Fecal Suspension Research Articles

Hydrogen peroxide vapour treatment inactivates norovirus but has limited effect on post-treatment viral RNA levels

Background: Hydrogen peroxide vapour is used as a room disinfectant. Its activity against murine norovirus, a surrogate viability marker for human norovirus, indicates that it is also active against human norovirus.Aim: The aim of this study is to assess how this effect on viability is reflected in measurements of RNA by quantitative PCR (qPCR).Methods: Faeces suspensions of two human norovirus field strains, genogroup I and II and one cultured murine norovirus strain, (genogroup V) were dried on plastic plates, and underwent hydrogen peroxide vapour treatment or were mock treated. The influence of hydrogen peroxide on RNA was measured on genogroups I, II and V by qPCRs and for the cultivable murine norovirus also for viability by cell culture. Virucidal activity on murine norovirus was measured by endpoint titrations as the 50% tissue culture infectious dose, based both on cytopathic effect and on presence of replicating intracellular minus strand RNA.Results: The mean impact on the human norovirus qPCRs was 0.4 log10. The murine norovirus qPCR changed by 1.7 log10 but by an alternative qPCR only by 0.4 log10. These minor changes contrasted the 4.5-5.0 log10 murine norovirus viability reduction after treatment measured by cytopathic effect or intracellular negative-strand RNA.Conclusion: Inactivation of murine norovirus viability by hydrogen peroxide vapour was not reflected in qPCR levels. This finding might be extrapolated to the related human norovirus genogroups. We further found that cellular minus strand murine norovirus PCR was an observer-independent marker to study reduction of murine norovirus viability.

Plasma quercetin metabolites are affected by intestinal microbiota of human microbiota-associated mice fed with a quercetin-containing diet.

Protective effect of quercetin on high-fat diet-induced non-alcoholic fatty liver disease in mice has been reported. Recent research has revealed that several intestinal bacteria metabolize quercetin. We hypothesize that the difference in composition of intestinal microbiota affects quercetin absorption from the intestine. Germ-free BALB/cA female mice (18 weeks old) were randomly divided into four groups and orally administered with fecal suspension from four human individuals (HF1, HF2, HF3, HF4) to produce the human microbiota-associated mice. All mice were fed the 0.05% quercetin-containing pelleted diet for four weeks. Significant differences were observed in plasma total cholesterol and cecal microbiota among the four groups. Plasma quercetin concentration was significantly higher in the HF3 group than in the HF1 group. The plasma isorhamnetin/quercetin ratio showed significant negative correlation with visceral fat levels (r = −0.544, p = 0.013). Positive correlation was observed between the Log10 Enterobacteriaceae count and the plasma quercetin metabolites. Principal component analysis showed that all groups were distributed in different regions by using the correlation diagram with the second and third principal component. This study indicates that intestinal microbiota of human microbiota-associated mice inoculated with different fecal suspensions react to dietary quercetin in different ways.

Early intervention with faecal microbiota transplantation: an effective means to improve growth performance and the intestinal development of suckling piglets

Recent studies indicate that early postnatal period is a critical window for gut microbiota manipulation to optimise the immunity and body growth. This study investigated the effects of maternal faecal microbiota orally administered to neonatal piglets after birth on growth performance, selected microbial populations, intestinal permeability and the development of intestinal mucosal immune system. In total, 12 litters of crossbred newborn piglets were selected in this study. Litter size was standardised to 10 piglets. On day 1, 10 piglets in each litter were randomly allotted to the faecal microbiota transplantation (FMT) and control groups. Piglets in the FMT group were orally administrated with 2ml faecal suspension of their nursing sow per day from the age of 1 to 3 days; piglets in the control group were treated with the same dose of a placebo (0.1M potassium phosphate buffer containing 10% glycerol (vol/vol)) inoculant. The experiment lasted 21 days. On days 7, 14 and 21, plasma and faecal samples were collected for the analysis of growth-related hormones and cytokines in plasma and lipocalin-2, secretory immunoglobulin A (sIgA), selected microbiota and short-chain fatty acids (SCFAs) in faeces. Faecal microbiota transplantation increased the average daily gain of piglets during week 3 and the whole experiment period. Compared with the control group, the FMT group had increased concentrations of plasma growth hormone and IGF-1 on days 14 and 21. Faecal microbiota transplantation also reduced the incidence of diarrhoea during weeks 1 and 3 and plasma concentrations of zonulin, endotoxin and diamine oxidase activities in piglets on days 7 and 14. The populations of Lactobacillus spp. and Faecalibacterium prausnitzii and the concentrations of faecal and plasma acetate, butyrate and total SCFAs in FMT group were higher than those in the control group on day 21. Moreover, the FMT piglets have higher concentrations of plasma transforming growth factor-β and immunoglobulin G, and faecal sIgA than the control piglets on day 21. These findings indicate that early intervention with maternal faecal microbiota improves growth performance, decreases intestinal permeability, stimulates sIgA secretion, and modulates gut microbiota composition and metabolism in suckling piglets.

