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Celiac disease (CD) is an immune-mediated enteropathy triggered by the ingestion of cereal gluten proteins. This disorder is associated with imbalances in the gut microbiota composition that could be involved in the pathogenesis of CD. The aim of this study was to characterize the composition and diversity of the cultivable duodenal mucosa-associated bacteria of CD patients and control children. Duodenal biopsy specimens from patients with active disease on a gluten-containing diet (n = 32), patients with nonactive disease after adherence to a gluten-free diet (n = 17), and controls (n = 8) were homogenized and plated on plate count agar, Wilkins-Chalgren agar, brain heart agar, or yeast, Casitone, and fatty acid agar. The isolates were identified by partial 16S rRNA gene sequencing. Renyi diversity profiles showed the highest diversity values for active CD patients, followed by nonactive CD patients and control individuals. Members of the phylum Proteobacteria were more abundant in patients with active CD than in the other child groups, while those of the phylum Firmicutes were less abundant. Members of the families Enterobacteriaceae and Staphylococcaceae, particularly the species Klebsiella oxytoca, Staphylococcus epidermidis, and Staphylococcus pasteuri, were more abundant in patients with active disease than in controls. In contrast, members of the family Streptococcaceae were less abundant in patients with active CD than in controls. Furthermore, isolates of the Streptococcus anginosus and Streptococcus mutans groups were more abundant in controls than in both CD patient groups, regardless of inflammatory status. The findings indicated that the disease is associated with the overgrowth of possible pathobionts that exclude symbionts or commensals that are characteristic of the healthy small intestinal microbiota.
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Published Ahead of Print 8 July 2013.
10.1128/AEM.00869-13.
2013, 79(18):5472. DOI:Appl. Environ. Microbiol.
Leonor Fernández-Murga and Yolanda Sanz
Ester Sánchez, Ester Donat, Carmen Ribes-Koninckx, Maria
with Celiac Disease in Children
Duodenal-Mucosal Bacteria Associated
http://aem.asm.org/content/79/18/5472
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Duodenal-Mucosal Bacteria Associated with Celiac Disease in Children
Ester Sánchez,
a
Ester Donat,
b
Carmen Ribes-Koninckx,
b
Maria Leonor Fernández-Murga,
a
Yolanda Sanz
a
Microbial Ecology and Nutrition Research Group, Institute of Agrochemistry and Food Technology, National Research Council (IATA-CSIC), Valencia, Spain
a
; Hospital
Universitario La Fe, Valencia, Spain
b
Celiac disease (CD) is an immune-mediated enteropathy triggered by the ingestion of cereal gluten proteins. This disorder is
associated with imbalances in the gut microbiota composition that could be involved in the pathogenesis of CD. The aim of this
study was to characterize the composition and diversity of the cultivable duodenal mucosa-associated bacteria of CD patients
and control children. Duodenal biopsy specimens from patients with active disease on a gluten-containing diet (n 32), patients
with nonactive disease after adherence to a gluten-free diet (n 17), and controls (n 8) were homogenized and plated on plate
count agar, Wilkins-Chalgren agar, brain heart agar, or yeast, Casitone, and fatty acid agar. The isolates were identified by partial
16S rRNA gene sequencing. Renyi diversity profiles showed the highest diversity values for active CD patients, followed by non-
active CD patients and control individuals. Members of the phylum Proteobacteria were more abundant in patients with active
CD than in the other child groups, while those of the phylum Firmicutes were less abundant. Members of the families Enterobac-
teriaceae and Staphylococcaceae, particularly the species Klebsiella oxytoca, Staphylococcus epidermidis, and Staphylococcus
pasteuri, were more abundant in patients with active disease than in controls. In contrast, members of the family Streptococ-
caceae were less abundant in patients with active CD than in controls. Furthermore, isolates of the Streptococcus anginosus and
Streptococcus mutans groups were more abundant in controls than in both CD patient groups, regardless of inflammatory sta-
tus. The findings indicated that the disease is associated with the overgrowth of possible pathobionts that exclude symbionts or
commensals that are characteristic of the healthy small intestinal microbiota.
C
eliac disease (CD) is a chronic intestinal disorder caused by a
deregulated immune response to gluten proteins from wheat,
rye, and barley and their cross-related varieties in genetically sus-
ceptible individuals. CD presents a set of diverse clinical features,
which typically includes fatigue, weight loss, diarrhea, and ane-
mia. Damage to the intestinal mucosa in patients with CD is char-
acterized by intraepithelial lymphocytosis, crypt hyperplasia, and
villous atrophy (1). In CD patients, the pathological response to
gluten proteins involves both adaptive and innate immunity. It is
known that gliadin-specific CD4
T cells develop an inflamma-
tory reaction by production of Th1 cytokines (e.g., gamma inter-
feron [IFN-]) at the mucosal level, which also induces CD8
cells to kill epithelial cells, contributing to tissue damage (2). In
addition, a new subset of T cells, termed Th17 cells, was shown to
contribute to CD pathogenesis by producing proinflammatory
cytokines (such as interleukin-17 [IL-17], IFN-, and IL-21), al-
though these cells can also produce mucosa-protective and regu-
latory factors (IL-22 and transforming growth factor )(3, 4).
Some gluten peptides that are not recognized by T cells can induce
tissue damage by activating components of innate immunity;
thus, peptide p31-43/49 activates the production of IL-15 and
natural killer cell receptor-mediated cytotoxicity by intraepithelial
lymphocytes, contributing to tissue injury (5–7). Improvement of
the pathological lesions occurring in the intestinal mucosa of
sensitive individuals is usually observed after gluten with-
drawal from the diet; however, compliance with this dietary
recommendation is complex, and other alternative strategies
are being investigated (8).
HLA class II molecules DQ2 and DQ8 are the major risk factors
predisposing individuals to CD and account for 35% of the genetic
risk (9). Although the role of these molecules has been well estab-
lished in the pathogenesis of CD, their frequency in the general
population is approximately 30%, whereas only 1 to 3% of indi-
viduals actually develop the disease (10). These data would suggest
that the presence of HLA molecules is a necessary factor but is not
sufficient alone for disease development. Although gluten is the
main environmental trigger of CD, its intake does not fully explain
disease development, and thus, other environmental factors are
thought to be involved. In recent years, early microbial infections
(11, 12) and imbalances in the composition of the gastrointestinal
microbiota (13–20) have also been associated with CD. Molecular
techniques have shown that, compared to the fecal and duodenal
microbiota of healthy individuals, the fecal and duodenal micro-
biota of CD patients is characterized by the presence of higher
numbers of Gram-negative bacteria (bacteroides and enterobac-
teria) and lower numbers of Gram-positive bacteria, like bifido-
bacteria (19, 20). In vitro assays have shown that this altered mi-
crobiota and some enterobacteria isolated from CD patients could
activate proinflammatory pathways, while some bifidobacteria
could inhibit the inflammatory or toxic effects induced by the
same isolated enterobacteria and gluten peptides (21–24). Altera-
tions in the intestinal microbiota are also involved in the patho-
genesis of chronic inflammatory bowel disease (IBD) (25, 26) and
other immune-related disorders (27–29). For instance, IBD pa-
tients have altered duodenal bacterial populations in comparison
to healthy controls (30–32). Nevertheless, neither the specific bac-
teria involved in pathologies affecting the small intestine nor their
possible pathogenic modes of action are fully understood.
Received 16 March 2013 Accepted 23 June 2013
Published ahead of print 8 July 2013
Address correspondence to Yolanda Sanz, yolsanz@iata.csic.es.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.00869-13
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This study was designed to establish whether live culture-de-
pendent bacteria associated with the duodenal mucosa of patients
with active and nonactive CD and controls differ in composition
and biodiversity, as reported in previous molecular studies, with a
view to exploring their potentially pathogenic features in the fu-
ture.
MATERIALS AND METHODS
Subjects. Biopsy samples from three groups of children were included in
this study: 32 from patients with active CD (mean age, 5.1 years; range, 2
to 14 years) on a normal gluten-containing diet, 17 from patients with
nonactive CD (mean age, 5.9 years; range, 3 to 8 years) after following a
gluten-free diet for at least 2 years, and 8 from control children (mean age,
6.9 years; range, 3 to 13 years) with no known gluten intolerance. The
control group consisted of children who were investigated for weight loss,
growth retardation, or functional intestinal disorders of non-CD origin;
and their non-CD status was confirmed by showing a normal villous
structure by examination of the biopsy specimen. CD was diagnosed ac-
cording to the criteria given by the European Society for Pediatric Gastro-
enterology, Hepatology and Nutrition (33). The children included in the
study had not been treated with antibiotics for at least 1 month before
sampling.
