Dietary nucleotides and fecal microbiota in formula-fed infants: a
randomized controlled trial1–3
Atul Singhal, George Macfarlane, Sandra Macfarlane, Julie Lanigan, Kathy Kennedy, Alun Elias-Jones,
Terence Stephenson, Peter Dudek, and Alan Lucas
ABSTRACT
Background: Dietary nucleotides are nonprotein nitrogenous
compounds that are thought to be important for growth, repair,
and differentiation of the gastrointestinal tract. A higher nucleo-
tide intake may also have favorable effects on the fecal microbial
composition and incidence of diarrhea in infancy. However, few
studies have tested this hypothesis with an experimental study
design.
Objective: We tested the hypothesis that nucleotide supplementa-
tion of infant formula has beneficial effects on fecal bacteriology.
Design: Oligonucleotide probes were used to measure bacterial
genus-specific 16S ribosomal RNA in stools of a subset of infants
(mean age: 20.4 wk) who were randomly assigned to nucleotide-
supplemented (31 mg/L; n ҃ 35) or control formula (n ҃ 37) from
birth until age 20 wk or were breastfed (reference group; n ҃ 44). The
microbial pattern was assessed as the ratio of Bacteroides-
Porphyromonas-Prevotella group (BPP) to Bifidobacterium spe-
cies.
Results: The ratio of BPP to Bifidobacterium spp. rRNA in infants
randomly assigned to the nucleotide-supplemented formula was
lower than in infants receiving the control formula (mean difference:
Ҁ118%; 95% CI: Ҁ203%, Ҁ34%; P ҃ 0.007), but it did not differ
in infants who were breastfed. The difference between randomized
formula-fed groups was independent of potential confounding fac-
tors (P ҃ 0.003).
Conclusions: Our data support the hypothesis that nucleotide sup-
plementation improves the composition of the gut microbiota in
formula-fed infants. Because this effect could contribute to previ-
ously described benefits of nucleotide supplementation for gastro-
intestinal tract and immune function, these findings have important
implications for optimizing the diet of formula-fed infants. Am
J Clin Nutr 2008;87:1785–92.
INTRODUCTION
Breast milk was shown to have a number of short- and long-
term benefits, but the most frequently cited benefit is for a lower
incidence of gastroenteritis in infants breastfed rather than
formula-fed (1, 2). Several factors were suggested to contribute
to this protective effect, including, eg, a higher concentration of
nucleotides in human milk compared with cow-milk-based for-
mula (3– 6). Nucleotides are nonprotein nitrogenous compounds
suggested to be essential nutrients in certain clinical conditions,
to modulate immune function, and to be important for growth,
repair, and differentiation of the gastrointestinal tract (7–12).
Am J Clin Nutr 2008;87:1785–92. Printed in USA. © 2008 American Society for Nutrition
However, although nucleotides have been added to some infant
formulas for many years, relatively few data from large-scale
randomized studies support their benefits and use in humans.
In formula-fed infants, dietary nucleotide supplementation
was shown to reduce the incidence of diarrhea (3– 6), possibly by
a favorable effect on the gastrointestinal microbiota. For in-
Downloaded from www.ajcn.org by guest on November 20, 2010
stance, in vitro studies suggest that nucleotides enhance the
growth of bifidobacteria (13–15), which by reducing stool pH
could reduce the growth of pathogenic bacteria and hence the
incidence of infectious diarrhea (7, 8, 10, 16). Bifidobacterium
spp. are found in higher proportions in the stools of breastfed
compared with formula-fed infants (16 –19), but whether this
effect is related to the higher concentration of nucleotides in
breast milk compared with formula is uncertain.
Data to support an effect of dietary nucleotides on the gut
microbiota are conflicting (20, 21). One study concluded that the
addition of nucleotides to infant formula resulted in a microbial
pattern in the stool that was more like that of breastfed infants
(20), whereas, in contrast, another investigation suggested that
dietary nucleotides discouraged the growth of bifidobacteria
(21). However, those previous studies were small and nonran-
domized and used bacterial culture rather than more robust mo-
lecular techniques to assess fecal microbiology (20, 21). Here,
we have investigated the effects of nucleotide supplementation
of infant formulas on fecal microbiota, incidence of diarrhea, and
stool characteristics with the use of an experimental study design.
We used genus-specific 16S ribosomal RNA (rRNA)–targeted,
oligonucleotide probes to test the hypothesis that a higher dietary
intake of nucleotides has beneficial effects on the fecal micro-
biota of formula-fed infants. Specifically, we assessed the ratio of
1
From the Childhood Nutrition Research Center, Institute of Child Health,
London, United Kingdom (AS, JL, KK, and AL); the Dundee University Gut
Group, Ninewells Hospital Medical School, Dundee, United Kingdom (GM
and SM); the Leicester General Hospital NHS Trust, Leicester, United King-
dom (AE-J); the Faculty of Medicine and Health Sciences, Academic Divi-
sion of Child Health, University of Nottingham, United Kingdom (TS); the
HJ Heinz Company Ltd, Kendal, United Kingdom (PD).
2
Supported by the Medical Research Council with a charitable contribu-
tion from the HJ Heinz Company Ltd. The trial formulas were generously
suppled by the HJ Heinz Company Ltd, Hayes Middlesex, United Kingdom.
3
Reprints not available. Address correspondence to A Singhal, Childhood
Nutrition Research Center, Institute of Child Health, 30 Guilford Street,
London, United Kingdom, WC1N 1EH. E-mail: a.singhal@ich.ucl.ac.uk.
Received December 11, 2007.
Accepted for publication February 28, 2008.
1785
1786 SINGHAL ET AL

