What Is Known/What Is New
What Is Known
- Bacterial colonization plays a major role in the development of postnatal gut function, with effects on gut health and host metabolism.
- Breastfeeding and vaginal delivery are among the most influential factors affecting the establishment of the gut microbiome in young infants.
What Is New
- The microbiome of human-reared infant monkeys is characterized by a higher ratio of Firmicutes-to-Bacteroidetes, a profile associated with metabolic disorders in humans.
- The stimulatory effect of breast milk on commensal propagation was more evident for Bifidobacteria than Lactobacilli.
- The gut microbiome during early infancy is predictive of future risk for Campylobacter jejuni infections in rhesus monkeys.
Birth is an abrupt event transitioning the neonate from relatively sterile uterine conditions into the external world. The full significance of the rapid microbial colonization that begins during delivery and continues postpartum through exposure to microorganisms from the mother and rearing environment is just beginning to be appreciated (1,2). These microbiota have a critical role supporting intestinal homeostasis, stimulating immunity, and influencing host metabolism, and may even contribute to the emergence of different behavioral phenotypes (3–5). Though the foundations of the microbiome can be influenced by prenatal conditions, the community structure of the microbiome is largely impacted by delivery mode and infant diet, especially by decisions about breast- or formula-feeding (6–9). Breast milk contains numerous proteins and prebiotic oligosaccharide substrates, and provides viable bacteria that compete with pathogens for adherence to the intestinal mucus and epithelial surfaces (10,11). It is important to more fully understand the ramifications of delivery mode and parental decisions about infant feeding because, despite recommendations from the World Health Organization, cesarean delivery is common and only 40% of American infants are exclusively breastfed until 6 months of age (12,13).
A nonhuman primate model was employed to investigate the benefits of exposure to the mother and breast milk for microbial colonization. We hypothesized that mother-reared (MR) infants would have a higher abundance of several commensal taxa as compared with infants initially reared in a nursery setting and fed formula (HR) (14,15). As the microbiome can affect digestive efficiency, host metabolism, and protect against enteric pathogens, we also looked for differences in growth and the later incidence of diarrheic episodes.
Fifty-four infant rhesus macaques (Macaca mulatta) were generated from healthy, multiparous mothers at the Harlow Center for Biological Psychology and Wisconsin Primate Research Center. Thirty-five were housed with their mothers and exclusively breastfed (MR). All were full-term vaginal deliveries. Nineteen were fed formula and reared by humans (HR) for 1 month in isolate incubators mimicking a Neonatal Intensive Care Unit (NICU) setting, with 13 delivered by cesarean-section (see text, Supplemental Digital Content 1, http://links.lww.com/MPG/B654, for further details of HR husbandry). Infants from both rearing conditions were randomly selected to be representative of the population born between 2016 and 2018. Mothers and older infants were fed a commercial biscuit diet, supplemented with fruit. Typically, MR infants may first sample biscuits between 2 and 4 weeks after birth. HR infants were introduced to chow at 2 weeks of age and were progressively weaned from formula by 2 months. All protocols were approved by the Institutional Animal Care and Use Committee and conducted in accordance with federal guidelines.
Rectal swabs were obtained at 2, 4, or 8 weeks of age. Genomic DNA was extracted and quantified using a Qubit 2.0 Fluorometer, and amplicon sequencing performed on Illumina MiSeq (See Text, Supplemental Digital Content 2, http://links.lww.com/MPG/B654, for further details of specimen collection, bacterial DNA isolation, and sequence analysis). Infant growth and health were closely monitored. No infants were treated with antibiotics during the sampling period; however, the mothers of 5 MR infants had been administered antibiotics perinatally. Infants were first weighed at a mean 3.65 (±0.61) days, and then regularly at 2-to-8-week intervals. Growth was indexed by weight, as well as growth rate between birth and 8 months. Diarrheic symptoms and treatments were recorded. In the majority of cases, to determine if Campylobacter was the cause, plates with selective media were inoculated (Campy CVA Medium and Charcoal Selective Medium), and streaked for isolation. Identification was made using a MALDI-TOF analyzer at the UW Veterinary Clinical Pathology Lab.
Sequence and Statistical Analysis
Significance was tested by implementations in Quantitative Insights into Microbial Ecology (QIIME) (16), PAST (PAleontological STatistics) (17), and R Statistics, including ANOVA, Analysis of Similarity (ANOSIM), nonparametric t-tests (Kruskal-Wallis), and adjustments for false discovery rates (FDR). Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) (18) was utilized to correct for variable 16S copy number. Taxonomic analyses were restricted to more abundant taxa present at a minimum 1% composition in total observations across samples and are presented at phylum and genus levels. To examine diversity indices, Faith's Phylogenetic Diversity (PD), Chao1 Index, and Observed Species were generated to examine richness (alpha diversity), and weighted UniFrac dissimilarity matrices examined for differences in microbial community structure (beta diversity) while accounting for both abundance and phylogenetic relatedness. Repeated measures ANOVA examined developmental changes in microbial composition, followed by pairwise comparisons adjusted for multiplicity.
