Adhesion Of Enterotoxigenic Escherichia Coli Research Articles

The colonization factor CS6 of enterotoxigenic Escherichia coli contributes to host cell invasion

Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children and travelers in low-income regions. The virulence of ETEC is attributed to its heat-labile and heat-stable enterotoxins, as well as its colonization factors (CFs). CFs are essential for ETEC adherence to the intestinal epithelium. However, its invasive capability remains unelucidated. In this study, we demonstrated that the CS6-positive ETEC strain 4266 can invade mammalian epithelial cells. The invasive capability was reduced in the 4266 ΔCS6 mutant but reintroduction of CS6 into this mutant restored the invasiveness. Additionally, the laboratory E. coli strain Top 10, which lacks the invasive capability, was able to invade Caco-2 cells after gaining the CS6-expressing plasmid pCS6. Cytochalasin D inhibited cell invasion in both 4266 and Top10 pCS6 cells, and F-actin accumulation was observed near the bacteria on the cell membrane, indicating that CS6-positive bacteria were internalized via actin polymerization. Other cell signal transduction inhibitors, such as genistein, wortmannin, LY294002, PP1, and Ro 32–0432, inhibited the CS6-mediated invasion of Caco-2 cells. The internalized bacteria of both 4266 and Top10 pCS6 strains were able to survive for up to 48 h, and 4266 cells were able to replicate within Caco-2 cells. Immunofluorescence microscopy revealed that the internalized 4266 cells were present in bacteria-containing vacuoles, which underwent a maturation process indicated by the recruitment of the early endosomal marker EEA-1 and late endosomal marker LAMP-1 throughout the infection process. The autophagy marker LC3 was also observed near these vacuoles, indicating the initiation of LC-3-associated phagocytosis (LAP). However, intracellular bacteria continued to replicate, even after the initiation of LAP. Moreover, intracellular filamentation was observed in 4266 cells at 24 h after infection. Overall, this study shows that CS6, in addition to being a major CF, mediates cell invasion. This demonstrates that once internalized, CS6-positive ETEC is capable of surviving and replicating within host cells. This capability may be a key factor in the extended and recurrent nature of ETEC infections in humans, thus highlighting the critical role of CS6.

Synergistic Effect of B-Type Lotus Seedpod Oligomeric Procyanidin and Probiotics against Adhesion of Enterotoxigenic Escherichia coli In Vitro

Enterotoxigenic Escherichia coli (ETEC) adhesion to gut epithelial cells is a prerequisite for diarrhea. Here, we studied the synergistic effect and potential mechanism of B-type lotus seedpod oligomeric procyanidin (LSPC) combined with probiotics against adhesion of ETEC. The results indicated that LSPC exhibited an effective anti-ETEC adhesion effect. LSPC showed signally synergistic effects with probiotics, e.g., Streptococcus thermophilus (ST) and Lactobacillus rhamnosus (LGG) in response to ETEC adhesion, with the combination of LSPC and LGG being the most efficacious. This may be attributed to the restoration of transmembrane resistance of cells, the increased expression of anti-inflammatory factors (IL-10, 1.89-fold), and the reduction of cellular inflammatory factor levels (TNF-α and IL-8), regulated by LGG-LSPC, resulting in a better enhancement of cellular immune defense and barrier function. In addition, the results of gas chromatography showed that LSPC, as a prebiotic, could significantly increase the total amount of short-chain fatty acids (especially, butyric acid) produced by probiotics (e.g., LGG), thus better maintaining intestinal health against ETEC infection. In conclusion, the synergistic effect of LSPC and probiotics (represented by LGG) against ETEC adhesion in epithelial cells may be achieved through the enhancement of cellular immune defense, cellular barrier function, and maintenance of homeostasis in the gut.

