebo during infancy. Lastly, we analyzed cross-sectional samples from HIV-uninfected and HIV-infected women. Results: The multiplex Luminex assay correlated well with singleplex ELISAs for all four analyzed proteins with correlation coefficients of 0.86, 0.90, 0.87 and 0.96 for PspA, PspC, PdB and PsaA respectively. Antibody titers to PspC, PdB, LytB, SP 0082, PcsB and StkP showed increases in titer with respect to increasing age. Prevailing nasopharyngeal pneumococcal colonization in young children was associated with higher antibody titers to PspA, PspC, PdB, SP 0082, LytB, IgA1-proteinase, PpmA, PcsB and StkP. Conversely higher antibody titers to PspC, PdB, LytB, SP 0082, PcsB and StkP were associated with lower prevalence of pneumococcal colonization in older children and adults. In children under two years of age, PCV vaccination was associated with lower antibody titers to PspA, PspC, LytB, PdB, IgA1-proteinase, PcsB and StkP as well as higher antibody titers against SP 0082 and PpmA at multiple time-points. In PCV-vaccinated children under two years of age, those who were HIV-unexposed , -uninfected had higher antibody titers to PspA, PspC, SP 0082, IgA1-proteinase, PpmA and StkP compared to HIV-exposed, uninfected children. Conclusion: There was an age-related increase in antibody titers to PspA, PspC, PdB, SP 0082, LytB, IgA1-proteinase, PpmA, PcsB, and StkP in children under two years of age. PCV immunization was, however, associated with lower antibody titers to PspA, PspC, LytB, PdB, IgA1-proteinase, PcsB and StkP in young children which was not attributed to differences in the prevalence of nasopharyngeal colonization. Furthermore, HIV-infection status in young children was associated with higher antibody responses to PspA, PspC, PdB, SP 0082, LytB, IgA1-proteinase, PpmA, PcsB and StkP proteins in HIV-unexposed uninfected children compared to HIV-exposed uninfected and HIV-exposed infected children. Higher antibody concentrations to Project ID: Pneumococcal protein antigens Student: Zanele Ditse Date: 04 October 2011 - 6 - PspC, PdB, LytB, SP 0082, PcsB and StkP was negatively associated with nasopharyngeal pneumococcal colonization in older children and adults; indicating a protective role against colonization and a potential role as vaccine candidates.

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Streptococcus pneumoniae (pneumococcus) is a major human pathogen and commensal organism of the nasopharynx. A major virulence factor of the pneumococcus is the cholesterol dependent, pore forming cytolysin pneumolysin. This toxin acts extracellularly, but the mechanism of release has not been well elucidated. Despite being a catalase negative organism, the pneumococcus produces up to millimolar concentrations of hydrogen peroxide through the activity of pyruvate oxidase. In all strains analyzed, deletion of the pyruvate oxidase gene yielded a significant reduction in the amount of PLY observed in the supernatant via western blot. A single strain, WU2 was also observed to have a significant (p<.05) reduction in the amount of PLY observed in the supernatant when treated with extracellular catalase. Furthermore, a significant correlation between hydrogen peroxide production and PLY release was observed in a panel of 15 clinical isolates.

Proper regulation of apoptosis during pneumococcal pneumonia is essential for resolution of infection. We hypothesized that reactive oxygen species (ROS) produced during infection causes sufficient DNA damage to alter expression of pro-apoptotic proteins. Despite inducing DNA damage, challenge with pneumococci did not cause alterations the expression of the pro-apoptotic protein Puma (p53 up-modulated regulator of apoptosis) at early (4 and 6 hour) and late (16 and 24 hour) time points tested in this study. Puma-dependent global expression patterns were assessed, and the data demonstrated significant changes in expression of various genes (Prdx2, Ripk1, Api5 and IL-10) involved in cell death and the inflammatory response. In conclusion, although the presence of Puma is necessary for normal apoptotic cellular death and host resolution of infection, Puma expression in bone marrow neutrophils and lung epithelial cells is not dependent on ROS produced during pneumococcal infection.

