NUTRITIONAL REGULATION OF INTESTINAL DEVELOPMENT AND MICROBIAL COLONIZATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1007824
• Project No.: ILLU-698-912
• Project Start Date: Oct 20, 2015
• Project End Date: Sep 30, 2020

Project Director
Donovan, SH, M.

Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801

Performing Department
Food Science & Human Nutrition

Non Technical Summary
The microbiota contributes pivotal nutritive, metabolic, immunological, and protective functions for the host. Dysbioses and lower microbial diversity in early life are associated with adverse health outcomes, thus, it is of critical importance to understand the factors that influence the development of the microbiome in infants in order to develop strategies to support optimal colonization. Despite the importance of initial colonization in establishing the microbiome, samples from infants were not included in the Human Microbiome Project, thus, a limitation of the existing literature using 16S rDNA-based sequencing methods is the small sample sizes, ranging from 1-18 infants, which hinders the generalizability of the findings. This research will systematically investigate how early life nutrition modulates microbial colonization in a large prospective cohort study of human infants in the first year of life. These findings will be compared with data in our laboratory describing the development of the gut microbiome in piglets and thus has application to animal agriculture as well.

Animal Health Component

0%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	1820	1010	60%
702	3450	1010	30%
702	3510	1010	10%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
3510 - Swine, live animal; 1820 - Soybean; 3450 - Milk;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

intestinal development

microbial colonization

early nutrition

microbiota

oligosaccharides

health

Goals / Objectives
The objective of this proposal is to apply a multi-omic approach to determine the impact of early nutrition on the neonatal microbiome and metabolome. Our central hypothesis is that HMO and prebiotics will differentially modulate gut microbiome structure and metabolic potential, which in turn will influence the metabolome. We further hypothesize that the actions of oligosaccharides will differ based on the feeding context (BF, FF, CF). To test this hypothesis, two specific aims are: Aim 1) determine the impact of diet (BF, FF and CF) on the composition of the microbiota in the first year of life by sequencing bacterial 16S rDNA amplicons, and Aim 2) determine the impact of diet (BF, FF and CF) and oligosaccharides on microbiota (Aim 1) and the microbial metatranscriptome and metabolome at 6 weeks of age. The data obtained from this research will be compared with existing data in the Donovan laboratory describing the development of the gut microbiome in piglets and thus has application to animal agriculture as well.

Project Methods
Human infants (n=150) will be exclusively breast-fed, exclusively formula-fed or fed both human milk and infant formula. Stool samples will be collected at 5 time points: 1 week, 6 weeks, 12 weeks, and 12 months postpartum and 1 month after the addition of solid food, which could occur between 4-6 months based on parent preferences. Human milk samples will also be collected at 6 weeks postpartum. Dietary intake and body weights will be obtained at all time points. For Aim 1, the composition of the microbiota will be determined by amplifying the V3-V4 16s rDNA and sequencing the products using MiSeq. Regression models will be used to explore associations between diet, types of oligo-saccharides, route of delivery, antibiotic use and alterations in the gut microbiome as measured by the sequencing data. Outcomes (response variables) to be examined will include alpha-diversity and the abundance of each bacterial taxon (phyla, order, class, family, genus and species). For Aim 2, human milk oligosaccharides will be measured by high performance liquid chromatography-chip time-of-flight mass spectrometry. The fecal metatranscriptome will be assessed by sequencing of RNA libraries by Illumina HiSeq2500. Lastly, the fecal metabolome will be evaluated MALDI FT-ICR MS. Multivariate relationships between the metatranscriptome and metabolome will be analytically quantified using Canonical Correlation Analysis.

Progress 10/20/15 to 09/30/20

Outputs
Target Audience:Practitioners interested in improving child health. Scientists interested in how early nutrition influences gut development. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The researchers mastered new techniques in conducting this research. In addition, several undergraduate students were trained and participated in the research. The graduate students had the opportunity to present the results at national and international conferences. How have the results been disseminated to communities of interest?Over the past five years, sevenmanuscripts were published in high quality journals, including a description of the STRONG Kids 2 cohort, which was published in Current Developments in Nutrition in 2019. A total of sevenabstracts were published and the results presented by graduate students at national and international conferences. Dr. Donovan also presented the findings as an invited speaker at universities and at national and international conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Over the course of this project, we have characterized the microbiome and metabolome of a subset of 150 infants who areparticipants in the STRONG Kids2 longitudinal cohort. Stool samples were collected at five time points over the first year of life from infants who were exclusively breastfed (BF; n=50), exclusively formula-fed (FF; n=50) or mixed-fed (MF; n=50).We have described the pattern of microbial colonization between 1 week and 12 months of age by sequencing the bacterial 16S rDNA gene. We showed that the fecal microbiota changed over time and differed between BF and FF or MF infants.We have also described the metabolic potential of the microbiome at sixweeks of age through sequencing the bacterial DNA (metagenomics) and measuring the fecal metabolites (metabolomics). Metabolomics identified metabolic variations induced by diet in infants. Distinct metabolite differences between BF and infants fed all (FF) or some (MF) formula. Microbial bile acid metabolism was sensitive for discerning amongst the feeding modes. We also measured the human milk oligosaccharides (HMO) in the milk of breastfeeding mothers and related the HMO composition to the fecal microbiota composition of their infants. Overall, these data showed that feeding mode was the primary factor influencing early microbiome development. However, the unexpected finding was that the FF and MF infants did not differ from each other and both groups were significantly different from BF infants. We had anticipated that the MF infants would be intermediate between the BF and FF infants. Thus, early life nutrition is critically important for establishing the gut microbiome composition and formula exerts a dominant role in microbiota composition and metabolic activity. This is important as the microbiota regulates infant intestinal, metabolic, immune and brain development. Studies have shown that early life fecal microbial composition is associated with life-long health outcomes. Furthermore, our work with HMO has relevance to animal agriculture for at least two reasons. First, some of the oligosaccharides in human milk are also present in bovine and porcine milk, thus, information gained from HMO/human infants has implications for animal health. Second, the oligosaccharides in bovine milk have been isolated for additional to human infant formula. Thus, identification of biological roles for oligosaccharides can provide new markets for components present in bovine milk.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Davis, E.C., Dinsmoor, A.M., Wang, M. and Donovan, S.M. 2020. Microbiome composition in pediatric populations from birth to adolescence: Impact of dietary and prebiotic and probiotic interventions. Digestive Diseases and Sciences 2020; 65: 706-722.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Dinsmore, A., Arthur, A.E., Fiese, B., Khan, N.A. and Donovan, S.M. 2020. Early life factors predictive of weight status in 2 year-olds. Current Developments in Nutrition 2020; 4(suppl 2): 977, https://doi.org/10.1093/cdn/nzaa054_049.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Davis, E.C., Wang, M. and Donovan, S.M. 2020. Bacterial co-occurrence patterns between human milk and microbial sites of breastfeeding dyads. Current Developments in Nutrition 2020; 4(suppl 2): 966. https://doi.org/10.1093/cdn/nzaa054_038
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Donovan, S.M. 2020. Evolution of the gut microbiome in infancy within an ecological context. Current Opinion in Clinical and Metabolic Care 2020; 23: 223-227.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Members of the target audience included practitioners interested in improving child health and scientists interested in how early nutrition influences gut development. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The researchers mastered new techniques in conducting this research. In addition, several undergraduate students were trained and participated in the research. The graduate student had the opportunity to present the results at a national conference. How have the results been disseminated to communities of interest?A description of the STRONG Kids 2 cohort was published in Current Developments in Nutrition. Abstracts were published and the results presented at the European Society for Pediatric Gastroenterology Hepatology and Nutrition 2019 meeting held in Glasgow, Scotland and at the Nutrition 2019 conference held in Baltimore, MD. Dr. Donovan also presented the findings as an invited speaker at universities and at national and international conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue to analyze samples from the 450-family STRONG kids cohort, including maternal and infant fecal samples and mother's milk. In addition to Hatch funding, Dr. Donovan received additional funding from the National Dairy Council, the Gerber Foundation, and the NIH to completely analyze infant and mother microbiome and infant intestinal cell gene expression, which will begin in the next reporting period.

Impacts
What was accomplished under these goals? Objectives and Study: The gut microbiota is a key regulator of infant gastrointestinal, immune, cognitive, and metabolic development. Its composition and metabolic function are mediated by early postnatal nutrition. Microbial metabolites are important mediators of microbial interactions with the host. Herein, the effect of nutrition on the fecal metabolome of human infants was investigated. Methods: Fecal samples were collected from six-week-old exclusively breast-fed (BF; n=25), formula-fed (FF; n=25) or mixed-fed (MF; n-25) participants in the STRONG Kids 2 longitudinal cohort. Within each diet group, infants were either delivered vaginally (n=13) or by Cesarean section (n=12). Fecal metabolite profiles were analyzed using ultra high-performance liquid chromatography/tandem accurate mass spectrometry methods (Metabolon, Durham, NC). Metabolite concentrations were compared by diet and delivery mode by two-way ANOVA. Results: A total of 804 known and 196 structurally unknown biochemicals were detected in feces. There were significant main effects of diet (582 compounds), delivery mode (139 compounds), and diet by delivery interaction (124 compounds) (p<0.05). Principal component analysis showed that infants receiving formula (FF and MF) clustered together and were significantly separated from BF. Comparisons between diet groups showed that compared to BF infants, FF and MF infants had a similar number of metabolites in differing in abundance (588 and 548, respectively). Only 98 metabolites differed between FF and MF. Amino acids, human milk oligosaccharides (HMO), and fatty acids were the main differentiating metabolites between the MF and FF and the BF infants. The levels of amino acids and unsaturated fatty acids between C5 and C12 were higher (p<0.05) in MF and FF than BF. In contrast, HMO, unsaturated fatty acids between C14 and C22 and long chain polyunsaturated fatty acids were in higher abundance in BF than MF and FF. Taurine-conjugated bile acids (taurocholate, taurochenodeoxycholate) and sulfated secondary bile acids (taurolithocholate 3-sulfate, taurocholenate sulfate) were greater in feces of BF than MF and FF infants. The abundance of other secondary bile acids differentiated MF from FF infants; being greater in FF than BF, but not MF. Conclusion: Metabolomics identified metabolic variations induced by diet in infants. Distinct metabolite differences between BF and infants fed all (FF) or some (MF) formula. Microbial bile acid metabolism was sensitive for discerning amongst the feeding modes. On-going work is integrating bacterial metagenomic sequences with these metabolomic findings.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Fiese, B.H., Musaad, S., Bost, K.K., McBride, B.A., Lee, S.Y., Teran-Garcia, M. and Donovan, S.M. 2019. The STRONG Kids 2 birth cohort study: A cell-to-society approach to dietary habits and weight trajectories across the first five years of life. Curr. Develop. Nutr. 2019;3(3):nzz007. doi: 10.1093/cdn/nzz007.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Davis, E.C., Monaco, M., Musaad, S. and Donovan, S.M. 2019. Early life nutrient intake is associated with weigh-for-length Z-scores at 3 and 12 months. Curr. Develop. Nutr. 2019;3(suppl 1): nzz048.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Donovan, S.M. and Wang, M. 2019. Fecal metabolite profiles of mixed-fed infants are more similar to formula-fed than breastfed infants. J Pediatr. Gastroenterol. Nutr. 2019; 68 (Suppl 1): 998.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Members of the target audience included practitioners interested in improving child health and scientists interested in how early nutrition influences gut development. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The researchers mastered new techniques in conducting this research. In addition, several undergraduate students were trained and participated in the research. The graduate student had the opportunity to present the results at one national and one international conference. How have the results been disseminated to communities of interest?Abstracts have been published and the results presented at the Nutrition 2018 meeting held in Boston, MA in June 2018 and at the International Society for Research in Human Milk and Lactation Conference in Kanagawa, Japan in October 2018. Dr. Donovan also presented the findings of the overall project at several national and international conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue to analyze samples from the 450-family STRONG kids cohort, including maternal and infant fecal samples and mother's milk. In addition to Hatch funding, Dr. Donovan received additional funding from the Gerber Foundation andan NIH grant to analyze infant and mother microbiome and infant intestinal cell gene expression, which will begin in the next reporting period.

