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1
Hecker, Kim Ione. "Bleach-It-Away Clostridium difficile." ScholarWorks, 2018. https://scholarworks.waldenu.edu/dissertations/5471.
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Hospital-associated infections (HAIs) are infections patients contract as a result of being hospitalized. HAI rates decreased for almost all pathogens in the past few years, with the exception of Clostridium difficile infections (CDIs), which have been steadily climbing, placing hospital-acquired CDI at the top of the HAI list. The Center for Disease Control and Prevention reported in 2010 almost a half a million people were infected with CDIs yearly in the United States, and CDIs claimed the lives of approximately 29,000 people, representing a 4-fold increase from 1993. To address the problem in the local hospital, a quality improvement initiative called Bleach-It-Away was initiated. The initiative involved nurses wiping down the high touch areas in the patient's medical intensive care (MICU) rooms once every shift. The purpose of this quantitative research project was to evaluate the effectiveness of the Bleach-It-Away practice. The project question asked if the Bleach-It-Away practice was effective in reducing CDI rates. Deidentified CDI rates were provided by the clinical practice site covering a period of 12 months prior to implementation and 12 months after implementation of the practice. An independent t-test was used to determine whether there were significant improvements in CDI rates in the MICU. No significant improvement was seen in the postimplementation total CDI rates (p=.07) compared to the preimplementation rates. While the process did not demonstrate a significant improvement, positive social change is possible as hospitals recognize the many factors contributing to CDIs and the need for collaboration from various disciplines to control the problem.
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Maulner, Stéphanie. "Les endolysines de Clostridium difficile : Potentiel thérapeutique pour traiter les infections à C. difficile (ICD)." Mémoire, Université de Sherbrooke, 2010. http://savoirs.usherbrooke.ca/handle/11143/4059.
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Clostridium difficile, un bacille à Gram positif anaérobie strict qui forme des spores, est un pathogène opportuniste responsable de simples diarrhées ou de colites pseudomembraneuses qui peuvent provoquer la mort. Le traitement de base réside en l'arrêt des antibiotiques qui ont détruit la flore de l'hôte et provoqué les symptômes, ou en la prescription de vancomycine et/ou de métronidazole. Malheureusement, l'efficacité de ces antibiotiques est variable et le nombre de rechutes est élevé. En outre, de plus en plus de souches deviennent résistantes aux antibiotiques. C'est pour cette raison qu'un besoin d'alternatives thérapeutiques s'est fait ressentir. Une des approches prometteuses est l'utilisation des endolysines, qui sont des enzymes hydrolytiques encodées par les bactériophages et qui se sont déjà révélées être efficaces contre plusieurs bactéries à Gram positif. Dans cette étude, nous avons démontré l'activité lytique d'endolysines encodées par des phages de Clostridium difficile sur des cellules vivantes. Différentes endolysines ont été clonées dans E. coli, exprimées et purifiées, puis leur activité a été vérifiée de plusieurs manières. Certains facteurs biochimiques propres à ces enzymes ont été étudiés, tels que les cofacteurs nécessaires pour une meilleure activité lytique, le pH optimal et le spectre d'efficacité sur différentes souches bactériennes. Finalement, l'étude de ces enzymes comme outil de diagnostic ou de biologie moléculaire est abordée. Les résultats de nos travaux indiquent que les endolysines PlyCD52 et PlyCD38-2 de C. difficile possèdent une faible activité lytique. L'activité des endolysines n'est pas influencée par les cofacteurs Tween 0,5%, Triton 0,1%, MgCl[indice inférieur 2] contrairement à l'EDTA qui inhibe celle-ci. Le pH optimum semble être compris entre 7 et 8,5 et ces enzymes agissent sur différentes souches de C. difficile à l'exception de la souche CD630. Malgré ces résultats encourageants, des travaux supplémentaires seront nécessaires afin de stabiliser les enzymes qui ont une forte tendance à précipiter et d'obtenir une meilleure activité.
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Taibi, Fatima. "Études des riborégulateurs c-di-GMP chez Clostridium difficile." Mémoire, Université de Sherbrooke, 2016. http://hdl.handle.net/11143/8785.
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Chez une bactérie, la régulation de l’expression génétique est essentielle afin de maintenir l’équilibre, s’assurer du bon fonctionnement des processus cellulaires et mieux s’adapter aux changements environnementaux. Elle peut s’effectuer à plusieurs niveaux (la transcription, la traduction et la synthèse ou la dégradation des protéines), et par le bais de différents mécanismes, dont les protéines, font le plus grand part de cette régulation. Cependant, au début des années 2000, une découverte fascinante a mis en évidence un nouveau mécanisme de régulation dont l’ARN est l’acteur principal. Ce sont les riborégulateurs (riboswitches). Ces derniers sont localisés dans la partie non traduite de certains ARNmessagers (ARNm) et capable de lier un ligand spécifique sans l’intervention des protéines, afin de réguler l’expression génique du gène d’intérêt. Aujourd’hui, plusieurs familles de riborégulateurs sont caractérisées, entre autres les riborégulateurs c-di-GMP. Ces derniers sont présents chez plusieurs espèces bactériennes notamment les bactéries pathogènes telles que Clostridium difficile, une bactérie nosocomiale opportuniste qui a causé des problèmes majeurs durant les dernières années, vu sa multirésistance aux antibiotiques. Le séquençage de son génome a révélé la présence de 66 riborégulateurs dont 16 sont des riborégulateurs c-di-GMP. Il a été proposé que parmi ces derniers, certains régulent l’expression des gènes impliqués dans deux phénotypes essentiels chez C. difficile : la motilité et la formation du biofilm.
La présente étude porte sur la caractérisation structurale et fonctionnelle de deux riborégulateurs c-di-GMP chez le C. difficile, le Cdi1-1 et Cdi1-12, qui se trouvent en amont du gène CD1990 et le gène CD2830 (ZmpI) respectivement. Au début, nous avons prédit les deux structures liées (en présence du ligand) et non liées (en absence du ligand). Nous avons ainsi démontré qu’un des deux riborégulateurs (Cdi1-12) est fonctionnel et capable de lier le c-di-GMP in vitro. Ensuite, nous avons caractérisé les changements structuraux potentiels lors de l’interaction riborégulateur Cdi1-12/ligand. Nous avons également caractérisé le mécanisme de régulation en cis du riborégulateur Cdi1-12 in vitro et nous avons constaté que c’est un riborégulateur transcriptionnel Rho-indépendant. À la fin de notre étude, nous avons confirmé le mode de régulation de ce riborégulateur in vivo dans la bactérie modèle Bacillus subtilis.
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Sundström, Joakim. "Clostridium difficile – ett växande problem : Om sjuksköterskans arbete för att förebygga spridning av C. difficile i slutenvården." Thesis, Mittuniversitetet, Avdelningen för omvårdnad, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:miun:diva-20170.
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Van, Tyle Kendall M. "The Molecular Epidemiology of Clostridium difficile: Description of Clostridium difficile Associated Diarrhea (CDAD) Following a Formulary Change From Levofloxacin to Gatifloxacin." The University of Arizona, 2006. http://hdl.handle.net/10150/624511.
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Class of 2006 AbstractBackground: 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.
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Wroe, Allison J. "Immune response to Clostridium difficile infection and an investigation of the mechanisms of moxifloxacin resistance in clinical C. difficile isolates." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4452.
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Clostridium difficile is an increasingly common cause of nosocomial infection. C. difficile infection (CDI) presents as a spectrum ranging from asymptomatic carriage to mild diarrhoea, pseudomembranous colitis, toxic megacolon and intestinal perforation. It is not yet fully understood why this spectrum is seen, however, it is believed that the immune response mounted by an individual plays an important role in determining the outcome of infection. This thesis comprises three studies. Firstly, a comparative study of immune cell populations within the lamina propria of colonic tissue not exhibiting pathological changes and taken from individuals with symptomatic CDI (cases); asymptomatic carriers; and non-colonised controls. Effector T cells, B cells, plasma cells and macrophages were enumerated by means of immunohistochemical staining of tissue sections. Secondly, a study to establish the prevalence within these three study groups of specific host single nucleotide polymorphisms (SNPs) in the TLR2, TLR5 and IL-8 genes by PCR genotyping and to determine whether an association existed between these genotypes and susceptibility to CDI. Thirdly, an examination of the mechanisms of moxifloxacin resistance in a collection of clinical isolates. This study also sought to determine whether the competitive advantage conferred by resistance to moxifloxacin influenced the fitness of C. difficile isolates, in particular growth and the expression of the virulence factors toxins A and B. Carriers were found to have fewer of all four immune cell types quantified than both cases and controls. However, in only one instance, that of plasma cells, was this difference statistically significant. Cases had fewer of all cell types than controls but these differences were not significant. These findings suggest that individuals who become infected, both symptomatically and asymptomatically, with C. difficile display altered mucosal immune cell populations when compared with those of uninfected individuals. The data regarding host polymorphisms are suggestive of an association between the presence of SNPs and increased susceptibility to CDI. The variant IL-8 and TLR2 genotypes were carried by cases and carriers while the variant TLR5 genotype was carried by cases only. No variant genotypes were present in control subjects. All moxifloxacin resistant isolates characterised in this study, with the exception of an isolate with intermediate resistance and a third-generation mutant with reduced susceptibility, carried the common gyrA mutation ACT→ATT (Thr82→Ile). Efflux pumps are known to play a role in multi-drug resistance in many bacterial species. Semiquantitative PCR analysis of expression of the putative efflux pumps cme and cdeA found no correlation between overexpression and moxifloxacin resistance, suggesting that these genes do not play a role. Three novel mutations in the putative promoter region of CD3197, a MerR family transcriptional regulator found immediately upstream of cme, were identified. No association between the presence of these mutations and overexpression of cme or resistance or sensitivity to moxifloxacin was found. The competitive advantage conferred by resistance to moxifloxacin does not influence the fitness of C. difficile isolates, as measured in terms of growth and toxin production.