Dextran and levan exopolysaccharides from tempeh-associated lactic acid bacteria with bioactivity against enterotoxigenic Escherichia coli (ETEC)

Soybean tempeh contains bioactive carbohydrate that can reduce the severity of diarrhea by inhibiting enterotoxigenic Escherichia coli (ETEC) adhesion to mammalian epithelial cells. Lactic acid bacteria (LAB) are known to be present abundantly in soybean tempeh. Some LAB species can produce exopolysaccharides (EPS) with anti-adhesion bioactivity against ETEC but there has been no report of anti-adhesion bioactive EPS from tempeh-associated LAB. We isolated EPS-producing LAB from tempeh-related sources, identified them, unambiguously elucidated their EPS structure and assessed the bioactivity of their EPS against ETEC. Pediococcus pentosaceus TL, Leuconostoc mesenteroides WA and L. mesenteroides WN produced both dextran (α-1,6 linked glucan; >1000 kDa) and levan (β-2,6 linked fructan; 650–760 kDa) in varying amounts and Leuconostoc citreum TR produced gel-forming α-1,6-mixed linkage dextran (829 kDa). All four isolates produced EPS that could adhere to ETEC cells and inhibit auto-aggregation of ETEC. EPS-PpTL, EPS-LmWA and EPS-LmWN were more bioactive towards pig-associated ETEC K88 while EPS-LcTR was more bioactive against human-associated ETEC H10407. Our finding is the first to report on the bioactivity of dextran against ETEC. Tempeh is a promising source of LAB isolates that can produce bioactive EPS against ETEC adhesion and aggregation.

A Polyvalent Adhesin-Toxoid Multiepitope-Fusion-Antigen-Induced Functional Antibodies against Five Enterotoxigenic Escherichia coli Adhesins (CS7, CS12, CS14, CS17, and CS21) but Not Enterotoxins (LT and STa).

The increasing prevalence and association with moderate-to-severe diarrhea make enterotoxigenic Escherichia coli (ETEC) adhesins CS7, CS12, CS14, CS17, and CS21 potential targets of ETEC vaccines. Currently, there are no vaccines licensed to protect against ETEC, a top cause of children's diarrhea and travelers' diarrhea. Recently, a polyvalent adhesin protein (adhesin MEFA-II) was demonstrated to induce antibodies that inhibited adherence from these five ETEC adhesins and reduced the enterotoxicity of ETEC heat-stable toxin (STa), which plays a key role in causing ETEC-associated diarrhea. To improve adhesin MEFA-II for functional antibodies against STa toxin and the other ETEC toxin, heat-labile toxin (LT), we modified adhesin MEFA-II by adding another STa toxoid and an LT epitope; we examined the new antigen immunogenicity (to five adhesins and two toxins) and more importantly antibody functions against ETEC adherence and STa and LT enterotoxicity. Data show that mice intramuscularly immunized with the new antigen (adhesin MEFA-IIb) developed robust IgG responses to the targeted adhesins (CS7, CS12, CS14, CS17, and CS21) and toxins (STa and LT). Mouse antibodies inhibited the adherence of ETEC strains expressing any of these five adhesins but failed to neutralize STa or LT enterotoxicity. In further studies, rabbits intramuscularly immunized with adhesin MEFA-IIb developed robust antigen-specific antibodies; when challenged with an ETEC isolate expressing CS21 adhesin (JF2101, CS21, and STa), the immunized rabbits showed a significant reduction in intestinal colonization by ETEC bacteria. These data indicate that adhesin MEFA-IIb is broadly immunogenic and induces functional antibodies against the targeted ETEC adhesins but not the toxins.

A blend of selected botanicals maintains intestinal epithelial integrity and reduces susceptibility to Escherichia coli F4 infection by modulating acute and chronic inflammation in vitro.

In the pig production cycle, the most delicate phase is weaning, a sudden and early change that requires a quick adaptation, at the cost of developing inflammation and oxidation, especially at the intestinal level. In this period, pathogens like enterotoxigenic Escherichia coli (ETEC) contribute to the establishment of diarrhea, with long-lasting detrimental effects. Botanicals and their single bioactive components represent sustainable well-recognized tools in animal nutrition thanks to their wide-ranging beneficial functions. The aim of this study was to investigate the in vitro mechanism of action of a blend of botanicals (BOT), composed of thymol, grapeseed extract, and capsicum oleoresin, in supporting intestinal cell health during inflammatory challenges and ETEC infections. To reach this, we performed inflammatory and ETEC challenges on Caco-2 cells treated with BOT, measuring epithelial integrity, cellular oxidative stress, bacterial translocation and adhesion, gene expression levels, and examining tight junction distribution. BOT protected enterocytes against acute inflammation: while the challenge reduced epithelial tightness by 40%, BOT significantly limited its drop to 30%, also allowing faster recovery rates. In the case of chronic inflammation, BOT systematically improved by an average of 25% the integrity of challenged cells (p < 0.05). Moreover, when cells were infected with ETEC, BOT maintained epithelial integrity at the same level as an effective antibiotic and significantly reduced bacterial translocation by 1 log average. The mode of action of BOT was strictly related to the modulation of the inflammatory response, protecting tight junctions' expression and structure. In addition, BOT influenced ETEC adhesion to intestinal cells (-4%, p < 0.05), also thanks to the reduction of enterocytes' susceptibility to pathogens. Finally, BOT effectively scavenged reactive oxygen species generated by inflammatory and H2O2 challenges, thus alleviating oxidative stress by 40% compared to challenge (p < 0.05). These results support the employment of BOT in piglets at weaning to help manage bacterial infections and relieve transient or prolonged stressful states thanks to the modulation of host-pathogen interaction and the fine-tuning activity on the inflammatory tone.