The study was conducted in accordance with the ethical rules of the
Helsinki Declaration (Hong Kong revision, September 1989), according
to EEC Good Clinical Practice guidelines (document 111/3976/88, July
1990), and under the guidelines of current Spanish law which regulates
clinical research in humans (Royal Decree 561/1993). The study protocol
was approved by the Committee on Ethical Practice from CSIC and the
Hospital Universitario La Fe (Valencia, Spain). Written informed consent
was obtained from the parents of the children included in the study. The
clinical characteristics of the children are shown in Table 1.
Sample preparation and bacterial isolation. Duodenal biopsy speci-
mens (approximately 10 mg) were obtained by capsule endoscopy, kept
under anaerobic conditions (AnaeroGen; Oxoid, Hampshire, United
Kingdom), and analyzed in less than2htoavoid alterations in bacterial
viability. Biopsy specimens were homogenized in 200 l of a phosphate-
buffered saline (PBS) solution (130 mM sodium chloride, 10 mM sodium
phosphate, 0.05% cysteine, pH 7.2) by pipetting and thorough agitation
in a vortex mixer (10 s). Each homogenized sample was randomly plated
on two different culture media (100 l).
The following media were used: plate count agar (PCA; Scharlau, Bar-
celona, Spain) (34), Wilkins-Chalgren agar (Scharlau, Barcelona, Spain)
(35), brain heart agar (BH; Scharlau, Barcelona, Spain) (36), and yeast,
Casitone, and fatty acid agar (YCFA) (37). PCA plates were incubated
under aerobic conditions at 37°C for 48 h, whereas Wilkins-Chalgren, BH,
and YFCA plates were incubated under anaerobic conditions at 37°C for
48 h using anaerobic jars and an AnaeroGen system (Oxoid, Hampshire,
United Kingdom), which generates an atmosphere of 1% oxygen sup-
plemented with carbon dioxide within 30 min, facilitating the culture of
fastidious and obligate anaerobes. All the viable and cultivable bacteria
recovered from duodenal biopsy samples (mucus and mucosa-associated
bacteria) were isolated and restreaked onto the same agar media. For
preliminary identification of the isolates, conventional microbiological
methods were used, including analysis of colony and cellular morphology
and Gram staining. All isolates were stored at 80°C in the presence of
glycerol (20%, vol/vol) until use for further characterization.
DNA extraction. For DNA extraction, bacterial isolates were grown in
the same isolation broth media and harvested at the late log growth phase.
The bacterial suspensions were centrifuged for 5 min at 6,000 g, the
pellets were resuspended in 100 l of suspension buffer (10 mM Tris-HCl,
1 mM sodium EDTA, pH 8.0) with lysozyme (50 mg/ml) (Sigma, St.
Louis, MO), and the homogenates were incubated at 37°C for 1 h. The
bacterial DNA extraction procedure was adapted from a standard cetylt-
rimethylammonium bromide (CTAB) DNA purification method (38).
DNA samples were stored at 20°C until used as the templates for PCR.
Identification of bacterial isolates. The bacterial DNA of each isolate
was partially amplified with 16S rRNA gene target primers 968f (5=-AAC
GCGAAGAACCTTA-3=) and 1401r (5=-CGGTGTGTACAAGACCC-3=)
(39). When necessary, complete 16S rRNA amplification and sequencing
were performed with the primers 27-f (5=-AGAGTTTGATCCTGGCTC
AG-3=)(40) and 1401r. Amplification reactions were carried out in a
50-l volume containing 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl
2
,1
M each primer, 200 M deoxynucleoside triphosphates, and 2.5 U of
Taq polymerase (Ecotaq; Ecogen, Spain). The amplification program was
1 cycle at 94°C for 5 min; 30 cycles at 94°C for 1 min, 50°C for 1 min, and
72°C for 2 min; and finally, 1 cycle at 72°C for 7 min. The amplification
products were subjected to gel electrophoresis in 1% agarose gels, purified
using GFX PCR DNA and a Gel Band DNA purification kit (GE Health-
care, Buckinghamshire, United Kingdom), and sequenced in an ABI
Prism-3130XL genetic analyzer (Applied Biosystems, CA). Search analy-
TABLE 1 Clinical characteristics of study subjects
a
Characteristic Active CD (n 32) Nonactive CD (n 17) Control (n 8)
Mean (SD) age (yr) 5.1 (3.2) 5.9 (1.2) 6.9 (4.2)
No. (%) of study subjects
Sex (M/F) 14 (43.7)/18 (56.3) 8 (47.1)/9 (52.9) 4 (50)/4 (50)
Symptoms
Abdominal pain 5 (15.6) 0 (0) 2 (25)
Diarrhea 3 (9.4) 0 (0) 5 (62.5)
Weight loss 5 (15.6) 3 (17.6) 1 (12.5)
Anemia 9 (28.1) 2 (11.8) 0 (0)
Iron deficiency 17 (53.1) 0 (0) 0 (0)
Presence of antigliadin antibodies (AGA
)
32 (100) 0 (0) 0 (0)
Presence of antitransglutaminase antibodies (tTG
)
32 (100) 0 (0) 0 (0)
Duodenal biopsy
b
M0-1 0 (0) 17 (100) 8 (100)
M3 32 (100) 0 (0) 0 (0)
HLA type DQ2 and DQ8 32 (100) 17 (100) NA
c
a
Data are expressed as absolute numbers (percentages related to the total numbers) for all characteristics except age, which is expressed as the mean (standard deviation). M, male;
F, female.
b
Modified Marsh classification of CD (1): M0, normal mucosa; M0-1, infiltrative lesions, seen in patients on a gluten-free diet (suggesting that minimal amounts of gliadin are
being ingested), patients with dermatitis herpetiformis, and family members of patients with CD; M2, hyperplastic type, occasionally seen in patients with dermatitis herpetiformis;
M3, 40 intraepithelial lymphocytes per 100 enterocytes, crypts increased, and villi with atrophy (partial or complete villous atrophy), seen in cases of typical CD.
c
NA, not applicable.
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ses to determine the closest relatives of the partial 16S rRNA gene se-
quences retrieved were conducted in GenBank using the Basic Local
Alignment Search Tool (BLAST) algorithm, and sequences with more
than 97% similarity were considered to be of the same species.
Data and statistical analyses. The Renyi diversity index was used to
explore differences in the mucosa-associated bacteria among active and
nonactive CD patients and control children. This index provides three
further diversity index values: species richness (S), the Shannon diversity
index (H=), and the Simpson dominance index (1-D), which were deter-
mined using Paleontological Statistics (PAST) software (41).
Differences in the relative abundance of the duodenal mucosa-associ-
ated bacteria (estimated as isolates belonging to a specific taxon related to
all isolates recovered from samples from each child group) were estab-
lished by applying chi-square tests and, when appropriate, the two-tailed
Fisher’s exact test. Analyses were carried out with Statgraphics software
(Manugistics, Rockville, MD), and statistical differences were established
at a P value of less than 0.05.
RESULTS
Subjects. The clinical characteristics of the groups of children in-
cluded in the study are shown in Table 1. No statistically signifi-
cant differences in the gender ratio representation in the study
were detected. Patients with active CD on a normal gluten-con-
taining diet showed clinical symptoms of the disease, positive CD
serology markers (antigliadin antibodies and antitransglutami-
nase antibodies), and signs of severe enteropathy by duodenal
biopsy examination classified as type 3 according to the Marsh
classification of CD (M3) (1). Patients with nonactive CD who
had been on a gluten-free diet for at least 2 years showed negative
CD serology markers and normal mucosa or infiltrative lesions
classified as type 0-1 according to the Marsh classification of CD.
The study included 32 biopsy specimens from children with active
CD (mean age, 5.1 years), 17 biopsy specimens from children with
nonactive CD (mean age, 5.9 years), and finally, 8 biopsy speci-
mens from children without known gluten intolerance (mean age,
6.9 years) who were included in the control group for comparative
purposes.
