Differences in faecal bacteria populations and faecal bacteria metabolism in healthy adults and celiac disease patients

Biochimie 94 (2012) 1724e1729

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Research paper

Differences in faecal bacteria populations and faecal bacteria metabolism in healthy adults and celiac disease patients Esther Nistal a, Alberto Caminero d, Santiago Vivas f, g, José M. Ruiz de Morales e, g, Luis E. Sáenz de Miera b, Leandro B. Rodríguez-Aparicio c, Javier Casqueiro a, d, * a

Área de Microbiología, Universidad de León, 24071 León, Spain Área de Genética, Universidad de León, 24071 León, Spain Área de Bioquímica, Universidad de León, 24071 León, Spain d Instituto de Biología Molecular, Genómica y Proteómica (INBIOMIC), Universidad de León, 24071 León, Spain e Departamento de Inmunología, Hospital de León, Altos de Nava s/n, 24071 León, Spain f Departamento de Gastroenterología, Hospital de León, Altos de Nava s/n, 24071 León, Spain g Instituto de Biomedicina (IBIOMED), Universidad de León, 24071 León, Spain b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 January 2012 Accepted 30 March 2012 Available online 20 April 2012

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 PCRdenaturing gradient gel electrophoresis (DGGE) and gaseliquid 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. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: PCR-DGGE SCFAs Intestinal microbiota

1. Introduction Celiac disease (CD) is a chronic inflammatory disorder of the small intestine caused by lack of oral tolerance to wheat gluten proteins and other related prolamins in genetically predisposed individuals [1]. This disease can manifest at any age and present a variety of clinical features, but it often does so in early childhood,

Abbreviations: CD, celiac disease; DGGE, denaturing gradient gel electrophoresis; GFD, gluten-free diet; SCFAs, short chain fatty acids; UPGMA, unweightedpair group method with arithmetic average. * Corresponding author. Área de Microbiología, Universidad de León, 24071 León, Spain. Tel.: þ34 987291504; fax: þ34 987 291409. E-mail address: [email protected] (J. Casqueiro). 0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2012.03.025

with small intestinal villous atrophy and signs of malabsorption [2,3]. The ingestion of gluten is responsible for the symptoms of CD [4]; however, other environmental factors, such as alterations in the gut microbiota, have been suggested to be associated with the presentation of this disorder in childhood. Indeed, differences in the composition of faecal short-fatty acids in child CD patients compared to healthy controls have been demonstrated [5]. Moreover, an imbalance in the composition of both the duodenal and faecal microbiota of children with CD have also been reported [4,6e9]. The only available treatment for CD is complying a glutenfree diet (GFD) for life [10]. It has been reported that CD children treated with GFD partially restore the normal microbiota both at the upper small intestine and in the colon [11].

