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1
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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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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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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Goh, Shan. "Phenotypic and genotypic characterisation of bacteriophages of Clostridium difficile." University of Western Australia. Microbiology Discipline Group, 2003. http://theses.library.uwa.edu.au/adt-WU2004.0018.
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Clostridium difficile is an important hospital-acquired pathogen causing C. difficile-associated diarrhoea (CDAD) in patients exposed to antibiotics. The lack of information on bacteriophages of C. difficile, and the potential of phages as therapeutic agents for the treatment of CDAD, prompted the isolation and characterisation of phages active against clinical isolates of C. difficile in order to determine the prevalence and significance of phages of this anaerobe. Three (5.4 %) of 56 clinical C. difficile isolates induced by mitomycin C yielded dsDNA phages C2, C5, C6 and C8. The four phages differed from previously described C. difficile phages in particle morphology, burst size and host range. C2, C5 and C8 particles were members of the family Myoviridae, while C6 belonged to Siphoviridae. The burst sizes were 5 for C2, 7 for C5, 19 for C6 and 33 for C8. C8 had the broadest host range, lysing 27 out of 56 (48 %) C. difficile isolates, followed by C6 (43 %), C5 (20 %) and C2 (20 %). Superinfection experiments, restriction enzyme analysis and Southern hybridisation showed C2 and C5 to be closely related with C8 somewhat related to them, however, C6 was distantly related to the other three phages. C2 was further characterised as a representative phage. Its genome did not possess cohesive ends, and was shown to integrate chromosomally via an attP site identified within a 1.9 kb HindIII fragment. However, an integrase gene, which is typically close to the attP region, was not located. Nine of 16 HindIII fragments of C2, including the 1.9 kb fragment, were cloned into pUC18. Approximately 9 kb of the estimated 43 kb genome of C2 was sequenced and analysed. Seven of the nine translated sequences were homologous to phage structural proteins, two sequences were not homologous to any relevant protein in the Genbank and EMBL databases, and one was homologous to proteins of Clostridium species. Nucleotide homology between the C2 sequences and the recently sequenced C. difficile strain CD630 was found in three regions within CD630 genome. Seven of the nine sequences, including the 1.9 kb fragment, were clustered in one region. These data suggest that the genes constitute a phage structural gene module. The presence of C2-like sequences in CD630, and Southern hybridisation of C. difficile strains using phage probes, suggested related prophage sequences may be commonly present in this bacterial species. An investigation was carried out to determine the presence of toxin genes tcdA and tcdB, and PaLoc-associated gene tcdE, in phage DNA. In addition, the effect of phage infection on toxin production of toxigenic C. difficile strains was studied. Of the three genes, tcdE only was detected in phages C2, C5 and C8, but not in C6. Strains that maintained phages in a stable manner (lysogens) were isolated and used in toxin studies. The amount of toxin B produced was measured by cytotoxic assays using Vero cells, and toxin A production was measured by ELISA. Although phages did not encode toxin A or B genes, there was a significant increase in toxin B production in some lysogens. There was no increase in toxin A production. Transcriptional analyses of tcdA and tcdB in lysogens and parental strains was performed by real-time RT-PCR and Northern hybridisation to determine whether phage was affecting regulation of toxin transcription. Phage did not appear to affect toxin gene transcription, although results from real-time RT-PCR and Northern hybridisation were conflicting. A phage induced from the highly toxigenic reference strain VPI 10463 was also briefly characterised and investigated for its effect on toxin production in VPI 10463. The phage, ΦCV, had similar particle morphology to C2, C5 and C8, and had some HindIII bands in common with C2 and C5. Two cured variant strains produced significantly less toxin B compared to VPI 10463. In conclusion, several important properties of C. difficile phages were characterised. It appears these temperate phages may play a role in toxin production making them unsuitable as therapeutic agents for the treatment of CDAD. However, C2 phage may have potential as the basis for an integrative vector that will add to the genetic tools available for clostridia.
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Mizrahi, Assaf. "Caractérisation de la réponse immune de l’hôte dans les infections à Clostridium difficile." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS294.
