Diarrhoeal disorders are still a primary cause of disease and mortality in children under five globally, especially in underdeveloped countries. In Iraq, diarrhea diseases form a fairly public health challenge, with a 3.8 episode per child 3.8 episodes per year between children under five [1]. Enterpathogenic Eschhericia coli (EPEC) is identified as an important bacterial pathogen involving chronic diarrhea in newborns and young children, characterized by the expansion symptoms and the risk of extended symptoms and malnutrition [2]. The Pathogenesis of EPEC is mainly due to the ability to generate and affiliated lesions attached and attached to the intestinal epithelium, which is lighter by the chromosomal pathogenesis, which is referred to as the location of the Enterocyte crisis [3]. Lee encodes for the outer membrane protein intimin (coded of EAE genes), which promotes bacterial adhesion near epithelial cells. Specific EPEC strains also contain EPEC Adhesion Factor (EAF) plasmids, coded points with bundle formation pills (BFP, BFP genes) that promote an early connection to host cells [4]. The intestinal epithelium consists of an essential barrier between luminal content and underlying tissues. Close cross, located in the apical region of the lateral membrane, is the essential elements of this obstacle that controls paraselular permeability. It is important to preserve bowel obstruction interaction by adding sender proteins to Zonula Occludens-1 (ZOD), a significant scaffolding protein of dense intersection, act incytoscale [5]. The resolution of Zo-1 and other dense cross proteins are associated with many gastrointestinal diseases, including infectious diarrhea. During the entrance to the intestinal epithelium, it is served as a guard system, which identifies harmful microorganisms and triggers inflammatory reactions through the secretion of pregnancy scytokines and chemochins. Interlucin -8 (IL-8), a powerful neutrophil chemist, is important in acute inflammatory response to bacterial infections of the colon [6]. The high generation results in recruitment and activation of neutrophilic, so contribute to the inflammatory reaction seen in infectious diarrhea. Pre -Research has shown that EPEC can affect the host cell signaling path through the effective protein sent through a type III -secretion system affects potentially dense cross integrity and inflammatory reactions [7]. The precise effect of EPEC infection on ZO-1 and IL-8 gene expression in paediatric patients with diarrhoea in Iraq remains inadequately explored. This study aimed to examine the prevalence of EPEC in paediatric diarrhoea cases in Iraq and assess its effect on ZO-1 and IL-8 gene expression in intestinal epithelial cells, thereby clarifying potential pathophysiological mechanisms of EPEC-induced diarrhoea in the local population.

Study Population and Sample Collection

This case-control study was conducted from October 2024 to February 2025 three paediatric hospitals in Iraq: Al-Dewaniyah Children's Hospital, Central Children's Hospital and Al-Zahraa Children's Hospital. The study protocol received permission from the Ethics Committee of the Faculty of Medicine, University of Baghdad (approval number: 2023/BMC/142) and informed consent was acquired from the parents or legal guardians of all participants. A total of 120 children aged 6 to 60 months with acute diarrhoea (defined as three or more loose or watery stools per day lasting less than 14 days) were included as cases. The exclusion criteria comprised: (1) antibiotic therapy within the preceding two weeks; (2) diagnosed immunodeficiency diseases; (3) severe malnutrition (weight-for-height Z-score <-3); and (4) persistent gastrointestinal illnesses. Furthermore, 40 age-matched healthy children, devoid of diarrhoea or gastrointestinal symptoms for a minimum of one month, were enlisted as controls during regular examinations at the same hospitals. Stool specimens were obtained from all participants in sterile containers and conveyed to the laboratory within 2 hours for microbiological examination. In patients having endoscopy for clinical purposes (n = 35 EPEC-positive cases and 20 controls), two supplementary colonic biopsy specimens were collected from the descending colon: One for RNA extraction and gene expression analysis.

Microbiological Analysis and EPEC Identification

Stool specimens were grown on MacConkey agar and Eosin Methylene Blue (EMB) agar (Oxoid, UK) and incubated aerobically at 37°C for 24 hours. Suspected E. coli colonies (pink colonies on MacConkey agar or green metallic sheen colonies on EMB agar) were subcultured onto nutritional agar for purification. Biochemical identification was conducted by established methodologies, including indole, methyl red, Voges-Proskauer and citrate utilisation assays. Confirmed E. coli isolates were serotyped utilising commercially available antisera (Denka Seiken, Japan). Molecular identification of EPEC was conducted by recognising the eae (intimin) and bfp (bundle-forming pilus) genes by multiplex PCR.

