Ivo Doskocil

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.

Evaluation of antibacterial potential and toxicity of plant volatile compounds using new broth microdilution volatilization method and modified MTT assay

With aim to develop effective proof-of-concept approach which can be used in a development of new preparations for the inhalation therapy, we designed a new screening method for simple and rapid simultaneous determination of antibacterial potential of plant volatiles in the liquid and the vapour phase at different concentrations. In addition, EVA (ethylene vinyl acetate) capmat™ as vapour barrier cover was used as reliable modification of thiazolyl blue tetrazolium bromide (MTT) assay for cytotoxicity testing of volatiles on microtiter plates. Antibacterial activity of carvacrol, cinnamaldehyde, eugenol, 8-hydroxyquinoline, thymol and thymoquinone was determined against Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae using new broth microdilution volatilization method. The cytotoxicity of these compounds was evaluated using MTT test in lung fibroblast cells MRC-5. The most effective antibacterial agents were 8-hydroxyquinoline and thymoquinone with the lowest minimum inhibitory concentrations (MICs) ranging from 2 to 128μg/mL, but they also possessed the highest toxicity in lung cell lines with half maximal inhibitory concentration (IC50) values 0.86-2.95μg/mL. The lowest cytotoxicity effect was identified for eugenol with IC50 295.71μg/mL, however this compound produced only weak antibacterial potency with MICs 512-1024μg/mL. The results demonstrate validity of our novel broth microdilution volatilization method, which allows cost and labour effective high-throughput antimicrobial screening of volatile agents without need of special apparatus. In our opinion, this assay can also potentially be used for development of various medicinal, agricultural, and food applications that are based on volatile antimicrobials.

Isolation of Amaryllidaceae alkaloids from Nerine bowdenii W. Watson and their biological activities

Twenty-two isoquinoline alkaloids (1–22) were isolated from fresh bulbs of Nerine bowdenii (Amaryllidaceae) by standard chromatographic methods. The chemical structures were elucidated by MS, and 1D and 2D NMR spectroscopic analyses, and by comparison with literature data. 6-O-Demethylbelladine (11) and 4′-O-demethylbelladine (12) are reported here for the first time. Compounds isolated in sufficient amounts were evaluated for their acetylcholinesterase, and butyrylcholinesterase inhibition activity using Ellmans method. In the prolyl oligopeptidase assay, Z-Gly-Pro-p-nitroanilide was used as substrate. Untested alkaloids were also screened for their cytotoxic activity against p53-mutated Caco-2 and HT-29 colorectal adenocarcinoma cells. At the same time, healthy small intestine cells FH-74 Int were used to determine overall toxicity against noncancerous cells. The crinine-type alkaloid buphanisine (7) demonstrated interesting cytotoxicity against both tested cancer cell lines with IC50 values of 8.59 ± 0.15 μM for Caco-2 and 5.32 ± 1.70 μM for HT-29.

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

Abstract 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. & M.A. Curtis) H. Jahn ex Ryvarden, Trametes gibbosa (Pers.) Fr., Piptoporus betulinus (Bull.) P. Karst, Neolentinus lepideus (Fr.) Redhead & 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.

EFFECT OF HYDROLYZED MILK ON THE ADHESION OF LACTOBACILLI TO INTESTINAL CELLS

Abstract Milk is an essential part of the human diet and is undoubtedly a major calcium source in human nutrition, accepted well by most individuals. Knowledge on how the components from dairy products support or reduce the adherence of probiotics to the intestinal epithelium is limited. The purpose of this study was to investigate the effect of acid-hydrolyzed milk on the adhesion ability of two potentially probiotic strains (Lactobacillus plantarum S2, Lactobacillus gasseri R) to in vitro human intestinal epithelial model consisting of Caco-2 and mucus-secreting HT29-MTX co-culture. The adhesion of our tested strains L. gasseri and L. plantarum was 4.74 and 7.16%, respectively, when using inoculum of 2 × 108 CFU ml–1. Addition of acid-hydrolyzed milk to co-culture decreased the adherence by 53.7% for L. gasseri R and by 62.2% for L. plantarum S2. The results of this study evidently indicate the potential importance of the food matrix as a factor influencing probiotic colonization of the gut.

Spatio-temporal dynamics of Varroa destructor resistance to tau-fluvalinate in Czechia, associated with L925V sodium channel point mutation: Spread of tau-fluvalinate resistance in Varroa

BACKGROUND Extensive application of pyrethroids to control Varroa destructor, an invasive mite devastating bee colonies, has resulted in a global spread of resistant mite populations. In this study, we analyzed the spatio-temporal dynamics of resistant V. destructor populations in Czechia, stemming from the L925V mutation. Mites were collected during 2011-2018 directly or from winter beeswax debris, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and densitometry was used to detect the L925V mutation. RESULTS Pooled samples of 10 mites were classified, based on their PCR-RFLP patterns, as tau-fluvalinate-sensitive (56%), resistant (9%), or mixed (35%), with the latter including sensitive and resistant homo- and heterozygotes. We identified two zones with higher frequencies of resistance, one in southern Moravia and the other in Bohemia. The mutant populations were evenly distributed throughout the monitored districts, with a few temporal and spatial local fluctuations. The greatest increase in resistance was observed in 2016, following massive losses of bee colonies in the winter of 2015. This event appeared to be closely associated with fluctuations in resistant mite populations and their dispersion. CONCLUSION Two outbreaks of resistance were detected in Czechia; however, the amount of applied tau-fluvalinate was not correlated with the frequency of resistance in mites. There was no remarkable increase in mite resistance in 2011-2018, although the use of tau-fluvalinate increased 40-fold between 2011 and 2015. PCR-RFLP analysis, performed on mites present in beeswax debris, is a suitable method for monitoring the L925V mutation in V. destructor.

Screening of medicinal plants traditionally used in Peruvian Amazon for in vitro antioxidant and anticancer potential

Abstract Plants mentioned in this study have numerous records in traditional Peruvian medicine being used in treatment of cancer and other diseases likely to be associated with oxidative stress. Amongst the eight plant species tested, only Dysphania ambrosioides exhibited combinatory antioxidant and anti-proliferative effect on a broad spectrum of cancer cells (DPPH and ORAC values = 80.6 and 687.3 μg TE/mg extract, respectively; IC50 against Caco-2, HT-29 and Hep-G2 = 129.2, 69.9 and 130.6, respectively). Alkaloids and phenolic compounds might significantly contribute to anticancer/antioxidant activity of this plant. The results justify the traditional medicinal use of this plant. Our findings further suggest that D. ambrosioides might serve as a prospective material for further development of novel plant-based antioxidant and/or anti-proliferative agents. Detailed analysis of chemical composition together with toxicology assessments and in vivo antioxidant/anti-proliferative activity of this plant should be carried out in order to verify its potential practical use.