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CD is diagnosed at similar rates in children and adults [3]; however, there is scarce knowledge regarding the intestinal microbiota of adults with CD. Recently, differences in the upper small intestinal microbiota between children and adults with CD have been described by our group, but no clear differences were observed between the microbiota of controls and CD patients, probably because the controls were not healthy subjects [12]. The study of the intestinal microbiota in healthy subjects is only possible when it is analysed in faecal samples. This study aimed at investigating the differences in the faecal microbiota between healthy adults and adults with CD. 2. Patients and methods 2.1. Subjects and faecal sampling Thirty-two subjects were included in this study; ten subjects were untreated CD patients on a normal gluten-containing diet (mean age 38.5 years old; range 13e60 years old); eleven subjects were treated CD patients on a GFD (mean age 40.4 years old; range 21e66 years old); and eleven subjects were healthy with no known food intolerances (mean age 30.9 years old; range 24e42 years old). Untreated CD patients showed clinical symptoms of the disease, positive celiac serology markers (anti-transglutaminase antibodies) and a degree of duodenal mucosal atrophy classified as type 3 according to the Marsh classification of CD based on an examination of duodenal biopsies. Treated CD patients were on a GFD for at least 2 years, showed negative serology markers, and displayed complete recovery of the initial villous atrophy (Marsh 0 or Marsh I in the biopsy control). The healthy subjects were symptom-free volunteers whose CD was ruled out based on normal serum tTGA levels and an HLA-DQ phenotype that was not DQ2 or DQ8. None of the participants included in the study had been treated with antibiotics for at least 1 month prior to the sampling time. The study was conducted according to the guidelines outlined in the Declaration of Helsinki, and all of the procedures involving human subjects were approved by the local ethics committee of our hospital. Written informed consent was obtained from all of the subjects. Fresh stools were collected from the three groups of subjects. From healthy subjects samples were taken in normal gluten diet and in ten of these healthy subjects samples were also taken after one week in GFD. All samples were homogenised and aliquoted within 3 h of defecation. The aliquots were stored at 80  C until analysis. 2.2. Faecal short chain fatty acids (SCFAs) identification and quantification SCFAs were analysed as described in Caminero et al. [13] Briefly, 1 g of faeces was suspended in 2 mL of water and homogenised. The suspension was centrifuged for 10 min at 12,000 rpm, and the pH of the supernatant was decreased by the addition of HCl (final concentration of 80 mM). The internal standard 4-methylvaleric acid solution was spiked into the supernatant (final concentration of 258 mM). Then, the supernatant was injected into a gaseliquid chromatography for analysis. Chromatography was performed at the LTI laboratory of the University of León. The SCFAs were identified on chromatograms according to the procedure described by Zhao et al. [14] To quantify the peak area in terms of concentration, the relative response factor was used following the methodology described by Ranilla et al. [15]. 2.3. DNA extraction from faecal samples and PCR amplification The extractions of genomic DNA from faecal samples were carried out using the QIAamp DNA Stool Mini kit (Qiagen, Hilden,

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Germany) according to the manufacturer’s instructions. The DNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (Saveen &Werner, Limhamn, Sweden). PCR fragments of 200 bp, representing the total faecal bacteria, were amplified with the universal primers HDA1-GC and HDA2 [16]. PCR fragments of 380 bp, representing Lactobacillus-related species, were amplified with the primers Lac1 and Lac2-GC [17]. PCR fragments of 596 bp, representing species of Bifidobacterium, were amplified with the primers g-BifidF and g-BifidR-GC [9]. 2.4. Analysis of faecal microbiota by denaturing gradient gel electrophoresis (DGGE) DGGE analysis of PCR amplicons was carried out on the DCode Universal Mutation Detection System (Bio-Rad, Richmond, CA). The linear denaturing gradients of urea and formamide used for the separation of amplicons from total bacteria, Bifidobacterium group and Lactobacillus-related species were 35e55%, 45e55% and 30e50%, respectively. A 100% denaturant corresponds to 7 M urea and 40% (v/v) formamide. The gels were stained with an ethidium bromide solution for 30 min and viewed by UV transillumination. The selected DGGE bands were excised from the denaturing gels and reamplified with the corresponding primers, but without the GC-clamp. The resulting PCR products were cloned with the StrataClone PCR cloning kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. Transformants were checked for the presence of an insert of the expected size. The sequences were obtained from the sequencing service at the University of León and were compared to those in the GenBank database with the BLASTn algorithm. To complete the molecular identification of these sequences, a phylogenetic analysis was carried out. Sequences with 97% identity to the sequences of known bacteria after the phylogenetic analysis were considered to be the same species. 2.5. Statistical and clustering analysis The similarities between the banding patterns generated by PCR-DGGE analysis were analysed using the Dice coefficient and the unweighted-pair group method with the arithmetic average (UPGMA) clustering algorithm and were shown graphically as a dendrogram. The number of bands for each subject in every DGGE profile was considered as an indicator of diversity of the faecal microbiota [7]. The differences in the diversity and in the faecal SCFAs content between the three groups of subjects (healthy, untreated CD and treated CD) were analysed by applying the KruskalleWallis test and the ManneWhitney U test with the Bonferroni correction as needed. The differences in species number between the groups were analysed using c2 tests (Yates-correction applied). In every case, statistical significance was established at p values <0.05. 3. Results 3.1. SCFAs are significantly more abundant in patients with untreated CD and treated CD than healthy adults both in normal gluten diet and in GFD Healthy adults in GFD showed the lowest concentration of faecal SCFAs, and treated CD patients showed the highest concentration of faecal SCFAs (Table 1); the differences were significant between healthy adults (both in GFD and in normal diet) and treated CD patients and between healthy adults and untreated CD patients. The highest concentrations were detected for acetic acid, propionic acid and butyric acid, and the concentrations of these SCFAs

