Escherichia coli (E. coli) is a gram negative, rods shaped bacteria, facultative anaerobic belong to Enterobacteriaceae family   that are most common found in the gut of humans and animals as commensals but some strain being pathogenic [2]. E. coli strains which are associated with occurrence of diseases, encode many of virulent factors and can be categorized into pathogenic strains [3]. It has the capability to cause the enteric illness, and extraintestinal illnesses, such as urinary tract infections and septicemia and meningitis.Pathogenic strains of E. coli cause a worldwide morbidity and mortality [4]. The exposure to endotoxin (lipopolysaacharide) of E. coli led to the incidence   of   edema   and     inflammation     of    tissues.                               

Infections with bacteria are one of the main diseases that worldwide intimidate human health [5-6]. In common, antibiotics are the initial choice for treating the bacterial infections. However, to elevate the efficacy of antibiotics, increase the dose or the hesitancy of administration, so causing multidrug resistant and side effects [7-8]. In addition to the treatment challenge of bacteria,it can speedily form biofilms which give it the complexity and hardness to cure by conventional antibiotics. To heading theses crucial challenge,A styling of new anti-bacterial medications to each enhance efficacy and decreases the side effects holds great importance to avoid antibiotic resistance by bacterial infections. In recent years, therapy based on nanomaterials demonstrates as  prospect  to fighting bacterial infections which are difficult to treat, with the capacity to evade acquired antibiotics resistance, as metal based nanomaterials [9]. Among them, Silica nanoparticles (SiNPs) consider a unparalleled type of in organic nano particles with vast discipline of effective characteristics beneficial for fighting infections of bacteria [10-12].

Silica nano materials Owns confirmed to be rising efficient at various levels involving targeting of bacteria and prevention of biofilm formation [13], and it’s using as a system to drug deliver permits to increase efficacy of antibiotics and decreasing dose which solved issue of antibiotic resistance [13]. That is the cause to its widespread use as an alternative to synthesis of novel antibiotics which needs a high exploitation of period and cost [14]. Silica nanoparticles have been manifested to be antidotal to biological systems while intended with suitable structural features and used in the precise doses [15]. Thus, this study aims to expose levels of MCP-1 and TNF-α and histological changes in some organs in rats infected with E. coli treated with silica nanoparticles.

Prepare Clinical Bacterial  Isolation

E. coli was isolated from UTI patients' urine in the laboratory of Al-Diwaniyah Teaching Hospital/Iraq, clinical specimens of E. coli were cultured in MacConkey agar and incubated at 37C˚ for 24 hr. Specific bacteria were identified according to [16] depending on classical laboratory methods. Followed by, the isolation of E. coli sub-cultured in Luria-Bertani (LB) broth until it reached (1106 CFU/ml), with an optical density of  0.4 at 600 nm [17]. rats induced infection with E. coli at a dose of 1x106 CFU/kg intraperitoneally According to [18]. 

Preparation of SiO2NP suspension

SiO2NPs (diameters 10 nm with purity 99%,) were obtained from (Sky Spring Nanomaterials, INC) for use in this study. To prepare the concentration (0.001mg/kg) of SiO2NPs suspended, (0.0001gm) of SiO2 NPs were dissolved in (1 L) of distal water. Before utilization, the silica NP suspension was vortexed for one minute and solicited for 5 minutes. Then SiO2NP suspension injected ip after 3 days from infection with E. coli for 14 days.

In Vivo Study

Trail Animal Preparation: In animal house of Al-Qadisiyah University/Science College, adult white rats weighing between 250 and 300 g and 9-10 weeks of age were bred after being obtained from the College of Veterinary Medicine. After arriving, all animals were given a week to adapt and were given free access to a typical feed and water.

