It is well known that bacterial microflora in the human gastrointestinal tract play an important role in maintaining human health and may also contribute to the pathogenesis of IBD[17
]. The data from animal models of IBD suggest that intestinal inflammation is dependent on the presence of intestinal microflora, although no specific pathogen has been identified and confirmed to be responsible for this process. Some studies performed on adult patients showed a strong correlation between disease activity and increased numbers of bacteria attached to the mucosa[18,19
]. Although in our study we did not observe a significant increase in microflora contained in the biopsy samples in IBD adolescents in relation to controls, we did find a higher number of bacteria in biopsies taken from UC patients, particularly in those with the most advanced stage of changes as estimated at endoscopy. Increasing disease activity was inversely related to the numbers of lactobacilli in samples from CD (but not from UC patients) and to a minor degree to Streptococcus
In various studies carried out on adult IBD patients, it is possible to find conflicting results relating to the numbers of specific groups or genera of bacteria with respect to inflammation. Some authors reported increased numbers of aerobic Gram-negative rods (E. coli
), while others found increased numbers of non-spore forming anaerobes (Proteobacteria
and decreased numbers of spore-forming anaerobes like Clostridium
]. There are also papers in which the authors found no differences between the bacterial flora in inflamed and non-inflamed tissue in the same patient[25,26
]. On the contrary, Sepehri et al[27
] using a PCR method showed significant differences in bacterial flora between the inflamed and non-inflamed mucosa, and an increase in microbial diversity in controls and the non-inflamed tissue from adult IBD patients.
We found differences in microflora composition between endoscopically inflamed and non-inflamed mucosal sites in newly diagnosed IBD adolescents which were specific for each form of the disease. In the controls and in the endoscopically non-inflamed mucosa of IBD children there were high numbers of bifidobacteria compared with decreased populations of these bacteria in inflamed sites of IBD patients. Thus, our observations are in concordance with the earlier findings of Seksik et al[28
] who also showed decreased numbers of Bifidobacterium
in adult patients with active and inactive CD. Our CD and UC patients also had decreased numbers of Bifidobacterium
in inflamed mucosal sites compared with controls. It should also be noted that the numbers of lactobacilli, known for their protective effect in IBD, were lower in samples from CD adolescents with severe inflammation in our study[29
]. Seksik et al[28
] showed that the numbers of Lactobacillus
strains were low in active and inactive CD adult patients.
Conte et al[9
] recently reported the first, to our knowledge, study on gut-associated bacterial microflora in children with IBD. They found higher numbers of aerobic and facultative anaerobic bacteria in biopsy specimens from IBD children in comparison to controls, but lower numbers of Bacteroides
in CD patients.
It should be noted that more than 50% of intestinal bacteria are not cultivable and this may represent a limitation of the study. Furthermore, molecular analytical methods have utility in discriminating between bowel microbiota of altered compositions and could be used in future studies in children. These techniques enable characterization as well as quantification of the microbiota; composition of the microbiota may be identified with clone libraries, fingerprinting techniques may be used to analyze microbial community structure and dot blot hybridization or fluorescent in situ
hybridization (FISH) may be used to analyze a multitude of given taxa[30-32
]. In our previous pilot study (results not included here) we used FISH to analyze colonic microflora in children with IBD, however, this approach was limited by low sensitivity of the method.
Considering the conflicting results on microflora in IBD presented in the literature one should remember that the patient populations as well as stages of the illness in various studies were different. Other factors that could strongly affect the results and cause obvious discrepancies were: different sites from which the biopsies were taken, the cleaning procedures and microbiological methods used to identify the microflora. Moreover, it is possible that gut bacterial flora in children is generally less stable than in adults. All these factors could be responsible for the observed variations.
We have carried out our investigation using samples of tissue and feces which were stored at -80°C during transport from the ward to the laboratory. According to Achá et al[33
] and Dan et al[34
], freezing does not influence viability of fecal samples.
Colonic mucus is an adherent, water insoluble gel that has several functions, including protection of the epithelium from mechanical trauma, toxins, allergens and from microbial invasion[35
]. Thus, the mucus layer plays a protective role in the intestinal mucosa[36,37
]. The gut mucus lubricates the passage of food and protects the epithelial cells from direct contact with bacterial flora. The mucus consists of glycoproteins called mucins, which are encoded by many MUC
genes - to date more than 20 types of MUC
genes have been described[38
]. Thickness of the mucus layer is related to the dynamic equilibrium between mucus secretion and its subsequent removal into the lumen. In UC, the absence of an adherent layer in some areas may be a result of inadequate secretion or excessive removal. The findings of various studies on mucus synthesis in colitis have suggested that the epithelial layer remains intact in the presence of mild to moderate inflammation. In the presence of severe inflammation or inactivity, however, it tends to be lower than normal[10
In our study we have shown that the thickness of the mucus layer lining the intestinal lumen of adolescents with IBD is about three times thinner in both CD and UC patients compared with the control group. A similar relationship was observed in biopsies taken from inflamed and non-inflamed mucosa in the same IBD patients and these differences were statistically significant. In addition, we have demonstrated that mucus layer thickness measured in tissue samples taken from normal sites in patients with IBD was only slightly thinner than that in the control group and the difference was not statistically significant.
At present, there is no data in the literature on the mucus layer in children and adolescents with CD. There is also no clear explanation of observed differences, however, the results of the latest studies are in accordance with our findings. The course of CD and localization of inflammatory lesions in childhood may be quite different from than in adults. The majority of CD adolescents (63.6%) in our study had colon involvement on endoscopic assessment, similar to UC which may have influenced the observed mucus layer depletion in this group of patients. Moreover, Gersemann et al[39
] using a real-time PCR method in adult IBD patients, showed that goblet cells were diminished in the intestinal biopsies of both UC and CD patients, however, in the CD group enhanced differentiation was found.
In addition, using immunostaining Ardesjö et al[40
] reported the presence of immunoreactive agents in the serum of IBD patients, which reacted with goblet cells in the intestine.
A reduction in mucus layer thickness leads to bacteria from the intestinal lumen having closer contact with the intestinal epithelium, also causing a different selection of microorganisms, which may induce an inflammatory process in the bowel[10
]. In adult patients suffering from IBD, the numbers of bacterial cells adhering to the intestinal mucus layer are much higher as compared to controls without IBD, and this dependence is proportional to the severity of the clinical course[19,21
]. Whether this is a result or cause of bowel inflammation still remains unclear, however, the protective property of the mucosa is significantly weakened by this process.
In summary, our results showed that non-inflamed mucosa in both IBD patients and controls was covered with a thick mucus layer with the attached microflora showing a predominance of Bifidobacterium. In contrast, in inflamed sites there was a reduction in mucus layer thickness with the prevalence of specific bacterial groups which were different for CD and UC: Streptococcus in CD and Lactobacillus in UC. However, numbers of these bacteria decreased proportionally with the intensity of inflammation. Thus, our results also support the idea of a disruption of the balance between the different protective and harmful intestinal bacteria in the gastrointestinal tract. These results also provide evidence which suggest that the therapeutic use of probiotics, especially those containing Bifidobacterium and Lactobacillus may have some positive effects in patients with IBD.