Human gut microbiome viewed across age and geography

Changes in the taxonomic/phylogenetic composition of fecal communities as a function of age and population

Many reports have examined the bacterial species content of the gastrointestinal tracts of infants and children within one population using culture-based methods. Far fewer studies have attempted to compare the gut communities of humans living in markedly different socio-economic, geographic, and cultural settings^11,12. Culture-independent techniques have been used to define the gut microbiota at various points in postnatal development^6,13, but have been limited either by the analytic methods used, by the low number of subjects examined, or by the scope of the populations surveyed. These studies have nonetheless provided important insights. Using 16S rRNA gene-based microarrays, Palmer et al¹⁴ observed considerable intra- and interpersonal variation in fecal bacterial community structures during the first year of life in 12 unrelated children and 1 twin pair. Interpersonal variation was less within the twin pair, and intrapersonal variation decreased as a function of age. Penders and coworkers¹⁵ performed a qPCR study of five bacterial taxa in the fecal microbiota of 1,032 Dutch infants at 1 month of age, and documented differences based on birth mode (Caesarian versus vaginal; also see ref 8).

We collected bacterial V4-16S rRNA data from 326 individuals aged 0–17 years (83 Malawian, 65 Amerindian and 178 residents of the USA) plus 202 adults aged 18–70 years (31 Malawians, 35 Amerindians, and 136 residents of the USA). 16S rRNA datasets were first analyzed using UniFrac, an algorithm that measures similarity between microbial communities based on the degree to which their component taxa share branch length on a bacterial tree of life¹⁶. There were several notable findings. First, the phylogenetic composition of the bacterial community evolved towards an adult-like configuration within the 3-year period following birth in all three populations (Fig. 1a, Fig. S1). Second, interpersonal variation is significantly greater between children than between adults; this finding is robust to geography (Fig. 1b; also see ref. 4). Third, there were significant differences in the phylogenetic composition of fecal microbiota between individuals living in the different countries, with especially pronounced separation occurring between USA compared to Malawian and Amerindian gut communities; this was true for individuals aged 0–3 years, 3–17 years, and for adults (Fig. 1b, Table S3). Unsupervised clustering using Principal Coordinates Analysis (PCoA) of UniFrac distance matrices indicated that age and geography/cultural traditions primarily explain the variation in our dataset, where USA microbiota clustered separately from non-USA along principal coordinate 1 (Fig. 1c, S2). However, within the non-USA populations, separation between Malawians and Amerindians was also observed (along principal coordinate 3 in the case of adults, Fig. S2f). We did not find any significant clustering by a village for Malawians and Amerindians or by region within the USA. Fourth, bacterial diversity increased with age in all three populations (Fig. 2a,b). The fecal microbiota of USA adults was the least diverse compared to the two other populations (Fig. 2c, p<0.005, ANOVA with Bonferroni post-hoc test): these differences were evident in children older than 3 years of age (p<0.005, ANOVA with Bonferroni post-hoc test), but not in younger subjects.

We next used the nonparametric Spearman rank correlation to determine which bacterial taxa change monotonically with increasing age within and between the 3 sampled populations. We only considered children who were breastfed and used datasets obtained from the V4 region of the 16S rRNA gene as well as datasets of shotgun pyrosequencing reads from the fecal microbiomes of the 110 sampled individuals [24 babies (0.6–5 months old), 60 children and adolescents (6 months to 17 years old) and 26 adults]. Shotgun reads were mapped to 126 sequenced human gut-derived microbial species (Table S4). The advantage of using these 126 gut microbes as a reference database is that spurious hits of shotgun microbiome reads to taxa that are not present in the gut are minimized. Nonetheless, when we repeated the entire analysis, blasting 1,280 genomes in KEGG, the results were similar (Fig. S3). Phylotypes belonging to Bifidobacterium longum exhibited significant decline in proportional representation with increasing age in all 3 populations (Fig. S3a). The majority (75±20%) of all shotgun and 16S rRNA-V4 sequences in all babies mapped to members of the genus Bifidobacterium. Bifidobacteria continued to dominate fecal communities throughout the first year of life although their proportional representation diminished during this period, in agreement with the results of several studies of small numbers of children^4,6,7 (Fig. S3). Table S5 lists the species-level bacterial taxa whose representation changes significantly with age in all three populations, as well as species that change in a population-specific manner as defined from analysis of the shotgun sequencing data that were available from 110 of the 531 individuals.