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Shaw, Claire M. "Inactivation of Clostridium difficile spores in the healthcare environment using hydrogen peroxide vapour." Thesis, Loughborough University, 2013. https://dspace.lboro.ac.uk/2134/12460.
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Healthcare-acquired infections (HAIs) cost the National Health Service (NHS) in England in excess of £1 billion per year. One of the main HAIs is caused by the endospore-forming bacterium Clostridium difficile. The most common cause of healthcare-acquired diarrhoea in the developed world, C. difficile was responsible for around 850 deaths in England and Wales in 2011. To help reduce the spread of the HAI-causing bacteria, terminal disinfection of isolation rooms and wards using hydrogen peroxide vapour is actively promoted. The key advantages of hydrogen peroxide vapour are its high oxidation potential which has been reported to inactivate bacteria, fungi and spores. An additional advantage of hydrogen peroxide vapour is that it is relatively environmentally friendly, breaking down into oxygen and water. Investigation into bacterial inactivation kinetics was undertaken at controlled, steady concentrations of hydrogen peroxide vapour in the range of 10 ppm to 90 ppm. An exposure chamber was designed whereby the bacterial spores could be exposed to constant concentrations of hydrogen peroxide for various exposure times. Bacterial spores (1-log10 to 8-log10 cfu) were filter deposited onto membranes to achieve an even layer for consistent exposure of the hydrogen peroxide vapour to the spores. Bacillus subtilis is often used for method development in bacterial studies; advantages are it has been shown to be highly resistant to hydrogen peroxide vapour and is not a human pathogen. Following the method development, different strains of C. difficile (ribotypes 014, 027, 103 and 220) were exposed to identify differences in resistance. Inactivation models (Chick-Watson, Series-Event, Weibull and Baranyi) were used to fit the data generated using the environmental chamber. Decimal reduction values (D-values) were calculated from the models for comparative studies regarding the inactivation achieved for the different bacteria and different hydrogen peroxide concentrations. The findings from this thesis revealed the Weibull model provides the best fit for most of the data. An initial shoulder period was identified for B. subtilis which was absent for C. difficile inactivation by hydrogen peroxide vapour; B. subtilis is therefore more resistant to hydrogen peroxide disinfection than C. difficile. Typical D-values for B. subtilis and C. difficile when exposed to hydrogen peroxide vapour at a concentration of 90 ppm were 140 and 1 min, respectively. C. difficile inactivation data were used to develop a model to estimate the log reduction that could be achieved during an inactivation cycle based on the concentration-time integral ( ). This model could be used to estimate the log reduction of commercially available hydrogen peroxide decontamination systems; these release a fixed amount of hydrogen peroxide into the room resulting in a peak concentration before decomposition to oxygen and water. Releasing the hydrogen peroxide into the room in this manner results in spatial and temporal variation; this could result in differences in bacterial inactivation in different areas within the room. Using the aforementioned regression model, the inactivation achieved at all locations within the room could be predicted, which could be used to optimise the current hydrogen peroxide decontamination cycles.
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Stehmer, Theresa, and Jackie Campbell. "Evaluation of combination therapy for Clostridium difficile infections at an academic hospital." The University of Arizona, 2012. http://hdl.handle.net/10150/623589.
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Class of 2012 AbstractSpecific Aims: The incidence of non-response, recurrence, relapse, and rate of complications of Clostridium difficile infections treated with combination of metronidazole and vancomycin versus vancomycin or metronidazole alone over a one-year period by treatment and strain type (i.e. NAP1/BI/027) were evaluated. The incidence of mortality in patients with moderate to severe Clostridium difficile associated diarrhea prescribed metronidazole, vancomycin, or combination metronidazole plus vancomycin as initial therapy was also determined. Additionally, significant factors associated with the use of combination vancomycin-metronidazole as initial therapy for moderate to severe CDAD were characterized. Methods: T This retrospective medical record review has been approved by the Institutional Review Board. Adult patients with stool specimens tested for detection of Clostridium difficile toxin B by PCR between April 2010 and March 2011 at a tertiary care, academic medical center were evaluated. Patients were included in the study if diagnosed with moderate to severe disease and received either monotherapy with metronidazole, monotherapy with oral vancomycin, or combination therapy with metronidazole and oral vancomycin for at least 80% of the first 10 days of treatment. Patients who are discharged alive within 72 hours of admission or who received therapy for less than 48 hours were excluded. Main Results: All patients (N=411) with laboratory evidence of Clostridium difficile during the study time period were evaluated. A total of 26 subjects who received oral vancomycin monotherapy and 56 subjects who received oral vancomycin along with metronidazole for at least 80% of the first 10 days of treatment were identified. Of the subjects who received oral vancomycin monotherapy during the first ten days of therapy, 5 (19%) were classified has a treatment failure or died within the first 21 days of therapy and 5 (19%) had either a recurrence or reappearance of Clostridium difficile associated diarrhea between 22 and 65 days post start of therapy. Of the subjects who received a combination of oral vancomycin and metronidazole during the first 10 days of therapy, 14 (25%) were classified has a treatment failure or died within the first 21 days of therapy and 22 (39%) had either a recurrence or reappearance of Clostridium difficile associated diarrhea between 22 and 65 days post start of therapy. In the combination therapy group, 5 (9%) were reported to have an ileus, toxic megacolon, or necrotic bowel during the first 10 days of therapy. Conclusions: In this study, the subjects who received a combination of oral vancomycin and metronidazole had higher rates of clinical failure, death, and recurrence than subjects who received monotherapy. Current guideline statements recommend combination therapy only in patients with an ileus with Clostridium difficile-associated diarrhea.
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Feliciano, Lisa. "Clostridium difficile Infection (CDI): Use of Preventive Bundle to Decrease CDI Incidences." ScholarWorks, 2018. https://scholarworks.waldenu.edu/dissertations/5188.
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The challenge of combating Clostridium difficile infections (CDI) is a major problem within many health care organizations as CDI adds to the cost of care and is an uncomfortable and sometimes fatal complication of hospitalization for the patient. The practice-focused question for this doctoral project was targeted at patients in hospital settings on a medical surgical floor and asked if clostridium difficile preventive bundles reduce the incidence of CDI compared with nonstandardized preventative methods. Using the plan-do-study-act framework, the purpose of this DNP project was to use a clostridium difficile bundle approach to study the effects of clostridium difficile incidence (CDI) decrease on a medical-surgical unit with high CDI incidences. Standardized environmental cleaning practices resulted in improvement of the patient environment. High-touch cleaning improved from 43.7% to 83.3%. Time between CDI events lengthened from 19.9 days to 30.2, environmental cleaning with the use of Dazo auditing improved from 33.4% to 81.6%, isolation practices improved from 62.7% to 90%, and with the implementation of the nurse-driven CD testing protocol, unnecessary testing improved. Results showed that the CDI incidence on an acute care medical surgical unit was reduced through the use of a clostridium difficile preventive bundle in this DNP project. Reducing the incidence of CDI is a significant contribution to social change as this unwanted complication of hospitalization causes discomfort and pain and adds unnecessary cost to health care.
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Soavelomandroso, Anna P., Françoise Gaudin, Sandra Hoys, Valérie Nicolas, Gayatri Vedantam, Claire Janoir, and Sylvie Bouttier. "Biofilm Structures in a Mono-Associated Mouse Model of Clostridium difficile Infection." FRONTIERS MEDIA SA, 2017. http://hdl.handle.net/10150/626057.
