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Streptomyces are of great interest in the pharmaceutical industry as they produce a plethora of secondary metabolites that act as antibacterial and antifungal agents. They may thrive on their own in the soil, or associate with other organisms, such as plants or invertebrates. Some soil-derived strains exhibit hemolytic properties when cultivated on blood agar, raising the question of whether hemolysis could be a virulence factor of the bacteria. In this work we examined hemolytic compound production in 23 β-hemolytic Streptomyces isolates; of these 12 were soil-derived, 10 were arthropod-associated, and 1 was plant-associated. An additional human-associated S. sp. TR1341 served as a control. Mass spectrometry analysis suggested synthesis of polyene molecules responsible for the hemolysis: candicidins, filipins, strevertene A, tetrafungin, and tetrin A, as well as four novel polyene compounds (denoted here as polyene A, B, C, and D) in individual liquid cultures or paired co-cultures. The non-polyene antifungal compounds actiphenol and surugamide A were also identified. The findings indicate that the ability of Streptomyces to produce cytolytic compounds (here manifested by hemolysis on blood agar) is an intrinsic feature of the bacteria in the soil environment and could even serve as a virulence factor when colonizing available host organisms. Additionally, a literature review of polyenes and non-polyene hemolytic metabolites produced by Streptomyces is presented.

Various micropollutants (including pharmaceuticals, UV filters, cosmetics, cleansers, etc.), are increasingly being detected in the environment because of their partial or incomplete removal from wastewater in wastewater treatment plants (WWTPs). These compounds can be taken up by plants if treated wastewater is used for irrigation or if biosolids are used for soil amendment. Previous studies focused on this subject were usually performed under greenhouse conditions. Therefore, the aim of this study was to evaluate a behavior of studied compounds under natural climatic conditions. Experiment was carried out directly in the wastewater treatment plant, where nine raised beds were installed, which contained soils taken from topsoil of two soil types Arenosol (two beds) and Cambisol (seven beds). Either maize or a mixture of different vegetables (lettuce, carrot and onion) was grown in these beds. Of the seven beds with the Cambisol, one of the beds containing either maize or vegetables was irrigated with tap water and other pair of beds (maize or vegetables) was irrigated with treated wastewater (i.e., WWTP effluent). In another pair of beds (maize or vegetables), composted sludge from WWTP Three beds containing both types of biosolids were irrigated with tap water. Only vegetables were grown in the beds with the Arenosol, which were irrigated with either tap water or treated wastewater. Climatic data, irrigation doses, drainage water volumes, soil water contents and plant growth were monitored during the experiment. Selected compounds concentrations were measured in WWTP effluent, both biosolids, drainage water, soils, and plant tissues. Fifty five of 77 analyzed compounds were quantified in WWTP effluent. Main compounds were pharmaceuticals (e.g., telmisartan, gabapentin, diclofenac, carbamazepine, and its metabolites), UV filters (e.g., phenylbenzimidazole sulfonic acid) and compounds used in anticorrosive paints (e.g., 1H-benzotriazole). In the case of both biosolids, the dominant compounds were telmisartan, sertraline, trazodone, citalopram, diclofenac (i.e., pharmaceuticals) and 1H-benzotriazole. Uptake of different compounds by plants depended on a plant and properties of organic molecules, which affected their sorption in soils and mainly their ability to be taken up and translocated in plants. For instance, pharmaceuticals carbamazepine, gabapentin tramadol and venlafaxine were mainly found in lettuce leaves. Acknowledgement: Study was supported by the Ministry of Agriculture of the Czech Republic, project "The fate of selected micropollutants, which occur in treated water and sludge from wastewater treatment plants, in soil" (No. QK21020080) and partly also by the European Structural and Investment Funds, projects NutRisk (No. CZ.02.1.01/0.0/0.0/16_019/0000845).

Streptomyces sp. TR1341 was isolated from the sputum of a man with a history of lung and kidney tuberculosis, recurrent respiratory infections, and COPD. It produces secondary metabolites associated with cytotoxicity and immune response modulation. In this study, we complement our previous results by identifying the genetic features associated with the production of these secondary metabolites and other characteristics that could benefit the strain during its colonization of human tissues (virulence factors, modification of the host immune response, or the production of siderophores). We performed a comparative phylogenetic analysis to identify the genetic features that are shared by environmental isolates and human respiratory pathogens. The results showed a high genomic similarity of Streptomyces sp. TR1341 to the plant-associated Streptomyces sp. endophyte_N2, inferring a soil origin of the strain. Putative virulence genes, such as mammalian cell entry (mce) genes were not detected in the TR1341’s genome. The presence of a type VII secretion system, distinct from the ones found in Mycobacterium species, suggests a different colonization strategy than the one used by other actinomycete lung pathogens. We identified a higher diversity of genes related to iron acquisition and demonstrated that the strain produces ferrioxamine B in vitro. These results indicate that TR1341 may have an advantage in colonizing environments that are low in iron, such as human tissue.

