Sa1982 Detecting Enterotoxigenic Bacteroides Fragilis Carriage in Pediatric Inflammatory Bowel Disease

samples from 8 IBD and 3 control patients were thawed and cultured on selective Bacteroides Bile Esculin (BBE) agar to help select for Bacteroides spp. Sixteen isolates were collected from each of the 4 IBD and 2 control samples that grew on BBE. Touchdown PCR performed on boiled water preps of each isolate were positive for bft in 2 of the 4 IBD patients (4 of 16 isolates and 5 of 16 isolates, respectively) and both controls (1 of 16 and 6 of 16, respectively). Our stool DNA results suggest that the ETBF quantity, if present in our IBD and control samples, is less than ~105CFU/gm stool, though ETBF spiking experiments using these bft-negative human stools are needed to account for possible fecal PCR inhibitors that may affect our assay sensitivity. Second, our data suggests that testing of bacterial isolates is a more sensitive method to detect bft+ Bacteroides. Testing of more samples is needed to distinguish if there is a statistically significant difference in the percentage of IBD patients vs. controls who carry ETBF or in the quantity of ETBF they carry. We are in the process of testing for human stool inhibitors, as well as testing other IBD and control samples for bft. We will analyze associated clinical data to determine if BFT carriage correlates with clinical manifestations of IBD, such as disease location, severity, or timing of flares. Sa1983 Characterization of Mucosally - Adherent Escherichia coli in Pediatric Inflammatory Bowel Disease Patients Rebecca Flint, Ward Jarvis, Matthew Overton, Robert Baldassano, Sandra C. Kim

Sa1981 Pre-Operative Nutritional Status and Immunosuppression as Predictors of Post-Operative Events in Pediatric Ulcerative Colitis Patients Melissa Rose, Vesta Salehi, Robbyn E. Sockolow, Aliza B. Solomon

Background: Children with inflammatory bowel disease (IBD) have unique characteristics not seen in adult patients, including growth and nutritional deficiencies and aggressive disease. Thus, a better understanding of factors that potentially perpetuate disease is crucial. Prior studies have shown that both adults and children with IBD, particularly Crohn's disease (CD), have increased mucosally - adherent Escherichia coli. However, the relationship between E. coli in pediatric IBD patients and bacterial - associated virulence genes has not been clearly delineated. Aim: Determine whether mucosally - associated E. coli isolated from pediatric CD patients is found in greater numbers and express more virulence genes compared to children with ulcerative colitis (UC) and healthy controls. Methods: Mucosal biopsy samples (2 - 3 per section) were obtained from the ileum (IL), cecum (CEC), and distal colon (DC) of pediatric pts undergoing routine procedures. Samples were processed using established lab protocols for culture (plated on MacConkey) and quantification. Individual colonies were selected from bacterial cultures and incubated. PCR was used to confirm the presence of E. coli and to test for E. coli - associated virulence genes (Table 1) from bacterial DNA extracted from the cultures. Bacterial DNA was extracted from biopsies for E. coli quantification by real time PCR (qPCR). Results: Mucosal biopsy samples were analyzed (n= 42 pts; 50% M, 50% F; 10-23 yrs, mean: 16.2 ± 3.0 yrs). There were 34 CD (15.9±2.9 yrs), 4 UC (18.3±3.4 yrs), 2 healthy control, and 2 indeterminate colitis (IC) pts. There were no statistically significant differences in E. coli-associated virulence genes between CD and UC pts or between different regions (IL/CEC/DC) within disease subsets, but some trends were seen. Virulence genes kpsmII and fliC were expressed in CD but not UC pts, and all IBD pt, but no control, samples expressed fimA and hcp. In the E. coli quantification data, we group samples by quartile. 100% of IL and DC and 66.7% of CEC samples in the upper quartiles were from CD pts. In the lower quartiles, only 55.6% of IL, 22.2% of CEC, and 55.6% of DC samples were from CD pts. Conclusion: E. coli adherence virulence genes may be necessary to induce and maintain the dysregulated response in IBD. Differences in virulence genes may affect E. coli mucosal adherence in different pediatric IBD pt subsets. Ongoing studies are focused on sample size expansion, antimicrobial resistance and correlating patient risk alleles associated with impaired bacterial sensing/processing to E. coli quantification/characteristics. A better understanding of the relationship between E. coli, in pediatric IBD patients, and antimicrobials may lead to targeted therapies including less toxic therapies which modulate specific components of the intestinal microbiota. Table 1: Virulence genes

