Higher Fecal Levels Research Articles

Effective Colonization by Nontoxigenic Clostridioides difficile REA Strain M3 (NTCD-M3) Spores following Treatment with Either Fidaxomicin or Vancomycin.

Colonization with nontoxigenic Clostridioides difficile strain M3 (NTCD-M3) has been demonstrated in susceptible hamsters and humans when administered after vancomycin treatment. NTCD-M3 has also been shown to decrease risk of recurrent C. difficile infection (CDI) in patients following vancomycin treatment for CDI. As there are no data for NTCD-M3 colonization after fidaxomicin treatment, we studied the efficacy of NTCD-M3 colonization and determined fecal antibiotic levels in a well-studied hamster model of CDI. Ten of 10 hamsters became colonized with NTCD-M3 after 5 days of treatment with fidaxomicin when NTCD-M3 was administered daily for 7 days after treatment discontinuation. The findings were nearly identical to 10 vancomycin-treated hamsters also given NTCD-M3. High fecal levels of OP-1118, the major fidaxomicin metabolite, and vancomycin were noted during treatment with the respective agents and modest levels noted 3 days after treatment discontinuation at the time when most of the hamsters became colonized. These findings support the ongoing development of NTCD-M3 for the prevention of recurrent CDI. IMPORTANCE NTCD-M3 is a novel live biotherapeutic, that has been shown in a Phase 2 clinical trial to prevent recurrence of C. difficile infection (CDI) when administered shortly after antibiotic treatment of the initial CDI episode. Fidaxomicin was not, however, in widespread use at the time this study was conducted. A large multi-center Phase 3 clinical trial is now currently in the planning stage, and it is anticipated that many patients eligible for this study will be treated with fidaxomicin. Since efficacy in the hamster model of CDI has predicted success in patients with CDI, we studied the ability of NTCD-M3 to colonize hamsters after treatment with either fidaxomicin or vancomycin.

Foods may modify responsiveness to cancer immune checkpoint blockers by altering both the gut microbiota and activation of estrogen receptors in immune cells

Estrogen receptor alpha positive (ERα+) breast cancers are refractory to immune checkpoint blocker (ICB) monotherapy, while ICBs are part of a standard of care for triple negative breast cancers (TNBCs). Besides tumor ERα expression, another difference between the two types of breast cancers is that only ERα+ patients exhibit elevated tumor estradiol (E2) levels, compared with surrounding normal tissue. Recent evidence suggests that inhibition of ERα or activation of ERβ or G protein-coupled estrogen receptor (GPER) in immune cells in the tumor microenvironment (TME) increases tumor CD8+ T cell infiltration and boosts cancer ICB response. Ovarian and adipose-produced estrogens activate all three ERs equally, but plant estrogens (phytochemicals) preferentially activate ERβ or GPER. The gut microbiota is a key player in determining response to ICBs, and high abundance of Firmicutes and high fecal levels of short chain fatty acids (SCFAs) that are mainly produced by Firmicutes, are linked to improved effectiveness of ICB therapy. Interestingly, the gut microbiota of ERα+ breast cancer patients contain significantly lower abundance of Firmicutes species than the gut microbiota of TNBC patients. Many factors modify the gut microbiota, especially diet. The gut microbiota altering diets include (i) foods high in ERβ and GPER activating plant phytochemicals or (ii) SCFAs producing fiber that also reduces circulating estrogen levels, (iii) estrogen levels reducing fasting/caloric restriction, or (iv) ketogenic diet which reduces fecal SCFA levels but increases hepatic production of SCFA receptor activating ketone bodies. It is thus possible that certain foods or dietary patterns can modify both the gut microbiota and activation of the estrogen receptors in the tumor immune cells, and consequently regulate the effectiveness of ICB therapy against cancers.

Evaluation of a Functional Craft Wheat Beer Fermented with Saccharomyces cerevisiae UFMG A-905 to treat Salmonella Typhimurium infection in mice.

Functional foods containing probiotics are generally administered as dairy products. Non-dairy beverages are another possibility, but probiotic functionality must be confirmed in such vehicles. In the present study, a craft wheat beer brewed with the probiotic yeast Saccharomyces cerevisiae UFMG A-905 (905) was evaluated in a murine model of Salmonella Typhimurium infection. Unfiltered or filtered beer brewed with 905, a commercial wheat beer used as a negative control, or saline were administered orally to mice before and during oral S. Typhimurium challenge. High fecal levels of yeast were only counted in mice treated with the unfiltered 905 beer, which also had reduced mortality and body weight loss due to S. Typhimurium infection. Increased levels of intestinal IgA, translocation to liver and spleen, liver and intestinal lesions, pro-inflammatory cytokines in liver and ileum, and hepatic and intestinal myeloperoxidase and eosinophilic peroxidase activities were observed in animals infected with S. Typhimurium. All these parameters were reduced by the treatment with unfiltered 905 beer. In conclusion, the results show that a craft wheat beer brewed with S. cerevisiae UFMG A-905 maintained the probiotic properties of this yeast when administered orally to mice challenged with S. Typhimurium.