Influence of culture media on bacterial taxa recovered. Four
different culture media, including PCA, Wilkins-Chalgren agar,
BH, and YFCA, were used for isolating bacteria from biopsy spec-
imens from the CD patients and controls. The same proportion of
biopsy specimens (50%) from patients with active CD, patients
with nonactive CD, and control children were cultured in each
medium, and therefore, the suitability of each medium to recover
duodenal bacteria could be analyzed independently of subject
health status. A total of 29 CFU was recovered in PCA (1.0 1.4
CFU/10 mg of biopsy specimen, on average), 52 CFU was recov-
ered in Wilkins-Chalgren agar (1.9 1.8 CFU/10 mg of biopsy
specimen), 141 CFU was recovered in BH (4.4 6.9 CFU/10 mg
of biopsy specimen), and 81 CFU was recovered in YFCA (2.6
3.8 CFU/10 mg of biopsy specimen).
The abundance of cultivable bacterial species associated with
the mucosa of the subjects included in this study is shown in Table
2. Some differences in the bacterial phyla, genera, and species iso-
lated from the different culture media were detected.
When the isolates were classified into different phyla, differ-
ences were found for Proteobacteria, whose members were more
frequently recovered in PCA, followed by YFCA, Wilkins-Chal-
gren agar, and BH; significant differences were detected between
PCA and BH (P 0.01) and between YCFA and BH (P 0.02).
Differences among the culture media were not detected for iso-
lates belonging to the phyla Actinobacteria and Firmicutes.
In relation to families and species, members of the family
Staphylococcaceae were more frequently isolated in PCA and
Wilkins-Chalgren agar than in BH (P 0.01) and YCFA (P 0.01
and P 0.03, respectively). Of the staphylococcal species, Staph-
ylococcus epidermidis was more frequently isolated in PCA and
Wilkins-Chalgren agar than in YCFA (P 0.01), and Staphylococ-
cus pasteuri was isolated significantly more frequently in PCA than
in BH (P 0.01).
Members of the family Streptococcaceae were more frequently
isolated in BH and YFCA than in PCA (P 01 and P 0.02,
respectively) and in BH than in Wilkins-Chalgren agar (P 0.01).
Within this family, the Streptococcus anginosus group was signifi-
cantly more abundant in biopsy samples cultured in BH than in
those cultured in Wilkins-Chalgren agar (P 0.02).
Finally, members of the Clostridiaceae family were more fre-
quently isolated in Wilkins-Chalgren agar than in BH (P 0.02),
and those of the Enterobacteriaceae family were more frequently
isolated in PCA and YFCA than in BH (P 0.03 and P 0.02,
respectively).
The species richness (S), Shannon species diversity (H=), and
Simpson species dominance (1-D) indexes were calculated for
PCA (S 11, H= 2.18, and 1-D 0.86), Wilkins-Chalgren agar
(S 15, H= 2.35, and 1-D 0.87), BH (S 27, H= 2.69, and
1-D 0.91), and YFCA (S 22, H= 2.71, and 1-D 0.91), in
order to apply the Renyi index. Renyi diversity profiles showed
that the use of PCA and Wilkins-Chalgren agar led to the recovery
of bacteria with lower species diversity than the use of either BH or
YFCA. Renyi diversity profiles also showed that the curves for
PCA and Wilkins-Chalgren agar intersected each other, and the
same was observed for the curves for BH and YFCA; therefore, the
diversity of these pairs could not be compared (data not shown).
Duodenal mucosa-associated bacteria in CD patients and
controls. The proportion of biopsy specimens inoculated in each
culture medium was similar (25%) for each group of individuals
(patients with active CD, patients with nonactive CD, and con-
trols), and therefore, the total number of bacteria recovered in the
different media was considered to represent the differences among
the study groups, regardless of the different culture media used. A
total of 146 isolates were recovered from biopsy specimens from
active CD patients (4.6 4.8 CFU/10 mg of sample, on average),
84 were recovered from biopsy specimens from nonactive CD
patients (5.1 4.1 CFU/10 mg of sample), and 71 were recovered
from biopsy specimens from the control group (8.9 11.7
CFU/10 mg of sample).
The relative abundance of cultivable bacteria associated with
the duodenal mucosa of the different child groups and the differ-
ences in abundance between groups are shown in Table 3.Inre-
lation to the phyla, members of the phylum Proteobacteria were
more abundant in biopsy samples from patients with active CD
than in those from controls (P 0.01) and nonactive CD patients
(P 0.01), while the relative abundance of members of the Fir-
micutes in biopsy samples from patients with active CD was less
than that in samples from controls (P 0.01) and nonactive CD
patients. Members of the phylum Actinobacteria were also more
abundant in biopsy samples from patients with active CD than in
samples from nonactive CD patients (P 0.02).
In relation to families, members of the Enterobacteriaceae were
more abundant in patients with active CD than in nonactive CD
Sánchez et al.
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TABLE 2 Cultivable bacterial taxa from active and nonactive CD patients and control subjects isolated in PCA, Wilkins-Chalgren, BH, and YCFA
Closest relative
No. (%) of clones
a
PCA (n 29) Wilkins-Chalgren agar (n 50) BH (n 142) YCFA (n 81)
Phylum Actinobacteria 2 (6.9) 1 (2.0) 12 (8.5) 6 (7.4)
Actinomycetaceae 0 0 7 (4.9) 4 (4.9)
Actinomyces odontolyticus 0 0 7 (4.9) 4 (4.9)
Corynebacteriaceae 0 0 1 (0.7) 0
Corynebacterium accolens 0 0 1 (0.7) 0
Micrococcaceae 2 (6.9) 1 (2.0) 3 (2.1) 1 (1.2)
Kocuria kristinae 2 (6.9) 1 (2.0) 1 (0.7) 1 (1.2)
Rothia mucilaginosa 0 0 2 (1.4) 0
Propionibacteriaceae 0 0 1 (0.7) 1 (1.2)
Propionibacterium acnes 0 0 1 (0.7) 1 (1.2)
Phylum Firmicutes 20 (69.0) 43 (86.0) 124 (87.3) 65 (79.0)
Carnobacteriaceae 0 1 (2.0) 5 (3.5) 1 (1.2)
Granulicatella adiacens 0 1 (2.0) 5 (3.5) 1 (1.2)
Clostridiaceae 0
AB
5 (10.2)
A
2 (1.4)
B
2 (2.5)
AB
Clostridium bifermentans 0 3 (6.0) 1 (0.7) 0
Clostridium butyricum 0 2 (4.0) 0 0
Clostridium perfringens 0 0 1 (0.7) 2 (2.5)
Enterococcaceae 1 (3.5) 0 1 (0.7) 0
Enterococcus faecalis 1 (3.5) 0 1 (0.7) 0
Lactobacillaceae 0 0 0 2 (2.5)
Lactobacillus fermentum 0 0 0 2 (2.5)
Staphylococcaceae 10 (34.5)
A
14 (28.0)
A
14 (9.9)
B
9 (11.1)
B
Staphylococcus aureus 0 0 0 3 (3.7)
Staphylococcus epidermidis 6 (20.7)
A
9 (18.0)
A
13 (9.2)
AB
3 (3.7)
B
Staphylococcus hominis 0 2 (4.0) 0 1 (1.2)
Staphylococcus pasteuri 4 (13.8)
A
3 (6.0)
AB
1 (0.7)
B
2 (2.5)
AB
Streptococcaceae 9 (31.0)
A
22 (44.0)
A
90 (63.4)
B
40 (49.4)
AB
Streptococcus anginosus group 0
AB
0
A
17 (12.0)
B
8 (9.9)
AB
Streptococcus australis 0 0 4 (2.8) 0
Streptococcus bovis group 0 0 2 (1.4) 1 (1.2)
Streptococcus gallolyticus 0 0 1 (0.7) 0
Streptococcus mitis group 6 (20.7) 8 (16.0) 21 (14.8) 8 (9.9)
Streptococcus mutans group 0 0 1 (0.7) 4 (4.9)
Streptococcus pneumoniae 0 0 11 (7.8) 3 (3.7)
Streptococcus salivarius group 1 (3.5) 11 (22.0) 23 (16.2) 18 (22.22)
Streptococcus sanguinis group 2 (6.9) 3 (6.0) 14 (9.9) 6 (7.4)
Streptococcus suis 0 0 2 (1.4) 0
Veillonellaceae 0 0 3 (2.1) 2 (2.5)
Veillonella atypical 0 0 0 1 (1.2)
Veillonella dispar 0 0 1 (0.7) 0
Veillonella parvula 0 0 2 (1.4) 1 (1.2)
Unclassified Bacillales 0 1 (2.0) 3 (2.1) 0
Gemella haemolysans 0 1 (2.0) 2 (1.4) 0
Gemella sanguinis 0 0 1 (0.7) 0
Phylum Proteobacteria 7 (24.14)
A
6 (12.0)
AB
5 (3.5)
B
10 (12.6)
A
Burkholderiaceae 1 (3.5) 0 0 0
Burkholderia cepacia 1 (3.5) 0 0 0
Neisseriaceae 1 (3.5) 0 0 0
Neisseria flavescens 1 (3.5) 0 0 0
Enterobacteriaceae 5 (17.2)
A
5 (10.0)
AB
5 (3.5)
B
10 (12.3)
A
Enterobacter cloacae 2 (6.9) 2 (4.0) 0 2 (2.5)
Escherichia coli 0 0 3 (2.1) 3 (3.7)
Klebsiella oxytoca 3 (10.3) 3 (6.0) 3 (2.1) 6 (7.4)
Pseudomonadaceae 0 1 (2.0) 0 0
Pseudomonas stutzeri 0 1 (2.0) 0 0
a
Data are expressed as absolute numbers of isolated clones belonging to one specific taxonomic group (phylum, family, or species) and, in parentheses, the percentage related to the
total number of isolates from each culture medium (PCA, Wilkins-Chalgren agar, BH, and YCFA). Different letters within a row denote statistically significant differences at P
0.05, estimated by using a two-by-two chi-square test and, when appropriate, Fisher’s exact test.