Modulation of Innate Immunity by G-CSF and Inflammatory Response by LBPK95A Improves the Outcome of Sepsis in a Rat Model.

Introduction Sepsis is the primary cause of death from infection. We wanted to improve the outcome of sepsis by stimulating innate immunity in combination with modulating the severity of inflammatory responses in rats. Method Sepsis was induced by the injection of feces suspension (control). A 5-day course of G-CSF treatment was given before the septic insult (G-CSF). The inflammatory response was decreased using various doses of the LPS-blocking peptide LBPK95A (5 mg/kg = 100% Combi group, 0.5 mg/kg = 10% Combi group, and 0.05 mg/kg = 1% Combi group). Survival rates were observed. Bacterial clearance, neutrophil infiltration, tissue damage, and the induction of hepatic and systemic inflammatory responses were determined 2 h and 12 h after the septic insult. Results High-dose LBPK95A (100% Combi) reduced the survival rate to 10%, whereas low-dose LBPK95A (10% and 1% Combi) increased the survival rates to 50% and 80%, respectively. The survival rates inversely correlated with multiorgan damage as indicated by the serum levels of ALT and urea. G-CSF treatment increased the white blood cell counts, hepatic neutrophil infiltration, and bacterial clearance in the liver, lung, and blood. The blockade of the LPS-LBP interaction decreased neutrophil infiltration, led to increased white blood cell count, and decreased hepatic neutrophil infiltration, irrespective of dose. However, bacterial clearance improved in the 1% and 10% Combi groups but worsened in the 100% Combi group. G-CSF increased TNF-α and IL-6 levels. Irrespective of dose, the blockade of the LPS-LBP interaction was associated with low systemic cytokine levels and delayed increases in hepatic TNF-α and IL-6 mRNA expression. The delayed increase in cytokines was associated with the phosphorylation of STAT3 and AKT. Conclusion Our results revealed that increasing innate immunity by G-CSF pretreatment and decreasing inflammatory responses using LBPK95A improved the survival rates in a rat sepsis model and could be a novel strategy to treat sepsis.

Establishment of porcine enterocyte/myofibroblast co-cultures for the growth of porcine rota- and coronaviruses

A stable culture of primary porcine enterocytes is necessary to study porcine enteric virus replication characteristics. Because the direct cultivation of primary porcine enterocytes is difficult, alternatives have to be considered. As subepithelial myofibroblasts secrete extracellular matrix and growth factors contributing to the attachment, proliferation and differentiation of epithelial cells, co-cultures of primary porcine enterocytes (ileocytes and colonocytes) with myofibroblasts were developed and evaluated for their susceptibility to enteric viruses. First, it was demonstrated that the co-cultured ileocytes and colonocytes were susceptible to an archival rotavirus strain RVA/pig-tc/BEL/RV277/1977/G1P[7] and different other rotavirus genotypes (fecal samples containing G5P[7], G5P[13], G9P[23], G4P[6]). Next, the TGEV Purdue strain infected both ileocytes and colonocytes whereas the Miller strain only infected ileocytes. Last, the PEDV CV777 Vero adapted and non-adapted (fecal suspension) strains could infect co-cultured ileocytes but not colonocytes. The infectivity of the CV777 Vero adapted strain was higher when the cells were cultured without fetal bovine serum and the CV777 fecal suspension only infected the ileocytes cultured without fetal bovine serum. In conclusion, a novel co-culture of porcine enterocytes with myofibroblasts was established, which can be used for the investigation of the replication of enteric viruses.