Acidic Activated Charcoal Prevents Obesity and Insulin Resistance in High-Fat Diet-Fed Mice.

Obesity is becoming a major public health problem worldwide. Making charcoal from wood (“Sumi-yaki”) has been a traditional activity in the southern part of Nagano Prefecture for centuries, with activated charcoal having reported detoxifying effects. However, it is unclear whether activated charcoal also possesses anti-obesity properties. Additionally, since activated charcoal is usually alkaline and might be affected by gastric juice, we evaluated the effect of acidic activated charcoal on high-fat diet (HFD)-induced obesity. This study demonstrated that co-treatment of acidic activated charcoal with a HFD significantly improved obesity and insulin resistance in mice in a dose-dependent manner. Metabolomic analysis of cecal contents revealed that neutral lipids, cholesterol, and bile acids were excreted at markedly higher levels in feces with charcoal treatment. Moreover, the hepatic expressions of genes encoding cholesterol 7 alpha-hydroxylase and hydroxymethylglutaryl-CoA reductase/synthase 1 were up-regulated by activated charcoal, likely reflecting the enhanced excretions from the intestine and the enterohepatic circulation of cholesterol and bile acids. No damage or abnormalities were detected in the gastrointestinal tract, liver, pancreas, and lung. In conclusion, acidic activated charcoal may be able to attenuate HFD-induced weight gain and insulin resistance without serious adverse effects. These findings indicate a novel function of charcoal to prevent obesity, metabolic syndrome, and related diseases.

S100A8 and S100A9 Are Important for Postnatal Development of Gut Microbiota and Immune System in Mice and Infants

After birth, the immune system matures via interactions with microbes in the gut. The S100 calcium binding proteins S100A8 and S100A9, and their extracellular complex form, S100A8-A9, are found in high amounts in human breast milk. We studied levels of S100A8-A9 in fecal samples (also called fecal calprotectin) from newborns and during infancy, and their effects on development of the intestinal microbiota and mucosal immune system. We collected stool samples (n= 517) from full-term (n= 72) and preterm infants (n= 49) at different timepoints over the first year of life (days 1, 3, 10, 30, 90, 180, and 360). We measured levels of S100A8-A9 by enzyme-linked immunosorbent assay and analyzed fecal microbiomes by 16S sRNA gene sequencing. We also obtained small and large intestine biopsies from 8 adults and 10 newborn infants without inflammatory bowel diseases (controls) and 8 infants with necrotizing enterocolitis and measured levels of S100A8 by immunofluorescence microscopy. Children were followed for 2.5 years and anthropometric data and medical information on infections were collected. We performed studies with newborn C57BL/6J wild-type and S100a9-/- mice (which also lack S100A8). Some mice were fed or given intraperitoneal injections of S100A8 or subcutaneous injections of Staphylococcus aureus. Blood and intestine, mesenterial and celiac lymph nodes were collected; cells and cytokines were measured by flow cytometry and studied in cell culture assays. Colon contents from mice were analyzed by culture-based microbiology assays. Loss of S100A8 and S100A9 in mice altered the phenotypes of colonic lamina propria macrophages, compared with wild-type mice. Intestinal tissues from neonatal S100-knockout mice had reduced levels of CX3CR1 protein, and Il10 and Tgfb1 mRNAs, compared with wild-type mice, and fewer T-regulatory cells. S100-knockout mice weighed 21% more than wild-type mice at age 8 weeks and a higher proportion developed fatal sepsis during the neonatal period. S100-knockout mice had alterations in their fecal microbiomes, with higher abundance of Enterobacteriaceae. Feeding mice S100 at birth prevented the expansion of Enterobacteriaceae, increased numbers of T-regulatory cells and levels of CX3CR1 protein and Il10 mRNA in intestine tissues, and reduced body weight and death from neonatal sepsis. Fecal samples from term infants, but not preterm infants, had significantly higher levels of S100A8-A9 during the first 3 months of life than fecal samples from adults; levels decreased to adult levels after weaning. Fecal samples from infants born by cesarean delivery had lower levels of S100A8-A9 than from infants born by vaginal delivery. S100 proteins were expressed by lamina propria macrophages in intestinal tissues from infants, at higher levels than in intestinal tissues from adults. High fecal levels of S100 proteins, from 30 days to 1 year of age, were associated with higher abundance of Actinobacteria and Bifidobacteriaceae, and lower abundance of Gammaproteobacteria-particularly opportunistic Enterobacteriaceae. A low level of S100 proteins in infants' fecal samples associated with development of sepsis and obesity by age 2 years. S100A8 and S100A9 regulate development of the intestinal microbiota and immune system in neonates. Nutritional supplementation with these proteins might aide in development of preterm infants and prevent microbiota-associated disorders in later years.

