Submitted by Renata Lopes (renatasil82@gmail.com) on 2018-06-26T10:52:38Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-06-27T15:27:12Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2018-06-27T15:27:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-05-25 / Introdução: A doença inflamatória intestinal (DII) é um grupo de doenças caracterizado por inflamação de natureza crônica e etiologia multifatorial, constituído principalmente pela doença de Crohn e pela retocolite ulcerativa. Tais doenças comprometem preferencialmente indivíduos jovens em fase produtiva, tem curso flutuante com fases de remissão e atividade, e frequentemente apresentam comportamento clínico agressivo, com impacto na qualidade de vida. O Clostridium difficile é um bastonete gram positivo, produtor de esporos, ubíquo na natureza e representa causa importante de diarreia associada ao uso de antimicrobianos, através da produção das toxinas A e B. A literatura tem relatado maior ocorrência da infecção por C. difficile (ICD) em pacientes com DII, o que aumenta o risco de recorrência da DII e pior evolução. Objetivo: Avaliar a prevalência, os aspectos epidemiológicos, os fatores associados e a evolução clínica da infecção por C. difficile em pacientes ambulatoriais com DII. Métodos: Neste estudo prospectivo longitudinal, realizado entre outubro de 2013 a julho de 2016, com 120 pacientes com DII (55% apresentando colite) e 40 controles não-DII foram avaliados para ICD, através de análise de amostras fecais para pesquisa de toxinas do C. difficile, através do teste de ELISA. Foi avaliado fatores de riscos associados a ICD. A regressão multivariada foi realizada para identificar preditores de ICD. Resultados: A proporção de pacientes com ICD foi significativamente maior em pacientes com DII em atividade que DII em remissão (28,8% vs. 5,6% vs. 0%, respectivamente, p = 0,001). Feminino (OR = 1,39, IC 95%, 1,13-17,18), idade mais jovem (OR = 0,77, IC 95%, 0,65-0,92), tratamento com esteróides (OR = 7,42, IC 95%, 5,17-40,20) e terapia com infliximabe (OR = 2,97, IC 95%, 1,99-24,63) foram independentemente associados com ICD. Houve aumento nas probabilidades de ter ICD em pacientes com prednisona. Conclusões: A ICD é uma condição altamente concorrente em pacientes com DII apresentando exacerbação de colite a nível ambulatorial. O sexo feminino, a idade mais jovem, a terapia com infliximabe e o uso de esteróides foram associados de forma independente à co-ocorrência de ICD. A maioria dos pacientes com ICD apresentaram doença leve a moderada e o tratamento com vancomicina foi muito efetivo, parecendo reduzir o risco de complicações graves relacionadas à ICD. / Introduction: Inflammatory bowel disease (IBD) comprises a group of diseases of multifactorial etiology, characterized by chronic and progressive inflammation of the gastrointestinal tract, consisting mainly of Crohn's disease and ulcerative colitis. Clostridium difficile is a gram-positive, spore-producing rod that is ubiquitous in nature and is an important cause of diarrhea associated with the use of antimicrobials and in immunocompromised patients. The literature has reported a higher occurrence of Clostridium difficile infection (CDI) in patients with IBD, leading to significant morbidity and mortality. Objective: To evaluate the prevalence, epidemiological aspects and clinical evolution of CDI in outpatients with recurrent IBD. Methods: In this prospective longitudinal study, conducted between October 2013 and July 2016, 120 patients with IBD (55% with colitis flare) and 40 non-IBD controls were evaluated for CDI by searching for toxins A and B in feces fresh by ELISA. All patients with CDI were treated with oral vancomycin and followed up for an additional six months. Multivariate regression was performed to identify predictors of CDI. Results: The proportion of patients with CDI was significantly higher in patients with active IBD than in those with IBD in remission or in non-IBD controls (28.8% vs. 5.6% vs. 0%, respectively, p = 0.001). Females (OR = 1.39, 95% CI, 1.13-17.18), younger age (OR = 0.77, 95% CI, 0.65-0.92), steroid treatment (OR = 7.42, 95% CI, 5.17-40.20) and infliximab therapy (OR = 2.97, 95% CI, 1.99-24.63) were independently associated with CDI. There was a dose-related increase in odds of having CDI in patients using prednisone. All patients treated with vancomycin responded to therapy, but 21% had recurrent CDI and 16% were hospitalized. Neither colectomy nor mortality was noticed.Conclusions: CDI is a highly concurrent condition in outpatients IBD with colitis flare. Females, younger age, infliximab therapy, and steroid use were independently associated with the cooccurrence of IBD. Most patients with CDI had mild to moderate disease and vancomycin treatment was very effective, seeming to reduce the risk of serious complications related to CDI.