Successful transplantation of guinea pig gut microbiota in mice and its effect on pneumonic plague sensitivity.

Microbiota-driven variations in the inflammatory response are predicted to regulate host responses to infection. Increasing evidence indicates that the gastrointestinal and respiratory tracts have an intimate relationship with each other. Gut microbiota can influence lung immunity whereby gut-derived injurious factors can reach the lungs and systemic circulation via the intestinal lymphatics. The intestinal microbiota’s ability to resist colonization can be extended to systemic infections or to pathogens infecting distant sites such as the lungs. Unlike the situation with large mammals, the microtus Yersinia pestis 201 strain exhibits strong virulence in mice, but nearly no virulence to large mammals (such as guinea pigs). Hence, to assess whether the intestinal microbiota from guinea pigs was able to affect the sensitivity of mice to challenge infection with the Y. pestis 201 strain, we fed mice with guinea pig diets for two months, after which they were administered 0.5 ml of guinea pig fecal suspension for 30 days by oral gavage. The stools from each mouse were collected on days 0, 15, and 30, DNA was extracted from them, and 16S rRNA sequencing was performed to assess the diversity and composition of the gut microbiota. We found that the intestinal microbiota transplants from the guinea pigs were able to colonize the mouse intestines. The mice were then infected with Yersinia pestis 201 by lung invasion, but no statistical difference was found in the survival rates of the mice that were colonized with the guinea pig’s gut microbiota and the control mice. This indicates that the intestinal microbiota transplantation from the guinea pigs did not affect the sensitivity of the mice to pneumonic plague.

Gut microbiota-mediated protection against influenza virus subtype H9N2 in chickens is associated with modulation of the innate responses

Commensal gut microbiota plays an important role in health and disease. The current study was designed to assess the role of gut microbiota of chickens in the initiation of antiviral responses against avian influenza virus. Day-old layer chickens received a cocktail of antibiotics for 12 (ABX-D12) or 16 (ABX-D16) days to deplete their gut microbiota, followed by treatment of chickens from ABX-12 with five Lactobacillus species combination (PROB), fecal microbial transplant suspension (FMT) or sham treatment daily for four days. At day 17 of age, chickens were challenged with H9N2 virus. Cloacal virus shedding, and interferon (IFN)-α, IFN-β and interleukin (IL)-22 expression in the trachea, lung, ileum and cecal tonsils was assessed. Higher virus shedding, and compromised type I IFNs and IL-22 expression was observed in ABX-D16 chickens compared to control, while PROB and FMT showed reduced virus shedding and restored IL-22 expression to levels comparable with undepleted chickens. In conclusion, commensal gut microbiota of chickens can modulate innate responses to influenza virus subtype H9N2 infection in chickens, and modulating the composition of the microbiome using probiotics- and/or FMT-based interventions might serve to promote a healthy community that confers protection against influenza virus infection in chickens.

Role of moxifloxacin-dexamethasone in cardiac histomorphometric findings among Wistar rats from infected mothers.

To investigate cardiac changes in young rats, whose mothers underwent autogenic fecal peritonitis, during organogenesis phase and to evaluate the role of intravenous administration of moxifloxacin and dexamethasone in preventing infection-related cardiac changes. A prospective histomorphometric study was performed on 29 hearts of Wistar four-month old rats. Animals were divided into three groups: Negative Control Group (NCG) included 9 subjects from healthy mothers; Positive Control Group (PCG) included 10 subjects from mothers with fecal peritonitis (intra-abdominal injection of 10% autogenic fecal suspension in the gestational period) and did not receive any treatment; and Intervention Group (IG), with 10 animals whose infected mothers received moxifloxacin and dexamethasone treatment 24 hours after induction of fecal peritonitis. Nuclear count was higher in the IG group as compared to PCG (p = 0.0016) and in NCG as compared to PCG (p = 0.0380). There was no significant difference in nuclear counts between NCG and IG. Induced autogenic fecal peritonitis in pregnant Wistar rats determined myocardial changes in young rats that could be avoided by the early administration of intravenous moxifloxacin and dexamethasone.

Standardized Preparation for Fecal Microbiota Transplantation in Pigs.