Gut metagenomic and short chain fatty acids signature in hypertension: a cross-sectional study

Hypertension is an independent and preventable risk factor for the development of cardiovascular diseases, however, little is known about the impact of gut microbiota composition in its development. We carried out comprehensive gut microbiota analysis and targeted metabolomics in a cross-sectional study of 29 non-treated hypertensive (HT) and 32 normotensive (NT) subjects. We determined fecal microbiota composition by 16S rRNA gene sequencing and bacterial functions by metagenomic analysis. The microbial metabolites analysed were short chain fatty acids (SCFA) both in plasma and feces, and trimethylamine N-oxide (TMAO) in plasma. The overall bacterial composition and diversity of bacterial community in the two groups were not significantly different. However, Ruminococcaceae NK4A214, Ruminococcaceae_UCG-010, Christensenellaceae_R-7, Faecalibacterium prausnitzii and Roseburia hominis were found to be significantly enriched in NT group, whereas, Bacteroides coprocola, Bacteroides plebeius and genera of Lachnospiraceae were increased in HT patients. We found a positive correlation between the HT-associated species and systolic and diastolic blood pressure after adjusted for measured confounders. SCFA showed antagonistic results in plasma and feces, detecting in HT subjects significant higher levels in feces and lower levels in plasma, which could indicate a less efficient SCFA absorption. Overall, our results present a disease classifier based on microbiota and bacterial metabolites to discriminate HT individuals from NT controls in a first disease grade prior to drug treatment.

Intestinal short-chain fatty acid composition does not explain gut microbiota-mediated effects on malaria severity.

Malaria is a devastating disease resulting in significant morbidity and mortality, especially in the developing world. Previously, we showed that the gut microbiome modulates severity of malaria in mice, though the exact mechanism was unknown. One well-studied mechanism by which the intestinal microbiota exerts an effect on host health is by synthesis of short-chain fatty acids (SCFAs). SCFAs have pleiotropic effects on the host, including modulating the immune system and altering susceptibility to pathogens. The objective of the current work was to explore if gut microbiota-mediated resistance and susceptibility to malaria in mice is through differential production of SCFAs. Of the eight detected SCFAs, only propionic acid (C3) was different between two groups of resistant and two groups of susceptible mice, with higher levels in feces of susceptible mice compared to resistant mice. Nevertheless, subsequent analysis revealed no robust correlation between malaria severity and levels of fecal propionic acid. In spite of the broad effect of SCFAs on host physiology, including host immunity, this study shows that gut microbiota-mediated modulation of malaria severity in mice is independent of fecal SCFA levels. Additionally, our data indicates that intestinal SCFAs do not function as biomarkers for prediction of malaria disease severity.

Eicosapentaenoic Acid Reduces Fecal Levels of Calprotectin and Prevents Relapse in Patients With Ulcerative Colitis

