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ACCESSION NO: 0208670  SUBFILE: HNRIMS; CRIS 

PROJ NO: ILLU-698-311 AGENCY: NIFA ILLU 
PROJ TYPE: HATCH PROJ STATUS: TERMINATED 
START: 01 OCT 2010 TERM: 30 SEP 2015 FY: 2015

INVESTIGATOR: Donovan, SH, M.

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

NUTRITIONAL REGULATION OF INTESTINAL DEVELOPMENT

NARRATIVE: Human milk contains high concentrations of oligosaccharides that are not found in infant
formulas and are proposed to contribute to the health benefits of human milk. The proposed studies will
determine how oligosaccharides affect the bacteria in the intestine and immune maturation of neonates.
This knowledge will allow for the development of nutritional ingredients to improve the quality of infant
formulas for babies who are not breast fed.

OBJECTIVES: Extending this currently-active project for one year to 09/30/2015.The goal is to determine
how nutrients impact the intestinal development of healthy neonatal piglets and piglets with intestinal
compromise. The objective is to define the oligosaccharide fermentation patterns produced by ileal and
colonic contents of sow-reared and formula-fed piglets. This knowledge will allow for the development of
nutritional ingredients to improve the quality of infant formulas for babies who are not breast fed.

APPROACH: Piglets will be either sow-reared or fed infant formula devoid of prebiotics. On days 9 and
18, contents collected from the ileum and ascending colon will be collected, diluted in anaerobic dilution
solution and immediately used for in vitro assays. Contents will be incubated with differing substrates
(human milk oligosaccharides, fermentable carbohydrates; 80 mg/tube) at 37C and samples will be
obtained at for 0, 2, 4, 8, and 12 hours and processed immediately for analyses of pH, gas, short chain
fatty acids, and lactate. The composition of the microbiota at all time points will be determined by
amplifying the V3 16s rDNA sequences and separating the bands by denaturing gradient gel
electrophoresis (DGGE). Bands that differ between substrates will be identified by cloning and
sequencing.

PROGRESS: 2010/10 TO 2015/09
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, three undergraduate
students participated in the research in the past year, learning valuable research skills to enhance their
professional development. How have the results been disseminated to communities of interest?One
paper was published in the past year. What do you plan to do during the next reporting period to
accomplish the goals? Nothing Reported

IMPACT: 2010/10 TO 2015/09
What was accomplished under these goals? The impact of diarrheal disease and prebiotics was studied
in piglets fed with formula (FF), or formula with 4 g/L human milk oligosaccharides (HMO) or prebiotics
(PRE; 9:1 of short chain galactooligosaccharides and long chain fructooligosaccharides). At day 10,
approximately half of the piglets were infected with 5x106 focus-forming units of group A porcine RV stain
OSU. Stool consistency was monitored 3 times daily. Ascending (Asc) and descending (Dsc) colonic
contents were collected at 5 days post-infection, and pH, dry matter (DM) and SCFA concentrations were
measured. Asc microbiota were analyzed by 16S rRNA gene pyrosequencing. HMO and PRE groups had
shorter duration of RV-associated diarrhea than FF. RV infection increased the pH and lowered the DM of
Asc and Dsc contents vs. non-infected pigs. HMO groups had higher Dsc pH than FF and PRE, while DM
of HMO and PRE groups was lower than FF. Infection increased Asc propionate concentration
independent of diet. Redundancy analysis showed that microbial structure differed among infected and
diet groups. Bacteroides was increased in infected pigs, and unclassified Lachnospiraceae was enriched
in HMO groups. In conclusion, the intraluminal environment and microbiota were altered by RV infection
and HMO supplementation. HMO and prebiotics reduced the duration of RV-induced diarrhea, likely in
part by modulating gut environment and microbiota.

PUBLICATIONS (not previously reported): 2010/10 TO 2015/09

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.

Item No. 2 of 3 

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.

