Enteropathogenic Escherichia coli Infection Induces Diarrhea, Intestinal Damage, Metabolic Alterations, and Increased Intestinal Permeability in a Murine Model

Enteropathogenic E. coli (EPEC) are recognized as one of the leading bacterial causes of infantile diarrhea worldwide. Weaned C57BL/6 mice pretreated with antibiotics were challenged orally with wild-type EPEC or escN mutant (lacking type 3 secretion system) to determine colonization, inflammatory responses and clinical outcomes during infection. Antibiotic disruption of intestinal microbiota enabled efficient colonization by wild-type EPEC resulting in growth impairment and diarrhea. Increase in inflammatory biomarkers, chemokines, cellular recruitment and pro-inflammatory cytokines were observed in intestinal tissues. Metabolomic changes were also observed in EPEC infected mice with changes in tricarboxylic acid (TCA) cycle intermediates, increased creatine excretion and shifts in gut microbial metabolite levels. In addition, by 7 days after infection, although weights were recovering, EPEC-infected mice had increased intestinal permeability and decreased colonic claudin-1 levels. The escN mutant colonized the mice with no weight loss or increased inflammatory biomarkers, showing the importance of the T3SS in EPEC virulence in this model. In conclusion, a murine infection model treated with antibiotics has been developed to mimic clinical outcomes seen in children with EPEC infection and to examine potential roles of selected virulence traits. This model can help in further understanding mechanisms involved in the pathogenesis of EPEC infections and potential outcomes and thus assist in the development of potential preventive or therapeutic interventions.

Antidiarrheal Effect of Sechang-Zhixie-San on Acute Diarrhea Mice and Network Pharmacology Deciphering Its Characteristics and Potential Mechanisms

Sechang-Zhixie-San (SCZX) is an ancient prescription used for pediatric diarrhea by the Yi people in China, which consists of Rodgersia sambucifolia Hemsley (known as Yantuo and abbreviated as YT) and Bentonite (BN). Now, it is also a Chinese patent medicine used in the clinic to treat infantile diarrhea. Besides evaluating the antidiarrheal effect of SCZX on diarrhea mice induced by Folium Sennae, the purpose of this study is to outline the characteristics of the antidiarrheal effect and reveal the potential mechanisms of SCZX through the analysis of the mechanism and active components of YT via network pharmacology and molecular docking, combined with the research progress of BN obtained from the literature. SCZX (3.12 and 12.48 g/kg) effectively inhibited diarrhea in mice, significantly lowering the loose stool rate (LSR), loose stool level (LSL), and loose stool index (LSI). Using network pharmacology, the “herb-compound-target-pathway-pharmacological action” network was mapped to indicate the antidiarrheal mechanism of YT. And the docking results revealed that 4 components of YT including quercetin, geranyl-1-O-α-L-arabinopyranosyl-(1 ⟶ 6)-β-D-glucopyranoside, 3α-O-(E)-p-hydroxy-cinnamoyl-olean-12-en-27-oic acid, and daucosterol showed significant docking activities with STAT3, EGFR, and SLC10A2, involving 11 pathways such as Th17 cell differentiation, Jak-STAT signaling pathway, ErbB signaling pathway, and HIF-1 signaling pathway. According to our research results and literature reports, the antidiarrheal could be summarized into five aspects: inhibiting intestinal inflammation, acting as a barrier to the intestinal mucosal, regulating water and ion transport, involving the purification of intestinal microorganisms, and intestinal transmission, which might be dependent on multiple proteins and intervention in multiple pathways.

Fluorescence Detection of Type III Secretion Using a Glu-CyFur Reporter System in Citrobacter rodentium

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea worldwide. EPEC and the closely related murine model of EPEC infection, Citrobacter rodentium, utilize a type III secretion system (T3SS) to propagate the infection. Since the T3SS is not essential for the bacteria to survive or propagate, inhibiting the virulence factor with a therapeutic would treat the infection without causing harm to commensal bacteria. Studying inhibitors of the T3SS usually requires a BSL-2 laboratory designation and eukaryotic host cells while not indicating the mechanism of inhibition. We have designed a BSL-1 assay using the murine model C. rodentium that does not require mammalian cell culture. This CPG2-reporter assay allows for more rapid analysis of secretion efficiency than Western blotting and is sensitive enough to differentiate between partial and total inhibition of the T3SS. Here we present our method and the results of a small collection of compounds we have screened, including known T3SS inhibitors EGCG, regacin, and aurodox and related quorum sensing inhibitors tannic acid and ellagic acid. We have further characterized EGCG as a T3SS inhibitor and established its IC50 of 1.8 ± 0.4 μM. We also establish tannic acid as a potent inhibitor of the T3SS with an IC50 of 0.65 ± 0.09 μM.