Methanogens represent the final decomposition guild in anaerobic degradation of organic matter, occurring in digestive tract of various invertebrates. However, factors determining their community structure and activity in distinct gut sections is still debated. In this study, we focused on the tropical millipede species Archispirostreptus gigas (Diplopoda, Spirostreptidae) and Epibolus pulchripes (Diplopoda, Pachybolidae), which release considerable amounts of methane. We aimed to characterize relationships between physicochemical parameters, methane production rates, and methanogen community structure in the two major gut sections, midgut and hindgut. Microsensor measurements revealed that both sections were strictly anoxic, with reducing conditions prevailing in both millipedes. Hydrogen concentration culminated in anterior hindgut of E. pulchripes In both species, the intestinal pH was significantly higher in the hindgut than in the midgut. An accumulation of acetate and formate in the gut indicated bacterial fermentation activities in the digestive tract of both species. Phylogenetic analysis of 16S rRNA genes showed a prevalence of Methanobrevibacter spp. (Methanobacteriales), accompanied by a small fraction of so far unclassified "Methanomethylophilaceae" (Methanomassiliicoccales), in both species, which suggests that methanogenesis is mostly hydrogenotrophic. We conclude that anoxic conditions, negative redox potential, and bacterial production of hydrogen and formate promotes the gut colonization by methanogens. The higher activities of methanogens in the hindgut are explained by the higher pH of this compartment and their association with ciliates, which are restricted to this compartment and present an additional source of methanogenic substrates.ImportanceMethane (CH4) is the second most important atmospheric greenhouse gas after CO2 and is believed to account for 17% of global warming. Methanogens are a diverse group of archaea and can be found in various anoxic habitats including digestive tract of plant-feeding animals. Termites, cockroaches, the larvae of scarab beetles, and millipedes are the only arthropods known to host methanogens and emit large amounts of methane. Millipedes are ranked third most important detritivores after termites and earthworms, and they are considered keystone species in many terrestrial ecosystems. Both methane producing and non-emitting species have been observed in millipedes, but what limits their methanogenic potential is not known. In the present study, we show that physico-chemical gut conditions and the distribution of symbiotic ciliates are important factors determining CH4 emission in millipedes. We also find great similarities to other methane-emitting arthropods, which might be associated with their similar plant-feeding habits.

Litter decomposition is an important source of dissolved organic matter (DOC). In peatlands with hardly degradable soil DOC, the input of DOC from vascular plant litter can represent an important source of nutrients and decomposable substrates for soil microorganisms. We established a laboratory incubation with the litters of three peatland plant dominants (Sphagnum fallax, Vaccinium myrtillus and Eriophorum vaginatum) for 200 days, aiming to study DOC production and its quality. The quality of the DOC leached from the litters was characterized by a distribution of C, N and P among molecular weight (MW) fractions ( 100 kDa), their aromaticity, composition of low molecular weight compounds (organic acids, sugars and amino acids) and DOC biodegradability. The leaves of vascular plants decomposed the fastest, releasing larger amounts of nutrients and easily degradable organic acids, sugars and amino acids to the leachate, when compared to their roots and Sphagnum. DOC distribution to the MW fractions did not differ among the litters. Neither the DOC distribution to the respective fractions nor leachate C/N/P stoichiometry were factors driving DOC biodegradability. Total dissolved P distribution to the MW fractions significantly differed among the litters, with Sphagnum being very specific: P was initially associated only with high molecular weight DOC, while later it was redistributed to the lower MW fractions and complexed with Al and Fe. The complexation may retard soluble reactive P leaching especially from Sphagnum litter. DOC biodegradability was higher for the vascular plant leaf litter than for the Sphagnum litter in the early stages of decomposition (20 days) but later decreased and became more uniform for all litters. These temporal differences (by decomposition stage) were more pronounced than those caused by litter origin. Our results indicate that mainly leaf litter of vascular plants can release significant amounts of DOC during the early stage of decomposition. This DOC is more aromatic with higher biodegradability and more nutrients (especially P) as compared to Sphagnum and can thus temporarily stimulate microbial activity in habitats dominated by the vascular plants.

International audience; Guano is a typical deposit found in caves derived from the excretions of bats and in minor cases of birds. These organic deposits decompose and form a series of acid fluids and gases that can interact with the minerals, sediments, and rocks present in the cave. Over sixty phosphates are known and described from caves, but guano decay also often leads to the formation of nitrates and sulfates. In this study twenty-two European caves were investigated for their guano-related secondary minerals. Using various analytical techniques, seventeen phosphates, along with one sulfate (gypsum), were recognized as secondary products of guano decay. Among those minerals, some are very rare and result from the interaction of guano leachates with clays, fluvial deposits, or pyrite. Some of these minerals are even found only in the studied caves (spheniscidite, robertsite). The most common minerals belong to the apatite group. The common mineral association present in fresh decaying guano is brushite-ardealite-gypsum, minerals that usually are not present in older deposits because of their higher solubility. Most minerals are in hydrated form because of the wet cave environment; however, some specific dry conditions may favor the presence of dehydrated minerals, such as berlinite, formed during guano combustion. Investigation on the acidity of guano piles shows pH values as low as 3.5 with an increase of acidity with age and depth. Finally, cave guano deposits should be better studied in the future because of their role in paleoenvironmental and paleoclimatic reconstructions and because it is important to better understand the origin of guano-related minerals, especially the phosphates and sulfates. Among all of the caves studied, Corona ’e sa Craba (Italy) and Domica-Baradla Cave (Slovakia-Hungary) are considered to be outstanding sites with respect to their phosphate mineralogy.

