Alginate oligosaccharide protects against enterotoxigenic Escherichia coli-induced porcine intestinal barrier injury.

Alginate oligosaccharide (AOS) possesses various pharmaceutical benefits, making it an attractive candidate for biomedical applications. In the present study, we prepared AOS by depolymerising alginate; its degree of polymerisation mainly ranged from 2 to 8. We confirmed the enteroprotective potential of AOS against enterotoxigenic Escherichia coli (ETEC)-induced intestinal barrier injury in weaned pigs. Next, we illustrated the mechanisms underlying this effect of AOS using the porcine small intestinal epithelial cell line IPEC-J2. AOS potently reduced the binding of the bacteria-deprived endotoxin lipopolysaccharide (LPS) to the IPEC-J2 cell surface. Moreover, it suppressed the LPS-induced production of pro-inflammatory cytokines and the nuclear translocation of nuclear factor-κB (NF-κB) p65 in IPEC-J2 cells. These results indicate that AOS protects the intestinal epithelium from ETEC-induced inflammatory injury by preventing the activation of NF-κB, implying that AOS could be used as an anti-inflammatory agent for treating inflammation-related intestinal diseases in mammals.

Long-read-sequenced reference genomes of the seven major lineages of enterotoxigenic Escherichia coli (ETEC) circulating in modern time.

Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen responsible for the majority of diarrheal cases worldwide. ETEC infections are estimated to cause 80,000 deaths annually, with the highest rates of burden, ca 75 million cases per year, amongst children under 5 years of age in resource-poor countries. It is also the leading cause of diarrhoea in travellers. Previous large-scale sequencing studies have found seven major ETEC lineages currently in circulation worldwide. We used PacBio long-read sequencing combined with Illumina sequencing to create high-quality complete reference genomes for each of the major lineages with manually curated chromosomes and plasmids. We confirm that the major ETEC lineages all harbour conserved plasmids that have been associated with their respective background genomes for decades, suggesting that the plasmids and chromosomes of ETEC are both crucial for ETEC virulence and success as pathogens. The in-depth analysis of gene content, synteny and correct annotations of plasmids will elucidate other plasmids with and without virulence factors in related bacterial species. These reference genomes allow for fast and accurate comparison between different ETEC strains, and these data will form the foundation of ETEC genomics research for years to come.

Molecular mechanism of Escherichia coli H10407 induced diarrhoea and its control through immunomodulatory action of bioactives from Simarouba amara (Aubl.).

Enterotoxigenic Escherichia coli (ETEC) infection is a major cause of death in children under the age of five in developing countries. ETEC (O78:H11:CFA/I:LT+:ST+) mechanism has been studied in detail with either heat labile (LT) or heat stable (ST) toxins using in vitro and in vivo models. However, there is no adequate information on ETEC pathogenesis producing both the toxins (LT, ST) in BALB/c mice model. In this study, female mice have been employed to understand ETEC H10407 infection induced changes in physiology, biochemical and immunological patterns up to seven days post-infection and the antidiarrhoeal effect of Simarouba amara (Aubl.) bark aqueous extract (SAAE) has also been looked into. The results indicate that BALB/c is sensitive to ETEC infection resulting in altered jejunum and ileum histomorphology. Withal, ETEC influenced cAMP, PGE2, and NO production resulting in fluid accumulation with varied Na+, K+, Cl-, and Ca2+ levels. Meanwhile, ETEC subverted expression of IL-1ß, intestine alkaline phosphatase (IAP), and myeloperoxidase (MPO) in jejunum and ileum. Our data also indicate the severity of pathogenesis reduction which might be due to attainment of equilibrium after reaching optimum rate of infection. Nevertheless, degree of pathogenesis was highly significant (p < 0.01) in all the studied parameters. Besides that, SAAE was successful in reducing the infectious diarrhoea by inhibiting ETEC H10407 in intestine (jejunum and ileum), and shedding in feces. SAAE decreased cAMP, PGE2, and fluid accumulation effectively and boosted the functional activity of immune system in jejunum and ileum IAP, MPO, IL-1ß, and nitric oxide.