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TABLE 3 Relative abundance of cultivable bacterial taxa from biopsy specimens from patients with active and nonactive CD and control subjects
isolated in PCA, Wilkins-Chalgren agar, BH, or YCFA
Closest relative
No. (%) of clones
a
Active CD (n 146) Nonactive CD (n 85) Control (n 71)
Phylum Actinobacteria 15 (10.6)
A
2 (2.4)
B
4 (5.6)
AB
Actinomycetaceae 9 (5.8)
A
0
B
2 (2.8)
AB
Actinomyces odontolyticus 9 (5.8)
A
0
B
2 (2.8)
AB
Corynebacteriaceae 1 (0.7) 0 0
Corynebacterium accolens 1 (0.7) 0 0
Micrococcaceae 5 (3.5) 0 2 (2.8)
Kocuria kristinae 3 (2.0) 0 2 (2.8)
Rothia mucilaginosa 2 (1.3) 0 0
Propionibacteriaceae 0 2 (2.4) 0
Propionibacterium acnes 0 2 (2.4) 0
Phylum Firmicutes 104 (73.2)
A
76 (91.6)
B
66 (93.0)
B
Carnobacteriaceae 2 (1.3) 4 (4.8) 1 (1.4)
Granulicatella adiacens 2 (1.3) 4 (4.8) 1 (1.4)
Clostridiaceae 4 (2.8) 2 (2.4) 3 (4.2)
Clostridium bifermentans 1 (0.7) 0 3 (4.2)
Clostridium butyricum 0 2 (2.4) 0
Clostridium perfringens 3 (2.0) 0 0
Enterococcaceae 2 (1.4) 0 0
Enterococcus faecalis 2 (1.4) 0 0
Lactobacillaceae 0 0 2 (2.8)
Lactobacillus fermentum 0 0 2 (2.8)
Staphylococcaceae 32 (22.5)
A
8 (9.6)
B
2 (2.8)
B
Staphylococcus aureus 3 (2.0) 0 0
Staphylococcus epidermidis 28 (18.2)
A
6 (7.1)
B
2 (2.8)
B
Staphylococcus hominis 1 (7.8) 2 (2.4) 0
Staphylococcus pasteuri 12 (6.9)
A
1 (1.2)
B
0
B
Streptococcaceae 59 (41.6)
A
58 (69.9)
B
58 (81.7)
B
Streptococcus anginosus group 0
A
0
A
25 (35.2)
B
Streptococcus australis 4 (2.6) 0 0
Streptococcus bovis group 0 3 (3.6) 0
Streptococcus gallolyticus 0 1 (1.2) 0
Streptococcus mitis group 14 (9.1)
A
21 (25.0)
B
8 (11.3)
A
Streptococcus mutans group 0
A
0
A
5 (7.0)
B
Streptococcus pneumoniae 7 (4.6) 6 (7.1) 1 (1.4)
Streptococcus salivarius group 25 (16.2) 19 (22.2) 9 (12.7)
Streptococcus sanguinis group 9 (5.8) 6 (7.1) 10 (14.1)
Streptococcus suis 0 2 (2.4) 0
Veillonellaceae 3 (2.1) 2 (2.4) 0
Veillonella atypica 1 (0.7) 0 0
Veillonella dispar 1 (0.7) 0 0
Veillonella parvula 1 (0.7) 2 (2.4) 0
Unclassified Bacillales 2 (1.4) 2 (2.4) 0
Gemella haemolysans 1 (0.7) 2 (2.4) 0
Gemella sanguinis 1 (0.7) 0 0
Phylum Proteobacteria 23 (16.2)
A
5 (6.0)
B
1 (1.4)
B
Burkholderiaceae 0 0 1 (1.4)
Burkholderia cepacia 0 0 1 (1.4)
Enterobacteriaceae 22 (15.5)
A
4 (4.8)
B
0
B
Enterobacter cloacae 6 (3.9) 0 0
Escherichia coli 5 (3.3) 0 0
Klebsiella oxytoca 11 (7.1)
A
4 (4.8)
AB
0
B
Neisseriaceae 0 1 (1.2) 0
Neisseria flavescens 0 1 (1.2) 0
Pseudomonadaceae 1 (0.7) 0 0
Pseudomonas stutzeri 1 (0.7) 0 0
a
Data are expressed as the absolute numbers of isolated clones belonging to one specific taxonomic group (phylum, family, or species) and, in parentheses, the percentage related to
the total number of isolates from each group of children (patients with active CD, patients with nonactive CD, and controls). Different letters within a row denote statistically
significant differences at P 0.05, estimated by using a two-by-two chi-square test and, when appropriate, Fisher’s exact test.
Sánchez et al.
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patients and control children (P 0.03 and P 0.01, respec-
tively). In particular, Klebsiella oxytoca isolates were more abun-
dant in patients with active CD than in control children (P
0.02). In addition, members of the family Staphylococcaceae were
more abundant in patients with active CD than in patients with
nonactive CD and control individuals (P 0.02 and P 0.01,
respectively). In particular, S. epidermidis and S. pasteuri isolates
were more abundant in patients with active CD than in patients
with nonactive CD (P 0.03 and P 0.04, respectively) and in
control children (P 0.01 and P 0.01, respectively). Further-
more, members of the family Streptococcaceae were less abundant
in patients with active CD than in patients with nonactive CD
and in control children (P 0.01). Statistically significant differ-
ences in the abundance of some particular Streptococcus groups
were also detected; thus, the S. anginosus and Streptococcus mutans
groups were more abundant in control individuals than in pa-
tients with active CD (P 0.01 and P 0.02, respectively) and
nonactive CD (P 0.01 and P 0.02, respectively), whereas
members of the Streptococcus mitis group were more abundant in
patients with nonactive CD patients than those with active CD
(P 0.01). In relation to the family Actinomycetaceae, the isolates
of the only species of that family identified (Actinomyces odonto-
lyticus) were more abundant in patients with active CD than in
those with nonactive CD patients (P 0.04).
The species richness (S), Shannon species diversity (H=), and
Simpson species dominance (1-D) indexes were different between
patients with active CD (S 27, H= 2.73, and 1-D 0.91),
patients with nonactive CD (S 17, H= 2.35, and 1-D 0.82),
and controls (S 13, H= 2.06, and 1-D 0.82), indicating
different species diversity between the child groups studied. Renyi
diversity profiles showed that active CD patients had the highest
biodiversity of duodenal cultivable bacteria, followed by nonac-
tive CD patients and controls (data not shown).