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Clostridium difficile infection (CDI) is a major healthcare-associated disease with high recurrence rates. Host colonization is critical for the infectious process, both in first episodes and in recurrent disease, with biofilm formation playing a key role. The ability of C. difficile to form a biofilm on abiotic surfaces is established, but has not yet been confirmed in the intestinal tract. Here, four different isolates of C. difficile, which are in vitro biofilm producers, were studied for their ability to colonize germ-free mice. The level of colonization achieved was similar for all isolates in the different parts of the murine gastrointestinal tract, but pathogen burden was higher in the cecum and colon. Confocal laser scanning microscopy revealed that C. difficile bacteria were distributed heterogeneously over the intestinal tissue, without contact with epithelial cells. The R20291 strain, which belongs to the Ribotype 027 lineage, displayed a unique behavior compared to the other strains by forming numerous aggregates. By immunochemistry analyses, we showed that bacteria were localized inside and outside the mucus layer, irrespective of the strains tested. Most bacteria were entrapped in 3-D structures overlaying the mucus layer. For the R20291 strain, the cell-wall associated polysaccharide PS-II was detected in large amounts in the 3-D structure. As this component has been detected in the extrapolymeric matrix of in vitro C. difficile biofilms, our data suggest strongly that at least the R20291 strain is organized in the mono-associated mouse model in glycan-rich biofilm architecture, which sustainably maintains bacteria outside the mucus layer.
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Thanki, Anisha M. "Development of a phage-based diagnostic test for the identification of Clostridium difficile." Thesis, Loughborough University, 2016. https://dspace.lboro.ac.uk/2134/20340.
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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.
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Hörnström, Eva. "The effect of low temperature and transportation time on Clostridium difficile viability." Thesis, Uppsala universitet, Institutionen för kvinnors och barns hälsa, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-295127.
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Anaerobe opportunist Clostridium difficile causes the majority of hospital-acquired antibiotic-associated diarrhea. Infections can be severe because of its ability to withstand many antibiotics, to sporulate and to produce toxins (A, B and binary). In Sundsvall Hospital C. difficile is detected with real-time PCR, which targets the sequences of toxin B, binary toxin and a regulatory gene deletion seen in the virulent ribotype 027. All positive samples are stored frozen for one month, available for further analysis or outbreak investigation. The aim of this study was to investigate if temperature and transportation time may affect the viability of C. difficile, and the PCR-result. Frozen feces samples were cultivated, identified with MALDI-TOF and analyzed with real-time PCR after at least one month of storage. To simulate the effect of transportation time, samples were stored at 4-8°C for three and seven days before cultivation and identification. Controls were cultivated after freezing for comparison. Ninety percent of the frozen samples contained viable C. difficile. Discrepancies between PCR-results were found for two of the oldest samples collected (six months), which turned negative. Fresh samples showed lower amount of viable C. difficile after three days (50 %) than after seven days (60 %) of storage, perhaps because of competition with other bacteria and sporulation. The frozen control group contained a higher viable amount, 75 %. The results indicate that C. difficile tolerates to be stored at low temperatures as practiced today at the laboratory. Transportation time seem to affect the outcome of cultivation, but not the PCR-result.
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Lloyd, Aaron, Vinay Pasupuleti, Priyaleela Thota, Chaitanya Pant, David D. K. Rolston, Adrian V. Hernández, Vicente A. Benítes-Zapata, Thomas G. Fraser, Curtis J. Donskey, and Abhishek Deshpande. "Accuracy of loop-mediated isothermal amplification for the diagnosis of Clostridium difficile infection: a systematic review." Elsevier B.V, 2015. http://hdl.handle.net/10757/345286.
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Loop-mediated isothermal DNA amplification (LAMP) are currently used as standalone diagnostic test for C. difficile infection (CDI). We assessed the diagnostic accuracy of LAMP for the diagnosis of CDI. We searched 5 databases to identify studies that compared LAMP with culture cytotoxicity neutralization assay or anaerobic toxigenic culture (TC) of C. difficile. We used the random-effects model to calculate pooled sensitivities, specificities, diagnostic odds ratios and their 95% confidence intervals (CIs). The search of the databases yielded 16 studies (6,979 samples) that met inclusion criteria. When TC was used as the gold standard (6,572 samples), bivariate analysis yielded a mean sensitivity of 0.95 (95%CI, 0.93-0.97; I2 = 67.4) and a mean specificity of 0.99 (95%CI, 0.96-1.00; I2 = 97.0). LAMP is a useful diagnostic tool with high sensitivity and specificity for detecting CDI. The results should however be interpreted only in the presence of clinical suspicion and symptoms of CDI.Revisión por pares
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Taori, Surabhi Kamal. "Clostridium difficile in south-east Scotland : an analysis of severe, recurrent and community-associated disease with a report on the emergence of PCR ribotype 078." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8054.
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Clostridium difficile infection (CDI) has proven to be a constantly evolving disease periodically posing new diagnostic and clinical dilemmas. Different regions of the world have reported specific local genomic characteristics of the infecting strains, which may be related to variation in disease presentation and outcome. This study was performed to determine the clinical and molecular features of severe, recurrent and community-associated disease in the Lothian region of Scotland, UK among patients diagnosed from August 2010-July 2011. Three hundred and thirty-five patients with laboratory confirmed CDI were studied for epidemiological features, clinical presentation, and laboratory markers. They were followed up for one year to determine recurrence and mortality. Four hundred and thirty-two episodes were recorded. Ribotypes, presence of toxin genes and MLVA subtypes of isolates from these episodes were determined. During the course of the study, PCR ribotype 078 was identified as an important emerging type and concerns of “hypervirulence” were raised when an outbreak was recorded in 2012. This ribotype was studied to compare its clinical and molecular characteristics with other endemic ribotypes and between its own outbreak-related and endemic subtypes. Asymptomatic children were also sampled to determine their role as pools of potential pathogens. Severe episodes accounted for 40.4% of total and 29.3% patients had multiple episodes on record. One-year mortality was 32.8% of which CDI was listed on 25.5% death certificates. Ribotype 078 was confirmed in 6.8% episodes. Community-associated disease was identified in 25.3% patients, which differed significantly from hospital-associated disease in the number of antibiotics and gastrointestinal manipulation prior to CDI. Endemic PCR ribotype 078 caused significantly less recurrent disease and more community- associated disease when compared to the most prevalent ribotype 001. Patients who died from ribotype 078 within 30d had a lower Charlson comorbidity index than ribotype 001 counterparts suggesting that the former may infect healthier patients. MLVA subtyping of ribotype 078 proved useful in identifying epidemiological relationships during the outbreak. CDI had contributed to the death of 50% of all patients infected with the outbreak related ribotype 078 strain compared to 14.3% of those infected with the endemic strains. This study documents the changing epidemiology of CDI in the region and demonstrates differences in epidemic and endemic disease.
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Orellana, Robert Charles. "Recurrent Clostridioides difficile infection: epidemiology and bedside scoring system analysis, 2014-2016." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543505897011863.
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Pantaleon, Véronique. "Le biofilm de C. difficile : rôle des protéines de surface." Thesis, Paris 11, 2015. http://www.theses.fr/2015PA114812/document.