In vitro mechanism of antibacterial action of a citrus essential oil on an enterotoxigenic Escherichia coli and Lactobacillus rhamnosus.

AIM: This study investigated the in vitro mechanism of action of a commercial citrus EO, Brazilian orange terpenes (BOT), on an enterotoxigenic Escherichia coli (ETEC) isolated from pig gut and on Lactobacillus rhamnosus. METHODS AND RESULTS: Firstly, bacteria were exposed sequentially to BOT every 3 h (three times) at sub-minimal inhibitory concentrations and results showed that sequential exposure to BOT provoked a higher reduction of bacteria viability than a single exposure and the reduction of ETEC viability was higher compared to that of L. rhamnosus. Then, evaluation of the BOT effects on the cell membrane permeability and integrity, indicated that BOT increased the membrane permeability and caused disruptive effects on the integrity of bacterial cells as reflected by an increase of the relative electric conductivity and the release of essential cell constituents. Interestingly, BOT effects were more pronounced on the ETEC than on L. rhamnosus. This was ratified by scanning electron microscopy, which showed more noticeable morphological damages and disturbances on ETEC cells than on the L. rhamnosus cells. Limonene was detected as the major compound in BOT by polar/nonpolar GC-MS (78·65%/79·38%). CONCLUSIONS: Results revealed that the probable mechanism of the selective antibacterial action of the citrus EO, BOT, can be described as altering more remarkable the permeability and integrity of the cytoplasmic membrane as well as the external structure of ETEC cells than L. rhamnosus cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information about the mechanism of antibacterial action displayed by a citrus EO, a by-product of the citrus processing industry, as a natural alternative to antibiotics used in pig production sector to combat pathogens such as ETECs.

Phenolics with Bactericidal Activity Alter Motility and Biofilm Formation in Enterotoxigenic, Enteropathogenic, and Enterohemorrhagic Escherichia coli.

Most Escherichia coli strains are innocuous to human beings; however, some strains can cause diarrhea and are grouped into pathotypes. Since current trends promote the use of natural-origin compounds to control bacteria, in this study, the effects of the phenolic compounds (PCs) tannic acid (TA), gallic acid (GA), methyl gallate (MG), and epigallocatechin gallate (EG) on the growth, swarming motility, biofilm formation, and expression of selected virulence genes of three E. coli pathotypes (enteropathogenic Escherichia coli [EPEC], enterohemorrhagic Escherichia coli [EHEC], and enterotoxigenic Escherichia coli [ETEC]) were evaluated. Minimum bactericidal concentrations (MBCs) were determined by using microtiter plates, and the effects of sublethal PC concentrations on swarming motility were evaluated on Luria-Bertani agar. Biofilm formation was assessed in microtiter plates via crystal violet staining, and the expression levels of genes involved in biofilm formation (flhC, fliA, fliC, and csgA) and swarming motility (csgD and cyaA) were evaluated via quantitative PCR. All PC were bactericidal with minimal bactericidal concentrations ranging from 0.07 to 2.1 mg/mL. At concentrations lower than the MBC, PCs decreased swarming motility (14.8-100%). GA reduced biofilm formation in all of the tested strains; however, TA, MG, and EG induced biofilm formation in some strains at specific concentrations. TA induced the overexpression of csgA, csgD, and cyaA, whereas the other PCs did not have any effects or reduced their expression levels. The PCs tested in this study showed potential to control E. coli strains belonging to the EHEC, ETEC, and EPEC pathotypes by affecting their growth, swarming motility, and virulence gene expression; however, proper concentrations must be used to avoid the induction of undesirable virulence factor genes.