Pharmaceuticals may enter soils due to the application of treated wastewater or biosolids. Their leakage from soils towards the groundwater, and their uptake by plants is largely controlled by sorption and degradation of those compounds in soils. Standard laboratory batch degradation and sorption experiments were performed using soil samples obtained from the top horizons of seven different soil types and 6 pharmaceuticals (carbamazepine, irbesartan, fexofenadine, clindamycin and sulfamethoxazole), which were applied either as single-solute solutions or as mixtures (not for sorption). The highest dissipation half-lives were observed for citalopram (average DT50,S for a single compound of 152 ± 53.5 days) followed by carbamazepine (106.0 ± 17.5 days), irbesartan (24.4 ± 3.5 days), fexofenadine (23.5 ± 20.9 days), clindamycin (10.8 ± 4.2 days) and sulfamethoxazole (9.6 ± 2.0 days). The simultaneous application of all compounds increased the half-lives (DT50,M) of all compounds (particularly carbamazepine, citalopram, fexofenadine and irbesartan), which is likely explained by the negative impact of antibiotics (sulfamethoxazole and clindamycin) on soil microbial community. However, this trend was not consistent in all soils. In several cases, the DT50,S values were even higher than the DT50,M values. Principal component analyses showed that while knowledge of basic soil properties determines grouping of soils according sorption behavior, knowledge of the microbial community structure could be used to group soils according to the dissipation behavior of tested compounds in these soils. The derived multiple linear regression models for estimating dissipation half-lives (DT50,S) for citalopram, clindamycin, fexofenadine, irbesartan and sulfamethoxazole always included at least one microbial factor (either amount of phosphorus in microbial biomass or microbial biomarkers derived from phospholipid fatty acids) that deceased half-lives (i.e., enhanced dissipations). Equations for citalopram, clindamycin, fexofenadine and sulfamethoxazole included the Freundlich sorption coefficient, which likely increased half-lives (i.e., prolonged dissipations).

Peatland vegetation is composed mostly of mosses, graminoids and ericoid shrubs, and these have a distinct impact on peat biogeochemistry. We studied variation in soil microbial communities related to natural peatland microhabitats dominated by Sphagnum, cotton-grass and blueberry. We hypothesized that such microhabitats will be occupied by structurally and functionally different microbial communities, which will vary further during the vegetation season due to changes in temperature and photosynthetic activity of plant dominants. This was addressed using amplicon-based sequencing of prokaryotic and fungal rDNA and qPCR with respect to methane-cycling communities. Fungal communities were highly microhabitat-specific, while prokaryotic communities were additionally directed by soil pH and total N content. Seasonal alternations in microbial community composition were less important; however, they influenced the abundance of methane-cycling communities. Cotton-grass and blueberry bacterial communities contained relatively more α-Proteobacteria but less Chloroflexi, Fibrobacteres, Firmicutes, NC10, OD1 and Spirochaetes than in Sphagnum. Methanogens, syntrophic and anaerobic bacteria (i.e. Clostridiales, Bacteroidales, Opitutae, Chloroflexi and Syntrophorhabdaceae) were suppressed in blueberry indicating greater aeration that enhanced abundance of fungi (mainly Archaeorhizomycetes) and resulted in the highest fungi-to-bacteria ratio. Thus, microhabitats dominated by different vascular plants are inhabited by unique microbial communities, contributing greatly to spatial functional diversity within peatlands.

Manure application to soil affects the composition of soil bacterial communities as well as levels of antibiotic resistance genes in a manner dependent on various environmental and agricultural factors. These effects have been widely studied in arable soils, while the situation in grasslands that also receive inputs of fresh animal manure is largely unknown. Here, we focused on grassland soils under three different management types (pristine meadow, pasture, wintering pasture) from the same locality and compared the response of their bacterial communities to fresh cattle manure spiked or not with chlortetracycline. Soil microcosms were established either with soils (controls), + manure, + manure and chlortetracycline (CTC, 0.2 mg kg−1), and sampled at the same day (t0) and after 28 days (t28). Bacterial community composition was assessed by 454-pyrosequencing of 16S rRNA genes and correlations between the abundance of bacterial taxa and chosen tetracycline resistance genes (measured in our previous study) were calculated. Control soil bacterial communities at t0 differed significantly, reflecting differences in soil management history. Despite these initial differences, the responses of soil bacterial communities to cattle manure amendment showed similar trends in all three soils. Fresh cattle manure amendments significantly modified bacterial community structure in all soils at t0, increasing the relative abundance of OTUs affiliated to Clostridia and Bacilli. At 28d, all manure-amended soils showed a partial resilience, though they remained significantly different from controls. The effect of additional CTC was comparatively lower and only significant at t0 in all three soils. Several members of Firmicutes showed strong positive correlation with the abundance of tetracycline resistance genes tet(O), tet(Q) and tet(W), indicating that these taxa are potentially involved in dissemination of tetracycline resistance in manure-treated soils. In addition, this study indicated certain soil-management specific taxa, such as Geothrix and uncultured Burkholderiaceae, which could explain the previously observed differences in persistence of tetracycline resistance genes among soils.