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Table 1 Faecal SCFAs concentrations in samples from untreated celiac disease (UCD) patients, treated celiac disease (TCD) patients, healthy adults in normal diet (H) and healthy adults in gluten-free diet (HGFD). The concentrations are expressed in mmol/kg faeces and are the mean of “n” faecal samples. Metabolite

UCD (n ¼ 11)

Acetic acid 39.03  13.98b Propionic acid 11.61  5.77b i-Butyric acid 1.35  0.75 n-Butyric acid 7.27  3.24b i-Valeric acid 1.68  1.09 n-Valeric acid 1.35  0.90 Hexanoic acid 0.41  0.50 Heptanoic acid 0.22  0.18 Total SCFAs 69.92  22.58b

TCD (n ¼ 10) 54.18 14.20 0.86 11.47 1.11 1.36 0.37 0.21 83.77

        

H (n ¼ 11)

HGFD (n ¼ 10)

25.18b 25.86  9.78 20.27  10.96 6.05b 7.14  3.07 5.72  2.37 0.34 1.01  0.38 0.83  0.36 5.85b 5.24  2.72 3.98  2.10 0.56 1.39  0.60 1.07  0.44 0.98  0.44 0.73  0.42 0.64a 0.44 0.41  0.33 0.34  0.34 0.26 0.11  0.11 0.10  0.09 35.55b 42.17  15.40 32.90  16.20

In H or HGFD significant differences vs. UCD and/or TCD are highlighted in bold. a Significant difference vs. healthy adults in gluten-free diet p < 0.05. b Significant differences vs. healthy adults in normal diet and vs. healthy adults in gluten-free diet: p < 0.05.

between healthy and CD patients were significantly different (Table 1). 3.2. Healthy adults have a different faecal microbiota from untreated CD patients The predominant faecal microbiota was studied by performing a PCR-DGGE analysis using universal primers targeting the bacterial V3 region. The results showed (Fig. 1) that the DGGE profiles were complex and unique for each of the 32 faecal samples. To analyse the relationships among the microbial communities, the UPGMA method based on the similarity indices of the DGGE profiles from healthy subjects in normal gluten diet, untreated CD patients and treated CD patients was carried out. The DGGE profiles showed 4 differentiated clusters (Fig. 1), and most of the faecal communities of the healthy individuals in normal gluten diet clustered together (cluster I) and were separated from most of the faecal communities of the untreated CD patients, which were dispersed along clusters II, III and IV. Most of the faecal communities of the treated CD were located in two clusters: cluster I and cluster II. The UPGMA method with the DGGE profiles from healthy subjects in GFD diet, untreated CD patients and treated CD patients showed similar clusters (Supplementary material Fig. S1). 3.3. Subjects located in cluster I have significant differences in the levels of branched SCFAs compared to subjects located in the other clusters The DGGE profiles of the subjects located in cluster I have similar predominant microbial communities. To determine whether these similarities are related to SCFAs, we analysed the SCFAs present in the cluster I subjects (18 subjects; 10 healthy in normal gluten diet, 6 treated CD patients and 2 untreated CD patients) compared to all of the other subjects included in clusters II, III and IV (14 subjects: 8 untreated CD, 5 treated CD and 1 healthy). The results showed (Table 2) significant differences between the groups in the content of branched SCFAs, including isobutyric acid and isovaleric acid. Similar results were obtained with healthy adults in GFD (Supplementary material Table S1). 3.4. DGGE analysis of the Lactobacillus group The DGGE profiles of the PCR amplicons obtained with Lactobacillus group-specific primers are shown in Fig. 2. The DGGE profiles of healthy individuals in normal gluten diet (mean 3.5 bands) and untreated CD patients (mean 3.2 bands) showed

