Prevotella intermedia/nigrescens group bacteria are Gram-negative, non-motile, anaerobic rods abundant in the subgingival microbiome of human periodontitis patients, and relatively sparse in persons with periodontal health. P. intermedia/nigrescens may be inadequately suppressed in periodontal pockets with conventional mechanical-surgical forms of periodontal therapy. Therefore, short-term systemic antibiotic therapy is often recommended in the treatment of recalcitrant (refractory) severe periodontitis patients where high numbers of P. intermedia/nigrescens persist in the subgingival microbiota. Limited available data suggests that the antibiotic sensitivity profile of periodontal P. intermedia/nigrescens has changed over time among severe periodontitis patients in the United States, with increasing levels of antibiotic resistance reported. These findings have potentially important clinical implications for dental professionals and their severe periodontitis patients which need further confirmation and clarification. To further expand knowledge on this clinically relevant issue, the purpose of the present study was to determine and compare over a 10-year period (2011 versus 2021) the prevalence of in vitro resistance of periodontal P. intermedia/nigrescens to the antibiotics amoxicillin, metronidazole, clindamycin, and doxycycline. / Oral Biology

Thesis--University of Helsinki, 1999. / Includes bibliographical references.

Thesis--University of Helsinki, 1999. / Includes bibliographical references.

Une classification et une nomenclature fiables et actualisées sont indispensables pour identifier et différencier les microorganismes pathogènes chez l'homme. Ces dernières années ont vu la création de nombreux nouveaux taxons bactériens ainsi que la reclassification d'espèces ou de genres déjà connus, notamment au sein des bactéries anaérobies. Afin de clarifier la position taxonomique de certains groupes de bactéries anaérobies pour lesquelles des phénotypes de résistance aux antibiotiques inhabituels étaient observés, une approche taxonomique mixte et consensuelle associant l'analyse de marqueurs phénotypiques, génotypiques, génomiques et phylogénétiques a été utilisée. Dans un premier temps, cette approche a été appliquée à des bactéries présentant un bas niveau de résistance à la vancomycine et appartenant aux Clostridiaceae, famille en plein remaniement taxonomique. Les résultats obtenus ont abouti à la description d'une nouvelle espèce de Tissierella et d'un nouveau genre, Flavonifractor, qui résulte du regroupement de deux espèces préexistantes. Appliquée dans le cadre d'une étude sur la résistance au métronidazole chez Prevotella spp., cette approche a conduit, dans un deuxième temps, à la découverte d'une nouvelle espèce, Prevotella nanceiensis, dont la résistance au métronidazole a été impliquée dans un échec thérapeutique. Un nouveau gène (nimI) codant une nitroimidazole réductase et a priori intrinsèque à l'espèce Prevotella baroniae a également été décrit. Ce travail souligne l'importance d'une taxonomie claire et fiable en bactériologie médicale et l'intérêt d'une approche mixte et consensuelle en taxonomie bactérienne. / A reliable and up to date taxonomy is essential to unambiguously identify and differentiate human pathogenic microorganisms. Numerous new bacterial taxa have been created over the last years, meanwhile already known species or genus were reclassified, particularly concerning anaerobic bacteria. To clarify the taxonomic position of specific anaerobic bacteria displaying unusual antibiotic resistance, a polyphasic taxonomic approach combining the analysis of phenotypic, genotypic and phylogenetic markers has been used. This approach has been first applied to bacteria displaying low level resistance to vancomycin and belonging to the Clostridiaceae family. It resulted in the description of a new species of Tissierella as well as a new genus, Flavonifractor sp., which arises from the combination of two pre-existing species. Applied in a second time in a study on metronidazole resistance in Pretovella spp., this approach led to the discovery of a new species, Prevotella nanceiensis, the resistance of which to metronidazole has been implicated in therapeutic failure. A new gene (niml) coding a nitroimidazole reductase that seems to be intrinsic to the species Prevotella baroniae has also been described. This study emphasizes the importance of a clear and reliable taxonomy in medical bacteriology as well as the benefit of a polyphasic approach in taxonomy.

