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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.

Thesis advisor: Tim Van Opijnen / Exposure to antibiotics has previously been shown to induce morphological changes to bacterial cells in Escherichia coli and Staphylococcus aureus . Response profiles to antibiotics representing various mechanisms of action provides as quick, reliable and cheap means of identifying the mechanism of action of novel antimicrobials. We evaluated whether similar cytological profiling was possible in the pathogenic bacteria Streptococcus pneumoniae and whether there were any strain specific differences in morphological changes resulting from antibiotic exposure. We evaluated antibiotics from various classes and with different mechanisms of action to develop strain specific models of phenotypic responses in order to identify clustering associated with particular mechanisms of action. Various antibiotics belonging to, cell wall synthesis inhibitors, protein synthesis inhibitors, and DNA synthesis inhibitors were evaluated using S. pneumoniae strains TIGR4 and 19F. Following exposure to high doses of antibiotics, cells were imaged for DNA and cell wall components and analyzed. Our data shows that antibiotics of the same mechanism of action induce similar morphological changes. While TIGR4 and 19F show similar changes there are strain specific differences between them. Our data shows that cytological profiling effectively indicates the mechanism of action through imaging in S. pneumoniae allowing this technique to be used to study novel antimicrobials as well as better understand bacterial responses to antibiotic stress. / Thesis (BS) — Boston College, 2019. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology.

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.

Nitrogenase-catalyzed nitrogen fixation is a biochemically and genetically complex process requiring the participation of a number of different nif (nitrogen fixation) gene products. The nifF (electron transport), nifA (nif gene regulation) and nifE (FeMo-cofactor biosynthesis) genes from <i>Azotobacter vinelandii</i> were genetically analyzed. The nucleotide sequence of the nifF gene, which encodes a flavodoxin, was determined. Specific mutation strains indicated that in <i>A vinelandii</i> flavodoxin is not the unique physiological electron donor to nitrogenase. The nifF gene appears to be constitutively expressed but under nitrogen fixing conditions nifF gene expression is stimulated. / Master of Science

Streptococcus pneumoniae (pneumococcus) is a commensal gram-positive colonizer of the human nasopharynx capable of causing diseases including otitis media, pneumonia, bacteremia, and meningitis. Although it is often a harmless colonizer, there is a high rate of mortality and morbidity among the immunocompromised, elderly, and young children. While these infections can often be treated with antibiotics, resistance to numerous antibiotics is increasing. Antibiotic resistance is a well-studied dilemma; however, little information is known of how bacteria take up certain antibiotics. Because most antibiotics cannot diffuse freely across the bacterial cell wall, we hypothesize that metabolite transport proteins participate in the uptake of certain classes of antibiotics.

Klebsiella pneumoniae is a Gram-negative bacterium commonly giving rise to nosocomial infections especially in immunocompromised individuals. The high occurrence of antibiotic resistant K. pneumoniae strains in combination with the bacterium’s ability to form biofilms that are naturally more tolerant to antibiotic treatment represents a burden for the healthcare system due to potential therapeutic failure. In this study, we were investigating the effect of gentamicin, cefotaxime and ciprofloxacin on biofilm formation and eradication of a multi-resistant ESBL-producing K. pneumoniae strain associated with an outbreak at the Uppsala University Hospital in Uppsala, Sweden, from 2005 to 2007. Multidrug resistance was encoded on the pUUH239.2 plasmid. We also studied how resistant bacteria are selected for in mixed biofilm populations consisting of both resistant and susceptible bacteria under antibiotic treatment. Biofilms were grown in vitro by using an in-the-lab developed peg model. Biofilms were either allowed to form in the presence of the antibiotics or pre- formed and then exposed to antibiotic treatment at different time points. Our results suggest that gentamicin, cefotaxime and ciprofloxacin inhibit biofilm formation at concentrations below the minimum inhibitory concentration (MIC) and that biofilms become more tolerant to antibiotic treatment with increasing maturation. We further observed that increasing antibiotic concentrations select for the presence of the plasmid in less mature biofilms. Overall, these findings highlight the need of early antibiotic treatment during infection and give insight into the dynamics of resistant and susceptible bacteria in mixed biofilm populations in the presence of antibiotics.

Background Mycoplasma pneumoniae and Chlamydia pneumoniae are atypical pathogens responsible for pneumonia and a leading cause of morbidity and mortality in low income countries. The study objective is to determine the prevalence of this pathogens in Peruvian children with acute respiratory infections. Methods A consecutive cross-sectional study was conducted in Lima, Peru from May 2009 to September 2010. A total of 675 children admitted with clinical diagnoses of acute respiratory infections were tested for Mycoplasma pneumoniae and Chlamydia pneumoniae detection by polymerase chain reaction (PCR), and clinical symptoms were registered by the attending physician. Results Mycoplasma pneumonia was detected in 25.19% (170/675) of nasopharyngeal samples and Chlamydia pneumonia in 10.52% (71/675). The most common symptoms in patients with these atypical pathogens were rhinorrhea, cough and fever. A higher prevalence of Mycoplasma pneumoniae cases were registered in summer, between December 2009 and March 2010. Conclusions Mycoplasma pneumoniae and Chlamydia pneumonia are a significant cause of morbidity in Peruvian children with acute respiratory infections (ARI). Further studies should evaluate the use of reliable techniques such as PCR in Peru in order to avoid underdiagnoses of these atypical pathogens.

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.