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Influence of Milk-Feeding Type and Genetic Risk of
Developing Coeliac Disease on Intestinal Microbiota of
Infants: The PROFICEL Study
Giada De Palma
1
, Amalia Capilla
2
, Esther Nova
3
, Gemma Castillejo
4
, Vicente Varea
5
, Tamara Pozo
3
, Jose
´
Antonio Garrote
6
, Isabel Polanco
7
, Ana Lo
´pez
8
, Carmen Ribes-Koninckx
8
, Ascensio
´n Marcos
3
, Marı
´a
Dolores Garcı
´a-Novo
9
, Carmen Calvo
6
, Luis Ortigosa
10
, Luis Pen
˜a-Quintana
11
, Francesc Palau
2
, Yolanda
Sanz
1
*
1Instituto de Agroquı
´mica y Tecnologı
´a de Alimentos, Consejo Superior de Investigaciones Cientı
´ficas (IATA-CSIC), Valencia, Spain, 2Instituto de Biomedicina de Valencia
(CSIC), CIBER de Enfermedades Raras (CIBERER), Valencia, Spain, 3Department Metabolismo y Nutricio
´n, ICTAN-CSIC, Madrid, Spain, 4Unidad de Gastroenterologı
´a
Pedia
´trica, Hospital Universitario Sant Joan de Reus, Tarragona, Spain, 5Gastroenterologı
´a, Nutricio
´n y Hepatologı
´a Pedia
´trica, Hospital Universitario Sant Joan de Deu and
Unidad de Gastroenterologı
´aPedia
´trica del Institut Dexeus, Barcelona, Spain, 6Unidad de Gastroenterologı
´a Pedia
´trica, Hospital Clı
´nico Universitario de Valladolid,
Valladolid, Spain, 7Servicio de Gastroenterologı
´a y Nutricio
´n Pedia
´trica, Hospital Universitario La Paz, Madrid, Spain, 8Unidad de Gastroenterologı
´a Pedia
´trica, Hospital
Universitario La Fe, Valencia, Spain, 9Unidad de Gastroenterologı
´a, Hospital Universitario Infantil Nin
˜o Jesu
´s, Madrid, Spain, 10 Unidad de Gastroenterologı
´a, Hepatologı
´a
y Nutricio
´nPedia
´trica, Hospital Universitario Nuestra Sen
˜ora de Candelaria, Santa Cruz de Tenerife, Canarias, Spain, 11 Unidad de Gastroenterologı
´a, Hepatologı
´ay
Nutricio
´n Pedia
´trica, Hospital Universitario Materno-Infantil de Canarias, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain
Abstract
Interactions between environmental factors and predisposing genes could be involved in the development of coeliac
disease (CD). This study has assessed whether milk-feeding type and HLA-genotype influence the intestinal microbiota
composition of infants with a family history of CD. The study included 164 healthy newborns, with at least one first-degree
relative with CD, classified according to their HLA-DQ genotype by PCR-SSP DQB1 and DQA1 typing. Faecal microbiota was
analysed by quantitative PCR at 7 days, and at 1 and 4 months of age. Significant interactions between milk-feeding type
and HLA-DQ genotype on bacterial numbers were not detected by applying a linear mixed-model analysis for repeated
measures. In the whole population, breast-feeding promoted colonization of C. leptum group, B. longum and B. breve, while
formula-feeding promoted that of Bacteroides fragilis group, C. coccoides-E. rectale group, E. coli and B. lactis. Moreover,
increased numbers of B. fragilis group and Staphylococcus spp., and reduced numbers of Bifidobacterium spp. and B. longum
were detected in infants with increased genetic risk of developing CD. Analyses within subgroups of either breast-fed or
formula-fed infants indicated that in both cases increased risk of CD was associated with lower numbers of B. longum and/or
Bifidobacterium spp. In addition, in breast-fed infants the increased genetic risk of developing CD was associated with
increased C. leptum group numbers, while in formula-fed infants it was associated with increased Staphylococcus and B.
fragilis group numbers. Overall, milk-feeding type in conjunction with HLA-DQ genotype play a role in establishing infants’
gut microbiota; moreover, breast-feeding reduced the genotype-related differences in microbiota composition, which could
partly explain the protective role attributed to breast milk in this disorder.
Citation: De Palma G, Capilla A, Nova E, Castillejo G, Varea V, et al. (2012) Influence of Milk-Feeding Type and Genetic Risk of Developing Coeliac Disease on
Intestinal Microbiota of Infants: The PROFICEL Study. PLoS ONE 7(2): e30791. doi:10.1371/journal.pone.0030791
Editor: Markus M. Heimesaat, Charite
´- Campus Benjamin Franklin, Germany
Received November 23, 2011; Accepted December 29, 2011; Published February 3, 2012
Copyright: ß2012 De Palma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by public grants AGL2007-66126-C03-01-03/ALI and Consolider Fun-C-Food CSD2007-00063 from the Spanish Ministry of
Science and Innovation. GDP was recipient of I3P scholarship from Consejo Superior de Investigaciones Cientı
´ficas (CSIC), Spain. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: yolsanz@iata.csic.es
Introduction
Celiac disease (CD) is a chronic inflammatory disorder of the small
intestine that presents in genetically predisposed individuals following
gluten consumption [1]. This disease often manifests in early childhood
with small intestinal villous atrophy and signs of malabsorption [1].
Currently, a strict gluten-free diet is the only available treatment for
patients but compliance with this dietary practice is extremely complex
and, therefore, preventive strategies are being investigated [2].
The major genetic risk factor in CD is represented by Human
Leukocyte Antigen (HLA)-DQ genes. Several studies have
documented that the HLA-DQA1*05 and DQB1*02 alleles,
encoding for particular DQ2 molecules, confer high susceptibility
to CD [3]. This heterodimer can be encoded both in cis or trans
forms. The susceptibility to CD is increased in homozygous
subjects with a cis haplotype or possessing a second HLA-
DQB1*02 allele [4]. In Europe, approximately 90% of patients
have these genetic markers, whereas most of the remaining cases
carry the HLA-DQA1*03 and DQB1*0302 alleles coding for
DQ8 molecules [5]. Gluten is the main environmental factor
responsible for the signs and symptoms of the disease but other
environmental elements are also thought to play a role in the
PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e30791
disease risk, including the type of milk-feeding, incidence of
infections and intestinal dysbiosis [6–8].
Gut colonization starts immediately after birth and depends on
multiple factors such as the type of delivery, contamination from
the environment, the type of milk-feeding and, possibly, the
genotype [9–11]. An adequate gut microbial colonization process
contributes to the physiological development of the gut and the
maturation of the immune system, thereby determining the risk of
developing disease later in life. The early stage of colonization is
characterized by the presence of higher levels of facultative
anaerobes (enterobacteria, enterococci and streptococci) than of
strictly anaerobic bacteria (e.g. bifidobacteria, bacteroides, clos-
tridia, etc.); however, these proportions are reversed within a week
following birth. In breast-fed infants Bifidobacterium spp. predom-
inate, representing up to 90% of the total faecal microbiota,
whereas in formula-fed infants the microbiota is more heteroge-
neous [9,12,13]. Breast-milk has been shown to be a continuous
source of commensal bacteria to the infant gut, including species of
the genera Lactobacillus and Bifidobacterium [14,15]. It also contains
prebiotic substances which are considered the main factors that
stimulate the growth of Bifidobacterium spp. [16]. Epidemiological
studies suggest that breast-feeding confers a protective effect
against the risk of CD development, particularly when gluten is
introduced in the diet while the infant is still breast-fed [1,17].