Bacteroides-Porphyromonas-Prevotella group (BPP) to Bi- TABLE 1

fidobacterium spp. rRNA because the relative abundance of Nutritional composition of infant formulas1
these bacteria are known to differ between breastfed and Amount
formula-fed infants (19, 22), are known to be affected by dietary
factors in infancy (16, 18), and are suggested to influence neo- Per 100 g Per 100 mL
natal health (22). powder as fed

Energy (kJ) 2190 284

Energy (kcal) 524 68
SUBJECTS AND METHODS
Protein (g) 11 1.5
Study population Whey protein 6.9 0.9
Casein protein 4.3 0.6
Participants were recruited prospectively from 4 hospitals (2 Carbohydrate as lactose (g) 53.6 7.0
each in Leicester and Nottingham) in the United Kingdom be- Fat (g) 29.4 3.8
tween 1999 and 2002. All mothers of healthy singletons 쏜 37 wk Linoleic acid (mg) 2700 350
of gestation and without congenital abnormalities were eligible ␣-Linolenic acid (mg) 340 44
to participate. For the formula-fed arm, only infants of mothers ␥-Linolenic acid (mg) 250 33
who had decided not to breastfeed and had commenced formula Long-chain polyunsaturates (mg) 200 26
Calcium (mg) 300 39
feeding were eligible (n ҃ 200). A reference group of breastfed
Chloride (mg) 310 40
infants was identified at birth, and the infants were eligible for the
Copper (␮g) 320 42
study if they were still breastfeeding at 8 wk of age (n ҃ 101). Iodine (␮g) 35 4.5
Informed and written consent was obtained from all participants,