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-04-19T18:55:50Z No. of bitstreams: 1 Ferreira, Thaís Gonçalves [Dissertação, 2012].pdf: 3520619 bytes, checksum: 0a743346b244c2aef9fcdf90c62f99ae (MD5) / Made available in DSpace on 2017-04-19T18:55:50Z (GMT). No. of bitstreams: 1 Ferreira, Thaís Gonçalves [Dissertação, 2012].pdf: 3520619 bytes, checksum: 0a743346b244c2aef9fcdf90c62f99ae (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro / Ministério da Ciência e Tecnologia (MTC/Pronex) / Ministério da Saúde do Brasil / INTRODUÇÃO: Clostridium difficile é um importante patógeno entérico e o agente etiológico da diarreia associada ao C. difficile (CDAD). Pacientes com CDAD excretam uma grande quantidade de células vegetativas e esporos em suas fezes, levando a contaminação do ambiente hospitalar e propagação deste patógeno. Este fato pode ser explicado pela resistência dos esporos a muitos desinfetantes utilizados na rotina de desinfecção hospitalar. O objetivo deste estudo foi avaliar a atividade de desinfetantes hospitalares contra os esporos e células vegetativas de C. difficile. Além disso, foram analisados os perfis de proteínas totais das células vegetativas das cepas de C. difficile, tratadas com os mesmos desinfetantes. MÉTODOS: Os agentes de limpeza hospitalar Virkon® (peroxigênio), Cloro-Rio® (agente liberador de cloro ativo), Peresal® (peroxigênio), Riohex® (biguanida), e Cidex Opa® (aldeído), comumente utilizados em hospitais brasileiros, foram testados contra os esporos das cepas de C. difficile HU17- ribotipo 133 e SJ1-ribotipo 135, ambos ribotipos encontrados exclusivamente no Brasil. A cepa hipervirulenta de C. difficile, BI/NAP1/027, foi utilizada para comparação. Os testes foram realizados de acordo com o método padrão para teste da atividade esporocida de desinfetantes dos Estados Unidos, ASTM E2414-05. Para a análise do perfil de proteínas totais, as mesmas cepas de C. difficile, incluindo também a ATCC 9689, foram crescidas na presença e na ausência de concentração subinibitória dos desinfetantes citados e submetidas a eletroforese em gel de SDS-PAGE. RESULTADOS: Os agentes Cloro-Rio® e Cidex Opa® eliminaram completamente os esporos de todas as cepas testadas, enquanto o Riohex® não exibiu qualquer redução significante. Por outro lado, o Virkon® reduziu significativamente a concentração de esporos para as cepas HU17-133 e BI/NAP1/027, mas o mesmo não foi observado com a cepa SJ1-135. O agente Peresal® eliminou completamente os esporos da cepa HU17-133, mas apenas reduziu a concentração inicial dos esporos para as cepas C. difficile SJ1-135 e BI/NAP1/027. Os desinfetantes Cloro- Rio® e Riohex® foram os que mais afetaram a expressão de proteínas em todas as cepas avaliadas. CONCLUSÕES: Em conjunto, nossos resultados mostraram que o Cloro-Rio® e Cidex Opa® foram os desinfetantes mais eficazes para eliminação dos esporos de C. difficile. O estudo das proteínas afetadas pelos desinfetantes poderia ajudar a elucidar a resistência do C. difficile frente a alguns agentes de limpeza e criar novas alternativas para eliminar e/ou diminuir a contaminação do ambiente hospitalar por esta bactéria / Introduction: Clostridium difficile is an important enteric pathogen and the etiological agent of C. difficile-associated diarrhea (CDAD). Patients with CDAD, excrete a large amount of vegetative cells and spores in their stools, leading to contamination of the hospital environment and spread of the pathogen. This fact can be explained by the resistance of spores to many disinfectants used in hospital routine. The aim of this study was to evaluate the activity of hospital disinfectants against C. difficile spores and vegetative cells. Furthermore were analyzed the whole proteins profiles of the vegetative cells of these strains treated with the same disinfectant. METHODS: The hospital cleaning agents Virkon® (peroxygen), Cloro-Rio® (chlorine), Peresal® (peroxygen), Riohex® (biguanide), and Cidex Opa® (aldehyde), usually used in Brazilian hospitals, were tested against C. difficile strains spores HU17-ribotype 133 and SJ1-ribotype 135, both exclusively Brazilian. BI/NAP1/ribotype 027 strain was also used for comparison. The tests were performed in accordance