Experimental Design 

Twenty eight experimental rats were randomly distribution into: two egalitarian categories (figure 1) each including 14 rats, and each group was further subdivided into two egalitarian subgroups 7 rats for subgroup As shown below:

Group A

Consisted of (14 uninfected rats) as control, including   two subgroups:

• Subgroup AI: 7 rats without Sio2 NP treatment a long trial period. 

• Subgroup AII: 7 rats with (0.001mg/kg) of Sio2 NP treatment daily by ip injection for a long trial period.

Group B

Included 14 rats infected with E. coli via ip injection, including two subgroups:

• Subgroup BI: 7 rats without Sio2 NP treatment a long trial period. 

• Subgroup BII: 7 rats with (0.001mg/kg) of Sio2 NP treatment daily by ip injection for a long trial period.

 At finishing of experiment period, rats were dissected and samples of blood were drawn from an abdominal vein for an estimate of concentration of inflammatory biomarkers (MCP-1 and TNF- α) in serum. Organs (kidney, lung, and liver) were removed immediately and kept in 10% formalin for histopathological studies.  

Figure 1: Schematic Diagram Explain the Trail Design

Serological Assays (MCP-1 and TNF- α)

Kits of ELISA Rat for MCP-1 and TNF-α supplied by Elabscience Biotechnology Inc were used.  A concentration of (MCP-1 and TNF- α) in serum was evaluated according to the guidelines of manufacture by using the ELISA technique.

Histopathological Study

Samples of rat tissues (lung, kidney, and liver) were dehydrated, then, embedded in wax of paraffin and paraffin blocks were sliced into (4-μm thick). After that, de-waxed sections and followed by stained with Hematoxylin and eosin (HandE) [19].

Data Analysis

The results of TNF- α and MCP-1 levels were analyzed using SPSS, to determine the significance difference, (ANOVA 2 way) was used, after that followed by Tukey's multiple comparisons experience to compare between groups. (P) value (p<0.05) was considered significant.

Serum Levels of MCP-1 and TNF-α

Data analysis of    MCP-1 and TNF-α levels in serum of rats are shown in (Figure 2). The results recorded important increases (P<0.05)  in concentrations of MCP-1 and TNF-α of subgroup BI which was infected with E. coli in comparison with the un-infected subgroups ( AI and AII) and infected with E. coli that were treated with Sio2 NP (BII), on the other hand, the results revealed that the concentration of both biomarkers MCP-1 and TNF-α significantly decreased (P<0.05) in   (BII) subgroup, with no significant difference (P>0.05) among AI, AII and BII. ) in the levels of MCP-1, which indicates the effects of Sio2 NP in lowering these biomarkers.

Histopathological Study

Histological sections obtained from studied organs which included (lungs, livers and kidneys) revealed that infection with E. coli  caused many histopathological changes in all these organs.

Histological examination of rats' pulmonary sections in subgroups controls which were untreated (AI) and treated with SIO2 NP (AII), showed normal texture, pulmonary alveoli appeared to have one endothelial layer with normal structure in the bronchial epithelial (Figure 3 A and B). While the lungs of rats infected ip with E. coli which include subgroups, untreated (BI) and treated with SIO2 NP (BII), investigated those histological alternations included thickness of the endothelial layer of alveoli sacs, therefore, increased distances between them .in addition to necrosis, haemorrhage and infiltration of inflammatory cells in pulmonary branchial tissues (Figure3C and D) with oedema as shown in Figure 3D.

Microscopy examination of liver tissues of rats in all control subgroups investigates normal architecture represents the regular organization of hepatocytes which surround the central vein Figure 4A and D, respectively, While the Liver sections of rats infected with E. coli appeared to lose normal architecture, in addition to degeneration of hepatocytes, the inflammatory cell was accumulating with congestion in a central vein (Figure 4 CandD). Also, there is fibrosis in some regions was noted in the liver section of subgroup BI as shown in Figure 4 D.