Anxiety disorders, including generalized anxiety disorder (GAD), are prevalent mental health conditions with a complex etiology. The gut microbiota and diet have emerged as important factors in modulating anxiety symptoms. This thesis aimed to investigate the relationship between gut microbiota composition and anxiety symptoms in the context of GAD. This involved an in silico analysis of a cohort of GAD patients and healthy controls, coupled with experiments using a microbiota-humanized mouse model. A comprehensive in silico analysis was conducted using basic statistics and machine learning techniques on the human cohort. The analysis explored the associations between anxiety symptoms and various factors, including demographics, dietary intake, gastrointestinal symptoms, inflammatory markers, stool metabolites, microbiota composition, and PICRUSt2 microbiota predicted function. GAD patients exhibited higher gut microbiota abundance of Bacteroides, which was positively associated to carbohydrate degradation pathways. Machine learning analysis identified abdominal pain as a key indicator for distinguishing GAD donors. Moreover, anxiety symptoms were negatively correlated with inulin intake in GAD patients, altogether suggesting an association between a carbohydrate degradation imbalance in the GAD microbiota, abdominal pain, and anxiety symptoms. To investigate further, germ-free mice were colonized with stool samples from healthy controls and GAD patients, and they were fed either a low or high-fiber (inulin) diet. Assessments included behavioral tests, microbiota analysis, colonic gene expression, and mucus degradation. Bacteroides abundance positively correlated with anxiety-like behavior, mucus degradation, and expression of colonic genes related to immune activation, pain, and intestinal permeability, further supporting the results observed in the donor cohort. Improvements in metabolic parameters were also observed in mice fed high-fiber diet. Furthermore, we found that inulin dosage played a crucial role in mediating the observed immune activation and anxiety-like behavior, with excessive inulin supplementation showing a detrimental effect. This study provides insights into the complex relationship between dietary fiber, gut microbiota composition, and anxiety symptoms in mice and humans. Further studies are needed to determine optimal dosages of inulin supplementation as a potential therapeutic approach for managing anxiety symptoms. / Dissertation / Doctor of Philosophy (Medical Science) / Generalized anxiety disorder (GAD) is a common anxiety disorder that has complex causes. There's growing evidence that gut bacteria, known as microbiota, along with diet, can impact anxiety symptoms. In our study, we explored this connection by examining a group of GAD patients and healthy individuals, and by conducting experiments using a mouse model. We found that GAD patients have a microbiota with higher levels of Bacteroides and higher ability to feed from carbohydrates compared to healthy controls. We believe this is associated to abdominal pain and higher anxiety symptoms. To explore this in more depth, we introduced the gut microbiota from GAD patients and healthy controls into mice with no previous microbiota. Because we also found that GAD patients who ate foods with more specific fiber (inulin) content had less anxiety symptoms, we fed them a low or high-inulin diet and assessed anxiety-like behavior. We found that Bacteroides levels were associated with high anxiety-like behavior and gene expression in the colon associated to inflammation and pain in mice, further supporting the results found in the humans. Although more research is still needed, this study helps us better understand how the interaction between dietary fiber and gut bacteria can affect anxiety.

Prevotella intermedia e Prevotella nigrescens são espécies comumente associadas Porphyromonas gingivalis. os objetivos foram verificar a co-agregação entre cepas de P. intermedia, P. nigrescens e P. gingivalis; quantificar a biomassa, avaliar a proporção dos microrganismos nos biofilmes mistos; verificar a interferência do co-cultivo pela técnica de dois compartimentos, avaliar os biofilmes em ensaios de Hibridização In Situ (FISH) e verificar o papel dos genes PINA0102 e PIN0398 de P. intermedia no biofilme. Assim, 9 isolados clínicos de P. intermedia e 5 de P. nigrescens, as cepas padrão P. intermedia 17, P. intermedia 25611, P. nigrescens 33563, P. gingivalis W83, P. gingivalis 33277, os mutantes Pi17D0398 e Pi17D0102 foram avaliados em variados consórcios. Os resultados mostraram que as interações investigadas são cepa específicas. / Prevotella intermedia and Prevotella nigrescens are commonly associated with periodontal diseases. The goals of this study were to determine the co-aggregation of strains of P. intermedia, P. nigrescens and P. gingivalis; to quantify the biomass of these associations, to evaluate the ratio of these microorganisms in heterotypic biofilms, to verify the modulation of biofilms in co-culture using a trans-well system; to evaluate the structure of the biofilms by Fluorescent Hybridization In Situ (FISH) and to determine the role of genes PINA0102 and PIN0398 of P. intermedia in the modulation of its biofilm. Therefore, 9 clinical isolates of P. intermedia, 5 of P. nigrescens, type strains P. intermedia 17, P. intermedia 25611, P. nigrescens 33563, P. gingivalis W83, P. gingivalis 33277 and mutant strains Pi17D0398 and Pi17D0102 were grown in consortia of two strains. Our data demonstrate that the associations of P. intermedia, P. nigrescens and P. gingivalis are strain-specific.

