Soils play an important role in the ecosystem of karstic landscapes both as a buffer zone and as a source of acidity to belowground water. Although the microbiota of karstic soils is known to have a great effect on karstification processes, the activity and composition of these communities are largely unknown. This study gives a comparative analysis of soil microbial profiles from different parts of a doline located at Aggtelek, Hungary. The aim was to reveal the relationships between the vegetation type and genetic fingerprints and substrate utilisation (multi-SIR) profiles of the soil microbiota. Soil samples were collected in early and late springs along a transect in a doline covered with different types of vegetation. Genetic fingerprints of bacterial communities were examined by denaturing gradient gel electrophoresis (DGGE) based on the 16S rRNA gene, along with multi-SIR profiles of the microbial communities measured by the MicroResp method using 15 different carbon sources. Genetic fingerprinting indicated that vegetation cover had a strong effect on the composition of soil bacterial communities. Procrustean analysis showed only a weak connection between DGGE and multi-SIR profiles, probably due to the high functional redundancy of the communities. Seasonality had a significant effect on substrate usage, which can be an important factor to consider in future studies.

• Klíčová slova
• Bacteria, DGGE, Karst soil, MicroResp, Multi-SIR,
• MeSH
• Bacteria klasifikace   genetika   metabolismus   MeSH
• denaturační gradientová gelová elektroforéza MeSH
• geologické jevy MeSH
• mikrobiota fyziologie   MeSH
• půda chemie   MeSH
• půdní mikrobiologie * MeSH
• RNA ribozomální 16S genetika   MeSH
• roční období MeSH
• shluková analýza MeSH
• uhlík metabolismus   MeSH
• veřejné parky * MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Maďarsko MeSH
• Názvy látek
• půda MeSH
• RNA ribozomální 16S MeSH
• uhlík MeSH

Complex samples are a challenge for sequencing-based broad-range diagnostics. We analysed 19 urinary catheter, ureteral Double-J catheter, and urine samples using 3 methodological approaches. Out of the total 84 operational taxonomic units, 37, 61, and 88% were identified by culture, PCR-DGGE-SS (PCR denaturing gradient gel electrophoresis followed by Sanger sequencing), and PCR-DGGE-RM (PCR- DGGE combined with software chromatogram separation by RipSeq Mixed tool), respectively. The latter approach was shown to be an efficient tool to complement culture in complex sample assessment.

• Klíčová slova
• Catheter, Complex sample, Mixed chromatogram, PCR denaturing gradient gel electrophoresis, RipSeq Mixed,
• MeSH
• Bacteria klasifikace   genetika   izolace a purifikace   MeSH
• denaturační gradientová gelová elektroforéza metody   MeSH
• diagnostické techniky molekulární metody   MeSH
• diagnostické techniky urologické MeSH
• DNA bakterií MeSH
• lidé MeSH
• moč chemie   mikrobiologie   MeSH
• močové katétry mikrobiologie   MeSH
• polymerázová řetězová reakce metody   MeSH
• software * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH

The bacterial and microeukaryotic biodiversity were studied using pyrosequencing analysis on a 454 GS FLX+ platform of partial SSU rRNA genes in terrestrial and aquatic habitats of the Sør Rondane Mountains, including soils, on mosses, endolithic communities, cryoconite holes and supraglacial and subglacial meltwater lenses. This inventory was complemented with Denaturing Gradient Gel Electrophoresis targeting Chlorophyta and Cyanobacteria. OTUs belonging to the Rotifera, Chlorophyta, Tardigrada, Ciliophora, Cercozoa, Fungi, Bryophyta, Bacillariophyta, Collembola and Nematoda were present with a relative abundance of at least 0.1% in the eukaryotic communities. Cyanobacteria, Proteobacteria, Bacteroidetes, Acidobacteria, FBP and Actinobacteria were the most abundant bacterial phyla. Multivariate analyses of the pyrosequencing data revealed a general lack of differentiation of both eukaryotes and prokaryotes according to habitat type. However, the bacterial community structure in the aquatic habitats was dominated by the filamentous cyanobacteria Leptolyngbya and appeared to be significantly different compared with those in dry soils, on mosses, and in endolithic habitats. A striking feature in all datasets was the detection of a relatively large amount of sequences new to science, which underscores the need for additional biodiversity assessments in Antarctic inland locations.