RNA Extraction and Quantitative Real-Time PCR

Total RNA was isolated from intestinal biopsy specimens utilising the RNeasy Mini Kit (Qiagen, Germany) in accordance with the manufacturer's instructions. The quality and quantity of RNA were evaluated utilising a NanoDrop spectrophotometer (Thermo Scientific, USA). Complementary DNA (cDNA) was generated from 1 μg of total RNA with the QuantiTect Reverse Transcription Kit (Qiagen, Germany). Quantitative real-time PCR (qRT-PCR) was conducted utilising the QuantiTect SYBR Green PCR Kit (Qiagen, Germany) on a StepOnePlus Real-Time PCR System (Applied Biosystems, USA). The following primers were used:

• ZO-1-F: 5'-GTCCAGAATCTCGGAAAAGTGC-3' (150 bp)

• ZO-1-R: 5'-CTTTCAGCGCACCATACCAA-3' (150 bp)

• IL-8-F: 5'-ACTGAGAGTGATTGAGAGTGGAC-3' (174 bp)

• IL-8-R: 5'-AACCCTCTGCACCCAGTTTTC-3' (174 bp)

• GAPDH-F: 5'-GAAGGTGAAGGTCGGAGTC-3' (226 bp)

• GAPDH-R: 5'-GAAGATGGTGATGGGATTTC-3' (226 bp)

PCR conditions were as follows: initial denaturation at 95°C for 15 minutes; 40 cycles of denaturation at 94°C for 15 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 30 seconds. Each sample was analyzed in triplicate and the relative gene expression was calculated using the 2^-ΔΔCt method, with GAPDH as the reference gene.

Statistical Analysis

Data analysis was conducted utilising SPSS version 26.0 (IBM Corp., USA). Categorical variables were expressed as frequencies and percentages and analysed using the chi-square test.

Demographic and Clinical Characteristics

The current study comprised 120 children with diarrhoea and 40 control subjects. The baseline demographic data exhibited no major disparities between the two groups. The average age of children in the diarrhoea group was 24.6±13.5 months, whereas in the control group it was 25.8±12.9 months (p = 0.614). The gender distribution was comparable across groups, with men representing 56.7% (n = 68) of diarrhoea   cases  and   52.5%  (n = 21)  of  controls  (p = 0.726). 

Residence patterns shown no significant variation, with urban residence comprising 68.3% (n = 82) of diarrhoea cases and 75.0% (n = 30) of controls (p = 0.582). Breastfeeding status shown a statistically significant difference between the groups (p = 0.042). Exclusive breastfeeding was less prevalent among children with diarrhoea compared to the control group (15.0% vs 30.0%, respectively). A significant proportion of children with diarrhoea (47.5%, n = 57) were not breastfed, in contrast to merely 30.0% (n = 12) in the control group. Partial breastfeeding was noted in 37.5% (n = 45) of diarrhoea cases and 40.0% (n = 16) of control subjects. Of the 120 children with diarrhoea, watery diarrhoea was the predominant manifestation, impacting 60.8% (n = 73) of cases. Mucoid diarrhoea was noted in 26.7% (n = 32) of children, but bloody diarrhoea was the least prevalent, occurring in 12.5% (n = 15) of instances. The median duration of diarrhoea was 5 days (interquartile range: 3-8 days), with a median frequency of 6 episodes per day (interquartile range: 4-8 episodes). The evaluation of hydration status indicated that a significant proportion of children with diarrhoea (56.7%, n = 68) had signs of mild dehydration. Dehydration was not recognised in 35.0% (n = 42) of instances, however severe dehydration was identified in 8.3% (n = 10) of children with diarrhea (Table 1).