Protective effect of Limosilactobacillus reuteri-fermented yogurt on mouse intestinal barrier injury induced by enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a pathogen that causes traveler's diarrhea, for which an effective vaccine is lacking. Previous studies showed that Limosilactobacillus reuteri could inhibit E. coli, effectively increase the expression of its tight junction protein, and reduce the adhesion of ETEC to the intestinal epithelial Caco-2 cell line. In this study, three kinds of yogurt with different starter cultures were first prepared: Lm. reuteri yogurt (fermented by Lm. reuteri alone), traditional yogurt (fermented by Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus at a ratio of 1:1) and mixed yogurt (fermented by Lm. reuteri, S. thermophilus and L. delbrueckii subsp. bulgaricus at a ratio of 1:1:1). The physiological properties, oxidative stress, intestinal barrier function, tight junction protein, pathological conditions and intestinal microbiota composition were investigated. The data showed that Lm. reuteri-fermented yogurt pregavage could effectively alleviate the intestinal barrier impairment caused by ETEC in mice. It alleviated intestinal villus shortening and inflammatory cell infiltration, decreased plasma diamine oxidase concentration and increased claudin-1 and occludin expression in the jejunum of ETEC-infected mice. In addition, Lm. reuteri-fermented yogurt significantly reduced the ETEC load in fecal samples, reversed the increase in Pseudomonadota abundance and decreased Bacteroidota abundance caused by ETEC infection. Furthermore, the composition of the intestinal microbiota could maintain a stable state similar to that in healthy mice. These findings indicate that Lm. reuteri-fermented yogurt could alleviate intestinal barrier damage, inhibit ETEC growth and maintain the stability of the intestinal microbiota during ETEC infection. © 2023 Society of Chemical Industry.

Anti-adhesion bioactivity of tempeh against enterotoxigenic Escherichia coli can be improved through the addition of Lactobacillus plantarum culture during fermentation

Diarrhoea is prevalently widespread in developing countries and can lead to death. This disease could be caused by a prerequisite attachment of Enterotoxigenic Escherichia coli (ETEC) on human intestinal epithelial cells and followed by toxin production. Several antibiotics have been developed to reduce diarrhoeal severity. However, the use of antibiotics could also disrupt the balance of gut microbiota. This research found that tempeh fermented with the addition of Lactobacillus plantarum culture could decrease the adhesion of ETEC to the intestinal epithelial cells. This study measured the ability of L. plantarum to produce exopolysaccharide (EPS) and examined the effect of L. plantarum addition to tempeh, particularly its effect on anti-adhesion activity against ETEC. By using yeast agglutination assay to observe anti-adhesion activity, the data showed that soybean tempeh fermented with the addition of L. plantarum at OD600 of 0.5 had an increase of anti-adhesion bioactivity against ETEC nearly three times compared to tempeh without the addition of L. plantarum culture. Moreover, the exclusion test showed that EPS from L. plantarum could act as a preventive agent against ETEC adhesion to eukaryotic cells. Our finding indicated that the addition of lactic acid bacteria culture can be considered to augment the anti-adhesion bioactivity in soybean tempeh against ETEC.