Objective: To evaluate the frequency of post-operative events in children with ulcerative colitis (UC) in relation to nutritional status and use of immunosuppressive medications prior to surgery. Patients/Methods: A case series was conducted of 9 pediatric patients with UC who underwent surgical intervention between 2007 and 2011. Age of diagnosis, age of first surgical intervention, type(s) of surgical intervention(s), pre-operative immunosuppressive medication use and nutritional status (BMI <5th percentile) were reviewed and evaluated in the context of post-operative events. Results: The average age of diagnosis was 10.74 years (median 11.16). The average age at first surgical intervention was 13.92 years (median 12.5), with the average time from diagnosis to first surgical intervention being 3.22 years (median 2.83). Procedures included four (44.4%) two-stage ileal-pouch anal anastomosis (IPAA), three (33.3%) three-stage IPAA, and two total abdominal colectomy with ileostomy with plans to complete a three-stage IPAA (22.2%). One patient had four subsequent surgical pouch revisions, one had a small bowel resection at stage 3 of her IPAA, and one had pouch failure requiring end ileostomy placement. Five patients had elective procedures and four had emergent procedures. Reasons for emergent surgery included acute failure of medical therapy (3/9) and acute toxic colitis (1/9). Five patients (55.5%) were on immunosuppressive medications at the time of initial surgery; two were on steroids/biologics, one on an immunomodulator (IM) and two on steroids alone. The four remaining patients had been off of immunosuppression for 4-8 weeks pre-operatively. Pre-operative BMI percentile ranged from 0.19-97.83% (median 51.69%) and two patients (22.2%) had a BMI <5th percentile. Shortterm (<30 days) post-operative events occurred in four patients and included readmission for pain, wound infection (both with normal BMI and on steroids/biologics); prolonged small bowel edema delaying second stage (normal BMI, on IM); and pouchitis with sinus tract formation (low BMI, on no immunosuppression peri-operatively). Long-term (30180 days) post-operative events occurred in two patients, one with recurrent pouchitis, anastomotic leak and stricture, abscess, sinus tract formation and infection (initial surgery occurred at another hospital, patient was on biologics at time of surgery and BMI was normal) and one with an incisional hernia after her initial surgery (on steroids, normal BMI) and later pouch failure at 6 months (on no immunosuppression, BMI 6%). All patients except for one (88.8%) were seen by our practice as a second opinion. Conclusions: Immunosuppression is more common than malnutrition in pediatric patients with ulcerative colitis who have post-operative events, however malnutrition also likely plays a role in outcomes. Pre-Operative Nutritional Status and Immunosuppression as Predictors of Post-Operative Events in Pediatric Ulcerative Colitis Patients

Sa1982 Detecting Enterotoxigenic Bacteroides Fragilis Carriage in Pediatric Inflammatory Bowel Disease Lea Ann Chen, Shervin Rabizadeh, Sankar Chirumamilla, Shehzad A. Saeed, Emilia Albesiano, Andrew Goodwin, Shaoguang Wu, Charles O. Elson, Maria Oliva-Hemker, Cynthia L. Sears Enterotoxigenic Bacteroides fragilis (ETBF) causes asymptomatic, chronic colitis in C57BL/6 mice and increased colon tumorigenesis in multiple intestinal neoplasia (MinApc/+) mice via its primary virulence factor the B. fragilis toxin (BFT). Studies suggest an association between ETBF infection and active inflammatory bowel disease (IBD), but only small studies in humans are available. Our study aims to develop a sensitive diagnostic touchdown PCR protocol for bft detection and to apply this protocol to determine ETBF carriage rates in pediatric IBD patients. We spiked known quantities of a serially diluted laboratory ETBF strain into sham mouse stool. ETBF detection limits range from 2.3 x 103 to 4.6 x 105 CFU/ mL for pure ETBF culture and 4.1 x 105 to 4.6 x 106 CFU/gm stool for spiked sham stool. To test for ETBF carriage in humans, we prospectively enrolled pediatric patients with ulcerative colitis or Crohn's disease from two academic IBD centers. Control samples were similarly collected from confirmed non-IBD patients presenting to the pediatric GI clinics at the same institutions. Extracted stool DNA from 25 IBD patients and 25 controls were tested by our touchdown PCR protocol for bft with no positive results. Next frozen fecal

Sa1984 Quantification and Characterization of Klebsiella pneumoniae in Pediatric Patients With Inflammatory Bowel Disease Matthew Overton, Ward Jarvis, Rebecca Flint, Robert Baldassano, Sandra C. Kim Background: Inflammatory Bowel Diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are chronic relapsing diseases resulting from a dysregulated host immune response against normal commensal bacteria. Members of the Enterobacteriaceae family, including Klebsiella spp, are known to be increased in IBD patients. Previous studies have shown an association between increased anti-Klebsiella antibodies in patients with Crohn's and ankylosing spondylitis. Furthermore, studies in rodent IBD models have shown a correlation between increased intestinal Klebsiella pneumoniae and the development of colitis.

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AGA Abstracts

AGA Abstracts

four patients (30.7%) had a BMI <5th percentile at the time of surgery. Short-term (<30 days) post-operative events occurred in two patients with recurrent perianal disease. Both had normal BMI: one was on IM and one on biologics peri-operatively. Long-term (30-180 days) post-operative events included repeat surgery (1/13, on steroids/biologics), and rectal bleeding requiring readmission (1/13, on biologis); both had normal BMI. One patient had an anastomotic stricture requiring repeat surgery, however this was at >2 years. Of note, 9/ 13 (69%) were seen by our practice as a second opinion. Conclusions: Immunosuppression is more common than malnutrition in pediatric patients with Crohn's disease who have post-operative events. Pre-Operative Nutritional Status and Immunosuppression as Predictors of Post-Operative Events in Pediatric Crohn's Disease Patients