Purpose: Beta-cell destruction results due to a failure of immune-regulation in type 1 diabetes (T1D). We investigated alterations in the intestinal microbiome composition as a potential biomarker for predicting the risk of developing T1D.

Methods: Female non-obese diabetic (NOD) littermate mice were divided into 4 groups: 4-5wks old non-diabetics (n=5, blood glucose BG 90-150mg/dL), 7-10wks old prehyperglycemic mice (BG 170-250mg/dL; n=6), 23 wks old diabetic (BG≥250mg/dL;n=7) and age-matched 23 wks old mice (n=4) that had not yet become diabetic. QIAamp DNA Stool Mini Kit was used to purify bacterial DNA from cecum contents, followed by further purification with a PCR clean-up kit. 16S rRNA libraries were prepared from samples and deep-sequenced using Illumina platform (Genewiz). Primers targeted the hypervariable V3, V4, and V5 regions of the 16S rRNA gene using next generation amplicon sequencing. Species identification and relative abundance calculation was performed. Principal Coordinate Analysis was used to visualize (KiNG software) dissimilarities between the groups.

Results: The genera Bacteroides, Acetivibrio and Butyrivibrio demonstrated significant increase with establishment of T1D. An apparent increase in Anaerostipes, Candidatus azobacteroides, Prevotella and Rosebura was also observed. In contrast, the genera Oscillospora, Anaeroplasma, Eubacterium and Paludibacter were significantly decreased. Prehyperglycemic mice largely followed the same trend as the diabetic group. The PCoA plot comparing differentially abundant bacterial families across the different groups demonstrated three clear-cut clusters representing differences between controls, prehyperglycemic and diabetic mice groups. The age-matched, 'yet to become diabetic' mice fell in the same cluster as the diabetic mice, predicting that these diabetes-prone mice would become diabetic in time. Experiments are currently underway using a larger sample size and a non-diabetes prone mouse model as control for age-associated changes in microbiome.

Conclusions: The identified changes in the intestinal microbiome composition have significant clinical potential for T1D risk prediction. The findings are also relevant in the context of defective immune-regulation in allograft rejection and development of immunologic unresponsiveness.

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