Extraction of Microbial and Host DNA, RNA, and Proteins from Oak Bark Tissue

The application of high-throughput nucleic acid and protein sequencing technologies is transforming our understanding of plant microbiomes and their interactions with their hosts in health and disease. However, progress in studying host-microbiome interactions in above-ground compartments of the tree (the phyllosphere) has been hampered due to high concentrations of phenolic compounds, lignin, and other compounds in tree bark that severely limit the success of DNA, RNA, and protein extraction. Here we present modified sample-preparation and kit-based protocols for the extraction of host and microbiome DNA and RNA from oak (Quercus robus and Quercus petraea) bark tissue for subsequent high-throughput sequencing. In addition, reducing the quantity of bark tissue used for an established protein extraction protocol yielded high quality protein for parallel analysis of the oak-microbiota metaproteome. These procedures demonstrate the successful extraction of nucleic acids and proteins from oak tissue using as little as 50 mg of sample input, producing sufficient quantities for nucleic acid sequencing and protein mass spectrometry of tree stem tissues and their associated microbiota. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.Peer reviewe

RNA-viromics reveals diverse communities of soil RNA viruses with the potential to affect grassland ecosystems across multiple trophic levels

The distribution and diversity of RNA viruses in soil ecosystems are largely unknown, despite their significant impact on public health, ecosystem functions, and food security. Here, we characterise soil RNA viral communities along an altitudinal productivity gradient of peat, managed grassland and coastal soils. We identified 3462 viral contigs in RNA viromes from purified virus-like-particles in five soil-types and assessed their spatial distribution, phylogenetic diversity and potential host ranges. Soil types exhibited minimal similarity in viral community composition, but with >10-fold more viral contigs shared between managed grassland soils when compared with peat or coastal soils. Phylogenetic analyses predicted soil RNA viral communities are formed from viruses of bacteria, plants, fungi, vertebrates and invertebrates, with only 12% of viral contigs belonging to the bacteria-infecting Leviviricetes class. 11% of viral contigs were found to be most closely related to members of the Ourmiavirus genus, suggesting that members of this clade of plant viruses may be far more widely distributed and diverse than previously thought. These results contrast with soil DNA viromes which are typically dominated by bacteriophages. RNA viral communities, therefore, have the potential to exert influence on inter-kingdom interactions across terrestrial biomes

Sediment Composition Influences Spatial Variation in the Abundance of Human Pathogen Indicator Bacteria within an Estuarine Environment

Faecal contamination of estuarine and coastal waters can pose a risk to human health, particularly in areas used for shellfish production or recreation. Routine microbiological water quality testing highlights areas of faecal indicator bacteria (FIB) contamination within the water column, but fails to consider the abundance of FIB in sediments, which under certain hydrodynamic conditions can become resuspended. Sediments can enhance the survival of FIB in estuarine environments, but the influence of sediment composition on the ecology and abundance of FIB is poorly understood. To determine the relationship between sediment composition (grain size and organic matter) and the abundance of pathogen indicator bacteria (PIB), sediments were collected from four transverse transects of the Conwy estuary, UK. The abundance of culturable Escherichia coli, total coliforms, enterococci, Campylobacter, Salmonella and Vibrio spp. in sediments was determined in relation to sediment grain size, organic matter content, salinity, depth and temperature. Sediments that contained higher proportions of silt and/or clay and associated organic matter content showed significant positive correlations with the abundance of PIB. Furthermore, the abundance of each bacterial group was positively correlated with the presence of all other groups enumerated. Campylobacter spp. were not isolated from estuarine sediments. Comparisons of the number of culturable E. coli, total coliforms and Vibrio spp. in sediments and the water column revealed that their abundance was 281, 433 and 58-fold greater in sediments (colony forming units (CFU)/100 g) when compared with the water column (CFU/100 ml), respectively. These data provide important insights into sediment compositions that promote the abundance of PIB in estuarine environments, with important implications for the modelling and prediction of public health risk based on sediment resuspension and transport

Decay rates of faecal indicator bacteria from sewage and ovine faeces in brackish and freshwater microcosms with contrasting suspended particulate matter concentrations

AbstractTo safeguard human health, legislative measures require the monitoring of faecal indicator bacteria (FIB) concentrations in recreational and shellfish waters. Consequently, numerous studies have focussed on FIB survival in the water column and more recently in estuarine sediments. However, there is a paucity of information regarding the influence of contrasting suspended particulate matter (SPM) concentrations on the survival of FIB in the water column of estuaries. Here, microcosms containing freshwater or brackish water with low, high and extreme SPM concentrations were inoculated with sewage and ovine faeces and the decay rate of Escherichia coli, coliforms and enterococci were determined by enumeration over five consecutive days. E. coli derived from ovine faeces proliferated and persisted at high levels in both freshwater and brackish microcosms (no decay), whereas ovine enterococci demonstrated a net decay over the duration of the experiment. Furthermore, SPM concentration had a significant effect on the decay rates of both E. coli and enterococci from ovine faeces in brackish microcosms, but decay rate was greater at low SPM concentrations for E. coli, whereas the opposite was observed for enterococci, whose decay rates increased as SPM concentration increased. E. coli, enterococci and coliforms derived from wastewater demonstrated a net decay in both freshwater and brackish microcosms, with contrasting effects of SPM concentration on decay rate. In addition, some FIB groups demonstrated contrasting responses (decay or proliferation) in the first 24h following inoculation into freshwater versus brackish microcosms. Overall, SPM concentrations influenced the proliferation and decay rates of FIB in brackish waters, but had minimal influence in freshwater. These results demonstrate that the survival rates of FIB in aquatic environments are system specific, species and source dependent, and influenced by SPM concentration. This study has important implications for catchment-based risk assessments and source apportionment of FIB pollution in aquatic environments

