Tu1714

Changes in Bacterial Composition Following Fecal Microbiota Transplantationfor Severe Clostridium Difficie InfectionAlexa Weingarden, Matthew J. Hamilton, Michael J. Sadowsky, Alexander Khoruts

Severe Clostridium difficile infection (CDI) is associated with high mortality even withsurgical intervention. Fecal microbiota transplantation (FMT) is a potential alternative treat-ment approach, but one that has not yet been mechanistically investigated. The primaryaim of this study was to measure changes in the bacterial composition of fecal microbiotafollowing FMT in treatment of severe CDI refractory to standard antibiotic therapy. Herewe report outcomes of four consecutive cases of severe CDI, refractory to antibiotic therapy,treated with FMT at our institution. Standardized frozen fecal microbiota material we haddescribed previously (Hamilton, et al., Am J Gastroenterol, 2012) was used in three out offour cases. FMT was performed by a colonoscopic route following antibiotic cessation for12-24 hours and purging intestinal contents with a polyethylene glycol based laxative. Gentlecolonoscope insertion technique without loop formation was used in all cases, and CO2was used for insufflation. Bacterial composition of distal gut microbiota was characterizedbefore and after FMT with frozen material using high throughput 16S rRNA gene sequenceanalyses . FMT resulted in initial clinical improvement in all four cases. However, thesubsequent course was characterized by recurrence of CDI. This was associatedwith instabilityof the bacterial composition of fecal microbiota, specifically increasing abundance of Entero-bacteriaceae and decreasing abundance of Lachnospiraceae and Bacteroidaceae family mem-bers. Optimal stabilization of the fecal microbiota composition and clearing of CDI wasachieved by use of the second FMT performed on an outpatient basis following an interveningcourse of antibiotics. In conclusion, FMT is a promising alternative to surgical treatment ofsevere CDI. However, a single FMT may not achieve sustained protection against recurrenceof CDI in this clinical situation. This problem may be solved by a double FMT protocol wedescribe here. The first FMT allows clinical stabilization of the patient. However, three daysafter FMT patients are started on another course of antibiotics such as vancomycin orfidaxomicin. The second FMT is performed after completion of the antibiotic course on anoutpatient basis. Availability of standardized frozen fecal microbiota material should allowperformance of urgent FMT, which is commonly required in the setting of severe CDI. Thestandardized FMT protocol may simplify future testing of this approach in randomizedclinical trials.

Tu1715

Candida Albicans Colonization and Anti-Glycan Antibodies in Active andQuiescent Crohn's DiseaseRomain Gerard, Boualem Sendid, Aurore G. Techy, Gwenola Vernier-Massouille, ThierryJouault, Nadine Francois, Jean-Frederic Colombel, Daniel Poulain

Background : Recent experimental data have further suggested that apart from bacteria, thefungal flora (mycobiome) may have a role in intestinal inflammation (1). Anti-glycan antibod-ies including ASCA, which are highly prevalent in Crohn's disease (CD), are directed againstcell wall yeast antigens (2). Aims: To perform a longitudinal study of yeast colonization inthe stools in parallel with determination of anti-glycan antibodies in sera during active andquiescent CD. Patients and Methods: Stool samples and sera were collected from 22 patientswith CD in acute (Harvey-Bradshaw index .6 and CRP . 6mg/l) and in remission phases(Harvey-Bradshaw index ,4 and CRP ,6mg/l). Yeasts were isolated from stools by usualmycological procedures (CHROMOGAR medium; Becton Dickinson, Paris, France) andidentified by API 32C system. The number of colony forming units (CFUs) was assessedsemi-quantatively as follows: low (1-5 CFU), medium (5-20 CFU), high (20-50 CFU), andvery high (.50CFU). Serum samples were tested for anti-glycan antibodies: ASCA, ALCA,ACCA and AMCA by enzyme liked immunosorbent assay (IBDX® panel, Glycominds, Lod,Israel), and for anti-Candida mannan antibodies (Platelia Candida Ab, Bio-Rad, Marnes laCoquette, France). Results: All together yeasts were detected in 68 % of patients during theacute phase and 66% during remission and 81% of patients were colonized at least onceduring the follow-up. C. albicans was the most commonly isolated yeast species representing96% of isolated yeast species in all patients. One patient was colonized by C. parapsilosisand 3 patients had mixed colonization associating C. albicans with C. kefyr and C. glabrata.During the acute phase, 8/22 (36%) patients had a very high colonization level (CFU>50)as compared to 2/22 (9%) during remission (p ,0.03). Seven out of the 8 patients whowere heavily colonized (CFU>50) during the active phase had a decreased colonization levelduring the remission phase. There was no difference as far as gender, disease duration,disease behavior and location between these 8 patients and the others. Anti-glycan and anti-Candida antibodies status were stable during active versus quiescent disease and anti-glycan antibodies levels did not change significantly. Conclusion: CD patients are frequentlycolonized by C. albicans and are more heavily colonized in active than in quiescent phaseof the disease while the antiglycan antibodies status is not influenced by disease activity.Colonization by C. albicans may participate in triggering intestinal inflammation in CD.References (1) Iliev ID, et al. Science 2012;336:1314-7. (2).Poulain D, et al. Dig Dis2009;27:104-10.

