Variation in honey bee gut microbial diversity affected by ontogenetic stage, age and geographic location

Social honey bees, Apis mellifera, host a set of distinct microbiota, which is similar across the continents and various honey bee species. Some of these bacteria, such as lactobacilli, have been linked to immunity and defence against pathogens. Pathogen defence is crucial, particularly in larval stages, as many pathogens affect the brood. However, information on larval microbiota is conflicting. Seven developmental stages and drones were sampled from 3 colonies at each of the 4 geographic locations of A. mellifera carnica, and the samples were maintained separately for analysis. We analysed the variation and abundance of important bacterial groups and taxa in the collected bees. Major bacterial groups were evaluated over the entire life of honey bee individuals, where digestive tracts of same aged bees were sampled in the course of time. The results showed that the microbial tract of 6-day-old 5th instar larvae were nearly equally rich in total microbial counts per total digestive tract weight as foraging bees, showing a high percentage of various lactobacilli (Firmicutes) and Gilliamella apicola (Gammaproteobacteria 1). However, during pupation, microbial counts were significantly reduced but recovered quickly by 6 days post-emergence. Between emergence and day 6, imago reached the highest counts of Firmicutes and Gammaproteobacteria, which then gradually declined with bee age. Redundancy analysis conducted using denaturing gradient gel electrophoresis identified bacterial species that were characteristic of each developmental stage. The results suggest that 3-day 4th instar larvae contain low microbial counts that increase 2-fold by day 6 and then decrease during pupation. Microbial succession of the imago begins soon after emergence. We found that bacterial counts do not show only yearly cycles within a colony, but vary on the individual level. Sampling and pooling adult bees or 6th day larvae may lead to high errors and variability, as both of these stages may be undergoing dynamic succession

Influence of human milk oligosaccharides on adherence of bifidobacteria and clostridia to cell lines

Adhesion of gut bacteria to the intestinal epithelium is the first step in their colonization of the neonatal immature gut. Bacterial colonization of the infant gut is influenced by several factors, of which the most important are the mode of delivery and breast-feeding. Breast-fed infants ingest several grams of human milk oligosaccharides (HMOs) per day, which can become receptor decoys for intestinal bacteria. The most abundant intestinal bacteria in vaginally delivered infants are bifidobacteria, whereas infants born by cesarean section are colonized by clostridia. The influence of HMOs on the adhesion of five strains of intestinal bacteria (three bifidobacterial strains and two clostridial strains) to mucus-secreting and non-mucus-secreting human epithelial cells was investigated. Bifidobacterium bifidum 1 and Bifidobacterium longum displayed almost the same level of adhesion in the presence and absence of HMOs. By contrast, adhesion of Clostridium butyricum 1 and 2 decreased from 14.41% to 6.72% and from 41.54% to 30.91%, respectively, in the presence of HMOs. The results of this study indicate that HMOs affect bacterial adhesion and are an important factor influencing bacterial colonization of the gut. Adhesion of the tested bacteria correlates with their ability to autoaggregate

New Broth Macrodilution Volatilization Method for Antibacterial Susceptibility Testing of Volatile Agents and Evaluation of Their Toxicity Using Modified MTT Assay In Vitro

In this study, a new broth macrodilution volatilization method for the simple and rapid determination of the antibacterial effect of volatile agents simultaneously in the liquid and vapor phase was designed with the aim to assess their therapeutic potential for the development of new inhalation preparations. The antibacterial activity of plant volatiles (β-thujaplicin, thymohydroquinone, thymoquinone) was evaluated against bacteria associated with respiratory infections (Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and their cytotoxicity was determined using a modified thiazolyl blue tetrazolium bromide assay against normal lung fibroblasts. Thymohydroquinone and thymoquinone possessed the highest antibacterial activity against H. influenzae, with minimum inhibitory concentrations of 4 and 8 µg/mL in the liquid and vapor phases, respectively. Although all compounds exhibited cytotoxic effects on lung cells, therapeutic indices (TIs) suggested their potential use in the treatment of respiratory infections, which was especially evident for thymohydroquinone (TI > 34.13). The results demonstrate the applicability of the broth macrodilution volatilization assay, which combines the principles of broth microdilution volatilization and standard broth macrodilution methods. This assay enables rapid, simple, cost- and labor-effective screening of volatile compounds and overcomes the limitations of assays currently used for screening of antimicrobial activity in the vapor phase