• Klíčová slova
• Antarctica, bacteria, cyanobacteria, eukaryotes, green algae, terrestrial biodiversity,
• MeSH
• Acidobacteria genetika   MeSH
• Actinobacteria genetika   MeSH
• Bacteroidetes genetika   MeSH
• biodiverzita MeSH
• Chlorophyta genetika   MeSH
• denaturační gradientová gelová elektroforéza MeSH
• ekosystém MeSH
• houby klasifikace   genetika   MeSH
• Proteobacteria genetika   MeSH
• půda chemie   MeSH
• půdní mikrobiologie MeSH
• RNA ribozomální genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sinice genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Antarktida MeSH
• Názvy látek
• půda MeSH
• RNA ribozomální MeSH

Bacterial and fungal biodiversity throughout different biostimulation and bioaugmentation treatments applied to an industrial creosote-polluted soil were analyzed by means of polyphasic approach in order to gain insight into the microbial community structure and dynamics. Pyrosequencing data obtained from initial creosote polluted soil (after a biopiling step) revealed that Alpha and Gammaproteobacteria were the most abundant bacterial groups, whereas Fusarium and Scedosporium were the main fungal genera in the contaminated soil. At the end of 60-days laboratory scale bioremediation assays, pyrosequencing and DGGE data showed that (i) major bacterial community shifts were caused by the type of mobilizing agent added to the soil and, to a lesser extent, by the addition of lignocellulosic substrate; and (ii) the presence of the non-ionic surfactant (Brij 30) hampered the proliferation of Actinobacteria (Mycobacteriaceae) and Bacteroidetes (Chitinophagaceae) and, in the absence of lignocellulosic substrate, also impeded polycyclic aromatic hydrocarbons (PAHs) degradation. The results show the importance of implementing bioremediation experiments combined with microbiome assessment to gain insight on the effect of crucial parameters (e.g. use of additives) over the potential functions of complex microbial communities harbored in polluted soils, essential for bioremediation success.

• Klíčová slova
• Creosote, HMW–PAH, Next generation sequencing (NGS), Non-ionic surfactant, Soil bioremediation, White rot fungi (WRF),
• MeSH
• Bacteria klasifikace   MeSH
• biodegradace MeSH
• biodiverzita MeSH
• denaturační gradientová gelová elektroforéza MeSH
• houby klasifikace   MeSH
• kreosot analýza   MeSH
• látky znečišťující půdu analýza   MeSH
• mezerníky ribozomální DNA genetika   MeSH
• polycyklické aromatické uhlovodíky analýza   MeSH
• povrchově aktivní látky chemie   MeSH
• průmysl MeSH
• půda chemie   MeSH
• půdní mikrobiologie * MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kreosot MeSH
• látky znečišťující půdu MeSH
• mezerníky ribozomální DNA MeSH
• polycyklické aromatické uhlovodíky MeSH
• povrchově aktivní látky MeSH
• půda MeSH
• RNA ribozomální 16S MeSH

Abundance and diversity of methanogenic archaea were studied at five localities along a longitudinal profile of a Sitka stream (Czech Republic). Samples of hyporheic sediments were collected from two sediment depths (0-25 cm and 25-50 cm) by freeze-core method. Methanogen community was analyzed by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and sequencing method. The proportion of methanogens to the DAPI-stained cells varied among all localities and depths with an average value 2.08 × 10(5) per g of dry sediment with the range from 0.37 to 4.96 × 10(5) cells per g of dry sediment. A total of 73 bands were detected at 19 different positions on the DGGE gel and the highest methanogen diversity was found at the downstream located sites. There was no relationship between methanogen diversity and sediment depth. Cluster analysis of DGGE image showed three main clusters consisting of localities that differed in the number and similarity of the DGGE bands. Sequencing analysis of representative DGGE bands revealed phylotypes affiliated with members belonging to the orders Methanosarcinales, Methanomicrobiales and Methanocellales. The knowledge about occurrence and diversity of methanogenic archaea in freshwater ecosystems are essential for methane dynamics in river sediments and can contribute to the understanding of global warming process.

• Klíčová slova
• Denaturing gradient gel electrophoresis (DGGE), Diversity, Fluorescence in situ hybridization (FISH), Hyporheic sediment, Methanogen,
• MeSH
• Archaea klasifikace   genetika   metabolismus   MeSH
• biodiverzita * MeSH
• denaturační gradientová gelová elektroforéza MeSH
• fylogeneze MeSH
• geologické sedimenty mikrobiologie   MeSH
• methan metabolismus   MeSH
• řeky * mikrobiologie   MeSH
• sekvenční analýza DNA MeSH
• životní prostředí MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• methan MeSH

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.

