Date of Award

2017

Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Marcus Claesson

Second Advisor

Prof. Roy Sleator

Third Advisor

Dr. Aisling O'Driscoll and Prof. Fergus Shanahan

Abstract

The microbiota, a community of micro-organisms in a particular environment, have been intensely researched in the past decade and interest appears to continue to increase. The human body is richly populated with bacterial cells (3.8x10'^) and alterations in these communities are associated with disease. The work presented in this thesis explores aspects of microbiota research methodologies with regard to optimisation and concerns over a lack of reproducibility. Additionally, we investigated the effects of medication, in particular Proton Pump Inhibitors, on the human gut microbiota, along with comparisons between the microbiota of patients with active and inactive Inflammatory Bowel Disease.

Microbiota research lacks standardised approaches to experimental design and data analysis due to rapidly evolving techniques and software. We conducted a comparison between various approaches in both amplicon and shotgun metagenomics sequencing using various primer binding regions and next generation sequencers. We found disparities between both 16S rRNA gene variable regions and metagenomic sequencing techniques, the latter of which was largely due to the taxonomic classifier. We finally emphasise the requirement for careful study design and caution when comparing microbiota studies.

Proton Pump Inhibitors are prescribed to prevent gastric reflux and duodenal ulcers but concern has been raised due to associations with C. difficile infection. We assessed the microbiota of long-term PPI users versus controls, but found no similarities to the microbiota of C. infection. However we did uncover microbial alterations including by a decrease in biodiversity and an increase in Holdemania filiformis along with a decrease in Pseudoflabonifractor capillosiis in the PPI microbiota. Although we found no microbiota associations with C. diff, caution should be taken when prescribing PPI medications due to the negative impacts on the gut microbiota.

Changes in the gut microbiota have been associated with IBD, although there is disparity between the alterations. We conducted the largest longitudinal IBD microbiota study to date, whilst also collecting a large quantity of metadata. We compared the microbiota of patients with IBD in both active and inactive disease states to controls. Although we found significant differences in biodiversity and

taxonomic composition between patients with IBD and controls, there was a lack of significant change between disease activity states. However, there was a significant decrease in microbiota stability in patients with altering disease activity. Age, diet, medications, resection and geographical location significantly impacted the gut microbiota in both diversity and composition. This study provides evidence that when confounding factors are controlled for through longitudinal sampling, the microbiota is subjected to little change when transitioning between states. We also show the importance of collecting metadata such as age, dietary habits and medication status as they were seen to influence the microbiota. Analysing the most comprehensive and largest longitudinal IBD cohort to date, we found that the gut microbiota composition of patients with IBD differs significantly from controls. However, separate to other cross-sectional studies using smaller sample sizes, we found no differences disease activity states.

The research conducted in this thesis provides evidence of stool microbiota alternations as a result of diet, medications and disease, while also highlighting potential methodological biases in sequencing analysis.

Access Level

info:eu-repo/semantics/openAccess

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