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Clostridium difficile is a major cause of health-care associated diarrhoea. It is a serious and costly public-health concern worldwide. The pathogenesis of C. difficile disease is mediated primarily by toxins A and B. However, different adhesins have been recently characterized as important colonization factors playing a role in the establishment of the first step of infection. This study investigated for the first time the role of flagella in virulence of two important C. difficile isolates; non-epidemic historical C. difficile 630 PCR-ribotype 012 and newly emerged hypervirulent C. difficile R20291 a BIINAPlI027 strain which caused large outbreaks of CDAD worldwide. Non-motile, paralyzed flagellated and non-flagellated mutant strains were created using ClosTron and Allelic-exchange mutagenesis systems. Variations in motility, flagellation and virulence phenotypes between the two strains were observed. The in vitro cell adherence model using the human colonic carcinoma Caco-2 cell- line and in vivo mouse models of colonization demonstrated that the flagellar filament structure functions as an adhesin, rather than serving as a motility device, to mediate C. difficile R20291 attachment to human colonic epithelial cells. Motile bacteria outcompete non-motile bacteria for colonizing axenic-mouse intestine in vivo. On the contrary, flagella of C. difficile 630 do not play a role in virulence (cell adhesion and intestinal colonization). The non-motile, non-flagellated mutants adhered more efficiently to Caco-2 cells and colonized mice as successfully as the wild-type. The hamster model of virulence demonstrated that the fliC mutant made in a 630 background was more virulent than the wild-type. Overall, findings from these studies indicate that the flagellum plays an important role as a virulence factor in contributing to the "hypervirulence" of C. difficile BI/NAPlI027 which causes more severe infections than historical strains of this organism.

This thesis investigated the aerial dissemination of Clostridium difficile (C. difficile) in the clinical environment and the contribution that this transmission route made to contamination levels. This thesis describes evidence for the aerial dissemination of C difficile, a finding which may help to explain why Clostridium difficile infection (CDI) is so persistent within hospitals and difficult to eradicate.

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.

In low GC Gram-positive bacteria, the phenomenon of carbon catabolite repression is dependent on a metabolite-activated bifunctional protein kinase/phosphorylase. In Clostridium acetobutylicum ATCC 824, the hprK gene encodes the bifunctional protein kinase/phosphorylase that is dependent on fructose 1,6-bisphosphate for activity. However, a putative glpX class II gene that might encode for fructose 1,6-bisphosphatase is located upstream of hprK and to date this is a unique gene arrangement. Therefore, the product of the putative glpX gene might have a vital role in regulating the activity of bifunctional protein kinase/phosphorylase by hydrolysing fructose 1,6-bisphosphate which is needed for the kinase activity. In the present work, experimental evidence is presented that the putative glpX gene encodes a fructose 1,6-bisphosphatase which can hydrolyse fructose 1,6-bisphosphate to fructose 6-phosphate and phosphate. FBPase activity was first demonstrated by cloning and transforming the glpX gene into an E. coli fbp mutant which cannot grow on gluconeogenic substrates such as glycerol. The transformed glpX gene was able to complement the fbp mutation of E. coli for growth on glycerol. GlpX was overexpressed as a GST-fusion protein and purified, and activity was demonstrated using fructose 1,6-bisphosphate as substrate. Activity was assayed at pH 8.0, and in the presence of Mn2+, but the enzyme was inhibited completely by 1 mM phosphate. A putative fbp class III gene was also overexpressed and the encoded protein was purified as a GST-fusion product in order to compare Fbp with GlpX. To our knowledge, this is the first study that was able to purify a Fbp class III enzyme. The Fbp protein showed almost the same behaviour towards inhibitors compared to GlpX, but had a considerably higher specific activity than GlpX under the conditions of the experiments. The glpX gene was shown by RT-PCR to be transcribed together with hprK on the same mRNA during growth on glucose, and this indicates that glpX is unlikely to be a gluconeogenic gene. The results suggest that GlpX may play a novel and specific role in regulating the kinase activity activity of bifunctional protein kinase/phosphorylase and carbon catabolite repression in C. acetobutylicum.

Clostridium difficile is a leading healthcare-associated infection, which causes diarrhoea, and is almost exclusively precipitated by antibiotic exposure. Traditionally C. difficile infection (CDI) has been considered predominantly transmitted within hospitals. However, endemic spread hampers identification of the source of infections, and therefore control and prevention of disease. A cohort of consecutive hospital and community CDI cases in Oxfordshire from September 2007 to March 2011 was investigated. For each case hospital admission, ward movement and demographic data were available allowing contact events between cases to be reconstructed. Initially 944 cases to March 2010 underwent multilocus sequence typing (MLST), subdividing the endemic cases into 69 distinct lineages and demonstrating unexpectedly that ward-based contact with known symptomatic CDI cases only accounts for <25% of disease. To better determine the extent of transmission arising from symptomatic patients, irrespective of the route transmission, isolates from 1223 cases to March 2011 underwent whole genome sequencing. Serially sampled patients with recurrent or on-going disease were used to estimate rates of C. difficile evolution and within-host diversity and to show 0-2 single nucleotide variants (SNVs) are expected between transmitted isolates obtained <124 days apart (95% prediction interval). Mixed infection with more than one strain was investigated, but probably plays only a minor role in onward transmission. In the Oxfordshire CDI cohort, 333/957 (35%) CDI from April 2008 – March 2011 were within 2 SNVs of ≥1 previous case since September 2007 (consistent with transmission). 428/957 (45%) were >10SNVs from all previous cases: these distinct subtypes continued to be identified consistently throughout the study, suggesting cases arise from a considerable reservoir of C. difficile. Surprisingly, declines in the incidence of genetically-related CDI were similar to those in genetically distinct CDI suggesting interventions not just targeting symptomatic individuals, e.g. antimicrobial stewardship, have played a significant role in recent CDI declines. Finally, the feasibility of studying asymptomatic inpatients as potential source of the unexplained transmission was investigated. This thesis provides convincing evidence, in a setting with typical CDI incidence and infection control practice, that only the minority of CDI arises from other symptomatic cases. It demonstrates that much CDI arises from genetically diverse reservoirs, with each exposure resulting in relatively few secondary cases. Future control strategies therefore need to focus on identifying these reservoirs, one of which is plausibly asymptomatic inpatients, and also on interventions that prevent the transition from exposure and colonisation to disease, such as antimicrobial stewardship.