IBD is a chronic disease affecting both males and females at young age, it has been identified as the most prevalent gastrointestinal disorders, with a higher incidence in newly industrialized countries since the beginning of the 21st century [1]. The incidence rate of IBD increases globally. The exact pathogenesis of IBD is not known and has yet to be discovered [2]. Many bacteriological studies of fecal microbiota in patients with IBD have showed that a varied spectrum of bacterial species, including E. coli, is more abundant in IBD patients’ feces [3, 4].

A comparative genomic investigation E. coli isolates of IBD confirmed that they belonged to a different population groups which looks like extraintestinal pathogenic E. coli (ExPEC) rather than typical diarrheagenic pathotypes [5]. Most of E. coli strains in IBD related to phylogenetic groups B2 and D, which are common to ExPEC strains [6]. Microcins are associated with successful gut colonization, facilitating translocation and invasion, leading to bacteremia, and other infections. In fact, Escherichia coli strains from the more invasive phylogroups (e.g., B2) are frequently microcinogenic [7].

Microcin B17 was first isolated from E. coli strains, and its expression was linked later to the pMccB17plasmid, which holds the mcb operon. The operon consists of seven genes: The essential gene mcbA which encodes a post-translationally peptide that is influenced by the synthases of microcin (mcbB, mcbC, mcbD), which modify serine and cysteine residues within the peptide sequence into oxazole and thiazole rings [8].

B17 microcin derivatives may cause intestinal inflammation by the mechanism of CD1d-dependent pathology and stimulation of both Th1 and Th2 cytokines from the colon tissues depending on a study by Iyer et al. [9], in vivo experiments. Microcin B17 leads to the formation of tryptophan-derived metabolites, which inhibit the responses of epithelial CD1d-restricted IL-10 production. The restricted production of IL-10 causes uncontrolled inflammatory responses and pathology [10]. The infection of Escherichia coli has been widely evaluated on various IBD animals models. In mice, colonization by virulent E. coli causes enhance inflammation with interrupted of T-cell homeostasis, in the other hand, LPS mutants of these E. coli avoid the inflammation [11].

Laboratory animals

Twenty female white rats, Albino rats were used, purchased from animal house of the College of Pharmacy - Karbala University, their ages ranged between 12-14 weeks, and their average weights ranged between (200-210) grams. They were placed in cages prepared for this purpose in the animal house of the College of Pharmacy. Animal room maintained at a temperature of (25\(\pm\)2\({}^\circ\)C), a lighting period of twelve hours of light and twelve hours of darkness throughout the duration of the experiment. The animals were left to acclimatize for two weeks before the start of the experiment.

Preparation for rats’ administration

The bacterial cell suspensions were prepared at concentration of \(1.5\times 10^8\) colony-forming units/ mL (CFU/ml) depending on McFarland turbidity standard N\({}^\circ\) 0.5. To do this, 18-hour-old overnight bacterial cultures on nutrient agar were cultured, and a few bacteria colonies were collected carefully with a sterile loop and placed into 10 ml of sterile 0.90% saline solution and homogenized by mixing.

The Experimental design and animals

The animals were divided to four groups as the following :first group was orally given (E. coli/+mB17) isolated from crohn’s patients stool, the second group was orally given (E. coli/+mB17) isolated from patients stool with ulcerative colitis, the third group was orally given non producing microcin B17 E. coli(E. coli/-mB17) isolated from patients stool with IBD and the last group received distilled water, as control.

Over three months a total 35 doses were administered to each rat, the animals were observed daily for development of clinical symptoms and for mortality. Body weight was measured, food consumption and stool texture were observed every 2-3 days.

Blood samples

The animals were anesthetized with chloroform and dissected by opening the abdominal cavity. Blood samples were collected 5 ml from each animal by drawing blood directly from the heart using the Heart Puncture method. To obtain the largest amount of blood, blood samples were collected using medical syringes (5 ml). the blood sample divided in two tubes (2ml) in EDTA tubes for assessing complete blood account and 3 ml of blood was placed in gel tubes free of anticoagulant for the purpose of obtaining a sufficient amount of serum which was kept frozen at -20\({}^\circ\)C for the purpose of measuring biochemical parameters.