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Clostridium difficile est responsable de vingt-cinq pour cent des diarrhées post-antibiotiques et de la majorité des cas de colite pseudomembraneuse. C'est un bacille anaérobie à Gram positif sporulant. La bactérie est recouverte par un réseau cristallin bidimensionnel appelé couche S. Elle est formée par deux sous-unités de haut et bas poids moléculaire issues du clivage du précurseur SlpA par la protéase Cwp84. Ces deux protéines sont codées par des gènes situés dans le locus cwp et sont sécrétées par le système de sécrétion de type SecA2. Elles sont impliquées dans l'adhésion/colonisation du côlon par C. difficile. L'adhésion est une étape commune avec la formation du biofilm par les bactéries. Le biofilm est une communauté bactérienne enchâssée et protégée par une matrice extracellulaire produite par les membres de la communauté. C'est le mode de vie principal des bactéries. Nous avons caractérisé la voie de sécrétion de type SecA2 de C. difficile. La protéine SecA2 (codée également par un gène du locus cwp) est essentielle pour la survie de C. difficile. Elle a une double localisation : le cytoplasme et la membrane intracellulaire. SecA2 de B. anthracis est capable de dimériser avec les protéines SecA1 et SecA2 de C. difficile. De plus, la complémentation du mutant secA2 de B. anthracis par le gène secA2 de C. difficile est fonctionnelle. Le rôle de la couche S, de la protéase Cwp84 et de la mobilité dans le biofilm de C. difficile ont été également étudiés. La souche 630∆erm forme un biofilm fin et fragile tandis que le mutant 630∆ermcwp84::erm forme un biofilm épais et robuste. Nous avons montré que l'activité protéolytique de Cwp84 était impliquée dans la formation du biofilm. De plus, une inhibition de la traduction de l'ARN slpA avec un ARN antisens spécifique permet une augmentation de la taille du biofilm formé par la souche 630∆erm. Similairement, l'expression de l’allèle secA2 muté dominant qui bloque au moins partiellement la voie de sécrétion de type SecA2 augmente la taille du biofilm. Nos résultats suggèrent que la couche S, la protéase Cwp84 et la protéine SecA2 sont impliquées dans la formation du biofilm de C. difficile. Par ailleurs nous avons testé un panel de souches dans leur capacité à former un biofilm. Les résultats montrent que les souches non mobiles ne seraient pas capables de former un biofilm épais. Enfin, nous avons étudié la capacité de C. difficile à se développer en aérobiose au sein d’un biofilm mixte avec Bacillus cereus. Nous avons mis en évidence un recrutement et une multiplication de C. difficile dans la pellicule formée à l'interface air/liquide par B. cereus. Un rapport optimal des spores des deux espèces est requis pour le développement de C. difficile dans ces conditions. La présence de spores de C. difficile dans la pellicule suggère que les biofilms de l'environnement pourraient être des réservoirs de spores de C. difficile, et à l'origine de contaminations humaine et animaleClostridium difficile is responsible for twenty-five percent of post-antibiotics diarrhea and for most cases of pseudomembranous colitis. It is an anaerobic, sporulating, Gram-positive bacillus. The bacterium is covered by a two-dimensional lattice called S-layer. It is formed by two subunits of high and low molecular weight after the cleavage of the SlpA precursor by the Cwp84 protease. These two proteins are encoded by genes located in the locus cwp and are secreted by the SecA2 secretion system. They are involved in colonic adhesion/colonization by C. difficile. Adhesion is a common step with biofilm formation by bacteria. The Biofilm is a microbial community embedded in and protected by an extracellular matrix produced by the community members. The biofilm is the main bacterial way of life.We have characterized the SecA2 secretory pathway of C. difficile. The SecA2 protein (as encoded by a gene locus cwp) is essential for the survival of C. difficile. It has a dual location: cytoplasm and intracellular membrane. SecA2 of B. anthracis is able to dimerize with SecA1 and SecA2 proteins of C. difficile. Moreover, the complementation of B. anthracis secA2 mutant with secA2 gene of C. difficile is functional.The role of the S-layer, of the Cwp84 protease and of the motility in the biofilm of C. difficile have also been studied. The 630∆erm strain forms a thin and weak biofilm while the 630∆ermcwp84::erm mutant forms a thick and robust biofilm. We have shown that proteolytic activity of Cwp84 was involved in biofilm formation. Furthermore, a decrease in the translation of slpA RNA with an antisense RNA specific permits an increase in the size of the biofilm of the strain 630∆erm strain. Similarly, the expression of the dominant mutated secA2 allele, which at least partially blocks the SecA2 secretory pathway, increases the biofilm size. Our results suggest that the S-layer, the Cwp84 protease and the SecA2 protein are involved in biofilm formation of C. difficile. On the other hand, we have tested a panel of strains in their capacity to form a biofilm. The results show that non-motile strains are unable to form a thick biofilm.Finally, we have studied the ability of C. difficile to grow aerobically in a mixed biofilm with B. cereus. We highlighted a recruitment and proliferation of C. difficile in the film formed at the air/liquid interface with B. cereus. An optimal ratio of spores of both species is required for the development of C. difficile in these conditions. The presence of C. difficile spores in the film suggests that the environment biofilms could be reservoirs of spores of C. difficile, and the source of human and animal contamination
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Youssef, D., Beth A. Bailey, Abbassi A. El, R. Copeland, Leslie G. Adebonojo, T. Manning, and Alan N. Peiris. "Healthcare Costs of Staphylococcus Aureus and Clostridium Difficile Infections in Veterans: Role of Vitamin D Deficiency." Digital Commons @ East Tennessee State University, 2010. https://dc.etsu.edu/etsu-works/6311.
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Clostridium difficile and staphylococcal infections are associated with increased morbidity, mortality and healthcare costs. Vitamin D deficiency may also contribute to increased healthcare costs. There is increasing evidence that vitamin D may have an antimicrobial role. We examined the relationship of serum 25(OH)D levels to staphylococcal and C. difficile infections to determine if vitamin D deficiency was associated with adverse outcomes. In the outpatient setting, vitamin D deficiency in patients with C. difficile and staphylococcal infections were associated with significantly increased total outpatients costs and fee-based consultation. Laboratory expenses had a trend towards higher costs in the vitamin D-deficient group but did not reach statistical significance. The differences were most clearly seen in the in-patient group with enhanced laboratory, pharmacy and radiology costs. These differences resulted in vitamin D-deficient patients with C. difficile or staphylococcal infections having costs more than five times higher than the non-deficient patients. The total length of hospital stay was four times greater in the vitamin D-deficient group. In addition, the total number of hospitalizations was also significantly greater in the vitamin D-deficient group. Surgery costs demonstrated a tendency to be higher in the vitamin D-deficient group but failed to reach statistical significance. Vitamin D deficiency is intimately linked to adverse health outcomes and costs in Veterans with staphylococcal and C. difficile infections in North East Tennessee. We recommend that vitamin D status be checked in patients with these infections and appropriate therapy be instituted to restore vitamin D level to normal in an expeditious manner.
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Chachaty, Elisabeth. "Evaluation de l'impact des antibiotiques sur la flore intestinale des sujets sainsnotamment sur la colonisation par "Clostridium difficile" ou par des entérobactéries résistantes : comparaison de méthodes de typage des souches de "C difficile"." Paris 11, 1993. http://www.theses.fr/1993PA114842.
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Smith, Amelia, Kathyrn Matthias, and Hanna Phan. "Evaluating Treatment Options for NAP1 Versus Non-NAP1 Strains of Clostridium Difficile Infection Among Pediatric Patients at an Academic Hospital." The University of Arizona, 2014. http://hdl.handle.net/10150/614169.
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Class of 2014 AbstractSpecific Aims: The incidence of Clostridium difficile (C. Diff) infections in pediatric patients has continually risen, which could be caused by the emergence of a hyper virulent strain, specifically NAP1/B1/027. The objectives of the study were to evaluate the incidence of strain type, compare treatment(s) prescribed, treatment duration, rate of infection recurrence based on strain and severity, rates of re-infection or recurrence, and treatment failures for patients less than 6 months and up to 18 years of age. Methods: A retrospective study of patients admitted to an academic medical center with detection of C. diff toxin was performed. Data analyses included descriptive and inferential statistics to examine demographics, strain type, infection severity, and treatment failure. Main Results: Fourty-five patients with C. Diff toxin detection were included in study analyses and the median age was 6.2 [0.31- 17.9 years]. Oral or intravenous metronidazole was prescribed as initial therapy in 89% of the patients. Strain type was available in 77% of patients, with NAP1/B1/027 detected in 31% of stool samples tested. Within 21 days after initial toxin detection, there was a 13% rate of clinical failure or death, although none directly associated with C. Diff. Within days 22 - 65 after initial toxin detection, there was a 16% rate of recurrence or reinfection. Initial therapy selection, therapy duration, and rate of recurrence or reinfection were not significantly associated with NAP1/B1/027 strain type. Conclusion: Despite variability in severity of infection, the majority of pediatric patients with C. Diff were treated with metronidazole and were infected with a non-B1/NAP1/027 strain.
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Bordeleau, Éric. "Régulation du c-di-GMP et rôle de ce messager secondaire dans la formation de pili de type IV chez Clostridium difficile." Thèse, Université de Sherbrooke, 2014. http://hdl.handle.net/11143/5385.
