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Investigation of adherent-invasive E. coli in patients with Crohn's disease

Background: Crohn's disease and Ulcerative colitis are known as inflammatory bowel disease with high morbidity which are as a result of increasing immune responses to intestinal microbiota in genetically susceptible individuals. The association of adherent invasive Escherichia coli with Crohn's disease in human has been discussed for decades. The principal aim of this study was to assess the relationship between adherent invasive Escherichia coli in Iranian patients with Crohn's disease. Methods: The presence of adherent invasive Escherichia coli DNA and viable adherent invasive Escherichia coli cells were identified through PCR and conventional culture methods, respectively. All the specimens were subsequently cultured in Hi Chrome Agar medium. Results: Using molecular assay, the invasive plasmid antigen H and invasion-association locus genes were detected from tissue samples confirming the presence of adherent-invasive Escherichia coli. The invasive plasmid antigen H was detected in 46.7% of CD and 13.3% of healthy peoples. The invasion-association locus gene was found in 36.7% of patients with Crohn's disease and 10% in individuals without IBD. Conclusion: This study demonstrated an increased frequency of adherent invasive E. coli with invasive plasmid antigen H and invasion-association locus genes from patients with CD in comparison to control individuals. Moreover, it was shown that adherent invasive E. coli with the invasive plasmid antigen H and invasion-association locus genes can act as a predisposing factor in the development of IBD.


Introduction
Inflammatory bowel disease (IBD) is a chronic inflammatory disorder divided into Ulcerative colitis (UC) and Crohn's disease (CD) (1,2).The rising trend of the disease has observed in Middle Eastern countries as well as in other parts of the world and statistics shows the incidence also is on the rises in North America and Europe as well as other parts of the world. Likewise, a recent study demonstrated that the prevalence of IBD is increasing dramatically in Iran (3).
The incidence and prevalence of ulcerative colitis and Crohn's disease have been increased all over the world, especially among children and adults. It is believed that several factors such as immunology, genetic factors, stress, diet, being an alcoholic or a smoker, as well as bacteria such as the adherent-invasive strains of Escherichia coli, Campylobacter spp., Clostridium  2 hence, dysbiosis of the intestinal microbiota is likely cause of IBD (8).
It is known that intestinal bacteria can cause CEA-CAM6 expression through inducing inflammatory cytokines. The expression can then leads to production of E. coli with AIEC phenotype. The AIEC can attach to epithelial cells by a mechanism involving polymerization of microtubules. In fact, AIEC adheres to GP2 (glycoprotein 2) upon M cells by LPF (long polar fimbriae) and enter to Peyer's patches and exacerbates inflammation in the intestine (9). Moreover, AIEC able to adhere epithelial cells and invade into the cytoplasmic eukaryotic infectious cells with type 1fimbriae (fimH), invasive plasmid antigen H (ipaH) and invasion-association locus (ial) (10).
However, E. coli with adherent-invasive phenotype should be regarded as a separate issue of pathogenicity of E. coli causing inflammation in the human intestine.
The principal purpose of this study was to investigate of E. coli with adherent-invasive phenotype and their relation to Crohn's disease.

Methods
In the present study, sixty subjects including thirty patients with lesions of Crohn's colitis and thirty people without IBD were collected during colonoscopy examinations. Samples with CD were obtained from lesions of colon, or terminal ileum. Moreover, the biopsy of control group was performed on normal areas. DNA was extracted from the tissue samples and PCR assay performed by targeting ipaH and ial genes. CD diagnosis based on clinical symptoms, laboratory evaluations, and colonoscopy finding was confirmed by histological assessment. The control subjects were taken from people with noninflammatory IBD (nIBD). None of the individuals presented in this study used antibiotics or probiotics for the 3 months prior to the study. The study was accepted by the ethics committee of Tehran University Medical Sciences.

Microbial identification
Samples were immediately transferred into sterile vials containing either thioglycolate broth or saline (Sigma-Aldrich, Hi Media) and stored at -20˚C. The tissue biopsy were homogenized and inoculated into Hi Chrome E. coli agar (Sigma-Aldrich, Hi Media) and incubated for 18-24h at 35±2°C. The bacteria were stored in TSB broth containing 30% glycerol at a temperature of -70°C for further analysis.

DNA Extraction
Tissue biopsies were crushed and DNA extracted through RTP ® Mycobacteria kit (Berlin, Germany).

PCR assay
All samples were tested for the presence of ipaH and ial genes using molecular PCR. The nucleotide sequence of primers (Macrogen, Pishgam) and production size (base pairs) for amplification of the ipaH and ial genes are shown in Table 1 (11,12). PCR was performed in 12.5μL comprising 5μL master mix (Amplicon, Pishgam), 0.5 μL of each primer, 2 μL of the DNA template (50 ng) and 4.5μL of ddH 2 O. Subsequently, the thermal cycling status was used: 300 seconds at 94°C and 25 cycles of amplification consisting of 60 seconds at 95°C, 55 seconds at 58°C, and 60 seconds at 72°C, with 300 seconds at 72°C for the final extension. PCR products were investigated by electrophoresis on a 1% agarose gel in 1X TBE buffer [10.8 g Tris and 5.5 g Boric acid, 0.5 M Na 2 EDTA (pH 8.0)] (13).

