Mo1765 High Level Rifaximin Resistance in Escherichia coli Associated With Inflammatory Bowel Disease Correlates With Prior Rifaximin use and Mutations in Rpob

LC3, a key component of the autophagic machinery. This down-regulation was STAT6dependent and could be recapitulated by treatment of macrophages with IL-4 and IL-13. Knock-down of LC3 significantly inhibited autophagic killing of Citrobacter, attesting to the functional importance of the H. polygyrus-mediated down-regulation of this protein. These observations reveal a new aspect of the immunosuppressive effects of helminth infection and provide mechanistic insights into our earlier finding that H. polygyrus significantly worsens the In Vivo course of Citrobacter infection and the bacterial associated intestinal injury. Mo1763 Clinical Risk Factors for Crohn's Disease Postoperative Recurrence are Reflected in Alterations in Mucosally Adherent Microbiota at Surgical Resection Aravinth U. Murugananthan, Phil Tozer, David Bernardo Ordiz, Ailsa Hart, Stella C. Knight, Kevin Whelan, Naila Arebi, Hafid O. Al-Hassi Introduction and Aims: Clinical risk factors for Crohn's disease (CD) recurrence after ileocaecal resection (ICR) include smoking status, perforating disease and >1 surgical resection. The underlying mechanisms contributing to clinical risk are unknown. We aimed to study the relationship between risk factors and gut microbiota. Methods: Samples of macroscopically inflamed and non-inflamed small bowel from patients undergoing surgical resection for CD were analysed. Samples were snap frozen in liquid nitrogen. Cryosections were cut and the frozen sections were hybridised with oligonucleotide probes targeting the microbial 16S rRNA of total bacteria, Escherichia coli, Bacteroides-Prevotella, Faecalibacterium prausnitzii, Clostrium coccoides- Eubacterium rectale and bifidobacteria. The hybridised mucosa associated microbiota (MAM) were identified and quantified. Patients with ≥1 risk factor were classified as high risk for disease recurrence. Results: Fifteen patients underwent ICR (10 female); 9 were high risk (6 smokers, 4 fistulating disease and 2 recurrent resection- 3 patients had multiple risk factors). Faecalibacterium prausnitzii numbers in inflamed operative samples were lower in smokers compared with non-smokers (p=0.036). High-risk patients had lower numbers of bifidobacteria in both inflamed (p=0.006) and non-inflamed (p=0.01) operative samples compared with low risk patients. Conclusions: The risk of post-operative CD recurrence may be predetermined at a pre-operative stage due to dysbiosis. The role of MAM as a tool to stratify risk requires further study. Drugs that modulate MAM may, in future, play a role in reducing post-operative recurrence.

Mo1761 The Potential Role of ELMO1 in the Internalization of Enteric Bacteria Soumita Das, Katherine A. Owen, Michael R. Elliott, Kodi S. Ravichandran, James E. Casanova, Peter B. Ernst Background: The World Health Organization estimates that each year as many as 4-6 million people die of enteric bacterial infections. After invading intestinal epithelial cells, bacteria gain access to the subepithelial tissue and the lamina propria where they encounter and infect phagocytes. However, little is known about how phagocytes interact and clear the vast, complex populations of enteric bacteria. Clearance of bacteria by phagocytes plays an essential role in the host response to infection. Bacterial internalization requires the rearrangement of actin cytoskeleton, extension of the plasma membrane and activation of small Rho GTPases like Rac1. It has been shown previously that the BAI1-ELMO1-Dock180 pathway stimulate Rac1 activity which has a pivotal role in producing membrane ruffle. ELMO1 was first identified as a protein required for EnguLfment and cell Motility and phagocytosis of apoptotic cells. Recently we found that BAI1 (Brain Angiogenesis Inhibitor 1) recognizes Gram-negative bacteria through their lipopolysaccharide (LPS) and helps in bacterial attachment. We hypothesized that recognition of Gram-negative bacteria by BAI1 leads to ELMO1-mediated signaling events that regulate bacterial internalization and the host inflammatory responses in the intestine. Method: ELMO1 was depleted either by siRNA in murine bone marrow derived macrophages (BMDM) or by shRNA in murine macrophage cell line J774. Control & ELMO1 shRNA cells were compared for bacterial internalization and for downstream signaling events for inflammatory responses. Invasion of Salmonella enterica serovar Typhimurium (SL1344) was determined by gentamicin protection assay and Rac1 activity was measured by pulldown assay. To demonstrate potential role of ELMO1 during enterobacterial infection wild type and ELMO1 Knock out (KO) mice were infected with Salmonella and bacterial load, inflammatory cytokines and histopathology was evaluated in the ileum, Peyer's patches (PP), mesenteric lymph nodes (MLN), liver and spleen. Results: Downregulation of ELMO1 in macrophages reduced internalization of Salmonella and decreased Rac1 activity following infection. Bacterial internalization was significantly ablated in intestinal macrophages isolated from ELMO1 KO mice. Interestingly, inhibition of ELMO1/ Rac function significantly impairs the pro-inflammatory response to infection. Moreover, ELMO1-deficient mice showed attenuated inflammatory responses in the ileum, spleen and cecum. Conclusion: The preliminary results indicated a novel role of ELMO1 in Rac1 activation to control bacterial internalization into macrophages and subsequent inflammation during enteric diseases.

