Intestinal microbiota profiles of captive-bred cynomolgus macaques reveal influence of biogeography and age

Abstract

Background: Age-associated changes to the intestinal microbiome may be linked to inflammageing and the development of age-related chronic diseases. Cynomolgus macaques, a common animal model in biomedical research, have strong genetic physiological similarities to humans and may serve as beneficial models for the effect of age on the human microbiome. However, age-associated changes to their intestinal microbiome have previously only been investigated in faecal samples. Here, we have characterised and investigated the effects of age in the cynomolgus macaque intestinal tract in luminal samples from both the small and large intestine.

Results: Whole metagenomic shotgun sequencing was used to analyse the microbial communities in intestinal content obtained from six different intestinal regions, covering the duodenum to distal colon, of 24 healthy, captive-bred cynomolgus macaques, ranging in age from 4 to 20 years. Both reference-based and assembly-based computational profiling approaches were used to analyse changes to intestinal microbiota composition and metabolic potential associated with intestinal biogeography and age. Reference-based computational profiling revealed a significant and progressive increase in both species richness and evenness along the intestinal tract. The microbial community composition also significantly differed between the small intestine, caecum, and colon. Notably, no significant changes in the taxonomic abundance of individual taxa with age were found except when sex was included as a covariate. Additionally, using an assembly-based computational profiling approach, 156 putative novel bacterial and archaeal species were identified.

Conclusions: We observed limited effects of age on the composition of the luminal microbiota in the profiled regions of the intestinal tract except when sex was included as a covariate. The enteric microbial communities of the small and the large intestine were, however, distinct, highlighting the limitations of frequently used faecal microbial profiling as a proxy for the intestinal microbiota. The identification of a number of putative novel microbial taxa contributes to knowledge of the full diversity of the cynomolgus macaque intestinal microbiome.

Keywords: Ageing; Assembly-based computational profiling.; Cynomolgus macaque; Intestinal biogeography; Metagenomics; Microbiome; Non-human primate; Reference-based computational profiling.

Fig. 1

Kingdom-level intestinal microbiota composition across different spatial regions in captive cynomolgus macaques. Age groups are defined as 4–7 years (Young), 8–12 years (Adult) and 13–20 years (Aged). Within each facet, individual bars represent samples from different animals, arranged in order of ascending age from left to right. D Duodenum, J Jejunum

Fig. 2

Diversity of intestinal microbiota composition across regions in captive cynomolgus macaques of differing ages. Alpha diversity is assessed using three metrics: A Chao1 index B Inverse Simpson’s index C Shannon’s index. D Tables summarizing statistically significant differences in alpha diversity between intestinal regions (* = p < 0.05). E NMDS plot based on Bray–Curtis dissimilarity, showing the clustering of samples by intestinal region and age group. Age groups are defined as 4–7 years (Young), 8–12 years (Adult) and 13–20 years (Aged). D Duodenum, J Jejunum, I Ileum, C Caecum, PC Proximal Colon, DC Distal Colon

Fig. 3

The effect of age on region-specific intestinal microbiota alpha diversity in captive cynomolgus macaques. Alpha diversity is assessed using three metrics: A Chao1 index B Inverse Simpson’s index C Shannon’s index. Where the correlation between age and alpha diversity reaches statistical significance (p < 0.05) the p-value is displayed on the plot. D Duodenum, J Jejunum, I  Ileum, CCaecum, PC Proximal Colon, DC Distal Colon

Fig. 4

Top 10 most abundant bacterial taxa in the captive cynomolgus macaque intestinal microbiota across regions. Taxa are shown at the A Phylum B Genus and C Species level in animals of differing ages. Age groups are defined as 4–7 years (Young), 8–12 years (Adult) and 13–20 years (Aged). Within each facet, individual bars represent samples from different animals, arranged in order of ascending age from left to right. D Duodenum, J Jejunum

Fig. 5

Relative abundance of Segatella species in the captive cynomolgus macaque intestinal microbiota across regions. Age groups are defined as 4–7 years (Young), 8–12 years (Adult) and 13–20 years (Aged). Within each facet, individual bars represent samples from different animals, arranged in order of ascending age from left to right. Only samples with greater than 0.1% relative abundance of Segatella are shown. D Duodenum, I Ileum

Fig. 6

Top 10 most abundant archaeal taxa in the captive cynomolgus macaque intestinal microbiota across regions. Taxa are shown at the A Phylum B Genus and C Species level in animals of differing ages. Age groups are defined as 4–7 years (Young), 8–12 years (Adult) and 13–20 years (Aged). Within each facet, individual bars represent samples from different animals, arranged in order of ascending age from left to right. Only samples with greater than 0.01% relative abundance of Archaea are shown

Fig. 7

Abundance of MetaCyc pathways across different regions of the captive cynomolgus macaque intestinal tract. Pathways are categorised into higher-level organisational terms. Age groups are defined as 4–7 years (Young), 8–12 years (Adult) and 13–20 years (Aged). Within each facet, bars represent individual samples from different animals, arranged by ascending age from left to right. Pathway abundance is normalised and expressed as copies per million (CoPM) to account for variation in sequencing depth. D Duodenum

Fig. 8

Taxonomic labels of putative novel species-level genome bins (SGBs). SGBs were obtained from the intestinal metagenomes of captive cynomolgus macaques of differing ages. Classifications are shown at the A Phylum level B Class level, C Order level and D Family level

Fig. 9

Abundance of putative novel species-level genome bins (SGBs) at the genus level. SGBs were obtained from the intestinal metagenomes of captive cynomolgus macaques across different age groups: 4–7 years (Young), 8–12 years (Adult) and 13–20 years (Aged). Within each facet, labels represent individual samples from different animals, arranged by ascending age from left to right. Abundance is normalised and expressed as counts per million (CPM), with white represents absent taxa. Abbreviations: D = Duodenum