However, the mechanisms underlying the beneficial effects of
breast-milk on CD risk and their relationship with the gut
microbiota are unknown. A preliminary study was previously
conducted to test whether the HLA-DQ genotype could influence
the composition of the gut microbiota, although the population
under study was composed by a small number of exclusively
breast-fed infants [18] and the aim was to establish the basis for
this long-term study, including a representative cohort of infants.
The objective of this study was to assess the gut microbial
colonization process during the first 4 months of life in breast-fed
and formula-fed babies at risk of developing CD, by using
quantitative PCR (qPCR). The ultimate purpose of our research is
to gain a better understanding of the effects of early events leading
to the acquisition of intestinal microbiota and their interactions
with predisposing genes on CD risk.
Methods
Subjects and study design
A prospective observational study was carried out with a cohort
of 164 healthy full-term newborns recruited between June 2006
and November 2010, who had a first-degree relative affected by
CD. Data of mode of delivery, size, weight, weeks of gestation and
type of feeding were recorded at birth and over the study period
(Table 1). Infants were grouped for factors that influence the gut
microbial colonization process, including age, genetic risk of CD
development (HLA-DQ status), and type of milk-feeding (admitted
randomly). The infants classified into the breast-fed group were
those that received exclusively breast-feeding during the first 7
days of life, during the first month of life or during the first 4
months of life. Infants classified into the formula-fed group were
those that received either exclusively formula or both formula and
breast-milk at each sampling time. Infants were also grouped
according the duration of breast-feeding (never breast-fed, breast-
fed less than 1 month, breast-fed more than 1 month but less than
4 months and breast-fed for the 4 months). The study was
approved by the ethics committees of Consejo Superior de
Investigaciones Cientı
´ficas (CSIC) and the Hospitals involved in
the study, including Hospital Universitario Sant Joan de Reus,
Hospital Universitario Sant Joan de Deu, Institut Dexeus, Hospital
Clı
´nico Universitario de Valladolid, Hospital Universitario La Paz,
Hospital Infantil Universitario La Fe, Hospital Universitario
Infantil Nin˜o Jesu´s, Hospital Universitario Ntra Sen˜ora de
Candelaria and Hospital Universitario Materno-Infantil de
Canarias and conducted in accordance with the Helsinki
Declaration of 1975 as revised in 1983. Written informed consent
was obtained from the parents of infants included in the study.
HLA-DQ Genotyping
DNA was extracted from yugal mucosa cells by scraping the
inner side of the infants’ cheek with sterile swabs (Copan
innovation, Sarstedt, Germany) and purified according to the
DNA IQ
TM
Casework Sample Kit for MaxwellH16 protocol
(Promega Biotech Iberica, Spain). Low-resolution HLA-DQB1
typing was performed by PCR-SSP (Polymerase Chain Reaction-
Sequence Specific Primers) analysis [19]. Each PCR reaction was
performed on about 20 ng of extracted DNA, 0.5 U of DNA
polymerase (BIOTOOLS B&M S.A, Spain), 16PCR Master Mix
(Dynal AllSet
+TM
SSP or Olerup SSP
TM
) containing nucleotides
(200 mmol L
21
each), PCR buffer, 5% glycerol and 100 mgmL
21
cresol red, 0?25 mmol L
21
of each allele- or group-specific primer
pair and 0?1mmol L
21
of internal positive control primer pair
matching a segment of the human growth hormone gene in a final
volume of 10 mL. An initial denaturation step at 94uC for 2 min
was followed by 10 two-temperature cycles (94uC for 10 s and
65uC for 60 s) and 20 three-temperature cycles (94uC for 10 s,
61uC for 50 s and 72uC for 30 s). Detection of amplified alleles
was carried out on 2% agarose gels after ethidium bromide
staining. HLA-DQA1 alleles were genotyped in a stepwise fashion
Table 1. Demographic data of infants under study.
Demographic data Total infants (n = 164)
Delivery
Vaginal 115/164
Caesarean 49/164
1
Size (cm) 49.97 (2.42)
1
Weight (g) 3331.08 (537.49)
1
Gestation (weeks) 39.10 (1.44)
2
Breast feeding
7 days 115
1 month 109
4 months 64
3
Formula feeding
7 days 44
1 month 55
4 months 78
4
Genetic risk of CD
High genetic risk 48
Intermediate genetic risk 69
Low genetic risk 47
1
Data are expressed as mean and standard deviation in brackets.
2
Infants who were exclusively breast-fed at each sampling time were included
in the breast-feeding group.
3
Infants who received either exclusively formula or both formula and breast-
milk were included in the formula-feeding group.
4
Genetic risk of developing CD was established according to the HLA-DQ
genotype (see Materials and Methods section for details).
doi:10.1371/journal.pone.0030791.t001
Gut Microbial Colonization and Coeliac Disease
PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e30791
for high resolution typing to hone the risk classification of each
individual.
Faecal sampling and DNA extractions
Stool samples were collected from every subject at 7 days, 1
month and 4 months of age and frozen at 220uC immediately.
Samples (1 g) were diluted 1:10 (w/v) in PBS (pH 7.2) and
homogenized by thorough agitation in a vortex. Aliquots were
used for DNA extraction using the QIAamp DNA stool Mini kit
(Qiagen, Hilden, Germany) following the manufacturer’s instruc-
tions. DNA extractions from different pure cultures of reference
strains were done following the same protocol.
Quantitative PCR (qPCR) analysis of faecal bacteria
qPCR was used to quantify the different bacterial groups of the
faecal microbiota using genus-, group- and species-specific primers
as previously described [20,21]. Briefly, PCR amplification and
detection were performed with an ABI PRISM 7000-PCR
sequence detection system (Applied Biosystems, UK) using SYBRH
Green PCR Master Mix (SuperArray Bioscience Corporation,
USA). The bacterial concentration from each sample was
calculated by comparing the Ct values obtained from standard
curves of reference strains. Standard curves were created using
serial 10-fold dilutions of pure culture DNA corresponding to 10
2
to 10
9
cells, as determined by microscopy counts after staining
with 49, 6 diamino-2-phenylindole in an epifluorescence micro-
scope (Olympus BX51, Tokio, Japan).