Evaluating microbial responses to pharmaceuticals in agricultural soils is essential to improve our fundamental understanding of the fate of micropollutants and their potential implications for the environment and human health. In this study, we focused on the immediate (1 d), short- (13 d) and long-term effects (61 d) of pharmaceutical amendment on microbial communities in seven soils differing in physical chemical properties. Basal respiration was used to indicate microbial activity, while phospholipid fatty acids were used to determine microbial biomass and community structure. We identified four microbial responses to pharmaceutical amendment: stimulation, inhibition, stress and dormancy, which were highly significant in the short-term. The largest stimulatory effect accompanied by shifts in the microbial community structure towards fungi and G- bacteria was detected for sulfamethoxazole. The inhibitory effect was mainly observed for citalopram, irbesartan and pharmaceutical mixture in Cambisol Dystric with minor alterations in microbial community structure compare to a non-amended control. The stress effect was detected for all pharmaceuticals in Arenosol and Cambisol Haplic. While the dormancy effect was mainly observed in Chernozem Siltic for most of the pharmaceuticals. Microbial responses were highly dependent on the soil type, pharmaceutical compound and time, highlighting the importance to consider these parameters including a resilience of soil microbial communities to micropollutants within a long-term agricultural soil management.

Observations of earthworm colonization on sites where they were absent previously, such as on post-mining heaps, indicate that they may substantially alter soil conditions as well as soil microbial communities. However, long-term effects of earthworms on microbial activity of soil ecosystems remains poorly understood. To improve this situation, we set up microcosm experiments with clay only, with clay and litter, and with clay, litter and earthworms. Microcosms were sampled after 3, 60, 180 and 395 days of cultivation and divided into three subsamples: clay, litter, and drilosphere (earthworm tunnels and associates casts), when present. Microbial respiration, microbial biomass carbon, species composition, metabolic activity using BIOLOG, PLFA concentration, isoprenoid quinones content and DGGE fingerprints were analysed in all microcosms and at all subsamples. Microbial biomass carbon and basal soil respiration were significantly affected by type of microcosms and time of sampling. Microbial biomass in drilosphere was significantly higher than in clay and litter. The CFU (colony forming units) were significantly affected by presence of earthworms having significantly higher CFU in drilosphere than in all other subsamples. Analysis of PLFA indicated highest relative biomass of fungi and bacteria in clay from microcosms with earthworms. The DGGE analysis showed various compositions of microbial communities among subsamples from various treatments taken at different time. Finally, our results revealed significant effect of earthworms on activity and biomass of soil microbial community under long-term laboratory incubation.

The effects of earthworms Eisenia spp. on microorganisms of three different habitat soil, compost, and vermiculture were studied. Microbial communities of gut and fresh faeces of earthworms and substrates, the worms were collected from, were analysed. Microbial biomass and composition of the total microbial community were examined using phospholipid fatty acid (PLFA) biomarkers. Archaeal and bacterial communities were studied by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE). The culturing methods were used for assessment of counts, species richness and growth strategy of bacteria. In comparison with the substrates, the viable microbial biomass and the group of non-ester-linked PLFAs indicative of anaerobes were higher in both the gut and faeces of all earthworm populations. The prokaryotic community evaluated using DGGE revealed that archaeal community structure in the gut and faeces of earthworms from populations differed from that in substrates, whereas the passage through the gut had less influence on the bacterial community structure, particularly in compost and vermiculture. The counts of culturable bacteria increased due to gut passage only in forest and vermiculture populations. The fast-growing bacteria increased due to gut passage only in forest soil population. Actinobacteria (Arthrobacter, Microbacterium, Lechevalieria and Nesterenkonia) and Firmicutes (Bacillus and Paenibacillus) were generally favoured in substrates and their species richness decreased with gut passage, whereas Gammaproteobacteria (Aeromonas, Enterobacter, Pseudomonas and Salmonella) dominated in gut contents. The impact of earthworm activity on the microbial community was higher in nutrient-poor forest soil than in nutrient-rich compost and vermiculture substrates.

We found well-preserved, rocky artefacts that had been buried in the healing mud (fango) for more than 1,500 years at the Roman archaeological site at Varaždinske Toplice. This Roman pool with fango sediments and artefacts is fed from hot sulphidic springs. The fango exhibited nearly neutral pH, a high level of organic C, an elevated concentration of heavy metals and a high total microbial biomass, greater than 10(8) cells per gram of dry weight. The dominant microbes, assessed by molecular profiling (denaturing gradient gel electrophoresis), were affiliated with Thiobacillus, Sulfuricurvum, Polaromonas, and Bdellovibrio. Polymerase chain reaction screening for microbial functional guilds revealed the presence of sulphur oxidizers and methanogens but no sulphate reducers. The dominance of four Proteobacterial classes (α-, β-, δ- and ε-Proteobacteria) was confirmed by fluorescence in situ hybridisation; Actinobacteria were less abundant. Cultivable bacteria represented up to 23.4 % of the total bacterial counts when cultivation media was enriched with fango. These bacteria represented the genera Acinetobacter, Aeromonas, Arthrobacter, Comamonas, Ewingella, Flavobacterium, Pseudomonas, Rahnella and Staphylococcus. This study showed that the heterogeneous nature of fango at neutral pH created various microniches, which largely supported microbial life based on sulphur-driven, autotrophic denitrification.