High fecal levels of calprotectin indicate mucosal inflammation and have been shown to predict relapse in patients with ulcerative colitis (UC). Eicosapentaenoic acid (EPA), the major component of n-3 fish oil, has anti-inflammatory properties in patients with chronic inflammatory disorders. We performed a placebo-controlled trial of patients with UC at risk of relapse to determine the ability of the free fatty acid form of EPA (EPA-FFA) to reduce intestinal inflammation, using fecal level of calprotectin as a marker. From June 2014 to May 2016, 60 patients with UC with a partial Mayo score < 2 and fecal calprotectin ≥150 μg/g, in stable therapy for at least the 3 previous months, were randomly assigned to groups (1:1) given either EPA-FFA (500 mg, twice daily) or placebo for 6 months. A colonoscopy was performed at baseline. Clinical assessments and measurements of fecal calprotectin were made at baseline, at study months 3 and 6, or the time of clinical relapse. Patients with a relapse of UC underwent a second colonoscopy. The primary end point was a 100-point reduction in fecal levels of calprotectin at 6 months from the baseline value; the secondary end point was maintenance of clinical remission at 6 months. The primary end point was achieved by 19 of 30 patients (63.3%) in the EPA-FFA group vs 4 of 30 patients (13.3%) in the placebo group (odds ratio, 12.0; 95% CI, 3.12-46.24; P < .001). The secondary end point was achieved by 23 of 30 patients (76.7%) in the EPA-FFA group vs 15 of 30 (50%) patients in the placebo group (OR, 3.29; 95% CI, 1.08-9.95; P= .035). No serious adverse events were observed. In a placebo-controlled trial of 60 patients with UC, we found 6 months' administration of EPA-FFA to reduce fecal levels of calprotectin with no serious adverse events. This agent might be used to induce and maintain symptom-free remission in patients with UC. ClinicalTrials.gov number: NCT02179372.

Seasonality of reproduction in wild boar (Sus scrofa) assessed by fecal and plasmatic steroids

The collection of biological samples through non-invasive techniques represents one way of monitoring in vivo physiological changes associated with reproductive activity. Such techniques are particularly important for the study of animal species in the wild.The goals of this study were 1) to evaluate fecal progestogen (P), estrogen (E), and androgen (A) by means of radioimmunoassays, in male and female wild boars culled in the Piedmont, Italy area; 2) to compare them with plasmatic concentrations and the animals’ reproductive status; and 3) to assess variations in reproductive seasonality between two populations of wild boars living in a mountainous vs. a plain habitat in Piedmont.The results demonstrate a positive correlation between fecal and plasmatic steroid concentrations (r=0.46, 0.58, and 0.45 for plasma P4 and P, E2 and E, and T and A; P<0.05). Moreover, high fecal levels of both P and E (>170ng/g and >100pg/g respectively) were found in 70.6% of pregnant sows and in none of the non-pregnant animals, thus supporting the use of this technique for detecting pregnancy status in wild boar.Similar birth patterns were displayed by the mountain and plain populations, but births peaked significantly only in the mountain population, in the spring (46%, P<0.05, vs. other seasons). A corresponding autumnal peak of plasma testosterone concentrations in males was displayed only by the mountain population (7.4 vs.<2.0ng/mL in the other seasons, P<0.05).The correlation between fecal and plasmatic steroid concentrations obtained in this study supports the applicability of this non-invasive sampling technique for monitoring reproductive status in wild boar, thus enabling a more informed and correct management of the species.

Antimicrobial Components of the Neonatal Gut Affected Upon Colonization

Antimicrobial peptides (AMP) produced throughout our body are important effectors in the defense barrier of innate immunity. Here, we have analyzed antimicrobial activity and polypeptide composition of meconium versus neonatal feces to address the development of antimicrobial defense of the neonatal gut. Extracts of meconium exhibited antimicrobial activity against Bacillus megaterium, Escherichia coli, and group B streptococci (GBS) but not against the yeast Candida albicans. Extracts of neonatal feces were found to possess low activity against E. coli, GBS, and C. albicans. However, the anti-B. megaterium activity was higher in the fecal extracts than in meconium. All activities were reduced or abolished when salt was added to the antimicrobial assay. The AMP cathelicidin LL-37, alpha-defensin HNP-1-2, alpha-defensin HD 5, and lysozyme were identified in both meconium and fecal extracts. In addition, HNP-3 and a fragment of azurocidin were found in meconium, whereas the holoprotein azurocidin was detected in feces. In meconium, histones H2A and H4 were isolated and identified by their antimicrobial activity. Notably, LL-37 and lysozyme were found at significantly higher levels in feces than in meconium. Our findings reveal that meconium and feces contain AMP, acting in the defense of the neonatal gut, and may be implicated in the control of the initial colonization.