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Iron (mg) 5.0 0.6
and the study was approved by both national and local ethics Magnesium (mg) 40 5.2
committees in each center. Manganese (␮g) 26 3.4
Phosphorus (mg) 210 27
Study design Potassium (mg) 440 57
Sodium (mg) 130 17
Formula-fed infants were randomly assigned within a few
Zinc (mg) 2.6 0.3
days of birth to a nucleotide-supplemented infant formula (31 Vitamin A (␮g) 800 100
mg/L; n ҃ 100) or a control formula with 쏝5 mg/L nucleotides Thiamine (␮g) 320 42
(n ҃ 100). A random permuted block design, stratified by center Riboflavin (␮g) 420 55
(Nottingham or Leicester), allocated by an independent statisti- Vitamin B-8 (␮g) 270 35
cian, and concealed by sealed opaque envelopes was used. In- Vitamin B-12 (␮g) 1.1 0.14
fants not withdrawn from the study continued to receive the Biotin (␮g) 7.9 1.0
assigned formula until 5 mo of age. All mothers and research staff Folic acid (␮g) 26 3.4
members were blinded to the identity of the formula. Niacin (mg) 5.3 0.7
Pantothenic acid (mg) 1.8 0.2
The nucleotide composition of supplemented formula was
Vitamin C (mg) 53 6.9
based on the concentration and composition of free nucleotides
Vitamin D (␮g) 8.0 1.0
and nucleosides in human milk as reflected in current European Vitamin E (mg) 3.7 0.5
regulations (23). The supplemented formula contained cytidine Vitamin K (␮g) 21 2.7
monophosphate (CMP; 15 mg/L), uridine monophosphate (5 Choline (mg) 37 4.8
mg/L), adenosine monophosphate (6 mg/L), guanosine mono- Taurine (mg) 39 5.0
phosphate (2 mg/L), and inosine monophosphate (3 mg/L), 1
Nucleotide composition of the nucleotide-supplemented formula was
whereas the control formula (Farley’s First Milk) had 쏝3 mg/L as follows: cytidine monophosphate (15 mg/L), uridine monophosphate (5
of measurable CMP. The study formulas, manufactured by mg/L), adenosine monophosphate (6 mg/L), guanosine monophosphate (2
HJ Heinz Company Ltd, Hayes Middlesex, United Kingdom, mg/L), and inosine monophosphate (3 mg/L). Measurable nucleotide in the
met European guidelines for the composition of infant formula control formula was cytidine monophosphate (3 mg/L).
and were the same except for their nucleotide concentration.
Their composition is given in Table 1.
Demographic, social, clinical (including details of the method
of delivery, birth weight, and gestational age), and anthropomet- any medication (eg, antibiotics), and consultations with health
ric data were collected at random assignment in formula-fed care professionals. Research nurses followed the study partici-
infants and at age 8 wk in breastfed infants. Weight was recorded pants in their homes at 8, 16, and 20 wk of age and verified the
with the use of electronic scales accurate to 1 g (Seca, Hamburg, recorded information. Mothers also recorded their infant’s vol-
Germany), length was recorded with the use of a Rollametre ume of milk intake (in formula-fed infants only), food intake,
(Raven, Dunmow, United Kingdom) accurate to 1 mm, and head bowel function, and stool characteristics for a 3-d period before
circumference was measured with the use of a nonstretchable each follow-up visit and the age at which foods other than milk
tape accurate to 1 mm. Social class was based on the occupation were first introduced.
of the parent providing the main financial support for the family Tolerance to the formulas was monitored throughout the
(or if both parents worked, the father’s occupation), according to study. Mothers were asked to record crying time per 24-h period,
the registrar general’s classification and coded into 6 categories. the number of nighttime waking periods, pacifier use, and epi-
Mothers were asked to record clinical information on their in- sodes of colic (according to the mother’s interpretation of crying
fants, including episodes of illness, any treatment given, use of symptoms).
 NUCLEOTIDES AND FECAL MICROBIOTA
FIGURE 1. Progress of trial to age 20 wk. 1Incidence of diarrhea collected prospectively from random assignment to age 5 months.
Fecal bacteriology
All mothers still participating in the study at the 20-wk home
visit were given collecting pots and asked to collect a fresh
sample of their infant’s stool by frequently checking the nappy
during the day (excluding the first sample in the morning). The
sample was collected by the research nurse (or taken by a courier)
and frozen at Ҁ20 °C within 1 h. The samples were subsequently
frozen at Ҁ80 °C for later analysis of fecal flora.
Stool samples were assessed for 4 microbial species previ-
ously suggested to be influenced by breastfeeding compared with
formula feeding (19) and by nucleotide supplementation of in-
fant formula (20). After mechanical disruption of the bacterial
cells with the use of a mini bead-beater, total RNA was extracted
from stool samples with the use of the phenol-chloroform method
described by Dore et al (24). The RNA was then blotted, hybrid-
ized, and washed with the use of procedures described by Hop-
kins et al (25), with minor modifications. The initial nucleic acid
concentrations were measured spectrophotometrically at 260 nm
(an absorption at 260 nm of 1.0 corresponded to an RNA con-
centration of 40 g/mL). The RNA was then denatured in a 0.5 (by
vol) glutaraldehyde solution, diluted to 1.5 ng/L, and blotted onto
Hybond XL nylon hybridization membranes (Amersham Phar-
macia Biotech Inc, Bucks, United Kingdom). Oligonucleotide
probes were used to quantify the abundance of RNA from bi-
fidobacteria [Bif1412: 5'-CCGGTTTTMAGGGATCC-3' (26)],
enterobacteria [Entero 1418: 5'-CTTTTGCARCCCACT-3'
(27)], the BPP group [Bacto1080: 5'-GCACTTAAGCCGAC-
ACCT-3' (24)], and lactic acid bacteria [Lacto 722: 5'-YCA-
CCGCTACACATGRAGTTCCACT-3' (28)], and the values
obtained were expressed as a percentage of the total bacterial
rRNA, which was quantified with the use of the probe [Eub338:
5'-GCTGCCTCCCGTAGGAGT-3' (29)]. The sequences were
5' end-labeled with 32P with the use of ␥-32P ATP and purified
1787
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with the use of Microspin G-25 columns (Amersham Pharmacia
Biotech Inc). The membranes were hybridized with the labeled
probes overnight, before the wash procedure at the designated
temperature for each probe. RNA abundance was quantified by
measuring the 32P signal with the use of an Instant Imager
(Canberra Packard, Pangbourne, Berks, United Kingdom) and
IMAGEQUANT software (Molecular Dynamics, Sunnyvale,
CA). Reference concentrations of RNA representative of the
bacterial group were also run on each gel, and standard curves
were calculated by linear regression to measure group-specific
RNA.
Diarrhea incidence and stool characteristics
Diarrhea or vomiting episodes were recorded as separate ill-
nesses. Diarrhea was defined as a discrete illness lasting 쏜48 h
(2, 30) with 쏜3 loose stools in a 24-h period (5) and distinguished
from chronic diarrheal disease, such as intolerance to cow milk
or malabsorption (30). Mothers also recorded the stool consis-
tency for 3 d before each home visit according to Weaver et al
(31).
Statistical analysis
Our a priori outcomes were the effect of nucleotide supple-
mentation on the ratio of BPP to Bifidobacterium spp. rRNA and
the incidence of diarrhea. Diarrheal illness was assessed both as
the total number of diarrheal episodes and as the proportion of
infants with 욷1 episode of diarrhea from birth to 20 wk of age.
Sample size was initially calculated to detect a 0.5 SD difference
in the number of diarrheal episodes between randomized
formula-fed groups with 80% power at 5% significance. How-
ever, successful recruitment meant that the trial was continued
1788 SINGHAL ET AL