Colabomycin E is a new member of the manumycin-type metabolites produced by the strain Streptomyces aureus SOK1/5-04 and identified by genetic screening from a library of streptomycete strains. The structures of colabomycin E and accompanying congeners were resolved. The entire biosynthetic gene cluster was cloned and expressed in Streptomyces lividans. Bioinformatic analysis and mutagenic studies identified components of the biosynthetic pathway that are involved in the formation of both polyketide chains. Recombinant polyketide synthases (PKSs) assembled from the components of colabomycin E and asukamycin biosynthetic routes catalyzing the biosynthesis of "lower" carbon chains were constructed and expressed in S. aureus SOK1/5-04 ΔcolC11-14 deletion mutant. Analysis of the metabolites produced by recombinant strains provided evidence that in both biosynthetic pathways the length of the lower carbon chain is controlled by an unusual chain-length factor supporting biosynthesis either of a triketide in asukamycin or of a tetraketide in colabomycin E. Biological activity assays indicated that colabomycin E significantly inhibited IL-1β release from THP-1 cells and might thus potentially act as an anti-inflammatory agent.

In areas used for cattle overwintering detrimental effects normally associated with grazing are intensified. Among the alterations observed, increases on the N availability and soil pH may highly influence structure of ammonia oxidizing microbes and thus influence nitrification pattern in soil. To evaluate this assumption, we assessed the abundance and diversity of ammonia oxidizing bacteria (AOB) and archaea (AOA) in three sites with different degrees of animal impact (severe, moderate or no impact) of an overwintering pasture by means of qPCR and T-RFLP of amoA genes. In areas where no animal impact could be identified AOA was dominating over AOB. However, AOB abundance increased as the degree of animal impact enhances, becoming most dominant in the severely impacted site. Interestingly, the diversity of AOB was the highest in the severely impacted area, where AOA diversity was the lowest. Obviously the pressure imposed by altered environmental conditions created by cattle husbandry lead to the selection of AOB and AOA populations, adapted to alkaline pH and higher ammonia concentration.

The effects of tree species differing in foliage and litter chemistry on the chemical, micro-morphological, and biological properties of soil were studied on post-mining sites afforested with one of six tree species (spruce, pine, larch, oak, lime, and alder) and also on sites left to natural succession (dominated by willow). The sites were located on a large colliery spoil heap that had been produced by the mining of coal in alkaline tertiary clays near the city of Sokolov, Czech Republic. Because no topsoil had been applied to the sites, soil development resulted from in situ interactions among the deposited overburden (spoil), trees, and soil biota. Soil formation differed markedly among sites afforested with different tree species. On sites with trees producing litter with a low C/N ratio (the deciduous species), the organic Oe layer was narrow or absent and a thick organomineral A layer was evident. On sites with trees producing litter with a high C/N ratio (the evergreen species), in contrast, a thick Oe layer and a thin A layer were evident. Besides C/N ratio, earthworm abundance and earthworm bioturbation activity (measured as the amount of earthworm casts in the topsoil) were the strongest predictors of A layer thickness and C accumulation in the mineral topsoil. Sites with higher C accumulation in mineral soil had higher microbial biomass and lower microbial respiration, which may have contributed to the higher C storage. The gradient of bioturbation was correlated with changes in the composition of the bacterial community and other soil biota, but partial correlation showed that the effects of litter quality and bioturbation were largely independent. Overall, the results indicate that the effect of tree species on soil development is substantially mediated by soil fauna activity and especially by earthworm bioturbation.

Rapidly growing mycobacteria (RGM) are common soil saprophytes, but certain strains cause infections in human and animals. The infections due to RGM have been increasing in past decades and are often difficult to treat. The susceptibility to antibiotics is regularly evaluated in clinical isolates of RGM, but the data on soil RGM are missing. The objectives of this study was to isolate RGM from four grassland soils with different impact of manuring, and assess their resistance to antibiotics and the ability to grow at 37°C and 42°C. Since isolation of RGM from soil is a challenge, a conventional decontamination method (NaOH/malachite green/cycloheximide) and a recent method based on olive oil/SDS demulsification were compared. The olive oil/SDS method was less efficient, mainly because of the emulsion instability and plate overgrowing with other bacteria. Altogether, 44 isolates were obtained and 23 representatives of different RGM genotypes were screened. The number of isolates per soil decreased with increasing soil pH, consistently with previous findings that mycobacteria were more abundant in low pH soils. Most of the isolates belonged to the Mycobacterium fortuitum group. The majority of isolates was resistant to 2-4 antibiotics. Multiresistant strains occurred also in a control soil that has a long history without the exposure to antibiotic-containing manure. Seven isolates grew at 37°C, including the species M. septicum and M. fortuitum known for infections in humans. This study shows that multiresistant RGM close to known human pathogens occur in grassland soils regardless the soil history of manuring.