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Malgré la découverte du c-di-GMP en 1987, ce n’est que durant la dernière décennie que l’importance de ce messager secondaire dans la régulation des phénotypes bactériens a été exposée. Synthétisé par des diguanylate cyclases (DGC) et dégradé par des phosphodiestérases spécifiques (PDE), le c-di-GMP est prédit pour être un messager secondaire très répandu chez les bactéries et pratiquement exclusif à celles-ci. Le c-di-GMP est particulièrement reconnu pour son rôle dans la transition des bactéries motiles et planctoniques vers la formation de biofilm chez les bactéries à Gram négatif telles qu’Escherichia coli, Pseudomonas aeruginosa et Vibrio cholerae. De plus, le c-di-GMP est impliqué dans la régulation de l’expression de certains facteurs de virulence chez certaines bactéries. Ainsi, il est possible de révéler les mécanismes de régulation de certains phénotypes importants par l’étude de la signalisation à c-di-GMP dans une bactérie donnée. Clostridium difficile est une bactérie pathogène causant des diarrhées nosocomiales, des colites et pouvant causer des décès chez l’Homme. Les phénotypes impliqués dans la pathogenèse de C. difficile et leur régulation demeurent en grande partie méconnus. Le génome de C. difficile 630 était prédit être capable de coder pour 37 DGC et PDE putatives, un nombre en apparence élevé pour une bactérie à Gram positif. L’objectif global de mon doctorat était de déterminer si la signalisation à c-di-GMP était fonctionnelle chez C. difficile puis de déterminer le rôle du c-di-GMP chez cette bactérie. Dans un premier projet, mes travaux de doctorat ont permis de démontrer que la majorité des 37 DCG et PDE putatives chez C. difficile 630 sont fonctionnelles. Les 31 DCG et PDE les plus conservées dans les différentes souches de C. difficile ont été exprimées dans V. cholerae afin d’évaluer indirectement leur capacité de synthèse et de dégradation du c-di-GMP en mesurant leur impact sur motilité et la formation de biofilm de V. cholerae. La surexpression d’une DGC chez V. cholerae réduit la motilité par flagelle et augmente la formation de biofilm, alors que l’inverse est observé lors de la surexpression d’une PDE. De plus, l’activité d’une DCG, CD1420 (renommée DccA, CD630_14200), et une PDE, CD0757 (renommée CD630_07570) a été démontrée plus explicitement par des essais enzymatiques in vitro. Ainsi, ce projet a exposé l’important potentiel de la signalisation à c-di-GMP chez C. difficile, jusqu’alors étudiée presque exclusivement chez les bactéries à Gram négatif. Dans un deuxième projet, mes travaux de doctorat ont permis de démontrer le rôle des pili de type IV (T4P) dans l’agrégation de C. difficile et la régulation de leur expression par un riborégulateur à c-di-GMP. Les riborégulateurs sont des structures ARN situées dans la région 5’UTR des gènes capables de réguler l’expression des gènes en aval en fonction de la liaison d’un métabolite spécifique. Parmi les 16 riborégulateurs à c-di-GMP prédits dans le génome de C. difficile 630, le riborégulateur c-di-GMP-II Cdi2_4 est situé en amont du locus principal de synthèse de T4P. Mes travaux ont permis de montrer que l’augmentation de la concentration de c-di-GMP intracellulaire se traduit par une augmentation de l’expression des gènes de T4P, la formation de T4P à la surface des cellules et l’agrégation dépendante des T4P. De plus, le mécanisme de régulation du riborégulateur Cdi2_4 a été démontré in vitro. La liaison du c-di-GMP au riborégulateur Cdi2_4 prévient la formation d’un terminateur transcriptionnel et favorise ainsi la transcription des gènes de T4P en aval. Depuis la mise en évidence de la signalisation à c-di-GMP chez C. difficile dans la première partie de mon doctorat, un certain nombre de phénotypes régulés par c-di-GMP chez cette bactérie ont pu être déterminés ou prédits. Notamment, le c-di-GMP inhibe la transcription des gènes des flagelles en se liant au riborégulateur c-di-GMP-I Cd1 en amont et inhibe indirectement la production des toxines TcdA et TcdB. La démonstration de l’effet positif du c-di-GMP sur l’agrégation des cellules via les T4P, dans la deuxième partie de mon doctorat, contribue à notre compréhension de la signalisation à c-di-GMP chez C. difficile. Il apparait que le c-di-GMP inhibe la motilité et favorise la formation de structures pluricellulaires chez C. difficile à l’instar de plusieurs bactéries, néanmoins par des mécanismes de régulation distincts.
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Parisien, Albert. "Large-Scale Production in 'Escherichia coli' TG1 and Purification of Llama Single Domain Antibody ToxA5.1 Against 'Clostridium difficile' Toxin A." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/26242.
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Drug resistant strains of Clostridium difficile are a major health concern with over 3 million cases costing over 1 billion $ per year in the United-States. The diseases associated with these bacteria (CDAD) are toxin-mediated which offers a mean of treating and lessening the severity of CDAD symptoms. Toxin inactivation via antibodies therapy can drastically reduce CDAD morbidity and this project was aiming at investigating the large-scale production and recovery of a novel llama single domain antibody (pSJF2H-ToxA5.1) in recombinant Escherichia coli TG1 targeting C. difficile enterotoxin A (TcdA). In order to achieve these objectives, the project was divided into four segments: 1) ToxA5.1 being an intracellular recombinant protein, obtaining a high biomass production was the first step towards large-scale production. To achieve HCDC, effects of initial glucose concentration and pH-stat feeding strategy were studied; 2) Upon achieving HCDC, effects of parameters such as temperature, induction timing and media supplementation with complex nitrogen sources were investigated; 3) Once large-scale production of ToxA5.1 was obtained, the recombinant protein needed to be recovered and a selective cell lysis scheme where synergistic lysis effects of Triton X-100 and temperature were studied. And finally 4) Single-step purification using nickel nanoparticles (NNP) synthesized via a modified polyol method was studied.
Combining the HCDC strategy with a temperature shift and yeast extract addition at the time of induction, ToxA5.1 concentration of 127 mg/L was obtained. Synergistic and selective cell lysis using Triton X-100 and temperature was achieved where 95% of the available ToxA5.1 was recovered and still functional while ToxA5.1 fraction in the resulting lysate increased to 27% in the cell lysate. Single-step purification was achieved using the synthesized NNP which proved to be highly selective and could be used up to five times. Diameter of the NNP synthesized was controlled by using various concentration of ranging from 131 ± 80 nm to 47 ± 20 nm. Using experimental data from binding isotherm, the ToxA5.1-NNP system was modeled.
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Cosmetatos, Isabelle Cosmetatos Isabelle. "Fecal isolation of "Clostridium difficile" and its toxins in horses with typhlo-colitis : Oral administration of neomycin and phthalylsulfathiazole in horses : effects on clinical, hematological and hematochemical parameters and influence on the isolation rate of "C. difficile" in feces /." [S.l.] : [s.n.], 1995. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Thorsell, Mikaela. "Evaluation of C. diff Quik Chek Complete® and comparison with GeneXpert to establish a new diagnostic algorithm." Thesis, Uppsala universitet, Institutionen för kvinnors och barns hälsa, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-390609.
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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.
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Hamdi, Cassandra. "Clostridium difficile : Rapid typing Clostridium difficile using MALDI-TOF MS analysis." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-17659.
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Caproni, Lisa J. "Antibiotics and Clostridium difficile." Thesis, University of Edinburgh, 2004. http://hdl.handle.net/1842/24132.
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The aims of this thesis were to investigate the antibiotic susceptibility patterns of C. difficile with relation to the S-type of the isolates over a period of 18 months. Detailed growth curves were performed on strains NCTC 11223, the sequenced strain 630 and an endemic isolate 338a. Toxin A was shown to be produced upon entry to stationary phase in agreement with other studies. OD600 was found to be a good predictor of growth phase and allowed this measurement to be used for subsequent experiments. MICs were performed on 186 random isolates of C. difficile collected during an 18-month epidemiological study to investigate the patterns of sensitivity to six different antibiotics. No evidence of resistance was seen to the two treatment antibiotics and all strains were resistant to cefoxitin (MICs 64-256mg/ml), the antibiotic used in most selective media. Most strains (98.9%) had intermediate resistance to ceftriaxone. The MIC50 and MIC90 of the strains to amoxicillin and clindamycin were very close (8 and 16 for amoxicillin and 16 for clindamycin) but the range of MICs was great. Clindamycin resistance was common with 67% of strains resistant (MICs of > 8mg/ml), 25% with intermediate resistance (MIC > 4mg/ml) and only 8% sensitive (MICs of < 2mg/ml). Twelve isolates from six different patients had very high resistance to clindamycin with MICs > 128mg/ml. Multiple isolates from the same patient, taken at different times, showed changes in susceptibility patterns over time. The only major change in susceptibility over the time period was in clindamycin resistance; some strains appearing to become more resistant while others became less resistant. No differences were apparent in the MIC50 and MIC90 of the different S-types of C. difficile identified, although some S-types were present in very small numbers. No links between antibiotics prescribed and susceptibility patterns were found. Three strains (NCTC 11223, strain 630 and endemic isolate 338a) were cultured in sub-lethal concentrations of the six antibiotics (1/2,1/4 and1/8 of the MIC) over 104 hours and growth and toxin A measured three times a day. The effects varied between strain and antibiotic. The most common effect on the growth of the strains was to increase the initial lag period by ca. 4h compared to the antibiotic-free controls through the clindamycin resistant strain NCTC 11223, (MIC >512mg/ml) showed not lag whatsoever in comparison to the controls when grown in this antibiotic. The most common effect on toxin A production was in the onset of toxin elaboration. Normally toxin began to appear in low levels in early stationary phase before accumulating to high levels by the start of decline.
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Båverud, Viveca. "Clostridium difficile in horses /." Uppsala : Dept. of Veterinary Microbiology, Swedish Univ. of Agricultural Sciences ([Institutionen för veterinärmedicinsk mikrobiologi], Sveriges lantbruksuniv.), 2002. http://epsilon.slu.se/avh/2002/91-576-6378-5.pdf.
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Cairns, Michelle Dawn. "Evolution of Clostridium difficile." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10060221/.
