Clostridium difficile is a Gram positive pathogen of significant importance in the UK, Europe and the USA. No vaccine has been developed and current treatments are focused on hospital management and the use of antibiotics. The disease is spread in hospitals in the spore form and the role of spores in C. difficile infecton is poorly understood. In this project spores of C. difficile have been characterised. The proteins from the outermost layers of the spore were identified and the genes cloned. Three of these surface proteins have unique enzymatic properties that maybe important for symptoms of disease. The ability of C. difficile spores to adhere to intestinal cells was found to be far greater than with live cells and through this we have identified that the spore may play an important role in colonisation. The regulation of spore coat gene expression during sporulation was also examined and temporal phases of genes expression identified. A major part of this project was to develop a mucosal vaccine to C. difficile. The approach used was to clone the C-terminus of toxin A onto the surface of Bacillus subtilis spores and use these recombinant spores to immunise mice and hamsters. We found that oral delivery of these spores conferred 75% protection to C. difficile infection in a hamster model of infection. Further, parenteral immunisation of the same antigens (toxin A and B) failed to generate mucosal responses and this showed that mucosal immunisation is critical for good protection. Finally, we found that antibodies to the C-terminus of toxin A were cross reactive to the C-terminus of toxin B. This showed that mucosal delivery of just the C-terminus of toxin A is sufficient to confer protection in an animal model of infection. The outcome of this work is that we have shown the parameters for successful immunisation and vaccination against C. difficile.

Clostridium difficile is the most common antibiotic related diarrhéa disease in Sweden. New recommendations from the Swedish public health authority and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) had led to that a more advanced diagnostic algorithm is of priority. Hence this study, whose purpose was to investigate whether the performance of the rapid test C. diff Quik Chek Complete® could enable the introduction of a new diagnostic algorithm for detection of toxin-forming C. difficile in laboratory medicine in Sundsvall, according to these new recommendations. In the study 119 patient stool-samples were analysed with both GeneXpert and C. diff Quik Chek Complete® and these two combined fulfils these new recommendations of detecting toxin A and B from toxigenic C. difficile together with the enzyme Glutamate Dehydrogenase (GDH) which is produced by all C. difficile stems. The results shows that C. diff Quik Chek Complete® is well matched with GeneXpert and that most of the samples would come to be answered immediately after analysis with C. diff Quik Chek Complete®. The laboratory will save both time and money to establish C. diff Quik Chek Complete® in their algorithm for diagnosing C. difficile infection.

Clostridium difficile est une bactérie pathogène responsable de diarrhées nosocomiales et de la plupart des colites pseudomembraneuses. Le principal facteur de risque est la prise d’antibiotiques qui altère la composition du microbiote intestinal, et favorise ainsi l’implantation de la bactérie au niveau colique. Après une étape de colonisation, la bactérie produit ses principaux facteurs de virulence, les toxines A et B. La colonisation est un processus multifactoriel, qui met en jeu différentes protéines de surface dont des adhésines et une cystéine protéase Cwp84.Dans une première partie, nous avons analysé le processus de maturation de la protéase Cwp84, ainsi que sa localisation dans la bactérie, afin de mieux comprendre son rôle dans la virulence de C. difficile. La protéase recombinante, purifiée sous forme de zymogène, présente un processus de maturation particulier comprenant des clivages successifs, qui aboutissent à la forme mature de 47 KDa. La protéase ainsi activée présente une activité protéolytique sur la fibronectine. Dans la bactérie, Cwp84 existe sous deux formes majoritaires, associées à la surface de la bactérie : une première forme, d’environ 80 KDa, associée aux protéines de la couche S, dont le rôle serait de cliver le précurseur des protéines de la couche S en deux protéines matures ; une deuxième forme, d’environ 50 KDa correspondant vraisemblablement à la forme mature de la protéase recombinante de 47 KDa, est retrouvée à la fois dans la fraction extracellulaire et associée à la surface de la bactérie. Nous avons montré que la protéase rélarguée est capable de se ré-associer sous sa forme mature de manière spécifique à la surface de C. difficile. Dans une deuxième partie, nous avons analysé l’impact de conditions environnementales mimant celles rencontrées par la bactérie au cours de son transit dans le tractus digestif de l’hôte, sur la modulation de facteurs de colonisation, dont la protéase. Nous avons