Mechanisms of reduced susceptibility and genotypic prediction of antibiotic resistance in Prevotella isolated from cystic fibrosis (CF) and non-CF patients

OBJECTIVES: To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cultured from patients with cystic fibrosis (CF), patients with invasive infection and healthy control subjects and to determine whether genotype can be used to predict phenotypic resistance.

METHODS: The susceptibility of 157 Prevotella isolates to seven antibiotics was compared, with detection of resistance genes (cfxA-type gene, ermF and tetQ), mutations within the CfxA-type β-lactamase and expression of efflux pumps.

RESULTS: Prevotella isolates positive for a cfxA-type gene had higher MICs of amoxicillin and ceftazidime compared with isolates negative for this gene (P < 0.001). A mutation within the CfxA-type β-lactamase (Y239D) was associated with ceftazidime resistance (P = 0.011). The UK CF isolates were 5.3-fold, 2.7-fold and 5.7-fold more likely to harbour ermF compared with the US CF, UK invasive and UK healthy control isolates, respectively. Higher concentrations of azithromycin (P < 0.001) and clindamycin (P < 0.001) were also required to inhibit the growth of the ermF-positive isolates compared with ermF-negative isolates. Furthermore, tetQ-positive Prevotella isolates had higher MICs of tetracycline (P = 0.001) and doxycycline (P < 0.001) compared with tetQ-negative isolates. Prevotella spp. were also shown, for the first time, to express resistance nodulation division (RND)-type efflux pumps.

CONCLUSIONS: This study has demonstrated that Prevotella isolated from various sources harbour a common pool of resistance genes and possess RND-type efflux pumps, which may contribute to tetracycline resistance. The findings indicate that antibiotic resistance is common in Prevotella spp., but the genotypic traits investigated do not reflect phenotypic antibiotic resistance in every instance.

A novel experimental approach to investigate radiolysis processes in liquid samples using collimated radiation sources

Here is detailed a novel and low-cost experimental method for high-throughput automated fluid sample irradiation. The sample is delivered via syringe pump to a nozzle, where it is expressed in the form of a hanging droplet into the path of a beam of ionising radiation. The dose delivery is controlled by an upstream lead shutter, which allows the beam to reach the droplet for a user defined period of time. The droplet is then further expressed after irradiation until it falls into one well of a standard microplate. The entire system is automated and can be operated remotely using software designed in-house, allowing for use in environments deemed unsafe for the user (synchrotron beamlines, for example). Depending on the number of wells in the microplate, several droplets can be irradiated before any human interaction is necessary, and the user may choose up to 10 samples per microplate using an array of identical syringe pumps, the design of which is described here. The nozzles consistently produce droplets of 25.1 ± 0.5 μl.

Yellow earthworms:Distinctive pigmentation associated with arsenic- and copper-tolerance in Lumbricus rubellus

Lumbricus rubellus Hoffmeister, inhabiting soil at the 19th century Devon Great Consols mine at Tavistock, Devon, UK, show high tolerance to Cu- and As-toxicity and frequently have a striking yellow coloration. Specimens from this site (mature and immature) and from an uncontaminated site on Lancaster University campus (mature) were photographed, and the slide images digitized and analyzed. All L. rubellus showed reddish-purple pigmentation of the body wall that declined in intensity posteriorly. The metal- and metalloid-resistant earthworms, whether mature or immature, showed yellowing in the posterior half of the body. The source of the coloration was intense yellow pigmentation of the chloragogenous tissue surrounding the alimentary canal. The yellow pigmentation is masked by reddish-purple body wall pigmentation anteriorly. Total As concentrations in tissues were determined for the anterior, middle and posterior sections of resistant and non-resistant L. rubellus. Highest concentrations were in the middle sections of the mature and immature resistant L. rubellus (36.17 ± 19.77 and 27.77 ± 9.02 mg As kg^-1, respectively). Resistant immature L. rubellus lost condition over 28 d in soil treated with 750 mg As kg^-1, possibly due to a higher metabolism, whilst there was no loss in condition for resistant mature L. rubellus in the treated soil. As far as the authors are aware, this is the first report of yellow pigmentation of this kind in earthworms. The pigmentation may provide a useful indicator of exposure/resistance to soil contamination. © 2002 Elsevier Science Ltd. All rights reserved.

Arbuscular mycorrhizal fungi confer enhanced arsenate resistance on Holcus lanatus

The role of arbuscular mycorrhizal fungi (AMF) in arsenate resistance in arbuscular mycorrhizal associations is investigated here for two Glomus spp. isolated from the arsenate-resistant grass Holcus lanatus. Glomus mosseae and Glomus caledonium were isolated from H. lanatus growing on an arsenic-contaminated mine-spoil soil. The arsenate resistance of spores was compared with nonmine isolates using a germination assay. Short-term arsenate influx into roots and long-term plant accumulation of arsenic by plants were also investigated in uninfected arsenate resistant and nonresistant plants and in plants infected with mine and nonmine AMF. Mine AMF isolates were arsenate resistant compared with nonmine isolates. Resistant and nonresistant G. mosseae both suppressed high-affinity arsenate/phosphate transport into the roots of both resistant and nonresistant H. lanatus. Resistant AMF colonization of resistant H. lanatus growing in contaminated mine spoil reduced arsenate uptake by the host. We conclude that AMF have evolved arsenate resistance, and conferred enhanced resistance on H. lanatus.

Survival and behaviour of the earthworms Lumbricus rubellus and Dendrodrilus rubidus from arsenate-contaminated and non-contaminated sites

Two arsenic- and heavy metal-contaminated mine-spoil sites, at Carrock Fell, Cumbria and Devon Great Consols Mine, Devon, were found to support populations of the earthworms Lumbricus rubellus Hoffmeister and Dendrodrilus rubidus (Savigny). L. rubellus and D. rubidus collected from the Devon site and an uncontaminated site were kept for 28 days in uncontaminated soil and in soil containing sodium arsenate (494 mg As kg^-1). The state of the specimens was recorded every 7 days using a semi-quantitative assessment of earthworm health (condition index, C. I.). The C. I. remained high for all specimens except those of L. rubellus and D. rubidus from uncontaminated sites, which displayed 60 and 10% mortality, respectively. L. rubellus collected from the Carrock Fell site, and L. rubellus and D. rubidus from an uncontaminated site, burrowed as rapidly into soil containing up to 1235 mg As kg^-1 in the form of sodium arsenate as into uncontaminated soil when placed on the soil surface. When earthworms were allowed a choice between uncontaminated soil and soil contaminated with sodium arsenate in concentrations of up to 1235 mg As kg^-1, the threshold concentration for avoidance of contaminated soil was lower for L. rubellus and D. rubidus from uncontaminated soil than for specimens from contaminated soil. There was no significant effect of pH on soil discrimination. The LC₅₀ concentration of As for L. rubellus from Devon Great Consols was significantly higher (P < 0.001) than for L. rubellus from the uncontaminated site: 1510 and 96 mg As kg^-1, respectively. © 2001 Elsevier Science Ltd. All rights reserved.

Arsenate resistance in the ericoid mycorrhizal fungus Hymenoscyphus ericae

• Differential resistance to arsenate (AsO₄ ^3-) is demonstrated here among populations of the ericoid mycorrhizal fungus Hymenoscyphus ericae isolated from Calluna vulgaris in natural heathland soils and soils contaminated with AsO₄ ^3-. • Isolates (c. 25) of the fungus from each of two As and Cu mine sites, and a natural heathland site, were screened for AsO₄ ^3- and Cu²⁺ resistance by growing isolates in media containing a range of AsO₄ ^3- and Cu²⁺ concentrations. • H. ericae populations from the mine sites demonstrated resistance to AsO₄ ^3- compared with the heathland population; the mine-site populations producing significant growth at the highest AsO₄ ^3- concentration (4.67 mol m^-3), whereas growth of the heathland population was almost completely inhibited. EC₅₀ values for mine-site isolates were estimated to be 5-41-times higher than the heathland population. All isolates produced identical responses to increasing Cu²⁺ concentrations, with no differences observed between mine-site and heathland isolates. • Populations of H. ericae on the contaminated mine sites have developed adaptive resistance to AsO₄ ^3-. By contrast, Cu²⁺ resistance appears to be constitutive.

Genetic diversity of root-associated fungal endophytes from Calluna vulgaris at contrasting field sites

A total of 107 putative ericoid mycorrhizal endophytes were isolated from hair roots of Calluna vulgaris from two abandoned arsenic/copper mine sites and a natural heathland site in southwest England. The endophytes were initially grouped as 14 RFLP types, based on the results of ITS-RFLP analysis using the restriction endonucleases Hinf I, Rsa I and Hae III. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with sequences for known ericoid mycorrhizal endophytes and selected ascomycetes. The majority of endophyte isolates (62-92%) from each site were identified as Hymenoscyphus ericae, but a number of other less common mycorrhizal RFLP types were also identified, all of which appear to have strong affinities with the order Leotiales. None of the less common RFLP types was isolated from C. vulgaris at more than one field site. Neighbour-joining analysis indicated similarities between the endophytes from C. vulgaris and mycorrhizal endophytes isolated from other Ericaceae and Epacridaceae hosts in North America and Australia.