Evaluation of Enzyme Immunoassay Method versus Real-time PCR for Diagnosis of Rotavirus Infection in Acute Infantile Diarrhea

Background: Acute infectious gastroenteritis is a common cause of fatality between children in the developing countries which is usually due to viral etiology. Rotavirus is a ds-RNA (60-80nm), non-enveloped virus with a segmented genome. Group (A) of the virus is an important human pathogen that accounts for (90%) of the isolates. An easy, rapid, non-expensive and sensitive method is needed to detect this virus for clinical controlling. The objective of this study is to evaluate Enzyme immunoassay technique versus Quantitative real-time PCR in the diagnosis of infection with Rotavirus in the children with acute diarrhea. Methodology: This study was conducted on (75) infants and young children, from The Pediatric Department at Tanta University Hospitals in the period from December 2019 to March 2020 and were diagnosed according to history and clinical examination using Vesikari scoring system for acute severe gastroenteritis. Also, 10 healthy infants and children were taken as a control group. Stool samples were obtained from the patients and the controls. These specimens were tested with ELISA and Quantitative real-time PCR for detection of Rotavirus in stool. Results: The study revealed that 62 patients (82.6 %) were positive by ELISA and 74 cases (98.6 %) were positive with real time RT-PCR. Additionally, all the control group gave negative results by the two techniques. Conclusion: Enzyme immunoassay is an accurate and suitable method as a routine diagnostic measure for Rotavirus that can run a large number of samples. But, it is expensive when used for a single sample. Quantitative real time PCR was more sensitive and specific measure that can detect Rotavirus RNA in too minimal amounts in stools.

Prevalence of TEM and SHV Genes in Diarrheaogenic Escherichia Coli Resistant to Beta Lactam Antibiotics in Egyptian Infants

Introduction/Objective Diarrhea is considered as the fifth leading cause of mortality in children worldwide. It kills 1 of 9 children according to CDC and causes morbidity in 1.7 billion pediatric cases every year. The most pathogenic strain of diarrheaogenic Escherichia coli (DEC) in Egypt is entero-hemorrhagic Escherichia coli (9.38%), while the most prevalent is entero-toxigenic Escherichia coli (15.63%). The aim of this study is to measure the percentage of TEM and SHV genes in diarrheagenic Escherichia coli (DEC) that are resistant to beta lactam antibiotics among infants. Indicating the essence of different antibiotics prescription for treatment in such cases. Methods Inclusion criteria included age between 2 months and 12 months. Stool samples from 196 cases of acute diarrhea were collected from inpatient and outpatient pediatric clinic in pediatric department at Zagazig University hospitals in Egypt. In 56 samples, diarrheaogenic Escherichia coli strains were confirmed by multiplex PCR after being biochemically identified. Culture and susceptibility tests were conducted. Resistant DEC strains were tested for the presence of TEM and SHV genes using universal primers for conventional PCR. Results By using culture and susceptibility test, 91.1% (51 cases) of isolated DEC strains were found to be resistant to amoxicillin-clavulanic acid. 78.6% (44 cases) resistant to ampicillin-sulbactam. 28.6% (16 cases) resistant to cefotaxime. 28.6% (16 cases) resistant to ceftazidime. 28.6% (16 cases) resistant to cefuroxime. 26.8% (15 cases) resistant to cefoxitin. 26.8% (15 cases) resistant to ceftriaxone. 14.3% (8 cases) resistant to cefepime. 10.7% (6 cases) resistant to aztreonam. 3.5% (2 cases) resistant to imipenem. By using conventional PCR, TEM gene was positive in 28.6% (16 cases) and SHV gene in 7.1% of (4 cases). Conclusion TEM gene was detected in 28.6% (16 out of 56 true positive DEC cases) and SHV gene in 7.1% (4 out of 56 true positive DEC cases). Around 28.6% of diarrheaogenic Escherichia coli or about 8% of infantile diarrhea (16 out of 196 total cases) were found to be resistant to different beta lactam antibiotics due to presence of TEM and SHV genes. This reflects the usefulness of other type antibiotics for treatment in such percentage.