Role of mucus-bacteria interactions in Enterotoxigenic Escherichia coli (ETEC) H10407 virulence and interplay with human microbiome

The intestinal mucus layer has a dual role in human health constituting a well-known microbial niche that supports gut microbiota maintenance but also acting as a physical barrier against enteric pathogens. Enterotoxigenic Escherichia coli (ETEC), the major agent responsible for traveler’s diarrhea, is able to bind and degrade intestinal mucins, representing an important but understudied virulent trait of the pathogen. Using a set of complementary in vitro approaches simulating the human digestive environment, this study aimed to describe how the mucus microenvironment could shape different aspects of the human ETEC strain H10407 pathophysiology, namely its survival, adhesion, virulence gene expression, interleukin-8 induction and interactions with human fecal microbiota. Using the TNO gastrointestinal model (TIM-1) simulating the physicochemical conditions of the human upper gastrointestinal (GI) tract, we reported that mucus secretion and physical surface sustained ETEC survival, probably by helping it to face GI stresses. When integrating the host part in Caco2/HT29-MTX co-culture model, we demonstrated that mucus secreting-cells favored ETEC adhesion and virulence gene expression, but did not impede ETEC Interleukin-8 (IL-8) induction. Furthermore, we proved that mucosal surface did not favor ETEC colonization in a complex gut microbial background simulated in batch fecal experiments. However, the mucus-specific microbiota was widely modified upon the ETEC challenge suggesting its role in the pathogen infectious cycle. Using multi-targeted in vitro approaches, this study supports the major role played by mucus in ETEC pathophysiology, opening avenues in the design of new treatment strategies.

Feasibility of avian antibodies as prophylaxis against enterotoxigenic escherichia coli colonization.

This research aims to evaluate the feasibility of using avian immunoglobulins (IgY) raised against adhesion factors of enterotoxigenic Escherichia coli (ETEC) as prophylaxis of diarrheal illness caused by these pathogens. ETEC requires adhesion to human intestinal epithelial cells as a primary step in establishing enteric infection. Therefore, inhibition of adhesion may prevent such infections and reduce clinical burdens of diarrheal illness. IgY samples were prepared from eggs of hens immunized with an adhesin-tip multiepitope fusion antigen (MEFA), developed against nine adhesin tip epitopes derived from clinically relevant ETEC strains. The resulting IgY was evaluated for its ability to inhibit adhesion of ETEC to cell-surface targets. Potential impacts of anti-MEFA IgY on growth of both pathogenic and commensal E. coli isolates were also evaluated. Enzyme linked immunosorbent assay (ELISA) titers were achieved for IgY targeting each of the nine individual epitopes included in the adhesin-tip MEFA. Furthermore, anti-MEFA titers exceeding 1:219 were sustained for at least 23 weeks. All ETEC strains used in design of the adhesin-tip MEFA, and five of five clinical ETEC strains were significantly (P < 0.05) inhibited from adhesion to mammalian cells in culture. These findings demonstrate that IgY targeting ETEC adhesin-tip MEFA have the potential to disrupt in vitro adherence of ETEC. A formulation containing adhesin-tip MEFA IgY can be considered a potential candidate for in vivo evaluation as prophylaxis of diarrheal diseases. Animal studies of this formulation are planned.

Dispersal of pathogen-associated multispecies biofilm by novel probiotic Bacillus subtilis in a contact-dependent manner.

Biofilms are involved in pathogenesis of various bacterial infections. Treatment of biofilm-related bacterial infection remains a major challenge due to the reduced efficacy of antibiotics and associated antibiotic resistance. Given the high prevalence of Enterotoxigenic Escherichia coli (ETEC), Salmonella Typhimurium (S. Typhimurium) and methicillin-resistant Staphylococcus aureus (MRSA)-related infections and associated drug resistance, it is imperative to develop alternative strategies for treatment and prevention. The current study investigated antibiofilm activity of a recently isolated Bacillus subtilis (B. subtilis-9) against these pathogens. Crystal violet staining showed that treatment with B. subtilis-9 significantly reduced biofilm biomass of ETEC (60%-80%), S. Typhimurium (68%-73%) and MRSA (66%-82%). In addition, B. subtilis-9 significantly reduced pre-formed biofilm biomass of ETEC (59%), S. Typhimurium (62%), MRSA (65%) and multispecies (58%). Fluorescence microscopy revealed that B. subtilis-9 treatment significantly reduced the thickness of biofilm and viability of the embedded bacteria. Additionally, B. subtilis-9 significantly reduced planktonic cell growth of ETEC (92%), S. Typhimurium (94%) and MRSA (93%). Interestingly, transwell assay showed that B. subtilis-9 exhibited antibiofilm properties in a cell-to-cell contact-dependent manner and significantly reduced mRNA expression of biofilm-related genes, bssS, luxS and ihfB in ETEC. Novel B. subtilis-9 exhibits a strong inhibitory activity against ETEC, S. Typhimurium and MRSA biofilm formation and adhesion to abiotic surfaces. With further investigations, our study could bring forward a novel Bacillus-based probiotic intervention strategy to combat pathogenic biofilms, in clinical and agricultural settings. Probiotic bacteria propose a potential alternative in combating biofilm-related infections, however, data on the efficacy and strain selection are limited. Data from this study are critical in further developing Bacillus-based novel probiotic applications that may reduce the use of antibiotics in biofilm-related infections in humans and animals.