Fecal Lactoferrin and Calprotectin After Ileocolonic Resection for Crohn's Disease

This study was designed to assess the role of fecal lactoferrin and calprotectin as markers of intestinal inflammation in patients with Crohn's disease who have undergone ileocolonic resection. Sixty-three patients who had undergone ileocolonic resection for Crohn's disease with a median follow-up of 40.5 (range, 5-102) months were enrolled. Clinical examination and blood test were performed, and fecal lactoferrin and calprotectin levels were dosed. The predictors for fecal lactoferrin and calprotectin levels that resulted to be significant at the univariate analyses were included in two multiple regression analysis models. The mean lactoferrin level was 21 +/- 3.9 microg/g and the mean calprotectin fecal level was 247 +/- 22.7 ng/ml. C-reactive protein levels (P < 0.01), calprotectin levels (P < 0.01), and the presence of clinical recurrence (P = 0.04) resulted to be independent predictors of lactoferrin levels. Only lactoferrin levels resulted to be an independent predictor for calprotectin fecal levels (P < 0.01). Crohn's disease patients maintain high fecal levels of lactoferrin and calprotectin at long-term follow-up after resection of the diseased bowel even in case of clinical remission. The significant correlation between the two fecal markers may be the expression of the ongoing intestinal inflammation. Only lactoferrin significantly correlated with C-reactive protein and showed a reliable threshold value for systemic inflammation. Lactoferrin fecal levels may be a reliable indicator for intestinal inflammation influencing the systemic inflammatory status. The third predictor of lactoferrin fecal level was the presence of episodes of clinical recurrence during the postoperative follow-up.

Colonization Resistance Induced in Mice Orogastrically Dosed with Human Faecal Homogenates from Different Donors

Human faecal samples from various donors were assessed for the capacity to provide protection against Salmonella typhimurium both in vitro and in vivo . Faecal material was collected from four healthy human subjects. Populations of anaerobic bacteria and enterics were enumerated using selective media and the isolates screened for the capacity to inhibit the in vitro growth of S. typhimurium . Isolates with inhibitory activity against S. typhimurium were detected in two out of four subjects. These isolates accounted for 8 and 2% of the total culturable faecal population, which corresponded to 6 x 10 8 and 2 x 10 8 CFU per g, respectively. Ex-germ-free mice (Balb/C) were dosed with faecal material and then challenged with S. typhimurium . Protection against infection was monitored by enumerating the faecal levels of the pathogen. Human flora associated mice had high faecal levels of anaerobes (10 10 CFU per g). Faecal levels of enterics in these animals ranged from 10 4 to 10 8 CFU per g. Animals dosed with human faecal material gained protection against gastrointestinal colonisation by S. typhimurium . In these animals, the faecal levels of Salmonella ranged from 1 x 10 2 to 5 x 10 5 CFU per g, compared with the levels of greater than 5 x 10 6 CFU per g in animals not previously associated with the human flora. Human faecal material containing the highest levels of inhibitory isolates provided the best protection in mice, against colonisation by S. typhimurium . Keywords: colonization resistance, germ-free.

Ofloxacin and the gastrointestinal tract: a potential role in the treatment of bacterial enteritis

Ofloxacin is highly active against most pathogens causing bacterial enteritis. High faecal levels are achieved readily following a single oral dose and may persist for up to five days despite partial binding by faeces. In addition, adequate ofloxacin levels persist in pancreatic secretions and bile for 12 to 14 h following oral administration. Clinical data from various centres demonstrate a prompt response when ofloxacin is administered once-daily for shigellosis, salmonellosis and various other enteric pathogens. These theoretical observations and clinical data suggest a potential for once-daily oral ofloxacin therapy for bacterial diarrhoea.

Quinolones and the gastrointestinal tract.

A number of studies have evaluated the efficacy of the new fluoroquinolones for therapy of bacterial enteric diseases and for prevention of gram-negative sepsis in granulocytopenic patients. The success of the quinolones in these settings is related to several special features of these agents, including their spectrum of activity and high fecal levels, which are in turn reflected in their effect on the gastrointestinal flora. Other factors that are important, particularly for invasive disease such as typhoid fever and shigellosis, include good intracellular and bowel wall penetration, and lymph node and systemic drug concentrations many times higher than the MICs of the causative organisms. This article reviews the factors that contribute to the changes in fecal flora, and the results of clinical studies in patients with diarrhea, granulocytopenic patients, and patients with selected other infections of, or related to, the gastrointestinal tract.

Impact of fluoroquinolones on the gastrointestinal flora.

A number of studies have evaluated the effect of fluoroquinolones on fecal flora. The results of these studies have consistently shown marked suppression or elimination of gram-negative facultative bacteria. The effect on gram-positive bacteria is more variable, with slight to moderate suppression reported most frequently. Total fecal anaerobes have generally been unchanged, although some anaerobic species have been moderately suppressed. The effect on facultative gram-negative bacteria can be explained by high fecal levels of quinolones and very low MICs for most of the organisms. The general lack of effect of anaerobes may be due to the higher MICs and an inoculum effect on the MBCs that is caused by the large number of organisms (10(9)-10(11) colony-forming units/g) characteristically found in feces.