Histological examination of kidney tissues in control subgroups AI and AII observe normal renal structure representing normal urinary units including glomeruli and its capillaries in addition to the normal organization to tubules of urinary (Figure 5 A and B). Sections of renal rats infected ip with E. coli for two subgroups B1 andB11 showed necrosis and congestion with increased infiltration of cells of inflammatory in urinary tubules and there is dilation in some tubules in Figure 5 C and D. Also, the collapsing of glomeruli was observed in renal tissues of B1 subgroups as shown in Figure 5 C.

The effects of treating with Sio2 NP on the concentrations of TNF-α and MCP-1 in the rats' serum injected ip with E. coli. subgroups AI   and AII represent uninfected groups as control untreated and treated with Sio2 NP respectively. subgroups BI and BII  which injected ip with E. coli) represent untreated and treated with Sio2 NP respectively. Values indicate means ±standard error. Different letters represent significant 

Figure 2: Data analysis of    MCP-1 and TNF-α levels in serum of rats

Figure 3: Histological examination of rats' pulmonary sections in subgroups controls

Figure 4: Microscopy examination of liver tissues of rats in all control subgroups

differences at level (p<0.05). A similar letter indicates no significant difference at the level (p>0.05). The effects of treating with Sio2 NP on rats' liver tissues that were injected ip with E. coli. Subgroups AI   and AII   represent uninfected subgroups as control untreated and treated with Sio2 NP in (AandB) respectively. Subgroups BI and BII which injected ip with E. coli.  represent untreated and treated with Sio2 NP in (CandD) respectively. AI and AII appear normal structure and organization of hepatocytes in (AandB) respectively. Infected subgroups show Disorganization with degenerate  hepatocytes  (blackhead), Congestion, 

Figure 5: Histological examination of kidney tissues in control subgroups AI and AII

inflammatory cell aggregation (yellow arrows), and fibrosis in some liver regions (blue arrows) (EandH. stain 100X).

Oxidative Stress Biomarker

Escherichia coli (E. coli) is a famous to be a partialy of normal intestinal flora but can be also cause of intestinal and external intestinal disease in humans. There are hundreds of identified E. coli descents, resulting in a spectrum of disease from mild, self-limited gastroenteritis to renal failure and septic shock. Its virulence lends to E. coli’s capability to avoid host defenses and progress resistance to diffuse antibiotics. This review will split E. coli infections to those causing intestinal disease and those causing extra intestinal disease 

Intestinal diseases will be characterized   by causative E. coli subtypes, including enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), which is also known as Shiga toxin-producing Escherichia coli (STEC) and will be referred to as EHEC/STEC, enteroinvasive Escherichia coli (EIEC), enteropathogenic Escherichia coli (EPEC), and enteroaggregative Escherichia coli (EAEC). In the present investigation, the blood levels of TNF-a and MCP-1 rose in infected rats that were not treated with SiO2 NP, but they diminished in rats that received SiO2 NP treatment [20].

Enhanced production of specifically pro-inflammatory cytokines, such as necrotic factor-kappa B (NF-kB) and tumor necrosis factor-a (TNF-a), and an increase in the levels of acute phase proteins [21-23].   In reaction to bacterial infection, several organs and cell types generate TNF-a [24]. Fever and shock are caused by acute phase proteins that are induced by TNF-a. A prototype of CC chemokines, monocyte chemoattractant protein (MCP-1) has been shown to be a potent chemoattractant to monocytes, macrophages, and T cells in the fight against bacterial infection. [25-26]. Numerous cells, including fibroblasts, monocytes/macrophages, endothelial cells, and epithelial cells, generate MCP-1 [27]. The intriguing function of MCP-1 in monocyte/macrophage-mediated host defense against bacterial infections has been clarified by earlier research [28]. Additionally, MCP-1 has been shown to significantly increase the lifespan of mice infected with Salmonella enterica serotype Typhimurium and Pseudomonas aeruginosa. It also enhances macrophage bacterial death [26,29]. While the reduction in TNF-a and MCP-1 levels in the rats treated with SiO2 NP may be related to the fact that many drug-resistant pathogens are simultaneously targeted and combated by silica nanoparticles (NPs), which are regarded as a potent class of nanocarriers for delivering antimicrobial drugs due to their biocompatibility and porous surface. These NPs may also be an effective solution for controlling multi-species biofilms. [30-31]. Inaddition, SiNPs have a high roughness and penetrability, enabling them to effectively penetrate biofilms and eliminate pathogens more effectively than other nanoparticles with lower roughness. They are equally effective against both vegetative and dormant bacterial cells, bacterial fragments, viruses, and fungi [31-32].