We also used Random Forests, a supervised machine learning technique¹⁷, and the V4-16S rRNA datasets obtained from all 528 individuals to identify bacterial species-level operational taxonomic units (OTUs) that differentiate fecal community composition in children and adults within and between the three populations. The purpose of a classifier such as Random Forests is to learn a function that maps a set of input values or predictors (here, relative OTU abundances in a community) to a discrete output value (here, USA versus non-USA microbiota). Random Forests is a particularly powerful classifier that can exploit non-linear relationships and complex dependencies between OTUs. The measure of the method’s success is its ability to correctly classify unseen samples, estimated by training it on a subset of samples, and using it to classify the remaining samples (cross-validation). The cross-validation error is compared to the baseline error that would be achieved by always guessing the most common category. As an added benefit, Random Forests assigns an importance score to each OTU by estimating the increase in error caused by removing that OTU from the set of predictors. In our analysis, we considered an OTU to be highly predictive if its importance score was at least 0.001; all error estimates and OTU importance scores were averaged over 100 rarefactions at the same sample size for each community (305,631 sequences) in order to control for sequencing effort.

Random Forests analysis confirmed the dominance of Bifidobacterium in the baby microbiota (Table S6a). For adults, Random Forests revealed distinct community signatures for Western (USA) and non-Western individuals (baseline error=0.289, cross-validation error=0.011 ± 0.000). Of the 92 highly predictive species-level OTUs shown in Table S6b, 73 were over-represented in non-USA adults, and 23 of the 73 were assigned to the genus Prevotella. Malawians and Amerindians could also be distinguished from each other, although the difference was less extreme than the USA versus non-USA comparison (baseline error=0.407, cross-validation error=0.018 ± 0.009, 56 highly predictive OTUs, Table S6c). Only 28 OTUs distinguished USA and non-USA infants (Table S6d). Intriguingly, three OTUs assigned to genus Prevotella were overrepresented in the USA infant microbiota, unlike the result observed in adults (Table S6d). Twenty-one OTUs discriminated Malawian and Amerindian baby microbiomes, 18 of which were overrepresented in the former: the majority belonged to family Enterococcaceae (Table S6d). Thus, a Western (USA) lifestyle appears to affect the bacterial component of the gut microbiota systematically, although this influence is subtle compared to the high degree of variability observed in infants and children within each population (perhaps analogous to human genetic variability, in which variation among populations is small compared to variation within populations).

Confirming the importance of Prevotella as a discriminatory taxon, a recent study also showed that this genus was present in higher abundance in the fecal microbiota of children living in West Africa (Burkina Faso) compared to children living in Europe (Italy)¹¹. Additionally, a member of this genus is one of three bacterial species that, in European adults, distinguishes strongly among three clusters, or enterotypes, of gut microbiota configurations that are claimed to be reproducible across Western adult populations¹⁸. Therefore, we asked whether the fecal microbiota of infants and adults in each of our three geographically distinct populations fell into natural discrete clusters¹⁹. We did not find strong evidence for discrete clustering (see Methods), but rather for variation driven in adults by a trade-off between Prevotella and Bacteroides (Fig. S4a). Including infants introduces a new, strongly supported gradient driven by Bifidobacteria, generally orthogonal to the Bacteroides/Prevotella trade-off (Fig. S4b). Clustering of sub-populations of increasing minimum age indicates that adult cluster membership is generally consistent, but that children between 0.6 years and 1 year of age may be clustered with adults or with younger children, depending on whether the younger children are included in the analysis (Fig. S4c).

