Exploring the Effects of an Alfalfa Leaf-Derived Adsorbent on Microbial Community, Ileal Morphology, Barrier Function, and Immunity in Turkey Poults during Chronic Aflatoxin B1 Exposure.

This article follows-up on our recently published work, which evaluated the impact of the addition of an alfalfa leaf-derived adsorbent in the aflatoxin B1 (AFB1)-contaminated diet in regard to the production parameters, blood cell count, serum biochemistry, liver enzymes, and liver histology of turkey poults. This paper presents complementary results on microbial community, ileal morphology, barrier function, and immunity. For this purpose, 350 1-day-old female turkey poults were randomly distributed into five groups: (1) Control, AFB1-free diet; (2) AF, AFB1-contaminated diet at 250 ng/g; (3) alfalfa, AFB1-free diet + 0.5% (w/w) adsorbent; (4) alfalfa + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) adsorbent; and (5) YCW + AF, AFB1-contaminated diet at 250 ng/g + 0.5% (w/w) commercial yeast cell wall-based adsorbent (reference group). In general, in the AF group, the growth of opportunistic pathogens was promoted, which lead to gut dysbacteriosis, mainly influenced by Streptococcus lutetiensis. Conversely, a significant increase in beneficial bacteria (Faecalibacterium and Coprococcus catus) was promoted by the addition of the plant-based adsorbent. Moreover, the AF group had the lowest villus height and a compromised barrier function, as evidenced by a significant (p < 0.05) increase in fluorescein isothiocyanate dextran (FITC-d), but these negative effects were almost reversed by the addition of the alfalfa adsorbent. Furthermore, the AF + YCW and alfalfa + AF groups exhibited a significant increase in the cutaneous basophil hypersensitivity response compared to the rest of the experimental groups. Taken together, these results pointed out that the alfalfa counteracts the adverse effects of AFB1 in poults, facilitating the colonization of beneficial bacteria and improving the barrier function of the turkey poults.

Intestinal microbiota diversity from broilers with runting and stunting syndrome performed by metagenomics.

Runting and stunting syndrome (RSS) is an enteric viral disease in commercial poultry that directly affects gut health; however, its influence on gut microbiota remains unknown. This study aimed to investigate the compositional changes in the bacterial community of the ileum of 7-day-old broiler chicks naturally affected or not affected by RSS, using next-generation sequencing (NGS) technology. Twenty-one samples were obtained from the ileal contents and mucosa of 11 chicks with RSS and 10 healthy chicks, raised in a dark house system located on a farm in the state of Minas Gerais, Brazil. The results revealed overall changes in the gut microbiota of the chicks with RSS, including a decrease in microbial richness and diversity. In particular, there was a decrease in Lactobacillus and an increase in Candidatus Arthromitus and Clostridium sensu stricto 1. These results indicate a relationship between viral infection and the gut microbial composition, which can cause gut dysbiosis and may influence inflammation in this organ.RESEARCH HIGHLIGHTS RSS causes dysbiosis of the gut microbiota of the ilea of chicks.A difference was found in gut microbiota between chicks with or without RSS.Candidatus Arthromitus was predominant in chicks with RSS.Clostridium sensu stricto 1 was strictly associated with chicks with RSS.

Colonoscopy aspiration lavages for mucosal metataxonomic profiling of spondylarthritis-associated gastrointestinal tract alterations.

The study of the GI-tract microbiota of spondylarthritis (SpA) patients has focused on the analysis of feces samples, that picture mostly the luminal microbiota. The aim of this study was to determine the contribution of mucosal and luminal microbiome to the gut dysbiosis in SpA, using colonoscopy aspiration lavages (CAL), a recent alternative for regional studies of the GI-tract. We analyzed 59 CAL (from sigmoid colon and distal ileum), and 41 feces samples, from 32 SpA patients and 7 healthy individuals, using 16S rRNA gene-targeted metataxonomic profiling. It was found high prevalence of GI-tract manifestations among SpA patients (65.3%). Metataxonomic profiling, confirmed CAL samples from the lower GI tract (colon or ileum) presented a distinctive and undifferentiated bacteriome and separate from that found in feces' samples or in the beginning of the GI tract (oral cavity (OC)). Lower GI-tract samples and feces of SpA patients exhibited similar behavior to the microbiota of IBD group with reduced microbial richness and diversity, comparing to the healthy controls. Interestingly, it was found increase in proinflammatory taxa in SpA patients, such as Enterobacteriaceae family (mostly in the ileum), Succinivibrio spp. and Prevotella stercorea. Conversely, SpA patients presented significant decrease in the SCFA producers Coprococcus catus and Eubacterium biforme. Our data support the value of CAL samples for the regional study of GI-tract and contribute with information of potential "disruptor taxa" involved in the GI-tract associated disorders observed in SpA patients.