Mo1764 Abundance and Distribution of Mucosa-Associated Hydrogenotrophs in the Healthy Colon and in Inflamed and Non-Inflamed Tissues of IBD Patients Franck Carbonero, Jennifer Croix, Ann Benefiel, Eugene Greenberg, H. Rex Gaskins Stool samples have been used extensively for gut microbiota composition studies, but they do not adequately represent the mucosa-associated microbiota. An even greater challenge is to characterize low abundant microbial communities from colonic tissues such as hydrogenotrophic microbiota. Hydrogenotrophic microbes, including acetogens, sulfate-reducing bacteria (SRB) and methanogenic archaea (MA) are of particular interest to intestinal disease. Breath methane has and is still suspected to be a biomarker of intestinal homeostasis, and hydrogen sulfide, the end-product of SRB metabolism, is a potent genotoxic and inflammatory agent. Here, biopsies from healthy and inflammatory bowel disease (IBD) patients were used to examine mucosa-associated acetogens, SRB and MA, and assess their potential link with chronic inflammation. The abundance of the three groups was quantified through qPCR targeting hydrogenotrophic functional genes and 16S rRNA genes for different SRB genera. The three hydrogenotrophic groups were ubiquitously associated with the colonic mucosa in right-colon, left-colon and rectum of all subjects. Biopsies, in some instances taken from inflamed and adjacent non-inflamed tissue, from eleven Crohn's disease (CD) and seven ulcerative colitis (UC) patients were quantified for the abundance of hydrogenotrophic microbes. A 10-fold greater abundance of SRB, in particular the genus Desulfotomaculum, was detected in inflamed tissue of CD and UC. This higher abundance of SRB in inflamed regions also correlated with a lower abundance of methanogens, which may reflect local competition for hydrogen. In contrast to the healthy control subjects in which SRB abundance decreased in a proximal to distal gradient, SRB were more consistently abundant in the three colonic regions for IBD patients. This increased prevalence of SRB in IBD patients is consistent with the proinflammatory properties of hydrogen sulfide and chronic inflammation. This study thus highlights the potential role of metabolites from under-represented microbes in the etiology of IBD, and their potential use as biomarkers and targets for therapeutic intervention.

Mo1762 Intestinal Helminth Infection Impairs the Ability of Murine Macrophages to Kill Bacterial Enteropathogens by Autophagy During Infectious Colitis Chien-wen Su, Mei Zhang, Jess L. Kaplan, Ramnik J. Xavier, Allan Walker, Bobby J. Cherayil, Hai Ning Shi

