Our results demonstrate that the gut microbiome of infants is influenced by ethnicity, infant age, weight gain, and breastfeeding. The gut microbiome has been proposed to influence the progression of chronic diseases and has been associated with adverse health outcomes [42]. Development of the microbiome within the first years of life may influence long-term health, and can be affected by perinatal, genetic, and dietary factors, including solid foods and milk diet.

The distribution of a number of maternal and infant parameters differed between white Caucasian and South Asians (i.e., vegetarian status, gestational diabetes mellitus prevalence, timing of introduction of solid foods, antibiotic use during pregnancy, mode of delivery, etc.) and thus seemed likely candidates to explain the microbiome differences by ethnicity. However, when these variables were added as independent predictors of the gut microbiome composition in the multivariable model, none except breastfeeding status at the time of sampling, infant age, and weight gain in the first year improved the fit of the model (ethnicity R2 = 0.084 versus R2 = 0.082 with all additional variables; breastfeeding status R2 = 0.040 versus R2 = 0.032 with all additional variables). This suggests that these variables were largely captured by the higher order variables of interest, i.e., ethnicity and breastfeeding. Next, after taking into account these significant predictors (breastfeeding status, infant age at 1-year stool, and weight gain in the first year of life) we found that groups of bacterial genera which are phylogenetically distinct (i.e., within the order Lactobacillales versus Clostridales) were present at different abundances within each ethnic group. This suggests that different metabolic strategies are at work within the gut microbiome of South Asian and white Caucasian infants. Additionally, these bacterial taxa are good candidates to predict diet-related influences on the microbiome, microbial influences on host metabolism, and bacterial stimulation of the host immune system [43].

Several members of the lactic acid bacteria (LAB), specifically Bifidobacterium, Lactococcus, Streptococcus, and Enterococcus, were more abundant within South Asians after taking into account breastfeeding status at the time of collection, infant age, and weight gain in the first year. LAB break down mainly carbohydrates that are not absorbed by the host to produce acetate and lactate, both of which are used as energy sources by other microbial groups [43, 44]. Also the abundance of members of the Atopobium cluster of Actinobacteria (i.e., genera such as Collinsella and Atopobium) was higher in South Asians. This group of bacteria are saccharolytic (i.e., they break down small sugars) [45] and have been seen to decrease in abundance in the microbiome of individuals with a diet rich in whole grains [46]. These genera have also been associated with higher levels of low-density lipoprotein in humans [47] and, along with other members of the Actinobacteria, have been associated with high hepatic levels of triglycerides and low hepatic levels of glycogen and glucose in mice [48]. It is of interest to note that these observations are based on v3 16S rRNA gene data. Several studies of the infant gut microbiome, which employ amplification and sequencing of other variable regions of the same gene often report very low levels of Actinobacteria [6, 9, 49]. Members of this phylum, such as the Bifidobacteria, have been shown to dominate the infant gut microbiome [4, 50, 51], suggesting a possible primer bias against this group.

In contrast, white Caucasians showed higher abundances of members of the Firmicutes from the order Clostridiales, which have been shown to be increased in response to diets rich in animal protein [52] and high in fat [53]. Products of bacterial fermentation of acetate and lactate, mentioned above, as well as non-digestible fiber and oligosaccharides by members of the Clostridiales seen here (Ruminococcus, Lachnospiraceae, and Oscillospira) include short chain fatty acids like butyrate, which is used by host cells as an energy source and can signal increased barrier function [43]. Though also proposed to be chemoprotective, the relationship between luminal butyrate exposure and colorectal cancer in humans has been examined only indirectly in case-control studies [54]. Nevertheless, these findings suggest different metabolic processes and immune stimuli at work within the South Asian and white Caucasian infant gastrointestinal tract, some of which may be explained by their heterogeneous diets.

When switching from a milk-based diet to a solid food diet, prior studies have shown a decrease in the abundance of Bifidobacterium along with an increase in members of the Firmicutes (such as Clostridium sp.) and Bacteroidetes [12, 20]. One study suggests that it is the cessation of breastfeeding that is required for maturation of the gut microbiota to occur with a decrease in Bifidobacterium and an increase in members of the Clostridiales only occurring after weaning [9]. As expected, after adjustment for ethnicity, infant age, and weight gain in the first year, Bifidobacterium and Lactobacillus were significantly associated with breastfeeding. Additionally, an increase in the abundance of several genera within the phylum Firmicutes were associated with not being breastfed at the time of sampling.

Bifidobacteria, along with the LAB, are known to be abundant members of the microbiome of breastfeeding infants [55], whereas genera within the order Clostridiales are known to be more abundant within the gut of adults [56]. Here bacterial profiles indicative of a breast milk diet were common among South Asians even those that were not breastfeeding at the time of collection, suggesting that these infants retain more of a breastfeeding microbiome than do white Caucasians of the same age. The reasons for this are unclear; however, dietary differences may be contributing. Our data show that equal proportions of infants in both groups were breastfed in the first year but does not capture breastfeeding frequency. It also shows that there was a much higher rate of formula use and an earlier introduction to solid food within South Asian than within white Caucasian infants. Because self-reported vegetarianism was more frequent in South Asians, it is possible that meat consumption hastens, or non-meat diets delay, changes induced within the infant gut microbiome during the switch to a solid food diet. It is important to note, however, that to our knowledge an analysis of the adult South Asian microbiome has not been reported, nor has a description of the maturation of the South Asian infant microbiome toward an adult-like composition; thus, our data must be interpreted within the context of the study, i.e., of South Asian infants born in Canada who consume a South Asian diet.

The underlying construct of “Ethnicity” brings together several biological and cultural factors, and it can be characterized using a number of different parameters (e.g., dietary habits, ancestral country of origin, etc.). In our multivariate model, ethnicity and breast feeding status remained independent and significant predictors of differences in the overall microbial communities (beta diversity), whereas vegetarian diet did not, which implies that the impact of ethnicity which incorporates some unique dietary patterns is not wholly explained by these dietary differences, as it also reflects other differences between the groups. After ensuring that these additional factors were potentially accounted for (i.e., years living in Canada, antibiotic use, timing of solid food introduction, etc.) we observed that breastfeeding, infant age, and weight gain in the first year significantly influenced the infant gut microbiome.

Strengths of our study include its relatively large size of nearly 200 infants from each of two different ethnic groups who have diverse dietary intakes; the availability of stool samples collected at similar times using similar methods; the high quality deep sequencing of the 16S rRNA gene for bacterial identification; a reliability analysis to demonstrate reproducibility of our methods; and detailed measurement of maternal and infant covariates. Limitations include incomplete data on maternal weight gain during and prior to pregnancy, which limits our ability to assess the influence of this important covariate on the infant gut microbiome; ethnicity in this study refers to the group a person self-identifies with and reflects a mix of cultural factors, including language, diet, religion, and ancestry—thus, ethnicity is a multidimensional construct which includes some within-group heterogeneity, and differences attributable to ethnicity may reflect a broad range of factors which are not purely biological; and the lack of a direct measure of infant dietary intake beyond feeding type at the time of stool collection.