Understanding the distribution of taxa and associated traits across different environments is one of the central questions in microbial ecology. High-throughput sequencing (HTS) studies are presently generating huge volumes of data to address this biogeographical topic. However, these studies are often focused on specific environment types or processes leading to the production of individual, unconnected datasets. The large amounts of legacy sequence data with associated metadata that exist can be harnessed to better place the genetic information found in these surveys into a wider environmental context. Here we introduce a software program, seqenv, to carry out precisely such a task. It automatically performs similarity searches of short sequences against the “nt” nucleotide database provided by NCBI and, out of every hit, extracts–if it is available–the textual metadata field. After collecting all the isolation sources from all the search results, we run a text mining algorithm to identify and parse words that are associated with the Environmental Ontology (EnvO) controlled vocabulary. This, in turn, enables us to determine both in which environments individual sequences or taxa have previously been observed and, by weighted summation of those results, to summarize complete samples. We present two demonstrative applications of seqenv to a survey of ammonia oxidizing archaea as well as to a plankton paleome dataset from the Black Sea. These demonstrate the ability of the tool to reveal novel patterns in HTS and its utility in the fields of environmental source tracking, paleontology, and studies of microbial biogeography. To install seqenv, go to: https://github.com/xapple/seqenv.

The effects of earthworms on soil CH4 emissions remain ambiguous, as previous studies revealed both positive and negative effects on net rates of CH4 production. These differences may be linked to land-use history such as pasturing intensity, as the treading of livestock and the input of faeces will affect physical and chemical soil characteristics that are important to methanogenic and methanotrophic bacteria. In order to concomitantly measure the effects of earthworms on the activities of both of these bacterial groups, we performed an isotope dilution assay to measure gross CH4 production and consumption rates. Samples from three soil types, differing mainly in pasture intensity over the past 15 years, were incubated for 8 weeks in the presence or absence of the vermicomposting species Eisenia andrei. Following the incubation, earthworms had a positive effect on gross process rates in the non-pasture control soil, had no effect in the moderate pasture soil, and had a negative effect in the intensive pasture soil. Phospholipid fatty-acid profiles and chemical characteristics of the fresh soil samples suggested that the non-pasture soil would likely produce less CH4 than the control soil. Likewise, methanogenic specific mcrA gene abundance following the incubation was two orders of magnitude higher in the two pasture soils than in the control soil. The isotope dilution assay revealed, however, higher gross CH4 production rates in the control soil. It was also found that gross CH4 transformation rates were neither correlated to net production rates nor to mcrA gene abundance. We discuss the value and limitations of the isotope dilution assay for understanding the factors and underlying mechanisms governing net CH4 emissions rates from soils.

The aim of the study was to evaluate the impact of cattle overwintering husbandry on composition of upland grassland soil archaeal, bacterial, and fungal communities by comparative fingerprinting (SSU rRNA denaturing gradient gel electrophoresis (DGGE)) of total (tDNA) and extracellular DNA (eDNA) extracted from three differently impacted soils (severely, moderately, and non-impacted soil) and cattle excrements. Cattle excrements carried a significant amount of viable microorganisms and eDNA, and as a result of its high external returns, the amounts of extractable eDNA in soil increased with cattle impact, being positively correlated with soil microbial biomass and activity. The soil eDNA fraction (2.8 to 5.7 μg g−1 dw) significantly contributed to the soil metagenome, representing 18–31 % of soil tDNA. The largest shift in soil community structure was observed for Archaea, followed by fungi and bacteria, indicating that soil bacteria possess the highest resilience to cattle-induced changes. Cattle excrements showed more diverse bacterial than archaeal and fungal communities. The specific DGGE bands of cattle excrements were also observed in community profiles of cattle-impacted soils, confirming the effect of cattle husbandry on composition of soil microbial communities, probably as a result of introduced rumen-borne microbes. Similar changes were also reflected in the eDNA-derived DGGE profiles, suggesting a higher contribution of bacteria to soil extracellular metagenome than Archaea and fungi. This study provided first evidences about the extracellular mobilome in cattle-impacted soils, carrying also genetic information of Archaea, and its susceptibility to impact by outdoor cattle husbandry.

Cattle treading accompanied by a high input of organic matter was previously found to favour methanogenesis in soils at a site used as winter pasture in outdoor cattle husbandry. In this current study, the phylogenetic microarray AnaeroChip revealed high methanogenic diversity in a cattle-impacted soil with predominance of Methanosarcina, and presence of Methanoculleus, Methanobacterium, Methanocalculus, Methanobrevibacter, Methanosaeta, Methanothermobacter, Methanogenium, Methanohalobium, and Methanolobus. The bioturbation effects of an epigeic earthworm, Eisenia andrei, on the methanogenic microbial community in cattle-impacted soil were studied in a 6-month laboratory microcosm experiment. The microarray showed that the methanogenic community was changed by addition of earthworms to the soil. The abundance of 16S rRNA Methanosarcina gene copies decreased two fold in soil with worms compared to soil without worms after 2 months of incubation and decreased three fold after 4 and 6 months of incubation. The biomass of anaerobic microorganisms, as determined by unsubstituted non-ester-linked phospholipid fatty acid analysis, decreased in soil incubated for 4 and 6 months with worms. The abundance of the methyl-coenzyme M reductase (mcrA) gene, which is involved in CH4 production and is present in all methanogens, was not, however, changed by worms, and addition of worms even increased the rate of methane production. This study provides the first data concerning interactions between earthworms and methanogens in cattle-impacted pasture soil. The results of this laboratory microcosm experiment indicate that E. andrei changes the composition of the soil methanogenic community but does not reduce the total abundance of the methanogenic community and methane production rate.