Adaptação de Escherichia coli enterotoxigênica ao eugenol / Adaptation of enterotoxigenic Escherichia coli to eugenol

O objetivo do trabalho foi verificar a capacidade de adaptação de ETEC- ATCC 35401 ao antimicrobiano eugenol. Para isso, a Concentração Mínima Bactericida (CMB) de eugenol foi determinada e em seguida,as células de ETEC foram expostas a concentrações subletais de eugenol (CMB/4, CMB/8 e CMB/16). Posteriormente testadas frente a diferentes concentrações do composto (CMB/2; CMB; 1,2CMB; 1,4CMB; 1,6CMB; 1,8CMB e 2CMB), estas foram incubadas e plaqueadas em TSA (Ágar Triptona de Soja) empregando a técnica de microgotas. As células de ETEC foram classificadas como capazes de se adaptarem quando essas cresceram em placas após cultivo em presença do componente em concentração igual ou maior que a CMB do eugenol (1,0% (v/v)). As células de ETEC apresentaram a capacidade de adaptação por crescerem na CMB, após exposta a CMB/16. Os resultados demonstram a necessidade de obtenção da concentração de uso adequado de eugenol a fim de evitar a adaptação.

Protecting intestinal epithelial cells against deoxynivalenol and E. coli damage by recombinant porcine IL-22.

Pigs suffer enteritis induced by pathogenic bacteria infection and toxins in the moldy feed, which cause intestinal epithelial damage and diarrhea through the whole breeding cycle. Interleukin-22 (IL-22) plays a critical role in maintaining intestinal mucosal barrier function through repairing intestinal epithelial damage. However, little was known about the effects of IL-22 against apoptosis caused by toxins and infection of intestinal pathogens in the intestinal epithelium, especially in pigs. In this study, we had successfully used prokaryotic expression system to produce recombinant porcine interleukin-22. Meanwhile, purified rIL-22 could activate STAT3 signal pathway and have been demonstrated to be safe to IPEC-J2 cells by increasing E-cadherin expression, without proinflammatory cytokines changes. Furthermore, rIL-22 reversed apoptosis induced by deoxynivalenol (DON) and played a vital part in repairing the intestinal injury. We also found that rIL-22 stimulated epithelial cells to secrete pBD-1 against enterotoxigenic E. coli (ETEC) K88 infection, as well as alleviating apoptosis ratio. This study provided a theoretical basis for curing intestinal inflammation caused by ETEC infection and epithelial apoptosis induced by DON with rIL-22 in pigs.

Antimicrobial peptide KR-32 alleviates Escherichia coli K88-induced fatty acid malabsorption by improving expression of fatty acid transporter protein 4 (FATP4)1.

Bacterial infection causes nutrient malabsorption in small intestine. KR-32, a kind of synthetic antimicrobial peptide, has the bacteriostatic effect. In the present study, 2 experiments were designed to analyze the effects of KR-32 on fat absorption of piglets with or without Escherichia coli infection. In Exp. 1, 12 weaning piglets (21 d old) were allocated to 2 groups: piglets with an intraperitoneal (i.p.) injection of antimicrobial peptide KR-32 (APK) and piglets with an i.p. injection of an equivalent volume (1 mL) of phosphate-buffered saline (PBS) (CON-1). Results showed that after 7 d of growth, KR-32 did not significantly change growth performance and apparent total tract digestibility (ATTD) of feed nutrients of normal pigs. To confirm whether KR-32 affects those of enterotoxigenic Escherichia coli (ETEC) K88-challenged pigs, we performed Exp. 2, in which 18 piglets (28 d old) were divided into the following 3 groups: 1) piglets orally challenged with 1 × 1010 cfu ETEC K88 on day 1 followed by an i.p. injection of 0.6 mg/kg KR-32 (K88 + APK); 2) piglets orally challenged with 1 × 1010 cfu ETEC K88 on day 1 followed by an i.p. injection of an equivalent volume (1 mL) of PBS (K88); and 3) piglets with an oral administration of fresh Luria-Bertani broth (50 mL) followed by an i.p. injection of an equivalent volume of PBS (CON-2). Results showed that ETEC K88 challenge led to poor ADFI, ADG, and G:F in piglets; decreased ATTD of feed nutrients, especially CP and ether extract (EE); and intestinal morphology disorder. After i.p. injection of KR-32, ADG and ATTD of CP and EE were greatly increased, G:F was significantly reduced (P < 0.05), and, especially, ATTD of EE returned to a normal level compared with group CON-2. Fatty acid absorption also highly increased after KR-32 injection. Then we focused on fat digestion and fatty acid uptake. The pH in the intestine and pancreas lipase showed no difference among the 3 treatment groups, whereas fatty acid transporter protein 4 (FATP4) expression was remarkably improved (P < 0.05) and the epithelial barrier was recovered after i.p. injection of KR-32. In conclusion, KR-32, given to ETEC K88-challenged piglets, improved growth performance, ATTD of EE, fatty acid absorption, and intestinal morphology, which indicated that KR-32 was likely to improve the expression of FATP4 and by repairing the epithelial barrier, thereby alleviating fatty acid malabsorption.