TABLE 2
Subject characteristics

Randomized formula-fed groups1

Control Nucleotide supplemented Breastfed reference P for comparison of 3

Variable (n ҃ 100) (n ҃ 100) (n ҃ 101) dietary groups

At birth
Male sex [n (%)] 56 (56) 61 (61) 51 (51) 0.42
Weight (kg) 3.5 앐 0.53 3.5 앐 0.6 3.5 앐 0.5 0.94
Weight z score 0.3 앐 1.0 0.1 앐 1.2 0.1 앐 1.0 0.64
Gestation (wk) 39.2 앐 1.3 39.5 앐 1.4 39.6 앐 1.1 0.14
Social class: nonmanual [n (%)] 39 (39) 41 (41) 81 (81) 쏝 0.0012
Mothers with degree [n (%)] 8 (8) 8 (8) 52 (52) 쏝 0.0012
Cesarean delivery [n (%)] 40 (40) 32 (32) 31 (31) 0.42
At randomization
Age (d) 7.5 앐 2.8 7.1 앐 2.6 —
Weight (kg) 3.4 앐 0.5 3.4 앐 0.6 —
Weight z score Ҁ0.6 앐 1.0 Ҁ0.5 앐 1.2 —
Length (cm) 50.3 앐 2.2 50.5 앐 2.8 —
Length z score Ҁ0.7 앐 1.1 Ҁ0.5 앐 1.4 —
Head circumference (cm) 35.2 앐 1.3 35.3 앐 1.5 —

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Head circumference z score Ҁ0.3 앐 1.0 Ҁ0.1 앐 1.1 —
1
There were no significant differences between the randomly assigned formula-fed groups analyzed with the Student’s t test and the chi-square test.
2
Determined by the chi-square test. There was a slight loss of n for some variables (쏝2%).
3
x៮ 앐 SD (all such values).
4
Determined by ANOVA.