Rapidly growing mycobacteria (RGM) inhabit soil and water but certain strains represent a health risk for human and animals. Both clinical and soil RGM may be under selection pressure for resistance to tetracycline (TET) antibiotics, since tetracyclines are administrated to humans and farm animals, and TET residues enter soil through manuring; however, resistance to TET and the presence of TET-resistance genes have been assessed only in clinical isolates. We were therefore interested in comparing soil and clinical RGM in terms of TET resistance and the presence of TET-resistance genes. We used 44 RGM from grasslands with different exposure to animal manure, and 38 clinical RGM from Czech hospitals. There was no difference between the clinical and soil isolates in TET resistance, with >50% resistant isolates in both groups. otr(A), otr(B), tet(K), tet(L) or tet(M) were not detected in any soil or clinical isolate. In contrast, most isolates harbored tet(V) and tap, both encoding mycobacterial efflux pumps, including species where these genes have never been evidenced before. The phylogeny of tet(V) correlated with isolates’ BOX-PCR profiles, suggesting that this gene evolved along with mycobacterial genomes as a part of the intrinsic resistome. In certain cases, tet(V) and/or tap were found in TET-sensitive isolates, or inversely, were not found in resistant strains. Concluding, intrinsic efflux pumps may be more important for TET resistance than horizontally transferred genes in both soil and clinical RGM. Their simple presence, however, does not attest to resistance, and therefore their diversity, function and expression merit further research.

The genetic diversity of streptomycetes in colliery spoil heaps (Sokolov, Czech Republic) was investigated by restriction pattern analysis of 16S-internal transcribed spacer rDNA and 16S sequences. We sampled freshly excavated Miocene sediment (17-19-million-year-old) and four sites of primary succession (initial, early, middle, and late stages; aged 1-44 years) on the same sediment. Active bacteria were present even in fresh Miocene sediment, and the relative proportion of actinomycetes among total bacterial and their genetic diversity increased significantly with the age of the sampling site. The replacement of pioneer species by late succession species during succession was observed. Plate assays of Streptomyces strains revealed 27% antibiotic-producing strains. Screening for nonribosomal peptide synthases and type I polyketide synthases systems suggested that 90% and 55% streptomycetes, respectively, are putative producers of biologically active compounds. The frequencies of tetracycline-, amoxicillin-, and chloramphenicol-resistant streptomycetes were 6%, 9%, and 15%, respectively. These findings document the occurrence of genetic elements encoding antibiotic resistance genes and the production of antibiotics by streptomycetes located in pristine environments. Our results indicate key roles for ancient streptomycetes related to S. microflavus, S. spororaveus, and S. flavofuscus in pioneering community development in freshly excavated substrates.

We compared two denaturing gradient gel electrophoresis (DGGE) systems—DCode (Biorad, Hercules, CA, USA) and PhorU (Ingeny, Leiden, NL), performing community level 16S and 18S rRNA gene fragment-PCR-DGGE with total DNA extracted from upland pasture soil used for outdoor cattle husbandry. The methodological evaluation of the DGGE apparatus as parameter influencing DGGE fingerprinting, based on cluster analysis of soil bacterial and fungal community fingerprints, was made in terms of the resulting information about microbial community structures and their response to different degrees of cattle impact. Although the comparative DGGE analysis with different DGGE systems provided similar clustering of microbial community structures in correlation with the degree of cattle impact, our results suggest the DGGE system to be a factor influencing DGGE analysis. To our knowledge this is the first attempt to investigate the hypothetical impact of the DGGE system due to different technical characteristics, recommending the use of one and the same DGGE apparatus throughout an experiment, if the monitoring of microbial community structures requires multiple gel-to-gel analysis.

The molecular diversity of Archaea in a bat guano pile in Cave Domica (Slovakia), temperate cave ecosystem with significant bat colony (about 1600 individuals), was examined. The guano pile was created mainly by an activity of the Mediterranean horseshoe bat (Rhinolophus euryale) and provides a source of organic carbon and other nutrients in the oligotrophic subsurface ecosystem. The upper and the basal parts of guano surface were sampled where the latter one had higher pH and higher admixture of limestone bedrock and increased colonization of invertebrates. The relative proportion of Archaea determined using CARD-FISH in both parts was 3.5-3.9 % (the basal and upper part, respectively). The archaeal community was dominated by non-thermophilic Crenarchaeota (99 % of clones). Phylogenetic analysis of 115 16S rDNA sequences revealed the presence of Crenarchaeota previously isolated from temperate surface soils (group 1.1b, 62 clones), deep subsurface acid waters (group 1.1a, 52 clones) and Euryarchaeota (1 clone). Four of the analyzed sequences were found to have little similarity to those in public databases. The composition of both archaeal communities differed, with respect to higher diversity of Archaea in the upper part of the bat guano pile. High diversity archaeal population is present in the bat guano deposit and consists of both soil- and subsurface-born Crenarchaeota.