The bile salt glycocholate induces global changes in gene and protein expression and activates virulence in enterotoxigenic Escherichia coli.

Pathogenic bacteria use specific host factors to modulate virulence and stress responses during infection. We found previously that the host factor bile and the bile component glyco-conjugated cholate (NaGCH, sodium glycocholate) upregulate the colonization factor CS5 in enterotoxigenic Escherichia coli (ETEC). To further understand the global regulatory effects of bile and NaGCH, we performed Illumina RNA-Seq and found that crude bile and NaGCH altered the expression of 61 genes in CS5 + CS6 ETEC isolates. The most striking finding was high induction of the CS5 operon (csfA-F), its putative transcription factor csvR, and the putative ETEC virulence factor cexE. iTRAQ-coupled LC-MS/MS proteomic analyses verified induction of the plasmid-borne virulence proteins CS5 and CexE and also showed that NaGCH affected the expression of bacterial membrane proteins. Furthermore, NaGCH induced bacteria to aggregate, increased their adherence to epithelial cells, and reduced their motility. Our results indicate that CS5 + CS6 ETEC use NaGCH present in the small intestine as a signal to initiate colonization of the epithelium.

Protective Ability of Biogenic Antimicrobial Peptide Microcin J25 Against Enterotoxigenic Escherichia Coli-Induced Intestinal Epithelial Dysfunction and Inflammatory Responses IPEC-J2 Cells.

Poison of intestinal induce severe health problems in human infants and young animals due to contaminating foods and feedstuffs. With the emergence of public health concerns and high-speed diffuse of drug-opposition of bacteria, the adoption of antimicrobial peptides as potential candidates in treating pathogen infections raised up. Nature Microcin J25 (MccJ25), a class of lasso peptides separated from a fecal strain of E. coli, has been replied to display powerful antimicrobial behavior. Herein, the study was to assess the usefulness of biogenic MccJ25 in the prophylaxis of ETEC K88 infection in IPEC-J2 cells. In vitro antimicrobial activity against ETEC K88 and cytotoxicity of biogenic MccJ25 were determined first. To further understand how biogenic MccJ25 mediates its impact, ETEC K88 adhesion in cells, membrane permeability [as indicated by reduced release of lactate dehydrogenase (LDH)], transepithelial electrical resistance (TEER), barrier function, and proinflammatory cytokines levels were determined in IPEC-J2 cells after treatment with biogenic MccJ25 and challenge with ETEC K88. Biogenic MccJ25 had a minimum inhibitory concentration of 0.25 µg/mL against ETEC K88, decreased ETEC K88 adhesion in cells and did not cause cytotoxicity toward cells. Furthermore, biogenic MccJ25 protects against ETEC-induced barrier dysfunction by increasing the TEER, decreasing the LDH and promoting tight junction proteins (TJPs) by promoting the assembly of occludin and claudin-1 in the tight junction complex. Biogenic MccJ25 was further found to relieve inflammation responses through modulation of interleukine-6, IL-8 and tumor necrosis factor-α levels via inhibition of mitogen-activated protein kinase (MAPK) and nuclear factor κB activation. In summary, biogenic MccJ25 can protects against ETEC K88-induced intestinal damage and inflammatory response, recommend the hidden adoption of biogenic MccJ25 as a novel prophylactic agent to reduce pathogen infection in animals, food or humans.