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Clostridium difficile est une bactérie présente sous formes de spores dans l’environnement qui vont être ingérées par l’hôte puis germer en formes végétatives dans le tube digestif. Les formes végétatives vont coloniser l’hôte grâce à différents facteurs de colonisation. Ensuite, C. difficile va produire des toxines A et B qui vont être responsables des signes cliniques de l’infection.Après ingestion et colonisation par une souche toxinogène, les présentations cliniques sont variables d’un individu à l’autre. Certaines personnes vont rester porteuses asymptomatiques et d’autres peuvent déclarer des formes menaçant le pronostic vital. De plus, un caractère particulier de l’infection à C. difficile (ICD) est la survenue de récidives. Cette variabilité inter-individuelle de réponses de l’hôte à la colonisation par C. difficile est probablement multifactorielle mais elle paraît reposer largement sur le développement d’une réponse immune efficace de l’hôte.De nombreux travaux ont évalué l’intérêt de la réponse immune développée contre les toxines de C. difficile aboutissant à l’élaboration de thérapeutiques par immunisations ciblant les toxines.Cependant, la réponse immune dirigée contre les toxines n’est pas exclusive. Au vu des principaux résultats de l’équipe et de la littérature récente, il semble que le précurseur des protéines de la couche S SlpA et la flagelline FliC soient d’une importance particulière puisque ces protéines interagissent activement avec les cellules de l’immunité innée via les TLR (Toll Like Receptor) et sont immunogènes chez l’Homme.Nous avons donc voulu reposer les bases du développement d’une réponse immune chez un individu naïf en utilisant un modèle murin d’ICD.Nous avons montré qu’après une infection dans ce modèle aussi bien qu’après deux infections itératives, les animaux développaient une réponse de type IgM dirigée contre les toxines avec une commutation de classe en IgG au niveau sérique. Cependant, bien que nous ayons également observé la production d’IgM spécifiquement dirigées contre SlpA ou FliC, les souris n’avaient paradoxalement pas développé d’IgG dirigés contre SlpA ou FliC.Nous avons ensuite décidé de mener des essais d’immunisation chez la souris en se focalisant sur SlpA. Après des immunisations répétées, en présence de la toxine cholérique utilisée comme adjuvant, nous avons mis en évidence une réponse spécifique objectivée par une augmentation des IgG sériques et des IgA mucosales anti-SlpA dans les deux modèles. Cette réponse était associée à une diminution significative des taux de colonisation et un retard au décès des hamsters.Nous avons ensuite montré sur une première cohorte prospective que les patients atteints d’une ICD simple avaient significativement plus d’IgG spécifiques anti-SlpA que les patients récidivant de l’ICD entre 5 et 25 jours après l’infection.Enfin, nous avons également constitué une deuxième cohorte de patients sur la base d’une étude cas-témoins avec des objectifs plus ambitieux notamment de constitution de collections biologiques cliniquement documentée. Parmi les objectifs, l’étude vise à déterminer de la valeur prédictive des anticorps dirigés contre les facteurs de colonisation et les toxines de C. difficile sur l’évolution de l’ICD et notamment les récidives. Les résultats préliminaires concernant les IgG dirigés contre SlpA sur les premiers couples de cas et de leurs témoins permettent de confirmer ceux qui avaient été observées sur la première cohorte. L’ensemble des collections biologiques et des données cliniques associées permettront très rapidement de générer de nombreux résultats pour chacun des antigènes d’intérêt et ouvrent à de nombreuses perspectives en termes de compréhension des processus physiopathologiques et d’études ancillaires sur la réponse immune cellulaire et/ou humorale ou l’étude de marqueurs biologiques prédictifs des formes sévères et/ou récidivantesClostridium difficile is a bacterium found in the environment as spores. It can be ingested by a host and germinate under vegetative forms in the digestive tract. These forms colonize hosts through colonization factors. C. difficile will then produce two toxins (A and B), responsible for clinical signs of infection.Following the ingestion and colonization by a toxigenic strain, a wide spectrum of clinical presentations can occur between individuals. Some will remain asymptomatic carriers when others will develop life-threatening infections. Besides, C. difficile infections (CDI) are specific as they can develop recurrences. These inter-individuals variabilities seem to be multifactorial, though, vastly depending on a host efficient immune response.Studies have assessed the interest of an immune response against C. difficile toxins, leading to immunization therapeutics targeting the toxins.However, immune response against toxins isn’t exclusive. Considering our team findings and recent literature, it seems that the S layer proteins precursor SlpA, as well as the FliC flagellin, have an important role. They indeed actively interact with the innate immunity cells via the TLR (Toll Like Receptor) and are immunogen in Humans.We therefore aimed at laying the foundations of an immune response development in a naïve individual, using a CDI mice model.We demonstrated within this model that further to one or two iterative infections, the animals developed an IgM response against the toxins, with a commutation in IgG at the serum level. However, despite the production of IgM specifically targeting SlpA or FliC, mice didn’t develop IgG against SlpA or FliC.We then decided to conduct immunization assays in mice, focusing on SlpA. After repetitive immunizations with the choleric toxin as an adjuvant, we noticed a specific response, objectified by a growth of serum IgG and anti-SlpA mucosal IgA in both models, as well as a significant decrease in colonization levels and a delay in the hamsters’ death.We showed on a first prospective cohort that patients with simple CDI had significantly more specific anti-SlpA IgG than patients with a CDI recurrence, occurring 5 to 25 days post infection.Finally, we constituted a second cohort of patients with a case-control study. It had more ambitious objectives, including the collection of clinically documented biologic samples. Among the objectives, this study aims at assessing the predictive value of antibodies against C. difficile colonization factors and toxins on the CDI evolution, notably on the recurrences.Preliminary results of IgG against SlpA on the first case-control couples confirm the first cohort observations. The biological collections and associated clinical data will soon enable to generate results for each antigen of interest. It opens new perspectives in terms of understanding of pathophysiological processes, and ancillary studies on the cellular and/or humoral immune response, as well as the study of predictive biological markers of severe forms or recurrences
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Couchman, Edward. "Investigating the Type IV pili of Clostridium difficile and Clostridium sordellii." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/48055.
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Type IV pili (T4P) are the only type of bacterial pili known to be produced by both Gram-negative and Gram-positive organisms. Though the main pilus shaft consists primarily of only one protein (the major pilin), T4P are unusual in their complexity, requiring multiple (10 or more) different protein components for assembly. Like most types of pili, T4P often function as virulence factors. In particular, T4P frequently operate as adhesins, enabling bacteria on which they are present to stick to each other (to form a biofilm or suchlike) or to adhere directly to host cells. Many T4P systems are able to retract, in which case the T4P may mediate flagella-independent motility. Most research into T4P has historically been performed on Gram-negative organisms, with T4P-encoding genes only being identified in Gram-positive organisms more recently. In particular, all sequenced species of the genus Clostridium are known to encode T4P, but only minimal investigation of these systems has been performed to date. In this study, the T4P of Clostridium difficile were investigated. C. difficile is an important pathogen, being the leading cause of antibiotic-associated diarrhoea in the developed world and thus a considerable burden on Western healthcare systems. By investigating the T4P of this species it was hoped to further elucidate its mechanisms of pathogenicity. Data is presented demonstrating the control of T4P expression by cyclic-di-GMP, and identifying which genes are essential for T4P production in C. difficile. Additionally, a genomic analysis of the related pathogen Clostridium sordellii was performed, using the first high quality genome sequence produced for this species. Genes encoding T4P were identified, analysed and investigated. Furthermore, plasmids carrying the genes encoding the species’ key virulence factors (Lethal Toxin, TcsL, and in some cases haemorrhagic toxin, TcsH) were identified. These plasmids appear to be unstable, a fact with significant implications for diagnosis of C. sordellii disease.