The intestine of pigs harbors a mass of microorganisms which are essential for intestinal homeostasis and host health. Intestinal microbial disorders induce enteric inflammation and metabolic dysfunction, thereby causing adverse effects on the growth and health of pigs. In the human medicine, fecal microbiota transplantation (FMT), which engrafts the fecal microbiota from a healthy donor into a patient recipient, has shown efficacy in intestinal microbiota restoration. In addition, it has been used widely in therapy for human gastrointestinal diseases, including Clostridium difficile infection, inflammatory bowel diseases, and irritable bowel syndrome. Given that pigs share many similarities with humans, in terms of anatomy, nutritional physiology, and intestinal microbial compositions, FMT may also be used to restore the normal intestinal microbiota of pigs. However, feasible procedures for performing FMT in pigs remains unclear. Here, we summarize a standardized preparation for FMT in pigs by combining the standard methodology for human FMT with pig production. The key issues include the donor selection, fecal material preparation, fecal material transfer, stool bank establishment, and the safety for porcine FMT. Optimal donors should be selected to ensure the efficacy of porcine FMT and reduce the risks of transmitting infectious diseases to recipients during FMT. Preparing for fresh fecal material is highly recommended. Alternatively, frozen fecal suspension can also be prepared as an optimal choice because it is convenient and has similar efficacy. Oral administration of fecal suspension could be an optimal method for porcine fecal material transfer. Furthermore, the dilution ratio of fecal materials and the frequency of fecal material transfer could be adjusted according to practical situations in the pig industry. To meet the potential large-scale requirement in the pig industry, it is important to establish a stool bank to make porcine FMT readily available. Future studies should also focus on providing more robust safety data on FMT to improve the safety and tolerability of the recipient pigs. This standardized preparation for porcine FMT can facilitate the development of microbial targeted therapies and improve the intestinal health of pigs.

Recovery of Nonpolio Enteroviruses from L20B Cell Line with Non-reproducible Cytopathic Effect

Samples showing cytopathology (CPE) on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in L20B or RD are considered negative for both poliovirus and nonpolio enteroviruses (NPEVs). The phenomenon is termed non-reproducible CPE. Its occurrence is usually ascribed to the likely presence of reoviruses, adenoviruses and other non-enteroviruses. This study aimed to investigate the likelihood that NPEVs are also present in cases with non-reproducible CPE. Twenty-six (26) cell culture suspensions were analyzed in this study. The suspensions were collected from the WHO National Polio Laboratory, Department of Virology, College of Medicine, University of Ibadan. The suspensions emanated from 13 L20B cell culture tubes that showed cytopathology within 5 days of inoculation with fecal suspension from AFP cases. However, on passage into one each of RD and L20B cell lines, the CPE was not reproducible. All samples were subjected to RNA extraction, cDNA synthesis, the WHO recommended VP1 RT-seminestedPCR assay, species resolution PCR assay, sequencing and phylogenetic analysis. Six (6) samples were positive for the VP1 RT-seminested PCR assay. Only four of which were positive by the species resolution PCR assay. The four amplicons were sequenced, however, only three (3) were successfully identified as Coxsackievirus A20 (2 isolates) and Echovirus 29 (1 isolate). The results of this study unambiguously showed the presence of NPEVs (particularly CVA20 and E29) in cell culture supernatants of samples with CPE on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in RD cell line. Therefore, like reoviruses, adenoviruses and other non-enteroviruses, NPEVs can also be recovered in cases with non-reproducible CPE.

Investigation of Enteric Viruses in the Feces of Neotropical Migratory Birds Captured on the Coast of the State of Pará, Brazil

ABSTRACT Migratory birds can become long-distance vectors for a wide range of microorganisms and can cause human disease, being the Brazilian coast a gateway for northern migratory birds. These animals are considered natural reservoirs of different viruses that cause important diseases, being relevant research of viral pathogens in migratory birds to epidemiology surveillance. The objective of the study was to investigate the presence of avian rotavirus (AvRV), avian reovirus (ARV) and picobirnavirus (PBV) in Neotropical migratory birds captured on the coast of Brazil. A total of 23 individual fecal samples of the migratory birds species Calidris pusilla (20 birds), Numenius phaeopus (1 bird) and Charadrius semipalmatus (2 birds) were collected. Fecal suspensions were prepared from the collected samples for subsequent extraction of double-stranded RNA (dsRNA), which was subjected to polyacrylamide gel electrophoresis (PAGE) and reverse transcription polymerase chain reaction (RT-PCR). The electrophoretic profiles were not detected by PAGE, and the amplification for the studied viruses PBV, ARV and AvRV (specie D, gene VP6 and NSP4) were negative. Positivity for AvRVD, VP7 gene was of 4.35% (1/23) for the migratory bird Calidris pusilla. After sequencing and building the tree of phylogenetic relationships avian Rotavirus Group D identified in this study was phylogenetically related and grouped into one branch, together to previously reported AvRVD from Brazil in chicken flocks with 99.8% nucleotide and 100% amino acid similarities.