Table 1:  Baseline Characteristics and Clinical Presentation of Study Participants

*Statistically significant at p<0.05; IQR: Interquartile Range; NA: Not Applicable

Clinical Features Associated with EPEC Infection

Among the 120 children with diarrhoea, 35 (29.2%) were positive for enteropathogenic Escherichia coli (EPEC), whereas 85 (70.8%) were negative for EPEC. The average age was marginally lower in EPEC-positive children (22.1±12.7 months) than in EPEC-negative children (25.6±13.8 months), although this difference was not statistically significant (p = 0.198). Multiple clinical characteristics demonstrated substantial correlations with EPEC infection. There was a significant difference in the kind of diarrhoea across groups (p = 0.037), with watery diarrhoea occurring more frequently in EPEC-positive children (71.4% compared to 56.5%). In contrast, mucoid diarrhoea occurred less frequently in EPEC-positive cases (17.1% compared to 30.6%), although the incidence of bloody diarrhoea was comparable between the groups (11.4% versus 12.9%). Children positive for EPEC exhibited a markedly prolonged duration and increased frequency of diarrhoea. The median duration was 7 days (IQR: 5-10) for EPEC-positive children, in contrast to 4 days (IQR: 3-7) for EPEC-negative children (p<0.001). The median frequency of diarrhoeal episodes was greater in EPEC-positive children, with 7 episodes per day (IQR: 5-9), compared to 5 episodes per day (IQR: 4-7) in EPEC-negative children (p = 0.003). The dehydration condition shown a notable correlation with EPEC infection (p = 0.014). EPEC-positive children exhibited a higher incidence of severe dehydration (17.1% vs 4.7%) and a lower occurrence of no dehydration (20.0% vs 41.2%) in comparison to EPEC-negative children. Dehydration was seen in 62.9% of EPEC-positive children and 54.1% of EPEC-negative children. Other clinical manifestations, such as fever, vomiting and stomach pain, exhibited no significant disparities between the EPEC-positive and EPEC-negative cohorts. Fever occurred in 62.9% of EPEC-positive children and 49.4% of EPEC-negative children (p = 0.175). Vomiting was observed in 51.4% of EPEC-positive children and 43.5% of EPEC-negative children (p = 0.427). Abdominal pain was observed in 42.9% of EPEC-positive children and 34.1% of EPEC-negative children (p = 0.362) (Table 2).

Table 2: Clinical Features Associated with EPEC Infection

*Statistically significant at p<0.05; EPEC: Enteropathogenic Escherichia coli; IQR: Interquartile Range

Intestinal Barrier Function and Inflammatory Response

In Table 3 we evaluated the effect of EPEC infection on intestinal barrier integrity and inflammatory response by measuring the expression of the tight junction protein ZO-1 and the inflammatory marker IL-8 in intestinal biopsy samples. The research demonstrated notable disparities between EPEC-positive patients and controls, in addition to variations among several EPEC pathotypes. All EPEC-positive patients had significantly reduced ZO-1 expression relative to controls (0.42±0.15 vs 1.00±0.00 fold change, p<0.001), signifying impaired intestinal barrier function. 

Table 3: Intestinal Barrier Function and Inflammatory Response by EPEC Pathotype

*Values expressed as mean±standard deviation relative to control group; ZO-1: Zonula occludens-1; IL-8: Interleukin-8; EPEC: Enteropathogenic Escherichia coli; eae: E. coli attaching and effacing gene; bfp: bundle-forming pilus gene

Concurrently, IL-8 expression was markedly heightened in EPEC-positive patients (5.60±1.70 versus 1.00±0.00 fold change, p<0.001), indicating augmented intestinal inflammation. Subsequent investigation by EPEC pathotype identified unique patterns of barrier failure and inflammatory response. Typical EPEC (eae+/bfp+) induced greater barrier disruption than atypical EPEC (eae+/bfp-), with ZO-1 expression diminished to 0.35±0.12 fold change, in contrast to 0.54±0.14 fold change in atypical EPEC (p = 0.006). Both pathotypes exhibited a markedly decreased expression of ZO-1 in comparison to controls (p<0.001 for both). The inflammatory response varied among EPEC pathotypes. Typical EPEC elicited a greater IL-8 expression (6.40±1.80 fold change) than atypical EPEC (4.30±0.90 fold change), with this discrepancy being statistically significant (p = 0.002). Each pathotypes exhibited markedly increased IL-8 expression relative to controls (p<0.001 for each). The data indicate that EPEC infection is linked to significant intestinal barrier impairment and inflammatory response, with typical EPEC resulting in more severe changes than atypical EPEC in both aspects.

