Pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements

The high and sometimes inappropriate use of antibiotics has accelerated the development of antibiotic resistance, creating a major challenge for the sustainable treatment of infections world-wide. Bacterial communities often respond to antibiotic selection pressure by acquiring resistance genes, i.e. mobile genetic elements that can be shared horizontally between species. Environmental microbial communities maintain diverse collections of resistance genes, which can be mobilized into pathogenic bacteria. Recently, exceptional environmental releases of antibiotics have been documented, but the effects on the promotion of resistance genes and the potential for horizontal gene transfer have yet received limited attention. In this study, we have used culture-independent shotgun metagenomics to investigate microbial communities in river sediments exposed to waste water from the production of antibiotics in India. Our analysis identified very high levels of several classes of resistance genes as well as elements for horizontal gene transfer, including integrons, transposons and plasmids. In addition, two abundant previously uncharacterized resistance plasmids were identified. The results suggest that antibiotic contamination plays a role in the promotion of resistance genes and their mobilization from environmental microbes to other species and eventually to human pathogens. The entire life-cycle of antibiotic substances, both before, under and after usage, should therefore be considered to fully evaluate their role in the promotion of resistance.     

Truncating ribosomal protein S19 mutations and variable clinical expression in Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a rare constitutional erythroblastopenia characterized by a specific defect in erythroid differentiation. Recently, mutations in the gene encoding ribosomal protein (RP) S19 were found in a subset of patients with the disease. To characterize further RPS19 mutations and to investigate genotype-phenotype relationships, we screened this gene for mutations in patients with DBA by direct sequencing and Southern-blot analysis. Four novel mutations were identified. A G120A nonsense mutation resulting in a stop at codon 33, a C302T nonsense mutation introducing a premature stop at codon 84, and a 327delG which results in a frame shift at codon 103. A fourth and more complex mutation (TT157-158AA, 160insCT) resulting in a Leu45Gln and a frame shift from codon 47 was found in three affected family members with variable phenotypes. The different clinical expression for identical mutations suggest the presence of other modulating factors for the disease. The mutations presented here further support the role of RPS19 in erythropoietic differentiation and proliferation.     

Inference Procedures for Assessing Interobserver Agreement among Multiple Raters

We propose a new procedure for constructing inferences about a measure of interobserver agreement in studies involving a binary outcome and multiple raters. The proposed procedure, based on a chi-square goodness-of-fit test as applied to the correlated binomial model (Bahadur, 1961, in Studies in Item Analysis and Prediction, 158-176), is an extension of the goodness-of-fit procedure developed by Donner and Eliasziw (1992, Statistics in Medicine 11, 1511-1519) for the case of two raters. The new procedure is shown to provide confidence-interval coverage levels that are close to nominal over a wide range of parameter combinations. The procedure also provides a sample-size formula that may be used to determine the required number of subjects and raters for such studies.     

Stability of dissolved and soluble Fe(II) in shelf sediment pore waters and release to an oxic water column

Shelf sediments underlying temperate and oxic waters of the Celtic Sea (NW European Shelf) were found to have shallow oxygen penetrations depths from late spring to late summer (2.2–5.8 mm below seafloor) with the shallowest during/after the spring-bloom (mid-April to mid-May) when the organic carbon content was highest. Sediment porewater dissolved iron (dFe, <0.15 µm) mainly (>85%) consisted of Fe(II) and gradually increased from 0.4 to 15 μM at the sediment surface to ~100–170 µM at about 6 cm depth. During the late spring this Fe(II) was found to be mainly present as soluble Fe(II) (>85% sFe, <0.02 µm). Sub-surface dFe(II) maxima were enriched in light isotopes (δ⁵⁶Fe ?2.0 to ?1.5‰), which is attributed to dissimilatory iron reduction (DIR) during the bacterial decomposition of organic matter. As porewater Fe(II) was oxidised to insoluble Fe(III) in the surface sediment layer, residual Fe(II) was further enriched in light isotopes (down to ?3.0‰). Ferrozine-reactive Fe(II) was found in surface porewaters and in overlying core top waters, and was highest in the late spring period. Shipboard experiments showed that depletion of bottom water oxygen in late spring can lead to a substantial release of Fe(II). Reoxygenation of bottom water caused this Fe(II) to be rapidly lost from solution, but residual dFe(II) and dFe(III) remained (12 and 33 nM) after >7 h. Iron(II) oxidation experiments in core top and bottom waters also showed removal from solution but at rates up to 5-times slower than predicted from theoretical reaction kinetics. These data imply the presence of ligands capable of complexing Fe(II) and supressing oxidation. The lower oxidation rate allows more time for the diffusion of Fe(II) from the sediments into the overlying water column. Modelling indicates significant diffusive fluxes of Fe(II) (on the order of 23–31 µmol m^?2 day^?1) are possible during late spring when oxygen penetration depths are shallow, and pore water Fe(II) concentrations are highest. In the water column this stabilised Fe(II) will gradually be oxidised and become part of the dFe(III) pool. Thus oxic continental shelves can supply dFe to the water column, which is enhanced during a small period of the year after phytoplankton bloom events when organic matter is transferred to the seafloor. This input is based on conservative assumptions for solute exchange (diffusion-reaction), whereas (bio)physical advection and resuspension events are likely to accelerate these solute exchanges in shelf-seas.     

Rapid effects of progesterone on ciliary beat frequency in the mouse fallopian tube

Background  

The physiological regulation of ciliary beat frequency (CBF) within the fallopian tube is important for controlling the transport of gametes and the fertilized ovum. Progesterone influences gamete transport in the fallopian tube of several mammalian species. In fallopian tubes isolated from cows, treatment with 20 micromolar progesterone caused a rapid reduction of the tubal CBF. The aims of this study were to establish methodology for studying fallopian tube CBF in the mouse, as it is an important model species, and to investigate if progesterone rapidly affects the CBF of mice at nM concentrations.     

