Naděžda Vrchotová

Cow excrements enhance the occurrence of tetracycline resistance genes in soil regardless of their oxytetracycline content

Fertilizing soils with animal excrements from farms with common antibiotic use represents a risk of disseminating antibiotic resistance genes into the environment. In the case of tetracycline antibiotics, it is not clear, however, whether the presence of antibiotic residues further enhances the gene occurrence in manured soils. We established a microcosm experiment in which 3 farm soils that had no recent history of fertilization with animal excrements were amended on a weekly basis (9 times) with excrements from either an oxytetracycline-treated or an untreated cow. Throughout the study, the concentration of oxytetracycline in excrements from the treated cow was above 500 μg g(-1)dw, whereas no oxytetracycline was detected in excrements from the healthy cow. Both excrements contained tetracycline resistance (TC-r) genes tet(L), tet(M), tet(V), tet(Z), tet(Q) and tet(W). The excrements from the treated cow also contained the tet(B) gene, and a higher abundance of tet(Z), tet(Q) and tet(W). Three weeks after the last excrement addition, the individual TC-r genes differed in their persistence in soil: tet(Q) and tet(B) were not detectable while tet(L), tet(M), tet(Z) and tet(W) were found in all 3 soils. There were, however, no significant differences in the total number, nor in the abundance, of TC-r genes between soil samples amended with each excrement type. The oxytetracycline-rich and the oxytetracycline-free excrement therefore contributed equally to the increase of tetracycline resistome in soil. Our results indicate that other mechanisms than OTC-selection pressure may be involved in the maintenance of TC-r genes in manured soils.

Spread of tetracycline resistance genes at a conventional dairy farm

The use of antibiotics in animal husbandry contributes to the worldwide problem of increasing antibiotic resistance in animal and human pathogens. Intensive animal production is considered an important source of antibiotic resistance genes released to the environment, while the contribution of smaller farms remains to be evaluated. Here we monitor the spread of tetracycline resistance (TC-r) genes at a middle-size conventional dairy farm, where chlortetracycline (CTC, as intrauterine suppository) is prophylactically used after each calving. Our study has shown that animals at the farm acquired the TC-r genes in their early age (1–2 weeks), likely due to colonization with TC-resistant bacteria from their mothers and/or the farm environment. The relative abundance of the TC-r genes tet(W), tet(Q), and tet(M) in fresh excrements of calves was about 1–2 orders of magnitude higher compared to heifers and dairy cows, possibly due to the presence of antibiotic residues in milk fed to calves. The occurrence and abundance of TC-r genes in fresh excrements of heifers and adult cows remained unaffected by intrauterine CTC applications, with tet(O), tet(Q), and tet(W) representing a “core TC-resistome” of the farm, and tet(A), tet(M), tet(Y), and tet(X) occurring occasionally. The genes tet(A), tet(M), tet(Y), and tet(X) were shown to be respectively harbored by Shigella, Lactobacillus and Clostridium, Acinetobacter, and Wautersiella. Soil in the farm proximity, as well as field soil to which manure from the farm was applied, was contaminated with TC-r genes occurring in the farm, and some of the TC-r genes persisted in the field over 3 months following the manure application. Concluding, our study shows that antibiotic resistance genes may be a stable part of the intestinal metagenome of cattle even if antibiotics are not used for growth stimulation, and that smaller dairy farms may also contribute to environmental pollution with antibiotic resistance genes.

Non-cellulosic polysaccharides from the leaves of small balsam (Impatiens parviflora DC.).