Prevotella intermedia e Prevotella nigrescens são espécies comumente associadas Porphyromonas gingivalis. os objetivos foram verificar a co-agregação entre cepas de P. intermedia, P. nigrescens e P. gingivalis; quantificar a biomassa, avaliar a proporção dos microrganismos nos biofilmes mistos; verificar a interferência do co-cultivo pela técnica de dois compartimentos, avaliar os biofilmes em ensaios de Hibridização In Situ (FISH) e verificar o papel dos genes PINA0102 e PIN0398 de P. intermedia no biofilme. Assim, 9 isolados clínicos de P. intermedia e 5 de P. nigrescens, as cepas padrão P. intermedia 17, P. intermedia 25611, P. nigrescens 33563, P. gingivalis W83, P. gingivalis 33277, os mutantes Pi17D0398 e Pi17D0102 foram avaliados em variados consórcios. Os resultados mostraram que as interações investigadas são cepa específicas. / Prevotella intermedia and Prevotella nigrescens are commonly associated with periodontal diseases. The goals of this study were to determine the co-aggregation of strains of P. intermedia, P. nigrescens and P. gingivalis; to quantify the biomass of these associations, to evaluate the ratio of these microorganisms in heterotypic biofilms, to verify the modulation of biofilms in co-culture using a trans-well system; to evaluate the structure of the biofilms by Fluorescent Hybridization In Situ (FISH) and to determine the role of genes PINA0102 and PIN0398 of P. intermedia in the modulation of its biofilm. Therefore, 9 clinical isolates of P. intermedia, 5 of P. nigrescens, type strains P. intermedia 17, P. intermedia 25611, P. nigrescens 33563, P. gingivalis W83, P. gingivalis 33277 and mutant strains Pi17D0398 and Pi17D0102 were grown in consortia of two strains. Our data demonstrate that the associations of P. intermedia, P. nigrescens and P. gingivalis are strain-specific.

Bacteroides melaninogenicus strains 2D and K110 were characterized with regard to their pathogenic, collagenolytic, proteolytic, hemagglutinating and metabolic activities. Both strains were members of the subspecies 13. melaninogenicus ss. asaccharolyticus. They possessed a cell-bound oxygen-sensitive collagenase, a cell-bound and a soluble oxygen-sensitive hemagglutinin (HA), and a protease. Both strains produced butyric and phenylacetic acids and were infective in guinea pigs as characterized by their ability to produce necrotic lesions and to be transferred from one animal to another. Strain 2D required hemin for growth and its growth rate was influenced by the addition of free amino acids to the medium. The hemagglutinating and proteolytic activities of strain 2D were investigated further to determine their relationship to infection. The soluble HA was reversibly inhibited by Hg and activity was restored in the presence of reducing agents. Iodoacetic acid caused irreversible inhibition. The HA was sensitive to heat and pronase treatment. Treatment of the red blood cells (RBC) with neuraminidase enhanced HA activity while the presence of galactose in the reaction mixture inhibited it, suggesting the involvement of galactose residues on the RBCs in the reaction.. Adsorption of the HA to RBC followed by elution and gel filtration resulted in the recovery of 50% of the HA activity and a 52-fold purification. Protease production by _B. melaninogenicus strain 2D was dependent on the growth rate of the organism. The protease was reversibly inhibited by HgCl₂ and irreversibly inhibited by iodoacetamide and iodoacetic acid. The enzyme was insensitive to serine protease inhibitors and EDTA. The pH optimum for proteolytic activity was 7.0, which correlates with the pH of its natural environment, the gingival crevice. It is thus classified as a neutral sulfhydryl enzyme. A 774-fold purification of the cellular protease of 2D, with a 160% recovery of activity, was accomplished by precipitation with 60% ethanol, ultracentrifugation and gel filtration through Sephadex G-100 and Sepharose 2B in the presence of urea. Electrophoretic analysis of the protease on SDS-polyacrylamide gels revealed four distinct bands, each of which was shown to be associated with carbohydrate. In the absence of SDS only one band, which did not migrate into the gel, was obtained. Any attempts to further dissociate the protease resulted in the loss of activity. The protease was active against azocoll, azocasein, casein and N,N-dimethylcasein. No glycosidase, lipase, collagenase or HA activities were detected. Protein, carbohydrate and lipid were detected in the preparation. The soluble protease which amounted to 20% of the cellular protease of strain 2D was subjected to gel filtration on Sephadex G-100 and eluted in a single peak at the void volume. The properties of the soluble protease were identical to those of the cell associated enzyme, suggesting the presence of a single proteolytic enzyme which was released into the culture medium with cell lysis or due to shedding of outer membrane fragments. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

