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Abstract Background No licensed human vaccines are available against enterotoxigenic Escherichia coli (ETEC), a major diarrhoeal pathogen affecting children in low- and middle-income countries and foreign travellers alike. ETVAX®, a multivalent oral whole-cell vaccine containing four inactivated ETEC strains and the heat-labile enterotoxin B subunit (LTB), has proved promising in Phase 1 and Phase 1/ 2 studies. Methods We conducted a Phase 2b double-blinded, randomized, placebo-controlled trial amongst Finnish travellers to Benin, West Africa. This report presents study design and safety and immunogenicity data. Volunteers aged 18–65 years were randomized 1:1 to receive ETVAX® or placebo. They visited Benin for 12 days, provided stool and blood samples and completed adverse event (AE) forms. IgA and IgG antibodies to LTB and O78 lipopolysaccharide (LPS) were measured by electrochemiluminescence. Results The AEs did not differ significantly between vaccine (n = 374) and placebo (n = 375) recipients. Of the solicited AEs, loose stools/diarrhoea (26.7/25.9%) and stomach ache (23.0/20.0%) were reported most commonly. Of all possibly/probably vaccine-related AEs, the most frequent were gastrointestinal symptoms (54.0/48.8%) and nervous system disorders (20.3/25.1%). Serious AEs were recorded for 4.3/5.6%, all unlikely to be vaccine related. Amongst the ETVAX® recipients, LTB-specific IgA antibodies increased 22-fold. For the 370/372 vaccine/placebo recipients, the frequency of ≥2-fold increases against LTB was 81/2.4%, and against O78 LPS 69/2.7%. The majority of ETVAX® recipients (93%) responded to either LTB or O78. Conclusions This Phase 2b trial is the largest on ETVAX® undertaken amongst travellers to date. ETVAX® showed an excellent safety profile and proved strongly immunogenic, which encourages the further development of this vaccine.

•The killed oral ETEC vaccine ETVAX ± dmLT adjuvant was safe in Bangladeshi adults.•All vaccinees responded to all 5 primary vaccine antigens in ALS specimens.•A majority of vaccinees responded to ≥4 antigens in plasma specimens.•A sensitive electrochemiluminescence assay was established for small sample volumes.•ALS responses measured by electrochemiluminescence and ELISA assays correlated well. The safety and immunogenicity of the second generation oral enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX, consisting of inactivated recombinant E. coli strains over-expressing the colonization factors (CFs) CFA/I, CS3, CS5 and CS6 and the heat labile toxoid LCTBA, were evaluated in Bangladeshi volunteers. To enable analysis of antibody responses against multiple vaccine antigens for subsequent use in small sample volumes from children, a sensitive electrochemiluminescence (ECL) assay for analysis of intestine-derived antibody-secreting cell responses using the antibodies in lymphocyte secretions (ALS) assay was established using Meso Scale Discovery technology. Three groups of Bangladeshi adults (n = 15 per group) received two oral doses of ETVAX with or without double mutant LT (dmLT) adjuvant or placebo in the initial part of a randomized, double-blind, placebo-controlled, age-descending, dose-escalation trial. CF- and LTB-specific ALS and plasma IgA responses were analyzed by ECL and/or ELISA. ETVAX was safe and well tolerated in the adults. Magnitudes of IgA ALS responses determined by ECL and ELISA correlated well (r = 0.85 to 0.98 for the five primary antigens, P < 0.001) and ECL was selected as the ALS readout method. ALS IgA responses against each of the primary antigens were detected in 87–100% of vaccinees after the first and in 100% after the second vaccine dose. Plasma IgA responses against different CFs and LTB were observed in 62–93% and 100% of vaccinees, respectively. No statistically significant adjuvant effect of dmLT on antibody responses to any antigen was detected, but the overall antigenic breadth of the plasma IgA response tended to favor the adjuvanted vaccine when responses to 4 or more or 5 vaccine antigens were considered. Responses in placebo recipients were infrequent and mainly detected against single antigens. The promising results in adults supported testing ETVAX in descending age groups of children. ClinicalTrials.gov Identifier: NCT02531802.