Metabolism of Selected 2-Arylbenzofurans in a Colon In Vitro Model System

2-arylbenzofurans represent a small group of bioactive compounds found in the plant family Moraceae. As it has not been investigated whether these substances are stable during passage through the gastrointestinal tract, their biological effects may be altered by the metabolism of intestinal microbiota or cells. The aim of the present study was to investigate and compare mulberrofuran Y (1), moracin C (2), and mulberrofuran G (3) in an in vitro model of human intestinal bacterial fermentation and in an epithelial model using the Caco-2 cell line. The analysis of compounds by LC-MS-Q-TOF showed sufficient stability in the fermentation model, with no bacterial metabolites detected. However, great differences in the quantity of permeation were observed in the permeability assay. Moreover, mulberrofuran Y (1) and moracin C (2) were observed to be transformed into polar metabolites by conjugation. Among the test compounds, mulberrofuran Y (1) was mostly stable and accumulated in endothelial cells (85.3%) compared with mulberrofuran G (3) and moracin C (2) (14% and 8.2%, respectively). Thus, only a small amount of mulberrofuran Y (1) was conjugated. Moracin C (2) and mulberrofuran G (3) were metabolized almost completely, with only traces of the unchanged molecule being found on the apical and cellular sides of the system. Only conjugates of mulberrofuran Y (1) and moracin C (2) were able to reach the basolateral side. Our results provide the basic description of bioavailability of these three compounds, which is a necessary characteristic for final evaluation of bio-efficacy

Vapors of Volatile Plant-Derived Products Significantly Affect the Results of Antimicrobial, Antioxidative and Cytotoxicity Microplate-Based Assays

Volatile plant-derived products were observed to exhibit broad spectrum of biological effects. However, due to their volatility, results of conventional microplate-based bioassays can be significantly affected by the vapors. With aim to demonstrate this phenomenon, antimicrobial, antioxidant, and cytotoxic activities of three essential oils (Alpinia elegans, Cinnamomum iners, and Xanthostemon verdugonianus), one supercritical CO2 extract (Nigella sativa), and four plant-derived compounds (capsaicin, caryophyllene oxide, 8-hydroxyquinoline, and thymoquinone) were evaluated in series of experiments including both ethylene vinyl acetate (EVA) Capmat sealed and nonsealed microplates. The results clearly illustrate that vapor transition to adjoining wells causes false-positive results of bioassays performed in nonsealed microtiter plates. The microplate layout and a duration of the assay were demonstrated as the key aspects defining level of the results affection by the vapors of volatile agents. Additionally, we reported biological activities and chemical composition of essential oils from A. elegans seeds and X. verdugonianus leaves, which were, according to our best knowledge, analyzed for the first time. Considering our findings, certain modifications of conventional microplate-based assays are necessary (e.g., using EVA Capmat as vapor barrier) to obtain reliable results when biological properties of volatile agents are evaluated

Antioxidative properties and acrylamide content of functional wheat-flour cookies enriched with wild-grown fruits

This study aimed to evaluate the impact of widely grown fruits (wild roses, elderberries, sea buckthorns, rowans, chokeberries, and hawthorns) as a functional ingredient in wheat-flour cookie formulation on antioxidative properties with a simultaneous reduction of the carcinogen-like compound acrylamide. The organoleptic features of the cookies were assessed by a panel of consumers. The following parameters were measured: chemical composition, total polyphenols, polyphenolic profile, antioxidant activity, and acrylamide content. The overall ratings of the tested cookies with the addition of chokeberries, hawthorns, sea buckthorns, and elderberries were more than satisfactory, while wild rose and rowan cookies were the most widely accepted and best rated by the panelists. The antioxidant activity of the tested cookies was 1.1–15.22 μmol trolox·g(−1) dm and 2.46–26.12 μmol Fe (II)·g(−1) dm as measured by the ABTS and FRAP methods, respectively. All the fruit-enriched cookies had significantly higher antioxidative properties (p < 0.05) in comparison to the control cookies, but among the fruit-enriched cookies, there were differences in the quality and quantity of particular polyphenols. The acrylamide content was significantly decreased by 59% (hawthorn), 71% (rowan), 87% (wild rose), 89% (sea buckthorn), 91% (elderberry), and 94% (chokeberry) compared with the control cookies (p < 0.05). Cookies enriched with wild-grown fruits could constitute a promising novel snack food

In vitro antioxidant and anti-proliferative activity of Ethiopian medicinal plant extracts

Identification and characterization of natural products with antioxidant and anti-proliferative activity has received much interest over the past few years. Ethiopia is one of the developing countries which have enormous diversity of plants and yet majority stays scientifically neglected and undiscovered. In this study, the ethanol extracts of 18 Ethiopian wild medicinal plants were investigated for their in vitro antioxidant and anti-proliferative potential. For this purpose DPPH (2,2-diphenyl-1-picrylhydrazyl), ORAC (oxygen radical absorbance capacity) and TPC (total phenolic content) assays together with MTT cell viability assay (performed on Hep-G2 and MRC-5) were used. Extracts of Carissa spinarum, Dodonaea angustifolia, Jasminum abyssinicum, Rumex nepalensis, Rubus steudneri and Verbascum sinaiticum exhibited the most significant results. However, it was discovered that C. spinarum, J. abyssinicum and R. steudneri possessed considerable toxicity against normal MRC-5 cell line. Only extracts of D. angustifolia and R. nepalensis demonstrated significant combinatory antioxidant/anti-proliferative effect, while V. sinaiticum showed best selective anti-proliferative activity. Since aforementioned extracts also exerted low or minimal toxicity to normal cells, we suggest these as prospective material for further development of novel plant-based agents effective against oxidative stress related diseases