The upper respiratory tract (URT), including the nasal and oral cavities, is a reservoir for pathogenic and commensal microbial species, collectively known as the microbiota. Microbial colonization of the URT occurs right after birth, and URT microbial composition has been linked to development of respiratory infections, allergy, and asthma, though few direct mechanisms have been uncovered. Thus, I set out to establish animal models for characterizing the URT microbiota, and its role in infections. I found that nasal washes, a predominant method for measuring URT bacterial colonization, were insufficient for completely extracting the URT microbiota. The age and source of mice greatly affected the composition of the microbiota, which could be transferred to germ-free mice via cohousing. I also established that mice colonized with the Altered Schaedler’s Flora in the gut microbiota have no cultivable URT microbiota. To test the function of the URT microbiota, I colonized mice with Streptococcus pneumoniae, the leading cause of bacterial pneumonia worldwide. I show that the presence of a nasal microbiota increases permissiveness to pneumococcal infection in murine models. Addition of a single URT isolate, Actinomyces naeslundii, increased pneumococcal adherence to human respiratory epithelial cells in vitro and increased pneumococcal dissemination in vivo in a sialidase-dependent manner. The microbiota affects expression of several host genes throughout the respiratory tract involved in pneumococcal pathogenesis. Together, this work establishes new models for assessing the URT microbiota, and highlights the contribution of the URT microbiota to pneumococcal pathogenesis and identifies druggable targets to prevent and treat infections. / Dissertation / Doctor of Philosophy (PhD) / Bacteria living in the gut have been shown to benefit our health, but the role of bacteria living in our respiratory tract is relatively unknown. I describe the methods for characterizing the bacteria in the nose of a mouse as a model of the human nose. I found that pockets of the mouse nose are colonized by different bacteria. I also characterized a mouse model that had bacteria in the gut without nasal bacteria. I used this mouse model to understand infections with Streptococcus pneumoniae, the worldwide leading cause of bacterial pneumonia. The mice without nasal bacteria were protected from infections, which was due to a nasal bacteria helping S. pneumoniae escape from the nasal tissue. This work established new models for understanding how bacteria affect respiratory health, and identified new targets for protecting against infections.

Streptococcus pneumoniae, ou pneumococo, é um constituinte da microbiota humana, mas em alguns casos pode causar doenças, como pneumonia, bacteremia e meningite. Uma das principais formas de conter as infecções pneumocócicas foi o desenvolvimento de vacinas baseadas na indução de anticorpos contra o polissacarídeo capsular (PS). Como as vacinas pneumocócicas conjugadas (PCVs) possuem um número limitado de PSs e sua efetividade contra doenças não-invasivas ainda é controversa, o desenvolvimento de uma nova geração de vacinas que não sejam sorotipo-específicas continua sendo uma prioridade. Pneumococcal surface protein A (PspA) é um antígeno com grande potencial para uso em vacinas contra pneumococo. PspA apresenta certa variabilidade entre isolados e é dividida em família 1 (clados 1 e 2), família 2 (clados 3, 4 e 5) e família 3 (clado 6). O objetivo deste trabalho é avaliar a imunogenicidade e eficácia de nanopartículas (NPs) compostas do polímero poli(glicerol-adipato-co-&omega;-pentadecalactona) (PGA-co-PDL) contendo o antígeno PspA (PspA4Pro) e formuladas em partículas micrométricas (NP/NCMP PspA4Pro) em modelo murino de imunização pulmonar. Inicialmente, foram testadas três técnicas de inoculação para imunização pulmonar de camundongos. Tanto a utilização de um insuflador pulmonar para inoculação das NP/NCMPs sob a forma de pó quanto o uso de um microsprayer para inoculação das NP/NCMPs após ressuspensão em salina não foram capazes de induzir uma resposta de indução de IgG sérico de forma homogênea. Na terceira estratégia de imunização foi utilizada uma micropipeta para a imunização através de instilação por via nasal de duas doses da