a significantly higher diversity (p < 0.05) than the DGGE profiles of treated CD patients (mean 2.3 bands), probably because of the treatment with a GFD. No significant differences were observed with healthy individuals in GFD (mean 3.5 bands). Cluster analysis was not performed owing to the profile simplicity of each group. The PCR amplicon bands that were identified by sequencing are indicated in Fig. 2 and shown in Table 3. The PCR amplicon bands corresponding to Leuconostoc citreum were present in all of the samples analysed, and Weissella cibaria was present in all of the samples from healthy adults and most of the CD patients. Lactobacillus sakei PCR amplicon bands were present at significantly higher levels (p < 0.05) in healthy adults in normal gluten diet and untreated CD patients than in treated CD patients. 3.5. DGGE analysis of Bifidobacterium species The DGGE profiles of the PCR amplicons obtained with Bifidobacterium-specific primers are shown in Fig. 3. The DGGE profiles of treated CD patients (mean 1.9) showed a significantly lower diversity (p < 0.05) than the DGGE profiles of healthy individuals in normal gluten diet (mean 3.3) and untreated CD patients (mean 2.7), probably owing to the treatment with a GFD. No significant differences were observed with healthy individuals in GFD (mean 2.9 bands). The PCR amplicon bands that were identified by sequencing are indicated in Fig. 3 and shown in Table 3. The PCR amplicon bands corresponding to Bifidobacterium adolescentis were frequently present in the samples from healthy adults and CD patients. Significant differences were observed in the PCR amplicon bands corresponding to B. bifidum and showed a higher presence in faecal samples from untreated CD patients than in faecal samples from healthy adults in normal gluten diet (p < 0.05). Significantly more Bifidobacterium catenulatum was present in untreated CD patients than in treated CD patients, whereas the PCR amplicons corresponding to Bifidobacterium sp. were present at significantly higher levels in healthy adults (both in normal gluten diet and GFD) than in treated CD patients (p < 0.05). 4. Discussion Intestinal dysbiosis involving specific bacterial species is associated with CD in children [4,6,7,11,18,19]. Our group has previously shown that the bacterial community composition in the small intestine of CD patients differs between children and adults [12]. In adults, the bacterial communities of untreated CD patients clustered together separately from the clustered bacterial communities of treated CD patients; however, the bacterial communities of the controls were dispersed [12], probably because the controls were patients with intestinal symptoms not related to CD. In the present study, we used faecal samples to evaluate the differences in the faecal microbiota between healthy adults (in normal gluten diet and in GFD) and adults with CD (with or without treatment with a GFD). The study of faecal samples allowed us to use healthy volunteers. Our work shows that healthy adults have a predominant faecal microbial community that clusters together in the UPGMA; these bacterial communities are different from communities present in untreated CD patients. We also observed significant differences in the presence of B. bifidum between untreated CD and healthy adults in normal gluten diet. The differences in the faecal microbiota between healthy children and children with CD have been previously reported [5,7,9,11,20]. Children with CD have altered faecal SCFAs compared to healthy controls irrespective of dietary treatment [5]. In this study, we found similar results in adults; untreated CD and treated CD patients have significant higher SCFAs concentrations than healthy

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Fig. 1. Clustering of denaturing gradient gel electrophoresis (DGGE) profiles of total faecal bacteria of healthy (H) adults in normal gluten diet, untreated celiac disease (UCD) patients and treated celiac disease (TCD) patients using HDA1-GC and HDA2 universal primers based on Dice’s similarity index and the unweighted-pair group method with the arithmetic average clustering algorithm. The clusters are differentiated as I, II, III and IV.