In summary, this clustering analysis suggests that some features of normal variation in the bacterial composition of the gut microbiota, such as the Prevotella/Bacteroides trade-off, are highly reproducible even in human population subsets of reduced variability. However, a complete description of variation in the human gut microbiota will require a substantially broader cross-cultural and cross-age sampling. Importantly, the observed age- and geographic patterns were also detected with lower sequencing coverage (see Supplementary Results for a discussion of the influence of sequencing depth on performance of Random Forests and PCoA analyses (Fig. S5, S6), and our analysis of non-bacterial taxa that vary with age and population, Fig. S7).

Shared functional changes in the microbiome as children mature

Few studies have described changes in the gene content of the gut microbiome as a function of age: the largest study reported to date was carried out in 13 healthy Japanese individuals (5 children, the youngest 3 months old, plus 8 adults)⁴. Our shotgun sequencing dataset from 110 individuals allowed us to characterize the representation of functional gene groups [KEGG Orthology (KO) annotations and Enzyme Commission numbers (ECs)] in microbiomes representing broader age groups (youngest 3 weeks), and several distinct geographic locations/cultural traditions. We used Hellinger distance measurements to show that just as children are significantly more different from one another than are adults in terms of their fecal bacterial community phylogenetic structure, they are also more different in terms of their repertoires of microbiome-encoded functions, as defined by the proportional representation of EC and KO assignments. Moreover, as with UniFrac distances, Hellinger distances were greater between the USA and the other two populations at all ages sampled (Fig S8). Of interest is the concordance of patterns of covariance between the two data types: Procrustes analysis disclosed that the goodness of fit was significant (P<0.001 with 1,000 iterations) whether UniFrac (the most appropriate metric for 16S rRNA data) or Hellinger distances (for consistency with the method used on the KEGG EC and KO data) were used to reduce the OTU table (Fig. S9a,b plus data not shown). Annotation of shotgun reads from the microbiomes using the COG database produced similar concordance with 16S rRNA datasets (Fig. S9c).

When examining KEGG EC profiles across 110 fecal microbiomes, we obtained the remarkable result that there were no ECs identified as being unique to adults (n=26) or babies (less than 6 months old, n=24). Moreover, the total number of ECs found in adults was not significantly different compared to the total number of ECs scored in babies (sampling normalized to coverage in Fig. S10a). This finding was robust to culture/geography. The fraction of sequences with assignable KEGG EC annotations declined with increasing age in all 3 populations (Fig. S10b). This may be due to the increased complexity of the adult microbiome, with fewer representative species characterized by genome sequencing, genetic manipulation, or biochemically (also see Supplementary Results and Figs. S11, S12 for a comparison of our dataset to a published dataset of fecal microbiomes sampled from 124 adults living in Denmark and Spain²).

We used ShotgunFunctionalizerR²⁰, a software tool designed for metagenomic analysis and based on a Poisson model, to identify 1,008 ECs whose proportional representation in fecal microbiomes differed significantly between all sampled breast-fed babies and all adults irrespective of their geographic location; 530 were significantly higher in adults (p<0.0001, Table S7). A prominent example of these shared age-related changes involves the metabolism of vitamins B12 (cobalamin) and folate. In contrast to folate, which is synthesized by both microbes and plants, cobalamin is produced primarily by microbes²¹. The gut microbiomes of babies are enriched in genes involved in the de novo biosynthesis of folate, while those of adults have a significantly higher representation of genes that metabolize dietary folate and its reduced form tetrahydrofolate (THF, Fig. S13, Table S7). Unlike de novo folate biosynthetic pathway components, which decrease with age, the proportional representation of genes encoding the majority of enzymes involved in cobalamin biosynthesis increase with age (Fig. S14, S15, Table S7). The folate and cobalamin pathways are linked functionally: methionine synthase (EC 2.1.1.13); this E.C., whose representation also increases with age, catalyzes formation of THF from 5-Methyl-THF and L-homocysteine and requires cobalamin as a cofactor (Fig. S13).