• MeSH
• Bacteria klasifikace   genetika   izolace a purifikace   MeSH
• denaturační gradientová gelová elektroforéza MeSH
• DNA bakterií genetika   MeSH
• ekosystém MeSH
• gastrointestinální trakt mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• Lactobacillaceae genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• střevní mikroflóra * MeSH
• včely embryologie   růst a vývoj   mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• RNA ribozomální 16S MeSH

The variation in the diversity of methanogens in sediment depths from Sitka stream was studied by constructing a 16S rRNA gene library using methanogen-specific primers and a denaturing gradient gel electrophoresis (DGGE)-based approach. A total of nine different phylotypes from the 16S rRNA library were obtained, and all of them were clustered within the order Methanosarcinales. These nine phylotypes likely represent nine new species and at least 5-6 new genera. Similarly, DGGE analysis revealed an increase in the diversity of methanogens with an increase in sediment depth. These results suggest that Methanosarcinales phylotypes might be the dominant methanogens in the sediment from Sitka stream, and the diversity of methanogens increases as the depth increases. Results of the present study will help in making effective strategies to monitor the dominant methanogen phylotypes and methane emissions in the environment.

• MeSH
• denaturační gradientová gelová elektroforéza MeSH
• DNA archebakterií chemie   genetika   MeSH
• fylogeneze MeSH
• geologické sedimenty mikrobiologie   MeSH
• Methanosarcinales izolace a purifikace   MeSH
• molekulární sekvence - údaje MeSH
• řeky MeSH
• ribozomální DNA chemie   genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• shluková analýza MeSH
• společenstvo * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• DNA archebakterií MeSH
• ribozomální DNA MeSH
• RNA ribozomální 16S MeSH

The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile.

• Klíčová slova
• Bacterial diversity, Bacteroidetes, Extracellular DNA, Firmicutes, Intracellular DNA, Metabarcoding, PCR-DGGE, Q-PCR, Rumen fluid, Storage conditions,
• MeSH
• bachor mikrobiologie   MeSH
• Bacteroidetes klasifikace   genetika   izolace a purifikace   MeSH
• denaturační gradientová gelová elektroforéza MeSH
• DNA bakterií izolace a purifikace   MeSH
• fylogeneze MeSH
• grampozitivní bakterie klasifikace   genetika   izolace a purifikace   MeSH
• kryoprezervace * MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• odběr biologického vzorku metody   MeSH
• skot MeSH
• taxonomické DNA čárové kódování * MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH

Methane production by intestinal methanogenic Archaea and their community structure were compared among phylogenetic lineages of millipedes. Tropical and temperate millipedes of 35 species and 17 families were investigated. Species that emitted methane were mostly in the juliform orders Julida, Spirobolida, and Spirostreptida. The irregular phylogenetic distribution of methane production correlated with the presence of the methanogen-specific mcrA gene. The study brings the first detailed survey of methanogens' diversity in the digestive tract of millipedes. Sequences related to Methanosarcinales, Methanobacteriales, Methanomicrobiales and some unclassified Archaea were detected using molecular profiling (DGGE). The differences in substrate preferences of the main lineages of methanogenic Archaea found in different millipede orders indicate that the composition of methanogen communities may reflect the differences in available substrates for methanogenesis or the presence of symbiotic protozoa in the digestive tract. We conclude that differences in methane production in the millipede gut reflect differences in the activity and proliferation of intestinal methanogens rather than an absolute inability of some millipede taxa to host methanogens. This inference was supported by the general presence of methanogenic activity in millipede faecal pellets and the presence of the 16S rRNA gene of methanogens in all tested taxa in the two main groups of millipedes, the Helminthophora and the Pentazonia.

• MeSH
• biodiverzita * MeSH
• členovci mikrobiologie   MeSH
• denaturační gradientová gelová elektroforéza MeSH
• Euryarchaeota genetika   metabolismus   fyziologie   MeSH
• feces chemie   MeSH
• fylogeneze MeSH
• gastrointestinální trakt mikrobiologie   MeSH
• methan biosyntéza   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Rumunsko MeSH
• Slovenská republika MeSH
• Názvy látek
• methan MeSH
• RNA ribozomální 16S MeSH

Denaturant gradient gel electrophoresis (DGGE) enables insight into the diversity of the studied microbial communities on the basis of separation of PCR amplification products according to their nucleotide sequence composition. However, the success of the method is accompanied by the inherent appearance of various sequence artifacts that bias the impression of community structure by generating additional bands representing no virtual microbes. PCR-DGGE artifacts require optimization of the method when aiming at the phylogenetic identification of the selected DGGE bands. The aim of our study was to develop a procedure which will increase the reliability of the identification. Samples of rumen fluid were used for the optimization since they contain a complex microbial community that supports the generation of artifactual bands. An optimized procedure following band excision and elution of microbial DNA is proposed including nuclease treatment, selection of DNA polymerase with proofreading activity, and cloning prior to sequencing and identification analysis.

• MeSH
• bachor mikrobiologie   MeSH
• Bacteria klasifikace   genetika   izolace a purifikace   MeSH
• denaturační gradientová gelová elektroforéza metody   MeSH
• DNA bakterií genetika   MeSH
• fylogeneze MeSH
• techniky typizace bakterií metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH