2014 IBD Abstracts

P-217
Specic PCR Assays Using CRISPR Genes for Detection of AIEC in Fecal Samples
Gathungu Grace1, Zhang Yuanhao1, Rowehl Leahana1, Frank Daniel2, Boedeker Edgar3,
Parkinson John4, Li Ellen1
1
Stony Brook University, Stony Brook, New York, 2University of Colorado, Aurora,
Colorado, 3University of New Mexico, Albuquerque, New Mexico, 4University of
Toronto, Toronto, Canada

BACKGROUND: Adherent-invasive Escherichia coli (AIEC) are implicated in the

pathogenesis of Crohns disease (CD). Many studies have dened pathogenic mechanisms using AIEC reference strain LF82. The phenotype for this group of bacteria
includes their ability to adhere and invade intestinal epithelial cells and to survive
within macrophages in vitro. Identication of individuals infected with AIEC requires
phenotypic screening of cultured bacteria, a laborious and inefcient process. Loci
common to and underlying the AIEC phenotype remain unknown in part because
AIEC are clonally diverse and belong to distinct serotypes. The availability of specic
molecular tools to detect and quantify AIEC would enable us to conduct large-scale
epidemiologic studies to better understand the impact of this pathogen on CD
disease severity. Our aim was to design AIEC specic genomic probes that can be
applied to a non-invasive and high throughput assay. Here we present the design of
gene expression assays to detect AIEC using 4 genes belonging to the clustered
regularly interspaced short palindromic repeats region (CRISPR).
METHODS: Using BLAST, the genomes of 12 AIEC strains were compared to 10 nonpathogenic E. coli to identify genes present in the AIEC strains but not in
commensals. Four genes were identied and primers were designed. Banked fecal
samples stored in RNA stabilization solution were available through an IRB approved
protocol at Stony Brook University. RNA was extracted from 53 fecal samples
obtained from patients and from strains LF82 and MG1655. Twenty 2 subjects had
inammatory bowel diseases (IBD), including CD (n 15), ulcerative colitis (UC) (n
5), indeterminate colitis (n 2), and 31 were healthy controls. Real time PCR assays
were conducted using 16S rRNA as a reference, with positive results set at cycle
thresholds (CT) <35.
RESULTS: No single gene was unique to all 12 AIEC strains and absent from
commensal E. coli, but genes were identied in 6 AIEC strains and absent from all
non-pathogenic bacteria. The DCT (universal-E. coli) was lower (i.e., reecting greater
locus abundance) in IBD (212.75) compared to control (213.40) samples, although
this difference was not statistically signicant. Five CD samples, I UC sample and 2
control samples had positive CT values for 3 of the LF82 CRISPR genes. LF82 and
MG1655, as expected, were positive and negative for all 4 genes. All other samples
were negative for all 4 loci. Thirty three percent of CD samples were positive for LF82
CRISPR genes compared with 6.4% of controls (P 0.029, using a Chi-square and
Fishers exact test).
CONCLUSIONS: We identied 4 genes that are uniquely expressed in many AIEC
strains. In some bacteria, CRISPR genes participate in the evasion of host
recognition. PCR assays using these genomic regions can be optimized to
create a non-invasive multiplex assay for detecting CD patients infected or
colonized with AIEC.
P-218 YI
Increased Abundance of Colonic Mucosal Faecalibacterium Prausnitzii in
Pediatric Treatment-Naive Ulcerative Colitis
Shah Rajesh1, Cope Julia1, Nagy-Szakal Dorottya2, Dowd Scot3, Hollister-Branton Emily1,
Kellermayer Richard4
1
Baylor College of Medicine, Houston, Texas, 2Baylor College of Medicine, Houston,
Texas, 3MR DNA (Molecular Research), Shallowater, Texas, 4Baylor College of
Medicine, Section of Pediatric Gastroenterology, Houston, Texas

BACKGROUND: Ulcerative colitis (UC) pathogenesis likely involves a dysregulated

interaction between host genes, intestinal microbes and environment. Twenty
percent of UC presents in children, where the disease is usually more aggressive
than in adults. In regards to the microbiome, Faecalibacterium prausnitzii has been
observed to harbor anti-inammatory properties, and has been associated both with
Crohns disease (CD) and UC. Lower abundance of F. prausnitzii has been detected in
patients with ileal CD. The decreased abundance of this bacterium has been associated with an increased risk of post-surgical relapse in CD patients as well. In UC,
conicting observations have been made about F. prausnitzii abundance. One study
connected lower abundance of F. prausnitzii with shorter time in remission and
greater risk of UC are. However, most of these metagenomic observations have
limitations, such as restriction to adults, studying stool samples, and including treatment experienced patients. All of these factors may inuence microbiome composition, and limited mucosa associated metagenomic data exists in respect to
pediatric UC in untreated patients. However, such information is likely to carry
important information towards unraveling the pathogenesis of this UC subtype.
The aim of our study was to characterize the colonic mucosal microbiome of treatment-nave pediatric UC patients.