PSXII-19 Pediococcus Acidilactici and Lactobacillus Helveticas Inhibit Enterotoxigenic Escherichia Coli Adhesion to Porcine Ipec-J2 Intestinal Cells but do not Protect Paracellular Epithelial Permeability

Abstract Weaning is a critical period during swine production, as pigs endure multiple stressors. Stressors provide an opportunistic gut environment for development of enterotoxigenic Escherichia coli (ETEC) diarrheal disease. Probiotics are known to improve gut health, but the specific mechanisms to protect intestinal barrier function are unknown. The objective is to investigate protective effects of Pediococcus acidilactici (PA) and Lactobacillus helveticus (LH) against ETEC challenge using porcine IPEC-J2 intestinal epithelial cells. IPEC-J2 cells were seeded at a concentration of 1 ×105 cells/insert, grown for 4 d, then treated with PA or LH for 3 h at concentrations of 108, 109, 1010, and 1011colony forming units (CFU)/mL. Bacterial adherence assays were performed to determine probiotic CFU adhered to the cells. To assess probiotic inhibition of ETEC adherence, cells were treated with probiotics at previously defined concentrations and ETEC 3030-2: K88ac (multiplicity of infection 2:1) for 2 h. Following incubation ETEC adherence assays were conducted. Cell cytotoxicity was assessed by lactate dehydrogenase (LDH), and paracellular permeability was measured using 4-kDa fluorescein isothiocyanate-dextran (FITC-dextran). Data were analyzed according to a randomized block design using GLM procedures of SAS. Both probiotics exhibited a concentration-dependent ability to attach to epithelial cells (PA 5.05, 6.03, 6.96, 7.67 ± 0.08 log10CFU/cm2 P&amp;lt; 0.001; LH 5.54, 6.24, 7.38, 8.16 ± 0.19 log10CFU/cm2 P&amp;lt; 0.001). In the presence of ETEC, probiotic treatment linearly inhibited ETEC adherence (PA 23.49, 31.48, 4.59, 0.17 ± 6.85 % of control P&amp;lt; 0.001; LH 39.50, 28.0, 0.49, 0.020 ± 5.21 % of control P&amp;lt; 0.001). Increasing concentrations of probiotics modulated IPEC-J2 cellular cytotoxicity in the presence of ETEC (P&amp;lt; 0.05). However, the probiotics were not able to protect FITC-dextran flux across the epithelial cell barrier following ETEC challenge. Our findings indicate these probiotics have protective effects in preventing ETEC adhesion and modulating cell death, but do not reduce paracellular flux.

Glycomacropeptide from camel milk inhibits the adhesion of enterotoxigenic Escherichia coli K88 to porcine cells

Enterotoxigenic Escherichia coli (ETEC) are a major cause of childhood diarrhoea and of diarrhoea in piglets and calves. This study compared the anti-adhesive activities against ETEC of glycomacropeptide (GMP) from Bactrian camels and dromedaries with that of bovine GMP. GMP was purified by ion exchange chromatography and ultra-filtration. The monosaccharide content of GMP from Bactrian camels and dromedaries was about twice as high when compared with bovine GMP. Glycans from camels included fucose and N-acetylglucosamine, which were absent in bovine GMP. GMP from both camel species prevented ETEC adhesion to porcine blood cells at a concentration of 0.24 g L−1 to 0.28 g L−1 respectively, a concentration that is about 20-fold lower when compared with bovine GMP. This increased activity likely relates to the increased glycosylation and the density of glycan spacing, and/or to differences in the glycan composition.

Lentils and Yeast Fibers: A New Strategy to Mitigate Enterotoxigenic Escherichia coli (ETEC) Strain H10407 Virulence?