beyond that originally planned to give a power of 0.4 SD differ-
ence in outcomes between the randomized groups at 80% power
and P 쏝 0.05.
Randomized formula-fed groups were compared with the Stu-
dent’s t test for normally distributed variables, the Mann-
Whitney U test for not-normally distributed variables, and the
chi-square test for dichotomous variables. The ratio of BPP to
Bifidobacterium spp. was loge transformed and then multiplied
by 100 before statistical analyses (32). Therefore, for 100 loge-
transformed data the SD represents the CV, and the difference in
means between randomized groups represented the percentage
difference between groups (32). Multiple linear regression was
used to adjust for confounding factors that could potentially
affect stool microbial pattern (sex, age at stool analysis, age at
which complementary foods were introduced, socioeconomic
status, and mother’s education). Values for 16S rRNA from the
4 different groups of bacteria quantified in stool (expressed as a
percentage of total bacterial rRNA) could not be transformed to
normality; hence, nonparametric statistics were used.
In a secondary analysis, the ratio of BPP to Bifidobacterium spp.
in breastfed infants was compared with the 2 formula-fed groups
with the use of one-factor analysis of variance and Bonferroni cor-
rections. Statistical analyses were conducted with the use of SPSS
for WINDOWS (version 12.0; SPSS Inc, Chicago, IL).
RESULTS
Of the 301 infants recruited into this study, 88 of 100 nucleotide-
supplemented, 85 of 100 control formula-fed, and 96 of 101 breast-
fed infants were followed at age 5 mo (Figure 1). Infants dropped
out of the study usually for reasons that were not given or were
nonclinical, social reasons. Only 5 infants (4 from the control group
and 1 from the nucleotide formula group) dropped out because of
perceived problems with the milk such as vomiting (n ҃ 3) or
apparent hunger (n ҃ 2). These infants were changed by their moth-
ers to formulas completely different from those used in the study.
Mothers of infants dropping out of the study did not continue to
collect clinical data, allow follow-up visits, or provide stool samples,
so they were not included in the analyses at age 20 wk. The 2 trial
formulas were well tolerated, and the time spent crying, incidence of
colic, nighttime wake periods, and use of pacifiers did not signifi-
cantly differ between randomized groups (data not shown). No in-
fant from any dietary group had a diagnosis of chronic diarrhea or
other gastrointestinal illness.
Nucleotide-supplemented and control formula-fed groups
were closely matched for age at assessment, sex, socioeconomic
status, percentage of children born by cesarean delivery, and
anthropometric variables (Table 2). The age at which compli-
mentary feeding was started and the use of antibiotics during the
follow-up period were also not significantly different between
randomized formula-fed groups (Table 2). As expected, mothers
of breastfed infants were better educated and of a higher socio-
economic status than were the mothers of formula-fed infants
(data not shown). More breastfed infants completed the study
than did formula-fed infants, but no significant difference was
observed in the completion rate between nucleotide-
supplemented or control formula-fed infants (Table 3).
Stool microbiology
A stool sample was collected from 116 (43%) of 269 infants
remaining in the study at 20 wk of age. Formula-fed infants who
provided a stool sample (n ҃ 72) compared with infants who did not
(n ҃ 128) had a higher proportion of girls, but they did not differ
significantly in age, gestation, randomized formula-fed group, an-
thropometric characteristics at birth, socioeconomic status, and
mother’s education (data not shown). Nucleotide-fed infants (n ҃
37) had a lower ratio of fecal BPP to bifidobacterial rRNA than did
controls (n ҃ 35) (mean difference: Ҁ118%; 95% CI: Ҁ203%,
 NUCLEOTIDES AND FECAL MICROBIOTA 1789
TABLE 3
Stool bacteriology and episodes of diarrhea1

Formula-fed groups2

P for comparison P4 for comparison

Control Nucleotide supplemented between formula- Breastfed reference of 3 dietary
Variable (n ҃ 100) (n ҃ 100) fed groups3 (n ҃ 101) groups

Completed study [n (%)] 85 (85) 88 (88) 0.45 96 (96) 0.075

Age at final visit and stool collection 20.5 앐 1.36 20.4 앐 1.4 0.6 20.3 앐 1.4 0.8
(wk)
Age at which complementary foods 14.0 앐 2.3 13.6 앐 2.3 0.3 15.4 앐 1.8 쏝 0.001a,b
introduced (mo)
Courses of antibiotic [n (%)] 0.25 0.45
1 14 (14) 21 (21) 12 (12)
2 5 (5) 12 (12) 2 (2)
3 1 (1) 1 (1) 1 (1)
4 0 (0) 1 (1) 0 (0)
Fecal bacteriology7
Ratio of BPP to bifidobacteria8 20.2 (158) 6.2 (187) 0.007 3.6 (146) 쏝 0.001a,c
BPP (%)9 34.6 (20.2) 29.1 (31.9) 0.0210 17.5 (24.3) 0.00111
Bifidobacteria (%)9 1.8 (2.9) 2.8 (6.5) 0.0910 4.9 (6.7) 0.00311