The leaves of the annual plant Impatiens parviflora DC., a herbal medicine in Asian countries and invasive in managed forests and natural environments in Central Europe, have the potential as a source of bioactive phenolic compounds and polysaccharides. Extractives accounted for ∼22% of the leaves, whereby, the methanolic extract contains mainly caffeic acid, ferulic acid, kaempferol, and quercetin derivatives, and 1,2,4-trihydroxynaphthalene-1-O-glucoside. From the pre-extracted leaves, non-cellulosic polysaccharides were isolated by a five-step extraction procedure using as extractants cold water, 0.05 M EDTA, and DMSO in the first three steps, and 1% and 5% NaOH in the last two steps. The isolated polysaccharide fractions were characterized by chemical, physicochemical, and spectroscopic analyses (FTIR and (1)H NMR). The first three fractions contained mainly pectin and the alkali-extracted ones methylglucuronoxylan and arabinogalactan. The suggested structural features were confirmed using HSQC NMR and COSY experiments for polysaccharides of the EDTA-fraction and the Pronase-treated 5% NaOH-fraction. The EDTA fraction comprised a pectin with low degree of methyl-esterification (DM, 37%) with few rhamnogalacturonan RG I segments bearing β-1,4-galactan as side chains. The alkali-extracted fraction comprised a degraded methylglucuronoxylan and type II 3,6-arabinogalactan in about equal amounts.

Various Extraction Methods for Obtaining Stilbenes from Grape Cane of Vitis vinifera L.

Grape cane, leaves and grape marc are waste products from viticulture, which can be used to obtain secondary stilbene derivatives with high antioxidant value. The presented work compares several extraction methods: maceration at laboratory temperature, extraction at elevated temperature, fluidized-bed extraction, Soxhlet extraction, microwave-assisted extraction, and accelerated solvent extraction. To obtain trans-resveratrol, trans-ε-viniferin and r2-viniferin from grape cane of the V. vinifera variety Cabernet Moravia, various conditions were studied: different solvents, using powdered versus cut cane material, different extraction times, and one-step or multiple extractions. The largest concentrations found were 6030 ± 680 µg/g dry weight (d.w.) for trans-resveratrol, 2260 ± 90 µg/g d.w. for trans-ε-viniferin, and 510 ± 40 µg/g d.w. for r2-viniferin. The highest amounts of stilbenes (8500 ± 1100 µg/g d.w.) were obtained using accelerated solvent extraction in methanol.

Separation and Identification of 1,2,4-Trihydroxynaphthalene-1-O-glucoside in Impatiens glandulifera Royle

Methanolic extract from lyophilized roots of Impatiens glandulifera Royle was analyzed by high performance liquid chromatography using DAD and FLD detection and this revealed one dominant highly fluorescent very unstable substance. The stability of this derivative is strongly dependent on the plant material drying procedure and extraction procedure used. The structure of the substance was established as 1,2,4-trihydroxynaphthalene-1-O-glucoside (THNG) according LC-MS and NMR measurements. When lyophilized plant material was extracted with methanol an almost four times higher amount of THNG was found in the extract, compared to the amount of 2-hydroxy-1,4-naphthoquinone obtained, while in the case of the same lyophilized plant material extracted with water there was no THNG in the extract. The main compounds in this case was 2-hydroxy-1,4-naphthoquinone. In the plant material dried at the laboratory temperature and extracted by methanol there are only traces of THNG.

Separation and identification of highly fluorescent compounds derived from trans-resveratrol in the leaves of Vitis vinifera infected by Plasmopara viticola.

A method for identification of highly fluorescent compounds in vine leaves infected by Plasmopara viticola was developed using reversed phase liquid chromatography with simultaneous diode array and fluorometric detection. Fluorescent compounds were extracted from leaves with a methanol-water mixture (70:30). Separation by HPLC was performed using a C18 column and gradient elution with water-acetonitrile mixtures (20–80% of acetonitrile). The main unknown fluorescent compound was identified by line spectral comparison with a standard obtained by UV photoisomerization of trans-resveratrol glucoside, and its structure was confirmed by liquid chromatography-mass spectrometry. Identification and structural elucidation of the fluorescent compound in the leaves of Vitis vinifera allows early detection of Plasmopara viticola invasion.

