The gastrointestinal epithelial layer forms a physical and biochemical barrier that maintains the segregation between host and intestinal microbiota. The integrity of this barrier is critical in maintaining homeostasis in the body and its dysfunction is linked to a variety of illnesses, especially inflammatory bowel disease. Gut microbes, and particularly probiotic bacteria, modulate the barrier integrity by reducing gut permeability and reinforcing tight junctions. Probiotic Escherichia coli Nissle 1917 (EcN) is a good colonizer of the human gut with proven therapeutic efficacy in the remission of ulcerative colitis in humans. EcN positively modulates the intestinal epithelial barrier through upregulation and redistribution of the tight junction proteins ZO-1, ZO-2 and claudin-14. Upregulation of claudin-14 has been attributed to the secreted protein TcpC. Whether regulation of ZO-1 and ZO-2 is mediated by EcN secreted factors remains unknown. The aim of this study was to explore whether outer membrane vesicles (OMVs) released by EcN strengthen the epithelial barrier. This study includes other E. coli strains of human intestinal origin that contain the tcpC gene, such as ECOR63. Cell-free supernatants collected from the wild-type strains and from the derived tcpC mutants were fractionated into isolated OMVs and soluble secreted factors. The impact of these extracellular fractions on the epithelial barrier was evaluated by measuring transepithelial resistance and expression of several tight junction proteins in T-84 and Caco-2 polarized monolayers. Our results show that the strengthening activity of EcN and ECOR63 does not exclusively depend on TcpC. Both OMVs and soluble factors secreted by these strains promote upregulation of ZO-1 and claudin-14, and down-regulation of claudin-2. The OMVs-mediated effects are TcpC-independent. Soluble secreted TcpC contributes to the upregulation of ZO-1 and claudin-14, but this protein has no effect on the transcriptional regulation of claudin-2. Thus, in addition to OMVs and TcpC, other active factors released by these microbiota strains contribute to the reinforcement of the epithelial barrier.

Background Enteric pathogens have developed mechanisms to disrupt tight junctions and increase gut permeability. Many studies have analysed the ability of live probiotics to protect intestinal epithelial cells against tight junction damage caused by bacterial pathogens. Escherichia coli Nissle 1917 (EcN) is among the probiotics that positively modulates the intestinal epithelial barrier by regulating expression and distribution of tight junction proteins. We previously reported that regulation of ZO-1, claudin-14 and claudin-2 is mediated by EcN secreted factors, either free-released or associated with outer membrane vesicles (OMVs). Factors secreted by commensal ECOR63 elicited comparable effects in intact epithelial T-84 and Caco-2 cell monolayers. Results Here we analyse the ability of OMVs and soluble secreted factors to protect epithelial barrier function in polarized T-84 and Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). Transepithelial electrical resistance, paracellular permeability, mRNA levels and subcellular distribution of tight junction proteins were monitored in the absence or presence of EcN and ECOR63 extracellular fractions. EPEC downregulated expression of ZO-1 ZO-2, occludin and claudin-14 and altered the subcellular localization of ZO-1, occludin and F-actin cytoskeleton. OMVs and soluble factors secreted by EcN and ECOR63 counteracted EPEC-altered transepithelial resistance and paracellular permeability, preserved occludin and claudin-14 mRNA levels, retained ZO-1 and occludin at tight junctions in the cell boundaries and ameliorated F-actin disorganization. Redistribution of ZO-1 was not accompanied by changes at mRNA level. Conclusion This study provides new insights on the role of microbiota secreted factors on the modulation of intestinal tight junctions, expanding their barrier-protective effects against pathogen-induced disruption.

BackgroundEscherichia coli Nissle 1917 (EcN) is a probiotic used in the treatment of intestinal diseases. Although it is considered safe, EcN is closely related to the uropathogenic E. coli strain CFT073 and contains many of its predicted virulence elements. Thus, it is relevant to assess whether virulence-associated genes are functional in EcN. One of these genes encodes the secreted autotransporter toxin (Sat), a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs) that are secreted following the type V autotransporter pathway. Sat is highly prevalent in certain E. coli pathogenic groups responsible for urinary and intestinal infections. In these pathogens Sat promotes cytotoxic effects in several lines of undifferentiated epithelial cells, but not in differentiated Caco-2 cells.ResultsHere we provide evidence that sat is expressed by EcN during the colonization of mouse intestine. The EcN protein is secreted as an active serine protease, with its 107 kDa-passenger domain released into the medium as a soluble protein. Expression of recombinant EcN Sat protein in strain HB101 increases paracellular permeability to mannitol in polarized Caco-2 monolayers. This effect, also reported for the Sat protein of diffusely adherent E. coli, is not observed when this protein is expressed in the EcN background. In addition, we show that EcN supernatants confer protection against Sat-mediated effects on paracellular permeability, thus indicating that other secreted EcN factors are able to prevent barrier disruption caused by pathogen-related factors. Sat is not required for intestinal colonization, but the EcNsat::cat mutant outcompetes wild-type EcN in the streptomycin-treated mouse model. Analysis of the presence of sat in 29 strains of the ECOR collection isolated from stools of healthy humans shows 34.8 % positives, with high prevalence of strains of the phylogenetic groups D and B2, related with extra-intestinal infections.ConclusionsSat does not act as a virulence factor in EcN. The role of Sat in intestinal pathogenesis relies on other genetic determinants responsible for the bacterial pathotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0591-5) contains supplementary material, which is available to authorized users.