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Enterobacteria (%)9 0.7 (0.3) 0.8 (0.3) 0.210 0.6 (0.2) 0.111
Lactic acid bacteria (%)4 0.5 (1.0) 0.5 (1.0) 0.310 1.0 (2.3) 0.0111
Episodes of diarrhea
No. by age 20 wk 0 (0–3)12 0 (0–10) 0.210 0 (0–2) 0.0111
쏜1 episode of diarrhea by age 20 26 (31) 33 (38) 0.25 18 (19) 0.025
wk [n (%)]
Stool consistency by age13
8 wk 3.2 앐 0.5 3.0 앐 0.5 0.03 2.4 앐 0.6 쏝 0.001a,b
16 wk 3.3 앐 0.5 3.1 앐 0.5 0.09 3.1 앐 0.6 쏝 0.001a,b
20 wk 3.5 앐 0.7 3.4 앐 0.6 0.5 2.2 앐 0.4 0.016d
Stool frequency by age (movements/d)
8 wk 1.5 앐 1.0 1.6 앐 1.1 0.7 4.2 앐 2.3 쏝 0.001a,b
16 wk 1.4 앐 0.7 1.5 앐 0.9 0.3 2.1 앐 1.4 쏝 0.001a,b
20 wk 1.4 앐 0.8 1.5 앐 0.7 0.6 1.7 앐 1.0 0.02e
1
BPP, Bacteroides-Porphyromonas-Prevotella group. There was a slight loss of n for some variables (쏝2%).
2
Infants were randomly assigned to formula groups.
3
Analyzed with the Student’s t test.
4
Determined by ANOVA. Values with different superscript letters are post hoc comparisons with Bonferroni’s test; breastfed compared with control
formula-fed infants: a P 쏝 0.001, d P ҃ 0.02, e P ҃ 0.03; breastfed compared with nucleotide-supplemented formula-fed infants b P 쏝 0.001; nucleotide-
supplemented compared with control formula-fed infants: c P ҃ 0.01.
5
Determined by the chi-square test.
6
x៮ 앐 SD (all such values).
7
Stool microbiota were assessed in a subset of infants: 35 control formula-fed, 37 nucleotide-supplemented formula-fed, and 44 breastfed.
8
Values are geometric mean; CV in parentheses.
9
Values are median; interquartile range in parentheses.
10
Determined by the Mann-Whitney U test.
11
Determined by the Kruskal-Wallis test.
12
Median; range in parentheses (all such values).
13
Analyzed as hard (code 5), mushy soft (code 4), formed soft (code 3), runny (code 2), or watery (code 1).