Changes in blood parameters in the treatment of septic complications of diabetes with the use of ozone therapy

Objective. To study the influence of ozonotherapy on the course of purulent-inflammatory processes in diabetes mellitus in an experiment.Material and methods. Experimental studies were performed on 30 white old rats. The first group, which was administered subcutaneous fecal suspension (10 rats), the second group-fecal suspension and diabetes mellitus (10), the third group — simulated diabetes mellitus, fecal suspension and application of ozone (10). Diabetes mellitus was modeled by subcutaneous administration of alloxan (100 mg / kg body weight). Subcutaneously sterile 0.9% sodium chloride solution (1.0 ml per 100 g body weight) was injected to the control group (10). On the 14th day after administration of alloxan, animals were subcutaneously administered 10% fecal suspension (a fecal pool of 20 animals in 0.9% sodium chloride solution) at a dose of 0.5 ml per 100 g body weight. Rats of the first experimental group received subcutaneous administration of fecal suspension. Animals of the third group of the fecal mixture were subcutaneously injected and subsequently for three days, once a day, intraperitoneally administered 1.0 ml per 100 g of body weight of the animal once a day for 3 days, an ozonized 0.9% solution of sodium chloride was administered.Results. Application of ozonotherapy in the presence of soft tissue ulcers on the background of diabetes in the old rats was not accompanied by a significant improvement in the parameters of regulation of the aggregate state of blood. An increase in the manifestations of hypercoagulability was found: a decrease in the time of plasma recalcification, activated partial thromboplastin time, prothrombin time,thrombin time, Hageman dependent fibrinolysis, potential activity of plasminogen. The introduction of ozonized sodium chloride solution in purulent soft tissue processes against the background of diabetes in the old rats did not contribute to the tread properties, as indicated by the growth of lysis of azo-albumin, azocasein, total fibrinolytic activity, and Kunitz's protein activity in plasma. Ozonotherapy contributed to an increase in the degree of intoxication, in the presence of abscesses in diabetes in the old rats, as indicated by the increase in the concentration of medium molecules. Such changes in old rats can be considered as an aggravation of purulent-inflammatory process, an increase in intoxication, a decrease in tread properties, an increase in manifestations of hypercoagulation in the background of chronic CSD, caused by age-related changes. Conclusions. Application of ozonotherapy in conditions of purulent-inflammatory processes in diabetes mellitus in old rats does not show tread properties on hemostatic system and blood plasma proteolysis. Ozone therapy enhances manifestations of intoxication on the background of diabetes mellitus with suppurative and inflammatory proc treatmentesses in old rats, which causes the need for a restrained use of this method of treatment.

Особливості імунного профілю у крові щурів з гострим поширеним перитонітом на тлі гіпотиреозу