with the standard method for testing the sporicidal activity of disinfectants of the United States, ASTM E2414-05. For the analysis of whole proteins profile, the strains were grown in the presence and absence of sub inhibitory concentrations of the disinfectants and applied on SDS-PAGE gel. RESULTS: Cloro-Rio® and Cidex Opa® completely eliminated spores of all strains tested, while Riohex® did not show any significant reduction. On the other hand, Virkon® significantly reduced HU17 and BI/NAP1/027 spores, but the same was not observed with SJ1 strain. The agent Peresal® completely eliminate the spores of strain HU17-133, but only reduced the initial concentration of spores for strains SJ1-135 and BI/NAP1/027. The disinfectants Cloro-Rio® and Riohex® were the most affected protein expression in all strains evaluated. CONCLUSIONS: Taken together, our results show that Cloro-Rio® and Cidex Opa® are the most remarkable agents for eliminating spores. The study of proteins affected by the disinfectant might help to elucidate the resistance of C. difficile against some cleaning agents and create new alternatives to eliminate and/or reduce the contamination of the hospital by this bacterium

Clostridium difficile is the most common bacterial cause of infectious diarrhoea in healthcare environments and in 2014 was responsible for 13,785 infections in the UK. C. difficile infection (CDI) is spread via the faecal-oral route and by contact with contaminated surfaces. However, despite the healthcare concerns no tests are available to validate if sufficient cleaning has been conducted. In addition, Polymerase Chain Reaction (PCR) and Enzyme Immunoassays (EIAs)-based tests used to diagnose CDI lack sensitivity and specificity and hence false negative results are commonly obtained. To overcome these concerns the aim of the PhD research has been to develop the first diagnostic test that exploits the specific interactions of C. difficile bacteriophages (phages), viruses that specifically infect and kill C. difficile. In order to develop a C. difficile phage-based test, first suitable phages that can be used for the test were identified and this was conducted by screening 35 different C. difficile phages against 160 clinically relevant C. difficile isolates. Five phages were found to infect the most number of isolates and were investigated further to identify whether a phage-based diagnostic could be developed based on phages binding (adsorption) to different C. difficile subgroups. However, for all five phages, adsorption rates were not consistently high for C. difficile subgroups in comparison to other common bacteria found in similar locations to C. difficile. Therefore, to increase specificity of the phage-based diagnostic test a new approach was taken by tagging two phages with luminescence luxAB genes (reporter phages), which would be expressed once C. difficile cells were infected with the phages. To design the C. difficile reporter phages, non-essential phage genes were replaced with the luxAB genes, but this study revealed mutagenesis of C. difficile was troublesome and extensive optimisation was required. In addition, once the reporter phages had successfully been constructed the luxAB genes were unstable within the phage genome and were lost during phage replication. Despite extensive optimisation and due to time constrains the luxAB genes were not stabilised within the phages but future work will focus on stabilising the genes.

Community-acquired Clostridium difficile infections have recently been increasing in incidence and severity. Several studies have isolated C. difficile spores from livestock and retail meats, suggesting that food may play a role in transmission. No research has been done, however, on what food conditions might allow for the survival and/or growth of the bacterium. We therefore modelled the minimum thresholds for C. difficile growth under low pH, water activity (aw), and temperature. We also sampled retail ground meats, cheese, and milk for the presence of C. difficile spores and subtyped food isolates for comparison with clinical strains. We found that C. difficile growth could be prevented by refrigeration temperatures. C. difficile spores were also detected for the first time in Canada in ground lamb, ground turkey, ground chicken, cheese and milk. The majority of these food isolates were genetically similar to epidemic strain NAP7/078, suggesting that food may not be a direct vector for C. difficile transmission, but could still be clinically relevant.