Results are presented in 2 ways: first comparing the MR and HR infants at the 3 age points. Serial changes in community structure for the MR infants are then described (see text, Supplemental Digital Content 3, http://links.lww.com/MPG/B654).
Using GreenGenes 13_5-assigned operational taxonomic units (OTUs), PICRUSt was employed to predict microbial metagenomes from the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthologs in order to evaluate whether shifts in community structure influenced the functional potential of bacterial communities (19). Finally, linear discriminant analysis (LDA) effect size algorithm from LEfSe (20) identified microbial taxa that distinguished the 2 rearing conditions.
Analysis 1: Community Structure
One hundred two rectal swabs were collected from 35 MR infants and 19 HR infants at 2, 4, and 8 weeks of age (see Table, Supplemental Digital Content 4, http://links.lww.com/MPG/B654, for more details). The microbial diversity and composition for the 5 MR infants born to mothers administered perinatal antibiotics did not differ significantly from MR infants without antibiotic exposure. Rearing condition had a significant effect on the gut microbial community structure. Principle Coordinates Analysis for phylogenetic and abundance data revealed a distinctive clustering based on rearing evident at each age point (Fig. 1A), and permutational multivariate analysis of variance (PERMANOVA) indicated divergent microbiota at 2, 4, and 8 weeks (P = 0.001, P = 0.001, P = 0.002, respectively). Unpaired t-test comparisons of the mean dissimilarity distances for the microbial profiles indicated that sample-to-sample phylogenetic distances were larger in HR infants by 2 weeks of age (t = 2.99; P < 0.001), but the dissimilarity in community structure then shifted and was greater in MR infants at 4 and 8 weeks (t = 3.29; P < 0.01, t = 3.60; P < 0.005; Fig. 1B). Overall, divergence of the microbial community structure increased in MR infants across the first 2 months of life, whereas is decreasing among HR infants (P < 0.001 and P = 0.011, respectively). Averaged OTU counts for HR infants were higher than for MR infants (444.62 vs 404.89). There was, however, not a large difference in the within-group pairwise phylogenetic distances (PD index; Fig. 1C), sample diversity (Chao1), or total species richness (observed OTUs) at any age point age (see Table, Supplemental Digital Content 5, http://links.lww.com/MPG/B654, for alpha diversity metric results).
Differences were evident at all taxonomic levels, but most prominent at the phylum and genus levels (Fig. 2A and B). Similar to humans, the most abundant microbes were members of the Gram-positive Firmicutes and Gram-negative Bacteroidetes phyla. However, the Firmicutes-to-Bacteroidetes ratio was impacted by rearing, with Firmicutes being relatively more abundant in HR infants at 2, 4, and 8 weeks of age (P < 0.001, P = 0.05, P < 0.001, respectively; Fig. 2C). Within Bacteroidetes, Prevotella was consistently the most abundant genus in MR infants, constituting an average 9.9% compared with only 5% in HR infants at 2 weeks of age (P = 0.03). The highest Prevotella levels were seen in MR infants at 8 weeks (13%), an abundance similar to adult monkeys.
Conversely, the genus Blautia (within Firmicutes) was more enriched in HR infants at 4 and 8 weeks of age (10.5% vs 3.9%, P = 0.07; 9.4% vs 4.9%, P = 0.02, respectively). HR infants also had a lower relative abundance of the phyla Actinobacteria (Fig. 2B), reflecting a significantly lower abundance of the genus Bifidobacterium at 2, 4, and 8 weeks of age (P < 0.001, P = 0.007, P = 0.002, respectively). Moreover, Bifidobacteria diminished over time in HR infants; constituting only 0.2% and 0.15% at 4 and 8 weeks as compared with 1% at 2 weeks (P = 0.018). Lactobacilli were also lower in HR than MR infants, but this difference was only significant at 8 weeks (P = 0.017) and was small (2% vs 3.5% abundance). At 2 weeks, the proportion of Clostridium was twice as high in HR compared with MR infants (1.7% vs 0.8%), but the abundance decreased with age (P = 0.056). The carriage rate, or percentage of infants in which the typically pathogenic Staphylococcus was detected was also higher among HR as compared with MR infants at 2 weeks (75% and 47%, χ2 = 3.48, P = 0.063), but differences in prevalence diminished by 4 weeks of age (P = 0.34).