Dietary fibers exhibit well-known beneficial effects on human health, but their anti-infectious properties against enteric pathogens have been poorly investigated. Enterotoxigenic Escherichia coli (ETEC) is a major food-borne pathogen that causes acute traveler’s diarrhea. Its virulence traits mainly rely on adhesion to an epithelial surface, mucus degradation, and the secretion of two enterotoxins associated with intestinal inflammation. With the increasing burden of antibiotic resistance worldwide, there is an imperious need to develop novel alternative strategies to control ETEC infections. This study aimed to investigate, using complementary in vitro approaches, the inhibitory potential of two dietary-fiber-containing products (a lentil extract and yeast cell walls) against the human ETEC reference strain H10407. We showed that the lentil extract decreased toxin production in a dose-dependent manner, reduced pro-inflammatory interleukin-8 production, and modulated mucus-related gene induction in ETEC-infected mucus-secreting intestinal cells. We also report that the yeast product reduced ETEC adhesion to mucin and Caco-2/HT29-MTX cells. Both fiber-containing products strengthened intestinal barrier function and modulated toxin-related gene expression. In a complex human gut microbial background, both products did not elicit a significant effect on ETEC colonization. These pioneering data demonstrate the promising role of dietary fibers in controlling different stages of the ETEC infection process.

Orally active bivalent VHH construct prevents proliferation of F4+ enterotoxigenic Escherichia coli in weaned piglets

SummaryA major challenge in industrial pig production is the prevalence of post-weaning diarrhea (PWD) in piglets, often caused by enterotoxigenic Escherichia coli (ETEC). The increased use of antibiotics and zinc oxide to treat PWD has raised global concerns regarding antimicrobial resistance development and environmental pollution. Still, alternative treatments targeting ETEC and counteracting PWD are largely lacking. Here, we report the design of a pH, temperature, and protease-stable bivalent VHH-based protein BL1.2 that cross-links a F4+ ETEC model strain by selectively binding to its fimbriae. This protein inhibits F4+ ETEC adhesion to porcine epithelial cells ex vivo and decreases F4+ ETEC proliferation when administrated as a feed additive to weaned F4+ ETEC challenged piglets. These findings highlight the potential of a highly specific bivalent VHH-based feed additive in effectively delimiting pathogenic F4+ ETEC bacteria proliferation in piglets and may represent a sustainable solution for managing PWD while circumventing antimicrobial resistance development.

Positive correlation between the number of bacteria in soybean tempeh with the bioactivity of its extract against enterotoxigenic Escherichia coli (ETEC) adhesion to eukaryotic cells

Bioactive oligosaccharides from soybean tempeh can inhibit the adhesion of enterotoxigenic Escherichia coli (ETEC) to intestinal cells, thus reducing the severity of ETEC-mediated diarrhea. Bacteria are also present in tempeh but there has yet been any report regarding their effect on the anti-adhesion bioactivity of tempeh. In this research, the bacterial number in tempeh was quantified and the anti-adhesion bioactivity of tempeh extract was determined using yeast agglutination assay. Statistical analysis showed a moderately (R = 0.69) significant positive correlation (P &lt; 0.01) between the number of bacteria in tempeh and the anti-adhesion bioactivity of its extract. In conclusion, tempeh that contains more bacteria is more effective in inhibiting ETEC adhesion to eukaryotic cells. This could be due to a symbiosis between Rhizopus and bacteria in breaking down soy matrix polysaccharides to release bioactive oligosaccharides.

Saccharomyces Cerevisiae Var Boulardii CNCM I-1079 Reduces Expression of Genes Involved in Inflammatory Response in Porcine Cells Challenged by Enterotoxigenic E. Coli and Influences Bacterial Communities in an In Vitro Model of the Weaning Piglet Colon.