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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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Meader, Emma. "Exploiting bacteriophages to tackle Clostridium difficile infection." Thesis, University of East Anglia, 2013. https://ueaeprints.uea.ac.uk/48147/.
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Clostridium difficile infection (CDI) currently affects around 20,000 people each year, in healthcare institutions and in the community, and will often follow disruption of the gut microbiome. Current treatment strategies call for the use of further antibiotics, of which there is a limited choice. There is a need for additional remedial and prophylactic solutions with greater specificity and low levels of toxicity and resistance. This thesis describes the pathogenesis of CDI, the current treatment strategies and navigates the growing body of studies investigating the potential use of phage. The project involved extensive screening including faecal samples and environmental sources in an attempt to identify novel phages of C. difficile and documents efforts to improve the therapeutic capacity of a selected phage, ФCD27, by mutagenesis. No exclusively lytic phages were isolated or obtained following mutagenesis with ethylmethane sulphonate, hydroxylamine or sodium pyrophosphate. Batch fermentation models of CDI showed that a prophylactic approach to phage therapy of CDI offers a higher efficacy than a remedial regime. A continuous model of CDI in a colon model was successfully produced and demonstrated variable efficacy rates from no apparent decrease in the burden of C. difficile to a reduction to below the limit of detection by culture, with no detrimental effect on commensal microbiota. The lysogenic capacity of ФCD27 appeared to prevent clearance of C. difficile in the models, but some strains containing the prophage exhibited reduced toxin production phenotypically. A possible mechanism of this altered phenotype included the action of ФCD27 repressor proteins on the promoter regions of C. diffiicle toxin genes or regulatory elements, but affinity of a candidate repressor, ORF44, to PaLoc constituents was not demonstrated. Studies have also demonstrated the ability of ФCD27 to prevent outgrowth of germinating C. difficile spores, thus potential as an environmental decontaminant. iii The findings of the project and the future prospects of phage therapy as an agent against CDI are discussed.
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Almdni, Sabir M. Shakir. "Recombinant antibodies against Clostridium difficile toxin A." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/4727/.
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Clostridium difficile is a major cause of nosocomial intestinal infection. The pathogen possesses two potent toxins, Toxin A and Toxin B, both of which contribute to diarrhoea, intestinal inflammation and tissue damage. Antibiotics are effective against the disease, however around 20 % of patients on treatment relapse after the termination of antibiotic therapy. The binding of Toxin A to a receptor on human intestinal epithelial cells initiates disease: this is considered the starting point from which the toxin elicits its effect. One feature of the carboxy-terminal domain of Toxin A is the presence of repeating units of amino acids that form a series of binding sites able to recognise disaccharides and trisaccharides on glycolipid and glycoprotein receptor molecules. Antibody response against the toxin can protect against C. difficile disease and efforts to generate vaccines have focused upon the carboxy-terminal, receptor binding domain. The aims of this project were to use phage display to isolate recombinant antibodies against those features of the carboxy-terminal domain of Toxin A thought to be responsible for receptor-binding and to assess if the antibodies were capable protecting against the action of Toxin A. Using published crystallographic data that has shown the interaction of Toxin A and trisaccharide, a region of about 113 amino acids from the carboxy-terminal region of Toxin A was expressed as a fusion to maltose-binding protein. The MBP fusion protein was expressed, purified on amylose resin, and characterised. The fusion protein was then used to isolate single chain antibodies from the Tomlinson libraries of scFvs, a synthetically diversified phage display library of single scaffold human antibodies. Conventional bio-panning methods were used in which the MBP fusion protein was bound to a plastic surface and the phage display libraries were pre-mixed with native MBP to inhibit the isolation of anti-MBP antibodies. Progressive enrichment of scFvs through 3 rounds of selection was observed. Those scFvs that showed strongest reaction against the target protein in ELISA but failed to react with native MBP were sequenced, expressed as soluble antibodies and purified on nickel chelating columns. While the resulting panel of scFvs showed similarities of sequence, none were identical. All were reactive with native, full-length Toxin A and appeared to bind to conformational (nonlinear) epitopes. Cross-reaction with Toxin B from C. difficile was also evident. A panel of truncation mutants were generated from the MBP fusion protein and using these in ELISA with the scFvs, reactivity appeared to be directed to features of a long repeat sequence of Toxin A. To assay whether the isolated scFvs possessed biological activity of significance, in vitro and in vivo protection assays were established. For experiments in vitro, the action of Toxin A upon cultured Vero cells was studied. Native Toxin A triggered a conversion of the cells from stellate to rounded morphology. When cells were exposed to 100 ng of toxin, this effect was evident within 60 minutes; at a 10-fold lower dose, the minimum quantity to which a response was detectable, virtually all cells had undergone rounding within 2 h. When individual scFvs were mixed with 10 ng of Toxin A prior to addition to Vero cells, there was a consistent delay in cytopathic activity that extended to 5 h. In this assay, the percentage of cells that had retained their stellate morphology 5 h post-challenge was dependent on the scFv used. To quantify the potency of this neutralising activity, the amount of each scFv required to achieve 50% protection during a 2 h challenge period was established. This revealed 3.5-fold difference between the most and the least effefctive scFv. The most potent scFv was used in an in vivo assay in which Toxin A was administered to the ligated intestinal loops of rats. Again, protective activity was evident. Overall, phage display technology enabled the assembly of a panel of scFv antibodies against the putative receptor binding site in the carboxy-terminal domain of Toxin A from C. difficile. The scFvs were able to protect against the cytopathic activity of Toxin A in vitro and in vivo and proposals are made about how these observations could be taken forward in a model of C. difficile infection that best mimics the human disease.
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Ogbu, H. I. "Amino acids utilisation by Clostridium difficile strains." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/37371/.
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The carbon and energy metabolism of the human pathogen Clostridium difficile is poorly understood. Amino acid metabolism by the Stickland reactions has previously been described as a primary source of energy in a number of Clostridium species, especially when grown in a medium containing only amino acids. Deeper insights may be gained by metabolic analyses using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) platforms, but such experiments are best performed in well-defined growth media. A number of C. difficile strains have been successfully cultivated on defined media but these media provide an excess of nutrients, particularly in terms of amino acid provision, resulting in undesirable background growth in the absence of glucose. To overcome these challenges, three variants of a defined medium were developed that contain the essential nutrients that support the growth of this bacterium, in the presence of a carbon and energy source such as glucose together with an LC-MS/MS method that will simultaneously measure all twenty amino acids. Since the focus in this study was amino acid utilisation, a comprehensive and most effective technique that will provide as much information as possible for understanding the metabolic requirements of eight Clostridium difficile strains (CD630∆erm, DH196, R20291, EK15, EK28, R12801, L26, O17 Serotype F) and two transposon mutants (CRG-2979, CRG-3887) was required. Due to high water solubility and the range of ionic characteristics of amino acids, an aqueous normal phase chromatographic method was considered to simultaneously separate a mixture of amino acids without derivatisation. Aqueous normal phase chromatographic method represents an important new technology with a capability of the silica-hydride-base stationary phases, offering a distinct advantage for practical application with a high degree of reproducibility and long-term stability of polar and non-polar compounds. The developed methods were subsequently adapted to study amino acid utilisation in the presence or absence of a fermentable carbohydrate and/or selenium. OD determinations were performed by measuring absorbance at 600 nm (or OD600) using a Biomate 3 spectrophotometer. The concentration of individual amino acids remaining in the spent C. difficile culture media was measured after 24 h and/or 48 h using the developed LC-MS/MS method in order to determine which amino acids were being utilised by these organisms and in which order. This analysis should give further insight on the importance of amino acids for the survival of this bacterium in the gut, which may possibly lead to discovery of novel fermentable products and metabolic pathways and/or eventually aid in the successful control of the disease. Data obtained for CD630∆erm, DH196, R20291, EK15, EK28, R12801, L26, O17 Serotype F strains showed that they all grew on the fully defined medium, with different growth profiles in terms of lag phase, growth rate, and maximum OD reached. The LC-MS/MS data generated suggest that cysteine, glutamine, isoleucine, leucine, serine, threonine and tyrosine are preferentially utilised, both in the presence and absence of glucose. Several other amino acids, including asparagine, glycine, phenylalanine, proline and valine were also utilised but to a lesser extent. Notable in this study was the lack of glutamate utilisation, except by strain L26, and the excretion of alanine after its initial uptake by most of the tested stains. The excretion of alanine may be due to the use of pyruvate as an amino acceptor during the degradation of preferentially fermented amino acids, whereas, glutamate is not a substrate for most C. difficile strains. Thus, LC-MS/MS profiling confirmed that these organisms derive most of their carbon and energy from the fermentation of a selected range of amino acids. Given the importance of selenium-dependent Stickland reactions to the growth of this bacterium, further studies were undertaken to evaluate the metabolism of amino acids by two different C. difficile strains (630∆erm, R20291) and two transposon mutants CRG-2979 (defective in hadB, encoding one of the two subunits of hydroxyisocaproyl-CoA dehydratase required for reductive degradation of leucine) and CRG-3887 (defective in selA encoding selenocysteine synthase) in the presence or absence of glucose/selenium. LC-MS/MS data reveal that amino acid utilisation was affected by the presence of selenite, notably proline utilisation, which could be explained by the presence of the enzyme proline reductase and the lack of glycine consumption, known to be selenium-dependent. The non-utilisation of glycine could be explained by the presence of proline which represses the formation of the necessary enzyme systems required for glycine degradation. Data generated for CRG-2979 reveals that this mutant could only thrive in the presence of selenium when glucose is present, possibly due to the presence of proline replacing leucine as the major Stickland acceptor. The results of the transposon mutant CRG-3887, were much of a surprise too, because this mutant was predicted to be deficient in proline and glycine breakdown which is also reliant on selenoenzymes. This suggests the presence of selenium independent proline fermentation pathway although this hypothesis is not supported by the existing literature. Experimental data provided further evidence about the ability of this bacterium to obtain its carbon and energy in the absence of a fermentable carbohydrate; by Stickland reactions and that the presence or absence of certain amino acids could repress the utilisation or biosynthesis of other amino acids.
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Islam, Jasmin. "Evaluating patient susceptibility in Clostridium difficile infection." Thesis, University of Brighton, 2013. https://research.brighton.ac.uk/en/studentTheses/059dba8c-ad37-4d2a-9f0e-13b94f843e0e.