ressuspensão das NP/NCMPs. A imunização com NP/NCMP PspA4Pro através dessa técnica mostrou-se adequada, com a indução de IgG anti-PspA4 Pro no soro e no lavado broncoalveolar (BALF). Anticorpos IgG induzidos pela imunização com NP/NCMP PspA4Pro mostraram ligação à superfície de bactérias intactas expressando PspA dos clados 3, 4 e 5 (família 2), mas não foi observada ligação a bactérias expressando PspA dos clados 1 e 2 (família 1). Camundongos imunizados foram então desafiados com as linhagens ATCC6303 (sorotipo 3, PspA de clado 5) ou EF3030 (sorotipo 19F, PspA de clado 1) e o BALF foi coletado após 12 e 24 horas, respectivamente. Foi observada uma redução da carga bacteriana no BALF após desafio com a linhagem ATCC6303, além de uma redução na concentração de IL-6, TNF- KC/CXCL1 e MIP-2/CXCL2 em camundongos imunizados com NP/NCMP PspA4Pro. Todavia, esta redução da carga bacteriana no BALF não foi observada após desafio com a linhagem EF3030. Foi realizado ainda um desafio para avaliar a sobrevivência final após desafio com a linhagem ATCC6303 e foi observado que a imunização com NP/NCMP PspA4Pro foi capaz de induzir proteção parcial. A imunização pulmonar NP/NCMP PspA4Pro foi, portanto, capaz de induzir anticorpos no soro e pulmões dos camundongos, conferindo proteção contra uma linhagem de pneumococo expressando PspA da mesma família. Deste modo, estudos futuros são necessários para garantir maior proteção vacinal. / Streptococcus pneumoniae, or pneumococcus, is part of the human microbiota, but in some cases it can cause diseases, such as pneumonia, bacteremia and meningitis. One of the main forms of controlling pneumococcal infections was the development of vaccines based on the induction of antibodies against capsular polysaccharide (PS). Since a limited number of serotypes are included in pneumococcal conjugate vaccines (PCVs) and their effectiveness against non-invasive diseases is still controversial, the development of a new generation of serotype-independent vaccines is still a priority. Pneumococcal surface protein A (PspA) is an antigen with great potential for vaccine use. PspA shows some variability between strains and is divided in family 1 (clades 1 and 2), family 2 (clades 3, 4 and 5) and family 3 (clade 6). The aim of this work is to evaluate the immunogenicity and efficacy of poly (glycerol-adipate-co-&omega-pentadecalactone) (PGA-co-PDL) nanoparticles (NPs) containing PspA (PspA4Pro) and formulated in micrometric particles (NP/NCMP PspA4Pro) in a murine model of pulmonary immunization. Initially, three inoculation techniques were tested for lung immunization of mice. Both the inoculation of the NP/NCMPs as dry powder using a pulmonary insufflator and inoculation of the resuspension of the NP/NCMPs in saline using a microsprayer did not induce IgG serum antibodies reproducibly. For the third immunization strategy, a micropipette was used for immunization through nasal instillation of two doses of the resuspension of the NP/NCMPs. Immunization with NP/NCMP PspA4Pro using this technique showed reproducible results, with the induction of anti-PspA4Pro IgG in serum and bronchoalveolar lavage (BALF). IgG antibodies induced by immunization with NP/NCMP PspA4Pro showed binding to the surface of intact bacteria expressing PspA from clades 3, 4 and 5 (family 2), but no binding was observed for bacteria expressing PspA from clades 1 and 2 (family 1). Immunized mice were then challenged with strains ATCC6303 (serotype 3, PspA clade 5) or EF3030 (serotype 19F, PspA1) and BALF was collected after 12 and 24 hours, respectively. A reduction in bacterial load in BALF was observed in mice challenged with ATCC6303. A reduction in IL-6, TNF-KC/CXCL1 and MIP-2 CXCL2 levels in BALF of mice immunized with NP/NCMP PspA4Pro was also observed. Reduction in bacterial load was not observed for mice challenged with strain EF3030 though. A challenge with strain ATCC6303 strain was also performed to evaluate overall survival and partial protection was observed for the group immunized with NP/NCMP PspA4Pro. In conclusion, lung immunization with NP/NCMP PspA4Pro is able to induce antibodies in serum and lungs, conferring protection against a pneumococcal strain expressing PspA from the same family. Future studies are thus necessary to guarantee broader vaccine protection.