Enterotoxigenic Escherichia coli (ETEC) is the main infectious agent responsible for piglet post-weaning diarrhea with high mortality rates. Antimicrobials represent the current principal strategy for treating ETEC infections in pig farms, but the occurrence of multi-resistant bacterial strains has considerably increased in the last decades. Thus, finding non-antibiotic alternatives becomes a real emergency. In this context, we investigated the effect of a live yeast strain, Saccharomyces cerevisiae var boulardii CNCM I-1079 (SB) in an in vitro model of the weaning piglet colon implemented with a mucus phase (MPigut-IVM) inoculated with ETEC and coupled with an intestinal porcine cell line IPI-2I. We showed that SB was able to modulate the in vitro microbiota through an increase in Bacteroidiaceae and a decrease in Prevotellaceae families. Effluents collected from the SB treated bioreactors were able to mitigate the expression level of genes encoding non-gel forming mucins, tight junction proteins, innate immune pathway, and pro-inflammatory response in IPI-2I cells. Furthermore, SB exerted a significant protective effect against ETEC adhesion on porcine IPEC-J2 intestinal cells in a dose-dependent manner and showed a positive effect on ETEC-challenged IPEC-J2 by lowering expression of genes involved in pro-inflammatory immune responses. Our results showed that the strain SB CNCM I-1079 could prevent microbiota dysbiosis associated with weaning and protect porcine enterocytes from ETEC infections by reducing bacterial adhesion and modulating the inflammatory response.

Chicken Egg Yolk Antibody (IgY) Protects Mice Against Enterotoxigenic Escherichia coli Infection Through Improving Intestinal Health and Immune Response.

Chicken egg yolk antibody (IgY), considered as a potential substitute for antibiotics, has been used for preventing pathogens infection in food, human and animals. This study investigated effects of IgY on growth, adhesion inhibitory and morphology of enterotoxigenic Escherichia coli (ETEC) K88 in vitro, and evaluated the protective effects of IgY on intestinal health and immune response of mice infected with ETEC in vivo. Sixty pathogen-free C57BL/6J (4-6 weeks of age) mice were divided into six treatments: control (neither IgY nor ETEC infection), ETEC infection, ETEC-infected mice treated with 250 μL of high-dose (32 mg/mL), medium-dose (16 mg/mL) or low-dose (8 mg/mL) anti-ETEC IgY, or ETEC-infected mice treated with 250 μL of non-specific IgY (16 mg/mL). Anti-ETEC IgY inhibited ETEC growth, reduced adherence of ETEC to intestinal epithelial cells J2 and damaged the morphology and integrity of ETEC cell. Oral administration of anti-ETEC IgY effectively ameliorated ETEC-induced clinical signs, reduced ETEC colonization and intestinal permeability, alleviated inflammatory response through reducing the production and expression of proinflammatory cytokines, improved intestinal morphology, and inhibited excessive activation of the mucosal immune response of challenged mice. The overall protective effects of high-dose and medium-dose anti-ETEC IgY against ETEC infection were more effective. These results suggest that anti-ETEC IgY may function as a promising novel prophylactic agent against enteric pathogens infection.

Antibacterial and Antibiofilm Activities of Novel Antimicrobial Peptides against Multidrug-Resistant Enterotoxigenic Escherichia Coli

Post-weaning diarrhea due to enterotoxigenic Escherichia coli (ETEC) is a common disease of piglets and causes great economic loss for the swine industry. Over the past few decades, decreasing effectiveness of conventional antibiotics has caused serious problems because of the growing emergence of multidrug-resistant (MDR) pathogens. Various studies have indicated that antimicrobial peptides (AMPs) have potential to serve as an alternative to antibiotics owing to rapid killing action and highly selective toxicity. Our previous studies have shown that AMP GW-Q4 and its derivatives possess effective antibacterial activities against the Gram-negative bacteria. Hence, in the current study, we evaluated the antibacterial efficacy of GW-Q4 and its derivatives against MDR ETEC and their minimal inhibition concentration (MIC) values were determined to be around 2~32 μg/mL. Among them, AMP Q4-15a-1 with the second lowest MIC (4 μg/mL) and the highest minimal hemolysis concentration (MHC, 256 μg/mL), thus showing the greatest selectivity (MHC/MIC = 64) was selected for further investigations. Moreover, Q4-15a-1 showed dose-dependent bactericidal activity against MDR ETEC in time–kill curve assays. According to the cellular localization and membrane integrity analyses using confocal microscopy, Q4-15a-1 can rapidly interact with the bacterial surface, disrupt the membrane and enter cytosol in less than 30 min. Minimum biofilm eradication concentration (MBEC) of Q4-15a-1 is 4× MIC (16 μg/mL), indicating that Q4-15a-1 is effective against MDR ETEC biofilm. Besides, we established an MDR ETEC infection model with intestinal porcine epithelial cell-1 (IPEC-1). In this infection model, 32 μg/mL Q4-15a-1 can completely inhibit ETEC adhesion onto IPEC-1. Overall, these results suggested that Q4-15a-1 may be a promising antibacterial candidate for treatment of weaned piglets infected by MDR ETEC.