Tu1716

Evaluation of the Impact of Different Commercially Available DNA ExtractionKits and Laboratories for Assessing Bacterial Community Structure in FecalSamples - Implications for IBD ResearchNicholas A. Kennedy, Alan Walker, UK IBD Microbiota Consortia, UK IBD GeneticsConsortia, Susan H. Berry, Sylvia H. Duncan, Freda Farquharson, Petra Louis, JackSatsangi, Harry J. Flint, Charlie W. Lees, Georgina L. Hold

Background:Determining the bacterial community structure in fecal samples through ampli-fication and sequence analysis of extracted DNA has become a very important facet of IBDresearch. The possible impact of different commercially available DNA extraction kits uponbacterial community structures has, however, received little attention. Limited evidence alsoexists on the possible influence of different laboratory environments on the data generated.

S-829 AGA Abstracts

Aim: To analyse bacterial communities in healthy volunteer and IBD patient fecal samplesextracted using the MoBio and Fastprep DNA extraction kits in two established gastrointesti-nal research laboratories. Methods: Fecal samples from two healthy volunteers and two IBDpatients with relapse were investigated. DNA extraction was undertaken using the MoBioand Fastprep DNA extraction kits in established molecular laboratories (AU and RINH).Two protocols were followed for processing samples using the Fastprep kit (Figure 1). EachDNA sample was then split and an aliquot transferred to the other lab. PCR amplificationfor Next generation sequencing (NGS) of bacterial 16S rRNA genes was performed in bothlaboratories on all samples. Quantitative PCR analysis (q-PCR) to validate NGS data wasalso performed. Hierarchical clustering was done using the Jaccard similarity coefficient onthe NGS data. Results: DNA extracted using methods FastPrep1, FastPrep2 and the MoBiokit yielded median DNA concentrations of 476 (interquartile range 290-518), 453 (IQR228-689) and 22 (IQR 9-36) ng/μL respectively. Those obtained with the MoBio kit weresignificantly lower than with the FastPrep kit (p ,0.0001), while the concentrations werecomparable between the two methods of the FastPrep kit. Hierarchical clustering of NGSdata revealed four clusters, with the samples clustering by patient. Within each patientcluster, the samples clustered by DNA extraction kit. Linear modelling of the effect of patientand kit on relative abundance of common bacterial classes revealed significant differencesbetween the MoBio kit and the FastPrep kit. Ruminococcaceae and Bacteroidetes weresignificantly increased in samples extracted with the MoBio kit, while Lachnospiraceae andEnterobacteriaceae were significantly reduced (p , 0.05 in each case). q-PCR revealed goodcorrelation with the NGS data, with R2 of 0.94, 0.82, 0.69 and 0.57 for Enterobacteriaceae,Bacteroides, Ruminococcaceae and Lachnospiraceae respectively. Conclusion: This studydemonstrates significant differences in DNA yield and bacterial DNA composition seen whencomparing DNA extracted from the same faecal sample with different extraction kits. Thishighlights the importance of ensuring that all samples to be analysed together are preparedwith the same DNA extraction method, and the need for caution when comparing studiesthat have used different methods.