Studying the histopathological changes in small and large intestine of rats

Histological sections were carried out as described by Humason [12]. Following rat sacrifice, the small and large intestines were removed for histological analysis. The organs were fixed quickly in 10% neutral buffered formalin, then dehydrated in a series graded of ethyl alcohol before being flooded in paraffin. 5 mm sections were cut and stained with hematoxylin and eosin. A Zeiss Axioscope photomicroscope was used to photograph histological slides. The reading was performed with an Olympus light microscope. Histopathology was determined based on severity of changes compared to control sections.

Evaluation of biochemical parameters Rat Interleukin 10, IL-10

This is an Enzyme-Linked Immunosorbent Assay (ELISA) kit from (BT LAB-China). Rat IL-10 antibody has been pre-coated on the plate. IL-10 found in the serum sample or in the standard binds to antibodies coated on the wells. The biotinylated rat IL10 Antibody is then added to the sample and links to IL-10. Streptavidin-HRP is then added, which binds to the biotinylated IL-10 antibody. Through the washing stage after incubation, unattached Streptavidin-HRP is washed away. After then, the solution of substrate is added, and the color changes in accordance to the amount of Rat IL-10. The reaction is stopped by adding an acidic stop solution, and absorbance at 450 nm is measured to determine the standard curve Figure 1.

Rat Tumor necrosis factor\(\alpha\)(TNF-\(\alpha\))

This kit from (SUNLONG BIOTECH-China). ELISA technique is used in this kit. The plate has been pre-coated with a TNF-\(\alpha\) antibody. Samples or standards are conjugated with the specific antibody in the corresponding wells. Then, in each well, a Horseradish Peroxidase (HRP)- bounded specific antibody for TNF-\(\alpha\) is added and incubated. The excessive components are washed away. Each well receives the solution of substrate. The wells containing TNF-\(\alpha\) and HRP bounded TNF-\(\alpha\) antibody will show blue and then convert to yellow after the added of stop solution. The optical density (OD) is defined spectrophotometrically at 450 nm, The OD value is proportional to TNF- concentration.

We can calculate the concentration of TNF-\(\alpha\) or IL10 in the samples by comparing the OD of the samples to the standard curve Figure 2 and Figure 1.

To determine the effect of different components in study parameters, the Statistical Analysis System- SAS (2012) application was utilized. In this study, the Least Significant Difference-LSD test (Analysis of Variation-ANOVA) was employed to compare means.

The research was carried out in accordance with the ethical principles outlined in the Helsinki Declaration. A local Ethics Committee reviewed and approved the study protocol, according to the document with the number 3727 in the date September 28, 2021.

Evaluation of serological parameters Serum interleukin-10(IL-10) level in rats after dosage

Table 1 illustrate the results of the IL-10 level in the serum of rats groups enrolled in this experiments. The highest level of interleukin was detected in the sera of group II rats with E. coli/+mB17 isolated from ulcerative colitis patients compared to control group I (110.9 vs. 93.3) pg/ mL . Although there are no significant differences remarked between groups p-value>0.05, but there are differences between group II and group I were statistically significant.

In Table 1, I is control, II is rats with UC isolates+ toxin, III is rats with CD isolates +toxin, IV is rats with IBD isolates without toxins and \(*\) is the mean difference is significant at the 0.05 level.

Rat Tumor necrosis factor-\(\alpha\) (TNF-\(\alpha\)) ELISA Kit

In Table 2 group II had the highest mean value of TNF-\(\alpha\) levels and there was a significant difference between all groups and between group I and the all other groups (p<0.05).

In Table 2, I is control, II is rats with UC isolates+ toxin, III is rats with CD isolates +toxin, IV is rats with IBD isolates without toxins and * is the mean difference is significant at the 0.05 level.

Measurement of blood parameters

In the Table 3 there was a significantly increase in the numbers of WBC and lymphocyte 19.7 (103 \(\mu\)L), 6.7 (103 \(\mu\)L) respectively, in group II, compared to control group and there was a decrease in the number of RBC and the amount of hemoglobin in the infected rat groups compared with control, especially group II represents the rats orally given E. coli isolated from UC. There is a statistically significant differences P<0.05 between groups in the mean numbers of RBC and HGB.

In Table 3, I is control, II is rats with UC isolates+ toxin, III is rats with CD isolates +toxin, IV is rats with IBD isolates without toxins and * is the mean difference is significant at the 0.05 level.

Histological changes

Figure 3 shows normal structure appearance of small and large intestine after administration of 1.5ml of normal saline in control rats intestine.