montré qu’un pH acide favorise à la fois l’expression et le processus de maturation de la protéase vers sa forme mature de 47 KDa. Des analyses protéomiques et transcriptomiques ont montré que d’autres protéines impliquées dans colonisation sont surexprimées dans un milieu avec glucose, cette régulation étant vraisemblablement liée à la diminution du pH résultant de la fermentation du glucose plutôt qu’à un effet direct de ce sucre. Cette régulation des facteurs de virulence par le pH acide est probablement un élément favorable au processus de colonisation de l’hôte. Ces différentes analyses ont également permis l’identification de facteurs de virulence potentiels, qui devront être caractérisés par la suite. / Clostridium difficile, a gram-positive spore-forming, anaerobic bacterium, is the etiological agent of pseudomembranous colitis and of many cases of nosocomial diarrhea. The main risk factor is the use of antibiotics that alters the intestinal microbiota, predisposing to C. difficile intestinal colonization. C. difficile pathogenicity is mediated mainly by its A and B toxins, secreted after host colonization that involves various surface proteins, including different adhesins and proteolytic enzymes as the cysteine protease Cwp84.We sought to analyze the localization and the maturation process of the proteaseCwp84. We showed that the recombinant protein Cwp8430-803, purified as zymogen form, presents a particular maturation process including consecutive cleavages, leading to the mature form of 47 kDa. This protease has a proteolytic activity against the fibronectin. Two identifiable forms of the protease were found to be associated in the bacteria: a form of about 80 kDa and a cleaved one of 47 kDa, identified as the mature protease. They were found mainly in the bacterial cell surface fractions, and weakly in the extracellular fraction. The 80 kDa protein was non covalently associated to the S-layer proteins, while the 47 kDa form was found to be tightly associated with the underlying cell wall. Our data supported that the anchoring of the Cwp84 47 kDa form is presumably due to a re-association of the secreted protein.We also studied the regulation of virulence factors depending of environmental conditions that mimic those encountered by the bacterium in the digestive tract. We showed that an acidic pH affects the expression and the proteolytic process of Cwp84. The mature form was only recovered with an acidic pH. Proteomic and transcriptomic analysis of some surface proteins involved in colonization revealed that their expression was increased in media containing glucose. However, this regulation is probably related to the decrease in pH resulting from fermentation of glucose, rather than a direct effect of glucose. The acidic pH could lead in vivo to modulation of virulence factors expression and is probably a favorable feature in the colonisation process. We also identified new surface associated-proteins, that could represent potential virulence factors; they will be characterized later.

Clostridium difficile colitis (CDC) is associated with a higher risk of acute death among hospitalized ulcerative colitis (UC) patients. However, the risk of colectomy with CDC in these patients has varied across studies. No study has assessed the long-term health impact of CDC in UC patients. Therefore, the present study evaluated the impact of CDC on five-year health outcomes of hospitalized UC patients based on Ontario health administrative data. No overall association was observed between CDC and five-year risks of colectomy or death in overall cohort. However, patients who were discharged from hospital without undergoing colectomy demonstrated marginally higher five-year risks of colectomy and hospital re-admission. Mortality risk and length of stay during index hospitalization were also higher in patients with CDC. Analysis of a parallel cohort of UC patients derived using a published case definition corroborated most of these results, but demonstrated a higher five-year mortality risk with CDC.

Clostridium difficile colitis (CDC) is associated with a higher risk of acute death among hospitalized ulcerative colitis (UC) patients. However, the risk of colectomy with CDC in these patients has varied across studies. No study has assessed the long-term health impact of CDC in UC patients. Therefore, the present study evaluated the impact of CDC on five-year health outcomes of hospitalized UC patients based on Ontario health administrative data. No overall association was observed between CDC and five-year risks of colectomy or death in overall cohort. However, patients who were discharged from hospital without undergoing colectomy demonstrated marginally higher five-year risks of colectomy and hospital re-admission. Mortality risk and length of stay during index hospitalization were also higher in patients with CDC. Analysis of a parallel cohort of UC patients derived using a published case definition corroborated most of these results, but demonstrated a higher five-year mortality risk with CDC.

