Effect of a Polyphenol-Based Additive in Pig Diets in the Early Stages of Growth.

The weaning period is a stressful period for the gastrointestinal tract (GIT) of piglets. This work aims to evaluate the effects of the commercial polyphenol-based product GreenFIS® on: (1) GIT health and performance of 60 weaned piglets; (2) digestibility in 18 growing pigs. Three diets were tested: a control diet (C), C plus 2.5 g of GreenFIS®/kg C (T1), and C plus 5 g of GreenFIS®/kg C (T2). After the post-weaning trial three piglets per treatment were sacrificed for the GIT histological analysis. No differences between diets were recorded in terms of growing performance or clinical and biochemical blood parameters. The GIT histological analysis did not show any indicators of inflammation for any of the groups. The feces of the two extreme treatments (C and T2) were analyzed for microbiota, revealing a greater presence of the Ruminococcus bromii group, positively associated with starch degradation, in T2. In the second experiment six pigs per treatment were randomly chosen for the digestibility study. The inclusion of GreenFIS® at both levels led to a higher fecal digestibility of gross energy (86.2%, 89.1%, and 89.5%, for C, T1, and T2, respectively) and crude protein (87.0%, 90.2%, and 90.0%). In conclusion, the additive did not improve, in the excellent experimental hygienic conditions, the gut health, but it did increase nutrient digestibility.

Adaptation of Microbial Communities to Environmental Arsenic and Selection of Arsenite-Oxidizing Bacteria From Contaminated Groundwaters.

Arsenic mobilization in groundwater systems is driven by a variety of functionally diverse microorganisms and complex interconnections between different physicochemical factors. In order to unravel this great ecosystem complexity, groundwaters with varying background concentrations and speciation of arsenic were considered in the Po Plain (Northern Italy), one of the most populated areas in Europe affected by metalloid contamination. High-throughput Illumina 16S rRNA gene sequencing, CARD-FISH and enrichment of arsenic-transforming consortia showed that among the analyzed groundwaters, diverse microbial communities were present, both in terms of diversity and functionality. Oxidized inorganic arsenic [arsenite, As(III)] was the main driver that shaped each community. Several uncharacterized members of the genus Pseudomonas, putatively involved in metalloid transformation, were revealed in situ in the most contaminated samples. With a cultivation approach, arsenic metabolisms potentially active at the site were evidenced. In chemolithoautotrophic conditions, As(III) oxidation rate linearly correlated to As(III) concentration measured at the parental sites, suggesting that local As(III) concentration was a relevant factor that selected for As(III)-oxidizing bacterial populations. In view of the exploitation of these As(III)-oxidizing consortia in biotechnology-based arsenic bioremediation actions, these results suggest that contaminated aquifers in Northern Italy host unexplored microbial populations that provide essential ecosystem services.

Structure/activity virtual screening and in vitro testing of small molecule inhibitors of 8-hydroxy-5-deazaflavin:NADPH oxidoreductase from gut methanogenic bacteria.

Virtual screening techniques and in vitro binding/inhibitory assays were used to search within a set of more than 8,000 naturally occurring small ligands for candidate inhibitors of 8-hydroxy-5-deazaflavin:NADPH oxidoreductase (FNO) from Methanobrevibacter smithii, the enzyme that catalyses the bidirectional electron transfer between NADP+ and F420H2 during the intestinal production of CH4 from CO2. In silico screening using molecular docking classified the ligand-enzyme complexes in the range between - 4.9 and - 10.5 kcal/mol. Molecular flexibility, the number of H-bond acceptors and donors, the extent of hydrophobic interactions, and the exposure to the solvent were the major discriminants in determining the affinity of the ligands for FNO. In vitro studies on a group of these ligands selected from the most populated/representative clusters provided quantitative kinetic, equilibrium, and structural information on ligands' behaviour, in optimal agreement with the predictive computational results.

Impacts of dietary silver nanoparticles and probiotic administration on the microbiota of an in-vitro gut model.