Table 2 Comparison of SCFAs in the faecal samples from the subjects in cluster I vs. samples from the subjects in clusters II, III and IV. The concentrations are expressed in mmol/ kg faeces and are the mean of “n” faecal samples. Metabolite

Cluster I (n ¼ 18)

Acetic acid Propionic acid i-Butyric acid n-Butyric acid i-Valeric acid n-Valeric acid Hexanoic acid Heptanoic acid Total SCFAs

38.48 9.83 0.86 7.71 1.12 1.07 0.43 0.19 59.66

a

        

23.99 5.60 0.34a 5.43 0.55a 0.49 0.39 0.24 34.64

Clusters II, III and IV (n ¼ 14) 39.23  15.92 12.17  5.98 1.330.66 8.18  3.87 1.730.96 1.42  0.87 0.39  0.47 0.18  0.11 64.64  24.72

Significant differences vs. clusters II, III and IV: p < 0.05.

controls (both in normal gluten diet and GFD). SCFAs are the principal anions resulting from bacterial fermentation of polysaccharide, oligosaccharide, protein, peptide and glycopeptide precursors in the colon [21]. Thus, the differences in the concentrations of SCFAs could be due to the differences in the microbiota, intestinal transit time or incoming substrates in the colon. We have shown that healthy adults have a different microbiota than untreated CD patients, which may explain the differences in the SCFAs concentrations between these groups. However, the bacterial communities from treated CD patients are located mainly in two clusters (cluster I, 6 out of 11 patients and cluster II, 4 out of 11 patients). Most of the treated CD patients (6 out of 11) have a faecal microbiota similar to healthy adults, suggesting at least a partial restoration of the microbiota in healthy individuals. Several studies have demonstrated that the faecal microbiota in children with untreated CD was only partially restored after a long-term

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Fig. 2. DGGE profiles of the 16S rRNA gene fragments of Lactobacillus spp. and the related lactic acid bacteria of faecal DNA from healthy adults in normal gluten diet (lanes 1e11), untreated celiac disease patients (lanes 12e21), treated celiac disease patients (lanes 22e32) and healthy adults in GFD (lanes 33e42) amplified with the primers Lac1 and Lac2-GC. The numbered bands were sequenced.

treatment with a GFD [11,22]. Interestingly, the SCFAs of subjects in cluster I (mostly healthy adults in normal gluten diet and treated CD patients) have significant lower concentrations in branched SCFAs compared to the SCFAs of subjects located in clusters II/III/IV. Similar results were obtained with healthy adults in GFD (Supplementary material). A strong correlation between isobutyric acid and isovaleric acid in healthy children and CD children was recently reported [5]. These data suggest that treated CD patients located in cluster I may have, at least in part, a restored “healthy microbiota” and also display a partial restoration in normal SCFAs metabolism. Complete restoration of the healthy microbiota seems difficult since it has been previously shown in healthy individuals that GFD influence the composition of the gut microbiota [23,24]. In

our study, DGGE analysis showed in treated CD patients a significant reduction in the diversity of both Lactobacillus and Bifidobacterium species, this reduction seems to be due to GFD because it is not observed in untreated CD patients. As well, in a previous study it was shown that GFD in healthy adults affects the faecal populations of both Lactobacillus and Bifidobacterium [23,24]. In summary, we found differences in the faecal microbiota of untreated CD patients that may be partially restored by a GFD and villous recovery. A GFD is associated with a reduction in the diversity of Lactobacillus and Bifidobacterium. Faecal SCFAs content is higher in CD subjects than in healthy individuals. Further investigations will explain the influence of these differences in CD pathogenesis and prognosis.

Table 3 The frequency of lactic acid bacteria and Bifidobacterium species identified by sequencing the DGGE bands amplified from the faecal DNA of healthy adults in normal gluten diet (H) and healthy adults in gluten-free diet (HGFD), untreated celiac disease (UCD) patients and treated celiac disease (TCD) patients using the Lactobacillus group-specific primers and the Bifidobacterium group-specific primers. Amplicon IDa

Closest relative (Accession number)b

Lactic acid bacteria L18 L31 L5, L12, L17, L37 L19 L15 L39 L11, L25, L42 L1, L9, L41

Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus

L3 L6, L14, L24, L28, L32, L33, L35, L38, L40 L27 L2, L8, L13, L26, L29, L30, L34 Bifidobacterium species Bf2, Bf5, Bf10, Bf13, Bf19, Bf24, Bf25, Bf30, Bf43, Bf46 Bf 46 Bf28, Bf45 Bf27 Bf6, Bf11, Bf17, Bf39, Bf41, Bf44 Bf1, Bf12, Bf20 Bf7, Bf8, Bf18 a b c

amylovorus (FR683089) delbrueckii (HM683089) gasseri (JF720007) mucosae (FR693800) paracasei (HQ423165) reuteri (GQ131183) ruminis (HQ022863) sakei (JF756332)

