Abstracts

INTRODUCTION

Celiac disease (CD) is a multifactorial disorder that involves interactions between genetic and environmental factors⁽²⁷27. Sanz Y, De Pama G, Laparra M. Unraveling the ties between celiac disease and intestinal microbiota. Int Rev Immunol. 2011;30:207-18.⁾. Environmental factors incriminated include early life gluten exposure, short duration of breastfeeding, intestinal infections and changes in microbiota⁽⁹9. Ghosh S. Advances in our understanding of the pathogenesis of celiac disease. Can J Gastroenterol. 2011;25:186.⁾.

Changes in the composition of intestinal microbiota in CD have been demonstrated in children⁽³3. Collado MC, Donat E, Ribes-Koninckx C, Calabuig M, Sanz Y. Imbalances in faecal and duodenal Bifidobacterium species composition in active and non-active coeliac disease. BMC Microbiol. 2008;232:1-9.^, 55. Di Cagno R, Rizzello CG, Gagliardi F, Ricciuti P, Ndagijimana M, Francavilla R, et al. Different fecal microbiotas and volatile organic compounds in treated and untreated children with celiac disease. Appl And Environ Microbiol. 2009;75:3963–71.^, 2121. Nadal I, Donat E, Ribes-Koninckx C, Calabuig M, Sanz Y. Imbalance in the composition of the duodenal microbiota of children with celiac disease. J Med Microbiol. 2007;56:1669–74.⁾. Meanwhile, we have no knowledge of studies evaluating the balance of microbiota in adults with CD under treatment (diet), which is presumed that there is an equal number of bifidobacteria in relation to individuals without CD.

The presence of bifidobacteria in the gastrointestinal tract has been associated with a variety of beneficial health effects^{(4, 16)}. The resistance provided by these bacteria to the host against pathogens represents its direct effect on human health. On the other hand, their indirect effect is observed through the interaction with the lymphoid tissue and villi⁽²⁶26. Sanders ME. Impact of probiotics on colonizing microbiota of the gut. J Clin Gastroenterol. 2011;45:S115–9.⁾.

Although the precise mechanisms of probiotics in the intestinal tract are not fully elucidated, they are targets of detailed in vivo and in vitro scientific research and seem to occur in a specie-specific form. Strains of theBifidobacterium genus have been shown to afford protection against the inflammatory response and mucosal damage caused by gliadin peptides. In vitro studies have indicated that bifidobacteria strains reduces the production of inflammatory cytokines (IFN-γ and TNF-α) and increases IL-10. Protective effects were seemed by increasing the number of goblet cells in the small intestine, which were reduced by the presence of gliadin, IFN-γ, and enterobacteria. In relation to cytokine production, B. bifidum enhanced the production of chemotactic factors (MCP-1) and inhibitors of metalloproteinases (TIMP-1), which could contribute to gut mucosal protection. In addition, strains of differentLactobacillus species exerted inductive rather than suppressive effects on both the innate and adaptive immunity. L. casei strain could enhance both mucosal and systemic T-cell-mediated responses to gluten. Furthermore to immunomodulatory effects, B. longum also showed to contribute to the hydrolysis of gliadin peptides during intestinal digestion by reducing the presence of toxic amino acid sequences and their adverse effects, reducing the cytotoxicity of gliadin peptides and their ability to activate the NF-kB subunits and TNF-α and IL-1β production. Another research group reported that a strain of B. lactis, but not of L. fermentum, inhibited the gliadin-induced increase of epithelial permeability in vitro⁽²⁶26. Sanders ME. Impact of probiotics on colonizing microbiota of the gut. J Clin Gastroenterol. 2011;45:S115–9.^{, 27}27. Sanz Y, De Pama G, Laparra M. Unraveling the ties between celiac disease and intestinal microbiota. Int Rev Immunol. 2011;30:207-18.⁾.

In this context, this study was designed to compare the concentration of fecal bifidobacteria and pH of patients with celiac disease and control subjects in order to identify if the imbalance on fecal microbiota still remains during the treatment of celiac disease and identify the necessity of dietary supplementation with pre- or probiotics.

METHODS

Subjects

Two groups of adults were included in this study: (Group 1) 14 celiac patients, with previous positive serology markers and with previous signs of enteropathy by duodenal biopsy examination, but actually on a gluten-free diet for at least 2 years; without showing clinical symptoms of the disease, (Group 2) 42 control healthy adults without known food intolerance. None of volunteers included in the study were treated with antibiotics, pre- or probiotic for at least 3 months before the sampling time.