The low relative abundance of ECs involved in cobalamin biosynthesis in the fecal microbiomes of babies correlates with the lower representation of members of Bacteroidetes, Firmicutes, and Archaea in their microbiota (see Fig. S16 for Spearman correlation coefficients). While the biosynthetic pathway for cobalamin is well represented in the genomes of these organisms (Fig. S16), Bifidobacterium, Streptococcus, Lactococcus, Lactobacillus which dominate the baby gut microbiota (Table S5, Fig. S3), are deficient in these genes (Fig. S16). In contrast, a number of these early gut colonizers contain ECs involved in folate biosynthesis/metabolism (Fig. S16). The traditional view of the developing infant gut is that the principal change is in the representation of Bifidobacteria. Although differences in representation of Bifidobacteria contribute to this effect, differences in vitamin metabolism among the rest of the bacteria remain even when all Bifidobacteria reads were excluded (data not shown). These changes in vitamin biosynthetic pathway representation in the microbiome correlate with published reports indicating that blood levels of folate decrease and cobalamin increase with age²².

Besides cobalamin and folate, the relative abundance of ECs involved in the biosynthesis of vitamins B7 (biotin) (biotin synthase, EC2.8.16) and B1 (thiamine) (thiamine-phosphate diphosphorylase, EC2.5.1.3) are significantly higher in adult microbiomes compared to the microbiomes of babies (Fig. S17, Table S7). Together, these findings suggest that the microbiota should be considered when assessing the nutritional needs of humans at various stages of development.

Random Forests analysis asks a somewhat different statistical question from ShotgunFunctionalizeR: i.e., which genes or species are most discriminatory among different class labels, rather than which are most over/underrepresented, and tends to identify fewer features than does ShotgunFunctionalizeR when applied to the same data. Random Forests analysis yielded 107 ECs that best discriminate the adult and baby microbiomes (Table S7). These predictive ECs were among the most significantly different ECs determined by ShotgunFunctionalizeR and included ECs involved in the metabolism of cobalamin and folate (Table S7). In addition, Random Forests revealed that ECs involved in fermentation, methanogenesis and metabolism of arginine, glutamate, aspartate and lysine were higher in the adult microbiomes, while ECs involved in the metabolism of cysteine and fermentation pathways found in lactic acid bacteria [acetolactate decarboxylase (EC4.1.1.5) and 6-phosphogluconate dehydrogenase (EC1.1.1.44)] were represented primarily in baby microbiomes (Fig. S17).

Comparison of the representation of KOs, between baby and adult microbiomes, yielded essentially the same results as those reported with ECs. The only novel finding was the overrepresentation of KOs assigned to a wide variety of ABC transporters in baby microbiomes (Table S7b).

Population- and age-specific differences in the representation of microbiome functions

ShotgunFunctionalizeR, Random Forests and Spearman rank correlation analyses were all used to compare EC representation in fecal microbiomes as a function of predefined categories of geographic location and age. 476 ECs were identified as being significantly different in the USA versus Malawian and Amerindian breast-fed babies (p<0.0001, ShotgunFunctionalizeR; Table S8). The most prominent differences involved pathways related to vitamin biosynthesis and carbohydrate metabolism. Malawian and Amerindian babies had higher representation of ECs that were components of the vitamin B2 (riboflavin) biosynthetic pathway (Figs. 3a, S18). These differences were not evident in adults (Table S7). Riboflavin is found in human milk and in meat and dairy products. We did not measure the levels of these vitamins in mothers and in their breast milk in the sampled populations, although it is tempting to speculate the observed differences in baby microbiomes may represent an adaptive response to vitamin availability.

Studies in gnotobiotic mouse models indicate that the ability of members of the microbiota to access host-derived glycans plays a key role in establishing a gut microbial community^23,24. As expected^4,5, compared to adults, baby microbiomes were enriched in ECs involved in foraging of glycans represented in mother’s milk and the intestinal mucosa (mannans, sialylated glycans, galactose, fucosyloligosaccharides; Table S7). A number of genes involved in utilizing these host glycans are significantly overrepresented in Amerindian and Malawian compared to USA baby microbiomes, most notably exo-alpha-sialidase and alpha-L-fucosidase (Fig. 3a, Table S8). These population-specific biomarkers may reflect differences in the glycan content of breast milk. In fact, the representation of these glycoside hydrolases decreases as Malawian and Amerindian babies mature and transition to a diet dominated by maize-, cassava-and other plant-derived polysaccharides. In contrast, alpha-fucosidase gene representation increases as USA infants age and are exposed to diets rich in readily absorbed sugars (Fig. S19d, Table S9).

