2017-04-17

Obesity and associated Type 2 Diabetes (T2D) are becoming a serious concern for global populations resulting in suffering, early mortality and a burden on Health Services. The aims of this study were to evaluate the role of gut faecal microbiota in these conditions in an animal model of type 2 diabetes and in a study of patients undergoing bariatric surgery. Our hypothesis was that gut bacterial communities play a role in determining the regulation of host metabolism and thus can contribute to obesity, T2D, metabolic syndrome and insulin resistance. In the animal model of T2D, animals were fed a high fat diet and treated with a low dose of streptozotocin (STZ; 30 mg/kg) (HFS group). Outcomes of this treatment were compared to control group fed with a normal diet (NDV) or maintained on a high fat diet only (HFV) or administered STZ only (NDS). Faecal samples were collected from individual rats (n=6 each group) and DNA isolated. In initial experiments a segment of the bacterial, 16S rDNA was amplified (primers C2 and C4) and the resulting amplicons cloned and clones sequenced by the Sanger method. Differences in the abundance of certain taxa were found but limitations on numbers based on this approach did not allow a more detailed analysis. Therefore, a Next Generation Sequencing (NGS) approach was adopted and bacterial identity within communities was established by Illumina MiSeq of the variable region (V3V5) between the conserved regions of the 16S rRNA gene. Sequence data were analysed with QIIME suite of tools to establish phylum, family and genus. Eleven phyla were identified, with Bacteriodetes and Firmicutes predominant and much lower abundance of the other phyla. The abundance of organisms in the phylum Bacteriodetes was decreased while Firmicutes was increased in the STZ-normal diet, high fat diet and STZ-high fat diet animals compared to controls. Differences of the bacterial communities at different phylogenetic levels were indicated by PCoA and by α-diversity between each of the experimental groups. There was a significant reduction of butyrate-producing bacteria and an increase in bacteria producing lipopolysaccharide (LPS) in both HFV and HFS groups compared to NDV. In the studies of the bariatric patients, treated by the Roux-en-Y Gastric Bypass procedure (RYGB) to treat obesity and T2D. The composition of gut microbiome and biochemical parameters of these obese/T2D patients were determined before and after surgery. The aims of this study were to evaluate gut faecal microbiota and host biochemical parameters (glucose, lipid profiles, LPS, short-chain fatty acids (SCFAs), bile acids (BAs), metabolic hormones and pro-inflammatory cytokine) in patients at 2, 6 and 13 weeks post operation compared to at 4 weeks pre-surgery (patient n=6). Bacterial identity was established by Illumina MiSeq sequencing of the variable region (V3-V5) of the 16S rRNA gene. Again the sequence data were analysed with the QIIME to establish phylum, and genus. Seven phyla were identified, with Firmicutes predominant and followed by Actinobacteria, Bacteroidetes, and Proteobacteria, and a lower abundance of Fusobacteria, Tenericutes and Verrucomicrobia. Extensive differences were found in the microbial communities between patients, both before and after surgery, as seen by identity of taxa present or absent and their abundances. The abundance trends of organisms in the phylum Firmicutes was decreased after surgery at W2, W6 and W13 while Proteobacteria was increased. Immediately after surgery, organisms in the Bacteroidetes phylum were decreased and Actinobacteria increased at W2 but these trends were reversed at W6 and W13. Significant decreases in Body Mass Index (BMI), and changes in biochemical profile, particularly decreased levels of HbA1c%, leptin, SCFAs and primary bile acid were observed. The data was analysed by PCoA and after surgery at W13, the microbiome of all patients clustered together in a group that was not apparent from the earlier times. This clustering was also seen when the biochemical and microbial data were combined into a single data set. In conclusion, from the studies of rat, the induction of obesity and diabetes is associated with distinct patterns of changes of microbial community that may be associated with decreased butyrate and increased LPS. It is postulated that resulting decreased mucin production and increased inflammation would lead to a loss of integrity of the gut barrier function. In the studies of human changes of the microbiome of the gut were associated with the development of a more uniform community structure as a result of surgery. Significant decreases in body weight with changes in microbial community and biochemical profile suggest a complex interaction between host homeostatic metabolic control and the microbiome.

2008-10-18

Pathological study of common carp (cyprinus carpio) fish infected with Aeromonas hydrophila