Comparative Analysis of Gut Microbiota and Host Phenotypic Characteristics Across Enterotype-like Clusters in Cynomolgus and Rhesus Macaques

Abstract

Microbiome has been increasingly recognized for its close association with host physiology and diseases. Due to their close genetic relatedness to humans and standardized environmental conditions, captive macaque species serve as the most evolutionarily comparable preclinical animal models for studying human microbiome research. However, the characterization of gut microbiota and host phenotypic traits within each enterotype-like cluster of macaque species remains poorly understood. We analyzed microbiome characteristics and host metadata within enterotype-like clusters of cynomolgus and rhesus macaques housed in the same facility but with different origins of birth. At the phylum level, Bacteroidota, Firmicutes, Spirochaetota, and Proteobacteria were predominantly observed in both species. Except for Fibrobacterota at the phylum level, no significant interspecies differences were observed in bacterial composition or alpha diversity across taxonomic levels. Based on a prevalence threshold of 90%, cynomolgus macaques were found to share 12.4% of genera, while rhesus macaques shared 18.2%. Based on the relative abundance patterns of the genera Prevotella 9, Rikenellaceae RC9 gut group, and Treponema, the fecal microbiome of cynomolgus macaques was classified into three enterotype-like clusters (cluster 1, cluster 2, and cluster 3) whereas that of rhesus macaques was classified into two enterotype-like clusters. Using linear mixed-effects models, we identified species-specific associations between enterotype-like clusters and host phenotypes. In cynomolgus macaques, clustering was primarily associated with hematological and selected biochemical parameters, whereas in rhesus macaques, enterotype-like clusters were limited to body weight and hemoglobin. Despite a standardized diet and shared environments, distinct clusters and pronounced microbial individuality associated with birthplace suggest that early-life colonization is a key determinant of long-term gut microbiome structure and host phenotypes in captive primates. Also, identifying enterotype-like clusters in NHPs prior to analysis is essential for accurate and relevant human microbiome modeling, since each cluster may correspond to distinct human enterotypes and phenotypic traits.