Introduction: Clostridium difficile infection (CDI) is a common hospital-acquired infection and a financial burden on the healthcare system. There is a need to reduce its impact on patients and the entire health system. More accurate estimates of the financial impact of CDI will assist hospitals in creating better CDI reduction strategies with limited resources. Previous research has not sufficiently accounted for the skewed nature of hospital cost data, baseline patient mortality risk, and the time-varying nature of CDI. Objective: We conducted a retrospective cohort study to estimate the cost impact of hospital-acquired CDI from the hospital perspective, using a number of analytical approaches. Method: We used clinical and administrative data for inpatients treated at The Ottawa Hospital to construct an analytical data set. Our primary outcome was direct costs and our primary exposure was hospital-acquired CDI. We performed the following analyses: Ordinary least square regression and generalized linear regression as time-fixed methods, and Kaplan-Meier survival curve and Cox regression models as time-varying methods. Results: A total of 49,888 admissions were included in this study (mean (SD) age of 64.6 ± 17.8 years, median (IQR) baseline mortality risk of 0.04 (0.01-0.14)). 360 (0.73%) patients developed CDI. Estimates of incremental cost due to CDI were substantially higher when using time-fixed methods than time-varying methods. Using methods that appropriately account for the time-varying nature of the exposure, the estimated incremental cost due to CDI was $8,997 per patient. In contrast, estimates from time-fixed methods ranged from $49,150 to $55,962: about a six fold difference. Conclusion: Estimates of hospital costs are strongly influenced by the time-varying nature of CDI as well as baseline mortality risk. If studies do not account for these factors, it is likely that the impact of hospital-acquired CDI will be overestimated.

Class of 2006 Abstract / Background: The processes’ underlying a recent rise in the rate of Clostridium difficile associated diarrhea (CDAD) at the Southern Arizona Veterans Administration Health Care System (SAVAHS) is unclear. Past changes to formulary in workhorse oral flouroquinolone from levofloxacin to gatifloxacin are under scrutiny. An infection-control component was also possible. Methods: 142 patients suspected of having CDAD had stool specimens submitted for toxin assay from late July to late Oct of 2004. A retrospective chart review was performed using the Veterans Administration Computerized Patient Record System (CPRS) to examine total antibiotic use in the three months prior to having specimens submitted for laboratory toxin analysis. A subset-analysis was performed on 100 specimens submitted for toxin analysis. Parallel culture was performed and 9 isolates of C. difficile were obtained for molecular analysis and fingerprinting. Results: Of the 142 patients sampled, 20 tested positive for C. difficile toxin with the remaining 122 patients testing negative. Antibiotic usage was categorized by total antibiotic use and gatifloxacin use. 98 patients received at least 1 antibiotic within the preceding 3 months with 44 patients receiving no antibiotic therapy of any kind. Of the 98 patients that received antibiotic therapy, 44 received gatifloxacin, however, all of these patients also received at least one other antibiotic. Of the nine isolates fingerprinted, two distinct genetic clusters were identified.

Clostridium difficile (CD) est l’entéropathogène le plus fréquemment responsable d'infections nosocomiales intestinales post-antibiotiques. L'apparition de cas graves liés à l'émergence de souches hypervirulentes ces dernières années a contribué à accroître la morbidité et la mortalité. Les toxines TcdA et TcdB contribuent directement aux lésions intestinales associées aux infections à CD (ICD), mais d'autres facteurs bactériens sont nécessaires à l’adhésion et la colonisation intestinale. Les flagelles de CD, qui confèrent la mobilité et la chimiotaxie, pourraient jouer un rôle dans la pathogenèse en contribuant à la réponse inflammatoire de l’hôte et aux lésions de la muqueuse. En effet, en activant le récepteur TLR5, les flagelles peuvent provoquer l'activation des cascades de signalisation cellulaire des MAPK et de NF-κB conduisant à la sécrétion de cytokines pro-inflammatoires. Notre objectif était d’étudier ce rôle potentiel des flagelles de CD in vitro et in vivo. Nous avons montré que l'interaction de la flagelline de CD avec le TLR5 active principalement la voie de NF-κB, et, dans une moindre mesure, la voie des MAPK, conduisant ainsi à la régulation de l'expression de gènes pro-inflammatoires et à la synthèse de médiateurs pro-inflammatoires. De plus, en utilisant un modèle murin d’ICD, nous avons démontré un effet synergique des flagelles et des toxines dans l’induction d’une réponse