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Metagenomics offers opportunities to advance our understanding of the complexities of the microbial communities that inhabit the human gastrointestinal tract (GIT). Whilst this research field has great potential, there are limitations and caveats that affect its impact, and the translation of the “microbiome into medicine”. In particular, the number of representative microbes that still remain uncultured is substantial and constrains our capacity to define the functional roles such microbes play in health and disease. New techniques to gain representative isolates of these microbes are required, and it would also be advantageous if these microbes are amenable to genetic manipulation. Techniques in bacterial genetics applied to a broader diversity of human GIT bacteria would allow us to fully assess the functional aspects of their potential interactions with a host, which would expand understanding of their relation to host health and well-being. With this in mind, the aims of my PhD project were to utilize a new approach developed in our lab referred to as metaparental mating, to expand our collection of genetically tractable bacteria considered relevant to GIT homeostasis, and then assess their immunomodulatory capacity. Based on these results and findings, I then chose to undertake a more detailed and integrated culture-based and genomic analysis of two “new” bacterial isolates assigned to poorly populated and relatively uncharacterised lineages within Clostridium Cluster IV: the genera Flavonifractor and Pseudoflavonifractor. Chapter 1 provides an overview of the current literature with a focus on the roles of the GIT microbiota and its roles in host health and well-being. I provide evidence and rationale for the basis of the research undertaken throughout my PhD studies based on the gaps in our knowledge of the roles specific members of the microbiota play in the GIT of humans Specific focuses are highlighted with regards to the increasing interest in Polyphenols and their beneficial impacts on the microbiota and the host. Chapter 2 describes my use of the metaparental mating technique to recover representative isolates of Firmicutes-affiliated bacteria. I validated the utility of the metaparental mating technique to recover a broad diversity of the bacteria present in human stool assigned to these lineages, and in particular the use of a plasmid that contains the evoglow-C-Bs2 bioluminescence reporter gene, which augmented antibiotic resistance selection and the identification of transconjugant strains. My phylogenetic assessment of the isolates I recovered shows the collection includes bacteria assigned to Enterococcus, Clostridium clusters IV, XIVa and XVIII. I then assessed 22 of my recovered isolates for their ability to inhibit lipopolysaccharide-stimulated NF-κB activation of the luciferase reporter gene using the RAW 264.7 mouse macrophage cell line. I was able to show that 7/22 of these iii isolates inhibit NF-κB activation of the reporter gene to a magnitude similar or greater than Faecalibacterium prausnitzii A2-165. Of particular interest to me was the isolation of two isolates assigned to Flavonifractor and Pseudoflavonifractor, and I chose to focus on these two isolates for the remainder of my PhD studies. Chapter 3 is focused on my assessment of Flavonifractor sp. AHG0014 with reference to the type strain, F. plautii strain DSMZ 4000T. In particular, my culture-based studies examined quercetin metabolism by both these strains. My results suggest that the growth of both strains does not proceed until quercetin per se is reduced to relatively low concentrations (~5 µM). The genome of strain AHG0014 was sequenced and found to be similar in size and G:C content to the four Flavonifractor genomes available. In addition, I was also able to retrieve genome sequences of four more strains that were “unassigned” but should now be considered as representatives of the Flavonifractor genus. My assessments of these 9 genomes showed that the chalcone isomerase (chi) gene implicated in quercetin metabolism is part of the core genome and is contained in a multi-gene locus that most likely encodes for both quercetin uptake and metabolism. Using a combination of the 16S rRNA gene and quercetin metabolism genes to screen metagenomics datasets, I found that these genes are significantly more abundant in a cohort of Crohn’s disease patients compared to healthy controls; suggesting the proposed association of Flavonifractor spp. and GIT homeostasis needs more detailed assessment. Chapter 4 focuses on my assessment of “Pseudoflavonifractor” sp. AHG0008. I first showed that similar to P. capillosus DSMZ 23940T, AHG0008 does not metabolise quercetin. Interestingly, and despite the similarity between these two bacteria based on 16S rRNA gene analysis, the genome of strain AHG0008 is much smaller and quite different when compared to P. capillosus DSMZ 23940T. I then used a combination of bioinformatics methods to recover more closely related genomes produced from metagenomics datasets (MAGs), and my assessment of the whole-genome-based phylogeny and Average Nucleotide Identity scores confirmed that strain AHG0008 is the first cultured isolate of a divergent branch of the presumptive Pseudoflavonifractor lineage. I also found that strain AHG0008 and the MAGs possess a relatively large percentage of genes encoding amino acid transport and metabolism. Chapter 5 provides my overview of the findings arising from my PhD research, which I believe has provided a greater awareness and new understanding of these diverse and underrepresented bacterial lineages. I also provide some perspective and suggestions with respect to future research that will provide new insights into the roles these bacteria play in human health and disease. |