Ingestion of silver nanoparticles (AgNPs) is inevitable linked to their widespread use in food, medicines and other consumer products. However, their effects on human microbiota at non-lethal concentrations remain poorly understood. In this study, the interactions among 1 µg mL-1 AgNPs, the intestinal microbiota, and the probiotic Bacillus subtilis (BS) were tested using in-vitro batch fermentation models inoculated with human fecal matter. Results from metagenomic investigations revealed that the core bacterial community was not affected by the exposure of AgNPs and BS at the later stage of fermentation, while the proportions of rare species changed drastically with the treatments. Furthermore, shifts in the Firmicutes/Bacteriodetes (F/B) ratios were observed after 24 h with an increase in the relative abundance of Firmicutes species and a decrease in Bacteroidetes in all fermentation cultures. The co-exposure to AgNPs and BS led to the lowest F/B ratio. Fluorescent in-situ hybridization analyses indicated that non-lethal concentration of AgNPs negatively affected the relative percentage of Faecalibacterium prausnitzii and Clostridium coccoides/Eubacterium rectales taxa in the fermentation cultures after 24 h. However, exposure to single and combined treatments of AgNPs and BS did not change the overall diversity of the fecal microflora. Functional differences in cell motility, translation, transport, and xenobiotics degradation occurred in AgNPs-treated fermentation cultures but not in AgNPs+BS-treated samples. Compared to the control samples, treated fecal cultures showed no significant statistical differences in terms of short-chain fatty acids profiles, cytotoxic and genotoxic effects on Caco-2 cell monolayers. Overall, AgNPs did not affect the composition and diversity of the core fecal microflora and its metabolic and toxic profiles. This work indicated a chemopreventive role of probiotic on fecal microflora against AgNPs, which were shown by the decrease of F/B ratio and the unaltered state of some key metabolic pathways.

Effects of sublethal concentrations of silver nanoparticles on Escherichia coli and Bacillus subtilis under aerobic and anaerobic conditions.

The present work is aimed at comparing the effects of sublethal concentrations of silver nanoparticles (AgNPs) on the growth kinetic, adhesion ability, oxidative stress, and phenotypic changes of model bacteria (Escherichia coli and Bacillus subtilis) under both aerobic and anaerobic conditions. Growth kinetic tests conducted in 96-well microtiter plates revealed that sublethal concentrations of AgNPs do not affect E. coli growth, whereas 1 µg/ml AgNPs increased B. subtilis growth rate under aerobic conditions. At the same concentration, AgNPs promoted B. subtilis adhesion, while it discouraged E. coli attachment to the surface in the presence of oxygen. As determined by 2,7-dichlorofluorescein-diacetate assays, AgNPs increased the formation of intracellular reactive oxygen species, but not at the highest concentrations, suggesting the activation of scavenging systems. Finally, motility assays revealed that 0.01 and 1 µg/ml AgNPs, respectively, promoted surface movement in E. coli and B. subtilis under aerobic and anaerobic conditions. The results demonstrate that E. coli and B. subtilis react differently from AgNPs over a wide range of sublethal concentrations examined under both aerobic and anaerobic conditions. These findings will help elucidate the behavior and impact of engineered nanoparticles on microbial ecosystems.

Effects of Cocoa Husk Feeding on the Composition of Swine Intestinal Microbiota.

A two-diet/two-period change over experiment was performed to investigate the effects of cocoa husks, as a source of dietary fiber and polyphenols, on pig intestinal microbial composition. Six pigs were fed a conventional cereal-based diet or a diet obtained by substitution of 7.5% of the conventional diet with cocoa husks for 3 weeks. Experimental diets were isoproteic and isoenergetic. At the end of each 3 week testing period, samples of fresh feces were collected and analyzed for microbial composition by fluorescence in situ hybridization. Cocoa husks did not affect feed intake, weight gain, and feed efficiency. Analysis of fecal microbial populations, grouped by phyla, showed a decrease of Firmicutes and an increase of Bacteroidetes in cocoa husk-fed pigs. Particularly, cocoa husks reduced fecal populations of the Lactobacillus-Enterococcus group and Clostridium histolyticum and increased the Bacteroides-Prevotella group and Faecalibacterium prausnitzii, suggesting a potential for cocoa husks in the improvement of intestinal microbial balance.

Rhizobacterial communities associated with spontaneous plant species in long-term arsenic contaminated soils.