Impacts
What was accomplished under these goals? Objective: Human milk (HM) is composed of a diverse profile of immune components, which are believed to influence infant innate and adaptive immunity. Factors such as maternal age, geography, gestational age, and lactation stage influence immune composition. However, the breadth of cytokines, chemokines, and growth factors (CCGFs) present in HM is not well-characterized, and whether breastfeeding exclusivity affects CCGF composition is unknown. The objective was to measure CCGFs in mature HM and assess differences in composition between exclusively breastfeeding (EBF) and mixed-feeding (MF) mothers. Methods: Milk samples (EBF=44; MF=25) collected atsix weeks postpartum from healthy mothers enrolled in the STRONG Kids 2 birth cohort study were utilized for analysis. All mothers vaginally-delivered their infants at term. Samples were analyzed for 41 CCGFs using a human cytokine/chemokine multiplex magnetic bead panel. Results: Forty components were detectable in the milk samples. Twelve CCGFs (EGF, TGF-α, GRO, MDC, PDGF-AA, IL-15, IL-4, IL-7, IL-8, IP-10, MCP-1, VEGF) were identified in more than 94% of samples. TNF-α, Fractalkine, and IL-1α were detected in more than 78% of samples. Among this subset of 15 analytes, IL-8 was the only CCGF, within limits of detection (LOD), which differed between groups, with greater concentrations in the milk of MF relative to EBF mothers (p=0.002). EGF and GRO were above the LOD (10,000 pg/ml) in most samples. Fisher exact tests were used to determine differences in frequency of detection for all CCGFs between EBF and MF mothers. The probability of detecting Fractalkine was greater among EBF compared to MF mothers (p=0.04). The probability of detecting IL-3 (p=0.009), IL-6 (p=0.02), IL-9 (p=0.02), and MIP-1β (p=0.006) was greater among MF compared to EBF mothers. Conclusion: While levels of many CCGFs have been shown to be higher in early lactation, results demonstrate that a wide range of immune components are present at detectable levels in mature HM. The high frequency of detection of a subset of CCGFs may point to a core immune composition in mature HM. Differences in CCGF components in HM of EBF and MF mothers suggests that breastfeeding exclusivity impacts the immune profile of HM, which may be related to differences in oral microbiota of EBF and MF infants.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Davis, E.C. and Donovan, S.M. 2018. Immune composition of mature human milk in exclusively breastfeeding and mixed-feeding mothers. Current Developments in Nutrition 2018; 2(11): 31.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Davis, E.C., Wang, M. and Donovan, S.M. 2018. Human milk microbiota in breastfeeding and mixed-feeding mothers. Breastfeeding Medicine 2018; 13: A48.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Members of the target audience include practitioners interested in improving child health and scientists interested in how early nutrition influences gut development. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The researchers mastered new techniques in conducting this research. In addition, several undergraduate students were trained and participated in the research. How have the results been disseminated to communities of interest?Papers have been published and the results presented at the Experimental Biology 2016 meeting held in Chicago, Illinois in April 2016. Dr. Donovan also presented the findings of theoverall projectat several national and international conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue to analyze samples from the 450-family STRONG kids cohort, including maternal and infant fecal samples and mother's milk. In July 2017, Dr. Donovan received additional funding through a five-year NIH grant to complete the analyses and also measure infant gene expression in exfoliated intestinal epithelial cells.

Impacts
What was accomplished under these goals? Feeding mode is known to impact an infant's growth pattern throughout the first year of life, which can be an important factor in the risk of childhood obesity. While breastfeeding has been shown to be protective against later overweight and obesity, the underlying mechanisms involved remain unclear. Feeding practices of young infants are hard to measure, but may provide valuable information for clinicians and researchers studying the long-term implications of early life growth trajectories. It is hypothesized that feeding mode affects self-regulation and infant satiety; however, few studies have investigated whether intake characteristics differ among infants who are exclusively breastfed (EBF), mixed-fed (MF), or exclusively formula-fed (EFF). The objective was to assess whether feeding frequency, average intake per feeding, and overall daily intake differ among infants of different feeding regimes, and whether these characteristics may be related to the infant's weight-for-length (WFL) Z-score and maternal BMI. Intake data was collected for 191 healthy, six-week old infants, enrolled in the STRONG Kids 2 cohort at the University of Illinois at Urbana-Champaign. Mothers weighed infants, before and after each feeding, over an average of 23.38 ± 2.26 hours, in order to measure intake volume. Feeding mode was assessed from a questionnaire completed by the mother. The overall intake weight (ml/kg BW) consumed across the entire weighing period was not significantly different between the three feeding methods. Average intake per feeding, across all feeding modes, was positively associated with overall ml/kg intake (P<0.0001) and negatively associated with feeding frequency (P<0.0001). Feeding frequency tended to differ by feeding mode (P=0.056). EBF infants (n = 151) fed on average 9.17 ± 1.87 times across the entire weighing period, which was significantly greater than the number of feeds of 7.92 ± 1.31 recorded for the EFF infants (n = 12). The feeding frequency of MF infants (n = 28) did not differ from that of EBF or EFF infants. Average intake per feeding was significantly higher among EFF infants (102.2 ± 34.0) compared to both EBF infants (84.4 ± 25.5) and MF infants (74.8 ± 29.4), which did not differ from each other. Further analysis was completed to evaluate differences among 132 EBF infants based on whether or not mothers indicated regular breast milk pumping. While the overall intake (ml/kg) and total number of feeds per day did not differ among the two groups of EBF infants, average intake per feeding tended to be greater (P=0.06) in infants whose mothers regularly pump breast milk. While none of the reported feeding characteristics were associated with infant WFL Z-score, feeding frequency was negatively associated with maternal BMI at six weeks postpartum. Average intake per feeding and overall intake (ml/kg) were not associated with maternal BMI. These findings suggest that intake characteristics of infants differ based on feeding mode and may provide context regarding differences in growth trajectories observed during later infancy.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Davis, E.C., Wang, M. and Donovan, S.M. 2017. The role of early life nutrition in the establishment of gut microbial composition and function. Gut Microbes 8:143-171.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Davis, E.C., Donovan, S.M. and Dong, P. 2017. Early life milk intake among infants of different feeding modes. FASEB Journal 31: 316.8.