Introduction. Peritonitis remains one of the most common complications of surgical diseases of the abdominal cavity [5, 11]. According to individual authors, in severe forms of disseminated purulent peritonitis, the mortality reaches 50–80 % [3, 13, 15]. All this points to the need for further improvement of the methods of diagnosis and treatment of this pathological process.The aim of the study – establishment of features of the immune system cellular and humoral units functioning in rats with acute widespread peritonitis on the background of experimental hypothyroidism.Materials and Methods. White Wistar rats were used in the work. Hypothyroidism was modeled by the administration of Mercazolil at a dose of 25 mg / kg for 21 days. Acute common peritonitis was modeled by injecting 0.5 ml of 10 % filtered fecal suspension into the abdominal cavity of the animals under study. Cellular (CD4+, CD8+, CD22+-lymphocyte) and humoral (IgA, IgM, IgG) immunity were measured. The animals were decapitated under thiopental anesthesia after 24 hours, on the 4th, 7th and 10th days from the beginning of peritonitis modeling.Results and Discussion. It was found that in animals with acute widespread peritonitis against hypothyroidism, there is a significant decrease in CD4+ lymphocytes, a violation of the ratio of the main subpopulations of lymphocytes (CD4+ and CD8+), which is accompanied by a significant decrease in the immunoregulatory index. The dynamics of changes in B-lymphocytes CD22+, which are producers of immunoglobulins, was indicative. These changes correlated with the state of specific humoral immunity – the concentration of immunoglobulins in the serum of the corresponding groups of animals.When simulating common peritonitis, euthyroid animals showed a decrease in IgG concentration and an increase in IgM and IgA. Against the background of hypothyroidism, we noted a decrease in the level of immunoglobulins of all classes after 24 hours of the duration of the pathological process with a further decrease to the tenth day. The indices of all classes of immunoglobulins were also significantly lower than those of euthyroid animals with acute widespread peritonitis.Conclusions. In animals with acute common peritonitis, hypothyroidism is significantly reduced CD4+ lymphocytes, a violation of the ratio of the main subpopulations of lymphocytes (CD4+ and CD8+), which is accompanied by a significant decrease in the immunoregulatory index. The insufficiency of the cellular part of the immune response was found to reflect an increase in the mutual negative effect of the inflammatory process in the peritoneum and systemic disorders of immune defense in conditions of concomitant hypothyroidism. There is a direct relationship between the reduced level of CD22+ lymphocytes and the indices of immunoglobulins of classes A and G.In acute widespread peritonitis against the background of mercazolil-induced hypothyroidism, there is a violation of the functionality of the immune system, manifested by an imbalance of immunoglobulins in the bloodstream and a decrease in the resistance of the humoral link of the immune system.

Exogenous Fecal Microbiota Transplantation from Local Adult Pigs to Crossbred Newborn Piglets.

This study was conducted to investigate the effect of exogenous fecal microbiota transplantation on gut bacterial community structure, gut barrier and growth performance in recipient piglets. Twelve litters of Duroc × Landrace × Yorkshire piglets of the same birth and parity were weighed and divided into two groups. One group (recipient piglets) was inoculated orally with fecal microbiota suspension of healthy adult Jinhua pigs daily from day 1 to day 11. The other (control) was given orally the same volume of sterile physiological saline at the same time. The experiment lasted 27 days. The results showed that the relative abundance of Firmicutes, Prevotellaceae, Lachnospiraceae, Ruminococcus, Prevotella, and Oscillospira in the colon of recipient piglets was increased. Proteobacteria, Fusobacteriaceae, Clostridiaceae, Pasteuriaceae, Alcaligenaceae, Bacteroidaceae, Veillonellaceae, Sutterella, Escherichia, and Bacteroides in the colon of recipient piglets were decreased. An average daily weight gain of recipient piglets was increased, and diarrhea incidence of the recipient was decreased during the trial. Intestinal morphology and tight junction barrier of recipient piglets were improved. The optical density of sIgA+ cells, the number of goblet cells and relative expressions of MUC2 in the intestinal mucosa of recipient piglets were enhanced. Protein expressions of β-defensin 2 and mRNA expressions of TLR2 and TLR4 in the intestinal mucosa of recipient piglets were also increased. These findings supported that the exogenous fecal microbiota had significant effects on animal’s growth performance, intestinal barrier function, and innate immune via modulating the composition of the gut microbiota.

An in vitro study to assess the impact of tetracycline on the human intestinal microbiome