inflammatoire de la muqueuse caecale. Dans ce modèle, l'absence de flagelles diminue considérablement le degré d'inflammation de la muqueuse caecale et la seule présence de toxines, sans flagelles, ne suffit pas à provoquer d’importantes lésions épithéliales. Ces résultats mettent en évidence le rôle des flagelles de CD dans l’induction d’une réponse inflammatoire intestinale en synergie avec l’action des toxines bactériennes sur l'épithélium. / Clostridium difficile (CD) is the most common enteropathogen responsible for intestinal nosocomial post-antibiotic infections. The appearance of severe cases related to the emergence of hypervirulent strains these last years has contributed to increased mortality and morbidity. The CD TcdA and TcdB toxins contribute directly to CD infection (CDI)-associated lesions of the gut, but other bacterial factors are needed for the bacteria to adhere and colonize the gut. The CD flagella, which confer motility and chemotaxis for successful intestinal colonization, could play an additional role in bacterial pathogenesis by contributing to the inflammatory response of the host and mucosal injury. Indeed, by activating the TLR5, flagella can elicit activation of the MAPK and NF-κB cascades of cell signaling, leading to the secretion of pro-inflammatory cytokines. Our objective was to study the potential role of CD flagella in vitro and in vivo. We reported that the interaction of CD flagellin-TLR5 predominantly activates the NF-κB, and, in a lesser degree, the MAPKs pathways, thus leading to up-regulation of pro-inflammatory gene expression and subsequent synthesis of pro-inflammatory mediators. Moreover, by using a mouse model of CDI, we demonstrated a synergic effect of flagella and toxins in eliciting an inflammatory mucosal response. In this model, the absence of flagella dramatically decreased the degree of mucosal inflammation in mice and the sole presence of toxins without flagella was not enough to elicit epithelial lesions. These results highlight the important role of CD flagella in eliciting mucosal lesions as long as the toxins exert their action on the epithelium.

Clostridium difficile est une bactérie enteropathogène responsable d'infections intestinales dont les manifestations cliniques varient d’une simple diarrhée à une colite pseudomembraneuse parfois mortelle. L’une des problématiques majeures rencontrées dans la prise en charge des infections à C. difficile (ICD) est la survenue de récidives. Chez de nombreuses espèces bactériennes, la formation de biofilm est associée à la chronicité de l’infection. Le biofilm est un mode de vie dans lequel les bactéries sont engluées dans une substance polymérique qu'elles secrètent elles-mêmes. L’aptitude de C. difficile à former un biofilm in vitro a été clairement établie depuis 2012. Cependant aucune étude n’a montré jusqu’ici s’il est capable de former un biofilm in vivo. L’objectif de cette thèse était d’une part d’étudier différents paramètres impliqués dans la formation du biofilm in vitro et d’autre part de déterminer si C. difficile est capable de former un biofilm in vivo. Dans un premier temps, nous avons analysé la capacité de différentes souches de C. difficile (souches cliniques et souches de laboratoires modifiées génétiquement) à former un biofilm in vitro. Parmi ces dernières, la souche mutée pour le gène cwp84, codant une protéase de surface responsable de la maturation de la couche S de C. difficile, présente un biofilm particulièrement robuste et épais comparé à la souche parentale 630∆erm. L’activité protéolytique de Cwp84 est donc impliquée dans la formation du biofilm et module certaines propriétés de surface de la bactérie telles que l’hydrophobicité. Nous avons également étudié la composition en sucre de la matrice du biofilm, après une étape de mise au point qui a permis de déterminer les conditions permettant d’obtenir suffisamment de matériel. Les conditions retenues ont été les suivantes : formation de biofilm sur un support en verre, en présence de glucose et en milieu renouvelé. La présence d’un sucre qui possède un profil proche du PSII (polysaccharide associé à la surface des cellules planctoniques de C. difficile) dans la matrice du biofilm a été détecté par spectroscopie infrarouge. Dans un second temps, afin d’étudier l’aptitude de C. difficile à former un biofilm in vivo, différents modèles animaux ont été utilisés : un modèle de souris monoxéniques dans lequel plusieurs souches de C. difficile ont été testées (souches 630∆erm, mutant cwp84, R20291, P30) et un modèle de souris dixénique pour étudier la formation de biofilm mixte (C. difficile/Finegoldia magna et C. difficile/Clostridium scindens). Dans le modèle monoxénique, quelles que soient les souches testées, C. difficile est distribué de manière hétérogène tout au long de la surface du tissu intestinal. Les bactéries sont majoritairement retrouvées isolées sauf pour la