Phosphorus nutrition of arsenate-tolerant and nontolerant phenotypes of velvetgrass

Velvetgrass (Holcus lanatus L.), also known as Yorkshire fog grass, has evolved tolerance to high levels of arsenate, and this adaptation involves reduced accumulation of arsenate through the suppression of the high affinity phosphate-arsenate uptake system. To determine the role of P nutrition in arsenate tolerance, inhibition kinetics of arsenate influx by phosphate were determined. The concentration of inhibitor required to reduce maximum influx (V(max)) by 50%, K₁, of phosphate inhibition of arsenate influx was 0.02 mol m^-3 in both tolerant and nontolerant clones. This was compared with the concentration where influx is 50% of maximum, a K(m), for arsenate influx of 0.6 mol m^-3 for tolerants and 0.025 mol m^-3 for nontolerants and, therefore, phosphate was much more effective at inhibiting arsenate influx in tolerant genotypes. The high affinity phosphate uptake system is inducible under low plant phosphate status, this increasing plant phosphate status should increase tolerance by decreasing arsenate influx. Root extension in arsenate solutions of tolerant and nontolerant tillers grown under differing phosphate nutritional regimes showed that indeed, increased plant P status increased the tolerance to arsenate of both tolerant and nontolerant clones. That plant P status increased tolerance again argues that P nutrition has a critical role in arsenate tolerance. To determine if short term flux and solution culture studies were relevant to As and P accumulation in soils, soil and plant material from a range of As contaminated sites were analyzed. As predicted from the short-term competition studies, P was accumulated preferentially to As in arsenate tolerant clones growing on mine spoil soils even when acid extractable arsenate in the soils was much greater than acid extractable phosphate. Though phosphate was much more efficient at competing with arsenate for uptake, plants growing on arsenate contaminated land still accumulated considerable amounts of As. Plants from the differing habitats showed large variation in plant phosphate status, pasture plants having much higher P levels than plants growing on the most contaminated mine spoil soils. The selectivity of the phosphate-arsenate uptake system for phosphate compared with arsenate, coupled with the suppression of this uptake system enabled tolerant clones of the grass velvetgrass to grow on soils that were highly contaminated with arsenate and deficient in phosphate.

Polymorphism and physiology of arsenate tolerance in Holcus lanatus L. from an uncontaminated site

The polymorphism of arsenate tolerance in a Holcus lanatus L. population from an uncontaminated soil was investigated and a high percentage of tolerant individuals (65%) was found in the population studied. Influx of arsenate was highly correlated to arsenate tolerance within the population, with the most tolerant individuals having the lowest rates of arsenate influx. Isotherms for the high affinity arsenate uptake systems were determined in six tolerant and six non-tolerant genotypes. Tolerant plants had the lowest rates of arsenate influx. This was achieved by adaptation of the V_max of arsenate influx with the V_max of the high affinity uptake system saturating at lower substrate concentrations in the tolerant plants. The polymorphism is discussed with relation to adaptation to the extreme environments to which the plants are subjected on mine-spoil soils. © 1992 Kluwer Academic Publishers.

An Evaluation of 1D Loss Model Collections for the Off-Design Performance Prediction of an Automotive Turbocharger Compressor

Abstract. Single-zone modelling is used to assess different collections of impeller 1D loss models. Three collections of loss models have been identified in literature, and the background to each of these collections is discussed. Each collection is evaluated using three modern automotive turbocharger style centrifugal compressors; comparisons of performance for each of the collections are made. An empirical data set taken from standard hot gas stand tests for each turbocharger is used as a baseline for comparison. Compressor range is predicted in this study; impeller diffusion ratio is shown to be a useful method of predicting compressor surge in 1D, and choke is predicted using basic compressible flow theory. The compressor designer can use this as a guide to identify the most compatible collection of losses for turbocharger compressor design applications. The analysis indicates the most appropriate collection for the design of automotive turbocharger centrifugal compressors.