Fecal pH and Bifidobacteria

For isolation and quantification of bifidobacteria and fecal pH determination, the participants collected stool samples in airtight plastic bags to prevent the ingress of air, then those samples were analyzed within 8 hours after collection. One gram of feces of each participant was weighed and diluted in sterile deionized water for pH analysis. One gram of feces of each participant was also weighed and diluted in 9 mL of 0.31 mM phosphate buffer. From this dilution (10^-1), decimal dilutions were performed up to 10^-7 and 100 µL of each dilution was spread-plated on selective medium for bifidobacteria BIM-25⁽²⁰20. Muñoa FJ, Pares R. Selective medium for isolation and enumeration of Bifidobacterium spp. Appl Environ Microbiol. 1988;54:1715-8.⁾. The plates were incubated at 37ºC for 72 hours under anaerobic conditions (Anaerobac of Probac^®). At a later stage, bacterial population (bifidobacteria) count was calculated as colony forming units (CFU) per gram of fecal sample weight. Gram stain, catalase test, and fructose-6-phosphate phosphoketolase test according to Orban and Patterson (2000)⁽²⁴24. Orban JI, Patterson JA. Modification of the phosphoketolase assay for rapid identification of bifidobacteria. J Microbiol Meth. 2000;40:221-4.⁾ were carried out for all types of isolated colonies in order to confirm Bifidobacterium spp genus.

Statistical analysis

The statistical analysis was performed in the statistical program GraphPad Prism^® version 4.0 in 2003. For analysis of data distribution, it was applied the normality test D’Agostino and Pearson Omnibus Normality Test. Nonparametric Student t test was performed and it was adopted the significance level of 95% (P<0.05).

Ethical Adherence

This study protocol was approved by Ethics Committee in Human Beings of the Federal University of Santa Catarina (Process: 772, title page: 333218 – May 31, 2010). Adults were enrolled in the study after a written informed consent was obtained.

RESULTS

Among the 42 healthy control subjects, 21 were female and 21 male, ages between 21 and 58 years, mean 27 years. Among the 14 celiac patients evaluated, ten were female and four male. The mean age was 40 years, but it was analyzed individuals from 12 to 70 years. The majority (92.8%) of celiac was white. All celiac (100%) had confirmation of the diagnosis by small intestine biopsy and the main clinical symptoms of the disease before treatment are described in Table 1.

Regarding the stool samples analysis, the macro and the micromorphology of the bifidobacteria colonies were similar in both groups of celiac patients and control that presented in accordance with the characteristics described in the literature⁽³¹31. Thitaram SN, Siragusa GR, Hinton A Jr. Bifidobacterium-selective isolation and enumeration from chicken caeca by a modified oligosaccharide antibiotic-selective agar medium. Lett Appl Microbiol. 2005;41:355-60.⁾. The concentration of bifidobacteria in stool samples from celiac disease patients and controls are shown in Figure 1. The celiac patients showed significantly smaller amount of CFU of bifidobacteria per gram compared to controls.

The average fecal pH of celiac patients was 7.19 ± 0.521 and 7.18 ± 0.522 of control subjects. There was no significant difference between groups.

DISCUSSION

The improvement of screening methods for CD, that became more sensible and specific, allowed the realization of many populational studies, which resulted in revealing celiac disease rates much higher than previously imagined⁽⁷7. Freeman HJ, Chopra A, Clandinin MT, Thomson ABR. Recent advances in celiac disease. World J Gastroenterol. 2011;17:2259-72.⁾. CD is now considered the most common food intolerance in the world⁽²²22. Nobre SR, Silva T, Pina Cabral JE. Doença Celíaca Revisitada. J Port Gastrenterol. 2007;14:184-93.⁾. Some studies accomplished in different regions of Brazil, showed prevalence of CD in adults 1/123⁽¹⁸18. Melo SB, Fernandes MI, Peres LC, Troncon LE, Galvão LC. Prevalence and demographic characteristics of celiac disease among blood donors in Ribeirao Preto State of Sao Paulo, Brazil. Dig Dis Sci. 2006;51:1020–5.⁾ and 1/214⁽²³23. Oliveira RP. High prevalence of celiac disease in Brazilian blood donor volunteers based on screening by IgA antitissue transglutaminase antibody. Eur J Gastroenterol Hepatol. 2007;19:43-9.⁾. The low prevalence can justify the reduced number of patients in the celiac group.