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Clostridium difficile infection (CDI) is the leading cause of nosocomial diarrhoea and causes substantial morbidity and mortality. Efforts to reduce the impact of CDI have succeeded in reducing rates through antibiotic stewardship, improved diagnostic testing and optimisation of infection control measures. Further reductions in CDI could be achieved through a better understanding of what makes patients susceptible to CDI. Such knowledge would support interventions targeting patients most at risk and help develop treatments to reduce susceptibility. The aim of this thesis was to further our understanding of patient susceptibility to CDI by investigation of three specific areas. The first study investigated the role of the probiotic Lactobacillus casei DN114001 in preventing antibiotic associated diarrhoea (AAD), including CDI, as part of a large multicentre, double-blind, randomised placebo-controlled trial. Probiotics are live microorganisms that may help restore antibiotic disruption to the host microflora and prevent C. difficile colonisation. The final results were not available at the time of writing this thesis and therefore a descriptive analysis of the first 650 blinded cases is provided. This is the largest probiotic study ever conducted and will contribute significantly to the existing literature in the field. The humoral immune response has been implicated in determining outcome in CDI. Previous studies have focused on recurrence of CDI and toxin A (TcdA), which was originally thought to be the most important virulence factor in CDI. However, recent studies have suggested toxin B (TcdB) may be essential for CDI pathogenesis. Therefore, the second study tested the hypothesis that antibodies to TcdB determine patient susceptibility in CDI. A case-control laboratory based study was conducted using a novel antibody ELISA and antibody responses to both toxins were assessed in two cohorts recruited in Brighton, UK and Michigan, USA. Lower antibody levels to TcdB, but not TcdA, were found in cases of acute CDI compared to controls. These novel findings are in contrast to previous studies and confirm the importance of TcdB in CDI pathogenesis. In addition, the antibody response to TcdB could be used as a surrogate marker for the efficacy of novel therapeutic agents. The third study sought to identify risk factors predicting recurrence of CDI. A longitudinal cohort study of 248 patients with confirmed CDI was conducted that confirmed the previously observed relationship between concomitant antibiotic treatment and risk of recurrence. The study also identified a novel risk factor namely that treatment on a cohort ward was associated with recurrence of CDI. This is likely to be a result of reinfection of patients who remain susceptible to CDI after treatment. This is the first study to demonstrate an association between cohorting of patients and recurrence of CDI and raises important questions about current infection control policies in hospitals. Efforts to combat CDI have focused on reducing exposure of patients to infection. The data presented here contribute to a rapidly emerging understanding that patient susceptibility is a crucial factor in determining risk of infection, risk of severe disease and risk of recurrence following treatment. In the near future interventions targeting susceptibility including probiotics, specific antibiotics such as fidaxomicin and immunotherapies such as vaccines may all have a role to play in combatting this devastating disease.
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Agoumellah, Fatiha. "Pathologie de Clostridium difficile chez le nourrisson de moins d'un an." Paris 5, 1998. http://www.theses.fr/1998PA05P199.
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Frey, Steven M. "The localization of two epitopes recognized by the monoclonal antibody PCG-4 on toxin A of Clostridium difficile." Thesis, This resource online, 1990. http://scholar.lib.vt.edu/theses/available/etd-05022009-040613/.
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Delamare, Olivier. "Diagnostic au laboratoire des colites à "Clostridium difficile" : à propos de quelques cas." Paris 5, 1988. http://www.theses.fr/1988PA05P016.
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Hussack, Greg. "Single-domain Antibody Inhibitors of Clostridium difficile Toxins." Thèse, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/20362.
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Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The Gram-positive bacterium produces two exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease (CDAD) and are targets for CDAD therapy. In this work, recombinant single-domain antibody fragments (VHHs) which target the cell receptor binding domains of TcdA or TcdB were isolated from an immune, llama phage display library and characterized. Four VHHs (A4.2, A5.1, A20.1, and A26.8) were potent neutralizers of the cytopathic effects of TcdA in an in vitro assay and the neutralizing potency was enhanced when VHHs were administered in combinations. Epitope mapping experiments revealed that some synergistic combinations consisted of VHHs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Binding assays revealed TcdA-specific VHHs neutralized TcdA by binding to sites other than the carbohydrate binding pocket of the toxin. The TcdB-specific VHHs failed to neutralize TcdB, as did a panel of human VL antibodies isolated from a synthetic library. To enhance the stability of the C. difficile TcdA-specific VHHs for oral therapeutic applications, the VHHs were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. The mutant VHHs were found to be well expressed, were non-aggregating monomers, retained low nM affinity for TcdA, and were capable of in vitro TcdA neutralization. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants without compromising inherent VHH trypsin resistance. Collectively, the second disulfide not only increased VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance. These are all desirable characteristics for the design of protein-based oral therapeutics. In conclusion, llama VHHs represent a class of novel, non-antibiotic inhibitors of infectious disease virulence factors such as C. difficile toxins.
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Aspinall, Steven Thomas. "The isolation, identification and detection of Clostridium difficile." Thesis, University of Central Lancashire, 1992. http://clok.uclan.ac.uk/20986/.