H N ¼ 11 (%)

HGFD N ¼ 10 (%)

UCD N ¼ 10 (%)

TCD N ¼ 11 (%)

0(0) 1(9) 3(27) 0(0) 0(0) 0(0) 1(9) 5(45)

0(0) 0(0) 1(10) 0(0) 0(0) 0(0) 1(10) 3(30)

1(10) 0(0) 5(50) 1(10) 2(20) 0(0) 1(10) 4(40)

0(0) 1(9) 3(27) 0(0) 1(9) 1(9) 1(9) 0(0)

Lactobacillus sanfranciscensis (HM162420) Leuconostoc citreum (AB572028)

2(8) 11(100)

0(0) 10(100)

0(0) 10(100)

0(0) 11(100)

Leuconostoc sp. (GU998855) Weissella cibaria (AB572037)

1(9) 11(100)

0(0) 10(100)

0(0) 9(90)

0(0) 8(73)

Bifidobacterium adolescentis (HQ259739)

8(73)

6(60)

7(70)

9(82)

Bifidobacterium animalis subsp. lactis (HQ293104) Bifidobacterium bifidum (HQ259744) Bifidobacterium kashiwanohense (AB491757) Bifidobacterium longum (HQ591348) Bifidobacterium catenulatum (AB507091) or B. pseudocatenulatum (NR037117) Bifidobacterium sp. (AB470316)

0(0)

0(0)

2(20)

2(18)

1(9) 0(0) 11(100) 4(36)

2(20) 0(0) 8(80) 3(30)

6(60) 0(0) 10(100) 8(80)

2(18) 1(9) 5(45) 2(18)

5(45)

4(40)

2(20)

The identification labels corresponding to the DGGE bands shown in Figs. 2 and 3. The sequence identity was 99%. Significant differences were established at p < 0.05 using the c2 test.

0(0)

Significant differencesc

H vs. TCD UCD vs. TCD

H vs. UCD

UCD vs. TCD H vs. TCD HGFD vs. TCD

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Fig. 3. DGGE profiles of the 16S rRNA gene fragments of Bifidobacterium spp. of faecal DNA from healthy adults in normal gluten diet (lanes 1e11), untreated celiac disease patients (lanes 12e21), treated celiac disease patients (lanes 22e32) and healthy adults in GFD (lanes 33e42) amplified with the primers g-BifidF and g-BifidR-GC. The numbered bands were sequenced.

Acknowledgements This research was supported by a grant from the Instituto de Salud Carlos III, Fondo de Investigación Sanitaria cofunded by FEDER (FIS PI10/02447) and by a grant from the Junta de Castilla y León, Consejería de Sanidad (Ref 520/A/10). Esther Nistal received a grant from the Junta de Castilla y León, cofunded by Fondo Social Europeo. Appendix A. Supplementary material Supplementary data related to this article can be found online at doi:10.1016/j.biochi.2012.03.025. References [1] Y. Sanz, G. De Pama, M. Laparra, Unraveling the ties between celiac disease and intestinal microbiota, Int. Rev. Immunol. 30 (2011) 207e218. [2] A. Fasano, C. Catassi, Coeliac disease in children, Best Pract. Res. Clin. Gastroenterol. 19 (2005) 467e478. [3] S. Vivas, J.M. Ruiz de Morales, M. Fernandez, et al., Age-related clinical, serological, and histopathological features of celiac disease, Am. J. Gastroenterol. 103 (2008) 2360e2365. [4] I. Nadal, E. Donat, C. Ribes-Koninckx, et al., Imbalance in the composition of the duodenal microbiota of children with coeliac disease, J. Med. Microbiol. 56 (2007) 1669e1974. [5] B. Tjellström, L. Stenhammar, L. Högberg, et al., Gut microflora associated characteristics in children with celiac disease, Am. J. Gastroenterol. 100 (2005) 2784e2788. [6] M.C. Collado, M. Calabuig, Y. Sanz, Differences between the fecal microbiota of coeliac infants and healthy controls, Curr. Issues Intest. Microbiol. 8 (2007) 9e14. [7] Y. Sanz, E. Sánchez, M. Marzotto, M. Calabuig, S. Torriani, F. Dellaglio, Differences in faecal bacterial communities in coeliac and healthy children as detected by PCR and denaturing gradient gel electrophoresis, FEMS Immunol. Med. Microbiol. 51 (2007) 562e568. [8] S. Schippa, V. Iebba, M. Barbato, et al., A distinctive “microbial signature” in celiac pediatric patients, BMC Microbiol. 10 (2010) 175. [9] R. Di Cagno, G.C. Rizzello, F. Gagliardi, et al., Different fecal microbiotas and volatile organic compounds in treated and untreated children with celiac disease, Appl. Environ. Microbiol. 75 (2009) 3963e3971.