Statistical Analysis
Data were analyzed using Pearson Chi-Square test, and P value below 0.05 considered as statistically significant.

Clinical information of patients and controls
The average age of patients with IBD and controls were 38.8 and 52.86 years old respectively. The participants included 14 males and 16 female patients with CD, and 18 males and 12 females without IBD. In addition, the age range for patients with CD and without IBD was 18-64 and 25-76 years old respectively.

Detection of adherent-invasive E. coli with ipaH and ial genes
Of 60 tissue samples collected, E. coli were isolated from 20 (66.6%) patients with CD and 22 (73.3%) from healthy people ( Table 2). All isolated bacteria were confirmed as AIEC by biochemical tests and PCR assay. The 3 ipaH, ial genes were amplified by utilizing particular primers and become visible as a band of approximately 619 and 320 kb on agarose gel respectively ( Figs. 1 and 2). The difference between patients with Crohn's disease and controls was not significant (p=0.59). Of 30 patients with CD, the positive and negative rate of ipaH gene was 46.7% (n=14) and 53.3% (n=16) respectively.
The percentage of ipaH among 30 individuals without IBD was 13.3% (n=4) but 86.6% (n=26) was not detected (Table 3). Furthermore, the positivity of ial gene were 36.7% (n=11) and 10% (n=3) in patients with CD and healthy people respectively (Table 4). Moreover, the presence of ipaH and ial genes in adherent-invasive E. coli in patients with CD was more than control populations.
Due to its high sensitivity, PCR assay was more reliable than cultivation. The difference between the correlation of adherent-invasive E. coli with ipaH and ial in CD patients with control populations was statistically significant (p<0.05). All positive amplified fragments were sequenced and high percentage of adherent-invasive E. coli was identified.
Furthermore, a chi square test was performed and the correlation between adherent-invasive E. coli with ipaH and ial genes in patients with CD found to be (N=30) =7.937, p= .005 and (N=30) =5.963, p=.015 respectively. Nonetheless all positive amplified fragments were sequenced and high percentage of adherent-invasive E. coli was detected.

Discussion
Crohn's disease is considered as a chronic inflammatory of GI disorder.
Although the etiology of inflammatory bowel disease is not still known (14), but numerous studies demonstrated that the mortality rate of this disease is high (15). Furthermore, it is demonstrated that several factors may play role in induction of the disease. In a research by Sartor RB et al, the intestinal microbiota effect on epithelial cells, and inflammation of the intestinal mucosa have shown to trigger the pathogenesis of both CD and UC (16). Moreover, CD patients demonstrated an altered intestinal microbial community, with different kind of bacteria in colonic and ileal areas (17).
Previous studies showed that Enterobacteriaceae especially E. coli could contributed the onset of IBD. Nevertheless other bacteria can trigger the pathogenesis of in-    (19). An emerging evidence indicated an association between adherent invasive E. coli with IBD and colorectal cancer (20, 21 and 22). The present study shows no difference in the incidence of E. coli positive culture between CD and control patients ( Table 2). The lack of E. coli in some tissue sample was probably due to consumption of antibiotics which in turn can cause dysbiosis in the human gastrointestinal tract (23). In some studies, using the culturebased method demonstrated that E. coli bacteria were found in the intestinal mucosa as well as in ulcers of both CD and UC patients compared to controls (24). In comparison with nIBD, our finding shows that the abundance of adherent invasive E. coli harboring ipaH and ial genes was higher in CD individuals. Additionally, we found that 46.7% and 36.7% of CD specimens harbored ipaH and ial, an increased proportion of AIEC, compared to control individuals. In spite of interesting debate, Dogan et al conducted a similar prevalence of AIEC in the ileum of patients with CD (25). Several studies showed a high proportion of AIEC harboring fimH, fimA and Cas genes in CD (26,27). According to the present study, in comparison with nIBD, PCR assay displayed a high abundance of ipaH and ial genes in CD patients with statistically significant difference. These data proposed that AIEC most likely harbored ipaH and ial genes which in turn could be effective in the initiation of the CD. Nonetheless, since other virulence factors for AIEC were not analyzed, so it may be taken into consideration in the future study.

Conclusion
Despite the role of several factors in the progression of IBD, the present study displays a high prevalence of AIEC with ipaH and ial from patients with CD. According to these findings, E. coli with AIEC phenotype could be a predisposing factor in the development of inflammatory bowel disease. In addition to AIEC, host immune response, genetics of patients and environmental factors play an important role in the initiation of IBD. Therefore the present study suggests that appropriate treatment against AIEC could be practically effective in patients with inflammatory bowel disease.