souche R20291 qui forme le plus souvent des petits agrégats. Pour cette souche, différents marquages immunohistochimiques réalisés sur des coupes de cecum et de colon ont montré que la majorité des bactéries sont enchâssées dans de petites structures en 3 dimensions adhérentes à la couche du mucus. Le polysaccharide PSII est détecté en grande quantité à l'intérieur de cette structure. Ce composé étant présent dans la matrice de biofilm de C. difficile formé in vitro, ces résultats suggèrent que la souche R20291 pourrait s’organiser en biofilm dans le modèle de souris monoxénique. En modèle de souris dixéniques, nous avons montré que la présence de F. magna n’influe pas sur le niveau de colonisation de C. difficile, alors que l'association avec C. scindens semble être bénéfique aux deux bactéries puisqu'une augmentation de la population globale de deux espèces est observée comparé à la population présente dans chaque modèle mono-espèce. En conclusion, une souche productrice de biofilm in vitro semble être capable de s’organiser en une structure biofilm in vivo. Le rôle du biofilm dans l'étape de colonisation du colon par C. difficile et les rechutes des ICD devra être analysé. / Clostridium difficile is an enteropathogenic bacterium responsible for intestinal infections, the clinical symptoms vary from moderate diarrhea to pseudomembranous colitis, sometimes fatal. One of the major problems encountered in the management of C. difficile infections (DCI) is the occurrence of recurrences. In many bacterial species, biofilm formation is associated with the chronicity of infection. Biofilm is a way of life in which bacteria are entrapped in a polymeric substance secreted by the bacteria themselves. The ability of C. difficile to form an in vitro biofilm has been clearly established since 2012. However, no study has so far shown whether it is capable of forming a biofilm in vivo. The objective of this thesis was to analyze different parameters involved in the formation of biofilm in vitro and to determine whether C. difficile is able to form a biofilm in vivo.We first analyzed the ability of different strains of C. difficile to form a biofilm in vitro (clinical strains and strains genetically modified laboratories). Among the latter, the mutant strain for the cwp84 gene, encoding a surface-associated protease responsible for the maturation of the S layer of C. difficile, forms a particularly robust and thick biofilm compared to the 630Δerm parental strain. The proteolytic activity of Cwp84 is involved in the formation of biofilm and modulates certain surface properties of the bacterium such as hydrophobicity. We have also studied the polysaccharide composition of the biofilm matrix, after a development stage that allowed us to determine the optimal conditions for obtaining sufficient material. The conditions retained were the following: formation of biofilm on a glass support in the presence of glucose and in a renewed medium. We were able to determine by infrared spectroscopy the presence of a sugar which has a similar profile than the PSII (polysaccharide associated with the surface of C. difficile planktonic cells) in the matrix of the biofilm.Second, in order to study the ability of C. difficile to form a biofilm in vivo, different animal models were used: a monoxenic mouse model in which we tested several strains of C. difficile (strains 630Δerm, mutant cwp84, R20291, P30) and a dixenic mouse model to study the formation of mixed biofilm (C .difficile/Finegoldia magna and C. difficile/Clostridium scindens). In the monoxenic model, regardless of the strains tested, C. difficile is distributed heterogeneously throughout the intestinal tissue surface. The bacteria are mostly found isolated except for C. difficile R20291 which usually forms small aggregates. For this strain we have shown, thanks to various immunohistochemical labeling performed on cecum and colon sections, that the majority of the bacteria are embedded in a small 3-dimensional structures overlaying the mucus layer. The PSII polysaccharide is present in a large amount in this structure. As this compound has been detected in the in vitro C. difficile biofilm matrix, these results suggest strongly that the R20291 strain could be organized into biofilm structures in the monoxenic mouse model. In the dixenic mouse model, we have shown that the presence of F. magna does not influence the level of colonization of C. difficile, whereas the association with C. scindens seems to be beneficial to both bacteria since an increase of the global population is observed in this model compared to the population present in each single-species model.To conclude, an in vitro biofilm producing strain appears to be able to organize in a biofilm structure in vivo. The role of biofilm in the colonization step of the intestinal tract by C. difficile and in the occurrence of recurrences should be further analyzed.