The microbial community composition in three soil fractions (bulk soil, rhizosphere and rhizoplane) of the root-soil system of a thistle, Cirsium arvense, and of a tufted hair grass, Deschampsia caespitosa, was investigated. The two spontaneous wild plant species were predominant in two Italian lands contaminated since centuries by arsenic and at present show high levels of arsenic (from 215 to 12,500 mg kg(-1)). In order to better understand how the rhizobacterial ecosystem responds to a long-term arsenic contamination in term of composition and functioning, culture-independent techniques (DAPI counts, fluorescence in situ hybridization and denaturing gradient gel electrophoresis analysis) along with cultivation-based methods were applied. Microbial community structure was qualitatively similar in the two root-soil systems, but some quantitative differences were observed. Bacteria of the α-, ß-, and γ-subclasses of the Proteobacteria were dominant in all fractions, while the subdominant groups (Cytophagaceae, gram-positive spore-forming, and filamentous bacteria) were significantly more abundant in the root-soil system of D. caespitosa. As regards to arsenic resistant strains, Firmicutes, Actinobacteria, Enterobacteria and γ-Proteobacteria were isolated from soil system of both plants. Our results suggest that the response to a high level of arsenic contamination governed the rhizosphere microbial community structure together with the soil structure and the plant host type effects. Data from this study can provide better understanding of complex bacterial communities in metal-polluted soils, as well as useful information of indigenous bacterial strains with potential application to soil remediation.

Effects of a selection of additives on in vitro ruminal methanogenesis and in situ and in vivo†NDF digestibility.

The effects of 18 essential oils, yeast, Quebracho tannin and Quillaja saponin on ruminal methane and gas production (GP) were studied in vitro. A lactating cow diet was incubated with rumen inoculum. Doses of the additives (mg/L) were: essential oils = 500; tannin = 67; yeast = 8.35 and 16.7; and saponin = 300. Lemongrass, estragole, eugenol, geraniol, limonen, thyme oil and thymol produced less gas (overall mean 33.8 mL/200 mg dry matter (DM)) than control (43.6 mL/200 mg DM; P < 0.001). Methane produced (mL/200 mg DM) by guaiacol (10.7), lemongrass (9.6), limonene (11.4), thyme oil (10.9) and thymol (2.1) was lower than control (12.5) (P < 0.001). Methane percentage on total GP was lower (P < 0.001) for cinnamic alcohol (25.4), guaiacol (24.5), thymol (19.7) and vanillin (26.3) than control (28.8). In a second experiment, thymol, guaiacol and yeast were added to the diet of dry fistulated cows to determine in situ neutral detergent fiber digestibility (NDFD) of six forages and in vivo dietary NDFD. Thymol and yeast decreased in situ NDFD after 24 h (33.9% and 33.5% vs. 38.1%; P = 0.008). Thymol decreased in vivo total tract NDFD (40.8% vs. 51.4%; P = 0.02). Differences in GP and methane levels were registered within classes of additives. A careful selection of additives may allow for the manipulation of ruminal fermentation.

Arsenite oxidation in Ancylobacter dichloromethanicus As3-1b strain: detection of genes involved in arsenite oxidation and CO2 fixation.

The aim of this study was to characterize a facultative chemolithotrophic arsenite-oxidizing bacterium by evaluating the growth and the rate of arsenite oxidation and to investigate the genetic determinants for arsenic resistance and CO(2) fixation. The strain under study, Ancylobacter dichloromethanicus As3-1b, in a minimal medium containing 3 mM of arsenite as electron donor and 6 mM of CO(2)-bicarbonate as the C source, has a doubling time (t(d)) of 8.1 h. Growth and arsenite oxidation were significantly enhanced by the presence of 0.01 % yeast extract, decreasing the t(d) to 4.3 h. The strain carried arsenite oxidase (aioA) gene highly similar to those of previously reported arsenite-oxidizing Alpha-proteobacteria. The RuBisCO Type-I (cbbL) gene was amplified and sequenced too, underscoring the ability of As3-1b to carry out autotrophic As(III) oxidation. The results suggest that A. dichloromethanicus As3-1b can be a good candidate for the oxidation of arsenite in polluted waters or groundwaters.

Prebiotic potential and gastrointestinal effects of immature wheat grain (IWG) biscuits.

