Prevalence and control of Clostridium difficile in patients with cystic fibrosis

The aim of this thesis was to investigate the high prevalence of Clostridium difficile in patients with cystic fibrosis (CF), and to control its dissemination. To determine the carriage rate of C. difficile in CF patients, 60 patients were tested for C. difficile and its toxin. In total, 50% of patients were found to be asymptomatic carriers of C. difficile despite toxin being detected in 31.66% of patients. Ribotyping of the C. difficile isolates revealed 16 distinct ribotypes, including the hyper virulent RT078. All isolates were sensitive to both Vancomycin and Metronidazole. The effect of CF and its treatment on the gut microbiota of CF patients was assessed by 16s sequencing of the gut microbiota of 68 CF patients. When compared to a healthy control group, CF patient gut microbiota was found to be less diverse and had an increased Firmicutes to Bacteriodetes ratio. Interestingly, CF patients who were carriers of C. difficile had a less diverse gut microbiota than C. difficile negative CF patients. Multilocus sequence typing was found to be comparable to PCR-ribotyping for typing C. difficile isolates from high risk patient groups. The sequence type ST 26 is potentially associated with CF patients as all seven isolates were found in this group and this sequence type has been previously reported in CF patients in a geographically distinct study. The bacteriophage ФCD6356 was assessed as a targeted antimicrobial against C. difficile in an ex-vivo model of the human distal colon. Despite reducing viable C. difficile by 1.75 logs over 24 hours, this bacteriophage was not suitable due to its lysogenic nature. Following treatment, all surviving C. difficile were immune to reinfection due to prophage integration. However, the ФCD6356 encoded endolysin was capable of reducing viable C. difficile by 2.9 over 2 hours in vitro after being cloned and expressed in Escherichia coli.

Treatment of Clostridium difficile infection: a national survey of clinician recommendations and the use of faecal microbiota transplantation

Adherence to Clostridium difficile infection treatment guidelines is associated with lower recurrence rates and mortality as well as cost savings. Our survey of Irish clinicians indicates that patients are managed using a variety of approaches. FMT is potentially underutilised despite its recommendation in national and European guidelines.

Effect of room temperature transport vials on DNA quality and phylogenetic composition of faecal microbiota of elderly adults and infants

Background: Alterations in intestinal microbiota have been correlated with a growing number of diseases. Investigating the faecal microbiota is widely used as a non-invasive and ethically simple proxy for intestinal biopsies. There is an urgent need for collection and transport media that would allow faecal sampling at distance from the processing laboratory, obviating the need for same-day DNA extraction recommended by previous studies of freezing and processing methods for stool. We compared the faecal bacterial DNA quality and apparent phylogenetic composition derived using a commercial kit for stool storage and transport (DNA Genotek OMNIgene GUT) with that of freshly extracted samples, 22 from infants and 20 from older adults. Results: Use of the storage vials increased the quality of extracted bacterial DNA by reduction of DNA shearing. When infant and elderly datasets were examined separately, no differences in microbiota composition were observed due to storage. When the two datasets were combined, there was a difference according to a Wilcoxon test in the relative proportions of Faecalibacterium, Sporobacter, Clostridium XVIII, and Clostridium XlVa after 1 week's storage compared to immediately extracted samples. After 2 weeks' storage, Bacteroides abundance was also significantly different, showing an apparent increase from week 1 to week 2. The microbiota composition of infant samples was more affected than that of elderly samples by storage, with significantly higher Spearman distances between paired freshly extracted and stored samples (p