Validation of Broth Macrodilution Volatilization Method for Testing of Essential Oils in Liquid and Vapor Phase: Chemical Composition, Cytotoxicity, and Antibacterial Effect of Indian Medicinal Plants against Pneumonia-Causing Pathogens

Essential oils (EOs) have great potential in inhalation therapy for the treatment of respiratory infections. However, innovative methods for evaluation of antimicrobial activity of their vapors are still needed. The current study reports validation of the broth macrodilution volatilization method for assessment of the antibacterial properties of EOs and shows the growth-inhibitory effect of Indian medicinal plants against pneumonia-causing bacteria in liquid and vapor phase. Among all samples tested, Trachyspermum ammi EO exhibits the strongest antibacterial effect against Haemophilus influenzae, with minimum inhibitory concentrations of 128 and 256 µg/mL in the liquid and vapor phases, respectively. Furthermore, Cyperus scariosus EO is found to be nontoxic to normal lung fibroblasts assessed by modified thiazolyl blue tetrazolium bromide assay. Chemical analysis performed using gas chromatography–mass spectrometry identified α-citral, cyperotundone, and thymol as the main constituents of Cymbopogon citratus, C. scariosus, and T. ammi EOs, respectively. In addition, β-cymene is identified as the major compound of T. ammi EO vapors when analyzed using solid-phase microextraction and gas-tight syringe sampling techniques. This study demonstrates the validity of the broth macrodilution volatilization method for antimicrobial screening of volatile compounds in the vapor phase and suggests the therapeutic potential of Indian medicinal plants in inhalation therapy

Functional Properties of <i>Dunaliella salina</i> and Its Positive Effect on Probiotics

The unicellular green microalga Dunaliella is a potential source of a wide range of nutritionally important compounds applicable to the food industry. The aim of this study was to assess the effect of Dunaliella salina dried biomass on the growth and adherence of 10 strains of Lactobacillus, Lacticaseibacillus, and Bifidobacterium. The immunomodulatory, antioxidant, and cytotoxic effects of D. salina on human peripheral mononuclear cells and simulated intestinal epithelial cell lines Caco-2 and HT-29 were evaluated. Furthermore, the hypocholesterolemic effects of the microalgae on lipid metabolism in rats fed a high-fat diet were analyzed. The addition of D. salina biomass had a positive effect on the growth of nine out of 10 probiotics and promoted the adherence of three bifidobacteria strains to human cell lines. The antioxidant and immunomodulatory properties of D. salina were concentration-dependent. The inflammatory cytokines (TNF-α and IL-6) were significantly increased following Dunaliella stimulation at the lowest concentration (0.5% w/v). Eight week supplementation of D. salina to the diet of hypercholesteromic rats significantly decreased the serum concentrations of LDL-C, VLDL, IDL-B, and IDL-C. D. salina is not cytotoxic in intestinal cell models; it promotes adherence of selected bifidobacteria, it affords immunomodulatory and antioxidant effects, and its addition to diets may help decrease atherosclerosis risk factors

In vitro immunomodulatory activity, cytotoxicity and chemistry of some central European polypores

Context: Some mushrooms of the order Polyporales are known for their immunomodulatory actions. Objective: The objective of this study is to evaluate the in vitro phagocytic and cytotoxic effects of extracts from polyporales native to Central Europe. Materials and methods: The effects of ethanol extracts from 27 polypore species on opsonized zymosan-induced phagocytosis of isolated human neutrophils were tested by a chemiluminescence method. Colon epithelial cell lines, Caco-2 and HT-29, were used for cytotoxicity assays, and extracts were chemically characterized in terms of total phenolic and β-glucan content. Results: We observed phagocytosis or respiratory burst enhancing activity in 17 extracts, of which five species, namely Aurantiporus fissilis (Berk. &#38; M.A. Curtis) H. Jahn ex Ryvarden, Trametes gibbosa (Pers.) Fr., Piptoporus betulinus (Bull.) P. Karst, Neolentinus lepideus (Fr.) Redhead &#38; Ginns, Polyporus squamosus (Huds.) Fr., significantly increased phagocytosis in granulocytes by 205, 181, 158, 155 and 141%, respectively. The β-glucan content of the three most potent extracts was 58, 42 and 74 mg/g, respectively, and the polyphenol content was 155.6, 133.5 and 155.2 μmol of gallic acid equivalent/g, respectively. Some extracts showed cytotoxic activity, with higher cytotoxicity in Caco-2 than in HT-29 cells. Pycnoporus cinnabarinus (Jacq.) P. Karst. extract was cytotoxic to both cell lines, with IC50 values of 81 and 31 μg/mL, respectively. Discussion and conclusion: The most promising extracts were from N. lepideus and Polyporus squamosus, which are edible species and may be considered safe. Our findings support their use as culinary preparations or food supplements for various immunological gut disorders