Co-occurrence of mcr-1, mcr-4 and mcr-5 genes in multidrug-resistant ST10 Enterotoxigenic and Shiga toxin-producing Escherichia coli in Spain (2006-2017).

Colistin is an antimicrobial polypeptide commonly employed for controlling and treating neonatal and post-weaning diarrhoea (PWD) diseases caused by Enterotoxigenic and Shiga toxin-producing Escherichia coli (ETEC and STEC). The plasmid-mediated colistin resistance gene, mcr-1 was first described in late 2015 and, since then, multiple studies have reported its global distribution. In addition, five different mcr genes have been identified. The aim of this study was to characterise the colistin-resistant E. coli clonal groups implicated in PWD in farms of intensive pig production. Of 186 ETEC and STEC isolated in Spain from 2006 to 2017, 76.9% showed resistance to colistin. Of those, 102 were mcr-4 carriers, 37 mcr-1 and 5 mcr-5, with co-occurrence of mcr-1/mcr-4, mcr-1/mcr-5 and mcr-4/mcr-5 in five isolates. Three different mcr-4 variants were detected, including the new mcr-4.4 and mcr-4.5 described here. Interestingly, the clonal group ST10-A (CH11-24) appears to be primarily responsible for the spread of mcr-4. In summary, our results show that the pig industry is an important reservoir of colistin-resistant E. coli, carriers of other additional risk genes, such as blaESBL. These food-producing animals might be spreading a cocktail of multiple resistances, posing a worrisome threat to human health.

Maillard neoglycans as inhibitors for in vitro adhesion of F4+ enterotoxigenic Escherichia coli to piglet intestinal cells.

Adhesion of enterotoxigenic (ETEC) E. coli to host intestinal cells is mediated by lectin-like fimbriae that bind to specific glycan moieties on the surfaces of enterocytes. To prevent in vitro binding of E. coli F4 fimbriae (F4 ETEC+) to piglet enterocytes, neoglycans were synthesized by the Maillard reaction conjugating lactose (Lac), galacto-oligosaccharides (GOS) or chitin oligosaccharides (Ochit) to porcine serum albumin (PSA). Neoglycans were characterized by SDS-PAGE, intrinsic tryptophan fluorescence and recognition by plant lectins, as well as by F4 ETEC variants. Electrophoretic patterns suggested the binding to PSA of 63, 13 and 2 molecules of Lac, GOS and Ochit, respectively. All neoglycans displayed quenching of tryptophan fluorescence consistent with the degree of glycation estimated by SDS-PAGE. Plant lectins recognized the neoglycans according to their specificity, whereas antigenic variants of F4 ETEC (ab, ac and ad) recognized PSA-Ochit and PSA-Lac with higher affinity than that for GOS. Neoglycans partially hindered the in vitro binding of F4+ ETEC to piglet enterocytes in a dose-dependent manner. The most effective blocking was observed with PSA-Lac that partially inhibited the adhesion of bacteria to enterocytes in a dose dependent manner, as quantified by flow cytometry. Increased production of the cytokines IL-6 and TNF-α was observed in response to F4+ ETEC infection of enterocytes and production was reduced in the presence of PSA-Ochit and PSA-GOS. These results suggest that neoglycans synthesized by the Maillard reaction could be useful in the prophylaxis of diarrhea in piglets.

Synthetic Porcine Hepcidin Exhibits Different Roles in Escherichia coli and Salmonella Infections.