Statistical analyses
Data were analysed using the SPSS 19.0 software for Windows
(SPSS Inc, Chicago, IL, USA). The demographic characteristics of
the study subjects are given as mean values (standard deviations
[SDs]) for continuous variables and as numbers and proportions
for categorical variables. Differences in demographic characteristic
measures between the study groups were assessed using the Chi-
Square Pearson test for categorical variables and ANOVA and post
hoc LSD test for continuous variables. Microbiological data were
transformed from exponential numbers into logarithms to adjust
to normal and, in the tables, are expressed as mean values of log
cells/g faeces and standard deviations (SDs). A mixed model with
repeated measures with three fixed factors [genetic risk, type of
milk-feeding and age (repeated measure)] was applied to
determine the effects of genetic risk and type of feeding on
bacterial counts. Interactions (magnitudinal) between these three
factors were also analysed and not detected. Interactions between
faecal microbial counts and type of delivery were not found and,
therefore, data from infants with different type of delivery were
grouped for statistical analyses. Differences between bacterial
numbers at each sampling time (age) were analysed by ANOVA
and post hoc LSD test. Correlations between factors (bacterial
counts, age and genetic risk) were determined by Pearson
correlation coefficients and correlations between bacterial counts
and type of milk-feeding were tested by applying the Chi-Square
Pearson test. In all cases, p-values less than 0.050 were considered
statistically significant.
Results
Subjects and genetic risk of CD
The demographic characteristics of the infants under study are
shown in Table 1. All newborns were full-term and most were
delivered naturally (115 of 164). The size and weight of the infants
at the moment of delivery were within standard parameters.
Infants were classified into three main groups according to their
HLA-DQ genotype, and probabilities of developing CD were
estimated according to previous studies [22,23]. The first group
included those individuals carrying the DQ2 haplotype in both cis
(DQA1*0501-DQB1*0201 in homozygosis) and trans conforma-
tions (DQA1*0201-DQB1*0202 with DQA1*0505-DQB1*0301
in heterozygosis). The second group included those subjects
carrying the DQ2 haplotype in cis conformation along with any
other haplotype, as well as subjects carrying the DQ8 haplotype
(DQA1*0301-DQB1*0302) in homozygosis. The third group
included those individuals with other common genotypes not
associated with CD. Of the 164 infants under study, 28.40% were
classified in the first risk group, with the highest probability
(.20%) of developing CD (the high genetic risk group) and
42.59% in the second group, with a .7% probability of
developing CD (the intermediate genetic risk group). The
remaining 29.01% of infants comprised the third group, with
the lowest probability (,1%) of developing CD (the low genetic
risk group).
Influence of milk-feeding type on faecal microbiota of
infants at risk of developing CD
The effect of milk-feeding type on the composition of the faecal
microbiota, irrespectively of genotype, was evaluated over the
study period. The type of milk feeding significantly influence
different bacterial group counts by analysing data with the linear
mixed model with time sampling as the repeated measure. C.
leptum group (P= 0.005), B. longum (P= 0.050), and B. breve
(P= 0.008) numbers were significantly higher in breast-fed than
in formula-fed infants, whereas Bacteroides fragilis group (P= 0.004),
C. coccoides-E. rectale group (P,0.001), E. coli (P = 0.026), and B.
lactis (P= 0.002) numbers were higher in formula-fed infants than
in breast-fed infants.
Correlations between milk-feeding type and several bacterial
group counts at each sampling time were also analysed. Increased
numbers of C. leptum group and B. breve correlated with breast-
feeding at 7 days of age (r = 20.255, P= 0.012; r = 20.197,
P= 0.036, respectively). Moreover, formula-feeding correlated
with increased numbers of E. coli,C. coccoides-E. rectale group and
B. lactis at 1 month of age (r = 0.218, P= 0.006; r = 0.217,
P= 0.011; r = 0.574, P = 0.002, respectively), and with increased
numbers of C. coccoides-E. rectale and Bacteroides fragilis groups at 4
months of age (r = 0.280, P= 0.001; r = 0.267, P= 0.004, respec-
tively).
When analysing the cumulative effect of breast-feeding on the
microbiota composition of 4-month-old infants, statistically
significant negative correlations were established between in-
creased Bacteroides fragilis and C. coccoides-E. rectale group counts and
longer breast-feeding duration (r = 20.218, P= 0.020; r = 20.245,
P = 0.003, respectively).
Influence of HLA-DQ genotype on the fecal microbiota of
infants at risk of developing CD
The influence of HLA-DQ genotype, irrespective of milk-
feeding type, on fecal microbiota composition was established by
applying a linear mixed-model analysis with sampling time as the
repeated measure. According to this analysis, the effect of the
genetic risk on bacterial numbers was significant for Bifidobacterium
spp. (P,0.001) and B. longum (P,0.001), whose numbers increased
when genetic risk of CD decreased. The effect of genetic risk was
also significant for Staphylococcus spp. (P= 0.010) and Bacteroides
fragilis group (P= 0.050), whose counts were higher when infants’
genetic risk was greater. The influence of HLA-DQ genotype on
Gut Microbial Colonization and Coeliac Disease
PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e30791
fecal microbiota composition at each sampling time (7 days, 1 and
4 months) is also shown in Tables 2, 3 and 4.
Correlations between genetic risk and several bacterial group
counts at different infant ages (sampling time) were also
established. Statistically significant correlations were found
between increased numbers of both Bifidobacterium spp.
(r = 0.235, P= 0.007 at 7 days; r = 0.268, P= 0.001 at 1 month;
r = 0.257, P= 0.001 at 4 months) and B. longum (r = 0.209,
P= 0.013 at 7 days; r = 0.207, P= 0.011 at 1 month; r = 0.200,
P= 0.016 at 4 months) and reduced genetic risk during the whole
sampling period. In contrast, increased numbers of Staphylococcus
spp. at 1 and 4 months (r = 20.465, P= 0.001 at 1 month;
r=20.278, P= 0.038 at 4 months) and B. lactis at 4 months
(r = 20.332, P= 0.026) correlated with increased genetic risk.
Influence of the HLA-DQ genotype in the faecal
microbiota of either breast-fed or formula fed infants
The microbiota composition of infants grouped according to
milk-feeding type was also analysed as a function of HLA-DQ
genotype over the study period to eliminate the effects of milk-
feeding type. In breast-fed infants, the effect of genetic risk of CD
was significant on bacterial counts of Bifidobacterium spp.
(P= 0.046), which decreased when the genetic risk was increased,
and on bacterial counts of Staphylococcus spp. (P= 0.030), C. leptum
group (P= 0.047), B. adolescentis ((P= 0.028) and B. dentium
(P= 0.009), which increased when the genetic risk was also
increased, according to linear mixed-model analysis with sampling
time (age) as the repeated measure. The differences in mean
bacterial numbers according to the genetic risk of developing CD
in breast-fed infants analysed at each sampling point are also
shown in Tables 5, 6 and 7.
Correlations between genetic risk and bacterial counts were also
analysed and these were significant between increased genetic risk
of CD development and increased C. leptum group counts in breast-
fed infants (r = 20.408, P= 0.004).
In formula-fed infants, the effect of genetic risk on Bifidobacterium
spp. and B. longum counts was found significant (P,0.001 and
P,0.001, respectively) by applying a linear mixed model analysis
with sampling time as the repeated measure, and those infants
with reduced genetic risk had increased numbers of these bacterial
groups. The effect of genetic risk on numbers of B. fragilis group
and Staphylococcus spp. was also significant, following the opposite
trend (P= 0.008 and P= 0.004, respectively). The differences in
mean bacterial numbers according to the genetic risk of
developing CD in formula-fed infants analysed at each sampling
point are also shown in Tables 8, 9 and 10.