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We have found that infection of mice with the intestinal helminth parasite Heligmosomoides polygyrus exacerbates the colitis caused by concurrent infection with Citrobacter rodentium, a Gram-negative rodent pathogen similar to human enteropathogenic E. coli. The severity of the colitis in the co-infected mice correlated with high Citrobacter loads in the gut, translocation of the bacteria into mucosal and systemic immune compartments and uncontrolled bacterial growth. Subsequent investigations from our laboratory showed that the increased bacterial translocation and replication was associated with significantly impaired killing of Citrobacter by macrophages from helminth-infected animals, an abnormality that was dependent on Th2 responses. Autophagy is an important mechanism used by macrophages to kill intracellular pathogens. The results reported here demonstrate that autophagy is also involved in the macrophage killing of the extracellular enteropathogen C. rodentium following phagocytosis. The process was significantly impaired in macrophages isolated from mice chronically infected with the helminth parasite H. polygyrus. The H. polygyrus-mediated inhibition of autophagy was Th2-dependent since it was not observed in macrophages isolated from helminth infected STAT6-deficient mice. Moreover, autophagy of Citrobacter was inhibited by treating macrophages with IL-4 and IL-13. The effect of H. polygyrus on autophagy was associated with down-regulation, at the level of both protein and mRNA, of

High Level Rifaximin Resistance in Escherichia coli Associated With Inflammatory Bowel Disease Correlates With Prior Rifaximin use and Mutations in Rpob Belgin Dogan, Vishesh Kothary, Ellen J. Scherl, Zhi-Dong Jiang, Herbert L. DuPont, Josee Harel, Kenneth W. Simpson BACKGROUND: Escherichia coli is implicated in the pathogenesis of inflammatory bowel disease (IBD). Rifaximin, a semi synthetic non-absorbable derivative of rifampicin developed to treat travelers' diarrhea, is reported to improve symptoms in patients with mild-tomoderate IBD. However, rifaximin has a low level of resistance selection and may select stable highly resistant mutants in a single step In Vitro (J. Antimicrob Chemother. 2008 61:1016-9). Therefore, we examined the association of rifaximin use and resistance in mucosal E. coli from patients with IBD. METHODS: We examined rifaximin resistance of 39 E. coli strains isolated from the ileum of 24 patients with CD (8 with a prior history of rifaximin treatment; 33 E. coli strains total) and 5 patients with ulcerative colitis (UC) (2 with a prior history of rifaximin treatment; 6 E. coli strains total). Minimum inhibitory concentration of rifaximin was determined and the relationship of resistance to mutations

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deep 16S rRNA gene sequencing approach to compare the mucosa-associated microbiota from inflamed sites of the intestine collected from dogs with IBD and from non-IBD healthy controls. Animals: 18 dogs diagnosed with moderate-to-severe IBD (Canine IBD Activity Index [CIBDAI] score > 5) and 6 healthy control dogs. Methods: Clinical disease severity in IBD dogs was scored using the CIBDAI. Total RNA extracted from endoscopic intestinal biopsies was transcribed into cDNA and bacterial tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) based upon the V1-V3 region of the 16S rRNA gene was performed on cDNA aliquots from each dog. Differences in microbial communities between dog groups were investigated using the phylogeny-based unweighted Unifrac distance metric, which provides a measure of similarity among microbial communities in different biologic specimens. Results: All IBD dogs had CIBDAI > 5 (mean=6.8) accompanied by marked endoscopic abnormalities to the intestinal mucosa. Gastrointestinal signs and intestinal inflammation in IBD dogs were accompanied by significant differences in composition of the intestinal microbiota. The proportions of sequences belonging to the members of Bacteroidales and Clostridiales, including the genera Faecalibacterium, Ruminococcus, and Dorea within the Clostridium clusters IV and XIVa, were significantly reduced in the IBD dogs (P<0.01) compared to healthy control dogs. In contrast, the proportions of Proteobacteria, including β-, γ-, and δ-Proteobacteria were significantly higher in the IBD dogs (P=0.012), with butyrate reducing Diaphorobacter and sulfate-reducing Syntrophobacterales being significantly associated with canine IBD. Conclusions: Dogs with active IBD exhibit alterations in microbial diversity which share similarities to dysbiosis reported in humans with chronic intestinal inflammation. Most notably, we observed an increase in sequences belonging to Proteobacteria, and a decrease in Bacteroidetes, Fusobacteria, and the Clostridiales. These bacterial groups may serve as useful targets for diagnosis and potentially the development of better treatment modalities for both human and canine IBD.