In our study, the largest number of female celiac participants corroborates with the higher prevalence of celiac disease in this sex⁽¹⁵15. Kneepkens CM, von Blomberg BM. Clinical practice: Coeliac disease. Eur J Pediatr. 2012;16.⁾. In addition, in a previous Brazilian study, 97.6% of celiac patients participants were white⁽²⁹29. Sdepanian VL, Morais MB, Fagundes-Neto U. Celiac disease: clinical characteristics and methods used in the diagnosis of patients registered at the Brazilian Celiac Association. J Pediatr. 2001;131-8.⁾, which is in agreement with what was observed in our study (92.8%). Additionally, the main symptoms displayed by celiac patients of this study before treatment was similar to those presented by celiac residents in another region of Brazil⁽²⁷27. Sanz Y, De Pama G, Laparra M. Unraveling the ties between celiac disease and intestinal microbiota. Int Rev Immunol. 2011;30:207-18.⁾ and those described in reviews in the literature⁽⁷7. Freeman HJ, Chopra A, Clandinin MT, Thomson ABR. Recent advances in celiac disease. World J Gastroenterol. 2011;17:2259-72.^{, 15}15. Kneepkens CM, von Blomberg BM. Clinical practice: Coeliac disease. Eur J Pediatr. 2012;16.^{, 17}17. Lionetti E, Catassi C. New clues in celiac disease epidemiology, pathogenesis, clinical manifestations, and treatment. Int Rev Immunol. 2011;30:219–31.^{, 22}22. Nobre SR, Silva T, Pina Cabral JE. Doença Celíaca Revisitada. J Port Gastrenterol. 2007;14:184-93.⁾.

In the control group represented just by adults, the composition of the intestinal microbiota at the species level is relatively stable. This stability is reduced by factors as disease and old age. Ageing itself has relatively little effect on the overall gastrointestinal function but, due to the decreased adaptive capacity of the gastrointestinal tract, elderly people may not recover from illnesses as quickly as young adults⁽³²32. Tiihonen K, Ouwehand AC, Rautonen N. Human intestinal microbiota and healthy ageing. Ageing Res Rev. 2010;9:107-16.⁾. Some studies have compared the composition of the faecal microbiota of different age groups. Hopkins and collaborators (2001)⁽¹²12. Hopkins MJ, Sharp R, Macfarlane GT. Age And disease related changes in intestinal bacterial populations assessed by cell culture 16S rRNA abundance, and community cellular fatty acid profiles. Gut. 2001;48:198-205.⁾ observed that levels ofBifidobacterium were lower in elderly subjects compared to younger adults, so the results in the celiac group can have suffered some influence by age.

The currently available treatment for CD consists in dietary exclusion of grains and derivatives containing gluten called as gluten-free diet (GFD) throughout the life⁽¹⁷17. Lionetti E, Catassi C. New clues in celiac disease epidemiology, pathogenesis, clinical manifestations, and treatment. Int Rev Immunol. 2011;30:219–31.⁾. The compliance to a gluten-free diet is an extremely challenging task, given the problems related to cross-contamination, lack of clear food labeling policies, and poor information on minimal toxic amounts of gliadins for celiac subjects. Even so, patients adhering to a GFD report an improvement in their life quality. The response to therapy is poor in up to 30% of patients, and dietary non-adherence is the chief cause of persistent or recurrent symptoms⁽¹¹11. Green PHR, Cellier C. Celiac Disease. N Engl J Med. 2007;357:1731-43.⁾. While CD serology became negative in 83% of celiac patients with Marsh lesions on a GFD, mucosal biopsy histology normalized in only 8%⁽⁷7. Freeman HJ, Chopra A, Clandinin MT, Thomson ABR. Recent advances in celiac disease. World J Gastroenterol. 2011;17:2259-72.⁾. So the complete normalization of duodenal lesions is exceptionally rare in adult celiac patients despite adherence to GFD, symptoms disappearance and negative CD related serology⁽⁷7. Freeman HJ, Chopra A, Clandinin MT, Thomson ABR. Recent advances in celiac disease. World J Gastroenterol. 2011;17:2259-72.⁾. Because of this, also due to the refractory disease cases and the instability of fecal samples, our number of celiac patients was so small.