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Clostridium difficile continues to be a leading cause of healthcare-associated infections in the developed world. Increased detection of C. difficile infection (CDI) and development of typing schemes to differentiate between strains is primarily due to the recognition of global outbreaks of a single strain, BI/NAP1/027 which is characterised by three common typing techniques; restriction endonuclease analysis (REA), pulsed-field gel electrophoresis (PFGE) and PCR ribotyping. Phylogenetic analysis using multilocus sequence typing (MLST) divides C. difficile into five phylogenetic lineages which align the well-known PCR ribotypes; 027, 023, 017, 078 and a lineage containing diverse PCR ribotypes. MLST data in this thesis confirmed the five phylogenetic lineages were maintained after testing a larger collection of isolates from varied sources with further micro-diversity within the individual lineages. MLST investigation did not identify a lineage exclusive to nonhuman strains or any correlation between sequence type and geographical location. Data in this thesis also supports the notion that PCR ribotyping and REA do not correspond as well as previously considered. This may result in phylogenetically similar strains being designated as a different type or variant. The toxin A-B+ PCR ribotype 017 strain that forms a predominant lineage is little investigated. Through whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis, a historical clone of PCR ribotype 017 was identified from a London hospital ward. Although no phenotype exclusive to the clonal strain was characterised, this is the first report in the UK investigating the phylohistory of isolates from hospitalised patients with CDI due to PCR ribotype 017. Further investigation of PCR ribotype 017 with a larger and global collection of strains revealed two distinct sub-lineages containing multiple independent clonal expansions, antimicrobial resistant SNP determinants, deletions and insertions which were well distributed geographically and temporally. The data suggests transmission between humans and animals and findings support a USA origin with multiple, global transmission events. The key findings of this thesis are that C. difficile as a species is continually evolving with the appearance of divergent sub-lineages. WGS is superior to routine typing methodologies for tracking this evolution and will have significant impacts for outbreak investigation, understanding the phylohistory and phylogeography of C. difficile and other pathogens that are a threat to human health.
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Wheeldon, Laura J. "Studies on Clostridium difficile." Thesis, Aston University, 2008. http://publications.aston.ac.uk/15406/.
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Clostridium difficile Is the major cause of nosocomial diarrhoea in the UK and is associated with high morbidity and mortality rates. There has been a large increase in cases of C. difficile associated disease (CDAD) in the last decade and It is thought that the emergence of the hypervirulent strain (ribotype 027) has contributed towards this rise. A major factor in the control and prevention of the disease is adequate cleaning of the clinical environment and disinfection, usually with chlorine based agents. However, the spores of C. difficile are highly resistant to many disinfectants.
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Karlsson, Sture. "Toxin production in Clostridium difficile /." Stockholm : Karolinska institutet, 2004. http://diss.kib.ki.se/2004/91-77349-812-2/.
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Permpoonpattana, Patima. "Clostridium difficile : infection and immunity." Thesis, Royal Holloway, University of London, 2013. http://repository.royalholloway.ac.uk/items/33009ec4-7815-0803-d39b-f968c8d9cdbb/7/.
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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.
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Underwood, Sarah. "Sporulation initiation in Clostridium difficile." Thesis, University of Leeds, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505066.
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Clostridium difficile is a leading cause of hospital-acquired diarrhoea, responsible for over 30% of cases of antibiotic-associated colitis, nearly all cases of pseudomembranous colitis and costs the NHS over 200 million per year. This bacterium is able to persist in the hospital environment to cause recurrent infection by the formation of stable spores, refractile to current decontamination procedures. A more comprehensive understanding of the sporulation signal transduction pathway is essential for the design of a decontamination regime effective in removing the spores from the nosocomial environment and the logical design of novel antimicrobial agents. This project aimed to elucidate the mechanism of sporulation initiation . regulation and the role of sporulation-associated proteins in other C. difficile virulence processes, such as toxin production and colonisation. Analysis of sporulation in response to various hospital cleaning agents showed that the combination of a neutral detergent (such as Hospec) with EDTA is a more effective cleaning agent than the chlorine-based agents currently used, as the combination product is uniquely able to both kill vegetative cells and inhibit spore formation. A variety of molecular approaches were used to elucidate information regarding the C. difficile sporulation initiation pathway and the relationship between sporulation and toxin production. Three putative C. difficile sporulation-associated sensor histidine kinases (CD1A, CD2A and CD3B) were identified and shown to be independently involved in sporulation initiation. Furthermore, CD3B has been shown to directly phosphorylate the master response regulator SpoOA, strongly suggesting that this pathway is a two-component system, as opposed to the extended phosphore lay pathway found in B. subtilis. Previous studies on bacteria capable of both toxin production and endospore formation have described links between the two processes. Data presented here indicates SpoOA has a role in indirectly regulating C. difficile toxin A and B production, as the protein is capable of specifically binding promoter regions of the toxin regulatory genes tcdC and tcdD. Inoculation of a triple-stage continuous-culture chemostat that modelled the human gut with C. difficile spoDA- mutant provided further evidence that SpoDA has a key role in both colonisation a!1d toxin production. Overall, this work adds to the growing body of evidence that SpaDA is a master global regulator and has a crucial role in the pathogenicity of C. difficile, making it an excellent target for future novel antimicrobial therapies.
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Martins, Luís Filipe Pires. "Clostridium difficile uma ameaça renovada." Master's thesis, Instituto de Ciências Biomédicas Abel Salazar, 2008. http://hdl.handle.net/10216/21105.
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César, Artur Jorge Fernandes. "Clostridium difficile - prevenção e controlo." Master's thesis, Faculdade de Medicina da Universidade do Porto, 2009. http://hdl.handle.net/10216/50362.
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César, Artur Jorge Fernandes. "Clostridium difficile - prevenção e controlo." Dissertação, Faculdade de Medicina da Universidade do Porto, 2009. http://hdl.handle.net/10216/50362.
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Martins, Luís Filipe Pires. "Clostridium difficile uma ameaça renovada." Dissertação, Instituto de Ciências Biomédicas Abel Salazar, 2008. http://hdl.handle.net/10216/21105.
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Ticchi, Laurence. "Clostridium difficile et ses toxines : prévalence de "Clostridium difficile" et de la toxine A asymptomatique." Paris 5, 1990. http://www.theses.fr/1990PA05P183.
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Chilton, Caroline Hazel. "Comparative proteomic analysis of Clostridium difficile." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5960.
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The recent increase in availability of next generation sequencing methodologies has led to extensive analysis of the genome of Clostridium difficile. In contrast, protein expression analysis, crucial to the elucidation of mechanisms of disease, has severely lagged behind. In this study, in-depth proteomic analysis of three strains of varying virulence, demonstrated previously in an animal model, has been undertaken against a background of the sequenced genomes. Strain B-1 is a historic, virulent, ribotype 005 clone, strain A represents the emerging hypervirulent 027 ribotype, while strain Tra5/5, ribotype 001, is of low virulence. To undertake a comprehensive overview of the expressed proteome, both 1D and 2D gel electrophoresis were used to separate and display the protein content of each isolate. This was coupled to MALDI-TOF and LC-MS/MS mass spectrometry for protein identification. A total of 888 different proteins were characterised by comparative analysis of isolates grown in parallel for 64 hours on blood agar. Of these, only 38% were shared between all isolates. An additional 350, 243 and 398 proteins were detected from broth cultures, and the use of a hexapeptide bead library, designed to capture low abundance proteins, led to the detection of a further 148, 127, and 171 proteins in strains A, B-1 and Tra5/5 respectively. Relative differential expression was investigated using Differential In Gel Electrophoresis (DIGE), and five proteins were shown to have a statistically higher concentration in strain A, twelve in strain B-1 and eight in strain Tra5/5. A number of these were surface proteins, with selected S-layer proteins found to be up-regulated in each strain, and the flagellar protein, FliC, up-regulated in both A and B-1. Furthermore, differential post-translation modification events were seen in flagellar and S-layer proteins. In-vivo expression of these proteins was mapped using Western blotting. Immunodetection of the majority of these, including FliC and the high molecular weight S-layer protein, were conserved between the three strains, but a notable series of immunoreactive protein spots were present in strains A and Tra5/5 but not B-1, most likely corresponding to an additional S-layer protein present in the genomes these strains, but not that of B-1. Protein expression differences for a number of previously proposed virulence proteins were evident between strains, including toxin B, sporulation, flagella and the S-layer proteins, metabolic enzymes, stress response proteins and ABC transporters. This study strongly supports the view that the virulence of Clostridium difficile is multifactorial, and that a number of related factors, although not directly required for pathogenicity, may serve to modulate the virulence of individual strains.
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Kirby, Jonathan M. "The pathogenesis of Clostridium difficile infection." Thesis, University of Bath, 2011. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.545329.
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Clostridium difficile is a major problem as the aetiological agent of antibiotic associated diarrhoea. The mechanism by which the bacterium colonises the gut is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The aims of this study were to further define roles for selected surface proteins using a knockout approach, to evaluate the feasibility of surface protein based immunotherapeutics and to obtain structural information using X-ray crystallography. Mutants of cell wall-binding domain (PFam04122) containing proteins CD1036, CD2735, CD2784, Cwp66, CD2791, Cwp84, CD2795 and the flagella cap (FliD) were created. Mutants were characterised with regard to growth, sporulation, toxin production, adhesion in vitro, and, for the Cwp84 mutant, using the in vivo hamster model. The surface-located cysteine protease, Cwp84, was found to play a key role in maturation of the C. difficile S-layer, yet the Cwp84 mutant still caused disease with a similar pathology to the wildtype. Culture supernatant levels of toxin A were increased in CD2735, Cwp66, CD2791, CD2795 and particularly in Cwp84 and FliD 24 hr cultures, while CD2735, Cwp66, CD2791, CD2795 mutants also showed reduced adherence to Caco-2 cells compared to the wild-type. Passively administered immunotherapy, generated to low pH surface protein extracts of the C. difficile R20291 strain, did not protect hamsters from challenge with the cognate strain. Structural studies were undertaken on the surface proteins CD2791, Cwp66 and CD2767. Crystallisation conditions were identified for a recombinant N-terminal domain of CD2767 and an X-ray data set collected to 2 Å, although the structure was not solved by molecular replacement. Together these results further our knowledge of C. difficile surface proteins, although further work is required to identify which surface proteins play key roles in vivo during infection.