Supplementary fructooligosaccharides (FOS) were evaluated on growth, survival, gut morphology, digestive enzyme activity and expression of intestinal barrier genes in tropical gar (Atractosteus tropicus). Four treatments were designed to include FOS (5, 10, 15 and 20 g/Kg) and were compared with a control (0 g/Kg) and the current commercial trout diet. Ten fish were stocked (0.25 ± 0.01 g and 4.2 ± 0.4 cm total length) per experimental unit by triplicate in a 45-day experiment. Fish fed with 5 and 10 g/Kg of FOS had the highest average weight and total average length. Fish fed 5 g/Kg FOS showed the best growth indexes (absolute weight gain: 2.16; specific growth rate: 4.96; and feed conversion rate: 1.23) and survival (96.66%) and also had the highest acid protease, chymotrypsin and leucine aminopeptidase activities. Fish fed 5 g/Kg FOS had a high absorption area and accumulation of lipids in the liver. Finally, relative overexpression of occluding (occ), nucleotide-binding oligomerization domain-2 (nod2) and mucin 2 (muc2) was detected in fish fed 10 and 15 g/Kg FOS. The supplementation between 5 and 10 g/Kg FOS in diets for A. tropicus juveniles could be beneficial for growth, digestive enzyme activities, gut morphology and intestinal barrier function.

No abstract

Halotolerant bacteria with biosurfactant (BS) and bioemulsifiers (BE) activity can coexist in Karstic sinkholes with marine influence. Two sinkholes in the Yucatan peninsula were selected to isolate bacteria with BE and BS activity stable in NaCl. The optimal time, the effect of nitrogen and carbon source in the medium, and the conditions (agitation, pH and salinity) for the production of BS and BE compounds in planktonic and sessile (stimulate the formation of biofilms in cell roller) culture were determined. Eighty strains showed the highest emulsification activity (EI24 ≥ 50%) and drop-collapse ≥ 4 mm. 87% of the strains are moderately halotolerant, and 21% bordered the limit of extreme halotolerance. Twenty-four strains maintained or improved their BS and BE activity under salinity conditions at 5% and 10%, being the most active genera Bacillus, Paenibacillus and Lysinibacillus, identified by sequencing of the 16S rRNA gene. The results show that the nitrogen source positively affects the BS and BE activity, regardless of the type of culture. The sessile culture markedly stimulated BS activity with significant differences. However, we did not find a greater influence on the culture conditions. The results suggest that halotolerant bacteria from sinkholes could be implemented in bioremediation and other biotechnological applications.

Reports surrounding the role of resistant starch (RS) on postprandial lipemia in humans are scarce. The aim of the present study is to examine the effects of resistant starch on the postprandial lipemic response, subjective measures of appetite, and energy intake in overweight and obese subjects. In a randomized, single-blind, crossover study, 14 overweight/obese participants ate a high-fat breakfast (679 kcal, 58% from fat) and a supplement with native banana starch (NBS), high-amylose maize starch (HMS), or digestible maize starch (DMS) on three separate occasions. All supplements provided were matched by the available carbohydrate content, and the RS quantity in NBS and HMS supplements was identical. Appetite was estimated using visual analogue scale (VAS) and an ad libitum test meal. Postprandial glycemia, triglycerides, cholesterol, high-density lipoprotein (HDL) cholesterol, and insulin excursions did not differ between treatments. Subjective appetite measures of satiety were significantly increased after HMS; however, no effects on energy intake were observed during the ad libitum test meal. These findings suggest that a single acute dose of RS cannot be expected to improve postprandial lipemia in subjects with overweight or obesity on a high-fat meal. However, the potential benefits of long-term supplementation should not be ruled out based on these results.

Mannan-oligosaccharides (MOS) are non-digestible carbohydrates, and their use in aquaculture as prebiotics is well documented. The objective of this work was to test whether MOS supplemented in the diet of A. tropicus larvae (2, 4, and 6 g kg−1) influence growth parameters, the activity of digestive enzymes, and the expression of genes related to the intestinal barrier. The highest total length was observed in larvae fed 6 g kg−1 MOS compared to control larvae. Trypsin activity increased with the addition of MOS to the diets, but leucine aminopeptidase activity only increased with 6 g kg−1 MOS. Lipase and α-amylase activities increased in larvae fed with 2 and 4 g kg−1 MOS. The expression of zo-2 was higher with the 6 g kg−1 MOS treatment. The cl-3 transcripts were lower with 2 g kg−1 MOS but higher with 6 g kg−1 MOS. All tested concentrations of MOS increased the expression of muc-2. In this study, incorporating mannan-oligosaccharides into the diet of A. tropicus larvae had a positive effect, and the concentration of 6 g kg−1 produced the best results. Therefore, including this prebiotic in the diets for the culture of A. tropicus larvae is suitable.