In this study the effects of immature wheat grains (IWG), a natural source of fructo-oligosaccharides (FOS), on intestinal microbiota and gastrointestinal function were evaluated. Ileal effluents from three ileostomates were collected for 8 h after IWG-enriched or Control biscuit administration, and then fermented in vitro with human faecal inoculum. On fermentation broths, microbial counts and SCFA were measured. Moreover, we investigated the effect of IWG biscuits on gastric emptying. Twelve healthy volunteers underwent ultrasound measurement of gastric emptying of a standard meal consumed 5 h after a breakfast composed by lactose-free milk and IWG or Control biscuits; hunger and satiety sensations were also evaluated in this period. Bifidobacteria and lactic acid bacteria counts were higher (P < 0.05) in IWG than in Control cultures, supporting the prebiotic effects of IWG, probably linked to its FOS content; moreover IWG fermentation produced more acetate. In vivo IWG biscuits delayed gastric emptying of the next meal (P < 0.05), decreased hunger (P < 0.001) and increased satiety (P < 0.005). Therefore in vitro results substantiate the potential of IWG as a prebiotic ingredient and in vivo data suggest an effect of IWG on gastric emptying and on persistent satiety. These preliminary data tend to support the use of IWG in formulating functional prebiotic products suitable to promote satiety.

Arsenic-resistant bacteria associated with roots of the wild Cirsium arvense (L.) plant from an arsenic polluted soil, and screening of potential plant growth-promoting characteristics.

A rhizobacterial community, associated with the roots of wild thistle Cirsium arvense (L.) growing in an arsenic polluted soil, was studied by fluorescence in situ hybridization (FISH) analysis in conjunction with cultivation-based methods. In the bulk, rhizosphere, and rhizoplane fractions of the soil, the qualitative picture obtained by FISH analysis of the main phylogenetic bacterial groups was similar and was predominantly comprised of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. The arsenic-resistant isolates belonged to 13 genera, the most abundant being those of Bacillus, Achromobacter, Brevundimonas, Microbacterium, and Ochrobactrum. Most bacteria grew in the presence of high arsenic concentrations (over 100mM arsenate and 10mM arsenite). Most strains possessed the ArsC, ArsB and ACR3 genes homologous to arsenate reductase and to the two classes of arsenite efflux pumps, respectively, peculiar to the ars operon of the arsenic detoxification system. ArsB and ACR3 were present simultaneously in highly resistant strains. An inconsistency between 16S rRNA phylogenetic affiliations and the arsenate reductase sequences of the strains was observed, indicating possible horizontal transfer of arsenic resistance genes in the soil bacterial community. Several isolates were able to reduce arsenate and to oxidise arsenite. In particular, Ancylobacter dichloromethanicum strain As3-1b possessed both characteristics, and arsenite oxidation occurred in the strain also under chemoautotrophic conditions. Some rhizobacteria produced siderophores, indole acetic acid and 1-amino-cyclopropane-1-carboxylic acid deaminase, thus possessing potential plant growth-promoting traits.

Metabolism of stevioside and rebaudioside A from Stevia rebaudiana extracts by human microflora.

Stevia rebaudiana standardized extracts (SSEs) are used as natural sweeteners or dietary supplements in different countries for their content of stevioside or rebaudioside A. These compounds possess up to 250 times the sweetness intensity of sucrose, and they are noncaloric and noncariogenic sweeteners. The aim of this study was to investigate the in vitro transformation of stevioside and rebaudioside A after incubation with human microflora, the influence of these sweeteners on human microbial fecal community and which specific groups metabolize preferentially stevioside and rebaudioside A. The experiments were carried out under strict anaerobic conditions in batch cultures inoculated with mixed fecal bacteria from volunteers. The hydrolysis was monitored by HPLC coupled to photodiode array and mass spectrometric detectors. Isolated bacterial strains from fecal materials incubated in selective broths were added to stevioside and rebaudioside A. These sweeteners were completely hydrolyzed to their aglycon steviol in 10 and 24 h, respectively. Interestingly, the human intestinal microflora was not able to degrade steviol. Furthermore, stevioside and rebaudioside A did not significantly influence the composition of fecal cultures; among the selected intestinal groups, bacteroides were the most efficient in hydrolyzing Stevia sweeteners to steviol.