Figure 3: Histological Sections of (A) Small and (B) Large Intestine of Uninfected Rats (Control) H & E Stain by Using Magnification 10X

Histopathological examination of small and large intestine in Figure 4 of rats were orally given E. coli/+mB17 isolated from CD patients stool. We can see normal small intestine but there is moderate to mild inflammation and reactive lymphoid tissue in the large intestine.

Figure 4: Histological Sections of (A) Small and (B) Large Intestine of Rats with E. coli/+mB17 Isolated from CD Patients Stool , H & E Stain by Using Magnification 10X

In the Figure 5, the rats were orally given E. coli/+mB17 isolated from UC patients stool we can notice there are no noticeable changes in small intestine but in large intestine (B) there is development of edema with inflammatory cells infiltration in mucosa and submucosa.

Figure 5: Histological Sections of (A) Small Intestine, 4 X and (B) Large Intestine, 10 X of Rats with E. coli/+mB17 Isolated from UC Patients Stool, H & E Stain

In the last group (Figure 6) rats orally given E. coli/-mB17 isolated from IBD patients stool, these isolates nonproducing toxin micricin B17 but the effects clear also in the large intestine with the beginning of congestion in sub mucosa and inflammation in the mucosa but the small intestine was normal.

Figure 6: Histological Sections of (A) Small Intestine and (B) Large Intestine of Rats with E. coli/-mB17 Isolated from IBD Patients Stool, H & E Stain by Using Magnification 10X

Limited data are available about the pathogenic roles of microcin B17 in natural hosts or experimental animal models. Recent researches have linked between Escherichia coli producing microcinB 17 (E. coli/+mB17) and the development of inflammatory bowel disease (IBD) [9]. Experimental studies in rodents have led to a better understanding of the role played by many inflammatory mediators in the development and progression of colitis [13]. Tables 1 and 2 showed increase in the level of IL-10 and TNF-\(\alpha\) in all infected groups compared with the control, these results indicate that these bacteria have caused immunological responses in experimental animals.

Anti- inflammatory Interleukin-10 (IL-10) is a key immunoregulatory cytokine that suppresses and terminates inflammatory immunological responses, primarily by inhibiting monocyte and macrophage activity [14], the patients with IBD have an elevated rate of IL10 compered to healthy persons [15, 16, 17] IL-10 inhibits immune responses and reduces the damage caused by inflammation. Schmitz et al. [18] developed colitis in IL-10 deletion gene mice, when he infected them with adherent and invasive Escherichia coli (AIEC) they developed chronic inflammation in the small and large intestines. In another study IL-10-deficient mice (IL-10(-/-)) spontaneously develop intestinal inflammation characterized by discontinuous transmural lesions affecting the small and large intestines [19]. Meta-analysis study illustrated that the serum IL-10 level increased in UC patients unlike in the control group, and it is contributing in the pathogenesis and progression of disease in these patients [20]. Loss-of-function of IL-10 by mutations in IL-10 or IL-10 receptor (IL10R) genes in some people develop severe, medical-refractory, infantile-onset inflammatory bowel disease (IBD) [21, 22]. In a study by Tomoyose et al. [23] found that after the induction of colitis in mice, there was a time-dependent increase in tissue TNF-\(\alpha\) level, followed by a peak of the IL-10 level. E. coli was a stronger inducer of IL-10 in animal’s models with colitis [24]. However, a massive production of IL-10 at early stages of acute inflammation may result in inhibition of TNF- and may be responsible for ineffective elimination of infectious agents [24]. The results came in consistence to a study by Abdul-Zahraa et al. [25] reported that the administration of microcin to mice, elevated Serum level of TNF-\(\alpha\) to 21.86 (pg/ml) when compared to control 14.32(pg/ml) and IL-10 was 27.24 (pg/ml) compared to control 19.54(pg/ml).

On the other hand, proinflammatory cytokine tumor necrosis factor-\(\alpha\) (TNF-\(\alpha\)) have the role in the development of intestinal inflammation and important in the pathophysiology of inflammatory bowel disease (IBD) and tumor formation [26, 27].

Increased levels of the proinflammatory cytokine tumor necrosis factor-\(\alpha\) (TNF-\(\alpha\)) in the gut are linked to disease activity and severity. In a study by Gunasekera et al. [28] found that patients with IBD have an elevated levels of TNF-\(\alpha\) in which UC patients had 1.18 pg/ml and in CD patients 3.12 pg/ml compared to control 0.61pg/ml. It was found elevated the level of TNF-\(\alpha\) in rate’s serum after injected them with lipopolysaccharide (LPS) extracted from E. coli [29]. An increased level of TNF-\(\alpha\) were observed in other studies with induced animals colitis [30, 31]. The results of this study come accordance with a study by Yu et al. (2018) in which he reported that oral administration of microcin J25 (MccJ25) infected mice significantly increased the pro-inflammatory cytokines TNF-\(\alpha\) secretion levels when compared to the control group [32].