Nosocomial infections, which do not often relate to the diseases are increasing nowadays. Clostridium difficile belongs to the frequent nosocomial infections and it is known as post-antibiotic colitis. The main reason of colitis is the usage of antibiotics, especially broad-spectrum antibiotics. The thesis is divided into the theoretical part and practical, as well. Theoretical part describes the division of the nosocomial infections, infection of the intestinal tract, anatomy, physiology of the intestines and infectious diarrhoeal diseases. The thesis is subsequently aimed to the clostridial infections and precautions against the spread of the disease. Practical part is aimed to the knowledge of the nurses, skills and attitude towards this issue.The thesis uses quantitative investigation and technique of the questionnaires, hidden observation of the nurses working on the selected wards and additional interviews with head nurses. The research was conducted in hospital in Tábor, a.s. The questionnaires were distributed on the surgery, orthopaedics, surgical JIP, ARO, ONP, infective ward, rehabilitative ward, TRN, cardio JIP, internal ward-cardio, internal ward-gastro. The thesis was formed from 143 questionnaires and 171 questionnaires were distributed. Hidden observation was made by head nurses from individual wards and it was logged to the relevant observation sheets.From existing findings we can say that there exist specifics of nursing care at the patient with the clostridium difficile. Among to these specifics we can cite the barrier nursing care where we can include the isolation of the patient, disinfection and hygiene of hands,using protectors, appropriate usage of laundries and infectious waste, location of the patient according to the epidemiological perpective and individualization of the tools for the patients. From another investigation ensue that the nurses keep barrier nursing care, superficial disinfecion, decontamination of the tools. From the results is evident that the nurses do not know the methods of the transmission of the clostridial infection. On the base of another investigations we have found out that the nurses do not know principles of the barrier nursing care. In conclusion is it possible to say that the nurses do not have so extensive information, that are essential for care for the patiens with clotridium difficile. In order to care for these patients in right way is neccessary to know principles of the barrier nursing care and keep them all. Keeping the principles of the barrier nursing care is crucial step in preventing the transmission nosocomial infections. The results will be provided to the officials of the individual hospital´s wards as an option of improvement in caring for the patiens with clostridial infection. The results were partially presented at a conference in Tabor´s hospital in May 2014. We recommend to re-train the staff of the hospital, which would be specifically aimed towards the principles of the barrier nursing care and towards the disinfection and decontamination in related to the nosocomial infections. On the base of these findings was made a proposal of the nursing care standard, which would specify and unite the care for the patiens with clostride infection. Subsequently, it would be apropriate to repeat the research in 1 2 years and than both researches compare together.

La proline est un acide aminé unique qui se caractérise par un cycle pyrrolidine qui lui confère des propriétés physiques et chimiques spécifiques comparé aux autres 20 acides aminés acycliques retrouvés de façon majoritaire dans le protéome humain. Cette différence se retrouve également dans les motifs polyproline et est utilisée par la Nature en de nombreuses façons comme le repliement des protéines, les mécanismes régulatoires liés à l’activité de certaines protéases, la formation de structures secondaires, le biomarquage de peptides substrats des oligopeptidases, et le clivage des motifs diproline par les proline-proline endopeptidases. La proline a par conséquent été un centre d’intérêt pour les chimistes médicinaux lors du développement d'agents thérapeutiques. Cette thèse se propose d’étudier plusieurs composés biologiquement actifs dérivés de la proline. La première étude relate les avancements liés au composé SH-BC-893, un azacycle contraint dérivé de FTY720 développé au sein du groupe Hanessian. SH-BC-893 est un agent anticancéreux qui permet l’affamement des cellules cancéreuses au travers de la disruption de l’apport des nutriments cellulaires externes et internes. Par son activation de la protéine phosphatase 2A, il permet d’internaliser les récepteurs transmembranaires d’acides aminés et de glucose et de réguler à la baisse les récepteurs de lipides à faible densité. Son action permet également la perturbation de la dégradation de nutriments internes provenant de la macropinocytose et de l’autophagie en prévenant la fusion entre le lysosome et les endosomes respectifs. Afin d’augmenter l’activité de SH-BC-893, deux séries de composés ont été synthétisés et testés en vue d’une activité