Aims: To identify specific gut bacteria associated with coeliac disease (CD) at diagnosis and after treatment with a gluten-free diet (GFD) in a paediatric population.
Methods: 30 and 18 faecal samples from untreated and treated CD patients and 25 and 8 biopsy samples from untreated and treated CD patients, respectively, were analysed. In addition, 30 faecal and 8 biopsy samples from control children were evaluated for comparative purposes. Gut bacterial groups were quantified by real-time PCR.
Results: Bacteroides and Clostridium leptum groups were more abundant in faeces and biopsies of CD patients than in controls regardless of the stage of the disease. E coli and Staphylococcus counts were also higher in faeces and biopsies of non-treated CD patients than in those of controls, but their levels were normalised after treatment with a GFD. Bifidobacterium levels were lower in faeces of both groups of CD patients and in biopsies of untreated CD patients compared to controls. Similar bacterial groups were related to CD in biopsies and faeces, indicating that faecal microbiota partly reflects that of the small intestine in CD patients, and could constitute a convenient biological index of this disorder.
Conclusions: Duodenal and faecal microbiota is unbalanced in children with untreated CD and only partially restored after long-term treatment with a GFD, constituting a novel factor linked to this disorder.
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Both duodenal and faecal microbiota are unbalanced in untreated children with coeliac disease and only partially restored after long-term adherence to a gluten-free diet.
Therefore, specific components of the gut microbiota could be viewed as novel factors contributing to the disease and as possible faecal indexes of this disorder.
Imbalances in the intestinal microbiota are novel features of coeliac disease.
Coeliac disease (CD) is a chronic inflammatory disorder of the small intestine characterised by a permanent intolerance to dietary gluten. This disease can manifest at any age with a variety of clinical features, but typical cases often presents in early childhood.1 In patients, gluten peptides elicit a Th1-type inflammatory immune response that causes intestinal villous atrophy, crypt hyperplasia and infiltration of intraepithelial lymphocytes. The ingestion of gluten is responsible for the signs and symptoms of CD, and its removal from the diet is currently the only available treatment. Moreover, other factors such as imbalances in the intestinal microbiota have been suggested to be associated with this disorder.2–4 Infections could be involved in CD pathogenesis by both impairing mucosal barrier function and favouring the access of dietary gluten to sub-epithelial lymphoid tissue, and promoting a defensive Th1-mediated immune response with production of pro-inflammatory cytokines.5 So far, differences in the microbiota composition and related metabolites between CD patients and healthy controls have been reported mainly in faeces,3 6 7 whereas less information is available on the mucosa-associated microbiota.2 4 A rod-shape bacterium has been found in most biopsy specimens of adult CD patients but it has not been identified.2 More recently, specific studies have been carried out to characterise the duodenal microbiota of CD patients4; however, comparisons between mucosal and faecal microbiota in patients with active disease and treated with a gluten-free diet (GFD) are not available in order to define which are the bacterial groups relevant to the disease. Other reports have described differences between the microbiota composition of mucosal and faecal samples in healthy subjects8 as well as in allergy and inflammatory bowel disease (IBD) patients.9 10
The objective of this study was to compare the composition of the duodenal and faecal microbiota of CD patients (untreated and treated with a GFD) and healthy age-matched controls, as well as to establish possible relations between biopsy and faecal associated bacteria and this disorder by the use of real-time PCR.
PATIENTS AND METHODS
Three groups of children were included in this study: (1) untreated CD patients on a normal gluten-containing diet (56.4–60.6 months old); (2) treated CD patients who had been on a GFD for at least 2 years (63.5–57.8 months old); and (3) control children without gluten intolerance (45.0–49.2 months old). The following faecal samples and duodenal biopsy specimens were analysed: 30 faecal and 25 biopsy samples from untreated CD patients; 18 faecal and 8 biopsy samples from treated CD patients; and 30 faecal and 8 biopsy samples from control children. None of the children included in the study was treated with antibiotics for at least 1 month before the sampling time.
Sample preparation and DNA extraction
Faecal and biopsy samples were frozen immediately at −20°C and kept until processing. Duodenal biopsy specimens were obtained by upper intestinal capsule or endoscopy. Faeces (1 g) and duodenal biopsy samples (10–15 mg) were diluted 1:10 (w/v) in phosphate buffered saline (pH 7.2), homogenised and used for DNA extraction. DNA from pure cultures of reference bacterial strains and faecal and biopsy samples were extracted using the QIAamp DNA stool Mini kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions.