Clostridium difficile (CD) représente la première cause d'infections digestives nosocomiales dans les pays développés. Les infections à CD (ICD) induisent une inflammation intestinale importante qui se manifeste principalement par des colites pseudomembraneuses ainsi que par un taux de mortalité élevé. Les facteurs majeurs de virulence sont les toxines TcdA et TcdB. Dans la première partie des travaux de cette thèse, nous avons validé le rôle in vivo d'un autre facteur bactérien, les flagelles, dans un modèle murin d'ICD, en montrant que les flagelles induisent une réponse inflammatoire au niveau de la muqueuse caecale en synergie avec les toxines. Nous avons ensuite montré une régulation de de cette réponse par un microARN (miARN) exerçant un rôle anti-inflammatoire en modulant l'activation de la voie de signalisation de NF-KB. Le traitement des souris infectées par ce miARN réduit l'inflammation intestinale apportant la preuve du concept pour une nouvelle approche thérapeutique. / Clostridium difficile (CD) is the leading cause of nosocomial digestive infections in developed countries. CD infections (CDI) induce significant intestinal inflammation that is manifested primarily by pseudomembranous colitis and a high mortality rate. The major virulence factors are TcdA and TcdB toxins. In the first part of the work of this thesis, we validated the in vivo role of another bacterial factor, the flagella, in a mouse model of CDI, showing that the flagella induce an inflammatory response in ceacal mucosa in synergy with toxins. We then showed a regulation of this response by a microRNA (miRNA) exerting an anti-inflammatory role by modulating the activation of the NF-KB signaling pathway. The treatment of mice infected with this miRNA reduces intestinal inflammation providing proof of concept for a new therapeutic approach.

Clostridium difficile est une bactérie anaérobie stricte responsable de diarrhées consécutives à une antibiothérapie et de colites pseudomembraneuses. La destruction du microbiote intestinal de barrière favorise l’implantation de C. difficile, qui se multiplie, adhère aux cellules épithéliales et produit les toxines TcdA et TcdB. Les infections à C. difficile sont devenues un problème majeur de santé publique. En particulier les nombreuses rechutes posent un problème thérapeutique. L’immunisation active est une des approches permettant de prévenir les rechutes et réduire l’incidence des infections. Plusieurs essais de vaccination ciblant les toxines sont en cours de développement mais sont sans action sur la première étape de l'infection à C. difficile, la colonisation intestinale. Notre objectif a été de développer une stratégie vaccinale par voie muqueuse pour lutter contre la colonisation intestinale. Des protéines de surface de C. difficile ainsi que la toxine TcdB ayant un rôle dans la pathogénicité ont été sélectionnées et utilisées comme cibles vaccinales. Certains candidats vaccinaux ont été encapsulés afin de pouvoir délivrer les antigènes au niveau de la muqueuse intestinale par voie orale. De plus, pour induire une réponse immunitaire localisée au niveau de la muqueuse intestinale, différents adjuvants ont été testés. Nous avons analysé après vaccination la réponse immunitaire induite au niveau local et systémique. Pour identifier de nouveaux candidats vaccin, le rôle de différentes protéines de surface dans la colonisation intestinale de C. difficile a été caractérisé. L'ensemble des essais in vivo a été mené dans deux modèles animaux de référence pour les infections à C. difficile : la souris et le hamster, pour suivre respectivement la colonisation intestinale par C. difficile et le taux de survie après infection. / Clostridium difficile is an anaerobic bacterium that is responsible for post-antibiotic diarrhea and pseudomembranous colitis. The disruption of the intestinal microbiota barrier effect promotes the establishment of C. difficile, which multiplies, adheres to epithelial cells, and produces the toxins TcdA and TcdB. C. difficile infections are considered as a major public threat. In particular, the multiple recurrences are difficult to treat. Active immunization is one of the new approaches to reduce recurrences and the incidence of these infections. Several vaccine targeting toxins are in development. However, these vaccines have no effect on the first step of C. difficile infection, the host colonization. The objective was to develop a mucosal vaccine strategy to act on intestinal colonization. Surface proteins of C. difficile and TcdB involved in the pathogenicity process were selected and used as vaccine targets. These antigens were produced and encapsulated to deliver them to the intestinal mucosa. Furthermore, to induce a gut mucosal immunity, different adjuvants were tested. After vaccination, we analyzed the local and systemic immune response by immunoassays. Finally, in order to characterize new vaccine candidates, the role of different surface proteins in C. difficile colonization was evaluated. These vaccine trials were conducted in two animal models of reference for C. difficile infections: the mouse and the hamster models, which permit to follow the colonization and the survival rate after infection, respectively.