Content of selected biologically active compounds in tea infusions of widely used European medicinal plants

Content of selected biologically active compounds in tea infusions of widely used European medicinal plants Herbal tea infusions are a very important source of flavonoids and other biologically active compounds in human medicine and nutrition. Melissa officinalis, Agrimonia eupatoria, Sambucus nigra, Achillea millefolium, Filipendula ulmaria, Betula pendula and Glechoma hederacea were selected as common European medicinal herbs and sources for tea infusion preparations. Quercetin, rutin, catechin, chlorogenic acid, and squalene were determined in the prepared infusions. Free quercetin was not found in any of the infusions, but tea infusions did contain rutin and other quercetin glycosides, the content of which was, after acid hydrolysis, determined as quercetin. The highest levels of total quercetin were found in infusions from Filipendula ulmaria and Sambucus nigra (120 and 108 mg L-1, respectively) corresponding to the content of rutin found also in these two infusions (25.2 and 194 mg L-1, respectively). The Sambucus nigra infusion contained the largest content of chlorogenic acid (166 mg L-1), and infusions from Melissa officinalis, Agrimonia eupatoria, Betula pendula and Glechoma hederacea contained only small amounts of squalene.

Verification of presence of caprolactam in sprouted achenes of Fagopyrum esculentum Moench and its influence on plant phenolic compound content

The presence of caprolactam, a precursor of Nylon-6, among those synthetic polymers which are widely-spread throughout the environment, could be the reason for its being found in plants. The aim of this work was to confirm the previously described presence of caprolactam in dry and sprouted achenes, as well as in achene exudates of common buckwheat (Fagopyrum esculentum Moench). When the lyophilized sprouted and dry buckwheat achenes, along with exudates from growth experiments, with caprolactam-free medium were analysed by HPLC, no caprolactam was found. After addition of caprolactam into the growth medium, we confirmed the uptake of caprolactam in the lyophilized sprouted buckwheat achenes. The uptake of caprolactam is also a function of light conditions during the growth experiments. Caprolactam also inhibits the content of phenolic compounds; especially rutin, vitexin, isovitexin, orientin, and homoorientin in buckwheat plants.

Thin-layer chromatographic separation of selected polyaromatic hydrocarbons on copper phthalocyanine dye impregnated plates

The separation of anthracene, phenathrene, pyrene, benzo[ghi]perylene, triphenylene, perylene and fluoranthene on aluminium oxide thin-layer chromatography plates, coated with hydrolyzed copper phthalocyanine dye Ostazin turquoise V-G, has been studied. The elution order reflects planarity of the molecules or carcinogenity. Based on the measurement of the adsorption isotherms of the hydrolyzed copper phthalocyanine dye, it has also been shown that different aluminium oxide surfaces have different affinities, a phenomenon important for the preparation of the thin-layer chromatography plates.

Musts with an increased content of lignans from added spruce knot chips

Novotná P., Tříska J., Híc P., Balík J., Vrchotová N., Strohalm J., Houška M. (2016): Musts with an increased content of lignans from added spruce knot chips. Czech J. Food Sci., 34: 318–324. Red and white musts were enriched with the lignan hydroxymatairesinol, which is the main lignan contained in spruce knots. Chips from the milled spruce knots were then used to enrich grape musts. After enrichment, the musts were stored and samples were taken in 1, 5, 9, and 12 months. The samples were subjected to a variety of analyses and sensory evaluations. Analyses included hydroxymatairesinol and alpha-conidendrin content, antioxidant activity (determined by the FRAP method), content of total polyphenols, sensory evaluation (intensity of woody aroma, intensity of bitterness and astringent taste, and consumer acceptability), and must antimutagenicity. The analysis of variance allowed predicting which factors such as grape type, quantity of added wood chips, sugar addition, method of preservation, and storage time had the most significant influence on the analytical parameters (lignan content, antioxidant activity, and total polyphenol content). In all cases lignan content in the musts was significantly influenced by the addition of spruce wood chips. Total polyphenol content in the musts was significantly affected by the type of musts and by heat treatment (time of thermomaceration). Evaluation of must antimutagenicity showed that all samples, except the sample of white musts after thermomaceration without holding at temperature and without adding chips (10 g/20 kg mash), inhibited mutagenicity.