Materials EXCLUSION CRITERIA INCLUSION CRITERIA

Transcription

1 Bifidobacteria numbers in colon of children with inflammatory bowel disease (IBD). Gosiewski T 1, Kochan P 1,StrusM 1, Brzychczy-Wloch M 1, Drzewiecki A 1, Kowalska-Duplaga K 2, Wedrychowicz A 2, Jedynak-Wasowicz U 2, Sladek M 2, Pieczarkowski S 2, Fyderek K 2, Heczko P.B 1 1 Chair of Microbiology, 2 Clinic of Paediatrics, Gastroenterology and Nutrition Medical College, Jagiellonian University What is IBD? IBD (Inflammatory Bowel Disease) is inflammatory disease of the G.I. tract with undetermined aetiology. Crohn`s Disease (CD) Ulcerative colitis (UC) Indeterminate colitis (IC) Pathological changes are present throughout the GI tract, but they usually involve only fragments of the bowel Inflammation relates to the whole thickness of the GI tract wall Fistula formation is common Loss of body weight Pathological changes are present only in the colon and these changes are continuous. Inflammation relates only to mucosa Blood in stools No fistula formation Loss of body weight only if the course is severe Intermediate form between CD and UC.

2 IBD pathogenesis BACTERIAL INTESTINAL MICROFLORA IBD GALT GENETIC PREDISPOSITIONS Factors which predispose to IBD. IBD epidemiology Higher incidence of IBD in developed countries and smaller in developing countries Growing number of IBD cases in countries in Asia, South America and Eastern Europe, including Poland (dynamic civilization development and economic growth).

3 IBD epidemiology IBD epidemiology in children 8 7 frequency per IBD CD UC IC Europe Poland Aims of the study Estimation of changes in the total bacterial flora from the GI tract of children with primary diagnosis of IBD in comparison to the control group.

4 Materials Number of subjects in the study: n=64, children, 1 18 years-old hospitalized in the Clinic of Paediatrics, Gastroenterology and Nutrition of the Polish-American Institute of Paediatrics, Jagiellonian University Medical College Study group: (n=37) IBD patients with primary diagnosis of: 1. CD: n= UC: n= IC: n= 3 Control group: (n=27) patients with functional GI tract disturbances. INCLUSION CRITERIA Bloody diarrhoea Occult blood in stools Chronic diarrhoea Abdominal pain and/or low body weight Histopathological confirmation of CD, UC or IC EXCLUSION CRITERIA Age: < 1 year of life Antibiotic therapy 30 days prior to symptoms Confirmed infectious aetiology Exclusively breastfed children Children with immunodefficiencies Children whos parents/legal guardians did not give their consent for the child to participate in the study

5 Methods Phenotypic methods quantitative microbiological cultures of stool and tissue samples Culture media: blood agar; McConkey s agar; Enterococcosel agar; MRS agar; BL agar; Wilkins Chalgren-supl. Agar; Schaedler`s agar Molecular methods Quantitative FISH from stool samples: Probes: EUB338, STRP, ECOLI, Lab158, Bif164, BAC303, Erec428 Estimation of the mucus thickness produced by the goblet cells Fluorescently labeled MAA lectin, showing affinity to human mucus polysaccharides. Quantitative cultures of the respective stool fractions Content of bacteria in 1 g of faecal matter in the Ist fraction of stool 100% 90% 80% Streptococcus 70% Lactobacillus 60% Enterococcus 50% 40% Enterobacteriaceae 30% Bifidobacterium 20% Bacteroides 10% 0% CD UC IC control group χ 2 =6,4491*10 8, p <0,0001 Comparison of the average (%) bacterial content in 1 g of faecal matter in the Ist fraction of stool collected from patients with IBD and from the control group. Content of bacteria in 1 g of faecal matter in the 2nd fraction of stool 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% CD UC IC control group Streptococcus Lactobacillus Enterococcus Enterobacteriaceae Bifidobacterium Bacteroides χ 2 =2,0761*10 9, p <0,0001 Comparison of the average (%) bacterial content in 1 g of faecal matter in the IInd fraction of stool collected from patients with IBD and from the control group. Content of bacteria in 1 g of faecal matter in the 3rd fraction of stool 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% CD UC IC control group Streptococcus Lactobacillus Enterococcus Enterobacteriaceae Bifidobacterium Bacteroides χ 2 =1,3052*10 9, p <0,0001 Comparison of the average (%) bacterial content in 1 g of faecal matter in the IIIrd fraction of stool collected from patients with IBD and from the control group.

6 Quantitative FISH Examples of FISH photographs - combination of Bif164+EUB probes. Magnification 1000x. (A) stool sample; (B) tissue sample. Estimation of the mucus thickness MAA lectin labeled by FITC Photos showing the thickness of the mucus layer lining the intestinal wall. Magnification 200x. (A) intestinal tissue sample from CD patient; (B) intestinal tissue sample from UC; (C) intestinal tissue sample from control group.

7 Conclusions The bacterial populations in the intestinal lumen in children with IBD, were more numerous than these in children without IBD. In children with IBD, there were, in comparison to controls, increased numbers of bacteria of Enterobacteriaceae family, including E.coli and also those of Streptococcus genus, but lower numbers of anaerobic bacteria and especially these of Bifidobacterium genus. The inflammatory process taking place in the intestines of children with IBD, caused a thinning of the mucus layer lining the intestinal wall.