Comparing benthic biogeochemistry at a sandy and a muddy site in the Celtic Sea using a model and observations

Results from a 1D setup of the European Regional Seas Ecosystem Model (ERSEM) biogeochemical model were compared with new observations collected under the UK Shelf Seas Biogeochemistry (SSB) programme to assess model performance and clarify elements of shelf-sea benthic biogeochemistry and carbon cycling. Observations from two contrasting sites (muddy and sandy) in the Celtic Sea in otherwise comparable hydrographic conditions were considered, with the focus on the benthic system. A standard model parameterisation with site-specific light and nutrient adjustments was used, along with modifications to the within-seabed diffusivity to accommodate the modelling of permeable (sandy) sediments. Differences between modelled and observed quantities of organic carbon in the bed were interpreted to suggest that a large part (>90%) of the observed benthic organic carbon is biologically relatively inactive. Evidence on the rate at which this inactive fraction is produced will constitute important information to quantify offshore carbon sequestration. Total oxygen uptake and oxic layer depths were within the range of the measured values. Modelled depth average pore water concentrations of ammonium, phosphate and silicate were typically 5–20% of observed values at the muddy site due to an underestimate of concentrations associated with the deeper sediment layers. Model agreement for these nutrients was better at the sandy site, which had lower pore water concentrations, especially deeper in the sediment. Comparison of pore water nitrate with observations had added uncertainty, as the results from process studies at the sites indicated the dominance of the anammox pathway for nitrogen removal; a pathway that is not included in the model. Macrofaunal biomasses were overestimated, although a model run with increased macrofaunal background mortality rates decreased macrofaunal biomass and improved agreement with observations. The decrease in macrofaunal biomass was compensated by an increase in meiofaunal biomass such that total oxygen demand remained within the observed range. The permeable sediment modification reproduced some of the observed behaviour of oxygen penetration depth at the sandy site. It is suggested that future development in ERSEM benthic modelling should focus on: (1) mixing and degradation rates of benthic organic matter, (2) validation of benthic faunal biomass against large scale spatial datasets, (3) incorporation of anammox in the benthic nitrogen cycle, and (4) further developments to represent permeable sediment processes.     

A position-effect control for gene transposition: state of expression of yeast mating-type genes affects their ability to switch

Mating-type switches of the yeast Saccharomyces cerevisiae occur by unidirectional transposition of copies of unexpressed mating-type genetic information, residing at HML and HMR loci, into the expressed MAT locus. The HML and HMR loci remain unchanged. In contrast, in appropriate strains where the silent loci are also allowed to express, for example in mar mutants, efficient switches of HML and HMR are shown to occur at rates equivalent to those observed for MAT. Thus the position-effect control on the direction of transposition is affected by the state of expression of the locus under study the expressed loci switch regardless of their location.     

Development and evaluation of a core genome multilocus sequence typing scheme for Paenibacillus larvae,the deadly American foulbrood pathogen of honeybees

Paenibacillus larvae is the causative agent of the fatal American foulbrood disease in honeybees (Apis mellifera). Strain identification is vital for preventing the spread of the disease. To date, the most accessible and robust scheme to identify strains is the multilocus sequence typing (MLST) method. However, this approach has limited resolution, especially for epidemiological studies. As the cost of whole-genome sequencing has decreased and as it becomes increasingly available to most laboratories, an extended MLST based on the core genome (cgMLST) presents a valuable tool for high-resolution investigations. In this study, we present a standardized, robust cgMLST scheme for P. larvae typing using whole-genome sequencing. A total of 333 genomes were used to identify, validate and evaluate 2419 core genes. The cgMLST allowed fine-scale differentiation between samples that had the same profile using traditional MLST and allowed for the characterization of strains impossible by MLST. The scheme was successfully used to trace a localized Swedish outbreak, where a cluster of 38 isolates was linked to a country-wide beekeeping operation. cgMLST greatly enhances the power of a traditional typing scheme, while preserving the same stability and standardization for sharing results and methods across different laboratories.     

Energetics and Kinetics of the Prebiotic Synthesis of Simple Organic Acids and Amino Acids with the FES-H2S/FES2 Redox Couple as Reductant

The thermodynamics of the FeS-H2S/FeS2 redox couple and a select number of reactions critical to the synthesis of simple carboxylic acids and amino acids have been evaluated as a function of temperature. This thermodynamic evaluation shows that the reducing power of the FeS-H2S/FeS2 redox couple decreases drastically with temperature. By contrast the equilibria describing the reduction of CO2 and the formation of simple carboxylic acids and amino acids require an increasingly higher reducing power with temperature. Given these two opposite trends, the thermodynamic driving force for CO2 reduction and amino acid formation with the FeS-H2S/FeS2 redox couple as reductant diminishes with increasing temperature. An evaluation of the mechanism of CO2 reduction by the FeS-H2S/FeS2 couple suggests that the electron transfer from pyrrhotite to CO2 is hindered by a high activation energy, even though the overall reaction is thermodynamically favorable. By comparison the electron transfer from pyrrhotite to either CS2, CO, or HCOOH are far more facile. This theoretical analysis explains the results of experimental work by Keefe et al. (1995), Heinen and Lauwers (1996) and Huber and Wächtershäuser (1997). The implication is that a reaction sequence involving the reduction of CO2 with the FeS-H2S/FeS2 couple as reductant is unlikely to initiate a proposed prebiotic carbon fixation cycle (Wächtershäuser, 1988b; 1990b, 1990a, 1992, 1993).