UC, The inter-individual degree of similarities were 64,7% (s,d, 20.5 ; 41.5-94,9) in control sublects, 517% (s,d. 175 ; 14,8-84,7) in patients with active CD and 73.7% (sA 18,5 ; 52 3~93,7) in patients with quiescent UC Conclusion: We observed a great stability ot the dominant mucosa associated flm'a of the ileum, right colon, leR colon and rectum in the same individual either in the control group or in the IBD group. On the other hand, the mucosa associated fiom varied from one individual to the other. No particular pattern of dominant mucosa associated flora could be observed in relation to disease state or location.

T1067

Profiling the Bacterial Community Associated with the Rectal Mucosa in Inflammatory Bowel disease (IBD) Using 16S rDNA Denaturing Gradient Gel Electrophoresis Graeme Green, Maria Mylonaki, David Pampton, Jonathan Brostoff, Nell Rayment, Barry Hudspith, Norman Staines, Kenneth Bruce

Background: Mucosa-associated bacteria are likely to play a pathogenic role in IBD We have apphed a Polymerase Chain Reaction (PCR) based-technique to investigate the bacterial community associated with zvctal mucosa in order to provide a less biased and more comprehensive approacCh than the use of methods such as selective culture, immunocyto- chemistry, fluorescent in situ hybridisation and electron microscopic examinatinn of biopsies. Ivlethods: Rectal mucosal biopsies (n = 28) were taken at routine colonoscopy from 5 patients with ukerative colitis (mactive 4), 2 patients with Crohns disease (inactive 1) and 11 controls with normal rectal mucosa, Biopsies were immediately snap-trozen prior m mechanical and chemical nucleic acid extraction. Phylogenetically informative 16S rDNA tiagmems were amplified by PCR, Bacterial species composition of the PCR products were identified either by sequence analysis and comparison to phylogenetic data bases (n = 3, 2 controls, i inactive UC), or through Denaturing Gradiem Gel Electrophoresis (DGGE) (n= 15), in wl'nch an electmphoretic profile of the dominant bacterial species is generated as a set of discrete bands which can be compared by Phoretix image anal}sis software.. Results: 16S rDNA clone libraries showed that biopsies contained a wide range of bacterial species from the genera Rumniocc~cus (l 1/3 bmpsies assessed), Fusobacterinm (7/3), and Bacteroides (7/3). When analysed using DGGE, bacterial community" profiles generated from the same DNA extract (n = 2) were identical, and those from adjacent rectal biopsies in the same patient (n = 8) vel T similar, in contrast, none of the 15 patiems biopsied had the same DGGE profile. 1"hem were no clear difli:rences in species profile between patients with IBD and controls in this preliminaI T senes. Conclusions: The bacterial communities of two gut biopsies, taken from adjacent locations, from the same individual were indistinguishable by DGGE profiling. This emphastses that DNA amplification with subsequent DGGE or sequence analysis and companson with done libraries gives highly reproducible results, This satisfies an essential requiremem for the comprehensive analysis of the bacteria that are associated with the rectal mucosa in 1BD aud healthy individuals. (Supported by the Christopher Reeves Charitable Trust)

T1068

Critical Role for Tumor Necrosis Factor-Alpha in Host Defense against Salmonella typhim~rium Hiren V. Muzumdar, Ming-Yu Wang, Beth McCormick, Marcm R. Saban, Ricardo Saban, Barry K, Wershil