Using chemical, microbial and fluorescence techniques to understand contaminant sources and pathways to wetlands in a conservation site

Nutrients and faecal contaminants can enter wetland systems in a number of ways, with both biological and potentially human-health implications. In this study we used a combination of inorganic chemistry, dissolved organic matter (DOM) fluorescence and Escherichia coli and total coliform (TC) count techniques to study the sources and multiple pathways of contamination affecting a designated sand dune site of international conservation importance, surrounded by agricultural land. Analysis of stream samples, groundwater and dune slack wetlands revealed multiple input pathways. These included riverbank seepage, runoff events and percolation of nutrients from adjacent pasture into the groundwater, as well as some on-site sources. The combined techniques showed that off-site nutrient inputs into the sand dune system were primarily from fertilisers, revealed by high nitrate concentrations, and relatively low tryptophan-like fulvic-like ratios < 0.4 Raman units (R.U.). The E. coli and TC counts recorded across the site confirm a relatively minor source of bacterial and nutrient inputs from on-site grazers. Attenuation of the nutrient concentrations in streams, in groundwater and in run-off inputs occurs within the site, restoring healthier groundwater nutrient concentrations showing that contaminant filtration by the sand dunes provides a valuable ecosystem service. However, previous studies show that this input of nutrients has a clear adverse ecological impact

The interaction of human microbial pathogens, particulate material and nutrients in estuarine environments and their impacts on recreational and shellfish waters

Anthropogenic activities have increased the load of faecal bacteria, pathogenic viruses and nutrients in rivers, estuaries and coastal areas through point and diffuse sources such as sewage discharges and agricultural runoff. These areas are used by humans for both commercial and recreational activities and are therefore protected by a range of European Directives. If water quality declines in these zones, significant economic losses can occur. Identifying the sources of pollution, however, is notoriously difficult due to the ephemeral nature of discharges, their diffuse source, and uncertainties associated with transport and transformation of the pollutants through the freshwater–marine interface. Further, significant interaction between nutrients, microorganisms and particulates can occur in the water column making prediction of the fate and potential infectivity of human pathogenic organisms difficult to ascertain. This interaction is most prevalent in estuarine environments due to the formation of flocs (suspended sediment) at the marine-freshwater interface. A range of physical, chemical and biological processes can induce the co-flocculation of microorganisms, organic matter and mineral particles resulting in pathogenic organisms becoming potentially protected from a range of biotic (e.g. predation) and abiotic stresses (e.g. UV, salinity). These flocs contain and retain macro- and micro- nutrients allowing the potential survival, growth and transfer of pathogenic organisms to commercially sensitive areas (e.g. beaches, shellfish harvesting waters). The flocs can either be transported directly to the coastal environment or can become deposited in the estuary forming cohesive sediments where pathogens can survive for long periods. Especially in response to storms, these sediments can be subsequently remobilised releasing pulses of potential pathogenic organisms back into the water column leading to contamination of marine waters long after the initial contamination event occurred. Further work, however, is still required to understand and predict the potential human infectivity of pathogenic organisms alongside the better design of early warning systems and surveillance measures for risk assessment purposes

Reionization: Characteristic Scales, Topology and Observability

Recently the numerical simulations of the process of reionization of the universe at z>6 have made a qualitative leap forward, reaching sufficient sizes and dynamic range to determine the characteristic scales of this process. This allowed making the first realistic predictions for a variety of observational signatures. We discuss recent results from large-scale radiative transfer and structure formation simulations on the observability of high-redshift Ly-alpha sources. We also briefly discuss the dependence of the characteristic scales and topology of the ionized and neutral patches on the reionization parameters.Comment: 4 pages, 5 figures (4 in color), to appear in Astronomy and Space Science special issue "Space Astronomy: The UV window to the Universe", proceedings of 1st NUVA Conference ``Space Astronomy: The UV window to the Universe'' in El Escorial (Spain

A comparison of precipitation and filtration-based SARS-CoV-2 recovery methods and the influence of temperature, turbidity, and surfactant load in urban wastewater

Wastewater-based epidemiology (WBE) has become a complimentary surveillance tool during the SARS-CoV-2 pandemic. Viral concentration methods from wastewater are still being optimised and compared, whilst viral recovery under different wastewater characteristics and storage temperatures remains poorly understood. Using urban wastewater samples, we tested three viral concentration methods; polyethylene glycol precipitation (PEG), ammonium sulphate precipitation (AS), and CP select™ InnovaPrep® (IP) ultrafiltration. We found no major difference in SARS-CoV-2 and faecal indicator virus (crAssphage) recovery from wastewater samples (n = 46) using these methods, PEG slightly (albeit non-significantly), outperformed AS and IP for SARS-CoV-2 detection, as a higher genome copies per litre (gc/l) was recorded for a larger proportion of samples. Next generation sequencing of 8 paired samples revealed non-significant differences in the quality of data between AS and IP, though IP data quality was slightly better and less variable. A controlled experiment assessed the impact of wastewater suspended solids (turbidity; 0–400 NTU), surfactant load (0–200 mg/l), and storage temperature (5–20 °C) on viral recovery using the AS and IP methods. SARS-CoV-2 recoveries were >20% with AS and  0.05), whilst surfactant and storage temperature combined were significant negative correlates (p < 0.001 and p < 0.05, respectively). In conclusion, our results show that choice of methodology had small effect on viral recovery of SARS-CoV-2 and crAssphage in wastewater samples within this study. In contrast, sample turbidity, storage temperature, and surfactant load did affect viral recovery, highlighting the need for careful consideration of the viral concentration methodology used when working with wastewater samples