Molecular detection of pathogenic bacteria in the colonic biopsies from patients with Ulcerative Colitis

Background/Aim Ulcerative Colitis (UC) is an inflammatory bowel disease which is common in many areas of the world including Egypt. A lot of controversy regarding the pathogenesis of UC exist. The current study is an attempt to detect some pathogenic bacteria in UC patients. Materials and methods Endoscopic colonic biopsies obtained from 40 patients with ulcerative colitis and 20 controls were analyzed by means of real-time PCR technique for the presence of Clostridium difficile, Helicobacter Pylori (H. pylori) and pathogenic Escherichia Coli (E. coli) which are positive for KPC and/or OXA-48. Results All patients and control samples were negative for Clostridium difficile. Three of the 40 patient samples (7.5%) and none of the 20 controls were positive for H. pylori with no significant difference between the two groups. KPC-positive E. coli were detected in 11 of the 40 patients (27.5%) and in none of the controls with a significant difference between the two groups (P=0.01). All patients and control samples were negative for OXA-48 positive E. coli. Conclusion Although this study does not support the claim that Clostridium difficile and/or H. pylori have a role in UC, it greatly suggests that pathogenic E. coli may be involved in one way or another in the course of UC.


Introduction
Ulcerative Colitis (UC) is an inflammatory bowel disease which occurs with different frequencies around the world with the highest incidence in Canada, United States, United Kingdom and Sweden 1, 2 . Although no accurate data about the exact prevalence of UC in the Middle East, some studies pointed out that the incidence of UC is increasing in this area of the world due to changes in the lifestyle 3, 4 .
The pathogenesis of UC has been a subject of much controversy. Several authors believed that UC may be induced by pathogenic bacteria. Shigella, Salmonella and Yersinia have been suggested as possible cause of UC 5 . More recent studies have argued that pathogenic Escherichia Coli (E. coli) belonging to the B2 and D subgroups play an important role in the pathogenesis of UC 6, 7 . Moreover, other investigators have shown that UC patients have a high risk of Clostridium difficile infection when compared with healthy individuals 8,9 . Also Hel-icobacter Pylori (H. pylori) was found colonizing to the gastric mucosa 10 . The relationship between H. pylori and UC is controversial. Some studies concluded that H. pylori has a protective role against UC [11][12][13] . Contradictory results have been reported by other investigators who claimed that H. pylori may have a causative role in UC 14,15 .
In the current study we investigated the presence of Clostridium difficile, H. pylori and pathogenic E. coli in the colonic tissue specimens from patients with UC.

• Study Subjects and specimen collection:
This is a retrospective study which was performed on formalin fixed, paraffin-embedded (FFPE) colonic tissue specimens obtained by endoscopy from 40 patients with ulcerative colitis (UC). They were 21 females and 19 males. Their median age was 40 years. Twenty two specimens obtained from rectum or rectosigmoid and 18 were obtained from left colon.
Diagnosis of UC was based on the clinical picture, endoscopic and histologic findings 16 . It is worth mentioning that 4 of 40 UC patients (10%) had helicobacter gastritis as proved by gastric biopsy done prior to the study. The disease activity was assessed histologically according to Gupta et al 17 . All patients received no antibiotics for at least 2 months before taking the biopsy. Twenty control specimens were obtained from age-matched individuals who underwent endoscopy be-cause of abdominal pain or discomfort. They showed normal endoscopic and histologic findings.
This work was approved by the institutional review board of Ain Shams faculty of Medicine which waived the requirement for informed consent because it was a retrospective study and the cases were analyzed in an anonymous way. • Real-time PCR procedure: Genomic DNA extraction from the 60 formation-fixed paraffin embedded colonic tissue specimens (40 UC patients and 20 controls) was performed using the DNA purification Kit (QIA amp DNA FFPE kit, Qiagen, Germany). The steps were done following the manufacturer protocol.