DISCUSSION
The study reported herein demonstrates that the microbiota asso-
ciated with the duodenal mucosa of CD patients has a character-
istic deviation from the normal microbiota structure, which may
characterize the disease. The alterations reported in the present
study are partly consistent with those previously detected by mo-
lecular techniques using specific primers or probes (19, 20). Thus,
our results support the hypothesis that normal components of the
microbiota are excluded and replaced by others that could act as
pathobionts in this specific disease environment. Although such
associations do not demonstrate causality between the altered mi-
crobial groups and the disease, they provide a rationale for further
studies on the possible pathogenic modes of action of such alter-
ations and specific bacteria in CD.
To obtain bacterial isolates that are representative of those in-
habiting the duodenal mucosa in both numbers and diversity,
four different culture media previously described in the literature
(34–37) were used. In general, the greatest species diversity and
quantitative recovery of mucosa-associated bacteria were ob-
tained using the BH and YFCA culture media. These differences
could be linked to the high nutritional requirements of duodenal
bacteria, which are better met by the compositions of these media;
incubation conditions may also have been more appropriate, as
they were more anaerobic than those used for PCA and Wilkins-
Chalgren agar. The diverse morphology of the small intestine fa-
vors a precise spatial relationship for strains within particular in-
testinal nutritional and microaerobic environments (42), and
therefore, it is rather complicated to completely reproduce the in
vivo environmental conditions. Also, even though the duodenum
environment is not strictly anaerobic, the possibility that some
anaerobic bacteria were lost due to oxygen exposure during sam-
ple manipulation cannot be disregarded.
We also analyzed whether some of the media used proved bet-
ter at isolation of specific bacteria. In this regard, PCA and
Wilkins-Chalgren agar seemed to favor the growth and isolation
of members of the family Staphylococcaceae but hindered the
growth of members of the family Streptococcaceae. BH medium
favored the growth of members of the family Streptococcaceae but
hampered that of members of the family Enterobacteriaceae.
Wilkins-Chalgren agar also favored the recovery of members of
the family Clostridiaceae compared to the other media. We con-
firm that none of the media or incubation conditions tested were
suitable for the recovery of all viable bacteria detected in the sam-
ples analyzed when used alone, and therefore, various media must
be used to improve the recovery of bacteria that are representative
of the live bacteria inhabiting the duodenum.
We observed an increased diversity of the cultivable mucosa-
associated bacteria recovered from CD patients compared to the
diversity of bacteria recovered from the controls, and these differ-
ences were restored after adherence to a gluten-free diet. In con-
cordance with this finding, denaturing or temperature gradient
gel electrophoresis (DGGE and TGGE, respectively) analysis of
duodenal samples showed a higher bacterial diversity associated
with the small intestinal microbiota of CD patients (13, 18). How-
ever, several recent molecular studies (43–46) have reported that
reduced mucosal bacterial diversity is associated with inflamma-
tory bowel disease (IBD), although the conditions and techniques
used were not comparable to those used in the present study and
the section of the intestinal tract studied was not the same.
Considering the isolates from all subject groups under study,
our results show that the most abundant were those belonging to
the phylum Firmicutes, followed by those of the phyla Proteobac-
teria and Actinobacteria. This is in concordance with the findings
of a previous culture-independent study, where the same three
phyla dominated the proximal small intestine of CD patients, fol-
lowed by other phyla, such as Bacteroidetes or Fusobacteria (47).
Although our previous culture-independent studies also detected
increased numbers of duodenal and fecal Bacteroides spp. in CD
patients compared with controls (19, 20, 48), this bacterial group
was not isolated with the culture conditions applied, probably due
to exposure to oxygen during the process of homogenization of
biopsy specimens and the use of nonselective media for Bacte-
roides, which could have helped to limit the growth of less anaer-
obic and less nutritionally demanding bacteria. Culture-indepen-
dent studies indicate that the members of the normal human gut
microbiota mainly belong to two phyla, Firmicutes and Bacte-
roidetes, with a smaller number of bacteria belonging to the Pro-
teobacteria and Actinobacteria, although these conclusions are
mainly based on analyses of the fecal microbiota composition (45,
49). Previous data also suggest that only 12% of the total species
richness was detected by applying both molecular and cultivation-
based approaches (50). Remarkably, with both approaches, Firmi-
cutes represented the most abundant group, Proteobacteria were
relatively poorly detected by molecular approaches, and Bacte-
roidetes were less abundant when they were assessed with cultiva-
tion-based approaches than with molecular techniques (49–51).
Duodenal-Mucosal Bacteria in Celiac Disease
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In relation to CD, differences in phylum representation were iden-
tified, and in particular, isolates belonging to the Proteobacteria
were more abundant in active CD patients than in nonactive CD
patients and controls. In this context, other studies have also as-
sociated an increase in the Proteobacteria and, in particular, an
increase in adherent-invasive Escherichia coli, Campylobacter con-
cisus, and enterohepatic Helicobacter with IBD (52).
In addition, active and nonactive CD seemed to be associated
with a decreased abundance of members of the family Streptococ-
caceae, specifically, the S. anginosus and S. mutans groups. The
active phase of the disease was also associated with increased pro-
portions of Enterobacteriaceae and Staphylococcaceae and, in par-
ticular, the species Klebsiella oxytoca, S. epidermidis, and S. pas-
teuri. In concordance with these observations, recent culture-
independent studies indicate that the duodenal and fecal
microbiotas of CD patients are characterized by higher numbers
or proportions of Escherichia coli and Staphylococcus species (19,
20). Furthermore, previous studies using cultured-dependent
techniques have shown increased levels of S. epidermidis (16)in
feces from both active and nonactive CD patients in comparison
with healthy controls and a lower prevalence of salivary S. mutans
in association with CD (53). It seems that dominant genera in the
normal microbiota of healthy individuals, which may act as sym-
bionts, like Streptococcus spp., are replaced in the CD patient mi-
crobiota by potential pathobionts, like Staphylococcus spp. (S. epi-
dermidis) and enterobacteria, which could contribute to breaking
down the normal dynamics and balance of the ecosystem.
To our knowledge, this is the first time that cultivable mucosa-
associated bacteria of patients with active and nonactive CD have
been studied, because previous studies were focused on the char-
acterization of CD microbiota using molecular tools, such as
DGGE and TGGE (13, 15, 54), fluorescence in situ hybridization
(FISH) (20), or real-time PCR (19). Culture-dependent studies
are intrinsically biased by the culture media used, the impact of
potential oxygen exposure, and the inability to detect viable but
noncultivable bacteria present in biological samples; notwith-
standing these limitations, the results obtained in the present
study are coherent with those of previous studies based on molec-
ular techniques, which overcome these limitations. Therefore, the
use of culture-dependent techniques has allowed the characteriza-
tion of the active fraction of the mucosal microbiota of CD pa-
tients and will facilitate future investigation into the possible
pathogenic role that isolated bacteria play in the development of
CD.
Conclusions. This study demonstrates that the duodenal-mu-
cosal microbiota of CD patients presents alterations in the diver-
sity and abundance of different cultivable bacterial taxa, which
could be a consequence of the pathogenesis of CD, which involves
massive destruction of the small bowel mucosa and the conse-
quent release of intracellular contents and serum into the gut. In
the active phase of the disease, the mucosa-associated microbiota
was characterized by a higher abundance of members of the phy-
lum Proteobacteria and the families Enterobacteriaceae and
Staphylococcaceae, apparently excluding members of the phylum
Firmicutes and the family Streptococcaceae, which are normal in-
habitants of the healthy small intestine. These alterations are at-
tenuated after long-term adherence to a gluten-free diet, but the
microbiota is not completely restored; in particular, a reduced
abundance of specific species of Streptococcus (S. anginosus and S.
mutans) also characterizes the microbiota of CD patients with
active and nonactive disease. These findings also suggest their po-
tential use as hallmarks of CD, regardless of inflammatory status.
ACKNOWLEDGMENTS
This work was supported by grants AGL2011-25169 and Consolider Fun-
C-Food CSD2007-00063 from the Spanish Ministry of Economy and
Competitiveness. The scholarship to E. Sánchez from the Institute
Danone is fully acknowledged.
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... (2) We found higher microbial diversity at the initial state of CD. Likewise, previous studies showed an increased diversity at the beginning of CD (Sanchez et al., 2013;Schippa et al., 2010). Schippa et al. suggested that mucosal alterations could be one of the reasons for the decreased diversity under GFD (Schippa et al., 2010). ...