double. La première série a eu pour but de cibler HDAC2 suite à une étude de Spiegel au sein de laquelle son inhibition a été rapportée pour FTY720. La seconde a visé à obtenir des composés activant d’autres portions ou d’autres homologues de PP2A via l’insertion de fragments tricycliques suite aux études rapportant l’activation de PP2A par des dérivés de composés neuroleptiques tricycliques du type de la perphénazine. La synthèse et les tests biologiques de ces composés sont également décrits. Le deuxième chapitre décrit la synthèse d’une série de morpholines pontées comportant le motif de l’acide gamma-aminobutyrique (GABA) contraint. GABA est un composant prépondérant de la régulation du système nerveux central au travers de son action inhibitrice sur les neurorécepteurs GABA-A, GABA-B, GABA-C. De nombreux agents thérapeutiques inspirés de GABA ont été développés avec les années, notamment baclofen. Les morpholines pontées rapportées ici possèdent un centre quaternaire stéréocontrôlé pour lequel un ensemble de substituants a été varié, incluant le groupement phényle para-chloré du baclofen ainsi que l’iso-butyle de la Pregabalin, autre composé actif dans le système nerveux central dérivé de GABA. La modélisation du dérivé du baclofen a été réalisée au sein du site actif de GABA-B. L’ensemble des composés ont également été modélisés sous forme d’acides aminés au sein de la base de données LLAMA afin de déterminer leur capacité à occuper l’espace de Lead-likeness et confirmer qu’ils satisfont les conditions de Lipinski. La troisième partie décrit la synthèse d’un nouveau mime de diproline, nommé ProCyp. Le dimère est composé d’un noyau pyrrolidine attaché à une unité cyclopentane à l’aide d’un pont méthylène hydroxylé. Les quatre isomères cyclopentanes trans dérivés de la (L)-proline ont été synthétisés et leur stéréochimie absolue a été déterminée par étude RMN et de cristallographie. Ces composés représentent la première forme de mimes de diproline de type ProCyp et peuvent être intégrés au sein de peptidomimétiques afin de mimer l’aspect structurel des diprolines ainsi que l’intermédiaire tétrahédral des ProPro endopeptidases. L’ensemble des isomères disponibles permet de choisir le plus opportun à l’appuis d’études de modélisation. Le dernier chapitre rapporte le développement de deux séries de composés incluant le dimère ProCyp. Les motifs polyproline sont de prime importance pour de nombreux mécanismes de reconnaissance par la Nature, en particulier dans les voies de signalisation dont le dérèglement peut entrainer une myriade de troubles de santé notamment inflammatoires, immunitaires et cancéreux. La première application est centrée sur la synthèse et les tests biologiques de peptidomimétiques du motif riche en proline du peptide p22phox, sous-unité de la NADPH oxidase 2 (NOX2) dont la reconnaissance par le domaine riche en proline de p47phox permet l’assemblage de NOX2 nécessaire à son activité. Lors de stress cellulaire externe, cet assemblage peut devenir excessif et engendrer un excès de production d’espèces réactives d’oxygènes, entrainant un ensemble de biomécanismes délétaires pour l’hôte. Les peptidomimétiques comportent un cœur triproline, dont deux des trois prolines ont été remplacées par le module ProCyp. Le meilleur isomère a été sélectionné sur la base de la modélisation moléculaire afin de mimer les angles de torsion de la triproline correspondant à une hélice de type polyproline II. La deuxième application concerne le développement de peptidomimétiques inhibiteurs de la ProPro endopeptidase (PPEP-1) de Clostridium difficile (C. diff). C. diff a été identifiée comme une des menaces nosocomiales majeures en raison de son caractère opportuniste lorsque la flore intestinale des patients est détruite suite à un traitement impliquant la prise soutenue d’antibiotiques. C. diff possède un arsenal de mécanismes d’évasion et de prolifération au sein de l’hôte incluant la formation de colonies recouvertes d’un biofilm protecteur sur la paroi epithéliale de l’hôte lors de la réponse immunitaire. Ces mécanismes sont complétés à l’aide de peptidases qui leurs permettent de couper les flagelles d’ancrages, dont PPEP-1. Le site de scission P1-P1’ de PPEP-1 implique deux prolines, flanquées par une séquence peptidique clairement identifiée. Une série de peptides basés sur cette séquence dont la diproline centrale a été remplacée par le dimère ProCyp a été synthétisée. L’isomère ProCyp a été choisi sur la base de son recouvrement avec l’heptapeptide cristallisé au sein d’un mutant de PPEP-1 par le Pr. Baumann. / Proline is a unique amino acid characterized by a cyclic side chain that gives it specific physical and chemical properties compared to the other 20 acyclic amino acids found in the human proteome. This difference is also found in polyproline motifs and is used by Nature in many ways such as protein folding, regulatory mechanisms related to the activity of certain proteases, formation of secondary structures, biomarking of peptide substrates of oligopeptidases, and cleavage of diproline motifs by proline-proline