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in RNA polymerase (the bacterial target for rifaximin ) and E. coli phylogroup (A,B1,B2,D), evaluated by sequencing a1027 bp fragment of rpoB and triplex PCR respectively. RESULTS: Resistance to rifaximin was present in E. coli from 9/29 patients with IBD (7/24 CD, 2/5 UC) and correlated with prior rifaximin use (P<0.0001: CD, 6/8 treated vs 1/16 untreated; UC 2/2 treated vs 0/3 untreated). All rifaximin resistant strains were highly resistant (MIC = 1024 μg/ml, threshold value = 32 μg /ml). E. coli phylogroup was not associated with resistance. Mutations in rpoB were identified in 8/10 rifaximin-resistant strains vs 0/17 sensitive strains (P<0.0001). Mutations in rpoB were encoded by single amino acid substitutions: D516N (2 strains), H526N (3 strains), H526L (1 strain), N518D (1 strain), S574F (1 strain), previously described In Vitro. CONCLUSION: E. coli isolated from 31% of 29 patients with IBD exhibited high level resistance to rifaximin. The presence of resistance was strongly associated with prior use of rifaximin and amino acid substitutions in rpoB. These findings indicate that E. coli associated with IBD are commonly resistant to rifaximin and have significant implications for treatment trials targeting IBD-associated E. coli.

conditions to CACDI. Younger patients are more likely to have HIV infection and IBD. Additionally, our populations had significantly less CACDI compared to previous studies. There was no significant difference in PPI and antibiotic exposure between the two groups. Mo1768 ZBP-89 Essential for TPH1 Expression and Innate Mucosal Immunity Bryan E. Essien, Helmut Grasberger, Arthur Tessier, Juanita L. Merchant Background: ZBP-89 is a ubiquitously expressed transcription factor originally cloned using a GC-rich DNA element from the gastrin promoter and subsequently shown to bind to the promoters of multiple genes. We have recently found that ZBP-89 is highly expressed in the serotonin (5HT)-producing enterochromaffin (EC) cells of the intestine and colon. Therefore, we hypothesized that ZBP-89 regulates production of 5HT by modulating expression of tryptophan hydroxylase 1 (TPH1), the rate-limiting biosynthetic enzyme. Aim: To determine whether ZBP-89 directly regulates TPH1 gene expression and whether modulating the enzyme affects tissue 5HT levels and subsequently mucosal defense against a pathogen. Methods: Mice conditionally-null for ZBP-89 were generated by breeding the ZBP-89LoxL/ Lox mice to VillinCre expressing mice on a C57BL/6 genetic background (ZBP-89dColon). Immunohistochemistry was used to co-localize ZBP-89 to 5HT-expressing enterochromaffin cell (EC) cells. Microarray analysis was performed using RNA extracted from mucosal scrapings from WT and ZBP-89dColon mouse colons. Morphometry was used to quantify EC numbers. EMSAs were used to demonstrate ZBP-89 binding to the TPH1 promoter. WT and ZBP-89dColon mice were gavaged with Salmonella typhimurium SL1344 strain (S. typhi) at 107cfu after streptomycin pre-treatment in the presence or absence of 5-hydroxytryptophan (5-HTP). The bacterial load in the gut lumen, spleen and liver was assessed by quantitative cultures. Results: Immunofluorescence confirmed ubiquitous nuclear expression of ZBP-89 protein that colocalized with 5HT in the mouse EC cells. ZBP-89dColon mice exhibited reduced numbers of 5HT-positive EC cells in the colon. Microarray analysis of WT versus ZBP-89dColon mice revealed a 4-fold decrease in TPH1, cryptdin-5/22 and chromogranin A mRNA consistent with ZBP-89 regulation of EC gene targets. Infecting the ZBP-89dColon mice with S. typhi resulted in greater weight loss and diarrhea than the infected WT mice. There was a greater (2-orders of magnitude) S. typhi bacterial load in the ileum, cecum and colons of the ZBP-89dColon compared to WT mice. The ZBP-89dColon exhibited greater bacterial loads in the spleen and liver (sepsis) and reduced survival (6 days) compared to WT mice (8 days). To determine if the increased susceptibility to S. typhi by the ZBP-89dColon mice was due to reduced TPH1 gene expression and subsequent 5HT production, we treated both groups of mice with 5HTP, the product of TPH1 tryptophan hydroxylation and found that it boosted 5HT tissue levels in both mouse groups and extended mouse survival by an additional 2 days (6 to 8 days). Conclusion: ZBP-89 is required for TPH1 gene expression and 5HT production during S. typhi infection. Reduced TPH1 gene expression in the ZBP-89dColon mice exacerbated the acute effects of a S. typhi infection due to less 5HT.