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Schack, Senta. "Clostridium-difficile-Infektion nach herzchirurgischem Eingriff." Doctoral thesis, Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-198482.
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Clostridium difficile ist einer der führenden Nosokomialkeime in Bezug auf postoperative Diarrhoe. Die Inzidenz ist steigend und der Verlauf bei fulminanter Infektion häufig fatal. Es besteht der Anspruch der Vermeidung schwerer Verläufe und der horizontalen Verbreitung des Erregers.
Ziel der Arbeit war, für den prä-, intra- und postoperativen Zeitraum Risikofaktoren zu identifizieren, welche Einfluss auf Ausprägung und Schwere der Infektion hatten.
Die vorliegende klinische Studie umfasst 2.823 Patienten mit Diarrhoe nach kardiochirurgischem Eingriff, darunter 1.256 Patienten mit Clostridium-difficile-Nachweis, welche im Herzzentrum Leipzig von April 1999 bis April 2011 operativ versorgt worden sind. Die Datenanalyse erfolgte retrospektiv an zuvor festgelegten Parametern, die mittels statistischer Verfahren analysiert wurden.
Besonderes Augenmerk wurde auf die Entwicklung gastrointestinaler Komplikationen und die Mortalität gelegt. Risikofaktoren für eine fulminante CDI waren u.a. männliches Geschlecht, kardiopulmonale Komorbiditäten, Diabetes mellitus Typ II, Verwendung von Assist-Systemen, perioperative Transfusionstherapie, sowie lange Operationszeiten und ein verlängerter Aufenthalt auf Intensivstation. Das Überleben bei fulminanter Infektion war mit einer Sterblichkeit von 63,4% bei einer 30-Tages-Mortalität von 21,6% deutlich schlechter als das der Vergleichsgruppen.
Die Identifikation der perioperativen Risikofaktoren soll eine individualisierte Stratifizierung und damit die optimale Überwachung von Hochrisikopatienten für einen frühen Therapiebeginn und im besten Falle eine Prävention möglich machen.
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Willing, Stephanie. "Assembling the surface of Clostridium difficile." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/45396.
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Clostridium difficile is a Gram-positive, spore forming, obligate anaerobe and pathogen of humans. C. difficile infection (CDI) is a potentially fatal gastrointestinal disease, typically acquired following antibiotic treatment. As a serious nosocomial pathogen, C. difficile also inflicts a large economic cost on healthcare systems. The symptoms of CDI are primarily the result of two toxins, and these have been the focus of much research. However, less well understood is how C. difficile manages to colonise the gastrointestinal tract. The surface of the vegetative cell is likely to prove very important in the colonisation process. The vegetative cell is covered by a paracrystalline array known as a surface layer. There are 28 paralogues of the surface layer protein, SlpA, found on the C. difficile 630 genome; these comprise the cell wall protein family. Each protein within this family has in common a 100 amino acid motif (CWB 2; PF04122) repeated in triplicate at either the N or C-terminus. This motif is found in many species of Gram-positive bacteria, but despite being widespread nothing is known about its anchoring mechanism. This study demonstrates that the CWB 2 repeats have likely evolved into a discrete pseudo-trimer domain such that each repeat is necessary for cell wall protein anchoring. Amino acids that are conserved in the CWB 2 sequence are investigated and residues important for cell wall anchoring identified. A cell wall associated polysaccharide is identified as a ligand of the CWB 2 repeats. A genetic locus that putatively encodes for the biosynthesis of this cell wall associated polysaccharide is investigated, and aberrations in the surface layer demonstrated when these genes are knocked-down. The potential of the CWB 2 repeats to mediate lateral interactions between the surface layer protein SlpA is analysed in experiments that suggest a role for Ca2+ in surface layer assembly. Additionally, a mutant with an unusual colony morphology obtained whilst attempting to inactivate a putative cell wall biosynthesis gene is investigated, revealing a link between a transcription coupled repair factor and toxin expression.
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Rihn, Bertrand. "Cytotoxine et enterotoxine de clostridium difficile." Strasbourg 1, 1991. http://www.theses.fr/1991STR13025.
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Clostridium difficile est une bacterie anaerobie stricte impliquee dans l'etiologie des diarrhees et des colites pseudomembraneuses survenant au decours d'antibiotherapies. Clostridium difficile secrete deux toxines proteiques: une enterotoxine (ou toxine a) et une cytotoxine (ou toxine b). La toxine b native, purifiee a l'homogeneite, presente une masse moleculaire de 290 kda. Quatre peptides trypsiques dont les extremites -cooh et -nh#2 terminales ont ete sequences et montrent une homologie tres importante avec les enolases (e. C. 4. 2. 1. 11). La toxine b penetre dans les cellules astrogliales sous l'action du calcium. Les effets cellulaires lies a la penetration de la toxine b consistent en une fragmentation des filaments d'actine sans alteration des filaments intermediaires ni des microtubules. La toxine b provoque une diminution de la synthese des proteines totales; la synthese des acides nucleiques est moins affectee. Un antiserum specifique et neutralisant son activite biologique a servi pour sa detection de la toxine b dans 210 selles de malades a l'aide d'un test immuno-enzymatique. Ce test est correle etroitement avec la cytotoxicite et la presence de clostridium difficile dans les produits pathologiques des malades. La toxine a de clostridium difficile a ete purifiee a l'homogeneite; elle se presente sous la forme d'un dimere a#1=415 kda et a#2=16 kda en electrophorese denaturante. Son pi est de 3,5. Elle provoque une accumulation de liquide dans l'anse ligaturee de lapin. Il a ete montre que les souches toxinogenes de clostridium difficile adherent aux cellules caco-2 alors que les souches non toxinogenes ne possedent pas cette activite biologique. La presence de ces deux proteines toxiques secretee par clostridium difficile ainsi que la capacite des souches toxinogenes a adherer aux cellules intestinales en culture expliquent la pathogenie des enterocolites a clostridi
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Smyth, Deborah. "Stress and survival of Clostridium difficile." Thesis, Ulster University, 2017. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.706470.
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CDI has been implicated in significant morbidity and mortality rates leading to complications such as pseudomembranous colitis, toxic megacolon and sepsis. Several classical symptoms include infectious and profuse diarrhoea, high grade fever, nausea and abdominal cramps. The aim of this thesis was to insert a functional dnaK heat shock gene in the ClosTron generated C. difficile 630Δerm::dnaK by using a pMTL80000 series plasmid to restore the wild- type phenotype thus correcting the growth rate and temperature sensitivity. The dnaK gene, under the influence of a strong /cZv promoter, conjugated successfully into C. difficile 630Δerm::dnaK. For comparison, a novel pyrE background method established at the University of Nottingham was used to create two further dnaK disruption mutants, C. difficile 630ΔermΔpyrE::dnaK and C. difficile R20291ΔpyrE: :dnaK. These pyrE~ dnaK disruption mutants were then complemented by the insertion of pMTL-YN series plasmids containing a functional dnaK gene. The process was approached in a logical fashion for construction and complementation of the new dnaK deletion mutants in the pyrE' background. It was verified by PCR that the dnaK gene successfully inserted into the pMTL-YN series plasmids and that these plasmids were successfully conjugated into the required C. difficile isolates. Results achieved from the growth curve experiments and motility assays showed that the dnaK deletion mutants in the pyrE' background and associated complements displayed no difference in their growth in comparison to the wild-type (C. difficile 630Δerm).
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Vajhi, Jafari N. "Defining innate immunity to Clostridium difficile." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1347959/.
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Clostridium difficile is a spore-forming anaerobic bacillus and a leading cause of nosocomial diarrhoea. C. difficile infection occurs often following antibiotic treatment leading to alteration of the gut microbiota enabling C. difficile to thrive and cause a range of symptoms from asymptomatic carriage to severe diarrhoea, PMC and death. C. difficile virulence is associated with production of toxins (A, B and CDT) nonetheless this bacteria harbours an array of other virulence factors. In the present study, C. difficile-mediated innate immunity response was investigated utilising four different strains including: R20291 a hypervirulent strain (A+B+, CDT+), 630 a fully sequenced strain (A+B+) and variant strains M68 and CF5 (A-B+). Initial studies showed that all strains shared comparable survival and sporulation capacity whereas strains R20291, M68, and CF5 achieved similar growth kinetics. R20291 showed significant adherence to IEC and was the most potent strain. 630 and M68 were found to be as cytotoxic leading to significant cell apoptosis, IEC TJ disruption and increased paracellular permeability. In contrast, CF5 had the least effect on cell death and IEC barrier integrity. Although all four strains induced antimicrobial immunity and pro-inflammatory cytokines, CF5 mediated the least pro-inflammatory responses. Similar findings were also found in an ex vivo model of infection utilising human colonic explants. C. difficile strains up-regulated murine DC maturation markers leading to a predominant anti-inflammatory IL-10 secretion, which is known to suppress IL-12 and IL-23 induction although C. difficile may generate a cytokine milieu that favours dual Th1/Th17 immunity. C. difficile toxin mutant strains showed DC activation and cytokine production in a toxin-dependent and independent manner and triggered an ASC-containing inflammasome causing the activation of caspase-1 and release of mature IL-1β. These findings indicated that multiple bacterial factors may play a role in initiating host innate immune responses to C. difficile infection.