endopeptidases. Proline has therefore been a focus of interest for medicinal chemists in the development of therapeutic agents. This thesis proposes to study several biologically active compounds derived from proline. The first study reports on the advances related to the compound SH-BC-893, a constrained azacycle derived from FTY720 developed within the Hanessian group. SH-BC-893 is an anti-cancer agent that allows the starvation of cancer cells through the disruption of external and internal cellular nutrient supply. Through its activation of protein phosphatase 2A, it internalizes transmembrane amino acid and glucose receptors and downregulates low-density lipid receptors. Its action also allows the disruption of internal nutrient degradation from macropinocytosis and autophagy by preventing fusion between the lysosome and the respective endosomes. To enhance the activity of SH-BC-893, two series of compounds were synthesized and tested for dual activity. The first set aimed at targeting HDAC2 following a study by Spiegel in which its inhibition was reported for FTY720. The second set aimed at obtaining compounds activating other portions or homologs of PP2A via the insertion of tricyclic fragments following studies reporting PP2A activation by derivatives of tricyclic neuroleptic compounds of the perphenazine type. The synthesis and biological tests of these compounds are also described. The second chapter describes the synthesis of a series of bridged morpholines with a constrained gamma butyric amino acid (GABA) motif. GABA is a major component of central nervous system regulation through its inhibitory action on GABA-A, GABA-B, GABA-C neuroreceptors. Many therapeutic agents inspired by GABA have been developed over the years, notably baclofen. The bridged morpholines reported here have a stereocontrolled quaternary center for which a variety of substituents have been used, including the para-chlorophenyl of baclofen and the iso-butyl of Pregabalin, another GABA-derived compound active in the central nervous system. The baclofen derivative was modeled within the GABA-B active site. All compounds were also modeled as amino acids within the LLAMA database to determine their ability to occupy the lead-likeness space and confirm that they satisfy the Lipinski conditions. The third part describes the synthesis of a new diproline mime, named ProCyp. The dimer is composed of a pyrrolidine ring attached to a cyclopentane unit by means of a hydroxylated methylene bridge. The four trans cyclopentane isomers derived from (L)-proline were synthesized and their absolute stereochemistry was determined by NMR and crystallographic studies. These compounds represent the first form of ProCyp-type diproline mimes and can be incorporated into peptidomimetics to mimic the structural aspect of diprolines as well as the tetrahedral intermediate of ProPro endopeptidases. The set of available isomers allows to choose the most appropriate one to support modeling studies. The final chapter reports the development of two series of compounds including the ProCyp dimer. Polyproline motifs are of prime importance for many of Nature's recognition mechanisms, particularly in signaling pathways whose dysregulation can lead to a myriad of health disorders including inflammatory, immune and cancerous. The first application is focused on the synthesis and biological testing of peptidomimetics of the proline-rich motif of the peptide p22phox, a subunit of NADPH oxidase 2 (NOX2) whose recognition by the proline-rich domain of p47phox allows the assembly of NOX2 necessary for its activity. During external cellular stress, this assembly can become excessive and lead to an excess of reactive oxygen species production, resulting in a series of deleterious biomechanisms for the host. The peptidomimetics comprise a triproline core, of which two of the three prolines have been replaced by the ProCyp module. The best isomer was selected on the basis of molecular modeling to mimic the torsion angles of the triproline corresponding to a polyproline II helix. The second application concerns the development of peptidomimetics that inhibit the ProPro endopeptidase (PPEP-1) of Clostridium difficile (C. diff). C. diff has been identified as one of the major nosocomial threats due to its opportunistic nature when the intestinal flora of patients is destroyed following treatment with sustained antibiotics. C. diff has an arsenal of mechanisms of escape and proliferation within the host including the formation of colonies covered by a protective biofilm on the host epithelial wall during the immune response. These mechanisms are complemented by peptidases that allow them to cleave anchoring flagella, including PPEP-1. The P1-P1' cleavage site of PPEP-1 involves two prolines, flanked by a clearly identified peptide sequence. A series of peptides based on this sequence with the central diproline replaced by the ProCyp dimer was synthesized. The ProCyp isomer was chosen on the basis of its overlap with the heptapeptide crystallized in a PPEP-1 mutant by Prof. Baumann.