P. gingivalis é um dos principais patógenos da doença periodontal, é encontrado em biofilmes orais com S. gordonii e P. intermedia e em células endoteliais da artéria coronária in vivo. P. gingivalis necessita de ferro em seu metabolismo e pode usar certas proteínas do hospedeiro como fontes deste íon em ambientes limitantes. Assim, este estudo investigou o papel dos genes PGN0741/PG0637 (receptor dependente de TonB) e PGN0531/PG1380 (fvW) de P. gingivalis na formação de biofilme em diferentes concentrações de ferro, em biofilmes mistos com S. gordonii e P. intermedia, e na adesão e invasão de células endoteliais da artéria coronária. Os resultados mostraram divergências no papel dos genes TonB e fvW na formação dos monobiofilmes e mistos e em diferentes concentrações de ferro, demonstrando uma relação cepa-dependente. Na adesão, fvW se mostrou importante para ambas cepas, mas na persistência apenas para P. gingivalis W83. Este trabalho enfatiza, assim, a necessidade do uso de mais de uma cepa de P. gingivalis no estudo do papel de genes em ensaios experimentais. / P. gingivalis is one of the major pathogens of periodontal diseases. It is found in oral biofilms associated with S. gordonii and P. intermedia, and inside of coronary artery endothelial cells in vivo. P. gingivalis requires iron for growth and can exploit iron-carrying proteins of the host as sources in limiting environments. Thus, this work aimed to study the role of genes PGN0741/PG0637 (TonB-dependent receptor) and PGN0531/PG1380 (fvW) of P. gingivalis in biofilm formation under different iron concentrations, in mixed biofilms with S. gordonii and P. intermedia, and in the adhesion and invasion of coronary artery endothelial cells. Our data showed discordance for the role of TonB and fvW in homo- and heterotypic biofilm formation and in different iron concentrations. The relevance of both genes was strain-dependent. Gene fvW was relevant for adhesion to endothelial cells, but only for strain W83 during persistence. Therefore, our study emphasizes the importance of using different strains for a better understanding of the role of genes in experimental assays.

P. gingivalis é um dos principais patógenos da doença periodontal, é encontrado em biofilmes orais com S. gordonii e P. intermedia e em células endoteliais da artéria coronária in vivo. P. gingivalis necessita de ferro em seu metabolismo e pode usar certas proteínas do hospedeiro como fontes deste íon em ambientes limitantes. Assim, este estudo investigou o papel dos genes PGN0741/PG0637 (receptor dependente de TonB) e PGN0531/PG1380 (fvW) de P. gingivalis na formação de biofilme em diferentes concentrações de ferro, em biofilmes mistos com S. gordonii e P. intermedia, e na adesão e invasão de células endoteliais da artéria coronária. Os resultados mostraram divergências no papel dos genes TonB e fvW na formação dos monobiofilmes e mistos e em diferentes concentrações de ferro, demonstrando uma relação cepa-dependente. Na adesão, fvW se mostrou importante para ambas cepas, mas na persistência apenas para P. gingivalis W83. Este trabalho enfatiza, assim, a necessidade do uso de mais de uma cepa de P. gingivalis no estudo do papel de genes em ensaios experimentais. / P. gingivalis is one of the major pathogens of periodontal diseases. It is found in oral biofilms associated with S. gordonii and P. intermedia, and inside of coronary artery endothelial cells in vivo. P. gingivalis requires iron for growth and can exploit iron-carrying proteins of the host as sources in limiting environments. Thus, this work aimed to study the role of genes PGN0741/PG0637 (TonB-dependent receptor) and PGN0531/PG1380 (fvW) of P. gingivalis in biofilm formation under different iron concentrations, in mixed biofilms with S. gordonii and P. intermedia, and in the adhesion and invasion of coronary artery endothelial cells. Our data showed discordance for the role of TonB and fvW in homo- and heterotypic biofilm formation and in different iron concentrations. The relevance of both genes was strain-dependent. Gene fvW was relevant for adhesion to endothelial cells, but only for strain W83 during persistence. Therefore, our study emphasizes the importance of using different strains for a better understanding of the role of genes in experimental assays.