This study examined the EPEC, ETEC and STEC pathotypes in E. coli isolates from water supplied to schools in a municipality in Maranhão, Brazil with a low human development index. For this, 57 bacterial strains isolated from 19 water samples were used. All strains were confirmed as belonging to the E. coli species, by Gram staining and phenotypic tests. From pure cultures of E. coli, DNA was extracted followed by characterization of the isolates by polymerase chain reaction (PCR). This is the first study carried out in the state of Maranhão on the research of the diarrheagenic strains EPEC, ETEC and STEC in water for human consumption offered in schools, with the detection of virulent genes characteristic of enterotoxigenic E. coli (26.31%; n = 15/57) and E. coli producing Shiga toxin (8.78%; n= 5/57). Of the strains identified, 8.78% (n= 5/57) corresponded to combinations of est + stx1 genes. It is concluded that the detection of the diarrheagenic strains ETEC and STEC in E. coli isolates from water samples for human consumption indicates that, in addition to the water being contaminated, it harbors strains with pathogenic potential to cause diarrheal infection in its users. Keywords: ETEC, school environment, STEC.

IntroductionEnterotoxigenic E. coli (ETEC) is a major pathogen causing piglet diarrhea. This study aimed to investigate the mechanism of porcine circular RNAs (circRNAs) in regulating intestinal immunity during ETEC infection.MethodsThe circRNA expression profiles were obtained in ETEC-infected and uninfected IPEC-J2 cells via RNA-sequencing. The stable covalently closed structure of circRNAs was validated using qRT-PCR and RNase R digestion methods. The potential circRNA/miRNA/mRNA interactions were analyzed using Miranda software, dual-luciferase reporter assay, knockdown and over-expression of the target gene or RNA. The expression of pyroptosis-related factors was assessed by qRT-PCR and Western blot. Flow cytometry was utilized to quantify pyroptotic cells, and transmission electron microscopy was used to observe cellular morphology.ResultsIn this study, a total of 328 differentially expressed circRNAs were identified in ETEC-infected versus uninfected IPEC-J2 cells, among which a novel circRNA named circ₀020647 was significantly upregulated post-infection. Circ₀020647, encoded by an intergenic sequence, forms a covalently closed loop structure. We demonstrated that circ₀020647 acts as a molecular sponge for miRNA ssc-mir-185 through direct binding, which in turn targets BRD4 mRNA. Following ETEC infection, circ₀020647 promoted pyroptosis in IPEC-J2 cells by increasing the expression of NLRP3, GSDMD, and caspase-1. Additionally, circ₀020647 was involved in ETEC-induced cell injury, characterized by LDH efflux, IL-1β and IL-18 secretion, formation of membrane pores, and mitochondrial abnormalities. We revealed that the role of circ₀020647 in regulating pyroptosis was mediated by the ssc-mir-185/BRD4 axis.ConclusionOur study constructed a novel circ₀020647/ssc-mir-185/BRD4 network that played an important role in the pyroptosis of IPEC-J2 cells induced by ETEC infection. Our findings imply that the circRNA/miRNA/mRNA network may be a novel biomarker and a potential therapeutic target for diarrhea in piglets caused by ETEC.

