Investigation of diet - microbiota interactions as affected by the carbohydrate intake of healthy human subjects

Abstract

In relation to human health and well-being, strategies that positively influence the composition and activity of the microbiota are keenly sought, and diet is widely accepted as being a major factor for altering the gastrointestinal microbiota. The overarching aim of my Ph.D. studies was to characterize how diets with varying profiles of carbohydrate content and intake affect the gut and the oral microbiota of healthy individuals. Two key groups of subjects were used in separate studies: elite male athletes (endurance walkers) and healthy conventional subjects. During my studies, advances in DNA sequencing (and related bioinformatics methods) have enabled transition from the taxonomic profiling of microbial communities, to the production of data representing their collective genetic potential (i.e. the metagenome). As such, I have used both approaches here, and provide new insights into the nature of the diet x microbiota interactions in healthy individuals.Chapter One reviews literature relating to diet x microbiota interactions with specific reference to how some diets (such as low FODMAP) affect the gut microbiota. Chapter Two provides the methodological details shared across the other research Chapters, and that samples either stored at sub-optimal temperatures, or that undergo repeated freeze-thaw cycles, are depleted of bacteria with a Gram-negative cell wall ultrastructure (e.g. Bacteroidetes, Proteobacteria). These results emphasized the importance of sample preservation and storage on these types of data generated from stool samples.Chapters Three and Four presents my results and conclusions about how the dietary pattern of elite race walkers during their period of intensified training affected their oral and stool microbiota, respectively. This research was undertaken in complement to the Supernova 1 study coordinated by Australian Institute of Sports. Stool and saliva samples were collected at the beginning and end of a three-week dietary intervention period, from elite male endurance race walkers choosing to consume either a High Carbohydrate (HCHO), High Carbohydrate-periodised (PCHO) or a Low Carbohydrate High Fat (LCHF) diet, and the microbial communities were examined using 16S rRNA amplicon sequencing. The results in Chapter Three show that the LCHF diet results in substantive changes in the oral microbiota, and in particular, reductions in the relative abundance of bacterial taxa known to be key nitrate-nitrite reducers (Haemophilus, Neisseria, and Prevotella) whereas increased the relative abundance of Streptococcus not known to be associated with nitrate reduction in the oral cavity. The Results in Chapter Four showed that the athletes could be stratified into either a Bacteroides-dominant or Prevotella-dominant “enterotypes” and while the diets consumed during intensified training did not disrupt these enterotypes, the LCHF diet significantly increased the relative abundance of Bacteroides and Dorea spp., whereas the relative abundance of Faecalibacterium spp. were reduced in athletes consuming the LCHF diet. Furthermore, the relative abundance of Bacteroides and Dorea following consumption of LCHF diet were found to be significantly negatively associated with fat oxidation and economy measures, respectively. Collectively, these results suggest that a ketogenic LCHF diet invokes profound changes in the oral and stool microbiota of athletes and can be associated with athlete performance measures during intensified training and simulating race conditions.Chapter Five presents my findings of how a diet prepared from foods to provide either a low (LP 1-3 g/day oligosaccharides; 0.50 g/day polyols) or moderate (MP 6-8 g/day oligosaccharides; 3.66 g/day polyols) daily intake of prebiotic carbohydrates affected the gut microbiota of healthy adults. The parent study was a single-blinded, randomised crossover study, managed by our collaborators with the Alfred Hospital (Monash University) Translational Nutrition program. Here, I first produced both 16S rRNA and ITS-2 gene amplicon profiles to characterise the prokaryote and fungal communities, respectively. These analyses showed that the prokaryote richness is reduced and fungal richness is increased by the MP diet as compared to the LP diet. The reduction in prokaryote richness was reflected in a significant increase in the relative abundance of Bifidobacterium spp. with the MP diet. Saccharomyces-related fungal lineages were the most abundant across the cohort with both diets, I did find different prokaryote-fungal relationships with the LP and MP diets. I then used these same DNA samples for shotgun metagenome sequencing (MGS) analyses that further confirmed an expansion of Bifidobacterial spp. and revealed significant increase in gene counts for the metabolism of sorbitol and mannitol and related phosphotransferase transport systems (PTS) pathways in response to the MP diet suggesting a bifidogenic effect of moderate amounts of sorbitol and mannitol.Chapter Six provides an integrated assessment and interpretation of the findings and potential impacts arising from my Ph.D. research. I believe my findings are novel and provide a better understanding of the diet x microbiota interactions in healthy individuals. I discuss these new insights with respect to what constitutes a healthy microbiota, and how a person’s diet can be rationally managed and personalised to sustain healthy gut function, nutrition, and well-being.