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Ransom, Eric M. "Clostridium difficile: shedding light on pathogenesis." Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/5828.
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Clostridium difficile is a strictly anaerobic, spore-forming bacterium that is linked to over 250,000 infections annually in the United States. One of the greatest challenges facing C. difficile research has been the lack of genetic tools. This limited repertoire is due, in part, to the anaerobic nature of C. difficile. For example, most fluorescent protein reporters require O2 for chromophore maturation. Here, we demonstrate that O2-dependent fluorescent proteins produced anaerobically can acquire fluorescence after cells are fixed with cross-linkers to preserve native patterns of protein localization. This was shown using the blue and the red codon-optimized fluorescent proteins, CFPopt and mCherryOpt, respectively.
Little is known about cell division in C. difficile. Here we identify and characterize a three-gene operon encoding cell division proteins found only in C. difficile and a small number of closely related bacteria. These proteins were named MldA, MldB, and MldC, for midcell localizing division proteins. MldA is predicted to be a membrane protein with coiled-coil domains and a peptidoglycan-binding SPOR domain. MldB and MldC are predicted to be cytoplasmic proteins; MldB has two predicted coiled-coil domains, while MldC lacks obvious conserved domains or sequence motifs. Mutants of mldA or mldB had morphological defects, including loss of rod shape (a curved cell phenotype) and inefficient separation of daughter cells (a chaining phenotype). Fusions of CFPopt to MldA, MldB, and MldC revealed that all three proteins localize sharply to the division site. Mutants lacking the Mld proteins are severely attenuated for pathogenesis in a hamster model of C. difficile infection. Because all three Mld proteins are essentially unique to C. difficile, they could be exploited as targets for antibiotics that combat C. difficile without disrupting the intestinal microbiome.
C. difficile pathogenesis is mediated primarily by two large exotoxins called Toxin A (TcdA) and Toxin B (TcdB). Transcription of tcdA and tcdB depends on TcdR, an alternative sigma factor for RNA polymerase. Previous studies have shown both toxins are produced upon entry into stationary phase, and that this response is mediated in part by the CodY repressor, which senses GTP and branched chain amino acids. Here we used mCherryOpt as a reporter of gene expression to visualize toxin expression at the level of individual cells. This approach led to the unexpected discovery that only a subset of cells in the population induces expression of tcdA (and tcdB under specific conditions). In other words, toxin production is a “bistable” phenotype. Further experiments indicated TcdR plays a central role in mediating bistability, while CodY makes a minor but still significant contribution to bistability. Why it is advantageous for only a subset of C. difficile cells to produce toxin is not known, but one interesting possibility is related to conflicting requirements for transmission to a new host. Some cells produce toxin to provoke diarrhea while other cells differentiate into spores that can survive exposure to air.
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Krajewski, Christina [Verfasser]. "Korrelation der Typisierung von Clostridium difficile Isolaten und klinischen Daten der Patienten mit Clostridium difficile Infektion / Christina Krajewski." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2018. http://d-nb.info/1168900581/34.
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Kerzmann, Amy N. "Mechanistic analysis of Clostridium difficile toxin A." [Bloomington, Ind.] : Indiana University, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3378359.
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Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2009.Title from home page (viewed on Jul 12, 2010). Source: Dissertation Abstracts International, Volume: 70-10, Section: B, page: 6182. Advisers: Andrew L. Feig; James T. Drummond.
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Mullan, Nivette K. "Mucosal cell responses to Clostridium difficile toxins." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/13217/.
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Colonic inflammation in C. difficile infection is mediated by released toxins A and B. I have investigated responses to C. difficile toxin A and B by primary human colonic myofibroblasts, which represent a distinct subpopulation of mucosal cells that are normally located below the intestinal epithelium and epithelial cell lines, Caco-2 and HT29. Myofibroblasts, isolated from normal human colonic mucosal specimens, Caco-2 and HT29 cells incubated with purified toxin A or B displayed a dose dependent response. Myofibroblast morphology changed to a stellate shaped cell, with processes that were immunoreactive for alpha smooth muscle actin. Most of the myofibroblasts remained viable, with persistent stellate morphology, despite exposure to high concentrations (up to 10 μ g/ml) of toxin A for 72 h. In contrast, a majority of the toxin B exposed myofibroblasts lost their processes prior to cell death over 24-72 h. Investigating toxin A+B on myofibroblasts, at low concentrations, toxin A provided protection against toxin B-induced cell death. Most of the intestinal epithelial, HT29 cells remained viable despite exposure to high concentrations of either toxin (up to 10 μ g/mi). By contrast, a significant loss in cell viability was observed in Caco-2 cells exposed to either toxin. Within 4 h, myofibroblast and epithelial cell types exposed to either toxin A or B lost expression of the non-glucosylated form of Racl, but total intracellular RhoA remained unchanged in myofibroblasts and Caco-2 cells. A time-dependent reduction in RhoA expression was seen in HT29 in response to toxin A or B. Active RhoA expression was lost within 4h in myofibroblasts exposed to either toxin. Despite pre-exposure to high concentrations of toxin A for 3 h, colonic myofibroblasts were able to recover their morphology and proliferative capacity during prolonged culture in medium. This was also shown when pre-exposure to toxin A was extended to 48 h. However, toxin B-pre-exposed myofibroblasts were not able to recover. In conclusion, primary human colonic mucosal myofibroblasts are resistant to toxin A (but not toxin B)-induced cell death. Responses by colonic myoflbroblasts may play an important role in mucosal protection, repair, and regeneration in colitis due to C. difficile infection. Investigation into the apparent resilience of HT29 cells has highlighted the importance of cell specific substrate specificity by C. difficile toxins.
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Johal, Shawinder Singh. "The pathogenesis of Clostridium difficile induced disease." Thesis, University of Nottingham, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.403400.
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Alabdali, Yasir. "Characterisation of antibiotic resistance in Clostridium difficile." Thesis, University of Sheffield, 2017. http://etheses.whiterose.ac.uk/18910/.
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Clostridium difficile is a Gram-positive, obligate anaerobe and an opportunistic pathogen that causes antibiotic associated diarrhoea. The incidence of C. difficile infection (CDI) increased dramatically in the early years of this century, an epidemic caused by the previously rare ribotype 027. In addition to causing large hospital outbreaks this lineage was also associated with seemingly more severe disease. Ribotype 027 strains have been reported to produce more spores and more toxin, perhaps going someway to explaining the efficient transmission and poor clinical outcome. We sought to understand the peptidoglycan biosynthetic pathways active in both vegetative cells and during sporulation, in order to identify proteins playing a role in resistance to cell wall targeting antibiotics. A total of 11 genes predicted to encode penicillin-binding proteins (PBPs) were identified in the genome of R20291, the UK prototypic ribotype 027 strain. Two putative PBPs were taken forward for further study: one class B PBP, SpoVD, required for both sporulation and cephalosporin resistance, and one class C PBP, Cwp20, that contributes to cephalosporin resistance. A ΔspoVD mutant showed two strong phenotypes: a sporulation defect and cephalosporin sensitivity. In addition, an interaction between SpoVD and SpoVE that appears to be crucial in both sporulation and cephalosporin resistance was demonstrated. A Δcwp20 mutant had a clear defect in cephalosporin resistance. Disruption of cwp20 in a strain that completely lacked the S-layer provided further evidence for a role cephalosporin resistance. Cwp20 was determined to be a class A β-lactamase. The third part of this thesis is devoted to identification of genes that are responsible for ceftazidime, cefoxitin and ciprofloxacin resistance. A total of 6,000 transposon mutants were screened for resistance to each antibiotic. Three genes with defects in resistance to these antibiotics were chosen for detailed analysis. SpoVE, a membrane protein and putative lipid II flippase, was found to be involved in both cefoxitin and ceftazidime resistance. A CD0398 mutant was found to have a defect under ceftazidime selection. Complementation restored ceftazidime resistance and CD0399 was identified as a class A β-lactamase. A CD0622 mutant was found to have a defect under ciprofloxacin selection and CD0622 was demonstrated to act as an efflux pump.
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He, Miao. "Genomic variation and evolution of Clostridium difficile." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609982.
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