Tu1717

Interaction Between Glycans and the Immune System: Do Glycans Play a Rolein Crohn's Disease?Liran Baram, Sarit Cohen-Kedar, Lior Spektor, Hofit Elad, Hagit Tulchinsky, Guzner-GurHanan, Iris Dotan

Introduction: Crohn's disease (CD) is characterized by loss of tolerance towards intestinalmicroorganisms, reflected by serologic responses towards fungal-related glycans such asmannan and beta-glucans. Objective: To explore glycan-induced immune responses andtheir correlation with intestinal inflammation. Methods: Peripheral blood mononuclear cells(PBMCs), isolated from CD and normal control patients, were stimulated by glycans or heatkilled (HK) yeasts. Mucosal cells were freshly isolated from surgical specimens. Mucosalbiopsies were obtained from CD and ulcerative colitis (UC) patients (inflamed/ non-inflamed)and controls. Expression of the beta-glucan receptor- Dectin1, and mannose receptor- MR,cytokine secretion, and signaling pathways were assessed using flow cytometry, immunofluo-rescence, and ELISA. Dectin1 (CLEC7A) and MR (MRC1) mRNA levels were assessed byreal time quantitative PCR. Results: Mannan induced significantly higher pro-inflammatorycytokine secretion by CD vs. normal PBMCs: IL-1-beta [1277.3 vs. 557.8 pg/ml], IL-23[52.1 vs. 1 pg/ml], (p,0.05). Significant inhibition of glycan-induced cytokine secretionwas observed using Syk and Src inhibitors (up to 90% inhibition). HK C. albicans inducedhigher TNF-α, while HK S. cerevisiae induced lower IL-10 secretion by CD vs. normalPBMCs. Dectin-1 is expressed in the lamina propria and by freshly isolated epithelial cells.Mucosal Dectin1 and MR expression was higher in inflamed CD, but not UC vs. controls.Conclusions: Glycans are capable of stimulating innate immune responses. Glycan receptorsare expressed by peripheral and mucosal immune cells and are enhanced in intestinalinflammation, specifically in CD. CD is characterized by hyper-responsiveness towards yeast-characteristic glycans. Thus, glycans may have a role in the pathogenesis of CD.

Tu1718

Gut Microbiota Transfer and Genetic Strain Differences Have Time-Dependent Effects on Colitis Severity in IL-10-Deficient Mice on 129SvEv andC57BL/6 BackgroundsBo Liu, Yoshiyuki Mishima, Michael T. Shanahan, Chang Soo Eun, Lisa C. Holt, CongWu, Ryan B. Sartor

Colitis susceptibility in IL-10 deficient (KO) mice depends on genetic background and gutmicrobiota. Berg et al. reported that KO mice on a 129SvEv (129) background developedmore aggressive inflammation than those on a C57BL/6 (B6) background and Gulati et al.showed that mouse strain influenced antimicrobial peptide expression and perhapsmicrobiota (PLoS ONE 2012). Aim: Compare the impact of 1) differential genetic background

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son severity of colitis induced by identical microbiota and 2) transfer of microbiota fromdifferent genetic backgrounds on induction of colitis in wild-type (WT) and KO mice ofeach strain. Methods: Germ- free KO or WT mice on B6 or 129 backgrounds were colonizedwith specific pathogen free feces (Helicobacter species-free) from 129 WT or B6 WT mice(Taconic) and evaluated 2 and 6 weeks post inoculation (PI). Colitis severity was evaluatedby blinded histological scores combined with measurement of cytokines secreted by MLNcells stimulated by cecal bacterial lysates (CBL). Lamina propria (LP) Th1/Th17 T cells wereanalyzed by flow cytometry (FACS). Results: No colitis was seen in B6WT or 129WTrecipients inoculated with B6 or 129 feces. At 2 weeks PI, KO mice on both B6 or 129backgrounds that were colonized with B6 feces developed more aggressive colitis comparedwith mild/moderate disease in those mice inoculated with 129 feces (p ,0.05, Table). B6feces induced more aggressive inflammation in B6KO than in 129 KO mice (p ,0.05)accompanied by higher levels of IL-17, IFNγ and IL-12/23p40 production by CBL- stimulatedMLN cells (p,0.05-0.005). Interestingly, B6KO and 129KO mice colonized with 129 fecesdisplayed no obvious differences in severity of colitis and production of IL-17 and IFN γ byMLN cells. FACS assay showed that frequency of IFN γ+- (2.4 vs. 1.8% of total LP cells) andIL-17+- CD4+ T cells (1.4 vs. 0.9%) in LP was higher in B6 feces →B6KO mice vs.129feces→B6KO. At 6 weeks PI, increased colitis and elevated cytokine secretion were seen in129 KO vs. B6KO mice inoculated with B6 feces (p ,0.05 & 0.005). The source of fecalcolonization did not consistently affect the intensity of chronic colitis or cytokine secretionin B6KO or 129KO recipients. Conclusions: IL-10-/- mice on the B6 background developedaggressive colitis earlier than mice on a 129 background and remained stable or evendecreased with passage of time in contrast to 129 KO mice, which displayed progressivelyincreased colitis that exceeded that of B6 KO mice by 6 weeks. Different sources of fecesinfluence the severity of disease in IL-10 KO mice, particularly early in disease course,perhaps before host genetic background alters the microbiota.