Statistically significant correlations were also established
between the genetic risk of CD development and bacterial group
numbers in formula-fed infants at different sampling times. At 7
days, reduced numbers of Bifidobacterium spp. correlated with
increased genetic risk of developing CD (r = 0.362, P=0.028). At
1 month, reduced numbers of Bifidobacterium spp. and B. longum
correlated with increased genetic risk of developing CD
(r = 0.511, P,0.001 and r = 0.454, P= 0.001, respectively) , and
increased numbers of Staphylococcus spp. correlated with increased
genetic risk of developing CD (r = 20.573, P= 0.013). At 4
months, reduced numbers of Bifidobacterium spp. and B. longum
correlated with increased genetic risk of developing CD
(r = 0.412, P,0.001 and r = 0.336, P= 0.002, respectively) , and
increased numbers of Staphylococcus spp. and B. lactis correlated
with increased genetic risk (r = 20.345, P= 0.040 and
r=20.442, P= 0.010, respectively).
Table 2. Faecal microbiota of infants with different HLA-DQ genotype at 7 days of age analysed by qPCR.
2
p-value
High risk Intermediate risk Low risk High- Intermediate- High-
n = 39 n = 67 n = 44 Intermediate Low Low
1
Mean SD Mean SD Mean SD Risk Risk Risk
Bacteroides fragilis
group 4.54 1.59 4.32 1.34 4.12 1.49 0.538 0.623 0.352
Staphylococcus
spp. 5.40 0.90 5.34 1.13 4.61 1.11 0.893 0.184 0.200
C. coccoides - E. rectale
group 5.39 1.53 5.20 1.56 5.17 1.46 0.583 0.917 0.548
C. leptum
group 4.36 1.21 4.44 1.47 4.23 1.54 0.841 0.563 0.749
Lactobacillus
group 6.50 1.13 6.69 1.05 6.52 1.02 0.373 0.417 0.911
E. coli
6.19 1.93 5.71 1.93 6.00 1.85 0.258 0.505 0.710
Bifidobacterium
spp. 6.38 1.79 6.99 1.66 7.43 1.42 0.087 0.200 0.007*
B. longum
5.27 1.29 6.12 1.58 6.21 1.72 0.012* 0.774 0.010*
B. breve
5.27 1.47 5.31 1.79 5.14 1.56 0.908 0.643 0.755
B. bifidum
4.47 0.97 4.67 1.45 4.89 1.27 0.523 0.432 0.206
B. adolescentis
4.74 0.79 4.90 1.50 5.23 0.45 0.788 0.575 0.410
B. catenulatum
4.69 1.38 5.41 1.61 5.11 1.85 0.193 0.523 0.487
B. angulatum
3.63 0.50 4.94 0.66 4.43 1.08 0.035* 0.260 0.184
B. infantis
5.62 0.58 5.78 1.91 4.80 0.69 0.819 0.215 0.300
B. lactis
3.91 0.48 3.89 0.55 4.22 0.63 0.959 0.244 0.229
B. dentium
4.42 0.22 4.27 0.61 4.21 - - - -
1
Data are expressed as mean and standard deviation of log cells/g faeces.
2
Statistical significant differences were calculated using ANOVA and post hoc LSD test. Significant differences were established at p,0.050.
doi:10.1371/journal.pone.0030791.t002
Gut Microbial Colonization and Coeliac Disease
PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e30791
Table 3. Faecal microbiota of infants with different HLA-DQ genotype at 1 month of age analysed by qPCR.
2
p-value
High risk Intermediate risk Low risk High- Intermediate- High-
n = 48 n = 69 n = 47 Intermediate Low Low
1
Mean SD Mean SD Mean SD Risk Risk Risk
Bacteroides fragilis
group 5.00 1.93 4.48 1.64 4.60 1.36 0.176 0.760 0.363
Staphylococcus
spp. 6.12 1.09 5.17 1.01 4.66 0.93 0.014* 0.149 0.001*
C. coccoides - E. rectale
group 5.75 1.20 5.55 1.70 5.53 1.36 0.517 0.950 0.512
C. leptum
group 4.69 1.79 4.83 1.40 4.52 1.61 0.731 0.383 0.679
Lactobacillus
group 6.72 1.08 6.93 1.04 6.79 0.97 0.291 0.475 0.746
E. coli
6.38 1.74 6.01 2.05 6.45 1.68 0.317 0.234 0.871
Bifidobacterium
spp. 6.47 1.47 7.19 1.26 7.49 1.45 0.010* 0.282 0.001*
B. longum
5.55 1.67 6.11 1.58 6.44 1.63 0.001* 0.305 0.011*
B. breve
5.33 1.65 5.53 1.72 5.29 1.61 0.596 0.511 0.908
B. bifidum
4.98 1.38 4.78 1.53 4.77 1.29 0.503 0.958 0.510
B. adolescentis
5.48 1.14 4.71 0.68 5.44 1.37 0.140 0.122 0.936
B. catenulatum
4.90 1.59 5.31 1.74 4.69 1.34 0.344 0.139 0.663
B. angulatum
4.71 0.41 4.59 0.78 4.58 0.38 0.705 0.966 0.663
B. infantis
5.57 0.90 6.23 1.64 4.95 0.52 0.260 0.055 0.307
B. lactis
4.27 0.91 3.87 0.65 4.02 0.40 0.237 0.648 0.503
B. dentium
4.51 0.60 4.86 0.62 5.38 1.38 0.590 0.522 0.207
1
Data are expressed as mean and standard deviation of log cells/g faeces.
2
Statistical significant differences were calculated using ANOVA and post hoc LSD test. Significant differences were established at p,0.050.
doi:10.1371/journal.pone.0030791.t003
Table 4. Faecal microbiota of infants with different HLA-DQ genotype at 4 months of age analysed by qPCR.
2
p-value
High risk Intermediate risk Low risk High- Intermediate- High-
n = 42 n = 65 n = 47 Intermediate Low Low
1
Mean SD Mean SD Mean SD Risk Risk Risk
Bacteroides fragilis
group 5.52 2.09 4.88 1.86 4.91 1.60 0.137 0.932 0.188
Staphylococcus
spp. 5.52 1.20 5.40 0.94 4.80 0.79 0.720 0.060 0.040*
C. coccoides - E. rectale
group 6.00 1.43 6.04 1.55 5.88 1.26 0.898 0.590 0.716
C. leptum
group 4.89 1.65 5.02 1.25 4.83 1.07 0.653 0.501 0.851
Lactobacillus
group 6.70 0.98 6.99 0.94 6.95 0.92 0.117 0.816 0.210
E. coli
6.48 1.62 6.61 1.94 6.60 1.60 0.726 0.977 0.767
Bifidobacterium
spp. 6.74 1.11 6.77 1.29 7.55 1.11 0.893 0.001* 0.002*
B. longum
6.06 1.40 6.22 1.32 6.76 1.31 0.570 0.040 0.019*
B. breve
5.99 1.73 5.94 1.66 5.98 1.62 0.886 0.901 0.982
B. bifidum
5.10 0.94 5.01 1.39 5.04 1.33 0.746 0.926 0.824
B. adolescentis
5.83 1.03 4.99 0.74 6.06 1.42 0.056 0.035* 0.648
B. catenulatum
5.65 1.67 5.42 1.74 5.57 1.32 0.608 0.722 0.866
B. angulatum
4.79 1.00 4.70 0.71 4.23 0.88 0.806 0.190 0.115
B. infantis
6.03 1.38 6.24 1.33 6.56 1.73 0.750 0.612 0.334
B. lactis
4.98 0.88 4.30 0.81 4.25 0.69 0.026 0.865 0.019*
B. dentium
4.54 0.48 4.37 0.71 4.74 - - - -
1
Data are expressed as mean and standard deviation of log cells/g faeces.