Hepcidin, an antimicrobial peptide, was discovered to integrate diverse signals from iron status and an infection threat and orchestrate a series of host-protective responses. Several studies have investigated the antimicrobial role of hepcidin, but the results have been controversial. Here, we aimed to examine the role of hepcidin in bacterial adherence and invasion in vitro We found that porcine hepcidin could decrease the amount of the extracellular pathogen enterotoxigenic Escherichia coli (ETEC) K88 that adhered to cells because it caused the aggregation of the bacteria. However, addition of hepcidin to macrophages infected with the intracellular pathogen Salmonella enterica serovar Typhimurium enhanced the intracellular growth of the pathogen through the degradation of ferroportin, an iron export protein, and then the sequestration of intracellular iron. Intracellular iron was unavailable by use of the iron chelator deferiprone (DFO), which reduced intracellular bacterial growth. These results demonstrate that hepcidin exhibits different functions in extracellular and intracellular bacterial infections, which suggests that different defense strategies should be taken to prevent bacterial infection.

Exopolysaccharides of Lactobacillus reuteri: Their influence on adherence of E. coli to epithelial cells and inflammatory response.

The aim of the study was to characterize exopolysaccharides (EPS) originated from Lactobacillus reuteri strain DSM 17938 (EPS-DSM17938) and L. reuteri strain L26 Biocenol™ (EPS-L26) and evaluate their influence on adherence of enterotoxigenic Escherichia coli (ETEC) to IPEC-1 cells and proinflammatory gene expression. Both EPS were d-glucan polysaccharides with higher molecular weight (Mw), but differing in spatial conformation and elicited variable cytokine profile. EPS-DSM17938, relatively linear polysaccharide with (1→4) and (1→6) glycosidic linkages, increased IL-1ß gene expression (0.1mg/mL; P<0.05), while EPS-L26, more branched polysaccharide with (1→3) and (1→6) glycosidic linkages, exerted slight but statistically significant up-regulation of NF-κB, TNF-α and IL-6 mRNA (P<0.05). The most significant finding is that preincubation of IPEC-1 cells with both EPS followed by ETEC infection inhibit ETEC adhesion on IPEC-1 cells (P<0.01) and ETEC-induced gene expression of proinflammatory cytokine IL-1ß and IL-6 (P<0.01).

[Efficacy of disinfection treatments using essential oils and ultrasound on tomato fruits inoculated with Escherichia coli and impact on antioxidant activity]. / Eficiencia de la desinfección con aceites esenciales y ultrasonido sobre Escherichia coli inoculada en frutos de tomate y el impacto sobre la actividad antioxidante.

Fresh produce often harbors a great number of microorganisms; hence, its growing demand may constitute a risk for consumers. The aim of this study was to evaluate the efficacy of several disinfection procedures against enterotoxigenic Escherichia coli (ETEC) inoculated on tomato fruits and the conservation of the antioxidant properties of these disinfected fruits. Fruits were immersed for 5 or 10min in oregano or thyme essential oil dispersions (5, 10ppm), with or without ultrasound treatment. Antioxidant activity of disinfected fruits was determined as the ability to scavenge 2,2-diphenyl-1-pricrylhydrazyl (DPPH) radicals and was reported as percentage of inhibition (%I). The most efficient disinfectant treatments showing significant differences (p≤.05) between the reductions log10 CFU/g (S) of ETEC were those using 10ppm oregano for 10min, with S=3.05 in individual treatments and S=4.03 in mixed treatments. The highest %I was obtained with individual sonication treatments (69.52 and 72.48), while in combined treatments the %I values increased with thyme oil 5ppm and ultrasound for 5min (51.27%) and 10min (53.31%).

Eficiencia de la desingección con aceites esenciales y ultrasonido sobre Escherichia coli inoculada en frutos de tomate y el impacto sobre la actividad antioxidante / Efficacy of disinfection treatments using essential oils and ultrasound on tomato fruits inoculated with escherichia coli and impact on antioxidant activity