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The aim of the study was to determine the feasibility of an enzyme linked immunosorbant assay (ELISA) for detection of Clostridium difficile in broth cultures and faeces, to develop a new selective isolation medium and rapid identification test for Cl.difficile, to investigate the use of SDS-PAGE for typing isolates and to determine the presence of antibodies against Cl.difficile antigens in the sera of infected patients. Detection of C1.difficile antigens using ELJSA: Using a competitive ELISA, a methodology was developed to determine the feasibility of the test for detection of Cl.difficile in broth cultures and faeces. Negative broth cultures produced no reduction in absorbance, whilst positive cultures resulted in >84% reduction. Negative faecal extracts yielded reductions of (18%, whilst positive samples produced >64% reduction (with a coefficient of variation of 5%). ELISA was thus found to be a feasible method but highly specific antibodies are required. Media development: Various culture media and supplements were investigated for optimal growth of Cl.difficile and minimum inhibitory concentration determinations were performed for several antibiotics against a range of faecal isolates. Using Cl.difficile agar base containing cysteine hydrochloride (0.5 gIl), moxalactam (32 pg/ml) and norfloxacin (12 pg/ml) as a new selective medium, the isolation rate of Cl.difficile from faeces was increased by 20% and the selectivity improved by 33% compared with the commercially available cycloserine (500 pg/mi), cefoxitin (16 pg/mi), fructose agar. Identification: By investigating several enzymic tests for various Clostridium spp., the use of prolyl aminopeptidase, p-galactosidase, leucine aminopeptidase, indole production and acid phosphatase allowed the rapid identification of 96.4% of strains of Cl.difficile (within 30 minutes). The remaining isolates required an additional test, whilst no other Clostridium spp. tested produced similar results. The incorporation of these tests into filter paper squares allowed all five tests to be combined onto a small plastic carrier strip for ease of use. SDS-PAGE typing: A typing system using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was developed for differentiation of strains of Cl.difficile. Although complex protein profiles were obtained, the method was found to be reproducible and produced ten different protein patterns from the isolates tested. There was no difference between the protein patterns of the isolates obtained from asymptomatic infants and those from symptomatic adults. Antibody detection: Using a Western blotting technique, the antibodies raised in twelve patients with Cl.difficile infection against the individual protein bands of the isolates after SDS-PAGE were found to be highly variable. No common antibodies were detected and some individuals with no recent history of infection produced antibodies which reacted with certain Cl.difficile antigens.
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Lim, Chien-Sen Jenson. "Functional studies of toxin A from Clostridium difficile." Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.407433.
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Nazari, Shirvan Ali. "Molecular analysis of surface proteins of Clostridium difficile." Thesis, University of Glasgow, 2012. http://theses.gla.ac.uk/3522/.
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Since the Gram positive anaerobe Clostridium difficile was first isolated and described, it has emerged as an increasingly important nosocomial pathogen in Europe, North America and elsewhere, and the prime causative agent of antibiotic associated dirrhoea and pseudomembranous colitis in humans. The two large toxins, A and B, are the main virulence factors, proteins that are expressed in the gastrointestinal tract after colonisation by C. difficile. The pathological symptoms mediated by these toxins include disruption of the integrity of the epithelium, fluid loss, intestinal inflammation and tissue destruction. Important as the toxins are to C. difficile pathogenesis, several other proteins are known to contribute to colonisation and other aspects of the disease process remain poorly understood. Immunological studies using antisera from the patients revealed a number of candidates and amongst these, proteins present, or thought to be present, at the bacterial surface contribute to adhesion, motility and other interactions with the human host. The aims of this study were to produce a number of surface proteins from C. difficile as recombinant products and to isolate antibodies against these targets via phage display. The goal was to assess if these antibodies could inhibit the normal function of these targets and to confirm their location in C. difficile. Of 11 clostridial proteins, expression and purification of 3 proved impossible (Cwp84, FbpA and Acd) but 8 others (CspA, GroEL, FliC, FliD, a putative sortase, Cwp66, and its amino and carboxy terminal regions) were used for antibody isolation along with recombinant and native forms of SlpA. Phage display yielded a large panel of specific single chain variable fragments (scFv) antibodies that were expressed, purified and characterised. Reaction between the scFvs and their targets took place in ELISA and Western blotting suggesting the recognition of linear rather than conformational epitopes. The binding of scFvs to SlpA and its components showed strain specificity 3 with good recognition of protein from C. difficile 630 but no reaction towards SlpA from R20291, and 027 ribotype. Binding of scFvs of a range of specificities to extracts from C. difficile M120 indicated that a component of the S layer from this strain might possess immunoglobulin binding activities in the manner of a superantigen. The scFvs against flagellar proteins FliC and FliD were able to inhibit bacterial motility and therefore there would be potential in testing whether other scFvs generated in this study were able to inhibit the biological activity of their targets. Some scFvs were tested in immunofluorescence microscopy. The positive results from these experiments showed that the reagents and the strategy pursued could be used to establish surface exposure of the targets and other components of the bacterial surface. Given the high specificity of the reagents, and in the case of Cwp66, the ability to isolate scFvs against defined regions of the protein, the strategy has the capacity to define the orientation of proteins in the bacterial surface. In contrast, the use of scFvs to locate their targets in electron microscopy using immunogold reagents was unsuccessful. As this approach has been successful in other studies, it deserves further investment of effort. Overall, expression of proteins from C. difficile in an E.coli host was generally successful and phage display provided a rapid, highly efficient method for the isolation of specific immunological reagents. These have the potential to explore the location, orientation and activity of proteins from the pathogen.
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Heeg, Daniela. "Spore formation and spore germination of Clostridium difficile." Thesis, University of Nottingham, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.594825.