Mo1766 Short Chain Fatty Acids and Bile Salts Modulate the Growth of Crohn'sAssociated Adherent and Invasive E. coli (AIEC) Deepali Herlekar, Katrina Stewart, Belgin Dogan, Craig Altier, Ellen J. Scherl, Kenneth W. Simpson Background: Adherent invasive Escherichia coli (AIEC) have been consistently isolated from the inflamed ileum of people with Crohn's disease (CD) and murine models of IBD, but are infrequently isolated from inflamed or healthy colon (Gastroenterology, 2004, 127:412). Differences in the chemical composition of the enteric microenvironment of the colon vs. the ileum, and the inflamed vs. the healthy ileum could account for the differential isolation of AIEC. Aim: Assess the impact of short chain fatty acids (SCFA) and bile acids on the growth of CD associated AIEC. Methods: The growth of ileal CD AIEC strains 541-15, 5411, LF82 and 576-1 (phylogroups A, B1, B2 and D respectively) and E. coli DH5-alpha (control) was evaluated in a Bioscreen apparatus for 24 hrs at 37°C, with OD600 read every 15 mins. For bile salt experiments E. coli was grown in HEPES-LB pH7.4 supplemented with Na-salts of cholic, taurocholic, glycocholic, chenodeoxycholic and deoxycholic acid (0-10mM). NaCl was used to control for osmolality. For SCFA experiments E. coli was grown in media designed to simulate ileal (LB-HEPES, pH7.4, acetate 8 mM,propionate 1.5 mM,butyrate 2.5 mM) or colonic conditions (LB-MOPS, pH6.7, acetate 65 mM,propionate 29 mM,butyrate 29 mM). Control media was supplemented with NaCl at 13 or 123 mM. Maximal OD600 and time to half-maximal OD600 (T0.5) were evaluated by paired statistical analysis. Results: Growth of AIEC and DH5-alpha in SCFA-colonic conditions (T0.5= 669±95 mins mean±SE) was slower (P<0.01) than in control media without SCFA (339±38 mins). Growth of AIEC and DH5-alpha in SCFA-ileal conditions (T0.5 357±37mins) was similar to growth in control media without SCFA (357±36 mins). There was no difference in maximal OD600 in colonic or ileal conditions. Growth (T0.5) of AIEC was stimulated by sodium salts of cholic (2-5mM, 258±13 vs control 311±22 mins, P<0.05), taurocholic (15 mM, 225±12 vs control 281±19 mins, P<0.05) and glycocholic (2-5 mM, 240±12.3 vs. control 296±14 mins, P<0.01), but not deoxycholic (236±17 vs control 200±36 mins, P= 0.07) or chenodeoxycholic acid (218±14 vs. control 221±12.8 mins, P=0.39). In contrast, T0.5 for DH5-alpha was similar in bile salt-supplemented and control media, with the exception of Na-deoxycholate which suppressed growth. Conclusions: Our results indicate that SCFA at proportions, concentrations and pH reported in the colon, but not the ileum, suppress the growth of AIEC and DH5-alpha. Conversely, free and conjugated salts of cholic acid at concentrations similar to those anticipated in the inflamed, bile salt malabsorbing distal ileum, stimulate the growth of AIEC, but not DH5-alpha. Our results support the concept that differences in the enteric microenvironment of the colon vs. the ileum, and the inflamed vs. the healthy ileum may account for the differential isolation of AIEC.