Correlation between Molecular Markers and Clinical Severity

To elucidate the association between intestinal barrier failure, inflammation and clinical illness severity, we conducted correlation studies between gene expression levels and clinical indicators. The findings demonstrated robust and statistically significant correlations that elucidate the pathophysiological mechanisms driving EPEC-induced diarrhoea. ZO-1 expression exhibited significant negative relationships with the severity of clinical illness. Reduced ZO-1 expression (signifying increased barrier dysfunction) was substantially correlated with prolonged diarrhoea length (r = -0.67, p < 0.001) and elevated frequency of diarrhoeal episodes (r = -0.59, p<0.001). The data indicate that patients with greater intestinal barrier impairment experienced more persistent and frequent diarrhoeal symptoms. In contrast, IL-8 expression had robust positive associations with clinical indicators. Elevated IL-8 levels, indicative of heightened intestinal inflammation, were strongly associated with prolonged diarrhoea length (r = 0.62, p<0.001) and an increased frequency of diarrhoeal bouts (r = 0.54, p = 0.001). This suggests that patients exhibiting more pronounced inflammatory responses experienced more severe clinical manifestations. The association between barrier function and inflammation was substantiated by the significant negative correlation between ZO-1 and IL-8 expression (r = -0.71, p<0.001). This inverse association illustrates that a decline in intestinal barrier integrity (reduced ZO-1) correlates with an escalation in inflammatory response (elevated IL-8), thereby demonstrating a definitive pathophysiological connection between barrier failure and inflammation in EPEC infection. The results present strong evidence that the molecular changes seen in EPEC infection directly correlate with clinical disease severity, with barrier failure and inflammation as critical factors influencing symptom duration and frequency (Table 4).

Table 4: Correlation between Gene Expression and Clinical Parameters

ZO-1: Zonula Occludens-1; IL-8: Interleukin-8; Correlation coefficients calculated using Pearson Correlation Analysis

