ACCESSION NO: 1007824  SUBFILE: CRIS 

PROJ NO: ILLU-698-912 AGENCY: NIFA ILLU 

PROJ TYPE: HATCH PROJ STATUS: REVISED 
START: 20 OCT 2015 TERM: 30 SEP 2020 FY: 2018

INVESTIGATOR: Donovan, SH, M.

PERFORMING INSTITUTION: 
UNIVERSITY OF ILLINOIS 
2001 S. Lincoln Ave. 
URBANA, ILLINOIS 61801

NUTRITIONAL REGULATION OF INTESTINAL DEVELOPMENT AND MICROBIAL COLONIZATION

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.

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.

APPROACH: 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: 2018/10 TO 2019/09
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.

IMPACT: 2018/10 TO 2019/09
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 (not previously reported): 2018/10 TO 2019/09

1. 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.
2. 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.
3. 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.