Besides, anti-TNF- therapy is systemically administered and effective in the treatment of IBD [33] TNF-\(\alpha\) expression in patients with active CD was increased compared to controls (35.5% vs 25.7%, ), and was significantly decreased in anti-TNF treated CD patients (26.2%) [33].

We evaluated some of blood parameters as shown in table 3 such as the account of white blood cell (WBC), lymphocyte , red blood cell and hemoglobin(Hgb), the results shows increase in WBC and lymphocyte and decreased in RBC and hemoglobin (Hgb) compared with the control.

The function of white blood cells (leucocytes) is to protect the body against invading infections. Leucocytes are significantly less numerous than erythrocytes, yet their numbers rise during an infection. Leucocytes, which are classified as granulocytes (neutrophils, eosinophils, and basophils) or agranulocytes (monocytes and lymphocytes), can recognize foreign material and either engulf cells or emit membrane-disrupting substances that can kill the organism. Lymphocytes are vital in the immune response to diseases because they monitor the internal environment and produce antibodies against infections [34].

The decrease in the number of RBC and the hemoglobin indicate the beginning of anemia which is correlated with the increase production of pro-inflammatory cytokines, such as IL-6, IL-17, and TNF-\(\alpha\), these cytokines caused anemia either by increase hepcidin expression (a liver-derived peptide regulator of iron homeostasis) Hepcidin, in turn, acts as a negative regulator of intestinal iron absorption and macrophage iron release [35]. Or by decrease expression of erythropoietin (Erythropoietin is a hormone produced naturally by our kidneys to stimulate the synthesis of red blood cells). The treatment with anti-TNF-alpha agents has been shown to improve iron deficiency by improving erythropoiesis [36].

Anatomically Crohn’s disease typically affected any part of the intestine in a patchy manner. In contrast, ulcerative colitis affects the rectum and can spread continuously across the entire colon or only a portion of it. The inflammation in ulcerative colitis is only present in the mucosa and submucosa with cryptitis and crypt abscesses in the large intestine only in contrast to the thickened submucosa, transmural inflammation, fissuring ulceration, and granulomas that are histologically present in Crohn’s disease [37].

E. coli with or with out microcin B17 producing, which are used in the study showed the same histological changes in the rat’s large intestine only. Edema, congestion and inflammatory cells infiltration in mucosa and submucosa are the most important changes. Studies on the of pathogenic E.coli in UC patients (obtained from UC patients’ stool specimens and rectal biopsies) indicated that E. coli isolates adhered more strongly to buccal epithelial cells, causing mucosal damage similar to that seen with enteropathogenic E. coli (EPEC) [38] MicrocinB17 as a oxazole structure was sufficient to induce intestinal inflammation in vivo. Intra-rectal administration of mice lead to increased weight loss and pathology characterized by superficial inflammation of the gut wall, neutrophil accumulation, and ulceration of the epithelial layer of the colon [9].

In a study by Yu et al. (2018) in which the microcin J25 showed effects and changes in the intestine of murine models, the mice-treated with 18.2 mg/kg of microcin J25 (MccJ25) significantly reduced the V/C and it has significant influence on the villous height (V) and crypt depth (C) and the V/C in jejunum in jejunum compared with control group [32]. And in another study by Hammad and Obaid, the genotoxic effects of crude bacteriocin detected on albino mice bone marrow cells in vivo, The results showed an acute dose-dependent toxic effect of the crude bacteriocin ; The higher doses (150 and 300 mg/kg) caused a significant increase in the micronuclei frequency in the bone marrow cells. Furthermore, DNA damage increased significantly and proportionally to higher bacteriocin doses [39]. Purified toxin was used in these two studies and that may be the cause of its effectiveness.

E. coli producing microcin B17 with many virulence factor which was isolated from IBD patients stool has the ability to induce the inflammatory responses and histological changes in the rats’ intestine. Beside, the microbiota in the large intestine of the rats may have the role to enhance the pathogenicity of these isolates.

The authors declare no conflicts of interest.

All authors contributed equally to this paper. They have all read and approved the final version.

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