... Schippa et al. suggested that mucosal alterations could be one of the reasons for the decreased diversity under GFD (Schippa et al., 2010). On the other hand, potential harmful bacterial growth may be another explanation for this pattern (Caminero et al., 2015;Sanchez et al., 2013). In the present study, we showed less abundant genera such as unidentified Lachnospiraceae, F I G U R E 2 (a, b) Relative bacterial abundances of CD.P (a) and CD.T (b) groups. ...
... However, because genetic and environmental factors highly shape the gut microbiota, it is a great challenge to compare children with CD and healthy pairs. In most studies, comparisons of CD patients under GFD with a control group who did not have CD, however, needed endoscopy for some reason (functional dyspepsia, gastroesophageal reflux, etc.) (Cheng et al., 2013;Sanchez et al., 2013;Schippa et al., 2010). In addition to the methodological differences between the studies, the dietary pattern, which was not evaluated in almost any study, may also be effective in not determining a CD-specific gut microbiota. ...
Article
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Celiac disease is a chronic inflammatory condition that is not well understood in relation to the microbiome. Our objective was to demonstrate changes in the microbiota and the relationships between nutrients in children with celiac disease (CD) who followed a gluten‐free diet (GFD). A group of 11 children who were recently diagnosed with CD, ranging in age from 3 to 12, were monitored for a period of 6 months. GFD is designed based on the individual's specific energy and nutrient needs, with strict control over dietary adherence. Food consumption, blood, and fecal samples were taken. Fecal samples were put through 16s rRNA sequencing. Microbial modifications were demonstrated using alpha diversity, beta diversity, nonmetric multidimensional scaling analysis (NDMS), t‐test, and metastats. Mean age was 6.4 ± 2.66 years and 54.5% were male participants. Serological parameters were negative after 6 months. Both unweighted (p = .019) and weighted (p = .021) Unifrac distances were higher before GFD, and differences were reliable according to NDMS analysis (stress = 0.189). The abundance of Bacteroides ovatus was increased (p = .014), whereas unidentified Lachnospiraceae, Paeniclostridium, Paraclostridium Peptostreptococcus, and Dielma were decreased after GFD (p < .001). Associations between nutrients and several genera and species were identified. The presence of genus Bifidobacterium and Bifidobacterium adolescentis was inversely associated with fat intake after GFD (p < .01). Microbiota changes became evident over a period of 6 months. The presence or absence of small bacteria may play a role in the development of CD. Modifying the children's dietary intake can potentially influence the microbial composition.
... However, this approach can be confounded if a patient remains free of symptoms for months or years. A causal role of antibiotic-associated gut dysbiosis can nevertheless be suspected when the disease is more intense after exposure to broad-spectrum antibiotics (compared with narrow-spectrum antibiotics) and shows an antibiotic dose dependence (68)(69)(70). Lastly, one cannot rule out direct, negative effects (i.e., not mediated by the gut microbiota) of antibiotics. Some classes of antibiotic affect myocytes and neurons directly; for example, aminoglycosides, capreomycin, and macrolides are ototoxic (71,72). ...
... People with active celiac disease have dysbiosis, with greater abundances of Enterobacteriaceae, Proteobacteria, Staphylococcaceae, and Proteobacteria (70). Furthermore, a reduced abundance of FIGURE 2 In industrial and postindustrial societies, a number of environmental factors have detrimental consequences for a vulnerable human microbial ecosystem; these factors include exposure to antibiotics and xenobiotics, sanitation of the living space, Western-type diets, sedentariness, and pollution. ...
Article
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Antibiotics are safe, effective drugs and continue to save millions of lives and prevent long-term illness worldwide. A large body of epidemiological, interventional and experimental evidence shows that exposure to antibiotics has long-term negative effects on human health. We reviewed the literature data on the links between antibiotic exposure, gut dysbiosis, and chronic disease (notably with regard to the “developmental origins of health and disease” (“DOHaD”) approach). Molecular biology studies show that the systemic administration of antibiotic to infants has a rapid onset but also often a long-lasting impact on the microbial composition of the gut. Along with other environmental factors (e.g., an unhealthy “Western” diet and sedentary behavior), antibiotics induce gut dysbiosis, which can be defined as the disruption of a previously stable, functionally complete microbiota. Gut dysbiosis many harmful long-term effects on health. Associations between early-life exposure to antibiotics have been reported for chronic diseases, including inflammatory bowel disease, celiac disease, some cancers, metabolic diseases (obesity and type 2 diabetes), allergic diseases, autoimmune disorders, atherosclerosis, arthritis, and neurodevelopmental, neurodegenerative and other neurological diseases. In mechanistic terms, gut dysbiosis influences chronic disease through direct effects on mucosal immune and inflammatory pathways, plus a wide array of direct or indirect effects of short-chain fatty acids, the enteric nervous system, peristaltic motility, the production of hormones and neurotransmitters, and the loss of intestinal barrier integrity (notably with leakage of the pro-inflammatory endotoxin lipopolysaccharide into the circulation). To mitigate dysbiosis, the administration of probiotics in patients with chronic disease is often (but not always) associated with positive effects on clinical markers (e.g., disease scores) and biomarkers of inflammation and immune activation. Meta-analyses are complicated by differences in probiotic composition, dose level, and treatment duration, and large, randomized, controlled clinical trials are lacking in many disease areas. In view of the critical importance of deciding whether or not to prescribe antibiotics (especially to children), we suggest that the DOHaD concept can be logically extended to “gastrointestinal origins of health and disease” (“GOHaD”) or even “microbiotic origins of health and disease” (“MOHaD”).
... In summary, the pathogenetic changes observed in celiac disease include release of pro-inflammatory cytokines, activation of IEL and the production of specific antibodies (schematically illustrated by Figure 1). This succession of events results ultimately in inflammatory changes in the mucosa of the small intestine leading to enterocytes' apoptosis, increased IEL (>25/100 enterocytes), crypt hyperplasia, and villous atrophy, all well-known landmarks of celiac disease [12,[14][15][16]. What then, if any, is the role of microbiota in this series of events? ...
... Sanchez et al. [16] found higher diversity values for patients with active CeD compared to controls, as well as increased phylum Proteobacteria and decreased phylum Firmicutes. Members of the Enterobacteriaceae and Staphylococcaceae, particularly the species Klebsiella oxytoca, Staphylococcus epidermidis, and Staphylococcus pasteuri, were more abundant in patients with active disease than in controls. ...
Article
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Celiac disease (CeD) is an autoimmune disease with a strong association with human leukocyte antigen (HLA), characterized by the production of specific autoantibodies and immune-mediated enterocyte killing. CeD is a unique autoimmune condition, as it is the only one in which the environmental trigger is known: gluten, a storage protein present in wheat, barley, and rye. How and when the loss of tolerance of the intestinal mucosa to gluten occurs is still unknown. This event, through the activation of adaptive immune responses, enhances epithelial cell death, increases the permeability of the epithelial barrier, and induces secretion of pro-inflammatory cytokines, resulting in the transition from genetic predisposition to the actual onset of the disease. While the role of gastrointestinal infections as a possible trigger has been considered on the basis of a possible mechanism of antigen mimicry, a more likely alternative mechanism appears to involve a complex disruption of the gastrointestinal microbiota ecosystem triggered by infections, rather than the specific effect of a single pathogen on intestinal mucosal homeostasis. Several lines of evidence show the existence of intestinal dysbiosis that precedes the onset of CeD in genetically at-risk subjects, characterized by the loss of protective bacterial elements that both epigenetically and functionally can influence the response of the intestinal epithelium leading to the loss of gluten tolerance. We have conducted a literature review in order to summarize the current knowledge about the complex and in part still unraveled dysbiosis that precedes and accompanies CeD and present some exciting new data on how this dysbiosis might be prevented and/or counteracted. The literature search was conducted on PubMed.gov in the time frame 2010 to March 2024 utilizing the terms “celiac disease and microbiota”, “celiac disease and microbiome”, and “celiac disease and probiotics” and restricting the search to the following article types: Clinical Trials, Meta-Analysis, Review, and Systematic Review. A total of 364 papers were identified and reviewed. The main conclusions of this review can be outlined as follows: (1) quantitative and qualitative changes in gut microbiota have been clearly documented in CeD patients; (2) intestinal microbiota’s extensive and variable interactions with enterocytes, viral and bacterial pathogens and even gluten combine to impact the inflammatory immune response to gluten and the loss of gluten tolerance, ultimately affecting the pathogenesis, progression, and clinical expression of CeD; (3) gluten-free diet fails to restore the eubiosis of the digestive tract in CeD patients, and also negatively affects microbial homeostasis; (4) new tools allowing targeted microbiota therapy, such as the use of probiotics (a good example being precision probiotics like the novel strain of B. vulgatus (20220303-A2) begin to show exciting potential applications.