Quantitative real-time PCR (qPCR) analysis
Quantitative real-time PCR was used to characterise the gut microbiota by use of specific primers targeting different bacterial groups and the SYBR Green PCR Master Mix (SuperArray Bioscience Corporation, Frederick, Maryland, USA), as previously described (Table 1). PCR amplification and detection were performed with an ABI PRISM 7000-PCR sequence detection system (Applied Biosystems, UK). Bacterial concentration from each sample was calculated by comparing the Ct values obtained from standard curves that were created by using serial 10-fold dilution of pure cultures of DNA corresponding to 102 to 109 cells. The following strains were used as references: Bifidobacterium longum subsp. longum CECT 4503, Bacteroides fragilis DSMZ 2451, Clostridium coccoides DSMZ 933, C leptum DSMZ 935, Staphylococcus aureus CECT 86, Lactobacillus casei ATCC 393, E coli CECT 45 and Akkermansia muciniphila strain MucT (ATCC BAA-835T).
Statistical analysis was done using SPSS V.11.0 (SPSS, Chicago, Illinois, USA). Due to non-normal distribution, microbial data are expressed as medians with interquartile ranges (IQR); differences between two groups of samples were determined by applying the Mann–Whitney U test. Correlations between variables were determined by applying the Spearman rank correlation coefficient. Differences in prevalence of bacterial groups were established by applying the χ2 test. A value of p<0.050 was considered statistically significant.
Table 2 shows the clinical characteristics of the different groups of CD children included in the study.
Untreated CD patients were on a normal gluten-containing diet, and showed clinical symptoms of the disease, positive coeliac serology markers (anti-gliadin antibodies and anti-transglutaminase antibodies) and signs of severe enteropathy by duodenal biopsy examination classified as type 3 according to the Marsh classification of CD.11 Treated CD patients were on a gluten-free diet for at least 2 years, and showed negative coeliac serology markers and normal mucosa or infiltrative lesion classified as type 0–1 according to the Marsh classification.
Faecal microbiota composition in CD patients and control children
⇓Tables 3 and 4 show the prevalence and bacterial counts of faecal samples from the three groups of children under study assessed by qPCR. The prevalence of E coli was significantly higher in untreated (p = 0.012) and treated CD patients (p = 0.031) than in control children (table 3). Staphylococcus was less prevalent in controls than in untreated CD patients (p = 0.026) and treated CD patients (p = 0.085).
Total bacterial counts were significantly lower in control children than in untreated CD (p = 0.009) and treated CD patients (p = 0.039) (table 4). Bacteroides and C leptum groups were also present in significantly higher numbers in both untreated (p = 0.028 and 0.012, respectively) and treated CD patients (p = 0.015 and <0.001, respectively) compared to controls (table 4). E coli counts were also higher in both untreated and treated CD patients than in controls although the differences were only significant between untreated CD patients and controls (p = 0.011).Total bacteria, Bacteroides, C leptum and E coli group counts were not significantly different between untreated and treated CD patients (p>0.05). Staphylococcus levels were significantly higher in untreated CD patients than in treated CD patients (p = 0.007) and controls (p = 0.031). Bifidobacterium counts were significantly higher in control children than in untreated (p = 0.014) and treated CD patients (p = 0.002). Lactobacillus group levels were significantly different only between treated CD patients and controls (p = 0.035).
Duodenal microbiota composition in CD patients and control children
⇑Tables 3 and 4 show the prevalence and bacterial counts of duodenal biopsies and faeces from the three studied groups of children (untreated and treated CD patients and controls). The prevalence of the C coccoides group was higher in healthy controls than in untreated (p = 0.038) and treated CD patients (p = 0.154). Lactobacillus prevalence was significantly higher in untreated than in treated CD patients (p = 0.002); it was also higher in controls than in treated CD patients (p = 0.038). A muciniphila prevalence was significantly higher (p = 0.011) in untreated than in treated CD patients.
Untreated CD patients showed the highest total bacterial counts in biopsies, followed by treated CD patients and controls, although the differences were not significant (table 4). Bacteroides and C leptum groups were present in significantly higher numbers in both untreated (p = 0.002 and p = 0.040, respectively) and treated (p = 0.030 and p = 0.003, respectively) CD patients than in controls. Staphylococcus and E coli group were present in higher levels in untreated CD patients than in treated patients (p = 0.025 and 0.030, respectively) and controls (p = 0.038 and 0.030, respectively), whereas significant differences were not found between treated CD samples and control samples. Bifidobacterium counts were significantly higher in control children than in untreated CD patients (p = 0.009), although no differences were found comparing controls with treated CD patients (p = 0.461). Lactobacillus group levels were lower in treated CD patients (p<0.001) and controls (p<0.001) than in untreated CD patients, whereas no differences were detected between controls and treated CD patients (p = 0.214).