The human intestinal microbiome, a generally stable ecosystem, could be potentially altered by the ingestion of antimicrobial drug residues in foods derived from animals. Data and the scientific published literature on the effects of antimicrobial residues on the human intestinal microbiome are reviewed by national regulatory authorities as part of the human food safety evaluation of veterinary antimicrobial agents used in food-producing animals. In this study, we determined if tetracycline, at low residue concentrations, could impact the human intestinal microbiome structure and the resistance-gene profile, following acute and subchronic exposure. The effects of 0.15, 1.5, 15, and 150 μg/ml of tetracycline, after 24 h and 40 days of exposure, in 3% human fecal suspensions, collected from three individuals (A, B, and C) were investigated using in vitro batch cultures. Results were variable, with either no change or minor changes in total bacterial 16S rRNA gene copies after exposure of fecal samples to tetracycline, because of the inter-individual variation of human gastrointestinal tract microbiota. Bacterial community analysis using rRNA-based pyrosequencing revealed that Firmicutes and Bacteroidetes were the predominant phyla in the three fecal samples; the ratio of phylotypes varied among individuals. The evaluation of bacterial community changes at the genus level, from control to tetracycline-treated fecal samples, suggested that tetracycline under the conditions of this study could lead to slight differences in the composition of intestinal microbiota. The genus Bacteroides (of the Bacteroidetes) was consistently altered from 1.68 to 5.70% and 4.82–8.22% at tetracycline concentrations of 0.15 μg/ml or above at both time points for individual A, respectively, and increased 5.13–13.50% and 10.92–22.18% for individual B, respectively. Clostridium family XI increased 3.50–25.34% in the presence of tetracycline at 40 days for individual C. Principal Component Analysis (PCA) confirmed the pyrosequencing findings of inter-individual variability of the ratio of phylotypes and the effect of tetracycline. Among the 23 tetracycline resistance genes (TRGs) screened, four tet genes (tetO, Q, W, and X) were major TRGs in control and tetracycline-dosed fecal samples. A variable to slight increase of copy number of TRGs appeared to be related to tetracycline treatment, interindividual variability and duration of exposure. Despite, the inherent variability of the intestinal microbiota observed among or within individuals, this pilot study contributes to the knowledge base of the impact of low residue concentrations of tetracycline on the human intestinal microbiome on the potential for antimicrobial resistance.

A Combined LC-MS Metabolomics- and 16S rRNA Sequencing Platform to Assess Interactions between Herbal Medicinal Products and Human Gut Bacteria in Vitro: a Pilot Study on Willow Bark Extract.

Herbal preparations are complex mixtures of natural products, many of which are able to reach the distal gut due to low oral bioavailability. There, they can influence the microbial communities, and can be metabolized into potentially absorbable bioactive compounds by the intestinal bacteria. This aspect has often been disregarded when searching for the active principles of medicinal plants and herbal medicinal products. The aim of this study was to establish an interdisciplinary platform to unravel interactions of herbal medicine and intestinal microbiota, using a combined LC-MS metabolomics and 16S rRNA microbiome sequencing approach. Willow bark extract (WBE), a herbal medicinal product with a long history of traditional use and a well-established anti-inflammatory activity, was incubated with human fecal suspension under anoxic conditions. Samples were taken after 0.5, 4, and 24 h of incubation. Microbiome analyses revealed that incubation with WBE had a marked effect on microbial community composition and functions. For example, the proportion of Bacteroides sp. was clearly enhanced when the fecal sample used in this study was incubated with WBE. LC-MS analysis showed that WBE constituents were readily metabolized by fecal bacteria. Numerous microbial metabolites could be annotated, allowing the construction of putative microbial degradation pathways for the main groups of WBE constituents. We suggest that studies of this type help to increase the knowledge on bioactive principles of medicinal plants, since gut microbial metabolites might have been underestimated as a source of bioactive compounds in the past.

Quercetin metabolism by fecal microbiota from healthy elderly human subjects.

Quercetin is a polyphenol found in food that has numerous health benefits. This study investigated the relationship between quercetin metabolism, gut microbiota composition, and dietary intake in elderly Japanese subjects. A food frequency questionnaire was used to assess dietary intake during the week prior to stool sample collection. Fecal suspensions from 56 subjects were anaerobically incubated with quercetin and fecal microbiota composition was analyzed by next-generation sequencing. Inter-individual variations in quercetin concentration and fecal microbiota composition at family level suggested differences in microbial quercetin metabolism. The abundance of Sutterellaceae (r = −0.292) and Oscillospiraceae (r = −0.334) was negatively correlated whereas that of Fusobacteriaceae (r = 0.361) and Enterobacteriaceae (r = 0.321) was positively correlated with quercetin concentration. Niacin (r = −0.313), vitamin B6 (r = −0.297), vitamin B12 (r = −0.266), vitamin D (r = −0.301), and ratio of animal protein to total protein (r = −0.27) were also negatively correlated with quercetin concentration. Bacterial abundance was positively or negatively related to intake of food components. This is the first report describing the relationship between fecal quercetin metabolism, human microbiota, and dietary intake in the elderly.

