A comparative analysis of the genus Faecalibacterium and the utilisation of an enrichment based strategy for the isolation of gut phage

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Date
2020-12-11
Authors
Fitzgerald, Cormac Brian
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University College Cork
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Abstract
Faecalibacterium prausnitzii is a Gram negative member of the Ruminococcaceae family and is highly oxygen sensitive. F. prausnitzii has been shown to have the ability to reduce inflammation in the human gut. It can produce a number of anti- inflammatory substances, including butyrate. A reduction in the abundance of F. prausnitzii has also been identified in patients with CRC, type 2 diabetes and IBD. This thesis describes the most extensive comparative analysis done to date on F. prausnitzii. Chapter 3 reports the analysis of all 31 sequenced strains of F. prausnitzii, including 11 strains that were sequenced specifically for this project. We were able to confirm the two phylogroups of F. prausnitzii that had previously been determined by 16s rRNA analysis. We also examined the differential abundance of COG categories between phylogroups. A striking lack of synteny between strains of F. prausnitzii was also described in Chapter 3. In Chapter 4 we increased the number of F. prausnitzii strains to 78 which confirmed the presence of a third phylogroup. In this chapter questions regarding the lack of synteny between strains was also addressed. In order to determine if the genomic rearrangement of the species happened frequently or only over many generations, F. prausnitzii strain A2-165 was grown in duplicate and isolates were sequenced before and following 13 passages in broth. A large inversion was observed in one of the duplicates. We next determined whether rearrangements could be detected in individual reads discarded by the assembler. BLAST was employed to view alignments of all reads against the assembled genomes. The results from this analysis showed that a significant number of reads had been rearranged across the genome. When this is compared to Bacteroides it was possible to even more clearly note that the rearrangements of reads in F. prausnitzii were dispersed throughout the genome whereas in Bacteroides they were much more localised. Chapter 5 briefly explores the varying levels of synteny across 240 different bacterial genera. We see in this chapter that F. prausnitzii is indeed on the lower end of the spectrum with relation to synteny. We can also see that there is a wide variety of different levels of synteny present for the genera that were included in the analysis. Chapter 6 explores a non-plaque based method for the identification and propagation of novel bacteriophage from the intestinal tract. The isolation strategy involved two stages. The first required the pooling of 20 faecal filtrates (viromes) and propagating them on purified bacterial hosts obtained from culture collections as well as a number of strains that were isolated in the laboratory. This protocol was modified slightly for the second stage of the isolation procedure where three faecal filtrates were processed from faecal samples and potential bacterial hosts were isolated from the same samples from which the filtrates were created. Several previously unknown phage were isolated using this method including the discovery of the first cultured representative of the CrAssphage family of viruses. The work described in this thesis contributes to the body of knowledge relating to the important gut commensal, F. prausnitzii. In addition, a protocol was successfully developed for the isolation of bacteriophage from the gut which resulted in the description of the first culturable representative of the CrAssphage family.
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Faecalibacterium , Genomics , Bacteriophage , Comparative , Prausnitzii
Citation
Fitzgerald, C. B. 2020. A comparative analysis of the genus Faecalibacterium and the utilisation of an enrichment based strategy for the isolation of gut phage. PhD Thesis, University College Cork.