Changes in the gut microbiota appear to be involved with a reduction of healthy bacteria and increase in potentially pathogenic species. These changes are generally described in gastrointestinal disorders and infections⁽³⁰30. Shanahan F. The colonic microflora and probiotic therapy in health and disease. Curr Opin Gastroenterol. 2011;27:61-5.⁾. In this context, there are recommendations for the use of probiotics in necrotizing enterocolitis, childhood diarrhea, inflammatory bowel disease, irritable bowel syndrome, allergy, and to improve the immune response⁽⁶6. Floch MH, Walker A, Madsen K, Sanders ME, Macfarlane GT, Flint HJ, et al. Recommendations for probiotic use-2011 update. J Clin Gastroenterol. 2011;45:S168–71.⁾. The roles of probiotic bacteria for patient’s health also led Sanz (2009)⁽²⁸28. Sanz Y. Novel perspectives in celiac disease therapy. Mini Rev Med Chem. 2009;9:359–67.⁾ to propose the use of probiotics as a nutritional strategy to improve the quality of life of celiac patients.

In this context, our results showed a significantly lower concentration of bifidobacteria in the feces of celiac patients compared with controls, even in FGD, and absence of typical symptoms.

Some studies have also reported that allergic children and patients with atopic diseases are often colonized by a reduced number of bifidobacteria in comparison with healthy subjects, demonstrating a strong correlation between the concentration of bifidobacteria and the host immune disorders⁽¹³13. Kalliomäki M, Kirjavainen P, Eerola E, Kero P, Salminen S, Isolauri E. Distinct patterns of neonatal gut microflora in infants in whom atopy was and was not developing. J Allergy Clin Immunol. 2001;107:129-34.⁾.

The imbalance in duodenal biota from celiac patients has already described in children⁽²¹21. Nadal I, Donat E, Ribes-Koninckx C, Calabuig M, Sanz Y. Imbalance in the composition of the duodenal microbiota of children with celiac disease. J Med Microbiol. 2007;56:1669–74.⁾, and it was also observed a reduction in concentration of species of Bifidobacterium in feces and intestinal biopsy specimens of children⁽³3. Collado MC, Donat E, Ribes-Koninckx C, Calabuig M, Sanz Y. Imbalances in faecal and duodenal Bifidobacterium species composition in active and non-active coeliac disease. BMC Microbiol. 2008;232:1-9.^{, 5}5. Di Cagno R, Rizzello CG, Gagliardi F, Ricciuti P, Ndagijimana M, Francavilla R, et al. Different fecal microbiotas and volatile organic compounds in treated and untreated children with celiac disease. Appl And Environ Microbiol. 2009;75:3963–71.⁾. However, the children’s intestinal biota is variable and depends on the type and how long they were breastfeed. Differences in gut microbiota composition exist between breast-fed and formula-fed infants⁽²⁵25. Parracho H, McCartney AL, Gibson GR. Probiotics and prebiotics in infant nutrition. Proc Nutr Soc. 2007;66:405-11.⁾. In addition, it is difficult to specify when the intestinal microbiota becomes adult.

As Collado and collaborators⁽³3. Collado MC, Donat E, Ribes-Koninckx C, Calabuig M, Sanz Y. Imbalances in faecal and duodenal Bifidobacterium species composition in active and non-active coeliac disease. BMC Microbiol. 2008;232:1-9.⁾ observed in celiac children, we observed that celiac adult patients with non active disease showed less amount of bifidobacteria than control group in fecal samples. Therefore, this imbalance appeared to be independent of disease activity and of fecal pH.

The association of CD with intestinal dysbiosis and the evidences supporting the roles for microbiota and specific bacteria in regulating key aspects of innate and acquired immunity have led to the evaluation of possible benefits of potentially probiotics⁽²⁷27. Sanz Y, De Pama G, Laparra M. Unraveling the ties between celiac disease and intestinal microbiota. Int Rev Immunol. 2011;30:207-18.⁾.

In this sense, the analysis of the fecal bifidobacteria concentration, better than pH, could be a routine screening in clinical laboratories to evaluate the necessity of dietary supplementation for the use of pre- and/or probiotics on celiac patients.

For fecal analysis of intestinal, contents should be taken in mind variables such as the intestinal motility, the total quantity of ingested fiber, intestinal secretions, such as the production of short chain fatty acids, and duration of dietary intervention. In this sense, the fecal pH may not accurately reflect the pH of the colon⁽⁸8. Garro MS, Aguirre L, Savoy de Giori G. Biological activity ofBifidobacterium longum in response to environmental pH. Appl Microbiol Biotechnol. 2006;70:612-7.^{, 14}14. Kleessen B, Sykura B, Zunfit HJ, Blaut M. Effect of inulin and lactose on fecal microflora, microbial activity, and bowel habit in elderly constipated persons. Am J Clin Nutr. 1997;65:1397-402.⁾.