This study examined the incidence of EPEC in paediatric patients with diarrhoea in Iraq and assessed its effect on ZO-1 and IL-8 gene expression in intestinal epithelial cells. Our data indicate that EPEC infection is linked to a substantial downregulation of ZO-1 and an overexpression of IL-8, which coincide with clinical indicators of illness severity. The prevalence of EPEC in children with diarrhoea in our study (29.2%) aligns with rates documented in other developing nations, such as Brazil (25.6%), India (31.2%) and other African countries (20-40%) [8-10]. The elevated prevalence of typical EPEC (62.9%) relative to atypical EPEC (37.1%) in our diarrhoeal cases aligns with the epidemiological trends documented in most poor nations, where typical EPEC continues to be a notable pathogen. Atypical EPEC has become a more prevalent cause of diarrhoea in affluent nations [11]. It may indicate a difference in hygiene practice, socio-economic conditions and the health care system between the areas of epidemic. It turned out that the most important EPECs were O111, O126 and O55, which formed 57.1% of all isolates. These findings match previous data from nearby countries such as Iran and Jordan, which reflect regional similarities in Epec Serogrup distribution [12,13]. Identification of separate large serogrops can lead to the guidance of targeted monitoring and vaccination initiatives in the area. In our examination, the clinical features of EPEC -positive diarrhea, longer periods, height frequency and characterized by the greater severity of dehydration, recognized principles of EPEC patogenesis. Pre -Research has shown that EPEC can induce frequent diarrhea in some examples, resulting in significant illness and potential malnutrition [14]. EPEC-positive individuals (71.4%) adjust the prevalence of water diarrhea with the pathophysiological system of EPEC, interfering with the intestinal epithelial function, resulting in expanded parametic permeability and ion secretion. Z-1 is an essential scaffolding protein connecting tight cross-transmembrain proteins to actincytoscalatone, thus retaining epithelial barrier integrity [15]. In vitro probes show that EPEC can cause tight intersection solution for multiple mechanisms, including cytoskeletal rehabilitation, which is provided by ESPF and MAP of Effercor -Protein, distributed in host cells through Type III Secretion System [16]. Our findings make these comments wider for clinical samples from pediatric patients and confirm that the EPEC-induced disintegration of ZO-1 is a relevant in the pathogenesis of diarrhea in children. The more significant downturns of the Z-1 in typical EPEC infections, which, unlike the atapical EPEC infections, may indicate the variation in the virta capacity between the two patotypes. Specific EPEC is characterized by the presence of LI-coded Type III-Secretion System and EAF-Plasodet BundleFormat Pilgrimage, which shows a promotional capacity for early compliance to host cells and subsequent connections, which can ease the compromise with narrow intersections [17]. Along with this, with the decline in ZO-1, we noted a sufficient increase in IL-8 MRNA in epithelial cells infected with EPEC. IL-8 is a powerful neutrophilic chemoothant required in acute inflammatory reaction to bacterial colon infections [18]. EPEK-EL-8 expression in infected samples indicates activation of pregnancy signal paths in response to the infection. This discovery indicates previous research that it indicates that EPEC can stimulate IL-8 synthesis in intestinal epithelial cells through many mechanisms, including NF-κB activation and MAPK signal road [19]. Atypical EPEC forward the higher height of IL-8 and a height of specific EPEC strains in specific EPEC infections in relation to infections. The existence of EAF plasmids in traditional EPECs can increase inflammatory reactions through supplementary virus factors or improve better transmission of protein proteins. The clear negative correlation between Z-1 and IL-8 expression levels in our study indicates a possible relationship between intestinal barrier loss and inflammatory reactions in EPEC-induced diarrhea. This observation is in accordance with the assumption that obstacles of obstacles can promote bacterial transloor and increase the host -immun cell exposure to microbial antigens, then intensifies inflammatory reactions [20]. Prinflametry cytokines, such as IL-8, can affect the construction and function of tight intersections, aggravated obstacle solution [21]. The clinical importance of Z-1-red regulation and IL-8 expression are exposed to the duration and frequency of diarrhea of ​​their sufficient associations. The reverse relationship between Z-1 expression and the severity of the disease confirms the concept that the loss of bowel obstruction function is an important mechanism in EPEC-induced diarrhea. The strong relationship between IL-8 expression and the severity of the disease indicates that inflammation plays a role in the clinical symptoms of EPEC infection. These findings provide molecular insight into EPEC-related diarrhea pathophyziology and outline potential therapeutic goals for intervention. There are some limitations in this study that should be recognized. Cross-sectional design causes determination of the order of link or events. If changes in Z-1 and IL-8 expressions succeed in tracking these changes at the front or at the beginning of the diarrhea and during the entire healing process, it will be important to detect the longitudinal probe. The study focuses on two special indicators for intestinal barriers and inflammation, although the pathophyziology of EPEC-induced diarrhea may include other variables and routes. Subsequent research should detect a wide selection of inflammatory arbitration to increase the knowledge of the extra dense cross proteins (eg chlaudin, ocludin) and the molecular system involved. Despite these obstacles, our research provides innovative insight into molecular etiology of EPEC-induced diarrhea in children in Iraq. The results emphasize the importance of ZO-1 down ingredients and IL-8 abroad in EPEC development, reflecting potential therapeutic goals for intervention. Future research should investigate methods for maintaining intestinal obstacle integrity and regulating inflammatory reactions in EPEC infections, allowing clinical consequences in affected pediatric patients.

In conclusion, our research indicates that EPEC is a major contributor to acute diarrhoea in paediatric patients in Iraq, with typical EPEC strains being predominant. EPEC infection is linked to a notable downregulation of ZO-1 and an overexpression of IL-8 in intestinal epithelial cells, correlating with clinical indicators of disease severity. These molecular modifications may facilitate the development of EPEC-induced diarrhoea by impairing intestinal barrier integrity and provoking inflammatory responses. Our research elucidates the molecular pathways behind EPEC disease and identifies possible therapeutic targets for intervention. Future research should concentrate on formulating ways to maintain intestinal barrier integrity and regulate inflammatory responses in EPEC infections, hence potentially enhancing clinical outcomes in affected paediatric patients.

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