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METHODS: Left sided colonic biopsy samples from 9 treatment naive UC patients and
13 controls (no endoscopic or histologic evidence of UC) were studied. The Illumina
MiSeq sequencing platform was used to interrogate the V1-V3 regions of the bacterial
16s rRNA gene. Processing of sequence data, assignment of operational taxonomic
units (OTUs) and calculation of alpha-diversity were performed in QIIME (Quantitative
Insights Into Microbial Ecology). STAMP (Statistical analysis of taxonomic and
functional proles) was used to analyze the high-throughput metagenomic data
set. Clinical information, including Montreal classication of disease extent, physicians global assessment of severity and medications were reviewed.
RESULTS: Pediatric UC patients had a comparable Shannon diversity index
compared to controls. There were no signicant differences between the groups
when analyzed at the phylum, class, order, family, genus or species levels. There was,
however, a trend towards a higher abundance of F. prausnitzii in UC patients
compared to controls (P 0.069). Signicantly (Fischer exact test: P 0.027) more
UC patients (7/9 77.8%) had .3% F. prausnitzii abundance in the colonic mucosa
than controls (3/13 23.1%). F. prausnitzii abundance at diagnosis did not predict
short term (within 6 months) clinical outcomes in this small cohort.
CONCLUSIONS: Our study demonstrated limited, but detectable differences
between the colonic mucosal microbiome of treatment naive UC patients and
controls in spite the small sample sizes. Signicantly more UC patients had increased
abundance of F. prausnitzii than controls. These results warrant further studies on
larger groups to clarify the role of F. prausnitzii in pediatric UC.
P-219
HTLV-1 Infected Regulatory T-Cell Expansion in Duodenum is Highly Associated
with a Complicated Clinical Course in HTLV-1/Strongyloides Co-Infection
Malpica Luis1, Lopez Mariano2, Zavala Soa2, White Clinton Jr,3 Montes Martin2,
Antnez-de Mayolo Eleazar2, Gotuzzo Eduardo2
1
Jackson Health System, University of Miami, Miami, Florida, 2Universidad Peruana
Cayetano Heredia, Lima, Peru, 3University of Texas Medical Branch at Galveston,
Galveston, Texas

BACKGROUND: Strongyloides stercoralis (SS) is an intestinal nematode unique in its

ability to replicate in the human host, allowing ongoing cycles of autoinfection,
persisting for decades within the same host. Although usually asymptomatic, overwhelming infections can occur in SS and HTLV-1 co-infected individuals. SS infection
might promote the clonal expansion of HTLV-1 infected cells. During infection
regulatory T cells (Treg) serve as a vital mechanism of negative regulation of specic
Th2 responses necessary to control the parasite. HTLV-1 in turn leads to an increase
of regulatory T cells (Treg), which blunt the immune response to the parasites and
remain as chronic or persistent infections. We have shown that the proportion of
Treg in blood is higher in patients with Strongyloidiasis co-infected with HTLV-1
than in those with only SS (PLos. Neglected Tropical Diseases. 2009; 3 (6): e456). We
hypothesized that duodenal tissue with SS is a modulated immune site where the
clonal expansion of HTLV-1 infected Treg cells is promoted.
METHODS: We compared the HTLV-1 proviral load/slide levels and proportion of
CD3+, CD8+, IgE+ and FoxP3+ cells in 2 groups of patients: (1) Non-parasitic chronic
duodenitis subjects with no history of Strongyloidiasis and negative HTLV-1 serology
(NP) (n 3) and (2) patients with a history of HTLV-1/Strongyloides co-infection
(HSS) (n 10). Parafn embedded duodenal biopsies were isolated both to measure
HTLV-1 proviral load/slide by qPCR and to count CD3+, CD8+, IgE+ and FoxP3+ cells
per 0.35 mm2 by Immunohistochemistry. Four patients with Strongyloidiasis who