Background: Mice with mutations in the c-kit gene exhibit impaired immunity to Salmonella typhimurium int~:ction manifested as a significant increase in mortality. Bone marrow" trans- plantation of c-ki>deficient mice does not alter the mortality associated with Salmonella intection, Objectives: To determine and evaluate potential molecular mechanisms revolved in the impaired host response to Salmonella typhimuriam infection in c-kit-deficient mice, Methods: c-kit-deficient and normal ( + / + ) mice (n=8/group) were administered either 15 x 107 Salmondla typhimurium (strain X3306) or saline by gavage. After 3 days, the mice were sacrificed, and RNA isolated fi'om the distal small intestine, RNA was then pooled into 4 groups and reverse trans<ribed, 1"he cDNAs obtained were hybridized to the Atlas TM Plastic Mouse 5K Microarray and gene expression levels between the 4 groups were determined. Ratios of gene expression in Salmonella- to saline<hallenged mice were calculated. A greater than three4bld increase in gene expression levels comparing these ratios between normal arid c-kit-deficient mice was considered significant. Based on the results of the micmarray analysis, tumor necrosis factor (TNF)-~-deficient and wild-t)`Ne mice (n= 12/group) were inkcted with Salmonella typhimurium, The survival rate was then determined. Results: A total of 240 genes exhibited increased expression ni + / + compared with c-kit-deficient mice, Included among these genes, were several associated with TNF-eL-dependent pathways. The mortality rate in TNF-cc-deticient mice was 100% as compared with no deaths in tbe wild-type (control) group (p < 0 0001). Conclnsions: TNF-cr plays a critical role in host ddense to Salmonella typhimurium intection in mice, and may contribute to impaired host detense in c-kit-deficient mice. Gene array analysis is a useful approach to identify molecular targets of fiost detense to microbial pathogens,

T1069

Role of Luminal Bacteria In Stress Induced Intestinal Pathophysiology and Inflammation Jennifer Jury, Kathene Johnson-Henry, Mehri Zareie, Ping-Chang Yang, Johan D. Soderholm Philip M Sberman, Derek M. McKay, Mary H Perdne

Background Our previous studies have shown that in na{ve rats chronic psychological stress causes intestinal mucosal barrier dysfunction, bacterial adhesion and penetration into epithelial cells, as well as an increase in recrintment and activation of immune cells. Although microbes have been inlpiicated in the pathogenesis of chronic inflammation, the sequence of events and the tactors which hnk inlpaired bamer timction, enteric flora and inflammation are unclear. This study attempts to determine the role of commensal hacterla in the mucosal changes and intlammation resnlting from chronic psychological stress by modil~4ng gut flora

with antibiotics Methods: Male rats were treated with a combination of antibiotics (AB): imipenum and vancomyein (50/kg/day) for 5 days, while controls were not treated (NT). AB and NT rats were then subjected to 1 h of water avoidance stress (W.ad) for 10 consecutive days (AB continued during stress period). Subsequently, segments of ileum and colon were prepared for light and 'electron microscopy (to assess inflammation and pathology), and Ussing chamber studies (to assess mucosal ion transport and barrier hmction). Resuks: Rats in both groups lost weight during the stress period. Colonic motility (as indicated by increased pellets expelled/h of WAS) was not different between the AB treated and NT groups, lon secretion and macmmolecukar transport were increased by WAS in both the ileum and colon and AB did not significantly alter these responses. However, AB did reduce epithelial damage and the numbers of inflammatory cells (mast cells, eosinophils and mononudear ceils) recruited to the mucosa during WAS. In addition, there were fewer bacteria adhering to the epithelium and penetrating into epithehal cells in AB vs NT stressed rats Bacterial cultures of the colon suggested that the critical adhering microbes were anaerobic bacteria. Conclusions: This stud), shows that altering the composition of intralumi- nal bacteria with broad spectrum antibiotics attenuates the effects of psychological stress on the intestinal mucosa, reducing 1) epithelial damage, 2) mucosal inflammation and 3) enhanced bacterial internalization. However, antibiotics do not abrogate stress-induced gut pathophysiology'.