Predicted Shifts in Functional Pathways
PICRUSt predictions indicated that the different community structures would be accompanied by significant shifts in several metabolic pathways, including environmental and genetic information processing, cellular processes, and metabolism (Table 1). At Level 2 of the KEGG pathway analysis, functional predictions for HR infants indicated pathway increases related to energy, carbohydrate and lipid metabolism, and xenobiotic biodegradation and metabolism. Conversely, MR infants evinced increases in gene mapping to glycan biosynthesis and pathways associated with metabolism of vitamins, terpenoids and polyketides, and amino acids. KEGG predictions also projected that genes related to environmental adaption and immunity would be downregulated in HR infants, while biochemical pathways implicated in human infectious disease would be enhanced.
Infant Growth and Health Trajectories
Growth curve analysis was used to analyze weight gain through 8 months of age. The overall growth trajectory of HR infants was faster [F(1,78.77) = 14.52, P < 0.001; Fig. 3]. Despite having similar birth weights (P = 0.22), HR infants weighed an average 188 g more at 2 months [F(1, 51) = 38.76, P < 0.001] and 88 g more at 4 months [F(1, 51) = 7.60, P < 0.01]. Weight differences diminished by 6 months after infants had transitioned to solid food. The phylogenetic richness of the microbial community structure (PD index) at 8 weeks tended to be positively associated with infant weight at the time of sampling (r = 0.350, P = 0.08), and was significantly correlated with subsequent weights at 4, 6, and 8 months of age (r = 0.433, P = 0.027; r = 0.408, P = 0.043; and r = 0.418, P = 0.034, respectively). More microbial richness at 8 weeks was also associated with a faster growth trajectory (r = 0.404, P = 0.041). When comparing gut health between rearing conditions, more incubator-reared and formula-fed infants were treated for both acute and chronic diarrhea by 6 months of age (7 of 19 HR vs 3 of 35 MR, χ2 = 6.53, P = 0.011; see Figure, Supplemental Digital Content 6, http://links.lww.com/MPG/B654). Differences in vulnerability to enteric pathogens, identified as Campylobacter jejuni in 70% of cultured stool, continued to persist after infants were weaned to solids (6 of 19 HR vs 2 of 35 MR, χ2 = 4.69, P = 0.03).
Our findings concur with prior research demonstrating that bacterial colonization is strongly affected by early rearing conditions. Vaginally born infants were colonized by microbial populations more closely related to the maternal reproductive tract and gut, and dominated by Prevotella spp and Lactobacillus spp (8,21). This natural colonization was perturbed if delivered by cesarean-section and instead the infants show a delayed colonization by Bacteroides and Bifidobacterium and may have been enriched in skin microbiota, including Staphylococcus, thus resembling the gut microbial composition of human infants exposed to a NICU (22–24).
Breast-feeding is a second major factor influencing the establishment of the microbiome during the neonatal period. It favored the growth of commensal Bifidobacteria and Lactobacillus, whereas the formula-fed infant monkeys had an overrepresentation of Clostridium(25–27). However, whereas Bifidobacteria species can constitute nearly 81% of the gut microbiota in vaginally born and breastfed human infants (7), levels were considerably lower in the monkey (28). This species difference may be because of the higher concentrations of Bifidobacterium-promoting oligosaccharides present in human milk. The ratio of oligosaccharides to lactose is 1 : 1.26 in humans, whereas it is only 1 : 6 in the rhesus monkey (29,30). We expected a higher abundance of Lactobacilli in MR infants because of vertical transmission and stimulation from prebiotic constituents in breast milk. However, other studies have found that this effect is not always clearly evident (8,31). In addition, the absence of a larger difference when compared with Lactobacilli abundance in the HR infant may reflect the recent addition of prebiotic glycans to the particular formula they consumed. Fructooligosaccharides, including those present in Similac Sensitive formula, can significantly increase both Lactobacillus and Bifidobacterium. Lactobacilli growth is, however, less dependent on oligosaccharides (32) and a dose-dependent stimulating effect on growth has been seen only for Bifidobacteria(33). The effect of early feeding regimens on Lactobacilli may also be delayed. A prior study on nursery-reared rhesus infants fed formula with prebiotics indicated that they ultimately had substantially lower levels of Lactobacillus, but not until after they were weaned onto solid foods (26).
Despite significant differences in overall composition and individual taxa, we did not see large differences in phylogenetic richness between the 2 rearing conditions. However, phylogenetic diversity was predictive of weight gain in both conditions as the infants began to consume solid foods. Other studies have found diminished diversity in HR infant monkeys at weaning and during the transition to solids (26), so it is possible that a diminished community richness only becomes apparent later. Our HR infants did, however, evince a higher ratio of Firmicutes-to-Bacteroidetes, due in part to an abundance of Blautia, a profile associated with more efficient absorption of calories and metabolic disorders in humans (14,15,34). This difference resulted in a corresponding shift in predicted metabolic pathways based on functions attributed to these microbes, including increased genes associated with carbohydrate and lipid metabolism. KEGG predictions were substantiated by larger and faster weight gains in HR infants. A differential effect of early rearing on growth rate has been reported previously for nursery-reared monkeys as well as in formula-fed human infants (28,35). Though this difference was diminished after weaning, rapid weight gain during infancy has been correlated with later risk for adult obesity, dyslipidemia, and insulin resistance in humans (36).