Pasture soils used for cattle overwintering may represent significant sources of N 2 O emissions from soils. Therefore, the long-term effect of cattle overwintering on the abundance and activity of a denitrifying community was explored. The study was performed at a cattle overwintering area in South Bohemia (Czech Republic), where three sites differing in the degree of animal impact were selected: severely impacted (SI) and moderately impacted (MI), as well as a control site with no impact (NI). N 2 O flux measurement and soil sampling were performed in spring and fall of 2005. The activity was measured in terms of potential denitrification activity. Bacterial nirK , nirS and nosZ genes were used as functional markers of the denitrifying communities; abundance was analyzed using a real-time PCR assay. Surprisingly, in situ N 2 O emissions were the highest in spring at MI and significantly differed from those at SI and NI, while in autumn, rates of emissions generally decreased. In contrast potential denitrification rates were highest at SI, followed by MI, and the lowest at NI. An overall significant shift in N 2 O/N 2 molar ratio was shown in cattle impacted sites. The highest abundance of all genes measured at both sampling times was found at site SI, whereas at site MI increased numbers were observed only in spring. Our results indicate a strong influence of cattle on the abundance as well as the activity of microbes involved in denitrification.

A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike “classical” primary metabolism ALAS, the C5N unit-forming cALAS (cyclizing ALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of “classical” ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87 % putatively encoding cALAS. Phylogenetic analysis of the hemA homologues revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GeneBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

Archaea and bacteria are important drivers for nutrient transformations in soils and catalyse the production and consumption of important greenhouse gases. In this study, we investigate changes in archaeal and bacterial communities of four Czech grassland soils affected by outdoor cattle husbandry. Two show short-term (3 years; STI) and long-term impact (17 years; LTI), one is regenerating from cattle impact (REG) and a control is unaffected by cattle (CON). Cattle manure (CMN), the source of allochthonous microbes, was collected from the same area. We used pyrosequencing of 16S rRNA genes to assess the composition of archaeal and bacterial communities in each soil type and CMN. Both short- and long- term cattle impact negatively altered archaeal and bacterial diversity, leading to increase of homogenization of microbial communities in overwintering soils over time. Moreover, strong shifts in the prokaryotic communities were observed in response to cattle overwintering, with the greatest impact on archaea. Oligotrophic and acidophilic microorganisms (e.g. Thaumarchaeota, Acidobacteria, and α-Proteobacteria) dominated in CON and expressed strong negative response to increased pH, total C and N. Whereas copiotrophic and alkalophilic microbes (e.g. methanogenic Euryarchaeota, Firmicutes, Chloroflexi, Actinobacteria, and Bacteroidetes) were common in LTI showing opposite trends. Crenarchaeota were also found in LTI, though their trophic interactions remain cryptic. Firmicutes, Bacteroidetes, Methanobacteriaceae, and Methanomicrobiaceae indicated the introduction and establishment of faecal microbes into the impacted soils, while Chloroflexi and Methanosarcinaceae suggested increased abundance of soil-borne microbes under altered environmental conditions. The observed changes in prokaryotic community composition may have driven corresponding changes in soil functioning.

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.

Acid sulphate (AS) soils along the Baltic coasts contain significant amounts of organic carbon and nitrogen in their subsoils. The abundance, composition, and activity of microbial communities throughout the AS soil profile were analysed. The data from a drained AS soil were compared with those from a drained non-AS soil and a pristine wetland soil from the same region. Moreover, the potential production of methane, carbon dioxide, and nitrous oxide from the soils was determined under laboratory conditions. Direct microscopic counting, glucose-induced respiration (GIR), whole cell hybridisation, and extended phospholipid fatty acid (PLFA) analysis confirmed the presence of abundant microbial communities in the topsoil and also in the deepest Cg2 horizon of the AS soil. The patterns of microbial counts, biomass and activity in the profile of the AS soil and partly also in the non-AS soil therefore differed from the general tendency of gradual decreases in soil profiles. High respiration in the deepest Cg2 horizon of the AS soil (5.66 μg Cg(-1)h(-1), as compared to 2.71 μg Cg(-1)h(-1) in a top Ap horizon) is unusual but reasonable given the large amount of organic carbon in this horizon. Nitrous oxide production peaked in the BCgc horizon of the AS and in the BC horizon of the non-AS soil, but the peak value was ten-fold higher in the AS soil than in the non-AS soil (82.3 vs. 8.6 ng Ng(-1)d(-1)). The data suggest that boreal AS soils on the Baltic coast contain high microbial abundance and activity. This, together with the abundant carbon and total and mineral nitrogen in the deep layers of AS soils, may result in substantial gas production. Consequently, high GHG emissions could occur, for example, when the generally high water table is lowered because of arable farming.