Progress 10/20/15 to 09/30/16

Outputs
Target Audience:Practitioners interested in improving child health and scientists interested in how early nutrition influences gut development. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The researchers mastered new techniques in conducting this research. In addition, several undergraduate students were trained and participated in the research. How have the results been disseminated to communities of interest?Papers have been published and the results presented at the Experimental Biology 2015 meeting held in Boston, Massachusetts in April 2015. What do you plan to do during the next reporting period to accomplish the goals?We will complete analysis of the samples collected in the study and publish remaining data. Additional funding will be sought from NIH.

Impacts
What was accomplished under these goals? This study tested the hypothesis that the fecal bacterial genera of breast-fed (BF) and formula-fed (FF) infants differ and that human milk oligosaccharides (HMOs) modulate the microbiota of BF infants. Fecal samples were obtained from BF (n = 16) or FF (n = 6) infants at 3-month postpartum. Human milk samples were collected on the same day when feces were collected. The microbiota was assessed by pyrosequencing of bacterial 16S ribosomal RNA genes. HMOs were measured by high-performance liquid chromatography-chip time-of-flight mass spectrometry. The overall microbiota of BF differed from that of FF (P = 0.005). Compared with FF, BF had higher relative abundances of Bacteroides, lower proportions of Clostridium XVIII, Lachnospiraceae incertae sedis, Streptococcus, Enterococcus, and Veillonella (P < 0.05). Bifidobacterium predominated in both BF and FF infants, with no difference in abundance between the 2 groups. The most abundant HMOs were lacto-N-tetraose + lacto-N-neotetraose (LNT + LNnT, 22.6%), followed by 2'-fucosyllactose (2'FL, 14.5%) and lacto-N-fucopentaose I (LNFP I, 9.5%). Partial least squares regression of HMO and microbiota showed several infant fecal bacterial genera could be predicted by their mothers' HMO profiles, and the important HMOs for the prediction of bacterial genera were identified by variable importance in the projection scores. These results strengthen the established relation between HMO and the infant microbiota and identify statistical means whereby infant bacterial genera can be predicted by milk HMO. Future studies are needed to validate these findings and determine whether the supplementation of formula with defined HMO could selectively modify the gut microbiota.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wang, M., Li, M., Wu, S., Lebrilla, C.B., Chapkin, R.S., Ivanov, V. and Donovan, S.M. 2015. Fecal microbiota composition of breast-fed infants differs from formula-fed and is correlated with human milk oligosaccharides consumed. Journal Pediatric Gastroenterology and Nutrition 60: 825-833.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, M., Monaco, M.H. and Donovan, S.M. 2016. Impact of early gut microbiota on immune and metabolic development and function. Seminars in Fetal and Neonatal Medicine 21:380-387.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Berding, K.A. and Donovan, S.M. 2016. Microbiome and nutrition in autism spectrum disorder: Current knowledge and research needs. Nutrition Reviews 74:723-736.