No description available.

There is a pandemic of low-value clinical care that threatens the sustainability of our publicly funded healthcare systems. Over 30% of the health services provided to patients provide no benefit or may actually result in harm. Health services research is needed to critically evaluate our clinical practices and programs to ensure we create systems that consistently deliver high-value care. Unlike drug trials, health services research is complicated by enormous heterogeneity across cultures, environments, behaviours and systems. Ideally, local research communities should devise and conduct health services research to ensure that both the research questions and outcomes are relevant to community members, and thus more likely to result in sustainable healthcare systems. Embedded researcher models are emerging as a viable approach to supporting local research activities. Embedded researchers are part of the community they serve, provide research expertise to local investigators and community members, and help develop local research systems that facilitate health services research activities. While they may still collaborate with academic partners, this is not necessary for their research success. This thesis documents the transformation of a large community hospital in Ontario into a learning health centre through the use of an embedded researcher model. The first part of the thesis is focused on the results of incorporating an embedded research plan into the hospital’s new antimicrobial stewardship program. The research that emerges from this work contributes new knowledge about the value of antimicrobial stewardship to important patient outcomes such as reduced lengths of hospital stay and rates of Clostridium difficile infections. The thesis concludes with a discussion of the implementation of all the necessary components needed to support a learning health centre and how an embedded researcher model facilitated this transformation and could be used by any similar organization to achieve the same result. / Thesis / Doctor of Philosophy (PhD) / Over 30% of the health services provided by our healthcare systems does not benefit and may actually harm patients. Health services research is therefore a necessary activity required to reduce this waste. In Ontario, over 65% of patients receive their acute care in large community-based hospitals, and yet, these hospitals have minimal research activity and capacity despite repeated attempts by the academic research community to engage these institutions through a variety of collaborative models such as integrated knowledge translation. This thesis provides a blueprint for the transformation of a large community hospital into a learning health centre through the use of a locally created, locally relevant, embedded researcher model. Starting with a proof of concept through the systematic evaluation of an antimicrobial stewardship program, the thesis ends with a ‘how to’ guide for the implementation of the foundational elements needed to support health services research in similar organizations.

Clostridium difficile is a major cause of infectious diarrhea in hospitalized patients. Clostridium difficile-associated disease (CDAD) is of gaining importance now due to its increasing incidence and severity. However, little is known about the C. difficile infections in the Czech Republic. The aim of the study was to characterize C. difficile strains recently isolated (2008 to 2011) from patients hospitalized with gastrointestinal disease in four Prague health care institutions using molecular typing methods; PCR toxinotyping, PCR ribotyping and MLVA (multilocus variable number tandem repeat analysis). Among 273 C. difficile strains, we identified 8 toxinotypes (0, III, IV, V, VI, VIII, IX a XXIII) and 63 ribotypes, of which ribotypes 596 (23,4 % patient), 017 (13,9 %) and 176 (7 %) were the most frequent. According to PCR ribotyping, the situation in the Czech Republic is the most similar to the situation in Poland. Within ribotypes 017, 017/1 and 017/2 and ribotypes 596 and 596/1, 5 and 4 distinct clusters were identified by MLVA, none of which was institution-specific. Additionally, pathogenic C.difficile were isolated from piglet faeces (63,3 %) in a single piglet farm, evaluating the role of C. difficile as an emerging animal pathogen. All piglet isolates belonged to the toxinotype 0 and the ribotype...