La creciente demanda de frutos frescos puede constituir un riesgo para la salud de los consumidores, teniendo en cuenta la gran variedad de microorganismos que estos suelen albergar. El objetivo del presente estudio fue evaluar la eficacia de varios procedimientos de desinfección sobre Escherichia coli enterotoxigénica (enterotoxigenic E. coli [ETEC]) inoculada en tomate y la conservación de las propiedades antioxidantes de los frutos desinfectados. Los frutos fueron sumergidos durante 5 o 10min en dispersiones de aceites esenciales de orégano o tomillo (5 o 10ppm), combinados o no con la aplicación de ultrasonido. La actividad antioxidante se determinó por la neutralización del radical 2,2-difenil-1-pricrilhidrazil (DPPH) y se reportó como porcentaje de inhibición (%I). Los tratamientos de desinfección más eficaces para una significativa reducción log10 UFG/g (S) de ETEC fueron con 10ppm de aceite de orégano durante 10min, con reducciones S=3,05 en tratamientos individuales y S=4,03 en mixtos. Los %I más altos se lograron con tratamientos individuales con sonicación (69,52 y 72,48) y en tratamientos combinados con aceite de tomillo 5ppm y ultrasonido durante 5 y 10min, con valores de 51,27 y 53,31%, respectivamente

Fresh produce often harbors a great number of microorganisms; hence, its growing demand may constitute a risk for consumers. The aim of this study was to evaluate the efficacy of several disinfection procedures against enterotoxigenic Escherichia coli (ETEC) inoculated on tomato fruits and the conservation of the antioxidant properties of these disinfected fruits. Fruits were immersed for 5 or 10min in oregano or thyme essential oil dispersions (5, 10ppm), with or without ultrasound treatment. Antioxidant activity of disinfected fruits was determined as the ability to scavenge 2,2-diphenyl-1-pricrylhydrazyl (DPPH) radicals and was reported as percentage of inhibition (%I). The most efficient disinfectant treatments showing significant differences (p≤.05) between the reductions log10 CFU/g (S) of ETEC were those using 10ppm oregano for 10min, with S=3.05 in individual treatments and S=4.03 in mixed treatments. The highest %I was obtained with individual sonication treatments (69.52 and 72.48), while in combined treatments the %I values increased with thyme oil 5ppm and ultrasound for 5min (51.27%) and 10min (53.31%)

Effects of chitosan on intestinal inflammation in weaned pigs challenged by enterotoxigenic Escherichia coli.

The aim of this study was to investigate whether supplementation with chitosan (COS) could reduce diarrhea and to explore how COS alleviates intestinal inflammation in weaned pigs. Thirty pigs (Duroc×Landrace×Yorkshire, initial BW of 5.65±0.27) weaned at age 21 d were challenged with enterotoxigenic Escherichia coli during a preliminary trial period, and then divided into three treatment groups. Pigs in individual pens were fed a corn-soybean meal diet, that contained either 0 (control), 50 mg/kg chlortetracycline, or 300 mg/kg COS for 21 days. The post-weaning diarrhea frequency, calprotectin levels and TLR4 protein expression were decreased (P<0.05) in both the COS and chlortetracycline groups compared with control. Simultaneously, supplemental COS and chlortetracycline had no effect on the mRNA expression of TNF-α in the jejunal mucosa, or on the concentrations of IL-1ß, IL-6 and TNF-α in serum. However, COS supplementation improved (P<0.05) the mRNA expression of IL-1ß and IL-6 in the jejunal mucosa. The results indicate that supplementation with COS at 300 mg/kg was effective for alleviating intestinal inflammation and enhancing the cell-mediated immune response. As feed additives, chitosan and chlortetracycline may influence different mechanisms for alleviating inflammation in piglets.

Investigation into in vitro and in vivo models using intestinal epithelial IPEC-J2 cells and Caenorhabditis elegans for selecting probiotic candidates to control porcine enterotoxigenic Escherichia coli.