Ҁ34%; P ҃ 0.007). This difference remained significant after ad-
justment of potential confounding factors (age, sex, age at which
complementary feeds were introduced, socioeconomic status, and
mother’s education) (mean difference: Ҁ131%; 95% CI: Ҁ217%,
Ҁ45%; P ҃ 0.003). The abundance of BPP was significantly lower
in nucleotide-supplemented formula-fed infants than in controls
(Table 3). However, the abundance of bifidobacteria, lactic acid
bacteria, and enterobacteria did not differ significantly between ran-
domized formula-fed groups (Table 3).
In a secondary analysis that used analysis of variance, the ratio
of BPP to Bifidobacterium spp. RNA statistically differed in the
3 dietary groups (P 쏝 0.001) and between the 2 randomized
formula-fed groups (P ҃ 0.01). The ratio of BPP to bifidobac-
teria in breastfed infants was lower than in infants fed control
formula (P 쏝 0.001), but it was not significantly different from
infants assigned to the nucleotide-supplemented formula (P ҃
0.5). In the whole study population, the percentage of rRNA from
bifidobacteria correlated with both BPP (r ҃ Ҁ0.3, P ҃ 0.004)
and lactic acid bacteria (r ҃ 0.2, P ҃ 0.04).
Diarrhea incidence, stool frequency, and stool
characteristics
The total number of diarrheal episodes or presence of 욷1
episode up to 20 wk of age did not differ significantly between
1790 SINGHAL ET AL
randomized formula-fed groups (Table 3). Nucleotide-
supplemented infants produced softer stools at 8 wk of age but
not at 16 or 20 wk of age, but stool frequency did not differ
significantly between randomly assigned formula-fed infants at
any age. As expected, breastfed infants had significantly fewer
diarrheal episodes during the first 20 wk of age and also between
8 and 20 wk of age (during which comparable, prospective col-
lected data were available in both formula-fed and breastfed
infants) (data not presented). Breastfed infants also had a higher
stool frequency and softer stools than did formula-fed infants
(Table 3). The ratio of BPP to Bifidobacterium spp. did not
significantly correlate with the number of episodes of diarrhea in
the whole study population and when the analysis was confined
to formula-fed infants.
DISCUSSION
The addition of nucleotides to infant formula was suggested to
have beneficial effects on health (3–12), but relatively few ex-
perimental studies have tested this hypothesis in humans. In a
prospective randomized trial, we found that supplementation of
infant formula with 31 mg nucleotides/L had advantages for the
balance of intestinal microbiota as measured by the ratio of BPP
to Bifidobacterium spp. Bifidobacterium spp. predominate in the
large intestine of breastfed infants (where they are suggested to
confer health benefits by protecting against infection from en-
teropathogenic organisms), whereas formula-fed infants are
more frequently colonized by higher amounts of species such as
BPP (19). Our data therefore support the hypothesis that nucle-
otides have a prebiotic effect on colonic microbiology (10) and
that higher concentrations of nucleotides in human milk could
contribute to the gastrointestinal benefits of breastfeeding com-
pared with formula feeding. Consequently, the addition of nu-
cleotides to infant formulas is probably important for optimizing
the diet of formula-fed infants.
Only 2 previous studies have investigated the effect of dietary
nucleotides on stool microbiota in infants. Gil et al (20) found
that infants fed nucleotide-supplemented formula (n ҃ 11) had a
higher percentage of Bifidobacterium spp. in stools at 4 wk of age
than did controls (n ҃ 12). In contrast, a larger and more recent
study showed that bifidobacterial counts were reduced in
nucleotide-supplemented (n ҃ 32) compared with control
formula-fed (n ҃ 33) infants at 2 wk of age (21). Nonrandom
dietary assignment, difficulties in accurate quantification of stool
bacterial counts with the use of culture techniques, differences in
both age at which the fecal microflora was assessed, and the
amount of nucleotide supplementation could all potentially ex-
plain these discrepant findings. Difficulties in culturing some
BPP and bifidobacteria may also be important in the accurate
assessment of fecal microflora in infancy (19). However, unlike
previous reports, we investigated the effect of nucleotides on
stool microbiology with the use of an experimental design and
molecular rather than culture techniques. In accordance with
earlier in vitro (13–15) and clinical studies (20), we found that
nucleotide supplementation of infant formula was associated
with a stool microbial composition more like that of a breastfed
infant. Indeed, in terms of the equilibrium between BPP and
bifidobacteria, the fecal microbial composition of breastfed in-
fants did not significantly differ from infants given nucleotide-
supplemented formula, but it was markedly different from in-
fants given control formula.
In contrast to previous reports (3– 6), we did not find an effect
of nucleotide supplementation on the incidence of diarrhea. One
possible explanation for this is that, unlike findings from a de-
veloping country (3), a low incidence of diarrhea in a more-
developed setting could have reduced our study’s power to detect
small but clinically important differences between nucleotide-
supplemented and control formula-fed infants. Another potential
explanation is a low concentration of nucleotides (31 mg /L) in
our supplemented formula, which, although in line with Euro-
pean regulations (울33.5 mg/L) (23), was lower than that used in
most randomized studies that showed a benefit of nucleotides on
diarrheal illness (72 mg /L) (4 – 6). The latter concentration is an
