الفهرس 
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Abstract
Colorectal cancer (CRC) is the worlds’ third most common cancer and the second leading cause of cancer death. Because of its high morbidity and mortality rate in both (males and females), CRC is an important public health issue. Most cases of are sporadic, and numerous studies have suggested that gut microbiota may play a crucial role in CRC development
According to previous studies, the gut microbiota is thought to exert its effects on cancer susceptibility through its metabolic activities and its profound effects on immune cell function. Increasing evidence indicates that imbalances in the normal gut microbiota can promote inflammatory states by producing carcinogenic secondary metabolites that lead to neoplasia. The increased secretion of bacterial toxins and carcinogenic metabolites and decreased secretion of beneficial metabolites compromise intestinal barrier function, leading to immune dysregulation and further cellular proliferation, which can contribute to CRC.
Escherichia coli is a member of the gut microbiota frequently reported to be associated with colorectal tumors. CRC-associated E. coli strains are known to harbor the pks genomic island. This island is responsible for the synthesis of colibactin genotoxin Colibactin-producing E. coli (E. coli clb+) increased the number of tumors in different CRC mouse models. It induces DNA damage such as inter-strand crosslinks, which leads to double-strand breaks, cell cycle arrest and cellular senescence.
The goal of the present study was to assess the possible link between pks+ E. coli genotoxin and colorectal cancer. To attain this goal, fresh tissue biopsy samples were collected from subjects of the study and then QIAamp Mini Spin Column were used to extract purified DNA. Syber Green Real Time PCR was performed on the extracted DNA first to detect total bacterial DNA depending on the 16S rRNA and then to detect the presence of pks island associated with toxigenic E. coli strains.
In the present study, a total of 150 patients (65 males and 85 females) were recruited from the Surgical Endoscopy Unit in the Medical Research Institute hospital, Alexandria University. Then they were divided into two groups, group I including; 100 neoplastic patients (benign and malignant), and group II including; 50 non-neoplastic patients (inflammation (IBD AND non-IBD) and non-inflammatory control). The age of the participants ranged from 18 to 98 years.
Regarding the pathological diagnosis reports, it was found that the neoplastic group was subdivided into 46% benign and 54% malignant patients. On the other hand, the non-neoplastic group was categorized into 40% non-inflammatory control group and 40% non-IBD patients and 20% IBD patients
Concerning the sex of the participants in this study, no statistically significant difference was observed among the study groups (p = 0.059). Males dominated in the malignant neoplastic group, while females dominated in the non-malignant neoplastic group.
Regarding patient`s age which ranged from 18 to 98 years old, there was a higher incidence of malignant neoplastic diseases in patients above 45 years old. The age of malignant patients was significantly higher than non-malignant patients (p value = <0.001).
In order to find if there is an association between CRC and E. coli as a part of normal flora, the current study detected first the presence of 16S rRNA genes to insure the presence of total bacterial DNA in the extracted biopsy samples. The 16S rRNA gene was amplified by Syber Green Real Time PCR using a primer set universal for bacteria. This gene was detected in the whole 100 neoplastic and 50 non-neoplastic patients’ samples.
In the current study, the 16S rRNA bacterial DNA cycle threshold amplification revealed that, no statistically significant difference in the mean Ct value among neoplastic (17.83± 2.28), non-neoplastic inflammation groups (17.90 ± 3.19), and non-inflammatory control group (18.08± 0.95), (p value = 0.906)
The genotoxic E. coli strains were identified as pks+ E. coli, by amplifying clbB gene. In order to use clbB gene as a surrogate marker of the whole pks island, we examined clbQ and clbA genes, which are located close to the 5` and 3`terminals of the pks island, respectively. All samples that were positive for clbB gene in this study were also positive for both clbQ and clbA genes.
The mean Ct values for clbB, clbA, and clbQ were 36.29, 34.81 and 34.16 in benign neoplastic group while they were 36.07, 31.82 and 33.0 in malignant neoplastic group respectively. Regarding non-neoplastic group, the mean Ct values for clbB, clbA, and clbQ were 34.27, 38.27 and 37.79 in IBD, and 37.03, 37.37 and 38.96 in non-IBD and 33.06, 36.04 and 35.22 in non-inflammatory control group; all respectively.
The comparison between the patients’ numbers that were positive for clbB gene amplification among the different pathological groups according to low (less than 30 cycles) and high (more than 30 cycles) Ct values for clbB gene didn’t show any statistically significant difference. (MCp =0.423). On the other hand, when the comparison was based on the total number of patients (n=150) including the number of patients having negative Ct values, a highly statistically significant difference was found between benign and malignant groups (MCp <0.001) and also between neoplastic and non-neoplastic groups (p= 0.005).
In the present study, among 100 neoplastic patients,42 (42%) malignant patients were positive for pks E. coli, while 15 (15%) benign patients tested positive. Whereas among 50 non-neoplastic subjects, in inflammation non-IBD there were 7 (14%) positive and in IBD patients 5 (10%) were also positive for pks E. coli. On the other hand, pks
E. coli was found positive in 3 of 20 (6%) non-inflammatory control individuals
The pks+ E. coli was found to be significantly higher in biopsy samples of malignant patients (42%) than benign patients (15%) (p<0.001), in addition to a significant difference in the presence of the pks island between inflammatory group (85%) and non-inflammatory control group (15%), (p <0.001). There is also an overall significant difference between the prevalence of pks island in neoplastic patients (57%) compared to non-neoplastic patients (30%) (p. <0.001)
Though the findings of this study strongly support the suggestion that pks+ E. coli plays an important role in the pathogenesis of colorectal cancer, more researches are needed to be conducted in order to pave the way for stronger scientific evidence on the role of the microbiota in CRC development.