2
Statistical significant differences were calculated using ANOVA and post hoc LSD test. Significant differences were established at p,0.050.
doi:10.1371/journal.pone.0030791.t004
Gut Microbial Colonization and Coeliac Disease
PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e30791
Table 5. Faecal microbiota of breast-fed infants with different HLA-DQ genotype at 7 days of age analysed by qPCR.
2
p-value
High risk Intermediate risk Low risk High- Intermediate- High-
n = 27 n = 50 n = 31 Intermediate Low Low
1
Mean SD Mean SD Mean SD Risk Risk Risk
Bacteroides fragilis
group 4.19 1.58 4.43 1.32 3.99 1.42 0.570 0.378 0.710
Staphylococcus
spp. 5.40 0.94 5.51 1.01 4.94 1.19 0.826 0.460 0.580
C. coccoides - E. rectale
group 5.28 1.67 5.19 1.62 4.98 1.29 0.819 0.625 0.514
C. leptum
group 4.38 1.30 4.70 1.49 4.38 1.36 0.471 0.418 0.995
Lactobacillus
group 6.55 1.26 6.64 1.07 6.41 0.94 0.727 0.360 0.632
E. coli
6.03 1.93 5.54 1.79 6.18 1.80 0.315 0.199 0.780
Bifidobacterium
spp. 6.61 1.89 7.14 1.64 7.52 1.51 0.224 0.362 0.057
B. longum
5.49 1.31 6.18 1.66 6.42 1.83 0.098 0.522 0.039*
B. breve
5.44 1.41 5.81 1.78 5.07 1.69 0.430 0.106 0.447
B. bifidum
4.52 1.00 4.88 1.54 4.91 1.39 0.334 0.920 0.324
B. adolescentis
5.36 0.64 5.14 1.54 5.16 0.54 0.792 0.972 0.824
B. catenulatum
4.77 1.63 5.72 1.69 5.54 2.20 0.212 0.787 0.358
B. angulatum
3.40 0.42 4.92 0.59 4.27 1.08 0.048* 0.207 0.229
B. infantis
5.59 0.64 5.80 2.13 5.23 0.04 0.827 0.656 0.778
B. lactis
3.81 0.54 4.02 0.59 4.07 0.68 0.535 0.890 0.464
B. dentium
4.42 0.22 4.35 0.65 - - - - -
1
Data are expressed as mean and standard deviation of log cells/g faeces.
2
Statistical significant differences were calculated using ANOVA and post hoc LSD test. Significant differences were established at p,0.050.
doi:10.1371/journal.pone.0030791.t005
Table 6. Faecal microbiota of breast-fed infants with different HLA-DQ genotype at 1 month of age analysed by qPCR.
2
p-value
High risk Intermediate risk Low risk High- Intermediate- High-
n = 28 n = 53 n = 30 Intermediate Low Low
1
Mean SD Mean SD Mean SD Risk Risk Risk
Bacteroides fragilis
group 4.87 1.72 4.56 1.67 4.87 1.37 0.480 0.497 0.988
Staphylococcus
spp. 5.59 0.82 5.16 0.97 4.64 1.03 0.430 0.223 0.121
C. coccoides - E. rectale
group 5.57 1.41 5.37 1.75 5.22 1.44 0.636 0.717 0.461
C. leptum
group 4.81 2.21 4.99 1.48 4.69 1.43 0.735 0.483 0.824
Lactobacillus
group 6.66 1.18 6.85 1.08 6.73 1.05 0.449 0.625 0.804
E. coli
6.39 1.49 5.56 1.93 6.52 1.66 0.064 0.030 0.792
Bifidobacterium
spp. 6.74 1.57 7.20 1.32 7.45 1.40 0.200 0.468 0.077
B. longum
5.83 1.96 5.95 1.63 6.24 1.52 0.768 0.477 0.373
B. breve
5.55 1.82 5.29 1.71 5.26 1.73 0.594 0.952 0.580
B. bifidum
5.34 1.41 4.51 1.34 4.72 1.41 0.030* 0.550 0.135
B. adolescentis
5.68 1.35 4.64 0.76 5.23 1.11 0.122 0.272 0.493
B. catenulatum
5.15 1.97 5.11 1.88 4.81 1.64 0.953 0.644 0.660
B. angulatum
4.69 0.55 4.76 0.94 4.52 0.45 0.900 0.637 0.700
B. infantis
5.58 0.96 6.55 1.55 5.40 0.19 0.156 0.257 0.848
B. lactis
3.87 0.30 3.46 0.42 3.96 0.40 0.254 0.064 0.769
B. dentium
4.14 0.14 4.86 0.62 6.35 - - - -
1
Data are expressed as mean and standard deviation of log cells/g faeces.
2
Statistical significant differences were calculated using ANOVA and post hoc LSD test. Significant differences were established at p,0.050.
doi:10.1371/journal.pone.0030791.t006
Gut Microbial Colonization and Coeliac Disease
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Table 7. Faecal microbiota of breast-fed infants with different HLA-DQ genotype at 4 months of age analysed by qPCR.
2
p-value
High risk Intermediate risk Low risk High- Intermediate- High-
n = 11 n = 39 n = 21 Intermediate Low Low
1
Mean SD Mean SD Mean SD Risk Risk Risk
Bacteroides fragilis
group 4.47 1.93 4.77 1.77 4.51 1.67 0.636 0.640 0.956
Staphylococcus
spp. 4.52 0.33 5.42 0.90 4.27 0.51 0.102 0.041 0.712
C. coccoides - E. rectale
group 5.81 1.84 5.47 1.35 5.45 1.09 0.485 0.969 0.502
C. leptum
group 5.70 1.28 4.90 1.09 4.08 0.76 0.075 0.020* 0.002*
Lactobacillus
group 6.51 1.00 6.92 0.90 6.96 0.88 0.190 0.876 0.188
E. coli
6.15 1.46 6.43 1.90 6.32 1.71 0.662 0.832 0.814
Bifidobacterium
spp. 7.23 1.22 6.80 1.32 7.33 1.29 0.347 0.156 0.833
B. longum
6.54 1.38 6.25 1.29 6.37 1.32 0.532 0.741 0.740
B. breve
6.46 2.48 5.95 1.71 6.14 1.65 0.508 0.691 0.691
B. bifidum
5.41 1.31 4.95 1.38 4.96 1.24 0.335 0.971 0.384
B. adolescentis
6.68 1.83 4.71 0.55 6.70 0.94 0.015 0.006 0.975
B. catenulatum
5.49 1.60 5.07 1.76 5.38 0.93 0.591 0.598 0.897
B. angulatum
4.46 1.15 4.33 0.69 4.30 - - - -
B. infantis
4.92 - 6.54 1.77 6.89 1.88 - - -
B. lactis
4.09 0.23 4.12 0.85 4.00 0.66 0.953 0.819 0.883
B. dentium
4.29 - 4.14 0.66 - - - - -
1
Data are expressed as mean and standard deviation of log cells/g faeces.