DNA amplification and detection was performed using
Quanti-tech SYBR Green PCK Kit (Qiagen, Germany). A PCR reaction was done to amplify the primers as shown in (Table 1). Ten microliter of the template DNA was added to the reaction mixture following the instructions of the manufactures. PCR was performed using Roter Gene 5 plex machine (Qiagen, Germany). The amplification protocol included initial denaturation step at 94oC for 15 min, followed by 45 cycles which consisted of annealing at 55oC for 30 seconds and extension at 72oC for 30 seconds 18 .
An additional test was performed to detect Carbapenem resistant E. coli (MDR) using the KPC and OXA-48 resistant gene primers according to Monterio et al. 2012 18 as shown in (Table 1). The same conditions and reagents quantities were applied. The results were interpreted by melting curve analysis. It is an assessment of the dissociation characteristics of double-stranded DNA during heating. As the temperature is raised, the double strand begins to dissociate leading to a rise in the absorbance intensity. The temperature at which 50% of DNA denaturated is known as melting point 19 .

Statistical analysis
Descriptive data were presented as count and percentages. Quantitative variables were given as median. The statistical analysis was performed using Fisher's exact test and the statistical software SPSS, version 16, A value of P ≤ 0.05 was considered significant.
They were classified into 12(30%) having active disease and 28 (70%) having quiescent disease. Inflammation was marked in 15 patients (including the 12 with active disease), moderate in 9 and mild in 16 cases. Low grade dysplasia was detected in 5 cases (12.5%). No case had high grade dysplasia. All tissue specimens from patients and controls were negative for Clostridium difficile. H. pylori were identified in 3 of the 40 UC specimens (7.5%). They were absent in all 20 control tissue specimens (Figure 1) (Table 3).
There was no significant association between KPC positively and patient age, gender or disease activity (P>0.05) (       In the current study we investigated the presence of Clostridium difficile, H. pylori and pathogenic E. coli in the colonic tissue from patients with UC, using real-time PCR technique. We could not detect Clostridium difficile in all specimens from patients and controls. This results contra-dicts that of Lin et al. and Shoaei et al. 20,21 who detected Clostridium difficile in 17.6% and 29.4% of UC patients respectively. In fact, this contradiction was expected since the previous two studies were performed on fecal samples while our study was performed on colonic tissue. It is well known that stool analysis for bacteria may give much higher false positive results than tissue analysis since most bacteria live within the intestinal lumen and don't enter the mucosa 22 . That is why, Lin et al concluded that microbial analysis of colonic tissue samples may give more solid data than stool analysis 24,25 . Amre et al. reported that the low prevalence of H. pylori infection in patients with inflammatory bowel disease may explain the role of the hygienic hypothesis in the development of this disease. The authors speculated that inadequate microbial stimulation of the gut-associated lymphoid tissue leads to maturation of the mucosal immunity 26 . This speculation is supported by the study of Koloski et al. who reported that a clean environment decreases the incidence of common infections as H. pylori. This leads to autoimmunity and increased susceptibility to certain autoimmune diseases as UC 27 . The limitation of the current study is the small number patients included and using FFPE tissue samples. However, it has the advantage of using colonic tissue, rather than stool sample, thus giving more reliable data. Also, we used real-time PCR technique which is an accurate technique to reassure the amplicon by monitoring the accumulation of specific product during each cycle and discriminating it from outliers through melting curve analysis. Moreover, amplification and detection was performed in the same tube (close tube system). This decreases the possibility of contamination and false positive result 34 . At the same time, the primers used in the study were species specific. So, they only anneal to the templates from one species, thus increasing the accuracy of the results 35-37 .

Conclusion
The present study suggests that Clostridium difficile and H. pylori seem to have no role in UC. More importantly, the detection of pathogenic E. coli in 27.5% of our UC patients suggests that these bacteria may be involved in the process of inflammation in one way or another. This point of research is still in its beginning, and a lot of future studies are recommended to clarify the relationship between E. coli and UC.

Financial support
The authors received no financial support for the research, authorship, and/or publication of this article.

Conflict of interest
No potential conflict of interest relevant to this article was reported.