Diarrhoeal disease attributable to enterotoxigenic Escherichia coli (ETEC) causes substantial morbidity and mortality predominantly in paediatric populations in low- and middle-income countries. In addition to acute illness, there is an increasing appreciation of the long-term consequences of enteric infections, including ETEC, on childhood growth and development. Provision of potable water and sanitation and appropriate clinical care for acute illness are critical to reduce the ETEC burden. However, these interventions are not always practical and may not achieve equitable and sustainable coverage. Vaccination may be the most cost-effective and equitable means of primary prevention; however, additional data are needed to accelerate the investment and guide the decision-making process for ETEC vaccines. First, to understand and quantify the ETEC disease burden, additional data are needed on the association between ETEC infection and physical and cognitive stunting as well as delayed educational attainment. Furthermore, the role of inappropriate or inadequate antibiotic treatment of ETEC-attributable diarrhoea may contribute to the development of antimicrobial resistance (AMR) and needs further elucidation. An ETEC vaccine that mitigates acute diarrhoeal illness and minimizes the longer-term disease manifestations could have significant public health impact and be a cost-effective countermeasure. Herein we review the ETEC vaccine pipeline, led by candidates compatible with the general parameters of the Preferred Product Characteristics (PPC) recently developed by the World Health Organization. Additionally, we have developed an ETEC Vaccine Development Strategy to provide a framework to underpin priority activities for researchers, funders and vaccine manufacturers, with the goal of addressing globally unmet data needs in the areas of research, product development, and policy, as well as commercialization and delivery. The strategy also aims to guide prioritization and co-ordination of the priority activities needed to minimize the timeline to licensure and use of ETEC vaccines, especially in in low- and middle-income countries, where they are most urgently needed.

Porcine Post Weaning Diarrhoea (PWD) is one of the most important swine disease worldwide, caused by Enterotoxigenic Escherichia coli (ETEC) strains able to provoke management, welfare and sanitary issues. ETEC is determined by proteinaceous surface appendages. Numerous studies conducted by now in pigs have demonstrated, at the enterocytes level, that, the genes mucin 4 (MUC4) and fucosyltransferase (FUT1), coding for ETEC F4 and F18 receptors respectively, can be carriers of single nucleotide polymorphisms (SNPs) associated with natural resistance/susceptibility to PWD. The latter aspect was investigated in this study, evaluating the SNPs of the MUC4 and FUT1 genes in slaughtered pigs reared for the most in Central Italy. Genomic DNA was extracted from 362 swine diaphragmatic samples and then was subjected to the detection of known polymorphisms on MUC4 and FUT1candidate target genes by PCR-RFLP. Some of the identified SNPs were confirmed by sequencing analysis. Animals carrying the SNPs associated with resistance were 11% and 86% for the FUT1 and MUC4 genes respectively. Therefore, it can be assumed that the investigated animals may be an important resource and reservoir of favorable genetic traits for the breeding of pigs resistant to enterotoxigenic E.coli F4 variant.

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high production costs. The rise of antibiotic selective pressure together with ongoing limitations on their use, demands new strategies to tackle this pathology. The pertinence of using bacteriophages as an alternative is being explored, and in this work, the efficacy of phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC EC43-Ph (serotype O9:H9 expressing the enterotoxin STa and two adhesins F5 and F41) was assessed. Foreseeing the oral application on piglets, FJ1 was encapsulated on calcium carbonate and alginate microparticles, thus preventing phage release under adverse conditions of the simulated gastric fluid (pH 3.0) and allowing phage availability in simulated intestinal fluid (pH 6.5). A single dose of encapsulated FJ1, provided to IPEC-1 cultured cells (from intestinal epithelium of piglets) previously infected by EC43, provided bacterial reductions of about 99.9% after 6 h. Although bacteriophage-insensitive mutants (BIMs) have emerged from treatment, the consequent fitness costs associated with this new phenotype were demonstrated, comparatively to the originating strain. The higher competence of the pig complement system to decrease BIMs' viability, the lower level of colonization of IPEC-1 cells observed with these mutants, and the increased survival rates and health index recorded in infected Galleria mellonella larvae supported this observation. Most of all, FJ1 established a proof-of-concept of the efficiency of phages to fight against ETEC in piglet intestinal cells. Keywords: Swine colibacillosis, ETEC, bacteriophage, pig neonatal cell line, BIMs