Another biomarker that distinguishes microbiomes based on age and geography is urease (EC3.5.1.5). Urease gene representation is significantly higher in Malawian and Amerindian baby microbiomes and decreases with age in these two populations, unlike in the USA where it remains low from infancy through adulthood (Fig. 3a, Fig. S19e). Urea comprises up to 15% of the nitrogen present in human breast milk²⁵. Urease releases ammonia that can be used for microbial biosynthesis of essential and nonessential amino acids^26,27. Furthermore, urease plays a major role in nitrogen recycling, particularly when diets are deficient in protein^28,29. Under conditions where dietary nitrogen is limiting, the ability of the microbiome to utilize urea would presumably be advantageous to both the microbial community and host. Urease activity has been characterized previously in Streptococcus thermophilus³⁰. While most attribute urease to Helicobacter and Proteus spp., the relative abundance of members of these two genera was low (<0.05%) and not significantly different between the three populations. Our analysis of shotgun reads that matched to the 126 gut genomes revealed that the representation of five species that possess EC3.5.1.5 (Bacteroides cellulosilyticus WH2, Coprococcus comes, Roseburia intestinalis, Streptococcus infantarius and S. thermophilus) was significantly higher in Malawian and Amerindian compared to USA baby microbiomes (Table S5).

Additional support of the role of diet in shaping the infant gut microbiome comes from the differences detected between the breast-fed and formula-fed babies who were part of the USA infant twin cohort (see Supplementary Results and Figs. S2c, S8c, and S20).

Effects of kinship on the microbiome across countries

Differences in social structures may influence the extent of vertical transmission of the microbiota and the flow of microbes and microbial genes among members of a household. Differences in cultural tradition also affect food, exposure to pets and livestock, and many other factors that could influence how and from where a gut microbiota/microbiome is acquired. We previously observed that adult MZ twins are no more similar to one another in terms of their gut bacterial community structure than adult DZ twins³². This result suggests that the overall heritability of the microbiome is low. We confirmed that the phylogenetic architecture of the fecal microbiota of MZ Malawian co-twins ≤3 years of age is not more similar than the microbiota of similarly aged DZ co-twins (n=15 MZ and 6 DZ twin pairs). We found that this is also true for MZ and DZ twin pairs aged 1–12 months of age (n=16 twin pairs), as well as teenaged twins (13–17 years-old; n=50 pairs) living together in the USA (Fig. 4).

Although biological mothers are in a unique position to transmit an initial inoculum of microbes to their infants during and following birth, our analysis of mothers of teenage USA twins showed that their fecal microbiota were no more similar to their children than were those of biological fathers, and that genetically unrelated but co-habiting mothers and fathers were significantly more similar to one another microbially than were members of different families (Fig. 4; note that no fathers were sampled in Malawi and only 4 fathers in the Amerindian cohort). These latter observations emphasize the importance of a history of numerous common environmental exposures in shaping gut microbial ecology. Moreover, the similarity in overall pattern of the effects of kinship on microbial community structure suggests that despite the large influence of cultural factors on which microbes are present in both children and adults in each population, the bases for the degree of similarity among members of a family are consistent across the three populations studied.

Prospectus

Our results emphasize that it is essential to sample a broad population of healthy humans over time, both in terms of their age, geography and cultural traditions, in order to discover features of gut microbiomes that are unique to different locations and lifestyles. In addition, we need to understand how the pressures of westernization are changing the microbial parts of our genetic landscape - changes that potentially mediate the suite of pathophysiological states correlated with Westernization. Finally, given the need for global policies about sustainable agriculture and improved nutrition, it will be important to understand how we can match these policies not only to our varying cultural conditions but also to our varied gut microbiomes.

METHODS SUMMARY

Supplementary Material