CEACAMs serve as toxin-stimulated receptors for enterotoxigenic Escherichia coli

The enterotoxigenic Escherichia coli (ETEC) are among the most common causes of diarrheal illness and death due to diarrhea among young children in low-/middle-income countries (LMICs). ETEC have also been associated with important sequelae including malnutrition and stunting, placing children at further risk of death from diarrhea and other infections. Our understanding of the molecular pathogenesis of acute diarrheal disease as well as the sequelae linked to ETEC are still evolving. It has long been known that ETEC heat-labile toxin (LT) activates production of cAMP in the cell, signaling the modulation of cellular ion channels that results in a net efflux of salt and water into the intestinal lumen, culminating in watery diarrhea. However, as LT also promotes ETEC adhesion to intestinal epithelial cells, we postulated that increases in cAMP, a critical cellular "second messenger," may be linked to changes in cellular architecture that favor pathogen-host interactions. Indeed, here we show that ETEC use LT to up-regulate carcinoembryonic antigenrelated cell adhesion molecules (CEACAMs) on the surface of small intestinal epithelia, where they serve as critical bacterial receptors. Moreover, we show that bacteria are specifically recruited to areas of CEACAM expression, in particular CEACAM6, and that deletion of this CEACAM abrogates both bacterial adhesion and toxin delivery. Collectively, these results provide a paradigm for the molecular pathogenesis of ETEC in which the bacteria use toxin to drive up-regulation of cellular targets that enhances subsequent pathogen-host interactions.

Antimicrobial and antiadhesive effects of Lactobacillus isolates of healthy human gut origin on Enterotoxigenic Escherichia coli (ETEC) and Enteroaggregative Escherichia coli (EAEC)

PurposeDiarrhea is one of the five leading causes of mortality in children under the age of five, especially in developing countries. Nowadays, by increasing the resistance of pathogens to antibiotics, employment of probiotics as novel therapeutic method, could be considered as a necessity.The aim of this study was to examine the features and antagonistic action of Lactobacillus strains, against the growth and adhesion of Enterotoxigenic Escherichia coli (ETEC) and Enteroaggregative Escherichia coli (EAEC) strains creating diarrhea in children. Then, we introduced new strains of Lactobacillus as probiotic candidates, to prevent diarrheal infections in children. MethodsStool samples were collected from healthy individuals, and Lactobacillus strains were isolated. The antimicrobial effect of the isolates against ETEC and EAEC strains investigated by agar well diffusion method and their resistance to acidic and bile conditions. The potency of selected isolates in adhesion to HT-29 epithelial cells and their ability to inhibit the adhesion of ETEC and EAEC strains to this cell were measured. At the end, identification of the optimally efficient Lactobacillus isolates was performed by 16S rDNA sequencing and making Phylogenetic tree using MEGA (version 4.0) software. ResultsIn total, 157 isolates suspected to Lactobacillus were isolated from 115 stool samples. In antimicrobial activity test, ETEC and EAEC growth was inhibited by 132 and 84 isolates respectively, while 17 isolates showed resistance to Bile. Of 17 Bile resistant Lactobacillus isolates, 15 isolates were resistant to pH: 3.2. Further, among 15 isolates, only two isolates, were resistant to pH: 2.5. In the adhesion assay, five isolates had more adhesion tendency to HT-29 epithelial cells than L. rhamnosus GG, which was considered as a positive control. Investigation of isolates that inhibit adhesion of ETEC and EAEC strains to HT-29 cells showed that four isolates were able to inhibit ETEC adhesion. However, only one out of four isolates was relatively able to have an impact on EAEC adhesion. ConclusionIn conclusion, three species of Lactobacillus including L. paracasei (two strain), L. fermentum (two strain) and L. plantarum showed good probiotic properties compared to other isolates that were identified by sequencing. In this study, strain L. fermentum 61.1 had the highest adhesion ability to HT-29 cells and strain L. paracasei 47.2 had the highest potency to inhibit ETEC adhesion to HT-29 cells. These isolates have good probiotic properties and are likely to be effective in preventing or treating diarrheal infections, especially in children.