Tu1719

CD103+ Dcs Contribute to the Manifestation of Citrobacter RodentiumInduced ColitisVera Kitowski, Markus F. Neurath, Raja Atreya, Kai HIldner

Dendritic cells (DC) are crucial orchestrators of adaptive immune responses during mucosalinflammatory conditions and infections. Furthermore, previous data under participation ofour group showed that certain pathogens specifically target subpopulations of dendritic cells(DC) to gain access to the infected host. For example, after i.v. administration Listeriamonocytogenes specifically infect CD8a+ DCs, which results in the transport of bacteriafrom the marginal zone into the T-cell zone of the white pulp. Mice deficient for thetranscription factor Batf3 lack CD8a+ and related CD103+ DC and are therefore protectedagainst i.v. Listeria infection. Here we seek to examine the contribution of mucosal CD103+DCs to intestinal inflammation and infection in the Citrobacter rodentium colitis modelusing Batf3-/- mice lacking CD103+CD11b- DCs. Oral Citrobacter rodentium (C. r.) infectioninduces colitis in mice and is widely accepted as a colitis model for human-pathogenic E.coli infections like EHEC. Following up the course of infection for more than 21 days afterp.o. infection of wildtype or Batf3-/- mice with a bioluminescent strain of C. r by in vivobioluminescence imaging, bacterial counts increased in wildtype mice and peaked at day7-10. In contrast, Batf3-/- mice were virtually protected against the expansion of C. r. invivo and displayed significantly reduced bacterial counts compared to wildtype control micethroughout the 21 day observation period. The caecum and here a specific immunologicstructure, the caecal patch (CP), are crucial anatomic sites where C. r. infection first accumu-lates within hours after p.o. infection followed by the spread of the bacteria anterogradethrough the colon over the next days. This implies that the CP might be a privileged nichethat is permissive for C. r. infection. Hence, we sought to test the hypothesis that the CPis involved in the diminished infection of Batf3-/- mice. Therefore we performed time courseexperiments comparing the bacterial burden at the CP in Batf3+/+ and Batf3-/- mice. 5hafter oral infection we did not detect any differences in bioluminescence between bothgroups. However, after 24h and especially after 72h Batf3+/+ mice showed significant higherbacterial counts at the CP compared to Batf3-/- mice. In summary, our data suggest a crucialrole of CD103+ DCs during the pathogenesis and establishment of the Citrobacter rodentiumcolitis and infection model. Further experiments are under way to further investigate thecontribution of CD103+ CD11b- DC to the relative protection of Batf3-/- mice after C.r. infection.

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Tu1720

High Plasma Level of Lipopolisaccharyde and Anti-Flagellin Antibodies AmongPatients With Irritable Bowel Syndrome- Signs of Bacterial Translocation?Aldona Dlugosz, Piotr Nowak, Jessica Nyström, Samir Abdurahman, Greger Lindberg

A role of immune activation in the pathophysiology of irritable bowel syndrome (IBS) issupported by a number of observations. Mediators released by immune cells impact on thefunction of enteric and sensory afferent nerves as well as on epithelial tight junctionscontrolling mucosal barrier in cell culture systems. Innate immune responses to conservedmicrobial products such as lipopolysaccharide (LPS) and flagellin are likely to be importantin the microbial-host interactions and intestinal homeostasis. We hypothesized that bacterialtranslocation and activation of mucosal immunity against common microbial antigens mightbe involved in the development of IBS. We therefore compared serum levels of LPS, CD14and flagellin antibodies between patients with different subtypes of IBS and healthy controls.Methods: We analyzed serum obtained from 91 patients (74 females) aged 19-(43)-73 yearsand 106 healthy volunteers (77 females) aged 19-(38)-62 years. Diarrhoea-predominant IBS(D-IBS) was present in 32 patients (35%), 22 patients (24%) had constipation-predominantIBS (C-IBS) and 37 patients (41%) had non-C-non-D IBS. We used ELISA for sCD14 andantibodies and limulus amebocyte assay (LAL) for LPS. Results: We found a significantlyhigher plasma level of LPS in patients with D-IBS compared to controls (p=0.0096). Thelevel of antibodies to flagellin was significantly higher among patients with IBS than amongcontrols (p=0.0003, mainly driven by higher levels in D-IBS). Since we used an in-houseanti-flagellin specific IgG ELISA we also determined the ratio Flagellin IgG/Total IgG.Obtained results confirmed the higher level of anti-flagellin antibodies among IBS patients(p=0.0014). The levels of soluble CD14 was lower among IBS-D patients compared tocontrols (p=0.0284). Conclusion: Our results support the concept that immune reactivityto luminal antigens may have a role in the development of IBS, especially in D-IBS.