Impact of prenatal antimicrobial treatment on fetal brain damage due to autogenous fecal peritonitis in Wistar rats: A Histomorphometric Study

Purpose: To investigate brain neuronal density in newborn rats whose mothers were subjected to fecal peritonitis and compare findings between rats born to mothers treated and not treated with antimicrobials.
 Methods: Peritonitis was induced with a 10% fecal suspension (4mL/kg) in 2 pregnant rats. Of these, 1 received antimicrobial treatment 24 hours after peritonitis induction: moxifloxacin and dexamethasone plus 2 mL of the inner bark of the Schinus terebinthifolius raddi extract. One pregnant rat underwent no intervention and served as a control.
 Results: The newborn brains of rats born to mothers with fecal peritonitis were significantly smaller and of less firm consistency. Brain neuronal density was lower in the untreated group than in the control and treated groups (P<0.01).
 Conclusions: Untreated peritonitis caused brain damage in the offspring, which was averted by effective early antimicrobial treatment. This approach may provide an early avenue for translation of such therapy in humans.
 Keywords: peritonitis, brain injuries, rats

Синдром ендогенної інтоксикації в щурів із гострим поширеним перитонітом на тлі мерказоліл-індукованого гіпотиреозу

Introduction. The growth of endogenous toxicosis occurs when the admission intensity of toxic metabolic products increased sharply, or when the activity of antitoxic systems or elimination of toxic metabolites inhibits. The syndrome of endogenous intoxication develops at various pathological conditions, when the number of natural waste products of the body, which appeared in large quantities in biological environments, as well as aggressive components exceeds the capacity of biotransformation. The aim of the study – to learn the effect of reduced production of thyroid hormones on the indices of endogenous intoxication of rats with acute widespread peritonitis.Research Methods. The study used white Wistar rats. Hypothyroidism was simulated by the administration of Mercazolil at a dose of 25 mg / kg for 21 days. Acute widespread peritonitis was simulated by injecting 0.5 ml of 10 % filtered fecal suspension into the abdominal cavity of the studied animals. The determination of molecules of average weight, erythrocyte and leukocyte indices of intoxication, level of circulating immune complexes and catepsin D was carried out.Results and Discussion. It was established that the presence of hypothyroidism in experimental animals to which was simulated acute widespread faecal peritonitis was accompanied by, more expressed than in euthyroid rats, increase in endotoxicosis – medium molecular weight peptides, erythrocyte and leukocyte indices of intoxication, circulating immune complexes and worse dynamics of their normalization. Phase changes in cathepsin D activity were observed.Conclusions. Deficiency of iodine-containing thyroid hormones is accompanied by a reliable increase in the indices characterizing the syndrome of endogenous intoxication in comparison with the euthyroid animals. This may be one of the reasons for the development of multiple organ failure in these conditions.

Особливості кисень-залежних процесів у щурів з гострим поширеним перитонітом на тлі мерказоліл-індукованого гіпотиреозу

Мета роботи: дослідження впливу зниженої продукції тиреоїдних гормонів на стан кисень-залежних процесів.Матеріали і методи. У роботі використовували білих щурів лінії Вістар. Гіпотиреоз моделювали уведенням мерказолілу в дозі 25 мг/кг протягом 21-ї доби. Гострий поширений перитоніт моделювали уведенням 0,5 мл 10 % профільтрованої калової суспензії в черевну порожнину досліджуваних тварин. Визначали співвідношення фагоцитуючих клітин у місці ураження, продукування активних форм кисню системою фагоцитуючих мононуклеарів, спонтанний та індукований тест із нітросинім тетразолієм (НСТ-тест).Результати досліджень та їх обговорення. Проведеними дослідженнями встановлено, що наявність гіпотиреозу в експериментальних тварин, яким моделювали гострий поширений каловий перитоніт, супроводжувалась порушенням співвідношення кількості фагоцитуючих клітин у місці ушкодження, менш вираженим, ніж в еутиреоїдних щурів, зростанням активних форм кисню, а також показників спонтанного та індукованого НСТ-тесту і, як наслідок, зменшенням інтенсивності продукування ними активних форм кисню. За умов гіпотиреозу знижується кисень-залежна біоцидність нейтрофілів та зменшуються функціональні резерви фагоцитуючих клітин.