Relations between microbiota composition of biopsy and faecal samples
Figure 1 shows the correlations between bacterial counts of faeces and biopsies. Correlations were detected between the faecal and biopsy levels of Bifidobacterium in untreated CD patients (r = 0.86, p<0.001), treated CD patients (r = 0.67, p<0.001) and controls (r = 0.68, p<0.001). Correlations were also detected between the faecal and biopsy levels of Bacteroides (r = 0.86, p<0.001), Staphylococcus (r = 0.80, p<0.001), C coccoides (r = 0.83, p<0.001), C leptum (r = 0.77, p<0.001), Lactobacillus (r = 0.83, p<0.001), E coli (r = 0.82, p<0.001) and A muciniphila (r = 0.78, p<0.001) in untreated CD patients. Similarly, correlations were detected for Bacteroides (r = 0.71 and r = 0.68, p<0.001), Staphylococcus (r = 0.61, p = 0.004 and r = 0.44, p = 0.017, respectively), C coccoides (r = 0.71 and r = 0.68, p<0.001), C leptum (r = 0.67 and r = 0.85, p = 0.001), Lactobacillus (r = 0.67, p = 0.001 and r = 0.69, p<0.001), E coli (r = 0.86, p = 0.05 and r = 0.66, p = 0.020) in treated CD patients and controls. A muciniphila correlation was only detected in controls (r = 0.51, p = 0.003).
This is the first comparative study on the relations between the faecal and duodenal mucosa-associated microbiota of CD patients, untreated and treated by a long-term GFD, providing new insights into the possible role of bacteria in CD pathogenesis at different stages of the disease.
Bacteroides and C leptum groups were significantly more abundant in faeces and biopsies of both groups of CD patients than in healthy controls, regardless the stage of the disease. Significantly increased levels of Bacteroides have also previously been detected in faeces and biopsies of untreated CD patients compared to controls by in situ hybridisation and flow cytometry techniques.3 4 Moreover, significant differences have been found between treated and healthy controls in both faeces and biopsies in the present study, providing sounder links between Bacteroides and the disease. With a few exceptions,10 increased levels of Bacteroides have also been detected in the gut microbiota of patients suffering from Crohn’s disease, which is also characterised by Th1-type polarised immune response.12 In IBD patients the concentration of Bacteroides associated with the mucosa was shown to be higher and increased with the severity of the disease.13 The obtained results are also in agreement with the microbial differences found between faecal samples from two population groups with high and low incidence of allergies and CD (Swedish and Estonian infants), characterised by increased levels of Bacteroides and Clostridium in the former population group.14 The C leptum group has also been identified as the clostridia probably contributing to increases in total Clostridium levels previously detected in faeces of untreated CD patients compared with controls.3 Information on the significance of this bacterial group in duodenal microbiota of CD patients is newly reported here.
The prevalence of E coli and Staphylococcus was significantly or almost significantly higher in faeces of both groups of CD patients than in those of controls. In addition, E coli and Staphylococcus counts were significantly higher in faeces and biopsies of non-treated CD patients than in those of controls, but levels were normalised after treatment with a GFD in parallel to the remission of the mucosal lesion. Therefore, these two bacterial groups seemed to be associated with the active phase of the disease and their increases could be a secondary consequence of the inflammatory milieu trigger by gluten ingestion. In IBD patients, numbers of E coli associated with the mucosa were also increased.15 In addition, an adherent invasive E coli pathovar was identified in the mucosa of patients with Crohn’s disease, which was able to replicate and induce tumour necrosis factor α production in macrophages.16 Increased levels of Staphylococcus in duodenal biopsies of untreated CD patients compared with controls were detected, in agreement with a previous study of faeces collected only from untreated CD patients.3
Increased levels of Staphylococcus and Enterobacteriaceae were also higher in infants with allergies compared to healthy infants, suggesting a relationship between these bacterial groups and immune dysregulation.17
Bifidobacterium levels were significantly lower in faeces of both groups of CD patients as well as in biopsies of untreated CD patients compared to control children. A similar trend was detected in biopsy and faecal samples of untreated CD patients and controls but the differences were not significant.3 4 These results suggest that either Bifidobacterium could protect against CD, or inherent features of the CD intestine influence Bifidobacterium colonisation. Bifidobacterium numbers in the mucosa of IBD patients were also reduced compared to controls.18 19 The same trend was reported for allergic infants compared to healthy infants,17 constituting the basis for the proposed use of probiotic bacteria in the management of this disorder.20
Significant correlations were found between the main bacterial group counts detected in biopsies and faeces. Similar bacterial groups were related to CD in biopsies and faeces, indicating that faecal microbiota partly reflects that of the small intestinal mucosa in CD patients, and could constitute a convenient biological marker of this disorder.
Akkermansia muciniphila strain MucT ( = ATCC BAA-835T = CIP 107961T) was kindly provided by Molecular Microbial Ecology Group, Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Wageningen, The Netherlands.
Funding: This work was supported by grants AGL-2005-05788-C02-01 and Consolider Fun-C-Food CSD2007-00063 from the Spanish Ministry of Science and Innovation. I3P-CSIC Postdoctoral Contract from the European Social Fund to MCC is fully acknowledged.
Competing interests: None.
Ethics approval: Ethics approval was obtained from the ethics committee of CSIC and the hospitals taking part in the study (Hospital Universitario La Fe and Hospital General Universitario, Valencia, Spain).
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