A numerical study of automotive turbocharger mixed flow turbine inlet geometry for off design performance

Mixed flow turbines represent a potential solution to the increasing requirement for high pressure, low velocity ratio operation in turbocharger applications. While literature exists for the use of these turbines at such operating conditions, there is a lack of detailed design guidance for defining the basic geometry of the turbine, in particular, the cone angle – the angle at which the inlet of the mixed flow turbine is inclined to the axis. This investigates the effect and interaction of such mixed flow turbine design parameters.
Computational Fluids Dynamics was initially used to investigate the performance of a modern radial turbine to create a baseline for subsequent mixed flow designs. Existing experimental data was used to validate this model.
Using the CFD model, a number of mixed flow turbine designs were investigated. These included studies varying the cone angle and the associated inlet blade angle.
The results of this analysis provide insight into the performance of a mixed flow turbine with respect to cone and inlet blade angle.

Spatial and temporal variability of the freshwater reservoir effect (FRE) in lacustrine sediments in a cold climate zone, Northwest Territories (Canada)

Climate variability along the 600 km Tibbitt to Contwyoto Winter Road (TCWR) in central Northwest Territories is poorly understood. With the transportation of goods from Yellowknife to the mines projected to increase significantly as new mines open, it is critical that planners and mine developers have reasonable data on the future viability of the road, as alternative transportation costs (e.g. air transport) are prohibitively high.

The research presented here is part of a paleoclimate study based on the analysis of multiple proxy data derived from freeze cores in lakes along the TCWR.

Fast Rule Mining over Multi-dimensional Windows

Association rule mining is an indispensable tool for discovering
insights from large databases and data warehouses.
The data in a warehouse being multi-dimensional, it is often
useful to mine rules over subsets of data defined by selections
over the dimensions. Such interactive rule mining
over multi-dimensional query windows is difficult since rule
mining is computationally expensive. Current methods using
pre-computation of frequent itemsets require counting
of some itemsets by revisiting the transaction database at
query time, which is very expensive. We develop a method
(RMW) that identifies the minimal set of itemsets to compute
and store for each cell, so that rule mining over any
query window may be performed without going back to the
transaction database. We give formal proofs that the set of
itemsets chosen by RMW is sufficient to answer any query
and also prove that it is the optimal set to be computed
for 1 dimensional queries. We demonstrate through an extensive
empirical evaluation that RMW achieves extremely
fast query response time compared to existing methods, with
only moderate overhead in pre-computation and storage

VentBase:Developing a consensus among stakeholders in the deep-sea regarding environmental impact assessment for deep-sea mining-A workshop report

Mining seafloor massive sulfides for metals is an emergent industry faced with environmental management challenges. These revolve largely around limits to our current understanding of biological variability in marine systems, a challenge common to all marine environmental management. VentBase was established as a forum where academic, commercial, governmental, and non-governmental stakeholders can develop a consensus regarding the management of exploitative activities in the deep-sea. Participants advocate a precautionary approach with the incorporation of lessons learned from coastal studies. This workshop report from VentBase encourages the standardization of sampling methodologies for deep-sea environmental impact assessment. VentBase stresses the need for the collation of spatial data and importance of datasets amenable to robust statistical analyses. VentBase supports the identification of set-asides to prevent the local extirpation of vent-endemic communities and for the post-extraction recolonization of mine sites. © 2013.

A biological survey method applied to seafloor massive sulphides (SMS) with contagiously distributed hydrothermal-vent fauna

Strategies for mitigation of seafloor massive sulphide (SMS) extraction in the deep sea include establishment of suitable reference sites that allow for studies of natural environmental variability and that can serve as sources of larvae for re-colonisation of extracted hydrothermal fields. In this study, we characterize deep-sea vent communities in Manus Basin (Bismarck Sea, Papua New Guinea) and use macrofaunal data sets from a proposed reference site (South Su) and a proposed mine site (Solwara 1) to test the hypothesis that there was no difference in macrofaunal community structure between the sites. We used dispersion weighting to adjust taxa-abundance matrices to down-weight the contribution of contagious distributions of numerically abundant taxa. Faunal assemblages of 3 habitat types defined by biogenic taxa (2 provannid snails, Alviniconcha spp. and Ifremeria nautilei; and a sessile barnacle, Eochionelasmus ohtai) were distinct from one another and from the vent peripheral assemblage, but were not differentiable from mound-to-mound within a site or between sites. Mussel and tubeworm populations at South Su but not at Solwara 1 enhance the taxonomic and habitat diversity of the proposed reference site. © Inter-Research 2012.