2
Statistical significant differences were calculated using ANOVA and post hoc LSD test. Significant differences were established at p,0.050.
doi:10.1371/journal.pone.0030791.t007
Table 8. Faecal microbiota of formula-fed infants with different HLA-DQ genotype at 7 days of age analysed by qPCR.
2
p-value
High risk Intermediate risk Low risk High- Intermediate- High-
n = 11 n = 17 n = 13 Intermediate Low Low
1
Mean SD Mean SD Mean SD Risk Risk Risk
Bacteroides fragilis
group 5.39 1.34 3.79 1.37 4.06 1.63 0.046* 0.723 0.103
Staphylococcus
spp. 5.40 1.04 4.82 1.46 4.40 1.27 0.560 0.673 0.377
C. coccoides - E. rectale
group 5.65 1.19 5.25 1.46 5.55 1.84 0.541 0.640 0.890
C. leptum
group 4.32 1.08 3.59 1.02 3.44 2.37 0.290 0.852 0.314
Lactobacillus
group 6.37 0.74 6.84 1.01 6.82 1.20 0.241 0.973 0.280
E. coli
6.60 1.97 6.11 2.25 5.47 2.05 0.578 0.513 0.303
Bifidobacterium
spp. 5.73 1.35 6.61 1.72 7.23 1.23 0.164 0.287 0.029*
B. longum
4.81 1.17 5.95 1.39 5.79 1.32 0.031* 0.758 0.087
B. breve
4.79 1.63 4.38 1.45 5.32 1.20 0.515 0.125 0.450
B. bifidum
4.28 0.92 4.13 1.03 4.84 0.94 0.763 0.090 0.308
B. adolescentis
4.12 0.12 3.70 - 5.37 0.27 0.692 0.431 0.808
B. catenulatum
4.53 0.83 4.77 1.24 4.37 0.68 - - -
B. angulatum
4.09 - 5.01 1.14 5.42 - - - -
B. infantis
5.74 - 5.69 - 4.06 0.08 - - -
B. lactis
4.15 0.14 3.52 0.07 4.48 0.54 0.109 0.032* 0.317
B. dentium
- - 3.88 - 4.21 - - - -
1
Data are expressed as mean and standard deviation of log cells/g faeces.
2
Statistical significant differences were calculated using ANOVA and post hoc LSD test. Significant differences were established at p,0.050.
doi:10.1371/journal.pone.0030791.t008
Gut Microbial Colonization and Coeliac Disease
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Table 9. Faecal microbiota of formula-fed infants with different HLA-DQ genotype at 1 month of age analysed by qPCR.
2
p-value
High risk Intermediate risk Low risk High- Intermediate- High-
n = 18 n = 16 n = 17 Intermediate Low Low
1
Mean SD Mean SD Mean SD Risk Risk Risk
Bacteroides fragilis
group 5.28 2.42 4.46 1.70 3.94 1.18 0.317 0.537 0.126
Staphylococcus
spp. 6.43 1.16 5.23 1.20 4.70 0.86 0.069 0.446 0.018*
C. coccoides - E. rectale
group 6.00 0.84 6.02 1.59 6.12 0.99 0.958 0.822 0.780
C. leptum
group 4.57 1.28 4.37 1.15 4.20 1.96 0.751 0.774 0.554
Lactobacillus
group 6.81 0.92 7.10 0.99 6.91 0.86 0.376 0.569 0.753
E. coli
6.38 2.11 7.66 1.55 6.20 1.74 0.053 0.031* 0.788
Bifidobacterium
spp. 6.06 1.24 7.39 0.75 7.73 1.40 0.002* 0.400
,
0.001*
B. longum
5.14 1.04 6.68 1.30 6.87 1.82 0.003* 0.701 0.001*
B. breve
5.05 1.41 5.89 1.52 5.34 1.39 0.126 0.338 0.590
B. bifidum
4.53 1.24 5.62 1.83 4.85 1.09 0.041 0.145 0.544
B. adolescentis
5.23 1.01 4.84 0.55 6.28 2.53 0.698 0.231 0.391
B. catenulatum
4.62 1.08 5.70 1.43 4.57 1.00 0.041* 0.025* 0.922
B. angulatum
4.74 0.32 4.51 0.84 4.73 0.03 0.644 0.696 0.986
B. infantis
5.51 - 4.32 - 4.64 0.57 - - -
B. lactis
4.43 1.05 4.45 0.29 4.38 - - - -
B. dentium
5.25 0.30 - - 4.40 - - - -
1
Data are expressed as mean and standard deviation of log cells/g faeces.
2
Statistical significant differences were calculated using ANOVA and post hoc LSD test. Significant differences were established at p,0.050.
doi:10.1371/journal.pone.0030791.t009
Table 10. Faecal microbiota of formula-fed infants with different HLA-DQ genotype at 4 months of age analysed by qPCR.
2
p-value
High risk Intermediate risk Low risk High- Intermediate- High-
n = 30 n = 26 n = 27 Intermediate Low Low
1
Mean SD Mean SD Mean SD Risk Risk Risk
Bacteroides fragilis
group 6.05 2.00 5.13 2.01 5.30 1.48 0.132 0.791 0.218
Staphylococcus
spp. 5.75 1.22 5.38 1.05 4.91 0.81 0.410 0.301 0.043*
C. coccoides - E. rectale
group 6.06 1.28 6.81 1.50 6.20 1.30 0.049* 0.112 0.718
C. leptum
group 4.66 1.69 5.18 1.43 5.31 0.96 0.214 0.762 0.122
Lactobacillus
group 6.76 0.98 7.08 1.01 6.94 0.96 0.227 0.594 0.510
E. coli
6.61 1.69 7.00 1.92 6.79 1.52 0.421 0.659 0.715
Bifidobacterium
spp. 6.56 1.03 6.81 1.18 7.71 0.95 0.364 0.003*
,
0.001*
B. longum
5.89 1.39 6.15 1.41 7.04 1.25 0.500 0.022* 0.003*
B. breve
5.88 1.54 5.92 1.61 5.86 1.62 0.930 0.905 0.969
B. bifidum
4.98 0.75 5.09 1.44 5.10 1.42 0.752 0.981 0.737
B. adolescentis
5.64 0.83 5.45 0.82 5.57 1.65 0.751 0.865 0.916
B. catenulatum
5.71 1.74 5.84 1.68 5.72 1.58 0.826 0.849 0.980
B. angulatum
4.89 1.00 5.22 0.33 4.23 0.92 0.489 0.044* 0.093
B. infantis
6.11 1.40 6.00 1.03 6.32 1.66 0.884 0.690 0.735
B. lactis
5.28 0.80 4.39 0.82 4.31 0.71 0.016* 0.785 0.007*
B. dentium
4.56 0.50 4.85 0.72 4.74 - - - -
1
Data are expressed as mean and standard deviation of log cells/g faeces.