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Clostridium difficile is the major underlying cause of antibiotic-associated diarrhoea and poses a risk for healthcare systems worldwide. Endospores produced during sporulation are widely regarded to be the infectious agent of C. difficile associated diarrhoea. These spores are able to withstand a variety of antimicrobial agents and industrial cleaning products and are therefore able to reside on surfaces in healthcare settings for prolonged periods of lime. In order to cause disease in susceptible individuals, spores need to abjure dormancy and return to vegetative cell growth through germination. Sporulation and germination have been studied extensively in Bacillus spp. Knowledge about the sporulation and germination pathways in C. difficile, however, remains incomplete. Here, forward and reverse genetics methods were employed to analyse sporulation and germination phenotypes of C. dfficile. Using forward genetics, 19 mutants with potential sporulation and/or germination phenotypes were isolated, three of which were completely deficient for sporulation. In an attempt to explore the use of transposon suicide vectors, a protocol for the successful transformation of C. difficile was developed. A reverse genetic mutant in the germination specific lytic transglycosylase Slee created by ClosTron mutagenesis was used to study spore germination in vivo. This study is the first report of the use of a germination mutant in vivo. The sporulation characteristics of 52 clinical C. difficile isolates have been analysed indicating that a variation in the rate of sporulation is not associated with molecular type. The germination characteristics of 37 clinical C. difficile isolates were examined, indicating that different isolates exhibit varying germination characteristics in response to bile salts.
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So, Yung-chun, and 蘇雍竣. "Molecular epidemiology of toxigenic Clostridium difficile in HongKong." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46632220.
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Craggs, Joanna K. "Structure-function relationships of Clostridium difficile toxin A." Thesis, University of Nottingham, 1999. http://eprints.nottingham.ac.uk/12047/.
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Ten overlapping fragments covering the entire Clostridium difficile toxin A gene were cloned and expressed in Escherichia coli. Eight fragments (a', a2, b, c, d, e, f and g) represented the first 5.55kb of the gene whereas two fragments (hl and h2) each spanned the entire C-terminal repeat region of the molecule. All activities relating to binding to carbohydrates (i. e. cold haemagglutination of rabbit erythrocytes), binding to bovine thyroglobulin and non-specific binding to a murine monoclonal antibody were restricted solely to peptides H1 (amino acids [aa] 1834-2683) and H2 (aa 1832-2683). Peptide H2 alone also displayed the ability to bind to cells and to be internalised into endosome-like compartments within the cells. Taken together with the observation that peptide H2 caused a cytopathic effect on Vero cells which was atypical of the holotoxin, these results may indicate that the repeat region of toxin A stimulates intracellular signalling pathways prior to Rho glucosylation. Peptide A2 (aa 1-536) glucosylated recombinant RhoA (rRhoA) in vitro, whereas peptides A'(aa 1-205), B (aa 542-859), C (aa 114-859), D (aa 869-1330), E (aa 542- 1161), G (aa 869-1830) and H2 (aa 1832-2683) did not. The results obtained for peptides A', A2 and C indicate that the first 536 as encompass the catalytic domain for this activity, that more than the first 205 as alone are needed for expression of enzymic activity, and that for a peptide to be active it must not lack the first 113 aa. The first 113 as of the holotoxin are probably essential for the correct folding of the catalytic domain and expression of its activity. These studies were also the first to locate the toxin A ATP binding site to a peptide spanning as 542-859 (peptide B) of the holotoxin. Antibody reaction profiles of antiserum to holotoxin A against toxin A peptides and of antiserum to the peptides against holotoxin A indicate that this region is unexposed in the native state. Also of interest was the observation that the only peptides, which contained the nucleotidebinding site (B and E), lacked the ability to glucosylate rRhoA. Further peptide A2, which possessed glucosyltransferase activity, lacked the nucleotide-binding site. These studies therefore, suggest that a nucleotide-binding site is not required for in vitro glucosylation of rRhoA by toxin A, and fail to identify a role for the toxin A nucleotide binding site. An engineered truncated form of toxin A, consisting of the first 539 as of the holotoxin (encompassing glucosyltransferase activity) fused to the 852 as C-terminal peptide H2 (repeat end binding portion) caused a conventional cytopathic effect (CPE), but was 1,400 fold less cytotoxic to Vero cells than the holotoxin. Peptide A2 (aa 1-536) alone had no effect on Vero cells or in rabbit ileal loops suggesting that peptide H2 aided delivery of the glucosyltransferase molecule into cells leading to a CPE. The truncated toxin lacked the nucleotide binding site and the putative membrane-translocating domain (internal hydrophobic region). The reduced activity of the truncated toxin suggests that although not essential for cytotoxic activity, the nucleotide-binding site and the internal hydrophobic region are important for stability and/or efficient translocation of the holotoxin into the cytosol.
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Banerji, Oishik. "Structural studies of the Clostridium difficile surface layer." Thesis, University of Sheffield, 2017. http://etheses.whiterose.ac.uk/19162/.
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Peltier, Johann. "Structure et autolyse du peptidoglycane de Clostridium difficile." Rouen, 2011. http://www.theses.fr/2011ROUES032.