The Histological study showed that the lungs of rats infected ip with E. coli which include subgroups, untreated (BI) and treated with SIO2 NP (BII), investigated that histological alternations included thickness of the endothelial layer of alveoli sacs, therefore, increased distances between them .in addition to necrosis, haemorrhage and infiltration of inflammatory cells in pulmonary branchial tissues (Figure3C and D) with oedema as shown in (Figure 3D) , Several studies on E. coli endotoxin in small animals have shown that E. coli endotoxin induces an inflammatory cascade, either through its direct action on the microvascular endothelium or on neutrophils, with accumulation of inflammatory cells in the interstitium, microcirculation, and air spaces of the lung [33-35]. It also causes hypoxia and pulmonary hypertension, with bronchoconstriction, neutrophil sequestration, and increased flow of protein-rich lymph into the lung [36].

Histological examination of kidney tissues in control subgroups AI and AII observe normal renal structure representing normal urinary units including glomeruli and its capillaries in addition to the normal organization of urinary tubules. On the other hand, the renal sections of rats infected ip with E. coli for two subgroups B1 andB11 showed necrosis and congestion with increased infiltration of inflammatory cells in urinary tubules and there is dilation in some tubules. Also, the collapsing of glomeruli was observed in renal tissues of B1 subgroups, The causes of these changes can be attributed to the pathological effects caused by colonic bacteria, as proven by [37]. study demonstrated varying degrees of mitochondrial swelling and autophagy in spleen, kidney, and lung tissue under a transmission electron microscope. Mitochondrial swelling indicates cell damage or death. The study suggested that Mice infected with E. coli experience pathological tissue damage as a result of elevated inflammatory factors. In mice, E. coli can cause inflammation and oxidative stress, which can harm the kidney, lung, and spleen tissues in different ways. [37].

Microscopy examination of liver tissues of rats in all control subgroups investigates normal architecture represents the regular organization of hepatocytes which surround the central vein, respectively, While the Liver sections of rats infected with E. coli appeared to lose normal architecture, in addition to degeneration of hepatocytes, the inflammatory cell was accumulating with congestion in a central vein. Also, there is fibrosis in some regions was noted in the liver section of subgroup BI The reason can be attributed to that Following infection, intestinal bacteria release toxins into the intestine, which then translocate across the intestinal epithelium into the bloodstream, and then target additional host organ cells as well as capillary endothelial cells in the renal glomerulus. Then, apoptosis (programmed cell death), vascular damage, and protein synthesis inhibition harm these cells. Bacterial toxins that affect vascular endothelial cells cause hemolytic uremic syndrome (HUS), which ultimately leads to multiorgan thrombosis. One of the most aggressive substances that can harm microvascular tissue is a toxin. Microthrombi occur as a result of the endothelial cells' usual anticoagulant pattern being changed to a procoagulant one. This could be related to hepatocyte damage by suppressing protein synthesis, leading to death, and vascular damage during HUS is a result of the impact of toxins on vascular endothelial cell. [38-39], further demonstrated that blood AST, ALT, and bilirubin levels significantly increased in 6-hour E. coli-induced sepsis, indicating liver impairment. Microscopic analysis of the liver, which showed a number of pathological alterations in the liver tissue, corroborated these conclusions [40].

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