Microbiome features differentiating unsupervised-stratification based clusters of patients with abnormal glycometabolism

The alteration of gut microbiota structure plays a pivotal role in the pathogenesis of abnormal glycometabolism. However, the microbiome features identified in patient groups stratified solely based on glucose levels remain controversial among different studies.. In this study, we stratified 258 participants (discovery cohort) into three clusters according to an unsupervised method based on 16 clinical parameters involving the levels of blood glucose, insulin, and lipid. We found 67 cluster-specific microbiome features (i.e., amplicon sequence variants, ASVs) based on 16S rRNA gene V3-V4 region sequencing. Specifically, ASVs belonging to Barnesville and Alistipes were enriched in Cluster 1, in which participants had the lowest blood glucose levels, high insulin sensitivity, and high-fecal short-chain fatty acid concentration. ASVs belonging to Prevotella copri and Ruminococcus gnavus were enriched in Cluster 2, which was characterized by a moderate level of blood glucose, serious insulin resistance, and high levels of cholesterol and triglyceride. Cluster 3 was characterized by a high level of blood glucose and insulin deficiency, enriched with ASVs in P. copri and Bacteroides vulgatus. In addition, machine learning classifiers using the 67 cluster-specific ASVs were used to distinguish individuals in one cluster from those in the other two clusters both in discovery and testing cohorts (N = 83). Therefore, microbiome features identified based on the unsupervised stratification of patients with more inclusive clinical parameters may better reflect microbiota alterations associated with the progression of abnormal glycometabolism. IMPORTANCE The gut microbiota is altered in patients with type 2 diabetes (T2D) and prediabetes. The association of particular bacteria with T2D, however, varied among studies, which has made it challenging to develop precision medicine approaches for the prevention and alleviation of T2D. Blood glucose level is the only parameter in clustering patients when identifying the T2D-related bacteria in previous studies. This stratification ignores the fact that patients within the same blood glucose range differ in their insulin resistance and dyslipidemia, which also may be related to disordered gut microbiota. In addition to parameters of blood glucose levels, we also used additional parameters involving insulin and lipid levels to stratify participants into three clusters and further identified cluster-specific microbiome features. We further validated the association between these microbiome features and glycometabolism with an independent cohort. This work highlights the importance of stratification of patients with blood glucose, insulin, and lipid levels when identifying the microbiome features associated with the progression of abnormal glycometabolism.