Tu1721

Shigella Spp. Adversely Affect Paracellular Permeability in In Vitro and Ex-Vivo Models of Intestinal MucosaMaria R. Fiorentino, Karen M. Lammers, Marcelo Sztein, Alessio Fasano

Background: Shigellosis is a leading cause of diarrheal disease worldwide particularly indeveloping countries where it is estimated that 163 million cases with more than one milliondeaths occur annually. The pathogenesis of Shigella is based on the bacteria's ability to invadeand replicate within the colonic epithelium, which results in severe intestinal inflammatoryresponse and epithelial destruction. Aim: Because vaccines against shigellosis are urgentlyneeded, we are characterizing the pathogenic effects caused by wild type and potentialvaccine strains of Shigella flexneri and dysenteriae-1 in in vitro and ex-vivo models ofintestinal mucosa. Methods: To model the interactions of Shigella with the intestinal mucosa,we apically exposed monolayers of human colonic Caco2 cells and mouse jejeunum mucosato bacterial cultures. We monitored changes in paracellular permeability by measuring thetransepithelial electric resistance (TEER) and the passage of FITC-labeled molecules fromthe apical to the basolateral side. Additionally, we examined the pro-inflammatory responseof epithelial cells exposed to bacteria. Results: Inoculation of Shigella into human Caco2cell monolayers and murine mucosa mounted on snapwells, caused severe mucosal damage,which was apparent as a drastic reduction of the TEER, accompanied by a sharp increaseof paracellular passage of FITC-dextran (4kDa) through the epithelial layer. This decreasein epithelial permeability can be accounted for a breakdown of the tight junction integrity.Indeed, infected Caco2 cells showed the disruption of tight junction components at the cell-cell boundary and no adverse effect on cellular viability. Shigella infection of Caco2 cellsinduced the secretion of IL-8. The attenuated vaccine strains of Shigella (CVD1208S andCVD 1256) elicited a pro-inflammatory response with no damage of the intestinal barrierintegrity. Conclusions: Both the in vitro and ex-vivo systems are effective means to studythe interaction of Shigella with intestinal epithelial cells. Our experiments show that Shigellainterferes with the mucosal barrier function by disrupting the role of components of tightjunctions. In addition, the preliminary data with attenuated strains make them very attractiveas candidate vaccines.

Tu1722

Small Intestinal Microbiota of Colonic IBD Patients May Give Rise to anAltered Gut Microenvironment Enabling Selective Enrichment of BacterialPopulations: Implications of Microbiome Analysis on Ileal EffluentToru Kono, Mitsue Nishiyama, Atsushi Kaneko, Masahiro Yamamoto, Nobuhiro Ueno,Yutaka Kohgo, Yasuhito Uezono

Background & Aim: Recent analysis of the human microbiome indicates that an imbalancebetween harmful and protective bacteria in the intestine is largely responsible for the etiologyof inflammatory bowel diseases (IBD). Although our knowledge of the diversity of the humancolonic microbiota has expanded considerably over the past decade, the composition of themicrobiota in the small intestine remains largely unknown. Here we present the results ofa microbiome study on the ileal effluent collected from ileostomy patients treated with atraditional Japanese herbal medicine daikenchuto (TU-100). TU-100, a prescribed drugwidely used in Japan for the treatment of Crohn's disease and postoperative ileus, has beenshown to alter the intestinal microbiota in experimental animals. Methods: The studyinvolved 11 individuals with colorectal cancers (7 patients) or inflammatory bowel diseases(2 ulcerative colitis and 2 Crohn's disease patients) that had undergone ileostomy aftersurgical resection of the diseased area. All patients were apparently free of inflammatorylesions in the small intestines. Ileal effluent was collected before and four hours afteroral administration of TU-100 (5 g/person). DNA was isolated from each sample and thecomposition of the microbial community was determined using 454 pyrotag sequencing.Results: A total of 761886 16S rRNA sequences with acceptable quality were obtained. Weused a 2% dissimilarity as an indicator of phylotype. In total, 488 phylotypes were foundin the 11 individuals. The profiles of the microbial community markedly differed betweenindividuals and between pre- and post-TU-100 treatment even in the same individual.