... [57]. These findings almost aligned with those reported by Sanchez et al. [64], which found that microbes of the Proteobacteria phylum were more prevalent in CeD patients HCs, whereas Firmicutes showed the opposite. Deeply, certain families, such as Enterobacteriaceae (Klebsiella spp.) and Staphylococcaceae (Staph. ...
... epidermidis and Staph. pasteuri), were more abundant in CeD [64]. ...
Article
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There is increasing evidence indicating that changes in both the composition and functionality of the intestinal microbiome are closely associated with the development of several chronic inflammatory diseases, with celiac disease (CeD) being particularly noteworthy. Thanks to the advent of culture-independent methodologies, the ability to identify and quantify the diverse microbial communities residing within the human body has been significantly improved. However, in the context of CeD, a notable challenge lies in characterizing the specific microbiota present on the mucosal surfaces of the intestine, rather than relying solely on fecal samples, which may not fully represent the relevant microbial populations. Currently, our comprehension of the composition and functional importance of mucosa-associated microbiota (MAM) in CeD remains an ongoing field of research because the limited number of available studies have reported few and sometimes contradictory results. MAM plays a crucial role in the development and progression of CeD, potentially acting as both a trigger and modulator of the immune response within the intestinal mucosa, given its proximity to the epithelial cells and direct interaction. According to this background, this review aims to consolidate the existing literature specifically focused on MAM in CeD. By elucidating the complex interplay between the host immune system and the gut microbiota, we aim to pave the way for new interventions based on novel therapeutic targets and diagnostic biomarkers for MAM in CeD.
... Pseudomonadota were also linked to persistent symptoms despite CeD patients following a gluten-free diet [9]. Gram-positive bacteria belonging to Staphylococcaceae family have also been reported in higher prevalence in duodenal biopsies and stool samples of active CeD patients compared with non-CeD control groups [10,11]. Studies have also shown differences in microbiota compositions in genetically susceptible children who end up developing CeD compared to those who remain healthy [12][13][14]. ...
Article
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Background Celiac disease is an autoimmune disorder triggered by dietary gluten in genetically predisposed individuals that primarily affects the small intestine. Studies have reported differentially abundant bacterial taxa in the gut microbiota of celiac patients compared with non-celiac controls. However, findings across studies have inconsistencies and no microbial signature of celiac disease has been defined so far. Results Here, we showed, by comparing celiac patients with their non-celiac 1st-degree relatives, that bacterial communities of related individuals have similar species occurrence and abundance compared with non-relatives, regardless the disease status. We also found in celiac patients a loss of bacterial species associated with fiber degradation, and host metabolic and immune modulation, as ruminiclostridia, ruminococci, Prevotella, and Akkermansia muciniphila species. We demonstrated that the differential abundance of bacterial species correlates to different dietary patterns observed between the two groups. For instance, Ruminiclostridium siraeum, Ruminococcus bicirculans, and Bacteroides plebeious, recognized as fiber-degraders, appear more abundant in non-celiac 1st-degree relatives, which have a vegetable consumption pattern higher than celiac patients. Pattern of servings per day also suggests a possible link between these species’ abundance and daily calorie intake. Conclusions Overall, we evidenced that a kinship approach could be valuable in unveiling potential celiac disease microbial traits, as well as the significance of dietary factors in shaping microbial profiles and their influence on disease development and progression. Our results pave the way for designing and adopting novel dietary strategies based on gluten-free fiber-enriched ingredients to improve disease management and patients' quality of life.
... Several studies showed intestinal dysbiosis (altered gut microbiota composition or function) in patients with CD, either untreated or treated with a gluten-free diet [10][11][12][13][14][15][16][17]. Gut microbiota affects gut permeability and gut inflammatory activity (both directly and via the release of metabolites), which are suspected to play a role in increasing the risk of autoimmune disorders [18]. ...
Article
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Background Studies have indicated an association between cesarean section (CS), especially elective CS, and an increased risk of celiac disease (CD), but the conclusions of other studies are contradictory. The primary aim of this study (CD-deliver-IT) was to evaluate the rate of CS in a large population of CD patients throughout Italy. Methods This national multicenter retrospective study was conducted between December 2020 and November 2021. The coordinating center was the Pediatric Gastroenterology and Liver Unit of Policlinico Umberto I, Sapienza, University of Rome, Lazio, Italy. Eleven other referral centers for CD have participated to the study. Each center has collected data on mode of delivery and perinatal period of all CD patients referring to the center in the last 40 years. Results Out of 3,259 CD patients recruited in different Italian regions, data on the mode of delivery were obtained from 3,234. One thousand nine hundred forty-one (1,941) patients (60%) were born vaginally and 1,293 (40%) by CS (8.3% emergency CS, 30.1% planned CS, 1.5% undefined CS). A statistically significant difference was found comparing median age at time of CD diagnosis of patients who were born by emergency CS (4 years, CI 95% 3.40–4.59), planned CS (7 years, CI 95% 6.02–7.97) and vaginal delivery (6 years, CI 95% 5.62–6.37) (log rank p < 0.0001). Conclusions This is the first Italian multicenter study aiming at evaluating the rate of CS in a large population of CD patients through Italy. The CS rate found in our CD patients is higher than rates reported in the general population over the last 40 years and emergency CS seems to be associated with an earlier onset of CD compared to vaginal delivery or elective CS in our large nationwide retrospective cohort. This suggests a potential role of the mode of delivery on the risk of developing CD and on its age of onset, but it is more likely that it works in concert with other perinatal factors. Further prospective studies on other perinatal factors potentially influencing gut microbiota are awaited in order to address heavy conflicting evidence reaming in this research field.
Chapter
The gut lumen is a complex environment that contains a variety of substances, including food antigens, food-borne pathogens, commensal microorganisms, and their metabolites. The mucosal and intestinal barriers usually prevent these substances from leaking out of the gut. However, dysbiosis, which is a decline in commensal microbes and an increase in pathogenic bacteria, can weaken these barriers and lead to leaky gut syndrome. When the gut barrier is compromised, hostile gut antigens can infiltrate the lamina propria, circulation, and other organs, including the brain. This infiltration can result in immunological inflammation and the emergence of autoimmunity, such as multiple sclerosis (MS). However, research suggests that restoring normal gut microbiota can improve mucosal and intestinal barrier development and immune response modulation, which may aid in preventing autoimmunity and MS. This book chapter discusses the role of gut microbiota in the onset of MS and explores the potential for gut flora normalization as a therapy for this condition. By improving our understanding of the relationship between the gut microbiome and MS, we may be able to develop more effective treatments and preventative measures for this autoimmune disorder.
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Gut microbiota has a significant role in maintaining the overall health in humans and higher animals. Balanced diet, genetic makeup, and use of antibiotics highly influence the microbial population in the gastrointestinal tract. This review article presents a detailed background on the gut microbiota, including its composition and the various factors influencing its diversity and stability. Strategies for maintaining a healthy gut microbiota are explored, along with an examination of the role of neurotransmitters in regulating gut-brain communication. Research shows that machine learning has a huge potential in elucidating the gut microbiome. Wellbeing and health is directly associated with gut microbiome.
Chapter
Microbial organisms intricately synthesize myriad metabolites, whose remarkable chemical properties have a substantial impact within the domains of agriculture and health care. Being an essential source of nutrition, secondary metabolites (SMs) or biomolecules such as antibiotics and growth hormones have enormous potential in human health. Bioactive molecules are synthesized by activating a specific subset of genes that are actively expressed under favorable conditions, hence augmenting chemical diversity within microbial populations. In the past few decades, a significant contribution has been made toward understanding how efficiently secondary metabolites could be used in health care. These biomolecules are routinely produced due to secondary metabolism and are categorically divided into phenols and polyphenols, terpenoids, sulfur-containing compounds, and nitrogen-containing compounds. The production of these metabolites is essential for fighting against abiotic stresses by enhanced reactive oxygen species (ROS) scavenging and stress tolerance. These metabolites are regulated by low-molecular-weight compounds, sigma factors, transfer RNAs, and often gene products from development processes. Besides strengthening the adaptability of plants, secondary metabolites are utilized as antiviral, antibacterial, and antitumoral agents and as vasodilators or constrictors and laxatives for health provision. The current state of metabolomics exhibits immense potential in identifying and developing novel plant-based secondary metabolites.