Mo1769 Westernalized Diet on Ceabac10 Mice Induces a Specific Dysbiosis With Increased Escherichia coli, Modulates Innate Immune Gene Expression and Favors AIEC Colonization Promoting Inflammation Margarita Martinez-Medina, Jérémy Denizot, Arlette Darfeuille-Michaud, Nicolas Barnich Background: Abnormal expression of CEACAM6 on ileal epithelium in Crohn's disease (CD) patients allows Adherent-Invasive Escherichia coli (AIEC) to colonize gut mucosa, leading to inflammation, and epidemiologic evidence incriminates westernized diet as risk factor for inflammatory bowel disease. Our aim was to evaluate the effects of high fat-high sugar (HF/ HS) westernalized diet on CEABAC10 mice expressing human CEACAM6 infected with AIEC strain LF82 in terms of intestinal permeability, colonic dysbiosis, AIEC colonization and inflammation. Methods: Ten weeks old CEABAC10 and WT mice were treated with conventional or HF/HS diet for 7 to 11 weeks. A subset of each condition was infected with AIEC strain LF82. Intestinal permeability was assessed by FITC-dextran quantification in serum 5 hours after intragastric administration. Microbiota associated to colonic mucosa was studied by 16S rDNA based denaturing gradient gel electrophoresis, quantitative PCR of specific bacterial groups and deep sequencing with 454-GS-FLX of Roche. After infection, AIEC colonization was quantified in feces after 1, 2 and 3 days of post-infection by colony counting. Inflammation was assessed by histological inflammatory score and cytokine release (ELISA quantification). Host expression genes related to intestinal barrier function and innate immunity was evaluated by RT-qPCR. Results: HF/HS diet during a 7 week period significantly increased intestinal permeability in CEABAC10 mice. HF/HS diet also induced important alterations in the whole mucosa-associated microbial community of both genotypes, with increased numbers of E. coli and Bacteroides spp and depletion of Segmented Filamentous Bacteria (SFB). E. coli population was specially favored in CEABAC10 background. Interestingly, ceacam6, nod2 and tlr5 mRNA levels were increased in colonic mucosa of CEABAC10 mice with HF/HS diet compared to conventional diet, whereas tff3 mRNA level was decreased. In addition, orally AIEC-infected HF/HS-treated CEABAC10 mice showed increased gut persistence of LF82 compared to CEABAC10 receiving conventional food, what correlated with increased inflammation as observed by increased histological scores and higher secretion of TNF-α in colonic mucosa. Conclusion: These results strongly support the multifactorial theory of CD etiology and gives weight to westernalized diet, which modulates innate immune gene expression and microbiota composition favoring AIEC colonization leading to gut inflammation.

Mo1767 Risk Factors for Community Acquired vs Hospital Acquired Clostridium difficile Infection Suraj Naik, Stanley Giddings, Ravi Kottoor, Xiaoyu Li BACKGROUND:The incidence of C. difficile Infections has been rising over the past ten years. Moreover there have been recent studies that have shown that community acquired C Difficile infections have increased (CACDI) and account for 33-41% of the cases of C. difficile Infection (CDI). There have been a number of epidemiological studies using hospital derived reports and administrative databases, however, there have been few studies that study and characterize CACDI. A previous study identified CACDI patients being younger, having less antibiotic exposure, and proton pump inhibitor exposure. AIMS: The aims of our study were to determine risk factors and outcomes of community acquired CDI vs hospital acquired CDI. We also tried to determine whether there were any particular risk factors that predisposed younger patients (<50) versus older patients (>50) to CDI. METHODS: We performed a retrospective cohort study of all patients that were seen at Shands Hospital, (Jacksonville) including the outpatient facilities, with a confirmed diagnosis of C. difficile infection from 1/07- 12/10. Patients were identified by a using a database of confirmed laboratory data. We abstracted demographic, historical, laboratory and outcome data. RESULTS: There were a total of 666 patients identified with CDI of which 564 were admitted. 87 (13%) of these were determined to be community acquired. 39/87 (44%) were ages less then 50 vs 151/579 (22%) p<0.05 supporting previous conclusions that younger patients were more likely to develop CACDI. Additional risk factors that differentiated CACDI from hospital acquired CDI included inflammatory bowel disease (IBD) (12% vs 1%) and steroid use (17& vs 9%). Risk factors for hospitalized patients, included cardiac failure, use of quinolones, and from a nursing home. With regards to age, factors that predisposed to patients less than age 50 to getting CDI included comorbid conditions (Hypertension, Cardiac Failure), antibiotic use, nursing home, inflammatory bowel disease and HIV infection. Patients older than 50 had risk factors that included comorbid conditions, (Hypertension, Cardiac Failure). Autoimmune Disease and antibiotic use, steroid use and nursing home facility visit. Overall mortality was 8.65% with death attributed to CDI at 1.65% which correlates to current studies. CONCLUSION: IBD and steroid use are predisposing

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