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.

Genomic diversity of Lactobacillus salivarius

Lactobacillus salivarius is unusual among the lactobacilli due to its multireplicon genome architecture. The circular megaplasmids harboured by L. salivarius strains encode strain-specific traits for intestinal survival and probiotic activity. L. salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. In terms of probiotic strain selection, it is important to have an understanding of the level of genomic diversity present in this species. Comparative genomic hybridization (CGH) and multilocus sequence typing (MLST) were employed to assess the level of genomic diversity in L. salivarius. The wellcharacterised probiotic strains L. salivarius UCC118 was employed as a genetic reference strain. The group of test strains were chosen to reflect the range of habitats from which L. salivarius strains are frequently recovered, including human, animal, and environmental sources. Strains of L. salivarius were found to be genetically diverse when compared to the UCC118 genome. The most conserved strains were human GIT isolates, while the greatest level of divergence were identified in animal associated isolates. MLST produced a better separation of the test strains according to their isolation origins, than that produced by CGHbased strain clustering. The exopolysaccharide (EPS) associated genes of L. salivarius strains were found to be highly divergent. The EPS-producing phenotype was found to be carbonsource dependent and inversely related to a strain's ability to produce a biofilm. The genome of the porcine isolate L. salivarius JCM1046 was shown by sequencing to harbour four extrachromosomal replicons, a circular megaplasmid (pMP1046A), a putative chromid (pMP1046B), a linear megaplasmid (pLMP1046) and a smaller circular plasmid (pCTN1046) which contains an integrated Tn916-like element (Tn6224), which carries the tetracycline resistance gene tetM. pLMP1046 represents the first sequence of a linear plasmid in a Lactobacillus species. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable, and the identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications. In summary, this thesis used a comparative genomics approach to examine the level of genotypic diversity in L. salivarius, a species which contains probiotic strains. The genome sequence of strain JCM1046 provides additional insight into the spectrum of extrachromosomal replicons present in this species.