2
Statistical significant differences were calculated using ANOVA and post hoc LSD test. Significant differences were established at p,0.050.
doi:10.1371/journal.pone.0030791.t010
Gut Microbial Colonization and Coeliac Disease
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The cumulative effect of breast-feeding on microbiota compo-
sition was also evaluated at 4 months of age and some significant
correlations were established. When infants had never been breast-
fed, significant correlations between increased genetic risk and low
numbers of Bifidobacterium spp., and B. longum were detected
(r = 0.343, P = 0.026; r = 0.455, P = 0.003, respectively). Similarly,
when infants were breast-fed for more than 1 month and less than
4 months, low numbers of Bifidobacterium spp. correlated with high
genetic CD risk (r = 0.408, P = 0.031), whereas high B. lactis
numbers correlated with high genetic CD risk (r = 20.697,
P = 0.006). On the other hand, when breast-feeding was exclusive
until the fourth month of age, the high genetic risk correlated with
increased numbers of C. leptum group (r = 20.454, P = 0.001).
Discussion
This is the first report on the effects of both milk feeding type
and HLA-DQ genotype on the gut microbial colonization process
of healthy full-term infants with a family history of CD. In the
present study, milk-feeding type influenced the composition of the
microbiota, partially in agreement with previous studies [24].
Breast-feeding favoured C. leptum group, B. longum and B. breve gut
colonization, while formula-feeding favoured that of B. lactis,E.
coli,C. coccoides-E. rectale group and B. fragilis group. Similar trends
were detected when considering the cumulative effect of breast-
feeding particularly for C. coccoides-E. rectale and B. fragilis groups.
In addition, specific features of fecal microbiota were associated
with the genetic risk of developing CD, based on HLA-DQ
genotype, when considered irrespectively of milk-feeding type.
Increased numbers of Bifidobacterium spp. and B. longum were
characteristic of microbiota of infants with the lowest genetic risk,
whereas increased numbers of Staphylococcus spp. and B. fragilis
group were characteristic of that of infants with the highest genetic
CD risk. To date, there is limited evidence of a correlation
between genotype and intestinal microbiota composition in
humans. In previous human studies, monozygotic twins were
demonstrated to have more similar faecal bacterial DNA profiles
than unrelated individuals [25] and monozygotic twins more so
than dizygotic twins [10]. More recently a strong bond between
genotype, phenotype and changes in gut microbiota has been
reported in patients with Cohn’s disease, demonstrating that
specific gene alterations (e.g. NOD2 and ATG16L1) can have an
impact on intestinal microbiota composition [26]. Animal and
human studies indicate that the host genotype may influence
factors such as the repertoire of mucins, which act as bacterial
adhesion sites in the intestinal mucosa, as well as the immune
responses. Together, these can contribute to modulating the
colonization of certain microorganisms [27]. In this context, our
results suggest that HLA-DQ genotype influences the microbial
colonization pattern early in life and, therefore, could be an
additional factor influencing the risk of developing CD later in life.
Enterocytes, which are in close proximity with intestinal content
and bacteria, can express HLA class II molecules of the MHC to a
certain extent and are able to act as antigen presenting cells [28].
Moreover, HLA class II molecules are primarily expressed by
dendritic cells present in the lamina propria that can sample the
mucosal surface for microbial antigens, which can be presented to
naı
¨ve B and T cells after processed to peptides that are loaded on
MHC class I and class II molecules. This is a critical step in
triggering the mucosal innate immune response, which could
restrict bacterial colonization and influence disease risk [29]. In
particular, the increased Staphylococcus counts detected in the high
CD genetic risk group of infants could favour the activation of a
robust T-cell response by the preferential interaction of certain
superantigens, such as staphylococcal superantigen A, with HLA-
DQ molecules, thereby enhancing the risk of T-cell mediated
diseases [30], such as CD.
The influence of the HLA-DQ genotype was also analysed in
subgroups of either breast-fed or formula fed infants to gather
more information about the respective effects of each variable
(genotype and milk-feeding type) on the microbiota of the infants
under study. In the whole infant population, formula feeding
favoured the presence of increased numbers of B. fragilis group,
which were also higher in infants with higher genetic risk of
developing CD in the whole population and in the subgroup of
formula-fed infants, suggesting that the colonization of this
bacterial group is greatly defined by type of milk-feeding.
Nevertheless, increased counts of Staphylococcus spp. were associated
with increased genetic risk of developing CD in the whole
population and in both breast- and formula-fed infant subgroups,
but their colonization was not favoured by formula feeding,
suggesting that the HLA-DQ genotype plays a more prominent
role in the colonization of this bacterial group. Notably, reduced
numbers of Bifidobacterium spp. were associated with an increased
risk of developing CD in the whole population and in both breast-
and formula-fed infants, and the colonization of species of this
genus was also favoured by breast-feeding. Therefore, the findings
suggest that bifidobacterial numbers can be influenced by both the
HLA-DQ genotype and the milk-feeding type. Moreover, low
counts of the species B. longum were found in infants of higher risk
to develop CD in the whole population and in the subgroup of
formula-fed infants but not in the subgroup of breast-fed infants
indicating that the breast-feeding is providing to the infant’s gut
microbiota certain Bifidobacterium spp. [31,16], which could
partially explain the protective role attributed to breast feeding
in the risk of developing CD in previous epidemiological studies
[17].
These and other commensal bacteria are recognized as
constituting major stimuli for the adequate development of immune
functions and oral tolerance [6], which could also be related to the
risk of developing CD. In another prospective study, a reduced
ratio of Bifidobacterium to Clostridium counts in the faecal microbiota
of infants was shown to precede the development of atopic diseases
later in life, indicating that the relative proportions of these
bacterial groups may favour or protect against the development of
immune-related disorders [32]. Moreover, Bifidobacterium spp. and
B. longum levels in both biopsies and faeces have been reported
lower in CD patients than in healthy controls [31,33]. However,
breast feeding also favoured the presence of increased numbers of
C. leptum group when this factor was considered alone, and this
bacterial group was associated with an increased risk of developing
CD in the subgroup of breast-fed infants. C. leptum group numbers
were also reported to be higher in the fecal and duodenal
microbiota of CD patients than in healthy controls [34].
Overall, this study demonstrates that the milk-feeding type and
the HLA-DQ genotype differently influence the bacterial coloni-
zation pattern of the newborn intestine during the first 4 months of
life and, therefore, could also influence the risk of developing CD
in later life. Breast-feeding reduced the genotype-related differ-
ences in microbiota composition, which could partly explain the
protective role attributed to breast milk in this disorder. Further
studies are underway to reveal additional evidence of the role
played by early intestinal colonization patterns in CD develop-
ment in this cohort of infants.
Acknowledgments
We thank Dr. Laura Barrios and Fernando Santoven˜a for statistical advice.
Gut Microbial Colonization and Coeliac Disease
PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e30791
Author Contributions
Conceived and designed the experiments: YS. Performed the experiments:
GDP AC. Analyzed the data: GDP AC YS. Contributed reagents/
materials/analysis tools: FP EN GC VV TP AJG IP AL CR-K AM ADGN
CC LO LPQ. Wrote the paper: GDP AC YS.
References
1. Fasano A, Catassi C (2005) Coeliac disease in children. Best Pract Res Clin
Gastroenterol 19: 467–478.