Zinc oxide (ZnO) at pharmacological doses is extensively employed in the pig industry as an effective tool to manage post-weaning diarrhea (PWD), a condition that causes huge economic losses because of its impact on the most pivotal phase of a piglet’s production cycle. In a multifactorial way, ZnO exerts a variety of positive effects along the entire gastrointestinal tract by targeting intestinal architecture, digestive secretions, antioxidant systems, and immune cells. ZnO also has a moderate antibacterial effect against Escherichia coli F4 (K88), the main causative agent of PWD. However, the environmental impact of ZnO and new emerging threats are posing serious questions to the sustainability of its extensive utilization. To work towards a future free from pharmacological ZnO, novel nutritional approaches are necessary, and many strategies have been investigated. This review article provides a comprehensive framework for ZnO utilization and its broad mode of action. Moreover, all the risks related to pharmacological ZnO levels are presented; we focus on European institutions’ decisions subsequently. The identification of a novel, complete solution against PWD should be accompanied by the adoption of holistic strategies, thereby combining good management practices to feeding approaches capable of mitigating Escherichia coli F4 (K88) infections and/or lowering ZnO utilization. Promising results can be obtained by adjusting diet composition or employing organic acids, natural identical compounds, polyphenol-rich extracts, prebiotics, and probiotics.

Intestinal infection with enterotoxigenic Escherichia coli (ETEC) is an important disease in swine resulting in significant economic losses. Knowledge about the epidemiology, the diagnostic approach and methods of control are of fundamental importance to tackle the disease. The ETEC causing neonatal colibacillosis mostly carry the fimbriae F4 (k88), F5 (k99), F6 (987P) or F41, while the ETEC of post-weaning diarrhoea carry the fimbriae F4 (k88) and F18. These fimbriae adhere to specific receptors on porcine intestinal brush border epithelial cells (enterocytes), starting the process of enteric infection. After this colonization, the bacteria produce one or more enterotoxins inducing diarrhoea, such as the heat stable toxin a (STa), the heat stable toxin b (STb), and the heat labile toxin (LT). A role in the pathogenesis of the disease was demonstrated for these toxins. The diagnosis of enteric colibacillosis is based on the isolation and quantification of the pathogenic E.coli coupled with the demonstration by PCR of the genes encoding for virulence factors (fimbriae and toxins). The diagnostic approach to enteric colibacillosis must consider the differential diagnosis and the potential different causes that can be involved in the outbreak. Among the different methods of control of colibacillosis, the use of antimicrobials is widely practiced and antibiotics are used in two main ways: as prophylactic or metaphylactic treatment to prevent disease and for therapeutic purposes to treat diseased pigs. An accurate diagnosis of enteric colibacillosis needs an appropriate sampling for the isolation and quantification of the ETEC responsible for the outbreak by using semi-quantitative bacteriology. Definitive diagnosis is based on the presence of characteristic lesions and results of bacteriology along with confirmation of appropriate virulence factors to identify the isolated E.coli . It is important to confirm the diagnosis and to perform antimicrobial sensitivity tests because antimicrobial sensitivity varies greatly among E. coli isolates. Growing concern on the increase of antimicrobial resistance force a more rational use of antibiotics and this can be achieved through a correct understanding of the issues related to antibiotic therapy and to the use of antibiotics by both practitioners and farmers.

Enterotoxigenic Escherichia coli (ETEC) is a significant contributor to diarrhea. To determine whether ETEC-catecholamine hormone interactions contribute to the development of diarrhea, we tested the effects of catecholamine hormones acting on ETEC in vitro. The results showed that in the presence of norepinephrine (NE) and epinephrine (Epi), the growth of 9 out of 10 ETEC isolates was promoted, the MICs of more than 60% of the isolates to 6 antibiotics significantly increased, and the biofilm formation ability of 10 ETEC isolates was also promoted. In addition, NE and Epi also significantly upregulated the expression of the virulence genes feaG, estA, estB, and elt. Transcriptome analysis revealed that the expression of 290 genes was affected by NE. These data demonstrated that catecholamine hormones may augment the diarrhea caused by ETEC.