In addition to influencing host metabolism, gut bacteria can sustain infant health by providing protection against pathogens (3,4). When clinical records were reviewed at both 6 and 12 months, the HR monkeys exhibited more diarrheic symptoms with verified C jejuni infections during and subsequent to the transition to solid foods, confirming the benefits of breast-feeding for providing sustained protection against enteric pathogens (37). C jejuni is associated with gut dysbiosis and the abundance of Campylobacter is predictive of the overall microbial composition, even in asymptomatic infant and juvenile monkeys (38–40). Further demonstrating the importance of maternal influences, a prior study documented that a high-fat maternal diet before birth had a protracted effect on the abundance of Campylobacter species present without symptoms in infant monkeys (9). Previous studies have also documented differences in the immune responses of MR and HR monkeys (28,41), which are likely associated with the protective qualities of lactic acid bacteria and Bifidobacterium, commensal strains that were more abundant in MR infants and known to enhance intestinal epithelial barrier function (42,43). Oligosaccharides in breast milk can selectively stimulate the propagation of these microbes, as seen in the predicted upregulation of genes involved in the metabolism of host glycans in the MR infants (32,44). Through these mechanisms, breast milk can be protective against diarrheal disease. Specifically, it has been shown that probiotics can discourage the growth and intestinal adhesion of C jejuni, Clostridium, and other pathogenic organisms (45). Moreover, Prevotella, the predominant genera in MR monkeys, may also have a protective role as it contributes to the production of fermentation enzymes responsible for short-chain fatty acids (SCFA) (46). These enzymes and SCFA are critically important for the regulation of immune responses. Collectively, these findings reaffirm the view that there is a critical early window for initiating the trajectory to gut health.
Although our findings concur with many prior studies, limitations should be acknowledged. The HR condition differed in more than 1 way from the MR condition, because it involved both formula-feeding and early rearing in an incubator. However, both factors can co-occur in human infants. Infants born premature or delivered through cesarean-section are more likely to be admitted to the NICU and their mothers are less likely to breast-feed, or to delay breast-feeding initiation (47,48). Many of the HR infants were being reared in this manner because the biological mother had a cesarean delivery. There were repeated attempts to reunite the HR infant with its mother during the first week of life, a practice that could be considered to mimic the bacterial exposure that might occur during the ‘skin-to-skin contact’ encouraged for preterm NICU infants (49). Future research will have to more selectively vary the dietary and social variables that differed between the 2 rearing conditions. Ours was designed to discern more maximal microbial differences that might occur in the absence of the mother and breast milk. For this type of investigation, a monkey is preferable to rodent models because breast milk composition and gut maturity are different in species with altricial neonates (29,30). Though ours is one of the larger studies to examine factors influencing the infant microbiome in monkeys, the sample size did limit statistical power. It precluded stratified analyses to identify interactions between delivery mode and feeding method. We were also underpowered to conduct a serial analysis of the changing microbiome in individual HR infants, but did have a sufficient number of MR infants for repeated measures analysis (see text, Supplemental Digital Content 3, http://links.lww.com/MPG/B654). Finally, the gut microbiome is not the only outcome known to differ between MR and HR monkeys; the mother also stimulates neural and behavioral development (50).
In summary, breastfed infant rhesus exposed continuously to maternal sources of bacteria more quickly acquired microbiota typical of adults and had higher levels of several beneficial symbionts. The findings concur with the view that there is a biological expectancy that a mother will be present to provide a sustained microbial inoculation. Many clinical, personal, and economic factors contribute to obstetrical decisions about delivery mode and parental decisions about feeding regimens after birth, but the influence on the infant's gut microbiome should be taken into consideration. Although the improved composition of formula, including prebiotic factors, now allow it to more closely resemble mother's milk, we still need to advance our understanding of its prebiotic functions. The initial community structure of the infant's microbiome can have long-term metabolic and physiologic effects influencing the developmental trajectory to adult health in animals and humans.
Acknowledgements are due to the staff of the WNPRC who provided specimens from the HR infants (P51OD011106) and to Alexandra Proctor for help with DNA extractions and advice on the 16S sequence analysis. All bacterial DNA was extracted and processed by the laboratory of G.J.P. and M.L. at ISU.
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