2. Sanz Y (2009) Novel Perspectives in Celiac Disease Therapy. Mini-Reviews in
Medicinal Chemistry 9: 359–367.
3. Sollid LM (2002) Coeliac disease: dissecting a complex inflammatory disorder.
Nat Rev Immunol 2: 647–655.
4. Dubois PC, van Heel DA (2008) Translational mini-review series on the
immunogenetics of gut disease: immunogenetics of coeliac disease. Clin Exp
Immunol 153: 162–173.
5. Kagnoff MF (2007) Celiac disease: pathogenesis of a model immunogenetic
disease. J Clin Invest 117: 41–49.
6. Sanz Y, Sa´ nchez E, De Palma G, Medina M, Marcos A, et al. (2008) Indigenous
gut microbiota, probiotics, and coeliac disease. In: Overton LT, Ewente MR,
eds. Child Nutrition & Physiology Nova Science Publishers Inc, NY. pp
210–224.
7. Plot L, Amital H (2009) Infectious associations of Celiac disease. Autoimmunity
Reviews 8: 316–319.
8. Plot L, Amital H, Barzilai O, Ram M, Nicola B, et al. (2009) Infections May
Have a Protective Role in the Etiopathogenesis of Celiac Disease. pp 670–674.
9. Salminen S, Isolauri E (2006) Intestinal colonization, microbiota, and probio tics.
Journal of Pediatrics 149: S115–S120.
10. Stewart JA, Chadwick VS, Murray A (2005) Investigations into the infl uence of
host genetics on the predominant eubacteria in the faecal microflora of children.
J Med Microbiol 54: 1239–1242.
11. Bezirtzoglou E, Romond C (1990) Effect of the feeding practices on the
establishment of bacterial interactions in the intestine of the newborn delivere d
by cesarean section. J Perinat Med 18: 139–143.
12. Bezirtzoglou E (1997) The intestinal microflora during the first weeks of life.
Anaerobe 3: 173–177. S1075-9964(97)90102-5 [pii];10.1006/anae.1997.0102
[doi].
13. Bezirtzoglou E, Tsiotsias A, Welling GW (2011) Microbiota profile in feces of
breast- and formula-fed newborns by using fluorescence in situ hybridization
(FISH). Anaerobe;S1075-9964(11)00033- 3 [pii];10.1016/j.anaerobe.2011.03.
009 [doi].
14. Martin R, Jimenez E, Heilig H, Fernandez L, Marin ML, et al. (2009) Isolation
of bifidobacteria from breast milk and ass essment of the bifidobacterial
population by PCR-denaturing gradient gel electrophoresis and quantitative
real-time PCR. Appl Environ Microbiol 75: 965–969.
15. Reviriego C, Eaton T, Martin R, Jimenez E, Fernandez L, et al. (2005)
Screening of virulence determinants in Enterococcus faecium strains isolated
from breast milk. J Hum Lact 21: 131–137.
16. Martin R, Langa S, Reviriego C, Jiminez E, Marin ML, et al. (2003) Human
milk is a source of lactic acid bacteria for the infant gut. J Pediatr 143: 754–758.
17. Ivarsson A, Hernell O, Stenlund H, Persson LA (2002) Breast-feeding protects
against celiac disease. Am J Clin Nutr 75: 914–921.
18. De Palma G, Capilla A, Nadal I, Nova E, Pozo T, et al. (2010) Interplay
Between Human Leukocyte Antigen Genes and the Microbial Colonization
Process of the Newborn Intestine. Current Issues in Molecular Biology 12: 1–10.
19. Olerup O, Aldener A, Fogdell A (1993) HLA-DQB1 and -DQA1 typing by PCR
amplification with sequence-specific primers (PCR-SSP) in 2 hours. Tissue
Antigens 41: 119–134.
20. Matsuki T, Watanabe K, Fujimoto J, Miyamoto Y, Takada T, et al. (2002)
Development of 16S r RNA-gene-targeted group-specific primers for the
detection and identification of predominant bacteria in human feces. Appl
Environ Microbiol 68: 5445–5451.
21. Malinen E, Kassinen A, Rinttila T, Palva A (2003) Comparison of real-time
PCR with SYBR Green I or 59-nuclease assays and dot-blot hybridization with
rDNA-targeted oligonucleotide probes in quantification of selected faecal
bacteria. Microbiology 149: 269–277.
22. Donat E, Planelles D, Capilla-Villanueva A, Montoro JA, Palau F, et al. (200 9)
Allelic distribution and the effect of haplotype combination for HLA type II loci
in the celiac disease population of the Valencian community (Spain). Tissue
Antigens 73: 255–261.
23. Bourgey M, Calcagno G, Tinto N, Gennarelli D, Margaritte-Jeannin P, et al.
(2007) HLA related genetic risk for coeliac disease. Gut 56: 1054–1059.
24. Penders J, Thijs C, Vink C, Stelma FF, Snijders B, et al. (2006) Factors
influencing the composition of the intestinal microbiota in early infancy.
Pediatrics 118: 511–521.
25. Zoetendal EG (2001) The Host Genotype Affects the Bacterial Community in
the Human Gastronintestinal Tract. Microb Ecol Health Dis 13: 129–134.
26. Frank DN, Robertson CE, Hamm CM, Kpadeh Z, Zhang T, et al. (2011)
Disease phenotype and genotype are associated with shifts in int estinal-
associated microbiota in inflammatory bowel diseases. Inflamm Bowel Dis 17:
179–184.
27. Sanz Y, Nadal I, Sanchez E (2007) Probiotics as drugs against human
gastrointestinal infections. Recent Pat Antiinfect Drug Discov 2: 148–156.
28. Kaiserlian D, Vidal K (1993) Antigen presentation by intestinal epithelial cells.
Immunol Today 14: 144.
29. Hapfelmeier S, Muller AJ, Stecher B, Kaiser P, Barthel M, et al. (2008) Microbe
sampling by mucosal dendritic cells is a discrete, MyD88-independent step in
DeltainvG S. Typhimurium colitis. J Exp Med 205: 437–450.
30. Rajagopalan G, Polich G, Sen MM, Singh M, Epstein BE, et al. (2008)
Evaluating the role of HLA-DQ polymorphisms on immune response to
bacterial superantigens using transgenic mice. Tissue Antigens 71: 135–145.
31. Newburg DS (2005) Innate immunity and human milk. J Nutr 135: 1308–13 12.
32. Kalliomaki M, Kirjavainen P, Eerola E, Kero P, Salminen S, et al. (2001)
Distinct patterns of neonatal gut microflora in infants in whom atopy was and
was not developing. J Allergy Clin Immunol 107: 129–134.
33. Nadal I, Donat E, Ribes-Koninckx C, Calabuig M, Sanz Y (2007) Imbalance in
the composition of the duodenal microbiota of children with coeliac disease.
J Med Microbiol 56: 1669–1674.
34. Collado MC, Donat E, Ribes-Koninckx C, Calabuig M, Sanz Y (2009) Specific
duodenal and faecal bacterial groups associated with paediatric coeliac disease.
J Clin Pathol 62: 264–269.
Gut Microbial Colonization and Coeliac Disease
PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e30791