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The unique anatomy and physiology of the intestine in conjunction with its microbial content create the steepest oxygen gradients in the body, which plunge to near anoxia at the luminal midpoint. Far from static, intestinal oxygen gradients ebb and flow with every meal. This in turn governs redox effectors nitric oxide, hydrogen sulfide and reactive oxygen species of both host and bacterial origin. The review will illustrate how the intestine and microbes utilize oxygen gradients as a backdrop toward mechanistically shaping redox relationships and a functional coexistence.
Article
Cereal Chem. 85(1):1–13 Celiac disease (CD) is an inflammatory disorder of the upper small in-testine triggered by the ingestion of wheat, rye, barley, and possibly oat products. The clinical feature of CD is characterized by a flat intestinal mucosa with the absence of normal villi, resulting in a generalized malabsorption of nutrients. The prevalence of CD among Caucasians is now thought to be in a range of 1:100–300. There is a strong genetic asso-ciation with human leukocyte antigens (HLA-)DQ2 and DQ8 and cur-rently unknown non-HLA genes. During the last decade, intense bio-chemical studies have contributed to substantial progress in understanding the general principles that determine the pathogenesis of CD. The precipi-tating factors of toxic cereals are the storage proteins, termed gluten in the field of CD (gliadins and glutenins of wheat, secalins of rye, and hordeins of barley). There is still disagreement about the toxicity of oat avenins. The structural features unique to all CD toxic proteins are sequence domains rich in Gln and Pro. The high Pro content renders these proteins resistant to complete proteolytic digestion by gastrointestinal enzymes. Consequently, large Pro-and Gln-rich peptides are cumulated in the small intestine and reach the subepithelial lymphatic tissue. Depending on the amino acid sequences, these peptides can induce two different immune responses. The rapid innate response is characterized by the secretion of the cytokine interleukin-15 and the massive increase of intraepithelial lymphocytes. The slower adaptive response includes the binding of gluten peptides (native or partially deamidated by tissue transglutaminase) to HLA-DQ2 or -DQ8 of antigen presenting cells and the subsequent stimu-lation of T-cells accompanied by the release of proinflammatory cyto-kines such as interferon-γ and the activation of matrix metalloproteinases. Both immune responses result in mucosal destruction and epithelial apop-tosis. Additionally, stimulated T-cells activate B-cells that produce serum IgA and IgG antibodies against gluten proteins (antigen) and tissue trans-glutaminase (autoantigen). These antibodies can be used for noninvasive screening tests to diagnose CD. The current essential therapy of CD is a strict lifelong adherence to gluten-free diet. Dietetic gluten-free foods produced for CD patients underlie the regulations of the Codex Alimen-tarius Standard for Gluten-Free Foods. The "Draft Revised Codex Stan-dard" edited in March 2006 proposes a maximum level of 20 mg of glu-ten/kg for naturally gluten-free foods (e.g., based on rice or corn flour) and 200 mg/kg for foods rendered gluten-free (e.g., wheat starch). Nu-merous analytical methods for gluten determination have been developed, mostly based on immunochemical assays, mass spectrometry, or poly-merase chain reaction. So far, only two enzyme-linked immunosorbent assays have been successfully ring-tested and are commercially available. During the last decade, future strategies for prevention and treatment of CD have been proposed. They are based on the removal of toxic epitopes by enzymatic degradation or gene engineering and on blocking parts of the immune system. However, any alternative treatment should have a safety profile competitive with gluten-free diet.
Chapter
Most protocols for the preparation of bacterial genomic DNA consist of lysis, followed by incubation with a nonspecific protease and a series of extractions prior to precipitation of the nucleic acids. Such procedures effectively remove contaminating proteins, but are not effective in removing exopolysaccharides which can interfere with the activity of enzymes such as restriction endonucleases and ligases. In this unit, however, the protease incubation is followed by a CTAB extraction whereby CTAB complexes both with polysaccharides and with residual protein, effectively removing both in the subsequent emulsification and extraction. This procedure is effective in producing digestible chromosomal DNA from a variety of gram-negative bacteria, all of which normally produce large amounts of polysaccharides. If large amounts of exceptionally clean DNA are required, the procedure can be scaled up and the DNA purified on a CsCl gradient, as described in the alternate protocol.
Article
To determine whether intestinal Staphylococcus spp. and their pathogenic features differed between coeliac disease (CD) patients and healthy controls. 60 children, including active CD (n=20) and non-active CD (n=20) patients and healthy controls (n=20), were studied. Staphylococci were isolated from faeces and identified by PCR and DNA sequencing. The carriage of virulent genes, including adhesion (atlE and fbe), cell aggregation (icaD), global regulatory (agr and sar) and methicillin-resistant (mecA) genes, was analysed by PCR. Staphylococcus epidermidis was more abundant in the microbiota of active and non-active CD patients than in controls. Staphylococcus haemolyticus was more abundant in active CD patients than in control subjects. Staphylococcus aureus was less abundant in active CD patients than in the other child groups. Staphylococcus spp. diversity was higher in active CD patients than in non-active CD patients and controls. The presence of the mecA gene and the simultaneous presence of both the mecA and atlE genes were higher in S. epidermidis clones isolated from CD patients, with active and non-active disease, than in those from control subjects. The individual presence of the other virulent genes was lower in S. epidermidis from active CD patients than in those from the other -child- groups. Increased abundance of S. epidermidis carrying the mecA gene, in active and non-active CD patients, most likely reflects increased exposure of these subjects to opportunistic pathogens and antimicrobials.
Article
Unlabelled: Differences in the intestinal microbiota between children and adults with celiac disease (CD) have been reported; however, differences between healthy adults and adults with CD have not been clearly demonstrated. The aim of this study was to evaluate the differences in the intestinal microbiota between adults with CD and healthy individuals. Microbial communities in faecal samples were evaluated by PCR-denaturing gradient gel electrophoresis (DGGE) and gas-liquid chromatography of short chain fatty acids (SCFAs). The study group included 10 untreated CD patients, 11 treated CD patients and 11 healthy adults (in normal gluten diet and in GFD). UPGMA clustered the dominant microbial communities of healthy individuals together and separated them from the dominant microbial communities of the untreated CD patients. Most of the dominant microbial communities of the treated CD patients clustered together with those of healthy adults. The treated CD patients showed a reduction in the diversity of Lactobacillus and Bifidobacterium species. The presence of Bifidobacterium bifidum was significantly higher in untreated CD patients than healthy adults. There was a significant difference between untreated CD patients and healthy adults, as well as between treated CD patients and healthy adults, regarding acetic acid, propionic acid, butyric acid, and total SCFAs. In conclusion: healthy adults have a different faecal microbiota from that of untreated CD patients. A portion of the treated CD patients displayed a restored "normal" microbiota. The treated CD patients significantly reduce the Lactobacillus and Bifidobacterium diversity. Healthy adults have a different faecal SCFAs content from that of CD patients.
Chapter
This compendium of techniques coverage of state-of-the-art developments in molecular biology, with over 600 pages of information contributed by a wide range of authorities.
Article
The gastrointestinal microbiota has come to the fore in the search for the causes of IBD. This shift has largely been driven by the finding of genetic polymorphisms involved in gastrointestinal innate immunity (particularly polymorphisms in NOD2 and genes involved in autophagy) and alterations in the composition of the microbiota that might result in inflammation (so-called dysbiosis). Microbial diversity studies have continually demonstrated an expansion of the Proteobacteria phylum in patients with IBD. Individual Proteobacteria, in particular (adherent-invasive) Escherichia coli, Campylobacter concisus and enterohepatic Helicobacter, have all been associated with the pathogenesis of IBD. In this Review, we comprehensively describe the various associations of Proteobacteria and IBD. We also examine the importance of pattern recognition in the extracellular innate immune response of the host with particular reference to Proteobacteria, and postulate that Proteobacteria with adherent and invasive properties might exploit host defenses, drive proinflammatory change, alter the intestinal microbiota in favor of dysbiosis and ultimately lead to the development of IBD.