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La première partie de ce travail a porté sur l'analyse de la structure et la biosynthèse du peptidoglycane (PG) de Clostridium difficile. Le PG montre une architecture originale avec une N-déacétylation très marquée des résidus N-acétylglucosamine des chaînes glycanes ainsi qu'une proportion prédominante de pontages interpeptidiques de type DAP3→DAP3 inhabituels générés par L,D-transpeptidation. La formation de ces liaisons n'est pas affectée par l'ampicilline et implique au moins trois L,D-transpeptidases. Deux d'entre elles ont pu être caractérisées chez C. Difficile. Une autre particularité potentielle de la biosynthèse du PG de C. Difficile est liée à la présence d'un opéron de type vanG chez C. Difficile 630. Cet opéron est largement prévalent dans l'espèce mais il ne confère aucune résistance à la vancomycine. Les gènes composant cet opéron sont transcrits et largement induits en présence de vancomycine. L'étude des précurseurs du PG est en cours et permettra de mieux comprendre pourquoi l'opéron ne confère pas la résistance. La biosynthèse et l'hydrolyse du PG fonctionnent selon un équilibre qui conditionne la structure du PG. Le rôle de deux peptidoglycane hydrolases (PGH) majeures chez C. Difficile a été étudié. Un double mutant de ces deux PGH présente un défaut de séparation des cellules modéré et de l'autolyse bactérienne. Cette étude met ainsi en avant la forte redondance fonctionnelle des nombreuses PGH de C. DifficileThe first part of this work focused on peptidoglycan (PG) structure analysis and biosynthesis in Clostridium difficile. The PG has an original architecture with high level of N-acetylglucosamine deacetylation of glycan chains and a predominant proportion of unusual A2pm3→A2pm3 type cross-links generated by L, D-transpeptidation. The formation of these bonds is not affected by ampicillin and involves at least three L, D-transpeptidases. Two of them were characterized in C. Difficile. Another potential feature of PG biosynthesis in C. Difficile is due to the presence of a vanG-like operon in C. Difficile 630. This operon is widely prevalent in the species but does not confer resistance to vancomycin. The genes of this operon are efficiently transcribed and strongly induced in the presence of vancomycin. The study of the precursors of PG is in progress and will contribute to understand why the operon did not confer resistance. PG biosynthesis and hydrolysis operate on a balance that determines the structure of PG. The role of two major peptidoglycan hydrolases (PGH) in C. Difficile was studied. A double mutant of these two PGH has a moderate def"ect in cell separation and bacterial autolysis. This study puts forward the strong functional redundancy of many PGH in C. Difficile
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El, Meouche Imane. "Etude du régulateur transcriptionnel SigmaD chez Clostridium difficile." Rouen, 2014. http://www.theses.fr/2014ROUES008.
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La principale partie de ce travail porte sur l'étude du facteur SigD de C. Difficile et de son implication dans la régulation de l'autolyse, de la mobilité et de la production de ses deux facteurs majeurs de virulence, les toxines A et B. Nous avons pu montrer, par inactivation du gène sigD, que SigD régule positivement la mobilité de C. Difficile mais n'affecte pas, ou peu son autolyse. Le régulon global de SigD a été ensuite déterminé par une analyse transcriptonique en microarrays. Parmi les gènes sous-exprimés chez le mutant du gène sigD, nous retrouvons les gènes flagellaires tardifs, les gènes des toxines A et B et le gène de leur régulateur TcdR. Nous avons pu identifier des promoteurs SigD-dépendants notamment en amont des gènes de la flagelline FliC, de l'anti-SigD FlgM et deTcdR. De plus, nous avons prouvé que SigD se lie avec l'ARN polymérase pour démarrer la transcription de tcdR, dont il est un régulateur direct. Par ailleurs, nous avons identifié une séquence consensus propre au régulateur SigD chez C. Difficile. Enfin, nous avons déterminé le rôle antagoniste de FlgM, l'anti-SigD, dans la répression des gènes SigD dépendants. SigD s'avère être un régulateur positif et direct de la mobilité et de la synthèse des toxines chez C. Difficile. Une partie complémentaire du travail s'intéresse aux autolysines Acd et Cwp19 de C. Difficile. Des mutants simples des gènes acd et cwp19 ont permis de montrer que seule Cwp19 intervient dans l'autolyse de C. Difficile en présence du TritonX-100. Cette autolysine est impliquée dans la lyse à long terme chez C. Difficile. Un double mutant acd-cwp19 semble avoir le même profil autolytique que le simple mutant cwp19. La glucosaminidase Acd ne semble donc pas avoir une implication majeure dans l'autolyse de C. Difficile. Des analyses complémentaires en cours de réalisation permettront de déterminer l'activité enzymatique de l'autolysine Cwp19The main part of this work focuses on the characterization of the C. Difficile SigD factor and its role in the regulation of autolysis, motility and production of the two major virulence factors, toxins A and B. After the inactivation of sigD, we show that SigD factor positively controls C. Difficile motility whereas its contribution to the autolysis, if any, is modest. The global regulon of SigD was then determined by transcriptonic analysis using microarrays. Among the down-regulated genes in the sigD mutant strain, we find the late flagellar genes, the genes encoding toxins A and B and the gene encoding their regulator TcdR. We could identify SigD-dependent promoters upstream many genes including those encoding the flagellin FliC, the anti-SigD FlgM, and TcdR. In addition, we proved that SigD binds with RNA polymerase to initiate the transcription of tcdR. Furthermore, we identified a specific SigD-dependent consensus sequence in C. Difficile. Finally, we determined the antagonistic role of FlgM, the anti-SigD, in the repression of SigD-dependent genes. We prove that SigD is a positive and direct regulator of motility and toxin synthesis in C. Difficile. Another part of the work focuses on the autolysins Cwp19 and Acd of C. Difficile. Single mutants for acd and cwp19 genes showed that only Cwp-19 is involved in the long-term lysis of C. Difficile. A double-mutant acd-cwp19 seems to have the same autolytic profile as cwp19 single mutant. The glucosaminidase Acd does not seem to have a major involvement in autolysis of C. Difficile. Additional analyzes are in progress to determine the enzymatic activity of the Cwp19 autolysin
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Rexach, Carmen Elisabeth. "The epidemiology of Clostridium difficile in pediatric patients /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2002. http://uclibs.org/PID/11984.
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Etscheid, Susanne [Verfasser]. "Umgebungskontamination durch Clostridium difficile im Krankenhaus / Susanne Etscheid." Ulm : Universität Ulm, 2017. http://d-nb.info/1147484627/34.
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