Prophylactic and therapeutic supplementation using fructo-oligosaccharide improves the intestinal homeostasis after mucositis induced by 5- fluorouracil.

The beneficial effects of prebiotic, such as fructo-oligosaccharides (FOS), in intestinal inflammation have been demonstrated in several studies. Herein, we evaluate whether joint treatment with FOS, both before and during mucositis, had additional beneficial effects and investigated the mechanisms underlying in the action of FOS on the intestinal barrier. BALB/c mice were randomly divided into five groups: CTR (without mucositis + saline solution), FOS (without mucositis + 6 % FOS), MUC (mucositis + saline solution), PT (mucositis + 6 % FOS supplementation before disease induction), and TT (mucositis + 6 % FOS supplementation before and during disease induction). Mucositis was induced by intraperitoneal injection (300 mg/kg) of 5-fluorouracil (5-FU). After 72 h, the animals were euthanized and intestinal permeability (IP), tight junction, bacterial translocation (BT), histology and morphometry, and immunoglobulin A secretory (sIgA), inflammatory infiltrate, and production of short-chain fatty acids (acetate, butyrate and propionate) were evaluated. The MUC group showed an increase in the IP, BT, and inflammatory infiltrate but a decrease in the tight junction expression and butyrate and propionate levels (P < 0.05). In the PT and TT groups, FOS supplementation maintained the IP, tight junction expression, and propionate concentration within physiologic levels, increased butyrate levels, and reduced BT and inflammatory infiltrate (P < 0.05). Total treatment with FOS (TT group) was more effective in maintaining histological score, morphometric parameters, and sIgA production. Thus, total treatment (prophylactic and therapeutic supplementation) with FOS was more effective than pretreatment alone, in reducing 5-FU-induced damage to the intestinal barrier.

Genomic characterization of enteropathogenic Escherichia coli (EPEC) of avian origin and rabbit ileal loop response; a pet macaw (Ara chloropterus) as a possible zoonotic reservoir.

Enteropathogenic Escherichia coli (EPEC) constitutes one of the main causes of mortality in children in low- to medium-income countries. Diverse animal species have been linked as reservoirs, including birds. The aim of this study was to describe the genomic and phylogenetic features of an EPEC recovered from a pet macaw and further characterizing the macro and microscopic lesion in a rabbit ileal loop experimental model. The isolate was whole-genome sequenced (WGS) obtaining its genotypic and phenotypic in silico characteristics and inoculated in a rabbit experimental model with subsequently evaluating the strain's pathogenicity by scanning electron microscopy (SEM) and histopathology. The isolate was characterized as O109:H21-B1-ST40 typical EPEC, harboring several virulence factors of diarrheagenic E. coli. The macaw EPEC genome was located in a monophyletic clade of human and animal ST40 EPEC sequences. In vivo inoculation demonstrated severe hemorrhage with SEM and histopathological analysis confirming these lesions to be associated with intra-epithelial lymphocytes. Therefore, the isolate not only shared several genotypic and phylogenetic similarities with EPEC that affects humans and animals, but was able to induce severe tissue injury in a mammal model. These findings highlight the underrated role of pet birds as zoonotic reservoirs and the diversity in virulence factors being unraveled by new WGS studies.

Milk Fermented by Lactobacillus paracasei NCC 2461 (ST11) Modulates the Immune Response and Microbiota to Exert its Protective Effects Against Salmonella typhimurium Infection in Mice.

Probiotics form a promising strategy to maintain intestinal health. Milks fermented with probiotic strains, such as the Lactobacillus paracasei ST11, are largely commercialized in Brazil and form a low-cost alternative to probiotic pharmaceutical formulations. In this study, we assessed the probiotic effects of milk fermented by L. paracasei ST11 (administered through fermented milk) in a Salmonella typhimurium infection model in BALB/c mice. We observed in this murine model that the applied probiotic conferred protective effects against S. typhimurium infection, since its administration reduced mortality, weight loss, translocation to target organs (liver and spleen) and ileum injury. Moreover, a reduction in the mRNA expression of pro-inflammatory cytokines such as IFN-γ, IL-6, TNF-α and IL-17 in animals that received the probiotic before challenge was observed. Additionally, the ileum microbiota was better preserved in these animals. The present study highlights a multifactorial protective aspect of this commercial probiotic strain against a common gastrointestinal pathogen.

Mucosal Exposure to Cigarette Components Induces Intestinal Inflammation and Alters Antimicrobial Response in Mice.

The main environmental risk factor associated with the development of Crohn's disease (CD) is cigarette smoking. Although the mechanism is still unknown, some studies have shown that cigarette exposure affects the intestinal barrier of the small bowel. Among the factors that may be involved in this process are Paneth cells. These specialized epithelial cells are located into the small intestine, and they are able to secrete antimicrobial peptides, having an essential role in the control of the growth of microorganisms. Alterations in its function are associated with inflammatory processes, such as CD. To study how cigarette components impact ileum homeostasis and Paneth cells integrity, we used intragastric administration of cigarette smoke condensate (CSC) in mice. Our results showed that inflammation was triggered after mucosal exposure of CSC, which induced particular alterations in Paneth cells granules, antimicrobial peptide production, and a reduction of bactericidal capacity. In fact, exposure to CSC generated an imbalance in the fecal bacterial population and increased the susceptibility of mice to develop ileal damage in response to bacterial infection. Moreover, our results obtained in mice unable to produce interleukin 10 (IL-10-/- mice) suggest that CSC treatment can induce a symptomatic enterocolitis with a pathological inflammation in genetically susceptible individuals.

Toxoplasma gondii promotes changes in VIPergic submucosal neurons, mucosal intraepithelial lymphocytes, and goblet cells during acute infection in the ileum of rats.

BACKGROUND: The intestinal mucosa plays an important role in the mechanical barrier against pathogens. During Toxoplasma gondii infection, however, the parasites invade the epithelial cells of the small intestine and initiate a local immune response. In the submucosal plexus, this response promotes an imbalance of neurotransmitters and induces neuroplasticity, which can change the integrity of the epithelium and its secretory function. This study evaluated the submucosal neurons throughout acute T. gondii infection and the relationship between possible alterations and the epithelial and immune defense cells of the mucosa. METHODS: Forty Wistar rats were randomly assigned to 8 groups (n = 5): 1 control group, uninfected, and 7 groups infected with an inoculation of 5000 sporulated T. gondii oocysts (ME-49 strain, genotype II). Segments of the ileum were collected for standard histological processing, histochemical techniques, and immunofluorescence. KEY RESULTS: The infection caused progressive neuronal loss in the submucosal general population and changed the proportion of VIPergic neurons throughout the infection periods. These changes may be related to the observed reduction in goblet cells that secret sialomucins and increase in intraepithelial lymphocytes after 24 hours, and the increase in immune cells in the lamina propria after 10 days of infection. The submucosa also presented fibrogenesis, characterizing injury and tissue repair. CONCLUSIONS AND INFERENCES: The acute T. gondii infection in the ileum of rats changes the proportion of VIPergic neurons and the epithelial cells, which can compromise the mucosal defense during infection.

Transforming Growth Factor ß1/SMAD Signaling Pathway Activation Protects the Intestinal Epithelium from Clostridium difficile Toxin A-Induced Damage.

Clostridium difficile, the main cause of diarrhea in hospitalized patients, produces toxins A (TcdA) and B (TcdB), which affect intestinal epithelial cell survival, proliferation, and migration and induce an intense inflammatory response. Transforming growth factor ß (TGF-ß) is a pleiotropic cytokine affecting enterocyte and immune/inflammatory responses. However, it has been shown that exposure of intestinal epithelium to a low concentration of TcdA induces the release of TGF-ß1, which has a protective effect on epithelial resistance and a TcdA/TGF-ß signaling pathway interaction. The activation of this pathway in vivo has not been elucidated. The aim of this study was to investigate the role of the TGF-ß1 pathway in TcdA-induced damage in a rat intestinal epithelial cell line (IEC-6) and in a mouse model of an ileal loop. TcdA increased the expression of TGF-ß1 and its receptor, TßRII, in vitro and in vivo TcdA induced nuclear translocation of the transcription factors SMAD2/3, a hallmark of TGF-ß1 pathway activation, both in IEC cells and in mouse ileal tissue. The addition of recombinant TGF-ß1 (rTGF-ß) prevented TcdA-induced apoptosis/necrosis and restored proliferation and repair activity in IEC-6 cells in the presence of TcdA. Together, these data show that TcdA induces TGF-ß1 signaling pathway activation and suggest that this pathway might play a protective role against the effect of C. difficile-toxin.

Ex vivo model of rabbit intestinal epithelium applied to the study of colonization by enteroaggregative escherichia coli / Modelo ex vivo de epitélio intestinal de coelho aplicado ao estudo de colonização por Escherichia coli enteroagregativa

ABSTRACT BACKGROUND The diarrheal syndrome is considered a serious public health problem all over the world and is considered a major cause of morbidity and mortality in developing countries. The high incidence of enteroaggregative Escherichia coli in diarrheal syndromes classified as an emerging pathogen of gastrointestinal infections. After decades of study, your pathogenesis remains uncertain and has been investigated mainly using in vitro models of adhesion in cellular lines. OBJECTIVE The present study investigated the interaction of enteroaggregative Escherichia coli strains isolated from childhood diarrhea with rabbit ileal and colonic mucosa ex vivo, using the in vitro organ culture model. METHODS The in vitro adhesion assays using cultured tissue were performed with the strains co-incubated with intestinal fragments of ileum and colon over a period of 6 hours. Each strain was tested with three intestinal fragments for each region. The fragments were analysed by scanning electron microscopy. RESULTS Through scanning electron microscopy we observed that all strains adhered to rabbit ileal and colonic mucosa, with the typical aggregative adherence pattern of “stacked bricks” on the epithelium. However, the highest degree of adherence was observed on colonic mucosa. Threadlike structures were found in greater numbers in the ileum compared to the colon. CONCLUSION These data showed that enteroaggregative Escherichia coli may have a high tropism for the human colon, which was ratified by the higher degree of adherence on the rabbit colonic mucosa. Finally, data indicated that in vitro organ culture of intestinal mucosa from rabbit may be used to elucidate the enteroaggregative Escherichia coli pathogenesis.

RESUMO CONTEXTO A síndrome diarréica é considerada um grave problema de saúde pública em todo o mundo e é considerada uma das principais causas de morbidade e mortalidade nos países em desenvolvimento. A elevada incidência de Escherichia coli enteroagregativa nas síndromes diarreicas a classificou como um patógeno emergente de infecções gastrintestinais. Depois de décadas de estudo, sua patogênese ainda é incerta e tem sido investigada usando principalmente modelos in vitro de adesão em linhagens celulares. OBJETIVO O presente estudo investigou a interação de cepas de Escherichia coli enteroagregativa isoladas de diarreia infantil com mucosa ileal e colônica de coelho ex vivo, utilizando o modelo de cultura de órgão in vitro. MÉTODOS Os ensaios de adesão in vitro utilizando tecido cultivado foram realizados com as cepas co-incubadas com fragmentos intestinais de íleo e de cólon durante um período de 6 horas. Cada cepa foi testada em três fragmentos intestinais para cada região. Os fragmentos foram analisados por microscopia eletrônica de varredura. RESULTADOS Através da microscopia eletrônica de varredura observamos que todas as cepas aderiram a mucosa ileal e colônica de coelho, com o padrão de aderência agregativo típico de “tijolos empilhados” no epitélio. Entretanto, o maior grau de adesão foi observado na mucosa do cólon. Estruturas filiformes foram encontradas em maior número no íleo em comparação com o cólon. CONCLUSÃO Esses dados mostraram que Escherichia coli enteroagregativa pode ter um maior tropismo para o cólon humano, o que foi ratificado pelo maior grau de aderência na mucosa do cólon de coelho. Finalmente, os dados indicaram que a cultura de órgão in vitro da mucosa intestinal de coelho pode ser utilizado para elucidar a patogênese de Escherichia coli enteroagregativa.

EX VIVO MODEL OF RABBIT INTESTINAL EPITHELIUM APPLIED TO THE STUDY OF COLONIZATION BY ENTEROAGGREGATIVE ESCHERICHIA COLI.

BACKGROUND: The diarrheal syndrome is considered a serious public health problem all over the world and is considered a major cause of morbidity and mortality in developing countries. The high incidence of enteroaggregative Escherichia coli in diarrheal syndromes classified as an emerging pathogen of gastrointestinal infections. After decades of study, your pathogenesis remains uncertain and has been investigated mainly using in vitro models of adhesion in cellular lines. OBJECTIVE: The present study investigated the interaction of enteroaggregative Escherichia coli strains isolated from childhood diarrhea with rabbit ileal and colonic mucosa ex vivo, using the in vitro organ culture model. METHODS: The in vitro adhesion assays using cultured tissue were performed with the strains co-incubated with intestinal fragments of ileum and colon over a period of 6 hours. Each strain was tested with three intestinal fragments for each region. The fragments were analysed by scanning electron microscopy. RESULTS: Through scanning electron microscopy we observed that all strains adhered to rabbit ileal and colonic mucosa, with the typical aggregative adherence pattern of "stacked bricks" on the epithelium. However, the highest degree of adherence was observed on colonic mucosa. Threadlike structures were found in greater numbers in the ileum compared to the colon. CONCLUSION: These data showed that enteroaggregative Escherichia coli may have a high tropism for the human colon, which was ratified by the higher degree of adherence on the rabbit colonic mucosa. Finally, data indicated that in vitro organ culture of intestinal mucosa from rabbit may be used to elucidate the enteroaggregative Escherichia coli pathogenesis.

High Incubation Temperature and Threonine Dietary Level Improve Ileum Response Against Post-Hatch Salmonella Enteritidis Inoculation in Broiler Chicks.

This study assessed the effect of both embryonic thermal manipulation and dietary threonine level on the response of broilers inoculated with Salmonella Enteritidis, considering bacterial counts in the cecal contents, intestinal morphology, mucin and heat shock protein 70 gene expression, body weight and weight gain. Thermal manipulation was used from 11 days of incubation until hatch, defining three treatments: standard (37.7°C), continuous high temperature (38.7°C) and continuous low temperature (36.7°C). After hatch, chicks were distributed according to a 3x2+1 factorial arrangement (three temperatures and two threonine levels and one sham-inoculated control). At two days of age, all chicks were inoculated with Salmonella Enteritidis, except for the sham-inoculated control group. There was no interaction between the factors on any analyses. High temperature during incubation was able to reduce colonization by Salmonella Enteritidis in the first days, reducing both Salmonella counts and the number of positive birds. It also increased mucin expression and decreased Hsp70 expression compared with other inoculated groups. High temperature during incubation and high threonine level act independently to reduce the negative effects associated to Salmonella Enteritidis infection on intestinal morphology and performance, with results similar to sham-inoculated birds. The findings open new perspectives for practical strategies towards the pre-harvest Salmonella control in the poultry industry.

Accuracy of the Timed Up and Go test for predicting sarcopenia in elderly hospitalized patients