T1070

Specific detection of the probiotic Escberichia coil strain Nissfe 1917 in fecal samples Gabriele Blum-Oehler, Sibylle Oswald, Karin Eiteljoerge, Corinne Enders, Ulrich Sonnenbom, Wolfgang Kmis, Joerg Hacker

The non-pathogenic Escherichia coli strain Nissle 1917 (EcN) is the active agent of the probiotic drug Mutaflor which has been shown in clinical trials to beneficially affect the course of disease in ulcerative colitis and Crohn's disease patients. EcN produces fimbrial adhesins, siderophores, and at least two microcins. Furthermore, EcN carries two unique smaU cr),ptic plasmids, pMUT1 and pMUT2. So tar, methods to follow colonization of patients with BeN were solely" based on classical microbiological methods. Although efficient these methods are time-consuming, and detection limits vary greatly depending on the overall amount of similar microorganisms in the sample. To overcome these problems, we have developed five PCR assays in order to establish a specific detection system for EcN. The PCRs are based on the chromosomaily encoded major fimbrial subunit genes of type 1 fimbriae (limA) and F1C fimbriae 0bcA) as well as on specific DNA sequences of plasmids pMUT1 and pMUT2. For validation we have screened 354 different pathogenic and non- pathogenic E. coli strains from various origins and could demonstrate that the four primers based on plasmid pMUT2 (Muta 7/8 and 9/10) were most specific for EcN To assess the usefulness and sensitivity of the pMUT2-based PCR assays, fecal specimens from health)' humans and ammals (pigs and calves), who had never been treated with EcN, were spiked with 10-fold dilutions of an overnight culture of EcN DNA extracts were prepared from the fecal samples (without prior cultivation). The PCR results obtained demonstrated an excellent sensitMty of the assays, since even in the presence of normal gut bacteria, with primers Muta 7/8 gcN could still be detected at a level of 10.000 CFU/ml, while the detection limit with primers Muta 9/10 was even 1.000 CFU/ml. Application of the pfasmid-based PCR assays to fecal samples from patients suffering from ulcerative colitis, treated or not treated with EcN, confirmed the utility' and sensitMty of the method. The detection limits in clinical specimens were comparable to those obtained during the validation procedure. The PCR assays presented here are useful tools for effective and accurate identification of EcN, not only from pure cultures, but also, after oral application of EcN, from fecal samples in the presence of other E. coli strains. The new detection assays will help saving time and materials and may also allow us to gain nmre insight into colonization efficienaies of strain EcN in humans and animals.

T1071

Differences, in Human, between the Bacter/al Communities of the Cecum and the Feces: a Study using Direct Analysis of 16S rRNA Genes Antonia Suau, Philippe Marteau, Joel Dote, Matthew David Collins, Philippe Pochart

Backgrounds/Aims : The human intestinal tract harbors a complex microbial community which plays a key role not only in nutrition and colonic physiology, but also in the pathogenesis of some colonic diseases. The composition of the cecal microflora is poorly known in human. Culture methods have shown that it differs from the fecal microt]ora. However, the limit of such methods is that 60 to 80% of the observable fecal bacteria cannot be cultivated. The phylogenetic analysis of bacterial 16S rRNA genes, amplified directly from complex communities, provides an efficient strategy" for exploring the biodivemity of a particular biota, and has been largely used to describe the fecal flora. The aim of the present stud3," was m corapare the composition of the cecal and the tecal flora of an healthy adult using this strategy,. Methods : A sample of cecal fluid from a health)," sul~ject was collected using a long tube which had been positioned on the day before sampling. Feces were collected on the same day. Total DNA was extracted from intestinal samples. Then 16S rRNA genes were amplified using PCR, cloned and sequenced. Sequences were compared one to another and with sequences available in public databases in order to derive a phylogenetic picture of the two ecosystems. Results : There were 24 distinct molecular species among the 78 clones fi-om the cecal sample and 30 among the 74 from the tecal sample. Potentially new species accuumed for 29% of the molecular species in the cecum compared to 43% in the feces. The B!fidobacteriam and Streptococcus groups were more important in the cecum (44 and 11%, respectively) than in the feces (13 and 1%, respectively). Furthermore, E. coil could be detected only in the cecum. On the other hand, some phylogenetic groups appeared to be more important in the feces compared to the cecum both by the munber of clones and the species diversity : Clostridium coccoides (42 and 18 %, respectively), Clostridium leptum (16 and 1%, respec- tively) and Atopobium (11 and 5 %, respectively).

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