The colonic fermentation of remaining substrates, held by bacteria such as bifidobacteria, is a complex process. In the cecum and proximal colon the fermentation is higher due to the intense supply of nutrients, production of high short-chain fatty acids, causing a decrease in pH⁽⁵5. Di Cagno R, Rizzello CG, Gagliardi F, Ricciuti P, Ndagijimana M, Francavilla R, et al. Different fecal microbiotas and volatile organic compounds in treated and untreated children with celiac disease. Appl And Environ Microbiol. 2009;75:3963–71.^{, 16}16. Leahy SC, Higgins DG, Fitzgerald GF, van Sinderen D. Getting better with bifidobacteria. J Appl Microbiol. 2005;98:1303-15.⁾, and resulting in rapid bacterial growth. In the distal colon, the substrates are less available, the pH approaches neutral (6.0 to 7.0) and bacterial growth occurs more slowly⁽¹⁰10. Gibson GR, Roberfroid MB. Dietary modulation of human colonic microbiota: introducing the concept of the prebiotics. J Nutr. 1995;125:1401-12.⁾.

In general, there is an increase in fecal concentration of bifidobacterias and a decrease in fecal pH after consumption of pre- or probiotic⁽¹⁹19. Mohan R, Koebnick C, Schildt J, Mueller M, Radke M, Blaut M. Effects of Bifidobacterium lactis Bb12 supplementation on body weight, fecal pH, acetate, lactate, calprotectin, and IgA in preterm infants. Pediatr Res. 2008;64:418-22.⁾. However, Bouhnik et al.⁽¹1. Bouhnik Y, Flourié B, D’Agay-Abensour L, Pochart P, Gramet G, Durand M, Rambaud JC. Administration of transgalacto-oligosaccharides increases fecal bifidobacteria and modifies colonic fermentation metabolism in healthy humans. J Nutr. 1997;127:444-8.⁾ demonstrated that the intake of prebiotics increased the contents of bifidobacteria in feces, but no changes were observed in total amount of aerobic microorganisms, concentrations of bile salts and fecal pH.

Despite the difference in fecal pH between control and celiac groups was not statistically significant, it is noteworthy that the pH of control subjects, with higher contents of fecal bifidobcteria, was slightly more acid than the celiac patients, probably due to fermentation performed by these bacteria⁽²2. Búrigo T, Fagundes RLM, Trindade EBSM, Franz HCF. Bifidogenic effect of fructooligosaccharides in the intestinal flora of patients with hematological neoplasia. Rev Nutr 2007;20:491-7. ⁾.

At the same time, the results demonstrate that the smallest amount of bifidobacteria in the feces of celiac patients probably is not related to the fecal pH, but with the pathogenesis of DC. Therefore, the relationship between the amount of bifidobacteria and DC should be elucidated. The smallest amount of bifidobacteria in the feces of celiac might be due to the absence of some kind of food that stimulates their growth. Or due to the intestinal inflammatory process that can be present even in celiacs under GFD, probably due to small contaminations that occur in foods referred as gluten free.

According to all demonstrations of probiotic health benefits⁽²⁷27. Sanz Y, De Pama G, Laparra M. Unraveling the ties between celiac disease and intestinal microbiota. Int Rev Immunol. 2011;30:207-18.⁾ bifidobacteria has shown a great therapeutic potential and may improve the prognosis of CD. In this sense, we suggest that the intestinal biota could be evaluated more frequently by laboratory tests and if necessary carry out a dietary supplementation with probiotics or prebiotics to improve the quality of life of celiac patients.

However, more in-depth studies investigating the interrelationships among intestinal bacteria, diet and CD are desperately needed to justify why celiac patients have fewer bifidobacteria in controlled phase of the disease (non-active celiac disease), i.e. under gluten-free diet and no clinical symptoms. For example, more studies are needed in patients with negative biopsy to answer if the biota’s imbalance is related to inflammation or villi’s damage that are still present in a patient under the GFD, or if it is a consequence of contamination of less than 20 ppm of gluten in diet called gluten free, or if the GFD may be failing to provide substrates required for bifidobacteria’s growth. Once the answers to these fundamental questions are available, it should be possible to develop specific dietary recommendations for CD based on modification of the composition or activities of the colon’s commensal microbiota.

REFERENCES

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