OBJECTIVES: The ability of the Timed Up and Go test to predict sarcopenia has not been evaluated previously. The objective of this study was to evaluate the accuracy of the Timed Up and Go test for predicting sarcopenia in elderly hospitalized patients. METHODS: This cross-sectional study analyzed 68 elderly patients (≥60 years of age) in a private hospital in the city of Salvador-BA, Brazil, between the 1st and 5th day of hospitalization. The predictive variable was the Timed Up and Go test score, and the outcome of interest was the presence of sarcopenia (reduced muscle mass associated with a reduction in handgrip strength and/or weak physical performance in a 6-m gait-speed test). After the descriptive data analyses, the sensitivity, specificity and accuracy of a test using the predictive variable to predict the presence of sarcopenia were calculated. RESULTS: In total, 68 elderly individuals, with a mean age 70.4±7.7 years, were evaluated. The subjects had a Charlson Comorbidity Index score of 5.35±1.97. Most (64.7%) of the subjects had a clinical admission profile; the main reasons for hospitalization were cardiovascular disorders (22.1%), pneumonia (19.1%) and abdominal disorders (10.2%). The frequency of sarcopenia in the sample was 22.1%, and the mean length of time spent performing the Timed Up and Go test was 10.02±5.38 s. A time longer than or equal to a cutoff of 10.85 s on the Timed Up and Go test predicted sarcopenia with a sensitivity of 67% and a specificity of 88.7%. The accuracy of this cutoff for the Timed Up and Go test was good (0.80; IC=0.66-0.94; p=0.002). CONCLUSION: The Timed Up and Go test was shown to be a predictor of sarcopenia in elderly hospitalized patients. .

Effect of Trimetaphosphate and Fluoride Association on Hydroxyapatite Dissolution and Precipitation In Vitro

The present study analyzed the action of sodium trimetaphosphate (TMP) and/or fluoride on hydroxyapatite. Hydroxyapatite powder was suspended in different solutions: deionized water, 500 µg F/mL, 1,100 µg F/mL, 1%TMP, 3%TMP, 500 µg F/mL plus 1%TMP and 500 µg F/mL plus 3%TMP. The pH value of the solutions was reduced to 4.0 and after 30 min, raised to 7.0 (three times). After pH-cycling, the samples were analyzed by X-ray diffraction and infrared spectroscopy. The concentrations of calcium fluoride, fluoride, calcium and phosphorus were also determined. Adding 1% or 3% TMP to the solution containing 500 µg F/mL produced a higher quantity of calcium fluoride compared to samples prepared in a 1,100 µg F/mL solution. Regarding the calcium concentration, samples prepared in solutions of 1,100 µg F/mL and 500 µg F/mL plus TMP were statistically similar and showed higher values. Using solutions of 1,100 µg F/mL and 500 µg F/mL plus TMP resulted in a calcium/phosphorus ratio close to that of hydroxyapatite. It is concluded that the association of TMP and fluoride favored the precipitation of a more stable hydroxyapatite.

O presente estudo avaliou a ação do trimetafosfato de sódio (TMP) e/ou fluoreto sobre a hidroxiapatita. Pó de hidroxiapatita foi suspenso em diferentes soluções: água deionizada, 500 µg F/mL, 1100 µg F/mL, 1%TMP, 3%TMP, 500 µg F/mL adicionado a 1%TMP e 500 µg F/mL associado a 3%TMP. O pH das soluções foi reduzido para 4,0 e depois de 30 min, elevado para 7,0 (três vezes). Depois do processo de ciclagem de pH, as amostras foram analisadas por difração de raios-X e espectroscopia por infravermelho. As concentrações de fluoreto de cálcio, fluoreto, cálcio e fósforo também foram determinadas. A adição de 1% ou 3% TMP na solução contendo 500 µg F/mL produziu uma maior quantidade de fluoreto de cálcio comparado às amostras tratadas com uma solução de 1100 µg F/mL. A respeito da concentração de cálcio, amostras tratadas com soluções de 1100 µg F/mL e 500 µg F/mL adicionado ao TMP foram estatisticamente similares e mostraram maiores valores. Soluções de 1100 µg F/mL e 500 µg F/mL adicionado ao TMP resultaram em uma proporção molar Ca/P mais próxima à da hidroxiapatita. Conclui-se que a associação de TMP e F favoreceu a precipitação de uma hidroxiapatita mais estável.

Avian extraintestinal Escherichia coli exhibits enterotoxigenic-like activity in the in vivo rabbit ligated ileal loop assay.