AIMS: To identify a fast, economic and reliable method for preselecting lactic acid-producing bacterial (LAB) isolates to control enterotoxigenic Escherichia coli (ETEC). METHODS AND RESULTS: Two assays with porcine intestinal epithelial IPEC-J2 cells or Caenorhabditis elegans for selecting effective probiotic candidates were compared. Both assays were based on measuring death of cells or worms caused by ETEC strain JG280. Six of 13 LAB isolates showed ≥50% protection in each assay, among which only four isolates (≥50% protection) were consistently selected by both assays. Isolate CL9 (Lactobacillus reuteri) was further studied. It reduced gene expression of estA, estB and elt in JG280 in both assays. Furthermore, the isolate protected IPEC-J2 and C. elegans from cell and worm death caused by STa, STb or LT enterotoxin expressed in E. coli DH5α. CL9 also promoted host defensive responses by decreasing IL-8 and increasing IL-10 production in IPEC-J2 cells and expression of antimicrobial peptide genes clec-60, clec-85 in C. elegans. CONCLUSIONS: Caenorhabditis elegans is useful for preselecting probiotic candidates to control ETEC after initial screening with IPEC-J2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: A combination of IPEC-J2 cell and C. elegans assays can improve the effectiveness in preselecting probiotic candidates.

The suppressive activities of six sources of medicinal ferns known as gusuibu on heat-labile enterotoxin-induced diarrhea.

Diarrheal disease is one of the most important worldwide health problems. Enterotoxigenic Escherichia coli (ETEC) is the most frequently isolated enteropathogen in diarrheal diseases. In developing countries, a very large number of people, especially children, suffer from diarrhea. To combat this problem, World Health Organization has constituted the Diarrhea Diseases Control Program which guides studies on traditional medicinal practices and preventive measures. Gusuibu, a traditional folk medicine, has been claimed to heal certain types of diarrhea. However, so far no scientific study has been carried out on the anti-diarrheal mechanism of Gusiubu. The present study was performed to examine the suppressive activities of ethanol extracts of six sources of folk medicinal ferns used as Gusuibu on heat-labile enterotoxin (LT)-induced diarrhea. Inhibitory effects of six sources were evaluated on the ETEC LT subunit B (LTB) and monosialotetrahexosylganglioside (GMI) interaction by GM1-enzyme linked immunosorbent assay and patent mouse gut assay. Our results indicated that Drynaria fortunei had no anti-diarrheal effect, while, among the remaining five folk medicinal ferns, four belonging to family Davalliaceae had significant abilities on both the blocking of LTB and GM1 interaction and the inhibition of LT-induced diarrhea. In conclusion, these findings suggested the potential application of Gusuibu as an anti-diarrheal remedy.

Selection of Escherichia coli heat-labile toxin (LT) inhibitors using both the GM1-ELISA and the cAMP Vero cell assay.

Weaned piglets are very susceptible to diarrhea caused by enterotoxigenic Escherichia coli. In the past, various natural components were proposed to have beneficial effects by reducing the effects of diarrheal infectious diseases in humans and animals, and thus may represent an alternative for the use of (prophylactic) antibiotics. Alternatives may inactivate enterotoxigenic Escherichia coli heat-labile toxin (LT) by interfering with toxin binding to the cellular receptor GM1. In this study, various plants and other natural substances were tested for inhibitory properties, in the GM1 binding assay, and in the LT-induced cAMP production in Vero cells. The toxic dose of each compound was determined in a cell viability assay, and the highest nontoxic concentrations were used in the GM1 and cAMP assays. Results demonstrated that only d-(+)-galactose, lactose, N-acetyl-d-galactosamine, and two tea extracts were able to inhibit the binding of LT to its GM1 receptor. In the cAMP assay, only the two tea extracts showed inhibitory activity. This shows that d-(+)-galactose, lactose, and N-acetyl-d-galactosamine can indeed inhibit LT binding to GM1 based on structural homology with GM1 in the absence of living cells. However, in the cAMP assay, d-(+)-galactose, and lactose, N-acetyl-d-galactosamine are apparently metabolized to below their effective inhibitory concentration, likely predicting limited practical applicability in vivo. Both tea extracts maintained their activity in the presence of cells. The active compounds in both are probably polyphenols, which are not easily metabolized, and most likely work by aggregating the toxin. In conclusion, the combination of methods used here is a convenient and fast method for preselecting natural substances containing potentially toxin-binding compounds. Furthermore, if antidiarrhea activity is attributed to compounds found inactive here, their activity is unlikely based on interference with toxin binding.