estimate of both free and enzymatically liberated nucleosides in
human milk (ie, total potentially available nucleosides) and is
higher than the free nucleotide and nucleoside content alone (11,
33). Our study therefore supports the hypothesis that nucleotide
supplementation of formula to a higher concentration, more sim-
ilar to the total available to breastfed infants, is required for a
protective effect against diarrhea. Theoretically, differences in
the pattern of nucleotide supplementation could also explain
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differences in study findings, although, to our knowledge, no
evidence supports this hypothesis.
We considered a number of study limitations. First, we did not
measure live bacterial counts and, like most studies that used 16S
rRNA, we could only estimate the proportions of bacterial groups
and not specific species or strains. Our study therefore makes the
assumption that the group-specific percentage of bacterial rRNA
in feces reflects the viable bacterial counts in the colon. In addi-
tion, changes in abundance of fecal bacterial population ex-
pressed as a proportion of total rRNA could represent shifts in
total community composition and ribosomal abundance and not
necessarily changes in absolute amounts (25). Nevertheless, 16S
rRNA measurements were shown to correlate well with viable
bacterial counts (25) and to provide a good measure of the gut
microbial composition, which is associated with both neonatal
intestinal health (18, 22) and infant feeding (19). Furthermore,
the previous observation that nucleotides may affect the relative
proportions of bifidobacteria rather than absolute amounts (20)
supports our assessment in terms of microbial patterns, rather
than absolute bacterial counts.
Second, the effect of nucleotides on bifidobacteria did not
reach statistical significance, possibly because the percentage of
rRNA from Bifidobacterium spp. in our infants aged 앒143 d was
lower than that seen previously in infants aged 쏝 20 d (19), but
more similar to amounts seen in older infants (85–225 d) (24) or
adults (쏝1%) (34). However, this difference was not unexpected
in view of the fall in fecal bifidobacterial numbers that occurs
with the introduction of solid feeding and with the transition from
infant to adult stool microbiota in the first year of life (16 –18).
Furthermore, the percentage of BPP rRNA in the present study
was similar to a previous report (앒30%) (24), which supports the
validity of our 16S rRNA assay. We assessed fecal microbiology
at 5 mo of age to allow the investigation of associations between
nucleotide supplementation, diarrheal disease, and fecal micro-
biology. Nonetheless, the advantage of nucleotide supplementa-
tion for fecal microbiology was independent of the age at which
complementary foods were introduced, which itself did not differ
between nucleotide-supplemented or control formula-fed in-
fants. The fact that differences between randomized formula-fed
 NUCLEOTIDES AND FECAL MICROBIOTA
groups were seen in older infants, after the introduction of com-
plementary foods and therefore large amounts of dietary nucle-
otides, raises the possibility that the effect of nucleotides on fecal
microflora could be even greater in neonates, a hypothesis that
requires further testing.
Finally, our study could not address mechanisms. Although
쏜90% of ingested nucleotides are absorbed as nucleosides in
the upper intestine (7, 8), some probably pass into the colon,
where they could act as cofactors for the growth of bifidobac-
teria. For instance, the separate addition of adenosine mono-
phosphate, CMP, guanosine monophosphate, uridine mono-
phosphate, and inosine monophosphate was shown to
stimulate the in vitro growth of bifidobacteria, whereas the
simultaneous addition of these nucleotides had an even
greater effect (15). Thus, nucleotide supplementation could
have a direct nutritional or prebiotic effect as suggested pre-
viously (10) and as supported by findings from in vitro studies
(13–15). Dietary nucleotides were also suggested to promote
intestinal mucosal and epithelial growth (9), which could
provide another nutrient source for colonic bacteria (22). Fi-
nally, nucleotides could directly inhibit the growth of specific
bacterial groups (eg, BPP), thereby altering the overall mi-
crobial pattern, although little evidence supports this hypoth-
esis. A more likely explanation for the lower percentage of
BPP in nucleotide-supplemented infants is the inhibition of
BPP growth by the bifidobacteria-mediated increase in intes-
tinal acidity (7–9), a hypothesis supported by the negative
correlation between the percentage of rRNA from BPP and
Bifidobacterium spp. in the present study.
Evidence that the neonatal gut microbiota have long-term
effects on immune function (22) and atopic disease (35) raises
the possibility that the benefits of nucleotide supplementation
of infant formula for immune function could be mediated in
part through an effect on the gut microbiota (4, 10). Further
research is required to test this hypothesis and the potential
long-term benefits for nucleotide supplementation of infant
formula.
We thank Helen Clough and Wendy Jenkins for technical assistance and
the families who participated in this study.
The author’s responsibilities were as follows—AS (principal investiga-
tor): was the main author; GM and SM: provided expertise in the assessment
of fecal microbiota; JL and KK: supervised data collection. All authors
contributed to study design and preparation of the final manuscript for sub-
mission. None of the authors had a personal or financial conflict of interest.
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