Avian pathogenic Escherichia coli (APEC) strains harbor a number of virulence genes and cause extraintestinal diseases, such as septicemia, swollen-head syndrome, salpingitis, and omphalitis in poultry. APEC strains are not known to cause intestinal diseases. Herein, for the first time, it is reported that APEC strains were able to induce an enterotoxigenic-like effect in rabbit ligated ileal loops. Strain SEPT362 caused cell detachment of the intestinal villi, which also showed a flattened and wilted appearance, but the integrity of the tight junctions was maintained. Additionally, this strain did not adhere to enterocytes in vivo, although adhesin encoding genes ( fimH, csgA, lpfA2-3, and ECP) were present while other lpfA types, sfa, afa, papC, and ral genes were not. This enterotoxigenic-like activity was conserved after thermal treatment of the supernatant at 65°C but not at 100°C. Moreover, experiments based on filtering with different molecular weight cut-off (MWCO) pore sizes demonstrated that the component associated with the observed biological effect has a molecular weight >100 kDa. Blast search and polymerase chain reaction assays for known E. coli virulence factors showed that strain SEPT362 harbors the gene encoding for the toxin EAST-1 and the serine protease autotransporter (SPATE) Tsh, but is negative for genes encoding for the toxins LT-I, STh, STp, Stx1, Stx2, CNF-1, CNF-2, CDT and the SPATEs Sat, Pic, Vat, SigA, SepA, EatA, EspP, or EspC. A cloned copy of the tsh gene in E. coli K-12 was also tested and was shown to have an enterotoxic effect. These results suggest that APEC might induce fluid accumulation in the rabbit gut. The Tsh autotransporter seems to be one of the factors associated with this phenotype.

Escherichia coli isolates from inflammatory bowel diseases patients survive in macrophages and activate NLRP3 inflammasome.

Crohn's disease (CD) is a multifactorial pathology associated with the presence of adherent-invasive Escherichia coli (AIEC) and NLRP3 polymorphic variants. The presence of intracellular E. coli in other intestinal pathologies (OIP) and the role of NLRP3-inflammasome in the immune response activated by these bacteria have not been investigated. In this study, we sought to characterize intracellular strains isolated from patients with CD, ulcerative colitis (UC) and OIP, and analyze NLRP3-inflammasome role in the immune response and bactericidal activity induced in macrophages exposed to invasive bacteria. For this, intracellular E. coli isolation from ileal biopsies, using gentamicin-protection assay, revealed a prevalence and CFU/biopsy of E. coli higher in biopsies from CD, UC and OIP patients than in controls. To characterize bacterial isolates, pulsed-field gel electrophoresis (PFGE) patterns, virulence genes, serogroup and phylogenetic group were analyzed. We found out that bacteria isolated from a given patient were closely related and shared virulence factors; however, strains from different patients were genetically heterogeneous. AIEC characteristics in isolated strains, such as invasive and replicative properties, were assessed in epithelial cells and macrophages, respectively. Some strains from CD and UC demonstrated AIEC properties, but not strains from OIP. Furthermore, the role of NLRP3 in pro-inflammatory cytokines production and bacterial elimination was determined in macrophages. E. coli strains induced IL-1ß through NLRP3-dependent mechanism; however, their elimination by macrophages was independent of NLRP3. Invasiveness of intracellular E. coli strains into the intestinal mucosa and IL-1ß production may contribute to CD and UC pathogenesis.

Treatment with Saccharomyces boulardii reduces the inflammation and dysfunction of the gastrointestinal tract in 5-fluorouracil-induced intestinal mucositis in mice.