Item No. 3 of 3 

ACCESSION NO: 1024037  SUBFILE: CRIS 
PROJ NO: ILLU-698-979 AGENCY: NIFA ILLU 
PROJ TYPE: HATCH PROJ STATUS: NEW 
START: 01 OCT 2020 TERM: 30 SEP 2023

INVESTIGATOR: Donovan, SH, M.

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

NUTRITIONAL REGULATION OF MICROBIAL COLONIZATION AND POSTNATAL GROWTH

NON-TECHNICAL SUMMARY: Over the past decade, cataloguing the composition of the human
microbiome across the life span has emerged as one of the most compelling areas of human health. The
microbiota contributes nutritive, metabolic, immunological, and protective functions for the host. The early
neonatal period is a critical phase of the lifespan for establishing the microbiome. This early microbial
colonization proceeds in succession through a process of "seeding and feeding" and the human milk
microbiota and human milk oligosaccharides (HMO) are key factors to supporting a health microbiota.
Dysbiosis, or abnormal microbiome composition, can occur due to formula feeding or antibiotic use and is
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
microbial colonization. The proposed research will systematically investigate how early life nutrition
modulates microbial colonization and child outcomes, including growth trajectories in the first three years
of life. This work will leverage an ongoing prospective cohort study of human infants (STRONG Kids) at
the University of Illinois and funding from NIH and the National Dairy Council.A main goal of this study is
to identify microbes associated with growth trajectories. Thus, this outcome will also have direct
applications to animal agriculture. For example, microbes that are associated with more rapid growth
trajectories in children could be viewed as potential probiotics to administer to young piglets, particularly
during the weaning transition. We could test these microbes in other piglet research that is on-going in Dr.
Donovan's laboratory, including studies with gnotobiotic piglets, which enables mechanistic studies of
host-microbe interactions.

OBJECTIVES: The objective of the proposed research is to apply a multi-omic approach to determine the
impact of early nutrition and HMO on the composition and function of the neonatal microbiome to relate
those factors to growth in the first three years of life. Our central hypothesis is that differences in
breastmilk HMO composition will differentially modulate gut microbiome structure and metabolic potential,
which in turn will be associated with infant growth trajectories. To test this hypothesis we will apply
multivariate regression models as well as mediation and moderation analyses. Macroecological dynamics
of the gut microbiota over time will be tested using nonlinear regression models, including power-laws
models. Additionally, we will test more sophisticated statistical methods to infer causality between
simplified components of microbiome and HMO composition and growth, including structural equation
modeling (SEM).

APPROACH: Subjects (n=450) will be recruited from the ongoing STRONG Kids 2 cohort. As infants,
they will have been exclusively breast-fed, exclusively formula-fed, or fed both human milk and infant
formula. Stool samples will be used forfivetime points in the first year of life (1 week, 6 weeks, and 3, 6
and 12 months of age) and at 18, 24 and 36 months age for microbiome analysis by sequencing theV3-
V4 region of the 16S rDNA. Human milk samples were collected at sixweeks postpartum and human milk
oligosaccharide (HMO) composition has been measured by high performance liquid chromatography-chip
time-of-flight mass spectrometry. Mothers will complete questionnaires with health history and child
dietary intake and infant/child heights and weights will be measured at all time points. Regression models
will be used to explore associations between HMO, 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 andabundance of each bacterial taxon (phyla, order, class, family, genus and
species). We will check for non-constant variance, influential points, and multicolinearity using standard
methods to insure that model assumptions are met. For continuous measures (such as abundance of
each taxon) standard linear regression will be used after consideration of potential non-linearities and
parametric transformations. Mediation and moderation analysis will be conducted to determine the
interactions between HMO composition, microbiome diversity, and composition and growth parameters.
In addition, we will apply newer methods to investigate the macroecological dynamics of the gut
microbiota over time using nonlinear regression models, including power-laws models. Lastly, we will
apply more sophisticated statistical methods to infer causality between simplified components of
microbiome and HMO composition and growth, including structural equation modeling (SEM). We
anticipate that these statistical analyses will uncover novel relationships between early life nutrition,
microbiota composition and infant growth.