[10] M.M.P. da Silva Neves, M.B. González-Garcia, H.P.A. Nouws, et al., Celiac disease diagnosis and gluten-free food analytical control, Anal. Bioanal. Chem. 397 (2010) 1743e1753. [11] M.C. Collado, E. Donat, C. Ribes-Koninckx, et al., Specific duodenal and faecal bacterial groups associated with paediatric coeliac disease, J. Clin. Pathol. 62 (2009) 264e269. [12] E. Nistal, A. Caminero, A.R. Herrán, et al., Differences of small intestinal bacteria populations in adults and children with/without celiac disease: effect of age, gluten diet, and disease, Inflamm. Bowel Dis. (2011) doi:10.1002/ ibd21830. [13] A. Caminero, E. Nistal, L. Arias, et al., A gluten metabolism study in healthy individuals shows the presence of faecal glutenasic activity, Eur. J. Nutr. (2011) doi:10.1007/s00394-011-0214-3. [14] G. Zhao, M. Nyman, J.A. Jönsson, Rapid determination of short-chain fatty acids in colonic contents and faeces of humans and rats by acidified waterextraction and direct-injection gas chromatography, Biomed. Chromatogr. 20 (2006) 674e682. [15] M.J. Ranilla, M.D. Carro, S. López, et al., Influence of nitrogen source on the fermentation of fibre from barley straw and sugarbeet pulp by ruminal microorganisms in vitro, Br. J. Nutr. 86 (2001) 717e724. [16] J. Walter, G.W. Tannock, A. Tilsala-Timisjarvi, et al., Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers, Appl. Environ. Microbiol. 66 (2000) 297e303. [17] J. Walter, C. Hertel, G.W. Tannock, et al., Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis, Appl. Environ. Microbiol. 67 (2001) 2578e2585. [18] M.C. Collado, E. Donat, C. Ribes-Koninckx, et al., Imbalances in faecal and duodenal Bifidobacterium species composition in active and non-active coeliac disease, BMC Microbiol. 8 (2008) 232. [19] E. Sánchez, E. Donat, C. Ribes-Koninckx, et al., Intestinal Bacteroides species associated with coeliac disease, J. Clin. Pathol. 63 (2010) 1105e1111. [20] G. De Palma, I. Nadal, M. Medina, et al., Intestinal dysbiosis and reduced immunoglobulin-coated bacteria associated with coeliac disease in children, BMC Microbiol. 10 (2010) 63. [21] J.M.W. Wong, R. de Souza, C.W.C. Kendall, et al., Colonic health: fermentation and short chain fatty acids, J. Clin. Gastroenterol. 40 (2006) 235e243. [22] R. Di Cagno, M. De Angelis, I. De Pasquale, et al., Duodenal and faecal microbiota of celiac children: molecular, phenotype and metabolome characterization, BMC Microbiol. 11 (2011) 219. [23] G. De Palma, I. Nadal, M.C. Collado, et al., Effects of a gluten-free diet on gut microbiota and immune function in healthy adult human subjects, Br. J. Nutr. 102 (2009) 1e7. [24] Y. Sanz, Effects of a gluten-free diet on gut microbiota and immune function in healthy adult human subjects, Gut Microbe. 1 (2010) 135e137.