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2014 IBD Abstracts

died with an unknown HTLV-1 serology were also included in order to detect proviral sequences in duodenal tissue by PCR in situ. Statistical analysis was based on
non-parametric tests.
RESULTS: There were differences in the distribution of duodenal intraepithelial CD3+
and CD8+ cells ([NP: 44.67, HSS: 6.38, P 0.007] and [NP: 48.67, HSS: 6.9, P 0.013]
respectively). Presence of IgE+, CD3+ and CD8+ cells in parasite adjacent areas were
diminished [1 way ANOVA, P < 0.0001]. The proportion of FoxP3+ cells was higher in
the coinfection group [NP: 2.67, HSS: 23.9, P 0.022], which weakly correlated with
the HTLV-1 proviral load/slide [Spearman r 0.4939, P 0.2405]. PCR in situ could
detect sequences of provirus in the tissue of every HSS patient and turned out
positive in 3/4 patients with a fatal outcome. These 3 had the highest levels of
HTLV-1 proviral load/slide and also presented gastrointestinal bleeding. Interestingly, 2 patients from the HSS group with the highest count of FoxP3+ cells in
duodenal biopsies also had gastrointestinal bleeding.
CONCLUSIONS: Our data shows increased FoxP3+ cell counts and a modulation of
the anti-helminthic response in the duodenum, specically at the parasite adjacent
areas. Detection and quantication of HTLV-1 in duodenal tissue by PCR could show
a clonal expansion of HTLV-1 infected cells, most likely Treg phenotype. Altogether,
this suggests that higher local counts of HTLV-1 infected FoxP3+ cells are capable of
downregulating anti-helminthic responses, facilitate tissue invasion and lead to
complicated courses of Strongyloidiasis.
P-220
Escherichia coli Displaying Virulence Features of Multiple Diarrheogenic
Pathotypes Detected in a Crohns Disease Patient
da Silva Santos Ana CarolinaSantos1, Rodrigues Josias2, Romeiro Fernando3,
Sassaki Ligia4
1
UNESP/IBB, Botucatu, Brazil, 2Microbiology UNESP, Botucatu, Brazil, 3FMB- UNESP,
Botucatu, Brazil, 4UNESP/FMB, Botucatu, Brazil

BACKGROUND: The number of Escherichia coli in the gut of Crohns disease (CD)
patients is higher than that of normal subjects, but the virulence potential of these

bacteria is not fully known. Previous studies have shown that these E. coli are closely
related to extraintestinal pathogenic categories (ExPEC), are able to invade epithelial
cells, and usually do not produce exotoxins. We report here the detection, in a CD
patient, of an E. coli which belongs to a classical enteropathogenic (EPEC) serotype
and displays virulence markers of enteroinvasive (EIEC), enteroaggregative (EAEC)
and enterohemorrhagic (EHEC) pathotypes.
METHODS: The E. coli strain was isolated, in 2009, by classical bacteriological procedures from a 56 year old woman who underwent ileo-terminal resection 1 year
before, due to intestinal obstruction. The bacterial characterization was carried
out by in vitro adhesion and invasion assays to cultured epithelial cells and macrophages and screening by PCR to identify virulence genetic markers of diarrheogenic
E. coli (DEC) and to detect one of the gene combinations which dene the phylogroups of the E. coli reference (EcoR) collection. The strain was also tested for the
ability to produce biolm and shiga cytotoxins and had its whole genome
sequenced by Ion Torrent Sequencing Technology.
RESULTS: The studied strain, which was detected both in ileum biopsies and
the stools of the patient, displayed the aggregative adherence (AA) phenotype
to Hep-2 cells and an ability to enter Caco-2 cells 3x as high as that of
EIEC reference strain and 89% of that of the prototype AIEC LF82 strain.
Although it could invade cultured macrophages, the strain was unable to
replicate inside these cells. PCR screening revealed the presence of eae,
aggR and stx1. Tests with bacterial culture supernatants in Vero cells demonstrating cytotoxicity suggested the production of Stx1. In addition, the strain
revealed to be a strong biolm producer, belonged to the B2 EcoR phylogroup,
to the O126:H27 serogroup and to the multilocus sequencing type (MLST)
ST3057. The 2 later features were deduced from the whole genome sequence
of the strain.
CONCLUSIONS: The characterization of this E. coli isolate from a CD patient revealed a combination of virulence markers of distinct DEC pathotypes, namely eae
and stx1 of EHEC, AA, aggR and biolm formation of EAEC, and invasiveness of
EIEC. These features along with its serotype and phylogroup identity seem to
suggest a potential to be involved in CD, an observation which should be tested
with additional studies.