Intestinal mucositis is an important toxic side effect of 5-fluorouracil (5-FU) treatment. Saccharomyces boulardii is known to protect from intestinal injury via an effect on the gastrointestinal microbiota. The objective of the present study was to evaluate the effect of S. boulardii on intestinal mucositis induced by 5-FU in a murine model. Mice were divided into saline, saline (control)+5-FU or 5-FU+S. boulardii (16 × 109 colony-forming units/kg) treatment groups, and the jejunum and ileum were removed after killing of mice for the evaluation of histopathology, myeloperoxidase (MPO) activity, and non-protein sulfhydryl group (mainly reduced glutathione; GSH), nitrite and cytokine concentrations. To determine gastric emptying, phenol red was administered orally, mice were killed 20 min after administration, and the absorbance of samples collected from the mice was measured by spectrophotometry. Intestinal permeability was measured by the urinary excretion rate of lactulose and mannitol following oral administration. S. boulardii significantly reversed the histopathological changes in intestinal mucositis induced by 5-FU and reduced the inflammatory parameters: neutrophil infiltration (control 1·73 (SEM 0·37) ultrastructural MPO (UMPO)/mg, 5-FU 7·37 (SEM 1·77) UMPO/mg and 5-FU+S. boulardii 4·15 (SEM 0·73) UMPO/mg); nitrite concentration (control 37·00 (SEM 2·39) µm, 5-FU 59·04 (SEM 11·41) µm and 5-FU+S. boulardii 37·90 (SEM 5·78) µm); GSH concentration (control 477·60 (SEM 25·25) µg/mg, 5-FU 270·90 (SEM 38·50) µg/mg and 5-FU+S. boulardii 514·00 (SEM 38·64) µg/mg). Treatment with S. Boulardii significantly reduced the concentrations of TNF-α and IL-1ß by 48·92 and 32·21 % in the jejunum and 38·92 and 61·79 % in the ileum. In addition, S. boulardii decreased the concentrations of chemokine (C-X-C motif) ligand 1 by 5-fold in the jejunum and 3-fold in the ileum. Interestingly, S. boulardii reduced the delay in gastric emptying (control 25·21 (SEM 2·55) %, 5-FU 54·91 (SEM 3·43) % and 5-FU+S. boulardii 31·38 (SEM 2·80) %) and induced the recovery of intestinal permeability (lactulose:mannitol ratio: control 0·52 (SEM 0·03), 5-FU 1·38 (SEM 0·24) and 5-FU+S. boulardii 0·62 (SEM 0·03)). In conclusion, S. boulardii reduces the inflammation and dysfunction of the gastrointestinal tract in intestinal mucositis induced by 5-FU.

Effects of nitric oxide synthase inhibition on glutamine action in a bacterial translocation model.

Glutamine may be a precursor for NO synthesis, which may play a crucial role in bacterial translocation (BT). The goal of the present study was to investigate the potential effects of glutamine on BT and the immunological response in an experimental model of NO synthase inhibition by NG-nitro-L-arginine methyl ester (l-NAME). Mice were randomly assigned to four groups: sham; intestinal obstruction (IO); IO+500 mg/kg per d glutamine (GLN); IO+GLN plus 10 mg/kg per d l-NAME (GLN/LN). The groups were pretreated for 7 d. BT was induced by ileal ligation and was assessed 18 h later by measuring the radioactivity of 99mTc-Escherichia coli in the blood and organs. Mucosal damage was determined using a histological analysis. Intestinal permeability (IP) was assessed by measuring the levels of 99mTc-diethylenetriaminepentaacetic acid in the blood at 4, 8 and 18 h after surgery. IgA and cytokine concentrations were determined by ELISA in the intestinal fluid and plasma, respectively. BT was increased in the GLN/LN and IO groups than in the GLN and sham groups. IP and intestinal mucosa structure of the sham, GLN and GLN/LN groups were similar. The GLN group had the highest levels of interferon-γ, while IL-10 and secretory IgA levels were higher than those of the IO group but similar to those of the GLN/LN group. The present results suggest that effects of the glutamine pathway on BT were mediated by NO. The latter also interferes with the pro-inflammatory systemic immunological response. On the other hand, IP integrity preserved by the use of glutamine is independent of NO.

Denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA to monitor changes in mouse gut bacterial communities during Salmonella enterica serovar Enteritidis latent infection.

Changes in intestinal microbial flora during a 4-week period of Salmonella enterica serovar Enteritidis colonization in resistant mice (latent carrier animals) were evaluated using a culture independent method involving denaturing gradient gel electrophoresis. The contents of the ileocecal portion of the intestines produced 26 bands. Fifty-seven percent of the bands were expressed in more than 80% of the samples. Forty percent of the bands present in the negative control were common to all samples, and 60% differed from those obtained 12 h and 1, 5, 10, and 28 days post-inoculation (PI). A dendrogram distinguished the negative control as the external group, and 2 clusters were formed with 76% similarity, separating the 12-h PI and 3-day PI time points from the others. These groupings were also revealed through multivariate analysis in a principal component analysis and the Venn diagram. The production of interferon γ 12 h and 3 days PI may explain this brief imbalance in microbiota that was quickly reversed in the subsequent days. These findings demonstrate that S. enterica serovar Enteritidis can colonize the gut and persist in balance with the microbiota of resistant hosts.